Hirsutella Rhossiliensis Research Articles

Parasitism of Hirsutella rhossiliensis on Different Nematodes and Its Endophytism Promoting Plant Growth and Resistance against Root-Knot Nematodes.

The endoparasitic fungus Hirsutella rhossiliensis is an important biocontrol agent of cyst nematodes in nature. To determine the potential parasitism of the fungus on a non-natural host, the pinewood nematode (Bursaphelenchus xylophilus) living in pine trees and the endophytic ability of the fungus on plants, in this paper, we first constructed and utilized a green fluorescent protein (GFP)-tagged H. rhossiliensis HR02 transformant to observe the fungal infection process on B. xylophilus and its colonization on Arabidopsis roots. Then, we compared the fungal parasitism on three species of nematodes with different lifestyles, and we found that the fungal parasitism is correlated with nematode species and stages. The parasitic effect of H. rhossiliensis on adults of B. xylophilus is similar to that on second-stage juveniles (J2) of the root-knot nematode Meloidogyne incognita after 24 h of inoculation, although the virulence of the fungus to second-stage juveniles of M. incognita is stronger than that to those of B. xylophilus and Caenorhabditis elegans. Moreover, the endophytism of H. rhossiliensis was confirmed. By applying an appropriate concentration of H. rhossiliensis conidial suspension (5 × 106 spores/mL) in rhizosphere soil, it was found that the endophytic fungus can promote A. thaliana growth and reproduction, as well as improve host resistance against M. incognita. Our results provide a deeper understanding of the fungus H. rhossiliensis as a promising biocontrol agent against plant-parasitic nematodes.

Application of Nematophagous Fungi and Salicylic Acid as Biological Control Agents against Root-Knot Nematode, Meloidogyne incognita Infected Gladiolus: A Valuable Export Plant

This study aims to use safety agents to control the root-knot nematode, Meloidogyne incognita infected gladiolus plants (Gladiolus grandiflorus) in pots under field conditions, directly by using two nematophagous fungi, Purpureocillium lilacinumwhich parasite on nematode eggs and Hirsutella rhossiliensis which parasite on second stage juvenile of nematode , and indirectly by using treatment of salicylic acid (SA). The applied agents applied either individually or in combination, reduced nematode numbers and improved gladiolus vegetative and flowering characteristics. Tri-treatment (SA + P. lilacinum + H. rhossiliensis) achieved the best results aptly, leading to increase reduction percentage of nematode juveniles to 80.2%, decreased egg masses production and root gall index to 91.3 and 83.3%, respectively, compared with bionematicide Tervigo (Abamectin) which gave reduction percentage of nematode juveniles as 78.9%, decreased egg masses production and root gall index (80.0, 66.7%, respectively). Also, the previous tri-treatment improved flowering traits as it led to an increment proportion of flowering stem length (58.6%); number of florets/spike (46.2%) and fresh weight of inflorescence (67.9%) compared with Tervigo (43.3, 41.8 and 54.2%, respectively). In addition, tri-treatment (SA + P. lilacinum + H. rhossiliensis) significantly improved chemical properties of N, P, K and total carbohydrate (76.8, 52.4, 47.4 and 31.4%, respectively) compared with Tervigo (42.7, 23.8, 6.3 and 4.5%, respectively). The obtained results can be concluded that, both nematophagous fungi complement each other, and adding SA led to their superiority in its effectiveness for M. incognita control over the nematicide Tervigo (Abamectin). Therefore, the use of chemical nematicide in which causes harmful effects to environment and humans should be reduced or substituted with clean biological control.

A Comparative Analyses of the Complete Mitochondrial Genomes of Fungal Endosymbionts in Sogatella furcifera, White-Backed Planthoppers.

Sogatella furcifera Horvath, commonly known as the white-backed planthoppers (WBPH), is an important pest in East Asian rice fields. Fungal endosymbiosis is widespread among planthoppers in the infraorder Fulgoromorpha and suborder Auchenorrhyncha. We successfully obtained complete mitogenome of five WBPH fungal endosymbionts, belonging to the Ophiocordycipitaceae family, from next-generation sequencing (NGS) reads obtained from S. furcifera samples. These five mitogenomes range in length from 55,390 bp to 55,406 bp, which is shorter than the mitogenome of the fungal endosymbiont found in Ricania speculum, black planthoppers. Twenty-eight protein-coding genes (PCGs), 12 tRNAs, and 2 rRNAs were found in the mitogenomes. Two single-nucleotide polymorphisms, two insertions, and three deletions were identified among the five mitogenomes, which were fewer in number than those of four species of Ophiocordycipitaceae, Ophiocordyceps sinensis, Hirsutella thompsonii, Hirsutella rhossiliensis, and Tolypocladium inflatum. Noticeably short lengths (up to 18 bp) of simple sequence repeats were identified in the five WBPH fungal endosymbiont mitogenomes. Phylogenetic analysis based on conserved PCGs across 25 Ophiocordycipitaceae mitogenomes revealed that the five mitogenomes were clustered with that of R. speculum, forming an independent clade. In addition to providing the full mitogenome sequences, obtaining complete mitogenomes of WBPH endosymbionts can provide insights into their phylogenetic positions without needing to isolate the mtDNA from the host. This advantage is of value to future studies involving fungal endosymbiont mitogenomes.

Corn and Soybean Host Root Endophytic Fungi with Toxicity Toward the Soybean Cyst Nematode.

Although fungal endophytes are commonly investigated for their ability to deter microbial plant pathogens, few studies have examined the activity of fungal root endophytes against nematodes. The soybean cyst nematode (SCN; Heterodera glycines), the most severe yield-limiting pathogen of soybean (Glycine max), is commonly managed through rotation of soybean with corn (Zea mays), a nonhost of the SCN. A total of 626 fungal endophytes were isolated from surface-sterilized corn and soybean roots from experimental plots in which soybean and corn had been grown under annual rotation and under 1, 3, 5, and 35 years of continuous monoculture. Fungal isolates were grouped into 401 morphotypes, which were clustered into 108 operational taxonomic units (OTUs) based on 99% sequence similarity of the full internal transcribed spacer region. Morphotype representatives within each OTU were grown in malt extract broth and in a secondary metabolite-inducing medium buffered with ammonium tartrate, and their culture filtrates were tested for nematicidal activity against SCN juveniles. A majority of OTUs containing isolates with nematicidal culture filtrates were in the order Hypocreales, with the genus Fusarium being the most commonly isolated nematicidal genus from corn and soybean roots. Less commonly isolated taxa from soybean roots included the nematophagous fungi Hirsutella rhossiliensis, Metacordyceps chlamydosporia, and Arthrobotrys iridis. Root endophytic fungal diversity in soybean was positively correlated with SCN density, suggesting that the SCN plays a role in shaping the soybean root endophytic community.

Effect of additives on the efficacy of microencapsulated Hirsutella rhossiliensis controlling Heterodera schachtii on sugar beets

The nematophagous fungus Hirsutella rhossiliensis is a common antagonist of a number of important plant-parasitic nematodes including the beet cyst nematode Heterodera schachtii. Although H. rhossiliensis is highly pathogenic, slow growth characteristics and weak competiveness under field conditions limit its biocontrol potential. Cellulose-based microcapsules were tested for their ability to improve fungal competitiveness in the soil and nematode parasitism. In a first approach microcapsules containing 15% fungal mycelium plus 15% corn gluten and 0.5% yeast extract as additives gave low levels of H. schachtii control. Fungal parasitism was not observed and indicated that nematode mortality was most likely caused by non-specific accumulation of nematicidal compounds associated with decomposition of the additives in the microcapsules. Removal of the corn gluten additive resulted in a minor increase of 20% parasitism of H. schachtii juveniles. Furthermore, the yeast extract that acted as nutrient source for the fungus diffused through the semi-permeable membrane of the capsules during the formulation process and was ineffective. Replacing the corn gluten and yeast extract with 3% autoclaved baker’s yeast and using finely dispersed mycelium instead of pelletized mycelium increased juvenile parasitism in the soil up to 90% and decreased number of juveniles per cm root length by over 80%. Cellulose-based microcapsules supplemented with autoclaved baker’s yeast, therefore, proved to be an effective delivery system for H. rhossiliensis to control H. schachtii.

Synthesis of glycosphingolipids from the fungus Hirsutella rhossiliensis

The total synthesis of two neutral glycosphingolipids (GSLs) from the fungus Hirsutella rhossiliensis has been achieved. The GSLs possess a common neogala-core (Galβ1-6Gal) and have the following sequence: α-d-Manp(1 → 3)-β-d-Galp(1 → 6)-β-d-Galp(1 → 6)-β-d-Galp(1↔1)Cer (1) and α-d-Manp(1 → 3)-β-d-Galp(1 → 6)[α-d-Glcp(1 → 4)]-β-d-Galp(1 → 6)-β-d-Galp(1↔1)Cer (2). Our efficient synthetic strategy uses the different reactivity of the hydroxyl groups of galactose and the α-orienting solvent effect of dioxane-toluene to generate GSLs (1) and (2).

Amazonian Dark Earth and Its Black Carbon Particles Harbor Different Fungal Abundance and Diversity

AbstractAmazonian Dark Earth (ADE) is a highly fertile soil of anthropogenic origin characterized by high levels of charred black carbon (BC). It is considered a model of fertility; however, knowledge on the fungal community structure and diversity inhabiting ADE and its BC particles is scarce. Fungal community structure and diversity of ADE and its BC from four sites under different land uses (three agricultural systems and a secondary pristine forest) in the Brazilian Central Amazon were evaluated by 18S rRNA gene pyrosequencing. Fungal communities in ADE and BC were dissimilar and showed differential abundances of fungal operational taxonomic units (OTUs). Estimated fungal species richness (abundance-based coverage estimate and Chao-1 index) and diversity estimators (Shannon and Simpson's reciprocal indices) were higher in ADE than in BC in all agricultural areas. No differences were observed in those parameters in ADE and BC samples from the secondary forest. Pezizomycotina fungi and OTUs assigned to Cordyceps confragosa, Acremonium vitellinum, Camarops microspora, and Hirsutella rhossiliensis were more abundant in BC particles than in ADE. These findings represent a breakthrough in our understanding of fungal communities in BC particles from ADE, and will be valuable in future studies considering biochar application in soil.

SMRT Sequencing Revealed Mitogenome Characteristics and Mitogenome-Wide DNA Modification Pattern in Ophiocordyceps sinensis.

Single molecule, real-time (SMRT) sequencing was used to characterize mitochondrial (mt) genome of Ophiocordyceps sinensis and to analyze the mt genome-wide pattern of epigenetic DNA modification. The complete mt genome of O. sinensis, with a size of 157,539 bp, is the fourth largest Ascomycota mt genome sequenced to date. It contained 14 conserved protein-coding genes (PCGs), 1 intronic protein rps3, 27 tRNAs and 2 rRNA subunits, which are common characteristics of the known mt genomes in Hypocreales. A phylogenetic tree inferred from 14 PCGs in Pezizomycotina fungi supports O. sinensis as most closely related to Hirsutella rhossiliensis in Ophiocordycipitaceae. A total of 36 sequence sites in rps3 were under positive selection, with dN/dS >1 in the 20 compared fungi. Among them, 16 sites were statistically significant. In addition, the mt genome-wide base modification pattern of O. sinensis was determined in this study, especially DNA methylation. The methylations were located in coding and uncoding regions of mt PCGs in O. sinensis, and might be closely related to the expression of PCGs or the binding affinity of transcription factor A to mtDNA. Consequently, these methylations may affect the enzymatic activity of oxidative phosphorylation and then the mt respiratory rate; or they may influence mt biogenesis. Therefore, methylations in the mitogenome of O. sinensis might be a genetic feature to adapt to the cold and low PO2 environment at high altitude, where O. sinensis is endemic. This is the first report on epigenetic modifications in a fungal mt genome.

The mitochondrial genome of the nematode endoparasitic fungus Hirsutella rhossiliensis

In this study, we report the complete mitochondrial genome of Hirsutella rhossiliensis (Ophiocordycipitaceae, Hypocreales, Ascomycota). We construct the mitochondrial DNA genome organization of 62 483 bp in length of H. rhossiliensis by using the whole-genome resequencing method. Conserved genes including the large and small rRNA subunits, 26 tRNA and 14 protein-coding genes are identified. These protein-coding genes utilize ATG, GTG or TTG as initiation codons and TAA or TAG as termination codons. Moreover, we detect 10 group I introns and one unclassified intron in six genes (rnl, cob, cox1, cox3, nad1 and nad5) encoding ORFs of ribosomal protein S3 and GIY-YIG/LAGLIDADG endonucleases or hypothetical proteins. This mitochondrial genome will be useful in understanding the distribution and genetic diversity of this species.

Functional response of the fungus Hirsutella rhossiliensis to the nematode, Heterodera glycines.

Functional response is a key index in determining the population fluctuation in predation. However, the lack of operable research system limits the studies on functional response of fungal predators. Hirsutella rhossiliensis is a dominant parasite of the soybean cyst nematode, Heterodera glycines. In a soil microcosm bioassay, we determined fungal biomass at different days within 21 days after inoculation, and parasitism rate of H. glycines by the fungus was determined. The functional response of H. rhossiliensis to H. glycines was established and found to be Holling's type III, which was influenced by mycelial densities. Meanwhile, we conducted anti-fungal analysis of metabolic fractions extracted from H. rhossiliensis to explain the potential mechanism of the intraspecific competition illustrated by functional response. The result of anti-fungal experiments indicated that the fungal predators had more complicated interaction at population level than expected, which might be regulated by self-inhibition metabolite(s). This study was the first functional response study of fungal predators in microcosm. With the increasing recognition of emerging fungal threats to animal, plant, and ecosystem health, the methodologies and hypotheses proposed in this study might inspire further research in fungal ecology.

Parasitism of secondary-stage juvenile of Heterodera glycines and four larva stages of Caenorhabditis elegans by Hirsutella spp

The fungi Hirsutella rhossiliensis and Hirsutella minnesotensis generally parasitize only plant-parasitic nematodes in nature but parasitize the bacterivorous nematode Caenorhabditis elegans on agar plates. To establish a model system for studying the interaction between fungi and nematodes, we compared the parasitism of the first- to fourth-stage larvae (L1–L4) of C. elegans and second-stage juvenile (J2) of Heterodera glycines by twenty isolates of Hirsutella spp. Although parasitism differed substantially among isolates, both H. minnesotensis and H. rhossiliensis parasitized a higher percentage of H. glycines J2s than of C. elegans larvae. Parasitism of C. elegans L1s was correlated with parasitism of H. glycines J2s. Parasitism of C. elegans by H. rhossiliensis and H. minnesotensis was negatively correlated with larva size and motility, i.e., parasitism was higher for the younger stages. The C. elegans L1 is recommended for studying parasitism of nematodes by H. rhossiliensis and H. minnesotensis.

Interactions between nematodes and their microbial enemies in coastal sand dunes

European foredunes are almost exclusively colonised by Ammophila arenaria, and both the natural succession and the die-out of this plant have been linked to populations of plant-parasitic nematodes (PPN). The overarching aim of this study was to investigate top-down control processes of PPN in these natural ecosystems through comparative analyses of the diversity and dynamics of PPN and their microbial enemies. Our specific aims were, first, to identify and quantify PPN microbial enemies in European sand dunes; second, to assess their life history traits, their spatial and temporal variation in these ecosystems, and third, to evaluate their control potential of PPN populations. This was done by seasonal sampling of a range of sites and making observations on both the nematode and the microbial enemy communities in rhizosphere sand. Nine different nematode microbial enemies belonging to different functional groups were detected in European sand dunes. Their high diversity in these low productivity ecosystems could both result from or lead to the lack of dominance of a particular nematode genus. The distribution of microbial enemies was spatially and temporally variable, both among and within sampling sites. Obligate parasites, either with low host-specificity or having the ability to form an environmentally resistant propagule, are favoured in these ecosystems and are more frequent and abundant than facultative parasites. Three microbial enemies correlated, either positively or negatively, with PPN population size: Catenaria spp., Hirsutella rhossiliensis and Pasteuria penetrans. Microbial-enemy supported links in the food-web may be involved in the control of PPN populations through indirect effects. The endospore-forming P. penetrans was the most successful top-down control agent, and was implicated in the direct control of Meloidogyne spp. and indirect facilitation of Pratylenchus spp. Overall, our findings suggest strong and diverse top-down control effects on the nematode community in these natural ecosystems.

Fermentation and microencapsulation of the nematophagous fungus Hirsutella rhossiliensis in a novel type of hollow beads

In this work, fermentation and formulation aspects of the nematophagous fungus Hirsutella rhossiliensis BBA were investigated. When incubated in 2% (w/w) glucose and 0.5% (w/w) yeast extract medium in a 1-L Erlenmeyer flask without baffles, heavy pellet formation was observed. Only 40% of the mycelium had a size less than 500 μm. When a flask with three baffles was used, the portion of mycelium <500 μm rose to 95%. In the next step, the influence of aeration rate and stirrer speed on production of finely dispersed mycelium in a stirred tank reactor was investigated. The best fermentation results were obtained at 0.4 vvm and 400 rpm stirrer speed with 90% mycelium <500 μm and 5 g/L biomass. Then, mycelium was microencapsulated in hollow beads based on sulfoethylcellulose (SEC). Experiments on the capsule nutrient reservoir showed that 15% (w/w) corn gluten and 0.5% (w/w) yeast extract could be replaced with 3% (w/w) autoclaved baker's yeast which was never used as capsule additive before. Radial growth of mycelium out of dried hollow beads containing 1% (w/w) biomass and 3% (w/w) baker's yeast was faster than for alginate beads containing equivalent amounts of biomass and yeast indicating a higher bio-control potential.

Novel neogala-series glycosphingolipids with terminal mannose and glucose residues from Hirsutella rhossiliensis, an aureobasidin A-resistant ascomycete fungus

Hirsutella rhossiliensis, a nematophagous fungus belonging to the Ascomycota, is resistant to aureobasidin A (AbA). In this fungus, the biosynthetic pathway leading to mannosylinositolphosphoceramides, which is inhibited by AbA, was not detected. Instead, this fungus contains neutral complex glycosphingolipids (GSLs) and monoglycosylceramides. Except for monoglycosylceramides, neutral GSLs share a neogala-series core structure, Galbeta1-6Galbeta1-Cer. Among the GSLs of H. rhossiliensis, three novel GSLs with terminal Man and Glc residues on the sugar chain were elucidated. We analyzed GSL structure using compositional sugar, fatty acid, and sphingoid analyses, methylation analysis, matrix-assisted laser desorption ionization time-of-flight/mass spectrometry (MALDI-TOF MS), and (1)H nuclear magnetic resonance spectroscopy (NMR). The following structures were determined: Manalpha1-3Galbeta1-6Galbeta1-6Galbeta1-Cer; Glcalpha1-2Galbeta1-6Galbeta1-6Galbeta1-Cer; and Manalpha1-3Galbeta1-6(Glcalpha1-4)Galbeta1-6Galbeta1-Cer. In the ceramides, the fatty acids were predominantly saturated h24:0-acids and the sphingoids were predominately t18:0- or t18:1-sphingoids. In contrast, the ceramides of Glcbeta1-Cer contained d18:2- and d19:2-sphingoids. These findings indicate the presence of a novel biosynthetic pathway of neogala-series GSLs in fungi.

Effectiveness of Hirsutella minnesotensis and H. rhossiliensis in control of the soybean cyst nematode in four soils with various pH, texture, and organic matter

The parasitism of soybean cyst nematode, Heterodera glycines, by the fungi Hirsutella rhossiliensis and Hirsutella minnesotensis and their biocontrol effectiveness against the nematode were investigated in four soils with various pH, texture, and organic matter. Fungal parasitism was assayed in the soils in 25 mL vials. As expected, percentage of H. glycines second-stage juveniles (J2) parasitized by either fungus increased with increasing number of fungus-colonized J2 initially added into the soils. Parasitism of J2 by the fungi was negatively related with soil pH. Both positive and negative relationships with fungal parasitism were observed for soil sandiness and organic matter. In greenhouse study, both fungi at 0.2–0.8 g fresh mycelium of liquid culture per 0.3 L pot and 1% corn-grits culture effectively reduced nematode population density. The relationship between biocontrol effectiveness and the soil factors depended on fungal species and inoculation levels. In general, percentage reduction of egg population density in the soil was negatively correlated with soil pH and positively correlated with sandiness. There was no or weak correlation between egg reduction and organic matter. The percentage of J2 parasitized by the fungi 2 months after planting did not correlate with the soil factors. Plant growth was better in the two soils with intermediate pH and sand than the soil with high pH and low sand or with low pH and high sand. It appeared that soil pH and/or texture are important in influencing biocontrol effectiveness, but further studies are needed to determine the effect of individual factors because they are correlated.

Purification, characterization, and gene cloning of an alkaline serine protease from a highly virulent strain of the nematode-endoparasitic fungus Hirsutella rhossiliensis

Hirsutella rhossiliensis OWVT-1 has substantial potential as a biocontrol agent against plant-parasitic nematodes. Serine proteases have emerged as a potentially useful factor in the nematode-fungus interactions. When grown in liquid culture with the nematode Panagrellus redivivus as the sole nitrogen source, an extracellular alkaline protease (Hasp) was produced by the OWVT-1. The purified Hasp killed the juveniles of the soybean-cyst nematode (Heterodera glycines) and degraded proteins of the nematode cuticle. The molecular mass of Hasp was estimated to be 33 kDa. The optimum pH and temperature for enzyme activity were pH 9 and 75 degrees C. The amino acid sequence obtained by the N-terminal sequence analysis was applied for the primer design to isolate the Hasp cDNA gene, which consists of 1170 bp open reading frame. Analysis of the cDNA and corresponding genomic sequence revealed that Hasp included four exons (279, 186, 513, and 192 bp) divided by three introns (65, 99, and 93 bp). Southern blotting showed that Hasp was a single-copy gene in the genome. The deduced amino acid sequence was very similar to other serine proteases of endoparasitic and egg-parasitic fungi of nematodes and of entomopathogenic fungi but was less similar to the serine proteases of nematode-trapping fungi. In a phylogenetic analysis of the amino acid sequences of serine proteases, the serine protease of H. rhossiliensis OWVT-1 clustered with the serine proteases of parasites of nematode eggs rather than with those of the trapping fungi.

The living strategy of nematophagous fungi

The infection structures, trophism, and ecological character of nematophagous fungi are reviewed in this article on the basis of data extracted from the literature and the most recent experiments conducted in this area. Traditionally, nematophagous fungi are classified into four groups according to their modes of attacking nematodes: nematode-trapping fungi using adhesive or mechanical hyphal traps, endoparasitic fungi using their spores, eggparasitic fungi invading nematode eggs or females with their hyphal tips, and toxin-producing fungi immobilizing nematodes before invasion. In the present review, we focus on the first two groups. The living strategies of these nematophagous fungi depend on the diversity of their infection structures, such as different traps and spore types, which determine the modes of infecting nematodes. The diversity of trophic modes of nematophagous fungi is an important prerequisite for fungal survival and activity in soil. The abundance and activity of Hirsutella rhossiliensis and H. minnesotensis, representatives of endoparasites and potential biocontrol agents against nematodes, are highly dependent on environmental factors. Comprehensive understanding of the survival and activity of nematophagous fungi in soil is fundamental for the exploitation of these fungi as successful biocontrol agents.

Population dynamics and biocontrol efficacy of the nematophagous fungus Hirsutella rhossiliensis as affected by stage of the soybean cyst nematode

Monitoring the population dynamics of a biocontrol agent in soil is important for understanding, predicting and increasing its efficacy. In this study, the population dynamics and the efficacy of a promising biocontrol agent against nematode, the fungus Hirsutella rhossiliensis, were investigated in greenhouse experiments with quantitative real-time polymerase chain reaction (PCR) and bioassay. To explore the effects of the fungus on nematode inoculum, soil infested with eggs or second-stage juveniles (J2) of Heterodera glycines was used. The results showed that the quantity of H. rhossiliensis DNA based on real-time PCR decreased over time, regardless of eggs or J2 as inoculum. The quantity of H. rhossiliensis DNA [femtogram (fg)/g soil] was highest (3.83 ± 1.96 × 10 8 fg/g soil) when the fungus was first added to soil and then decreased rapidly to 3.24 ± 1.22 × 10 7 fg/g in egg-infested soil and 7.34 ± 2.94 × 10 7 fg/g in J2-infested soil 17 days after planting, and then declined gradually between 17 and 59 days after planting. The data based on the bioassay showed that the percentage of J2 parasitized by H. rhossiliensis decreased throughout the experiment in both egg- and J2-infested soils. H. rhossiliensis controlled H. glycines more effectively in J2-infested soil than in egg-infested soil. At the end of the experiments (59 days after planting), nematode suppression and plant growth promotion in J2-infested soil were 79% and 55%, respectively, which were higher than the 34% and 2% in egg-infested soil. In soil that was not inoculated with the fungus, the number of H. glycines was 10 times higher in J2-infested soil than in egg-infested soil, indicating the greater nematode inoculum potential in soil infested with J2. DNA yield of H. rhossiliensis was statistically higher in J2-infested soil than in egg-infested soil in earlier period (prior to day 31) ( p < 0.05), which is consistent with the hypothesis that the numbers of J2 present during and soon after fungal inoculation are critical for maintaining the population density and biocontrol efficiency of the fungus.

Purification and characterization of a neutral serine protease with nematicidal activity from Hirsutella rhossiliensis

Serine protease plays an important role in fungal infection to invertebrate hosts. An extracellular protease (Hnsp) was detected in liquid culture of Hirsutella rhossiliensis OWVT-1 with nematodes (Panagrellus redivivus) as the unique nitrogen source and purified to homogeneity by ammonium sulphate precipitation, anion exchange chromatography and gel filtration. Its molecular mass was about 32 kDa, and the optimal reaction pH value and temperature were pH 7 and 40 degrees C, respectively. The Hnsp activity was stable at pH 6-8 and decreased radically at 50 degrees C for 10 min. Hnsp was highly sensitive to inhibitor of PMSF and well decomposed the substrate N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide, suggesting that it belonged to the chymotrypsin/subtilisin of serine proteases. The N-terminal amino acid sequence of Hnsp was SVTDQQGADCGLARISHRE, which showed high homology with other serine proteases from nematophagous fungi. Ability to kill nematode and degrade its cuticle in vitro indicated that Hnsp could be involved in the infection of nematode.

Suppression of nematodes in a coastal grassland soil

The nematophagous fungi Arthrobotrys oligospora and Myzocytiopsis glutinospora increase to large numbers (>103 propagules/g of soil) when moth larvae killed by entomopathogenic nematodes are added to soil microcosms. In spite of these increases, it is unclear how effective these nematophagous fungi are in suppressing nematodes. We measured nematode mortality in microcosms with small numbers of assay nematodes, and we examined assay nematodes recovered at the end of the experiment for signs of fungal parasites. Because the microcosms did not have a moat or other refuge, the assay nematodes remained vulnerable for the 3 days that they were in the soil. Mortality in this experiment was not substantially increased compared to a previous experiment, which measured the mortality of a larger number of assay nematodes in microcosms surrounded by a moat. Mortality, however, increased from 34 to 50% when recovered assay nematodes were examined and when those with conidia of the nematophagous fungus Hirsutella rhossiliensis were considered dead. The zoosporic fungus M. glutinospora was not detected, perhaps because the soil water potential was too low. Contrary to our expectations, there was no evidence of negative feedback on nematodes (i.e., no evidence of density-dependent mortality) because the addition of dead moth larvae greatly increased numbers of resident nematodes and A. oligospora but did not greatly affect the probability of nematode mortality.