Optimization of the Fermentation Media and Parameters for the Bio-control Potential of Trichoderma longibrachiatum T6 Against Nematodes

Abstract

The cereal cyst nematode Heterodera avenae is one of the important soil-borne pathogens of cereal crops and causes high yield losses worldwide. Trichoderma spp. formulations are applied as commercial bio-control agents against soil-borne plant pathogens such as H. avenae. However, the relationship between Trichoderma longibrachiatum fermentation parameters and its bio-control potential against H. avenae has not been exclusively established. In the present study, the effect of 10 different fermentation media and conditions on the nematicidal activity of T. longibrachiatum T6 (T6) was evaluated with a single-factor method and a Plackett-Burman design, and the interaction between different fermentation parameters was investigated by a Box-Behnken design. The variables for enhancing the nematicidal activity of T6 culture filtrates were explored and optimized using response surface methodology (RSM). The Minor Medium (MM) plus wheat bran-2 medium was found to be the most effective fermentation medium for T6 culture filtrates against the second stage juveniles (J2s) of H. avenae. The maximum mortality of the J2s was obtained using the T6 culture filtrates under the following fermentation conditions: initial pH 6, 28°C culture temperature, 180 rpm rotating speed, 60 ml of fermentation media, 7 days of incubation time, and 1 ml of inoculation volumes. Among these parameters, the initial pH, inoculation volume, and incubation day were identified as the most significant parameters and critical independent variables for enhancing the nematicidal activity of the T6 culture filtrates. After further optimizations based on statistical predictions, the highest nematicidal activity (92.42%) was obtained with the T6 culture filtrates fermented under an initial pH of 6.06, an inoculation volume of 1.62 ml, and an incubation time of 7.15 days. The nematicidal activity was increased approximately by as high as 1.07% compared with that before optimization. Bio-control efficacy of T6 culture filtrates was 83.88% at the 70^th day after wheat seeds sowing in greenhouse experiments. The results from the validation experiments agreed with the model predictions. Our study has improved the bio-control potential of Trichoderma spp. against the plant-parasitic nematodes H. avenae and provided a cost-efficient bio-resource in the future development of novel bio-control agents.

Keywords: Box–Behnken design; Heterodera avenae; Plackett–Burman design; Trichoderma species; fermentation media and conditions; nematicidal activity; response surface methodology.

FIGURE 1

Effect of different fermentation parameters on the nematicidal activity of Trichoderma longibrachiatum T6 culture filtrates against H. avenae. The mortalities are shown under the parameters of (i) initial pH, (ii) incubation temperature, (iii) inoculation volume, (iv) liquid volume, (v) rotating speed, and (vi) incubation day. T represents T6 culture filtrate treatment. PC represents the J2s that were treated with the liquid fermentation medium but not with T. longibrachiatum T6 (positive control). NC represents the J2s that were treated with sterilized water (negative control). The keys are T-24 h, T-48 h, and T-72 h for T6 culture filtrate treatments, and PC-24 h, PC-48 h, and PC-72 h for the positive controls at 24 h, 48 h, and 72 h after the treatments, respectively. The data are grouped by the parameter, and there are six groups (i–vi). The statistical analysis was applied in each group on the mortality for each treatment (T-24 h, PC-24 h, T-48 h, PC-48 h, T-72 h, and PC-72 h). Data are means of six replicates ± standard error. The line bars represent the standard errors of the means. Different letters denote significant difference at p < 0.05 based on Duncan’s new multiple range tests using one-way ANOVA for a range of each fermentation parameter.

FIGURE 2

The mortality response to the effects of residuals, absolute standardized effect, and standardized effect on the nematicidal activity of Trichoderma longibrachiatum T6 against H. avenae as presented with (A) normal plot, (B) half normal plot, and (C) pareto chart presentation, with (i) initial pH, (ii) incubation temperature, (iii) inoculation volume, (iv) liquid volume, (v) rotating speed, and (vi) incubation day.

FIGURE 3

The mortality response to the interactive effects of pHs and incubation days on the nematicidal activity of Trichoderma longibrachiatum T6 against H. avenae as presented with (A) contour plots and (B) the 3D response surface presentation.

FIGURE 4

The mortality response to the interactive effects of inoculation volumes and incubation days on the nematicidal activity of Trichoderma longibrachiatum T6 against H. avenae as presented with (A) contour plots and (B) the 3D response surface presentation.

FIGURE 5

The mortality response to the interactive effects of pHs and inoculation volumes on the nematicidal activity of Trichoderma longibrachiatum T6 against H. avenae as presented with (A) contour plots and (B) the 3D response surface presentation.