Analysis of Tomato Potato Psyllid on sticky traps caught during for presence of Ca. Liberibacter solanacearum and Ca.Phytoplasma australiense

Transcription

1 Analysis of Tomato Potato Psyllid on sticky traps caught during for presence of Ca. Liberibacter solanacearum and Ca.Phytoplasma australiense Berry NA, Scott I, Thompson S, Drayton G, Beard S September 2010 A report prepared for: Horticulture New Zealand NA Berry, I Scott, S Thompson, G Drayton, S Beard Plant & Food Research, Lincoln SPTS No PFR Client Report No PFR Contract No

2 DISCLAIMER Unless agreed otherwise, The New Zealand Institute for Plant & Food Research Limited does not give any prediction, warranty or assurance in relation to the accuracy of or fitness for any particular use or application of, any information or scientific or other result contained in this report. Neither Plant & Food Research nor any of its employees shall be liable for any cost (including legal costs), claim, liability, loss, damage, injury or the like, which may be suffered or incurred as a direct or indirect result of the reliance by any person on any information contained in this report. The New Zealand Institute for Plant & Food Research Limited has exercised reasonable skill, care and diligence in the work described in this report, but shall not be liable for the commercial performance of any products or for any losses arising from the use of the information contained herein. By listing the chemicals, we do not endorse their use in any way in covered or outdoor crops outside of label claims. This report has been prepared by The New Zealand Institute for Plant & Food Research Limited (Plant & Food Research), which has its Head Office at 120 Mt Albert Rd, Mt Albert, Auckland. This report has been approved by: Nadine Berry Senior Entomologist, Vegetable, Arable & Southern Entomology Team Date: September 2010 Louise Malone Science Group Leader, Applied Entomology Group Date: September 2010

3 Contents 1 Introduction 1 2 Method 2 3 Results Hawke s Bay Tomatoes Potatoes Waikato Potatoes Manawatu Potatoes Pukekohe Potatoes North Canterbury Potatoes Mid Canterbury Potatoes South Canterbury Potatoes Discussion/Recommendations 20 5 Acknowledgements 21 6 References 22

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5 Executive summary Analysis of Tomato Potato Psyllid on sticky traps caught during for presence of Ca. Liberibacter solanacearum and Ca.Phytoplasma australiense Nadine Berry, September 2010, SPTS No The impact of the Tomato Potato Psyllid (Bactericera cockerelli) and associated pathogen(s) has threatened the future production of potatoes, tomatoes, capsicums and tamarillos in New Zealand. Seasonal variation in Tomato Potato Psyllid (TPP)/Ca. Liberibacter solanacearum /Ca. Phytoplasma australiense levels may provide an opportunity to guide the selection of control measures applied during higher risk periods. The objective of the current study was to determine the prevalence of Ca. Liberibacter solanacearum and Ca. P. australiense in TPPs caught on sticky traps in commercial potato and field tomato sites during the growing season. Data from sticky traps in Pukekohe, Waikato, Hawke s Bay and the Manawatu regions of the North Island and South, Mid and North Canterbury regions of the South Island were included in this analysis. Molecular screening of TPP DNA from stored traps of representative sites from all monitoring locations has shown that Ca. L. solanacearum is present at all sites over most monitoring periods, while Ca. P. australiense is present less frequently at some monitoring sites and during some periods. The bulk extraction and screening of DNA from up to five individuals on a trap was used to detect Ca. L. solanacearum and Ca. P. australiense. Bulk extraction enabled a reduction in sample number and cost. Although a pathogen-free period was not established, further analysis of the proportion of infected versus uninfected TPP individuals between monitoring sites/dates could increase our understanding of the variation in disease incidence between monitoring sites. For further information please contact: Nadine Berry The New Zealand Institute for Plant & Food Research Ltd Plant & Food Research Lincoln Canterbury Agricultural and Science Centre Private Bag 4704 Christchurch Mail Centre 8140 NEW ZEALAND Tel: Fax: Nadine.Berry@plantandfood.co.nz Retrospective analysis report NB8 Sept Page i

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7 1 Introduction Since 2006 solanaceous crops grown in New Zealand have been affected by an exotic insect pest, the Tomato Potato Psyllid (Bactericera cockerelli) (Sulc), (Hemiptera, Triozidae) (TPP). Subsequent studies in New Zealand, the United States and Mexico identified a new to science bacterial pathogen, Candidatus Liberibacter solanacearum, that was associated with the zebra chip disorder, which reduces potato yield and quality (Liefting et al. 2008, 2009; Hansen et al. 2008; Lin et al. 2008) and is vectored by the TPP (Munyaneza et al. 2007). In 2009, another bacterial species, Candidatus Phytoplasma australiense, was discovered in potatoes in New Zealand (Liefting et al. 2009). This micro-organism is thought to be associated with psyllid yellow-like/zebra chip disease symptoms. The impact of TPP and associated pathogen(s) has threatened the future production of potatoes, tomatoes, capsicums and tamarillos in New Zealand. It is also having a substantial effect on the yield, quality and economics of potato and process tomato production in New Zealand. For example, Potatoes NZ has recently estimated that the complex has directly and/or indirectly cost the industry NZ$150 million over the season. Studies by Munyaneza et al. (2008) suggest that not all TPP populations induce zebra chip symptoms. Other US studies have suggested that there may be seasonal variation in the occurence of Ca. L. solanacearum) in TPP populations (Crosslin et al. 2010). In New Zealand, numbers of TPP caught on sticky traps in and were highly variable between regions and monitoring sites (Berry & Jorgensen, unpubl. data). In addition, the prevalence of visual zebra chip symptoms varied between sites and regions during these seasons. If the percentage of TPP carrying Ca. L. solanacearum /Ca. P. australiense varies between season, there may be an opportunity to apply control measures during higher risk periods. The identification of lower risk periods may provide opportunities to establish predators and parasitoids. Furthermore, data on the incidence of Ca. P. australiense in TPP populations will extend our knowledge of the distribution Ca. P. australiense in New Zealand. Therefore, the objective of the current study was to determine the incidence of Ca. L. solanacearum and Ca. P. australiense in TPPs caught on sticky traps using data gathered during the growing season. Retrospective analysis report NB8 Sept Page 1

8 2 Method TPP numbers were monitored weekly using yellow sticky traps in commercial potato and field tomato sites throughout New Zealand from mid October 2009 until April Four double-sided yellow sticky traps (100 x 250 mm, Bug-Scan, Biobest Biological Systems) were placed at canopy height in most crops, one trap in each quarter of the crop (N, S, W and E location), 3 5 m in from the middle of each crop edge. Each trap was attached at canopy height and secured to a metal stake. Traps were replaced weekly and stored in clear plastic A4 sleeves for ease of transport and psyllid identification. TPP adults were identified and counted using a 40x binocular microscope. Traps were then stored at less than 4 o C for subsequent molecular screening of TPP DNA for the presence of Ca. Liberibacter solanacearum and Ca. Phytoplasma australiense. Adult TPPs were identified, removed from sticky traps and placed individually into 1.7 ml clear microtubes, each containing 100% propylene glycol. Sticky trap monitoring locations, site numbers and host plants are listed in Table 1. Representative sites from all monitoring locations were selected for inclusion in the analysis. Page 2 Retrospective analysis report NB8 Sept

9 Table 1. TPP Monitoring locations and sites sampled for Ca. L. solanacearum and Ca. P. australiense analysis and their respective monitoring periods Region Crop Number of monitoring sites Number of monitoring sites sampled for Ca. L. Solanacearum & Ca. Phytoplasma australiense analysis Monitoring period for screened sites Pukekohe Potatoes /10/ /03/2010 Manawatu Potatoes /11/ /4/2010 Waikato Potatoes /12/ /03/2010 Hawke s Bay Field tomatoes /11/ /04/2010 Hawke s Bay Potatoes /11/ /03/2010 North Canterbury Potatoes 2 2 Site 1: 23/11/ /02/2010 Mid Canterbury Potatoes 21 3 Site 1: Site 2: 28/12/ /03/ /10/ /04/2010 Site 2: 18/01/ /03/2010 Site 3: 04/01/ /03/2010 South Canterbury Potatoes 13 2 Site 1: 08/12/ /02/2010 Site 2: 28/12/ /03/2010 A preliminary trial was conducted to determine the limits of detection/sensitivity for the bulk extraction and the numbers of TPP samples that would subsequently be screened. These trial results indicated that the bulking and extraction of DNA from 5 TPPs was sufficient to reliably detect Ca. L. solanacearum and Ca. P. australiense. The bulk extraction of TPP individuals was carried out to: reduce sample number and cost, improve the probability of detecting Ca. L. solanacearum and Ca. P. australiense, and in order to discover a pathogen-free period during the growing season. Retrospective analysis report NB8 Sept Page 3

10 TPPs were removed from individual traps from the same location and monitoring period, and were sorted into groups of up to 5 individuals for DNA extraction. TPP DNA samples contained either bulked (maximum of 5 individuals) or individual TPP. DNA was extracted from the TPP using a modified CTAB protocol (IAW Scott, pers. comm.). This protocol has previously been demonstrated to maximise DNA recovery from both insect and plant samples. A DNA sample comprised of up to 5 individual TPPs caught on a trap. DNA was tested to determine extraction efficiency. The presence of potential PCR inhibitors was confirmed using primers designed to amplify insect nuclear ITS or mitochondrial COI sequences. Appropriate positive and negative controls were used. Semi-nested PCR protocols were used to test insect DNA samples for the presence of Ca. L. solanacearum and Ca. P. australiense DNA. The specificity of the tests had previously been confirmed by DNA sequence analyses of PCR fragments amplified when the tests were developed and optimised (IAW Scott, pers. comm.). In the case of Ca. P. australiense, this followed specificity testing against a panel of other Phytoplasma DNAs. Appropriate controls were used during PCR screening of insect DNAs; positive plant control material was sourced from both MAF BNZ (tomato, capsicum) and previously tested potato material, including that collected during a Potatoes NZ Liberibacter/Phytoplasma survey in 2009 or by PFR staff during the last season. PCR amplification products were separated on agarose gels alongside DNA size markers and visualised using ethidium bromide. Digital images of each gel were taken and scored for presence/absence and checked to ensure that when bands were present they were of the correct size. Figures 1 and 2 illustrate examples of an agarose gel of DNA amplified with Ca. P. australiense and Ca. Liberibacter solanacearum primers respectively. Page 4 Retrospective analysis report NB8 Sept

11 249 bp Figure 1. Example of an agarose gel: Bands at 249 bp indicate Ca. P. australiense target DNA amplified from TPP DNA samples. The TPP were taken from sticky traps at monitoring sites in Hawke s Bay, Pukekohe, South Canterbury and North Canterbury. Retrospective analysis report NB8 Sept Page 5

12 579 bp Figure 2. Example of an agarose gel: Bands at 579 bp indicate Ca. Liberibacter solanacearum target DNA amplified from TPP DNA samples. The TPP were taken from sticky trips at monitoring sites in the Manawatu and the Waikato. Page 6 Retrospective analysis report NB8 Sept

13 3 Results Initiation of sticky trap monitoring of TPP adults at each site was dependent on crop planting/plant emergence date (16/10/ /01/2010). In mid-december 2009, TPP adults first appeared in low numbers on sticky traps in potato crops at the Waikato, Manawatu and Pukekohe monitoring sites. At field tomato and potato monitoring sites in Hawke s Bay, TPP adults appeared in low numbers in early November and early December respectively. Depending on planting and trapping commencement, TPP adults first appeared in low numbers between late December and mid January in North, Mid and South Canterbury potato monitoring sites. TPP adult numbers rose considerably in early-mid January in all North Island monitoring sites and had declined by early April in most regions. In North and Mid Canterbury sites numbers of TPP adults rose significantly between February and March and were declining by early April. Low numbers of TPP were captured in South Canterbury potato monitoring sites. Molecular screening results of TPP DNA from stored traps for the presence of Ca. Liberibacter solanacearum and Ca. Phytoplasma australiense in eight New Zealand regions (12 sites) are described overleaf. Retrospective analysis report NB8 Sept Page 7

14 3.1 Hawke s Bay Tomatoes Of the 42 samples of TPP DNA samples screened, 31 (73.81%) were positive for Ca. L. solanacearum and 5 (11.90%) were positive for Ca. P. australiense. Ca. L. solanacearum and Ca. P. australiense positive TPP DNA samples (bulked and/or individual TPP) were first detected from sticky traps in field tomatoes in the first week of trap monitoring (1st TPP caught, week ending 2/11/2009 (1 TPP/5 traps) (Figure 3). On 11 of the 17 `trap out dates (when traps removed from the field included TPP, 100% of the TPP samples screened were Ca. Liberibacter positive. Ca. L. solanacearum positive TPP were present at all 17 trap out dates during the field tomato monitoring period. Conversely, TPP DNA samples were positive for Ca. P. australiense on only 6 of 17 trap out dates. Figure 3. Percentage of TPP DNA samples (bulked and/or individual TPP) positive for Ca. Liberibacter solanacearum and/or Ca. Phytoplasma australiense during the trapping period of early November 2009 early March 2010 in field tomatoes, Hawke s Bay. Numbers of TPP from trap out dates 4/1/10 1/3/10 were high so two groups (2 x 5 TPP) of TPPs have been extracted and screened to date. Page 8 Retrospective analysis report NB8 Sept

15 3.1.2 Potatoes Of the 113 samples of TPP DNA samples screened, 76 (67.23%) were positive for Ca. L. solanacearum and 11 (9.73%) were positive for Ca. P. australiense. Ca. L. solanacearum positive TPP DNA samples were first detected from sticky traps in potatoes (Site 1) in the 3 rd week of trap monitoring (week ending 1/12/2009, Figure 4). Ca. P. australiense positive TPP DNA samples were first detected on sticky traps in potatoes in the 6 th week of trap monitoring (week ending 5/1/2010, Figure 4). On 15 of 17 trap out dates (where TPP were caught) at least some of TPP DNA samples tested Ca. L. solanacearum positive. On 5 of those 15 trap out dates all TPP DNA samples tested Ca. L. solanacearum positive. In contrast, some TPP DNA samples tested positive for Ca. P. australiense on only 5 of 17 trap out dates. Figure 4. Percentage of TPP DNA samples (bulked and/or individual TPP) positive for Ca. Liberibacter solanacearum and/or Ca. P. australiense during the trapping period of early December 2009 late March 2010 in potatoes, Hawke s Bay. Numbers of TPP from trap out dates 28/1/10 1/4/10 were high so two groups (2 x 5 TPP) of TPPs have been extracted and screened to date. Retrospective analysis report NB8 Sept Page 9

16 3.2 Waikato Potatoes Of the 42 samples of TPP DNA samples screened, 31 (73.81%) were positive for Ca. L. solanacearum and none was positive for Ca. P. australiense. Ca. L. solanacearum positive TPP DNA samples were first detected from sticky traps in potatoes in the 2 nd week of trap monitoring (week ending 8/12/2009, Figure 5). No Ca. P. australiense positive TPP DNA samples were detected on sticky traps in the Waikato potato monitoring site (Figure 5). Some Ca. L. solanacearum positive TPP DNA samples were detected on every trap out date (15/15). On 10 of 15 trap out dates (where TPP were caught) all TPP DNA samples were Ca. L. solanacearum positive. Figure 5. Percentage of TPP DNA samples (bulked and/or individual TPP) positive for Ca. Liberibacter solanacearum and/or Ca. Phytoplasma australiense during the trapping period of early December 2009 early April 2010 in potatoes, Waikato. Numbers of TPP from trap out dates 04/3/10 08/4/10 were high so two groups (2 x 5 TPP) of TPPs have been extracted and screened to date. Page 10 Retrospective analysis report NB8 Sept

17 3.3 Manawatu Potatoes Of the 73 samples of TPP DNA samples screened, 68 (93.15%) were positive for Ca. L. solanacearum and 10 (13.70%) were positive for Ca. P. australiense. Ca. L. solanacearum positive TPP DNA samples were first detected from sticky traps in potatoes in the 7 th week of trap monitoring (1 st TPP caught, week ending 23/12/2009; Figure 6). Ca. P. australiense positive TPP DNA samples were first detected on sticky traps in potatoes in the 8 th week of trap monitoring (week ending 30/12/2009, Figure 6). On 14 of 16 trap out dates (where TPP were caught) at least some TPP DNA samples tested were Ca. L.solanacearum positive. On 12 of those 14 trap out dates all samples tested positive for Ca. L. solanacearum. In contrast, some samples tested positive for Ca. P. australiense at only 6 of 16 trap out dates (where TPP were caught). Figure 6. Percentage of TPP DNA samples (bulked and/or individual TPP) positive for Ca. Liberibacter solanacearum and/or Ca. Phytoplasma australiense during the trapping period of mid December 2009 early April 2010 in potatoes, Manawatu. The numbers of TPP from trap out dates 05/2/10 01/4/10 were high so two groups (2 x 5 TPP) of TPPs have been extracted and screened to date. Retrospective analysis report NB8 Sept Page 11

18 3.4 Pukekohe Potatoes Of the 55 samples of TPP DNA samples screened, 41 ( %) were positive for Ca. L. solanacearum and 3 (5.45%) were positive for Ca. P. australiense. Ca. L. solanacearum positive TPP DNA samples were detected from sticky traps in potatoes in the 3 rd week of trap monitoring (1 st TPP caught, week ending 15/12/2009; Figure 7). Ca. P. australiense positive TPP DNA samples were first detected on sticky traps in potatoes in the 8 th week of trap monitoring (week ending 19/1/2010, Figure 7). On 14 of 15 trap out dates (where TPP were found) at least some TPP DNA samples tested Ca. L. solanacearum positive. On 6 of those 14 trap out dates all samples were Ca. L. solanacearum positive. Conversely, samples taken from only 2 of the 14 trap out dates (where TPP were found) tested positive for Ca. P. australiense. Figure 7. Percentage of TPP DNA samples (bulked and/or individual TPP) positive for Ca. Liberibacter solanacearum and/or Ca. Phytoplasma australiense during the trapping period of mid December 2009 mid March 2010 in potatoes, Pukekohe. Numbers of TPP from trap out dates 26/1/10 16/3/10 were high so two groups (2 x 5 TPP) of TPPs have been extracted and screened to date. Page 12 Retrospective analysis report NB8 Sept

19 3.5 North Canterbury Potatoes Of the 28 samples of TPP DNA samples screened, 15 (53.57%) were positive for Ca. L. solanacearum and 8 (28.57%) were positive for Ca. P. australiense. Ca. L. solanacearum positive TPP DNA samples were first detected from sticky traps in potatoes (Site 1, see Table 1) in the 6 th week of trap monitoring (week ending 4/1/2010, Figure 8). Ca. P. australiense positive TPP DNA samples were first detected on sticky traps in potatoes (Site 1) in the 6 th week of trap monitoring (week ending 4/1/2010, Figure 8). On 7 of 8 trap out dates (where TPP were found) at least some samples tested positive for Ca. L. solanacearum. On 3 of those 7 trap out dates, all samples tested positive for Ca. L. solanacearum. In contrast, samples tested positive for Ca. P. australiense at only 4 of the 8 trap out dates (where TPP were found). Figure 8. Percentage of TPP DNA samples (bulked and/or individual TPP) positive for Ca. Liberibacter solanacearum and/or Ca. Phytoplasma australiense during the trapping period of late December 2009 mid February 2010 in potatoes, North Canterbury (Site 1). Numbers of TPP from trap out dates 15/2/10 22/2/10 were high so two groups (2 x 5 TPP) of TPPs have been extracted and screened to date. Retrospective analysis report NB8 Sept Page 13

20 At the second North Canterbury site, of the 10 samples of TPP DNA samples screened, 7 (70%) were positive for Ca. L. solanacearum and 1 (10%) was positive for Ca. P. australiense. Ca. L. solanacearum positive TPP DNA samples were first detected from sticky traps in potatoes (Site 2, see Table 1) in the 5 th week of trap monitoring (1 st TPP caught, week ending 4/1/2010, Figure 9). Ca. P. australiense positive TPP DNA samples were first detected on sticky traps in potatoes (Site 2, see Table 2) in the 12 th week of trap monitoring (week ending 22/2/2010, Figure 9). Samples tested positive for Ca. L. solanacearum at 5 of 6 trap out dates (where TPP were found) and at 3 of these dates all samples tested positive. Again, Ca. P. australiense positive TPP DNA samples were detected on traps from only 1 of the 6 trap out dates (where TPP was found). Figure 9. Percentage of TPP DNA samples (bulked and/or individual TPP) positive for Ca. Liberibacter solanacearum and/or Ca. Phytoplasma australiense during the trapping period of early January 2010 early March 2010 in potatoes, North Canterbury (Site 2). Page 14 Retrospective analysis report NB8 Sept

21 3.6 Mid Canterbury Potatoes Of the 37 samples of TPP DNA samples screened, 27 (72.97%) were positive for Ca. L. solanacearum and 6 (16.22%) were positive for Ca. P. australiense. Ca. L. soalancearum positive TPP DNA samples were first detected from sticky traps in potatoes (Site 1, see Table 1) in Week 14 of trap monitoring (week ending 22/1/2010, Figure 10). Ca. P. australiense positive samples were first detected from sticky traps in potatoes (Site 1, see Table 1) in Week 14 of trap monitoring (week ending 22/1/2010, Figure 10). On 13 of 14 trap out dates (where TPP were found) at least some samples tested positive for Ca. L. soalancearum. On 10 of those dates all samples tested positive. In contrast, samples tested positive for Ca. P. australiense for only 4 of 14 trap out dates (where TPP were found). Figure 10. Percentage of TPP DNA samples (bulked and/or individual TPP) positive for Ca. Liberibacter solanacearum and/or Ca. Phytoplasma australiense during the trapping period of mid January 2010 mid April 2010 in potatoes, Mid Canterbury (Site 1). Numbers of TPP from trap out dates 26/2/10 23/4/10 were high so two groups (2 x 5 TPP) of TPPs have been extracted and screened to date. Retrospective analysis report NB8 Sept Page 15

22 At the second Mid Canterbury site, of the 26 samples of TPP DNA samples screened, 12 (46.15%) were positive for Ca. L. solanacearum and 3 (11.54%) were positive for Ca. P. australiense. Ca. L. solanacearum positive TPP DNA samples were first detected from sticky traps in potatoes (Site 2, see Table 1) in Week 12 of trap monitoring (1 st TPP caught, week ending 1/2/2010, Figure 11). Ca. P. australiense positive samples were first detected from on sticky traps in potatoes (Site 2, see Table 1) in Week 17 of trap monitoring (week ending 1/3/2010, Figure 11). On 7 of the 10 trap out dates (where TPP were found) at least some of the samples tested positive for Ca. L. solanacearum. On two of those dates all samples tested positive. Samples that tested positive for Ca. P. australiense came from traps on only 2 of the 10 trap out dates (where TPP were found). Figure 11. Percentage of TPP DNA samples (bulked and/or individual TPP) positive for Ca. Liberibacter solanacearum and/or Ca. Phytoplasma australiense during the trapping period of end January 2010 end March 2010 in potatoes, Mid Canterbury (Site 2). Page 16 Retrospective analysis report NB8 Sept

23 At the third Mid Canterbury site, of the 31 samples of TPP DNA samples screened, 30 (96.77%) were positive for Ca. L. solanacearum and 3 (9.68%) were positive for Ca. P. australiense. Ca. L. solancearum positive TPP DNA samples were first detected from sticky traps in potatoes (Site 3, see Table 1) in the 7 th week of trap monitoring (1 st TPP found, week ending 11/1/2010, Figure 12). Ca. P. australiense positive DNA samples were first detected from sticky traps in potatoes (Site 3, see Table 1) in the 12 th week of trap monitoring (week ending 15/2/2010, Figure 12). Ca. L. solanacearum positive samples were detected on all 12 trap out dates (where TPP were found) and on 11 of those trap out dates all samples tested positive. However, samples tested positive for Ca. P. australiense on only 3 of the 12 trap out dates (where TPP were found). Figure 12. Percentage of TPP DNA samples (bulked and/or individual TPP) positive for Ca. Liberibacter solanacearum and/or Ca. Phytoplasma australiense during the trapping period of early January 2010 end March 2010 in potatoes, Mid Canterbury (Site 3). Numbers of TPP from trap out dates 08/3/10 29/3/10 were high so two groups (2 x 5 TPP) of TPPs have been extracted and screened to date. Retrospective analysis report NB8 Sept Page 17

24 3.7 South Canterbury Potatoes Of the 8 samples of TPP DNA samples screened, 4 (50%) were positive for Ca. L. solanacearum and 2 (25%) were positive for Ca. P. australiense. Ca. L. solanacearum positive TPP DNA samples were first detected from sticky traps in potatoes (Site 1, see Table 1) in the 6 th week of trap monitoring (1 st TPP found, week ending 20/1/2010, Figure 13). Ca. P. australiense was first detected in samples collected on sticky traps in potatoes (Site 1, see Table 1) in the same week (Figure 13). Ca. L. solanacearum was detected in DNA samples from 3 of 4 trap out dates (where TPP were found), but on only one of these dates was Ca. L. solanacearum detected in all samples. Similarly, Ca. P. australiense was detected in samples from only 1 of the 4 trap out dates (where TPP were found). Figure 13. Percentage of TPP DNA samples (bulked and/or individual TPP) positive for Ca. Liberibacter solanacearum and/or Ca. P. australiense during the trapping period of mid January 2010 mid February 2010 in potatoes, South Canterbury (Site 1). Page 18 Retrospective analysis report NB8 Sept

25 At the second South Canterbury site, of the 11 samples of TPP DNA samples screened, 9 (81.81%) were positive for Ca. L. solanacearum and none was positive for Ca. P. australiense. Ca. L. solanacearum positive TPP DNA samples were first detected from sticky traps in potatoes (Site 2, see Table 1) in the 10 th week of trap monitoring (week ending 11/1/2010, Figure 14). No Ca. P. australiense was detected. On 7 of 8 trap out dates (where TPP were found) at least some of the samples tested positive for Ca. L. solanacearum and all samples tested positive at 6 of those sites. Figure 14. Percentage of TPP DNA samples (bulked and/or individual TPP) positive for Ca. Liberibacter solanacearum and/or Ca. Phytoplasma australiense during the trapping period of early January 2010 end March 2010 in potatoes, South Canterbury (Site 2). Retrospective analysis report NB8 Sept Page 19

26 4 Discussion/Recommendations Sticky trap monitoring of TPP populations from mid October 2009 to the end of April 2010 in commercial potato and field tomato sites throughout New Zealand provided important information on the presence and abundance of TPP populations during the season. Numbers of TPP were highly variable between sites and regions during the monitoring period (Berry 2010). TPP numbers rose significantly in early January and declined by early April in most regions. Numbers of TPP found on traps were higher at North Island monitoring sites than at South Island sites (Berry 2010). Molecular screening of TPP DNA samples has shown that Ca. Liberibacter solanacearum was present in TPP populations from the first appearance of TPP on sticky traps in North and South Island monitoring regions. A proportion of Ca. L. solanacearum positive TPP DNA samples were detected from sticky traps over most monitoring sites and dates. In contrast, Ca. Phytoplasma australiense positive TPP DNA samples were detected less frequently in all monitoring regions and over all monitoring dates. No Ca. P. australiense positive TPP DNA samples were detected at 2 of the 12 monitoring locations (Waikato, Site 1; South Canterbury, Site 2). Bulking and extraction of DNA from up to five TPPs was sufficient to reliably detect Ca. L. solanacearum and Ca. P. australiense in this study. The method of bulking TPP DNA enabled a large number of TPP to be screened from a number of sites over the growing season. Further information could be gained by estimating the proportion of infected versus uninfected TPP individuals caught on sticky traps during the monitoring period or between regions. Such information could help us to determine the effect of TPP population dynamics on disease expression at different sites and regions. The relationship between plant symptoms believed due to Ca. L. solanacearum and/or Ca. P. australiense and TPP infestations is poorly understood and may be attributed to or affected by a range of factors, including climate, plant defence mechanisms, plant cultivar, host plant reservoirs and insect pest management strategies. In the US, Li et al. (2009) reports on the variability in the severity of zebra chip within a field and between years, hypothesising that this may be due to differences in weather conditions. Munyaneza et al. (2009) also reported that despite the presence of Ca. L. solanacearum positive TPPs in Washington, no observable zebra chip symptoms were detected in commercial potato fields. Stored sticky traps from additional monitoring sites and TPP not yet analysed from the current study may provide the opportunity to test individual insects in order to gain baseline information on the proportion of Ca. L. solanacearum positive TPPs. As previously mentioned, Ca. P. australiense positive TPP DNA samples were detected less frequently in all monitoring regions and over all monitoring dates. The detection of Ca. P. australiense in TPP DNA samples merely indicates the ability of TPP to acquire the pathogen, probably following feeding, but does not indicate its ability to propagate the bacterium or to transmit the bacterium to other hosts. In addition, the pathogenicity status of Ca. P. australiense in potatoes has yet to be established and is currently being investigated. In New Zealand the organism is already associated with lethal diseases in a number of native plant species (e.g. Cordyline australis (Agavaceae), Coprosma robusta (Rubiaceae), Phormium tenax, Phormium cookianum (Phormiaceae)) and strawberry (Fragaria ananassa (Rosaceae)) (Andersen et al. 2006). In summary, results of the current study have shown that Ca. L. solanacearum is present in all sites over most monitoring periods while Ca. P. australiense is present less frequently at some monitoring sites and periods. Further analysis of the proportion of infected versus uninfected Page 20 Retrospective analysis report NB8 Sept

27 TPP individuals between monitoring sites/dates could increase our understanding of the extent of variation in disease incidence between monitoring sites. 5 Acknowledgements Thanks to EuroGrow Potatoes Ltd, Fruitfed Services, Heinz Watties, Wilcox Ltd and McCains Ltd for their contributions to the regional monitoring carried out in the Sustainable TPP Management Sustainable Farming Fund. We thank Marsha Stevens for removing TPPs from sticky traps. This research was funded by the Sustainable Farming Fund extension. Retrospective analysis report NB8 Sept Page 21

28 6 References Andersen MT, Newcomb RD, Liefting LW, Beever RE Phytopathology 96(8): Berry N National Tomato Potato Psyllid monitoring Psyllid News, July P Potatoes New Zealand. Crosslin JM, Munyaneza JE, Brown JK, Liefting LW Potato zebra chip disease: A phytopathological tale. Online. Plant Health Progress doi: /php RV. Hansen AK, Trumble JT, Stouthamer R, Paine TD A new huanglongbing species, Candidatus Liberibacter psyllaurous. Found to infect tomato and potato, is vectored by the psyllid Bactericera cockerelli (Sulc). Applied Environmental Microbiology 74: Leifting LW, Perez-Egusquiza ZC, Clover GRG, Anderson JAD A new Candidatus Liberibacter species in Solanum tuberosum in New Zealand. Plant Disease 92: Leifting LW, Sutherland PW, Ward LI, Paice KL, Weir BS, Clover GRG A new Candidatus Liberibacter species associated with diseases of Solanaceous crops. Plant Disease 93: Li W, AbadJA, Fench-Monar RD, Rascoe J, Wen A, Gudmestad NC, Secor GA, Lee I-M, Levy L Multiplex real-time PCR for detection, identification and quantification of Candidatus Liberibacter solancearum in potato plants with zebra chip. Journal of Microbiological Methods 78: Lin H, Doddapanei, H, Munyaneza JE, Civerolo EL, Sengoda VG, Buchman JL, Stenger DC Molecular characterization and phylogenetic analysis of 16S rrna from a new Candidatus Liberibacter solanacearum strain associated with zebra chip disease of potato (Solanum tuberosum L.) and the potato psyllid (Bactericera cockerelli Sulc). Journal of Plant Pathology 91: Munyaneza JE, Crosslin JM, Upton JE Association of Bactericera cockerelli (Homoptera: Psyllidae) with zebra chip, a new potato disease in southwestern United States and Mexico. Journal of Economic Entomology 100: Munyaneza JE, Buchman JL, Upton JE, Goolsby JA, Crosslin JM, Bester G, Miles GP, Segoda VG Impact of different potato psyllid populations on zebra chip disease incidence, severity and potato yield. Subtropical Plant Science 60: Munyaneza JE, Crosslin JM, Buchman JL Seasonal occurrence and abundance of the Potato Psyllid, Bactericera cockerelli, in South Central Washington. American Journal of Potato Research 86: Page 22 Retrospective analysis report NB8 Sept