Isolation and identification of causative agent of potato common scab and characterization of effective antagonistic bacteria as biological control of disease.

Abstract

Potato common scab (PCS) is an economically important and worldwide crop problem. PCS produces visible blemish lesions on the surface of tubers causing diminished quality and low market value. The severity of the lesions can be ranged from superficial raised brown to corky dark brown color with pustules or deep pitted scars which were few millimeters inside the tubers. The lesions may be small, discrete or covering large area of the tubers. Based on the severity of the lesions, common scab (CS) infected tubers sometimes unable for human consumption therefore causing severe economic loss to the growers. Common scab is caused by gram positive Streptomyces. The causal agent of PCS is Streptomyces scabies. Several other Streptomyces species including Streptomyces acidiscabies, Streptomyces europaeiscabies, Streptomyces turgidiscabies, Streptomyces stelliscabies and Streptomyces bottropensis are also reported as major PCS pathogens. In this study, CS infected tubers were collected from potato growing regions of Pakistan (Potato Research Institute, PRI Sahiwal and Lahore), Spain (A Coruna, Ourense, San Esteban and Lugo) and USA (Presque Isle and Orono, Maine). From Spain, almost one hundred and twenty-four bacterial isolates were isolated from CS infected tubers. Similarly, eighteen and ten bacterial isolates were isolated from CS infected tubers collected from USA and Pakistan, respectively. Among the bacterial isolates, twentyeight, thirteen and seven isolates were confirmed as scab-related Streptomyces spp. from Spain, USA and Pakistan, respectively after 16S rRNA analysis. Further identification of selected isolates was based on species specific primers PCR amplification. 16S-23S ITS region amplification, RFLP and phylogenetic analysis revealed four, two and one isolates belonged to S. turgidiscabies, S. scabies and S. stelliscabies, respectively from Pakistan. Similarly, from Spain, sixteen isolates were confirmed as S. europaeiscabies, six, four and two isolates were confirmed as S. turgidiscabies, S. bottropensis and S. stelliscabies, respectively and thirteen isolates were confirmed as S. scabies from USA. For antagonistic bacterial isolates, CS suppressive soil was collected from agriculture potato farms of Okara, Sahiwal and Lahore having no previous CS history over last five to ten years. From suppressive soil samples, five bacterial strains were isolated (A-1, A-2, A-4, A1RT and AC12AB) and screened on the basis of their antagonistic activity assessed by disk diffusion assay against CS isolates (S. scabies). Two best antagonistic bacterial isolates namely A1RT and AC12AB were selected on the basis of strong antagonistic activity against CS pathogens and promising results for plant growth promoting attributes including IAA (Indole-3-acetic acid) production, siderophores production, nitrogen fixation, potassium and phosphate solubilization. Antagonistic bacterial strains A1RT and AC12AB were evaluated to produce IAA quantitatively and qualitatively by colorimetric and HPLC methods, respectively. After 16S rRNA analysis bacterial isolate A1RT and AC12AB were identified as Streptomyces hygroscopicus and Streptomyces violaceusniger, respectively. CS pathogenic Streptomyces strains were analyzed for the presence of pathogenicity islets (PAI) including txtAB, tomA and nec1 genes by PCR amplification. Most of the pathogenic Streptomyces strains screened in this study were found to contain all three pathogenicity related genes like txtAB, nec1 and tomA. Methanolic extracts of Streptomyces hygroscopicus strain A1RT and Streptomyces violaceusniger strain AC12AB were purified with different chromatographic techniques including thin layer chromatography (TLC), silica gel chromatography, Sephadex column chromatography, solid phase extraction and preparative HPLC. The structure of purified compound was elucidated with help of one-dimensional (1H-NMR, 13C-NMR) and twodimensional NMR (1H/1H-COSY, HMQC and HMBC) techniques. The NMR experiment reveled the structure of bioactive compound as Isatropolone C and Azalomycin from antagonistic Streptomyces hygroscopicus strain A1RT and Streptomyces violaceusniger strain AC12AB, respectively. The plant experiments were conducted to check the in-vitro effect of antagonistic bacterial strains. Greenhouse assays were designed in Pakistan, Spain and USA which resulted significant decrease in CS disease severity and increase in number of shoots, shoot length, tuber weight and number of tubers. Finally, field trials were conducted in Pakistan with antagonistic Streptomyces hygroscopicus strain A1RT, Streptomyces violaceusniger strain AC12AB and combination of strain A1RT and AC12AB used as inoculum into the potato plants. About 84, 83, and 86.1% (p<0.05) PCS disease reduction was observed in field trials when S. scabies used with Streptomyces hygroscopicus strain A1RT, Streptomyces violaceusniger strain AC12AB and a combination of Streptomyces hygroscopicus strain A1RT and Streptomyces violaceusniger, strain AC12AB respectively and selected consortia also attributed significant increase in number of shoots, length of shoots, number of tubers and yield per hectare (p<0.05).In conclusion, the selected bacteria used in this study as a consortium, A1RT (Streptomyces hygroscopicus) and AC12AB (Streptomyces violaceusniger) may be used as an excellent biological control agent for decreasing the severity of CS infection and enhancing the growth of potato plants.