Erwinia carotovora ssp.carotovora Infection Induced“Defense Lignin”Accumulation and Lignin Biosynthetic Gene Expression in Chinese Cabbage(Brassica rapa L.ssp.pekinensis)

Erwinia carotovora subsp. carotovora (Ecc) infects and causes soft rot disease in hundreds of crop species includingvegetables, flowers and fruits. Lignin biosynthesis has been implicated in defensive reactions to injury and pathogeninfection in plants. In this work, variations of lignin content and gene expression in the molecular interaction betweenChinese cabbage and Ecc were investigated. H_2O_2 accumulation and peroxidase activity were detected by 3, 3-Dimethoxybenzidine staining at mocked and Ecc-inoculated sites of Chinese cabbage leafstalks. Mason lignin contentin inoculated plants increased by about 7.84%, 40.37%, and 43.13% more than that of the mocked site at 12, 24 and 72 hafter inoculation, respectively. Gas chromatography detected more p-coumaryl (H) and less coniferyl (G) and sinapyl (S)monolignins in leafstalks of Chinese cabbage. All three monomers increased in Ecc-infected leafstalks, and the Ecc-induced"defense lignin" were composed of more G and H monolignins, and less S monolignin. After searching the expressedsequence tags (EST) data of Chinese cabbage, 12 genes putatively encoding enzymes involved in lignin biosynthesis wereselected to study their expression. All of these genes could be induced by mock inoculation and Ecc infection, while thegene expression lasted for several more hours in the infected samples than in mocked and untreated plants. Our resultsindicated that "defense lignin" was different from the developmental lignin in composition; G and S monolignins weresignificantly induced in plants in response to the soft rot Ecc; thus, lignin biosynthesis was differentially regulated andplayed a role in plant response to the soft rot Ecc.     

Abiotic and Biotic Stresses and Changes in the Lignin Content and Composition in Plants

Lignin is a polymer of phenylpropanoid compounds formed through a complex biosynthesis route,represented by a metabolic grid for which most of the genes involved have been sequenced in several plants,mainly in the model-plants Arabidopsis thaliana and Populus.Plants are exposed to different stresses,which may change lignin content and composition.In many cases,particularly for plant-microbe interactions,this has been suggested as defence responses of plants to the stress.Thus,understanding how a stressor modulates expression of the genes related with lignin biosynthesis may allow us to develop study-models to increase our knowledge on the metabolic control of lignin deposition in the cell wall.This review focuses on recent literature reporting on the main types of abiotic and biotic stresses that alter the biosynthesis of lignin in plants.     

The defense-responsive genes showing enhanced and repressed expression after pathogen infection in rice (Oryza sativa L.)

Despite large numbers of studies about defense response, processes involved in the resistance of plants to incompatible pathogens are still largely uncharacterized. The objective of this study was to identify genes involved in defense response by cDNA array analysis and to gain knowledge about the functions of the genes involved in defense response. Approximately 20000 rice cDNA clones were arrayed on nylon filters. RNA samples isolated from different rice lines after infection with incompatible strains or isolates of Xanthomonas oryzae pv. oryzae or Pyricularia grisea, respectively, were used to synthesize cDNA as probes for screening the cDNA arrays. A total of 100 differentially expressed unique sequences were identified from 5 pathogen-host combinations. Fifty-three sequences were detected as showing enhanced expression and 47 sequences were detected as showing repressed expression after pathogen infection. Sequence analysis revealed that most of the 100 sequences had various degrees of homology with     

Physiological and Molecular Features of the Pathosystem Arabidopsis thaliana L.-Sclerotinia sclerotiorum Libert

The fungal pathogen Sclerotinia sclerotiorum Libert causes rot diseases on many crops worldwide and large economic losses occur frequently because of a lack of resistant varieties. The pathogenesis of S. sclerotiorum and the molecular basis of plant responses to the pathogen are poorly understood. In the present investigation, the process of S. sclerotiorum infection in Arabidopsis thaliana L., a plant that is highly susceptible to this fungus, was analysed. In addition, the defense activation in the host was investigated. A convenient inoculation method using millet grain was developed for S. sclerotiorum in Arabidopsis. The fungus rapidly infected the plants, probably through ball- or cushion-like infection structures. Visible symptoms developed within 24 h and plants were killed 72 h after inoculation. Cellulase, the main enzyme that caused host tissues to rot, was secreted by S. sclerotiorum in a pH-dependent manner. Oxalic acid, another pathogenic factor secreted by the fungus, induced necrotic lesions on the leaves, infection with S. sclerotiorum strongly induced the production of the pathogenesis-related （PR） proteins β-1,3-glucanase and chitinase in Arabidopsis. Furthermore, the PR gene PDF. 1 was induced, but not PR1, indicating that the pathogen activated basal defense of jasmonic acid/ethylene dependence, which is consistent with its necrotrophic characteristics. This pathosystem for Arabidopsis-S. sclerotiorum could provide an approach for the analysis of the interactions between S. sclerotiorum and other crops, thereby facilitating genetic manipulation techniques for controlling this pathogen.     

Cell Wall Lignin is Polymerised by Class Ⅲ Secretable Plant Peroxidases in Norway Spruce

Class Ⅲ secretable plant peroxidases occur as a large family of genes in plants with many functions and probable redundancy. In this review we are concentrating on the evidence we have on the catalysis of lignin polymerization by class Ⅲ plant peroxidases present in the apoplastic space in the xylem of trees. Some evidence exists on the specificity of peroxidase isozymes in lignin polymerization through substrate specificity studies, from antisense mutants in tobacco and poplar and from tissue and cell culture lines of Norway spruce (Picea abies) and Zinnia elegans. In addition, real time (RT-)PCR results have pointed out that many peroxidases have tissue specific expression patterns in Norway spruce. Through combining information on catalytic properties of the enzymes, on the expression patterns of the corresponding genes, and on the presence of monolignols and hydrogen peroxide in the apoplastic space, we can show that specific peroxidases catalyze lignin polymerization in the apoplastic space of Norway spruce xylem.     

Phenotypic and Genetic Diversity of Erwinia carotovora ssp. carotovora Strains from Asia

A collection of 87 strains of the soft rot pathogen Erwinia carotovora ssp. carotovora (Ecc) isolated from various host plants in Japan, Korea and Thailand was characterized by bacteriological, pathological and genetic properties. On the basis of pathogenicity on the potato, tomato, onion and cucumber, strains were divided into four groups. They were also characterized by PCR‐restriction fragment length polymorphisms (RFLP) of 16S ribosomal DNA (rDNA), 16S‐23S rDNA intergenic spacer regions (ISRs) and a pel gene encoding pectate lyase. By analysis of 16S rDNA RFLP generated by Hinf I, Ecc strains were differentiated into two groups where it was discovered that most strains from Korea and Japan belonged to the same group. In the analysis of ISRs RFLP with MboI, two patterns were found. All Thai strains showed the same pattern. In the analysis of the pel gene RFLP with Sau3AI, all strains were separated into two independent patterns except for one strain. The strain (MAFF 301937) isolated from the mulberry showed a unique RFLP pattern of the pel gene. In cluster analysis based on 26 phenotypic characters, Ecc strains were composed of two groups, A and B. Group A contained typical Ecc strains which provided negative reactions in testing the production of reducing substances from sucrose and acids from α‐methyl glucoside. All Thai strains and most of the Korean strains belonged to group A, whereas group B contained atypical Ecc strains, which were isolated in Japan and Korea; the properties of this group were similar to those of E. carotovora ssp. atroseptica. The research reported here was undertaken to provide information on the strains of E. carotovora ssp. carotovora in Asia.     

Potential of natamycin in enhancing the antagonistic activity of Bacillus amyloliquefaciens BGP20 against post-harvest bacterial soft rot of green pepper

Bacillus amyloliquefaciens BGP20 is a promising antagonist in controlling post-harvest bacterial soft rot of vegetables caused by Erwinia carotovora subsp. carotovora (Ecc). The objective of this study was to screen a kind of natural and safe additive which could enhance the bio-control activity of BGP20 against post-harvest bacterial soft rot of green pepper. The results of this study indicated that the additive natamycin had stronger inhibition against the pathogen Ecc compared with bamboo vinegar and chitosan in the 2× Yeast extract and Tryptone (2YT) medium. However, natamycin had a slight negative effect on the growth of BGP20 in the 2YT medium. In preventative treatments, natamycin significantly improved the bio-efficacy of BGP20, and enhanced its competitive position against Ecc in the wounds of green pepper. Compared with the treatment with BGP20 alone, the viable count of BGP20 after 72?h of incubation increased by 115.8% in the wounds of green pepper treated with BGP20 and 0.1% natamycin, while that of Ecc decreased by 92.1%. In addition, natamycin remarkably promoted the flocculation of Ecc cells in the 2YT medium, while promoting the dispersion of BGP20. Natamycin had no negative effects on the spore germination of BGP20 and its shelf life. These results indicated that natamycin had perfect compatibility with the antagonist BGP20, and it had a great potential in enhancing the bio-control activity of BGP20 against post-harvest bacterial soft rot of green pepper in preventative treatments.     

Bacterial Stem Rot in Greenhouse Pepper (Capsicum annuum L.) in Sardinia (Italy): Occurrence of Erwinia carotovora subsp. carotovora

An unusual bacterial disease was observed in pepper plants during research carried out in greenhouses in central‐north Sardinia. The characteristics were: the presence of lesions and exudates on stems, soft rot of the pith, and a brownish‐black colour in the petioles and leaf‐veins. Only two isolates of 21 were pathogens. One was obtained from exudate present on the stem and the other from pith. Experimental infections revealed that the bacterial isolates were particularly aggressive in the stems and fruit of pepper and tomato. Biochemical, physiological and serological tests in conjunction with fatty acid profile analysis confirmed that they were Erwinia carotovora subsp. carotovora (Jones) Bergey et al. The product of 434 bp polymerase chain reaction (PCR) enabled a preliminary identification of isolates to be made. Restriction fragment length polymorphism (RFLP) analysis of amplification products showed that the isolates DPP 23_ef and DPP 24_m, strain type CFBP 2046 and DPP 28₁, isolated from pepper fruit, belonged to the RFLP group 12, whereas DPP 29, also isolated from pepper fruit, was included in RFLP group 1. Measures to prevent and control this recently introduced disease are suggested in the conclusion of this paper.     

Differentiation of Erwinia carotovora subsp. atroseptica and carotovora by RAPD-PCR

Six different 10-mer oligonucleotide primers were used to differentiate Erwinia carotovora subsp. atroseptica (Eca) and carotovora (Ecc) using RAPD-PCR. All primers gave different banding patterns for Eca and Ecc indicating their value for identification. UPGMA clustering analysis clearly showed two separate clusters, one for Eca and the other for the Ecc group. Similarity within Eca strains was very high, over 85% among most isolates but within the Ecc group extensive genetic diversity was found and many of the Ecc strains were no more than 50% similar. Similarity between the 10 Eca and 10 Ecc strains was generally only 10–25% based on the results from six primers. Three RAPD fragments from Eca group, which were amplified by three different RAPD primers, were isolated and used as probes for Southern hybridisation to test, if homologous fragments were amplified from Ecc strains. All these probes hybridised only with Eca isolates indicating that these fragments could be useful in order to develop a PCR-based detection system for Eca strains.     

Use of TnphoA to enrich for extracellular enzyme mutants of Erwinia carotovora subspecies carotovora

Soft rot Erwinia species secrete a range of enzymes into the extracellular environment. Therefore, the genetically amenable Erwinia system is a useful model for the study of protein secretion by Gram-negative bacteria. We have used a λ-sensitive derivative of Erwinia carotovora subspecies carotovora (Ecc) and the transposon TnphoA, to isolate a range of extracellular enzyme mutants. The use of TnphoA provides an enrichment for extracellular enzyme mutants over other transposon-based systems. In these mutants, the alkaline phosphatase activity of the hybrid protein Is found in the periplasm, and is under the control of the Ecc promoters. Three TnphoA-induced extracellular enzyme mutants were studied in detail. One proved to be an enzyme structural gene mutant, whilst the other two appeared to be secretory mutants.     

Complementation of an Erwinia carotovora subsp. carotovora protease mutant with a protease-encoding cosmid

Summary We report the complementation of a genetic lesion in the genome of Erwinia carotovora subsp. carotovora (Ecc), a pathogenic bacterium that incites soft rot of plants. A Sau3AI genomic library of Ecc was constructed using the conjugal cosmid pLAFR-3 as a vector. Sixteen cosmid clones encoding various plant tissue-degrading enzymes were identified, including a proteolytic clone, five cellulolytic clones, and ten pectolytic clones. We detected a mutant of Ecc with no proteolytic activity following transposon mutagenesis with an unstable Tn5-carrying plasmid. Conjugal transfer of the protease-encoding cosmid to this mutant restored near-wildtype extracellular protease production. Further manipulation and study of genes encoding pathogenic determinants in Ecc will be possible using this system.     

Molecular cloning of ompRS, a regulatory locus controlling production of outer membrane proteins in Erwinia carotovora subsp. carotovora

Summary A locus, ompRS, controlling synthesis of outer membrane proteins was cloned from Erwinia carotovora subsp. carotovora (Ecc) by complementation of an Escherichia coli ompR—envZ mutant. The Ecc ompRS locus was both structurally and functionally similar to the ompR—envZ operon controlling porin gene expression in E. coli as shown by DNA hybridization and complementation of E. coli ompR and envZ mutants. Furthermore, introduction of ompRS into E. coli (ompR—envZ) strains restored the osmoregulation of the major outer membrane protein genes ompC and ompF Maxicell analysis of ompRS-carrying plasmids suggested that proteins similar in size to the E. coli ompR and envZ gene products were encoded by the Ecc ompR and ompS genes, respectively. Introduction of an ompRS transposon mutant onto the Ecc chromosome by marker exchange mutagenesis showed that ompRS is essential for production of a major outer membrane porin in Ecc. This mutational defect could be complemented by clones carrying Ecc ompRS or E. coli ompR envZ. The lack of the porin did not, however, compromise the virulence of these Ecc mutants.