Omenan kloroottisen lehtilaikkuviruksen testaaminen virolaisista luumulajikkeista

Suomessa noudatetaan Maa- ja metsätalousministeriön vuonna 2006 antamaa asetusta varmennetusta taimituotannosta, jonka mukaan kasvien tulee olla puhtaita taudinaiheuttajista. MTT Laukaa on vastuussa ydinkasvien ylläpidosta ja valiotaimien asetuksen mukaisesta tuotannosta. MTT Laukaaseen oli toimitettu vuonna 2007 Varsinais-Suomen maaseutuoppilaitoksen alueelta Tuorlasta neljä eri virolaista luumulajiketta. Lajikkeet olivat ’Ave’, ’Liisu’, ’Mari’ ja ’Renklod Haritonovoi’. Lajike ’Liisu’ kuitenkin kuoli mikrolisäyksessä Laukaassa melko pian. Muut lajikkeet antoivat vuonna 2008 DAS ELISA -testauksissa positiivisia tuloksia omenan kloroottiselle lehtilaikkuvirukselle (ACLSV). Luumulajikkeita ei siksi voinut ottaa tuotantoon. Opinnäytetyön tarkoituksena oli testata ACLSV:ta sekä MTT Laukaan kasveista että Tuorlan alkuperäisistä luumupuista. Testausmenetelminä käytettiin DAS ELISA- ja PCR-menetelmiä sekä mehutestausta. Eri menetelmien luotettavuutta vertailtiin. Osa sekä MTT Laukaan että Tuorlan luumuista antoivat positiivisia tuloksia DAS ELISA- ja PCR-menetelmillä, mutta ei mehutestauksella. Tutkimustuloksissa todettiin vaihteluita eri menetelmien välillä. Erityisesti mehutestauksen huono luotettavuus nousi esille. Todettiin että ainakin mehutestaus- ja joissakin tapauksissa myös DAS ELISA -tulokset tulisi varmistaa herkemmällä menetelmällä (PCR). Itse asiassa mehutestauksen mielekkyys kasvien testausmuotona kyseenalaistettiin.In Finland certified plant production follows the statute given by the Finnish Ministry of Agriculture and Forestry in 2006. MTT Plant Production Research at Laukaa is responsible for nuclear plant maintenance and elite plant production of certified production. According to the statute plants have to be free from certain plant pathogens. In 2007 four Estonian plum cultivars ‘Ave’, ‘Liisu’, ‘Mari’ and ‘Renklod Haritonovoi’ were sent to MTT Laukaa from the Countryside College of Southwest Finland Tuorla. However the cultivar ‘Liisu’ died in the very early stage of micropropagation at MTT Laukaa. The other plum cultivars gave positive results for apple chlorotic leaf spot virus (ACLSV) by the DAS-ELISA method in 2008. Because of this the cultivars could not be taken to the certified production. The purpose of the Bachelor’s Thesis was to test ACLSV from the Estonian plum samples taken from MTT Laukaa and from the mother trees in Tuorla. Virus testing methods were DAS-ELISA, PCR and the sap inoculation test. The reliability of different methods was compared. Some of the studied samples gave positive results of ACLSV by DAS-ELISA and PCR, but not by the sap inoculation test. The results of the present study indicate that there exists variation between the testing methods. Especially the unreliability of the sap inoculation test came up. The sap inoculation results, likewise in some cases the DAS-ELISA results should be confirmed by the more sensitive method (PCR). In addition the usability of the sap inoculation in overall as a testing method of ACLSV was questioned

Use of reverse transcription-polymerase chain reaction (RT-PCR) for Cymbidium mosaic virus (CyMV) detection in orchids

The reverse transcription-polymerase chain reaction CRT-PCR) was adapted for detection of Cymbidium mosaic virus CCyMV) in orchids. The oligonucleotide primers used were selected from the predicted homologous coat protein region of CyMV and other Potexviruses which enabled to amplify approximately 313 bp and 227 bp fragments using optimum reaction conditions of 2.5 mM MgCh and 30 cycles of amplification. The RT-PCR allowed the detection of CyMV RNA and virion in purified fonns as well as in crude tissue extracts of orchid. Direct CyMV RNA detection was possible in leaves, shoots, stems, roots and petals. The detection limits of RNA in purified CyMV and virion by RT-PCR described were 10 ng and 2 ng, respectively. The PCR amplified fragments were confinned to be CyMV-specific by dotblot hybridization with DIG-labelled CyMV cDNA probe. The suitability of the RT-PCR in routine testing of CyMV was detennined and compared with those of DAS-ELISA. Thirty samples of leaf tissues representing various genera or hybrids of cultivated local orchid from glasshouse and commercial nurseries were tested for CyMV by RT-PCR and DAS-ELISA. Among 15 samples that tested positive for CyMV infection by DAS-ELISA, only 7 samples gave the expected amplification fragments when subjected in RTPCR assays. The equal detection limit on purified CyMV virion by RT-PCR and DAS-ELISA and lower sensitivity of RT-PCR in detecting CyMV in a field indexing trial suggested that RT-PCR is unsuitable to replace DAS-ELISA for routine testing of CyMV in local orchids

The use of attenuated isolates of Pepino mosaic virus for cross-protection

Pepino mosaic virus (PepMV) has recently emerged as a highly infectious viral pathogen in tomato crops. Greenhouse trials were conducted under conditions similar to commercial tomato production. These trials examined whether tomato plants can be protected against PepMV by a preceding infection with an attenuated isolate of this virus. Two potential attenuated isolates that displayed mild leaf symptoms were selected from field isolates. Two PepMV isolates that displayed severe leaf symptoms were also selected from field isolates to challenge the attenuated isolates. The isolates with aggressive symptoms were found to reduce bulk yields by 8 and 24% in single infections, respectively. Yield losses were reduced to a 0–3% loss in plants that were treated with either one of the attenuated isolates, while no effects were observed on the quality of the fruits. After the challenge infection, virus accumulation levels and symptom severity of the isolates with aggressive symptoms were also reduced by cross-protection. Infection with the attenuated isolates alone did neither affect bulk yield, nor quality of the harvested tomato fruits

Detection of Cherry Leaf Roll Virus in intensively managed grafted English (Persian) walnut trees in Italy

Blackline disease, caused by Cherry leaf roll virus (CLRV), is considered a serious threat limiting English walnut (Juglans regia L.) production in Italy and the EU if walnut species other than J. regia are used as rootstock. In spring 2014, canopy decline or death of several walnut trees associated with presence of a necrotic strip at the rootstock-scion junction was observed on plants grafted onto \u2018Paradox' (J. hindsii 7 J. regia) in a commercial orchard located in the Veneto region (north-eastern Italy). To ascertain the presence of CLRV in this orchard and in other walnut intensively managed orchards located in the same region, a monitoring was carried out in 2014- 2015

Genes involved in barley yellow dwarf virus resistance of maize

KEY MESSAGE: The results of our study suggest that genes involved in general resistance mechanisms of plants contribute to variation of BYDV resistance in maize. ABSTRACT: With increasing winter temperatures in Europe, Barley yellow dwarf virus (BYDV) is expected to become a prominent problem in maize cultivation. Breeding for resistance is the best strategy to control the disease and break the transmission cycle of the virus. The objectives of our study were (1) to determine genetic variation with respect to BYDV resistance in a broad germplasm set and (2) to identify single nucleotide polymorphism (SNP) markers linked to genes that are involved in BYDV resistance. An association mapping population with 267 genotypes representing the world’s maize gene pool was grown in the greenhouse. Plants were inoculated with BYDV-PAV using viruliferous Rhopalosiphum padi. In the association mapping population, we observed considerable genotypic variance for the trait virus extinction as measured by double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) and the infection rate. In a genome-wide association study, we observed three SNPs significantly [false discovery rate (FDR) = 0.05] associated with the virus extinction on chromosome 10 explaining together 25 % of the phenotypic variance and five SNPs for the infection rate on chromosomes 4 and 10 explaining together 33 % of the phenotypic variance. The SNPs significantly associated with BYDV resistance can be used in marker assisted selection and will accelerate the breeding process for the development of BYDV resistant maize genotypes. Furthermore, these SNPs were located within genes which were in other organisms described to play a role in general resistance mechanisms. This suggests that these genes contribute to variation of BYDV resistance in maize. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00122-014-2400-1) contains supplementary material, which is available to authorized users

A survey of cherry leaf roll virus in intensively managed grafted english (Persian) walnut trees in Italy

Blackline disease, caused by Cherry leaf roll virus (CLRV), is considered a serious threat limiting English walnut (Juglans regia) production in Italy and the EU if walnut species other than J. regia e.g. \u2018Paradox\u2019 hybrid (J. regia 7 J. hindsii), French hybrid (J. regia 7 J. major or J. regia 7 J. nigra) or northern California black walnut (J. hindsii) are used as the rootstock. The virus transmissibility by pollen as well as latent infections can result in the spread of CLRVcontaminated propagative material, which is a major means of the virus dispersal by human activities. In 2014 and 2015 to ascertain the presence and the distribution of blackline symptoms in commercial orchards and to provide a description of the symptomatology, visual inspections and double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) analyses were carried out on 1,684 walnut trees in four different intensively managed grafted English walnut orchards in northeast Italy (Veneto Region). Trees with clear blackline symptoms at the scion-rootstock junction, often associated with general decline of the plant, were found only in one commercial orchard in northeast Italy on trees older than ten years of cvs. \u2018Tulare\u2019 and \u2018Chandler\u2019, grafted onto \u2018Paradox\u2019 rootstock. To our knowledge this is the first report of CLRV (blackline) decline and death in a commercial walnut orchard in Italy

Pyramiding of Ryd2 and Ryd3 conferring tolerance to a German isolate of Barley yellow dwarf virus-PAV (BYDV-PAV-ASL-1) leads to quantitative resistance against this isolate

Barley yellow dwarf virus (BYDV) is an economically important pathogen of barley, which may become even more important due to global warming. In barley, several loci conferring tolerance to BYDV-PAV-ASL-1 are known, e.g. Ryd2, Ryd3 and a quantitative trait locus (QTL) on chromosome 2H. The aim of the present study was to get information whether the level of tolerance against this isolate of BYDV in barley can be improved by combining these loci. Therefore, a winter and a spring barley population of doubled haploid (DH) lines were genotyped by molecular markers for the presence of the susceptibility or the resistance encoding allele at respective loci (Ryd2, Ryd3, QTL on chromosome 2H) and were tested for their level of BYDV-tolerance after inoculation with viruliferous (BYDV-PAV-ASL-1) aphids in field trials. In DH-lines carrying the combination Ryd2 and Ryd3, a significant reduction of the virus titre was detected as compared to lines carrying only one of these genes. Furthermore, spring barley DH-lines with this allele combination also showed a significantly higher relative grain yield as compared to lines carrying only Ryd2 or Ryd3. The QTL on chromosome 2H had only a small effect on the level of tolerance in those lines carrying only Ryd2, or Ryd3 or a combination of both, but the effect in comparison to lines carrying no tolerance allele was significant. Overall, these results show that the combination of Ryd2 and Ryd3 leads to quantitative resistance against BYDV-PAV instead of tolerance

Distribution and incidence of viruses in Irish seed potato crops

peer-reviewedVirus diseases are of key importance in potato production and in particular for the production of disease-free potato seed. However, there is little known about the frequency and distribution of potato virus diseases in Ireland. Despite a large number of samples being tested each year, the data has never been collated either within or across years. Information from all known potato virus testing carried out in the years 2006–2012 by the Department of Agriculture Food and Marine was collated to give an indication of the distribution and incidence of potato virus in Ireland. It was found that there was significant variation between regions, varieties, years and seed classes. A definition of daily weather data suitable for aphid flight was developed, which accounted for a significant proportion of the variation in virus incidence between years. This use of weather data to predict virus risk could be developed to form the basis of an integrated pest management approach for aphid control in Irish potato crops

Deteksi Penyakit Bacterial Fruit Blotch pada Melon Menggunakan ELISA

Bacterial fruit blotch (BFB) caused by Acidovorax citrulli is a serious seedborne disease in Cucurbitaceae causing 90-100% yield losses. The aim of this study was to explore BFB symptom on melon and also to detect A. citrulli infection in commercial seed and symptomatic fruits from the field in Yogyakarta Special Region province and its surrounding using DAS-ELISA method. Samples include melon from Sleman, Bantul, Kulon Progo, Gunung Kidul, Magelang, Purworejo regencies while commercial seeds i.e. Action 434, Glamour and Mai 116 were collected. DASELISA detection method used reagent set from Agdia. Based on the field observation, this study found melon commercial fruit shares similar symptom with BFB, which showed discrete oily dark green spots, while the netting failed to develop over necrotic areas, resulting in smooth sunken spots. DAS-ELISA detection revealed that samples collected from Jetak village, district of Mungkid, Magelang and from Bligo village, district of Ngluwar, Magelang and in commercial seed Mai 116 were positively infected by A. citrulli.INTISARIBacterial fruit blotch (BFB) merupakan penyakit penting pada famili Cucurbitaceae yang disebabkan oleh Acidovorax citrulli. Penyakit ini dilaporkan dapat menurunkan hasil mencapai 90-100%. Tujuan dari penelitian ini adalah untuk mengetahui gejala penyakit BFB pada melon dan deteksiA. citrulli pada benih komersial dan sampel buah bergejala dengan metode DAS-ELISA di DIY dan sekitarnya. Pengambilan sampel dilakukan di kabupaten Sleman, Bantul, Kulon Progo, Gunung Kidul, Purworejo dan Magelang. Selain dari lapangan, diuji pula benih melon komersial yaitu Action-434, Glamour dan Mai 116. Metode deteksi dengan ELISA menggunakanreagent set dari Agdia. Dari hasil pengamatan di lapangan ditemukan buah melon dengan gejala yang mirip dengan gejala BFB yaitu adanya becak berwarna hijau tua kebasahan pada permukaan buah, jaring tidak terbentuk sempurna dan pada bagian daging buah di bawah becak tadi membusuk. Hasil deteksi dengan DAS-ELISA mengindikasikan bahwa A. citrulli terdeteksi pada sampel yang berasal dari desa Bligo, kecamatan Ngluwar dan desa Jetak, kecamatan Mungkid, kabupaten Magelang, serta pada benih komersial MAI 116

Attempts to eradicate two Pelargonium viruses (PFBV and PLPV) by meristem culture and shoot-tip cryotherapy

Attempts to eradicate the Pelargonium flower break virus (PFBV) and Pelargonium line pattern virus (PLPV) by meristem culture and apex “droplet-vitrification” cryopreservation was carried out using 5 different cultivars. A simple meristem culture did not permit to eliminate PFBV and only 15% of Pelargonium x hortorum ‘Stellar Artic’ plants regenerated from meristems was PLPV-ELISA-negative. Plants regenerated from cryopreserved apices were tested by DAS-ELISA after a 3-month growing period. Viruses were not detected in 25 and 50% of the tested plants for PFBV and PLPV respectively. Immunolocalisations were carried out for virus localisation in apices from greenhouse plants (control) and vitroplants regenerated after meristem culture or cryopreservation. Immunolocalisations realised on control explants excised from DAS-ELISA positive plants showed that PFBV and PLPV were present in the apices, even in the meristematic dome. However, viral particles were more numerous in the cells of the basal zone than in the more meristematic ones. Immunolocalisations realised on apices from the DAS-ELISA negative cryoregenerated plants showed the viruses were still present. Our results firstly demonstrated that PFBV and PLPV are even present inside meristematic cells and secondly that cryopreservation could decrease their amount in Pelargonium plants but without eliminating them totally. More knowledge on virus behaviour during cryopreservation processes could optimize the management of genetic resources using this conservation method