Diversity of bacteriophages infecting Ralstonia solanacearum and their application as bio control agents

Abstract

newlineBacterial wilt caused by Ralstonia solanacearum is one of the most destructive plant diseases of the world that is very difficult to manage. Sustainable management of R. solanacearum using bacteriophages is one of the most promising yet unexplored areas of biological control in India. In the present study, 50 R. solanacearum isolates were collected from various bacterial wilt infected fields in 5 districts of Upper Brahmaputra Valley Zone (UBVZ) of Assam namely Golaghat, Jorhat and Sivsagar, Dibrugarh and Tinsukia from several crops like bhut jolokia, chilli, capsicum, tomato, brinjal, wild brinjal, potato and ginger. This was followed by isolation of seven lytic phages of R. solanacearum from soil from bhut jolokia fields of Jorhat, Assam. After six rounds of purification of the phages, the plaque morphology, phage titre and the multiplicity of infection (MOI) was determined where it was observed that phage RSP1 had the biggest plaque size (3.28 ± 0.40 nm) and phage RSP4 had the highest titre (1.2E+09 PFU/ml). The adsorption time, latent period, rise period as well as the burst size of the isolated phages was also determined. Physiochemical characterization of the phages revealed that the phages were stable from 4and#8451;-50 and#8451;, a pH range of 4-9 and except RSP1; all other phages were stable 1% and 5% chloroform. Morphological classification using TEM revealed that all the phages had an icosahedral head with a very short non- contractile tail indicating that they may belong to either Podoviridae and/or Autographiviridae family of order Caudovirales. From the lysis dynamics curves of the individual phages, 3 phage cocktails were formed and cocktail-3 having all the seven phages gave the best results. Study on host range of the phages stated that all the phages were multi strain specific in nature and phage RSP4 had the highest host range. In vitro suppression of R. solanacearum in soil using phage cocktail-3 revealed that the cocktail was effective in managing R. solanacearum population in soil when used as a prophylactic manner. Finally, from the study on long term viability of phages at different storage temperatures it can be recommended that these seven phages could be stored in glycerol stocks at -45 and#8451; or -20 and#8451; and in SM buffer in +4 and#8451; without losing viability for a year.