Structure and temporal shifts in virulence of Pseudoperonospora cubensis populations in the Czech Republic

The structure and temporal dynamics of the virulence of Pseudoperonospora cubensis (causal agent of cucurbit downy mildew) were studied in pathogen populations in the Czech Republic from 2001 to 2010. A total of 398 P. cubensis isolates collected from Cucumis (Cm.) sativus, Cm. melo, Cucurbita (Cr.) maxima, Cr. pepo, Cr. moschata and Citrullus lanatus were analysed for variation in virulence (pathotypes). Virulence was evaluated on a differential set of 12 genotypes of cucurbitaceous plants. All isolates of P. cubensis were characterized by their level of virulence (classified according the number of virulence factors, VF; low VF = 1–4, medium VF = 5–8, high VF = 9–12): high (75%), medium (24%) and low (1%). The structure and dynamics of virulence in the pathogen populations were expressed by pathotypes using tetrad numerical codes and a total of 67 different pathotypes of P. cubensis were determined. The most susceptible group of differentials was Cucumis spp., while the lowest frequency of virulence was recorded on Cr. pepo ssp. pepo, Ci. lanatus and Luffa cylindrica. A high proportion (c. 90%) of isolates was able to infect cucurbit species Benincasa hispida and Lagenaria siceraria, which are not commonly cultivated in the Czech Republic or elsewhere in central Europe. In the recent pathogen populations (2008–2010) there was prevailing frequency (70–100%) of isolates with high numbers (9–12) of virulence factors. ‘Super pathotype’ 15.15.15 was often observed in the study within the pathogen populations and was one of the four most frequently recorded pathotypes. Pseudoperonospora cubensis populations shifted to a higher virulence over time. From 2009 the pathogen population changed dramatically and new pathotypes appeared able to establish natural and serious infection of Cucurbita spp. and Ci. lanatus, which was not observed in 2001–2008. Generally, virulence structure and dynamics of P. cubensis populations are extremely variable in the Czech Republic.     

Characterization of Pseudoperonospora cubensis isolates from Europe and Asia using ISSR and SRAP molecular markers

Downy mildew caused by Pseudoperonospora cubensis is a major disease of cucurbits worldwide. New genotypes of the pathogen have recently appeared in the USA, EU and Israel causing breakdown of genetic resistance, expansion of host range, and the appearance of a new A2 mating type. Seventy-eight P. cubensis isolates were collected during 1996–2011 from cucurbits fields in different regions of Turkey, Israel and the Czech Republic and genetic diversity was analysed using highly polymorphic ISSR and SRAP molecular markers. The data acquired showed remarkable genetic diversity within and among the isolates. While isolates from Turkey and Czech Republic exhibited uniform genetic background, the isolates from Israel were clearly distinguished from the others. The results may indicate on migration and/or frequent sexual reproduction of the pathogen in Israel. Moreover the selected markers can be suggested for monitoring genetic diversity within P. cubensis isolates in further studies.     

Pathogenic variability of Plasmopara halstedii infecting sunflower in the Czech Republic

Plasmopara halstedii was isolated from diseased sunflowers collected from eight locations in the Czech Republic from 2007 to 2014. Races of the pathogen were determined based on 84 isolates collected during the study. In total, eight races of P. halstedii were detected using a set of nine sunflower differential lines. Races 700, 704, 705, 710, 714 and 715 were proven by soil drench inoculation, and two additional races (730 and 770) proposed by the previously applied leaf disc inoculation method. Race 700 was the most dominant in the Czech P. halstedii populations, with race 710 being the second most frequent. Races 704 and 714 were found over three seasons, while other races were recorded only in one growing season (race 730 in 2010, and the new races 705 and 715 in 2014). A comprehensive study was further conducted for isolates collected in 2013–14 using an extended differential set consisting of 15 sunflower lines. According to the latter methodology which marks races with five‐digit virulence codes, races 70060, 70471, 70571, 71060, 71461 and 71571 were recorded. The growing complexity of P. halstedii pathogenicity exhibited by the ability to infect higher numbers of differential genotypes and resulting in determination of the new pathogen races (virulence profiles) 70571, 71461 and 71571 is alarming. Although the limited number of isolates studied cannot characterize the entire pathogen diversity in the Czech Republic, the trend towards more diverse virulence in P. halstedii populations is clearly demonstrated by the new records of races 704, 705, 714 and 715, all capable of overcoming the resistance gene Pl₆.     

The morphology,behaviour and molecular phylogeny of Phytophthora taxon Salixsoil and its redesignation as Phytophthora lacustris sp. nov.

Since its first isolation from Salix roots in 1972, isolates of a sexually sterile Phytophthora species have been obtained frequently from wet or riparian habitats worldwide and have also been isolated from roots of Alnus and Prunus spp. Although originally assigned to Phytophthora gonapodyides on morphological grounds, it was recognized that these isolates, informally named P. taxon Salixsoil, might represent a separate lineage within ITS Clade 6. Based on phylogenetic analyses and comparisons of morphology, growth‐temperature relationships and pathogenicity, this taxon is formally described here as Phytophthora lacustris sp. nov. Isolates of P. lacustris form a clearly resolved cluster in both ITS and mitochondrial cox1 phylogenies, basal to most other Clade 6 taxa. Phytophthora lacustris shares several unusual behavioural properties with other aquatic Clade 6 species, such as sexual sterility and tolerance of high temperatures, that have been suggested as adaptations to riparian conditions. It appears to be widespread in Europe and has also been detected in Australia, New Zealand and the USA. It was shown to be weakly or moderately aggressive on inoculation to Alnus, Prunus and Salix. The extent of P. lacustris’ activity as a saprotroph in plant debris in water and as an opportunistic pathogen in riparian habitats needs further investigation. Its pathogenic potential to cultivated fruit trees also deserves attention because P. lacustris has apparently been introduced into the nursery trade.     

Host switching between native and non‐native trees in a population of the canker pathogen Chrysoporthe cubensis from Colombia

The purpose of this study was to test the hypothesis that Chrysoporthe cubensis on native trees in South America could be the source of the pathogen that causes severe stem cankers and often mortality in commercially propagated Eucalyptus trees. This was done by investigating populations originating from two adjacent Eucalyptus (Myrtaceae) plantations in Colombia, and wild Miconia rubiginosa trees (Melastomataceae) growing alongside these stands. Polymorphic microsatellite markers were used to quantify allele sizes in 20 and 39 isolates from the two Eucalyptus stands and 32 isolates from adjacent M. rubiginosa trees. Gene and genotypic diversities were calculated from these data, and population differentiation and assignment tests were performed to ascertain whether the populations were genetically different. Results showed that there were no differences between any of the populations using these techniques, and that they can be treated as a single population. Therefore, the results support the hypothesis that host switching has occurred in C. cubensis in Colombia.     

Population genetic structure of Mycosphaerella graminicola and Quinone Outside Inhibitor (QoI) resistance in the Czech Republic

Damage caused by the wheat pathogen Mycosphaerella graminicola increased rapidly during the last two decades in the Czech Republic. We collected isolates from naturally infected fields in seven wheat-growing locations and analysed these using eight microsatellite markers. All markers were highly polymorphic. We found a high degree of genetic diversity and low clonality within all sampled Czech populations. We identified 158 unique multilocus haplotypes among 184 isolates. Field populations showed weak genetic structure but we detected more differentiation between climatic regions within the Czech Republic. We compared the Czech field populations to populations from the United Kingdom, Germany and Switzerland and found a marked differentiation between Czech populations and Western European populations. We hypothesize that decades of different agricultural practices, including the use of different wheat cultivars, may explain this genetic differentiation. We detected a rapid increase in QoI fungicide resistance during the sampling period from 2005 to 2011, coinciding with the widespread application of this class of fungicides in the Czech Republic. M. graminicola populations in the Czech Republic underwent a rapid adaptive evolution from sensitivity to resistance similar to what was described earlier in Western Europe.     

Pathogenic and molecular comparison of Puccinia kuehnii isolates and reactions of sugarcane varieties to orange rust

Sugarcane orange rust, a disease caused by Puccinia kuehnii, was first reported in Brazil in 2009. There are no studies comparing the Brazilian P. kuehnii collections and the reaction of important sugarcane varieties under controlled conditions. This work compared the reaction of seven sugarcane varieties inoculated with six different P. kuehnii isolates from Brazilian sugarcane areas and verified the pathogenic and genetic variability of these isolates. The incubation (I) and latency (L) disease periods, disease severity (SEV), total number of lesions (TNL), total number of sporulating lesions (TNSL), and percentage of sporulating lesions (%SL) were evaluated. Furthermore, ITS1 and IGS ribosomal sequences of all P. kuehnii isolates used in this study were compared with pathogen sequences from 13 different countries. The disease incubation ranged from 7 to 10 days and the latency ranged from 10 to 21 days. SEV and TNL showed large variations and few significant differences between the reaction of the varieties to P. kuehnii, in contrast with the variables TNSL and %SL. The P. kuehnii isolates did not compose different virulent races, but the isolate from one site (Araras) was a more aggressive race. The ITS1 and IGS ribosomal sequences of six P. kuehnii isolates were identical with each other and to most P. kuehnii American sequences deposited at GenBank. The studied sequences of P. kuehnii isolates differed from the sequences from Asia, Tahiti and Oceania.     

Pineapple heart rot isolates from Ecuador reveal a new genotype of Phytophthora nicotianae

Pineapple heart rot disease, caused by Phytophthora nicotianae (syn. P. parasitica), is responsible for significant annual reductions in crop yield due to plant mortality. In Ecuador, new infections arise during the rainy season and increase production costs due to the need for biocontrol and fungicide applications. Studies of P. nicotianae population structure suggest that certain genetic groups are associated with host genera; however, it is not clear how many host‐specific lineages of the pathogen exist or how they are related. The objectives of this study were to determine the level of genetic variation in the P. nicotianae population causing heart rot disease of pineapple in Ecuador and compare the genotypes found on pineapple to those previously reported from citrus, tobacco and ornamentals. Thirty P. nicotianae isolates collected from infected pineapple leaves from four farms were genotyped using nine simple sequence repeat loci. In addition, the DNA sequences of mitochondrial loci cox2 + spacer and trnG‐rns were analysed. Together, these loci supported a single clonal lineage with two multilocus genotypes differing in a single allele and low mitochondrial diversity. This lineage was distinct but closely related to isolates collected from vegetables and ornamentals in Italy. The results support the hypothesis of host specialization of P. nicotianae in intensive cropping systems and contribute to the understanding of population structure of this important pathogen.     

Occurrence and molecular characterization of azoxystrobin resistance in cucumber downy mildew in Shandong province of China

Cucumber downy mildew caused byPseudoperonospora cubensis (Berk. and Curt.) Rostov. limits crop production in Shandong Province of China. Since management of downy mildew is strongly dependent on fungicides, a rational design of control programs requires a good understanding of the fungicide resistance phenomenon in field populations of the pathogen. A total of 106 and 97 isolates ofP. cubensis were obtained in 2006 and 2007, respectively. The EC₅₀ values for the growth of all the 106 isolates collected in 2006 were 0.0063–0.0688μg ml⁻¹ (average: 0.0196±0.0048μg ml⁻¹) azoxystrobin and these were therefore considered sensitive isolates. However, 57 field isolates ofP. cubensis of the 97 collected in 2007 with EC₅₀ values that ranged from 0.609 to >51.2μg ml⁻¹ were considered resistant to azoxystrobin. Fragments of the fungicide-targeted mitochondrial cytochromeb gene from total pathogen DNA were amplified using polymerase chain reaction and their sequences analyzed to elucidate the molecular mechanism of resistance. A single point mutation (GGT to GCT) in the cytochromeb gene, resulting in substitution of glycine by alanine at position 143, was found in the three selected azoxystrobin-resistant isolates of downy mildew. This substitution in cytochromeb exhibited different resistance levels, with the resistance factor from 21.15 to greater than 2618.9. In addition, the different resistance levels seemed to appear within 1 year (between 2006 and 2007). Therefore, growers of Shandong Province in China now are faced with a challenge in managing the azoxystrobin resistance in cucumber downy mildew. http://www.phytoparasitica.org posting March 10, 2008.     

Mating type and sexual reproduction of <Emphasis Type="Italic">Pseudoperonospora cubensis</Emphasis>, the downy mildew agent of cucurbits

The oomycete Pseudoperonospora cubensis is a leaf pathogen causing severe damage to members of the Cucurbitaceae, especially cucumber and melon. It propagates clonally by sporangia. Oospores of P. cubensis were previously observed in nature but their formation in the laboratory was never reported nor their germination or infection. Here we report on the sexual reproduction of P. cubensis under controlled conditions in the laboratory. When field isolates were inoculated singly onto detached leaves of cucurbits in growth chambers no oospores were produced. However, when pairs of selected isolates were mixed and inoculated onto detached leaves, oospores were formed in the mesophyll within 6–11 days, suggesting that P. cubensis is heterothallic, having two opposite mating types, A1 and A2. Isolates belonging to pathotype 3 were all A1 whereas isolates belonging to the new pathotype 6 were either A1 or A2. Oospores were spherical, ~40 μm in diameter, hyaline to red-brown in color. Oospores were produced regularly, in large numbers, in Cucumis sativum and Cucumis melo, very seldom and in very small numbers in Cucurbita pepo, Cucurbita maxima and Citrullus lanatus, and not in Cucurbita moschata. Oospores were formed at 12.5–21°C but not at 25°C. Under moisture-saturated atmosphere oospores were also produced in leaves of intact plants. Oospores inoculated onto detached leaves in growth chambers produced F1 downy mildew lesions at 6–21 days after inoculation, many in Cucumis sativum, Cucumis melo and Cucurbita moschata, very few in Cucurbita pepo or Citrullus lanatus, and none in Cucurbita maxima. This report shows that P. cubensis is heterothallic, having A1 and A2 mating types which can cross and enable sexual reproduction in cucurbits. A preliminary report on part of the results has been published earlier.     

Genetic differentiation of the wheat leaf rust fungus Puccinia triticina in Europe

The objective of this study was to determine whether genetically differentiated groups of Puccinia triticina are present in Europe. In total, 133 isolates of P. triticina collected from western Europe, central Europe and Turkey were tested for virulence on 20 lines of wheat with single leaf rust resistance genes, and for molecular genotypes with 23 simple sequence repeat (SSR) markers. After removal of isolates with identical virulence and SSR genotype within countries, 121 isolates were retained for further analysis. Isolates were grouped based on SSR genotypes using a Bayesian approach and a genetic distance method. Both methods optimally placed the isolates into eight European (EU) groups of P. triticina SSR genotypes. Seven of the groups had virulence characteristics of isolates collected from common hexaploid wheat, and one of the groups had virulence characteristics of isolates from tetraploid durum wheat. There was a significant correlation between the SSR genotypes and virulence phenotypes of the isolates. All EU groups had observed values of heterozygosity greater than expected and significant fixation values, which indicated the clonal reproduction of urediniospores in the overall population. Linkage disequilibria for SSR genotypes were high across the entire population and within countries. The overall values of R_ST and F_ST were lower when isolates were grouped by country, which indicated the migration of isolates within Europe. The European population of P. triticina had higher levels of genetic differentiation compared to other continental populations.     

Lack of host specificity of Colletotrichum spp. isolates associated with anthracnose symptoms on mango in Brazil

The aim of the present study was to analyse the genetic and pathogenic variability of Colletotrichum spp. isolates from various organs and cultivars of mango with anthracnose symptoms, collected from different municipalities of São Paulo State, Brazil. Colletotrichum gloeosporioides isolates from symptomless citrus leaves and C. acutatum isolates from citrus flowers with post‐bloom fruit drop symptoms were included as controls. Sequencing of the ITS region allowed the identification of 183 C. gloeosporioides isolates from mango; only one isolate was identified as C. acutatum. amova analysis of ITS sequences showed larger genetic variability among isolates from the same municipality than among those from different populations. fAFLP markers indicated high levels of genetic variability among the C. gloeosporioides isolates from mango and no correlation between genetic variability and isolate source. Only one C. gloeosporioides mango isolate had the same genotype as the C. gloeosporioides isolates from citrus leaves, as determined by ITS sequencing and fAFLP analysis. Pathogenicity tests revealed that C. gloeosporioides and C. acutatum isolates from either mango or citrus can cause anthracnose symptoms on leaves of mango cvs Palmer and Tommy Atkins and blossom blight symptoms in citrus flowers. These outcomes indicate a lack of host specificity of the Colletotrichum species and suggest the possibility of host migration.     

Resistance to a highly aggressive isolate of Sclerotinia sclerotiorum in a Brassica napus diversity set

Sclerotinia stem rot (SSR) of oilseed rape (OSR, Brassica napus), caused by Sclerotinia sclerotiorum, is a serious problem in the UK and worldwide. As fungicide‐based control approaches are not always reliable, identifying host resistance is a desirable and sustainable approach to disease management. This research initially examined the aggressiveness of 18 Sclerotinia isolates (17 S. sclerotiorum, one S. subarctica) on cultivated representatives of B. rapa, B. oleracea and B. napus using a young plant test. Significant differences were observed between isolates and susceptibility of the brassica crop types, with B. rapa being the most susceptible. Sclerotinia sclerotiorum isolates from crop hosts were more aggressive than those from wild buttercup (Ranunculus acris). Sclerotinia sclerotiorum isolates P7 (pea) and DG4 (buttercup), identified as ‘aggressive’ and ‘weakly aggressive’, respectively, were used to screen 96 B. napus lines for SSR resistance in a young plant test. A subset of 20 lines was further evaluated using the same test and also in a stem inoculation test on flowering plants. A high level of SSR resistance was observed for five lines and, although there was some variability between tests, one winter OSR (line 3, Czech Republic) and one rape kale (line 83, UK) demonstrated consistent resistance. Additionally, one swede (line 69, Norway) showed an outstanding level of resistance in the stem test. Resistant lines also had fewer sclerotia forming in stems. New pre‐breeding material for the production of SSR resistant OSR cultivars relevant to conditions in the UK and Europe has therefore been identified.     

The gene flow and mode of reproduction of Dothistroma septosporum in the Czech Republic

Since 1911, dothistroma needle blight, caused by Dothistroma septosporum, has been recorded in most European countries. In the Czech Republic, the fungus has become an important disease of pines since 2000, especially Austrian pines in plantations of Christmas and ornamental trees. The aim of this study was to analyse the population structure, gene flow and mode of reproduction of this pathogen. Microsatellite and mating‐type markers were analysed in a Dothistroma population in the southeastern part of the country using reference isolates from other European countries. The haplotypic diversity was high, with 87 unique and 13 shared haplotypes (probable clones) identified in 121 samples. Based on structure analysis, the isolates were divided into two populations, with an uneven distribution over the sampling sites. The grouping of the sites to the populations did not follow a geographical pattern because certain isolates that were sympatrically co‐occurring at the same site were placed in different populations. Tests for random mating (the index of association and a parsimony tree‐length permutation test) showed a significant clonal mode of reproduction in most cases, but the intrapopulation haplotypic diversity is unexpectedly high. Although a teleomorphic stage of D. septosporum has not been previously observed in the Czech Republic, the high intrapopulation haplotypic diversity can be explained by infrequent sexual reproduction consistent with the occurrence of both mating types.     

Genetic variation and evolutionary forces shaping Cucumber vein yellowing virus populations: risk of emergence of virulent isolates in Europe

The genetic variation and evolutionary mechanisms shaping Cucumber vein yellowing virus (CVYV) populations were investigated by analysis of nucleotide sequences coding for P1b, P1b/P3 and coat proteins (CP) from isolates collected in different countries. The complete genome sequence of isolate ISM from Israel was also determined and compared to those of isolates Jor from Jordan and ALM32 from Spain. This isolate had overall nucleotide identities of 94·23 and 94·96% with ALM32 and Jor, respectively. Nucleotide variation among isolates was not homogeneously distributed, with the 5′ half of the genome being more variable than the 3′ half. A Bayesian phylogenetic tree of the CP showed that CVYV isolates clustered into two main clades: isolates from the Middle East region (Lebanon, Israel and Jordan) clustered in both clades whereas the isolate from Tunisia clustered in clade I and the European isolates clustered as a homogeneous phylogroup in Clade II. A similar topology was observed for P1b but with incongruences with respect to the CP, suggesting genetic exchange among virus isolates, which were confirmed with recombination algorithms. The low genetic diversity within the European phylogroup with respect to the other isolates, neutralist tests and genetic differentiation analyses suggest that the Middle East region is the cradle of CVYV and that a unique virus introduction event occurred in Europe, where the virus spread rapidly. Taken together, these findings indicate a risk of emergence of virulent CVYV isolates in Europe either through migration from the Middle East or by genetic changes of the European isolates.     

Phenotypic and phylogenetic studies associated with the crucifer white leaf spot pathogen,Pseudocercosporella capsellae,in Western Australia

Pseudocercosporella capsellae (white leaf spot disease) is an important disease on crucifers. Fifty‐four single‐conidial isolates collected from Brassica juncea (Indian mustard), B. napus (oilseed rape), B. rapa (turnip), and Raphanus raphanistrum (wild radish) across Western Australia were investigated for differences in pathogenicity and virulence using cotyledon screening tests, genetic differences using internal transcribed spacer (ITS) sequencing and phylogenetic analysis, and growth rates on potato dextrose, V8 juice and malt extract agars. All isolates from the four crucifer hosts were pathogenic on the three test species: B. juncea, B. napus and R. raphanistrum, but showed differences in levels of virulence. Overall, isolates from B. juncea, B. napus and B. rapa showed greatest virulence on B. juncea, least on R. raphanistrum and intermediate virulence on B. napus. Isolates from R. raphanistrum showed greatest virulence on B. juncea, least on B. napus and intermediate virulence on R. raphanistrum. Growth and production of a purple‐pink pigment indicative of cercosporin was greatest on malt extract agar and cercosporin production on V8 juice agar was positively correlated with virulence of isolates on B. juncea and B. napus. ITS sequencing and phylogenetic analysis showed that isolates collected from B. napus, B. juncea and B. rapa, in general and with few exceptions, had a high degree of genetic similarity. In contrast, isolates from R. raphanistrum were clearly differentiated from isolate groups collected from Brassica hosts. Pseudocercosporella capsellae reference isolates from other countries generally grouped into a single separate cluster, highlighting the genetic distinctiveness of Western Australian isolates.     

Genetic diversity of Botrytis in New Zealand vineyards and the significance of its seasonal and regional variation

Species‐ and population‐specific differences in fungicide resistance and aggressiveness within Botrytis makes basic data on genetic diversity important for understanding disease caused by this fungus. Genetic diversity of Botrytis was surveyed between 2008 and 2012 from grapes from five New Zealand wine‐growing regions. A total of 1226 isolates were gathered from symptomless flower buds at the start of the growing season and 1331 isolates from diseased fruit at harvest. Two species were found, B. cinerea and B. pseudocinerea. Botrytis pseudocinerea was common in both Auckland vineyards sampled, and infrequent elsewhere. However, even in Auckland, it was rarely isolated from diseased fruit. The presence of the Boty and Flipper transposons was assessed. Isolates with all four transposon states (Boty only, Flipper only, both Boty and Flipper, no transposons) were found for both species. Both vineyards in the Auckland region had high numbers of Flipper‐only isolates at flowering; both vineyards from the Waipara region had high numbers of Boty‐only isolates at flowering. Most isolates from diseased fruit at harvest contained both transposons. These observations suggest that B. pseudocinerea, and isolates with one or both of the transposons missing, may be less aggressive than B. cinerea, or than isolates with both transposons present. Two clades were resolved within B. pseudocinerea, only one of which has been reported from European vineyards. Phylogenetic diversity within B. cinerea in New Zealand was similar to that known from Europe, including isolates that appear to match Botrytis ‘Group S’. The taxonomic implications of this genetic diversity are discussed.     

Identity and variability of Pythium species associated with yield decline in aerobic rice cultivation in the Philippines

The cultivation of aerobic rice in the tropics enables farmers to save water without lowering productivity. Unfortunately, this system suffers from declining yields due to a disease complex involving nematodes, pathogenic Pythium spp. and nutrient deficiencies. Assessing the impact of each underlying factor can contribute to efficient disease control measures. This study therefore investigated pathogenic and genotypic variability among Pythium species from affected aerobic rice fields in the Philippines using pathogenicity assays and sequence information from the internal transcribed spacer (ITS) region and β‐tubulin gene. Three closely related Pythium spp., P. arrhenomanes, P. graminicola and P. inflatum, were recovered from affected aerobic rice fields. All P. arrhenomanes isolates reduced rice seedling growth, whereas only a few P. graminicola isolates and no P. inflatum isolates were pathogenic, indicating that P. arrhenomanes is probably the most important species affecting rice. Both P. arrhenomanes and P. graminicola isolates showed little genetic variation, despite the observed pathogenic variation within P. graminicola. Intraspecific variation was higher among P. inflatum isolates, but again no correlation was observed with phenotype. When screening P. arrhenomanes isolates from other hosts such as sugarcane, maize and several grasses, a link between pathogenic and genetic variability was detected. However, rice and maize isolates seemed to lack host specificity, and therefore crop rotation with maize might be a risky strategy to manage yield decline in Philippine aerobic rice fields.     

Characterization and epidemiology of Colletotrichum acutatum sensu lato (C. chrysanthemi) causing Carthamus tinctorius anthracnose

In 2012, Colletotrichum isolates were collected from field‐grown safflower (Carthamus tinctorius) crops in central Italy from plants exhibiting typical anthracnose symptoms. Colletotrichum isolates were also collected from seed surfaces and from within seeds. The genetic variability of these isolates was assessed by a multilocus sequencing approach and compared with those from Colletotrichum chrysanthemi and Colletotrichum carthami isolates from different geographic areas and other Colletotrichum acutatum sensu lato‐related isolates. Phylogenetic analysis revealed that all of the strains isolated from C. tinctorius belonged to the species described as C. chrysanthemi, whereas all of the strains belonging to C. carthami had been isolated from Calendula officinalis. Phenotypic characterization of isolates was performed by assessing growth rates at different temperatures, morphology of colonies on potato dextrose agar (PDA) and the size of conidia. All C. chrysanthemi isolates from safflower had similar growth rates at different temperatures, comparable colony morphologies when grown on PDA and conidial sizes consistent with previously described C. chrysanthemi isolates. Pathogenicity tests were performed by artificially inoculating both seeds and plants and confirmed the seedborne nature of this pathogen. When inoculated on plants, C. chrysanthemi caused the typical symptoms of anthracnose on leaves. This is the first record of this pathogen on C. tinctorius in Italy, and it presents an updated characterization of Colletotrichum isolates pathogenic to safflowers in Europe.     

Occurrence of azoxystrobin‐resistant isolates in Passalora fulva,the pathogen of tomato leaf mould disease

Since 2007, serious damage to tomato from leaf mould caused by Passalora fulva has frequently been observed in commercial greenhouses in Gifu Prefecture, Japan. One of the factors relating to this damage was suspected to be a decrease in azoxystrobin sensitivity of the pathogen. Biological and molecular studies were conducted to characterize fungicide resistance. In in vitro sensitivity tests using mycelial homogenate placed on fungicide‐amended medium, the minimum inhibitory concentrations (MIC) of azoxystrobin for mycelial growth of the isolates divided into two ranges, 0.031–0.5 mg L^?1 and 8–32 mg L^?1. Isolates with MICs within the two ranges were considered as sensitive and resistant, respectively, to azoxystrobin because, in in vivo tests, the percentage protection conferred by this fungicide (100 mg a.i. L^?1) against these isolates was 89.7–100% and 4.5–31.1%, respectively. Resistant isolates had a replacement of phenylalanine with leucine at codon 129 (F129L) in cytochrome b. Forty‐five percent of the 271 isolates collected from 63 tomato greenhouses from 2007 to 2008 were resistant to azoxystrobin. In many greenhouses where the isolation frequency of resistant isolates was 80% or more, azoxystrobin had been used twice per crop for approximately 6 years. In 2012, 27% of the 405 isolates collected were resistant to azoxystrobin, and there was a marked difference in the frequency of occurrence of resistant isolates in the field populations between the three locations sampled. The occurrence of azoxystrobin‐resistant P. fulva isolates (F129L mutants) inflicted considerable damage on greenhouse tomatoes.