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• 2. Maintaining Biosecurity Category
  • Studies on the distribution and taxonomy of Arhopalus spp. In Australia and New Zealand
  • Incidence of stained growth rings in elm trees growing in Waikato
  • Sensitivity and reliability of methyl bromide sachets used to monitor and verify fumigation parameters
  • Clonal status of Salmonella. Typhimurium DT 160
  • Development of a synthetic pheromone for use in the gum leaf skeletoniser eradication programme.
  • DNA diagnostic procedures for the identification of selected species and populations of Lymantria and Orgyia moths from intercepted egg masses
  • Investigation of the Suitability of NZ Forest flora for reproduction and development of Lynmantriid Species
  • Lophodermium identification tools project.
  • Destroying feral bee colonies

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2. Maintaining Biosecurity Category

2.1 MBS 330

Programme Title:	Studies on the distribution and taxonomy of Arhopalus spp. In Australia and New Zealand
Programme Leader:	Dr Q. Wang
Institution:	Institute of Natural Resources, Massey University

Summary

The goal of this project is to provide exact identification of, and al illustrated key to Arhopalus species occurring in Australia and New Zealand and to define their current geographic distribution in both countries.

We obtained Australasian Arhopalus specimens from all known Australasian insect collections, key European and American museums, and MAF woodborer/bark beetle survey in 2000/2001. Specimens of each species were compared with types or authentic specimens from Europe and North America. Thirty specimens for each sex of every Australasian Arhopalus species were measured under a stereomicroscope using ocular micrometers. Images were photographed with a digital camera. Distributional records were from collection notes of each specimen examined and literature.

Three species have been found in Australasian region, all of which were accidentally introduced from Europe: Arhopalus rusticus (Linnaeus, 1758), A. ferus (Mulsant, 1893), and A. syriacus (Reitter, 1895). An illustrated key to, and a morphometrics of, these species were provided. In Australasia, A. ferus occurs only in New Zealand, distributed throughout the country except central and Southwest Otago and Stewart Island, A. rusticus is only found in Melbourne, and A. syriacus in NSW State forests around Sydney.

Goal:

To provide exact identification of, and an illustrated key to Arhopalus species occurring in Australia and New Zealand and to define their current geographic distribution in both countries.

BACKGROUND

The taxonomy of Arhopalus was in a state of confusion and it was likely that the species previously recorded in Australasia were in fact misidentified, causing difficulties in timber trade between these two countries as well as between Australia-New Zealand and other countries. Prior to this project, no reliable key for identifying Australian-New Zealand Arhopalus beetles was available and the current geographic distribution of these species was not know precisely. These problems have made it difficult for New Zealand authorities to respond quickly to detection of Arhopalus on imported material and to assess the risk posed to New Zealand by Australian timber. Therefore, there was an urgent need for a reliable and user-friendly key to identify Australian and New Zealand Arhopalus beetles and for information on their current distribution in these two countries.

APPROACH & OUTCOMES

We obtained Australasian Arhopalus specimens from all known Australasian insect collections, key European and American museums, and MAF woodborer/bark beetle survey in 2000/2001. Specimens of each species were compared with types or authentic specimens from Europe and North America. An illustrated key to Australasian Arhopalus species was made using these specimens.

To determine sexual dimorphism in external morphology and to enable correction identification of both complete and incomplete specimens (such as specimens without head, abdomen, or antennae), we made measurements of 30 specimens for each sex of every Australasian Arhopalus species under a stereomicroscope (Leica, Germany) using ocular micrometers. Images were photographed with Nikon Coolpix 995 digital camera through a phototube attached Leica Stereomicroscope. Automontage Software was used to collate layered images of microstructures. Distributional records were from collection notes of each specimen examined, and literature, if considered reliable.

Three species have been found in Australasian region, all of which were accidentally introduced from Europe: Arhopalus rusticus (Linnaeus, 1758), A. ferus (Mulsant, 1893), and A. syriacus (Reitter, 1895). These species can be easily identified using the following key (Illustrations are provided in full report):

1. Third segment of tarsus incised apically to about ½ its total length; elytron with sutural angles rounded; male eighth tergite deeply emarginate at apex………………….A ferus

Third segment of tarsus incised almost to base…………………………………………2

2. Terminal segment of maxillary palpus strongly securiform, with length 1 to 1.26 times its apical width; elytron with sutural angles always rounded; male eighth tergite slightly emarginate at apex……………………………………………………………..A. syriacus

Terminal segment of maxillary palpus slightly widened apically, with length 1.34-1.39 times its apical width; elytron with sutural angles usually angulate, sometimes with a weak spine; male eighth tergite rounded at apex…………………………………….A. rusticus

Proportional relationships between body parts were found to be very stable between species and/or between sexes and are particularly useful for sex and species identification when specimens are incomplete or specimen examiners are not familiar with Arhopalus. A statistically analysed morphometrics was given.

Distribution maps of these three species in Northern Hemisphere were given. In Australasia, A.ferus occurs only in New Zealand, distributed throughout the country except central and Southwest Otago and Stewart Island, A. rusticus is only found in Melbourne, and A. syriacus in NSW State forests around Sydney. A distribution map of A. ferus in New Zealand was also provided.

PUBLICATIONS:

In preparation

2.2 MBS 332

Programme Title:	Incidence of stained growth rings in elm trees growing in Waikato
Programme Leader:	Mr Lindsay Bulman
Institution:	Forest Research

SUMMARY

The project was undertaken to determine if the incidence of staining in the inner growth rings of elms growing in Waikato differed significantly from that found in Auckland where Dutch elm disease is present. From 945 elms, four branches that had at least 8 years growth, one branch from each cardinal point, were cut using pole pruners from high in the crown of each tree. The cut surface was examined for signs of stain; the branch was then sectioned at 500 mm intervals and each cut surface examined. If stained material was found, a 200 mm sample of the stained material from each branch was collected and sent to Forest Research for diagnosis. Isolations were made onto 3% malt agar and all fungi growing on the plates were identified, where possible. Data on elm size, species, location, and condition were recorded. A binomial generalised linear model and a Bayesian analysis were used to determine if the incidence of staining in Waikato differed significantly from that found in Auckland.

The incidence of staining was significantly lower in Waikato (4%) compared with that found in Auckland (123%). This effect was consistent across all elm types sampled. The incidence of staining in Waikato was lower than was first assumed and accordingly 945 elms were sampled. Ophiostoma novo-ulmi was not isolated, and the mix of fungi isolated was broadly similar to that found in Auckland elms. In Waikato, green elms (6.3%) had significantly higher incidence of staining than golden elms and variegated elms (1.9%). The respective incidence of staining found in Auckland was 20.5% and 7.3%. The posterior probability that the incidence of staining in golden and green elms growing in Waikato is greater than or equal to those growing in Auckland was less than 0.00001. The majority of staining (68% of stained growth rings) was found in growth rings of the current 2-year-old wood. In the Auckland surveys, 37% of staining was found in wood of this age. Insect damage was associated with 13% of stained growth rings in both Waikato and Auckland.

Goal:

The aim of this programme was to determine the incidence of staining in the inner growth rings of elms growing in an area where Dutch elm disease is not present.

BACKGROUND

The fungus that causes Dutch elm disease (Ophisostoma novo-ulmi) may be confined to the wood invaded in the year of infection and become buried by tissue free of infection as the years go by. Such trees may show localised symptoms typical of the disease in the first year of infection, but in subsequent years they often appear healthy or display just minor dieback. The detection of O. novo-ulmi in inner growth rings of asymptomatic elms forms an important part of the Dutch elm disease eradication campaign whereby almost 5,000 elms in the Auckland region have been sampled, and 13% had staining in one or more growth rings. However, O.novo-ulmi was recovered from only 3 trees. Without determining the incidence of staining in a disease-free region (i.e., Waikato) the significance of this result remains in doubt. If the incidence of staining in the two regions is not significantly different then we have evidence that agents other than O.novo-ulmi were responsible for almost all the stain in trees in Auckland. If the incidence of staining in Auckland is significantly greater than that in Waikato then the implication is that some of the staining was caused by O.novo-ulmi and the sampling technique presently used is not intensive enough to detect it.

APPROACH & OUTCOMES

It was assumed that the incidence of staining in Waikato would be one third less than that found in Auckland, which required 1,320 elms to be sampled to give a 95% surety of detecting a difference with 95% confidence. After permission to sample elms had been granted from five local authorities in Waikato, sampling started in late October 2001 and was completed in mid December. Four branches were cut from each tree and all cut surfaces were examined for staining. When staining was found samples were taken and sent for diagnosis. The incidence of staining found in Waikato was compared with that found in Auckland.

The incidence of staining in Waikato was significantly less than found in Auckland. The sampling method used in both regions was identical. In both studies elms were sampled from a variety of locations (residential properties, parks and reserves, rural areas, urban roadsides). The climate of both regions is similar. There were no identifiable environmental or biotic factors that could account for the difference in staining between the two regions except that Dutch elm disease is present in Auckland and has been known there since late 1989, but has never been found in Waikato. Infection by O.novo-ulmi results in staining of elm wood but its recovery can be extremely variable. Of the 5 infected elms sampled in a study by Forest Research, recovery varied from 8% to 74% of branches sampled from individual trees. It is likely that O.novo-ulmi was responsible for some of the staining found in asymptomatic elms in Auckland, but the fungus was either no longer viable or insufficient samples were taken to guarantee its recovery. This finding has implications for the Dutch elm disease eradication campaign in Auckland and it is probable that a more intensive examination of selected trees will be necessary to accomplish the objective of the asymptomatic survey there.

PUBLICATIONS:

None intended at this stage.

2.3 MBS 334

Programme Title:	Sensitivity and reliability of methyl bromide sachets used to monitor and verify fumigation parameters
Programme Leader:	Dr Zheng Zhang
Institution:	Crop & Food Research

SUMMARY

The Cross Check methyl bromide sachets were effective in indicating the completion of methyl bromide fumigation when the fumigation temperature did not fall below 21^oC. The development of a pinkish colour in the sachets, indicating the end-point of fumigation, is very temperature-sensitive. When temperature was not controlled, the development of the end-point was affected by variation in the ambient temperature during the fumigation operation.

The size of the sachet determines the area into which methyl bromide may diffuse and the even thickness of the packing plastic around the sachets also affects the diffusion rate. Therefore, good control of these two parameters is recommended.

Goal:

To determine the reliability of methyl bromide sachets used to monitor and verify fumigation parameters.

BACKGROUND:

We evaluated one brand of methyl bromide sachets, Cross Check, for its sensitivity and reliability and then the potential for it to be used in commercial-scale methyl bromide fumigation operations. We examined the colour-display of methyl bromide sachets for indicating the end-point of fumigation at the designed CT value, at under the designed CT value as well as after an overdose. We evaluated the affect of fumigation temperature on the sensitivity of colour-display.

APPROACH & OUTCOMES

Experimental trials were carried out in a 100 cubic feet (2.83 m3) vacuum chamber. For each fumigation configuration two replicate tests were conducted. Within each chamber, three sachets were compared. The three sachets were positioned in the chamber randomly, but not less than 60cm away from the inlet of methyl bromide gas.

For sensitivity of end-point colour-display trials we carried out sixteen incomplete (shortened) fumigation configurations, three types of the full CT value fumigation (24, 150 and 1000 h g m-3) and 10% and 20% overdosed fumigation (of all three types of sachets). 1/40 N Ammonium thiocyanate was used to titrate the sachet solution to determine the actual degree of completion of fumigation comparing to the calculated one. The relationships between observed and calculated CT values were obtained.

The Cross Check methyl bromide sachets have proved to be useful and very effective at indicating the completion of methyl bromide fumigation when the fumigation temperature is above 21^oC.

The indication of completion of fumigation with a pinkish end-point colour appears to be very temperature-sensitive. When temperature is not controlled, the successful fumigation indication depends on the ambient temperature variation during the fumigation operation. The CT value of incompletely fumigated sachets can be determined by titration. With different fumigation configurations, we obtained a good correlation (non-linear) between the absorbance of bromide ions by the sachets and their CT values. The results were reproducible. The non-linear correlation was caused by:

• Fluctuating ambient temperatures;
• Physical or chemical properties of the plastic packaging material;
• Decreasing concentrations of fumigant due to sachet absorbance; and
• Undetectable minor leaks of fumigant.

The methyl bromide sachets absorb methyl bromide from the headspace of a fumigator. Even with the application of the same amount of methyl bromide into the same fumigation space, the concentration of methyl bromide in the headspace may vary due to different commodity absorption rate of methyl bromide or different ratio between headspace and void space volume. We recommend that methyl bromide sachets should be designed for fumigating specific commodities with a recommended headspace/void space ratio.

PUBLICATION

One paper is in preparation for future publication.

2.4 MBS 335

Programme Title:	Clonal status of Salmonella. Typhimurium DT 160
Programme Leader:	Carolyn Nicol
Institution:	E.S.R.

Summary

In New Zealand S. typhimurium DT 160 was first reported as the cause of human disease in 1998 (1 case) followed by three cases in 1999, all from the Canterbury area. Since the beginning of 2000 a rise in the number of cases has occurred both in humans and animals (predominantly in birds). This strain has been responsible for a large number of sparrow deaths by septicaemia rather than by gastro-enteritis as in other animal species. By September 2000, 25% of human Salmonella isolates belonged to this phage type and disease was associated with increased hospitalisation rates.

Previous PFGE analysis of 14 human and 30 non-human DT 160 isolates collected during 2000 showed that isolates were indistinguishable using the enzyme, Xbal. Allied research has shown the isolates were fully sensitive to antibiotics and therefore, there is little risk of antibiotic resistance.

This study will determine whether overseas isolates belong to the same clone and attempt to identify the origin of the New Zealand strain. PFGE methods have been used previously to indicate the original of microbes from overseas sources (Sulakvelidze et al., 2000). This information will aid MAF in the development of import health standards for animals and animal products.

Goal:

Determine the relationship between New Zealand and overseas strains of Salmonella typhimurium DT 160 using pulsed field gel electrophoresis (PFGE).

BACKGROUND:

The aim of this objective was to examine the clonality of overseas isolates of S. Typhimurium DT 160 and determine their relationship to New Zealand isolates. This information may indicate the origin of the New Zealand strain and would aid in the development of health standards for humans, animals and animal health products. Isolates from several non-clinical sources and from cases of human infection were obtained from selected countries where this strain had been identified recently. The serotype and phage type was verified and isolates were characterised by PFGE. Gel analysis software was used to compare PFGE patterns of overseas isolates with New Zealand isolates and to establish strain clonality.

APPROACHES AND OUTCOMES

Salmonellosis is a notifiable disease in New Zealand. All salmonellae isolated from human cases are referred to the Enteric Reference Laboratory (ERL) for serotyping and if confirmed as S. Typhimurium (STM) they are phage typed. In addition, a considerable number of isolates from animal health laboratories, poultry and meat producer laboratories are typed. In this manner it is possible to monitor which serotypes and phage types are endemic and to detect when a novel type is introduced.

Salmonellosis occurred in New Zealand at the rate of 49.7 cases per 100,000 population in 2001. The majority (60%) of isolates were STM. All isolates of STM are phage typed, and a definitive type (DT) is reported for approximately 96%.

Table 1 lists the distribution of the six most frequently occurring DTs identified in the period 1998 to 2001, and compares them to the number of DT 160 isolates in the same period.

Table 1. Distribution of frequently occurring Definitive Types in humans by year

The first isolate of DT160 in New Zealand was isolated in November 1998, from a one-year old child living near Christchurch. There was no history of overseas travel and the source of the infection was not determined.

DT160 was not isolated again until November 1999, when another isolate was received from the Christchurch area followed by a further two isolations in December. Again no epidemiological link was identified among the three cases. From December 1999, cases continued to occur at the rate of one or two per month, all in Christchurch area. In July 2000 DT 160 was isolated from a case of human infection in Invercargill for the first time, and over the following winter months, cases started to occur in the North Island in Palmerston North and Wellington.

By the end of the year 2000, cases of DT160 infection were occurring throughout the country, increasing in frequency from one to two per month to over 40 per month. By 2001, DT160 was the phage type of STM isolated most frequently from human cases of salmonellosis.

Worldwide DT160 has previously been associated with illness in birds, usually sparrows, but has infrequently been observed in humans. DT160 was isolated from nine to 60 sparrows trapped in Guelph, Canada in 1979 (Tizard et al., 1979), and was associated with epizootic illness and mortality among sparrows in the south of England between 1966 and 1972 (McDonald et al., 1980). Prior to the emergence of DT160 in New Zealand, the only published report of infection with this phage type among humans was an institutional outbreak in the United Kingdom in 1979 (Penfold et al., 1979). During that investigation DT160 was isolated from sparrows present in the hospital kitchen. Inpatients were reported to have been exposed to infection through contamination of uncovered food by sparrow droppings.

The first animal isolate in New Zealand was from a domestic cat from Christchurch in May 2000. Isolates from a horse, two cockatoos, and a sparrow followed soon after. Although it is usual for the sparrow population to die off slightly during winter (Mackereth G., personal communication) during the winter 2000 sparrow deaths increased markedly. An investigation into the cause of this phenomenon established that the birds had died from salmonella septicaemia (Alley et al 2000) and DT160 was isolated from both the blood and internal organs of the birds. DT160 has been found subsequently in many other animals and birds (Table 2),. And has become an important cause of animal morbidity, causing septicaemia in wild birds, leg joint and bone disease in horses, and enteritis in dogs and cats.

Table 2. Non human Isolates of DT160 (confirmed by ERL) by year.

Species	2000	2001
Alpaca		1
Avian (mainly sparrows)	60	74
Bovine	7	28
Canine	1	11
Cervine	1	2
Equine	8	13
Feline	7	53
Monkey	1	1
Otter		1
Ovine	2
Porcine		2
Rabbit	1	1
Shellfish		3
Food		3
Environment	15	25
Poultry*	51	110
Misc	3	7
Total	157	335
*Including feed and environment

A representative collection of fifty-two isolates of DT160 was analysed in this study. The collection included nineteen humans and nineteen non-humans New Zealand isolates, twelve American isolates and 2 Danish isolates. All isolates were subjected to cluster analysis of MLFP profiles after PFGE using Bionumerics software. PFGE technique is widely acknowledged to be highly discriminatory and epidemiologically relevant making it one of the most reliable genotyping procedures currently available (Heir et al, 2002).

The combination of both phenotypic and genotypic methodology in this study complemented each other and provided a robust hierarchy for discrimination of disparate isolates. New Zealand isolates, matched by date of isolation and linked to geographical place of isolation, belong to the same clonal grouping and are closely related to the overseas isolates included in the study.

Results from the MBS336 study also indicate that the New Zealand strain of DT160 is of clonal origin and is closely related to the overseas isolates tested.

There are very little published data of human infection with DT160 worldwide but in New Zealand there is compelling evidence that this pathogen has been widely disseminated by wild birds. A case – control study of sporadic human infections carried out between May and August 2001 identified contact with birds as a significant risk factor (Thornley et al., 2002). Isolates of DT160 will continue to be so monitored in the future and any further evolution of the clonal group will be described.

2.5 MBS 336

Programme Title:	Molecular and animal-based studies on the New Zealand Salmonella typhimurium DT160 clone to examine epidemiology, pathogenicity and immunogenicity.
Programme Leader:	Dr Gail Greening
Institution:	Institute of Environmental Science and Research/Massey University

Summary

Salmonella typhimurium DT160 became an important cause of human disease in New Zealand during the three-year period 1999-2001. The epidemic of DT160 infection among humans developed in parallel with an episode of illness due to the same pathogen among animals, in particular wild birds. The causes of the epidemic among humans have not been fully identified, although other studies have suggested potential zoonotic and foodborne transmission.

Goal:

Conduct molecular, animal-based studies and epidemiological analysis to examine the epidemiology, pathogenicity and immunogenicity of the New Zealand Salmonella Typhimurium DT160 clone.

Background

Molecular methods were used to analyse the virulence markers and other strain characters of New Zealand Salmonella Typhimurium DT160 isolates. A comparative epidemiological analysis of S.Typhimurium DT160 infection in human and animal populations was carried out to determine if DT160 presented a more serious threat to human health than other salmonella, to identify explanations for the origin and spread of DT160 in New Zealand, and to provide information to assist with prevention and control. Continued investigation of significant outbreaks of DT160 in birds and other animals, and field studies to determine the rate of carriage of DT160 by sparrows and poultry were used to identify possible infection sources.

All DT160 isolates have been indistinguishable by molecular typing methods, indicating a common point source of origin. Possible sources could be either contaminated animal feed or migratory birds. Wild birds appear to play an important role in DT160 transmission. Contact with wild birds, including dead birds, bird droppings, and food, water and environments that may have been contaminated with bird droppings should be avoided.

Approach & Outcomes

The research was carried out under 3 objectives by ESR and Massey University staff.

Objective 1 used a range of molecular methods to analyse the virulence markers and other strain characteristics of New Zealand Salmonella typhimurium DT160 isolates. Isolates were selected using geographical and temporal factors and by their association with disease in humans, animals and avian species.

Objective 2 investigated the comparative epidemiology of S.typhimurium DT160 infection in human and animal populations. The analysis described the emergence of DT160 as a significant human pathogen in NZ, compared the human and animal pattern of DT160 infection, assessed whether DT160 presented a more serious threat to human health than other salmonella, identified explanations for the origin and spread of this organism in NZ, and identified potential areas for improving prevention and control of this infection.

Objective 3 investigated reported isolations of Salmonella typhimurium DT160 in bird and other animal populations to identify possible sources of infection, and continued the collation and investigation of reported isolations of DT160 in bird and other populations. This was achieved by a) an in-depth investigation of significant outbreaks of DT160 in birds and other animals and b) field studies to determine the rate of carriage of DT160 by sparrows and poultry where possible.

During 2000 there was an explosive emergence of DT160 infection among both human and animal populations in New Zealand. The surveillance data do not identify the origin of DT160, but the organism could have either been introduced from overseas or evolved within New Zealand. All DT160 isolates have been indistinguishable by molecular typing methods. This suggests that the epidemic originated from a point source and that the organism was of recent origin. Molecular analyses demonstrate close similarities with isolates from overseas and imply that DT160 was introduced into New Zealand from an unknown overseas area. The epidemiological data suggest that possible modes of introduction could include migratory birds or imported contaminated animal feed.

The epidemiological analysis highlighted limitations in the New Zealand surveillance of animal Salmonella infection. Surveillance currently relies on voluntary submission of isolates for laboratory typing. Surveillance of Salmonella contamination of food and environments is less systematic. Improved integration of surveillance information would provide a solid basis for identification and control of future epidemics.

The data support efforts to reduce the incidence of DT160 infection and salmonellosis generally. Although the source of the initial human and sparrow DT160 infections in New Zealand remains unresolved, wild birds appear to play an important role in its transmission. Contact with wild birds, including dead birds, bird droppings, and food, water and environments that may have been contaminated with bird droppings should be avoided. Contact with infected or carrier animals or their faeces is also a potential source of human infection. Public information on DT160 and preventative measures is required. The information generated in this project can be used towards mitigating future impacts of this emerging pathogen.

PUBLICATIONS:

Alley, M.R., Connolly, J.H., Fenwich, S.G., et al. An epidemic of salmonellosis caused by S.typhimurium DT160 in wild birds and humans in New Zealand. Submitted to the NZ Veterinary Journal, June 2002.

Baker, M., Thornley, C., Gilmore, K., Bennett, J., Nicol, C., Greening, G., Connolly, J. Large New Zealand outbreak of Salmonella STM160 in humans linked to outbreak in birds. [Draft paper to be submitted to Emerging Infectious Diseases].

2.6 MBS 337

Programme Title:	Development of a synthetic pheromone for use in the gum leaf skeletoniser eradication programme.
Programme Leader:	Dr Max Suckling
Institution:	HortResearch

Summary

Gum leaf skeletoniser is a native to Australia and is considered a serious defoliator of eucalypt trees. Gum leaf skeletoniser was first detected on 12 June 1997 within the Tauranga District, and in 2001 in Auckland. Attempts to eradicate it appear to have been successful. However, the insect is still considered a significant biosecurity risk to MAF.

Goal:

The goal of this programme is to develop a synthetic pheromone for the gum leaf skeletoniser (Uraba lugens), in order to help MAF to provide a more effective monitoring tool for detecting any residual (or future) population and indicating where to apply treatment measures to increase the probability of successful eradication.

Background

The identification of the pheromone of this Australian noctuid moth presents a technical challenge requiring close co-operation between Australia and New Zealand research teams. The identification was achieved by pheromone extraction, identification, and synthesis, followed by laboratory and field validation through trapping in Australia.

Approach & Outcomes

Pheromone extracts were obtained from a colony established after the discovery of the insect in Auckland. Coupled gas-chromatography – electroantennogram studies, followed by GC-mass spectrometry confirmed the presence of four electrophysiologically-active compounds.

The two minor peaks were identified: Z9-11:14Ac and Z11-16:Ac. Male gum leaf skeletoniser antennae gave good responses to synthetic standards of both of these compounds. The two major peaks were hypothesised to be a hexadecadienyl (Mr238.41) and a hexadecadienyl acetate (Mr 280.45). Both dienic compounds have a range of possible isomers. The range was narrowed with the confirmation that the positions of the double bonds in the two major compounds are at positions C10 and C12. Dienes with E,Z configuration were obtained.

Lures were prepared with four replicates of 12 blends plus a blank, and tested in HortResearch delta traps in Australia, at a rural side 3 hr north of Brisbane (Goomeri), where the grazier had indicated outbreaks to have been reported previously. Insect damage potentially caused by U.lugens was found, but no larvae were located and light trapping was unsuccessful. Traps were also deployed closer to Brisbane. Additional traps were sent to Brisbane, but lures labelled “Research value only” were returned by Australian customs. A second shipment was also significantly delayed. Despite these problems, the project has made recent and significant progress.

A total of 11 specimens of U.lugens (Noctuidae:Nolinae) have now been caught at Goomeri, Queensland and at Brisbane Forest Park, and the genitalia are consistent with specimens from Tasmania and Western Australia. Significant numbers (n-175) of a second moth genus (“Nola” spp., also in the family Nolinae) has also been caught at one site, and the genitalia mounted for taxonomic identification. This result indicates the potential for identification of pheromones of Australian moths by operating traps with a range of likely baits.

The results for U.lugens indicate that

1. A blend of the pheromone containing necessary and sufficient components has been identified, but probably not yet optimised.
2. The low numbers suggest that trapping effort has generally not been conducted where the insect is present in high density and/or in the correct life stage. Trapping with the existing lures is continuing.

The next stage of the project requires further testing of the alternative isomers and ratios.

2.7 MBS 361

Programme Title:	DNA diagnostic procedures for the identification of selected species and populations of Lymantria and Orgyia moths from intercepted egg masses
Programme Leader:	Dr Karen Armstrong
Institution:	Lincoln University

Summary

This programme aimed to validate a previously developed DNA diagnostic test for identifying immature life stages intercepted at the border and investigate extending the capability to distinguish populations of gypsy moth (L. dispar), pink gypsy moth (L.mathura) and the nun moth (L.monacha) that have different quarantine attributes.

Genetic markers for O. thyellina were determined by DNA sequence analysis and PCR-RFLP. Method validation followed all procedures in the manual (Version 1, MBS 301, 1999/2000) and used specimens intercepted by MAF at the major ports. Population genetic markers were investigated using PCR at four microsatellite loci plus sequence and PCR-RFLP analysis of the ITS1, ITS2, 168 and COI DNA regions.

The DNA diagnostic procedure to identify lymantria species from egg masses has been updated to include markers for O. thyellina and procedures for poor quality specimens. The method is now fully operational. Since August 2000, 93% of Lymantriid interceptions on Japanese used car imports have been identified to the species level, which effectively compares to zero prior to this research. The data confirm that L. dispar is the most common species from Japan to reach the New Zealand border. Population-level variation was detectable in mitochondrial and nuclear DNA regions for L. dispar, L. monacha and L. mathura, but as yet there is no clear correlation with ‘race’ or geographic origin. Further specimen collection for development of the population genetic database is necessary before statistical multilocus assignment tests can be employed to determine population origins. The main recommendations from this research are that:

• The revised version of the manual (VII/2001) be adopted in place of the original
• The molecular identification of Lymantriid species be extended to include selected Southern Hemisphere species.
• Collection and analysis of Asian and European moths is continued to take advantage of the genetic markers identified here for evaluating populations of quarantine significance

Goal:

To validate the previously developed DNA diagnostic test for identification of immature life stages intercepted at the border and investigate extending the capability to distinguish populations of gypsy moth (L.dispar), pink gypsy moth (L.mathura) and the nun moth (L.monacha).

BACKGROUND

Previous research in this programme (MBS301, 1999/2000) resulted in the design of a rapid molecular method for identifying egg masses from six high-risk Northern Hemisphere lymantria species. Markers were required for a seventh species, O. thyellina, for which accurately identified specimens had not been obtainable. A validation period was also desirable before implementing the method as a routine diagnostic tool. A second objective was to determine if populations important to quarantine and risk analysis processes could be genetically distinguished particularly Asian and European forms of L. dispar and the recently observed different pheromone types of L. mathura and L. monacha.

Approach & Outcomes

Genetic markers for O. thyellina were determined by DNA sequence analysis and PCR-RFLP. Method validation followed all procedures in the manual for PCR-RFLP of the ITS1 nrDNA (Version 1, MBS 301, 1999/2000) and used specimens intercepted by MAF at the major ports. Population genetic markers were investigated using PCR at four microsatellite loci plus sequence and PCR-RFLP analysis of the ITS1, ITS2, 16S and COI DNA regions.

Completion of markers and method validation: Diagnostic PCR-RFLP patterns for O. thyellina were determined and a validation process undertaken with specimens found on imported Japanese used cars during August-December 2000. Of fifty-six immature life stage specimens, the majority were easily identified (40 L. dispar and one O. thyellina) without modifications. The remainder were larval exuviae, pupal cases and eggs in very poor condition that failed to PCR amplify, presumably due to extreme degradation or lack of DNA. A set of species-specific PCR primers for small amplicons was therefore designed and the level of identifications to the species level improved from 75% during the validation period to 93% to date (n=151); this compares to zero identification to the species level prior to this research. The remaining unidentified specimens were exuviae or of other species not characterised during method development. The DNA diagnostic procedure is now fully operational and the data confirm that L.dispar is the most common species from Japan to reach the New Zealand border. This procedure is easily upgradeable to include other species, for example from the Southern Hemisphere.

Population markers: Seventy five L.dispar specimens were successfully typed at four microsatellite loci using a novel and simple, non-polyacrylamide electrophoresis system. Some cross-species reactivity was observed for L.monacha and L. mathura at three of the loci. Polymorphisms in the ITS1, ITS2, COI and 16S that fell on a restriction site were screened across populations; others that could be analysed using SSCP were not evaluated during this programme. Using the available markers there was no clear distinction between Asian and European gypsy moths; this is consistent with the growing body of information on their overlapping genetic, morphological and behavioural characteristic. There were also no clear differences between L. mathura from Japan compared to Korea or L. monacha from Japan compared to Europe. Further specimen collection for development of the genetic database is necessary before statistical multilocus assignment tests can be considered for determining population origins, Collections may be possible through USDA-ARS collaboration, but dependency on other outside agencies in the past has hindered progress in this programme. An organised collection from New Zealand, targeting appropriate geographic regions would be more effective.

2.8 MBS 362

Programme Title:	Soil attached to shipping containers is a potential biosecurity risk to New Zealand – the case for Fusarium Oxysporum
Programme Leader:	Dr John Marshall
Institution:	NZ Institute for Crops & Food Research Ltd

Summary

In an earlier study (Marshall & Varney 2000) Crop & Food Research assessed the quarantine risks posed by the soil attached to shipping containers entering New Zealand. The work involved collecting samples of soil from containers, analysing the samples to detect a predetermined range of organisms, and comparing results to similar organisms in New Zealand soils. It was concluded that soil attached to shipping containers is a potential pathway for pests and diseases to enter New Zealand.

In this continuation of the first study, a single fungal species considered to be a representative and highly variable fungal pathogen of New Zealand agriculture, Fusarium oxysporum, was selected for further examination. The extent of genetic diversity between F. oxysporum isolates from soil attached to incoming shipping containers (from the 2000 study) and F. oxysporum isolated from New Zealand soils in this study were compared by molecular analysis (RAPD and ITS nucleotide sequence comparison).

Four major grouping of F. oxysporum were determined by ITS genetic analysis, with each major group containing both shipping container isolates and New Zealand soil isolates. This suggests that there is little genetic variability between F.oxysporum isolates from shipping containers and New Zealand soils based on ITS genetic analyses.

RAPD analysis was carried out to provide clearer resolution of ITS sequence analysis. RAPD results showed more genetic variability between the shipping container isolates and New Zealand isolates than was evident from ITS genetic analyses. Phylogenetic analysis of RAPD results showed distinct groupings of DNA from both shipping container F. oxysporum isolates and New Zealand soil F. oxysporum. This observation suggests that the shipping container isolates contain some genetic material not presently found in New Zealand F. oxysporum isolates.

To determine whether this new genetic variability could contain potential virulence factors, pathogenicity assays were carried out on F. oxysporum isolates from both shipping containers and New Zealand soils. Results of inoculating F. oxysporum isolates onto pea seedling roots indicated that both New Zealand and shipping container isolates of this organism exhibited the potential to cause disease symptoms (root rot). The results of this study indicate that F. oxysporum species arriving in New Zealand on shipping containers showed similar disease potential to New Zealand soil isolates and, therefore, that soil attached to shipping containers is unlikely to pose an immediate biosecurity risk to New Zealand

Background

The rolled towel bioassay was sourced from an MSc thesis that dealt with root rot of pea. The researchers in this case were attempting to re-isolate Alpanomyces eutiches from roots showing disease symptoms, but found that F. oxysporum was recovered at a high rate when diseased tissue was plated on agar. Results of the rolled towel bioassay carried out in this case revealed root rot symptoms. Pea seedlings were examined for symptoms of both root rot and wilt disease. Cross sections of roots were examined rather than external symptoms such as wilting and discoloration of leaves in order to rule out confounding factors such as environmental stress or the presence of alternative pathogens. Mycelia adherence to root systems during seedling inoculation may have contributed to higher than actual values being recorded in the case of the subjective root rot analysis. Those roots, which were assigned a value of three, had lesions as well as discoloration and, therefore, it was clearer that disease symptoms were present. However, roots assigned a value of two were only scored on the basis of discoloration. Adherence of coloured mycelia may have made these appear more discoloured than they actually were.

It is evident from this assay that F. oxysporum isolates from soil associated with shipping containers are potentially pathogenic.

Approach & Outcomes

Fusarium oxysporum was selected for examination because it is considered a representative and highly variable fungal pathogen of New Zealand agriculture. The phylogenetic analyses created from ITS sequence and RAPD data revealed that there was genetic diversity between New Zealand and shipping container isolates of F. oxysporum. In light of this, organisms entering New Zealand in soil on the bottom of shipping containers may be pathogens with new genetic characteristics.

Pathogenicity testing revealed that isolates from both New Zealand and shipping container soils are capable of causing disease symptoms in pea plants. This suggests that organisms entering this country in soil associated with shipping containers have the potential to cause disease, but are not noticeably more pathogenic than F. oxysporum isolates already present in New Zealand.

The results of this study suggest that some F. oxysporum isolates entering New Zealand in soil associated with shipping containers from foreign ports may be genetically different to New Zealand isolates. However, they were not shown to have marked increase in disease potential over New Zealand isolates and, therefore, may not pose an immediate biosecurity risk to New Zealand agriculture.

2.9 MBS 331

Programme Title:	Investigation of the Suitability of NZ Forest flora for reproduction and development of Lynmantriid Species
Programme Leader:	Malcolm Kay
Institution:	Forest Research

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

2.10 MBS 333

Programme Title:	Lophodermium identification tools project.
Programme Leader:	Dr Peter Johnston
Institution:	Landcare Research

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

2.11 MBS 345

Programme Title:	Destroying feral bee colonies
Programme Leader:	Mark Goodwin
Institution:	HortResearch

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

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