DOI: 10.5061/dryad.b2rbnzsfr
title: Effects of inoculation concentration, photoperiod and temperature on growth, conidiation and conidial germination of Bionectria ochroleuca

description: 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.