Strawberry Pathotype Research Articles

Comparison of Parasitic Fitness between Two Pathotypes of Alternaria alternata to Japanese Pear.

Attempts were made to examine whether the strawberry pathotype of A. alternata could inhabit in the orchards of Japanese pear cv. Nijisseiki as a causal agent of black spot of the pear. First, black necrotic lesions that naturally occurred on Nijisseiki leaves were collected from the 3 pear orchards in Aichi Prefecture in 1983 and 1984. Field isolates from the 1219 lesions were assayed for their pathogenicity to leaves of Japanese pear and strawberry, but no isolate identified as the strawberry pathotype was detected. Secondly, the pathogen-free nursery plants of strawberry cv. Morioka-16, which were from the meristem-tip cultures, were transplanted in a Nijisseiki pear orchard as effective traps of air-borne spores of the strawberry pathotype during May, 1983 to September, 1984. From lesions that appeared on some leaves of the strawberry plants, an Alternaria fungus was isolated and identified as the strawberry pathotype, suggesting that the strawberry pathotype is inhabiting in Nijisseiki pear orchards at much lower density than the Japanese pear pathotype. In a simulation model experiment, parasitic fitness of the two pathotypes to the pear leaves was compared using highly virulent strains of the respective pathotypes. The results showed that the strawberry pathotype was inferior to the Japanese pear pathotype in parasitic fitness, judged by lesion number and lesion size, on the pear leaves under laboratory, phytotron and field conditions.

Resistant Plants to Alternaria alternata Strawberry Pathotype Selected from Calliclones of Strawberry Cultivar Morioka-16 and Their Characteristics

To breed an improved 'Morioka-16' which is resistant to Alternaria black spot, caused by Alternaria alternate strawberry pathotype, culture-caused mutagenesis was used. Calliclones induced by shoot apex culture of cv. Morioka-16 were inoculated with the pathogen by three screening methods.Using one of these methods, three plants resistant to the disease were successfully selected from 1196 calliclones which had been inoculated with the pathogen.These three mutants were named 'M16-AR 1', 'M16-AR 2' and 'M16-AR 3' and their plant and fruit characters were examined. No differences between cv. Morioka-16 and the three mutants were found in terms of plant height, leaf number, flowering time, harvesting time, yield, and fruit characters.

Slightly Damaged Type of Plasma Membrane Modification in Strawberry Leaves Treated with AF-Toxin I Produced by Alternaria alternata Strawberry Pathotype.

Plasma membrane modifications in AF-toxin I-treated susceptible strawberry leaves were similar to those in AK-toxin I-treated susceptible Japanese pear leaves, but the former was slightly different in ultrastructural features from the latter. The modifications due to AF-toxin I had the characteristic features in ultrastructure as follows: 1) the specific occurrence of small plasmalemmal invaginations at plasmodesmata, 2) the accumulation of scanty polysaccharide-components and a small amount of membranous fragments into the invaginated spaces between the cell walls and the plasma membranes, 3) the appearance of a moderate number of exocytotic Golgi vesicles, and 4) the relatively low incidence of the invaginations. The AF-toxin I-induced modifications were recognized as the slightly damaged type of plasma membrane modifications while the AK-toxin I-induced modifications as the severely damaged type. The results suggested that the effects of AF-toxin I on susceptible strawberry cells were different in some ways from those of AK-toxin I on susceptible Japanese pear cells. In addition, the leakage-sites of tissue-ions were examined in AF-toxin I-treated strawberry leaves, using both antimonate-fixation method and analytical electron microscopy. As the results, it was proved that Na and Mg ions were leaked from the plasma membranes in the vicinity of plasmodesmata at which the modifications specifically occurred in AF-toxin I-treated susceptible cells. This indicated that the slightly damaged type of plasma membrane modifications caused by AF-toxin I in susceptible strawberry leaves was closely associated with the electrolyte-leakage as well as the severely damaged type of plasma membrane modifications caused by AK-toxin I in susceptible Japanese pear leaves.

Gene Analysis of Mutants Resistant to Alternaria alternata Strawberry Pathotype Selected from Calliclones of Strawberry Cultivar Morioka-16.

Gene analysis of the disease-resistant selections, 'M16-AR 1', 'M16-AR 2' and 'M16-AR3' was made. These calliclones were derived by culturing the shoot apices of 'Morioka-16' which is susceptible to Alternaria alternata strawberry pathotype. The analysis is based on the results of disease reactions of their progenies from selfing (S1) and hybrid crosses (F1)with 'Morioka-16' and with 'Hokowase', a recessive homozygote with respect to the susceptible gene. The following results were obtained:S1 progenies of each line showed resistance to the pathogen. F1 progenies of reciprocal crosses between 'M16-AR 1, 2 or 3' × 'Morioka-16' (heterozygous in relation to the susceptible gene) and between 'M16-AR 1, 2 or 3' × 'Hokowase' (homozygous recessive in relation to the susceptible gene) resulted in the following segregation ratios of susceptibility (S) and resistance (R) to the pathogen.'M16-AR 1, 2 or 3' × 'Morioka-16' and vice versa l (S) : 1 (R)'M16-AR 1, 2 or 3' × 'Hokowase' and vice versa 0 (S) : 1 (R)The results described above suggest that the genotype of each of the three lines exhibited variation from the genotype of 'Morioka-16', namely, heterozygote to recessive homozygote with respect to the susceptible gene.

Effect of Host-Specific AF-Toxins from Alternaria alternata Strawberry Pathotype on Viability of Strawberry Cultured Cells.

An assay system using strawberry cultured cells was established for assessing the biological activities of AF-toxins produced by Alternaria alternata strawberry pathotype, causal agent of Alternaria black spot of strawberry. MTT [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide]-colorimetric assay, in which cell viability was quantitatively determined by spectrophotometric measurement of cell extracts containing MTT-formazan generated in viable cells, was employed to assess the host-selective toxicity of AF-toxins to strawberry cultured cells. Cultured cells of three genotypes of strawberry, whose leaves are super-susceptible, susceptible and resistant to the pathogen and AF-toxin I, exhibited the same genotype-specific responses to AF-toxin I as their leaves. However., the sensiivity of susceptible cultured cells to the toxin was higher than that of their leaves: damage of the cultured cells was induced by the toxin treatment at much lower concentrations than was leaf necrosis. Protoplasts derived from their cultured cells also showed the similar reactions to the toxin. Experiments exposing cultured cells to AF toxin I for definite times demonstrated that a 2-min exposure of super-susceptible cells to the toxin apparently caused the cell damage, and that a 10-min exposure affected the cells as intensely as a 6-hr continuous exposure. These results indicate that a toxic quality can be absorbed or taken up in a relatively short time: continuous exposure of cells to the toxin is not necessary for causing cell damage.

イチゴ黒斑病菌に対するイチゴ品種の感受性の遺伝

This work was undertaken to elucidate the heritability of susceptibility in strawberry cultivars to Alternaria alternata strawberry pathotype and the origin of susceptible cultivars. To resolve the first question, SI strawberry seedlings derived from self pollination of 37 cultivars and 13 strains and F1 strawberry seedlings obtained by means of diallel cross among susceptible cultivars (S), 'Morioka-16' derived from ('Fairfax' × 'Ettersburg') × 'Chiyoda' and 'Robinson' from 'Howard-17' × 'Washington', and resistant cultivars (R), 'Hokowase' and 'Donner', were inoculated with the spores (isolate OH-5). The following summarizes the results of disease reaction of SI and F1 seedlings to the pathogen.1. Regarding successive generation of the tested cultivars and strains, the segregating ratio of susceptible plants to resistant plants was almost 3 : 1 in susceptible cultivars, 'Morioka-16' and 'Robinson'. The segregation ratio was 0 : 1 among all progenies resulting from the other 48 cultivars and strains resistant to the pathogen. These include progenies of 'Fairfax' and 'Chiyoda', cross parents of 'Morioka-16' and 'Howard-17', a cross parent of 'Robinson'.2. In the case of disease reaction of F1 seedlings obtained by diallel corss among 'Morioka-16', 'Robinson', 'Hokowase' and 'Donner' to the pathogen, the ratio of susceptible plants to resistant plants in the F1 generation was as follows.(1) 'Morioka-16' (S) × 'Robinson' (S) and vice versa 3 : 1(2) 'Morioka-16' (S) × 'HOltOwase' (R) and vice versa 1 : 1(3) 'Robinson' (S) × 'HOkawase' (R) and vice versa 1 : 1(4) 'Hokowase' (R) × 'Donner' (R) and vice versa 0 : 13. As to the origin of the susceptibility of 'Morioka-16' and 'Robinson' to the pathogen, it seems highly probable that 'Ettersburg' which transmitted the trait to 'Morioka-16', and 'Washington', which passed the trait to 'Robinson', are carriers of the dominant susceptible gene. We come to this conclusion because the cross parents, 'Fairfax', 'Chiyoda' and 'Howard-17' are resistant, recessive homozygote with respect to the susceptible gene.However, the origin of the susceptible cultivars, 'Morioka-16' and 'Robinson', could not exactly be traced, because 'Ettersburg' and 'Washington' are not available in Japan.

Synthesis of Methyl Esters of AF-Toxin IIa and IIc, Toxins to Japanese White Pear Produced by Alternaria alternata Strawberry Pathotype.

Synthesis of Methyl Esters of AF-Toxin IIa and IIc, Toxins to Japanese White Pear Produced by Alternaria alternata Strawberry Pathotype.

Determination of Host-Selective Toxin Production During Spore Germination ofAlternaria alternataby High-Performance Liquid Chromatography

The Japanese pear pathotype of Alternaria alternata, which causes black spot disease, produces multiple host-selective toxins (AK-toxins I and II) in culture; the strawberry pathotype, which causes black spot disease, also produces a series of analogous host-selective toxins (AF-toxins I, II, and III). Analysis by high-performance liquid chromatography of toxin production during spore germination showed that virulent isolate A85-10 of the Japanese pear pathotype produced about 0.02 pg of AK-toxin I per spore in the first 6 hr after germination and a trace amount of AK-toxin II after 24 hr (...)

わが国のイチゴ黒斑病に対する感受性品種

The purpose of this paper is to report susceptible strawberry cultivars and strains to Alternaria black spot of strawberry (Alternaria alternata strawberry pathotype) in Japan. The test for susceptible strawberry cultivars and strains to the pathogen was carried out by artificial inoculation with the spores (isolate OH-5) in June and September, 1988 and 1989. Natural infections under field conditions were also recorded.Among sixty Japanese and foreign strawberry cultivars and strains tested, only two cultivars, ‘Morioka-16’ previously known as susceptible cultivar in Japan and ‘Robinson’ from the United States of America, were susceptible to the pathogen in Japan. The other cultivars including ‘Chiyoda’ and ‘Fairfax’, used as crossing parents of ‘Morioka-16’, were completely resistant.

Structure of AF-toxin II, one of the host-specific toxins produced by [formula omitted][formula omitted] strawberry pathotype

AF-toxin II was a mixture of three stereo-isomers, whose structures were determined as 2a , 2b and 2c by means of chemical and spectroscopic methods.

Studies on host-specific AF-toxins produced by Alternaria alternata strawberry pathotype causing alternaria black spot of strawberry. 5 Effect of toxins on membrane potential of susceptible plants as assessed by electrophysiological method.

Plasma membrane dysfunction of strawberry and Japanese pear petiole cells treated with AF-toxins was studied by means of electrophysiological methods. The light-dependent and-independent components of membrane potential of susceptible strawberry were rapidly depolarized after AF-toxin I (3.2×10-6 M) treatment. AF-toxin II (2.6×10-6 M) did not cause any change in the membrane potential and its two components of susceptible straw-berry. Resistant strawberry has shown no depolarization by AF-tioxin I (3.2×10-6 M) treatment. The membrane potentials between xylem vessel and parenchyma symplast of strawberry and Japanese pear petioles were measured by the xylem perfusion method. AF-toxin I induced inhibition of the activity of electrogenic ion pump on both susceptible strawberry and Japanese pear, but AF-toxin II induced it only in susceptible pear. In each case, respiration-dependent membrane potential difference rapidly decreased. These two AF-toxins did not cause any change in membrane potential of strawberry and Japanese pear resistant to the disease. Prior treatment of susceptible strawberry tissuse with AF-toxin II, non-toxic to strawberry, protected them from AF-toxin I action as was evident in electrophysiological indices. Respiration-dependent component of membrane potential in protected tissues was not influenced for at least 2 hr after AF-toxin I treatment.

Studies on host-specific AF-toxins produced by Alternaria alternata strawberry pathotype causing Alternaria black spot of strawberry. 4. Protective effect of AF-toxin II on AF-toxin I-induced toxic action and fungal infection.

Chemically characterized pure host-specific toxins produced by Alternaria alternata strawberry pathotype (AF-toxins) were examined for their biological activities. AF-toxin I induced even at concentrations as low as 3.2×10-8M leaf veinal necrosis on both leaves of susceptible cv. Morioka 16 of strawberry and cv. Nijisseiki of Japanese pear. AF-toxin II was found to cause veinal necrosis only on leaves of the susceptible Japanese pear cv. Nijisseiki with a minimum concentration of 2.6×10-8M. AF-toxin III was toxic to susceptible strawberry leaves at 3.1×10-7M, and also toxic to Nijisseiki pear leaves at considerable high concentrations more than 1.6×10-5M. At concentrations up to 10-4M, there was no effect observed in any of the experiments with the strawberry cv. Hoko-wase and Japanese pear cv. Chojuro, immune to the disease. We also examined the ability of AF-toxin II to protect strawberry leaf tissues from AF-toxin I action and fungal infection. Detached leaves were exposed to AF-toxin II for 18hr, and then treated with AF-toxin I. AF-toxin II gave a remarkable protection against AF-toxin I-induced veinal necrosis and potassium leakage. Minimum ratio of AF-toxin II: AF-toxin I giving 80% protection was approximately 10:1. Pre-treatment with AF-toxin II reduced significantly the number of black necrotic spots on inoculated susceptible cv. Morioka-16 of strawberry. The present experiment provided the first evidence of protecting plant from disease by means of counteracting toxin action by a chemical structurally related to the host-specific toxin.

Studies on host-specific AF-toxins produced by Alternaria alternata strawberry pathotype causing alternaria black spot of strawberry. 3. Use of toxin for determining inheritance of disease reaction in strawberry cultivar Morioka-16.

The inheritance was determined for the reaction of strawberry to Alternaria alternate strawberry pathotype and its host-specific toxin (AF-toxin). Susceptible cv. Morioka-16 and resistant cv. Hokowase were used for the crosses. Susceptibility to the pathogen and sensitivity to the toxin were both inherited as a single locus with two alleles expressing incomplete dominance when heterozygous. Susceptible cv. Morioka-16 was found to be susceptible heterozygote and resistant cv. Hokowase was found to be resistant homozygote. Furthermore, susceptible homozygote selected from F1 progenies was more susceptible to the pathogen and also more sensitive to AF-toxin than cv. Morioka-16, susceptible heterozygote.

Studies on host-specific AF-toxins produced by Alternaria alternata strawberry pathotype causing alternaria black spot of strawberry. 2. Role of toxins in pathogenesis.

The role of AF-toxins in the interaction of A. alternata strawberry pathotype and its hosts, strawberry cv. Morioka-16 and Japanese pear cv. Nijisseiki, was investigated at the early stage of infection. Time course study of the production of AF-toxins from germinating spores elucidated that all three AF-toxins (I, II and III) were detectable early after inoculation and increased in quantity with incubation time. The leaves inoculated with pathogen were allowed to leak electrolytes by AF-toxin produced during the germination of spores. The electrolyte leakage was shown to have two peaks at different stages of the infection behaviors of the spores; the first was detected before the establishment of infection and the second was detected after the establishment of infection. The first peak suggests that the AF-toxins from germinating spores prior to fungal invasion could change the physiological potential related to the membrane system of the host cells to be desirable to the pathogen. The effect of AF-toxins on the establishment of infection by pathogenic spore was investigated by treating the leaves both the high molecular weight fraction “inducer” in germination fluids of spores and AF-toxins. The treatment with “inducer” rendered the susceptible leaves relatively resistant to the pathogen, while, the treatment with AF-toxins rendered the leaves susceptible even to non-pathogenic A. alternata. Concomitant treatment with the “inducer” and AF-toxins resulted in the suppression of the induction of host resistance. These data suggest that pathogenic spores of A. alternata strawberry pathotype produce AF-toxins during their germination, and thereby may suppress the resistance response through the disturbance of membrane integrity of the host cells, and consequently succeed in establishing infection.

Studies on host-specific AF-toxins produced by Alternaria alternata strawberry pathotype causing alternaria black spot of strawberry. 1. Production of host-specific toxins and their biological activities.

The causal pathogen of Alternaria black spot of strawberry was identified as a distinct pathotype of Alternaria alternata (Fries) Keissler. Only one strawberry cultivar Morioka-16 was susceptible to the pathogen. Several cultivars of Japanese pear including Nijisseiki which were susceptible to A. alternata Japanese pear pathotype were also diseased by this pathogen. This fungus produced at least three species of host-specific toxins (named AF-toxin I, II and III, respectively). AF-toxin I and II were considered the major toxins. AF-toxin I induced necrosis and potassium leakage on the susceptible cvs. Morioka-16 and Nijisseiki. AF-toxin II induced only potassium leakage from cv. Morioka-16 but did not induce necrosis. AF-toxin II as well as AF-toxin I caused necrosis and potassium leakege from the cv. Nijisseiki.