印度梨形孢(Piriformospora indica)对核盘菌(Sclerotinia sclerotiorum)生长的抑制作用

The Inhibition Effect of Piriformospora indica on the Growth of Sclerotinia sclerotiorum

探讨印度梨形孢对核盘菌的抑制作用及其机理,为利用印度梨形孢进行核盘菌生物防治奠定基础.通过PEG介导的原生质体转化法将绿色荧光蛋白编码基因GFP导入核盘菌“1980”基因组,获得了菌丝形态正常的绿色荧光蛋白转基因菌株“1980-GFP”.通过固体、液体对峙培养,明确印度梨形孢对核盘菌生长的影响;利用实时荧光定量PCR检测对峙培养过程中核盘菌防御与生长发育相关基因Sssod1,Sssmk1,Ssshk1及SsITL的表达水平.平板对峙培养结果表明:印度梨形孢可抑制核盘菌“1980-GFP”的生长,致使其菌落延伸半径降低了36.43%~53.56%,生长速度降低了84.76%,菌核数量降低了73.38%,干质量降低了64.16%.对峙培养后,核盘菌菌丝尖端生长异常,形成短而多的分叉,GFP荧光信号弱化.印度梨形孢发酵培养液致使核盘菌菌丝体干质量相较于PDB和1/2PDB培养液中分别降低了52.01%和25.52%.实时荧光定量PCR分析结果表明:对峙培养过程中,Sssmk1在48 h上调了3.89倍;Sssod1在72 h上调了2.78倍;SsITL在24~72 h内上调了2.94~4.71倍;96 h时,Sssod1,SsITL表达下调;Ssshk1在中后期表达下调.以上结果表明:对峙培养时,印度梨形孢能影响核盘菌的防御响应及生长发育相关基因的表达,并能显著抑制核盘菌的生长发育.

In this study,we explored the inhibitory effect of Piriformospora indica on Sclerotinia sclerotiorum to lay a foundation for the further application of P.indica as a biological control agent.The gene(GFP)encoding green fluorescent protein was transformed into the genome of wild-type S.sclerotiorum“1980”by PEG-mediated protoplast transformation to obtain“1980-GFP”.Under the confrontation cultivation with P.indica,the growth of S.sclerotiorum was investigated.Additionally,the expressions of the key genes including Sssod1,Sssmk1,Ssshk1 and SsITL,involved in defense and growth,were detected by quantitative real-time PCR.The confrontation cultivation showed that P.indica had direct antagonism against S.sclerotiorum.Cultivated on PDA,the colony radius of S.sclerotiorum was decreased by 36.43%~53.56%.Colony growth rate,dry weight and amount of sclerotium were decreased by 84.76%,64.16%and 73.38%,respectively.The hyphae of S.sclerotiorum was abnormal,forming lots of short bifurcations after confrontation cultivation.Additionally,the fermentation broth of P.indica could inhibit the growth of S.sclerotiorum.The dry weight of mycelium cultivated in fermentation broth of P.indica was decreased by 52.01%and 25.52%compared with that in PDB and 1/2PDB,respectively.The results of quantitative real-time PCR analysis showed that the expression of Sssmk1 was up-regulated 3.89 times at 48 h(hours post confrontation cultivation),Sssod1 was up-regulated 2.78 times at 72 h,SsITL was up-regulated 2.94~4.71 times at 24~72 h,Sssod1 and SsITL were down-regulated at 96 h,and Ssshk1 was down-regulated in the middle and late stages of confrontation.The results indicated that P.indica affects the expression of genes involved in growth and defense in S.sclerotiorum,and can significantly inhibit the growth of S.sclerotiorum.