Biological control of soil-borne cotton Verticillium wilt has gained recent increased interest both in China and abroad. Regarding the mechanism of using antagonistic bacteria to control plant diseases, previous studies have suggested that these bacteria mainly induce an inhibitory effect and have a competitive effect at infection sites (Dimkić et al. 2022; Ge et al. 2017). Therefore, the ideal biocontrol antagonistic bacteria not only require strong antagonistic ability and a wide antibacterial range, but also require strong viability and colonization in host plants. Under the condition of artificially configured medium, the application of screened bacteria often obtains positive results. However, under natural conditions, due to the competition of soil microorganisms or the influence of other adverse environmental factors, the growth and function of the introduced alien biocontrol bacteria are hindered, resulting in many unsatisfactory test results. Therefore, in order to induce play the stable and effective role in production of disease control bacteria, it is necessary to strengthen investigations pertaining to colonization theory.
In the use of endophytic bacteria to control soil-borne diseases, the effective colonization of endophytic bacteria in plants is one of the important prerequisites for the biocontrol effect. Therefore, the study of the colonization ability of endophytic bacteria has become an important part of the current postgraduate prevention mechanism (Adams et al. 2002; Li et al. 2012; Marag et al. 2018; Yang et al. 2013). In this study, the traditional rifampicin resistance labeling technique was used to recover and detect the number of colonization of BHZ-29R in cotton plants after root irrigation. The results showed that the strain BHZ-29 could colonize in cotton plants, and the number of colonization in the roots of cotton seedlings was greater than that in the stems, and the colonization time in the roots was longer than that in the stems, but the number of colonization in the roots and stems gradually decreased with time until it was not detected. The number of BHZ-29R colonized in the roots and stems of cotton seedlings remained relatively stable within 6 days after inoculation. If the inoculation could be strengthened again after 6 days of inoculation to maintain a certain population advantage, it might have a long-term protective effect on plants. The relationship between the number of colonization, inoculation amount and control effect of this strain needs further study. Studies have shown that plants can specifically attract microorganisms that are beneficial to their ecology and evolution. Just as bacteria in the rhizosphere have been selected, endophytic bacteria have also been selected and colonized within plants. Not all soil bacteria can enter the plant and become endophytic bacteria (Shi et al. 2021). In the screening process of biocontrol bacteria against watermelon Fusarium wilt, we obtained a strain SHT-2 isolated from soil with good antagonistic activity and pot control effect. Similarly, rifampicin resistance marker technology was used to detect the root and stem of cotton seedlings. The strain of rifampicin resistance marker was not detected, indicating that it could not enter the cotton plant. Previous studies have proposed that the biocontrol bacteria Paenibacillus polymyxa C5 can only form a biofilm on the root surface of tobacco, but can not enter the root tissue (Ren et al. 2012). Plants are equivalent to a device that filters soil microorganisms and can select those capable endophytic bacteria (Ren et al. 2012), but the specific mechanism is very complex and cannot be resolved at present.
Antagonistic bacteria not only colonize effectively in cotton, but also induce systemic resistance. In recent years, the role of biocontrol bacteria in inducing plant disease resistance has attracted much attention. Studies both in China and abroad have shown that a series of protective enzymes such as superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), polyphenol oxidase (PPO) and phenylalanine ammonia-lyase (PAL), which are involved in various physiological metabolic processes in plants, are closely related to plant defense response and disease resistance and are usually used as important indicators to assess the plant defense response (Prasannath et al. 2017). PAL and PPO are related to the formation of phenolic substances and lignin in plants and are key regulatory enzymes for obtaining systemic resistance. POD and CAT are closely related to the scavenging of active oxygen in plants and positively correlated with disease resistance of plants (Delledonne et al. 1998; Kim et al. 2014).
The content of MDA as the final product of membrane lipid peroxidation, can reflect the degree of membrane lipid peroxidation, and the degree of damage to the cell membrane that has been positively correlated (Zhang et al. 2019; Abdelaal et al. 2018). Ascorbic acid (Vc) is an antioxidant in plants, which can directly scavenge O2- and · HO, and convert H2O2 into water through ascorbic acid oxidase. When plants are damaged by pathogenic organisms and stress factors, these can reduce the toxicity of reactive oxygen species to plants and maintain the normal physiological functions of plant cells (Hasanuzzaman et al. 2021; Sachdev et al. 2021; Mansoor et al. 2022).
In the present study, the RIF-resistant BHZ-29 was obtained using the antibiotic labeling method, and the colonization of BHZ-29 in the roots, stems, and leaves of different resistant varieties of cotton. The results showed that BHZ-29 could be effectively colonized in the roots, stems and leaves of the three varieties of cotton, and the colonization ability was ranked as follows: root > stem > leaf. The maximum colonization of roots was 9.13 × 105 CFU/g in Xinluzao 61T at 20 d, the maximum colonization of stem was 1.82 × 105 CFU/g in 9T at 20 d, and the maximum colonization of leaf was 1.30 × 105 CFU/g in Xinluzao 36S at 15 d. The results showed that BHZ-29 could not only enter and survive in the roots of cotton by the initial root injury method, but also transmit upward with the growth of cotton, and then stably colonize in various parts of the plant to play a biocontrol role, which was consistent with previous studies on the colonization characteristics of antagonistic bacteria (Liu et al. 2019; Achari et al. 2019; Bodhankar et al. 2017; Sachdev et al. 2021; Mansoor et al. 2022). Gu et al. found that Bacillus amyloliquefaciens Lj1 could affect the activities of defense enzymes in cucumber plants and could induce resistance in cucumber to powdery mildew (Gu et al. 2013). A similar phenomenon was also found in this study, the activities of POD, CAT, SOD, PPO, PAL and the content of Vc in cotton inoculated with BHZ-29 and inoculated with BHZ-29 + VD were significantly increased, and the content of MDA was decreased, which indicated that BHZ-29 could induce disease resistance in cotton plants. This is thus expected to become an important biocontrol agent for the biological control of cotton Verticillium wilt and provide a theoretical basis for the development and application of biocontrol bacteria.