Study of a Mexican isolate of Arthrobotrys musiformis (Orbiliales): Predatory behavior and nematocidal activity of liquid culture filtrates against Haemonchus contortus (Trichostrongylidae), protein profile and myco-constituent groups.

A Mexican isolate of the nematophagous fungus Arthrobotrys musiformis was obtained from a soil sample from the Chapultepec ecological reserve zone, in Cuernavaca, Morelos, Mexico. This isolate demonstrated an important predatory activity (74.9%) against the parasitic nematode Haemonchus contortus (L3) and its fungal liquid culture filtrates (LCF) grown in two media showed the following highest nematocidal activities (NA): In Czapek-DoxBroth (CzDoxB) 80.66% and potato-dextrose broth (PDB) 49.84%. Additionally, two major compounds derived from carboxylic acids and two derivates from alkane group were identified by GC-MS. These compounds have been associated to many biological activities. On the other hand, the protein profile analysis by SDS-electrophoresis followed by a zymogram revealed a 10 kDa protein with protease activity. This study provides important information for future experiments focused to explore the potential use of this protein as well as the identified bioactive compounds presents in the LCF as potential candidates against sheep haemonchosis.

Nematode-Trapping Fungi Produce Diverse Metabolites during Predator-Prey Interaction.

Nematode-trapping fungi are natural antagonists of nematodes. These predatory fungi are capable of switching their lifestyle from a saprophytic to predatory stage in the presence of nematodes by developing specialized trapping devices to capture and consume nematodes. The biochemical mechanisms of such predator-prey interaction have become increasingly studied given the potential application of nematode-trapping fungi as biocontrol agents, but the involved fungal metabolites remain underexplored. Here, we report a comprehensive liquid-chromatography mass spectrometry (LC-MS) metabolomics study on one hundred wild isolates of nematode-trapping fungi in three different species, Arthrobotrys oligospora, Arthrobotrys thaumasia, and Arthrobotrys musiformis. Molecular networking analysis revealed that the fungi were capable of producing thousands of metabolites, and such chemical diversity of metabolites was notably increased as the fungi switched lifestyle to the predatory stage. Structural annotations by tandem mass spectrometry revealed that those fungal metabolites belonged to various structural families, such as peptide, siderophore, fatty alcohol, and fatty acid amide, and their production exhibited species specificity. Several small peptides (<1.5 kDa) produced by A. musiformis in the predatory stage were found, with their partial amino acid sequences resolved by the tandem mass spectra. Four fungal metabolites (desferriferrichrome, linoleyl alcohol, nonadecanamide, and citicoline) that were significantly enriched in the predatory stage were identified and validated by chemical standards, and their bioactivities against nematode prey were assessed. The availability of the metabolomics datasets will facilitate comparative studies on the metabolites of nematode-trapping fungi in the future.

Simultaneous exposure of nematophagous fungi, entomopathogenic nematodes and entomopathogenic fungi can modulate belowground insect pest control.

Entomopathogenic nematodes (EPNs) and fungi (EPF) are well known biological control agents (BCAs) against insect pests. Similarly, the nematophagous fungi (NF) are considered good BCA candidates for controlling plant parasitic nematodes. Because NF can employ EPNs as food and interact with EPF, we speculate that the simultaneous application of EPNs and EPF might result in higher insect mortality, whereas the triple species combination with NF will reduce the EPN and EPF activity by predation or inhibition. Here we evaluated single, dual (EPN + EPF, EPF + NF, EPN + NF) and triple (EPN + EPF + NF) combinations of one EPN, Steinernema feltiae (Rhabditida: Steinernematidae), one EPF, Beauveria bassiana (Hypocreales: Clavicipitaceae), and two NF, Arthrobotrys musiformis (Orbiliales: Orbiliaceae) and Purpureocillium lilacinum (Hypocreales: Ophiocordycipitaceae) under laboratory conditions. First, we showed that EPF reduced the growth rate of NF and vice versa when combined in both rich and limiting media, suggesting a negative interaction when combining both fungi. Three different fungal applications (contact with mycelia-conidia, immersion in conidial suspension, and injection of conidial suspension) were tested in single, dual and triple species combinations, evaluating Galleria mellonella (Lepidoptera: Pyralidae) larval mortality and time to kill. When mycelia was presented, the EPF appeared to be the dominant in combined treatments, whereas in immersion exposure was the EPN. In both types of exposure, NF alone did not produce any effect on larvae. However, when A. musiformis was injected, it produced larval mortalities >70% in the same time span as EPN. Overall, additive effects dominated the dual and triple combinations, with the exception of injection method, where synergisms occurred for both NF species combined with EPN + EPF. This study illustrates how differences in species combination and timing of fungal arrival can modulate the action of BCAs when augmented in the soil. Further studies are required to fine-tune these multitrophic interactions to provide successful, sustainable and resilient pest management in agroecosystems.

In vitro and in vivo studies of nematophagous fungi Arthrobotrys musiformis and Arthrobotrys robusta against the larvae of the trichostrongylides.

Six isolates of Arthrobotrys musiformis and five isolates of Arthrobotrys robusta were assessed in in vitro test regarding the capacity of prey larvae of the natural mixed trichostrongylides. In 5 isolates of A. robusta, the decrease percentage of infective larvae (L3) of trichostrongylides ranged from 97.71%-99.98% and for the isolates of A. musiformis, 5 isolates ranged from 97.99%-99.95% and only NF015 isolate 60.72%. In the following, the isolate (NPS045) of A. musiformis was selected to assess its excretion time in feces after oral administration of goats. Regarding L3 reduction rate, results demonstrated by NPS045 at each time point after fungal administration were 31.65% (12 h), 51.25% (24 h), 41.07% (48 h), 6.44% (72 h), 0% (96 h) and (120 h) (p<0.05) respectively, when compared to the control group. In the plates of the treated groups, the presence of the isolate (NPS045) was detected in samples at 12, 24 and 48 h after the fungus dose and 72 h later was not done. All native isolates of nematophagous fungi, including 6 isolates of A. musiformis and 5 isolates of A. robusta were assessed in vivo regarding the capacity of supporting the passage through goat gastrointestinal tract. The 3 isolates of A. musiformis could be able to pass through the digestive tract of goats without complete loss of ability of preying larvae of trichostrongylides in feces and their efficacies ranged from 47.60% to 55.93%. The two isolates of A. robusta survived the passage and the percentage reduction of L3 in feces were 41.96% and 66.97%, respectively. The remaining isolates were negative for both the efficacy of L3 reduction and the fungal examination in feces. In this study, the native isolates whose efficacies are good in vivo test have preliminarily demonstrated to be potential for the biological control of small ruminant parasite.

Predatory activity of Butlerius nematodes and nematophagous fungi against Haemonchus contortus infective larvae / Atividade predatória de nematóides Butlerius e fungos nematófagos contra larvas infectantes de Haemonchus contortus

Abstract The purpose of this study was to evaluate the predatory activity of the nematode Butlerius spp. and fungal isolates of Duddingtonia flagrans, Clonostachys rosea, Arthrobotrys musiformis and Trichoderma esau against H. contortus infective larvae (L3) in grass pots. Forty-eight plastic gardening pots containing 140 g of sterile soil were used. Panicum spp. grass seeds (200 mg) were sown into each pot and individually watered with 10 mL of tap water. Twelve days after seeding, the pots were randomly divided into 6 groups (n=8). Two thousand H. contortus infective larvae (L3) were added to each group. Additionally, the following treatments were established: Group 1 – 2000 Butlerius spp. larvae; group 2 – A. musiformis (1x107 conidia); group 3 – T. esau (1x107 conidia); group 4 – C. rosea (1x107 conidia), group 5 – D. flagrans (1x107conidia) and Group 6 – no biological controller (control group). The larval population of H. contortus exposed to Butlerius spp. was reduced by 61.9%. Population reductions of 90.4, 66.7, 61.9 and 85.7% were recorded in the pots containing A. musiformis, T. esau, C. rosea and D. flagrans, respectively. The results of this study indicate that the predatory nematode Butlerius spp. and the assessed fungi display an important predatory activity can be considered suitable potential biological control agents.

Resumo O objetivo deste estudo foi avaliar a atividade predatória do nematoide Butlerius spp. e isolados fúngicos de Duddingtonia flagrans, Clonostachys rosea, Arthrobotrys musiformis e Trichoderma esau contra larvas infectantes (L3) de Haemonchus contortus em vasos plantados com Panicum spp. Foram utilizados quarenta e oito potes plásticos de jardinagem contendo 140 g de solo estéril, 200 mg de sementes de Panicum spp.. Cultivar colonião, foi semeado em cada vaso e, diariamente, molhados com 10 mL de água da torneira. Doze dias após, os vasos foram divididos em 6 grupos (n = 8), e duas mil L3 de H. contortus foram adicionadas a cada vaso. Foram estabelecidos os seguintes tratamentos: Grupo 1 - 2.000 larvas de Butlerius spp.; Grupo 2 - A. musiformis (1x107 conídios); grupo 3 - T. esau (1x107 conídios); grupo 4 - C. rosea (1x107 conídios); grupo 5 - D. flagrans (1x107conidia); e Grupo 6 – somente L3 de H. contortus que serviu como controle negativo. A população de L3 de H. contortus expostas a Butlerius spp. foi reduzida em 61,9%. Redução populacional de 90,4, 66,7, 61,9 e 85,7% foram observadas nos vasos contendo A. musiformis, T. esau, C. rosea e D. flagrans, respectivamente. Os resultados deste estudo indicaram que o nematoide Butlerius spp. e os fungos avaliados exibiram importante atividade predatória e podem ser considerados como agentes de controle biológico.

Capacidad predadora in vitro de hongos nematófagos nativos de Cundinamarca sobre nematodos gastrointestinales de bovinos / Predatory Capacity in vitro of Native Nematophagous Fungi from Cundinamarca on Gastrointestinal Nematodes of Cattle / Capacidade predadora in vitro de fungos nematófagos nativos de Cundinamarca sobre nematódeos gastrointestinais de bovinos

La dependencia y el uso indiscriminado de antihelmínticos químicos como único método de control de los nematodos gastrointestinales (NGI) de bovinos ocasiona problemas en el medio ambiente, la salud pública y la productividad de la ganadería bovina. Es necesario desarrollar estrategias no químicas de control. Los hongos nematófagos pueden ser una alternativa viable y promisoria para el control de estos endoparásitos. El objetivo de este estudio fue aislar, identificar y evaluar in vitro el potencial nematófago de hongos de Cundinamarca sobre larvas L3 de NGI de bovinos. Se sembraron 60 muestras de suelo de fincas ganaderas en cajas de Petri con medio agar-agua para el atrapamiento de los hongos, y se identificaron mediante claves morfométricas tres cepas del hongo Arthrobotrys oligospora (L1, XVIII y XXI) y una de Arthrobotrys musiformis (XXIV). Se utilizaron 1 x 10(6) conidios o clamidosporas de cada hongo, los cuales se enfrentaron a 100 larvas de nematodos. El aislamiento XXIV (A. musiformis) demostró mayor capacidad predadora (96,8 %) que los aislamientos (A. oligospora) XVIII, L1 y XXI (69, 68, 71,1 y 87,62 %, respectivamente). No hubo diferencias estadísticamente significativas (p > 0,05) entre las cepas de mayor capacidad predadora. Este es el primer registro de identificación y evaluación in vitro de la capacidad predadora de los hongos A. oligospora y A. musiformis nativos de Cundinamarca. Los resultados sugieren que estos hongos podrían emplearse como agentes biocontroladores de nematodos de bovinos.

Dependence and indiscriminate use of chemical anthelmintics as the sole method for controlling gastrointestinal nematodes (GIN) of cattle causes problems in the environment, public health, and the productivity of cattle. It is important to develop non-chemical control strategies. Nematophagous fungi can be a viable and promising alternative for the control of these endoparasites. This study aimed to isolate, identify and evaluate in vitro the potential of nematophagous fungi from Cundinamarca on L3 larvae of gastrointestinal nematodes of cattle. 60 soil samples from cattle ranches were sown in Petri boxes containing agar-water for trapping fungi, and three strains of the fungus Arthrobotrys oligospora (L1, XVIII, and XXI) and one of Arthrobotrys musiformis (XXIV) were identified by morphometric keys. 1 x 10(6) conidia or chlamydospores of each fungi were used, which faced 100 nematode larvae. Isolate XXIV (A. musiformis) showed greater predatory capacity (96.8%) than isolates (A. olígospora) XVIII, LI, and XXI (69.68, 71.1, and 87.62%, respectively). There were no statistically significant differences (p > 0.05) among the strains with more predatory capacity. This is the first record of in vitro identification and evaluation of the predatory capacity of A. olígospora and A. musiformis, native fungi from Cundinamarca. The results suggest that these fungi could be used as biocontrol agents of nematodes in cattle.

A dependência e o uso indiscriminado de anti-helmínticos químicos como único método de controle dos nematódeos gastrointestinais (NGI) de bovinos ocasionam problemas no meio ambiente, a saúde pública e a produtividade da pecuária bovina. É necessário desenvolver estratégias não químicas de controle. Os fungos nematófagos podem ser uma alternativa viável e promissória para o controle destes endoparasitas. O objetivo deste estudo foi isolar; identificar e avaliar in vitro o potencial nematófago de fungos de Cundinamarca sobre larvas L3 de NGI de bovinos. Foram semeadas 60 amostras de solo de fazendas de gado em placas de Petri com ágar-água para a armadilhagem dos fungos, e se identificaram através de chaves morfométricas três cepas do fungo Arthrobotrys oligospora (L1, XVIII e XXI) e uma de Arthrobotrys musiformis (XXIV). Utilizaram-se 1 x 10(6) conidios ou clamidósporos de cada fungo, os quais se enfrentaram a 100 larvas de nematódeos. O isolamento XXIV (A. musiformis) demonstrou maior capacidade predadora (96,8 %) que os isolamentos (A. oligospora) XVIII, L1 e XXI (69, 68, 71,1 e 87,62 %, respectivamente). No houve diferenças estatisticamente significativas (p > 0,05) entre as cepas de maior capacidade predadora. Este é o primeiro registro de identificação e avaliação in vitro da capacidade predadora dos fungos A. oligospora e A. musiformis nativos de Cundinamarca. Os resultados sugerem que estes fungos poderiam empregar-se como agentes bio controladores de nematódeos de bovinos.