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Previous issue date: 2008-10-07 / Phytoplasmas are wall-less prokaryotes associated with diseases in numerous plant species
worldwide. In nature they are transmitted by phloem-sucking insects. Since phytoplasmas cannot
be cultured in vitro, molecular techniques are needed for their diagnosis and characterization. In
Brazil, chayote witches broom is a disease associated with ChWBIII phytoplasma, which can
reduce the quality of chayote fruits. ChWBIII has other natural plant hosts, in the family
Cucurbitaceae, and bitter melon (Momordica charantia) is the major alternative host.
The present work aimed at (a) determine the transmissibility of ChWBIII to periwinkle
(Catharanthus roseus) through experimental methods; (b) study the symptomatology exhibited
by C. roseus infected with ChWBIII phytoplasma; (c) evaluate two different strategies of plant
tissue dehydration, to detect phytoplasma in naturally diseased plants of chayote, bitter melon and
periwinkle; (d) study the putative presence and transmission of ChWBIII phytoplasma to seeds of
M. charantia; and (e) verify phytoplasma variability in corn, passionfruit, poinsetia and C.
roseus.
Pathogenicity and transmissibility of ChWBIII through dodder and grafting was verified by the
symptomatology developed in C. roseus, and by PCR with universal primer pairs specific to
phytoplasmas 16S rDNA. Afterwards, the identity of ChWBIII in donor plants of bitter melon, in
dodder and in C. roseus was confirmed by sequences analyses.
Samples from diseased plants of bitter melon, periwinkle and chayote, that were submitted to
desiccation with silica gel showed phytoplasmal 16S rDNA that was amplified with P1/P7 in
PCR and reamplified in nested PCR with F2n/R2 yielding products of approximately 1.2 kb.
Molecular assays demonstrated the presence of ChWBIII phytoplasma in seeds of symptomatic
fruits of M. charantia, collected from plants with witches broom symptoms. The use of primers
specific to 16SrIII group confirmed the identification of the phytoplasma associated with seeds as
belonging to this group, what was further confirmed through sequencing. Sequences analyses
revealed that the phytoplasma associated with bitter melon seeds is ChWBIII.
The presence of phytoplasmas in symptomatic plants of corn, passionfruit, poinsetia and C.
roseus was detected by PCR and nested PCR with universal primer pairs. Phytoplasmas isolated
from passionfruit, poinsetia and C. roseus were classified as belonging to 16SrIII group,
according to nested PCR with primers specific to this group. Analysis of the sequences of the
phytoplasmas from passion fruit and C. roseus confirmed the identity of these organisms,
revealing 99% homology with ChWBIII.
The results determined are the following: (a) the transmissibility of ChWBIII to the
experimental host C. roseus by inverse transmission through dodder, as well as by graft
transmission; (b) the symptomatology exhibited by C. roseus infected with ChWBIII
phytoplasma; (c) the viability of preserved tissue from chayote, bitter melon and periwinkle to
detect phytoplasma, indicating the possible use of this kind of sample, for other botanical species
infected with phytoplasmas; (d) the presence of phytoplasma in seeds of symptomatic fruits, from plants of bitter melon infected with ChWBIII, what suggests the putative transmission of the
phytoplasma from the mother plants to the seeds; (e) the diversity of phytoplasma host plants,
that was verified in corn, passionfruit, poinsetia and C. roseus.
The results obtained in the present study lead to a better epidemiolgical understanding of the
diseases caused by phytoplasmas, and may help to search for insect vectors and alternate plant
hosts, enabling the design of effective disease management strategies. / Fitoplasmas s?o procariotos sem parede celular associados com in?meras enfermidades de
esp?cies de plantas no mundo. Esses pat?genos na natureza s?o transmitidos por insetos que se
alimentam do floema das plantas. Como os fitoplasmas n?o podem ser cultivados in vitro
empregam-se t?cnicas moleculares para sua diganose e caracteriza??o No Brasil, o
superbrotamento do chuchuzeiro ? uma doen?a associada ao fitoplasma ChWBIII, que pode
causar redu??o da qualidade de frutos de chuchu. O ChWBIII tem outras esp?cies hospedeiras
naturais, da fam?lia Cucurbitaceae, sendo a principal dessas, mel?o-de-S?o-Caetano (Momordica
charantia).
O presente trabalho teve como objetivos: (a) determinar a transmissibilidade do ChWBIII por
m?todos experimentais para vinca (Catharanthus roseus); (b) estudar a sintomatologia exibida
por C. roseus infectado com o fitoplasma ChWBIII; (c) avaliar duas diferentes estrat?gias de
desidrata??o de tecido vegetal para a detec??o de fitoplasmas de plantas naturalmente doentes de
chuchuzeiro, mel?o-de-S?o-Caetano e vinca; (d) estudar a poss?vel presen?a e a transmiss?o do
fitoplasma ChWBIII ?s sementes de M. charantia; e, (e) verificar a variabilidade de fitoplasmas
em milho, maracuj?, poins?tia e C. roseus.
A patogenicidade e a transmissibilidade do ChWBIII, mediante Cuscuta e enxertia, foi
verificada pela sintomatologia em C. roseus e por PCR usando primers universais espec?ficos
para o 16S rDNA de fitoplasmas. Posteriomente, a identidade do ChWBIII em plantas doadoras
de mel?o-de-S?o-Caetano, em Cuscuta e em C. roseus foi confirmada por an?lise de seq??ncias.
Amostras de plantas doentes de mel?o-de-S?o-Caetano, vinca e chuchuzeiro submetidas ?
desseca??o com s?lica gel, continham 16S rDNA de fitoplasma, o qual foi amplificado por PCR
com primersP1/P7 e reamplificado em nested PCR com os primers F2n/R2, fornecendo produtos
de aproximadamente 1.2 kb.
Testes moleculares demonstraram a presen?a do fitoplasma ChWBIII em sementes de M.
charantia de frutos sintom?ticos, obtidos de plantas com superbrotamento do mel?o-de-S?o-
Caetano . O emprego de primers espec?ficos ao grupo 16SrIII confirmou a identifica??o do
fitoplasma associado ? sementes como pertencente a esse grupo, o que foi corroborado atrav?s
do seq?enciamento. A an?lise das seq??ncias revelou que o fitoplasma associado a sementes de
mel?o-de-S?o-Caetano ? o ChWBIII.
A presen?a de fitoplasmas em plantas sintom?ticas de milho, maracuj?, poins?tia e C. roseus
foi detectada atrav?s de PCR e nested PCR com iniciadores universais. Os fitoplasmas isolados
de maracuj?, poins?tia e C. roseus foram inseridos no grupo 16SrIII, de acordo com nested PCR
com primers espec?ficos a esse grupo. An?lise das seq??ncias dos fitoplasmas de maracuj? e de
C. roseus confrrmou a identidade dos mesmos, revelando 99% de homologia com o ChWBIII.
Com a presente pesquisa determinou-se: (a) a transmissibilidade de ChWBIII mediante a
transmiss?o inversa por Cuscuta para a hospedeira experimental C. roseus e, tamb?m mediante a
indexa??o biol?gica; (b) a sintomatologia exibida por C. roseus infectado com o fitoplasma ChWBIII; (c) a viabilidade do uso de tecidos preservados de chuchuzeiro, mel?o-de-S?o-Caetano
e vinca, para a detec??o de fitoplasmas, indicando a possibilidade de uso desse tipo de amostras
para outras esp?cies bot?nicas afetadas por fitoplasmoses; (d) a presen?a de fitoplasmas nas
sementes de frutos sintom?ticos provenientes de plantas de mel?o-de-S?o-Caetano infectadas
com o ChWBIII, o que sugere a poss?vel transmiss?o do fitoplasma da planta m?e para as
sementes; (e) a diversidade de plantas hospedeiras de fitoplasmas no Brasil, que foi verificada em
milho, maracuj?, poins?tia e C. roseus.
Os resultados obtidos no presente estudo possibilitam o maior entendimento epidemiol?gico
das enfermidades incitadas por fitoplasmas e podem auxiliar na descoberta de insetos vetores e de
plantas hospedeiras alternativas, favorecendo o estabelecimento de estrat?gias de controle dessas
doen?as.
| Identifer | oai:union.ndltd.org:IBICT/oai:localhost:tede/502 |
| Date | 07 October 2008 |
| Creators | Jim?nez, Nilda Zulema Albornoz |
| Contributors | Montano, Helena Guglielmi |
| Publisher | Universidade Federal Rural do Rio de Janeiro, Curso de P?s-Gradua??o em Fitossanidade e Biotecnologia Aplicada, UFRRJ, Brasil, Entomologia Aplicada |
| Source Sets | IBICT Brazilian ETDs |
| Language | Portuguese |
| Detected Language | English |
| Type | info:eu-repo/semantics/publishedVersion, info:eu-repo/semantics/masterThesis |
| Format | application/pdf |
| Source | reponame:Biblioteca Digital de Teses e Dissertações da UFRRJ, instname:Universidade Federal Rural do Rio de Janeiro, instacron:UFRRJ |
| Rights | info:eu-repo/semantics/openAccess |
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