Skip to main content
SpringerLink
Log in

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

  • Original Article
  • Published:
3 Biotech Aims and scope Submit manuscript

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8
Fig. 9

Similar content being viewed by others

References

  • Abbas A (1995) Variation in some cultural and physiological characters and host pathogen interaction of Fusarium oxysporum f. sp. lentis and inheritance of resistance to lentil wilt in Syria. Syria: University of Aleppo, Dissertation

  • Agrawal SC, Singh K, Lal SS (1991) Plant protection of lentil in India. In: Lentil in South Asia, New Delhi, India: ICAR-ICARDA seminar, 11–15 March, pp 147–167

  • Al-Husien NH, Hamwieh A, Ahmed S, Bayaa B (2017) Genetic diversity of Fusarium oxysporum f. sp. lentis population affecting lentil in Syria. J Phytopathol 165:306–312

    Article  Google Scholar 

  • Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215:403–410

    Article  CAS  Google Scholar 

  • Alves-Santos FM, Ramos B, Garcia-Sanchez MA, Eslava AP, Diaz-Minguez JM (2002) A DNA based procedure for in planta detection of Fusarium oxysporum f. sp. phaseoli. Phytopathology 92:237–244

    Article  CAS  Google Scholar 

  • Anonymous (2014) Agricultural statistics at a glance. Ministry of Agriculture and Farmer’s Welfare, Government of India

    Google Scholar 

  • Belabid L, Baum M, Fortas Z, Bouznad Z, Eujayl I (2004) Pathogenic and genetic characterization of Algerian isolates of Fusarium oxysporum f. sp. lentis by RAPD and AFLP analysis. Afr J Biotechnol 3:25–31

    Article  CAS  Google Scholar 

  • Bogale M, Wingfield BD, Wingfield MJ, Steenkamp ET (2006) Characterization of Fusarium oxysporum isolates from Ethiopia using AFLP, SSR and DNA sequence analyses. Fungal Divers 23:51–66

    Google Scholar 

  • Chaudhary RG, Amarjit K (2002) Wilt disease as a cause of shift from lentil cultivation in Sangod tehsil of Kota, Rajasthan. Indian J Pulses Res 15:193–194

    Google Scholar 

  • Cunha CMS, Hinz RH, Pereira A, Tcacenco FA, Paulino EC, Stadnik MJ (2015) A SCAR marker for identifying susceptibility to Fusarium oxysporum f. sp. cubense in banana. Sci Hortic-Amsterdam 191:108–112

    Article  CAS  Google Scholar 

  • Datta S, Choudhary RG, Shamim M, Singh RK, Dhar V (2011) Molecular diversity in Indian isolates of Fusarium oxysporum f. sp. lentis inciting wilt disease in lentil (Lens culinaris Medik). Afr J Biotechnol 10:7314–7323

    CAS  Google Scholar 

  • Dubey SC, Singh SR, Singh B (2010) Morphological and pathogenic variability of Indian isolates of Fusarium oxysporum f. sp. ciceris causing chickpea wilt. Arch Phytopathology PFL 43:174–189

    Article  Google Scholar 

  • Dubey SC, Priyanka K, Upadhyay BK (2014) Development of molecular markers and probes based on TEF-1α, β-tubulin and ITS gene sequences for quantitative detection of Fusarium oxysporum f. sp. ciceris by using real-time PCR. Phytoparasitica 42:355–366

    Article  CAS  Google Scholar 

  • Farahani S, Morid B, Maleki M, Saberi S (2015) Using SCAR molecular marker to detect resistance genes to Fusarium oxysporum f. sp. ciceris in chickpea cultivars and lines. Biol Forum Int J 7:1369–1376

    CAS  Google Scholar 

  • Geiser DM, Jimenez-Gasco MM, Kang S, Makalowska I, Veeraraghavan N, Ward TJ, Zhang N, Kuldau GA, O’Donnell K (2004) Fusarium-ID v. 1.0: a DNA sequence database for identifying Fusarium. Eur J Plant Pathol 110:473–479

    Article  CAS  Google Scholar 

  • Hall TA (1999) BIOEDIT; a user friendly biological sequences alignment, editor and analysis of program for windows 95/98/NT. Nucl Acid S 41:95–98

    CAS  Google Scholar 

  • Hill AL, Reeves PA, Larson RL, Fenwick AL, Hanson LE, Panella L (2011) Genetic variability among isolates of Fusarium oxysporum from sugar beet. Plant Pathol 60:496–505

    Article  Google Scholar 

  • Hiremani NS, Dubey SC (2018) Race profiling of Fusarium oxysporum f. sp. lentis causing wilt in lentil. Crop Prot 108:23–30

    Article  Google Scholar 

  • Huang X, Madan A (1999) CAP3: a DNA sequence assembly program. Genome Res 9:868–877

    Article  CAS  Google Scholar 

  • Jimenez-Gasco MM, Milgroom MG, Jimenez-Diaz RM (2002) Gene genealogies support Fusarium oxysporum f. sp. ciceris as a monophyletic group. Plant Pathol 51:72–77

    Article  CAS  Google Scholar 

  • Khare MN (1980) Wilt of lentil. First Technical Report: Project Pl-480. Jawaharlal Nehru Krishi Vishwa Vidyalaya, Jabalpur, India, pp 155

  • Koenig RL, Ploetz RC, Kistler HC (1997) Fusarium oxysporum f. sp. cubense consists of a small number of divergent and globally distributed clonal lineages. Phytopathology 87:915–923

    Article  CAS  Google Scholar 

  • LoBuglio KF, Pitt JI, Taylor JW (1993) Phylogenetic analysis of two ribosomal DNA regions indicates multiple independent losses of asexual alaromyces state among sexual alaromyces state among asexual Penicillium species in subgenus Biverticullm. Mycologia 85:592–604

    Article  CAS  Google Scholar 

  • Luongo L, Vitale S, Haegi A, Belisario A (2012) Development of SCAR markers and PCR assay for Fusarium oxysporum f. sp. melonis race 2-specific detection. J Plant Pathol 94:193–199

    Google Scholar 

  • Mendel M, Higa A (1970) Calcium dependent bacteriophages DNA. J Mol Bio 53:159–162

    Article  Google Scholar 

  • Mohammadi N, Goltapeh EM, Dolatabadi HK, Ahari AB, Pouralibaba H (2011) The genetic diversity of Iranian isolates causing Fusarium wilt of lentil. Int J Agric Technol 7:1809–1822

    Google Scholar 

  • Murray MG, Thompson WF (1980) Rapid isolation of high molecular weight plant DNA. Nucleic Acids Res 8:4321–4326

    Article  CAS  Google Scholar 

  • Mutlu N, Demirelli A, Ilbi H, Ikten C (2015) Development of codominant SCAR markers linked to resistant gene against the Fusarium oxysporum f. sp. radicis-lycopersici. Theor Appl Genet 128:1791–1798

    Article  CAS  Google Scholar 

  • Nourollahi K, Madahjalali M (2017) Analysis of population genetic structure of Iranian Fusarium oxysporum f. sp. lentis isolates using microsatellite markers. Australas Plant Path 46:35–42

    Article  Google Scholar 

  • O’Donnell K (2000) Molecular phylogeny of the Nectria haematococca- Fusarium solani species complex. Mycologia 92:919–938

    Article  Google Scholar 

  • O’Donnell K, Kistler HC, Cigelnik E, Ploetz RC (1998) Multiple evolutionary origins of the fungus causing Panama disease of banana: concordant evidence from nuclear and mitochondrial gene genealogies. P Natl Acad Sci USA 95:2044–2049

    Article  Google Scholar 

  • Pouralibaba HR, Rubiales D, Fondevilla S (2016) Identification of pathotypes in Fusarium oxysporum f. sp. lentis. Eur J Plant Pathol 144:539–549

    Article  CAS  Google Scholar 

  • Pouralibaba HR, Šatović Z, Cobos MJ, Rubiales D, Fondevilla S (2018) Genetic diversity and structure of Fusarium oxysporum f.sp. lentis isolates from Iran, Syria and Algeria. Eur J Plant Pathol 15:10. https://doi.org/10.1007/s10658-018-01617-7

    Article  Google Scholar 

  • Prasanthi LR, Reddy BVB, Rani KR, Prasad YS, Rajeshwari T, Reddy KR (2015) Development of sequence characterized amplified region (SCAR) marker for Fusarium wilt resistant gene in pigeonpea (Cajanus cajan L. Millsp.). Int J Plant Breed 3:134–138

    Google Scholar 

  • Reddy JM, Raoof MA, Ulaganathan K (2012) Development of specific markers for identification of Indian isolates of Fusarium oxysporum f. sp. ricini. Eur J Plant Pathol 134:713–719

    Article  CAS  Google Scholar 

  • Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGA6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol 30:2725–2729

    Article  CAS  Google Scholar 

  • Ullmann A, Jacob F, Monod J (1967) Characterization by in vitro complementation of a peptide corresponding to an operator-proximal segment of the beta-galactosidase structural gene of Escherichia coli. J Mol Biol 24:339–343

    Article  CAS  Google Scholar 

  • Untergasser A, Cutcutache I, Koressaar T, Ye J, Faircloth BC, Remm M, Rozen SG (2012) Primer3—new capabilities and interfaces. Nucleic Acids Res 40(15):e115

    Article  CAS  Google Scholar 

  • White TJ, Bruns T, Lee S, Taylor J (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for Phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ (eds) PCR protocols: a guide to methods and applications. Academic Press, New York, pp 315–322

    Google Scholar 

  • Wunsch MJ, Baker AH, Kalb DW, Bergstrom GC (2009) Characterization of Fusarium oxysporum f. sp. loti forma specialis nov., a monophyletic pathogen causing vascular wilt of birdsfoot trefoil. Plant Dis 93:58–66

    Article  CAS  Google Scholar 

Download references

Acknowledgements

The authors are thankful to Dr R. G. Chaudhary (IIPR, Kanpur) and Dr D. R. Saxena (RAK College, Sehore) for providing the Fol cultures. The first author is also thankful to Indian Agricultural Research Institute, New Delhi and UGC, New Delhi for Rajiv Gandhi National Fellowship. Thanks are also due to the advisory committee Drs. R. K. Jain, Pratibha Sharma, H. K. Dikshit and T. R. Sharma for their support and suggestions; also to Drs. B. K. Upadhyay and Birendra Singh for technical help and support. Further, the authors declare that they do not have conflict of interest of any kind.

Author information

Author notes

  1. Neelakanth S. Hiremani

    Present address: Division of Crop Protection, ICAR- Central Institute for Cotton Research, Nagpur, 440010, India

  2. Sunil C. Dubey

    Present address: Division of Plant Quarantine, ICAR-National Bureau of Plant Genetic Resources, New Delhi, 110012, India

Authors and Affiliations

  1. Division of Plant Pathology, ICAR-Indian Agricultural Research Institute, New Delhi, 110012, India

    Neelakanth S. Hiremani & Sunil C. Dubey

Authors
  1. Neelakanth S. Hiremani
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Sunil C. Dubey
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

SCD- Planning and supervision of the experiment, correction of the manuscript. NSH- Carried out the experiment, analysed the data and prepared the manuscript.

Corresponding author

Correspondence to Sunil C. Dubey.

Ethics declarations

Conflict of interest

All the help and financial assistance has been duly acknowledged. The authors declare that they have no conflict of interest.

Electronic supplementary material

Below is the link to the electronic supplementary material.

13205_2019_1734_MOESM1_ESM.pdf

Online Resource 1. Isolates of Fusarium oxysporum f. sp. lentis representing major lentil growing areas of India (PDF 197 kb)

13205_2019_1734_MOESM2_ESM.pdf

Online Resource 2. The plates showing the blue/ white colonies after spreading (a) and only white colonies after streaking (b) a single white colony on the Luria agar plate amended with IPTG, X-gal and ampicillin after overnight incubation at 37 °C. The arrow indicates a white colony and plate B served as master plate (PDF 408 kb)

13205_2019_1734_MOESM3_ESM.pdf

Online Resource 3. The plate showing the white colonies after streaking a single colony from master plate on the Luria agar plate amended with IPTG, X-gal and ampicillin (sub-plate) after overnight incubation at 37 °C (a), the arrows indicate single white transformed colonies. The gel picture (b) shows the amplification of inserted DNA through colony polymerase chain reaction for MS 2 marker. Lanes M- 100 bp ladder, 1- FLS 23 and 2- FLS 75 (PDF 397 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Hiremani, N.S., Dubey, S.C. Phylogenetic relationship among Indian population of Fusarium oxysporum f. sp. lentis infecting lentil and development of specific SCAR markers for detection. 3 Biotech 9, 196 (2019). https://doi.org/10.1007/s13205-019-1734-4

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1007/s13205-019-1734-4

Keywords

Search

Navigation