Potential biocontrol activity of Arthrobotrys oligospora and Trichoderma harzianum BI against Meloidogyne javanica on tomato in the greenhouse and laboratory studies

In this investigation, the biological control activity of Arthrobotrys oligospora and Trichoderma harzinum BI against the root-knot nematode, Meloidogyne javanica, infecting tomato, was assessed both in in vitro and in in vivo experiments. In greenhouse experiments, tomato seedlings at six-leaf stage were inoculated with 10⁶?spores/ml of A. oligospora and T. harzianum BI and number of 2000 nematode eggs per individual seedling. In in vitro assays, the per cent inhibition of nematode eggs hatching, the death per cent of second-stage juvenile (J2) and proteolytic activity on casein hydrolysis was evaluated. Results showed that A. oligospora and T. harzianum BI decreased the mean numbers of galls, eggmasses and egg per eggmass significantly (p?<?0.05) compared with control. Percentage hatching inhibition of M. javanica treated with A. oligospora and T. harzianum BI was 25 and 52%, respectively. Moreover, A. oligospora and T. harzianum BI significantly increased (p?<?0.05) the mortality rate of M. javanica (J2) after two and four days (74, 85 and 53, 63%, respectively). A. oligospora and T. harzianum BI had a proteolytic activity of 3.9 (U/min per ml) and 2.4 (U/min per ml) at pH 5.0, respectively. Our data suggest that the application of these two fungi in tomato rhizosphere infected with root-knot nematode M. javanica had antagonistic effects on the infection and reproduction of this nematode and the ability to control its population.     

Effects of Nematophagous Fungi on Numbers and Death Rates of Bacterivorous Nematodes in Arable Soil

In a series of microcosm experiments with an arable, sandy loam soil amended with sugarbeet leaf, the short-term (8 weeks) dynamics of numbers of nematodes were measured in untreated soil and in γ-irradiated soil inoculated with either a field population of soil microorganisms and nematodes or a mixed population of laboratory-propagated bacterivorous nematode species. Sugarbeet leaf stimulated an increase in bacterivorous Rhabditidae, Cephalobidae, and a lab-cultivated Panagrolaimus sp. Differences were observed between the growth rates of the nematode population in untreated and γ-irradiated soils, which were caused by two nematophagous fungi, Arthrobotrys oligospora and Dactylaria sp. These fungi lowered the increase in nematode numbers due to the organic enrichment in the untreated soil. We estimated the annually produced bacterivous nematodes to consume 50 kg carbon and 10 kg nitrogen per ha, per year, in the upper, plowed 25 cm of arable soil.     

Intracellular Proteolytic Activity in Mycelia of Arthrobotrys oligospora Bearing Mycoparasitic or Nematode Trapping Structures

Persson, Y., and Friman, E. 1993. Intracellular proteolytic activity in mycelia of Arthrobotrys oligospora bearing mycoparasitic or nematode trapping structures. Experimental Mycology 17, 182-190. The fungus Arthrobotrys oligospora parasitizes other fungi with the aid of coils and captures and digests nematodes by means of adhesive traps. We have compared proteolytic activities of mycelial extracts from coils and traps with those of vegetative hyphae. A. oligospora produced a number of proteases active at both alkaline and acidic pH. Coil extract had significantly higher proteolytic activity than extracts of vegetative hyphae. Several coil culture-specific bands were found after substrate gel electrophoresis. Pepstatin-sensitive proteolytic activity at acidic pH was higher in coil extract than in normal mycelial extracts, although the total proteolytic activity was the same. No proteolytic activity was connected solely to mycelial extracts with traps and no enhancement of proteolytic activity was observed during infection of nematodes.     

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.     

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.     

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.     

Microbial parasites associated with Tylenchulus semipenetrans in citrus orchards of Catalonia,Spain

Citrus orchards in Catalonia, Spain were surveyed to identify microbial parasites of Tylenchulus semipenetrans, and their distribution and density. Of 62 orchards, 48 were positively infested with the citrus nematode. Fungal strains were isolated from single eggs, females or second-stage juveniles of the citrus nematode in 69% of the infested orchards. The fungi identified in order of occurrence were Paecilomyces lilacinus, Fusarium solani, Fusarium spp., Cylindrocarpon cylindroides, Verticillium fungicola, Cladosporium cladosporioides, F. oxysporum, Veronaea botryosa, Sepedonium chrysospermum, Volutella ciliata, Exophiala pisciphila and Acremonium sp. Fungal parasitism was related directly to the number of females g^?1 of root and magnesium in the soil, and inversely, to the number of eggs g^?1 of root and phosphorus in the soil (R ²=0.8654; P<0.0001). Nematode trapping fungi were isolated from soil samples in 29% of the orchards, and Monacrosporium elegans, Arthrobotrys dactyloides, A. javanica, A. superba and A. oligospora var. microspora, were the species present. Endospores of the hyperparasite Pasteuria adhering to vermiform stages of T. semipenetrans were found in 50% of the orchards infested with the citrus nematode.     

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.     

The influence of saprophytic competition on nematode predation by nematode-trapping fungi

Competivive stress imposed by common soil saprophytes may cause an increase in predation by the nematode-trapping fungi, Arthrobotrys oligospora and Monacrosporium cionopagum, on the bacteria-feeding nematode Acrobeloides buetschli. Nematode-trapping species grown with saprophytic competitors in an artificial soil substrate increased their trapping activity compared to control cultures. The results support the hypothesis that competition stimulates the predatory activity of nematode-trapping fungi as an adaptation to overcome their low competitive saprophytic ability.     

Structure-based virtual screening of highly potent inhibitors of the nematode chitinase CeCht1

Nematode chitinases play vital roles in various physiological processes, including egg hatching, larva moulting, and reproduction. Small-molecule inhibitors of nematode chitinases have potential applications for controlling nematode pests. On the basis of the crystal structure of CeCht1, a representative chitinase indispensable to the eggshell chitin degradation of the model nematode Caenorhabditis elegans, we have discovered a series of novel inhibitors bearing a (R)-3,4-diphenyl-4,5-dihydropyrrolo[3,4-c]pyrazol-6(2H)-one scaffold by hierarchical virtual screening. The crystal structures of CeCht1 complexed with two of these inhibitors clearly elucidated their interactions with the enzyme active site. Based on the inhibitory mechanism, several analogues with improved inhibitory activities were identified, among which the compound PP28 exhibited the most potent activity with a K_i value of 0.18 μM. This work provides the structural basis for the development of novel nematode chitinase inhibitors.     

Control of root-knot nematode (Meloidogyne javanica) with combination of Arthrobotrys oligospora and salicylic acid and study of some plant defense responses

Root-knot nematodes (RKN) (Meloidogyne spp.) are economically the most important pathogens of agricultural products. The aim of the present study was to control Meloidogyne javanica by using Arthrobotrys oligospora and salicylic acid (SA) and to analyse the kinetics of enzymes, phenylalanine ammonia lyase (PAL), peroxidase (POX), polyphenol oxidase (PPO) and phenolic compounds accumulation in the root system of tomato after inoculation with M. javanica, A. oligospora and SA. The ability of A. oligospora to produce extracellular proteases was also examined. In greenhouse studies, we used soil drenching of A. oligospora (10⁶ spores/ml) and soil drenching or leaf spraying of SA (5 mM) in six-leaf stage, separately and in combination. Experiments were performed in a completely randomised design. The efficiencies of treatments were appraised by using diameter of galls, number of galls per plant, number of egg masses per plant, number of eggs per egg mass, root and foliage fresh weight. The results showed that the combined application of A. oligospora and SA provided the best nematode control. The activity of the enzymes and phenolic compounds increased in comparison with the control. The nematophagous fungus A. oligospora produced extracellular proteases in the broth culture. Using A. oligospora and SA could be effective in control of M. javanica in tomato.     

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.     

Fungal parasitism of the cyst nematode Heterodera schachtii: infection and enzymatic activity

Abstract Fungal egg parasites isolated from eggs of the cyst nematode Heterodera avenae in Sweden were investigated with respect to their ability to infect cyst nematode eggs of H. schachtii in vitro. The infection was studied by interference phase contrast microscopy of whole cysts and of cryosections of cysts exposed to the fungi on agar plates.
Verticillium suchlasporium was the most effective parasite, infecting 53% of the nematode eggs, while V. chlamydosporium infected 12% of the eggs. The fungi Paecilomyces lilacinus, Cylindrocarpon destructans or Fusarium oxysporum did not parasitize nematode eggs; nor did Arthrobotrys oligospora , a nematode trapping fungus nor Penicillium viridicatum which served as a control fungus.
The ability of the fungi to infect eggs was correlated with their lytic enzyme activity. Fungi that readily infected eggs also showed chitinase activity and presence of proteolytic activity. The Verticillium species had an activity between 3.7 and 14.6 μmol N -acetyl-glucosamine per mg protein per hour (CU) while it was 4.5 CU or lower for P. lilacinus . Other isolates did not shown any chitinase activity.     

Evaluation of biocontrol potential of Arthrobotrys oligospora against Meloidogyne graminicola and Rhizoctonia solani in Rice (Oryza sativa L.)

The nematode trapping and mycoparasitic potential of Arthrobotrys oligospora was tested in vitro against Meloidogyne graminicola and Rhizoctonia solani, respectively. Five isolates of A. oligospora were isolated from different locations of India. Diversity of the trapping structures is large and highly dependent on the environmental condition and nature of the fungus. In A. oligospora, a three-dimensional adhesive net (in response to nematode) and hyphal coils developed around the hyphae of R. solani. In vitro trap formation and predacity were tested against second-stage juveniles of M. graminicola (J₂) and the interactions between A. oligospora and R. solani were recorded. Under field conditions, we demonstrated the biocontrol potential of A. oligospora against R. solani causing sheath blight of rice (Oryza sativa) for the first time. All the isolates of A. oligospora parasitized and killed M. graminicola and R. solani. Application of A. oligospora, isolate VNS-1, in soil infested with M. graminicola and R. solani reduced the number of root knot by 57.58–62.02%, sheath blight incidence by 55.68–59.32% and lesion length by 54.91–66.66% under green house and miniplot (field) conditions. Applications of A. oligospora to the soil increased plant growth: shoot length by 56.4–68.8%, root length by 44.0–54.55%, fresh weight of shoot and root by 62.91–65.4% and 38.9–44.19%, respectively, as compared to the plants grown in nematode infested soil.     

Do Organic Amendments Enhance the Nematode-Trapping Fungi Dactylellina haptotyla and Arthrobotrys oligospora?

Soil cages (polyvinyl chloride pipe with mesh-covered ends) were used to determine how the quantity of two organic amendments affected the nematode-trapping fungi Dactylellina haptotyla and Arthrobotrys oligospora, which were studied independently in two different vineyards. Each cage contained 80 cm³ of field soil (120 g dry weight equivalent), fungal inoculum (two alginate pellets, each weighing 1.9 mg and containing assimilative hyphae of one fungus), and dried grape or alfalfa leaves (0, 360, or 720 mg equivalent to 0, 4,500, or 9,000 kg/ha) with a C:N of 28:1 and 8:1, respectively. Cages were buried in the vineyards, recovered after 25 to 39 days, and returned to the laboratory where fungus population density and trapping were quantified. Dactylellina haptotyla population density and trapping were most enhanced by the smaller quantity of alfalfa amendment and were not enhanced by the larger quantity of alfalfa amendment. Arthrobotrys oligospora population density was most enhanced by the larger quantity of alfalfa amendment, but A. oligospora trapped few or no nematodes, regardless of amendment. Trapping and population density were correlated for D. haptotyla but not for A. oligospora.     

Functional Coupling of a Nematode Chemoreceptor to the Yeast Pheromone Response Pathway

Sequencing of the Caenorhabditis elegans genome revealed sequences encoding more than 1,000 G-protein coupled receptors, hundreds of which may respond to volatile organic ligands. To understand how the worm''s simple olfactory system can sense its chemical environment there is a need to characterise a representative selection of these receptors but only very few receptors have been linked to a specific volatile ligand. We therefore set out to design a yeast expression system for assigning ligands to nematode chemoreceptors. We showed that while a model receptor ODR-10 binds to C. elegans Gα subunits ODR-3 and GPA-3 it cannot bind to yeast Gα. However, chimaeras between the nematode and yeast Gα subunits bound to both ODR-10 and the yeast Gβγ subunits. FIG2 was shown to be a superior MAP-dependent promoter for reporter expression. We replaced the endogenous Gα subunit (GPA1) of the Saccharomyces cerevisiae (ste2Δ sst2Δ far1Δ) triple mutant (“Cyb”) with a Gpa1/ODR-3 chimaera and introduced ODR-10 as a model nematode GPCR. This strain showed concentration-dependent activation of the yeast MAP kinase pathway in the presence of diacetyl, the first time that the native form of a nematode chemoreceptor has been functionally expressed in yeast. This is an important step towards en masse de-orphaning of C. elegans chemoreceptors.     

Production of Chitinases and β-1,3-Glucanases by Stachybotrys elegans, a Mycoparasite of Rhizoctonia solani

The in vitro production of chitinases and β-1,3-glucanases by Stachybotrys elegans, a mycoparasite of Rhizoctonia solani, was examined under various culture conditions, such as carbon and nitrogen sources, pH, and incubation period. Production of both enzymes was influenced by the carbon source incorporated into the medium and was stimulated by acidic pH and NaNO₃. The activity of both enzymes was very low in culture filtrates from cells grown on glucose and sucrose compared with that detected on chitin (for chitinases) and cell wall fragments (for β-1,3-glucanases). Protein electrophoresis revealed that, depending on the carbon source used, different isoforms of chitinases and β-1,3-glucanases were detected. S. elegans culture filtrates, possessing β-1,3-glucanase and chitinase activities, were capable of degrading R. solani mycelium.     

Life-History Traits of the Model Organism Pristionchus pacificus Recorded Using the Hanging Drop Method: Comparison with Caenorhabditis elegans

The nematode Pristionchus pacificus is of growing interest as a model organism in evolutionary biology. However, despite multiple studies of its genetics, developmental cues, and ecology, the basic life-history traits (LHTs) of P. pacificus remain unknown. In this study, we used the hanging drop method to follow P. pacificus at the individual level and thereby quantify its LHTs. This approach allowed direct comparisons with the LHTs of Caenorhabditis elegans recently determined using this method. When provided with 5×10⁹ Escherichia coli cells ml^–1 at 20°C, the intrinsic rate of natural increase of P. pacificus was 1.125 (individually, per day); mean net production was 115 juveniles produced during the life-time of each individual, and each nematode laid an average of 270 eggs (both fertile and unfertile). The mean age of P. pacificus individuals at first reproduction was 65 h, and the average life span was 22 days. The life cycle of P. pacificus is therefore slightly longer than that of C. elegans, with a longer average life span and hatching time and the production of fewer progeny.     

Linoleic acid — The nematicidal principle of several nematophagous fungi and its production in trap-forming submerged cultures

Linoleic acid was shown to be the only detectable nematicidal agent in the mycelial extracts of several predacious fungi of the genus Arthrobotrys. Although the compound is present in saprophytic cultures, induction of trap formation by nematodes or phenylalnyl-valine caused a significant increase in its production. In submerged cultures, the number of traps formed by Arthrobotrys conoides and Arthrobotrys oligospora was directly correlated to the increase of the concentration of linoleic acid. In A. conoides, the ratio of ergosterol to linoleic acid decreased from 2.6 in saprophytic cultures to 1.1 in trap-forming cultures induced with nematodes. Linoleic acid exhibited nematicidal activities towards the free-living nematode Caenorhabditis elegans with an LD₅₀ value of 5 g/ml.