Antagonistic activity of the fungus Pochonia chlamydosporia on mature and immature Toxocara canis eggs.

In vitro tests were performed to evaluate the ability of 6 isolates of the nematophagous fungus Pochonia chlamydosporia to infect immature and mature Toxocara canis eggs on cellulose dialysis membrane. There was a direct relationship between the number of eggs colonized and the increase in the days of interaction, as well as between the number of eggs colonized and the increase in the concentration of chlamydospores (P<0.05). Immature eggs were more susceptible to infection than mature eggs. The isolate Pc-04 was the most efficient egg parasite until the 7th day, and showed no difference in capacity to infect mature and immature eggs in comparison to Pc-07 at 14 and 21 days of interaction, respectively. Isolate Pc-04 was the most infective on the two evolutionary phases of the eggs at most concentrations, but its ability to infect immature eggs did not differ from that presented by the isolates Pc-07 and Pc-10 at the inoculum level of 5000 chlamydospores. Colonization of infective larvae inside or outside the egg was observed in treatments with the isolates Pc-03, Pc-04, Pc-07 and Pc-10. The isolate Pc-04 of P. chlamydosporia has great biological capacity to destroy immature and mature T. canis eggs in laboratory conditions.

The biological control of Ancylostoma spp. dog infective larvae by Duddingtonia flagrans in a soil microcosm.

Experiments to evaluate the potential ability of the nematode-trapping fungus Duddingtonia flagrans (Isolate CG768) to prey on the Ancylostoma spp. dog infective larvae (L(3)) in pasteurized soil were performed through several laboratory assays. A microcosm approach was used with increasing fungal concentrations in an inoculum of a chlamydospore water suspension. The highest fungal concentrations provide a more consistent larval reduction than the lowest concentrations, but no difference was observed from 10,000 to 25,000 chlamydospores per grain of soil. When using D. flagrans in a water suspension, in white rice and in milled maize, there were reductions in the larval population of 72.0%, 78.4% and 79.4%, respectively, but there was no difference between white rice and milled maize (p<0.05). To evaluate the nematode control by D. flagrans inoculated in milled maize at 10,000 chlamydospores per grain of soil under greenhouse conditions, observations were performed at 10, 15, 20, 25 and 30 days after inoculation and the percent reduction in the larval population was 61.4%, 73.2%, 70.8%, 64.5% and 57%, respectively (p<0.05). There was an inverse relationship between the number of L(3) recovered from the soil and the total days of exposure to the fungus (p<0.05). These results showed that D. flagrans could present some potential to be used as a non-chemotherapeutic alternative for regulation of Ancylostoma spp. populations in the environment.

Duddingtonia flagrans, Monacrosporium thaumasium and Pochonia chlamydosporia as possible biological control agents of Oxyuris equi and Austroxyuris finlaysoni.

The action of four fungal isolates of the species Duddingtonia flagrans (AC001), Monacrosporium thaumasium (NF34a) and Pochonia chlamydosporia (VC1 and VC4) on eggs of Oxyuris equi and Austroxyuris finlaysoni was evaluated in two assays (A and B). Eggs of O. equi (Test A) and A. finlaysoni (Test B) were plated on Petri dishes with 2% water-agar with grown fungal isolates and control without fungus. After 5, 10 and 15 days, 100 eggs were collected and classified according to the following parameters: type 1 effect, physiological and biochemical effect without morphological damage to the eggshell; type 2 effect, lytic effect with morphological alteration of the eggshell and embryo; and type 3 effect, lytic effect with morphological alteration of the eggshell and embryo, hyphal penetration and internal egg colonization. Pochonia chlamydosporia isolates VC1 and VC4 showed ovicidal activity for type 1, 2 and 3 effects on eggs of O. equi and eggs of A. finlaysoni. In vitro assays A and B showed that P. chlamydosporia had a negative influence on eggs of O. equi and A. finlaysoni and can be considered as a potential biological control agent of nematodes.

Predation of Ancylostoma spp. dog infective larvae by nematophagous fungi in different conidial concentrations.

In the present work, it was evaluated the in vitro effect of 12 isolates from the fungal species Arthrobotrys, Duddingtonia, Nematoctonus and Monacrosporium genera in different conidial concentrations on the capture of Ancylostoma spp. dog infective larvae (L(3)), on 2% water-agar medium at 25 degrees C, at the end of a period of 7 days. The concentrations used for each nematophagous fungus were 1000, 5000, 10,000, 15,000 and 20,000conidia/Petri dish plated with 1000 Ancylostoma spp. L(3). All nematode-trapping fungi isolates tested reduced the averages of the uncaptured Ancylostoma spp. L(3) recovered, with the increase of the fungal inoculum concentration, in comparison to the fungus-free control (p<0.05). The adhesive network producing species were better predators than the constricting ring or adhesive knob producing species. Duddingtonia flagrans (Isolate CG768) was the most effective, reducing the averages of the uncaptured Ancylostoma spp. L(3) recovered in 92.8%, 96.3%, 97.5%, 98.3% and 98.9%, respectively in five fungal inoculum concentrations established. Other effective nematophagous fungi were Arthrobotrys robusta (Isolate I31), which reduced the averages of the uncaptured Ancylostoma spp. L(3) recovered in 85.4%, 88.3%, 90.7%, 92.5% and 95.2%, and Arthrobotrys oligospora (Isolate A183), with reductions of 66.6%, 79.8%, 86.8%, 89.5% and 90.8%, respectively for both, in the five fungal inoculum concentrations established. No difference was found between Isolates A183 and I31 in the conidial concentrations of 15,000/Petri dish. Nematoctonus robustus (Isolate D1) and Arthrobotrys bronchophaga (Isolate AB) had the smallest percentages of reduction among the tested isolates and showed the lowest predacious activity. The Isolates CG768, I31 and A183 were considered potential biological control agents of Ancylostoma spp. dog free-living stages, being directly influenced by the fungal inoculum concentration.

Scanning electron microscopy of Ancylostoma spp. dog infective larvae captured and destroyed by the nematophagous fungus Duddingtonia flagrans.

The interaction between the nematode-trapping fungus Duddingtonia flagrans (isolate CG768) against Ancylostoma spp. dog infective larvae (L(3)) was evaluated by means of scanning electron microscopy. Adhesive network trap formation was observed 6h after the beginning of the interaction, and the capture of Ancylostoma spp. L(3) was observed 8h after the inoculation these larvae on the cellulose membranes colonized by the fungus. Scanning electron micrographs were taken at 0, 12, 24, 36 and 48 h, where 0 is the time when Ancylostoma spp. L(3) was first captured by the fungus. Details of the capture structure formed by the fungus were described. Nematophagous Fungus Helper Bacteria (NHB) were found at interactions points between the D. flagrans and Ancylostoma spp. L(3). The cuticle penetration by the differentiated fungal hyphae with the exit of nematode internal contents was observed 36 h after the capture. Ancylostoma spp. L(3) were completely destroyed after 48 h of interaction with the fungus. The scanning electron microscopy technique was efficient on the study of this interaction, showing that the nematode-trapping fungus D. flagrans (isolate CG768) is a potential exterminator of Ancylostoma spp. L(3).

Ovicidal activity of Paecilomyces lilacinus on Moniezia sp. eggs.

The ovicidal activity of Paecilomyces lilacinus was evaluated on Moniezia sp. eggs. Eggs of Moniezia sp. were incubated on plates with 2% agar-water inoculated with grown fungal isolates and a control treatment without fungus. After 5, 10 and 15 days post-inoculation, the eggs were removed and classified according to the following parameters: effect type 1, lytic effect without morphological damage to eggshells; effect type 2, lytic effect with morphological change in embryos and eggshells; and effect type 3, lytic effect with morphological change in embryos and eggshells, with hyphal penetration and internal colonization of eggs. Paecilomyces lilacinus showed percentages for ovicidal activity (P < 0.01), mainly type 3 effect, of 19, 20 and 23% on eggs of Moniezia sp., after 5, 10 and 15 days post-inoculation, respectively. Therefore P. lilacinus can be considered as a potential biological control agent for this cestode.

Interaction between the nematophagous fungus Duddingtonia flagrans and infective larvae of Haemonchus contortus (Nematoda: Trichostrongyloidea).

The interaction between Duddingtonia flagrans and infective larvae of Haemonchus contortus was studied in vitro under optical and scanning electron microscopy. Trap formation by the fungus started 9 hours after inoculation and first larvae were found 11 hours after larval inoculation on colonies grown on the surface of dialysis membranes. Scanning electron micrographs were taken 12, 24, 36 and 48 h after larval predation. Details of predation structures and fungus-larvae interaction are described. A mucilaginous substance occurred at the points of adherence of traps to nematode cuticle. Bacteria were also found at some points of interaction between fungus and larval cuticle. Cuticle penetration by fungus hyphae occurred only 48 h after predation.

Viabilidade de formulação peletizada do fungo nematófago Monacrosporium sinense, no controle biológico de nematóides parasitos gastrintestinais de bezerros / Viability of pellet formulation of Monacrosporium sinense as biological control of gastrointestinal nematodes of calves

A viabilidade de uma formulação do fungo Monacrosporium sinense foi avaliada no controle de nematóides parasitos gastrintestinais de bovinos. Dois grupos de 10 bezerros cada um, mestiços Holandês x Zebu, de seis a nove meses de idade, foram colocados em pastagem de Brachiaria brizantha. Em um dos grupos, cada animal recebeu 20g de péletes em matriz de alginato de sódio, contendo massa miceliana do fungo M. sinense via oral, duas vezes por semana, durante seis meses, com início no mês de outubro; no outro grupo, controle, os bezerros não receberam esse tratamento. As contagens de ovos por grama de fezes (OPG) e de larvas infectantes por kg de matéria seca foram maiores (P<0,05) no grupo-controle, e a diferença entre o OPG dos dois grupos, no final do experimento, foi de 79 por cento. A viabilidade dos péletes em germinar e a atividade predatória do fungo após o encapsulamento foram avaliadas in vitro. A porcentagem de péletes com cultivo positivo para o fungo variou entre 90 e 100 por cento, e o percentual de redução de larvas infectantes in vitro variou entre 90,6 e 100 por cento. A aplicação de péletes de M. sinense na dosagem e periodicidade usadas são eficazes no biocontrole de nematóides parasitos gastrintestinais de bovinos.

The viability of a formulation of the fungus Monacrosporium sinense was evaluated as control of bovine gastrointestinal nematodes parasites. Two groups were used and they were made up of 10 Holstein X Zebu crossbred, six to eight-month-old. They were grazing on Brachiaria brizantha pasture. In the treated group, each animal received orally, twice a week 20g of pellets of sodium alginate containing mycelial of the fungus M. sinense, during six months, with the onset in October. In the control group, the calves did not receive that treatment. The counting of eggs per gram of faeces (EPG) and the counting of infective larvae per kg of dry matter were higher (P<0.05) in the control group than in the treated group. The difference of the EPG between the groups at the end of the experimental period was 79 percent. The viability of the pellets germination and the predatory activity of the fungus after the encapsulation were evaluated in vitro. The percentage of pellets with positive culture for fungus varied between 90-100 percent and the percentage of reduction of infective larvae varied between 90.6-100 percent. The use of that dose and the periodic application of M. sinense pellets were efficient as control of bovine gastrointestinal nematode parasites.

Efeito antagônico de fungos predadores dos gêneros Monacrosporium, Arthrobotrys e Duddingtonia sobre larvas infectantes de Cooperia sp. e Oesophagostomum sp. / Antagonistic effect of nematophagous fungi Monacrosporium, Arthrobotrys and Duddingtonia on infective Cooperia sp. and Oesophagostomum sp. larvae

Seis isolados dos fungos nematófagos Monacrosporium thaumasium (isolado NF 34A), Monacrosporium sinense (isolado SF 470), Monacrosporium appendiculatum (isolado CGI), Arthrobotrys robusta (isolado I 31), Arthrobotrys cladodes (isolado CG 719) e Duddingtonia flagrans (isolado CG 768) foram avaliados em laboratório quanto à capacidade de predar larvas infectantes de Cooperia sp. e Oesophagostomum sp. Nos testes in vitro, os fungos foram eficientes em predar os nematóides (P<0,05), e não houve variação na capacidade predatória entre os fungos testados (P>0,05) durante os cinco dias do ensaio. Estruturas reprodutivas (conídios) foram encontradas em todos os isolados no quinto dia. Todos os fungos testados são promissores para serem utilizados no controle biológico de Cooperia sp. e Oesophagostomum sp., parasitos de bovinos.

Six isolates of nematophagous fungi Monacrosporium thaumasium (isolate NF 34A), Monacrosporium sinense (isolate SF 470), Monacrosporium appendiculatum (isolate CGI), Arthrobotrys robusta (isolate I 31), Arthrobotrys cladodes (isolate CG 719) and Duddingtonia flagrans (isolate CG 768) were evaluated under laboratory conditions regarding the capacity to entrap infective Cooperia sp. and Oesophagostomum sp. larvae. In the in vitro tests the fungi tested were equally efficient to prey the nematodes (P<0.05) during the five days of the experiment. Reproductive structures (conidia) from all isolates were visualized in 5th day. All fungal isolates were efficient in the control of bovine Cooperia sp. and Oesophagostomum sp. parasites.

Controle biológico de tricostrongilídeos (Nematoda: Trichostrongyloidea) gastrintestinais de bovinos pelo fungo Monacrosporium sinense / Biological control of gastrointestinal trichostrongylids (Nematoda: Trichostrongyloidea) of cattle by pellets of Monacrosporium sinense

Avaliou-se a resistência do fungo nematófago Monacrosporium sinense (isolado SF 470) em passar pelo trato gastrintestinal de bovinos e testou-se sua capacidade predatória sobre larvas infectantes de tricostrongilídeos após a passagem. Cem gramas de péletes formulados em matriz de alginato de sódio do isolado fúngico de M. sinense foram administrados, por via oral (VO), a dois bezerros mestiços holandês zebu de seis meses de idade. Para o controle utilizaram-se dois outros animais, de igual padrão, aos quais foram administrados 100g de péletes, VO, livre do isolado fúngico. Amostras fecais, coletadas nos tempos de 12h, 18h, 24h, 48h, 72h e 96h após os tratamentos, foram acondicionadas em placas de Petri e em coproculturas a 25ºC por 15 dias. Houve redução (P<0,05) no número de larvas infectantes de Cooperia spp. e Haemonchus spp. nos animais tratados em relação ao grupo-controle. A diferença ao final do experimento foi de 69,9 por cento nas placas e 61,3 por cento nas coproculturas. O fungo M. sinense demonstrou ser efetivo no controle de larvas infectantes de Cooperia spp e Haemonchus spp em condições de laboratório.

Aplicação de formulação do fungo predador de nematóides Monacrosporium thaumasium (Drechsler, 1937) no controle de nematóides de bovinos / Control of bovine gastrointestinal nematodes using formulation of the nematode-trapping fungus Monacrosporium thaumasium (Drechsler, 1937)

A viabilidade de uma formulação do fungo Monacrosporium thaumasium (Drechsler, 1937) foi avaliada no controle biológico de nematóides parasitos gastrintestinais de bovinos. Dois grupos de sete bezerras cada, mestiças Holandês x Zebu, de quatro a seis meses de idade, foram colocados em pastagens de Cynodon dactilon. No grupo A, cada animal recebeu 20g de pellets (formulação granulada) de M. thaumasium via oral, duas vezes por semana, durante quatro meses, com início na estação chuvosa (outubro, 2001). No grupo B (controle), as bezerras não receberam nenhum tratamento. As contagens de ovos por grama de fezes (OPG) e das larvas infectantes encontradas na pastagem do grupo B foram significativamente maiores (P<0,05) do que as do grupo A e a diferença entre o OPG dos animais dos grupos A e B, no final do experimento, foi de 88,8 por cento. O gênero Cooperia foi o mais prevalente em ambas as pastagens. Conclui-se que a aplicação de pellets de M. thaumasium na dosagem e periodicidade de aplicação usadas foi eficiente no controle de nematóides parasitos gastrintestinais de bovinos.