Advertisement

Microbial Ecology

, Volume 77, Issue 3, pp 651–663 | Cite as

Genomic and Functional Characterization of the Endophytic Bacillus subtilis 7PJ-16 Strain, a Potential Biocontrol Agent of Mulberry Fruit Sclerotiniose

  • Wei-fang Xu
  • Hui-shuang Ren
  • Ting Ou
  • Ting Lei
  • Jun-hong Wei
  • Chuan-shu Huang
  • Tian Li
  • Gary Strobel
  • Ze-yang ZhouEmail author
  • Jie XieEmail author
Plant Microbe Interactions

Abstract

Bacillus sp. 7PJ-16, an endophytic bacterium isolated from a healthy mulberry stem and previously identified as Bacillus tequilensis 7PJ-16, exhibits strong antifungal activity and has the capacity to promote plant growth. This strain was studied for its effectiveness as a biocontrol agent to reduce mulberry fruit sclerotiniose in the field and as a growth-promoting agent for mulberry in the greenhouse. In field studies, the cell suspension and supernatant of strain 7PJ-16 exhibited biocontrol efficacy and the lowest disease incidence was reduced down to only 0.80%. In greenhouse experiments, the cell suspension (1.0 × 106 and 1.0 × 105 CFU/mL) and the cell-free supernatant (100-fold and 1000-fold dilution) stimulated mulberry seed germination and promoted mulberry seedling growth. In addition, to accurately identify the 7PJ-16 strain and further explore the mechanisms of its antifungal and growth-promoting properties, the complete genome of this strain was sequenced and annotated. The 7PJ-16 genome is comprised of two circular plasmids and a 4,209,045-bp circular chromosome, containing 4492 protein-coding genes and 116 RNA genes. This strain was ultimately designed as Bacillus subtilis based on core genome sequence analyses using a phylogenomic approach. In this genome, we identified a series of gene clusters that function in the synthesis of non-ribosomal peptides (surfactin, fengycin, bacillibactin, and bacilysin) as well as the ribosome-dependent synthesis of tasA and bacteriocins (subtilin, subtilosin A), which are responsible for the biosynthesis of numerous antimicrobial metabolites. Additionally, several genes with function that promote plant growth, such as indole-3-acetic acid biosynthesis, the production of volatile substances, and siderophores synthesis, were also identified. The information described in this study has established a good foundation for understanding the beneficial interactions between endophytes and host plants, and facilitates the further application of B. subtilis 7PJ-16 as an agricultural biofertilizer and biocontrol agent.

Keywords

Bacillus subtilis Control efficiency Plant growth promotion Genome sequence Antimicrobial metabolites 

Notes

Acknowledgments

The authors acknowledge the financial support of the National Natural Science Foundation of China (31601678) to Jie Xie, the Fundamental Research Funds for the Central Universities (XDJK2018D020) to Ting Ou, and the Natural Science Foundation of Chongqing (cstc2015jcyjys80001) to Jie Xie.

Supplementary material

248_2018_1247_MOESM1_ESM.docx (4.6 mb)
ESM 1 (DOCX 4714 kb)

References

  1. 1.
    Gray E, Gray RE (1987) Observations on popcorn disease of mulberry in south central Kentucky. Castanea 52:47–51Google Scholar
  2. 2.
    Ju WT, Kim HB, Sung GB, Park KY, Kim YS (2016) Mulberry popcorn disease occurrence in Korea region and development of integrative control method. Int J Ind Entomol 33:36–40Google Scholar
  3. 3.
    Kuai YZ, Wu FA (2012) A review on pathogens of mulberry fruit sclerotiniosis and its control technology. Sci Seric (In Chinese) 38:1099–1104Google Scholar
  4. 4.
    Lü RH, Zhao AC, Li J, Wang XL, Yu YS, Lu C, Yu MD (2013a) Biological study of hypertrophy sorosis scleroteniosis and its molecular characterization based on LSU rRNA. Afr. J. Microbiol. Res. 7:3405–3411CrossRefGoogle Scholar
  5. 5.
    Lü RH, Zhao AC, Yu J, Wang CH, Liu CY, Cai YX, Yu MD (2017) Biological characteristics and epidemiological analysis of the pathogen of hypertrophy sorosis scleroteniosis, Ciboria shiraiana. J Microbiol (In Chinese) 57:388–398Google Scholar
  6. 6.
    Hong SK, Kim WG, Sung GB, Nam SH (2007) Identification and distribution of two fungal species causing sclerotial disease on mulberry fruits in Korea. Mycobiology 35:87–90CrossRefGoogle Scholar
  7. 7.
    Hu JH, Cai YX, Zhou SJ, Zhang JC, Zhang HL, Chen YB, Li PM, Ying GM (2011) Diversity of mulberry sclerotiniose pathogen and ITS analysis. J Ningbo Univ (In Chinese) 24:20–23Google Scholar
  8. 8.
    Whetzel HH, Wolf FA (1945) The cup fungus, Ciboria carunculoides, pathogenic on mulberry fruits. Mycologia 37:476–491CrossRefGoogle Scholar
  9. 9.
    Sultana R, Ju HJ, Chae JC, Kim K, Lee KJ (2013) Identification of Ciboria carunculoides RS103V, a fungus causing popcorn disease on mulberry fruits in Korea. Res Plant Dis 19:308–312CrossRefGoogle Scholar
  10. 10.
    Lü RH, Zhao AC, Jin XY, Du YW, Wu WB, Wang XL, Yu MD (2011) A primary experiment on the control of mulberry fruit sclerotiniosis using herbicide glyphosate. Sci Seric (In Chinese) 37:907–913Google Scholar
  11. 11.
    Lü RH, Ding ZY, Wang XL, Wu WB, Zhao AC, Li J, Yu MD (2013b) An experiment on killing mulberry fruit sclerotiniosis ascospores with the infrared high-temperature thermistor. J Southwest Univ (In Chinese) 35:10–14Google Scholar
  12. 12.
    Ye MQ, Yue HL, Luo GQ, Yang Q, Kuang ZS (2014) Effect of a fungal pathogen, Trichoderma hamatum, on growth and germination of Ciboria carunculoides under laboratory conditions. Pak J Zool 46:1377–1384Google Scholar
  13. 13.
    Sultana R, Kim K (2016) Bacillus thuringiensis C25 suppresses popcorn disease caused by Ciboria shiraiana in mulberry (Morus australis L.). Biocontrol Sci Tech 26:145–162CrossRefGoogle Scholar
  14. 14.
    Choudhary DK, Johri BN (2009) Interactions of Bacillus spp. and plants with special reference to induced systemic resistance (ISR). Microbiol. Res. 164:493–513CrossRefGoogle Scholar
  15. 15.
    Gond SK, Bergen MS, Torres MS, James F, White J (2015) Endophytic Bacillus spp. produce antifungal lipopeptides and induce host defence gene expression in maize. Microbiol Res. 172:79–87CrossRefGoogle Scholar
  16. 16.
    Ji XL, Lu GB, Gai YP, Zheng CC, Mu ZM (2008) Biological control against bacterial wilt and colonization of mulberry by an endophytic Bacillus subtilis strain. FEMS Microbiol. Ecol. 65:565–573CrossRefGoogle Scholar
  17. 17.
    Shrestha BK, Karki HS, Groth DE, Jungkhun N, Ham JH (2016) Biological control activities of rice-associated Bacillus sp. strains against sheath blight and bacterial panicle blight of rice. PLoS One 11:1–18Google Scholar
  18. 18.
    Stein T (2005) Bacillus subtilis antibiotics: structures, syntheses and specific functions. Mol. Microbiol. 56:845–857CrossRefGoogle Scholar
  19. 19.
    Sun GZ, Yao T, Feng CJ, Chen L, Li JH, Wang L (2017) Identification and biocontrol potential of antagonistic bacteria strains against Sclerotinia sclerotiorum, and their growth-promoting effects on Brassica napus. Biol. Control 104:35–43CrossRefGoogle Scholar
  20. 20.
    Gatson JW, Benz BF, Chandrasekaran C, Satomi M, Venkateswaran K, Hart ME (2006) Bacillus tequilensis sp. nov. isolated from a 2000-year-old Mexican shaft-tomb, is closely related to Bacillus subtilis. Int J Syst Evol Microbiol 56:1475–1484CrossRefGoogle Scholar
  21. 21.
    Fox GE, Wisotzkey JD, Jurtshuk JR (1992) How close is close: 16S rRNA sequence identity may not be sufficient to guarantee species identity. Int J Syst Bacteriol 42:166–170CrossRefGoogle Scholar
  22. 22.
    Pan HQ, Li QL, Hu JC (2017) The complete genome sequence of Bacillus velezensis 9912D reveals its biocontrol mechanism as a novel commercial biological fungicide agent. J Biotechnol 247:25–28CrossRefGoogle Scholar
  23. 23.
    Xie J, Ren HS, Tang CM, Zuo WD, Chen J, Huang CS, Wang ZJ, Dai FW, Zhou ZY (2015) Identification of a mulberry endophytic bacterium and its antagonistic activity on Scleromitula shiraiana. Sci Seric (In Chinese) 41:815–824Google Scholar
  24. 24.
    Glickmann E, Dessaux Y (1995) A critical examination of the specificity of the salkowski reagent for indolic compounds produced by phytopathogenic bacteria. Appl Environ Microbiol 61:793–796Google Scholar
  25. 25.
    Xie J, Shu P, Strobel GA, Chen J, Wei JH, Xiang ZH, Zhou ZY (2017) Pantoea agglomerans SWg2 colonizes mulberry tissues, promotes disease protection and seedling growth. Biol Control 113:9–17CrossRefGoogle Scholar
  26. 26.
    Chin CS, Alexander DH, Marks P, Klammer AA, Drake J, Heiner C, Clum A, Cpoeland A, Huddleston J, Eichler EE, Turner SW, Korlach J (2013) Nonhybrid, finished microbial genome assemblies from long-read SMRT sequencing data. Nat Methods 10:563–569CrossRefGoogle Scholar
  27. 27.
    Besemer J, Lomsadze A, Borodovsky M (2001) GeneMarkS: a self-training method for prediction of gene starts in microbial genomes. Implications for finding sequence motifs in regulatory regions. Nucleic Acids Res 29:2607–2618CrossRefGoogle Scholar
  28. 28.
    Krzywinski M, Schein J, Birol I, Connors J, Gascoyne R, Horsman D, Jones SJ, Marra MA (2009) Circos: an information aesthetic for comparative genomics. Genome Res 19:1639–1645CrossRefGoogle Scholar
  29. 29.
    Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGA6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol 30:2725–2729CrossRefGoogle Scholar
  30. 30.
    Jasim B, Joseph AA, John CJ, Mathew J, Radhakrishnan EK (2013) Isolation and characterization of plant growth promoting endophytic bacteria from the rhizome of Zingiber officinale. 3 Biotech 4:197–204CrossRefGoogle Scholar
  31. 31.
    Patagundi BI, Shivasaran CT, Kaliwal BB (2014) Isolation and characterization of cellulose producing bacteria from soil. Int J Curr Microbiol App Sci 3:59–69Google Scholar
  32. 32.
    Chen H, Xiao X, Wang J, Wu LJ, Zheng ZM, Yu ZL (2008) Antagonistic effects of volatiles generated by Bacillus subtilis on spore germination and hyphal growth of the plant pathogen, Botrytis cinerea. Biotechnol. Lett. 30:919–923CrossRefGoogle Scholar
  33. 33.
    Chun J, Bae KS (2000) Phylogenetic analysis of Bacillus subtilis and related taxa based on partial gyrA gene sequences. Antonie Van Leeuwenhoek 78:123–127CrossRefGoogle Scholar
  34. 34.
    Peypoux F, Bonmatin JM, Wallach J (1999) Recent trends in the biochemistry of surfactin. Appl Microbiol Biotechnol 51:553–563CrossRefGoogle Scholar
  35. 35.
    Zouari I, Jlaiel L, Tounsi S, Trigui M (2016) Biocontrol activity of the endophytic Bacillus amyloliquefaciens strain CEIZ-11 against Pythium aphanidermatum and purification of its bioactive compounds. Biol Control 100:54–62CrossRefGoogle Scholar
  36. 36.
    Raaijmakers JM, De BI, Nybroe O, Ongena M (2010) Natural functions of lipopeptides from Bacillus and Pseudomonas: more than surfactants and antibiotics. FEMS Microbiol Rev 34:1037–1062CrossRefGoogle Scholar
  37. 37.
    Cawoy H, Debois D, Franzil L, De-Pauw E, Thonart P, Ongena M (2015) Lipopeptides as main ingredients for inhibition of fungal phytopathogens by Bacillus subtilis/amyloliquefaciens. Microb Biotechnol 8:281–295CrossRefGoogle Scholar
  38. 38.
    Vanittanakom N, Loeffler W, Koch U, Jung G (1986) Fengycin--a novel antifungal lipopeptide antibiotic produced by Bacillus subtilis F-29-3. J Antibiot 39:888–901CrossRefGoogle Scholar
  39. 39.
    Borriss R, Chen XH, Rueckert C, Blom J, Becker A, Baumgarth B, Fan B, Pukall R, Schumann P, Sproer C, Junge H, Vater J, Pühler A, Klenk HP (2011) Relationship of Bacillus amyloliquefaciens clades associated with strains DSM 7T and FZB42T: a proposal for Bacillus amyloliquefaciens subsp. amyloliquefaciens subsp. nov. and Bacillus amyloliquefaciens subsp. plantarum subsp. nov. based on complete genome sequence comparisons. Int J Syst Evol Microbiol 61:1786–1801CrossRefGoogle Scholar
  40. 40.
    Kenig M, Abraham EP (1976) Antimicrobial activities and antagonists of bacilysin and anticapsin. J Gen Microbiol 94:37–45CrossRefGoogle Scholar
  41. 41.
    Khochamit N, Siripornadulsil S, Sukon P, Siripornadulsil W (2015) Antibacterial activity and genotypic-phenotypic characteristics of bacteriocin-producing Bacillus subtilis KKU213: potential as a probiotic strain. Microbiol Res 170:36–50CrossRefGoogle Scholar
  42. 42.
    Liu T, Chen YP, Li QJ, Gu ZF (2014) Cloning and functional analyses of TasA, an antimicrobial protein gene from Bacillus pumilus DX01. J Shanghai Jiaotong Univ (In Chinese) 32:48–52 58Google Scholar
  43. 43.
    Liu GQ, Kong YY, Fan YJ, Geng C, Peng DH, Sun M (2017) Whole-genome sequencing of Bacillus velezensis LS69, a strain with a broad inhibitory spectrum against pathogenic bacteria. J Biotechnol 249:20–24CrossRefGoogle Scholar
  44. 44.
    Chen Y, Gao X, Chen Y, Qin H, Huang L, Han Q (2014) Inhibitory efficacy of endophytic Bacillus subtilis EDR4 against Sclerotinia sclerotiorum on rapeseed. Biol Control 78:67–76CrossRefGoogle Scholar
  45. 45.
    Lam B, Strobel GA, Harrison L, Lam S (1987) Transposon mutagenesis and tagging of fluorescent Pseudomonas: antimycotic production is necessary for control of Dutch elm disease. Proc Natl Acad Sci 84:6447–6451CrossRefGoogle Scholar
  46. 46.
    Hu XJ, Roberts DP, Xie LH, Maul JE, Yu CB, Li YS, Jiang M, Liao XS, Che Z, Liao X (2014) Formulations of Bacillus subtilis BY-2 suppress Sclerotinia sclerotiorum on oilseed rape in the field. Biol Control 70:54–64CrossRefGoogle Scholar
  47. 47.
    Zhao YJ, Selvaraj JN, Xing FG, Zhou L, Wang Y, Song HM, Tan XX, Sun LC, Sangare L, Folly YM, Liu Y (2014) Antagonistic action of Bacillus subtilis strain SG6 on Fusarium graminearum. PLoS One 9:e92486CrossRefGoogle Scholar
  48. 48.
    Pereira SIA, Monteiro C, Vega AL, Castro PML (2016) Endophytic culturable bacteria colonizing Lavandula dentata L. plants: isolation, characterization and evaluation of their plant growth-promoting activities. Ecol Eng 87:91–97CrossRefGoogle Scholar
  49. 49.
    Shen SY, Fulthorpe R (2015) Seasonal variation of bacterial endophytes in urban trees. Front Microbiol 6:1–13Google Scholar
  50. 50.
    Dunlap CA, Bowman MJ, Schisler DA (2013) Genomic analysis and secondary metabolite production in Bacillus amyloliquefaciens AS 43.3: a biocontrol antagonist of Fusarium head blight. Biol Control 64:166–175CrossRefGoogle Scholar
  51. 51.
    Chan YK, McCormick WA, Seifert KA (2003) Characterization of an antifungal soil bacterium and its antagonistic activities against Fusarium species. Can J Microbiol 49:253–262CrossRefGoogle Scholar
  52. 52.
    Lin D, Qu LJ, Gu H, Chen Z (2001) A 3·1-kb genomic fragment of Bacillus subtilis encodes the protein inhibiting growth of Xanthomonas oryzae pv. oryzae. J Appl Microbiol 91:1044–1050CrossRefGoogle Scholar
  53. 53.
    Earl AM, Losick R, Kolter R (2008) Ecology and genomics of Bacillus subtilis. Trends Microbiol 16:269–275CrossRefGoogle Scholar
  54. 54.
    Zheng ZY, Yang Y, Ren JQ, Zhang MH, Zeng XL (2016) Biological control test of mulberry fruit sclerotiniosis with two kinds of Trichoderma preparations. Sci Seric (In Chinese) 42:168–170Google Scholar
  55. 55.
    Zhang SM, Wang YX, Meng LQ, Li J, Zhao XY, Cao X, Chen XL, Wang AX, Li JF (2012) Isolation and characterization of antifungal lipopeptides produced by endophytic Bacillus amyloliquefaciens TF28. Afr J Microbiol Res 6:1747–1755Google Scholar
  56. 56.
    Torres MJ, Pérez-Brandan C, Sabaté DC, Petroselli G, Erra-Balsells R, Audisio MC (2017) Biological activity of the lipopeptide-producing Bacillus amyloliquefaciens PGPBacCA1 on common bean Phaseolus vulgaris L. pathogens. Biol Control 105:93–99CrossRefGoogle Scholar
  57. 57.
    Radhakrishnan R, Lee IJ (2016) Gibberellins producing Bacillus methylotrophicus KE2 supports plant growth and enhances nutritional metabolites and food values of lettuce. Plant Physiol Biochem 109:81–189CrossRefGoogle Scholar
  58. 58.
    Dorra BA, Olfa FG, Slim T (2018) Rizhospheric competence, plant growth promotion and biocontrol efficacy of Bacillus amyloliquefaciens subsp. plantarum strain 32a. Biol Control.  https://doi.org/10.1016/j.biocontrol.2018.01.013
  59. 59.
    Rooney AP, Price NP, Ehrhardt C, Swezey JL, Bannan JD (2009) Phylogeny and molecular taxonomy of the Bacillus subtilis species complex and description of Bacillus subtilis subsp. inaquosorum subsp nov. Int J Syst Evol Microbiol 59:2429–2436CrossRefGoogle Scholar
  60. 60.
    Cai XC, Liu CH, Wang BT, Xue YR (2017) Genomic and metabolic traits endow Bacillus velezensis CC09 with a potential biocontrol agent in control of wheat powdery mildew disease. Microbiol Res 196:89–94CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Wei-fang Xu
    • 1
    • 2
  • Hui-shuang Ren
    • 1
    • 2
  • Ting Ou
    • 1
    • 2
  • Ting Lei
    • 3
  • Jun-hong Wei
    • 1
    • 2
  • Chuan-shu Huang
    • 3
  • Tian Li
    • 1
    • 2
  • Gary Strobel
    • 4
  • Ze-yang Zhou
    • 1
    • 2
    • 5
    Email author
  • Jie Xie
    • 1
    • 2
    Email author
  1. 1.State Key Laboratory of Silkworm Genome Biology, College of BiotechnologySouthwest UniversityChongqingPeople’s Republic of China
  2. 2.Key Laboratory of Sericultural Biology and Genetic Breeding, Ministry of Agriculture, College of BiotechnologySouthwest UniversityChongqingPeople’s Republic of China
  3. 3.Institute of Sericulture Science and Technology ResearchChongqingPeople’s Republic of China
  4. 4.Department of Plant SciencesMontana State UniversityBozemanUSA
  5. 5.College of Life ScienceChongqing Normal UniversityChongqingPeople’s Republic of China

Personalised recommendations