Differential Responses of Antagonistic Microbes in Controlling Bacterial Wilt (Ralstonia solanacearum) in Potato Seed Production

The research was carried out in the greenhouse of the West Java Province Potato Seed Center in Sukamanah Village, Pangalengan District, Bandung Regency, from January to April 2023. The microbial solution was made in the BPTPH Cianjur disease laboratory. The research design was a factorial randomized block design with six replications. The first factor was the kinds of antagonistic microbes in four treatments, and the second factor was the wilt concentration of Ralstonia solanacearum in two treatments (Table 1). The microbes used were the results of microbial exploration in research by Dianawati et al. [2]. The potato (Solanum tuberosum L.) cultivar used was ‘Granola’. One experimental unit consisted of one tray. One tray consisted of 105 plants.

Table 1. Treatment of kinds of antagonism microbe and Ralstonia solanacearum concentration

The four microbial types treated were microbes A and B identified as Bacillus subtilis, microbe C was Pseudomonas fluorescens, while D was a consortium of B and C. Microbe A was Bacillus subtilis strain H15, from a storage collection from Padjadjaran University with a host plant of potatoes, while microbes B and C were a collection of the National Research and Institute Agency (NRIA) obtained from farmers with a host plant of bamboo roots [2]. Microbes were propagated in nutrient broth medium, which was incubated in an orbital shaker for 42 hours. The density of the microbial suspension used was adjusted to a concentration of 10⁸ CFU mL^-1. The Ralstonia solanacearum isolate came from the collection of the Disease Laboratory of the Ornamental Crops Research Institute, Indonesia, and its concentration depended on each treatment (10⁴ and 10⁸ CFU mL^-1).

The Ralstonia solanacearum-resistant microbial selection test was carried out in a tray with a substrate of burnt husks and compost that had been sterilized by steaming. Each tray that had been planted with one-week plantlet cuttings was soaked in a microbial suspension for one minute. Each tray that had been planted with one-week plantlet cuttings was soaked in a microbial suspension for one minute. Tray immersion was carried out in a plastic box containing a microbial suspension. One week later, each tray was inoculated with Ralstonia solanacearum at the concentration according to the treatment using a handsprayer to the seedlings at the ages of 2, 3, 4, and 5 weeks after cutting (WAC). Inoculation volume per tray by handsprayer was one liter for each concentration treatment of Ralstonia solanacearum. The Ralstonia solanacearum suspension was sprayed evenly over all parts of the plant at a spray distance of approximately 10-20 cm. Spraying was carried out until evenly wet but not excessively dripping. Spraying was carried out in the morning when the dew had disappeared around 7 a.m. with a temperature of around 20-22 ℃ and air humidity of 85-90%. Each tray was separated with plastic mulch to avoid contamination between treatments. The sample observed was 10 plants per experimental unit, randomly selected without involving borders. If the seeds were affected by wilting, they were removed and not counted again to prevent the spread of the wilt attack to the surrounding area. Plants were maintained for up to 8 WAC with watering to maintain media humidity. Pest and disease control was carried out if there were indications of an attack.

The variable observed in this study was the number of wilted plants every week until the age of 8 WAC. Criteria for determining wilt were wilted shoots, and then verified by immersing the tissue in water, and a white, mist-colored bacterial mass was formed [2]. The percentage of wilted plants was the percentage of the number of wilted plants compared to the total number of plants observed. The cumulative percentage of wilting was the sum of the percentage of wilted plants up to the last week of observation. Inhibition rate was the percentage of control wilted plants compared to the treatment and then divided by the percentage of control wilted plants [2]. Harvest observations were the fresh weight of stover, roots, and total plants.

Inhibition rate $=\frac{\mathrm{Pt}-\mathrm{Pc}}{\mathrm{Pc}} \times 100 \%$

Pt = percentage of wilted plants of the treatment.

Pc = percentage of wilted plants of the control.

Data were analyzed using variance analysis, and if there were significant differences, continued with the Duncan test and Orthogonal Contrast test with a confidence level of 95% (p < 0.05) and the SAS program version 9.4. Testing between observed variables used the Pearson correlation test and Principal Component Analysis (PCA) test with the R Studio program version 4.4.3. The calculation of the PCA value is the sum of dim1 (PCA1) and dim2 (PCA2) [2].

4.1 The control effect of antagonistic microorganisms on bacterial wilt

Preparing healthy seeds is very necessary so that they can grow well in the field and can withstand the threat of biotic and abiotic stress. Healthy seedlings can be conditioned by applying antagonistic microorganisms in the form of non-pathogenic bacteria from the start so that systemically induced resistance occurs, or what is known as induced systemic resistance (ISR) [25]. The study [10] stated that ISR causes plants to be able to survive in the presence of pathogens, whereas in the absence of microbes, the plants would die. Providing antagonistic microbes at the beginning of growth on sterile media will cause the microbes to grow quickly without competition with other unwanted microbes. However, to test the effectiveness of this microbial antagonism, it is necessary to assess it by giving the Ralstonia solanacearum pathogen at low (10⁴ CFU mL^-1) and high (10⁸ CFU mL^-1) concentrations to study its response to the growth of potato cuttings as a systemic acquired resistance (SAR) response. The study [25] stated that the combination of ISR and SAR could increase prevention against pathogen attacks and expand the spectrum of prevention against various pathogens.

All cuttings in this study experienced Ralstonia solanacearum attack at both low and high Ralstonia solanacearum concentrations (Figures 1 and 2). If Ralstonia solanacearum had entered the plant's environment through water, then Ralstonia solanacearum would easily infect plants because Ralstonia solanacearum could enter plants in various ways. The way in which Ralstonia solanacearum enters was through plant wounds, root movement, or the natural opening, and then it entered the xylem tissue and developed in the vessels of the plant stem [26, 27]. Extracellular polysaccharides (EPS) produced by Ralstonia solanacearum in the xylem would block the movement of water and nutrients in plant stems, causing the plant to wilt and ending with the plant dying as a whole [27].

The higher Ralstonia solanacearum concentration given would accelerate the occurrence of Ralstonia solanacearum attacks at 3 WAC compared to low Ralstonia solanacearum concentrations at 6 WAC (Figure 1). This was because high Ralstonia solanacearum concentration in this study could increase wilt attacks by 50.57%. Lowe-Power et al. [27] stated that with a low Ralstonia solanacearum inoculation of 10⁴ CFU mL^-1 on Ralstonia solanacearum-resistant cultivar plants, the plants could survive without showing symptoms of wilting. However, at a high concentration of Ralstonia solanacearum 10⁸ CFU mL^-1, the plants would immediately show wilting symptoms. The rapid response of plants to Ralstonia solanacearum at high Ralstonia solanacearum concentration showed that the virulence of Ralstonia solanacearum in this study was relatively high due to the repeated application of Ralstonia solanacearum four times during growth. Kashyap et al. [28] reported that the Ralstonia solanacearum population increased rapidly after inoculation 48 hours later.

In environmental conditions polluted with Ralstonia solanacearum, the application of all types of microbes could help inhibit wilt attacks at both low and high Ralstonia solanacearum concentrations of 55-76% (Figure 1). The percentage of inhibition of wilting attacks was higher than in research conducted by Raza et al. [17], which ranged from 26-38%. The results of the orthogonal contrast test also showed that the wilting percentage and cumulative wilting percentage of the microbial treatment were higher than those of the control (Table 3). Even in this study, the cumulative percentage of wilting in various types of microbes at low Ralstonia solanacearum concentrations could be reduced below 6% (Figure 1(a)). Apart from that, the inhibition of Ralstonia solanacearum wilt attacks due to the application of microbes in this study was quite effective, with a delay of wilt attacks of around 2-3 weeks, namely up to 6 WAC for microbes C and D and 7 WAC for microbes A and B (Figure 1(b)). This delay was longer than in the study [28] research which was only delayed 10-14 days. Thus, this research showed that both Bacillus subtilis and Pseudomonas fluorescens microbes could inhibit Ralstonia solanacearum by various mechanisms, including niche and nutritional competition, antibiosis, resistance induction, parasitism, and VOC products. Sun et al. [5] reported that lipopeptides produced from Bacillus subtilis strain 31 played an important role in inhibiting Ralstonia solanacearum. Several studies report VOC that could inhibit Ralstonia solanacearum produced by Bacillus subtilis, such as pyrazine and benzothiazole [29], while by Pseudomonas fluorescens were 13-tetradecadien-1-ol, 2-butanone, and 2-methyl-n-1-tridecene [30]. These VOCs could damage the DNA of pathogens and induce changes in the expression of enzyme ontogenesis levels, which changed the target organisms to abnormal ones. Tahir et al. [7] reported abnormalities in this pathogen in the form of rupture and detachment of cell walls, movement of cytoplasm outward, and deformed cell shapes.

4.2 Differential response mechanisms of different microorganisms

The difference in inhibition rate against Ralstonia solanacearum attacks occurred between microbes, namely microbes A and B, respectively 76.86 and 72.31%, followed by microbes D and C at 54.87 and 50.82% (Table 3). The study [7] reported almost the same inhibition rate of Pseudomonas fluorescens and Bacillus subtilis, 49 and 47%, respectively, while Kashyap et al. [31] reported the respective inhibition rate of Pseudomonas fluorescens and Bacillus subtilis microbes of 72 and 69%. The inhibition rate of microbes A and B, namely Bacillus subtilis, in this study appeared to be higher than that of microbes C (Pseudomonas fluorescens) and D (Bacillus subtilis + Pseudomonas fluorescens), indicating that Bacillus subtilis was more effective at inhibiting Ralstonia solanacearum than Pseudomonas fluorescens. Initially, Pseudomonas fluorescens could inhibit Ralstonia solanacearum up to 6 WAC, but repeated application of Ralstonia solanacearum up to four times caused an increase in wilting attacks (Figure 1), so that Pseudomonas fluorescens might not be able to control Ralstonia solanacearum. In fact, Pseudomonas fluorescens was well known to have a high level of virulence [20], but it had a weakness, namely, it did not produce spores, so it was easily damaged [32]. The study [17] reported that the effectiveness of VOCs from Pseudomonas fluorescens in inhibiting Ralstonia solanacearum was determined by the ability of the bacteria to colonize roots. Apart from that, the high virulence of Pseudomonas fluorescens also needs to be considered to get the right concentration to avoid the reverse mechanism effect. The study [2] reported that soaking Pseudomonas fluorescens for 10 and 15 minutes could reduce the effectiveness of Pseudomonas fluorescens compared to soaking for 5 minutes because of its higher concentration. Meanwhile, Bacillus sp could produce endospores so that it was resistant to extreme conditions [7, 33] and could grow quickly in various media [34]. This made Bacillus easy to maintain, formulate, and store, and it can survive well in the soil without disturbing other bacterial populations [32]. This characteristic caused Bacillus subtilis in this study to appear superior to Pseudomonas fluorescens.

The difference response of microbe A and B of Bacillus subtilis with differences in strain may be attributed to strain-specific variation in the production of bioactive secondary metabolites, particularly lipopeptides and VOC. The study [35] demonstrated that different Bacillus strains produce distinct profiles and quantities of lipopeptides such as surfactin, iturin, and fengycin, which play key roles in antifungal and antibacterial activity. Likewise, variations in VOC composition can influence the degree of pathogen inhibition through direct antimicrobial effects or indirect mechanisms such as ISR activation [36]. Therefore, the higher inhibition rate observed in microbe A may indicate that Bacillus subtilis UNPAD produced a more effective combination or higher concentration of these bioactive compounds compared to Bacillus subtilis Garut. These findings suggest that differences in metabolite profiles among strains are likely a contributing factor to the observed variation in antimicrobial efficacy and should be further investigated through metabolomic or molecular analyses.

The low percentage of wilting and cumulative wilting due to the application of antagonistic microbes in this study did not always result in high plant growth (Tables 3 and 4). This was indicated by the low Pearson correlation value between the variables percentage wilting and cumulative wilting with the variables plant height, number of leaves, wet and dry weight of plants (Table 5), and the position of the variables plant growth and plant weight in different quadrants with the variables percentage wilting and cumulative wilting (Figure 2(a)). These conditions showed that each microbe tested had different characteristics in responding to wilting attacks and plant growth, so that two large groups of microbes or five clusters were obtained, as in Figure 2(b). Two large groups of microbes in response to wilting and plant growth, namely microbes with a high percentage of wilting and cumulative wilting with low plant growth, namely controls, microbes B and C, while microbes A and D had a low percentage of wilting and cumulative wilting with high plant growth. Identifying the characteristics of the four microbes used by PCA value of 69.4% was a novelty from this research. High inhibition rate on microbe B did not necessarily produce high plant growth, while low inhibition rate on microbe D still could increase plant growth. Meanwhile, microbes A and C showed the same positive and negative tendencies between inhibition rate and plant growth (Figure 2).

Microbes A and B were Bacillus subtilis, which could overcome wilting at both low and high Ralstonia solanacearum concentrations (Figure 1). However, the influence of these two microbes on plant growth was very different. The study [37] stated that different Bacillus subtilis strains would produce different hydrolytic enzymes, including cellulase, protease, and β-glucanases, so that they would produce different plant growth responses. The high ability to inhibit wilting by microbial A was in line with the high plant height at 5 WAC, the number of leaves at 4, 5, and 6 WAC, the fresh weight of shoot and total plant, dry weight of shoot, root, and total plant (Table 3). This showed that Bacillus subtilis in treatment A could not only inhibit wilting, but could also increase plant growth (Table 3). Apart from producing compounds that could kill pathogens, Bacillus subtilis could also produce plant growth-promoting rhizobacteria (PGPR), which could help plant growth [28]. The study [5] reported that Bacillus subtilis strain R31 could indirectly inhibit the growth of Ralstonia solanacearum and the bacterial community within it, thereby restoring the abundance of other normal bacterial communities that contributed to metabolic functions that influence plant growth.

Meanwhile, microbe B could only inhibit wilt attacks, but could not increase plant growth because plant growth was the same as the control (Table 3). The Bacillus subtilis Garut strain of microbe B may not produce enough PGPR to increase plant growth, so it was only able to inhibit Ralstonia solanacearum. This was the same as research by Istifadah et al. [38] in chilies, where Bacillus subtilis could suppress Ralstonia solanacearum wilt, but did not increase plant growth. Soesanto et al. [18] stated that although microbes could inhibit pathogen attacks, this was not always followed by increased plant growth because the production of PGPR, which played a role in growth, was not optimal. This was because microbes to produce PGPR take time [16] and need to be applied early and repeatedly [5, 7, 39].

Microbe C had a lower percentage of inhibition rate than microbes A and B (Table 3). Thus, C microbes at both high and low Ralstonia solanacearum concentrations had low growth (Figure 2). This showed that Pseudomonas fluorescens in this study was no better than the control for increasing plant growth. Pseudomonas fluorescens' inability to overcome Ralstonia solanacearum in the 7th week was thought to have disrupted plant growth.

Microbe D had a lower percentage of inhibition rate than microbes A and B, but plant height growth and plant dry weight were higher than controls and microbes B and C, and were not different from microbe A (Table 3). This could be explained because the plant seeds affected by wilt in this study were observed and then removed and not counted again, so that the remaining plants were plants that could survive the wilt attack. Yuliar et al. [6] stated that isolates taken from the rhizospheres of diseased plants could suppress disease more than those from the rhizospheres of healthy plants. Plants that could survive wilt attacks would induce SAR by producing certain hormones to fight Ralstonia solanacearum attacks so that plant growth was better [40]. Success in inducing this resistance could occur due to the combination of compatible microbes such as Bacillus subtilis and Pseudomonas fluorescens in the microbes D. The study [41] stated that although each microbe in a microbial consortium has a different mode of action, combining various compatible microbes in a consortium would broaden the spectrum of activity against diverse pathogens. In addition, consortia of microbes could contribute to PGPR and/or increase the activity of other microorganisms [42]. Thus, microbial consortia were not only related to virulence stability, but also to the application spectrum [14]. Several studies also reported the success of the Bacillus subtilis and Pseudomonas fluorescens consortium microbes in various plants such as chilies [43], potatoes [9], tomatoes [44], and garlic [45].

4.3 Implications for potato seed

The results of this research showed that the response of plant seeds to various types of microbes was very diverse. Bacillus subtilis UNPAD and consortia Bacillus subtilis + Pseudomonas fluorescens were the microbes with the best plant growth. However, Pseudomonas fluorescens alone had a low inhibition rate and plant growth. Even though the type of microbe was the same, namely Bacillus subtilis, its effect on the growth of cuttings could be different depending on the strain. Bacillus subtilis, which could inhibit wilt attacks, may not necessarily increase plant growth as happens with microbe B, and this phenomenon was common in the world of microbiology [46]. Meanwhile, the microbial consortium D, which was initially able to inhibit Ralstonia solanacearum but then Ralstonia solanacearum increased, showed better growth because it induced a SAR by utilizing the excess properties of Bacillus subtilis and Pseudomonas fluorescens. Thus, Bacillus subtilis UNPAD and the microbial consortium are recommended as effective antagonistic agents for potato seed production.