The effects of different I2-KI solutions and times on the staining results
As shown in Fig. 1, as the treatment duration and concentration increased, the blue stain gradually became darker. The 0.1% (a) and 0.25% (b) I2-KI solutions did not complete their staining in any of the tested treatment times (Fig. 1A–F). The 0.75% (d) and 1.0% (e) I2-KI solutions resulted in an uneven dark or light color under long or short treatment times, respectively. The banana fruit slices stained with 0.5% I2-KI solution for 150 s obtained a more uniform staining effect that allowed for easier identification.
The effects of different ratios of pTRV2-MaBAM9b with pTRV1 on the silencing results
As shown in Fig. 2A, when the ratio of pTRV2-MaBAM9b with pTRV1 reached 1:2, the blue stain of I2-KI became darker. The color became darkest at a ratio of 1:3 compared to the control (CK). This result indicated that ratios of 1:2 and 1:3 could efficiently suppress starch degradation. Further investigation found that the contents of soluble sugars, including glucose, fructose, and sucrose, were greatly decreased by 62.8%, 61.9%, and 62.6% compared to CK, respectively, at a ratio 1:3 (Fig. 2B). The β-amylase activities were also significantly decreased compared to CK, decreasing by 18.7%, 62.7%, and 76.7% at ratios of 1:1, 1:2, and 1:3, respectively (Fig. 2C). In contrast, the total starch contents at ratios of 1:1, 1:2, and 1:3 were 339.7, 398.8, and 436.2, indicating a 2.7%, 20.5%, and 31.9% increase compared to CK, respectively (Fig. 2D). The expression of endogenous MaBAM9b was greatly suppressed, decreasing by 28.0%, 42.0%, and 63.0% at ratios of 1:1, 1:2, and 1:3, respectively. These results indicated that the ratio of pTRV2-MaBAM9b together with pTRV1 at 1:3 obtained the best silencing efficiency.
The effects of different bacterial fluid OD values on the silencing results
As shown in Fig. 3A, with the increase in OD600 value of the bacterial fluid from 0.4 to 0.6, the I2-KI staining gradually became darker compared to CK. When the OD600 value reached 0.8, the stain was darkest. However, when the OD600 value increased to 1.0 or 1.2, the staining became weaker. This result demonstrated that an OD600 of 0.8 obtained the best results for suppressing starch degradation. Further investigation indicated that the total starch contents at OD600 values of 0.4, 0.6, 0.8, 1.0, and 1.2 were 235.7, 264.5, 326.0, 303.1, and 278.4, which were increased by 6.9%, 19.7%, 47.6%, 37.2%, and 26.0% relative to CK, respectively (Fig. 3B). Conversely, the β-amylase activities at OD600 values of 0.4, 0.6, 0.8, 1.0, and 1.2 were significantly decreased compared to CK, decreasing by 9.2%, 15.3%, 27.6%, 22.4%, and 9.2%, respectively (Fig. 3C). The contents of soluble sugars, including glucose, fructose, and sucrose, were also greatly reduced by 67.2%, 67.8%, and 74.8% compared to CK, respectively, at OD600 0.8 (Fig. 3D). The expression of endogenous MaBAM9b was greatly suppressed, decreasing by 29.0%, 49.0%, 79.0%, 68.0%, and 52.0%, respectively. These results suggested that an OD600 value of 0.8 obtained the best silencing efficiency.
The effects of different co-cultivation times on the silencing results
As shown in Fig. 4A, as the co-cultivation time progressed, the blue stain of I2-KI became gradually darker. When the co-cultivation time reached 5 d, the color was the darkest. However, when the co-cultivation time increased to 7 d, the staining became weaker. This result demonstrated that co-cultivation for 5 d was best for suppressing starch degradation. Further investigation found that the total starch contents at 3 d, 5 d, and 7 d were 198.4, 315.7, and 301.4, increasing by 12.5%, 79.1%, and 71.0% compared to 1 d, respectively (Fig. 4B). Conversely, the β-amylase activities at co-cultivation times of 3 d, 5 d, and 7 d were significantly reduced compared to 1 d, decreasing by 10.7%, 29.8%, and 27.4%, respectively (Fig. 4C). The contents of glucose, fructose, and sucrose also decreased greatly by 60.5%, 53.8%, and 64.1% at an co-cultivation time of 5 d, respectively (Fig. 4D). The expression of endogenous MaBAM9b was greatly suppressed by 26.0%, 58.0%, and 42.0%, respectively. These results suggested that 5 d of co-cultivation obtained the best silencing efficiency.