Variation in virulence of Greek isolates ofPhytophthora citrophthora as measured by their ability to cause crown rot on three peach rootstocks

The virulence ofPhytophthora citrophthora isolated from various host-plants on three peach rootstocks (GF677, PR204, KID I) was examined. There was no significant difference among the rootstocks with respect to their susceptibility to testedP. citrophthora isolates. The most virulent isolate originated from sycamore (Acer pseudoplatanus); isolates from pistachio trees (Pistacia vera) also showed high virulence but were significantly less virulent than the sycamore isolate. Isolates originating from plum (Prunus domestica), almond (Prunus amygdalus) and lemon (Citrus limon) trees were moderately virulent on peach rootstocks; those from cyclamen (Cyclamen persicum) showed the lowest virulence of those tested. There was thus great variation in virulence among the testedP. citrophthora isolates. It is possible that the isolates ofP. citrophthora from sycamore, pistachio, plum, almond and lemon trees are a threat to peach trees, whereas the low virulence of the isolates from cyclamen hosts suggests that these pathogens are not a serious threat to peach trees. http://www.phytoparasitica.org posting Jan. 3, 2002.     

Testing variability in pathogenicity ofPhytophthora cactorum, P. citrophthora andP. syringae to apple, pear, peach, cherry and plum rootstocks

The relative virulence ofPhytophthora cactorum andP. syringae originating from almond trees, and ofP. citrophthora originating from citrus, to apple, pear, peach, cherry and plum rootstocks, was studiedin vivo andin vitro. Results of the different experiments were in good agreement. All testedPhytophthora isolates showed little virulence to pear rootstocks-causing only minor crown rot symptoms - and no virulence at all to apple rootstocks. In contrast, they were highly virulent to stone fruit rootstocks, causing crown rot disease. The non-pathogenicity of these isolates to pome rootstocks could be interpreted as strict host specificity.     

Phytophthora root and crown rot of raspberry in Bulgaria

Root and crown rot of raspberry (Rubus idaeus L.) was observed in a plantation at the experimental station of small fruits in Kostinbrod, Bulgaria. Isolates ofPhytophthora spp. were obtained from diseased plants. Colony morphology, growth rates, features of asexual and sexual structures were studied and as a result twoPhytophthora species were identified:Phytophthora citricola Saw. andPhytophora citrophthora (R.E. Sm. & E.H. Sm.) Leonian. Their pathogenicity was confirmed in artificial inoculation experiments. The isozyme (-esterase) patterns ofP. citrophthora andP. citricola isolates from raspberry and from the collection of the CBS, Baarn the Netherlands were compared, using micro-gel electrophoresis. Both species are reported for the first time as pathogens of raspberry in Bulgaria. This is only the second report in phytopathological literature ofP. citrophthora on raspberry, the first being from Chile [Latorre and Munoz, 1993].     

Intraspecific Variation in Phytophthora citrophthora from Citrus Trees in Eastern Corsica

Isolates of Phytophthora pathogenic to citrus crops on Eastern Corsica and associated with gummosis were identified by PCR-RFLP of internal transcribed spacers (ITS) sequences and characterized by the random amplified microsatellites (RAMS) technique. A sample of 114 isolates collected from diseased trunks and fruits, and from soil, were overwhelmingly Phytophthora citrophthora. Further analysis indicated that the P. citrophthora population was not homogeneous in citrus groves. There were two groups, with a few (4%) atypical isolates in two marginal groups. The major groups have been re-examined in the light of mating behaviour, RFLPs of mitochondrial DNA and sequence comparisons of ITS regions of rDNA. They were found distinct with all these criteria and perhaps constitute distinct taxa. The results indicate that important modifications occurred in the population structure of P. citrophthora over time in Corsican groves. These changes may have impact on the recent outbreaks of gummosis.     

Phytophthora boehmeriae boll rot: a new threat to cotton cultivation in the mediterranean region

A boll rot of cotton (Gossypium hirsutum L.) was observed for the first time in Greece in August 1993 in Larissa and Volos counties, and in August and September 1995 in Trikala and Phthiotis counties. Fungi of the genusPhytophthora were isolated from diseased plants. Morphological characteristics of the pathogen were recorded on mounts made directly from the infected tissues or after growth of the isolated fungus on corn meal agar or sterile distilled water. Colony morphology, growth rates, features of asexual and sexual structures and maximum growth temperatures were examined. APhytophthora species new to Europe,Phytophthora boehmeriae Sawada, attacking cotton bolls, was identified. The pathogenicity of the isolates was confirmed by artificial inoculations of detached cotton bolls. Analysis of α-esterase isozymes revealed unique banding patterns for isolates ofP. boehmeriae compared with those ofP. cactorum andP. parasitica, which arePhytophthora species with similar morphology.     

Evaluation of morphology of infection structures in distinguishing between different Allium rust fungi

Fifteen isolates of rust fungi were collected in the United Kingdom and in the Netherlands from severalAllium species. The samples represented three putative rust species. The morphology of the telia, teliospores and urediniospores was investigated. From urediniospores infection structures were induced on leek, and their morphology was described. Telia and teliospores were not available on every sample. Morphology of the infection structures clearly differentiated between ‘leek’ type and ‘chive’ type isolates. The morphology of infection structures ofPuccinia mixta is very similar to that ofUromyces ambiguus, but clearly distinct fromPuccinia allii sensu Jennings from leek. Quantitative differences in urediniospore characters differentiated between the putative species, but there was overlap between the taxa. We conclude that morphology of infection structures of urediniosporelings is a useful trait for identification of rust isolates fromAllium. This is especially true where more than one rust species may occur on the same host species, as withAllium fistulosum.     

Characterization of theErwinia amylovora population in Israel

A collection of 205 strains ofErwinia amylovora isolated in Israel over a period of 12 years has been established. The strains were isolated from different varieties of pear, apple, loquat and quince grown in Israel, and collected from different locations in the country. They were characterized in respect to degree of virulence on several hosts and serological and molecular characters. Pathogenicity tests carried out on flowering branches of pear and apple, shoots of pears, and on trees of pear and loquat grown in containers outdoors, revealed no significant differences in the severity of blossom blight or shoot blight among the various strains. ELISA and immunofluorescence assays revealed no serotypic groups among the Israeli strains. Genomic diversity was studied by random amplified polymorphic DNA (RAPD) analysis using 24 arbitrary 10-base primers. All the strains examined (45 Israeli and 11 from Egypt, Cyprus and Greece) produced the same RAPD patterns with each of the primers used. Amplification patterns were indistinguishable from those produced by strains isolated from the neighboring countries. Results presented in this study suggest that the population ofE. amylovora in Israel is homogenous.     

Enhancement of Postharvest Disease Resistance in Ya Li Pear (Pyrus bretschneideri) Fruit by Salicylic Acid Sprays on the Trees during Fruit Growth

The Ya Li pear (Pyrus bretschneideri) trees were sprayed three times with 2.5 mM salicylic acid (SA) around 30, 60 and 90 days after full flowering. The fruit were harvested at commercial maturity (about 120 days after full flowering), inoculated with Penicillium expansum, and incubated at 20 °C, 95–100% RH. The results showed that resistance to the pathogen of the mature pear fruit was remarkably enhanced by the SA sprays. Disease incidence in the SA-treated fruit was 58.0% or 26.5%, and lesion diameter on SA-treated fruit was 58.4% or 29.0% lower than that in/on fruit without SA treatment (control) on day 12 or 17 after incubation, respectively. The SA spray applied to the trees around 30 days after full flowering notably enhanced accumulation of hydrogen peroxide in the young fruit. Meanwhile, activities of defense enzymes, including peroxidase, phenylalanine ammonia-lyase (PAL), chitinase or β-1,3-glucanase in the young fruit from SA-treated trees was 29.5%, 60.0%, 24.4% or 35.7% higher than that in the control fruit 4 days after the SA spraying. Furthermore, after harvest, activities of PAL, chitinase and β-1,3-glucanase were still significantly higher in the mature pear fruit from the trees sprayed three times with SA than those of the control fruit. Activities of the antioxidant enzymes including catalase and ascorbate peroxidase in the young fruit were significantly reduced by SA spraying. However, the activity of another antioxidant enzyme, glutathione reductase in the young fruit was significantly enhanced by SA spraying. These results suggest that enzymes exerting their functions in different ways may be coordinately regulated by SA in the pear fruit. Our study indicates that treatment of SA sprays on the trees may provide further protection against postharvest disease of Ya Li pear fruit in practice and could be used as an alternative and economical approach to reduce application of chemical fungicides.     

Preliminary evaluation of nine fungicides for control ofPhytophthora cactorum andP. citrophthora associated with crown rot in peach trees

Excised twig assay and excised stem inoculation were used to evaluate nine fungicides (metalaxyl, fosetyl-Al, copper hydroxide, copper sulfate, copper oxychloride, captan, quintozene, propamocarb and chlorothalonil) againstPhytophthora cactorum andP. citrophthora associated with crown rot in peach trees. Segments were soaked in fungicide solutions at different concentrations and then inserted vertically intoP. cactorum orP. citrophthora cultures growing on cornmeal agar plus antibiotics, or inoculated by inserting a mycelium-bearing agar plug directly into the cambium. Following incubation, the bark was scraped off and length of necrosis was measured. Metalaxyl was the only fungicide that inhibited canker development on segments at the manufacturer-recommended concentration. Fosetyl-Al, captan, copper hydroxide and copper sulfate inhibited canker development at 3, 4, 4 and 8 gl^-1, respectively. The other fungicides did not affect canker length significantly compared with non-treated twigs, with the exception of propamocarb, which reduced the development ofP. cactorum on excised stems. The tested methods enabled rapid and effective evaluation of a large number of chemicals to prevent crown rot diseases caused byPhytophthora in the laboratory. http://www.phytoparasitica.org posting Dec. 5, 2001.     

Detection and Identification of <Emphasis Type="Italic">Phytophthora</Emphasis> Species in Southern Italy by RFLP and Sequence Analysis of PCR-amplified Nuclear Ribosomal DNA

In four neighbouring regions of southern Italy, Basilicata, Campania, Apulia and Calabria, pepper and zucchini plants showing Phytophthora blight symptoms, tomato plants with either late blight or buckeye rot symptoms, plants of strawberry showing crown rot symptoms and declining clementine trees with root and fruit rot were examined for Phytophthora infections by means of polymerase chain reaction (PCR) assays, using primers directed to nuclear ribosomal DNA (rDNA) repeat sequences. All diseased plants and trees examined tested positive. The detected fungal-like organisms were differentiated and characterized on the basis of primer specificity as well as through extensive restriction fragment length polymorphism (RFLP) and sequence analysis of PCR-amplified rDNA. Phytophthora capsici was identified in diseased pepper and zucchini plants, P. infestans was identified in tomato with late blight symptoms whereas buckeye rot-affected tomatoes and diseased strawberry plants proved to be infected by P. nicotianae and P. cactorum, respectively. Declining clementine trees were infected with P. citrophthora and P. nicotianae in about the same proportion. Also, thirty-one pure culture-maintained isolates of Phytophthora which had previously been identified in southern Italy by traditional methods but were never examined molecularly, were examined by RFLP and sequence analysis of PCR-amplified nuclear rDNA. Among these, an isolate from gerbera which had previously been identified by traditional methods only at genus level, was assigned to P. tentaculata. For the remaining pure culture-maintained isolates examined, the molecular identification data obtained corresponded with those delineated by traditional methods. Most of the diseases examined were already known to occur in southern Italy but the pathogens were molecularly detected and fully characterized at nuclear rDNA repeat level only from other geographic areas, very often outside Italy. A new disease to southern Italy was the Phytophthora blight of zucchini. This is also the first report on the presence and molecular identification of P. tentaculata from Italy.     

Monitoring of <Emphasis Type="Italic">Phytophthora</Emphasis> species on fruit trees in Bulgaria

Disease on fruit trees in Bulgaria caused by Phytopthora cactorum and P. citrophthora was found in the period 1998–1999. Leaves of some trees become reddish during July, and later in the season fall off. Infected trees die during the same season, or the next season. Observations on symptom development and spread of Phytophthora root and crown rot of fruit trees was undertaken from 1999 to 2009. Disease incidence is between 2% and 14% in some gardens and nurseries. The disease was registered in the regions of Plovdiv, Kjustendil, Sliven, Yambol, Karnobat, Bourgas and Svishtov. Samples from infected plant tissues were taken and isolations were done on selective PARP media, or by applying a baiting bioassay. Based on morphological and cultural characteristics and temperature requirements the following Phytophthora species have been identified: Phytophthora cactorum, P. citrophthora, P. drechsleri, P. cryptogea, hybrid and Pythium. Pathogenicity of the isolates was tested on green apple fruits or one-year-old apple rootstocks. Laboratory studies of the effect of temperature on mycelia growth showed that most isolates can grow from 5° up to 30°C, with an optimum from 18° to 25°C. Only three strains grew at 35–36°C, two developed slowly, one grew well. The optimal pH for mycelia development was tested. Aiming at control of disease, in vivo pot trials have been carried out for studying resistance of rootstocks to P. cactorum. At the end of the growing season a good level of resistance has been shown in the rootstocks M29C, Gizela 6, and MAXMA 14.     

Vegetative Compatibility amongVerticillium dahliae Isolates from Watermelon in Greece

Vegetative compatibility among 17 isolates ofVerticillium dahliae obtained from watermelon, originating from eight regions of Greece, was investigated using complementation tests between nitrate-nonutilizing(nit) mutants. Among 529 chlorate-resistant sectors obtained, only 107 werenit mutants. These mutants were paired with tester strains (from Greece and other countries) of previously described vegetative compatibility groups (VCGs), and also were paired in many combinations among themselves. All isolates were self-compatible. Sixteen isolates were found to belong to VCG2. Only isolate V75 could not be assigned to a VCG, because the threenit mutants obtained from it showed negative reactions with the tester strains of four VCGs and with complementary mutants from other isolates. Based on this sample, we conclude that the population ofV. dahliae from watermelon in Greece is homogeneous in respect to VCG.     

Characterization of <Emphasis Type="Italic">Pectobacterium carotovorum</Emphasis> subsp. <Emphasis Type="Italic">carotovorum</Emphasis> causing soft-rot disease on <Emphasis Type="Italic">Pinellia ternata</Emphasis> in China

Pinellia ternata is a traditional Chinese herb which has been used in China for over 1,000 years. A soft-rot disease characterized by water-soaked lesions and soft-rot symptoms with a stinking odour was commonly observed in cultivated fields of this plant, and Pectobacterium-like bacteria were consistently isolated from the infected tissues. Two typical strains (SXR1 and ZJR1), isolated from Shanxi and Zhejiang, respectively, were identified. Pathogenicity tests revealed that these strains were virulent to P. ternata and induced the same symptoms as observed in the field. Characterization involving fatty acid profile, metabolic and physiological properties, 16S rDNA sequence and PCR-RFLP identified both isolates as P. carotovorum subsp. carotovorum (Pcc). The 16S rDNA of both isolates shared 97–99% sequence similarity with that of Pcc strains. The phylogenetic trees showed that both isolates were clustered in the group of Pcc and P. carotovorum subsp. odorifera and both PCR-RFLP profiles were consistent with the pattern E produced by the minority of Pcc strains. Thus, isolates SXR1 and ZJR1 were characterized as Pcc in spite of some differences. This is the first report that Pcc has been proven as a causal agent of soft-rot disease on P. ternata.     

<Emphasis Type="Italic">Neofusicoccum parvum</Emphasis> associated with fruit rot and shoot blight of peaches in Greece

Shoot blights and fruit rots comprise the most serious diseases of peaches in Greece. In this study, the importance of the fungus Neofusicoccum parvum as a casual agent of a fruit rot and shoot blight of peach trees in Greece was investigated. This pathogen was isolated from both immature and mature peach fruit of the cultivar “Catherine” and later on from mature fruit of the peach cultivars “Andross”, “RedHaven”, “Sun Crest” and “Sun Cloud”. In the first year of investigation, N. parvum was found causing preharvest fruit rot and shoot blights of peach trees only at the location “Ammos-Mesi-Meliki Verias” in the prefecture of Imathia (the main peach production area of Greece) at incidences of 30 and 8%, respectively. However, in 2006 N. parvum was isolated from more locations such as Diavatos, Veria, Kopanos and Agia Marina in the prefecture of Imathia, but only at less than 3% of the total surveyed rotted peach fruit and blighted shoots. The pathogen overwintered as sub-epidermal pycnidia in blighted shoots or mummified fruit that remained on peach trees. This study also showed that the optimum temperature for mycelial growth and conidial germination of N. parvum was 25 oC. Pathogenicity tests using peach fruit showed that isolates of N. parvum and Diplodia seriata (isolated from pistachio grown in the same region) showed no significant differences in their virulence. In laboratory inoculation tests using detached shoots from 25 peach and nectarine cultivars, N. parvum isolates obtained from rotted peaches caused different size cankers on these cultivars. The cultivar Big Top was the most susceptible while the cultivar Maria Bianca the least susceptible.     

Real-time detection of <Emphasis Type="Italic">Phytophthora nicotianae</Emphasis> and <Emphasis Type="Italic">P. citrophthora</Emphasis>in citrus roots and soil

Two primers, specific for Phytophthora nicotianae (Pn6) and P. citrophthora (Pc2B), were modified to obtain Scorpion primers for real-time identification and detection of both pathogens in citrus nursery soils and roots. Multiplex PCR with dual-labelled fluorogenic probes allowed concurrent identification of both species ofPhytophthora among 150 fungal isolates, including 14 species of Phytophthora. Using P. nicotianaespecific primers a delayed and lower fluorescence increase was also obtained from P. cactorumDNA. However, in separate real-time amplifications, the aspecific increase of fluorescence from P. cactorum was avoided by increasing the annealing temperature. In multiplex PCR, with a series of 10-fold DNA dilutions, the detection limit was 10 pg l^-1 for P. nicotianaeand 100 pg l^–1 for P. citrophthora, whereas in separate reaction DNA up to 1 pg l^-1 was detected for both pathogens.Simple and rapid procedures for direct DNA extraction from soil and roots were utilised to yield DNA whose purity and quality was suitable for PCR assays. By combining these protocols with a double amplification (nested Scorpion-PCR) using primers Ph2-ITS4 amplifying DNA from the main Phytophthora species (first round) and PnB5-Pn6 Scorpion and Pc2B Scorpion-Pc7 (second round), it was possible to achieve real-time detection of P. nicotianaeand P. citrophthora from roots and soil. The degree of sensitivity was similar to that of traditional detection methods based on the use of selective media. The analyses of artificially and naturally infested soil showed a high and significant correlation between the concentration of pathogen propagules and the real-time PCR cycle threshold.     

Nitrogen effect on competition between winter cereals and littleseed canarygrass

Field experiments were conducted in northern Greece during 1994, 1995, and 1996 to study the effect of nitrogen fertilization on competition between littleseed canaryglass (Phalaris minor Retz.) and wheat (Triticum aestivum L.), barley (Hordeum vulgare =distichum L.) or triticale (Triticosecale). The presence of 400P. minor plants per square meter until early March did not have an adverse effect on dry weight of any crop. However, their further presence significantly reduced dry weight of wheat and triticale, but not that of barley. Grain yield of wheat and triticale was reduced 48% and 47%, respectively, by season-long competition ofP. minor, whereas the corresponding reduction for barley was only 8%. Crop yield reduction due toP. minor competition resulted mainly from reduction in ear number and less from reduction in 1000-grain weight. Nitrogen fertilization (150 kg N ha⁻¹), compared with control (0 kg N), slightly increased yield of all crops grown without weed competition. The same treatment also increased dry weight and competitive ability ofP. minor against wheat and triticale, compared with that of control (0 kg N); the split application of nitrogen (50 kg N ha⁻¹ before crop sowing and 100 kg N ha⁻¹ in early March) caused a slightly greater increase inP. minor dry weight than did 150 kg N ha⁻¹ applied once before crop sowing. Dry weight ofP. minor grown with barley was not affected by nitrogen fertilization, but it was severely reduced compared with that ofP. minor grown with wheat or triticale. http://www.phytoparasitica.org posting April 22, 2003.     

<Emphasis Type="Italic">Phoma glomerata</Emphasis> (Corda) Wollenw. &; Hochapfel a new threat causing cankers on shoots of peach trees in Greece

Shoot blights are common diseases of peach trees in Greece. This study is the first report of a shoot blight and canker disease of peach in Greece caused by the fungus Phoma glomerata (Corda) Wollenw. & Hochapfel. The pathogen caused distinct cankers with abundant gumming on shoots of peach trees. The rate of development of P. glomerata in vitro was reduced as temperatures increased from 25°C to 30°C, decreased from 25°C to 15°C, and was totally inhibited at 35 and 10°C. The rate of conidial germination and the germ tube elongation in vitro was reduced as temperatures increased from 25°C to 35°C, decreased from 25°C to 10°C, and was totally inhibited at 2–4°C. Pathogenicity tests showed that 24 peach and nectarine cultivars grown in Imathia Perfecture, Greece, were equally susceptible to P. glomerata. The fungicides thiophanate methyl, carbendazim, and tebuconazole were evaluated against the development of P. glomerata and disease symptoms. All fungicides inhibited the growth and conidial germination of P. glomereta and disease symptoms, and all 30 isolates tested were sensitive to the above fungicides. The disease caused by P. glomerata could be a threat to peach cultivation in Greece and its management should be investigated in the field.     

Genetic variation and host specificity of <Emphasis Type="Italic">Phytophthora citrophthora</Emphasis> isolates causing branch cankers in Clementine trees in Spain

Considerable tree losses have been observed during the past few years in Spain due to Phytophthora branch canker of clementines caused by Phytophthora citrophthora. The emergence of this disease led to the speculation that either the pathogen has evolved increasing its aggressiveness or specificity to clementines. A total of 134 isolates of P. citrophthora collected from 2003 to 2005 in 135 citrus orchards in Spain and 22 reference isolates were analyzed genotypically and phenotypically to determine the structure of the population. Genotypic diversity was evaluated by means of Inter-Simple Sequence Repeat (ISSR) markers. Among the phenotypic characteristics examined, sporangial characters, sexual behavior, growth rates and colony morphology of the isolates at different temperatures were studied. The aggressiveness and host-specificity of selected isolates were evaluated by pathogenicity tests on sweet oranges and clementines under field conditions. Phytophthora branch canker of clementines was associated mainly with one genotype (P-1), which included 88% of the isolates obtained from branches. Strains isolated years before the first disease outbreak clustered also with this major genotype, thus it may be considered as a predominant population. Thirteen other minor genotypes were determined, but most contained only one isolate. Although there was wide variation in the morphological and physiological characters, all Phytophthora isolates obtained from branch cankers were sexually sterile and showed a characteristic petalloid colony pattern. As in previous greenhouse studies, pathogenicity tests under field conditions demonstrated that clementines and their hybrids were more susceptible to P. citrophthora than sweet oranges. However, no evidence was found to support the hypothesis that the emergence of the disease was associated with more aggressive or host-specific forms of P. citrophthora.     

Seasonal susceptibility of citrus scions to <Emphasis Type="Italic">Phytophthora citrophthora</Emphasis> and <Emphasis Type="Italic">P. nicotianae</Emphasis> and the influence of environmental and host-linked factors on infection development

Three citrus scions were evaluated to determine seasonal changes in susceptibility to infections by Phytophthora citrophthora and Phytophthora nicotianae. In a period of 24 months, the Clementine mandarin cv. Hernandina, the hybrid Fortune mandarin and the sweet orange cv. Lane-Late were branch-inoculated under field and laboratory conditions. Field studies showed that the cultivars inoculated with P. citrophthora developed the highest lesion areas during March–June (spring) and September–October (autumn) and with P. nicotianae from June to August (summer). However, lesion areas on detached citrus branches did not show a definite pattern of infection because lesion sizes fluctuated irregularly during the study. The lesion area caused by P. nicotianae in different citrus scions correlated significantly with the monthly mean maximum values of temperature, relative humidity, and the percentage of the relative water content in the 24-month period of inoculations. In contrast, there was no correlation between these variables and the extent of colonisation by P. citrophthora. Nevertheless, a significant relationship was observed between lesion areas caused by P. citrophthora from October to May of each year and the same variables that were significant in inoculations with P. nicotianae. Seasonal changes in the susceptibility of citrus cultivars to P. citrophthora and P. nicotianae may facilitate timing of disease control measures to coincide with periods when disease development is greatest.     

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.