Análise comparativa da região codificadora para a proteína capsidial de isolados de PepYMV e PVY coletados em pimentão

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

The development of a serological-based diagnostic test for Dasheen mosaic potyvirus (DsMV)

Dasheen mosaic potyvirus (DsMV) is an important virus affecting taro. The virus has been found wherever taro is grown and infects both the edible and ornamental aroids, causing yield losses of up to 60%. The presence of DsMV, and other viruses,prevents the international movement of taro germplasm between countries. This has a significant negative impact on taro production in many countries due to the inability to access improved taro lines produced in breeding programs. To overcome this problem, sensitive and reliable virus diagnostic tests need to be developed to enable the indexing of taro germplasm. The aim of this study was to generate an antiserum against a recombinant DsMV coat protein (CP) and to develop a serological-based diagnostic test that would detect Pacific Island isolates of the virus. The CP-coding region of 16 DsMV isolates from Papua New Guinea, Samoa, Solomon Islands, French Polynesia, New Caledonia and Vietnam were amplified,cloned and sequenced. The size of the CP-coding region ranged from 939 to 1038 nucleotides and encoded putative proteins ranged from 313 to 346 amino acids, with the molecular mass ranging from 34 to 38 kDa. Analysis ofthe amino acid sequences revealed the presence of several amino acid motifs typically found in potyviruses,including DAG, WCIE/DN, RQ and AFDF. When the amino acid sequences were compared with each other and the DsMV sequences on the database, the maximum variability was21.9%. When the core region ofthe CP was analysed, the maximum variability dropped to 6% indicating most variability was present in the N terminus. Within seven PNG isolates ofDsMV, the maximum variability was 16.9% and 3.9% over the entire CP-coding region and core region, respectively. The sequence ofPNG isolate P1 was most similar to all other sequences. Phylogenetic analysis indicated that almost all isolates grouped according to their provenance. Further, the seven PNG isolates were grouped according to the region within PNG from which they were obtained. Due to the extensive variability over the entire CP-coding region, the core region ofthe CP ofPNG isolate Pl was cloned into a protein expression vector and expressed as a recombinant protein. The protein was purified by chromatography and SDS-PAGE and used as an antigen to generate antiserum in a rabbit. In western blots, the antiserum reacted with bands of approximately 45-47 kDa in extracts from purified DsMV and from known DsMV -infected plants from PNG; no bands were observed using healthy plant extracts. The antiserum was subsequently incorporated into an indirect ELISA. This procedure was found to be very sensitive and detected DsMV in sap diluted at least 1:1,000. Using both western blot and ELISA formats,the antiserum was able to detect a wide range ofDsMV isolates including those from Australia, New Zealand, Fiji, French Polynesia, New Caledonia, Papua New Guinea, Samoa, Solomon Islands and Vanuatu. These plants were verified to be infected with DsMV by RT-PCR. In specificity tests, the antiserum was also found to react with sap from plants infected with SCMV, PRSV-P, PRSV-W, but not with PVY or CMV -infected plants.

Perunan Y-viruksen testaaminen korkeassa happi-hiilidioksidipitoisuudessa idätetyistä perunoista

Perunalla (Solanum tuberosum L.) tällä hetkellä maailmanlaajuisesti eniten sato- ja laatutappioita aiheuttaa perunan Y-virus (PVY). Vaikka pelkän Y-viruksen aiheuttamaa satotappiota on vaikea mitata, on sen arvioitu olevan 20-80 %. Viruksen tärkein leviämistapa on viroottinen siemenperuna. Korkealaatuinen siemenperuna on edellytys ruoka-, ruokateollisuus- ja tärkkelysperunan tuotannolle. Kasvuston silmämääräinen tarkastelu aliarvioi yleensä Y-viruksen esiintyvyyttä. Laboratoriotestauksen avulla saadaan tarkempi tieto pellolta korjatun sadon saastunta-asteesta. Ongelmana Y-viruksen testaamisessa on, että sitä ei havaita dormanssissa olevista perunoista otetuista näytteistä yhtä luotettavasti kuin jo dormanssin ohittaneista perunoista testattaessa. Erilaisia menetelmiä kemikaaleista (Rindite, bromietaani) kasvihormoneihin (mm. gibberelliinihappo) ja varastointiolosuhteiden muutoksiin (kylmä- ja lämpökäsittely) on kokeiltu perunan dormanssin purkamiseen, mutta tulokset ovat olleet vaihtelevia. Tässä tutkielmassa perunan dormanssin purkamiseen käytettiin happi-hiilidioksidikäsittelyä (O2 40 % ja CO2 20 %) eripituisina käsittelyaikoina. Tarkoituksena oli selvittää, vaikuttaako käsittely perunan itämiseen ja dormanssin luontaista aikaisempaan purkautumiseen tai Y-viruksen havaitsemiseen. Lisäksi haluttiin selvittää, voiko Y-viruksen määrittämisen ELISA-testillä (Enzyme Linked Immunosorbent Assay) tehdä yhtä luotettavasti myös muista kasvinosista (mukula, itu), kuin tällä hetkellä yleisesti käytetystä perunan lehdestä. Idätyskäsittelyn vaikutuksista dormanssin purkautumiseen saatiin vaihtelevia, eikä kovinkaan yleistettäviä tuloksia. Käsittelyn ei myöskään havaittu vaikuttavan PYY-viroottisuuden havaitsemiseen eri näytemateriaaleilla testattaessa. Kun eri kasvinosien toimivuutta testissä vertailtiin, mukulamateriaalin todettiin aliarvioivan PVY-viroottisuutta kaikissa kokeissa. Myös itumateriaali aliarvioi pääsääntöisesti PVY-viroottisuutta ELISA:lla tehdyissä määrityksissä. Luotettavin testimateriaali oli perunan lehti.

马铃薯Y病毒外壳蛋白基因的导入以及转基因马铃薯植株的大田攻毒

应用四种不同的马铃薯试管微型薯诱导体系生产试管微型薯，通过比较建立了—种有效的试管微型薯诱导系统。这种诱导系统使用液体培养基，具有试管微型薯发生频率高、薯块体积大和微型薯形成早的特点。同时，此系统使用的培养基成分成本低、方法简便以及所用设备简单，适用于容器内大批量生产马铃薯试管微型薯以及马铃薯种质资源的保存。 对银离子在马铃薯叶片组织培养过程中对愈伤组织诱导或芽分化和再生的影响作了研究。结果表明银离子通过抑制叶片组织培养过程中形成的乙烯与其受体的结合从而促进芽再生，但是其对叶片愈伤组织的诱导无显著效果。银离子的这些作用在通过同时应用2，4-D而明显表现出来。2，4-D通过促进乙烯的生物合成而降低银离子的促进作用，两者则通过对乙烯的调节而影响马铃薯叶片的愈伤组织诱导和芽分化再生。 将马铃薯Y病毒外壳蛋白基因通过根癌农杆菌双元载体系统导入马铃薯品种Desiree、K4和Favorita，获得了若干转基因株系。除了K4品种中—转化株系具有非正常生长形态外，其余转基因植株都生长发育正常。由此表明以根癌农杆菌介导的马铃薯转化中，构建于双元载体上的外源目的基因是随机进入并整合到受体细胞的染色体上。具有畸形生长性状的转基因植株的产生说明了PVY CP基因的整合可能干扰了控制正常生长发育、尤其是形态建成的基因表达。 在转化试验中，应用了试管微型薯薄片、茎切段和叶片三种外植体作为转化材料。对转化过程中农杆菌对外植体的侵染时间、共培养时间、外植体的类型以基因型对转化频率的影响作了比较研究。发现以试管微型薯薄片和茎切段作为受体的最佳侵染时间是十分钟，而叶片则为五分钟，三种外植体的最佳共培养时间皆为四天。在各种处理的最佳条件下，Desiree比K4具有相对较高的转化频率，表明马铃薯Desiree比K4在转化反应上更温和或顺从。 通过比较几种由不同统计得出的农杆菌介导的转化频率，认为使用“净转换频率”(Net Transformation Frequency)能更精确地表达马铃薯的转化效率。而在以前的报导中还没有—种统一的、并且能被广泛接受和使用的表达转化效率的参数或指标。． 以叶片作为起始材料的转化具有较高的转化频率。在转化外植体的植株再生过程中应用了2，4-D和AgN03两种乙烯调节剂分别于愈伤组织诱导和芽分化再生阶段，使其产生高频的植株再生。尤其是它的净化频率明显高于其它外植体的转化频率，并且无显著品种之间的差异，具有高效马铃薯转化系统的特征。 以聚合酶链式反应(PCR)检测转化再生植株得到的结果与DNA杂交(Southern blot analysis)的鉴定结果比较，结论是相同的。由此表明在以农杆菌介导的马铃薯转化试验中，PCR可被用于证实外源目的DNA的导入，它以简便、迅速的特点帮助节省时间以及提供及时的转化证据。 对三个马铃薯品种的一系列转基因株系在大田条件下进行了攻毒试验．最后从Favorita 品种中筛选出了两个抗性较强的无性系，它们具有明显较低的病毒侵染发生频率以及正常的生长发育性状，具有很大潜力成为生产上推广应用的抗病新品种。

A classification of Pepper yellow mosaic virus isolates into pathotypes

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Caracterização de um isolado de Bidens mosaic virus proveniente de alface

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Seleção de clones de batata para microclimas de altitude no Planalto Central

Iniciou-se em 1986, em Anápolis, um programa de desenvolvimento de cultivares de batata adaptadas ao clima de altitude do Brasil Central, partindo-se de 15.000 genótipos, resultantes de 200 famílias obtidas pela Empresa Brasileira de Pesquisa Agropecuária (EMBRAPA) Hortaliças em 1985 e 1986. No primeiro ciclo, em 1986, foram selecionados 5.000 genótipos, considerando-se aspectos fenológicos, incidência de doenças, qualidade dos tubérculos e potencial de produção. Esses mesmos critérios foram adotados nas gerações posteriores, selecionando-se, anualmente, 15-20% de genótipos superiores. em 1990 avaliaram-se 52 destes clones, tendo como testemunhas as cultivares Achat e Bintje; Destes foram selecionados 28 clones promissores que foram submetidos à cultura de ápices caulinares e à indexação para os vírus PLRV, PVY e PVX na Empresa Brasileira de Pesquisa Agropecuária (EMBRAPA) Hortaliças. No período de 1995 a 1997 foram avaliados em Goiás, nos municípios de Anápolis, Morrinhos, Pirenópolis e Urutaí, e em Jaboticabal. Os dados foram submetidos à análise de variância, e aqueles referentes a 14 genótipos em 7 ambientes, à regressão pelo método de Eberhart & Russell. Os clones BAT 2, BAT 3, BAT 4, BAT 19, BAT 27 e BAT 28 destacaram-se entre os mais produtivos, considerando-se, também, as características de tubérculos para o consumo. Os genótipos responderam proporcionalmente à melhoria do ambiente. O clone BAT 19 foi o mais estável.

Variabilidade de potyvirus infectando Capsicum spp. no estado de São Paulo

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Análise molecular da proteína HC-Pro (Helper Component-Proteinase) e seu papel na relação patógeno-hospedeiro

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Caracterização e ocorrência de bidens mosaic virus (bimv) em regiões produtoras de alface no Estado de São Paulo

Pós-graduação em Agronomia (Proteção de Plantas) - FCA

Solanum americanum: reservoir for Potato virus Y and Cucumber mosaic virus in sweet pepper crops

Weeds can act as important reservoirs for viruses. Solanum americanum (Black nightshade) is a common weed in Brazil and samples showing mosaic were collected from sweet pepper crops to verify the presence of viruses. One sample showed mixed infection between Cucumber mosaic virus (CMV) and Potato virus Y (PVY) and one sample showed simple infection by PVY. Both virus species were transmitted by plant extract and caused mosaic in tomato (Solanum lycopersicum cv. Santa Clara), sweet pepper (Capsicum annuum cv. Magda), Nicotiana benthamiana and N. tabaccum TNN, and local lesions on Chenopodium quinoa, C. murale and C. amaranticolor. The coat protein sequences for CMV and PVY found in S. americanum are phylogenetically more related to isolates from tomato. We conclude that S. americanum can act as a reservoir for different viruses during and between sweet pepper crop seasons.

Studio dei meccanismi molecolari coinvolti nel determinismo sintomatologico di piante infette da virus e virus-satelliti

The aim of our work was to study the molecular mechanisms involved in symptoms appearance of plants inoculated either with a virus or with a virus-satellite complex. In the first case, we tried to set up a reliable method for an early identification of PVYNTN strains present in Italy and causing potato tuber necrosis. This, to prevent their spread in the field and to avoid severe yield losses, especially in seed potato production. We tried to localize the particular genomic region responsible for tuber necrosis. To this purpose, we carried out RT-PCR experiments using various primer combinations, covering PVY genomic regions larger than those previously used by other authors. As the previous researchers, though, we were not able to differentiate all NTN from others PVY strains. This probably because of the frequent virus variability, due to both genomic mutations and possible recombination events among different strains. In the second case, we studied the influence of Y-sat (CaRNA5 satellite) on symptoms of CMV (Cucumber mosaic virus) in Nicotiana benthamiana plants: strong yellowing appearance instead of simple mosaic. Wang et al (2004), inoculating the same infectious complex on tobacco plants transformed with a viral suppressor of plant silencing (HC-PRO), did not experience the occurrence of yellowing anymore and, therefore, hypotesized that changes in symptoms were due to plant post transcriptional gene silencing (PTGS) mechanism. In our case, inoculation of N. benthamiana plants transformed with another PTGS viral suppressor (p19), and other plants defective for RNA polymerase 6 (involved in systemic silencing), still resulted in yellowing appearance. This, to our opinion, suggests that in our system another possible mechanism is involved.

Transcriptional changes and oxidative stress associated with the synergistic interaction between potato virus X and potato virus Y and their relationship with symptom expression.

Many virus diseases of economic importance to agriculture result from mixtures of different pathogens invading the host at a given time. This contrasts with the relatively scarce studies available on the molecular events associated with virus---host interactions in mixed infections. Compared with single infections, co-infection of Nicotiana benthamiana with Potato virus X (PVX) and Potato virus Y (PVY) resulted in increased systemic symptoms (synergism) that led to necrosis of the newly emerging leaves and death of the plant. A comparative transcriptional analysis was undertaken to identify quantitative and qualitative differences in gene expression during this synergistic infection and correlate these changes with the severe symptoms it caused. Global transcription profiles of doubly infected leaves were compared with those from singly infected leaves using gene ontology enrichment analysis and metabolic pathway annotator software. Functional gene categories altered by the double infection comprise suites of genes regulated coordinately, which are associated with chloroplast functions (downregulated), protein synthesis and degradation (upregulated), carbohydrate metabolism (upregulated), and response to biotic stimulus and stress (upregulated). The expressions of reactive oxygen species?generating enzymes as well as several mitogen-activated protein kinases were also significantly induced. Accordingly, synergistic infection induced a severe oxidative stress in N. benthamiana leaves, as judged by increases in lipid peroxidation and by the generation of superoxide radicals in chloroplasts, which correlated with the misregulation of antioxidative genes in microarray data. Interestingly, expression of genes encoding oxylipin biosynthesis was uniquely upregulated by the synergistic infection. Virus-induced gene silencing of ?-dioxygenase1 delayed cell death during PVX?PVY infection.