We carried out a quantitative detection of Candidatus Liberibacter asiaticus, the bacterium associated with the disease of huanglongbing, in the vector psyllid Diaphorina citri by using a TaqMan real-time PCR assay. The concentration of the bacterium was monitored at 5-day intervals for a period of 20 days after psyllids were exposed as fifth instar nymphs or adults to a Ca. L. asiaticus-infected plant for an acquisition access period of 24 h. When adults fed on Ca. L. asiaticus-infected plant, the concentration of the bacterium did not increase significantly and the pathogen was not transmitted to any citrus seedlings. In contrast, when psyllids fed on infected plant as nymphs, the concentration of the pathogen significantly increased by 25-, 360-and 130-fold from the initial acquisition day to 10, 15 and 20 days, respectively. Additionally, the pathogen was successfully transmitted to 67% of citrus seedlings by emerging adults. Our data suggested that multiplication of the bacterium into the psyllids is essential for an efficient transmission and show that it is difficult for adults to transmit the pathogen unless they acquire it as nymphs.

Turnip mosaic virus (TuMV), a species of the genus Potyvirus, occurs worldwide. Seventy-six isolates of TuMV were collected from around the world, mostly from Brassica and Raphanus crops, but also from several non-brassica species. Host tests grouped the isolates into one or other of two pathotypes ; Brassica (B) and Brassica-Raphanus (BR). The nucleotide sequences of the first protein (P1) and coat protein (CP) genes of the isolates were determined. One-tenth of the isolates were found to have anomalous and variable phylogenetic relationships as a result of recombination. The 5h-terminal 300 nt of the P1 gene of many isolates was also variable and phylogenetically anomalous, whereas the 380 nt 3h terminus of the CP gene was mostly conserved. Trees calculated from the remaining informative parts of the two genes of the non-recombinant sequences by neighbour-joining, maximum-likelihood and maximum-parsimony methods were closely similar, and so these parts of the sequences were concatenated and trees calculated from the resulting 1150 nt. The isolates fell into four consistent groups ; only the relationships of these groups with one another and with the outgroup differed. The ' basal-B ' cluster of eight B-pathotype isolates was most variable, was not monophyletic, and came from both brassicas and non-brassicas from southwest and central Eurasia. Closest to it, and forming a monophyletic subgroup of it in most trees, and similarly variable, was the ' basal-BR ' group of eight BR pathotype Eurasian isolates. The third and least variable group, the ' Asian-BR ' group, was of 22 BR-pathotype isolates, all from brassicas, mostly Raphanus, and all from east Asia mostly Japan. The fourth group of 36 isolates, the ' world-B ' group, was from all continents, most were isolated from brassicas and most were of the B-pathotype. The simplest of several possible interpretations of the trees is that TuMV originated, like its brassica hosts, in Europe and spread to the other parts of the world, and that the BR pathotype has recently evolved in east Asia.

Members of the Fusarium graminearum species complex are important cereal pathogens worldwide and belong to one of at least nine phylogenetically distinct species. We examined 298 strains of the F. graminearum species complex collected from wheat or barley in Japan to determine the species and trichothecene chemotype. Phylogenetic analyses and species-diagnostic polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLPs) revealed the presence and differential distribution of F. graminearum sensu stricto (s. str.) and F. asiaticum in Japan. F. graminearum s. str. is predominant in the north, especially in the Hokkaido area, while F. asiaticum is predominant in southern regions. In the Tohoku area, these species co-occurred. Trichothecene chemotyping of all strains by multiplex PCR revealed significantly different chemotype compositions of these species. All 50 strains of F. graminearum s. str. were of a 15- or 3-acetyl deoxynivalenol type, while 173 (70%) out of 246 strains of F. asiaticum were of a nivalenol type. The possibility of gene flow between the two species was investigated by use of 15 PCR-RFLP markers developed in this study. However, no obvious hybrids were detected from 98 strains examined, including strains collected from regions where both species co-occur.

Potyviruses have variable single-stranded RNA genomes and many show clear evidence of recombination. This report studied the distribution of recombination sites in the genomes of 92 isolates of the potyvirus Turnip mosaic virus (TuMV); 42 came from the international gene sequence databases and an additional 50 complete genomic sequences were generated from field samples collected in Europe and Asia. The sequences were examined for evidence of recombination using seven different sequence comparison methods and the exact position of each site was confirmed by sequence composition analysis. Recombination sites were found throughout the genomes, except in the small 6K1 protein gene, and only 24 of the genomes (26 %) showed no evidence of recombination. Statistically significant clusters of recombination sites were found in the P1 gene and in the CI/6K2/VPg gene region. Most recombination sites were bordered by an upstream (59) region of GC-rich and downstream (39) region of AU-rich sequence of a similar length. Correlations between the presence and type of recombination site and provenance, host type and phylogenetic relationships are discussed, as is the role of recombination in TuMV evolution.

The genomes of a representative world-wide collection of 32 Turnip mosaic virus (TuMV) isolates were sequenced and these, together with six previously reported sequences, were analysed. At least one-fifth of the sequences were recombinant. In phylogenetic analyses, using genomic sequences of Japanese yam mosaic virus as an outgroup, the TuMV sequences that did not show clear recombination formed a monophyletic group with four well-supported lineages. These groupings correlated with differences in pathogenicity and provenance; the sister group to all others was of Eurasian B-strain isolates from nonbrassicas, and probably represents the ancestral TuMV population, and the most recently 'emerged' branch of the population was probably that of the BR-strain isolates found only in east Asia. Eight isolates, all from east Asia, were clear recombinants, probably the progeny of recent recombination events, whereas a similar number, from other parts of the world, were seemingly older recombinants. This difference indicates that the presence of clear recombinants in a subpopulation may be a molecular signature of a recent 'emergence'.