Protein extraction for Cocos nucifera with and without lethal yellowing phytoplasma: gel-based proteomics

CELSO REYES MARTINEZ DANAE MORALES ANGELES MARIA DEL SOCORRO NARVAEZ CAB JUAN ALBERTO OSUNA CASTRO DANIEL ZIZUMBO VILLARREAL CARLOS MARIANO OROPEZA SALIN ELPIDIO PEÑA BELTRAN (2007)

Two-dimensional gel electrophoresis (2-DE) is one of the two methodologies to separate and identify gene expression at the proteome level. However, the separation of proteins from plant tissue extract is often complicate, because it is necessary to modify the protocols depending upon the type of tissue and upon the presence of different interfering compounds. In this work it is established a routine procedure for the application of proteomic analysis to study interaction between palm coconut and lethal yellowing (LY) phytoplasma. The characteristics of protocol include: (i) cleanup of pigments and contaminants of the trunk palm coconut tissue, using a solution of acetone with TCA and 2-mercaptoethanol. (ii) Phenol extraction of proteins in presence of methanol with ammonium acetate. 2-DE analysis revealed differences in palm coconut with and without LY agent and similitude between palm ecotypes.

Article

COCOS NUCIFERA PHYTOPLASMA PHENOLS PROTEOMICS ECOTYPES GEL ELECTROPHORESIS PLANT EXTRACTS METHANOL PIGMENTS BIOLOGÍA Y QUÍMICA BIOLOGÍA Y QUÍMICA

Importancia de las arvenses en la dispersión del amarillamiento letal del cocotero

NELBA TERAN VILLANUEVA (2014)

Tesis (Maestría en Ciencias, especialista en Producción Agroalimentaria en el Trópico).- Colegio de Postgraduados, 2014.

El cocotero (Cocos nucifera) uno de los cultivos tropicales de mayor importancia económica por su contribución sustancial a la seguridad alimentaria, se ha visto afectado por la enfermedad conocida como amarillamiento letal del cocotero (ALC), una de las más devastadores y que se ha localizado en todas las regiones productoras de México. El objetivo de la presente investigación fue analizar el papel que juegan las arvenses en la dispersión del ALC; para ello se realizó un registro de la flora asociada a la plantación, identificándose taxonómicamente las especies de plantas (arvenses); se hicieron análisis moleculares para detectar la presencia del fitoplasma de dicha enfermedad en las arvenses en ocho sitios dentro de la plantación. Posteriormente se realizó un análisis de componentes principales para conocer la similaridad ecológica de los sitios. Para la determinación molecular de la presencia de fitoplasma se consideraron las siguientes especies: Digitaria abyssinica, Digitaria insularis, Conyza canadensis, Eustachys petraea, Emilia sonchifolia, Tridax procumbens, por ser las más frecuentes dentro de la plantación, Vernonia cinerea, Portulaca pilosa, Sida acuta y Panicum laxum; de ellas se colectaron muestras completas para la extracción de ADN que fueron analizadas mediante la técnica molecular de PCR en tiempo real usando primers generales (P1/P7) y específicos (LY16sr/LY16sf) para Candidatus Phytoplasma palmae. La composición de la flora asociada a la plantación resultó en 93 especies correspondientes a 34 familias y 75 géneros. El análisis de componentes principales destacó de manera general, la disimilaridad de cuatro sitios (1, 2, 4 y 8), que resaltan la heterogeneidad florística de la comunidad de arvenses en la plantación de estudio, además de un grupo homogéneo formado por los otros cuatro. Los análisis de PCR en tiempo real revelaron a las arvenses: D. abyssinica, D. insularis, E. petraea y P. pilosa, como posibles reservorios de fitoplasma. _______________ IMPORTANCE OF THE WEEDSIN THEDISPERSIÓN COCONUT LETHAL YELLOWING. ABSTRACT: The coconut (Cocos nucifera L.), one of the tropical crops of major economic importance for their substantial contribution to food security, has been affected by the disease known as lethal yellowing (CLY), one of the most devastating and which has been located in all producing regions of México. The aim of this research was to analyze the role of weeds in the dispersion of the CLY; for this a record of the flora associated with planting and the taxonomical identification of plants species (weeds) was performed; Molecular analyzes were carried out to detect the presence of phytoplasma disease on weeds at eight sites within the plantation. Subsequently, a principal component analysis was performed to determine the ecological similarity of the sites. For the molecular identification of the presence of phytoplasma, the following species were considered: Digitaria abyssinica, Digitaria insularis, Conyza canadensis, Eustachys petraea and Tridax procumbens, which was being the most common in the plantation, the remaining five were selected for having been reported or have close relatives reported as reservoirs of phytoplasma, in this case were Emilia sonchifolia, Vernonia cinerea, Portulaca pilosa, Sida acuta and Panicum laxum; DNA extract of samples were analyzed by molecular technique real-time PCR using general primers (P1/P7) and specific (LY16sr/LY16sf) for Candidatus Phytoplasma palmae. The composition of the flora associated with planting resulted in 93 species corresponding to 34 families and 75 genera. The principal component analysis emphasized, in general, the dissimilarity of four sites (1, 2, 4 and 8), highlighting the floristic diversity of weed community in planting study, as well as a homogeneous group formed by the other four. The real-time PCR analysis revealed the weed: D. abyssinica, D. insularis, E. petraea and P. pilosa as potential reservoirs of phytoplasma.

Master thesis

Fitoplasma PCR en tiempo real Flora asociada ALC Myndus crudus Phytoplasma Real-time PCR Associated flora Producción Agroalimentaria en el Trópico Maestría CIENCIAS AGROPECUARIAS Y BIOTECNOLOGÍA

First Report of a 16SrIV Group Phytoplasma Associated with Declining Coyol Palms in Honduras

CARLOS MARIANO OROPEZA SALIN (2006)

Lethal yellowing (LY) of coconut palm (Cocos nucifera L.) caused by a subgroup 16SrIV-A phytoplasma has been present along the northern coast and adjacent Bay Islands of Honduras since 1996. In the southern municipalities of San Esteban and Guanaco, approximately 150 km from the Atlantic coast, substantial numbers of coyol palm (Acrocomia aculeata (Jacq.) Lodd. ex Mart.) and several coconut palms growing nearby were either dead or in an advanced stage of decline during 2005. Declining palms of both species exhibited loss of fruit, withered inflorescences, and dried, discolored (grayish brown) leaves. Most or all leaves on declining palms had collapsed and hung downward around the stem. Samples (3 to 5 g) of tissue were excised from stems of 20 coyol and 2 coconut palms. DNA was extracted from each sample using a previously described protocol (2) and assayed for phytoplasma DNA using polymerase chain reaction (PCR) employing phytoplasma universal rRNA primer pair P1 (1) and P7 (4). A product of expected size (approximately 1.8 kb) was amplified from 12 of 20 diseased coyol palms, 2 of 2 diseased coconut palms and from DNA of a Florida-grown Chinese fan palm (Livistona chinensis(Jacq.) R. Br. ex Mart.) with LY symptoms included as a known positive control. Amplified P1/P7 products used in a nested PCR assay with 16SrIV-specific primer pair LY16Sf (2) and LY16Sr (5′-GCTTACGCA-GTTAGGCTGTC-3′) yielded a product of approximately 1.39 kb. Neither primary nor nested PCRs generated a product from DNA of healthy coconut palm. Poor quality of tissues within stems of declining palms may have contributed to lack of amplification of any product from eight coyol samples. A reevaluation of DNA samples using PCR employing nonribosomal primer pair LYF1/LYR1, which specifically detects subgroup 16SrIV-A phytoplasmas (3), failed to amplify an expected 1-kb product from any palm sample other than the LY positive control. Digestion of nested amplification products (1.39 kb) with AluI endonuclease and electrophoresis of digests through 8% polyacrylamide gels revealed the same three-fragment restriction pattern for all phytoplasmas detected in coyol and coconut palms. The pattern differed from the five-fragment profile of LY phytoplasma rDNA included for comparative purposes. These results indicate that coyol palm is a new phytoplasma host and that decline symptoms on coyol and adjacent coconuts were not a consequence of natural dispersion of subgroup 16SrIV-A phytoplasmas from t

Article

COYOL PALMS PHYTOPLASMA  BIOLOGÍA Y QUÍMICA

In situ PCR detection of phytoplasma DNA in embryos from coconut palms with lethal yellowing disease

IVAN ISIDRO CORDOVA LARA Phil Jones Nigel A. Harrison Carlos Mariano Oropeza Salín (2003)

DNA of the lethal yellowing (LY) phytoplasma was detected in 13 of 72 embryos from fruits of four diseased Atlantic tall coconut palms by polymerase chain reaction (PCR) assays employing phytoplasma universal rRNA primer pair P1/P7, nested LY group‐specific rRNA primer pair 503f/LY16Sr or LY phytoplasma‐specific nonribosomal primer pair LYF1/R1. Phytoplasma distribution in sectioned tissues from six PCR positive embryos was determined by in situ PCR and digoxigenin‐11‐deoxy‐UTP (Dig) labelling of amplification products. Dig‐labeled DNA products detected by colourimetric assay were clearly evident on sections from the same three embryos investigated in detail by in situ PCRs employing primer pairs P1/P7 or LYF1/R1. Deposition of blue–green stain on sections as a result of each assay was restricted to areas of the embryos corresponding to the plumule and cells ensheathing it. By comparison, similarly treated embryo sections derived from fruits of a symptomless Atlantic tall coconut palm were consistently devoid of any stain. Presence of phytoplasma DNA in embryo tissues suggests the possible potential for seed transmission which remains to be demonstrated.

Article

BACTERIA (MICROORGANISMS) COCOS NUCIFERA PHYTOPLASMA PROKARYOTA BIOLOGÍA Y QUÍMICA CIENCIAS DE LA VIDA BIOLOGÍA CELULAR CITOGENÉTICA CITOGENÉTICA

Molecular characterization of Yucatan tomato phytoplasma (Group 16Sr III)

RAUL TAPIA TUSSELL ALBA PRISCILIA SUASTE DZUL ALBERTO CORTES VELAZQUEZ CLAUDIA GUADALUPE TORRES CALZADA ANDRES FELIPE DE JESUS QUIJANO RAMAYO RODOLFO MARTIN MEX ANGEL NEXTICAPAN GARCEZ DAISY DE LA CARIDAD PEREZ BRITO (2012)

Tomato (Lycopersicon esculentum) is an important vegetable crop in Mexico. Recently, a phytoplasma associated with leaf yellowing and curling, severe stunting and little leaf in tomato plant was identified as Yucatan tomato phytoplasma (16SrIII group). DNAs extracted from tomato leaves with symptoms were examined for the presence of this phytoplasma by nested polymerase chain reaction (PCR). Positive results were obtained in 44% of samples, yielding an rDNA product of 1.25 kb. In vitro and in silico restriction fragment length polymorphism (RFLP) patterns obtained with endonucleases HpaII, MseI, RsaI and TaqI were characteristics of group 16SrIII, according to the classification scheme of phytoplasmas. The pattern with AluI and HaeIII discriminated between these phytoplasmas and the members of 16SrIII group. Molecular characterization of the causal agent of Yucatan tomato phytoplasma will facilitate the study of this disease’s epidemic aspects and its phytosanitary management. In addition, it will contribute to a greater knowledge of the genetic diversity of phytoplasmas present in Mexico.

Article

DIAGNOSTICS PHYTOPLASMA 16S RDNA PCR-RFLP BIOLOGÍA Y QUÍMICA

Occurrence of a 16SrIV group phytoplasma not previously associated with palm species in Yucatan, Mexico

ROBERTO CARLOS VAZQUEZ EUAN MARIA DEL SOCORRO NARVAEZ CAB CARLOS MARIANO OROPEZA SALIN (2011)

The occurrence of 16SrIV group phytoplasmas in palm species Sabal mexicana and Pseudophoenix sargentii is reported here for the first time. Palm trees showed leaf decay and leaf yellowing syndromes, respectively. An amplification product (1.4 kb) was obtained in symptomatic S. mexicana (18 of 21) and symptomatic P. sargentii (1 of 1) palm trees sampled in different locations in Yucatan State, Mexico; five of the positive S. mexicana and the positive P. sargentii trees died. The identity of the phytoplasmas from these species was determined by restriction fragment length polymorphism profiling with restriction enzymes AluI and HinfI, showing there could be two phytoplasma strains of the 16SrIV group. In one S. mexicana palm, the profile was the same as observed with these enzymes for phytoplasmas of 16SrIV-A subgroup, previously associated with Cocos nucifera palm trees and, in the rest of the trees, including the P. sargentii palm, the profile was for phytoplasmas of the 16SrIV-D subgroup. These identities were supported by analyses of the amplicons obtained by nested polymerase chain reaction by nucleotide-nucleotide BLAST analysis. Geographical distribution of the association S. mexicana/16SrIV group phytoplasmas was found widely dispersed in Yucatan State. A potential role of S. mexicana palm trees as a permanent source ofphytoplasma inoculum is suggested. In addition to P. sargentii, other palm species (Thrinax radiata and C. nucifera) coexisting with S. mexicana trees were also sampled and analyzed.

Article

CANDIDATUS PHYTOPLASMA COCOS NUCIFERA PSEUDOPHOENIX SARGENTII SABAL SABAL MEXICANA THRINAX RADIATA BIOLOGÍA Y QUÍMICA

‘Candidatus Phytoplasma trifolii' (16SrVI) en chile mirasol (Capsicum annuun L.) cultivado en Zacatecas, México

‘Candidatus Phytoplasma trifolii' (16SrVI) in mirasol chili pepper (Capsicum annuun L.) cultivated in Zacatecas, México

JORGE ARMANDO MAURICIO CASTILLO SILVIA SALAS MUÑOZ RODOLFO VELASQUEZ VALLE Salvador Ambriz LUIS ROBERTO REVELES TORRES (2015)

"Plantas de chile (Capsicum annuum L.) para secado tipo Mirasol que mostraban síntomas de yema grande, amarillamiento y enrollamiento foliar, fueron colectadas en parcelas comerciales en el municipio de Calera de Víctor Rosales, Zacatecas, México. Se analizaron mediante la reacción en cadena de la polimerasa (PCR) anidada para determinar la presencia de fitoplasmas, con el par de oligonucleótidos universales P1/Tint, seguido del par R16F2n/R16R2, y del análisis de fragmentos del ADNr 16S mediante la obtención de bandas polimórficas a partir de patrones de restricción (RFLP). La presencia de dos patrones de restricción diferentes indicó la existencia de dos nuevas cepas de ‘Candidatus Phytoplasma trifolii', grupo 16SrVI. La primera secuencia de fitoplasma (ChZac4F3) correspondió al subgrupo 16SrVI-A; en cambio la segunda secuencia de fitoplasma (ChZac5F1) se clasificó dentro de un nuevo subgrupo (16SrVI-J). Ambas secuencias mostraron una similitud de 99.2 y 99.1 %, respectivamente, al ser comparadas con la cepa de referencia de ‘Candidatus Phytoplasma trifolii' (AY390261) al analizarlas con el programa iPhyClassifier. Este es el primer reporte de dos nuevas cepas de ‘Candidatus Phytoplasma trifolii' asociadas con la sintomatología de yema grande, amarillamiento y enrollamiento foliar en chile Mirasol en Zacatecas, México."

"Dry chili pepper Mirasol type plants (Capsicum annuum L.) exhibiting foliar yellowing, big bud, and rolled-up margins symptoms were collected from commercial pepper fields in Calera de Victor Rosales country, Zacatecas, México. They were examined to determine the presence of phytoplasms by nested polymerase chain reaction (PCR) using the universal primer pair P1/Tint, followed by the primer pair R16F2n/R16R2 and restriction fragment length polymorfism (RFLP) analysis of 16S rDNA sequences. The presence of two different RFPL patterns in clones from different samples indicated the presence of two phytoplasmas isolates. Each was classified as strains of ‘Candidatus Phytoplasma trifolii', that belong to group 16SrVI. The first ChZac4F3 phytoplasma sequence belongs to subgroup 16SrVI-A, while the second ChZac5F1 phytoplasma sequence was classified into a new subgroup (16SrVI-J). ChZac4F3 and ChZac5F1 phytoplasma sequences showed a similarity of 99.2 and 99.1 %, respectively, with the reference strain of ‘Candidatus Phytoplasma trifolii' (AY390261), when both were analyzed via the iPhyClassifier program. This is the first report of two new strains of ‘Candidatus Phytoplasma trifolii' associated to foliar yellowing, big bud and rolled-up margins symptoms in Mirasol chili pepper in Zacatecas, México."

Article

‘Candidatus Phytoplasma trifolii' Capsicum annuum Diagnóstico molecular PCR CIENCIAS AGROPECUARIAS Y BIOTECNOLOGÍA

Caracterización molecular de begomovirus y fitoplasmas asociados a una infección mixta en calabacita Cucurbita pepo L. en B.C.S., México.

MAYELA VARGAS SALINAS (2017)

"Desde 2010, en la región agrícola de La Paz, BCS, México, en el cultivo de calabacita (Cucurbita pepo) se han observado síntomas que se han asociado a una enfermedad mixta en la que pudieran estar involucrados dos patógenos (begomovirus y fitoplasmas) ya detectados en otros cultivos de la región, tales como: hojas pequeñas, severo retraso de crecimiento y deformación de frutos. Relacionados con begomovirus, se han detectado problemas de enrollamiento de la hoja, mosaicos y engrosamiento de venas; y atribuidos a fitoplasmas, amarillamiento y alta proliferación de brotes axilares. Adicionalmente, se observaron chicharritas y altas poblaciones de mosquitas blancas, insectos reportados como vectores de estos patógenos. El objetivo del presente trabajo fue identificar, caracterizar y realizar el estudio filogenético de los agentes causales de esta enfermedad, estimar el daño de la infección y llevar a cabo la identificación de los vectores. En el caso de los begomovirus, se realizó PCR con cebadores degenerados que amplifican la cubierta proteica y la región intergénica. Se obtuvo la secuencia completa del genoma con la técnica de círculo rodante, que analizada por alineamiento múltiple con otros begomovirus disponibles en GenBank, se comprobó una identidad entre el 98.8-99.1%. El aislado de BCS pertenece al begomovirus Squash leaf curl virus SLCuV-[MX:BCS:La Paz:17] GenBank MF187211A. La caracterización molecular del virus mostró que el SLCuV-BCS es un begomovirus bipartita, con componentes genómicos (A y B) de 2,638 y 2,608 pb; con 5 marcos de lectura abiertos en A y dos en B. El análisis filogenético mostró que SLCuV-[MX:BCS:La Paz:17] se establece entre dos grupos filogenética y geográficamente bien definidos como el clado SLCuV de Medio Oriente y SLCuV de Estados Unidos. La detección preliminar de fitoplasmas se hizo con MEB, donde se confirmó la presencia del patógeno de acuerdo a las características morfológicas observadas en los tejidos vasculares. La identificación y caracterización se realizó por PCR anidada amplificando la región del gen 16S ARNr de 1.2 kb. El análisis de alineamiento múltiple de las secuencias nt arrojó una similitud del 98.6% con 'Candidatus Phytoplasma pruni'. Se observó la firma genética ´CAAGAYBATKATGTKTAGCYGGDCT´, que establece la identidad de especie del fitoplasma. Los análisis filogenéticos ubicaron el aislado de BCS dentro del grupo 16SrIII..."

Symptoms have been observed in the cultivation of the squash Cucurbita pepo in the agricultural region of La Paz, Baja California Sur (BCS), Mexico, which could be associated with a mixed disease where two pathogens (begomovirus and Phytoplasmas) were involved before and detected in other crops in the region since 2010. The symptoms associated with the mixed infection were: small leaves, severe stunted growth and fruit deformation. In addition, curled leaf, mosaic and vein thickening symptoms have been observed related to begomovirus and yellowing and high proliferation of axillary buds to Phytoplasmas. Moreover, leafhoppers and high populations of whitefly insects reported as vectors of these pathogens were observed. Thus the objective of this work was to identify, characterize and carry out a phylogenetic study of the causative agents of this disease to estimate infection damage and the identification of the vectors. In the case of begomoviruses, PCR was performed with degenerate primers that amplify the protein coat and the intergenic region. The complete genome sequence was obtained with the rolling circle technique, which by analyzing multiple alignments with other available GenBank begomoviruses obtained an identity from 98.8-99.1%. The isolate of BCS belonged to the begomovirus Squash leaf curl virus SLCuV-[MX:BCS:La Paz:17] GenBank MF187211A. The molecular characterization of the virus showed that SLCuV-BCS is a bipartite begomovirus with genomic components (A and B) of 2,638 and 2,608 bp, with 5 open reading frames in A and two in B. The phylogenetic analysis showed that SLCuV-[MX:BCS:La Paz:17] was established between two phylogenetic and geographically well defined groups such as the SLCuV clade of the Middle East and SLCuV of the United States of America. The preliminary detection of phytoplasmas was performed with scanning electron microscope where the presence of the pathogen was confirmed according to the morphological characteristics observed in the vascular tissues. The identification and characterization was performed by nested PCR amplifying the region of the 16S rRNA gene of 1.2 kb. The multiple alignment analysis of the nt sequences indicated a similarity of 98.6% with 'Candidatus Phytoplasma pruni'. The genetic signature 'CAAGAYBATKATGTKTAGCYGGDCT' was observed, establishing the identity of the phytoplasma species. Phylogenetic analyzes located the BCS isolate within the 16SrIII group..."

Master thesis

SLCuV, ‘Ca. Phytoplasma pruni’, 16SrIII CIENCIAS AGROPECUARIAS Y BIOTECNOLOGÍA CIENCIAS AGRARIAS FITOPATOLOGÍA FITOPATOLOGÍA