We have identified a family of dispersed repetitive DNA sequences in the genome of a Magnaporthe grisea isolate from finger millet (Eleusine coracana). Southern blot analyses showed that this element is present in a moderate copy number (30-40 copies) in the genome of blast isolates from wheat (Triticum aestivum). DNA sequence data suggested that this element contains a region highly homologous to the reverse transcriptase domain of MGR583, a poly A-type retrotransposon. Using the reverse transcriptase domain of this element as a molecular probe, the genetic structure of the wheat blast population was examined. DNA fingerprinting analyses revealed that the wheat-infecting isolates constitute a separate, single lineage of their own, suggesting that they are derived from a single origin.
Phylogenetic relationships among 43 bacterial strains of Xanthomonas and two strains of Stenotrophomonas maltophilia were clarified by PCR-restriction fragment length polymorphism (RFLP) analyses of three DNA fragments corresponding to 16S, 23S rDNA and the 16S-23S rRNA internal transcribed spacer region (ITS) using ten restriction enzymes. From the data of the 16S rDNA RFLPs, seven genotypes were detected, while eight genotypes were observed based on the data of the 23S rDNA RFLPs among Xanthomonas strains tested. When the data of the 16S and 23S rDNA RFLPs were combined, the Xanthomonas strains were divided into thirteen rDNA genotypes. Cluster analysis based on rDNA genotypes showed that they were divided into two groups designated as subgroups 1 and 2, which were distinguishable from the related genus Stenotrophomonas. On the basis of the ITS RFLPs, 20 types were observed, and two groups were formed. The members of each group were the same as those based on the rDNAs. The data suggests that this genus is phylogenetically grouped into at least two lineages.
Chlortetracycline (CTC) is known to fluoresce when interacting with Ca2+ in hydrophobic environments such as membranes. CTC fluorescence in potato tubers was rapidly reduced by hyphal wall component-elicitor (HWC-elicitor) from Phytophthora infestans in a dose-dependent manner, suggesting that Ca2+ in the plasma membrane was released by the elicitor. Pre-treatments of the tuber slices with verapamil and EGTA, a Ca2+ channel blocker and an extracellular Ca2+ chelator, respectively, suppressed the reduction of CTC fluorescence induced by the HWC-elicitor. The reduced fluorescence was preceded by an oxidative burst observed in the tuber tissues loaded with luminol after stimulation with HWC-elicitor. Application with HWC-elicitor also immediately reduced fluorescence of the CTC-loaded, Ca2+-bound plasma membrane fraction in vitro. Moreover, RNA gel blot analysis showed that verapamil and EGTA dramatically suppressed the accumulation of transcript for phenylalanine ammonia-lyase induced by the elicitor. These results demonstrate that extracellular Ca2+ is rapidly released from the plasma membrane by the treatment with HWC-elicitor and plays a key role in signal transduction leading to the oxidative burst and the activation of a defense gene.
Cladosporium phlei as well as Epichloë typhina are natural fungal pathogens of timothy grass (Phleum pratense). An increase of resistance of the grass against C. phlei in the presence of E. typhina was already shown in several experimental designs. However, E. typhina can grow endophytically for long periods without causing external symptoms which makes the detection of infected plants and, thus, an exact evaluation of the resistance effect in the field difficult. New or modified methods were developed and used for the effective detection of the endophyte in field samples and for the isolation of both fungi. The relevance of a protective effect of E. typhina in timothy grass against infection with C. phlei was clearly established in field samples as well as in growth chamber and in vitro experiments.
To investigate the effect of non-host plants on the multiplication of Erwinia carotovora subsp. carotovora (Ecc) in field soil and the incidence of soft rot in Chinese cabbage, a field which had previously been used for continuous cropping of Chinese cabbage was dressed with sandy soil. The effects of intercropping Chinese cabbage and wheat and of a mixed cropping of Chinese cabbage and oats were then studied for three years. Ecc could be detected in the soil near the roots of spring-sown Chinese cabbage within the first year. In summer-sown Chinese cabbage, regardless of the type of non-host plants, the multiplication rate of the bacteria was the same as that in continuously cropped Chinese cabbage. The results of the third year were similar. However, in the second year, the bacteria were not detected before the Chinese cabbage was harvested. In contrast to multiplication rates, the incidence of soft rot in both the first year spring and summer-sown Chinese cabbage was the same as that for the continuously cropped field. However, disease severity did not increase with successive cropping. Instead, the onset of the disease was somewhat delayed, the disease was less severe, and disease severity tended to decline. Cultivating Chinese cabbage with a non-host plant to enable intercrossing between the root systems of the two species for three years; the non-host plants appeared to have no effect on the multiplication of Ecc or on the incidence of soft rot.
We investigated the distribution of Kouyawarabi plants (Onoclea sensibilis L.) infested with Pseudomonas plantarii in Yamagata prefecture and confirmed survival activity of the overwintered bacterium in the plants. Of 239 samples collected from 19 locations in the prefecture, 77 leaf samples were infested with the bacterium. The leaf samples collected from Mogami-machi were highly infested with P. plantarii. High infestation rate of bacterial seedling blight of rice was also observed in Mogami-machi area. The plants were artificially inoculated with the organism and exposed outdoors in summer. In the following spring, the bacteria were re-isolated from the leaves, stems and rhizomes of the plants. The pathogenic bacteria were successfully re-isolated from the overwintered whole plants at the level of 1.2×104-3.4×106cfu/g. Because the organism appears to survive in Kouyawarabi plants under natural conditions in Yamagata prefecture, the plants are considered to be an important infection source of the disease.
In November 1997, prairie gentian plants with necrotic yellow symptom were found in Iwate Prefecture, Japan. A spherical virus with a diameter of ca. 80nm was isolated. The virus infected 22 plant species of 11 families after mechanical inoculation. By serological tests, the virus was closely related to tomato spotted wilt virus (TSWV). In RT-PCR with TSWV specific primers, a fragment of about 550bp was amplified. The fragment had a high degree of sequence homology to TSWV-RNA. Based on these results, the virus isolated from prairie gentian was identified as TSWV. This is the first report of TSWV on prairie gentian in Japan.
Detection of Japanese yam mosaic potyvirus (JYMV) in Chinese yam (Dioscorea opposita Thunb. cv. ichoimo) by the immunocapture polymerase chain reaction (IC-PCR) and differentiation of virulent and attenuated isolates using restriction fragment length polymorphism (RFLP) of IC-PCR products using Tsp 509 I were investigated. JYMV was detected by IC-PCR from diseased leaves, bulbils, tubers or diseased leaves frozen at -40°C for 6 months with a sensitivity of 108, 105, 106 and 107-fold dilutions, respectively. The sensitivity of IC-PCR for the detection of JYMV from diseased leaves was about 8000-fold over ELISA and 10, 000-fold over RT-PCR. PCR-RFLP revealed different banding patterns for the virulent and attenuated isolates of JYMV. This will be useful for distinguishing virulent and attenuated isolates from Chinese yam (cv. ichoimo).
A vascular wilt disease of sunflower (Helianthus annuus L.) caused by Verticillium sp. was found in Hokkaido Prefecture, Japan, in 1996 and 1997. The diseased plants showed symptoms of wilting, leaf chlorosis, defoliation, stunting and vascular discoloration of the stem. Based on size of conidia, morphological characteristics of microsclerotia and extracellular polyphenol oxidase activity, the Verticillium wilt pathogen of sunflower belongs to Verticillium dahliae Klebahn.