| Plant endophytes are a group of microorganisms that are widely present in plant tissues,but do not cause disease symptoms.They are interdependent and adaptive with plants,and form a harmonious and mutually beneficial relationship with plants in the process of long-term evolution.Many plant endophytes have multiple biological functions,such as helping plants to absorb nutrients,thereby promoting plant growth,inducing plant resistance,thereby enhancing plant immunity,and preventing biotic(e.g.plant pathogens and pests)and abiotic stresses(e.g.drought).Exploitation of plant endophytes in agricultural use becomes a hot research area in recent years.Cotton is an important economic crop.Cotton Verticillium wilt(CVW)caused by Verticillium dahliae is considered as‘cancer’of cotton,as it causes substantial yield losses for cotton industry.CVW is a typical vascular disease.Certain endophytes of cotton inhabiting in the vascular tissue of cotton may produce suppressive effect on infection by V.dahliae.Thus,a study was done to isolate endophytic fungi and bacteria from healthy plants of cotton,selected fungi and bacteria were evaluated for control of CVW under greenhouse and field conditions,and characterized for interaction with V.dahliae and cotton.The results achieved were described as follows:1.Ninety-three isolates of endophytic fungi were isolated from 180 cotton samples,including 70 strains from cotton stems and 23 strains from cotton roots.The fungi belonged to 20 species in 16 genera.The dominant fungal genera include Acrocalymma,Alternaria,Diaporthe,Fusarium,Phomopsis and Plectosphaerella.2.Based on the results about spore production ability as well as the control efficacy against CVW in greenhouse,five isolates with a significant and consistent suppressive effect were screened out.They were three isolates of Fusarium proliferatum(10R-2,10R-7,10R-9)and two isolates of Fusarium solani(6R-1,17R-8).Under the greenhouse and field conditions,the average control efficacy against CVW reached up to 50%.3.The mode of action of F.proliferatum 10R-7 against CVW was preliminarily explored.Isolate 10R-7 was detected capable to produce volatiles with antifungal activity against V.dahliae.The culture filtrates from the cultures of 10R-7 in Czapek-Dox broth medium(CDM)were detected to able to inhibit mycelial growth and conidial germination of V.dahliae.When the concentration of the 10R-7 cultural filtrates supplemented in PDA(potato dextrose agar)medium reached to 4%(v/v),growth of V.dahliae was completely inhibited.The antifungal substances in the culture filtrates of10R-7 were identified.The results showed that the fusaric acid was the main component.The antifungal activity of pure fusaric acid was verified,and it could effectively inhibit growth of V.dahliae.When the concentration of fusaric acid reached to 20μg/m L,growth of V.dahliae was completely inhibited.However,fusaric acid at the concentrations ranging from 2.5 to 80.0μg/m L failed to suppress infection by V.dahliae,suggesting that fusaric acid produced by F.proliferatum 10R-7 is probably not the main factor for its suppression of CVW.On the other hand,isolate 10R-7 was transformed with the green fluorescent protein gene(GFP)and a derivative isolate 10R-7GFP was obtained.Microscopic observation showed that isolate 10R-7GFP could colonize the vascular tissue in cotton roots.This result suggests that isolate 10R-7 may prevent infection by V.dahliae by occupying the ecological site and the subsequent competition.At the same time,we designed a dose effect test in a greenhouse experiment to determine influence of the doses of isolate 10R-7 on its biocontrol effect.The results showed that the biocontrol effect of isolate 10R-7 was positively correlated with the conidial concentration of isolate 10R-7.This result further illustrates the importance of competition in the interaction between isolate 10R-7 and V.dahliae.4.Fifty-nine isolates of endophytic bacteria were isolated from healthy cotton plants,of which 44 isolates were isolated from cotton stems and 15 isolates were isolated from cotton roots.After multiple rounds of greenhouse screening,five bacterial isolate with a consistent and significant control effect were obtained.They were Agrobacterium sp.HB3S-13,Pseudomonas putida HB3S-20,Serratia sp.3R-4,Staphylococcus sp.4R-1and Bacillus cereus 2S-16.Isolate HB3S-20 had the control efficacy as high as 55.94%against CVW and showed a significant growth promotion effect on cotton plants.Three methods(root irrigation at seedling stage,root irrigation in seedling bed and seed coating)were applied respectively for isolate HB3S-20 under the field conditions.The results showed that root irrigation at seedling stage was the proper application method regarding control of CVW.5.The mode of action of P.putida HB3S-20 was determined.The results showed that isolate HB3S-20 could produce siderophores and enhance the activity of defense-related enzymes(superoxide dismutase and peroxidase)in cotton plants in addition to the antifungal activity.The strain HB3S-20 was transformed with the GFP gene to obtain the derivative isolate HB3S-20GFP.Colonization of HB3S-20GFP in cotton roots was observed by laser confocal microscope when it was inoculated on the roots.The results showed that HB3S-20GFP may suppress the V.dahliae infection by colonization in the vascular tissue of cotton roots.6.A rifampicin-resistant derivative HB3S-20Rif~r was obtained by using rifampicin to induce mutation of HB3S-20.The selective isolation and colony-counting technique was used to determine the bacterial population of the mutant in the roots and stems(1 cm and 3 cm above the stem base).The results showed that HB3S-20Rif~r could be detected in roots and stems of cotton,indicating that HB3S-20Rif~r could colonize the cotton roots,and spread to the stems.In a word,the results from the pesent study above enriched understanding of the resources of endophytic fungi and bacteria in cotton,and laid a foundation for use of endophytes to control CVW. |
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