First Report of Streptomyces scabies Causing Potato Common Scab in Punjab, Pakistan

Abstract

Common scab (CS) is caused by gram positive, soilborne, filamentous Actinobacteria in the genus Streptomyces. Potato common scab is very prevalent in the potato growing region of Pakistan (Anwar et al. 2013). In this study, 14 CS-infected potato samples were collected from three different potato growing fields of Punjab, Pakistan, during 2015. The samples were heavily infected with a superficial corky layer to pitted scars of CS. The procedure for isolating Streptomyces spp. from the scabby portion of potatoes was adopted as mentioned by Song et al. (2004). One isolate was identified as Streptomyces scabies after morphological and physiological characterization (Wanner 2006). The Streptomyces isolate was grown in yeast maltose broth for 3 days at 28°C. The pellet was used for genomic DNA extraction by using Gene Jet genomic DNA purification kit (Thermo Scientific, USA) and DNA quantity was checked by NanoDrop system (Thermo Scientific). High quality DNA was used for PCR amplification of 16S rRNA gene using primer pair pA/pH (Edwards et al. 1989). The amplicon was sequenced and submitted to NCBI to obtain an accession number. Species and strains specific 16s rRNA gene amplification was performed with PCR. This Streptomyces strain, designated AC-46, gave positive PCR band amplification with primer pairs of S. scabies scab1m/scab2m and S. europaeiscabiei scab1/scab2m (Wanner 2006). To definitely determine whether AC-46 was S. scabies or S. europaeiscabiei, internal transcribed spacer (ITS) region was amplified using primer pair ITSL/ITSR (Song et al. 2004). The amplification was subjected to digestion with restriction enzyme Hpy99I (New England Biolabs). After restriction fragment length polymorphism (RFLP) analysis, Streptomyces strain AC-46 was confirmed as S. scabies with accession no. KU560917. Although Anwar et al. (2013) previously reported the occurrence of S. scabies from CS-infected potato tubers in Pakistan, the methodologies used did not enable definitive identification as S. scabies. Genes responsible for pathogenesis including txtAB, nec1, and tomA were also analyzed. PCR amplification confirmed the presence of txtAB gene with primer pairs txtAB1/txtAB2 (Wanner 2006), nec1 gene with primer pair nf/nr (Bukhalid et al. 1998), and tomA gene with primer pair tom3/tom4 (Wanner 2006), respectively. Pathogenicity of S. scabies strain AC-46 was evaluated in a pot assay. Medium sized (8 liter) pots were filled with substrate (Autoclaved Compo-Sana Universal, Germany). Potatoes (cv. Astrax) were grown at 25 to 28°C in pots for 3 weeks until they germinated. S. scabies strain AC-46, S. scabies (kindly provided by Dr. J. Leiminger, Germany, DSMZ41658) as positive control, and nonpathogenic Streptomyces spp. (KU560918, txtAB negative) as negative control were grown in yeast malt broth in five separate pots in duplicate. Pellets of bacterial suspension were dissolved in 100 ml of sterile normal saline to obtain 106 CFU/ml concentration. One hundred milliliters of vermiculite (bacterial suspension) was drenched into germinated potato plants, separately. After 12 weeks, plants were harvested and disease symptoms were recorded based on percentage of tubers affected by S. scabies. The disease symptoms varied from mild to severe deep pitted scabs (Wanner 2006) without netted scab while no visible CS symptoms were observed for negative controls. Disease severity calculated for S. scabies, AC-46, and negative control was 34.6, 57, and 0%. Pathogenic Streptomyces strain was reisolated from infected tubers to confirm Koch’s postulates.