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Effects of inoculation concentration, photoperiod and temperature on growth, conidiation and conidial germination of Bionectria ochroleuca

Citation

Zheng, Yi; Xie, Yichun; Xie, Yan; Yu, Shixiao (2022), Effects of inoculation concentration, photoperiod and temperature on growth, conidiation and conidial germination of Bionectria ochroleuca, Dryad, Dataset, https://doi.org/10.5061/dryad.b2rbnzsfr

Abstract

The short-term stock of Bionectria ochroleuca ochroleuca at 4°C was activated, sub-cultured, and grown at 25°C on PDA medium for 10 days to obtain a working culture. To determine the effect of temperature on fungal growth, the plates were incubated at 15, 20, 25, and 30°C under continuous darkness or an 8 h light/16 h dark regime for 10 days. To investigate the effect of photoperiod on fungal growth and reproduction, fungal cultures were incubated at 25°C with 0, 8, 16, and 24 h light exposure per day for 10 days. The cultures were exposed to full light induced by fluorescent lamps with 800–1200 lx. Mycelial growth was measured as the mean of two randomly selected orthogonal diameters of the colony on day 10 with a ruler. The colonies were in the shape of near-perfect circles, with differences between two diameters less than 1%. The conidia were then washed out using a spreader with 10 ml ddH2O. A spore suspension was transferred to sample tubes with a pipette and quantified using a hemacytometer. To investigate the effect of nutrients, temperature, and light on conidial germination, freshly grown conidia were collected 5 days post-inoculation from colonies grown on PDA. Conidia were washed off PDA with ddH2O using a spreader, and the spore suspension was adjusted  to approximately 107 spores mL-1 for the following tests. To test the impact of nutrient concentration on conidial germination, the conidia suspension was mixed with different concentrations of potato dextrose broth (PDB) to achieve a final nutrient concentration of 0, 0.1, 1, 2, and 5% and was then incubated at 25°C under continuous darkness. To determine the optimal temperature for conidial germination, conidia were incubated in 1% PDB at 4, 15, 20, 25, and 30°C under continuous darkness. To investigate the effect of light on conidial germination, conidia were cultured in 1% PDB at 25°C under continuous light or darkness. All assays had a final spore concentration of 5 × 106 conidia/mL. All the above incubation treatments were performed by placing 25 mL of the freshly mixed spore-PDB/H2O suspension onto each slide and placing the slides in a Petri dish with a moistened filter paper. Conidial germination was examined under a light microscope (400×) 3, 6, 9, 12, 24, and 36 h after incubation. These assays were performed on three technical replicates, and five fields were examined for each slide. When over 85% of the conidia in the observation field on a slide germinated, measurements on that slide were terminated. A conidium was considered germinated when the germination tube exceeded one half of the largest dimension of the conidium. In order to confirm that inoculation concentration does not have strong influence on mycelial growth and conidiation, we inoculated the PDA plates with 0.5 μl spore suspension of two different concentrations (105 μl-1 and 104 μl-1) and incubated them in 25 ℃ with full-time light or darkness for 10 days. We compared colony sizes and the number of conidia (log transformed) between the groups of different inoculation concentrations, separately for the full-light group and full-darkness group, and vice versa.

Funding

National Natural Science Foundation of China, Award: 31830010

National Key Research and Development Program of China, Award: 2017YFA0605100