Biological Characteristics of Beauveria majiangensis Strain MJ1015 and Optimization of Solid Medium Technology for Sporulation

B. majiangensis strain MJ1015 was isolated from the larva of H. scrobiculata on a blueberry farm in Guizhou, China (Chen et al. 2018), and is stored in the Species Preservation Room at Guizhou Institute of Biology (preservation number CGMCC No. 15090).

Aerial conidia of MJ1015 were obtained from a stock culture growing on a potato dextrose agar (PDA) plate by suspending them in sterile water containing 0.05% Tween-80. The number of conidia in the suspension was counted using a hemocytometer (Yue Cheng Trading Co., Ltd., China) and then diluted to 1.0 × 10¹⁰ conidia/l with 0.05% Tween 80 for use in subsequent experiments.

Media plates were inoculated in the center with 1 μl of spore suspension using a pipette and then incubated in the dark in a climate chamber with 90 ± 5% relative humidity for 14 days. The colony growth rate and spore production were measured.

To assess the effects of different media on colony growth rate and sporulation, four different media were inoculated with MJ1015 spore suspension: Sabouraud dextrose agar with yeast extract (SDAY), which comprised 10 g/l of tryptone, 40 g/l of dextrose, 20 g/l of agar, 10 g/l of yeast extract, and, with a pH 6.0 ± 0.1; PDA medium; ¼ Sabouraud dextrose agar with yeast extract (¼SDAY, 2.5 g/l of tryptone, 10 g/l of dextrose, 20 g/l of agar, 5 g/l of yeast extract); and base medium (CZM), which comprised 30 g/l of dextrose, 2 g/l NaNO₃, 0.5 g/l MgSO₄·7H₂O, 1 g/l K₂HPO₄, 0.5 g/l KCl, 0.001 g/l FeSO₄ · 7H₂O, 20 g/l of agar. The inoculated plates were cultivated in a climate chamber at 26.5 ± 1°C, with at least three replications of each treatment.

To assess the effects of different temperatures on colony growth rate and sporulation, plates of PDA medium were inoculated with spore suspension and then incubated in the climate chamber at 20 ± 1°C, 23 ± 1°C, 26 ± 1°C, 29 ± 1°C, or 30 ± 1°C, with at least three replications of each temperature treatment.

To assess the effects of different pH values on colony growth rate and sporulation, plates of PDA medium were prepared with pH values adjusted to pH 4, 5, 6, 7, 9, 11, or 13 with 0.1 mol/l HCl or 0.1 mol/l NaOH under sterile conditions. The PDA plates were inoculated with spore suspension and cultured at 29°C, with at least three replications of each pH value.

Based on the results of the single-factor screening experiments, we used a Plackett-Burman design (N = 12) to assess five factors: two carbon sources, a nitrogen source, and two inorganic salts. Three dummy variables were used to estimate the error. For each factor, two levels were assessed: high (+1) and low (-1) (Table I). The high level was 1.25 times that of the low level. Colony growth rate and sporulation measurements were taken as response value Y. The design code and the coding values are shown in Table I. The culture conditions and the number of replicates were the same as those used in the carbon source screening experiment.

Based on the main factors promoting sporulation identified by the Plackett-Burman method, the steepest ascent method was designed using rice, NaNO₃, and K₂HPO₄ · 3H₂O. The step size and the direction of each significant factor were determined by the coefficient of sporulation regression equation of the Plackett-Burman design model. The culture conditions and the number of replicates were the same as those used in the carbon source screening experiment.

Based on our analysis of the significance factors and levels obtained using the Plackett-Burman test and steepest ascent method, we performed a response surface analysis experiment comprising three factors and five levels on a solid medium, which was designed using a central combination experimental design. The design is shown in Table II. The culture conditions and the number of replicates were the same as those used in the carbon source screening experiment.

Colony growth rate (mm/d) and sporulation (conidia/l) data are presented as means ± the standard error (SE) and were statistically analyzed by performing a one-way analysis of variance (ANOVA) or Welch’s ANOVA using SPSS 21.0. SigmaPlot 14 software was used to plot the experimental results. Calculation of growth rate – colony growth diameter was measured with vernier caliper crossovers after 14 days of culture and recorded: Colonygrowthrate(mmd)= Colonygrowthdiameter (mm) culturetime (d)$$Colony{\rm{ }}growth{\rm{ }}rate{\rm{}}\left( {{{mm} \over d}} \right) = {{Colony{\rm{ }}growth{\rm{ }}diameter(mm)} \over {culture{\rm{ }}time(d)}}$$

Calculation of spore number – the sterilized hole punch of 1 cm² was used to take bacteria cakes from the center of the colony ½ distance from the edge, and three bacteria cakes were taken from each treatment. The cakes were placed in 20 ml sterile water containing 0.05% Tween-80 and fully oscillated and mixed on the vortex oscillator to make spore suspension.

The spore concentration was measured with the blood cell counting plate and the number of spores (spores/l) was calculated: Sporenumber l=N5×25×10×106×dilutionratio$${{Spore{\rm{ }}number} \over l} = {N \over 5} \times 25 \times 10 \times {10^6} \times {\rm{}}dilution{\rm{ }}ratio$$ where N is the total number of spores in the 5 squares in the middle.

The colony growth rate of strain MJ1015 on PDA was significantly faster (3.37 mm/d; Fig. 1a), and sporulation was significantly higher (7.73 × 10¹⁰ conidia/l; Fig. 1b) than on the other three media. Although there was no significant difference in the colony growth rate on SDAY and ¼ SDAY, sporulation was significantly higher on SDAY than on ¼ SDAY. The colony growth rate on CZM was significantly slower (2.84 mm/d), and sporulation was significantly lower (4.80 × 10⁹ conidia/l) than on the other media.

Fig. 1.

Colony growth and sporulation were observed under all the different temperature treatments (i.e., from 20°C to 30°C; Fig. 1c). The growth rate increased with increasing temperature up to 29°C. The optimal temperature for mycelial growth was 29°C and spore production was significantly higher than at 23°C, 26°C, or 30°C, but was not significantly different from that at 20°C (Fig. 1d). Moreover, the lowest level of sporulation occurred at 30°C, and the growth rate was significantly lower than at 26°C or 29°C.

Colony growth and sporulation were observed under all the different pH treatments (i.e., from pH 4 to 13; Fig. 1e and 1f). However, the optimal pH for mycelial growth (3.23 mm/d) and sporulation (1.32 × 10⁹ conidia/l) was pH 5.

When we replaced the 40 g/l of dextrose in the SDAY medium with one of 13 different carbon sources, the colony growth rate of strain MJ1015 was either not significantly different or was better than that on SDAY (Fig. 2a and 2b). It appears that the growth rate on a 1:1 ratio of rice and wheat (3.26 ± 0.03 mm/d) was not significantly different from that of corncob, bran flour, wheat, rice straw powder, chaff, rice, and potato. Moreover, a 1 : 1 ratio of rice and wheat was the optimal carbon source for promoting sporulation ((4.11 ± 0.22) × 10⁹ conidia/l), significantly higher than that produced on media with other carbon sources.

Fig. 2.

To determine the optimal proportion of rice and wheat in the medium for colony growth and sporulation, we assessed 11 different rice: wheat ratios (Fig. 2c and 2d). A significantly higher growth rate (3.02 ± 0.03 mm/d) and greater sporulation ((1.66 ± 0.02) × 10¹⁰ conidia/l) were achieved on medium with rice: wheat ratio of 2 : 1 compared with the other treatments.

When we replaced the yeast extract powder and tryptone in the SDAY medium with one of 11 different nitrogen sources, the colony growth rate of strain MJ1015 was significantly better than that on SDAY (Fig. 2e and 2f). When strain MJ1015 was grown on media containing NaNO₃, the mycelial growth rate (3.81 ± 0.04 mm/d) was significantly faster, and sporulation was significantly higher ((25.83 ± 0.80) × 10⁹ conidia/l) than on other media, suggesting that NaNO₃ was the optimal nitrogen source for the growth and sporulation of strain MJ1015.

When strain MJ1015 was grown on media containing rice: wheat ratio of 2 : 1, NaNO₃, and either 0.10% K₂HPO₄ · 3H₂O or CaCO₃, the average mycelial growth rate (4.34 ± 0.03 mm/d or 4.12 ± 0.04 mm/d, respectively) was significantly higher than that on other media (Fig. 2g and 2h). In addition, significantly higher sporulation (41.01 ± 1.56) × 10⁹ conidia/l) was achieved on media containing CaCO₃ than on other media.

Based on the test results shown in Table III, multiple regression equation fitting and ANOVA of sporulation by MJ1015 colonies were performed using Design-Expert 12 software (Table IV). The regression equation for colony sporulation was: Y(ln( sporulation ))=22.64+0.7365×X1+0.0313×X2−−0.4789×X4+0.0448×X5−0.4468×X7$$\matrix{ {{Y_{(\ln ({\rm{sporulation}}))}}} \hfill & = \hfill & {22.64 + 0.7365 \times {X_1} + 0.0313 \times {X_2} - } \hfill \cr {} \hfill & - \hfill & {0.4789 \times {X_4} + 0.0448 \times {X_5} - 0.4468 \times {X_7}} \hfill \cr } $$

Regression analysis showed that the Plackett-Burman model (Table IV) was highly significant (p < 0.01), indicating that the model could be used to analyze significant factors of B. majiangensis MJ1015 sporulation. The results of the regression analysis (Table IV) showed that rice was positively correlated with sporulation (p < 0.01), increasing its content promoted the sporulation of strain MJ1015, whereas NaNO₃ and K₂HPO₄ · 3H₂O were negatively correlated with sporulation (p < 0.05). Increasing the NaNO₃ and K₂HPO₄ · 3H₂O content of solid media could inhibit sporulation. However, the other factors were not significant (Table IV). Because the added reagents did not affect the colony growth rate in our laboratory, the colony growth rate was not analyzed here or in sub-sequent experiments.

Steepest ascent tests of rice, NaNO₃, and K₂HPO₄ · 3H₂O were carried out with sporulation as the main factor from Plackett-Burman result. Based on the coefficient of the sporulation regression equation of the Plackett-Burman design model, the step size and direction of each significant factor were determined, as shown in Table V. Colony spore production peaked at the “origin + 10Δ” factor level and then decreased. The response interval of the maximum value was around factor level 10, which was selected as the center point.

According to the Plackett-Burman test results and steepest ascent path, a three-factor and five-level response surface analysis was conducted on rice, NaNO₃ and K₂HPO₄ · 3H₂O using a central combination design. The central combination design and its levels are shown in Table II, and the experimental results are shown in Table VI.

Multiple regression equation fitting and ANOVA with Table VI test data using Design-Expert 12 software yielded the regression equation: Y(Sporulation (conidia/l)) =(4.508+0.246×A−0.303×B−−0.093×C−0.045×AB−0.130×AC−−0.020×BC−0.769×A2−0.964×B2− −0.700×C2 )×1010$$\matrix{ {{Y_{({\rm{Sporulation (conidia/l))}}}}} \hfill & { = (4.508 + 0.246 \times A - 0.303 \times B - } \hfill \cr {} \hfill & { - 0.093 \times C - 0.045 \times AB - 0.130 \times AC - } \hfill \cr {} \hfill & { - 0.020 \times BC - 0.769 \times {A^2} - 0.964 \times {B^2} - } \hfill \cr {} \hfill & {\left. { - 0.700 \times {C^2}} \right) \times {{10}^{10}}} \hfill \cr } $$

The regression equation results (Table VII) revealed that the regression model has relevance (p < 0.0001) and that the model has a high goodness of fit value (R² = 0.9348), indicating that the model is a good reflection of the relationship between the investigated factors and response values. The colony spore yield lack of fit was not significant (p = 0.6030), indicating that the model did not lose fit; that is, the modified model could be used for the theoretical prediction of solid spore yield optimization of strain MJ1015. According to the response surface diagrams showing the effects of rice (A), NaNO₃ (B), and K₂HPO₄ · 3H₂O (C), on sporulation (Fig. 3), A, B, and C have maximum values.

Fig. 3.

In summary, based on changes in sporulation with nutrient composition, combined with response surface optimization experiments, Design-Expert 12 software was used to analyze spore production as the primary response value and indicated that the optimal medium conditions for MJ1015 sporulation were 64.70 g/l of rice, 0.30 g/l of NaNO₃, and 0.36 g/l of K₂HPO₄ · 3H₂O. The spore yield of strain MJ1015 when grown on the optimal medium, was predicted to be 4.56 × 10¹⁰ conidia/l.

The average growth rate of strain MJ1015 on the optimized solid phase medium was (4.46 ± 0.04) mm/d, 85% and 96% faster than that on SDAY and PDA, respectively. Furthermore, the sporulation of strain MJ1015 ((4.54 ± 0.16) × 10¹⁰ conidia/l) was 43.90 times and 9.65 times that on SDAY and PDA, respectively.