软腐病是导致辣椒采后贮运期间腐烂损失最严重的细菌性病害。为了降低辣椒因软腐病而造成的损失,该研究从腐烂辣椒中分离病原菌并筛选拮抗菌以控制该病原菌引起的软腐病。结果显示腐烂辣椒中分离的病原菌XC1为胡萝卜软腐病果胶杆菌巴西亚种(Pectobacterium carotovorum subsp. brasiliense,Pcb),Pcb XC1具有强致病能力,104 CFU/mL及以上浓度在室温条件下(25 ℃)3 d内能够导致辣椒100%腐烂。进一步,采用双层琼脂平板法筛选出一株拮抗菌1151,经16S rDNA鉴定为枯草芽孢杆菌。体外抑菌结果表明枯草芽孢杆菌1151菌体及所产抗菌肽均对Pcb XC1有抑制作用。辣椒体内控病结果表明枯草芽孢杆菌1151菌体及所产抗菌肽均能降低辣椒的腐烂率并减小病斑直径,其中抗菌肽处理效果更佳。抗菌肽能够减少辣椒体内99%的Pcb XC1数量。扫描电镜结果也表明抗菌肽处理后的辣椒表面软腐病症更少,无明显细胞壁降解引起的褶皱和碎片。一方面从蛋白层面,经LC-MS/MS鉴定到了BHP、SP-B和Hb 98-114共3个抗菌肽。另一方面从基因层面,经BAGEL4数据库从枯草芽孢杆菌1151基因组中鉴定到了一个细菌素基因簇,含有2个细菌素(Subtilosin A和Subtilosin Sbox)。该研究表明枯草芽孢杆菌1151能够减轻辣椒由软腐病引起的腐烂损失,其所产抗菌肽发挥了重要作用,对辣椒采后的贮藏保鲜方法具有重要参考意义。
Soft rot is the most serious bacterial disease causing rot loss of postharvest pepper during storage and transportation. In order to reduce the loss of pepper caused by soft rot, pathogen was isolated from rotten pepper and antagonist was screened to control the soft rot caused by this pathogen. The result showed that the pathogen XC1 isolated from rotten pepper was Pecobacterium carotovorum subsp. brasiliense (Pcb). Pcb XC1 had strong pathogenicity, with concentrations of 104 CFU/mL and above, it could cause 100% rot of pepper within 3 days at room temperature (25 ℃). Furthermore, an antagonistic strain 1151 was screened by double-layer agar plate method, which was identified to be Bacillus subtilis by 16S rDNA. In vitro antibacterial result showed that cells and antimicrobial peptide of B. subtilis 1151 had inhibitory activity against Pcb XC1. In vivo result of pepper disease control showed that cells and antimicrobial peptide of B. subtilis 1151 could reduce the rotting rate of pepper and its lesion diameter, among which antimicrobial peptide performed better. The antimicrobial peptide could reduce the number of Pcb XC1 in pepper by 99%. The result of scanning electron microscope also showed that the soft rot symptom of pepper surface treated by antimicrobial peptide was less, and there were no obvious folds and debris caused by cell wall degradation. On one hand, from the protein level, 3 antimicrobial peptides were identified by LC-MS/MS which were BHP, SP-B and Hb 98-114. On the other hand, from the gene level, a bacteriocin gene cluster was identified from the genome of B. subtilis 1151 by BAGEL4 database, containing two bacteriocins Subtilisin A and Subtilisin Sbox. This study showed that B. subtilis 1151 could reduce the rot loss of pepper caused by soft rot, and its antimicrobial peptides played an important role. It has important reference significance for the storage and preservation method of postharvest pepper.
[1] CHUAH A M, LEE Y C, YAMAGUCHI T, et al.Effect of cooking on the antioxidant properties of coloured peppers[J].Food Chemistry, 2008, 111(1):20-28.
[2] 王立浩, 张宝玺, 张正海, 等.“十三五”我国辣椒育种研究进展、产业现状及展望[J].中国蔬菜, 2021(2):21-29.
WANG L H, ZHANG B X, ZHANG Z H, et al.Status in breeding and production of Capsicum spp.in China during‘the thirteenth five-year plan’Period and future prospect[J].China Vegetables, 2021, (2):21-29.
[3] KIM W-R, KIM E O, KANG K, et al.Antioxidant activity of phenolics in leaves of three red pepper (Capsicum annuum) cultivars[J].Journal of Agricultural & Food Chemistry, 2014, 62(4):850-859.
[4] CERVANTES-HERNÁNDEZ F, ALCALÁ-GONZÁLEZ P, MARTÍNEZ O, et al.Placenta, pericarp, and seeds of Tabasco chili pepper fruits show a contrasting diversity of bioactive metabolites[J].Metabolites, 2019, 9(10):206.
[5] FABELA-MORÓN M F, CUEVAS-BERNARDINO J C, AYORA-TALAVERA T, et al.Trends in capsaicinoids extraction from Habanero chili pepper (Capsicum chinense jacq.):Recent advanced techniques[J].Food Reviews International, 2020, 36(2):105-134.
[6] 谷会, 朱世江, 詹儒林.辣椒采后软腐病拮抗细菌的分离及生防效果[J].热带作物学报, 2012, 33(7):1 276-1 280.
GU H, ZHU S J, ZHAN R L.Isolation and biocontrol efficacy of antagonistic bacteria against bacterial soft rot of pepper after harvest[J].Chinese Journal of Tropical Crops, 2012, 33 (7):1 276-1 280.
[7] 谷会, 朱世江, 谢江辉.我国北运辣椒贮运保鲜存在的问题及对策[J].广东农业科学, 2011, 38(7):107-110.
GU H, ZHU S J, XIE J H.Existing issues and countermeasures in preservation during storing and transporting of peppers transported to Northern China[J].Guangdong Agricultural Sciences, 2011, 38(7):107-110.
[8] 金苹. 辣椒疮痂病、黑霉病、软腐病、病毒病、猝倒病和白绢病的识别与防治[J].农业灾害研究, 2013,3(52):6-9.
JIN P.Identification and control of scab, black mould disease, soft rot, virus disease, damping-off and southern blight of pepper[J].Journal of Agricultural Catastrophology, 2013,3(52):6-9.
[9] 胡毅, 王微, 兰吉玉, 等.黔东南州辣椒农药残留膳食摄入风险评估[J].世界农药, 2021, 43(9):49-56.
HU Y, WANG W, LAN J Y, et al. Risk assessment of dietary intake of pesticide residues in pepper in Qiandongnan prefecture[J]. World Pesticide, 2021, 43(9):49-56.
[10] NANTAWANIT N, CHANCHAICHAOVIVAT A, PANIJPAN B, et al.Induction of defense response against Colletotrichum capsici in chili fruit by the yeast Pichia guilliermondii strain R13[J].Biological Control, 2010, 52(2):145-152.
[11] YI L H, DANG J, ZHANG L H, et al.Purification, characterization and bactericidal mechanism of a broad spectrum bacteriocin with antimicrobial activity against multidrug-resistant strains produced by Lactobacillus coryniformis XN8[J].Food Control, 2016, 67:53-62.
[12] GOH H F, PHILIP K.Isolation and mode of action of bacteriocin BacC1 produced by nonpathogenic Enterococcus faecium C1[J].Journal of Dairy Science, 2015, 98(8):5 080-5 090.
[13] ASGHAR M A, YOUSUF R I, SHOAIB M H, et al.Antibacterial, anticoagulant and cytotoxic evaluation of biocompatible nanocomposite of chitosan loaded green synthesized bioinspired silver nanoparticles[J].International Journal of Biological Macromolecules, 2020, 160:934-943.
[14] LI T T, LIU Q W, WANG D F, et al.Characterization and antimicrobial mechanism of CF-14, a new antimicrobial peptide from the epidermal mucus of catfish[J].Fish & Shellfish Immunology, 2019, 92:881-888.
[15] LI L, YUAN L F, SHI Y X, et al.Comparative genomic analysis of Pectobacterium carotovorum subsp.brasiliense SX309 provides novel insights into its genetic and phenotypic features[J].BMC Genomics, 2019, 20(1):486.
[16] NAAS H, SEBAIHIA M, ORFEI B, et al.Pectobacterium carotovorum subsp.brasiliense and Pectobacterium carotovorum subsp.carotovorum as causal agents of potato soft rot in Algeria[J].European Journal of Plant Pathology, 2018, 151(4):1 027-1 034.
[17] ONKENDI E M, RAMESH A M, KWENDA S, et al.Draft genome sequence of a virulent Pectobacterium carotovorum subsp.brasiliense isolate causing soft rot of cucumber[J].Genome Announcements, 2016, 4(1).DOI:10.1128/genomeA.01530-15.
[18] QI T, WANG S P, DENG L L, et al.Controlling pepper soft rot by Lactobacillus paracasei WX322 and identification of multiple bacteriocins by complete genome sequencing[J].Food Control, 2021, 121(6):107629.
[19] HUANG Y F, LIU C Y, WANG H, et al.Bioinformatic analysis of the complete genome sequence of Pectobacterium carotovorum subsp.brasiliense BZA12 and candidate effector screening[J].Journal of Plant Pathology, 2019, 101(1):39-49.
[20] BHAT K A, MASOOD S D, BHAT N A, et al.Current status of post harvest soft rot in vegetables:A review[J].Asian Journal of Plant Sciences, 2010, 9(4):200-208.
[21] JOKO T, SUBANDI A, KUSUMANDARI N, et al.Activities of plant cell wall-degrading enzymes by bacterial soft rot of orchid[J].Archives of Phytopathology and Plant Protection, 2014, 47(10):1 239-1 250.
[22] SURIANI, DJAENUDDIN N, MUIS A.Utilization of antagonistic bacteria Bacillus subtilis to control Fusarium verticilloides on corn[J].IOP Conference Series Earth and Environmental Science, 2020, 484(1):012100.
[23] KUMAR A, RABHA J, JHA D K.Antagonistic activity of lipopeptide-biosurfactant producing Bacillus subtilis AKP, against Colletotrichum capsici, the causal organism of anthracnose disease of chilli[J].Biocatalysis and Agricultural Biotechnology, 2021, 36:102133.
[24] JOHNSON E M, JUNG D Y G, JIND Y Y, et al.Bacteriocins as food preservatives:Challenges and emerging horizons[J].Critical Reviews in Food Science and Nutrition, 2018, 58(16):2 743-2 767.
[25] COMPAORÉ C S, NIELSEN D S, OUOBA L I I, et al.Co-production of surfactin and a novel bacteriocin by Bacillus subtilis subsp.subtilis H4 isolated from Bikalga, an African alkaline Hibiscus sabdariffa seed fermented condiment[J].International Journal of Food Microbiology, 2013, 162(3):297-307.
[26] KAWULKA K E, SPRULES T, DIAPER C M, et al.Structure of subtilosin A, a cyclic antimicrobial peptide from Bacillus subtilis with unusual sulfur to alpha-carbon cross-links:Formation and reduction of alpha-thio-alpha-amino acid derivatives[J].Biochemistry, 2004, 43(12):3 385-3 395.
