Three novel Ambrosia Fusarium Clade species producing clavate macroconidia known (F. floridanum and F. obliquiseptatum) or predicted (F. tuaranense) to be farmed by Euwallacea spp. (Coleoptera: Scolytinae) on woody hosts.

The Ambrosia Fusarium Clade (AFC) comprises at least 16 genealogically exclusive species-level lineages within clade 3 of the Fusarium solani species complex (FSSC). These fungi are either known or predicted to be farmed by Asian Euwallacea ambrosia beetles (Coleoptera: Curculionidae: Scolytinae) in the tribe Xyleborini as a source of nutrition. To date, only 4 of the 16 AFC lineages have been described formally. In the absence of Latin binomials, an ad hoc nomenclature was developed to distinguish the 16 species lineages as AF-1 to AF-16. Herein, Fusarium species AF-3, AF-5, and AF-7 were formally described as F. floridanum, F. tuaranense, and F. obliquiseptatum, respectively. Fusarium floridanum farmed by E. interjectus on box elder (Acer negundo) in Gainesville, Florida, was distinguished morphologically by the production of sporodochial conidia that were highly variable in size and shape together with greenish-pigmented chlamydospores. Fusarium tuaranense was isolated from a beetle-damaged Para rubber tree (Hevea brasiliense) in North Borneo, Malaysia, and was diagnosed by production of the smallest sporodochial conidia of any species within the AFC. Lastly, F. obliquiseptatum was farmed by an unnamed ambrosia beetle designated Euwallacea sp. 3 (E. fornicatus species complex) on avocado (Persea americana) in Queensland, Australia. It uniquely produces some clavate sporodochial conidia with oblique septa. Maximum likelihood analysis of a multilocus data set resolved these three novel AFC taxa as phylogenetically distinct species based on genealogical concordance. Particularly where introduced into exotic environments, these exotic mutualists pose a serious threat to the avocado industry, native forests, and urban landscapes in diverse regions throughout the world.

AM fungi facilitate the competitive growth of two invasive plant species, Ambrosia artemisiifolia and Bidens pilosa.

Invasive species often cause enormous economic and ecological damage, and this is especially true for invasive plants in the Asteraceae family. Arbuscular mycorrhizal fungi (AMF) play an important role in the successful invasion by exotic plant species because of their ability to promote growth and influence interspecific competition. However, few studies have evaluated the effects of invasive Asteraceae species on AMF diversity and how feedback mechanisms during competition with native species subsequently affect the accumulation of nutrient resources. Two exotic Asteraceae, Ambrosia artemisiifolia and Bidens pilosa, were monitored during competition with a native grass species, Setaria viridis, which is being replaced by these exotic species in natural areas around the study site. From these species continuously maintained in a field plot for 5 years, we collected the rhizosphere soil and cloned and identified soil AMF. Furthermore, AM fungal spores were isolated from rhizosphere soil of the two invasive species and used as inoculum in greenhouse experiments, to compare growth and nutrient accumulation during competition. The results indicate that although the AMF diversity in the rhizosphere soil of A. artemisiifolia and B. pilosa differed, the three most abundant species (Septoglomus viscosum, Septoglomus constrictum, Glomus perpusillum) were identical. The addition of AMF inoculum changed the competition between the plants, increasing the competitive ability of the invasives and decreasing that of the native. The results show a similar AMF community composition between A. artemisiifolia and B. pilosa, increased AMF root colonization of the invasive species during competition, AMF-enhanced N accumulation, and AMF-facilitated competitive growth of the invasive species.

Deciphering the biology of Cryptophyllachora eurasiatica gen. et sp. nov., an often cryptic pathogen of an allergenic weed, Ambrosia artemisiifolia.

A little known, unculturable ascomycete, referred to as Phyllachora ambrosiae, can destroy the inflorescences of Ambrosia artemisiifolia, an invasive agricultural weed and producer of highly allergenic pollen. The fungus often remains undetectable in ragweed populations. This work was conducted to understand its origin and pathogenesis, a prerequisite to consider its potential as a biocontrol agent. The methods used included light and transmission electron microscopy, nrDNA sequencing, phylogenetic analyses, artificial inoculations, and the examination of old herbarium and recent field specimens from Hungary, Korea, Ukraine and USA. The Eurasian and the North American specimens of this fungus were to represent two distinct, although closely related lineages that were only distantly related to other lineages within the Ascomycota. Consequently, we describe a new genus that includes Cryptophyllachora eurasiatica gen. et sp. nov. and C. ambrosiae comb. nov., respectively. The pathogenesis of C. eurasiatica was shown in A. artemisiifolia. No evidence was found for either seed-borne transmission or systemic infection. Two hypotheses were developed to explain the interaction between C. eurasiatica and A. artemisiifolia: (i) as yet undetected seed-borne transmissions and latent, systemic infections; or (ii) alternative hosts.

Wood decay fungus Flavodon ambrosius (Basidiomycota: Polyporales) is widely farmed by two genera of ambrosia beetles.

The ambrosia fungus Flavodon ambrosius is the primary nutritional mutualist of ambrosia beetles Ambrosiodmus and Ambrosiophilus in North America. F. ambrosius is the only known ambrosial basidiomycete, unique in its efficient lignocellulose degradation. F. ambrosius is associated with both native American beetle species and species introduced from Asia. It remains unknown whether F. ambrosius is strictly a North American fungus, or whether it is also associated with these ambrosia beetle genera on other continents. We isolated fungi from the mycangia and galleries of ambrosia beetles Ambrosiodmus rubricollis, Ambrosiodmus minor, Ambrosiophilus atratus, and Ambrosiophilus subnepotulus in China, South Korea, and Vietnam. Phylogenetic analyses suggest that all Asian and North American isolates represent a single haplotype. These results confirm Flavodon ambrosius as the exclusive mutualistic fungus of multiple Ambrosiodmus and Ambrosiophilus beetle species around the world, making it the most widespread known ambrosia fungus species, both geographically and in terms of the number of beetle species. The Flavodon-beetle symbiosis appears to employ an unusually strict mechanism for maintaining fidelity, compared to the symbioses of the related Xyleborini beetles, which mostly vector more dynamic fungal communities.

An affinity-effect relationship for microbial communities in plant-soil feedback loops.

Feedback loops involving soil microorganisms can regulate plant populations. Here, we hypothesize that microorganisms are most likely to play a role in plant-soil feedback loops when they possess an affinity for a particular plant and the capacity to consistently affect the growth of that plant for good or ill. We characterized microbial communities using whole-community DNA fingerprinting from multiple "home-and-away" experiments involving giant ragweed (Ambrosia trifida L.) and common sunflower (Helianthus annuus L.), and we looked for affinity-effect relationships in these microbial communities. Using canonical ordination and partial least squares regression, we developed indices expressing each microorganism's affinity for ragweed or sunflower and its putative effect on plant biomass, and we used linear regression to analyze the relationship between microbial affinity and effect. Significant linear affinity-effect relationships were found in 75 % of cases. Affinity-effect relationships were stronger for ragweed than for sunflower, and ragweed affinity-effect relationships showed consistent potential for negative feedback loops. The ragweed feedback relationships indicated the potential involvement of multiple microbial taxa, resulting in strong, consistent affinity-effect relationships in spite of large-scale microbial variability between trials. In contrast, sunflower plant-soil feedback may involve just a few key players, making it more sensitive to underlying microbial variation. We propose that affinity-effect relationship can be used to determine key microbial players in plant-soil feedback against a low "signal-to-noise" background of complex microbial datasets.

Are communities of microbial symbionts more diverse than communities of macrobial hosts?

In this study, fungal viruses (mycoviruses) of plant-associated fungi were used to test the general assertion that communities of parasitic and mutualistic symbionts may be more species-diverse than communities of their hosts. Mycoviruses are poorly studied in general, but can affect the fitness and ecology of the fungi and plants with which they associate. To date, mycovirus incidence and diversity in natural communities remain largely unaddressed. Here, we compared the incidence and diversity of fungi associated with tallgrass prairie plants to the diversity and incidence of mycoviruses within those fungi. Specifically, we sampled viruses from fungi associated with a parasitic plant (Cuscuta cuspidata) and its most frequent host plant (Ambrosia psilostachya) in a tallgrass prairie habitat in Oklahoma. For each plant sample we cultured fungal endophytes from surface-sterilized above-ground tissues. From the cultured fungi we extracted DNA to identify fungi, and extracted double-stranded RNA (dsRNA) to detect mycoviruses. Mycoviruses were further characterized using reverse transcription-PCR and sequence analyses. We found at least 25 fungal taxa associated with the two plants, and 10 % of these fungi contained readily detectable viruses. Several mycovirus types were shared among fungal taxa, indicating that mycoviruses may be less specialized than originally thought. Although the virus community was not as diverse as the fungal endophyte community (16 taxa), species accumulation rates of mycoviruses (inferred from rescaled rarefaction curves) may be higher than those of their associated fungal hosts. Thus, mycoviruses represent a further layer of undocumented biodiversity in ecological communities.

Isolations from the redbay ambrosia beetle, Xyleborus glabratus, confirm that the laurel wilt pathogen, Raffaelea lauricola, originated in Asia.

The laurel wilt pathogen Raffaelea lauricola was hypothesized to have been introduced to the southeastern USA in the mycangium of the redbay ambrosia beetle, Xyleborus glabratus, which is native to Asia. To test this hypothesis adult X. glabratus were trapped in Taiwan and on Kyushu Island, Japan, in 2009, and dead beetles were sent to USA for isolation of fungal symbionts. Individual X. glabratus were macerated in glass tissue grinders, and the slurry was serially diluted and plated onto malt agar medium amended with cycloheximide, a medium semiselective for Ophiostoma species and their anamorphs, including members of Raffaelea. R. lauricola was isolated from 56 of 85 beetles in Taiwan and 10 of 16 beetles in Japan at up to an estimated 10 000 CFUs per beetle. The next most commonly isolated species was R. ellipticospora, which also has been recovered from X. glabratus trapped in the USA, as were two other fungi isolated from beetles in Taiwan, R. fusca and R. subfusca. Three unidentified Raffaelea spp. and three unidentified Ophiostoma spp. were isolated rarely from X. glabratus collected in Taiwan. Isolations from beetles similarly trapped in Georgia, USA, yielded R. lauricola and R. ellipticospora in numbers similar to those from beetles trapped in Taiwan and Japan. The results support the hypothesis that R. lauricola was introduced into the USA in mycangia of X. glabratus shipped to USA in solid wood packing material from Asia. However differences in the mycangial mycoflora of X. glabratus in Taiwan, Japan and USA suggest that the X. glabratus population established in USA originated in another part of Asia.

Evidence for a new lineage of primary ambrosia fungi in Geosmithia Pitt (Ascomycota: Hypocreales).

Geosmithia is a genus of mitosporic filamentous fungi typically associated with phloeophagous bark beetles world-wide. During this study, the fungal associates of ambrosia beetles Cnesinus lecontei, Eupagiocerus dentipes, and Microcorthylus sp. from Costa Rica, were studied using morphology and DNA sequences. Fungal associates belonged to four undescribed Geosmithia species. Geosmithia eupagioceri sp. nov. and G. microcorthyli sp. nov. are evidently primary ambrosia fungi of their respective vectors E. dentipes and Microcorthylus species. They both have convergently evolved distinct morphological adaptations including the production of large, solitary and globose conidia, and yeast-like cells. Tunnels of C. lecontei contained an undescribed Geosmithia species, but its nutritional importance for its vector is unclear. An auxiliary ambrosia fungus, Geosmithia rufescens sp. nov., was found associated with both G. eupagioceri and the Geosmithia species associated with C. lecontei. G. microcorthyli is genetically quite similar to the phloem-associated Geosmithia sp. 8 from Europe. Large differences in morphology between these two species suggest the rapid co-evolution resulting from the close symbiosis of the former with its beetle host. The ITS rDNA sequences of G. microcorthyli and Geosmithia sp. 8 were not diagnostic, suggesting that alternative markers such as EF-1α, IGS rDNA or ß-tubulin should be used, together with morphological and ecological data, for species delimitation in this genus. The primary ambrosia fungi described here are derived from phloem-associated ancestors, and represent two independent lineages of ambrosia fungi in the Hypocreales and a new ecological strategy within Geosmithia.

Multigene phylogeny of filamentous ambrosia fungi associated with ambrosia and bark beetles.

Most 'ambrosia' fungi are members of a heterogeneous group of ophiostomatoids that includes the anamorph genera Ambrosiella, Raffaelea and Dryadomyces. The taxonomy of these fungi based on morphological features has been complicated by these features being poorly descriptive and having evolved convergently. In this work we report maximum parsimony and Bayesian phylogenetic analysis of a multigene dataset (nSSU rDNA, nLSU rDNA and beta-tubulin gene) from sixty-seven taxa that include members of genera Ambrosiella, Raffaelea and Dryadomyces and a diverse set of ophiostomatoid relatives. We discuss the phylogenetic status of genus Ambrosiella and its relationships with representatives of Ophiostomatales teleomorph and anamorph genera. Our analysis shows that ten of the thirteen species that had been assigned to the genus Ambrosiella are related to the teleomorph genera Grosmannia or Ophiostoma, within the Ophiostomatales. The multigene analysis and expanded taxon samplings provide a higher resolution for the species phylogeny and clarify detailed relationships between Ambrosiella associates of ambrosia and bark beetles and the closely related species of genera Raffaelea and Dryadomyces. We discuss difficulties in using the morphology of conidiophores and the mode of conidiogenesis to re-define the phylogenetic classification of Ambrosiella species. Finally, we report a correlation between the molecular classification of Ophiostomatales-related species of Ambrosiella and Raffaelea and their ecological niches.

Initial genetic analysis of Xylella fastidiosa in Texas.

Xylella fastidiosa is the causative agent of Pierce's Disease of grape. No published record of X. fastidiosa genetics in Texas exists despite growing financial risk to the U.S. grape industry, a Texas population of the glassy-winged sharpshooter insect vector (Homalodisca vitripennis) now spreading in California, and evidence that the bacterium is ubiquitous to southern states. Using sequences of conserved gyrB and mopB genes, we have established at least two strains in Texas, grape strain and ragweed strain, corresponding genetically with subsp. piercei and multiplex, respectively. The grape strain in Texas is found in Vitis vinifera varieties, hybrid vines, and wild Vitis near vineyards, whereas the ragweed strain in Texas is found in annuals, shrubs, and trees near vineyards or other areas. RFLP and QRT PCR techniques were used to differentiate grape and ragweed strains with greater efficiency than sequencing and are practical for screening numerous X. fastidiosa isolates for clade identity.

Which role can arbuscular mycorrhizal fungi play in the facilitation of Ambrosia artemisiifolia L. invasion in France?

Ambrosia artemisiifolia L. (common ragweed), an annual invasive plant, was introduced more than 100 years ago from North America to Europe. Like the majority of other invasive plants in Europe, it develops in open, disturbed areas such as fields, wastelands, roadsides, and riverbanks. Recently, arbuscular mycorrhizal fungi (AMF) have been suspected to play a role in some plant invasion processes. As the common ragweed is known to be colonized by AMF in its native range, the intensity of mycorrhizal root colonization was studied in 35 natural populations in eastern France. About 94% of the A. artemisiifolia populations sampled were mycorrhizal. Root colonization levels varied from 1 to 40% depending on the ecological sites, with lower levels for agricultural habitats and higher levels in disturbed sites, such as wastelands or roadsides. A subsequent greenhouse experiment showed positive impacts of AMF on the growth and development of A. artemisiifolia. It is proposed that the spread of this invasive plant species could be facilitated by AMF, underlining the need to integrate symbiotic interactions in future work on invasive plant processes.

Dryadomyces amasae: a nutritional fungus associated with ambrosia beetles of the genus Amasa (Coleoptera: Curculionidae, Scolytinae).

During surveys of woodlands in Taiwan a previously undescribed fungus Dryadomyces amasae gen. sp. nov., was found in the sapwood of dead angiosperm timbers in the gallery systems of the scolytine ambrosia beetle Amasa concitatus. The fungus grows predominantly in the immediate vicinity of the feeding beetle larvae and serves as a nutritional ambrosia fungus. The transmission of D. amasae to new breeding substrates is ensured by an oral mycetangium, a paired organ which was found in A. concitatus and A. aff. glaber near the mandibles and contained multiple cells of the fungus. Morphologically D. amasae resembles species of Ambrosiella. However, phylogenetic analysis based on partial nucSSU rDNA placed the fungus with certain species of the genus Ambrosiella within the Ophiostomatales, whereas the type species of Ambrosiella, A. xylebori, was assigned to the Microascales. A. xylebori, as well as A. ferruginea and A. hartigii, demonstrated phialidic conidial development, leading to an emendation of the description of the genus Ambrosiella. In contrast, Dryadomyces exhibited conidial development by apical, sympodial wall formation with prominent denticles bearing conidia. Other, previously described Ambrosiella species exhibited non-phialidic conidiogenesis, but lacked denticles on the conidiophores. Consequently, their classification needs further revision.