Observations on the pathogenicity, epidemiology and control of plant pathogenic pseudomonads with special reference to Pseudomonas syringae pv.

actinidiae (Psa-V)

Pseudomonas syringae pv. actinidiae (Psa-V) in golden kiwifruit (Actinidia chinensis) is one of the most serious bacterial pathogen-host combinations known. Relatively few studies have been made of Psa-V but preliminary conclusions about this pathogen can be draw from the many studies made of related pathogens. Systematic field studies of bacterial epidemiology, management and control were at their height in the 1960s 1980s. Questions concerning infection processes, disease development at the population levels, epidemiology, and the effectiveness of control measures, were all the subject of publication and discussion at international conferences in that period. Psa-V was first observed in Italy in 2008, where it has subsequently caused severe canker losses, particularly to gold kiwifruit (Actinidia chinensis). It was first observed in Te Puke in November, 2010. Unless the past season in the Te Puke region has atypically favoured the pathogen, then it is clear that gold kiwifruit will be as or more susceptible to Psa-V here than in Italy and the management and control of the pathogen using available methods will be problematic. Strict attention to the regulation of movement of plant material from infected regions may slow the spread of the pathogen to other regions, but individual escapes will be very difficult to prevent. A breeding programme to introduce resistance into suitable kiwifruit cultivars is the only measure likely to give a satisfactory outcome in the long term. Aetiology 1. When a bacterial epidemic follows a recent introduction of a pathogen, the population can be considered to be clonal or nearly so, and it follows that all cells will share similar virulence. 2. It was established in the 1970s that a single pathogenic cell, homologous with its host, was capable of causing infection. Heterologous reactions (HR) resulted from local infections by more than one cell. 3. The application of very high levels of inocula in susceptible combinations sometimes fails to induce any reaction, except a hypersensitive reaction (HR) because inoculation conditions do not result in penetration of inoculum into the susceptible infection courts. 4. The three major environmental factors affecting primary infection are moisture, temperature and light. Temperature and light-controlled humidity cabinets should be suitable for successful inoculation. Extended moisture periods following inoculation may induce an HR. Temperatures below 200C will favour disease development, and most success should be obtained by 16/8 day/night cycles. When leaves transpire, the stomata are so numerous and open so widely that the lower leaf surface offers practically no barrier to gas exchange or bacterial infection. Light is probably the most critical factor involved in stomatal opening. 5. Quorum sensing (QS) is a well-established phenomenon in which bacteria are stimulated to specific metabolic action only when physically closely associated cells act in concert. It has been shown that QS influences many metabolic processes including several disease steps in P. syringae. However, the available data indicates that the expanded populations develop more slowly after inoculation (one or more days) and after the multiplication of individual pathogenic cells (a few hours) have already begun. Evolutionary considerations suggest that it would be fatal for a single-celled pathogen to require the multiple invasions of infection sites for development. Systemic infection 1. It has been claimed that the angular lesions in the leaf parenchyma are major points of entry into the vascular system, spread following into the vasculature of leaf veins, petioles and canes. This has led to recommendations to remove all visibly infected leaves to prevent infection of canes. Examination of leaves from Italy and New Zealand show that lesions are confined to leaf

parenchyma by the veins, a characteristic of most bacterial pathogens. No instance of lesions spreading into veins was observed.

Figure showing invasion of leaf parenchyma delimited by veins (Photo: J.P. Wilkie) 2. There is little evidence of significant movement of individual bacterial cells either in the parenchyma or vasculature. Cells multiply at lesion margins, pushing young cells into the adjacent tissue, thus advancing the lesion.

Pruning 1. Pruning to control bacterial pathogens has been a routine recommendation for a century. However, it was the subject of several conference sessions and workshops in the early 1970s when the consensus was that pruning was not an effective control measure. Numerically, there are many more natural infection sites than pruning wounds and, although the latter were more susceptible, they made a lesser contribution to infection. Clearly Psa-V does infect through pruning cuts, but trials in Italy have recently shown that the cuts are, for practical purposes, impossible to sterilize, even by cauterizing wounds with a blow-lamp immediately after pruning. It is clear that the continuing recommendation in New Zealand to create quarantine zones around infected vines or blocks by pruning has no efficacy and is made at a cost. Bactericidal Control 1. Attempts at bacterial control with in-season copper sprays have been made for more than a century. It has been shown that many plants, including kiwifruit (unpublished), suffer from severe phytotoxicity, making the cure as harmful as the disease. Once the pathogen has penetrated leaves, whether or not symptoms are expressed, it is impossible for control sprays, coppers or antibiotics, to affect them. Streptomycin is now cleared for spray application on kiwifruit. Past trials have given highly variable results and other P. syringae pathovars in lab tests develop absolute resistance in a single step. Sprays applied to cover leaf scars as infection sites in autumn have proved efficacious with other pseudomonads, but have not yet been tested against Psa-V in kiwifruit. 2. Concern has been expressed that tolerance to copper and streptomycin may render sprays ineffective. However, confirmation that Psa-V isolated from infected tissue is copper tolerant and that these isolates form a preponderance of infections. 3. Past studies of injection of antibiotics into kiwifruit trunks resulted in the direct transmission of the antibiotic to the leaves where it caused chlorosis with severe stunting of the leaf margins (unpublished). Application to orchards without formal trials is highly inadvisable. Biocontrol 1. Systematic trials of biocontrol have been reported since the 1960s. The methods are invariably based on the same approach. Saprobes are screened in culture for antagonism to the target pathogen. In glass-house trials, antagonistic strains are briefly inhibitory if the antagonist is applied in numbers at least an order of magnitude greater than the pathogen and immediately prior to, or at the same time as inoculation of the pathogen. The closing sentence of such reports is usually that field trials with be conducted, but nothing more is heard. 2. The reasons for failure of this approach are now well-understood. The characteristic of pathogens is to induce leakage in the host cell wall, releasing the cellular contents as nutrients for their multiplication and secondarily to saprobes. Although pathogens grow more slowly than saprobes in culture, the latter are dependent on the prior activity of the pathogenic cells for growth in the plant. Saprobes survive in or on leaves for some hours or days. If the populations are examined over extended periods, it will be found that the saprobes decline except under unusual circumstances, but the pathogen multiplies exponentially after a resting phase of a few hours. It follows that the saprobe can have little influence on the pathogenic population. Sporeforming bacteria as biocontrol agents are also inactive in their plant association but they instantly form resting spores in adverse conditions, which is why high populations can be recorded after copper sprays. 3. Bacteriophages (phages are viruses that multiply in bacteria and kill them) were tested exhaustively in the 1970s - 80s without success. The reason is probably that phage particles are immobile and will be ineffective in reaching bacterial populations in plants until this particular problem is solved.

Dispersal 1. It has been known for decades that bacterial pathogens are dispersed for hundreds of metres, perhaps kilometres in clouds of inoculum generated as aerosols in wind-driven rain, which spread between blocks and orchards. The implications for control have been forgotten. Where a local infection site is observed, then it follows that the pathogen is already present in all surrounding vines and blocks. The recommended pruning measures can therefore have no influence on epidemic development. 2. Localized expression, sometimes only in one part of a single vine, is due to the highly specific environmental conditions required for epidemic development. Fundamental to an understanding of bacterial epidemiology is that epidemics only occur, sometimes after years of bacterial association with the host at sub-clinical levels. Given the virulence of PsaV, epidemics may be more regular than those occurring in weaker pathogens, but absence of symptoms of the pathogen is certainly not evidence of its absence. Containment 1. Only significant physical breaks between infected and uninfected orchards will delay further spread of the pathogen, with rigorous attention to preventing the movement of infected material between regions. The recent identifications of Psa-V near Tauranga, Katikati, Waihi, and Wakatane, most probably on inadequately disinfected vehicles. However, attempts at active containment in other crops in the past have never been effective (citrus canker in Florida using draconian measures being the exception) and it is now clear attempts to contain Psa by attempts at eradication in identified containment zones is unrealistic. Epilogue Until 1992, scientific research in New Zealand was fully funded within the Department of Scientific Research (DSIR). Scientists were expected to anticipate and attempt to answer questions considered to be of the most social or economic importance. A new model of science management was introduced then, with the creation of independent Crown Research Institutes. These are partially government-funded, scientists in CRIs being expected to contract extra funds for specific projects from the private sector and other interest groups. It has been an inevitable consequence of the model that many areas of no immediate interest have been under-funded and have lost their critical mass in scientific expertise with dismissals and attrition of staff. Now, little time is allowed for scientists to maintain a depth of understanding from the past literature or from exploratory experimentation. Many important concepts, once thoroughly understood in the science community, have been forgotten. As well, individual scientists and groups have become secretive, sharing information and resources with colleagues only on the basis of exclusive collaborations by which they hope to capture funds. It is now common for individuals to lay claims to expertise that they do not have and to make research proposals to clients that do not serve their interest. In the case of Psa-V, specific lines of research are proposed by scientists but recommendations for control appear to be largely in the hands of the industry without adequate expert advice. There are more than 20 staff employed by KVH, of whom 5-6 are scientists, none of whom has expertise in the epidemiology, management and control of plant pathogenic bacteria. The very effective communications between DSIR and MAF were severed with the formation of the CRIs, with MAF being given powers of regulation but lacking any expertise to make sensible decisions. These factors have already resulted in substantial loss and waste which, presumably, will continue unabated for the foreseeable future. J. M. Young MSc (Hons1) PhD Mt Albert Auckland youngj@clear.net.nz 3 October 2011

John Young was recruited into Plant Diseases Division, DSIR, in 1967, where, for the next 20 years, he studied the epidemiology, management and control of plant pathogenic bacteria, including those of kiwifruit. Subsequently he transferred to Landcare Research to study the molecular relationships of pathogens, towards their rapid identification for quarantine purposes. For a period, he was Chair of the Bacteriology Section of the International Society for Plant Pathology. He retired in 2006, having published 123 notes and papers on bacterial pathogens, and then was engaged with Landcare as a Research Associate, publishing 13 papers, two of which have raised considerable interest in the international community. He is a member of the EUs Committee On Science and Technology (COST), in the subgroup specificly studying the epidemiology of bacterial diseases of fruit trees. In 2009, he was invited to a COST meeting in Italy to give a review of his work on the epidemiology, management and control of pathovars of Pseudomonas syringae, and in 2010 in Serbia, was one of four experts to lead a group of 12 graduate students and scientists to explain all important aspects of bacterial plant pathology. He has continued as a Research Associate to lead three molecular studies, one of which is to show the relationships of all bacterial pathogens known in kiwifruit. He continues to review manuscripts for several international journals.