Pathogen eradication using the pistachio dieback bacterium as a model.

Abstract

Pistachio dieback is a bacterial disease involving internal staining, trunk and limb lesions, decline, dieback and, in some instances, death of trees. The causal agent is Xanthomonas translucens pv. pistaciae (Xtp), a vascular pathogen that provides a local model to assess the effectiveness of existing eradication strategies for systemic bacterial pathogens of woody perennials which are likely to be introduced into Australia. Burning and burial are two accepted means of disposal of diseased plant material for eradication purposes. However, there is little or no information on the survival of bacterial pathogens following burning or burial of infected wood. The aim of the project was to evaluate the efficacy of burning and burial as means of safe disposal of diseased wood. Burning of pistachio wood, naturally infected with Xtp, was conducted twice in field conditions. Controlled laboratory experiments with pure cultures of Xtp and with naturally and artificially infected pistachio wood were performed to support the results of the burns. Viable Xtp was detected in some non-burned wood, but not in charcoal, ash or partially burned wood. In liquid culture, 65°C was lethal to Xtp whereas survival at 60°C or less varied with culture medium and duration of exposure. In infected wood Xtp survived exposure to 40 - 55°C for at least 60 min in vitro but was killed by exposure to 60°C for 15 min or more. These data corroborated the burning experiment. Survival of Xtp in infected pistachio wood placed on the soil surface or buried 10 cm deep was evaluated in an open environment at the University of Adelaide, Waite Campus orchard, South Australia. The experiment was conducted from August 2008 to March 2011 using naturally infected pistachio wood segments and mulched wood, and was partially repeated in 2010 over 5 months using naturally infected pistachio wood segments and artificially infected twigs. Viable Xtp was isolated from branch segments and from mulched wood buried for 31 and 23 months, respectively. Viable Xtp was not detected in branch segments placed on the soil surface at any time, but was detected in one mulched wood sample at 20 months and in artificially infected twigs for up to 3 months after the placement of wood on the soil surface. Prevailing dry weather conditions during the study might have contributed to the quick decline in Xtp population in the wood on the soil surface. Infrequent isolation of Xtp from buried materials might have been due to the entry of the pathogen into a dormant state, such as viable but nonculturable, in response to changing environments during burial. The ability of copper to induce Xtp to become viable but nonculturable was investigated. Copper induced nonculturability in Xtp at 0.05 mM but this effect was not obvious at 0.005 or 0.01 mM. Xtp exhibited some ability to adapt to the presence of copper at 0.05 mM and there was some indication that spontaneous mutants existed in the Xtp population prior to exposure to copper. Further research is required to confirm the existence of the viable but nonculturable state in Xtp as well of spontaneous copper-resistant mutants in the population. In conclusion, burning is an appropriate eradication technique to dispose of infected debris, providing the pathogen is exposed to a temperature of 60°C or greater for at least 15 minutes. Decomposition of woody material and loss of viability of the pathogen were slow and influenced by environmental conditions. In addition, the pathogen might enter a nonculturable state or evolve in response to changing conditions during burial and become a possible source of inoculum for new infections. Overall, knowledge gained from this study provides information to support and extend existing eradication response strategies for newly introduced or emerging pathogens.