Genome-assisted development of nuclear intergenic sequence markers for entomopathogenic fungi of the Metarhizium anisopliae species complex.

Entomopathogenic fungi in the genus Metarhizium are useful for biological control programmes against economically important arthropod pests worldwide. However, understanding the true diversity and ecology of these organisms is hampered by convergent morphologies between species. The application of molecular techniques has enabled greater resolution of species than allowed by morphology alone. In particular, the commonly used biocontrol agent M. anisopliae was found to be a species complex composed of nine species. This prior work was conducted with commonly used markers in fungal phylogenetics (BTUB, RPB1, RPB2 and TEF), which likely under-represent diversity in the M. anisopliae complex. Using sequence data from nuclear genomes of M. acridum and M. robertsii we identified regions of conserved gene synteny and developed primers to amplify intergenic regions of seven loci. Using ex-type and authenticated tissue specimens for species in the M. anisopliae complex, we demonstrate that sequence data derived from intergenic loci is more variable and phylogenetically informative than previously available markers. These new markers will facilitate investigations at or below the species level for the M. anisopliae complex. The method of marker development employed here should be extendable to any group with sufficiently divergent genome data available.

A PCR-based tool for cultivation-independent detection and quantification of Metarhizium clade 1.

The entomopathogenic fungus Metarhizium anisopliae and sister species are some of the most widely used biological control agents for insects. Availability of specific monitoring and quantification tools are essential for the investigation of environmental factors influencing their environmental distribution. Naturally occurring as well as released Metarhizium strains in the environment traditionally are monitored with cultivation-dependent techniques. However, specific detection and quantification may be limited due to the lack of a defined and reliable detection range of such methods. Cultivation-independent PCR-based detection and quantification tools offer high throughput analyses of target taxa in various environments. In this study a cultivation-independent PCR-based method was developed, which allows for specific detection and quantification of the defined Metarhizium clade 1, which is formed by the species Metarhizium majus, Metarhizium guizhouense, Metarhizium pingshaense, Metarhizium anisopliae, Metarhizium robertsii and Metarhiziumbrunneum, formerly included in the M. anisopliae cryptic species complex. This method is based on the use of clade-specific primers, i.e. Ma 1763 and Ma 2097, that are positioned within the internal transcribed spacer regions 1 and 2 of the nuclear ribosomal RNA gene cluster, respectively. BLAST similarity searches and empirical specificity tests performed on target and non-target species, as well as on bulk soil DNA samples, demonstrated specificity of this diagnostic tool for the targeted Metarhizium clade 1. Testing of the primer pair in qPCR assays validated the diagnostic method for specific quantification of Metarhizium clade 1 in complex bulk soil DNA samples that significantly correlated with cultivation-dependent quantification. The new tool will allow for highly specific and rapid detection and quantification of the targeted Metarhizium clade 1 in the environment. Habitat with high Metarhizium clade 1 densities can then be analyzed for habitat preferences in greater detail using cultivation-dependent techniques and genetic typing of isolates.

Asperisporium and Pantospora (Mycosphaerellaceae): epitypifications and phylogenetic placement.

The species-rich family Mycosphaerellaceae contains considerable morphological diversity and includes numerous anamorphic genera, many of which are economically important plant pathogens. Recent revisions and phylogenetic research have resulted in taxonomic instability. Ameliorating this problem requires phylogenetic placement of type species of key genera. We present an examination of the type species of the anamorphic Asperisporium and Pantospora. Cultures isolated from recent port interceptions were studied and described, and morphological studies were made of historical and new herbarium specimens. DNA sequence data from the ITS region and nLSU were generated from these type species, analysed phylogenetically, placed into an evolutionary context within Mycosphaerellaceae, and compared to existing phylogenies. Epitype specimens associated with living cultures and DNA sequence data are designated herein. Asperisporium caricae, the type of Asperisporium and cause of a leaf and fruit spot disease of papaya, is closely related to several species of Passalora including P. brachycarpa. The status of Asperisporium as a potential generic synonym of Passalora remains unclear. The monotypic genus Pantospora, typified by the synnematous Pantospora guazumae, is not included in Pseudocercospora sensu stricto or sensu lato. Rather, it represents a distinct lineage in the Mycosphaerellaceae in an unresolved position near Mycosphaerella microsora.

Barcoding generalist predators by polymerase chain reaction: carabids and spiders.

Identification of arthropod predators is challenging when closely related species are found at a given locality. Identification of the immature stages is especially problematic, because distinguishing morphological features are difficult to use or have not been described. We used polymerase chain reaction (PCR) to distinguish closely related carabids and spiders, and to match eggs and larvae (or nymphs) with identified adult parents. Within the Carabidae, we amplified species-specific mitochondrial cytochrome oxidase I (COI) fragments for three species each in the genera Poecilus and Harpalus, and two each in Chlaenius and Bembidion. Within the Araneae, we amplified species-specific COI fragments for two Hibana species (Anyphaenidae), Pardosa milvina and Rabidosa rabida (Lycosidae), Frontinella communis and Grammonota texana (Linyphiidae), and Cheiracanthium inclusum (Miturgidae). We are able to correctly identify all immature stages tested--eggs, larvae (or nymphs) and pupae--by comparison of the amplified fragments with those of the adults. Using COI markers as species identifiers is a tenet of the Barcode of Life initiative, an international consortium to provide a molecular identifier for every animal species.

Partial incompatibility between ants and symbiotic fungi in two sympatric species of Acromyrmex leaf-cutting ants.

We investigate the nature and duration of incompatibility between certain combinations of Acromyrmex leaf-cutting ants and symbiotic fungi, taken from sympatric colonies of the same or a related species. Ant-fungus incompatibility appeared to be largely independent of the ant species involved, but could be explained partly by genetic differences among the fungus cultivars. Following current theoretical considerations, we develop a hypothesis, originally proposed by S. A. Frank, that the observed incompatibilities are ultimately due to competitive interactions between genetically different fungal lineages, and we predict that the ants should have evolved mechanisms to prevent such competition between cultivars within a single garden. This requires that the ants are able to recognize unfamiliar fungi, and we show that this is indeed the case. Amplified fragment length polymorphism genotyping further shows that the two sympatric Acromyrmex species share each other's major lineages of cultivar, confirming that horizontal transfer does occasionally take place. We argue and provide some evidence that chemical substances produced by the fungus garden may mediate recognition of alien fungi by the ants. We show that incompatibility between ants and transplanted, genetically different cultivars is indeed due to active killing of the novel cultivar by the ants. This incompatibility disappears when ants are force-fed the novel cultivar for about a week, a result that is consistent with our hypothesis of recognition induced by the resident fungus and eventual replacement of incompatibility compounds during force-feeding.

First Report of Phytophthora heveae and Pythium spp. on Tropical Tree Seedlings in Panama.

Damping-off was observed on experimentally planted seedlings of Ana-cardium excelsum (wild cashew), a timber tree, and Tetragastris panamensis, a canopy tree, within lowland tropical rain forest in Panama. Disease impact was greatest during the wet season (May through December). During the 1995 wet season, 40.7% (572/1,404) of T. panamensis seedlings died due to damping-off disease. Sixty-eight percent (703/1,034) of A. excelsum seed lings died due to damping-off during the 1996 wet season. Symptoms included leaf, cotyledon, and stem necrosis. Phytophthora heveae sporangia were observed on both host species, and oospores were found within stems of T. panamensis. Plating of diseased A. excelsum seedlings on potato dextrose agar with rifampicin (25 mg/ml) and pimaricin (10 mg/ml) produced cultures of Phytophthora heveae and Pythium from 27.4% (110/402) and 44.5% (179/402) of seedlings, respectively. Pythium isolates included P. vexans, P. splendens, and P. chamaehyphon species types, but P. vexans species types accounted for 70% of the Pythium isolates. Disease symptoms on experimental seedlings also were evident on naturally occurring seedlings. Mycelial plugs from six A. excelsum isolates of Phytophthora heveae were used to separately inoculate stems of three A. excelsum seedlings each. Of 18 seedlings inoculated, 88.8% developed characteristic symptoms and died in an average of 8.7 ± 1.0 (standard error [SE]) days. Nine Pythium isolates were used to separately inoculate stems of one to three A. excelsum seedlings each; three of these isolates were known to be P. vexans species types. All of the 20 seedlings inoculated with a Pythium isolate developed characteristic symptoms and died in an average of 6.1 ± 0.3 (SE) days. Both Phytophthora heveae and Pythium isolates were reisolated readily from diseased seedlings. Cotyledons and stems of seven to eight T. panamensis seedlings per isolate were inoculated with two Phytophthora heveae isolates originating from T. panamensis. Necrotic lesions on cotyledons consistent with field symptoms developed on 33.3% of 15 seedlings, but disease did not spread within the stem. Measurements of key morphological structures and cardinal temperatures of four Phytophthora heveae isolates from A. excelsum were consistent with published species descriptions (1), except (i) sporangia with two apices were present, although infrequent; (ii) chlamydospores were produced; and (iii) antheridia were narrower and often shorter than published measurements (7 to 12 m long; 2 to 6 m wide). Internal transcribed spacer (ITS) sequences from Phytophthora heveae isolates cultured from A. excelsum and T. panamensis were matched to reference sequences of Phytophthora heveae with only 3-bp differences (2). ITS sequences for isolates of Pythium vexans, P. splendens, and P. chamaehyphon species types clustered within clades of reference strains of these species (C. A. Lévesque, personal communication). Phytophthora heveae and Pythium spp. have been reported from the tropics. However, this is the first report of these pathogens on seedlings of A. excelsum and T. panamensis. Reference: (1) D. C. Erwin and O. K. Ribeiro. 1996. Phytophthora Diseases Worldwide. The American Phytopathological Society, St. Paul, MN, pp. 100-107, 336-337. (2) D. E. L. Cook et al. A molecular phylogeny of Phytophthora and related Oomy-cetes. Fungal Genet. & Biol. In press.

Phylogenetic relationships of agaric fungi based on nuclear large subunit ribosomal DNA sequences.

Phylogenetic relationships of mushrooms and their relatives within the order Agaricales were addressed by using nuclear large subunit ribosomal DNA sequences. Approximately 900 bases of the 5' end of the nucleus-encoded large subunit RNA gene were sequenced for 154 selected taxa representing most families within the Agaricales. Several phylogenetic methods were used, including weighted and equally weighted parsimony (MP), maximum likelihood (ML), and distance methods (NJ). The starting tree for branch swapping in the ML analyses was the tree with the highest ML score among previously produced MP and NJ trees. A high degree of consensus was observed between phylogenetic estimates obtained through MP and ML. NJ trees differed according to the distance model that was used; however, all NJ trees still supported most of the same terminal groupings as the MP and ML trees did. NJ trees were always significantly suboptimal when evaluated against the best MP and ML trees, by both parsimony and likelihood tests. Our analyses suggest that weighted MP and ML provide the best estimates of Agaricales phylogeny. Similar support was observed between bootstrapping and jackknifing methods for evaluation of tree robustness. Phylogenetic analyses revealed many groups of agaricoid fungi that are supported by moderate to high bootstrap or jackknife values or are consistent with morphology-based classification schemes. Analyses also support separate placement of the boletes and russules, which are basal to the main core group of gilled mushrooms (the Agaricineae of Singer). Examples of monophyletic groups include the families Amanitaceae, Coprinaceae (excluding Coprinus comatus and subfamily Panaeolideae), Agaricaceae (excluding the Cystodermateae), and Strophariaceae pro parte (Stropharia, Pholiota, and Hypholoma); the mycorrhizal species of Tricholoma (including Leucopaxillus, also mycorrhizal); Mycena and Resinomycena; Termitomyces, Podabrella, and Lyophyllum; and Pleurotus with Hohenbuehelia. Several groups revealed by these data to be nonmonophyletic include the families Tricholomataceae, Cortinariaceae, and Hygrophoraceae and the genera Clitocybe, Omphalina, and Marasmius. This study provides a framework for future systematics studies in the Agaricales and suggestions for analyzing large molecular data sets.

Molecular determination of species boundaries in corals: genetic analysis of the Montastraea annularis complex using amplified fragment length polymorphisms and a microsatellite marker.

Analyses of DNA have not been widely used to distinguish coral sibling species. The three members of the Montastraea annularis complex represent an important test case: they are widely studied and dominate Caribbean reefs, yet their taxonomic status remains unclear. Analysis of amplified fragment length polymorphisms (AFLPs) and a microsatellite locus, using DNA from sperm, showed that Montastraea faveolata is genetically distinct. One AFLP primer yielded a diagnostic product (880 bp in M. faveolata 920 bp in M. franksi and M. annularis) whose homology was established by DNA sequencing. A second primer revealed a 630 bp band that was fixed in M. faveolata, and rare in M. franksi and M. annularis; in this case homologies were confirmed by Southern hybridizations. A tetranucleotide microsatellite locus with several alleles exhibited strong frequency differences between M. faveolata and the other two taxa. We did not detect comparable differences between M. annularis and M. franksi with either AFLPs (12 primers screened) or the microsatellite locus. Comparisons of AFLP patterns obtained from DNA from sperm, somatic tissues, and zooxanthellae suggest that the technique routinely amplifies coral (animal) DNA. Thus analyses based on somatic tissues may be feasible, particularly after diagnostic differences have been established using sperm DNA.

Pathogenic and Molecular Characterization of Three Phomopsis Isolates from Peach, Plum, and Asian Pear.

Three isolates of Phomopsis, causing shoot blight of peach, shoot tissue necrosis of plum, or bud death of Hosui Asian pear, respectively, were evaluated for their pathogenicity on apple, pear, peach, and plum. Current year's shoots of 1-year-old Stayman Winesap apple, Barlett pear, Babygold-7 peach, and Bruce plum trees were inoculated with each isolate by wounding a bud and applying agar blocks bearing young hyphae. The length of cankers on shoots was measured 10, 17, and 24 days after inoculation. Cankers developed on shoots of all hosts inoculated with the peach isolate and on peach shoots inoculated with plum and Asian pear isolates. No cankers developed on apple, pear, or plum shoots inoculated with plum and Asian pear isolates. In the first experiment, 10 days after inoculation, the length of cankers on apple trees (56.0 mm) inoculated with the peach isolate was not significantly different (P ≥ 0.05) from that on peach (42.8 mm), but was significantly greater than that on plum (25.3 mm) and pear (13.1 mm). The cankers on peach were significantly longer than those on pear, but not on plum. Cankers on all four hosts were significantly different from one another 17 and 24 days after inoculation. There was no significant difference between the length of cankers on peach shoots inoculated with plum and Asian pear isolates, and they were significantly smaller than those inoculated with the peach isolate. None of the control trees developed cankers. The three isolates differed in colony morphology, and appearance of conidiomata, conidiogenous cells, and α-conidia on potato-dextrose agar. None of the isolates produced ß-conidia in culture. Multi-locus DNA fingerprint analysis and internal transcribed spacer sequence comparisons revealed similarities between the plum and Asian pear isolates but a significant difference between these two and the peach isolate. The results indicate that the Phomopsis sp. that causes shoot blight of peach has the potential to cause disease on other stone and pome fruits, and peach may also be susceptible to isolates of Phomopsis from different tree fruit hosts.

Evolutionary history of the symbiosis between fungus-growing ants and their fungi.

The evolutionary history of the symbiosis between fungus-growing ants (Attini) and their fungi was elucidated by comparing phylogenies of both symbionts. The fungal phylogeny based on cladistic analyses of nuclear 28S ribosomal DNA indicates that, in contrast with the monophyly of the ants, the attine fungi are polyphyletic. Most cultivated fungi belong to the basidiomycete family Lepiotaceae; however, one ant genus, Apterostigma, has acquired a distantly related basidiomycete lineage. Phylogenetic patterns suggest that some primitive attines may have repeatedly acquired lepiotaceous symbionts. In contrast, the most derived attines have clonally propagated the same fungal lineage for at least 23 million years.

Phylogeny of mycoplasmalike organisms (phytoplasmas): a basis for their classification.

A global phylogenetic analysis using parsimony of 16S rRNA gene sequences from 46 mollicutes, 19 mycoplasmalike organisms (MLOs) (new trivial name, phytoplasmas), and several related bacteria placed the MLOs definitively among the members of the class Mollicutes and revealed that MLOs form a large discrete monophyletic clade, paraphyletic to the Acholeplasma species, within the Anaeroplasma clade. Within the MLO clade resolved in the global mollicutes phylogeny and a comprehensive MLO phylogeny derived by parsimony analyses of 16S rRNA gene sequences from 30 diverse MLOs representative of nearly all known distinct MLO groups, five major phylogenetic groups with a total of 11 distinct subclades (monophyletic groups or taxa) could be recognized. These MLO subclades (roman numerals) and designated type strains were as follows: i, Maryland aster yellows AY1; ii, apple proliferation AP-A; iii, peanut witches'-broom PnWB; iv, Canada peach X CX; v, rice yellow dwarf RYD; vi, pigeon pea witches'-broom PPWB; vii, palm lethal yellowing LY; viii, ash yellows AshY; ix, clover proliferation CP; x, elm yellows EY; and xi, loofah witches'-broom LfWB. The designations of subclades and their phylogenetic positions within the MLO clade were supported by a congruent phylogeny derived by parsimony analyses of ribosomal protein L22 gene sequences from most representative MLOs. On the basis of the phylogenies inferred in the present study, we propose that MLOs should be represented taxonomically at the minimal level of genus and that each phylogenetically distinct MLO subclade identified should represent at least a distinct species under this new genus.