Screening， Identification and Efficacy Determination of Biocontrol Bacteria against Pear Fire Blight

doi:10.13560/j.cnki.biotech.bull.1985.2025-0862

Abstract

Abstract:

Objective Pear fire blight, caused by Erwinia amylovora, is a destructive bacterial disease causing a significant threat to pear and apple industries. This study aimed to screen strains with strong antagonistic activity against E. amylovora from the branches of Crataegus cuneata and Malus sieversii, providing microbial resources for the biological control of E. amylovora. Method The plate confrontation method was applied to screen the antagonistic strains against E. amylovora. The physiological and biochemical tests, 16S rDNA and rpoB gene sequencing were to determine the taxonomic status of these antagonistic strains. The methanol extractionwas to obtain in vitro antibacterial substances inhibiting E. amylovora. Thin-layer chromatography (TLC) was used to separate active components, and liquid chromatography-mass spectrometry (LC-MS) was used to identify the lipopeptide types in the fermentation broth. Bioinformatics analysis was conducted on the whole-genome data of KXI strain to explore its secondary metabolite gene clusters. The greenhouse control efficacy was tested on detached leaves, twigs, and young fruits of Korla fragrant pear (Pyrus sinkiangensis Yü). Result Seven strains with strong antagonistic activity against E. amylovora were isolated, among which KX1 showed the strongest antagonistic effect. Based on physiological and biochemical characteristics, 16S rDNA and rpoB gene sequence analysis, strain KX1 was identified as Bacillus velezensis. Preliminary detection revealed that the methanol extract of the B. velezensis KX1 contained lipopeptides, and TLC and LC-MS analysis revealed that surfactins were the major component of the lipopeptides. The crude lipopeptide extract retained antibacterial activity after treatments with different temperatures, pH values, storage times. The genome of strain KX1 was 4.1 Mb in size with a GC content of 46.33%. antiSMASH predicted that one secondary metabolite gene cluster shared 80% homology with the gene cluster related to fengycin. Greenhouse control efficacy tests showed that the disease index of immature Korla fragrant pear young fruits, leaves, and twigs in the protective experiment significantly reduced after inoculation with strain KX1. The disease prevention effects was 100%, 55.33%, and 70.65%, respectively, with protective efficacy being superior to therapeutic efficacy. Conclusion Bacillus velezensis KX1 effectively inhibit the activity of E. amylovora and has potential for biological control applications.

Key words: pear fire blight, antagonistic bacteria, Bacillus velezensis, biological control, control efficacy

ZHANG Man, DANG Jing-bo, JIANG Yuan, WEI Jie, XING Jie, WANG Zhe, SUN Li. Screening， Identification and Efficacy Determination of Biocontrol Bacteria against Pear Fire Blight[J]. Biotechnology Bulletin, 2026, 42(1): 279-293.

Figures/Tables 13

Table 1 List of primers used in this study

项目

Item

引物名称Primer name

引物序列

Primer sequence （5′-3′）

目的片段大小

Length of target fragment （bp）

16S rDNA^［32］

27F

1492R

AGTTTGATCMTGGCTCAG

GGTTACCTTGTTACGACTT

1 500

rpoB^［33-34］

2292F

3354R

AGGTCAACTAGTTCAGTATGGAC

AAGAACCGTAACCGGCAACTT

500

枯草杆菌蛋白酶

Subtilisin

Qk1F

Qk1R

CTTAAACGTCAGAGGCGGAG

ATTGTGCAGCTGCTTGTACG

704

表面活性素

Surfactin

srfAAF

srfAA R

GAAAGAGCGGCTGCTGAAAC

CCCAATATTGCCGCAATGAC

273

丰原素

Fengycin

fenDF

fenDR

CCTGCAGAAGGAGAAGTGAAG

TGCTCATCGTCTTCCGTTTC

293

伊枯草菌素

Iturin

ituCF

ituCR

TTCACTTTTGATCTGGCGAT

CGTCCGGTACATTTTCAC

575

枯草菌素

Subtilin

spasF

spasR

GGTTTGTTGGATGGAGCTGT

GCAAGGAGTCAGAGCAAGGT

375

假定蛋白

Yndj

yndjF

yndjR

CAGAGCGACAGCAATCACAT

TGAATTTCGGTCCGCTTATC

212

Fig. 1 Screening of antagonistic bacteria and the inhibitory effect against E. amylovora C1A: Antagonism of seven strains against E. amylovora C1. B: Diameters of inhibition zones of seven strains. Different letters indicate significant different (P<0.05). The same below

Table 2 Physiological and biochemical characteristics of antagonistic bacteria

菌株Strain	形状Shape	菌落颜色Color of colony	能动性Motility	革兰氏染色Gram staining	淀粉水解Starch hydrolysis	V-P试验V-P test	MR试验MR test	硝酸盐还原试验Nitrate reduction test	靛基质试验Indole test	接触酶试验Catalase test
KX1	杆状Rod	白色White	+	+	+	-	+	-	-	+
KX2	杆状Rod	白色White	+	+	+	-	+	-	-	+
KX3	杆状Rod	白色White	+	+	+	+	-	-	-	+
P1	杆状Rod	白色White	+	+	+	-	+	-	-	+
P2	杆状Rod	白色White	+	+	+	-	+	-	-	+
P3	杆状Rod	白色White	+	+	+	-	+	-	-	+
Z4	杆状Rod	白色White	+	+	+	+	-	-	-	+

Fig. 2 Phylogenetic tree construction of 7 antagonistic strains based on 16S rDNA and rpoB genes

Fig. 3 Antibacterial effects of seven antagonistic supernatants against E. amylovora C1A: Antagonistic activities of supernatants from 7 antagonistic strains against E. amylovora C1. B: Diameters of inhibition zones of 7 antagonistic supernatants

Fig. 4 Antagonistic effects of crude extract from strain KX1 against E. amylovora C1EM: Extracted with methanol; CS: cell-free supernatant; CM: methanol control; BC: bacterial solution; BD: cell disruption

Fig. 5 Identification of KX1 crude extractA: Results of functional gene amplification of strain KX1 crude extract. B: TLC results of crude extract. M: DL 2000 plus DNA marker. NC: Negative control; 1, 2, 3, 4, 5, 6 indicate genes related to antibiotic synthesis fenD, Qk1, yndJ, spas, srfAA, and ituC, respectively

Fig. 6 LC-MS analysis of lipopeptides from strain KX1

Fig. 7 Stability of strain KX1 crude extractA: Inhibition effect of strain KX1crude extract on E. amylovora C1under different temperature treatment. B: Inhibition effect of KX1 crude extract on E. amylovora C1 under different pH treatment. C: Inhibition effect of KX1crude extract on E. amylovora C1 under different storage days. There is no significant difference between values of the same letter (P>0.05)

Fig. 8 Whole genome sequence analysis of strain KX1A: Circular genome circle map of strain KX1, the circular map showed CDS of DNA in the forward direction, sequence in the reverse direction, rRNA and tRNA, GC skew graph, and GC ratio graph; B: KEGG function classification of strain KX1; C: putative gene clusters in biosynthesis of secondary metabolites of strain KX1

Fig. 9 Effects of B. velerensis KX1 on control of E. amylovora C1 in Pyrus sinkiangensis Yü leaves (A), twigs (B), and immature fruits (C)NC: Negative control; PC: positive control; TrC: treatment control; PTr: protective treatment; CTr: curative treatment. The same below

Fig. 10 Disease index of E. amylovora C1 in Pyrus sinkiangensis Yü leaves (A), twigs (B) and immature fruits (C)

Table 3 Efficacy of B. velerensis KX1 on fire blight disease

处理 Treatment		叶片 Leaves		枝条 Twigs		幼果 Fruit
处理 Treatment		病情指数 Disease index	防病效果 Control efficacy （%）	病情指数 Disease index	防病效果 Control efficacy （%）	病情指数 Disease index	防病效果 Control efficacy （%）
阴性对照 Negative control		2.56±0.56^h		0.00±0.00^f		0.00±0.00^d
阳性对照 Positive control		69.23±0.74^a		62.96±1.5^a		75.00±0.85^a
KX1处理 Treatments by KX1	TrC	9.64±1.2^g		3.70±0.88^e		0.00±0.00^d
	PTr	30.92±1.1^d	55.33±1.36^a	18.52±1.9^c	70.65±2.11^b	0.00±0.00^d	100^a
	CTr	31.62±0.64^c	54.33±2.02^a	25.93±0.89^b	58.82±6.01^c	62.50±0.75^b	16.67±7.12^c
链霉素处理 Treatments by streptomycin	TrC	10.26±0.41^f		0.00±0.00^f		0.00±0.00^d
	PTr	30.85±1.3^e	55.44±3.33^a	7.40±0.89^d	88.25±4.70^a	37.50±1.25^c	40.00±9.46^b
	CTr	37.76±1.4^b	45.46±3.01^b	29.63±0.75^b	52.94±3.33^b	62.50±0.79^b	16.67±8.24^c

References 55

[1]	Klee SM, Sinn JP, McNellis TW. The apple fruitlet model system for fire blight disease [M]//Plant Innate Immunity. New York, NY: Springer New York, 2019: 187-198.
[2]	杨金花, 徐叶挺, 张校立. 梨火疫病研究进展 [J]. 分子植物育种, 2022, 20(3): 1003-1013.
	Yang JH, Xu YT, Zhang XL. Advances of fire blight in pear [J]. Mol Plant Breed, 2022, 20(3): 1003-1013.
[3]	Slack SM, Zeng Q, Outwater CA, et al. Microbiological examination of Erwinia amylovora exopolysaccharide ooze [J]. Phytopathology, 2017, 107(4): 403-411.
[4]	Thomson SV. The role of the stigma in fire blight infections [J]. Phytopathology, 1986, 76(5): 476.
[5]	Koczan JM, Lenneman BR, McGrath MJ, et al. Cell surface attachment structures contribute to biofilm formation and xylem colonization by Erwinia amylovora [J]. Appl Environ Microbiol, 2011, 77(19): 7031-7039.
[6]	Sun WB, Gong PJ, Zhao YC, et al. Current situation of fire blight in China [J]. Phytopathology, 2023, 113(12): 2143-2151.
[7]	刘凤权, 明亮, 赵延存, 等. 中国梨火疫病发生与防控进展 [J]. 落叶果树, 2023, 55(6): 1-7.
	Liu FQ, Ming L, Zhao YC, et al. Progress in the occurrence and control of pear fire blight in China [J]. Deciduous Fruits, 2023, 55(6): 1-7.
[8]	Ryu DK, Adhikari M, Choi DH, et al. Copper-based compounds against Erwinia amylovora: response parameter analysis and suppression of fire blight in apple [J]. Plant Pathol J, 2023, 39(1): 52-61.
[9]	McManus PS, Stockwell VO, Sundin GW, et al. Antibiotic use in plant agriculture [J]. Annu Rev Phytopathol, 2002, 40: 443-465.
[10]	Mikiciński A, Puławska J, Molzhigitova A, et al. Bacterial species recognized for the first time for its biocontrol activity against fire blight (Erwinia amylovora) [J]. Eur J Plant Pathol, 2020, 156(1): 257-272.
[11]	Din IU, Hu LN, Jiang Y, et al. Bacterial lipopeptides are effective against pear fire blight [J]. Microorganisms, 2024, 12(5): 896.
[12]	Halbwirth H, Fischer TC, Roemmelt S, et al. Induction of antimicrobial 3-deoxyflavonoids in pome fruit trees controls fire blight [J]. Z Für Naturforschung C, 2003, 58(11/12): 765-770.
[13]	Lee J, Jung WK, Ahsan SM, et al. Identification of Pantoea ananatis strain BCA19 as a potential biological control agent against Erwinia amylovora [J]. Front Microbiol, 2024, 15: 1493430.
[14]	Nguyen LTT, Park AR, Van Le V, et al. Exploration of a multifunctional biocontrol agent Streptomyces sp. JCK-8055 for the management of apple fire blight [J]. Appl Microbiol Biotechnol, 2024, 108(1): 49.
[15]	Johnson KB, Stockwell VO. Management of fire blight: a case study in microbial ecology [J]. Annu Rev Phytopathol, 1998, 36: 227-248.
[16]	Mechan Llontop ME, Hurley K, Tian L, et al. Exploring rain as source of biological control agents for fire blight on apple [J]. Front Microbiol. 2020, 11: 199.
[17]	Kim IY, Lew B, Zhao YF, et al. Biocontrol of fire blight via microcapsule-mediated delivery of the bacterial antagonist Pantoea agglomerans E325 to apple blossoms [J]. BioControl, 2022, 67(4): 433-442.
[18]	Ghimire B, Orellana R, Chowdhury SR, et al. Assessing biofungicides and host resistance against Rhizoctonia large patch in zoysia grass [J]. Pathogens, 2024, 13(10): 864.
[19]	白雪莹, 韩剑, 孙博源, 等. 梨火疫病和梨腐烂病生防潜力粘细菌的筛选鉴定及室内防效评价 [J]. 中国生物防治学报, 2023, 39(6): 1384-1397.
	Bai XY, Han J, Sun BY, et al. Screening and identification of biocontrol potential myxobacteria strains against fire blight and pear canker diseases and evaluation of indoor control efficacy [J]. Chin J Biol Control, 2023, 39(6): 1384-1397.
[20]	江星彤, 柏晓玉, 冯玉莲, 等. 极端耐旱链霉菌D67对梨火疫病原菌毒力因子的抑制作用 [J]. 微生物学通报, 2025, 52(7): 3165-3175.
	Jiang XT, Bai XY, Feng YL, et al. Inhibitory effects of extreme drought-resistant Streptomyces D67 on the virulence factors of Erwinia amylovora [J]. Microbiol China, 2025, 52(7): 3165-3175.
[21]	林胜楠, 吴梓菲, 王宁, 等. 梨火疫病生防菌的筛选及抑菌机理初探 [J]. 西北农业学报, 2025, 34(7): 1346-1355.
	Lin SN, Wu ZF, Wang N, et al. Screening of biocontrol bacteria against fire blight and preliminary study on its biocontrol mechanism [J]. Acta Agric Boreali Occidentalis Sin, 2025, 34(7): 1346-1355.
[22]	Sundin GW, Wang N. Antibiotic resistance in plant-pathogenic bacteria [J]. Annu Rev Phytopathol, 2018, 56: 161-180.
[23]	Sundin GW, Werner NA, Yoder KS, et al. Field evaluation of biological control of fire blight in the eastern United States [J]. Plant Dis, 2009, 93(4): 386-394.
[24]	Quan Zeng JP. Early events in fire blight infection and pathogenesis of Erwinia amylovora [J]. J Plant Pathol, 2021, 103: S13-S24.
[25]	Ngugi HK, Lehman BL, Madden LV. Multiple treatment meta-analysis of products evaluated for control of fire blight in the eastern United States [J]. Phytopathology, 2011, 101(5): 512-522.
[26]	车建美, 郑雪芳, 王阶平, 等. 一株产纤维素酶菌株的筛选、鉴定及全基因组分析 [J]. 生物技术通报, 2025, 41(3): 294-307.
	Che JM, Zheng XF, Wang JP, et al. Screening, identification and whole genome analysis of a cellulase producing strain [J]. Biotechnol Bull, 2025, 41(3): 294-307.
[27]	Anon. Diagnostics PM 7/20 (2) Erwinia amylovora [J]. EPPO Bull, 2013, 43(1): 21-45
[28]	马云涛, 胡丽娜, 孙文婧, 等. 梨火疫病拮抗菌JK2的筛选鉴定及发酵条件优化 [J]. 生物技术通报, 2024, 40(11): 202-213.
	Ma YT, Hu LN, Sun WJ, et al. Screening and identification of antagonistic bacterium JK2 against fire blight disease and the optimization of its fermentation conditions [J]. Biotechnol Bull, 2024, 40(11): 202-213.
[29]	方中达. 植病研究方法 [M]. 3版. 北京: 中国农业出版社, 1998.
	Fang ZD. Research methods of plant diseases [M]. 3rd ed. Beijing: China Agriculture Press, 1998.
[30]	Krieg NR, Staley JT, Brown DR, et al. Bergey’s manual^® of systematic bacteriololgy[M]. USA: The Williams &Wilkins Co, 1986.
[31]	东秀珠, 蔡妙英. 常见细菌系统鉴定手册 [M]. 北京: 科学出版社, 2001.
	Dong XZ, Cai MY. Handbook of identification of common bacterial systems [M]. Beijing: Science Press, 2001.
[32]	Turner S, Pryer KM, Miao VPW, et al. Investigating deep phylogenetic relationships among cyanobacteria and plastids by small subunit rRNA sequence analysis [J]. J Eukaryot Microbiol, 1999, 46(4): 327-338.
[33]	Case RJ, Boucher Y, Dahllöf I, et al. Use of 16S rRNA and rpoB Genes as molecular markers for microbial ecology studies [J]. Appl Environ Microbiol, 2007, 73(1): 278-288.
[34]	Ki JS, Zhang W, Qian PY. Discovery of marine Bacillus species by 16S rRNA and rpoB comparisons and their usefulness for species identification [J]. J Microbiol Meth, 2009, 77(1): 48-57.
[35]	Joshi R, McSpadden Gardener BB. Identification and characterization of novel genetic markers associated with biological control activities in Bacillus subtilis [J]. Phytopathology^® , 2006, 96(2): 145-154.
[36]	Chen XH, Scholz R, Borriss M, et al. Difficidin and bacilysin produced by plant-associated Bacillus amyloliquefaciens are efficient in controlling fire blight disease [J]. J Biotechnol, 2009, 140(1/2): 38-44.
[37]	Cui Z, Hu L, Zeng L, Meng W, et al. Isolation and characterization of Priestia megaterium KD7 for the biological control of pear fire blight [J]. Front Microbiol. 2023, 14:1099664.
[38]	Medhioub I, Cheffi M, Tounsi S, et al. Study of Bacillus velezensis OEE1 potentialities in the biocontrol against Erwinia amylovora, causal agent of fire blight disease of rosaceous plants [J]. Biol Control, 2022, 167: 104842.
[39]	李晓妹, 韩丽丽, 何亚南, 等. 20个苹果品种(类型)对梨火疫病菌的抗病性评价 [J]. 植物检疫, 2022, 36(4): 6-12.
	Li XM, Han LL, He YN, et al. Evaluation of resistance of 20 apple varieties (types) to Pyrus Pyricularia [J]. Plant Quar, 2022, 36(4): 6-12.
[40]	Stockwell VO, Johnson KB, Sugar D, et al. Mechanistically compatible mixtures of bacterial antagonists improve biological control of fire blight of pear [J]. Phytopathology, 2011, 101(1): 113-123.
[41]	刘思慧, 明珂, 陈国庆, 等. 一种抗稻曲病菌的摩氏假单胞菌JP2-207及其抗性机制初探 [J]. 中国稻米, 2024, 30(2): 13-17.
	Liu SH, Ming K, Chen GQ, et al. A preliminary study on the mechanism of Pseudomonas mosselii JP2-207 against rice false smut fungus Ustilaginoidea virens [J]. China Rice, 2024, 30(2): 13-17.
[42]	Wang J, Xie XY, Li B, et al. Complete genome analysis and antimicrobial mechanism of Bacillus velezensis GX0002980 reveals its biocontrol potential against mango anthracnose disease [J]. Microbiol Spectr, 2025, 13(6): e02685-24.
[43]	Tola SD, Muleta D, Assefa F, et al. Characterization and identification of hot pepper-associated endospore-forming bacteria with potential applications as biofertilizers and in biocontrol of pepper wilt pathogens [J]. BMC Microbiol, 2025, 25(1): 198.
[44]	Ongena M, Jacques P. Bacillus lipopeptides: versatile weapons for plant disease biocontrol [J]. Trends Microbiol, 2008, 16(3): 115-125.
[45]	贺旭, 韩剑, 盛强, 等. 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 X, Han J, Sheng Q, et al. Screening of antagonistic bacterium FX1 against Erwinia amylovora and its control effect of the antibacterial substances on fire blight [J]. Acta Hortic Sin, 2023, 50(5): 1118-1129.
[46]	杨瑞先, 刘萍, 王祖华, 等. 牡丹根腐病原菌拮抗细菌抑菌活性物质分析 [J]. 生物技术通报, 2022, 38(2): 57-66.
	Yang RX, Liu P, Wang ZH, et al. Analysis of antimicrobial active metabolites from antagonistic strains against Fusarium solani [J]. Biotechnol Bull, 2022, 38(2): 57-66.
[47]	付克剑, 苏友波, 崔永和, 等. 荧光假单胞菌YG-1对烟草根黑腐病菌的体外拮抗 [J]. 中国烟草科学, 2024, 45(1): 39-47.
	Fu KJ, Su YB, Cui YH, et al. In vitro antagonism of Pseudomonas fluorescens YG-1 against Thielaviopsis basicola [J]. Chin Tob Sci, 2024, 45(1): 39-47.
[48]	张雯, 卞丹, 沈燕秋, 等. 枯草芽胞杆菌抑菌活性物质鉴定、抑菌特性及发酵条件优化 [J]. 中国食品学报, 2017, 17(12): 105-115.
	Zhang W, Bian D, Shen YQ, et al. Identification and characterization of antibacterial metabolites and optimization of cultural conditions for Bacillus subtilis [J]. J Chin Inst Food Sci Technol, 2017, 17(12): 105-115.
[49]	Soussi S, Essid R, Hardouin J, et al. Utilization of grape seed flour for antimicrobial lipopeptide production by Bacillus amyloliquefaciens C5 strain [J]. Appl Biochem Biotechnol, 2019, 187(4): 1460-1474.
[50]	周池, 周诗晶, 陶禹, 等. 贝莱斯芽胞杆菌XY40-1: 全基因组特征分析及对辣椒疫病的生物防治效果评价 [J]. 微生物学报, 2024, 64(12): 4882-4901.
	Zhou C, Zhou SJ, Tao Y, et al. Bacillus velezensis XY40-1: whole genomic characteristics and biocontrol effects on pepper Phytophthora blight [J]. Acta Microbiol Sin, 2024, 64(12): 4882-4901.
[51]	张雨薇, 唐培培, 刘子琦, 等. 一株抑制烟草疫霉的耐受盐芽胞杆菌(Bacillus halotolerans)的分离、鉴定及全基因组测序分析 [J]. 微生物学通报, 2025, 52(4): 1760-1774.
	Zhang YW, Tang PP, Liu ZQ, et al. Isolation, identification, and whole genome sequencing of a Bacillus halotolerans strain inhibiting Phytophthora nicotianae [J]. Microbiol China, 2025, 52(4): 1760-1774.
[52]	Fazle Rabbee M, Baek KH. Antimicrobial activities of lipopeptides and polyketides of Bacillus velezensis for agricultural applications [J]. Molecules, 2020, 25(21): 4973.
[53]	Hamley IW. Lipopeptides: from self-assembly to bioactivity [J]. Chem Commun, 2015, 51(41): 8574-8583.
[54]	刘振亚, 苏宣乐, 唐丽, 等. 梨火疫病抗性评价体系的建立及其种质资源抗性鉴定 [J]. 福建农业学报, 2024, 39(5): 609-614.
	Liu ZY, Su XL, Tang L, et al. An evaluation protocol for fire blight resistance of pear cultivars [J]. Fujian J Agric Sci, 2024, 39(5): 609-614.
[55]	Farkas Á, Mihalik E, Dorgai L, et al. Floral traits affecting fire blight infection and management [J]. Trees, 2012, 26(1): 47-66.