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.     

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.     

Ecology of nematophagous fungi: Effect of the soil nutrients N,P and K,and seven major metals on distribution

The effects of the major soil nutrients and seven common metals on the distribution of nematophagous fungi were evaluated by comparing the concentration of the elements in soils with and without nematophagous fungi. Mineral nutrients, which were all positively correlated with nematode density, were the most important elements determining the presence of such fungi. Endoparasites producing adhesive conidia were independent of all the elements tested, while those species forming ingested conidia were isolated from soils high in nutrients, indicating a strong dependence on nematode density. Knob forming predators which rely on their ability to spontaneously produce traps to obtain nutrients were isolated from soils with low concentrations of mineral nutrients, while species with constricting rings were isolated from richer soils containing a greater density of nematodes. Net-forming species were largely independent of soil fertility, although generally they were isolated from soils with limited nutrientsd, especially K. Like other fungi, nematophagous fungi are generally not found in soils containing elevated concentrations of heavy metals. The results indicate that even concentrations of heavy metals which naturally occur in Irish soils can restrict the distribution of this fungal group. However the endoparasiteMeria coniospora is tolerant to all metals and to Cu in particular.     

Control of Root-Knot Nematodes on Tomato by the Endoparasitic Fungus Meria coniospora

The endoparasitic nematophagous fungus Meria coniospora reduced root-knot nematode galling on tomatoes in greenhouse pot trials. The fungus was introduced to pots by addition of conidia at several inoculum levels directly to the soil or addition of nematodes infected with M. coniospora to the soil; both methods reduced root galling by root-knot nematodes. These studies represent a part of a recently initiated effort to evaluate the potential of endoparasitic nematophagous fungi for biocontrol of nematodes.     

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.     

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.     

Endoparasitic nematode-trapping fungi of Mason State Forest

An initial survey involving the endoparasitic namatode-trapping fungi of Mason State Forest in Illinois was conducted from 15 May to 26 July 1971. Three endoparasitic forms were isolated from the soil samples collected; one non-endoparasitic form was also isolated. Soil pH's and soil nutrient levels were determined for the investigation period.     

Nematophagous fungi: Endoparasites ofRhabditis terricola

Three techniques were compared for the recovery of endoparasitic nematophagous fungi from soil. The Baermann funnel technique (32 species) was superior to differential centrifugation (19 species) or soil sprinkling (21 species) in terms of number of species found. The main advantage of the Baermann funnel was in the recovery of lower fungi, especially those with flagellate states. In all, 40 species of endoparasites (70% of the known flora) were recorded. Thirty-two of those were found capable of attacking the soil nematodeRhabditis terricola Dujardin.     

Attraction of Pinewood Nematode to Endoparasitic Nematophagous Fungus Esteya vermicola

The investigations on attraction of nematodes to nematophagous fungi have mostly dealt with the nematode-trapping species. Esteya vermicola is the endoparasitic fungus of pinewood nematode (PWN) with high infection activity. In the present study, the attraction of PWNs to E. vermicola was investigated. It was confirmed that the living mycelia and exudative substances of E. vermicola were attractive to PWN. Compared with the nematode-trapping fungus A. brochopaga as well as nematode-feeding fungus B. cinerea, E. vermicola showed the significantly strongest attraction ability to nematode. It therefore appeared that the attraction ability reflects the dependence of the fungi on nematodes for nutrients. Furthermore, a new method was developed and used in the study to confirm the effect of volatile substances for the attraction of nematode to fungi. The results suggested that the attractive substances were consisted of avolatile exudative and volatile diffusing compounds.     

Diversity of nematode destroying fungi in Taita Taveta,Kenya

The diversity of nematode destroying fungi in Taita Taveta, Wundanyi division, Coast Province, Kenya, was investigated between May 2006 and December 2007 aiming at harnessing their potential in the biological control of plant parasitic nematodes in the area. Given that the intensity of land cultivation is continually increasing in the study area, it is prudent to document the status of the nematode destroying fungi before the remaining forest habitats are ultimately disrupted. Soil samples were collected from forest, maize/bean, napier grass, shrub and vegetable fields, which represented the main land use types in the study area. The soil sprinkle technique method was used to isolate the nematode destroying fungi from the soil. The fungi were identified to species level. Eighty-five isolates, distributed in eight genera and 14 taxa were identified as nematode destroying fungi. The species identified were Arthrobotrys dactyloides, Arthrobotrys oligospora, Arthrobotrys superba, Acrostalagamus obovatus, Dactyllela lobata, Harposporium aungulilae, Harposporium liltiputanum, Harposporium spp, Haptoglosa heterospora, Monacrosporium asterospernum, Monacrosporium cianopagum, Myzocytium, spp, Nematoctonus georgenious and Nematoctonus leptosporus. Vegetable land use had the highest diversity of nematode destroying fungi. The results show that the study area is rich in nematode destroying fungi with A. oligospora being widespread and a possible candidate for biological control of plant parasitic nematodes.     

Fungi recovered from root-knot nematodes infecting vegetables under protected cultivation

A survey was conducted in root-knot nematode-infested plastic houses to determine the diversity and frequency of occurrence of fungi associated with the nematode. The relationships between percentage fungal parasitism and physicochemical properties of soil were also investigated. Fifty-nine plastic houses were sampled in southeastern Spain, 42 treated with nematicides and 17 left untreated. Eleven fungal genera and unidentified fungi were isolated from nematode eggs or juveniles. Fungal parasitism occurred more frequently in untreated (82.4%) than treated (50%) soils. The species richness in untreated soils ranged from 0 to 5, the Shannon–Wiener diversity index (a measurement of how many different fungi there are in site taking into account how evenly they are distributed among the site) from 0 to 2.01, and the evenness index from 0.46 to 0.99. In treated soils, species richness ranged from 0 to 4, the Shannon–Wiener diversity index from 0 to 1.61, and the evenness index from 0.81 to 1. Of the sites with nematophagous fungi, Arthrobotrys dactyloides (34%), Cylindrocarpon sp., Neosartoria hiratsukae (17%), and Fusarium solani (14%) were the fungi most frequently found. Physicochemical properties of soil were similar in nematicide treated and untreated soils. Percent fungal parasitism in untreated soils correlated positively with lime, silt and carbonate content of soil.     

Nematophagous Fungi Associated with Root Galls of Rice Caused by Meloidogyne graminicola and its Control by Arthrobotrys dactyloides and Dactylaria brochopaga

Root galls of rice caused by Meloidogyne graminicola were examined for natural colonization by nematophagous fungi from four fields with different nematode infestations. Old galls from severely infested fields had a higher frequency of Monacrosporium eudermatum and Stylopaga hadra than young galls. The frequency of Arthrobotrys oligospora, Arthrobotrys dactyloides, Dactylaria brochopaga and Monacrosporium gephyropagum was lower. A greater proportion (%) of root galls were colonized by nematophagous fungi in those fields in which rice roots had a greater root gall index. This indicated that disease severity supported the colonization of galls by nematophagous fungi. In vitro predacity tests of four fungi showed that A. dactyloides was most effective in capturing and killing J₂ of Mel. graminicola followed by D. brochopaga and Mon. eudermatum. Application of inocula of A. dactyloides and D. brochopaga in soil infested with Mel. graminicola, respectively, reduced the number of root galls by 86% and of females by 94%, and eggs and juveniles by 94%. The application of these fungi to soil increased plant growth: shoot length by 42.7% and 39.8%, root length by 45.5% and 48.9%, fresh weight of shoot by 59.9% and 56.7% and fresh weight of root by 20.3% and 25.1%, respectively, compared to these parameters for plants grown in nematode‐infested soil.     

Ecology of nematophagous fungi: vertical distribution in a deciduous woodland

Summary The distribution of nematophagous fungi in soil collected from a deciduous woodland is compared to various biotic and abiotic soil factors. The microfungi are isolated at all depths down to a maximum of 35 cm. Predators forming constricting rings, adhesive branches and adhesive knobs are restricted to the upper litter and humus layers. The net forming predators and endoparasites are isolated at all depths, although they are significantly more abundant in the lower mineral rich soils. A much greater species diversity of nematophagous fungi is recorded in the upper organic zones.Preliminary soil analysis indicates thatCephalosporium balanoides is independent of all soil variables, while predators able to form traps spontaneously are restricted to the organic soils which are rich in nematodes. Non-spontaneous trap forming predators, which are excellent saprophytes, are isolated from the deeper soils which are low in nutrients. The ecological significance of these results is discussed.     

In vitro soil receptivity assays to egg-parasitic nematophagous fungi

Soil application of nematophagous fungi for the biological control of plant-parasitic nematodes often fails, and in many cases it has been difficult to reisolate the agent delivered to the soil. A reason for these results could be the inability of the fungi to proliferate in soil. We used a soil–membrane technique to study the capacity of several isolates of the nematophagous fungi Pochonia chlamydosporia and Paecilomyces lilacinus to grow and establish in sterilized and nonsterilized sandy soils from SE Spain and Western Australia. Growth of all fungi tested was inhibited in nonsterilized soil, although there was intraspecific variability in sensitivity among isolates of the same species. With respect to hyphal density, P. chlamydosporia isolate 5 (from Italy) was the least inhibited in nonsterilized soil from both sites. Relative growth analyses confirmed this result for soil from SE Spain, while with this method, P. chlamydosporia isolate 4624 (from Australia) appeared to be least inhibited in the Australian soil. The results indicate that a soil can be more receptive to its indigenous isolates than to nonindigenous isolates. Apparently, soil microbiota can determine the ability of nematophagous fungi to proliferate in soil.     

Repulsion of Meloidogyne incognita by Alginate Pellets Containing Hyphae of Monacrosporium cionopagum,M. ellipsosporum,or Hirsutella rhossiliensis

The responses of second-stage juveniles (J2) of Meloidogyne incognita race 3 to calcium alginate pellets containing hyphae of the nematophagous fungi Monacrosporiura cionopagum, M. ellipsosporum, and Hirsutella rhossiliensis were examined using cylinders (38-mm-diam., 40 or 72 mm long) of sand (94% <250-μm particle size). Sand was wetted with a synthetic soil solution (10% moisture, 0.06 bar water potential). A layer of 10 or 20 pellets was placed 4 or 20 mm from one end of the cylinder. After 3, 5, or 13 days, J2 were put on both ends, on one end, or in the center; J2 were extracted from 8-ram-thick sections 1 or 2 days later. All three fungal pellets were repellent; pellets without fungi were not. Aqueous extracts of all pellets and of sand in which fungal pellets had been incubated were repellent, but acetone extracts redissolved in water were not. Injection of CO₂ (20 μl/minute) into the pellet layer attracted J2 and increased fungal-induced mortality. In vials containing four randomly positioned pellets and 17 cm³ of sand or loamy sand, the three fungi suppressed the invasion of cabbage roots by M. javanica J2. Counts of healthy and parasitized nematodes observed in roots or extracted from soil indicated that, in the vial assay, the failure of J2 to penetrate roots resulted primarily from parasitism rather than repulsion. Data were similar whether fungal inoculum consisted of pelletized hyphae or fungal-colonized Steinernema glaseri. Thus, the results indicate that nematode attractants and repellents can have major or negligible effects on the biological control efficacy of pelletized nematophagous fungi. Factors that might influence the importance of substances released by the pellets include the strength, geometry, and duration of gradients; pellet degradation by soil microflora; the nematode species involved; and attractants released by roots.     

The phylogenetic relationship and non‐specific symbiotic habit of mycorrhiza fungi from a terrestrial orchid (Cymbidium)

To investigate the specificity of the symbiotic relationship between Cymbidium plants and their mycorrhiza fungi, thirty mycorrhiza fungi were isolated from roots of six terrestrial Cymbidium species. The internal transcribed spacer (ITS) region of nuclear ribosomal DNA (rDNA) were amplified by polymerase chain reaction (PCR) with universal fungal primers ITS1/ITS4. All fungal strains isolated from natural roots of orchids were inoculated into the rhizomes of in vitro Cymbidium goeringii. Phylogenetic analysis indicated fungal isolates of different cluster could be obtained from a special terrestrial Cymbidium species. Observation of light microscope and scanning electron microscope showed that fungi entered the cortical tissue by destroying cell wall of epidermal cells, where they formed hyphal knots in the cortical cells and were digested gradually. A large number of small protuberances were visible on cross sections of the rhizome. There was no strict inter‐species specificity between the isolated mycorrhiza fungi and terrestrial Cymbidium.     

Isolation,identification, and in vitro predatory activity of nematophagous fungus Arthrobotrys musiformis and its interaction with nematodes using scanning electron microscopy

We isolated and identified nematophagous fungus Arthrobotrys musiformis from materials derived from cattle and sheep. We also molecularly characterised the native fungal isolates and evaluated the nematophagous activity of the isolates. A total of 19 isolates of A. musiformis were isolated from 1532 samples, and the detection rate of A. musiformis in all samples was 1.24%. These isolates were identified using a light microscope and their 5.8S rDNA, 18S rDNA, 28S rDNA, and the internal transcribed spacers 1 and 2 region. Interaction of the isolate (NPS045) with the nematode targets of the infective larvae (L₃) of Haemonchus contortus and the free-living nematode Caenorhabditis elegans was observed by scanning electron microscopy (SEM). SEM result showed that the two species of nematodes were initially captured at 5?h after being added to the isolates. L₃ was penetrated at 22?h after capture and completely destroyed by the fungus at 68?h. C. elegans was penetrated at 14?h post-capture and was completely digested by the fungus at 24?h. In vitro experimental assay of samples in 24-well plates showed that for the three fungal isolates, the L₃s of Trichostrongylus colubriforms were reduced by 94.80%, 90.17%, and 89.02%.     

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.     

Actinomycetes and fungi isolated from plant-parasitic nematode infested soils: screening of the effective biocontrol potential,indole-3-acetic acid and siderophore production

Root-knot nematodes are serious pathogens that severe damage to major crops. They damage plant root system that caused significant yield losses. Moreover, the predisposition of nematode-infected plants is secondary infection from fungal plant pathogen that additional adverse effects on plant growth. Our target is to find the antagonist for control nematode, and secondary infection agents and stimulate plant growth. Twenty-three plant-parasitic nematode infested soils were taken from some provinces in the northern and center of Thailand and actinomycetes and fungi were isolated. Eighty-three isolates belong to actinomycete and 67 isolates were fungi. The predominant actinomycete taxa was Streptomyces (97.6%). The predominant fungal taxa were Penicillium (37.3%) and Fusarium (32.8%). All actinomycete and fungal isolates were subjected for primary screening in vitro for their effects on egg hatching and juvenile mortality of Meloidogyne incognita. Secondary screening was evaluated for antagonist effect on plant pathogenic fungi collected from nematode-infected plant, plant growth hormone (indole-3-acetic acid; IAA) and siderophore production. From primary screening, 7 actinomycete and 10 fungal isolates reduced egg hatching and kill juveniles of M. incognita after 7 days incubation. In secondary screening, 10 nematophagous microbes produced IAA and 9 isolates produced hydroxamate siderophore. Streptomyces sp. CMU-MH021 was selected as a potential biocontrol agent. It reduced egg hatching rate to 33.1% and increased juvenile mortality rate to 82% as contrasted to the control of 79.6 and 3.6%, respectively. This strain had high activity to against tested fungi and high ability on IAA (28.5 μg ml⁻¹) and siderophore (26.0 μg ml⁻¹) production.