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.     

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.     

Investigation of a phage resistantSerratia entomophila strain (BC4B), establishment of generalised transduction and construction ofS. entomophila RecA mutants

ArecA clone was isolated from a cosmid library ofSerratia entomophila constructed in theEscherichia coli strain HB101. Subcloning and transposon mutagenesis were used to identify a 1.36 kb fragment containing therecA gene. A clonedrecA mutation, generated by transposon mutagenesis and the replacement of a portion of therecA gene with an antibiotic resistance cassette, was introduced into the chromosome via a marker exchange technique. TherecA 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 ofS. entomophila recA strains have been demonstrated.     

Survival and Phospholipid Fatty Acid Profiles of Surface and Subsurface Bacteria in Natural Sediment Microcosms

Although starvation survival has been characterized for many bacteria, few subsurface bacteria have been tested, and few if any have been tested in natural subsurface porous media. We hypothesized that subsurface bacteria may be uniquely adapted for long-term survival in situ. We further hypothesized that subsurface conditions (sediment type and moisture content) would influence microbial survival. We compared starvation survival capabilities of surface and subsurface strains of Pseudomonas fluorescens and a novel Arthrobacter sp. in microcosms composed of natural sediments. Bacteria were incubated for up to 64 weeks under saturated and unsaturated conditions in sterilized microcosms containing either a silty sand paleosol (buried soil) or a sandy silt nonpaleosol sediment. Direct counts, plate counts, and cell sizes were measured. Membrane phospholipid fatty acid (PLFA) profiles were quantified to determine temporal patterns of PLFA stress signatures and differences in PLFAs among strains and treatments. The Arthrobacter strains survived better than the P. fluorescens strains; however, differences in survival between surface and subsurface strains of each genus were not significant. Bacteria survived better in the paleosol than in the nonpaleosol and survived better under saturated conditions than under unsaturated conditions. Cell volumes of all strains decreased; however, sediment type and moisture did not influence rates of miniaturization. Both P. fluorescens strains showed PLFA stress signatures typical for gram-negative bacteria: increased ratios of saturated to unsaturated fatty acids, increased ratios of trans- to cis-monoenoic fatty acids, and increased ratios of cyclopropyl to monoenoic precursor fatty acids. The Arthrobacter strains showed few changes in PLFAs. Environmental conditions strongly influenced PLFA profiles.     

The effect of nitrogen fertilizer and irrigation on black point incidence in soft white spring wheat

Agronomic studies were conducted to examine the effect of fertilizer N on black point incidence in Fielder soft white spring wheat (Triticum aestivum L. em Thell.). Black point incidence rose with increases in the amount of N supplied either as fertilizer applied during the growing season in irrigation water or as soil N, specifically nitrate, from fertilizer N application in previous years. A comparison of four different irrigation regimes demonstrated that black point incidence was highest under frequent irrigation (irrigate to field capacity at 75% available moisture) and lowest under conventional irrigation (irrigate to field capacity at 50% available soil moisture). In each irrigation regime, disease incidence increased as N rates were raised from 0 to 120 kg ha^-1. A residual fertilizer-N study demonstrated in 1985 and 1986 that black point incidence generally rose with increasing levels of nitrogen from either preplant applications in the spring or soil nitrate from the previous year. However, additions of fertilizer N were shown to slightly reduce black point incidence at soil nitrate levels above 150 kg ha^-1. A two-year fertilizer N study demonstrated that in treatments receiving the same amount (90 kg ha^-1) of fertilizer N, the amount broadcast as a preplant treatment versus the amount applied in irrigation water in a fertigation treatment had no effect on black point incidence, but all fertilized treatments had significantly higher levels of disease than the unfertilized check.Contribution no. 3879016     

Non‐Native Grass Removal and Shade Increase Soil Moisture and Seedling Performance during Hawaiian Dry Forest Restoration

Invasive non‐native species can create especially problematic restoration barriers in subtropical and tropical dry forests. Native dry forests in Hawaii presently cover less than 10% of their original area. Many sites that historically supported dry forest are now completely dominated by non‐native species, particularly grasses. Within a grass‐dominated site in leeward Hawaii, we explored the mechanisms by which non‐native Pennisetum setaceum, African fountain grass, limits seedlings of native species. We planted 1,800 seedlings of five native trees, three native shrubs, and two native vines into a factorial field experiment to examine the effects of grass removal (bulldozed vs. clipped plus herbicide vs. control), shade (60% shade vs. full sun), and water (supplemental vs. ambient) on seedling survival, growth, and physiology. Both grass removal and shade independently increased survival and growth, as well as soil moisture. Seedling survival and relative growth rate were also significantly dependent on soil moisture. These results suggest that altering soil moisture may be one of the primary mechanisms by which grasses limit native seedlings. Grass removal increased foliar nitrogen content of seedlings, which resulted in an increase in leaf‐level photosynthesis and intrinsic water use efficiency. Thus in the absence of grasses, native species showed increased productivity and resource acquisition. We conclude that the combination of grass removal and shading may be an effective approach to the restoration of degraded tropical dry forests in Hawaii and other ecologically similar ecosystems.     

Abiotic edaphic factors affecting the growth of a threatened North American Sunflower, Helianthus paradoxus (Asteraceae)

This study evaluated the relationships among soil moisture, soil salinity, and soil oxygen on the growth of Helianthus paradoxus (Asteraceae), a threatened inland salt marsh species of western North America. The study was conducted in large growth boxes (1×2×0.3 m) tilted at an angle to achieve a saturated to dry water gradient similar to that found in the marsh. This experimental design allowed the evaluation of major abiotic factors (soil moisture and soil salinity) which have been shown to be potentially important for this species, while removing major biotic factors, such as competition from other community dominants. Maximum aboveground biomass occurred in the middle rows of the boxes, where surface soil water was reduced and subsurface soil water was intermediate in the gradient. Regression analyses indicated that H. paradoxus would grow best where surface soil water is approximately 5%, subsurface soil water ranges from 20 to 30%, and where surface soil salinity is less than 0.5 g kg⁻¹. Edaphic variables, particularly soil moisture and soil salinity, affect the growth of H. paradoxus. Data presented here suggest that the survival of this species depends on maintenance of the hydrologic regime.     

Formulation and Delivery of the Bacterial Antagonist Bacillus subtilis for Management of Lentil Vascular Wilt Caused by Fusarium oxysporum f. sp. lentis

Different formulations of Bacillus subtilis were prepared using standard laboratory protocols. Bacillus subtilis survived in glucose and talc powders at 8.6 and 7.8 log₁₀ CFU/g, respectively, for 1 year of storage at room temperature compared with 3.5 log₁₀ CFU/g on a peat formulation. Glasshouse experiments using soil and seed treatments were conducted to test the efficacy of B. subtilis for protecting lentil against the wilt disease caused by Fusariumoxysporum f. sp. lentis. Seed treatments with formulations of B. subtilis on glucose, talc and peat significantly enhanced its biocontrol activity against Fusarium compared with a treatment in which spores were applied directly to seed. The formulations decreased disease severity by reducing colonization of plants by the pathogen, promoting their growth and increased the dry weight of lentil plants. Of these treatments the glucose and talc‐based powder formulations were more effective than the peat formulation and the spore application without a carrier. It was shown that the B. subtilis spores applied with glucose were viable for longer than those applied with other carriers. Seed treatment with these formulated spores is an effective delivery system that can provide a conducive environment for B. subtilis to suppress vascular wilt disease on lentil and has the potential for utilization in commercial field application.     

Encapsulation of R. planticola Rs-2 from alginate-starch-bentonite and its controlled release and swelling behavior under simulated soil conditions

The plant growth-promoting bacteria (PGPR) Raoultella planticola Rs-2 was encapsulated with the various blends of alginate, starch, and bentonite for development of controlled-release formulations. The stability and release characteristics of these different capsule formulations were evaluated. The entrapment efficiency of Rs-2 in the beads (capsules) was more than 99%. The diameter of dry beads ranged from 0.98 to 1.41 mm. The bacteria release efficiency, swelling ratio, and biodegradability of the different bead formulations were enhanced by increasing the starch or alginate contents, but were impeded by higher bentonite content. The release kinetics of viable cells from capsules and the swelling ratio of capsules were studied in simulated soil media of varying temperature, moisture, pH, and salt content. The release of loaded Rs-2 cells and swelling of capsules are greatly affected by moisture, temperature, pH and salt content of the release medium. The release of viable Rs-2 cells from capsules was positively associated with the swelling properties of the capsules. The release of Rs-2 cells occurred through a Case II diffusion mechanism. In summary, this work indicates that alginate-starch-bentonite blends are a viable option for the development of efficient controlled-release formulations of Rs-2 biofertilizer, and which could have a promising application in natural field conditions.     

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.     

Expression of the Antifeeding Gene anfA1 in Serratia entomophila Requires RpoS

The rpoS gene of Serratia entomophila BC4B was cloned and used to create rpoS-mutant strain BC4BRS. Larvae of the New Zealand grass grub Costelytra zealandica infected with BC4BRS became amber colored but continued to feed, albeit to a lesser extent than infected larvae. Subsequently, we found that expression of the antifeeding gene anfA1 in trans was substantially reduced in BC4BRS relative to that in the parental strain BC4B. Our data show that a functional rpoS gene is vital for full expression of anfA1 and for development of the antifeeding component of amber disease.     

Stage‐dependent synergism using Metarhizium anisopliae and Serratia entomophila against Costelytra 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.     

Fluidized-bed drying and storage stability of Cryptococcus flavescens OH 182.9, a biocontrol agent of Fusarium head blight

A method to produce dried granules of Cryptococcus flavescens (formerly Cryptococcus nodaensis) OH 182.9 was developed and the granules evaluated for storage stability. Small spherical granules were produced and dried using a fluidized-bed dryer. A drying and survival curve was produced for the process of fluidized-bed drying at 30°C. The granules were dried to different moisture contents (4, 7, 9 and 12%) and evaluated for storage stability at 4°C for up to a year. These different moisture contents granules had the following respective water activities (0.22, 0.38, 0.47 and 0.57 a _w). The results show the storage stability varied significantly across this moisture content range. The 9% moisture content sample had the best short-term stability (up to 4 months), while 4% moisture content had the best long-term survival (1 year). A desorption isotherm of C. flavescens was determined and modeled. The results of the storage stability and drying studies are interpreted in context of the desorption isotherm.     

Antecedent Conditions Influence Soil Respiration Differences in Shrub and Grass Patches

Quantifying the response of soil respiration to past environmental conditions is critical for predicting how future climate and vegetation change will impact ecosystem carbon balance. Increased shrub dominance in semiarid grasslands has potentially large effects on soil carbon cycling. The goal of this study was to characterize the effect of antecedent moisture and temperature conditions on soil respiration in a grassland now dominated by shrubs. Continuous measurements of soil respiration, soil temperature, and soil moisture were made over the entire summer of 2005 within distinct vegetation microsites in this shrubland community—under grasses, shrubs, and in open spaces. We analyzed these data within a Bayesian framework that allowed us to evaluate the time-scale over which antecedent conditions influence soil respiration. The addition of antecedent conditions explained an additional 16% of the variation in soil respiration. High soil moisture during the preceding month increased respiration rates in both the grass and shrub microsites. However, the time period over which antecedent soil moisture influenced the temperature sensitivity of soil respiration was shorter in the shrub compared to the grass microsites (1 vs. 2 weeks, respectively). The depth of moisture was important; for example, for respiration under shrubs, near-surface moisture was more influential on the day of the respiration measurement but subsurface moisture was more influential on the antecedent time scale. Although more mechanistic studies are required, this study is the first to reveal that shrub encroachment changes the time scales over which soil moisture and temperature affect soil respiration.     

The Importance of Soil Characteristics in Determining Survival of First‐Year Cottonwood Seedlings in Altered Riparian Habitats

Most major rivers in the southwestern United States have been hydrologically altered to meet human needs. Altered hydrological regimes have been associated with declines in native riparian forests. Today, many riparian areas have little or no regeneration of native riparian species and are now dominated by exotic Saltcedar (Tamarix chinensis Lour.). Success of riparian restoration efforts at least partially depends on the number of seedlings surviving the first growing season. Seedling survival is influenced by many abiotic and biotic factors including competition from other plants and available soil moisture, which is partially dependent on soil texture. In this study, we evaluated the relative importance of four soil categories (sandy loam, loam, silt, and clay), rate of soil moisture decline, salinity, beginning‐ and end‐season Saltcedar density, initial Cottonwood (Populus deltoides Marshall subsp. wislizenii (Wats.) Eckenw.) seedling density, percent vegetation cover by potential dominant competitors Pigweed (Amaranthus L.) and Barnyard grass (Echinochloa crusgalli L., Beauv.), and average total vegetation height to Cottonwood seedling survival. Factors influencing seedling survival differed among the four soil types. Rate of moisture decline was important in sandy soils, whereas vegetation height influenced seedling survival in loamy soils. Overall, models of seedling survival in all the four soil types indicated rate of moisture decline as the single most important variable influencing Cottonwood survival. High initial densities of Saltcedar were correlated to higher survival in Cottonwood seedlings. Therefore, it is important to identify soil texture and understand soil moisture decline rates when proposing riparian Cottonwood restoration.     

Towards greener pastures—Pathogens and pasture pests

Two of New Zealand's most important insect pests, grass grub and porina, are endemic species which have successfully colonised improved pastures. Population densities of these insects within this new environment are far greater than in the native plant systems in which they evolved. Within these high populations diseases have flourished, and high numbers of diseases are recorded from each of these pests. These include bacteria, fungi, nematodes, viruses and protozoa. Diseases have been frequently associated with population collapses in both grass grub and porina, and the role of diseases in natural population regulation is discussed. Insect diseases can also be applied artificially and can have a useful role in pest management.     

Growth and competition in annual legume—perennial grass pasture in a dry monsoonal climate

The seasonal growth characteristics of a perennial (sabi grass (Urochloa mosambicensis))-annual (Townsville stylo (Stylosanthes humilis)) pasture ecosystem in the dry monsoonal region of norther Australia are described. The phases during which the botanical composition of the ecosystem may change are dry season plant survival, germination, establishment, growth and propagation. A conceptual model with general applicability for a pasture of perennial and annual species is presented and the inter-relationships between the identified phases are indicated. For the example studied, there was a large decline in the proportion of the annual legume in the establishment phase, due to competition for restricted soil moisture. This explains the trend to grass dominance observed in sabi grass-Townsville stylo swards, and is considered to apply generally in a perennial-annual pasture in a dry monsoonal environment. Two distinct environmental periods occurred during the season and were reflected in the observed growth characteristics. During the first, the growth rate of Townsville stylo was slow and plant density fluctuated sharply. The growth rate of sabi grass also fluctuated sharply due to high temperature and radiation, and variable but limiting soil moisture. In the second period temperatures were lower and soil moisture was higher. During this period growth rate of sabi grass and tiller production declined but both the plant density of sabi grass and the growth rate of Townsville stylo increased. As sabi grass has a high initial growth rate and nitrogen and phosphorus contents which are nutritionally acceptable during the transition between the dry and wet seasons, monospecific sabi grass swards are suggested for grazing during this period.     

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.     

Field trials against Hoplia philanthus (Coleoptera: Scarabaeidae) with a combination of an entomopathogenic nematode and the fungus Metarhizium anisopliae CLO 53

The white grub, Hoplia philanthus Füessly (Coleoptera: Scarabaeidae), is a major pest of turf and ornamental plants in Belgium. Previously, the combination of lethal concentration of the entomopathogenic nematodes Heterorhabditis megidis or Steinernema glaseri with the entomopathogenic fungus Metarhizium anisopliae (strain CLO 53) caused additive or synergistic mortality to third-instar H. philanthus in the laboratory and greenhouse. In this present study, we examined this interaction under field conditions and compared a combination of a commercial formulation of Heterorhabditis bacteriophora (Nema-green^®) and M. anisopliae. Controls were M. anisopliae, chlorpyrifos (Dursban 5 Granules) and H. bacteriophora. Field applications (surface or subsurface) were made against a mixed population of second/third-instar H. philanthus at a sport field and lawn infested in the province of West-Flanders. In both trials, the combination of M. anisopliae with H. bacteriophora at 5 × 10¹² conidia/ha +2.5 × 10⁹ infective juveniles/ha resulted in additive or synergistic effects, causing more than 95% grub mortality when the nematodes was applied 4 weeks after the application of fungus. However, application of nematode, chlorpyrifos or fungus alone provided 39–66%, 42–60% (surface) and 33–76%, 82–100% or 37–65%, (subsurface) control of H. philanthus. We concluded that the pathogen combinations we tested are compatible elements of integrated pest management and are likely to improve control of H. philanthus larvae and perhaps other insect pests beyond what is expected from single application of the pathogen.     

A model of insect—pathogen dynamics in which a pathogenic bacterium can also reproduce saprophytically

We developed a model of the population dynamic interaction between an insect and a pathogenic bacterium motivated by study of Serratia entomophila, a commercially exploited pathogen of the New Zealand grass grub (Costelytra zealandica). The bacterium is able to reproduce saprophytically, though it competes for saprophytic substrates with non-pathogenic strains, which appear to be superior competitors, probably because they lack a plasmid that carries genes required for pathogenicity. The effect of saprophytism and competition on the invasion criterion (R₀), short-term dynamics and long-term dynamics are described. Saprophytism can reduce (possibly to zero) the host threshold at which the pathogen can invade, though this reduction is less when there is competition with non-pathogenic strains. In a model of short-term population dynamics designed to mimic the application of bacteria to a host epizootic, saprophytism enhances the reduction in host density, though again this is tempered by competition with non-pathogens. In the long term, a pathogen that can develop saprophytically can drive its host to extinction in the absence of competition with non-pathogens. When the latter are present, host extinction is prevented. The addition of saprophytic reproduction can stabilize an otherwise unstable host–pathogen model, but we were unable to find a stable equilibrium given the further addition of a wholly saprophytic bacterial strain. The model suggests that enhancing or selecting for saprophytic ability could be a way of improving biological control.