Endophytic Beauveria bassiana increases galling of 'Rutgers' tomato roots with Meloidogyne incognita.

Beauveria bassiana is endophytic in many plant species and has been shown to protect host plants against insect pests and plant pathogens. However, less is known about its activity against plant-parasitic nematodes. In vitro and plant assays were conducted to determine the effect of B. bassiana 11-98 (Bb) on Meloidogyne incognita (root-knot nematode; RKN). Beauveria bassiana was confirmed as an endophyte in 'Rutgers' tomato and colonization patterns of Bb in 'Rutgers' (highly susceptible to RKN) were compared with those in 'Mountain Spring' (less susceptible to RKN). In greenhouse tests with 'Rutgers' at 30 and 60 days after treatment (DAT) with RKN and Bb, there were few differences in plant growth variables among treatments in repeated trials. However, RKN root galling and egg count/root system were enhanced in plants treated with Bb at 60 DAT. In an in vitro assay with egg masses from greenhouse tests, the percentages of hatched eggs, and mobile and immobile nematodes did not differ significantly for RKN and RKN+Bb treatments. The presence of viable Bb from roots was confirmed by collecting egg suspensions from root galls and plating them on selective medium. Colonies of Bb were verified on agar medium, but no parasitism of RKN eggs was observed. Research is needed to investigate factors responsible for increased galling by RKN in the presence of endophytic Bb in 'Rutgers' tomato.

Soil indigenous microbiome and plant genotypes cooperatively modify soybean rhizosphere microbiome assembly.

BACKGROUND: Plants have evolved intimate interactions with soil microbes for a range of beneficial functions including nutrient acquisition, pathogen resistance and stress tolerance. Further understanding of this system is a promising way to advance sustainable agriculture by exploiting the versatile benefits offered by the plant microbiome. The rhizosphere is the interface between plant and soil, and functions as the first step of plant defense and root microbiome recruitment. It features a specialized microbial community, intensive microbe-plant and microbe-microbe interactions, and complex signal communication. To decipher the rhizosphere microbiome assembly of soybean (Glycine max), we comprehensively characterized the soybean rhizosphere microbial community using 16S rRNA gene sequencing and evaluated the structuring influence from both host genotype and soil source. RESULTS: Comparison of the soybean rhizosphere to bulk soil revealed significantly different microbiome composition, microbe-microbe interactions and metabolic capacity. Soil type and soybean genotype cooperatively modulated microbiome assembly with soil type predominantly shaping rhizosphere microbiome assembly while host genotype slightly tuned this recruitment process. The undomesticated progenitor species, Glycine soja, had higher rhizosphere diversity in both soil types tested in comparison to the domesticated soybean genotypes. Rhizobium, Novosphingobium, Phenylobacterium, Streptomyces, Nocardioides, etc. were robustly enriched in soybean rhizosphere irrespective of the soil tested. Co-occurrence network analysis revealed dominant soil type effects and genotype specific preferences for key microbe-microbe interactions. Functional prediction results demonstrated converged metabolic capacity in the soybean rhizosphere between soil types and among genotypes, with pathways related to xenobiotic degradation, plant-microbe interactions and nutrient transport being greatly enriched in the rhizosphere. CONCLUSION: This comprehensive comparison of the soybean microbiome between soil types and genotypes expands our understanding of rhizosphere microbe assembly in general and provides foundational information for soybean as a legume crop for this assembly process. The cooperative modulating role of the soil type and host genotype emphasizes the importance of integrated consideration of soil condition and plant genetic variability for future development and application of synthetic microbiomes. Additionally, the detection of the tuning role by soybean genotype in rhizosphere microbiome assembly provides a promising way for future breeding programs to integrate host traits participating in beneficial microbiota assembly.

Agricultural intensification and urbanization negatively impact soil nematode richness and abundance: a meta-analysis.

Human activity has extensively transformed the land surface by agricultural intensification and urbanization. In soil, nematodes are the most abundant invertebrates. The effect of human interventions was assessed on overall richness, overall abundance, richness and abundance of nematodes of each trophic group and colonizer-persister (c-p) guild by comparing urban, agriculture and disturbed grassland (DGL) with natural grassland (NGL) and forest ecosystems. Meta-analyses were conducted to generate quantitative summaries from 111 published articles that met the inclusion criteria, 91 expressed data in grams and 20 expressed data in cm3. Results from data expressed per 100 g of soil indicated that overall richness was higher in forest than in NGL, DGL, urban, and agriculture ecosystems. The richness of all c-p guilds and of all trophic groups except herbivores was highest in forest ecosystems. In contrast, overall abundance was highest in DGL, agriculture and forest ecosystems. The abundance of c-p 1, c-p 2 and c-p 3 guilds and bacterivores, fungivores and herbivores was highest in disturbed ecosystems, while the abundance of c-p 4 and c-p 5 guilds and predators and omnivores was highest in relatively undisturbed ecosystems. Results from data expressed as nematodes per 100 cm3 of soil indicated that abundance followed a similar pattern, but richness often differed between the two methodologies. These meta-analyses strengthen the concept that human interventions adversely impact both richness and abundance using nematodes as soil health bioindicators.Human activity has extensively transformed the land surface by agricultural intensification and urbanization. In soil, nematodes are the most abundant invertebrates. The effect of human interventions was assessed on overall richness, overall abundance, richness and abundance of nematodes of each trophic group and colonizer-persister (c-p) guild by comparing urban, agriculture and disturbed grassland (DGL) with natural grassland (NGL) and forest ecosystems. Meta-analyses were conducted to generate quantitative summaries from 111 published articles that met the inclusion criteria, 91 expressed data in grams and 20 expressed data in cm3. Results from data expressed per 100 g of soil indicated that overall richness was higher in forest than in NGL, DGL, urban, and agriculture ecosystems. The richness of all c-p guilds and of all trophic groups except herbivores was highest in forest ecosystems. In contrast, overall abundance was highest in DGL, agriculture and forest ecosystems. The abundance of c-p 1, c-p 2 and c-p 3 guilds and bacterivores, fungivores and herbivores was highest in disturbed ecosystems, while the abundance of c-p 4 and c-p 5 guilds and predators and omnivores was highest in relatively undisturbed ecosystems. Results from data expressed as nematodes per 100 cm3 of soil indicated that abundance followed a similar pattern, but richness often differed between the two methodologies. These meta-analyses strengthen the concept that human interventions adversely impact both richness and abundance using nematodes as soil health bioindicators.

Enhancing plant productivity while suppressing biofilm growth in a windowfarm system using beneficial bacteria and ultraviolet irradiation.

Common problems in a windowfarm system (a vertical and indoor hydroponic system) are phytopathogen infections in plants and excessive buildup of biofilms. The objectives of this study were (i) to promote plant health by making plants more resistant to infection by using beneficial biosurfactant-producing Pseudomonas chlororaphis around the roots and (ii) to minimize biofilm buildup by ultraviolet (UV) irradiation of the water reservoir, thereby extending the lifespan of the whole system with minimal maintenance. Pseudomonas chlororaphis-treated lettuce grew significantly better than nontreated lettuce, as indicated by enhancement of color, mass, length, and number of leaves per head (p < 0.05). The death rate of the lettuce was reduced by ∼ 50% when the lettuce was treated with P. chlororaphis. UV irradiation reduced the bacteria (4 log reduction) and algae (4 log reduction) in the water reservoirs and water tubing systems. Introduction of P. chlororaphis into the system promoted plant growth and reduced damage caused by the plant pathogen Pythium ultimum. UV irradiation of the water reservoir reduced algal and biofilm growth and extended the lifespan of the system.

Homeowner attitudes and practices towards residential landscape management in Ohio, USA.

This study describes the results of a survey of 432 homeowners in Ohio, USA concerning their perceptions and practices regarding management of residential landscapes. The results reveal that outdoor residential environments are extremely important to homeowners, who tend to view their yards as serving multiple functions: a place to observe nature and to socialize as well as a place of beauty and recreation. Use of a lawn care company to apply chemicals is reported by 22 % of respondents, while 40 % either apply chemicals themselves or have someone other than a lawn care company do it. Logistic regressions reveal that factors influencing a homeowner's decision to employ a lawn care company or to apply chemicals themselves include: household income (+), perceived impacts on the environment (-), whether the next door neighbor does it (+), and type of residential environment (rural -, suburban and urban +). A theme that emerges throughout the study is the perceived importance of the role of the lawn in residents' sense of social status or acceptance in the neighborhood. This perception can be viewed as a positive in ensuring that residential environments are well maintained, but also as a negative resulting in environmental degradation or presenting a barrier to creativity in the development of alternative residential environments. Specific policy implications of these findings are that efforts aimed at educating homeowners about the environmental impacts of their lawn care choices are likely to have more success if they are directed at neighborhood groups rather than individuals, show that alternatives are easy to adopt, affordable, and can produce the characteristics of lawns that homeowners seek.

purL gene expression affects biofilm formation and symbiotic persistence of Photorhabdus temperata in the nematode Heterorhabditis bacteriophora.

Extensive studies of the well-known legume and rhizobium symbiosis model system suggest that the purine metabolic pathway plays a key role in microbe-plant interactions, although the exact mechanism is unknown. Here, we report the impact of a key purine metabolic gene, purL, on the symbiotic interaction between the bacterium Photorhabdus temperata and its nematode partner Heterorhabditis bacteriophora. Real-time PCR assays showed that the purL gene was upregulated in P. temperata in the nematode infective juvenile compared with artificial media. Mutation of the purL gene by in-frame deletion dramatically decreased the capacity of the bacterium to persist in infective juveniles and its ability to form biofilm in vitro. It was further demonstrated that purL gene expression was positively related to bacterial biofilm formation and the symbiotic persistence of the bacterium in nematode infective juveniles. A ΔpurL mutant lost the ability to support infective juvenile formation in the media which weakly supported biofilm formation, suggesting that a critical level of biofilm formation is required by the bacteria to support infective juvenile formation and thus establish their partnership. In addition, the defects in both biofilm formation and symbiotic ability due to the disruption of the purL gene could be partially restored by the addition of 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), an intermediate of the purine biosynthesis pathway. Overall, these data indicate that the purine metabolic pathway is important in microbe-animal symbioses, and that it may influence symbiotic interactions at the level of biofilm formation.

Phylogenetic and cophylogenetic relationships of entomopathogenic nematodes (Heterorhabditis: Rhabditida) and their symbiotic bacteria (Photorhabdus: Enterobacteriaceae).

Mutualistic association between entomopathogenic Photorhabdus bacteria and Heterorhabditis nematodes represents one of the emerging model systems in symbiosis studies, yet little is known about this partnership from a coevolutionary perspective. Herein, we investigated phylogenetic and cophylogenetic relationships of Heterorhabditis and Photorhabdus strains using molecular markers Internal Transcribed Spacer and gyrase B gene sequences, respectively. The phylogenies presented consistent, well supported, monophyletic groups in the parsimonious and likelihood analyses for both the nematode and bacterial strains and supported the placement of currently recognized taxa, from which a potentially new Heterorhabditis species represented by a Thailand strain MP68 was identified. While the nematode strains with distant geographic distributions showed no detectable phylogenetic divergence within H. bacteriophora or H. georgiana monophyletic groups, their respective symbiotic bacteria speciated into two Photorhabdus species: P. luminescens and P. temperata, indicating the occurrence of duplication. Although such evolutionary process reduces the phylogenetic congruence between Heterorhabditis nematodes and Photorhabdus bacteria, global cophylogenetic tests using ParaFit detected a highly significant correlation between the two phylogenies (ParaFitGlobal = 0.001). Further, the associations between H. zealandica, H. indica and H. megidis strains and their symbiotic bacteria exhibited significant contribution to the overall cophylogenetic structure. Overall, this study reveals evidence of coevolution between Photorhabdus bacteria and Heterorhabditis nematodes and provides a framework for further examination of the evolution of these associations.

Photorhabdus luminescens subsp. kleinii subsp. nov. (Enterobacteriales: Enterobacteriaceae).

Association between bacteria Photorhabdus and their nematode hosts Heterorhabditis represents one of the emerging models in symbiosis studies. In this study, we isolated the bacterial symbionts of the nematode Heterorhabditis georgiana. Using gyrB sequences for phylogenetic analysis, these strains were shown to be part of the species of Photorhbdus luminescens but with clear separation from currently recognized subspecies. Physiological properties and DNA-DNA hybridization profiles also supported the phylogenetic relationship of these strains. Therefore, a new subspecies, Photorhabdus luminescens subsp. kleinii subsp. nov., is proposed with the type strain KMD37(T) (=DSM 23513 =ATCC =NRRL B-59419).

Susceptibility of the Adult Japanese Beetle, Popillia japonica to Entomopathogenic Nematodes.

To build upon prior research demonstrating the potential of entomopathogenic nematode dissemination by infected adult Japanese beetle, Popillia japonica, we evaluated susceptibility of the adult beetles to 20 strains of Steinernema and Heterorhabditis under laboratory conditions. The nematodes were applied at a rate of 10,000 infective juveniles per 10 adult beetles in 148 mL plastic cups containing autoclaved sand and sassafras leaves as a source of food for the beetles. All strains infected the beetles and caused 55% to 95% mortality. The most virulent strains that caused 50% beetle mortality in less than 5 days included a strain of H. georgiana (D61), three strains of Steinernema sp. (R54, R45, and FC48), and two strains of S. carpocapsae (All and D60). The ability of two strains of Steinernema sp. (R45 and R54) and two strains of Heterorhabditis bacteriophora (D98 and GPS11) to infect and reproduce in the beetle was further examined to assess the potential of infected beetles to disseminate nematodes upon their death. All four strains infected and killed the beetles, but only Steinernema strains reproduced in the cadavers. We conclude that both Heterorhabditis and Steinernema strains are able to cause mortality to adult Japanese beetle, but Steinernema strains may be effectively disseminated due to their reproduction in the beetle.

Photorhabdus temperata subsp. stackebrandtii subsp. nov. (Enterobacteriales: Enterobacteriaceae).

The bacterial symbiont of the entomopathogenic nematode Heterorhabditis bacteriophora strain GPS11 was characterized by 16S rRNA gene sequence and physiological traits. The phylogenetic tree built upon 16S rRNA gene sequences clustered the GPS11 bacterial isolate with Photorhabdus temperata strains which have been previously isolated from Heterorhabditis species. The phylogenetic tree further identified four subgroups in P. temperata, and the relationships among these subgroups were confirmed by gyrase subunit B (gyrB) gene sequence analysis. The subgroup containing the GPS11 bacterial isolate differs from other subgroups in sequences of 16S rRNA and gyrB gene, physiological traits, nematode host species, and geographic origin. Therefore, the subgroup comprising the GPS11 bacterial isolate is proposed here as a new subspecies: Photorhabdus temperata subsp. stackebrandtii subsp. nov. (type strain GPS11). The type strain has been deposited in ATCC and DSMZ collections.

Comparative in vivo gene expression of the closely related bacteria Photorhabdus temperata and Xenorhabdus koppenhoeferi upon infection of the same insect host, Rhizotrogus majalis.

BACKGROUND: Photorhabdus and Xenorhabdus are Gram-negative, phylogenetically related, enterobacteria, forming mutualism with the entomopathogenic nematodes Heterorhabditis and Steinernema, respectively. The mutualistic bacteria living in the intestines of the nematode infective juveniles are pathogenic to the insect upon release by the nematodes into the insect hemolymph. Such a switch needs activation of genes that promote bacterial virulence. We studied in vivo gene expression in Photorhabdus temperata and Xenorhabdus koppenhoeferi upon infection of the white grub Rhizotrogus majalis using selective capture of transcribed sequences technique. RESULTS: A total of 40 genes in P. temperata and 39 in X. koppenhoeferi were found to be upregulated in R. majalis hemolymph at 24 h post infection. Genomic presence or upregulation of these genes specific in either one of the bacterium was confirmed by the assay of comparative hybridization, and the changes of randomly selected genes were further validated by quantitative real-time PCR. The identified genes could be broadly divided into seven functional groups including cell surface structure, regulation, virulence and secretion, stress response, intracellular metabolism, nutrient scavenging, and unknown. The two bacteria shared more genes in stress response category than any other functional group. More than 60% of the identified genes were uniquely induced in either bacterium suggesting vastly different molecular mechanisms of pathogenicity to the same insect host. In P. temperata lysR gene encoding transcriptional activator was induced, while genes yijC and rseA encoding transcriptional repressors were induced in X. koppenhoeferi. Lipopolysaccharide synthesis gene lpsE was induced in X. koppenhoeferi but not in P. temperata. Except tcaC and hemolysin related genes, other virulence genes were different between the two bacteria. Genes involved in TCA cycle were induced in P. temperata whereas those involved in glyoxylate pathway were induced in X. koppenhoeferi, suggesting differences in metabolism between the two bacteria in the same insect host. Upregulation of genes encoding different types of nutrient uptake systems further emphasized the differences in nutritional requirements of the two bacteria in the same insect host. Photorhabdus temperata displayed upregulation of genes encoding siderophore-dependent iron uptake system, but X. koppenhoeferi upregulated genes encoding siderophore-independent ion uptake system. Photorhabdus temperata induced genes for amino acid acquisition but X. koppenhoeferi upregulated malF gene, encoding a maltose uptake system. Further analyses identified possible mechanistic associations between the identified gene products in metabolic pathways, providing an interactive model of pathogenesis for each bacterium species. CONCLUSION: This study identifies set of genes induced in P. temperata and X. koppenhoeferi upon infection of R. majalis, and highlights differences in molecular features used by these two closely related bacteria to promote their pathogenicity in the same insect host.

Transcriptomic analysis of the entomopathogenic nematode Heterorhabditis bacteriophora TTO1.

BACKGROUND: The entomopathogenic nematode Heterorhabditis bacteriophora and its symbiotic bacterium, Photorhabdus luminescens, are important biological control agents of insect pests. This nematode-bacterium-insect association represents an emerging tripartite model for research on mutualistic and parasitic symbioses. Elucidation of mechanisms underlying these biological processes may serve as a foundation for improving the biological control potential of the nematode-bacterium complex. This large-scale expressed sequence tag (EST) analysis effort enables gene discovery and development of microsatellite markers. These ESTs will also aid in the annotation of the upcoming complete genome sequence of H. bacteriophora. RESULTS: A total of 31,485 high quality ESTs were generated from cDNA libraries of the adult H. bacteriophora TTO1 strain. Cluster analysis revealed the presence of 3,051 contigs and 7,835 singletons, representing 10,886 distinct EST sequences. About 72% of the distinct EST sequences had significant matches (E value < 1e-5) to proteins in GenBank's non-redundant (nr) and Wormpep190 databases. We have identified 12 ESTs corresponding to 8 genes potentially involved in RNA interference, 22 ESTs corresponding to 14 genes potentially involved in dauer-related processes, and 51 ESTs corresponding to 27 genes potentially involved in defense and stress responses. Comparison to ESTs and proteins of free-living nematodes led to the identification of 554 parasitic nematode-specific ESTs in H. bacteriophora, among which are those encoding F-box-like/WD-repeat protein theromacin, Bax inhibitor-1-like protein, and PAZ domain containing protein. Gene Ontology terms were assigned to 6,685 of the 10,886 ESTs. A total of 168 microsatellite loci were identified with primers designable for 141 loci. CONCLUSION: A total of 10,886 distinct EST sequences were identified from adult H. bacteriophora cDNA libraries. BLAST searches revealed ESTs potentially involved in parasitism, RNA interference, defense responses, stress responses, and dauer-related processes. The putative microsatellite markers identified in H. bacteriophora ESTs will enable genetic mapping and population genetic studies. These genomic resources provide the material base necessary for genome annotation, microarray development, and in-depth gene functional analysis.

Transcriptional profiling of trait deterioration in the insect pathogenic nematode Heterorhabditis bacteriophora.

BACKGROUND: The success of a biological control agent depends on key traits, particularly reproductive potential, environmental tolerance, and ability to be cultured. These traits can deteriorate rapidly when the biological control agent is reared in culture. Trait deterioration under laboratory conditions has been widely documented in the entomopathogenic nematode (EPN) Heterorhabditis bacteriophora (Hb) but the specific mechanisms behind these genetic processes remain unclear. This research investigates the molecular mechanisms of trait deterioration of two experimental lines of Hb, an inbred line (L5M) and its original parental line (OHB). We generated transcriptional profiles of two experimental lines of Hb, identified the differentially expressed genes (DEGs) and validated their differential expression in the deteriorated line. RESULTS: An expression profiling study was performed between experimental lines L5M and OHB of Hb with probes for 15,220 ESTs from the Hb transcriptome. Microarray analysis showed 1,185 DEGs comprising of 469 down- and 716 up-regulated genes in trait deteriorated nematodes. Analysis of the DEGs showed that trait deterioration involves massive changes of the transcripts encoding enzymes involved in metabolism, signal transduction, virulence and longevity. We observed a pattern of reduced expression of enzymes related to primary metabolic processes and induced secondary metabolism. Expression of sixteen DEGs in trait deteriorated nematodes was validated by quantitative reverse transcription-PCR (qRT-PCR) which revealed similar expression kinetics for all the genes tested as shown by microarray. CONCLUSION: As the most closely related major entomopathogen to C. elegans, Hb provides an attractive near-term application for using a model organism to better understand interspecies interactions and to enhance our understanding of the mechanisms underlying trait deterioration in biological control agents. This information could also be used to improve the beneficial traits of biological control agents and better understand fundamental aspects of nematode parasitism and mutualism.

Moraxella osloensis gene expression in the slug host Deroceras reticulatum.

BACKGROUND: The bacterium Moraxella osloensis is a mutualistic symbiont of the slug-parasitic nematode Phasmarhabditis hermaphrodita. In nature, P. hermaphrodita vectors M. osloensis into the shell cavity of the slug host Deroceras reticulatum in which the bacteria multiply and kill the slug. As M. osloensis is the main killing agent, genes expressed by M. osloensis in the slug are likely to play important roles in virulence. Studies on pathogenic interactions between bacteria and lower order hosts are few, but such studies have the potential to shed light on the evolution of bacterial virulence. Therefore, we investigated such an interaction by determining gene expression of M. osloensis in its slug host D. reticulatum by selectively capturing transcribed sequences. RESULTS: Thirteen M. osloensis genes were identified to be up-regulated post infection in D. reticulatum. Compared to the in vitro expressed genes in the stationary phase, we found that genes of ubiquinone synthetase (ubiS) and acyl-coA synthetase (acs) were up-regulated in both D. reticulatum and stationary phase in vitro cultures, but the remaining 11 genes were exclusively expressed in D. reticulatum and are hence infection specific. Mutational analysis on genes of protein-disulfide isomerase (dsbC) and ubiS showed that the virulence of both mutants to slugs was markedly reduced and could be complemented. Further, compared to the growth rate of wild-type M. osloensis, the dsbC and ubiS mutants showed normal and reduced growth rate in vitro, respectively. CONCLUSION: We conclude that 11 out of the 13 up-regulated M. osloensis genes are infection specific. Distribution of these identified genes in various bacterial pathogens indicates that the virulence genes are conserved among different pathogen-host interactions. Mutagenesis, growth rate and virulence bioassays further confirmed that ubiS and dsbC genes play important roles in M. osloensis survival and virulence, respectively in D. reticulatum.

Identification of two down-regulated genes in entomopathogenic nematode Heterorhabditis bacteriophora infective juveniles upon contact with insect hemolymph.

Entomopathogenic nematode Heterorhabditis bacteriophora infective juveniles (IJs) employ multiple strategies to combat with insect innate immune system after invasion. We employed suppressive subtractive hybridization (SSH) technique to study the gene expression in the IJs upon contact with the insect hemolymph in vitro. We identified two clones having higher expression levels in the IJs than IJs treated with insect hemolymph. The differential expression levels were confirmed by Northern blot hybridization with reference to the constitutive expression level of Heterorhabditis bacteriophora actin2 gene. Clone HbGPS11.C1G02 encoded a phosphofructokinase (PFK) with a 2.5 kb transcript and clone HbGPS11.C4C08 corresponded to a 2.1 kb transcript encoding a protein that had weak similarity to trans-sialidase from Trypanosoma cruzi. The differential expression of PFK in H. bacteriophora IJs before and during recovery process represented a switch from active movement in search of insect hosts to a state of combating insect innate immune system. This first report of H. bacteriophora differential gene expression provides a glimpse at the gene expression profile of H. bacteriophora IJ recovery process.

Expressed sequence tag analysis of gene representation in insect parasitic nematode Heterorhabditis bacteriophora.

We compared Heterorhabditis bacteriophora GPS11 expressed sequence tags (ESTs) to the ESTs of animal-parasitic, human-parasitic, plant-parasitic, and free-living nematodes. We identified 127 previously nondescribed ESTs of which 119 had homologs in ESTs and 8 had homologs in proteins of free-living nematodes. These ESTs were assigned putative functions in transcription, signal transduction, cell cycle control, metabolism, information processing, and cellular processes, thereby providing better insight into H. bacteriophora metabolism, sex determination, and signal transduction. We also identified 36 H. bacteriophora ESTs that had significant similarities to ESTs of parasitic nematodes, but not to ESTs or proteins of free-living nematodes species. Among these are the ESTs encoding a centrin, an ankyrin-repeat containing protein, and a nuclear hormone receptor. Our analysis also revealed that parasitic nematode-specific ESTs in this H. bacteriophora data set had more homologs in animal-parasitic nematodes than those parasitizing humans or plants.

Biological control of terrestrial molluscs using Phasmarhabditis hermaphrodita--progress and prospects.

Phasmarhabditis hermaphrodita Schneider (Nematoda: Rhabditidae) is a nematode that parasitises a wide range of slug and snail species. It has been formulated into a biological control agent (Nemaslug) and was commercialised in 1994. It is now available in fourteen European countries. A review is given of all research on P. hermaphrodita, including basic biology, mass cultivation, formulation, host range, application strategies, field efficacy and effects on non-target organisms. The many critical gaps in present knowledge are highlighted, and future research is proposed that will lead to greater understanding of this unusual parasite and may enable its more widespread use in the management of mollusc pests.

Comparative analysis of the expressed genome of the infective juvenile entomopathogenic nematode, Heterorhabditis bacteriophora.

We report the first cDNA-sequencing project of the entomopathogenic nematode, Heterorhabditis bacteriophora. A total of 1246 expressed sequence tags (ESTs) were generated by random sequencing of clones from a cDNA library of the infective juvenile stage. The ESTs were annotated resulting in 1072 useful ESTs that were categorized into functional categories according to Kyoto Encyclopedia of Genes and Genomes. Approximately 459 of 1072 ESTs (43%) had significant similarities to annotated sequences in GenBank. Of these, 417 had significant similarities to the free-living nematode Caenorhanditis elegans proteins. Most ESTs (18%) belonged to the genetic information processing category followed by metabolism (15% ESTs) and environmental information processing (15%) pathways. Several interesting ESTs were found that may have roles in the infectivity and survival of infective juveniles. These included proteases, dauer pathway genes (akt-1, pdk-1 & daf-7) and aging and stress resistance genes such as superoxide dismutase (sod-4), heat shock genes (hsp-4 & hsp-6), and eat genes, and signaling proteins like G-protein coupled receptors, regulators of G-protein signaling (rgs), and serine/threonine kinases. Other interesting ESTs include systemic RNAi defective protein (sid-1), ribonuclease III family members (rnh-2 &rnc) and transposase gene (Tc3A). About 67% of the ESTs did not find matches in any of the searched databases suggesting potentially novel genes in this enomopathogenic nematode. Note: Sequences described in this paper have been deposited in Genbank under the accessions DN 152655-DN 152999, and DN 153000-DN 153726.

Genetic variation and relationships between isolates and species of the entomopathogenic nematode genus Heterorhabditis deciphered through isozyme profiles.

We studied variation in isozyme patterns of 8 metabolic enzymes in 5 species of Heterorhabditis (H. bacteriophora, H. indica, H. marelata, H. megidis, and H. zealandica) comprising 18 isolates. Isozyme banding patterns of all the 8 enzymes were species specific; however, 3 enzymes, i.e., arginine kinase, fumarate hydratase, and malate dehydrogenase, displayed distinct patterns among all the 18 isolates. Phylogenetic analysis of the isozyme patterns produced dendrograms depicting a high degree of genetic variation among Heterorhabditis species, with the average pairwise distance of 0.2000. Trees constructed using different phylogenetic methods showed a relatively close genetic relationship between H. megidis and H. zealandica and between H. bacteriophora and H. indica. Also, H. bacteriophora HP88 was the most distant species from H. megidis (UK isolate), H. marelatus (Oregon isolate), and H. zealandica (X1 isolate) with pairwise distance of 0.1957, 0.2228, and 0.2120, respectively. Phylogenetic analysis also revealed genetic variation among H. bacteriophora isolates with the average pairwise distance of 0.1507. GPS2 and GPS3 were the most closely related isolates with the average distance of only 0.0870, followed by GPS1 and GPS2 with average distance of 0.1087. In contrast, KMD19 and HP88, OH25, and HP88, and OH25 and Acows isolates were the most divergent populations with a pairwise distance of 0.2011 and 37 character differences. Pairwise distance analysis also revealed that genetic divergence among populations of H. bacteriophora is relatively independent of geographic distance. Overall, these results demonstrate strong subspecies structuring in H. bacteriophora.

Infection Behavior and Overwintering Survival of Foliar Nematodes, Aphelenchoides fragariae, on Hosta.

We studied the pathogenicity and overwintering survival of the foliar nematode, Aphelenchoides fragariae, infecting Hosta spp. Nematodes applied to either lower or upper sides of noninjured and injured hosta leaves were able to infect and produce typical symptoms on nine cultivars. Leaves of only four cultivars (Borschi, Fragrant Blue, Patomic Pride, and Olive Bailey Langdon) showed no symptoms of nematode infection. The nematodes overwintered as juveniles and adults in soil, dry leaves, and dormant buds, but not in roots. Nematode winter survival was higher in dormant buds and soil from the polyhouse than in an open home garden. Of the nematodes found in the dormant buds, 35% to 79% were located between the first two outside layers of the buds. The nematodes tolerated 8 hr exposure to 40 degrees C and -80 degrees C in leaf tissues. Relative humidity influenced nematode migration from soil to leaves. The presence of nematodes only on the outer surface of foliage (leaves and petioles) confirmed the migration of A. fragariae on the surface of the plants. Of the total number of nematodes found on the foliage, 25% to 46% and 66% to 77% were alive at 90% and 100% relative humidity, respectively, suggesting that high moisture is required for the survival and upward movement of nematodes. We conclude that A. fragariae can overwinter in soil, infected dry leaves, and dormant buds and migrate in films of water on the outer surface of the plant during spring to leaves to initiate infection.