Primer pairs and nucleic acid preparations were used with RT-PCR to detect peach latent mosaic viroid (PLMVd) from stone fruits collected across Japan. The viroid was detected from 94.3% of the peach samples, 5.3% of Japanese plum samples, 4.4% of mume samples and 3.7% of sweet cherry samples, but was not detected from European plums and apricots in local orchards in Japan. It was also detected from many peach cultivars at the National Institute of Fruit Tree Science (NIFTS), including all peach yellow mosaic- and oil blotch-infected cultivars. Symptom expression was also monitored on the RT-PCR-assayed stone fruit trees maintained at NIFTS. Only one of the peach cultivars positive for PLMVd expressed mosaic symptoms on leaves; however, the other cultivars had no symptoms. Two PLMVd isolates from peach cultivars Nakatsu Hakutou and Akatsuki in Japan were sequenced; they consisted of 337 nucleotides. The Nakatsu Hakutou isolate differed from a French isolate at 24 sites in the sequence, including 24 base exchanges, whereas the Akatsuki isolate differed at 28 sites, including 26 base exchanges, one insertion and one deletion. Two isolates showed 91 and 92% sequence homology with the PLMVd from France.
The growth of Xanthomonas oryzae pv. oryzae (Xoo), causal agent of bacterial leaf blight and typical vascular pathogen, was investigated in tissues and guttation fluids of rice leaves to elucidate the mechanisms of susceptibility in the host-parasite interaction. Light and scanning electron-microscopic observation showed that, in the compatible cultivar-bacterial strain combination, bacterial masses were abundant in the lumen of xylem vessels, but were absent in other vascular elements. The inner surface of spiral and ring vessels where the bacterium multiplied appeared to be digested by the causal organism. Histochemical tests also revealed that the xylem vessel walls were degraded after infection with Xoo. Based on the histological study, the growth of Xoo was examined in guttation fluids collected from rice leaves. Unexpectedly, the growth rate of Xoo was very low in an aqueous solution of freeze-dried guttation fluid. When 0.5% sucrose was added, however, the bacterium could grow extensively in a solution containing freeze-dried guttation fluids. In addition, the growth of Xoo was also promoted by substances in the pectic fraction extracted from cell walls of rice leaves. The results suggested that nutritional factors such as pectic substances from affected xylem vessel walls were involved in the multiplication of Xoo in the lumen of xylem vessels.
DNA fingerprinting with multilocus probes, MAGGY and MGR586, was conducted to investigate the population structure of the rice blast fungus Magnaporthe grisea in Japan. Two-hundred seventy-eight field isolates collected during 1993-1997 and 22 archival isolates collected in 1976 from various cultivars and locations were examined. Two fingerprint groups were identified at the 70% similarity level in each collection. A one-to-one correspondence was found between the groups in field isolates and the groups in archival isolates. These results suggest that two discrete lineages exist in the rice blast population. They were temporarily designated as JL1 and JL2 (Japanese Lineage). JL1 was a predominant lineage representing 97% and 77% of the field and archival isolates, respectively, and present throughout Japan. In addition, these two lineages corresponded to two of the five lineages previously detected in collections made before 1960, suggesting that the structure of the rice blast population in Japan had drastically changed during 1960-1976. The two lineages showed a very similar virulence spectrum, and no relationship could be recognized between lineages and pathotypes. MAGGY revealed robust groups which were similar to those revealed by MGR586, although the two elements differ in character and structure, suggesting that MAGGY could be used as an alternative probe for monitoring the population dynamics of the rice blast pathogen.
The circular DNA plasmid pAAT56 has been isolated from strain T88-56 of the Japanese pear pathotype of Alternaria alternata. The pAAT56 is 5354bp and possibly encodes two long open reading frames, designated ORF1 (1290bp) and ORF2 (1653bp). We have previously observed negative biological effects of pAAT56 on fungal phenotypes: reduction in pathogenicity and occasionally in vegetative growth. Here we analyzed the intracellular localization of pAAT56 and its expression at the RNA and protein levels. Mitochondria and nuclei were prepared from mycelia of strain T88-56 by subcellular fractionation. Total cellular DNA, mitochondrial DNA and nuclear DNA were subjected to gel blot analysis with pAAT56 probe. The analysis detected pAAT56 in total DNA and nuclear DNA, but not in mitochondrial DNA, indicating that the plasmid is not mitochondrial. RNA gel blot analysis with pAAT56 probe detected the presence of three RNAs of about 1.7, 2.7 and 5.4kb. Use of strand-specific RNA probes in RNA gel blot analysis demonstrated that all the transcripts are sense strands for ORF1 and ORF2. An ORF1-fusion protein was expressed in an Escherichia coli system and used for preparation of antiserum. Antiserum raised against the ORF1 product cross-reacted with six proteins (about 38, 49, 52, 62, 70 and 73kDa) in the pAAT56-carrying strain, but not in its pAAT56-cured derivatives. One of the proteins was identical in size to the predicted ORF1 product (49kDa). This result implies that the ORF1-related proteins are produced from pAAT56 in fungal cells, probably accompanied with posttranslational processing and/or interaction with fungal cellular protein (s).
We compared the structure and homology of the hrp gene cluster and its neighboring region for grouping Pseudomonas syringae pathovars. Cosmid clones selected from genomic libraries constructed for P. syringae pv. glycinea, pv. maculicola and pv. tabaci covered a genomic region of more than 80kb that was homologous to hrp genes and neighboring regions in pv. phaseolicola. Comparison of restriction sites and homologies of this large genomic region revealed that the structures of the hrp gene clusters of pv. glycinea, pv. phaseolicola and pv. tabaci closely resemble each other, whereas that of pv. maculicola is distinct from those of the other three pathovars. Hybridization probes derived from pv. phaseolicola and pv. maculicola were used for grouping 32 pathovars of P. syringae by Southern hybridization analysis. Patterns of hybridization signal strength suggested that P. syringae pathovars can be classified into at least the following three groups: strains having strong homologies to probes from pv. phaseolicola (group I), strains having strong homologies to probes from pv. maculicola (group II) and strains having moderate to no homology to probes from either of the pathovars (group U).
From 1993-96, the sensitivity to isoprothiolane in field isolates of Pyricularia oryzae was monitored using EC50 values for mycelial growth on agar medium. Sensitivity to organophosphorus tiolate (PTL) fungicides, IBP and EDDP, was also monitored as EC50 values, because mutants resistant to PTL fungicides (in vitro mutants) obtained by in vitro selection showed resistance to isoprothiolane. EC50 values of isoprothiolane and IBP were compared with those obtained from 1977-83. The sensitivity to Isoprothiolane apparently fell into one group that was as sensitive as the sensitive stock isolates in each monitoring period. From 1977-83, the sensitivity to IBP was recognized as two groups: One was sensitive, and the other was the moderately resistant (MR) isolates which consisted of a less resistant cluster at a high incidence and a more resistant one at a low incidence. From 1993-96, the sensitivity of isolates belonging to the less IBP-resistant MR cluster increased, but those in the more resistant MR cluster were unchanged. From 1993-96, the sensitivity to EDDP was near that of the sensitive stock isolates. No highly resistant isolates like the in vitro mutants were found in the field isolates at both monitoring periods. The difference in the resistance level of IBP-MR isolates and their sensitivity to Isoprothiolane and EDDP was not correlated. Preventive foliar applications revealed that IBP-sensitive and -MR isolates were sensitive to isoprothiolane and EDDP both at their practical doses and at half the practical doses. These results suggest that IBP-MR isolates in fields do not show cross-resistance to isoprothiolane and EDDP.
This study was carried out to clarify the effect of true resistance genes in rice cultivars on the occurrence of pathogenic variants of the rice blast fungus, Pyricularia oryzae Cavara, during proliferation of avirulent isolates on panicles of the rice plants using isogenic lines of the Sasanishiki multiline. Re-isolates from blast-infected panicles of five rice cultivars and two lines which harbored different true resistance genes were tested to determine the Japanese races. The number of blast races and the rate of variation in the re-isolates from panicles of resistant rice cultivars were clearly higher than those from the susceptible rice. Pathogenic variation of the rice blast fungus was stimulated when the avirulent isolate proliferated on the panicles of resistant rice cultivars. Some pathogenic variants could break down the same true resistant genes in the re-isolates from the panicle of rice cultivars. However, many variants could break down other rice cultivars with different true resistance genes. From these results, the direction of pathogenicity of variants of this fungus was not influenced by the kind of true resistant genes in rice cultivars. The variants which could overcome the Pi-ta, Pi-i and Pi-z genes appeared frequently, whereas those overcoming the Pi-k and Pi-b genes were found less often. However, few or no variants could break down the Pi-km, Pi-ta2 and Pi-zt genes. Three types of avirulence genes in rice blast fungus are, therefore, considered likely, those which were highly unstable, moderately unstable or relatively stable.
Cotyledons of Japanese radish, oil seed and broccoli were inoculated with four isolates [Japanese radish (Rs), oil seed (Bc), broccoli (Bo) and shepherd's purse (Cb)] of Peronospora parasitica Pers. ex Fr. after heat treatment with 47.5°C or 50°C for 30sec. The treatment induced conidiation by avirulent isolates except by the Cb isolate on all plant species. However, these isolates formed fewer conidia on the treated cotyledons than that on those of the virulent isolate. Cytological observations revealed that formation of the haustorial sheath and death of haustoria-containing cells were suppressed in the treated cotyledons inoculated with avirulent isolates without the Cb isolate. Suppression of papilla formation and promotion of hyphal development were observed in heat-treated cotyledons inoculated with the Cb isolate. By 24 to 48hr after inoculation, however, death of cells with haustoria and formation of sheaths were observed. The period of suppression was different for each combination of isolate and plant species. These results suggest that pre-inoculation heat treatment of cruciferous plant cotyledons suppresses defense reactions against P. parasitica, and that pathogenicity of the Cb isolate differs considerably from the other three isolate. The continuous suppression of the plant defense reaction after formation of haustoria may be necessary for success of infection by the fungus.
Glomerella cingulata (Stoneman) Spaulding et Schrenk (anamorph: Colletotrichum gloeosporioides (Penzig) Penzig et Saccardo) has been known as the pathogen of ripe rot of grapevine (Vitis vinifera L.). In addition to this fungus, another Colletotrichum species was isolated from fruits of grape cultivar ‘Sekirei’ with ripe rot in Shimane Prefecture in 1994. Colonies of the isolate on potato dextrose agar (PDA) were reddish to pale brown. Its conidia on PDA were one-celled, hyaline, ellipsoid to fusiform with acute ends and 12-17×4-5μm. Appressoria formed on potato carrot agar were pale grayish brown, ellipsoid, rarely irregular in shape, and 8-11×4-7μm. The isolate grew slower than that of G. cingulata isolates from grape fruits on PDA at the optimum temperature. These morphological and physiological characteristics agreed with those of Colletotrichum acutatum Simmonds ex Simmonds. The ripe rot symptom was reproduced on grapevine fruits after inoculation with the isolate of C. acutatum. These results lead us to propose that C. acutatum, as well as G. cingulata, causes ripe rot of grapevine.
Diseases of statice (Limonium sinuatum), causing symptoms including dwarfing, yellowing and witches' broom, occurred at Chiba and Saga Prefectures during the autumn of 1996. Electron microscopy revealed the presence of numerous phytoplasma particles in the sieve tubes in diseased plants. In transmission tests using Macrosteles striifrons and Scleroracus flavopictus, M. striifrons transmitted the phytoplasma agent. Host range of the two phytoplasma isolates was similar.