All the 38 isolates showed distinctive morphological variation in their appearance. Colony morphology varied from dark brown to light brown and light tan colored colonies (Fig. 1c), sclerotia and presence of aerial hyphae (Table 1). Correlation analysis showed a positive correlation within the isolates of same crops. The isolates from sugar beet showed a positive correlation with sugar beet isolates and dry beans with dry bean isolates (Fig. 2). There was high positive correlation within the isolates of sugar beets, RZ_SB16, RZ_SB56, RZ_SB373, RZ_SB387, RZ_SB389, RZ_SB364, RZ_391, RZ_332 (r = 1.00 / 1.00, p <0.0001). Similarly, the isolates from dry beans R_DB10, RZ-DB305, RZ_DB386, RZ_DB116, RZ_DB336, RZ_DB360, RZ_DB379 showed significantly high correlation with each other (r = 1.00 / 1.00, p < 0.0001). Though most of the isolates showed morphological correlation within the isolates of same crop there was also positive cross crop correlation with statistical significance but at a lower degree. Isolates RZ_DB22 showed correlation with RZ_SB358 (r = 0.77/1.00, p < 0.01) and RZ_SB359 (r = 0.63/1.00, p < 0.05), RZ_DB305 with RZ-SB375 (r = 0.67/1.00, p < 0.05) (Fig. 2) (Supplementary file: Table: T1). There was no correlation between the isolates from the same geographical origin and year of origin.
A total of 50 UBC primers were screened and 19 primers were selected based on their 100% polymorphism index. A total of 396 alleles were identified from 41 isolates (representative gel image at Fig. 3). An average number of loci per primer was 20.84 and band size ranged from 1 – 3 kb. The primer UBC 889 produced the highest number of polymorphic loci (29) followed by UBC 808 (27), UBC 809 (26) and UBC 812(25) (Table 2). The Shannon information index (I) varied from 0.235 – 0.280 with an average of 0.251. The percentage of polymorphic loci (% P) ranged from 43.26% - 92.88%. The highest % polymorphic loci (92.88%) was observed within the dry bean isolates (population 2) (Table 3). The Nei genetic distance within the population ranged from 0.033 – 0.083 with an average of 0.51.
Cluster analysis based on the UPGMA method had produced three distinct clusters. The first clusters majorly represented the isolates from the dry beans (RZ_DB 116, RZ_386, RZ_DB336, RZ_DB10, RZ_DB305, and RZ_DB360) showing their genetic similarity. However, four sugar beets isolates (RZ_SB373, RZ_374, RZ_375, and RZ_SB38) also showed significant similarities with the dry bean isolates in the first cluster. The second cluster included only sugar beet the isolates viz. RZ_SBC51, RZ_SB349, RZ_B358, RZ_SB37, RZ-SB1, RZ_SB389, RZ_SB391, RZ_SB16, RZ_SB56, RZ_SB338, RZ_SBC23, RZ_SB359, RZ_SB387, RZ_SB332, RZ_SB364. While the third cluster showed cross relation among the dry bean and sugar beet isolates (Fig. 4). Dry bean isolates like RZ_DB22, RZ_DB222, and RZ_DB379 showed genetic relatedness with RZ_SB330, RZ_SB188, and RZ_SB54 in the third cluster. Isolates like RZ_SB374 and RZ_SB375 (with au = 99% and bp = 97%) which were isolated in the year of 2008 from Scottsbluff showed similarity in genetic makeup. Isolates like RZ_SB332 (2005) showed similar genetics with isolates RZ_SB364 (2006) (au = 99% and bp = 96%) (#22).
The AMOVA analysis based on ΦPT values indicated that most of the genetic diversity occurred within the populations (87%, P <0.023) while variability among the population only contributed 13% (P <0.023) (Table 4). Statistically significant genetic differentiation was observed among the isolates.