Occurrence of Viable But Non-Culturable Fire Blight in Apple Calyces

Transcription

1 Occurrence of Viable But Non-Culturable Fire Blight in Apple Calyces Satish Wimalajeewa Apple & Pear Australia Limited Project Number: AP06048

2 AP06048 This report is published by Horticulture Australia Ltd to pass on information concerning horticultural research and development undertaken for the apple and pear industry. The research contained in this report was funded by Horticulture Australia Ltd with the financial support of the apple and pear industry. All expressions of opinion are not to be regarded as expressing the opinion of Horticulture Australia Ltd or any authority of the Australian Government. The Company and the Australian Government accept no responsibility for any of the opinions or the accuracy of the information contained in this report and readers should rely upon their own enquiries in making decisions concerning their own interests. ISBN Published and distributed by: Horticultural Australia Ltd Level 1 50 Carrington Street Sydney NSW 2000 Telephone: (02) Fax: (02) horticulture@horticulture.com.au Copyright 2007

3 AP06048 Occurrence of Viable But Non-Culturable Fire Blight in Apple Calyces Project Completion Date 31 November Final report by Alma Reynolds and Satish Wimalajeewa

4 AP06048: Occurrence of Viable But Non-Culturable Fire Blight in Apple Calyces Report prepared by: Alma Reynolds Industry Services Manager, APAL PO Box 612 Mooroopna Victoria 3629 Ph: Purpose of this report This report outlines the outcomes of a visit to specialist Spanish researchers to explore the possibility of carrying out specific research into the character and behaviour of Erwinia amylovora bacteria under specific conditions. The visit and discussion was conducted by Dr Satish Wimalajeewa. Any recommendations contained in this publication do not necessarily represent current HAL policy. No person should act on the basis of the content of this publication, whether as to matters of fact or opinion or other content without first obtaining specific independent professional advice in respect of the matters set out in this publication. This project was funded by Horticulture Australia Limited under the auspices of Apple & Pear Australia Limited

5 Introduction: As part of the on-going issues surrounding the technical aspects of the New Zealand apple Import Risk Analysis (IRA), the apple and pear industry has decided it is desirable to carry out further research on recently documented specific behaviours of Erwinia Amylovora bacteria. Accordingly the apple and pear Industry Advisory Committee set aside a budgeted sum to carry out the research. This project provided a small amount of funding support to allow one of Australia s foremost fire blight authorities to visit researchers in Spain to ascertain whether or not they are an appropriate team to carry out the required research. Purpose of the Project The purpose of this project is for a person with the required skill and experience to seek out a reputable group of plant scientists to carry out research into the ability of Erwinia Amylovora bacteria to enter a state whereby they are viable but unable to be cultured in the normal way. This state means that the bacteria are live and able to initiate infection but cannot be detected using the standard tests. One of the main issues in finding the appropriate team of scientists was to avoid those countries that have a vested interest in exporting apples from areas where fire blight is an endemic disease. Clearly this is not an easy task and so it was felt necessary to visit the team first. The final outcome of the project was to try to develop a proposal for appropriate research to be carried out as part of the industry s overall technical response plan. Operation of the Project The industry selected Dr Satish Wimalajeewa, an Australian plant pathologist with an international reputation for his expertise in fire blight, to carry out this project. Dr Wimalajeewa was on a private visit to the UK but agreed to spend three days of his time to travel to Spain and meet with the scientific team at Instituto Valenciano de Investigaciones Agrarias (IVIA). The purpose of the meeting was to discuss the nature of any research that may be carried out and to ascertain the international independence of the team. Dr Wimalajeewa travelled to Valencia on 4 th September and returned to London on 6 th September having met with the team leader Dr Maia Lopez and the rest of the IVIA team for a number of hours over two days. Outcome of the project The outcome of this project is that Dr Lopez and IVIA together with Dr Wimalajeewa has developed a project proposal that will soon be presented to HAL for funding out of the apple and pear budgeted amount set aside for the purpose. The outline of the proposal can be found as attachment 1. Budget The budget for this project was $2,600 being $1,525 for air fares and $1,075 for accommodation, meals and ground transport. The total expenditure for the project was $1,580.30, so the final result was a project completed well below budget. The full budget and expenditure account can be found as attachment 2. Media Summary A recent visit to Spain by Dr Satish Wimalajeewa has been counted as a complete success when he returned to recommend to the Australian apple and pear industry that they work closely with a team of Spanish researchers to try to resolve one of the unanswered questions about fire blight. Recent research has shown that the bacteria that cause fire blight can enter a form of hibernation which means that they are still capable of causing infection but cannot be detected by the regular tests. This state is known as viable but non-culturable

6 or VBNC. The Australian apple and pear industry wanted to find a suitable team of scientists to undertake this research and Dr Wimalajeewa returned to recommend Dr Maria Lopez and the team from Instituto Valenciano de Investigaciones Agrarias (IVIA). The outcome Attachment of this project 1 is that, in the near future, a further submission for a three-year research project into VBNC fire blight will be presented to Horticulture Australia Limited. PROPOSED STUDIES ON THE OCCURRENCE OF THE VBNC STATE IN E. amylovora IN APPLE FRUIT CALYCES TO BE CONDUCTED AT INSTITUTO VALENCIANO DE INVESTIGACIONES AGRARIAS (IVIA) IN SPAIN Following assessment of costs for the project by IVIA, the proposal will be submitted by Satish Wimalajeewa to Apple and Pear Australia Ltd for their consideration for funding. The scientists involved in this work will be Maria M. López, Elena G. Biosca (University of Valencia), Ester Marco-Noales and Mónica Ordax. Since these workers have been able to induce the VBNC (viable but non-culturable) state in vitro in E. amylovora and also to resuscitate and prove its pathogenicity it is proposed that the following work be undertaken by them. Briefly, the experiments will be as follows: 1. Preliminary experiments. 1.1 Determination of virulence of selected strains (CFBP1430 and NCPPB 2080) on apple fruits. 1.2 Introduction of a gfp-plasmid phc60 in one of the selected E. amylovora strains. 1.3 Comparison of traits of the gfp-marked strain with the wild strain. 1.4 Determination of optimal concentration of copper to induce the VBNC state. 1.5 Optimization of enrichment protocols for the resuscitation of VBNC E. amylovora cells. 2. Survival experiments with inoculated apple material. They will be done with plant material collected from fire blight free orchards of two cultivars (Gala and Golden Delicious). Plant material will be inoculated by pipetting a bacterial suspension into the calyces, or by spraying on the flowers, using the two strains mentioned above. Inoculation will be done on: 2.1 Detached fruits. 2.2 Attached fruits on potted trees or on cut branches. 2.3 Flowers on potted trees. Plant material will be inoculated with E. amylovora after determining the minimal dose (cfu/ml) causing symptoms, which will be the positive control and inoculum doses for survival monitoring (without causing symptoms). Negative controls (sterile water) will be also included. The survival and induction of the VBNC state will be followed in fruits by total, viable and culturable counts, as previously described (Ordax et al., 2006) and by optimized protocols in this study. The experimental period will be: a) two weeks for detached fruits (2.1); b) three weeks for attached fruits (2.2); and c) on flowers initially until fruit development (approximately one month) and from then on every ten days until harvest, and after harvest every ten days under standard storage conditions (2.3). 3. Survival experiments with copper treated apple material after inoculation. These experiments will be done with a predetermined optimal concentration of copper solution to induce the VBNC state, which will be introduced into the calyces about h after fruit inoculation with E. amylovora. Inoculations and survival monitoring will be done as described above (section 2). 4. Survival experiments with fruits from naturally infected trees. Fruits collected from naturally diseased apple trees will be tested at intervals of two weeks after harvest to evaluate the proportion of fruits with culturable, VBNC, and dead E. amylovora cells, using techniques optimized in this study. From that point onwards they will be checked again periodically at intervals of one week during the first month and from then on once a month for six months for the presence of VBNC E. amylovora cells.

7 In all experiments, resuscitated VBNC cells will be tested for pathogenicity. All the experiments will be repeated at least twice in independent assays, and the results will be statistically analyzed. It is anticipated that results from the above experiments will show whether VBNC state does occur in apple in NATURE, and, if so as to how long the bacteria remain viable and pathogenic though it survived in a VBNC state in the assayed conditions. Attachment 2 AP06048 Occurrence of Viable But Non-Culturable Fire Blight in Apple Calyces Budget Actual Income HAL $ 2, $ 2, (not yet paid) Expenditure Air fares $ 1, $ 1, Accommodation meals and on-ground transport $ 1, $ $ 2, $ 1, Balance $ - $ 1,019.70