<Emphasis Type="Italic">Paecilomyces lilacinus</Emphasis>, a potential biocontrol agent on apple rust mite <Emphasis Type="Italic">Aculus,schlechtendali</Emphasis> and interactions with some fungicides <Emphasis Type="Italic">in vitro</Emphasis>

The apple rust mite Aculus schlechtendali (Nal.) (Acari: Eriophyidae), is a main pest in apple-growing areas in Ankara, Turkey, and chemical control applications have some limitations. Entomopathogenic fungi have a potential for biological control of mites. In this study, an entomopathogenic fungus, Paecilomyces lilacinus (Thom) Samson (Deuteromycota: Hyphomycetes), was first isolated from the mite cadavers on Japanese crab apple leaves and pathogenicity of the fungus was observed in different inoculum densities and relative humidities. The pathogen caused up to 98.22% mortality of the mite population. The effects of some fungicides on the entomopathogenic fungus were determined in in vitro studies. Carbendazim, penconazole and tebuconazole were the most effective fungicides on mycelial growth of P. lilacinus, with EC₅₀ values under 3 μg ml⁻¹. In spore germination tests, captan, mancozeb, propineb were the most effective fungicides, followed by tebuconazole, penconazole, nuarimol and chlorothalonil. Sulphur could not inhibit the conidia germination totally at 5,000 μg ml⁻¹. Copper oxychloride and fosetyl-al prevented conidia formation at concentrations above 1,000 μg ml⁻¹.     

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.     

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.     

Relationship between the incidence of latent infections caused by <Emphasis Type="Italic">Monilinia</Emphasis> spp<Emphasis Type="Italic">.</Emphasis> and the incidence of brown rot of peach fruit: factors affecting latent infection

Five field experiments were performed in commercial orchards located in Lleida (Spain) over three growing seasons, 2000–2002, in order to estimate the relationship between the incidence of latent infection caused by Monilinia spp. in peaches and the incidence of post-harvest brown rot. No latent infection was recorded at popcorn and the maximum incidence occurred pre-harvest; in some orchards a second peak was detected during the pit hardening period. Monilinia laxa is the most prevalent species isolated from peaches with brown rot. There was a positive correlation between the incidence of latent infection and that of post-harvest brown rot. The average incidence of latent infection during the crop season explained 55% of the total variation in the incidence of post-harvest brown rot. The effect of temperature (T) and duration of wetness (W) on the incidence of latent infection in peach and nectarine orchards was analysed using multiple regression. The regression analysis indicated that T and W jointly explained 83% of the total variation in the incidence of latent infection. The model predicts no latent infections when T < 8°C, and >22 h wetness are required when T = 8°C but only 5 h at 25°C are necessary for latent infection to occur. The incidence of brown rot and latent infection of peaches caused by M. laxa under controlled experimental conditions were also affected by T and W, as well as by fruit maturity and inoculum concentration. Latent infections were produced in fruit when T was not suitable for the development of brown rot symptoms. In these experiments more than 4–5 h of daily wetness were required after embryo growth in fruit sprayed to run-off with an inoculum concentration higher than 10⁴ conidia ml⁻¹ of M. laxa for brown rot and latent infections to develop. The fitted model obtained from the field data was able to predict the observed data obtained under controlled environmental conditions.     

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.     

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.     

Efficacy and dose–response relationship in biocontrol of Fusarium disease in maize by Streptomyces spp.

Two isolates of Streptomyces spp. DAUFPE 11470 and DAUFPE 14632 were evaluated to determine the antagonist–pathogen inoculum concentration relationship under greenhouse conditions. Pathogen and antagonist concentration, significantly (P < 0.05) affected development of Fusarium disease in maize with a significant interaction between pathogen and antagonist concentration. Dose–response relationship also differed significantly (P < 0.05) between the two isolates, but both isolates demonstrated effective control of Fusarium disease, regardless of pathogen concentration. The isolate DAUFPE 11470 provided the most effective control. The lowest value for disease incidence occurred at low pathogen (10³ chlamydospore g⁻¹ soil) and high antagonist concentration (10⁶ cfu ml⁻¹) for both isolates. The disease incidence for control plants ranged from 19% to 76%. However, in relation to control the lowest disease reduction occurred at low pathogen (10³ chlamydospore g⁻¹ soil) and high antagonist concentrations (10⁶ cfu ml⁻¹). These reductions were 10.6% and 13% for DAUFPE 14632 and DAUFPE 11470, respectively. The highest disease reductions, in relation to control plants, occurred at high pathogen (10⁶ chlamydospore g⁻¹ soil) and antagonist (10⁶ cfu ml⁻¹) concentrations for both isolates. These values were 55% and 62.2% for DAUFPE 14632 and DAUFPE 11470, respectively. The chlamydospore germination of Fusarium moniliforme was affected by glucose addition, antagonist isolates and type of inoculum. The lowest chlamydospore germination was observed with bacterial suspensions of the isolates, for all glucose additions. The results suggested that both Streptomyces spp. isolates were effective at different doses as biocontrol agents against F. moniliforme. Also, there was evidence for at least two mechanisms of biocontrol and that apparently, both isolates showed the same mechanisms of biocontrol action related to production of bioactive compounds and competition for carbon. Further studies will be developed to improve the level and effectiveness of control by these isolates.     

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.     

Erwinia amylovora can pass through the abscission layer of fruit-bearing twigs and invade apple fruit during fruit maturation

Invasion of apple fruit by Erwinia amylovora from fruit-bearing twigs through the abscission layer at fruit maturation was examined. Erwinia amylovora (ca. 10⁵ cfu) tagged with bioluminescence genes from Vibrio fischeri was deposited in artificial wounds on fruit-bearing twigs of apple trees grown in a containment greenhouse on September 22, 27, or October 5, 2004. On October 22, 176 apples were harvested and cut horizontally in half. The upper halves were stamped on plates of selective medium, and the lower halves were flooded with iodine solution to assess maturity. All fruit were symptomless and fully mature. The pathogen was recovered from 19 (10.8%) apples. The result showed that if at least ca. 10⁵ cfu of E. amylovora are present in fruit-bearing twigs at the time of fruit maturation, the bacteria can pass through the abscission layer into the fruit, even though the mature fruit lack symptoms.     

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.     

Use of sclerotia of<Emphasis Type="Italic">Sclerotinia sclerotiorum</Emphasis> for efficient production of conidia of<Emphasis Type="Italic">Coniothyrium minitans</Emphasis> in liquid culture

A study was conducted to determine the feasibility of using sclerotia ofSclerotinia sclerotiorum for producing conidia ofConiothyrium minitans in liquid culture. The medium (SST) was made of water containing 2.0, 1.5, 1.0 or 0.5% (w/v) ground sclerotia ofS. sclerotiorum and 100 μgl ⁻¹ thiamine hydrochloride (HCl). One ml of conidial suspension (2 × 10⁷ conidia ml⁻¹) ofC. minitans LRC 2534 was inoculated into 100 ml of SST medium or control (thiamine HCl in water) and incubated at 20 ± 2°C on a shaker at 200 rpm. Subsamples were removed periodically and examined under a compound microscope. Conidia in the SST media germinated within 24 h, developed into branched hyphae within 48 h, produced pycnidia after 3–4 days, and the pycnidia released mature conidia after 7 days. Production of conidia varied with the concentration of sclerotia in the SST medium. It was lower (3.6 × 10⁶ conidia ml⁻¹) at 0.5% but higher (1.2 × 10⁸ conidia ml⁻¹) at 2%. The new conidia were viable and the colonies developing from them showed the original morphological characteristics. It was concluded that using SST liquid medium as a substrate for mass production of conidia ofC. minitans has potential for use in commercial development of this mycoparasite as a biocontrol product. http:www.phytoparasitica.org posting Jan. 23, 2007.     

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.     

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.     

Effects of temperature,free moisture duration and inoculum concentration on infection of sweet cherry by<Emphasis Type="Italic">Pseudomonas syringae</Emphasis> pv.<Emphasis Type="Italic">syringae</Emphasis>

The effects of temperature, free moisture duration and inoculum concentration on infection caused byPseudomonas syringae pv.syringae (Pss), on sweet cherry (Prunus avium) were investigated. Epiphytic populations ofPss are an important source of inoculum for bacterial canker and it has been demonstrated that a cyclic pattern exists during the year, from undetectable during the warm and dry periods to large populations following cool and wet periods. The effects of temperature and inoculum concentration on the infection caused byPss on immature fruits and 1-yr-old twigs were significant (P<0.001). Fruit and twig infection increased linearly in proportion to the logarithm ofPss when bacterial concentrations were higher than 10³ cfu ml⁻¹ and temperatures were between 5 and 20°C. Regardless of the inoculum concentration and the free moisture duration, fruit and twig infection was either absent or low at 5°C but it increased linearly as temperature increased from 5 to 20°C. Growth ratein vitro was very slow (0.03–0.04 cfu h⁻¹) at 5°C and fast (0.21–0.23 cfu h⁻¹) at 20°C. Therefore, it is possible that multiplication of the epiphytic populations may be significantly reduced in the field with air temperatures below 5°C. A significant (P<0.001) effect of free moisture was obtained only when a low inoculum concentration (10³ cfu ml⁻¹) was used, and a significant linear response between free moisture and disease incidence was obtained only at 10°C. An apparent threshold population ofPss higher than 10³ cfu ml⁻¹ was needed to infect immature fruits and 1-yr-old twigs of sweet cherry. http://www.phytoparasitica.org posting July 10, 2002.     

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.     

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.     

<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.     

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 Sclerotinia Diseases of Rapeseed by Aerial Applications of the Mycoparasite Coniothyrium minitans

Indoor and field experiments were conducted to evaluate the efficacy of applying the mycoparasite Coniothyrium minitans to the aerial parts of rapeseed plants at the flowering stage to control sclerotinia diseases caused by Sclerotinia sclerotiorum. Under controlled conditions, a petal inoculation technique was used to determine the effect of conidial suspensions of C. minitans on suppression of sclerotinia leaf blight. Results showed that C. minitans was effective in inhibiting infection initiated by ascospores of S. sclerotiorum on flower petals by restricting mycelial growth of the pathogen. Suppression of lesion development was related to the conidial concentration of C. minitans, with larger lesions at low concentration (5×10³conidia ml⁻¹), but smaller lesions at high concentration (5×10⁴ conidia ml⁻¹ or higher). When C. minitans-treated rapeseed leaves were inoculated with mycelia of S. sclerotiorum, C. minitans failed to prevent infection of leaves, but caused a significant reduction in number of sclerotia produced on the diseased leaves. No significant difference in efficacy was detected between the two isolates of C. minitans, LRC 2137 and Chy-1, on the two rapeseed cultivars, Westar (spring type) and Zhongyou 821 (winter type). Results of field trials showed a significant reduction of stem rot of rapeseed in four (1997, 1999, 2003 and 2004) out of five years by aerial application of C. minitans, compared with controls. No significant difference in suppressive efficacy was observed between the treatments of C. minitans (10⁶ conidia ml⁻¹), C. minitans (10⁶ conidia ml⁻¹) + benomyl (50 μg ml⁻¹) and benomyl (100 μg ml⁻¹) in 2003, and between the treatments of C. minitans (10⁶ conidia ml⁻¹), C. minitans (10⁶ conidia ml⁻¹) + vinclozolin (100 μg ml⁻¹) and vinclozolin (500 μg ml⁻¹) in 2004. Sclerotia of S. sclerotiorum collected from diseased plants in plots treated with C. minitans in 1999, 2000 and 2003, or with C. minitans + benomyl in 2003 were infected by C. minitans at frequencies ranging from 21.3 to 54.5%. This study concludes that aerial spraying of C. minitans is an effective method for controlling sclerotinia diseases of rapeseed.     

A molecular mechanism of resistance to streptomycin in <Emphasis Type="Italic">Xanthomonas oryzae</Emphasis> pv. <Emphasis Type="Italic">oryzicola</Emphasis>

Xanthomonas oryzae pv. oryzicola, the causal agent of rice leaf streak disease, was found to be sensitive to streptomycin (an aminocyclitol glycoside antibiotic), by inhibition of protein synthesis resulting from interference with translational proofreading. This study aimed to determine the molecular resistance mechanism of X. oryzae pv. oryzicola to streptomycin. Seven streptomycin-resistant mutants were obtained by UV induction or streptomycin selection. These mutants can grow at 100 μg ml⁻¹ of streptomycin while the wild-type strain (RS105) cannot grow at 5 μg ml⁻¹. Sequencing indicated that the rpsL gene encoding ribosomal protein S12 has 375 bp encoding 125 amino acid residues. In all resistant strains, a mutation in which AAG was substituted for AGG (Lys→Arg) occurred either at codon 43 or 88. Two plasmids, pUFRRS and pUFRRX, were constructed by ligating the rpsL gene into the cosmid pUFR034. The plasmids pUFRRS and pUFRRX containing the Lys→Arg mutation of the rpsL gene conferred streptomycin resistance to the sensitive wild-type strain by electroporation. Both transformants, RS1 and RS2, could grow in the medium containing 50 μg ml⁻¹ of streptomycin. A mutation at codon 43 or 88 in rpsL can result in resistance of Xanthomonas oryzae pv. oryzicola to streptomycin.