Influence of the preservation period in silica-gel on the predatory activity of the isolates of Duddingtonia flagrans on infective larvae of cyathostomins (Nematoda: Cyathostominae)

The continued maintenance of nematophagous fungi predatory activity under laboratory conditions is one of the basic requirements for a successful biological control. The purpose of this study was to evaluate the influence of time on the preservation of the fungus Duddingtonia flagrans (AC001 and CG722) stored in silica-gel for 7 years and their subsequent predatory activity on cyathostomin L₃ larvae in 2% water-agar medium (2% WA). Samples of the isolates AC001 and CG722, originating from vials containing grains of silica-gel sterilized and stored for 7 years, were used. After obtaining fungal conidia, the predation test was conducted over 7 days on the surface of 9.0 cm Petri dishes filled with 2% WA. In the treated groups each Petri dish contained 500 cyathostomin L₃ and conidia of fungal isolates in 2% WA. In the control group (without fungi) the plates contained 500 L₃ in 2% WA. The experimental results showed that isolated AC001 and CG722 were efficient in preying on cyathostomin L₃ (p < 0.01) compared to control (without fungus). However, no difference was observed (p > 0.01) in the predatory activity of the fungal isolates tested. Comparing the groups, there was a significant reductions of cyathostomin L₃ (p < 0.01) of 88.6% and 78.4% on average recovered from the groups treated with the isolates AC001 and CG722, respectively, after 7 days. The results of this test showed that the fungus D. flagrans (AC001 and CG722) stored in silica-gel for at least 7 years maintained its predatory activity on cyathostomin L₃.     

Occurrence of Bursaphelenchus mucronatus (Nematoda: Aphelenchoididae) and the microhabitat distribution of fungi in declining pine trees in a locale in Korea

A total of 33 pine trees with symptoms of decline were collected in Jeonnam Province, South Korea, and were examined for the presence of nematodes. About 20% of the trees sampled were positive with Bursaphelenchus species. All Bursaphelenchus species were found in recently dead or dying trees. Based on morphological observations, the nematode extracted from the declining pine trees was identified as B. mucronatus. The highly pathogenic pine wood nematode B. xylophilus was not found in any pine trees sampled. B. mucronatus was easily reared on fungus Botrytis cinerea. Twenty one fungal isolates were isolated from dead trees, fallen twigs, and healthy pine trees. The fungal isolates belonged to Trichoderma genus and were dominant in the wood of partially declining pines. The blue‐stain fungi transmitted by the Monochamus beetle were not detected. The B. mucronatus population decreased markedly on Auxarthron reticulatum DY‐2 isolated from soils. The number of nematodes also reduced on Verticillium saksenae A‐1, a nematophagous fungus, and Beauveria bassiana, an entomopathogenic fungus. This observation suggested the fungal production of nematicidal activity against B. mucronatus. When the fungal culture filtrates were also used for nematicidal activity on B. mucronatus, the culture filtrates of A‐1, DY‐2 and B. bassiana showed over 50% mortality within 48 h exposure. The fungi BC4, BC5 and BC6 isolated from declining pine trees inhibited the reproduction of B. mucronatus, and their culture filtrates also expressed nematicidal activity, indicating a possible interaction between the fungi in pine trees and nematodes at microhabitat level.     

Predatory activity and passage of six nematophagous fungi species in gastrointestinal tract of trichostrongylide-infected sheep*

With the development of anthelmintic resistance of parasitic nematodes, screening the potential of predatory fungi candidates by in vitro and in vivo assay is necessary for biocontrol of parasitic nematodes in sheep. Fifteen native isolates of fungi species of six predators were evaluated through in vitro and in vivo experiment to evaluate the capacity of passing through the gastrointestinal tract of sheep. The results of the in vitro experiment showed that the reduction percentages of the third-stage larvae (L3) of trichostrongylides in faeces were 97.02–98.49% for five isolates of Arthrobotrys (Monacrosporium) sinense; 83.47–99.12% for three isolates of Arthrobotrys oviformis; 80.00–97.41% for four isolates of Arthrobotrys conoides; and 99.18%, 77.56%, and 75.72% for one isolate of Arthrobotrys microscaphoides, Arthrobotrys salina, and Dactylellina leptospora, respectively. In the in vivo assay, the results exhibited that the larval recoveries in faeces were significantly decreased (p?D. leptospora. After fungal treatment (within 24?h) of one isolate of A. salina, although the efficacy against L3 was 43.83%, tested fungus was detected in the faeces. The remaining isolates, except one isolate of D. leptospora and one isolate of A. conoides, were positive for either L3 reduction or fungal survival in faeces. The present study showed that nematophagous fungi could survive in the passage through the alimentary tract of sheep and should be potential candidates as a feed supplement.     

Four endoparasitic nematode destroying fungi isolated from Sand Ridge State Forest soil

A survey to determine the endoparasitic nematode destroying fungi located within Sand Ridge State Forest of Illinois was conducted from 1 January to 3 April 1973. A total of seven nematode destroying fungal species were isolated from the collected soil samples. Harposporium helicoides, H. crassum, H. lilliputanum are endoparasitic nematophagous fungi that have been isolated previously from the forest soil. Acrostalagmus gonoides, A. obovatus, Cephalosporium balanoides, and Monacrosporium cionopagum are nematophagous fungal species that had not been isolated previously from Illinois soil. Soil pH's and soil nutrient levels were not important in the isolation frequency of the collected endoparasitic nematode destroying fungi.     

In vitro salt and thermal tolerance of fungal endophytes of Nicotiana spp. growing in arid regions of north-western Australia

Abstract

Endophytic fungal strains isolated from indigenous Nicotiana plants naturally growing in dry and hot regions of north-western Australia were characterised based on their tolerance to salinity and temperature. Sixty-eight fungal isolates were tested on eight levels (0.5 M, 1.0 M, 1.5 M, 2.0 M, 2.5 M, 3.0 M, 3.5 M and 4.0 M) of five different of salts solutions NaCl, KCl, MgCl₂, CaCl₂ and MgSO₄ and at various temperatures (25–50?°C). The salt adaptation test indicated that the fungal strains namely Aspergillus niger (E-202), A. ochraceous-A (E-134), Aurantiporus sp. (E-135), Cladosporium halotolerance (E-128), Pleurostomophora richardsiae (E-13) and Trichoderma sp. (E-185.1) were tolerant to higher concentrations of various salts. The most growth-limiting salt turned out to be MgCl₂ followed by the chaotrope CaCl₂. Responses to temperature tolerance revealed that most fungi tested could grow at 30?°C. About 50% all the fungi did not show any growth when the temperature was raised above 30?°C. When the temperature was raised up to 50?°C all the fungi failed to grow but the fungus Rasamsonia piperina (E-172). Endophyte strains identified could be promising candidates for future research in investigating the fungus–plant interactions and their roles in plant adaptation to inhospitable environments.     

A new model system for the study of the animal innate immune response to fungal infections

The association of the model organism Caenorhabditis elegans and the fungus Pleurotus ostreatus gives the possibility to study the molecular and genetic mechanisms of the early stages of the spatial and temporal interactions of animals with fungal pathogens. We identified the stages of the infection process of P. ostreatus on the nematode C. elegans. We found that prior to penetration inside a worm a fungal toxin paralyzed and immobilized, but did not kill C. elegans. This finding opens the possibility for the further study of the effect of paralyzing toxins on host organisms. The membrane permeability of paralyzed worms increased dramatically and leakage products initiated the growth of directional hyphae towards the nematodes. The hyphae penetrated into live C. elegans animals either through natural openings or directly by piercing the cuticle. Upon contact with the nematode cuticle, P. ostreatus attached to it, formed appressoria-like structures and infection pegs, piercing the cuticle and penetrating inside the nematode body. The small zones around the penetration loci are of special interest for the evaluation of initial contacts between two organisms and for the study of the C. elegans local defense response against fungal infection.     

Growth and survival of Verticillium chlamydosporium goddard,a parasite of nematodes,in soil

Selective media for the isolation of the nematophagous fungus Verticillium chlamydosporium, are described. These enabled densities > 500 colony forming units (CFU) g^‐1 soil to be reliably estimated. However, there was little relationship between estimates of the Verticillium biomass in a sterilized soil and the numbers of CFU which developed on the selective media. The growth and survival of the fungus infield soils were studied and estimates of the numbers of CFU in soils in which cyst‐nematode multiplication was suppressed were greater than in those in which the nematode multiplied. Isolates of the fungus differed in their ability to proliferate in soil, but some increased rapidly from applications of chlamydospores or a mixture of hyphae and conidia in alginate granules containing wheat bran. The energy source (wheat bran) was essential for the establishment of the fungus from granular applications. Numbers of CFU greatly exceeded those of chlamydospores, but there was considerable variation in the relationship in different soils. Some isolates of V. chlamydosporium proliferated in soil and survived in considerable numbers for at least 3 months. Hence, pre‐cropping applications of the fungus should survive long enough to kill nematode eggs and females that develop on roots of spring‐sown crops.     

Efficacy of entomopathogenic nematode,Steinernema carpocapsae against the diamondback moth,Plutella xylostella (L.) in laboratory condition

In vitro studies were carried out on the diamondback moth, Plutella xylostella larvae using an insect entomopathogenic nematode isolate, Steinernema carpocapsae obtained from the Koppert company, the Netherlands. Larvae of P. xylostella were collected from cabbage farms around Mashhad city of Iran. During the study, the responses of larvae at 25?°C for three periods of 24, 48 and 72?h with different concentrations of 0, 5, 10, 20, 40, 80, 160 and 320 third instar larvae of nematode (infective stage?=?IJs) per insect into 10?cm Petri dishes containing filter paper soaked with 1?ml of nematodes suspension were compared. Maximum mortality caused by S. carpocapsae nematode was 88% at 24?h, and it was 100% at 48 and 72 h. With increasing nematode population level and exposure time (ET in hour), mortality of P. xylostella larvae was increased. Based on probit analysis, LC₅₀ values of S. carpocapsae nematode in three test periods were 45.61, 12.02 and 40.80 IJs per insect, respectively. Initial ANOVA was performed for S. carpocapsae nematode. The effect of both nematode population levels (IJ) and ET on third instar larvae of the diamondback moth, P. xylostella and interaction between IJ and ET were significant. In general, it is recommended to apply this nematode in suitable condition for controlling diamondback moth.     

Endophytic Colonisation of Opium Poppy, Papaver somniferum, by an Entomopathogenic Beauveria bassiana Strain

Beauveria bassiana strain EABb 04/01-Tip isolated from stem-borer larvae of Timaspis papaveris (Hymenoptera: Cynipidae), a serious pest of opium poppy in Spain, was shown to be able to become established endophytically in this pharmaceutical crop. Microbiological, molecular and light and electron microscopic methods were used to study fungal colonisation and to describe its mode of penetration. After inoculation with a foliar spray of conidia, microbiological methods showed 100% of plants examined 24, 48, 72 and 144 h after treatment to be colonised endophytically by the fungus, although the percentage of previously surface sterilised leaf pieces showing fungal growth was 100% at 24 and 48 h, and 80 and 75% at 72 and 144 h after treatment, respectively. The fungus was also observed in leaf pieces obtained from newly formed leaves, indicating that it could spread from treated leaves to leaves formed after fungal application. For molecular studies, a polymerase chain reaction (PCR) protocol was used to amplify the ITS1-5.8S-ITS2 regions of the rDNA of the plant and the fungus. This procedure allowed the detection of the fungus on the surface of the leaves and also endophytically, but only at 72 h after treatment. A nucleotide BLAST search revealed that the ITS1-5.8S-ITS2 sequence of strain EABb 04/01-Tip showed 100% homology with a similar sequence from Cordyceps bassiana. SEM images revealed that although numerous conidia were observed on the leaf surface, few germinated and penetrated. Intracellular colonisation by B. bassiana was not observed, but hyphae were detected growing into the xylem vessels. The fungus was found to colonise 40.5 ± 4.3% of seedlings (with two cotyledons and the two first real leaves) from seeds dressed with a fungal spore suspension. These results may have implications in the biological control of T. papaveris, including the possible systemic protection of the plant against this cynipid.     

Effects of culture filtrates from the nematophagous fungus Verticillium leptobactrum on viability of the root-knot nematode Meloidogyne incognita

Filtrates of three isolates of the nematophagous fungus Verticillium leptobactrum were evaluated for their nematicidal activity against the root-knot nematode Meloidogyne incognita. The filtrates inhibited egg hatching, with maximum toxicity observed for isolate HR21 at 50% (v:v) dilution, after 7 days exposure. Filtrates also inactivated second-stage juveniles (J2) at 10-50% dilutions. A scanning electron microscopy study of treated eggs showed severe alterations caused by the filtrate of isolate HR43 on M. incognita eggs, which appeared collapsed and not viable, suggesting the production of chitin-degrading enzymes or other active compounds.     

Comparative Genomic Analysis of Drechmeria coniospora Reveals Core and Specific Genetic Requirements for Fungal Endoparasitism of Nematodes

Drechmeria coniospora is an obligate fungal pathogen that infects nematodes via the adhesion of specialized spores to the host cuticle. D. coniospora is frequently found associated with Caenorhabditis elegans in environmental samples. It is used in the study of the nematode’s response to fungal infection. Full understanding of this bi-partite interaction requires knowledge of the pathogen’s genome, analysis of its gene expression program and a capacity for genetic engineering. The acquisition of all three is reported here. A phylogenetic analysis placed D. coniospora close to the truffle parasite Tolypocladium ophioglossoides, and Hirsutella minnesotensis, another nematophagous fungus. Ascomycete nematopathogenicity is polyphyletic; D. coniospora represents a branch that has not been molecularly characterized. A detailed in silico functional analysis, comparing D. coniospora to 11 fungal species, revealed genes and gene families potentially involved in virulence and showed it to be a highly specialized pathogen. A targeted comparison with nematophagous fungi highlighted D. coniospora-specific genes and a core set of genes associated with nematode parasitism. A comparative gene expression analysis of samples from fungal spores and mycelia, and infected C. elegans, gave a molecular view of the different stages of the D. coniospora lifecycle. Transformation of D. coniospora allowed targeted gene knock-out and the production of fungus that expresses fluorescent reporter genes. It also permitted the initial characterisation of a potential fungal counter-defensive strategy, involving interference with a host antimicrobial mechanism. This high-quality annotated genome for D. coniospora gives insights into the evolution and virulence of nematode-destroying fungi. Coupled with genetic transformation, it opens the way for molecular dissection of D. coniospora physiology, and will allow both sides of the interaction between D. coniospora and C. elegans, as well as the evolutionary arms race that exists between pathogen and host, to be studied.     

Occurrence and colonization of nematophagous fungi in different substrates,agricultural soils and root galls

Seventeen species of nematophagous fungi were recorded, three species were endoparasitic and fourteen species were predacious fungi. Among the predacious fungi Arthrobotrys oligospora, Dactylaria brochopaga and Monacrosporium eudermatum were very frequent, whereas others were recorded at lower frequency. Twelve species of nematophagous fungi from compost as well as cow dung manure, 15 species from leaf litter and only eight species from agricultural soils were recorded. In general, substrate colonization by nematophagous fungi was higher in leaf litter, compost and cow dung manure. The agricultural soil amended with FYM (farm yard manure) recorded nine species of nematophagous fungi while unamended soil recorded only seven species. Thirteen species of nematophagous fungi were recorded from soils under banyan tree. Of all these fungi unidentified net-forming fungus, M. eudermatum, A. cladodes, D. brochopaga, S. hadra, A. oligospora and A. dactyloides had higher percentage of soil colonization. In soil collected under pipal tree only eight species were recorded, of which A. oligospora, A. cladode and an unidentified fungus were more predominant as their percentage colonized in soil samples was higher. Few studies have examined root galls as a substratum for colonization of nematophagous fungi. Of all the root gall samples, okra root galls recorded maximum colonization by predacious fungi. Maximum percentage of root gall colonization was recorded for M. eudermatum followed by A. oligospora and M. ellipsosporum. M. eudermatum was also most predominant colonizer of balsam, brinjal and rice root galls.     

Isolation,identification and characterisation of the nematophagous fungus Arthrobotrys thaumasia (Monacrosporium thaumasium) from China

The objective of this study is to select a native isolate of Arthrobotrys thaumasia (Monacrosporium thaumasium), a nematophagous fungus that shows potential for use in biocontrol programmes for ruminants. First, we looked for native isolates of A. thaumasia and characterised them using light microscopy and molecular markers. Then, we determined the effect of temperature, pH and nutrition on the growth rate and trap formation of a representative isolate. Of the 1532 samples of different types related to sheep and cattle, 11 isolates of A. thaumasia were isolated, and their occurrence frequency in the samples was 0.71% (11/1532). We sequenced the rDNA internal transcribed spacer of isolate NBS005, submitted it to GenBank (ID: KX640093) and then sequenced it using BLAST. The NBS005 could not grow at 37.5°C but could grow from 11°C to 35°C, and it exhibited its optimum growth at 30°C on 1% corn meal agar (CMA). Over 4 days, the fungus did not grow in the pH interval from 1 to 3 or from 13 to 14 but did grow in the pH interval from 4 to 12 and exhibited its optimum growth between pH 9 and 10 on 2% CMA. The factors responsible for the trap formation of NBS005 in liquid culture were identified. Trap formation was induced only by contact with L3 lysate. The concentrations of sucrose, ammonium chloride and tryptone of 0.4, 0.2 and 0.2%, respectively, promoted trap formation, and there were higher numbers of trapping nets in 2% wheat bran liquid medium containing L3 lysate.     

An improved method for light- and electron microscopial studies of nematode/fungal interactions

A method is presented that enables studies to be made of single nematode-fungal interactions under conditions where fungal growth at the expense of external nutrients is prevented. The nematophagous fungus Arthrobotrys ologospora was used as a model organism in these studies. The method is based on removal of the traps from the vegetative mycelium, immediately after a nematode was captured and transfer of the trap with the captured nematode into a droplet of sterile distilled water placed in a moisture chamber. In the absence of external nutrients, such isolated traps of A. oligospora were fully effective in penetrating and subsequently digesting the captured nematode. Solely vegetative mycelium was formed at the expense of the digested nematode; this developed from the trap that originally had captured the nematode. One advantage of the present method is that studies on various stages of the nematode-fungal interaction can now be performed under conditions that exclude major influences of external nutrients which otherwise could be communicated to the trap cells by way of the vegetative mycelium.     

Effects of Metarhizium anisopliae on Meccus pallidipennis (Hemiptera: Reduviidae) over different types of wall surfaces

The entomopathogenic fungus Metarhizium anisopliae is virulent for the insect triatomine Meccus pallidipennis. To evaluate the functionality of a fungal formulation (vegetable oil and emulsifiers) of this fungus, virulence was assayed by insect mortality on the pronotum of third instar nymphs (N3) M. pallidipennis in laboratory conditions and ST₅₀ was calculated. Mortality was evaluated directly: 100%, 97.33% and 98.66% mortalities were caused by formulation, emulsified formulation and fungal conidia, respectively, at day 8 of insect infection. Another bioassay was carried out in simulated external conditions (peridomicility) using red and gray brick walls, a stone fence and mountain soil (experimental units). These simulated conditions were infected with 10?ml of a 1?×?10⁹?conidia/ml emulsified formulation by means of a manual sprinkler prior to the placement of the nymphs. Ten N3 M. pallidipennis were deposited in each experimental unit and insect mortality was monitored every 12?h for 22 days. Each treatment was replicated four times. With the red brick wall, a mortality of 90% at day 22 and a ST₅₀ of 15 days were obtained on N3 M. pallidipennis; with the gray brick wall, 100% mortality and a ST₅₀ of 13 days; and with the stone fence, 88.33% mortality and a ST₅₀ of 21 days. The results obtained in this research work indicate that the formulation with conidia of the M. anisopliae strain EH-473/4 may be auxiliary in the development of strategies for the control of Chagas disease insect transmitters such as M. pallidipennis.     

Culture medium characteristics on the predatory activity of the nematophagous fungus Duddingtonia flagrans

The influence of casein and pH on the activity of the nematophagous fungus Duddingtonia flagrans (AC001) on trichostrongylide larvae was evaluated. A ‘positive influence’ was observed contributing to the reduction of 63% in the average number of recovered L₃ in the media supplemented with casein and pH 7.0.     

Detection of the nematophagous fungus Hirsutella rhossiliensis in soil by real-time PCR and parasitism bioassay

Hirsutella rhossiliensis, a nematophagous fungus, has shown potential in biocontrol of plant-parasitic nematodes. Monitoring the population dynamics of a biocontrol agent in soil requires comprehensive techniques and is essential to understand how it works. Bioassay based on the fungal parasitism on the juveniles of soybean cyst nematode, Heterodera glycines, can be used to evaluate the activity of the fungus but fails to quantify fungal biomass in soil. A real-time polymerase chain reaction (PCR) assay was developed to quantify the fungal population density in soil. The assay detected as little as 100 fg of fungal genomic DNA and 40 conidia g⁻¹ soil, respectively. The parasitism bioassay and the real-time PCR assay were carried out to investigate the presence, abundance and activity of H. rhossiliensis in soil after application of different inoculum levels. Both of the percentage of assay nematodes parasitized by H. rhossiliensis based on the parasitism bioassay and the DNA yield of the fungus quantified by real-time PCR increased significantly with the increase of the inoculum levels. The DNA yield of the fungus was positively correlated with the percentage of assay nematodes parasitized by H. rhossiliensis. The combination of the two is useful for monitoring fungal biomass and activity in soil.     

Ecology of nematophagous fungi: distribution and habitat

A survey of 161 samples of soil and plant material collected from sites throughout Ireland has shown that nematophagous fungi exist in a wide range of habitats. 205 isolations were made consisting of 11 endoparasitic and 20 predatory species. The commonest endoparasites and their frequency of occurrence were Myzocytium spp. (9·3%), Acrostalagmus obovatus (6·8%) and Harposporium anguillulae (6·2%). Predators were equally abundant, the commonest being Dactylella bembicodes (8·7%), D. mammillata (6·8%) and D. ellipsospora (6·2%). Arthrobotrys oligospora and A. musiformis both had a highly restricted distribution. Adhesive knobs and constricting rings were significantly more abundant than the other trapping mechanisms. All the samples were classified into one of 10 broad habitat types. Of these temporary agricultural pasture, coastal vegetation and coniferous leaf litter had the greatest percentage of sites from which nematophagous fungi were isolated. A number of species showed distinct habitat associations, in particular Arthrobotrys robusta, A. musiformis and Dactylella cionopaga.     

The influence of fungicides on Arthrobotrys oligospora in simulated putting green soil

Plant‐parasitic nematodes are destructive pests in bentgrass putting greens. Few chemical or nonchemical approaches for nematode management exist. Studies were conducted to determine: the in vitro tolerance of the nematophagous fungus Arthrobotrys oligospora, to the fungicides chlorothalonil and myclobutanil used to manage diseases on putting greens; the concentration of fungicides obtained from simulated putting green soil; and the ability of the fungus to reduce populations of the ring nematode, Criconemella ornata. Both fungicides reduced in vitro hyphal growth and germination of conidia above 10 mg kg^‐1. Soil concentrations of chlorothalonil were less than 5 mg kg^‐1 and concentrations of myclobutanil were below detection limits. Nematode populations were not affected by A. oligospora in simulated greens but nematode populations were lowest in pots inoculated with A. oligospora and receiving fungicide treatments. Results of these studies indicate that applications of chlorothalonil and myclobutanil used to manage fungal diseases of bentgrass may not adversely affect A. oligospora; however, the fungus may not reduce nematode populations below desired thresholds.