不同环境和营养条件对捕食线虫真菌圆锥节丛孢菌生长、产孢及几丁质酶表达的影响

doi:10.16431/j.cnki.1671-7236.2024.02.027

摘要/Abstract

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。

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

Abstract: 【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.

Key words: nematode-trapping fungi; Arthrobotrys conoides; temperature; pH; carbon-nitrogen resource; chitinase

中图分类号:

S852.66

孙殿明, 张慧梅, 李柠杏, 孙焱森, 李若冰, 乔军, 才学鹏, 孟庆玲. 不同环境和营养条件对捕食线虫真菌圆锥节丛孢菌生长、产孢及几丁质酶表达的影响[J]. 中国畜牧兽医, 2024, 51(2): 709-718.

SUN Dianming, ZHANG Huimei, LI Ningxing, SUN Yansen, LI Ruobing, QIAO Jun, CAI Xuepeng, MENG Qingling. Effects of Different Environmental and Nutritional Conditions on Growth Characteristics,Spore Production and Chitinase Expression of Nematode-trapping Fungi Arthrobotrys conoides[J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(2): 709-718.

参考文献

[1] BARONE C D,WIT J,HOBERG E P,et al.Wild ruminants as reservoirs of domestic livestock gastrointestinal nematodes[J].Veterinary Parasitology,2020,279:109041.
[2] 赵乾明,齐萌,徐泽立,等.规模舍饲羊场羊消化道及环境寄生虫情况调查[J].中国畜牧兽医,2023,50(5):2156-2165. ZHAO Q M,QI M,XU Z L,et al.Investigation of digestive tract and environmental parasites in sheep on sheep farm[J].China Animal Husbandry&Veterinary Medicine,2023,50(5):2156-2165.(in Chinese)
[3] 张克勤,莫名和.中国真菌志[M].北京:科学出版社,2006. ZHANG K Q,MO M H.Chinese Mycology[M].Beijing:Science Press,2006.(in Chinese)
[4] 刘杏忠,裘维蕃.食线虫真菌的研究进展[A].中国虫生真菌研究与应用[C].1991. LIU X Z,QIU W F.Research progress of nematode fungi[A].Research and Application of Insect Fungi in China[C].1991.(in Chinese)
[5] 徐镔蕊,秦泽荣,缪作清.节丛孢属食线虫真菌对体外奥斯特线虫幼虫的捕食效果[J].中国兽医杂志,1999,25(2):8-9. XU B R,QIN Z R,MIAO Z Q.Predation effect of Arthropodus nematode fungi on in vitro Osteria larvae[J].Chinese Journal of Veterinary Medicine,1999,25(2):8-9.(in Chinese)
[6] NOURANI S L,GOLTAPEH E M,SAFAIE N,et al.Enhancing the pathogenicity of Arthrobotrys conoides and A.oligospora against Meloidogyne javanica J2 by transferring of protease (Ac1) gene and evaluation of antagonistic capability of transgenic isolates[J].Biological Control,2018,122:127-135.
[7] YURIDIA A,MANUEL V,AGUILAR L,et al.Morphological and molecular characterization,predatory behaviour and effect of organic extracts of four nematophagous fungi from Mexico[J].Fungal Ecology,2021,49:101004.
[8] 李思媛.新疆乌鲁木齐地区三株捕食性真菌的分离鉴定及捕食效率研究[D].乌鲁木齐:新疆农业大学,2022. LI S Y.Isolation and identification of three predatory fungi and study on predation efficiency in Urumqi,Xinjiang[D].Urumqi:Xinjiang Agricultural University,2022.(in Chinese)
[9] 王利平,代林远,李鹏.苏云金芽孢杆菌研究进展[J].中国畜牧兽医,2011,38(9):224-227. WANG L P,DAI L Y,LI P.Research progress on Bacillus thuringiensis[J].China Animal Husbandry&Veterinary Medicine,2011,38(9):224-227.(in Chinese)
[10] GOMAA E Z.Microbial chitinases:Properties,enhancement and potential applications[J].Protoplasma,2021,258(4):695-710.
[11] 钟文强,孟庆玲,乔军,等.少孢节丛孢菌新疆分离株几丁质酶AO-14基因的克隆表达[J].畜牧与兽医,2017,49(11):118-123. ZHONG W Q, MENG Q L, QIAO J, et al.Cloning and expression of the chitinase AO-14 gene from a Xinjiang isolate of Arthrobotrys oligospora[J].Animal Husbandry and Veterinary Medicine,2017,49(11):118-123.(in Chinese)
[12] 贡莎莎,孟庆玲,乔军,等.少孢节丛孢菌XJ-A1几丁质酶基因AO-190的克隆、表达及其酶活性的测定[J].华北农学报,2018,33(4):67-74. GONG S S, MENG Q L, QIAO J, et al.Cloning, expression and activity of XJ-A1 chitinase gene AO-190 from Arthrospora oligospora[J].Acta Agriculturae Boreali-Sinica, 2018, 33(4):67-74.(in Chinese)
[13] 钟文强,孟庆玲,乔军,等.少孢节丛孢菌胞外几丁质酶AO-379基因克隆及其表达分析[J].南方农业学报,2018,49(1):148-154. ZHONG W Q, MENG Q L, QIAO J, et al.Cloning and expression analysis of extracellular chitinase AO-379 gene from Arthrospora oligospora[J].Journal of Southern Agriculture, 2018,49(1):148-154.(in Chinese)
[14] 贡莎莎,孟庆玲,乔军,等.少孢节丛孢菌XJ-A1几丁质酶AO-483基因的克隆及生物学活性[J].浙江农业学报,2019,31(2):222-228. GONG S S, MENG Q L, QIAO J, et al.Cloning and biological activity of XJ-A1 chitinase AO-483 gene from Arthrospora oligospora[J].Acta Agriculturae Zhejiangensis,2019,31(2):222-228.(in Chinese)
[15] YANG J K,YU Y,LI J,et al.Characterization and functional analyses of the chitinase-encoding genes in the nematode-trapping fungus Arthrobotrys oligospora[J].Archives of Microbiology,2013,195:453-462.
[16] CHARLIER J,HOGLUND J,MORGAN E R,et al.Biology and epidemiology of gastrointestinal nematodes in cattle[J].Veterinary Clinics of North America:Food Animal Practice,2020,36(1):1-15.
[17] RODRIGUES J A,ROQUE F L,ÁLVARES F B V,et al.Efficacy of a commercial fungal formulation containing Duddingtonia flagrans (Bioverm^®) for controlling bovine gastrointestinal nematodes[J].Revista Brasileira de Parasitologia Veterinaria,2021,30(2):e026620.
[18] ELMSLY T A,DIXON J.Growth rates of ripe rot fungi at different temperatures[R].New Zealand Avocado Growers'Association Annual Research Report,2008.
[19] 王逢会,王波波,徐春兰,等.温度和pH对捕食性线虫真菌分离株少孢节丛孢菌生长的影响及生物学特性观察[J].甘肃农业大学学报,2015,50(2):100-105. WANG F H,WANG B B,XU C L,et al.Effects of temperature and pH on growth and biological characteristics of fungal isolates of predatory nematodes and biological characteristics of Arthrobotrys oligospora[J].Journal of Gansu Agricultural University,2015,50(2):100-105.(in Chinese)
[20] ROUSK J,BROOKES P C,BAATH E.Contrasting soil pH effects on fungal and bacterial growth suggest functional redundancy in carbon mineralization[J].Applied and Environmental Microbiology,2009,75(6):1589-1596.
[21] ABUBAKAR A,SUBERU H A,BELLO I M,et al.Effect of pH on mycelial growth and sporulation of Aspergillus parasiticus[J].Journal of Plant Sciences,2013,1(4):64-67.
[22] 秦泽荣,缪作清,刘杏忠,等.温度和pH值对节丛孢属食线虫真菌生长的影响观察[J].华北农学报,1999,3:141-144. QIN Z R,MIAO Z Q,LIU X Z,et al.Effects of temperature and pH on the growth of C.elegans fungi[J].Acta Agriculturae Boreali-Sinica,1999,3:141-144.(in Chinese)
[23] SADIQ R M A,AHMED J F.Effect of some physical factors on growth of five fungal species[J].European Academic Research,2017,2:1069-1078.
[24] 王瑞,杨晓野,杨莲茹,等.不同碳氮源及其比例和培养基初始pH值对少孢节丛孢菌菌丝生长的影响[J].黑龙江畜牧兽医,2008,9:11-14. WANG R,YANG X Y,YANG L R,et al.Effects of different carbon and nitrogen sources,their ratios and initial pH values of medium on the growth of mycelium of Arthrobotrys oligospora[J].Heilongjiang Animal Science and Veterinary Medicine,2008,9:11-14.(in Chinese)
[25] 李军燕.Duddingtonia flagrans生长特性和捕食线虫活性物质的研究[D].呼和浩特:内蒙古农业大学,2014. LI J Y.Study on growth characteristics and predatory nematode active substances of Duddingtonia flagrans[D].Hohhot:Inner Mongolia Agricultural University,2014.(in Chinese)
[26] LONARDO D P,WAL A,HARKES P,et al.Effect of nitrogen on fungal growth efficiency[J].Plant Biosystems,2020,154:433-437.
[27] NAVARATHNA D H,HARRIS S D,ROBERTS D D,et al.Evolutionary aspects of urea utilization by fungi[J].FEMS Yeast Research,2010,10(2):209-213.
[28] STROPE P K,NICKERSON K W,HARRIS S D,et al.Molecular evolution of urea amidolyase and urea carboxylase in fungi[J].BMC Evolutionary Biology,2011,11:80.
[29] PORIA V,RANA A,KUMARI A,et al.Current perspectives on chitinolytic enzymes and their agro-industrial applications[J].Biology,2021,10(12):1319.
[30] SZILAGYI M,MISKEI M,KARANYI Z,et al.Transcriptome changes initiated by carbon starvation in Aspergillus nidulans[J].Microbiology Reading,2013,159(1):176-190.