Pseudomonas fluorescens SP007s enhances defense responses against the soybean bacterial pustule caused by Xanthomonas axonopodis pv. glycines

Dusit Athinuwat; Wannaporn Thepbandit; Supatcharee Siriwong; Sutruedee Prathuangwong

doi:10.1515/opag-2022-0267

Article Open Access

Pseudomonas fluorescens SP007s enhances defense responses against the soybean bacterial pustule caused by Xanthomonas axonopodis pv. glycines

Dusit Athinuwat , Wannaporn Thepbandit , Supatcharee Siriwong and Sutruedee Prathuangwong

Published/Copyright: July 18, 2024

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From the journal Open Agriculture Volume 9 Issue 1

Abstract

A bacterial pustule caused by Xanthomonas axonopodis pv. glycines (Xag) is one of the main diseases of soybeans in Thailand. Beneficial microbes crucial to sustainable plant production were examined in this study. Soybean plants were sprayed with Pseudomonas fluorescens SP007s strain three times before Xag infection. The results showed a significant reduction in bacterial pustule disease severity by up to 85%, increased leaf accumulation of salicylic acid with 134% during the infection process of Xag. Furthermore, the Xag population size in soybean leaves was reduced by priming with SP007s. The mechanism of SP007s in the chemical structure of mesophyll was characterized by using the synchrotron radiation-based Fourier transform infrared (SR-FTIR) analysis. The SR-FTIR spectral changes from the mesophyll showed higher integral area groups of polysaccharides (peak of 900–1,200 cm⁻¹). These biochemical changes were involved with the primed resistance of the soybean plants against the bacterial pustule disease as well as the polysaccharide signals that were linked to hypersensitive responses leading to a rapid death of plant cells to effectively restrict the growth of pathogens at the infected site. Therefore, we consider that SP007s can be a promising biocontrol agent by activating immunity of soybean plants.

Keywords: biocontrol; plant disease; induced resistance; plant immune

1 Introduction

Soybean (Glycine max) is one of the most important economic crops that supply large sources of fatty acids, proteins, vitamins, minerals, and other nutrients [1] for humans and animals. Soybean is also used in the manufacturing of industrial feedstocks and combustible fuels, among other non-food applications [2]. However, insect pests and pathogens cause important diseases globally, especially the soybean bacterial pustule caused by Xanthomonas axonopodis pv. glycines (Xag), which can significantly reduce the production of soybeans by causing symptoms such as leaf spots, pustules, and defoliation. Therefore, the development and improvement of bioagents inducing resistance against bacterial pustules is one of the main objectives for soybean production. In biocontrol agents, plant growth-promoting rhizobacteria (PGPR) provide an environmentally friendly way to control plant diseases in agriculture [3]. The PGPR penetrates and produces biofilms on the outer surface of plant roots, which produce antimicrobial substances, such as antibiotic metabolites and bacteriocins. The inhibitory effects of such chemicals on pathogens help to reduce plant diseases by directly inhibiting pathogens or making the plant resistant to them. Pseudomonas species have been extensively studied for their beneficial effects on plants. The key findings are condensed in a number of thorough studies showing that increasing the availability and absorption of mineral nutrients via mineral solubilization results in plant development and yield increase. Many Pseudomonas strains can effectively inhibit pathogen attacks through direct and indirect mechanisms. In addition, Pseudomonas species can activate the salicylic acid (SA)-dependent signaling system that provides disease resistance to multiple plant species such as rice, vegetables, tomato, and grape [4,5,6,7,8,9,10]. P. fluorescens SP007s is a bacterium with significant advantages in biocontrol, particularly in managing plant diseases and promoting sustainable agriculture. One of the primary benefits of using SP007 is its strong antifungal and antibacterial properties. The bacterium produces various secondary metabolites, including antibiotics such as pyoluteorin, 2,4-diacetylphloroglucinol (DAPG), and phenazine, which are effective against a broad spectrum of plant pathogens [8]. Research has shown that specific formulations of SP007 are highly effective in controlling soft rot disease cause by Pectobacterium carotovorum and promoting growth in kale [9]. This highlights its potential to enhance crop yield and quality in vegetable production. Additionally, bioformulations of SP007s have proven effective against dirty panicle disease of rice cause by Trichoconis padwickii Ganguly, Cercospora oryzae, Curvularia lunata, Fusarium semitectum, Bipolaris oryzae, and Sarocladium oryzae and also increased gamma-aminobutyric acid (GABA) levels in treated seeds, suggesting that this microorganism may enhance the plant’s defense against stress conditions [10].

The SA-dependent mechanism involves the production of SA, which is linked to various physiological processes in plants, including defense responses that are induced by plant pathogen infections [11]. Priming is a crucial process in induced resistance, in which plants demonstrate a more rapid and robust production of defense mechanisms in response to bacterial pathogen infection [12]. The main regulators of signaling networks that are involved in induced plant defense include the following plant hormones: SA, jasmonic acid, and ethylene [13]. P. fluorescens produces a range of secondary metabolites, enzymes, and volatile organic compounds that help in inhibiting the growth and colonization of harmful pathogens. These mechanisms can induce host-defense responses and enhance the resistance of the host organism against infections [14]. Upon pathogen recognition, plants can generate reactive oxygen species (ROS) through an oxidative burst. ROS, such as hydrogen peroxide and superoxide radicals, act as signaling molecules and contribute to the activation of defense responses. They can also induce programmed cell death (PCD) in the infected area, limiting the pathogen spread. PCD in the infected area is a fundamental component of the hypersensitive response (HR) in plant defense. HR initiates SAR extending the localized defense systemically throughout the plant, providing long-term protection, and enhancing plant resistance to pathogens [15]. P. fluorescens is known for its ability to trigger HR in plants, which restricts the spread of pathogens. P. fluorescens isolates use their type III injector systems to deliver effector proteins to the host plant cells, triggering the HR and thereby contributing to the plant defense against pathogens [16]. This type of injector system is commonly denominated as type III secretion system (T3SS) referencing complex molecular mechanisms. The T3SS enables the bacteria to inject effector proteins directly into the cytoplasm of host cells [17]. The effector proteins are usually enzymes or signaling molecules that can modulate multiple cellular processes, including membrane trafficking, cell signaling, and immune response [18]. Thus, the initial contact with P. fluorescens initiates an HR response, which triggers systemic resistance involving SA as the signal. One of the responses is the formation of callose, which can be detected by Fourier-transform infrared (FTIR) microspectroscopy. FTIR microspectroscopy may also detect other metabolites involved in defense by identifying characteristic absorption bands related to functional groups such as proteins, lipids, and carbohydrates [19]. Monitoring changes in these absorption bands can provide insights about the production and accumulation of defense-related metabolites.

The main objective of the present study was to explore the potential of P. fluorescens SP007s for controlling the bacterial pustule caused by Xag through stimulation of the plant defense intermedia, including endogenous SA. Moreover, the biochemical changes were analyzed, having a special focus on phospholipids, pectins, amides, lignins, and polysaccharides, which are associated with defense mechanisms in soybean plants during Xag infection.

2 Materials and methods

2.1 Plant material, bacterial strains, and inoculum preparation

The soybean seeds of the susceptible cultivar (CV.SJ.4) were used in this study. The seeds were sterilized with 10% Clorox for 1 min, rinsed with sterile water three times [20], and sown in 10-in diameter pots containing natural sterile soil and fertilizer.

P. fluorescens SP007s strain and Xag were provided by the Plant Pathology Section of Thammasat University, Thailand. Cultures were revived by streaking the stock culture (−20°C). Then, it was re-cultured on nutrient agar (NA) medium and incubated at 28 ± 2°C for 48 h. P. fluorescens SP007s was prepared in 500 mL of Luria–Bertani (LB) broth, and Xag was prepared in 500 mL of nutrient broth for 48 h at 180 rpm, 28 ± 2°C. Afterward, the cultures were centrifuged at 12,000 g for 20 min to remove the broth compounds. The cell biomass was preserved at 4°C and adjusted to 1 × 10⁸ cfu mL⁻¹ with sterile distilled water before being used for further experimentation.

2.2 Priming of plant and Xag inoculation

The experiment with four treatments, including SP007s, SA, copper hydroxide, and sterile distilled water, was conducted in a completely randomized design (CRD) with four replications, three plant per replication. The plants were sprayed with 30 mL of SP007s (1 × 10⁸ cfu mL⁻¹) at 7, 14, and 21 days post-sowing (dps) as a priming stage. The procedure was carried out in the same manner, with 1 mM SA and copper hydroxide 77% WP in separate as a positive control and sterile distilled water as a negative control. At 45 dps, the soybean plants in all treatments were inoculated with a density of Xag suspension at 1 × 10⁸ cfu mL⁻¹ by foliar spraying, covered with plastic bags, and incubated for 24 h. Next, the plants were moved from the plastic bags to the greenhouse. The pots were kept under greenhouse conditions under 12 h of photoperiod, 28 ± 4°C, and 60–75% humidity for further experimentation.

2.3 Detection of endogenous SA

Soybean leaf samples from each treatment (0.5 g) were homogenized in liquid nitrogen mixed with 1 mL of extraction buffer, 90% methanol, 9% glacial acetic acid, and 1% water. Next, they were centrifuged at 14,000 g at 4°C for 15 min. About 0.5 mL of the sample was mixed with 0.5 mL of 0.02 M ferric ammonium sulfate solution and incubated at 30°C for 5 min. Two hundred microliters of each sample were transferred to a 96-well plate. The absorbance was read at 530 nm using a microplate reader (Bio-Tek, Winooski, VT, USA) and compared with the SA (Sigma, USA) standard curve in order to determine the amount of endogenous SA in the sample.

2.4 PCD of SP007

The leaf samples were collected at 24 h post-inoculation to detect PCD, HR associated cell death in response to Xag infection. The leaf samples were decolorized by boiling them in trypan blue–lactophenol (2 mg trypan blue, 25% lactic, 23% phenol, 25% glycerol) at 95°C for 5 min and soaked in 25% chloral hydrate overnight. Subsequently, the leaf samples were washed with water, soaked in 0.1% aniline blue for 1 min, washed with water again, and mounted on a Petri dish with 30% glycerol in water. The stained cells on the epidermis were counted as a cell death event.

2.5 Rapid detection of biochemical changes using synchrotron radiation-based Fourier-transform infrared (SR-FTIR) microspectroscopy

The leaf samples were collected and fixed in the optimal cutting temperature compound (Scigen Tissue-Plus™, England) with liquid nitrogen to embed tissue samples prior to frozen sectioning on a microtome-cryostat. The samples were sliced at 7 μm by using a Wetzlar cryostat microtome (Germany) and then laid on a 13 mm × 2 mm window. The spectral data were collected at the beamline 4.1 Synchrotron Light-IR Spectroscopy station. The measurement was identified by mapping an aperture size of 10 μm × 10 μm and a spectral resolution mode of 4 cm⁻¹ with 64 scans. The spectral derivative was generated with OPUS 7.2 software (Hanau, Germany), and the data were analyzed with the CytospecTM software and the unscramblerX 10.0 software (NJ, USA) [19]. The average spectra of each sample were explained by references of band assignments and the functional groups of FTIR spectra (Table S1).

2.6 Disease rating

The bacterial pustule disease symptoms that appeared on the studied soybean leaves after 14 days post-inoculation (dpi) were analyzed based on the scale developed by Zhao et al. [21] (Table 1, Figure S1)

Table 1

Bacterial pustule disease symptoms scaled by the disease spot numbers at five levels

Disease scale	Infection of plant
1	Symptoms are found on less than 20 disease spots in the leaf area
3	21–50 irregular disease spots
5	>50 small lesion, deformed disease spots covering 10–25% of the leaf area
7	A large lesion covering the surface of 25–50% of the soybean leaf
9	A large lesion covering the surface of more than 50% of the soybean leaf

The disease severity index and the control efficacy were calculated as follows (formulas (1) and (2)) [22]:

(1) Σ ( Disease rating × Number of plants of this rating ) Total soybean plants × M aximum rating scale × 100

(2) Control treatment − Treated treatment Control treatment × 100

2.7 Evaluation of Xag growth in the soybean plants

The experiments, including SP007s-treated and SP007s-untreated, were conducted in CRD with four replications. The SP007s-treated and SP007s-untreated soybean leaves were observed at 0, 1, 2, 3, 7, and 14 dpi with Xag. Leaves were cut into 8 mm diameter leaf disks by using a cork borer, and they were homogenized in 1 mL of sterile 10 mM MgCl₂. The leaf homogenate was diluted in a serial dilution and spread on NA plates. Next, the samples were incubated in the growth chamber for 24 h. The colonies that appeared on NA plates were counted as the metric for each sample.

2.8 Statistical analysis

The obtained experimental data were analyzed using the one-way ANOVA software version 20 by SPSS (SPSS Inc., Chicago, USA). The mean difference values among groups were divided according to Duncan’s multiple range test with a significant test result (p ≤ 0.05).

3 Results

3.1 Detection of endogenous SA

An increase in endogenous SA content was observed in soybean leaves at the pre-inoculation stage and 24 h post-inoculation (hpi) with Xag. The results revealed that soybean plants treated with SP007s three times before inoculation increased the content of endogenous SA from 40.13 to 93.86 µg g⁻¹ FW and in the SA-treated samples (100.30 µg g⁻¹ FW), followed by the water-mock and the copper hydroxide-treated plants, which increased to 55.90 and 48.15 µg g⁻¹ FW, respectively (Figure 1).

Figure 1

Effects of pre-foliar spraying of soybean plant with Pseudomonas fluorescens SP007s, SA, copper hydroxide 77% WP (20 g L⁻¹) (commercial pesticide), and water-mock treatment (negative control) three times before Xanthomonas axonopodis pv. glycines inoculation on the concentration of endogenous SA. Values are presented as the mean of four replicates, three plants per replication. Mean values in the graph followed by different letters are significantly different according to DMRT at p ≤ 0.05.

3.2 Biochemical change analysis in soybean mesophyll

The average spectra of the chemical compounds and the structure of the plant cells in the soybean tissue at the mesophyll level were observed in the range of 3,000–900 cm⁻¹. The averaged spectra from SR-FTIR microspectroscopy showed the characteristic features of C═O stretching from lipids and pectins exhibited peak at 1,730 cm⁻¹, C–N and N–H stretching of protein amide I at 1,650 cm⁻¹ and amide II at 1,530 cm⁻¹, C–C ring from cellulose at 1,150 cm⁻¹, C–C and C–O–H consisting of polysaccharides and polycarbohydrates at 1,100–1,000 cm⁻¹ as shown in Figure 2. Such spectral peaks can be used to characterize the biochemical components of the samples in each treatment.

Figure 2

The average original spectra of soybean tissue (mesophyll) from the water-mock treated samples (control), Pseudomonas fluorescens SP007s-treated (SP007s), SA treated, and copper hydroxide (Cu(OH)₂) treated were measured by SR-FTIR microspectroscopy.

Principal component analysis (PCA) was used to classify the groups of the treatment distributions, as shown in Figure 3a. The results showed three clearly distinguishable groups: the SP007s- and SA-treated groups, the copper hydroxide-treated group, and the water-mock group. Such groups were separated by PC1 and PC2, represented by 55 and 28% of the variance, respectively. The PC1 loading plot showed the disparate variables between the SP007s- and SA-treated samples from the copper hydroxide-treated samples and the water-mock treated samples.

Figure 3

(a) The categorization of soybean tissue on each treatment using PCA score plots. (b) Loading factors of PC1 and PC2 correspond to the PCA score plots of SR-FTIR microspectroscopy.

The PC1 loading plot indicated that the phospholipids peaked at 2,925 cm⁻¹, C═O, C–N, and N–H of amide I and amide II exhibited peaks at 1,704 and 1,533 cm⁻¹, and the C–O–C stretching of hemicellulose and polysaccharides peaked at 1,114 and 1,060 cm⁻¹, which corresponds to the copper hydroxide-treated samples and the water-mock treated samples. The divergence of the water-mock treated samples (control) from the copper hydroxide-treated samples, the SP007s-treated samples, and the SA-treated samples was identified by the PC2 loading plot. The PC2 loading plot showed peaks at the phospholipid section at 2,867 cm⁻¹, and at the proteins amide I and II sections at 1,654 and 1,538 cm⁻¹, respectively (Figure 3b).

The comparison among the spectra of the soybean leaf tissues in the range of 3,000–900 cm⁻¹ is shown in Figure 4a. Biochemical composites in the SP007s treatment spectrum of the CO–CC polysaccharide section showed higher values than those from the other treatments at the vibrational peak at 1,114 cm⁻¹. The alpha helix structure of the amide I protein exhibited peak at 1,654 cm⁻¹ in the water-mock treated samples spectrum, which was the highest. However, the stacking of the second derivative of the average spectra was not clear for each range of biochemical composition. Thus, we integrated the area under the IR peak based on the separation into six groups of the chemical components, including phospholipid (3,000–2,800 cm⁻¹), pectin (1,740 cm⁻¹), amide I (1,700–1,600 cm⁻¹), amide II (1,600–1,500 cm⁻¹), lignin (1,513 cm⁻¹), and polysaccharide (1,200–900 cm⁻¹), as shown in Figure 4b. The results revealed that the integral areas of phospholipid, amide I, amide II, and lignin from the mesophyll soybean leaf tissue in the SP007s-treated samples were significantly lower compared to the other treatments. Polysaccharides from the SP007s-treated samples were observed to be significantly higher than those from the other treatments.

Figure 4

(a) Overlay of the second derivative of the average spectra from the soybean mesophyll-treated sample, including Pseudomonas fluorescens SP007s-treated (SP007s), SA-treated, and copper hydroxide-treated (Cu(OH)₂) compared with water-mock treated (control). (b) Integral areas of absorbance between 3,000 and 900 cm⁻¹. Mean values in the graph followed by different letters are significantly different according to DMRT at p ≤ 0.05.

3.3 Detection of PCD of SP007s

Trypan blue staining of the leaf tissues at 24 hpi was used to observe rapid PCD in water-mock treated and SP007s-treated leaf samples, as shown in Figure 5. Water-mock treated leaves showed lower cell death spots (black arrows) compared to the SP007s-treated samples.

Figure 5

Observed program cell death events in treated leaves at 16 h post-inoculation on the upper, middle, and lower parts of the soybean plant (left to right): (a) water-mock treated and (b) Pseudomonas fluorescens SP007s-treated.

3.4 Disease rating

The SP007s was assessed for its ability to induce plant resistance by foliar spraying at 7, 14, and 21 dps. The results showed that the plants treated with SP007s significantly reduced the disease severity of bacterial pustule at 14 dpi compared to the samples treated with water-mock. The disease severities of the soybean plants treated with SP007s had an average ratio of 11%, followed by the samples treated with SA (17%) and the copper hydroxide-treated samples (44%). These measured disease severities were significantly lower than the ones obtained from the water-mock-treated samples, which was 72% (Table 2 and Figure 6).

Table 2

Efficacy of Pseudomonas fluorescens SP007s on the severity and reduction of the soybean bacterial pustule caused by Xanthomonas axonopodis pv. glycines under greenhouse conditions

Treatment¹	Disease severity (%)	Reduction of disease severity compared with control (%)
SP007s	11c	85
SA	17c	77
Copper hydroxide	44b	38
Water-mock	72a

¹The plants were induced by the foliar spraying technique for each treatment at 7, 14, and 21 dps before Xanthomonas axonopodis pv. glycines inoculation. The mean in the column followed by the same letter (a, b, c) is a non-significance difference according to Duncan’s multiple range test at p ≤ 0.05.

Figure 6

Disease symptoms in infected and treated leaves at 14 dpi: (a) Pseudomonas fluorescens SP007s-treated, (b) SA-treated, (c) copper hydroxide-treated, and (d) water-mock treated samples.

3.5 Assessment of Xag growth in the soybean plants

The population sizes of Xag between SP007s-treated leaves and the control leaves were compared. At 7 dpi, the population of Xag in soybean leaves treated with SP007s had grown to 3.3 × 10⁶ cfu leaf⁻¹ and reached a maximum of 6.1 × 10⁶ cfu leaf⁻¹ at 14 dpi. However, the Xag population in the leaves treated with distilled water multiplied to 3.1 × 10¹⁰ cfu leaf⁻¹ at 14 dpi. The Xag population size on the leaves treated with SP007s samples was half of the leaves treated with distilled water, indicating that the application of SP007s in foliar spray reduced the growth of Xag (Figure 7).

Figure 7

Growth curves of Xanthomonas axonopodis pv. glycines in the soybean plant samples treated and non-treated with Pseudomonas fluorescens SP007s, which were inoculated with Xag under greenhouse conditions. 0 days is before inoculation. The mean in the column followed by the same letter (a, b) is a non-significance difference according to DMRT at p ≤ 0.05.

4 Discussion

Pseudomonas strains have been extensively utilized due to their growth promotion and biological control activities. Several reports have highlighted the capacity of Pseudomonas spp. to suppress the proliferation of plant pathogens through various mechanisms, such as direct interaction and induced resistance. In this study, the PGRP activity of SP007s was confirmed, and its biological control potential was demonstrated. SP007s significantly decreased bacterial pustule on soybeans caused by Xag with detected changes in the biochemical components. The crucial roles of SP007s in plant immunological interactions were explored by examining the alterations in the intermediate biochemical composition of the host defense system. This included investigating the changes in SA, which is associated with enhanced resistance against soybean bacterial pustules. Spraying the SP007s three times before pathogen inoculation resulted in a significant increase in endogenous SA contents of a 134%. During pathogen invasion and infection, the contents of endogenous SA are always increasing in order to prevent further infection. However, resistant plants often exhibit a more rapid and higher increase in endogenous SA levels [11]. This heightened response is due to the presence of specific resistance genes that facilitate a robust defense signaling cascade, which can modulate the activity of plant defense mechanisms, leading to changes in the physiological, biochemical, and molecular states of the plant [23,24,25]. The SA pathway stimulates the production of antimicrobial proteins, phytoalexins, and other defense compounds [26]. SA acts as a signaling molecule to activate SAR, which increases the plant resistance to subsequent pathogen attacks [27,28]. The priming effect induced by microbial bioagents as elicitors results in enhanced readiness and responsiveness of plant defense mechanisms, allowing plants to respond more rapidly and effectively when they encounter pathogen attacks [29]. Plants detect and activate defense signals early during the pathogen infection process. Plants produce damage-associated molecular patterns (DAMPs), such as extracellular protein fragments, peptides, nucleotides, and amino acids. Lakkis et al. reported that P. fluorescens is an inducer of ISR in grapevine, which increased endogenous SA and 1-aminocyclopropane carboxylic acid contents after a Botrytis cinerea infection [6]. Similarly, Suresh et al. [5] demonstrated that the P. fluorescens VSMKU3054 can induce systemic resistance in tomatoes against Ralstonia solanacearum by increasing the amount of defense-related enzymes, including peroxidase, polyphenol oxidase, lipoxygenase, phenylalanine ammonia lyase, and the total phenol content that involves SA-dependent signaling and SA pathway.

The resistance activity of these intermedia enzymes can contribute to changes in their biochemical components. In this research, the biochemical changes in soybean mesophyll tissue were investigated by using the SR-FTIR technique to describe the defense mechanism of the SP007s-treated samples. The results showed that the spectral range of 1,200–900 cm⁻¹, which corresponded to the C–O stretching, C–H bonds, and C–O ring vibrations from the SP007s-treated samples revealed a significantly higher agglomeration of polysaccharides in comparison to the water-mock treated samples. Degradation often involves chemical reactions that modify the structure of the polysaccharide formed as degradation products, leading to additional absorption bands in the FTIR spectrum. Polysaccharides are complex carbohydrates that occur naturally and consist of chains of sugar residues linked by glycosidic bonds. They play regulatory roles in various biological processes. It is an important component of a plant defense strategy against biotic stress, as well as for protecting the plant from attacks by pathogens [30]. Plant cell walls are composed of various polysaccharides, which serve as informational components that initiate the production of different defense compounds. These include phytohormones, pathogenesis-related (PR) proteins, and secondary metabolites [31,32]. Multiple reviews have indicated that the polysaccharides found in the plant cell wall mainly contribute to growth, and disease resistance, and are engaged in different biotransformation processes [33]. In response to pathogen attacks, plants can produce enzymes that facilitate the addition or removal of specific sugars from the polysaccharides. This enzymatic activity can modify the properties of the cell wall, making it more resistant to pathogen attacks. At the same time, Kozieł et al. [33] demonstrated that the constituent plant cell wall metabolism can alter the transmission of viruses and trigger apoplast and symplast resistance mechanisms, thereby contributing to the complex network of the plant defense system. In addition, polysaccharides are involved in plant PCD. PCD is a natural and tightly regulated process that occurs in plants as well as in other multicellular organisms that are involved in various developmental processes, responses to environmental cues, and defense mechanisms. However, not all tissues can respond to the same extent with increased PCD. PCD is more prevalent in specific parts of the plant and younger leaves (Figure 5). PCD is an essential process in plant development, defense, and response to various stresses. It involves a coordinated series of biochemical and morphological events that lead to the selective elimination of cells. One form of PCD in plants is HR, which occurs during defense responses against pathogens. To facilitate host colonization, phytopathogenic bacteria secrete immune-suppressive effectors into host cells via the T3SS [34]. Biocontrol agent, P. fluorescens, secretes a variety of effector proteins into the host plant cells. These effectors can manipulate host cell processes to promote bacterial colonization [35]. However, some of these effectors can also be recognized by plant immune receptors, leading to the activation of defense responses, where their recognition triggers the HR [36,37,38].

De Vleesschauwer et al. [4] demonstrated that the application of P. fluorescens WCS374r as an inducer enhanced basal defense against the leaf blast pathogen Magnaporthe oryzae by an SA-independent mechanism, suggesting alternative pathways might be involved in the plant’s immune response. The rapid recruitment of phenolic compounds, triggering reiterative hydrogen peroxide (H₂O₂) microbursts, and causing rapid HR-associated cell death in response to fungal infection consisting of rapid recruitment of phenolic compounds at sites of pathogen entry, concerted expression of a diverse set of structural defenses, and a timely hyperinduction of hydrogen peroxide formation putatively driving cell wall fortification. HR often can induce SAR-enhanced resistance to pathogenic infection through the SA pathway in which the entire plant becomes more resistant to subsequent infections [39,40]. Similarly, Gruau et al. demonstrated that P. fluorescens PTA-CT2 triggers local HR and systemic immunity, which integrates SA signaling in grapevine [41]. During root colonization by PTA-CT2, the grapevine roots trigger some induction of localized HR-like cell death in roots after pathogen infection as rapid induction of the hypersensitive related gene, a known marker for HR, at 24 hpi. Subsequently, the expression patterns of the SA pathway. Callose is a specific type of polysaccharide that is a β-1,3-glucan polysaccharide. It is a major component of the plant cell wall and is involved in various physiological processes, including defense responses [42]. Callose deposition often occurs in response to pathogen infection or other environmental stresses, and it is an SA-pathway marker in reinforcing cell walls [43,44]. During PCD, plants increase callose deposition. It is a common phenomenon initiated at early, intermediate, and late defense responses within the host cell.

The cell wall serves as the initial defense mechanism in plants, contributing to the specificity of disease resistance and to the overall fitness [45]. Biochemical profiling using synchrotron-based spectroscopy visualizes the changes in the biochemical spectrum of cell wall components in relation to host defense responses [46,47]. Our results showed an increased peak intensity in polysaccharides, suggesting that these structural components have a role in the response to pathogen infection. Increasing the levels of polysaccharides in plants is one of the strategies employed by the plant to enhance its defense against pathogens [48]. DAMPs, microbe-associated molecular patterns, and lipopolysaccharides are involved in plant defense mechanisms, particularly in the context of the polysaccharides present in the plant cell wall [49]. These molecular patterns serve as signals that the plant uses to detect and respond to cellular damage or the presence of potential pathogens, activating immune mechanisms to protect the plant [50]. P. fluorescens can produce pectinolytic enzymes, including pectinases such as polygalacturonases and pectate lyases [49]. These enzymes can degrade pectin, a major component of the plant cell wall, by breaking down its various constituents, including homogalacturonan (HG) [51]. The release of pectin fragments, such as DAMPs and oligogalacturonides (OGs), can trigger defense responses in plants, and the modification of HG can contribute to the reinforcement of the cell wall [52]. In accordance with the investigations of Benedetti et al., and Gramegna et al. [53,54] reported that the breakdown products of HG, which include OGs, are considered DAMPs that are produced in response to plant pathogen attacks and physical damage. OGs, derived from the degradation of HG, are considered DAMPs that serve as both a defense response against pathogen attacks and a local signal to alert the plant about physical damage. Davidsson et al. [55] reported that OGs can enhance the resistance of Arabidopsis thaliana, which induces pathogen resistance-associated gene expressions. Similarly, Gravino et al. [31] stated that OGs can activate a complicated signaling route that leads to the creation of numerous defenses, including phytohormones, secondary metabolites, and PR proteins. Similarly, Yu et al. [56] stated that the beneficial microbes can increase the levels of SA, leading to the transcriptional activation of genes encoding PR proteins. Moreover, P. fluorescens also has a direct mode of action. This direct action includes the secretion of antimicrobial compounds, siderophores, and enzymes that degrade pathogenic cell walls and can inhibit competing microorganisms as well as plant pathogens [57,58]. Additionally, the bacterium’s ability to colonize plant roots effectively ensures that it can exert these biocontrol activities precisely where they are needed most [59]. The combination of direct antimicrobial action and the induction of plant defenses underscores the multifaceted approach of P. fluorescens to promoting plant health and protecting crops from a variety of pathogens. Through its diverse modes of action, P. fluorescens not only protects plants from pathogens but also promotes healthier plant growth, contributing to increased agricultural productivity [60] as shown in Figure S2 and Table S2. Therefore, SP007s can be used as a biocontrol agent against this specific pathogen for a sustainable crop protection strategy in agriculture.

5 Conclusions

This study highlights the potential of a spray-induced treatment with SP007s as an environmentally sustainable solution for controlling the soybean bacterial pustule caused by Xag. The findings indicate that SP007s effectively stimulate plant defense responses, resulting in significant changes in the biochemical components within the mesophyll, including an increased accumulation of SA and polysaccharides. These metabolites play a crucial role in inhibiting the growth of Xag, leading to an important reduction in disease severity. In order to fully explore the practical application of SP007s as a priming agent for soybeans, additional research is required, particularly through field-scale experiments.

Acknowledgments

The authors would like to thank the Synchrotron Light Research Institute (SLRI) in Thailand, which provided the FTIR instruments and the beamline 4.1 Infrared Spectroscopy and Imaging systems.

Funding information: This research was supported by Center for Advanced Studies for Tropical Natural Resources (CASTNaR), Kasetsart University, that has been granted by the Office of the Higher Education Commission.
Author contributions: All authors have accepted responsibility for the entire content of this manuscript and consented to its submission to the journal, reviewed all the results and approved the final version of the manuscript. DA and SP: conceptualization, DA, WT, SS experiment investigation, DA and WT arranging the results, discussion, and writing the original draft. DA, WT, and SP: revised the manuscript. All authors contributed to the article and approved the submitted version.
Conflict of interest: Authors state no conflict of interest.
Supplementary materials: Figure S1, Figure S2, Table S1 and Table S2.
Data availability statement: The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

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Received: 2023-07-12

Revised: 2023-12-28

Accepted: 2024-01-30

Published Online: 2024-07-18

This work is licensed under the Creative Commons Attribution 4.0 International License.

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https://doi.org/10.1515/opag-2022-0267

Keywords for this article

biocontrol; plant disease; induced resistance; plant immune

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BY 4.0

Pseudomonas fluorescens SP007s enhances defense responses against the soybean bacterial pustule caused by Xanthomonas axonopodis pv. glycines

Article

Abstract

1 Introduction

2 Materials and methods

2.1 Plant material, bacterial strains, and inoculum preparation

2.2 Priming of plant and Xag inoculation

2.3 Detection of endogenous SA

2.4 PCD of SP007

2.5 Rapid detection of biochemical changes using synchrotron radiation-based Fourier-transform infrared (SR-FTIR) microspectroscopy

2.6 Disease rating

2.7 Evaluation of Xag growth in the soybean plants

2.8 Statistical analysis

3 Results

3.1 Detection of endogenous SA

3.2 Biochemical change analysis in soybean mesophyll

3.3 Detection of PCD of SP007s

3.4 Disease rating

3.5 Assessment of Xag growth in the soybean plants

4 Discussion

5 Conclusions

Acknowledgments

References

Supplementary Material

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