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Article

Native Phosphate Solubilizing Bacteria Mitigate the Effect of the Phytopathogen Sclerotium rolfsii on Peanut (Arachis hypogaea L.) Plants in a P-Deficient Environment

by
Ana Laura Gentile
1,2,
Maria Soledad Figueredo
2,
Maria Soledad Anzuay
1,2,
Maria Laura Tonelli
1,2,
Adriana Fabra
1,2,
Tania Taurian
1,2 and
Liliana Ludueña
1,*
1
Instituto de Investigaciones Agrobiotecnológicas (INIAB) Río Cuarto, Río Cuarto 5800, Argentina
2
Departamento de Ciencias Naturales, Facultad de Ciencias Exactas, Físico-Químicas y Naturales, Universidad Nacional de Río Cuarto, Agencia Postal 3, Río Cuarto 5800, Argentina
*
Author to whom correspondence should be addressed.
Agronomy 2025, 15(10), 2278; https://doi.org/10.3390/agronomy15102278
Submission received: 25 August 2025 / Revised: 22 September 2025 / Accepted: 24 September 2025 / Published: 26 September 2025
(This article belongs to the Special Issue Research Progress of Beneficial Microorganisms in Controlling Crop Pathogens)
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Abstract

Phosphorus (P) deficiency and soil-borne fungal diseases are major constraints to peanut (Arachis hypogaea L.) production. Phosphate-solubilizing bacteria (PSB) can improve P availability in the soil, thereby promoting plant growth. However, their potential to improve plant resistance against pathogens under P-limited conditions remains poorly understood. In this study, we first evaluated the ability of two PSB strains, Enterobacter sp. J49 and Serratia sp. S119, to induce systemic resistance (ISR) in peanut plants against the fungal pathogen Sclerotium rolfsii. Results showed that strain S119 reduced disease severity by 40%, whereas strain J49 reduced both incidence (30%) and severity (40%). The protective effect produced by strain J49 was mediated by ISR, as evidenced by the early increase in phenolic compounds accumulation (48 h) and total peroxidase activity (72 h) in inoculated plants. Under P-deficient conditions, the J49 strain was also able to protect peanut plants against S. rolfsii, as demonstrated by a significant reduction in disease severity (55%). These findings highlight the potential of multifunctional bacterium Enterobacter sp. J49 to enhance sustainable peanut production by simultaneously improving P acquisition and strengthening plant defense mechanisms.

1. Introduction

It is estimated that the global population will reach nearly 10 billion by 2050, demanding a significant increase in agricultural production to ensure food security [1]. Achieving this goal represents a major challenge, particularly under the current scenario of global climate change, which is already exerting profound impacts in agroecosystems through the increased frequencies of extreme weather events (such as drought, flooding, and heat waves) as well as rising soil salinity and declining nutrient availability. In addition, soil-borne and foliar pathogens are becoming more prevalent and aggressive, further threatening crop productivity and food security worldwide [2,3]. Among them, soil-borne pathogens represent a particularly severe constraint, as they can persist in the soil for long periods, spread rapidly under favorable conditions, and cause substantial yield losses [4].
Peanut (Arachis hypogaea L.) is one of the main oilseeds produced worldwide [5]. It is a legume rich in protein and oil, and it is used for direct human food consumption (confectionery peanuts), indirect food production (butter, oil, etc.), or as animal feed (pellet, fodder) [6]. Argentina is the world’s seventh largest producer of peanuts and stands out as the leading peanut exporter by exporting more than 70% of its production [7]. Worldwide, peanut production is commonly affected by various biotic and abiotic factors. Among the biotic factors, diseases caused by bacteria, viruses, nematodes, and fungi become particularly relevant depending on the specific environmental and climatic conditions of each year. One of the main rhizoplane pathogens that cause severe effects on peanut productivity is Sclerotium rolfsii, the causal agent of stem wilt [8,9]. The yield losses caused by stem wilt range from 25% to 80% in years with a high disease severity [10]. The development of stem wilt is favored by periods of high humidity and high temperatures (>30 °C), conditions that frequently occur in peanut cultivating areas [8]. Typically, fungicides and agronomic practices such as crop rotation with non-host species or deep plowing to bury infected residues are applied for disease control. However, these methods are not completely effective in mitigating its impact [11,12]. Moreover, the use of synthetic fungicides entails significant economic and environmental costs. Agrochemicals have been reported to leave harmful residues in the soil, water, and atmosphere, and their overuse can lead to the development of resistance in phytopathogenic strains [13,14].
In soil microbiome, some microorganisms associated with plants or the metabolites secreted by them are able to stimulate a plant defense response, providing an improvement in its defensive capacity against a broad spectrum of pathogens. This mechanism elicited by plant growth-promoting bacteria (PGPB) is called the induction of systemic resistance (ISR) and it is characterized by not causing symptoms in the host plant [15]. Unlike other protection mechanisms, direct interaction between the PGPB and the pathogen is not necessary. This systemic defense response is elicited spatially and temporally separated from the phytopathogen attack [15]. PGPB induces a “priming” state in the plant in which its capacity to mobilize the defensive cellular response is increased. Then, when a pathogen attack occurs, plants respond more quickly and/or effectively, resulting in local and systemic immunity [16]. ISR is primarily manifested through the activation of biochemical and mechanical defense mechanisms [17]. In particular, ISR elicited by PGPB against soil-borne fungal pathogens has been associated with ultrastructural modifications of the cell wall that prevents the entry of the pathogen’s mycelium into the vascular zone. These structural modifications involve the deposition of newly formed callose and the accumulation of phenolic compounds (PCs) at the site of penetration of the invading hyphae [18,19]. That is why the content of total PCs or their derived polymers, is one of the most studied parameters when evaluating ISR [20,21,22]. Peroxidase (PX) enzymes have also been associated with ISR as they play an important role in limiting the spread of cellular infection by establishing structural barriers [23]. This makes them key components of plant resistance against broad-host-range necrotrophic pathogens such as S. rolfsii.
On the other hand, peanut cultivation is also affected by different abiotic stresses such as drought, frost, salinity, high temperatures, floods, and nutrient deficiencies [9]. In recent years, it has been observed that the intensification of agricultural practices has generated a decrease in the nutritional quality of soils, presenting low levels of nutrients, predominantly phosphorus (P) [24,25,26]. Although P is an abundant element in soil forming organic and inorganic compounds, its availability is reduced by its low solubility and great fixation to soil components [27]. Plants can only use a small proportion of the available soil P and the main form directly absorbable by them is inorganic orthophosphate ions (HPO4−2 and H2PO4−) [28]. A P deficient environment affects photosynthesis, signal transduction, macromolecule biosynthesis, and biological N2 fixation, among other processes [29]. Thus, the deficiency of this nutrient in agricultural soils represents a serious limitation for the growth and yield of crops such as peanuts. Currently, farmers apply synthetic phosphate fertilizers to address this issue [30]. However, the use of these chemical fertilizers has long-term environmental impacts, including water eutrophication, depletion of soil fertility, and loss of biodiversity [31,32]. Furthermore, 75–90% of the applied P is precipitated through the formation of metal cation complexes and rapidly becomes fixed in the soil, leaving only a small fraction available for plant uptake [33]. These environmental consequences have prompted the search for sustainable P supplementation strategies that can simultaneously enhance crop productivity and preserve soil quality [34]. In the rhizosphere, phosphate-solubilizing bacteria (PSB) represent an important group within PGPBs. These microorganisms are capable of solubilizing inorganic (Pi) and mineralizing organic (Po) insoluble phosphate compounds, thereby making P more available to plants [35]. Phosphate solubilization is the process of releasing orthophosphate ions from soil Pi compounds (Fe-P, Al-P and Ca-P) [36,37]. PSB carry out this mechanism primarily through the secretion of acids, which are mainly organic and, less frequently, inorganic, leading to the acidification of the extracellular environment [38]. Organic acids are typically low-molecular-weight compounds containing one or more carboxyl groups, which chelate cations bound to P (Fe3+, Al3+, Ca2+), thereby converting it into soluble forms [36,37]. The mineralization of Po occurs through the action of enzymes such as acid phosphatases and phytases [39].
The native PSB strains Serratia sp. S119 and Enterobacter sp. J49, selected for this study, belong to a PGPB collection previously obtained from peanut plants [40]. These strains are highly efficient in solubilizing inorganic and mineralizing Po, respectively, and also exhibit phytohormone production (indole-3-acetic acid), ACC deaminase activity, and high root colonization under P-deficient conditions [41]. Moreover, both strains have been shown to promote the growth of peanut and maize plants in microcosm and field assays [24,25]. Regarding their biocontrol traits, Serratia sp. S119 and Enterobacter sp. J49 have previously been evaluated only for their in vitro antagonistic activity against the peanut pathogens Sclerotinia sclerotiorum and Sclerotinia minor [40], with antibiosis observed exclusively for strain S119 against the former fungus.
Although P deficiency and the incidence of fungal diseases frequently occur simultaneously in natural environments, studies evaluating the effect of PGPB inoculation under combined stress conditions remain scarce. In particular, research on PSB-induced systemic resistance under the dual stress of P deficiency and pathogen challenge is especially limited and represents a critical knowledge gap that our study aims to address. This underscores the importance of investigating the interaction between multiple stresses, as it more accurately reflects real field scenarios. To date, no studies have addressed the peanut–PSB interaction under simultaneous P deficiency and fungal pathogen pressure. Considering the imminent increase in the frequency and intensity of combined stress events due to climate change, there is a clear need to develop sustainable strategies to enhance crop tolerance to these challenges.
The main objective of this study was to evaluate whether inoculation with native PSB can mitigate the effects of simultaneous abiotic (phosphorus deficiency) and biotic (fungal pathogen) stresses in peanut plants. All experiments were conducted under controlled conditions to determine whether PSB inoculation could reduce the detrimental effects of the phytopathogen Sclerotium rolfsii through ISR in a P-deficient environment.

2. Materials and Methods

2.1. Bacterial Strains, Culture Conditions, and Inoculum Preparation

Two phosphate-solubilizing bacteria (PSB) from a previous laboratory collection obtained from peanut plants cultivated in the central and southern regions of Córdoba were used in this study [40]. These strains, Serratia sp. S119 and Enterobacter sp. J49, were isolated from root nodules and were selected for their effective phosphate-solubilizing capacity as well as other plant growth-promoting activities. Both strains have also been shown to promote peanut and maize growth in field assays [24,25]. Selected strains were grown on LB medium (Luria-Bertani, Sigma—Aldrich, St. Louis, MO, USA) at 28 °C [42]. For long-term storage, strains were kept in 20% glycerol at −80 °C, and for short-term storage, in 40% glycerol at −20 °C.
For inoculum preparation, bacterial cultures (~108 CFU mL−1) were centrifuged at 2500 rpm for 5 min at room temperature, and the cells were resuspended in a sterile 0.85% NaCl solution. The number of viable cells was determined following the method described by Somasegaran and Hoben [43].

2.2. Fungal Phytopathogen Strain Culture Conditions and Inoculum Preparation

The fungal phytopathogen S. rolfsii was obtained from infected peanut plants (Córdoba, Argentina) and grown on Potato Dextrose Agar (PDA) (Britania, Buenos Aires, Argentina) according to Mena et al. [44] at 28 °C for 7 days. For long-term storage the phytopathogen was kept in 15% glycerol at −20 °C and sclerotia were maintained at room temperature.
For fungal inoculum preparation, sterile wet wheat seeds contained in a Petri dish were infected with a 5 mm diameter S. rolfsii mycelial plug. They were maintained at 28 °C for 7–10 days approximately until abundant mycelium growth was observed [44].

2.3. In Vitro Inhibitory Effect of Phosphate-Solubilizing Bacterial Strains on Fungal Peanut Pathogen Growth (Antibiosis)

To assess the ability of the selected PSB to inhibit the growth of the S. rolfsii, a 5 mm mycelial plug of the fungal pathogen from a 7-day old culture was placed in the center of a PDA plate following the methodology proposed by Tonelli et al. [45]. Serratia sp. S119 or Enterobacter sp. J49 growing in LB medium were streaked on both sides of the plate and incubated at 28 °C for 6 days. The plates with only a fungal culture served as the control. Fungal growth inhibition was assessed by measuring the mycelial radial growth and compared to control growth. Dual culture assays were carried out in triplicate.

2.4. Peanut Seeds Surface Disinfection and Germination

Seeds of Arachis hypogaea L. (var. Runner, cv. Granoleico) were surface-disinfected and germinated following the protocol of Vincent [46]. In short, seeds were immersed in 96% ethanol for 30 s, treated with 30% H2O2 (Anedra, Buenos Aires, Argentina) for 20 min, and rinsed six times with sterile distilled water. Subsequently, disinfected seeds were placed in the dark at 28 °C on sterile Petri dishes containing a layer of Whatman No. 1 filter paper and moistened cotton, where they were allowed to germinate until radicles reached about 2 cm in length.

2.5. Evaluation of Induction of Systemic Resistance

To prevent direct contact between the bacteria and the fungi, plants were grown following the methodology described by Figueredo et al. [47]. Briefly, two plastic cups filled with a mixture of sterile perlite and sand (2:1) were placed one above the other and connected by a hole made in the base of the upper cup. A germinated peanut seed was sown in the upper plastic cup so that the root reached the bottom plastic cup through the hole that connected both cups.
Peanut seedling radicles (placed in the bottom cup) were inoculated with 3 mL of the phosphate-solubilizing bacterial suspensions (108 CFU mL−1). After seven days, plants were exposed to the pathogen by depositing a wheat seed colonized with S. rolfsii mycelium (20 mg) in the crown zone (upper cup). To promote conditions conducive to disease development, seedlings were covered with nylon bags for 72 h. The treatments included in this experiment were as follows: 1—plants inoculated with the PSB Serratia sp. S119, 2—plants inoculated with the PSB Serratia sp. S119 and challenged with phytopathogen S. rolfsii, 3—plants inoculated with the PSB Enterobacter sp. J49, 4—plants inoculated with the PSB Enterobacter sp. J49 and challenged with phytopathogen S. rolfsii, 5—pathogen-inoculated plants, and 6—untreated control plants. Plants were grown under a controlled environment (16 h light and 28 °C/8 h darkness and 22 °C, 50% relative humidity), watered regularly and supplied once a week with Hoagland solution [48].
At 30 days after bacterial inoculation, the assessment of disease incidence (measured as % of diseased plants) and disease severity (evaluated by shoot and root dry weights after dried at 60 °C for 10 days until constant weight) were recorded. The experiment was conducted three times, with 10 plants per treatment in each replicate.

2.6. Combined Stress Assay

To evaluate the effect of PSB inoculation on peanut plants under combined stress conditions, plants were grown either under phosphorus-sufficient conditions (with a soluble P source, SOLP) or under phosphorus-deficient conditions (with an insoluble P source, tricalcium phosphate, TCP (Sigma—Aldrich, St. Louis, MO, USA)). The following eight treatments were applied: 1—pathogen-inoculated plants + SOLP, 2—pathogen-inoculated plants + TCP, 3—plants inoculated with Enterobacter sp. J49 and challenged with S. rolfsii + SOLP, 4—plants inoculated with Enterobacter sp. J49 and challenged with S. rolfsii + TCP, 5—plants inoculated with Enterobacter sp. J49 + SOLP, 6—plants inoculated with Enterobacter sp. J49 + TCP, 7—untreated control plants + SOLP, and 8—untreated control plants + TCP.
The same growth system described previously was used, but irrigation was performed with Hoagland solution containing tricalcium phosphate (Ca3(PO4)2, 5 g L−1) as the sole phosphorus source in the bottom plastic cup. For the phosphorus-sufficient controls (SOLP), an unmodified Hoagland solution was used. Plants were grown under a controlled environment (16 h light and 28 °C/8 h darkness and 22 °C, 50% relative humidity).
At 30 days after bacterial inoculation, the assessment of disease incidence (measured as % of diseased plants) and disease severity (evaluated by shoot and root dry weights after dried at 60 °C for 10 days until constant weight) were recorded. The experiment was conducted three times, with 10 plants per treatment in each replicate.

2.7. Total Peroxidase (PX) Activity Determination

For peroxidase (PX) activity assays, 0.1 g of peanut stems were ground in liquid nitrogen with a mortar and pestle in an extraction buffer (50 mM potassium phosphate, 1 mM EDTA, pH 7.4, Anedra, Buenos Aires, Argentina) supplemented with 1% polyvinylpyrrolidone (PVP) (Sigma–Aldrich, St. Louis, MO, USA). The homogenate was centrifuged at 9500× g for 20 min at 4 °C, and the resulting supernatant was stored at −20 °C until analysis. The protein concentration of each sample was determined by the method described by Bradford [49], using bovine albumin (1 mg mL−1) as standard. Total PX activity was determined by measuring increase in absorbance at 470 nm according to Sosa Alderete [50]. Activity was calculated applying Lambert–Beer law using molar extinction coefficient 11.3 mM−1cm−1. One unit (U) of PX was defined as the amount of enzyme that catalyzes the formation of 1 µmol of product in 1 min of reaction. PX activity was expressed as U mg−1 protein. The experiment was conducted three times, with 10 plants per treatment in each replicate.

2.8. Phenolic Compounds (PC) Determination

Phenolic compounds were extracted following Ainsworth et al. [51] with minor modifications. Briefly, 0.1 g of peanut stems were ground in liquid nitrogen, suspended in 95% methanol, and incubated 48 h at room temperature in the dark. After centrifugation (9500× g, 5 min), supernatants were stored at −20 °C. Quantification was performed by mixing the extract with a Folin–Ciocalteu reagent and Na2CO3 (both from Anedra, Buenos Aires, Argentina), and absorbance was measured at 765 nm. Results were expressed as gallic acid equivalents (GAE) per gram of dry weight. The assay was repeated three times with 10 plants per treatment.

2.9. Quantification of Phosphorus Content in Vegetal Tissue

Total phosphorus in aerial tissue of peanut plants was determined using the vanadomolybdo-phosphoric acid yellow color method [52] with modifications in which only nitric acid was used for the digestion of vegetable tissues. The experiment was conducted three times, with 10 plants per treatment in each replicate.

2.10. Statistical Analysis

Data were subjected to analysis of variance (ANOVA). Statistical significance was determined by the LSD Fisher test (p ≤ 0.05), using Infostat software version 2014 [53]. For the comparison between the two treatments we applied a Student t-test.

3. Results

3.1. In Vitro Evaluation of the Antimicrobial Activity of Serratia sp. S119 and Enterobacter sp. J49 Against the Pathogen Sclerotium rolfsii

In order to evaluate in vitro direct antagonistic effects (antibiosis) of bacterial strains Serratia sp. S119 and Enterobacter sp. J49 on S. rolfsii, the diameter of the fungal growth halo was assessed in the presence and absence of each strain individually. Obtained results indicated that none of the evaluated strains were capable of directly inhibiting S. rolfsii growth in vitro, as no inhibition halos were observed (Figure 1).

3.2. Induction of Systemic Resistance (ISR) in Peanut Plants by the PSB Strains Serratia sp. S119 and Enterobacter sp. J49

To evaluate whether the bacterial strains can protect peanut plants against S. rolfsii through the ISR mechanism, disease incidence and severity were assessed in plants inoculated with the PSB Serratia sp. S119 or Enterobacter sp. J49 and subsequently challenged with the fungal pathogen.
Plants inoculated with Serratia sp. S119 and challenged with S. rolfsii showed no statistically significant differences in disease incidence with respect to those plants treated with S. rolfsii only (Figure 2). In contrast, plants inoculated with Enterobacter sp. J49 and challenged with the pathogen showed a statistically significant reduction in disease incidence (30%) compared to pathogen-inoculated plants (Figure 2).
To assess disease severity, plant biomass was measured at harvest (30 days’ post-bacterial inoculation), distinguishing between shoot and root dry weight. In plants inoculated with Serratia sp. S119 and challenged with S. rolfsii, a statistically significant increase in shoot dry weight (40%) was observed compared to pathogen-inoculated plants (Figure 3). As expected, the highest shoot dry weight values were recorded in untreated control plants and in those only inoculated with Serratia sp. S119. Regarding root dry weight, no statistically significant differences were detected between plants inoculated with Serratia sp. S119 and challenged with S. rolfsii and the pathogen-inoculated plants.
On the other hand, plants inoculated with Enterobacter sp. J49 and challenged with the pathogen showed a statistically significant increase in shoot (40%) and root (66%) dry weight compared to pathogen-inoculated plants (Figure 4). The values of dry weight tissues observed in plants inoculated with the J49 strain and then challenged with S. rolfsii were similar to those recorded in untreated control plants and in those plants inoculated only with the bacterial strain (without the pathogen).
Based on the overall results, Enterobacter sp. J49 was chosen for further investigation, as it was the sole strain capable of protecting peanut plants through the ISR mechanism.

3.3. Elicitation of Early Total Peroxidase Activity and Phenolic Compounds Accumulation in Peanut Plants by Enterobacter sp. J49 Inoculation

To evaluate whether Enterobacter sp. J49 elicits the plant defense system, total PX enzyme activity and the accumulation of PC were measured at 24, 48, and 72 h post-S. rolfsii challenge at the infection site. This sampling time frame was selected based on previous studies showing that these time points are critical for peanut defense responses against S. rolfsii [47]. Results at 24 h indicated that inoculation with Enterobacter sp. J49 did not modify total PX enzyme activity nor PC accumulation, showing values similar to those of control plants in both cases (Figure 5 and Figure 6). Similarly, total PX activity levels of pathogen-inoculated plants were comparable to those of control plants and those inoculated only with Enterobacter sp. J49. At 48 h, PC accumulation was significantly higher in plants inoculated with the J49 strain and challenged with S. rolfsii with respect to the other treatments, while total PX activity showed no significant difference among treatments. Finally, at 72 h, plants inoculated with Enterobacter sp. J49 and challenged with S. rolfsii showed a statistically significant increase in total PX activity compared to all the treatments including pathogen-inoculated plants. In addition, a significant increase in PC content was observed in these plants and in the pathogen-inoculated plants, relative to the control plants (Figure 5 and Figure 6).

3.4. Inoculation of Native Phosphate Solubilizing Bacteria Enterobacter sp. J49 to Mitigate the Simultaneous Effect of Pathogen S. rolfsii (Biotic) and P Deficiency (Abiotic) Stresses

To evaluate if Enterobacter sp. J49 can protect peanut plants simultaneously exposed to S. rolfsii (biotic stress) and P deficiency (abiotic stress), initially the incidence of fungal disease (IFD) was assessed under these conditions. Results indicated that regardless of P deficiency (TCP) or P sufficiency (SOLP), and the presence or absence of Enterobacter sp. J49, all peanut plants challenged with S. rolfsii showed disease symptoms. It was possible to observe that plants inoculated with Enterobacter sp. J49 and exposed to both stresses (challenged with S. rolfsii and P deficiency) presented a lesser IFD than pathogen-inoculated and P deficiency plants (20.3%) even though this difference was not statistically different (Figure 7). Under phosphorus-sufficient conditions (SOLP), plants inoculated with Enterobacter sp. J49 and challenged with S. rolfsii, showed a significant reduction in IFD compared to the pathogen-inoculated plants grown with SOLP.
Subsequently, disease severity was assessed by measuring plant biomass, distinguishing between shoot and root dry weight at the time of harvest (30 days post-bacterial inoculation). Under phosphate-deficient conditions (TCP), plants inoculated with the J49 strain and challenged with S. rolfsii exhibited reduced disease severity, as evidenced by a significant increase in shoot dry weight compared to pathogen-inoculated plants (Figure 8). Under phosphate-available conditions (SOLP), inoculated and challenged plants showed increased shoot and root dry weight relative to the pathogen-inoculated plants.
In pathogen-inoculated plants, no statistically significant differences in shoot or root dry weight were observed between phosphate-deficient (TCP) and phosphate-available (SOLP) conditions. Both untreated control plants and those inoculated only with Enterobacter sp. J49 displayed similar shoot and root dry weight values, which were higher than those observed in the pathogen-inoculated plants (Figure 8).
Considering Enterobacter sp. J49 is a highly effective PSB, we evaluated if its inoculation on peanut plants challenged with the fungal pathogen in a phosphate deficient environment allowed bacteria to express this plant-promoting ability and enhance P content. Aerial P content was quantified in plants simultaneously exposed to S. rolfsii and phosphate deficiency (PTC) at harvest time (30 days post-bacterial inoculation). Results indicated that plants inoculated with Enterobacter sp. J49 but not challenged with the pathogen, showed no statistically significant differences in P content compared to untreated control plants (Figure 9). On the other hand, a statistically significant increase in P content was observed in the aerial tissue of plants challenged with S. rolfsii, regardless of the presence of Enterobacter sp. J49, compared to untreated control plants and those inoculated only with the PGPB strain (Figure 9).

4. Discussion

Peanut production worldwide is constrained by the high incidence of soil-borne fungal pathogens and the widespread occurrence of nutritional deficiency, particularly phosphorus (P) limitation. Traditionally, these problems have been managed through chemical inputs; however, their long-term use raises concerns regarding environmental safety and sustainability. The PGPB, particularly PSB, represent a promising alternative, as they can simultaneously enhance P availability and stimulate plant defense mechanisms. Despite the fact that P deficiency and pathogen pressure often co-occur in field conditions, little is known about how PSB performs under these combined stress scenarios in peanut plants. In particular, the ability of PSB to trigger ISR in the presence of nutrient stress and pathogen challenge remains largely unexplored. Recently, it has been reported that PSB, in addition to their role in making phosphorus available to plants, also have potential as biocontrol agents [54,55].

4.1. Native Phosphate Solubilizing Bacteria as Inducers of Peanut Systemic Resistance Against S. rolfsii

In this study, the native peanut strains Serratia sp. S119 and Enterobacter sp. J49, both PSBs, were evaluated for their ability to protect peanut plants against the phytopathogen S. rolfsii. Even though the S119 strain has previously demonstrated antagonistic activity against Sclerotinia sclerotiorun [40], none of the strains were able to inhibit the growth of S. rolfsii in a dual culture assay. In contrast, Zhang et al. [56] reported that Enterobacter sp. V1 inhibits the phytopathogen Verticillium dahliae through the production of an antifungal protein. Additionally, other studies have described antifungal activity within the Serratia genus; for instance, Serratia marcescens MSU97 and strains of Serratia plymuthica have shown in vitro antagonistic effects against plant-pathogenic oomycetes [57].
Besides antagonistic activity, biocontrol bacteria can enhance plant defense mechanisms by promoting the accumulation of structural barriers in plant cells and triggering biochemical defense responses in the host. This process, known as ISR, provides broad-ranging protection against a variety of pathogens [58]. ISR has been described as a more effective approach than direct antagonism for managing crop diseases [59,60]. To evaluate whether the PSB strains Serratia sp. S119 and Enterobacter sp. J49 can protect peanut plants against S. rolfsii through ISR, disease incidence and severity were assessed. In this study, the S119 strain was able to diminish disease severity but no significant decrease in the incidence was detected. Previous reports have documented ISR-mediated protection by members of the Serratia genus in rice and wheat [61,62]. The limited and less consistent protection observed here could be related to the specificity of ISR induction, which may vary depending on the host–microbe interaction and the pathogen involved. In addition, variability in the magnitude and timing of plant defense activation could also contribute to the weaker effect observed, as previously highlighted for other PGPR–plant systems [60]. The results obtained in the present study suggest that S119 may have some potential to protect peanut plants against this pathogen, but further studies are needed to clarify its role.
On the other hand, peanut plants inoculated with the strain Enterobacter sp. J49 and challenged with the same pathogen displayed a significantly reduced disease incidence and severity. Values of growth parameters (shoot and root dry weight) under this condition were similar to those observed in untreated control plants and in plants inoculated with the bacterium alone (in the absence of S. rolfsii). Thus, based on these results and considering the plant growth system used in these experiments, it is possible to suggest that Enterobacter sp. J49 protects peanut plants against S. rolfsii through ISR. These results are consistent with the findings of Son et al. [63], who demonstrated that Enterobacter asburiae and Enterobacter cancerogenus were able to reduce the severity of gray leaf spot disease caused by Stemphylium solani through ISR in pepper plants.
It is well established that biocontrol agents—both bacterial and fungal—can activate plant defense mechanisms through induced systemic resistance (ISR) by priming the plant’s immune system. Upon pathogen attack, ISR is manifested through a wide range of genetic, biochemical, physiological, and structural modifications [23]. Among structural defenses, the most frequently reported responses include reinforcement of the cell wall via lignification, suberization, callose deposition, and the accumulation of phenolic compounds (PC) at infection sites. Phenolic derivatives play multiple roles: some contribute structurally to cell wall strengthening, while others exhibit antimicrobial or antioxidant activity [64]. In addition to PC, peroxidases (PX) are key enzymes commonly associated with ISR and are frequently used as markers of its activation. Plants possess a wide array of PX isoforms that perform diverse physiological functions, including auxin catabolism, lignin and suberin biosynthesis, cell wall cross-linking, pathogen defense, and cell elongation. In this study, we demonstrated that Enterobacter sp. J49 increased total PX activity at 72 h and PC accumulation at 48 h post-challenge with S. rolfsii. These results suggest that Enterobacter sp. J49 enhances the plant’s defense system against S. rolfsii, triggering a faster ISR-mediated response. This is consistent with the concept of priming [60], in which prior exposure to a beneficial microorganism leads to quicker activation of defense pathways upon pathogen attack. Comparable findings have been reported by Jetiyanon et al. [65] and Arora et al. [66], where Enterobacter asburiae RS83 inoculation increased PX and superoxide dismutase (SOD) activity in lettuce challenged with Pectobacterium carotovorum. Similarly, Enterobacter bugandensis WRS7 enhanced defense-related enzyme activity in wheat, providing protection under drought stress conditions.
In this work, we used the two-pot system described by Figueredo et al. [47]. This approach ensures spatial separation between the PSB and the phytopathogen—an essential requirement to experimentally demonstrate ISR [67]. In addition, PSB and phytopathogen inoculation were carried out seven days apart, providing temporal separation between the two. Spatial separation guarantees that the observed effect is not the result of direct antagonism between the biocontrol bacterium and the pathogen (initially discarded in this study), while temporal separation allows the biocontrol bacterium to prime the plant’s defense system prior to pathogen attack. Taken together, the absence of in vitro antagonism against S. rolfsii and the evidence of a priming response strongly suggest that the protection exerted by Enterobacter sp. J49 in peanut plants against this phytopathogen is mediated by ISR.

4.2. The Phosphate Solubilizing Bacterium Enterobacter sp. J49 Mitigates the Effect of the Phytopathogen S. rolfsii on Peanut Plants Growing Under P-Deficient Conditions

It is known that climate change can increase the frequency, complexity, and intensity of several stress combinations that affect crops, soils, and microbial communities [3]. While there is substantial information regarding plant responses to individual stresses, data on how plants respond to combined abiotic and biotic stresses remain scarce [68,69,70]. This is one of the first studies to evaluate the ability of PGPB strains to alleviate the effects of simultaneous P deficiency and fungal pressure stresses in peanut plants.
In order to evaluate whether the native PGPB Enterobacter sp. J49 is capable of protecting peanut plants simultaneously exposed to S. rolfsii and P deficiency, disease incidence and severity caused by this fungal pathogen were assessed in peanut plants grown under phosphorus-deficient (PTC) and phosphorus-sufficient (PSOL) conditions. Under P-deficient conditions, plants inoculated with the J49 strain and challenged with S. rolfsii displayed lower disease severity, as indicated by a significant increase in shoot dry weight compared to pathogen-inoculated plants. These results confirm the ability of Enterobacter sp. J49 to protect peanut plants from the phytopathogen S. rolfsii by ISR in a P-deficient environment. Several previous studies [24,25,71] have already proposed the application of Enterobacter sp. J49 in phosphorus-deficient soils as a strategy to alleviate this abiotic stress. The ability of this strain to protect peanut crops against S. rolfsii through ISR in a P-deficient environment adds to the positive attributes of this PGPB.
Considering that Enterobacter sp. J49 is a highly effective PSB, we evaluated whether its inoculation in peanut plants challenged with the fungal pathogen under P-deficient conditions would enable the bacterium to maintain its phosphate-solubilizing activity and enhance P uptake. Although no statistically significant differences in P content were observed between plants inoculated with Enterobacter sp. J49 and untreated control plants, these results do not rule out the occurrence of phosphate solubilization and its contribution to plant nutrition. PSB activity may increase the levels of soluble phosphate in the soil and/or result in rapid phosphorus uptake by the plant, followed by its incorporation into various metabolic pathways, which is reflected in plant growth parameters such as shoot dry weight [24]. On the other hand, although a significant increase in P content was not observed in plants inoculated with strain J49, it is important to highlight the other PGP properties of this bacterium. Thus, its ability to provide iron through the production of siderophores, combined with its capacity to produce phytohormone-like molecules, could explain the beneficial effect observed in plants inoculated with this PSB.
Interestingly, a statistically significant increase in P content was detected in the aerial tissues of plants challenged with S. rolfsii, regardless of the presence of Enterobacter sp. J49, when compared to both untreated control plants and those inoculated only with the PSB strain. In this regard, a possible explanation was reported by Mendes et al. [72], these authors informed an increase in the solubilization of rock phosphate present in the soil in the presence of filtrates from Aspergillus niger and S. rolfsii. Their study determined that oxalic acid secreted by these fungi was mainly responsible for the observed phosphate solubilization. This finding suggests that the presence of the pathogen may contribute to the increase in P content or in its redistribution in aerial tissue of peanut plants, to favor the pathogenesis process [73,74].

5. Conclusions

In the face of increasing combined abiotic and biotic stresses driven by climate change, this study highlights the potential of the native strain Enterobacter sp. J49 as a sustainable bioinoculant for peanut cultivation. The strain effectively reduced disease severity caused by S. rolfsii under phosphorus-deficient conditions, likely through the activation of ISR. These findings support the use of native PSB as a promising strategy to enhance crop resilience under simultaneous biotic and abiotic stress conditions.

Author Contributions

Conceptualization, L.L. and M.S.F.; methodology, M.S.F., L.L. and M.S.A.; formal analysis, M.S.F., L.L., T.T. and M.L.T.; investigation, A.L.G., M.S.F. and L.L.; writing—original draft preparation, M.S.A., M.S.F., T.T. and M.L.T.; writing—review and editing, A.F.; funding acquisition, M.S.F. and L.L. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by Agencia Nacional de Promoción de la Investigación, el Desarrollo Tecnológico y la Innovación: PICT-2020-SERIEA-02940 and Secretaría de Ciencia y Técnica de la Universidad Nacional de Río Cuarto (SECYT-UNRC).

Data Availability Statement

The original contributions presented in the study are included in the article, further inquiries can be directed to the corresponding author.

Acknowledgments

L.L., M.S.A., M.S.F., T.T., M.L.T. and A.F. are members of the research center of CONICET, Argentina. A.L.G. is a student of CONICET. During the preparation of this manuscript, the authors used ChatGPT version GPT-5 (chat.openai.com accessed on 25 August 2025) to correct the spelling and grammar in the abstract and the results section. The authors have reviewed and edited the output and take full responsibility for the content of this publication.

Conflicts of Interest

The authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

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Figure 1. In vitro antimicrobial activity (antibiosis) of the PSBs Serratia sp. S119 and Enterobacter sp. J49 against Sclerotium rolfsii growing on Petri dishes with APG medium after three (a) and six (b) days of incubation. (A) Control (S. rolfsii), (B) Enterobacter sp. J49 (S. rolfsii + J49), and (C) Serratia sp. S119 (S. rolfsii + S119). Experiments were repeated three times with n = 5.
Figure 1. In vitro antimicrobial activity (antibiosis) of the PSBs Serratia sp. S119 and Enterobacter sp. J49 against Sclerotium rolfsii growing on Petri dishes with APG medium after three (a) and six (b) days of incubation. (A) Control (S. rolfsii), (B) Enterobacter sp. J49 (S. rolfsii + J49), and (C) Serratia sp. S119 (S. rolfsii + S119). Experiments were repeated three times with n = 5.
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Figure 2. Disease incidence of peanut plants inoculated with Serratia sp. S119 (left) or Enterobacter sp. J49 (right) against S. rolfsii. Values are the mean ± standard error (S.E.). The experiment was repeated three times with 10 replicates per treatment. Different letters indicate significant differences according to Student’s t-test (p < 0.05). S. rolfsii indicates pathogen-inoculated plants. S119: S. rolfsii indicates plants inoculated with bacterial strain Serratia sp. S119 and then challenged with S. rolfsii. J49: S. rolfsii indicates plants inoculated with bacterial strain Enterobacter sp. J49 and then challenged with S. rolfsii.
Figure 2. Disease incidence of peanut plants inoculated with Serratia sp. S119 (left) or Enterobacter sp. J49 (right) against S. rolfsii. Values are the mean ± standard error (S.E.). The experiment was repeated three times with 10 replicates per treatment. Different letters indicate significant differences according to Student’s t-test (p < 0.05). S. rolfsii indicates pathogen-inoculated plants. S119: S. rolfsii indicates plants inoculated with bacterial strain Serratia sp. S119 and then challenged with S. rolfsii. J49: S. rolfsii indicates plants inoculated with bacterial strain Enterobacter sp. J49 and then challenged with S. rolfsii.
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Figure 3. Plant growth parameters of peanut plants inoculated with Serratia sp. S119. Values are the mean ± S.E. The experiment was repeated three times with 10 replicates per treatment. Different letters indicate significant differences according to the ANOVA LSD Fisher test (p < 0.05). S. rolfsii indicates pathogen-inoculated plants. S119: S. rolfsii indicates plants inoculated with bacterial strain Serratia sp. S119 and then challenged with S. rolfsii. S119 indicates plants inoculated only with Serratia sp. S119. Control indicates untreated control plants.
Figure 3. Plant growth parameters of peanut plants inoculated with Serratia sp. S119. Values are the mean ± S.E. The experiment was repeated three times with 10 replicates per treatment. Different letters indicate significant differences according to the ANOVA LSD Fisher test (p < 0.05). S. rolfsii indicates pathogen-inoculated plants. S119: S. rolfsii indicates plants inoculated with bacterial strain Serratia sp. S119 and then challenged with S. rolfsii. S119 indicates plants inoculated only with Serratia sp. S119. Control indicates untreated control plants.
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Figure 4. Plant growth parameters of peanut plants inoculated with Enterobacter sp. J49. Values are the mean ± S.E. The experiment was repeated three times with 10 replicates per treatment. Different letters indicate significant differences according to the ANOVA LSD Fisher test (p < 0.05). S. rolfsii indicates pathogen-inoculated plants. J49: S. rolfsii indicates plants inoculated with the bacterial strain Enterobacter sp. J49 and then challenged with S. rolfsii. J49 indicates plants inoculated only with Enterobacter sp. J49. Control indicates untreated control plants.
Figure 4. Plant growth parameters of peanut plants inoculated with Enterobacter sp. J49. Values are the mean ± S.E. The experiment was repeated three times with 10 replicates per treatment. Different letters indicate significant differences according to the ANOVA LSD Fisher test (p < 0.05). S. rolfsii indicates pathogen-inoculated plants. J49: S. rolfsii indicates plants inoculated with the bacterial strain Enterobacter sp. J49 and then challenged with S. rolfsii. J49 indicates plants inoculated only with Enterobacter sp. J49. Control indicates untreated control plants.
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Figure 5. Total peroxidase activity in peanut plants inoculated with Enterobacter sp. J49 at different times post-inoculation with S. rolfsii. Values are the mean ± S.E. The experiment was repeated three times with 10 replicates per treatment. Different letters indicate significant differences according to the ANOVA LSD Fisher test (p < 0.05). Control indicates untreated control plants. Enterobacter sp. J49 indicates plants inoculated only with this strain. S. rolfsii indicates pathogen-inoculated plants. Enterobacter sp. J49: S. rolfsii indicates plants inoculated with bacterial strain Enterobacter sp. J49 and then challenged with S. rolfsii.
Figure 5. Total peroxidase activity in peanut plants inoculated with Enterobacter sp. J49 at different times post-inoculation with S. rolfsii. Values are the mean ± S.E. The experiment was repeated three times with 10 replicates per treatment. Different letters indicate significant differences according to the ANOVA LSD Fisher test (p < 0.05). Control indicates untreated control plants. Enterobacter sp. J49 indicates plants inoculated only with this strain. S. rolfsii indicates pathogen-inoculated plants. Enterobacter sp. J49: S. rolfsii indicates plants inoculated with bacterial strain Enterobacter sp. J49 and then challenged with S. rolfsii.
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Figure 6. Phenolic compounds content in peanut plants inoculated with Enterobacter sp. J49 at different times post-inoculation with S. rolfsii. Values are the mean ± S.E. The experiment was repeated three times with 10 replicates per treatment. Different letters indicate significant differences according to the ANOVA LSD Fisher test (p < 0.05). Control indicates untreated control plants. Enterobacter sp. J49 indicates plants inoculated only with this strain. S. rolfsii indicates pathogen-inoculated plants. Enterobacter sp. J49: S. rolfsii indicates plants inoculated with bacterial strain Enterobacter sp. J49 and then challenged with S. rolfsii.
Figure 6. Phenolic compounds content in peanut plants inoculated with Enterobacter sp. J49 at different times post-inoculation with S. rolfsii. Values are the mean ± S.E. The experiment was repeated three times with 10 replicates per treatment. Different letters indicate significant differences according to the ANOVA LSD Fisher test (p < 0.05). Control indicates untreated control plants. Enterobacter sp. J49 indicates plants inoculated only with this strain. S. rolfsii indicates pathogen-inoculated plants. Enterobacter sp. J49: S. rolfsii indicates plants inoculated with bacterial strain Enterobacter sp. J49 and then challenged with S. rolfsii.
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Figure 7. Percentage of disease incidence caused by S. rolfsii in peanut plants growing with sufficient P (SOLP) or insoluble P (TCP). Values are the mean ± S.E. The experiment was repeated three times with 10 replicates per treatment. Different letters indicate significant differences according to the ANOVA LSD Fisher test (p < 0.05). S. rolfsii indicates pathogen-inoculated plants. J49: S. rolfsii indicates plants inoculated with bacterial strain Enterobacter sp. J49 and then challenged with S. rolfsii.
Figure 7. Percentage of disease incidence caused by S. rolfsii in peanut plants growing with sufficient P (SOLP) or insoluble P (TCP). Values are the mean ± S.E. The experiment was repeated three times with 10 replicates per treatment. Different letters indicate significant differences according to the ANOVA LSD Fisher test (p < 0.05). S. rolfsii indicates pathogen-inoculated plants. J49: S. rolfsii indicates plants inoculated with bacterial strain Enterobacter sp. J49 and then challenged with S. rolfsii.
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Figure 8. Plant growth parameters. Shoot dry weight (left); root dry weight (right) in peanut plants growing with soluble P (SOLP) or insoluble P (TCP) and challenged with S. rolfsii. Values are the mean + S.E. The experiment was repeated three times with 10 replicates per treatment. Different letters indicate significant differences according to the ANOVA LSD Fisher test (p < 0.05). Control indicates untreated control plants. Enterobacter sp. J49 indicates plants inoculated only with this strain. S. rolfsii indicates pathogen-inoculated plants. Enterobacter sp. J49: S. rolfsii indicates plants inoculated with bacterial strain Enterobacter sp. J49 and then challenged with S. rolfsii.
Figure 8. Plant growth parameters. Shoot dry weight (left); root dry weight (right) in peanut plants growing with soluble P (SOLP) or insoluble P (TCP) and challenged with S. rolfsii. Values are the mean + S.E. The experiment was repeated three times with 10 replicates per treatment. Different letters indicate significant differences according to the ANOVA LSD Fisher test (p < 0.05). Control indicates untreated control plants. Enterobacter sp. J49 indicates plants inoculated only with this strain. S. rolfsii indicates pathogen-inoculated plants. Enterobacter sp. J49: S. rolfsii indicates plants inoculated with bacterial strain Enterobacter sp. J49 and then challenged with S. rolfsii.
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Figure 9. Phosphorus (P) content in the aerial tissue of peanut plants grown with tricalcium phosphate (TCP). Data represent the mean ± S.E. of three experiments (n = 10 each). Different letters indicate statistically significant differences according to Fisher’s LSD test (p < 0.05). Control indicates untreated control plants. Enterobacter sp. J49 indicates plants inoculated only with this strain. S. rolfsii indicates pathogen-inoculated plants. Enterobacter sp. J49: S. rolfsii indicates plants inoculated with bacterial strain Enterobacter sp. J49 and then challenged with S. rolfsii.
Figure 9. Phosphorus (P) content in the aerial tissue of peanut plants grown with tricalcium phosphate (TCP). Data represent the mean ± S.E. of three experiments (n = 10 each). Different letters indicate statistically significant differences according to Fisher’s LSD test (p < 0.05). Control indicates untreated control plants. Enterobacter sp. J49 indicates plants inoculated only with this strain. S. rolfsii indicates pathogen-inoculated plants. Enterobacter sp. J49: S. rolfsii indicates plants inoculated with bacterial strain Enterobacter sp. J49 and then challenged with S. rolfsii.
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MDPI and ACS Style

Gentile, A.L.; Figueredo, M.S.; Anzuay, M.S.; Tonelli, M.L.; Fabra, A.; Taurian, T.; Ludueña, L. Native Phosphate Solubilizing Bacteria Mitigate the Effect of the Phytopathogen Sclerotium rolfsii on Peanut (Arachis hypogaea L.) Plants in a P-Deficient Environment. Agronomy 2025, 15, 2278. https://doi.org/10.3390/agronomy15102278

AMA Style

Gentile AL, Figueredo MS, Anzuay MS, Tonelli ML, Fabra A, Taurian T, Ludueña L. Native Phosphate Solubilizing Bacteria Mitigate the Effect of the Phytopathogen Sclerotium rolfsii on Peanut (Arachis hypogaea L.) Plants in a P-Deficient Environment. Agronomy. 2025; 15(10):2278. https://doi.org/10.3390/agronomy15102278

Chicago/Turabian Style

Gentile, Ana Laura, Maria Soledad Figueredo, Maria Soledad Anzuay, Maria Laura Tonelli, Adriana Fabra, Tania Taurian, and Liliana Ludueña. 2025. "Native Phosphate Solubilizing Bacteria Mitigate the Effect of the Phytopathogen Sclerotium rolfsii on Peanut (Arachis hypogaea L.) Plants in a P-Deficient Environment" Agronomy 15, no. 10: 2278. https://doi.org/10.3390/agronomy15102278

APA Style

Gentile, A. L., Figueredo, M. S., Anzuay, M. S., Tonelli, M. L., Fabra, A., Taurian, T., & Ludueña, L. (2025). Native Phosphate Solubilizing Bacteria Mitigate the Effect of the Phytopathogen Sclerotium rolfsii on Peanut (Arachis hypogaea L.) Plants in a P-Deficient Environment. Agronomy, 15(10), 2278. https://doi.org/10.3390/agronomy15102278

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