苦瓜黑腐病菌的分离鉴定及其高效生防芽孢杆菌筛选

doi:10.7668/hbnxb.20195444

摘要/Abstract

摘要：

明确苦瓜黑腐病病原并筛选高效生防芽孢杆菌,为科学防治苦瓜黑腐病提供理论基础。采用组织分离法对苦瓜黑腐病菌的病原进行分离纯化,并进一步依据柯赫法则验证分离病原菌的致病性。通过形态学观察和分子系统发育分析对病原菌进行鉴定。以代表菌株NCKG8.2-4为供试病原,通过拮抗试验比较枯草芽孢杆菌斯氏亚种TEB-1、解淀粉芽孢杆菌 SWB-2、地衣芽孢杆菌 SWB-1、贝莱斯芽孢杆菌 SB023、多粘类芽孢杆菌 SWP-1对苦瓜黑腐病菌的拮抗效果,进一步通过无细胞发酵液梯度抑菌试验,评价拮抗效果最佳菌株发酵液对病原菌的抑菌效果。结果表明,NCKG8.2-4为引起苦瓜黑腐病的病原菌,其ITS、β-Tubulin和GAPDH片段与瓜拟多隔孢的一致性均为100%;且基于β-Tubulin或GAPDH序列的系统发育分析,将其分别与瓜拟多隔孢聚在一支。拮抗试验结果显示,贝莱斯芽孢杆菌SB023对苦瓜黑腐病菌NCKG8.2-4的菌落生长抑制区域最大;同时平板抑菌效果随SB023无细胞发酵液含量的增加而增加,PDA中添加9%的无细胞发酵液对病原菌NCKG8.2-4的抑菌率高达93.7%,其EC₅₀为3.48%。明确了苦瓜黑腐病的病原为瓜拟多隔孢,贝莱斯芽孢杆菌SB023菌株对该病病原具有高效抑菌效果。

关键词: 苦瓜, 黑腐病, 瓜拟多隔孢, 芽孢杆菌, 生物防治

Abstract:

The purport was to identify the black rot pathogen of bitter gourd (Momordica charantia L.) and screen highly effective biocontrol Bacillus spp.,so as to provide a theoretical basis for scientific prevention and control of black rot of bitter gourd.The pathogen of black rot was isolated and purified by tissue separation method from postharvest bitter gourd,and the pathogenicity of isolated pathogens was further verified according to Koch's rule.The pathogen was identified by observing morphological characteristics combined with molecular phylogenetic analysis.The representative strain NCKG8.2-4 was used as the test pathogen,and antagonistic experiments were conducted to compare the effects of Bacillus subtilis subsp. spizizenii TEB-1,Bacillus amyloliquefaciens SWB-2,Bacillus licheniformis SWB-1,Bacillus velezensis SB023 and Bacillus polymyxa SWP-1 on black rot pathogen of bitter gourd.The inhibition effect of the best antagonistic strains on test pathogen was further evaluated by the gradient inhibition experiment of the cell-free supernatant (CFS) from its fermentation broth.The results showed that NCKG8.2-4 strain was pathogen causing black rot of bitter gourd,and the ITS,β-Tubulin and GAPDH fragments sequences were 100% consistent with Stagonosporopsis cucurbitacearum.And it was respectively clustered with S.cucurbitacearum on a branch by phylogenetic analysis based on β-Tubulin or GAPDH sequences.The results of antagonistic experiment showed that the inhibitory area of SB023 against colony growth of black rot pathogen NCKG8.2-4 was the largest.At the same time,the inhibitory effect increased with the Mcrease of SB023 CFS content,and the inhibitory rate of 9% CFS for NCKG8.2-4 growth was up to 93.7% and the EC₅₀ of SB023 CFS was 3.48% to NCKG8.2-4 on PDA.The pathogen of black rot of bitter gourd was identified as S.cucurbitacearum,and Bacillus velezensis SB023 had a high inhibitory effect on the pathogen NCKG8.2-4.

Key words: Momordica charantia L., Black rot, Stagonosporopsis cucurbitacearum, Bacillus spp., Biocontrol

中图分类号:

S432植物病害及其防治

常艳华, 刘华峰, 刘小红, 辛原仪, 文国琴, 宋波, 张廷富. 苦瓜黑腐病菌的分离鉴定及其高效生防芽孢杆菌筛选[J]. 华北农学报, 2025, 40(4): 194-202. doi: 10.7668/hbnxb.20195444.

CHANG Yanhua, LIU Huafeng, LIU Xiaohong, XIN Yuanyi, WEN Guoqin, SONG Bo, ZHANG Tingfu. Isolation and Identification of Black Rot Pathogen on Bitter Gourd and Screening of Bacillus spp.for Hyper-Effective Biocontrol[J]. Acta Agriculturae Boreali-Sinica, 2025, 40(4): 194-202. doi: 10.7668/hbnxb.20195444.

http://www.hbnxb.net/CN/Y2025/V40/I4/194

图/表 8

图1 采后苦瓜黑腐病的症状 A.市售苦瓜黑腐采样瓜堆;B.发病的苦瓜果实。

Fig.1 Symptoms of black rot of postharvest bitter gourd A.The sampling pile of black rot of bitter gourd in market;B.Fruits of diseased bitter gourd.

图2 代表菌株NCKG8.2-4在PDA培养基上的菌落及菌丝形态 A、B.PDA上培养4 d菌落的正反面;C.菌丝。

Fig.2 Morphological characteristics of colony and hyphae from representative strain NCKG8.2-4 A,B.Both sides of the colony cultured for 4 days on PDA;C.Hyphae.

图3 代表菌株NCKG8.2-4对苦瓜的致病性 A.灭菌PDA菌饼对照组(上),代表菌株NCKG8.2-4菌饼接种试验组(下);B.对照组局部特写(上),试验组局部特写(下)。

Fig.3 Pathogenicity of the representative strain NCKG8.2-4 to bitter gourd A.Control group inoculated with sterilized PDA cakes (top),experimental group inoculated cakes with representative strains NCKG8.2-4 (bottom); B.Local close-up of control group (top) and experimental group (bottom).

图4 NCKG8.2-1、NCKG8.2-4和NCKG9.19-1菌株的PCR扩增产物电泳检测 M.DS 5000 DNA Marker;1-3.NCKG8.2-1、NCKG8.2-4和NCKG9.19-1的GAPDH片段扩增产物;4-6.NCKG8.2-1、NCKG8.2-4和NCKG9.19-1的β-Tubulin片段扩增产物;7-9.NCKG8.2-1、NCKG8.2-4和NCKG9.19-1的ITS扩增产物。

Fig.4 The electrophoretic detection of PCR products amplified from NCKG8.2-1,NCKG8.2-4 and NCKG9.19-1 strains M.DS 5000 DNA Marker;1-3.GAPDH fragment amplification products of NCKG8.2-1,NCKG8.2-4 and NCKG9.19-1;4-6.β-Tubulin fragment amplification products of NCKG8.2-1,NCKG8.2-4 and NCKG9.19-1;7-9.ITS amplification products of NCKG8.2-1,NCKG8.2-4,and NCKG9.19-1.

表1 NCKG8.2-4的ITS、β-Tubulin和GAPDH与拟多隔孢属相关序列Blast结果

Tab.1 Blast results of ITS,β-tubulin and GAPDH from NCKG8.2-4 with sequences related to Stagonosporopsis spp.

物种名称 Scientific name	ITS			β-Tubulin			GAPDH
物种名称 Scientific name	覆盖率/% Query Cover	一致性/ % Identity	GenBank 登录号 Accession number	覆盖率/% Query Cover	一致性/ % Identity	GenBank 登录号 Accession number	覆盖率/% Query Cover	一致性/ % Identity	GenBank 登录号 Accession number
瓜拟多隔孢S.cucurbitacearum	100	100.00	MK690410.1	100	100.00	OP236414.1	100	100.00	OP236413.1
木瓜拟多隔孢S.caricae	100	100.00	MW149263.1	67	100.00	MW149265.1	-	-	-
黄瓜拟多隔孢S.cucumeris	100	99.80	OP788059.1	66	97.71	OP810569.1	-	-	-
西瓜拟多隔孢S.citrulli	99	99.60	OQ793641.1	100	98.47	OQ791174.1	-	-	-
壳多孢霉S.rhizophila	100	99.01	OR879693.1	69	90.88	OR861534.1	-	-	-
莴苣缬草拟多隔孢S.valerianellae	96	98.16	GU237819.1	57	96.00	GU237705.1	-	-	-
细叶百脉根拟多隔孢S.loticola	100	98.81	MH861380.1	58	90.55	MT005564.1	-	-	-
菜豆拟多隔孢S.hortensis	100	98.62	MH854795.1	57	94.68	MN984062.1	-	-	-
花园土拟多隔孢S.stuijvenbergii	93	98.52	MN823449.1	54	93.36	MN824623.1	-	-	-
黄芪拟多隔孢S.astragali	100	98.42	MH854839.1	64	93.47	GU237677.1	-	-	-
意大利风铃拟多隔孢S.trachelii	100	98.42	MN732904.1	57	96.01	MT013364.1	-	-	-
羽扇豆拟多隔孢S.lupini	96	98.36	GU237844.1	57	94.97	GU237700.1	-	-	-
菊蒿花拟多隔孢S.tanaceti	96	97.96	JQ897475.1	57	95.02	JQ897493.1	97	89.35	KR780037.1
菊蒿花拟多隔孢S.tanaceti	-	-	-	-	-	-	97	89.35	LC683195.1
菊花拟多隔孢S.chrysanthemi	100	97.83	OM346748.1	99	94.78	OM945724.1	-	-	-

图5 基于ITS、 β-Tubulin和GADPH序列构建的系统发育树

Fig.5 Phylogenetic trees constructed based on ITS,β-Tubulin and GADPH sequences

图6 5种生防细菌对苦瓜黑腐病菌NCKG8.2-4平板拮抗效果 A.TEB-1、SWB-2、SWB-1、SB023和SWP-1菌株对NCKG8.2-4的拮抗效果;B.TEB-1、SWB-2、SWB-1、SB023和SWP-1菌株对NCKG8.2-4菌丝形态的影响。

Fig.6 Antagonistic effects of five biocontrol bacteria on black rot pathogens NCKG8.2-4 of bitter gourd A.Antagonistic effects of strains TEB-1,SWB-2,SWB-1,SB023 and SWP-1 on NCKG8.2-4,respectively; B.Influence of TEB-1,SWB-2,SWB-1,SB023 and SWP-1 on mycelial morphology of NCKG8.2-4,respectively.

图7 贝莱斯芽孢杆菌SB023发酵液CFS对苦瓜黑腐病菌的抑制效果 A.SB023菌株不同浓度CFS对NCKG8.2-4抑菌效果;B.SB023菌株不同浓度CFS对NCKG8.2-4抑菌率。

Fig.7 Inhibitory effect of B.velezensis SB023 CFS on black rot pathogens of bitter gourd A.The inhibitory effects of SB023 strain with different concentrations of cell-free supernatant on NCKG8.2-4;B.Inhibitory rate of SB023 strain with different concentrations of cell-free supernatant on NCKG8.2-4.

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