Nematocidal activity and biocontrol efficacy of endophytic Bacillus velezensis Pt-RP9 from Pinus tabuliformis against pine wilt disease caused by Bursaphelenchus xylophilus

Pine wilt disease(PWD)is a globally significant quarantine forest disease caused by Bursaphelenchus xylophilus(PWN),resulting in substantial ecological and economic losses.Traditional nematode management practices are neither cost-effective nor environmentally friendly,prompting the exploration of biocontrol as a promising alternative for managing this devastating forest disease.Obtaining novel and specific biocontrol agents is extremely crucial for the effective and precise control of PWD.In the present study,a total of 136 endophytic isolates were obtained from various tissues of P.tabuliformis trees in the Qinling Mountains of China,comprising 65 isolates from roots,41 from stems,and 11 from needles,which were then subjected to nematocidal activity assay against PWN in vitro.Among which,nine endophytic bacterial isolates exhibited exceptionally strong nematocidal capacity,with a corrected mortality rate exceeding 90%,which were then identified as the genus of Bacillus through morphological features,endospore staining,and 16S rDNA sequencing,with one strain as B.mycoides,two as B.cereus,and six as B.velezensis.Additionally,the inhibition effects of the three Bacillus species on the reproduction of PWN in vitro was assessed using an original detection model.Overall,the bacterial fermentation suspension exhibited stronger inhibition compared to the bacteria-free fermentation filtrate.Among the three Bacillus isolates,B.velezensis Pt-RP9 displayed the highest level of inhibition,with significantly lower values of reproduction rate represented a 244.4-fold reduction,the egg amounts represented a 1069.7-fold reduction.Subsequently,the biocontrol efficacy of B.velezensis Pt-RP9 against PWD was evaluated in greenhouse experiments.Pt-RP9 demonstrated significant biocontrol effectiveness against PWD,with control efficiencies ranging from 31.25%to 68.89%across all treatments,particularly showing improved efficacy when pine seedlings were pretreated with Pt-RP9 before PWN inoculation.Besides,the density of PWN in the seedlings treated with Pt-RP9 was significantly lower than that of PWN group,indicating that B.velezensis Pt-RP9 can effectively inhibit PWN reproduction in vivo.Furthermore,to delve deeper into the protective mechanisms of B.velezensis Pt-RP9 against PWD,the MDA content and activities of defensive enzymes in P.tabuliformis seedlings were assayed.The pine seedlings treated with Pt-RP9 exhibited significantly reduced lipid peroxidation levels in cell membranes compared to the control groups,along with increased activities of peroxidase,catalase,and polyphenol oxidase,but superoxide dismutase,Chitinase,and Phenylalanine ammonialyase showed no significant differences among all treatment groups.To our knowledge,this study is the first to showcase the nematocidal activity of endophytes from P.tabuliformis against PWN and their biocontrol efficacy against PWD,marking a significant advancement in the field.The findings highlight the potential of B.velezensis Pt-RP9 as a crucial biological control agent against PWD,presenting a novel and sustainable disease management approach for pine forests.