Characterization and pathogenicity of Colletotrichum species associated with anthracnose on chilli (Capsicum spp.) in Thailand

Fungal isolates from chilli ( Capsicum spp.) fruits in Thailand that showed typical anthracnose symptoms were identified as Colletotrichum acutatum , C . capsici and C . gloeosporioides . Phylogenetic analyses from DNA sequence data of ITS rDNA and β-tubulin ( tub 2) gene regions revealed three major clusters representing these three species. Among the morphological characters examined, colony growth rate and conidium shape in culture were directly correlated with the phylogenetic groupings. Comparison with isolates of C . gloeosporioides from mango and C . acutatum from strawberry showed that host was not important for phylogenetic grouping. Pathogenicity tests validated that all three species isolated from chilli were causal agents for chilli anthracnose when inoculated onto fruits of the susceptible Thai elite cultivar Capsicum annuum cv. Bangchang. Cross-infection potential was shown by C . acutatum isolates originating from strawberry, which produced anthracnose on Bangchang. Interestingly, only C . acutatum isolates from chilli were able to infect and produce anthracnose on PBC 932, a resistant genotype of Capsicum chinense . This result has important implications for Thai chilli breeding programmes in which PBC 932 is being hybridized with Bangchang to incorporate anthracnose resistance into chilli cultivars.     

Effects of antagonistic microorganisms on the post-harvest development of Colletotrichum gloeosporioides on mango

Potential microbial antagonists of Colletotrichum gloeosporioides were isolated from blossom, leaves and fruit of mango and screened using a series of assay techniques. In total 648 microorganisms, including bacteria, yeasts and filamentous fungi, were isolated and tested for their inhibition of growth of C. gloeosporioides on malt extract agar. In vitro , 121 organisms inhibited the fungus and were tested further for their ability to affect conidial germination. Of these isolates, 45 bacteria and yeasts inhibited germination. These were inoculated onto mangoes artificially infected with C. gloeosporioides and assessed for their potential to reduce the development of anthracnose lesions. Seven isolates were chosen for use in a trial in the Philippines using freshly harvested fruit. This final screening procedure yielded two potential candidates for further trials, isolate 204 (identified as Bacillus cereus ) and isolate 558 (identified as Pseudomonas fluorescens ). In post-harvest trials under commercial conditions, isolates 204 and 558 were both tested in combination with different application methods including the addition of adhesive material, peptone, fruit wax or sucrose polyester. Application of isolate 204 did not reduce disease development, whereas application of 558 significantly reduced anthracnose development. No additional benefit was achieved by incorporating the bacteria in adhesive material, peptone, fruit wax or sucrose polyester.     

Molecular identification of Colletotrichum gloeosporioides causing yam anthracnose in Nigeria

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.     

Stomatal penetration of cowpea (Vigna unguiculata) leaves by a Colletotrichum species causing latent anthracnose

The infection process of a Colletotrichum species causing latent infection and anthracnose in cowpea ( Vigna unguiculata ) was studied in seedlings by light and confocal microscopy. Leaf surfaces were extensively colonized by an anastomosing network of germ-tubes and conidia. This epiphytic mycelium produced abundant secondary conidia on short conidiophores. Although melanized appressoria were developed, the host surface was not penetrated directly. The fungus only gained ingress into leaves through stomatal openings, by means of undifferentiated germ-tubes, and slowly colonized the mesophyll by intercellular hyphae, without initially producing visible symptoms. Anthracnose lesions with multisetate acervuli appeared on senescent leaves after a prolonged symptomless period of host colonization lasting > 2 weeks. Analysis of the nucleotide sequences of the amplified D2 and ITS-2 regions of rDNA revealed close similarities (95–96%) between this cowpea pathogen and isolates of C . gloeosporioides from Aeschynomene virginica, Stylosanthes scabra and Mangifera indica. These results, in addition to other morphological and growth attributes, identify this endophytic anthracnose pathogen of cowpea as a Colletotrichum species distinct from C. capsici and C. destructivum .     

Identification of Subpopulations of Colletotrichum acutatum and Epidemiology of Almond Anthracnose in California

ABSTRACT In recent years, almond anthracnose has developed into a major problem for the California almond industry. The identification of the causal pathogen as Colletotrichum acutatum was confirmed using species-specific primers and restriction fragment length polymorphisms of ribosomal DNA in comparative studies with isolates of C. acutatum from strawberry and C. gloeosporioides from citrus. Two distinct clonal subpopulations among the almond isolates of C. acutatum were identified. These two subpopulations differed in their colony appearance (pink versus gray cultures), conidial morphology, virulence in laboratory inoculation studies, temperature relationships for growth, and molecular fingerprints using random and simple-repeat primers in polymerase chain reactions. Both subpopulations were commonly isolated from the same orchard or even the same fruit. In other orchards, one subpopulation predominated over the other subpopulation. Using random, simple-repeat, and species-specific primers, isolates of the almond anthracnose pathogen from Israel were very similar to the California isolates that produce gray colonies. In addition to fruit, the pathogen was isolated from blighted blossoms, water-soaked or necrotic leaf lesions, symptomless peduncles, and spurs and wood from branches showing dieback symptoms, indicating that the amount of tissue that may be infected is more extensive than previously considered. Overwintering fruit mummies were identified as inoculum sources for early spring infections. Growth studies using almond kernels with different moisture contents indicated that postharvest damage of stored kernels likely originates from preharvest field infections.     

Studies of Colletotrichum gloeosporioides disease on yam, Dioscorea alata, in Solomon Islands

A detailed account of symptoms produced by Colletotrichum gloeosporioides on susceptible and resistant cultivars of yam ( Dioscorea alata ) in Solomon Islands is given. A superficial blackening of upper leaf surfaces of some cultivars is unusual and worthy of further study. In pathogenicity tests with isolates of C. gloeosporioides from various sources, those that were virulent on D. alata were all from D. alata except one from D. esculenta and one from Cucumis sativus but only nine of 30 isolates from D. alata were virulent on this host. Since all four isolates of the yam pathogen tested on D. nummularia and D. trifida were virulent, it is inappropriate to retain the epiphet f. sp. alatae proposed by earlier authors. Considerable variation in cultural morphology existed among virulent isolates, the only consistent character being production of dark pigment, although this was also produced by some saprophytic isolates. Most reisolations of C. gloeosporioides from lesions resulting from inoculation yielded isolates identical with the original inoculum but also some reisolates had a different morphology.     

Phenotypic,molecular and pathogenic characterization of Phlyctema vagabunda,causal agent of olive leprosy

Olive leprosy, caused by the fungus Phlyctema vagabunda, is a classic fruit rot disease widespread in the Mediterranean basin. From 2009 to 2013, new disease symptoms consisting of small circular necrotic leaf lesions, coin branch canker and shoot dieback were observed in Spanish and Portuguese olive orchards showing intense defoliation. Phlyctema‐like anamorphs were consistently isolated from leaves and shoots with symptoms. Representative isolates from affected leaves, shoots and fruits were characterized based on morphology of colonies and conidia, optimum growth temperature and comparison of DNA sequence data from four regions: ITS, tub2, MIT and rpb2. In addition, pathogenicity tests were performed on apple and olive fruits, and on branches and leaves of olive trees. Maximum mycelial growth rate ranged between 0.54 and 0.73 mm per day. Conidia produced on inoculated apple fruits showed slight differences in morphology among the representative fungal isolates evaluated. Phylogenetic analysis clustered all of the Phlyctema‐like isolates in the same clade, identifying them as Phlyctema vagabunda. On fruits, influence of wounding, ripening and cultivar resistance was studied, with cv. Blanqueta being the most susceptible cultivar. On branches, a mycelial‐plug inoculation method reproduced olive leprosy symptoms and caused shoot dieback. On leaves, Koch's postulates were fulfilled and the pathogen caused characteristic necrotic spots and plant defoliation. This is the first time that the pathogenicity of P. vagabunda in olive leaves has been demonstrated.     

Etiology and Population Genetics of Colletotrichum spp. Causing Crown and Fruit Rot of Strawberry

ABSTRACT Isolates of Colletotrichum spp. from diseased strawberry fruit and crowns were evaluated to determine their genetic diversity and the etiology of the diseases. Isolates were identified to species using polymerase chain reaction primers for a ribosomal internal transcribed spacer region and their pathogenicity was evaluated in bioassays. Isolates were scored for variation at 40 putative genetic loci with random amplified polymorphic DNA and microsatellite markers. Only C. acutatum was recovered from diseased fruit. Nearly all isolates from crowns were C. gloeosporioides. In crown bioassays, only isolates of C. gloeosporioides from strawberry caused collapse and death of plants. A dendrogram generated from the genetic analysis identified several primary lineages. One lineage included isolates of C. acutatum from fruit and was characterized by low diversity. Another lineage included isolates of C. gloeosporioides from crowns and was highly polymorphic. The isolates from strawberry formed distinctive clusters separate from citrus isolates. Evaluation of linkage disequilibrium among polymorphic loci in isolates of C. gloeosporioides from crowns revealed a low level of disequilibrium as would be expected in sexually recombining populations. These results suggest that epidemics of crown rot are caused by Glomerella cingulata (anamorph C. gloeosporioides) and that epidemics of fruit rot are caused by C. acutatum.     

Glomerella cingulata on camellia

Glomerella cingulata was shown to be associated with a leaf blotch, canker and dieback of imported camellia plants. It was particularly damaging on hybrids of Camellia saluenensis such as C. x williamsii cv. Donation. Pathogenicity tests, using whole plants and detached leaves of cv. Donation, distinguished between virulent and a virulent strains of the fungus. The virulent strain did not cause severe damage when inoculated into a range of other woody ornamentals. Conversely, Colletotrichum spp., including G. cingulata , isolated from other hosts were not virulent to Camellia cv. Donation. Apart from a loss of virulence associated with a mutation to paleness of colonies in culture, virulent isolates with dark-grey colony centres were morphologically identical to avirulent isolates from camellia, including tea plant ( Camellia sinensis ), and to some isolates from other hosts. A form of Colletotrichum acutatum isolated from camellia and readily distinguished from G. cingulata by bright pink colonies and conidial shape was shown to be avirulent on Camellia cv. Donation. It is proposed that the virulent form on ornamental camellia be described as Glomerella cingulata f.sp. camelliae.     

Detection of Eutypa lata and Eutypella vitis in Grapevine by Nested Multiplex Polymerase Chain Reaction

ABSTRACT Two fungi were isolated from grapevines in Michigan vineyards with Eutypa dieback symptoms: Eutypa lata and Eutypella vitis. These fungi are difficult to distinguish morphologically but are genetically distinct as determined by sequencing of the internal transcribed spacer (ITS) regions. The ITS regions of 25 Eutypa lata and 15 Eutypella vitis isolates were sequenced. Eutypa lata sequences were more variable than those of Eutypella vitis. Polymerase chain reaction (PCR) primers were designed for each species and evaluated against isolates of both fungi as well as 11 closely related Diatrypaceous fungi and 23 isolates of other fungi representing various pathogenic, saprophytic, and endophytic genera on grape and other small fruit crops. The primers were specific for their intended species. A nested multiplex PCR protocol was developed and used to successfully detect these fungi in wood samples from cankers with and without stromata from naturally infected vines as well as in artificially inoculated, potted canes. The primers developed in this study will assist in our abilities to diagnose and study the roles of Eutypa lata and Eutypella vitis in Eutypa dieback development.     

Genetic and Pathogenic Analyses of Colletotrichum gloeosporioides Isolates from Strawberry and Noncultivated Hosts

ABSTRACT Colletotrichum crown rot of strawberry in Florida is caused primarily by Colletotrichum gloeosporioides. To determine potential inoculum sources, isolates of Colletotrichum spp. from strawberry and various noncultivated plants growing in the areas adjacent to strawberry fields were collected from different sites. Species-specific internal transcribed spacer primers for C. gloeosporioides and C. acutatum were used to identify isolates to species. Random amplified polymorphic DNA (RAPD) markers were used to determine genetic relationships among isolates recovered from noncultivated hosts and diseased strawberry plants. Selected isolates also were tested for pathogenicity on strawberry plants in the greenhouse. In all, 39 C. gloeosporioides and 3 C. acutatum isolates were recovered from diseased strawberry crowns, and 52 C. gloeosporioides and 1 C. acutatum isolate were recovered from noncultivated hosts. In crown inoculation tests, 18 of the 52 C. gloeosporioides isolates recovered from noncultivated hosts were pathogenic to strawberry. Phylogenetic analysis using RAPD marker data divided isolates of C. gloeosporioides from noncultivated hosts into two separate clusters. One cluster contained 50 of the 52 isolates and a second cluster contained 2 isolates that were homothallic in culture. Isolates from strawberry were interspersed within the cluster containing the 50 isolates that were recovered from noncultivated hosts. The results are not inconsistent with the hypothesis that C. gloeosporioides isolates obtained from strawberry and noncultivated hosts adjacent to strawberry fields are from the same population and that noncultivated hosts can serve as potential inoculum sources for Colletotrichum crown rot of strawberry.     

Identification of different isolates of Cassava brown streak virus and development of a diagnostic test

An RT-PCR based detection method for Cassava brown streak virus (CBSV)-infected cassava has been developed. The RT-PCR detection method described includes RNA extraction methods for cassava leaves, a distinct primer set for the virus and RT-PCR conditions. The primers were designed to the virus coat protein gene and generate a virus-specific product of 231 bp from infected cassava. The test can detect the virus in the new growth of cassava sticks before any disease symptoms are visible. This test was used successfully with infected cassava from both Tanzania and Mozambique. Three isolates from Tanzania were found to exhibit different symptoms on the secondary host plants Nicotiana benthamiana and N. tabacum SR1. They have nucleotide sequence variation within the coat protein region of up to 8% and amino acid differences of up to 6%.     

Molecular Characterization and Diagnosis of QoI Resistance in Cucumber and Eggplant Fungal Pathogens

ABSTRACT The molecular mechanism of QoI fungicide resistance was studied using isolates of cucumber Corynespora leaf spot fungus (Corynespora cassiicola) and the eggplant leaf mold (Mycovellosiella nattrassii). In both pathogens, a mutation at position 143 from glycine to alanine (G143A) was detected in the cytochrome b gene that encodes for the fungicide-targeted protein. Moreover, the nucleotide sequence at amino acid position 143 was converted from GGT or GGA in sensitive (wild-type) to GCT or GCA in resistant (mutant-type) isolates. The methods of polymerase chain reaction restriction fragment length polymorphism commonly used for QoI resistance monitoring were employed successfully, leading to the amplified gene fragment from resistant isolates being cut with the restriction enzyme ItaI. However, heteroplasmy (the coexistence of wild-type and mutated alleles) was found when the resistant isolates of C. cassiicola, M. nattrassii, and Colletotrichum gloeosporioides (strawberry anthracnose fungus) were subcultured in the presence or absence of QoI fungicides. QoI resistance of cucumber powdery and downy mildew isolates persisted for a few years following the removal of the selection pressure imposed by the fungicide under both laboratory and commercial greenhouse conditions. The proportion of mutated sequences in cytochrome b gene decreased over time in the pathogen population. The protective efficacy of the full dose of azoxystrobin decreased when the populations of powdery and downy mildews contained resistant isolates at 10%. Using FMBIO, a fluorescence bio-imaging analyzer, the mutant allele from the QoI-resistant isolates could be detected at the level of 1%, whereas the detection sensitivity of ethidium-bromide-stained gels was approximately 10 times lower.     

Identification of the causal agent of pistachio dieback in Australia

Symptoms associated with pistachio dieback in Australia include decline (little or no current season growth), xylem staining in shoots two or more years old, trunk and limb lesions (often covered by black, superficial fungal growth), excessive exudation of resin, dieback and death of the tree. Bacteria belonging to the genus Xanthomonas have been suggested as the causal agent. To confirm the constant association between these bacteria and the disease syndrome, the absence of other pathogens and the identity of the pathogen, we performed a series of isolations and pathogenicity tests. The only microorganism consistently isolated from diseased tissue was a bacterium that produced yellow, mucoid colonies and displayed morphological and cultural characteristics typical of the genus Xanthomonas. Database comparisons of the fatty acid and whole-cell protein profiles of five representative pistachio isolates indicated that they all belonged to X. translucens, but it was not possible to allocate the isolates to pathovar. Pathogenicity tests on cereals and grasses supported this identification. However, Kochs postulates have been only partially fulfilled because not all symptoms associated with pistachio dieback were reproduced on inoculated two-year-old pistachio trees. While discolouration was observed, dieback, excessive resinous exudate and trunk and limb lesions were not produced; expression of these symptoms may be delayed, and long-term monitoring of a small number of inoculated trees is in progress.     

Characterization of Colletotrichum acutatum Isolates Causing Anthracnose of Almond and Peach in California

ABSTRACT The causal organism responsible for the recent outbreak of almond and peach anthracnose in California was identified and characterized as Colletotrichum acutatum. Isolates of C. acutatum from almond were found to be similar to California strawberry isolates and South Carolina peach and apple isolates of C. acutatum based on conidial morphology, temperature relationships, fungicide sensitivity, and polymerase chain reaction (PCR) methods using DNA species-specific primers. On almond, blossoms and immature or mature fruit were affected by the disease, causing direct losses of crop. On peach, the disease was observed only on mature fruit. Pathogenicity of almond and peach isolates of C. acutatum was demonstrated on wound- and nonwound-inoculated almond or peach fruit by fulfilling Koch's postulates. Conidial morphology of isolates was variable, depending on the medium or substrate used to culture the isolates. Isolates of C. acutatum from strawberry, almond, and peach were grouped together based on a similar response to temperature, with an optimal growth rate at 25 degrees C (generally less than 10 mm/day), whereas isolates of C. gloeosporioides from citrus and papaya had an optimal growth rate at 30 degrees C (generally greater than 10 mm/day). In fungicide disk assays, isolates of C. acutatum from strawberry, peach, and apple, as well as almond and peach isolates from California, were less sensitive to benomyl at 300, 600, or 1,200 mug/ml. In contrast, C. gloeosporioides isolates from citrus and papaya were very sensitive to benomyl at all concentrations evaluated. All isolates of both species were sensitive to captan (300, 600, or 1,200 mug/ml). Oligonucleotide primers were synthesized for C. acutatum, C. fragariae, or C. gloeosporioides using published DNA sequences from the internal transcribed spacer 1 region of ribosomal DNA. Thirty-two Colletotrichum isolates from almond fruit produced DNA products with a C. acutatum primer (CaInt-2) that matched products and approximate molecular weight of known C. acutatum isolates. No PCR products were produced with primers for C. gloeosporioides or C. fragariae. Isolates from citrus and papaya produced DNA products only with primers from C. gloeosporioides or C. fragariae. Thus, worldwide, anthracnose of almonds may be caused by either C. gloeosporioides, as previously reported, or by C. acutatum, as indicated in this study.     

Clarification of the Etiology of Glomerella Leaf Spot and Bitter Rot of Apple Caused by Colletotrichum spp. Based on Morphology and Genetic, Molecular, and Pathogenicity Tests

ABSTRACT Morphological characteristics and vegetative compatibility groups (VCGs) of 486 isolates of Glomerella cingulata, Colletotrichum gloeosporioides, and C. acutatum collected from apple leaves with Glomerella leaf spot (GLS) symptoms and fruit with bitter rot symptoms in the United States and Brazil were studied. From this collection, 155 isolates of G. cingulata (93 from fruit, 61 from leaves, and 1 from buds), 42 isolates of C. gloeosporioides from fruit, and 14 isolates of C. acutatum (10 from fruit and 4 from leaves) were studied using mitochondrial (mt)DNA restriction fragment length polymorphism (RFLP) haplotypes. A subset of 24 isolates was studied by examining the sequence of a 200-bp intron of the glyceraldehyde 3-phosphate dehydrogenase (GDPH) nuclear gene. In addition, 98 isolates were tested for pathogenicity on leaves of cvs. Gala and Golden Delicious in the greenhouse, and 24 isolates were tested for pathogenicity on fruit of cv. Gala in growth chambers. In total, 238 and 225 isolates of G. cingulata were separated into four distinct morphological types and six VCGs, respectively. Five morphological types and six VCGs were identified among 74 and 36 isolates of C. gloeosporioides, respectively. Three morphological types and four VCGs were identified among 74 and 23 isolates of C. acutatum, respectively. Seven different mtDNA RFLP haplotypes were observed within isolates of G. cingulata, two within isolates of C. gloeosporioides, and two within isolates of C. acutatum. Phylogenetic trees, inferred based on maximum likelihood and maximum parsimony methods using the intron sequence, produced similar topologies. Each species was separated into distinct groups. All isolates tested were pathogenic on fruit, though only isolates with specific VCGs and haplotypes were pathogenic to leaves. Vegetative compatibility was a better tool than molecular characters for distinguishing isolates of G. cingulata pathogenic on both leaves and fruit from the ones pathogenic only on fruit. Isolates of G. cingulata capable of causing both GLS and bitter rot were included in haplotypes and groups based on the sequence analysis of the 200-bp intron that also included isolates capable of causing bitter rot only. Additionally, isolates of G. cingulata from the United States and Brazil which cause GLS were included in different haplotypes and sequence analysis groups. Therefore, one hypothesis is that isolates of G. cingulata from the United States capable of causing both GLS on foliage and bitter rot on fruit may have arisen independently of Brazilian isolates of G. cingulata capable of causing both GLS and bitter rot, and the two groups of isolates may represent distinct populations.     

Detection of <Emphasis Type="Italic">Xanthomonas axonopodis</Emphasis> pv. <Emphasis Type="Italic">manihotis</Emphasis>, the causal agent of cassava bacterial blight diseases in cassava (<Emphasis Type="Italic">Manihot esculenta</Emphasis>) in Ghana by polymerase chain reaction

Xanthomonas axonopodis pv. manihotis (Xam) is the causal agent of cassava bacterial blight (CBB) disease. CBB is a major constraint to cassava cultivation in Ghana. In this study, a survey was conducted in eight regions of Ghana to assess the presence of CBB disease. Out of the eight regions visited, CBB, though at different prevalence, was observed in five regions. Cassava plants samples showing suspected bacterial blight symptoms were collected for analysis by Polymerase Chain Reaction (PCR). The results of the analysis showed that Ashanti region had the highest prevalence in percentage of CBB, which recorded (70%), followed by Volta region (60%); Brong Ahafo region (40%); Eastern region (40%) and Greater Accra region (20%). Morphological examination of the putative pathogen was carried out on Cefazolin trehalose agar (CTA) and Nutrient agar (NA) media. The isolates were subjected to conventional PCR using Xanthomonas genus specific primer, RST2/RST3, Xam specific Variable Number Tandem Repeat (VNTRs) loci, XaG1_67F/R and X-gumD primers, which produced 840, 446 and 402 bp, respectively. The isolates also tested positive with SYBR Green fluorescent dye, using Real-time PCR. The resulting PCR products were sequenced and analyzed using a BLASTn program, which revealed homology between 93 and 100% with several other Xam strains retrieved from GenBank nucleotide database. The pathogenicity test of the isolates on the susceptible Esam cassava variety produced symptoms typical of Xam and the pathogen was consistently re-isolated from the inoculated cassava plants and thereby satisfying the Koch’s postulates.     

Root Rot of Miniature Roses Caused by Pythium helicoides

Miniature roses growing in an ebb-and-flow watering system developed dieback during the summer growing season of 1996 in Gifu Prefecture. The main diagnostic symptoms were chlorosis of leaf followed by blight, and a brown, water-soaked root rot followed by dieback. Pythium isolates were recovered from the rotted root. The isolates form proliferous ellipsoidal papillate sporangia, spherical smooth oogonia, elongate antheridia, and aplerotic oospores. The optimum temperature for hyphal growth was 35°C with a growth rate of 34 mm/24 hr. Optimum temperature of zoospore formation (25-30°C) was lower than that of mycelial growth, and zoospores were produced even at 10°C. The isolates were identified as P. helicoides on the basis of these characteristics. In pathogenicity tests disease severity was highest at the highest tested temperature (35°C) at which the disease naturally occurred in summer. Four days after inoculation, the leaves turned yellow and the roots had a water-soaked rot, followed by leaf blight and root dieback after 7 days. The disease transmission test showed that diseased plants were found throughout the bench after 10 days. Received 4 July 2001/ Accepted in revised form 10 October 2001     

Colonization of roots, stolons, tubers and stems of various potato (Solanum tuberosum) cultivars by the black-dot fungus Colletotrichum coccodes

In an attempt to better understand the importance of tuber-borne inoculum in black dot development, several potato cultivars were inoculated with various Colletotrichum coccodes isolates. Symptoms developed first on underground organs (starting 2 weeks after inoculation on roots, and later on stolons and tubers) of inoculated plants; stem infections developed only after 7–10 weeks, depending on the cultivar. Infection with C. coccodes resulted in a reduction in numbers of stolons and tubers in cv. Bintje, but not in the later maturing cv. Roseval. Significant isolate by cultivar interactions were detected from the analysis of root symptoms after inoculation of three potato cultivars (Bintje, Spunta and Desiree) with five C. coccodes isolates. Such an interaction was also detected for stolon/tuber symptoms at the latest scoring date (98 days after inoculation), but not at earlier dates (58, 70 and 84 days after inoculation). These results suggest that protocols based on root colonization might be used for investigating cultivar response to black dot and pathogenicity of C. coccodes isolates, and that some specificity exists in the reaction of potato genotypes to this pathogenic fungus.