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Phylogenetic relationship among Indian population of Fusarium oxysporum f. sp. lentis infecting lentil and development of specific SCAR markers for detection.

ABSTRACT: The present study was taken up to understand the phylogenetic relationship using ITS and TEF markers among 22 isolates of Fusarium oxysporum f. sp. lentis (Fol) causing lentil wilt belonging to eight races isolated from different geographic locations of India and to develop specific markers for its detection. The nucleotide sequences of ITS region varied from 490 to 560 bp whereas, 670-725 bp for TEF 1α. The phylogeny analysis revealed that the isolates were more than 98% similar based on the neighbour joining analysis and were grouped into two major clusters in both ITS and TEF. The first major cluster of ITS had twenty isolates whereas for TEF, there were 15 isolates. Two sets of SCAR markers MS1 (162 bp) and MS2 (125 bp) were designed and synthesised. These markers were used against race representative Fol isolates for amplification. While, MS 1 marker was able to detect the genomic DNA up to 0.1 ng, MS 2 could detect the Fol genomic DNA up to 0.05 ng. The specificity of these two markers to detect Fol and their inability to amplify most common lentil pathogens (Rhizoctonia solani, R. bataticola, Sclerotium rolfsii, Sclerotinia sclerotiarum, and Aschochyta rabiei) makes them a reliable tool for detection. The phylogenetic analysis is helpful in the understanding of variability in Fol populations and the SCAR markers help in rapid and reliable detection of an important pathogen of lentil.

SUBMITTER: Hiremani NS

PROVIDER: S-EPMC6499849 | biostudies-literature | 2019 May

REPOSITORIES: biostudies-literature

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Phylogenetic relationship among Indian population of Fusarium oxysporum f. sp. lentis infecting lentil and development of specific SCAR markers for detection.

Hiremani Neelakanth S NS Dubey Sunil C SC

3 Biotech 20190503 5

The present study was taken up to understand the phylogenetic relationship using ITS and TEF markers among 22 isolates of Fusarium oxysporum f. sp. lentis (Fol) causing lentil wilt belonging to eight races isolated from different geographic locations of India and to develop specific markers for its detection. The nucleotide sequences of ITS region varied from 490 to 560 bp whereas, 670-725 bp for TEF 1α. The phylogeny analysis revealed that the isolates were more than 98% si ...[more]

PMID: 31065496

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Project description:Fusarium wilt caused by Fusarium oxysporum f. sp. lentis (Fol) is the most devastating disease of lentil present worldwide. Identification of multi-race fusarium wilt resistance genes and their incorporation into existing cultivars will help to reduce yield losses. In the present study, 100 lentil germplasms belonging to seven lentil species were screened against seven prevalent races of Fol, and accessions IC201561 (Lens culinaris subsp. culinaris), EC714243 (L. c. subsp. odemensis), and EC718238 (L. nigricans) were identified as resistant. The typical R gene codes for the nucleotide-binding site and leucine-rich repeats (NBS-LRR) at the C terminal are linked to either the Toll/interleukin 1-like receptor (TIR) or coiled coil (CC) at the N terminal. In the present study, degenerate primers, designed from the NBS region amplifying the P-loop to the GLPLA motif, isolated forty-five resistance gene analogues (RGAs) from identified resistant accessions. The sequence alignment identified both classes of RGAs, TIR and non-TIR, based on the presence of aspartate (D) and tryptophan (W) at the end of the kinase motif, respectively. The phylogenetic analysis grouped the RGAs into six classes, from LRGA1 to LRGA6, which determined the diversity of the RGAs present in the host. Grouping of the RGAs identified from Lens nigricans, LnRGA 2, 9, 13 with I2 revealed the structural similarity with the fusarium resistance gene. The similarity index ranged from 27.85% to 86.98% among the RGAs and from 26.83% to 49.41% among the known R genes, I2, Gpa2, M, and L6. The active binding sites present along the conserved motifs grouped the RGAs into 13 groups. ADP/ATP, being the potential ligand, determines the ATP binding and ATP hydrolysis activity of the RGAs. The isolated RGAs can be used to develop markers linked to the functional R gene. Furthermore, expression analysis and full-length gene isolation pave the path to identifying the molecular mechanism involved in resistance.

Project description:Fusarium wilt caused by Fusarium oxysporum f. sp. cubense (Foc), is the most lethal soil-borne fungal pathogen infecting bananas. Foc race 1 (R1) and 4 (R4) are the two most predominant races affecting the economically important Cavendish group of bananas in India. A total of seven vegetative compatibility groups (VCGs) from three pathogenic races were isolated during our field survey and were found to be highly virulent towards cv. Grande Naine. According to comparative genome analyses, these Indian Foc VCGs were diverse in genomic organization and effector gene profiles. As a result, false-positive results were obtained with currently available molecular markers. In this context, the study has been initiated to develop PCR-based molecular markers for the unambiguous identification of Indian Foc R1 and R4 isolates. Whole-genome sequences of Foc R1 (GCA_011316005.3), Foc TR4 (GCA_014282265.3), and Foc STR4 (GCA_016802205.1), as well as the reference genomes of Foc (ASM799451v1) and F. oxysporum f. sp. lycopersici (Fol; ASM14995v2), were aligned to identify unique variable regions among the Foc races. Using putative chromosome and predicted gene comparison, race-specific unique Foc virulence genes were identified. The putative lineage-specific identified genes encoding products secreted in xylem (SIX) that may be necessary for disease development in the banana. An in silico analysis was performed and primers were designed from a region where sequences were dissimilar with other races to develop a specific marker for Foc R1, R4, TR4, and STR4. These race-specific markers allowed target amplification in the characterized highly virulent Foc isolates, and did not show any cross-amplification to any other Foc races, VCGs or banana pathogens, Fusarium species, and non-pathogenic Fusarium oxysporum isolates. The study demonstrated that the molecular markers developed for all the three Foc races of India could detect the pathogen in planta and up to 0.025 pg µL-1 DNA levels. Thus, the markers developed in this study are novel and could potentially be useful for the accurate diagnosis and detection of the Indian Foc races which are important for the effective management of the disease.

Dataset Information

Phylogenetic relationship among Indian population of Fusarium oxysporum f. sp. lentis infecting lentil and development of specific SCAR markers for detection.

Publications

Phylogenetic relationship among Indian population of <i>Fusarium oxysporum</i> f. sp. <i>lentis</i> infecting lentil and development of specific SCAR markers for detection.

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