柑橘衰退病毒含量对其蚜传效率的影响

为明确毒源植株和蚜虫中柑橘衰退病毒(Citrus tristeza virus,CTV)的发生情况与蚜虫传播CTV能力的关系,将褐色橘蚜、棉蚜、橘二叉蚜和绣线菊蚜置于分别感染了4个CTV分离株的锦橙上取食24 h后,运用巢式RT-PCR和实时RT-PCR检测蚜虫和锦橙的带毒情况,并分析蚜虫的传毒能力。结果显示,蚜虫中CTV的平均带毒率为0.76~0.84,其中棉蚜的最高,其次为绣线菊蚜、褐色橘蚜和橘二叉蚜。锦橙中各CTV分离株的含量差异不大,与蚜虫传毒效率间无显著相关性;也未发现蚜虫带毒率与其传播CTV能力之间存在相关性。蚜虫体内CTV的含量为5.36×103±2.33×103~2.01×106±3.67×105拷贝,褐色橘蚜中含量最高,其次为棉蚜、橘二叉蚜和绣线菊蚜;且高蚜传能力CTV分离株在褐色橘蚜体内的含量远高于低蚜传能力分离株。表明蚜虫体内CTV的含量可能与蚜虫传毒能力有关。     

Pathogenicity of <Emphasis Type="Italic">Beauveria bassiana</Emphasis> and <Emphasis Type="Italic">Metarhizium anisopliae</Emphasis> against <Emphasis Type="Italic">Galleria mellonella</Emphasis>

The greater wax moth Galleria mellonella L. (Lepidoptera: Pyralidae) is occasionally found in beehives and is a major pest of stored wax. Entomopathogenic fungi have recently received attention as possible biocontrol elements for certain insect pests. In this study, 90 isolates of Beauveria bassiana and 15 isolates of Metarhizium anisopliae were screened for proteases and lipases production. The results showed significant variations in the enzymatic action between the isolates. In the bioassay, the selected isolates evinced high virulence against the 4th instar of the G. mellonella larvae. The isolates BbaAUMC3076, BbaAUMC3263 and ManAUMC3085 realized 100% mortality at concentrations of 5.5 × 10⁶ conidia ml⁻¹, 5.86 × 10⁵ conidia ml⁻¹, and 4.8 × 10⁶ conidia ml⁻¹, respectively. Strong enzymatic activities in vitro did not necessarily indicate high virulence against the tested insect pest. The cuticle of the infected larvae became dark and black-spotted, indicating direct attack of fungus on the defense system of the insects. The LC₅₀ values were 1.43 × 10³, 1.04 × 10⁵ and 5.06 × 10⁴ for Bba3263AUMC, Bba3076AUMC and Man3085AUMC, respectively, and their slopes were determined by computerized probit analysis program as 0.738 ± 0.008, 0.635 ± 0.007 and 1.120 ± 0.024, respectively.     

Survival of <Emphasis Type="Italic">Ralstonia solanacearum</Emphasis> in infected tissues of <Emphasis Type="Italic">Capsicum annuum</Emphasis> and in soils of the state of Pernambuco,Brazil

The survival of Ralstonia solanacearum A1-9^Rif race 1 phylotype I was studied in ten different soil types in the absence of the host plant as well as in infected tissues of the stem and root of bell peppers buried in the soil at 0, 5, and 15 cm. The survival time of R. solanacearum A1-9^Rif in the ten soil types ranged from 42 up to 77 days. Among the chemical and physical characteristics of the soil, clay content, residual moisture, and available water were positively correlated, and pH was negatively correlated, with survival time, population size at 42 days, and area under the population curve. The pathogen survival differed significantly in relation to the plant tissues, but not with respect to the incorporation depth of the infected tissues. The root tissue of bell pepper supported a larger bacterial population at 7 and 21 days (5 × 10⁴ and 3.1 × 10⁴ CFU g⁻¹ tissue, respectively) compared with the stem tissue (0.35 × 10⁴ and 0.48 × 10⁴ CFU g⁻¹ tissue, respectively) and also had a larger area under the population curve. On the other hand, the stem tissues presented a greater decomposition rate and pH compared with the roots. In conclusion, the different types of studied soils as well as the infected bell pepper tissues were considered potential primary sources of R. solanacearum inocula, but only for a short period.     

Growth,population dynamics,and diversity of <Emphasis Type="Italic">Fusarium equiseti</Emphasis> in ginseng fields

Fusarium equiseti is prevalent in ginseng soil, straw mulch and in ginseng root tissues and is the cause of a root surface discolouration on ginseng grown in British Columbia. Population levels of the fungus in ginseng fields ranged from 3.8 × 10³ cfu g⁻¹ soil to 1.4 × 10⁴ cfu g⁻¹ soil and were highest at 0–5 cm soil depths compared to 10–15 cm. Soil population levels were negatively correlated with S content in soil and positively correlated with Zn levels. Barley or wheat straw added to soil significantly increased population levels under laboratory conditions. Mycelial growth in culture was highest at 26–30°C and at pH 7.2–7.8. Samples of flowers and berries, and harvested seed, contained DNA of F. equiseti detected using a Fusarium-specific DNA array and the fungus was isolated from these tissues on agar medium. A high degree of genetic variation in the EF-1 alpha gene sequence was present among 52 isolates of F. equiseti which originated from ginseng fields. At least seven clades were identified. Inoculum dispersal from straw mulch used in ginseng gardens can result in seed contamination by the fungus. In addition, fungal growth near the soil surface under warm summer conditions can result in infection and crown discolouration of ginseng roots.     

Effect of <Emphasis Type="Italic">Beauveria bassiana</Emphasis> mixed with diatomaceous earth on mortality,mycosis and sporulation of <Emphasis Type="Italic">Rhyzopertha dominica</Emphasis> on stored wheat

A laboratory assay was designed to determine the insecticidal efficacy of Beauveria bassiana (Balsamo) Vuillemin (Hyphomycetes: Moniliales) and diatomaceous earth (Diafil 610) against Rhyzopertha dominica (F.) (Coleoptera: Bostrychidae). The fungus B. bassiana was applied at 2.23 × 10⁷, 2.23 × 10⁸ and 2.23 × 10⁹ conidia kg⁻¹ of wheat individually as well as mixed with 200 and 400 ppm of Diafil 610. The conditions for the trials were 30 ± 2^oC with 55% r.h. and the counts for mortality were made after 8, 16 and 24 d. All the dead adults were removed after each count and the vials were kept for the next 60 d to assess the emergence of the F₁ generation. The findings from these studies proved that the extended exposure interval and the highest combined dose rate of the entomopathogenic fungus and the diatomaceous earth gave the maximum mortality of the beetles. The emergence of the progeny was also highly suppressed where the maximum dose rate of the synergized treatments was applied. The rate of mycosis and sporulation in the cadavers of R. dominica was maximum where the low dose rates of B. bassiana were applied.     

Effect of botanical extracts on the biological activity of granulosis virus against <Emphasis Type="Italic">Pieris brassicae</Emphasis>

A granulosis virus strain infecting Pieris brassicae (PbGV) was isolated from the dry temperate region of northwestern Himalayas as a potential microbial agent for its management. The effect of different botanicals (having insecticidal action against P. brassicae) on the bioefficacy of PbGV was evaluated under laboratory conditions using leaf disc bioassays on cabbage for improving the insecticidal performance of the PbGV. The synergistic action of different botanical extracts was evident in terms of reduction in LC₅₀ values against different botanical extracts. Among different extracts, petroleum-ether extract of neem seed kernel (NSK) when combined with PbGV resulted in maximum reduction of LC₅₀ value (4.39 × 10² occlusion bodies [OBs] ml⁻¹) followed by methanolic extract (7.38 × 10² OBs ml⁻¹) and aqueous extract (9.36 × 10³ OBs ml⁻¹) as compared with PbGV alone (1.85 × 10⁴ OBs ml⁻¹) for 2nd instar larvae of the test insect. These trends were found analogous in cases of 3rd and 4th instars of P. brassicae with different solvent extracts of NSK. The other botanicals evaluated, viz., Eupatorium and Artemesia, also resulted in reduction of LC₅₀ values for 2nd, 3rd and 4th instars as compared with PbGV alone when different extracts were combined with virus for bioassays. The studies suggest that the PbGV in combination with botanical pesticides could be more useful as a bio-pesticide against cabbage butterfly (P. brassicae) in IPM programs.     

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.     

Assessment of <Emphasis Type="Italic">Pichia anomala</Emphasis> (strain K) efficacy against blue mould of apples when applied pre- or post-harvest under laboratory conditions and in orchard trials

The yeast Pichia anomala strain K was selected in Belgium from the apple surface for its antagonistic activity against post-harvest diseases of apples. The efficacy of this strain against P. expansum was evaluated in the laboratory in three scenarios designed to mimic practical conditions, with different periods of incubation between biological treatment, wounding of fruit surface, and pathogen inoculation. Higher protection levels and higher final yeast densities were obtained when the applied initial concentration was 1 × 10⁸ cfu ml⁻¹ than when it was only 1 × 10⁵ cfu ml⁻¹. The protection level correlated positively with the yeast density determined in wounds and was influenced by apple surface wetness. In orchard trials spanning two successive years, biological treatment against P. expansum, based on a powder of P. anomala strain K (1 × 10⁷ cfu ml⁻¹), β-1,3-glucans (YGT 2 g l⁻¹), and CaCl₂.2H₂0 (20 g l⁻¹), was applied to apples pre- or post-harvest under practical conditions and its effect compared with standard chemical treatments. The first year, the highest reduction (95.2%) against blue decay was obtained by means of four successive fungicide treatments and the next-highest level (87.6%) with pre-harvest high-volume spraying of the three-component mixture 12 days before harvest. The second year, the best results were obtained with post-harvest Sumico (carbendazim 25% and diethofencarb 25%) treatment and post-harvest biological treatment, both by dipping the apples, 88.3 and 56.3% respectively. A density threshold of 1 × 10⁴ cfu cm⁻² of strain K on the apple surface seemed to be required just after harvest for high protective activity, whatever the method and time of application. In the case of pre-harvest biological treatments, variations in meteorological conditions between the 2 years may have considerably affected strain K population density and its efficacies.     

Conventional PCR and real time quantitative PCR detection of <Emphasis Type="Italic">Phytophthora cryptogea</Emphasis> on <Emphasis Type="Italic">Gerbera jamesonii</Emphasis>

A conventional PCR and a SYBR Green real-time PCR assays for the detection and quantification of Phytophthora cryptogea, an economically important pathogen, have been developed and tested. A conventional primer set (Cryp1 and Cryp2) was designed from the Ypt1 gene of P. cryptogea. A 369 bp product was amplified on DNA from 17 isolates of P. cryptogea. No product was amplified on DNA from 34 other Phytophthora spp., water moulds, true fungi and bacteria. In addition, Cryp1/Cryp2 primers were successfully adapted to real-time PCR. The conventional PCR and real-time PCR assays were compared. The PCR was able to detect the pathogen on naturally infected gerbera plants and on symptomatic artificially infected plants collected 21 days after pathogen inoculation. The detection limit was 5 × 10³ P. cryptogea zoospores and 16 fg of DNA. Real-time PCR showed a detection limit 100 times lower (50 zoospores, 160 ag of DNA) and the possibility of detecting the pathogen in symptomless artificially infected plants and in the re-circulating nutrient solution of closed soilless cultivation systems.     

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.     

Genetic diversity and differentiation of <Emphasis Type="Italic">Helicoverpa armigera</Emphasis> nuclear polyhedrosis virus isolates from India

India has a rich biodiversity of microbes. Soil is the major source for isolation of entomopathogens, after the infected insects themselves. Four isolates of Helicoverpa armigera nuclear polyhedrosis virus (HearNPV) were obtained from the samples collected one each from Anand, Surat and Junagadh of Gujarat and Patancheru of Andhra Pradesh. All the HearNPV isolates appeared as clear, irregular six-sided objects with rounded edges, phase-bright under phase contrast. Junagadh, Surat, Patancheru and Anand isolates gave 27.47–42.80%, 36.83–51.32%, 26.05–43.76% and 42.99–54.85% mortality, respectively, when the percent mortality was pooled over period. The least number of HearNPV polyhedral inclusion bodies (PIBs) (5.1 × 10⁷) of the Anand isolate were required to kill 50% of the H. armigera population within 120 h. The Anand isolate was fastest (90.30 h), followed by Surat (120.26 h), Junagadh (139.53 h) and Patancheru (143.10 h) in killing 50% of the H. armigera population at a dose of 10⁹ PIBs ml⁻¹. RAPD analysis of all 15 arbitrary oligonucleotide primers generated 353 scorable bands with 201 loci. A total of 181 polymorphic bands were obtained, ranging in size from 141 to 1,873 base pairs. The percentage of polymorphic loci was 90.19%. The mean polymorphism information content (PIC) value for 15 primers was found to be 0.99. The similarity coefficient values based on 15 RAPD markers ranged from 0.235 to 0.407. The four isolates were grouped into two clusters: one cluster consisted of Junagadh and Anand and the second cluster consisted of Surat and Patancheru.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation as a useful tool for the molecular genetic study of the phytopathogen <Emphasis Type="Italic">Curvularia lunata</Emphasis>

In order to explore the molecular mechanisms of virulence and genetic variance of Curvularia lunata in maize, an ATMT (Agrobacterium tumefaciens-mediated transformation) system was established in order to create a wide range of insertional transformants of C. lunata. Our results showed that the germinating conidia were the ideal starting material for transformation. Based on our optimised transformation condition, the transformation efficiency of C. lunata with T-DNA was improved greatly, and the average transformation frequency was as high as 84 ± 5 transformants per 1 × 10⁶ germlings. Southern blotting results of 39 randomly-selected transformants showed a unique hybridisation pattern and predominant single-copy insertions. An ATMT library containing approximate 3000 transformants was generated, and four types of transformants were screened in terms of growth rate, sporulation, mycelial pigmentation, and toxin production in vitro. This library will be used to identify genes involved in the virulence of the pathogen.     

<Emphasis Type="Italic">Pseudomonas</Emphasis> sp. LSW25R,antagonistic to plant pathogens,promoted plant growth,and reduced blossom-end rot of tomato fruits in a hydroponic system

A Gram-negative rhizobacterial isolate (LSW25) antagonistic to Pseudomonas corrugata, a vein necrosis pathogen of tomato, and promotes the growth of tomato seedlings was isolated from surface-sterilised tomato roots. A spontaneous rifampicin-resistant mutant (LSW25R) was selected to facilitate its tracking, and identified as Pseudomonas sp. and named as Pseudomonas sp. LSW25R (LSW25R), based on its sequences of the internal transcribed spacer (ITS) region and 16 S rRNA gene. LSW25R inhibited mycelial growth of 12 other plant fungal pathogens such as Botrytis cinerea on V8 agar plates. By using a scanning electron microscope, LSW25R colonised not only the root surface around the natural aperture of tomato radicles but also under epidermal cells like endophytic bacteria. LSW25R successfully colonised the roots of tomato, eggplant and pepper seedlings, significantly promoted the fresh weight, height and dry matter of tomato plants at 10⁸ cfu·ml⁻¹, and increased the plant growth of eggplants and peppers at 10⁴ cfu·ml⁻¹, suggesting that the optimal population density of LSW25R for growth promotion varies from species to species. Moreover, densities of LSW25R inside roots and the lowest leaf of tomato plants were > 9.3 × 10³ cfu·g⁻¹. Although the growth promotion of tomato by LSW25R was observed under N- or Ca-deficient conditions as well as a standard nutrient condition, the uptake of calcium was increased only under the standard nutrient condition. In a hydroponic system, LSW25R not only successfully colonised the rhizosphere during cultivation due to its broad spectrum of antifungal activity and endophytic colonisation, but also reduced blossom-end rot of tomato fruits presumably through increasing calcium uptake.     

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.     

球孢白僵菌孢子萌发促进剂的筛选及其对杀蚜效果的增强作用

以生物学相容性较好的聚乙二醇辛基苯基醚为乳化剂,从五大类生化营养基质中筛选出海藻糖、葡萄糖、蛋白胨、酪氨酸和菜籽油作为孢子萌发促进剂的基本成份,采用5因子正交旋转组合设计并实施了36个组配试验以优化配方。将所获孢子萌发促进剂加入球孢白僵菌(Beauveria bassiana)孢子乳悬液中喷雾接种桃蚜(Myzus persicae)无翅成蚜,在20℃和12L:12D的条件下饲养并逐日观察发病死亡数。所获数据经时间-剂量-死亡率模型模拟分析,桃蚜接种后第4～6天为死亡高峰,在10~6～10~8个孢子/ml的浓度下喷雾处理6天后的累计死亡率为62.9％～88.7％,LT_(50)估计值为3.8～4.8天,接种后第4、5和6天的LD_(50)估计值分别为3.75×10~7、3.73×10~5和2.08×10~5个孢子/ml。在连续6天的观察中,接种成蚜日均产若蚜数比对照分别下降了40.7％～85.0％,下降幅度随孢子乳悬液浓度提高而增大。这些结果表明,以上孢子萌发促进剂具有作为球孢白僵菌孢子制剂添加剂的应用潜力。     

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.     

Investigation of biological properties, double-stranded RNA patterns and antigen concentration in citrus species infected with citrus tristeza virus

Biological properties and dsRNA patterns of one Cyprus and three Turkish isolates of citrus tristeza virus (CTV) were investigated. In addition, CTV antigen concentration and effect of tissue sampling time from naturally infected Shamouti sweet orange trees grown in the field of Icel Province, Turkey, were also determined. The Cyprus isolate showed vein clearing symptoms on grapefruit, ‘Madam Vinous’ and Mexican lime and stem pitting symptoms on Mexican lime. The three Turkish isolates showed only vein clearing symptoms on Mexican lime. All four isolates showed a full-length major double-stranded RNA (dsRNA) band of 13.3 × 10⁶ Da mol. wt in extracts from infected Madam Vinous sweet orange trees, and major or minor dsRNA bands with 2.0. 0.8 and 0.5 × 10⁶ mol.wt. All seven different citrus varieties inoculated with the Igdir (D) strain contained full-length dsRNA. The additional two dsRNA of 0.8 and 0.5 × 10⁶ mol.wt were also detected as clearly as full-length dsRNA in these hosts, but were weaker inCitrus exelsa and ‘Interdonat’ lemon. Madam Vinous, rough lemon and Mexican lime were the best hosts for dsRNA analysis. ELISA values were highest in April (OD_405nm =0.476), decreased steadily until August, and then increased gradually through December. ELISA values were lowest in July and August (OD_405nm =0.157 and 0.141, respectively). dsRNA recovery from a field tree infected with isolate Igdir D was good in March, April and May and poor in January and February. No dsRNA band was detected in August or September. http://www.phytoparasitica.org posting July 9, 2002.     

Vascular blackening of wasabi rhizomes caused by <Emphasis Type="Italic">Pectobacterium carotovorum</Emphasis> subsp. <Emphasis Type="Italic">carotovorum</Emphasis>

Wasabi (Wasabia japonica) is grown for its highly-valued rhizome which is used as a condiment in Japanese food. Symptoms of vascular blackening in the rhizome were first observed in 2005 in plants grown in British Columbia, Canada. Microscopic observations and microbial isolation from infected tissues revealed that most of the xylem tracheid cells were blackened and bacteria were consistently associated with symptomatic plants. The bacterium most frequently recovered was identified as Pectobacterium carotovorum subsp. carotovorum (Pcc) using BioLog™ and sequencing of a specific ~510 bp IGS region. Pathogen-free plants obtained using meristem-tip micropropagation were inoculated with a wasabi isolate of Pcc. Vascular blackening symptoms developed in the rhizome after 8 weeks when the rhizome was first wounded by stabbing or cutting, or if the roots were pre-inoculated with Pythium species isolated from rhizome epidermal tissues, followed by inoculation with Pcc at 1 × 10⁸ cells ml⁻¹. Xylem tracheid cells were blackened and Pcc was reisolated from all diseased tissues. The highest frequency of rhizome vascular blackening occurred at 22°C and 27°C and these tissues occasionally succumbed to soft rot at higher temperatures, but not when inoculated tissues were incubated at 10°C. The rooting medium used by growers for vegetative propagation of wasabi was shown to contain Pcc but the pathogen was not recovered from the irrigation water. Entry of Pcc through wounds on wasabi rhizomes and the host tissue response result in symptoms of vascular blackening.     

Genetic analysis of lethal tip necrosis induced by <Emphasis Type="Italic">Clover yellow vein virus</Emphasis> infection in pea

Clover yellow vein virus (ClYVV) elicits lethal tip necrosis in the pea line PI 118501. Pea line PI 118501 develops necrotic lesions and veinal necrosis on inoculated leaves, followed by systemic necrosis, leading to plant death. To understand the genetic basis of this lethal tip necrosis, we crossed lines PI 226564 and PI 250438, which develop mosaic symptoms in response to ClYVV inoculation. In reciprocal crosses of PI 118501 with PI 226564, all F₁ plants had mosaic symptoms with slight stem necrosis and early yellowing of upper leaves. Essentially the same symptom was manifested in PI 118501 × PI 250438 F₁ plants. In F₂ populations from the cross between PI 118501 and PI 226564, the observed ratios of necrosis, mosaic with slight stem necrosis, and mosaic fit the expected 1 : 2 : 1 ratio. These results indicate that a single incompletely dominant gene confers the induction of necrosis in PI 118501. This locus in pea, conferring necrosis induction to ClYVV infection, was designated Cyn1 (Clover yellow vein virus-induced necrosis). A linkage analysis using 100 recombinant inbred lines derived from a cross of PI 118501 and PI 226564 demonstrated that Cyn1 was located 7.5 cM from the SSR marker AD174 on linkage group III.     

Shift of sensitivity of <Emphasis Type="Italic">Botrytis cinerea</Emphasis> to azoxystrobin in greenhouse vegetables before and after exposure to the fungicide

From 2004 to 2006, 213 isolates of Botrytis cinerea never exposed to Q_O center inhibitors (Q_OIs) were collected to determine the baseline sensitivity to azoxystrobin. In the absence of salicylhydroxamic acid (SHAM), the mean EC₅₀ values were 10.49 ± 13.12 and 0.36 ± 0.48 mg l⁻¹ for inhibiting mycelial growth and conidium germination, respectively. In the presence of SHAM, the mean EC₅₀ values were 2.24 ± 1.29 and 0.22 ± 0.11 mg l⁻¹. In 2010, five azoxystrobin-resistant isolates were detected with the resistance frequency of 2.25% in greenhouse tomatoes after 4 years of continuous exposure. These resistant isolates showed cross-resistance to other Q_OIs but not to boscalid. In addition, these resistant isolates had comparable growth, sporulation and pathogenicity ability as sensitive isolates and maintained resistance in plants and the presence of SHAM. The G143A point mutation predicted to cause a change from glycine to alanine at codon 143 of cyt b gene was found in all resistant isolates.