Thesis (MSc)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Phytoplasma diseases have caused disastrous effects in vineyards around the world. Therefore, the recent discovery of phytoplasmas in South African vineyards could be highly detrimental to the local wine industry. Antimicrobial peptides (AMPs) are small molecules expressed by almost all organisms as part of their non-specific defence system. These peptides can offer protection against a wide variety of bacterial and fungal pathogens in plants. Due to the fact that phytoplasmas lack an outer membrane and cell wall, AMPs are considered to be perfect candidates to confer resistance to this phytopathogen. The current study intends to explore the in planta activity of AMPs against the grapevine pathogen aster yellows phytoplasma (AYp) through Agrobacterium-mediated transient expression. The AMPs, Vv-AMP1, D4E1 and Snakin1 (isolated from potato and grapevine) were selected to be tested for their in planta effect against AYp. Cauliflower mosaic virus 35S expression vectors containing four different AMP-encoding sequences were therefore constructed. As an alternative method to observe the effect Vv-AMP1 might have on AYp in planta, grafting of Vv-AMP1 transgenic Vitis vinifera cv "Sultana‟ plant material was used. To allow assumptions about AMP efficacy in this transient expression system, attempts were made to describe the spatial distribution and pathogen titre of AYp in V. vinifera cv "Chardonnay‟ material. Additionally, transmission experiments were carried out to infect Catharanthus roseus and Nicotiana benthamiana with AYp through the insect vector Mgenia fuscovaria. Material was screened for AYp infection by a nested-PCR procedure using universal primers described by Gundersen and Lee (1996). For quantification of AYp infection, a semi-quantitative real-time PCR (qPCR) protocol was optimized, using the SYBR Green-based system. In total, 86 V. vinifera cv "Chardonnay‟ plantlets were screened for AYp infection two-, three-, four-, seven- and eleven weeks after introduction into in vitro conditions. No AYp infection could however be detected and plantlets displayed a "recovery phenotype‟. To examine the distribution of AYp in canes of an infected V. vinifera cv "Chardonnay‟ plant, leaf and the corresponding node material from five canes were screened by a nested-PCR procedure. It can be concluded, that AYp was found predominantly in the nodes when compared to leaf material in the late season of the year. It is also highly unlikely for leaf material to show phytoplasma infection, if in the corresponding node no AYp could be detected. As AYp-infected grapevine material could not be maintained in vitro, the effect of VvAMP-1 transgenic grapevine against AYp could not be tested. Infection of C. roseus and N. benthamiana plants with AYp was successfully achieved by insect vector transmission experiments. Transient expression assays were conducted on AYp-infected N. benthamiana material. Quantification of phytoplasma in this material showed a decrease of AYp in both the AMP treatment groups and the control groups. This study optimized a qPCR procedure to detect and quantify AYp in infected plant material. The Agrobacterium-mediated transient expression system used during this study was not reliable, as no significant effect of the AMPs on AYp titre could be observed. This study showed, that AYp cannot be established and maintained in in vitro cultured V. vinifera cv "Chardonnay‟ material, and tissue culture itself might therefore be a way to eradicate AYp in this cultivar. To our knowledge, this study is the first to report on the spatial distribution of AYp in canes of an infected V. vinifera cv "Chardonnay‟ vine. / AFRIKAANSE OPSOMMING: Fitoplasma siektes veroorsaak ramspoedige gevolge in wingerde oor die hele wêreld. Dus kan die onlangse ontdekking van fitoplasma in Suid-Afrikaanse wingerde baie nadelige gevolge vir die plaaslike wynbedryf beteken. Antimikrobiese peptiede (AMPe) is klein molekules wat in amper al organismes as deel van hulle nie-spesifieke verdedegingsstelsel tot uitdruk kom. Hierdie peptiede kan beskerming aanbied teen ʼn wye verskeidenheid van bakteriële en swampatogene in plante. As gevolg van die feit dat fitoplasmas geen buitenste membraan en selwand het nie, word AMPe oorweeg as middle om weerstand te verleen teen hierdie fitopatogene. Die huidige studie beoog om die in planta aktiwiteit an AMPe teen die wingerdstok patogeen aster geel fitoplasma (AYp) deur middle van Agrobakteriumbemiddelde tydelike uitdrukkingssiteme, te ondersoek. Die AMPe, Vv-AMP1, D4E1 en Snakin1 (geïsoleer van aartappel en wingerd plante) is gekies om getoets te word vir hul in planta effek teen AYp. Blomkoolmosaïek-viruse 35S uitdrukkingsvektore met vier verskillende AMP-kodering rye, is dus ontwikkel. As ʼn aternatiewe method om die moontlike effek van Vv-AMP1 op AYp in planta in ag te neem, is oorplantings van die Vv-AMP1 transgeniese Vitis vinifera cv "Sultana‟ plantmateriaal gebruik. Om voorsiening te maak vir AMPe se doeltreffenheid in hierdie tydelike uitdrukkingsvektore, is pogings aangewend om die ruimlike verspreiding en patogeen konsentrasie van AYp in V. vinifera cv "Chardonnay‟ te beskryf. Addisioneel is transmissie eksperimente uitgevoer om Catharanthus roseus en Nicotania benthamiana te besmet met AYp deur die insekvektor, Mgenia fuscovaria. Plantmateriaal is getoets vir AYp deur van ʼn PCR protokol gebruik te maak met universele inleiers (grondlae) soos beskyf deur Grundersen en Lee (1996). Vir kwantifiseering van die AYp infeksie, is n semi-kwantitatiewe qPCR protokol geoptimaliseer, met hulp van die SYBR Groen-gebaseerde stelsel. In geheel is 86 V. vinifera cv "Chardonnay‟ planties getoets vir AYp infeksie – twee-, drie-, vier-, sewe- en elf weke na die bekendstelling aan die in vitro voorwaardes. Geen AYp infeksie kon egter opgespoor word en die plante het “herstel fenotipe‟ vertoon. Om die verspreiding van AYp in stingelknope van ʼn besemtte V. vinifera cv "Cardonnay‟ plant, blaar en ooreenstemmende stingelknope uit vyf stingels te ondersoek, is hulle getoets deur ʼn PCR protokol. Daar kon afgelei word dat AYp hoofsaaklik in die stingelknop in vergelyking met die blaarmaterial laat in die season, gevind word. Dit is hoogs oonwaarskynlik om fitoplasma infeksies in blaarmaterial te vind, as in die ooreenstemmende stingelknop daar geen AYp oopgespoor kon word nie. As gevolge daarvan dat die AYp-geinfekteerde wingerdmateriaal nie in vitro gegroei kon word nie, kon die effek van VvAMP-1 transgeniese wingerd teen AYp nie getoets word nie. Infeksies van C. roseus en N. benthamiana plante met AYp is suksesvol bereik deur transmissie eksperiemente. met ʼn insekvektor. Tydellike uitdrukkingvektore toetse is uitgevoer op die AYp besmette N. benthamiana material. Kwantifisering van fitoplasma in hierdie material het die afname van AYp in altwee, die AMP behandelings groepe en die kontrole groepe getoon. Hierdie studie het ʼn qPCR geoptimaliseer om besmette plantmaterial met AYp op te spoor en dit te kwantifiseer. Die Agrobacterium-bemiddelde tydelike uitdrukingsvektore wat in hierdie studie gebruik is, was nie vertroubaar genoeg, want geen beduidelike effek van die AMPe op AYp konsentrasie kon waargeneen word nie. Hierdie studie het bewys dat AYp nie vasgestel is en in stand gehou kan word deur in vitro aankweeking van V. vinifera cv "Chardonnay‟ material nie, en weefselkulture kan dus ʼn manier wees om AYp in hierdie kultivar uit te roei. Tot kennis, is hierdie studie die eerste studie om die ruimtelike verspreiding van AYp in stingelknope van ʼn besmette V. vinifera cv "Chardonnay‟ wingerstok te rapporteur. / Winetech and DAAD

A subject of the diploma thesis was the optimization of a procedure of detecting a phytoplasma Candidatus Phytoplasma mali using woody indicator Golden Delicious in greenhouse conditions as an alternative method to the phytoplasma presence testing by biological indexing in field conditions. To control the presence of phytoplasma in the experimental plants, molecular method of real-time PCR was used. The experiment was set up on the demonstration area of the Department of Breeding and Propagation of Horticultural Plants in the grounds of Mendel University in Brno. Three different ways of transferring of the phytoplasma infection were compared: method of parallel grafting of the tested variety and of the indicator on the seedling in the period of dormancy (method number 1), method of summer budding of the tested variety on indicator grafted on the seedling in the period of dormancy (method number 2), and method of double summer budding of the indicator and of the tested variety on the seedling (method number 3). On a woody indicator Golden Delicious, varieties Virgin Czech, Canadian rennet, Gascoigne, Ribston and Boskoop were tested, 10 plants of each variety. The experiment lasted 10 months. A total of 150 tested plants were assessed by a visual inspection of the symptoms presence. During the monitoring period, none of the tested plants showed typical symptoms of the apple proliferation. Presence of the Candidatus Phytoplasma mali was verified using real-time PCR method in 60 tested (indicator) plants. The infection was confirmed in 12 of the indicator plants. The detected quantities of the phytoplasma in the tissues however reached very low values. The highest number of the infected plants was found in tests of the Gascoigne variety, established by the method of double summer budding of the indicator and of the tested variety on the seedling (method number 3). This method provided the most successful inoculation of the indicator by the phytoplasma. Based on the results obtained during the presented study, it can be declared that biological testing of the phytoplasma presence using short-term greenhouse tests is not reliable. When using testing with the indicator ´Golden Delicious´, it is recommended to realize the experiments for at least two growing seasons, as it is in the field tests. Due to the fact that during the time-limited realization period of the experiment in this study it was found that the phytoplasma was not sufficiently propagated, the final results of the comparison of the indicator plants inoculation methods will be available at the end of autumn of 2017.

The aim of this thesis was to optimize the method of detection of phytoplasmas using DNA-microarray. It consisted of testing an appropriate method of genetic material isolation, development and optimization of PCR to amplify different groups of phytoplasmas, optimization of detection of DNA at a microarray, and sequence analysis of phytoplasma in order to design more suitable probes. PCR was first optimized for collection isolates, then also for natural samples. All 16Sr groups from the collection were sequenced and phytoplasmas were detected in them by hybridization. Phytoplasmas were detected also in natural samples: oilseed rape (species Brassica napus), red clover (Trifolium pretense), purple coneflower (Echinacea purpurea), and apple tree (Malus domestica). Using the DNA from insect vectors, only sample 202/6 from the group 16Sr-XII was positive. The sequence of red clover and oilseed rape correspond with the database samples in the group 16Sr-I "Aster yellows".

Fitoplasmas, procariotos sem parede celular e habitantes de floema, têm causado danos consideráveis em diversas culturas comercialmente importantes. A associação desses fitopatógenos com várias doenças que ocorrem em espécies hortícolas têm sido comumente registradas tanto no território brasileiro como em outras regiões do mundo. Em áreas de plantio comercial, na região de Piracicaba-SP e Bragança Paulista-SP, plantas de tomate e berinjela apresentando porte reduzido, clorose foliar acentuada, produção exagerada de pequenos ramos, desenvolvimento anormal do cálice, encurtamento de entre-nós, redução no tamanho de folhas, flores e frutos, que podem ser traduzidos em sintomas de enfezamento, foram observadas evidenciando infecção por fitoplasma. Através da técnica de duplo PCR utilizando os oligonucleotídeos universais R16 mF1/mR2 e R16 F2n/R2, fragmentos de DNA de 1,2 Kb foram amplificados de amostras sintomáticas, demonstrando a presença de fitoplasma nos tecidos das plantas. Nenhum fragmento foi detectado em plantas sadias. O exame de tecidos do floema de plantas sintomáticas, submetidos ao microscópio eletrônico de transmissão, revelou a presença de corpúsculos pleomórficos no interior dos vasos, corroborando com os resultados da detecção através de PCR. O uso de oligonucleotídeos específicos para identificação demonstrou a ocorrência de fitoplasmas afiliados ao grupo 16SrIII, tanto em tomate quanto em berinjela. Análises de RFLP, usando as enzimas de restrição AluI, MseI, HpaII, KpnI, RsaI, HhaI e MboI, confirmaram a ocorrência de fitoplasmas do referido grupo em ambas as espécies vegetais. Para confirmar os resultados de RFLP, os fragmentos de DNA de 1,2 Kb dos fitoplasmas presentes em tomate e berinjela, amplificados com o par de iniciadores R16 F2n/R2, foram clonados em Escherichia coli para a determinação de suas seqüências de nucleotídeos. Os fragmentos clonados sequenciados foram comparados, por homologia de nucleotídeos, entre si e com isolados cujas seqüências já haviam sido depositadas no “GenBank”. O resultado do sequenciamento revelou heterogeneidade das seqüências do gene 16S rDNA ocorrendo em alguns isolados, demonstrando e confirmando a variabilidade genética de organismos do grupo 16SrIII presentes no Brasil. / Phytoplasmas are cell wall-less prokaryotes and phloem- inhabitants. They caused considerable damages in some commercially important crops in the Brazilian territory and others regions of the world. It has been observed in the region of Piracicaba and Bragança Paulista, São Paulo State, commercial fields of eggplant and tomate showing reduced size, extensive chlorosis, proliferation of axillary buds, abnormal development of calyx, internode shortening and small leaves, flowers and fruits. These symptoms can be translated by stunting, making clear the phytoplasma infection. Presence of phytophasm was confirmed by nested PCR assay with the universal primer R16 F2N/R2 where DNA fragments of 1.2 kb were amplified from symptomatic samples. No fragments was observed in healthy plants. Analysis of symptomatic plant’s phloem tissue when submitted to electron microscope of transmission, disclosed the presence of pleomorphic bodies inside the tissues, corroborating with the results of detection by PCR assay. The use of specific primers for groups demonstrated the phytoplasmas occurrence associated to the 16SrIII group, in both tomato and eggplant. RFLP analyses using AluI, MseI, HpaII, KpnI, RsaI, HhaI and MboI restriction enzymes confirmed the occurrence of phytoplasmas on 16SrIII group in both plant species with typical stunting symptoms. The products of 1.2 kb of tomato and eggplant isolates amplified with the pair of primers R16 F2n/R2, were cloned in Escherichia coli to determine nucleotides sequence and to confirm the RFLP results. The nucleotide sequences were compared by homology and with isolates whose sequence have been already deposited in the “GenBank”. The results revealed 16S rRNA sequence heterogeneity occurring in some isolates showing the genetic variability of group 16SrIII phytoplasma in Brazil.

A cultura da ameixeira vem despertando a atenção de produtores brasileiros, pois oferece alta rentabilidade, possibilita a prática da agricultura familiar, emprega mão de obra, recebe incentivos de programas oficiais e conta com um mercado altamente promissor. No entanto, a cultura ainda necessita de cultivares com melhor adaptação climática, qualidade de frutos e resistência às doenças. Atualmente, dentre as doenças, o \"amarelo das fruteiras de caroço\", associada a um fitoplasma, vem causando sérios problemas. Em 2013, em pomares comercias de ameixeira (Prunus salicina), instalados em Paranapanema (SP), foram observadas plantas portadoras de sintomas tipicamente induzidos por fitoplasmas, caracterizados por superbrotamento de ramos, redução no comprimento de entrenós, além de amarelecimento, deformação e redução do tamanho de folhas. Nestas plantas foi identificado um fitoplasma pertencente ao grupo 16SrI-B (Candidatus Phytoplasma asteris). Visando aumentar os conhecimentos sobre este patossistema, o objetivo desse trabalho foi estudar a dinâmica da colonização de plantas comerciais de ameixa infectadas pelo fitoplasma. Para isso, em dois pomares, amostras da parte aérea e raiz de plantas sintomáticas, pertencentes às variedades Gulfblaze e Azteca, foram coletadas mensalmente, durante um ano. O DNA total foi extraído e submetido ao PCR em tempo real para quantificar o fitoplasma nos tecidos do hospedeiro. Nessas reações foram utilizados os iniciadores universais UniRNAForward/UniRNAReverse. O fitoplasma foi detectado em todas as amostras, tanto aquelas da parte aérea quanto de raízes, em ambas as variedades estudadas. A concentração do patógeno nos tecidos do hospedeiro variou de 5,3 x 103 a 4,54 x 106 e 3,28 x 103 a 1,28 x 106 número de cópias/100ng de DNA total, na parte aérea e nas raízes, respectivamente. Os resultados mostraram uma flutuação sazonal na concentração do fitoplasma, onde as maiores concentrações foram encontradas nas épocas mais quentes do ano, principalmente no mês de dezembro, e uma redução na concentração do patógeno foi observada nas épocas mais frias, embora o fitoplasma tenha permanecido na parte aérea da planta durante a fase de dormência do hospedeiro. A variedade Gulfblaze apresentou maior concentração do patógeno comparada com a variedade Azteca. O fitoplasma associado ao amarelo da ameixeira também foi encontrado em maior concentração na copa da planta. Com base nos resultados, amostras retiradas da parte aérea e nas épocas mais quentes do ano são as mais indicadas para os procedimentos de detecção do patógeno, visando uma diagnose mais confiável. / Plum is a crop that has aroused the attention of Brazilian growers due to high profitability, family farming, incentive arrangement and promising market. However the plum culture still need of varieties with good climate adaptation, best quality fruit, and disease resistance. Currently, among the diseases, stone fruit yellows, associated with a phytoplasma have caused serious problems. In 2013, in commercial orchards of plum (Prunus salicina) located in Paranapanema (SP), plants were observed exhibiting symptoms typically induced by phytoplasmas, characterized by witches broom and shortened internodes. Besides, the leaves were yellowish, malformed, and reduced in size. In these plants was identified a phytoplasma belonging to the group 16SrI-B (Candidatus Phytoplasma asteris). The aim of the present work was to study the dynamics of colonization of commercial plants plum by the phytoplasma, in order to better understanding of the disease. Thus, in two orchards, samples from aerial parts and roots of symptomatic plants belonging to Gulfblaze and Azteca varieties were collected monthly, during a year. Total DNA was extracted and submitted to the Real Time PCR to quantify the phytoplasma in host tissues. In these reactions the universal primers UniRNAForward/UniRNAReverse were used. The phytoplasma was detected in all samples, both aerial parts and roots of the two varieties. The concentration of the pathogen in host tissues ranged from 5,3 x 103 to 4,54 x 106 and 3,28 x 103 to 1,28 x 106 number of copies/100 ng of total DNA in aerial parts and roots, respectively. The results showed a seasonal fluctuation in the concentration of phytoplasma. The highest concentrations were found in the warmer seasons of the year, especially in December, and a reduction in pathogen concentration was observed when the weather was milder, although the phytoplasma remained in aerial part of the plant during the dormant phase of the host. The Gulfblaze variety showed a higher concentration of the pathogen compared with the Azteca variety. In addition, the phytoplasma associated with the disease was found in highest concentration in the aerial part of plant. Based on the results, samples collected from the aerial part and in the warmer seasons of the year are the most recommended for pathogen detection procedures for safe diagnosis.

O brócolis (Brassica oleraceae var. italica) é uma das hortaliças mais importantes do país, cujo volume de comercialização na CEAGESP é de aproximadamente 13 mil toneladas por ano. Recentemente, uma nova doença tem causado perdas relevantes para as culturas instaladas na maior região produtora do Estado de São Paulo. Os sintomas característicos da doença são expressos pelo enfezamento da planta e necrose dos vasos de floema. Devido ao fato destes sintomas indicarem a presença de fitoplasmas nas culturas de repolho e couve-flor, localizadas na mesma região geográfica onde foi observada esta nova doença, levantou-se a suspeita de que estes mesmos agentes patogênicos pudessem estar associados com as plantas doentes de brócolis. Assim, o DNA total de plantas de brócolis sintomáticas foi analisado por PCR com primers específicos para a região 16S rDNA de fitoplasmas. Os resultados revelaram que estes patógenos estavam associados com as plantas doentes. Através das técnicas de RFLP do sequenciamento de nucleotídeos desta mesma região genômica, os fitoplasmas foram identificados como pertencentes aos grupos 16SrI, 16SrIII e 16SrXIII. Através de análise de RFLP, fitoplasmas também foram identificados em diversas espécies de plantas daninhas e em cigarrinhas da família Cicadellidae coletadas em áreas adjacentes a campos de produção de brócolis. Fitoplasmas do grupo 16SrIII foram identificados em plantas daninhas das espécies Agetarum conyzoides (mentrasto), Crotalaria lanceolata (crotalária), Lepidium virginicum (mentruz), Nicandra physalodes (juá-de-capote), Paulicourea marcgravii (erva-de-rato), Ricinus communis (mamona), Sida rhombifolia (guanxuma), Sonchus oleraceae (serralha amarela), Bidens pilosa (picão preto), Erigeron bonariensis (buva), Emilia sonchifolia (falsa serralha), Leonorus sibiricus (rubim), enquanto que fitoplasmas do grupo 16SrVII foram encontrados as últimas quatro espécies citadas. Com relação aos insetos, fitoplasmas foram detectados em indivíduos das subfamílias Deltocephalinae, Agalliinae e Typhlocybinae. Dentro da subfamília Deltocephalinae, a cigarrinha Balclutha hebe portava fitoplasma do grupo 16SrI, enquanto que cigarrinhas das espécies Atanus nitidus, Planicephalus flavicosta e Schapytopius fuliginosus abrigavam fitoplasmas do grupo 16SrIII. Nos tecidos de duas cigarrinhas da subfamília Agalliinae e uma da Typhlocybinae, as quais não foram identificadas quanto a espécie, foram encontrados fitoplasmas do grupo 16SrIII. As análises epidemiológicas revelaram um padrão espacial agregado de plantas doentes e a ocorrência de um maior progresso da doença nos bordos dos campos de cultivo de brócolis, que estão localizados nas proximidades de áreas com a presença de plantas daninhas. / Broccoli (Brassica oleraceae var. italica) is one of the most important vegetables in Brazil, whose trading volume in CEAGESP is approximately 13 000 tons per year. Recently, a new disease has caused significant losses in this crop cultivated in the largest producing region of the São Paulo State. The characteristic symptoms of the disease are expressed by plant stunting and necrosis of phloem vessels. Because these symptoms indicate the presence of phytoplasmas in cabbage and cauliflower crops, grown in the same geographical region, it was suspected that the same pathogens could be associated with the affected broccoli plants. Therefore, the total DNA from symptomatic plants of broccoli was analyzed by PCR with specific primers for the 16S rDNA of phytoplasmas. Through the techniques of RFLP and nucleotide sequencing of the same genomic region, the phytoplasmas were identified as belonging to the groups 16SrI, 16SrIII and 16SrXIII. Through RFLP analysis, phytoplasmas were also identified in several species of weeds and leafhoppers in the family Cicadellidae collected in adjacent areas of broccoli fields. Phytoplasmas belonging of the 16SrIII group were identified in the weeds belonging to the species Agetarum conyzoides, Crotalaria lanceolata, Lepidium virginicum, Nicandra physalodes, Paulicourea marcgravii, Ricinus communis, Sida rhombifolia, Sonchus oleraceae, Bidens pilosa, Erigeron bonariensis, Emilia sonchifolia, Leonorus sibiricus, while phytoplasmas of the 16SrVII group were found in the last four mentioned species. In respect to insects, phytoplasmas were detected in individuals from subfamilies Deltocephalinae, Agalliinae and Typhlocybinae. Within the subfamily Deltocephalinae, the leafhopper Balclutha hebe carried phytoplasmas of the 16SrI group, while that of the species Atanus nitidus, Planicephalus flavicosta e Schapytopius fuliginosus harbored phytoplasmas of the 16SrIII group. In the tissues of two leafhoppers of the subfamily Agalliinae and one of the Typhlocybinae, which were not identified as specie, were found phytoplasmas of the 16SrIII group. The epidemiological analysis revelead an aggregated pattern of the diseased plants and a higher progress of the diseased in the border of the broccoli fields, whitch were located nearby areas where the presence of weeds was abundant.

O enfezamento vermelho do milho, causado por um fitoplasma, é uma das mais importantes doenças da cultura. Os danos ocasionados são relevantes, resultando em perdas econômicas significativas. Aspectos relacionados à interação milho-fitoplasma têm sido pouco investigados e, por consequência, são escassas as informações sobre alterações fisiológicas e bioquímicas que ocorrem no hospedeiro. O objetivo do presente trabalho foi justamente estudar as alterações bioquímicas que ocorrem em plantas de milho infectadas pelo agente causal do enfezamento vermelho. O trabalho foi desenvolvido em duas etapas. Na primeira, foram determinadas as alterações no conteúdo de proteínas e fenóis e na atividade das enzimas ß-1,3 glucanase e quitinase em nove híbridos comerciais de milho, experimentalmente inoculados com o fitoplasma. Na segunda etapa, dentre os nove híbridos, foram selecionados o mais resistente e o mais suscetível, sendo feita avaliação das alterações de proteínas, fenóis, clorofilas, açúcares redutores, peroxidase, ß-1,3 glucanase e quitinase em plantas infectadas pelo fitoplasma. Os ensaios foram conduzidos em casas teladas, sendo as plantas inoculadas aos 10 dias após a semeadura, através de 7-10 insetos infectivos da cigarrinha Dalbulus maidis por planta. A partir do aparecimento dos sintomas, amostras de folhas foram analisadas periodicamente para as determinações dos parâmetros bioquímicos. Para os nove híbridos, foi demonstrado um aumento médio de 20% no teor de proteínas, 90% de fenóis, 140% de quitinase e de 160% de ß-1,3 glucanase, em plantas inoculadas. Nos ensaios com os híbridos resistente e suscetível inoculados, os resultados mostraram que houve um aumento em todos os parâmetros bioquímicos avaliados, exceção feita à clorofila, cujo teor foi reduzido em função da infecção pelo patógeno. De modo geral, os valores obtidos para os diferentes parâmetros foram mais elevados no híbrido suscetível quando comparados ao híbrido resistente. O aumento das concentrações de proteínas, açúcares redutores e fenóis em plantas inoculadas revela alteração nos processos metabólicos do hospedeiro decorrentes da infecção pelo patógeno. Alterações nas atividades enzimáticas parecem fazer parte de uma resposta inespecífica de defesa. A redução na quantidade de clorofila total indica que o fitoplasma pode interferir no mecanismo fotossintético, podendo levar ao aceleramento do processo de senescência foliar. / Maize bushy stunt , caused by a phytoplasma, is an important disease of corn crop. The damages are relevant, resulting in significant economic losses. Aspects related to corn-phytoplasma interaction has been little investigated and consequently information about physiological and biochemical alterations that occur in the host are rare. Thus, the objective of the present work was to study biochemical alterations that occur in corn plants infected by the maize bushy stunt phytoplasma. The work was carried out on two steps. First, using 9 commercial hybrids, alterations in the levels of protein and phenol and in the activities of the enzymes b-1,3 glucanase and chitinase, in plants inoculated with the phytoplasma were determinated. In the second step, among the 9 hybrids the most resistant and the most susceptible one was selected and changes in protein, phenol, clorophyl, reducing sugar, peroxidase, b-1,3 glucanase and chitinase in infected plants. were evaluated. The experiments were carried out inside greenhouse, and the corn plants were inoculated by using infective Dalbulus maidis leafhoppers 10 days after seeding. When symptoms started to appear, leaf samples were harvested at different times for the biochemical analyses. It was shown an average increase of 20% in protein and 90% in phenol content and increases around 140% and 160% in the activities of chitinases and b–1,3-glucanases, respectively, in all 9 hybrids tested. When only the most susceptible and resistant hybrids were inoculated, the results showed an increase in all biochemical parameters evaluated, with exception for the chlorophyll content that decreased. In a general way, the values observed for the compounds and enzyme activities were higher in the susceptible hybrid when compared to the resistant one. The increases in protein, reducing sugar and phenol contents in inoculated plants point out changes in host metabolism due to the phytoplasma. The changes in enzyme activity seems to be an unspecific response of the plant to the pathogen and the reduction in chlorophyll content shows that the phytoplasma can interfere in photosynthesis and perhaps speedy senescence in the leaf tissue.

Uma doença, denominada de enfezamento, de etiologia desconhecida, tem sido observada nos campos de cultivo de repolho da região do cinturão verde de São Paulo. A doença tem causado sérios danos à cultura nos últimos anos. A sintomatologia apresentada pelas plantas afetadas tem sido caracterizada por clorose foliar, avermelhamento das nervuras e do limbo, enfezamento generalizado, proliferação de brotos e má formação da cabeça, de folhas e órgãos florais. A presença destes sintomas levou à suspeita da ocorrência de fitoplasma associado à doença. Com o objetivo de investigar o possível agente etiológico, plantas de repolho naturalmente infectadas foram coletadas em São Paulo e também nos Estados do Paraná e Rio Grande do Sul, onde a doença, recentemente, tem ocorrido com alta intensidade. Cigarrinhas que ocorrem em cultivos comerciais também foram amostradas em campos de Ibiúna/SP. Após a extração, o DNA total foi submetido ao teste de duplo PCR empregando-se os pares de primers universais P1/Tint e R16F2n/R2 e os pares específicos para identificação de fitoplasmas. Análises de PCR mostraram a amplificação consistente de fragmentos de DNA de 1,2 kb, evidenciando a associação constante de fitoplasma com tecidos das plantas sintomáticas e de insetos. O uso de primers específicos para identificação demonstrou a ocorrência de fitoplasmas afiliados ao grupos 16SrI e 16SrIII, tanto em repolho como nas cigarrinhas. Análises de RFLP, usando as enzimas de restrição AluI, Bsh 12361, HhaI HpaII, KpnI, MboI, MseI e RsaI confirmaram a ocorrência de fitoplasmas pertencentes aos referidos grupos no material vegetal e nos insetos. O padrão espacial de plantas sintomáticas no campo foi do tipo agregado e não houve evidência da disseminação planta a planta, indicando um papel mais importante do comportamento do vetor no arranjo espacial da doença do que de influências do patógeno ou do hospedeiro. / A disease called stunt, of unknown etiology, has occurred in cabbage crops located in the green belt region of the São Paulo State (Brazil). The disease has caused serious yield losses in the last years. The symptomatology exhibited by the affected plants has been characterized by foliar chlorosis, intense red coloration of leafs, general stunt, shoot proliferation and malformation of heads, leaves and floral parts. The presence of those symptoms suggested the occurrence of phytoplasma associated with the disease. In order to investigate the possible agent of the disease, naturally infected cabbage plants were collected from the States of São Paulo, and also from Paraná and Rio Grande do Sul, where the disease has occurred recently with level of intensity. Leafhoppers present in cabbage fields were sampled from Ibiúna, in São Paulo State. Total DNA was extracted and submitted to nested PCR with the universal primer pairs P1/Tint and R16 F2n/R2 and specific primers for phytoplasmas identification. PCR assays revealed the amplification of DNA fragments of 1.2kb, demonstrating consistently the presence of phytoplasma in the tissues from symptomatic plants and insects. Specific primers revealed the occurrence of phytoplasmas affiliated with the groups 16SrI and 16SrIII, both cabbage plants and leafhoppers. RFLP analyses using the restriction enzymes AluI, Bsh 12361, HhaI, HpaII, KpnI, MboI, MseI and RsaI confirmed the occurrence of phytoplasmas belonging to the same groups in plants and insects. Spatial pattern of symptomatic plants in the field was aggregated and there was no evidence of the spread plant-to-plant. This indicates a more important role of the vector behavior on spatial pattern than influences of the pathogen or host.

Fitoplasmas são procariotos sem parede celular, pertencentes à classe dos Mollicutes, que habitam os vasos de floema e causam doenças de relevância econômica em uma ampla gama de espécies vegetais. A videira (Vitis sp) é a terceira fruteira mais cultivada no mundo e apresenta elevada importância econômica no Brasil. Aspectos fitossanitários se constituem em fatores limitantes da produção, com destaque para as doenças causadas por fitoplasmas, denominadas de amarelos. Essas doenças estão amplamente disseminadas nos continentes europeu e americano, ocorrendo em tradicionais países produtores. Plantas exibindo sintomas característicos da doença têm sido também observadas em cultivos comerciais localizados em algumas áreas do território brasileiro. Visando investigar este tipo de doença, plantas sintomáticas foram amostradas em parreirais comerciais dos Estados de São Paulo e Paraná e mantidas em casa de vegetação. O presente estudo visou demonstrar a associação entre fitoplasmas e os amarelos, identificar e analisar filogeneticamente os fitoplasmas detectados, demonstrar sua transmissão para plantas sadias e determinar o melhor estádio fenológico da planta para amostragem de tecidos, para confirmação da diagnose. Para isto, durante 45 meses, amostras foram coletadas de plantas doentes envasadas sendo usado o método de PCR, análise de RFLP, sequenciamento das bases nucleotídicas e enxertia de tecidos entre plantas de videira. Os produtos amplificados em PCR evidenciaram a presença de fitoplasmas em todas as plantas portadoras de amarelos. Estes fitoplasmas foram identificados como representantes dos grupos 16SrI-B e 16SrIII, os quais ocorreram em infecções simples ou mistas. Os fitoplasmas apareceram em todas as fases de desenvolvimento da planta, porém com maior freqüência nas amostras coletadas no estádio fenológico correspondente à brotação de ramos e aparecimento de folhas novas. Esta fase se mostrou a mais indicada para as amostragens, visando à detecção de fitoplasmas para fins de diagnose. A transmissão dos fitoplasmas por enxertia foi alta, demonstrando que estes são os agentes causais da doença e que inspeções devem ser adotadas como medida de controle para evitar sua disseminação via material de propagação vegetativa. / Phytoplasmas are wall-less prokaryotes, phloem-limited plant pathogens, belonging to Mollicute class. They are agents of diseases that cause serious damage to a diversity of cultivated especies. Grapevine (Vitis sp) is the third most cultivated fruit crop in the world and it has high economical impact in Brazil. Phytossanitary aspects are important production constraint factors, especially regarding diseases caused by phytoplasmas, which are generally known as grapevine yellows. These diseases are widely spread throughout main producer countries, located in Europe and North America. Plants exhibiting typical symptoms of the disease have also been observed in crops located in some areas of Brazilian territory. In order to investigate this kind of malady, symptomatic plants were sampled from commercial vineyards located in the States of São Paulo and Paraná, Brazil and kept in greenhouse conditions. The present study aimed to: demonstrate the association between phytoplasmas and yellows; identify and analyse phylogenetically the detected phytoplasmas; demonstrate their transmission to healthy plants and determinate optimal phenologycal stage for phytoplasma detection to diagnosis confirmation. Samples were collected from diseased plotted plants grown during 45 months. PCR method, RFLP analyses, sequencing of nucleotide bases from 16S rRNA, and grafting technique were used in this study. Amplified genomic fragments revealed the presence of phytoplasmas in all plants with symptoms of yellows. Those phytoplasmas were identified as representatives of the groups 16SrI-B and 16SrIII, occurring in simple and mixed infections. Phytoplasmas were detected in all phenological stages evaluated, but they occurred with high frequences in material collected during budburst. These stage may be considered as the most indicated for sampling to confirm diagnosis based on symptomatology. Phytoplasmas transmission by grafting of healthy scion grafts onto infected rootstocks was high, demonstrating that they are the disease agents and inspection is a control practice that must be adopted to prevent their dissemination through propagative material.

No Brasil, o cultivo de ameixeira tem atingido significativa importância econômica em termos de rentabilidade. Embora rentável, a cultura exige cuidados constantes em relação aos danos provocados por doenças. Entre elas, o \'amarelo\', causado por fitoplasmas, tem se mostrado como uma doença de relevância nos países produtores desta fruta. Em pomares comerciais localizados em Paranapanema-SP, foram observadas ameixeiras (Prunus salicina) exibindo sintomas típicos de \'amarelos\', caracterizados por superbrotamento de ramos, redução no comprimento de entrenós, além de amarelecimento, deformação e redução do limbo foliar. Com objetivo de identificar o fitoplasma e determinar o modelo de colonização do hospedeiro pelo patógeno foi desenvolvido o presente estudo. Para isto, em três pomares, foram amostradas plantas sintomáticas, assintomáticas e sadias, pertencentes às variedades Gulfblaze e Azteca. O DNA total foi extraído de folhas e ramos e usado em duplo PCR com os iniciadores R16mF2/mR1 e R16F2n/R2, visando a detecção do fitoplasma. Os resultados confirmaram a presença de fitoplasma pela amplificação de fragmentos de DNA de 1,2 kb do gene 16S rRNA. Os produtos de PCR gerados por dez isolados de fitoplasmas de cada variedade foram clonados e três clones de cada isolado foram sequenciados. Uma vez que nenhum polimorfismo foi encontrado, uma sequência consenso foi selecionada para cada isolado e um isolado foi escolhido como representativo para cada variedade. Estas sequências foram designadas por PlY-BR01 e PlY-BR02, com 1.246 pb, e depositadas no GenBank sob os números de acesso KF499086 e KF499087, respectivamente. A sequência do 16S rRNA do fitoplasma da ameixeira apresentou 100% de identidade com o fitoplasma representante do grupo 16SrI-B (AY265208). A análise de RFLP virtual e o cálculo do coeficiente de similaridade, permitiram classificar o fitoplasma da ameixeira no grupo 16SrI-B. A análise filogenética revelou que o mesmo está estritamente relacionado ao subgrupo 16SrI-B. No estudo da colonização das plantas pelo patógeno, amostras da copa e raiz foram coletadas mensalmente, durante um ano, de plantas infectadas das variedades Gulfblaze e Azteca. O DNA total foi extraído e submetido ao PCR em Tempo Real com os iniciadores UniRNAForward/UniRNAReverse, para quantificar o fitoplasma nos tecidos do hospedeiro. O fitoplasma foi detectado tanto em amostras de copa como de raiz, em todas as árvores infectadas de ambas as variedades. A concentração do mesmo nos tecidos vegetais variou de 5,77x105 a 1,93x109 e 6,18x105 a 3,92x109 N°cópias/100 ng de DNA total, na copa e na raiz, respectivamente. O experimento mostrou uma flutuação sazonal na concentração do fitoplasma, sendo as maiores concentrações encontradas no verão, embora o fitoplasma tenha permanecido na parte aérea do hospedeiro mesmo no período mais frio, que compreende a fase de dormência da planta. Com base nestes resultados, ficou evidente que as amostras retiradas da copa e coletadas durante o período mais quente do ano são as mais adequadas para os procedimentos de detecção do fitoplasma, visando confirmar a diagnose feita com base na sintomatologia. / In Brazil, the cultivation of plum trees has achieved significant economic importance in terms of profitability. Although profitable, the culture demands attention in relation to damages provoked by diseases. Among them, the \'yellow\', caused by phytoplasmas, is a relevant disease present in the plum producing countries. In commercial orchards located in Paranapanema-SP region, were observed plum trees (Prunus salicina) exhibiting typical symptoms of \'yellow\', characterized by intense shoot proliferation, shortened internodes, generalized yellowing, leaf malformation and small leaves. The present study was conducted in order to identify the phytoplasma associated with symptomatic plants and determine the pattern of host colonization by the pathogen. Therefore, were sampled symptomatic, asymptomatic and healthy plants of the varieties Gulfblaze and Azteca, grown in three commercial orchards. Total DNA was extracted from leaves and shoots and submitted to nested PCR primed with R16mF2/mR1 and R16F2n/R2, in order to detect phytoplasma. The results confirmed the presence of phytoplasma by amplification of DNA fragments of 1.2 kb from 16S rRNA gene. The PCR products generated by ten phytoplasma isolates of each variety were cloned and three clones of each isolate were sequenced. Since no polymorphism was found, a consensus sequence was selected for each isolate and an isolate was chosen as representative for each variety. These sequences were designated by PlYBR01 and PlY-BR02, with 1,246 bp, and deposited in GenBank under the accession numbers KF499086 and KF499087, respectively. The sequence of the 16S rRNA of the phytoplasma founded in plum trees showed 100% identity with the phytoplasma representative of 16SrI-B group (AY265208). The virtual RFLP analysis and the calculation of the similarity coefficient, allowed classify the phytoplasma present in plum trees as a member of 16SrI-B group. Phylogenetic analysis supported that this phytoplasma is closed related to the 16SrI-B subgroup. In the study about plant colonization, the samples from leaves, shoots and root were monthly collected, for one year, from the infected plants of the varieties Gulfblaze and Azteca. Total DNA was extracted and submitted to the Real Time PCR with primers UniRNAForward/UniRNAReverse, in order to quantify phytoplasma in host tissues. The phytoplasma was detected in both aerial parts and roots in all infected trees of the two varieties. The concentration of the pathogen in plant tissues ranged from 5.77 x105 to 1.93 x109 and 6.18 x105 to 3.92 x109 N°copies/100ng total DNA in aerial parts and roots, respectively. The experiment showed a seasonal fluctuation in the concentration of the phytoplasma in plants, with the highest concentrations found in summer, although the phytoplasma have remained in the shoots of the host even in the coldest period, comprising the dormant phase of the plant. Based on these results, it was evident that the samples collected from the aerial parts and during the hottest period of the year are the most appropriate for detection of phytoplasma to confirm the diagnosis based on symptomatology.