Serratia plymuthica HK9-3 enhances tomato resistance against Phytophthora capsici by modulating antioxidant defense systems and rhizosphere micro-ecological condition.

Tomatoes are frequently challenged by various pathogens, among which Phytophthora capsici (P. capsici) is a destructive soil-borne pathogen that seriously threatens the safe production of tomatoes. Plant growth-promoting rhizobacteria (PGPR) positively induced plant resistance against multiple pathogens. However, little is known about the role and regulatory mechanism of PGPR in tomato resistance to P. capsici. Here, we identified a new strain Serratia plymuthica (S. plymuthica), HK9-3, which has a significant antibacterial effect on P. capsici infection. Meanwhile, stable colonization in roots by HK9-3, even under P. capsici infection, improved tomato growth parameters, root system architecture, photosynthetic capacity, and boosted biomass. Importantly, HK9-3 colonization significantly alleviated the damage caused by P. capsici infection through enhancing ROS scavenger ability and inducing antioxidant defense system and pathogenesis-related (PR) proteins in leaves, as evidenced by elevating the activities of peroxidase (POD), superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), phenylalanine ammonia lyase (PAL), polyphenol oxidase (PPO), and chitinase, ß-1,3-glucanase, and increasing the transcripts of POD, SOD, CAT, APX1, PAL1, PAL2, PAL5, PPO2, CHI17 and ß-1,3-glucanase genes. Notably, HK9-3 colonization not only effectively improved soil microecology and soil fertility, but also significantly enhanced fruit yield by 44.6% and improved quality. Our study presents HK9-3 as a promising and effective solution for controlling P. capsici infection in tomato cultivation while simultaneously promoting plant growth and increasing yield, which may have implications for P. capsici control in vegetable production.

Plant growth-promoting rhizobacteria Pseudomonas aeruginosa HG28-5 improves salt tolerance by regulating Na+/K+ homeostasis and ABA signaling pathway in tomato.

Salinity stress badly restricts the growth, yield and quality of vegetable crops. Plant growth-promoting rhizobacteria (PGPR) is a friendly and effective mean to enhance plant growth and salt tolerance. However, information on the regulatory mechanism of PGPR on vegetable crops in response to salt stress is still incomplete. Here, we screened a novel salt-tolerant PGPR strain Pseudomonas aeruginosa HG28-5 by evaluating the tomatoes growth performance, chlorophyll fluorescence index, and relative electrolyte leakage (REL) under normal and salinity conditions. Results showed that HG28-5 colonization improved seedling growth parameters by increasing the plant height (23.7%), stem diameter (14.6%), fresh and dry weight in the shoot (60.3%, 91.1%) and root (70.1%, 92.5%), compared to salt-stressed plants without colonization. Likewise, HG28-5 increased levels of maximum photochemical efficiency of PSII (Fv/Fm) (99.3%), the antioxidant enzyme activities as superoxide dismutase (SOD, 85.5%), peroxidase (POD, 35.2%), catalase (CAT, 20.6%), and reduced the REL (48.2%), MDA content (41.3%) and ROS accumulation in leaves of WT tomatoes under salt stress in comparison with the plants treated with NaCl alone. Importantly, Na+ content of HG28-5 colonized salt-stressed WT plants were decreased by15.5% in the leaves and 26.6% in the roots in the corresponding non-colonized salt-stressed plants, which may be attributed to the higher K+ concentration and SOS1, SOS2, HKT1;2, NHX1 transcript levels in leaves of colonized plants under saline condition. Interestingly, increased abscisic acid (ABA) content and upregulation of ABA pathway genes (ABA synthesis-related genes NCED1, NCED2, NCED4, NECD6 and signal genes ABF4, ABI5, and AREB) were observed in HG28-5 inoculated salt-stressed WT plants. ABA-deficient mutant (not) with NCED1 deficiency abolishes the effect of HG28-5 on alleviating salt stress in tomato, as exhibited by the substantial rise of REL and ROS accumulation and sharp drop of Fv/Fm in the leaves of not mutant plants. Notably, HG28-5 colonization enhances tomatoes fruit yield by 54.9% and 52.4% under normal and saline water irrigation, respectively. Overall, our study shows that HG28-5 colonization can significantly enhance salt tolerance and improved fruit yield by a variety of plant protection mechanism, including reducing oxidative stress, regulating plant growth, Na+/K+ homeostasis and ABA signaling pathways in tomato. The findings not only deepen our understanding of PGPR regulation plant growth and salt tolerance but also allow us to apply HG28-5 as a microbial fertilizer for agricultural production in high-salinity areas.

High red/far-red ratio promotes root colonization of Serratia plymuthica A21-4 in tomato by root exudates-stimulated chemotaxis and biofilm formation.

Effective colonization on plant roots is a prerequisite for plant growth promoting rhizobacterias (PGPR) to exert beneficial activities. Light is essential for plant growth, development and stress response. However, how light modulates root colonization of PGPR remains unclear. Here, we found that high red/far red (R/FR) light promoted and low R/FR light inhibited the colonization and growth enhancement of Serratia plymuthica A21-4 (S. plymuthica A21-4) on tomato, respectively. Non-targeted metabolomic analysis of root exudates collected from different R/FR ratio treated tomato seedlings with or without S. plymuthica A21-4 inoculation by UPLC-MS/MS showed that 64 primary metabolites in high R/FR light-grown plants significantly increased compared with those determined for low R/FR light-grown plants. Among them, 7 amino acids, 1 organic acid and 1 sugar obviously induced the chemotaxis and biofilm formation of S. plymuthica A21-4 compared to the control. Furthermore, exogenous addition of five artificial root exudate compontents (leucine, methionine, glutamine, 6-aminocaproic acid and melezitose) regained and further increased the colonization ability and growth promoting ability of S. plymuthica A21-4 on tomato under low R/FR light and high R/FR light, respectively, indicating their involvement in high R/FR light-regulated the interaction of tomato root and S. plymuthica A21-4. Taken together, our results, for the first time, clearly demonstrate that high R/FR light-induced root exudates play a key role in chemotaxis, biofilm formation and root colonization of S. plymuthica A21-4. This study can help promote the combined application of light supplementation and PGPR to facilitate crop growth and health in green agricultural production.

Accumulation and metabolism of pyroxasulfone in tomato seedlings.

Pyroxasulfone (PYS) is an isoxazole herbicide favored for its high activity. However, the metabolic mechanism of PYS in tomato plants and the response mechanism of tomato to PYS are still lacking. In this study, it was found that tomato seedlings had a strong ability to absorb and translocate PYS from roots to shoots. The highest accumulation of PYS was in the apex tissue of the tomato shoots. Using UPLC-MS/MS, five metabolites of PYS were detected and identified in tomato plants, and their relative contents in different parts of tomato plants varied greatly. The serine conjugate, DMIT [5, 5-dimethyl-4, 5-dihydroisoxazole-3-thiol (DMIT)] &Ser, was the most abundant metabolites of PYS in tomato plants. In tomato plants, the conjugation of thiol-containing metabolic intermediates of PYS to serine may mimic the cystathionine ß-synthase-catalyzed condensation of serine and homocysteine (in the pathway sly00260 sourced from KEGG database). This study ground breakingly proposed that serine may play an important role in plant metabolism of PYS and fluensulfone (whose molecular structure is similar to PYS). PYS and atrazine (whose toxicity profile is similar to PYS but not conjugate with serine) produced different regulatory outcomes for endogenous compounds in the pathway sly00260. Differential metabolites in tomato leaves exposed to PYS compared with the control, including amino acids, phosphates, and flavonoids, may play important roles in tomato response to PYS stress. This study provides inspiration for the biotransformation of sulfonyl-containing pesticides, antibiotics and other compounds in plants.

Luffa rootstock enhances salt tolerance and improves yield and quality of grafted cucumber plants by reducing sodium transport to the shoot.

Soil salinity severely limits crop yield and quality. Grafting onto tolerant rootstocks is known as an effective means to alleviate salt stress. The present study was planned to find out the potential roles, mechanisms and applications of luffa rootstock to improve salt tolerance of grafted cucumber plants. Here, we screened a highly salt-tolerant luffa rootstock by evaluating the growth, photosynthetic performance, antioxidant defense and the accumulation of Na+ and K+ under salt stress. Reciprocal grafting between cucumber and luffa showed that luffa rootstock significantly improved the salt tolerance of cucumber plants, as evidenced by higher fresh weight, photochemical efficiency (Fv/Fm), and lower relative electrical conductivity (REC), which was closely associated with the decreased accumulation of Na+ and increased the accumulation of K+ in shoots of luffa grafted cucumber seedlings, leading to a lower Na+:K+ ratio in shoot when compared with self-grafted cucumber. Furthermore, grafting with intermediate stock of luffa also sufficiently alleviated cucumber salt stress by reducing Na+ accumulation in shoot and the whole plant but increasing Na+ accumulation in interstock and root under salt stress, fully proving the salt tolerance depending on the capacity of luffa interstock to limit the transport of Na+ from the root to the shoot. More importantly, luffa rootstock improved the growth, yield and quality of grafted cucumber plants grown in pots in solar greenhouse as revealed by increased net photosynthetic rate, plant height, leaf number, yield, Vitamin C and soluble sugar but decreased titratable acid under both salinity and normal conditions. Together, these results, for the first time, clearly demonstrated that luffa,a new highly salt-tolerant rootstock, enhances salt tolerance and improves yield and quality of grafted cucumber plants by reducing sodium transport to the shoot.

The transcription factor SlNAP1 increases salt tolerance by modulating ion homeostasis and ROS metabolism in Solanum lycopersicum.

NAC transcription factors (TFs) play an important role in the plant resistant response to biotic and abiotic stresses. However, the functions of the most NAC TFs are still unknown, especially in tomato. Here, we identified and functionally characterized an NAC TFs, SlNAP1, in tomato, and found that SlNAP1 was significantly induced by salt stress. Under 150 mM NaCl treatments, morphological indexes of SlNAP1 over-expressed (SlNAP1-OE) transgenic tomato lines were significantly better than the wild-type (WT) plants. The content of Na+ in leaves and roots of SlNAP1-OE transgenic plants decreased, while the K+ content in leaves, roots, and stems increased compared with WT plants. The expression of the salt stress-related genes (NHX1, HKT1;2 and SOS1) in SlNAP1-OE plants were also significantly up-regulated under salt stress. The SOD, POD and CAT activities and the expression level of antioxidant oxidase synthesis genes of SlNAP1-OE lines were significantly increased. In addition, the SlNAP1-OE lines accumulated less MDA, H2O2 and O2•-, improved antioxidant defense systems which contributed to increase salt tolerance. In summary, our data suggest that SlNAP1 positively regulates salt tolerance in tomato by regulating ion homeostasis and ROS metabolism.

Dissection of Paenibacillus polymyxa NSY50-Induced Defense in Cucumber Roots against Fusarium oxysporum f. sp. cucumerinum by Target Metabolite Profiling.

To gain insights into the roles of beneficial PGPR in controlling soil-borne disease, we adopted a metabolomics approach to investigate the beneficial impacts of P. polymyxa NSY50 on cucumber seedling roots under the pathogen of Fusarium oxysporum f. sp. cucumerinum (FOC). We found that NSY50 pretreatment (NSY50 + FOC) obviously reduced the production of reactive oxygen species (ROS). Untargeted metabolomic analysis revealed that 106 metabolites responded to NSY50 and/or FOC inoculation. Under FOC stress, the contents of root osmotic adjustment substances, such as proline and betaine were significantly increased, and dehydroascorbic acid and oxidized glutathione (GSH) considerably accumulated. Furthermore, the contents of free amino acids such as tryptophan, phenylalanine, and glutamic acid were also significantly accumulated under FOC stress. Similarly, FOC stress adversely affected glycolysis and the tricarboxylic acid cycles and transferred to the pentose phosphate pathway. Conversely, NSY50 + FOC better promoted the accumulation of α-ketoglutaric acid, ribulose-5-phosphate, and 7-phosphosodiheptanone compared to FOC alone. Furthermore, NSY50 + FOC activated GSH metabolism and increased GSH synthesis and metabolism-related enzyme activity and their encoding gene expressions, which may have improved redox homoeostasis, energy flow, and defense ability. Our results provide a novel perspective to understanding the function of P. polymyxa NSY50, accelerating the application of this beneficial PGPR in sustainable agricultural practices.

Comparative Transcriptome Analysis and Genetic Methods Revealed the Biocontrol Mechanism of Paenibacilluspolymyxa NSY50 against Tomato Fusarium Wilt.

Fusarium wilt caused by Fusarium oxysporum f. sp. lycopersici (Fol) is a common disease that affects tomatoes, which can cause the whole plant to wilt and seriously reduce the production of tomatoes in greenhouses. In this study, the morphological indexes, photosynthetic performance and incidence rate of NSY50 under Fol infection were evaluated. It was found that NSY50 could improve the growth of tomato seedlings and significantly reduce the incidence rate of Fusarium wilt. However, the molecular mechanism of NSY50 that induces resistance to Fusarium wilt is still unclear. We used transcriptomic methods to analyze NSY50-induced resistance to Fol in tomatoes. The results showed that plant defense related genes, such as PR and PAL, were highly expressed in tomato seedlings pretreated with NSY50. At the same time, photosynthetic efficiency, sucrose metabolism, alkaloid biosynthesis and terpene biosynthesis were significantly improved, which played a positive role in reducing the damage caused by Fol infection and enhancing the disease tolerance of seedlings. Through transgenic validation, we identified an important tomato NAC transcription factor, SlNAP1, which was preliminarily confirmed to be effective in relieving the detrimental symptoms induced by Fol. Our findings reveal that P. polymyxa NSY50 is an effective plant-growth-promoting rhizosphere bacterium and also a biocontrol agent of soil-borne diseases, which can significantly improve the resistance of tomato to Fusarium wilt.

Melatonin Alleviates Copper Toxicity via Improving ROS Metabolism and Antioxidant Defense Response in Tomato Seedlings.

The excessive accumulation of copper (Cu2+) has become a threat to worldwide crop production. Recently, it was revealed that melatonin (MT) could play a crucial role against heavy metal (HM) stresses in plants. However, the underlying mechanism of MT function acted upon by Cu2+ stress (CS) has not been substantiated in tomatoes. In the present work, we produced MT-rich tomato plants by foliar usage of MT, and MT-deficient tomato plants by employing a virus-induced gene silencing methodology and exogenous foliar application of MT synthesis inhibitor para-chlorophenylalanine (pCPA). The obtained results indicate that exogenous MT meaningfully alleviated the dwarf phenotype and impeded the reduction in plant growth caused by excess Cu2+. Furthermore, MT effectively restricted the generation of reactive oxygen species (ROS) and habilitated cellular integrity by triggering antioxidant enzyme activities, especially via CAT and APX, but not SOD and POD. In addition, MT increased nonenzymatic antioxidant activity, including FRAP and the GSH/GSSG and ASA/DHA ratios. MT usage improved the expression of several defense genes (CAT, APX, GR and MDHAR) and MT biosynthesis-related genes (TDC, SNAT and COMT). Taken together, our results preliminarily reveal that MT alleviates Cu2+ toxicity via ROS scavenging, enhancing antioxidant capacity when subjected to excessive Cu2+. These results build a solid foundation for developing new insights to solve problems related to CS.

Red and blue light function antagonistically to regulate cadmium tolerance by modulating the photosynthesis,antioxidant defense system and Cd uptake in cucumber(Cucumis sativus L.).

Cadmium (Cd) is highly toxic to both plants and humans.Light plays crucial roles in plant growth, development and stress responses, but how light functions in plant Cd response remain unclear.Here,we found that Cd treatment significantly induced the expression of PHYB but not PHYA and CRY1 in leaves and roots of cucumber. Correspondingly,compared with white light (W) during Cd stress,red light(R) increased Cd sensitivity,whereas blue light (B) enhanced Cd tolerance as evidenced by decreased Cd-induced chlorosis, growth inhibition, photosynthesis inhibition and chloroplast ultrastructure damage.Furthermore,B markedly increased the transcripts and activities of the antioxidant enzymes including ascorbate peroxidase (APX),catalase (CAT),superoxide dismutase (SOD) and glutathione reductase (GR),as well as glutathione (GSH) content and GSH1 expression, resulting in hydrogen peroxide (H2O2) and superoxide (O2.-) reduction,but R treatment showed the opposite trend. Moreover, R and B markedly up-regulated and down-regulated the expression levels of Cd uptake and transport genes including IRT1, NRAMP1 and HMA3, leading to more and less Cd accumulation than the W-treated plants in both shoots and roots, respectively under Cd stress. Collectively, our data clearly demonstrate that R and B function antagonistically to regulate Cd tolerance in cucumber via modulating the photosynthesis, antioxidant defense system and Cd uptake, providing a novel light quality control strategy to enhance crop Cd tolerance and food safety.

S-Glycosylation of Fluensulfone in Tomatoes: An Important Way of Fluensulfone Metabolism.

Fluensulfone (FSF) becomes increasingly popular because of its nonfumigation application method. However, studies on the metabolic mechanism of FSF in plants are lacking. Here, tomato seedling was cultivated in hydroponic media to investigate the connection among FSF's metabolism in tomato, the regulation of tomato endogenous glycosides, and the elimination of hydrogen peroxide in tomato cells. The accumulation of FSF was only detected in the lower stems of tomatoes; FSF was mainly metabolized into S-glycosylated conjugates in the roots, and the roots were the tissues with the highest metabolite content; and no FSF and metabolites were detected in the upper leaves. In response to FSF stress (2 mg/L for 7 d), the content of sugar and glycosides in the stems of tomato seedlings significantly increased. The amount of some compounds on the pathway related to glucose was affected by FSF. The three precursor compounds (homomethioine, isoleucine, and l-tyrosine) in the pathway of glucosinolate biosynthesis increased significantly under the stress of FSF, which indicates that FSF may compete with them for UGT74B1. Besides, FSF-induced flavonoid glycosides may play a role in the process of removing hydrogen peroxide. This research provides inspiration for the fate of many xenobiotics containing sulfonyl groups in plants.

Gamma-aminobutyric acid improves phenanthrene phytotoxicity tolerance in cucumber through the glutathione-dependent system of antioxidant defense.

Phenanthrene (PHE), a typical organic pollutant, has drawn attention in recent years due to its toxicity to plants and human health. Gamma-aminobutyric acid (GABA) induce plant tolerance to diverse stresses. However, the role and regulatory mechanisms of GABA in PHE stress responses in plants remains largely uncharacterized. Here, we showed that GABA content increased by 44.5%, 89.2%, 160% and 39.2% under 50, 100, 200 and 300 µM PHE treatment, respectively compared with mock. GABA treatment alleviated PHE-induced growth inhibition in a dose-dependent manner, with the most effective concentration of 50 mM GABA. Although exogenous GABA could not influence the accumulation of PHE in cucumber, it significantly mitigated photosynthetic inhibition and enhanced the transcripts and activities of the antioxidant enzymes such as ascorbate peroxidase (APX), catalase (CAT), peroxidase (POD) and superoxide dismutase (SOD), resulting in less accumulation of hydrogen peroxide (H2O2) and superoxide (O2.-). Importantly, timecourse analyses of glutathione (GSH) homeostasis showed that GABA markedly increased GSH content and GR activity as well as the transcripts of GSH biosynthesis-related genes GSH1, GSH2 and GR during PHE stress. Conversely, pretreatment with GSH biosynthesis inhibitor buthionine sulfoximine (BSO) abolished the GABA-induced changes in PHE stress. Together, these results suggest that GABA enhances tolerance to PHE stress via a GSH-dependent system of antioxidant defense in cucumber.

Gamma-aminobutyric acid enhances tolerance to iron deficiency by stimulating auxin signaling in cucumber (Cucumis sativusL.).

Iron deficiency severely affects crop yield and quality. Gamma-aminobutyric acid (GABA) plays a vital role in plant responses to multifarious stresses. However, the role of GABA in Fe deficiency responses and the potential mechanisms remain largely unknown in cucumber. Here, we found that Fe deficiency raised the GABA levels in leaves and roots of cucumber. To probe the role of GABA in Fe deficiency, the seedlings were subjected to five levels of GABA concentrations (0, 5, 10, 20 and 40 mmol L-1) for 7 days under Fe deficiency. The results demonstrated that 20 mM GABA in alleviating the Fe deficiency-induced stress was the most effective. GABA pretreatment reduced the Fe deficiency-induced chlorosis and inhibition of photosynthesis and growth, and significantly enhanced the contents of iron in shoots and roots. Exogenous GABA significantly decreased the pH of nutrient solution and increased ferric-chelate reductase (FCR) activity induced by Fe deficiency and the transcript levels of Fe uptake-related genes HA1, FRO2 and IRT1 in roots. GABA also increased the content of auxin (IAA) and expression of auxin biosynthesis (YUC4), response (IAA1), and transport (PIN1) genes under Fe deficiency. Furthermore, exogenous the auxin transport inhibitor 1-naphthylphthalamic acid (NPA) application abolished the GABA-induced changes in Fe deficiency. In summary, we found that GABA improves tolerance to iron deficiency via an auxin-dependent mechanism in cucumber.

Proteomic analysis of heat stress resistance of cucumber leaves when grafted onto Momordica rootstock.

Various biotic and abiotic stresses threaten the cultivation of future agricultural crops. Among these stresses, heat stress is a major abiotic stress that substantially reduces agricultural productivity. Many strategies to enhance heat stress tolerance of crops have been developed, among which is grafting. Here, we show that Momordica-grafted cucumber scions have intrinsically enhanced chlorophyll content, leaf area, and net photosynthetic rate under heat stress compared to plants grafted onto cucumber rootstock. To investigate the mechanisms by which Momordica rootstock enhanced cucumber scions heat stress tolerance, comparative proteomic analysis of cucumber leaves in response to rootstock-grafting and/or heat stress was conducted. Seventy-seven differentially accumulated proteins involved in diverse biological processes were identified by two-dimensional electrophoresis (2-DE) in conjunction with matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry (MALDI-TOF/TOF MS). The following four main categories of proteins were involved: photosynthesis (42.8%), energy and metabolism (18.2%), defense response (14.3%), and protein and nucleic acid biosynthesis (11.7%). Proteomic analysis revealed that scions grafted onto Momordica rootstocks upregulated more proteins involved in photosynthesis compared to scions grafted onto cucumber rootstocks under heat stress and indicated enhanced photosynthetic capacity when seedlings were exposed to heat stress. Furthermore, the expression of photosynthesis-related genes in plants grafted onto Momordica rootstocks significantly increased in response to heat stress. In addition, increased high-temperature tolerance of plants grafted onto Momordica rootstock was associated with the accumulation of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and oxygen-evolving enhancer protein 1 (OEE1). Taken together, the data indicated that Momordica rootstock might alleviate growth inhibition caused by heat stress by improving photosynthesis, providing valuable insight into enhancing heat stress tolerance in the global warming epoch.

The effect of exogenous calcium on cucumber fruit quality, photosynthesis, chlorophyll fluorescence, and fast chlorophyll fluorescence during the fruiting period under hypoxic stress.

BACKGROUND: Plants often suffer from hypoxic stress during waterlogging and hydroponic culturing. This study investigated the response of cucumber (Cucumis sativus L.) plant growth parameters, leaf photosynthesis, chlorophyll fluorescence, fast chlorophyll a fluorescence transient (OJIP), and fruit quality parameters to hypoxic stress alleviated by exogenous calcium. During the fruiting period, cucumber plants were exposed to hypoxia and hypoxia + Ca2+ treatment (4 mM Ca2+) for 9 d. RESULT: Exogenous calcium application enhanced the biomass and fruit quality of hypoxic stressed cucumber and also increased the net photosynthesis rate, stomatal conductance, intercellular CO2 concentration, maximum quantum efficiency of photosystem II photochemistry, actual photochemical efficiency of PSII, photochemical quenching coefficient, and non-photochemical quenching coefficient. Additionally, measurement of chlorophyll a fluorescence transients showed the positive K- and L-bands were more pronounced in leaves treated with hypoxia compared with those with hypoxia + Ca2+, indicating that hypoxic treatment induced uncoupling of the oxygen-evolving complex and inhibited electron transport beyond plastoquinone pool (Qa, Qb) including possible constraints on the reduction of end electron acceptors of photosystem I. Exogenous calcium can reduce these stress-induced damages in cucumber. CONCLUSION: This research focused the effect of exogenous calcium on cucumber photosynthesis during the fruiting period under hypoxic stress. Hypoxic stress might impair the photosynthetic electron-transport chain from the donor side of PSII up to the reduction of end acceptors of PSI, and exogenous calcium enhanced electron transport capacity and reduced hypoxic damage of cucumber leaves.

Isolation of a potential biocontrol agent Paenibacillus polymyxa NSY50 from vinegar waste compost and its induction of host defense responses against Fusarium wilt of cucumber.

Fusarium wilt caused by Fusarium oxysporum f. sp. cucumerinum (FOC) is one of the major destructive soil-borne diseases infecting cucumber. In this study, we screened 60 target strains isolated from vinegar waste compost, from which 10 antagonistic strains were identified to have the disease suppression capacity of bio-control agents. The 16S rDNA gene demonstrated that the biocontrol agents were Paenibacillus polymyxa (P. polymyxa), Bacillus amyloliquefaciens (B. amyloliquefaciens) and Bacillus licheniformis (B. licheniformis). Based on the results of antagonistic activity experiments and pot experiment, an interesting strain of P. polymyxa (named NSY50) was selected for further research. Morphological, physiological and biochemical characteristics indicated that this strain was positive for protease and cellulase and produced indole acetic acid (22.21±1.27µg mL-1) and 1-aminocyclopropane-1-carboxylate deaminase (ACCD). NSY50 can significantly up-regulate the expression level of defense related genes PR1 and PR5 in cucumber roots at the early stages upon challenge with FOC. However, the gene expression levels of a set of defense-related genes, such as the plant nucleotide-binding site (NBS)-leucine-rich repeat (LRR) gene family (e.g., Csa001236, Csa09775, Csa018159), 26kDa phloem protein (Csa001568, Csa003306), glutathione-S-transferase (Csa017734) and phenylalanine ammonia-lyase (Csa002864) were suppressed by pretreatment with NSY50 compared with the single challenge with FOC after nine days of inoculation. Of particular interest was the reduced expression of these genes at disease progression stages, which may be required for F. oxysporum dependent necrotrophic disease development.

Paenibacillus polymyxa NSY50 suppresses Fusarium wilt in cucumbers by regulating the rhizospheric microbial community.

Paenibacillus polymyxa (P. polymyxa) NSY50, isolated from vinegar residue substrate, suppresses the growth of Fusarium oxysporum in the cucumber rhizosphere and protects the host plant from pathogen invasion. The aim of the present study was to evaluate the effects of NSY50 application on cucumber growth, soil properties and composition of the rhizospheric soil microbial community after exposure to Fusarium oxysporum. Bacterial and fungal communities were investigated by Illumina sequencing of the 16S rRNA gene and the internal transcribed spacer (ITS) regions (ITS1 and ITS2). The results showed that NSY50 effectively reduced the incidence of Fusarium wilt (56.4%) by altering the soil physico-chemical properties (e.g., pH, Cmic, Rmic, total N and Corg) and enzyme activities, especially of urease and ß-glucosidase, which were significantly increased by 2.25- and 2.64-fold, respectively, relative to the pathogen treatment condition. More specifically, NSY50 application reduced the abundance of Fusarium and promoted potentially beneficial groups, including the Bacillus, Actinobacteria, Streptomyces, Actinospica, Catenulispora and Pseudomonas genera. Thus, our results suggest that NSY50 application can improve soil properties, shift the microbial community by increasing beneficial strains and decreasing pathogen colonization in the cucumber rhizosphere, and reduce the occurrence of cucumber Fusarium wilt, thereby promoting cucumber growth.

Proteomic Analysis Reveals the Positive Roles of the Plant-Growth-Promoting Rhizobacterium NSY50 in the Response of Cucumber Roots to Fusarium oxysporum f. sp. cucumerinum Inoculation.

Plant-growth-promoting rhizobacteria (PGPR) can both improve plant growth and enhance plant resistance against a variety of environmental stresses. To investigate the mechanisms that PGPR use to protect plants under pathogenic attack, transmission electron microscopy analysis and a proteomic approach were designed to test the effects of the new potential PGPR strain Paenibacillus polymyxa NSY50 on cucumber seedling roots after they were inoculated with the destructive phytopathogen Fusarium oxysporum f. sp. cucumerinum (FOC). NSY50 could apparently mitigate the injury caused by the FOC infection and maintain the stability of cell structures. The two-dimensional electrophoresis (2-DE) approach in conjunction with MALDI-TOF/TOF analysis revealed a total of 56 proteins that were differentially expressed in response to NSY50 and/or FOC. The application of NSY50 up-regulated most of the identified proteins that were involved in carbohydrate metabolism and amino acid metabolism under normal conditions, which implied that both energy generation and the production of amino acids were enhanced, thereby ensuring an adequate supply of amino acids for the synthesis of new proteins in cucumber seedlings to promote plant growth. Inoculation with FOC inhibited most of the proteins related to carbohydrate and energy metabolism and to protein metabolism. The combined inoculation treatment (NSY50+FOC) accumulated abundant proteins involved in defense mechanisms against oxidation and detoxification as well as carbohydrate metabolism, which might play important roles in preventing pathogens from attacking. Meanwhile, western blotting was used to analyze the accumulation of enolase (ENO) and S-adenosylmethionine synthase (SAMs). NSY50 further increased the expression of ENO and SAMs under FOC stress. In addition, NSY50 adjusted the transcription levels of genes related to those proteins. Taken together, these results suggest that P. polymyxa NSY50 may promote plant growth and alleviate FOC-induced damage by improving the metabolism and activation of defense-related proteins in cucumber roots.

Proteomic and Physiological Analyses Reveal Putrescine Responses in Roots of Cucumber Stressed by NaCl.

Soil salinity is a major environmental constraint that threatens agricultural productivity. Different strategies have been developed to improve crop salt tolerance, among which the effects of polyamines have been well-reported. To gain a better understanding of the cucumber (Cucumis sativus L.) responses to NaCl and unravel the underlying mechanism of exogenous putrescine (Put) alleviating salt-induced damage, comparative proteomic analysis was conducted on cucumber roots treated with NaCl, and/or Put for 7 days. The results showed that exogenous Put restored the root growth inhibited by NaCl. Sixty-two differentially expressed proteins implicated in various biological processes were successfully identified by MALDI-TOF/TOF MS. The four largest categories included proteins involved in defense response (24.2%), protein metabolism (24.2%), carbohydrate metabolism (19.4%), and amino acid metabolism (14.5%). Exogenous Put up-regulated most identified proteins involved in carbohydrate metabolism, implying an enhancement in energy generation. Proteins involved in defense response and protein metabolism were differently regulated by Put, which indicated the roles of Put in stress resistance and proteome rearrangement. Put also increased the abundance of proteins involved in amino acid metabolism. Meanwhile, physiological analysis showed that Put could further up-regulated the levels of free amino acids in salt stressed-roots. In addition, Put also improved endogenous polyamines contents by regulating the transcription levels of key enzymes in polyamine metabolism. Taken together, these results suggest that Put may alleviate NaCl-induced growth inhibition through degradation of misfolded/damaged proteins, activation of stress defense, and the promotion of carbohydrate metabolism to generate more energy.

Vinegar residue compost as a growth substrate enhances cucumber resistance against the Fusarium wilt pathogen Fusarium oxysporum by regulating physiological and biochemical responses.

Fusarium wilt caused by the fungus Fusarium oxysporum f. sp. cucumerinum (FOC) is the most severe soil-borne disease attacking cucumber. To assess the positive effects of vinegar residue substrate (VRS) on the growth and incidence of Fusarium wilt on cucumber, we determined the cucumber growth parameters, disease severity, defense-related enzyme and pathogenesis-related (PR) protein activities, and stress-related gene expression levels. In in vitro and pot experiments, we demonstrated the following results: (i) the VRS extract exhibited a higher biocontrol activity than that of peat against FOC, and significantly improved the growth inhibition of FOC, with values of 48.3 %; (ii) in response to a FOC challenge, antioxidant enzymes and the key enzymes of phenylpropanoid metabolic activities, as well as the PR protein activities in the roots of cucumber, were significantly increased. Moreover, the activities of these proteins were higher in VRS than in peat; (iii) the expression levels of stress-related genes (including glu, pal, and ethylene receptor) elicited responses to the pathogens inoculated in cucumber leaves; and (iv) the FOC treatment significantly inhibited the growth of cucumber seedlings. Moreover, all of the growth indices of plants grown in VRS were significantly higher than those grown in peat. These results offer a new strategy to control cucumber Fusarium wilt, by upregulating the activity levels of defense-related enzymes and PR proteins and adjusting gene expression levels. They also provide a theoretical basis for VRS applications.