Pine wilt disease (PWD) caused by pine wood nematode (PWN, Bursaphelenchus xylophilus) originated in North America and has since spread to Asia and Europe. PWN is currently a quarantine object in 52 countries. In recent years, pine wilt disease has caused considerable economic losses to the pine forest production industry in China, as it is difficult to control. Thus, one of the key strategies for controlling pine wilt disease is to identify epidemic points as early as possible. The use of hyperspectral cameras mounted on drones is expected to enable PWD monitoring over large areas of forest, and hyperspectral images can reflect different stages of PWD. The trend of applying hyperspectral techniques to the monitoring of pine wilt disease is analyzed, and the corresponding strategies to address the existing technical problems are proposed, such as data collection of early warning stages, needs of using unmanned aerial vehicles (UAVs), and establishment of models after preprocessing.
ObjectiveTo generate recombinant Bacillus subtilis (B. subtilis) engineered for expression of porcine β-defensin-2 (pBD-2) and cecropin P1 (CP1) fusion antimicrobial peptide and investigate their anti-bacterial activity in vitro and their growth-promoting and disease resisting activity in vivo.MethodsThe pBD-2 and CP1 fused gene was synthesized using the main codons of B. subtilis and inserted into plasmid pMK4 vector to construct their expression vector. The fusion peptide-expressing B. subtilis was constructed by transformation with the vector. The expressed fusion peptide was detected with Western blot. The antimicrobial activity of the expressed fusion peptide and the recovered pBD-2 and CP1 by enterokinase digestion in vitro was analyzed by the bacterial growth-inhibitory activity assay. To analyze the engineered B. subtilis on growth promotion and disease resistance, the weaned piglets were fed with basic diet supplemented with the recombinant B. subtilis. Then the piglets were challenged by enteropathogenic Escherichia coli (E. coli). The weight gain and diarrhea incidence of piglets were measured after challenge.ResultsThe recombinant B. subtilis engineered for expression of pBD-2/CP1 fusion peptide was successfully constructed using the main codons of the B. subtilis. Both expressed pBD-2/CP1 fusion peptide and their individual peptides recovered from parental fusion peptide by enterokinase digestion possessed the antimicrobial activities to a variety of the bacteria, including gram-negative bacteria (E. coli, Salmonella typhimurium, and Haemophilus parasuis) and gram-positive bacteria (Staphylococcus aureus). Supplementing the engineered B. subtilis to the pig feed could significantly promote the piglet growth and reduced diarrhea incidence of the piglets.ConclusionThe generated B. subtilis strain can efficiently express pBD-2/CP1 fusion antimicrobial peptide, the recovered pBD-2 and CP1 peptides possess potent antimicrobial activities to a variety of bacterial species in vitro. Supplementation of the engineered B. subtilis in pig feed obviously promote piglet growth and resistance to the colibacillosis.
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