Evaluation of Sporidiobolus pararoseus strain YCXT3 as biocontrol agent of Botrytis cinerea on post- by Eduardo Santiago

Biological Control 62 (2012) 53–63

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Evaluation of Sporidiobolus pararoseus strain YCXT3 as biocontrol agent of Botrytis cinerea on post-harvest strawberry fruits R. Huang a,b, H.J. Che a, J. Zhang a, L. Yang a, D.H. Jiang a, G.Q. Li a,⇑ a b

State Key Laboratory of Agricultural Microbiology and Key Laboratory of Plant Pathology of Hubei Province, Huazhong Agricultural University, Wuhan 430070, China Plant Protection Institute, Jiangxi Academy of Agricultural Sciences, Nanchang 330200, Jiangxi Province, China

h i g h l i g h t s

g r a p h i c a l a b s t r a c t

" We evaluate Sporidiobolus pararoseus as biocontrol agent of Botrytis cinerea. " The yeast could not inhibit growth of B. cinerea in dual cultures on agar medium. " The yeast cells effectively suppressed B. cinerea in infection of strawberry fruits. " The yeast produces volatiles highly effective in suppression of B. cinerea. " We conclude that S. pararoseus is a promising biocontrol agent against B. cinerea.

a r t i c l e

i n f o

Article history: Received 6 December 2011 Accepted 23 February 2012 Available online 3 March 2012 Keywords: Sporidiobolus pararoseus (strain YCXT3) Botrytis cinerea Strawberry Volatile organic compounds Biological control

a b s t r a c t This study evaluated Sporidiobolus pararoseus (Sp) strain YCXT3 as biocontrol agent of Botrytis cinerea (Bc), the causal agent of strawberry gray mold disease. Efﬁcacy of live yeast cells and volatile organic compounds (VOCs) of Sp in suppression of Bc on strawberry fruits was determined. Results showed that in dual cultures of Sp and Bc on potato dextrose agar at 20 °C, Sp did not inhibit mycelial growth of Bc. However, inoculation of the yeast cell suspensions of Sp (1 105 or 1 106 yeast cells ml 1) on strawberry fruits resulted in reducing the disease incidence from 96–100% in the control treatment to 39–50% in the Sp treatment and the disease severity index from 5.1–7.0 in the control treatment to 1.1–1.9 in the Sp treatment. We found that the VOCs from the Sp cultures on yeast extract peptone dextrose agar were highly effective in inhibiting both the conidial germination and the mycelial growth of Bc. A total of 39 VOCs, including 2-ethyl-1-hexanol, were identiﬁed in cultures of Sp using GC–MS. Authentic 2-ethyl-1hexanol was found to have strong anti-fungal activity against Bc with the IC50 values of 1.5 and 5.4 ll l 1 for conidial germination and mycelial growth, respectively. The VOCs from the Sp cultures were effective in suppression of gray mold disease under the air-tight conditions. This study suggests that the strain YCXT3 of Sp is a promising agent for control of Bc and production of VOCs is a valid biocontrol mechanism for this yeast strain. Ó 2012 Elsevier Inc. All rights reserved.

1. Introduction Botrytis cinerea Pers.: Fr. (teleomorph: Botryotinia fuckeliana (de Bary) Whetzel) is a is a plant pathogenic fungus distributed

world-wide and causes gray mold disease on both pre-harvest and post-harvest plant tissues (Williamson et al., 2007). It infects more than 200 species of plants comprising many economically important crops such as cucumber (Cucumis sativus L.), table grape (Vitis vinifera L.), tomato (Lycopersicon esculentum Mill.) and strawberry (Fragaria ananassa Duch.) (Williamson et al., 2007). Yield loss caused by B. cinerea on these crops can be severe under cool and humid conditions (Yu et al., 2006; Droby and Lichter, 2007).

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Previous studies showed that B. cinerea is a necrotrophic pathogen. It infects plant tissues through multiple mechanisms, including secretion of cell wall-degrading enzymes (e.g. cutinase and polygalacturonases) and phytotoxic metabolites (e.g. botrydial and oxalic acid) (Choquer et al., 2007). So far, cultivars highly resistant to infection by B. cinerea are not available in germplasm of many crops including strawberry (Droby and Lichter, 2007). Control of gray mold disease is mainly dependent on the use of chemical fungicides (Blacharski et al., 2001; Mertely et al., 2002). However, there are several public concerns over the large scale use of fungicides to control gray mold disease, as the frequent use of fungicide may generate some negative side effects, including residues in plant produce and induction of fungicide-resistance in B. cinerea (Hunter et al., 1987; Yourman and Jeffers, 1999; Diánez et al., 2002). Therefore, it is imperative to exploit alternative measures, such as biological control, to suppress gray mould disease on strawberry, as well as on many other crops (Sutton et al., 1997; Paulitz and Bélanger, 2001; Fravel, 2005; Droby and Lichter, 2007; Sharma et al., 2009). Many microbial agents have been found to be effective in suppression of B. cinerea on numerous crops under both pre-harvest and post-harvest conditions (Paulitz and Bélanger, 2001; Fravel, 2005; Sharma et al., 2009). The promising biocontrol agents include Bacillus amyloliquefaciens (Mari et al., 1996), Bacillus subtilis (Hang et al., 2005), Paenibacillus polymyxa (Helbig, 2001), Candida oleophila (Lima et al., 1997), Clonostachys rosea (=Gliocladium roseum) (Sutton et al., 1997), Metschnikowia fructicola (Karabulut et al., 2004; Blachinsky et al., 2007), Ulocladium atrum (Boff et al., 2002), Pichia guilliermondii (Wszelaki and Mitcham, 2003), Streptomyces spp. (Wan et al., 2008; Li et al., 2011) and Trichoderma harzianum (Elad et al., 1993). Commercial products based on C. oleophila, such as Aspire (strain I-182), on Metschnikowia fructicola, such as ‘‘Shemer’’, and on T. harzianum, such as Trichodex (strain T39), are now available for protection of greenhouse vegetables or post-harvest fruits from infection by B. cinerea (Fravel, 2005; Blachinsky et al., 2007). Several mechanisms, including antibiosis, competition for limited nutrients and space, induction of plant defense response, suppression of sporulation of B. cinerea, and mycoparasitism, have been proposed to be responsible for biological control of B. cinerea by these microbial agents (Sutton et al., 1997; Filonow, 1998; Jijakli and Lepoivre, 1998; Paulitz and Bélanger, 2001; Janisiewicz and Korsten, 2002). Sporidiobolus pararoseus Fell and Tallman is a ballistosporous yeast belonging to Basidiomycota. It is known to exist on the phylloplane and the fruit surface of many terrestrial plants (Nakase, 2000; Allen et al., 2004; Li et al., 2010; Janisiewicz et al., 2010; Machado and Bettiol, 2010; Raspor et al., 2010). Sharma et al. (2008) reported that a strain of S. pararoseus isolated from kinnow fruits (Citrus nobilis C. deliciosa) showed effective inhibition against several phytopathogenic fungi, including Alternaria alternata, Botryodiplodia theobromae, Geotrichum candidum, Penicillium digitatum and Penicillium italicum. Competition for limited nutrients and space was proposed to be the major mechanism for S. pararoseus in suppression of these pathogens (Sharma et al., 2008). Janisiewicz et al. (2010) indicated that S. pararoseus isolated from nectarines (Prunus persica var. nectarine) could effectively suppress brown rot of nectarine fruits caused by Monilinia fructicola Honey. Raspor et al. (2010) found that 26 isolates of S. pararoseus from grapevine (V. vinifera) or fermented grape musts could inhibit mycelial growth of B. cinerea on agar medium, although the inhibitory activity of S. pararoseus was lower than that of Aureobasidium pullulans, Metschnikowia pulcherrima, Metschnikowia reukauﬁi and P. guilliermondii. Machado and Bettiol (2010) showed that S. pararoseus could inhibit sporulation of B. cinerea on leaf discs of lilies (Lilium spp.). However, information about the efﬁcacy of the yeast

cells and volatile organic compounds of S. pararoseus in suppression of B. cinerea infection on plant tissues, including post-harvest strawberry fruits, is not available in the literature. Volatile organic compounds (VOCs) are low molecular weight substances (usually <300 Da) with low polarity, but with high vapor pressure (Vespermann et al., 2007). Antimicrobial VOCs produced by bacteria such as B. subtilis (Chen et al., 2008), actinomyces such as Streptomyces spp. (Wan et al., 2008; Li et al., 2011), filamentous fungi such as Muscodor albus (Mercier and Manker, 2005), yeasts such as Candida intermedia (Huang et al., 2011) and higher plants such as ‘Isabella’ grapes (Vitis labrusca) (Kulakiotu et al., 2004a,b) were reported to be effective in suppressing both the conidial germination and the mycelial growth of B. cinerea on agar media and in suppressing Botrytis diseases on plant tissues. However, whether or not S. pararoseus produces VOCs inhibitory either to the conidial germination or to the mycelial growth of B. cinerea remains unknown. A strain of S. pararoseus designated as strain YCXT3 was isolated from a healthy strawberry leaf in our previous study (Huang, 2011). It was used in the current study to address the following objectives: (i) to detect the interaction between S. pararoseus and B. cinerea in dual cultures and suppression of B. cinerea by the volatile organic compounds (VOCs) of S. pararoseus; (ii) to determine the composition of the VOCs of S. pararoseus; and (iii) to detect the efficacy of the live yeast cells and the VOCs of S. pararoseus in suppression of strawberry gray mold disease under controlled conditions. 2. Materials and methods 2.1. Fungal strains and culture media The yeast strain YCXT3 of S. pararoseus was isolated from a healthy leaf of strawberry (F. ananassa) grown in Yi Chang City of Hubei Province, China. It was identified on the basis of morphological and physiological characteristics and the DNA sequence of the internal transcribed spacer (ITS) region (ITS1-5.8S rDNA-ITS2) (GenBank Acc. No. GQ913347) (Huang, 2011). The strain SB-1 of B. cinerea was isolated from a diseased fruit of strawberry collected from Wuhan of China (Huang et al., 2011). The two fungal strains were maintained on Potato Dextrose Agar (PDA) at 4 °C. Working cultures were prepared by transferring yeast cells of S. pararoseus or mycelia of B. cinerea on PDA in Petri dishes, which were then incubated at 20 °C. Conidia of B. cinerea were harvested from 14day-old PDA cultures (Li et al., 2002). The yeast cells of S. pararoseus were harvested from 5-day-old PDA cultures by washing the cultures with sterile distilled water. The resulting yeast cell suspensions of S. pararoseus (1 108 yeast cells ml 1) and the conidial suspensions of B. cinerea (1 106 conidia ml 1) were used as inoculum for the experiments in this study. The percentage of germinated conidia of B. cinerea was higher than 90% (20 °C, 12 h). Three culture media, including PDA, Water Agar (WA) and Yeast Extract Peptone Dextrose agar (YEPDA), were used in this study. PDA was made of fresh potato tubers (boiled water extract of 200 g peeled potato tuber, 20 g D-glucose, 20 g agar and 1000 ml water). WA was prepared with agar in water (2%, w/v). YEPDA contained 10 g yeast extract, 20 g peptone, 20 g D-glucose, 20 g agar and 1000 ml water. 2.2. Dual cultures Inhibition of mycelial growth of B. cinerea by S. pararoseus was tested according to the method described by Raspor et al. (2010) with minor modifications. For the dual culture treatment (S. pararoseus + B. cinerea), a loop of yeast cells of S. pararoseus from

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3-day-old cultures on YEPDA (20 °C) was streaked on a PDA plate (9 cm diam.) at the distance of 2 cm from the rim of that plate. An agar plug (6 mm diam.) containing mycelial mats of B. cinerea was removed from the margin area of a 2-day-old PDA culture of B. cinerea (20 °C) was inoculated on the same plate at the distance of 5 cm from the yeast line. For the single culture treatment, a PDA plate was inoculated with a mycelial agar plug of B. cinerea alone. There were three plates serving as three replicates for both the dual culture treatment and the single culture treatment. The plates were incubated at 20 °C for 4 and 6 days for daily observation of the mycelial growth of B. cinerea in each plate. 2.3. Suppression of mycelial growth of B. cinerea by the VOCs of S. pararoseus Two trials, the simultaneous incubation (SI) trial and the nonsimultaneous incubation (NSI) trial, were conducted in this experiment. There were three treatments in each trial: (i) control; (ii) S. pararoseus; (iii) S. pararoseus + activated carbon (AC). AC acts as the VOC absorbent (Vespermann et al., 2007; Wan et al., 2008; Huang et al., 2011). In the SI trial, mycelial agar plugs (MAPs) of B. cinerea were inoculated in the center of Petri dishes each containing 20 ml PDA, one MAP per dish. For the control treatment, a PDA dish with B. cinerea was covered face-to-face above a YEPDA dish containing un-inoculated YEPDA (fresh and sterile) to make a DDC; For the treatment of S. pararoseus, a PDA dish with B. cinerea was covered face-to-face above a YEPDA dish with S. pararoseus (ca. 1 107 yeast cells per dish) to make a DDC; for the treatment of S. pararoseus + AC, a piece of cotton gauze (Warp Weft = 21S 21S, Nanchang Xiangyi Medical Apparatus Co., Ltd., Nanchang, Jiangxi Province, China) with the average opening size of about 1 mm2 was covered above the YEPDA dish with S. pararoseus (ca. 1 107 yeast cells per dish). TT™ AC particles (ca. 1 cm diam.) (Guangzhou Huana Automobile Products Co., Ltd., Guang Dong, China) were placed on the gauze, 6 g per dish. Then, a PDA dish with B. cinerea was covered face-to-face above the gauze and the YEPDA dish to make a DDC. All the DDCs were individually sealed with Paraﬁlm™ (Chicago, IL, USA). For each treatment, there were nine DDCs, which were divided into three groups, three DDCs (replicates) per group. Finally, the three groups of DDCs were incubated at 4 °C for 6 days, at 10 °C for 4 days and at 20 °C for 3 days, respectively. The colony diameter of B. cinerea in each DDC was determined. In order to understand the difference of the VOCs from the cultures of S. pararoseus at 4, 10 and 20 °C in suppression of the mycelial growth of B. cinerea, the yeast cells of S. pararoseus in on the YEPDA medium in each DDC were collected by washing with water (10 ml per dish). The concentration of the resulting yeast cell suspensions was determined using a haemocytometer and a compound light microscope. The experiment was repeated three times with three replicates for each treatment each time. The NSI trial was done using the procedures similar to those described in the SI trial of this experiment. There was only one difference, namely S. pararoseus was incubated on YEPDA in Petri dishes at 20 °C for 24 h. Then, the PDA dishes with B. cinerea (1 MAP per dish) were face-to-face covered above the YEPDA dishes with S. pararoseus and above the cotton gauze with AC particles on the YEPDA dishes with S. pararoseus for the treatments of S. pararoseus and S. pararoseus + AC, respectively. The bioassay was repeated three times with three replicates for each treatment each time. 2.4. Suppression of conidial germination of B. cinerea by the VOCs of S. pararoseus Three treatments, namely control, S. pararoseus and S. pararoseus + AC, were included both in the SI trial and in the NSI trial of

this experiment. The procedures for this experiment were similar to those for testing the effect of the VOCs of S. pararoseus on mycelial growth of B. cinerea. In both trials, aliquots of the conidial suspension of B. cinerea (1 106 conidia ml 1) were pipetted on WA in Petri dishes, 100 ll per dish, and plated. In each trial, the WA dishes with B. cinerea conidia were individually covered face-toface above dishes containing un-inoculated YEPDA, the YEPDA cultures of S. pararoseus (0 or 24 h old) and YEPDA cultures (0 or 24 h old) of S. pararoseus (0 or 24 h old) + AC for the treatments of control, S. pararoseus and S. pararoseus + AC, respectively, to make DDCs. Then, the DDCs were individually sealed with Parafilm™ and incubated at 4 °C for 48 h, at 10 °C for 48 h or at 20 °C for 24 h for determination of the percentage of germinated conidia and the length of germ tubes of B. cinerea in each WA dish. Meanwhile, the yeast cells of S. pararoseus in each YEPDA dish were washed with sterile distilled water and counted under microscope for determination of the yeast biomass. The experiment was repeated three times with three replicates for each treatment in each trial. 2.5. Suppression of strawberry gray mold disease by the live yeast cells of S. pararoseus In this bioassay, we conducted three experiments to determine the efficacy of the live yeast cells of S. pararoseus in suppression of strawberry gray mold disease. The first experiment was done on the strawberry fruits artificially inoculated with B. cinerea. There were two treatments in this experiment: B. cinerea alone and S. pararoseus + B. cinerea. Mature healthy fruits of strawberry (F. ananassa var. Feng Xiang No. 5) were harvested in a greenhouse in Wuhan of China and those with the size of 4.0–4.5 3.0– 3.5 cm (length width, 16–20 g per fruit) were selected for this experiment. Three lots of strawberry fruits, 11 fruits per lot, were surface-sterilized in 70% ethanol for 30 s, washed in sterile distilled water and blotted on paper towels to remove the water remains on the fruit surface. One lot of the fruits was used for the treatment of B. cinerea alone and the other two lots of fruits were used for the treatment of S. pararoseus + B. cinerea. In the treatment of B. cinerea alone, the 11 fruits were placed in a plastic container (27 20 7 cm, length width height) were individually inoculated with B. cinerea by pipetting aliquots of the conidial suspension of B. cinerea (1 106 conidia ml 1) on the fruit surface, 50 ll per fruit (Huang et al., 2011). For the treatment of S. pararoseus + B. cinerea, the two lots of the fruits were dipped in two yeast cell suspensions of S. pararoseus (1 105 and 1 106 yeast cells ml 1), respectively, for 5 s. Then, the fruits of each lot were taken out, placed on moisturized paper towels in a plastic container and individually inoculated with the conidial suspension of B. cinerea, 50 ll per fruit (Huang et al., 2011). The containers for these treatments were individually sealed with 0.1-mm-thick transparent plastic films (Gold Mine Plastic Industry Ltd., Jiangmen, China) to maintain high humidity and placed in a growth chamber at 20 °C for 7 days. Disease severity for each fruit was rated using the numerical rating scale of 0–8 as described by Huang et al. (2011). The data were used for calculation of the disease incidence (the percentage of diseased fruits) and the disease severity index with the formula also described by Huang et al. (2011). The experiment was repeated three times. The second experiment was done on strawberry fruits naturally infected with B. cinerea and other fungi, including Mucor sp., Penicillium sp. and Rhizopus sp. The selected strawberry fruits were directly treated either with the yeast cell suspension of S. pararoseus (1 105 or 1 106 yeast cells ml 1) or with water (control), 11 fruits per treatment. The treated fruits for each treatment were then placed on moisturized paper towels in a plastic container, which was immediately sealed with a piece of transparent plastic

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film and placed in an incubator at 20 °C for 7 days. The fruits were then individually rated for the incidence and the severity of fruit rot (Huang et al., 2011). The experiment was repeated three times. The third experiment was conducted to determine the effect of the time intervals between inoculations with S. pararoseus and B. cinerea on the suppressive efficacy of S. pararoseus against B. cinerea. There were five time-intervals: 0, 1, 3, 5 and 7 days. For each time interval, there were two treatments, S. pararoseus + B. cinerea and B. cinerea alone. Ten lots of strawberry fruits, 11 fruits per lot, were selected, five fruit lots for S. pararoseus + B. cinerea and the remaining five fruit lots for B. cinerea alone. The fruits of each lot were surface-sterilized with 70% ethanol, washed in sterile distilled water and blotted on paper towels and finally dipped in the yeast cell suspension of S. pararoseus (1 107 cells ml 1) or water for 5 s and placed in a plastic container. For the 0-day-interval, the strawberry fruits in each container were immediately inoculated with the conidial suspension of B. cinerea (1 106 conidia ml 1), 50 ll per fruit. The container was sealed with a piece of transparent plastic film and placed in an incubator at 20 °C for 7 days. Finally, the fruits were individually rated both for the incidence and for the severity index of gray mold disease (Huang et al., 2011). For the remaining time-intervals, the S. pararoseus- or water-treated fruits were placed in plastic containers, 11 fruits per container. The containers were individually sealed with transparent plastic films and placed in an incubator (20 °C). After incubation for 1, 3, 5 and 7 days, the fruits in each container were inoculated with the conidia suspension of B. cinerea (1 106 conidia ml 1), 50 ll per fruit. After that, the containers were re-sealed with the transparent plastic films and incubated at 20 °C for another 7 days. The fruits were individually rated for the incidence and the severity index of gray mold disease (Huang et al., 2011). This whole experiment was repeated three times. 2.6. Efficacy of the VOCs from S. pararoseus in control of strawberry gray mold disease The experiment was done in closed glass desiccators (18 23 cm, diameter height, ca. 5.8 L in volume) without loading of any drying agent. There were three treatments: (i) B. cinerea alone (Bc); (ii) B. cinerea + S. pararoseus (Bc + Sp); and (iii) B. cinerea + S. pararoseus + AC (Bc + Sp + AC). In the treatment of Bc, two un-covered Petri dishes containing the un-inoculated YEPDA medium were placed at the bottom space of a desiccator. Fourteen fruits were surface-sterilized, individually inoculated with B. cinerea and placed on the perforated ceramic mesh above the un-inoculated YEPDA dishes in the desiccator. In the treatment of Bc + Sp, 2, 4, 6, 8 or 10 un-covered YEPDA dishes containing 24-h-old cultures of S. pararoseus were placed at the bottom of a desiccator as the fumigation source. Then, 14 fruits were surface-sterilized, inoculated with B. cinerea and placed on the mesh in the desiccator. In the treatment of Bc + Sp + AC, ten un-covered YEPDA dishes containing 24-h-old cultures of S. pararoseus and 100 g AC powder (Lu Yuan Commodity Manufacturing Company, Huang Gang, China) were placed at the bottom of a desiccator. Then, 14 fruits were surface-sterilized, inoculated with B. cinerea (1 105 conidia per fruit) and placed on the mesh in the desiccator. There were three desiccators (replicates) for each treatment. The desiccators were covered and placed in an incubator at 20 °C for 7 days. The fruits in each desiccator were individually rated for the incidence and the severity index of gray mold disease (Huang et al., 2011). 2.7. GC–MS analysis of the volatile organic compounds of S. pararoseus The strain YCXT3 of S. pararoseus was inoculated on YEPDA medium in Petri dishes (9 cm diameter, 20 ml medium per dish)

at about 1 107 yeast cells per dish. The YEPDA medium without inoculation of S. pararoseus was used as control. Both the S. pararoseus-inoculated dishes and the control dishes were placed in an incubator at 20 °C for 24 h. Then, two S. pararoseus dishes and two control dishes were selected and the YEPDA medium (with or without S. pararoseus) in each dish was all transferred to a 100-ml extraction glass vial. The vial was sealed with a silicon rubber mat and incubated at 40 °C for 15 min to equilibrate the solid phase and the phase of volatile organic compounds (VOCs) in the vial. A 50/30-lm solid-phase micro-extraction fiber containing divinylbenzene/carboxen/polydimethylsiloxane (DVB/PDMS/CAR) (Supelco, Bellfonte, PA, USA) was inserted into the vial for extraction of the VOCs from the yeast culture or the control YEPDA at 40 °C for 40 min. Then, the fiber was pulled out from the vial and inserted into the injection port of a gas chromatography-mass spectrometry (GC–MS) machine (6890N-5975B, Agilent Technologies Inc., CA, USA) equipped with a J & WHP-5MS fused-silica capillary column (30 m 0.25 mm ID, 0.25 lm thickness film) (Agilent Technologies Inc., CA, USA) for analysis of the VOC composition. The GC machine was operated in splitless mode with the injector temperature being set at 250 °C. The column temperature was programmed as follows: 40 °C for 3 min, then increased at 3 °C per minute to 160 °C, held at 160 °C for 2 min, continued to increase at 8 °C per minute to 220 °C and finally held at 220 °C for 3 min. Ultra-high purity helium was used as the carrier gas, which was set at the flow rate of 1.2 ml per minute. The MS machine was operated in electron ionization mode at 70 eV and 230 °C. Mass spectra were obtained using a scan modus with the total ion counts within the range of 50–550 m/z. Identification of the VOCs was done by comparing the mass spectra and the retention times for the detected compounds with the related data for the standard compounds deposited in the database of the National Institute of Standards and Technology (NIST)/EPA/NIH library (NIST05) and the Wiley Registry of Mass Spectral Database (Wiley 7.0) in the mass spectrometry. Analysis of VOCs in the sample of the S. pararoseus culture or the control YEPDA was repeated twice. The VOCs appearing both in the cultures of S. pararoseus and in the control YEPDA were not considered to be produced by S. pararoseus. The values for the areas of all the detected VOC peaks from the GC– MS output data were added to get the total peak area. The percentage of the peak area for a VOC in the total peak area was calculated to estimate the abundance of that VOC in the total detected VOCs. 2.8. Antifungal activity of some synthetic VOCs Eight VOCs identified in YEPDA cultures of S. pararoseus, including 2-ethyl-1-hexanol, 1-chloro-octadecane, 2-dodecen-1yl( )succinic anhydride, 1-decene, tetradecane, 2,6,10-trimethyldodecane, pentadecane, and 2-hexyl-1-decanol, were selected for testing their antifungal activity against B. cinerea. The compound 2-ethyl-1-hexanol was purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). The remaining seven compounds were purchased from Sigma–AldrichÒ Co. (St. Louis, MS, USA). The authentic VOCs were individually tested for suppressing both the conidial germination and the mycelial growth of B. cinerea. The remaining 31 VOC components in the VOC profile of S. pararoseus were not individually tested for inhibition of B. cinerea, as the authentic compounds for these VOCs were not available for us. In the test for suppressing the mycelial growth, a mycelial agar plug (MAP) removed from the colony margin of a 2-day-old PDA culture of B. cinerea (20 °C) was inoculated in a Petri dish containing 20 ml PDA. Meanwhile, a filter paper piece (FPP) (1.2 1.2 cm, length width) was placed in the center of another Petri dish. The solution of a given authentic VOC was pipetted at 1, 2.5, 5, 10 or 20 ll onto the FPP in the dish. After removal of the cover dish,

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Fig. 1. Effects of the volatile organic compounds (VOCs) produced by Sporidiobolus pararoseus strain YCXT3 and the VOCs of S. pararoseus plus activated carbon (AC) on mycelial growth of Botrytis cinerea at 4, 10 and 20 °C. (A) and (B) Effects of the VOCs of S. pararoseus and the VOCs of S. pararoseus + AC on mycelial growth of B. cinerea in the simultaneous incubation (SI) trial and in the non-simultaneous incubation (NSI) trials, respectively; (C) and (D) the yeast biomass of S. pararoseus on yeast extract peptone dextrose agar in the SI trial and the NSI trial, respectively. Results in each histogram are expressed as means ± standard errors (n = 9). Mean values for different treatments in each histogram labeled with different letters indicate signiﬁcant difference (P < 0.01) according to Fisher’s protected least signiﬁcant difference test.

the PDA dish inoculated with B. cinerea was covered face-to-face above the FPP dish with the given dose of an authentic VOC to make a double-dishes chamber (DDC), which was sealed using double layers of Parafilm™ to make a closed DDC (ca. 180 cm3 in volume). For the control treatment, a PDA dish with B. cinerea was covered on a dish with a blank FPP (without addition of any authentic VOC) to make a control DDC, which was also sealed with Parafilm™. There were three DDCs (replicates) for each treatment. The DDCs were placed in an incubator at 20 °C for 3 days. The diameter of the colony of B. cinerea in each DDC was measured. The percentage of inhibition of the mycelial growth of B. cinerea by a given authentic VOC at each dose was calculated on the basis of the difference of the colony diameter between the treatments of control and the VOC at the given dose. The concentration value for 50% inhibition of the mycelial growth (IC50) expressed as ll l 1 was inferred from the data about the inhibition percentages and the VOC doses applied in the DDCs (Huang et al., 2011). In the test for suppressing conidial germination, aliquots of a conidial suspension (1 106 conidia ml 1) were pipetted onto the WA medium in Petri dishes (9 cm diameter), 100 ll conidial suspension per dish. The conidial suspension drop in each dish was evenly spread on the surface of the WA medium. After removal of the cover dish, a WA dish with the B. cinerea conidia was covered face-to-face above a control dish with a blank FPP or on a dish with a FPP soaked with a given authentic VOC at 1, 2.5, 5, 10 or 20 ll to make a DDC, which was immediately sealed using Parafilm™ to make a closed DDC. There were three DDCs for the control treat-

ment and the treatment of VOC at each dose. The DDCs were placed in an incubator at 20 °C for 12 h. The frequency of the conidial germination of B. cinerea was determined by randomly counting at least 150 conidia in each WA dish under a compound light microscope. A conidium was considered to have germinated when the length of the germ tube was equal to, or greater than, length of that conidium. The effect of VOC on percentage of suppression of conidial germination of B. cinerea was calculated as above. 2.9. Statistical analysis All the statistical analyses in this study were conducted using SAS/STATÒ software (SAS Institute, Cary, NC, version 8.0, 1999). Data on colony diameter, percentage of germinated conidia (arcsine-transformed), length of germ tubes, and on disease incidence (arcsine-transformed) and the disease severity index in different repetitions were pooled for analysis, as they were not signiﬁcantly different (P > 0.01) between experiments according to the F-tests in analysis of variance (ANOVA). The procedure UNIVARIATE was used to analyze the data on disease incidence and disease severity index between the treatments of B. cinerea alone and S. pararoseus at each time interval. Mean values of each parameter was compared with the Student’s t-test (P < 0.01). The procedure ANOVA was used to analyze the data in the remaining experiments. Means of each parameter for different treatments in each experiment were compared using Fisher’s Protected Least Signiﬁcant Difference Test (P < 0.01).

R. Huang et al. / Biological Control 62 (2012) 53–63

3. Results

3.2. Suppression of mycelial growth of B. cinerea by the VOCs of S. pararoseus

3.1. Dual cultures Results of the dual cultural experiment showed that S. pararoseus did not inhibit mycelial growth of B. cinerea. In the control treatment (B. cinerea alone), B. cinerea colonized the entire plates after incubation at 20 °C for 4 days (data not shown). In the dual culture treatment (S. pararoseus + B. cinerea), B. cinerea colonized most part of the medium in each plate after incubation for 4 days with the colony front contacting the yeast colony. Formation of inhibitory zones between colonies of S. pararoseus and B. cinerea was not observed. After incubation for 6 days, B. cinerea grew over the colonies of S. pararoseus and colonized the remaining part of the medium in each dual culture plate.

The volatile organic compounds (VOCs) from the YEPDA cultures of S. pararoseus were effective in inhibiting mycelial growth of B. cinerea. After incubation at 4 °C for 6 days, 10 °C for 4 days and 20 °C for 3 days, the average colony diameter of B. cinerea reached 87–90 mm in both trials (SI and NSI) for the control treatment (Fig. 1). For the treatment of S. pararoseus, due to the presence of the VOCs from the cultures of S. pararoseus, the average colony diameter of B. cinerea was signiﬁcantly (P < 0.01) reduced to 39, 36 and 29 mm at 4, 10 and 20 °C, respectively, in the SI trial, and to 6, 8 and 14 mm at these temperatures, respectively, in the NSI trial (Fig. 1). However, the suppressive effect of the VOCs of S. pararoseus on mycelial growth of B. cinerea was partially or com-

Fig. 2. Effects of the volatile organic compounds (VOCs) produced by Sporidiobolus pararoseus strain YCXT3 and the VOCs of S. pararoseus plus activated carbon (AC) on conidial germination and germ-tube extension of Botrytis cinerea on water agar (WA) at 4, 10 and 20 °C. (A), (B) and (C) Data on percentages of germinated conidia, length of germ tubes of B. cinerea on WA and the yeast biomass of S. pararoseus on yeast extracts peptone dextrose agar (YEPDA), respectively, in the simultaneous incubation trial; (D), (E) and (F) data on percentages of germinated conidia, length of germ tubes of B. cinerea on WA and the yeast biomass of S. pararoseus on YEPDA, respectively, in the nonsimultaneous incubation trial. Results in each histogram are expressed as means ± standard errors (n = 9). Mean values for different treatments at each temperature labeled with different letters indicate signiﬁcant difference (P < 0.01) according to Fisher’s protected least signiﬁcant difference test.

R. Huang et al. / Biological Control 62 (2012) 53–63

Fig. 3. Effect of the live yeast cells of Sporidiobolus pararoseus strain YCXT3 on incidence and severity index of strawberry gray mold disease (20 °C, 7 days). Results in each histogram are expressed as means ± standard errors. Mean values for different treatments labeled with different letters indicate signiﬁcant difference (P < 0.01) according to Fisher’s protected least signiﬁcant difference test.

pletely nullified by the presence of activated carbon (AC) in the treatment of S. pararoseus + AC. The average colony diameter of B. cinerea in this treatment reached 85–90 mm in the SI trial and 67–90 mm in the NSI trial (Fig. 1). 3.3. Suppression of conidial germination of B. cinerea by the VOCs of S. pararoseus The VOCs from the YEPDA cultures of S. pararoseus were effective in inhibiting conidial germination of B. cinerea (Fig. 2). After incubation at 4 °C for 48 h, 10 °C for 48 h and 20 °C for 24 h, the percentage of the germinated conidia of B. cinerea reached 100% in the control treatment (Fig. 2). The average length of germ tubes of B. cinerea reached 124, 451 and 391 lm at 4, 10 and 20 °C, respectively. In the treatment of S. pararoseus, the yeast biomass of S. pararoseus was 0.3–3.3 109 yeast cells per dish in the SI trial, lower than that of 4.4–6.9 109 yeast cells per dish in the NSI trial. As a result, in the SI trial, the percentages of the germinated conidia of B. cinerea were higher than 95% without any significant differences (P > 0.01) from the percentages of the germinated conidia in the control treatment. In the NSI trial, however, the percentages of the germinated conidia of B. cinerea were lower than 60% with significant differences (P < 0.01) from the percentages of the germinated conidia in the control treatment (Fig. 2). The average length of germ tubes of B. cinerea was 67, 150 and 168 lm, respectively, in the SI trial, whereas 0, 16 and 18 lm, respectively, in the NSI trial. All these values were significantly lower (P < 0.01) than the corre-

Fig. 4. Effect of the live yeast cells of Sporidiobolus pararoseus strain YCXT3 on fruit rot incidence and fruit rot severity index of strawberry naturally infected by Botrytis cinerea, Mucor sp., Rhizopus sp. and Penicillium sp. (20 °C, 7 days). Results in each histogram are expressed as means ± standard errors. Mean values for different treatments labeled with different letters indicate signiﬁcant difference (P < 0.01) according to Fisher’s protected least signiﬁcant difference test.

sponding values in the control treatment. In the treatment of S. pararoseus + AC, the suppressive effects of the VOCs of S. pararoseus on conidial germination and length of germ tubes of B. cinerea were partially or completely nullified by the presence of AC. The average percentages of the germinated conidia of B. cinerea at 4, 10 and 20 °C reached 99–100% in the SI trial and 96–99% in the NSI trial (Fig. 2). These values were not significantly different (P > 0.01) from those in the control treatment. The average length of germ tubes reached 106–386 lm in the SI trial and 68–290 lm in the NSI trial (Fig. 2). These values were significantly (P < 0.01) higher than those in the treatments of S. pararoseus, although they were still significantly (P < 0.01) lower than those in the control treatment. 3.4. Suppression of strawberry gray mold disease by the live yeast cells of S. pararoseus Results of the three experiments in this bioassay showed that the live yeast cells of S. pararoseus were effective in suppression of strawberry gray mold disease (Fig. 3). In the first experiment, the fruit rot incidence reached 100% and the fruit rot severity index reached 7.0 in the treatment of B. cinerea alone after incubation at 20 °C for 7 days. However, in the treatments of B. cinerea + S. pararoseus at 1 105 and 1 106 yeast cells ml 1, the fruit rot incidence was significantly (P < 0.01) reduced to 39% and 48%, respectively (Fig. 3). The fruit rot severity index was significantly (P < 0.01) reduced to 1.1 in both treatments (Fig. 3). In the second experiment, after incubation at 20 °C for 7 days, the fruit rot incidence reached 96% and the fruit rot severity index

R. Huang et al. / Biological Control 62 (2012) 53–63

incidence was significantly (P < 0.01) reduced to 50% and the fruit rot severity index was significantly (P < 0.01) reduced to 1.1 and 1.9, respectively. Occurrence of Rhizopus sp., Mucor sp. and Penicillium sp. was rarely observed on the strawberry fruits in these two S. pararoseus treatments (Fig. 4). In the third experiment, the fruit rot incidence reached 82– 100% and the fruit rot severity index reached 6.1–7.3 in the treatment of water + B. cinerea at the time intervals of 0–7 days (Fig. 5). However, in the treatment of S. pararoseus + B. cinerea at the time intervals of 0–7 days, the fruit rot incidence was significantly (P < 0.01) decreased to 55–77% and the fruit rot severity index was significantly (P < 0.01) reduced to 2.6–3.8 (Fig. 5). 3.5. Efficacy of the VOCs from S. pararoseus in control of strawberry gray mold disease

Fig. 5. Effect of time intervals between inoculations with the live yeast cells of Sporidiobolus pararoseus strain YCXT3 (1 107 yeast cells ml 1) and conidia of Botrytis cinerea (5 104 conidia per fruit) on strawberry gray mold disease (20 °C, 7 days). Results in each histogram are expressed as means ± standard errors. ‘‘⁄⁄’’ indicates signiﬁcant difference (P < 0.01) between the treatments of B. cinerea alone and S. pararoseus + B. cinerea at each time interval according to Student’s t-test.

The VOCs from the YEPDA cultures of S. pararoseus were effective in suppression of strawberry gray mold disease under the controlled air-tight conditions (Fig. 6). After incubation at 20 °C for 7 days, all the strawberry fruits inoculated with B. cinerea alone showed soft rot and gray moldy symptoms with the fruit rot severity index reaching 7.9 (Fig. 6). In the treatment of B. cinerea + S. pararoseus, however, the fruit rot incidence and/or the fruit rot severity index were significantly (P < 0.01) decreased and the degree of the decrease was positively correlated to the amount of the yeast biomass of S. pararoseus used for fumigation. With the increase of the yeast biomass of S. pararoseus from 1 1010 yeast cells per desiccator to 5 1010 yeast cells per desiccator, the fruit rot incidence decreased from 100% to 29% and the fruit rot severity index decreased from 3.8 to 0.3 (Fig. 6). The suppressive effect of the VOCs of S. pararoseus on both the fruit rot incidence and the fruit rot severity index was completely nullified by the presence of AC in the treatment of B. cinerea + S. pararoseus + AC (Fig. 6). The fruit rot incidence reached 100% and the fruit rot severity index reached 7.4 in this treatment (Fig. 6). 3.6. Identification of the VOCs from S. pararoseus A total of 39 volatile organic compounds (VOCs) from the 1day-old YEPDA cultures (20 °C) of S. pararoseus were identified (Table 1). These compounds fell into eight chemical classes, including 21 alkanes, 10 organic acids, 2 alcohols, 2 ketones, 1 alkene, 1 anhydride, 1 amine and 1 furan. The most abundant VOC produced by S. pararoseus was N-phenyl-2-naphthalenamine with the relative peak area (RA) accounting for 6.96%. Twenty-four VOCs, including 2-ethyl-1-hexanol, 2-hexyl-1-decanol, 2,6,10-trimethyldodecane, pentadecane; tetradecane; and 1-chloro-octadecane, were moderately abundant with the RA values ranging from 1.07% to 3.71%. The remaining 14 VOCs were the least abundant with the RA values lower than 1.0%. 3.7. Antifungal activity of selected authentic VOCs

Fig. 6. Effect of the volatile organic compounds from Sporidiobolus pararoseus strain YCXT3 on suppression of strawberry gray mold disease (20 °C, 7 days). Sp = S. pararoseus; AC = activated carbon. Results in each histogram are expressed as means ± standard errors. Mean values for different treatments with the same letters in each histogram are not signiﬁcantly different (P > 0.01) according to Fisher’s protected least signiﬁcant difference test.

reached 5.1 in the control treatment (Fig. 4). Other fungi, including Rhizopus sp., Mucor sp. and Penicillium sp., frequently occurred on the strawberry fruits in this treatment. However, in the treatments of S. pararoseus at 1 105 and 1 106 yeast cells ml 1, the fruit rot

Eight VOCs appearing in the cultures of S. pararoseus were selected and tested for antifungal activity against B. cinerea. Results showed that 2-ethyl-1-hexanol was effective in suppression of both conidial germination and mycelial growth of B. cinerea with the IC50 values being 1.5 and 5.4 ll l 1, respectively (Table 2). The remaining seven VOCs were not effective in suppression of B. cinerea with the IC50 values higher than 700 ll l 1 (Table 2). 4. Discussion S. pararoseus has been documented as a plant epiphytic yeast in previous studies (Nakase, 2000; Allen et al., 2004; Li et al., 2010;

R. Huang et al. / Biological Control 62 (2012) 53–63 Table 1 Volatile organic compounds released from 1-day-old cultures of Sporidiobolus pararoseus strain YCXT3 on YEPDA.

a b c d

RT (min)a

Possible compoundb

RA (%)c

MW (Da)d

6.75 17.71 24.21 26.56 27.83 28.03 28.33 28.84 28.94 28.95 29.09 29.11 29.64 29.65 29.78 30.31 30.58 31.64 31.89 32.20 32.38 32.51 33.48 34.49 34.87 34.96 35.13 35.52 35.86 36.66 37.38 37.77 38.56 38.85 40.67 40.86 44.36 52.23

Heptane, 3-methylene1-Hexanol, 2-ethylDecane, 3,8-dimethylCyclohexane, 2-butyl-1,1,3-trimethyl1-Decene Undecane, 2,6-dimethylUndecane, 4,8-dimethylTetradecane, 6,9-dimethyl1-Iodo-2-methylundecane Tridecane, 7-methyl1-Decanol, 2-hexyl1.alpha.,2.beta.,3.alpha.,4.beta.-Tetramethylcyclopentane Oxalic acid, cyclohexylmethyl nonyl ester Cyclohexanone, 5-(1-hydroxy-2-propenyl)-2,2-dimethyl-, (R⁄,S⁄)-(.+-.)1,7-Dimethyl-4-(1-methylethyl)cyclodecane Oxalic acid, cyclobutyl hexadecyl ester Oxalic acid, 6-ethyloct-3-yl heptyl ester Sulfurous acid, butyl heptadecyl ester Oxalic acid, cyclobutyl pentadecyl ester Cyclododecane, ethylDodecane, 2,5-dimethylTridecane, 5-methylDodecane, 2,6,10-trimethylTetradecane Sulfurous acid, 2-propyl tetradecyl ester Octadecane, 1-chloroTritetracontane 3-Methyl-2-(2-oxopropyl)furan Tetrapentacontane, 1,54-dibromoTetradecane, 5-methylOxalic acid, cyclobutyl heptadecyl ester 2-Dodecen-1-yl( )succinic anhydride Pentadecane Methoxyacetic acid, 2-octyl ester 2,11-Dioxabicyclo[4.4.1]undeca-3,5-dien-10-one,1,3,7,7-tetramethylOxalic acid, allyl octadecyl ester Tetracontane, 3,5,24-trimethylHexadecenoic acid, Z-11-

1.15 1.36 1.68 0.87 0.90 3.71 2.60 2.73 0.81 2.07 1.69 0.93 1.07 0.84 0.53 1.54 3.36 1.39 1.20 1.46 2.86 2.35 2.68 1.79 2.38 1.61 3.55 0.74 0.66 0.80 0.58 0.48 1.47 1.67 0.84 0.55 0.34 1.72

112 130 170 182 140 184 184 226 296 198 242 126 312 182 210 368 328 376 354 196 198 198 212 198 321 288 605 138 917 212 382 266 212 202 222 382 605 254

RT = retention time detected in the GC–MS analysis. See ‘‘Section 2’’ for details about the parameters of the detection column and the operating conditions in GC–MS. The VOCs of Sporidiobolus pararoseus with the relative peak areas of less than 0.1% are not included in this table. RA = relative peak area; the value for a VOC represented the percentage of the area of the peak for that VOC in the total area of peaks for all the detected VOCs. MW = molecular weight.

Table 2 Values of the 50% inhibition concentration (IC50) of nine synthetic volatile organic compounds for suppression of the conidial germination and the mycelial growth of Botrytis cinerea. Compound

IC50 value (mean ± standard error) (ll l For conidial germination

2-Ethyl-1-hexanol 1-Chloro-octadecane 2-Dodecen-1-yl( )succinic anhydride 1-Decene Tetradecane 2,6,10-Trimethyl-dodecane Pentadecane 2-Hexyl-1-decanol

1.5 ± 0.6 >1000.0 >1000.0 >700.0 >1000.0 >1000.0 >1000.0 >1000.0

)

For mycelial growthb 5.4 ± 0.1 >700.0 >800.0 >1000.0 >1000.0 >1000.0 >1000.0 >1000.0

a Conidial germination of B. cinerea was determined on water agar after incubation at 20 °C for 12 h. For each investigated VOC, the average percentages of the germinated conidia of B. cinerea in different dose treatments were individually compared with the average percentage of the germinated conidia of B. cinerea in the control treatment for estimating inhibitory efficacy. The efficacy values were analyzed with the corresponding VOC doses for inferring the IC50 value. b Mycelial growth of B. cinerea was determined on potato dextrose agar after incubation at 20 °C for 72 h. For each investigated VOC, the average colony diameters of B. cinerea in different dose treatments were compared with the average colony diameter of B. cinerea in the control treatment for estimating the inhibitory efficacy values and the IC50 value.

R. Huang et al. / Biological Control 62 (2012) 53–63

Janisiewicz et al., 2010; Machado and Bettiol, 2010; Raspor et al., 2010). We isolated S. pararoseus strain YCXT3 from a healthy strawberry leaf, suggesting that S. pararoseus might be an indigenous yeast member on strawberry leaves. Huang (2011) showed that the strain YCXT3 of S. pararoseus could rapidly colonize both the wounded areas and the non-wounded areas on strawberry fruits. In the current study, we found that the live yeast cells of strain YCXT3 of S. pararoseus can effectively suppress infection of post-harvest strawberry fruits by B. cinerea. All these findings indicate that S. pararoseus is a potential agent for control of B. cinerea on strawberry fruits. Further evaluations of the biocontrol efficacy of S. pararoseus against B. cinerea on strawberry under pre- and post-harvest conditions are necessary. Previous studies showed that S. pararoseus can effectively suppress A. alternata, B. theobromae, G. candidum, P. digitatum, P. italicum and Monilinia fructicola on fruits of kinnow and nectarine (Sharma et al., 2008; Janisiewicz et al., 2010). In the present study, we found that on strawberry fruits naturally infected with B. cinerea, S. pararoseus could not only suppress B. cinerea, but also suppress other fungi, including Mucor sp., Penicillium sp. and Rhizopus sp. The results of these studies suggest that S. pararoseus may have potential to suppress a wide range of decay fungi on post-harvest fruits. Sharma et al. (2008) indicated that S. pararoseus outcompeted A. alternata, B. theobromae, G. candidum, P. digitatum and P. italicum on kinnow fruits through competition for limited nutrients and space. Raspor et al. (2010) reported that S. pararoseus generated slight inhibitory effect on the mycelial growth of B. cinerea on Nutrient Yeast Extract Dextrose Agar (NYDA). In this study, we observed that S. pararoseus strain YCXT3 did not inhibit the mycelial growth of B. cinerea on PDA. These findings suggest that S. parariseus might be incapable of accumulating highly-toxic substances in NYDA and PDA inhibitory to B. cinerea, thus supporting the previous proposal made by Sharma et al. (2008) that S. pararoseus might be a competitor for limited nutrients and space. Previous studies indicated that production of 1,3-b-exoglucanase by Pichia species (Jijakli and Lepoivre, 1998; Masih and Paul, 2002) and induction of plant defense response by Candida spp., Cryptococcus laurentii and Metschnikowia fructicola (Droby et al., 2002; El-Ghaouth et al., 2003; Tian et al., 2007; Hershkovitz et al., 2011) are involved in biological control of B. cinerea by these yeast species. Whether or not these two mechanisms occur in biological control of B. cinerea by S. pararoseus strain YCXT3 on strawberry fruits remains unknown and needs additional clarifications. Previous studies on the antimicrobial volatile organic compounds (VOCs) from the filamentous fungus M. albus have set a good example for development of fungal biofumigant to control of post-harvest fruit diseases under air-tight conditions (Mercier and Smilanick, 2005; Schnabel and Mercier, 2006; Mlikota Gabler et al., 2006). In this study, we observed that the VOCs produced by strain YCXT3 of S. pararoseus at 4, 10 and 20 °C can effectively inhibit both the conidial germination and the mycelial growth of B. cinerea on agar media. We also found that the VOCs from the cultures of S. pararoseus can effectively suppress strawberry gray mold disease. These findings suggest that production of the antifungal VOCs by S. pararoseus is an effective mechanism in suppression of B. cinerea on post-harvest strawberry fruits. Therefore, S. pararoseus has potential to be developed as a biofumigant to control gray mold disease of strawberry stored in fruits-containing cartons. Huang (2011) showed strain YCXT3 of S. pararoseus is not fastidious in nutrient requirements and proliferated rapidly both on agar media and on the surface of strawberry fruits. These biological and physiological characteristics make S. pararoseus strain YCXT3 suitable to be developed as a biofumigant. Further studies to evaluate the biofumigation efficacy of the VOCs from S.

pararoseus in suppression of strawberry gray mold disease under the commercial storage conditions are warranted. In this study, we found that S. pararoseus can produce 2-ethyl-1hexanol. Production of this VOC compound has been reported in bacteria such as Pseudomonas spp. (Fernando et al., 2005) and Bacillus (Chen et al., 2008), and in fungi such as Cladosporium (Schuchardt and Kruse, 2009). Moreover, higher plants such as wheat (Triticum aestivum L.) and lettuce (Lactuca sativa L.) were detected capable of releasing 2-ethyl-1-hexanol (Batten et al., 1995; Lonchamp et al., 2009). These results suggest that 2-ethyl-1-hexanol can be synthesized by diverse taxonomic groups of organisms. We found that 2-ethyl-1-hexanol is effective in suppression of both conidial germination and mycelial growth of B. cinerea. Therefore, 2-ethyl-1-hexanol might be one of the active components in the VOC profile of S. pararoseus responsible for suppression of B. cinerea. Vaughn et al. (1993) reported that 1-hexanol, a relative of 2ethyl-1-hexanol, released from strawberry fruits during ripening can effectively inhibit mycelial growth of several decay fungi including B. cinerea. Production of 2-ethyl-1-hexanol and 1-hexanol by some plants and the antifungal activity of these two compounds against B. cinerea suggest that the two compounds may play a role in plant defense against infection by B. cinerea. Whether or not the two VOC compounds can induce plant defense response to fungal infection remains unknown and needs further clarification. Norbäck et al. (2000) reported that occurrence of human asthma symptoms is related to the emission of 2-ethyl-1-hexanol from dampness-related alkaline degradation of plasticiser di (ethylhexyl)-phthalate in polyvinyl chloride floor material. This result suggests that 2-ethyl-1-hexanol might be toxic to human beings causing asthma disease. Production of 2-ethyl-1-hexanol by S. pararoseus strain YCXT3 implies that use of this yeast strain in biological control may have risk. Further studies are warranted to evaluate the potential risk of this particular strain of S. pararoseus as biocontrol agent in biological control gray mold disease of strawberry and other economic crops. Acknowledgments This research was funded by the Natural Science Foundation of China (Grant No. 31070122). We thank Dr. Gang Fan at College of Food Science and Technology, Huazhong Agricultural University, Wuhan, China, for help in the analysis of volatile organic compounds produced by S. pararoseus. References Allen, T.W., Quayyum, H.A., Burpee, L.L., Buck, J.W., 2004. Effect of foliar disease on the epiphytic yeast communities of creeping bentgrass and tall fescue. Canadian Journal of Microbiology 50, 853–860. Batten, J.H., Stutte, G.W., Wheeler, R.M., 1995. Effect of crop development on biogenic emissions from plant populations grown in closed plant growth chambers. Phytochemistry 39, 1351–1357. Blachinsky, D., Antonov, J., Bercovitz, A., Elad, B., Feldman, K., Husid, A., Lazare, M., Marcov, N., Shamai, I., Keren-Zur, M., Droby, S., 2007. Commercial applications of ‘‘Shemer’’ for the control of pre- and post-harvest diseases. IOBCWPRS Bulletin 30, 75–78. Blacharski, R.W., Bartz, J.A., Xiao, C.L., Legard, D.E., 2001. Control of post-harvest Botrytis fruit rot with pre-harvest fungicide applications in annual strawberry. Plant Disease 85, 597–602. Boff, P., Köhl, J., Jansen, M., Horsten, P.J.F.M., Lombaers-van der Plas, C., Gerlagh, M., 2002. Biological control of gray mold with Ulocladium atrum in annual strawberry crops. Plant Disease 86, 220–224. Chen, H., Xiao, X., Wang, J., Wu, L.J., Zheng, Z.M., Yu, Z.L., 2008. Antagonistic effects of volatiles generated by Bacillus subtilis on spore germination and hyphal growth of the plant pathogen, Botrytis cinerea. Biotechnology Letters 30, 919–923. Choquer, M., Fournier, E., Kunz, C., Levis, C., Pradier, J.M., Simon, A., Viaud, M., 2007. Botrytis cinerea virulence factors: new insights into a necrotrophic and polyphageous pathogen. FEMS Microbiology Letters 277, 1–10. Diánez, F., Santos, M., Blanco, R., Tello, J.C., 2002. Fungicide resistance in Botrytis cinerea isolates from strawberry crops in Huelva (Southwestern Spain). Phytoparasitica 30, 529–534.

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