Complementary auxotrophic nitrate-nonutilizing (nit) mutants were used to investigate vegetative compatibility within 27 strains of Verticillium dahliae isolated from several hosts originating from Africa, Asia, Europe and the United States. Using about 500 nit mutants generated from these strains, three vegetative compatibility groups, 1, 2 and 4, were identified. Simultaneously, virulence of each strain was assessed on cultivars of Gossypium hirsutum, G. barbadense and G. arboreum, based upon Foliar Alteration Index (FAI) and Browning Index (BI) estimation. The strains in VCG1 were of both the cottondefoliating pathotype and race 3 (on cotton) but were non pathogenic on tomato; those in VCG2 and VCG4 were of the nondefoliating pathotype and belonged to different races on cotton and on tomato. Hyaline mutants deriving from parental wild-type strain showed differences in pathogenicity but were always assigned to the parental VCG. A relationship was established between VCGs and the taxonomic position of host plants. Data from nit pairings indicated that the sub-populations of V. dahliae (VCGs) may not be completely isolated genetically.
The first two authors contributed equally to this work.
SummaryLipid peroxidation, often associated with hypersensitive cell death, may be initiated either by active oxygen species (AOS) or lipoxygenases (LOX). Here we report a detailed analysis of this oxidative process in both incompatible and compatible interactions between the cotton cultivar Reba B50 and Xanthomonas campestris pv. malvacearum (Xcm). The hypersensitive reaction (HR) was characterized by a massive production of polyunsaturated fatty acid (PUFA) hydroperoxides together with typical tissue dehydration. Among these, isomers peroxidized on carbon 9, largely predominant, were chiral, showing an excess in the S enantiomer. The HR process was accompanied by an increase in 9S-LOX activity and preceded by transcription of a LOX gene (GhKLox1). These results showed that: (i) AOS produced during the oxidative burst were not involved in PUFA peroxidation during HR; and (ii) as previously described in elicited leaves of tobacco, the massive enzymatic lipid peroxidation was closely associated with hypersensitive cell death. During disease development in this cotton cultivar, the 9-lipoxygenation of PUFAs was late, weak, preceded by a faint accumulation of GhKLox1 transcripts, and associated with chlorosis but not with necrosis. Consequently, the main difference between incompatible and compatible interactions was in the precocity and intensity of the oxidative process, rather than in its nature. These data provide the evidence for a correlation between lipid peroxidation and hypersensitive cell death induced by pathogens.
Cotton cotyledons displayed a hypersensitive reaction (HR) in the resistant cultivar Reba B50 after infiltration with the avirulent race 18 of Xanthomonas campestris pv. malvacearum (Xcm). Generation of active oxygen species during the HR was studied biochemically and cytochemically. O2·¯ was detected in cotyledon disks by the cytochrome c reduction assay 3 h after inoculation. This activity was inhibited by superoxide dismutase (SOD) and by the peroxidase inhibitors salicylhydroxamic acid (SHAM) and KCN but not by the NADPH oxidase inhibitor diphenyleneiodonium chloride (DPI). Strong NADH oxidation activity also was found 3 h after inoculation in crude extracts or in apoplastic washing fluid and was dramatically decreased after treatment with SHAM or KCN. NADH oxidation was activated by 2,4- dichlorophenol and MnCl2, indicating the involvement of a peroxidase. Activity of cationic peroxidase isoforms (pI 9 to 9.5) constitutively expressed in cotyledons was found to be enhanced 3 h after inoculation in the resistant cultivar. Activities of apoplastic peroxidase(s) and H2O2 accumulation were observed cytochemically, 3 and 4 h post inoculation, respectively. When digitonin, a O2·- elicitor, was infiltrated into cotyledons of resistant and susceptible cultivars, generation of O2·¯ radicals was shown to be reduced by SOD and inhibited by SHAM and KCN as observed after infection, and also by DPI. Our results strongly suggest that cotton cotyledons contain two O2·¯- generating systems and that cells undergoing the HR in response to an avirulent race of Xcm produce O2·¯ through the activation of an apoplastic peroxidase.
Stems of susceptible and resistant cassava plants have been cytologically investigated for their defense reactions to an aggressive strain of Xanthomonas campestris pv. manihotis. Histochemistry, in conjunction with gold cytochemistry, revealed that in susceptible and resistant plants, phloem and xylem parenchyma cells displayed a wide range of responses that limited the bacterial growth within the infected plants. Lignification and suberization associated with callose deposition were effective mechanisms that reinforced host barriers in the phloem. In the infected xylem, vessels were plugged by a material of pectic and (or) lignin-like origin. Flavonoids have been seen to be incorporated in secondary cell wall coatings. These reactions occurred at a higher intensity in the resistant plants. The number of phoem and xylem cells producing autofluorescent compounds was higher in infected resistant plants than in susceptible plants. Reactions have been observed in the resistant variety only, such as secretion of phenol-like molecules by tyloses and hyperplasic activity of phloem cells that compartmentalized bacterial lysis pockets, which are potent secondary inoculum sources.Key words: lignin, suberin, callose, phenol, tylose, flavonoid, pectin.
We analyzed the production of reactive oxygen species, the accumulation of salicylic acid (SA), and peroxidase activity during the incompatible interaction between cotyledons of the cotton (Gossypium hirsutum) cv Reba B50/Xanthomonas campestris pv malvacearum (Xcm) race 18. SA was detected in petioles of cotyledons 6 h after infection and 24 h post inoculation in cotyledons and untreated leaves. The first peak of SA occurred 3 h after generation of superoxide (O 2 ⅐؊ ), and was inhibited by infiltration of catalase. Peroxidase activity and accumulation of SA increased in petioles of cotyledons and leaves following H 2 O 2 infiltration of cotyledons from 0.85 to 1 mM. Infiltration of 2 mM SA increased peroxidase activity in treated cotyledons and in the first leaves, but most of the infiltrated SA was rapidly conjugated within the cotyledons. When increasing concentrations of SA were infiltrated 2.5 h post inoculation at the beginning of the oxidative burst, the activity of the apoplastic cationic O 2 ⅐؊ -generating peroxidase decreased in a dose-dependent manner. We have shown that during the cotton hypersensitive response to Xcm, H 2 O 2 is required for local and systemic accumulation of SA, which may locally control the generation of O 2 ⅐؊ . Detaching cotyledons at intervals after inoculation demonstrated that the signal leading to systemic accumulation of SA was emitted around 3 h post inoculation, and was associated with the oxidative burst. SA produced 6 h post infection at HR sites was not the primary mobile signal diffusing systemically from infected cotyledons.
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