| Metarhizium anisopliae is a widely distributed soil-inhabiting fungus that can infect approximately 200 species of insect and some arthropods,but it is not infectious or toxic to mammals.Thus,M.anisopliae have been explored as an important biocontrol agent to control insect pests of plants.In this study,the tomato-plant pathogen interaction system was used to evaluate the ability of M.anisopliae control plant disease,including root knot nematode disease caused by Meloidogyne incognita and gray mold caused by Botrytis cinerea.In addition,the disease controlling mechanism of M.anisopliae was analyzed.The major findings were concise as below:1. M.anisopliae can parasite root-knot nematode.M.incognita is an important soil-borne nematode infecting many vegetables and field crops worldwide.The environment safely options to control M.incognita remain limited.The biocontrol potentiality of M.anisopliae strain IBCCM321.93 was evaluated against M.incognita.Both scanning electron microscopy(SEM)and transmission electron microscopy(TEM)observation revealed that M.anisopliae can parasite nematode eggs.The GFP-labeled-M.anisopliae was also evaluated to monitoring eggs parasitism,showed that mycelia of M.anisopliae can parasite nematode eggs.In addition,the parasitism of nematode adult females by M.anisopliae was observed by light microscope.In an in vitro bioassay,the 1×10~8conidia/ml of M.anisopliae inhibited 75.5%of nematode eggs hatching at 8 days post inoculation(8dpi)compared to the water control.2. Metabolites of M.anisopliae possessed the potentiality against M.incognita.Culture filtrates of M.anisopliae reduced egg hatching and increased second?stage juvenile(J2)mortality.The 95%concentration of filtrates inhibited 97.6%of egg hatch at11 dpi and caused 100%J2 mortality at 12 h after treatment.Moreover,after 12 h incubation culture filtrate showed 97.68%juvenile mortality at 95%concentration when it was boiled at 100oC for 15 min and this value was statistically similar as recorded in non-boiled culture filtrate.3. M.anisopliae can colonize on tomato roots and promoted plant growth.The tomato seedlings were inoculated with GFP-labelled-M.anisopliae strain,the laser confocal microscopic observation exhibited that M.anisopliae can colonize on tomato roots.In addition,molecular detection by PCR and isolation of fungal strain from inoculated tomato roots also confirmed root colonization of M.anisopliae.Besides,M.anisopliae colonized plants root endophytically and promoted plant growth.Tomatoes treated with M.anisopliae was1.95 times and 1.26 times higher than water control after 4weeks of inoculation in pot and field soil,respectively.4. M.anisopliae has potential as a new agent in the biocontrol of M.incognita.In the pot experiment,the results revealed that the application of 1×10~8M.anisopliae conidia per g dry soil in the tomato pot reduced approximately 48%-54%root galls,49%-58.3%egg masses per g root and 56%-61%number of eggs in root tissue,as well as 54%-62%number of J2 in the soil.In addition,M.anisopliae suppressed nematode infection when it was challenged against naturally M.incognita infested soil in greenhouse field.M.anisopliae reduced 16.6%root galls and 22.78%number of eggs per root in greenhouse when plants were infected by nematode before treated with fungal conidia.In addition,the culture filter of M.anisopliae significantly reduced nematode infection of tomato roots,including 60%biocontrol potentiality in preventive and 35%in curative application.5. RNA-seq was used to understand the molecular mechanism of M.anisopliae on root-knot nematode eggs infection.Genes of M.anisopliae were significantly up-regulated or down-regulated at 12 hpi and 24 hpi were screened.Results demonstrated that genes related to serine-type peptidase,serine-type endopeptidase and serine-type hydrolase were enrichment and most of which had homologhs in the PHI database.It suggests that serine peptidases of M.anisopliae play important role at early stage eggs infection.In addition,genes related to lectins,cell wall protein,extracellular membrane protein CFEM were up-regulated at both of 12 hpi and 24 hpi hypothesized to be involved in recognition,adhesion and cuticle penetration process of nematode eggs infection.The expression level of signal transduction genes particularly G protein-coupled receptor,c AMP-mediated signaling protein and protein kinase at 24 hpi suggesting that MAPK singal pathway might be play important roles at this stage.Chitinase,transporter,CYPs(Cytochrome P450)and biosynthesis of secondary metabolites genes were up-regulated at 12 hpi and 24 hpi.Therefore,these genes could be putitavely responsible for eggs infection.However,eight appressoria forming specific genes were up-regulated at 24 hpi and only 3 were upregulated at 12 hpi.Moreover,38 novel genes with fold change higher than 4 at 24 hpi and 15genes at 12 hpi were also upregulated with unknown function might be important during infection process.Furthermore,the enrichment and expression profiles of peptidase,signal transdruction,transporter and biosynthesis of secondary metabolites related genes demonstrate that nematode eggs infection and insect infection patterns of M.anisopliae is more or less similar.6. B.cinerea significantly controlled by M.anisopliae.Gray mold disease caused by Botrytis cinerea is a devastating disease that leads to serious financial loss.In this study,the entomopathogenic fungus M.anisopliae that acts against the gray mold pathogen B.cinerea was evaluated.M.anisopliae produced a significant inhibition zone in front of the B.cinerea colony in the dual culture test.In addition,volatile organic compounds generated by M.anisopliae were shown to have an inhibitory effect on B.cinerea mycelia growth and reduced 41%of gray mold severity of postharvest tomatoes.The 10%concentration of the culture filtrate of M.anisopliae inhibited 88.62%of colony radial growth as well as 63.85%of sclerotia germination and all conidia germination of B.cinerea.Furthermore,the culture filtrate of M.anisopliae retained its inhibitory effect against the radial growth of B.cinerea even after heating for 15 min at 100°C.Feasible mechanisms of M.anisopliae involved in the control of B.cinerea were explored,and it was demonstrated that the plasma membrane of B.cinerea conidia was damaged by the product of metabolism of M.anisopliae.In addition,after treating with culture filtrate of M.anisopliae,the B.cinerea phenotype was shown to be abnormal,and cell organelles of B.cinerea mycelia were damaged significantly.A significant control efficacy of M.anisopliae against tomato gray mold was detected on both the detached leaf assay(84.24%)as well as the whole plant(72.38%).In addition,a 78%reduction in tomato fruit mold was detected at a 10%treated concentration of M.anisopliae.These findings suggest that M.anisopliae possesses potential as a biocontrol agent against tomato gray mold in the greenhouse and during the postharvest stage.In summary,M anisopliae has a good control effect on tomato root knot nematode and gray mold.M anisopliae is known as an important insect biocontrol resource.Therefore,it has a good potential in combined prevention and control of nematode diseases,fungal diseases and insects.In addition,the disease controlling mechanism of M.anisopliae was analyzed and the mechanism of some genes associated with egg parasitism was screened out.This study broadens the application range of M anisopliae,and has certain leading significance for improving the biocontrol effect of this strain. |
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