First report of pod wart disease of peanut caused by Streptomyces spp. in the Western Hemisphere

In August 2018 pods with wart-like symptoms similar to those reported by Kritzman et al. (1996) were collected from peanut (Arachis hypogaea cv. Georgia-06G) plants at the University of Georgia, Bowen Farm, Tifton, Georgia, USA (Fig. 1). Putative pathogenic Streptomyces spp. were isolated from peanut pericarp as previously described (Wanner, 2004). Individual bacterial colonies originating from a single wart-like lesion were spread onto water agar media. Plates were incubated at 30°C for 48 hrs, and bacteria from a single colony were picked with a sterile inoculation loop and transferred to obtain pure culture.

Bacterial colonies were grey in colour with aerial and substrate mycelium similar to typical pathogenic strains of Streptomyces spp. (Loria et al., 1997). Scanning electron and light microscopy revealed structures typical of Streptomyces spp., that is rough, cylindrical spores (Figs. 2-3) in flexuous chains. Genomic DNA was extracted from cultures grown on oat bran agar media at 30°C. Mycelia were scraped from the agar surface with a sterile toothpick and DNA extracted using the DNeasy Power Soil Kit (Qiagen Inc., USA) according to the manufacturer's instructions. DNA-based analyses targeting the 16S rRNA, recombinase A (recA), RNA polymerase b-subunit (rpoB) and thaxtomin synthase (txtAB) were used. Partial sequences of the 16S rRNA (1,219 bp), recA (913 bp), rpoB (994 bp) and txtAB (385) genes were amplified using primers previously reported (Wanner, 2006; Guo, 2008). PCR products were sequenced and submitted to GenBank (Accession Nos. MK630207.1, MK737925 and MK749839 respectively). BLASTn analysis of 16S rRNA (LC207997.1), recA (MF925465.1) and rpoB (KX503547.1) sequences revealed 99% to 100% identity with Streptomyces spp. and had the pathogenicity gene encoding txtAB.

Three plants of cv. Talbert Small Red were each inoculated with a 50 ml (10⁶ CFU/ml) bacterial suspension using a soil drench method to confirm pathogenicity. Non-inoculated plants served as controls. Plants were grown in the greenhouse under a 16 hour photoperiod. Peanuts were harvested 133 days after planting and assessed for the presence of wart symptoms (Fig. 4). Pods recovered from non-inoculated controls were asymptomatic. Streptomyces spp. were re-isolated and recovered from inoculated symptomatic peanut pericarps as described above. The recovered bacteria were subsequently identified using the 16S rRNA, recA and rpoB coding sequences. Thus, the host plant inoculation coupled with DNA sequencing analyses of re-isolated bacteria fulfilled Koch's postulates.

Peanut wart has been reported in Israel (Kritzman et al., 1996) and South Africa (de Klerk, et al., 1997). However, to our knowledge, this is the first report of Streptomyces spp.causing pod wart disease of peanut in the United States and the Western Hemisphere.