Characteristics of immunofluorescence microscopy and of dilution-plating to detect Pseudomonas syringae pv. phaseolicola in bean seed lots and for risk assessment of field incidence of halo blight

Routine laboratory testing of 710 bean seed lots from various origins forPseudomonas syringae pv. phaseolicola (Psp) with immunofluorescence microscopy (IF) showed that 27.5% of the seed lots (five subsamples of 1000 seeds tested per sample) contained two or more IF-positive cells in a total of 500 microscope fields (magnification 500×). Simultaneously performed dilution-platings of IF-positive subsamples on King's medium B confirmed presence of Psp for one-third of these IF-positive seed lots. The grey area of disagreement between both laboratory tests was studied by comparison of test data and by field trials.The number of IF-positive cells per subsample was positively correlated with isolation and identification of Psp (R=0.85). The detection level of IF was ca. 10² Psp cells per ml of undiluted subsample extract. The detection level of Psp by isolation on King's medium B was variable, being inversely related with the saprophyte to Psp ratio. The high sensitivity of IF was in part due to high percentages of dormant or dead IF-positive cells in the sample extract. Field trials over two years with 10 000 seeds per seed lot, showed disease incidence for 9 of the 22 seed lots. Of ten IF-positive lots with five positive subsamples per sample, nine were positive in the field test plot (the negative lot gave primary infection spots of Psp when used for commercial growing). By isolation, seven of these ten IF-positive lots were positive. Of the five IF-positive lots with two or less positive subsamples, isolation and field trial were both negative. Based on data on seed transmission from literature, field incidence was unlikely for these five samples in a 10 000 seeds field trial. All seven IF-negative lots were negative in the field trial. The value of IF and isolation for indexing bean seed lots for Psp is discussed.This study was carried out at the Centre for Plant Breeding and Reproduction Research (CPRO-DLO), Binnenhaven 1, P.O. Box 16, 6700 AA Wageningen the Netherlands, to which address correspondence should be addressed.     

Polymorphism of 14α-demethylase Gene (CYP51) in the Cereal Eyespot Fungi Tapesia acuformis and Tapesia yallundae

Cereal eyespot fungi Tapesia acuformis and Tapesia yallundae are closely related species which show different behaviours upon treatment with sterol 14-demethylase inhibitors (DMIs). T. acuformis is naturally resistant to DMIs belonging to the triazole family and susceptible to the imidazole ones, whilst T. yallundae is sensitive to both inhibitors. Cloning of the target enzyme gene, CYP51, from the two species revealed an important polymorphism between them. Further sequencing of CYP51 from sixteen T. acuformis and eleven T. yallundae strains with different phenotypes with regards to resistance to DMIs confirmed that at least eleven variations are species related. Among them, a conserved phenylalanine residue at position 180, found both in T. yallundae and in all known CYP51 proteins from filamentous fungi and yeast, was replaced in T. acuformis by a leucine. Therefore, a leucine at 180 could be possibly involved in natural resistance of T. acuformis to triazoles. Other mutations were observed in some resistant strains, sometimes simultaneously, but in contrast to what was reported for other filamentous fungi, where a mutation at the 136 position of the CYP51 gene product seemed to correlate with resistance to DMIs, we did not find a clear relationship between a given mutation and a particular phenotype. This result suggests that resistance to DMIs could have a polygenic nature in Tapesia. We took advantage of species-related variations to develop a PCR-based assay allowing rapid and easy discrimination between field strains of the two species.     

Control of seed-borne pathogens on legumes by microbial and other alternative seed treatments

Greenhouse trials were carried out in order to test the efficacy of different seed treatments as alternatives to chemicals against Colletotrichum lindemuthianum cause of anthracnose on bean and Ascochyta spp. cause of Ascochyta blights on pea, respectively. Resistance inducers, commercially formulated microorganisms, non-formulated selected strains of different microorganisms (fungi, bacteria and yeasts) and plant extracts were applied as dry or liquid seed treatments on naturally infested seeds. Seedling emergence and disease incidence and/or severity were recorded. Almost all seed treatments turned out to be ineffective in controlling the Ascochyta infections, which is in line with the literature stating that these pathogens are difficult to control. The only alternative treatments that gave some control of Ascochyta spp. were thyme oil and a strain of Clonostachys rosea. The resistance inducers tested successfully controlled infections of bean by C. lindemuthianum. Among the formulated microorganisms, Bacillus subtilis-based formulations provided the best protection from anthracnose. Some strains of Pseudomonas putida, a disease-suppressive, saprophytic strain of Fusarium oxysporum and the mustard powder-based product Tillecur also proved to be effective against bean anthracnose. However, among the resistance inducers as well as among the other groups, certain agents caused a significant reduction of plant emergence. Different alternative seed treatments can therefore be used for the control of C. lindemuthianum on bean, while on pea only thyme oil and a strain of Clonostachys rosea showed some effectiveness against Ascochyta spp.     

Efficacy of Different Control Methods Applied Separately and in Combination in Managing Root-knot Nematodes (Meloidogyne spp.) in Common Beans

Investigations on the efficacy of various methods of managing root-knot nematodes in microplots and under field conditions revealed that soil solarization, Furadan 5G and Tagetes erecta applied separately or in combination with other control methods, were the most effective in reducing the numbers of three root-knot nematodes, and root gall and egg mass indices. These management methods also resulted in significant increases (P0.05) in number of pods per plant, number of seeds per pod and 100-seed weight, and increased seed yield by up to 96.7 percent. Farmyard manure and Crotalaria ochroleuca were the least effective treatments. The use of T. erecta was the most economical root-knot nematode control method.     

Root rot pathogens in field soil,roots and seeds in relation to common bean (Phaseolus vulgaris), disease and seed production

The interrelationships among bean productivity, prevalence of pathogens in roots, seeds and soil, and root rot disease were described at the pod maturity stage in 13 commercial fields. The soil population and frequency of pathogens isolated from seeds varied by pathogen species and field location. Fusarium solani was the most prevalent fungus isolated from bean seeds and field soil compared to Rhizoctonia solani, Macrophomina phaseolina and F. oxysporum. Principal component analysis revealed that the first component explaining 32% of the total variance was correlated with the root rot index. PC1 was more strongly linked to root and seed infections in comparison with soil populations of pathogens. Based on a correlation between PC2 (accounting for 23% of the total variance) and the number of seeds per bean plant, charcoal, Fusarium and Rhizoctonia root rots were recognized as more important determinants of seed losses to root rot disease. There were correlations among the major pathogens infecting either roots or seeds of beans. These findings provide useful information for future experimental plans to optimize management strategies for bean root rots.     

Suitability of Ten Plant Baits for the Rapid Detection of Pathogenic Pythium Species in Hydroponic Crops

Ten types of plant baits were tested in the laboratory to assess their capacity to detect pathogenic Pythium species. These were orange tree leaves, tomato leaves, pepper leaves, geranium leaves, Bermuda grass leaves, pine needles, immature carnation petals, hemp-seed cotyledons, pepper and cucumber fruits. The Pythium spp. tested were P. aphanidermatum, P. irregulare and Pythium group F from hydroponic market garden crops in the Poniente region of Almería (south-east Spain). The test consisted of observing the velocity at which five baits were colonized and the day of colonization of the first bait. Results indicated that the slowest baits to be infected were immature carnation petals and pine needles. These two, together with Bermuda grass leaves, were also the baits infected in lowest number, such that practically no further infection was produced in the baits after the fifth day of contact with the inoculated water. The other plant baits tested were equally suitable for detection of Pythium spp. over the first two days, although only orange leaves and hemp-seed cotyledons were infected on the first day.     

Analysis of protease activity in <Emphasis Type="Italic">Aspergillus flavus</Emphasis> and <Emphasis Type="Italic">A. parasiticus</Emphasis> on peanut seed infection and aflatoxin contamination

Aspergillus flavus and A. parasiticus are aflatoxin-producing fungi that can infect peanut seeds in field crops. An association between A. parasiticus proteolytic enzyme activities and peanut fungal infection was examined. For this study, a model of inductive and non-inductive culture media to produce A. parasiticus extracellular protease before infection was used. These A. parasiticus cultures were used to infect peanut seeds of cultivars resistant and susceptible to aflatoxin contamination. Peanut seeds of both cultivars exposed to fungi grown on casein medium (inductive medium) showed higher internal and external infection and a higher fungal protease content than those observed on potato dextrose agar (PDA) and sucrose medium (non-inductive media). A further study showed higher fungal colonisation and aflatoxin contamination in seeds of the resistant cultivar pre-incubated with Aspergillus extracellular proteases than in those incubated without proteases. Moreover, protease activities affected the viability of non-infected resistant cultivar seeds, inhibiting germination and radicle elongation and enhancing seed tissue injury. The results strongly suggest that protease production by A. parasiticus is involved in peanut seed infection and aflatoxin contamination resulting in seed tissue damage, affecting seed viability and facilitating the access of fungi through the testa. The analysis of fungal extracellular proteases formed on peanut seed during infection showed that A. flavus and A. parasiticus produced metallo and serine proteases; however, there were differences in the molecular masses of the enzymes between both species. The greatest activity in both species was by serine protease, that could be classified as subtilase.     

Interactions between Plasmopara helianthi, Glomus mosseae and Two Plant Activators in Sunflower Plants

Interactions between Plasmopara helianthi, Glomus mosseae and two plant activators DL--amino-n-butyric acid (BABA) and CGA 245704 (acibenzolar-S-methyl (BTH)) in sunflower plants susceptible to downy mildew were studied in four experiments using different methods of treatment and pathogen inoculation. Both chemicals were applied as soil drenches and foliar sprays, whereas P. helianthi infection was obtained by root and cotyledon inoculations of the seedlings. Soil drenches at the rates of 50 and 100mgkg^–1 soil of BABA and BTH given 1 and 3 days before P. helianthi inoculation, respectively to mycorrhizal plants, provided moderate protection against the pathogen (about 50–55%). Morphological changes and decrease in mycorrhizal colonization in roots of BTH-treated plants and in BTH-treated mycorrhizal plants were also observed. Delay in the emergence and reduction of the root systems were more evident at the highest concentration but decreased with time. These effects were absent with the BABA treatment.Foliar spray treatment of BABA and BTH, applied at 4000 and 200µgml^–1, respectively (1 day post-inoculation) to mycorrhizal plants provided good protection (about 80%) against P. helianthi foliar infections. No effects on mycorrhizal colonization or on root systems were observed. In vitro tests on the effect of the compounds on the mycorrhizal fungus showed that the germination of G. mosseae sporocarps increased with BABA treatment whereas it was greatly inhibited by BTH treatment.     

Characterization of a New Nepovirus Infecting Apricot in Southeastern France: Apricot Latent Ringspot Virus

A pathogen was transmitted from apricot trees showing symptoms of viral infection to GF305 peach seedlings which reacted by stunting, shortened internodes and chlorotic mottling. The agent was transmitted to cherry, apricot, peach and plum by grafting and to several herbaceous hosts by mechanical inoculation. Isometric nepovirus-like particles of 30–31 nm diameter extracted from infected Chenopodium quinoa sedimented as two peaks in sucrose gradients. These particles contained two single stranded RNAs of approximately 5.9 and 7.9 kb, and a single coat protein subunit of 53.7 kDa. No cross-reactions were observed with a number of nepoviruses infecting fruit trees. Inoculation of purified particles to herbaceous or woody hosts reproduced the same symptoms caused by the original isolate. Sequencing of a 2.2 kbp cDNA clone covering the 3 end of the small genomic RNA identified an open reading frame encoding a 317 aa N-truncated protein exhibiting significant similarities with the coat protein of nepoviruses. The 1257 nt long 3 non-coding region showed up to about 65% homology to the equivalent region of members of the subgroup C of nepoviruses. The properties of this pathogen do not match those of any previously described nepovirus. It should therefore be considered as a new member of the subgroup C of nepoviruses, for which the name of Apricot latent ringspot virus (ALRSV) is proposed.The nucleotide sequence reported in this work has been deposited in the EMBL databank under the accession number AJ278875.     

Organic Seed-treatment as a Substitute for Chemical Seed-treatment to Control Common Bunt of Wheat

Common bunt of wheat, caused by Tilletia laevis and T. tritici, is a major seed and soil-borne disease in West Asia and North Africa. The use of resistant cultivars and chemical seed-treatments are the current control measures used to combat this disease. The aim of this study was to investigate alternative control measures to chemical seed-treatments that are environmentally friendly to support cultivar resistance. Several organic nutrients [skimmed milk powder, hucket (local skimmed milk) and wheat flour] at a concentration of 160g per kg of seeds were used as seed-treatments on two susceptible bread and durum wheat cultivars (Bau and Sebou, respectively) to examine their effectiveness in controlling the disease. Field trials over four years showed that skimmed milk powder, hucket, and wheat flour reduced common bunt infection levels by 96%, 93% and 62%, respectively. In most cases, the effectiveness of the skimmed milk powder and hucket was equal to the chemical seed-treatment; thus, these organic nutrients offer an effective and environmentally safe alternative to chemical treatments. However, a study of their economic value as well as of their effect on seed germination, and field emergence is needed.     

氯虫苯甲酰胺拌种对稻纵卷叶螟的防治效果及安全性评价

为减少水稻抽穗前田间稻纵卷叶螟的化学防治次数及用药量，降低对天敌的伤害及农药残留，最终实现稻纵卷叶螟的简约化防控，采用氯虫苯甲酰胺对水稻种子进行拌种处理，室内测定了药剂拌种对种子发芽的安全性，并结合不同栽培模式下的田间试验，评价了不同用量下氯虫苯甲酰胺拌种对稻纵卷叶螟的控制时长及保叶效果，明确了药剂拌种对天敌蜘蛛的影响及最终残留情况。结果表明:当氯虫苯甲酰胺按有效成分用量30~120 g/hm2拌种时，对种子的发芽率及出苗均无影响。直播稻模式下，氯虫苯甲酰胺按有效成分60 g/hm2拌种，播种后110 d，其保叶效果仍高达97.13%；旱育手栽秧模式下，氯虫苯甲酰胺按有效成分90 g/hm2拌种，播种后108 d保叶效果为82%以上，旱育机插秧模式下按有效成分90~120 g/hm2拌种，播种后90 d保叶效果仍在95%以上；3种模式下拌种处理的保叶效果及持效期均远优于常规喷雾防治。研究表明，利用氯虫苯甲酰胺对水稻种子进行拌种处理，可有效控制水稻前期及中期稻纵卷叶螟的为害，降低后期田间虫口基数，并且对田间天敌蜘蛛安全性高，同时在收获的稻谷中未检出氯虫苯甲酰胺残留(残留量低于定量限0.000 8 mg/kg)。在江苏省丘陵地区粳稻区应用氯虫苯甲酰胺拌种处理，可确保水稻直至抽穗初期无需再进行稻纵卷叶螟的防治，并可显著减少后期稻纵卷叶螟虫源基数，实现稻纵卷叶螟的简约化防治。     

The effect of a mixture of seed-borne Microdochium nivale var. majus and Microdochium nivale var. nivale infection on Fusarium seedling blight severity and subsequent stem colonisation and growth of winter wheat in pot experiments

Greenhouse experiments were conducted in order to determine the impact of seed-borne Microdochium nivale var. nivale and var. majus inoculum, and seed treatment with a carboxin+thiram mixture, on the development of seedling blight, and on subsequent stem colonisation and growth of winter wheat (cv. Cadenza). Experiments were conducted at temperatures favourable (3°C) and unfavourable (22°C) to M. nivale. Seed-borne inoculum resulted in seedling blight symptom development when plants were grown at 3°C, but not when plants were grown at 22°C. For seedlings grown at 3°C, plants arising from heavily blighted seedlings developed more severe symptoms of stem colonisation, when compared with those arising from seedlings from carboxin+thiram treated seeds. In addition, the vigour of such plants (assessed by determining the number of tillers and ears per plant, stem length, green leaf area, dry weight and yield) was also significantly lower than for plants arising from carboxin+thiram treated seeds. Microdochium nivale var. majus and var. nivale appeared to have little effect on plant vigour from seedlings grown at 22°C. This is the first recorded incidence of seedling blight affecting subsequent plant growth. Microdochium nivale var. majus and var. nivale stem colonisation increased from growth stage (GS) 40–49 to harvest in plants raised from seedlings grown at both temperatures. Microdochium nivale var. majus and var. nivale were isolated from the second node at GS 40–49 and the third node at harvest of plants from seedlings grown at 3°C. For plants from seedlings raised at 22°C, M. nivale var. majus and var. nivale were isolated from the first node at GS 40–49 and the second node at harvest. Carboxin+thiram seed treatment decreased the extent and severity of stem colonisation on plants from seedlings grown at 22°C.     

Application of enzymes during bulb tissue extraction for detection of lily symptomless virus by ELISA in Lilium spp

Lily symptomless virus (LSV) was readily detected with the enzyme-linked immunosorbent assay (ELISA) in leaves ofLilium Mid-Century hybrids cvs Enchantment and Destiny andLilium speciosum cv. Brabander. In bulbs of cv. Enchantment LSV could be detected without any additional treatment of the extracts, but for reliable results in cv. Destiny it was necessary to pre-incubate the bulb extracts with cellulase or hemicellulase.Samenvatting In de bladeren van secundair geïnfecteerde planten van de leliecultivars Enchantment, Destiny en Brabander kon LSV met ELISA gemakkelijk worden aangetoond. In bladextracten van Enchantment kon nog ca. 100 ng LSV/ml extract worden teruggevonden. Bij toepassing van de standaard-methode van extraheren (2 g weefsel malen in 4 ml extractiebuffer) kon ook in de bolschubben van Enchantment LSV worden aangetoond; bij Destiny gelukte dat aanzienlijk minder goed. Wijzigingen in de extractie-procedure of een speciale behandeling van het standaard-extract (dialyseren, verhitten tot 50°C etc.) leidde niet tot een betere aantoonbaarheid van het virus met ELISA. Enzymen als hemicellulase, cellulase en pectinase, toegevoegd aan het standaard-extract van bolschubben van Destiny vóór de toetsing (incubatie gedurende 18 h bij kamertemperatuur), verbeteren de aantoonbaarheid van het LSV aanzienlijk.     

Characterisation of <Emphasis Type="Italic">Phoma tracheiphila</Emphasis> by RAPD-PCR,microsatellite-primed PCR and ITS rDNA sequencing and development of specific primers for <Emphasis Type="Italic">in planta</Emphasis> PCR detection

Thirty six isolates of Phoma tracheiphila from Italy, the causal agent of the mal secco disease on Citrus species, were characterised by different molecular tools in comparison with representative isolates of other phytopathogenic Phoma species. These included analysis of the distribution of RAPD and microsatellite markers and sequencing of the internal transcribed spacer (ITS) region of the nuclear rRNA genes. The results obtained with 12 RAPD primers (92 markers) and 7 microsatellite primers (56 markers) suggest that Italian isolates of P. tracheiphila are genetically homogeneous, leading to identical patterns upon amplification with all the tested primers. Accordingly, ITSI-5.8S-ITS2 sequences were highly conserved (98–100% identity along a 544-characters alignment) among all the isolates of P. tracheiphila. A neighbor-joining analysis of ITS sequences of P. tracheiphila in comparison with those of other Phoma species, as well as with alignable sequences from anamorphic and teleomorphic taxa retrieved in BLAST searches, revealed a close relationship between P. tracheiphila and Leptosphaeria congesta. A pair of P. tracheiphila-specific primers was designed on the consensus sequence (555 residues) obtained from the alignment of the newly generated P. tracheiphila ITS sequences. A PCR-based specific assay coupled to electrophoretic separation of amplicons made it possible to detect P. tracheiphila in naturally infected Citrus wood tissue collected from both symptomatic and symptomless plants. The limit of detection was 10 pg of genomic DNA and 5 fg of the ITS target sequence.     

Role of chemical characteristics of the seed coat in the resistance of selected cowpea varieties to Callosobruchus maculatus (F.) (Coleoptera: Bruchidae) in Nigeria

The role of seed coat chemical factors in the resistance of the cowpea varieties, Kanannado, IT89KD-391 and Borno brown, to the cowpea seed bruchid Callosobruchus maculatus (F.), was investigated under laboratory conditions (30-35C and 65-67% RH) in Maiduguri, Nigeria. Significantly higher numbers of eggs were laid on de-coated than on intact Kanannado seeds whereas significantly smaller numbers of eggs were laid on de-coated than on intact IT89KD-391 or Borno brown seeds. Susceptibility was higher in de-coated than in intact Kanannado seeds (susceptibility indices [SI] 3.4 and 0.0, respectively). Egg-hatch was significantly reduced in seeds with intact seed coats by 88.6%, while the proportion of eggs that failed to hatch in de-coated seeds was 31.9%. Treatment of Borno brown seeds especially with 32 and 64 mg of extracts from Kanannado and IT89KD-391 seed coats, reduced oviposition by 61.9% and 95.2%, respectively. Treatment with 32 mg of the seed coat extracts reduced egg-hatch by 49.2%. Identical dosages (32 and 64 mg) of these seed coat extracts also significantly reduced susceptibility ofBorno brown to C. maculatus (SI values 6.7 and 1.5, respectively). Comparable SI values for Borno brown treated with 16 mg of the seed coat extracts or extract-free acetone were 14.9 and 14.0, respectively.     

A Botrytis cinerea Putative 3-keto Reductase Gene (ERG27) that is Homologous to the Mammalian 17β-Hydroxysteroid Dehydrogenase type 7 gene (17β-HSD7)

Botrytis cinerea (anamorph of Botryotinia fuckeliana) is a filamentous ascomycete that causes grey mould on grapevine. We had previously described two distinct populations, named HydR1 and non-HydR1, that comprise two distinct genetic entities based on genetic polymorphism, natural resistance towards the fungicide hydroxyanilide fenhexamid, and vegetative incompatibility between them. Here, we used PCR to isolate the 3-keto reductase gene ERG27 by virtue of sequence homology with Saccharomyces cerevisiae ERG27. The gene product was longer than the yeast's enzyme but possessed the main characteristic features of reductases. It displayed striking homology with mammalian 17-HSD7, therefore confirming the hypothesis of a common function between Erg27p like protein and 17-HSD7 in sterol biosynthesis (i.e. cholesterol, ergosterol). On the other hand, we analysed the polymorphism of the B. cinerea gene product and found a dozen of amino-acid differences between strains of HydR1 and non-HydR1 types that could underlie HydR1 natural resistance to fenhexamid. First, this polymorphism analysis showed that HydR1 strains form a homogeneous group distinct from the non-HydR1 group of strains. These results support our hypothesis that HydR1 and non-HydR1 strains constitute two different species. Second, Erg27p like protein sequence analysis showed that a high resistant phenotype to fenhexamid, HydR3, found in treated populations of non-HydR1 strains, had two mutations (usually found in mammalian 17-HSD7) that could be useful as population markers.     

The β-tubulin Gene is a Useful Target for PCR-based Detection of an Albino Ophiostoma piliferum Used in Biological Control of Sapstain

The potential of Cartapip, an albino Ophiostoma piliferum, as a biocontrol agent against sapstain in logs has been tested in Germany. To detect the albino strain in field-tested wood, the usefulness of the -tubulin gene as a target region for developing PCR-based assays was evaluated with 102 strains of O. piliferum and 31 strains of other wood-inhabiting species. A partial -tubulin gene sequence of O. piliferum strains from different geographic origins was amplified by PCR and analyzed by restriction enzyme digestions and DNA sequencing. Variation in size and nucleotide sequences was found in intron regions indicating that intraspecific variation is present in the -tubulin gene. Consequently, -tubulin gene-derived PCR methods using PCR–RFLP patterns generated by HinfI and SpeI and sequence-specific primers Cat1 and Cat2, were developed and their specificity for Cartapip was accessed with field-tested logs and lumber. The -tubulin gene-based PCR methods were found to be valuable tools for rapid and reliable identification of Cartapip in field-tested logs and lumber in Germany. Specificity tests against other wood-inhabiting species and wild type O. piliferum strains from diverse nations showed that the Cat1 and Cat2 primers have potential to be used in other European countries, New Zealand, Alberta and British Columbia.     

Analysis of genetic relations between <Emphasis Type="Italic">Broad bean wilt virus 1</Emphasis> and <Emphasis Type="Italic">Broad bean wilt virus 2</Emphasis>

We determined the complete nucleotide sequence of RNA-1 and the 5-terminal region of RNA-2 from Broad bean wilt virus 1 (BBWV-1) isolate PV132. This report is the first analysis of the genome organization of BBWV-1. We also determined the complete nucleotide sequence of RNA-1 from Broad bean wilt virus 2 (BBWV-2) isolate IP and analyzed the genetic relations between BBWV-1 and BBWV-2. Similar to the BBWV-2 isolates, both RNAs of PV132 encoded a single large polyprotein, which was predicted to contain some functional proteins in a manner similar to those of comovirus. With respect to the deduced amino acid sequences of the mature proteins, PV132 and IP had only 20%–40% homology to comovirus. On the other hand, IP was 73%–98% homologous to BBWV-2 isolates, but PV132 was 39%–67% homologous to the isolates. Although the extent of the homologies differed, the homologies were limited between BBWV-1 and BBWV-2 not only for the coat protein but also for the other proteins. These results clearly support the placement of BBWV-1 and BBWV-2 in the genus Fabavirus as distinct species, proposed on the basis of double immunodiffusion tests.The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession numbers AB084450 (RNA-1 of isolate PV132), AB084451 (RNA-2 of isolate PV132), and AB023484 (RNA-1 of isolate IP)     

Correlation between field disease and latent infection by Glomerella cingulata of strawberry plants as revealed by a simple diagnostic method using ethanol immersion

Leaves of strawberry plants growing in fields were collected and assayed by ethanol immersion treatment (SDEI) to detect latent infection by Glomerella cingulata and Dendrophoma obscurans. SDEI revealed that 83% of the plants in growers fields were latently infected by G. cingulata and 58% by D. obscurans. In such fields, only 0.8%–2.5% of the plants later became wilted or died because of G. cingulata. When the latently infected plants from naturally infested fields were kept under optimal conditions for disease development for 5 weeks, 70.0%–83.3% of them wilted or died from G. cingulata. However, only 5.6%–6.7% of the plants diagnosed by SDEI as being without latent infection wilted or died. The results indicate the importance of selecting healthy plants, suggesting that SDEI is an effective method for detecting latent infection and reducing losses from G. cingulata.     

First Report of <Emphasis Type="Italic">Pepper mottle virus</Emphasis> on <Emphasis Type="Italic">Capsicum annuum</Emphasis> in Japan

Pepper mottle virus, genus Potyvirus, was first identified in Japan based on particle morphology, host range, aphid transmission, and molecular classification using the nucleotide sequence of the coat protein gene and 3-untranslated region.