68% NaCl containing 0.05% Tween 80. The fish in this experiment were artificially grazed by one end of a wire netting in advance (this process was done by the same person).

Three groups of these wounded yellow catfish were then immersed in FM07 suspension containing 106, 107 and 108 CFU mL−1, respectively, for 2 days and one group of fish used as control was immersed in oxygenated water. Three groups of yellow catfish were immersed in FM07 suspension containing 106, 107 and 108 CFU mL−1, respectively, and one group of fish used as control was immersed in oxygenated water. All the fish were healthy and no AZD2281 chemical structure wounds were found. These experimental infections lasted 8 weeks. Mortalities were recorded during the experimental infections. All fish were examined for gross pathological changes. Any dead or moribund fish were checked for the presence of the fungal pathogen. Live and moribund fish were killed with an overdose of tricaine methanesulfonate (MS-222). The tissues of the diseased fish from Niushan Lake and those with artificial infection were excised and fixed in Bouin’s fluid. Samples from healthy yellow catfish were also fixed in Bouin’s fluid as control. Part of the musculature was decalcified with formic acid–sodium citrate

solution. All tissues were dehydrated with ethanol, embedded in paraffin wax, and blocks sectioned at 6 μm with a rotary microtome. Slides were stained with Harris’ hematoxylin and eosin. The stained sections on the slide were covered with Canada balsam and photographed under a microscope (Zeiss Axioplan 2 imaging and Axiophot see more Dehydratase 2). The hyphae in the necrotic tissue were nonseptate, broad and branched. The color of the pure culture was white initially and soon became grayish brown. Microscopic examination revealed globose sporangia, measuring 30–78 μm in diameter (Fig. 1a). Columellae were subglobose to obovoid and collarettes were conspicuous

(Fig. 1b). Sporangiophores were either long and branched sympodially or shorter with slightly recurved lateral branches. Sporangiospores were hyaline, ellipsoidal to slightly asymmetrical or obovoidal and measured 5.0–7.0 μm in length and 3.2–5.5 μm in width. The sporangiospore walls were finely ornamented (Fig. 1c). Chlamydospores were produced in the basal mycelium, which were thick-walled, subglobose, oval or irregularly shaped, measuring 45 μm in length and 30 μm in width (Fig. 1d). Rhizoids and stolons were absent. The optimum growth temperature was 30 °C and there was no growth at 40 °C. There were no zygospores produced in test mating and this procedure was repeated with the same results. A 638-bp ITS rRNA gene fragment was amplified from the fungi and was deposited in GenBank under the accession number GQ415044. Compared with the sequences of ITS rRNA gene available in GenBank, the amplified nucleotide sequence showed 100% homology with the ITS rRNA gene sequence of M. circinelloides (accession number EF583641) (Fig. 2).