Real-time Scorpion-PCR detection and quantification of <Emphasis Type="Italic">Erwinia amylovora</Emphasis> on pear leaves and flowers

A specific primer couple (E3–E4) amplifying a single DNA fragment of 111 bp from plasmid pEA29 was designed to identify, detect and quantify Erwinia amylovora by real-time Scorpion-PCR. Specificity of primers and probe was assessed both by means of BLAST analyses and by using genomic DNA from a large number of E. amylovora isolates and other bacteria. In Scorpion-PCR, the limit of detection was of 1 pg of total DNA and a high correlation (r = 0.999) was achieved between target DNA quantity and cycle threshold (Ct). Combining two sequential amplifications with conventional reported primers (PEANT1–PEANT2) and Scorpion primers (E3 Scorpion-E4) the detection limit was of 1 fg (nested Scorpion-PCR). Using serial dilution of the bacterial suspensions the limit of detection was 3.2 × 10⁴ CFU ml⁻¹ in Scorpion-PCR and 2.8 × 10² CFU ml⁻¹ in nested Scorpion-PCR. Real-time PCR combined with effective procedures for DNA extraction enabled the detection and the quantification of the epiphytic population of E. amylovora in the washings of flowers and leaves of artificially inoculated pear. A significant correlation (r = 0.92) was achieved between pathogen CFU on semi-selective media and the corresponding target DNA concentration evaluated by real-time PCR.     

Occurrence of bacterial black shoot disease of European pear in Yamagata Prefecture

Black lesions on shoots of European pear trees observed in an orchard in Yamagata Prefecture in May 2007 were suspected to be caused by a bacterial pathogen. The surface of the colonies isolated on a high sucrose medium did not have the crater morphology that is characteristic of E. amylovora bvs. 1–3, and a specific DNA fragment was amplified from the isolates in the PCR using the EprpoD primer set. The partial sequences of the 16S rRNA gene placed the isolates in the genus Erwinia. The isolates differed serologically from E. amylovora biovars and E. pyrifoliae in an Ouchterlony double-diffusion test although their bacterial properties suggested that they are closely related to E. amylovora biovars and E. pyrifoliae. In a DNA–DNA hybridization test, the relatedness between the isolates and E. amylovora biovars or E. pyrifoliae did not exceed 70% level, indicating that they are independent species. Thus, the isolates belongs to the genus Erwnia but are not E. amylovra or E. pyrifoliae. After succulent pear shoots were injected with bacterial suspensions (10⁹, 10⁸, 10⁷ and 10⁶ cfu/ml) of the isolates, lesions formed with 10⁹ and 10⁸ cfu/ml, but the disease incidence with 10⁸ cfu/ml was much lower than with E. amylovora and E. pyrifoliae. Virulence of the present isolates is thus thought to be very weak. On the basis of these results, we consider that this is a new shoot disease of European pear. In the 2007 season, all affected trees were pulled out after harvest. No symptoms have been observed in field surveys since the fruitlet season in 2007.     

玫烟色虫草和球孢白僵菌对朱砂叶螨和二斑叶螨的致病力评价

为探寻具有杀螨潜力的生防真菌,采用喷雾法测定分析玫烟色虫草Cordyceps fumosorosea IF-1106菌株和球孢白僵菌Beauveria bassiana BB-1339菌株对朱砂叶螨Tetranychus cinnabarinus和二斑叶螨Tetranychus urticae卵、幼螨及雌成螨的致病力。结果表明,感染玫烟色虫草IF-1106菌株和球孢白僵菌BB-1339菌株后螨类的形态特征不一致,感染IF-1106菌株后形成棉絮状菌丝,而感染BB-1339菌株后则形成羊毛状菌丝。IF-1106菌株和BB-1339菌株对朱砂叶螨和二斑叶螨卵的LC₅₀分别为2.38×10⁷、8.26×10⁷CFU/mL和4.48×10⁷、1.21×10⁸CFU/mL,对朱砂叶螨和二斑叶螨幼螨的LC₅₀分别1.97×10⁷、8.26×10⁷CFU/mL和7.65×10⁶、8.99×10^5...     

Contamination of bean seeds by <Emphasis Type="Italic">Xanthomonas axonopodis</Emphasis> pv. <Emphasis Type="Italic">phaseoli</Emphasis> associated with low bacterial densities in the phyllosphere under field and greenhouse conditions

Xanthomonas axonopodis pv. phaseoli and its variant fuscans are the causal agents of common bacterial blight of bean. Production of seeds is recommended in arid climates with the use of pathogen-free seeds. However, contamination of seeds still occurs in these seed production areas. To verify if low contamination levels of sown seeds could explain these field contaminations, we used seeds that were naturally contaminated with CFBP4834-R, a rifamycin-resistant X. axonopodis pv. phaseoli fuscous strain, to contaminate field plots at different rates. We also inoculated seeds to verify some parameters of plant colonization and seed transmission. In growth chambers, seedling contamination was always successful from seeds contaminated with CFBP4834-R having population sizes greater than 1 × 10³ CFU seed⁻¹ and were not successful below 1 × 10² CFU seed⁻¹. In the greenhouse, the efficiency of contamination of seeds was not significantly different between contaminated plants that had a low or a high CFBP4834-R population size and reached between 40% and 52% whatever the origin of the inoculum (aerial or seed-borne). In field experiments, under low relative humidity, plots with 0.1–0.003% contamination rates or plots sown with seeds that were inoculated with low CFBP4834-R population sizes (1 × 10² and 1 × 10⁴ CFU seed⁻¹) led to an asymptomatic colonization of bean during the entire growing season with low CFBP4834-R population sizes. Seeds were contaminated both in primary and secondary foci. The contamination of seeds without symptom expression during the growing season represents a risk for eventual disease outbreaks.     

Sensitivity of <Emphasis Type="Italic">Botrytis cinerea</Emphasis> to propamidine: in vitro determination of baseline sensitivity and the risk of resistance

The baseline sensitivity of Botrytis cinerea to propamidine and assessment of the risk of propamidine resistance in vitro are presented in this article. The baseline sensitivities of 41 wild-type strains were distributed as a unimodal curve with EC₅₀ values of mycelial growth ranging from 0.182 to 1.460 μg ml⁻¹, with a mean of 0.79 ± 0.27 μg ml⁻¹. A total of 10 resistant mutants, obtained from one parental strain, were induced by UV irradiation and selected for resistance to propamidine with an average frequency of 1.98 × 10⁻⁹ and 0.025 respectively. These mutants were divided into three classes of resistant phenotypes with low (LR), moderate (MR) and high (HR) levels of resistance, determined by the EC₅₀ values of 5.0–15.0 μg ml⁻¹, 15.1–75.0 μg ml⁻¹ and more than 75.0 μg ml⁻¹ respectively. Neither positive cross-resistance nor negative cross-resistance was detected between propamidine and the fungicides, benzimidazole carbendazim, anilino-pyrimidine pyrimethanil, dicarboximide iprodione or procymidone. All 10 propamidine-resistant mutants showed reduced mycelial growth in vitro, sporulation, spore germination and pathogenicity when compared with the parental strain. These studies demonstrated that propamidine possesses a low risk of resistance developing. However, as B. cinerea is a high-risk pathogen, appropriate precautions against resistance development should be taken.     

Efficacy of bacterial antagonists and different commercial products against Fusarium wilt on rocket

Seven experimental trials were carried out to evaluate the efficacy of the bacterial strains Achromobacter xylosoxydans AM1 and Serratia sp. DM1 obtained from suppressive soils and from soilless used rockwool substrates (Pseudomonas putida FC6B, Pseudomonas sp. FC7B, Pseudomonas putida FC8B, Pseudomonas sp. FC9B and Pseudomonas sp. FC24B) against Fusarium wilt on rocket caused by Fusarium oxysporum ff. spp. raphani and conglutinans. Along with these strains, two commercial bioproducts (RootShield—Trichoderma harzianum T22; Cedomon—Pseudomonas chlororaphis MA342) were also tested. Different application strategies such as soil treatment (trials I to VI; 10⁷ and 10⁸ CFU ml⁻¹) and root dipping (trial VII; 10⁸ and 10⁹ CFU ml⁻¹) were performed in a glasshouse in order to test the efficacy of the bacterial strains against Fusarium oxysporum ff. spp. raphani and conglutinans. The lowest disease incidence (16.7%) was observed with the application of Achromobacter sp. AM1, Serratia sp. DM1 at 10⁸ CFU ml⁻¹ and Pseudomonas sp. FC9B at 10⁷ CFU ml⁻¹ against F. oxysporum f. sp. conglutinans (experiment I). Maximum plant biomass (5.0 g/plant) was registered in Serratia sp. DM1 at 10⁸ CFU ml⁻¹ treated plants in trial IV. The trials against F. oxysporum f. sp. raphani (experiment II) showed that the application of Pseudomonas sp. FC7B, P. putida FC8B at 10⁸ CFU ml⁻¹ and P. chlororaphis MA342 at 7.5 × 10⁶ CFU ml⁻¹ significantly reduced disease incidence to values ranging between 87% and 92%. The highest plant biomass was recorded with the application of Achromobacter sp. AM1 and P. putida FC6B at 10⁷ CFU ml⁻¹ (3.9 to 4.2 g) carried out 7 days before the artificial inoculation of the pathogens (trial IV). The present study showed the potential biocontrol activity of the bacterial strains Achromobacter sp. AM1, Serratia sp. DM1 and Pseudomonas sp. FC9B against F. oxysporum f. sp. conglutinans and of Pseudomonas sp. FC7B, P. putida FC8B, P. chlororaphis MA342, Achromobacter sp. AM1 and P. putida FC6B against F. oxysporum f. sp. raphani. Growth-promoting activity of biocontrol bacteria used during the trials was observed.     

Improved control of moldy-core decay (<Emphasis Type="Italic">Alternaria alternata</Emphasis>) in Red Delicious apple fruit by mixtures of DMI fungicides and captan

The sterol biosynthesis inhibitors bromuconazole and difenoconazole and tank mixes of each fungicide with captan were applied to apples and evaluated as controls for moldy-core and fruit decay caused by Alternaria alternata. Effectiveness of a mixture of bromuconazole and captan in controlling colonization by the fungus was also evaluated. Decay formation by A. alternata on mature detached fruits was partially inhibited by bromuconazole at 0.5 μg ml⁻¹ and was completely inhibited at 50 μg ml⁻¹; it was significantly affected by either bromoconazole at 5 μg ml⁻¹ or captan at 1,250 μg ml⁻¹, and was completely inhibited by their mixture. In general, three foliar applications of bromuconazole or difenoconazole in the field, during the bloom period, reduced the numbers of infected fruits by 40–60% compared with untreated control trees. However, tank mixes of either fungicide with captan improved control of moldy-core in fruits at harvest. Tank mixtures of bromuconazole and captan also significantly reduced the percentage of fruits colonized by A. alternata when sampled at various days after full bloom. Artificial inoculations in the orchard at full bloom did not change the inhibitory effects of the tank mixtures. Large-scale demonstration trials in commercial orchards supported these findings. The inhibitory effects of tank mixes on decay development in detached fruits, and on moldy-core in the field indicate that a control programme based on mixtures of either bromuconazole or difenoconazole with captan during the bloom period can effectively reduce moldy-core on Red Delicious apples.     

Effect of seed priming with <Emphasis Type="Italic">Serratia plymuthica</Emphasis> and <Emphasis Type="Italic">Pseudomonas chlororaphis</Emphasis> to control <Emphasis Type="Italic">Leptosphaeria maculans</Emphasis> in different oilseed rape cultivars

The efficacy of a seed treatment of oilseed rape (OSR) (Brassica napus) with the rhizobacteria Serratia plymuthica (strain HRO-C48) and Pseudomonas chlororaphis (strain MA 342) applied alone or in combination against the blackleg disease caused by Leptosphaeria maculans was tested with different cultivars. Seeds were soaked in bacterial suspensions (bio-priming) to obtain log₁₀ 6–7 CFU seed⁻¹. Cotyledons were inoculated with a 10 ul droplet of L. maculans spore suspension of log₁₀ 7 spores ml⁻¹ and the disease index (size of lesions) was evaluated 14 days later. A mean disease reduction of 71.6% was recorded for S. plymuthica and of 54% for P. chlororaphis. The combined treatment was not superior to the treatment with S. plymuthica alone. The reduction of the disease caused by S. plymuthica was independent of the cultivar’s susceptibility, whereas the control effect recorded with P. chlororaphis increased with decreasing cultivar resistance to blackleg disease. The bacterial colonization of OSR was restricted to the roots and hypocotyl. No significant difference in bacterial colonization of the rhizosphere was observed between different cultivars, nor between single or combined bacterial seed treatments.     

Genetic resistance to bacterial blight disease in Persian walnut

Bacterial blight disease of Persian walnut (Juglans regia, L.), caused by Xanthomonas arboricola pv. juglandis (Xaj), leads to significant nut losses in northern, central and western areas of Iran. To identify the natural sources of resistance to disease in the endemic walnut genotypes of Iran, sixteen walnut genotypes, collected from different areas of Hamedan province, were inoculated with Xaj in a randomized complete block design with five replicates for each genotype. Two-year old genotypes were gently sprayed with a suspension of bacteria adjusted to approximately 2 × 10⁹ cfu ml⁻¹ of distilled water in May. Infected leaves were rated for disease 28 and 42 days after inoculation, using a 0 to 5 severity scale, based on the number, size and distribution of lesions on the leaves. Data analyses showed that there were variations among genotypes in response to pathogen. Upon inoculation by bacterial suspension genotype 94 showed the highest resistance to both disease incidence and its progress after 4–6 weeks of infection. Genotype 65 showed high susceptibility to disease and genotype 69 showed high susceptibilities both to disease incidence and its progress after 4–6 weeks of infection.     

Biological control of pythium root rot of chrysanthemum in small-scale hydroponic units

The capacity of several strains of root-colonizing bacteria to suppressPythium aphanidermatum, Pythium dissotocum and root rot was investigated in chrysanthemums grown in single-plant hydroponic units containing an aerated nutrient solution. The strains were applied in the nutrient solution at a final density of 10⁴ CFU ml⁻¹ and 14 days later the root systems were inoculated withPythium by immersion in suspensions of 10⁴ zoospores ml⁻¹ solution. Controls received no bacteria, noPythium, or one of thePythium spp. but no bacteria. Strain effectiveness was estimated based on percent roots colonized byPythium and area under disease progress curves (AUDPC). In plants treated respectively withPseudomonas (Ps.)chlororaphis 63-28 andBacillus cereus HY06 and inoculated withP. aphanidermatum, root colonization by the pathogen was 83% and 72% lower than in the pathogen control, and AUDPC values were reduced by 61% and 65%. ForP. dissotocum, the respective strains reduced root colonization by 87% and 91%, and AUDPC values by 70% and 90%. In plants treated respectively withPs. chlororaphis Tx-1 andComamonas acidovorans C-4-7-28, root colonization byP. aphanidermatum was 84% and 80% lower than in the controls and AUDPC values were reduced by 66% and 57%; these strains did not suppressP. dissotocum. Burkholderia gladioli C-2-74 andC. acidovorans OCR-7-8-38, respectively, suppressed colonization of roots byP. dissotocum by 74% and 86%, and reduced AUDPC values by 60% and 70%, but were ineffective againstP. aphanidermatum. C. acidovorans OCR-7-8-39 reduced colonization and AUDPC values ofP. aphanidermatum by 57% and 42%, respectively.Pseudomonas corrugata 13,Ps. fluorescens 15 and JZ12, and three additional strains ofC. acidovorans were weakly or nonsuppressive againstP. aphanidermatum. Strains that reduced AUDPC values forP. aphanidermatum orP. dissotocum when applied at 10⁴ CFU ml⁻¹ were 11%–39% less effective at 10³ CFU ml⁻¹. Four tested strains (Ps. chlororaphis 63-28,Ps. chlororaphis Tx-1,B. cereus HY06, andB. gladioli C-7-24) in most instances suppressed root colonization and lowered AUDPC values ofP. aphanidermatum when applied at 14, 7 or 0 days before inoculation, but reduction of the respective variables was generally greater when the strains were applied at 14 days (63%–87% and 75%–78%) or 7 days (44%–47% and 31%–88%) than at 0 days (14%–31% and 23%–62%) before inoculation.Ps. chlororaphis Tx-1,Ps. chlororaphis 63-28 andB. cereus HY06 significantly suppressedP. aphanidermatum whether the temperature of the nutrient solution was high (32°C) or moderate (24°C). Taken together, the observations suggest thatPs. chlororaphis 63-28,B. cereus HY06,Ps. chlororaphis Tx-1,B. gladioli C-2-74 andC. acidovorans OCR-7-8-38 have the potential for controlling Pythium root rot in hydroponic chrysanthemums. http://www.phytoparasitica.org posting Jan. 24, 2007.     

Mutational analysis of type III effector genes from <Emphasis Type="Italic">Xanthomonas citri</Emphasis> subsp. <Emphasis Type="Italic">citri</Emphasis>

Xanthomonas citri subsp. citri, the causal agent of citrus canker, relies extensively on a type III secretion system for infection by delivering type III effectors into host cells. In the genus Xanthomonas, two major regulators, HrpG and HrpX, are involved in the expression of genes encoding the type III secretion system. Twenty-three candidate type III effectors were identified as targets for analysis. The involvement in pathogenicity of 20 candidate effector genes in X. citri strain 306 (Xcc-306) was investigated using site-directed mutagenesis. Pathogenicity assays in grapefruit of 19 genes using site-directed mutagenesis revealed that none of the mutants demonstrated to have reduced ability to cause canker disease. A mutation in the TAL effector pthA4 ^– resulted in loss of hypertrophy although no changes were observed in bacterial growth in leaves. Mutations in hrpG, hrpX, or hrpA genes displayed a complete loss of pathogenicity. Moreover, all mutants maintained the ability to trigger a hypersensitive response (HR) in non-host tomato. In contrast to previous studies, hrpG ^– , hrpX ^– and hrpA ^– mutants also retained the ability to elicit an HR in tomato, indicating the presence of an Hrp independent elicitor in Xcc-306.     

Weed hosts of <Emphasis Type="Italic">Verticillium dahliae</Emphasis> in cotton fields in Turkey and characterization of <Emphasis Type="Italic">V. dahliae</Emphasis> isolates from weeds

A weed survey conducted in 2004 and 2005 in Aydin province of Turkey showed that Solanum nigrum, Xanthium strumarium, Amaranthus retroflexus, Portulaca oleracea, Sonchus oleraceus and Datura stramonium were the most prevalent weeds in the cotton fields exhibiting Verticillium wilt. Verticillium dahliae Kleb. was recovered from A. retroflexus and X. strumarium in those cotton fields. This is the first report of V. dahliae occurring naturally in A. retroflexus in Turkey. Pathogenicity tests on cotton and weeds showed that the virulence of V. dahliae isolates from weeds was higher on cotton plants than on weeds, with the disease severity ranging from 31.7% to 98.0%. Disease severity of V. dahliae isolates was 54.7–93.9% on eggplant, 23.7–51.6% on cucumber and 11.0–16.4% on tomato, whereas it did not cause any disease symptoms, or only low levels, on pepper and bell pepper. Two vegetative compatibility groups (VCGs) were identified among seven tested weed isolates: VCG2A (two isolates) and VCG2B (three isolates) using international reference strains.     

Aggressiveness and mycotoxin production of eight isolates each of <Emphasis Type="Italic">Fusarium graminearum</Emphasis> and <Emphasis Type="Italic">Fusarium verticillioides</Emphasis> for ear rot on susceptible and resistant early maize inbred lines

Fusarium graminearum and F. verticillioides are among the most important pathogens causing ear rot of maize in Central Europe. Our objectives were to (1) compare eight isolates of each species on two susceptible inbred lines for their variation in ear rot rating and mycotoxin production across 3 years, and (2) analyse two susceptible and three resistant inbred lines for potential isolate x line interactions across 2 years by silk-channel inoculation. Ear rot rating, zearalenone (ZEA) and deoxynivalenol (DON) concentrations were evaluated for all F. graminearum isolates. In addition, nivalenol (NIV) concentrations were analysed for two NIV producers. Fumonisin (FUM) concentrations were measured for all F. verticillioides isolates. Mean ear rot severity was highest for DON producers of F. graminearum (62.9% of the ear covered by mycelium), followed by NIV producers of the same species (24.2%) and lowest for F. verticillioides isolates (9.8%). For the latter species, ear rot severities differed highly among years (2006: 24%, 2007: 3%, 2008: 7%). Mycotoxin concentrations among isolates showed a broad range (DON: 100–284 mg kg⁻¹, NIV: 15–38 mg kg⁻¹, ZEA: 1.1–49.5 mg kg⁻¹, FUM: 14.5–57.5 mg kg⁻¹). Genotypic variances were significant for isolates and inbred lines in all traits and for both species. Isolate x line interactions were significant only for ear rot rating (P < 0.01) and DON concentration (P < 0.05) of the F. graminearum isolates, but no rank reversals occurred. Most isolates were capable of differentiating the susceptible from the resistant lines for ear rot severity. For resistance screening, a sufficiently aggressive isolate should be used to warrant maximal differentiation among inbred lines. With respect to F. verticillioides infections, high FUM concentrations were found in grains from ears with minimal disease symptoms.     

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.     

Effects of garlic (<Emphasis Type="Italic">Allium sativum</Emphasis>) juice containing allicin on <Emphasis Type="Italic">Phytophthora infestans</Emphasis> and downy mildew of cucumber caused by <Emphasis Type="Italic">Pseudoperonospora cubensis</Emphasis>

The volatile antimicrobial substance allicin (diallylthiosulphinate) is produced in garlic when the tissues are damaged and the substrate allicin (S-allyl-l-cysteine sulphoxide) mixes with the enzyme alliin-lyase (E.C.4.4.1.4). Allicin undergoes thiol-disulphide exchange reactions with free thiol groups in proteins and it is thought that this is the basis of its antimicrobial action. At 50 μg ml^-1, allicin in garlic juice inhibited the germination of sporangia and cysts and subsequent germ tube growth by Phytophthora infestans both in vitro and in vivo on the leaf surface. Disease severity in P. infestans-infected tomato seedlings was also reduced by spraying leaves with garlic juice containing allicin over the range tested (55–110 μg ml⁻¹) with an effectiveness ranging from approximately 45–100%. Similarly, in growth room experiments at concentrations from 50–1,000 μg ml⁻¹, allicin in garlic juice reduced the severity of cucumber downy mildew caused by Pseudoperonospora cubensis by approximately 50–100%. These results suggest a potential for developing preparations from garlic for use in specialised aspects of organic farming, e.g. for reducing pathogen inoculum potential and perhaps for use under glass in horticulture.     

EMS and UV irradiation induce unstable resistance against CAA fungicides in <Emphasis Type="Italic">Bremia lactucae</Emphasis>

Wild type (WT) field isolates of Bremia lactucae failed to germinate in vitro or infect lettuce leaves in the presence of CAA (carboxylic acid amide) fungicides. Minimal inhibitory concentrations (MIC) for mandipropamid, dimethomorph, benthiavalicarb and iprovalicarb were 0.005, 0.5, 0.5 and 5 μg ml⁻¹, respectively. Mutagenesis experiments showed that spores exposed to EMS (ethyl methane sulphonate) or UV irradiation (254 nm) could infect lettuce leaves in the presence of up to 100 μg ml⁻¹ CAA. The proportion of infected leaves relative to the number of spores inoculated (infection frequency) was inversely related to the concentration of CAA used, ranging between 0 and 160 per 1 × 10⁶ spores. Resistant mutants (RM) lost their resistance within 1–14 reproduction cycles on CAA-treated plants. Crosses were made between RMxWT isolates and RMxRM isolates with an attempt to obtain stable homozygous resistant off-springs. Such crosses yielded few resistant but unstable progeny isolates. Mutagenic treatments given to hybrid isolates also failed to produce stable resistance. Previous gene sequencing data showed that stable resistance to CAAs is based on a single SNP in the cellulose synthase 3 (CesA3) gene of Plasmopara viticola. Therefore, we sequenced a 582 bp DNA fragment of Ces3A of WT, RM and hybrid isolates of B.lactucae. No mutation in this gene fragment was found. We conclude that mutagenic agents like EMS or UV may induce resistance to CAA in Bremia lactucae but this resistance is not stable and not linked to mutations in CesA3 gene.     

Effect of the endophyte <Emphasis Type="Italic">Neotyphodium lolii</Emphasis> on susceptibility and host physiological response of perennial ryegrass to fungal pathogens

The effect of the endophyte Neotyphodium lolii on susceptibility of perennial ryegrass (Lolium perenne) to ten fungal pathogens in detached leaves was studied. The pathogens were Alternaria alternata, Ascochyta leptospora, Bipolaris sorokiniana, Curvularia lunata, Fusarium acuminatum, F. avenaceum, F. chlamydosporum, F. solani, F. oxysporum, and Gliocladium roseum. In addition, the effect of the endophyte on four pathogens (A. alternata, B. sorokiniana, Curvularia lunata and F. avenaceum) in living plants was studied, and changes in host superoxide dismutase (SOD) or peroxidases (POD) activity were examined. The total lengths of lesions on detached leaves were greater (P < 0.05) on E- plants than on E+ plants except for A. leptospora although differences between E+ and E- were not consistently significant at all sample times (days after inoculation).The numbers of lesions were greater (P < 0.05) and the lesions were larger (P < 0.05) on intact E- plants than on intact E+ plants for all of the four pathogens. SOD enzyme activity was significantly greater (P < 0.05) in E+ plants than in E- plants only for A. alternata, C. lunata, and F. avenaceum. POD enzyme activity was significantly greater (P < 0.05) in E+ plants than in E- plants only for C. lunata, B. sorokiniana and the uninoculated control.     

Composition of culture medium influences zoosporogenesis and differentiation of <Emphasis Type="Italic">Aphanomyces cochlioides</Emphasis>

A modified medium was used to culture mycelium and produce a large quantity of zoospores of Aphanomyces cochlioides, a principal pathogen of damping-off disease of sugar beet and root rot disease of spinach. The semisolid medium consisted of 17 g corn meal agar (CMA) added with 4 g of yeast extract (YE) per liter of 50 mM phosphate buffer (pH 6.8–7.0). This medium supported the production of ca. 10⁶ zoospores ml⁻¹ in 6-day-old cultures, approximately 11-fold higher than the commonly used CMA (17 g CMA per liter of water, pH 6.0 ± 0.2). Although morphological characters of the zoospores produced from the hyphae grown on CMA and CMA + YE were almost similar, they contrasted their developmental strategy after encystment induced by mechanical agitation. Cystospores originating from the zoospores on CMA regenerated zoospores (>80%), while those from CMA + YE germinated (ca. 80%) and produced hyphae. Furthermore, 4–10% of the germinated cystospores on CMA + YE had double germ tubes. The soluble protein profiles of zoospores produced on CMA and on CMA + YE demonstrated that several proteins were either different or expressed differently. Our results suggest that the culture medium directly influences zoosporogenesis in A. cochlioides hyphae and the developmental strategy of the produced zoospores.     

Selection,characterization and genetic analysis of laboratory mutants of <Emphasis Type="Italic">Botryotinia fuckeliana</Emphasis> (<Emphasis Type="Italic">Botrytis cinerea</Emphasis>) resistant to the fungicide boscalid

Resistance to the fungicide boscalid in laboratory mutants of Botryotinia fuckeliana (Botrytis cinerea) was investigated. The baseline sensitivity to boscalid was evaluated in terms of colony growth (EC₅₀ = 0.3–3 μg ml⁻¹; MIC = 10–30 μg ml⁻¹) and conidial germination (EC₅₀ = 0.03–0.1 μg ml⁻¹; MIC = 1–3 μg ml⁻¹) tests. Mutants were selected in vitro from wild-type strains of the fungus on a fungicide-amended medium containing acetate as a carbon source. Mutants showed two different levels of resistance to boscalid, distinguishable through the conidial germination tests: low (EC₅₀ ∼ 0.3 μg ml⁻¹, ranging from 0.03 to 1 μg ml⁻¹; MIC > 100 μg ml⁻¹) and high (EC₅₀ > 100 μg ml⁻¹) resistance. Analysis of meiotic progeny from crosses between resistant mutants and sensitive reference strains showed that resistant phenotypes were due to mutations in single major gene(s) inherited in a Mendelian fashion, and linked with both the Daf1 and Mbc1 genes, responsible for resistance to dicarboximide and benzimidazole fungicides, respectively. Gene sequence analysis of the four sub-units of the boscalid-target protein, the succinate dehydrogenase enzyme, revealed that single or double point mutations in the highly conserved regions of the iron-sulphur protein (Ip) gene were associated with resistance. Mutations resulted in proline to leucine or phenylalanine replacements at position 225 (P225L or P225F) in high resistant mutants, and in a histidine to tyrosine replacement at position 272 (H272Y) in low resistant mutants. Sequences of the flavoprotein and the two transmembrane sub-units of succinate dehydrogenase were never affected.