| Apple Valsa canker is the most devastating apple disease caused by ascomycete fungus Valsa mali.The disease kills trees,destroys orchards and causes severe economic losses.Currently,the control of this disease still mainly relies on chemical agents,but misuse of chemicals pesticide could cause plenty of problems such as pesticide residues in fruits,ecological environment pollution in orchards and drug resistance of pathogen.Therefore,it is of great theoretical significance and potential application value to excavate and develop biocontrol microbial resources for green control of apple Valsa canker.Beneficial fungal Endophyte is a hot spot in the field of biological control because of the antibacterial activity and the ability to enhance plant disease resistance.Based on this situation,an endophytic fungi with obvious antibacterial activity on V.mali were isolated from the bark of Chaenomeles cathayensis and named MG2 in this study.Then,its taxonomic status was clarified by morphological observation and molecular biological identification.Further,the antibacterial activity of its mycelium and fermentation broth was determined by plate confrontation test.Its control effect on apple Valsa canker was confirmed by branch inoculation test in vitro and field test,and its biocontrol mechanism was explored by RNA-sequencing.The specific results are as follows:1.A potential endophytic biocontrol fungus MG2 with obvious antibacterial activity on V.mali was isolated from the bark of C.cathayensis.The colony was pale yellow at the initial stage,and olive green or taupe at the later stage.The plate confrontation test showed that the inhibition rate of MG2 to V.mali colony growth was 98.65% after pre-inoculation of MG2 strain for 2 days;the inhibition rate was 95.95% after pre-inoculation of MG2 strain for 1 day;and the inhibition rate was 82.21% when MG2 and V.mali were inoculated at the same time.By observing the shape and color of cleistothecium and ascospores,and the attachment of appendage,combined with polygene phylogenetic analysis,the endophytic fungus MG2 was finally identified as Chaetomium globosum.2.According to the antibacterial spectrum analysis,MG2 could only inhibit the mycelial growth of Valsa pathogenic fungi,such as V.pyri,V.malicola,V.salicina and V.leucostoma,and the inhibition rates were 80.22%,76.57%,76.91% and 79.97% respectively.Scanning electron microscope observation showed that the mycelium of V.mali became thicker and grew disorderly and irregularly after being inhibited by MG2.It was found that MG2 didn’t produce any volatile substances with significant antibacterial activity against the mycelial growth of V mali through the plate butt test.In vitro branch and leaf inoculation test showed that MG2 had no pathogenic effect on apple trees.MG2 has certain protective and therapeutic effects on apple Valsa canker,with relative control effects of72.46% and 21.33% respectively.The analysis of colonization ability showed that the colonization rate was 47.80% after inoculating MG2 to apple branches for 3 days,was92.78% after 6 days of inoculation,and was 98.48% after 9 days of inoculation.The black cleistothecium of MG2 were produced in the wound after 9 days of inoculation,which indicated that MG2 could be planted in apple branches and existed stably.3.Furthermore,the antibacterial and disease prevention effects of MG2’s fermentation broth were evaluated.It was found that the higher the content of fermentation broth in the culture medium,the stronger the antibacterial effect.When the content of fermentation broth was 10.0%,5.0%,2.0%,1.0% and 0.5%,the inhibition rates of V.mali mycelium growth were 99.68%,96.53%,91.76%,84.83% and 78.91% respectively.Scanning electron microscope observation showed that the fermentation broth could cause the hypha of V.mali to thicken,grow disorderly and become more branched.The fermentation broth could obviously inhibit the germination of V.mali conidia.When the content of fermentation broth in the culture medium was 10.0%,5%,2.0%,1.0% and 0.5%,the conidia will be deformed,irregularly expanded and the protoplasm will be extravasated.In vitro branch text showed that the fermentation broth have a good protective and therapeutic effect on apple Valsa canker,with relative control effects of 65.07% and 32.70% respectively.The antibacterial stability test of fermentation broth showed that it had good thermal stability and ultraviolet stability,and it could still maintain good antibacterial activity under the conditions of 100℃treatment for 30 min and ultraviolet irradiation for 3 h.The field test results showed that the fermentation broth have a good protective and therapeutic effect on apple Valsa canker,and the relative control effects were 55.14% and 39.29% respectively.4.By analyzing and comparing the genes expressed level of V.mali inhibited by C.globosum MG2 with control,1674 differentially expressed genes were identified,among which 788 genes in V.mali were significantly up-regulated and 886 genes were significantly down-regulated.GO and KEGG function enrichment analysis showed that the carbohydrate metabolism ability of V.mali was seriously affected,and a large number of hydrolytic glycosidic bonds,hydrolytic enzymes of carbonyl compounds,cellulose binding and carbohydrate binding genes were down-expressed under biotic stress,thus affecting carbohydrate metabolism.Down-regulated expression of genes involved in pyruvate metabolism and branched-chain amino acids(valine,leucine and isoleucine)metabolism leads to the influence of TCA cycle,insufficient energy supply,and inhibition of the process of synthesizing non-essential substances for life and storing secondary metabolites.Down-regulated expression of peripheral genes,extracellular domain genes and plasma membrane genes involved in V.mali cells affected the formation of new cell wall,normal function of cell membrane system and mitosis of cells.It was speculated that Chaetomium globosum MG2 might produce chitinase,cellulose and glucanase,damaging V.mali cell wall.On the other hand,V.mali made several responses to the inhibitory stress of MG2,such as supplying energy through the metabolism of galactose,fatty acids and sphingolipids,enhancing the metabolism of glycine,serine and threonine,maintaining the normal TCA cycle,and then maintaining the balance of protein,sugar and fat metabolism.By enhancing transmembrane transport,respiration and the synthesis of enzymes containing metal ions,the cell membrane potential,osmotic pressure,coordinated transport of substances can be maintained,and the damaged biofilm system can be repaired. |
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