Temperature and plant age drive downy mildew disease epidemics on oilseed <Emphasis Type="Italic">Brassica napus</Emphasis> and <Emphasis Type="Italic">B. juncea</Emphasis>

Studies were undertaken on the effects of temperature (14/10 °C and 22/17 °C day/night) and plant age (15, 23, 31 and 40 day-old-plants) on the severity of downy mildew (Hyaloperonospora parasitica) on oilseed Brassica cultivars (temperature: Brassica juncea Montara, B. napus Atomic, ATR-Hyden, Hyola 432, Hyola 450 TT, Thunder TT; plant age: B. juncea Dune, B. napus Surpass 402 and Hyola 450 TT). For temperature studies, there were significant (P?<?0.001) effects of temperature, cultivar, and cultivar x temperature interaction. On cotyledons of susceptible cultivars (B. napus Hyola 450 TT and Thunder TT), plants were symptomatic at 22/17 °C by 48 h post inoculation (hpi) and with abundant sporulation evident by 72 hpi, and with all cotyledons of B. napus Thunder TT collapsed by 7 days post inoculation (dpi). However, at 14/10 °C, there were no symptoms on the same cultivars until 5 dpi, and sporulation only observed at 7 dpi. Percent disease index values (DI%) at 22/17 °C of B. juncea Montara and B. napus ATR-Hyden, Hyola 432, Atomic, Hyola 450 TT and Thunder TT were 4.5, 49.0, 51.4, 65.8, 86.3 and 96.0, respectively, with all except B. juncea Montara having significantly lower (P?<?0.001) disease at 14/10 °C with DI% values of 2.8, 30.4, 27.9, 31.1, 44.4 and 76.4, respectively. For plant age studies, there were significant (P?<?0.001) effects of plant age, cultivar, and cultivar x plant age interaction. DI% was significantly higher at 15 compared to 40 day-old-plants (dop) across all cultivars. B. juncea Dune showed greatest resistance, particularly on 40 dop, with DI% values of 25.8, 24.6, 22.9 and 7.5, for 15, 23, 31 and 40 dop, respectively. B. napus Surpass 402 showed high susceptibility on cotyledons of 15 dop but moderate resistance on leaves of other ages, with DI% values of 59.0, 31.2, 27.1 and 26.2 for 15, 23, 31 and 40 dop, respectively. B. napus Hyola 450 TT showed very high susceptibility at the cotyledon stage on 15 dop, but some resistance on 23 dop and more so on 31 and 40 dop, with DI% values of 84.0, 41.2, 35.4 and 32.9 for 15, 23, 31 and 40 dop, respectively. Together, these findings explain for the first time why development of downy mildew epidemics on susceptible cultivars occurs early in the growing season when warmer seasonal temperatures in autumn coincide with presence of seedlings; in contrast to later in the growing season on less susceptible older plants coinciding with cooler and less favourable winter temperatures. Increasing maximum and minimum temperatures associated with climate change have likely fostered the increased severity of downy mildew over the past 15 years.     

Multifarious activity of bioformulated <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis> PS1 and biocontrol of <Emphasis Type="Italic">Sclerotinia sclerotiorum</Emphasis> in Indian rapeseed (<Emphasis Type="Italic">Brassica campestris</Emphasis> L.)

PGPR strain Pseudomonas fluorescens PS1 was evaluated to formulate carrier based bioformulations. The viability of P. fluorescens PS1 was monitored at different time intervals during the period of storage at room temperature in different carriers such as soil, charcoal, sawdust and sawdust-soil. Sawdust-soil was found to be the most efficient carrier material for P. fluorescens PS1 followed by other carriers. After 1 year of storage, P. fluorescens PS1 was re-isolated and assayed for its antifungal activity against Sclerotinia sclerotiorum a phytopathogenic fungus causing stem blight in Indian mustard, Brassica campestris. Results of scanning electron microscopy exhibited that P. fluorescens PS1 caused morphological alteration in mycelia of S. sclerotiorum as evident by hyphal perforation, and fragmented lysis. Seed bacterization of B. campestris with P. fluorescens PS1 induced enhanced seed germination, increased overall plant growth as well as reduced stem blight in mustard with improved yield. These findings demonstrate that P. fluorescens PS1 has significant potential to raise disease-free crops due to the presence of a wide array of PGP characteristics.     

Potential of <Emphasis Type="Italic">Trichoderma</Emphasis> spp. and <Emphasis Type="Italic">Talaromyces flavus</Emphasis> for biological control of potato stem rot caused by <Emphasis Type="Italic">Sclerotinia sclerotiorum</Emphasis>

Sixteen isolates belonging to 11 species of Trichoderma (T. asperellum, T. ceramicum, T. andinensis, T. orientalis, T. atroviride, T. viridescens, T. brevicompactum, T. harzianum, T. virens, T. koningii and T. koningiopsis) were evaluated for biological control of potato (Solanum tuberosum) stem rot caused by Sclerotinia sclerotiorum. In dual culture tests, all antagonists significantly reduced sclerotia formation, and were able to inhibit radial growth of the pathogen. Growth inhibition by production of volatile and non-volatile inhibitors was also measured in in vitro tests. In screening the most efficient species of Trichoderma, establishment of mycelium on sclerotia and sclerotia lysis were also considered as important biocontrol qualities. Excluding T. asperellum, T. brevicompactum, T. andinensis and T. harzianum, all tested Trichoderma species were able to lyse sclerotia. The sclerotia-destroying species of Trichoderma and one isolate of Talaromyces flavus were tested in greenhouse tests and during 2 years of field experimentation during the 2007 and 2008 cropping seasons. After one aerial application of spore suspension in greenhouse trials, T. koningii, T. virens, T. ceramicum and T. viridescens were the most effective bio-agents and reduced significantly disease severity, and the least biocontrol efficacy was observed in T. flavus. Under field conditions and after five soil and foliar applications of spore suspension, all tested antagonists reduced significantly disease incidence. T. viridescens followed by T. ceramicum showed the best results. T. flavus and T. orientalis were less effective than other tested antagonists in both field trials.     

Potential inhibitors against <Emphasis Type="Italic">Sclerotinia sclerotiorum</Emphasis>, produced by the fungus <Emphasis Type="Italic">Myrothecium</Emphasis> sp. associated with the marine sponge <Emphasis Type="Italic">Axinella</Emphasis> sp.

Sclerotinia sclerotiorum is a worldwide ascomycete fungal plant pathogen, which causes enormous yield losses on major economic crops such as crucifers, grain legumes and several other plant families. The objective of this research was to isolate and characterise some bioactive products from cultures of fungi associated with the marine sponge Axinella sp. In total, nine fungal isolates were obtained from the marine sponge Axinella sp. collected from the South China Sea. A group of test strains, including two G⁺ strains (Bacillus subtilis and Staphylococcus aureus), two G⁻ strains (Escherichia coli and Pseudomonas aeruginosa) and three fungi including two plant pathogenic fungi Sclerotinia sclerotiorum and Magnaporthe grisea and Saccharomyces cerevisiae, were employed as the indicator organisms for bioactivity screening. Using antagonistic tests and bioactive screening of the ethyl acetate (EtOAc) extracts of the corresponding cultures, fungal isolate JS9 showed the stronger efficacy against the test indicator strains, especially the indicator fungal pathogens. Isolate JS9 was further identified as Myrothecium sp. by a combination of morphological features and 18S rDNA BLAST on GenBank. Two macrocyclic trichothecenes, roridin A (compound 1) and roridin D (compound 2) were purified by tracking the activity of the EtOAc extract fractions and characterised with spectral analyses including MS, ¹H-NMR, ¹³C-NMR and disortionless enhancement by polarization transfer (DEPT). In vitro antifungal tests showed that the two macrocyclic trichothecenes were bioactive against S. cerevisiae, M. grisea and S. sclerotiorum with minimal inhibitory concentrations of 31.25, 125 and 31.25 μg ml⁻¹ for roridin A, and 62.5, 250 and 31.25 μg ml⁻¹ for roridin D, respectively. The present investigation demonstrated that two antifungal trichothecenes including roridin A and roridin D produced by the fungus Myrothecium sp. isolated from the marine sponge Axinella sp. could be potential inhibitors against the plant pathogen S. sclerotiorum. Lian Wu Xie and Shu Mei Jiang contributed equally to this work.     

Genetic diversity and differentiation of <Emphasis Type="Italic">Sclerotinia sclerotiorum</Emphasis> populations in sunflower

Ninety-six isolates of sunflower Sclerotinia sclerotiorum (Lib.) de Bary from Inner Mongolia (IM) in China, from Canada and the United Kingdom (UK) were sampled to investigate the genetic diversity and structure using Sequence-Related Amplified Polymorphism. A total of 123 polymorphic bands were obtained, ranging in size from 100 to 500 base pairs. The five populations of S. sclerotiorum isolated from the three countries showed various levels of genetic variability. The percentage of polymorphic loci varied from 30.89% in the UK population to 97.56% in the Middle IM population. The values of Shannon index (i) varied from 0.1876 in the UK population to 0.5301 in the West IM population. The heterozygosity of the five geographic populations obtained by estimating allele frequency varied from 12.91% in the UK population to 35.44% in the West IM population. The genetic identity, as indicated by the Nei unbiased identity index, ranged from 0.9744 between populations from Canada and East IM to 0.6477 between populations from West IM and UK. UPGMA cluster analysis using Nei’s genetic distance gave distances ranging from 0.0259 to 0.4343. The rates of gene flow among five geographic populations ranged from 1.5406 between West IM and UK populations to 18.4149 between West IM and Middle IM populations. The four populations from West IM, Middle IM, East IM and Canada were clustered into one subgroup in which the isolates from West and Middle IM belonged to one population, whereas those from East IM and Canada essentially were another population. The isolates from the UK formed a population that was significantly distinct from other populations.     

Occurence of sclerotinia stem rot of fenugreek caused by <Emphasis Type="Italic">Sclerotinia trifoliorum</Emphasis> and <Emphasis Type="Italic">S. sclerotiorum</Emphasis> in Tunisia

Fenugreek is an annual leguminous crop grown for hay and grains in Tunisia. It is also considered a valuable rotation crop with cereals. Sclerotinia rot was observed in production fields since 2010. The survey conducted in 2013 revealed that the incidence of diseased plants varied between 5 and 20%. The identification of isolates of Sclerotinia obtained from fenugreek plants with symptoms of stem rot was determined using morphological and molecular criteria. The size, shape and abundance of sclerotia in potato dextrose agar (PDA) cultures were used to classify isolates as S. sclerotiorum or S. trifoliorum. A comparison of colony diameter on PDA after 24, 48 and 72 h at 25 °C, showed that one isolate grew faster (36 mm/day) than the other 10 isolates (14.8 mm/day). There was a significant difference in sclerotial size between the fast and the slow growing isolates, but there was no significant difference in the number of sclerotia produced after 3 weeks on PDA. Two of the slow growing isolates exhibited ascospore dimorphism, whereas the fast growing isolate did not. PCR amplification with the primer pair ITS5/ITS4 produced a fragment of 560 base pairs from the fast growing isolate and 1000 base pairs from all of the slow growing isolates. The ITS sequences of the fast growing isolate had 100% homology with S. sclerotiorum, whereas those of the slow growing isolates had 100% homology with S. trifoliorum. Isolates of both species were pathogenic on fenugreek seedlings in the greenhouse assay and there was no significant difference in the percentage of dead plants two weeks after inoculation between the two species.     

Reaction of glucosinolate-myrosinase defence system in <Emphasis Type="Italic">Brassica</Emphasis> plants to pathogenicity factor of <Emphasis Type="Italic">Sclerotinia sclerotiorum</Emphasis>

The glucosinolate-myrosinase defence system, specific to Brassicales plants, produces toxic volatile compounds during mechanical injury or pathogen attack. The reaction of this system to oxalic acid, known as a pathogenicity factor of Sclerotinia sclerotiorum, is not fully understood. The hydrolysis of glucosinolates was studied at varying conditions in the presence of oxalic acid in the substrate. In a bioassay, colonies of the pathogen were exposed to volatiles from hydrated mustard powder used as a myrosinase and glucosinolate source. The glucosinolate-myrosinase (GSL-M) system was activated in the presence of oxalic acid at a concentration and pH similar to that expected in vivo. Volatile production was inhibited only when the pH fell to 3 or below. It is unlikely that oxalic acid plays a significant role in disarming the GSL-M system during infection of Brassica hosts.     

Epidemiology of root rot caused by <Emphasis Type="Italic">Leptosphaeria maculans</Emphasis> in <Emphasis Type="Italic">Brassica napus</Emphasis> crops

The infection of above-ground tissues of Brassica napus by Leptosphaeria maculans is well understood. However, root infection (root rot) under field conditions, the development of root rot over time and its relationship to other disease symptoms caused by L. maculans has not been described. A survey of B. napus crops was conducted in Australia to investigate the incidence and severity of root rot. Additionally, the pathway of root infection was examined in field experiments. Root rot was present in 95% of the 127 crops surveyed. The severity and incidence of root rot was significantly correlated with that of crown canker; however, the strength of this relationship was dependent on the season. Root rot symptoms appeared before flowering and increased in severity during flowering and at maturity, a pattern similar to crown canker suggesting that the infection of the root is an extension of the crown canker phase of the L. maculans lifecycle. All isolates of L. maculans tested in glasshouse experiments caused root rot and crown canker in B. napus and Brassica juncea. In the field, the main pathway of root infection is via invasion of cotyledons or leaves by airborne ascospores, rather than from inoculum in the soil. Root rot was present in crops in fields that had never been sown to B. napus previously, in plants grown in fumigated fields, and in glasshouse-grown plants inoculated in the hypocotyl with L. maculans.     

Population genetic structure of <Emphasis Type="Italic">Sclerotinia sclerotiorum</Emphasis> on canola in Iran

The genetic structure of 276 Sclerotinia sclerotiorum isolates representing 37 field populations from four provinces in northern Iran were analysed with six polymorphic microsatellite loci. In total, 80 haplotypes were detected with 19 haplotypes (23.7%) shared amongst at least two regional populations. None of the haplotypes were shared among all four regional populations. Of the 80 haplotypes, 32 haplotypes (40%) occurred in low frequencies represented by only one isolate. Moderate levels of gene diversity (H = 0.51 to 0.61) and genotypic diversity (Ĝ = 12.0 to 22.0; clonal fraction = 0.39 to 0.67) for regional populations were observed. Genotypic diversities (Ĝ) did not differ significantly among populations. All regional populations were in linkage equilibrium indicating the occurrence of outcrossing. Low to moderate levels of population subdivision (0.03 to 0.07), were observed among regional populations. Only one large panmictic population was inferred by Structure, indicating no significant population structure. A Mantel test showed no significant isolation by distance (r = −0.43; P = 0.18), indicating anthropogenic movement of inoculum. The results demonstrated that S. sclerotiorum populations in northern Iran, are randomly mating and have a number of shared haplotypes among regional populations; this possibly represents recent founder populations and/or a high occurrence of anthropogenic migration of infected plant material among populations.     

ABA signaling inhibits oxalate-induced production of reactive oxygen species and protects against <Emphasis Type="Italic">Sclerotinia sclerotiorum</Emphasis> in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Oxalic acid is an essential virulence factor of Sclerotinia sclerotiorum that elicits wilting symptoms in infected host plants. Foliar wilting in response to oxalic acid is known to be dependent on an increase in stomatal conductance. To determine whether stomatal regulation controls susceptibility to S. sclerotiorum, abscisic acid-insensitive and open stomata mutants of Arabidopsis thaliana were analyzed. Whereas abscisic acid-insensitive mutants were hypersusceptible to S. sclerotiorum, open stomata mutants were as susceptible as wild type. It was concluded that stomatal regulation does not control susceptibility to S. sclerotiorum because open stomata mutants are known to only impair guard cells whereas abscisic acid-insensitive mutants also affect other cell types. Guard cell-independent processes also control sensitivity to oxalic acid because oxalic acid was more toxic to abscisic acid-insensitive mutants than to open stomata mutants. To explore a possible mechanism of toxicity, production of reactive oxygen species was measured in plant cells after exposure to oxalic acid. Oxalic acid was found to elicit reactive oxygen species production independently of abscisic acid. Nevertheless, cancellation of reactive oxygen species elicitation after co-stimulation of wild-type guard cells with oxalic acid and abscisic acid provided evidence for antagonistic interaction between both molecules.     

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.     

Assessing the phenotypic and genotypic diversity of <Emphasis Type="Italic">Sclerotinia sclerotiorum</Emphasis> in France

White mould caused by the ascomycete Sclerotinia sclerotiorum affects the production of many economically important crops. The incidence of this disease has recently increased in France, especially in melon crops, which were not affected much in the past. One possible explanation for this situation is the emergence of strains with particular characteristics, including increased aggressiveness to melon. To test this hypothesis, 200 isolates of S. sclerotiorum were collected from six host crops (bean, brassica oilseed rape, carrot, lettuce, melon, witloof chicory) in different regions. They were genotyped with 16 microsatellites markers. A subsample of 96 isolates were assessed for their aggressiveness on melon leaves. Overall, the isolates from melon did not show higher aggressiveness on melon leaves than those which originated from other host plants. Moreover, the melon isolates did not present distinctive genetic characteristics in comparison with those from other crops and shared several of the 128 identified multilocus haplotypes with isolates collected from carrot, witloof chicory and oilseed rape. Furthermore the Bayesian analysis of the genetic structure indicated that the host plant is not a structuring factor of the three genetic clusters identified, and it suggested instead the occurrence of an isolation-by-distance process. Possible consequences of these results for the management of white mould and alternative hypotheses to explain the recent changes in disease incidence are presented.     

Pathogenicity,colony morphology and diversity of isolates of <Emphasis Type="Italic">Guignardia citricarpa</Emphasis> and <Emphasis Type="Italic">G. mangiferae</Emphasis> isolated from <Emphasis Type="Italic">Citrus</Emphasis> spp.

In the present study, the pathogenicity of 36 isolates of Guignardia species isolated from asymptomatic ‘Tahiti’ acid lime fruit peels and leaves, ‘Pêra-Rio’ sweet orange leaves and fruit peel lesions, and a banana leaf were characterized. For pathogenicity testing, discs of citrus leaves colonized by Phyllosticta citricarpa under controlled laboratory conditions were kept in contact with the peels of fruit that were in susceptible states. In addition, pathogenicity was related to morphological characteristics of colonies on oatmeal (OA) and potato dextrose agar (PDA). This allowed the morphological differentiation between G. citricarpa and G. mangiferae. Polymerase chain reactions (PCRs) were also used to identify non-pathogenic isolates based on primers specific to G. citricarpa. A total of 14 pathogenic isolates were detected during pathogenicity tests. Five of these were obtained from leaf and fruit tissues of the ‘Tahiti’, which until this time had been considered resistant to the pathogen. Given that the G. citricarpa obtained from this host was pathogenic, it would be more appropriate to use the term insensitive rather than resistant to categorize G. citricarpa. A non-pathogenic isolate was obtained from lesions characteristic of citrus black spot (CBS), indicating that isolation of Guignardia spp. under these conditions does not necessarily imply isolation of pathogenic strains. This also applied to Guignardia spp. isolates from asymptomatic citrus tissues. Using fluorescent amplified fragment length polymorphism (fAFLP) markers, typically pathogenic isolates were shown to be more closely related to one another than to the non-pathogenic forms, indicating that the non-pathogenic isolates display higher levels of genetic diversity.     

<Emphasis Type="Italic">Leptosphaeria maculans</Emphasis>, a fungal pathogen of <Emphasis Type="Italic">Brassica napus</Emphasis>, secretes a subtilisin-like serine protease

Leptosphaeria maculans,a fungal pathogen of Brassica napus, secretes large amounts of a 28kDa protein (SP2) in liquid culture. This protein shows high sequence similarity to secreted serine proteases from other ascomycetes and is the major component of culture filtrate with protease activity, as analysed on casein zymogels. The sp2 gene is expressed during infection of B.napuscotyledons when L. maculans hyphae are growing between mesophyll cells, as well as at later stages when the fungus invades the vascular tissue.     

Genetic structure of <Emphasis Type="Italic">Pyrenophora teres</Emphasis> f. <Emphasis Type="Italic">teres</Emphasis> and <Emphasis Type="Italic">P. teres</Emphasis> f. <Emphasis Type="Italic">maculata</Emphasis> populations from western Canada

Infection by Pyrenophora teres f. teres (Ptt) or P. teres f. maculata (Ptm), the causal agents of the net and spot forms of net blotch of barley, respectively, can result in significant yield losses. The genetic structure of a collection of 128 Ptt and 92 Ptm isolates from the western Canadian provinces of Alberta (55 Ptt, 27 Ptm), Saskatchewan (58 Ptt, 46 Ptm) and Manitoba (15 Ptt, 19 Ptm) were analyzed by simple sequence repeat (SSR) marker analysis. Thirteen SSR loci were examined and found to be polymorphic within both Ptt and Ptm populations. In total, 110 distinct alleles were identified, with 19 of these shared between Ptt and Ptm, 75 specific to Ptt, and 16 specific to Ptm. Genotypic diversity was relatively high, with a clonal fraction of approximately 10 % within Ptt and Ptm populations. Significant genetic differentiation (PhiPT = 0.230, P = 0.001) was found among all populations; 77 % of genetic variation occurred within populations and 23 % between populations. Lower, but still significant genetic differentiation (PhiPT = 0.038, P = 0.001) was detected in Ptt, with 96 % of genetic variation occurring within populations. No significant genetic differentiation (PhiPT = 0.010, P = 0.177) was observed among Ptm populations. Isolates clustered in two distinct groups conforming to Ptt or Ptm, with no intermediate cluster. The high number of haplotypes observed, combined with an equal mating type ratio for both forms of the fungus, suggests that P. teres goes through regular cycles of sexual recombination in western Canada.     

<Emphasis Type="Italic">Eremothecium coryli</Emphasis> and <Emphasis Type="Italic">E.</Emphasis><Emphasis Type="Italic">ashbyi</Emphasis> cause yeast spot of azuki bean

Yeast-like fungi were isolated from lesions on azuki bean (cv. Shin-Kyotodainagon) seeds that had been sucked by bean bugs in Kyoto Prefecture, Japan. On the basis of morphological and physiological characteristics and sequence data of the internal transcribed spacer (ITS) regions including the 5.8S rDNA, these yeasts were identified as Eremothecium coryli and E. ashbyi. Pathogenicity of those yeasts was confirmed by a reinoculation test. To our knowledge, this is the first report of the occurrence of yeast spot in azuki bean in Japan. The nucleotide sequence data reported are available in the GeneBank/EMBL/DDBJ database as accessions AB478291–AB478309 for E. coryli AZC1–19 and AB478310–AB478317 for E. ashbyi AZA1–8.     

Germination and invasion by ascospores and pycnidiospores of <Emphasis Type="Italic">Leptosphaeria maculans</Emphasis> on spring-type <Emphasis Type="Italic">Brassica napus</Emphasis> canola varieties with varying susceptibility to blackleg

The infection processes of ascospores and pycnidiospores of Leptosphaeria maculans were studied on cotyledons of six cultivars of spring-type Brassica napus: one with resistance controlled by a single dominant gene (cv. Surpass 400), three with polygenic resistance (cvs. Dunkeld, Grouse, and Outback), and two susceptible cultivars (Westar and Q2). On all cultivars, ascospore germination, penetration, and development of symptoms on cotyledons were much earlier than that with pycnidiospores. At 2h after inoculation ascospores began to germinate, by 4h about 50% had germinated, and by 6–8h 85%–90% had germinated. In contrast, pycnidiospores began to germinate 1 day after inoculation (dai) and reached only 50% germination by 3 dai. Ascospores began germinating from terminal cells and then later from the interstitial cells. Pycnidiospores germinated predominantly from one end and sometimes from both ends. Germ tubes from ascospores penetrated stomata as early as 4h after inoculation, whereas those from pycnidiospores penetrated at 2 dai. Symptom development with ascospores was 2 days earlier than that with pycnidiospores. Symptoms on Surpass 400 were evident as early as 3–5 dai with ascospores and 5–7 dai with pycnidiospores. However, on other cultivars, symptoms were not evident until 10 dai with ascospores and 12 dai with pycnidiospores. This report is the first on differences in the infection processes by the two spore types. Ascospore and pycnidiospore attachment, germination, and penetration did not differ between resistant and susceptible cultivars, but there were major differences after penetration. Under high humidity, 80%–90% of stomata of susceptible Westar and Q2 had aerial hyphae emerging from stomatal pores. However, fewer stomata (5%–10%) had aerial hyphae on Surpass 400 by 10 dai with ascospores and 12 dai with pycnidiospores, but even these were usually poorly developed. Host differences in spring-type B. napus in relation to production of aerial hyphae have not previously been reported. In Surpass 400, rapid necrosis of guard cells occurred within a few hours of penetration by either type of spore, and subsequently one or a few cells immediately adjacent to the penetration site died. This necrosis then spread to the cells around the penetration site to form a hypersensitive response (in the form of a small, dark lesion) to both ascospores and pycnidiospores. This is the first detailed report on interactions between spring-type B. napus and L. maculans in relation to single dominant gene-based resistance. Neither the cultivars with polygenic resistance nor the susceptible cultivars had such a response.     

Characterization of <Emphasis Type="Italic">Inago1</Emphasis> and <Emphasis Type="Italic">Inago2</Emphasis> retrotransposons in <Emphasis Type="Italic">Magnaporthe oryzae</Emphasis>

From the genome of a Japanese field isolate of the rice blast fungus, Magnaporthe oryzae, we newly identified Inago1 and Inago2 LTR retrotransposons. Both elements were found to be Ty3/gypsy-like elements whose copies were dispersed within the genome of Magnaporthe spp. isolates infecting rice and other monocot plants. Southern hybridization patterns of nine re-isolates derived from conidia of the strain Ina168 produced after a methyl viologen treatment were not changed, indicating that the insertion pattern of Inago elements is relatively stable.     

First report of blueberry red ringspot disease caused by <Emphasis Type="Italic">Blueberry</Emphasis><Emphasis Type="Italic">red</Emphasis><Emphasis Type="Italic">ringspot</Emphasis><Emphasis Type="Italic">virus</Emphasis> in Japan

Virus-like symptoms—red ringspots on stems and leaves, circular blotches or pale spots on fruit—were found on commercial highbush blueberry (Vaccinium corymbosum) cultivars Blueray, Weymouth, Duke and Sierra in Japan. In PCR testing, single DNA fragments were amplified from total nucleic acid samples of the diseased blueberry bushes using primers specific to Blueberry red ringspot virus (BRRV). Sequencing analysis of the amplified products revealed 95.7–97.7% nucleotide sequence identity with the BRRV genome. This paper is the first report of blueberry red ringspot disease caused by BRRV in Japan. The nucleotide sequence data reported in this paper are available in the GenBank/EMBL/DDBJ database as accessions AB469884 to AB469893 for BRRV isolates from Japan.