| Streptomyces are among the most widely distributed group of microorganisms with extensive practical use and enormous economic value in nature. About 50% antibiotics are the secondary metabolites of Streptomyces in ten thousands activity substances that have been found in microbiology. Streptomyces have been evaluated as biocontrol agents against various plant pathogens since they produce an array of secondary metabolites such as enzyme inhibitors, herbicides and large number of antibiotics. In July 2007, a small contaminant colony appeared in a PDA plate of Magnaporthe grisea in a plant pathology laboratory at Huazhong Agricultural University, and it showed strong inhibition of the fungus. The colony was transferred onto a fresh plate, grown for identification using morphological and molecular methods, and named Streptomyces globisporus JK-1. And then, biocontrol potential and mode of action of S. globisporus JK-1 were also studied in the present paper. The main results are listed as follows:1. Based on morphological, cultural, biochemical and physiological characteristics, strain JK-1 was identified as Streptomyces globisporus JK-1.2. The effect of medium components (i.e. carbon and nitrogen sources) and other culture conditions on production of antifungal substances of S. globisporus JK-1 was investigated. The results showed that among the seven tested medium, the best medium for growth and antifungal substances production were LB and potato sucrose broth (PSB), respectively; for the growth of S. globisporus JK-1 on medium PSB, the temperature range was 10 to 35℃, with optimum of 25℃; the dry weight and the antifungal substances production reached the highest level after incubation at 25℃for 6 days; the favorable pH value for growth and antifungal substance production was 5-11, with the optimum of pH 6-8. Among the 12 tested carbon and nitrogen sources in basal mineral salts medium, glycerol and tyrosine were the most favorable for its growth; maltose and ammonium sulfate were the most favorable for its antifungal substances production.3. To determine the property of the antifungal substances, the culture filtrates and the crude antifungal substances were treated in various ways. Antifungal activity was stable after incubation at 120℃for 30 min or treated with Proteinase K at 37℃for 60 min. The antifungal activity of the antifungal substances was stable at acid conditions while inactive in strong alkalinity (pH≥12). No antifungal activity was tested in the supernate of the culture filtrates, and the most antifungal activity was tested in the precipitate after adding the ammonium sulfate. The results of ultrafiltration of crude antifungal substances showed that the molecular weight was above 10 KD.4. Culture filtrates of JK-1 inhibited mycelial growth of M. oryzae, and histological investigations showed that conidial germination and appressorial formation of M. oryzae were suppressed on detached rice leaves. When applied at 15μl per 5-cm-long detached leaf, washed cells of JK-1 at 108 CFU/ml could suppress disease incidence of rice blast caused by M. oryzae co-inoculated at 5×105 spores/ml, but disease incidence was not reduced when washed cells of JK-1 were used at 106 or 107 CFU/ml. Applying the culture filtrate on rice seedlings in the greenhouse at 2 hours post inoculation (HPI) with M. oryzae showed 88.3% disease reduction of rice blast, while culture filtrate application before pathogen inoculation showed even higher rates of disease reduction. Suppression of the infection process of M. oryzae on detached rice leaves was observed by light and scanning electron microscopy, showing inhibited conidial germination and reduced appressorial formation on rice leaves sprayed with culture filtrate before 3 HPI. Application of JK-1 as undiluted culture broth or filtrates to plant tissues did not cause any noticeable negative effects. These results indicate that the antifungal substance(s) in the culture filtrate of S. globisporus JK-1 can exhibit an inhibitory effect on M. oryzae and a suppressive effect on rice blast disease.5. Antifungal activity against Botrytis cinerea of volatile substances from S. globisporus JK-1 grown on autoclaved wheat seed was studied in vitro and in vivo. The volatiles suppressed mycelial growth of various plant pathogens in vitro, especially that of B. cinerea and Sclerotinia sclerotiorum. Conidial germination and sporulation of B. cinerea were suppressed in the presence of volatiles. Disease incidence and severity on fungal-inoculated tomato fruit (Lycopersicon esculentum) were inhibited when fumigated with 120 g/L wheat seed culture of S. globisporus JK-1. Suppression of the infection process of B. cinerea on tomato fruit was observed via scanning microscopy, showing inhibition of conidial germination and of infection hyphae formation on tomato fruit, as well as abnormal morphology of infection hyphae and conidia exposed to the volatiles. The viability of the conidia obtained from the volatile-treated and non-treated disease lesions was tested with the vital stains fluorescein diacetate (FDA) and propidium iodide (PI). Conidia fumigated with 30 g/L,60 g/L or 120 g/L wheat seed culture of S. globisporus JK-1 at 20℃for 6 days showed 59.4%,76.7%, or 85.3% reduction in viability, respectively. Transmission electron microscopy of fumigated and untreated B. cinerea showed excessive vesiculation or thickened cell walls in exposed conidia and increased vesiculation or strong retraction of plasma membrane in exposed hyphae. The results of present study provide a better understanding of the mode of action of volatiles from JK-1 on B. cinerea. The inhibition growth of B. cinerea both in vitro and in vivo studies showed that volatiles from S. globisporus JK-1 have potential for control of postharvest grey mold of tomato fruits through fumigant action.6. Antifungal activity against Penicillium italicum of volatile substances from S. globisporus JK-1 grown on autoclaved wheat seed was studied in vitro and in planta. Fungal spore germination and mycelial growth of P. italicum cultures as well as sporulation and disease incidence on fungal-inoculated fruit were suppressed in the presence of the volatiles. For naturally infected fruit, disease incidence was reduced from 25% to 7.5%. Suppression of the infection process of P. italicum on Shatang Mandarin fruits (Citrus microcarpa) was observed via scanning electronic microscopy, showing inhibited spore germination on the Shatang Mandarin, and abnormal morphology for conidiophores and hyphae exposed to the volatiles. Based on gas chromatography/mass spectrophotometric analyses,41 volatile organic compounds were identified from the volatiles of S. globisporus JK-1, and the most abundant compound was trans-1,10-dimethyl-trans-9-decalol (geosmin), an earthy smelling substance. Among these, technical grade formulations of eight were chosen for further study:phenylethyl alcohol, caryophyllene, dimethyl disulfide, dimethyl trisulfide, acetophenone, d-Iimonene, isoledene, and aromadendrene. D-limonene, isoledene and aromadendrene showed no observable antifungal activity in vitro and in planta at tested concentrations. Both phenylethyl alcohol and caryophyllene showed weak inhibitory activity in vitro but no significant efficacy against P. italicum on Shatang Mandarin. Dimethyl disulfide or dimethyl trisulfide showed antifungal activity in vitro and efficacious control on Shatang Mandarin at a concentration of 100μL per liter of airspace in treatment containers. Acetophenone showed antifungal activity in vitro at a concentration of 100μL L-1 and efficacious control on Shatang Mandarin at the highest concentration of 1000μL L-1. Volatiles from S. globisporus JK-1 have potential for control of blue mold of citrus species through fumigant action.
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