Ecology of Xanthomonas campestris pv. juglandis bacteriophages isolated from walnut orchards in New Zealand

An increasing problem in walnut orchards is the inability to fully control the blight caused by Xanthomonas campestris pv. juglandis. Recently, studies on the use of phages as agents of control of bacterial diseases have been explored worldwide with great enthusiasm. The aim of this research project was to gain a better understanding of the ecology and characteristics of phages that would potentially be useful in the control of X campestris pv. juglandis in walnut orchards in New Zealand. Plant material from 326 samples was screened for the presence of phages around New Zealand between 2000 and 2002. Twenty-six phage isolates able to infect the bacterium were characterized by host-range, plaque and particle morphology, stability under various storage conditions and restriction enzyme patterns. Additionally, testing lysogens within X campestris pv. juglandis populations showed a number of phageresistant bacteria coexisting in these ecosystems. Phages were categorized as lytic, lysogenic or inactive and considered specific to the species level of Xanthomonas. Bacteriophages presented a restricted host range when seven different genera and 13 species where tested. Only фP16 and фP19 were able to infect different genera. Morphologically, the bacteriophages belonged to the Podoviridae and Siphoviridae families and therefore correspond to double stranded DNA bacteriophages. Time was crucial for phage survivability under storage conditions. In general, walnut bacteriophages high titres (HTs) declined more than 50% over a 12-month period. In addition, deep-freezing temperatures (-34°C) favoured survival and chloroform negatively affected phage activity. Phage genome sizes were estimated to be about 44.5 ± 4.4 kb. PCR of a common 400 kb fragment revealed that phages belonging to two different families exhibited a 100% DNA identity which is probably the result of acquisition of genetic material from a global phage pool, by horizontal gene transfer. HindIII profiles from X campestri pv. juglandis phages showed high number of common bands suggesting that phages may be close related. From the wild populations of X campestris pv. juglandis isolates, a number of phage-resistant bacteria were recovered showing inability of the phage to be adsorbed by the bacteria. PCR analysis confirmed the absence of phage DNA inclusion into the bacteria genome, suggesting that these colonies were phage-resistant bacteria emerging from the wild population. It is concluded that these types of studies are helpful for finding phages active to X campestris pv. juglandis from which the most virulent and most resistant ones to environmental stress may be selected for future biocontrol programmes.