Paraburkholderia sp. GD17 improves tomato plant growth and resistance to Botrytis cinerea-induced disease

doi:10.21203/rs.3.rs-2205450/v1

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Research Article

Paraburkholderia sp. GD17 improves tomato plant growth and resistance to Botrytis cinerea-induced disease

https://doi.org/10.21203/rs.3.rs-2205450/v1

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Journal Publication

published 24 Jan, 2023

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Background and aims

The plant growth promoting rhizobacteria have been repeatedly addressed in improving plant growth and resistance against pathogens. This study explored the role of Paraburkholderia sp. GD17 in improving tomato plant growth and resistance to Botrytis cinerea.

Methods

Tomato roots were inoculated with GD17 strain, and then the leaves of well-colonized plants were infected with B. cinerea. Physiological and biochemical parameters, and gene expression were analyzed.

Results

In the absence of B. cinerea, GD17 efficiently improved plant growth, and increased photosynthetic efficiency. In the presence of B. cinerea, GD17-bacterized plants exhibited an enhanced resistance, as indicated by 67% of disease index in non-bacterized plants, while by 24% in bacterized ones. In response to B. cinerea, the defense reaction was reinforced in bacterized plants, as shown by enhanced antioxidative capacity and mitigated oxidative damage, as well as increased PR gene expression in bacterized plants compared with control. Photosynthesis was inhibited by B. cinerea, while it was substantially attenuated in bacterized plants. In the presence of B. cinerea, contents of soluble sugar significantly increased in non-bacterized plants, while it was controlled in bacterized plants. The carbohydrate catabolism-related genes, including starch degradation, photorespiration, and pentose phosphate pathway, generally presented a higher expression in bacterized plants under B. cinerea attack.

Conclusions

GD17 strain improved tomato plant growth by increasing the photosynthetic efficiency. GD17 enhanced plant resistance against B. cinerea-induced disease by increasing defense and alleviating oxidative damage. Additionally, GD17 optimized the trade-off between plant growth and defense by strengthening carbohydrate metabolic regulation.

Tomato

Botrytis cinerea

Paraburkholderia

Antioxidative defense

Pathogenesis-related gene

Trade-off between growth and defense

Botrytis cinerea (Bc) is an airborne necrotrophic fungal phytopathogen infecting over 200 crops worldwide, causing gray mold disease leading to serious yield reduction and quality deterioration, especially on greenhouse-grown vegetables such as tomatoes (Williamson et al. 2007). Bc naturally parasitizes in plant aerial parts with the infection way either through wound sites or direct penetration, and feeds on killed saprophytic tissues including leaves, petioles, stems, flowers, or fruits. Additionally, due to a long-term viability of Bc as mycelia and/or conidia or sclerotia on crop debris and in the soil even after a growing season, it is hard to control (Elmer and Michailides 2007). In tomato planting management, due to the lack of resistant varieties, it is always tough to control the occurrence of Bc-induced gray mold especially using a single measure (Williamson et al. 2007). The application of fungicides is the main means in traditional control of gray mold. However, with the increasing or abuse use of chemicals, it not only induces the development of fungicide-resistant Bc strains, but also causes serious environmental pollution, and even directly threats human health due to the residues on the surface of edible parts such as fruits (Rosero-Hernández et al. 2019). Therefore, it is very meaningful to develop more safe and effective methods in the prevention and control of gray mold.

With increasing attention to green agricultural products, biological control strategy using microbial agents is attracting a great deal of interest in agricultural management, among which plant growth-promoting rhizobacteria (PGPR) have been extensively applied as bio-fertilizers and bio-pesticides, with advantages of eco-friendly, sustainability, comprehensive and broad-spectrum effects, and also low cost (for a review see Syed Ab Rahman et al. 2018). PGPR affect plants by different ways. In a symbiotic way with plant roots, PGPR promote the host growth and disease resistance by bioactive substances synthesized by themselves or by stimulating the host, such as phytohormones, antibiotics, and toxins. In a free-living way, PGPR are beneficial to plants by regulating the rhizosphere suitability such as increasing the availability of essential nutrients (e.g. nitrogen, phosphorus, iron), recruiting or optimizing other rhizosphere microbial populations, creating a healthy soil environment by inhibiting soil-borne pathogenic growth, and so on (Syed Ab Rahman et al. 2018). Several lines of evidence have shown that Bc-induced tomato gray mold can be efficiently controlled by fungi (Barra-Bucarei et al. 2019; Sarven et al. 2020), and bacteria (Wang et al. 2020).

Many kinds of bacteria in the genus Burkholderia are important PGPR with extensive functions and distribution (Coenye and Vandamme 2003). Due to the huge differences in functionality, together with the molecular signatures and phylogenomic analysis, the genus Burkholderia is proposed to be reclassified as the emended genus Burkholderia only retaining the clinically pathogenic and phytopathogenic species, and a new genus Paraburkholderia gen. nov. mainly collecting environmental species (Sawana et al. 2014). Therefore, the strains of Burkholderia with the ability of promoting plant growth and tolerance to stress conditions reported in previous studies are belonged to the genus Paraburkholderia. Among this genus, the well-studied strain is the P. phytofirmans PsJN. It can efficiently enhance grapevine plant resistance against Bc-induced gray mold (Miotto-Vilanova et al. 2016). Our laboratory previously isolated a strain of Paraburkholderia (named as P. sp. GD17) from the roots of wild soybean, and it has the special features of PGPR, such as N₂-fixation, P-solubilization, production of indoleacetic acid, and inhibition of ethylene biosynthesis (Guo et al. 2018). This strain can efficiently colonize in rice roots promoting plantlet growth and tolerance under salt stress conditions (Zhu et al. 2021). In this study, the GD17 strain was used as a bio-control agent in tomato plant response to B. cinerea challenge, and it not only promoted plant growth under healthy conditions, but also efficiently prevented Bc-induced disease development on leaves.

Plant material and treatment

Seeds of tomato (Lycopersicon esculentum) cv. Castlemart were gifted by Prof. Howe (Michigan State University). The surface-sterilized seeds were germinated in dark at 25°C for 2 day by evenly placing on a moist filter paper. The uniformly germinated seeds were selected and sown in a pot (15-cm-diameter and 18 cm in height) filled with autoclaved mixture of vermiculite and soil (1:3, v:v), with one seed per pot. The controlled conditions in plant growth chamber were 14 h of light (150 µmol m^− 2 s^− 1) at 25°C and 10 h of dark at 18°C, and 70% of humidity. The seedlings were watered every two days (using 100 ml of Hoagland nutrient solution for the first watering). For the inoculation of GD17, 20-day-old plants were irrigated using 100 ml of bacterium suspension (10⁷ CFU ml^− 1) prepared with Hoagland nutrient solution. Control plants received Hoagland solution without GD17. Fifteen days post-inoculation of GD17, the plants were infected by spraying suspension of B. cinerea (10⁴ conidia ml^− 1) on the adaxial surface of leaf, and only ddH₂O was supplied for control. Therefore, the tested materials included control, + GD17, + Bc, and GD17 + Bc. Except as otherwise stated in the text, the analyses of parameters were carried out after 15 d of Bc infection.

Assessment of GD17 colonization efficiency and gray mold disease index

Colonization efficiency of GD17 in the root interior was evaluated according to the description by Zhu et al. (2021), and presented as the colony forming units (CFU) per gram of root fresh weight. Briefly, root samples were harvested from 5 plants at 0, 1, 7, and 15 d post-inoculation of GD17, respectively, and sterilized with 5% (v/v) sodium hypochlorite solution for 10 min and rinsed three times with sterile water. This disinfection protocol ensured no residual bacteria on the sample surface as detected by incubating on LB plate at 25°C for 24 h. The surface-disinfected roots were crushed with a sterile pestle, and then mixed with 20 ml of 0.9% NaCl solution. The suspension was vortexed for 3 min, and then let stand for the settlement of tissues debris. The supernatant was made into serial dilutions to be spread on LB plate. The plates were incubated at 28ºC for 2 days, and resulting colonies were counted. The gray mold disease index (DI) was assessed after 15 days of Bc infection according to the classification of disease grades (0–5) and the formula described by Sriram et al. (1997):

DI = (Numbers of diseased leaves × Average grade/Numbers of analyzed leaves × Maximum disease grade) × 100%

Measurement of plant growth

The fresh and dry weights of plant aerial parts and roots were measured to assess the effect of GD17 colonization, Bc infection, alone or in combination, on plant growth. Ten plants of each treatment were harvested after a total 50 d growth (such as the treatment of GD17 + Bc, 20-day-old plants inoculated with GD17 for 15 d, then infected by Bc for 15 d), and rinsed carefully with tap water. After blotting the surface with filter paper, aerial parts and roots were respectively collected and weighed. The dry mass was obtained after 48 h of drying in 80°C oven.

Determination of physiological and biochemical parameters

The extraction and determination of total chlorophyll were carried out following the method of Zhang and Qu (2003). Net photosynthetic rate, stomatal conductance, and intercellular CO₂ concentration were detected using a portable photosynthetic analyzer (Li-6200, Lincoln, NE, USA) under 200 µmol m^− 2 s^− 1 illumination at 25°C. Chlorophyll a fluorescence parameters were analyzed using fluorescent imaging system (FluorCam, PSI Ltd., Brno, Czech Republic) according to the manufacturer’s instructions and as described by Liu et al. (2020). Maximum quantum efficiency of PSII (Fv/Fm), actual quantum efficiency (Ф_PSII), non-photochemical fluorescence quenching (NPQ) and electron transport rate (ETR) were automatically generated by the instrument’s own software FluorCam 7.0. The extract and quantification of soluble sugars in leaves were performed using the anthrone colorimetric method following the description of Zhang and Qu (2003), and proline contents were measured by the method of Bates et al. (1973).

The extraction and activity determination of superoxide dismutase (SOD; EC 1.15.1.1), peroxidase (PRX; EC 1.11.1.7) and catalase (CAT; EC 1.11.1.6) were carried out according to the previous description (Zhu et al. 2021). In the preparation of extracts, the necrotic tissues (lesions) caused by Bc infection were excluded from the tested samples. The isoenzyme patterns of SOD and PRX were analyzed by native polyacrylamide gel electrophoresis according to the protocols described by Hao et al. (2012). The protein concentration in the crude enzyme extract was measured by the method of Bradford (1976). The reduced form of glutathione (GSH) was estimated following the method of Griffith and Meister (1979). The oxidized form of glutathione (GSSG) was the difference between the glutathione amounts of 1,4-dithiothreitol (DTT)-treated samples and the non-treated samples. Levels of hydrogen peroxide were estimated by quantifying the formation of titanium–hydro peroxide complex as described by He et al. (2014). Malondialdehyde (MDA) contents were analyzed according to Shalata and Tal (1998).

Histochemical detection of H₂O₂ and O₂˙ˉ

The accumulation of H₂O₂ in leaves was detected by in situ staining with 3,3′-diaminobenzidine tetrahydrochloride (DAB) following the description of Zhu et al. (2021). Briefly, detached fresh leaves were sucked into DAB solution (1 mg ml^− 1 DAB, 0.05% Tween 20, pH 3.8) by vacuuming, and then incubated for 45 min at room temperature under light. Superoxide anion (O₂˙ˉ) accumulation in leaves was assessed using nitroblue tetrazolium (NBT) staining. The detached fresh leaves were vacuum infiltrated with the mixture of NaN₃ (10 mM) and NBT (0.1% w/v), and then incubated for one hour at room temperature under light. All the stained leaves were soaked in 95% ethanol at 80°C to remove chlorophyll. The each staining experiment included several groups of leaves, and a similar tendency was found among them. Therefore, a representative group was presented in the text.

Gene expression analysis

Fresh leaves were collected from 10 plants of each treatment group, respectively, and ground in liquid nitrogen using a mortar and pestle. Total RNA was isolated using the RNA Isolation System (Promega), and the first-strand cDNA was synthesized using PrimeScript RT Reagen Kit (TaKaRa), both following the manufacturer's instructions. The transcript levels were determined by real-time quantitative PCR using Lightcycler 96 PCR system (Roche, Basel, Switzerland), and the reaction program was set as recommended by Qu et al. (2018). Relative gene expression was determined according to the 2^–ΔΔCT method. The Ct geometric average value of Actin 7 (ACT7; Solyc11g005330) and elongation factor 1-alpha (EF; Solyc06g005060) was used as an internal control. The short descriptions of the genes analyzed in this study and their primer sequences were presented in Supplementary Table S1 and S2, respectively.

Statistical analysis

All data came from three independent biological experiments, and presented as the mean ± SD. Statistical analysis was carried out using the software SAS (SAS Institute, Cary, NC, USA), taking p < 0.05 as significance according to Duncan's multiple range test.

Colonization of GD17, disease index of gray mold and plant growth

Substantial numbers of GD17 bacterium were found inside roots one day post-inoculation, and dramatically increased with the extension of inoculation duration as indicated by colony-forming units (CFU) (Fig. 1A). No GD17 strain was detected in non-inoculated plant roots.

To explore the systemic effect of GD17 symbiosis with roots on the leaf disease development induced by Bc, the well-colonized plants 15 days post-inoculation of GD17 were challenged with pathogens. After 15 days of Bc infection, the disease index (DI) was around 24% in the bacterized plants, while it reached 67% in control (non-bacterized) plants (Fig. 1B). Specifically, more necrotic plaques and larger areas were observed in control (Fig. 2). Furthermore, the onset of gray mold was delayed in the bacterized plants. For instance, there were obvious necrotic spots on non-bacterized plant leaves 7 d post-inoculation of Bc, while at this time, no disease symptoms and just pathogen hypha on leaf surface were observed in bacterized plants (Fig. 2). After 15 d of Bc infection, yellow-brown lesions were formed throughout the whole leaf of non-bacterized plant, but only a few spots were observed on the leaf veins of bacterized plant (Fig. 2). This indicated that the colonization of GD17 inside roots provided a systemic protection against the occurrence of gray mold.

The symbiosis of GD17 with roots promoted plant growth under healthy conditions. After a total of 50 days of growth, the fresh weight (FW) and dry weight (DW) of aerial part increased by 31% and 44%, and root FW and DW by 56% and 58%, respectively, in bacterized plants compared with non-bacterized ones (Fig. 3). The symbiosis also efficiently attenuated Bc-induced plant growth inhibition. After 15 d of Bc infection, the FW and DW of aerial part decreased by 19% and 25%, and root FW and DW by 14% and 7% in the bacterized plants, while the FW and DW of aerial part decreased by 46% and 46%, and root FW and DW by 22% and 21% in non-bacterized plants, respectively, when compared with their respective healthy plants (Fig. 3).

GD17-triggered defense response to Bc challenge

The plant resistance to necrotrophic pathogens is always coupled with an increased antioxidative defense. Under healthy conditions, the effect of GD17 was inconsistent on the activities of SOD, PRX and CAT, wherein SOD activity increased, PRX decreased, and CAT unchanged. In response to Bc challenge, the activity of all three antioxidases increased, in which a higher level for SOD and CAT, and a comparable level for PRX were detected in GD17-bacterized plants compared with non-bacterized ones (Fig. 4). The inoculation of GD17 elevated the content of GSH and the ratio of GSH/GSSG under healthy conditions. In response to Bc, the level of both decreased in all tested plants, but they were still substantially higher in the bacterized plants than in non-bacterized ones (Fig. 4). The changed patterns of SOD and PRX were also revealed by their isoenzyme profiles shown by polyacrylamide gel electrophoresis (Fig. 5).

Bc infection increased the production of H₂O₂ in leaves by 28% and 50% in bacterized and non-bacterized plants, respectively (Fig. 4). The increased production and distribution of reactive oxygen species (ROS) were also visualized by in situ staining reactions, with DAB specificity for H₂O₂, and NBT for superoxide anion (O₂˙ˉ) (Fig. 6). Correspondingly, Bc infection caused more serious oxidative damage to non-bacterized plants relative to the bacterized ones, where MDA and electrolyte leakage increased by 106% and 71% in the former, and by 40% and 20% in the latter, respectively, when compared with their respective basal level (Fig. 4).

The expression of several antioxidant-related genes was analyzed. Generally, the expression of most of the tested genes tended to be moderately up-regulated in bacterized plants in the absence of Bc (Fig. 7). However, in response to Bc challenge, all of the tested genes presented an obviously upregulated expression in bacterized plants, while a mixed expression pattern occurred in non-bacterized plants, such as upregulated for SOD and PRX, unchanged for CAT and APX, and down-regulated for GSH, GR and MDAR. This led to a higher accumulation of the transcripts for all analyzed genes in bacterized plants than in non-bacterized ones (Fig. 7).

The pathogenesis-related (PR) protein-mediated defense and the expression patterns of PR genes are extensively used to dissect plant response to pathogen challenge. In the absence of Bc, the expression of PR1 and PR3 was heavily down-regulated in bacterized plants. However, in the presence of Bc, all of the tested genes (PR1, -2 and − 3) presented an obviously upregulated expression in bacterized plants, while only mild upregulation for PR1, and significant down-regulation for PR2 and − 3 in non-bacterized plants, which resulted in a much higher accumulation of the transcripts for all three PR genes in bacterized plants than in non-bacterized ones (Fig. 8).

GD17-mediated regulation in photosynthesis-related processes and carbohydrate catabolism

Photosynthesis is a vital regulation target in trade-off between plant growth and defense. Under healthy conditions, application of GD17 improved photosynthesis-related performance, as indicated by an increase in total chlorophyll by 35%, net photosynthetic rate by 20%, stomatal conductance by 17%, and intercellular CO₂ concentration by 10%, respectively (Table 1). Bc infection reduced the levels of all these parameters to a greater degree in non-bacterized plants relative to the bacterized ones. This led to a much higher level for all these parameters in bacterized plants than in non-bacterized ones in response to Bc challenge (Table 1). The change patterns of photosynthesis were also revealed by a visualized imaging of chlorophyll a fluorescence parameters such as Fv/Fm, Ф_PSII, ETR and NPQ (Fig. 9 and Table 1).

To further understand the mechanism of GD17-mediated regulation on photosynthesis, the expression pattern of photosynthesis-related genes was analyzed. HEMA1, GSA, HEMD, HEMG, CHLH, and CHLM are important enzymes in chlorophyll biosynthesis. Under healthy conditions, most of their encoding genes presented a higher expression level in GD17-bacterized plants (Fig. 10A). In response to Bc challenge, they all expressed at a higher level in the bacterized plants, while a mixed expression pattern occurred in non-bacterized ones (Fig. 10A). In the absence of Bc, the effect of GD17 was inconsistent on the expression of PSI genes (PsaD, PsaN and Cab5) and PSII genes (PsbQ, PsbW and OEP1), with a moderate upregulation or down-regulation depending on individual genes. However, GD17 substantially prevented the Bc-induced expression down-regulation of these genes, and even a higher expression level was measured for individual genes in the presence than in the absence of Bc (Fig. 10B). Similarly, the regulatory effect of GD17 was also observed on the expression of photosynthetic electron transporter-encoding gene PETE and PETM, and ATP synthase-encoding gene ATPC and ATPD (Fig. 10C), as well as the Calvin cycle-related genes, such as RBCS1, FBA1, FRB and SBP (Fig. 10D).

The soluble sugars play an important role in balancing plant growth and defense. Under healthy conditions, soluble sugar increased by 24% in GD17-bacterized plants. However, in the presence of Bc, it was enhanced by around 92% in non-bacterized plants, while only by 8% in the bacterized ones, compared with their respective control level (Fig. 11). Proline content increased by 47% in GD17 plant under healthy conditions. Bc infection decreased proline level by 43% and 33% in non-bacterized and bacterized plants, respectively, compared with their non-infected control (Fig. 11).

Finally, the expression of carbohydrate catabolism-related genes was analyzed. Under healthy conditions, GD17 upregulated the expression level of starch degradation-related gene AMY, but did not affect the BMY3. In response to Bc challenge, the expression of AMY was not affected in non-bacterized plants, but was moderately down-regulated in the bacterized plants. The expression of BMY3 was inhibited by Bc infection in both plants, with a greater degree in non-bacterized plants (Fig. 12A). In the absence of Bc, GD17 slightly promoted the expression of photorespiration-related gene AGT, but did not influenced the SGT. Bc infection decreased the expression level of AGT and SGT, both to a greater degree in non-bacterized plants than in the bacterized ones (Fig. 12B). Similarly, the expression of pentose phosphate pathway-related gene G6PD and PGD was little impacted in bacterized plants under healthy conditions. However, in response to Bc, the expression of G6PD was significantly suppressed in non-bacterized plants, while it was obviously induced in bacterized ones. The expression of PGD was substantially inhibited by Bc infection in both plants, with a particularly severe degree in non-bacterized plants (Fig. 12C).

Mounting evidence has demonstrated that inoculation with PGPR can improve plant growth or inhibit disease development or both (Backer et al. 2018). This study showed that inoculation of Paraburkholderia sp. GD17 efficiently promoted tomato plant growth and resistance to Botrytis cinerea-induced gray mold. This not only provided an insight into the plant growth-promoting and phytopathogen-antagonistic properties of Paraburkholderia species, but also provided a scientific basis for the possible application of GD17 strain in tomato sustainable production. Among the Paraburkholderia species, the best studied strain is P. phytofirmans PsJN, which can clonize the internal tissues of a dozen plant species reported to promote plant growth or enhance resistance to plant pathogens (Miotto-Vilanova et al. 2016; Esmaeel et al. 2018). For instance, well-established testing systems showed that the PsJN strain acted as a remarkable biocontrol agent against B. cinerea-induced gray mold on grapevine (Ait Barka et al. 2002). However, little information is available about the role of Paraburkholderia species in the bio-control of tomato gray mold.

PGPR promotes plant growth by multi-facetted mechanisms, such as solubilizing mineral elements (P, K, Fe and Zn), fixing atmospheric nitrogen, synthesizing phytohormones (indole-3-acetic acid, gibberellins and cytokinin), and reducing ethylene production (For a review see Rana et al. 2020). In an earlier study, our laboratory isolated a strain, named as Paraburkholderia sp. GD17, and it has most of the PGPR properties (Guo et al. 2018). Further study revealed that application of GD17 can improve rice plant growth under both healthy and salt stress conditions by multiple mechanisms, such as promoting mineral element absorption and root-shoot transport, increasing plant antioxidative defense, and so on (Zhu et al. 2021). In this study, it was clearly demonstrated that the clonization of GD17 in tomato roots systemically improved the host photosynthetic efficiency as shown by increased net photosynthetic rate and other photosynthesis-related parameters (Table 1). The analysis of gene expression suggested that the increased photosynthetic efficiency in GD17-bacterized plants was associated with up-regulated expression of photosynthesis-related genes, especially in the chlorophyll biosynthesis, photosynthetic electron transport and phosphorylation, and Calvin cycle (Fig. 10A, 10C and 10D). This implied that the plant growth-promoting property of PGPR was directly correlated with improved carbon assimilation capacity probably by up-regulating the expression levels of photosynthesis-related genes.

Diverse mechanisms have been proposed to decipher the role of PGPR in systemically antagonizing pathogen-induced plant diseases. In this study, the expression patterns of PR1, -2 and − 3 strongly implied the involvement of pathogenesis-related (PR) proteins in GD17-conferred resistance in tomato plants against Bc assault (Fig. 8). On the basis of their biochemical and biological properties, PR proteins are classified into 17 subfamilies. They are generally present constitutively in various plant tissues and organs, while inducibly increased under stress conditions (Van Loon et al. 2006). The members of PR1 subfamily are extensively studied in plant response to biotic and abiotic stresses, especially as markers in the establishment of pathogen-induced systemic acquired resistance (SAR). Although the biological role of PR1 proteins in plant defense is poorly known, several studies have provided evidence demonstrating that PR1 proteins have antimicrobial activity in plants against pathogens. For instance, genetically transformed tobacco plants with a homologous gene of PR1 from Wasabia japonica showed a remarkable resistance to Botrytis cinerea (Kiba et al. 2007). Exogenous application of PR1 proteins not only inhibited in vitro zoospore germination of Phytophthora infestans, but also limited colonization of the pathogen in tomato leaf discs (Niderman et al. 1995). PR2 proteins as β-1,3-endoglucanases and PR3 as endochitinases are involved in plants against fungi. For instance, the expression level of PR2 was significantly increased in tomato plants challenged with B. cinerea (Crespo-Salvador et al. 2018). Over-expression of the maize PR2 in Arabidopsis efficiently improved plant resistance to B. cinerea infection (Xie et al. 2015). The evidence available suggests that PR proteins are involved in PGPR-mediated plant resistance to fungus pathogens. For example, PR1 and PR2 presented higher expression levels in P. phytofirmans PsJN-bacterized grapevine plants than non-bacterized ones after B. cinerea challenge (Miotto-Vilanova et al. 2016).

The combat between oxidative stress and antioxidative defense is ubiquitous in the interaction between plants and pathogens. Reactive oxygen species (ROS), agents for oxidative stress especially under adverse conditions, can be produced in various subcellular compartments by distinct mechanisms, such as through electron leakage in the electron transfer chain of chloroplasts and mitochondria, photorespiration in peroxisomes, enzymatic reaction mediated by NADPH oxidases (Respiratory burst oxidase homolog, Rboh) and class III peroxidase (PRX) in plasma membranes and cell walls, and so on (Nadarajah 2020). ROS have a dual function in plant response to pathogens. For instance, the RbohB-silencing tomato plants accumulated less H₂O₂, and correspondingly showed an increased sensitivity to B. cinerea challenge, suggesting that a normal production of H₂O₂ (amount and site) can act as a messenger to trigger plant immunity to the pathogen infection (Li et al. 2015). However, Trichoderma asperellum-conferred tomato leaf resistance to B. cinerea was correlated with a decreased H₂O₂ (Herrera-Téllez et al. 2019). Similar observations also occurred in other species of plants. For example, the resistant cultivar of grapevine generated less ROS (H₂O₂ and O₂ˉ˙) than did the susceptible one under B. cinerea challenged (Wan et al. 2015). This suggests that the homeostasis of ROS between production and scavenging is important in plants coping with pathogens especially those with necrotrophic lifestyle. This was reflected in the present study where the Bc-induced increases of H₂O₂ and O₂ˉ˙ were efficiently limited in GD17-bacterized plant leaves, as indicated by spectrophotometer-measured contents of H₂O₂ in leaves (Fig. 4), and also by the histochemical detection with DAB-mediated brown coloration staining for H₂O₂ and NBT-mediated dark blue staining for O₂ˉ˙ (Fig. 6). Generally, ROS-associated oxidative burst is one of the major mechanisms responsible for the control of biotrophic pathogens-induced diseases, such as by establishing hypersensitive response (HR) leading to the formation of necrotic plaque around the penetration site with limited pathogen development, but it does not prevent the necrotrophic pathogen-induced disease development, and even boosts this process (Temme and Tudzynski 2009). For example, a successful infection of B. cinerea on plants is based on production of ROS and the formation of necrotic lesion (Govrin et al. 2006). B. cinerea kills host cells not only by secreting toxins such as botrydial (Colmenares et al. 2002), but also by producing ROS by itself and/or by stimulating host cells (Choquer et al. 2007). Therefore, antioxidative defense might be an important mechanism in plants against necrotrophic pathogen-induced disease development. In this study, the changed patterns of enzymatic activity (SOD, PRX and CAT) and non-enzymatic antioxidant (GSH) content, as well as the expression patterns of several antioxidative enzyme-encoding genes (APX, GSH, GR and MDAR) indicated that GD17-conferred tomato resistance to Bc-induced disease was associated with an increased antioxidative defense. This was agreed with some observations available on PGPR-conferred plant resistance to pathogens. For instance, the well-studied Bacillus-bacterized plants against pathogens is always correlated with an increased antioxidative defense (Rais et al. 2017; Hashem et al. 2019). Furthermore, the changed pattern of class III plant peroxidases (PRXs; EC 1.11.1.7) isoenzymes attracted attention. Based on the special coloration reaction using polyacrylamide gel electrophoresis (PAGE), both the number and activity of PRX isoenzymes were dramatically increased in plant leaves upon Bc challenge, despite no significant difference between GD17-bacterized and non-bacterized plants (Fig. 5). This further confirmed that PRXs were induced by fungus pathogens. PRXs are multigene family proteins belonging to the PR-protein 9 subfamily (van Loon et al. 2006). They are involved in plants against pathogen attack by multiple mechanisms, such as strengthening the structure of cell wall, a physical barrier for pathogen invasion and spread, by promoting the formation of lignin and suberin, and the cross-linking of cell wall components, establishing highly toxic environments by massively generating ROS, and so on (Passardi et al. 2005; Almagro et al. 2009).

Although decreased photosynthetic activity is considered as an active response in plant coping with pathogens, the impact of photosynthetic products (carbohydrates) on plant resistance to pathogens experiences complex mechanisms, among which more attention has been given to the regulation of source-sink allocation of sugars by sugar transport machinery, and the relative proportion of various sugars (sucrose, glucose and fructose) (Kanwar and Jha 2019; Courbier et al. 2020). However, little attention was received regarding to the effect of enhanced soluble sugar levels on disease development. Generally, high-sugar concentrations in plant tissues are conducive to the establishment of defense systems. For example, sugars as carbon and energy sources are involved in the synthesis of defense-related chemicals, as signal molecules inducing the expression of defense genes such as MAPK or PR, as antioxidants participating in plant response to oxidative stress (for a review see Moghaddam and Van Den Ende 2012). However, high sugar levels also benefit the growth of pathogens. For instance, evidence available showed that B. cinerea-induced disease development on tomato leaves was positively correlated with the concentrations of soluble sugar in leaves (Courbier et al. 2020). This implied that maintaining a moderate range of sugar concentrations in plant tissues might be necessary to plant resistance against disease development. This can be inferred in the present study, where the concentration of soluble sugar was substantially lower in GD17-bacterized plants relative to non-bacterized ones after Bc infection (Fig. 11), which might be beneficial to limit disease development. Nevertheless, the concentration of soluble sugar was still higher in the bacterized plants under Bc challenge than under healthy conditions (Fig. 11), which might be favourable for the establishment of a stronger defense. Besides as a component of proteins, proline has multiple biological functions as a free form. Evidence available has shown that proline-rich proteins displayed antifungal activity to pathogens including B. cinerea (Li et al. 2012; Cao et al. 2015). Intracellular free proline can regulate osmotic balance, stabilize biomacromolecules, scavenge radicals to maintain redox balance (Szabados and Savoure 2010). Furthermore, proline metabolisms (synthesis and catabolism) have been extensively implicated in the plant-pathogen interaction (For a review see Alvarez et al. 2022). In this study, although Bc infection decreased the content of proline in leaves, its absolute value was still substantially higher in the bacterized plants than in non-bacterized ones (Fig. 11), suggesting that proline might be involved in GD17-conferred resistance to the pathogen.

Photosynthesis and innate immunity are two closely related fundamental processes throughout the whole life cycle of a plant. An increased photosynthesis helps plants resist pathogens by providing energy and carbon skeleton to synthesize defense-related substances, while it also produces more carbohydrates supporting pathogen growth (Swarbrick and Lefert 2006). This suggested that a moderate photosynthetic efficiency might be favourable for plant coping with pathogen challenge. In this study, Bc infection significantly damped the photosynthetic efficiency, while it was substantially alleviated in GD17-bacterized plants, as indicated by the parameters measured by a photosynthetic analyzer and a chlorophyll a fluorescent imaging system (Table 1, and Fig. 9), also reflected on the expression patterns of photosynthesis-related genes including those encoding the enzymes in chlorophyll synthesis, the structural and function proteins of PSI and PSII, the components of photosynthetic electron transport chain, the subunits of ATP synthase complex, and the enzymes in Calvin cycle (Fig. 10). Together with the resistant phenotype of GD17-bacterized plants, it was speculated that the photosynthetic efficiency and carbohydrate content (as shown by soluble sugar content in Fig. 11) in Bc-infected GD17-plants might be suitable for plant coping with pathogen challenge probably by both limiting carbon availability for pathogen growth and building resistant pathways as described before (Walters and Boyle 2005). Additionally, the transcription regulation of the related genes might be one of the major targets responsible for GD17-conferred disease resistance. This was also explicitly reflected on the expression of carbohydrate catabolism-related genes. For example, both α- and β-amylase-encoding genes expressed at a higher level in the bacterized plants relative to non-bacterized ones in response to the pathogen (Fig. 12A). This meant that starch degradation was improved in the bacterized plants in response to Bc. The involvement of starch degradation in plant response to pathogens is not only limited to providing soluble sugar, but also correlated with other defense pathways. For example, β-amylase-mediated starch degradation contributed to callose deposition in plant cell wall, which prevented pathogen colonization, spread and disease initiation and development in plant tissues (Gamir et al. 2018). Furthermore, starch degradation plays an important role in diminishing the “photosynthetic acclimation” caused by starch-induced block of CO₂ diffusion from the intracellular space to the stroma (Jauregui et al. 2018).

The expression patterns of two key photorespiratory genes AGT (encoding alanine:glyoxylate aminotransferase) and SGT (encoding serine-glyoxylate transaminase) implied that the photorespiration was engaged in the bacterized plant resistance against Bc. Although not much is known about the physiological significance of photorespiration, it is explicit that the photorespiration can mitigate stress-induced photosynthetic impairment by releasing CO₂ and consuming excessive light energy (Wingler et al. 2000). Furthermore, photorespiration has been definitely implicated in plant-pathogen interaction, as suggested by globally analyzing the expression of related genes, as well as utilizing the genetically modified plant mutants (knockout) or transgenic lines (overexpression) of photorespiration-related genes (for a view see Sørhagen et al. 2013). For instance, in comparison of a pathogen-resistant melon line with its susceptible ones, the expression levels of AGT, SGT, and GOX (encoding glycolate oxidase in photorespiration), and their enzymatic activities were much higher in the former than in the latter (Taler et al. 2004).

Pentose phosphate pathway (PPP) is an important primary metabolic process in plants especially under stress conditions by supplying plants with carbon skeletons for the synthesis of many necessary compounds including defense chemicals, and producing a large amount of NADPH. Additionally, due to the metabolic intermediates and enzymes in PPP being shared by Calvin cycle, thus the PPP is an important complement for CO₂ assimilation and carbohydrate production particularly under pathogen challenge where the photosynthetic efficiency is suppressed. In this study, the expression patterns of PPP-related gene G6PD and PGD suggested that the PPP might be involved in GD17-bacterized plant resistance against Bc-induced disease. Glucose-6-phosphate dehydrogenase (G6PD) and phosphogluconate dehydrogenase (PGD) are only two enzymes catalyzing substrate dehydrogenation to generate NADPH in oxidative pentosephosphate pathway (OPPP). The G6PD activity contributed to plant resistance to pathogens probably by oxidative bursts, callose deposition, and metabolic regulation. For instance, overexpression of G6PD increased tobacco resistance to Phytophthora nicotianae, while knockout mutants exhibited a susceptible phenotype (Scharte et al. 2009). With Rhizoctonia solani challenge, a higher activity of both G6PD and PGD was detected in the resistant variety of rice than in susceptible one (Danson et al. 2000).

The inoculation with Paraburkholderia sp. GD17 in tomato roots improved plant growth, in which an increased photosynthetic efficiency might be a major contributor. In response to B. cinerea challenge, GD17-bacterized plants presented an enhanced resistance, as indicated by a decreased number and size of necrotic plaques on leaves, also by ameliorated plant growth inhibition and photosynthetic impairment, compared with control (non-bacterized) plants. The involved mechanism was correlated with the initiation of defense responses including the antioxidative defense and pathogenesis-related gene expression. Furthermore, the metabolic regulation for carbohydrate synthesis and catabolism were implicated in the GD17-conferred resistance against Bc infection, where the photosynthetic efficiency and soluble sugar accumulation were controlled at moderate levels, which might play a crucial role in the trade-off between the building of defense system and the limitation of pathogen growth. Future research will focus on the effect of GD17 strain in greenhouse or field assays to inspect whether the bacterial application can increase tomato yield under healthy conditions or enhance plant resistance to pathogens.

Acknowledgments This research was supported by the National Natural Science Foundation of China (Grant No. 31572213 to HL).

Author contributions HL and LG designed and carried out the research. GA, ZD, LH and FW contributed to carry out the physiological, biochemical and the qRT-PCR analyses. HL and FW analyzed the data and wrote the manuscript. All authors read and approved the manuscript.

Conflict of interest The authors declare that they have no conflict of interest.

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Paraburkholderia sp. GD17 improves tomato plant growth and resistance to Botrytis cinerea-induced disease

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