Detection of Pantoea spp., an Emerging Pathogen of Rice through Multiplex PCR System

Author: Biswajit Jena, A.K. Senapati and S.K. Behera

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Abstract

The recent fluctuations in climate have introduced a novel challenge to rice cultivation, characterized by the occurrence of panicle blight and incomplete grain filling. A new causative agent was classified under the taxonomic genus Pantoea have been confirmed as the causative agents of leaf and grain blight disease in different rice growing regions of Odisha. A multiplex polymerase chain reaction (PCR) assay was developed with the purpose of differentiating various species within the Pantoea genus. A set of polymerase chain reaction (PCR) primers was designed to selectively detect the housekeeping genes, namely in fB, gyrB, and rpoB, in species P. agglomerans, P. ananatis, and P. stewartii. These primers were designed to have varying base pair compositions. A separate set of primers was developed to facilitate the identification of the at pD gene, a gene that is typically found in all Pantoea spp. Additionally, a universal set of primers was designed to amplify the 16S rRNA region, a region that is common among all the bacterial species. In the mPCR system, a total of five isolates were taken, namely BA1, BAL1, BP1, which were identified as Pantoea agglomerans, NP1, identified as Pantoea dispersa, and one Xoo bacterium. The polymerase chain reaction (PCR) was conducted, resulting in the formation of discrete bands measuring 1.5 kilobases (kb) for each of the isolates. Distinct bands were observed at a length of 330 base pairs (bp) during the amplification of the at pD gene in the four Pantoea isolates, with the exception of Xoo. The amplification of the inf B gene was conducted on the isolates of Pantoea agglomerans, resulting in the formation of distinct bands at 730 base pairs (bp) for the isolates BA1, BAL1, and BP1. The method employed in this study involved the utilisation of multiplex polymerase chain reaction (mPCR) to simultaneously amplify bacterial DNA using multiple sets of primers. The resulting distinct bands observed in the gel electrophoresis analysis allowed for the differentiation of various isolates, as well as the identification of Pantoea bacteria at the species level.

Keywords

Pantoea agglomerans, Pantoea dispersa, mPCR, Xoo (Xanthomonas oryzae pv. oryzae

Conclusion

A new multiplex PCR scheme was developed to diagnose plant-pathogenic Pantoea spp. This tool enabled the efficient confirmation of the presence of Pantoea species mainly the Pantoea agglomerans in Odisha, which affecting rice production and productivity. When PCR was conducted, separate bands at 1.49 kb developed for each isolate. The atpD gene was amplified in distinct bands at 330 bp for all four Pantoea isolates except Xoo. Pantoea agglomerans isolates were amplified for the inf B gene, and separate bands for isolates BA1, BAL1, and BP1 appeared at 730 bp. In this manner, using mPCR, multiple sets of primers were utilized to concurrently amplify the bacterial DNA and separate bands were generated, differentiating Pantoea bacteria at the species level. This novel molecular diagnostic tool will aid in the correct identification of important Pantoea plant-pathogenic species. It will be particularly valuable for plant protection services and epidemiological monitoring of these major crop-threatening bacteria due to its reliability, specificity, sensitivity, and cost effectiveness.

References

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How to cite this article

Biswajit Jena, A.K. Senapati and S.K. Behera (2023). Detection of Pantoea spp., an Emerging Pathogen of Rice through Multiplex PCR System. Biological Forum – An International Journal, 15(1): 674-677.