Showing papers in "Plant Pathology in 2002"

Potato diseases caused by soft rot erwinias : an overview of pathogenesis

Abstract: Three soft rot erwinias, Erwinia carotovora ssp. carotovora, E. carotovora ssp. atroseptica and E. chrysanthemi are associated with potatoes causing tuber soft rot and blackleg (stem rot). Latent infection of tubers and stems is widespread. As opportunistic pathogens, the bacteria tend to cause disease when potato resistance is impaired. Pathogenesis or disease development in potato tubers and stems is discussed in terms of the interaction between pathogen, host and environment, microbial competition and recent findings on the molecular basis of pathogenicity. Emphasis is placed on the role of free water and anaerobiosis in weakening tuber resistance and in providing nutrient for erwinias to multiply. Blackleg symptoms are expressed when erwinias predominate in rotting mother tubers, invade the stems and multiply in xylem vessels under favourable weather conditions. Soft rot erwinias tend to out-compete other bacteria in tuber rots because of their ability to produce larger quantities of a wider range of cell wall-degrading enzymes. However, despite extensive studies on their induction, regulation and secretion, little is known about the precise role of the different enzymes in pathogenesis. The putative role of quorum-sensing regulation of these enzymes in disease development is evaluated. The role certain pathogenicity-related characters, including motility, adhesion, siderophores, detoxifying systems and the hrp gene complex, common to most bacteria including symbionts and saprophytes, could play in latent and active infections is also discussed.

Disease complexes involving plant parasitic nematodes and soilborne pathogens

Abstract: This review discusses the mechanisms underlying synergistic interactions between phytophagous nematodes and soilborne pathogens, and identifies biotic and abiotic factors affecting these interactions. Approaches towards the resolution and management of nematode‐pathogen complexes are considered and discussed.

Clonality and long‐distance migration of Puccinia striiformis f.sp. tritici in north‐west Europe

Abstract: The biotrophic fungus Puccinia striiformis f.sp. tritici, a basidiomycete that causes yellow rust on wheat, is spread by wind-dispersed spores. Analysis of amplified fragment length polymorphism (AFLP) variation showed that the fungus frequently migrates between the UK, Germany, France and Denmark. There is no biological evidence for sexual or parasexual reproduction under natural conditions, and this was supported by the lack of recombination, as revealed by AFLP, over the time and area represented by the samples in this study. A phylogeographic analysis revealed that there was effectively a single, clonal population in the four countries, up to 1700 km apart, consistent with a ‘continent-island’ model in which Denmark is the recipient of migrants from other countries. In five cases, specific pathogen clones were dispersed between the UK and Denmark, and on at least two recent occasions clones were also spread from the UK to Germany and France, causing outbreaks of yellow rust on wheat cultivars that were previously resistant to the disease in these countries. The agronomic consequences of migration were enhanced because of the limited genetic diversity for yellow rust resistance in wheat cultivars in the area. These results demonstrate that long-distance migration of pathogen clones, coupled with low diversity in the host species, may cause previously useful resistance genes to become ineffective for disease control on a continental scale.

Evolution of gene‐for‐gene systems in metapopulations: the effect of spatial scale of host and pathogen dispersal

Abstract: The concept of gene-for-gene coevolution is a major model for research on disease resistance in crop plants. However, few theoretical or empirical studies have examined such systems in natural situations, and as a consequence, there is little knowledge of how spatial effects are likely to influence the evolution of host resistance and pathogen virulence in gene-for-gene interactions. In this work, a simulation approach was used to investigate the epidemiological and genetic consequences of varying host and pathogen dispersal in metapopulation situations. The results demonstrate clear impacts of dispersal distance on the total number of host and pathogen genotypes that are maintained, as well as on genetic variation at individual host resistance and pathogen virulence loci. Several other important results also emerged from this study. In contrast to the predictions of many earlier nonspatial models, so-called ‘super-races’ of pathogens do not always evolve and dominate, indicating that it is not necessary to assume costs of resistance or virulence to maintain high levels of polymorphism in biologically realistic situations. The rate of evolution of both resistance and virulence depend on the scale of dispersal, with greater mixing (as a function of dispersal scale) resulting in a faster approach to a dynamic endpoint. The model in this paper also predicts that, despite the greater total genotypic diversity of pathogens across the metapopulation, variation in host resistance will generally be greater than variation in pathogen virulence within local populations.

Development of conventional and quantitative real‐time PCR assays for the detection and identification of Rhizoctonia solani AG‐3 in potato and soil

Abstract: A specific and sensitive PCR assay was developed for the detection and identification of Rhizoctonia solani AG-3, the main causal pathogen of stem canker and black scurf of potato. A conventional primer set (Rs1F2 and Rs2R1) was designed from the nuclear ribosomal internal transcribed spacer (ITS1 and ITS2) regions of R. solani. Following PCR amplification, a 0·5-kb product was amplified from DNA of all isolates of AG-3 using primers Rs1F2 and Rs2R1. No product was amplified when DNA from isolates belonging to a range of other R. solani anastomosis groups or from a selection of other potato pathogens was tested, confirming the specificity of the primers for AG-3 only. Rhizoctonia solani AG-3 was also detected in potato tissue with varying black scurf severity, and in soil inoculated with sclerotia of R. solani to a minimum detection level of 5 × 10−4 g sclerotia/g soil. In addition, specific primers RsTqF1 (based on the Rs1F2 sequence) and RsTqR1, and a TaqMan™ fluorogenic probe RQP1, were designed to perform real-time quantitative (TaqMan) PCR. The conventional PCR and real-time PCR assays were compared and combined with direct DNA extraction from soil and a seed-baiting method to determine the most reliable method for the detection and quantification of AG-3 in both artificially inoculated field soil and naturally infested soils. It was shown that direct DNA extractions from soil could be problematic, although AG-3 was detectable using this method combined with the real-time PCR assay. The amplification of Rhizoctonia solani by seed baiting increased the sensitivity of the assay compared with direct extraction of DNA from the soil, and AG-3 was detectable in artificially inoculated and naturally infested soils when seed baiting was combined with either the conventional PCR or the real-time PCR assay. The potential for using these rapid and quantitative AG-3-specific assays to address epidemiological questions and as tools for decision-making in disease management is discussed.

Phenotypic and genotypic structure of Phytophthora infestans populations on potato and tomato in France and Switzerland.

Abstract: A total of 134 isolates of Phytophthora infestans were collected from potato and 42 from tomato fields in Switzerland and France in 1996 and 1997, and compared with isolates from other countries. The structure of the populations was analysed phenotypically and genotypically, and associated to geographical, seasonal and host origin. Phenotype characteristics of the isolates included mating type; sensitivity to phenylamide fungicides; virulence on potato differentials; and pathogenic fitness. Genotypes were assessed for mitochondrial DNA haplotype with restriction fragment length polymorphism-polymerase chain reaction (RFLP-PCR) as well as amplified fragment length polymorphism (AFLP) and simple sequence repeats or microsatellites (SSR). The majority (96%) of isolates originating from potato were the A1 mating type, whereas half the isolates collected from tomato were A2 mating type. Isolates with sensitive, intermediate and resistant responses to the phenylamide fungicide metalaxyl-M were detected in the populations. Isolates from potato represented races with highly complex virulence spectra, whereas those from tomato belonged to simple races. The pathogenic fitness of isolates was highest on the host of origin, and was significantly reduced for isolates from potato on tomato. One of the four haplotypes, Ia, dominated the population (93% of isolates). Among isolates collected from potato, 15 different SSR genotypes were detected of which two, A-03 and A-06, dominated the population. From tomato, 11 SSR genotypes were found of which four, A-03, B-03, D-03 and F-01, formed the major proportion of the population. Every ninth and fourth isolate from potato and tomato, respectively, represented a different SSR genotype. Four genotypes were isolated from both hosts, whereas 11 and seven genotypes, respectively, originated exclusively from either potato or tomato. The SSR genotype D-02, represented by the ‘old’ Ib haplotype, was still detected in a few isolates in the current population, and in older reference isolates from different countries. The SSR genotype was not associated with mating type or sensitivity to phenylamide fungicides. A total of 40 AFLP genotypes were distinguished among the isolates, every second isolate representing another genotype. The diverse phenotypic and genotypic structure of the current field populations in Europe suggests that they may have evolved from local processes including sexual recombination, host preference and selection rather than through long-distance migration.

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

Abstract: 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 genusspecific 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 .

In vitro inhibition of soil microorganisms by 2-phenylethyl isothiocyanate

Abstract: Isothiocyanates (ITCs) are produced by the enzymatic hydrolysis of glucosinolates in cruciferous plants and can kill fungi, oomycetes and bacteria. The effect of 2-phenylethyl ITC (2-PE ITC), the main ITC liberated from the roots of canola, was tested in vitro on a range of fungi, oomycetes and bacteria. Bacteria were generally more tolerant than the eukaryotic pathogens to 2-PE ITC, although both groups showed considerable variability in response (ED90 ranging from 0·005 to 1·5 mm for eukaryotes, and from 0·33 to greater than 3·34 mm for complete inhibition of bacterial growth). While intraspecies variability was low, interspecies variability was high within some genera. Amongst the eukaryotes, Trichoderma spp. were the most tolerant to 2-PE ITC, while other genera, including Aphanomyces, Gaeumannomyces, Phytophthora and Thielaviopsis, were very sensitive. A range of responses was exhibited by Pythium spp. and the different anastomosis groups of Rhizoctonia solani. A simple laboratory screening may be an effective way to identify candidate soilborne pathogens for control in rotation cropping systems which include a Brassica crop phase.

Following the dynamics of strobilurin resistance in Blumeria graminis f.sp. tritici using quantitative allele-specific real-time PCR measurements with the fluorescent dye SYBR Green I

Abstract: Strobilurin-resistant isolates of Blumeria (Erysiphe) graminis f.sp. tritici, the cause of wheat powdery mildew, were more than 10-fold less sensitive to azoxystrobin than sensitive isolates. In all resistant isolates, a mutation resulting in the replacement of a glycine by an alanine residue at codon 143 (G143A) in the mitochondrial cytochrome b gene was found. Allele-specific primers were designed to detect this point mutation in infected wheat leaves. Using quantitative fluorescent allele-specific real-time polymerase chain reaction (PCR) measurements, strobilurin-resistant A143 alleles could be detected amongst strobilurin-sensitive G143 alleles at a frequency of at least 1 in 10 000, depending on the amount of target and nontarget DNA. Most isolates tested were dominant homoplasmic for either the A143 or G143 allele, although mixed populations of alleles could be detected in some isolates. In some of these isolates, strobilurin resistance was not always stable when they were maintained for many generations in the absence of selection. The allele-specific real-time PCR assay was also used to follow the dynamics of A143 alleles in field populations of B. graminis f.sp. tritici before and after application of fungicides. As expected, the A143 allele frequency only increased under selection pressure from a strobilurin fungicide. After three sprays of azoxystrobin, a pronounced selection for the strobilurin-resistant allele, with an increase in average frequency from 2·2 to 58%, was measured. The use of quantitative real-time PCR diagnostics for early detection of fungicide resistance genes at low frequency, coupled with risk evaluation, will be invaluable for further resistance risk assessment and validation of antiresistance strategies.

Antifungal vapour-phase activity of allyl-isothiocyanate against Penicillium expansum on pears

Abstract: The fungitoxicity of allyl-isothiocyanate (AITC) vapour against Penicillium expansum, the causal agent of blue mould on pears (cvs Conference and Kaiser), was evaluated. The best control of blue mould was obtained by exposing fruits for 24 h in a 5 mg L−1 AITC-enriched atmosphere, the extent of control depending on the inoculum density. Lesion diameter was inversely related to AITC concentration. In treated fruits the percentage of infected wounds increased with conidial concentration, with fewer than 20% affected at 1 × 103 conidia mL−1 to almost 80% at 1 × 106 conidia mL−1. In comparison, >98% of wounds were infected in untreated fruits irrespective of conidial concentration. AITC treatments were effective up to 24 h after inoculation for Conference and 48 h for Kaiser. AITC treatments also controlled a thiabendazole-resistant strain of P. expansum, reducing the incidence of blue mould by 90% in both cultivars. The use of AITC produced from pure sinigrin or from Brassica juncea defatted meal may be an economically viable alternative to synthetic fungicides against P. expansum.

Sugarcane yellow leaf virus and sugarcane yellows phytoplasma: elimination by tissue culture

Abstract: Yellow leaf syndrome (YLS) is a recently reported disease of sugarcane, characterized by yellowing of the leaves. Two pathogens: a virus, Sugarcane yellow leaf virus (SCYLV); and a phytoplasma, sugarcane yellows phytoplasma (SCYP), are associated with the disease. The use of tissue culture was investigated as a means to eliminate both SCYLV and SCYP from exotic varieties undergoing quarantine in Mauritius. Of 43 varieties in quarantine, 28 were infected with SCYLV and 19 with SCYP when checked by RT–PCR and nested PCR, respectively. Seventeen varieties were coinfected with both pathogens. Thirty infected varieties were induced to form callus in vitro using leaf rolls as explants. After two subcultures, 19 varieties were successfully regenerated and tested for SCYLV and SCYP. No pathogen could be detected in any regenerated plantlets. All the regenerated plants remained free from both SCYLV and SCYP over a period of 1 year in the glasshouse, confirming that the pathogens had been eliminated by tissue culture.

Induction of systemic disease resistance and pathogen defence responses in Asparagus officinalis inoculated with nonpathogenic strains of Fusarium oxysporum

Abstract: The ability of nonpathogenic isolates of Fusarium oxysporum (np Fo ) to induce systemic resistance and defence responses against subsequent challenge with a pathogenic strain of F. oxysporum f. sp. asparagi ( Foa ) was examined in Asparagus officinalis . In a split-root experiment, roots inoculated with np Fo exhibited a hypersensitive response and those subsequently inoculated with Foa displayed resistance. Induction of systemic resistance in np Fo -treated plants led to significantly fewer necrotic lesions ( P = 0·05) and reduced Foa disease severity compared with plants not treated with np Fo . In hyphal-sandwich root inoculation experiments, activities of peroxidase and phenylalanine ammonia-lyase and lignin content were higher in np Fo -treated plants and increased more rapidly than in np Fo -untreated plants after Foa inoculation. Antifungal activity (inhibition of fungal spore germination and germ-tube growth) from exudates of roots inoculated with Foa were observed for np Fo -treated plants but not for np Fo -untreated plants. Thus, isolates of np Fo may function as inducers of systemic acquired resistance (SAR) and defence responses against Foa invasion in A. officinalis .

First report of the pitch canker fungus, Fusarium circinatum, on pines in Chile

Abstract: Pitch canker, caused by Gibberella circinata (Fusarium circinatum), is currently regarded as one of the most serious diseases of pines in the world. The disease was first discovered in the south-eastern United States in the early 1900s and subsequently, has caused sporadic occurrences of severe disease in that area. More recently, the disease appeared in California where it is currently causing a serious epidemic on native Pinus radiata, which is of international concern (Gordon et al., 2001). Pinus radiata forms the basis of extensive areas of exotic forest plantation in many countries in the Southern Hemisphere. For this reason, pitch canker is viewed as one of the greatest threats to globally relevant forest industries in these areas. Consequently, various international programmes have been established in an attempt to reduce this threat (Devey et al., 1999). During the course of recent surveys in the Concepcion area of Chile, dying P. radiata plants have been discovered in both containerized and open rooted clonal hedges. Plants appear to die rapidly and typically display resin exudation from the root collar areas and pitch-soaked wood associated with these lesions. Isolations from symptomatic tissue have consistently yielded cultures of Fusarium belonging to the Fusarium subglutinans species complex. Isolates have sterile hyphal coils strongly reminiscent of F. circinatum. Single conidial cultures paired with mating tester strains of the ‘H’ mating population (Britz et al., 1999) consistently showed sexual compatibility. To further confirm their identity, a PCR-RFLP test based on histone H3 gene sequences (Steenkamp et al., 1999) was used on six isolates to compare them with members of the F. subglutinans species complex. This technique reliably distinguishes F. circinatum from other species in this complex. Results confirmed morphological comparisons and mating compatibility tests showing that the fungus associated with dying plants in Chile is F. circinatum. Although F. circinatum is causing damage to P. radiata plants in Chilean nurseries, it has as yet, not been found on trees in plantations. In this sense, the situation is similar to that in South Africa, where the pathogen causes serious damage to seedlings in nurseries but is not associated with typical symptoms of pitch canker. The fact that P. radiata is highly susceptible to infection by F. circinatum and that this tree forms the basis of the local forestry is of significant concern. This concern is heightened by the fact that various insect pests of P. radiata are present in Chile that are capable of providing infection sites for the pitch canker fungus. Consequently, various strategies linked to tree breeding and selection have been established to minimise the long-term threat of pitch canker in Chile.

Effects of calcium on biocontrol activity of yeast antagonists against the postharvest fungal pathogen Rhizopus stolonifer

Abstract: Calcium chloride (2% w/v) significantly inhibited the growth of the pathogen Rhizopus stolonifer, but did not affect the colony-forming units (CFU) of yeasts Candida guilliermondii and Pichia membranefaciens in potato dextrose broth. The concentration of yeast suspension influenced spore germination and germ tube growth of R. stolonifer in vitro, as well as disease incidence and lesion development in fruits. There were significant negative relationships between the suspension concentrations of the yeasts and the growth as well as infectivity of the pathogen. The addition of calcium resulted in lower spore germination rates and slower growth of germ tubes in vitro, as well as in lower disease incidences and smaller lesion diameters compared with treatments with yeast antagonists alone. When yeast cell suspensions reached a concentration of 5 × 108 CFU mL−1, growth of the pathogen was completely limited in vitro, and no infection was found in peach and nectarine fruits treated with or without calcium.

The recent history of Puccinia striiformis f.sp. tritici in Denmark as revealed by disease incidence and AFLP markers

Abstract: Disease observations and amplified fragment length polymorphism (AFLP) markers were used to study recent developments in the Puccinia striiformis f.sp. tritici population in Denmark. The fungus appeared spontaneously at 10 locations in Denmark in 1997 after it was not observed under natural conditions in 1996. The pattern of disease development and prevailing winds suggested that the fungus reappeared by airborne spores from the south or west. In 1998, disease incidence was more evenly distributed throughout the country. Forty-eight single lesion isolates were collected from most crops where the disease was observed in these years; all except one from 1997 belonged to two pathotypes that were not previously detected in the country, and both possessed the newly discovered Yr17 virulence. The isolates were characterized with AFLP markers together with 28 isolates representing eight of 13 pathotypes observed prior to 1996. Initial screening of 240 PstI/MseI AFLP primer combinations on four isolates showed that a primer combination, on average, revealed 0·4 polymorphisms between any isolate pair. A selection of 21 primer combinations resulted in 28 AFLP markers, which revealed 16 AFLP phenotypes among all 76 isolates. The two Yr17-virulent pathotypes consisted of three AFLP phenotypes, which were observed in both 1997 and 1998; the two most frequent AFLP phenotypes occurred at most sampling locations and often within the same crop. AFLP diversity was larger among samples collected prior to 1996, and also in this period most AFLP phenotypes were observed at different sampling locations. These results are consistent with the features of an entirely asexually reproducing pathogen dispersed by aerial spores across large areas.

Colonization of winter oilseed rape tissues by A/Tox+ and B/Tox0 Leptosphaeria maculans (phoma stem canker) in France and England

Abstract: The colonization of winter oilseed rape plants and epidemiology of phoma stem canker differed between A/Tox+ and B/Tox0Leptosphaeria maculans. In France and England, where plant colonization was investigated during two and three growing seasons, respectively, there was a difference in timing of leaf infection; A/Tox+L. maculans was predominant on leaves in the autumn (October/November) but there was an increase in the incidence of B/Tox0 in the winter (January/February). In May, June and July both species could be isolated from all external parts of the plant (root to the upper stem) and all crown (stem base) tissues, although they differed in their distribution. At the root and crown, A/Tox+L. maculans was predominant and was located throughout the cortex, wood and pith tissues, but the rarer B/Tox0 was located mainly in the cortex. Approximately equal numbers of A/Tox+ and B/Tox0 isolates were obtained from the upper stem – there was a greater proportion of B/Tox0 isolates than at the crown. In England, after harvest in 1999 and 2000, pseudothecia on the lignified tap root and crown tissues produced predominantly A/Tox+ ascospores (94%), while pseudothecia higher up the stem produced more B/Tox0 ascospores (60%) than A/Tox+ ascospores (40%). The timing of the onset of leaf spotting, earlier in the season for A/Tox+ than B/Tox0L. maculans, and the predominance of mycelium of A/Tox+ at the crown are consistent with the assumption that A/Tox+ is more likely to cause the most damaging stem cankers than B/Tox0L. maculans. Identification as A/Tox+ or B/Tox0 by cultural characteristics differed only slightly (2·3%) from identification by molecular techniques.

Biochemical and molecular characterization of Bacillus amyloliquefaciens, B. subtilis and B. pumilus isolates with distinct antagonistic potential against Xanthomonas campestris pv. campestris

Abstract: Fifty-one Bacillus isolates were characterized by fatty acid methyl ester (FAME) analysis; universal primer polymerase chain reaction (UP-PCR) fingerprinting; production of secondary metabolites and antagonistic activity against Xanthomonas campestris pv. campestris (causal agent of black rot in cabbage) in vitro and in vivo. Based on FAME analysis and/or PCR fingerprinting, the isolates were clustered into three different groups, named as Bacillus amyloliquefaciens, B. subtilis and B. pumilus. Seed treatment with Bacillus spp. generally reduced germination of seeds and incidence of black rot, but no relationship was found between the results of in vitro and in vivo experiments. The B. amyloliquefaciens group contained isolates that were generally the most effective at reducing attack of black rot in vivo. The metabolic profiles of these isolates suggested that they produced surfactin, iturin, bacillomycine and/or azalomycin F. Isolates belonging to the B. subtilis group were mostly able to synthesize surfactin and arthrobactin. Surfactin, amphomycin, arthrobactin and valinomycin were generally found in culture extracts of isolates belonging to the B. pumilus group. No effect on growth of the pathogen was detected when the activity of filtered culture extracts and selected metabolites produced by the three different Bacillus species was tested in vitro against X. c. pv. campestris. However, inhibition was seen when bacterial liquid cultures were used. When the ability to colonize cabbage endophytically was examined for seven selected isolates with different antagonistic potential against black rot, it was found that the ability was related to the species and not to the antagonistic activity of the isolates.

Bayesian analysis of plant disease prediction

Abstract: Rule-based systems for the prediction of the occurrence of disease can be evaluated in a number of different ways. One way is to examine the probability of disease occurrence before and after using the predictor. Bayes's Theorem can be a useful tool to examine how a disease forecast (either positive or negative) affects the probability of occurrence, and simple analyses can be conducted without knowing the risk preferences of the targeted decision makers. Likelihood ratios can be calculated from the sensitivity and specificity of the forecast, and provide convenient summaries of the forecast performance. They can also be used in a simpler form of Bayes's Theorem. For diseases where little or no prior information on occurrence is available, most forecasts will be useful in that they will increase or decrease the probability of disease occurrence. For extremely common or extremely rare diseases, likelihood ratios may not be sufficiently large or small to substantially affect the probability of disease occurrence or make any difference to the actions taken by the decision maker.

Gene genealogies support Fusarium oxysporum f. sp. ciceris as a monophyletic group

Abstract: Fusarium oxysporum f. sp. ciceris (Foc), the causal agent of fusarium wilt of chickpea, consists of two pathotypes ( yellowing and wilting) and eight races (races 0, 1B/C, 1A and 2-6) of diverse geographical distribution. Six Foc isolates, one each of races 0, 1B/C, 1A, 4, 5 and 6, representing the two pathotypes and the geographical range of the pathogen, showed identical sequences in introns of the genes for translation elongation factor la (EF1α), β-tubulin, histone 3, actin and calmodulin. Eleven additional Foc isolates representative of all races, pathotypes and geographical range, and three isolates of F. oxysporum (Fo) nonpathogenic to chickpea were further analysed for sequence variation in the EF1α gene. All isolates pathogenic to chickpeas shared an identical EF1 a gene sequence, which differed from that shared by the three Fo isolates nonpathogenic to chickpea. EF1α gene sequences from the 17 Foc isolates and the three Fo isolates were compared with 24 EF1α gene sequences in GenBank from isolates of 11 formae speciales of F. oxysporum by parsimony analysis. Foc isolates formed a grouping distinct from other formae speciales and nonpathogenic isolates. These results indicate that F. oxysporum f. sp. ciceris is monophyletic.

Molecular identification of Colletotrichum gloeosporioides causing yam anthracnose in Nigeria

Abstract: Four forms of Colletotrichum representing three distinct virulence phenotypes were found associated with foliar anthracnose of yam in Nigeria: the highly virulent (= severity of disease) slow-growing grey (SGG); the moderately virulent fast-growing salmon (FGS); the weakly virulent fast-growing grey (FGG); and the moderately virulent fast-growing olive (FGO) morphotype. Isolates of the four forms were identified as C. gloeosporioides, based on morphology. The reaction of monoconidial cultures on casein hydrolysis medium (CHM), PCR-RFLP and sequence analysis of the internal transcribed spacer region of the ribosomal DNA (ITS1-5·8S-ITS2) were used to establish the identity of the yam anthracnose pathogen(s). All yam isolates were distinguished from C. acutatum by the absence of protease activity on CHM. On ITS PCR and enzymatic digestion of PCR products, all FGS, FGO and SGG isolates produced RFLP patterns identical to those of C. gloeosporioides reference isolates, while FGG isolates revealed unique ITS RFLP banding patterns. Sequence analysis of the ITS1 region and of the entire ITS region revealed that SGG, FGS and FGO isolates were highly similar (98–99% nucleotide identity) and showed 97–100% identity to C. gloeosporioides. Less than 93% similarity of these fungal isolates to reference C. acutatum and C. lindemuthianum isolates was observed. The molecular study confirmed that foliar anthracnose of yam is caused by C. gloeosporioides. While a high similarity was found among most C. gloeosporioides fungi from yam, isolates of the FGG form did not cluster with any previously described Colletotrichum species, and probably represent a distinct species.

Induction of resistance in cocoa against Crinipellis perniciosa and Verticillium dahliae by acibenzolar-S-methyl (ASM)

Abstract: The benzothiadiazole compound acibenzolar-S-methyl (ASM) was assessed as an inducer of resistance against Crinipellis perniciosa, agent of witches’ broom, and Verticillium dahliae, agent of vascular wilt, both on cocoa. ASM induced a reduction in incidence of witches’ broom of up to 84·5% when sprayed 30 days before inoculation on cocoa seedlings of cv. Catongo. ASM also induced a reduction in severity of Verticillium wilt to 55·4% on cv. Theobahia. For both pathosystems, effects of dose on disease were not clearly observed. The efficacy of the inducer increased with the interval between sprayings and the respective inoculations with the pathogens. In another experiment, the effect of ASM on the control of witches’ broom on cocoa seedlings was compared with that of cuprous oxide and tebuconazole, all sprayed 15 days before inoculation. ASM reduced disease incidence by 60·1% compared with the inoculated control. ASM was superior to tebuconazole, and there was also a tendency for ASM to be better than cuprous oxide. To understand the mechanism of action of ASM as an inducer of resistance, alterations in the levels of total phenolics, polyphenol oxidases and peroxidases were evaluated 3, 15 and 30 days after spraying of seedlings of cv. Catongo. Enzyme activities from seedlings of cv. Theobahia were evaluated 30 days after spraying. On cv. Catongo, no significant differences in total phenolic content and polyphenol oxidase activity were detected after spraying. However, an increase in peroxidase activity was detected at all times of evaluation. On cv. Theobahia, significant increases in activities of peroxidase and polyphenol oxidase were detected, indicating that defence responses due to ASM were dependent on host genotype.

Foliar aggressiveness of Northern Ireland isolates of Phytophthora infestans on detached leaflets of three potato cultivars

Abstract: 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.

Effects of timing of Leptosphaeria maculans ascospore release and fungicide regime on phoma leaf spot and phoma stem canker development on winter oilseed rape (Brassica napus) in southern England

Abstract: At Rothamsted during 1997/98, 1998/99 and 1999/2000 winter oilseed rape growing seasons, numbers of air-borne ascospores of Leptosphaeria maculans were often > 4 m−3 from autumn (September/October) to spring (April/May), while few or no ascospores were detected during the summer. Mature pseudothecia were generally not observed on debris of the previous crop until September. One-year-old debris (harvested in July 1998) had 95% discharged and 5% mature pseudothecia in August 1999, but by 15 September new pseudothecia (of which 30% were mature) were observed and the first increase in air-borne ascospores (> 4 m−3) occurred. Phoma leaf spotting appeared in untreated field plots 14–25 days after the first increase in air-borne ascospores in autumn. The fungicide mixture difenoconazole plus carbendazim decreased the incidence of new leaf lesions for 1 month after application in autumn and for 2 months in mid-winter. When L. maculans was isolated from infected leaves, the growth rate of isolates from leaves to which fungicide was applied was less than that of those from untreated leaves. Foliar applications of fungicide to field plots in the autumn and winter not only decreased the incidence of crown cankers but also reduced the rate of canker development on stem bases in the spring and early summer (when severity of crown cankers increased linearly with time). In untreated crops, when phoma leaf spots appeared early in the autumn, crown cankers developed early in the spring but only became severe enough before harvest to reduce yield greatly in 1997/98. Yield loss was associated with crown cankers that girdled more than half of the stem by harvest (mean severity > 3 on a 0–5 scale). Infections of new leaves produced after stem extension, from January onwards, led to phoma stem lesion development above the crown. In the three seasons, phoma stem lesions became moderately severe (> 2) by harvest only in untreated plots in 1997/98.

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

Abstract: 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 (PVYO), the necrotic strain (PVYN), the C strain (PVYC) 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.

Plant defence reactions against fusarium wilt in chickpea induced by incompatible race 0 of Fusarium oxysporum f.sp. ciceris and nonhost isolates of F. oxysporum

Abstract: Germinated seeds of ‘kabuli’ chickpea cv. ICCV 4 were inoculated with a conidial suspension of the incompatible race 0 of Fusarium oxysporum f.sp. ciceris (Foc) or of nonhost F. oxysporum resistance ‘inducers’, and 3 days later were challenged by root dip with a conidial suspension of highly virulent Foc race 5. Prior inoculation with inducers delayed the onset of symptoms and/or significantly reduced the final amount of fusarium wilt caused by race 5. However, the extent of disease suppression varied with the nature of the inducing agent; the nonhost isolates of F. oxysporum were more effective at disease suppression than the incompatible Foc race 0. Inoculation with the inducers gave rise to synthesis of maackiain and medicarpin phytoalexins in inoculated seedlings; these did not accumulate in plant tissues but were released into the inoculum suspension. Inoculation with inducers also resulted in accumulation of chitinase, β -1,3-glucanase and peroxidase activities in plant roots. These defence-related responses were induced more consistently and intensely by nonhost isolates of F. oxysporum than by incompatible Foc race 0. The phytoalexins and, to a lesser extent, the antifungal hydrolases, were also induced after challenge inoculation with Foc race 5. However, in this case the defence responses were induced in both preinduced and noninduced plants infected by the pathogen. It is concluded that the suppression of fusarium wilt in this study possibly involved an inhibitory effect on the pathogen of preinduced plant defences, rather than an increase in the expression of defence mechanisms of preinduced plants following a subsequent challenge inoculation.

Effects of arbuscular mycorrhizal fungi on severity of root rot of bananas caused by Cylindrocladium spathiphylli

Abstract: The interaction between four arbuscular mycorrhizal (AM) fungi, Glomus sp., G. proliferum, G. intraradices and G. versiforme, and the root-rot fungus Cylindrocladium spathiphylli, and subsequent effects on growth and phosphorus nutrition of banana (Musa acuminata, AAA, cv. Grande Naine) were investigated under glasshouse conditions. Overall, root infection by C. spathiphylli reduced the growth of banana plants, but preinoculation with AM fungi significantly attenuated this detrimental effect. Lower disease severity, stimulation of growth and increase of shoot P content were observed for the plants inoculated with one of the four AM fungi. Glomus sp. and G. proliferum induced the largest increase in growth parameters and shoot P content as compared to G. intraradices and G. versiforme, in the presence as well as in the absence of C. spathiphylli. Root damage caused by C. spathiphylli was decreased in the presence of A-M fungi, but the inoculation of mycorrhizal plants with C. spathiphylli also decreased the intensity of AM fungal root colonization, indicating a clear interaction between the two organisms.

Pathogenicity and race characterization of Fusarium oxysporum f. sp. phaseoli isolates from Spain and Greece

Abstract: Virulence (≡ severity of disease) and physiological specialization of nine isolates of Fusarium oxysporum f. sp. phaseoli recovered in El Barco de Avila (Castilla y Leon, west-central Spain) and of two isolates from Chryssoupolis (Greece) were determined. The susceptibility/resistance response showed by a differential set of common bean cultivars (Phaseolus vulgaris) selected at the Centro Internacional de Agricultura Tropical (CIAT) delineated the isolates into two new races: races 6 and 7. The results of pathogenicity tests did not show any significant differences in virulence among the isolates. However, the reactions of several Spanish common bean cultivars indicated the presence of two groups of isolates, highly virulent and weakly virulent, among the Spanish isolates analysed. These results indicate that isolates classified in the same race are not homogeneous with respect to virulence, and suggests that race analysis using the CIAT differential cultivars is insufficient to describe the physiological specialization of F. oxysporum f. sp. phaseoli.

Survival of Clavibacter michiganensis ssp. michiganensis in infected tomato stems under natural field conditions in California, Ohio and Morocco

Abstract: The survival and half-life of Clavibacter michiganensis ssp. michiganensis (C. michiganensis), the causal agent of bacterial canker of tomato, were determined in infected plant debris under natural field conditions in California, Ohio and Morocco using a semiselective agar medium. The organism survived significantly longer in tomato stems left on the soil surface than in stems buried in the soil at all locations studied. The pathogen was recovered in high amounts from tomato stems left on the soil surface for 314 days in Ohio and California, USA, and for 194 and 132 days in Melk Zhar and Ait Melloul, Morocco, respectively; it was recovered from stems buried in the soil for up to 314 days in Ohio, up to 240 days in California, and up to 60 days in Ait Melloul and Melk Zhar. The half-life of the pathogen in stems left on the soil surface ranged from 23·2 to 24·8 days in the USA, and from 7·8 to 12·3 days in Morocco, whereas the half-life in buried stems ranged from 14·0 to 16·7 days in the USA and from 3·7 to 9·5 days in Morocco. Based on the half-life data, the predicted survival times of C. michiganensis in stems on the soil surface in Ohio, California, Melk Zhar and Ait Melloul would be up to 822, 770, 424 and 261 days, respectively, while the predicted survival times in stems buried in the soil would be 541, 497, 305 and 128 days, respectively. These results show that the survival and half-life of C. michiganensis in plant debris are relatively long and are influenced by both tissue exposure and geographic location.

Genetic diversity in the mango malformation pathogen and development of a PCR assay

Abstract: Malformation is a destructive disease of mango, Mangifera indica Its causal agent possesses the morphological features of Fusarium subglutinans, a species whose taxonomy and nomenclature has recently been in a state of flux Genetic diversity was examined among 74 F subglutinans-like isolates from malformed mango in Brazil, Egypt, Florida (USA), India, Israel and South Africa With nitrate-nonutilizing (nit) auxotrophic mutants, seven vegetative compatibility groups (VCGs) were identified Three of the VCGs were found in a single country, and VCG diversity was greatest in Egypt and the USA where, respectively, four and three different VCGs were found RAPD profiles generated with arbitrary decamer primers were variable among isolates in different VCGs, but were generally uniform for isolates within a VCG In PCR assays, a 20-mer primer pair that was developed previously to identify F subglutinans from maize (mating population [MP]-E of the Gibberella fujikuroi complex) also amplified a specific 448 bp fragment for isolates of F sacchari from sugarcane (MP-B) and what was probably F circinatum (pine, MP-H) With the exception of three isolates from Brazil, it did not amplify the fragment from F subglutinans-like isolates from mango A second pair of 20-mer primers was developed from a unique fragment in the RAPD assays It amplified a specific 608 bp fragment for 51 of 54 isolates from mango (all but the three Brazilian isolates) It also amplified a smaller, 550 bp fragment from isolates of F nygamai (MP-G), but did not amplify DNA of isolates of any other taxon of Fusarium that was tested