Genetic diversity and virulence variability of Sclerotinia sclerotiorum in Eastern and Northeastern India

Abstract

Sclerotinia sclerotiorum, the necrotrophic cosmopolitan fungus, has become an emerging and re-emerging pathogen in the subtropical regions. Genetic diversity of 36 isolates of the fungus isolated from infected samples collected from the eastern and North eastern states was carried out using UP-PCR and SSR. Virulence variability was analysed based on four different measures. Among the eight UP-PCR primers and various combinations used, L-21, 3-2 and AA2M2-AS4 generated maximum number of fingerprints (13, 13 and 12, respectively) ranging from 100bp to 1kb. The isolates exhibited varied level of aggressiveness; majority (77.78%) were moderately virulent, 8.33% (22.22% of Assam and 6.67% of West Bengal) isolates were highly virulent, and 13.89% were less virulent. Several amplification products viz., 500bp generated by AA2M2-AS4, 150bp by AA2M2-L-21 and 100bp by L-21-3-2 were positively correlated with disease severity grading at 5% level of significance, whereas, 600bp band generated by AA2M2-3-2 was correlated at 1% level of significance. This indicates presence of these bands in highly virulent isolates. Out of the eight SSR primers, TATG9 did not generate any amplification and the isolates were divided into two major groups; the group II contained single isolate from Nagaland (NG4) indicating it to be genetically diverse from rest of the isolates. The subgroup A of the major group I was the largest and most diverse group with 11 members indicating genetic admixture within different geographic populations with different levels of similarity (70-100%). Genetic diversity based on SSR banding pattern showed highest value of Nei's gene diversity and Shannon's index of diversity (%pb = 61.11; h = 0.219; I = 0.330) for the Nagaland population with 9 members followed by West Bengal population with 15 members. Nei's genetic distance of all the tested populations was low, ranging from 0.0014 to 0.2350; however, genetic identity was high ranging from 0.7905 to 0.9986. The findings suggest that the pathogen populations of eastern and North eastern region were predominantly clonal with some evidence of infrequent out crossing.

Fig 1. UP-PCR based dendrogram of S. sclerotiorum isolates, coloured according to the pathogenicity of the isolates as- green colour indicate highly virulent isolates whereas blue and yellow colours show moderate and low virulent isolates, respectively.

Fig 2. Polygenetic map of different geographic populations of S. sclerotiorum based on Nei’s coefficients and group average hierarchical clusters produced by the POPGENE 1.32.

Fig 3. UPGMA dendrogram showing the genetic relationship between the 36 isolates of S. sclerotiorum based on SSR marker.

Fig 4. UPGMA polygenetic map of different S. sclerotiorum populations based on average hierarchical clusters produced by the POPGENE 1.32.

Fig 5

Distruct output by CLUMPAK representing admixture analysis of population structure for the isolates of S. sclerotiorumfrom West Bengal (WB), Assam (AS), Nagaland (NG), Mizoram (MZ) and Sikkim (SK).