Molecular taxonomic characterization and infra-specific diversity of entomopathogenic Beauveria bassiana fungi from Argentina.

It has been the aim of this study to molecular-taxonomically identify 15 Beauveria isolates collected from different geographical regions and insect hosts in Argentina and to investigate the levels of inter- and intra-specific diversity across this set of isolates. Based on phylogenetic analyses of EF1A-RPB1-RPB2 concatenated genes and BLOC markers, all Beauveria strains were identify as Beauveria bassiana. Within the B. bassiana clades of both phylogenies, isolates from Argentina were not clustered according to geographic origin or host. The 15 fungal isolates were further analyzed by PCR amplification of the intron insertion hot spot region of the nuclear 28S rRNA encoding sequence. By intron sequence and position, seven different group-I intron combinations termed variants A, B1, B2, C, D, E and F were found in the 15 isolates under study. Variants B1/B2 consisting of a single 28Si2 intron were found in ten isolates, whereas variant A occurred twice and variants C through F were unique across the set of isolates under study. The determination of the different introns and intron combinations in the 28S rRNA gene is a powerful tool for achieving infraspecific differentiation of B. bassiana isolates from Argentina.

Species Discrimination within the Metarhizium PARB Clade: Ribosomal Intergenic Spacer (rIGS)-Based Diagnostic PCR and Single Marker Taxonomy.

(1) Background: The entomopathogenic fungus Metarhizium anisopliae sensu lato forms a species complex, comprising a tight cluster made up of four species, namely M. anisopliae sensu stricto, M. pinghaense, M. robertsii and M. brunneum. Unambiguous species delineation within this "PARB clade" that enables both the taxonomic assignment of new isolates and the identification of potentially new species is highly solicited. (2) Methods: Species-discriminating primer pairs targeting the ribosomal intergenic spacer (rIGS) sequence were designed and a diagnostic PCR protocol established. A partial rIGS sequence, referred to as rIGS-ID800, was introduced as a molecular taxonomic marker for PARB species delineation. (3) Results: PARB species from a validation strain set not implied in primer design were clearly discriminated using the diagnostic PCR protocol developed. Using rIGS-ID800 as a single sequence taxonomic marker gave rise to a higher resolution and statistically better supported delineation of PARB clade species. (4) Conclusions: Reliable species discrimination within the Metarhizium PARB clade is possible through both sequencing-independent diagnostic PCR and sequencing-dependent single marker comparison, both based on the rIGS marker.

Exploring Bacterial and Fungal Biodiversity in Eight Mediterranean Olive Orchards (Olea europaea L.) in Tunisia.

A wide array of bacteria and fungi are known for their association with pests that impact the health of the olive tree. The latter presents the most economically important cultivation in Tunisia. The microbial diversity associated with olive orchards in Tunisia remains unknown and undetermined. This study investigated microbial diversity to elucidate the microbial interactions that lead to olive disease, and the bio-prospects for potential microbial biocontrol agents associated with insect pests of economic relevance for olive cultivation in the Mediterranean area. Bacterial and fungal isolation was made from soil and olive tree pests. A total of 215 bacterial and fungal strains were randomly isolated from eight different biotopes situated in Sfax (Tunisia), with different management practices. 16S rRNA and ITS gene sequencing were used to identify the microbial community. The majority of the isolated bacteria, Staphylococcus, Bacillus, Alcaligenes, and Providencia, are typical of the olive ecosystem and the most common fungi are Penicillium, Aspergillus, and Cladosporium. The different olive orchards depicted distinct communities, and exhibited dissimilar amounts of bacteria and fungi with distinct ecological functions that could be considered as promising resources in biological control.

Characterization, identification and virulence of Metarhizium species from Cuba to control the sweet potato weevil, Cylas formicarius Fabricius (Coleoptera: Brentidae).

AIMS: Entomopathogenic Metarhizium fungi are widely recognized for their biological control potential. In Cuba, several fungus-based bio-insecticides have been developed and are produced as part of integrated pest management (IPM) programmes for economically relevant agricultural pests. Screening of fungal isolates from the INISAV strain collection was used for the development of bio-insecticides against important pest insects as, for example the sweet potato weevil, Cylas formicarius. METHODS AND RESULTS: Six fungal isolates from Cuba were microscopically, morphologically and molecular-taxonomically characterized using marker sequences ef1a, rpb1 and rpb2, and the 5TEF region of the ef1a gene. Five isolates were assigned to the species Metarhizium anisopliae sensu stricto and one isolate to Metarhizium robertsii. The pathogenic potential was evaluated against adults of C. formicarius, and growth and conidial production on different nutritional media were determined. Metarhizium anisopliae strain LBM-267 displayed pronounced virulence against the sweet potato weevil and abundant conidia production on several culture media. CONCLUSIONS: Entomopathogenic fungal isolates from Cuba were assigned to the taxonomic species M. anisopliae sensu stricto and M. robertsii. Virulence assessment with respect to C. formicarius led to the identification of two M. anisopliae isolates holding biocontrol potential. Isolate LBM-11 has previously been developed into the bio-insecticide METASAVE-11 that is widely used to control several species of plant pathogenic weevils, Lepidoptera and thrips in Cuba. Isolate LBM-267 has not been employed previously but is as virulent against C. formicarius as LBM-11; its growth and conidial production capacities on different nutritional media will likely facilitate economically feasible bio-insecticide development. SIGNIFICANCE AND IMPACT OF THE STUDY: Metarhizium anisopliae isolate LBM-267 has been selected as a promising candidate for biocontrol of the sweet potato weevil, an economically important agricultural pest in Cuba, and for further R&D activities within the framework of the Biological Control Program of Cuba.

COI Haplotyping and Comparative Microbiomics of the Peach Fruit Fly, an Emerging Pest of Egyptian Olive Orchards.

The peach fruit fly, Bactrocera zonata (Tephritidae), is economically relevant as a highly polyphagous pest infesting over 50 host plants including commercial fruit and horticultural crops. As an invasive species, B. zonata was firmly established in Egypt and holds potential to spread further across the Mediterranean basin. The present study demonstrated that the peach fruit fly was found multiplying in olive orchards at two distant locations in Egypt. This is the first report of B. zonata developing in olives. COI barcoding has revealed evidence for high diversity across these peach fruit fly populations. These data are consistent with multiple rather than a single event leading to both peach fruit fly invasion to Egypt and its adaptation to olive. Comparative microbiomics data for B. zonata developing on different host plants were indicative for microbiome dynamics being involved in the adaptation to olive as a new niche with a potential adaptive role for Erwinia or Providencia bacteria. The possibility of symbiont transfer from the olive fruit fly to the peach fruit fly is discussed. Potentially host switch relevant bacterial symbionts might be preferred targets of symbiosis disruption strategies for integrated pest management or biological control of B. zonata.

Genomic analysis of the nomenclatural type strain of the nematode-associated entomopathogenic bacterium Providencia vermicola.

BACKGROUND: Enterobacteria of the genus Providencia are mainly known as opportunistic human pathogens but have been isolated from highly diverse natural environments. The species Providencia vermicola comprises insect pathogenic bacteria carried by entomoparasitic nematodes and is investigated as a possible insect biocontrol agent. The recent publication of several genome sequences from bacteria assigned to this species has given rise to inconsistent preliminary results. RESULTS: The genome of the nematode-derived P. vermicola type strain DSM_17385 has been assembled into a 4.2 Mb sequence comprising 5 scaffolds and 13 contigs. A total of 3969 protein-encoding genes were identified. Multilocus sequence typing with different marker sets revealed that none of the previously published presumed P. vermicola genomes represents this taxonomic species. Comparative genomic analysis has confirmed a close phylogenetic relationship of P. vermicola to the P. rettgeri species complex. P. vermicola DSM_17385 carries a type III secretion system (T3SS-1) with probable function in host cell invasion or intracellular survival. Potentially antibiotic resistance-associated genes comprising numerous efflux pumps and point-mutated house-keeping genes, have been identified across the P. vermicola genome. A single small (3.7 kb) plasmid identified, pPVER1, structurally belongs to the qnrD-type family of fluoroquinolone resistance conferring plasmids that is prominent in Providencia and Proteus bacteria, but lacks the qnrD resistance gene. CONCLUSIONS: The sequence reported represents the first well-supported published genome for the taxonomic species P. vermicola to be used as reference in further comparative genomics studies on Providencia bacteria. Due to a striking difference in the type of injectisome encoded by the respective genomes, P. vermicola might operate a fundamentally different mechanism of entomopathogenicity when compared to insect-pathogenic Providencia sneebia or Providencia burhodogranariea. The complete absence of antibiotic resistance gene carrying plasmids or mobile genetic elements as those causing multi drug resistance phenomena in clinical Providencia strains, is consistent with the invertebrate pathogen P. vermicola being in its natural environment efficiently excluded from the propagation routes of multidrug resistance (MDR) carrying genetic elements operating between human pathogens. Susceptibility to MDR plasmid acquisition will likely become a major criterion in the evaluation of P. vermicola for potential applications in biological pest control.

Bacillus velezensis strain MBY2, a potential agent for the management of crown gall disease.

The reduction of the use chemical pesticides in agriculture is gaining importance as an objective of decision-makers in both politics and economics. Consequently, the development of technically efficient and economically affordable alternatives as, e.g., biological control agents or practices is highly solicited. Crown gall disease of dicotyledonous plants is caused by ubiquitous soil borne pathogenic bacteria of the Agrobacterium tumefaciens species complex, that comprises the species Agrobacterium fabrum and represents a globally relevant plant protection problem. Within the framework of a screening program for bacterial Agrobacterium antagonists a total of 14 strains were isolated from Tunisian soil samples and assayed for antagonistic activity against pathogenic agrobacteria. One particularly promising isolate, termed strain MBY2, was studied more in depth. Using a Multilocus Sequence Analysis (MLSA) approach, the isolate was assigned to the taxonomic species Bacillus velezensis. Strain MBY2 was shown to display antagonistic effects against the pathogenic A. fabrum strain C58 in vitro and to significantly decrease pathogen populations under sterile and non-sterile soil conditions as well as in the rhizosphere of maize and, to a lower extent, tomato plants. Moreover, the ability of B. velezensis MBY2 to reduce C58-induced gall development has been demonstrated in vivo on stems of tomato and almond plants. The present study describes B. velezensis MBY2 as a newly discovered strain holding potential as a biological agent for crown gall disease management.

Isolation, Identification, and Characterization of the Nematophagous Fungus Arthrobotrys oligospora from Kyrgyzstan.

PURPOSE: Predatory fungi have been the subject of fundamental studies and their potential as biological control agents against parasitic plant nematodes has been assessed. The aim of the present study was to isolate and identify predatory fungi, performing in vitro and in vivo screening to select highly active strains to control parasitic nematodes. METHODS: Different nutrient media were used to isolate predatory fungi and determine their morphological and cultural properties. Identification was performed by classical and molecular biology methods. In vitro and in vivo screening was conducted to select highly active strains. RESULTS: Twelve isolates of Arthrobotrys oligospora (Orbiliomycetes) found in nature were investigated for their predaceous efficacy against garlic stem nematodes (Ditylenchus dipsaci). The effect of temperature and pH on the growth rate and trap formation of representative isolates was determined and isolates were characterized by light microscopy and molecular markers. BLAST was used to sequence the rDNA internal transcribed spacer of A. oligospora isolate KTMU-7. The optimum growth of A. oligospora strains was achieved at 20-25 °C on 1-2% corn meal agar (CMA) within the pH range of 5.6-8.6. The factors responsible for the trap formation of these fungal strains were identified. In vitro and in vivo experiments were performed to evaluate the nematicidal activity of local predatory fungal isolates against soil nematodes. CONCLUSIONS: Preliminary studies proved A. oligospora to be a potentially effective biological control agent, immobilizing 85.7 ± 2.19% of garlic stem nematodes in soil from the rhizosphere of potato plants.

Identification of fungi in Tunisian olive orchards: characterization and biological control potential.

BACKGROUND: Olive production is the main agricultural activity in Tunisia. The diversity of fungi was explored in two different olive groves located in two distant geographical zones in Sfax (Tunisia) with different management practices. RESULTS: Fungal isolation was made from soil and the major olive tree pests, namely the Olive fly, Bactrocera oleae Gmelin (Diptera: Tephritidae), and the Olive psyllid, Euphyllura olivina Costa (Homoptera: Psyllidae). A total of 34 fungal isolates were identified according to their phenotypic, genotypic, biochemical and biological activities. Twenty fungal species were identified belonging to six different genera (Alternaria, Aspergillus, Cladosporium, Fusarium, Lecanicillium and Penicillium) by the analysis of their ITS1-5.8S-ITS2 ribosomal DNA region. Different bioassays performed in this work revealed that 25/34 (73.5%) of the identified fungal isolates showed an entomopathogenic and/or antagonistic activity, 9/34 (26.5%) of them displayed phytopathogenic features. CONCLUSIONS: Fungal species that showed entomopathogenic and/or antagonistic potentialities and that are non-phytopathogenic, (17/34; 50%) of our fungal isolates, could be explored for olive protection against fungal diseases and pests, and might have a future application as biocontrol agents.

A new species of Herpomyces (Laboulbeniomycetes: Herpomycetales) on Periplaneta fuliginosa (Blattodea: Blattidae) from Argentina.

We are becoming increasingly aware of the dazzling diversity of fungi-new taxa are being discovered from poorly sampled habitats and integrative approaches point at (near-)cryptic species in many groups. The class Laboulbeniomycetes, which is composed of three orders (Herpomycetales, Laboulbeniales, Pyxidiophorales), is no exception. However, still, in the orders Herpomycetales and Laboulbeniales, species are predominantly described based on morphology alone. Here, we present a new species of Herpomyces from Argentina parasitic on Periplaneta fuliginosa, the smokybrown cockroach. Herpomyces spegazzinii, sp. nov., is characterized based on morphology and molecular data. Phylogenetic inference based on internal transcribed spacer (ITS, consisting of ITS1-5.8S-ITS2) barcode region supports the status of this fungus as a separate species, sister to the recently described H. shelfordellae.

Providencia entomophila sp. nov., a new bacterial species associated with major olive pests in Tunisia.

Bioprospection for potential microbial biocontrol agents associated with three major insect pests of economic relevance for olive cultivation in the Mediterranean area, namely the olive fly, Bactrocera oleae, the olive moth, Prays oleae, and the olive psyllid, Euphyllura olivina, led to the isolation of several strains of readily cultivable Gram-negative, rod-shaped bacteria from Tunisian olive orchards. Determination of 16S ribosomal RNA encoding sequences identified the bacteria as members of the taxonomic genus Providencia (Enterobacterales; Morganellaceae). A more detailed molecular taxonomic analysis based on a previously established set of protein-encoding marker genes together with DNA-DNA hybridization and metabolic profiling studies led to the conclusion that the new isolates should be organized in a new species within this genus. With reference to their original insect association, the designation "Providencia entomophila" is proposed here for this hypothetical new taxon.

Natural occurrence in Argentina of a new fungal pathogen of cockroaches, Metarhizium argentinense sp. nov.

The aim of this study was to search for entomopathogenic fungi that infect wild cockroaches in forest ecosystems in two protected natural areas of Argentina. Two isolates of Metarhizium argentinense were obtained and identified from wild cockroaches (Blaberidae: Epilamprinae) through the use of morphological characteristics and molecular phylogenetic analyses. This novel species was found in Argentina and is a member of the Metarhizium flavoviride species complex. Phylogenetic analyses, based on sequence similarity analysis using internal transcribed spacer (ITS) and a set of four protein-coding marker sequences (EF1A, RPB1, RPB2 and BTUB), supported the status of this fungus as a new species. In addition, we tested the biological activity of the new species through assays against Blattella germanica nymphs and found that the two evaluated isolates were pathogenic. However, isolate CEP424 was more virulent and caused a confirmed mortality of 76 % with a median lethal time of 7.2 d. This study reports the southernmost worldwide location of a Metarhizium species that infects cockroaches and will help expand the knowledge of the biodiversity of pathogenic fungi of Argentine cockroaches.

Prevalence of Beauveria pseudobassiana among entomopathogenic fungi isolated from the hard tick, Ixodes ricinus.

Human and animal disease-transmitting hard ticks (Acari: Ixodidae) are of great concern for public health and animal farming. Alternatives to tick control by chemical acaricides are urgently needed, and one intensively evaluated biocontrol strategy is based on the use of tick-pathogenic filamentous fungi. An indispensable prerequisite for the development of tick-derived fungal isolates into registered myco-acaricides is their sound taxonomic characterisation. A set of fungal strains isolated from ixodid ticks in the Republic of Moldova was genetically characterised at the genus and species level together with further tick-derived fungal isolates from different geographic locations in Europe and North America. In a previous study, the same isolates had been assigned to the species Beauveria bassiana. Using a recent molecular taxonomic approach based on phylogenetic reconstruction from both internal transcribed spacer (ITS) and protein-encoding gene sequences, all fungi investigated were conclusively assigned to one of the two "hyphomycete" genera, Beauveria or Isaria (Ascomycota; Hypocreales; Cordycipitaceae). Within the genus Isaria, two species, Isaria farinosa and Isaria fumosorosea, were equally represented. Within the genus Beauveria, the comparatively rare species Beauveria pseudobassiana was found to strongly prevail among the isolates from Moldova, and one of the two tick-derived Beauveria strains from North America could be assigned to this species as well. In particular, the previous classification as B. bassiana could not be confirmed for any of the characterised tick pathogens from Europe and North America. The data presented here lend support to the hypothesis that within the genus Beauveria specific adaptation to ticks might have occurred within the species B. pseudobassiana. To test this hypothesis, a more extensive molecular taxonomic survey carefully reconsidering previous taxonomic assignments of tick-derived fungal isolates is needed.

Ultrastructural characterization and multilocus sequence analysis (MLSA) of 'Candidatus Rickettsiella isopodorum', a new lineage of intracellular bacteria infecting woodlice (Crustacea: Isopoda).

The taxonomic genus Rickettsiella (Gammaproteobacteria; Legionellales) comprises intracellular bacteria associated with a wide range of arthropods including insects, arachnids and crustaceans. The present study provides ultrastructural together with genetic evidence for a Rickettsiella bacterium in the common rough woodlouse, Porcellio scaber (Isopoda, Porcellionidae), occurring in Germany, and shows that this bacterium is very closely related to one of the same genus occurring in California that infects the pill bug, Armadillidium vulgare (Isopoda, Armadillidiidae). Both bacterial isolates displayed the ultrastructural features described previously for crustacean-associated bacteria of the genus Rickettsiella, including the absence of well-defined associated protein crystals; occurrence of the latter is a typical characteristic of infection by this type of bacteria in insects, but has not been reported in crustaceans. A molecular systematic approach combining multilocus sequence analysis (MLSA) with likelihood-based significance testing demonstrated that despite their distant geographic origins, both bacteria form a tight sub-clade within the genus Rickettsiella. In the 16S rRNA gene trees, this sub-clade includes other bacterial sequences from woodlice. Moreover, the bacterial specimens from P. scaber and A. vulgare are found genetically or morphologically different from each of the four currently recognized Rickettsiella species. Therefore, the designation 'Candidatus Rickettsiella isopodorum' is introduced for this new lineage of isopod-associated Rickettsiella bacteria.

Multilocus sequence analysis (MLSA) of 'Rickettsiella agriotidis', an intracellular bacterial pathogen of Agriotes wireworms.

Wireworms, the polyphagous larvae of click beetles belonging to the genus Agriotes (Coleoptera: Elateridae) are severe and widespread agricultural pests that affect numerous crops globally. A new bacterial specimen identified in diseased wireworms had previously been shown by microscopy and 16S ribosomal RNA (rRNA) gene-based phylogenetic reconstruction to belong to the taxonomic genus Rickettsiella (Gammaproteobacteria) that comprises intracellular bacteria associated with and typically pathogenic for a wide range of arthropods. Going beyond these earlier results obtained from rRNA phylogenies, multilocus sequence analysis (MLSA) using a four marker scheme has been employed in the molecular taxonomic characterization of the new Rickettsiella pathotype, referred to as 'Rickettsiella agriotidis'. In combination with likelihood-based significance testing, the MLSA approach demonstrated the close phylogenetic relationship of 'R. agriotidis' to the pathotypes 'Rickettsiella melolonthae' and 'Rickettsiella tipulae', i.e., subjective synonyms of the nomenclatural type species, Rickettsiella popilliae. 'R. agriotidis' forms, therefore, part of a Rickettsiella pathotype complex that most likely represents the species R. popilliae. As there are currently no genetic data available from the R. popilliae type strain, the respective assignment cannot be corroborated directly. However, an alternative taxonomic assignment to the species Rickettsiella grylli has been positively ruled out by significance testing. MLSA has been shown to provide a more powerful tool for taxonomic delineation within the genus Rickettsiella as compared to 16S rRNA phylogenetics. However, the limitations of the present MLSA scheme for the sub-species level classification of 'R. agriotidis' and further R. popilliae synonyms has been critically evaluated.

A Rickettsiella bacterium from the hard tick, Ixodes woodi: molecular taxonomy combining multilocus sequence typing (MLST) with significance testing.

Hard ticks (Acari: Ixodidae) are known to harbour intracellular bacteria from several phylogenetic groups that can develop both mutualistic and pathogenic relationships to the host. This is of particular importance for public health as tick derived bacteria can potentially be transmitted to mammals, including humans, where e.g. Rickettsia or Coxiella act as severe pathogens. Exact molecular taxonomic identification of tick associated prokaryotes is a necessary prerequisite of the investigation of their relationship to both the tick and possible vertebrate hosts. Previously, an intracellular bacterium had been isolated from a monosexual, parthenogenetically reproducing laboratory colony of females of the hard tick, Ixodes woodi Bishopp, and had preliminarily been characterized as a "Rickettsiella-related bacterium". In the present molecular taxonomic study that is based on phylogenetic reconstruction from both 16 S ribosomal RNA and protein-encoding marker sequences complemented with likelihood-based significance testing, the bacterium from I. woodi has been identified as a strain of the taxonomic species Rickettsiella grylli. It is the first time that a multilocus sequence typing (MLST) approach based on a four genes comprising MLST scheme has been implemented in order to classify a Rickettsiella-like bacterium to this species. The study demonstrated that MLST holds potential for a better resolution of phylogenetic relationships within the genus Rickettsiella, but requires sequence determination from further Rickettsiella-like bacteria in order to complete the current still fragmentary picture of Rickettsiella systematics.

Multilocus sequence typing (MLST) for the infra-generic taxonomic classification of entomopathogenic Rickettsiella bacteria.

The genus Rickettsiella comprises intracellular bacterial pathogens of a wide range of arthropods that are currently classified in four recognized species and numerous further pathotypes. However, both the delineation of and the synonymization of pathotypes with species are highly problematic. In the sequel of a previous phylogenomic study at the supra-generic level, nine selected genes - the 16S and 23S rRNA genes and the protein-encoding genes dnaG, ftsY, gidA, ksgA, rpoB, rpsA, and sucB - were evaluated for their potential as markers for the generic and infra-generic taxonomic classification of Rickettsiella-like bacteria. A methodological approach combining phylogenetic reconstruction with likelihood-based significance testing was employed on the basis of sequence data from the species Rickettsiella grylli and Rickettsiella popilliae, pathotypes 'Rickettsiella melolonthae' and 'Rickettsiella tipulae'. This study provides the first multilocus sequence typing (MLST) data for the genus Rickettsiella and identifies two new genetic markers, gidA and sucB, for the infra-generic classification within this taxon. In particular, aforesaid genes were found more reliable and informative markers than the corresponding 16S rRNA-encoding sequences that failed to produce strictly significant infra-generic taxonomic assignments. However, gidA- and sucB-based phylogenies were consistent with the currently accepted view of species delineation and species-pathotype synonymization within the genus Rickettsiella.

Genetic and electron-microscopic characterization of Rickettsiella bacteria from the manuka beetle, Pyronota setosa (Coleoptera: Scarabaeidae).

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

Genetic and electron-microscopic characterization of 'Rickettsiella agriotidis', a new Rickettsiella pathotype associated with wireworm, Agriotes sp. (Coleoptera: Elateridae).

Wireworms, the polyphagous larvae of click beetles belonging to the genus Agriotes (Coleoptera: Elateridae), are severe and widespread agricultural pests affecting numerous crops. A previously unknown intracellular bacterium has been identified in a diseased Agriotes larva. Microscopic studies revealed the subcellular structures characteristic of Rickettsiella infections. Molecular phylogenetic analysis based on 16S ribosomal RNA and signal recognition particle receptor (FtsY) encoding sequences demonstrates that the wireworm pathogen belongs to the taxonomic genus Rickettsiella. Therefore, the new pathotype designation 'R. agriotidis' is proposed to refer to this organism. Moreover, genetic analysis makes it likely that--on the basis of the currently accepted organization of the genus Rickettsiella--this new pathotype should be considered a synonym of the nomenclatural type species, Rickettsiella popilliae.

Comparative genomics using microarrays reveals divergence and loss of virulence-associated genes in host-specific strains of the insect pathogen Metarhizium anisopliae.

Many strains of Metarhizium anisopliae have broad host ranges, but others are specialists and adapted to particular hosts. Patterns of gene duplication, divergence, and deletion in three generalist and three specialist strains were investigated by heterologous hybridization of genomic DNA to genes from the generalist strain Ma2575. As expected, major life processes are highly conserved, presumably due to purifying selection. However, up to 7% of Ma2575 genes were highly divergent or absent in specialist strains. Many of these sequences are conserved in other fungal species, suggesting that there has been rapid evolution and loss in specialist Metarhizium genomes. Some poorly hybridizing genes in specialists were functionally coordinated, indicative of reductive evolution. These included several involved in toxin biosynthesis and sugar metabolism in root exudates, suggesting that specialists are losing genes required to live in alternative hosts or as saprophytes. Several components of mobile genetic elements were also highly divergent or lost in specialists. Exceptionally, the genome of the specialist cricket pathogen Ma443 contained extra insertion elements that might play a role in generating evolutionary novelty. This study throws light on the abundance of orphans in genomes, as 15% of orphan sequences were found to be rapidly evolving in the Ma2575 lineage.