Microbes, models and management of the New Zealand grass grub,

[First paragraph]Improved grasslands pose particular problems in pest management. The areas are extensive and the returns per hectare from grassland agriculture are much less than those from intensive cropping or horticulture, but pastures are usually sown to last for a number of years and have a much more stable ecology than in cropping or horticulture. For these reasons, biological controls and plant resistance have long been the preferred options for managing pasture pests. Within this context, the role of diseases in pasture pest population dynamics has received increasing attention, especially their ability to control pest outbreaks. Diseases are common within our major pasture pest species but their role in population regulation is often difficult to define. One of New Zealand’s major pasture pest species, the grass grub Costelytra zealandica, is widespread and often damaging throughout the country. The insect is an endemic species that has adapted to an introduced pasture system, dominated by exotic plant species (Lolium perene/Trifolium repens), where it can reach populations ten-fold higher than in its native habitat. Such high densities favour disease transmission and it is not surprising that a wide array of pathogens have been recorded from this insect (Glare et al., 1993). But the insect can still be highly damaging and can cause total loss of sown species within 3–4 years from sowing in grass grub prone regions. Probably the most important of these diseases, found throughout New Zealand, is amber disease caused by strains of the bacteria Serratia entomophila and S. proteamaculans. This is an unusual disease, controlled by a bacterial plasmid which has only been found in New Zealand bacterial isolates (Jackson et al., 2001).     

Impact of historical changes in land-use on the soil fauna

Replacing native forests and grasslands with plantations, pastures and crops has resulted in both contraction of ranges and exploitation of modified habitats by native species, and both general and restricted dispersal of introduced species of soil fauna. Contraction is shown by native earthworms, land snails, ring nematodes and various arthropods, while the areas with changed land use suggest certain native insects are more numerous than 150 years ago. Damage to pastures by grass grub and porina show clearly how native species can exploit modified habitats. Introduced Lumbricidae make a positive contribution to soil processes in pastures throughout New Zealand. The distribution of cyst nematodes reflects the distribution and intensity of cultivation of the hosts with which they arrived. Changes in soil structure, such as with the elimination or introduction of earthworms, can have marked effects on other soil structure, such as with the elimination or introduction of earthworms, can have marked effects on other soil animals. The faunal changes found are consistent with changes in land use; habitat protection for floristic and scenic reasons will help preserve both known and unknown elements of the soil fauna.     

Autochthonous bacterial flora indicated by PCR-DGGE of 16S rRNA gene fragments from the alimentary tract of Costelytra zealandica (Coleoptera: Scarabaeidae)

Aims: To locate and identify putative autochthonous bacteria within the grass grub gut that may have a role in symbiosis. Methods and Results: Polymerase chain reaction-denaturing gradient gel electrophoresis (DGGE) fingerprinting was used to investigate bacterial diversity in the grass grub larval gut. The microbial community profiles from five geographically distinct populations were compared and the influence of feeding was analysed. Bacterial community in the midgut was highly variable between locations and was affected by feeding. The hindgut contained a more diverse but stable bacterial community that was less affected by external conditions. Forty-seven distinct DGGE bands, representing different bacterial genotypes, could be distinguished from all samples, with 34 different bands occurring in the hindgut. The 22 most common bands were isolated and DNA was sequenced. Sequence analysis revealed that most bacteria (16/22) were affiliated to the Clostridiales with the predominant bacteria affiliated to the genus Clostridium. The remaining bacteria were aligned to the Betaproteobacteria, Deltaproteobacteria and Bacteroidetes. Conclusions: The grass grub larva has an autochthonous microflora with predominance of Clostridium spp. in the hindgut. Significance and Impact of the Study: Occurrence of an autocthonous microflora in the grass grub hindgut suggests a symbiotic relationship which could help explain the ability of larval scarabs to feed on recalcitrant organic matter.     

Selection,development and testing of phage‐resistant strains of Serratia entomophila for grass grub control

Use of the bacterium Serratia entomophila as an inundative biological control agent for the New Zealand grass grub (Costelytra zealandica) depends on the consistent production of high yields of the bacterium in liquid fermentation. Following the phage related failure of several S. entomophila fermentations, a programme was initiated to isolate phage‐resistant strains. No naturally occurring strains were found to be resistant to eight stocks of phage isolated from either grass grub larvae or the fermenter. Therefore, ethylmethane sulphonate was used to generate phage‐resistant mutants. Strains which showed cross resistance to all eight phage test stocks were tested for pathogenicity towards grass grub. Several strains showing unimpaired pathogenicity were selected for further tests. Four of these phage‐resistant strains produced high cell yields, even when grown in the presence of high numbers of fermenter‐derived phage. Phage‐resistant strains have subsequently been produced in bulk in industrial fermenters, for use in large‐scale field trials throughout New Zealand.     

Native insects as targets and non-target species of biological control

The use of population models for predicting desirable and undesirable outcomes of biological control are described using three case studies from New Zealand. The first reviews the models of Barlow and colleagues used to understand and improve the control of native grass grub Costelytra zealandica populations by augmentative application of pathogenic Serratia entomophila bacteria. A variety of modelling approaches have been used to predict grass grub outbreaks and thus the cost-effectiveness of applying bacteria. Models have also been developed to understand the competitive interactions between pathogenic and non-pathogenic forms of the bacteria. The other two case studies show how retrospective modelling has been used to quantify the non-target impact of introduced biological control agents. The parasitoid Microctonus aethiopoides was introduced to control the lucerne pest Sitona discoideus, but was found to disperse outside of the target habitat and attack several native weevil species in the Entiminae family. Retrospective modelling suggests that, given average parasitism levels of 15%, native Irenimus spp. and Nicaeana spp. weevil populations are likely to have been reduced by 8% due to non-target parasitism. Similarly, population models have shown that native red admiral butterfly (Bassaris gonerilla) populations are likely to have been reduced by 5% due to non- target parasitism by Pteromalus puparum, which was introduced to New Zealand for the control of the cabbage white butterfly (Pieris rapae).     

Use of the green fluorescent protein to monitor the fate of Serratia entomophila causing amber disease in the New Zealand grass grub, Costelytra zealandica

A series of constitutive green fluorescent protein (pGFPuv) derivatives of the bacterium Serratia entomophila (Enterobacteriaceae) were constructed, allowing the fate of cells causing amber disease ingested by the New Zealand grass grub (Costelytra zealandica, Coleoptera: Scarabaeidae) to be monitored. Examination of tissue and contents of the alimentary tract over time from ingestion, under fluorescence microscopy, revealed that the major site of S. entomophila colonisation in the grass grub is intestinal particulate matter. Visual examinations showed that wild type pathogenic strain persisted in high numbers in the grass grub intestinal tract, notably in the area of the hindgut, but the S. entomophila pADAP-free strain 5.6RC and the pADK mutant derivatives (pADK-4, -10, -13) that gave a non-feeding without gut clearance phenotype, were unable to colonise the gut. The indiscriminate colonisation of the intestinal tract particulate matter by pathogenic bacteria, rather than the colonisation of a specific site of activity, suggests that the bacterial toxins are induced and released from the bacteria while they live freely in the grass grub intestinal tract.     

An iridescent virus and a rickettsia from the grass grub Costelytra zealandica (Coleoptera: Scarabaeidae)

Natural iridescent virus and rickettsia infections of Costelytra zealandica (White) and Odontria sp. indet. larvae were studied at a site in the upper Pareora Gorge scenic reserve, S. Canterbury. By sequentially sampling the site, it was found that neither the iridescent virus nor the rickettsiae appear to give rise to host mortalities that significantly alter the population density. Many larvae were found that appeared healthy, but carried inapparent iridescent virus infections. These diseases are not suitable for biological control of the grass grub.     

Lack of interactions between fire ant control products and white grubs (Coleoptera: Scarabaeidae) in turfgrass

Insecticides are widely used to manage turfgrass pest such as white grubs (Coleoptera: Scarabaeidae). Red imported fire ants, Solenopsis invicta (Buren) are important predators and pests in managed turfgrass. We tested the susceptibility of white grub life stages (adults, egg, and larvae) to predation by S. invicta and determined if insecticides applied for control of S. invicta would result in locally greater white grub populations. Field trials over 2 yr evaluated bifenthrin, fipronil, and hydramethylnon applied to large and small scale turfgrass plots for impacts on fire ant foraging and white grub populations. Coincident with these trials, adults, larvae, and eggs of common scarab species were evaluated for susceptibility to predation by S. invicta under field conditions. Field trials with insecticides failed to show a significant increase in white grub populations resulting from treatment of turfgrass for fire ants. This, in part, may be because of a lack of predation of S. invicta on adult and larval scarabs. Egg predation was greatest at 70% but < 20% of adults and larvae were attacked in a 24 h test. Contrary to other studies, results presented here suggest that fire ants and fire ant control products applied to turfgrass have a minimal impact on white grub populations.     

Use of herbivore‐induced plant volatiles as search cues by Tiphia vernalis and Tiphia popilliavora to locate their below‐ground scarabaeid hosts

Japanese beetle, Popillia japonica Newman, and oriental beetle, Anomala orientalis (Waterhouse) (both Coleoptera: Scarabaeidae) are considered invasive species and have been reported as key pests of urban landscapes in the Northeastern USA. Tiphia vernalis Rohwer and Tiphia popilliavora Rohwer (Hymenoptera: Tiphiidae) were introduced as biocontrol agents against these beetles. These parasitic wasps burrow into the soil and search for grubs. When a host is found, the wasp attaches an egg in a location that is specific for the wasp species. It is unknown if these wasps can detect patches of concealed hosts from a distance above ground and what role, if any, herbivore‐induced plant volatiles play in their host location. This study evaluated the responses of female T. vernalis and T. popilliavora to grub‐infested and healthy plants in Y‐tube olfactometer bioassays. Also the effect of root herbivory on the composition of turfgrass (Poaceae) volatile profiles was investigated by collecting volatiles from healthy and grub‐infested grasses. Tiphia wasps were highly attracted to volatiles emitted by grub‐infested tall fescue (Festuca arundinacea Schreb.) and Kentucky bluegrass (Poa pratensis L.) over healthy grasses. In contrast, wasps did not exhibit a significant preference for grub‐infested perennial ryegrass (Lolium perenne L.) as compared with the control plants. The terpene levels emitted by grub‐infested Kentucky bluegrass and tall fescue were greater than that of control plants. Low levels of terpenes were observed for both test and control perennial ryegrass. The elevated levels of terpenes emitted by grub‐infested Kentucky bluegrass and tall fescue coincided with the attractiveness to the tiphiid wasps. Here, we provide evidence that plant exposure to root‐feeding insects P. japonica and A. orientalis resulted in an increase in terpenoid levels in turfgrasses, which strongly attracts their above‐ground parasitoids.     

Plasmid transfer among several members of the family Enterobacteriaceae increases the number of species capable of causing experimental amber disease in grass grub

Abstract Ability to cause amber disease in the New Zealand grass grub, Costleytra zealandica (Coleoptera: Scarabaeidae), by Serratia entomophila and S. proteamaculans (Enterobacteriaceae), is dependent on the presence of a large plasmid in bacterial strains. Transfer of the plasmid alone to several other Enterobacteriaceae resulted in the ability to cause the disease in grass grub larvae. No species other than S . entomophila or S . proteamaculans has previously been recorded causing amber disease.     

Surface coating aids survival of Serratia entomophila (Enterobacteriaceae) in granules for surface application

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 improve survival. Existing formulations require subsurface application limiting the area that can be treated. Formulations that allow delivery by broadcast methods are desirable to reduce application costs and increase the potential for aerial application to inaccessible areas. Two formulations were prepared for use in experiments examining the persistence and movement of inoculum through soil. When granules were applied to the soil surface, bacterial survival was negligible in uncoated core, but improved with increasing thickness of the coating. Both survival of bacteria and release into the soil were influenced by soil moisture content. Granules at <12% soil moisture showed high bacterial mortality and reduced delivery to the soil, while at 28% soil moisture most bacteria were released to the soil. There was a high level of survival of the applied bacteria within granules at 20% and 28% soil moisture. The formulations maintained viability of S. entomophila in granules stored under ambient conditions for more than 6 months. In laboratory and field tests, the application of granules caused disease in the target grass grub larvae, whether application was applied to the surface or subsurface. In field trials, broadcast applied granules could produce equivalent disease to thin-coat granules drilled into the soil, but these levels of disease were associated with the occurrence of precipitation shortly after application.     

Impact of Japanese beetle (Coleoptera: Scarabaeidae) feeding on seashore paspalum

Ten cultivars of seashore paspalum, Paspalum vaginatum Swartz, were compared for their response to Japanese beetle, Popillia japonica Newman, larval root feeding. Cultivars of Bermuda grass, Cynodon sp., and zoysiagrass, Zoysia sp., also were included for comparison. Turf grown in pots in the greenhouse was infested with second and third instars in this 2-yr study. Grub survival and weight gain, foliar growth, and root loss were compared among turfgrass species and cultivars. Few species-related differences were identified. Differences in grub tolerance were, however, observed to be a function of turfgrass cultivar. Some turf types demonstrating tolerance to grub feeding had rapid root growth and high root mass in control pots, but this was not consistent for all cultivars showing enhanced ability to maintain foliar growth despite grub feeding. The paspalum cultivars that seemed most tolerant of grub feeding were '561-79', 'Sea Isle 2000', 'Durban', 'HI-10', 'Kim-1', 'Sea Dwarf', and 'Sea Spray'.     

Cloning Serratia entomophila antifeeding genes--a putative defective prophage active against the grass grub Costelytra zealandica

Serratia entomophila and Serratia proteamaculans (Enterobacteriaceae) cause amber disease in the grass grub Costelytra zealandica (Coleoptera: Scarabaeidae), an important pasture pest in New Zealand. Larval disease symptoms include cessation of feeding, clearance of the gut, amber coloration, and eventual death. A 155-kb plasmid, pADAP, carries the genes sepA, sepB, and sepC, which are essential for production of amber disease symptoms. Transposon insertions in any of the sep genes in pADAP abolish gut clearance but not cessation of feeding, indicating the presence of an antifeeding gene(s) elsewhere on pADAP. Based on deletion analysis of pADAP and subsequent sequence data, a 47-kb clone was constructed, which when placed in either an Escherichia coli or a Serratia background exerted strong antifeeding activity and often led to rapid death of the infected grass grub larvae. Sequence data show that the antifeeding component is part of a large gene cluster that may form a defective prophage and that six potential members of this prophage are present in Photorhabdus luminescens subsp. laumondii TTO1, a species which also has sep gene homologues.     

Quantification and kinetics of the decline in grass grub endopeptidase activity during initiation of amber disease

Amber disease in the grass grub (Costelytra zealandica White) (Coleoptera: Scarabaeidae), caused by strains of the bacteria Serratia entomophila or S. proteamaculans, is characterised by cessation of feeding and clearance of the midgut. Analysis of the midgut enzyme activity in diseased grass grub larvae showed that proteolytic activity was reduced to low levels. The endopeptidases, trypsin, elastase, and chymotrypsin, were all markedly reduced in activity whereas the exopeptidases (leucine-aminopeptidase and carboxypeptidase A and B) were much less affected. There was no effect on the non-proteolytic enzymes, esterase and alpha-amylase. Sequential analysis of enzyme levels in the gut during onset of disease showed that proteolytic activity dropped after cessation of feeding and preceded gut clearance. In starved, uninfected larvae enzyme activity levels remained high, indicating that decline in enzyme activity is not associated with absence of food and cessation of feeding, but with the onset of disease.     

Diversity and Genetic Population Structure of Fungal Pathogens Infecting White Grub Larvae in Agricultural Soils

White grub larvae are important soil-dwelling pests in many regions of Mexico as they attack many important crops such as maize. The use of synthetic chemicals is currently the main control strategy, but they are not always effective; thus, other alternatives are needed. Microbial control using entomopathogenic fungi represents an important alternative strategy, and species within the genera Beauveria and Metarhizium are considered amongst the most promising candidates. Seventeen Beauveria spp. and two Metarhizium spp. isolates were obtained in surveys of white grub larvae from different regions of Guanajuato, Mexico. All isolates were capable of infecting healthy larvae of the white grub Phyllophaga polyphilla in laboratory assays, but mortality never exceeded 50 %. Isolates were identified using morphological and molecular methods. Based on elongation factor1-α and ITS partial gene sequence data, all Beauveria isolates were identified as Beauveria pseudobassiana. Elongation factor1-α and β-tubulin sequence data identified the Metarhizium isolates to be Metarhizium pingshaense. In contrast, three additional Metarhizium isolates obtained the previous year in the same region were identified as M. pingshaense, Metarhizium anisopliae and Metarhizium robertsii. Microsatellite genotyping showed that all B. pseudobassiana isolates were the same haplotype. Enterobacterial Repetitive Intergenic Consensus fingerprinting information confirmed no significant variation amongst the B. pseudobassiana isolates. The ecological role of these isolates and their impact on white grub larvae populations are discussed.     

Antioxidative Responses and Morpho-anatomical Alterations for Coping with Flood-Induced Hypoxic Stress in Grass Pea (<Emphasis Type="Italic">Lathyrus sativus</Emphasis> L.) in Comparison with Pea (<Emphasis Type="Italic">Pisum sativum</Emphasis>)

Flooding stress constrains crop growth and yield because most agricultural species are flood-sensitive. However, many of the plant species that live in permanently or temporarily flooded habitats have evolved specific traits to cope with these harsh conditions. Grass pea (Lathyrus sativus L.) is a legume that tolerates stresses such as drought, diseases, and pests; however, it is unclear whether grass pea has a tolerance mechanism for flooding stress. To understand if grass pea tolerates hypoxia and how it deals with hypoxic stress, the effects of hypoxia on root tip death, physiological, and morpho-anatomical alterations in grass pea and pea (Pisum sativum), which is sensitive to hypoxia, were compared. The results showed that activities of antioxidant enzymes, namely superoxide dismutase, catalase, ascorbate peroxidase, and glutathione content in grass pea were greater than in pea during hypoxia, which protected the root tip from oxidative damage and reduced ion leakage, which helped maintain membrane integrity. Furthermore, aerenchyma and lateral root development accompanied by ethylene production, moderate ROS accumulation-mediated cell death, and Ca²⁺ spatial-temporal heterogeneity developed well in grass pea compared to pea, which may not only facilitate internal gas diffusion but also promote removal of toxic by-products under hypoxic conditions. These results demonstrate that grass pea is more tolerant to hypoxic stress induced by flooding than garden pea seedlings. This discovery not only provides significant information for understanding the hypoxia-tolerant mechanisms in plants, but also promotes the usability of grass pea in flood-prone areas.     

Food habits of pasture wireworm,Conoderus exsul (Coleoptera: Elateridae)

Abstract

The food preferences and rates of growth of the omnivorous pasture wireworm, Conoderus exsul (Sharp) were studied. Larvae of soil-living insects were preferred to germinating maize seed, and wireworm growth rate was greater on insect compared with plant food. In glasshouse pot trials, wireworms increased their attack rate as the density of grass grub (Costelytra zealandica (White)) or white-fringed weevil (Graphthognathus leucoloma Boheman) larvae was increased. In field experiments that compared grass grub numbers in cages accessible to wireworms or protected from predators, predation varied with season from a low of 15% in late summer up to 82% in late autumn. White-fringed weevil larval mortality was increased by addition of wireworms in two field experiments, but not affected in a third trial where initial white-fringed weevil density was low. Grass grub mortality was increased by the presence of wireworms in field plots. Adult C. exsul were observed feeding on sweetcorn pollen.     

Identification of a Serratia entomophila genetic locus encoding amber disease in New Zealand grass grub (Costelytra zealandica).

Serratia entomophila UC9 (A1MO2), which causes amber disease in the New Zealand grass grub Costelytra zealandica, was subjected to transposon (TnphoA)-induced mutagenesis. A mutant (UC21) was found to be nonpathogenic (Path-) to grass grub larvae in bioassays and was shown, by Southern hybridization, to contain a single TnphoA insertion. This mutant failed to adhere to the gut wall (Adn-) of the larvae and also failed to produce pili (Pil-). A comparative study of the total protein profiles of wild-type S. entomophila UC9 and mutant UC21 revealed that the mutant lacked an approximately 44-kDa protein and overexpressed an approximately 20-kDa protein. Transfer of cosmids containing homologous wild-type sequences into mutant strain UC21 restored wild-type phenotypes (Path+, Pil+, and Adn+). One of the complementing cosmids (pSER107) conferred piliation on Pil- Escherichia coli HB101. The TnphoA insertion in UC21 was mapped within an 8.6-kb BamHI fragment common to the complementing cosmids, and we designated this gene locus amb-1. Six gene products with molecular masses of 44, 36, 34, 33, 20, and 18 kDa were detected in E. coli minicells exclusive to the cloned 8.6-kb fragment (pSER201A). The 44-kDa gene product was not detected in E. coli minicells containing the cloned mutant fragment. Saturation mutagenesis of this fragment produced four unlinked insertional mutations with active fusions to TnphoA. These active fusions disrupted the expression of one or more gene products encoded by amb-1. The 8.6-kb fragment cloned in the opposite orientation (pSER201B) expressed only a 20-kDa protein. We propose that these are the products of structural and/or regulatory genes involved in adhesion and/or piliation which are prerequisites in the S. entomophila-grass grub interaction leading to amber disease.     

Dedicated Herbaceous Biomass Feedstock Genetics and Development

Biofuels and bio-based products can be produced from a wide variety of herbaceous feedstocks. To supply enough biomass to meet the needs of a new bio-based economy, a suite of dedicated biomass species must be developed to accommodate a range of growing environments throughout the USA. Researchers from the US Department of Agriculture’s Agricultural Research Service (USDA-ARS) and collaborators associated with the USDA Regional Biomass Research Centers have made major progress in understanding the genetics of switchgrass, sorghum, and other grass species and have begun to use this knowledge to develop new cultivars with high yields and appropriate traits for efficient conversion to bio-based products. Plant geneticists and breeders have discovered genes that reduce recalcitrance for biochemical conversion to ethanol and drop-in fuels. Progress has also been made in finding genes that improve production under biotic and abiotic stress from diseases, pests, and climatic variations.     

Isolation and enumeration of Serratia entomophila—a bacterial pathogen of the New Zealand grass grub, Costelytra zealandica

Several agar media were tested for their use in a selective isolation and identification scheme for Serratia entomophila , a bacterium causing amber disease of the New Zealand grass grub, Costelytra zealandica (White). Soil dilutions were plated on caprylate thallous agar (CTA), selective for Serratia spp. Most strains of Ser. entomophila grew well on CTA; the mean efficiency of colony formation on CTA was 94 ± 3% of that on a non-selective medium. The identity of colonies growing on CTA was determined on the basis of their growth reactions on DNase-toluidine blue agar, adonitol agar and itaconate agar. Serratia entomophila could be distinguished from other Serratia spp. found in New Zealand soils, in particular Ser. proteamaculans , another causal agent of amber disease of grass grub. The identification scheme allowed the selective recovery of Ser. entomophila from field soils containing a diverse microflora.