Screening of Antagonistic Bacterium FX1 Against Erwinia amylovora and Its Control Effect of the Antibacterial Substances on Fire Blight

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

Acta Horticulturae Sinica ›› 2023, Vol. 50 ›› Issue (5): 1118-1129.doi: 10.16420/j.issn.0513-353x.2022-0048

• Plant Protection • Previous Articles Next Articles

Screening of Antagonistic Bacterium FX1 Against Erwinia amylovora and Its Control Effect of the Antibacterial Substances on Fire Blight

HE Xu¹, HAN Jian¹^,², SHENG Qiang³, LUO Ming¹^,²^,^*(), BAO Huifang⁴, HUANG Wei³

¹College of Agriculture，Xinjiang Agricultural University，Urumqi 830052，China
²Key Laboratory of Monitoring and Safety Control of Agricultural and Forestry Pests，Universities of Xinjiang Uygur Autonomous Region，Urumqi 830052，China
³Xinjiang Bazhou Academy of Agricultural Sciences，Korla，Xinjiang 841003，China
⁴Institute of Applied Microbiology，Xinjiang Academy of Agricultural Sciences，Urumqi 830091，China

Received:2022-11-09 Revised:2023-01-23 Online:2023-05-25 Published:2023-05-31
Contact: LUO Ming E-mail:luomingxjau@sina.com

Abstract

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

CLC Number:

S661.2

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.

Figures/Tables 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

Screening of Antagonistic Bacterium FX1 Against Erwinia amylovora and Its Control Effect of the Antibacterial Substances on Fire Blight

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处理 Treatment	稀释倍数Dilution	7 d		15d		平均防效/% Average efficacy
处理 Treatment	稀释倍数Dilution	病情指数 Disease index	防效/% Control efficacy	病情指数 Disease index	防效/% Control efficacy	平均防效/% Average efficacy
发酵液 Fermentation broth	1	6.67 ± 0.00 c	70.57 ± 0.00 ab	18.33 ± 11.14 abc	41.49 ± 0.36 ab	56.03 ± 14.54 abc
除菌发酵滤液 Filtered fermentation broth	1	8.89 ± 3.14 bc	60.78 ± 0.14 abc	17.78 ± 8.31 abc	43.26 ± 0.27 ab	52.02 ± 8.76 abc
除菌发酵滤液	5	9.68 ± 4.96 bc	57.28 ± 0.22 abc	20.16 ± 2.53 abc	35.67 ± 0.08 b	46.48 ± 10.81 abc
Filtered fermentation broth	10	10.00 ± 1.63 bc	55.88 ± 0.07 bc	21.33 ± 4.99 abc	31.91 ± 0.16 b	43.90 ± 11.99 abc
	50	13.33 ± 14.40 abc	41.18 ± 0.64 cd	22.22 ± 8.31 abc	29.07 ± 0.27 b	35.13 ± 6.06 bc
	100	19.67 ± 3.68 ab	31.40 ± 0.13 d	25.67 ± 4.92 ab	18.09 ± 0.16 b	24.75 ± 6.66 c
脂肽类物质粗提液 Crude extract of lipopeptide	1	6.00 ± 2.83 c	73.53 ± 0.12 ab	15.78 ± 5.34 bc	49.65 ± 0.17 ab	61.59 ± 11.94 ab
	5	9.00 ± 6.48 bc	61.80 ± 0.27 abc	16.67 ± 2.36 abc	46.81 ± 0.08 ab	54.30 ± 7.50 abc
	10	10.00 ± 4.08 bc	55.88 ± 0.18 bc	20.00 ± 10.80 abc	36.17 ± 0.34 b	46.03 ± 9.86 abc
	50	10.28 ± 4.53 bc	54.65 ± 0.20 bc	20.22 ± 3.81 abc	35.46 ± 0.12 b	45.06 ± 9.60 abc
	100	10.33 ± 4.64 bc	54.41 ± 0.20 bc	20.33 ± 3.68 abc	35.11 ± 0.12 b	44.76 ± 9.65 abc
农用链霉素 Agricultural streptomycin	4 000	4.33 ± 0.47 c	80.88 ± 0.02 a	8.67 ± 0.94 c	72.34 ± 0.03 a	76.61 ± 4.24 a
H₂O对照 Control		22.67 ± 3.77 a		31.33 ± 3.40 a

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