Morphological variability, molecular phylogeny, and biological characteristics of the nematophagous fungus Duddingtonia flagrans.

This study aims to investigate the molecular phylogenetic analysis, morphological variability, nematode-capturing ability, and other biological properties of Chinese Duddingtonia flagrans isolates. We isolated 13 isolates of D. flagrans and found features that have never been reported before, such as two to three septa incluing club-shaped conidia. Meanwhile, we conducted molecular phylogenetic analysis of the seven isolates and tested the radical growth of the isolates under different pH values, temperatures, and media. The capturing ability against infective larvae (L3) of Cooperia spp. in yak was detected in vitro. Finally, one isolate was selected for scanning electron microscopy (SEM) to investigate the trap formation process. The fungal sequence was obtained and submitted to GenBank (Accession no. KY288614.1, KU881774.1, KP257593.1, KY419119.1, MF488979.1, MF488980.1, and MF488981.1), and the tested isolates were identified as D. flagrans. Except for three isolates, the radial growth of the other isolates on 2% corn meal agar and 2% water agar exhibited faster growth than on other media. The fungus could not grow at 10 and 40°C but grew within 11 to 30°C. Moreover, it did not grow at pH 1-3 and 13-14, but instead at pH 4-12. In the in vitro experimental, L3s were reduced by 94.36%, 88.15%, and 91.04% for SDH035, DH055, and F088, respectively. SEM results showed that at 8 hr post addition of nematodes, some of the latter were captured. In the later stages of the interaction of the fungus with nematodes, a large number of chlamydospores were produced, especially on the predation trap. Results of the present study provided information about the molecular phylogenetic analysis, morphological variability, nematode-capturing ability, and other biological properties of Chinese Arthrobotrys flagrans isolates before administering them for biocontrol.

Isolation and Characterization of China Isolates of Duddingtonia flagrans, a Candidate of the Nematophagous Fungi for Biocontrol of Animal Parasitic Nematodes.

The nematophagous fungus Duddingtonia flagrans has been investigated as a biological agent for the control of gastrointestinal nematodes infecting domestic animals in other countries. However, D. flagrans has not been detected in China. In this study 1,135 samples were examined from 2012 to 2014; 4 D. flagrans isolates (SDH 035, SDH 091, SFH 089, SFG 170) were obtained from the feces of domestic animals and dung compost. The 4 isolates were then characterized morphologically. The SDH 035 strain was characterized by sequencing the ITS1-5.8S rDNA-ITS2 region. A BLAST search showed that the SDH 035 strain (GenBank KP257593) was 100% identical to Arthrobotrys flagrans (AF106520) and was identified as D. flagrans. The morphological plasticity of the isolated strain and the interaction of this strain with the nematode targets were observed by subjecting the infected trichostrongylide L3 to scanning electron microscopy. At 6 and 8 hr after trichostrongylide L(3) was added, hyphal ramifications were observed and L(3) were captured, respectively. Scanning electron micrographs were obtained at 0, 6, 12, 18, 24, 30, 36, 42, and 48 hr, where 0 is the time when trichostrongylide L(3) were first captured by the fungus. The details of the capture process by the fungus are also described. Chlamydospores were observed in the body of L(3) in the late stage of digestion. A sticky substance and bacteria could be observed in contact areas between predation structures and nematode cuticle.

Kinetics of capture and infection of infective larvae of trichostrongylides and free-living nematodes Panagrellus sp. by Duddingtonia flagrans.

Duddingtonia flagrans, a nematode-trapping fungus, has been investigated as an agent for biological control against infective larvae of gastrointestinal nematode parasites of production animals. The initial process of nematode-trapping fungi infection is based on an interaction between the trap structure of the fungus and the surface of the nematode cuticle. This report investigates by light and scanning electron microscopy the kinetics of capture and infection during the interaction of D. flagrans with the infective larvae (L(3)) of trichostrongylides and the free-living nematode Panagrellus sp. D. flagrans was cultivated for 7 days in a Petri dish containing agar-water. L(3) and Panagrellus sp. were inoculated in the Petri dishes and the samples consisting of agar-L(3)-fungi and agar-Panagrellus sp.-fungi were collected after 10, 20, 30, 40, 50, 60, and 70 min and 3, 4, 5, 10, 15, 20, and 25 h of interaction. All samples were observed by light microscopy. The samples with 1, 5, 15, and 25 h of interaction were also analyzed by scanning electron microscopy. The interaction was monitored up to 25 h. An initial differentiation of predation structures was observed after 30 min of interaction. The presence of traps and of captured L(3) or Panagrellus sp. occurred after 70 min. The live captured nematodes were observed up to 3 h of interaction. However, after 4 h, all Panagrellus sp. were dead. It took 15 h of interaction for the fungus to invade the L(3), and the presence of hyphae inside the nematode near the region of penetration was evident. At this time, the hyphae had filled the whole body of Panagrellus sp. The complete occupation of the body of L(3) occurred at 20 h of interaction and with 25 h the nematode was completely damaged except for the cuticle. Although the double cuticle of L(3) slows the penetration of D. flagrans, it was possible to verify that the process of trap formation and capture occurs quickly when both nematodes were tested, suggesting that the organisms would eventually be killed once in contact with the fungi encouraging the use of the fungus as a biological control agent.