ENDOPHYTIC BACTERIA ISOLATED FROM PLANTS AND THEIR ANTAGONISTIC AND SYNERGISTIC INTERACTIONS

ABSTRACT

This study is aimed at creating a complex biological compound based on endophytic bacteria isolated from various plants, as well as analyzing the interactions between different bacterial strains. One of the key objectives of the research is to characterize the method used to study and evaluate the antagonistic and synergistic activity of endophytic bacteria in relation to one another. To determine the nature of strain interactions, a simple, reproducible, and informative Cross-streaking method was employed. This method makes it possible to identify various types of microbial interactions, including growth inhibition, neutral interactions, and mutual stimulation. Based on the results obtained, it was determined that the bacterial strains Pseudomonas putida KoPr129, Priestia endophytica KoPr131, Bacillus subtilis CrEw1018, and Bacillus amyloliquefaciens HAPH2 are able to develop and exhibit synergistic interactions within the same medium. In the future, it is planned to develop a complex biopreparation based on these bacterial strains and to apply it in agricultural crops.

АННОТАЦИЯ

Данное исследование направлено на создание комплексного биологического соединения на основе эндофитных бактерий, выделенных из различных растений, а также на анализ взаимодействия разных бактериальных штаммов. Одной из ключевых задач исследования является характеристика метода, применяемого для изучения и оценки антагонистической и синергической активности эндофитных бактерий по отношению друг к другу. Для определения характера взаимодействия штаммов был использован простой, воспроизводимый и информативный метод Cross-streak. Данный метод позволяет выявить различные типы микробных взаимодействий, включая ингибирование роста, нейтральные взаимодействия и взаимную стимуляцию. На основе полученных результатов было установлено, что бактериальные штаммы Pseudomonas putida KoPr129, Priestia endophytica KoPr131, Bacillus subtilis CrEw1018 и Bacillus amyloliquefaciens HAPH2 способны развиваться и проявлять синергетические взаимодействия в одном питательном среде. В дальнейшем планируется разработка комплексного биопрепарата на основе этих бактериальных штаммов и его применение на сельскохозяйственных культурах. Данный метод позволяет выявить различные типы микробных взаимодействий, включая ингибирование роста, нейтральные взаимодействия и взаимную стимуляцию. На основе полученных результатов было установлено, что бактериальные штаммы Pseudomonas putida KoPr129, Priestia endophytica KoPr131, Bacillus subtilis CrEw1018 и Bacillus amyloliquefaciens HAPH2 способны развиваться и проявлять синергетические взаимодействия в одном питательном среде. В дальнейшем планируется разработка комплексного биопрепарата на основе этих бактериальных штаммов и его применение на сельскохозяйственных культурах.

Keywords: Endophytic bacteria, strains, methods, complex consortium.

Ключевые слова: Эндофитные бактерии, штаммы, методы, сложный консорциум.

Introduction

Among microorganisms, endophytic bacteria are regarded as a promising biopotential tool for broad agricultural applications. These microorganisms colonize plant tissues and significantly stimulate plant growth through various direct and indirect mechanisms. Such beneficial mechanisms include biological nitrogen fixation, phosphate solubilization, and the production of siderophores [1, p.5]. The analysis of endophytic bacteria involves studying the simultaneous growth of different strains in the same medium, which enables the evaluation of their direct interactions. Subsequently, the growth of indicator organisms is assessed and compared with a control to confirm the activity of specific bacterial strains [2, p. 4861]. In co-culture analysis, different strains are inoculated together in supportive growth media, which allows for the reproduction of microbial interactions and provides a more accurate evaluation of antagonistic activity compared to other methods [3, p. 2785]. Endophytic bacteria constitute a diverse group of microorganisms that colonize internal plant tissues without causing disease and play a crucial role in regulating plant physiology, growth, and stress tolerance. Numerous studies have shown that endophytes enhance nutrient uptake, improve resistance to abiotic stress factors, and stimulate plant growth through the production of phytohormones and bioactive metabolites (4, p. 314-330; 5, p. 45-58). Their natural interaction with host plants contributes significantly to bioprotection processes, including competition with pathogens and the induction of systemic resistance mechanisms (6, p. 127-135; 7, p. 1279–1288). Given the increasing demand for eco-friendly agricultural technologies, investigating the antagonistic and synergistic interactions among endophytic bacteria has become highly relevant for biotechnology, agriculture, and environmental sustainability. Antagonistic interactions often involve the production of antimicrobial compounds, siderophores, lytic enzymes, and other inhibitory metabolites that suppress phytopathogens or competing microorganisms (8, p. 223–228; 9, p. 4857–4867). Conversely, synergistic interactions among bacterial strains may enhance collective metabolic activity, improve colonization efficiency, and strengthen plant growth-promoting effects (10, p. 7204–7209; 11, p. 973–979). Understanding these complex intermicrobial relationships is essential for selecting promising endophytic strains as biopreparations and assessing their functional potential in plant protection and growth promotion systems. Such knowledge can facilitate the development of effective microbial consortia with optimized antagonistic or synergistic profiles (12, p. 10–12; 13, p. 440–444). Based on this, the aim of the present study is to evaluate the antagonistic and synergistic properties of selected endophytic bacterial strains, assess their biological activity, and explore their potential application as bioagents in sustainable agriculture.

Materials and methods

In the research work, endophytic bacterial strains preserved in the collection of the Molecular Biotechnology Laboratory at Samarkand State University were used (Table 1).

Table 1.

Endophytic bacterial strains used in the study

To evaluate the growth and development of different endophytic bacterial strains in the same medium, the cross-streak method was used. This method involves streaking two microorganisms across each other on LB agar plates. The isolates were incubated at 37 °C for 24 hours. The antagonistic and synergistic interactions between isolates were assessed at the intersections of the streak lines: the absence of a zone indicated synergistic interaction, while the presence of a zone suggested antagonistic activity.

Results and discussions

The LB agar medium and the cross-streak method revealed both antagonistic and synergistic interactions among the studied endophytic bacterial strains.

Table 2.

Growth of endophytic bacterial strains in a single medium

Note: (+) – able to grow together; (–) – unable to grow together.

Table 2, strains marked with a (+) sign are said to be able to grow and develop in the same medium. Strains marked with a (-) sign are said to be unable to grow and develop in the same medium.

Figure 1. Interactions among endophytic bacterial strains

Note: The strains in the figure are numbered as follows: 1-KoPr101, 2-KoPr113, 3-KoPr118, 4-KoPr129, 5-KoPr131, 6-CrEw1004, 7-CrEw1015, 8-CrEw1018, 9-CrEw1021, 10-HAST17, 11-HAPH2, 12-SSU4.

Table 3. 

Antagonistic interactions were observed among several strains

Note: Antagonistic intercommunications were analyzed for 12 strains

Table 3 shows that 12 promising endophytic bacterial strains isolated from plants exhibit antagonistic intercommunications with each other. Among the bacterial strains with antagonistic relationships, the highest index was shown by SSU4, KoPr 118 bacterial strains. Among the 12 bacterial strains, there were 26 antagonistic relationships, indicating that these bacterial strains cannot grow in the same environment.

Table 4.

 Synergistic relationships of endophytic bacterial strains were identified among the following strains.

Note. Synergistic intercommunications were analyzed for 12 strains

Table 4, each of the 12 endophytic bacterial strains analyzed was analyzed separately, and we can see that these strains exhibit synergistic relationships with the indicated bacterial strains. These bacterial strains KoPr 129, KoPr 131, CrEw 1018, and HAPH2 can also grow synergistically in the same environment with the 12 strains mentioned above. Based on the results, 4 strains among the 12 strains have synergistic interactions with all strains.

Discussion.

In the research work conducted, antagonistic and synergistic intercommunications between 12 collection strains were analyzed. The experiments conducted were evaluated using the Cross Streak method. Based on the results of the research, 23 antagonistic and 40 synergistic intercommunications were found between 12 strains. In the work of Norma et al. (2019), when evaluating the antagonistic and synergistic activity of 6 strains, six synergistic and twenty antagonistic intercommunications were found between the strains. Based on the six synergistic intercommunications obtained as a result of their work, it was noted that these strains were developed in the same environment and used as a complex combination [12, p. 442]. Tsigarida et al. (2003) in the process of studying the antagonistic and synergistic intercommunications of bacterial strains Pseudomonas sp., S. putrefaciens and B. thermosphacta admitted that there is an antagonistic relationship between Pseudomonas sp., S. putrefaciens. They stated that these strains cannot be used in the same environment with each other [13, p. 7207]. Júnior et al. (2011) studied the antagonistic and synergistic monostables of bacterial strains L. paracasei, F. nucleatum and P. prevotii against other types of bacterial strains Clostridium butyricum, Prevotella intermedia and Gemella morbillorum. They studied that bacterial strains F. nucleatum and P. prevotii, despite the production of antagonistic substances, did not cause antagonistic activity against bacterial strains L. paracasei. G. morbillorum [14, p. 976]. Based on our research, it was determined that all strains can grow and develop in the same environment based on the synergistic intercommunications of bacterial strains in Table 4 above. In the future, it was determined to use Pseudomonas putida KoPr129, Priestia endophytica KoPr131, Bacillus subtilis CrEw1018, and Bacillus amyloliquefaciens HAPH2 strains as a complex combination based on the synergistic intercommunications.

Conclusion.

At present, various agricultural crops are increasingly exposed to different diseases as a result of external environmental factors. Complex formulations developed on the basis of diverse endophytic bacteria are considered effective agents against pathogens that cause these diseases. To develop a complex preparation using the cross-streak method, the antagonistic and synergistic interactions of 12 endophytic bacterial strains were investigated under identical culture conditions, and the strains capable of co-developing in the same medium were analyzed. Among them, the strains KoPr129, KoPr131, CrEw1018, and HAPH2 exhibited synergistic interactions, indicating their potential for combined use in future applications. The strongest antagonistic interactions were observed in strains SSU4 and KoPr118, which collectively demonstrated 26 antagonistic interactions with other bacterial strains. The identified antagonistic and synergistic interactions for each strain will serve as a basis for the future development of bacterial preparations.

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