Bacillus subtilis 34 and water-retaining polymer reduce Meloidogyne javanica damage in tomato plants under water stress

1 Introduction

Tomato (Solanum lycopersicum L.) is among the main vegetable crops grown worldwide. Tomato crop areas reached 5 million hectares in 2020, with an average production of 187 million Mg [1]. The world’s largest tomato-producing countries are China, USA, India, Turkey, Egypt, Italy, Iran, Spain, and Brazil [1]. Tomato is grown in approximately 52,000 hectares in Brazil, where the main producing states are Goiás, São Paulo, and Minas Gerais [2]. In Minas Gerais, tomato crops are concentrated in the regions of Jaíba, Araguari, and Patos de Minas [3]. Despite the low rainfall conditions, the average yield in the Jaíba region, northern Minas Gerais, reached 73.63 Mg ha⁻¹ [3] when grown under irrigation.

Water deficit can cause morphological, biochemical, and physiological disorders, affecting vital cellular processes and resulting in crop yield losses [4]. Damage to the photosynthetic apparatus and oxidative injuries to proteins and lipids in cell membranes are among the deleterious effects of drought on tomato plants [5]. Several studies have reported that decreases in tomato crop yield are directly proportional to reductions in water use efficiency (WUE) [6,7]. Therefore, formulating strategies for improving WUE in tomato plants is essential.

Root-knot nematodes of the genus Meloidogyne can infect a wide range of host plants worldwide. The species M. incognita and M. javanica are among the main plant parasitic nematodes [8]. Parasitism of Meloidogyne species has resulted in annual losses of approximately $70 billion [9]. Yield losses in tomato crops range from 25 to 100%, depending on the Meloidogyne species infesting the crop, population density, and tomato cultivar [10].

Parasitism of M. javanica in tomato roots may also cause plant wilting, and the same symptom results from the plant’s response to water deficit. Meloidogyne species are sedentary endoparasites that infect plant roots and establish feeding sites (giant cells) within the vascular cylinder, causing parenchyma cells to become multinucleated [11]. These cells provide water and nutrients to the nematode during its life cycle [12]. Simultaneously with the formation of giant cells, parenchyma cells undergo hyperplasia, resulting in the formation of galls on the roots.

The use of specific bacteria has shown positive effects on nematode control in recent decades. Studies have shown that Bacillus subtilis has an important function in controlling M. javanica and promoting tomato plant growth [13,14]. B. subtilis regulates nematode behavior by competing for nutrients and interfering with host recognition [15]. Additionally, B. subtilis can maintain this nematicidal effect even under high temperatures, making it a potential biocontrol agent for use in greenhouse vegetable growing to suppress M. javanica populations.

Water-retaining polymers (WRPs) absorb, store, and release water molecules [16]. They are used in arid and semiarid regions to mitigate negative impacts of water deficit and improve WUE, consequently contributing to plant growth. Although the use of B. subtilis or WRPs may improve plant performance under drought conditions, no information on the effects of combining these two techniques is currently found in the literature.

Considering that water stress and parasitism of Meloidogyne spp. can cause damage and losses to tomato crops and the lack of information on combining WRPs and rhizobacteria to control phytonematodes, the objective of this study was to assess the effects of the combined application of Bacillus subtilis 34 (BS34) and WRP on the control of Meloidogyne javanica in tomato crops under water-deficit conditions.

2 Materials and methods

Experiments were conducted at Hydraulic and Phytopathology Laboratories and in a greenhouse of the State University of Montes Claros, Janaúba campus, Minas Gerais, Brazil (15°49′47″S, 43°16′05″W, and altitude of 533 m). Seedlings were produced by planting three tomato seeds (Santa Cruz group, cultivar Kada Gigante) in plastic trays filled with a commercial substrate (Bioplant^®). Thinning was performed after plant emergence, leaving one plant per cell.

Bacillus subtilis isolate 34 (BS34) (isolated from banana roots in an area infested with Panama disease) was cultured in a rice medium following the methodology described by Lopes et al. [13]. A 100 µL of bacterial suspension stored in saline solution at room temperature was added to 300 mL glass flasks containing 50 mL of rice medium for inoculation. These flasks were kept under shaking (220 rpm) on an orbital shaker at 28°C for 32 h to reach a concentration of 6.14 × 10⁸ CFU mL⁻¹ [14].

Tomato seedlings of the Santa Cruz group, cultivar Kada Gigante, were transplanted to 3 dm³ pots containing autoclaved sandy soil 21 days after sowing. The treatments with WRP contained 50 g of a WRP (Polyter^®) placed in the planting hole before transplanting the seedlings. Five grams of the product were immersed in 1 L of distilled water for 7 h for hydration. The treatments with BS34 consisted of 50 mL of the bacterial suspension applied to the soil around the seedling at 7, 9, and 11 days after transplanting (DAT); 50 mL of distilled water was applied to plants in the control treatment.

The species-level identification of M. javanica was performed based on the phenotypes of the esterase enzyme. The plant parasitic nematode was cultivated on Santa Cruz tomato plants, cultivar Kada Gigante, for 3 months. After this period, eggs were extracted from the roots according to the methodology of Hussey and Barker [21], modified by Boneti and Ferraz [22]. Subsequently, 7.5 mL of an aqueous suspension containing 5,000 eggs and possible second-stage juveniles (J2) of M. javanica was applied at 10 DAT. The nematode suspension was applied to three holes around each tomato plant at a depth of approximately 3 cm. Control treatment plants were treated with 7.5 mL of distilled water.

The seedlings were maintained under soil moisture corresponding to field capacity (10 kPa) until 15 DAT. Then, they were subjected to different soil water tensions (10, 25, 40, 55, and 70 kPa) through irrigation, maintaining the soil moisture at field capacity. Irrigation was based on readings of soil water tension twice a day (07 and 17 h) in each treatment, using tensiometers installed in the pots. Tension readings were converted to matric potential and then used in the soil water retention equation (Figure 1) to calculate the soil moisture level.

Figure 1

Soil water retention curve.

These soil moisture levels and soil volume in the pots were used to calculate the daily water volume to be applied [17]; the water volumes applied to each experimental unit during the experiment were recorded.

The following agronomic variables were assessed at 65 DAT: shoot and root fresh weights (RFWs), shoot dry weight, carotenoid content, chlorophyll a and b contents, and WUE. Fresh and dry weights were measured on a digital scale (Balmak^®); fresh weight was determined immediately after harvesting; the material was then dried in an air oven at 65°C until constant weight to obtain the dry weight. Contents of chlorophyll a and b and carotenoids were analyzed following the methodology proposed by Scopel et al. [18] and adjusted using the equation described by Arnon [19]. WUE was calculated by determining the ratio between shoot dry weight (g) and water consumption (L).

Regarding the nematode evaluation, galls, egg masses, eggs per gram of root, and J2 per 200 cm³ of soil were counted; the reproduction factor (RF) was determined using the formula RF = (final population/initial population/number of egg masses + J2 applied to the soil). Egg masses were counted after washing the roots in running water and immersing them in a phloxine B solution (15 mg L⁻¹) [20]. Eggs were extracted using the technique described by Hussey and Barker [21] modified by Boneti and Ferraz [22]; J2 was extracted from the soil using the technique described by Jenkins [23]. Egg suspensions and J2 of M. javanica were placed in a counting chamber and counted under an inverted microscope.

The experiment was conducted in a randomized block design with eight replications, using a 4 × 5 factorial arrangement consisting of four growing environments with different applications of BS34 and WRP (control, WRP alone, BS34 alone, and BS34 + WRP) and five soil water tensions (10, 25, 40, 55, and 70 kPa). The obtained data were subjected to analysis of variance at a 5% significance level using the F-test. Soil water tension data were subjected to regression analysis, and the mean values obtained for BS34 and WRP applications were compared using Tukey’s test. All statistical analyses were performed using the R software [24].

3 Results

The effect of the interaction between the growing environment (applications of BS34 and WRP) and soil water tension was significant for the following tomato agronomic variables: shoot fresh and dry weights, RFW, chlorophyll a and b contents, carotenoid content, and WUE (p < 0.05; Table 1). All treatments resulted in increased shoot fresh weight (SFW) compared to the control treatment, regardless of soil water tension.

The application of BS34 + WRP and BS34 alone to the soil at water tensions of 25, 40, and 55 kPa resulted in SFW. Treatments with the application of BS34 and WRP at a soil water tension of 10 kPa resulted in similar SFW. Applications of BS34 + WRP, BS34, and WRP at a soil water tension of 70 kPa resulted in similar SFW (Table 1). Tomato plants under application of BS34 + WRP at soil water tensions of 10, 25, 40, and 55 kPa resulted in higher shoot dry weights (SDW). BS34 applied alone resulted in higher SDW compared to the application of WRP and the control treatment, regardless of soil water tension (Table 1).

RFW was higher when applying BS34 + WRP to the soil, regardless of soil water tension (Table 2). BS34 applied alone resulted in higher RFW compared to the application of WRP and the control treatment, regardless of soil water tension. However, WRP applied alone yielded higher RFW means at soil water tensions of 10, 40, 55, and 70 kPa than the control treatment (Table 2). BS34 + WRP and BS34 alone at soil water tensions of 10 and 25 kPa resulted in higher chlorophyll a contents. WRP applied alone at soil water tensions of 40, 55, and 70 kPa resulted in higher chlorophyll contents than the control treatment (Table 2). Applying BS34 + WRP and BS34 alone at a soil water tension of 25 kPa resulted in higher chlorophyll b contents. Regarding the other soil water tensions, BS34 + WRP yielded better results. The application of WRP alone and the control treatment at soil water tensions of 10, 25, 40, and 55 kPa resulted in similar chlorophyll b contents (Table 2).

Carotenoid contents were higher in control treatment at soil water tensions of 25, 40, 55, and 70 kPa. The lowest carotenoid contents were found for BS34 + WRP, regardless of the soil water tension. BS34 applied alone resulted in lower carotenoid content than WRP applied alone (Table 2).

WUE was higher when applying BS34 + WRP, regardless of the soil water tension. Treatments with BS34 + WRP, BS34, and WRP, and the control treatment at a soil water tension of 10 kPa yielded 3.12, 2.58, 2.08, and 1.64 g of SDW, respectively, for each liter of water. BS34 applied alone at soil water tensions of 10, 40, 55, and 70 kPa resulted in a higher WUE than applying WRP alone; however, WRP alone yielded higher WUE than the control treatment, regardless of the soil water tension (Table 2).

SFW decreased as the soil water tension was increased, regardless of the growing environment (Figure 2). SFW decreased by 0.4342 g for each 1 kPa increase in the soil water tension when BS34 + WRP was applied. The smallest decrease (0.2617 g) was found when applying WRP alone. Applying BS34 alone resulted in a decrease of 0.3217 g, whereas the control treatment resulted in a higher decrease (0.4533 g). Treatments with BS34 alone resulted in a 0.35% decrease in SFW from the soil water tension of 10 to 25 kPa. The decreases in SFW found for BS34 + WRP, control treatment, and WRP alone were 0.73, 0.94, and 1.94%, respectively.

Figure 2

Shoot fresh weight of tomato plants grown under different soil water tensions and application of WRP and BS34 to the soil. *Significant at a 5% significance level using the t-test.

SDW decreased by 0.1392 g for each 1 kPa increase in soil water tension when applying BS34 + WRP (Figure 3). When applied alone, BS34 and WRP resulted in decreases of 0.12 and 0.1058 g in SDW, respectively. The control treatment resulted in a decrease of 0.2183 g for each 1 kPa increase in soil water tension. SDW decreased by 1.75% from the soil water tension of 10–25 kPa when BS34 + WRP was applied and decreased by 29% from the lowest to the highest soil water tension (10–70 kPa).

Figure 3

Shoot dry weight of tomato plants grown under different soil water tensions and application of WRP and BS34 to the soil. *Significant at a 5% significance level by using the t-test.

RFW decreased by 0.9175 g for each 1 kPa increase in the soil water tension when applying BS34 + WRP (Figure 4). When applied alone, BS34 and WRP resulted in decreases of 2.2683 and 2.2750 g in RFW, respectively. The control treatment resulted in a decrease of 2.2825 g for each 1 kPa increase in soil water tension. RFW decreased by 1.59% and 3.20% from the soil water tension of 10–25 kPa when applying BS34 + WRP and BS34 alone, respectively, and decreased by 2.58% (control treatment) and 8.67% (WRP applied alone).

Figure 4

Root fresh weight of tomato plants grown under different soil water tensions and application of WRP and BS34 to the soil. *Significant at a 5% significance level by using the t-test.

Chlorophyll a content decreased by 0.1118 µg cm⁻² for each 1 kPa increase in soil water tension when applying BS34 + WRP (Figure 5). When applied alone, BS34 and WRP resulted in decreases of 0.1414 and 0.1827 µg cm⁻², respectively. The control treatment resulted in a decrease of 0.2230 µg cm⁻² for each 1 kPa increase in soil water tension (Figure 5). Chlorophyll a content decreased by 0.46, 0.74, and 2.64% from the soil water tension of 10–25 kPa when applying BS34 + WRP, BS34, and WRP, respectively, and decreased by 3.64% in the control treatment.

Figure 5

Chlorophyll a content in tomato plants grown under different soil water tensions and application of WRP and BS34 to the soil. *Significant at a 5% significance level using the t-test.

Chlorophyll b content decreased by 0.0682 µg cm⁻² for each 1 kPa increase in soil water tension when applying BS34 + WRP (Figure 6). When applied alone, BS34 and WRP resulted in decreases of 0.0901 and 0.0881 µg cm⁻², respectively. The control treatment resulted in a decrease of 0.0909 µg cm⁻² for each 1 kPa increase in the soil water tension (Figure 6).

Figure 6

Chlorophyll b content in tomato plants grown under different soil water tensions and application of WRP and BS34 to the soil. *Significant at a 5% significance level using the t-test.

Carotenoid content increased by 0.0305 µg cm⁻² for each 1 kPa increase in soil water tension when BS34 + WRP was applied (Figure 7). WRP applied alone, and the control treatment resulted in increases of 0.0209 and 0.0270 µg cm⁻², respectively. The lowest increase in the carotenoid content (0.014 µg cm⁻²) was found when applying BS34 alone.

Figure 7

Carotenoid contents in tomato plants grown under different soil water tensions and application of WRP and BS34 to the soil. *Significant at a 5% significance level by the t-test.

WUE increased by 0.0298 g L⁻¹ for each 1 kPa increase in soil water tension when applying BS34 + WRP (Figure 8). When applied alone, BS34 and WRP resulted in increases of 0.0198 and 0.0176 g L⁻¹, respectively. However, the control treatment resulted in a decrease of 0.0039 g L⁻¹ in WUE for each 1 kPa increase in the soil water tension.

Figure 8

Water use efficiency in tomato plants grown under different soil water tensions and application of WRP and BS34 to the soil. *Significant at a 5% significance level using the t-test.

Regarding the control of Meloidogyne javanica, the interaction effect between the factors (growing environment and soil water tension) was significant for the following nematode variables: number of galls, number of egg masses, number of eggs per gram of root, and reproduction factor (Table 3). All treatments resulted in decreased numbers of galls and egg masses per gram of root compared to the control treatment, regardless of the soil water tension. Considering the soil water tension of 10 kPa, the highest decreases in the number of galls per gram of root were found for BS34 + WRP treatment (82.52%), followed by BS34 (74.82%) and WRP (10.49%) treatments, and the highest decreases in number of egg masses per gram of root were found for BS34 + WRP (80.69%), followed by BS34 (66.90%) and WRP (13.10%).

The number of eggs per gram of root varied among the growing environments. The control treatment showed a higher number of eggs, regardless of the soil water tension. Applications of BS34 + WRP and BS34 alone resulted in a lower number of eggs (Table 3). Considering the soil water tension of 10 kPa, the treatments with BS34 + WRP and BS34 alone resulted in decreases greater than 90% in the reproduction factor compared to the control treatment. Regarding the other water tensions, all treatments resulted in a decreased reproduction factor (Table 3).

The application of BS34 + WRP increased to 0.0002 galls g⁻¹ for each 1 kPa increase in soil water tension (Figure 9). When applied alone, BS34 and WRP resulted in increases of 0.0006 and 0.0020 galls g⁻¹, respectively, whereas the control treatment increased to 0.0022 galls g⁻¹ (Figure 9). BS34 + WRP and BS34 alone increased to 0.0007 egg masses g⁻¹ for each 1 kPa increase in soil water tension (Figure 10). WRP applied alone increased to 0.0012 egg masses g⁻¹, whereas the highest increase (0.0020 egg masses g⁻¹) was found for the control treatment. Egg masses increased by 9.67 and 12.5% from the soil water tension of 10–25 kPa when applying BS34 + WRP and WRP alone, respectively.

Figure 9

Number of galls per gram of root of tomato plants grown under different soil water tensions and application of WRP and BS34 to the soil. *Significant at a 5% significance level by using the t-test.

Figure 10

Number of egg masses of Meloidogyne javanica per gram of root of tomato plants grown under different soil water tensions and application of WRP and BS34 to the soil. *Significant at a 5% significance level by the t-test.

BS34 + WRP and BS34 alone resulted in increases of 0.04575 and 0.036158 eggs g⁻¹, respectively, for each 1 kPa increase in soil water tension (Figure 11). WRP applied alone increased to 0.04360 eggs g⁻¹, whereas the control treatment resulted in the highest increase (0.6385 eggs g⁻¹). The number of eggs increased by 2.54 and 22.42% from the soil water tension of 10–25 kPa for the application of BS34 + WRP and the control treatment, respectively (Figure 11).

Figure 11

Number of eggs of Meloidogyne javanica per gram of root of tomato plants grown under different soil water tensions and application of WRP and BS34 to the soil. *Significant at a 5% significance level by using the t-test.

The reproduction factor increased by 0.0069 and 0.0047 when applying BS34 + WRP and BS34 alone, respectively, for each 1 kPa increase in soil water tension (Figure 12). WRP alone increased to 0.0337, whereas the control treatment resulted in the highest increase (0.0464) in the reproduction factor (Figure 12).

Figure 12

Reproduction factor for Meloidogyne javanica in tomato plants grown under different soil water tensions and application of WRP and BS34 to the soil. *Significant at a 5% significance level by the t-test.

The interaction effect was not significant for the number of second-stage juveniles (J2) of M. javanica in the soil (p > 0.05); therefore, the individual effects of the factors were assessed (Table 4; Figure 13). The lowest numbers of J2 were found for BS34 and BS34 + WRP. The treatment with the application of WRP alone did not differ from the control treatment (Table 4). The number of J2 increased by 0.7346 for each 1 kPa increase in the soil water tension (Figure 13).

Figure 13

Number of second-stage juveniles (J2) of Meloidogyne javanica as a function of different soil water tensions. *Significant at a 5% significance level by using the t-test.

4 Discussion

The combined application of BS34 and a WRP), overall improved the development of tomato plants, regardless of soil water tension, as the results found for the evaluated agronomic variables were better compared to the control treatment (Tables 1 and 2). The use of BS34 can stimulate plant growth through solubilization of phosphate, nitrogen fixation, and production of indole-3-acetic acid, siderophores, and phytohormones [25,26,27].

The control of Meloidogyne javanica may have indirectly promoted the growth of tomato plants due to several factors. The B. subtilis isolates used in the present study showed promising results for controlling M. javanica and M. incognita in other crops [14,15,28,29,30]).

WRP can store large amounts of water and release it to plants as the soil dries, promoting a favorable microclimate for microbial activity [31]. The application of BS34 + WRP increased RFW as the soil water tension was increased compared to the control treatment (Table 2). RFW increased by 36.75, 38.68, 49.50, 60.07, and 59.00% for soil water tensions of 10, 25, 40, 55, and 70 kPa, respectively.

The water released by polymers mitigates water stress in plants. WRPs become highly hydrophilic in the soil, as they have carboxylic groups that facilitate water retention and subsequent release of water to plants [32,33], mitigating the effects of droughts, mainly in sandy soils in arid and semiarid regions [34]. Increasing water availability by applying WRP protects the root system against dehydration, reduces nutrient leaching, and improves soil aeration and drainage, thus contributing to root and shoot development [35,36,37].

The application of BS34 may have produced compounds that contributed to the development of tomato plants. B. subtilis not only mobilizes nutrients but also produces a wide range of compounds that directly affect plant growth, as it can alter the homeostasis of plant growth hormones, promoting cell division and plant growth [38].

Normal plant growth depends on maintaining a balance in water metabolism, as the main challenge for plant survival is the soil water content. The application of WRP to the soil reduced the stress caused by water deficit in the evaluated tomato plants, probably due to increased soil water availability, as WUE was higher in plants subjected to the combined application of WRP and bacteria (Table 2; Figure 8). The plants utilized 1 L of water to produce 3.12 g of dry matter when BS34 and WRP were applied to the soil and 1 liter of water to produce 1.64 g of dry matter in the control treatment.

Photosynthetic pigment contents, such as chlorophylls, in tomato plants can affect the amount of radiation absorbed by the plant during its development. The negative results found for pigments in treatments with high soil water tensions and the absence of BS34 or WRP resulted in lower dry weights. Tomato plants grown under the application of BS34 + WRP presented higher chlorophyll a and b contents, which resulted in better vegetative development (Table 2; Figures 5 and 6).

Carotenoid contents increased as the water deficit was increased (increases in soil water tension), regardless of the growing environment (Figure 7). This may be attributed to a plant strategy to minimize damage to the photosynthetic apparatus, as carotenoids act as a filter of ultraviolet radiation, absorbing visible radiation and providing photoprotection by rapidly quenching the excitation of chlorophyll, thus preventing photooxidation [39].

The numbers of galls, egg masses, and eggs per gram of root, the number of J2 in the soil, and the reproduction factor of M. javanica increased as the soil water tension was increased (Table 3; Figure 13); this may be attributed to decreases in the root system development caused by water deficit, resulting in higher numbers per gram of root. These results are consistent with those reported by Khanizadeh et al. [40], who found higher reproduction of M. hapla in strawberry plants for the highest evaluated water deficit.

The reproduction factor of M. javanica, which represents the nematode reproductive capacity, was less than 1 when applying BS34 + WRP or BS34 alone, regardless of soil water tension. This is a similar result to that found for plants resistant to this nematode [41]. The application of BS34 controlled the development of M. javanica (Table 3; Figure 12). Nematicidal activity of B. subtilis has been reported in other studies [13,14,42]. B. subtilis damages Meloidogyne spp. by regulating their behavior, competing for nutrients, and interfering with host recognition [43]. Furthermore, B. subtilis can maintain the nematicidal effect, even under high temperatures, explaining the reduction of M. javanica when applying BS34 + WRP, even at high soil water tensions.

During the infective stage (J2), approximately 30% of the body weight of M. javanica consists of lipids, which are the main energy source used during the penetration and parasitism of the host [44]. BS34 has been described as a lipase producer [45]. Lipase enzymes degrade lipid molecules in nematodes. Lipases have an important function in controlling M. javanica by degrading its energy reserves and acting on lipid membranes [46]. This may explain the low parasitism of M. javanica in tomato plants grown in soils subjected to the application of BS34.

Lipids are essential for the survival of phytonematodes. Additionally, decreases in lipid reserves are associated with reduced infectivity and motility and delayed juvenile development [47]. Lipid losses in M. javanica can affect the nematode infectivity, resulting in its death. Losses of 50–60% in lipid reserves in Meloidogyne species result in loss of infectivity for the nematode [48].

5 Conclusions

The combined application of BS34 and a WRP to the soil improves plant growth and results in efficient control of Meloidogyne javanica nematodes in tomato plants grown under soil water tensions of 10, 25, 40, 55, and 70 kPa.

Water deficit increases M. javanica population. Soil water tensions higher than 10 kPa decrease tomato plant development.