Costelytra Zealandica Research Articles

Tall fescue use on dairy farms

Tall fescue (Festuca arundinacea Schreb.) is now well proven on New Zealand dairy farms as a pasture species capable of producing high levels of milk production. Compared with perennial ryegrass (Lolium perenne) it offers benefits of higher annual and summer growth, higher clover contents in pasture, higher green leaf to stem ratios in summer, reduced plant pulling, better animal health, grass grub (Costelytra zealandica) and Argentine stem weevil (Listronotus bonariensis) tolerance, tolerance of wet soils, and drought tolerance and growth. Tall fescue can be recommended for dairy farmers in areas where summer growth and quality of ryegrass is reduced by moisture stress or high temperatures, and for those willing to adjust their grazing and establishment practices to suit tall fescue. Keywords: dairy farms, Festuca arundinacea, tall fescue

Predicting Canterbury grass grub outbreaks: the role of temperature

Probability (Markov) matrices and graphical analysis suggest that inclusion of mean summer temperature (Dec - Feb) significantly improves predictions of grass grub (Costelytra zealandica) outbreaks in Canterbury based on current density and disease levels. Outbreaks tend not to occur (7% chance) following a warm/dry summer. If the summer is cool, outbreaks are most likely if the density is currently high (irrespective of disease), or if both the current density and the disease level are low (43% chance if any of these conditions are met). The fact that high current density is one of the predisposing conditions suggests that with favourable summers, grub densities may stay high for more than one year or at least decline only slowly from very high levels.

Improved Sequential Probability Ratio Tests for Negative Binomial Populations

SUMMARY The negative binomial family of distributions is indexed by two parameters, m, the mean, and k, where 1/k is a measure of aggregation. In constructing sequential probability ratio tests (SPRTs) concerning m, it has been found necessary to assume a common value of k for the null and alternative hypotheses. A method is shown of assessing the performance of SPRTs without making this assumption, using a model of the dependence of k on m. Truncation of the tests is also allowed for. A test can be adjusted to have given error probabilities under the assumptions of varying k and truncation. The power and average sample number of a test are calculated. The method is illustrated by performing sequential hypothesis tests concerning infestation by grass-grub larvae. The accuracy of Wald's approximations for constructing tests having given error probabilities is assessed and found to be low. Wald (1947) describes a method for finding sequential tests of hypotheses which are known as sequential probability ratio tests (SPRTs) and which have certain optimality properties. The negative binomial distribution can be defined in terms of two parameters, m, the mean, and k where 1/k is a measure of aggregation or clumping. In applying Wald's method to constructing tests of hypotheses concerning the mean of a negative binomial distribution, it has been necessary to assume a value of k common to the null and alternative values of m (Oakland, 1950). Wetherill (1975), in describing a sequential sampling scheme for negative binomial data, comments on the assumption of a fixed value for k and states that Variations in [k] will alter the properties of the SPRT, and the effect of such variations does not appear to have been investigated. In addition there are two general sources of inaccuracy in calculations for sequential tests. First, Wald's formulas for constructing a test with given Type I and Type II error probabilities are only approximations. Second, the sample size in the SPRTs constructed by Wald's method is unbounded whereas in practice the tests are truncated. In this paper a method is shown whereby tests constructed by Wald's method for the negative binomial distribution, assuming a constant value of k, can be assessed after truncation and without assuming a constant value of k, with error probabilities being calculated. The test is then adjusted so that it has the desired error probabilities to a high degree of accuracy under these assumptions. The method is applied to the sequential sampling of grass grub (Costelytra zealandica). The dependence of k on m is explored by calculating maximum likelihood estimates, mn and k, of m and k, for a number of data sets. A model of this dependence is built by regressing k on mh. Then, in calculating error probabilities and average sample numbers for the sequential hypothesis test, k is assumed to have a distribution whose mean varies with m. A discrete approximation to this distribution is used in the calculations. The power and average sample number are calculated and compared with those calculated under an assumption of constant k.

Investigation of a phage resistant Serratia entomophila strain (BC4B), establishment of generalised transduction and construction of S. entomophila RecA mutants.

A recA clone was isolated from a cosmid library of Serratia entomophila constructed in the Escherichia coli strain HB101. Subcloning and transposon mutagenesis were used to identify a 1.36 kb fragment containing the recA gene. A cloned recA mutation, generated by transposon mutagenesis and the replacement of a portion of the recA gene with an antibiotic resistance cassette, was introduced into the chromosome via a marker exchange technique. The recA strains created were deficient in DNA repair, homologous recombination and both the spontaneous and UV induction of prophages. S. entomophila recA strains showed continued pathogenicity towards the New Zealand grass grub, Costelytra zealandica. Simple procedures for further construction of S. entomophila recA strains have been demonstrated.

Gut pH and amylase and protease activity in larvae of the New Zealand grass grub (Costelytra zealandica; Coleoptera: Scarabaeidae) as a basis for selecting inhibitors

Using a purpose-built antimony silver-silver chloride microelectrode we determined the pH of gut contents of field-collected third instars of the pasture pest Costelytra zealandica (White) (Coleoptera: Scarabaeidae) (grass grub), pH was 8.4 ± 0.69 (mean ± SD) ( n = 7) in the anterior pre-caecal portion of the midgut; it rose to 10.8 ±1.28 ( n = 19) in the anterior part of the remaining midgut, and 10.9 ± 0.95 ( n = 17) in the middle of the midgut, before dropping again to 9.3 ± 0.68 ( n = 19) at the posterior end of the midgut. The pH was 8.2 ± 0.35 ( n = 17) in the large hindgut. These data correct a previous suggestion that pH was approximately neutral in the grass grub gut. The data were then used to determine the functional localization of amylase and protease activities within the gut, by conducting assays at physiological pH values rather than the pH optima of the enzymes. Highest amylase activity was in the pre-caecal portion of the midgut; the hindgut showed relatively little activity. No activity was detected in the main part of the midgut because of that region's high pH. Protease activity was detected throughout the midgut. The pH data were also used to select putative inhibitors of the grass grub amylase. At pH 8.5 (the pH of the pre-caecal midgut), inhibitors from wheat and other Gramineae were active but a purified inhibitor from kidney bean showed no activity. These results indicate the importance of determining gut pH in studies of insect digestive enzymes.

The amb2 locus from Serratia entomophila confers anti-feeding effect on larvae of Costelytra zealandica (Coleoptera: Scarabaeidae)

Serratia entomophila (Se) causes amber disease in the soil-dwelling pest, Costelytra zealandica (Cz). The disease presents two main signs: anti-feeding effect (AFE) and development of amber coloration (AC). To identify the genetic loci involved in pathogenicity, non-pathogenic (Path −) Se mutants were created by transposon (Tn phoA) mutagenesis [Upadhyaya et al., J. Bacteriol. 174 (1992) 1020–1028]. The mutant UC24 lost the ability to produce amber disease signs and it was shown to contain a single Tn phoA insertion. The Tn phoA insertion site was mapped in a 5.3-kb DNA fragment, which was named amb2 locus. Cosmids containing amb2 fully restored AFE and partially restored AC in UC24. Escherichia coli (Ec) HB101 bearing the amb2 locus was able to cause AFE in a multiple-dose bioassay. SDS-PAGE analysis of the amb2 gene products produced in minicells showed the synthesis of two proteins of 16 and 19.5 kDa, named AnfA and AnfB. The genes encoding these proteins were mapped by deletion analysis. Pathogenicity tests with insect larvae fed with bacteria carrying the anfA and anfB gene regions separately showed that both regions are essential for AFE. It is proposed that the AnfA and AnfB proteins are virulence factors (toxin-like proteins) causing AFE in Cz.

Characterization of Serratia entomophila Bacteriophages and the Phage-Resistant Mutant Strain BC4B

Successful large-scale fermentations of the bacterium Serratia entomophila for use in biological control of the soil-dwelling insect Costelytra zealandica has required the development of a phage-resistant mutant, BC4B. We report our investigations into S. entomophila phages and the nature of the phage resistance mechanism of strain BC4B. The parental strain of BC4B, A1MO2, was found to contain two previously unidentified prophages, (phi)9A and (phi)9B, which were UV inducible and also released spontaneously in large numbers. BC4B was shown to be completely cured of (phi)9A. Single lysogens of (phi)9A and (phi)9B were not homoimmune to any other S. entomophila phages. However, on the basis of DNA-DNA homology, all S. entomophila phages except (phi)CW3 were shown to have significant regions of homology and also packaged their DNA via pac-like mechanisms. The failure of phage particles to adsorb was identified as the basis of phage resistance in BC4B. In addition, it was demonstrated that all known S. entomophila phages are naturally temperature sensitive.

Screening white clover for resistance to grass grub in a controlled environment

Abstract Controlled environment conditions were used to compare growth of individual third instar grass grub (Costelytra zealandica) larvae on white clover (Trifolium repens) lines and genotypes. There were repeatable differences in weight gain of grass grub larvae between different white clover lines in one soil with the highest weight gains being on ‘Grasslands Kopu’ grown in Opiki humic silty clay loam. However, selection of genotypes supporting high or low grub weight gains and re‐screening them for grub weight gains was not successful. Root type had no effect on grub weight gain, but the differences in weight gain between two soil types was of comparable magnitude to that observed for line differences. Weight gain correlated negatively with feeding deterrent activity and positively with soluble sugar content of root extracts.

Genes Essential for Amber Disease in Grass Grubs Are Located on the Large Plasmid Found in Serratia entomophila and Serratia proteamaculans

The bacteria Serratia entomophila and S. proteamaculans cause amber disease in the grass grub, Costelytra zealandica (Coleoptera: Scarabaeidae), an important pasture pest in New Zealand. Disease symptoms include rapid cessation of feeding and amber coloration of larvae. A 105-kb plasmid (designated pADAP) has consistently been found only in pathogenic isolates of both species. Investigations into the involvement of pADAP in amber disease have been hindered by the lack of both a selectable marker on the plasmid and a reliable transposon delivery system. Kanamycin-resistant transposon insertions into three cloned HindIII fragments (9.5, 9.6, and 10.6 kb) were isolated and introduced into pADAP by shuttle mutagenesis. Inserts into the 9.5-and 9.6-kb HindIII fragments on pADAP did not alter disease-causing ability. When plasmids with inserts into the 9.6-kb region were conjugated into plasmid-minus, nonpathogenic isolates of S. entomophila and S. proteamaculans, all of them became pathogenic. Transposon insertions into two regions of the 10.6-kb HindIII fragment continued to cause cessation of feeding but failed to produce amber coloration. Further analysis of a mutant from each amber-minus region (pADK-10 and pADK-13) demonstrated that the antifeeding effect was produced only at dosages higher than that of the wild-type strain. Complementation with the wild-type HindIII fragment restored full-blown disease properties for pADK-13, but not for pADK-10.

DNA Probes for Two Microsporidia, Nosema bombycis and Nosema costelytrae

Two DNA fragments which hybridize specifically with DNA of Nosema bombycis and Nosema costelytrae, respectively, were obtained from genomic DNA of each microsporidian species and sequenced. Neither fragment hybridized with genomic DNA from four other microsporidian isolates tested: Nosema apis, Vairimorpha sp. from cabbage white butterfly (Pieris rapae), and two isolates of Vavraia oncoperae, one from New Zealand grass grubs, Costelytra zealandica, and another from porina caterpillars, Wiseana spp. The probe for N. bombycis did not hybridize with genomic DNA from N. costelytrae or with DNA from silkworms (Bombyx mori), the primary insect host of this microsporidium. Likewise, the probe for N. costelytrae did not hybridize with genomic DNA from N. bombycis or with DNA from grass grubs (C. zealandica). Both fragments were AT-rich (59 and 79% of total bases, respectively), had G+C/A+T ratios of 0.70 and 0.25, respectively, and represented repeated sequences dispersed throughout the genome.

Bacillus and Serratia species for Scarab control

Few microorganisms are commercially available for use against white grubs (larvae of Scarabaeidae). Entomopathogenic bacteria, particularly Bacillus popilliae, have been used the longest for white grub suppression. Other bacteria, namely B. thuringiensis and Serratia spp. offer promise for future control. This papes examines two genera of bacteria (Bacillus and Serratia) from the historical and current perspective. Bacillus popilliae, the firs microbial control agent registered in the United States, has a long history of use in suppressing populations of the Japanese beetle, Popillia japonica. However, lack of in vitro production and the slow and sporadic nature of its activity, severely limits its utilization. B. thuringiensis, the most widely used microbial pesticide, has not been used for scarab, control. However, strains with scarab activity have recently been discovered. Scarab larvae have been collected in the United States with signs and symptoms similar to those characteristic of amber disease (caused by Serratia entomophila) in the New Zealand grass grub, Costelytra zealandica. A total of 147 bacteria have been obtained from the digestive tracts of larvae of the Japanese beetle and masked chafers, Cyclocephala spp., as well as from larvae and soil collected in Japan and China. Seventy five of these have been identified as Serratia spp. Most (40) of the remaining bacteria are in the genus Enterobacter. A majority of the bacteria (73) and of the Serratia (38) came from P. japonica.

Amber Disease Reduces Trypsin Activity in Midgut of Costeltyra zealandica (Coleoptera; Scarabaeidae) Larvae

Amber disease of the New Zealand grass grub Costelytra zealandica (Coleoptera: Scarabaeidae) is caused by ingestion of pADAP plasmid carrying isolates of Serratia entomophila or Serratia proteamaculans (Enterobacteriaceae) and causes infected larvae to cease feeding and clear their midgut to a pale amber colour where midgut serine protease activities are virtually eliminated. Using bacterial strains and mutants expressing combinations of the anti-feeding (afp) and gut clearance (sep) gene clusters from pADAP, we manipulated the disease phenotype and demonstrated directly the relationship between gene clusters, phenotype and loss of enzyme activity. Treatment with afp-expressing strains caused cessation of feeding without gut clearance where midgut protease activity was maintained at levels similar to that of healthy larvae. Treatment with strains expressing sep-genes caused gut clearance followed by a virtual elimination of trypsin and chymotrypsin titre in the midgut indicating both the loss of pre-existing enzyme from the lumen and a failure to replenish enzyme levels in this region by secretion from the epithelium. Monitoring of enzymatic activity through the alimentary tract during expression of disease showed that loss of serine protease activity in the midgut was matched by a surge of protease activity in the hindgut and frass pellets, indicating a flushing and elimination of the midgut contents. The blocking of enzyme secretion through amber disease appears to be selective as leucine aminopeptidase and α-amylase were still detected in the midgut of diseased larvae.

Winter management of grass grub(Costelytra zealandia(white))

Abstract Rolling pasture in May‐early June depressed grass grub (Costelytra zealandica (White)) densities by crushing larvae feeding close to the surface. The relationship between rolling intensity and decreasing grass grub density was generally exponential. This initial depression (25–90%) was followed by a further population depression during the pre‐pupal and pupal stages. Rolling and pasture‐treading by dairy cows in late July and August when larvae were deep in the soil profile, also depressed pupal densities. Marked reductions in grass grub densities were recorded following insecticide treatment (63–73%) and differing rolling intensities (28–73%) carried out in June. When both methods were combined, the reductions obtained were additive. Depressions in pest densities resulting from treading (69%) in early July and rolling (65%) in early August were similar and as with the combination of insecticide and rolling, were additive (93%). Losses in pasture production and changes in botanical composition we...

Artificial Diets for Third Instar Japanese Beetle (Coleoptera: Scarabaeidae)

Of four diets tested, one based on lima beans and casein was selected as the best and easiest for rearing third instars of Japanse beetles, Popillia japonica Newman. The diet gave the best survival and heaviest pupae and was previously used for rearing larvae of the New Zealand grass grub, Costelytra zealandica (White).

Assessment of grass grub (Costelytra zealandica(White)) (Coleoptera: Scarabaeidae) damage in ryegrass‐white clover pasture in Southland

Abstract The effects of grass grub density on pasture quality and quantity under sheep grazing were examined at three sites near Riversdale, central Southland, New Zealand. Using insecticide for 2–3 years enabled pastures with low levels of grass grub and those with natural levels to be compared. Grass grub reduced pasture production in autumn, winter, and spring by 6.4, 8.0, and 4.4% per 100 larvae/m2 in April‐May, respectively. Although grass grub caused occasional reductions in the proportion of ryegrass (up to 20% of sward) and white clover (up to 7% of sward), during spring‐summer, the long‐term effect on pasture composition was minor. This information can be used to predict the value of pasture lost and the economics of adopting a particular control option.

Stage‐dependent synergism usingMetarhizium anisopliaeandSerratia entomophilaagainstCostelytra zealandica

Larvae of the New Zealand grass grub (Costelytra zealandica) were treated with the entomopathogenic fungus, Metarhizium anisopliae, and the bacterium, Serratia entomophila, singly and in combination. The mortality of second instar larvae up to 41 days in treatments with both pathogens together was significantly greater than the additive mortalities of single pathogen treatments, and therefore synergistic. Treatment of third instar larvae with both pathogens did not increase mortality compared with the fungus alone. Second instar larvae were more resistant to M. anisopliae than third instar larvae. S. entomophila causes a chronic disease and bacterial treatments alone resulted in disease, but little mortality for either instar within 5 weeks. In both fungus alone and fungus/bacteria treatments, less than half the cadavers supported fungal sporulation. The use of a dual pathogen system for control of grass grub larvae is discussed.

Biological control of pastoral pests using Acremonium spp. endophytes

Although the role of the endophytic fungus Acremonium lolii in protecting the host plant from attack by insect pests, particularly Argentine stem weevil (Listronotus bonariensis), is often described in terms of plant resistance, it is a case of classical biological control. Endophytes in commercially available perennial ryegrass cultivars are acting as biological control agents against at least four pest species in New Zealand. Generally, endophyte adversely affects insect stages that feed on the above-ground parts of the plant, particularly those stages that feed near the base of the plant. It is apparent from insect feeding tests that there is no one universal anti-insect metabolite that will adversely affect all pest populations. It is therefore likely that an endophytelgrass combination that produces a cocktail of metabolites at low concentrations may be more useful than a combination that produces a very narrow suite of metabolites. Keywords: Acremonium spp., Listronotus bonariensis, Heteronychus arator, Costelytra zealandica, Wiseana spp., biological control, endophyte

Bacteriophage Can Inhibit the Development of Amber Disease in the New Zealand Grass Grub, Costelytra zealandica (White)

Bacteriophage Can Inhibit the Development of Amber Disease in the New Zealand Grass Grub, Costelytra zealandica (White)

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.

Association of a Large Plasmid with Amber Disease of the New Zealand Grass Grub, Costelytra zealandica, Caused by Serratia entomophila and Serratia proteamaculans

Amber disease, a chronic disease of Costelytra zealandica (Coleoptera: Scarabaeidae) larvae, is caused by some isolates of the bacteria, Serratia entomophila and Serratia proteamaculans (Enterobacteriaceae). A large plasmid was visualized in some isolates of these two Serratia species. Further investigation showed that 18 of 19 pathogenic isolates of Serratia spp. were found to have a megaplasmid, estimated to be 105 kb. The one virulent isolate lacking a 105-kb plasmid had two smaller plasmids. The 105-kd plasmid was present in only 1 of 22 naturally occurring, nonpathogenic isolates examined. Plasmid DNA isolated from pathogenic isolates of each species showed identical restriction patterns. Heat-treated derivatives of pathogenic S. entomophila and S. proteamaculans isolates, which lost disease-causing ability, also lost the 105-kb plasmid. Among isolates of 7 other species of Serratia, single isolates of Serratia grimesii and Serratia ficaria were found to have a similarly sized plasmid, but caused no disease symptoms. The implications of the association between the plasmid and amber disease are discussed.