First report of Pandora neoaphidis resting spore formation in vivo in aphid hosts

The entomopathogenic fungus Pandora neoaphidis is a recognized pathogen of aphids, causes natural epizootics in aphid populations, and interacts and competes with aphid predators and parasitoids. Survival of entomophthoralean fungi in periods of unsuitable weather conditions or lack of appropriate host insects is accomplished mainly by thick-walled resting spores (zygospores or azygospores). However, resting spores are not known for some entomophthoralean species such as P. neoaphidis. Several hypotheses of P. neoaphidis winter survival can be found in the literature but so far these hypotheses do not include the presence of resting spores. Resting spores were found in an aphid population where P. neoaphidis was the only entomophthoralean fungus observed during surveys conducted in organic horticultural crops in greenhouses and open fields in Buenos Aires province, Argentina. This study sought to use molecular methods to confirm that these resting spores were, in fact, those of P. neoaphidis while further documenting and characterizing these resting spores that were produced in vivo in aphid hosts. The double-walled resting spores were characterized using light and transmission electron microscopy. The Argentinean resting spores clustered together with P. neoaphidis isolates with bootstrap values above 98 % in the small subunit ribosomal RNA (SSU rRNA) sequence analysis and with bootstrap values above 99 % the Internal Transcribed Spacer (ITS) II region sequence analysis. This study is the first gene-based confirmation from either infected hosts or cultures that P. neoaphidis is able to produce resting spores.     

Comparing two methods for quantifying soil-borne Entomophaga maimaiga resting spores

To improve usability of methods for quantifying environmentally persistent entomophthoralean resting spores in soil, we modified and tested two methods using resting spores (azygospores) of the gypsy moth pathogen Entomophaga maimaiga. Both methods were effective for recovering resting spores at concentrations >100 resting spores/g dry soil. While a modification of a method originally described by Weseloh and Andreadis (2002) recovered more resting spores than a modified method based on Percoll density gradients, the ability to estimate true densities from counts was similar for both methods. Regression equations are provided for predicting true resting spore densities from counts, with R² values for both methods ?0.90.     

Detection and quantification of Entomophaga maimaiga resting spores in forest soil using real-time PCR

Environmental sampling to monitor entomopathogen titre in forest soil, a known reservoir of insect pathogens such as fungi and viruses, is important in the evaluation of conditions that could trigger epizootics and in the development of strategies for insect pest management. Molecular or PCR-based analysis of environmental samples provides a sensitive method for strain- or species-based detection, and real-time PCR, in particular, allows quantification of the organism of interest. In this study we developed a DNA extraction method and a real-time PCR assay for detection and quantification of Entomophaga maimaiga (Zygomycetes: Entomophthorales), a fungal pathogen of the gypsy moth, in the organic layer of forest soil. DNA from fungal resting spores (azygospores) in soil was extracted using a detergent and bead mill homogenization treatment followed by purification of the crude DNA extract using Sephadex–polyvinylpolypyrrolidone microcolumns. The purification step eliminated most of the environmental contaminants commonly co-extracted with genomic DNA from soil samples but detection assays still required the addition of bovine serum albumin to relieve PCR inhibition. The real-time PCR assay used primers and probe based on sequence analysis of the nuclear ribosomal ITS region of several E. maimaiga and two E. aulicae strains. Comparison of threshold cycle values from different soil samples spiked with E. maimaiga DNA showed that soil background DNA and remaining co-extracted contaminants are critical factors determining detection sensitivity. Based on our results from comparisons of resting spore titres among different forest soils, estimates were best for organic soils with comparatively high densities of resting spores.     

Soil Fungicides for Controlling Chytridiaceous Mycoparasites of Gigaspora margarita and Glomus fasciculatum

The percentage of azygospores of Gigaspora margarita with zoosporangia of chytridiaceous fungi (CF) was reduced significantly after agitating them in fenaminosulf before incubation in soil. Fenaminosulf did not affect zoosporangia development on chlamydospores of Glomus fasciculatum. Metalaxyl and ethazol were not effective against CF on spores of either mycorrhizal fungus. Azygospores of G. margarita were treated with fenaminosulf and used as the inoculum for pot cultures. After 19 weeks, the percentage of azygospores containing CF was reduced significantly by this treatment, whereas root colonization and sporulation by the mycorrhizal fungus were unaffected. Pot cultures of G. margarita, either drenched with fenaminosulf or not, did not differ in the percentage of azygospores containing CF. However, root colonization and sporulation by the mycorrhizal fungus was temporarily delayed when pots were drenched with fenaminosulf.     

Development,Distribution, and Host Studies of the Fungus Macrobiotophthoira vermicola (Entomophthorales)

The life cycle and host range of Macrobiotophthora vermicola were studied. Secondary spores produced from forcibly ejected primary spores adhered to the cuticle of Cruznema tripartitum, germinated, and penetrated the cuticle within 30 minutes. New primary spores were produced within 24 hours of initial spore adhesion. In a host range study, species of Rhabditidae, Diplogasteridae, and Aphelenchoidea were hosts, but not species of Bunonematidae, Tripylidae, Cephalobida, or Tylenchina. Numbers of second-stage Meloidogyne incognita juveniles were not decreased when added to soil seeded with infected C. tripartitum. In six Tennessee soybean fields, Macrobiotophthora vermicola was the most commonly encountered nematode-destroying fungus, followed by a sterile, nonseptate fungus and Arthrobotrys conoides. Nematophagous fungi were isolated more frequently from silt loam soils than from clay soils. Addition of C. tripartitum to soil extract plates as a bait nematode did not increase isolations of nematophagous fungi.     

First record of Entomophaga maimaiga (Entomophthorales: Entomophthoraceae) in Slovakia

The entomopathogenic fungus Entomophaga maimaiga was found for the first time in Slovakia in 2013. Late instar larvae of gypsy moth, Lymantria dispar, from two sites with different population densities were dissected to evaluate the presence of pathogens. The presence of conidia and resting spores of E. maimaiga in gypsy moth cadavers was confirmed from both sites.     

Highly diversified fungi are associated with the achlorophyllous orchid Gastrodia flavilabella

Background

Mycoheterotrophic orchids are achlorophyllous plants that obtain carbon and nutrients from their mycorrhizal fungi. They often show strong preferential association with certain fungi and may obtain nutrients from surrounding photosynthetic plants through ectomycorrhizal fungi. Gastrodia is a large genus of mycoheterotrophic orchids in Asia, but Gastrodia species’ association with fungi has not been well studied. We asked two questions: (1) whether certain fungi were preferentially associated with G. flavilabella, which is an orchid in Taiwan and (2) whether fungal associations of G. flavilabella were affected by the composition of fungi in the environment.

Results

Using next-generation sequencing, we studied the fungal communities in the tubers of Gastrodia flavilabella and the surrounding soil. We found (1) highly diversified fungi in the G. flavilabella tubers, (2) that Mycena species were the predominant fungi in the tubers but minor in the surrounding soil, and (3) the fungal communities in the G. flavilabella tubers were clearly distinct from those in the surrounding soil. We also found that the fungal composition in soil can change quickly with distance.

Conclusions

G. flavilabella was associated with many more fungi than previously thought. Among the fungi in the tuber of G. flavilabella, Mycena species were predominant, different from the previous finding that adult G. elata depends on Armillaria species for nutritional supply. Moreover, the preferential fungus association of G. flavilabella was not significantly influenced by the composition of fungi in the environment.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1422-7) contains supplementary material, which is available to authorized users.     

An Assay for Hirsutella rhossiliensis Spores and the Importance of Phialides for Nematode Inoculation

A spore assay was developed to measure the relative density of spores of the nematophagous fungus Hirsutella rhossiliensis in soil. Orchard soil containing H. rhossiliensis-parasitized Criconemella xenoplax was placed in vials and incubated for 0-120 days before the addition of probe nematodes, Heterorhabditis heliothidis juveniles. After 18 hours, H. heliothidis were extracted from the soil and examined for adhering spores of H. rhossiliensis. No spores were detected when H. heliothidis were added to freshly mixed soil, but the percentage of H. heliothidis with spores increased rapidly if soil was incubated undisturbed. Because mixing soil detaches spores from phialides, the results indicate that spores must be attached to phialides to adhere to nematodes. The spore assay was compared with a plate assay that measures the population density of H. rhossiliensis-parasitized C. xenoplax. Results from the two assays were highly correlated, suggesting that spores occur in three phases: reserves in nematodes that may be converted into spores; spores on phialides and therefore capable of adhering to nematodes; and spores detached from phialides and thus incapable of adhering to nematodes.     

A new species of Gigaspora from desert soils of Rajasthan,India

A new species of the endogonaceous fungus Gigaspora, isolated from the Indian semi-arid region, is described. The fungus, named G. tuberculata, produces rusty-brown azygospores with septate subtending hypha. The azygospores bear warts all over the outer wall. The shape, size and general appearance of these spores resemble those of Scutellospora persica.Neeraj and A.K. Varma are with the Microbiology Unit, School of Life Sciences, Jawaharlal Nehru University, New Delhi 110 067, India; K.G. Mukerji is with the Applied Mycology Laboratory, Botany Department, University of Delhi, Delhi 110 006, India. B.C. Sharma is with the Department of Textile Engineering, Indian Institute of Technology, New Delhi 110 016, India.     

Variability in azygospore production among Entomophaga maimaiga isolates

This study describes in vitro and in vivo azygospore production by nine isolates of Entomophaga maimaiga, a fungal pathogen of the gypsy moth, Lymantria dispar. The three E. maimaiga isolates that consistently produced azygospores in vitro were also strong producers of azygospores in vivo. However, two additional isolates that were strong azygospore producers in vivo did not produce azygospores in vitro. Isolates that produced azygospores in vitro produced both conidia and azygospores more frequently in vivo than isolates not producing azygospores in vitro. In vitro azygospore production varied over time as well as by isolate. After >2 years of cold storage, while three isolates continued in vitro azygospore production, three isolates no longer produced azygospores in vitro.     

Parasitic and Saprophytic Abilities of the Nematode-Attacking Fungus Hirsutella rhossiliensis

The ability of Hirsutella rhossiliensis to colonize various substrates in sterile and nonsterile soil was measured. Hirsutella rhossiliensis was recovered from 67% and 77% of living, inoculated Criconemella xenoplax incubated in sterile and nonsterile soil, respectively. In contrast, the fungus was recovered from 100% and 18% of heat-killed, inoculated nematodes incubated on sterile and nonsterile soil, respectively. Hirsutella rhossiliensis was readily recovered from inoculated, autoclaved wheat seeds incubated in sterile soil but not from seeds incubated in nonsterile soil. Autoclaved peach roots were a poor substrate for the fungus. Germination of H. rhossiliensis spores incubated on agar disks above soil was about 90% regardless of soil treatment. However, germ tube length was greatly suppressed by nonsterile soil. Our results suggest that H. rhossiliensis is a better parasite than saprophyte and that the fungus may be specialized for attacking nematodes.     

Phylogenetic evaluation of Geomyces and allies reveals no close relatives of Pseudogymnoascus destructans, comb. nov., in bat hibernacula of eastern North America

White-nose syndrome (WNS)  of bats, caused by the fungus previously known as Geomyces destructans, has decimated populations of insectivorous bats in eastern North America. Recent work on fungi associated with bat hibernacula uncovered a large number of species of Geomyces and allies, far exceeding the number of described species. Communication about these species has been hindered by the lack of a modern taxonomic evaluation, and a phylogenetic framework of the group is needed to understand the origin of G. destructans and to target closely related species and their genomes for the purposes of understanding mechanisms of pathogenicity. We addressed these issues by generating DNA sequence data for the internal transcribed spacer (ITS) region, nuclear large subunit (LSU) rDNA, MCM7, RPB2, and TEF1 from a diverse array of Geomyces and allies that included isolates recovered from bat hibernacula as well as those that represent important type species. Phylogenetic analyses indicate Geomyces and allies should be classified in the family Pseudeurotiaceae, and the genera Geomyces, Gymnostellatospora, and Pseudogymnoascus should be recognized as distinct. True Geomyces are restricted to a basal lineage based on phylogenetic placement of the type species, Geomyces auratus. Thus, G. destructans is placed in genus Pseudogymnoascus. The closest relatives of Pseudogymnoascus destructans are members of the Pseudogymnoascus roseus species complex, however, the isolated and long branch of P. destructans indicates that none of the species included in this study are closely related, thus providing further support to the hypothesis that this pathogen is non-native and invasive in eastern North America. Several conidia-producing isolates from bat hibernacula previously identified as members of Pseudeurotium are determined to belong to the genus Leuconeurospora, which is widespread, especially in colder regions. Teberdinia hygrophila is transferred to Pseudeurotium as Pseudeurotium hygrophilum, comb. nov., in accordance with the one name per fungus system of classification, and two additional combinations are made in Pseudogymnoascus including Pseudogymnoascus carnis and Pseudogymnoascus pannorum. Additional sampling from other regions of the world is needed to better understand the evolution and biogeography of this important and diverse group of fungi.     

Suppression of Cyst Nematode by Natural Infestation of a Nematophagous Fungus

Penetration of cabbage roots by Heterodera schachtii was suppressed 50-77% in loamy sand naturally infested with the nematophagous fungus Hirsutella rhossiliensis. When Heterodera schachtii was incubated in the suppressive soil without plants for 2 days, 40-63% of the juveniles had Hirsutella rhossiliensis spores adhering to their cuticles. Of those with spores, 82-92% were infected. Infected nematodes were killed and filled with hyphae within 2-3 days. Addition of KCl to soil did not increase infection of Heterodera schachtii by Hirsutella rhossiliensis. The percentage of infection was lower when nematodes were touched to two spores and incubated in KCl solution than when nematodes naturally acquired two spores in soil.     

温郁金内生真菌Chaetomium globosum L18对植物病原菌的抑菌谱及拮抗机理

通过体外培养法,研究了药用植物温郁金内生真菌Chaetomium globosum L18对几种常见的植物病原菌的抑菌谱及其拮抗机理。结果表明,Chaetomium globosum L18对多种植物病原真菌和细菌均有不同程度的抑制作用,具有较广的抑菌谱,但对不同植物病原菌的抑制作用具有显著性差异(P<0.05),抑制率最高可达到92.9%;抑菌机制结果显示,竞争作用和重寄生作用是其主要的拮抗机制之一;发酵产物抑制作用测定发现,内生真菌Chaetomium globosum L18能够分泌产生抗菌物质抑制病原菌菌丝的生长和孢子萌发,可引起病原菌菌丝菌丝膨大成串珠状,分枝增多,分枝顶端膨胀后细胞壁破裂,原生质外溢,产生溶菌作用;使分生孢子萌发畸形,萌发率降低。     

Biotic and abiotic regulation of resting spore formation in vivo of obligate aphid pathogen Pandora nouryi: Modeling analysis and biological implication

Entomophthoralean fungus Pandora nouryi is an obligate aphid pathogen that enables to produce resting spores (azygospores) for surviving host absence. To explore possible mechanisms involved in the regulation of resting spore formation in vivo, host cohorts consisting of 40-60 nymphs of green peach aphid Myzus persicae produced within 24 h on cabbage leaf discs in petri dishes were exposed to spore showers of P. nouryi at the concentrations (C) from a very few to nearly 2000 conidia/mm² and then reared for 7-11 days at the regimes of 10-25 °C (T) and 8-16 h daylight (H_L) or ambient (17.5 ± 3.1 °C, 13:11 L:D). Aphid mortalities observed from 35-83 cohorts (showered separately) at each regime showed typical sigmoid trend and fit well a general logistic equation (0.79 ? r² ? 0.88), yielding similar LC₅₀ estimates of 1.7-6.1 conidia/mm². The proportions (P) of cadavers forming resting spores in the cohorts also fit the same equation (0.73 ? r² ? 0.85) at all tested regimes except at 10 °C, a low temperature for the host-pathogen interaction. This indicates the dependence of resting spore formation on the spore concentration. The effects of T and H_L on P over C were well elucidated by the fitted modified logistic equations P = 0.578/{1 + exp[1.710 − (0.136 − 0.0053T)C]} and P = 0.534/{1 + exp[1.639 + (0.034 − 0.0053H_L)C]} (both r² = 0.79). Our results highlight that the resting spore formation in vivo of P. nouryi is regulated primarily by the concentration of host-infecting conidia discharged from cadavers and facilitated by lower temperature and longer daylight.     

Interaction of Vesicular-arbuscular Mycorrhizal Fungi and Phosphorus with Meloidogyne incognita on Tomato

The influence of two vesicular-arbuscular mycorrhizal fungi and phosphorus (P) nutrition on penetration, development, and reproduction by Meloidogyne incognita on Walter tomato was studied in the greenhouse. Inoculation with either Gigaspora margarita or Glomus mosseae 2 wk prior to nematode inoculation did not alter infection by M. incognita compared with nonmycorrhizal plants, regardless of soil P level (either 3 μg [low P] or 30 μg [high P] available P/g soil). At a given soil P level, nematode penetration and reproduction did not differ in mycorrhizal and nonmycorrhizal plants. However, plants grown in high P soil had greater root weights, increased nematode penetration and egg production per plant, and decreased colonization by mycorrhizal fungi, compared with plants grown in low P soil. The number of eggs per female nematode on mycorrhizal and nonmycorrhizal plants was not influenced by P treatment. Tomato plants with split root systems grown in double-compartment containers which had either low P soil in both sides or high P in one side and low P in the other, were inoculated at transplanting with G. margarita and 2 wk later one-half of the split root system of each plant was inoculated with M. incognita larvae. Although the mycoorhizal fungus increased the inorganic P content of the root to a level comparable to that in plants grown in high P soil, nematode penetration and reproduction were not altered. In a third series of experiments, the rate of nematode development was not influenced by either the presence of G. margarita or high soil P, compared with control plants grown in low P soil. These data indicate that supplemental P (30 μ/g soil) alters root-knot nematode infection of tomato more than G. mosseae and G. margarita.     

Parasitism of Xiphinema rivesi and X. americanum by Zoosporic Fungi

Living Xiphinema americanum (Xa) and X. rivesi (Xr) extracted from soil samples and stored for 1-5 days at 4 or 20 C contained aseptate fungal hyphae. The fungi directly penetrated the nematode''s cuticle from spores encysted near the head. Penetration through the stoma, vulva, or anus was rare. Catenaria anguillulae (Cat), Lagenidium caudatura (Lag), Aphanomyces sp. (Aph), and Leptolegnia sp. (Lep) were isolated into pure culture from infected nematodes. The pathogenicity of these zoosporic fungi was determined by incubating mixed freshly extracted Xa and Xr in 2% soil extract (pH = 6.7, conductivity = 48 μmhos, 20 ± 2 C) containing zoospores obtained from single-spore isolates. After 4 days, Cat, Lag, Aph, and Lep had infected 78, 18, 13, and 22%, respectively, of the nematodes. Both Xa and Xr were infected by every fungus; however, the relative susceptibility of Xa and Xr to these fungi was not determined. All noninoculated control nematodes remained uninfected and alive. In a second experiment, parasitism of Xa and Xr by Aph and Lep was increased when nematodes were incubated in 2% soil extract for 4 days before exposure to zoospores. In a third experiment, parasitism of Xa and Xr by Cat was greater in diluted saturation soil extract (conductivity = 100-400 μmhos) than in undiluted saturation extract (conductivity = 780 μmhos). Cat produced small zoospores (4-μm-d), bulbous infection hyphae, and assimilative hyphae of varying diameters in nematodes, whereas Lag, Aph, and Lep produced large zoospores (8-μm-d) and tubular, uniform infection and assimilative hyphae in nematodes.     

Rhizosphere Colonization and Control of Meloidogyne spp. by Nematode-trapping Fungi

The ability of nematode-trapping fungi to colonize the rhizosphere of crop plants has been suggested to be an important factor in biological control of root-infecting nematodes. In this study, rhizosphere colonization was evaluated for 38 isolates of nematode-trapping fungi representing 11 species. In an initial screen, Arthrobotrys dactyloides, A. superba, and Monacrosporium ellipsosporum were most frequently detected in the tomato rhizosphere. In subsequent pot experiments these fungi and the non-root colonizing M. geophyropagum were introduced to soil in a sodium alginate matrix, and further tested both for establishment in the tomato rhizosphere and suppression of root-knot nematodes. The knob-forming M. ellipsosporum showed a high capacity to colonize the rhizosphere both in the initial screen and the pot experiments, with more than twice as many fungal propagules in the rhizosphere as in the root-free soil. However, neither this fungus nor the other nematode-trapping fungi tested reduced nematode damage to tomato plants.     

Use of spent mushroom substrates from Agaricus subrufescens (syn. A. blazei, A. brasiliensis) and Lentinula edodes productions in the enrichment of a soil-based potting media for lettuce (Lactuca sativa) cultivation: Growth promotion and soil bioremediation

This study aimed to assess physicochemical and microbiological properties of fresh spent mushroom substrates (SMSs) – without post-crop heat treatment – from Agaricus subrufescens and Lentinula edodes production to optimize the use of these residues in the soil enrichment for lettuce growth promotion and soil remediation. Organic matter and C content of both SMSs were high. Fresh A. subrufescens SMS was a good source of N, P and K. On the other hand, L. edodes SMS presented a lower concentration of these nutrients and a high level of immaturity. Both SMSs presented high electric conductivity values (2.5–3.4 mS/cm). Microbiological analysis, based upon enumeration of culturable bacteria (thermophilic and mesophilic) and fungi, and also evolution of CO₂, showed that SMSs played higher microbial diversity than soil control. Laccase activity from A. subrufescens SMS tended to remain constant during a 2-month period, while L. edodes SMS presented low laccase activity throughout the same period. Agaricus subrufescens and L. edodes were able to grow on a PDA (Potato Dextrose Agar) media supplemented with different concentrations of atrazine (1–50 μg/ml), degraded the herbicide, attaining rates of 35% and 26%, respectively. On experiments of lettuce growth promotion using a soil-based potting media with different SMS rates, 5% and 10% (dw) rates of A. subrufescens SMS resulted in higher lettuce aerial dry weights than the rates of 25% and 40%, the chemical fertilization (NPK) and the control (soil). At 10% supplementation, lettuce aerial dry weight increased 2.2 and 1.3 times compared to the control and the NPK treatment, respectively. Protein content increased along with SMS rates. Fresh A. subrufescens SMS was an excellent supplement for lettuce growth promotion and showed potential for remediation of biocides possibly due to improved microbial diversity and enzymatic activity. Fresh L. edodes SMS was not a good fertilizer, at least under the conditions tested. However, microbiological analysis showed that promising results may be achieved when using fresh L. edodes SMS for soil remediation.