Tomato Pathotype Research Articles

Assessment of vegetative compatibility of race-2 tomato wilt isolates of Verticillium dahliae in Japan

Verticillium dahliae race-2 can invade the resistant cultivars of tomato possessing theVe gene. This new race was recently found in several regions in Japan, and 10 isolates ofV. dahliae race-2 from these regions were used in our study. Pathogenicity tests identified these isolates as the tomato pathotype (B). We examined the vegetative compatibility of 8 of these 10 Japanese isolates ofV. dahliae race-2 to estimate their genetic relatedness with the testers of Japanese vegetative compatibility group previously proposed (VCGJ) usingnit mutants. Compatiblenit1 and NitM mutants were obtained from allV. dahliae race-2 isolates. Selected representativenit1 and NitM mutants of eachV. dahliae race-2 isolates were paired with VCGJ testers. All isolates ofV. dahliae race-2 showed a strong reaction with VCGJ2, i.e., tomato pathotype. All isolates ofV. dahliae race-2 except for isolate To22 reacted weakly to VCGJ1 and J3. Japanese isolates ofV. dahliae race-2 were assigned as VCGJ2 and were hence vegetatively closely related with those ofV. dahliae race-1. The origin of Japanese isolates ofV. dahliae race-2 was discussed.

AAL-Toxin-Deficient Mutants of Alternaria alternata Tomato Pathotype by Restriction Enzyme-Mediated Integration

ABSTRACT Host-specific toxins are produced by three pathotypes of Alternaria alternata: AM-toxin, which affects apple; AK-toxin, which affects Japanese pear; and AAL-toxin, which affects tomato. Each toxin has a role in pathogenesis. To facilitate molecular genetic analysis of toxin production, isolation of toxin-deficient mutants utilizing ectopic integration of plasmid DNA has been attempted. However, the transformation frequency was low, and integration events in most transformants were complicated. Addition of a restriction enzyme during transformation has been reported to increase transformation frequencies significantly and results in simple plasmid integration events. We have, therefore, optimized this technique, known as restriction enzyme-mediated integration (REMI), for A. alternata pathotypes. Plasmid pAN7-1, conferring resistance to hygromycin B, with no detectable homology to the fungal genome was used as the transforming DNA. Among the three restriction enzymes examined, HindIII was most effective, as it increased transformation frequency two-to 10-fold depending on the pathotype, facilitating generation of several hundred transformants with a 1-day protocol. BamHI and XbaI had no significant effect on transformation frequencies in A. alternata pathotypes. Furthermore, the transforming plasmid tended to integrate as a single copy at single sites in the genome, compared with trials without addition of enzyme. Libraries of plasmid-tagged transformants obtained with and without addition of restriction enzyme were constructed for the tomato pathotype of A. alternata and were screened for toxin production. Three AAL-toxin-deficient mutants were isolated from a library of transformants obtained with addition of enzyme. These mutants did not cause symptoms on susceptible tomato, indicating that the toxin is required for pathogenicity of the fungus. Characterization of the plasmid integration sites and rescue of flanking sequences are in progress.

Random Amplified Polymorphic DNA Analysis of Japanese Isolates ofVerticillium dahliaeandV. albo-atrum

Japanese isolates of Verticillium dahliae and V. albo-atrum have been differentiated into four and two pathogenicity groups, respectively. Japanese isolates, 29 isolates of V. dahliae and 15 isolates of V albo-atrum, representing different pathogenicity groups were analyzed by means of the random amplified polymorphic DNA (RAPD) method using polymerase chain reaction (PCR). V. dahliae could be differentiated into three subclusters and V. albo-atrum into two subclusters. In V. dahliae, the first subcluster (RAPD type I) included isolates in group A (eggplant pathotype), group C (sweet pepper pathotype), and another group with pathogenicity not determined. The second subcluster (RAPD type II) included group B isolates (tomato pathotypes). The third subcluster (RAPD type III) contained four diploid isolates (group D, brassica pathotype) and one haploid isolate (84111). In V. albo-atrum, the isolates were divided into two subclusters. The first subcluster (RAPD type IV) included isolates in only the alfalfa pathotype. The second subcluster (RAPD V) included one alfalfa pathotype isolate (Vaa-s02), three potato pathotype isolates, and three undetermined pathotype isolates from alfalfa. Similarities and differences in banding patterns obtained by RAPD could be a useful molecular tool in identification and phylogenetic studies of the pathogenicity groups.

A Rapid and Sensitive Procedure for the Quantitative Detection of AL-toxin by Fluorescence Derivatization and Separation by High Performance Liquid Chromatography.

An improved high performance liquid chromatographic (HPLC) method was developed for the detection and quantitation of host-specific AL-toxin produced by Alternaria alternata tomato pathotype. Precolumn derivatization of the toxin with o-phthalaldehyde (OPA) plus mercaptoethanol or 4-fluoro-7-nitrobenzofurazan (NBD-F) yielded highly fluorescent products showing well-resolved peaks on reversed phase HPLC. The minimum amounts of AL-toxins detectable by this method were approximately 1ng. This method seems to offer advantages over conventional techniques, because prechromatographic derivatization of the toxin with OPA or NBD-F is rapid and easy, and fluorescent derivatives permit excellent detection sensitivity. Toxin production in culture filtrates and spore-germinated fluids of the pathogen was quantitatively analyzed using this procedure.

Cultivar-specific and Non-specific Responses in Tomato Cell Cultures to AL-toxin from Alternaria alternata Tomato Pathotype.

Cultivar-specific and Non-specific Responses in Tomato Cell Cultures to AL-toxin from Alternaria alternata Tomato Pathotype.

Difficulty to establish vegetative compatibility of Japanese isolates of Verticillium dahliae Kleb. using melanin-synthesis deficient mutants.

Japanese isolates of Verticillium dahliae were formerly designated as tomato pathotype and non-tomato pathotype by pathogenicity tests. We attempted to classify 18 Japanese isolates based on vegetative compatibility. Only three isolates of the tomato pathotype induced compatible alm-brm mutants. A non-secreting brm mutant was not induced from isolate of the non-tomato pathotype. Complementation was not achieved by a pair of mutants from the tomato pathotype and those from the non-tomato pathotype. From these results, Japanese isolates of V. dahliae could not be separated into any vegetative compatibility groups using melanin synthesis-deficient mutants.

Inhibitory Effect of AL-toxin Produced by Alternaria alternata Tomato Pathotype on the Biosynthesis of Phopshatidylethanolamine in Tomato Leaves Susceptible to the Fungus.

AL-toxin, a host specific toxin produced by Alternaria alternata tomato pathotype, had been reported to cause accumulation of ethanolamine (EA) and phosphoethanolamine (PEA) in susceptible tomato leaves. In this connection, the effect of AL-toxin on the metabolism of phospholipids, particularly on the biosynthetic pathway of phosphatidylethanolamine (PtdEA) was investigated. [14C] EA was fed in leaf discs in both susceptible and resistant cultivars, Mie First and Saturn, respectively. The incorporation of [14C] EA into both tomato leaf discs similarly increased with incubation time, regardless of AL-toxin treatment, while significant inhibition of its incorporation into PtdEA was only found in susceptible leaf discs treated with the toxin. In a comparative study on the effect of AL-toxin on the biosynthesis of phosphatidylcholine (PtdC), the inhibition of incorporation of [14C] choline into PtdC in susceptible leaf discs was very low as compared to that of [14C] EA into PtdEA. Results from the present work indicate that AL-toxin have a greater inhibitory effect on the biosynthesis of PtdEA through EA in the leaves of susceptible cultivar, but not in resistant one.

Effect of AL-toxin Produced by Alternaria alternata Tomato Pathotype on Cultured Roots of Tomato.

The effect of AL-toxin produced by Alternaria alternata tomato pathotype on susceptible and resistant plants was investigated by employing a quantitative, simple and easy assay system using cultured roots of tomato. Root growth and root cell viability were examined by weighing and MTT [3-(4, 5-dimethylthiazol-2-yl)-2, 5-Biphenyl tetrazolium bromide] colorimetric assay, respectively. Cultured roots of susceptible cv. Mie First incubated in culture media containing different concentrations of AL-toxin I showed a detectable reduction and complete inhibition of the growth at toxin concentrations of 0.01 and 1μg/ml, respectively. The growth of cultured roots of resistant tomato cv. Saturn was significantly and completely reduced at toxin concentrations of 0.1 and 10μg/ml, respectively. When the root viability was determined by the colorimetric assay using MTT, the viability of susceptible cultivar was significantly reduced at a toxin concentration of 1μg/ml, two days after treatment, while reduction in the viability of resistant cultivar was not detected even at a toxin concentration of 10μg/ml. These results indicate that the use of cultured roots of tomato may contribute to a quantitative and qualitative determination for evaluating the host specific mode of action of AL-toxin.

Changes in Amino Acid and Amine Contents of Susceptible Tomato Cultivars Infected with Alternaria alternata Tomato Pathotype or Treated with AL-toxin.

The changes in amino acid and amine contents of susceptible and resistant tomato cultivars infected with Alternaria alternata tomato pathotype or treated with its host-selective AL-toxin were investigated. AL-toxin has been known as a potential inhibitor of aspartate carbamoyltransferase (ACTase) in susceptible tomato plants. Inhibition of ACTase should result in the accumulation of aspartic acid in the susceptible plant treated with AL-toxin. In this study, however, no difference was observed in the amount of aspartic acid and other amino acids between toxin-treated and non-treated leaves of the susceptible and resistant cultivars until at least 24hr after treatment. In the susceptible tomato plants inoculated with spores at the base of the stem, amino acid contents in leaflets showing necrotic symptom were similar to that in corresponding leaflets without symptom. Aspartic acid content in symptom-developed leaflets was rather lower than that in symptom-less ones. These results do not support the hypothesis that AL-toxin inhibits ACTase. On the other hand, two amines, ethanolamine and phosphoethanolamine, markedly accumulated in susceptible tomato leaves 12 to 24hr after toxin treatment, but not in resistant ones. The increase in these amines was also observed in symptom-developing leaflets but not in symptomless ones of plants inoculated with the spores of the pathogen. These results suggest that AL-toxin induces disorder of metabolism related to these amines in susceptible tomato cells.

Effect of AL-toxin Produced by Alternaria alternata Tomato Pathotype on Viability of Cultured Tomato Cells Determined by MTT-Colorimetric Assay.

Effect of AL-toxin Produced by Alternaria alternata Tomato Pathotype on Viability of Cultured Tomato Cells Determined by MTT-Colorimetric Assay.