Nematophagous Fungus Hirsutella Rhossiliensis Research Articles

Effect of additives on the efficacy of microencapsulated Hirsutella rhossiliensis controlling Heterodera schachtii on sugar beets

The nematophagous fungus Hirsutella rhossiliensis is a common antagonist of a number of important plant-parasitic nematodes including the beet cyst nematode Heterodera schachtii. Although H. rhossiliensis is highly pathogenic, slow growth characteristics and weak competiveness under field conditions limit its biocontrol potential. Cellulose-based microcapsules were tested for their ability to improve fungal competitiveness in the soil and nematode parasitism. In a first approach microcapsules containing 15% fungal mycelium plus 15% corn gluten and 0.5% yeast extract as additives gave low levels of H. schachtii control. Fungal parasitism was not observed and indicated that nematode mortality was most likely caused by non-specific accumulation of nematicidal compounds associated with decomposition of the additives in the microcapsules. Removal of the corn gluten additive resulted in a minor increase of 20% parasitism of H. schachtii juveniles. Furthermore, the yeast extract that acted as nutrient source for the fungus diffused through the semi-permeable membrane of the capsules during the formulation process and was ineffective. Replacing the corn gluten and yeast extract with 3% autoclaved baker’s yeast and using finely dispersed mycelium instead of pelletized mycelium increased juvenile parasitism in the soil up to 90% and decreased number of juveniles per cm root length by over 80%. Cellulose-based microcapsules supplemented with autoclaved baker’s yeast, therefore, proved to be an effective delivery system for H. rhossiliensis to control H. schachtii.

Fermentation and microencapsulation of the nematophagous fungus Hirsutella rhossiliensis in a novel type of hollow beads

In this work, fermentation and formulation aspects of the nematophagous fungus Hirsutella rhossiliensis BBA were investigated. When incubated in 2% (w/w) glucose and 0.5% (w/w) yeast extract medium in a 1-L Erlenmeyer flask without baffles, heavy pellet formation was observed. Only 40% of the mycelium had a size less than 500 μm. When a flask with three baffles was used, the portion of mycelium <500 μm rose to 95%. In the next step, the influence of aeration rate and stirrer speed on production of finely dispersed mycelium in a stirred tank reactor was investigated. The best fermentation results were obtained at 0.4 vvm and 400 rpm stirrer speed with 90% mycelium <500 μm and 5 g/L biomass. Then, mycelium was microencapsulated in hollow beads based on sulfoethylcellulose (SEC). Experiments on the capsule nutrient reservoir showed that 15% (w/w) corn gluten and 0.5% (w/w) yeast extract could be replaced with 3% (w/w) autoclaved baker's yeast which was never used as capsule additive before. Radial growth of mycelium out of dried hollow beads containing 1% (w/w) biomass and 3% (w/w) baker's yeast was faster than for alginate beads containing equivalent amounts of biomass and yeast indicating a higher bio-control potential.

Population dynamics and biocontrol efficacy of the nematophagous fungus Hirsutella rhossiliensis as affected by stage of the soybean cyst nematode

Monitoring the population dynamics of a biocontrol agent in soil is important for understanding, predicting and increasing its efficacy. In this study, the population dynamics and the efficacy of a promising biocontrol agent against nematode, the fungus Hirsutella rhossiliensis, were investigated in greenhouse experiments with quantitative real-time polymerase chain reaction (PCR) and bioassay. To explore the effects of the fungus on nematode inoculum, soil infested with eggs or second-stage juveniles (J2) of Heterodera glycines was used. The results showed that the quantity of H. rhossiliensis DNA based on real-time PCR decreased over time, regardless of eggs or J2 as inoculum. The quantity of H. rhossiliensis DNA [femtogram (fg)/g soil] was highest (3.83 ± 1.96 × 10 8 fg/g soil) when the fungus was first added to soil and then decreased rapidly to 3.24 ± 1.22 × 10 7 fg/g in egg-infested soil and 7.34 ± 2.94 × 10 7 fg/g in J2-infested soil 17 days after planting, and then declined gradually between 17 and 59 days after planting. The data based on the bioassay showed that the percentage of J2 parasitized by H. rhossiliensis decreased throughout the experiment in both egg- and J2-infested soils. H. rhossiliensis controlled H. glycines more effectively in J2-infested soil than in egg-infested soil. At the end of the experiments (59 days after planting), nematode suppression and plant growth promotion in J2-infested soil were 79% and 55%, respectively, which were higher than the 34% and 2% in egg-infested soil. In soil that was not inoculated with the fungus, the number of H. glycines was 10 times higher in J2-infested soil than in egg-infested soil, indicating the greater nematode inoculum potential in soil infested with J2. DNA yield of H. rhossiliensis was statistically higher in J2-infested soil than in egg-infested soil in earlier period (prior to day 31) ( p < 0.05), which is consistent with the hypothesis that the numbers of J2 present during and soon after fungal inoculation are critical for maintaining the population density and biocontrol efficiency of the fungus.

Suppression of nematodes in a coastal grassland soil

The nematophagous fungi Arthrobotrys oligospora and Myzocytiopsis glutinospora increase to large numbers (>103 propagules/g of soil) when moth larvae killed by entomopathogenic nematodes are added to soil microcosms. In spite of these increases, it is unclear how effective these nematophagous fungi are in suppressing nematodes. We measured nematode mortality in microcosms with small numbers of assay nematodes, and we examined assay nematodes recovered at the end of the experiment for signs of fungal parasites. Because the microcosms did not have a moat or other refuge, the assay nematodes remained vulnerable for the 3 days that they were in the soil. Mortality in this experiment was not substantially increased compared to a previous experiment, which measured the mortality of a larger number of assay nematodes in microcosms surrounded by a moat. Mortality, however, increased from 34 to 50% when recovered assay nematodes were examined and when those with conidia of the nematophagous fungus Hirsutella rhossiliensis were considered dead. The zoosporic fungus M. glutinospora was not detected, perhaps because the soil water potential was too low. Contrary to our expectations, there was no evidence of negative feedback on nematodes (i.e., no evidence of density-dependent mortality) because the addition of dead moth larvae greatly increased numbers of resident nematodes and A. oligospora but did not greatly affect the probability of nematode mortality.

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.

DIFFUSION AND EFFECTIVENESS OF THE NEMATOPHAGOUS FUNGUS HIRSUTELLA RHOSSILIENSIS IN CONTROL OF THE CYST NEMATODE HETERODERA DAVERTI UNDER FIELD CONDITIONS

An epidemiological survey on 176 soil samples, collected from carnation fields in the Vesuvius area (Naples, Italy) revealed, in more than 90% of samples, the occurrence of second stage juveniles of the cyst nematode Heterodera daverti parasitized by the nematophagous fungus Hirsutella rhossiliensis. The diffusion of the fungus over the area of cultivation of carnation could account for the substantial decrease in severity of nematode attacks, which were observed over the last five years. The influence of various factors on H. rhossiliensis-mediated suppressiveness in soils subjected to repeated treatments with fumigants is discussed.

Nutritional Requirements of the Nematophagous Fungus Hirsutella rhossiliensis

Six natural media were examined for growth and sporulation of six isolates of the nematophagous fungus Hirsutella rhossiliensis , using solid and/or liquid culture. Twenty carbohydrates, 19 nitrogen (N) compounds, and nine vitamins were also tested for their effects on growth, sporulation, and spore germination of a further three isolates (ATCC46487, OWVT-1 and JA16-1). Variations in nutritional requirements existed among the fungal isolates. In general, V-8 juice agar (VA), cornmeal agar and potato dextrose agar were good media for growth, and malt extract agar, VA and yeast dextrose agar were good for sporulation of all six isolates. Glycogen was the best and sucrose, inulin, D- ( + ) - trehalose and soluble starch were also good carbon (C) sources for growth and spore germination of the three isolates ATCC46487, OWVT-1 and JA16-1 in both liquid and solid culture. None of the isolates utilized D- ( + )xylose as a C source. L- sorbose, D- ribose, citric acid and D- fructose were poor for growth of all isolates. The best C source for sporulation was D- ( + )-trehalose for ATCC46487, D- sorbitol for OWVT-1 and D- ( + )-cellobiose for JA16-1. Casein was the best N source for growth of ATCC46487 and OWVT-1, while peptone was best for JA16-1. L- asparagine, L- proline, and peptone were also good for growth of all three isolates. L - cystine was not utilized by H. rhossiliensis and DL- methionine inhibited growth of all isolates. Spore germination of all isolates was well supported by most N compounds examined but was inhibited by L- cystine. No significant difference in sporulation of ATCC46487 was observed among the N sources. DL- threonine was the best N source for spore production by OWVT-1 and L- phenylalanine was best for JA16-1. Vitamins generally enhanced fungal growth and sporulation, with thiamine having the greatest influence. Excluding some vitamins individually from the medium containing all other test vitamins sometimes increased growth and/or sporulation of certain isolates.

Soil pH and the Activity of a Pelletized Nematophagous Fungus

ABSTRACT When formulated as assimilative hyphae in alginate pellets, the nematophagous fungus Hirsutella rhossiliensis was more active (i.e., parasitized more assay nematodes) in an acidic vineyard soil than in a neutral vineyard soil. To determine whether soil pH explained the difference, fungus activity was measured in soil from the neutral site that had been acidified (by adding sulfuric acid) and in soil from the acidic site that had been neutralized (by adding calcium hydroxide). As hypothesized, the activity of pelletized Hirsutella rhossiliensis was negatively correlated with soil pH. Maximum activity occurred at pH(calcium chloride) 4.5, and activity gradually declined to near zero as the pH increased to 6.5 and rapidly declined to near zero as the pH dropped below 4.0. Assays performed on leached soil samples indicated that the effects of sulfuric acid and calcium hydroxide were largely due to pH rather than to specific ions or osmotic potential. The effect of pH, however, was indirect. Heating the neutral soil to 60 degrees C for 2 h did not alter soil pH or electrical conductivity but increased fungus activity to levels equivalent to those in acidified soil. We conclude that, in these two soils, heat treatment or low soil pH suppresses soil organisms that otherwise interfere with growth of Hirsutella rhossiliensis from alginate pellets.

Enchytraeids and nematophagous fungi in soil microcosms

In small containers of soil in the laboratory, the enchytraeid Enchytraeus crypticus destroyed colonies of the nematophagous fungus Hirsutella rhossiliensis growing from pelletized hyphae, and reduced biological control of the root-knot nematode Meloidogyne javanica. The enchytraeid also reduced the population densities of the nematode-trapping fungi Monacrosporium gephyropagum, Arthrobotrys thaumasia, and A. haptotyla. Because addition of pelletized hyphae caused rapid and large increases in numbers of E. crypticus, the enchytraeid apparently interfered with biological control by ingesting and consuming the fungi.

Trophic interactions between bacterial-feeding nematodes in plant rhizospheres and the nematophagous fungus Hirsutella rhossiliensis to suppress Heterodera schachtii

Trophic exchanges in soil food webs may suppress populations of pest organisms. We hypothesize that the suppressive condition of soils might be enhanced by manipulating components of the food web. Specifically, by enhancing populations of bacterial-feeding nematodes, propagule density of the nematophagous fungus Hirsutella rhossiliensis should increase and constrain populations of Heterodera schachtii, a plant-parasitic nematode. The rhizospheres of Crotalaria juncea and Vicia villosa stimulated population growth of the bacterial-feeding nematode, Acrobeloides bodenheimeri, but not of the nematodes Caenorhabditis elegans or Rhabditis cucumeris. The rhizospheres of Tagetes patula, Eragrostis curvula, and Sesamum indicum had no effect on any of the bacterial-feeding nematodes investigated. Acrobeloides bodenheimeri was most susceptible to parasitism by the nematophagous fungus H. rhossiliensis with 35% of individuals being parasitized in a laboratory assay. In three separate trials, parasitism of H. schachtii by H. rhossiliensis was not enhanced when populations of A. bodenheimeri were amplified in a suitable rhizosphere.

Suppression of potato cyst nematode root penetration by the endoparasitic nematophagous fungusHirsutella rhossiliensis

The endoparasitic nematophagous fungusHirsutella rhossiliensis was tested for its ability to suppress root penetration and cyst formation by the potato cyst nematode speciesGlobodera pallida. Isolates ofH. rhossiliensis were obtained from infected potato cyst nematode juveniles from different starch potato fields in The Netherlands. The isolates showed no difference in spore adhesion to juveniles on agar plates (adhesion rate: ±90%). The most rapid growing isolate, CBS 108.94, was used for experiments. Vegetative mycelial colonies ofH. rhossiliensis CBS 108.94, grown in potato dextrose broth, were used as soil inoculum. During submerged cultivation the mycelial colonies produced phialides (spore-bearing cells) but no spores. Exposed to the air, however, spores were rapidly formed. The effect of different soil inoculum densities of mycelial colonies on root penetration byGlobodera pallida was examined in an experiment in 250-ml pots. Up to a mycelial colony concentration representing a potential spore density of 104 g−1 soil no suppression occurred. At approximated densities of 2.5×104 and 105 spores g−1 soil the numbers of juveniles which penetrated roots were reduced by 30% and 34%, respectively. The distribution of the inoculum could be improved by fragmentation of the mycelial colonies before soil inoculation. Using mycelial fragments, again no suppression of root penetration was observed up to a potential spore density of 104 g−1 soil, but at densities of 105 and 106 g−1 a suppression of 54% and 88%, respectively, was measured. In a greenhouse experiment, soil inoculation with mycelial colonies with a potential spore production of 2.5×105 g−1 soil resulted in a suppression of root penetration of 37% and 51% after 5 and 6 weeks, respectively, but the number of newly formed cysts after 18 weeks in soil was not different for control and inoculated pots. It is concluded thatH. rhossiliensis may be useful for the reduction of root damage caused by juveniles of potato cyst nematodes, but the usefulness for population control is doubtful.

Infection of Second Stage Juveniles of Potato Cyst Nematodes By the Nematophagous Fungus Hirsutella Rhossiliensis in Dutch Potato Fields

The infection of second stage juveniles of potato cyst nematodes (PCN) by fungal parasites was studied in populations extracted from soil by centrifugal flotation. Soil was collected from five experimental plots which had annual crops of potatoes for at least nine years. The percentage of fungus-infected juveniles ranged from 10 to 60%. After 12 weekly additions of Globodera pallida juveniles, the percentage of infected individuals increased to 45-90%. The endoparasitic nematophagous fungus Hirsutella rhossiliensis was the only fungal parasite found. The fungal infection of juveniles of PCN was also studied in soil samples from 20 fields in a starch potato growing area in northeastern Netherlands where potatoes are intensively grown (once every two years). Infection by H. rhossiliensis was observed in 17 of the 20 field populations of free-living juveniles, with an average infection percentage of 15%. After 12 weekly additions of G. pallida juveniles, the average infection percentage increased to 25% and extended to 100% of the samples. Again H. rhossiliensis was the predominant parasite. In a laboratory experiment most of the juveniles added to pasteurized soil at weekly intervals were recovered after 12 weeks when H. rhossiliensis was not present but few were recovered when H. rhossiliensis was present, probably because of the rapid disintegration of infected juveniles. Detached conidia of H. rhossiliensis did not adhere to the juvenile cuticle on agar plates, but newly formed conidia, attached to conidiophores, adhered to and infected juveniles. The possible role of H. rhossiliensis in the biological control of potato cyst nematodes and other plant parasitic nematodes is discussed.

Variability among isolates of the nematophagous fungus Hirsutella rhossiliensis

Morphological, pathogenic and genetic variability among 25 isolates of the nematophagous fungus Hirsutella rhossiliensis was quantified and compared. Genetic variability was assessed with 36 RAPD markers that were generated with eight random decamer primers. Cluster analysis of the PCR products indicated substantial genetic variability among isolates of this species. Out of 300 possible isolate-isolate combinations, 193 had less than 50% similarity (shared polymorphisms). In contrast, most of the isolates were similar morphologically and pathogenically, regardless of host or geographical origin. We suggest that, in spite of substantial molecular divergence in isolated populations of this fungus, the environment selects for similar characteristics associated with pathogenicity. Isolates from nematodes in the Hoplolaimidae (Rotylenchus robustus and Hoplolaimus galeatus), however, grew slower on agar, produced larger conidia, and were less pathogenic towards nematodes than were isolates from other hosts.

Alginate Pellet Formulation of Hirsutella rhossiliensis for Biological Control of Plant-Parasitic Nematodes

Abstract Vegetative colonies of the nematophagous fungus Hirsutella rhossiliensis were grown in shake culture, rinsed free of media, macerated, and pelletized in 1% alginate. Nondried or dried (1 day at 22°C) pellets were added to 17 cm 3 of loamy sand and packed into vials. After 14 days at 20°C, assay nematodes (healthy second-stage juveniles of Heterodera schachtii ) were added to each vial, recovered after 66 h, and examined to determine the percentage with at least one fungal spore attached (transmission). Averaged over five experiments, transmission with four nondried or dried pellets per vial was 62 or 73%, respectively. Dried pellets were 1.7 mm in diameter, weighed 0.58 mg, and contained 0.14 mg dry wt of hyphae. Whereas transmission with dried pellets stored at 5°C for 13-195 days remained high, transmission decreased if dried pellets were stored at 22°C for longer than 13 days; this decrease was more rapid in nonheated than in heated soil, and in soil at −8 kPa than at −28 kPa. Transmission with dried pellets doubled if pellets were in soil for 28 days rather than 14 days before assay nematodes were added. Transmission with dried pellets was low when hyphal density was low or when hyphae were stored and macerated in water. Addition of dried pellets to soil suppressed invasion of roots by H. schachtii .

Population biology and biological control of nematodes.

We studied the population biology of the nematophagous fungus Hirsutella rhossiliensis to understand its potential as a biological control agent. Because the fungus is an infectious and transmissible parasite, we framed our study within an epidemiological context. Field observations, theory, and experiments demonstrated that (i) parasitism of nematodes by H. rhossiliensis is dependent on nematode density, (ii) local populations of the fungus will go extinct unless supplied with some minimum number of nematodes (the host threshold density), and (iii) natural epidemics of this fungus in populations of nematodes develop slowly and only after long periods of high host density. Additional in-depth research on population biology is needed to explain other biological control systems and to guide future research. The most effective research will combine field observation, theory, and experimentation.

Density‐Dependent Host‐Pathogen Dynamics in Soil Microcosms

Temporal density—dependent parasitism and a host threshold density are important features of disease induced by infectious parasites in populations of aboveground, macroscopic organisms. We determined whether these features also occur in soil microcosms containing a microscopic host (the nematode Heterodera schachtii) and its parasite (the nematophagous fungus Hirsutella rhossiliensis). Soil microcosms are especially interesting because (1) the environment and scale are quite different from conventional host—parasite systems and may result in considerably different disease dynamics, (2) the small size of the soil microcosms, although biologically appropriate, facilitates experimentation and parameter estimation, and (3) some soil—borne, microscopic organisms (such as H. schachtii) are important agricultural pests. Temporal density—dependent parasitism was directly assessed with laboratory experiments in which host density and environment were controlled. A theory, which complements and extends the experiments, was developed to enable direct comparison of observed and predicted dynamics and to provide a stringent test of our understanding of processes underlying the dynamics. The theory was simple, yet explicitly described the essential biology. Parameters for the theory were measured with short—term experiments. We found that the disease dynamics in soil microcosms exhibited both temporal density—dependent parasitism and a host threshold density. However, epidemics were slow to develop. Observed and predicted dynamics were quite similar, indicating that our understanding of the underlying biology was correct.