解蛋白芽孢杆菌Bacillus proteolyticus提高银杉抗落针病的生防机制研究

[Objective]Cathaya argyrophylla[Chun et Kuang.]is a key wild plant under first-degree protection in China.As a unique relict plant in China,C.argyrophylla is rare in number and has high protection and scientific research value.In June 2022,needle cast was observed in approximately 30%of C.argyrophylla(n=200)at Dashahe National Nature Reserve in Daozhen County,Guizhou,China.This disease severely restricts the growth of C.argyrophylla,interferes with the return of C.argyrophylla seedlings to the wild,and exacerbates its endangered situation.To identify the pathogen of Needle Cast of C.argyrophylla and to find targeted control methods for this disease.[Method]We collected C.argyrophylla needles afflicted with Needle Cast.We then employed the tissue isolation method to isolate and purify the pathogen,corroborated its pathogenicity through the application of Koch's postulates,and identified the pathogen through the utilisation of morphological and molecular biological techniques.Tissue isolation method was employed to isolate endophytic bacteria from healthy C.argyrophylla needles.An in vitro double culture was utilised to screen the biocontrol bacteria with an inhibitory effect on C.argyrophylla needles disease.The biocontrol effect was evaluated by spraying the biocontrol bacteria on the C.argyrophylla seedlings.A preliminary study was conducted to elucidate the mechanism of the biocontrol bacteria in improving the resistance of C.argyrophylla to needles disease.[Result]The results showed that the pathogen of Needle Cast of C.argyrophylla was Neofusicoccum parvum;A strain of the bacterium X6-1(Bacillus proteolyticus)was isolated from within the needles of C.argyrophylla,which could secrete protease and β-glucanase and had a better bacterial inhibitory activity against N.parvum in vitro,and could achieve 59.13%of the efficacy against Needle Cast of C.argyrophylla seedlings;The results of the effects of strain X6-1 on the physiological system and the composition of the phyllosphere microbiome community of C.argyrophylla showed that the inoculation of B.proteolyticus was able to alleviate the damage to cell membranes and the destruction of the needle tissue structure due to the invasion of pathogens,and to increase the photosynthesis,the content of the hormones JA,GA,ABA,and IAA,and the activity of antioxidant enzymes of C.argyrophylla,as compared with the treatment of the pathogenic bacteria alone.Furthermore,the application of Strain X6-1 resulted in alterations to the composition of the phyllosphere microbiome community.This was evidenced by an increase in the abundance of beneficial microorganisms and a reduction in the abundance of Neofusicoccum spp.By transcriptome analysis,inoculation with B.proteolyticus induced the expression of 8,890 differential genes(1,604 genes significantly up-regulated and 7,286 genes significantly down-regulated),which were mainly involved in metabolic pathways such as photosynthesis,ribosomes,Glycolysis/Gluconeogenesis、Glutathione metabolism and TCA cycle.[Conclusion]In conclusion,we revealed that the application of endophytic bacteria associated with C.argyrophylla seedlings can regulate the physiological response of C.argyrophylla seedlings and change the composition of the phyllosphere microbiome,so as to inhibit the occurrence of needle fall disease in C.argyrophylla seedlings.