Incidence of Soil-borne Wheat Mosaic Virus in Mixtures of Susceptible and Resistant Wheat Cultivars

The multiplication of Soil-borne wheat mosaic virus (SBWMV) was studied in mixtures of two winter wheat (Triticum aestivum) cultivars, one susceptible (Soissons) and the other resistant (Trémie). Two seed mixtures of susceptible and resistant varieties in ratios of 1 : 1 and 1 : 3 and their component pure stands, i.e. each variety grown separately, were grown in a field infected with SBWMV. The presence of the virus was detected using DAS-ELISA from January to May. The resistant cultivar Trémie showed no foliar symptoms nor could the virus be detected in the leaves or roots. In May, about 88% of plants of susceptible cultivar Soissons grown in pure stands were infected. At this time, the disease reduction relative to pure stands was 32.2% in the 1 : 1 mixture and 39.8% in the 1 : 3 mixture. Optical density (OD) values from ELISA of the infected plants in the two mixtures were consistently lower than that of the infected plants in cultivar Soissons in pure stands. The ELISA index (EI) calculated using three scales of OD values was 65.5% in the susceptible cultivar in pure stands. The value for this index was 19.1% in the 1 : 1 mixture and 7.9% in the 1 : 3 mixture. The plants of the resistant cultivar Trémie infected in the same field and transferred in January to a growth cabinet at 15 °C multiplied the virus and produced viruliferous zoospores. These results show that the resistant cultivar Trémie plays a role in disease reduction in the cultivar mixtures in field conditions. Possible reasons for this are discussed.     

Different Classes of Resistance to Turnip Mosaic Virus in Brassica rapa

Pathotype-specific and broad-spectrum resistance to turnip mosaic virus (TuMV) have been identified in the diploid A genome brassica species Brassica rapa. The pathotype-specific resistance is effective against pathotype 1 isolates of TuMV, which are the most common in Europe. It is almost identical in its specificity to that of a mapped resistance gene (TuRB01) present in the A genome of the amphidiploid species Brassica napus. A mutant of a pathotype 1 isolate of TuMV (UK 1M) that is able to overcome TuRB01 also overcame the B. rapa resistance. This, combined with the fact that a single-nucleotide mutation in the cylindrical inclusion gene of TuMV that has been shown to induce a change from avirulence to virulence against TuRB01, had an identical effect on the B. rapa resistance, suggest that the two resistances are conditioned by the same gene. A second source of resistance in B. rapa prevented systemic spread of all TuMV isolates tested. A third source of resistance that appears to provide immunity to, or severely restrict replication of most isolates of TuMV has been characterised. This resistance source also prevented systemic spread of all TuMV isolates tested. Prior to this study, no resistance to pathotype 4 or pathotype 12 isolates of TuMV had ever been identified. For each of these three resistance sources, plant lines that are not segregating for some of the resistance phenotypes and that are presumably homozygous for the genes controlling these phenotypes have been generated. Strategies for further characterising and deploying these resistances in different Brassica species are described.     

The Use of Molecular Beacons Combined with NASBA for the Sensitive Detection of Sugarcane Yellow Leaf Virus

Sugarcane yellow leaf virus (ScYLV) is widely distributed in Brazil and other sugarcane producing countries causing significant yield losses. Due to the high incidence of the aphid vector, the virus is widespread in the field and in parental clones used in sugarcane breeding programmes. Aiming to present a sensitive and reliable detection of ScYLV, we have adapted an AmpliDet RNA system, compared it with the currently available detection methods and discussed its applicability for routine diagnosis. AmpliDet RNA consists of nucleic acid sequence-based amplification (NASBA) of the target RNA with specific primers and simultaneous real-time detection of the amplification products with molecular beacons. The results showed that the system produced a detection level of at least 100fg of purified virus. Virus was readily detected in plant tissues with low levels of infection (without the need of previous RNA extraction) and in the hemolymph of aphids. The method showed to be virus-specific, testing negative for other species of the Luteoviridae. In conclusion, the system has potential to become a diagnostic method for the detection of sugarcane viruses.     

Southern bean Mosaic Virus the Causal Agent of a New Disease of Phaseolus vulgaris beans in Spain

Southern bean mosaic virus (SBMV) has been identified as the cause of a new disease in greenhouse-cultivated common bean (Phaseolus vulgaris), in the south-east of Spain. The identification was based on host range comparisons, morphological and serological characteristics of the virus, the size of its dsRNA species and the nucleotide sequence of an 810-bp fragment from ORF2. The virus could be clearly discriminated from the related sobemovirus Southern cowpea mosaic virus. This is the first report of SBMV in Spain.     

Identification of genes expressed during spore germination of Mycosphaerella pinodes

Mycosphaerella blight, caused by Mycosphaerella pinodes, is one of the major diseases of cultivated pea (Pisum sativum L.). To isolate the genes that are up- and down-regulated during spore germination, suppression subtraction hybridization (SSH) was performed between ungerminated and germinated spores. The 232 and 128 clones from forward and reverse libraries, respectively, were collected, sequenced, and analyzed with a BLASTX homology search. About 95% of the 32 selected clones were expressed during spore germination on a paper sheet and during infection of pea leaves. We discuss the applicability of the SSH libraries for analyzing M. pinodes genes involved in the early stage of infection.     

Molecular analysis and virus transmission tests place Olpidium virulentus, a vector of Mirafiori lettuce big-vein virus and tobacco stunt virus, as a distinct species rather than a strain of Olpidium brassicae

The rDNA-ITS sequences of ten single-sporangium isolates of Olpidium virulentus (a noncrucifer strain of Olpidium brassicae), which transmits Mirafiori lettuce big-vein virus (MLBVV) and tobacco stunt virus (TStV), were compared with those of six single-sporangium isolates of O. brassicae. The sequence similarity within isolates of O. virulentus or O. brassicae was almost identical (98.5%–100.0%), but was low between the two species (79.7%–81.8%). In a phylogenetic analysis of the rDNA-ITS region, O. virulentus and O. brassicae fell into two distinct clusters, indicating that O. virulentus, a vector of MLBVV and TStV, is a distinct species rather than a strain of O. brassicae.     

An epidemiological survey of Chrysanthemum chlorotic mottle viroid in Akita Prefecture as a model region in Japan

In epidemiological surveys on Chrysanthemum chlorotic mottle viroid (CChMVd) in various chrysanthemums cultivated in Akita Prefecture, Japan, in 2002–2005, approximately 20% of cultivars harbored CChMVd, including more symptomatic types than nonsymptomatic. Large-flowered cultivars were less frequently infected than small-flowered and spray types. The number of CChMVd-infected chrysanthemums is increasing, and the disease is found throughout major chrysanthemum-producing districts. Chrysanthemums infected only with CChMVd, in general, had no noticeable symptoms of disease. Most of those dually infected with known viruses and/or viroid also had no symptoms characteristic of chlorotic mottle disease. The lack of noticeable symptoms in major Japanese cultivars may have resulted in the unnoticed spread of the viroid.     

Scab Response and Moniliformin Accumulation in Kernels of Oat Genotypes Inoculated with Fusarium avenaceum in Poland

Inoculation experiments with 14 genotypes of oats (10 cultivars and 4 lines) were performed during 1996, 1997 and 1998 in Sitaniec, South-Eastern Poland. Panicles of oats were inoculated with a conidial suspension of Fusarium avenaceum, which caused a reduction in yield by 33% and in 1000 kernel weight (TKW) by 21%. During the period between inoculation and harvest, F. avenaceum was able to accumulate moniliformin (MON) in kernels at an average level of 0.13mgkg^–1 (gg^–1). The highest reduction of yield components caused by the F. avenaceum inoculation was found for cv. Santor, followed by lines CHD 1171, STH 2795 and cvs: Kwant and Farys, while cvs Slawko, Dukat, Borys and Komes exhibited the highest resistance to the disease in terms of TKW and yield reductions after inoculation.     

Identification of a new pathotype of Puccinia hordei with virulence for the resistance gene Rph7

Barley leaf rust resistance gene Rph7, derived from barley accession Cebada Capa, is the most effective R-gene for resistance to Puccinia hordei. Virulence for this gene was known in the USA, Israel and Morocco but not yet in Europe. We found an unexpected leaf rust infection in the field at Córdoba, Spain in 2004 on Rph7 carrying lines. This virulence for Rph7 was confirmed in growth chamber experiments, being the first report of Rph7 virulence in European populations of P. hordei. A collection of 680 barley accessions was screened for resistance against this new isolate. Twelve accessions showed segregation with individual plants showing resistance based on hypersensitivity (low infection type). These individual resistant plants were selected and grown in the greenhouse to obtain seeds.     

Rapid Identification of Agrobacterium Species By Staircase Electrophoresis of Low Molecular Weight RNA Profiles

Several plant species are infected by different species of the genus Agrobacterium. One problem is that no rapid and sensitive method is available for the identification of isolates of Agrobacterium at the species level. The usefulness of LMW RNA profiles for the identification of Agrobacterium species was examined. The profiles of strains belonging to the proposed species were identical to those of the type strain of each species except in two cases. In A. radiobacter, two groups of strains with different tRNA profiles were detected and in A. rhizogenes two groups with different 5S rRNA zones were found. Nevertheless, with the LMW RNA profiles it was possible to assign any isolate to one of each group within these species. The results obtained showed that all isolates studied here can be assigned to a species of Agrobacterium and hence that LMW RNA profiles offer a suitable method for the identification of tumor-inducing bacteria.     

Rapid Identification of Clavibacter michiganensis Subspecies Sepedonicus based on the Stable Low Molecular Weight RNA (LMW RNA) Profiles

Clavibacter michiganensis subsp. sepedonicus causes potato ring rot disease. The identification process for this bacterium is complex and long. This work demonstrates that the stable low-molecular-weight (LMW) RNA profiles allow their rapid identification. Staircase electrophoresis in polyacrylamide gels was used to analyze the LMW RNA profiles of 54 strains of C. michiganensis subsp. sepedonicus from different geographic origins. The profiles of several strains of other subspecies of C. michiganensis and other pathogens of potatoes were also analyzed. All the strains of C. michiganensis subsp. sepedonicus had the same LMW RNA profile. They had a band in class 2 of tRNA that was absent in the other subspecies of the species C. michiganensis. Also, the LMW RNA of C. michiganensis subsp. sepedonicus was different with respect to the LMW RNA profiles of other pathogens of potato. The results indicate the possible utilization of LMW RNA profiles in identification of the bacteria causing potato ring rot disease.     

Identification of divergent variants of Grapevine virus A

Eight isolates of Grapevine virus A (GVA), which induced different symptoms in leaves of Nicotiana benthamiana, were recovered from various grapevines. The dsRNA patterns of two isolates, which consistently induced mild vein clearing (referred here as mild isolates of GVA) were similar, but different from those of other isolates of GVA. Analysis based on overall nucleotide (nt) sequence identity in the 3 terminal part of the GVA genome, comprising part of ORF3 (putative movement protein, MP), entire ORF4 (capsid protein, CP), entire ORF5 and part of 3 UTR, revealed that GVA isolates separate into three groups (I, II, III), sharing 91.0–99.8% nt sequence identity within groups and 78.0–89.3% nt sequence identity between groups. Mild isolates of the virus were group III and shared only 78.0–79.6% nt sequence identity with the other isolates. The comparison of predicted amino acid sequences for MP and CP revealed many amino acid alterations, revealing distinct local net charges of these proteins for mild isolates of the virus. Based on both conserved and divergent nt regions in the CP and ORF5, oligonucleotide primers were designed for the simultaneous RT-PCR detection of all GVA isolates and for the specific detection of the most divergent virus variants represented here by mild isolates of the virus.     

Identification and pathogenicity of Pythium species causing damping-off of kidney bean

Five Pythium species (Pythium irregulare, P. mamillatum, P. myriotylum, P. spinosum and P. ultimum var. ultimum) were isolated from the hypocotyls and roots of kidney bean plants with damping-off from a commercial field and from experimental plots that have undergone either continuous cropping with kidney bean or rotational cropping with arable crops. In inoculation tests, all five Pythium species were pathogenic to kidney bean. This is the first report of damping-off of kidney bean caused by Pythium species; we named this disease damping-off of kidney bean. The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under accession numbers AB291811, AB291944 and AB291945.     

Virus transmission by aphids in potato crops

This paper reviews the contribution of vector activity and plant age to virus spread in potato crops. Determining which aphid species are vectors is particularly important for timing haulm destruction to minimize tuber infection by potato virus Y (PVY). Alate aphids of more than 30 species transmit PVY, and aphids such asRhopalosiphum padi, that migrate in large numbers before flights of the more efficient vector,Myzus persicae, appear to be important vectors. Differences in methodology, aphid biotypes and virus strains prevent direct comparisons between estimates of vector efficiencies obtained for aphids in different countries in north western Europe. M. persicae is also the most efficient vector of potato leafroll virus (PLRV), but some clones ofMacrosiphum euphorbiae transmit PLRV efficiently toNicotiana clevelandii and potato test plants. The removal of infected plants early in the season prevents the spread of PLRV in cool regions with limited vector activity. The proportion of aphids acquiring PLRV from infected potato plants decreases with plant age, and healthy potato plants are more resistant to infection later in the season. Severe symptoms of secondary leafroll developed on progeny plants of cv. Maris Piper derived from mother plants inoculated with PLRV in June or July of the previous year. Progeny plants derived from mother plants inoculated in August showed only mild symptoms, but the concentration of PLRV in these plants was as high as that in the plants with severe symptoms.     

Identification and Characterisation of Bacteria Causing Soft-rot in Agave tequilana

Agave tequilana is the raw material for the production of the alcoholic beverage tequila. A bacterial disease has affected the A. tequilana crop in recent years. Previous reports based on colony and cell morphology, Gram stain and potato rot indicated that Erwinia sp. is the main pathogen. We isolated a several bacterial isolates capable of producing soft-rot symptoms in greenhouse pathogenicity assays. An extensive characterisation involving pathogenicity tests, fatty acid profile, metabolic and physiological properties, ribosomal DNA sequence and intergenic transcribed spacer amplification (ITS-PCR) and restriction banding pattern (ITS-RFLP) was made of each isolate. Three different species: Erwinia cacticida, Pantoea agglomerans and Pseudomonas sp. were identified. Fatty acid and metabolic profiles gave low similarity values of identification but 16S rDNA sequence, ITS-PCR and ITS-RFLP confirmed the identification of E. cacticida. In the phylogenetic tree, E. cacticida from blue agave was grouped neither with E. cacticida type strains nor with Erwinia carotovora. This is the first report that associates E. cacticida with A. tequilana soft-rot symptoms.     

Sequence Analysis Shows that a Dwarfing Disease on Rice, Wheat and Maize in China is Caused by Rice Black-streaked Dwarf Virus

Isolates of plant reoviruses causing severe stunting and dark leaf symptoms on wheat in Hebei province, on maize in Hubei province and on rice in Zhejiang province, China have been characterized. Four of the ten genome segments corresponding to Rice black-streaked dwarf virus (RBSDV) S7, S8, S9, S10 were amplified by RT-PCR from the Hebei and Zhejiang isolates and sequenced. Sequences of S9 and S10 were also obtained from the Hubei isolate. Sequences of corresponding segments of the Chinese isolates were very similar to each other (94.0–99.0% identical nucleotides and 96.3–100% identical amino acids) and were closer to those of a previously reported Japanese RBSDV isolate (90.0–94.9% identical nucleotides and 91.1–98.6% identical amino acids) than to those of an Italian Maize rough dwarf virus isolate (85.1–88.1% identical nucleotides and 85.5–94.3% identical amino acids). The Chinese isolates should be classified as RBSDV.