Isolation and Identification of Black Rot Pathogen on Bitter Gourd and Screening of Bacillus spp.for Hyper-Effective Biocontrol

doi:10.7668/hbnxb.20195444

Abstract

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

CLC Number:

S432植物病害及其防治

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.

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Figures/Tables 8

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.

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.

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).

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.

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	-	-	-

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

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.

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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