Bacteriophages For Biocontrol Of Pectolytic Bacteria Infecting Potatoes In Nakuru County,Kenya

Pectolytic bacteria associated with blackleg disease of potatoes and soft rot in vital crops are an impediment to quality food production and contribute to economic loss.This study aims at identification of pectolytic bacterial phytopathogens infecting potatoes in Nakuru county,Kenya,and isolation of bacteriophages infecting them for experimental tests to determine suitable phage cocktails for use as biocontrol agents against potato soft rot.Potato tubers showing symptoms of soft rot were collected from different farms in Molo and Mau Narok regions within Nakuru county.Isolation was done using crystal violet pectate medium（CVPM）.Out of 71 isolates that showed growth on crystal violet pectate medium,pathogenicity tests revealed that 36（50.7%）of them had the ability to macerate tissues of potato tubers.All the isolates yielded a fragment of approximately 1500bp after 16S rDNA amplification.Using the BIOLOG microbial identification system,20 bacterial isolates were identified as Pectobacterium carotovorum subsp.carotovorum,7 of the isolates were Pseudomonas fluorescens B while 9 were Pseudomonas fluorescens A.Y1/Y2 primers were used to confirm the identity of Pectobacterium carotovorum strains and successfully amplified pectate lyase（pel）encoding gene,approximately 434bp,in all the 20Pectobacterium carotovorum species.Isolated Pectobacterium spp.were used in isolation of bacteriophages from soil and water samples,collected in Wuhan,China.Morphological characterization revealed that seven of the phages belong to the family Myoviridae while four belong to the family Siphoviridae.Lytic activity was assessed by monitoring change in optical density(OD₆₀₀)of phage/bacteria suspension for 12 h using a micro-plate reader and one step growth experiments.Phage Therapy was tested using potato tuber slices and whole tubers.All the phages were found to be lytic with latency periods ranging from 1 to 1.5 h in LB broth at 28^oC and displayed biocontrol potential against P.carotovorum,reducing the incidence of soft rot disease by over 50%.However,the best results were obtained when high titres,1 x 10⁹ PFU ml^-1,of individual phages and the phage cocktail were used causing≥90%bacteria mortality.Spot-on-lawn tests showed that they could lyse 20 strains of P.carotovorum but had no antibacterial activity against the tested strains of Pseudomonas fluorescens while only one phage,pcp5f,showed antibacterial activity against Pectobacterium atrosepticum.Two phage resistant strains of P.carotovorum were also found to be susceptible to pcp5f infection.This study reveals the identity of pectolytic bacterial species from two genera,Pectobacterium and Pseudomonas,as causative agents of potato soft rot in Nakuru county,Kenya,and provides a basis for the development of an eco-friendly biocontrol agent,bacteriophage cocktail,effective in reducing the impact of soft rot and blackleg diseases of potatoes.