During an investigation of mycoparasitic fungi on sclerotia of Sclerotinia sclerotiorum in China, Gliocladium species was consistently encountered and isolated from natural soils collected over the country. Colonizati...During an investigation of mycoparasitic fungi on sclerotia of Sclerotinia sclerotiorum in China, Gliocladium species was consistently encountered and isolated from natural soils collected over the country. Colonization frequencies on sclerotia of S. sclerotiorum by the fungi ranged from 40% to 100% when the sclerotia were introduced into soils and coincubated at 22-24 ℃ for 4 weeks. Identification showed that G. roseum, G. virens and G. catenulatum were the dominant species among the 300 isolates gained. Molecular taxonomy of some Gliocladium isolates analysed by rDNA ITS sequence used ITS1 and ITS4 as primers was demonstrated to be identical with morphological classification. Reinoculation tests by placing surface-sterilized sclerotia onto colony of Gliocladium isolates for 7 days and then surface-sterilized again resulted in 100% sclerotia colonized. Dipping sclerotia with spores suspension of isolates for 5 min and incubated the sclerotia in Petri dishes at 25 ℃ for 24 h resulted in infection of the sclerotia. Microscopic observations indicated that Gliocladium isolates grew along the host hyphae, coiled around, formed appressorium-like structures, penetrated and degraded hyphae of S. sclerotiorum when dual-cultured in slides. Paraffin-section showed that sclerotial tissue collapsed when infected by Gliocladium isolates. Host range test demonstrated that the isolates suppressed hyphal growth of several plant pathogenic fungi, including S. sclerotiorum, Botrytis cinerea, Fusarium oxysporum, Alternaria tenuis, and Rhizoctonia solani. Activities of fungal cell wall degrading enzymes in cultural filtrates and parasitized sclerotia were detected. Results indicated that chitinase and glucanase were important factors involved in the mycoparasitism. A 51 kDa chitinase was isolated and purified from G. catenulatum HL-1-1 culture filtrate. A part of amino acid sequences of the enzyme has been analysed. It was found to have a high homogeneity to chitinase A from Stenotrophomonas maltophilia, while no homogeneity to chitinases from Trichoderma and other fungi. It was considered to be a new chitinase quite different from those produced by other mycoparasitic fungi. Optimum temperature and pH for the enzyme activity were 60 ℃ and 6.0. respectively. Ca 2+. promoted the activity, while Fe 3+., Cu 2+. and Ag+ inhibited it. Km of the chitinase was 2.832. The chitanase significantly inhibited the growth of hyphae, germination of conidia and sclerotia of some plant pathogenic fungi. Gene encoding this chitinase is being cloned from the total RNA of HL-1-1 isolates by RT-PCR. Biological characteristics of some Gliocladium isolates were studied. Results showed that temperature ranged from 22 ℃ to 30 ℃ was optimum for isolates hyphae growth and spore germination, while they could endure high temperature. Spores could not germinate below 5 ℃ or over 40 ℃ and lethal temperature of spores was 52 ℃. Optimum pH was 5.0 for hyphae growth and 6.0 for sporulation. Dark condition enhanced hyphae growth and light promoted good for sporulation. Glucose, sucrose and mannose were optimum C source for hyphal growth while glucose, xylose, soluble starch and chitin optimum for sporulation. Soybean cake powder, yeast extract and casein hydrolysate were good for hyphal growth while soybean cake powder, beef extract, urea and NaNO 3 were good N sources for sporulation. Extract of sclerotia promoted spore germination of the isolate,but D-glucose inhibited. Greenhouse tests confirmed that Gliocladium isolates GJ-1-1, SS-1-1, HL-1-1, SH-1-1, GW-1-1 promoted the seedling growth of soybean, cucumber, wheat and rice. Our primary studies showed that some Gliocladium isolates are of great potential in biocontrol of plant fungal diseases, such as soybean stem rot caused by S. sclerotiorum and cucumber root disease caused by R. solani.展开更多
文摘During an investigation of mycoparasitic fungi on sclerotia of Sclerotinia sclerotiorum in China, Gliocladium species was consistently encountered and isolated from natural soils collected over the country. Colonization frequencies on sclerotia of S. sclerotiorum by the fungi ranged from 40% to 100% when the sclerotia were introduced into soils and coincubated at 22-24 ℃ for 4 weeks. Identification showed that G. roseum, G. virens and G. catenulatum were the dominant species among the 300 isolates gained. Molecular taxonomy of some Gliocladium isolates analysed by rDNA ITS sequence used ITS1 and ITS4 as primers was demonstrated to be identical with morphological classification. Reinoculation tests by placing surface-sterilized sclerotia onto colony of Gliocladium isolates for 7 days and then surface-sterilized again resulted in 100% sclerotia colonized. Dipping sclerotia with spores suspension of isolates for 5 min and incubated the sclerotia in Petri dishes at 25 ℃ for 24 h resulted in infection of the sclerotia. Microscopic observations indicated that Gliocladium isolates grew along the host hyphae, coiled around, formed appressorium-like structures, penetrated and degraded hyphae of S. sclerotiorum when dual-cultured in slides. Paraffin-section showed that sclerotial tissue collapsed when infected by Gliocladium isolates. Host range test demonstrated that the isolates suppressed hyphal growth of several plant pathogenic fungi, including S. sclerotiorum, Botrytis cinerea, Fusarium oxysporum, Alternaria tenuis, and Rhizoctonia solani. Activities of fungal cell wall degrading enzymes in cultural filtrates and parasitized sclerotia were detected. Results indicated that chitinase and glucanase were important factors involved in the mycoparasitism. A 51 kDa chitinase was isolated and purified from G. catenulatum HL-1-1 culture filtrate. A part of amino acid sequences of the enzyme has been analysed. It was found to have a high homogeneity to chitinase A from Stenotrophomonas maltophilia, while no homogeneity to chitinases from Trichoderma and other fungi. It was considered to be a new chitinase quite different from those produced by other mycoparasitic fungi. Optimum temperature and pH for the enzyme activity were 60 ℃ and 6.0. respectively. Ca 2+. promoted the activity, while Fe 3+., Cu 2+. and Ag+ inhibited it. Km of the chitinase was 2.832. The chitanase significantly inhibited the growth of hyphae, germination of conidia and sclerotia of some plant pathogenic fungi. Gene encoding this chitinase is being cloned from the total RNA of HL-1-1 isolates by RT-PCR. Biological characteristics of some Gliocladium isolates were studied. Results showed that temperature ranged from 22 ℃ to 30 ℃ was optimum for isolates hyphae growth and spore germination, while they could endure high temperature. Spores could not germinate below 5 ℃ or over 40 ℃ and lethal temperature of spores was 52 ℃. Optimum pH was 5.0 for hyphae growth and 6.0 for sporulation. Dark condition enhanced hyphae growth and light promoted good for sporulation. Glucose, sucrose and mannose were optimum C source for hyphal growth while glucose, xylose, soluble starch and chitin optimum for sporulation. Soybean cake powder, yeast extract and casein hydrolysate were good for hyphal growth while soybean cake powder, beef extract, urea and NaNO 3 were good N sources for sporulation. Extract of sclerotia promoted spore germination of the isolate,but D-glucose inhibited. Greenhouse tests confirmed that Gliocladium isolates GJ-1-1, SS-1-1, HL-1-1, SH-1-1, GW-1-1 promoted the seedling growth of soybean, cucumber, wheat and rice. Our primary studies showed that some Gliocladium isolates are of great potential in biocontrol of plant fungal diseases, such as soybean stem rot caused by S. sclerotiorum and cucumber root disease caused by R. solani.
