Interaction of nematodes with nematophagus fungi: induction of trap formation, attraction and detection of attractants

Induction of trap formation in Arthrobotrys oligospora, A. conoides and Monacrosporium cystosporum was studied during a 24 h period in the presence of the free-living nematodes Panagrellus redivivus on various nutrient media. A definite pattern of trap formation was observed in these fungi. Low nutrient mineral salt medium had the most pronounced effect on trap formation. Attraction and repulsion of P. redivivus towards these three nematophagous and three non-nematophagous fungi was studied. The nematodes were attracted towards three nematophagous and one non-nematophagous fungi, the other two repelled them. Attractants of the nematophagous fungi were determined by thin-layer chromatography. In the case of A. oligospora and M. cystosporum, four spots were traced, whereas in A. conoides, five spots were detected.     

Thioredoxin1 regulates conidia formation,hyphal growth,and trap formation in the nematode-trapping fungus Arthrobotrys oligospora

Arthrobotrys oligospora, a model nematophagous fungus that produces specific adhesive networks to capture nematodes, has been proposed as a potentially effective biological agent to control harmful plant-parasitic nematodes. Although thioredoxin has been characterized as playing important roles in many cellular processes in other species, its function in nematophagous fungi has not been studied. Here, the function of a thioredoxin homolog, Aotrx1, was investigated in A. oligospora. The encoding gene of Aotrx1 in the nematophagous fungus A. oligospora was knocked out by homologous recombination; strain growth was assessed. The ΔAotrx1 strain of A. oligospora showed a significant decrease in growth rate on different media (PDA, CMY, and TG), a 70% decrease of conidia production, and a lower germination rate compared with the wild type. The mutant strain was unable to form traps to capture nematodes and was more sensitive to SDS and H2O2. Thioredoxin is involved in conidia development, trap formation, normal mycelial growth, and resistance to environmental stresses in the nematode-trapping fungus A. oligospora.     

GPH1 is involved in glycerol accumulation in the three-dimensional networks of the nematode-trapping fungus <Emphasis Type="Italic">Arthrobotrys oligospora</Emphasis>

Turgor is very important for the invasive growth of fungal pathogens. Glycerol, a highly osmotic solvent, is considered to play an important role in turgor generation. The nematophagous fungus Arthrobotrys oligospora mainly lives as a saprophyte. In the presence of nematodes, A. oligospora enters the parasitic stage by forming three-dimensional networks (traps) to capture nematodes. In A. oligospora, we found that glycerol accumulated during nematode-induced trap formation. We demonstrated that deleting gph1, which encodes glycogen phosphorylase, decreased the glycerol content, compared with that of a wild-type strain. Although the number of traps induced by nematodes was not affected in the Δgph1 mutant, the capture rate was lower. Meanwhile, deleting gph1 also affected the growth rate and conidiation capacity of the fungus. These results indicate that glycerol derived from GPH1 is essential for the full virulence of A. oligospora against nematodes.     

The NADPH oxidase AoNoxA in <Emphasis Type="Italic">Arthrobotrys oligospora</Emphasis> functions as an initial factor in the infection of <Emphasis Type="Italic">Caenorhabditis elegans</Emphasis>

Reactive oxygen species (ROS) produced by NADPH oxidases can serve as signaling molecules to regulate a variety of physiological processes in multi-cellular organisms. In the nematophagous fungus Arthrobotrys oligospora, we found that ROS were produced during conidial germination, hyphal extension, and trap formation in the presence of nematodes. Generation of an AoNoxA knockout strain demonstrated the crucial role of NADPH oxidase in the production of ROS in A. oligospora, with trap formation impaired in the AoNoxA mutant, even in the presence of the nematode host. In addition, the expression of virulence factor serine protease P186 was up-regulated in the wild-type strain, but not in the mutant strain, in the presence of Caenorhabditis elegans. These results indicate that ROS derived from AoNoxA are essential for full virulence of A. oligospora in nematodes.     

Toward Practical Biological Control of Parasitic Nematodes in Domestic Animals

In a series of laboratory and field experiments where the nematophagous fungus Arthrobotrys oligospora was mixed directly with feces it has been demonstrated that it is possible to use nematophagous fungi for biological control of animal parasitic nematodes. A procedure used for selection of nematophagous fungi that can pass the digestive tract of ruminants, horses, and pigs is described. The selected fungus, Duddingtonia flagrans, has been used in further field experiments, and the results have confirmed that by the addition of D. flagrans to feed supplement it is possible to reduce the parasitic burden significantly.     

Antagonists of Plant-parasitic Nematodes in Florida Citrus

In a survey of antagonists of nematodes in 27 citrus groves, each with a history of Tylenchulus semipenetrans infestation, and 17 noncitrus habitats in Florida, approximately 24 species of microbial antagonists capable of attacking vermiform stages of Radopholus citrophilus were recovered. Eleven of these microbes and a species of Pasteuria also were observed attacking vermiform stages of T. semipenetrans. Verticillium chlamydosporium, Paecilomyces lilacinus, P. marquandii, Streptomyces sp., Arthrobotrys oligospora, and Dactylella ellipsospora were found infecting T. semipenetrans egg masses. Two species of nematophagous amoebae, five species of predatory nematodes, and 29 species of nematophagous arthropods also were detected. Nematode-trapping fungi and nematophagous arthropods were common inhabitants of citrus groves with a history of citrus nematode infestation; however, obligate parasites of nematodes were rare.     

Heavy trap formation by Arthrobotrys oligospora in liquid culture

Abstract The nematode-trapping fungus Arthrobotrys oligospora was grown in liquid culture in modified separatory funnels with vigorous air bubbling. The growth medium was a dilute soya peptone broth supplemented with a trap-inducing substance. The dipeptide l -phenylalanyl- l -valine or its constituents phenylalanine and valine were used as inducers of trap formation. Trap formation started within 2 days after inoculation and the traps increased in number and size during a 7-day period. The traps formed in liquid culture were fully functional in trapping nematodes. At the ultrastructural level they were characterized by the presence of many electron-dense microbodies similar to those in trap cells grown on solid media. Biomass increase, amounting to 6–7 mg dry weight · ml⁻¹, and trap formation were highly synchronized. The mycelial mass was homogeneously dispersed in the growth vessel during the entire growth period. Heavy trap formation in liquid culture by this fungus has not been reported previously.     

An endogenous rhythm of trap formation in the nematophagous fungus Arthrobotrys oligospora

In the predacious fungus Arthrobotrys oligospora Fres., the number and distribution of traps formed after the addition of living nematodes to the colonies were determined. At 21°C the traps were formed periodically; the mean period was 42.3±0.8 h. The periodicity was independent of light-dark (LD) cycles of 24 h (10:14). Temperature influenced the hyphal elongation but did not affect the periodic trap formation; at lower temperatures the peaks of trap formation were close together, showing partial overlapping. Induction of rhythmic mycelial growth and conidiation by chemical means was effective only in LD-cycles. The latter diurnal rhythm was weakly correlated with the trap formation and did not affect the endogenous period of approximately 42 h.Abbreviations LD light-dark - DD continuous darkness - LNM low-nutrient medium - CMA corn meal agar     

Taxonomy and ecology ofDactylella iridis: Its redescription as an entomogeneous and nematode-capturing hyphomycete

Trinacrium iridis, Dactylella ramiformis, and our own isolates obtained from a dead midge were proved to be a single species by means of morphological, ecological and DNA homology studies. The simple-pore septal ultrastructure and Q-10 (2H) ubiquinone of this fungus revealed its ascomycetous affinity. Morphological and ecological characteristics of this species led it to be accomodated in the genusDactylella. The fungus is redescribed here, because the combined nameD. iridis has been defectively issued. We have corrected nomenclatural errors and described all characteristics of this species.Dactylella iridis produces three types of conidia; Y-shaped aquatic, cylindrical aerial or terrestrial, and ellitical secondary conidia. The production of the former two types of conidia is enhanced by wetting mycellia grown on a sterilized midge body on silkworm, chrysalis extract agar plates. All the strains examined have a predatory behavior on nematodes. Thus, the fungus has versatile talents for aquatic, terrestrial, saprophytic, entomogenous and nematophagous lives.     

Factors responsible for transition of the <Emphasis Type="Italic">Duddingtonia flagrans</Emphasis> carnivorous fungus from the saprotrophic to the zootrophic nutrition type

Quantitative investigation of the factors responsible for trap formation in the nematophagous fungus Duddingtonia flagrans F-882 in submerged liquid culture was carried out. The data obtained suggest a complex program for the regulation of zootrophic nutrition in D. flagrans. Optimal concentrations of such carbon and nitrogen sources as sucrose (0.4%), ammonium ions (0.2%), and tryptone (0.2%) promote trap formation in the case of contact with the nematodes Panagrellus redivivus. Increased concentrations of these compounds, however, inhibit trap formation. The sensitivity of the mycelium to nematode excreta depends on the state of the culture and is increased under limitation by certain nutrient components or in the course of prolonged starvation. A direct correlation was found between the number of caught nematodes and the number of chlamydospores formed on the mycelium. The nutrients obtained from the nematode biomass are used for formation of additional chlamydospores (on average, about 20 chlamydospores per nematode). Environmental and evolutionary aspects of the role of zootrophic nutrition in carnivorous fungi are discussed.     

Development and fate of electron-dense microbodies in trap cells of the nematophagous fungusArthrobotrys oligospora

The development of electron-dense microbodies in cells of capture organs of the nematophagous fungus Arthrobotrys oligospora was studied with different ultrastructural techniques. Kinetic experiments revealed that the synthesis of these microbodies started in a very early stage of trap formation; the organelles originated from special regions of endoplasmic reticulum by budding. Mature organelles were surrounded by a single membrane of approximately 9 nm (KMnO4-fixation) and lacked crystalline inclusions. The presence of the electron-dense microbodies was independent of the conditions during which the traps had developed. The organelles remained intact during aging of the trap cells. They were also observed in the trophic hyphae after capture and penetration of nematodes. However, the distribution patterns of these organelles in the trophic hyphae, which were identical to those observed after germination of isolated traps on different cultivation media, suggested that their presence must be explained by dilution of organelles in newly formed cells.     

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.     

Ion Beam Mutagenesis in Arthrobotrys oligospora Enhances Nematode-Trapping Ability

The nematode-trapping fungus Arthrobotrys oligospora is able to produce extracellular protease that degrades the body walls of parasitic nematode larvae found in livestock and immobilizes the nematodes. Our aim was to obtain a strain of A. oligospora with a strong ability to trap nematodes by production of high levels of extracellular protease. A wild type strain of A. oligospora was subjected to mutagenic treatments involving low-energy ion beam implantation to generate mutants. Among these mutants, A. oligospora N showed high efficiency in trapping nematodes and was also able to secrete more extracellular protease, helping it to penetrate and digest the body walls of larvae. This work represents the first application of low-energy ion beams to generate mutations in a nematode-trapping fungus, and provides a new method of obtaining a fungus with high potential application.     

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.     

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.     

Production of nematode-attracting and nematicidal substances by predacious fungi

Arthrobotrys conoides Drechsler,Arthrobotrys oligospora Fressenius andMonacrosporium rutgeriensis R. C. Cooke, Pramer belong to the peculiar group of predactious fungi which trap and kill nematodes. We have found that these cultures produce nematode-attracting and nematicidal substances the production of which is potentiated in the presence of nematodes. Our method of nematode attraction assay is also described.     

Steinernema feltiae (DD-136) and S. glaseri: Persistence in Soil and Bark Compost and Their Influence on Native Nematodes

Infective juveniles (J3) of the entomogenous nematodes Steinernema feltiae DD-136 (ca. 10,000 J3/100 ml) and S. glaseri (ca. 2,500 J3/100 ml) were incubated in steam-sterilized and nonsterilized sandy soil and bark compost for 8 weeks at 25 C. The nematodes were recovered by a two-step extraction procedure at 1-week intervals, and their infectivity to lepidopterous larvae (Spodoptera litura and Galleria mellonella) and their effect on the population and community of native nematodes in soil were determined. Survival of inoculated nematodes and mortality of insects were enhanced in sterilized media. Nonsterilized bark compost proved to be equally as suitable a medium as sterilized compost. In nonsterilized soil, the survival curve of S.feltiae declined more rapidly than that or S. glaseri which was less infective to insects despite its greater persistence even in nonsterilized soil. Soon after the addition of steinernematids to soil, the population of native nematodes showed a fluctuation with an increase in rhabditids and a decrease in other kinds of nematodes.     

不同培养基上繁殖的昆虫病原线虫格氏线虫表皮蛋白的差异

表皮蛋白(surface coat proteins, SCPs)是格氏线虫Steinernema glaseri克服寄主免疫系统的关键因素, 能够抑制昆虫免疫反应, 促进线虫的侵染。为了进一步研究表皮蛋白抑制昆虫免疫的作用机理和线虫与寄主间的相互关系, 我们比较分析了不同培养基繁殖的格氏线虫表皮蛋白的差异。我们用人工培养基和大蜡螟Galleria mellonella末龄（5龄）幼虫分别培养得到格氏线虫侵染期幼虫, 利用酒精提取表皮蛋白。SDS-PAGE和非变性PAGE分析显示, 大蜡螟来源的格氏线虫的表皮蛋白具有更多的蛋白条带。体外和体内的裂解血细胞实验表明, 大蜡螟来源的格氏线虫的表皮蛋白表现出强烈的裂解血细胞活性, 而人工培养基来源的格氏线虫的表皮蛋白活力不明显; 且具有裂解血细胞活性的表皮蛋白为诱导表达型蛋白。不同培养基来源的格氏线虫的表皮蛋白组成的差异和活性的不同, 表明格氏线虫表皮蛋白的产生受线虫培养条件影响较大。