梨火疫病拮抗细菌FX1及其抑菌物质的防病作用

doi:10.16420/j.issn.0513-353x.2022-0048

摘要/Abstract

摘要：

以前期从植物酵素液中初筛出的梨火疫病菌（Erwinia amylovora）拮抗细菌为供试菌株，采用打孔抑菌圈法、共培养法复筛出对梨火疫病菌具有强抑菌活性的贝莱斯芽孢杆菌（Bacillus velezensis）FX1菌株。FX1菌株的除菌发酵滤液对梨火疫病菌的抑菌圈直径为26.11 mm，与梨火疫病菌共培养的抑菌率为99.99%。比较不同方法提取的FX1菌株发酵滤液粗提物的抑菌活性，结果显示：酸沉淀法和硫酸铵饱和沉淀法得到的粗提物具有显著的抑菌作用，酸沉淀法提取物抑菌活性更强。利用特异性引物在FX1菌株中扩增检测到脂肽类（bmyB、fenD、ituC、srfAA、srfAB、bioA、yngG和yndJ）和抑菌蛋白类（TasA）抑菌物质合成相关基因，表明该菌株可能具有合成脂肽类和抑菌蛋白类物质的能力。利用库尔勒香梨（Korla Fragrant Pear）离体花序和杜梨（Pyrus betulaefolia）盆栽苗测定FX1菌株抑菌物质的防病效果显示，喷施FX1菌株脂肽类物质粗提液、除菌发酵滤液和菌体发酵液对香梨花序3 ~ 5 d的保护性防效分别为79.07%、56.85%和50.02%；对杜梨苗7 ~ 15 d的保护性防效分别为72.64%、72.85%和71.54%，均达到了与农用链霉素（82.33%）接近的效果，治疗性防效分别为61.59%、52.02%和56.03%。B. velezensis FX1菌株的胞外抑菌物质对梨火疫病具有较好的预防和治疗作用，有进一步深入研究和开发应用的潜力。

关键词: 梨火疫病菌, 拮抗细菌, 抑菌物质, 生物防治

Abstract:

The antagonistic bacteria against Erwinia amylovorawhich were isolated from plant fermentation were used as the test strains，among which Bacillus velezensis FX1 rescreened out by using perforation-inhibition zone method and co-cultivation method respectively. The results showed that the filtered fermentation broth of FX1 had the strongest antibacterial activity against E. amylovora，and the diameter of inhibition zone was 26.11 mm，after co-culture with the bacteria，the antibacterial rate was 99.99%. The antimicrobial activity of crude extracts from fermentation filtrate of FX1 extracted by different methods were compared. The acid precipitation，ammonium sulphate saturation precipitation and n-butanol extract methods were used for the crude extracts of this strain，and the crude extracts obtained by the acid precipitation and ammonium sulphate saturation precipitation methods exhibited significant antibacterial activity，with the acid precipitation extract showing the strongest antibacterial activity. The biocontrol functional genes were amplified by specific primers，and the gene fragments of bmyB，fenD，ituC，srfAA，srfAB，bioA，yngG，yndJ and TasA were amplified from genomic DNA of the FX1，which showed that the strain may produce lipopetide and antibacterial protein metabolites. The preventive efficacy of spraying FX1 strain lipopeptide crude extract，filtered fermentation broth and fermentation broth in vitro inflorescences of Korla Fragrant Pear for 3-5 d were 79.07%，56.85%，50.02%，respectively. The preventive and therapeutic effects on Pyrus betulaefolia seedlings for 7-15 days were 72.64%，72.85% and 71.54%，61.59%，52.02% and 56.03%，respectively. And the preventive effects was similar to that of agricultural streptomycin（82.33%）. The antibacterial substances of B. velezensis FX1 showed obvious preventive and therapeutic effect on E. amylovora，and has the potential for future in-depth research，development，and application in biocontrol fire blight disease.

Key words: Erwinia amylovora, antagonistic bacteria, antibacterial substance, biological control

中图分类号:

S661.2

贺旭, 韩剑, 盛强, 罗明, 包慧芳, 黄伟. 梨火疫病拮抗细菌FX1及其抑菌物质的防病作用[J]. 园艺学报, 2023, 50(5): 1118-1129.

HE Xu, HAN Jian, SHENG Qiang, LUO Ming, BAO Huifang, HUANG Wei. Screening of Antagonistic Bacterium FX1 Against Erwinia amylovora and Its Control Effect of the Antibacterial Substances on Fire Blight[J]. Acta Horticulturae Sinica, 2023, 50(5): 1118-1129.

图/表 6

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菌株 Strain	分离来源 Sampling source	抑菌圈直径/mm The inhibition zone diameters	共培养Co-culture
			梨火疫病菌E. amylovora		抑菌率/% Inhibitive rate
			OD₆₀₀	活菌数/（CFU · mL^-1） Number of viable bacteria	抑菌率/% Inhibitive rate
FX68	西瓜Watermelon	14.10 ± 0.92 g	1.792	（1.75 ± 1.15）× 10⁹ a	58.92 ± 0.04 c
FJ13	苹果Apple	14.18 ± 1.08 g	1.778	（5.55 ± 2.70）× 10⁸ b	86.97 ± 0.05 b
FX25	香葱Scallion	14.57 ± 0.83 g	1.727	（7.69 ± 1.83）× 10⁷ c	98.19 ± 0.00 a
FX69	西瓜Watermelon	14.68 ± 1.75 g	1.715	（7.50 ± 0.56）× 10⁷ c	98.23 ± 0.00 a
FJ20	苹果Apple	20.75 ± 0.93 f	1.564	（7.45 ± 2.75）× 10⁷ c	98.25 ± 0.01 a
SN37	香梨Banana	20.96 ± 0.72 f	1.516	（4.95 ± 0.05）× 10⁷ c	98.84 ± 0.00 a
FX52	牛奶Milk	21.37 ± 2.12 ef	1.480	（4.00 ± 0.56）× 10⁷ c	99.06 ± 0.00 a
FN10	苹果Apple	21.91 ± 2.29 def	1.447	（1.50 ± 0.42）× 10⁷ c	99.64 ± 0.00 a
FN7	瓜类Melons	22.05 ± 1.47 def	1.414	（1.20 ± 3.53）× 10⁷ c	99.71 ± 0.01 a
HN89	香梨Banana	22.16 ± 0.37 def	1.402	（9.50 ± 0.00）× 10⁶ c	99.77 ± 0.01 a
FN9	苹果Apple	22.42 ± 0.59 cdef	1.367	（8.20 ± 0.28）× 10⁶ c	99.81 ± 0.00 a
FX59	蒲公英Dandelion	23.48 ± 0.00 bcde	1.333	（7.10 ± 0.70）× 10⁶ c	99.83 ± 0.01 a
FX43	大葱Leek	23.69 ± 2.15 bcd	1.332	（6.50 ± 2.82）× 10⁶ c	99.85 ± 0.01 a
FX70	西瓜Watermelon	23.70 ± 0.56 bcd	1.312	（4.87 ± 0.14）× 10⁶c	99.89 ± 0.00 a
FX51	牛奶Milk	23.72 ± 0.79 bcd	1.303	（3.65 ± 1.20）× 10⁶ c	99.91 ± 0.01 a
FX42	大葱Leek	23.76 ± 2.15 bcd	1.265	（3.60 ± 0.70）× 10⁶ c	99.92 ± 0.00 a
FN12	瓜类Melons	23.82 ± 1.14 bcd	1.239	（3.15 ± 0.21）× 10⁶ c	99.93 ± 0.02 a
FX74	西瓜Watermelon	24.06 ± 0.64 abcd	1.233	（2.95 ± 0.21）× 10⁶ c	99.93 ± 0.00 a
FX41	大葱Leek	24.61 ± 0.91 abc	1.224	（1.70 ± 0.00）× 10⁶ c	99.96 ± 0.01 a
FN11	苹果Apple	25.31 ± 0.65 ab	1.202	（5.85 ± 0.77）× 10⁵ c	99.98 ± 0.01 a
FX72	西瓜Watermelon	25.37 ± 1.94 ab	1.181	（3.50 ± 1.06）× 10⁵ c	99.99 ± 0.00 a
FX1	香葱Scallion	26.11 ± 0.40 a	1.116	（3.20 ± 0.42）× 10⁵ c	99.99 ± 0.01 a
E. amylovora			1.801	（4.26 ± 0.90）× 10⁹ a

菌株 Strain	分离来源 Sampling source	抑菌圈直径/mm The inhibition zone diameters	共培养Co-culture
			梨火疫病菌E. amylovora		抑菌率/% Inhibitive rate
			OD₆₀₀	活菌数/（CFU · mL^-1） Number of viable bacteria	抑菌率/% Inhibitive rate
FX68	西瓜Watermelon	14.10 ± 0.92 g	1.792	（1.75 ± 1.15）× 10⁹ a	58.92 ± 0.04 c
FJ13	苹果Apple	14.18 ± 1.08 g	1.778	（5.55 ± 2.70）× 10⁸ b	86.97 ± 0.05 b
FX25	香葱Scallion	14.57 ± 0.83 g	1.727	（7.69 ± 1.83）× 10⁷ c	98.19 ± 0.00 a
FX69	西瓜Watermelon	14.68 ± 1.75 g	1.715	（7.50 ± 0.56）× 10⁷ c	98.23 ± 0.00 a
FJ20	苹果Apple	20.75 ± 0.93 f	1.564	（7.45 ± 2.75）× 10⁷ c	98.25 ± 0.01 a
SN37	香梨Banana	20.96 ± 0.72 f	1.516	（4.95 ± 0.05）× 10⁷ c	98.84 ± 0.00 a
FX52	牛奶Milk	21.37 ± 2.12 ef	1.480	（4.00 ± 0.56）× 10⁷ c	99.06 ± 0.00 a
FN10	苹果Apple	21.91 ± 2.29 def	1.447	（1.50 ± 0.42）× 10⁷ c	99.64 ± 0.00 a
FN7	瓜类Melons	22.05 ± 1.47 def	1.414	（1.20 ± 3.53）× 10⁷ c	99.71 ± 0.01 a
HN89	香梨Banana	22.16 ± 0.37 def	1.402	（9.50 ± 0.00）× 10⁶ c	99.77 ± 0.01 a
FN9	苹果Apple	22.42 ± 0.59 cdef	1.367	（8.20 ± 0.28）× 10⁶ c	99.81 ± 0.00 a
FX59	蒲公英Dandelion	23.48 ± 0.00 bcde	1.333	（7.10 ± 0.70）× 10⁶ c	99.83 ± 0.01 a
FX43	大葱Leek	23.69 ± 2.15 bcd	1.332	（6.50 ± 2.82）× 10⁶ c	99.85 ± 0.01 a
FX70	西瓜Watermelon	23.70 ± 0.56 bcd	1.312	（4.87 ± 0.14）× 10⁶c	99.89 ± 0.00 a
FX51	牛奶Milk	23.72 ± 0.79 bcd	1.303	（3.65 ± 1.20）× 10⁶ c	99.91 ± 0.01 a
FX42	大葱Leek	23.76 ± 2.15 bcd	1.265	（3.60 ± 0.70）× 10⁶ c	99.92 ± 0.00 a
FN12	瓜类Melons	23.82 ± 1.14 bcd	1.239	（3.15 ± 0.21）× 10⁶ c	99.93 ± 0.02 a
FX74	西瓜Watermelon	24.06 ± 0.64 abcd	1.233	（2.95 ± 0.21）× 10⁶ c	99.93 ± 0.00 a
FX41	大葱Leek	24.61 ± 0.91 abc	1.224	（1.70 ± 0.00）× 10⁶ c	99.96 ± 0.01 a
FN11	苹果Apple	25.31 ± 0.65 ab	1.202	（5.85 ± 0.77）× 10⁵ c	99.98 ± 0.01 a
FX72	西瓜Watermelon	25.37 ± 1.94 ab	1.181	（3.50 ± 1.06）× 10⁵ c	99.99 ± 0.00 a
FX1	香葱Scallion	26.11 ± 0.40 a	1.116	（3.20 ± 0.42）× 10⁵ c	99.99 ± 0.01 a
E. amylovora			1.801	（4.26 ± 0.90）× 10⁹ a

处理Treatment	稀释倍数Dilution ratio	3 d		5 d		平均防效/% Mean of the control efficacy
处理Treatment	稀释倍数Dilution ratio	花腐率/% Flower rot rate	防效/% Control efficacy	花腐率/% Flower rot rate	防效/% Control efficacy	平均防效/% Mean of the control efficacy
发酵液Fermentation broth	—	18.74 ± 0.11 bcde	61.50 ± 0.23 cde	52.11 ± 0.07 b	38.54 ± 0.08 c	50.02 ± 11.48 abc
除菌发酵滤液 Filtered fermentation broth	—	16.59 ± 0.02 cdef	65.94 ± 0.05 bcd	44.30 ± 0.08 bc	47.76 ± 0.10 bc	56.85 ± 9.09 abc
	5	22.96 ± 0.02 bcde	52.84 ± 0.04 cde	44.61 ± 0.06 bc	47.40 ± 0.07 bc	50.12 ± 2.72 abc
	10	23.49 ± 0.02 bcd	51.77 ± 0.05 cde	44.87 ± 0.04 bc	47.09 ± 0.05 bc	49.43 ± 2.34 abc
	50	27.21 ± 0.09 bc	44.12 ± 0.18 de	45.30 ± 0.09 bc	46.58 ± 0.11 bc	45.35 ± 1.23 bc
	100	29.18 ± 0.08 b	40.09 ± 0.17 e	58.52 ± 0.09 b	30.99 ± 0.11 c	35.54 ± 4.55 c
脂肽类物质粗提液 Crude extract of lipopeptide	—	4.89 ± 0.02 g	89.95 ± 0.04 ab	26.98 ± 0.02 d	68.18 ± 0.03 a	79.07 ± 10.89 ab
	5	5.26 ± 0.04 fg	89.20 ± 0.08 ab	33.07 ± 0.06 cd	61.00 ± 0.08 ab	75.10 ± 14.10 ab
	10	5.86 ± 0.04 fg	87.96 ± 0.10 ab	33.43 ± 0.08 cd	60.57 ± 0.09 ab	74.27 ± 13.70 ab
	50	11.51 ± 0.05 efg	76.36 ± 0.10 abc	44.53 ± 0.01 bc	47.49 ± 0.01 bc	61.93 ± 14.44 abc
	100	12.05 ± 0.01 defg	75.26 ± 0.02 abc	45.42 ± 0.09 bc	46.44 ± 0.10 bc	60.85 ± 14.41 abc
农用链霉素 Agricultural streptomycin	4 000	3.18 ± 0.01 g	93.47 ± 0.01 a	23.04 ± 0.03 d	72.83 ± 0.03 a	83.15 ± 10.32 a
H₂O对照 Control		48.68 ± 0.09 a		84.82 ± 0.07 a

处理Treatment	稀释倍数Dilution ratio	3 d		5 d		平均防效/% Mean of the control efficacy
处理Treatment	稀释倍数Dilution ratio	花腐率/% Flower rot rate	防效/% Control efficacy	花腐率/% Flower rot rate	防效/% Control efficacy	平均防效/% Mean of the control efficacy
发酵液Fermentation broth	—	18.74 ± 0.11 bcde	61.50 ± 0.23 cde	52.11 ± 0.07 b	38.54 ± 0.08 c	50.02 ± 11.48 abc
除菌发酵滤液 Filtered fermentation broth	—	16.59 ± 0.02 cdef	65.94 ± 0.05 bcd	44.30 ± 0.08 bc	47.76 ± 0.10 bc	56.85 ± 9.09 abc
	5	22.96 ± 0.02 bcde	52.84 ± 0.04 cde	44.61 ± 0.06 bc	47.40 ± 0.07 bc	50.12 ± 2.72 abc
	10	23.49 ± 0.02 bcd	51.77 ± 0.05 cde	44.87 ± 0.04 bc	47.09 ± 0.05 bc	49.43 ± 2.34 abc
	50	27.21 ± 0.09 bc	44.12 ± 0.18 de	45.30 ± 0.09 bc	46.58 ± 0.11 bc	45.35 ± 1.23 bc
	100	29.18 ± 0.08 b	40.09 ± 0.17 e	58.52 ± 0.09 b	30.99 ± 0.11 c	35.54 ± 4.55 c
脂肽类物质粗提液 Crude extract of lipopeptide	—	4.89 ± 0.02 g	89.95 ± 0.04 ab	26.98 ± 0.02 d	68.18 ± 0.03 a	79.07 ± 10.89 ab
	5	5.26 ± 0.04 fg	89.20 ± 0.08 ab	33.07 ± 0.06 cd	61.00 ± 0.08 ab	75.10 ± 14.10 ab
	10	5.86 ± 0.04 fg	87.96 ± 0.10 ab	33.43 ± 0.08 cd	60.57 ± 0.09 ab	74.27 ± 13.70 ab
	50	11.51 ± 0.05 efg	76.36 ± 0.10 abc	44.53 ± 0.01 bc	47.49 ± 0.01 bc	61.93 ± 14.44 abc
	100	12.05 ± 0.01 defg	75.26 ± 0.02 abc	45.42 ± 0.09 bc	46.44 ± 0.10 bc	60.85 ± 14.41 abc
农用链霉素 Agricultural streptomycin	4 000	3.18 ± 0.01 g	93.47 ± 0.01 a	23.04 ± 0.03 d	72.83 ± 0.03 a	83.15 ± 10.32 a
H₂O对照 Control		48.68 ± 0.09 a		84.82 ± 0.07 a

处理 Treatment	稀释倍数Dilution ratio	7 d		15 d		平均防效/% Mean of the control efficacy
处理 Treatment	稀释倍数Dilution ratio	病情指数 Disease index	防效/% Control efficacy	病情指数 Disease index	防效/% Control efficacy	平均防效/% Mean of the control efficacy
发酵液Fermentation broth	—	4.34 ± 1.17 de	80.27 ± 0.05 ab	12.28 ± 1.43 cd	62.80 ± 0.04 ab	71.54 ± 8.74 ab
除菌发酵滤液 Filtered fermentation broth	—	4.17 ± 0.83 de	81.05 ± 0.04 ab	11.67 ± 1.67 cd	64.65 ± 0.05 ab	72.85 ± 8.20 ab
	5	5.40 ± 1.80 cde	75.47 ± 0.08 abc	17.25 ± 10.34 bc	47.73 ± 0.31 abc	61.60 ± 13.87 ab
	10	6.51 ± 2.06 cde	70.42 ± 0.09 abc	17.25 ± 6.03 bc	47.73 ± 0.18 abc	59.08 ± 11.35 ab
	50	6.59 ± 2.44 cde	70.03 ± 0.11 abc	20.44 ± 6.00 bc	38.05 ± 0.18 bc	54.04 ± 15.99 ab
	100	7.22 ± 2.08 cd	67.17 ± 0.09 bc	20.56 ± 3.42 bc	37.71 ± 0.10 bc	52.44 ± 14.73 ab
脂肽类物质粗提液 Crude extract of lipopeptide	—	4.21 ± 0.58 de	80.85 ± 0.03 ab	11.74 ± 1.24 cd	64.42 ± 0.04 ab	72.64 ± 8.22 ab
	5	4.72 ± 0.28 de	78.54 ± 0.01 abc	16.39 ± 1.39 bcd	50.34 ± 0.04 abc	63.81 ± 13.47 ab
	10	8.52 ± 2.86 bc	61.26 ± 0.13 cd	16.46 ± 6.54 bcd	50.12 ± 0.20 abc	55.69 ± 5.57 ab
	50	8.61 ± 0.28 bc	60.86 ± 0.01 cd	18.61 ± 3.06 bc	43.60 ± 0.09 bc	52.23 ± 8.63 ab
	100	11.43 ± 0.00 b	48.05 ± 0.00 d	25.71 ± 0.00 ab	22.08 ± 0.00 c	35.07 ± 12.99 b
农用链霉素 Agricultural streptomycin	4 000	3.36 ± 1.19 e	84.73 ± 0.05 a	6.63 ± 0.73 d	79.92 ± 0.02 a	82.33 ± 2.41 a
H₂O对照 Control		22.00 ± 0.05 a		33.00 ± 0.16 a