Endosymbiotic Fungi Research Articles

Trichomycetes from Governor's Lake and Lake Micmac within the Halifax Regional Municipality, Nova Scotia, Canada

Trichomycetes are endosymbiotic fungi and protists that attach to the gut lining of insects and other arthropods. The vast majority of trichomycetes have been reported from lotic habitats (primarily flowing streams), whereas lentic habitats are relatively unexplored. Despite being largely understudied, lentic waters should be viable habitats for trichomycetes because insect types commonly colonized by trichomycetes in lotic systems are also found in lentic habitats. As part of a larger study, two lakes in separate watersheds within the Halifax Regional Municipality, Nova Scotia, Canada, were sampled. Six species of trichomycetes were found, including three new species, Smittium guttisporum sp. nov. R.T. William and Strongman, Stachylina serpula sp. nov. R.T. William and Strongman, and Stachylina zeppelin sp. nov. R.T. William and Strongman, all isolated from dipteran (Chironomidae) hosts. A new continental record for Smittium bulbosporophorus L.G. Valle & Santam., previously only reported from Spain, is documented from Governor's Lake, Timberlea, Nova Scotia. Smittium dipterorum Lichtw., known from Costa Rica, Dominican Republic, and Mexico was also collected from the Governor's Lake site, establishing a new Canadian record.

Aphid genotypes vary in their response to the presence of fungal endosymbionts in host plants

Genetic variation for fitness-relevant traits may be maintained in natural populations by fitness differences that depend on environmental conditions. For herbivores, plant quality and variation in chemical plant defences can maintain genetic variation in performance. Apart from plant secondary compounds, symbiosis between plants and endosymbiotic fungi (endophytes) can produce herbivore-toxic compounds. We show that there is significant variation among aphid genotypes in response to endophytes by comparing life-history traits of 37 clones of the bird cherry-oat aphid Rhopalosiphum padi feeding on endophyte-free and endophyte-infected tall fescue Lolium arundinaceum. Clonal variation for life-history traits was large, and most clones performed better on endophyte-free plants. However, the clones differed in the relative performance across the two environments, resulting in significant genotype x environment interactions for all reproductive traits. These findings suggest that natural variation in prevalence of endophyte infection can contribute to the maintenance of genetic diversity in aphid populations.

Optimization and production of a biosurfactant from the sponge-associated marine fungus Aspergillus ustus MSF3

Marine endosymbiotic fungi Aspergillus ustus (MSF3) which produce high yield of biosurfactant was isolated from the marine sponge Fasciospongia cavernosa collected from the peninsular coast of India. Maximum production of biosurfactant was obtained in Sabouraud dextrose broth. The optimized bioprocess conditions for the maximum production was pH 7.0, temperature 20 °C, salt concentration 3%, glucose and yeast extract as carbon source and nitrogen sources respectively. The response surface methodology based analysis of carbon and nitrogen ratio revealed that the carbon source can increase the biosurfactant yield. The biosurfactant produced by MSF3 was partially characterized as glycolipoprotein based on the estimation of macromolecules and TLC analysis. The partially purified biosurfactant showed broad spectrum of antimicrobial activity. The strain MSF3 can be used for the microbially enhanced oil recovery process.

Control of Clavicipitaceous Anamorphic Endophytes with Fungicides, Aerated Steam and Supercritical Fluid CO2-Seed Extraction

The effects of soil drenches with systemic fungicides on viability of clavicipitaceous anamorphic endophytes, non-choke inducing endosymbiotic fungi of the genus Neotyphodium that systemically infect grasses, were tested in endophyte-infected seedlings of Hordeum brevisubulatum subsp. violaceum, Lolium perenne and Festuca arundinacea germplasm. None of the endophytes of these grasses were sensitive to benomyl at 3-5 ppt. The endophyte of H. brevisubulatum was sensitive only to propiconazole. The viability of the F. arundinacea endophyte (N, coenophialum) was reduced, but not completed eliminated by prochloraz, imazalil and propiconazole in the 2-3 ppt range. Aerated-steam seed treatments at 60 C for 5 min were effective in reducing viability of the perennial ryegrass endophyte (N. lolii) by 83%, although some reduction in seed germination and negative growth effects were observed. A novel disease-control method, supercritical fluid carbon dioxide extraction (SFE-Cod, for the elimination of Neotyphodium-endophytes in grass seeds is reported here for the first time. The endophyte N. lolii was completely controlled in seeds of L. perenne cv. Ellett by extracting the seeds at 400 atmospheres for 10 or 20 min. Only minor reductions in seed germination resulted from the SFE-CO, seed-extraction treatment and no appreciable effects on growth of seedlings or mature plants were observed. Several potential applications of these disease-control technologies for seedborne fungal pathogens are discussed.

Removal of Fungal Contaminants and their DNA from the Surface of Diaphorina citri (hemiptera: psyllidae) Prior to a Molecular Survey of Endosymbionts

biology (Dale & Moran 2006), and molecular sur veys to detect arthropod-associated microbes have bypassed the necessity of culturing them in vitro for identification (Darby & Welburn 2006). To date, no consistent method has been developed to eliminate microbial DNA from the external surfaces of arthropods prior to conducting molec ular surveys of endosymbionts. This is problem atic because resolving the relationships between a host and its endosymbionts can be complicated by polymerase chain reaction (PCR) amplification of DNA from contaminating external microbes (Thornhill et al. 2008). A variety of techniques that may kill microbes on arthropod surfaces have been reported, including washing with ace tone (Fukatsu & Nikoh 1998), ethanol (Davidson et al. 1994; Van Borm et al. 2002), bleach (David son et al. 1994), formaldehyde (Duperchy & Zim mermann 2003), exposure to ultraviolet light (Van Borm et al. 2002), radiation (Duperchy & Zimmermann 2003) and antibiotic treatment (Duperchy & Zimmermann 2003). However, the effectiveness of these methods for eliminating DNA from external microbes so that it is not am plified in subsequent PCRs has not been evalu ated. DNA from contaminating external fungi should be eliminated, if possible, prior to deter mining if there are endosymbiotic fungi associ ated with a laboratory colony of the Asian citrus psyllid Diaphorina citri Kuwayama (Hemiptera: Psyllidae). Examples of fungal endosymbionts have been reported in association with other hemipteran insects, including aphids and plan thoppers (Noda et al. 1994; Fukatsu & Ishikawa 1995; Hongoh & Ishikawa 2000). A laboratory col ony of D. citri (Skelley & Hoy 2004) was used to evaluate a variety of solutions (Table 1) for their ability to kill surface-inhabiting fungi and to eliminate their DNA so that they could not be de tected using a high-fidelity PCR protocol. Adult D. citri in this colony support an abundance of exter nal fungal growth, primarily on the ventral por tion of their abdomens. This is due to being reared 702 Florida Entomologist 91(4) December 2008

Low Resource Availability Differentially Affects the Growth of Host Grasses Infected by Fungal Endophytes

Grasses infected with endosymbiotic ascomyceteous fungi (family Clavicipitaceae) often show increased growth and vigor; however, when resources are limited, the costs of being infected may outweigh the advantages. We grew multiple genotypes of two grasses species (Lolium perenne and Lolium arundinaceum) infected (or uninfected) with Neotyphodium sp. under limited light or water availability and measured both biomass and relative growth rates. There was a significant interaction between water stress and infection in L. perenne, while in L. arundinaceum, there were significant differences between infected and uninfected plants but no significant interaction. In both species, there were significant interactions between light level and infection. At full sun, infected plants were larger, while under reduced light, uninfected plants produced as much biomass as or more than infected plants. Although infected plants have increased vigor under high resource levels, infection will not necessarily result in increas...

Endosymbiotic fungi structurally integrated with leaves reveals a lichenous condition of C4 grasses

This paper addresses the assumed autonomy of vascular plants by revealing the presence of an obligate fungus structurally integrated with leaf anatomy of C4 grasses. We examined leaf surfaces of 26 species representing 14 genera of C4 grasses. In all species, we found similarities between leaf surface microhair-like structures and Uredomycete teliospores. These bicellular structures produced hyphae and spores, confirming they were fungal, rather than plant tissue. The plant-fungus structural morphology was also observed in Bouteloua eriopoda plants regenerated from embryonic meristem cells. The conserved symbiosis between fungi and C4 grasses suggests a lichenous association with evolutionary significance. The structural integration of endosymbiotic fungi with cells and tissues offers novel and unexplored approaches to developing physiological, ecological, and systematic models of C4 grasses.

Confirmation and identification of parasitic stages of obligate endobionts ( Harpellales) in blackflies ( Simuliidae) by means of rRNA sequence data

Over the last 35 y, the life cycle of endosymbiotic gut fungi ( Harpellales) has been expanded to include cyst stages associated with the developing ovaries. Ovarian cysts (chlamydospore stages) have been identified after germination and production of asexual trichospores in vitro, but germination is not always successful, and spores exhibit morphological variation. Sequence data (for partial 18S and 28S rRNA genes) were generated for these putative stages of Harpellales using ungerminated cysts from adult blackflies and germinated cysts associated with field-collected blackfly egg masses. Cladistic analyses of the 18S and 28S rRNA sequences confirmed that ovarian cysts are stages in the life cycle of Harpellales. Ungerminated cysts, from a blackfly collected from New York state were identified as Pennella simulii and two samples from Newfoundland Prosimulium mixtum adults were identified as Harpella melusinae. Cysts with bipolar germ tubes, associated with field-collected Simulium egg masses from Newfoundland, were also identified as H. melusinae. Two other samples of cysts could not be matched with available sequences of gut fungi from larval hosts. The potential use of this approach to identify pathogenic stages associated with adult ovaries or field-collected egg masses among other host groups is highlighted and promoted as a tool to test the hypothesis that ovarian cysts are a dispersal stage common to all genera of Harpellales.