Biotecnología

 

Serological and molecular tools for strain discrimination of Citrus tristeza virus isolates from Nuevo Leon, Mexico

 

Herramientas serológicas y moleculares para la discriminación de aislamientos del virus tristeza de los cítricos del Estado de Nuevo León, México

 

María M. Iracheta–Cárdenas¹, Isidro H. Almeyda–León², Bayarm Cevik³, Charles L. Niblett⁴, Richard F. Lee⁵ and Mario A. Rocha–Peña^2,*

 

¹ Instituto de Biotecnología. Facultad de Ciencias Biológicas. Universidad Autónoma de Nuevo León. Pedro de Alba s/n. Cd. Universitaria. San Nicolás de los Garza, Nuevo León.

² Unidad de Investigación en Biología Celular y Molecular, INIFAP/UANL. Apartado Postal 128–F. 66450. Cd. Universitaria. 66450. San Nicolás de los Garza, Nuevo León, México. *Autor for correspondence: (mrocha@fcb.uanl.mx).

³ Faculty of Agriculture. Department of Plant Protection. Süleyman Demirel University. 32260. Isparta, Turkey.

⁴ Horticultural Sciences Department, University of Florida, P. O. Box 110780, Gainesville, FL 32611–0780 USA.

⁵ National Clonal Germplasm Repository for Citrus and Dates, USDA–ARS.1060 Martin Luther King Blvd. Riverside, California 92507. USA.

 

Received: September, 2009.
Approved: April, 2010.

 

ABSTRACT

Citrus tristeza virus (CTV) is an important issue to the Mexican citrus industry. CTV infected plants have been reported in 20 citrus producing states of the country as symptomless virus infections, even on plants grafted on the sensitive sour orange rootstock. The detection of severe CTV isolates is mandatory to reinforce the operation of the CTV campaign nationwide. In this study, 13 CTV isolates from Nuevo León and one from Tamaulipas states were characterized for activity against the strain discriminatory MCA13 monoclonal antibody, bi–directional (BD) PCR, and hybridization with specific DNA probes, with RT–PCR amplicons, of the p25 coat protein gene. Ten out of 13 CTV isolates from Nuevo León and the Tamaulipas isolate gave a positive reaction to the MCA13 monoclonal antibody. BD–PCR tests yielded a cDNA fragment of 300 bp characteristic of decline–inducing CTV strains with eight of 13 isolates from Nuevo León and the Tamaulipas isolate. Hybridization with strain group specific DNA probes showed reactivity of eight CTV isolates with Probe II associated with decline inducing CTV strains, and three CTV isolates with Probe VII, associated to mild CTV strains. As a conclusion, it was shown the usefulness of the MCA13 monoclonal antibodies, BD–PCR and the hybridization with strain group specific DNA probes (SGSP) for the discrimination of CTV isolates, and the discovering of severe CTV isolates in the state of Nuevo León. Both BD–PCR and hybridization with SGSP were more suitable for strain discrimination of CTV isolates, than the MCA13 monoclonal antibody. BD–PCR was able to detect mixtures of both mild and decline inducing CTV isolates in the sample; hybridization with SGSP was suitable for detecting either mild, decline inducing and stem pitting CTV isolates.

Key words: bi–directional PCR, DIBA, hybridization, MCA 13 monoclonal antibody.

 

RESUMEN

El virus de la tristeza (Citrus tristeza virus = CTV) es un problema de importancia para la industria citrícola mexicana. Se han reportado plantas infectadas por el CTV en 20 estados productores de cítricos del país, en forma de infecciones asintomáticas, incluso en plantas injertadas en el patrón hipersensible naranjo agrio. La detección de aislamientos severos de CTV es necesaria para reforzar la operación de la campaña de CTV en todo el país. En el presente estudio se caracterizaron 13 aislamientos del CTV del estado de Nuevo León y uno del estado de Tamaulipas por su actividad contra el anticuerpo monoclonal MCA13, PCR bi–direccional (BD) e hibridación con sondas específicas para el gen p25 de la proteína de cápside. Diez de 13 aislamientos del CTV de Nuevo León y el aislamiento de Tamaulipas dieron una reacción positiva al anticuerpo monoclonal MCA 13. Las pruebas BD–PCR dieron un fragmento de ADNc de 300 pares de bases característico de razas tipo decaimiento en ocho de los 13 aislamientos de Nuevo León y del aislamiento de Tamaulipas. En la hibridación con sondas ADN específicas, dieron reacción con ocho aislamientos (CTV) con la Sonda II asociada con linajes CTV del tipo decaimiento y tres aislamientos mostraron reactividad con la Sonda VII asociada con linajes de tipo débil. Como conclusión, se mostró la utilidad del anticuerpo monoclonal MCA13, del PCR bi–direccional y de la hibridación con sondas específicas de (SGSP) para la discriminación de aislamientos de CTV, así como el descubrimiento de aislamientos severos de CTV en el estado de Nuevo León. Tanto el PCR–BD y la hibridación con sondas específicas (SGSP) fueron más adecuados para la discriminación de la aislamientos de CTV, que el anticuerpo monoclonal MCA13. El PCR–BD pudo detectar mezclas de aislamientos débiles y del tipo decaimiento del CTV en una misma muestra; la hibridación con SGSP fue adecuada para detectar aislamientos CTV ya sea de tipo débil, tipo decaimiento y picado de tallo.

Palabras clave: PCR bi–direccional, DIBA, hibridación, anticuerpo monoclonal MCA 13.

 

INTRODUCTION

Citrus tristeza virus (CTV) is distributed worldwide and it is the causal agent of the most economically important viral disease of citrus (Rocha–Peña et al., 1995a). It has been documented (Garnsey et al., 2005) that CTV occurs in the field as a mixture of strains or isolates which differ in biological properties, such as symptoms in the field (Rocha–Peña et al., 1995a), reaction on indicator plants (Garnsey et al., 2005) and aphid transmissibility (Rocha–Peña et al., 1995b; Yokomi et al., 1994). This phenomenon frequently leads to apparent latent infections of diverse CTV isolates for many years, even in plants grafted on the sensitive sour orange rootstock without any noticeable disease symptoms (Bar–Joseph, 1978; Rocha–Peña et al., 1998). Over the years, the occurrence of unexpected declining CTV infected trees in the field may result if no phytosanitary measures are accomplished against the virus on a timely basis (Rocha–Peña et al., 1998).

Several serological and molecular approaches have been developed as an effort to differentiate CTV isolates (Niblett et al., 2000). These approaches include the use of the strain discriminatory MCA 13 monoclonal antibody (Permar et al., 1990), serological probes for orange stem pitting CTV isolates (Nikolaeva et al., 1998) single strand conformation polymorphism (SSCP) addressed to several CTV genes (Niblett et al., 2000), bidirectional PCR (Cevik et al., 1996), several hybridization approaches with specific group DNA probes (Cevik, 1997^[6]; Narváez et al., 2000), and several approaches using restriction analysis patterns with RT–PCR amplicons (Marques et al., 2006; Roy et al., 2003), among others.

In México, CTV occurs as symptomless virus infections, even in plants grafted on sour orange rootstock (Rocha–Peña et al., 2005); however, the occurrence of severe CTV isolates was determined by Silva–Vara et al. (2001). The citrus industry of México is considered to be at immediate risk of CTV epidemics because sour orange is the predominant rootstock throughout the country and the presence of the most efficient vector, Toxoptera citricida (Kirkaldy), commonly called the brown citrus aphid (Rocha–Peña et al., 2005; SAGARPA, 2006). The objectives of this study were to evaluate some of the molecular methods available and to determine whether some CTV isolates collected from Nuevo León and Tamaulipas states, México, are mild or severe.

 

MATERIALS AND METHODS

Thirteen CTV isolates collected from orange trees in the state of Nuevo León, (Silva–Vara et al., 2001) and one originally collected from the neighbouring state of Tamaulipas (González–Garza, 1983^[7]) (Table 1) were individually maintained in planta in at least two Mexican lime (c. aurantifolid) plants. Citrus samples were used either as fresh or dry tissue. Dry citrus tissue of type CTV isolates T36, T66 (declining, MCA+) and T55 (mild, MCA13–) from Florida (Permar et al., 1990; Rocha–Peña et al., 1995b) were used as positive controls, for decline and mild isolates.

Serological strain discrimination

Serological reactivity of citrus extracts from plants infected with CTV isolates was evaluated against the strain discriminatory MCA13 monoclonal antibody in dot–immunobinding assay (DIBA) (Rocha–Peña et al., 1991). The MCA13 monoclonal antibody, from Nokomis Corp. (Florida, USA), was used at a 1:5000 (v/v) dilution and goat anti–mouse IgG labeled with alkaline phosphatase (SIGMA A–3688) at 1:30000 (v/v). A duplicate of the membranes containing the extracts of CTV infected and healthy citrus samples were run aside by using the CTV specific rabbit C3 antibodies (0.2 mg mL^–1) (Iracheta–Cárdenas et al., 2008); goat anti–rabbit enzyme conjugate (Sigma A–8025) was used at 1:30000 (v/v) dilution.

Nucleic acid extraction

RNA extraction was performed according to the method of Pappu et al. (1993). Infected tissue (250 mg) was ground to powder with liquid nitrogen and homogenized with 500 μL of TES buffer [0.1 M Tris HCl, pH 8.0; 2 % SDS (w/v); 2 mMEDTA], 250 μL phenol, 250 μLof a solution of chloroform–isoamylic alcohol (24:1). Then it was heated at 70 °C for 5 min, and centrifuged at 5000 rpm in a clinical centrifuge with a swinging bucket rotor (CCSBR), at 4 °C during 5 min. The supernatant was then placed into a Sephadex G–100–50 column, the column was centrifuged again in a CCSBR at 1500 rpm during 4 min at 4 °C, and the extract was recuperated in a microcentrifuge tube.

Reverse transcription (RT) and polymerase chain reaction (PCR)

The RT–PCR components were run in a final volume of 25 μLas follows: 2.5 μLPCR mix 10X, 0.75 μLMgCl₂ (1.5 mM), 2.5 μLof dithiotreitol (0.1 M), 2.0 μL(25 picomols) of each CN119 (5'–AGATCTACCATGGACGACGAAAC AAAG–3') /CN120 (5'–GAATTCGCGGCCCGTCAAC GTGTGTTAAATTCC–3') primers, 1.0 μLAMLV reverse transcriptase (200 units), 0.5 μLTaq DNA polymerase (2.5 units), 0.5 μLRNasin, 5 μLof the viral RNA extract, 2.0 μLdNTP's (200 μM) and 6.25 μLof bidistilled water. The thermal programme was one cycle of 45 min at 42 °C, followed by 35 cycles of 1 min at 94 °C, 1 min at 55 °C, 1 min at 72 °C, and a final extension of 10 min at 72 °C. The combination of CN119/CN120 primers yields a product size of approximate 700 bp.

Bidirectional polymerase chain reaction

Bidirectional PCR (BD–PCR) was conducted according to Cevik et al. (1996). The protocol consists of one single two steps RT–PCR runs with two external (CN118 and CN 119) and two internal [CN218 (5'–TTTGGACTGACGTCGTGTT–3') and CN219 (5'–TTCGTCGACGACGACAGGTA–3')] set of primers. BD–PCR cocktail mixture and thermal program were conducted according to Cevik et al. (1996). The combination of CN218/CN120 and CN119/CN219 yield product sizes of approximate 300 and 400 bp, for decline inducing and mild CTV strains, (Cevik et al., 1996).

Hybridization with strain group specific DNA probes

Molecular hybridization was conducted according to Cevik, 1997) and Halbert et al. (2003) with a set of specific DNA probes that have unique sequences for a strain discrimination of CTV isolates. The unique sequences of these strain specific probes are the subject of U.S. Patent # 6140046 (USP, 2000). Probe 0 contains a nucleotide sequence conserved in the CP gene of all known strains of CTV and hence it is a universal probe for CTV. Probe I hybridizes with decline–inducing strains; Probes II, III, IV and V hybridize with different groups of stem–pitting strains from various geographical origins throughout the world; Probes VI and VII hybridizes with mild strains from Florida and from Orient countries; Probe VIII hybridizes with all mild strains (Nibblet et al., 2000). The cDNA from the well characterized T36 (decline on plants grafted on sour orange, seedling yellows), T30 (mild strains from Florida), B53, B185, B249 (decline on plants grafted on sour orange, seedling yellows, stem pitting on grapefruit or sweet orange or both), CTV isolates (Niblett et al., 2000) was used as control. The amplified PCR products of the p25 gene of each CTV isolate was blotted on nylon membranes and assayed by hybridization using the CTV strain group specific probes labeled at the 5' end with biotin to react with a strepavidin conjugated alkaline phosphatase for detection of the hybridized amplicons by chemiluminescence (Cevik, 1997).

 

RESULTS AND DISCUSSION

The serological test performed with the C3 rabbit polyclonal antibodies gave a positive reaction with all the CTV isolates evaluated. CTV isolates T36 and T66 (decline) and T55 (mild) from Florida used as positive controls, gave positive reactions with the C3 rabbit polyclonal antibodies also (Table 2). The tests performed with the specific strain MCA13 monoclonal antibody gave a positive reaction with CTV isolates MX01 through MX07, MX11, MX13, MX14, and with the decline CTV isolates T36 and T66 CTV from Florida. The MCA 13 monoclonal antibody gave a negative reaction with CTV isolates MX08, MX09, MX10, and MX12 from México and with the mild CTV isolate T55 from Florida. No positive reaction was obtained with healthy controls in either test (Table 2).

The RT–PCR yielded an expected 700 bp cDNA fragment for all the CTV isolates from México. The same 700 bp cDNA fragment was also obtained for the decline isolates T36, T66 and the mild isolate T55 CTV from Florida used as positive controls (data not shown). When the BD–PCR (Figure 1) was performed, a cDNA fragment of300 bp characteristic of decline of CTV isolates (MCA 13 + ) was obtained with isolates MX01 through MX08, MX13 and MX14, also, with Florida decline isolates T36 and T66 included as a control. A cDNA fragment of 400 bp cDNA characteristic of mild isolates of CTV (MCA 13—) was obtained with samples MX08, MX09, and MX14 from México and also with mild isolate T55 from Florida (Table 2).

The detection of both 400 and 300 bp amplicons in samples MX08 and MX14 can be interpreted as a mix–infection of both mild and decline inducing CTV isolates in the same sample. The occurrence of mix–infection of several CTV isolates is a common phenomenon in field samples (Brlansky et al., 2003) and has been reported with the use of BD–PCR (Cevik et al., 1996; Huang et al., 2004; Korkmaz et al., 2008).

The BD–PCR system for strain discrimination was developed on the basis of differences at position 371 of the p25 coat protein gene of MCA13 reacting ( + ) decline inducing or stem–pitting severe and mild (MCA13 —) CTV isolates (Cevik et al., 1996). Severe CTV isolates yield a 300 bp amplicons, while mild CTV isolates produce 400 bp amplicons (Cevik et al., 1996). The BD–PCR has been used in some extent for strain discrimination purposes of CTV isolates (Huang et al., 2004; Kormaz et al., 2008; Nibblet et al., 2000). There are some other RT–PCR based systems for detection of CTV isolates (Hung et al., 2000; Ruiz–Ruiz et al., 2009); however, the use of real–time PCR reported for strain discrimination of CTV isolates (Ruiz–Ruiz et al., 2009) is relatively expensive and involves several different regions in the CTV genome, other than the p25 coat protein gene. Likewise, the use of RT–PCR in a single step reported by Hung et al. (2000) do not discriminate between mild from severe isolates.

The tests conducted with strain group specific biotin–labeled DNA probes are shown in Table 3. All CTV isolates tested positive with the Probe 0 universal for all CTV isolates. Likewise, cDNA from T36, B53, B185, T3, B249, and T30, used as reference, reacted positively with their corresponding strain specific probes (Table 3). The CTV isolates MX02 through MX07, and MX10 reacted with Probe II. The CTV isolates MX08, MX11 and MX12 reacted with Probe VIII and CTV isolate MX 13 reacted with both Probe VI and VIII. CTV isolate MX14 reacted with Probe V. The Probe VII specific to CTV isolates from Asiatic countries was not included in this study.

According to these results, the reaction of samples MX02 through MX07, and MX10 from Nuevo León with Probe II identified the presence of decline inducing CTV isolates in the samples analyzed. The reaction of sample MX14 with Probe V indicated the presence of a stem–pitting isolate in the CTV sample originally collected in Tamaulipas.

The strain group specific biotin–labeled DNA probes were designed on the basis of differences in the nucleotide sequences of the coat protein p25 gene of a number of CTV isolates with a range of different biological properties and from diverse geographical origins (Cevik, 1997; USP, 2000). These have been used for strain discrimination purposes of CTV isolates by several research groups (Gen<j, 2004; Halbert et al., 2004; Herron et al., 2005). There has been another reported hybridization based system with cDNA specific probes for strain discrimination of CTV isolates (Narváez et al., 2000); however, this system was not available for comparison in this study.

The information generated from this study showed the usefulness of the MCA13 monoclonal antibody, the BD–PCR, and the biotin–labeled strain group specific cDNA probes to discriminate CTV isolates from México. These three methods were efficient to detect the presence of severe CTV isolates in Nuevo León (Table 2, 3 and Figure 1).

Nevertheless, the three methods were not 100 % coincident in results for all the CTV isolates tested, particularly between the MCA13 monoclonal antibody and the other two molecular methods tested. The BD–PCR yielded cDNA fragments of 300 and 400 bp for both MX08 and MX14 isolates (Figure 1, Table 2). This indicates a possible mix–infection in the original MX08 and MX14 samples with mild (MCA13—) and decline (MCA13 + ) CTV isolates being present. The detection of the 300 bp fragment in BD–PCR of the MX08 sample indicates that a severe isolate of CTV is also present. But, the lack of reactivity with the MCA13 can be attributed to a probable lower titer of this severe isolate, not detected by the serological assay. The higher sensitivity of BD–PCR, as compared to serological assay has been reported for the detection of CTV field isolates (Huang et al., 2004). This feature of potential failure to detect the presence of low titer severe decline inducing CTV isolates is a disadvantage of the MCA13 monoclonal antibody over the BD–PCR. The amplification of the 400 bp product indicates that a mild isolate of CTV is also present in the same MX08 sample. Likewise, CTV isolate MX10 performed as a MCA13— in the serological tests (Table 2), but reacted with Probe II (Table 3) which is specific for severe decline inducing CTV isolates (Cevik, 1997; Nibblet et al., 2000).

Both BD–PCR and hybridization with specific DNA probes were almost a 100 % coincident with the results obtained (Tables 2, 3).

The lack of a 100 % coincidence mostly with the use of the MCA13 antibody over the molecular methods has been reported, and it can be attributed to the higher sensibility of the molecular methods (Huang et al., 2004) and to the presence of mixed isolates of CTV in the original field sample after the passage through several citrus hosts (Nickel et al., 1996).

The reactivity of the MCA13 monoclonal antibody, along with the amplification of 300 bp cDNA fragments in the BD–PCR test (Figure 1, Table 2), and hybridization with Probe II with samples MX02 through MX08, MX11, MX13, and with Probe V by MX14 (Table 3) provide evidence of the presence of severe CTV isolates in the samples collected from Nuevo León and Tamaulipas. The detection of severe CTV isolates by molecular methods has also been documented for the states of Veracruz and Puebla by BD–PCR (Almeyda–León et al., 2007).

The finding of severe CTV isolates in the samples analyzed in this study, is of interest even in the absence of decline symptoms in field trees. The CTV has been a regulated pathogen in México since 1997 and up to 2006, and 4468 symptomless CTV infected plants were detected in the 20 citrus producing states (SAGARPA, 2006). Although the original field CTV infected plants in Nuevo León (Silva–Vara et al., 2001) and elsewhere in México (SAGARPA, 2006) have been removed from the field, the occurrence of symptomless severe CTV isolates should not be ignored. These findings reinforce the need for continuing the CTV campaign nationwide. It has been documented that latent severe CTV isolates infections can be present in a citrus industry for many years, and even decades without any noticeable evidence of disease symptoms, until the unexpected decline due to CTV occurs when efficient aphid vectors either increase their populations or become established in new locations (Bar–Joseph, 1978; Rocha–Peña et al., 1995a, 1998). The brown citrus aphid (Toxoptera citricida) and the melon aphid (Aphis gossypii) are the most important aphid vectors of CTV (Rocha–Peña et al., 1995a; Yokomi et al., 1994), and both species are present in México (Rocha–Peña et al., 2005; SAGARPA, 2006; Silva–Vara et al., 2001). The T. citricida transmits CTV isolates 6 to 25 times more efficiently than A. gossypii (Yokomi et al., 1994). Wherever the T. citricida has been introduced, severe decline strains of CTV have appeared, and in some instances, stem–pitting strains of CTV (Halbert et al., 2004; Rocha–Peña et al., 1995a, 1998).

 

CONCLUSIONS

It can be concluded the usefulness of the serological and molecular tools for the discrimination of CTV isolates. Both BD–PCR and hybridization with specific DNA probes were more suitable for strain discrimination of CTV isolates, than the MCA13 monoclonal antibody. The BD–PCR was able to detect mixtures of mild and severe CTV isolates, while hybridization with specific DNA probes was suitable for detecting either mild, decline inducing and stem pitting CTV isolates. In the state of Nuevo León severe CTV isolates were discovered.

 

ACKNOWLEDGMENTS

This research had support by Fundación Produce Nuevo León, A. C. grant No. 1322, CONACYT–SIREYES Project No. 032/95, both Institutions from México, and by the USDA/University of Florida Specific Cooperative Research Agreement and the Binational Agricultural Research and Development with Israel (BARD). Journal Series Number INIFAP/CIRNE/A–447.

 

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NOTES

⁶ Cevik, B. 1997. Molecular differentiation of strains of Citrus tristeza virus using the coat protein gene sequences. MSc. Thesis. University of Florida. Gainesville. 112 p.

⁷ González–Garza, R. 1983. Identificación y control de las virosis de los cítricos en los estados de Nuevo León y Tamaulipas. Informe de Labores. CAEGET/INIA/SARH. Convenio Cooperativo de Investigación en Virología Vegetal INIFAP–UANL. Monterrey, Nuevo León, México (Mimeo). 175 p.