Parasite Of Nematode Eggs Research Articles

Cyclophilin acts as a ribosome biogenesis factor by chaperoning the ribosomal protein (PlRPS15) in filamentous fungi.

The rapid transport of ribosomal proteins (RPs) into the nucleus and their efficient assembly into pre-ribosomal particles are prerequisites for ribosome biogenesis. Proteins that act as dedicated chaperones for RPs to maintain their stability and facilitate their assembly have not been identified in filamentous fungi. PlCYP5 is a nuclear cyclophilin in the nematophagous fungus Purpureocillium lilacinum, whose expression is up-regulated during abiotic stress and nematode egg-parasitism. Here, we found that PlCYP5 co-translationally interacted with the unassembled small ribosomal subunit protein, PlRPS15 (uS19). PlRPS15 contained an eukaryote-specific N-terminal extension that mediated the interaction with PlCYP5. PlCYP5 increased the solubility of PlRPS15 independent of its catalytic peptide-prolyl isomerase function and supported the integration of PlRPS15 into pre-ribosomes. Consistently, the phenotypes of the PlCYP5 loss-of-function mutant were similar to those of the PlRPS15 knockdown mutant (e.g. growth and ribosome biogenesis defects). PlCYP5 homologs in Arabidopsis thaliana, Homo sapiens, Schizosaccharomyces pombe, Sclerotinia sclerotiorum, Botrytis cinerea and Metarhizium anisopliae were identified. Notably, PlCYP5-PlRPS15 homologs from three filamentous fungi interacted with each other but not those from other species. In summary, our data disclosed a unique dedicated chaperone system for RPs by cyclophilin in filamentous fungi.

Pochonia chlamydosporia Isolate PC-170-Induced Expression of Marker Genes for Defense Pathways in Tomatoes Challenged by Different Pathogens.

Pochonia chlamydosporia is a fungal parasite of nematode eggs. Studies have shown that some strains of Pochonia chlamydosporia can promote plant growth and induce plants’ systemic resistance to root-knot nematodes by colonizing in their roots. This study aimed to verify the effect of the PC-170 strain on tomato growth and systemic resistance. Split-root experiments were conducted to observe the systemic resistance induced by PC-170. To explore the defense pathway that was excited due to the colonization by PC-170, we tested the expression of marker genes for defense pathways, and used mutant lines to verify the role of plant defense pathways. Our results showed that PC-170 can colonize roots, and promotes growth. We found a role for jasmonic acid (JA) in modulating tomato colonization by PC-170. PC-170 can activate tomato defense responses to reduce susceptibility to infection by the root-knot nematode Meloidogyne incognita, and induced resistance to some pathogens in tomatoes. The marker genes of the defense pathway were significantly induced after PC-170 colonization. However, salicylic acid (SA)- and jasmonic acid (JA)-dependent defenses in roots were variable with the invasion of different pathogens. Defense pathways play different roles at different points in time. SA- and JA-dependent defense pathways were shown to cross-communicate. Different phytohormones have been involved in tomato plants’ responses against different pathogens. Our study confirmed that adaptive JA signaling is necessary to regulate PC-170 colonization and induce systemic resistance in tomatoes.

Chitosan modulates Pochonia chlamydosporia gene expression during nematode egg parasitism.

SummaryClimate change makes plant‐parasitic nematodes (PPN) an increasing threat to commercial crops. PPN can be managed sustainably by the biocontrol fungus Pochonia chlamydosporia (Pc). Chitosan generated from chitin deacetylation enhances PPN parasitism by Pc. In this work, we investigate the molecular mechanisms of Pc for chitosan resistance and root‐knot nematode (RKN) parasitism, using transcriptomics. Chitosan and RKN modify the expression of Pc genes, mainly those involved in oxidation–reduction processes. Both agents significantly modify the expression of genes associated to 113 GO terms and 180 Pc genes. Genes encoding putative glycoproteins (Pc adhesives) to nematode eggshell, as well as genes involved in redox, carbohydrate and lipid metabolism trigger the response to chitosan. We identify genes expressed in both the parasitic and endophytic phases of the Pc lifecycle; these include proteases, chitosanases and transcription factors. Using the Pathogen—Host Interaction database (PHI‐base), our previous RNA‐seq data and RT‐PCR of Pc colonizing banana we have investigated genes expressed both in the parasitic and endophytic phases of Pc lifecycle.

Chitosan Increases Tomato Root Colonization by Pochonia chlamydosporia and Their Combination Reduces Root-Knot Nematode Damage.

The use of biological control agents could be a non-chemical alternative for management of Meloidogyne spp. [root-knot nematodes (RKN)], the most damaging plant-parasitic nematodes for horticultural crops worldwide. Pochonia chlamydosporia is a fungal parasite of RKN eggs that can colonize endophytically roots of several cultivated plant species, but in field applications the fungus shows a low persistence and efficiency in RKN management. The combined use of P. chlamydosporia with an enhancer could help its ability to develop in soil and colonize roots, thereby increasing its efficiency against nematodes. Previous work has shown that chitosan enhances P. chlamydosporia sporulation and production of extracellular enzymes, as well as nematode egg parasitism in laboratory bioassays. This work shows that chitosan at low concentrations (up to 0.1 mg ml-1) do not affect the viability and germination of P. chlamydosporia chlamydospores and improves mycelial growth respect to treatments without chitosan. Tomato plants irrigated with chitosan (same dose limit) increased root weight and length after 30 days. Chitosan irrigation increased dry shoot and fresh root weight of tomato plants inoculated with Meloidogyne javanica, root length when they were inoculated with P. chlamydosporia, and dry shoot weight of plants inoculated with both P. chlamydosporia and M. javanica. Chitosan irrigation significantly enhanced root colonization by P. chlamydosporia, but neither nematode infection per plant nor fungal egg parasitism was affected. Tomato plants cultivated in a mid-suppressive (29.3 ± 4.7% RKN egg infection) non-sterilized clay loam soil and irrigated with chitosan had enhanced shoot growth, reduced RKN multiplication, and disease severity. Chitosan irrigation in a highly suppressive (73.7 ± 2.6% RKN egg infection) sterilized-sandy loam soil reduced RKN multiplication in tomato. However, chitosan did not affect disease severity or plant growth irrespective of soil sterilization. Chitosan, at an adequate dose, can be a potential tool for sustainable management of RKN.

Yellow Pigment Aurovertins Mediate Interactions between the Pathogenic Fungus Pochonia chlamydosporia and Its Nematode Host

Nematophagous fungi are globally distributed soil fungi and well-known natural predators of soil-dwelling nematodes. Pochonia chlamydosporia can be found in diverse nematode-suppressive soils as a parasite of nematode eggs and is one of the most studied potential biological control agents of nematodes. However, little is known about the functions of small molecules in the process of infection of nematodes by this parasitic fungus or about small-molecule-mediated interactions between the pathogenic fungus and its host. Our recent study demonstrated that a P. chlamydosporia strain isolated from root knots of tobacco infected by the root-knot nematode Meloidogyne incognita produced a class of yellow pigment metabolite aurovertins, which induced the death of the free-living nematode Panagrellus redivevus. Here we report that nematicidal P. chlamydosporia strains obtained from the nematode worms tended to yield a total yellow pigment aurovertin production exceeding the inhibitory concentration shown in nematicidal bioassays. Aurovertin D was abundant in the pigment metabolites of P. chlamydosporia strains. Aurovertin D showed strong toxicity toward the root-knot nematode M. incognita and exerted profound and detrimental effects on the viability of Caenorhabditis elegans even at a subinhibitory concentration. Evaluation of the nematode mutation in the β subunit of F1-ATPase, together with the application of RNA interference in screening each subunit of F1FO-ATPase in the nematode worms, demonstrated that the β subunit of F1-ATPase might not be the specific target for aurovertins in nematodes. The resistance of C. elegans daf-2(e1370) and the hypersensitivity of C. elegans daf-16(mu86) to aurovertin D indicated that DAF-16/FOXO transcription factor in nematodes was triggered in response to the aurovertin attack. These findings advance our understanding of the roles of aurovertin production in the interactions between nematodes and the pathogen fungus P. chlamydosporia.

Comparative gene expression profiles of Trichoderma harzianum proteases during in vitro nematode egg-parasitism

The parasitism of nematodes by Trichoderma harzianum involves the production and co-ordinated secretion of various eggshell-degrading enzymes. While several protease-encoding genes of Trichoderma spp. have been characterized under different biocontrol conditions, their functional role in the nematode egg-infection process has been poorly understood to date. The present study was carried out to investigate the role and importance of different proteases implicated in nematode egg-parasitism. Parallel expressional characterization of 13 peptidase-encoding genes clearly suggests that the highly transcribed genes encoding acidic serine protease pra1; aspartic protease p6281 and p9438; metalloendopeptidase p7455 and the most prominently induced sedolisin serine protease p5216 were co-expressed and they may play pivotal roles in the egg-parasitic process. In addition, gene expression profiles indicated similarities (e.g. p1324) as well as characteristic differences (e.g. p5216, p7455, p9438, p5431, prb1, SS10 and SA76) between egg-parasitism and other biocontrol-related processes. These results underline the diversity and the functional specialization of fungal protease genes and reveal new insights into the host-pathogen interaction between nematode eggs and T. harzianum.

Biology, growth parameters and enzymatic activity of Pochonia chlamydosporia isolated from potato cyst and root-knot nematodes

Pochonia chlamydosporia, a widespread fungal parasite of potato cyst nematodes (PCN), Globodera spp., and root-knot nematodes (RKN), Meloidogyne spp., has been studied as a biological control agent. Three Portuguese isolates (Pc1, Pc2, Pc3) obtained from PCN eggs and two non-native isolates (Vc10, Pc280) were characterised using ERIC-PCR and screened by in vitro assays for their ability to produce chlamydospores, parasitise eggs of Globodera rostochiensis and Meloidogyne chitwoodi and colonise the rhizosphere of barley. The effects of temperature on growth, sporulation, parasitism and enzymatic activity were also evaluated. Isolates Pc1 and Pc3, despite their different geographical origins, had identical molecular profiles. Pc2 produced the higher numbers of chlamydospores in solid medium (1.15 × 107 chlamydospores g−1), whereas Pc3 produced the least (3 × 105 chlamydospores g−1). These isolates extensively colonised the rhizosphere of barley (>90% root fragments) and the proportion of parasitised eggs, detected on agar plates, was low (<60% for RKN and <55% for PCN), Pc1 being the best parasite against both nematode species. The influence of temperature was similar for all isolates: no growth was observed at 10, 33 and 35°C. Spores/hyphal fragments remained viable for nearly 1 month at 10 and 33°C and isolates resumed growth after incubation at 25°C, although chlamydospores and conidia production, viability and nematode egg parasitism were affected. Exposure to 35°C was lethal for isolates Pc1, Pc2 and Pc280. When grown in liquid media all isolates produced esterases, but protease activity was only observed in Pc2 and Vc10. The highest enzymatic activity was detected in isolate Pc2 in low nutrient media. Enzymatic activity decreased in the presence of nematode eggs for both Pc2 and Vc10. Molecular, biochemical and biological analyses, including biotic and abiotic factors, are important in the screening of potential biological control agents, particularly in the case of P. chlamydosporia, due to the high variability among isolates.

Gene cloning, molecular modeling, and phylogenetics of serine protease P32 and serine carboxypeptidase SCP1 from nematophagous fungiPochonia rubescensandPochonia chlamydosporia

The fungi Pochonia chlamydosporia and Pochonia rubescens are parasites of nematode eggs and thus are biocontrol agents of nematodes. Proteolytic enzymes such as the S8 proteases VCP1 and P32, secreted during the pathogenesis of nematode eggs, are major virulence factors in these fungi. Recently, expression of these enzymes and of SCP1, a new putative S10 carboxypeptidase, was detected during endophytic colonization of barley roots by these fungi. In our study, we cloned the genomic and mRNA sequences encoding P32 from P. rubescens and SCP1 from P. chlamydosporia. P32 showed a high homology with the serine proteases Pr1A from the entomopathogenic fungus Metarhizium anisopliae and VCP1 from P. chlamydosporia (86% and 76% identity, respectively). However, the catalytic pocket of P32 showed differences in the amino acids of the substrate-recognition sites compared with the catalytic pockets of Pr1A and VCP1 proteases. Phylogenetic analysis of P32 suggests a common ancestor with protease Pr1A. SCP1 displays the characteristic features of a member of the S10 family of serine proteases. Phylogenetic comparisons show that SCP1 and other carboxypeptidases from filamentous fungi have an origin different from that of yeast vacuolar serine carboxypeptidases. Understanding protease genes from nematophagous fungi is crucial for enhancing the biocontrol potential of these organisms.

Effects on plant growth and root-knot nematode infection of an endophytic GFP transformant of the nematophagous fungus Pochonia chlamydosporia

Pochonia chlamydosporia (Pc123) is a fungal parasite of nematode eggs which can colonize endophytically barley and tomato roots. In this paper we use culturing as well as quantitative PCR (qPCR) methods and a stable GFP transformant (Pc123gfp) to analyze the endophytic behavior of the fungus in tomato roots. We found no differences between virulence/root colonization of Pc123 and Pc123gfp on root-knot nematode Meloidogyne javanica eggs and tomato seedlings respectively. Confocal microscopy of Pc123gfp infecting M. javanica eggs revealed details of the process such as penetration hyphae in the egg shell or appressoria and associated post infection hyphae previously unseen. Pc123gfp colonization of tomato roots was low close to the root cap, but increased with the distance to form a patchy hyphal network. Pc123gfp colonized epidermal and cortex tomato root cells and induced plant defenses (papillae). qPCR unlike culturing revealed reduction in fungus root colonization (total and endophytic) with plant development. Pc123gfp was found by qPCR less rhizosphere competent than Pc123. Endophytic colonization by Pc123gfp promoted growth of both roots and shoots of tomato plants vs. uninoculated (control) plants. Tomato roots endophytically colonized by Pc123gfp and inoculated with M. javanica juveniles developed galls and egg masses which were colonized by the fungus. Our results suggest that endophytic colonization of tomato roots by P. chlamydosporia may be relevant for promoting plant growth and perhaps affect managing of root-knot nematode infestations.

Production of extracellular enzymes by different isolates of Pochonia chlamydosporia

For the first time, the specific activities of chitinases, esterases, lipases and a serine protease (VCP1) produced by different isolates of the nematophagous fungus Pochonia chlamydosporia were quantified and compared. The isolates were grown for different time periods in a minimal liquid medium or media supplemented with 1 % chitin, 0.2 % gelatin or 2 % olive oil. Enzyme-specific activities were quantified in filtered culture supernatants using chromogenic p-nitrophenyl substrates (for chitinases, lipases and esterases) and a p-nitroanilide substrate (to measure the activity of the proteinase VCP1). Additionally, information on parasitic growth (nematode egg parasitism) and saprotrophic growth (plant rhizosphere colonisation) was collected. Results showed that the production of extracellular enzymes was influenced by the type of medium ( p < 0.05) in which P. chlamydosporia was grown. Enzyme activity differed with time ( p < 0.05), and significant differences were found between isolates ( p < 0.001) and the amounts of enzymes produced ( p < 0.001). However, no significant relationships were found between enzyme activities and parasitic or saprotrophic growth using Kendall's coefficient of concordance or Spearman rank correlation coefficient. The results provided new information about enzyme production in P. chlamydosporia and suggested that the mechanisms which regulate the trophic switch in this fungus are complex and dependent on several factors.

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

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

PCR Primers with Enhanced Specificity for Nematode-Trapping Fungi (Orbiliales)

Nematode-trapping fungi, a monophyletic lineage within the Orbiliales (Ascomycota), use specialized structures to capture and consume nematodes in soil, leaf litter, and other substrates. These fungi have been studied both because of their unique predatory life history and because they are potential control agents of important plant- and animal-parasitic nematodes. Ecological studies of nematode-trapping fungi have primarily used culture-based methods, but molecular detection techniques are now available and should be useful for studying this group. We developed Orbiliales-specific PCR primers for the ITS and 28s rDNA to directly detect nematode-trapping fungi without culturing and also to screen fungal isolates for phylogenetic placement in the Orbiliales. We used these primers to selectively amplify, clone, and sequence Orbiliales DNA extracted from soil, litter, and wood, and we compared the results of molecular detection with those obtained using a culture-based method. Of the eight species of nematode-trapping Orbiliales detected with the culture-based assay, only three were detected with PCR. The molecular assay, however, detected 18 species of uncultured Orbiliales, many of which are closely related to nematode-trapping fungi and fungal parasites of nematode eggs. Our results suggest that the combined use of Orbiliales-specific primers and culture-based techniques may benefit future studies of nematophagous fungi.

Evaluating Pochonia chlamydosporia in a double‐cropping system of lettuce and tomato in plastic houses infested with Meloidogyne javanica

The effect of Pochonia chlamydosporia, a facultative fungal parasite of nematode eggs, alone or in combination with oxamyl was evaluated in a double‐cropping system of lettuce and tomato in unheated plastic houses infested with Meloidogyne javanica at two sites for two consecutive growing seasons. An additional treatment of methyl bromide fumigation was included to compare crop yield in nematode‐free vs. nematode‐infested soil. Final population densities, reproductive rate, root gall rating, and egg production were determined after each crop. Pochonia chlamydosporia was isolated from nematode eggs up to nine months after application to soil. The fungus survived in the rhizosphere for the entire growing season at one site, but only at low densities. Final population densities of M. javanica decreased after cultivation of lettuce and increased after tomato, and this pattern of population fluctuation was unaffected by treatment, experiment or site. The reproductive rate on lettuce was equal to or below 1, and it was similar among treatments in both experiments at both sites. Eggs were not found on lettuce roots. On tomato, the reproductive rate in the fungus + oxamyl treatment was significantly lower (P &lt; 0·05) than other treatments in experiment 1 at both sites. Fungus + oxamyl consistently reduced root gall ratings on tomato in all cases, but numbers of eggs per g root varied depending on treatment. Methyl bromide‐treated plots remained free of M. javanica at the end of the 2‐year study.

Nutritional requirements of Pochonia chlamydospora and ARF18, fungal parasites of nematode eggs.

Twenty carbohydrates (C), 18 nitrogen compounds (N), and 9 vitamins were examined for their effects on the growth and conidiation of the nematode-egg-parasitic fungi Arkansas Fungus 18 (ARF18, isolate 908) and Pochonia chlamydospora var. chlamydospora in solid and liquid cultures. Glycogen was the best, and inulin, D-(+)-galactose, and soluble starch were good C sources for the growth of ARF18 in both solid and liquid cultures. ARF18 could not utilize alpha-cellulose in liquid; alpha-lactose and D-mannitol in solid; and D-(+)-xylose, L-(-)-sorbose, and D-(-)-ribose in both solid and liquid cultures. Casein was the best N source for ARF18 growth in both solid and liquid cultures and L-aspartic acid, DL-glutamic acid, peptone, and L-histidine were good in solid culture. ARF18 could not utilize L-cystine and L-tyrosine in solid culture, and L-cystine, DL-methionine, peptone, L-proline, and ammonium nitrate in liquid culture. Supplement of vitamins appeared to be unnecessary for ARF18 to grow. For P. chlamydospora var. chlamydospora growth, all test C sources, except L-(-)-sorbose, alpha-cellulose, citric acid, and D-(+)-glucose, were good in both solid and liquid cultures. Most N compounds were good for P. chlamydospora var. chlamydospora growth with casein and peptone the best. Vitamins had limited effect on P. chlamydospora var. chlamydospora growth. P. chlamydospora var. chlamydospora conidiation was well supported by D-(-)-ribose, D-(-)-fructose, melibiose, and D-(+)-galactose as C sources and by L-aspartic acid, DL-glutamic acid, and L-arginine as N sources. Excluding myo-inositol from the medium containing all other test vitamins increased P. chlamydospora var. chlamydospora conidiation, while excluding pyridoxine appeared to reduce its conidiation.

Biological control of clover cyst nematode (&lt;i&gt;Heterodera trifolli&lt;/i&gt;) with fungi parasitic to nematodes or their eggs

The ability of four fungi to suppress nematode reproduction and ameliorate yield loss of white clover (Trifolium repens) in soil infested with clover cyst nematode (Heterodera trifolii) was examined in a pot experiment. Three fungal parasites of nematode eggs (two sterile fungi and Scolecobasidium constrictum) and a parasite of nematodes (Hirsutella rhossiliensis) were added to soil in the form of alginate beads. At eight weeks after sowing, the mean number of cysts which developed per pot was reduced by Scolecobasidium constrictum, one of the sterile fungi and H. rhossiliensis. H. rhossiliensis also reduced the number of cysts per gram of plant dry matter and maintained dry matter yield per pot and per plant at a level not significantly different from that of control pots (without nematodes) or pots treated with H. rhossiliensis alone. The number of eggs which developed within each cyst was increased in the presence of H. rhossiliensis. Results suggest that fungi such as H. rhossiliensis may have the potential to enhance the establishment of white clover.

Fungi and Actinomycetes Associated With Cysts of Heterodera Trifolii Goffart (Nematoda: Tylenchida) in Pasture Soils in New Zealand

A survey of ten pastures sites in the North Island of New Zealand was conducted to identify micro-organisms colonising cysts of clover cyst nematode (Heterodera trifolii). A total of 707 isolates of fungi and actinomycetes was recovered from 488 surface-sterilised cysts plated onto water agar Ninety-three percent of cysts were colonised, with 39% containing more than one organism. The most commonly isolated organisms (and their frequency of occurrence) were species of actinomycetes (30% of cysts), Fusarium (26%), Verticillium (16%), Gliocladium (14%), Mortierella (9%), Paecilomyces (8%), Phialophora (7%), Penicillium (6%), Exophiala (5%), Acremonium (3%), Cylindrocarpon (2%) and Trichocladium (2%). Predominant species of these included Fusarium oxysporum, F. culmorum, F. solani, Verticillium chlamydosporium, Gliocladium roseum, Mortierella alpina, Paecilomyces lilacinus, an unidentified Paecilomyces sp., Exophiala pisciphila and Trichocladium opacum. Young white cysts were most commonly colonised by actinomycete species (38%), Fusarium spp. (12%) and Gliocladium spp. (10%); older (brown) cysts by actinomycete spp. (24%), Verticillium spp. (23%) and Fusarium spp. (20%), and very old (black) cysts by Fusarium spp. (41%), actinomycete spp. (31%) and Gliocladium spp. (19%). Many of the species isolated are known colonists of the roots of white clover (Trifolium repens) and some are known parasites of nematode eggs.

Nematophagous fungi: Rhopalomyces elegans

Rhopalomyces elegans is known to be a parasite of nematode eggs. This species is now found to be capable of capturing and parasitizing both the larval and adult stages of a species of Rhabditis. Penetration from an appressorium is followed by considerable subcuticular growth before death and colonization of the host. The presence of functional anastomoses is demonstrated in the vegetative hyphae of this mucoralean fungus.