Abstract
Hirsutella rhossiliensis and H. minnesotensis are endoparasitic fungi of the second-stage juvenile (J2) of the soybean cyst nematode (Heterodera glycines) in nature. They also parasitize both H. glycines J2 and Caenorhabditis elegans on agar plates. Agrobacterium tumefaciens-mediated transformation conditions were established for these Hirsutella spp. The resulting transformants were similar to the corresponding wild-type strains. The infection processes of H. glycines J2 and C. elegans second larval stage (L2) by H. minnesotensis expressing ZsGreen were microscopically analyzed. Conidia of H. minnesotensis adhered to passing nematodes within 8 h post-inoculation (hpi), formed an infection peg between 8 and 12 hpi, and penetrated the nematode cuticle between 12 and 24 hpi for C. elegans L2 and between 12 and 32 hpi for H. glycines J2. Hyphal proliferation inside of the nematode coelom was observed at approximately 32 hpi for C. elegans L2 and at approximately 40 hpi for H. glycines J2. The fungus consumed the whole body and grew out to produce conidia at approximately 156 and 204 hpi for C. elegans L2 and H. glycines J2, respectively. The efficient transformation protocol and a better understanding of infection process provide a solid foundation for studying the molecular and cellular mechanisms underlying fungal parasitism of nematodes.
Highlights
Rotylenchus and soil mites[11,12]
Enhanced understanding of the infection process of H. glycines by H. minnesotensis is crucial to exploit this fungus as an effective biocontrol agent and to study the molecular mechanisms underlying its interaction with nematodes
The minimum concentrations of hygromycin B and geneticin resulting in complete growth inhibition were 150 μ g/ml and 400 μ g/ml, respectively, for H. minnesotensis AS3.9869 and 100 μ g/ml and 400 μ g/ml, respectively, for H. rhossiliensis AS6.0004
Summary
Rotylenchus and soil mites[11,12]. H. minnesotensis has been found in the U.S, Germany, Poland and China[12,13], and it infects diverse nematodes in the genera Aphelenchoides, Heterodera, Mesocriconema, Belonolaimus, Hoplolaimus, Steinernema, and Heterorhabditis[12,14] and Collembola[15]. The parasitism of bacteria-feeding nematodes, such as C. elegans by H. minnesotensis has not been detected in nature, the successful infection of C. elegans, by this fungus at different larval stages on agar plates has been documented[22], suggesting that this model nematode can potentially help enhance the current understanding of the molecular and cellular mechanisms underlying H. minnesotensis–nematode interactions. To realize this potential, it is critical to develop genetic manipulation tools for H. minnesotensis. One H. minnesotensis transformant was used to investigate the manner by which it infects H. glycines J2s and C. elegans L2s using fluorescence microscopy
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