Rearing system for the predatory phytoseiid <Emphasis Type="Italic">Euseius concordis</Emphasis> (Acari: Phytoseiidae)

Several species of predatory mites, especially those of the family Phytoseiidae, are potentially useful for the control of pest mites and insects. Among the phytoseiids, Euseius concordis (Chant), a species commonly found in South America, has been studied for possible use as biological control agent of pest mites. Given that Euseius species are known to feed on plant leaves and on pollen, the objective of this study was to determine plant species and pollen sources suitable to establish a pilot method of production of that species, based on a set of comparisons in the laboratory. Out of four plant species evaluated in the study, higher survivorship of E. concordis females in the absence of supplementary food was observed on leaflets of Canavalia ensiformis L. Out of two pollen types, higher survivorship was obtained on pollen of Typha domingensis L. In a subsequent step, the population of E. concordis increased 19.3× within 21 days when that pollen of T. domingensis was offered to the predator on plantlets of C. ensiformis, at 25.0 ± 1 °C, 70.0 ± 10% RH and 12 h photophase. Future studies may confirm the economic viability of this setup for the mass production of E. concordis.     

A qPCR assay that specifically quantifies <Emphasis Type="Italic">Tricholoma matsutake</Emphasis> biomass in natural soil

Tricholoma matsutake is an ectomycorrhizal basidiomycete that produces prized, yet uncultivable, “matsutake” mushrooms along densely developed mycelia, called “shiro,” in the rhizosphere of coniferous forests. Pinus densiflora is a major host of this fungus in Japan. Measuring T. matsutake biomass in soil allows us to determine the kinetics of fungal growth before and after fruiting, which is useful for analyzing the conditions of the shiro and its surrounding mycorrhizosphere, predicting fruiting timing, and managing forests to obtain better crop yields. Here, we document a novel method to quantify T. matsutake mycelia in soil by quantifying a single-copy DNA element that is uniquely conserved within T. matsutake but is absent from other fungal species, including close relatives and a wide range of ectomycorrhizal associates of P. densiflora. The targeted DNA region was amplified quantitatively in cultured mycelia that were mixed with other fungal species and soil, as well as in an in vitro co-culture system with P. densiflora seedlings. Using this method, we quantified T. matsutake mycelia not only from shiro in natural environments but also from the surrounding soil in which T. matsutake mycelia could not be observed by visual examination or distinguished by other means. It was demonstrated that the core of the shiro and its underlying area in the B horizon are predominantly composed of fungal mycelia. The fungal mass in the A or A₀ horizon was much lower, although many white mycelia were observed at the A horizon. Additionally, the rhizospheric fungal biomass peaked during the fruiting season.     

Persistence of ecto- and ectendomycorrhizal fungi associated with <Emphasis Type="Italic">Pinus montezumae</Emphasis> in experimental microcosms

Ectomycorrhizal (ECM) and ectendomycorrhizal fungal species associated with Pinus montezumae were recorded in 8 year-old trees established in microcosms and compared with those associated with 2 year-old trees, in order to determine their persistence over the long-term. Mycorrhizal root tips were morphologically and anatomically characterized and sequenced. The extension of extramatrical mycelium of ECM fungi with long exploration strategies was evaluated. In total, 11 mycorrhizal species were registered. Seven mycorrhizal species were detected on both 2 and 8 year-old pines: Atheliaceae sp., Rhizopogon aff. fallax, R. aff. occidentalis, Suillus pseudobrevipes, Tuber separans, Wilcoxina mikolae and Wilcoxina rehmii. One species, Thelephora terrestris, was exclusively associated with two year–old seedlings, while Cenococcum geophilum, Pezizaceae sp. and Pyrenomataceae sp. were exclusively found on 8 year-old trees. Atheliaceae sp. was the ECM fungal species that presented the most abundant mycelium. Finally, we report one new fungal species of Pezizaceae occurring as a symbiont of P. montezumae.     

Preservation affects the vegetative growth and fruiting body production of <Emphasis Type="Italic">Cordyceps militaris</Emphasis>

Cordyceps militaris is a model species of Cordyceps fungi, and has been traditionally used as an edible and medicinal fungus due to its richness of bioactive and pharmacological metabolites. The fruiting bodies of this fungus are widely used as healthy food and nutrition supply. In industrial production, fruiting bodies are cultivated on artificial media, but their yield and quality are usually affected by the quality of fungal strains. In this study, the effect of colony growth rate of the fungal strains, fungal age and repeated subculturing on the fungal biomass accumulation was investigated. The results indicated that the fungal biomass was positively correlated with the colony growth rate and not affected by fungal age and the repeated subculturing. The preservation conditions for stock cultures, including choice of cultures, lyophilization, temperature and protective agents were optimized based on the mycelial formation and conidia production in artificial inoculum. The development of fruiting bodies from the fungal strains stored under the optimized preservation conditions were further analyzed to determine the ideal time period of preservation. Results indicated that storing the fungus at 4 °C could maintain the fungal vitality and fruiting body producing capacity for at least 12 months. This study established practical criteria of fungal inoculum for artificial cultivation of fruiting body and provided a simple and efficient preservation method for C. militaris. The results may shed light on preservation for other Cordyceps species and other edible fungi.     

The ectomycorrhizal status of a tropical black bolete, <Emphasis Type="Italic">Phlebopus portentosus</Emphasis>, assessed using mycorrhizal synthesis and isotopic analysis

Phlebopus portentosus is one of the most popular wild edible mushrooms in Thailand and can produce sporocarps in the culture without a host plant. However, it is still unclear whether Phlebopus portentosus is a saprotrophic, parasitic, or ectomycorrhizal (ECM) fungus. In this study, Phlebopus portentosus sporocarps were collected from northern Thailand and identified based on morphological and molecular characteristics. We combined mycorrhizal synthesis and stable isotopic analysis to investigate the trophic status of this fungus. In a greenhouse experiment, ECM-like structures were observed in Pinus kesiya at 1 year after inoculation with fungal mycelium, and the association of Phlebopus portentosus and other plant species showed superficial growth over the root surface. Fungus-colonized root tips were described morphologically and colonization confirmed by molecular methods. In stable isotope measurements, the δ¹³C and δ¹⁵N of natural samples of Phlebopus portentosus differed from saprotrophic fungi. Based on the isotopic patterns of Phlebopus portentosus and its ability to form ECM-like structures in greenhouse experiments, we conclude that Phlebopus portentosus could be an ECM fungus.     

Diversity of fungi associated with roots of <Emphasis Type="Italic">Calanthe</Emphasis> orchid species in Korea

While symbiotic fungi play a key role in the growth of endangered Calanthe orchid species, the relationship between fungal diversity and Calanthe species remains unclear. Here, we surveyed root associated fungal diversity of six Calanthe orchid species by sequencing the internal transcribed spacer (ITS) region using 454 pyrosequencing. Our results revealed that Paraboeremia and Coprinopsis are dominant fungal genera among Calanthe species. In terms of overall relative abundance, Paraboeremia was the most common fungal genus associated with Calanthe roots, followed by Coprinopsis. Overall fungal diversity showed a significant degree of variation depending on both location and Calanthe species. In terms of number of different fungal genera detected within Calanthe species, C. discolor had the most diverse fungal community, with 10 fungal genera detected. This study will contribute toward a better understanding of those fungi that are required for successful cultivation and conservation of Korean Calanthe species.     

Exploring virulence of new and less studied species of <Emphasis Type="Italic">Metarhizium</Emphasis> spp. from Brazil for two-spotted spider mite control

The two-spotted spider mite Tetranychus urticae is an important pest of strawberry crops in Brazil and many other countries. Focus for biocontrol studies involving entomopathogenic fungi has been on three species from the genus Metarhizium: M. anisopliae sensu stricto (s.s.), M. brunneum and M. robertsii. Also, the species Beauveria bassiana has been studied for spider mite control and one isolate (ESALQPL63) is commercially available in Brazil. New and undescribed Metarhizium species have been found recently in Brazil and provide a pool of isolates with potential for biocontrol in Brazil and probably also elsewhere. The mortality of adult females of T. urticae when exposed to four new Brazilian species of Metarhizium was compared to the mortality when exposed to M. anisopliae s.s., M. brunneum, M. pingshaense, M. robertsii and Beauveria bassiana ESALQPL63. Fungal suspensions were sprayed onto mites at 10⁷ conidia/mL with 0.05% Tween 80 in laboratory bio-assays. We measured total mortality and percentage sporulating cadavers 10 days after exposure and calculated median lethal time (LT₅₀). The lowest LT₅₀ (4.0 ± 0.17) was observed for mites treated with Metarhizium sp. Indet. 1 (ESALQ1638), which also performed well with respect to mortality after 10 days and capacity to sporulate from cadavers. Among the other little studied species tested, M. pingshaense (ESALQ3069 and ESALQ3222) and Metarhizium Indet. 2 (ESALQ1476) performed well and were comparable to B. bassiana (ESALQPL63). The new Metarhizium isolates and species thus showed potential for biological control.     

<Emphasis Type="Italic">Rhizoglomus venetianum</Emphasis>, a new arbuscular mycorrhizal fungal species from a heavy metal-contaminated site,downtown Venice in Italy

Rhizoglomus venetianum, a new arbuscular mycorrhizal fungal species, has been isolated and propagated from a heavy metal-contaminated site in Sacca San Biagio island, downtown Venice, Italy. Interestingly, under the high levels of heavy metals occurring in the site, the new fungus was able to grow only intraradically. In greenhouse trap and single species cultures under low heavy metal levels, the fungus produced innumerous spores, clusters, and sporocarps extraradically, which were formed terminally on subtending hyphae either singly, in small spore clusters, or, preferably, in loose to compact non-organized sporocarps up to 2500?×?2000?×?2000 μm. Spores are golden-yellow to bright yellow brown, globose to subglobose to rarely oblong, 75–145?×?72–140 μm in diameter, and have four spore wall layers. Morphologically, the new fungus is similar to R. intraradices, and phylogenetically, it forms a monophyletic clade next to R. irregulare, which generally forms irregular spores and lacks, like R. intraradices, the flexible innermost wall layer beneath the structural/persistent third wall layer. A key for the species identification is presented comprising all 18 Rhizoglomus species, so far described or newly combined.     

Suitability of edaphic arthropods as prey for <Emphasis Type="Italic">Proctolaelaps bickleyi</Emphasis> and <Emphasis Type="Italic">Cosmolaelaps brevistilis</Emphasis> (Acari: Mesostigmata: Melicharidae,Laelapidae) under laboratory conditions

Soils are often complex habitats inhabited by a wide range of organisms, some harmful to plants and others beneficial, for example by attacking harmful organisms. Beneficial organisms include predatory mites, some of which have been commercialized for biological control of pest insects and mites. The objective of this work was to evaluate under laboratory condition the suitability of representative soil insect and mite pests, especially Aceria tulipae (Keifer), as prey to the soil-inhabiting predatory mites Proctolaelaps bickleyi (Bram) and Cosmolaelaps brevistilis (Karg). Predation, oviposition and survivorship of recently molted adult females of the predators were assessed in the dark in rearing chambers at 25 ± 1 °C and 75 ± 3% RH. Predation rate by P. bickleyi on A. tulipae was significantly higher than that by C. brevistilis (196.3 vs. 71.0 specimens/day). About 482 A. tulipae were preyed by each P. bickleyi at each day, when 500 A. tulipae were made available daily to the predator. Oviposition rate on that prey was also higher for P. bickleyi (4.2 eggs/day). For C. brevistilis, the highest level of oviposition was on Caliothrips phaseoli (Hood) (1.2 eggs/day). Survivorship was always higher for C. brevistilis (≥ 70%), given its ability to remain alive relatively long even in the absence of prey. High rates of survivorship of P. bickleyi were observed on A. tulipae, Bradysia matogrossensis (Lane) and Protorhabditis sp. Promising results were obtained for P. bickleyi on A. tulipae and even on other prey, justifying the conduction of complementary studies under field condition.     

The role of pre-symbiotic auxin signaling in ectendomycorrhiza formation between the desert truffle <Emphasis Type="Italic">Terfezia boudieri</Emphasis> and <Emphasis Type="Italic">Helianthemum sessiliflorum</Emphasis>

The ectendomycorrhizal fungus Terfezia boudieri is known to secrete auxin. While some of the effects of fungal auxin on the plant root system have been described, a comprehensive understanding is still lacking. A dual culture system to study pre mycorrhizal signal exchange revealed previously unrecognized root–fungus interaction mediated by the fungal auxin. The secreted fungal auxin induced negative taproot gravitropism, attenuated taproot growth rate, and inhibited initial host development. Auxin also induced expression of Arabidopsis carriers AUX1 and PIN1, both of which are involved in the gravitropic response. Exogenous application of auxin led to a root phenotype, which fully mimicked that induced by ectomycorrhizal fungi. Co-cultivation of Arabidopsis auxin receptor mutants tir1-1, tir1-1 afb2-3, tir1-1 afb1-3 afb2-3, and tir1-1 afb2-3 afb3-4 with Terfezia confirmed that auxin induces the observed root phenotype. The finding that auxin both induces taproot deviation from the gravity axis and coordinates growth rate is new. We propose a model in which the fungal auxin induces horizontal root development, as well as the coordination of growth rates between partners, along with the known auxin effect on lateral root induction that increases the availability of accessible sites for colonization at the soil plane of fungal spore abundance. Thus, the newly observed responses described here of the root to Terfezia contribute to a successful encounter between symbionts.     

Effects of selenium oxyanions on the white-rot fungus <Emphasis Type="Italic">Phanerochaete chrysosporium</Emphasis>

The ability of Phanerochaete chrysosporium to reduce the oxidized forms of selenium, selenate and selenite, and their effects on the growth, substrate consumption rate, and pellet morphology of the fungus were assessed. The effect of different operational parameters (pH, glucose, and selenium concentration) on the response of P. chrysosporium to selenium oxyanions was explored as well. This fungal species showed a high sensitivity to selenium, particularly selenite, which inhibited the fungal growth and substrate consumption when supplied at 10 mg L^?1 in the growth medium, whereas selenate did not have such a strong influence on the fungus. Biological removal of selenite was achieved under semi-acidic conditions (pH 4.5) with about 40 % removal efficiency, whereas less than 10 % selenium removal was achieved for incubations with selenate. P. chrysosporium was found to be a selenium-reducing organism, capable of synthesizing elemental selenium from selenite but not from selenate. Analysis with transmission electron microscopy, electron energy loss spectroscopy, and a 3D reconstruction showed that elemental selenium was produced intracellularly as nanoparticles in the range of 30–400 nm. Furthermore, selenite influenced the pellet morphology of P. chrysosporium by reducing the size of the fungal pellets and inducing their compaction and smoothness.     

<Emphasis Type="Italic">Cryptococcus gattii</Emphasis> Infection Presenting as an Aggressive Lung Mass

Cryptococcus gattii is an endemic fungus predominantly isolated in the tropical and subtropical regions, causing predominantly pulmonary disease with a predilection for the central nervous system. Herein, we report a case of rapidly progressing C. gattii pneumonia in an immune-deficient but virologically suppressed host with underlying human immunodeficiency viral (HIV) infection, exhibiting various fungal morphologies from bronchoalveolar lavage (BAL) cytological specimens. A 51-year-old Chinese male with known HIV disease was admitted to the Singapore General Hospital for evaluation of functional decline, febrile episodes, and a left hilar mass on chest radiograph. Computed tomography (CT) showed consolidation in the apical segment of the left lower lobe. He underwent bronchoscopy and BAL. Positron emission tomography-computed tomography done 10 days after the initial CT showed approximate doubling of the pulmonary lesion. Cytological examination of the fluid revealed yeasts of varying sizes. Subsequent fungal culture from BAL fluid grew C. gattii 10 days later.     

Proven Invasive Pulmonary Aspergillosis in Stem Cell Transplant Recipient Due to <Emphasis Type="Italic">Aspergillus sublatus</Emphasis>, a Cryptic Species of <Emphasis Type="Italic">A. nidulans</Emphasis>

Invasive fungal disease represents one of the severe complications in haematopoietic stem cell transplant recipients. We describe a case of a patient treated for relapse of chronic lymphoblastic leukaemia 6 years after HSCT. The patient was treated for invasive pulmonary aspergillosis but died 3 months later from multiple organ failures consisting of haemorrhagic necrotizing fungal pneumonia, refractory chronic hepatic graft versus host disease and cytomegalovirus hepatitis. Autopsy samples revealed histopathological evidence of fungal hyphae and an unusual Aspergillus nidulans-like species was isolated in pure culture. More precise identification was achieved by using scanning electron microscopy of ascospores and sequencing of calmodulin gene, and the isolate was subsequently re-identified as A. sublatus (section Nidulantes) and showed good in vitro susceptibility against all classes of antifungals. Commonly used ITS rDNA region and β-tubulin gene fail to discriminate A. sublatus from related pathogenic species, especially A. quadrilineatus and A. nidulans. Although this is the first case of proven IPA attributed to A. sublatus, we demonstrated that at least some previously reported infections due to A. quadrilineatus were probably caused by this cryptic species.     

<Emphasis Type="Italic">Melampsora salicis-bakko</Emphasis>, a new species on willows in Japan evidenced by morphological and molecular phylogenetic analyses

Through the morphological and molecular examinations of Melampsora species on willows, we clarified the taxonomic identity of the rust specimens on Salix bakko, S. hultenii and S. leucopithecia from Japan and described the following rust fungus as a new species, Melampsora salicis-bakko. This rust fungus resembled M. caprearum in morphology of teliospores, but it differed from M. caprearum mainly in the density of spines on the urediniospores. Molecular phylogenetic analyses using the rDNA ITS region (complete ITS1, 5.8S rRNA gene and ITS2) revealed that M. salicis-bakko was monophyletic, and that this rust fungus was distinct from other Melampsora species, including M. caprearum.