ISOLATION, PURIFICATION AND IDENTIFICATION OF MICROORGANISMS (FUNGI, BACTERIA, AND ACTINOMYCETES) FR by Saurabh Kumar Gautam

ISOLATION, PURIFICATION AND IDENTIFICATION OF MICROORGANISMS (FUNGI, BACTERIA, AND ACTINOMYCETES) FROM SOIL AND VERMICOMPOST

A MINOR PROJECT

Submitted to Gautam Budh Technical University for Partial fulfillment for the award of the degree of Bachelor’s of Technology IN BIOTECHNOLOGY 2013 Under the guidance of

Biotechnology and Bioresources Division Centre For Mycorrhizal Research

The Energy And Resources Institute (TERI) Indian Habitat Centre, New Delhi

Submitted By SAURABH KUMAR GAUTAM B.Tech Biotechnology DEPARTMENT OF BIOTECHNOLOGY COLLEGE OF ENGINEERING & TECHNOLOGY IILM ACADEMY OF HIGHER LEARNING GREATER NOIDA

ISOLATION, PURIFICATION AND IDENTIFICATION MICROORGANISMS (FUNGI, BACTERIA AND ACTINOMYCETES) FROM SOIL AND VERMICOMPOST A MINOR PROJECT Submitted by Saurabh Kumar Gautam (0915054044) In partial fulfilment for the Award of the degree Of BACHELOR’S OF TECHNOLOGY In Biotechnology

DEPARTMENT OF BIOTECHNOLOGY COLLEGE OF ENGINEERING & TECHNOLOGY IILM ACADEMY OF HIGHER LEARNING GREATER NOIDA

DECLARATION

I, Saurabh Kumar Gautam, student of Bachelorâ&#x20AC;&#x2122;s of Technology in Biotechnology (7th semester),College Of Engineering & Technology, IILM Academy Of Higher Learning,Greater Noida, hereby, declare that the work presented in the training report entitled, " ISOLATION, PURIFICATION AND IDENTIFICATION OF MICROORGANISMS (FUNGI, BACTERIA AND ACTINOMYCETES) FROM SOIL AND VERMICOMPOST " is an authentic record of my work done from 25rdJune 2012 to 25th august 2012, under the supervision of Dr. R. K. Mishra, researcher, Center of Mycorrhizal Research, Biotechnology and Bioresources Division, The Energy And Resources Institute (TERI).

Saurabh Kumar Gautam (0915054044)

CERTIFICATE

This is to certify that Saurabh Kumar Gautam has carried out his training work entitled " ISOLATION, PURIFICATION AND IDENTIFICATION

OF BENEFICIAL

MICROORGANISMS (FUNGI, BACTERIA AND ACTINOMYCETES) FROM SOIL AND VERMICOMPOST " of Bachelorâ&#x20AC;&#x2122;s of Technology in Biotechnology (7th semester), College Of Engineering & Technology, IILM Academy Of Higher Learning,Greater Noida from 25thJune 2012 to 25th August 2012 in the Centre for Mycorrhizal Research, Biotechnology and Bioresources Division, TERI under myguidance and supervision.

Dr. Biotechnology and Bioresources Division Centre for Mycorrhizal Research The Energy And Resources Institute (TERI), New Delhi

ACKNOWLEDGEMENT I humbly bow my head before lord almighty for blessing me with the power to complete this endeavor successfully. I express deep sentiments of gratitude to Dr. R.K.Pauchauri, Director General, The Energy and Resources Institute (TERI), New Delhi, for granting me permission to work in this esteemed institute. I put my respects and honor for Dr. Alok Adholeya Director, Biotechnology and Bioresources Division the granting work in his division For me, it was great honor to work under with my guide Dr. R.K. Mishra, Biotechnology and Bioresources Division &Centre for Mycorrhizal Research, TERI New Delhi. I am very thankful to him for the precious advise, consistent encouragement and moral support, critical and fruitful indulgence despite of busy schedule during knotty hours during my work period and without whom I would have never been able to submit my work. I put on record the timely suggestion and guidance received from Dr. Reena Singh for eliciting novel, supportive and perspectives towards the whole approach of the project. I extend my sincere thanks to Dr. Sashidhar Burla, Dr. Manab Das, Dr.Mandira and Mr. Sachin Rastogi for their valuable support. I am equally grateful to the CMR team: Ms Amrit Preet Kaur, Ms Rita, and Ms Poornima Saraswat and Ms Yeti for their moral support and guidance which greatly contributed to the success of the present study. They had been very kind and patient while suggesting me the outlines of this project and correcting my doubts and nurturing a comfortable atmosphere. With deep respect, I express my sincere gratitude to Dr. Sonia Advani (Head of Department) and other faculty members), College Of Engineering & Technology, IILM Academy Of Higher Learning, for their valuable suggestion and encouragement. I sincerely Thank Ms Nazz , Ms Aditi And Ms Charu Mr. Sandeep, Ms. Sreeparna, Ms. Amrit, Ms. Shilpi, Ms. Ankita, Ms. Rita, Mr. Pavan, Mr. Sachin, Mr Amit, and, Mr. Akhilesh Mr. Pavan Sunkireddy from the core of my heart. I also express my truthful thanks to Mr. Joshi. I would also like to make note of the immense support of Mr. Akhilesh I have no words to express the moral support shared with my friends during my course work. Thanks are also due to the entire management and staffs who have directly and indirectly helped me to complete my work. Last, but not least, I owe my gratitude to my parents and teachers for all the love and affection they have been showered upon me. Though mere words could not convey it all, still it is an attempt to express my heartfelt gratitude to everybody who made the project successful.

Saurabh Kumar Gautam

CONTENTS List of Figures

List of Tables

List of Abbreviations

Chapter 1 Introduction

Chapter 2 Literature

Chapter 3 Theory

Chapter 4 Materials and methods

Chapter 5 Methods 1

Chapter 6 Other methods used

Chapter 7 Results and discussions for Punjab

Chapter 8 Results and discussions for Haryana

Chapter 9 Results for vermicompost samples

Chapter 10 Conclusions

Chapter 11 References 35

LIST OF FIGURES Figure 1

Soil web and position of fungi and bacteria in it

Figure 2

Figure: showing morphology of the bacteria

Figure 3

Serial dilution

Figure 4

Microbial population/diversity in soil from Punjab

Figure 5

Microbial populations/diversity in soil from Haryana

Figure 6

Microscopic observations of fungi at 40X

Figure 7

Graph showing diversity in the readings

Figure 8

Microbial colonies on NA/PDA media plate

Figure 9

Microscopic observation of microorganisms(100X)

Figure 10

Field Trial result of vermicomposting

Figure 11

Results of before and after application of vermicompost in different crops

LIST OF TABLES Table 1

Microbial population in soil from Punjab

Table 2

Microbial populations in soil from Haryana

Table 3

Microbial analysis of vermicompost from different sources

Table 4

Vermicomposting field trial result

Chapter 1 INTRODUCTION India has about 114 million hectares of land under cultivation and is also the second most populous country in the world. To meet its ever-rising demand for food, the dual measures of increasing land area under cultivation and improving productivity per square kilometre will have to be taken. This means that close to 55 million hectares of wasteland or fallow land will have to be brought under cultivation. The present study deals with an attempt in the identification of the microorganisms isolated from different samples collected from the different agricultural regions of India. The samples were collected from different agricultural region includes soil sample from Haryana and Punjab. For isolation of soil microbes different types of culture media including potato dextrose agar, Nutrient Agar and potato dextrose agar amended with penicillin and streptomycin were used. The isolated microorganisms were selected by morphology. The ability to exploit the natural resources constituting a major step towards economic prosperity for developing countries as chemical fertilizers are expensive, short and may cause the problems of environmental pollution. The research is active to develop an alternative technology to minimize the dependency on chemical fertilizers and encourage the use of biofertilizers in the countries where agriculture is the major source of income. For this the requirement of study of soil microbes is important. The excessive use of fertilizers in agriculture is generally assumed to be a major cause of nitrate pollution. Nitrates may leak through the soil and occur in excess levels in drinking water. The authors of this book provide a detailed scientific analysis of this controversial issue. They review the evidence which shows that many other features of contemporary farming practices have contributed to the problem and that limiting fertilizer use is an oversimplistic solution to the problem. Changing crop production practices to 'mop-up' excess nitrate from soil microbes may be a better response. Importance of agriculturally important microorganisms in vermicompost and soils Microorganisms are present almost everywhere. These are ubiquitous and at the same time promiscuous. The soil acts as a reservoir for millions of microorganisms, of which more than 85% are beneficial for plant life. Good and healthy soil consists of 93% mineral and 7% bio organic substances. Microorganisms are an integral living component in soil. It is widely being recognized that the presence and abundance of microbial wealth make soils healthy in terms of growth enhancement and protection against pests and diseases. Vermicompost rich in Microorganisms not only mineralize more actively, but also contribute to the buildup of stable soil organic matter like humus and other natural carbon complexes. By this way, nutrients are recycled faster and soil structure/texture is improved. Earthworms play a major role in affecting populations of soil organisms, especially in causing changes in the soil microbial community 8

Chapter 2 LITERATURE The ability to exploit the natural resources constituting a major step towards economic prosperity for developing countries as chemical fertilizers are expensive, short and may cause the problems of environmental pollution. The research is active to develop an alternative technology to minimize the dependency on chemical fertilizers and encourage the use of biofertilizers in the countries where agriculture is the major source of income in some samples TERI Mycorrhiza was also added.

2nd level

Figure 1: Soil web and position of fungi and bacteria in it

Diversity and AbundanceSoil biodiversity includes a plethora of life that ishidden from our everyday view: viruses; bacteria, actinomycetes; fungi; algae; the protozoans (single-celled eukaryotes); microscopic invertebrates such as rotifers, tardigrades (water bears), soil 9

planaria,(flatworms), and nematodes (roundworms); themicroarthropods, Acari (mites) and Collembola(springtails); and larger invertebrates, easily seen bythe naked eye such as terrestrial gastropods (snailsand slugs), isopods (pill bugs, sow bugs), Oligochaeta(enchytraeids and earthworms), spiders, scorpions,beetles, centipedes, millipedes, crustaceans, ants, andtermites. Because of this diversity, the invertebrates are frequently grouped by size (body width), micro-fauna, mesofauna, and macro fauna. Vertebrates also depend on the soil as a habitat, forexample, moles, prairie dogs, meercats, wombats, small rodents, and some species of lizards, snakes, frogs, and even birds. Many aboveground invertebrates may be temporary inhabitants (such asnematodes parasitic on insects; immature flies in the Diptera such as tipulid crane flies and tephritid fruit

Chapter 3 THEORY

BACTERIA: Bacteria are unicellular microorganisms found in every habitat on Earth. Nearly all have cell walls composed of peptidoglycan and reproduce by binary fission (cloning of cells).Although many of these microbes are harmless or beneficial to humans, others are pathogenic, causing infectious diseases. Identification of Bacteria Some of the first steps in identifying bacteria is to examine: 1. The shape of the individual bacteria. 2. Whether the bacteria exist in specific groupings. 3. The colony morphology (the appearance of a "colony"; a group of millions of bacteria that arose from one single parent cell). Bacterial Shapes Most bacteria are classified according to shape: Bacillus (pl. bacilli) =

rod-shaped

Coccus (pl. cocci sounds like cox-eye) = spherical Spirillum (pl. spirilla) =

spiral

Some bacteria have more unusual shapes: Coccobacilli =

elongated coccal form

Filamentous =

bacilli that occur in long threads

Vibrios =

short, slightly curved rods

Fusiform =

bacilli with tapered ends

Prokaryote Arrangement of Cells Bacteria sometimes occurs in groups, rather than singly, and the single cell's shape influences the cell arrangements that they form as the bacterial cells divide.

Bacilli divide along a single axis, and are sometimes seen in pairs or chains. Since they only divide along one axis, you will not find bacilli in clusters, such as those formed by Staphylococcal bacteria. Cocci divide on one or more planes, producing cells in: Pairs (diplococci) Chains (streptococci) Packets (sarcinae) Clusters (staphylococci) Size, shape and arrangement of cells are often the first clues in identification of bacteria. However, since there are many "look-alikes", methods other than microscopy must be used to determine the genus and species of an organism.

Bacterial Colony Morphology Bacterial populations grow extremely fast when they supplied with the nutrients and environmental conditions that allow them to thrive. Through this growth, different types of bacteria will sometimes produce colonies that are distinctive in appearance. Some colonies may be colored; some are circular in shape, while others are irregular. The characteristics of a colony (shape, size, colour, etc.) are termed the "colony morphology". Colony morphology is a way scientists can identify bacteria. There are a few basic characteristics of colony morphology that are typically evaluated. 1. Form - What is the basic shape of the colony? For example, circular, filamentous, etc. 2. Elevation - What is the cross-sectional shape of the colony? To see this, turn the Petri dish on end. 3. Margin - What is the magnified shape of the edge of the colony? 4. Surface - How does the surface of the colony appear? For example, smooth, glistening, rough, dull (opposite of glistening), rugose (wrinkled), etc. 5. Opacity - Is the colony transparent (clear), opaque, translucent (almost clear, but distorted vision, like looking through frosted glass), iridescent (changing colours in reflected light), etc. 6. Chromo genesis (pigmentation) - For example, white, buff, red, purple, etc.

Figure 2: Showing howing morphology of the bacteria

Chapter 4 MATERIAL AND METHODS Collection of soil samples Samples from the different agricultural region were collected following standard procedures. A total of fifteen (15) soil samples were collected from different field trial in Kaithal and Karnal districts of Haryana state and Total twenty one (21) soil sample have been collected from various field trials at Amritsar and Jalandhar district of Punjab. The samples were transferred to clean polythene bags (zip pouch bags) immediately after collection sealed and labeled. All the samples were brought to laboratory for further processing. In the laboratory, the samples were stored at 22-24째C until used. Types of media In an attempt a suitable media for the isolation of different, initially different media are tested. 1. Potato Dextrose Agar (PDA) (Riker and Riker, 1936) Potato (peeled and sliced)

200 g

Dextrose

20.0 g

Agar

15.0 g

Distilled water

1000 ml

7.0

2. Nutrient Agar (NA) medium (himedia) Peptic digest of animal tissue

5.0

Sodium chloride

5.0

Beef extract

1.0

Yeast extract

2.0

Agar

15.0

Final pH (at 25Â°C)

7.4Âą0.2

Distilled water

1000ml

The agar powder is used in all the above media to solidify the media. Antibiotics can also be added to only PDA medium to inhibit bacterial growth and make it more selective for the isolation of fungi only. Penicillin, streptomycin, chloramphenicol, cyclohexamide and other antibiotics can be used.

Chapter 5 METHODS 1 Isolation and Purification of microorganisms from soil and vermicompost samples 1) Spread plating of soil and vermicompost samples The standard plating method pour plate was evaluated for their suitability for routine isolation. In spread plate technique, 100 Âľl inoculum was uniformly spread on the solidified agar surface in the Petri plates. For each medium three replicates were prepared. 2) Isolation of microorganisms Fungi The pour plate technique was adopted for isolation. All the plates were incubated at 21+2OC. However, colonies started appearing from sixth day onwards (Zhao et al., 2004).Plates were examined for the appearance of fungi colonies from sixth day onwards and up to 30 days. The actinomycetes also appeared on the same plates. Bacteria The pour plate technique was adopted for isolation. All the plates were incubated at 27+3OC. However, colonies started appearing after 24 onwards (observed).Some bacterial colonies appeared on the second day with characteristics features and colour. 3) Purification of soil and vermicompost samples Fungi Many colonies with different morphological and cultural characteristics, generally colony appeared with a tough leathery or chalky texture; dry or folded appearance and branching filamentous with or without aerial mycelia were picked ( Mincer et al., 2002; Oskey et al., 2004) from the isolation plates and inoculated for purification. The pure cultures were transferred to PDA Plates and incubated for seven to ten days at 21+ 2oC. When sufficient

growth had occurred, the plates were stored at 4Â°C in a Cold room. The stock cultures were maintained and transferred to fresh media plates once in 10-15 days and stored at 4Â°C.

Soil samples (serial diluted)

Liquid portion (100ÂľL) spread onto plate

Incubate inoculated plates at desired temperature

Growth of isolates from outer mycelia

Visible growth of isolate transferred onto fresh media plates

Repeated subculture on fresh media Until pure isolate obtained

Pure isolate Bacteria Many colonies with different morphological and cultural characteristics, generally colony appeared with a filament or round; dry or convex and branching with or without characteristic were picked from the isolation plates and streaked for purification. The pure cultures were

transferred to NA Plates and incubated for 24 hours to 48 hours at 27+ 3oC. When sufficient growth had occurred, the plates were stored at 4°C in a Cold room. The stock cultures were maintained and transferred to fresh media plates once in 10-15 days and stored at 4°C.

Soil samples

Liquid portion (100µL) spread onto plate

Incubate inoculated plates at desired temperature

Growth of isolates from NA plates

Visible growth of isolate streaked onto fresh NA plates

Repeated subculture OR Streaking on fresh NA plates Until pure isolate obtained

Pure culture of bacteria Identification and characterization Under morphological characters, other parameters taken into consideration for the identification of microorganisms like, fungi, bacteria and actinomycetes are the following cultural characteristics 1. Growth behavior in nutrient medium 2. Growth and colony characters on nutrient agar plates 3. Type and nature of growth and sporulation on agar plates.

Chapter 6 OTHER METHODS USED Aim To study the bacterial cell morphology and determine the Gram character of bacteria by Differential staining Principle Gram stain is the most important differential stain used in bacteriology and this special staining Procedure was introduced in 1889 by a Danish physician named Christian Gram. It permits separation of bacteria into two kinds, the Gram positive organisms and the Gram negative Species which makes it an essential tool for classification and differentiation of microorganisms. Differential staining requires the use of at least three chemical reagents that are applied sequentially to a heat fixed smear. Requirements Gram staining requires 1. Aqueous Crystal Violet: Acts as a primary stain to impart colour to all the cells. 2. Gram’s Iodine: This reagent serves as a mordant and forms an insoluble complex by binding to the primary stain. 3. Decolorize: This acts as a lipid solvent and as a protein dehydrating agent and its action is determined by the lipid concentration of the microbial cell walls. 4. Counter Stain: Aqueous Safranin is the final reagent to stain red those cells that have not been previously decolourized. 1. Crystal violet (aqueous 1%) Crystal violet 1g Distilled water 100 ml Dissolve crystal violet in water, filter if necessary. 2. Gram’s Iodine Potassium Iodide 2 g in 100 ml distilled water 3.

Gram’s decolouriser Acetone 50%

Alcohol 50% 4. Safranin (aqueous 2%): 2 g in 100 ml of distilled water, mix well and filter if necessary. Methodology 1. Preparation of a fixed bacterial smear: put a drop of bacterial culture on the slide with the help an inoculation needle and spread it in the center of the slide. Allow it to dry. Intermittent gentle heating is sometimes done to prepare heat fixed smear. If the bacterial growth is taken from slant then first put drops of sterile saline (0.2%) distilled water on the slide and suspend bacterial growth in it on the slide and spread using inoculation needle. 2. Place the slide on the staining rack and flood the smear with crystal violet for about 1 min. 3. Wash the stain gently with iodine solution. Stain with a fresh iodine solution for 1 min. 4. Wash in tap water or by dipping in a beaker containing water. 5. Add a few drops of decolourizer and continue until colour ceases to come out of the preparation. This may take 5 seconds to 1 minute. 6. Wash gently with water as in step 4. 7. Counter stain with dilute carbol fuchsin or safranin for 10-30 seconds. 8. Wash with water and dry off most of the slide with absorbent paper and leave the smearto dry by evaporation. 9. Dry slide is a permanent preparation and is examined under the microscope directlywithout a cover slip first under the low power and then under higher magnification. Examine under the oil immersion lens using cover slip. Observation Gram positive cells are purple and Gram negative cells are pink or red. Some species represent borderline cases and are best classified as Gram variable.

Figure 3: Serial dilution techniques

For further techniques refer to trainee handbook for biotechnology prepared for training report

Chapter 7 RESULTS AND DISCUSSION PUNJAB Total twenty one (21) soil sample have been collected from various field trials at Amritsar and Jalandhar district of Punjab and analysed to see the microbial diversity in soil. Microorganisms were identified conventionally according to their morphological features. The dominant species of different types of bacteria like, Pseudomonas spp, Bacillus spp and fungi, like, Fusarium spp, Aspergillus niger, Aspergillus spp, Penicillium spp, Cercospora spp and some other genus were isolated from soil.

Figure 4: Microbial population/diversity in soil from Punjab

Table 1: Microbial population in soil from Punjab S.

Farmers Name

Fungi

Bacteria

Fusarium Aspergillusspp Penicilliumspp Cercosporaspp Others Pseudomonas Bacillus spp spp spp 1

MANJU SINGH

NIRMAL

KULBEER SINGH

MALKIT SINGH

GURMAN

MALKIT

AMRIK

MALKIT

BALBIR

HANSA

MALKIT

BALBIR

AMRIK

KULBEER

MALKIT

SUKHMATH

AGIPAL

AMRIK

LAKHMINDER -

The sign (+) indicates present, the minus sign (-) means absent

Chapter 8 RESULTS AND DISCUSSION HARYANA In present investigation, total fifteen (15) soil samples were collected from different field trial in Kaithal and Karnal districts of Haryana state. Samples were analyzed through serial dilution technique (Waksman, 1927) to see the microbial population and diversity in soil. During the experimentation several fungi like, Penicillium spp, Fusarium spp, Cercospora spp, Aspergillus niger and Aspergillus spp etc and bacteria like, Pseudomonas spp and Bacillus spp and some species of Actinomycetes were identified.In some field trial (Mr. ILAM SINGH) with vermicompost were also analyzed for maximum microbial diversity. Results indicated that, when vermicompost was added in the soil, microbial diversity is more as compared to control.

Table 2: Microbial populations in soil from Haryana S. No

Farmers Name

Fungi

Bacteria

Actinomy cetes

Fusariu m spp

Aspergill usspp

Penicilliu mspp

Cercospor aspp

Others

Pseudomon as spp

Bacillus spp

Streptomy ces spp

HARICHA ND

SRICHAN D

AMIT SINGH

RAMNIVA S

MOHINDE

R SINGH 6

RIKHI RAM

JAIMAL SINGH

BALJEET SINGH

UDAY KUMAR

DHARMPA L

TEJ PAL SINGH

ILAM SINGH

KARAM SINGH

NARESH CHAND

RAM KUMAR

The sign (+) indicates present, the minus sign (-) means absent

Figure 5: Microbial populations/diversity in soil from Haryana Table: 3 Microbial analysis of vermicompost from different sources Sources of Vermicompost

Microbial population

Fungi (CFU-g) Demonstration 4 x 105 unit

Microbial diversity

Bacteria (CFU-g)

Actinomycetes Fungi (CFU-g)

Bacteria

Actinomycete Others s

8 x 106

2 x 105

Pseudomona s spp

Streptomyces

Penicilliums pp

Aspergillussp Bacillus spp p And some Fusarium other group spp of bacteria were present Cercosporas pp

Some other unidentifi ed microbes have been observed

Beneficiary units

4 x 105

8 x 106

2 x 105

Penicilliums pp

Pseudomona s spp

Aspergillusni ger

Bacillus spp

Fusarium spp

Sate of Art

6 x 105

10 x 106

3 x 105

Moniliaspp Penicilliums pp Aspergillusni ger Fusarium spp

Streptomyces

Some other unidentifi ed microbes have been observed

Streptomyces

Some other unidentifi ed microbes have been observed

And some other group of bacteria were present

Pseudomona s spp Bacillus spp And some other group of bacteria were present

Trichoderma spp

Results indicate that the total microbial population and diversity were increased during vermicomposting process (Table: 3).

Figure 6: Microscopic observations of fungi at 40X

10 9 8 7 6 5 4 3 2 1 0

Demonstration unit Beneficiary units Sate of Art

Fungi (CFU-g)

Bacteria(CFU Bacteria(CFU-g)

Actinomycetes (CFU-g)

Figure 7: Graph raph showing diversity in the readings

Figure 8: Microbial diversity/populations on NA/PDA media plate

Figure 9:: Microscopic observation of Microorganisms (100x)

Chapter 9 RESULTS FOR VERICOMPOST SAMPLES

Table 4: Vermicomposting field trial result

S. No

Farmers Name

Fungi

Bacteria

Actinomycetes

Fusarium spp

Aspergillus spp

Penicillium spp

Cercospora spp

Others

Pseudomonas spp

Bacillus spp

Streptomyces spp

ILAM SINGH without VC

ILAM SINGH with VC

KARAM SINGH Without VC

KARAM SINGH with VC

0=ABSENT 1=PRESENT

Streptomyces spp

Bacillus spp

KARAM SINGH WITH VC KARAM SINGH WITHOUT VC ILAM SINGH WITH VC ILAM SINGH WITOUT VC Pseudomonas spp

Others

Cercospora spp

Penicillium spp

Aspergillus spp

Fusarium spp

1 0.8 0.6 0.4 0.2 0

Fungi Bacteria Actinomycetes

ILAM SINGH WITOUT VC ILAM SINGH WITH VC KARAM SINGH WITHOUT VC KARAM SINGH WITH VC

Figure 10: Field Trial rial result of vermicomposting Results indicated that, when crop was sown without fortified vermicompost, the microbial population and diversity was recorded less whereas in vermicompost treated plot the diversity was higher. In VC plot most of the microorganisms are beneficial in nature like, Pseudomonas, Bacillus, Aspergillus, Penicillium and Actinomycetes. It also reduces the incidence of soil borne pathogen

Figure 11: Before and after application of vermicompost

Chapter 10 CONCLUSIONS Conclusions: In both the areas, the population and diversity of bacteria was more as compare to fungi and actinomycetes. This increase in microbial diversity in soil and vermicompost indicated that vermicompost facilitates microbial proliferation in final stabilized product. It is concludes that, in vermicompost more number of microbial diversity and population is present and those are beneficial in nature and, in soil most of the microbes are pathogenic and saprophytic in nature on the basis of their growth and sporulation pattern/behaviour. The soils which were sandy in nature had less number of microbial diversity. The sample with vermicompost had more moisture containing capacity and hence increased microbial diversity. Further aspects: On the basis of the soil character and water retaining capacity and various temperature conditions of the atmosphere supported increase in microbial diversity benefitted seasonal growth of crops and through this we can extract some of the seasonal microbes and which will help in easy growth of off season agricultural crops. Further study Molecular characterization of the selected microbes which could be further used as abio fertilizer for improving soil qualities. Publication plan: 1. Isolation, Identification and Characterization of important microorganisms in soil and vermicompost from Haryana and Punjab

Chapter 11 REFERENCES 1)Farming, fertilizers and the nitrate problem.AuthorsAddiscott, T. M.; Whitmore, A. P.; Powlson, D. Farming, fertilizers and the nitrate problem. 1991 pp. 170 pp. 2)Bauman, R. (2005) Microbiology. Pearson BanjaminCummings. ParkTalaro, K. (2008) Foundations in Microbiology.McGraw-Hill. 3)BIODIVERSITY,D H Wall, Colorado State University, Fort Collins,CO, USA 2005, Elsevier Ltd. All Rights Reserved 4)Sustainable Farming Systems, pp. 13–22. Melbourne:CSIRO Press. Cook RJ and Baker KF (1983) The Nature and Practice of 5)Biological Control of Plant Pathogens. St Paul, MN: AmericanPhytopathological Society Press. Dowling DN and O’Gara F (1994) Metabolites of Pseudo6)Monas involved in the biocontrol of plant disease. Trends in Biotechnology 4: 239– 249.Hoitink HAJ and Boehm MJ (1999) Biocontrol within the 7)Context of soil microbial communities: a substrate-dependent phenomenon. Annual Review of Phytopathology 37: 427–446.Hokkanen HMT and Lynch JM (eds) (1995) Biological 8)Control: Benefits and Risks. Cambridge, UK: Cambridge University Press. 9)Hornby D (ed.) (1990) Biological Control of Soil-borne Plant Pathogens. Wallingford, UK: CAB International. 10)Kloepper JW (1993) Plant growth-promoting rhizobacteriaas biological control agents. In: Metting FB Jr. (ed.) Soil 11)Microbial Ecology – Applications in Agricultural for macromolecularcomparisons. Methods Enzymol. 234, 23. 12) Wilfinger, W.W., Mackey, M., andChomczynski, P. (1997) Effect of pH and ionic strength on the spectrophotometric assessment of nucleic acid purity. BioTechniques22, 474. 13)Sambrook, J., Fritsch, E.F., andManiatis, T. (1989) Molecular Cloning: A Laboratory Manual. 2nd ed. Cold Spring Harbor, NY:

LIST OF ABBREVATIONS NA: Nutrient agar PDA Potato dextrose agar TERI- The Energy And Resources Institute Spp: Species VC: Vermicompost CFU: Colony Forming Units MO: Microorganisms