Nusantara Bioscience vol. 4, no, 2, July 2012

Phytofabricated silver nanoparticles by using Hydrylla verticilata photo by Neilesh Sable

| Nus Biosci | vol. 4 | no. 2 | pp. 45-96| July 2012 | | ISSN 2087-3948 | E-ISSN 2087-3956 |

| Nus Biosci | vol. 4 | no. 2 | pp. 45-96 | July 2012 | | ISSN 2087-3948 | E-ISSN 2087-3956 | I S E A

J o u r n a l

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B i o l o g i c a l

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EDITORIAL BOARD: Editor-in-Chief, Sugiyarto, Sebelas Maret University Surakarta, Indonesia (sugiyarto_ys@yahoo.com) Deputy Editor-in-Chief, Joko R. Witono, Bogor Botanical Garden, Indonesian Institute of Sciences, Bogor, Indonesia (jrwitono@yahoo.com) Editorial Advisory Boards: Agriculture, Muhammad Sarjan, Mataram University, Mataram, Indonesia (janung4@yahoo.com.au) Animal Sciences, Freddy Pattiselanno, State University of Papua, Manokwari, Indonesia (pattiselannofreddy@yahoo.com) Biochemistry and Pharmacology, Mahendra K. Rai, SGB Amravati University, Amravati, India (pmkrai@hotmail.com) Biomedical Sciences, Afiono Agung Prasetyo, Sebelas Maret University, Surakarta, Indonesia (afieagp@yahoo.com) Biophysics and Computational Biology: Iwan Yahya, Sebelas Maret University, Surakarta, Indonesia (iyahya@uns.ac.id) Ecology and Environmental Science, Cecep Kusmana, Bogor Agricultural University, Bogor, Indonesia (cecep_kusmana@ipb.ac.id) Ethnobiology, Luchman Hakim, University of Brawijaya, Malang, Indonesia (lufehakim@yahoo.com) Genetics and Evolutionary Biology, Sutarno, Sebelas Maret University, Surakarta, Indonesia (nnsutarno@yahoo.com) Hydrobiology, Gadis S. Handayani, Research Center for Limnology, Indonesian Institute of Sciences, Bogor, Indonesia (gadis@limnologi.lipi.go.id) Marine Science, Mohammed S.A. Ammar, National Institute of Oceanography, Suez, Egypt (shokry_1@yahoo.com) Microbiology, Charis Amarantini, Duta Wacana Christian University, Yogyakarta, Indonesia (charis@ukdw.ac.id) Molecular Biology, Ari Jamsari, Andalas University, Padang, Indonesia (ajamsari@yahoo.com) Physiology, Xiuyun Zhao, Huazhong Agricultural University, Wuhan, China (xiuyunzh@yahoo.com.cn) Plant Science: Pudji Widodo, General Soedirman University, Purwokerto, Indonesia (pudjiwi@yahoo.com) Management Boards: Managing Editor, Ahmad D. Setyawan, Sebelas Maret University Surakarta (unsjournals@gmail.com) Associated Editor (English Editor), Wiryono, State University of Bengkulu (wiryonogood@yahoo.com) Associated Editor (English Editor), Suranto, Sebelas Maret University Surakarta Technical Editor, Ari Pitoyo, Sebelas Maret University Surakarta (aripitoyo@yahoo.co.id) Business Manager, A. Widiastuti, Development Agency for Seed Quality Testing of Food and Horticulture Crops, Depok, Indonesia (nusbiosci@gmail.com) PUBLISHER: Society for Indonesian Biodiversity CO-PUBLISHER: School of Graduates, Sebelas Maret University Surakarta FIRST PUBLISHED: 2009 ADDRESS: Bioscience Program, School of Graduates, Sebelas Maret University Jl. Ir. Sutami 36A Surakarta 57126. Tel. & Fax.: +62-271-663375, Email: nusbiosci@gmail.com ONLINE: biosains.mipa.uns.ac.id/nusbioscience

Society for Indonesia Biodiversity

Sebelas Maret University Surakarta

ISSN: 2087-3948 E-ISSN: 2087-3956

Vol. 4, No. 2, Pp. 45-49 July 2012

Phytofabrication of silver nanoparticles by using aquatic plant Hydrilla verticilata 1

NEILESH SABLE, SWAPNIL GAIKWAD, SHITAL BONDE, ANIKET GADE, MAHENDRA RAIď‚Š

Department of Biotechnology, Sant Gadge Baba Amravati University, Amravati-444602, Maharashtra India. Tel: +91-721-2662206 to 8, Fax: +91-7212662135, 2660949, ď‚Šemail: mkrai123@rediffmail.com Manuscript received: 20 June 2012. Revision accepted: 21 July 2012.

Abstract. Sable N, Gaikwad S, Bonde S, Gade A, Rai M. 2012. Phytofabrication of silver nanoparticles by using aquatic plant Hydrilla verticilata. Nusantara Bioscience 4: 45-49. In the context of current drive to developed new green technology in nanomaterials, synthesis of nanoparticles is of considerable importance. There has been considerable work done in the field of nanoscience and Nanotechnology during the last decade due to the introduction of various protocols for the synthesis of nanoparticles by using plants and microorganisms. Here we firstly report the extracellular phytosynthesis of silver nanoparticles (Ag-NPs) using aquatic plants Hydrilla verticilata. The characterization of the phytosynthesized Ag-NPs was done with the help of UV-Vis spectroscopy, FTIR, Nanoparticle Tracking Analysis (NTA), Zeta potential and SEM. The SEM micrograph revealed the synthesis of polydispersed spherical nanoparticles, with the average size of 65.55 nm. The phytofabricated Ag-NPs can be used in the field of medicine and agriculture, due to their antimicrobial potential.

Key words: phytofabrication, Hydrilla, Ag-NPs, SEM, FTIR Abstrak. Sable N, Gaikwad S, Bonde S, Gade A, Rai M. 2012. Fitofabrikasi nanopartikel perak menggunakan tumbuhan akuatik Hydrilla verticilata. Nusantara Bioscience 4: 45-49. Dalam konteks mendorong pengembangan teknologi hijau yang baru pada nanomaterial, sintesis nanopartikel sangat penting. Selama dekade terakhir terjadi perkembangan yang cukup pesat dalam bidang nanosains dan nanoteknologi karena diperkenalkannya berbagai protokol untuk mensintesis nanopartikel menggunakan tumbuhan dan mikroorganisme. Dalam penelitian ini, dilaporkan fitosintesis ekstraseluler nanopartikel perak (Ag-NP) menggunakan tumbuhan akuatik Hydrilla verticilata untuk pertamakalinya. Karakterisasi Ag-NP yang difitosintesis dilakukan dengan bantuan spektroskopi UV-Vis, FTIR, Analisis Pelacakan Nanopartikel (NTA), potensial Zeta dan SEM. Mikrograf SEM menunjukkan hasil sintesis nanopartikel berbentuk bulat yang tersebar, dengan ukuran rata-rata 65,55 nm. Fitofabrikasi Ag-NP dapat dimanfaatkan dalam bidang kedokteran dan pertanian, karena memiliki potensi antimikroba.

Kata kunci: fitofabrikasi, Hydrilla, Ag-NPs, SEM, FTIR

INTRODUCTION Nanotechnology is a relatively recent development in scientific research, the development of its central concepts happened over last decades. The development of experimental procedures for the synthesis of nanoparticles of different chemical compositions, sizes, shapes, and controlled polydispersity is vital for its advancement.

Currently, there is an ever-increasing need to develop environmentally benign processes in the field of nanoparticle synthesis, therefore focusing attention on biological systems. Nanobiotechnology is combination between nanotechnology and biology and which refers to the ability to create and manipulate biological and biochemical materials, devices, and systems at nano level (Kholoud et al. 2010).

Different microorganisms such as bacteria, fungi, and yeasts can be used as nanofactories for the biosynthesis of nanoparticles. It has been shown that fungi are good candidates for synthesis of metal and metal sulphides nanoparticles, near about 20 different fungi has been

investigated for the synthesis of metal nanoparticles.

Verticillum sp. reduces metal ions into Au and Ag nanoparticles (Mukherjee et al. 2002). Fusarium oxysporum produces high stable gold, silver and platinum nanoparticles (Mukherjee et al. 2002, Riddin et al. 2006).

Other reports of nanoparticles synthesis by fungi includes

by Aspergillus niger (Gade et al. 2008), Fusarium acuminatum (Ingle et al. 2009).

But, plants as a system for synthesis of nanoparticles are rapid and eco-friendly biosynthesis process. Plants are used to synthesize Nanoparticles either intracellularly or extracellularly (Bonde et al. 2012). Living plants (Torresdey et al. 2002, 2003) are used for synthesis of gold and silver nanoparticles, part of a plant like from geranium leaf broth (Shivshankar et al. 2003, 2004, 2005) or by fruits (Li et al. 2007) or even by sundried leaves (Huang et al.

2007).The rapid synthesis of silver nanoparticles by using different plant extracts of Pinus, Persimmon, Ginkgo, Magnolia and Platanus were used and compared for their extracellular metallic Ag-NPS synthesis (Song et al. 2008) and the other reports of utilization of plant for the synthesis

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of metal nanoparticle includes; Azadirachta indica (neem) (Shankar et al. 2004), Aloe vera (Chandran et al. 2006), Emblica officinalis (amla). (Amkamwar et al. 2005), Capsicum annuum (Li et al. 2007), Cinnamomum camphora (Huang et al. 2007), Gliricidia sepium Jacq.

(Raut et al. 2009), Carrica papaya (Mude et al. 2009), Opuntia ficus-indica (Gade et al. 2010), Murraya koenigii (Bonde et al. 2012), Ocimum sanctum (Mallikarjum et al.

2011), Saururus chinenis (Nagajyoti et al. 2011), Foeniculum vulgare (Bonde 2011).

The phytofabrication (fabrication by plants) of Ag-NPs from plant extracts has received some attention as a simple and viable alternative to bacterial and fungal system, also metal ions reduces much faster using plant system as compare to microbes (Rai et al. 2008). The reduction of metal ions is known to using enzyme extracted from the plant extract, owing to this property the plant is selected for bioreduction of silver ions in present study.

In the present study we have for the first time exploited aquatic plants for the synthesis of Ag-NPs. The aquatic weed Hydrilla verticilata was used for the synthesis of AgNPs using 1mM silver nitrate (AgNO3). The characterization of the phytofabricated Ag-NPs were carried out with the help of UV-Vis spectroscopy, FTIR, NTA, Zeta Potential and SEM. MATERIAL AND METHODS Extraction The 20 g Hydrilla verticilata (Figure 1) plant part was washed 2-3 times with sterilized distilled water to avoid any microbial contamination, and then surface sterilized by HgCl2 (0.1%) for 1 min, cut into small pieces and ground with 100 mL of sterilized distilled water in an omnimixer. Later, crude extract was filtered through muslin cloth and centrifuged at 10,000 g for 15 min to obtain clear leaf extract which was later used for the fabrication of AgNPs.

temperature for 24 hrs the colour of filtrate changes from light green to dark brown. This colour change indicates the formation Ag-NPs. UV-Visible Spectroscopy The preliminary detection of Ag-NPs was done with the help of UV-Visible spectrophotometer (Perkin-Elmer, Lambda 25) by scanning the absorbance spectra in the range of 250-800 nm wavelengths.

Fourier Transform Spectroscopy FTIR measurements of Ag-NPs synthesized from Hydrilla verticilata was carried out on a Perkin-Elmer FTIR Spectrophotometer in the range 450- 4000 cm-1 at resolution of 4cm-1. Scanning electron micrographs were taken using a JEOL 6380A instrument. The samples were fixed with 2.5% glutaraldehyde overnight at room temperature. The dehydration of fixed samples were carried out with gradient alcohol (10% to 95%), incubated for 20 min in each gradient and dipped in absolute alcohol for 2-5 min. The final specimen was prepared by placing a drop of dehydrated sample on a glass slide followed by coating with monolayer platinum for making the surface conducting.

NanoSight LM-20 analysis Liquid sample of Ag-NPs at the concentration range of 107-109/mL were introduced into a scattering cell through which a laser beam (approx. 40 mW at k = 635 nm) was passed. Particles present within the path of the laser beam were observed via a dedicated non- microscope optical instrument (LM-20, NanoSight Pvt. Ltd., UK) having CCD

Fabrication of Ag-NPs For the fabrication of Ag-NPs extract was challenged with AgNO3 (1 mM) solution and incubated at room temperature. Control (without treatment with AgNO3) (1 mM) i.e only extract) was also maintained.

Detection of Ag-NPs Visual observation In conical flask 99 mL of plant filtrate was taken and 1 mL of AgNO3 (100 mM) was added into it (final concentration becomes 1 mM).

After incubation of filtrate at room

Figure 1. Hydrilla verticilata plant

SABLE et al. – Synthesis of silver nanoparticles by using Hydrilla verticilata

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Figure 2. Control (left) and Ag-NPs (right) fabricated from Hydrilla verticilata Figure 3. UV-Vis spectra of (A) leaf extract (control) and (B) Ag-NPs showing absorbance at about 428 nm.

Figure 4. FTIR spectrum for extract (control) and experimental after treatment with 1mM silver nitrate solution.

5A

5B

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Figure 5. A. Particle size/concentration of Ag-NPs, B. Particle populations of Ag-NPs using NanoSight LM-20 Figure 6. Particle size distribution of Ag-NPs by intensity with Zeta Analyzer.

7A

7B

Figure 7. A. SEM micrograph of Ag-NPs (65.55 nm) (scale bar-100nm). B. A particle size distribution determined from the SEM images.

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camera. The motion of the particles in the field of view (approx. 100 X 100 µm) was recorded (at 30 fps) and the subsequent video and images were analyzed.

Particle size measurement Particle sizing experiments were carried out by means of laser diffractometry, using Zetasizer nano series (Malvern). Measurements were taken in the range range between 0.1-1000µm.

Scanning electron microscopy Scanning electron microscopy of Ag-NPs was carried out by fixation of 2.5% glutaraldehyde overnight at room temperature. Then cell filtrate was dehydrated with gradient alcohol (10% to 95%) and incubated for 20 min. for each gradient. It was dipped in absolute alcohol for 2-5 min. A drop of dehydrated sample placed on glass slide (1 cm x 1 cm ). The sample was coated with monolayer platinum. The slide was observed under scanning electron microscope.

RESULTS AND DISCUSSION The change in colour of plant extract from light green to dark brown when challenged against silver ions (1 mM AgNO3) at room temperature. The colour change in the extract was noted by visual observation (Figure 2). The characterization of Ag-NPs fabrication was done by using UV-visible spectrophotometer which confirms the presence of the absorbance peak at 428 nm (Figure 3).

Further characterization was done by Fourier Transform Infrared Spectroscopy (FTIR) measurements to identify the possible biomolecules responsible for the reduction of the Ag+ ions and capping of the bioreduced Ag-NPs by protein.

The amide linkages between amino acid residues in proteins give rise to the well-known signatures in the infrared region of the electromagnetic spectrum (Basavaraja et al. 2007). FTIR spectrum showed peaks in the range 1000-2000cm-1. Representative spectra of obtained nanoparticles manifest absorption peaks of respective functional groups and indicated the presence of stabilized protein molecules (Figure 4).

Nanoparticle Tracking and Analysis (NTA) was used to measure the dispersion characteristics i.e. size and size distribution. In particular, it is the most recently developed system, NTA, was assessed in-depth due to its ability to see and size of particles individually on a particle-by-particle basis. NTA allows individual nanoparticles in a suspension to be microscopically visualized and their brownian motion to be separately but simultaneously analyzed and from which the particle size distribution can be obtained on a particle-by-particle basis (Figure 5A). The Figure 5b showed particle populations by size and intensity. The distribution data were mean 64 nm, mode 21 nm and standard deviation 43 nm. This result corroborates the results obtained by Montes-Burgos et al. 2010.

Particle size determination of the formulated nanoparticles was shown under different categories like

size distribution by volume, by intensity (Figure 6). The average zeta potential of peak was found to be -40.1 mV, area 100% and width 8.51 mV. The formed Ag-NPs are well distributed with respect to volume and intensity is an indication of the formation of well built Ag-NPs and their monodispersity.

Scanning Electron Microscopy (SEM) study reveals the synthesis of spherical polydispersed Ag-NPs in the reaction mixtur e, which showed the spherical nanoparticles of size of 65.55 nm (Figure 7A). The particle size histogram showed average size of Ag-NPs (Figure 7B). Ag-NPs analyzed in NTA and scanning electron microscopy corroborates in their size.

CONCLUSION It has been demonstrated that the extract of plant Hydrilla verticilata is capable of fabricating Ag-NPs and these Ag-NPs are quite stable in solution due to capping likely by the proteins present in the extract. This is an efficient, eco-friendly and simple process and more efficient with appreciable control over size, composition and even the shape of the nanoparticles. Ag-NPs have more applications as antimicrobial agents.

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Nagajyoti PC, Prasad TN, Shreekanth VM, Lee KD. 2011. Biofabrication of silver nanoparticles using leaf of Saururus chinenis. Digest J Nanomat Biostruct 6 (1): 121-133.

Rai M, Yadav A, Gade A. 2008. Current trends in phytosynthesis of metal nanoparticles. Crit Rev Biotechnol 28(4):277-284.

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Phytosynthesis of silver nanoparticle using Gliricidia sepium (Jacq.).

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ISSN: 2087-3948 E-ISSN: 2087-3956

Vol. 4, No. 2, Pp. 50-56 July 2012

Antibacterial activity of Thymus vulgaris essential oil alone and in combination with other essential oils 1

KATERYNA KON1,, MAHENDRA RAI2

Department of Microbiology, Virology, and Immunology, Kharkiv National Medical University, 61022, Pr. Lenina, 4, Kharkiv, Ukraine. Tel. +380507174771, e-mail: katerynakon@gmail.com 2 Department of Biotechnology, Sant Gadge Baba Amravati University, Amravati-444602, Maharashtra India Manuscript received: 16 July 2012. Revision accepted: 31 July 2012.

Abstract. Kon K, Rai M. 2012. Antibacterial activity of Thymus vulgaris essential oil alone and in combination with other essential oils.

Nusantara Bioscience 4: 50-56. Essential oils (EOs) from plants represent an alternative approach in combating antibiotic-resistant bacteria. One of the EOs with proven antibacterial properties is Thymus vulgaris EO. The purpose of the present work was to investigate in vitro antibacterial activity of T. vulgaris EO alone and in combination with other EOs. The activity of T. vulgaris EO was screened in combination with 34 EOs against Staphylococcus aureus and Escherichia coli by disk diffusion method; then the most effective combinations were evaluated by broth microdilution method. Against S. aureus the synergistic effect was found in combination of T. vulgaris and Cinnamomum zeylonicum EOs with fractional inhibitory concentration (FIC) index of 0.26; Juniperus communis and Picea abies EOs showed additive effect (FIC indexes were 0.74 and 0.78, respectively). Combination of T. vulgaris EO with Aniba rosaeodora and Melissa officinalis EOs demonstrated synergistic effect against E. coli (FIC indexes were 0.23 and 0.34, respectively); combination of T. vulgaris and Mentha piperita EOs was additive (FIC index 0.55). Therefore, combining T. vulgaris EO with other EOs has potential in further enhancing its antibacterial properties.

Key words: Thymus vulgaris, essential oils, combinations, Staphylococcus aureus, Escherichia coli.

Abstrak. Kon K, Rai M. 2012. Aktivitas antibakteri minyak atsiri Thymus vulgaris tunggal atau campuran dengan minyak atsiri lain.

Bioscience Nusantara 4: 50-56. Minyak atsiri tumbuhan merupakan senyawa alternatif untuk melawan bakteri resisten antibiotik. Salah satu minyak atsiri yang terbukti bersifat antibakteri adalah minyak atsiri Thymus vulgaris. Penelitian ini bertujuan untuk mengetahui aktivitas in vitro antibakteri minyak atsiri T. vulgaris tunggal atau campuran dengan minyak atsiri lain. Aktivitas antibakteri minyak atsiri T. vulgaris dan campurannya dengan 34 minyak atsiri lain terhadap Staphylococcus aureus dan Escherichia coli ditapis dengan metode cawan difusi, kemudian campuran yang paling efektif diuji dengan metode mikrodilusi kaldu. Efek sinergis terhadap S. aureus ditemukan pada campuran antara minyak atsiri T. vulgaris dan Cinnamomum zeylonicum dengan indeks konsentrasi hambat fraksional (FIC) 0,26; minyak atsiri Juniperus communis dan Picea abies menunjukkan efek aditif (indeks FIC masing-masing adalah 0,74 dan 0,78). Campuran minyak atsiri T. vulgaris dengan Aniba rosaeodora dan Melissa officinalis menunjukkan efek sinergis terhadap E. coli (indeks FIC masing-masing adalah 0,23 dan 0,34); campuran minyak atsiri T. vulgaris dengan Mentha piperita menunjukkan efek aditif (indeks FIC 0,55). Oleh karena itu, campuran minyak atsiri T. vulgaris dengan minyak atsiri lainnya memiliki potensi untuk meningkatkan sifat antibakteri.

Kata kunci: Timus vulgaris, minyak atsiri, kombinasi, Staphylococcus aureus, Escherichia coli

INTRODUCTION Wide spread of antibiotic resistance remains a serious clinical problem, which stimulates studies for search of new methods for coping with drug resistance or renews interest in traditionally used and forgotten methods, such as treatment with antibacterial plant extracts and essential oils (EOs) (Ríos and Recio 2005; Fisher and Phillips 2009).

Combined therapy is traditionally used to increase antimicrobial activity and reduce toxic effects of agents (Houghton 2009).

Thyme plant is used since ancient times to achieve healing, antiseptic fumigator, food preservation and other useful effects (Stahl-Biskup and Sáez 2002). Nowadays, Thymus vulgaris EO belongs to EOs with the most

pronounced antimicrobial activity (Iten et al. 2009). It was shown to be active against many bacteria, viruses and fungi. High antimicrobial activity of thyme oil and its components, first of all thymol and carvacrol, was demonstrated against Staphylococcus aureus (Al-Bayati 2008; Soković et al. 2010; Lević et al. 2011), including methicillin-resistant isolates (Tohidpour et al. 2010), S. epidermidis (Soković et al. 2010), Enterococcus faecalis (Lević et al. 2011), Bacillus cereus (Al-Bayati, 2008), Vibrio cholerae (Rattanachaikunsopon and Phumkhachorn, 2010), Escherichia coli (Lević et al. 2011), Proteus mirabilis (Soković et al. 2010; Lević et al. 2011), P. vulgaris (Al-Bayati, 2008), Pseudomonas aeruginosa (Soković et al. 2010), Salmonella enteritidis (Soković et al.

2010), S. choleraesuis (Lević et al. 2011), S. typhimurium

KON & RAI – Antibacterial activity of Thymus vulgaris essential oil

(Soković et al. 2010), and other microorganisms.

In spite of many studies devoted to thyme oil, its combinations with other EOs have not been paid much attention. Gutierrez et al. (2009) studied combinations composed of thyme and oregano EOs against B. cereus, E. coli, Listeria monocytogenes and P. aeruginosa by checkerboard method and found that thyme-oregano EO combination had additive effect against B. cereus and P. aeruginosa, and indifferent effect against E. coli and L. monocytogenes. Furthermore, against L. monocytogenes the authors studied five more thyme EO combinations – with basil, lemon balm, marjoram, rosemary, and sage EOs. The results showed that basil, rosemary and sage EOs with thyme oil had additive effect, while lemon balm and marjoram EOs were indifferent.

The analysis of available literature shows that EO combinations, including combinations with thyme EO, represent perspective approach in antimicrobial treatment and prevention, however, in contrast to combinations of traditional antibiotics, this topic is not still well studied and requires further investigations.

The main goal of the present study was to investigate antimicrobial activity of thyme EO in combination with different EOs against S. aureus and E. coli.

MATERIALS AND METHODS Essential oils. We used commercial EO of Thymus vulgaris (purchased from NPF Zarstvo Aromatov, Sudak, Ukraine) and 34 different EOs (purchased from Aroma Inter, Mykolaiv, Ukraine; Aromatika, Kiyiv, Ukraine; NPF Zarstvo Aromatov, Sudak, Ukraine) (Table 1).

Strains, preparation of inocula. We used reference strains Staphylococcus aureus (ATCC 25923) and Escherichia coli (ATCC 25922). The cultures of bacteria were maintained in meat peptone agar slants at 4°C throughout the study and used as stock cultures. For preparation of inocula, cultures were grown until logarithmic phase, and then bacterial density was adjusted to approximately 108 colony forming units (CFU) per mL for disk diffusion method and 105 CFU/mL for microdilution method with sterile saline solution. Bacterial counts were confirmed by plating out on meat-peptone agar, plates were incubated at 37°C for 24 h.

Disk diffusion method. This method was used for the screening of EOs for increase of antibacterial activity in the presence of thyme oil. Bacterial suspension was spread over the plates 85 mm in diameter containing MuellerHinton agar using a sterile cotton swab in three directions in order to get a uniform microbial growth. Under aseptic conditions empty sterile disks were impregnated with 5 μl of EO. Disks were left for 5 min at room temperature for better oil absorption and were then placed on inoculated agar surface. A standard disc with ciprofloxacin (10 μg/disc) was used as a reference control. The Petri dishes were left for 30 min at room temperature (20-22°C) for better oil diffusion and were then placed to an incubator at 37°C for 24 h. After an incubation period diameters of inhibition zones around the disks with EOs were measured.

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We assessed diameter of inhibition zones around the disks with EOs mixtures. For this purpose, we prepared blends of EOs in sterile eppendorf tubes by mixing 50 µl of thyme oil with 50 µl of correspondent second oil. Paper disks were then impregnated with 5 µl of appropriate mixture of EOs. Results of disk diffusion assay for study of EO mixture were assessed by comparing the experimental inhibition zone area of oils mixture with theoretical inhibition zone area of indifferent combinatory effect (calculated as ½ of inhibition zone area for thyme oil + ½ of inhibition zone area for the second oil).

Minimal inhibitory concentration (MIC) test. We prepared serial doubling dilutions of each plant EO in 96well microtiter plates in volume 50 µL of Muellor Hinton Broth to give a range of concentrations from 0.0025% to 5% (volume/volume). After preparations of suspension of tested cultures 50 µL were added to oil dilutions to produce total volume of 100 µL. The resulting suspensions were then mixed with a micro-pipettor. Two controls were used: positive (50 µL of medium and 50 µL of culture), and negative (100 µL of medium). All microtiter plates with microorganisms were incubated at 37°C for 24 h. Inhibition of bacterial growth in the wells containing test oil was judged by comparison with growth in negative control well. The MICs were determined by measuring optical density at 570 nm and defined as the concentration of oil at which there was a sharp decline in the absorbance value.

MICs determination of mixtures of EOs. Mixture of thyme and different EOs in ratios 1:1 were tested for determinations of MICs by broth microdilution method.

In order to assess results of MICs of EOs in mixtures we calculated fractional inhibitory concentrations (FIC) with FIC indexes (Houghton 2005). Because mixtures were used in ratio 1:1, individual MIC of EO in blend was calculated as ½ of MIC of blend. Accordingly to this, FIC indexes were calculated as the following: FIC of thyme oil = (1/2 MIC of blend)/ (MIC of thyme oil alone); FIC of second oils = (1/2 MIC of blend)/ (MIC of second oil alone); FIC index = (FIC of thyme oil) + (FIC of second oil), where second oil is the EO which was tested in combination with thyme oil.

FIC indexes were interpreted as following: synergy, FIC < 0.5; addition, 0.5≤FIC≤1; indifference, 1<FIC≤4; antagonism, FIC>4 (Gutierrez et al. 2009).

Chemical composition. The main components of EOs were identified by mass-spectrometry analysis. The relative amount of individual components of the total oil was expressed as percentage peak area relative to total peak area. Qualitative identification of the constituents was performed by comparison of their relative retention times and mass spectra with those stored in NIST library or with mass spectra from literature (Stein et al., 2002).

Statistical analysis of data. All experiments were repeated in triplicates, and then mean values for diameters of inhibition zones, geometric mean MICs and accordingly to them FICs were calculated. Results were analysed using statistical software SPSS (version 20.0). The results are expressed as mean value ± standard deviation or as

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geometric mean. Comparison of groups was performed by U test Mann-Whitney and Kruskal-Wallis 1-way analysis of variance (ANOVA); differences were considered as statistically significant at p<0.05.

RESULTS AND DISCUSSION Antibacterial activity of essential oils alone The antibacterial activity of thyme oil and 34 EOs is summarized in the Table 1. The results proved that thyme EO had significant activity against S. aureus and E. coli with diameters of inhibition zones 22.74±1.56 mm and 22.46±5.48 mm, respectively. Furthermore, the majority of EOs possessed antimicrobial activity, but in very wide ranges. In general, activity of EOs was higher against S. aureus than against E. coli.

Multivariate analysis showed presence of significant differences between activity of EOs from different plant families (p=0.036). The highest activity against both tested strains was demonstrated by EOs of plants from Lamiaceae family with the mean inhibition zone 21.7±17.0 mm against S. aureus and 13.2±10.3 mm against E. coli. Rather high activity was also present in Lauraceae plant EOs against S. aureus (13.7±10.0 mm) and Myrtaceae plant EOs against E. coli (12.4±6.2 mm). Activity of Pinaceae and Rutaceae plant EOs against both strains was rather low.

S. aureus did not show any sensitivity to two EOs – eucalyptus and lemon. We found weak activity in juniper berry, rosemary, silver fir, grapefruit, pontica wormwood, and camphor white EOs. High antistaphylococcal activity was found in lavender, ylang-ylang, clary sage, clove, cedarwood, geranium, and especially in cinnamon EO. Against E. coli total absence of activity was noticed in eight EOs: calamus, camphor white, cedarwood, juniper berry, patchouli, sandalwood, Satsuma mandarin, and silver fir. Seven more EOs showed very weak antimicrobial activity with diameter of inhibition zone not exceeding 7 mm: thuja, bitter orange, grapefruit, lime, bay laurel, ylangylang and dill. Interestingly, among these EOs without antimicrobial effect against E. coli some EOs possessed high activity against S. aureus, such as cedarwood, which did not inhibit growth of E. coli but had inhibition zone against S. aureus 28.4±14.1 mm; ylang-ylang EO had inhibition zones 7.0±0.9 mm against E. coli and 21.7±8.0 mm against S. aureus; patchouli and sandalwood EOs also did not inhibit growth of E. coli but had inhibition zones against S. aureus 16.9±2.8 mm and 15.3±5.1 mm, respectively.

Along with high activity of thyme EO against E. coli, high sensitivity of this strain was also shown only to two more EOs – clove and cinnamon (diameters of inhibition zones were 22.0±1.8 mm and 37.4±4.0 mm, respectively).

Moderate level of activity against E. coli was demonstrated by lemon balm, peppermint and tea tree EOs with diameters of inhibition zones 10.4±1.3 mm, 10.8±1.3 mm, and 15.0±1.6 mm, respectively.

Twenty one of 35 studied EOs had significant differences in antibacterial activity against S. aureus and E.

coli, and 17 of these oils (basil, clary sage, lavender, patchouli, bay laurel, camphor white, cedarwood, silver fir, bitter orange, lime, Satsuma mandarin, calamus, dill, geranium, sandalwood, thuja, and ylang-ylang) had higher activity against S. aureus. Interestingly, peppermint, eucalyptus, tea tree and lemon EOs were more active against E. coli. Such differences in spectrum of antibacterial activity may be a good basis for further assessment of combinations between EOs. Antibacterial activity of essential oils in combination with thyme oil: results of disk diffusion method EOs exhibited wide range of interaction effects with thyme oil from strong antagonism to strong synergism against both tested strains. In general, enhancing effect with thyme EO was more noticeable against S. aureus than against E. coli: mean change of inhibition zone areas compared with theoretical area of indifferent interaction was (32.3±60.0)% against S. aureus, while against E. coli it was (-13.5±42.5)% (p < 0.001). Therefore, against S. aureus, in general, interactions between thyme and other EOs were synergistic, while against E. coli – antagonistic.

Compared with EOs alone, in combination with thyme oil a smaller number of EOs demonstrated significant differences in activity against tested strains: 14 EOs (basil, clary sage, lemon balm, patchouli, cedarwood, clove, siberian cedar, neroli, Satsuma mandarin, geranium, pontica wormwood, sandalwood, thuja, and ylang-ylang) were significantly more active against S. aureus than against E. coli. EOs, which alone were significantly more active against E. coli (peppermint, eucalyptus, tea tree and lemon), in combination with thyme oil demonstrated equal activity against both strains.

Against S. aureus the highest level of enhancing effect by using disk diffusion method was detected in Norway spruce EO: diameter of zone inhibition was changed from 8.6±1.5 mm without thyme oil to 32.1±13.7 mm in the mixture with thyme oil. Therefore, area of inhibition zone of mixture of thyme and Norway spruce oils was bigger than theoretical area of indifferent combination by 275.4%.

High enhancing effect with thyme oil was also characteristic for juniper berry EO (Figure 1). Interestingly, that with almost absent antibacterial activity alone, in combination with thyme oil inhibition zone area increased by 145.1% compared with theoretical area of indifferent interaction. Significant enhancing effect with thyme oil was also demonstrated by thuja oil (inhibition zone area increased by 95.2%), clove (93.5%), cinnamon (77.0%), and Siberian cedar EOs (76.2%). It is worth to mention that eucalyptus and lemon EOs, which did not show antibacterial activity, in combination with thyme oil demonstrated noticeable increase in inhibition zone areas – by 55.1% and 56.1% respectively. Near 50% increase in inhibition areas was also found in lavender and lemon balm oils combined with thyme EO. Among 34 studied EOs 9 had antagonistic interactions with thyme oil: bay laurel, bitter orange, peppermint, camphor white, patchouli, silver fir, myrtle, rosemary, and especially calamus EO.

KON & RAI – Antibacterial activity of Thymus vulgaris essential oil

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Table 1. Diameters of inhibition zones of essential oils alone and in mixture with thyme oil

Diameter of inhibition zone alone (Mean±SD)

Essential oils

Fold increase (%) Diameter of inhibition zone of inhibition area in combination with thyme comparing with oil (Mean±SD) theoretical area of indifference S. aureus E. coli p S. aureus E. coli 26.7±16.0 16.7±6.3 0.07 20.3±5.6 9.4±1.0 0.05 2.3% -54.9% 64.4±6.6 29.9±6.9 0.08 77.0% -9.7% 26.4±1.0 16.1±0.9 0.05 27.0% 36.4% 27.7±15.6 12.3±0.8 0.13 50.1% -60.6% 25.0±4.9 18.6±1.8 0.05 50.7% 65.9% 18.3±1.8 12.4±1.2 0.05 -20.8% -38.8%* 16.5±6.4 18.9±1.0 0.51 -12.0% 67.5% 14.6±3.5 15.7±2.3 0.83 -29.8% -35.5%

English name Latin name S. aureus E. coli p 21.7±17.0 13.2±10.3 0.15 Lamiaceae Basil Ocimum basilicum 15.8±3.0 8.9±0.6 0.05 Cinnamon Cinnamomum zeylonicum 64.2±2.3 37.4±4.0 0.08 Clary sage Salvia sclarea 23.3±6.7 8.4±0.4 0.05 Lavender Lavandula anqustifolia 21.5±19.5 7.2±0.1 0.05 Lemon balm Melissa officinalis 16.4±8.2 10.4±1.3 0.13 Patchouli Pogostemon patchoulу 16.9±2.8 0.04 Peppermint Mentha piperita 7.7±0.5 10.8±1.3 0.05 Rosemary Rosmarinus officinalis 7.0±0.3 7.4±0.5 0.28 Thyme Thymus vulgaris 22.7±1.6 22.5±5.5 0.85 13.7±10.0 7.1±1.6 0.08 21.0±6.6 17.8±5.3 0.25 Lauraceae Bay laurel Laurus nobilis 10.9±1.6 6.9±0.5 0.05 17.6±3.5 16.2±3.1 0.83 -8.5% Camphor white Cinnamomum camphora 7.4±0.4 0.03 16.2±4.2 17.4±5.9 0.83 -14.8% Cedarwood Juniperus virginiana 28.4±14.1 0.03 30.6±8.8 12.6±1.0 0.05 36.9% Rosewood Aniba rosaeodora 7.9±0.8 9.5±3.4 0.51 19.4±3.4 25.2±5.2 0.28 21.5% 10.8±7.7 12.4±6.2 0.35 21.0±6.6 16.3±1.8 0.12 Myrtaceae Cajuput Melaleuca cajeputi 7.7±0.9 8.1±0.6 0.28 17.4±2.9 14.7±1.2 0.13 -2.0% Clove Eugenia caryophyllata 24.6±7.2 22.0±1.8 0.83 32.3±6.4 16.7±2.9 0.05 93.5% Eucalyptus Evcalyptus globulus 6.0±0.0 9.5±0.7 0.04 19.9±4.6 17.9±0.2 0.51 55.1%* Myrtle Myrtus communis 7.8±2.1 7.3±0.4 0.83 15.4±5.7 14.1±1.9 0.83 -23.4% Tea tree Melaleuca alternifolia 8±1.0 15.0±1.6 0.05 20.0±4.3 17.9±0.2 0.51 47.6% 8.2±1.0 6.8±0.7 0.13 23.7±8.4 15.0±1.2 0.13 Pinaceae Norway spruce Picea abies 8.6±1.5 7.5±1.1 0.48 32.1±13.7 16.1±0.5 0.13 275.4% Siberian cedar Pinus sibirica 8.9±0.9 7.4±1.4 0.28 23.7±4.9 15.1±1.5 0.05 76.2% Silver fir Abies sibirica 7.1±0.7 0.04 15.3±2.1 13.7±1.6 0.51 -22.9% 8.3±1.8 7.4±1.5 0.30 19.0±1.3 15.7±1.8 0.01 Rutaceae Bitter orange Citrus aurantium (fruits) 8.2±0.1 6.6±1.0 0.05 16.9±1.0 16.4±0.3 0.83 -8.7% Grapefruit Citrus paradisi 7.2±0.2 6.6±1.0 0.51 18.0±4.9 12.7±2.8 0.28 5.4% Lemon Citrus limon 6.0±0.0 8.8±0.5 0.04 20.0±5.3 18.3±0.5 0.51 56.1%* Lime Citrus aurantifolia 10.0±1.2 6.8±0.7 0.05 18.9±3.0 15.8±2.3 0.28 8.5% Neroli C. aurantium (flowers) 10.6±2.9 9.8±1.0 0.83 20.1±1.5 15.8±2.2 0.05 20.3% Satsuma mandarin Citrus unshiu 7.7±0.5 0.04 19.9±0.3 15.2±4.2 0.05 28.3% Other Calamus Acorus calamus (Araceae) 13.1±3.3 0.04 13.1±2.7 9.3±2.5 0.28 -53.4% Dill Anethum graveolens (Apiaceae) 9.1±0.7 7.0±0.8 0.05 18.4±5.1 16.8±3.6 0.83 5.3% Geranium Pelargonium roseum (Geraniaceae) 29.2±5.6 8.3±0.5 0.05 25.7±2.5 14.1±1.2 0.05 -1.0% Juniper berry Juniperus communis (Cupressaceae) 6.7±0.6 0.12 25.3±4.6 20.4±5.9 0.28 145.1% Pontica wormwood Artemisia pontica (Asteraceae) 7.3±0.6 7.9±0.6 0.27 21.2±3.0 13.5±0.8 0.05 46.2% Sandalwood Santalum album (Santalaceae) 15.3±5.1 0.04 21.6±8.1 10.5±0.6 0.05 31.6% Thuja Thuja occidentalis (Cupressaceae) 9.7±1.6 6.5±0.8 0.05 25.2±1.2 13.7±1.1 0.05 95.2% Ylang-ylang Cananga odorata (Annonaceae) 21.7±8.0 7.0±0.9 0.05 26.7±6.9 11.5±1.6 0.05 38.4% Control Ciprofloxacin 28.8±1.7 38.7±0.2 Note: * In the absence of bacterial growth inhibition zones, the disks’ diameters (6 mm) were used to calculate fold increase, %

1.A

1.B

2.A

1.3% -19.1%* -37.2%* 128.6% -44.3% -47.3% -4.5% -23.9% -20.2% -17.2% 26.1% -49.4%* 5.8% -36.5% 2.3% -3.0% -11.4% -8.2%* -76.8%* 9.6% -48.9% 12.0%* -2.0% -64.4%* -50.2% -48.9%

2.B

Figure 1. Inhibition zones around the disk with juniper berry essential oil alone (left) and mixture of juniper berry and thyme essential oils (right) (A); inhibition zone around the disk with thyme essential oil alone (B) against Staphylococcus aureus Figure 2. Inhibition zones around the disk with rosewood essential oil alone (left) and mixture of rosewood and thyme essential oils (right) (A); inhibition zone around the disk with thyme essential oil alone (B) against Escherichia coli

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Against E. coli rosewood EO showed significant enhancing effect in combination with thyme oil (Figure 2) – inhibition zone area increased by 128.6% compared with theoretical area of indifferent interaction. High enhancing effect with thyme oil was also demonstrated by peppermint and lemon balm EOs: zones of inhibition increased by 67.5% and 65.9%, respectively. Several more EOs (clary sage, Siberian cedar, juniper berry, dill, and bitter orange) had some enhancing effect in ranges from 36.4% for clary sage to 5.8% for bitter orange EO. Eucalyptus, lime, pontica wormwood, bay laurel and lemon EOs were indifferent to the presence of thyme oil, while majority of EOs (21 of 34) exhibited antagonistic interactions with thyme oil from mild (decrease of inhibition zone by 9.7% for cinnamon oil) to strong antagonism in lavender, sandalwood and calamus EOs (zones of inhibition decreased by 60.6%, 64.4%, and 76.8%, respectively).

Interestingly, that calamus EO showed significant antagonistic effect with thyme oil against both tested strains, furthermore, antagonism was more noticeable against E. coli: decrease of inhibition zone area was 76.8% against E. coli and 53.4% against S. aureus.

Antibacterial activity of essential oils in combination with thyme oil: results of microdilution method For several EOs which showed high synergistic effect with thyme oil in disk diffusion method, we determined MICs alone and in mixture with thyme oil (Tables 2 and 3).

Table 2. Susceptibility of Staphylococcus aureus to essential oils alone and in blends

EOs Thyme Norway spruce Juniper berry Cinnamon

Geometric mean minimal Fractional inhibitory concentrations, % inhibitory (mg/mL) concentration In blend with Alone index thyme oil (1:1) 0.4 (4.0) 1.3 (11.2) 0.5 (4.5) 0.78 10.0 (86.7) 0.6 (5.5) 0.74 0.02 (0.2) 0.01 (0.1) 0.26

Table 3. Susceptibility of Escherichia coli to essential oils alone and in blends

EOs Thyme Peppermint Rosewood Lemon balm

Geometric mean minimal Fractional inhibitory concentrations, % inhibitory (mg/mL) concentration In blend with Alone index thyme oil (1:1) 0.3 (2.8) 3.2 (28.5) 0.3 (2.7) 0.55 0.4 (3.3) 0.1 (0.7) 0.23 10.0 (91.4) 0.2 (1.8) 0.34

The microdilution method demonstrated general agreement with disk diffusion method. Thyme EO showed high activity against both tested strains: MIC was 4.0 mg/mL against S. aureus and 2.8 mg/mL against E. coli (p = 0.884, so differences between susceptibility of S. aureus

and E. coli are not statistically significant). Among activity of three studied EO combinations against S. aureus the most active was cinnamon EO alone with MIC 0.2 mg/mL and cinnamon-thyme EO combination with MIC 0.1 mg/mL. This combination also demonstrated the highest synergistic effect with FIC index of 0.26. Norway spruce EO alone was less active than cinnamon oil; juniper berry EO alone inhibited S. aureus only at high concentration: MICs were 11.2 mg/mL for Norway spruce and 86.7 mg/mL for juniper berry EOs. However in combination with thyme oil activity was higher and MICs of these oils achieved 4.5 and 5.5 mg/mL, respectively. But, in general, interactions with thyme oil were additive: FIC indexes were 0.8 for Norway spruce and 0.7 for juniper berry EOs. In combination with thyme oil against E. coli the best synergistic effect was demonstrated by rosewood EO: FIC index was 0.2 and final MIC of combination was 0.7 mg/mL. Lemon balm EO also showed synergistic effect with thyme oil and high activity against E. coli: FIC index was 0.3 and MIC of combination achieved 1.8 mg/mL. Peppermint oil interacted with thyme oil in an additive manner with FIC index of 0.6. Activity of peppermintthyme EO combination was also rather high against E. coli with MIC 2.7 mg/mL. Therefore, all studied combinations can be used in order to inhibit growth of S. aureus and E. coli.

Chemical composition of thyme essential oil The major components of thyme EO were carvacrol, γterpinene and para-cymene (62.3%, 15.8% and 6.0%, respectively), therefore, the present thyme oil belongs to carvacrol chemotype. Thymol and α- terpinene were present in small amount (2.5% and 1.7%, respectively).

Minor components were α- pinene (0.8%), α- terpineole (0.4%), camphene (0.4%) and camphor (0.2%).

Discussion High prevalence of antibiotic resistance among bacteria causing infectious processes of different location has lead to revitalization of interest in EOs. Combined use of EOs has obvious advantages such as increasing activity of both agents, reduction of toxicity and minimizing adverse sensory effect of EOs in case of application of them as food preservatives. In many studies, EO of T. vulgaris demonstrated good antimicrobial properties; however, activity of thyme oil in combinations with other EOs is not well investigated. In the present study, we investigated activity of combinations of thyme oil with different EOs against representatives of two major bacterial groups – gram-positive S. aureus and gram-negative E. coli.

The results proved high antimicrobial activity of thyme EO and also demonstrated general higher susceptibility of S. aureus to EOs than E. coli in disk diffusion method.

Based on these preliminary results of enhancing activity in disk diffusion method, we chose several EOs for more detailed evaluation in micro-broth dilution method – Norway spruce, juniper berry and cinnamon EOs. For all these EOs, combinations with thyme oil were either synergistic or additive which demonstrated general agreement between disk diffusion and microdilution

KON & RAI – Antibacterial activity of Thymus vulgaris essential oil

methods. However, some differences were present as the best synergistic effect was seen in thyme-cinnamon combination, while two other combinations were additive.

Against E. coli, according to disk diffusion method, the most noticeable increase in antibacterial activity was present in combinations of thyme EO with rosewood, peppermint and lemon balm EOs. These three EOs were then studied by microdilution method which proved that the presence of beneficial effect between these EOs: synergism was detected in the combinations between thyme and rosewood, and between thyme and lemon balm EOs, while thyme-peppermint EOs combination was additive.

Effect of interactions between EOs depends on interactions of their components. Polymorphic variations in monoterpene production, characteristic for T. vulgaris (Thompson et al. 2003), make it important to determine the phenotype of studied thyme oil. In the present study, according to the major component, thyme oil belonging to carvacrol chemotype. Carvacrol is the substance with phenolic structure in which hydroxyl group plays an important role.

EO components with phenolic structure, such as thymol, carvacrol, and eugenol, possess high antimicrobial activity demonstrated in many studies (Soković et al. 2010; Bassolé et al. 2010). Several mechanisms have been proposed to explain their mechanism of action. Hydroxyl group on eugenol may react with proteins and inhibit action of enzymes; hydrophobic thymol and carvacrol may damage the outer membrane of gram-negative bacterial cell wall releasing lipopolysaccharides (Gómez-Estaca et al.

2010).

Bassolé et al. (2010) demonstrated synergistic interactions against E. coli between carvacrol and eugenol, carvacrol and thymol, carvacrol and linalool, carvacrol and menthol, menthol and eugenol, eugenol and thymol, and eugenol and linalool. Synergy between carvacrol of thyme oil and menthol of peppermint oil may be responsible for the additive effect between these EOs against E. coli demonstrated in our study. The main component of rosewood and lemon balm EOs, according to manufactures instructions, is linalool. Although linalool mechanism of action is not well understood, its documented synergistic interactions with carvacrol may play a key role in synergy between thyme and rosewood EOs and between thyme and lemon balm EOs against E. coli.

Against S. aureus, the present study has demonstrated synergistic effect between thyme and cinnamon EOs, the main component of which is cinnamaldehyde. Its mechanism of action includes inhibition of energy metabolism and interaction with bacterial cell membrane leading to its disruption and dispersion of the proton motive force by small ions leakage (Gill and Holley, 2004).

Interactions of EO components against S. aureus in general are less studied. Synergism between thyme and cinnamon EOs may be caused either by not well understood interactions between cinnamaldehyde and thyme EO components, or by already documented synergistic interactions against other gram-positive bacterium L. monocytogenes between carvacrol of thyme

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oil and eugenol of cinnamon oil, between thymol of thyme oil and eugenol, and between thymol and linalool of cinnamon oil (Bassolé et al. 2010).

Delgado et al. (2004) showed synergistic effect between thymol and cymene, present in different EOs, on B. cereus and proposed an explanation for it. Thymol and cymene have similar structure but, in contrast to thymol, cymene lacks the hydroxyl group. Both compounds are hydrophobic and accumulate preferentially in the cell membranes; after this the action of one compound may facilitate uptake of another into the lipid bilayer of cytoplasmic membrane, causing the observed synergistic effect. Cymene, which is present in juniper berry and cinnamon EOs, may be responsible for beneficial interactions with carvacrol or thymol of thyme oil.

CONCLUSION Combinations of EOs provide an effective and economically feasible approach in combating antibioticresistant bacteria. However, unlike studies on antibioticantibiotic combinations, combinations of EOs are not so widely investigated and future studies should be devoted to evaluation of EO combinations against clinical isolates of multidrug-resistant bacteria, and to study combined effect of different EO components including also oil components present in small proportions.

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Stein S, Mirokhin D, Tchekhovskoi D, Mallard G. 2002. The NIST Mass Spectral Search Program for the NIST/EPA/NIH Mass Spectra Library; Standard Reference Data Program of the National Institute of Standards and Technology. Gaithersburg, MD, USA. Thompson JD, Chalchat JC, Michet A, Linhart YB, Ehlers B. 2003.

Qualitative and quantitative variation in monoterpene co-occurrence and composition in the essential oil of Thymus vulgaris chemotypes. J Chem Ecol 29 (4): 859-880.

Tohidpour, A., Sattari, M., Omidbaigi, R., Yadegar, A., Nazemi, J. (2010) Antibacterial effect of essential oils from two medicinal plants against Methicillin-resistant Staphylococcus aureus (MRSA). Phytomedicine 17 (2): 142-145.

ISSN: 2087-3948 E-ISSN: 2087-3956

Vol. 4, No. 2, Pp. 57-61 July 2012

Adult mangrove stand does not reflect the dispersal potential of mangrove propagules: Case study of small islets in Lampung, Sumatra AGUNG SEDAYUâ&#x2122;Ľ, NOVITA FARAH ISYADINYATI, DIANA VIVANTI SIGIT

Department of Biology, Faculty of Mathematics and Natural Sciences, State University of Jakarta. Jl. Pemuda 11 Rawamangun, Jakarta Timur 13220.

Tel. +62-21-4894909. Fax. +62-21-4894909. â&#x2122;Ľemail: goeng93@yahoo.com Manuscript received: 26 December 2010. Revision accepted: 19 February 2011.

Abstract. Sedayu A, Isyadinyati NF, Sigit DV. 2011. Adult mangrove stand does not reflect the dispersal potential of mangrove propagules: Case study of small islets in Lampung, Sumatra. Nusantara Bioscience 4: 57-61. Most mangrove species are dispersed by water current with distance being a major constraint. We tried to demonstrate that distance is indeed the dispersal limiting factor in mangrove, and perhaps other marine plant species. Secondly, we also tried to clarify whether landmass is a real blockade for mangrove dispersal. Lastly, we argued that in order to study plant dispersal potential, one should not study the later stage of plant population, as normally plant ecologist would do, rather at their early life stage. Cluster analyses were used to test those hypotheses and confirmed our research hypotheses.

Key words: biogeography, dispersal, mangrove, propagules.

Abstrak. Sedayu A, Isyadinyati NF, Sigit DV. 2011. Tegakan mangrove dewasa tidak mencerminkan potensi penyebaran propagul mangrove: Studi kasus pulau-pulau kecil di Lampung, Sumatera. Nusantara Bioscience 4: 57-61. Sebagian besar jenis mangrove tersebar oleh arus air dengan jarak sebagai kendala utamanya. Penelitian ini mencoba untuk menunjukkan bahwa jarak menjadi faktor pembatas dalam penyebaran mangrove, dan jenis tumbuhan pantai lainnya. Kedua, penulis juga mencoba untuk mengklarifikasi apakah daratan adalah secara nyata membatasi penyebaran mangrove. Terakhir, penulis memperdebatkan bahwa untuk mempelajari potensi dispersal tumbuhan, seseorang tidak harus mempelajari tahap akhir dari populasi tanaman, sebagaimana banyak dilakukan para ahli ekologi tumbuhan, namun dapat pula pada tahap awal kehidupannya. Analisis klaster digunakan untuk menguji hipotesis tersebut dan dikonfirmasi dengan penelitian ini.

Kata kunci: biogeografi, penyebaran, mangrove, propagul.

INTRODUCTION Coconut tree, most probably originally from PolynesiaMelanesian, is naturally distributed pantropically on most beach areas, with the help of its floatable fruits. However, being an ethnobotanically ancient important crop, its limited distribution range in some places like South America, especially Panama, is mostly caused by preindustrial human migration (Ward and Brookfield 1992).

On the other hand, mangrove, with similar dispersal capability, had no economic importance to prehistoric human, hence their almost identical worldwide distribution to coconut tree is solely attributed to their own capability to colonize adjacent area Many of mangrove species are known to spread by floatable propagules. Some propagules, such as in Rhizophora, are dispersed by viviparous seed/embryo,; while others with their floatable non-viviparous fruits/seeds. The survival, including dispersal, recruitment and growth of the propagules depends on many inherent (genetic traits) and external (environmental) factors.

Initial propagule characters such as weight, shape, orientation, time of shoot emergence, and buoyancy, and

early growth, such as (time??) and numbers of plants with initiated roots and shoots are important traits determining the dispersal and recruitment of mangrove species along tidal area (Rabinowitz 1978a, b). These traits interact with external/environmental factors, such as salinity, water turbulence, water depth, temperature, tidal amplitude, water current and light exposure, disturbance, predatory and competition (McMillan 1971; Smith and Duke. 1987; Osborne and Smith 1990; Jimenez and Sauter 1991; Sousa et al. 2007). The interaction of such factors has resulted in the existing mangrove population stands along the pantropic.

For tidal species, water current and distance from mother tree (genetic source or original population) are particularly important in propagule dispersal. For land plants, water bodies such as seas, lakes, oceans or rivers act as physical barriers of natural distribution. On the contrary, for mangroves, landmasses virtually act as physical barriers of their distribution.

Using the natural mangrove stands at differing life stages at Teluk Lampung, Sumatra, we aimed to (i) understand the dispersal potential of mangrove species in terms of predicting the long distance travel of propagules from bigger island to smaller satellite islets and

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confirming whether the landmass are actual dispersal barriers for mangroves; (ii) test which life stages of mangroves (seedling, sapling and tree) are best to detect the mangrove dispersal potential.

MATERIALS AND METHODS Six stations on the western coast of Lampung had been chosen for this study. Two of which, Suamalu (05.724o S, 105.207o E) and Kalangan (05.645o S, 105.207o E) are situated on the coast of main Island, Sumatra, while the other four are on two small islets just across the former

two. Two stations are situated at Puhawang islet with one station (Puhawang Barat; 05.674o S, 105.207o E) is facing directly toward Sumatra and the other one (Puhawang Timur; 05.672o S, 105.235o E) facing Sunda Strait. The last two stations are situated at Kelagian islet, with one station (Kelagian 05.630o S, 105.213o E) is facing Sumatra and the other one (Goreng; 05.617o S, 105.222o E) facing Sunda Strait (Figure 1.). At each station, a line transect was set from the sea, landward, starting from where the outermost mangrove stands was located. The length of transects depended on how thick the mangrove stand was, about 60 m to 100 m each.

Figure 1. The study sites in Teluk Lampung; the symbols on each site depict their relative similarity as depicted in Figure 2.

SEDAYU et al. â&#x20AC;&#x201C; Mangrove of small islets in Lampung

At each transect, three nested quadrats were laid, the smallest one, 1 x 1 m, was designated for seedlings, the 5 x 5 m quadrat for saplings and the 10 x 10 m for trees. We counted for each quadrat the number of species, frequency and basal area in order to calculate the importance value of each species (Cox 1972). For identification, specimens of unknown individuals were taken and once identified were kept at the herbarium of UNJ (JUNJ). Data from each transects were treated as one to portray each station as one entity, therefore there were six figures of importance values of all species surveyed representing six stations, thus assembling a matrix of 6 x number of all species (Table 2).

The matrix was analyzed for its similarity index, using program PRIMER (Plymouth Routines in Multivariate Ecological Research) version 5.1.2., and the results were drawn as dendrograms.

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The sapling data plotted on figure 2B shows a distinct cluster between Kelagian at Pulau Kelagian and Puhawang Barat at Pulau Puhawang. Kelagian which is located on the closest end of Pulau Kelagian to Sumatra which has a distinct similarity with Puhawang Barat, which is also located at the closest end of Pulau Puhawang to Sumatra.

Other study sites are clumped together in an above cluster, consisting of four sites, however with unclear information with regards to its geographical position.

The seedling data on Figure 2C showed two big clusters, each forming an interesting grouping where sites on small islets adjoining the bigger main island (Sumatra) have the greatest similarity index, as well as those distal to Sumatra. The sites on Sumatra are not joined, interestingly, to each other, but clusters to sites facing the main island or afar from the main island.

Table 1. Composition of species in combined study areas.

RESULTS AND DISCUSSION Three dendrograms were produced representing three life stages of mangrove, seedling, sapling and tree. Figure 2A shows that the tree similarity indexes between sites is almost incongruous in the biogeographical point of view, since each site does not reflect its close affinity based on geographical distances. The mangrove on t h e furthest south site on Suamalu, which is located in Sumatra is the closest according its importance value similarity index to our northernmost site at Goreng on Pulau Kelagian. In the sense of biogeography, the closer the areas, the more similar their species component. Trees tend to form random stands, without a distinct pattern between places.

Family Bombacaceae Euphorbiaceae Meliaceae Rhizophoraceae

Sonneratiaceae

Species Camptostemon schultzii Excoecaria agallocha Xylocarpus granatum Bruguiera cylindrica Bruguiera gymnorrhiza Ceriops tagal Rhizophora apiculata R. x lamarckii R. mucronata Sonneratia alba

Composition (%) 0.36 0.12 0.36 2.65 4.45 6.5 58.24 16.97 7.7 2.65 100

Figure 2. The dendrogram of similarity between sites; (A) Tree; (B) Sapling; (C) Seedling. For information about symbols and names of places see Figure 1.

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Table 2. Importance values of each mangrove species matrix.

Kelagian Puhawang Barat Puhawang Timur Tr Sa Se Tr Sa Se Tr Sa Se Bruguiera cylindrica 27.1 32.08 45 0 0 0 0 0 0 Bruguiera gymnorrhiza 40.8 45.8 84.4 32.23 0 23.01 0 0 0 Camptostemon schultzii 16.44 0 0 0 0 0 0 0 0 Ceriops tagal 0 34.7 28 0 43.17 17.35 0 0 0 Excoecaria agallocha 0 0 0 0 0 0 0 0 0 Rhizophora apiculata 162.8 75.5 129,75 160.1 184.4 211 49.9 300 261.2 Rhizophora mucronata 0 27.34 0 107.6 72.42 47.91 0 0 0 Rhizophora x lamarckii 23.4 71.14 12.88 0 0 0 250.1 0 38.8 Sonneratia alba 29 0 0 0 0 0 0 0 0 Xylocarpus granatum 0 12.96 0 0 0 0 0 0 0 Note: Numbers are in percentage (%); Sa: Sapling; Se: Seedling; Tr: Tree.

Species

All species within our study area are species with floatable propagules. Rhizophoraceae (Rhizophora and Bruguiera) are species with highest important values, and the most common in all sites (Table 1 and 2), are equipped with viviparous propagules. Other species seed types are not viviparous, but buoyant. Sonneratia has edible arillate fruits known being eaten by bats and macaques, but the dispersal mode of this species is solely by floating, since the fruit has outer floatable tissue and too big to be swollen in whole. Excoecaria agallocha and Xylocarpus granatum have exploding capsules and fruits, and the shooting seeds which also have buoyancy potential. Camptostemon schultzii has floatable a capsule which, when splits, releases the seeds, having potential to disperse by water as well as wind (Noor et al.1999). We did not test whether the viviparous species thrives more successfully compared to the non-viviparous species, but this character seems to be a crucial feature in determining why species of Rhizophoraceae (all with viviparous fruits in our study site) were much more common in all three life stages surveyed.

Other investigators such as Smith and Sneadaker (2000) confirmed that the vivipary of Rhizophoraceae has a significant effect on its distribution along tropical and subtropical coastal areas. This explains why viviparous species is much more common than non- viviparous species, although they have similar means of distribution, water floatable propagules. Traits related to establishment were stronger predictors of distribution than those associated with dispersal (Clarke et al. 2001).

The distance between sites is the best explanation of the pattern shown in Figure 2.C., where the location adjacent to genetic source (i.e. bigger landmass, like Sumatra) has the largest similarity to that landmass, where the propagules presumably originated. Clarke (1993) observed that propagules of Avicennia marina was best transplanted within only 500 m afar from its point of release (mother tree), and the success slightly decreased at a distance of 1 km and was the least at 10 km, resulting restricted gene variation between populations and very slow recovery when mass mortality occurred. That explains why the sites distal to Sumatra landmass had much different importance values from those proximal to Sumatra. The immigration of mangrove propagules to sites secluded by land (i.e. opposing the small islets), from the genetic source is

Suamalu Tr Sa 0 27.8 0 0 0 0 0 21 0 0 164.4 177.9 61.5 43.6 0 0 62.3 29.7 11.3 0

Se 0 0 0 78.65 0 221.3 0 0 0 0

Goreng Tr Sa 0 0 0 0 0 0 0 0 0 0 50.85 148 0 0 180 120.6 68.31 31.4 0 0

Kalangan Se Tr Sa Se 0 0 0 0 0 65 17.71 0 0 0 0 0 0 0 19.33 158 0 0 24.9 0 49 0 238 142 0 0 0 0 251 235 0 0 0 0 0 0 0 0 0 0

inevitably much lower, as the landmass acts as physical barrier for the water transported propagules (Duke et al.

1988).

In both tree and sapling dendrograms (Figure 2 A, B), the pattern of dispersal potential of mangrove by water current is not obvious. In fact, the dendrograms produced in Fig. 2 A is almost illogical. In the Figure 2 B, at least the locations distal to Sumatra (Kelagian, 3 and Puhawang Barat, 5) are grouped in one cluster, showing that seedlings in those areas have higher similarity in species importance values, however the rest of clustering give no information in terms of biogeographical distribution of mangrove species. Both irrelevant dendrograms most likely reflect the later development of each mangrove population. Saplings and especially trees suffer from longer period of both inherent, genetic and environment pressures. Pinzon et al.(2003) demonstrated that natural mortality, humaninduced mortality, diseases and natural predations produce gaps in natural population. (Osborne and Smith 2003).

This research study implies that biogeographical studies focused on plant dispersal potential should focus at the plantâ&#x20AC;&#x2122;s early stages, when stands of juveniles are less likely affected by environment, competition, predation or habitat modification, leading to individual mortality.

Analysis for such purposes with later stages of plant development as sapling and tree may introduce bias in the analysis, as those stages are exposed to many factors leading to mortality for a longer period of time, hence afflicting the distribution of plants in a certain site.

CONCLUSIONS Most mangrove species are dispersed by water current with distance as a major constraint. We tried to demonstrate that distance is indeed the dispersal limiting factor in mangrove, and perhaps other marine plant species.

Secondly, we also tried to clarify whether landmass is a real blockade for mangrove dispersal. Lastly we argued that in order to study plant dispersal potential, one should not study the latter stage of plant population, as normally plant ecologist would do, rather at their early life stage.

Cluster analyses were used to test those hypotheses and confirmed our research hypotheses.

SEDAYU et al. – Mangrove of small islets in Lampung

ACKNOWLEDGEMENTS We gratefully acknowledge the Research Center for Oceanology, Indonesian Institute of Science (P3O LIPI), Jakarta for their cooperation in collecting field data. We thank our field assistants during the data collections in Lampung, and The State University of Jakarta’s staffs for their valuable discussions and supports.

REFERENCES Clarke PJ. 1993. Dispersal of grey mangrove (Avicennia marina) propagules in southeastern Australia. Aquat Bot 45: 195-204.

Clarke PJ, Kerrigan RA, Westphal CJ. 2001. Dispersal potential and early growth in 14 tropical mangroves: Do early life history traits correlate with patterns of adult distribution? J Ecol 89 (4): 648-659.

Cox GW. 1972. Laboratory manual of general ecology. W.M.C. Brown Company Publishers, Iowa.

Duke NC, Ball MC, Ellison JC. 1998. Factors influencing biodiversity and distributional gradients in mangroves. Global Ecol Biogeograph Lett 7 (1): 27-47.

Jimenez JA, Sauter K. 1991. Structure and dynamics of mangrove forests along a flooding gradient. Estuaries 14 (1): 49-56.

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McMillan C. 1971. Environmental factors affecting seedling establishment of the black mangrove on the Central Texas Coast.

Ecology 52 (5): 927-930.

Noor YR, Khazali M, Suryadiputra INN. 1999. Panduan Pengenalan Mangrove di Indonesia. Ditjen PKA, Dephut, Wetland InternationalIndonesia Programme, Bogor.

Osborne K, Smith III TJ. 1990. Differential predation on mangrove propagules in open and closed canopy forest habitats. Vegetatio 89 (1): 1-6.

Pinzón ZS, Ewel KC, Putz FE. 2003. Gap formation and forest regeneration in a Micronesian mangrove forest. J Trop Ecol 19 (2): 143-153.

Rabinowitz D. 1978a. Dispersal properties of mangrove diaspores.

Biotropica 10, 47-57.

Rabinowitz D. 1978b. Early growth of mangrove seedlings in Panama and an hypothesis concerning the relationship of dispersal and zonation. J Biogeograph 5: 113-133.

Smith III TJ, Duke NC. 1987. Physical determinants of inter-estuary variation in mangrove species richness around the tropical coastline of Australia. J Biogeograph 14 (1): 9-19.

Smith SM, Snedaker SC. 2000. Hypocotyl function in seedling development of the red mangrove, Rhizophora mangle L. Biotropica 32 (4a): 677-685.

Sousa WP, Kennedy PG, Mitchell BJ, Ordóñez L. 2007. Supply-side ecology in mangroves: Do propagule dispersal and seedling establishment explain forest structure? Ecol Monograph 77 (1): 53-76 Ward RG, Brookfield M. 1992. The dispersal of the coconut: Did it float or was it carried to Panama? J Biogeograph 19 (5): 467-480.

ISSN: 2087-3948 E-ISSN: 2087-3956

Vol. 4, No. 2, Pp. 62-75 July 2012

Patterns of fertility in the two Red Sea Corals Stylophora pistillata and Acropora humilis 1

MOHAMMED S.A. AMMAR1,♥ , AHMED H. OBUID-ALLAH2, MONTASER A.M. AL-HAMMADY3

National Institute of Oceanography and Fisheries (NIOF), Suez, P.O. Box 182, Egypt. Tel. (Inst.) +20 62 3360015. Fax. (Inst.) +20 62 3360016. email: shokry_1@yahoo.com 2 Department of Zoology,Faculty of Science, Assiut University, Egypt 3 National Institute of Oceanography and Fisheries, Hurghada, Egypt Manuscript received: 10 November 2011. Revision accepted: 22 December 2011.

Abstract. Ammar MSA Obuid-Allah AH, Al-Hammady MAM. 2012. Patterns of fertility in the two Red Sea Corals Stylophora pistillata and Acropora humilis. Nusantara Bioscience 4: 62-75. Patterns of fertilities (total testes and total eggs) for the hermatypic coral Acropora humilis were lower than those in Stylophora pistillata at the four studied sites. Site 3 (El-Hamraween harbor), the site impacted with phosphate, recorded the highest annual mean of testes number and egg number in the two studied species Acropora humilis and S. pistillata. However, site 1, the site impacted with oil pollution and fishing activities, recorded the lowest annual mean of testes number, total testes, egg number and total egg for the two studied species. Thus, phosphorus enrichment seems to be considerably less destructive than oil pollution, and thus may represent an ‘eco-friendly’. Testes were observed full of sperms during winter season in the sectioned polyps of A. humilis, while eggs were detected during autumn and winter. However, the lack of eggs in S. pistillata occurred only during spring season at all the studied sites. In the studied coral species, the ova were developed first before spermeria.

The breeding season of coral reefs differs in both different localities and different species extending from December to July in the northern Red Sea, Gulf of Aqaba and Southern Red Sea (the present study). While in A. humilis, the breeding season extend from February to June in the Great Barrier Reef, in the Gulf of Aqaba and in Hurghada (the present study). Tough control, public awareness and continuous shore patrolling to the activities of oil pollution and fishing activities at the vicinity of site 1 (Ras El-Behar) are urgent.

Although existing corals may continue to grow and survive in an area with elevated nutrients levels, it is essential to maintain water quality on coral reefs within ecologically appropriate limits to ensure successful reproduction of coral and provide recruits for the longterm maintenance and renewal of coral populations.

Key words: fertility, Red Sea, corals, Stylophora pistillata, Acropora humilis Abstrak. Ammar MSA Obuid-Allah AH, Al-Hammady MAM. 2012. Pola kesuburan pada dua karang dari Laut Merah Stylophora pistillata dan Acropora humilis. Nusantara Bioscience 4: 62-75. Pola fertilitas (testis total dan telur total) untuk karang hermatypic Acropora humilis lebih rendah daripada Stylophora pistillata di empat lokasi penelitian. Situs 3 (Pelabuhan El-Hamraween), lokasi yang terdampak dengan fosfat, tercatat memiliki rata-rata tahunan tertinggi jumlah testis dan jumlah telur pada dua spesies yang dipelajari A. humilis dan S. pistillata. Namun, situs 1, lokasi yang terdampak dengan pemcemaran minyak dan kegiatan penangkapan ikan, tercatat memiliki rata-rata tahunan terendah dari jumlah testis, testis total, jumlah telur dan telur total untuk dua spesies yang dipelajari. Dengan demikian, pengayaan fosfor tampaknya jauh kurang destruktif daripada pemcemaran minyak, sehingga dapat dianggap 'ramah lingkungan'. Testis yang diamati penuh sperma selama musim musim dingin pada polip A. humilis yang dibelah, sedangkan telur terdeteksi selama musim gugur dan musim dingin. Namun, ketiadaan telur pada S. pistillata hanya terjadi selama musim semi di semua lokasi penelitian. Pada spesies karang yang diteliti, ovum terbentuk lebih dahulu sebelum spermeria. Musim perkembangbiakan terumbu karang berbeda baik katrena perbedaan lokasi maupun spesies, terjadi dari Desember hingga Juli di Laut Merah bagian utara, Teluk Aqaba dan Laut Merah bagian selatan (lokasi penelitian ini). Sementara pada A. humilis, musim perkembangbiakan terjadi dari Pebruari hingga Juni di Great Barrier Reef, Teluk Aqaba dan di Hurghada (lokasi penelitian ini). Kontrol yang ketat, kesadaran masyarakat dan patroli pantai terus menerus terhadap kegiatan pencemaran minyak dan penangkapan ikan di sekitar lokasi 1 (Tanjung Ras El-Behar) sangat mendesak. Meskipun karang yang ada dapat terus tumbuh dan bertahan hidup di daerah dengan tingkat nutrisi yang meningkat, adalah penting untuk mempertahankan kualitas air pada terumbu karang dalam batas-batas ekologis yang tepat untuk menjamin keberhasilan reproduksi karang dan memberikan sumber bibit untuk pemeliharaan jangka panjang dan pembaharuan populasi karang.

Kata kunci: kesuburan, Laut Merah, karang, Stylophora pistillata, Acropora humilis

INTRODUCTION Studies of reproduction in hermatypic corals have largely provided information on the sexual biology (Fadlallah 1983; Harrison and Booth 2007; Kenyon 2008)

and life history of coral (Harriott 1983; Moorsel 1983; Babcock 1984; Kojis 1984; Chaves-Romo and ReyesBonilla 2007; Glynn and Colley 2009). Few researchers have studied deleterious environmental factors affecting coral reproduction (Loya and Rinkevich 1979; Markey et

AMMAR et al. â&#x20AC;&#x201C; Fertility pattern of Stylophora pistillata and Acropora humilis

al. 2007; Mangubhai and Harrison 2009; Vize 2009), but none have examined the natural variability of coral fecundity. Relatively few studies have concentrated on quantitative data on reproductive output, usually expressed as potential fecundity (Ward and Harrison 2000; Goffredo et al. 2006).

Recent research on sexual reproduction in scleractinian corals has been instrumental in the reconsideration of a number of hypotheses which attempted to relate the mode of coral reproduction to habitat (Stimson 1978; Harrison and Both 2007; Cairns 2007), coral morphology (Rinkevich and Loya 1979b; Flot et al. 2008) and ecology (Loya 1976). However, only a few workers have attempted to assess the effects of anthropogenic activities on coral reproduction (Loya and Rinkevich 1979; Markey et al.

2007; Humphrey et al. 2008; Randall and Szmant 2009) or to provide comparative data on natural variability of coral fecundity (Kojis and Quinn 1984). Furthermore, most of these studies have been restricted to the effects of oil pollution (Peters et al. 1981; Negri et al. 2005; Markey et al. 2007) and thermal stress (Jokiel and Guinther 1978; Glynn et al. 2008; Glynn and Colley 2009) on the reproductive biology of the corals.

More studies on real or potential fecundity at all levels within and between populations are therefore needed to assess the suitability of fecundity as an index of stress in corals. Reduced fecundity is a form of partial reproductive failure. Several mechanisms have been demonstrated in corals to date, including oocyte abortion (Loya and Rinkevich 1979; Kenyon 2008), planula abortion (Jokiel 1985; Harrison 2006; Graham et al. 2008), and incomplete spawning followed by rapid oocyte resorption of unspawned oocytes (Rinkevich and Loya 1979b; Kenyon 2008). Of these studies, only that of Rinkevich and Loya (1979c) observed reproductive failure in a natural situation of ill-defined stress.

Various forms of reproductive failure (oocyte abortion, planula abortion, reduced fecundity etc.) can be induced in corals by stress (Harrison and Wallace 1990; Krupp et al.

2006; Yakovleva et al. 2009). Therefore, it has been suggested that variation in fecundity could be useful as a measure of sub-lethal stress on reefs (Harrison and Wallace 1990). Measures of fecundity such as the number of eggs or the number of planulae per polyp can provide a useful index of reproductive effort and useful indicator of the health of a coral (Kojis and Quinn 1984; Harrison and Wallace 1990). Consequently, changes in these measures can be an indication of sublethal stress in corals. However, fecundity alone can underestimate the reproductive effort of corals and is better combined with other reproductive measures such as size of eggs and the volume of testis material (Harrison and Wallace 1990; Bassim and Sammarco 2003; Fabricius 2005).

Waller and Tyler (2005) reported two main reproductive patterns in marine invertebrates, the production of small numbers of large oocytes, and the production of large numbers of small oocytes. Scleractinian corals have a similar pattern of gametogenesis to that of other cnidarians (Giese and Pearse 1974; Gorbunov and Falkowski 2002; Holstein et al. 2003), and either broadcast

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spawn gametes for external fertilization and development or brood larvae within polyps, and may be hermaphroditic or gonophoric (Fadlallah 1983; Harrison and Wallace 1990; Huwang et al. 2009; Baird et al. 2009). In hermaphroditic coral species, eggs and spermaries may develop on the same mesentries (as in the faviids and mussids), on different mesentries within the same polyp (most pocilloporids and acroporids), in different polyps within the same colony, or rarely, at different times within the same colony (Fadlallah 1983; Harisson and Wallace 1990; Harrison 2006; Harrison and Hakeem 2007).

The breeding season of coral reefs differs in both different localities and different species (Nozawa et al.

2006; Mezaki et al. 2007). It extends from December to July in the northern Red Sea, Gulf of Aqaba (Shlesinger and Loya 1985). While in Acropora humilis, the breeding season extend from February to June in the Great Barrier Reef (Bothwell 1981), in the gulf of Aqaba (Shlesinger and Loya 1985). However, Baird et al. (2010) conclude that the major spawning season of corals on shallow-inshore reefs in the Dampier Archipelago is autumn, although taxa that spawn in spring and summer include Porites spp., Acropora spp., possibly T. mesenterina that are numerically dominant at many of these sites. Consequently, management initiatives to limit the exposure of coral spawn to stressors associated with coastal development may be required in up to five months per year. The gonads development in Stylophora pistillata is differing from A. humilis. However, female gonads in S. pistillata need approximately six months for maturation (July to December) while male gonads require only three months (October to December) (Baird et al. 2010). Humter (1989) reported that, the reproduction process in A. humilis and S. pistillata in the Red Sea occurs during spring and summer.

However, corals in the inner and eastern Gulf of Thailand spawned following the full moons of February/March, whereas spawning in the southwestern Gulf of Thailand and the Andaman Sea occurred 1 month later following the full moons of March/April (Kongjandtre et al. 2010). In Western Australia coral spawning occurs predominantly in the austral autumn in contrast to the Great Barrier Reef on Australiaâ&#x20AC;&#x2122;s east coast where most spawning occurs in spring (Baird et al. 2010).

Shlesinger and Loya (1985) found that the female gonads require about five months (October to February), while the male gonads require only two months (January and February) for maturity. The same authors also reported that, A. humilis release gametes during late spring and early summer. Guest (2004) and Waller et al. (2005) have sequential patterns of egg and sperm development in Diploastrea heliopora. Kongjandtre et al. (2010) illustrations partially conflict that, the male gonads of A. humilis starting their development at the end of January and February and completed their development in April, while female gonads started their development at the end of October before the male gonads become well developed at April. However, Gylnn et al. (2011) repeated that, reproduction of corals takes place mainly from March to May when seasonally high sea temperatures and rainfall prevailed in the GalĂĄpagos Islands.

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The aim of the study is to examine the fertility patterns as biomonitors for environmental threats on the two Red Sea corals A. humilis and S. pistillata, outlining the degree of harm of each impact, the possibility of recovery and putting the possible scientific solutions.

MATERIALS AND METHODS All field work was performed using SCUBA diving equipments, throughout the period from December 2007 to January 2009.

Study area A preliminary visual survey of the Egyptian Red Sea coast, using snorkelling and SCUBA led to selection of four sites at four widely geographically separated areas along the western coast of the Red Sea (Figure 1). These sites are Ras El-Behar (northern Hurghada), Middle reef (Hurghada), Kalawy bay (Safaga) and El-Harnraween

harbor (El-Quseir). Three of these sites are subjected to different human activities and the fourth one is considered as a control site. Ras El-Behar (Site 1) lies at the northern part of the Red Sea, at a distance of about 60 km northern to Hurghada city, between latitudes 27° 43' 12" N and 27°43' 51" N and longitudes 33°33' 12" E and 33°33' 04" E. This site is impacted by petroleum oil pollution coming fiom the nearby petroleum fields and oil tankers.

Furthermore, the co mme rcial fishing activities are impacting the same site. Middle reef (Site 2) is located 200 m off shore between the northern reef and crescent reef, directly in front of National Institute of Oceanography and Fisheries (NIOF). This location is about 5 km northern to Hurghada city, between longitudes 27°17.13' N and 27°17.09' N and latitudes 33 °46.43' E and 33 °46.47' E. The middle reef is situated in the area that has been subjected to land filling which is associated with high sedimentation rate. El-Hamraween harbor (Site 3) is located about 60 km southern of Safaga 20 km northern of

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Figure 1. Location map of the studied site: 1. Ras El-Behar (northern Hurghada), 2. Middle reef (Hurghada), 3.Kalawy bay (Safaga) and 4. Al-Hamraween harbor (El-Quseir).

AMMAR et al. – Fertility pattern of Stylophora pistillata and Acropora humilis

El-Qusier City and about 120 from the Capital City of the Red Sea governorate (Hurghada). It is dominated between latitudes 26°15' 02" N and 26°15'17" N and longitudes 34°12' 07" E and 34°12'00" E. The site is impacted by heavy load of phosphate due to preparation and shipment operations of phosphate are in El-Hamraween harbor.

Kalawy area (site 4) lies between latitudes 26°30'32" N and 26°30' 35"N and longitudes 34°03' 59" E and 34°04' 00" E. It lies about 30 km south Safaga City. This site is a pristine area, difficult to be accessed by fishermen because of the heavy wave breaking in addition to the tough patrolling in the area, thus it is considered as a control site in this study.

Selection of studied species Two coral species from two families commonly dominating coral assemblages in the Red Sea were chosen for this study; S. pistillata (Esper 1795) and A. humilis (Dana 1846). The choice of coral species was mainly based on the morphological and behavioral properties. The studied species have a digitate to branching growth form and comparable size of polyp (1-2 mm diameter). The use of branching rather than massive growth forms ensure that, a small branch can be removed easer from the mother colony without damaging neither the corals living tissue nor the remainder of the colony or other reef benthos. The choice of the studied species has proved useful for examining patterns of fertility.

Field sampling and maintenance Skirt fragments (<5 cm fragment) from three separate colonies of the studied species were seasonally collected at each of the three depth zones (reef flat, 3 m depth and 8 m depth). Only one terminal portion of the branch was sampled per coral colony, using a long nosed bone cutter.

Samples were divided into two portions. The first part of sample was kept in dark by wrapping them in aluminium foil and placed in whirl-package under water. On the deck, water was removed from the bags and immediately transferred to foam box filled with ice waiting for transportation to NIOF laboratories for analysis of chlorophyll concentration and zooxanthellae densities. The second part of the collected samples was stored in 10% formaldehyde in sea water for histological investigations.

More than 128 polyps of A. humilis and S. pistillata ranging between 1.2 mm to 2.2mm in diameter were histologically investigated.

Fertility measurements After 24 h in 10% formaldehyde sea water preservation, in the laboratory, pieces from the preserved branches were rinsed under the tap water for 15 min then decalcified with a solution of formic acid and sodium citrate for about 24-48 hours. The formic acid and sodium citrate were made up by adding the same volume of both formic acid and distilled water to make the formic acid solution, then sodium citrate salt was made up by dissolving 20 g of sodium citrate in 100 ml of distilled water. Finally, the two solutions were mixed in a ratio of 1:1. The mixed solution was poured into a beaker containing the samples till the decalcification

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process was completed. This method was preferred, since it was faster and easier to handle (Rinkevich and Loya 1979b). The decalcification process was continued until no skeletal materials remained. Samples were then preserved in Bouin's solution (Humason 1972) in plastic vials.

For histological analyses, tissue samples (0.75 to 1.5 cm2) were taken from mid-branch (Rinkevich and Loya 1979b). All tissue samples were dehydrated in an ethanol series (Humason 1972), cleared in toluene and embedded in tissue prep. The prepared blocks were sectioned horizontally (i.e. parallel to the surface) at 10µm using an automatic microtome instrument. All tissue sections were stained with Harris Hematoxylin and counterstained with Eosin.

Procedures of staining The sections were passed throughout the following stages according to Hassan (1980). Xylene 1 10 -12 min, Xylene 2 5 min, 100 %, 90%, 80%, 70% ethyl alcohol 5 min each, distilled water 1-2 min each, haematoxylin (HX) 3 min, distilled water 2 min, eosin 3 min, 80%, 90% and 100 alcohol 3 min each, 100% Butyl alcohol 3 min, Xylene 3 min. Mounting using Canada balsam or DPX on the section and cover the slide and put in oven at 60-70 ˚C Haematoxylin (HX) staning. HX 0.5 g, mercuric oxide 0.25 g, glacial acetic acid 4 ml, potas. alum 10 g, absolute alcohol 5 ml, distilled water 100 ml, dissolve the HX in alcohol, and the Pots. Alum in water, then mix them and heat to boiling, then add the mercuric oxide. When the solution becomes deep purple, stop heating, then cool and add the acetic acid to be used after 24 hours.

Eosin. Eosin yellow 1 g, distilled water, 100 ml, glacial acetic acid 0.5 ml. Each polyp was histologically examined under stereo binocular microscope (X20) then photographed using microscope camera, finally the eggs and testes sizes were measured by microscope eyepieces.

For the quantitative analyses, six pairs of mesenteries were exposed. Six mesenteries contained strings of eggs and six mesenteries developed testis, the number and size of eggs were scored. Two measurements were taken for each egg, a “length” and a “width” measurement using a calibrated eyepiece micrometer in the dissecting microscope. The egg size was the mean of these two measures. The polyp egg size was the mean of the individual egg sizes. “Egg total” was calculated by adding all the individual egg sizes (or by multiplying the polyp egg size by the polyp egg number). Testes were counted for the testes number. The length and width of each testis were measured and the mean of these measurements was called the testes size. Total testes were calculated by multiplying the polyp testis size by the polyp testes number.

RESULTS AND DISCUSSION Both of A. humilis and S. pistillata are simultaneous hermaphroditic species as appeared in the sections (Figure 2). Each of the male gonads (testes) and female ones (ova) are located in the same polyp. Testes and ovaries develop

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A

B

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Figure 2. Transverse section of the lower part of a mature hermaphrodite polyp showing male and female gonad in one polyp. A). S. pistillata. B). A. humilis; EC ectoderm; M mesentery; O female gonad; S male gonad.

Figure 3. A general histological structure of A. humilis polyp in the stomodael portion showing six pairs of mesenteries: A. actinopharynx; CE columnar epithelial; EC ectoderm; EN endoderm; G glandular cell; M mesentery; S siphonoglyph; Z zooxthanthela.

Figure 4. A general histological structure of S. pistillata polyp in the stomodael portion showing six pairs of mesenteries: A. actinopharynx; CE columnar epithelial; EC ectoderm; EN endoderm; G glandular cell; M mesentery; S siphonoglyph; Z zooxthanthela.

Figure 5. Transverse section of A. humilis (A): Male and female gonads during summer season at site 1. (B). Female gonad containing mature egg during Winter season at site 1. EC, ectoderm, M mesentery, O, female gonad, S, male gonad, SP, sperm, E, egg.

Figure 6. Transverse section of A. humilis, (A): Testes full of sperms and ovary containing egg during winter season at site 2. (B).

Testes and ovary during Summer season at site 2. EC ectoderm,M mesentery, O female gonad, S male gonad, E egg.

Figure 7. Transverse section of A. humilis (A): Testes during autumn season at site 3. (B). Male gonad and female one containing mature egg during winter season at site 3. EC ectoderm, M mesentery, O female gonad, S male gonad, E egg.

AMMAR et al. â&#x20AC;&#x201C; Fertility pattern of Stylophora pistillata and Acropora humilis

A

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Figure 8. Transverse section of A. humilis (A): Testes full of sperms during winter season at site 4. (B). Female gonad containing mature egg during winter season at site 4. EC ectoderm, M mesentery, O female gonad, S male gonad, Sp sperm, E egg.

Figure 9. Transverse section of S. pistillata (A): Female gonad containing mature egg during winter season at site 1. (B). Male gonad and Female gonad during spring season at site 1. EC ectoderm, M mesentery, O female gonad, S male gonad, E egg.

Figure 10. Transverse section of S. pistillata (A): Testes full of sperms during winter season at site 2. (B). Female gonad containing mature egg during winter season at site 2. EC ectoderm, M mesentery, O female gonad, S male gonad, Sp sperm, E egg.

Figure 11. Transverse section of S. pistillata (A): Testes full of sperms during winter season at site 3. (B). Female gonad containing mature egg during winter season at site 3. EC ectoderm, M mesentery, O female gonad, S male gonad, Sp sperm, E egg.

Figure 12. Transverse section of S. pistillata (A): Testes full of sperms during winter season at site 4. (B). Female gonad containing mature egg during spring season at site 4. EC ectoderm, M , mesentery, O female gonad, S male gonad, Sp sperm, E egg.

Figure 13. Transverse section of S. pistillata (A): Ovary containing mature egg and Testes full of sperms during winter season at site 4.

(B). Female gonad having no egg during summer season at site 4. EC ectoderm, M mesentery, O female gonad, S male gonad, E egg.

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Table 1. Annual means of total testes (µm) and total eggs of the two studied species A. humilis and S. pistillata at the four studied sites.

Species

Sites

Testes number

Testes size

Total testes

Egg number

Egg size

Total eggs

A. humilis

Site 1 Site 2 Site 3 Site 4 Site 1 Site 2 Site 3 Site 4

1.37±0.71 1.75±1.29 3.31±1.92 2.68±1.07 1.5±0.96 2.12±1.2 3.4±1.29 2.37±1.31

222.73±103.34 249.37±150.28 233.15±76.72 324.53±11 254.65±118.64 370.06±112.93 289.79±38.37 408.68±143.67

331.45±227.67 588.18±567.81 880.53±622.79 960.4±691.06 450.65±330.24 844.71±594.17 1021.67±490.83 1106.03±813.03

0.562±0.81 1.5±1.75 2.12±2.14 1.31±1.77 0.87±1.02 1.81±1.51 2.75±2.14 1.68±1.66

64.5±86.58 91.4±95.26 51.03±53.06 86.78±102.29 86.65±93.37 119.34±88.73 78.9±55.68 147.03±121.23

95.65±137.47 281.4±338.86 220.15±258.29 269.34±377.42 158.75±208.87 330.46±300.77 320.96±262.36 366.9±94.44

S. pistillata

Table 2. Seasonal testes number, testis size (µm) (testis length x testis width) and total testes of A. humilis at the studied sites respectively (X`+ SD).

Seasons Autumn

Winter

Spring

Summer

Sites

Egg number

Egg width

Egg length

Egg size

Total eggs

Site 1 Site 2 Site 3 Site 4 Site 1 Site 2 Site 3 Site 4 Site 1 Site 2 Site 3 Site 4 Site 1 Site 2 Site 3 Site 4

1.5±0.577 1.25±0. 95 1.5±1.29 2±0.81 1.75±95 3.25±0.95 5.75±0.5 4.25±0.5 1.25±0.5 1.75±0.95 4±0.816 2.5±0.577 1±0.81 0.75±0.95 2±0.81 2±0

199±10.42 164±109.38 142±95.31 218.75±16.37 331.25±33.78 419±13.49 302.75±51.37 445.5±45.58 256.75±8.22 286.5±8.88 262.5±24.11 340.75±16.62 145.25±97.003 103.5±119.68 215.25±15.77 262±26.64

215±14.44 171±114.07 145.25±97.31 182.75±105.38 353.75±37.24 453±9.3 301.5±6.65 525.25±62.56 264.5±7.41 294.25±10.99 272±19.51 352.25±13.17 96.25±111.15 103.75±119.95 224±14.94 269±24.58

207±12.36 167.5±111.72 143.625±96.3 200.75±59.18 342.5±35.5 436±7.03 302.125±25.12 485.375±46.1 260.625±6.82 290.375±9.84 267.25±21.68 346.5±14.76 80.83±61.029 103.625±119.82 219.625±15.22 265.5±25.57

310.25±121.15 280.375±216.8 292.375±258.49 397.25±210.9 583±266.48 1412±397.29 1742.375±256.43 2053.37±197.18 326±131.5 506.875±279.64 1056.875±146.77 860±164.2 113.33±110.5 153.5±192.5 430.5±148.74 531±51.15

Table 3. Seasonal egg number, egg size (µm) (egg length x egg width) and total eggs of A. humilis at the studied sites (X`+ SD).

Seasons Autumn

Winter

Spring

Summer

Sites

Egg number

Egg width

Egg length

Egg size

Total eggs

Site 1 Site 2 Site 3 Site 4 Site 1 Site 2 Site 3 Site 4 Site 1 Site 2 Site 3 Site 4 Site 1 Site 2 Site 3 Site 4

0.75±0.95 2.25±0.95 3±0.81 1.25±0.95 1.5±0 .57 3.75±0.95 5.5±0.57 4±0.81 0 0 0 0 0 0 0 0

69.25±79.98 159.25±9.06 92.5±4.9 132.25±88.32 173.75±7.71 188.75±7.58 105.25± 9.77 204±6.05 0 0 0 0 0 0 0 0

77.75±89.82 174.25±10.93 100.25±2.21 136.5±91.01 190.75±6.39 209±5.35 110.25±9.74 221.5±4.79 0 0 0 0 0 0 0 0

75.75±87.68 166.75±9.91 96.375±3.14 134.375±89.64 182.2±7.02 198.87±5.49 107.75±9.75 212.75±4.94 0 0 0 0 0 0 0 0

111.75±139.37 381.375±174.55 287.5±71.2 225.125±174.41 270.87±96.87 744.25±183.18 593.125±87.98 852.25±180.58 0 0 0 0 0 0 0 0

AMMAR et al. – Fertility pattern of Stylophora pistillata and Acropora humilis

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Table 4. Seasonal testes number, testis size (µm) (testis length x testis width) and total testes of S. pistillata at the studied sites (X`+SD).

Seasons Autumn

Winter

Spring

Summer

Sites

Egg number

Egg width

Egg length

Egg size

Total eggs

Site 1 Site 2 Site 3 Site 4 Site 1 Site 2 Site 3 Site 4 Site 1 Site 2 Site 3 Site 4 Site 1 Site 2 Site 3 Site 4

1.25±1.25 1.75±0.95 2.75±0.95 2±0.81 1.75±1.25 3±0.816 4.75±0.5 4±0.81 1.75±0.95 2.75±1.25 4.25±0.5 2.25±0.95 1.25±0.5 1±0.81 2±0.7 1.25±0.95

190.75±127.26 386.25±16.007 259.5±10.47 418±19.91 290± 199.28 519.75±12.25 323.5±7.76 553.5±21.7 257 9.83±9.83 318.75±28.33 287.75±11.87 390±20.7 245.75±4.03 223±16.57 249.25±5.49 220.75±147.26

197±131.33 406.5±8.81 267.25±10.68 435.5±17.36 330± 223.035 535.5±8.18 378.25±13.59 591±8.3 267.5±6.55 333±24.46 307.25±5.85 421.25±17.74 259.25±2.87 237.75±14.45 259.5±2.69 319±159.63

193.875±129.28 396.375±12.37 263.375±10.56 426.75±18.63 310±210.98 527.625±8.11 350.875±6.38 572.375±14.9 262.25±7.96 325.875±26.27 297.5±8.86 405.625±19.06 252.5±1.22 230.375±14.77 254.375±3.89 230±153.39

324.625±329.65 695.375±386.3 718.25±224.68 843.75±311.72 709.125±481.58 1584±432.52 1667.875±191.23 2296.37±507.93 453.25±234.07 872.125±360.85 1263.125±136.91 901.125±360.77 315.62±126.255 227.375±176.34 506±172.19 382.875±292.62

Table 5. Seasonal egg number, egg size (µm) (egg length x egg width) and total eggs of S. pistillata at the studied sites (X`+SD).

Seasons Autumn

Winter

Spring

Summer

Sites

Egg number

Egg width

Egg length

Egg size

Total eggs

Site 1 Site 2 Site 3 Site 4 Site 1 Site 2 Site 3 Site 4 Site 1 Site 2 Site 3 Site 4 Site 1 Site 2 Site 3 Site 4

1±0.81 3±0.81 4±0.81 1.75±0.95 1.75± 1.25 3.25±0.5 5±0.81 4±0.81 0 0 0 0 0.75±0.95 1±0.81 2±1.41 1±0.81

123.25±82.5 163.5±4.79 115.25±6.94 145.5±5.91 156.25± 104.44 212.75± 9.77 116.25± 5.43 208.75±5.43 0 0 0 0 59±68.17 92.25±61.57 72.25±48.47 76.75±51.33

129.5±86.43 168.5±5.06 119.5±4.65 150.25±7.27 161± 112.41 220.25± 6.18 130.5±4.04 219± 6.05 0 0 0 0 63.75±73.61 97.5±65.01 77.5±51.9 80.25±53.61

126.375±84.43 166±4.77 117.375±5.76 295.75±13.14 158.87± 107.3 216.5± 7.87 123.37±4.71 213.87±5.67 0 0 0 0 61.375±70.88 94.875±63.28 74.875±50.18 78.5±52.47

166.625±131.62 495.25±122.2 466.25±75.31 509.75±257.19 376.75± 260.29 700.87± 81.2 619.37± 123.29 853.62± 160.43 0 0 0 0 91.625±116.16 125.75±100.87 198.25±137.79 104.25±84.19

within mesentries between the retractor muscle and mesentrial filaments. The mesentries were all perfect and arranged in up to 6 pairs (12 mesentries) (Figures 3 and 4).

Some mesenteries of polyp were unpaired. Testes usually consist of one to six spermaries, each spermary being separated from the others by a thin membrane, probably of mesogleal origin. The ovaries usually contain one ovum at maturation that is surrounded by a thick endodermal layer during early oogensis.

Annual patterns of fertilities (average of the four seasons are shown in Table 1. Generally, the annual patterns of fertilities (total testes and total eggs) of the hermatypic coral A. humilis were lower than those in S. pistillata at the four studied sites. However, site 3 (ElHamraween harbor), the site impacted with phosphate, recorded the highest annual mean of testes number and egg number in the two studied species A. humilis and S. pistillata. On the other hand, the highest annual mean of total testes of A. humilis and S. pistillata were recorded at site 4 (control site), having value of 960.4±691.06 µm and

1106.03±813.03 µm for both species respectively. The highest annual mean of total eggs of A. humilis were recorded in site 2 (281.4± 338.86 µm) while that of S. pistillata was found in site 4 (366.9±94.44 µm). The present study detected that site 1, the site impacted with oil pollution and fishing activities, recorded the lowest annual mean of testes number, total testes, egg number, and total egg for each of the two studied species.

Fertility patterns of A. humilis Data concerning the average seasonal testes number, total testes (µm) (testis length x testis width), egg number and total eggs (µm) (egg length x egg diameter) in for A. humilis at the study sites are shown in Tables (2, 3). The seasonal mean of testes number, testes size and total testes at site 1 (Ras El-Behar) recorded their highest value in winter while the lowest values were recorded during summer. During winter season, testes were observed full of sperms in the sectioned polyps of A. humilis (Figure 5).

Male gonads of A. humilis started their development at the

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end of January and February and completed their development in April, while female gonads started their development at the end of October, becoming well developed in April. Eggs were detected only during autumn (average egg number = 0.75±0.95, egg size = 75.75±87.68 µm and average total eggs = 111.75±139.37 µm) and winter (average egg number = 1.5±0.57, egg size = 182.2±7.02 µm and average total eggs = 270.87±96.87 µm) at site 1.

At site 2 (Middle reef - NIOF) the maximum average of each of testes number, testes size and total testes of A. humilis were recorded during winter season, while the minimum values were recorded during summer season (Table 2, Figure 6). The average testes number during winter season was 3.25±0.95, and during summer season was 0.75±0.95 while the average total testes during winter season was 1412±397.29 µm, and during summer was 153.5±192.5 µm. The annual average of testes number was 1.75±1.29 and the annual average of total testes was 588.18±567.81 µm at site 2. The maximum average eggs number and total eggs of A. humilis at site 2 were recorded during winter season while the minimum values were recorded during autumn season.

The average testes number and total testes during winter season at site 3 (El-Hamraween harbor) were 5.75±0.5 and 1742.375±256.43 µm, respectively while in summer, the recorded values were 2±0.81 and 430.5±148.74 µm, respectively. However, the average egg number at site 3 were 3±0.81 in autumn and 5.5±0.57 in winter while the average total eggs were 287.5±71.2 µm in autumn and 593.125±87.98 µm in winter. Moreover, the annual average egg number was 2.12±2.14 and annual average total eggs was 220.15±258.29 µm at site 3 (Table 1).

In site 4 (Kalawy bay) the average testes number ranged from 2±0.81 during autumn and 2±0 during summer to 4.25±0.5 during winter season, with an average of 2.68±1.07 for the four seasons. The average total testes ranged from 397.25±210.9 µm during autumn to 2053.375±197.18 µm during winter season, with an average of 960.4±691.06 µm for the four seasons.

However, the average egg number were 1.25±0.95 during autumn and 4±0.81 during winter, with an average of 1.31±1.77 for the four seasons. The average total eggs were 225.125±174.41 µm during autumn and 852.25±180.58 µm during winter, with an average of 269.34±377.42 µm during the four seasons (Table 3, Figure 8).

In general, the highest average of testes number, testes size and total testes of A. humilis were recorded during winter season at all sites (Table 2) (Figures 5, 6, 7, 8). On the contrary, the lowest values were recorded during summer season at sites 1 and 2 and during autumn at sites 3 and 4. Site 3 (the site impacted with phosphate) had the highest average testes number (5.75±0.5, winter), however site 4 (control site) had the highest average testes size (485.375±46.1 µm, winter) and total testes (2053.375±197.18 µm, winter). In contrast, the minimum average testes number was found at site 2 (0.75±0.95, summer) while the minimum average testes size was recorded at site 1 (80.83±61.029 µm, summer). Also, the

lowest average total testes was recorded ate site 1 (113.33±110.5 µm, summer). On the other hand, the highest average eggs number was recorded at sit 3 (5.5±0.57, winter) while the highest average total eggs was recorded at sit 4 (control site) (852.25±180.52 µm, winter) (Table 3). No eggs were detected during spring and summer season.

Fertility patterns of S. pistillata Data concerning the average seasonal testes number, total testes (µm) (testis length x testis width), egg number and total eggs (µm) (egg length x egg diameter) in S. pistillata in the study sites are shown in Tables (4, 5).

Testes number of S. pistillata at site 1 during autumn season (1.25±1.25) and summer season (1.25±0.5) were less than those recorded during winter season (1.75±1.25) and spring season (1.75±0.95). Site 1 has an annual average testes number of 1.5±0.96 during the four seasons (Table 1). Moreover, the average testes size (310±210.98 µm) and total testes (709.125±481.58 µm) at site 1 during winter were higher than those during autumn (testes size = 193.875±129.28 µm and total testes = 324.625±329.65 µm) (Table 4) (Figure 9). The annual testes size and total testes at site 1 were 254.65±118.64 µm and 450.65±330.24 µm respectively. On the other hand, the lack of eggs in S. pistillata occurred only during spring season (breeding season) (Table 5) (Figure 9). The highest average egg number at site 1 was recorded during winter season (1.75±1.25) while the lowest average egg number was recorded during summer (0.75±0.95). Also, the maximum average total eggs at site 1 (376.75±260.29 µm) was recorded in winter while the minimum average total eggs (91.625±116.16 µm) was detected during summer. The average annual egg number, egg size and total eggs at site 1 were 0.87±1.02, 86.65±93.37 µm and 158.75±208.87 µm, respectively.

At site 2 (Middle reef - NIOF), the maximum seasonal testes number of S. pistillata (3±0.81) was recorded in winter while the minimum one (1±0.81) was recorded during summer (Table 4), with an annual average of 2.12±1.2 (Table 1). However, the highest average of each value of testes size (527.625±8.11 µm) and total testes (1584±432.52 µm) were measured in winter (Table 4, Figure 10) while the lowest values (230.375±14.77 µm and 227.375±176.34 µm) were recorded in summer. The annual testes size was 370.06±112.93 µm while the annual total testes was 844.71±594.17 µm. On the other hand, the seasonal egg numbers at site 2 were 3±0.81, 3.25±0.5 and 1±0.81 during autumn, winter and summer, respectively (Table 5), with an annual value of 1.81±1.51 (Table 1).

However, the seasonal total eggs at site 2 were 495.25±122.2 µm in autumn, 700.87±81.2 µm in winter and 125.75±100.87 µm in summer, with an annual value 330.46±300.77 µm.

The highest seasonal values of each of testes number (4.75±0.5), testes size (350.875±6.38 µm) and total testes (1667.875±191.23 µm) at site 3 (El-Hamraween harbor) were recorded during winter. (Table 4) (Figure 11).

However, the minimum seasonal testes number (2±0.7), testes size (254.375±3.89 µm) and total testes (506±172.19

AMMAR et al. – Fertility pattern of Stylophora pistillata and Acropora humilis

µm) were recorded during summer season, with annual value of 3.4±1.29, 289.79±38.37 µm and 1021.67±490.83 µm respectively. The seasonal egg numbers at site 3 were 4±0.81, 5±0.81 and 2±1.41 during autumn, winter and summer, respectively. Similarly, the seasonal egg sizes were 117.375±5.76 µm, 123.37±4.71 µm, 74.875±50.18 µm during autumn, winter and summer, respectively. The seasonal total eggs were 466.25±75.31 µm, 619.37±123.29 µm and 198.25±137.79 µm in autumn, winter and summer respectively. However, the annual egg numbers and total eggs were 2.75±2.14 and 320.96±262.36 µm respectively.

In site 4 (Kalawy bay) the seasonal testes number ranged from 1.25±0.95 during summer to 4±0.81 during winter season, with an annual value of 2.37±1.31. In a similar manner, the average total testes ranged from 382.875±292.62 µm during summer to 2296.37±507.93 µm during winter, with an annual value of 1106.03±813.03 µm. The seasonal total eggs at site 4 were 509.75±257.19 µm, 853.62±160.43 µm and 104.25±84.19 µm during autumn, winter and summer, respectively while, eggs number were 1.75±0.95, 4±0.81, and 1±0.81 during the same seasons respectively (Table 5, Figures 12 and 13).

Generally, the highest seasonal testes number, testes size and total testes of S. pistillata were recorded during winter season at all sites while the lowest values were recorded during summer (Tables 4). The highest seasonal testes number was recorded at site 3 (the site impacted with phosphate) (4.75±0.5, winter) while the highest seasonal testes size (572.375±14.9 µm) and total testes (2296.37± 507.93 µm) were recorded at site 4 (the control site) during winter. In contrast, the minimum average of each of testes number (1±0.81) and total testes (227.375±176.34 µm) were recorded at site 2 during summer while the minimum average testes size (193.875±129.28 µm) was recorded at site 1 during autumn. On the other hand, the highest average eggs number was recorded at sit 3 (5±0.81, winter) while the highest average egg size was recorded at site 4 (295.75±13.14 µm, autumn). Moreover, the maximum total eggs (853.62± 160.43µm) were recorded at site 4 (control site) during winter season, reporting no eggs during spring season.

Discussion In the present study, histological methods were used to assess testes and egg size in A. humilis and S. pistillata.

The dissection method could not be conducted as the polyps were too small to easily dissect without damaging gonads or losing oocytes. Histology has disadvantages including possible inaccuracies in oocyte sizemeasurements because of tissue shrinkage estimated to be 20 to 30% by Harriott (1983) and underestimating oocyte numbers by failing to section whole gonads or polyps (Harrison and Wallace 1990). Serial sectioning and the study of each section, while laborious will overcome the latter problem but it may still have a possible 20 to 30% of tissue shrinkage. Histological processing tends to cause oocyte shrinkage by dissolving the lipids despite the reasonably accurate mean seasonal values of total eggs and total testes.

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Both of A. humilis and S. pistillata are simultaneous hermaphroditic species as appeared in the sections (Figure 2). This result coincide with that of Rinkevich and Loya (1979a), Richmond and Hunter (1990), Wolstenholme (2004), Nishikawa et al. (2003) and Zakai et al. (2006).

Measures of fecundity in the number of eggs or the number of planulae per polyp can provide a useful index of reproductive effort, in turn a useful indicator of the health of a coral (Kojis and Quinn 1984; Harrison and Wallace 1990; Albright et al. 2008; Jokiel et al. 2008).

Consequently, changes in these measures can be taken as indication of sublethal stress in corals. However, fecundity alone can underestimate the reproductive effort of the corals and is better combined with other reproductive measures such as size of eggs and volume of testes material (Harrison and Wallace 1990; Kolinski and Cox 2003; Glynn and Colley 2009; Harrison 2011). What was mentioned in the present study for the pattern of reproduction of A. humilis and S. pistillata, is generally consistent with that mentioned by Rinkevich and Loya (1979b), Richmond and Hunter (1990), Nishikawa et al.

(2003), Wolstenholm (2004), Zakai et al. (2006) that each of these species is a simultaneous hermaphroditic species.

According to Fadlallah (1983), in the hermaphroditic species of the branching coral, the ovaries and spermaries may develop on different mesenteries within the same polyp in the same colony at the same time (most acroporids and pocilloporids). In contrast, Kojis and Quinn (1981, 1982), Ayre and Miller (2006), and Okubo et al. (2007) reported that, the same species have different reproductive seasons within one population.

In the present histological investigation, patterns of fertilities (total testes and total eggs) for the hermatypic coral A. humilis were lower than those in S. pistillata at the four studied sites. This apparent of disparity between the two species could be attributed to the differences in the polyp size of colonies of the two species or perhaps the larger total testes and total eggs of S. pistillata will result in the differences in reproductive mode (Rinkevich and Loya, 1979b; Fadlallah and Pearse 1982; Fadlallah 1985; Goffredo and Chadwic-Furman 2003; Zakai et al. 2006).

Szmant et al. (1980) provided further evidence for the lack of relationship between egg size, polyp size and mode of development in corals. Carroll et al. (2006) and Rinkevich and Loya (1979b) indicated that, A. humilis is a spawning species where the fertilization takes place externally while S. pistillata is a brooder species releasing planula larvae after the fertilization process.

The present investigation showed that, site 3, the site impacted with phosphate, recorded the highest annual mean of testes number and egg number in the two studied species A. humilis and S. pistillata. This agrees with the laboratory finding of Ward and Harrison (2000) who found that corals exposed to elevated phosphorus resulted in producing more but smaller eggs, and more testes material. Waller and Tyler (2005) reported two main reproductive patterns in marine invertebrates, the production of small numbers of large oocytes, and the production of large numbers of small oocytes. The present data also recorded that, site 3 had the lowest egg size of each of A. humilis and S. pistillata.

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Eutrophication causes significant problems for coral reefs, and can result in degradation of reef ecosystems (Pastorok and Bilyard 1985). Pollution may disrupt reproductive cycles in corals and inhibit larval settlement and post settlement survival (Tomascik and Sander 1987; Albright et al. 2008; Jokiel et al. 2008), and the coral abundance and diversity of recruits decreased with increasing eutrophication (Hunte and Wittenberg 1992). There was a significant but small decrease in egg size (430 μm in control eggs to 408 μm in eggs from ammonium enrichment treatments), but no differences in total fecundity or fertilization success. This may be related to the presence of zooxanthellae in the eggs of M. capitata, in contrast to changes in reproduction in Acropora species, whose eggs do not contain zooxanthellae (Cox and Ward 2002).

In shallow reef-building scleractinians, Harrison and Wallace (1990), Harrison (2006), Harrison (2008), and Harrison (2011) demonstrated an inverse relationship between oocyte size and fecundity. In the bathyal scleractinians from the NE Atlantic, sampled Lophelia pertusa produces relatively large numbers of small oocytes whereas Madrepora oculata produces small numbers of large oocytes. L. pertusa’s pattern is similar to that observed in Oculina varicosa which also produces large numbers of small oocytes (Brooke 2002). The present study detected that site 1, the site impacted with oil pollution and fishing activities, recorded the lowest annual mean of testes number, total testes, egg number and total egg in the two studied species A. humilis and S. pistillata.

This agrees with the laboratory finding of Rinkevich and Loya (1979a) who found that, chronic oil pollution damages the reproduction ability of S. pistillata. However, Guzmán and Holst (2003) evaluated the sublethal effects of oil on coral reproduction using healthy and injured colonies of the reef-building coral Siderastrea siderea at heavily oiled and un oiled reefs. Number of reproductive colonies and number of gonads per polyp were not sensitive to the level of oiling, but gonads were significantly larger at unoiled than oiled reefs during spawning periods. Oil pollution adversely affects sexual reproduction in coral (Harrison and Wallace 1990; Lane and Harrison 2002).

Rinkevich and Loya (1977) found that the fecundity of colonies of the brooding coral S. pistillata was four times greater on an unpolluted reef compared with colonies on a reef subjected to chronic oil pollution near Eilat, Red Sea. Guzman and Holst (1993), and Lane and Harrison (2002) found that corals from the oiled reefs had smaller gonads than those from unaffected areas. Stress could also reduce reproductive output (Brown and Howard 1985; Fabricius et al. 2003; Vermeij et al. 2006, 2010; Torres et al. 2008; Humphery et al. 2008; Randall and Szemant 2009; Harrison 2011) and even cause death in some cases (Ward 1995; Yakovleva et al. 2009).

In the present histological investigation, testes were observed full of sperms during winter season in the sectioned polyps of A. humilis, while eggs were detected during autumn and winter. This coincide with the investigations of Shlesinger and Loya (1985) that the female gonads require about five months (October to

February), while the male gonads require only two months (January and February) for maturity. Different synchronization occurred among hundreds of colonies of 20 Acropora species on equatorial reefs in Kenya where spawning occurred over 2-5 months within populations of different species, and gamete release in Acropora and some faviid species extended over a 9-month period from August to April (Mangubhai and Harrison 2006, 2009; Mangubhai 2009).

The same authors also reported that, A. humilis release gametes during late spring and early summer. Kongjandtre et al. (2010) illustrations partially conflict with the investigations mentioned before, as he reported that the male gonads of A. humilis start their development at the end of January and February and complete their development in April, while female gonads start their development at the end of October before the male gonads become well developd at April. However, Gylnn et al.

(2011) stated that reproduction of corals takes place mainly from March to May when seasonally high sea temperatures and rainfall prevailed in the Galápagos Islands.

In the studied coral species, the ova were developed first before spermeria. Rinkevich and Loya (1979a,b) found that, spermeria have never been found alone but always in association with large number of ova. Oogenesis and spermatogenesis started in different periods, with spermaries appearing in approximately the eighth month of ovary development and lasting about 3 months (Pires et al.

1999; Harrison et al. 2011). The lack of eggs in S. pistillata in the present study occurred only during spring season at all the studied sites. This could be explained by the fact that, the period between December and June was spent in the reproduction process releasing planulae where planulae show distinctive appearance at early spring (Rinkevich and Loya 1979a, b; Shlensinger and Loya 1985; Baird et al.

2010). The gonads development in S. pistillata is differing from A. humilis. However, female gonads in S. pistillata need approximately six months for maturation (July to December) while male gonads require only three months (October to December) (Baird et al. 2010). Richmond and Humter (1990) reported that, the reproduction process in A. humilis and S. pistillata in the Red Sea occurs during spring and summer. However, corals in the inner and eastern Gulf of Thailand spawned following the full moons of February/March, whereas spawning in the southwestern Gulf of Thailand and the Andaman Sea occurred 1 month later following the full moons of March/April (Kongjandtre et al. 2010). In Western Australia, coral spawning occurs predominantly in autumn in contrast to the Great Barrier Reef on Australia’s east coast where most spawning occurs in spring (Baird et al. 2010).

The breeding season of coral reefs differs in both different localities and different species (Baired et al. 2002; Wolstenholme 2004; Rosser and Gilmour 2008; Gilmour et al. 2009; Rosser and Baired 2009). It extends from December to July in the northern Red Sea, Gulf of Aqaba (Shlesinger and Loya 1985) and Southern Red Sea (the present study). While in A. humilis, the breeding season extend from February to June in the Great Barrier Reef (Bothwell 1981), in the gulf of Aqaba (Shlesinger and Loya

AMMAR et al. – Fertility pattern of Stylophora pistillata and Acropora humilis

1985) and in Hurghada (the present study). However, Baird et al. (2010) concluded that the major spawning season of corals on shallow-inshore reefs in the Dampier Archipelago is autumn, although taxa that spawn in spring and summer include Porites sp., Acropora sp., possibly T. mesenterina that are numerically dominant in many of these sites.

Consequently, management initiatives to limit the exposure of coral spawn to stressors associated with coastal development may be required in up to five months per year.

CONCLUSIONS AND RECOMMENDATIONS Patterns of fertilities (total testes and total eggs) for the hermatypic coral A. humilis were lower than those in S. pistillata at the four studied sites. Site 3, the site impacted with phosphate, recorded the highest annual mean of testes number and egg number in the two studied species A. humilis and S. pistillata. However, site 1, the site impacted with oil pollution and fishing activities, recorded the lowest annual mean of testes number, total testes, egg number and total egg for the two studied species. Thus, phosphorus enrichment semms to be considerably less destructive than oil pollution, and thus may represent an ‘eco-friendly’.

Testes were observed full of sperms during winter season in the sectioned polyps of A. humilis, while eggs were detected during autumn and winter. However, the lack of eggs in S. pistillata occurred only during spring season at all the studied sites. In the studied coral species, the ova were developed first before spermeria. The breeding season of coral reefs differs in both different localities and different species extending extending from December to July in the northern Red Sea, Gulf of Aqaba and Southern Red Sea (the present study). While in A. humilis, the breeding season extend from February to June in the Great Barrier Reef, in the Gulf of Aqaba and in Hurghada (the present study).

Tough control, public awareness and continuous shore patrolling to the activities of oil pollution and fishing activities at the vicinity of site 1 are urgent. Site 4 is already virgin, and needs more guarding to keep that site always pristine. Precise review on the effect of phosphate on corals is necessary as results of the present field study is different from most other literatures in that regard.

Although existing corals may continue to grow and survive in an area with elevated nutrients levels, it is essential to maintain water quality on coral reefs within ecologically appropriate limits to ensure successful reproduction of coral and provide recruits for the long-term maintenance and renewal of coral populations. Management strategies to limit the exposure of coral spawn to stressors associated with coastal development is urgent. Scientists should participate in policy debates to improve coral reef legislation and implementation.

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ISSN: 2087-3948 E-ISSN: 2087-3956

Vol. 4, No. 2, Pp. 76-80 July 2012

Effects of foliar application herbicides to control semi-parasitic plant Arceuthobium oxycedri MOHAMMAD REZA KAVOSI, FERIDON FARIDI, GOODARZ HAJIZADEH♥

Department of Forest Sciences, Gorgan University of Agricultural Sciences and Natural Resources, Beheshti St. 386, Gorgan, Golestan, Iran. Tel. /Fax. +98 171 2227867, ♥email: goodarzhajizadeh@gmail.com Manuscript received: 19 June 2012. Revision accepted: 30 July 2012.

Abstract. Kavosi MR, Faridi F, Hajizadeh G. 2012. Effects of foliar application herbicides to control semi-parasitic plant Arceuthobium oxycedri. Nusantara Bioscience 4: 76-80. Epiphytes are plants growing on the stem and branches of other growing plants. Dwarf mistletoe (Arceuthobium oxycedri) is one of the important macro epiphytes or semi-parasitic plant, is able to damage Junipers and provide favorable conditions for bother damaging factors such as pest, disease, rodent animals and vulnerability to unfavorable climate conditions. In this study used herbicides in three concentrations (1.35, 1.93, 2.7 g of Roundup, 0.675, 0.964, 1.35 g of Basagran, and 0.9, 1.28, 1.8 g of Gramoxone in1000 mL water) and three replications to examine the impact of each herbicide on A. oxycedri at Junipers forests in areas located at the East Gorgan state region, North of Iran. The results from Basagran on 95.55% dwarf mistletoe indicated that the plant was dried completely up. Roundup dried 61.67% of dwarf mistletoe. Gramoxone cause the lowest percent of dryness (23.89%). By analyzing results about the impact of herbicides on percentage of measuring dryness, there is difference in level significant 1% between of herbicides. The impact of each herbicide on A. oxycedri showed that concentration-3 has more impact increasing dwarf mistletoe dryness but with concentration-2 it has less difference. Ultimately concentration-2 Basagran can be used to reduce costs in order control dwarf mistletoe.

Key words: herbicide, Arceuthobium oxycedri, epiphyte, dwarf mistletoe, semi-parasitic plant Abstrak. Kavosi MR, Faridi M, Hajizadeh G. 2012. Pengaruh aplikasi herbisida daun untuk memgendalikan tumbuhan semi parasit Arceuthobium oxycedri. Bioscience Nusantara 4: 76-80. Epifit adalah tumbuhan yang hidup pada batang dan cabang tumbuhan lain.

Benalu kerdil (Arceuthobium oxycedri) merupakan salah satu tumbuhan makro-epifit penting atau semi parasit, yang dapat merusak tanaman juniper dan mendorong hadirnya faktor perusak seperti hama, penyakit, hewan pengerat dan keretanan terhadap kondisi iklim yang tidak menguntungkan. Dalam penelitian ini, herbisida dibuat dalam tiga konsentrasi (1,35, 1,93, 2.7 g Round-up, 0,675, 0,964, 1.35 g Basagran, dan 0,9, 1,28, 1.8 g Gromoxone dalam 1000 mL air) dan tiga ulangan untuk mengetahui dampak masing-masing herbisida pada A. oxycedri di hutan juniper Gurgan Timur, Iran. Aplikasi Bazargaran pada benalu kerdil menghasilkan kematian sebesar 95,55% menunjukkan bahwa tumbuhan tersebut mengering seluruhnya. Round-up menyebabkan mengeringnya 61,67% dari benalu kerdil.

Gromoxone menghasilkan persentasre pengeringan terendah (23,89%). Dengan menganalisis dampak herbisida terhadap persentase pengeringan, terdapat perbedaan pada taraf signifikansi 1% di antara herbisida. Dampak masing-masing herbisida pada A. oxycedri menunjukkan bahwa konsentrasi 3 lebih berdampak pada pengeringan benalu kerdil, namun hanya berbeda sedikit dengan konsentrasi 2.

Pada akhirnya konsentrasi 2, Basagran dapat dipilih untuk mengurangi biaya dalam pengendalian benalu kerdil.

Kata kunci: herbisida, Arceuthobium oxycedri, epifit, benalu kerdil, semi-parasit tanaman.

INTRODUCTION Epiphytes are growing on branch and stems of other growing plants. Some of them are parasites; they are not abundant in Iran, because of climate conditions comparing with moderate areas and rainforests (Iranshahr 1999).

Three genus and four species of these semi-parasitic plants has been reported at different areas of Iran, namelyViscum album, Loranthus europae, Loranthus grewinkii, and Arceuthobium oxycedri (Kavosi 2009). Dwarf mistletoes are parasite of different conifers as Pinaceae family including Abies, Keteleeria, Larix, Picea, Pinus, Pseudotsuga, Tsuga and Cupressaceae family including Juniperus (Hawksworth et al. 2002). Dwarf mistletoe (Arceuthobium oxycedri) is one of the important semi-

parasitic plants, acts privately from the host and grows only on conifers (Kamp et al. 2003). This semi-parasitic plant is 20cm high and growing on branches of Junipers, at a glance it confused with moss. Its branches are smooth and articulate. Its leaves are scale-like, triangular and locate opposite on the branches. It is easy to differentiate male and female basis of this color in nature. Males are greenyellow and females are dark green. This plant’s seeds are sticky and splitting the ripe fruit, throw and sit on the host tree’s branch. This seed begins to grow in favorable condition and produce such organism, grows between bark and woods of the host tree’s brand and make new base.

This plant is a parasite of Juniper, spread in Spain, Northern African, Turkey, and North part of Iran (Mousavi and Shimy 1997).

KAVOSI et al. – Herbicide application to control Arceuthobium oxycedri Arceuthobium oxycedri in Iran is parasite of native conifers; it can damage trees in stage pole and small pole (Stewart and Ross 2007). This semi-parasitic plant reduces the crop, produce of seed and wood quality and makes some problems to this tree (attack of pest, fungi and rodents) (Hawksworth et al. 2002). Finally, contamination from dwarf mistletoe will lead to produce compressed mass known as magic broom, which deforms branches. Intensive contamination lead tree to die completely (Hawksworth et al. 1996).

Herbicide selection is the first stage to control dwarf mistletoe. There are some challenges to find herbicide which easily available and are able to ruin dwarf mistletoe and not negative impact on host or other species (Hawksworth and Wiens 1996; Shamoun and Dewald 2002). Some killer herbicides have been tested to control mistletoes (Gill 1956; Quick 1964; Scharpf 1972; Dorji 2007). Most of chemical studies has used different formulas of herbicides such as 2,4,5-T and 2,4-D (amine salt). These herbicides not only kill mistletoes but also exert negative impact on the host and damage it. In low concentrations in which the host is secure parasite not be ruined and growing occur (Shamoun and Dewald 2002).

This research aimed at examines types and concentration of herbicides to kill Arceuthobium oxycedri and protect the host from damaging. In this way, efficient management will achieve about this semi-parasitic plant.

MATERIALS AND METHODS Regions involved in this study are Juniper Forest areas (Juniperus polycarpus) located at the East of Gorgan state region, North of Iran and 2200 m altitude. Conifer species in this area except J. polycarpus are J. sabina and J. communis. Dwarf mistletoe has not been observed on these two species. This showed that A. oxycedri appears only on the Juniperus polycarpus and not on the broad-leave tree.

In this study three types of herbicides (Roundup, Basagran, and Gramoxone) were used to evaluate the effects of each on the Arceuthobium oxycedri. Roundup is a systematic herbicide with SL 41% formula, after weeds sprout, it is used as spread on aired parts of them. This herbicide is taken by leaves and other aired organisms and Transferred to every part of weeds through vessels along with sap, cause weeds to death. This herbicide destroys chloroplast and gradually makes the weed yellow. The central shoot section is twisted but the plant isn’t wounded because the central shoot is exalted. Plant will die 2-4 weeks later. This period is depends on the content of herbicide and plant activity. Bentazone is commercially known as Basagran by SL 48% that is selective herbicide and efficient on weeds after growing. Third of herbicide is Paraquat commercially known as Gramoxone by SL 20%, Local–contact herbicide lead aired organisms to scorch. It is not able to transfer through underground organism. This herbicide is used as a boring against more than one–year– old weeds. Its consumption depends on the weeds mass and varies between 2 and 4 liter in hectare. Gramoxone destroy cellular wall and exit cellular juice, plant seems dew

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(Mousavi 2008). After characterizing scattered organisms of Junipers contaminated by semi-parasitic plants (Arceuthobium oxycedri) in Gorgan, spring (2009) some contaminated basis were chosen. In this study used herbicides in three concentrations (1.35, 1.93, 2.7 g of Roundup, 0.675, 0.964, 1.35 g of Basagran, and 0.9, 1.28, 1.8 g Gramoxone in 1000 mL water) and three replications (Table 1). Semi-parasitic plants were controlled chemically using two methods pre-sprouting and post-sprouting (Rashed-Mohassel and Mousavi 2006). We consider post– sprouting method. Herbicides were exerted to contaminate dwarf mistletoe and two or three weeks later their impacts were appeared. This was impact in clued color, changing leaf falling and Juniper stems and drying rate. In addition to these impacts, by their impacts on host trees, they were recorded to all treatments.

Table 1. Concentrations used of Roundup, Basagran and Gramoxone to control Arceuthobium oxycedri in study location.

Herbicides Roundup Basagran Gramoxone

Concentrations × 1000 mL 1 2 3 1.35 1.93 2.7 0.675 0.964 1.35 0.9 1.28 1.8

RESULTS AND DISCUSSION The drying percentage of Arceuthobium oxycedri using three types herbicides with three different concentrations were obtained by analyzing as seen in Table 2. Results suggest that, Basagran in every concentration 1, 2 and 3 bring about high dying percent but Roundup in concentration-3 has maximum effect (76.67%) and Gramoxone in concentration-2 has the maximum dryness (30%). Basagran dried 95.55% of dwarf mistletoe, which among this herbicide has the maximum impact on this semi-parasitic plant. Also, Gramoxone cause the lowest percent of dryness (23.89%) (Figure 1 and 2).

Table 2. Drying percentage mean of dwarf mistletoe by using three type’s herbicides and three different concentrations.

Herbicides Roundup

Basagran

Gramoxone

Total

Concentration

Mean

SD

N

1 2 3 Total 1 2 3 Total 1 2 3 Total 1 2 3 Total

51.67 56.67 76.67 61.67 93.33 95.00 98.33 95.55 15.00 30.00 26.67 23.89 53.33 60.55 67.22 60.37

18.9297 12.5831 5.7735 16.3936 5.7735 5.0000 2.8867 4.6398 8.6602 5.0000 5.7735 8.9365 35.6195 29.2024 32.1239 31.6813

3 3 3 9 3 3 3 9 3 3 3 9 9 9 9 27

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A

B

C

Figure 1. Efficacy of several herbicides on dwarf mistletoe. A. Basagran, B. Round-up, C. Gromoxone

system and prevent Arceuthobium oxycedri to sprout on the host a year after controlling. The impact of three concentration on drying rate showed that concentration-3 has more impact increasing dwarf mistletoe dryness and it has significant difference with concentration-1 at level significant 5%, but concentration-2 has not significant difference with concentration-1 and 3 (Figure 3).

Table 3. Results of variance analysis in dry Arceuthobium oxycedri that was treatment by using herbicides in different concentrations.

Source Figure 2. Efficacy of herbicides concentrations on dwarf mistletoe

with

three

different

According to Figure 2, it can be seen that Basagran in all concentrates dry Arceuthobium oxycedri in high percentage. The interesting point about this herbicide is that it dries the dwarf mistletoe to almost 50 cm near the poisoning dwarf mistletoe. Variance analysis from Table 3 showed that impacts of type herbicides and their concentration, there is considerable difference among all kind of herbicides at level significant 1% and every concentration at level significant 5%.

The experiment showed that Basagran has the maximum impact and is significant difference at level significant 1% with other herbicides (Figure 3). Based on results from this study, it has priority to others in fighting chemically against dwarf mistletoe. As it dry 100% of this semi-parasitic plant and also it dwarf mistletoe near poisoning branches, this guarantee that sprouting doesnâ&#x20AC;&#x2122;t occur again because the herbicide influence in entophytic

df

SS

MS

F

Sig.

Herbicide 2 23135.185 11567.593 138.811 0.000** Concentration 2 868.519 434.259 5.211 0.016* Herbicide Ă&#x2014; 4 592.593 148.148 1.778 0.177 ns concentration Error 18 1500.000 83.333 Total 26 26096.296 Note: Asterisks (*P < 0.05, **P < 0.01) indicate significant differences between the treatments. Asterisks (nsP > 0.05) indicate not significant differences between the treatments.

Since twentieth century, chemical control of semiparasitic plants has been an innovation. This method is beneficial to reduce these plants development by chosen herbicides (Fathi and Arjmand 1999). Developing chemistry applications in the second half of 19 century and some chemicals entered the selected control phenomenon into the fight against parasite weeds (Rashed-Mohassel and Mousavi 2006). Using herbicides especially in developed countries has been successful and it was examined that this element could solve the problems semi-parasitic plants and weeds (Finney 1988).

KAVOSI et al. â&#x20AC;&#x201C; Herbicide application to control Arceuthobium oxycedri

A

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B

Figure 3. Comparison of types (A) and concentration (B) of herbicides to kill Arceuthobium oxycedri and protect the host from damaging.

In this study, chemical toxins were used to control Arceuthobium oxycedri in order to identify the best toxin results from experiment showed that Basagran prevents dwarf mistletoe to spread because it has no negative impact on Juniper trees as its appearance remain fresh and green but it dried dwarf mistletoe completely and poisoning in seepage entophytic system of plant, prevent it from growing. This is important, because in previous studies; most of herbicides caused the host to dry (Gill 1956; Scharpf 1972; Dorji 2007). But this was not true about Basagran. Similar studies were carried out in which toxins lave not negative impact though they dried some semiparasitic plants, but they couldnâ&#x20AC;&#x2122;t prevent Arceuthobium oxycedri to sprout on the host a year after controlling (Quick 1964; Hawksworth and Wiens 1996). The results showed that concentration-3 has more impact on Juniper dryness and it differ from concentration-1 in level significant 5% but concentration-2 has less difference with concentrations-1 and 3 because of this it can be concluded that it is better to use concentration-2 in Basagran to reduce cost. In such situation lower values are sufficient to reach successful controlling (Griffin et al. 1992; Sullivan and Bouw 1993; Defelice and Kendig 1994). In this study, every herbicide brought about different reacts in lost and semi-parasitic plant. Roundup in various concentrations changed the dwarf mistletoe color (light brown to yellow) after two weeks and Arceuthobium oxycedri separated in stew bands and begun to fall gradually. This herbicide is used to prevent this plant fertility and production because without affecting lost tree it only cause the leaves and stews if A. oxycedri to fall. Basagran in most concentrations and treatments lead dwarf mistletoe to dry not falling from trees and it color became dark brown.

Other impacts of this herbicide dry growing and productive of host (without affecting wood structure) to distance 3050cm from dwarf mistletoe locution to the end of branch and after this distance branch will be fresh and green but Gramoxone in most con concentration has less impact with

little changed color (yellow-green) on productive and growing A. oxycedri and on the host because it delay the plant growth by destroying cellular wall and exiling cellular juice bored the semi-parasitic plant.

Juniperus polycarpus resistance to hand climate conditions and heavy falling snow in standing regions is because of their symmetric and mass crown. A. oxycedri change the crown to broomy form and change their symmetric form and severe trees dryness in contaminated regions by wind snow and cold weather (Hawksworth et al.

1996). Using herbicides, not only they kill dwarf mistletoe, but also return the crown to its normal form and made the plant resistant to hard climate conditions.

One interesting point in this study was that, because A. oxycedri is fresh all the times, it is easier to fight with this semi-parasitic plant. Trees make A. oxycedri poisoning with lowest pollution in environment. To use herbicides, mechanical or combustion machines are applied, but it is better to used mechanical to reduce cost, because junipers are spread in mountainous areas. Using herbicide instead of mechanic deletion to control weeds help to prevent soil erosion by returning material back to it (Rashed-Mohassel and Mousavi 2006). Effective management and analysis on A. oxycedri would control this semi-parasitic plant in coming years and herbicides consumption will be less that now and costs will be minimal (Burnside et al. 1986).

CONCLUSION The effectiveness of three herbicides at various dosages was determined, in study carried out in Gorgan state region, North of Iran during 2009 for the control of dwarf mistletoe, Arceuthobium oxycedri, an aerial parasite of Juniperus oxycedri. Results improved with Basagran dried of dwarf mistletoe, which among this herbicide has the maximum impact on this semi-parasitic plant and Gramoxone cause the lowest percent of dryness.

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REFERENCES Burnside OC, Moomaw RS, Roeth FW, Wicks GA, Wilson RG. 1986.

Weed seed demise in soil in weed free corn. Production across Nebraska 34: 248-251.

Defelice M, Kendig A. 1994. Using reduced herbicide rates for weed control in soybeans. University Extension MP686, University of Missouri, Columbia 5: 834-840.

Dorji S. 2007. Himalayan dwarf mistletoe (Arceuthobium minutissimum) and the leafy mistletoe Taxillus kaempferi on Blue pine (Pinus wallichiana) â&#x20AC;&#x201C; a case study in Western Bhutan. Institute of Forest Entomology, Forest Pathology and Forest Protection.

Fathi G, Arjmand A. 1999. Herbicides and plant physiology. Ferdowsi University Press.

Finney JR. 1988. World crop protection prospects: demisting the crystal ball. Brighton Crop Protection Conference, Pests and Diseases 1: 314.

Gill LS. 1956. Summary of chemicals tested for controlling western species of dwarf mistletoes (Arceuthobium spp). Spokane, Wash 3: 18-25.

Griffin JL, Reynolds DB, Vidrine PR, Saxton AM. 1992. Common cocklebur control with reduced rates of soil and foliar- applied imazaquin. Weed Technol 6: 847-851.

Hawksworth FG, Wiens D. 1996. Dwarf mistletoes: biology, pathology, and systematic. USDA, Forest Service, Agricultural Handbook.

Washington, DC. Hawksworth FG, Wiens D, Geils BW. 2002. Arceuthobium in North America. Department of Agriculture, Forest Service, Rocky Mountain Research Station 4: 29-56.

Hawksworth FG, Wiens D, Geils BW, Rebecca RG. 1996. Dwarf Mistletoes: Biology, Pathology, and Systematic. USDA Forest Service. Washington, DC.

Iranshahr M. 1999. Parasitic and semi-parasitic flower plants of Iran.

Agricultural Research and Education Organization. Tehran.

Kamp VD, Bart J, Shamoun SF. 2003. Biological control of western hemlock dwarf mistletoe. University of Columbia FIA. Kavosi MR. 2009. Damaging of lichen on tree and identification semiparasite of new hosts in Golestan forests. Third Forest National Congress. University of Tehran, Karaj. May 12-14.

Mousavi MR. 2007. Control of weeds (principle and method). Marz Danesh Press.

Mousavi MR, Shimy P. 1997. Parasitic weed of the world. Islamic Azad University Press.

Quick CR. 1964. Experimental herbicidal control of dwarf mistletoe on some California conifers. USDA, Forest Service. Washington, DC. Rashed-Mohassel MH, Mousavi SK. 2006. Principles in weed management. Ferdowsi University Press.

Scharpf RF. 1972. Summation of tests for chemical control of dwarf mistletoe. Medford, Oreg 19: 80-83.

Schweitzer EE, Lybecker DW, Zimdahl RL. 1988. Systems approach to weed management in irrigated crops. Weed Sci 36: 840-845.

Shamoun SF, Dewald LE. 2002. Management strategies for Dwarf Mistletoes: biological, chemical, and genetic approaches. USDA Forest Service Gen. Tech. Rep. RMRS-GTR, 98: 75-82.

Stewart CD, Ross CM. 2007. Embryological and phonological comparison between Arceuthobium americanum (the lodgepole pine dwarf mistletoe) growing on Pinus contorta var. latifolia in British Columbia and on P. banksiana in Manitoba. Department of Biological Sciences, Thompson Rivers University 17 (4): 107-115.

Sullivan J, Bouw WJ. 1993. Reduced rates of postemergence herbicides for weed control in sweet corn. Weed Technol 7: 995-1000.

ISSN: 2087-3948 E-ISSN: 2087-3956

Vol. 4, No. 2, Pp. 81-85 July 2012

Synthesis, characterization and physiological activity of some novel isoxazoles

1

VINAYSINGH J. HUSHARE1, PRITHVIRAJSINGH R. RAJPUT2, MANOJKUMAR O. MALPANI2, NITIN G. GHODILE2

Department of Chemistry, GVISH Amravati, Amravati-444602, Maharashtra India, Tel. +91 0721-2662740 Fax. +91 0721-2662740, email: momalpani@gmail.com 2 Department of Chemistry, Vidya Bharati Mahavidyalaya, C. K. Naidu Road, Camp., Amravati-444602, Maharashtra India Manuscript received: 25 June 2012. Revision accepted: 31 July 2012.

Abstract. Hushare VJ, Rajput PR, Malpani MO, Ghodile NG. 2012. Synthesis, characterization and physiological activity of some novel isoxazoles. Nusantara Bioscience 4: 81-85. A series of chlorosubstituted 4-aroylisoxazoles have been synthesized by refluxing chlorosubstituted-3-aroylflavones and 3-alkoylchromone with hydroxylamine hydrochloride in dioxane medium containing 0.5 mL piperidine. Chlorosubstituted-3-aroylflavones and chlorosubstituted-3-alkoylchromone were prepared by refluxing them separately with iodine crystal in ethanol. Initially chlorosubstituted-3-aroylflavanones and 3-alkoylchromanone were prepared by the interaction of different aromatic and aliphatic aldehydes with 1-(2’-hydroxy-3’,5’-dichlorophenyl)-3-phenyl-1,3-propanedione. Constitutions of synthesized compounds were confirmed on the basis of elemental analysis, molecular weight determination, UV-Visible, I.R. and 1HNMR spectral data. The titled compounds were evaluated for their growth promoting activity on some flowering plants viz. Papaver rhoeas, Calendula officinalise, Gladiola tristis, Gaillardia aristata, Dianthus chinensis, and Iberis sp. (candytuft). The results indicate that applicated plants had higher shoots and more number of leaves.

Key words: chlorosubstituted 3-aroylflavanones, 3-alkoylchromanone, 3-aroylflavones, 3-alkoylchromone, isoxazoles.

Abstrak. Hushare VJ, Rajput PR, Malpani MO, Ghodile NG. 2012. Sintesis, karakterisasi dan aktivitas fisiologis beberapa isoxazoles baru. Nusantara Bioscience 4: 81-85. Serangkaian chlorosubstituted-4-aroylisoxazoles telah disintesis dengan mencampur chlorosubstituted-3-aroylflavones dan 3-alkoylchromone dengan hidroksilamin hidroklorida dalam medium dioksan yang mengandung 0.5 mL piperidine. Chlorosubstituted-3-aroylflavones dan chlorosubstituted-3-alkoylchromone dibuat dengan cara mencampur keduanya secara terpisah dengan yodium kristal dalam etanol. Awalnya, chlorosubstituted-3-aroylflavanones dan 3-alkoylchromanone dibuat melalui interaksi antara aldehida aromatik dan alifatik yang berbeda dengan 1-(2'-hidroksi-3',5'-Dikhlorofenil)-3-fenil-1,3-propanedione.

Senyawa yang terbentuk diuji berdasarkan analisis unsur, berat molekul, serta data spektrum UV-Vis, IR dan 1H-NMR. Senyawa tersebut dievaluasi aktivitas fisiologisnya dalam mendorong pertumbuhan beberapa tanaman berbunga, yaitu: Papaver rhoeas, Calendula officinalise, Gladiola tristis, Gaillardia aristata, Dianthus chinensis, dan Iberis sp. (candytuft). Hasilnya, tanaman yang diaplikasi dengan senyawa tersebut lebih tinggi dan jumlah daunnya lebih banyak.

Kata kunci: chlorosubstituted 3-aroylflavanones, 3-alkoylchromanone, 3-aroylflavones, 3-alkoylchromone, isoxazoles.

INTRODUCTION Heterocyclic compounds are widely distributed in nature and are essential for the sustainable of life on earth (Joule and George 1978). The azoles containing one oxygen and one nitrogen atom at 1, 2-position are designated as isoxazoles. Isoxazole derivatives have been found to possess a broad spectrum of biological activities such as anti-inflammatory (Balvi and Anzaldi et. al. 2005), antibacterial (Magar et al. 2011), anticonvulsant (Muthukumar et al. 2011), antibiotic (Doyle et al. 1961), anti-tubercular (Haripara et al. 2004) anxiolytic (Wagner et al. 2004), antiepileptic (Schwate and Willmre 2001), PPAR- (Epple et al. 2006), agonists, acetylcholineesterase (Mantelingu et al. 2004) and erosive inhibiter (Gupta et al. 2005), antifungal (Flores et al. 2006), antitumer (Ramana and Reddy 2011), analgesic (Osman et al.

2012), chemotherapy (Hamada and Sharshira 2011), antiarthritic (Patterson et al. 1992), antiviral (Goda et al.

2003), anesthetic, anticancer, hypolipidermic (Argyropoulou et al. 2006), antiarrhythmic (Inoue et al.

1991), insect antifeedant (Soni et al. 2006), glycogen phosphorylaseinhibitor (Benltifa et al. 2006), antipsychotic (Markourtz et al. 1999), anticoagulant (Qan et al. 1999), acetylcholine esterase inhibitor (Mantelingu et al. 2004), CNS active (Griesbeck et al. 2011), isoxazole derivatives controlled botrytis cinera on cucumbers (Shionogi and Co. Ltd. 1983) and thrombosis (Padmavathi et al. 2000). One of the isoxazole derivatives was found to have antiviral properties against herpes type-2 virus (Sterling Drug Inc 1982), whereas some isoxazole derivatives also reported to have corrosion inhibitor properties for fuels and lubricants (Marzinzik and Felder 1997).

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The newly synthesized compounds successfully screened for their growth promoting impact on some flowering plants, viz. Papaver rhoeas, Calendula officinalise, Gladiola tristis, Gaillardia aristata, Dianthus chinensis, and Iberis sp.

MATERIALS AND METHODS This research includes synthesis, characterization and physiological activity test of some novel isoxazoles on some flowering plants. The melting points of all synthesized compounds were recorded using hot paraffin bath and are uncorrected. Chemicals used were of A.R. Grade. 1H NMR spectra using acetone. I.R. spectra were recorded on Perkin-Elmer spectrophotometer in the range 4000-400 cm-1 in nujol mull and as KBr pellets. UV-VIS spectrums were recorded in nujol medium.

RESULTS AND DISCUSSION Synthesis of new isoxazoles Synthesis pathway of novel compounds of isoxazoles is showed in Figure 1.

Synthesis of 2-benzoyloxy-3, 5-dichloroacetophenone (3a). 2-Hydroxy-3, 5-dichloroacetophenone (2a) (0.04 mol) and benzoyl chlorides (0.05 mol) were dissolved in 10% NaOH (30 mL). The reaction mixture was shacked for about half an hour. The products thus separated was filtered washed with water followed by sodium bicarbonate (10%) washing and then again with water. The solid product thus separated was crystallized from ethanol to get the compound m.p. 64oC. Synthesis of 1-(-2’-hydroxy-3’, 5’-dichlorophenyl)-3phenyl-1, 3-propanedione (4a). 2-Benzoyloxy-3, 5dichloroacetophenone (3a) (0.05 mol) was dissolved in dry pyridine (40 mL). The solution was warmed up to 60°C and pulverized KOH (15 g) was added to it slowly with constant stirring. Then it was kept for overnight and acidified by adding ice cold HCl (10%). The brownish yellow solid product thus separated was filtered, washed with sodium bicarbonate solution (10%) and finally again with water. It was then crystallized from ethanol to get the compound (4a) m.p. 110oC. Synthesis of 3-benzoyl-2-(4’-nitrophenyl-)-6, 8dichloroflavanone (5a). A mixture of 1-(2’-hydroxy-3’,5’dichlorophenyl)-3-phenyl-1,3-propanedione (4a) (0.01 mol) and p-nitrobenzaldehyde (0.02mol) was refluxed in dioxane (25 mL) containing 0.5 mL piperidine for 15 -20 min. After cooling the reaction mixture was acidified with dil. HCl (20%). The product thus separated was crystallized from ethanol to get the compound (5a). Similarly 3benzoyl-2-(4’-chlorophenyl)-6,8-dichloro-flavanone (5b) and 3-benzoyl-2-butyl-6,8-dichlorochroma-none (5c) were synthesized separately from the compound (4a) by using pchlorobenzaldehyde and valeraldehyde respectively.

Synthesis of 3-benzoyl-2-(4’-nitrophenyl-)-6, 8dichloroflavone (6a). 3-Benzoyl-2-(4’-nitrophenyl)-6, 8dichloroflavanone (5a) (0.01 mol) was refluxed for about

10 minutes with crystal of iodine in ethanol (20 mL). After cooling the reaction mixture was diluted with water. The solid product thus separated was filtered, washed with sodium bicarbonate solution and then with water. Finally it was crystallized from ethanol to get the compound (6a).

Similarly compounds 3-benzoyl-2-(4’-chlorophenyl-)-6, 8dichloroflavone (6b) and 3-benzoyl-2-butyl-6, 8dichlorochromone (6c) were synthesized separately from the compounds (5b) and (5c) respectively.

Synthesis of 3-(2’-hydroxy-3’, 5’-dichlorophenyl)-4benzoly-5-(4’’-nitrophenyl)-isoxazole (7a). A mixture of 3-benzoyl-2-(4’-nitrophenyl)-6,8-dichloroflavone (6a) (0.01 mol) and hydroxylamine hydrochloride (0.02 mol) was refluxed in dioxane (20 mL) containing 0.5 mL piperidine for two hours. After cooling the reaction mixture was diluted with water. The product thus separated was filtered crystallized from ethanol to get the compound (7a).

Similarly other compounds 3-(2’-hydroxy-3’,5’dichlorophenyl)-4-benzoly-5-(4’’-chlorophenyl)-isoxazole (7b) and 3-(2’-hydroxy-3’,5’-dichlorophenyl)-4-benzoly-5butyl-isoxazole (7c) were synthesized separately from the compounds (6b) and (6c) respectively.

Characterization of titled compounds The newly synthesized compounds were characterized on the basis of elemental analysis, molecular determination, U.V., I.R., N.M.R. spectral analysis Compound (5a)-Yield 75%, M.P. 90°C. Elemental analysis for C22H13O5NCl2: Found C = 59.68, H = 2.86, N = 3.10. Calculated C = 59.72, H = 2.94, N = 3.16%) UV spectrum (EtOH), λmax-364 nm ( n  *) IR (cm-1) 3050.5 (C-H stretching in Ar) 1611.4 (C=O), 1473.9 (CNO2), 1278 (-C-O), 1182.6 (-C-O) 717.7 cm-1 (-C-Cl). 1H NMR spectrum (400 MHz, CDCl3), δppm 6.75 (1H, d, CH-CH) 6.9 (1H, d, -CH-CH), 6.875 -8.712 (11H, m, ArH) (Silverstein, Bassler and Morril 1991).

Compound (5b)-Yield 70%, M.P. 120°C. Elemental analysis for C22H13O3Cl3: Found C = 61.10, H = 2.96,.

Calculated C = 61.18, H = 3.01, %. UV spectrum (EtOH), λmax -319 nm ( n  *) IR (cm-1) 2928.6 (C-H stretching in Ar) 1612.6 (C=O), 1427.7 (C-H in Ar), 1086.5 (C-O), 764.6 cm-1 (-C-Cl). 1H NMR spectrum (400 MHz, CDCl3), δppm 6.76 (1H, d, -CH-CH) 6.79 (1H, d, -CH-CH), 6.9268.315 (11H, m, Ar-H).

Compound (5c)-Yield 75%, M.P. 155°C. Elemental analysis for C20H18O3Cl2: Found C = 63.54, H = 4.50, Calculated C = 63.66, H = 4.77, %. UV spectrum (EtOH), λmax -363 nm ( n  *) IR (cm-1) 3068.7 (C-H stretching in Ar) 2866.4 (C-H stretching in Ar), 2866.4 (C-H in (CH2)3-), 1689.5 (C=O), 1605.8 (C-H stretching in Ar) 1457.7 (-CH3 Bending), 1286.3 (C -O in ether), 705.4 cm-1 (-C-Cl). 1H NMR spectrum (400 MHz, CDCl3), δppm 0.90 (3H, t, (CH2)3-CH3) 1.25 (2H, m (CH2)2-CH2 -CH3) 1.50 (2H, t, -CH2-CH2-CH2-CH3) 2.65 (2H, t, CH2-(CH2)2-CH3) 6.88 (1H, d, C-H), 6.93 (1H, d, C-H) 7.00 -8.225 (7H, ,m, Ar-H).

Compound (6a)-Yield 80%, M.P. 164°C. Elemental analysis for C22H11O5NCl2: Found C = 59.00, H = 2.30, N = 3.00, Calculated, C = 60.00, H = 2.50, N = 3.18, %).

HUSHARE et al. – Synthesis, characterization and physiological activities of some novel isoxazoles OCOCH3

OH

AlCl3

(CH3CO)2O CH3COONa

Cl

83

1200 C

Cl II

I

Cl OH

OH

C6H5COCl Cl

COCH3

Cl

NaOH 10%

Cl2 in Acetic acid

COCH3 ( 2a)

(Ia ) Cl

Cl OCOC6H5

OH R-CHO

KOH / BVT Pyridine Cl

COCH3

COCH2COC6H5

Cl

Ethanol, Pyridine

( 4a)

( 3a )

Cl

Cl O

R

O

I2 Ethanol, COC6H5

Cl

R

COC6H5

Cl O

O

(6a--6c )

(5a---5c )

Cl OH COC6H 5

NH 2OH.HCl Ethanol, piperidine

Cl

R

N

O

( 7a---7c )

Where R= C6H5-NO2, C6H5-Cl, (CH2)3-CH3 Figure 1. Synthesis of novel compounds of isoxazoles

Compound (6b)-Yield 75%, M.P. 168oC. Elemental analysis for C22H11O3Cl3: Found C =61.14, H = 2.20 Calculated C =61.18, H = 2.56%. UV spectrum (EtOH), λmax—345 nm ( n  *) IR (cm-1), 2917.8 (C-H stretching in Ar), 1639.5 (C=O),1604.1 (>C=O), 1091.1 (C-O in ether ), 768.2 cm-1 (-C-Cl). 1H NMR spectrum (400 MHz, CDCl3), δppm 6.848-8.319 (11H, m, Ar-H).

Compound (6c)-Yield 80%, M.P. 162°C. Elemental analysis for C20H16O3Cl2: Found C = 63.60, H = 4.10, Calculated C = 64.00, H = 4.27, %. UV spectrum (EtOH), λmax 301nm ( n  *) , IR (cm-1) 2942.5 (Ar -CH), 2851.7 (CH str. in alkane), 1637.9 (>C=O), 1431.7 (CH2bending),1345.8 (-CH3 bending) 1250.2 (-C-O in ether) 765.5 cm-1 (-C-Cl). 1H NMR spectrum (400 MHz, CDCl3), δppm 1.285 (3H, s, -(CH2)3-CH3), 2.050 (2H,qui, (-CH2)2-CH2-CH3), 2.808 (2H,m,H2-CH2-CH2-CH3), 2.839 (2H,t, CH2-(CH2)2-CH3) Compound (7a)-Yield 80%, M.P. 148oC. Elemental analysis for C22H12O5N2Cl2: Found C =57.80, H =2.40,

N=6.00, Calculated C = 58.02, H = 2.64, N=6.15 %).

Compound (7b)-Yield 85%, M.P. 1600C. Elemental analysis for C22H12O3NCl3:Found C =59.15, H =2.50, ,N=3.00, Calculated C =59.39, H =2.69, ,N=3.14 %. UV spectrum (EtOH), λmax 312nm, IR (cm-1) 3021 (O-H stretching), 1623 (>C=O), 1464.1 (>C=N), 1275.1 (C-ON), 763 cm-1 (-C-Cl). 1H NMR spectrum (400 MHz, acetone), δppm 6.915-8.129 (11H, m, Ar-H).

Compound (7c)-Yield 80%, M.P. 165°C. Elemental analysis for C20H17O3NCl2:Found C = 61.40, H = 4.20, ,N=3.40 Calculated C = 61.53, H = 4.35, N=3.60, %. UV spectrum (EtOH), λmax 303nm ( n  *) , IR (cm-1) 3351.4 (O-H streraching), 2938.5 (C-H in alkane), 1641.4 (>C=O), 1444.1 (-CH2 bending), 1351.5 (-CH3 bending), 1219 (C-O-N),768.7 cm-1 (-C-Cl). 1H NMR spectrum (400 MHz, acetone), δppm 1.282 ( 3H, t, -(CH2)3-CH3), 2.050 (2H, s, -CH2-CH2-CH2-CH3), 2.845 (2H, s, -CH2-CH2CH2-CH3 ), 2.874 ( 2H, s, -CH2-CH2-CH2-CH3), 6.910 to 8.122 ( 8H, m, Ar-H).

84

4 (2): 81-85, July 2012

Growth promoting activities on some flowering plants The experimental set up of the study was divided into seed treatment and field experiment. (i) Seed treatment: with a view to safeguard dormant seed’s potential from harmful external agencies, the seed of the test plants were treated by test compounds before sowing. (ii) Field experiment: pre-germinated quality seeds of P. rhoeas, C. officinalise, G. tristis, G. aristata, D. chinensis and Iberis sp. were procured from genuine agricultural agencies.

The beds of black cotton soil, 2.5 x 2.5m size were prepared on an open field. The sowing of seeds of all six flowering plants under examination were done in beds and in earthen pots separately by conventional methods and irrigated as and when required. The plants from each bed and pot were divided into two groups, i.e. A and B which were designated as ‘control’ and ‘treated’ group plants respectively. The plants from group B were sprayed with the solution of test compounds at fortnightly intervals. The field experiments were conducted to compare the treated plants of group B with untreated plants of controlled group

A. In this context, the observation were recorded on 15, 30, 45, 60, 75 and 90 days after sowing; corresponding to early vegetative, late vegetative, flowering, pod filling and pod maturation stages, with special reference to number of leaves and height of shoots. The results of field’s experiments with test compounds are tabulated in Table 1.

Data presented in Table 1 clearly indicates that shoot heights of P. rhoeas, C. officinalise, G. tristis, G. aristata, D. chinensis and Iberis sp. up to 45 days did not show any significant increase. However, the shoot heights increased to a remarkable extent from 60 days onwards after sowing and it continued with the same drift up to 90 days in almost all the treated plants as compared to the plants of control group. This trend of substantial increase in shoot height geared up in P. rhoeas and C. officinalise right from the second week of the experiment. It has been found that control group plants overshadowed this trend in the third observation only in the plants P. rhoeas and C. officinalise treated with compound 4a.

Periodicity of the observation (in days)

Table 1. Activity of the test compounds (4a), (5a), (6a), (7a) Poppy (P. rhoeas)

Calendula (C. officinalise)

Gladiolus (G. tristis)

Gaillardia (G. aristata)

Pink (D. chinensis)

Candytuft (Iberis sp)

Shoot height

No. of leaves

Shoot height

No. of leaves

Shoot height

No. of leaves

Shoot height

No. of leaves

Shoot height

No. of leaves

Shoot height

No of leaves

C

C

C

C

C

C

C

C

C

T

C

T

C

C

Activity of the test compound 1-(2’-hydroxy-3’, 5’-dichlorophenyl)-3-phenyl-1, 3-propanedione (4a) 15 3 3 9 10 2 1 9 4 2 1 1 1 4 3 4 5 2 30 7 5 15 15 4 7 18 18 6 5 2 2 6 8 15 8 5 45 10 10 20 25 8 10 30 30 10 9 4 2 8 15 30 20 7 60 12 12 22 30 10 16 55 55 15 16 5 3 12 18 50 38 10 75 16 17 30 47 13 20 80 85 20 20 6 4 16 20 70 60 13 90 20 23 50 78 15 21 100 115 22 21 6 4 20 21 105 95 18

2 4 8 12 16 20

4 10 15 25 34 50

4 3 2 9 8 6 4 15 16 8 7 45 29 12 11 60 41 15 15 100 63 18 19 130

5 20 40 70 105 150

Activity of the test compound 3-benzoyl-2-(4’-nitrophenyl)-6, 8-dichloroflavanone (5a) 15 3 3 9 7 2 2 9 12 2 2 1 1 4 2 4 30 7 6 15 10 4 5 18 28 6 5 2 2 6 5 15 45 10 12 20 20 8 11 30 40 10 9 4 2 8 8 30 60 12 18 22 25 10 15 55 60 15 16 5 5 12 10 50 75 16 22 30 38 13 20 80 100 20 21 6 5 16 12 70 90 20 26 50 48 15 22 100 130 22 25 6 5 20 18 105

5 8 20 38 60 80

2 5 7 10 13 18

2 4 7 9 12 17

4 10 15 25 34 50

2 3 4 9 6 6 8 15 12 8 12 45 18 12 15 60 26 15 19 100 45 18 24 130

10 27 62 100 145 205

Activity of the test compound 3-benzoyl-2-(4’nitrophenyl)-6, 8-dichloroflavone (6a) 15 3 3 9 8 2 3 9 14 2 2 1 1 4 4 30 7 7 15 15 4 6 18 30 6 5 2 2 6 6 45 10 12 20 38 8 10 30 50 10 11 4 3 8 10 60 12 16 22 50 10 14 55 70 15 15 5 3 12 14 75 16 20 30 60 13 18 80 90 20 21 6 5 16 18 90 20 24 50 100 15 20 100 120 22 23 6 5 20 21

6 2 8 5 27 7 50 10 80 13 140 18

3 7 10 15 20 21

4 10 15 25 34 50

5 3 3 9 12 6 7 15 18 8 12 45 29 12 15 60 45 15 19 100 70 18 23 130

8 18 56 98 135 200

T

T

T

T

T

T

T

4 15 30 50 70 105

T

Activity of the test compound 3-(2’-hydroxy-3’, 5’-dichlorophenyl)-4-benzoyl-5-(4’’-nitrophenyl) isoxazole (7a) 15 3 2 9 7 2 2 9 10 2 3 1 1 4 3 4 6 2 2 4 3 30 7 4 15 12 4 5 18 22 6 10 2 3 6 6 15 14 5 4 10 15 45 10 8 20 20 8 8 30 48 10 12 4 3 8 10 30 40 7 8 15 25 60 12 14 22 45 10 13 55 77 15 16 5 5 12 15 50 60 10 12 25 49 75 16 18 30 60 13 17 80 135 20 20 6 6 16 19 70 90 13 16 34 76 90 20 24 50 95 15 20 100 180 22 23 6 7 20 23 105 135 18 20 50 120 Note: C = control, T = treated

3 6 8 12 15 18

T

T

4 9 10 6 15 30 11 45 50 18 60 120 21 100 172 23 130 210

HUSHARE et al. – Synthesis, characterization and physiological activities of some novel isoxazoles

The treatment of compound 5a showed very positive results in the increase of shoot height in all test plants as compared to control group plants except in the D. chinensis plant that has been mentioned in the 2nd observation. The treatment of compound 6a promoted shoot height of test plants to a great extent except in P. rhoeas whereas this plant started responding to the treatment of titled compound 6a after 6th week onward only. The same trend was retained by the test plants on treatment with the titled compound 7a.

The data about the foliage of the test plants depicted in Table 1 revealed that there was a remarkable increase in the number of leaves in treated plants as compared to control group plants. However, in the first phase of the experiment there was no significant difference between treated and control group plants of P. rhoeas, and C. officinalise. But the fifth week onwards the foliage density gradually increases and it shoots up in the later phase of the study to a considerable extent as compared to the control group plants.

CONCLUSION The synthesized compounds were screened for their growth promoting activity on some flowering plants viz: Papaver rhoeas, Calendula officinalise, Gladiola tristis, Gaillardia aristata, Dianthus chinensis and Iberis sp. The efforts have been made to examine and analyze the morphology of treated plants. When the comparison of morphological characters was made between those of treated and control groups plants, it was interesting to note that all the plants exhibited significant shoot growth, and considerable increase in the number of leaves as compared to those of untreated ones.

ACKNOWLEDGEMENTS The authors are thankful to SAIF, CDRI, Lucknow for providing the spectral data. We also thank to the eminent faculty members of the Dr. Panjabrao Deshmukh Krishi Vidyapeeth (Dr. P.D.K.V.) Akola, India, namely Prof. Dr. R.M. Gade and M.S. Gaikwad from Department of Plant Pathology, Dr. D.H. Paithankar from Department of Horticulture, and Dr. Ashish U. Nimkar from Department of Forestry for providing the necessary help for the completion of interdisciplinary part of the present work.

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ISSN: 2087-3948 E-ISSN: 2087-3956

Vol. 4, No. 2, Pp. 86-96 July 2012

Review: Mycoendophytes in medicinal plants: Diversity and bioactivities 1

MAHENDRA RAI1,, ANIKET GADE1, DNYANESHWAR RATHOD1, MUDASIR DAR1, AJIT VARMA2

Department of Biotechnology, Sant Gadge Baba Amravati University, Amravati-444602, Maharashtra India. Tel: +91-721-2662206 to 8, Fax: +91-7212662135, 2660949, email: mkrai123@rediffmail.com 2 Amity Institute of Microbial Technology, Amity University, Sector 125, Noida, Uttar Pradesh, India Manuscript received: 23 June 2012. Revision accepted: 16 July 2012.

Abstract. Rai M, Gade A, Rathod D, Dar M, Varma A. 2012. Review: Mycoendophytes in medicinal plants: Diversity and bioactivities.

Nusantara Bioscience 4: 86-96. Endophytes are microorganisms that reside in internal tissues of living plants without causing any negative effect. These offer tremendous potential for the exploitation of novel and eco-friendly secondary metabolites used in medicine, the pharmaceutical industry and agriculture. The present review is focused on diversity of endophytes, current national and international bioactive secondary metabolite scenario and future prospects. Endophytic fungi as novel source of potentially useful medicinal compounds are discussed along with the need to search for new and more effective agents from endophytes to combat disease problems Key words: fungal diversity, mycoendophyte, medicinal plants, secondary metabolites.

Abstrak. Rai M, Gade A, Rathod D, Dar M, Varma A. 2012. Review: Mikoendofit pada tumbuhan obat: Keanekaragaman dan bioaktivitasnya. Nusantara Bioscience 4: 86-96. Endofit adalah mikroorganisme yang tinggal di dalam jaringan internal tumbuhan hidup tanpa menimbulkan efek negatif. Hal ini berpotensi sangat besar untuk menghasilkan metabolit sekunder baru dan ramah lingkungan yang digunakan dalam bidang kedokteran, industri farmasi dan pertanian. Ulasan ini difokuskan pada keanekaragaman endofit, perkiraan kebutuhan bioaktif metabolit sekunder secara nasional dan internasional pada saat ini dan prospeknya di masa depan. Fungi endofit sebagai sumber baru senyawa obat potensial berguna dibahas bersama dengan perlunya menemukan agen endofit baru dan lebih efektif untuk memerangi masalah penyakit.

Kata kunci: keanekaragaman fungi, mikoendofit, tumbuhan obat, metabolit sekunder

INTRODUCTION Endophytes are microbes (fungi or bacteria) that live within the plant tissues without causing any noticeable symptoms of disease (Tejesvi et al. 2007). We propose a new term “Mycoendophytes” for fungi living as endophytes in plants. According to Rodriguez et al. (2009) endophytes are classified into four classes. Class 1 endophytes are clavicipitaceous while class 2-4 are nonclavicipitaceous. Class 1 endophytes of grasses were first reported by European investigators in the late 19th century in seeds of Lolium temulentum, L. arvense, L. linicolum and L. remotum. From their first finding, investigators hypothesized that an association to toxic syndromes experienced by animals that consume infected tissues.

However, these hypotheses were largely untested until Bacon et al. (1977) linked the endophyte Neotyphodium coenophialum to the widespread occurrence of ‘summer syndrome’ toxicosis in cattle grazing tall fescue (Festuca arundinaceae) pastures. Class 2-4 which includes nonclavicipitaceous endophytes, showed the ability to colonize asymptomatically and confer habitat-adapting fitness benefits on genetically distant host species that may include both monocots and dicots. Most of these fungal endophytes

belongs to Ascomycota, with a marginal of Basidiomycota.

Class 2 endophytes within the Basidiomycota are members of the Agaricomycotina and Pucciniomycotina. Class 2 endophytes are dissimilar from the previous nonclavicipitaceous (NC) endophytes; because in general they colonize roots, stems and leaves and are capable of forming extensive infections within plants. Class 2 endophytes are transmitted via seed coats and or rhizomes; having low abundance in the rhizosphere Class 2 endophytes have confer habitat-adapted fitness benefits in addition to nonhabitat-adapted benefits; and typically have high infection frequencies (90-100%) in plants growing in highstress habitats (Rodriguez et al. 2009). Class 3 endophytes comprise the hyper diverse endophytic fungi associated with leaves of tropical trees, moreover the highly diverse associates of above-ground tissues of nonvascular plants, seedless vascular plants, conifers and woody and herbaceous angiosperms in biomes ranging from tropical forests to boreal and Arctic/Antarctic communities (Rodriguez et al. 2009). Class 4 endophytes are mainly ascomycetous fungi that are conidial or sterile and that form melanized structures such as inter- and intracellular hyphae and microsclerotia in the roots (Rodriguez et al.

2009).

RAI et al. â&#x20AC;&#x201C; Mycoendophytes in medicinal plants

Mycoendophytes have been found in healthy tissues of all the plant taxa studied to date. Endophytes invade the tissues of living plants and reside in the tissues between living plant cells (Vanessa and Christopher 2004). Some form a mutually beneficial relationship (symbiosis) with the host plants while others are opportunistic pathogens.

Petrini et al. (1992) reported that there may be more than one type of mycoendophytes found within a single plant.

For example, thirteen taxa of mycoendophyte were isolated from the leaf, stem and root tissues of Catharanthus roseus (Kharwar et al. 2008).

Herre et al. (2007) demonstrated that mycoendophyte plays potentially important mutualistic role by augmenting host defense response against pathogens. Endophytes may contribute to host protection by increasing the expression of intrinsic host defense mechanisms and or providing additional sources of defense extrinsic to those of the host.

There has been immense interest in the prospecting for these microbial endophytes as source of novel bioactive natural products. Endophytes do show much chemical diversity: alkaloids, peptides, steroids, terpenoids, isocoumarins, quinones, phenylpropanoids, lignans, phenols, phenolic acids, aliphatic compounds, lactones, and others. After the discovery of taxol produced by Taxomyces andreanea from Taxus brevifolia, interest in endophyte research has increased at a fast pace. T. brevifolia is a member of family Taxaceae and native to the north-western United States. The natural product of T. brevifolia taxol has been used in the treatment of cancer treatments. Isolation of taxol from Pestalotiopsis microspora, an endophyte of Taxus wallichiana and phytohormone gibberellin from Gibberella fusikuroi in rice plants underlines the potential of endophytes as a source of useful metabolites (Gehlot et al. 2008). The screening of microbial culture filtrates for the presence of secondary metabolites is an established method for the identification of biologically active molecules (Hamayun et al. 2009).

DIVERSITY OF MYCOENDOPHYTES Mycoendophytes are the most frequent endophytes isolated from medicinal plants. Dreyfuss and Chapela (1994) estimated that there may be at least 1 million species of mycoendophytes. Shipunov et al. (2008) tested the co-introduction and host-jumping hypotheses in plant communities by comparing endophytes isolated from the invasive spotted knapweed (Centaurea stoebe, Asteraceae) in its native and invaded ranges. Shipunov and his group (2008) indicated that endophytes can affect the competitiveness of C. stoebe. As both co-introduction and host-jumping of endophytes align with hypotheses of plant invasion that are based on enhanced competitiveness.

Kharwar et al. (2008) reported 183 mycoendophytes representing 13 fungal taxa isolated from leaf, stem and root tissues of C. roseus from two sites representing two different ecosystems in North India. The leaf tissues showed more diversity of endophytes like Drechslera, Curvularia, Bipolaris, Alternaria and Aspergillus spp. Wei et al. (2009) studied the colonization frequencies of

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endophytic Pestalotiopsis species diverse with host plants, ages, tissues and sites. Ya-li et al. (2010) reported 49 endophytic fungi which were recovered from Saussurea involucrata and identified using morphological and molecular techniques. Among theses fungi Cylindrocarpon sp. was the dominant species followed by Phoma sp. and Fusarium species. Li and Shun (2009) reported the recovery of 300 isolates in which 172 isolates were from Dracaena cambodiana and 128 from Aquilaria sinensis. Proksch et al. (2009) reported bioactive marine natural products isolated from marine sponges and marine derived fungi.

The maximum biological diversity in terrestrial ecosystems is in tropical and temperate rainforests; interestingly, they also have the greatest number of mycoendophytes. These ecosystems cover only 1.44% of the landâ&#x20AC;&#x2122;s surface, yet they harbour more than 60% of the worldâ&#x20AC;&#x2122;s terrestrial biodiversity (Strobel and Daisy 2003).

Hazalin et al. (2009) isolated 300 endophytes from various parts of plants collected from the National Park, Penang in Malaysia. Some of these endophytes demonstrated cytotoxic activity against the murine leukemic P388 cell line and 1.7% against a human chronic myeloid leukemia cell line K562 (Hazalin et al. 2009).

Strobel (2002) reported that fungal endophytes residing within plants could also produce metabolites similar to or with more activity than those of their respective hosts.

Therefore, the search for novel compounds should be directed towards plants that are used by indigenous populations for medicinal purposes, or plants growing in extreme environments, or those that are endemic. These are most likely to harbour novel endophytes that may produce unique metabolites (Strobel and Daisy 2003; Zhang et al.

2006; Deshmukh and Verekar 2008). Survey of literature provides an evidence of increasing research on endophytes and their secondary metabolites. Many endophytes produce important secondary metabolites, which play protective role against insect herbivores or are of industrial importance (Hawksworth et al. 1995; Arnold et al. 2003) (Table 1).

Isolation of endophytic fungi from coffee plants (Coffea arabica and C. robusta) was shown to have antimicrobial activity against various human pathogenic bacteria (Sette et al. 2006). Coffea arabica of family Rubiaceae is luxuriantly cultivated in Southern India, Madras, Mysore, Coorg, Travancore and Cochin. C. arabica is being used as an Analgesic, cardiotonic, CNS stimulant, nervine, stimulant and hypnotic. Large differences have been observed amongst the isolates with respect to their ability to produce metabolites having antimicrobial activity (Pelaez et al. 1998). At present, there is an urgent need to search for endophytic metabolite that can be developed as safe, effective antifungal agents that are non-petrochemical, eco-friendly and easily obtained (Liu et al. 2006).

Bacon et al. (1977) demonstrated the correlation between the presence of the mycoendophyte, Epichloe typhina isolated from Festuca arundinacea (Tall fescue) and the toxicity of its host to herbivorous domestic mammals. Further, they observed that several toxins produced by endophytic fungi and conferred host protection against different herbivores. Mycoendophytes were isolated from the toxic locoweeds Astragalus mollissimus,

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Table 1. Endophytes isolated from different medicinal plants Use of plant in traditional medicinal Alternaria alternata Vitis vinifera Blood circulation, eye (Grapevine) problems Alternaria sp., Opuntia (Cactus species) Wound healing Phoma spp.

Aspergillus flavus, Calotropis procera Asthma, leprosy, and in Phoma sp., (Milkweed, chronic eczema Penicillium Rubber bush) Botryosphaeria Bidens pilosa Reduces swelling, headache, rhodina (Spanish needle) Clears heat and toxins Endophytes

Host

Location

Bioactivity

Reference

Europe

Antifungal

Musetti et al. 2006

America

Antiviral

North Africa

Antimicrobial

Suryanarayanan et al.2005 Khan et al. 2007

Tropical America

Antifungal, cytotoxic and antiproliferative Antimalarial

Colletotrichum Artemisia annua gloeosporioides (Chinese wormwood) Colletotrichum sp., Plumeria rubra Phyllosticta sp.

(Red Frangipani)

Malaria

Colletotrichum species Curvularia lunata

Banana, Ginger, (Jamaica ginger) Azadirachta indica (Neem) Cylindrocarpon sp., Saussurea involucrata Phoma sp., Fusarium sp.

Entrophospora Nothapodytes foetida infrequens (Stinking tree) Fusarium Dianthus caryophyllus oxysporum (Carnation, Divine flower) Fusarium Juniperus recurva. oxysporum (Himalayan juniper) Fusarium solani Apodytes dimidiata (White pear)

Blood, cholesterol thinning, in heart disease Skin disease diabetic

South Asia

Antibacterial

India

Antifungal

rheumatoid arthritis, cough with cold, stomachache

Asia, Europe

Cancer

India

gastrointestinal system, improve heart health

Mediterranean region

Guignardia sp.

Eye inflammation, diarrhea, venereal diseases

Southern Africa Anticancer (topotecan and irinotecan America Antiviral, Antibacterial

Cinnamomum zeylanicum (Cinnamon) Muscodor albus Guazuma ulmifolia (Bastard cedar) Muscodor crispans Ananas ananassoides (Wild pineapple) Mycorrhizal fungi Rhododendron tomentosum (Labrador tea) Neotyphodium sp. Festuca arundinacea (Tall fescue) Nigrospora sp., Aegle marmelos Alternaria sp.

(Bel, Siriphal) Paecilomyces sp.

Torreya grandis (Chinese nutmeg yew)

Oldest herbal medicines

Sri Lankan

Antifungal Antibacterial

Weight loss, bleeding, childbirth, cold, cough Gastric pain

America

Antibacterial.

Antifungal Anti-tuberculosis

Penicillium commune Penicillium crysogenum, Aspergillus fumigates Pestalotiopsis microspora Pestalotiopsis pauciseta Phoma capitulum

Spondias mombin (Java plum)

Muscodor albus

China

Venereal disease rheumatism, Central America Antimicrobial diarrhoea, and leprosy

For long-continued vomiting Pakistan Intestinal parasites ear inflammation

Bolivia, Brazil

Randa et al. 2010 Tan and Zou 2001 Suryanarayanan and Thennarasan 2004 Photita et al. 2005

Verma and Kharwar 2006 Antimicrobial, anti- Ya-li et al. 2010 Antileukaemia and antitumor Antimicrobial

Amna et al. 2006

-

Kour et al. 2008

Postma and Rattink 1991

Shweta et al. 2010 RodriguesHeerklotz et al. 2001 Strobel 2006 Strobel et al. 2007 Mitchell et al.

2010 Kajula et al. 2010

coughs, dyspepsia, dysentery, North America leprosy, itch

antibacterial and antioxidant

-

Europe

-

Wound healer, scurvy.

India

Antimicrobial

Skin infections

China

Huang et al. 2001

Hibiscus tiliaceus

Fevers, coughs

Southeast Asia

Antitumor, antifungal, antiinflammation Antimicrobial

Catharanthus roseus (Sadabahar)

Blood clotting, eyewash, diabetes

India

Anticancer

Kharwar et al.

2008

Terminalia morobensis

anti-oomycetic

Antifungal, antioxidant Expectorant, antimicrobial Antispasmodic,

Harper et al. 2003

Tabebuia pentaphylla Justicia gendarussa

Papua New Guinea flu, cold and easing smoker's Mexico cough Cough, fever China

Sugawara et al.

2004 Gond et al. 2007

Yan et al. 2010

Vennila et al.

(2010) Gangadevi and

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Phoma eupyrena, Cladosporium cladosporioides Phoma medicaginis Phoma sorghina

Azadirachta indica (Neem)

treatment of pains in the head, paralysis Skin disease diabetic, antibacterial, antiviral

Medicago sp.

Tithonia diversifolia (Mexican sunflower)

digestive tract and kidneys Sprains, bone fractures, hepatitis

Italy West Africa

Antimicrobial Antimicrobial

Phoma sp.

Fagonia cretica (Dhamsha)

Alicante, Spain

Antifungal, algicidal Krohn et al. 2007

Phoma sp.

Saurauia scaberrinae (Guinea plant) Coffea arabica

Fever, vomiting, dysentery, typhoid, toothache, stomach troubles, skin diseases Fever and in holistic health care Stimulant and hypnotic.

Analgesic, cardiotonic Skin disease

Australia

Antibacterial

India

Antimicrobial

Hoffman et al.

2008 Sette et al. 2006)

Japan

Antimicrobial

Hata et al. 2002

Childbirth, wound healing, diarrhea and dysentery asthma Poisonous snake bites, boils and ulcers fever, headache Cardiac remedy

Tropical America

Antimicrobial

Srinivasan et al.

2010

China

Zhao et al. 2010

Europe

Antibacterial activity Anti cancer

Cancer

North America

Anticancer

Bashyal et al.1999 Stierle et al.1993

Cancer

North America

Anticancer, lung cancer

Wiyakrutta et al.

2004

Phomapsis Phyllosticta sp., Colletotrichum sp.

Phyllosticta sp.

Pasania edulis (Japanese oak) Guazuma tomentosa (Mutamba)

Pichia guilliermondii Seimatoantlerium nepalense Taxomyces andreanae Taxomyces andreanae

Paris polyphylla (Satuwa) Taxus wallichiana (Himalayan yew) Taxus sp.

(Yew plant) Taxus brevifolia (Pacific yew)

Oxytropis lambertii and Oxytropis sericea. It is native of northern China and Mongolia. Astragalus mollissimus has been used for anaemia and blood disorders, blenorrhea (profuse mucous discharge from the vagina or urethra), as an ointment for burns and to stimulate the immune system and suppress tumors. Moreover, it is used to treat chronic weakness of the lungs with shortness of breath, collapse of energy, prolapse of internal organs, spontaneous sweating, chronic lesions and deficiency edema. These produce the alkaloid swainsonine that causes locoism, a disease of ruminant animals (Braun et al. 2003). Thus, some endophytic fungi produce novel secondary metabolites of industrial potential (Schulz et al. 2002; Worapong et al.

2002) while others enhance the fitness of their host plants (Redman et al. 2002). The mycoendophyte Taxomyces andreanae, which produces taxol in vitro, was isolated from Taxus sp. (Stierle et al.1993). Vennila et al. (2010) studied the effect of taxol extracted from the endophytic fungus Pestalotiopsis pauciseta recovered from Tabebuia pentaphylla Hems. T. pentaphylla (family Bignoniaceae) is distributed in northern Mexico, Southern Florida and Cuba.

The generic name is derived from the words used for the trees by the indigenous peoples of Brazil. It is used against flu, cold and easing smoker's cough. Apparently it acts as expectorant, by promoting the lungs to cough up and free deeply embedded mucus and contaminants.

Zhou et al. (2010) summarized the recent advances in taxol-producing endophytic fungi all over the world. Kajula et al. (2010) studied the extracellular siderophore production as well as production of antibacterial and antioxidant compounds by endophytic fungi of Scots pine (Pinus sylvestris L.) and Labrador tea (Rhododendron

carminative

Muthumary 2008

India

Antimicrobial

Mahesh et al. 2005 Weber et al. 2004 Borges and Pupo 2006

tomentosum Harmaja). The pinolenic acid contained in pine nut oil helps curb appetite. It is used as a pain reliever in arthritis, aches, pains and sore muscles. It is important remedy for bladder, kidney, and rheumatic affections, diseases of the mucous membrane and respiratory complaints; externally in the form of liniment plasters and inhalants. Labrador tea is useful in coughs, dyspepsia, and irritation of the membranes of the chest. An infusion of the tea has been used to soothe irritation in infectious, feverish eruptions, in dysentery, leprosy and itch, etc.

Yang et al. (1994) reported that the phenol and phenolic acids, detected in culture medium of endophytes, often have pronounced biological activities. 2-Hydroxy-6methylbenzoic acid was isolated from endophytic Phoma species which showed remarkable antibacterial activity.

Phoma medicaginis exists as a prolonged asymptomatic infection of its host plant (Medicago species). In early Chinese medicines, physicians used young leaves of Medicago species to treat disorders related to the digestive tract and the kidneys. It produces significant levels of the toxin Brefeldin, during and after switching from the endophytic to the saprotrophic phase when the host dies (Weber et al. 2004). Suryanarayanan et al. (2005) studied the cactus Cylindropuntia fulgida for its endophytic diversity. The stem of certain Cacti have been investigated for the treatment of type II diabetes, reductions in nausea, dry mouth, and loss of appetite as well as less risk of a severe hangover.

Karsten et al. (2007) reported herbicidal and algaecidal activity in ethyl acetate extract of an endophytic Phoma sp. isolated from Fagonia cretica, F. cretica is used against fever, thirst, vomiting, dysentery, asthma, urinary

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discharges, liver trouble, dropsy, delirium, typhoid, toothache, stomach troubles, and skin diseases. Randa et al.

(2010) isolated a mycoendophyte (Botryosphaeria rhodina) from the stem of the medicinal plant Bidens pilosa (Asteraceae) that showed anti-inflammatory, antiseptic and antifungal effects. Bidens pilosa is used as a medicinal plant in many regions of Africa, Asia and tropical America.

The extract of Botryosphaeria rhodina also had significant cytotoxic and antiproliferative effects against several cancer cell lines.

Borges and Pupo (2006), has reported two novel hexahydroanthraquinone derivatives, dendryol E and F isolated from Phoma sorghina, which was found as an endophyte associated with a medicinal plant Tithonia diversifolia. The dried leaves of T. diversifolia showed anti-inflammatory and analgesic activities. Schwarz et al.

(2004) optimized the culture conditions of Phoma species and reported highest nematicidal activity in yeast malt glucose medium. Also, the secondary metabolites produced on Czapeck yeast autolysate and Yeast extract sucrose media by several Phoma species,separated by thin layer chromatography clarified the systematics of the genus (Montel et al. 1991). Phomodione, 2,6-diacetyl-7-hydroxy4a,9-dimethoxy-8,9b-dimethyl-4a.9b-dihydrodibenzo furan-1,3, an usnic acid derivative, were isolated from culture broth of a Phoma species which was an endophyte in the Guinea plant (Saurauia scaberrinae). Usnic acid and two of its derivatives, cercosporamide and phomodione, were also isolated from this fungus (Hoffman et al. 1998) Smith et al. (2008) provided direct evidence from bioassays of endophytes isolated from tropical plants and bioinformatic analyses, that gives a novel chemistry of potential value.

Raviraja (2005) studied eighteen species of mycoendophytes, isolated from bark, stem and leaf segments of five medicinal plant species growing within the Kudremukh range in the Western Ghats of India. The most common endophytic fungi were Curvularia clavata, C. lunata, C. pallescens and Fusarium oxysporum. The greatest species richness and frequency was found in the leaf segments, rather than the stem and bark segments of the host plant species. Thus, if endophytes could produce the same rare and important bioactive compounds as their host plants, this would not only reduce the need to harvest slow-growing and possibly rare plants but also help to preserve the worldâ&#x20AC;&#x2122;s ever-diminishing biodiversity.

PIRIFORMOSPORA INDICA: A NOVEL GROWTH PROMOTING ENDOPHYTE In 1998, Varma and colleagues discovered Piriformospora indica in the desert soil of Rajasthan and proved that it belongs to Basidiomycotina. The formation of pyriform chlamydospores is an important feature of this fungus. P. indica has tremendous capacity to enhance growth of host plant through its root-colonization (Rai et al. 2001). It is similar to arbuscular mycorrhizal fungi in many respects (Rai and Varma 2005; Deshmukh et al. 2006). Inoculation with fungus or with culture filtrates can enhance host plant

biomass. P. indica acts as multifunctional fungus because of its role as a biofertilizer, bioprotector, growth regulator, or it can increase draught tolerance (Sun et al. 2010).

Recently, Yadav et al. (2010) reported that a phosphate transporter gene (PiPT) plays an important role during the transport of phosphate from this fungus to the host plant.

Hence, the endophyte, P. indica has provided a new insight for understanding the mechanism of phosphate transfer in host plants. There are many advantages of using P. indica in agriculture and forestry (Singh et al. 2003; Yadav et al.

2010). P. indica can be used as a biological control agent against soil-borne root pathogens. The severity of the disease caused by Pseudocercosporella herpotrichoides and Fusarium culmorum were reduced when roots of winter wheat were colonized with P. indica. Kumar et al. (2009) also studied bioprotection potential of P. indica against Fusarium verticillioides, a root parasite and showed improvement in biomass, root length and number as compared with controls. Antioxidant enzyme activities were reduced by the presence of P. indica which helped the host plant.

Piriformospora indica has also proved to be a valuable tool for biological hardening of micropropagated plantlets.

Regenerated plantlets of tobacco subjected to two different biological hardening techniques showed 88-94% survival when inoculated as a root endophyte with P. indica, but only 62% survival in uninoculated controls. Thus, P. indica enhances plant growth, and survival via a positive impact on the nutritional and water status of the plant. It increases the reproductive potential, improves root performance, and provides natural defense against invaders, including pests and pathogens (Prasad et al. 2008).

ENDOPHYTES: THE NEW AND ECO-FRIENDLY DRUG PRODUCERS The number of eco-friendly drugs produced by mycoendophytes is large as compared to endophytic bacteria.

Natural products from fungal endophytes can be grouped into several categories, including alkaloids, steroids, terpenoids, isocoumarins, quinones, phenylpropanoids and lignans, phenol and phenolic acids, aliphatic metabolites, lactones, etc.

A novel amide, characterized as a ras-farnesyltransferase inhibitor was extracted from the culture broth of an endophytic Phoma species (Ishii et al. 2000). Mycoendophytes are being increasingly accepted as an ecological group of micro-organisms that may provide sources for new secondary metabolites with useful biological activities.

An array of active principle has been isolated and characterized from endophytes and many of these have diverse bioactivities (anti-cancerous, anti-oxidants, antifungal, anti-bacterial, anti-viral, anti-insecticidal and immune suppressants).Yang et al. (2006) reported two new 12-membered ring lactones isolated from the mycelial extracts of Cladosporium tenuissimum. Additional poliketides 12-membered macrolides have been produced by the endophytic C. tenuissimum of Maytenus hookeri (Silky bark). Mycoendophyte Nodulisporium species

RAI et al. â&#x20AC;&#x201C; Mycoendophytes in medicinal plants

associated with Juniperus cedre (Juniper), produced seven new metabolites. Chaetominine, an alkaloid with a new framework, produced by endophytic Chaetomium species was isolated from Adenophora axiliflora. The cytotoxic effect shown by the Chaetominine, against the human leukemia K562 and colon cancer SW1116 cell lines was higher than the drug 5-fluorouracil (Jiao et al. 2006).

Phaeosphoramides and two new carbon skeleton derivatives were isolated from the endophytic Phaeosphaeria avenaria.

Phaeosphoramide was found to be an inhibitor of the signal transducer and activator of transcription. This plays a vital role in regulating cell growth and survival, constituting a target for anticancer therapy (Maloney et al. 2006). Sumarah et al. (2010) identified the fungal endophytes from Picea rubens (red spruce) needles, isolated the active principles, and evaluated their toxicity. P. rubens is a species of spruce native to eastern North America. Three strains were toxic to the forest pest Choristoneura fumiferana (eastern spruce budworm). Leafy red spruce twigs are boiled for making spruce beer.

ANTICANCER AND ANTI-TUBERCULOSIS COMPOUNDS Cancer is a group of diseases characterized by unregulated growth and spread of abnormal cells, which can result in death if not controlled (Pimentel et al. 2010).

It has been considered as one of the major causes of death worldwide (about 13% of all deaths). Evidences are present about bioactive compounds produced by endophytes and could be an alternative approach for discovery of novel drugs, since many natural products from plants, microorganisms, and marine sources were identified as anticancer agents (Firakova et al. 2007). The anticancer properties of several secondary metabolites from endophytes have been investigated recently. The first anticancer agent produced by endophytes was Taxol and its derivatives. Taxol is a highly functionalized diterpenoid, isolated from yew (Taxus) species (Bacon and White 1994). The mode of action of Taxol is to prevent tubulin molecules from depolymerisation during the processes of cell division (Tan and Zou 2001). Camptothecin another potent antineoplastic agent, was firstly isolated from the wood of Camptotheca acuminata Decaisne (Nyssaceae) in China (Wall et al. 1966). Camptothecin and 10hydroxycamptothecin are two important precursors for the synthesis of the clinically useful anticancer drugs, topotecan, and irinotecan (Uma et al. 2008). The products were obtained from the endophytic fungi Fusarium solani isolated from Camptotheca acuminate (Kusari et al. 2009).

Ergoflavin, is an another dimeric xanthene linked in position 2 compound, belongs to the class ergochromes and is described as a novel anticancer agent isolated from an endophytic fungi growing on the leaves of an Indian medicinal plant Mimusops elengi (Sapotaceae) (Deshmukh et al. 2009). Another compound Secalonic acid D, a mycotoxin belonging to ergochrome class, is known to have potent anticancer activities, was isolated from the mangrove endophytic fungus demonstrated high

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cytotoxicity on HL60 and K562 cells by inducing leukemia cell apoptosis (Zhang et al. 2009). More novel 22-oxacytochalasins (Figure 1.A) (anti-cancer) drugs are also required worldwide to combat this scourge. These compounds have antitumor activity (Bills et al. 1996).

Crude Extracts of endophytic fungus Alternaria alternata, isolated from Coffea Arabica L., showed moderate cytotoxic activity to HeLa cells in vitro, when compared to the dimethyl sulfoxide (DMSO) treated cells (Fernandes et al. 2009). Tripterygium wilfordii is used in traditional Chinese medicine for the treatment of fever, chills, edema and carbuncle. There is a need to search for new antimicrobial agents because infectious diseases are still a global problem due to the development and spread of drugresistant pathogens.

Rukachaisirikul et al. (2007) reported endophytic Phomopsis species which produces secondary metabolites like phomoenamide, phomonitroester and Deacetylphomoxanthone, and showed antibacterial activity against Mycobacterium tuberculosis. Gordien et al. (2010) studied extracts from Scottish plants, lichens and mycoendophyte which were screened for activity against Mycobacterium aurum and M. tuberculosis. The greatest activity against M. aurum was shown by extracts of Juniperus communis roots, of the lichen Cladonia arbuscula and of a mycoendophyte isolated from Vaccinium myrtillus (Gordien et al. 2010). It is obvious that mycoendophytes serve as a source of potentially useful medicinal compounds. For example, 3-Nitropropionic acid was isolated from Phomopsis species which inhibited Mycobacterium tuberculosis and harbors anti-tuberculosis activity (Copp and Pearce 2007).

Alkaloids: These are useful anticancer agents that are often found in endophytic fungi. Wagenaar et al. (2000) isolated three novel cytochalasins from endophytic Rhinocladiella species which demonstrated antitumor activity. Most of the alkaloids have been detected in the cultures of grass-associated mycoendophyte, such as sexual Epichloe spp. and asexual Neotyphodium species.

Although metabolite production can be influenced by environmental factors, it seems to depend mostly on the strain or genotype of the endophytic species and less on the host grass genotype (Siegel et al. 1990). The alkaloids from mycoendophyte includes amines and amides, indole derivatives, pyrrolizidines and quinazolines (Figure 1 B).

Amines and amides are common substances produced by mycoendophyte from tall fescue, perennial ryegrass and many temperate grasses (Wilkinson et al. 2000). The ergot alkaloids are the second group of amine and amide alkaloids discovered in cultures of Neotyphodium endophytes, all are being characterized previously from ergot sclerotia (Tan and Zou 2001). These metabolites were later demonstrated to be neurotoxic to insects and mammal herbivores. Ergovaline and other structurally related ergopeptides are likely responsible for the toxicosis of livestock that consume endophyte-infected tall fescue. The biosynthesis of ergot alkaloids such as ergovaline is better understood in the ergot fungus Claviceps purpurea.

Steroids and terpenoids: Steroids have many important physiological effects, and some are found in

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mycoendophyte. A novel ergosterol derivative, 4a-homo22-hydroxy-4-oxaergasta-7, was isolated from a strain of Gliocladium sp., an endophyte on Taxus chinensis (Chinese yew). In addition to four cytochalasins, eleven novel sesquiterpenoids were isolated from cultures of the mitosporic fungus Geniculosporium species an endophyte associated with the red alga Polysiphonia species (Krohn et al. 2005).

Quinones, phenylpropanoids and lignans: Highly functionalised cyclohexenone epoxides, jesterone and hydroxyjesterone, were characterized from a newly identified endophyte Pestalotiopsis jesteri present in Fragraea bodenii. Guignardic acid is the first member of a novel class of natural products which were detected in the culture broth of Guignardia species obtained from Spondias mombin (Golden apple) (Rodrigues et al. 2001), which is a member of family Anacardiaceae native to the tropical America, including the West Indies. In past, it had been used as a febrifuge, diuretic, for leprosy, severe cough causing relief through vomiting.

Phenols, phenolic acids and antioxidants: Phenols and phenolic acids from fungal endophytes usually have pronounced biological and antioxidant activities. Pestacin and isopestacin are two novel dihydroisobenzofurancarrying phenols possessing antifungal and antioxidant activities (Figure 1 C and D). These were extracted from endophytic Pestalotiopsis microspora isolated from Terminalia morobensis (Harper et al. 2003). Both pestacin and isopestacin showed antimicrobial and antioxidant activity confirmed by electron spin resonance spectroscopy measurements. They are able to scavenge superoxide and hydroxyl free radicals in solution (Lewis et al.1997).

Aliphatic compounds and lactones: Chaetomellic acid, a potent and highly specific inhibitor of farnesylprotein transferase was characterised from the endophyte Chaetomella acutisea. Seven lactones were also characterized from an unidentified ascomycete endophyte isolated from Cistus salviifolius (White Rockrose) in Chile (Kopcke et al. 2002).

ANTIOXIDANT COMPOUNDS Antioxidant activity of a compound is actually the effectiveness of the compound against damage caused by reactive oxygen species (ROSs) and oxygen-derived free radicals, which contribute to a variety of physiological and pathological effects, for instance, DNA damages, carcinogenesis, and cellular degeneration (Haung et al.

2007; Seifried et al. 2007). Antioxidants are considered promising therapy for prevention and treatment of ROSlinked diseases as cancer, cardiovascular disease, atherosclerosis, hypertension, ischemia/reperfusion injury, diabetes mellitus, neurodegenerative diseases like Alzheimer and Parkinson diseases, rheumatoid arthritis, and ageing (Valko et al. 2007).

Naturally occurring antioxidant compounds are commonly found in vegetables, fruits and medicinal plants.

However, it has been observed that endophytes are also a potential source of novel natural antioxidants. Endophytic Xylaria sp. isolated from the medicinal plant Ginkgo

biloba, contain compounds showing antioxidant activities (Lui et al. 2007). Pestacin and isopestacin (1,3-dihydro isobenzofurans), were obtained from the endophytic fungus Pestalotiopsis microspora isolated from Terminalia morobensis a plant growing in the Papua New Guinea (Strobel et al. 2002; Harper et al. 2003). These compounds mainly isopestacin possess antioxidant activity by scavenging both superoxide and hydroxy free radicals in solution, added to the fact that isopestacin is structurally similar to the flavonoids (Strobel et al. 2002).

Graphislactone A, a phenolic compound isolated from the endophytic fungus Cephalosporium sp. residing in Trachelospermum jasminoides, demonstrated to have free radical-scavenging and antioxidant activities in vitro stronger than the standards, butylated hydroxytoluene (BHT) and ascorbic acid, coassayed in the study (Song et al. 2005).

ANTIBIOTICS, ANTIFUNGAL AND ANTIVIRAL COMPOUNDS Antibiotics are defined as low-molecular-weight organic natural products made by microorganisms that are active at low concentration against other microorganisms (Moon et al. 2002). Antibiotics from endophytic microbes have been reported to inhibit a variety of pathogens. For example, Cryptocandin is a unique antimycotic peptide isolated from Cryptosporiopsis quercina (Mohali et al. 2005) (Figure 1 E, F, and G). Similarly, Pestalotiopsis microspora, isolated from Torreya taxifolia produces several antifungal compounds. These include pestaloside, an aromatic glucoside, and two pyrones: pestalopyrone and hydroxyp-estalopyrone. T. taxifolia, is a rare and endangered species found in the Southeastern United States. It is used for the treatment of cancer. Other novel sesquiterpenes produced by endophytic fungi are 2hydroxydimeninol and a highly functionalized humulane (Surette et al. 2003).

Endophytic Muscodor albus was isolated from small limbs of Cinnamomum zeylanicum (Strobel 2006). This xylariaceaous (non-spore producing) fungus inhibits and kills certain other fungi and bacteria by producing a mixture of volatile compounds. Two novel human cytomegalovirus protease inhibitors, cytonic acids, have been isolated from the endophytic Cytonaema sp. (Schmid et al. 1993). Guo et al. (2008) studied the new antimicrobial metabolites isolated and extracted from the culture of Colletotrichum species from Artemisia annua, which is a traditional Chinese herb. It is well recognized for its synthesis of artemisinin (an antimalarial drug). These metabolites demonstrated activity against fungi and bacteria.

Endophytic fungi of the genera Xylaria, Phoma, Hypoxylon, and Chalara are producers of a group of substances known as the cytochalasins, of which over 20 are known. Many of these compounds possess antibiotic activities, but because of their cellular toxicity they have not been developed into pharmaceutical drugs. Three novel cytochalasins have recently been reported from a Rhinocladiella sp. as an endophyte on Tripterygium wilfordii.

RAI et al. â&#x20AC;&#x201C; Mycoendophytes in medicinal plants

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G F

Figure 1. Structure of compounds produced by endophytes (A) Cytochalasins antitumor compound from an endophyte Rhinocladiella sp., (B) Pyrrolizidines, (C) Isopestacin (D) Pestacin an antioxidant produced by an endophytic Pestalotiopsis microspora strain recoverd from Terminalia morobensis (E) Oocydin (F) Periconicin, (G) Cryptocandin

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FUTURE PERSPECTIVES AND CONCLUSIONS

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The need for new bioactive to overcome the growing problems of drug resistance in microorganisms and the appearance of new diseases is of increasing importance.

The capability of fungi to produce bioactive metabolites has encouraged researchers to isolate and screen fungi from diverse habitat and environments to search for novel bioactive metabolites.

Some endophytes produce phytochemical that were originally thought of as characteristic of the host plant. It appears that genetic interaction between the endophyte and the host has occurred over evolutionary time (Tan and Zou 2001). This concept was proposed to explain why Taxomyces andreanae produce taxol. The cultured endophytes, can be induced to produce the same rare and important bioactive compounds as when associated with their host plants, it would reduce the need to harvest slowgrowing and possibly rare plants. It would also help to preserve the world’s ever-diminishing biodiversity.

Furthermore, a microbial source of a high-value product is an economical way to produce a metabolite in a bulk quantity and thereby reduce its market price.

Researchers are searching for new antibiotics, chemotherapeutic agents, and agrochemicals that are highly effective, possess low toxicity, and have a minor environmental impact. This search is driven by the development of resistant infectious microorganisms e.g., species of Mycobacterium, Streptococcus and Staphylococcus. Furthermore, new diseases, like AIDS and respiratory syndrome, need the invention and development of novel active drugs to fight them. Diseases such as AIDS require drugs that target them specifically and also new therapies for treating the ancillary infections which are the consequence of a weakened immune system. New drugs are needed for immunocompromised cancer patients and those receiving organ transplants and those who are at risk of opportunistic pathogens, such as Aspergillus spp., Cryptococcus spp. and Candida spp. Finally, a number of synthetic agricultural products have been removed from the market due to safety and environmental problems so there is also a need to discover an alternative to control crop pests and pathogens.

To overcome the infectious disease, there is need for a variety of novel antimicrobial compounds of biological origin. The mycoendophytes hold enormous potential as sources of antimicrobials. These endophytes may open new vistas for the development of new drugs and agricultural products. The multi-drug resistance problem in microbes underscores the need for further research on novel metabolites obtained from mycoendophytes.

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Zhao J, Mou Y, Shan T, Li Y, Zhou L, Wang M, Wang J. 2010.

Antimicrobial Metabolites from the endophytic fungus Pichia guilliermondii isolated from Paris polyphylla var. yunnanensis.

Molecules 15 (11): 7961-7970 Zhou X, Zhu H, Liu L, Lin J, Tang K. 2010. A review: recent advances and future prospects of taxol-producing endophytic fungi. Chem Matl Sci 86 (6): 1707-1717.

Guidance for Authors Aims and Scope Nusantara Bioscience (Nus Biosci) is an official publication of the Society for Indonesian Biodiversity (SIB). The journal encourages submission of manuscripts dealing with all aspects of biological sciences that emphasize issues germane to biological and nature conservation, including agriculture, animal science, biochemistry and pharmacology, biomedical science, ecology and environmental science, ethnobiology, genetics and evolutionary biology, hydrobiology, microbiology, molecular biology, physiology, and plant science. Manuscripts with relevance to conservation that transcend the particular ecosystem, species, genetic, or situation described will be prioritized for publication.

Article The journal seeks original full-length research papers, short research papers (short communication), reviews, monograph and letters to the editor about material previously published; especially for the research conducted in the Islands of the Southeast Asian reign or Nusantara, but also from around the world.

Acceptance The acceptance of a paper implies that it has been reviewed and recommended by at least two reviewers, one of whom is from the Editorial Advisory Board. Authors will generally be notified of acceptance, rejection, or need for revision within 2 to 3 months of receipt.

Manuscript is rejected if the content is not in line with the journal scope, dishonest, does not meet the requiredquality, written in inappropriate format, has incorrect grammar, or ignores correspondence in three months.

The primary criteria for publication are scientific quality and biological or natural conservation significance. The accepted papers will be published in a chronological order.

Copyright Submission of a manuscript implies that the submitted work has not been published before (except as part of a thesis or report, or abstract); that it is not under consideration for publication elsewhere; that its publication has been approved by all co-authors. If and when the manuscript is accepted for publication, the author(s) agree to transfer copyright of the accepted manuscript to Nusantara Bioscience. Authors shall no longer be allowed to publish manuscript without permission.

Authors or others are allowed to multiply article as long as not for commercial purposes. For the new invention, authors are suggested to manage its patent before published.

Submission The journal only accepts online submission, through e-mail to the managing editor at unsjournals@gmail.com. The manuscript must be accompanied with a cover letter containing the article title, the first name and last name of all the authors, a paragraph describing the claimed novelty of the findings versus current knowledge, and a list of five suggested international reviewers (title, name, postal address, email address). Reviewers must not be subject to a conflict of interest involving the author(s) or manuscript(s). The editor is not obligated to use any reviewer suggested by the author(s).

Preparing the Manuscript Please make sure before submitting that: The manuscript is proofread several times by the author (s); and is criticized by some colleagues. The language is revised by a professional science editor or a native English speaker. The structure of the manuscript follows the guidelines (sections, references, quality of the figures, etc). Abstract provides a clear view of the content of the paper and attracts potential citers. The number of cited references complies with the limits set by Nus Biosci (around 20 for research papers).

Microsoft Word files are required for all manuscripts. The manuscript should be as short as possible, and no longer than 7000 words (except for review), with the abstract < 300 words. For research paper, the manuscript should be arranged in the following sections and appear in order: Title, Abstract, Key words (arranged from A to Z), Running title (heading), Introduction, Materials and Methods, Results and Discussion, Conclusion, Acknowledgements, and References. All manuscripts must be written in clear and grammatically correct English (U.S.). Scientific language, nomenclature and standard international units should be used. The title page should include: title of the article, full name, institution(s) and address(es) of author(s); the corresponding authors detailed postal and email addresses, and phone and fax numbers.

References Author-year citations are required. In the text give the authors name followed by the year of publication and arrange from oldest to newest and from A to Z. In citing an article written by two authors, both of them should be mentioned, however, for three and more authors only the first author is mentioned followed by et al., for example: Saharjo and Nurhayati (2006) or (Boonkerd 2003a, b, c; Sugiyarto 2004; El-Bana and

Nijs 2005; Balagadde et al. 2008; Webb et al. 2008). Extent citation as shown with word “cit” should be avoided. Reference to unpublished data and personal communication should not appear in the list but should be cited in the text only (e.g., Rifai MA 2007, personal communication; Setyawan AD 2007, unpublished data). In the reference list, the references should be listed in an alphabetical order. Names of journals should be abbreviated. Always use the standard abbreviation of a journal’s name according to the ISSN List of Title Word Abbreviations (www.issn.org/222661-LTWA-online.php). The following examples are for guidance.

Journal: Saharjo BH, Nurhayati AD. 2006. Domination and composition structure change at hemic peat natural regeneration following burning; a case study in Pelalawan, Riau Province. Biodiversitas 7: 154-158.

The usage of “et al” in long author lists will also be accepted: Smith J, Jones M Jr, Houghton L et al. 1999. Future of health insurance. N Engl J Med 965: 325–329 Article by DOI: Slifka MK, Whitton JL. 2000. Clinical implications of dysregulated cytokine production. J Mol Med. Doi:10.1007/s001090000086 Book: Rai MK, Carpinella C. 2006. Naturally occurring bioactive compounds.

Elsevier, Amsterdam.

Book Chapter: Webb CO, Cannon CH, Davies SJ. 2008. Ecological organization, biogeography, and the phylogenetic structure of rainforest tree communities. In: Carson W, Schnitzer S (eds) Tropical forest community ecology. Wiley-Blackwell, New York.

Abstract: Assaeed AM. 2007. Seed production and dispersal of Rhazya stricta. 50th annual symposium of the International Association for Vegetation Science, Swansea, UK, 23-27 July 2007.

Proceeding: Alikodra HS. 2000. Biodiversity for development of local autonomous government. In: Setyawan AD, Sutarno (eds) Toward mount Lawu national park; proceeding of national seminary and workshop on biodiversity conservation to protect and save germplasm in Java island. Sebelas Maret University, Surakarta, 17-20 July 2000.

[Indonesia] Thesis, Dissertation: Sugiyarto. 2004. Soil macro-invertebrates diversity and inter-cropping plants productivity in agroforestry system based on sengon.

[Dissertation]. Brawijaya University, Malang. [Indonesia] Online document: Balagadde FK, Song H, Ozaki J, Collins CH, Barnet M, Arnold FH, Q u a k e S R , Y o u L . 2 0 0 8 . A s yn t h e t i c E s c h e r i c h i a c o l i p r e d a t o r - p r e y e c o s y s t e m . M o l S ys t B i o l 4 : 1 8 7 . www.molecularsystemsbiology.com Tables should be numbered consecutively and accompanied by a title at the top. Illustrations Do not use figures that duplicate matter in tables.

Figures can be supplied in digital format, or photographs and drawings, which can be ready for reproduction. Label each figure with figure number consecutively.

Uncorrection proofs will be sent to the corresponding author by e-mail as .doc or .docx files for checking and correcting of typographical errors. To avoid delay in publication, proofs should be returned in 7 days.

A charge The cost of each manuscript is IDR 250,000,- plus postal cost or IDR 150,000,- for SIB members. There is free of charge for non Indonesian author(s), but need to pay postal cost for hardcopy.

Reprints Two copies of journal will be supplied to authors; reprint is only available with special request. Additional copies may be purchased by order when sending back the uncorrection proofs by e-mail.

Disclaimer No responsibility is assumed by publisher and co-publishers, nor by the editors for any injury and/or damage to persons or property as a result of any actual or alleged libelous statements, infringement of intellectual property or privacy rights, or products liability, whether resulting from negligence or otherwise, or from any use or operation of any ideas, instructions, procedures, products or methods contained in the material therein.

NOTIFICATION: All communications are strongly recommended to be undertaken through email.

| Nus Biosci | vol. 4 | no. 2 | pp. 45-96 | July 2012| | ISSN 2087-3948 | E-ISSN 2087-3956 | I S E A

J o u r n a l

o f

B i o l o g i c a l

S c i e n c e s

Phytofabrication of silver nanoparticles by using aquatic plant Hydrilla verticilata NEILESH SABLE, SWAPNIL GAIKWAD, SHITAL BONDE, ANIKET GADE, MAHENDRA RAI

45-49

Antibacterial activity of Thymus vulgaris essential oil alone and in combination with other essential oils KATERYNA KON, MAHENDRA RAI

50-56

Adult mangrove stand does not reflect the dispersal potential of mangrove propagules: Case study of small islets in Lampung, Sumatra AGUNG SEDAYU, NOVITA FARAH ISYADINYATI, DIANA VIVANTI SIGIT

57-61

Patterns of fertility in the two Red Sea Corals Stylophora pistillata and Acropora humilis MOHAMMED S.A. AMMAR, AHMED H. OBUID-ALLAH, MONTASER A.M. AL-HAMMADY

62-75

The effects of foliar application herbicides to control semi-parasitic plant Arceuthobium oxycedri MOHAMMAD REZA KAVOSI, FERIDON FARIDI, GOODARZ HAJIZADEH

76-80

Synthesis, characterization and physiological activity of some novel isoxazoles VINAYSINGH J. HUSHARE, PRITHVIRAJSINGH R. RAJPUT, MANOJKUMAR O. MALPANI, NITIN G. GHODILE Review: Mycoendophytes in medicinal plants: Diversity and bioactivities MAHENDRA RAI, ANIKET GADE, DNYANESHWAR RATHOD, MUDASIR DAR, AJIT VARMA

81-85 86-96

Society for Indonesian Biodiversity

Sebelas Maret University Surakarta

Published three times in one year PRINTED IN INDONESIA

ISSN 2087-3948

E-ISSN 2087-3956