Isolation and identification of endophytic bacteria, optimization of fermentation conditions, and inhibition of cotton wilt pathogen in shidoulan
Endophytic bacteria in plants refer to microorganisms such as bacteria, fungi, and actinomycetes that parasitize the living tissues and organs of plants. Almost every plant contains abundant microbial material, and endophytic bacteria gradually form a symbiotic relationship with the host through long-term evolution, without causing obvious external infection symptoms of the host plant. Research has shown that secondary metabolites of endophytic bacteria can not only inhibit pathogenic microorganisms, but also activate the host plant’s immune response system, enhance the expression profile of plant defense related genes, and are an important source of natural medicine. Microbial pesticides have become a new pesticide industry in the 21st century due to their advantages of green environmental protection and minimal impact on the quality of agricultural by-products, representing the direction of plant protection. Orchid plants have more abundant endophytic bacteria in their habitat. Chen et al. isolated 241 endophytic bacteria from five medicinal plants in the Orchid family (Paeonia lactiflora, Panax ginseng, Dendrobium nobile, Dendrobium chrysotoxum, and Dendrobium chrysotoxum). The author found during the in vitro culture of Bulbophyllum sp. that its tissues were rich in endophytic bacteria, and some bacteria could symbiotically coexist with the host’s in vitro tissues. Wilt disease is a common disease during the cotton planting period, which occurs widely in major cotton producing areas across the country and causes serious harm. It is estimated that 100 million kilograms of cotton are lost each year due to wilt disease. Fusarium wilt is caused by Fusarium oxysporum, which is classified into different specialization types based on host specificity, such as cotton specialization, cucumber specialization, banana specialization, etc. Some specialization types are further divided into different physiological races based on host varieties, forming unique physiological races different from other countries. The wilt disease caused by different specialized types and physiological races requires different prevention and control methods, so it is particularly important to identify the biocontrol agents for different specialized types and physiological races of Fusarium oxysporum. This experiment used Fusarium oxysporum cotton wilting specialization as the indicator bacterium, and searched for biocontrol bacteria with inhibitory effects from endophytic bacteria of Solanum genus plants. Fermentation conditions were optimized, and the main antibacterial substances, lipopeptides, were observed under electron microscopy for their effects on the morphology of pathogenic bacteria. This experiment lays a practical foundation for the preparation of antibacterial substances and the biological control of cotton wilt disease.

Fusarium oxysporum can cause plant root rot, wilt, and yellowing diseases. The main biocontrol bacteria used for cotton wilt disease include Trichoderma harzianum TH1, Trichoderma viride GY20, Marine Streptomyces ZW-1, Streptomyces spectacular SC11, Bacillus subtilis, Bacillus amyloliquefaciens SN06, Bacillus cereus YUPP-10, etc. Due to the existence of different specialized types and physiological races of Fusarium oxysporum, the pathogen used in this study was isolated from diseased cotton plants in our school’s experimental field, belonging to the cotton wilting specialized type. The screening target was to inhibit the antagonistic bacteria of this pathogen. A bacterium with good inhibitory effect was selected from plants of the genus Cymbidium and named BBs-27. Molecular identification showed the highest similarity with Bacillus subtilis subspecies. Many species of Bacillus have broad-spectrum antibacterial activity, including Bacillus subtilis, Bacillus licheniformis, Bacillus cereus, Bacillus amyloliquefaciens, and Bacillus cereus.
The fermentation of antibacterial substances is greatly influenced by the culture medium and environmental factors. Optimizing the composition of the culture medium and fermentation conditions can increase the yield of antibacterial substances. Liu et al. fermented Mohewei J7 with optimized medium, and the yield of antibacterial substances increased by 38.89%. Chen et al. optimized the fermentation medium of Bacillus amyloliquefaciens SC1150 strain, and the diameter of the antibacterial zone against physiological race 4 of banana wilt disease increased from 23.31 to 28.33. In this study, except for the medium containing 2.5% to 3.0% xylose, which did not produce any antibacterial substances, all other sugar additions were able to promote the production of antibacterial substances; LB only contains organic nitrogen, and adding 0.05% inorganic nitrogen source (NH4) 2HPO4 can improve the antibacterial activity. After optimization, the antibacterial rate increased from 51.57% to 88.06%, and the yield of lipopeptides was 0.6 g (dry weight)/L, which was higher than that of Bacillus subtilis HS-A38 and the mutant strain. Zhang et al The conditions for producing lipopeptides in Subtilis ATCC2133 were optimized, and the content was determined to be 4.885g/L by HPLC, which may be related to the strain and detection method.
There are various microorganisms that produce antimicrobial lipopeptides, such as bacteria, actinomycetes, fungi, etc. Among them, Bacillus is the genus that has been found to produce the most lipopeptides. Different microorganisms produce lipopeptides with different structures, characteristics, and antibacterial effects. The antibacterial substances produced by BBs-27 used in this study have some high temperature resistance and some deactivation at high temperatures, indicating that there are multiple types of antibacterial substances, but the main antibacterial substance is resistant to high temperature treatment at 121 ℃. Based on the physicochemical properties of lipopeptides and the antibacterial effect of lipopeptides in this experiment, it is speculated that the antibacterial substance produced by BBs-27 is mainly lipopeptides. Another part of the research that has been conducted suggests that other high-temperature inactivated antibacterial substances may be proteins, and further research on antibacterial proteins and peptide structures is needed.