Nanshan Du

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.

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 β-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.

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.