Abstract: In order to determine the role of ascorbate peroxidase, an antioxidant enzyme, in the cellular responses to oxidative stress and pathogens, transgenic tobacco plants were generated, using the Capsicum annuum ascorbate peroxidase-like 1 gene (CAPOA1), under the control of the CaMV 35S promoter. High levels of CAPOA1 gene expression were observed in the transgenic plants, with a 2-fold increase in total peroxidase activity. The constitutive expression of CAPOA1 in the tobacco exhibited no morphological abnormalities, while significantly increased growth was observed in transgenic plants, as compared to control plants. The CAPOA1-overexpressed plants exhibited increased tolerance to methyl viologen-mediated oxidative stress, and also enhanced resistance to the oomycete pathogen, Phytophthora nicotianae. However, the transgenic plants were not found to be resistant to the bacterial pathogen, Pseudomonas syringae pv. tabaci, but were weakly resistant to Ralstonia solanacearum. Our results suggested that the overproduction of ascorbate peroxidase increased peroxidase activity that enhances active oxygen scavenging system, leading to oxidative stress tolerance and oomycete pathogen resistance.
Abstract: A comparative proteome analysis was initiated to systematically investigate the physiological response of tomato (Solanum lycopersicum) to infection with Ralstonia solanacearum, causal agent of bacterial wilt. Plants of the susceptible tomato recombinant inbred line NHG3 and the resistant NHG13 were either infected or not infected with R. solanacearum and subsequently used for proteome analysis. Two-dimensional isoelectric focussing/sodium dodecyl-sulphate polyacrylamide gel electrophoresis (2-D IEF/SDS-PAGE) allowed the separation of about 650–690 protein spots per analysis. Twelve proteins were of differential abundance in susceptible plants in response to bacterial infection, while no differences were observed in the resistant genotype. LC-MS/MS analysis of these spots revealed 12 proteins, six of which were annotated as plant and six as bacterial proteins. Among the plant proteins, two represent pathogenesis related (PR) proteins, one stress response protein, one enzyme of carbohydrate and energy metabolism, and one hypothetical protein. A constitutive difference between resistant and susceptible lines was not found.
Abstract:Solanum aethiopicum is reported to carry resistance to bacterial wilt disease caused by Ralstonia solanacearum, which is one of the most important diseases of eggplant (Solanum melongena). These two species can sexually be crossed but the fertility of their progeny is very low. In order to transfer the resistance and improve the fertility, somatic hybrids between S. melongena cv. Dourga and two groups of S. aethiopicum were produced by electrical fusion of mesophyll protoplasts. Thirty hybrid plants were regenerated. When transferred to the greenhouse and transplanted in the field, they were vigorous and showed intermediate morphological traits. Their ploidy level was determined by DNA analysis through flow cytometry, and their hybrid nature was confirmed by examining isozymes and RAPDs patterns. Chloroplast DNA microsatellite analysis revealed that 18 hybrids had the chloroplasts of the eggplant and 12 those of the wild species. The parents and 16 hybrids were evaluated in the field for their fertility and resistance to bacterial wilt using a race 1, biovar 3 strain of R. solanacearum. All hybrids were fertile and set fruit with viable seeds. Their yield was either intermediate or as high as that of the cultivated eggplant. Both groups of S. aethiopicum were found tolerant to R. solanacearum, as about 50% of plants wilted after 8 weeks. The cultivated eggplant was susceptible with 100% of wilted plants 2 weeks after inoculation. All somatic hybrids tested were as tolerant as the wild species, except six hybrids showing a better level of resistance.
Abstract: In previous research, a mixture of Bacillus amyloliquefaciens strain IN937a and Bacillus pumilus strain IN937b consistently provided systemic protection against multiple diseases in various crops. The objective of this study was to investigate defense-related enzyme responses in plants induced by a mixture of IN937a and IN937b against different pathosystems. Four plant/pathosystems, tomato with Sclerotium rolfsii and Ralstonia solanacearum and pepper with S. rolfsii and Colletotrichum gloeosporioides, were used to test the efficacy of the mixture in greenhouse assays. Treatments consisted of non-challenged healthy control, nonbacterized pathogen control, and bacterized with a mixture of IN937a and IN937b and challenged later with pathogens. Total superoxide dismutase (SOD) and peroxidase (PO) activities were investigated. Before pathogen inoculation, higher levels of SOD and PO activities were observed in plants treated with a mixture of IN937a and IN937b compared with non-challenged healthy and nonbacterized pathogen controls. After challenge with all pathogens, plants treated with the bacterial mixture had SOD and PO activity levels 25–30% greater than the nonbacterized pathogen control. Additionally, significant disease protection in each plant pathosystem was observed with the bacterial mixture. Low levels of natural SOD and PO activities in the non-challenged healthy control occurred during the assay. In conclusion, a mixture of IN937a and IN937b induced similar responses of SOD and PO activities against different pathogens, and these physiological changes were associated with disease protection with all the tested pathogens.
Abstract: Pepper (Capsicum annuum L.) plants sprayed with DL-beta-amino-n-butyric acid (BABA) were protected against Phytophthora capsici infection. BABA treatment induced the synthesis and accumulation of beta-1,3-glucanases and chitinases in the stem tissues of pepper plants. Their accumulation was very pronounced in the stems challenge-inoculated with P. capsici after BABA treatment. Several beta-1,3-glucanase and chitinase isoforms accumulated in BABA treated P. capsici. When analysed by immunoblot of the denatured proteins, the 20 kDa beta-1,3-glucanase and 32 kDa chitinase were found in pepper stems treated with BABA and/or infected by P. capsici. BABA treatment did not stimulate capsidiol production in pepper stems, but prior treatment led to high accumulation in P. capsici-infected ones. Unlike capsidiol production, BABA treatment triggered a dramatic increase in the endogenous levels of salicylic acid (SA) in pepper stems. The increase in endogenous SA was much pronounced in P. capsici infected stems after BABA treatment. In conclusion, the induction of resistance to P. capsici in pepper plants by BABA treatment positively correlated with the accumulation of certain beta-1,3-glucanase and chitinase isoforms, and SA. These results suggest strongly that SA may act as an endogenous signal responsible for activating particular components of resistance to P. capsici and the induction of pathogenesis-related proteins such as beta-1,3-glucanase and chitinase.
Abstract: Plant chitinases have been of particular interest since they are known to be induced upon pathogen invasion. Inoculation of Piper colubrinum leaves with the foot rot fungus, Phytophthora capsici leads to increase in chitinase activity. A marked increase in chitinase activity in the inoculated leaves was observed, with the maximum activity after 60h of inoculation and gradually decreased thereafter. Older leaves showed more chitinase activity than young leaves. The level of chitinase in black pepper (Piper nigrum L.) upon inoculation was found to be substantially high when compared to P. colubrinum. RT–PCR using chitinase specific primers revealed differential accumulation of mRNA in P. colubrinum leaves inoculated with P. capsici. However, hyphal extension assays revealed no obvious differences in the ability of the protein extracts to inhibit growth of P. capsiciin vitro.
Abstract: The activities of phenylalanine ammonia lyase (PAL) and 61,3 glucanase in both leaf and root tissues of three black pepper varieties (tolerant P24; and susceptible Panniyur and Subhakara) were determined in healthy and
Phytophthora capsici infected tissues. Infection generally enhanced both of these enzyme activities . SDS-PAGE study revealed the induction of PR-proteins in the infected tissues. Western blotting with anti-tobacco 61,3 glucanase antibody confirmed the presence of these isoforms in the leaf extract . Among the three varieties studied, the Phytophthora tolerant P24 expressed higher rate of these defense-related enzymes/proteins.
Abstract: Etiolated hypocotyls of the resistant soybean (Glycine max [L.] Merr.) cultivar Harosoy 63 became susceptible to Phytophthora megasperma (Drechs.) f.sp. glycinea (Hildeb.) Kuan and Erwin race 1 after treatment with abscisic acid. Susceptibility was expressed by increases in lesion size and a major decrease in
accumulation of the isoflavonoid phytoalexin, glyceollin. In untreated hypocotyls, activity of phenylalanine ammonia-lyase and accumulation of mRNA for this enzyme increased rapidly after infection, but these increases were suppressed in abscisic acidtreated hypocotyls. The results suggest the possibility that biosynthesis of glyceollin in the resistance response of soybeans may be controlled at the transcriptional level by changes in abscisic acid concentrations caused by infection.
Abstract:Pseudomonas aeruginosa CMG103 was isolated from a metal-polluted river in Pakistan and displayed a high level of Zn and Cd resistance. An V-Km transposon mutant of strain CMG103, which showed a substantial decrease in resistance to Zn and Cd, was obtained. A 12.8 kb region determining Zn and Cd resistance in strain CM103 was cloned by complementing the mutant strain, and its nt sequence was determined. Five genes, czrSRCBA, involved in Zn and Cd resistance, were identified. The predicted gene products of czrCBAshow a significant similarity with the proteins encoded by the plasmid borne metal resistant determinants czc, cnr and ncc of Ralstonia strains, which determine a chemiosmotic cation-antiporter eZux system. The predicted CzrS and CzrR proteins show a significant similarity to the sensor and regulatory protein, respectively, of two component regulatory systems, such as CopS/CopR and PcoS/PcoR involved in the regulation of plasmid-borne Cu-resistant determinants, and CzcS/CzcR involved in the regulation of czc. The cloned czr region contained downstream of czrCBA additional ORFs whose predicted gene products are similar to proteins involved in catabolism of aromatic compounds. DNA–DNA hybridization indicated strong conservation of czr in other enviromental P. aeruginosa isolates and in the P. aeruginosatype strain PAO1, a clinical isolate. This was confirmed by a comparison of the sequence of the CMG103 czr region with the currently available genome sequence of strain PAO1. A high sequence identity (till 99% at the nt level ) and organizatory conservation of the czr region of CMG103 was found in PAO1 as well regarding coding sequences as intervening sequences between ORFs. The czr locus was localized between coordinates 2400 and 2550 kb on the physical map of the chromosome of PAO1.
Abstract: Pitch canker, caused by Gibberella circinata Nirenberg & O Donnell, is a problem for pines in both native and planted stands. The aerial phase of the disease results in shoot and canopy dieback, whereas soil or seedborne inoculum can cause damping off of emerging seedlings. Based on the extent of lesion development on inoculated shoots, families of Pinus radiata have been shown to differ significantly in resistance to pitch canker. This study was undertaken to determine if these same families also differ in mortality caused by G. circinata at the seedling emergence stage. For this purpose, seeds treated with a suspension of G. circinata spores were planted in a greenhouse and rated for pitch canker induced mortality. Variation between families, in mortality of emerging seedlings, was significant but the observed variation was not significantly correlated with measures of resistance based on stem inoculation tests. This suggests that mechanisms limiting the development of stem lesions do not confer measurable resistance to the seedling phase of the disease and therefore that early exposure to the pathogen may compromise selection for resistance to pitch canker in stands of Pinus radiata.
Abstract