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Characterization and biocontrol measures of Pseudomonas syringae pv. syringae associated with citrus blast disease

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Abstract

The present study was conducted to identify the two Pseudomonas syringae pv. syringae strains attained from the citrus blast infected tissues and also to find out the antimicrobial potentiality of plant extracts and antagonistic bacteria Bacillus spp. both under in vitro and in vivo conditions. Based on the microscopic observation and biochemical features including colouring, magnitude, size, and form of colonies displayed on LB medium, we speculate that ps17a and ps18a strains were similar to P. syringae pv syringae. For molecular identification, 16S rRNA and multi-locus sequence analysis (MLSA) with two housekeeping gene (rpoD and gyrB) sequences indicated an identical pattern to the corresponding sequences of other P. syringae pv. syringae strains which confirmed that a genetically homologous species of P. syringae pv. syringae was responsible for bacterial blast disease of citrus cultivars. Later, PCR amplification using two primer pairs (B1 and B2) yielded 752-bp fragments to confirm that syrB gene present on both isolated P. syringae pv syringae and reference strains (Pss16) can synthesize syringomycin, a potent toxin for plant pathogenicity induction. Based on the pathogenicity tests, two strains were similar in terms of virulence activity, however, were not analogous thus, having the same level of severity effect to induce bacterial blast. Moreover, antibiotic sensitivity assay showed disproportionate result for both strains which resemble susceptible strains determination. From the in vitro investigation of six plant extracts and three Bacillus isolates against ps17a and ps18a, the ethanolic extract of Allium sativum, Moringa oleifera, and Azadirachta indica showed the highest antibacterial activity against two isolated bacterial strains up to inhibition zone 23.4, 24.4 and 18.2 mm in diameter, respectively while Bacillus isolates proved to be better natural antimicrobial agents against the disease. Finally, based on in vivo experiment, the most effective plant extracts and antagonistic isolates BS5 exhibited significant necrosis growth reduction after 10 weeks of inoculation by ps17a up to 67.48 and 52.88% as compared to 65.42 and 55.3% by ps18a, respectively. Thus, six selected ethanolic plant extracts and three isolates of Bacillus spp. have a broad-spectrum antimicrobial activity which can be used as effective biocontrol agent.

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Abbreviations

PCR:

Polymerase chain reaction

MLSA:

Multi-locus sequence analysis

SDW:

Sterilized distilled water

LB:

Luria–Bertani

KN:

King’s B

SIM:

Sulfur–indole-motility

KIA:

Kligler iron agar

TSI:

Triple sugar iron

MSA:

Mannitol salt agar/multiple sequence alignment

OD:

Optimum density

CFU:

Colony-forming unit

DAI:

Days after incubation

CTAB:

Cetyl-trimethyl-ammonium-bromide

ANOVA:

Analysis of variance

LSD:

Least significance difference

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Acknowledgements

The authors are grateful to Professor Joarder DNA and Chromosome Research Laboratory, Dept. of Genetic Engineering and Biotechnology, the University of Rajshahi for providing lab facilities and guidance.

Funding

This study was funded by the Ministry of Education, People’s Republic of Bangladesh, Project No. 58, 2016–2017.

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  1. Md. Samiul Islam and Razia Sultana contributed equally in the paper and hence will be considered as the first author.

Authors and Affiliations

  1. Department of Plant Pathology, College of Plant Science and Technology and the Key Lab of Crop Disease Monitoring & Safety Control in Hubei Province, Huazhong Agricultural University, Wuhan, 430070, China

    Md. Samiul Islam

  2. Department of Genetic Engineering and Biotechnology, Faculty of Life and Earth Science, University of Rajshahi, Rajshahi, 6205, Rajshahi, Bangladesh

    Md. Samiul Islam, Razia Sultana, Md. Amit Hasan, Md. Shah Alam, Biswanath Sikdar & Md. Asadul Islam

  3. State Key Laboratory of Agricultural Microbiology, Department of Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan, 430070, People’s Republic of China

    Razia Sultana

  4. Department of Plant Pathology, College of Plant Protection, Nanjing Agricultural University and Key Laboratory of Integrated Management of Crop Diseases, Ministry of Education, Nanjing, 210095, People’s Republic of China

    Md. Kamaruzzaman

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Islam, M., Sultana, R., Hasan, M. et al. Characterization and biocontrol measures of Pseudomonas syringae pv. syringae associated with citrus blast disease. Vegetos 33, 555–569 (2020). https://doi.org/10.1007/s42535-020-00138-1

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