| Cotton plants materials sampled from12different cotton varieties (or lines) which were ranked according to their resistance to Verticillium wilt with the developing stage of full bloom. Using surface-sterilization method to isolate the endophytic fungi which are inhabit in cotton tissue segments of root,stem and leaf.In total642endophytic fungi were isolated.Preliminary classification is through colony morphology, such as morphological characteristics and microscopic observation,then combined with molecular biology method as the feature of ITS sequences to do a thorough appraisal. Analyzed the diversity, specificity and population distribution of endophytic fungi in different varieties (or lines) among different tissues.To make good use of these endophytic fungi resources, representative strains were selected to evaluate their inhibition activity against the target of cotton verticillium wilt virulent strains Verticillium dahliae(Vd080) in vitro. The antifungal activity of isolated endophytic fungi against V.dahliae growth was evaluated by three different methods. These were dual culture, volatile and nonvolatile metabolite bioassays,screening the strains which could inhibit V.dahliae strongly. In the greenhouse, using a method named vermiculate and sand in bottomless paper pot dipping in quantitative spores, through seed treatments and matrix inoculated with endophytic fungi to analysis the prevention role of endophytic fungi antagonism with verticillium wilt.Finally measured its control effect of the relevant bio-control strains against verticillium wilt in disease nursery and field.Conclusions are as follows:(1) The isolates can be divided into16groups through the colony morphology.Based on the ITS phylogenetic analysis,642endophytic fungi were classified into27different generas. Alternaria exhibited the highest colonization frequency (7.9%), followed by Acremonium (6.6%) and Gibberella (5.8%).(2) No significant difference in the abundance of isolated endophytes was found among resistant cotton varieties. Unlike tolerant varieties, resistant and susceptible ones had similar endophytic fungal population compositions. Fungal endophytes from the genus Alternaria were most frequently isolated, followed by Gibberella and Penicillium.(3) The endophytic fungi harbored in the cotton roots, stems and leaves exhibited a diverse population structure.Each tissues contained both their unique and conmmon species. Some endophytic fungi have strong organizational structure selectivity and specificity. More endophytic fungi appeared in leaf (391) compared with root (140) and stem (111) sections.The endophytic fungi harbored in the cotton roots, stems are similar, Gibberella exhibited highest colonization frequency (5.2%) and isolation recovery (22.5%), followed by Penicillium (CF=4.8%, IR=20.7%) and Acremonium (CF=4.0%, IR=17.1%).The group Alternaria was the largest population in the fungal endophytes isolated from cotton leaves (CF=20.7%, IR=31.7%). The second most dominant species was Acremonium with13.3%CF and20.5%IR. The concentration of dominance or Simpson’s dominance of endophytic fungi was highest in the leaf tissues (0.1797).The evenness of the stem tissue endophytic community (0.702) was comparatively more uniform than the other two tissues.(4) The results of the dual culture antagonistic bioassay showed seven endophytic fungi exhibited strong antifungal activity against Vdahliae with high growth inhibition rates (≥75%).CEF-714, which was identified as Leptosphaeria sp., exhibited maximal inhibition ability with the mean growth inhibition rate being88.1%. V. dahliae growth was also strongly inhibited by CEF-642, Talaromyces flavus (GI=83.8%). In addition, the isolates CEF-026(Gibberella intermedia), CEF-193(Acremonium sp.), CEF-325(Fusarium solani), CEF-818(Penicillium simplicissimum) and CEF-109(Ascomycete sp.) belonged to this strongly antagonistic group.CEF-193volatile substances exhibited the most inhibition activity, having a growth inhibition rate of94.3%. CEF-642also strongly reduced the growth of the targeted pathogen with an81.6%inhibition rate. CEF-818, CEF-714and CEF-325also produced volatile metabolites which greatly inhibited Vdahliae growth.The results of the nonvolatile antibiotic assay revealed that CEF-818, CEF-325, CEF-714and CEF-642secreted the highest amounts of Vdahliae resistant nonvolatile substances. The Vdahliae mycelia did not spread normally on the PDA plates containing the antibiotic substances mentioned above. The inhibition rate reached to100%.(5) In the greenhouse, CEF-818, CEF-714and CEF-193treated cotton seed can significantly decrease verticillium wilt disease index, the control effect could above50%.(6) Matrix inoculation experiment in disease nursery,disease index CEF-818is very low (9.6), the control effect against Verticillium Wilt could up to72%.CEF-193and CEF-714performance good control effect(64%,54%),too(7) In the field, the control effect of CEF-818on Verticillium wilt can reach above30%, CEF-714could reduce the disease index of Verticillium Wilt,too.In conclusion,CEF-818and CEF-714are selected as target endophytic fungi. And they have very good application value in biological control of cotton verticillium wilt research. |
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