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The grass grub endemic to New Zealand, Costelytra giveni (Coleoptera: Scarabaeidae), and the manuka beetle, Pyronota festiva and P. setosa (Coleoptera: Scarabaeidae), are prevalent pest species. Through assessment of bacterial strains isolated from diseased cadavers of these insect species, 19 insect-active Serratia proteamaculans variants and a single Serratia entomophila strain were isolated. When independently bioassayed, these isolates differed in host range, the rate of disease progression, and 12-day mortality rates, which ranged from 60 to 100% of the challenged larvae. A Pyronota spp.-derived S. proteamaculans isolate caused a transient disease phenotype in challenged C. giveni larvae, whereby larvae appeared diseased before recovering to a healthy state. Genome sequence analysis revealed that all but two of the sequenced isolates contained a variant of the S. entomophila amber-disease-associated plasmid, pADAP. Each isolate also encoded one of seven distinct members of the toxin complex (Tc) family of insect-active toxins, five of which are newly described, or a member of the extracellular contractile injection (eCIS) machine family, with a new AfpX variant designated SpF. Targeted mutagenesis of each of the predicted Tc- or eCIS-encoding regions abolished or attenuated pathogenicity. Host-range testing showed that several of the S. proteamaculans Tc-encoding isolates affected both Pyronota and C. giveni species, with other isolates specific for either Pyronota spp. or C. giveni. The isolation of several distinct host-specific pathotypes of Serratia spp. may reflect pathogen-host speciation. IMPORTANCE New pathotypes of the insect pathogen Serratia, each with differing virulence attributes and host specificity toward larvae of the New Zealand manuka beetle and grass grub, have been identified. All of the Serratia proteamaculans isolates contained one of seven different insect-active toxin clusters or one of three eCIS variants. The diversity of these Serratia-encoded virulence clusters, resulting in differences in larval disease progression and host specificity in endemic scarab larvae, suggests speciation of these pathogens with their insect hosts. The differing virulence properties of these Serratia species may affect their potential infectivity and distribution among the insect populations. Based on their differing geographic isolation and pathotypes, several of these Serratia isolates, including the manuka beetle-active isolates, are likely to be more effective biopesticides in specific environments or could be used in combination for greater effect.

An innovation systems approach involving agribusiness representatives, researchers and farmers identified that damage caused by grass grub (Costelytra giveni) was a key factor contributing to areas of reduced yield within high-producing pastures. Using a recognised yield mapping technique, areas of ‘Low’ and ‘High’ pasture height were identified in different paddocks over 3 years; ‘Low’ areas had significantly higher numbers of grass grub larvae than ‘High’ areas. Pasture production was measured for the ‘Low’ and ‘High’ pasture height areas, and the difference was calculated to be 6800 kg DM/ha for 2018/19. This difference persisted after grass grub larvae were no longer active. Farm systems modelling analysis estimated this ‘yield gap’ led to a $650/ha/year difference in profit. A survey of farmer perceptions of grass grub damage on 23 central Canterbury dairy farms estimated 19% of pastures were affected by grass grub, with 11% of the area in those paddocks being damaged. Econometric modelling showed differences in farmer perceptions of grass grub damage. Data from the survey and the pasture measurements were combined with the farm systems modelling results to estimate a $1,870,000/year regional-scale impact of the ‘yield gap’. An integrated pest management approach is suggested to control grass grub damage.

Some species in the genus Pyronota are known to be serious pests of pasture. The establishment of new dairy pastures on recently flipped soils on Cape Foulwind, Buller provided a unique opportunity to investigate the flight behaviour and biology of Pyronota festiva and P. setosa from 2008—2012. Flight behaviour was monitored by fixed-vane interruption traps and by field observations. Adults of both species flew through late spring/early summer (Nov—Dec) of each year and seasonal flight timing was consistent between years. Pyronota festiva flight timing and duration were similar to those previously reported in the literature and were slightly female dominated. Pyronota setosa flights started one to two weeks in advance of P. festiva and were dominated by males (>95%). Female P. setosa were rarely observed flying and were rarely caught in sweep-net sampling. Pyronota setosa beetles were never observed swarming or congregating and feeding on the foliage of mānuka. These differences in flight behaviour may help to explain the field observations that P. festiva can rapidly invade new pastures while P. setosa populations appear to spread more slowly.

The nonspore-forming bacterium Serratia entomophila may be used to control the New Zealand grass grub (Costelytra giveni) but is sensitive to environmental stress and must be formulated to ...

BACKGROUND Porina is the common name used to describe moths and caterpillars of the seven endemic species and three haplotypes of Wiseana in New Zealand. Several species have adapted to eating introduced pasture plants, however a paucity of defining morphological characteristics has meant that porina are grouped as indistinguishable species within a complex. This study aimed to develop non-sequencing identification methods for porina species. RESULTS We sequenced 1, 718 bp of the mitochondrial cytochrome oxidase genes for all Wiseana species and haplotypes using 75 specimens collected from 42 sites throughout New Zealand. Two identification methods were developed; the high resolution melt method uses 106 bp of the cytochrome oxidase I gene, and the restriction fragment polymorphism method uses a larger 700 bp region from the same gene. Validation was performed on a further 275 specimens. Melt curve profiles varied more with population heterogeneity than digest results. In both methods, W. mimica and W. jocosa were inseparable. W. fuliginea grouped with W. mimica and W. jocosa in the restriction method, but the melt curve profile differed. CONCLUSION Until now, ecological studies of porina at the species level have been implausible. Our non-sequencing based methods allow rapid identification of moths and caterpillars to species and haplotype level, paving the way for ecological studies of pasture pest species and the development of species-specific mitigation strategies.

BACKGROUND Endemic New Zealand grass grub Costelytra zealandica is a pest of introduced pasture that uses phenol as a sex pheromone. The pheromone could be used to monitor and manage grass grub populations, but the irritating properties and toxicity of phenol for human handlers, as well as the possible ecotoxicological effects, pose obstacles to the deployment of the pheromone. This study aimed to limit the use of phenol by dose-response studies and investigation into alternative attractants and synergists to phenol. RESULTS No difference in trap catch was seen across the range of 1-100 mg of phenol, while rates below this (0.001-0.1 mg) caused a large drop in catches. Our results indicated that 1 mg loading in lures was enough to indicate beetle presence over 1 week. 4-Hydroxybenzaldehyde and p-cresol proved unattractive in this study, both as single attractants and as synergists with phenol. Phenyl acetate, phenyl benzoate and diphenyl carbonate all formed phenol under hydrolytic conditions to act as successful propheromones, while phenyl acetate was found to be as attractive as phenol on its own. CONCLUSION This study described several ways to reduce or avoid the use of phenol in the field while maintaining lure effectiveness. © 2017 Society of Chemical Industry.

The bacterial biocontrol agent Serratia entomophila and the insecticide diazinon were applied as separate granular formulations with ryegrass seed and compared with a seedonly control treatment on three pastures of different ages and composition on the North Island volcanic plateau In the first 2 years diazinon and S entomophila significantly reduced healthy grass grub populations compared with the control However by the third year populations in the diazinon treatments had recovered and were significantly higher than in S entomophila or control plots Grass grub populations were reduced by disease outbreaks after S entomophila was applied which infected >40 of grass grub larvae in the treated plots in year two Bacterial extraction from soil a year after application confirmed establishment and persistence of S entomophila in treated plots Visual positive pasture growth responses were noted in both the S entomophila and diazinontreated plots

Birds and other forms of wildlife are a major issue for airport authorities worldwide, as they can create hazards to operating aircraft. Wildlife "strikes," the majority caused by birds, can cause damage to operating aircraft and in severe cases lead to a loss of human life. Many airfields contain large areas of ground cover herbage alongside their runways that consist of mixtures of grasses, legumes, and weeds that can harbor many invertebrates. Many airfields use insecticides to control insect populations; however, mounting pressure from regional councils and water boards aim to reduce this practice due to ground water runoff and contamination concerns. Avanex Unique Endophyte Technology, a product specifically developed to reduce the attractiveness of airports and surrounding areas to birds, is based on a novel association between a selected strain of Epichloë endophyte and a turf-type tall fescue cultivar. This grass-endophyte association acts through a direct mechanism whereby a negative response in birds is created through taste aversion and postingestion feedback as well as an indirect mechanism by deterring many invertebrates, a food source of many bird species.

Ostenia robusta (Hutton) is an endemic dolichopodid fly that sometimes occurs in the soil under agricultural crops and pastures. Little is known about its biology and the role it may play in agro-ecosystems. A population of these flies near Southbridge, Canterbury, was observed from 2013 to 2015. Adult flies emerged between December and March each year and were monitored with yellow sticky traps placed in wheat, ryegrass, radish and white clover crops. Adults will drink water and feed on 10% honey solution under laboratory conditions but no predatory behaviour was observed and attempts to induce oviposition were unsuccessful. From dissection, maximum female egg load was approximately 60-65 eggs. Very few O. robusta larvae were found in 2014 and none were associated with pupae of Costelytra zealandica (White), a result contradictory to 2012 and 2013. The diet range of larval O. robusta remains unknown, but this predator appears unlikely to play a significant role in the control of C. zealandica.

Outbreaks of insect pests can cause major losses in pasture productivity, but farmers are often poorly equipped to evaluate risk factors, predict pest impacts and determine appropriate control actions. Decision tools are described for pasture pest management using three case studies. For grass grub, "at risk" paddocks are identified based on factors such as time since sowing, soil type and larval damage the previous autumn/ winter. Targeted populations measurements can then be taken and a decision to control grass grub made using this objective measurement and/or an assessment of the pasture value, termed the insurance approach. For manuka beetle, pesticide application decisions are based on damage scores of individual paddocks and the correlation between these scores and larval densities. With porina, information on moth flight times, larval populations and pasture damage are used to make key control decisions. For all pests, decisions to control should be made in conjunction with other farm systems information, including the costs of lost forage production and long-term impacts on pasture persistence. Keywords: grass grub (Costelytra zealandica), porina (Wiseana spp.), manuka beetle (Pyronota spp.), farmer decision-making

African black beetle, Heteronychus arator (Scarabaeidae), is an exotic pest of pastures in northern New Zealand. Both adults and larvae feed on pasture grasses. Adults disperse by walking (short range) or flying (long range). Dispersal flights are triggered by warm night temperatures in spring and autumn. Short range adult dispersal in search of mates, food or oviposition sites is poorly understood. This study investigated walking activity of H. arator adults over three seasons in New Zealand pastures. Adult walking activity was monitored using pitfall traps along fence lines and in pasture plots on a dairy farm in Waikato, New Zealand, in spring 2013, spring 2014, and autumn 2015. Beetle populations were reduced by application of a biopesticide bait to compare walking activity between treated and control plots for up to 26 days post-treatment. Marked beetles were released into the pasture plots to measure the distance traveled by recaptured individuals. Trap catches along the fence lines were correlated with air temperatures in 2013. Trap catches were male biased in spring 2014 compared with autumn 2015. Trap numbers in the control plots were nearly double that of treated plots in both seasons. More beetles were caught in the pitfall traps at the edges of the treated plots than in the center. Trap catches were consistent throughout the control plot in spring 2014, but in autumn 2015 more beetles were caught in the center of the control plot than at the edges. Few marked beetles were recaptured with dispersal rates estimated as

Costelytra zealandica (Coleoptera: Scarabeidae) is a univoltine endemic species that has colonised and become a major pest of introduced clover and ryegrass pastures that form about half of the land area of New Zealand. Female beetles were previously shown to use phenol as their sex pheromone produced by symbiotic bacteria in the accessory or colleterial gland. In this study, production of phenol was confirmed from the female beetles, while bacteria were isolated from the gland and tested for attractiveness towards grass grub males in traps in the field. The phenol-producing bacterial taxon was identified by partial sequencing of the 16SrRNA gene, as Morganella morganii. We then tested the hypothesis that the phenol sex pheromone is biosynthesized from the amino acid tyrosine by the bacteria. This was shown to be correct, by addition of isotopically labelled tyrosine ((13)C) to the bacterial broth, followed by detection of the labelled phenol by SPME-GCMS. Elucidation of this pathway provides specific evidence how the phenol is produced as an insect sex pheromone by a mutualistic bacteria.

The diversity of Coleoptera communities in tussock grassland at two sites in Otago, one in Canterbury and one in the central North Island, was compared. The impact of agricultural disturbance on the communities was compared between native tussock, tussock oversown with exotic pasture species and cultivated sown pasture. Coleoptera were heat-extracted from turf samples taken in summer in two consecutive years. In native and oversown treatments, Staphylinidae and Curculionidae predominated in abundance at three of the four sites, and in species richness at all sites and treatments. Carabidae were more species-rich than Curculionidae in cultivated treatments at most sites. The mean density of Coleoptera in native tussock treatments ranged between 654/m2 and 97/m2. Carnivores were the predominant trophic group followed by herbivores. Species diversity was higher in the native or oversown treatments and the Otago sites were the most diverse overall, and the least disturbed. A total of 19 exotic specie...

The entomopathogenic bacterium Yersinia entomophaga MH96 (Ye MH96) was produced by aerobic fermentation and applied in laboratory and field bioassays to control bronze beetle (Eucolaspis sp.). In the laboratory, bacteria were applied to apple leaves at a range of cell densities using a Potter Tower. After 6 days of exposure, the LC50 of field- collected beetles was calculated as approximately 5.0×10 6 cells/cm 2 of leaf surface with high concentrations killing all treated adults. Time to death was inversely correlated with dose rate with the LT50 at the highest dose being 3.6 days. Marked apple shoots were sprayed in the field, each shoot enclosed in a sleeve cage and healthy beetles added. After 7 days, beetle mortality averaged 42% following a single application and 66% after multiple treatments. Leaf damage was reduced by bacterial application. The results show that Ye MH96 has potential for development as a biopesticide against this intractable pest of organic apple production.

New Zealand pastures are host to a range of native and exotic invertebrates. Many of these are pests that feed on the sown plant species, often causing plant death and deterioration in the productivity and persistence of the sward. While most research has focused on pest biology and control, studies of plant productivity show dramatic acute (short-medium term) and chronic (longterm) effects. For example, grass grub has been reported to cause 50% losses to ryegrass swards and Argentine stem weevil can cause a 20% loss of productivity, but the long term effects through loss of the sown species and changes in composition can be even more severe. Insect damage can be exacerbated by other biotic (e.g. weed invasion, grazing animals, plant pathogens) or abiotic factors (e.g. climate, soil fertility) to further reduce plant persistence. Some biotic and abiotic factors may interact with insect pests to have positive effects on pasture persistence, e.g. biocontrol agents that reduce pest density or addition of fertiliser to enable plants to recover from insect damage. For many pasture pests, knowledge of their ecology is based on research from over 20 years ago that, while still valid, needs to be updated to account for modern intensive pasture management practices, such as intensive grazing with a lack of reseeding, and new cultivars. To improve pasture persistence, integrated research programmes are needed that examine the relative importance of all factors contributing to plant survival and develop strategies to mitigate their effects. Keywords: Argentine stem weevil, black beetle, clover root weevil, drought, grass grub, grazing pressure, manuka beetle, nematodes, pasture persistence, pasture quality, pasture yield, porina, soil fertility, striped chafer.

An outbreak of the pasture insect pest black beetle began in the Waikato and Bay of Plenty in 2007/8 and has persisted. The extent and severity of damage caused by black beetle during the current outbreak has focused farmer and researcher attention on methods to maintain persistent pasture now and in future outbreaks. This paper reviews previous research in combination with data from the current outbreak and relates these to current pasture management practice. The possibility of being able to predict the distribution, occurrence and duration of black beetle outbreaks is explored while actual and potential means of controlling black beetle are outlined. We conclude that there are methods available to successfully renew pastures in the presence of black beetle but that outbreak situations increase risks and may limit subsequent pasture persistence. There are fewer readily available options to maintain an existing pasture and more research is urgently needed to provide these options. Keywords: Heteronychus arator, paspalum, pasture, pasture renewal, pest resistance, pest tolerance, ryegrass

Manuka beetles (Pyronota sp.; Scarabaeidae) are serious and persistent pests of dairy pastures on Cape Foulwind, Westport. When a selection of scarab-active fungal isolates were tested against 3 rd instar larvae of two Pyronota species, a locally sourced Beauveria brongniartii (F636) isolate consistently achieved the fastest and highest levels of larval mortality. Topical application of F636 caused an average of 80% larval mortality 6 weeks post-treatment. Mortality was shown to be dose rate dependent for both Pyronota species. When treatments were applied by incorporating rice grains colonised by the fungus into soil, simulating field application, F636 again produced the fastest and highest levels of larval mortalities, averaging 70% 6 weeks post-application. Mortalities of both Pyronota species reached 100% after 8 weeks when the assay medium was a grey sand based soil (ex Cape Foulwind). Isolate F636 shows promise as a biological control agent for this pest and field trials have been carried out in the autumn of 2010.

The entomopathogenic bacterium, Serratia entomophila, is an alternative to chemical control of grass grub (Costelytra zealandica) and is applied in a granule formulation to established pastures. Treatment of seed with microbial inoculants is an ideal mechanism for delivery and establishment of microbial control agents into the plant root zone where soil dwelling pests, such as grass grub, are located. Seed treatment with S. entomophila was tested in three glasshouse pot trials, for its ability to protect germinating ryegrass seedlings from grass grub damage. A range of larval densities was used and microbial seed treatment was compared with the insecticide imidacloprid. At medium larval densities (equivalent to 70 larvae/m 2 ), use of S. entomophila-coated seed resulted in 85% seedling establishment in comparison with 82% emergence from imidacloprid-treated seed. At a high larval density of 300/m 2 , where there was no establishment of untreated seed, 35-51% of seedlings established from S. entomophila-treated seed. Results suggest there is potential for seed coating to aid ryegrass establishment in autumn-sown pastures.

Phenol the attractant pheromone of adult males of the native New Zealand grass grub Costelytra zealandica (White) is produced in the beetles as the result of bacterial degradation of tyrosine A lure consisting of a resin impregnated with phenol has been widely used to monitor male beetle flight activity The present formulation is highly attractive for the first week in the field but then loses activity rapidly A number of phenolcontaining formulations were tested to improve the lure A new formulation gave lower catches that were more stable with time producing data more suitable for population density estimation Phenylacetaldehyde a bacterial metabolite of phenylalanine was tested as a possible synergist to phenol Field results showed that this floral compound exhibited no behaviourallyactive properties to grass grubs when tested together with phenol Catches with water traps were compared with those in sticky delta and flat delta traps in two vineyards and in pasture Water traps caught four times more beetles

Microfabricated systems have recently become useful for routing particles to precise locations in microfluidic channels. In this paper we discuss the modeling, fabrication and characterization of such a platform that combines acoustic forces and ac dielectrophoresis (DEP). This system integrates a bulk lead zirconate titanate (PZT) slab with substrate patterned microelectrodes for DEP manipulation of particles. Moreover, a one-dimensional transmission line model is presented to understand the coupling of the acoustic and dielectrophoretic transducers with the microdevice. While the acoustic model does not predict the lateral coupling in the system, it does provide some insight into axial (thickness-mode) frequencies of operation. Experiments are also conducted in which particles were routed into a large (0.75 mm wide) microchannel and preconcentrated and focused into coarse bundles by coupling an acoustic wave into the channel. Subsequently, particles are further focused into single file particle streams using interdigitated DEP electrodes. This system can be used for high throughput assays for which it is necessary to isolate and investigate small bundles of particles and single particles.

In the course of a series of investigations into the biodiversity of indigenous tussock grasslands, penicillia and aspergilli were isolated from soil samples from four locations: Deep Stream and Mt Benger in Otago, Tukino in the central North Island and Cass in Canterbury. Samples were collected in the summers of 2003 and 2005 and, at each locality, were taken from sites under indigenous tussock grassland, under oversown tussock grassland and under pasture cultivated from tussock grassland. Isolations were made on potato-glucose-chlortetracycline agar plates inoculated with soil suspensions. Thirty-six Penicillium species were recovered, all cosmopolitan, and of the 12 most common, 10 species: P. canescens, P. janthinellum, P. janczewskii, P. jensenii, P. lividum, P. loliense, P. melinii, P. miczynskii, P. sclerotiorum and P. simplicissimum were isolated from samples from all four locations but P. montanense and P. novae-zeelandiae were not recovered from Deep Stream or Mt Benger. P. lividum and P. sclerotiorum were recovered mainly from samples from indigenous sites and P. jensenii from oversown or cultivated sites. In other countries, some of these species have been associated with forest-grassland rather than grassland soils and it was thought possible that they were relics associated with pre-human forest vegetation which may have covered the locali- ties. At the subgenus, not species, level, however, the pattern was typical of grassland, not forest, soils, with the subgenus Furcatum dominant. Six of the common species are known to have the potential to produce tremorgens, neurotoxins known to cause incoor- dination when ingested by mammals but with little investigated effects on invertebrates. Aspergillus cervinus was the only Aspergillus species recovered, only from Tukino and principally from samples from the indigenous sites.

In a microfluidic ultrasonic separator device, the microchannel geometry is inherently angular due to the anisotropic etch processes used. A CFD model is used to investigate eddy formation induced by this angular geometry. Results are applicable to various microfluidic devices and show that certain etch patterns significantly reduce eddy formation.

An ultrasonic microfluidic particle manipulator has been modelled and its experimentally measured separation performance has been compared with the modelled results for 1um latexparticles and yeast particles in water.

The capture of Bacillus subtilis var. niger spores on an antibody-coated surface can be enhanced when that coated surface acts as an acoustic reflector in a quarter wavelength ultrasonic (3 MHz) standing wave resonator (Hawkes, J.J., Long, M.J., Coakley, W.T., McDonnell, M.B., 2004. Ultrasonic deposition of cells on a surface. Biosens. Bioelectron. 19, 1021–1028). Immunocapture in such a resonator has been characterised here for both spores and 1 μm diameter biotinylated fluorescent microparticles. A mean spatial acoustic pressure amplitude of 460 kPa and a frequency of 2.82 MHz gave high capture efficiencies. It was shown that capture was critically dependent on reflector thickness. The time dependence of particle deposition on a reflector in a batch system was broadly consistent with a calculated time of 35 s to bring 95% of particles to the coated surface. A suspension flow rate of 0.1 ml/min and a reflector thickness of 1.01 mm gave optimal capture in a 2 min assay. The enhancement of particle detection compared with the control (no ultrasound) situation was ×70. The system detects a total of five particles in 15 fields of view in a 2 min assay when the suspending phase concentration was 10 4 particles/ml. A general expression for the dependence of minimum concentration detectable on; number of fields examined, sample volume flowing through the chamber and assay time shows that, for a practical combination of these variables, the threshold detection concentration can be two orders of magnitude lower.

Soil samples were taken from around the base of tussock plants and paired areas of inter‐tussock vegetation at four native tussock grassland sites across New Zealand, in three consecutive summers. Seventy nematode taxa were identified with the plant associated nematode trophic group being the most abundant at three of the four sites. Of the plant parasitic nematodes, Criconema, Pratylenchus, and Helicotylenchus spp. were found at all sites, with Hemicycliophora, Longidorus, a putative Punctodera, and Rotylenchus found at only one site each. The observation of mermithid nematodes at three of the four sites, coupled with absence of insect parasitic Steinernema or Heterorhabditis sp. from microscopic observations and soil baiting suggests that the insect parasitism niche is occupied largely by mermithids in the tussock grassland habitats studied. A range of community indices were calculated, with Shannon‐Weiner (2.10–2.39), Simpson dominance (0.129–0.205), Maturity Index (2.67–2.92), and Structure I...

Bacterial and fungal communities in indigenous tussock grassland soils were studied at four locations, Mt Benger and Deep Stream (Otago), Cass (Canterbury) and Tukino (central North Island). Soil samples collected from inter‐tussock and tussock areas were used to enumerate total culturable bacteria (colony forming units (CFU)) as well as a number of specific groups of bacteria, fungal types and diversity, and soil microbial functional diversity. Soil microbial biomass carbon (C) and nitrogen (N) as well as extractable C and N were also determined. Fungal populations were lowest at Mt Benger while fluorescent Pseudomonas was lowest at Cass. In these indigenous soils, bacterial CFU were c. 30 times lower and fungal populations c. 10 times greater than in developed New Zealand pastoral soils. CFU of bacteria in complex media (r‐strategists) were similar in magnitude to bacteria growing on weak CA medium (K‐strategists). The microbial biomass C in indigenous grassland soils were also significantly gr...

This contribution introduces a short series of papers on biodiversity of indigenous tussock grassland at four sites in New Zealand. A survey of invertebrates of tussock grassland sites was carried out in summer of 3 successive years, 2001–03. The sites included two in Otago in snow tussock grassland at Deep Stream and Mt Benger, a site at Cass in inland Canterbury, and one at Tukino in the central North Island. Sampling was carried out by taking turf samples from each site, and extracting the invertebrates with heat extractors. The invertebrates were divided into 30 major groups. This quantitative sampling method allowed the densities of invertebrate groups to be calculated. Total macro‐invertebrate density for all sites ranged between c. 1600 and 5600 m–2. Total invertebrate abundance was highest at the central North Island site, but this was very much dominated by Formicidae. Across all sites, Formicidae, Coleoptera, and Pseudococcidae were consistently the most abundant groups represented.

The bacterium Serratia entomophila is a naturally occurring pathogen causing amber disease of the New Zealand grass grub (Costelytra zealandica) A novel granular formulation of S entomophila Bioshieldtrade; was applied to 18 pasture sites in a largescale programme to demonstrate efficacy against grass grub No significant difficulties were encountered in application of the granules through conventional machinery There were high populations of the applied bacteria in soil within the first week of application and within 6 weeks of application there was an average of 3 x 104 viable S entomophila of the applied strain per gram of soil This resulted in a significant (Plt;005) 20 increase in the incidence of amber disease in the treated grass grub populations compared to untreated populations Successful establishment of the bacteria in the soil and target population following Bioshieldtrade; granule application was demonstrated on a wide range of sites under various farming conditions

Grass grub (Costelytra zealandica) population density, mean larval weight and visible damage were lower in meadow fescue (Festuca pratensis) infected with the endophyte Neotyphodium uncinatum (E+) than in uninfected meadow fescue (E‐) in an unreplicated field trial. In two bioassays, third instar grass grubs ate all meadow fescue E‐ roots but significantly less of the E+ roots. Larvae fed E+ roots lost weight at the same rate as unfed control larvae. Larvae given a choice between maize and either E+ or E‐ meadow fescue in a pot trial consumed 33% more of the maize in the E+ treatment than in the E‐ treatment. Weight gain of larvae in E+ treatments was significantly less than in E‐ in both the choice and no-choice pot trials, but survival was the same. It was concluded that meadow fescue infected with N. uncinatum deters grass grub larval feeding but has no major toxic effects.

Microbial control agents targeting soildwelling organisms need to be compatible with commonly used fertilisers The bacterium Serratia entomophila is used as a microbial control agent for control of the New Zealand grass grub Costelytra zealandica and Beauveria bassiana is an entomopathogenic fungus used to control a range of insect pests These biocontrol agents were formulated into granules and applied to pots together with five fertilisers commonly used on pastures throughout New Zealand Compatibility with S entomophila was also assessed in a field trial where treatments were applied by direct drilling and surface application There appeared to be no deleterious effect from the application of the fertiliser treatments on the establishment and survival of either S entomophila or B bassiana On the contrary there was a suggestion that some nitrogenous fertilisers may lead to an increase in numbers of the bacterial biocontrol agent

The bacterium Serratia entomophila has been incorporated into prill formulations to improve distribution and application of this insect pathogen to pasture for biological control of the New Zealand grass grub Costelytra zealandica While bacteria can survive in ambient conditions for long periods within the prills their field efficacy depends on successful release of bacteria from the prills and colonisation of the surrounding soil Laboratory experiments were conducted to determine the effects of soil moisture on rate of release of bacteria from prill formulations Prills were held in soil adjusted to four soil moisture contents (ranging from field capacity to wilting point) and the release of bacteria was determined by dilution plating Bacteria were released most rapidly at high soil moistures and populations were maintained at 103104 colony forming units per g soil in most treatments Measurement of release of S entomophila from prills in soil cores subjected to various watering regimes demonstrated that free soil water is important for distributing bacterial inoculum throughout soil profile

Porina (Wiseana spp. Viette (Lepidoptera: Hepialidae)), regularly damage pastures on the South Island’s West Coast but local information useful for mitigation strategies is lacking. Flight activity of porina moths was monitored on 11 farms in the region using light traps over three summers from 2014—2017. Pastures on the same farms were sampled during winter (2015—17) to collect porina larvae. Porina moth flights typically began in October and continued through into March with peak flight activity from early December to mid-January. Wiseana copularis comprised >85% of moths captured. Wiseana umbraculata was caught regularly, representing about 10% of total catch. Observed flight patterns indicated that diflubenzuron for larval control should be applied from late February to early or mid-March for maximum impact on porina populations but exact timing should be driven by local not regional flights. Wiseana copularis was the most common species collected in pastures (265 of 267 larvae collected). One W. jocosa larva was found in 2015 and one W. umbraculata larva in 2016. No W. cervinata adults or larvae were identified. This suggests the species primarily responsible for damaging these pastures is W. copularis.

Larvae of manuka beetles, Pyronota spp. (Coleoptera: Scarabaeidae) cause pasture damage in New Zealand by feeding on the roots of grasses. Surveys for potential biocontrol agents revealed a putative disease, expressed as whitened larvae of one of the outbreak species, Pyronota setosa. Microbial diagnosis indicated an intracoelomic, intracellular infection, and intracellular bacteria have been identified with subcellular structures characteristic of infection by Rickettsiella-like microorganisms. These bacteria were rod-shaped, often slightly bent with a mean of 628 nm in length and 220 nm in width. Numerous associated protein crystals of variable size and shape occurred within round to oval shaped “giant bodies” either singly or as clusters of smaller crystals. Molecular phylogenetic analysis based on 16S ribosomal RNA and signal recognition particle receptor (FtsY) encoding sequences demonstrates that the manuka beetle pathogen belongs to the taxonomic genus Rickettsiella. Therefore, the pathotype designation ‘Rickettsiella pyronotae’ is proposed to refer to this organism. Moreover, genetic analysis makes it likely that – on the basis of the currently accepted organization of the genus Rickettsiella – this new pathotype should be considered a synonym of the nomenclatural type species, Rickettsiella popilliae.

Ewes with lambs, and does with kids, were grazed together or separately at two stocking rates on irrigated perennial ryegrass (Lolium perenne) and white clover (Trifolium repens) pastures over three seasons (October—March). At both stocking rates, mixed grazing in a 1: 1 ratio produced lambs that were 2-3 kg heavier at weaning than those from a sheep-only system (P < 0.05) and lambs and ewes that were 4-6 kg heavier in March (P < 0.05). Does and kids grazed with sheep at the lower stocking rate, grew at the same rate as those grazed separately, but at the higher stocking rate, mixed-grazed does were 3-4 kg lighter. Fibre yields of sheep and goats were not significantly affected by mixed or separate grazing. Productivity per ha was greatest under high stocking. “Goat only” pastures were always the tallest and in summer had the most herbage mass and white clover. Stocking rate affected height and herbage mass but not botanical composition.

A series of devices have been investigated which use acoustic radiation forces to concentrate micron sized particles. These multi-layered resonators use a quarter wavelength resonance in order to position an acoustic pressure node close to the top surface of a fluid layer such that particles migrate towards this surface. As flow through devices, it is then possible to collect a concentrate of particulates by drawing off the particle stream and separating it from the clarified fluid and so can operate continuously as opposed to batch processes such as centrifugation. The methods of construction are described which include a micro-fabricated, wet-etched device and a modular device fabricated using a micro-mill. These use silicon and macor, a machinable glass ceramic, as a carrier layer between the transducer and fluid channel, respectively. Simulations using an acoustic impedance transfer model are used to determine the influence of various design parameters on the acoustic energy density within the fluid layer and the nodal position. Concentration tests have shown up to 4.4-, 6.0- and 3.2 fold increases in concentration for 9, 3 and 1 ?m diameter polystyrene particles, respectively. The effect of voltage and fluid flow rates on concentration performance is investigated and helps demonstrate the various factors which determine the increase in concentration possible.

Several approaches have been described for the manipulation of particles within an ultrasonic field. Of those based on standing waves, devices in which the critical dimension of the resonant chamber is less than a wavelength are particularly well suited to microfluidic, or "lab on a chip" applications. These might include pre-processing or fractionation of samples prior to analysis, formation of monolayers for cell interaction studies, or the enhancement of biosensor detection capability. The small size of microfluidic resonators typically places tight tolerances on the positioning of the acoustic node, and such systems are required to have high transduction efficiencies, for reasons of power availability and temperature stability. Further, the expense of many microfabrication methods precludes an iterative experimental approach to their development. Hence, the ability to design sub-wavelength resonators that are efficient, robust and have the appropriate acoustic energy distribution is extremely important. This paper discusses one-dimensional modelling used in the design of ultrasonic resonators for particle manipulation and gives example of their uses to predict and explain resonator behaviour. Particular difficulties in designing quarter wave systems are highlighted, and modelling is used to explain observed trends and predict performance of such resonators, including their performance with different coupling layer materials.

A series of devices have been investigated which use acoustic radiation forces to concentrate micron sized particles. These multi-layered resonators use a quarter-wavelength resonance in order to position an acoustic pressure node close to the top surface of a fluid layer such that particles migrate towards this surface. As flow-through devices, it is then possible to collect a concentrate of particulates by drawing off the particle stream and separating it from the clarified fluid and so can operate continuously as opposed to batch processes such as centrifugation. The methods of construction are described which include a micro-fabricated, wet-etched device and a modular device fabricated using a micro-mill. These use silicon and Macor, a machinable glass ceramic, respectively, as a carrier layer between the transducer and fluid channel. Simulations using an acoustic impedance transfer model are used to determine the influence of various design parameters on the acoustic energy density within the fluid layer and the nodal position. Concentration tests have shown up to a 6-fold increase in concentration for 3 and 9 μm diameter polystyrene particles and up to 4-fold for 1 μm particles. The effect of voltage and fluid flow rates on concentration performance is investigated and helps demonstrate the various factors which determine the increase in concentration possible.

The use of acoustic radiation forces for the manipulation of particles, bacteria and cells is drawing increasing interest. Resonators using quarter-wavelength sized fluid chambers have been developed, where an acoustic pressure node is positioned near the reflector layer surface such that particles may be pushed close to or onto this surface. The thickness of the reflector layer has already been shown to greatly influence the performance of such resonators. These devices have applications in bio-sensing where a concentrate of particulates can be collected or particulates can be contacted with a sensor surface directly. A coupling layer is usually incorporated into the device and is located between the transducer and fluid layer. The ability of this layer to act as an acoustic matching layer depends on its acoustic properties and thickness. Here, the performance of various carrier layer materials in a near quarter-wavelength device is investigated and compared using modelled and experimental data. Results indicate that where the carrier layer thickness is selected for optimum performance, the resonator performance is comparable for most high Q factor carrier layer materials. However, for some materials the performance of the resonator, is highly sensitive to the coupling layer thicknesses and in turn can constrain the flexibility of the acoustic design.

Within an acoustic standing wave particles experience acoustic radiation forces, a phenomenon which is exploited in particle or cell manipulation devices. When developing such devices, one-dimensional acoustic characteristics corresponding to the transducer(s) are typically of most importance and determine the primary radiation forces acting on the particles. However, radiation forces have also been observed to act in the lateral direction, perpendicular to the primary radiation force, forming striated patterns. These lateral forces are due to lateral variations in the acoustic field influenced by the geometry and materials used in the resonator. The ability to control them would present an advantage where their effect is either detrimental or beneficial to the particle manipulation process. The two-dimensional characteristics of an ultrasonic separator device have been modelled within a finite element analysis (FEA) package. The fluid chamber of the device, within which the standing wave is produced, has a width to height ratio of approximately 30:1 and it is across the height that a half-wavelength standing wave is produced to control particle movement. Two-dimensional modal analyses have calculated resonant frequencies which agree well with both the one-dimensional modelling of the device and experimentally measured frequencies. However, these two-dimensional analyses also reveal that these modes exhibit distinctive periodic variations in the acoustic pressure field across the width of the fluid chamber. Such variations lead to lateral radiation forces forming particle bands (striations) and are indicative of enclosure modes. The striation spacings predicted by the FEA simulations for several modes compare well with those measured experimentally for the ultrasonic particle separator device. It is also shown that device geometry and materials control enclosure modes and therefore the strength and characteristics of lateral radiation forces, suggesting the potential use of FEA in designing for the control of enclosure modes in similar particle manipulator devices. 2006 Elsevier B.V. All rights reserved.

An ultrasonic resonator has been microfabricated from layers of silicon and Pyrex. A fluid channel of approximately 200?m in depth is etched into the Pyrex and allows particles within the fluid to be moved by acoustic radiation forces into the pressure nodal planes of the ultrasonic standing wave. Depending on the required application this can be used to generate a particle-free fluid sample, to concentrate particles prior to analysis, or to move particles to a surface within the resonator to aid analysis. In previously published work this resonator has been driven using a thickness mode bulk piezoceramic. While this has provided reasonable performance, the adhesion of the piezoceramic plate to the silicon has proved both the least repeatable and the least reliable element of the fabrication process. It has also been a factor in the long-term failure of test devices. To overcome these issues, multilayer thickfilm printed actuators have been developed to replace the bulk piezoceramic. Thick-film processing offers an effective means of depositing active materials onto substrates, and the technique is compatible with the microfabrication process, allowing multiple actuators to be printed onto a wafer comprising multiple devices. A variety of structures has been tested on ceramic substrates and shown to provide acceptable acoustic outputs when compared with bulk transducers mounted on identical substrates. A two layer actuator provides a good performance without excessive complexity and this configuration has been used on the resonator. Further acoustic and flow modeling of the device is described, and this has been used both to improve the channel geometry and to select better operating conditions for the system. It is shown that the thick-film actuated device working at the new operating conditions provides significantly improved performance when compared with the bulk piezoceramic device, and in particular is able to offer a five-fold reduction in concentration for 1?m latex particles, which had previously proved difficult to manipulate successfully.

This paper describes a technique for replacing the conventional bonded bulk PZT transducer, commonly used in MEMS devices, with a screen printed equivalent. Previously, the piezoelectric activity available from screen printed PZT has been considerably lower than the bulk material, but recent developments in material composition and device structure have allowed screen printed structures to deliver powers equivalent to bulk devices. An actuator using the multilayer screen printed technique was designed for use in an ultrasonic flow-through separator, and its successful use is reported.

Ultrasonic standing waves can be used to generate forces on particles within a fluid. Recent work has concentrated on developing devices that manipulate the particles so that they are concentrated near the centre of the cavity. It is also possible to design a device that concentrates the particles at the wall of a cavity. This paper describes a device that has the capability of operating in several modes to allow concentration of particles at either the cavity wall or the centre of the cavity, depending on the driving frequency.

Within an ultrasonic standing wave particles experience acoustic radiation forces causing agglomeration at the nodal planes of the wave. The technique can be used to agglomerate, suspend, or manipulate particles within a flow. To control agglomeration rate it is important to balance forces on the particles and, in the case where a fluid/particle mix flows across the applied acoustic field, it is also necessary to optimise fluid flow rate.To investigate the acoustic and fluid forces in such a system a particle model has been developed, extending an earlier model used to characterise the 1-dimensional field in a layered resonator. In order to simulate fluid drag forces, CFD software has been used to determine the velocity profile of the fluid/particle mix passing through the acoustic device. The profile is then incorporated into a MATLAB model. Based on particle force components, a numerical approach has been used to determine particle paths. Using particle coordinates, both particle concentration across the fluid channel and concentration through multiple outlets are calculated.Such an approach has been used to analyse the operation of a microfluidic flow-through separator, which uses a half wavelength standing wave across the main channel of the device. This causes particles to converge near the axial plane of the channel, delivering high and low particle concentrated flow through two outlets, respectively. By extending the model to analyse particle separation over a frequency range, it is possible to identify the resonant frequencies of the device and associated separation performance.This approach will also be used to improve the geometric design of the microengineered fluid channels, where the particle model can determine the limiting fluid flow rate for separation to occur, the value of which is then applied to a CFD model of the device geometry.

The New Zealand grass grub (Costelytra zealandica) is distributed throughout the South Island but surprisingly has not been recorded west of Reefton In 2006 pasture damage from rootfeeding scarab larvae on the West Coast initially attributed to grass grub was found to be caused by manuka beetles Pyronota spp Winter surveys during 20082012 between Karamea and Hokitika confirmed that most damage patches were caused by manuka beetle larvae but there was a small localised population of C zealandica associated with Westport airport and golf course In 2012 a network of phenolbaited pheromone traps was established around this epicenter during the grass grub flight season with traps spaced at approximately 05 km intervals Traps within the identified zone of grass grub infestation caught 15 beetles per night Single male beetles were trapped up to 75 km from the epicenter but with no evidence of established populations from larval sampling It is likely that the localised grass grub population became established after an accidental introduction of insects with soil or plant material to the modified and drained airport and golf course environments and may act as an infestation source for other areas Pastures on the nearby newlyflipped land of Cape Foulwind may also be suitable for grass grub and should be regularly inspected to anticipate and prevent outbreaks

Manuka beetles (Pyronota festiva and P setosa) have become a major problem in dairy pastures developed on flipped soils on the South Islands West Coast The beetles rapidly invaded these new pastures and signs of damage appeared within 13 seasons Damage is caused by beetle larvae feeding on the roots of grasses and clovers creating damage patches of dead and dying plants A visual damage scale has been prepared to assist farmers to grade damage and provide a basis to make control decisions Damage rankings were estimated by assessing both the proportion of the paddock with obvious damage patches and the overall vigour of the pasture Increasing damage severity was shown to be strongly correlated with numbers of beetle larvae in the soil Pastures invaded by manuka beetles had lower autumn production and if untreated overall pasture production declined to very low levels within 23 seasons due to poor pasture composition and low pasture covers It is estimated that milk yields on affected farms have been depressed by 30 where no controls have been implemented It is recommended that pastures with high damage gradings should either be treated with insecticide or renewed

The entomopathogenic fungus Metarhizium anisopliae (Ascomycota: Hypocreales) has recently been found capable of producing compact hyphal aggregates (microsclerotia) under specific culture conditions that become pigmented during growth. The microsclerotia can be formulated and are stable when dried for storage. Upon rehydration, hyphae grow from the microsclerotes, develop into a fungal pellet and produce large quantities of conidia. In this study, microsclerotia were produced using New Zealand, USA and Mexican strains of M. anisopliae and characterised for production requirements, stability and conidial production. Microsclerotia were produced in shake flasks and fermenter vessels, and differences were observed in growth and production according to strain and C:N ratio. The potential of microsclerotia formulations for biocontrol of red-headed cockchafer (Adoryphorus couloni, Coleoptera: Scarabaeidae) is currently under evaluation.

The Australian pasture pest Adoryphorus couloni (redheaded cockchafer RHCC) continues to slowly spread from the Port Hills and Banks Peninsula through Christchurch towards productive agricultural land on the Canterbury Plains There are currently no products chemical or biological registered in New Zealand to control this pest In Christchurch several parks used extensively for human recreation were badly damaged by RHCC grubs in the autumn/early winter of 2008 and had to be treated with chemical insecticides (diazinon) Laboratory trials were thus carried out to assess the susceptibility of New Zealand populations of RHCC to a microbial biocontrol agent Metarhizium anisopliae DATF001 (ChaferGuard) registered in Australia Fungal activity was directly influenced by temperature and mode of application Infection and mortality occurred faster at 20C than 15C High mortality levels (90100 after 7 weeks) were obtained when larvae were treated by topical application (105 conidia/grub) or exposure to the dry ChaferGuard formulation in soil; >80 of the cadavers in these treatments were mycosed Direct incorporation of conidia into soil was the least effective treatment Grass grub (Costelytra zealandica) was unaffected by the fungus This trial confirmed the efficacy of DATF001 and its potential for use against New Zealand populations of RHCC