Abstract
Spot blotch disease of wheat caused by Bipolaris sorokiniana Boerma (Sacc.) is an emerging problem in South Asian countries. Whole genome of a highly virulent isolate of B. sorokiniana BS112 (BHU, Uttar Pradesh; Accession no. GCA_004329375.1) was sequenced using a hybrid assembly approach. Secreted proteins, virulence gene(s), pathogenicity-related gene(s) were identified and the role of ToxA gene present in this genome, in the development of disease was recognized. ToxA gene (535 bp) was analyzed and identified in the genome of B. sorokiniana (BS112) which revealed 100% homology with the ToxA gene of Pyrenophora tritici repentis (Accession no. MH017419). Furthermore, ToxA gene was amplified, sequenced and validated in 39 isolates of B. sorokiniana which confirmed the presence of ToxA gene in all the isolates taken for this study. All ToxA sequences were submitted in NCBI database (MN601358-MN601396). As ToxA gene interacts with Tsn1 gene of host, 13 wheat genotypes were evaluated out of which 5 genotypes (38.4%) were found to be Tsn1 positive with more severe necrotic lesions compared to Tsn1-negative wheat genotypes. In vitro expression analysis of ToxA gene in the pathogen B. sorokiniana using qPCR revealed maximum upregulation (14.67 fold) at 1st day after inoculation (DAI) in the medium. Furthermore, in planta expression analysis of ToxA gene in Tsn1-positive and Tsn1-negative genotypes, revealed maximum expression (7.89-fold) in Tsn1-positive genotype, Agra local at 5th DAI compared to Tsn1-negative genotype Chiriya 7 showing minimum expression (0.048-fold) at 5th DAI. In planta ToxA–Tsn1 interaction studies suggested that spot blotch disease is more severe in Tsn1-positive genotypes, which will be helpful in better understanding and management of spot blotch disease of wheat.
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Data availability
Data are available in a publically accessible repository. The data presented in this study are openly available at https://www.ncbi.nlm.nih.gov.
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Acknowledgements
Authors are highly thankful to the ICAR-Consortium Research Platform (CRP) on Genomics (ICAR-G/CRP-Genomics/2015-2720/IARI-12-151) for funding this work. The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. We are very thankful to the Director and Joint Director (Research) of the ICAR-Indian Agricultural Research Institute, New Delhi, the Head, Division of Plant Pathology, ICAR-IARI, New Delhi, and the Director of ICAR-NIPB, New Delhi for providing facilities.
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RA, SA and SS conceived and designed the experiment and wrote the manuscript; RA, BMB, MSG and MSS contributed in corrections and suggestions; ARR and SS helped in performing bioinformatics analysis; RA, SA and PJ analyzed the data and conducted the wet lab experiments. All the authors read and approved the final manuscript.
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13205_2022_3213_MOESM3_ESM.xls
Supplementary file3 (XLS 323 KB) Annotation of fungal hybrid assembly in terms of GO proteins – Biological Process, Cellular Component, Molecular Function of B. sorokiniana
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Supplementary file4 (JPG 1290 KB) a PCR amplification profile of 39 isolates of B. sorokiniana harbouring ToxA (535 bp amplicon) gene. M (100 bp bioprep marker), 1-BS1, 2-BS3, 3-BS4, 4-BS6, 5-BS11, 6-BS14, 7-BS27, 8-BS29, 9-BS30, 10-BS32, 11-BS50, 12-BS54, 13-BS65, 14-BS66, 15-BS68, 16-BS69, 17-BS72, 18-BS75, 19-BS88, 20-BS91, 21-BS94, 22-BS112, 23-BS124, 24-BS125, 25-BS126, 26-BS127, 27-BS128, 28-BS129, 29-BS130, 30-BS131, 31-BS132, 32-BS133, 33-BS134, 34-BS135, 35-BS136, 36-BS137, 37-BS138, 38-BS139, 39-BS140. b Phylogenetic tree constructed based on ToxA gene sequences of different isolates of B. sorokiniana, P. tritici-repentis, P. avenaria and P. nodorum. The evolutionary distances were computed using the Maximum Composite Likelihood method and are in the units of the number of base substitutions per site. Evolutionary analyses were conducted in MEGA X
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Supplementary file6 (JPG 9362 KB) Alignment of nucleotide sequences of B. sorokiniana ToxA gene from India (BSToxA) and Australia (AusBSToxA1 and AusBSToxA2) aligned to P. tritici-repentis (ptr_ToxA) and P. nodorum (Sn4_ToxA)
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Supplementary file7 (JPG 5644 KB) Pathogenicity assay symptoms on different wheat genotypes inoculated with BS112. 1-WH 542, 2-WL 711, 3-Agra Local, 4-Suzoe, 5- HD 29, 6- PBW 343, 7-HD 3086, 8-Chiriya 7, 9-HD 30, 10-Milan, 11-HD 2329, 12- Ning 8139, 13-HD 2967
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Aggarwal, R., Agarwal, S., Sharma, S. et al. Whole-genome sequence analysis of Bipolaris sorokiniana infecting wheat in India and characterization of ToxA gene in different isolates as pathogenicity determinants. 3 Biotech 12, 151 (2022). https://doi.org/10.1007/s13205-022-03213-3
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DOI: https://doi.org/10.1007/s13205-022-03213-3