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Pochonia chlamydosporia is a nematophagous fungus with high potential for the biological control of root-knot nematodes (Meloidogyne spp.). The fungus is commonly produced in colonised rice substrate, but non-staple and more affordable substrates may encourage the production and use of this biological control agent. An in vitro study was conducted to screen different organic materials, including green and animal manures and industrial by-products as substrates for local production of viable fungal chlamydospores. Chlamydospore yield from sugarcane filter mud, bean straw, sunn hemp, goat manure, African marigold, wild sunflower, maize cobs, and velvet beans substrates was equal to or significantly greater than production using rice (standard substrate). In addition, the percentage viability of chlamydospores produced in most organic substrates was greater than 50%. The use of non-staple substrates deserves further investigation as a viable, and economical local option to rice substrate for mass production of P. chlamydosporia inoculum in Sub-Saharan Africa.

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.

Lecanicillium attenuatum is an important nematophagous fungus with potential as a biopesticide for control of plant-pathogenic nematodes. However, relatively low fungicide-tolerance limits its application in the field. To improve the propamocarb-tolerance of L. attenuatum , a low-energy N + ion implantation-based mutagenesis system was established. In the first round of N + ion implantation, a proper conidial lethality rate (87.2–88.8%) and the highest positive mutation rate (11.2–11.4%) were achieved, and the highest propamocarb-tolerant mutant LA-I-R1-T7-M1 and LA-I-R1-T7-M2 were isolated in the experiment 1 and experiment 2 respectively, having a median effective concentration (EC 50 ) value of 1223.6 and 1173.6 μg/ml respectively. Multiple rounds of N + ion implantation were further employed, but fungal propamocarb-tolerance had not been significantly enhanced until the fifth-round of ion implantation. The LA-I-R5-M1 was the highest tolerant to propamocarb among all the mutants, whose EC 50 value was increased 2.54-fold compared to wild-type strain, reaching to the 1298.7 μg/ml. Its EC 50 value was significantly increased with continuous passaging, which was stable from the fifth- to fifteenth-generation, and then declined. However the EC 50 value of the twentieth-generation strain was still higher than the EC 50 (1298.7 μg/ml) of the first-generation strain. The colony growth, conidia yield and conidial germination of mutants on plates, and their parasitism in the nematode eggs of Meloidogyne incognita and Heterodera glycines were not significantly changed in both single- and multiple-round of N + ion implantation. In conclusion, we had succeeded in improving the propamocarb-tolerance of L. attenuatum through this low-energy N + ion implantation-based mutagenesis system. The improved strain LA-I-R5-M1 could be potentially applied in combination with propamocarb in the field.

An emerging organic citrus industry in Florida could benefit greatly from effective, non-conventional methods to mitigate losses from pests and diseases. We studied part of a soil food web in an organic orchard to learn ways to conserve and enhance biological control of insect pests by native entomopathogenic nematodes (EPNs). We evaluated two OMRI (Organic Materials Review Institute) approved cultural practices: (i) a mulch of commercially pelleted chicken manure, (ii) a commercial formulation of Purpureocillium lilacinus, and (iii) an un-amended control. Several soil nutrients (i.e. nitrogen, phosphate, and potassium) were affected by the amendments, but initial equilibrium values (T0) were restored by the last sampling time (T12). The plant parasitic nematode Tylenchulus semipenetrans increased in both treatments compared to the untreated control at T3 (. P

Options for control of northern root-knot nematode (NRKN, Meloidogyne hapla ) on vegetables are very limited currently. In this study, we characterized the parasitism of Myrothecium verrucaria strain X-16, a new nematophagous fungus, on NRKN at the stages of eggs, J2, and adult females and evaluated its biocontrol efficacy in the greenhouse. Strain X-16 produced conidia that geminated and invaded in 80 h after in contact with eggs, causing the shrinkage and depression of egg shell and blastocolysis of the embryo. The strain also attacked 2nd-stage juveniles by producing developing surface networks of hyphae on the nematode body wall. Strain X-16 attacked adult females by producing dense networks of hyphae on the nematode body wall in 120 h. Strain X-16 had lethal effects (22–71% mortality) against NRKN J2 at the concentration as low as 3.1 × 10 7 conidia/ml and with the incubation treatment time as short as 24 h. The lethal effects linearly increased with the increase of conidial concentration, with the estimated LC 50 values as low as 1.0 × 10 8 conidia/ml. Soil treatments with strain X-16 at 1%, 2% or 4% (wt/wt) induced significant reductions in J2 nematode counts in 100 g of dry soil, Pf/Pi ratios and root-know index in cucumber in the greenhouse evaluations. These studies are the first to demonstrate that M . verrucaria is able to parasitize NRKN and strain X-16 can be a potential biocontrol agent for management of NRKN.

The nematophagous fungus Dactylella oviparasitica is considered the primary cause of a sugar beet cyst nematode ( Heterodera schachtii ) population suppression in a field at the Agricultural Operations, University of California, Riverside. Parasitism of H. schachtii by the ascomycete D. oviparasitica was studied using both Arabidopsis thaliana (type Landsberg erecta) and cabbage as host plants in gnotobiotic agar culture. Suitability of Arabidopsis as a host for H. schachtii was confirmed using seedlings grown with the nematode in axenic sand culture. Both developing males and females of H. schachtii broke through the Arabidopsis root surface during late juvenile stages and both were susceptible to D. oviparasitica parasitism. In contrast to Arabidopsis, developing juvenile males remained in nearly all observed cases enclosed within the cabbage root tissues while the larger body expansion of the female juveniles caused the root cortex to split; consequently only the latter ones were accessible to the fungus. In the presence of D. oviparasitica , the number of females with eggs was reduced by more than 95% and the number of eggs per female by almost 60% as compared to females developing on plates without the fungus. Viable eggs were not susceptible to parasitism while more than 90% of heat- or cold-killed eggs were rendered susceptible. These observations suggest that parasitism of developing juveniles may be the essential mode of action in the population suppression of H. schachtii .

The biocontrol potential of Pochonia chlamydosporia , a fungus with parasitic activity against economically important plant-parasitic nematodes, can be influenced by abiotic factors such as water availability. The objective of this study was to evaluate the effects of different water stress regimes on in vitro growth, sporulation, germination and parasitism of P . chlamydosporia isolates . The osmotic water potential of 1.7% corn meal agar (CMA) was modified by addition of potassium chloride (KCl) or glycerol, and the matric water potential was modified using polyethylene glycol (PEG 8000). The fungus was able to grow over a range of potentials but radial growth rates decreased with the increase of osmotic and matric stress. No growth was observed at −10 MPa on 1.7% CMA amended with glycerol and at −7.1 MPa on medium with PEG 8000 but all isolates were able to resume growth when transferred onto unmodified 1.7% CMA. The production of chlamydospores was repressed in both osmotic and matric modified media. Although the production of conidia increased in medium modified with KCl, the germination rate was lower. Spores/hyphal fragments remained viable in all isolates that were previously inoculated onto media with growth-limiting water potential (−10 MPa on 1.7% CMA amended with glycerol and −10 MPa on medium with PEG 8000). The percentage of viable conidia produced on 1.7% CMA, after inoculation under osmotic or matric stress conditions for 25 days, was over 74.5% in all isolates (osmotic stress) and ranged from 1% (Pc1) to 65.8% (Pc280) (matric stress). The in vitro infection of potato cyst nematodes, Globodera rostochiensis eggs by P. chlamydosporia isolates, grown under these limiting conditions, was studied using a standard bioassay. The percentage of parasitized eggs was significantly higher under osmotic stress except for isolates Pc2 and Pc3. P. chlamydosporia spores/hyphal fragments can remain viable at water potentials limiting for growth, for prolonged periods of time, suggesting that the osmoregulation mechanisms, used to compensate water stress, affect in vitro sporulation and increased pathogenicity. Knowledge on water requirements of P . chlamydosporia enables a better understanding of its survival and growth strategies in the soil environment and could aid the development of effective strategies to increase the production and quality of inoculum, thus contributing to the implementation of biosafe, sustainable management strategies against plant-parasitic nematodes.

The fallow period can represent an opportunity to farmers if the field can be trimmed to receive finishing cattle. Nematode control remains important at this stage, and the use of biological agents such as nematophagous fungi is desirable. Nonetheless, the indiscriminate use of fungicides on agricultural crops may affect the development and survival of fungal propagules. Therefore, we evaluated the in vitro activity of 14 agricultural fungicides against the Duddingtonia flagrans strain ARSEF 5701. The assays were based on CLSI M38-A2 broth microdilution and the agar diffusion method. The fungicides azoxystrobin, cyproconazole, cyprodinil and tebuconazole showed minimum inhibitory concentrations for D. flagrans below their respective maximum residue limits. Although complete radial growth inhibition was only observed for carbendazim at the maximum concentration tested, D. flagrans proved to be resistant to only two fungicides, cyprodinil and iprodione. The present study indicates that D. flagrans is sensitive to most of the common agricultural fungicides, and resistance depends upon the structure, i.e., hypha or chlamydospore. On the basis of these in vitro results, we concluded that the use of agricultural fungicides could seriously hamper the survival of the fungus D. flagrans in amended soils.

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 J2infested 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.

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.

Hirsutella rhossiliensis is a parasite of vermiform nematodes. In 1995, parasitism of the soybean cyst nematode second-stage juveniles (J2s) by this fungus was observed in a Minnesota field. A subsequent survey was conducted in 1996–1997 to determine the extent of infestation of soybean fields by this fungus in southern Minnesota. A total of 264 soil samples representing 237 fields in 27 counties was examined in three tests. In Test 1, 1000 J2s were added to 50 g of soil at days 0, 7, 14, and 21, and the nematodes were recovered at day 28. In Test 2, 2000 J2s were added to 50 cm3 of soil, and the nematodes were recovered at day 14. In Test 3, J2s were extracted from 50 cm3 of soil without addition of nematodes. The J2s were examined for fungal parasitism. Two species of Hirsutella, H. rhossiliensis and H. minnesotensis, were commonly encountered on the J2s. Combining data from all three tests, parasitism of J2s by Hirsutella species was observed in soil from 46% of the fields. Parasitism of J2s by H. rhossiliensis was observed in soil from 43% of fields and by H. minnesotensis in soil from 14% of fields. The highest percentage of J2s parasitized by Hirsutella spp. was 60%. Addition of J2s into soil increased efficiency of fungal detection.

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 .

The nematophagous fungus, Hirsutella rhossiliensis, was detected in 10 of 21 sugarbeet fields in California. Fewer than four parasitized nematodes per 100 cm3 soil were found in any field, and all parasitized specimens were the cyst nematode, Heterodera schachtii. Detection of the fungus was enhanced if soil samples were periodically inoculated with large numbers of healthy juveniles of H. schachtii prior to assay for the fungus. Detection efficiency was measured by assaying soil seeded with known numbers of parasitized H. schachtii. Detection was largely unaffected by soil type but declined from 63% at Time 0 to 17% after 2 days at 22°C. Thus, the failure to detect H. rhossiliensis in 11 fields could reflect the absence of the fungus or limitations of the assay. The results suggest that a one-time release of H. rhossiliensis with the intention of establishing the fungus in sugarbeet fields is not a viable alternative for implementation of biological control of H. schachtii.

Survival of the entomogenous nematodes Steinernema carpocapsae , S. glaseri , and Heterorhabditis bacteriophora was lower in nonsterilized soil than in sterilized soil. The nematode-parasitic fungus Hirsutella rhossiliensis was the dominant antagonist in the soil. In the nonsterilized soil, more S. glaseni than S. carpocapsae or H. bacteriophora were parasitized by H. rhossiliensis ; consequently, survival of S. glaseri was lower than that of the other two nematode species. Differential survival of the three nematode species in the presence of H. rhossiliensis may be due to differences in their retention of the second-stage (J2) cuticle, susceptibility to fungal adhesion, and motility. When H. bacteriophora with and without its protective J2 cuticle was placed in H. rhossiliensis -infested soil, only 22% of the nematodes without the cuticle survived after 4 days compared with 100% survival of those possessing the cuticle. The nematode species were not equally susceptible to adhesion of H. rhossiliensis conidia. When individual nematodes contacted five conidia, fewer conidia adhered to the J3 cuticle of S. carpocapsae than to the J3 cuticle of S. glaseri or H. bacteriophora . Highly motile nematodes will encounter more H. rhossiliensis conidia than will less motile nematodes. The relative motility of the nematode species in soil was S. glaseri > H. bacteriophora > S. carpocapsae . Entomogenous nematodes that retain their J2 cuticle, exhibit low motility, and are refractory to fungal adhesion may be suitable for controlling insects in habitats with abundant parasitic fungi such as H. rhossiliensis .