微生物学报

Primer name	Application	Primer sequences (5′→3′)	Fragment size (bp)
AU759f	Species-specific primers of R. solanacearum	GTCGCCGTCAACTCACTTTCC	280
AU760r		GTCGCCGTCAGCAATGCGGAATCG
Nmult21:1F	The forward primer for phylotype Ⅰ	CGTTGATGAGGCGCGCAATTT	144
Nmult21:2F	The forward primer for phylotype Ⅱ	AAGTTATGGACGGTGGAAGTC	372
Nmult23:AF	The forward primer for phylotype Ⅲ	ATTACSAGAGCAATCGAAAGATT	91
Nmult22:InF	The forward primer for phylotype Ⅳ	ATTGCCAAGACGAGAGAAGTA	213
Nmult22:RR	The reversed primer for phylotype identification	TCGCTTGACCCTATAACGAGTA
Endo-F	The primers for egl sequevar identification	ATGCATGCCGCTGGTCGCCGC	840
Endo-R		GCGTTGCCCGGCACGAACACC

Primer name	Application	Primer sequences (5′→3′)	Fragment size (bp)
AU759f	Species-specific primers of R. solanacearum	GTCGCCGTCAACTCACTTTCC	280
AU760r		GTCGCCGTCAGCAATGCGGAATCG
Nmult21:1F	The forward primer for phylotype Ⅰ	CGTTGATGAGGCGCGCAATTT	144
Nmult21:2F	The forward primer for phylotype Ⅱ	AAGTTATGGACGGTGGAAGTC	372
Nmult23:AF	The forward primer for phylotype Ⅲ	ATTACSAGAGCAATCGAAAGATT	91
Nmult22:InF	The forward primer for phylotype Ⅳ	ATTGCCAAGACGAGAGAAGTA	213
Nmult22:RR	The reversed primer for phylotype identification	TCGCTTGACCCTATAACGAGTA
Endo-F	The primers for egl sequevar identification	ATGCATGCCGCTGGTCGCCGC	840
Endo-R		GCGTTGCCCGGCACGAACACC

Pathogen of ginger bacterial wilt	Tuber of diseased ginger distribution number (×10³ CFU/g)	Rhizosphere soil of diseased ginger distribution number (×10³ CFU/g)
Data in the table are mean±SD, and the data are the mean of three replicates. Different lowercase and capital letters in the same column show values that are significantly different at the P<0.05 and P<0.01 level, respectively, by the least significant difference (LSD) test.
Enterobacter mori	1.33±0.58b	0.00±0.00B
Ralstonia solanacearum	5.67±2.08a	216.67±35.12A

Pathogen of ginger bacterial wilt	Tuber of diseased ginger distribution number (×10³ CFU/g)	Rhizosphere soil of diseased ginger distribution number (×10³ CFU/g)
Data in the table are mean±SD, and the data are the mean of three replicates. Different lowercase and capital letters in the same column show values that are significantly different at the P<0.05 and P<0.01 level, respectively, by the least significant difference (LSD) test.
Enterobacter mori	1.33±0.58b	0.00±0.00B
Ralstonia solanacearum	5.67±2.08a	216.67±35.12A

Strain number	Species	Antibacterial circle diameter against E. mori (mm)	Antibacterial circle diameter against R. solanacearum (mm)
Data in the table are mean±SD, and the data are the mean of five replicates.
FJAT-54970	Bacillus megaterium	0.00±0.00	0.00±0.00
FJAT-54974		0.00±0.00	0.00±0.00
FJAT-54978		0.00±0.00	0.00±0.00
FJAT-54986		0.00±0.00	0.00±0.00
FJAT-54989		0.00±0.00	0.00±0.00
FJAT-2349	Bacillus velezensis	0.00±0.00	0.00±0.00
FJAT-17531		0.00±0.00	0.00±0.00
FJAT-51047		0.00±0.00	0.00±0.00
FJAT-52631		0.00±0.00	0.00±0.00
FJAT-54560		16.11±0.15	0.00±0.00
FJAT-54972		0.00±0.00	0.00±0.00
FJAT-19700	Bacillus tequilens	0.00±0.00	0.00±0.00
FJAT-54984		0.00±0.00	0.00±0.00
FJAT-54973	Bacillus thuringiensis	0.00±0.00	0.00±0.00
FJAT-54975	Bacillus safensis	0.00±0.00	0.00±0.00
FJAT-54979	Bacillus licheniformis	0.00±0.00	0.00±0.00
FJAT-54981	Bacillus amyloliquefaciens	0.00±0.00	0.00±0.00
FJAT-54985	Bacillus aryabhattai	0.00±0.00	0.00±0.00
FJAT-54987	Bacillus pumilus	0.00±0.00	0.00±0.00
FJAT-20261	Brevibacterium frigoritolerans	0.00±0.00	0.00±0.00
FJAT-JK-2	Brevibacillus brevis	0.00±0.00	19.41±0.14
Streptomycin as control		19.56±0.32	23.14±0.46

Strain number	Species	Antibacterial circle diameter against E. mori (mm)	Antibacterial circle diameter against R. solanacearum (mm)
Data in the table are mean±SD, and the data are the mean of five replicates.
FJAT-54970	Bacillus megaterium	0.00±0.00	0.00±0.00
FJAT-54974		0.00±0.00	0.00±0.00
FJAT-54978		0.00±0.00	0.00±0.00
FJAT-54986		0.00±0.00	0.00±0.00
FJAT-54989		0.00±0.00	0.00±0.00
FJAT-2349	Bacillus velezensis	0.00±0.00	0.00±0.00
FJAT-17531		0.00±0.00	0.00±0.00
FJAT-51047		0.00±0.00	0.00±0.00
FJAT-52631		0.00±0.00	0.00±0.00
FJAT-54560		16.11±0.15	0.00±0.00
FJAT-54972		0.00±0.00	0.00±0.00
FJAT-19700	Bacillus tequilens	0.00±0.00	0.00±0.00
FJAT-54984		0.00±0.00	0.00±0.00
FJAT-54973	Bacillus thuringiensis	0.00±0.00	0.00±0.00
FJAT-54975	Bacillus safensis	0.00±0.00	0.00±0.00
FJAT-54979	Bacillus licheniformis	0.00±0.00	0.00±0.00
FJAT-54981	Bacillus amyloliquefaciens	0.00±0.00	0.00±0.00
FJAT-54985	Bacillus aryabhattai	0.00±0.00	0.00±0.00
FJAT-54987	Bacillus pumilus	0.00±0.00	0.00±0.00
FJAT-20261	Brevibacterium frigoritolerans	0.00±0.00	0.00±0.00
FJAT-JK-2	Brevibacillus brevis	0.00±0.00	19.41±0.14
Streptomycin as control		19.56±0.32	23.14±0.46

Treatments	Disease incidence (%)	Control efficiency (%)
The data are mean±SD, and the data are the mean of three replicates. −: Indicates unable to calculate.
TR1	30.55±4.81	69.45±4.81
TR2	38.89±4.82	61.11±4.82
CK1	100.00±0.00	−
CK2	100.00±0.00	−

Treatments	Disease incidence (%)	Control efficiency (%)
The data are mean±SD, and the data are the mean of three replicates. −: Indicates unable to calculate.
TR1	30.55±4.81	69.45±4.81
TR2	38.89±4.82	61.11±4.82
CK1	100.00±0.00	−
CK2	100.00±0.00	−

姜瘟致病菌的分离鉴定及其生防芽胞杆菌的筛选

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郑雪芳 ¹^,^# , 舒江霞 ²^,^# , 王阶平 ¹ , 杨晨 ³ , 车建美 ¹ , 肖荣凤 ¹ , 陈梅春 ¹ , 刘波 ¹^,^*

微生物学报 | 研究报告 2025,65(1): 136-149

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微生物学报 | 研究报告 2025, 65(1): 136-149

姜瘟致病菌的分离鉴定及其生防芽胞杆菌的筛选

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郑雪芳¹^,^#, 舒江霞²^,^#, 王阶平¹, 杨晨³, 车建美¹, 肖荣凤¹, 陈梅春¹, 刘波¹^,^*

作者信息

1 福建省农业科学院资源环境与土壤肥料研究所, 福建福州 350003

2 中福海峡平潭发展股份有限公司, 福建福州 350000

3 湖南农业大学植物保护学院, 湖南长沙 410125

Isolation and identification of the causal organisms of ginger bacterial wilt and screening of Bacillus strains for biocontrol

Xuefang ZHENG¹^,^#, Jiangxia SHU²^,^#, Jieping WANG¹, Chen YANG³, Jianmei CHE¹, Rongfeng XIAO¹, Meichun CHEN¹, Bo LIU¹^,^*

Affiliations

1 Institute of Resources, Environment and Soil Fertilizer, Fujian Academy of Agricultural Sciences, Fuzhou 350003, Fujian, China

2 Zhongfu Straits (Pingtan) Development Co., Ltd., Fuzhou 350000, Fujian, China

3 College of Plant Protection, Hunan Agricultural University, Changsha 410125, Hunan, China

出版时间: 2025-01-04 doi: 10.13343/j.cnki.wsxb.20240502

文章导航

摘要

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【目的】探究湖南省郴州市姜瘟发病情况，鉴定病原菌，筛选出拮抗生防菌，为姜瘟的有效控制提供依据。【方法】系统调查湖南省郴州市生姜主产区姜瘟发生情况，采集具有典型症状的姜块及其根系土壤，分离其病原菌，通过形态特征和分子鉴定确定病原菌的种属，通过回接姜苗确定其致病性；基于内源葡聚糖酶基因egl对分离的青枯雷尔氏菌进行序列变种鉴定；以分离鉴定到的病原菌为靶标菌筛选生防芽胞杆菌，并测定其防效。【结果】田间姜瘟平均发病率8.52%；从姜瘟病块分离到2株病原菌(FJAT-15492和FJAT-15494)，姜瘟根系土壤分离到3株病原菌(FJAT-15495、FJAT-15496和FJAT-15497)；经鉴定FJAT-15492为桑树肠杆菌(Enterobacter mori)，而其他4株菌为青枯雷尔氏菌(Ralstonia solanacearum)；致病性测定结果表明，分离的桑树肠杆菌和青枯雷尔氏菌均会引起姜瘟病害。病原菌分布特性分析显示，青枯雷尔氏菌在病姜块及其根系土壤中均有分布，而桑树肠杆菌只在病姜块中分布，且分布数量(1.33×10³ CFU/g)显著低于青枯雷尔氏菌的分布数量(5.67×10³ CFU/g)。演化型和序列变种鉴定结果表明，分离到青枯雷尔氏菌属于演化Ⅰ型，序列变种14；通过抑菌圈试验发现，短短芽胞杆菌(Bacillus brevis) FJAT-JK-2对青枯雷尔氏菌、贝莱斯芽胞杆菌(Bacillus velezensis) FJAT-54560对桑树肠杆菌有较强拮抗作用，抑菌圈直径分别为19.41 mm和16.11 mm，对姜瘟的室内防效分别为69.45%和61.11%。然而将2种生防菌发酵液按等体积混合对姜瘟的田间防效达52.57%。【结论】本研究鉴定出姜瘟的致病菌，并筛选出2株生防芽胞杆菌，为姜瘟的生物防治提供新的菌株资源。

关键词

姜瘟 / 桑树肠杆菌 / 青枯菌 / 芽胞杆菌 / 演化型 / 序列变种

Abstract

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[Objective] We investigated the occurrence of ginger bacterial wilt, identified the pathogens, and screened the antagonistic bacteria, aiming to provide a scientific basis for the control of the disease. [Methods] We systematically investigated the occurrence of bacterial wilt in the main ginger production areas in Chenzhou City, Hunan Province. The ginger tubers with typical symptoms of bacterial wilt and the rhizosphere soil were collected. Major pathogens were isolated and identified based on morphological characteristics and molecular evidence. The pathogenicity of the isolates was determined by inoculation of the isolates to ginger seedlings. Moreover, the sequevars of Ralstonia solanacearum isolates were identified based on the endogenous glucanase gene egl. Finally, the Bacillus strains for biocontrol of the pathogens were screened, and their biocontrol effects were measured. [Results] The average incidence of ginger bacterial wilt in the field was 8.52%. Two bacterial strains FJAT-15492 and FJAT-15494 were isolated from diseased ginger tubers, and three bacterial strains FJAT-15495, FJAT-15496, and FJAT-15497 were isolated from the rhizosphere soil of diseased ginger. The strain FJAT-15492 was identified as Enterobacter mori and the other four isolates were R. solanacearum. Both the isolates of E. mori and R. solanacearum could infect ginger seedlings and cause bacterial wilt. R. solanacearum strains existed in both diseased ginger tubers and rhizosphere soil, while E. mori only existed in diseased ginger tubers, with the count (1.33×10³ CFU/g) lower than that (5.67×10³ CFU/g) of R. solanacearum.Furthermore, the R. Solanacearum isolates were identified as phylotype Ⅰ and sequevar 14. Brevibacillus brevis FJAT-JK-2 demonstrated inhibitory effect on R. solanacearum, and Bacillus velezensis FJAT-54560 on E. mori, with inhibition zone diameters of 19.41 mm and 16.11 mm and indoor control effects of 69.45% and 61.11%, respectively. Moreover, the fermentation mixture of the two biocontrol strains had the field control effect of 52.57%. [Conclusion] This work identified the pathogens of ginger bacterial wilt and provided two new biocontrol strains against the disease.

Key words

ginger bacterial wilt / Enterobacter mori / Ralstonia solanacearum / Bacillus / phylotype / sequevar

引用本文

郑雪芳, 舒江霞, 王阶平, 杨晨, 车建美, 肖荣凤, 陈梅春, 刘波. 姜瘟致病菌的分离鉴定及其生防芽胞杆菌的筛选. 微生物学报, 2025 , 65 (1) : 136 -149 . DOI: 10.13343/j.cnki.wsxb.20240502

Xuefang ZHENG, Jiangxia SHU, Jieping WANG, Chen YANG, Jianmei CHE, Rongfeng XIAO, Meichun CHEN, Bo LIU. Isolation and identification of the causal organisms of ginger bacterial wilt and screening of Bacillus strains for biocontrol[J]. Acta Microbiologica Sinica, 2025 , 65 (1) : 136 -149 . DOI: 10.13343/j.cnki.wsxb.20240502

正文

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生姜是姜科植物的根茎，属多年生草本植物，性温、味辛，具有很高的营养价值和药用价值，既作为调味品又集食用、药用为一体，是当前效益较高的创汇农产品之一^[1]。我国是全球生姜产量最多的国家，主产区包括山东、河北、辽宁、湖南、四川、贵州、广西、湖北等。2021年中国生姜种植面积36.89万hm²，产量达1 219万t^[2]。姜瘟是一种重要土传病害，在热带和亚热带地区普遍发生，由于姜瘟逐年增加，严重影响了生姜的产量和质量，平常年份减产20%−30%，重病年减产可达50%−70%^[3-4]。

姜瘟通常被认为是由青枯雷尔氏菌(Ralstonia solanacearum，简称青枯菌)引起^[5-7]。然而，研究人员发现，因姜的品种和种植地域不同，感染姜瘟的致病菌也不同，如姚革等^[8]从四川省南充市南部县姜瘟土壤中分离出镰刀菌和核菌，而其他姜瘟地区则鉴定出瓜果腐霉、软腐欧式杆菌等致病菌；王胜利等^[9]从湖南省江永县姜瘟重病区的土壤分离到R. solanacearum和欧文氏杆菌(Erwinia spp.)；姜艳鹏等^[10]鉴定出山东省安丘市凌河镇姜瘟土样的致病菌为阴沟肠杆菌(Enterobacter cloacae)。

目前，姜瘟防治主要通过化学防治，但可用于化学防治的低毒、高效药剂较少，且存在环境和农产品污染等问题^[11]。土传病害的生物防治具有重要潜力^[12-13]。研究人员对姜瘟的生物防治作了大量研究，不同类型生防菌如木霉菌、假单胞菌、芽孢杆菌等被筛选并应用到姜瘟防治中^{[1, 14-16]}。芽胞杆菌(Bacillus sp.)是多种植物病原菌的拮抗菌，可以通过分泌抗菌物质、营养和位点竞争、诱导抗性等有效预防和控制作物病害^[17]，被普遍认为是一种对环境友好、经济有效的防治病害途径^[18]；芽胞杆菌能产生具有较强抗逆性的内生孢子，能耐盐、耐酸、耐高低温且易培养和保存、有效期长等特性，因而受到商家的青睐^[19-20]。贝莱斯芽胞杆菌^[21]、解淀粉芽胞杆菌^[22-23]、枯草芽孢杆菌^[24-25]、多黏芽孢杆菌^[26]、蜡状芽胞杆菌^[27]等均已成功用于作物青枯病的防治。

本研究采集湖南省郴州市姜瘟重病区的姜块和土壤样本进行病原菌的分离和鉴定，对分离到的青枯菌进一步进行演化型和egl的序列变种鉴定，并筛选对姜瘟致病菌具有拮抗作用的芽胞杆菌，为后期田间姜瘟的有效控制提供依据。

1 材料与方法

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1.1 病害调查、样本采集和病原菌分离纯化

田间试验选址在湖南省郴州市(24°53′− 26°50′N，112°13′−114°14′E，海拔150−160 m)属亚热带季风气候，年平均温度18.1 ℃，平均降雨量达1 397.4 mm，全市耕地面积22.71万hm²。2023年6月对湖南省郴州市姜瘟进行病害调查和样本采集。采取点面结合的调查方式调查姜瘟发病率，观察植株和姜块发病症状。选取姜瘟发病严重的田块，将具有典型发病症状植株连根拔起，去除根围土壤，将带有根系土壤和姜块的样本装至塑料袋中，带回实验室进行相关研究。

分别采用2, 3, 5-氯化三苯基四氮唑(2, 3, 5-triphenyltetrazolium chloride, TTC)培养基^[28]和营养琼脂(nutrient agar, NA)培养基^[29]进行青枯菌和其他细菌分离。将病姜切成大小相近的姜块，长宽约1 cm、厚度约0.1 cm，用75%乙醇浸30 s，再转入10%次氯酸钠溶液中浸3 min，无菌水漂洗3次，无菌滤纸吸干。将消毒后的姜块贴片于TTC和NA培养基，30 ℃培养48 h后，观察菌落的形态，姜块分离到2种致病菌(菌株编号分别为FJAT-15492和FJAT-15494)，将菌株纯化后加入终浓度20%的甘油，−80 ℃保存。

称取10 g土壤，加入90 mL无菌水中，180 r/min振荡30 min，10倍系列梯度稀释，将10⁻⁴、10⁻⁵、10⁻⁶稀释液涂布于TTC和NA培养基上，30 ℃培养48 h后，观察菌落的形态，从姜瘟根系土分离到3株疑似致病菌(菌株编号分别为FJAT-15495、FJAT-15496和FJAT-15497)，将菌株纯化后，加入终浓度20%的甘油，−80 ℃保存。

1.2 病原菌分离致病性测定及再分离

将上述分离的5株疑似致病菌在NA平板上活化后，接种营养肉汤(nutrient broth, NB)培养基(0.3%牛肉浸膏、0.5%蛋白胨和1%葡萄糖)，于30 ℃、180 r/min振荡培养48 h后，用清水稀释至菌浓度为1×10⁹ CFU/mL，用于致病力测定。

接种用品种为红皮南姜的生姜苗，将长势一致的姜苗移栽到盛满土的塑料小钵中(小钵直径15 cm、高10 cm)，1株/盆；采用灌根接种法，将上述菌悬液(浓度1×10⁹ CFU/mL)接种姜苗，接种量100 mL/盆，以清水为对照，每处理10株，置光照培养箱[(30±1) ℃，12 h光照培养，12 h暗培养，相对湿度80%]。接种后每天观察植株发病情况。

对发病植株的姜块及其根系土壤上的病原进行再分离鉴定，分离方法与1.1相同，完成柯赫氏法则验证。

1.3 病原菌的形态和分子鉴定

姜块和根系土分离到的菌株在NA或TTC平板上纯化，30 ℃培养48 h，观察菌株的菌落形态特征。

将分离到的病原菌采用16S rRNA基因通用引物27F和1492R^[30]进行PCR扩增。PCR反应体系：2×Easy Taq PCR SuperMix 12 μL，ddH₂O 9 μL，上、下游引物(10 mmol/L)各1 μL，模板DNA 2 μL。PCR反应条件^[30]：94 ℃ 5 min；94 ℃ 30 s，55 ℃ 1 min，72 ℃ 1.5 min，30个循环；72 ℃ 7 min。PCR产物经1.5%的琼脂糖凝胶电泳检测为目的条带后，送到福州尚亚生物技术有限公司进行序列测定。将获得序列提交GenBank数据库，登录号为PP218373.1，采用MEGA 6软件^[31]和邻接(neighbor joining, NJ) 法^[32]构建系统发育树，确定分离的姜瘟致病菌所属的种。

利用青枯菌的特异性检测引物pehA#6和pehA#3对分离的疑似青枯菌进行分子检测^[33]，PCR反应体系(25 μL)：2×Easy Taq PCR SuperMix 12 μL，ddH₂O 9 μL，上、下游引物(10 mmol/L)各1 μL，模板DNA 2 μL。PCR反应程序参考文献[33]。PCR产物经1.5%的琼脂糖凝胶电泳后，于凝胶成像仪(Bio-Rad公司)观察结果。

1.4 青枯菌的演化型鉴定

采用复合PCR法鉴定青枯菌演化型，复合PCR引物根据Fegan等的报道^[34]合成(表1)。PCR反应体系(25 μL)：2×Easy Taq PCR SuperMix 12.5 μL，引物Nmult22:RR (10 µmol/L) 4 μL，其他6个引物(10 µmol/L)各1 μL，DNA 1 μL，ddH₂O 1.5 μL。PCR反应程序参考文献[34]。

1.5 青枯菌的序列变种鉴定

利用引物Endo-F和Endo-R^[35] PCR扩增青枯菌的egl基因，引物序列见表1。PCR反应体系和反应程序参考文献[35]，PCR产物经1.5%的琼脂糖凝胶电泳检测为目的条带后，送到福州尚亚生物技术有限公司进行序列测定。从GenBank数据库下载每个变种代表性菌株的egl基因序列，用ClustalX软件，将供试菌株egl基因序列与不同变种的egl基因序列进行多重比较，NJ法构建系统发育树，确定供试青枯菌归属的序列变种。

1.6 姜瘟致病菌的生防芽胞杆菌筛选

以上述分离的姜瘟致病菌桑树肠杆菌和青枯菌为靶标菌，从已构建的芽胞杆菌资源库中筛选对这2种病原菌具拮抗作用的芽胞杆菌。制备含病原菌的双层培养基：先制备NA固体培养基(含1.8%琼脂)，然后将1 mL菌浓度为1×10⁹ CFU/mL的致病菌与9 mL NA半固体培养基(含0.8%琼脂)混合均匀后倒入NA固体培养基上；用无菌的直径为7 mm打孔器在凝固的平板上打孔。供试芽胞杆菌在NA培养基上活化后，接种NB培养基，30 ℃、180 r/min振荡培养48 h，培养液过0.22 μm滤膜后，加入双层培养基的孔中，以无菌水作为阴性对照，链霉素作为阳性对照，链霉素在桑树肠杆菌和青枯菌为靶标菌双层培养基中的添加浓度分别为100 mg/mL和200 µg/mL，设5个重复，30 ℃培养48 h后，测量抑菌圈直径。

1.7 生防芽胞杆菌对姜瘟防治效果

1.7.1 室内防效测定

将上述筛选到的生防芽胞杆菌(FJAT-JK-2对青枯菌具有拮抗和FJAT-54560对桑树肠杆菌具有拮抗)在NA培养基上活化后，接种NB培养基，于30 ℃、180 r/min振荡培养24 h后，菌液稀释至10⁸ CFU/mL，设不同处理，处理1 (TR1)：灌根接种法预接种FJAT-JK-2于姜盆栽苗(2−3叶龄)上，3 d后接种青枯菌，生防菌与病原菌接种浓度均为10⁸ CFU/mL，接种量均为100 mL/盆；对照组1 (CK1)：姜苗未接种生防菌，3 d后接种青枯菌，100 mL/盆；处理2 (TR2)：灌根接种法预接种FJAT-54560 (菌浓度10⁸ CFU/mL)，3 d后接种桑树肠杆菌(菌浓度10⁸ CFU/mL)，100 mL/盆；对照组2 (CK2)：姜苗未接种生防菌，3 d后接种桑树肠杆菌100 mL/盆；上述各处理12株姜苗，设3个重复。接种后每天观察植株发病情况，统计发病率，并计算生防菌对姜瘟的防治效果。

1.7.2 田间防效测定

田间试验田块选取姜瘟样本采集地。将生防菌FJAT-JK-2和FJAT-54560的发酵液(制备方法与1.7.1相同)稀释至10⁸ CFU/mL，等体积混合后，灌根接种生姜植株(处理组)，500 mL/株，每10 d灌根处理1次，连续处理5次，清水为对照，处理组和对照组各3田块(初始发病率均约8.5%)，于最后1次接种处理后5 d，统计田间姜瘟发病率，并计算生防菌的田间防治效果。

(1)$D I=(d n / t n) \times 100 \%$

(2)$C E=\left(D I_{\mathrm{TR}}-D I_{\mathrm{CK}}\right) / D I_{\mathrm{CK}} \times 100 \%$

式中：DI为发病率，dn为发病株数，tn为调查株数，CE为防治效果，DI_TR为处理组发病率，DI_CK为对照组发病率。

2 结果与分析

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2.1 姜瘟田间发病调查、致病菌的分离、形态特征及致病性测定

2023年7月对湖南省郴州市汝城县姜瘟调查发现，生姜种植田块发病较为严重(随机抽查8个田块)，田间平均发病率8.52% (图1A)。发病姜块根部呈现出黄褐色，严重时内部组织发生软化腐烂，挤压会有白色浓液流出(图1B)。初发病时叶片边缘卷曲，黄化，至整个叶片退绿、枯萎。

从姜瘟病块样本分离到2株致病菌，菌株编号分别为FJAT-15492和FJAT-15494，从姜瘟根系土壤分离到3株致病菌，菌株编号分别为FJAT-15495、FJAT-15496和FJAT-15497。其中菌株FJAT-15492在NA培养基上培养2 d，菌落较小，圆形凸起、淡黄色，表面光滑、具有流动性(图1D)；菌株FJAT-15494、FJAT-15495、FJAT-15496和FJAT-15497在TTC平板上培养2 d，菌落与青枯菌菌落形态特征^[36]一致：菌落近圆形或不规则，表面湿润，具有流动性，中间粉红色，边缘白色(图1C)。

致病性测定结果表明，分离的疑似青枯菌接种姜盆栽苗12 d，植株开始发病，叶片边缘卷曲，黄化，直至整个叶片枯萎(图1F)，与姜瘟田间症状一致。另一种致病菌株FJAT-15492接种，姜苗也出现同样症状，但发病进程慢，接种20 d植株才开始出现病症(图1G)。

2.2 姜瘟致病菌的分子鉴定

根据形态特征初步鉴定为青枯菌的菌株FJAT-15494、FJAT-15495、FJAT-15496和FJAT-15497 (图2A)，进一步利用青枯菌特异检测引物pehA#6和pehA#3进行分子鉴定，结果表明，经的PCR扩增，4株疑似青枯菌与参比菌株GMI1000阳性对照均扩增出504 bp的特异性条带，无菌水阴性对照未扩增出相应条带(图2B)。因此，确定菌株FJAT-15494、FJAT-15495、FJAT-15496和FJAT-15497为青枯菌。

经16S rRNA基因鉴定，FJAT-15492与E. mori模式菌株D11^T的16S rRNA基因序列相似性最高，为98.20%。在系统发育树上，FJAT-15492与E. mori模式菌株D11^T聚在同一类群(图2C)。因此，经16S rRNA基因鉴定FJAT-15492为桑树肠杆菌E. mori。

2.3 姜瘟致病菌的分布特性

统计2种不同致病菌桑树肠杆菌和青枯菌在病姜块和病姜根系土壤中的分布数量，结果如表2显示，桑树肠杆菌和青枯菌在病姜块均有分布，但青枯菌分布数量(5.67×10³ CFU/g)显著高于桑树肠杆菌的分布数量(1.33×10³ CFU/g) (P<0.05)，表明姜瘟是由桑树肠杆菌和青枯菌混合侵染，且青枯菌是优势菌。在病姜根系土壤中只检测到青枯菌的分布(216.67×10³ CFU/g)，未检测到桑树肠杆菌。

2.4 青枯菌的演化型和序列变种鉴定

分离到4株青枯菌，编号分别为FJAT-15494、FJAT-15495、FJAT-15496和FJAT-15497，经演化型复合PCR扩增，均出现2条带，片段大小分别为144 bp和280 bp，其中144 bp条带为演化型Ⅰ的特异性条带，280 bp条带为青枯菌的特异性条带(图3A)，表明姜瘟分离到的4株青枯菌均属于演化型Ⅰ (亚洲分支)菌株。

基于egl基因序列对分离到的4株青枯菌进行序列变种鉴定，PCR扩增条带大小为840 bp (图3B)，4株青枯菌egl基因序列与GenBank已知不同序列变种进行同源比对，采用邻接法构建系统发育树。结果表明，供试4株青枯菌均属于序列变种14，而参比菌株GMI1000属于序列变种18 (图3C)。

2.5 姜瘟致病菌的生防芽胞杆菌筛选

供试的21株芽胞杆菌中，贝莱斯芽胞杆菌FJAT-54560对桑树肠杆菌FJAT-15492有较强拮抗作用，抑菌圈直径为16.11 mm；短短芽胞杆菌FJAT-JK-2对青枯菌FJAT-15494也具有较强抑菌活性，抑菌圈直径为19.41 mm；而对照链霉素对桑树肠杆菌和青枯菌的抑菌直径分别为19.56 mm和23.14 mm (表3)。

进一步研究生防菌FJAT-JK-2和FJAT-54560对姜瘟的室内防效，结果表明，对照组1 (CK1)姜苗在接种青枯菌12 d植株开始发病，23 d植株发病率100%；对照组(CK2)姜苗在接种桑树肠杆菌20 d植株开始发病，35 d植株发病率100%。处理组1 (TR1)预接种FJAT-JK-2，3 d后接种青枯菌，植株18 d开始发病，比CK1推迟发病6 d，CK1发病率100.00%时，TR1姜苗的发病率为30.55%，即FJAT-JK-2对姜瘟的防效达69.45%。处理组2 (TR2)预接种FJAT-54560，3 d后接种桑树肠杆菌，植株25 d开始发病，比CK2推迟发病5 d，CK2发病率100.00%时，TR2姜苗的发病率为38.89%，防效达61.11% (表4)。

2.6 生防芽胞杆菌对姜瘟的田间防治效果

生防菌FJAT-JK-2和FJAT-54560的发酵制剂，300倍稀释后等体积混合，灌根接种到姜瘟发病田块，每7 d用药1次，连续5次，结果表明，对照组姜瘟平均发病率达19.44%，处理组的姜瘟平均发病率为9.22%，平均防效达52.57% (图4)。

3 讨论与结论

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姜瘟病是危害生姜产量和品质的重要病害。长期以来，对姜瘟致病菌一直存在着不同的看法^[37]。本研究对姜瘟发病地块的姜和土壤样本进行致病菌分离纯化，根据16S rRNA基因和青枯菌特异性引物检测，确定姜瘟致病菌为E. mori和R. solanacearum。将分离到的2种致病菌回接姜苗，均能引起姜瘟病害且症状与田间姜瘟症状一致。姜艳鹏等也从姜瘟发病田块分离鉴定出其致病菌为阴沟肠杆菌^[10]。肠杆菌可作为生姜内生菌，在有利于细菌生存或宿主敏感条件下影响根茎的质量，造成其腐烂^[38]。此外，国内外众多研究表明，姜瘟主要是由青枯菌侵染引起的^{[37, 39]}。本研究发现，湖南省郴州市姜瘟可能是由E. mori和R. solanacearum混合侵染引起的，与前人研究报道不同，即首次发现姜瘟是2种病原菌混合感染，且以R. solanacearum为优势种。

2005年，Fegan等提出青枯菌种下分化的4个分类单元，即种、演化型、序列变种和克隆，并建立了相应的鉴定方法^[34]。许多研究表明，我国青枯菌以演化型Ⅰ为主^[40-42]。本研究采用演化型复合PCR法将姜瘟分离的4株青枯菌鉴定为演化型Ⅰ，与前人研究结果相吻合^[42]。基于egl基因序列，青枯菌被划分超过15个序列变种^[42-43]。研究表明，采集相同寄主、相同区域的青枯菌，往往具有相同的序列变种^[44]。然而，She等研究发现，广东沙姜上分离到的青枯菌存在5个不同的序列变种^[42]。本研究发现，从姜瘟土壤分离到的4株青枯菌与序列变种14的代表菌株Pss81聚在一类群，一致性达96%。参比菌株GMI1000则属于序列变种18；GMI1000与Pss81均分离自番茄寄主，其中GMI1000和Pss81分别来源法属圭亚那和中国台湾。说明青枯菌序列变种与寄主和地理来源无密切相关性。

芽胞杆菌属细菌因其具有生长快、抗逆性强，易分离培养等特性，成为目前生防菌剂生产常用菌种之一^[45]。芽胞杆菌已被广泛应用于青枯病防治中，蜡状芽胞杆菌^[46]、枯草芽胞杆菌^[47]、贝莱斯芽胞杆菌^[21]等对姜瘟致病菌均具有显著的抑制作用，是该病害生物防治的优良菌种资源。田间应用表明芽胞杆菌生防菌剂在稳定性、与化学农药的相容性等方面明显优于其他生防菌^[48]，本研究从21株不同芽胞杆菌中筛选出短短芽胞杆菌FJAT-JK-2对姜青枯菌、贝莱斯芽胞杆菌FJAT-54560对姜桑树肠杆菌具有拮抗作用的菌株，为姜瘟病的生物防治提供生防菌资源。

生防菌从实验阶段到田间应用是一个复杂的过程。本研究筛选到对姜瘟致病菌具有显著抑菌效果的2株生防菌株，其室内防效分别达69.45%和61.11%，但二者发酵混合液的田间防效仅为52.57%。许多研究表明，生防菌的田间防效低于室内防效^[49-50]，究其原因可能是田间环境复杂，不可控制因素多，使得生防菌的田间防效低且不稳定，而且随着地理环境、生态环境等不同，生防菌防效差异大，如易有金等发现，生防枯草芽胞杆菌(Bacillus subtilis) B-001对湖南省不同县的烟草青枯病防效介于40.03%−78.14%^[51]。对于姜瘟的防控，可充分利用不同防治方法的优点，如可尝试将低毒化学农药和生防菌剂复配施用来提高防治效果。化学农药和生防菌剂复配对植物病害增效作用已有许多报道，如陈长卿等将生防菌NJ13 [甲基营养型芽胞杆菌(Bacillus methylotrophicus)]分别与2种化学药剂(嘧菌环胺和苯醚甲环唑)复配，发现其对人参黑斑病田间防效具有增效作用^[52]；刘合昌等研究发现，生防芽胞杆菌b702与甲霜灵混剂对辣椒疫病的防治效果明显优于单剂^[53]。最后，姜瘟防治重在预防：生姜种植地应该选地势高、排水良好的地块；在品种选择上，依据当地种植条件，选择抗病强的品种；在生姜生长期，雨后要及时排水，以防水淹，发现病株，要及时挖除等。后续研究中，需要进一步对筛选的生防菌进行定殖特性、菌株发酵技术、田间应用技术及生物安全性等研究，以期开发高效环保生防菌剂应用于实际生产。

基金

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福建省公益类科研院所专项(2021R1034003)
福建省自然科学基金(2024J01173)

参考文献

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文献

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doi: 10.13343/j.cnki.wsxb.20240502

接收时间：2024-08-14
首发时间：2026-03-21
出版时间：2025-01-04

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收稿日期：2024-08-14
录用日期：2024-10-29

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Special Fund for Scientific Research Institutes in the Public Interest of Fujian Province(2021R1034003)

福建省公益类科研院所专项(2021R1034003)

Natural Science Foundation of Fujian Province(2024J01173)

福建省自然科学基金(2024J01173)

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1 福建省农业科学院资源环境与土壤肥料研究所, 福建福州 350003

2 中福海峡平潭发展股份有限公司, 福建福州 350000

3 湖南农业大学植物保护学院, 湖南长沙 410125

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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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Primer name	Application	Primer sequences (5′→3′)	Fragment size (bp)
AU759f	Species-specific primers of R. solanacearum	GTCGCCGTCAACTCACTTTCC	280
AU760r		GTCGCCGTCAGCAATGCGGAATCG
Nmult21:1F	The forward primer for phylotype Ⅰ	CGTTGATGAGGCGCGCAATTT	144
Nmult21:2F	The forward primer for phylotype Ⅱ	AAGTTATGGACGGTGGAAGTC	372
Nmult23:AF	The forward primer for phylotype Ⅲ	ATTACSAGAGCAATCGAAAGATT	91
Nmult22:InF	The forward primer for phylotype Ⅳ	ATTGCCAAGACGAGAGAAGTA	213
Nmult22:RR	The reversed primer for phylotype identification	TCGCTTGACCCTATAACGAGTA
Endo-F	The primers for egl sequevar identification	ATGCATGCCGCTGGTCGCCGC	840
Endo-R		GCGTTGCCCGGCACGAACACC

Primer name

Application

Primer sequences (5′→3′)

Fragment size (bp)

AU759f

Species-specific primers of R. solanacearum

GTCGCCGTCAACTCACTTTCC

280

AU760r

GTCGCCGTCAGCAATGCGGAATCG

Nmult21:1F

The forward primer for phylotype Ⅰ

CGTTGATGAGGCGCGCAATTT

144

Nmult21:2F

The forward primer for phylotype Ⅱ

AAGTTATGGACGGTGGAAGTC

372

Nmult23:AF

The forward primer for phylotype Ⅲ

ATTACSAGAGCAATCGAAAGATT

Nmult22:InF

The forward primer for phylotype Ⅳ

ATTGCCAAGACGAGAGAAGTA

213

Nmult22:RR

The reversed primer for phylotype identification

TCGCTTGACCCTATAACGAGTA

Endo-F

The primers for egl sequevar identification

ATGCATGCCGCTGGTCGCCGC

840

Endo-R

GCGTTGCCCGGCACGAACACC

Pathogen of ginger bacterial wilt	Tuber of diseased ginger distribution number (×10³ CFU/g)	Rhizosphere soil of diseased ginger distribution number (×10³ CFU/g)
Data in the table are mean±SD, and the data are the mean of three replicates. Different lowercase and capital letters in the same column show values that are significantly different at the P<0.05 and P<0.01 level, respectively, by the least significant difference (LSD) test.
Enterobacter mori	1.33±0.58b	0.00±0.00B
Ralstonia solanacearum	5.67±2.08a	216.67±35.12A

Pathogen of ginger bacterial wilt

Tuber of diseased ginger distribution number (×10³ CFU/g)

Rhizosphere soil of diseased ginger distribution number (×10³ CFU/g)

Data in the table are mean±SD, and the data are the mean of three replicates. Different lowercase and capital letters in the same column show values that are significantly different at the P<0.05 and P<0.01 level, respectively, by the least significant difference (LSD) test.

Enterobacter mori

1.33±0.58b

0.00±0.00B

Ralstonia solanacearum

5.67±2.08a

216.67±35.12A

Strain number	Species	Antibacterial circle diameter against E. mori (mm)	Antibacterial circle diameter against R. solanacearum (mm)
Data in the table are mean±SD, and the data are the mean of five replicates.
FJAT-54970	Bacillus megaterium	0.00±0.00	0.00±0.00
FJAT-54974		0.00±0.00	0.00±0.00
FJAT-54978		0.00±0.00	0.00±0.00
FJAT-54986		0.00±0.00	0.00±0.00
FJAT-54989		0.00±0.00	0.00±0.00
FJAT-2349	Bacillus velezensis	0.00±0.00	0.00±0.00
FJAT-17531		0.00±0.00	0.00±0.00
FJAT-51047		0.00±0.00	0.00±0.00
FJAT-52631		0.00±0.00	0.00±0.00
FJAT-54560		16.11±0.15	0.00±0.00
FJAT-54972		0.00±0.00	0.00±0.00
FJAT-19700	Bacillus tequilens	0.00±0.00	0.00±0.00
FJAT-54984		0.00±0.00	0.00±0.00
FJAT-54973	Bacillus thuringiensis	0.00±0.00	0.00±0.00
FJAT-54975	Bacillus safensis	0.00±0.00	0.00±0.00
FJAT-54979	Bacillus licheniformis	0.00±0.00	0.00±0.00
FJAT-54981	Bacillus amyloliquefaciens	0.00±0.00	0.00±0.00
FJAT-54985	Bacillus aryabhattai	0.00±0.00	0.00±0.00
FJAT-54987	Bacillus pumilus	0.00±0.00	0.00±0.00
FJAT-20261	Brevibacterium frigoritolerans	0.00±0.00	0.00±0.00
FJAT-JK-2	Brevibacillus brevis	0.00±0.00	19.41±0.14
Streptomycin as control		19.56±0.32	23.14±0.46

Strain number

Species

Antibacterial circle diameter against E. mori (mm)

Antibacterial circle diameter against R. solanacearum (mm)

Data in the table are mean±SD, and the data are the mean of five replicates.

FJAT-54970

Bacillus megaterium

0.00±0.00

FJAT-54974

0.00±0.00

FJAT-54978

0.00±0.00

FJAT-54986

0.00±0.00

FJAT-54989

0.00±0.00

FJAT-2349

Bacillus velezensis

0.00±0.00

FJAT-17531

0.00±0.00

FJAT-51047

0.00±0.00

FJAT-52631

0.00±0.00

FJAT-54560

16.11±0.15

0.00±0.00

FJAT-54972

0.00±0.00

FJAT-19700

Bacillus tequilens

0.00±0.00

FJAT-54984

0.00±0.00

FJAT-54973

Bacillus thuringiensis

0.00±0.00

FJAT-54975

Bacillus safensis

0.00±0.00

FJAT-54979

Bacillus licheniformis

0.00±0.00

FJAT-54981

Bacillus amyloliquefaciens

0.00±0.00

FJAT-54985

Bacillus aryabhattai

0.00±0.00

FJAT-54987

Bacillus pumilus

0.00±0.00

FJAT-20261

Brevibacterium frigoritolerans

0.00±0.00

FJAT-JK-2

Brevibacillus brevis

0.00±0.00

19.41±0.14

Streptomycin as control

19.56±0.32

23.14±0.46

Treatments	Disease incidence (%)	Control efficiency (%)
The data are mean±SD, and the data are the mean of three replicates. −: Indicates unable to calculate.
TR1	30.55±4.81	69.45±4.81
TR2	38.89±4.82	61.11±4.82
CK1	100.00±0.00	−
CK2	100.00±0.00	−

Treatments

Disease incidence (%)

Control efficiency (%)

The data are mean±SD, and the data are the mean of three replicates. −: Indicates unable to calculate.

TR1

30.55±4.81

69.45±4.81

TR2

38.89±4.82

61.11±4.82

CK1

100.00±0.00

−

CK2

100.00±0.00

−