Identification Of The Pathogen Of Vegetable Pythium Damping-off And Screening And Application Of Novel High-efficiency Biocontrol Agents

Cucurbit seedling damping-off causes decay of germinating seeds and young seedlings,and it has been considered as one of the most serious problems for commercial cucurbit seedling industry.Many pathogens,including Pythium spp.,Fusarium spp.,and Rhizoctonia solani,have been reported to be responsible for damping-off.Currently,the pathogens for cucurbit damping-off in Hubei Province of China remains unclear.Chemical control is the main method to control damping-off in melons.However,overuse of fungicides can result in development of fungicide resistance in the pathogen populations,and also pollutes the environment,causing pesticide residues.Thus,there are increasing concerns about sustainable damping-off management strategies,including biological control.Therefore,this study was done to identify the pathogens for cucurbit damping-off in Hubei Province and to obtain the biocontrol agents for management of this disease.The results achieved were summarized below:(1)The pathogens for cucurbit damping-off in Hubei Province.A total of 413 oomycetous and fungal strains were isolated from cucurbit(watermelon,cucumber,wax gourd,loofah,pumpkin,melon)damping-off samples collected from various parts in Hubei Province,including 221 isolates of Pythium spp.,accounting for 53.5%;89 isolates of Fusarium spp.,accounting for 21.6%;65 isolates of Mucor spp.,accounting for 15.7%;20 isolates of Rhizoctonia solani,accounting for 4.8%;as well as 8 isolates of Sclerotinia sclerotiorum and Botrytis spp.,accounting for 4.3%.Pythium spp.were mainly isolated from watermelon(45%)and cucumber(30%).Pythium spp.isolated from soilless matrix and soil matrix accounted for 60%and 47%,respectively.Based on the morphological features(substrate mycelia,aerial mycelia,mycelial growth rates),221 isolates of Pythium spp.were divided into five categories(I-V).Representative isolates(3 to 5 isolates for each category)were selected for investigation of the morphological features for the specific structures(sporangia,antheridia,oogonia,oospores).The DNA sequences of ITS,Cox1 and Cox2 in the selected isolates were phylogentically analyzed to infer their taxonomic status.The results showed that the isolates for the categories Ⅰ,Ⅱ,Ⅲ,Ⅲ and Ⅴ belonged to P.aphanidermatum,P.irregular,P.spinosum,P.ultimum,and Pythium sp.,and these isolates accounted for 52%,9%,3%,30%,and 6%of the total isolates,respectively.By single and combined analysis of the ITS and Cox1 sequences,the isolates of the category V were found to be closely related to P.terrestris.Therefore,they were tentatively identified as P.terrestris.So far,there was no report about P.terrestris as well as P.irregular on cucurbit in China.(2)A strain(JKTJ-3)of Streptomyces murinus was obtained,it can produce many secondary metabolites and plant growth-promoting substances.The width of the inhibition zone of S.murinus JKTJ-3 against 13 pathogenic oomycetes or fungi(such as P.aphanidermatum)reached up to 23.2 mm.The inhibited hyphae of P.aphanidermatum showed spiral curling and swelling,and finally collapsed.The genome of Streptomyces JKTJ-3 was determined.It has the full length of 8.4 Mb and the G+C content of 71.81%,harboring 7,390 protein-encoding genes and 34 gene clusters for the secondary metabolites such as actinomycin D and pentamycin.Nine chitinase genes(Family No.18)and 10 glucanase genes(GH5,GH6,GH55)were identified in the genome of JKTJ-3.Production of actinomycin D by Streptomyces JKTJ-3 was confirmed by HPLC-MS analysis.Meanwhile,production of β-1,3-glucanase and chitinase were also detected in the filtrates of JKTJ-3 with the yields of 6.39 and 4.07 U/mL,respectively.Streptomyces JKTJ-3 showed the ability to dissolve phosphorus,to produce indoleacetic acid(IAA)and siderophores and to degrade lignin.After culturing in inorganic phosphorus liquid medium and organic phosphorus liquid medium for 4 days,the amounts of soluble inorganic phosphorus and organic phosphorus in the cultural filtrates were 57.7 and 35.6 mg/L,respectively.The amounts of IAA in the sterile filtrates of PDB and PDB amended with tryptophan(PDB-Trp)were 12.8 and 68.7 mg/L,respectively.These results indicate that Streptomyces JKTJ-3 can produce active substances(antagonistic substances,substances promoting plant growth)and fungal cell wall-degrading enzymes(β-1,3-glucanase and chitinase).(3)Streptomyces JKTJ-3 showed a high efficacy in suppression of cucurbit seedling damping-off and promotion of melon seedling growth.The results of the pot experiments showed that soaking watermelon seeds or treating soilless matrix with the cultures or the cultural filtrates of strain JKTJ-3 significantly reduced the incidence of P.aphanidermatum damping-off in watermelon.In the seeds-soaking test,the control effect of JKTJ-3 cultures and the cultural filtrates against watermelon damping-off were 39.2%and 50.7%,respectively;In the soilless matrix mixing test,the control effect of strain JKTJ-3 cultural filtrates(10～300 mL cultural filtrates per liter of soilless matrix)was 10.0%～51.4%,and the control effect of JKTJ-3 wheat grain cultures(10～160 g wheat grain cultures per liter of soilless matrix)was 53.2%～92.4%.In the inoculation interval test,the protective effect of JKTJ-3 sterile filtrates and wheat grain cultures treated the soilless matrix at 0 d,1 d,2 d and 3 d inoculated with P.aphanidermatum were 63.3%～100%.In vitro leaf test showed that the control efficacy values of strain JKTJ-3 cultures were 86.7%,88.2%and 91.8%,against pumpkin seedling blight,pumpkin damping off,and cucumber gummy stem blight,respectively.The plug seedling experiment showed that the seedling growth index of watermelon and cucumber seedlings increased by 18.1%and 12.5%,respectively,and the root activity was significantly improved by strain JKTJ-3 cultures(0.4%,v/v).Therefore,strain JKTJ-3 can significantly promote the growth of watermelon and cucumber in plug seedlings,and improve the quality of cucurbit seedlings.The above results provided the theoretical basis for the prevention and control of cucurbit damping-off disease,and the acquisition of S.murinus JKTJ-3 provides a technical support for cucurbit damping-off control.