Stero discovery v20

Isolates were cultured on PDA for 7 days at 25 °C (±1 °C) to observe the colony morphology [35 (link)]. Micromorphological features were observed from those cultured on synthetic nutrient-poor agar plates (SNA) [36 ]. The characteristics of sporulation formation, including the length of conidial chains, branching patterns of conidial chains and presence of secondary conidiophores, were captured with a Zeiss stereo microscope (SteRo Discovery v20) [35 (link)]. A ZEISS Axio Imager A2m microscope (Carl Zeiss, Göttingen, Germany) equipped with differential interference contrast (DIC) optics was used to capture conidial chains and conidia. Fifty mature conidia mounted in sterile water were measured at random under a light microscope at ×100 magnification.

One representative isolate was randomly selected from each Alternaria species for morphological research according to the method of Simmons (2007) . Mycelial plugs (5 mm) of purified cultures were transferred from the growing edge of 5-d-old cultures to the centre of 7-mm-diameter potato carrot agar (PCA) plates (Crous et al. 2009b ) in triplicate at 25 °C. Colony diameters were measured from 3 to 6 days to calculate mycelial growth rates (mm/d). Colony colour, size and density were also recorded. The morphology and size of conidial chains were studied and recorded using a Zeiss stereo microscope (SteRo Discovery v.20). The shape, colour and size of conidiophores and conidia were observed using a ZEISS Axio Imager A2m microscope (ZEISS, Germany) with differential interference contrast (DIC) optics. At least 30 measurements per structure were performed using Carl Zeiss Axio Vision software to determine their sizes, unless no or fewer individual structures were produced.

The isolates were plated in the center of potato dextrose agar medium (PDA), Czapek Dox agar (CZA), minimal methanol medium (MM), and complete medium (CM) and incubated at 25 °C with a 12/12 h light/dark cycle. Colony characteristics of the cultures were observed after 3 days. Acervuli and conidial masses were observed under a Zeiss stereo microscope (SteRo Discovery v20, Oberkochen, Germany). The morphological characteristics, such as shape, color, septation, and size of 30 conidia, conidiophores, acervuli, appressoria, and setae, were observed and measured under a Zeiss Axio Imager A2m microscope (Carl Zeiss, Oberkochen, Germany), respectively. Appressoria of the isolate were induced from conidia using a slide culture technique [26 ,55 (link)].
To determine the optimal growth temperature for the isolates, mycelial plugs (5 mm diam.) were placed on fresh PDA and incubated at 15, 20, 25, 30, and 35 °C, and colony diameters were recorded daily. Experiments were conducted at five temperatures, with each isolate having three replicates. The experiment was conducted two times.