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Genes name	Sequences of primer (5′→3′)	Functional description
actin	F: GAGCTGTTTTCCCTTCCATTGTC R: GACGACACCGTGCTCGATTGG	Actin
Gene_9536	F: TTCAGTTTCCCAGTTCA R: GCCATCAAGGTTCCCAC	Hexokinase
Gene_3455	F: CGTCCTACATTTATCAACA R: ATCCACGTCAAGATCACTA	Glycosyltransferase family 20
Gene_7772	F: GCCCGCAGTATGCCAACA R: GCGACGACAGCCCAAAGA	Phosphoglucomutase
Gene_5305	F: GGTCCAGATGTCATTCGT R: CCTCTTTGTTCACCCACT	Catalase-related immune-responsive
Gene_4935	F: CCAGTTGATTTCCAGCAT R: CTTCATAAAGGCGACGAG	Catalase
Gene_1287	F: GAATCGGTAGCAAACTCC R: CCGCCTTCTATCATCTCG	Peroxisome
Gene_13	F: ACATTGGTAGCCGTGGTT R: GTCATTGATGGCGTAGAA	Glucanosyltransferase; cellulase
Gene_11361	F: GCTCATCGCCGTCACTTA R: GGTCCACCGACATTCCAG	Chitin synthase 2
Gene_622	F: CGCTCGCAAGTGAAGTA R: GTGCCATTTGTCCCTAA	Cell wall

Genes name	Sequences of primer (5′→3′)	Functional description
actin	F: GAGCTGTTTTCCCTTCCATTGTC R: GACGACACCGTGCTCGATTGG	Actin
Gene_9536	F: TTCAGTTTCCCAGTTCA R: GCCATCAAGGTTCCCAC	Hexokinase
Gene_3455	F: CGTCCTACATTTATCAACA R: ATCCACGTCAAGATCACTA	Glycosyltransferase family 20
Gene_7772	F: GCCCGCAGTATGCCAACA R: GCGACGACAGCCCAAAGA	Phosphoglucomutase
Gene_5305	F: GGTCCAGATGTCATTCGT R: CCTCTTTGTTCACCCACT	Catalase-related immune-responsive
Gene_4935	F: CCAGTTGATTTCCAGCAT R: CTTCATAAAGGCGACGAG	Catalase
Gene_1287	F: GAATCGGTAGCAAACTCC R: CCGCCTTCTATCATCTCG	Peroxisome
Gene_13	F: ACATTGGTAGCCGTGGTT R: GTCATTGATGGCGTAGAA	Glucanosyltransferase; cellulase
Gene_11361	F: GCTCATCGCCGTCACTTA R: GGTCCACCGACATTCCAG	Chitin synthase 2
Gene_622	F: CGCTCGCAAGTGAAGTA R: GTGCCATTTGTCCCTAA	Cell wall

Item tested	Results
+: Positive (growth or reaction); –: Negative (no growth or no reaction).
Xylose	–
Mannitol	–
Glucose	+
Starch hydrolysis	+
Catalase	+
Nitrate reduction	+
Citrate	–
Voges-Proskauer test	+
Gelatin liquefaction	+

Item tested	Results
+: Positive (growth or reaction); –: Negative (no growth or no reaction).
Xylose	–
Mannitol	–
Glucose	+
Starch hydrolysis	+
Catalase	+
Nitrate reduction	+
Citrate	–
Voges-Proskauer test	+
Gelatin liquefaction	+

Pathogens	Inhibition rate (%)
The inhibition rate was calculated after 4 days at 25 ℃, three replicates are set for each treatment; Average ± standard deviation.
Botryotinia fuckeliana	78.11±2.20
Thanatephorus cucumeris	0.00±0.00
Verticillium dahliae	0.00±0.00
Colletotrichum gloeosporioides	54.63±3.00
Colletrichum lagenarium	63.69±0.44
Rhizoctonia solani	0.00±0.00
Cochiobolus sativus	67.30±2.61
Botrytis cinerea	68.22±2.41

Pathogens	Inhibition rate (%)
The inhibition rate was calculated after 4 days at 25 ℃, three replicates are set for each treatment; Average ± standard deviation.
Botryotinia fuckeliana	78.11±2.20
Thanatephorus cucumeris	0.00±0.00
Verticillium dahliae	0.00±0.00
Colletotrichum gloeosporioides	54.63±3.00
Colletrichum lagenarium	63.69±0.44
Rhizoctonia solani	0.00±0.00
Cochiobolus sativus	67.30±2.61
Botrytis cinerea	68.22±2.41

Treatment	Disease index	Control effect (%)
The control effects of B. subtilis C1R32 bacterial suspension and thiophanate-methyl were compared with pure water, and the control effect of B. subtilis C1R32 fermentation supernatant was compared with LB medium; Five replicates are set for each treatment; Average ± standard deviation.
Bacterial suspension	6.40±4.08	52.94
Fermentation supernatant	12.00±2.53	46.43
Thiophanate-methyl	0.00±0.00	100.00
LB	22.40±3.20	–
Water	13.60±4.08	–

Treatment	Disease index	Control effect (%)
The control effects of B. subtilis C1R32 bacterial suspension and thiophanate-methyl were compared with pure water, and the control effect of B. subtilis C1R32 fermentation supernatant was compared with LB medium; Five replicates are set for each treatment; Average ± standard deviation.
Bacterial suspension	6.40±4.08	52.94
Fermentation supernatant	12.00±2.53	46.43
Thiophanate-methyl	0.00±0.00	100.00
LB	22.40±3.20	–
Water	13.60±4.08	–

Contents of cell-free fermentation supernatant (%)	Inhibition rate (%)
The inhibition rate was calculated after 4 days at 25 ℃, three replicates are set for each treatment; Average ± standard deviation.
50	100.00±0.00
20	95.02±0.34
10	87.80±0.52
5	63.74±4.70
3	50.31±2.98
2	36.77±1.63

Contents of cell-free fermentation supernatant (%)	Inhibition rate (%)
The inhibition rate was calculated after 4 days at 25 ℃, three replicates are set for each treatment; Average ± standard deviation.
50	100.00±0.00
20	95.02±0.34
10	87.80±0.52
5	63.74±4.70
3	50.31±2.98
2	36.77±1.63

桑树内生枯草芽孢杆菌的分离鉴定及其对桑椹菌核病的生防机理

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李燕 ¹^,² , 欧婷 ¹^,² , 焦文莲 ¹^,² , 张克瑶 ¹^,² , 刘晓姣 ¹^,² , 谢洁 ¹^,²^,^*

微生物学报 | 研究报告 2024,64(9): 3253-3268

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微生物学报 | 研究报告 2024, 64(9): 3253-3268

桑树内生枯草芽孢杆菌的分离鉴定及其对桑椹菌核病的生防机理

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李燕¹^,², 欧婷¹^,², 焦文莲¹^,², 张克瑶¹^,², 刘晓姣¹^,², 谢洁¹^,²^,^*

作者信息

1 西南大学蚕桑纺织与生物质科学学院, 资源昆虫高效养殖与利用全国重点实验室, 重庆 400715

2 西南大学蚕桑纺织与生物质科学学院, 农业农村部蚕桑生物学与遗传育种重点实验室, 重庆 400715

Isolation and identification of an endophytic Bacillus subtilis from mulberry and preliminary exploration of its biocontrol mechanisms against mulberry fruit sclerotiniose

Yan LI¹^,², Ting OU¹^,², Wenlian JIAO¹^,², Keyao ZHANG¹^,², Xiaojiao LIU¹^,², Jie XIE¹^,²^,^*

Affiliations

1 State Key Laboratory of Resource Insects, College of Sericulture, Textile and Biomass Sciences, Southwest University, Chongqing 400715, China

2 Key Laboratory of Sericultural Biology and Genetic Breeding, Ministry of Agriculture and Rural Affairs, College of Sericulture, Textile and Biomass Sciences, Southwest University, Chongqing 400715, China

出版时间: 2024-06-14 doi: 10.13343/j.cnki.wsxb.20240100

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摘要

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【目的】从菌核病抗性桑树品种中筛选具有生防潜力的内生拮抗菌，为绿色防控桑椹菌核病提供优质菌种及有效策略。【方法】通过植物组织培养法及平板对峙培养法分离、筛选桑椹菌核病拮抗菌，根据形态学特征、生理生化特征和基于16S rRNA基因序列的系统发育分析对其进行菌种鉴定；分析拮抗菌抑菌谱及离体防效，评估其应用潜力；通过观察拮抗菌发酵上清液对病原菌菌丝生长的影响，检测拮抗菌作用后病原菌糖原和活性氧积累量变化，并测定病原菌基因表达变化，初步探究拮抗菌的抑菌机理。【结果】从健康桑枝中分离筛选出一株对桑椹菌核病菌(Sclerotinia sclerotiorum) PZ-2抑制作用强且稳定的内生细菌C1R32，其形态学和生理生化特征与芽孢杆菌一致，基于16S rRNA基因序列的系统发育分析显示该菌株与枯草芽孢杆菌聚在同一最小分支，将其鉴定为枯草芽孢杆菌(Bacillus subtilis)。B. subtilis C1R32对核盘菌在内的多种植物病原菌具有抑制作用；桑椹菌核病离体防效实验结果表明，B. subtilis C1R32菌悬液和发酵上清液处理组的防效分别为52.94%和46.43%；抑菌机理初探结果显示，B. subtilis C1R32发酵上清液能使S. sclerotiorum PZ-2菌丝膨大畸变，细胞壁破裂，胞质流出；该菌可通过减少S. sclerotiorum PZ-2糖原积累、促进活性氧迸发，影响其抗氧化相关基因的表达抑制病原菌。【结论】本研究从桑椹菌核病抗性品种中分离获得一株内生枯草芽孢杆菌，并初步探究其拮抗机理，为桑椹菌核病生物防治提供了潜在菌种资源。

关键词

桑树 / 内生枯草芽孢杆菌 / 桑椹菌核病 / 鉴定 / 生防机理

Abstract

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[Objective] To provide candidate strains and effective strategies for the control of mulberry fruit sclerotiniose, we screened out the endophytic bacteria with biocontrol potential for mulberry fruit sclerotiniose from a resistant mulberry cultivar. [Methods] The endophytic bacteria antagonistic to mulberry fruit sclerotiniose were isolated from mulberry plants by the tissue culture and confrontation culture methods. The antagonistic strain was identified based on morphological features, physiological and biochemical characteristics, and the phylogenetic relationship based on 16S rRNA gene sequences. The antimicrobial spectrum and control efficiency to detached mulberry fruits were determined to evaluate the application potential of the antagonistic strain. Furthermore, we observed the inhibitory effect of the fermentation supernatant of the strain on the mycelial growth of the pathogen, measured the variations in glycogen and reactive oxygen species accumulation of the pathogen treated with the antagonistic strain, and determined the expression of pathogen-related genes after treatment with the antagonistic strain to decipher the antagonistic mechanism of this strain. [Results] An endophytic bacterial strain C1R32 with strong and stable antagonistic activity on Sclerotinia sclerotiorum PZ-2 (the pathogen of mulberry fruit sclerotiniose) was isolated from a healthy mulberry branch. C1R32 showed similar morphological features and physiological and biochemical characteristics with Bacillus. The phylogenetic analysis based on 16S rRNA gene sequences revealed that C1R32 was located in the same clade with B. subtilis. Therefore, strain C1R32 was identified as B. subtilis. B. subtilis C1R32 had antagonistic activities against a variety of phytopathogens including S. sclerotiorum. The suspension and fermentation supernatant of B. subtilis C1R32 showed the control effects of 52.94% and 46.43%, respectively, on sclerotiniose of detached mulberry fruits. The cell-free fermentation supernatant of B. subtilis C1R32 caused the hypha swelling and distorting, cell wall breaking, and cytoplasm leakage of S. sclerotiorum PZ-2. Moreover, B. subtilis C1R32 inhibited S. sclerotiorum PZ-2 by reducing glycogen accumulation, promoting reactive oxygen species burst, and influencing the expression of genes associated with antioxidant activity. [Conclusion] We isolated an endophytic B. subtilis strain capable of controlling mulberry fruit sclerotiniose from a resistant mulberry cultivar and preliminarily explored its antagonistic mechanism, providing potential strain resources for the biocontrol of mulberry fruit sclerotiniose.

Key words

mulberry / endophytic Bacillus subtilis / mulberry fruit sclerotiniose / identification / biocontrol mechanism

引用本文

李燕, 欧婷, 焦文莲, 张克瑶, 刘晓姣, 谢洁. 桑树内生枯草芽孢杆菌的分离鉴定及其对桑椹菌核病的生防机理. 微生物学报, 2024 , 64 (9) : 3253 -3268 . DOI: 10.13343/j.cnki.wsxb.20240100

Yan LI, Ting OU, Wenlian JIAO, Keyao ZHANG, Xiaojiao LIU, Jie XIE. Isolation and identification of an endophytic Bacillus subtilis from mulberry and preliminary exploration of its biocontrol mechanisms against mulberry fruit sclerotiniose[J]. Acta Microbiologica Sinica, 2024 , 64 (9) : 3253 -3268 . DOI: 10.13343/j.cnki.wsxb.20240100

正文

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桑树(Morus L.)是桑科桑属植物，其果实桑椹为聚花果，极具药用和经济价值。桑椹中的活性化合物具有抗氧化、清除自由基、降糖降脂降血压、调节肠道微生物群、防治肿瘤等功效^[1-4]。以桑椹为主的鲜果销售和系列加工产品为各地桑农带来可观的经济收益，据统计，重庆市2015年果桑项目产值1 500万元以上，河南省2019年桑椹产值3亿元，四川省攀枝花市截至2021年底桑果累计收入1.71亿元，新疆维吾尔自治区阿克苏地区每年可实现桑椹收入2.5亿元^[5-8]。

桑椹菌核病是一种由桑实杯盘菌(Ciboria shiraiana)、核盘菌(Sclerotinia sclerotiorum)、桑椹核地杖菌(Scleromitula shiraiana)、肉阜状杯盘菌(Ciboria carunculoides)等病原真菌引起的桑椹病害，根据病原菌与病症的不同，可将桑椹菌核病分为肥大性菌核病、缩小性菌核病及小粒性菌核病^[9-13]。桑椹菌核病病原菌易于传播，若不妥善处理土壤中的病果及菌核，病原菌会逐年积累，给桑椹菌核病防治带来巨大困难^[14]。桑椹菌核病的防治措施有化学防治、农业防治、物理预防和生物防治。化学防治效果好，但存在农药残留、抗药性等问题；农业防治和物理预防绿色环保，但无法彻底清除病原菌，而且防治效果有限^[15-17]。近年来，生物防治因其绿色安全、高效持久等优点在桑椹菌核病防治方面备受关注。张健等^[18]的研究结果表明，田间喷洒哈茨木霉发酵液对桑椹菌核病的防治效果可达90%以上；Sultana等^[19]报道一株苏云金芽孢杆菌(B. thuringiensis) C25在田间条件下可减轻由C. shiraiana引起的桑椹菌核病；朱志贤等^[20]从桑椹中分离出一株贝莱斯芽孢杆菌(B. velezensis) Wh-1，其对S. shiraiana、C. carunculoides和C. shiraiana的抑菌率分别高达100.00%、100.00%和63.15%。

枯草芽孢杆菌作为芽孢杆菌属的典型代表，广泛分布于土壤、根际环境及植物各组织。近年来，因其对菌核病具有良好的生防潜力而受到广泛关注。朱孔艳等^[21]分离鉴定到一株向日葵菌核病生防内生菌B. subtilis Y116，该菌株在盆栽试验中可抑制向日葵发病。Monteiro等^[22]报道了一株对生菜菌核病有防治效果的B. subtilis，该菌株代谢物对S. sclerotiorum菌丝的生长有显著抑制作用。Cao等^[23]分离鉴定到一株B. subtilis RSS-1，其10⁸ CFU/mL悬浮液对油菜菌核病病原菌S. sclerotiorum的生物防治效果达到89.6%。本课题组前期从健康桑树中分离获得对S. shiraiana具有生防潜力的内生枯草芽孢杆菌，其展示了内生菌防治桑椹菌核病的巨大潜能^[12]。为获得对桑椹菌核病具有生防作用的优良菌株，丰富菌剂开发的菌种资源，本研究从桑树抗病品种‘川桑7637’中分离鉴定对核盘菌具有良好拮抗效果的内生菌B. subtilis C1R32，通过测定其抑菌谱及离体防效，并初步探究其抑菌机理，综合评估B. subtilis C1R32防治桑椹菌核病的潜力，为田间防治桑椹菌核病和开发生防制剂提供重要的菌种资源及研究基础。

1 材料与方法

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1.1 材料

1.1.1 植物和病原菌

桑枝：2022年秋采自重庆市蚕业科学技术研究院(29°48′58″N，106°24′51″E)，桑树品种为抗性品种‘川桑7637’。

病原菌：S. sclerotiorum PZ-2为本实验室分离保存，Botryotinia fuckeliana、Colletotrichum gloeosporioides、Colletrichum lagenarium、灰霉病菌(Botrytis cinerea)、旋孢腔菌(Cochiobolus sativus)、Thanatephorus cucumeri、大丽轮枝菌(Verticillium dahliae)、立枯丝核菌(Rhizoctonia solani)等为实验室收集保存菌株。

1.1.2 培养基

LB培养基(g/L)：胰蛋白胨10.0，酵母粉5.0，NaCl 10.0，琼脂15.0–20.0。

马铃薯葡萄糖琼脂(PDA)培养基(g/L)：马铃薯200.0，葡萄糖20.0，琼脂15.0–20.0。

PDB培养基(g/L)：马铃薯200.0，葡萄糖20.0。

胰酪大豆胨琼脂(tryptone soya agar, TSA)培养基(g/L)：胰蛋白胨15.0，大豆胨5.0，NaCl 5.0，琼脂15.0–20.0。

营养琼脂(nutrient agar, NA)培养基(g/L)：蛋白胨10.0，牛肉膏3.0，NaCl 5.0，琼脂15.0–20.0。

R2A琼脂培养基(g/L)：细菌蛋白胨0.5，可溶性淀粉0.5，酵母粉0.5，葡萄糖0.5，酸水解酪蛋白0.5，KH₂PO₄ 0.3，丙酮酸钠0.3，MgSO₄ 0.024，琼脂15.0–20.0。

以上培养基均采用121 ℃灭菌20 min。

1.1.3 主要试剂

KI-I₂染液：60 mg/mL KI溶液与10 mg/mL I₂溶液以体积比1:1混合。

200 mmol/L 4-羟乙基哌嗪乙磺酸(4-hydroxyethyl piperazine ethane sulfonic acid, HEPES)的配制：用1 mol/L NaOH溶液将1 mol/L HEPES溶液pH调至6.8–8.2，向20 mL 1 mol/L HEPES中加入80 mL水，得到200 mmol/L HEPES。

1.2 桑树内生拮抗菌的分离及筛选

1.2.1 内生菌分离纯化

利用植物组织培养法^[12]分离内生菌，具体方法：取健康的‘川桑7637’枝条，剪至5–6 cm长，置于75%乙醇中浸泡1–2 min，取出后于酒精灯火焰上燃尽表面乙醇，置于PDA培养基表面滚动数圈后28 ℃条件下培养，以验证表面灭菌是否彻底。随后用无菌刀片将其分三层，分别置于LB、TSA、NA、R2A培养基上28 ℃条件下培养，待植物组织周围长出菌落后，通过平板划线法分离纯化内生细菌。

1.2.2 桑椹菌核病拮抗菌初筛

利用平板对峙法^[24]筛选对S. sclerotiorum PZ-2有拮抗效果的内生菌。用打孔器取直径8 mm的S. sclerotiorum PZ-2菌饼，接种于培养基中央，在距中央2.5 cm处的4个方向分别划线接种不同内生菌，28 ℃培养，观察病原菌生长情况，筛选对S. sclerotiorum PZ-2具有拮抗效果的内生菌。

1.2.3 桑椹菌核病拮抗菌复筛

对初筛拮抗效果较好的拮抗菌进行平板对峙法复筛。用打孔器取直径5 mm的S. sclerotiorum PZ-2菌饼，接种于培养基中央，在距中央2 cm两侧对称处划线接种拮抗菌，以只接种病原菌的平板为对照，25 ℃培养4 d，测量与待测细菌划线垂直方向的病原真菌菌落直径，设置3个重复，并按公式(1)计算抑菌率。

(1)

1.3 桑椹菌核病拮抗菌的鉴定

1.3.1 形态学及生理生化特征鉴定

利用平板划线法将筛选获得的拮抗菌接种于PDA和LB培养基上，37 ℃培养12 h，观察其菌落形态。在LB上培养24 h和48 h后，分别进行革兰氏染色和芽孢染色。生理生化特征检测采用新型微生物微量生化鉴定管试剂盒(广东环凯微生物科技有限公司)。

1.3.2 基于16S rRNA基因序列的系统发育分析

参照文献[25]的方法提取目的菌株基因组，在1.5 mL离心管中加入30 μL DNA提取液(PrepMan Ultra Sample Preparation Reagent试剂盒) (Applied Biosystems公司)，挑取适量菌落与DNA提取液混合均匀，100 ℃金属浴10 min，之后12 000 r/min离心3 min，取20 μL上清液作为模板备用。

以目的菌株基因组为模板，利用16S rRNA基因通用引物27F (5′-AGAGTTTGATCCTGGC TCAG-3′)和1492R (5′-GGTTACCTTGTTACGA CTT-3′)扩增拮抗菌16S rRNA基因序列。PCR反应体系：上、下游引物(10 μmol/L)各1 μL，DNA模板1 μL，2×Rapid Taq Master Mix 12.5 μL，ddH₂O 9.5 μL。PCR反应条件：95 ℃ 3 min；95 ℃ 15 s，55 ℃ 15 s，72 ℃ 30 s，30个循环。将PCR扩增产物送至生工生物工程(上海)股份有限公司测序，将获得的基因序列在NCBI数据库(https://blast.ncbi.nlm.nih.gov/Blast.cgi)中进行比对，并利用MEGA 5.0软件基于16S rRNA基因序列构建系统发育树。

1.4 拮抗菌抑菌谱测定

以实验室保存的常见植物病原菌为靶标菌，用打孔器取直径为5 mm的菌饼，接种于培养基中央，在距离中央2 cm处左右两侧接种拮抗菌，每个处理设置3个重复，25 ℃培养，参照公式(1)计算抑菌率。

1.5 拮抗菌对桑椹菌核病的离体防效实验

参考文献[26-29]并微调后进行离体防效实验。

1.5.1 菌悬液及发酵上清液制备

挑取拮抗菌单菌落接种于10 mL LB液体培养基中，37 ℃、180 r/min培养过夜，将种子液接种至100 mL LB液体培养基，37 ℃、180 r/min培养过夜后，将发酵液12 000 r/min离心3 min收集菌体，用无菌水重悬到OD₆₀₀为0.6 (10⁸ CFU/mL)制成菌悬液。

将拮抗菌接种到LB液体培养基，37 ℃、180 r/min振荡培养4 d，取发酵液12 000 r/min离心30 min后，上清液过0.22 μm滤膜，获得无菌发酵上清液。

1.5.2 离体防效实验

将新鲜桑椹置于75%乙醇浸泡20–30 s，无菌水洗去乙醇，无菌滤纸吸干水分；无菌针头刺破桑椹后分别在纯水、LB液体培养基、拮抗菌菌悬液、拮抗菌发酵上清液、甲基硫菌灵(稀释750倍)中浸泡30 min，取出置于培养皿自然晾干，取3块直径为8 mm的S. sclerotiorum PZ-2菌饼接种在桑椹上针头刺破处，保鲜膜包裹后放入组培瓶，室温(15–20 ℃)静置培养^[26]。每个处理设置5个生物学重复，每个生物学重复包含5个桑椹。室温培养2 d后取下保鲜膜^[27]，去除菌饼，观察记录并按公式(2)^[28]计算病情指数，菌悬液处理组和甲基硫菌灵以纯水为对照，发酵上清液处理组以LB液体培养基为对照，均按公式(3)计算防效^[29]。

(2)

(3)

根据处理后桑椹发病时的表面菌丝覆盖率，将发病桑椹进行分级：0级，无发病桑椹；1级，桑椹表面菌丝覆盖率0%–25%；2级，桑椹表面菌丝覆盖率25%–50%；3级，桑椹表面菌丝覆盖率50%–75%；4级，桑椹表面菌丝覆盖率75%–100%。

1.6 拮抗菌抑菌机理初探

1.6.1 拮抗菌发酵上清液对病原菌菌丝生长抑制作用

将拮抗菌接种到PDB培养基中，37 ℃、180 r/min培养4 d后，取发酵液12 000 r/min离心30 min，随后上清液过0.22 μm滤膜，获得无菌发酵上清液。

利用菌丝生长速率法^[24]测定拮抗菌无菌发酵上清液对桑椹菌核病原菌S. sclerotiorum PZ-2的抑制效果。将病原菌S. sclerotiorum PZ-2分别接种至含有50%、20%、10%、5%、3%和2%拮抗菌无菌发酵上清液的PDA平板，25 ℃培养48 h，以未添加无菌发酵上清液的PDA平板为对照，参照公式(1)计算抑菌率，并在显微镜下观察S. sclerotiorum PZ-2菌丝形态。

1.6.2 拮抗菌对病原菌糖原积累的影响

通过糖原染色^[30]测定拮抗菌对病原菌糖原积累的影响。在铺有玻璃纸的PDA平板中央接种直径5 mm的S. sclerotiorum PZ-2菌饼，距中心2 cm处两侧接种拮抗菌，取25 ℃对峙培养1、2、3、5 d后的S. sclerotiorum PZ-2菌丝置于载玻片上，用KI-I₂染液染色1 min后盖上盖玻片，置于显微镜下观察，颜色越深代表菌丝糖原含量越多。以在PDA平板上培养相同时间的S. sclerotiorum PZ-2作为对照。

1.6.3 拮抗菌对病原菌胞内活性氧的影响

采用DCFH-DA活性氧荧光探针^[31-32]测定对峙培养1、2、3、5 d的S. sclerotiorum PZ-2菌丝内活性氧含量。将S. sclerotiorum PZ-2菌丝置于200 mmol/L HEPES缓冲液中静置浸润20 min后，转移至15 μmol/L的DCFH-DA溶液(北京鼎国昌盛生物技术有限责任公司)，25 ℃黑暗标记1 h，制片，置于荧光显微镜下观察，激发波长495 nm，发射波长515 nm。

1.6.4 拮抗菌对病原菌基因表达的影响

根据1.6.1、1.6.2和1.6.3实验结果，利用实时荧光定量PCR检测拮抗菌对S. sclerotiorum PZ-2基因组中糖代谢、抗氧化酶和细胞壁成分相关基因表达情况的影响。

按1.6.2方法收集病原菌菌丝，依据Omega Fungal RNA Kit R6840 (Omega Bio-Tek公司)说明书提取S. sclerotiorum PZ-2菌丝总RNA。

利用一步反转试剂盒(南京诺唯赞生物科技股份有限公司)将RNA反转为cDNA。反转体系：5×All-in-one qRT SuperMix 4 μL，Enzyme Mix 1 μL，RNA template 1 μg，RNase-free ddH₂O补足20 μL。反应程序：50 ℃ 15 min；85 ℃ 5 s。

参考NCBI数据库(https://www.ncbi.nlm.nih.gov/)中S. sclerotiorum基因组选取与糖代谢有关的基因Gene_9536 (己糖激酶)、Gene_3455 (糖基转移酶家族20)、Gene_7772 (葡萄糖磷酸变位酶)，与病原菌抗氧化相关的基因Gene_5305 (过氧化氢酶相关的免疫反应性)、Gene_4935 (过氧化氢酶)、Gene_1287 (过氧化物酶体)，与细胞壁组分合成相关的基因Gene_13 (糖基转移酶、纤维素酶)、Gene_11361 (几丁质合成酶2)、Gene_622 (细胞壁)。利用软件Primer 5设计以上基因引物，内参基因和各基因的引物序列如表1所示。

实时荧光定量PCR：每个样品设置3个生物学重复，每个生物学重复含3次技术重复。反应体系：TB Green Premix Ex Taq II (2×) 10 µL，正、反向引物(10 µmol/L)各0.8 µL，cDNA模板5 µL，RNase-free ddH₂O 3.4 µL。反应程序：95 ℃ 30 s；95 ℃ 10 s，40个循环；60 ℃ 30 s。

1.7 数据统计及分析

数据均采用Excel 2018进行分析计算，利用GraphPad Prism 8中t检验对相对基因表达量进行显著性分析并作图。

2 结果与分析

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2.1 桑椹菌核病拮抗菌的分离筛选

从‘川桑7637’枝条中分离纯化获得98株内生细菌，初筛结果显示有18株内生细菌对S. sclerotiorum PZ-2有拮抗效果，复筛发现内生细菌C1R32、C3T5、C3N15、C3N16、C1R38、C3R1-2对病原菌PZ-2有较好的抑制效果(图1A)，其中C1R32抑菌效果稳定且最佳，抑菌率达66.47% (图1A、1B)，因此选择C1R32进行后续研究。

2.2 桑椹菌核病拮抗菌的鉴定

2.2.1 形态学和生理生化特征

C1R32菌株在PDA和LB培养基中37 ℃培养12 h后，其生长形态无明显差别，均展现出快速生长特性，单菌落隆起，呈饱满的圆形，乳白色，边缘光滑，表面有光泽(图2A、2B)；革兰氏染色和芽孢染色结果显示C1R32菌株为革兰氏阳性杆状细菌(图2C)，产椭圆形芽孢(图2D)；生理生化指标鉴定结果显示C1R32能利用葡萄糖，水解淀粉、明胶，还原硝酸盐，有接触酶，Voges-Proskauer试验呈阳性(表2)，参照《常见细菌系统鉴定手册》^[33]初步将其鉴定为芽孢杆菌(Bacillus sp.)。

2.2.2 基于16S rRNA基因序列的系统发育分析

扩增Bacillus sp. C1R32的16S rRNA基因序列，测序获得长为1 397 bp的片段，其在NCBI数据库上BLAST比对结果显示，该片段与Bacillus subtilis菌株(登录号为OQ586633.1)的16S rRNA基因序列相似度达到了100%。基于16S rRNA基因序列的系统发育分析发现，Bacillus sp. C1R32与B. subtilis NBRC 13719 (NR112629.1)和B. subtilis JCM 1465 (NR113265.1)聚在同一最小分支(图3)，因此将C1R32鉴定为枯草芽孢杆菌，将其命名为B. subtilis C1R32。

2.3 拮抗菌抑菌谱测定

抑菌谱检测结果显示，B. subtilis C1R32对多种植物病原菌具有抑制作用，其对Botryotinia fuckeliana抑菌率高达78.11%，对Colletrichum lagenarium、Botrytis cinerea、Cochiobolus sativus抑制率超过60.00%，对Colletotrichum gloeosporioides抑制率也超过50.00%，表明B. subtilis C1R32具有一定的广谱抑菌能力(表3)。

2.4 拮抗菌对桑椹菌核病的离体防效实验

B. subtilis C1R32对桑椹菌核病的离体防效检测结果表明，接种病原菌后第2天，纯水和LB液体培养基对照处理的桑椹均发病严重。B. subtilis C1R32菌悬液处理组桑椹病情指数为6.40，以纯水处理组(病情指数为13.60)为对照计算其防效为52.94%。B. subtilis C1R32发酵上清液处理组病情指数为12.00，以LB液体培养基处理组(病情指数为22.40)为对照计算其防效为46.43%。整体而言，B. subtilis C1R32对桑椹菌核病具有良好离体防效，并且菌悬液防效优于发酵上清液防效(表4)。

2.5 拮抗菌抑菌机理初探

2.5.1 拮抗菌发酵上清液对病原菌菌丝生长抑制作用

病原菌在不同含量B. subtilis C1R32发酵上清液的PDA平板上均明显受到抑制，S. sclerotiorum PZ-2在含50%发酵上清液PDA平板上生长完全被抑制，虽然随着PDA平板中B. subtilis C1R32发酵上清液含量减少抑菌率逐渐下降，但含2%发酵上清液的PDA平板仍对S. sclerotiorum PZ-2有36.77%的抑制率(表5)。

光学显微镜观察B. subtilis C1R32发酵上清液对病原菌菌丝的生长抑制作用，结果显示，未用发酵上清液处理的病原菌菌丝粗壮，边缘整齐，尖端圆润，菌丝内容物均匀，菌丝呈现旺盛的生长趋势(图4A)。接种在含20%发酵上清液PDA平板上的S. sclerotiorum PZ-2菌丝变小，菌丝尖端膨大，内容物大量流出，菌丝生长滞缓(图4B)；接种在含10%发酵上清液PDA平板上的S. sclerotiorum PZ-2菌丝断裂，尖端不生长，出现菌丝膨大现象(图4C，黄色箭头所指)；接种在含5%发酵上清液PDA平板上的S. sclerotiorum PZ-2菌丝与对照组相比显著缩小，并呈现异常的弯折(图4D，蓝色箭头所指)；接种在含3%和2%发酵上清液PDA平板上的S. sclerotiorum PZ-2菌丝细小，显示出异常的弯折生长(图4E、4F，蓝色箭头所指)。不同浓度B. subtilis C1R32发酵上清液处理后的病原菌菌丝生长均被抑制，并且发酵上清液含量较高时菌丝损伤较严重。

2.5.2 拮抗菌对病原菌糖原积累的影响

利用KI-I₂染色评估B. subtilis C1R32对S. sclerotiorum PZ-2糖原积累的影响。结果显示，KI-I₂染色后，对照组PZ-2菌丝颜色随培养时间延长而加深(图5中A2)，对峙培养的PZ-2菌丝颜色随培养时间的延长越来越浅(图5中B2)，而且均比对照组颜色浅。此外，对峙培养的PZ-2菌丝细胞质减少，细胞破碎呈空壳状(图5，绿色箭头所指)，隔膜明显增宽(图5中B1，蓝色箭头所指)，并且随着对峙培养时间的延长，菌丝损伤程度逐渐增加，而对照组PZ-2菌丝并未出现明显损伤，结构完整，胞质均匀(图5中A1)。上述结果表明，B. subtilis C1R32影响S. sclerotiorum PZ-2糖代谢，使病原菌糖原积累量减少，同时影响S. sclerotiorum PZ-2细胞结构，使菌丝损伤。

2.5.3 拮抗菌对病原菌胞内活性氧的影响

通过DCFH-DA活性氧荧光探针染色测定B. subtilis C1R32对S. sclerotiorum PZ-2胞内活性氧的影响，结果显示，对照组菌丝呈现较弱绿色荧光(图6中A2)；与C1R32对峙培养1、2、3 d的PZ-2菌丝荧光强度均强于对照组(图6中B2)，对峙培养第2天的荧光最强，活性氧迸发后PZ-2菌丝出现损伤，呈现碎片状，至第5天则无法观察到荧光。此外，对照组菌丝均无明显破损(图6中A1)，而与C1R32对峙培养的PZ-2菌丝损伤(图6中B1)。活性氧探针染色结果表明在作用早期，B. subtilis C1R32促使S. sclerotiorum PZ-2活性氧水平升高，产生氧化胁迫，导致其细胞损伤。

2.5.4 拮抗菌对病原菌基因表达的影响

由于拮抗菌影响S. sclerotiorum PZ-2糖原合成、活性氧爆发和细胞壁结构，因此通过实时荧光定量PCR对S. sclerotiorum PZ-2糖代谢、抗氧化酶和细胞壁成分相关基因的相对表达情况进行分析。结果显示，对峙培养24 h后，S. sclerotiorum PZ-2的糖代谢相关基因有下调表达的趋势，Gene_9536 (己糖激酶)相对表达量为对照组的80.00%，Gene_7772 (葡萄糖磷酸变位酶)表达量仅为对照组的34.00%，出现显著下调(P < 0.001)；此外，病原菌的抗氧化相关基因会上调表达，其中Gene_5305 (过氧化氢酶相关的免疫反应性)表达量为对照组的3倍，Gene_4935 (过氧化氢酶)表达量为对照组的3.4倍，均显著上调(P < 0.05)，Gene_1287 (过氧化物酶体)的表达量显著(P < 0.001)下调为对照组的51.00%；S. sclerotiorum PZ-2细胞壁组分相关基因Gene_13 (糖基转移酶、纤维素酶)、Gene_622 (细胞壁)的表达量与对照组相比分别上调了47.15%和78.75%，Gene_11361 (几丁质合成酶2)的表达略有下调(图7)。基因表达量分析结果表明，B. subtilis C1R32会影响S. sclerotiorum PZ-2糖代谢相关基因表达，诱导S. sclerotiorum PZ-2抗氧化基因和细胞壁成分相关基因上调表达，这也与B. subtilis C1R32和S. sclerotiorum PZ-2对峙培养后的糖原染色和活性氧染色结果一致。

3 讨论与结论

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桑椹菌核病发病率高，防治困难，极大影响桑果产量与品质。利用健康桑树内生菌进行桑椹菌核病的绿色防控，不仅有利于促进产业健康发展，更能满足人们的食品安全要求。本研究分离获得一株对桑椹菌核病病原菌具稳定拮抗作用的内生细菌B. subtilis C1R32，该菌株发酵上清液可显著抑制桑椹菌核病病原菌S. sclerotiorum PZ-2生长，破坏病原菌细胞结构。桑椹离体防效实验结果表明，B. subtilis C1R32能抑制S. sclerotiorum PZ-2扩散生长，延缓桑果发病，具有良好的生防潜力(表4)。B. subtilis C1R32抑菌机理初探结果表明，B. subtilis C1R32通过促进病原菌活性氧增加、产生氧化胁迫，损伤细胞壁，并影响病原菌糖代谢基因表达，通过减少病原菌糖原合成等途径来抑制S. sclerotiorum PZ-2生长(图7)。

众多研究表明，内生菌可以通过影响病原真菌细胞结构、代谢物合成及氧化应激来抑制病原真菌，从而达到防治植物真菌病害的目的^[34-35]。细胞壁是病原真菌的重要细胞结构，生防菌破坏病原菌细胞壁是防控病害的重要策略。Wang等^[36]分离到一株内生莫海威芽孢杆菌(Bacillus mojavensis) BQ-33，该菌株对猕猴桃黑斑病菌(Didymella glomerata)有良好的拮抗作用，能破坏病原菌菌丝体细胞壁，使大量无机盐、蛋白质和核酸外渗。同样，本研究中B. subtilis C1R32处理S. sclerotiorum PZ-2后，病原菌纤维素酶相关基因上调表达，引起病原菌细胞壁破碎，导致细胞质大量流出。氧化应激是指机体活性氧产生和清除失衡，造成活性氧积累，从而诱导细胞产生细胞毒素，进而对细胞结构和生物大分子造成损伤，诱导病原菌发生氧化应激，其也为生防菌防治植物病害的重要机制^[34-35]。Lu等^[37]报道了一株B. subtilis BS45的甲醇提取物会引起小麦赤霉病菌(Fusarium graminearum)菌丝活性氧水平增加，进而诱导菌丝细胞氧化应激相关基因表达水平增加。这与本研究中B. subtilis C1R32对S. sclerotiorum PZ-2的抑制结果相同，B. subtilis C1R32促进S. sclerotiorum PZ-2活性氧迸发，产生氧化胁迫，促进病原菌凋亡。Han等^[38]从棉花中分离筛选出一株对棉花黄萎病菌(Verticillium dahliae)有拮抗效果的解淀粉芽孢杆菌(B. amyloliquefaciens) 41B-1，发现其代谢产物伊枯草菌素(iturin)可通过诱导V. dahliae活性氧升高从而达到杀菌作用。Su等^[39]报道侧孢短芽孢杆菌(Brevibacillus laterosporus) SN19-1无菌上清液处理水稻白叶枯病菌(Xanthomonas oryzae pv. Oryzae)后，病原菌胞内活性氧水平显著增加，致病力减弱。同时，生防菌还可以通过影响病原微生物代谢达到防治病害的作用。Hu等^[40]通过转录组测序发现，B. amyloliquefaciens A-1能通过紊乱黄曲霉(Aspergillus flavus) NRRL 3357的淀粉和蔗糖代谢途径基因表达来拮抗黄曲霉，其在减少梨、花生和玉米腐败方面表现出显著的潜力。张婷婷等^[41]报道，B. velezensis SX-45提取物可以显著减少人参根腐病原菌(Fusarium oxysporum)还原糖含量。本研究揭示了B. subtilis C1R32能够通过影响S. sclerotiorum PZ-2糖代谢相关基因表达，减少糖原积累，诱导S. sclerotiorum PZ-2氧化应激，以及损伤细胞结构等起到防治桑椹菌核病菌的作用，结果显示B. subtilis C1R32具有良好的生物防治潜能。后续研究将进一步鉴定B. subtilis C1R32菌株抑菌活性物质的主要成分，确定菌株的生物安全性及评估B. subtilis C1R32的田间防治效果的基础上，制备活菌制剂，将其应用于桑椹菌核病的生防实践。

基金

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国家自然科学基金(32371713)
西南大学大学生创新创业训练计划(X202310635221)
重庆市自然科学基金(CSTB2022NSCQ-MSX0536)

参考文献

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文献

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2024年第64卷第9期

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doi: 10.13343/j.cnki.wsxb.20240100

接收时间：2024-02-08
首发时间：2026-03-20
出版时间：2024-06-14

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出版历史

收稿日期：2024-02-08
录用日期：2024-06-12

基金

National Natural Science Foundation of China(32371713)

国家自然科学基金(32371713)

Southwest University College Students' Innovation and Entrepreneurship Training Program(X202310635221)

西南大学大学生创新创业训练计划(X202310635221)

Natural Science Foundation of Chongqing(CSTB2022NSCQ-MSX0536)

重庆市自然科学基金(CSTB2022NSCQ-MSX0536)

作者信息

1 西南大学蚕桑纺织与生物质科学学院, 资源昆虫高效养殖与利用全国重点实验室, 重庆 400715

2 西南大学蚕桑纺织与生物质科学学院, 农业农村部蚕桑生物学与遗传育种重点实验室, 重庆 400715

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2种不同金属材料的力学参数

科 Family	属数 Number of genus	种数 Number of species	占总种数比例 Percentage of total species (%)	属 Genus	种数 Number of species	占总种数比例 Percentage of total species (%)
鹅膏菌科Amanitaceae	2	11	5.26	鹅膏菌属 Amanita	10	4.78
小菇科 Mycenaceae	2	12	5.74	丝盖伞属 Inocybe	5	2.39
多孔菌科 Polyporaceae	8	14	6.70	蜡蘑属 Laccaria	5	2.39
红菇科 Russulaceae	3	23	11.00	小皮伞属 Marasmius	6	2.87
				小菇属 Mycena	11	5.26
				光柄菇属 Pluteus	5	2.39
				红菇属 Russula	17	8.13
				栓菌属 Trametes	5	2.39

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TxT

Genes name	Sequences of primer (5′→3′)	Functional description
actin	F: GAGCTGTTTTCCCTTCCATTGTC R: GACGACACCGTGCTCGATTGG	Actin
Gene_9536	F: TTCAGTTTCCCAGTTCA R: GCCATCAAGGTTCCCAC	Hexokinase
Gene_3455	F: CGTCCTACATTTATCAACA R: ATCCACGTCAAGATCACTA	Glycosyltransferase family 20
Gene_7772	F: GCCCGCAGTATGCCAACA R: GCGACGACAGCCCAAAGA	Phosphoglucomutase
Gene_5305	F: GGTCCAGATGTCATTCGT R: CCTCTTTGTTCACCCACT	Catalase-related immune-responsive
Gene_4935	F: CCAGTTGATTTCCAGCAT R: CTTCATAAAGGCGACGAG	Catalase
Gene_1287	F: GAATCGGTAGCAAACTCC R: CCGCCTTCTATCATCTCG	Peroxisome
Gene_13	F: ACATTGGTAGCCGTGGTT R: GTCATTGATGGCGTAGAA	Glucanosyltransferase; cellulase
Gene_11361	F: GCTCATCGCCGTCACTTA R: GGTCCACCGACATTCCAG	Chitin synthase 2
Gene_622	F: CGCTCGCAAGTGAAGTA R: GTGCCATTTGTCCCTAA	Cell wall

Genes name

Sequences of primer (5′→3′)

Functional description

actin

F: GAGCTGTTTTCCCTTCCATTGTC
R: GACGACACCGTGCTCGATTGG

Actin

Gene_9536

F: TTCAGTTTCCCAGTTCA
R: GCCATCAAGGTTCCCAC

Hexokinase

Gene_3455

F: CGTCCTACATTTATCAACA
R: ATCCACGTCAAGATCACTA

Glycosyltransferase family 20

Gene_7772

F: GCCCGCAGTATGCCAACA
R: GCGACGACAGCCCAAAGA

Phosphoglucomutase

Gene_5305

F: GGTCCAGATGTCATTCGT
R: CCTCTTTGTTCACCCACT

Catalase-related immune-responsive

Gene_4935

F: CCAGTTGATTTCCAGCAT
R: CTTCATAAAGGCGACGAG

Catalase

Gene_1287

F: GAATCGGTAGCAAACTCC
R: CCGCCTTCTATCATCTCG

Peroxisome

Gene_13

F: ACATTGGTAGCCGTGGTT
R: GTCATTGATGGCGTAGAA

Glucanosyltransferase; cellulase

Gene_11361

F: GCTCATCGCCGTCACTTA
R: GGTCCACCGACATTCCAG

Chitin synthase 2

Gene_622

F: CGCTCGCAAGTGAAGTA
R: GTGCCATTTGTCCCTAA

Cell wall

Item tested	Results
+: Positive (growth or reaction); –: Negative (no growth or no reaction).
Xylose	–
Mannitol	–
Glucose	+
Starch hydrolysis	+
Catalase	+
Nitrate reduction	+
Citrate	–
Voges-Proskauer test	+
Gelatin liquefaction	+

Item tested

Results

+: Positive (growth or reaction); –: Negative (no growth or no reaction).

Xylose

–

Mannitol

–

Glucose

Starch hydrolysis

Catalase

Nitrate reduction

Citrate

–

Voges-Proskauer test

Gelatin liquefaction

Pathogens	Inhibition rate (%)
The inhibition rate was calculated after 4 days at 25 ℃, three replicates are set for each treatment; Average ± standard deviation.
Botryotinia fuckeliana	78.11±2.20
Thanatephorus cucumeris	0.00±0.00
Verticillium dahliae	0.00±0.00
Colletotrichum gloeosporioides	54.63±3.00
Colletrichum lagenarium	63.69±0.44
Rhizoctonia solani	0.00±0.00
Cochiobolus sativus	67.30±2.61
Botrytis cinerea	68.22±2.41

Pathogens

Inhibition rate (%)

The inhibition rate was calculated after 4 days at 25 ℃, three replicates are set for each treatment; Average ± standard deviation.

Botryotinia fuckeliana

78.11±2.20

Thanatephorus cucumeris

0.00±0.00

Verticillium dahliae

0.00±0.00

Colletotrichum gloeosporioides

54.63±3.00

Colletrichum lagenarium

63.69±0.44

Rhizoctonia solani

0.00±0.00

Cochiobolus sativus

67.30±2.61

Botrytis cinerea

68.22±2.41

Treatment	Disease index	Control effect (%)
The control effects of B. subtilis C1R32 bacterial suspension and thiophanate-methyl were compared with pure water, and the control effect of B. subtilis C1R32 fermentation supernatant was compared with LB medium; Five replicates are set for each treatment; Average ± standard deviation.
Bacterial suspension	6.40±4.08	52.94
Fermentation supernatant	12.00±2.53	46.43
Thiophanate-methyl	0.00±0.00	100.00
LB	22.40±3.20	–
Water	13.60±4.08	–

Treatment

Disease index

Control effect (%)

The control effects of B. subtilis C1R32 bacterial suspension and thiophanate-methyl were compared with pure water, and the control effect of B. subtilis C1R32 fermentation supernatant was compared with LB medium; Five replicates are set for each treatment; Average ± standard deviation.

Bacterial suspension

6.40±4.08

52.94

Fermentation supernatant

12.00±2.53

46.43

Thiophanate-methyl

0.00±0.00

100.00

22.40±3.20

–

Water

13.60±4.08

–

Contents of cell-free fermentation supernatant (%)	Inhibition rate (%)
The inhibition rate was calculated after 4 days at 25 ℃, three replicates are set for each treatment; Average ± standard deviation.
50	100.00±0.00
20	95.02±0.34
10	87.80±0.52
5	63.74±4.70
3	50.31±2.98
2	36.77±1.63

Contents of cell-free fermentation supernatant (%)

Inhibition rate (%)

The inhibition rate was calculated after 4 days at 25 ℃, three replicates are set for each treatment; Average ± standard deviation.

100.00±0.00

95.02±0.34

87.80±0.52

63.74±4.70

50.31±2.98

36.77±1.63