Identification and characterization of biologically distinct isolates of Iris yellow spot virus (genus Tospovirus, family Bunyaviridae), a serious pathogen of onion

Iris yellow spot virus (IYSV) causes an economically important disease in onion bulb and seed crops. While considerable information on the genetic diversity of the virus is available, little is known about the biological variability of the virus. Using two experimental hosts, Nicotiana benthamiana and Datura stramonium, IYSV from naturally infected onion fields was evaluated to determine the existence of biologically different isolates using the following criteria: ability to establish infection and become systemic, and the severity of the disease caused in inoculated plants. Additionally, the nucleocapsid gene of these biologically distinct isolates of IYSY was characterized at the molecular level.     

PCR-based monitoring of recent isolates of tobacco blue mold from Europe reveals the presence of two genetically distinct phenotypes differing in fungicide sensitivity

Bioassays testing the fungicide sensitivity against metalaxyl of Peronospora tabacina isolates collected in German tobacco fields in 2005 revealed the presence of two phenotypes, resistant and sensitive. DNA fingerprints using SSR and minisatellite primers allowed separation of the samples into two groups. The differences in amplification patterns coincided with the sensitive and resistant reaction of the isolates in metalaxyl bioassays. New primers were developed which allowed PCR-based detection of P. tabacina and differentiation of the metalaxyl-sensitive and the metalaxyl-resistant phenotype, respectively. Screening of recent blue mold isolates from Germany and other European countries for metalaxyl sensitivity with leaf disk bioassays coincided completely with the PCR-based identification of the two phenotypes. In Germany, exclusively resistant isolates were found between 2002 and 2004. These still dominate. Since 2005 the co-occurrence of sensitive isolates has been shown. No similar monitoring has been done in any other European country. However, we found the resistant phenotype reaction in a French isolate of 2004 and in two out of three Italian isolates in 2007. Two isolates from Poland and Bulgaria in 2007 were sensitive to metalaxyl. For all 58 isolates tested since 2002 the metalaxyl bioassay-based resistance type and the PCR-based tests for sensitive and resistant genotype coincided. Using genotype-specific primers for future population studies may help to trace the sources of the pathogen from which yearly propagation starts.     

Incidence of Viruses Infecting Allium Spp. in Greece

A survey identified viruses infecting garlic, leek and onion crops and wild Allium species in Greece. Virus identification was based on ELISA, immunoelectron microscopy, and occasionally on RT-PCR. Samples of cultivated Allium species were collected from five districts, whereas samples of twenty-seven wild Allium species were also collected from all over Greece. Onion yellow dwarf virus (OYDV) and Leek yellow stripe virus (LYSV) were identified in 98.5% and 83.7% of all samples, respectively, and were found in all regions. Allexiviruses were also detected in all regions and their incidence ranged from 62.5% to 70.5% (depending on region and type of allexivirus). Garlic common latent virus (GCLV) was detected in samples from Arcadia (97.6%) and Evia (18.0%) and in one field in Larissa (23.0%). Shallot latent virus (SLV) was found only in two areas (Evros and Theva) and in fields planted with imported propagative material, from Iran and China. The incidence of virus-like symptoms in leek crops ranged from 10.0% to 90.0% in different regions and fields and all symptomatic plants were found to be infected by LYSV. Onion yellow dwarf virus was only found in seven symptomatic onion samples from southern Greece. Allium ampeloprasum spp. ampeloprasum and Allium flavum, were the only wild Allium species found to be infected with LYSV. Finally Turnip mosaic virus (TuMV) was found in A. sphaerocephalon, A. guttatum, A. subhirsutum, and A. neapolitanum.     

Auftreten und Nachweis von Fusarium oxysporum und F. proliferatum an Steck- und Saatzwiebeln

The pathogen Fusarium oxysporum f. sp. cepae inducing the Fusarium basal rot mainly spreads in warmer cultivation regions due to its adaptibility to high temperature. Meanwhile the pathogen occurs in Germany as well, especially in years with relatively high average temperature during the growing season. Phytopathological investigations of 300 symptomless onion bulbs showed a contamination rate of approximately 10% with regard to Fusarium spp, with F.?oxysporum proving to be the predominant species. Onion sets planted in these fields were latently infected with F.?oxysporum at rates of 19?C98%. Unexpectedly, the contaminated sets did not indispensably lead to a high occurrence of plants exhibiting characteristic symptoms of Fusarium basal rot such as wet and dry rot. Presumably, the development of symptoms is particularly affected by given climatic conditions. The results of pathogenicity tests of isolated Fusarium spp. isolates under controlled conditions support this assumption. The inoculation of the substrate with selected Fusarium spp. isolates resulted in a reduction of emergence by up to 70% under controlled conditions, which are suboptimal with regard to the cultivation of onions. The emergence of plants was not affected by Fusarium spp. under optimal cultivation of onions. However, under optimal cultivation conditions a reduction of plant growth occurred in a subsequent growth stage. Beside F.?oxysporum, F.?proliferatum could be detected in onion bulbs as well as seeds. The proportion of contaminated seeds accounted to 62%. Both species F.?oxysporum and F.?proliferatum proved to be pathogenic in onion although their isolates varied much in their virulence.     

Population studies on <Emphasis Type="Italic">Phytophthora infestans</Emphasis> on potatoes and tomatoes in southern Germany

Fifty-seven isolates of Phytophthora infestans from blighted potato foliage were collected in 1995 in southern Germany and analysed for mating type and sensitivity to metalaxyl. Fifty-six of them were characterised as A1 and one as A2 mating types. Resistance to metalaxyl was observed frequently: 53 isolates were resistant, three were partially sensitive, and one was sensitive. In a subsequent field study in 1999, 84 isolates collected from blighted potato and tomato foliage were analysed for mating type. Seventy-two were characterised as A1 and twelve as A2 mating types. The response of 76 isolates to metalaxyl and to propamocarb was tested. The majority (42) of the 76 isolates was classified as resistant to metalaxyl; 31 were partially sensitive and only three isolates were sensitive. The results with propamocarb were less discrete; 10 isolates were classified as resistant and three were clearly sensitive. AFLP fingerprinting was used to examine the genetic structure of the southern German P. infestans population collected in 1999 and indicated that the tested population can be sub-divided into a tomato group, a potato group and a mixed group containing isolates collected from both crops. The presence of Ia and IIa mitochondrial DNA haplotypes indicates that the German P. infestans isolates belong to the new pathogen population that has also been reported in neighbouring regions of Europe. The present study indicates that at the beginning of the season only a few genotypes were present, and the population became genetically more variable at the end of the growing season.     

Molecular comparisons amongst wheat bymovirus isolates from Asia, North America and Europe

To study the variation between wheat bymovirus isolates and to resolve uncertainties about the identity of the virus in some countries, leaves of infected plants were obtained from nine sites in China and from one each in Italy, Germany, USA and Canada. The German isolate was obtained from rye and the Canadian isolate was the type strain of wheat spindle streak mosaic virus (WSSMV). In RT-PCR, using primers designed from a partial sequence of a French isolate (tentatively described as WSSMV), genome fragments were obtained from the Italian and the French isolates but not from the Chinese ones. Conversely, products were consistently obtained from the Chinese isolates, but not from the Italian or French ones, when primers were designed from the sequence of a Japanese isolate of wheat yellow mosaic virus (WYMV). Nucleotide sequences were obtained from regions at or near the 3'-terminus of RNA1 of six Chinese isolates and the four from Europe and North America, usually including the coat protein. Nucleotide and amino acid sequence comparisons demonstrated that the European and North American isolates were extremely similar and were therefore WSSMV, while the Chinese isolates were close to the Japanese isolate and were thus WYMV.     

Analysis of Iris yellow spot virus replication in vector and non‐vector thrips species

Iris yellow spot virus (IYSV, genus Tospovirus) is a viral disease of bulb and seed onion crops and is transmitted by Thrips tabaci. Foliage damage of up to 75% has been reported in Kenya and Uganda. In this study, the rate of IYSV replication in the larva, pupa and adult stages of T. tabaci and other non‐vector thrips species and colour forms such as Frankliniella occidentalis, F. schultzei (dark) and F. schultzei (pale) was evaluated by monitoring relative levels of nucleocapsid (N) and non‐structural (NSs) proteins using N‐ and NSs‐specific antibodies. The effect of IYSV replication on mortality of thrips was also determined. N protein levels increased in all three stages of IYSV‐fed T. tabaci, indicating replication of IYSV. In IYSV‐fed non‐vector thrips, the increase of N protein levels in the larval stage was lower than IYSV‐fed T. tabaci but higher than their healthy counterparts. The N protein levels did not increase at pupal and adult stages. NSs protein was not detected in first instar of either vector or non‐vector thrips species. After a 4 h post‐acquisition period, a significant increase in NSs proteins was only observed in IYSV‐fed T. tabaci, clearly differentiating vectors and non‐vectors of IYSV. IYSV replication did not influence the survival of the vector thrips species, T. tabaci populations or the non‐vector thrips species. This study indicates the effectiveness of monitoring non‐structural proteins such as NSs, compared to nucleocapsid proteins, for differentiating vectors and non‐vectors of IYSV.     

Fusarium oxysporum schlecht. in Israel

Out of 208 isolates ofF. oxysporum, 158 produced toxic reactions when applied to rabbit skin. This fungus, ubiquitous in the soils of Israel, was the most prevalent component of theFusarium flora in the unirrigated (but not in the irrigated) soils sampled.F. oxysporum var.redolens was common only on unfertilized plots of heavy, unirrigated soils.F. oxysporum was a major constituent of theFusarium flora isolated from most of the 450 samples of 20 field and garden crops, but less common in 98 samples of citrus, avocado and mango fruits. It ranks among the most destructive pathogens of cucurbits (exceptCucurbita pepo), tomatoes, onions, and gladioli in Israel, but causes almost no damage to Brassicae, peas or cotton. In pathogenicity tests with 207 isolates from nine field and two fruit crops, numerous isolates caused seedling mortality, with watermelon, onion, cucumber, tomato and eggplant being the most susceptible. Spore measurements on 160 isolates from plants and 355 from soils, showed that spore size was not markedly affected by plant source or by irrigation, but did differ between soil samples taken at 5 and 20 cm depth, and from manured or unfertilized soils.     

A new large scale soil DNA extraction procedure and real-time PCR assay for the detection of Sclerotium cepivorum in soil

A real-time PCR assay specific for Sclerotium cepivorum, the causal agent of white rot in onions, was developed for use with a new DNA extraction method capable of processing up to 1?kg of soil in weight. The assay was specific for S. cepivorum when tested against 24 isolates representative of 14 closely related species and other pathogens of onion. The assay was highly sensitive when used with soil DNA extracted using the new DNA extraction procedure. In three different field soils tested, a good relationship between cycle threshold (Ct) and number of sclerotia was observed (R2?=?0.89). Twenty-nine soil samples from onion and leek crops were obtained and the pathogen was detected in four samples. All four positive samples were associated with current or past outbreaks of white rot of onion. Additional assays were also used on the 29 field soil samples, Botrytis aclada and Rhizoctonia solani AG8 were also detected, in one soil sample each. Rhizoctonia solani AG2-1 was more widespread and was detected in eight different soil samples. The method is therefore suitable to quantify levels of S. cepivorum in soil samples, with the added advantage that the technique allows other soil pathogens of interest to be assayed from the same DNA sample. The bulk soil DNA extraction method described here has the potential to be used to detect soil-borne pests and pathogens for other crops in a wide range of soil types.     

Occurrence and molecular characterization of Turkish isolates of Turnip mosaic virus

A total of 142 samples of plants showing symptoms of Turnip mosaic virus (TuMV) were collected from fields planted to Brassicaceae and non‐Brassicaceae crops in the southwest Marmora region of Turkey, during the 2004?06 growing seasons. Using enzyme‐linked immunosorbent assay (ELISA) TuMV was detected in the main brassica‐crop fields of Turkey, with an overall incidence of 13·4%. TuMV was detected in samples from Brussels sprouts, cabbage, wild mustard, radish and wild radish, but not cauliflower or broccoli. The full‐length sequences of the genomic RNAs of two biologically distinct isolates, TUR1 and TUR9, were determined. Recombination analyses showed that TUR1 was an intralineage recombinant, whereas TUR9 was a non‐recombinant. Phylogenetic analyses of the Turkish isolates with those from the rest of the world showed that the TUR1 and TUR9 isolates belonged to world‐Brassica and Asian‐Brassica/Raphanus groups, respectively. This study showed that TuMV is widely distributed in the Asia Minor region of Turkey.     

Recovery of fungicide-resistant <Emphasis Type="Italic">Botrytis</Emphasis> isolates from strawberry nursery plants

In a four-year survey of strawberry nursery plants, 340 Botrytis isolates were collected and examined for fungicide resistance. High percentages of isolates with resistance to members of all fungicide classes registered on strawberries in Germany were found, i.e. trifloxystrobin (90.3%), boscalid (53.8%), cyprodinil (41.5%), fludioxonil (28.2%) and fenhexamid (15.6%). Of these isolates, 10.3% possessed multiple resistance to all compounds. At 5.0%, resistance to the recently registered fungicide fluopyram was low. Dutch nursery material harboured significantly higher proportions of isolates with resistance to any or all fungicides than plants produced in Germany. These data point to nursery plants as a possible route of introducing fungicide-resistant Botrytis strains into commercial strawberry fields.     

Evaluation of morphology of infection structures in distinguishing between different Allium rust fungi

Fifteen isolates of rust fungi were collected in the United Kingdom and in the Netherlands from severalAllium species. The samples represented three putative rust species. The morphology of the telia, teliospores and urediniospores was investigated. From urediniospores infection structures were induced on leek, and their morphology was described. Telia and teliospores were not available on every sample. Morphology of the infection structures clearly differentiated between ‘leek’ type and ‘chive’ type isolates. The morphology of infection structures ofPuccinia mixta is very similar to that ofUromyces ambiguus, but clearly distinct fromPuccinia allii sensu Jennings from leek. Quantitative differences in urediniospore characters differentiated between the putative species, but there was overlap between the taxa. We conclude that morphology of infection structures of urediniosporelings is a useful trait for identification of rust isolates fromAllium. This is especially true where more than one rust species may occur on the same host species, as withAllium fistulosum.     

Genetic variation of Alternaria brassicae (Berk.) Sacc., causal agent of dark leaf spot of cauliflower and mustard in India

Cauliflower (Brassica oleracea L. var. botrytis) and mustard [Brassica juncea (L.) Czern] are important cruciferous crops facing serious yield and quality loss in India from dark leaf spot disease caused by Alternaria brassicae (Berk.) Sacc. Genetic variation among 32 pathogenic A. brassicae isolates from both crops was analyzed with random amplified polymorphic DNA and inter-simple sequence repeat primers in which the mean similarity coefficient was found to be 0.73 and 0.84, respectively. Further internal transcribed spacer analysis showed all isolates are 90–100 % similar to each other, indicating genetic similarity among different A. brassicae isolates that vary pathogenically.     

应用TaqMan MGB探针技术检测菜豆荚斑驳病毒

根据菜豆荚宽驳病毒(Beanpod mottle virus,BPMV)不同分离株外壳蛋白基因(coat protein,CP)的保守序列,设计了特异性引物与TaqMan MGB荧光探针,建立了BPMV的实时荧光RT-PCR检测方法.该方法与酶联免疫检测方法相比,灵敏度提高了100倍,具有快速、灵敏和高特异性的优点,适于对BPMV的快速检测.应用该方法,对来自美国不同地区的携带有BPMV的大豆样品进行检测,均能够得到BPMV的典型扩增曲线,表明引物与探针对BPMV不同分离株具有良好的简并性.     

Occurrence of Groundnut ringspot virus on Brazilian peanut crops

A high incidence of plants with mosaic, chlorotic spots, ringspots, necrosis, smaller leaves, and stunting was observed on peanut crops (Arachis hypogaea L.) in Itápolis, São Paulo State, Brazil. Transmission electron microscope examination of thin sections of infected leaves revealed the presence of spheroidal particles, ca. 80 nm in diameter, suggestive of Tospovirus. A DNA fragment of ~600 bp was amplified by RT-PCR from total RNA extracted from infected tissues using primers specific for the nucleocapsid gene of Groundnut ringspot virus (GRSV). Nucleotide and deduced amino acid sequences of the fragments showed high identities with known GRSV isolates.     

Unkrautflora in Mais

A weed monitoring conducted by Syngenta Agro GmbH in co-operation with the University of Hohenheim was carried out to evaluate the local and regional weed situation in maize in Germany. The fields were monitored at the two to six leaf stage of maize before weed control. A total of 2602 maize fields across Germany has been investigated from 2000 until 2004. Altogether 204 weed species (including volunteer crops) from 32 plant families have been recorded. The dicots were the predominant group with 166 species. The monocots were represented by 36 species and two species belonged to the family of Equisetaceae; volunteer crops were represented by 22 species. The most frequent species were Chenopodium spp. (79.7%) in particular Chenopodium album, followed by Stellaria media (61.0%), Fallopia convolvulus (55.7%), Echinochloa crus-galli (53.0%), Matricaria spp. (50.3%), Viola arvensis (47.8%), Polygonum aviculare (45.8%), Lamium spp. (41.6%), Galium aparine (39.7%) and Elytrigia repens (39.4%). The results demonstrate that with the exception of Echinochloa crus-galli, species usually considered for being typical for maize as e. g. Solanum nigrum, Amaranthus spp. and Galinsoga spp. are not dominating. Among the volunteer crops, rape was most abundant (17.2%). On average the weed density over all fields was 192 plants m^-2. The monitoring clearly shows that some weeds occur predominantly in certain areas as e. g. Solanum nigrum and Stellaria media in the north-west and Veronica spp. in the south of Germany. Beside climatic conditions differences in cropping systems and cultivation practices lead to a specific weed flora in maize. In comparison to similar investigations in the seventies of the last century, some species species as e. g. Veronica spp. and Viola arvensis have increased dramatically. This increase is mainly due to the use of sulfonylurea herbicides which are weak on these species. The increase of Geranium spp., weeds which are rather new in maize, is also linked to the low efficacy of the herbicides presently used on these species. All the species found during the five years monitoring are presented by their frequency of occurrence.     

Diversity in genetic structure and chemotype composition of <Emphasis Type="Italic">Fusarium graminearum</Emphasis> sensu stricto populations causing wheat head blight in individual fields in Germany

Fusarium head blight (FHB) is one of the most destructive diseases of wheat. Twelve small commercial wheat fields (size 1–3 hectares) were sampled in Germany for Fusarium populations at three spots per field with 10 heads each. PCR assays using generic primers confirmed 338 isolates as F.graminearum sensu stricto (s.s.) (64.9%) out of 521 Fusarium spp. that were further analyzed. Populations of F. graminearum s.s. in Germany contain three types of trichothecenes with a dominancy of 15-acetyldeoxynivalenol chemotype (92%) followed by 3-acetyldeoxynivalenol chemotype (6.8%) and a few isolates of nivalenol chemotype (1.2%). All these isolates were genotyped using 19 microsatellite loci. The 12 populations showed a high genetic diversity within the small scale sampling areas resulting in 300 different haplotypes. Genetic diversity within populations (71.2%) was considerably higher than among populations (28.8%) as shown by analysis of molecular variance. Gene flow (Nm) between populations ranged from 0.76–3.16. Composition of haplotypes of one population followed over 2 years changed considerably. No correlation between genetic and geographical distance was found. In conclusion, populations of F. graminearum s.s. in Germany display a tremendous genetic variation on a local scale with a restricted diversity among populations.     

Development of a simulation model for the population dynamics of the onion fly Delia antiqua in Germany*

Delia antiqua is a serious pest of many species of the genus Allium, including onions and chives. Over a period of two years (1997/1999), we have developed a model to simulate the population dynamics of D. antiqua based on an extended Leslie model. The model structure as well as the developmental functions for the different life stages of D. antiqua are presented. Using daily mean air and soil temperatures, the model calculates the population matrix for developmental stages of D. antiqua including eggs, larvae, pupae and adults. The simulation can be used to optimize the timing of control measurements against a specific life stage of D. antiqua. Flight activity can be simulated using wind speed as additional information. Simulated flight activity and the observed flight activity of D. antiqua monitored with water pan traps in three different regions of Germany in 1998 are presented and discussed. The model, which was programmed as part of the SWAT 3.5 package of programs to simulate the population dynamics of root‐fly pests in horticultural crops, will also be available via other expert systems such as PASO.