杉木内生细菌SM-1的鉴定及其对杉木赤枯病的防治效果

刘雨菁; 曾华龙; 梁炜康; 卢梦; 朱伟垚; 张蕾; 宋漳; 张清华

doi:10.19303/j.issn.1008-0384.2021.05.009

杉木内生细菌SM-1的鉴定及其对杉木赤枯病的防治效果

doi: 10.19303/j.issn.1008-0384.2021.05.009

刘雨菁^1,,
曾华龙¹,
梁炜康²,
卢梦¹,
朱伟垚¹,
张蕾³,
宋漳^1,,
张清华^1, ,

1.
福建农林大学林学院森林保护研究所，福建　福州　350002
2.
华南农业大学教育部天然农药与化学生物学重点实验室，广东　广州　510642
3.
四川省农业科学院植物保护研究所，四川　成都　610066

基金项目: 福建省自然科学基金项目（2019J041）；福建农林大学科技创新项目（KFA17054A）

详细信息

作者简介:
刘雨菁（1994−），女，硕士研究生，研究方向：森林病理学（E-mail：1457182642@qq.com）

通讯作者:
张清华（1986−），男，博士，讲师，研究方向：森林病理学（E-mail：fzhangqinghua@fafu.edu.cn）

中图分类号: S 763
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出版历程
- 收稿日期: 2020-11-09
- 修回日期: 2021-01-27
- 网络出版日期: 2021-04-20
- 刊出日期: 2021-05-31

Endophytic Bacteria for Biocontrol of Copper Blight on Cunninghamia lanceolate

1.
Institute of Forest Protection, Fujian Agriculture and Forestry University, Fuzhou, Fujian　350002, China
2.
Key Lab of Natural Pesticides and Chemical Biology, Ministry of Education/South China Agricultural University, Guangzhou, Guangdong　510642, China
3.
Institute of Plant Protection, Sichuan Academy of Agriculture, Chengdu, Sichuan　610066, China

More Information

Corresponding author: 张清华（1986-），男，博士，讲师，研究方向：森林病理学（E-mail：fzhangqinghua@fafu.edu.cn）

摘要

摘要: 目的杉木赤枯病是杉木的重要病害之一，严重威胁杉木苗木生产及幼树成林。为了更好保护杉木，防治杉木赤枯病，从杉木叶片分离到内生细菌SM-1，探讨其对杉木赤枯病菌（Pestalotiopsis apiculatus）的拮抗作用。方法经形态观察、生理生化特性观察、16S rRNA及Biolog系统鉴定其种类；含毒平板及牛津杯法检测SM-1无菌发酵液对赤枯病菌的抑制率，并观察SM-1对赤枯病菌菌丝形态的影响；离体枝条的叶片接种试验检测SM-1无菌发酵液的防病效果。结果 SM-1为萎缩芽孢杆菌（Bacillus atrophaeus），其无菌发酵液对赤枯病菌的抑制率可达58.80%；显微观察显示SM-1能够使赤枯病菌菌丝生长受到抑制，细胞膨大成球形；离体枝条的叶片接种试验揭示SM-1无菌发酵液对杉木赤枯病的抑制效率达95%，能够极显著减轻叶片发病程度（P＜0.01）。结论杉木内生芽孢杆菌SM-1对杉木赤枯病具有良好的生物防治潜力。
- 萎缩芽孢杆菌 /
- 杉木赤枯病 /
- 内生细菌 /
- 生物防治
Abstract: Objective Endophytic bacteria that could effectively control the copper blight, one of the most significant diseases that seriously infects the seedlings and saplings, of Chinese fir (Cunninghamia lanceolate) were investigated. Methods Candidate endophytic bacteria were cultured and isolated from leaves of Chinese fir followed by morphological, physiological, and biochemical analyses as well as 16S rRNA sequencing for identification. Pathogen inhibition against Pestalotiopsis apiculatus by the sterile fermentation filtrate of potential specimens was tested on toxic plate and Oxford cup and observed under a microscope. Effectiveness of the disease control by the selected bacteria was further verified in vitro on fir leaves. Results The endophytic bacterium coded SM-1 showed a significant antagonistic effect against P. apiculatus. It was isolated and identified as Bacillus atrophaeus. On the toxic plate and Oxford cup tests, the SM-1 fermentation filtrate yielded a P. apiculatus inhibition rate of 58.80%. Morphologically, the bacterium inhibited the mycelial growth and caused cellular swelling that changed the cells spherical in appearance of the pathogen. The efficacy of the SM-1 filtrate on copper blight on the fir leaves was 95% with significantly reduced disease severity (P＜0.01). Conclusion The SM-1, identified as B. atrophaeus, appeared to be a potential biocontrol agent to deter copper blight on Chinese fir.
- Bacillus atrophaeus /
- copper blight of Chinese fir /
- endophytic bacteria /
- biological control

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图 1 菌株SM-1革兰氏染色及形态显微观察（标尺10 μm）

Figure 1. Gram-stained cell and morphology of SM-1 under microscope (bar = 10 μm)

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图 2 菌株SM-1系统发育进化树

Figure 2. Phylogenetic tree of SM-1

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图 3 菌株SM-1对杉木赤枯病菌的抑制率

Figure 3. Inhibitory rate of SM-1 against P. apiculatus

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图 4 不同发酵时间发酵液对杉木赤枯病菌的抑制效果

Figure 4. Inhibitory effect on P. apiculatus by fermentation broth cultured for different length of time (Oxford cup method)

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图 5 不同含量发酵液的PDA平板对致杉木赤枯病菌的抑制效果

Figure 5. Inhibition effect on P. apiculatus by varied concentrations of fermentation broth in PDA plate test

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图 6 菌株SM-1对杉木赤枯病菌菌丝影响

Figure 6. Effect of SM-1 on P. apiculatus mycelial growth observed under optical microscope

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图 7 菌株SM-1无菌发酵液对杉木赤枯病的防治效果

Figure 7. Biocontrol efficacy of SM-1 fermentation broth on copper blight of Chinese fir

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表 1 生理生化鉴定结果

Table 1. Physiological and biochemical characteristics of SM-1

项目 Items	1	2	3	4	5	6	7
氧化酶活性 Oxidase activity	−	+	−	+	−	+	+
糖原 Glycogen	−	Nd	−	−	+	+	−
乳糖 Lactose	−	+	−	−	+	−	−
蜜二糖 Melibiose	−	+	−	−	−	−	+
甲基葡萄糖苷 Methyl α-d-glucoside	−	+	−	−	+	+	+
杜-杜拉诺糖 d-Turanose	−	+	−	−	−	−	+
注：① “+”表示阳性，“−”表示阴性； ②1. SM-1, 2. 解淀粉芽孢杆菌, 3.萎缩芽孢杆菌, 4.摩加夫芽孢杆菌, 5.暹罗芽胞杆菌, 6.贝莱斯芽孢杆菌, 7. B. subtilis subsp. Stercoris。 Note: ① ‘+’ means positive; ‘−’ means negative; ②1. SM-1, 2. B. amyloliquefaciens, 3. B. atrophaeus, 4. B. mojavensis, 5. B. siamensis, 6. B. velezensis, 7. B. subtilis subsp. Stercoris.

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表 2 与菌株SM-1最为相似的细菌种类

Table 2. Bacterial species with closest similarity to SM-1

种类 Species	菌株编号 Strain	16S 序列注册号 Accession number	相似性 Similarity/%
贝莱斯芽孢杆菌 Bacillus velezensis	CR-502	AY603658	99.29
B. subtilis subsp. Stercoris	D7XPN1	JHCA01000027	99.21
暹罗芽孢杆菌 B. siamensis	KCTC 13613	AJVF01000043	98.97
枯草芽孢杆菌枯草亚种 B. subtilis subsp. Subtilis	NCIB 3610	ABQL01000001	98.69
解淀粉芽孢杆菌 B. amyloliquefaciens	DSM 7	FN597644	98.69
B. nakamurai	NRRL B-41091	LSAZ01000028	98.69
B. subtilis subsp. inaquosorum	KCTC 13429	AMXN01000021	98.49
耐盐短杆菌 B. halotolerans	ATCC 25096	LPVF01000003	98.49
特基拉芽孢杆菌 B. tequilensis	KCTC 13622	AYTO01000043	98.49
萎缩芽孢杆菌 B. atrophaeus	JCM 9070	AB021181	98.49
莫海威芽孢杆菌 B. mojavensis	RO-H-1	JH600280	98.42
死亡谷芽孢杆菌 B. vallismortis	DV1-F-3	JH600273	98.42
枯草芽孢杆菌斯氏亚种 B. subtilis subsp. Spizizenii	NRRL B-23049	CP002905	98.35
B. glycinifermentans	GO-13	LECW01000063	97.45
副地衣芽孢杆菌 B. paralicheniformis	KJ-16	KY694465	97.38
B. licheniformis	地衣芽孢杆菌 ATCC 14580	AE017333	97.18
B. haynesii	NRRL B-41327	MRBL01000076	97.11

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表 3 Biolog系统对细菌菌株SM-1的鉴定

Table 3. Identification of bacterial strain SM-1 by Biolog system

排序 Rank	相似性 Similarity	位距 Distance	种类 Species
1	0.631	5.311	萎缩芽孢杆菌 Bacillus atrophaeus
2	0.163	5.752	解淀粉芽孢杆菌 B. amyloliquefaciens
3	0.062	6.336	摩加夫芽孢杆菌 B. mojavensis
4	0.046	6.511	简单芽胞杆菌 B. simplex

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