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松枯梢病(又名松梢枯病shoot blight of pine)是世界范围内针叶树种上分布最广、最常见的重要林木枝干病害之一[1]。自从我国自20世纪70年代末报道以来,松枯梢病已蔓延至黑龙江、吉林、辽宁、陕西、江苏、湖北、福建、安徽、江西、广东和广西等10余个省份[2],严重危害松属(Pinus)、冷杉属(Abies)、落叶松属(Larix)、崖柏属(Thuja)、雪松属(Cedrus)、刺柏属(Juniperus)、云杉属(Picea)和黄杉属(Pseudotsuga)约8属60多种针叶树种[3]。该病害病原存在基因型的分化,De Wet通过多基因系谱学和微卫星标记手段将松枯梢病原菌划分为3个不同类型,分别为A型Diplodia scrobiculata,B型Diplodia seriata和C型Sphaeropsis sapinea(同物异名:Diplodia pinea)[4]。在中国引起松枯梢病的病原菌是C型松球壳孢菌(Sphaeropsis sapinea)[5],该病原菌可危害大树和幼树,大树多为侧枝发病,小树上顶梢发病,导致顶芽枯死、枯梢、根腐,严重时会出现流脂现象[6],对松树人工林造成了严重危害。目前,对松枯梢病主要采取化学防治与营林措施相结合的防治技术[7]。但长期使用化学农药不仅会导致环境污染、农药残留、威胁人类健康,而且容易诱导病原菌产生抗药性[8],甚至会导致病害的再次流行。而营林措施见效慢,投入成本较大。为有效防控松枯梢病,必须寻求一种生态友好的防治策略,木霉菌由于特有的拮抗和促生作用,现已成为一类应用较多的生防真菌[9],它通过竞争、寄生或产生抗菌素等次生代谢产物,从而对病原菌起到抑制作用[10],并能促进植物生长[11],提高寄主抵抗性。常见的生防木霉有哈茨木霉(Trichoderma harzianum)、绿色木霉(T. viride)、棘孢木霉(T. asperellum)等。研究表明,哈茨木霉ES323可有效抑制番茄灰霉病菌灰葡萄孢菌菌丝生长,引起菌丝腔质液泡化并导致菌体裂解[12]。非洲哈茨木霉菌(Trichoderma afroharzianum)株NAIMCC-F-01938发酵液可使葡萄白粉病原菌分生孢子扭曲变形,可作为安全杀菌剂在田间使用,可使葡萄白粉病发病率降低43%[13]。接种棘孢木霉可使洋葱鳞茎内酚类化合物含量提高97.6%,提高了洋葱对叶枯病的抵御能力,对洋葱叶枯病病原菌Stemphylium vesicarium具有显著的防控效果[14]。内生木霉菌株V76-12可抑制油棕叶斑病原菌Curvularia oryzae菌丝生长,同时提高了油棕幼苗苯丙氨酸解氨酶PAL、过氧化物酶POD和多酚氧化酶PPO的活性[15],提高了寄主植物的抗性。在前期的研究中,本实验室以松枯梢病原松球壳孢菌(Sphaeropsis sapinea)为目标菌株,从土壤真菌中分离筛选到1株对松球壳孢菌具有良好抑菌效果的森吉木霉菌株M75。为明确森吉木霉M75对松球壳孢菌的抑制作用机理,本研究从森吉木霉M75对病原菌代谢系统酶活性和电导率、丙二醛含量等方面,探究生防菌森吉木霉M75发酵粗提液对松球壳孢菌的抑菌机制,为松枯梢病的生物防治提供理论依据。
森吉木霉M75发酵粗提液对松球壳孢菌的抑菌机制研究
Study on the Inhibition Mechanism of Trichoderma songyi Against Sphaeropsis sapinea
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摘要:
目的 研究森吉木霉M75发酵粗提液对松球壳孢菌Sphaeropsis sapinea代谢系统及生理生化的影响,探究其抑菌机制。 方法 采用分光光度计法,分别测定在不同处理时间段内森吉木霉M75发酵粗提液对松球壳孢菌代谢系统酶活性,以及对电导率、丙二醛含量的影响。 结果 森吉木霉M75发酵粗提液可显著影响松球壳孢菌代谢系统中各关键酶活性。其中超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、过氧化物酶(POD)、多酚氧化酶(PPO)在处理8~12 h内达到峰值,为75.25 U·(g·min)−1、0.066 U·(g·s)−1、5.8 U·(g·s)−1和12.33 U·(g·s)−1,分别是初始时的1.96、6.09、16.37和7.86倍;丙酮酸激酶(PK)、己糖激酶(HK)和乳酸脱氢酶(LDH)在处理12~24 h内显著下降,由初始时的0.55、0.066、2.35 U·(g·min)−1下降至0.18、0.03、0.66 U·(g·min)−1,后下降趋缓;琥珀酸脱氢酶(SDH)、苹果酸脱氢酶(MDH)和辅酶Ⅰ含量在处理12 h内呈快速下降趋势,由初始时的30.33 U·(g·min)−1、15.55 U·(g·min)−1、5.70 nmol·(g·min)−1分别下降至10.00 U·(g·min)−1、3.65 U·(g·min)−1、1.34 nmol·(g·min)−1,处理12 h后呈缓慢下降趋势,而对照组则不断升高;Na+,K+-ATP、Mg++-ATP和Ca++-ATP酶活性在处理8 h或12 h时达到峰值,分别为0.09、1.76和3.18 μmolPi·(g·h)−1;此后迅速下降,处理24 h后下降趋缓并逐渐趋于0;松球壳孢菌细胞膜电导率在处理12 h内显著升高,且处理组在各时间段的电导率值始终高于对照组;丙二醛含量在处理24 h内显著升高并达到峰值1.06 nmol·g−1,处理24 h至96 h内呈缓慢下降趋势,但在各处理时间段仍显著高于对照组。 结论 森吉木霉M75发酵粗提液通过破坏松球壳孢菌的防御系统,阻碍了病原菌正常的生理代谢路径,从而抑制病原菌的正常生长。 Abstract:Objecive To study the effects of crude fermentation extract of Trichoderma songyi M75 on the metabolism system, physiological and biochemical mechanisms against the pathogen Sphaeropsis sapinea of pine shoot blight. Method Spectrophotometer was used to determine the effect of the crude fermentation extract of T. songyi M75 on the metabolic system enzyme activity of S. sapinea, as well as the electrical conductivity and the content of malondialdehyde in different treatment periods. Result The crude fermentation extract of T. songyi M75 significantly reduced the activity of the key enzymes in the metabolic system of S. sapinea. The treatment of SOD, CAT, PPO and POD reached their peaks within 8 to 12 hours, which were 75.25 U·(g·min)−1, 0.066 U·(g·s) −1, 5.8 U·(g·s) −1 and 12.33 U·(g·s) −1, respectively, and were 1.96, 6.09, 16.37 and 7.86 times of the initial. PK, HK and LDH decreased significantly within 12 to 24 hours of treatment, from 0.55, 0.066, 2.35 U·(g·min)−1 at the initial time to 0.18, 0.03, 0.66 U·(g·min)−1 respectively, and then the downward trend gradually slowed down. The contents of SDH, MDH and coenzyme Ⅰ decreased rapidly from 30.33 U·(g·min)−1, 15.55 U·(g·min)−1 and 5.70 nmol·(g·min)−1 to 10.00 U·(g·min)−1, 3.65 U·(g·min)−1 and 1.34 nmol·(g·min)−1 respectively, and then the decline was slow and the control group was in the continuous increasing trend. The activities of Na+, K+-ATP, Mg++-ATP和Ca++-ATP peaked at 0.09, 1.76 and 3.18μmolPi·(g·h) −1 at the 8th or the 12th hours, after that, it decreased rapidly, and then decreased slowly and gradually to 0 after 24 hours of treatment. The conductivity increased significantly within 12 hours, and the value of the treatment group was always higher than that of the control. MDA increased significantly in 24 hours and reached the peak value of 1.06 nmol·g −1, and decline slowly from the 24th to the 96th hours, however, it was still significantly higher than that of the control in each treatment period. Conclusion The crude fermentation extract of T. songyi M75 can inhibit the normal growth of the S. sapinea by destroying the defense system and normal metabolic pathway of the fungus. -
Key words:
- Trichoderma songyi
- / extract
- / Sphaeropsis sapinea
- / antibacterial mechanism
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