2. Maintaining Biosecurity Category

2.1 MBS 320

Programme Title:

Green grafting

Programme Leader:

Marian McKenzie

Institution:

Crop & Food CRI

Summary

Goal: To develop the greengrafting technique for grapevine material in vivo and, using tissue culture, in vitro, and assess the technique as a method for detecting viral infection.

Background

Context of the project: Robust, sensitive, rapid and reliable techniques for assessing viral infection in grape clones entering New Zealand are an important requirement for a high performing grapevine industry based on healthy stock. Current viral indexing is based on graft-indexing of woody cuttings (which require a 2-3 year incubation period) and serological or molecular analyses (methods that are limited to known viruses for which antibodies and genome sequences are available). The limitations of these methods led to this assessment of the efficacy of viral indexing using two new grafting techniques, greengrafting and micrografting.

Approach & Outcomes

Approach: Preliminary tests were conducted to optimise the nutrient media for grapevine shoot production for use in greengrafting and micrografting and also to test an improved method of greengrafting (cleft verses splice). For both greengrafting and micrografting, Cabernet franc and Cabernet sauvignon scions infected with Grapevine Leafroll-associated Closterovirus III (GLRaV 3) were cleft-grafted on to virus-free indicator rootstocks LN 33 and Cabernet sauvignon in all four possible combinations in four replicates, each replicate having ten individual grafts and two control (no virus) grafts. Double Antibody Sandwich-Enzyme Linked Immunosorbent Assay (DAS-ELISA) was used to validate the visual symptoms of disease recorded at 2-4 weeks and 12 weeks.

Summary: Two new grafting methods, greengrafting and micrografting, were investigated for their potential use in virus indexing of grapevine stock. For both methods, Cabernet franc and Cabernet sauvignon scions infected with Grapevine Leafroll-associated Closterovirus III (GLRaV 3), were cleft-grafted on to virus-free indicator rootstocks LN 33 and Cabernet sauvignon. These two rootstocks and two scions were grafted in all four possible combinations in four replicates, each replicate having ten individual grafts and two control grafts (virus-free scion grafted on to virus-free rootstock). Leafroll symptoms were scored at three to four weeks and again at twelve weeks. Double Antibody Sandwich-Enzyme Linked Immunosorbent Assay (DAS-ELISA) was used to validate the visual symptoms recorded.

When maintained at saturated humidity in modified Huglin and Juliard solution, the machine-grafted green shoots produced roots and were ready for transfer to soil after three to four weeks with an overall success rate of 83.5%. At this time, reddening of leaves (30-62.5%), leaf rolling (22.5-55%) or interveinal yellowing (12.5-45%) could be observed on indicator rootstock. Overall, Cabernet sauvignon showed a significantly higher percentage of leafroll virus symptoms (80.0±7.2%) than LN 33 (56.3±8.5%) at the time of transfer to soil. Twelve weeks after greengrafting, Cabernet sauvignon recorded a significantly higher number of shoots (P < 0.001) with symptoms (90%) than LN 33 (32.5%). A tissue culture medium based on modified MS macro- and microelements and WP vitamins was successfully tested for in vitro growth of all three cultivars used. Micrografting was as successful as greengrafting, with an overall success rate of 73.6% for LN 33 rootstock and 81.9% for Cabernet sauvignon. When leafroll-infected scion material was micrografted on to virus-free rootstock, the rootstock leaf turned red (23.5-63.9%) or it showed leafrolling (22.6-34.4%) within 2-3 weeks. These symptoms were evident in more indicator rootstock after 12 weeks in culture (79.4-83.1%). The presence of the virus in 80% of the indicator rootstock with visual symptoms was confirmed by DAS-ELISA. Results indicate that both greengrafting and micrografting are effective methods for viral indexing of grapevine. Other rootstocks used in viral indexing need to be tested with different viruses to optimise the methods before they are widely adopted for viral indexing. Nevertheless, these methods can be used now in conjunction with wood indexing for post-entry quarantine to identify infected material and reject it much earlier than is currently possible.

Publications

Pathirana, R.; McKenzie, M. 2003: The use of greengrafting for the early detection of viruses in grapevine. Proceedings of Joint Convention of New Zealand Institute of Agricultural Science and New Zealand Society for Horticultural Science. 23 -24 June 2003, Albany, Auckland. Abstracts P. 23.

2.2 MBS 321

Programme Title:

Saprophytic fungi

Programme Leader:

Shaun Pennycook

Institution:

Landcare Research CRI

Summary

Goal: To improve the efficiency and effectiveness of border biosecurity by providing data that can be used to develop phytosanitary measures appropriate to the risk posed by individual fungal genera.

Background

Context of the project: Many of the fungi isolated from border interceptions by MAF

Quarantine Service are in genera such as penicillium, mucor, and aspergillus, in which: (a) species identification is difficult and expensive; yet (b) most species are harmless, ubiquitous saprophytes. A better assessment of the risk they pose to New Zealand biosecurity would facilitate more informed decisions on appropriate courses of action for dealing with border interceptions of such genera.

Approach & Outcomes

Approach: Nine target genera of fungi were identified for assessment: Acremonium, Aspergillus, Cladosporium, Curvularia, Epicoccum, Mucor, Paecilomyces, Penicillium, and Trichoderma. New Zealand records of these genera were collated from national databases and checklists. World species lists were compiled from internet databases, monographs, reviews, and recent primary literature, and subsidiary lists of plant pathogenic and animal/human pathogenic species were extracted. For each genus, an assessment was made of the risk posed by species not recorded from New Zealand, as an indication of whether species-level identification of border interceptions is required.

Outcomes: Species-level identifications are unnecessary for Epicoccum and Trichoderma, and probably unnecessary for Acremonium, Curvularia, Mucor, Paecilomyces, and Penicillium.

For Aspergillus, species-level identification is probably unnecessary except for animal/human health concerns.

For Cladosporium, species-level identifications may be necessary if any of the plant pathogenic species not recorded from New Zealand have host ranges that suggest they may represent a risk to New Zealand plants.

Methodology: Nine target genera of fungi-Acremonium, Aspergillus, Cladosporium, Curvularia, Epicoccum, Mucor, Paecilomyces, Penicillium, and Trichoderma-were identified for assessment because their species identification is difficult and expensive, yet most of their species are harmless, ubiquitous saprophytes. Lists of New Zealand records were compared with world species lists, and the risk posed by species not recorded from New Zealand was assessed.

Results: The requirement for species-level identification was assessed as:

Unnecessary: Epicoccum, Trichoderma.

Probably unnecessary: Acremonium, Curvularia, Mucor, Paecilomyces, and Penicillium.

Probably unnecessary, except for animal/human health concerns: Aspergillus.

Possibly necessary, because of potential plant disease risks: Cladosporium.

2.3 MBS 322

Programme Title:

Nucleic acid extraction

Programme Leader:

Gail Timmerman-Vaughan

Institution:

Crop & Food CRI

Summary

Goal: The goal of this programme is to develop nucleic acid extraction protocols and quality control assays to underpin the use of PCR and RT-PCR (reverse transcription PCR) assays to detect pathogens in the diverse plants that are imported into or grown in New Zealand.

Background

Context of the project: Molecular diagnostic methods, particularly those based on sequence detection using polymerase chain reaction (PCR), have tremendous potential to determine the presence of plant pathogens. The research in this programme will permit the increased use of nucleic acid-based diagnostic tests for plant pathogens to supplement and complement existing conventional methods. The research in this programme builds on research conducted at Crop & Food Research on genomes of a wide variety of plants, on development of PCR-based diagnostics, and on research on the molecular detection of plant pathogens.

Approach & Outcomes

Approach: Plant pathogens may have genetic material that consists of either DNA (e.g. fungi, bacteria and some viruses) or RNA (RNA plant viruses, viroids). Therefore, the extraction of RNA and/or DNA of adequate quality and quantity for PCR-based detection systems is a prerequisite. The range of plant species that is of interest for pathogen detection is very broad, and technical challenges to nucleic acid extraction can arise as a result of the composition of plant tissues, particularly for polysaccharides, phenolics and other secondary metabolites. We have assessed methods for extracting total nucleic acids (T-NA) that contain both DNA and RNA, and to extract RNA, for members of 32 diverse flowering plant families, represented by more than 57 genera. We have also developed a positive control assay for PCR and RT-PCR based on a conserved chloroplast sequence. The utility of this positive control assay has been demonstrated for both T-NA and RNA extractions derived from this list of diverse plant genera.

Outcomes: The research in this programme has been successful in developing methods for extracting T-NA and RNA from a diverse range of plant species. T-NA has been extracted from members of 32 diverse botanical families using one of three different methods, and the suitability of these T-NA extractions for use in PCR-based diagnostics has been demonstrated with a number of quality control assessments. RNA has been extracted from the same diverse range of plants using one of two methods and the quality and quantity of the RNA extracted has been characterised.

Difficulties in obtaining good quality nucleic acid extractions were encountered for some plant genera within the Proteaceae, Rosaceae, Ericaceae, Onagraceae, Theaceae, Myrtaceae, Labiatae, Pittosporaceae and Geraniaceae. These difficulties were largely overcome by applying other extraction methods that operate on different principles. In some cases, however, suitable quality extractions were never obtained (e.g. for vireya Rhododendron, while azalea type Rhododendron was extracted successfully). A positive control assay that is universally applicable across the angiosperms (and possibly across wider botanical groupings) was developed, and applied in both PCR and RT-PCR to characterise the quality of the nucleic acid extraction methods. While development of PCR assays for T-NA and RT-PCR assays for RNA extractions was straightforward, considerable optimisation was undertaken to attempt to develop RT-PCR assays that reliably amplify the processed RNA transcript sequences from T-NA. A particularly valuable characteristic of the quality control assay we have developed is that different sized products are produced from genomic DNA versus processed RNA transcripts, enabling T-NA and RNA extracts to be assessed in order to determine whether they contain RNA and/or DNA.

Summary: Molecular detection of nucleic acid sequences to determine the presence of pathogens in plant materials has the advantage of being quick, sensitive, and applicable to difficult problems. It also complements conventional pathogen detection methods. The research in this programme has developed nucleic acid extraction protocols and a positive control PCR assay that can underpin DNA and RNA sequence-based pathogen detection in the wide range of plants that are imported into or grown in New Zealand. A number of methods to isolate T-NA and RNA have been applied in order to overcome the technical barriers to nucleic acid extraction that can be encountered in plant species, such as the presence of polysaccharides, phenolics and other secondary metabolites. The positive control PCR assay has been developed and optimal conditions established for amplifying PCR products from T-NA and RT-PCR products from RNA. A Laboratory Manual for the use of molecular diagnostic laboratories has been produced.

2.4 MBS 323

Programme Title:

Plant viruses detection (Phase 1)

Programme Leader:

Mike Pearson

Institution:

Auckland Uniservices (Auckland University)

Summary

Work is currently in progress on the Closteroviridae, Luteoviridae and Tospoviruses (This is a 2-year project). As previously discussed work on the various groups was started in a staggered manner, rather than simultaneously. Consequently work on the different groups is reaching the various milestones at different times. A table indicating the progress for each group is attached (Appendix 1). Nepoviruses are to be the next group to target.

Background

1. Closteroviridae:

Published primers:

Karasev et al (1994) and Tian et al (1996) designed degenerate primers to conserved motifs of the heat shock protein 70 (hsp70). These primer sets were successfully used by Mezler et al (2001, Saldarelli et al (1998), and Routh et al (1998) to obtain PCR products from closteroviruses of unknown sequence. In this project we have tested the primers designed by Tian et al (1996) and have also designed and tested further primers to the optimal primer regions of hsp70 (Figure C1).

Design of degenerate primers across Closteroviridae:

The 2 regions of the Closteroviridae genome with sufficient sequences available for sensible analysis are the coat protein (CP) and the heat shock protein 70 (hsp70). We concluded, as had other workers previously, that the hsp70 was the most promising region for designing generic primers. The taxonomy of the family Closterovirdae was revised by Martelli et al. (2002). Our alignments were consistent with their taxonomy for the three genera Ampelovirus, Closterovirus, and Crinivirus.

Primer evaluation:

The following primers were evaluated: (Figure C2).

From phosphate-binding sequences 1+2 to amplify ~ 620bp fragments

Tian1 + Tian2

CCAF + CCAR (UA / HortRes)

CCACHF + CCACHR (UA / HortRes)

From phosphate binding sequence 1 and connect motif to amplify ~ 540bp fragments

CCAF + CLOSR (for Closterovirus genus) (UA / HortRes)

CCAF + CRINIR (for Crinivirus genus) (UA / HortRes)

CCAF + AMPELOR (for Ampelovirus genus) (UA / HortRes)

Specific primers to a 210 fragment of the coat protein of GLRaV-3 were also available in house (Hort Research), as a positive control for the presence of PCR competence of the nucleic acid extract.

Both one-step and two-step RT-PCR were tried.

One-step PCR

We initially used the Invitrogen one step RT-PCR kit (catalogue number 10928-042).

Template RNA was extracted from Sauvignon blanc infected with GLRaV-1 and GLRaV-3 (Ampelovirus) and from grapefruit infected with Citrus trizteza virus (Closterovirus). Beet pseudoyellows virus (Crinivirus) RNA supplied by MAF Lynfield was also used.

Various annealing temperatures were used either fixed throughout the PCR cycles or in a temperature gradient (touchdown or temperature gradient) through the PCR cycles. RT-PCR with low annealing temperatures (550C and 400C) was tested with each of the degenerate primer pairs, however, no detectable amplification of appropriate sized fragments was detected.

  • Touchdown RT-PCR, annealing temperature gradients and magnesium concentration gradients were tested for both the degenerate primers CCAF + CCAR and the hsp70-specific primers resulting in no detectable amplification of appropriate sized fragments.
  • The GLRaV-3 coat protein-specific primers functioned under all conditions and were used as positive controls to confirm that the PCR reactions were effective with each template.

Two-Step RT-PCR: (GLRaV-3)

The templates used were primed using random hexamers or the reverse primer of the GLRaV-3 coat protein primer pair, CPR. (Figure C1 and Figure C2). The touchdown PCR employed consisted of eight cycles with the annealing temperature decreasing from 600C to 460C (-20C per cycle) with a further 30 cycles of annealing temperature at 460C.

  • The hsp70-specific primers LR3F1 and LR3SR1 amplified fragments at the appropriate size of ~540bp from random hexamer-primed cDNA (Figure C2, faint band in lane 2) and with strong bands observed from magnesium concentrations 3.8 to 8.2 mM (Figure C3, lanes 2-10).
  • These bands were isolated, cloned (into E.coli DH5_ using the Promega pGEM-T easy vector) and sequenced. Both restriction enzyme digest of the insert (Figure C4A) and sequence information demonstrated that the expected size and sequence of GVLRaV-3 had been amplified.

Further experiments with LR3F1 and LR3SR1 using 629R-primed cDNA allowed optimization of these primers, with the best result achieved at an annealing temperature of 40_ and magnesium concentrations 6mm for 35 cycles. These primers may be useful for future experiments as positive controls to indicate that this region of the GLRaV-3 genome is present and can be amplified.

  • The degenerate primer pairs LR3F1 + LR3R1, LR3F1 + AMPELOR, LR3F1 + CCAR, and CCAF + AMPELOR combinations resulted in no detectable amplification of expected sized bands using this protocol.

The LR3F1 + AMPELOR combination resulted in amplification of ~ 300bp bands from magnesium concentrations 5mM-8mM rather than the expected ~540 bp size (Figure C2, lane 4). These bands were isolated and cloned as above. Restriction digest analysis demonstrated that inserts were of variable sizes (Figure C4B) and sequence analysis revealed that amplification of the 26S ribosomal RNA had been primed with the AMPELOR primer on both ends of the PCR product.

  • PCR with GLRaV-3 cDNA and the Tian primers at 400 with a magnesium concentration gradient from 3mM-8mM did not result in any detectable bands.

  • A touchdown PCR with annealing temperatures from 600 - 460 using GLRaV-3 cDNA with LR3F1 + CCAR did not result in any detectable bands (Figure C2, lanes 5 and 9).

Approach & Outcomes

Comments:

Amplification of expected bands using the hsp70-specific LR3F1 + LR3SR1 primers have been obtained and are reproducible using a Techne Touchgene PCR machine. Some of these experiments were first tried on other newer PCR machines (e.g. GeneAmp) without success. It is possible that the Techne Touchgene may have a slower ramping speed causing a slight time elongation for each temperature change, and this may have had an advantageous effect on the reactions.

The quantity of PCR product using the hsp70-specific LR3F1 + LR3SR1 primers was always much less than obtained with the coat protein-specific primers we routinely use to detect GLRaV-3. We anticipate that degenerate primers based on conserved regions of the hsp70 protein may never be as sensitive as primers to short regions of the coat protein gene and may not be useful for routine diagnostic purposes.

The Closterovirus primer (CLOSR) and the Crinivirus primers (CRINIR) have not yet been tested using a two-step RT-PCR.

Recommendations:

Given the lack of success with these primers we recommend that this work does not progress any further in the current project using the above technique. However, further work may be justified to test whether the utility of the degenerate hsp70 primers can be improved either by using purified viral dsRNA, or partially purified virus preparations as target. Although not as useful for routine diagnosis this may assist in the identification of unknown viruses.

2. Luteoviridae

Published primers:

There are a number of published primer sets for both individual species within the Lueoviridae or for particular groups/genera (Robertson and French, 1991; Du et al., 2000; Moon et al., 2000; Woo et al., 2001), but there appear to be no genuinely universal primers for the family.

Design of degenerate primers across Luteoviridae:

We attempted to align all available nucleotide sequences (both complete genomes and CPs only) but the sequences were too variable to a produce a sensible alignment. Nucleotide sequences for the 3 defined genera (Luteovirus, Polerovirus, and Enamovirus) and for the unassigned viruses were aligned separately.

  • For the all four of the groups, taken individually, the coat protein genes (ORF 3) gave the best alignments and looked most suitable for primer design.
  • From these alignments it looks possible to design separate primers for each genus.

Representative sequences from all four groups were selected for comparison of the RNA dRNA polymerase; movement protein, and aphid transmission genes (ORF1 and ORF2).

  • Neither nucleotide or amino acid sequences produced good alignments.

All available Luteoviridae amino acid sequences (500+) were then compared using phylogenetic trees constructed using PAUP. Duplicate sequences were then eliminated leaving 107 distinct sequences.

  • Allignment of the 107 sequences identified 3 distinct clades, which correspond to:
  •  
    • Enamovirus;
    • Luteovirus;
    • Polerovirus and unassigned species.
  • A single highly conserved region of 6 amino acids (just sufficient for one primer) was identified across all sequences. This has been edited in preparation for primer design (primer 1 on Fig L1).
  • Two other highly conserved regions were identified. One is common to Polerovirus, unassigned, and Luteovirus sequences (primer 2 on Fig L1), the other is common to Luteovirus and Enamovirus sequences (primer 3 on Fig L1).
  • From these alignments it looks possible to design primers for a multiplex PCR for the Luteoviridae using a single forward primer to the common region and a mixture of two reverse primers.

3. Tospoviruses:

Published primers:

Tospoviruses have tri-partite genome consisting of RNAs , L, M, and S. Mumford et al. (1996) described several sets of primers including universal tospovirus primers designed to RNA-S. Chu et al. (2001) subsequently published primers for based on sequences from RNA-L. Attempts by NPPRL to use the Mumford et al. (1996) primers have prooved unsuccessful (Ochoa, pers. comm.).

Primer design:

We are currently checking all available Tospovirus sequences to determine which regions look like possible candidates for degenerate primers. For the N (CP) gene of RNA-S all available amino acid sequences (c. 250) were compared using phylogenetic trees constructed using PAUP. Duplicate sequences were then eliminated leaving 73 distinct sequences representing a wide range of Tospovirus species.

· 73 sequences were aligned and show conserved areas that look suitable for primer design (Fig T2).

Primer evaluation:

Specific primers M27 + M26 to Tomato spotted wilt virus (TSWV) RNA polymerase to amplify ~ 256bp fragments (McDiarmid, pers. comm.).

Degenerate primers to TSWV RNA polymerase designed by Chu et al (2001):

gL3637 + gL4435 to amplify ~ 800bp fragment

gL3637 + gL4510 to amplify ~ 870bp fragment

Detection:

One-step PCR was used on template RNA extracted from Nicotiana benthamiana infected with TSWV.

Bands of the expected sizes resulted from the use of both sets of degenerate primers as well as the specific primers at a magnesium concentration of 3 to 8 mM with the following RT-PCR protocol: 420-1hr, 940-4min, 35 cycles of: 940 -30 sec, 500 -30 sec, 720-1min, and a final extension of 720-10min.

The bands obtained with the degenerate primer were strong and distinct although there was less product compared with the specific primer set (Figure T1).

Further work:

The sequence for Impatiens necrotic ringspot virus is very similar to TSWV in the region of the Chu et al (2001) primers; INRSV differs from TSWV by only 3 nucleotides in each of the two reverse primers. We have designed alternative reverse primers that require G-T matching in some instances. As a result the redundancy in the Chu et al (2001) reverse primers has been reduced from 48 and 24 fold redundancy to 2 and 4 fold respectively. We will test the effect of these changes on the optimal annealing temperature. Knowledge of the effect of G-T mismatch on annealing temperature and primer efficiency will be useful in the design of primers against other virus groups

Appendix 1. MAF Project 33: Generic detection of plant viruses - Progress summary March 2003

Virus Family / Genus

Primary literature search

Evaluation of literature

Sequence
BLAST

Sequence selection

Detailed Sequence
Analysis

Primers designed

Lab work

CLOSTEROVIRIDAE

              

Closterovirus

X

X

X

X

X

X

X

Crinivirus

X

X

X

X

X

X

X

(Ampelovirus)*

X

X

X

X

X

X

X

COMOVIRDAE

              

Nepovirus

X

IP

          

(Comovirus) *

              

(Fabavirus) *

              

LUTEOVIRIDAE

              

Luteovirus

X

X

X

X

X

    

(Polerovirus) *

X

X

X

X

X

    

(Enamovirus) *

X

X

X

X

X

    

GEMINIVIRIDAE

              

Begomovirus

X

            

BUNYAVIRIDAE

              

Tospovirus

X

X

X

IP

IP

IP

IP

UNASSIGNED GENERA

              

Furovirus

X

X

X

        

Pecluvirus

X

X

X

        

Pomovirus

X

X

X

        

Potexvirus

X

            

Tobamovirus

X

            

Tobravirus

X

            

* Not in original proposal X = completed IP = in progress

2.5 MBS 324

Programme Title:

Mouth & feet lesions

Programme Leader:

Hugh Black

Institution:

AgriQuality

Summary

Differential diagnosis for mouth and feet lesions in NZ sheep caused by infectious and non-infectious causes.

Background

To build a profile of the mouth and feet lesions that occur in NZ sheep that need to be considered within the differential diagnosis for foot and mouth disease.

Approach & Outcomes

Context of the Project: In the 2001 UK foot and mouth epidemic, diagnosis of this disease was made difficult by the presence of other erosive lesions in the mouths of sheep. These erosive lesions were labelled, "Ovine Mouth and Gum Obscure Disease" (OMAGOD). Providing information on erosive mouth lesions in NZ sheep was determined to be beneficial for veterinarians undertaking foot and mouth disease surveillance, should such an event occur in NZ.

Approach:

The structure of this research was divided into three parts:

  1. a literature review;
  2. a slaughter premise survey;
  3. assembling information for training/education.

Outcomes:

The study proved that lip and gum lesions similar to those seen in the UK during 2001 foot and mouth epidemic, also occur in New Zealand sheep. What was surprising was the high prevalence (3%) of these lesions in ewes, and the fact that they have rarely, if ever been reported as suspect FMD before. The prevalence in lambs was 0.3%.

The aetiology of lip and gum ulcers appears to be largely due to primary traumatic lesions. The trauma is probably associated with ovine flight behaviour when threatened by dogs or people, causing some sheep to run into wire fences or yard rails.

It also seems probable that some ulcers may arise from exposure to abrasive or irritant materials such as soil, mud, hard or sharp plant material, or possible chemical irritants such as salt blocks, which may penetrate or erode intact epithelium.

Following the completion of the literature review and slaughter premise survey, training material for Patrol Veterinarians and Initial Investigating Veterinarians (IIV's) was upgraded.

Summary:

The diagnosis of foot and mouth disease in sheep can be hampered by the presence of erosive mouth lesions that are due to other causes.

A survey of lesions in the mouths of lambs and ewes at two New Zealand slaughter premises (North Island and South Island) was undertaken.

The survey found that the prevalence of these lesions in ewes was high (3%). Outcomes from this survey suggest the main cause of ovine lip and gum ulcers in New Zealand sheep is trauma, with secondary bacterial infection, and foreign body penetration. Less frequently abrasive or irritant ingested materials may cause similar lesions.

The research has provided a significant opportunity to upgrade training material for veterinarians deployed during a foot and mouth disease investigation or epidemic.

Publications

External Publications: Clinical Communication to the New Zealand Veterinary Journal. Title: Lip and Gum Lesions in New Zealand Sheep.

2.6 MBS 325

Programme Title:

Contingent valuation

Programme Leader:

John Craig

Institution:

Auckland Uniservices (Auckland University)

Summary

Goal:

The programme uses the technique of contingent valuation to elicit household WTP (willingness to pay) for the avoidance of a range of reductions in the condition and quantity of urban tree estate. The elicited value may be used to estimate the economic impacts of those factors that cause damage to urban trees and to conduct cost-benefit analyses of response options or preventive measures.

Background

Context of Project:

The planning and management of the urban tree estate cannot be founded solely on narrow financial appraisal and natural sciences, but has to include human values more comprehensively. At present, there is little information on the type of values people attach to urban trees.

Approach & Outcomes

Approach:

Where actual market data are lacking, contingent valuation seeks to discover how people would value certain environmental changes by directly questioning a sample of the population concerned. These changes, and the markets in which they are to be valued, are hypothetical.

The survey instrument applied for this valuation exercise was a self-completion questionnaire with framing, attitudinal and ethical questions, information pack and payment scenario, debriefing questions as well as a request for socio-economic data. The sampling site included 15 New Zealand cities and the sample units were contacted in person and asked to participate in the survey.

A dichotomous choice bidding format was employed to elicit willingness to pay responses. This represents a simulated market where respondents are asked if they are willing to pay a predetermined amount, the amount of bid, for a specific resource (preventing a 20% reduction in the number of urban trees). Logistic regression was then used to analyse the data and estimate the willingness to pay.

Outcomes:

There is an estimated $ 116.7 million aggregated willingness to pay for the avoidance of a 20% quantitative reduction in the urban tree estate of the 15 New Zealand cities included in the study.

The need to measure value using a series of measures was reinforced by the finding that 58% of those who refused to pay the contingent bid said yes to contributing through volunteer work instead.

Income is not a significant predictor of the contingent choice.

There is a direct relationship between the willingness to pay for the avoidance of the reduction and the perceived seriousness of the loss. Half of the respondents perceived the 20% loss as very or extremely serious.

Those who find the supply and demand in disequilibrium represent 48 % of the population and indicate the imbalance in the favour of demand (95% want more trees) or supply (5% want fewer trees).

Aesthetics (beauty) is considered by many as a very important benefit of city trees, besides nature in the city, fresh air and habitat for wildlife. People also experience negative effects from trees; causing drainage problems was regarded as the most problematic, while dropping leaves everywhere registered the lowest importance score. If the support of the community for tree programs is the goal, both the reinforcement of benefits and the management of negative effects will be needed.

More than half of the population is expected to consider the significant benefits that trees provide as the strongest motivation for caring. Consideration for the benefits for future generations (28%) and trees as identity definers (13%) also play a significant role, while respect for the existence rights of trees motivates a smaller proportion (4%) of the population. These findings justify the usage of the concept of total value, defined as a sum of direct and indirect use values as well as non-use values.

Qualitative changes in the condition of trees impact on the provision of services; if the extent of this impact is revealed, the value of the damage may be approximated based on the perceived relative importance of the impacted benefits and the measured value of a quantitative change.

Instead of being measured on its own, the willingness to pay for the avoidance of the tree estate's reduction could be embedded as a part of a more inclusive package.

Valuing the ecological services that city trees provide using means such as the City Green 5 software would give additional insights and the communication of such estimates may influence public perceptions.

Summary:

The urban tree estate impacts on the quality of urban life through the provision of a series of benefits that are aesthetic, ecological, social and economic in nature. However, most of these benefits do not have a market price and information on the type of values people attach to urban trees is scarce.

In order to enhance a more comprehensive inclusion of human values into the planning and management of the urban tree estates, as well as to provide an input into the cost-benefit analysis of related policies and projects, the perceived value of a quantitative change in the urban tree estate of 15 New Zealand cities was measured by contingent valuation. The results reveal an estimated $ 116 million per year aggregated willingness to pay during a 3 year time period for the avoidance of a 20% reduction. Half of those surveyed were not happy with the current numbers of urban trees with the vast majority (95%) wanting more trees.

If the support of the community for tree programs is the goal, an understanding of the underlying motivations together with reinforcement of the benefits and management of the negative effects will be needed. If formulation of efficient land-use policies is intended, the comparison of benefits with the provision and management costs will be required.

2.7 MBS 326

Programme Title:

Pheromones for gumleaf skeletoniser (02/03)

Programme Leader:

Max Suckling

Institution:

HortResearch CRI

Summary

Goal:

This project targets the identification of a sex attractant for Uraba lugens (gum leaf skeletoniser), for MAF Biosecurity Authority.

Background

Context of project:

A synthetic sex pheromone provides a monitoring tool for cost-effectively detecting U. lugens populations, to indicate where and when to apply treatment measures to increase the probability of successful eradication or long term management. The project was spread over two years.

Approach & Outcomes

Approach:

Pheromone extracts were obtained from a colony established after the discovery of U. lugens in Auckland in December 2001. Coupled gas-chromatography - electroantennogram studies, followed by GC-mass spectrometry, and field trapping confirmed the identity of the pheromone.

Outcomes:

The pheromone was successfully identified and used for delimitation of the insect in Auckland during 2003.

Summary:

Progress in 2002/03 - Year 2

Australia

Further trapping in Australia failed to confirm the best lure ratio, with a total of only 32 U. lugens male moth caught in Queensland to 23 May of 2003. Traps deployed in Tasmania probably missed the flight of this single generation ("Highland") strain. The relationship of this "Highland" strain to the "Coastal" strain under scrutiny in New Zealand remains unclear.

Discovery of U. lugens larvae in Auckland in January 2003 led to both the opportunity to trap for the species in New Zealand, and more importantly, the requirement to deliver an optimised blend urgently for a delimitation survey, to underpin the serious consideration of an eradication response. In a single adult flight period (which proved to last only four weeks), the following progress was made:

Phenology in Auckland

Traps (n=18) were successfully deployed at each of three locations in Auckland from January 2003, to establish the flight timing of U. lugens males. Flight occurred from mid March-mid April.

Pheromone Blend ratio

A pheromone blend ratio trial (13 treatments, n=10 sites) was established to determine the optimum pheromone blend, building on the 2002 trapping data from Australia. Earlier preliminary results from Australia in year 1 were confirmed, with a three component blend being slightly superior to a two component blend.

Dose response

A pheromone loading trial (8 treatments, n=10 sites) was established to determine the potential for increasing trap sensitivity. The best loading was found to be 1 mg.

Vertical height transect

A vertical height transect (6 heights, n=10 trees) was established to determine the potential for increasing trap sensitivity. The trial was deployed too late in the flight of the insect, and therefore was not successful.

Insect distribution in Auckland

A survey across Auckland was conducted with AgriQuality to determine the distribution of the pest by pheromone traps, and to set the baseline for future work on the spread or decline of the population (if under eradication). The insect was found to be widespread in the south west of Auckland. Trap catches were correlated with ground finds.

Publications

Poster

Gibb, A.R., D.M. Suckling, S. Fielder, J.M. Allen, L. Jamieson, M. Larsen, and G. Walter. 2003. Development of a monitoring system for the Australian gum leaf skeletoniser Uraba lugens: pheromone identification and field trapping trials. 56th N.Z. Plant Prot. Conf. Abstract only.

Paper in prep.

Gibb, A.R., D.M. Suckling, S. Fielder, and J. Allen. Pheromone identification for the Australian gum leaf skeletoniser, Uraba lugens.

Report

Suckling, D.M, A.R. Gibb, S. Fielder, J. Allan , L. Jamieson, G. Clare & M. Lawson & G. Walter. 2003. Development of a synthetic pheromone for Uraba lugens, the gum leaf skeletoniser: Report II. MAF Client Report. HportResearch.

2.6 MBS 327

Programme Title:

Veterinary sentinel project (02/03)

Programme Leader:

Peter Davies (Now Nigel Perkins)

Institution:

Massey University

Goal:

(For first year)

Milestone 4: Create a palmtop data-capture method for commercial use in one private veterinary practice, based on a prototype design that has been worked out in consultation with veterinary practices.

Objective: Completion of functional VetPAD for use in first practice

Background

Notes on progress

VetPADbeta has a data capture front end via a palm pilot or pocket PC, that has been designed for veterinarians in private practice as a means of collecting data during the course of their day-to-day activities. VetPADbeta has a desktop PC back end with data transfer capabilities to and from the palmtop via cable or non-cable based transfer. Feedback from veterinary practitioners has been incorporated into design features of VetPADbeta.

In order to allow VetPADbeta to function in the way it is intended, there needs to be flow of data from VetPADbeta into practice software, and flow of data from practice software into VetPADbeta. Discussions have been held with companies marketing veterinary practice management software in New Zealand and hurdles have been identified in achieving data synchrony between VetPADbeta and currently marketed versions of practice software.

  • Veterinary practices in NZ appear to use a wide variety of software for practice management ranging from accounting software to custom built veterinary practice software.
  • There is no standard currently in existence for veterinary practice data eg data formats and coding of diagnoses and procedures.
  • Some current veterinary software products have no capability for data export/import. One current product has no immediate plans for developing data export or import capabilities.
  • One DOS based version of veterinary practice software that is still in use in NZ, and one Windows product, have data export capability but no data import.
  • There are no palmtop data entry vehicles in existence for current veterinary software so no existing data flow vehicle that can be easily accessed.
  • Software companies are understandably cautious about allowing VetPADbeta to have sufficient access to secure parts of their products in order to be able to implement two-way data flow between VetPADbeta and a commercial veterinary software product. Security and product stability are the major reasons for this reluctance. Ongoing discussions are being held with one of the major veterinary practice management products in NZ regarding potential avenues for either sharing data or incorporating VetPAD functionality into their product. There is unlikely to be a short term solution to this issue.

It was anticipated that VetPAD is unlikely to be adopted by veterinarians unless it is useful in their normal day-to-day activities and it reduces work load in recording/transferring billing and disease information. Design features of VetPAD therefore include collection of billing and client information for the benefit of the veterinarian as well as animal/disease information that can be exported for surveillance purposes.

It must be stressed that these hurdles were not unexpected. Development of EpiMAN in the early 1990s was associated with major issues relating data movement from sources such as Agribase into EpiMAN. It is extremely likely that hand held computers will play an increasingly important role in veterinary activities for billing at the client or farm level, and also for collecting animal health and treatment data for synchronisation with desktop systems. At the present moment integrated systems involving hand held computers have not appeared on the veterinary practice market.

The difficulties identified above have raised the importance of data standards in the development of a system that allows incorporation of disease data from multiple sources into one database for analysis and reporting. Several types of data transfer will be necessary for implementation of an effective practice based contribution to surveillance that involves handheld computers:

  • between a handheld computer and practice management software;
  • between a variety of practice management software packages;
  • between a variety of different practices using similar software but with variations on set up and coding methodology;
  • between practice management software and a national data collection and collation agency capable of storing, analysing and reporting on surveillance objectives.

Effective development of surveillance systems that incorporate semi-automated and automated data collection and analytical components would be greatly facilitated by the adoption of data standards for recording information and for data transfer. Our understanding is that such a standard is in existence for human disease coding (http://www.who.int/whosis/icd10/). There is also a standard developed by the American College of Pathologists (http://snomed.org), that is proposed as a veterinary standard. SNOMED® is currently implement in some form in a number of North American veterinary schools but we are not aware of it being incorporated in any commercial veterinary practice management software.

The difficulties identified above have meant that implementation of a working version of VetPADbeta into a practice has not been achieved. Discussions are being held with selected veterinary practices to determine if VetPADbeta can be utilised in a veterinary practice under a modified approach as follows:

  • VetPADbeta installed onto practice desktop PC(s) and onto palmtops used by veterinarians;
  • data capture at the level of the palmtop by veterinarians during their day-to-day activities. Data then transferred from palmtop to desktop versions of VetPADbeta and then printed out as hard copy for practice staff to use as they enter billing and disease information into their veterinary practice software;
  • periodic transfer of data from desktop versions of VetPADbeta to the EpiCentre for incorporation into this project.

Approach & Outcomes

It is the intention to install VetPADbeta in at least one veterinary practice to allow this milestone to be achieved as per the modified approach above. Final discussions are being held with two veterinary practices with this in mind. It is anticipated that this will provide valuable feedback on design and functionality of the product.

Discussions are continuing with one veterinary practice software product that is receptive to the concept of developing generic data transfer capability that would allow VetPADbeta to fully interact with their product (both data input and data export). Nontheless, it appears that given the difficulties in data compatibility and transfer between VetPADbeta and current commercial veterinary practice software (as described above), achievement of the next milestone may be affected.

2.7 MBS 329

Programme Title:

Contact rates for livestock

Programme Leader:

Robert Sanson

Institution:

AgriQuality

Research Programme not yet completed and reported at time of the preparation of this publication. Will be reported in next Research Results publication.

2.8 MBS 331

Programme Title:

Lymantriids

Programme Leader:

Nod Kay

Institution:

Forest Research CRI

Summary

To assess key elements of New Zealand's production and indigenous forest flora as hosts of potentially invasive Lymantriid defoliators.

Background

Context of tLymantriids are unrepresented in New Zealand's invertebrate fauna, but are recognised globally as invasive forest defoliators. New Zealand has a history of interception and localised establishment of these insects. Past assessments indicate that contrary to accepted paradigms New Zealand's indigenous flora is comparatively resistant to exotic defoliators, while the exotic plantation species are susceptible to indigenous defoliators. The project was undertaken both to assess the risk to New Zealand's forest and to validate an explanation of those results.

Approach:

Parameters of larval growth, development and mortality were measured for two species of Lymantriid exposed in no-choice trials to a range of indigenous an exotic host plants. Trials were undertaken in quarantine facilities in New Zealand and France and the host plant foliage was sourced from gardens and arboreta in New Zealand and Europe. Potential fecundity of adults reared from the trials was assessed by dissection. Results were correlated to host plant geographic range.

Approach & Outcomes

Outcomes:

Two populations of L. dispar were used as bioassays of host suitability of 22 species of understory, and eight taxa of dominant, New Zealand indigenous forest trees sourced from the gardens and arboreta of Europe. Other Lymantriid species were unavailable in Europe. However, T. anartoides was used to assess the same forest tree species collected from their natural environment in New Zealand. Both bioassay systems confirmed the relative resistance of New Zealand's indigenous flora to novel invertebrate defoliators. Two exotic production forest tree species, Pinus radiata and Pseudotsuga menziesii, were assessed as potential host of T. anartoides under quarantine conditions in New Zealand. Both were moderately susceptible to defoliation but unlikely to sustain epidemic populations. The efficacy of transgenic P. radiata was assessed for resistance to T. anartoides and shows some promise as a pest management tool.

An hypothesis developed as an explanation of the results and a re-evaluation of a national biosecurity strategy is discussed.

Summary:

Key elements of New Zealand's production and indigenous forest flora were trialed as potential hosts of invasive Lymantriid defoliators to assess the risk to New Zealand's forests and to explore an ecological explanation of ecosystem stability.

Parameters of larval growth, development and mortality were measured for two species of Lymantriid exposed in no-choice trials to a range of indigenous an exotic host plants. Trials were undertaken in quarantine facilities in New Zealand and France and the host plant foliage was sourced from gardens and arboreta in New Zealand and Europe.

In two bioassay systems a total of 52 indigenous plant species from 22 plant families proved resistant to two polyphagous invasive defoliators. Only a few representatives from families closely allied to those of the defoliators' primary hosts were acceptable as hosts. The results of both of the lymantriid bioassays with New Zealand indigenous plants show that the vast majority of these plants are unexpectedly resistant, according to accepted invasion paradigms. That the explanation may be intrinsically correlated to the host plants' geographic range is counter-intuitive, but explainable and highly relevant to the current debate as to the role of biodiversity in ecosystem stability. The results from the assessment of P. radiata are less clearcut but tend to support the existence of a resource allocation trade-off between growth and resistance.

Publications

  1. Kay, M. K. 2003. Macroecology and the prediction of invasive invertebrate guilds. - NZ Plant Protection Society Biosecurity Symposium, Rotorua, NZ. In press.
  2. Kay, M.K., Wratten, S.D. and Price, P.W. 2003: A reconciliation of biodiversity/function issues; top-down/bottom-up forces and a prediction of terrestrial cascades: an Island Resource Allocation hypothesis. Oikos `forum' submitted.

2.9 MBS 332

Programme Title:

Feral Bees Control - extension

Programme Leader:

Mark Goodwin

Institution:

HortResearch CRI

Summary

Goal: To determine whether eradicating feral honey bees from an area is achievable using a method developed by HortResearch.

Background

Varroa destructor presents a significant risk to New Zealand agriculture. The economic impact assessment of varroa in New Zealand carried out by the Ministry of Agriculture and Forestry (MAF) suggests that under beekeeper management alone varroa is likely to cost New Zealand's horticultural, pastoral, arable and beekeeping sectors between $400 and $900 million over the next 35 years. The surveillance programme currently established in the South Island aims to provide early detection of varroa so as to increase the likelihood of successful eradication, should an eradication attempt be made.

Approach & Outcomes

Approach:

Nine (4L capacity) bait stations each covered with a metal milk crate (0.5 x 0.5m) to exclude stock, were established 1km apart in a 4km2 site. Twenty, four-frame nucleus (nuc) colonies were situated at the site. All bait stations were filled with 50% white sugar syrup and sprayed with a highly aromatic honey solution. The area surrounding the bait stations (0.5m radius) were sprayed with approximately 1L of the honey solution. The stations were visited approximately every 90minutes to keep the sugar syrup topped up and to spray the honey solution. As the number of foraging bees increased the area being sprayed with honey solution was reduced to a radius approximately 0.2m from the bait stations. This process was repeated the following day to encourage the forager bees to return.

When more than 300 bees were simultaneously foraging at a bait station and had been foraging at this level during daylight hours, for the past 24 hours, the sugar syrup was replaced with sugar syrup containing 5% fipronil (0.05ml fipronil + 1L 60% sugar solution). The poisoned syrup was removed from the bait stations after 24 hours and the stations were refilled with fresh 50% sugar syrup and bracken and the area (0.5m radius) was sprayed again with 60% honey solution. The containers were refreshed every three days to prevent fermentation

Outcomes:

All 20 nucleus hives died within 13 days. A period of fine weather for at least 4 days is required to conduct a successful eradication. A constant source of fresh sugar syrup or poison must be available. A lack of interest in the bait stations may arise when more attractive or plentiful food sources are available. It may therefore be necessary to decrease the distance between bait stations. However, it is not known whether causing the bees to fly further distances would result in the bees succumbing to the poison before returning to their hive. It may also be possible to increase the distance between bait stations if foraging sources are limited.

The major factors that need to be considered if honey bees are to be eradicated from an area are:

  1. size and topography of the area that honey bees are to be eradicated from;
  2. seasonal variation / weather stability;
  3. size and behaviour of colonies due to season;
  4. land access;
  5. beekeeper support.

Summary:

Eradicating feral honey bees from an area was achieved using a method proposed by HortResearch. Suitable weather conditions and easy access to the bait stations were necessary. Bait stations were positioned 1km apart and filled with sugar syrup poisoned with fipronil. All 20 nucleus colonies were killed within 13 days post-poisoning.

3.0 MBS 333

Programme Title:

Lophodermium identification

Programme Leader:

Peter Johnston

Institution:

Landcare Research CRI

Summary

To enhance New Zealand's forest pest surveillance system by providing a simple, molecular-based diagnostic tool to allow rapid identification of Lophodermium seditiosum, a serious pathogen of pines that is not present in New Zealand.

Background

Many species of Lophodermium are known to occur on pines, but only Lophodermium seditiosum is considered a serious pathogen. It infects 2- and 3-needled pines in Europe and North America, killing needles in the first growing season. Establishment of L. seditiosum in New Zealand would cause considerable loss of wood volume in pine plantations. Two non-pathogenic species of Lophodermium occur on pines in New Zealand, and these are difficult to distinguish morphologically from L. seditiosum. A molecular-based detection technique will benefit New Zealand's border biosecurity by enhancing ability to rapidly and authoritatively recognise L. seditiosum.

Approach & Outcomes

Approach:

GeneDoc software was used to visualise putative RFLP patterns from sequence data we already had available from a wide range of pine-inhabiting Lophodermium species. We sequenced New Zealand isolates of L. pinastri and L. conigenum to check that the existing data (from North American isolates) were valid in the New Zealand situation. A combination of distinctness of banding patterns, technical ease, and availability and cost of restriction enzymes were used to select the best enzymes for this project.

Outcomes

The PCR/RFLP method targeting the ITS region provides a simple, reliable tool for distinguishing Lophodermium species from pines:

  • GeneDoc analysis of existing sequences suggested a double digest of the ITS region using the restriction enzymes HaeIII and HpaII would most effectively distinguish the species. A phylogenetic analysis divided the 15 putative species for which ITS sequences were available into 8 distinct clades. Each of these clades could be distinguished from the RFLP banding pattern
  • All isolates of L. pinastri (16 isolates), L. conigenum (14 isolates), and L. seditiosum (7 isolates) tested on gels showed a restriction pattern consistent with that predicted by GeneDoc, with no variation between isolates.
  • Lophodermium molitoris was recognised from New Zealand for the first time.
  • Two genetically distinct L. pinastri populations were recognised, one European and one North American. All New Zealand isolates are related to the population from the western USA, suggesting this is point of origin of this fungus for New Zealand.

To maximise the usefulness of this method as a quarantine tool for New Zealand's forestry industry there is a need to develop it so identifications can be made directly from infected plant tissue. Bypassing the step of first isolating the fungi, will allow identifications in a few hours, rather than the 3-4 weeks it may take if the fungus must first be isolated into pure culture.

Summary

The goal of this project was to enhance New Zealand's forest pest surveillance system by providing a simple, molecular-based diagnostic tool to allow the rapid identification of Lophodermium seditiosum, a serious pathogen of pines that is not present in New Zealand. The only 2 species of Lophodermium reported from pine in New Zealand are saprobes with an initial endophytic phase to their life cycle. The pathogenic North American species L. seditiosum is considered a quarantine threat to New Zealand's plantation forests. Because these fungi are difficult to distinguish morphologically, a simple molecular method has been developed to distinguish the Lophodermium species already known in New Zealand from L. seditiosum. The ITS region is digested using HaeIII and HpaII in a single reaction, resulting in unique RFLP banding patterns for each of the species treated. Gathering field samples for this project revealed a third Lophodermium species on pine in New Zealand, L. molitoris. This species has a similar biology to the two Lophodermium species already reported from New Zealand on pine. Two genetically distinct groups were recognised within Lophodermium pinastri, one North American and one European. All the New Zealand isolates of L. pinastri tested were identical to a group also known from the Pacific North West.

Publications

  1. Johnston, P.R.; Park, D.; Dick, M.A.; Ortiz-García, S.; Gernandt, D.S.; Cooper, J.A.; Smith, J. 2003: Lophodermium identification tools. 8th International Congress of Plant Pathology, Volume 2, Offered Papers, p.160 (abstract).
  2. Johnston, P.R.; Park, D.; Dick, M.A.; Ortiz-García, S.; Gernandt, D.S. Identifying pine-inhabiting Lophodermium species using PCR-RFLP (submitted to New Zealand Journal of Forest Pathology).

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