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Ciencia e investigación agraria

versión On-line ISSN 0718-1620

Cienc. Inv. Agr. vol.39 no.2 Santiago ago. 2012

http://dx.doi.org/10.4067/S0718-16202012000200010 

Cien. Inv. Agr. 39(2):339-346. 2012 www.rcia.uc.cl

CROP PROTECTION
RESEARCH NOTE

 

Molecular detection and classification of the phytoplasma that causes purple top in potatoes (Solanum tuberosum) in the State of Mexico

Detección y clasificación molecular del fitoplasma causante de la punta morada en papa (Solanum tuberosum), en el Estado de México

 

Ana T. Gutiérrez-Ibáñez1, Antonio Laguna-Cerda2, Reina Rojas-Martínez3, Ramiro González-Garza4, and Martha L. Salgado-Siclán2

1Programa Doctoral en Ciencias Agropecuarias y Recursos Naturales,
2Facultad de Ciencias Agrícolas, UAEMex, Campus Universitario Toluca-Ixtlahuaca, Km. 15 entronque al Cerrillo Toluca. Estado de México
CP 50200, México.
3Colegio de Posgraduados, Carretera México-Texcoco km 36.5 Montecillo. Texcoco CP 56230, México.
4BioCiencia S.A. de C.V. Agustín Melgar 2317 Nte, colonia Reforma CP 64550, Monterrey, Nuevo Leon, México.


Abstract

The Mexican potato (Solanum tuberosum L.) crop is affected by several diseases, including purple top (PPT). PPT is caused by phytoplasma and is transmitted by insects in the Cicadellidae family. In severe infections, this pathogen can cause yield losses of nearly 100%. The objective of this research was to identify and classify (at a molecular level) the causal agents of PPT. Potato plants were sampled in the state of Mexico (at Tenango del Valle, Zinacantepec, Atlacomulco and Donato Guerra) in the spring and summer of 2006. Approximately 40% of the plants tested positive for phytoplasma. The presence of plant DNA was determined by amplifying an endogenous gene with the 16s-1/16s-2 primers. To detect phytoplasma, the P1/P7 universal primers were used for PCR, the R16F2n/R16R2 primers were used for nested PCR, and the AluI, HpaII, and KpnI enzymes were used to perform RFLPs. The results indicated that the phytoplasma associated with the PPT disease belongs to the aster yellows (16Sr1) group and the I-B subgroup. The same phytoplasma was found in all three regions of study. When the sequence of the amplified fragment was compared with the registered Gene Bank (NCBI) sequences (using the BLAST package) a homology of 99% was found with the aster yellows (16Srl) group, "Candidatus Phytoplasma asteris".

Key words: Phytoplasma, potato diseases, potato purple top.


Resumen

Actualmente el cultivo de la papa (Solanum tuberosum L.) se ve afectado por la enfermedad conocida como punta morada (PMP), la cual es causada por un fitoplasma transmitido por insectos vectores de la familia Cicadellidae, ocasionando pérdidas de hasta un 100%. El objetivo de esta investigación fue la identificación y clasificación molecular del fitoplasma causante de dicha enfermedad. Durante la primavera y verano de 2006 se hizo un muestreo dirigido a plantas con síntomas en los municipios más importantes para la producción de papa en el Estado de México: Tenango del Valle, Zinacantepec, Atlacomulco y Donato Guerra, resultando el 40% de ellas positivas a fitoplasmas. Se verificó la presencia del ADN vegetal mediante la amplificación del gen endógeno, con los iniciadores 16s-1/16s-2. Para la detección de fitoplasmas se realizó un PCR - directo, con los partidores universales P1/P7, una PCR anidada con los partidores R16F2n/R16R2, y RFLP's del producto de PCR con las enzimas Alu I, Hpa II, KpnI. Se determinó que el fitoplasma, asociado con PMP pertenece al grupo aster yellows (16Sr1) subgrupo I-B, siendo el mismo fitoplasma para las tres regiones en estudio. Al comparar la secuencia del fragmento amplificado con las secuencias registradas en el Gen Bank (NCBI) y con el paquete BLAST, se encontró homología del 99% con el grupo del amarillamiento del aster (16Srl), "Candidatus Phytoplasma asteris".

Palabras clave: Fitoplasma, enfermedades de la papa, punta morada de la papa.


 

Introduction

Potato is the fifth main crop in Mexico (following wheat, rice, maize and barley). Approximately 67,000 ha are planted through seven regions that include 23 states. Approximately 1.35 million tons of potatoes are produced, which satisfies the domestic consumption demand. This surface covers different regions. The first region in which high-tech agriculture occurs is composed of valleys in which irrigation is used or a stable rain season occurs. This area makes up approximately 17, 000 hectares of the total potato plantations in the country, including those in the State of Mexico. The State of Mexico is third in potato production, with a planted area of 4,626.30 ha (SIAP, 2011). This potato crop presents different phytosanitary problems that have affected potato production. These problems are associated with the use of uncertified tuber seed in more than 60% of the total area of cultivated potatoes. In recent years, diseases caused by phytoplasmas that are transmitted by vector insects in the Cicadellidae family (such as potato purple top (PPT) and potato hair sprout (PHS)) have restricted potato production. These phytoplasmas affect between 70 and 100% of the national potato production. Overall, the damage varies from 20 to 100%, depending on the degree of infection (Cadena-Hinojosa, 1996). PPT occurs worldwide and is most likely found in some Asian countries. Recently, the occurrence of PPT has decreased in the United States; however, the cause of this decrease remains unknown (Maramorosh, 1998). In Mexico, PPT is one of the main pathogens for potato (Cadena-Hinojosa, 1996; Zavala-Quintana, 2002).

In most potato producing zones, PPT impacts potato quality and yields. Because of the necrosis induced in the interiors of the diseased tubers, PPT hinders or prevents the commercialization of potato crops for fresh consumption or for use in fried flakes. Because potato seeds are sold in Mexico before they sprout, the percentage of seeds that are infected remains unknown. PPT may cause economic losses when infected seeds are planted, which results in a population density that is lower than the target density (Zavala-Quintana, 2002). The observed symptoms vary depending on factors such as variety, the affected organ in the plant, the phenological state of the crop, the infection origin and time of onset, and the prevailing environmental conditions (Martínez-Soriano and López-Flores, 1999).

The plants from infected tubers may manifest the disease 20 days after emerging, depending on the nutrition and humidity conditions. The best-known symptoms include premature plant wilting, vascular necrosis in the stems, short in-ternodes, axillary sprouts that often produce air tubers, curling of recently emerged leaves, and purple leaves (which the disease is named for). The tubers from infected plants are small, low category tubers, and they often present different degrees of internal browning.

The relationship between PPT and the production of fine tuber sprouts is mentioned in previous reports (Nagaich and Giri, 1973; Martínez-Soriano et al, 1997). However, during harvest, the tubers that produce fine sprouts are indistinguishable from normal tubers based on their external and internal appearance. These tubers may only be identified by their elongated and weak sprouts, which occur weeks after harvest. The symptoms of this "ball of twine" disease may be gradual and range from the production of weak sprouts to a total lack of sprouting. Sprouts deficient in chlorophyll are also observed and are identified by white threads, which die and lead to seed necrosis. In some cases, supposedly healthy sprouts allow the emergence of an apparently healthy plant; however, the plant continues to infect and inoculate insects for the dispersion of PPT. These apparently healthy plants produce infected tubers that do not germinate or that germinate deficiently if they are not used as seed (Martínez-Soriano et al, 1997).

To date, the causal agent of PPT has not been identified. Different reports mention that PPT may be associated with the presence of fungi, phytoplasmas (Cadena-Hinojosa, 1996), viruses, or bacteria. In addition, PPT may be caused by the injection of a toxin by Bactericera cockerelli (Sulc.) insects during feeding. However, this toxin has not been isolated, and the metabolic disorder could be due to the presence of metabolites that are produced and introduced by the nymphs of the insect when feeding (Zavala, 2002).

The objective of this work was to identify and classify the phytoplasma that causes PPT in four municipalities that are important for potato production in the State of Mexico (Tenango del Valle, Zinacantepec, Atlacomulco and Donato Guerra).

Materials and methods

Samples were collected from 100 Alpha variety potato plants that showed PPT symptoms in the spring and summer of 2006. This collection was performed based on previous samplings by the State of Mexico's Comité Estatal de Sanidad Vegetal. Forty of the samples were collected in San Miguel Balderas (Tenango del Valle), 40 were collected in San Juan de las Huertas and San José Contadero (Zinacantepec), 10 were collected in the municipality of Atlacomulco, and 10 were collected in Donato Guerra (Table 1).

Samples from plants with purple-colored leaflets, flat stems (fasciation), air tubers, apical curling of leaves, or shortening of internodes and roots were collected for molecular analysis. The samples were processed in the Centro de Investigación de Estudios Avanzados en Fitomejoramiento (CIEAF) phytopathology laboratory in the Faculty of Agricultural Sciences at the Universidad Autónoma del Estado de México.

Plant DNA was collected with a commercial extraction kit (Plant DNAzol Reagent® (Invi-trogenTM)). A 0.8% agarose gel was dyed with ethidium bromide (0.1 μg mL-1) to demonstrate the integrity of the extracted DNA. The DNA bands were visualized with a GVM20 Syngene UV light transilluminator. In addition, the quality and concentration of the DNA were measured with an Eppendorf biophotometer. The extracted DNA was diluted in sterilized distilled water to obtain a final concentration of 20 ng μL-1. Finally, the diluted DNA was stored at -20 °C prior to use (Dellaporta et al., 1983).

Polymerase Chain Reaction (PCR)

The DNA from each of the samples was subjected to a PCR reaction. The PCR reaction was performed with 16s-1/16s-2 primers that are specific for chloroplasts (endogenous gene). This method amplifies a region of the 16S ribosomal gene that is specific for the chloroplast and generates a 315 bp product (Trejo-Saavedra, 2002). This process was used to determine if the vegetal material was free from inhibitor substances. The final PCR reaction concentrations were as follows: 2.5 mM of the PCR 1X buffer, 2.5 mM of MgCl2 and dNTPs, 10 pmol of each primer, and 1 unit of Taq DNA Polimerase. Each reaction mixture contained 80 ng of DNA and had a final volume of 25 μL. The samples were amplified in an automatic PTC-100 MJ Research thermal cycler programmed to perform the following steps in sequence: 1) a denaturalization cycle at 94 °C for 90 s, 2) 35 denaturalization cycles at 94 °C for 40 s, 3) annealing at 55 °C for 40 s, 4) polymerization at 72 °C for 60 s, and 5) a final extension at 72 °C for 180 s. The PCR products were analyzed by electrophoresis in 1.5% agarose gels that were dyed with ethidium bromide (0.1 μg mL-1) and observed by a GVM20 Syngene UV light transilluminator. The results were documented with the Gene Tools 3.1 software.



The obtained fragments were compared with the Ladder 100 pb (Invitrogen ®) molecular weight marker (Trejo-Saavedra, 2002).

Phytoplasma is not usually detected by direct PCR in hosts that have low phytoplasma concentrations. Thus, PCR-nesting was used for phytoplasma detection. The PCR-nesting primers were used to amplify a region of the RNAr 16S (Gundersen and Lee, 1996; Lee et al., 1993) gene. The oligonucleotides or universal primers for phytoplasma (P1/P7) were used in the first PCR reaction, which amplified an 1800 bp fragment (Smart et al, 1996). This reaction was performed at the previously described concentrations with a final volume of 50 μL. DNA from coconut plants (Cocos nucifera L.) infected with phytoplasmas that cause lethal yellowing was used as a positive control. The DNA from a healthy plant was used as a negative control. The procedure was performed for 35 cycles as follows: 1) denatur-ation at 94 °C for 2 min, 2) annealing at 54 °C for 1 min, and 3) polymerization at 72 °C for 5 min. The direct PCR products were diluted (1:30) with sterile deionized water (free of DNAses and ARNses (GIBCO®)). These dilutions were used to mold DNA for the PCR-nesting. The previously described conditions were used for 30 of the following cycles: 1) denaturalization at 94 °C for 5 min, 2) annealing at 52 °C for 1 min, and 3) polymerization at 72 °C for 2 min. The R16F2n/ R16R2 primers were used to amplify a 1200 bp fragment (Lee et al, 1993).

The final products from both reactions were run in agarose gel at 1 and 1.3%, respectively. The lengths of the obtained fragments were compared with the Ladder 100 pb (Invitrogen ®) molecular weight marker.

Amplifiedribosomalproduct (RFLPs) restriction analysis

Following the manufacturer's instructions, the PCR products obtained with the R16F2n/R16R2 primers were subjected to digestion with the following endonuclease restrictions (suggested by Lee et al., (1998): AluI, KpnI and HpaII (Invitrogen®). Five μL of PCR-nested product were added to each reaction with 2 μL of buffer and 1 μL of each enzyme. The final volume was 20 μL. This mixture was subjected to 2 h of incubation at 37 °C. The restriction fragments were separated by electrophoresis in a 3% agarose gel dyed with ethidium bromide.

The electrophoretic profile of the phytoplasma was compared with the previously reported patterns found in the literature (Lee et al., 1998).

Sequencing, analysis and comparison of sequences

The PCR products from the R16F2n/R16R2 primers were sent to the Laboratory of Molecular Biochemistry (Unidad de Biología and Prototipos, FES-Iztacala, of Universidad Nacional Autónoma de Mexico) for sequencing. The obtained sequences were compared with the sequences reported in the Bank of Genes (Genbank) of the National Center for Biotechnology Information (NCBI) (Centro Nacional para la Información en Biotecnología). The BLAST-N program was used on site to determine the existing genetic identity of the sequences with respect to those previously reported and deposited in the bank.

Results and discussion

Symptoms present in the 100 plant samples included flattening of the plant (reduced size and length of internodes), the presence of purple-colored leaflets, stem fasciation, sprouting of air tubers, and apical curling of the leaves (Figure 1).

Obtaining good quality DNA and detecting phy-topathogens with molecular techniques are highly important (Leyva-López et al., 2002). In all cases, the collection of total DNA by the Plant DNAzol Reagent® (Invitrogen TM) method provided good results. In addition, the electrophoresis showed a well defined band for all samples. The concentration and purity of DNA (determined by the biophotometer) were adequate for PCR (1.7-2.0); however, these values indicated contamination by proteins and RNA (indicated by the higher values) (Sambrook et al, 1989).



The absence of PCR inhibitors was verified by amplification of the 16S endogenous gene (Trejo-Saavedra, 2002) (Figure 2). For the positive control (C. nucifera) (and for the problem samples), the 315 bp fragment was amplified, and it indicated that the DNA was free from inhibitor PCR substances (such as ethanol, phenols, nucleases and other degrading compounds) (Ausubel et al., 1999). The negative control did not show amplification.

Detection ofphytoplasmas

Because amplification by direct PCR (using the two P1/P7 primers) was not observed, PCR-nesting was conducted for all analyzed samples (except for the positive control). The positive control DNA was extracted from infected coconut plants and characterized by high phytoplasma titers. Furthermore, amplification was not observed in the negative control.



A 1200 bp fragment was observed in the nested PCR for the positive control and the problem samples. Therefore, phytoplasma was detected in 40% of the plants analyzed (Figure 3). The negative remains could be associated with other pathogens, such as fungi, viruses or non cultivable bacteria (such as Candidatus Liberibacter solanacearum) (Secor et al., 2009).



The 16S DNAr sequence restriction fragment polymorphism length analysis with the R16F2n/ R16R2 primers and with the digestions with AluI, HpaII and KpnI enzymes (Figures 4 and 5) showed persistent patterns in the analyzed samples. The phytoplasma was associated with the aster yellows (16SrI) group and may be located in the I-B subgroup (according to the updated molecular classification of phytoplasmas by Seemüller et al. (1998)).

These results confirmed the presence of phytoplasma in the four study zones. Overall, 31% of the positive samples were collected from Tenango del Valle, 43% were collected from Zinacantepec, 19% were collected from Atlacomulco and 7% were collected from the municipality of Donato Guerra (Table 2).



The phytoplasma was localized indistinctively in the different organs analyzed (except in the root, which is in contrast with the findings of Almeyda et al. (2001). Previous authors determined the sensibility and consistency of the detection of this pathogen in small roots. The phytoplasma was detected in all of the sampled areas, with the highest number of positive samples occurring in the leaflets (70%), followed by the air tubers (25%) and the stems (5%). The phytoplasma was not detected in the roots.




The sequences obtained from the amplified fragment with the R16F2n/R16R2 primers were compared with the sequences reported previously in the GenBank data bank. The analysis of these sequences showed a 99% homology with the 16SrI (aster yellows) group (number of access: AF329315) (Lee et al., 1998; Seemüller et al, 1998; Leyva-López et al, 2002).

The similarity among the groups formed by direct PCR and by PCR-nesting and the patterns generated by the restriction enzymes supported the RFLP analysis, which characterizes and differentiates at a group level (Lee et al, 1998). These results correspond with the previous results from Cadena (1993) and Leyva et al. (2002), regarding phytoplasmas in Mexico.

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Received June 1, 2010. Accepted March 15, 2011.

Corresponding author: atarini@uaemex.mx

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