Asian Research Thesis Index

Abstract

Vascular wilt of lentil caused by various ecologically and phylogenetically diverse species of Fusarium is found in all the lentil growing areas of Pakistan and the disease could be visualized at both seedling and adult stages of plant growth. The disease is responsible for huge losses each year in Pakistan, yet, there is a scarcity and lack of literature and information regarding its occurrence, incidence, distribution, biology and management of wilt pathogens. Therefore, the study was planned keeping in view the national interests to avoid future losses caused by lentil wilt. The objectives of this study were to assess the wilt prevalence and incidence in major lentil growing districts of Punjab, morpho-molecular and pathogenic characterization of recovered wilt pathogens and the management through host plant resistance, biological control agents and fungicides. A two year field survey data (2011-12 and 2012-13) and laboratory isolations ascertained 213 isolates of Fusarium pathogen as associated wilt incidence identified in the fields. Disease was found widespread with 100% prevalence in all the major lentil growing districts of Punjab viz. Bhakkar, Layyah, Mianwali, Khushab, Sialkot, Narowal, Chakwal, Attock, Gujrat and Jhelum. The mean wilt incidence was found 28% with maximum incidence recorded at adult plant stage (32.4%) than at seedling (23.05%). Morphological characterization showed significant variation among the isolates and based on similar morphology, these were grouped into 67 type isolates for subsequent study. The in vitro pathogenicity testing through root dip method using line NARC-08-1 and cultivar Masoor-93 showed excellent production of wilt symptoms for pathogenic characterization. High pathogenic variability was revealed among the isolates. Based on disease reaction i.e. avirulent to highly virulent observed on most susceptible line NARC-08-1, isolates showed 0 to 100% disease incidence and severity index with significant (11.86 to 100%) reduction in yield. The isolates were grouped into four categories viz. highly virulent (13 isolates, 19.40%), moderately virulent (41, 61.19%), low virulent (8, 11.94%) and avirulent (5, 7.46%). The highly virulent isolates included FWC15, FWJ35, FWJ49, FWG1, FWS11, FWS13, FWN2, FWL2, FWL6, FWL9, FWL12, FWB10 and FWK2. Molecular characterization and DNA sequencing of isolates through PCR amplification of translation elongation factor TEF-1α gene region using primers ef1 and ef2 confirmed the identity of the Fusarium isolates at species level. The amplification produced a single DNA fragment of size 700bp in each of the isolates. Phylogenetic analysis of 67 morphologically and pathogenically diverse Fusarium isolates recovered from various lentil districts of the country revealed that the isolates belonged to different clades under five distinct species. The identified species included F. oxysporum, F. redolens, F. nygamai, F. commune and F. equiseti. This data supported the morphological variation observed among the isolates and divulged the association of these identified species in wilt disease incidence as reported in the major lentil producing region of the country. The findings revealed the highest prevalence of F. oxysporum (49.29%) in the region followed by F. redolens (29.57%), F. equiseti (10.79%) and F. commune (7.98%), while least prevalence was of F. nygamai (2.34%). The most virulent F. oxysporum isolate FWL12 (GenBank accession number KP297995) was selected for the management trials. Screening of the lentil germplasm revealed reduced wilt infection in five cultivars viz. Markaz-09, Masoor-86, Masoor-2006, Punjab Masoor-00518, Punjab Masoor-09 that showed 4.44 to 12.59% severity index, 20 to 46.67% incidence and 9.60 to 24.94% yield reduction. The biological management revealed the best efficiency of T. harzianum in reducing wilt infection on line NARC-08-1 and produced 8.9% disease severity index, 26.7% incidence with 16.27% yield reduction. Chemical seed treatment identified systemic fungicides as highly effective in disease management that resulted in improved 100% seed germination. Benomyl (6.7% incidence and 1.5% severity index) was found superior than Thiophanate methyl (13.3%, 3%). In conclusion, the results of this research study provide an overall current status of wilt disease in the country and high lightened the areas under current high risk of its spread. The findings also revealed the continuous reduction in the acreage of this crop in the major lentil region. The revealed association of five virulent and morpho-molecularly diverse species viz. F. oxysporum, F. redolens, F. nygamai, F. commune and F. equiseti with the wilt disease is reported for the first time in Pakistan. The screening suggests five cultivars viz. Markaz-09, Masoor-86, Masoor-2006, Punjab Masoor-00518 and Punjab Masoor-09 as an important source of resistance for lentil breeding against wilt. Moreover, T. harzianum proves an efficient biological control agent, while seed treatment suggests Benomyl and Thiophanate methyl as the most effective against the wilt pathogen.

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