EVALUATION OF EFFICACY OF PASTEURIA PENETRANS ALONE AND IN COMBINATION WITH VERTICILLIUM CHLAMYDOSPORIUM AGAINST MELOIDOGYNE JAVANICA

The potential control of Meloidogyne javanica using Pasteuria penetrans (Pp) alone and in combination with Verticillium chlamydosporium (Vc) was tested in earthen pots following a cropping sequence "tomato-tomato-tomato” over three crop cycles. After the final harvesting, analysis of variance showed significant effect of treatments (P0.01) regarding number of eggmasses, galls and nematode female populations. Similarly, significant effect of treatments (P0.01) was also recorded in case of infected nematode females with Pasteuria and number of eggs/eggmass while no significant effect (P 0.0 5) was observed in case of endospore production. Higher numbers of eggmasses (360) and root galling (6.2) was observed where biocontrol agents were absent (control). The treatments showed 46.58, 58.85 and 33.13 percent reduction in number of galls, eggmasses and nematodes in Pp alone and 43.34, 55.21 and 30.09 percent reductions in Vc+Pp treatments respectively. Numbers of females infected with the endospores of P. penetrans were recorded higher in Pp-3 alone treatment (13.2) followed by Vc+Pp combined treatment (13.0) and maize rotated treatment (10.4) respectively. Significantly lesser number of eggmasses, galls and nematodes were recorded in pots where tomato was rotated with maize (treatment 3) compared with control. Thus rotation prevented the buildup of nematode population and resulted in a 72% decrease in numbers of eggmasses, 38% in root galling and 46% regarding female populations over the control after the final harvest. Maximum colony forming units of V. chlamydosporium per gram of soil were recorded after its addition to the soil. The fungus established in the soil during the first crop and soil colonization of the fungus was also observed after final crop.

Hirsutella thompsonii and Pochonia chlamydosporia (Syn. Verticillium chlamydosporium) Mycelia Growth and Predation on Panagrellus redivivus

This research aimed to evaluate the nematophagous ability of 4077-Verticillium chlamydosporium var. chlamydosporium and 4466-Hirsutella thompsonii isolates and relate mycelia growth to the influence provoked by movement of nematodes. Each fungus grew in PDA (potato, dextrose, agar) medium end up to pure colonization. Then, ten mycelia plugs of 8 mm diameter were removed from colony borders and transferred to the center of ten Petri plates containing water-agar 2% medium. These plates were previously divided into four quadrants that received a number of 25 individuals of free-living nematodes (Panagrellus redivivus), composing a total of 100 nematodes per plate. Evaluations started after 24 hours of interaction, considering predation percentage and mycelia growth as stimuli of nematodes presence. Results showed growing predation performance to both isolates, being higher for V. chlamydosporium var. chlamydosporium since from first evaluation time, controlling more than 50% of nematode population initially added. Its predation potential was 39.2%, 38.4% and 48.35% higher than H. thompsonii at first, second and third evaluation day, respectively. Generally, nematodes did not stimulate mycelia growth, unless for H. thompsonii at 72 hours of interaction compared to control plates (without nematodes). Stress resulting from isolates transference from PDA to water-agar 2% resulted in sparse mycelia growth and it could have affected the predation performance of H. thompsonii that controlled nematodes in low levels throughout experiment. Independently of predation level, pictures revealed that both isolates has ability to control P. redivivus through hyphae penetration.

Fungus–benzimidazole interactions: a prerequisite to deploying egg-parasitic fungi Paecilomyces lilacinus and Verticillium chlamydosporium as biocontrol agents against fascioliasis and amphistomiasis in ruminant livestock

In vitro trials investigating the effects of albendazole and triclabendazole anthelmintics on the growth profiles of the egg-parasitic fungi Paecilomyces lilacinus and Verticillium chlamydosporium were undertaken. In addition, in vivo trials were conducted in goats fed on millet grain cultures of each fungus and administered albendazole and triclabendazole anthelmintics. In vitro growth revealed V. chlamydosporium to be more sensitive to albendazole compared to P. lilacinus. In contrast, triclabendazole had the least inhibitory effect on in vitro growth of both P. lilacinus and V. chlamydosporium. Similar to albendazole, growth of P. lilacinus was more vigorous at 0.5 ppm concentration of triclabendazole. Efforts to re-isolate these egg-parasitic fungi from faeces of goats fed on fungal millet grain cultures before and following single intraruminal administration of albendazole and triclabendazole showed that P. lilacinus was not able to be re-isolated from the faeces at any sampling period. In contrast, V. chlamydosporium was able to be re-isolated from the faeces at all of the sampling periods except for the samples taken at 8–18 h and 18–24 h after administration of albendazole and triclabendazole, respectively. Lack of fungal activity at these times coincided with peak plasma availability of anthelmintics and suggests faecal levels of drugs were also high at these times and impacted negatively on fungal viability.

Screening for Indian isolates of egg-parasitic fungi for use in biological control of fascioliasis and amphistomiasis in ruminant livestock

Wild isolates of the egg-parasitic fungiPaecilomyces lilacinusandVerticillium chlamydosporium, obtained from the organic environment of Durg, Chhattisgarh, India, were subjected to screening forin vitrogrowth using different media types, range of incubation temperature and pH, and their predatory activity to the eggs ofFasciola giganticaandGigantocotyle explanatum. Maximum growth ofP. lilacinuswas obtained in corn-meal agar compared to any other media types. The preferred medium for growth ofV. chlamydosporiumwas corn-meal agar, followed by potato-dextrose agar. After initial growth for 16 h of incubation, no growth was observed in water agar for both the fungi. Six different temperatures – 4°C, 10°C, 18°C, 26°C, 34°C and 40°C – were used to observe growth profiles of the fungi in corn-meal agar medium. While no and very little growth ofP. lilacinusandV. chlamydosporiumwas observed at 4°C and 10°C, respectively, growth profiles of both the fungi were optimal at 26–40°C. A range of pH (pH 4–8) supported growth of bothP. lilacinusandV. chlamydosporium. Full-grown plates of the fungi baited with viable eggs ofF. giganticaandG. explanatumrevealed thatV. chlamydosporiumwas more vigorous in its egg-parasitic ability compared toP. lilacinus. Distortion of the eggs started on day 2–3 of egg baiting in culture plates ofV. chlamydosporium, with complete distortion by day 7. On the contrary,P. lilacinusexhibited very limited egg-parasitic ability and some of the baited eggs even showed development of miracidia.

Observação in vitro da ação dos isolados fúngicos Duddingtonia flagrans, Monacrosporium thaumasium e Verticillium chlamydosporium sobre ovos de Ascaris lumbricoides (Lineu, 1758)

Observou-se a ação in vitro dos fungos nematófagos Duddingtonia flagrans, Monacrosporium thaumasium e Verticillium chlamydosporium sobre ovos de Ascaris lumbricoides. Após sete, dez e quatorze dias de interação, o fungo promissor a ser utilizado no controle biológico de Asaris lumbricoides foi o Verticillium chlamydosporium (26-30%). Os outros fungos não foram satisfatórios.

Quantification in Soil and the Rhizosphere of the Nematophagous Fungus Verticillium chlamydosporium by Competitive PCR and Comparison with Selective Plating

ABSTRACT A competitive PCR (cPCR) assay was developed to quantify the nematophagous fungus Verticillium chlamydosporium in soil. A γ-irradiated soil was seeded with different numbers of chlamydospores from V. chlamydosporium isolate 10, and samples were obtained at time intervals of up to 8 weeks. Samples were analyzed by cPCR and by plating onto a semiselective medium. The results suggested that saprophytic V. chlamydosporium growth did occur in soil and that the two methods detected different phases of growth. The first stage of growth, DNA replication, was demonstrated by the rapid increase in cPCR estimates, and the presumed carrying capacity (PCC) of the soil was reached after only 1 week of incubation. The second stage, an increase in fungal propagules presumably due to cell division, sporulation, and hyphal fragmentation, was indicated by a less rapid increase in CFU, and 3 weeks was required to reach the PCC. Experiments with field soil revealed that saprophytic fungal growth was limited, presumably due to competition from the indigenous soil microflora, and that the PCR results were less variable than the equivalent plate count results. In addition, the limit of detection of V. chlamydosporium in field soil was lower than that in γ-irradiated soil, suggesting that there was a background population of the fungus in the field, although the level was below the limit of detection. Tomatoes were infected with the root knot nematode (RKN) or the potato cyst nematode (PCN) along with a PCN-derived isolate of the fungus (V. chlamydosporium isolate Jersey). Increases in fungal growth were observed in the rhizosphere of PCN-infested plants but not in the rhizosphere of RKN-infested plants after 14 weeks using cPCR. In this paper we describe for the first time PCR-based quantification of a fungal biological control agent for nematodes in soil and the rhizosphere, and we provide evidence for nematode host specificity that is highly relevant to the biological control efficacy of this fungus.