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Bacillus thuringiensis C25 which is rich in cell wall degrading enzymes efficiently controls lettuce drop caused by Sclerotinia minor

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Abstract

Sclerotinia is a phytopathogenic genus of fungi that form sclerotia: black, hard, seed-like functioning bodies. Sclerotinia sclerotiorum and Sclerotinia minor are major threats to vegetable cultivation. In this study, we identified an efficient biocontrol agent against lettuce drop caused by S. minor. Initially, 43 bacterial strains isolated from Sclerotinia-infected field were screened for antifungal effects against S. minor. The selected bacterial strains (i.e. C5 and C25) conferred significant (P < 0.05) inhibition in mycelia growth, formation and viability of sclerotia of S. minor in vitro. Especially, strain C25 significantly (P < 0.05) suppressed the disease severity of lettuce drop caused by S. minor and Sclerotinia sclerotiorum. On the basis of biochemical and genetic analyses, strain C25 was identified as a subspecies of Bacillus thuringiensis. Strain C25 exhibited activities of variety of cell wall degrading enzymes such as proteases, β-1,3-glucanase, and chitinase. Scanning electron microscopy (SEM) implied that strain C25 induced degeneration, distortion, and rupture of hyphae of S. minor. Conclusively, our study was the first to report that Bacillus thuringiensis significantly suppressed sclerotial diseases by degrading the cell walls of pathogenic fungi, at least in part. Strain C25 would be a potential BCA (biocontrol agent) for the agricultural industry.

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Acknowledgments

This study was supported by the Cooperative Research Program for Agriculture Science and Technology Development (PJ009411) RDA, and Chonbuk National University, Korea.

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i) This manuscript is the original work and has not been submitted or published (partly or in full) anywhere.

ii) No data have been fabricated or manipulated (including images) to support our conclusions.

iii) The author declares no conflict of interest.

iv) Any human participants and/or animals were/was not involved in the current research.

v) The experiments were carried out following the current laws of the country where they were performed.

vi) Proper acknowledgment is given to the eligible organization or funding resource in our acknowledgment part.

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Correspondence to Kangmin Kim.

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Supplemental Table 1

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Supplemental Fig. 1

Screening of bacterial isolates showing antagonistic effect against S. minor by dual culture method. The experiment was performed with three replicates, buffer was used as control. The treatments are as labeled in table 1 in supplementary data (PDF 65 kb)

Supplemental Fig. 2

Different biological activities shown by isolated strains A) siderophore production, B) protease assay and C) phosphate solubilization. Buffer was used as control. The treatments are labeled in Table 1 (PDF 71 kb)

Supplemental Fig. 3

Amplification of the 16S rRNA23S rRNAgene region using group specific and specific primer to differentiate closely related species of Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis, where lane1, primer pair; NRUANT-RSI-F, ITS-B-r, lane 2, primer pair; BCF, BCR and lane 3 primer pair; ECO-CER-RSI-F, PST-CER-SI-R.M 1 kb Ladder and m 100 bp ladder. Arrow indicates the sizes amplified (PDF 36 kb)

Supplemental Fig. 4

Scanning electron microscopy of S. sclerotiorum hyphae in the absence or presence of selected bacteria. a-b) The hyphae of S. sclerotiorum grown on PDA media. c-d) Hyphae of S. sclerotiorum treated with B. subtilis GB03. e-f) Hyphae of S. sclerotiorum treated with C25. The arrows indicate degenerated fungal mycelia. Scale bars are presented in each panel (PDF 46 kb)

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Shrestha, A., Sultana, R., Chae, JC. et al. Bacillus thuringiensis C25 which is rich in cell wall degrading enzymes efficiently controls lettuce drop caused by Sclerotinia minor . Eur J Plant Pathol 142, 577–589 (2015). https://doi.org/10.1007/s10658-015-0636-5

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