Effects of relative humidity, light intensity and photoperiod on the colony development of Erysiphe sp. on Rhododendron

Growth and reproduction by powdery mildew pathogens is generally inhibited by decreasing relative humidity. With Erysiphe sp. on Rhododendron cv. Elizabeth, the initial stages of colony development were adversely affected by reducing the relative humidity from 100% to 70 and 85%. No significant effects on secondary or tertiary hyphal development were detected. Light intensity and photoperiod both had considerable effect on the induced resistance response of the host. Over the initial 5 days of colonization there were no significant differences between any of the treatments. After 13 days, however, expansion of fungal colonies at 180 photosynthetic active radiation (PAR) was limited solely to the area initially infested by primary hyphae. By comparison, in colonies grown at 80 PAR regardless of day length, secondary and tertiary hyphae had extended beyond the area first colonized. These effects resulted in differing morphologies, small colonies of densely packed hyphae formed at 180 PAR compared with open spreading colonies at 80 PAR.     

Genetic diversity in populations of the fungi Phaeomoniella chlamydospora and Phaeoacremonium aleophilum on grapevine in France

Isolates of Phaeomoniella chlamydospora ( Phc ) and Phaeoacremonium aleophilum ( Pha ), two haploid, deuteromycetous fungi, were obtained from vines showing symptoms of esca disease in different localities in two French regions, and within a single vineyard in one of these regions. The population genetic structure was determined in both fungi using random amplified polymorphic DNA (RAPD) analysis. Populations of Phc showed similar levels of diversity at local and regional levels. The most frequent Phc haplotypes were found in every population, and the frequencies of positive alleles of markers were similar across populations. The hypothesis that recombination had occurred was rejected for the full set of samples, but not for the samples reduced to haplotypes, indicating that Phc may be a recombining species. Different features were identified in Pha populations. First, the southern population of Pha appeared more diverse than the south-western populations. Second, genetic differentiation was identified between Pha populations from southern and south-western regions for several RAPDs. Finally, in the southern population of Pha no evidence for recombination was obtained, even by reducing the sample to haplotypes. Within the single vineyard surveyed, several haplotypes of both fungi were recovered and randomly distributed. Thus different infection events appeared to have occurred on a low spatial scale. Data from this study showed that haplotypes of both fungi were distributed over long distances geographically, and that most of the vineyards surveyed were infested by more than one haplotype of Phc and Pha .     

Biological and sequence comparisons of Potato virus Y isolates associated with potato tuber necrotic ringspot disease

The biological and molecular relationships between a large number of Potato virus Y (PVY) isolates were examined, concentrating mainly on isolates associated with potato tuber necrotic ringspot disease (PTNRD). Following detailed analysis of the coat-protein gene, four main groups were identified which broadly corresponded to the phenotype of the different isolates. The groups comprised the ordinary strain (PVY^O), the necrotic strain (PVY^N), the C strain (PVY^C) and a group of recombinant (between ordinary and necrotic) isolates. In the latter group, all members were associated with PTNRD. However, four nonrecombinant isolates were also identified which were associated with PTNRD or tuber necrosis. Three were from tubers showing PTNRD symptoms in the field, while the fourth originated from symptomless tubers, but could cause necrotic rings on tubers under glasshouse conditions. The results show that although coat-protein recombination is always found associated with the PTNRD phenotype, some nonrecombinant isolates have very similar biological properties.     

Detection of Colletotrichum coccodes from soil and potato tubers by conventional and quantitative real-time PCR

Colletotrichum coccodes is the causal agent of the potato blemish disease black dot. Two PCR primer sets were designed to sequences of the ribosomal internal transcribed spacer (ITS1 and ITS2) regions for use in a nested PCR. The genus-specific outer primers (Cc1F1/Cc2R1) were designed to regions common to Colletotrichum spp., and the species-specific nested primers (Cc1NF1/Cc2NR1) were designed to sequences unique to C . coccodes . The primer sets amplified single products of 447 bp (Cc1F1/Cc2R1) and 349 bp (Cc1NF1/Cc2NR1) with DNA extracted from 33 European and North American isolates of C. coccodes. The specificity of primers Cc1NF1/Cc2NR1 was confirmed by the absence of amplified product with DNA of other species representing the six phylogenetic groups of the genus Colletotrichum and 46 other eukaryotic and prokaryotic plant pathogenic species. A rapid procedure for the direct extraction of DNA from soil and potato tubers was used to verify the PCR assay for detecting C. coccodes in environmental samples. The limit of sensitivity of PCR for the specific detection of C. coccodes when inoculum was added to soils was 3·0 spores per g, or the equivalent of 0·06 microsclerotia per g soil, the lowest level of inoculum tested. Colletotrichum coccodes was also detected by PCR in naturally infested soil and from both potato peel and peel extract from infected and apparently healthy tubers. Specific primers and a TaqMan fluorogenic probe were designed to perform quantitative real-time (TaqMan) PCR to obtain the same levels of sensitivity for detection of C. coccodes in soil and tubers during a first-round PCR as with conventional nested PCR and gel electrophoresis. This rapid and quantitative PCR diagnostic assay allows an accurate estimation of tuber and soil contamination by C. coccodes .     

Comparison of viral RNA populations of pathogenically distinct isolates of Citrus tristeza virus : application to monitoring cross-protection

The population of sequence variants of Citrus tristeza virus (CTV) isolates of different geographic origins and pathogenicity properties was characterized by single-strand conformation polymorphism (SSCP) analysis of cDNA of the genes p18, p13, p20 and p23. The mild isolates analysed here usually yielded a SSCP profile with two DNA bands, suggestive of a predominant sequence variant, whereas the SSCP profile of the most virulent isolates contained more than two DNA bands, indicating that their viral populations are likely to be more complex. The set of SSCP profiles of the four genes allowed identification of individual isolates, but no profile characteristic of a geographic area or a biogroup was found. Sweet orange plants singly inoculated with a mild or with a severe isolate yielded the SSCP profile characteristic of each isolate, whereas the SSCP profile of plants successively inoculated with both isolates was a composite of the two individual profiles. The SSCP profile of plants singly inoculated remained constant, but the profile of doubly inoculated plants varied with time. Plants in which the SSCP profile of the severe isolate became predominant showed stem pitting, and those in which the predominant profile corresponded to the mild isolate remained symptomless. The results indicate that SSCP analysis can be used to study changes in RNA populations of doubly inoculated plants and to monitor cross-protection between mild and severe isolates.     

Detection of airborne inoculum of Leptosphaeria maculans and Pyrenopeziza brassicae in oilseed rape crops by polymerase chain reaction (PCR) assays

The potential use of DNA-based methods for detecting airborne inoculum of Leptosphaeria maculans and Pyrenopeziza brassicae , both damaging pathogens of oilseed rape, was investigated. A method for purifying DNA from spores collected using Hirst-type spore samplers and detecting it using polymerase chain reaction (PCR) assays is described. For both pathogens, the sensitivities of the DNA assays were similar for spore-trap samples and pure spore suspensions. As few as 10 spores of L. maculans or P. brassicae could be detected by PCR and spores of both species could be detected against a background of spores of six other species. The method successfully detected spores of P. brassicae collected using spore traps in oilseed rape crops that were infected with P. brassicae. Leptosphaeria maculans spores were detected using spore traps on open ground close to L. maculans -infected oilseed rape stems. The potential use of PCR detection of airborne inoculum in forecasting the diseases caused by these pathogens is discussed.     

Evaluation of resistance to Phytophthora cinnamomi in seed-grown trees and clonal lines of Eucalyptus marginata inoculated in lateral branches and roots

Seed-grown trees and six clonal lines of 3·5–4·5-year-old Eucalyptus marginata (jarrah) growing in a rehabilitated bauxite mine site in the jarrah forest were underbark-inoculated on lateral branches (1995) or simultaneously on lateral branches and lateral roots (1996) with isolates of Phytophthora cinnamomi in late autumn. Individual seedlings from which the clonal lines were derived had previously been assessed as either resistant (RR) or susceptible (SS) to P. cinnamomi . At harvest, the acropetal lesion and colonization lengths were measured. Overall, the length of colonization in roots and branches was more consistent as a measure of resistance than lesion length, because colonization length recorded the recovery of P. cinnamomi from macroscopically symptomless tissue ahead of the lesion which, on some occasions, was up to 6 cm. In both trials, one RR clonal line was able to contain the P. cinnamomi isolates consistently, as determined by small lesion and colonization lengths in branches and roots. In contrast, the remaining two RR clonal lines used in both trials were no different from the SS line in their ability to contain lesions or colonization. These latter two RR lines may therefore not be suitable for use in rehabilitation of P. cinnamomi -infested areas. Differences in lesion and colonization lengths among P. cinnamomi isolates occurred only in the 1995 trial. Colonization and lesion lengths in branches were up to eight times greater in 1996 than in 1995, but the relative rankings of clonal lines were consistent between trials. Although colonization was always greater in branches than roots, the relative rankings of the lines were similar between branch and root inoculations. Branch inoculations are a valid option for testing the resistance and susceptibility of young jarrah trees to P. cinnamomi .     

Movement of Xanthomonas campestris pv. vitians in the stems of lettuce and seed contamination

Xanthomonas campestris pv. vitians , the causal agent of bacterial leaf spot of lettuce (BLS), can be seedborne, but the mechanism by which the bacteria contaminates and/or infects lettuce seed is not known. In this study, the capacity of X. campestris pv. vitians to enter and translocate within the vascular system of lettuce plants was examined. The stems of 8- to 11-week-old lettuce plants were stab-inoculated, and movement of X. campestris pv. vitians was monitored at various intervals. At 4, 8, 12 and 16 h post-inoculation (hpi), X. campestris pv. vitians was recovered from 2 to 10 cm above (depending on stem length) and 2 cm below the inoculation site. Xanthomonas campestris pv. vitians was also recovered from surface-disinfested stem sections of spray-inoculated plants. Together, these results are consistent with X. campestris pv. vitians invading and moving systemically within the vascular system of lettuce plants. To investigate the mechanism of seed contamination, lettuce plants at the vegetative stage of growth were spray-inoculated with X. campestris pv. vitians and allowed to develop BLS. Seed collected from these plants had a 2% incidence of X. campestris pv. vitians external colonization, but no bacteria were recovered from within the seed.     

Genetic variability within Phaeoisariopsis griseola from Central America and its implications for resistance breeding of common bean

The genetic and virulence variability of 112 isolates of Phaeoisariopsis griseola , collected from various locations in Central America, were studied using seven random amplified polymorphic DNA (RAPD) primers and 12 common-bean differential genotypes. Broad molecular diversity ( H  = 0·92) among isolates was found using RAPD markers. Fifty pathotypes were identified on 12 differential bean genotypes, 29 of which were represented by only one isolate. Only 18 pathotypes were found in two or more countries. Pathotype 63-63 was the most virulent and caused leaf spots on all 12 common-bean differential genotypes. Comparison of virulence phenotypes and RAPD profiles to known Andean P. griseola isolates confirmed that all isolates belonged to the Mesoamerican group. Pairwise comparison between individual RAPD loci showed that the majority were in gametic phase linkage disequilibrium, revealing that P. griseola maintains a genetic structure that is consistent with asexual reproduction. The molecular and virulence diversities of P. griseola isolates from Central America imply that using single resistance genes to manage angular leaf spot is inadequate and stacking resistance genes may be necessary to manage the disease effectively.