灰飞虱体内类酵母共生菌异常毕赤酵母的分离培养及其对杀菌剂的敏感性

Isolation and sensitivity to fungicides of the yeast-like symbiont Pichia anomala( Hemiascomycetes: Saccharomycetaceae ) from Laodelphax striatellus( Hemiptera: Delphacidae)

【目的】随着杀虫剂等化学农药的大规模使用,灰飞虱Laodelphax striatellus的抗药性不断增强,防治效果下降,因此寻找新的防治方法尤为重要。本研究探索使用杀菌剂来减少灰飞虱体内共生菌的数量,以降低灰飞虱的活力,为"抑菌防虫"提供理论依据。【方法】对灰飞虱体内的类酵母共生菌(yeast-like symbiotes,YLS)进行离体培养并鉴定,测定离体培养YLS对不同浓度不同种类杀菌剂的敏感性,将对其抑制作用明显的杀菌剂喷施于麦苗,待灰飞虱取食后统计灰飞虱死亡率、体重,并通过荧光定量PCR检测体内共生菌的数量变化。【结果】离体培养得到1株YLS,经碳源同化实验和18S r DNA分子鉴定,将该菌株归为异常毕赤酵母Pichia anomala,以变性梯度凝胶电泳(denatured gradient gel electrophoresis,DGGE)技术验证了该菌株在灰飞虱体内的存在。对杀菌剂敏感性的测定结果显示,70%丙森锌和50%戊唑醇+25%肟菌酯对P.anomala抑制效果明显,而氟吡菌胺+霜霉威盐酸盐(687.5 g a.i./L)与40%嘧霉胺对P.anomala的抑制效果较差。用对P.anomala抑制效果明显的两种杀菌剂(70%丙森锌和50%戊唑醇+25%肟菌酯)以800 mg/L浓度喷施麦苗,灰飞虱取食喷施麦苗后的死亡率分别达到46.7%与63.3%,显著高于对照,而体重比对照组显著低。荧光定量检测结果表明,灰飞虱体内3种共生菌(Noda菌、季也蒙毕赤酵母和异常毕赤酵母)的数量在使用了各种杀菌剂后均显著降低。【结论】结果说明,体外喷施杀菌剂对灰飞虱体内共生菌有抑制作用,进而对灰飞虱的生长存活产生影响,这为杀菌剂与杀虫剂的协同使用防治灰飞虱奠定基础。

【Aim】With the extensive application of chemical pesticides,the small brown planthopper,Laodelphax striatellus has increased its resistance to pesticides so that the control effect of pesticides on L.striatellus is weakened. Therefore it is crucial to explore a new method for controlling this pest. In this study,we attempt to inhibit the abundance of symbiotes inside L. striatellus by fungicides to weaken the vitality of this pest,so as to provide a theoretical foundation for controlling L. striatellus through inhibiting yeast-like symbiont（ YLS）. 【Methods】The yeast-like symbiont was isolated from L. striatellus via in vitro culture and the susceptibility of the isolated YLS to different concentrations of fungicides was examined. The fungicides with apparently higher ability to inhibit the growth of YLS were applied on wheat seedlings used for feeding L. striatellus. The mortality and body weight of L. striatellus adults were measured and the variation of the amount of YLS inside L. striatellus was examined through real-time florescent quantitative PCR. 【Results】A strain of YLS was successfully isolated and identified as Pichia anomala based on the corresponding carbon assimilation experiment and 18 S r DNA phylogenic analysis.Denaturing gradient gel electrophoresis（ DGGE） result further confirmed the existence of this strain in L.striatellus. The test results of sensitivity to fungicides indicated that 70% propineb and 50% tebuconazole＋ 25% trifloxystrobin had obvious inhibitory effects on the growth of P. anomala while fluopicolide ＋propamocarb hydrochloride（ 687. 5 g a. i. / L） and 40% pyrimethanil had relatively less inhibitory effects on the growth of P. anomala. When two fungicides（ 70% propineb and 50% tebuconazole ＋ 25%trifloxystrobin with obvious inhibitory effect were applied on wheat seedlings at the concentration of 800 mg / L,the mortality of L. striatellus adults feeding the treated wheat seedlings reached 46. 7% and63. 3%,respectively,which was apparently higher than that of the control group,and the body weight of L. striatellus adults in the treatment group was also less than that of the control group. Real-time florescent quantitative PCR results indicated that the amount of YLS of three strains（ Hypomyces chrysospermus,Pichia guilliermondii, and P. anomala） inside L. striatellus dropped sharply after different fungicides were applied. 【Conclusion】The results suggest that the fungicides applied in vitro on L. striatellus have inhibitory effects on their YLS,thus impacting the survival of L. striatellus,and this validates the feasibility of ＂reduction in use of pesticides through use of fungicides＂strategy in controlling L. striatellus.