Antagonism of Streptomyces spp. on Ralstonia solanacearum causing bacterial wilt in potatoes

Ralstonia solanacearum isolate

The potato samples were collected from crops in the communities of San José and Congona, province of Cutervo. A total of 100 tubers with symptoms were collected, which were placed in Ziploc bags and transported to the laboratory, where the recommendation by Fornos et al . (2021) was followed., which consisted of washing five times with distilled water and then disinfecting with 70% alcohol. Subsequently, a cut was made in the tuber with a scalpel and an aliquot of the bacterial exudate was obtained from the affected area and seeded in plates with tetrazolium chloride (TZC) agar to be incubated at 30 °C for 48 h. The colonies of R. solanacearum were irregularly shaped, red, with white edges. Identification was made on the basis of morphophysiological characteristics (Phondekar et al ., 2020) by Gram staining, tests of 3% potassium hydroxide, catalase, oxidase, citrate, triple iron sugar (TSI), lysine iron (LIA) and motility, indole and ornithine (MIO) (Figure 1).

Figure 1

Figure 1. Isolation and identification of R. solanacearum. A and B) selection and collection of tubers; C) extraction of vascular ring flow; D) Gram-negative bacilli of vascular flow; E) colony-forming units in TZC and F) biochemical reaction.

Isolation of Streptomyces spp.

A total of 95 samples of potato cultivation soil were collected at a depth of 2 to 8 cm and deposited (5 to 10 g) in Ziploc bags. They were then processed according to the recommendations by Parada et al . (2017), making serial dilutions until obtaining 1x10^-3 g ml^-1 and pasteurization at 70 °C for 15 min. From each dilution, 100 µl was sown in Petri dishes with oat agar, and they were incubated at 30 °C for 7 days. For the identification, the following macroscopic characteristics of the colonies were considered: type, size, pigment diffusion, and aerial and vegetative mycelium coloration. In addition, Gram staining, a technique of microculture, and fermentation of sugars were performed (Figure 2).

Figure 2

Figure 2. Isolation and identification of Streptomyces spp. A) soil samples; B) dilutions; C) colony-forming units in oat agar; D) macroscopic appearance; E) microscopic observation and F) fermentation of sugars.

Evaluation of antagonism

The perpendicular streak methodology described by Hossain and Rahmanll (2014) was used, which consisted of seeding by depletion and streaking the strain of Streptomyces spp. in the central part of a Petri dish containing Mueller-Hinton agar and incubating it at 30 °C for seven days. The R. solanacearum strains were then seeded perpendicularly and incubated at 30 °C for 48 h. The reading was performed by measuring the inhibition halo, considering positive antagonism when the streak of R. solanacearum began to grow at least 5 mm away from the streak of Streptomyces spp.

Experimental design and statistical analysis

The antagonistic effect was evaluated by applying an analysis of variance (Anova), adjusting it to the 50 x 28 x 3 factorial design to demonstrate the differences between the fifty isolates of Streptomyces, twenty-eight of R. solanacearum, and three replications, generating 4 200 experimental units. The statistical analysis of the differences between the inhibition means was complemented with Tukey’s multiple comparisons test at 0.05% probability. To do this, the statistical software Statistica was used.

In vitro isolation of R. solanacearum

The three potato varieties: chaucha, molinera, and unica, were obtained from the communities of San José and La Congona, province of Cutervo. Forty-five percent of isolates were Gram-negative bacilli, of which 28% were R. solanacearum, with most of the isolates, 47.4%, belonging to the molinera variety (Table 1).

There are reports of isolates of R. solanacearum from pseudostems of banana, eggplant, and tomato (Ling et al ., 2020; Kaari et al ., 2022). Where nightshades are predisposed to substrates such as glutamine, asparagine, amino acids and sugars, which promote bacterial growth (Baroukh et al ., 2022). On the other hand, Vargas et al . (2023) state that the presence of R genes helps to recognize pathogenic proteins. In addition, a high transpiration rate and structure of the xylem favors the spread of the bacterium (Mamani, 2015).

The soil microbiota, as long as it is in balance, will prevent pathogens from reaching levels sufficient to cause diseases (Zhang et al ., 2022). However, the use of pesticides, nitrogen fertilizers, and phosphorus and potassium deficiency reduce beneficial microorganisms (Cao et al ., 2022). Ralstonia, on the other hand, spreads rapidly in ferralitic soils at pH between 5 and 7 and at temperatures between 25 and 35 °C. In addition, it uses virulence factors, such as type III secretion system (T3SS), motility, biofilms, and exopolysaccharides (EPS) to infect plants and obstruct xylem (Vailleau and Genin, 2023). Thus, it manipulates the metabolism of the host plant, where galacturonic acid and salicylic acid, released by the cell walls of the plants, are used for nutrition. For its part, L-glutamic acid promotes the production of virulence factors (Shen et al ., 2020).

Isolation and in vitro evaluation of the antagonism of Streptomyces spp.

Of 95 soil samples, 50 strains of Streptomyces spp. were isolated, 34% were pigmented and 64% did not present this characteristic; in addition, 22% showed antagonism (Table 2). Figure 3 shows the positive antagonism of strain 10 of Streptomyces spp., which managed to inhibit strains 16, 17, 19 and 26 of R. solanacearum.

Figure 3

Figure 3. Positive and negative antagonism of strain 10 of Streptomyces spp. against R. solanacearum by perpendicular streaks on Mueller-Hinton agar: A) reverse view of the culture and B) obverse view of the culture.

In the research, it was possible to obtain up to five different isolates of Streptomyces spp. in a single sample, and in others, no strain was obtained. Nevertheless, Espinosa et al . (2021) indicate that the presence and diversity of the bacterium depend on nutrients, soil pH, microbial competitors, and biological and chemical activity of the soil. On the other hand, the production of diffusible pigment was variable, similar to the report by Antido and Climacosa (2022), where 14 strains were gray, yellow and pink, which is attributed to synthesized pigments, such as melanin, actinorhodin, prodigiosin, carotenoids and polyketide compounds, whose production depends on environmental conditions, metabolism, and bacterial genetics (Abraham and Chauhan, 2018).

Twenty-two percent of Streptomyces spp. demonstrated antimicrobial activity, with strains 21, 10 and 16 standing out for their greater antagonism. Similar findings were reported by Zaki et al . (2022); Kaari et al . (2022), they obtained 9 and 4 antagonistic strains, respectively. Nonetheless, there is a difference in the radius of action as it is smaller than those reported by Ling et al . (2020), who obtained an inhibition zone of 30.5 and 32.8 mm. This difference depends on the interaction of factors, including genetic diversity, synthesis of peroxidase that degrades cell wall components and antibiotic production.

The antagonistic activity was evaluated through an analysis of variance, Anova, finding statistically significant differences between the treatments of the 50 strains of Streptomyces and 28 of R. solanacearum, as well as their interactions, where the p-value (0) demonstrates that there is antagonistic ability (Table 3).

According to Tukey’s discriminative test performed on the different treatments, it was shown that the antagonism averages of the strains S24, S47, S20, S39, S30, S33, S43, S18, S10, S16, and S21 of Streptomyces spp. showed different antagonistic activity. Strains of Streptomyces sp. (S24 and S47) showed a lower inhibition spectrum compared to strain 21 of Streptomyces sp. (Table 4).

The fact that R. solanacearum strains showed sensitivity to Streptomyces species indicates significant potential for biocontrol. However, it provided new information on strains with antagonistic spectrum with certain limitations, such as the variable effectiveness of the isolates. Therefore, more research is needed to understand the mechanisms of interaction and application in agricultural practices, generating ecological, sustainable, and productive agriculture.