The potential use of the ribosomal DNA internal transcribed spacer (ITS) sequences in understanding the phylogeny and systematics of Colletotrichum species has been evaluated. Sequence data from a limited number of isolates revealed that in Colletotrichum species the ITS 1 region (50.3% variable sites) shows a greater degree of intra- and inter-specific divergence than ITS 2 (12.4% variable sites). Nucleotide sequences of the ITS 1 region from 93 isolates representing 18 Colletotrichum species were determined. Data for 71 of these isolates where molecular and morphological identities concurred were used for phylogenetic analysis. The size of the ITS 1 region varied from 159 to 185 base pairs. Maximum intraspecific divergence was recorded with C. acutatum (5.8%), and C. capsici showed the greatest level of interspecific divergence (8.9-23.3%). Parsimony and distance analyses gave similar tree topologies. The bootstrapped consensus parsimony tree divided the 18 Colletotrichum species into six phylogenetic groups, designated 1-6. These groups, however, are not congruent with species clusterings based on spore shape. For example, the straight cylindrical spored species were represented both in groups 1 and 6; group 6 also included the falcate fusiform spored species C. capsici. The molecular evidence suggests refinement of the species concepts of some of the taxa examined. In group 6, divergence between C. gloeosporioides and C. fuscum (0.6-3.0%) or C. kahawae (0.6-3.0%) or C. fragariae (0.6-4.2%) overlap the divergence (3.6%) within C. gloeosporioides. It is suggested that C. fuscum as well as C. kahawae and C. fragariae fall within the group species C. gloeosporioides. ITS 1 data enabled clear distinction (7.1%) of Colletotrichum isolates from maize and sorghum into C. graminicola and C. sublineolum, respectively (group 2). Species such as C. acutatum, C. coccodes, C. dematium, and C. trichellum can be clearly distinguished based on ITS 1 sequence divergence, but C. destructivum cannot be confidently separated (98% homology) from C. linicola. Colletotrichum dematium f. truncatum is distinct (12.9%) from C. dematium and should probably be called C. truncatum.
An oligonucleotide primer (CaInt 2) was synthesized from the variable internal transcribed spacer (ITS) 1 region of ribosomal DNA (rDNA) from Colletotrichum acutatum. PCR with primers CaInt2 and ITS4 (from a conserved sequence of the rDNA) amplified a 490 bp fragment from several isolates of C. acutatum but not from other members of the genus Colletotrichum. Amplification of this fragment was achieved from 100 fg of fungal DNA. These primers amplified a fragment of the same size from DNA extracted from strawberry tissues infected by C. acutatum. Southern hybridization analysis confirmed the 490 bp fragment from C. acutatum DNA and infected strawberry to be identical. The species‐specific primer (CaInt2) developed in this work could be used for the accurate identification of C. acutatum and its detection on other host plants.
The aggressiveness of 20 Northern Ireland single-lesion isolates of Phytophthora infestans was compared following their inoculation onto detached leaflets of three potato cultivars chosen on the basis of their differing levels of race-nonspecific resistance to late blight: Bintje (highly susceptible); Cara (moderately resistant); and Stirling (more resistant). Five isolates from outside Northern Ireland were included for comparative purposes: two from the Republic of Ireland; two from the USA (representing the US-1 and US-8 clonal lineages); and one from Mexico. To control the variation between tests, a balanced incomplete block design was used, as opposed to either a complete block design or the method of inclusion of standard isolates, where such variation would have increased the error. Highly significant variation for disease parameters, including latent period, infection frequency, area under the lesion expansion curve (AULEC) and sporulation capacity, was found between isolates. These differences were much more marked on the cultivars exhibiting higher levels of race-nonspecific resistance. There was a significant interaction between isolate and cultivar for all parameters assessed and, overall, no one isolate was the most aggressive across all three potato cultivars. However, a group comprising seven of the 20 Northern Ireland isolates was consistently found to exhibit the highest levels of aggression towards all three cultivars for each of the disease parameters. These results demonstrate that significant variation for foliar aggressiveness exists within the Northern Ireland population of P. infestans , and indicate the importance of selecting appropriately aggressive isolates for evaluation of host resistance to late blight within breeding programmes.
Three nurseries produced apple rootstocks (M9) and budwood (cv. Royal Gala), which they exchanged at the end of the first year. Each nursery then budded its own budwood onto the rootstocks it had produced and that from the other two nurseries. Budded trees were grown on for a further year before being planted at HRI, East Malling in southern England; NIHPBS, Loughgall in Northern Ireland; and ADAS, Rosemaund in the West Midlands of England. Canker development was monitored twice a year. The position of the infected trees within the orchard was recorded, as was the position of the canker on each tree (main-stem or peripheral). Nectria galligena was isolated from representative cankers and analysed using molecular techniques. At the sites in Northern Ireland and HRI there was a strong positional effect, especially of peripheral cankers, indicating that most of the inoculum was external and had been spread from neighbouring orchards. There was little or no positional effect on main-stem cankers at any of the three sites. The proportions of different isolates taken from peripheral cankers was different in Northern Ireland from that in England, suggesting different populations associated with the geographic areas. In contrast, the populations of N. galligena obtained from main-stem cankers were very similar in England and Northern Ireland. It was concluded that a small proportion of trees developing canker were infected during propagation, with no symptom development until after planting. In a second trial it was demonstrated that trees infected during the propagation phase, and particularly at budding and heading back, could develop canker up to 3 years later. While it is clear that some canker developing in the orchard can be associated with the nursery of production, in climatic conditions conducive to the formation and dissemination of conidia, inoculum from surrounding infected orchards is the primary source of the pathogen. Aerial spread is therefore an essential element of the epidemiology of N. galligena , and its control is a crucial part of any canker-control programme.
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