Genetic diversity of Fusarium oxysporum and related species pathogenic on tomato in Algeria and other Mediterranean countries

In order to characterize the pathogen(s) responsible for the outbreak of fusarium diseases in Algeria, 48 Fusarium spp. isolates were collected from diseased tomato in Algeria and compared with 58 isolates of Fusarium oxysporum originating from seven other Mediterranean countries and 24 reference strains. Partial sequences of the translation elongation factor EF‐1α gene enabled identification of 27 isolates as F. oxysporum, 18 as F. commune and three as F. redolens among the Algerian isolates. Pathogenicity tests confirmed that all isolates were pathogenic on tomato, with disease incidence greater at 28°C than at 24°C. All isolates were characterized using intergenic spacer (IGS) DNA typing, vegetative compatibility group (VCG) and PCR detection of the SIX1 (secreted in xylem 1) gene specific to F. oxysporum f. sp. lycopersici (FOL). No DNA polymorphisms were detected in the isolates of F. redolens or F. commune. In contrast, the 27 Algerian isolates of F. oxysporum were shown to comprise nine IGS types and 13 VCGs, including several potentially new VCGs. As none of the isolates was scored as SIX1+, the 27 isolates could be assigned to F. oxysporum f. sp. radicis‐lycopersici (FORL). Isolates from Tunisia were also highly diverse but genetically distinct from the Algerian isolates. Several Tunisian isolates were identified as FOL by a PCR that detected the presence of SIX1. The results show that isolates from European countries were less diverse than those from Tunisia. Given the difference between Algerian populations and populations in other Mediterranean countries, newly emergent pathogenic forms could have evolved from local non‐pathogenic populations in Algeria.     

Effects of fusaric acid on cells from tomato cultivars resistant or susceptible toFusarium oxysporum f. sp.Lycopersici

Cell suspension cultures were set up from two tomato cultivars, one resistant, (Rio grande) and one susceptible (63.5) toFusarium oxysporum f. sp.lycopersici. Growth rates of the two cell cultures were comparable. Toxicity of fusaric acid, expressed as the fresh weight loss, was analyzed: It was significant in both cases after 10 h, but toxicity was twice as high for 63.5 suspension cells. In the same way, electrolyte leakage caused by fusaric acid was three times more important for 63.5 suspension cells. Moreover, fusaric acid treatment resulted in an acidification of the extracellular medium for 63.5 suspension cells (0.4 pH unit), whereas an alkalization was observed for Rio grande suspension cells (0.2 pH unit). Preliminary experiments suggest that fusaric acid was partially metabolized by Rio grande suspension cells, however, no detoxified forms of fusaric acid were detected either in cells or in culture filtrates. For these two tomato cultivars, the differences in sensitivity to fusaric acid of cultivated cells correspond to the differences in plant susceptibility toFusarium oxysporum f. sp.lycopersici.Abbreviations BAP 6-benzylaminopurine - conductivity - 2,4-D 2,4-dichlorophenoxyacetic acid - EtOAc ethyl acetate - FA fusaric acid - resistivity     

Involvement of a vascular hypersensitive response in quantitative resistance to Ralstonia solanacearum on tomato rootstock cultivar LS‐89

Ralstonia solanacearum causes bacterial wilt disease in Solanaceae spp. Expression of the Phytophthora inhibitor protease 1 (PIP1) gene, which encodes a papain‐like extracellular cysteine protease, is induced in R. solanacearum‐inoculated stem tissues of quantitatively resistant tomato cultivar LS‐89, but not in susceptible cultivar Ponderosa. Phytophthora inhibitor protease 1 is closely related to Rcr3, which is required for the Cf‐2‐mediated hypersensitive response (HR) to the leaf mould fungus Cladosporium fulvum and manifestation of HR cell death. However, up‐regulation of PIP1 in R. solanacearum‐inoculated LS‐89 stems was not accompanied by visible HR cell death. Nevertheless, upon electron microscopic examination of inoculated stem tissues of resistant cultivar LS‐89, several aggregated materials associated with HR cell death were observed in xylem parenchyma and pith cells surrounding xylem vessels. In addition, the accumulation of electron‐dense substances was observed within the xylem vessel lumen of inoculated stems. Moreover, when the leaves of LS‐89 or Ponderosa were infiltrated with 10⁶ cells mL^?1 R. solanacearum, cell death appeared in LS‐89 at 18 and 24 h after infiltration. The proliferation of bacteria in the infiltrated leaf tissues of LS‐89 was suppressed to approximately 10–30% of that in Ponderosa, and expression of the defence‐related gene PR‐2 and HR marker gene hsr203J was induced in the infiltrated tissues. These results indicated that the response of LS‐89 is a true HR, and induction of vascular HR in xylem parenchyma and pith cells surrounding xylem vessels seems to be associated with quantitative resistance of LS‐89 to R. solanacearum.     

Development of a Fusarium oxysporum f. sp. melonis functional GFP fluorescence tool to assist melon resistance breeding programmes

Fusarium wilt, caused by Fusarium oxysporum f. sp. melonis (Fom), is one of the most widespread and devastating melon diseases. This vascular disease is caused by the colonization of melon xylem vessels by any of the four Fom races reported (r0, r1, r2 and r1,2, subdivided into r1,2w and r1,2y). The macroscopic evaluation of disease symptoms (disease rating, DR) at several days post‐inoculation (dpi) with Fom spores has been the traditional method to determine the resistance of melon accessions to this fungal pathogen. In this study, one isolate from each Fom race was transformed by Agrobacterium tumefaciens to constitutively express the green fluorescent protein (GFP). Fom‐GFP transformants, as virulent as the corresponding wildtype races, were selected to develop an inoculation assay based on the non‐invasive evaluation of the fluorescence emitted by Fom‐GFP. It was determined that melon root neck was the appropriate area to follow Fom‐GFP and a fluorescence signal rating (FSR) was established in parallel to DR determination. This method allowed the evaluation of GFP signal in the root neck of inoculated melon seedlings at 11–15 dpi. The GFP signal was scored in 62 melon accessions/breeding lines inoculated with different Fom‐GFP, followed by evaluation of the macroscopic DR in the aerial part of melon seedlings at 20–28 dpi. Correlation analysis demonstrated a direct and significant relationship between FSR and DR. This method has shown to be an effective and reliable tool that can assist Fom resistance breeding programmes in melon.     

Variation in the response of melon genotypes to Fusarium oxysporum f.sp. melonis race 1 determined by inoculation tests and molecular markers

Screening of genotypes of melon ( Cucumis melo ) for resistance to wilt caused by Fusarium oxysporum f.sp. melonis is often characterized by wide variability in their responses to inoculation, even under carefully controlled conditions. The variability at the seedling stage of 17 genotypes susceptible to race 1 was examined in growth-chamber experiments. Disease incidence varied from 0 to 100% in a genotype-dependent manner. Using four combinations of light (60 and 90  µ E m⁻² s⁻¹) and temperatures of (27 and 31°C), only light intensity showed a statistically significant effect. Marker-assisted selection for fusarium resistance breeding using cleaved amplified polymorphic sequence (CAPS) and sequence-characterized amplified region (SCAR) markers were compared using a single set of genotypes that included 24 melon accessions and breeding lines whose genotype regarding the Fom-2 gene was well characterized. The practical value of the markers for discriminating a range of genotypes and clarifying the scoring of phenotypes was also tested using a segregating breeding population which showed codominant SCAR markers to be useful in marker-assisted selection.     

Persistence and translocation of a benzothiadiazole derivative in tomato plants in relation to systemic acquired resistance against Pseudomonas syringae pv tomato

A reproducible and accurate procedure, based on HPLC analysis, has been developed to determine simultaneously acibenzolar‐S‐methyl (CGA 245 704) and its acid derivative (CGA 210 007) in tomato leaves. The limit of detection and quantification of the method are 0.015 and 0.15 mg litre^?1 for CGA 245 704 and 0.030 and 0.30 mg litre^?1 for CGA 210 007. In tomato plants treated with 250 µM CGA 245 704, it was found that the inducer rapidly translocates from treated leaves (cotyledons, 1st and 2nd) to untreated leaves (3rd to 5th), with the maximum translocation (40% of the total quantity found) occurring 8 h after the treatment. CGA 245 704 residues decreased as time elapsed in both treated and untreated tomato leaves, reaching negligible values 72 h after treatment. The acid derivative, CGA 210 007, was formed in tomato plants as early as 2 h after CGA 245 704 treatment, albeit only in the treated leaves. CGA 210 007 residues decreased in treated tomato leaves with a trend similar to that observed for CGA 245 704. Treatment of tomato plants with CGA 245 704 or CGA 210 007 at 250 µM systemically protected the plants against Pseudomonas syringae pv tomato attacks, the causal agent of bacterial speak disease. Evidence of this were reductions in the degree of infection, the bacterial lesion diameter and the bacterial growth in planta. Since neither CGA 245 704 nor CGA 210 007 inhibited bacterial growth in vitro and the protection against bacterial speak of tomato was observed when the two compounds were completely degraded, the protection must be due to the activation of the plant's defence mechanisms. © 2001 Society of Chemical Industry     

Comparative analysis of pathogenic and nonpathogenic Fusarium oxysporum populations associated with banana on a farm in Minas Gerais,Brazil

Fusarium wilt is one of the most devastating diseases on banana. The causal agent, Fusarium oxysporum f. sp. cubense (Foc) is genetically diverse and its origin and virulence are poorly understood. In this study, pathogenic Foc isolates and nonpathogenic F. oxysporum isolates from Minas Gerais in Brazil were compared using EF‐1α and IGS sequences. This allowed the examination of the origin and evolutionary potential of Foc in a country outside the region of origin of the banana plant. Two different sequence types were found among Foc isolates. One appeared to be of local origin because it was identical to the sequence type of the largest group of nonpathogenic isolates. To explore if the ‘local’ Foc isolates had acquired pathogenicity either independently through coevolution with the host, or through horizontal gene transfer (HGT) of pathogenicity genes from other, probably introduced, Foc isolates, the presence and sequence of putative SIX effector genes were analysed. Homologues of SIX1, SIX3 and SIX8 were found. SIX1 sequences were identical and exclusively found in all pathogenic isolates, while variable ratios of sequences of multicopy gene SIX8 were found among nonpathogenic and different pathogenic isolates. This observation supports the HGT hypothesis. Horizontal transfer of genes between isolates of F. oxysporum has important implications for the development of reliable diagnostic tools and effective control measures. Full genome sequencing is required to confirm HGT and to further unravel the virulence mechanisms of forma specialis cubense.     

Induction of phytoalexins in pigeonpea (Cajanus cajan) in response to inoculation with Fusarium udum and other treatments

Cajanol accumulation was monitored in four cultivars of pigeonpea, Cajanus cajan (L) Millsp, after inoculation with 2 x 10(6) conidia ml-1 of Fusarium udum Butler. Rapid accumulation was observed only in wilt-resistant cultivar ICP 9145. Another wilt-resistant cultivar, ICP 8863, had low levels of cajanol similar to those in wilt-susceptible cultivars ICP 2376 and Malawi local, indicating that rapid cajanol accumulation is not positively correlated with resistance to wilt in pigeonpea. A comparison of various inducing agents showed live conidia of the pathogen to cause more rapid accumulation than the other agents.     

Localization of phytoalexin accumulation and determination of changes in lignin and carbohydrate composition in carnation (Dianthus caryophyllus L.) xylem as a consequence of infection with Fusarium oxysporum f. sp. dianthi,by pyrolysis-mass spectrometry

Minute pieces of xylem and other tissues from stems of healthy and fungus-infected plants of two carnation cultivars Novada and Lena were investigated for lignification (lignin/polysaccharide ratios) and lignin composition by means of pyrolysis mass spectrometry and pyrolysis gas chromatography mass spectrometry. This technique proved also very effective for the localization of dianthramide phytoalexins which accumulate in carnation after infection withFusarium oxysporum f.sp.dianthi. The composition of healthy tissues from both cultivars was practically the same. In the resistant cultivar Novada, infection induced a change from guaiacyl-syringyl lignin into a mainly guaiacyl lignin in the gum-occluded parts of the xylem. Considerable amounts of the phenolic amide dianthalexin and of other dianthramide phytoalexins were present in occluded xylem, but not in adjacent phloem, medulla or unoccluded xylem. Xylem from susceptible Lena suffering degradation was characterized by a loss of syringyl groups from the lignin and by demethylation of pectin in an early stage of infection. Small quantities of dianthalexin and other dianthramide phytoalexins were found in Lena when local defense responses (particularly occlusion) had occurred. In both cultivars evidence for degradation of hemicellulose was found.Samenvatting Kleine stukjes xyleem en andere weefsels van de stengels van gezonde en geïnoculeerde planten van een tweetal anjercultivars, Novada en Lena, werden onderzocht op mate van lignificatie (lignine/polysaccharide verhouding) en ligninesamenstelling met behulp van pyrolyse-massaspectrometrie en pyrolyse-gaschromatografie massaspectrometrie. Deze techniek bleek ook bruikbaar voor het localiseren van fytoalexinen van het dianthramide-type, welke in anjer accumuleren na infectie metFusarium oxysporum f.sp.dianthi. De samenstelling van gezonde weefsels was bij beide cultivars praktisch gelijk. Bij de resistente cultivar Novada leidde infectie tot een verandering van guaiacyl-syringyllignine in guaiacyl-lignine in door gommen afgesloten delen van het xyleem. Xyleem met gommen bevatte aanzienlijke hoeveelheden van het fenolamide dianthalexine en van de andere fytoalexinen van het dianthramide type. De fytoalexinen werden niet aangetroffen in aangrenzend floeem, merg of gezond ogend xyleem. Bij de vatbare cultivar Lena trad afbraak van het xyleem op, dat daarbij armer aan syringyllignine werd, terwijl tevens in een vroeg stadium pectine werd gedemethyleerd. In Lena werden slechts kleine hoeveelheden fytoalexinen gevonden, met name bij locaal opgetreden afweerreacties zoals gomvorming. Bij beide cultivars werden aanwijzingen voor afbraak van hemicellulosen gevonden.     

Expression of biotic stress response genes to Phytophthora infestans inoculation in White Lady,a potato cultivar with race-specific resistance to late blight

Expression changes of biotic stress response genes were analyzed during a 65 h period post inoculation with Phytophthora infestans in potato cultivar White Lady that possesses race-specific resistance to this pathogen. All analyzed respiratory burst oxidase homologs, the PR proteins, the serine-, cysteine- and aspartic protease inhibitors, as well as the Rpi-bt1 gene homolog were up-regulated in the biotrophic phase. The R1 and R2 gene homologs showed up-regulation only at 65 hpi, and interestingly, the R3a gene showed only a very slight expressional increase. It is concluded, that beside the constitutively expressed R genes a number of non-specific stress response genes contribute to the successful resistance response in race-specific defense.     

Visualization of <Emphasis Type="Italic">Ralstonia solanacearum</Emphasis> cells during biocontrol of bacterial wilt disease in tomato with <Emphasis Type="Italic">Pythium oligandrum</Emphasis>

The biocontrol agent Pythium oligandrum (PO) can suppress bacterial wilt caused by Ralstonia solanacearum (RS) in tomato. To understand the primary biocontrol mechanisms of bacterial wilt by PO, we pretreated tomato plants with sterile distilled water or preinoculated them with PO, followed by inoculation with RS, then observed PO and RS in fixed sections of tomato tissues using a confocal laser-scanning microscope and fluorescence labeling until 14 days after the inoculation with RS. Horizontal and vertical movement of RS bacteria was frequently observed in the xylem vessels of roots and stems of tomato plants (cv. Micro-Tom) that had not been inoculated with PO. In plants that were preinoculated with PO, the movement of RS was suppressed, and bacteria appeared to be restricted to the pit of vessels, a reaction similar to that observed in resistant rootstocks. PO colonization was mainly observed at the surfaces of taproots, the junctions between taproots and lateral roots, and the middle sections of the lateral roots. PO was not observed near wound sites or root tips where RS tended to colonize. However, RS colonization was significantly repressed at these sites in PO preinoculated plants. These observations suggest that the induction of plant defense reactions is the main mechanism for the control of tomato bacterial wilt by PO, not direct competition for infection sites.