Production Of Antifungal Substances Research Articles

Schauerella fraxinea gen. nov., sp. nov., a bacterial species that colonises ash trees tolerant to dieback caused by Hymenoscyphus fraxineus

The tolerance of ash trees against the pathogen Hymenoscyphus fraxineus seems to be associated with the occurrence of specific microbial taxa on leaves. A group of bacterial isolates, primarily identified on tolerant trees, was investigated with regard to their taxonomic classification and their potential to suppress the ash dieback pathogen. Examination of OGRI values revealed a separate species position. A phylogenomic analysis, based on orthologous and marker genes, indicated a separate genus position along with the species Achromobacter aestuarii. Furthermore, analysis of the ratio of average nucleotide identities and genome alignment fractions demonstrated genomic dissimilarities typically observed for inter-genera comparisons within this family. As a result of these investigations, the strains are considered to represent a separate species within a new genus, for which the name Schauerella fraxinea gen. nov., sp. nov. is proposed, with the type strain B3P038T (=LMG 33092 T = DSM 115926 T). Additionally, a reclassification of the species Achromobacter aestuarii as Schauerella aestuarii comb. nov. is proposed.In a co-cultivation assay, the strains were able to inhibit the growth of a H. fraxineus strain. Accordingly, a functional analysis of the genome of S. fraxinea B3P038T revealed genes mediating the production of antifungal substances. This potential, combined with the prevalent presence in the phyllosphere of tolerant ash trees, makes this group interesting for an inoculation experiment with the aim of controlling the pathogen in an integrative approach. For future field trials, a strain-specific qPCR system was developed to establish an efficient method for monitoring the inoculation success.

Optimized submerged batch fermentation for metabolic switching in Streptomyces yanglinensis 3-10 providing platform for reveromycin A and B biosynthesis, engineering, and production.

The cultivation system requires that the approach providing biomass for all types of metabolic analysis is of excellent quality and reliability. This study was conducted to enhance the efficiency and yield of antifungal substance (AFS) production in Streptomyces yanglinensis 3-10 by optimizing operation conditions of aeration, agitation, carbon source, and incubation time in a fermenter. Dissolved oxygen (DO) and pH were found to play significant roles in AFS production. The optimum pH for the production of AFS in S. yanglinensis 3-10 was found to be 6.5. As the AFS synthesis is generally thought to be an aerobic process, DO plays a significant role. The synthesis of bioactive compounds can vary depending on how DO affects growth rate. This study validates that the high growth rate and antifungal activity required a minimum DO concentration of approximately 20% saturation. The DO supply in a fermenter can be raised once agitation and aeration have been adjusted. Consequently, DO can stimulate the development of bacteria and enzyme production. A large shearing effect could result from the extreme agitation, harming the cell and deactivating its products. The highest inhibition zone diameter (IZD) was obtained with 3% starch, making starch a more efficient carbon source than glucose. Temperature is another important factor affecting AFS production. The needed fermentation time would increase and AFS production would be reduced by the too-low operating temperature. Furthermore, large-scale fermenters are challenging to manage at temperatures that are far below from room temperature. According to this research, 28°C is the ideal temperature for the fermentation of S. yanglinensis 3-10. The current study deals with the optimization of submerged batch fermentation involving the modification of operation conditions to effectively enhance the efficiency and yield of AFS production in S. yanglinensis 3-10.

Self-digestive solution of Lysobacter enzymogenes LE16 as a biofungicide to control plant powdery mildew

ABSTRACT The self-digestive solution (SDS) of the biocontrol bacterium Lysobacter enzymogenes LE16 shows strong antagonistic activities against multiple soil-borne phytopathogens but the positive evidence of this bacterium against plant foliar disease is still scanty. Thus, laboratory, greenhouse, and field experiments were carried out to estimate the efficacies of SDS, S-SDS (stored at room temperature for 12 months), and H-SDS (heated at 100°C for 30 min) against plant powdery mildew. This bacterium produced hydrolases (phosphatase, protease, lysozyme, chitinase, and β-1,3-glucanase) that degrade pathogen cell components and siderophores that compete for iron with phytopathogens. The top five antimicrobial metabolites identified in SDS were pyroglutamic acid, deoxycytidine, pyrrole-2-carboxylic acid, 13-oxo-9,11-tridecadienoic acid, and 3’-amino-3’-deoxythimidine. Among them, pyroglutamic acid may play a vital role in powdery mildew control. As a result, SDS, S-SDS, and H-SDS strongly inhibited the conidial germination of Erysiphe cichoracearum and Sphaerotheca fuliginea. The application of SDS significantly increased the activity of antioxidant enzymes in crop leaves and effectively controlled tobacco and cucumber powdery mildew in the greenhouse and the field. Therefore, L. enzymogenes LE16 can effectively control powdery mildew. The underlying mechanisms may be attributed to the induction of plant systemic resistance and the production of antifungal substances.

Identification of the biocontrol strain LB-2 and determination of its antifungal effects on plant pathogenic fungi

Biocontrol agents are key players in plant disease control. In this study, a potential biocontrol strain LB-2 was identified as Chaetomium globosum based on morphological observations and rDNA-ITS sequence analysis. The antifungal effect of LB-2 was determined with a dual culture assay showing that the mycelial growth of Botrytis cinerea, Fusarium oxysporum f. sp. cucumerinum, and Exserohilum turcicum was inhibited by LB-2, their hyphae at the colony contact surface being swollen and shrunken. Poison plate assay indicated that cell-free culture broth of LB-2 and its butanol and ethyl acetate (EtOAc) crude extracts exerted strong antifungal effects on the mycelial growth of the three tested phytopathogenic fungi, with the highest inhibition rate (89.99%) on E. turcicum induced by the butanol crude extract. Disease indices of northern corn leaf blight (caused by E. turcicum) in potted maize seedlings could significantly be reduced (P < 0.05) by foliar spraying of the cell-free culture broth, butanol and EtOAc crude extracts of LB-2. The highest disease control efficacy (53.47%) was shown by butanol crude extract. However, no antifungal effect was produced by the intracellular fluid of LB-2 in both in vitro and in vivo experiments. These results demonstrated that LB-2 is a promising Chaetomium spp. biocontrol agent against phytopathogenic fungi. Its antifungal effects might be achieved through a combination of living competition and production of antifungal substance, which are excreted by the mycelial cells.

Studies on “Effect of Organic Nitrogen Source on Production of Antifungal Substance from Clostridium Species”

Objective: In this study, antifungal substances were isolated from different soil bacteria like Clostridium species in the presence of different nitrogen sources. The isolated antifungal substances were then tested against Aspergillus and Trichoderma species to analyse their potential activity. Methodology and Results: Antifungal substances were isolated from different soil bacterial species and it was observed that only Clostridium species was suitable to isolate active antifungal substances against Aspergillus and Trichoderma species in the presence of different nitrogen sources. Peptone and yeast extract were good nitrogen sources to isolate most active antifungal substances from Clostridium species. Agar well diffusion method was used to measure the zone of inhibitions and the maximum zone of inhibition was found up to 22 mm. Conclusion: Yeast extract and Peptone were found to be good nitrogen sources for the growth of Clostridium sp. YLC and antibiotic production.

Medium Optimization for Antifungal Active Substance Production from Streptomyces Lydicus Using Response Surface Methodology

Response surface methodology was used to optimize the medium for antifungal active substance production from Streptomyces lydicus E12 in flask cultivation. Initially, the component factors, which influence antifungal substance production, were studied by varying one factor at a time. Starch, soybean cake powder, K2HPO4·3H2O and MgSO4·7H2O were found to have a significant effect on the production of antifungal substances by the traditional design. Then, a Box–Behnken design was applied for further optimization. A quadratic model was found to fit antifungal active substance production. The analysis revealed that the optimum values of the tested variable were starch 84.96 g/L, soybean cake powder 4.13 g/L, glucose 5 g/L, MgSO4·7H2O 1.23 g/L, K2HPO4·3H2O 2.14 g/L and NaCl 0.5 g/L. The test result of 67.44% antifungal inhibition agreed with the prediction and increased by 14.28% in comparison with the basal medium.

Optimisation of medium and culture conditions for the production of antifungal substances to Colletotrichum musae by Trametes elegans SR06

ABSTRACTAn endophytic fungus SR06 was isolated from a leaf of Amomum villosum Lour., which had a high antagonistic effect on Colletotrichum musae with an inhibition ratio of 41.20%. The antifungal substances could be secreted into fermentation broth, which had a high inhibitory activity. Strain SR06 was identified as Trametes elegans according to internal transcribed spacer sequence analysis. Response surface methodology (RSM) was used to optimise the process parameters of antifungal substances production. Using the Plackett–Burman design, three variables (glucose, yeast extract and MgSO4·7H2O) exerted significant effects on antifungal substances production. Then RSM experiments were conducted to further optimise the three variables. The optimal medium components were 26.45 g/L glucose, 10 g/L peptone, 14.96 g/L yeast extract and 1.49 g/L MgSO4·7H2O, and the optimal initial pH was 6.0, with a culture temperature of 28°C and a shaking speed of 180 rpm. Under the optimised conditions, a significant improvement in the production of antifungal substances by T. elegans SR06 was accomplished, and the inhibition zone diameter was up to 29.2 mm after culturing for 7d. The average control efficacy of the fermentation supernatant of SR06 against C. musae was 51.29% on banana fruits, which was significantly higher than that of the fungicide carbendazim.

Optimization of the cultural medium and conditions for production of antifungal substances by Streptomyces platensis 3-10 and evaluation of its efficacy in suppression of clubroot disease (Plasmodiophora brassicae) of oilseed rape

This study was conducted to optimize the cultural medium and conditions for production of antifungal substances by Streptomyces platensis 3-10 and to evaluate its efficacy in suppressing Plasmodiophora brassicae (PB), the causal agent for clubroot of oilseed rape. The optimization was done using the one-factor-at-a-time (OFAT) method, followed by orthogonal designing. The optimized medium contained starch at 3% (w/v), peptone at 0.75%, yeast extract at 0.025%, soybean meal at 1% (w/v) and minor/trace elements (K+, Mg2+, Mn2+, Zn2+), initial pH 6.5. After incubation in this medium at 28°C/150rpm for 72h, isolate 3-10 showed high antifungal activity. The cultural filtrates (CF3-10) produced an average inhibition-zone diameter of 50mm against Aspergillus niger. The live spores and metabolites of 3-10 in CF3-10 or its crude extracts (CE3-10) suppressed PB infection of oilseed rape roots by 3–83%, compared to the treatment with PB alone. The efficacy was positively correlated (correlation co-efficiency: 0.9526–0.9873, P<0.05) to the applied dose of each active component. Germination of PB resting spores was inhibited by 75% and 80% by CE3-10 (1000μg/mL, w/v) and CF3-10 (5%, v/v), respectively. These findings suggest that S. platensis 3-10 is a promising biocontrol agent against PB.

Culture Filtrates of Trichoderma Isolate H921 Inhibit &amp;lt;i&amp;gt;Magnaporthe oryzae&amp;lt;/i&amp;gt; Spore Germination and Blast Lesion Formation in Rice

Spore germination and appressorium formation of Magnaporthe oryzae spores was completely suppressed by an ethyl acetate extract of the culture filtrate from the H921 isolate (H921-EAE-CF). Production of antifungal substance(s) in the H921-EAE-CF began to increase up to 3 days after isolate H921 incubation. Furthermore, heat treatment (105°C or 121°C) of H921-EAE-CF did not alter its inhibitory effect on M. oryzae spore germination compared to non-heat-treated H921-EAE-CF. Blast lesion formation inhibition by H921-EAE-CF was dose-dependent. Internal transcribed spacer (ITS) region sequence analysis indicated that this isolate shared similarities with species of the genera Trichoderma. This study suggests that H921-EAE-CF contains some antifungal substances that could be promising candidates for control of rice blast disease.

Degradation of oxalic acid by the mycoparasite Coniothyrium minitans plays an important role in interacting with Sclerotinia sclerotiorum

Coniothyrium minitans (Cm) is a mycoparasite of the phytopathogenic fungus Sclerotinia sclerotiorum (Ss). Ss produces a virulence factor oxalic acid (OA) which is toxic to plants and also to Cm, and Cm detoxifies OA by degradation. In this study, two oxalate decarboxylase genes, Cmoxdc1 and Cmoxdc2, were cloned from Cm strain Chy-1. OA and low pH induced expression of Cmoxdc1, but not Cmoxdc2. Cmoxdc1 was partially responsible for OA degradation, whereas Cmoxdc2 had no effect on OA degradation. Disruption of Cmoxdc1 in Cm reduced its ability to infect Ss in dual cultures where OA accumulated. Compared with Chy-1, the Cmoxdc1-disrupted mutants had reduced expression levels of two mycoparasitism-related genes chitinase (Cmch1) and β-1,3-glucanase (Cmg1), and had no detectable activity of extracellular proteases in the presence of OA. On the other hand, the cultural filtrates of the Cmoxdc1-disrupted mutants in OA-amended media showed enhanced antifungal activity, possibly because of increased production of antifungal substances under acidic pH condition resulted from reduced Cmoxdc1-mediated OA degradation. This study provides direct genetic evidence of OA degradation regulating mycoparasitism and antibiosis of Cm against Ss, and sheds light on the sophisticated strategies of Cm in interacting with metabolically active mycelia and dormant sclerotia of Ss.

English

The antagonist bacterium&nbsp;Brevibacillus laterosporus&nbsp;ZQ2 with broad-spectrum antifungal activity was isolated from an apple rhizosphere at Mount Tai in China. The fermentation medium for the production of antifungal substances from ZQ2 was statistically optimized. In one-factor-at-a-time experiments, five components, including carbon sources, nitrogen sources, and inorganic salt, were selected. Two factors were determined as critical based on the Plackett-Burman design. Steepest ascent experiments were then used to determine the design space containing the optimum point, followed by application of the response surface methodology. A quadratic model fitting the actual production was established using a central composite design. The obtained optimum medium for the antifungal substances production consisted of the following (in g/L): glucose, 15.6; soybean meal, 8.2; starch, 20; ammonium sulfate, 2; and monopotassium phosphate, 1. Furthermore, the inhibition rate of the substances against&nbsp;Rhizoctonia solani&nbsp;increased by more than 132% compared with the control.&nbsp;B. laterosporus&nbsp;ZQ2 strongly inhibited the growth of many apple phytopathogens&nbsp;in vitro. The inhibition rates against different fungi ranged from 55.26 to 79.02%. As well, the strain significantly reduced the disease damage caused by&nbsp;R. solani&nbsp;in the&nbsp;in vivo&nbsp;tests. Maximum disease protection (46.89%) was recorded when the bacterium applied 3 days&nbsp;before pathogen inoculation. &nbsp; Key words:&nbsp;Brevibacillus laterosporus, antifungal activity, fermentation optimization.

Impact of beef extract and six carbon source on antifungal metabolites production by bacterium associated with entomopathogenic nematode against Fusarium oxysporum

A specific symbiotic Bacillus species isolated from a rhabditid entomopathogenic nematode, Rhabditis (Oscheius) sp., was found to produce a number of bioactive compounds. The present study was conducted to determine the effect of six different carbon sources in combination with beef extract on the production of antifungal substances by Bacillus sp. The yield of crude antimicrobial substances and antimicrobial activity against the test microorganism also differed significantly when the carbon sources in the fermentation media were changed. The highest yield was recorded for fructose plus beef extract (956 mg/l). The antifungal activity was significantly high in beef extract plus maltose (21 ± 1.5 mm) followed by beef extract plus glucose and beef extract plus fructose. Antifungal activity was significantly reduced in beef extract plus lactose and sucrose. High pressure liquid chromatography analysis of the crude antimicrobial substances revealed different peaks with different retention times indicating that they produced different compounds. When a carbon source was not included in the fermentation media, the antifungal production was substantially reduced. Carbon source in the fermentation medium plays a vital role in the production of antimicrobial substances. Beef extract and maltose as nitrogen and carbon sources in the fermentation medium produced maximum antifungal activity. It is concluded that Beef extract and maltose as nitrogen and carbon sources produced maximum activity which can effectively control the Fusarium oxysporum which causes vascular fusarium wilt in tomato, tobacco, legumes, cucurbits, sweet potatoes, banana, etc.

Influence of carbon and nitrogen sources on antifungal metabolite production by bacterium associated with entomopathogenic nematode against Penicillium expansum

A specific symbiotic Bacillus sp. isolated from a rhabditid entomopathogenic nematode, Rhabditis (Oscheius) sp. was found to produce large number of bioactive compounds. The present study was conducted to determine the effect of carbon and nitrogen sources for the production of antimicrobial substances by Bacillus sp. The yield of the crude antimicrobial substances and antimicrobial activity against the test micro-organism also differed significantly when carbon and nitrogen sources in the fermentation media were changed. The antifungal activity was significantly high in yeast extract plus fructose (46.5 ± 2.12 mm) followed by yeast extract plus maltose, beef extract plus fructose and meat infusion plus glucose. High pressure liquid chromatography analysis of the crude antimicrobial substances revealed different peaks with different retention time indicating that they produced different compounds. When the carbon source was not included in the fermentation media, the antimicrobial production was substantially reduced. The results indicate that carbon source in the fermentation media plays a vital role in the production of antifungal substances. It is concluded that yeast extract and fructose as nitrogen and carbon sources produced maximum activity, which can effectively control the blue mould caused by Penicillium expansum in apples and pears.

식물병원성 진균을 억제하는 근권세균의 항진균능과 식물생장촉진능

Since many pesticides cause various health and environmental problems, alternative measures to replace them are needed, and the bacteria producing the antifungal substances can be one of them. In this study, several rhizobacteria were isolated and their antifungal activities against some important plant pathogenic fungi were examined. Pseudomonas otitidis TK1 and Paenibacillus peoriae RhAn32 inhibited the growth of Fusarium oxysporum f. sp. niveum and F. oxysporum f. sp. lycopersici by 49.8% and 45.6%, and 45.1% and 48.3%, respectively compared to those of the control. P. peoriae RhAn32 also decreased the growth of F. oxysporum f. sp. raphani by 37.5%. This growth inhibition might be due to the production of antifungal substances, such as siderophore, hydrogen cyanide and chitinase, which were produced by these rhizobacteria. P. otitidis TK1 also produced plant growth hormones indole acetic acid and indole butyric acid at 293.41 μg/mg protein and 418.53 μg/mg protein, respectively. When P. otitidis TK1 and B. cereus TK2 were inoculated together with F. oxysporum f. sp. lycopersici to the 4 weeks grown tomato seedlings and incubated additional 8 weeks, the stem lengths of tomato increased up to 45.7% and 55.3% and root lengths were raised to 64.9% and 60.8%, respectively than those of the control group. The wet weights increased by 118% and 182%, respectively compared to the control group.

Characterization and application of microbial antagonists for control of Fusarium head blight of wheat caused by Fusarium graminearum using single and mixture strain of antagonistic bacteria on resistance and susceptible cultivars

Fusarium head blight (FHB) caused by Fusarium graminearum leads to significant yield losses of wheat (Triticum aestivum L.) in many provinces of Iran, especially in the northern provinces in the margin of Caspian Sea. One strategy for the control of FHB is to use antagonistic bacteria that are plant growth promoting. In order to assess the potential of phyllospheric microorganisms in biological control of FHB, one hundred and ninety isolates of Pseudomonas, Erwinia and Bacillus spp. were collected from phyllosphere of healthy and infected wheats. Among them, eight isolates were selected and purified with the most antagonistic ability against the growth of pathogenic fungal species (F. graminearum) using the dual culture method. According to the results of biochemical and physiological tests, they were identified as three biovar of Pseudomonas fluorescens, one isolate of them was Erwinia herbicola and two species of Bacillus such as Bacillus subtilis and Bacillus cereus. Production of antifungal substances and volatile metabolites, siderophores and secretion of lytic enzymes such as protease and cellulase were evaluated as the inhibitory mechanisms in vitro. Furthermore, the effects of antagonistic bacteria were studied on severity and incidence of disease caused by F. graminearum in greenhouse conditions. Statistical analysis of data indicated that wheat spikes treatment with antagonistic bacteria not only reduced the severity and incidence of disease compared with the control but also showed positive influence on growth and yield of wheat cultivars. In general, multiple isolates such as B. subtilis 1 and P. fluorescens bv. 1 and 4 together were determined as the most effective strains in reducing incidence of disease. It can be concluded that multiple antagonistic isolates have better effects of control than single isolate. Key words: Biological control, Fusarium head blight, Fusarium graminearum,Pseudomonas fluorescens, Erwinia herbicola and Bacillus subtilis.

Production of proteinaceous antifungal substances from Lactobacillus brevis NCDC 02

Forty standard Lactobacillus cultures were checked for their antifungal properties against nine standard fungi. Yeasts were found to be more resistant than moulds. Lactobacillus brevis NCDC 02 was selected for further studies due to the maximum antifungal activity and proteinaceous nature of antifungal substance production. The optimum production of antifungal substances was at 37°C for 48 h and at the initial pH of 6–7 of the medium. The antifungal substances were heat‐ and pH‐stable. The antifungal substances were found to be hydrophobic and their molecular weight was found to be more than 1 kDa and less than 5 kDa.

Comparative efficacy of diffusible and volatile compounds of tea rhizospheric isolates and thier use in biocontrol

An attempt was made to study in vitro production of antifungal substances by various strains of Bacillus subtilis and Pseudomonas corrugata. These strains were isolated from the rhizosphere of established tea Camellia sinensis L. located in North West Indian Himalayan regions viz Himachal Himalayas and Garhwal Himalayas. Selected strains were checked for their biocontrol potential against two phytopathogenic fungi Fusarium udum and Alternaria solani and the antagonists were found to cause inhibition in radial growth of the fungi. This diffusible effect was attributed to the production of diffusible and volatile compounds which were found to be potent antifungal in nature. The antifungal substances in bacterial strains were found to be extracellular, proteinacious in nature. The colony forming units (cfu) of pathogenic fungi were reduced by the diffusible and volatile compounds produced by antagonists. The inhibition in cfu was more by volatile compounds than by diffusible compounds. The volatile compounds included aldehydes, alcohols, ketones, sulphides and HCN. International Journal of Biological & Chemical Sciences Vol. 1 (1) 2007: pp. 28-34

Suppression of Sclerotinia sclerotiorum by antifungal substances produced by the mycoparasite Coniothyrium minitans

A study was conducted to investigate production of antifungal substances (AFS) by Coniothyrium minitans (Cm), a mycoparasite of Sclerotinia sclerotiorum (Ss), in modified Czapek-Dox (MCD) broth and potato dextrose broth (PDB), and effects of AFS of Cm on mycelial growth and germination of sclerotia and ascospores of Ss and incidence of leaf blight of oilseed rape caused by Ss. Results showed that mycelial growth of Ss was reduced by 41.6 and 84.5% on 3 day-old cultures grown on potato dextrose agar (PDA) amended with 10% (v v−1) of cultural filtrates of Cm grown in MCD (MCDcm) after incubation for 6 and 15 days, respectively, and by 2.7 and 15.7% on PDA amended with 10% (v v−1) of cultural filtrates of Cm grown in PDB for 6 and 15 days, respectively. In addition to retardation of mycelial growth, morphological abnormality of Ss such as hyphal swellings and cytoplasm granulation were also observed in colonies grown on PDA amended with cultural filtrates of MCDcm. Sclerotia of Ss soaked in the filtrates of MCDcm for 24 h remained viable, but their ability to undergo myceliogenic germination on PDA was delayed, compared to sclerotia treated with MCD. Germination of ascospores of Ss was unaffected on PDA amended with 10% of the filtrates of MCDcm. However, germ tubes of Ss were shortened and deformed by the formation of hyphal swellings in the treatment of MCDcm. Treatment of leaves of oilseed rape with cultural filtrates of MCDcm reduced incidence of leaf blight caused by Ss, compared to the controls (water or MCD). This study suggests that AFS produced by Cm plays an important role in the suppression of mycelial growth and germ-tube development of ascospores of Ss and that there is potential for using AFS of Cm to control leaf blight of oilseed rape caused by ascospores of Ss.

Grapes and Vineyard Soils as Sources of Microorganisms for Biological Control ofBotrytis cinerea

In the search for biological agents capable of controlling the phytopathogenic fungus <i>Botrytis cinerea</i>, winegrape cuticles of several <i>Vitis vinifera</i> varieties and different soils from Uruguayan vineyards were screened for microorganisms that could be antagonistic to the fungus. Among 223 isolates of yeasts and bacteria, eight non-<i>Saccharomyces</i> yeast strains and four bacteria showed greater than 50% effectiveness in vitro against <i>Botrytis cinerea</i> compared with the growth of the control. A species of <i>Bacillus</i> (isolate UYBC38) and an isolate of the yeast <i>Hanseniaspora uvarum</i> (isolate UYNS13) showed high in vitro antagonistic capability against the pathogen; both organisms were very effective (100% and 90%, respectively) in controlling rot development on grape clusters. These two isolates were selected for further laboratory and vineyard studies. In order to prevent fermentation problems, both strains were tested against the wine yeast M522 and other natural non-<i>Saccharomyces</i> strains before field trials were conducted. The possible mechanisms of antagonism against <i>B. cinerea</i> were studied. Yeast strain UYNS13 showed only competition for nutrients. Although the <i>Bacillus</i> sp. strain consumed significantly more nitrogen from grape juice than <i>B. cinerea</i>, the relevant mechanism of antagonism of this bacterium was the production of anti-fungal substances. These compounds completely inhibited germination of <i>B. cinerea</i> conidia in vitro by disrupting the conidial cell wall, resulting in leakage of protoplasm. Applications of UYBC38 in the vineyard showed that this strain could survive on leaves and fruits for 45 to 70 days after spraying. The strategy of increasing the natural antagonistic population present in vineyards to prevent fungal diseases is discussed as low-input viticulture.

Bacillus subtilis에서 분비되는 Colletotrichum gloeosporioides 생장 저해물질 생산에 미치는 배양조건의 영향

고추 탄저병균 Colletotrichum gloeosporioides의 생장을 저해하는 물질을 생산하는 세균인 Bacillus subtilis의 각종 배양조건을 달리하여 최적 생산 조건을 조사하였다. 병원균 생장 저해물질 생산을 위한 배지의 초기 산도는 pH 7.0이 최적이였으며, 탄소원으로는 glucose를 첨가했을 경우 다른 탄소원에 비해 2배 이상 높은 활성을 보였다. 질소원으로는 유기 질소원일 경우 비교적 높은 병원균 생장저해활성을 보였으나, 무기 질소원인 ammonium sulfate에서 가장 높은 활성을 보였다. 여러 가지 배지를 사용하여 조사한 결과, LB(Luria Bertani) 배지의 배양액에서 가장 높은 활성이 나타났다. The effect of culture parameters on the production of Colletotrichum gloeosporioides growth inhibitory substance from Bacillus subtilis was investigated. The maximal growth inhibition zone was observed in the medium of pH 7.0. Among the tested carbon sources, glucose showed the largest growth inhibition zone above two fold than other carbon sources. Ammonium sulfate and organic nitrogen sources were effective on the production of growth inhibitory substance. Luria Bertani (LB) medium was the best on the production of antifungal substance from B. subtilis.