摘要
[目的]研究温度、pH和不同营养条件对捕食线虫真菌圆锥节丛孢菌(Arthrobotrys conoides)生长及产孢特性的影响,分析其在碳氮饥饿条件下几丁质酶的表达水平,优化该菌的培养条件,为该菌的扩大培养提供参考依据。[方法]将圆锥节丛孢菌AC-shz1菌株接种于YPSSA培养基,分别置于不同温度(15、20、25、30和35℃)、pH(5.0、5.5、6.0、6.5、7.0、7.5和8.0)、碳源(果糖、半乳糖、葡萄糖、蔗糖、麦芽糖、乳糖、淀粉、纤维素、几丁质)、氮源(甘氨酸、苯丙氨酸、蛋白胨、酵母粉、硫酸铵、尿素)和碳氮比(1∶1、5∶1、10∶1、20∶1、40∶1)环境条件下进行培养,观察并记录菌丝的生长速度及形态,测定其产孢能力;在碳氮源饥饿条件下,采用实时荧光定量PCR方法对4种几丁质酶基因的表达水平进行分析。[结果]圆锥节丛孢菌在温度15~30℃均能生长,其中以25℃时菌丝生长最快且产孢能力最强;在pH为6.5~7.5时菌丝生长良好,其中以pH 7.0时产孢能力最强。圆锥节丛孢菌最适菌丝生长和产孢碳源为葡萄糖和蔗糖,其中含葡萄糖的培养基产孢量最高;最适氮源为酵母粉和蛋白胨,含酵母粉的培养基产孢量最高,以葡萄糖和酵母粉为碳氮源的最适碳氮比为5∶1。与对照组相比,在碳饥饿条件下,捕食线虫真菌圆锥节丛孢菌几丁质酶-14(AC-14,P<0.01)和捕食线虫真菌圆锥节丛孢菌几丁质酶-190(AC-190,P>0.05)基因转录水平上调,捕食线虫真菌圆锥节丛孢菌几丁质酶-379(AC-379,P>0.05)和捕食线虫真菌圆锥节丛孢菌几丁质酶-483(AC-483,P<0.01)基因转录水平下调;在氮饥饿条件下,上述4种几丁质酶基因转录水平均极显著下调(P<0.01)。[结论]菌丝生长和产孢最适温度为25℃,最适pH为7.0,最适碳氮源分别为葡萄糖和酵母粉,最适碳氮比为5∶1。本研究结果为进一步研发具有高效的捕食线虫真菌生物防控制剂奠定了基础。
[Objective]This experiment was conducted to understand the effects of temperature,pH and different nutritional conditions on the growth and spore production of the nematode-trapping fungi Arthrobotrys conoides(A.conoides),analyse the expression level of chitinase-encoding genes under carbon and nitrogen starvation,and optimise the culture conditions to provide a basis for the expansion culture of the fungi.[Method]The hyphae of A.conoides AC-shz1 were inoculated at different temperatures(15,20,25,30 and 35℃),pH(5.0,5.5,6.0,6.5,7.0,7.5 and 8.0),carbon resource(fructose,galactose,glucose,sucrose,maltose,lactose,starch,cellulose and chitin),nitrogen resource(glycine,phenylalanine,peptone,yeast powder,ammonium sulphate and urea)and carbon-nitrogen ratio(1∶1,5∶1,10∶1,20∶1 and 40∶1)of YPSSA medium,and the growth rate and morphology were observed and recorded,and its sporulation capacity was measured.The expression levels of four chitinase-encoding genes was also analyzed by Real-time quantitative PCR under carbon and nitrogen starvation.[Result]The results showed that A.conoides could grow at a temperature of 15-30℃,among which hyphae grew the fastest and had the strongest sporulation capacity at 25℃.When the pH was 6.5-7.5,the hyphae growth was the better and the sporulation capacity was the stronger at pH 7.0.It was found that the most suitable sources for hyphae growth and spore production were glucose medium and sucrose medium,among which glucose medium spore production was the highest.The most suitable nitrogen sources were yeast powder and peptone,among which yeast powder medium had the highest spore yield,and the optimal carbon-nitrogen ratio with glucose and yeast powder as carbon and nitrogen sources was 5∶1.Compared with control group,under carbon starvation,the transcript levels of the chitinase of the nematode-trapping fungi A.conoides-14(AC-14,P<0.01)and chitinase of the nematode-trapping fungi A.conoides-190(AC-190,P>0.05)genes were up-regulated,the chitinase of the nematode-trapping fungi A.conoides-379(AC-379,P>0.05)and chitinase of the nematode-trapping fungi A.conoides-483(AC-483,P<0.01)genes were down-regulated.Under nitrogen starvation,the transcript levels of all four chitinase-encoding genes were extremely significantly down-regulated(P<0.01).[Conclusion]The optimum temperature for hyphae growth and spore production was 25℃,the optimum pH was 7.0,and the optimum carbon-nitrogen sources were glucose and yeast powder,respectively,the optimum carbon-nitrogen ratio of 5∶1,which laid the foundation for the further development of highly effective nematode-trapping fungal biocontrol agents.
作者
孙殿明
张慧梅
李柠杏
孙焱森
李若冰
乔军
才学鹏
孟庆玲
SUN Dianming;ZHANG Huimei;LI Ningxing;SUN Yansen;LI Ruobing;QIAO Jun;CAI Xuepeng;MENG Qingling(College of Animal Science and Technology,Shihezi University,Shihezi 832003,China;China Institute of Veterinary Drug Control,Beijing 100081,China)
出处
《中国畜牧兽医》
CAS
CSCD
北大核心
2024年第2期709-718,共10页
China Animal Husbandry & Veterinary Medicine
基金
国家自然科学基金(32260888、32060801)
新疆维吾尔自治区研究生科研创新计划项目(XJGRI2015038)。
关键词
捕食线虫真菌
圆锥节丛孢菌
温度
pH
碳氮源
几丁质酶
nematode-trapping fungi
Arthrobotrys conoides
temperature
pH
carbon-nitrogen resource
chitinase
