E200mv

L-lysine-producing E. coli QDW, stored in glycerol, was streaked on an LB solid medium plate and cultured at 37 °C overnight in a constant temperature incubator. Single colonies were picked and transferred to an LB liquid medium in erlenmeyer flasks. The mixture was shaken at 37 °C for 24 h, and the bacterial concentration was recorded every 2 h. Finally, the growth curve of L-lysine-producing QDW was evaluated. In the middle and late period of the logarithmic growth phase of the bacteria, 10 mL of bacterial solution was taken and centrifuged at 2683.2 (× g) for 10 min at 4 °C. The bacteria were collected, and the SMM protoplast stable solution (pH = 6.5, 0.5 mol/L sucrose, 20 mmol/L anhydrous magnesium chloride, 0.02 mol/L maleic acid) was taken and centrifuged twice. The bacteria were suspended in 10 mL of SMM protoplast stable solution. The lysozyme solution was added and mixed well. The cells were incubated in a water bath at 37 °C, and the wall was broken by enzymatic hydrolysis. The separation process of protoplasts at 37 °C was observed under a microscope oil lens (E200MV, Nikon., Nanjing, China). Finally, the bacterial solution after enzymatic hydrolysis was centrifuged at 1509.3 (× g) for 15 min and washed once with SMM buffer; the protoplasts were then collected. The prepared protoplasts were stored in a refrigerator at 4 °C.

The inhibitory activities of Streptomyces extracts on spore germination of phytopathogenic fungi were observed using light microscopy (Nikon, E200MV, Japan). Each fungal strain was challenged in dose response to different extract concentrations (1/2 × EC₅₀, 1 × EC₅₀, or 2 × EC₅₀). The mixture (0.1 mL) of extracts and fungal spore suspension (10⁵ CFU/mL) at a ratio of 1:1 (v/v) was placed on a sterile glass slide and incubated in a moist chamber at 28°C for 20–24 h. For the control, we replaced extracts with sterile water. All experiments were performed in three replicates. We detected approximately 100 conidia in each field. The percentage of spore germination (PSG) was calculated as follows: PSG = (A – B)/A, where A and B represented the spore germination rate of control and treatment groups, respectively (Chen et al., 2018 (link)).

Conventional morphometric analysis was performed with the reticle of 100 points and 50 straight coupled to the microscope eyepiece (E200MV, Nikon Corporation, Tokyo, Japan) (Weibel, 2010 (link)). The total area of the reticulum was 10⁴ μm², which was fixed at the base of the epithelium and the positive cells were quantified. The positive cells at 1,000× magnification were counted in four airway fields, in at least three airways, and 10 random fields in the alveolar septa per animal. Based on the number of points that coincided with the positive cells within the lattice divided by the number of points coincident with the peribronchial area for the airways or alveolar walls (Leick-Maldonado et al., 2004 (link); Camargo et al., 2018 (link)).