蔬菜根结线虫生防芽胞杆菌的筛选及作用机理研究

doi:10.13560/j.cnki.biotech.bull.1985.2021-0409

摘要/Abstract

摘要：

芽胞杆菌是一类重要的植物病害生防因子。为挖掘防治蔬菜根结线虫的芽胞杆菌优良菌株,从根结线虫病抑制性土壤中分离到62株芽胞杆菌,测定不同菌株发酵滤液对二龄幼虫的致死作用和卵孵化的抑制作用初筛生防菌,然后利用室内盆栽试验测定生防菌株的防治效果,结果发现NB-04菌液灌根处理根结指数最低,防治效果达61.6%。进一步通过形态特征和16S rDNA序列比对,将NB-04菌株鉴定为甲基营养型芽胞杆菌Bacillus methylotrophicus。此外,测定了该菌株发酵滤液对根结线虫卵囊过氧化氢酶、乙酰胆碱酯酶和羧酸酯酶的活性以及卵囊总糖和总蛋白含量的影响,探讨其生防机理。结果显示NB-04菌株发酵滤液能够显著降低根结线虫卵囊过氧化氢酶、乙酰胆碱酯酶和羧酸酯酶的活性以及卵囊总糖和总蛋白含量。本研究为生物杀线剂的开发及利用提供依据。

关键词: 南方根结线虫, 生物防治, 甲基营养型芽胞杆菌, 生物防治机理

Abstract:

Bacillus sp. are a group of important biocontrol agents for the management of plant disease. In order to search for excellent Bacillus strain for vegetable root knot nematode,62 Bacillus sp. were isolated from the suppression soil of root knot nematode. The mortality of the second stage juvenile and the inhibition of egg hatch by the fermentation filtrates of the strains were conducted to primarily screen biocontrol strains,and pot experiments were used to determine the control effects of biocontrol strains. Results demonstrated that treatments with bactreial suspensions of strains NB-04 reduced root-knot index by 61.6%. Further based on morphological characteristics and 16S rDNA gene sequence analysis,strain NB-04 was identified as Bacillus methylotrophicus. To preliminary study the biocontrol mechanism of NB-04,the influence of fermention filtrates on M. incognita egg catalase,acetylcholinesterase,carboxylesterase,and the content of egg total sugar and protein were determined. The results showed that the activity of egg catalase,acetylcholinesterase,and carboxylesterase decreased significantly,as well as the content of total sugar and protein significantly declined. This study provides the basis for the research and development of bio-nematicide.

Key words: Meloidogyne incognita, biological control, Bacillus methylotrophicus, biocontrol mechanism

张洁, 夏明聪, 朱文倩, 梁娟, 孙润红, 徐文, 武超, 杨丽荣. 蔬菜根结线虫生防芽胞杆菌的筛选及作用机理研究[J]. 生物技术通报, 2021, 37(7): 175-182.

ZHANG Jie, XIA Ming-cong, ZHU Wen-qian, LIANG Juan, SUN Run-hong, XU Wen, WU Chao, YANG Li-rong. Screening of Bacillus sp. Against Vegetable Root-knot Nematode and Study on Its Biocontrol Mechanism[J]. Biotechnology Bulletin, 2021, 37(7): 175-182.

图/表 8

表1 不同细菌发酵滤液对根结线虫二龄幼虫的死亡率和卵孵化率的影响

Table 1 Effects of the bacterial fermentation filtrates on the mortality and egg hatch rates of M. incognita J2

处理 Treatment	死亡率 Mortality/%	校正死亡率 Adjusted mortality/%	卵孵化率 Egg hatch rate/%	孵化抑制率 Inhibition rate of egg hatch/%
NB-04	94.2±1.2 a	84.9 a	13.6±1.8 g	78.6 a
NB-36	90.4±2.5 ab	75.1 ab	12.0±1.5 g	81.1 a
NB-18	89.3±1.5 abc	72.4 abc	38.4±1.2 c	39.5 fg
NB-21	89.2±1.3 abc	72.1 abc	33.7±2.1 cd	46.9 ef
NB-12	88.6±0.4 abc	70.5 abc	13.5±1.6 g	78.7 a
NB-54	86.9±0.9 abcd	66.2 abc	28.5±3.3 de	55.1 de
NB-53	86.2±2.2 abcd	64.3 abcd	22.5±1.4 ef	64.6 bcd
NB-29	86.0±0.4 abcd	63.9 abcd	45.2±3.7 b	28.8 g
NB-15	85.7±0.1 abcd	63.1 abcd	18.5±1.9 fg	70.9 abc
NB-46	85.6±2.0 abcd	62.3 abcd	27.5±3.0 de	56.7 de
NB-17	85.2±2.0 bcd	61.8 abcd	27.8±1.7 de	56.2 de
NB-45	84.6±2.2 bcd	60.2 abcd	25.0±1.7 ef	60.6 cd
NB-31	81.4±4.1 cde	52.1 bcd	14.8±2.2 g	76.7 ab
NB-26	79.6±4.0 de	47.5 bcd	35.5±2.0 c	44.1 ef
NB-33	79.3±1.2 de	46.5 cd	27.8±2.0 de	56.2 de
NB-11	75.8±3.2 e	37.4 d	15.1±0.7 g	76.2 ab
对照 Control	61.2±6.0 f	/	63.5±4.3 a	/

表2 不同生防菌株对南方根结线虫的盆栽防治效果及番茄植株生长的影响

Table 2 Pot control effects of biocontrol strains against M. incognita and their influences on tomato plant growth

处理 Treatment	根结指数 Root-knot index	防治效果 Control effect/%	株高 Plant height/cm	鲜重 Fresh weight/g
NB-04	31.1±4.3 cd	61.6	52.0±4.5 a	23.9±1.0 a
NB-36	34.7±3.0 c	57.2	49.6±4.7 ab	21.5±2.9 a
NB-12	40.4±1.3 bc	50.1	46.1±3.7 ab	18.9±2.4 ab
NB-18	45.0±2.1 b	44.4	39.7±5.6 ab	16.4±2.5 ab
0.5% 阿维菌素 0.5% Avermectin	24.9±1.8 d	69.2	46.3±2.5 ab	19.8±2.0 ab
对照 Control	81.0±2.1 a	/	36.5±3.5 b	16.9±2.2 b

图1 NB-04的形态学特征 A：菌落;B：菌体;C：芽胞

Fig. 1 Mophological characteristics of NB-04 A：Colony morphology. B：Cell. C：Endospore

图2 NB-04菌株基于16S rDNA基因序列的系统发育树

Fig. 2 Phylogenetic tree of strain NB-04 based on 16S rDNA gene sequence

图3 NB-04菌株发酵液对根结线虫卵囊过氧化氢酶活性的影响

Fig. 3 Effects of NB-04 fermentation filtrates on the catalase activities of M. incognita egg masses

图4 NB-04菌株发酵液对根结线虫卵囊羧酸酯酶活性的影响

Fig. 4 Effects of NB-04 fermentation filtrates on the carboxylesterase activities of M. incognita egg masses

图5 NB-04菌株发酵液对根结线虫卵囊乙酰胆碱酯酶活性的影响

Fig. 5 Effects of NB-04 fermentation filtrates on the acetyl-cholinesterase activities of of M. incognita egg masses

图6 NB-04菌株发酵液对根结线虫卵囊总糖（A）和总蛋白（B）含量的影响

Fig. 6 Effects of NB-04 fermention on the content of total sugar（A）and protein（B）of of M. incognita eggs

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