TRABAJO ORIGINAL
USING A RIBOSOMAL PROBE FOR THE DETECTION OF THE PHYTOPLASMA ASSOCIATED WITH ´BUNCHY TOP SYMPTOM OF PAPAYA´ (BTS) IN PLANT AND INSECT HOST
USO DE UNA SONDA RIBOSOMAL PARA LA DETECCIÓN DE FITOPLASMA ASOCIADO CON LA ENFERMEDAD DEL SÍNTOMA DEL COGOLLO ARREPOLLADO (BTS) EN PLANTAS E INSECTOS HOSPEDANTES
Y. Arocha*, R. Almeida** and P. Jones*** ]]>
*National Centre for Animal and Plant Health (CENSA), Havana, Cuba. E-mail: yaimaarocha@yahoo.es
ABSTRACT
Recently, a phytoplasma of the 16SrII, Candidatus Phytoplasma aurantifolia group was associated with Bunchy Top disease (BTD) in eastern Cuba. Total DNA from more than 200 plant and insect leaf samples surveyed during 2005 and infected by the 16SrII phytoplasma was indexed by a non-radioactive nucleic acid hybridization assay (nrNAH). Phytoplasma 16S rDNA PCR products of selected samples were purified, labeled with alakaline phosphatase and used as a probe for the detection of such phytoplasma by the system of direct alkaline phosphatase labelling and chemiluminiscent detection (AlkPhos, Amersham LIFE SCIENCE, UK). The nrNAH assay detected the BTD phytoplasma in both papaya plants and Empoasca papayae Oman. The probe yielded hybridization signals reacting with reference controls and samples infected with the BTD phytoplasma. The nrNAH is considered a valuable diagnostic tool for the national phytosanitary surveillance, seed certification and breeding programs and a pathway to develop further specific assays.
Key words: phytoplasma; papaya; Empoasca papayae; Candidatus Phytoplasma aurantifolia; non-radioactive nucleic acid hybridization
RESUMEN
Recientemente, el grupo fitoplásmico 16SrII, Candidatus Phytoplasma aurantifolia fue asociado con la enfermedad similar al cogollo arrepollado, Bunchy Top disease (BTD) en el Este de Cuba. El ADN total de más de 200 muestras de plantas e insectos se muestreó durante el 2005, y las que resultaron infectadas por el fitoplasma BTD, fueron evaluadas mediante un ensayo de hibridación de ácidos nucleicos no radioactiva (nrNAH). Los productos de PCR correspondientes al ARN ribosomal 16S de muestras seleccionadas se purificaron, marcaron con fosfatasa alcalina y se utilizaron como sonda para la detección de este fitoplasma mediante el sistema de marcaje directo con fosfatasa alcalina y detección quimioluminiscente (AlkPhos, Amersham Life Science, UK). El ensayo nrNAH detectó el fitoplasma BTD tanto en plantas de fruta bomba como en Empoasca papayae Oman. La sonda arrojó señales de hibridación al reaccionar con los controles de referencia y las muestras infectadas con el fitoplasma BTD. El ensayo nrNAH se considera una valiosa herramienta diagnóstica para los programas de vigilancia fitosanitaria, certificación de semilla y mejoramiento genético, y una vía para desarrollar futuros ensayos específicos. ]]>
Palabras clave: fitoplasma; papaya; Empoasca papayae; Candidatus Phytoplasma aurantifolia; hibridación de ácidos nucleicos no radioactiva
INTRODUCTION
Papaya (Carica papaya L.) is widely cultivated in Cuba for the national consumption and export, with a production between 55 and 60000 t per year (5). It is distributed throughout the country representing 19% of the total of fruits produced, being Maradol Roja, the main variety (3).
Papaya is affected by various phytoplasma diseases (16) like mosaic and yellow crinkle associated with 16SrII group (Candidatus Phytoplasma aurantifolia) and dieback, with 16SrXII group (Stolbur).
Phytoplasmas present particular problems in the pursuit of Koch's postulates to establish them as the cause of a disease as they cannot be cultured in vitro (21). Therefore, molecular methods are the most feasible for their detection, identification and characterization in plant and insect vector hosts (1, 7, 9, 20).
Bunchy Top disease (BTD) was first reported in Cuba (3). Plants affected with the disease have shown a mix of symptoms similar to those caused by dieback, yellow crinkle and mosaic phytoplasmas in Austalia (16, 17), as well as those caused by a rickettsia associated with BTD in Puerto Rico (13), all of them related to losses over 70% (28). Although losses caused by BTD disease have not been yet quantified, the disease is widespread throughout all the provinces of the country (6). No vectors of such diseases have been identified, except the leafhopper Orosius argentatus Evans, which has been thought to be involved in epidemic outbreaks of dieback disease (28). However, Empoasca papayae Oman has been identified as the leafhopper candidate to vector BTD disease in Cuba (5).
Nucleic Acid Hybridization assays (NAH) have been widely used to differentiate phytoplasma species (4), being considered one of the most accepted methods for the diagnosis of phytoplasma diseases. There is a high tendency to use non radioactive nucleic acid hybridization methods (nrNAH) due to their research biosafety and technological advantages over PCR assays like minimizing contamination and speeding up the sample analysis (4, 33).
Recently, an nrNAH assay has been developed in Cuba for the generic diagnosis of phytoplasma diseases (4) by using a phytoplasma 16S ribosomal DNA (16S rDNA) probe. Regarding the potential menace of BTD for the Cuban papaya agriculture, it is required to evaluate the feasibility of this system for the field-scale diagnosis of the BTD phytoplasma in both plant and insect material. ]]>
In this paper, leaf and insect samples surveyed during 2005 and carrying the BTD phytoplasma were used to obtain a probe for the optimization of a non-radioactive nucleic acid hybridization for the detection of the phytoplasma associated with BTD in Cuba.
MATERIALS AND METHODS
Plant, insect material, and reference controls.
Total DNA from 230 papaya plants with (177) and without (53) BTD symptoms and 67 adult species of E. papayae leafhoppers, previously collected from papaya plantations of Guantánamo, Santiago de Cuba, Holguín, Camagüey and Granma, and identified as infected by the 16SrII phytoplasma (6) was evaluated by nrNAH. Phytoplasma DNA from Faba Bean phyllody (FBP, group 16SrII, Candidatus Phytoplasma aurantifolia), Sweet potato little leaf (SPLL, group 16SrII, Candidatus Phytoplasma aurantifolia) and Green Valley X (GVX, 16SrIII group, Candidatus Phytoplasma pruni) were used as reference controls.
Development of the ribosomal phytoplasma probe.
Two samples from each plant species and E. papayae were selected and evaluated by more than five repetitions of a nested PCR (nPCR) assay with generic phytoplasma primers P1 (14) and P7 (29) that amplify the conserved region of the 16S r DNA using a programmable thermocycler (MJ Research) and following PCR conditions previously described (5).
PCR products were purified from a 1% agarose gel according to the manufacturer's specifications (QIAquick Gel Purification kit (QIAgen, UK). Concentration of the purified DNA was quantified by a nanospectrophotometer (NanoDrop, UK) at 260 nm. Five mL of the PCR product (20 ng/mL) were llabelled as described by the kit of direct labelling of DNA probes with alkaline phosphatase and chemioluminiscent detection with CPD-Star (AlkPhos, Amersham Life Science, UK).
Nucleic acid hybridization analysis.
Ten microliters of samples and control DNAs were denatured at 100°C, during 5 minutes with denaturing solution (Sodium hydroxide, 0.5M NaOH and Sodium chloride, 1.5M NaCl) and neutralized with neutralizing solution (1M Tris-HCl, pH 8.0, and 0.5M NaCl) (4). DNAs were blotted into a nylon membrane (Hybond NX+, Amersham), previously treated with 2% Sodium Dodecyl Sulfate (SDS) during 10 minutes. ]]>
DNAs were fixed to the membrane at 80°C, during 2 hours (4). Hybridization was performed at 40°C. Both low and high stringency washes were performed at 40°C and room temperature, respectively, for 15 minutes each and using wash solutions previously described (4).The hybridization signal was detected by using the chemioluminiscent detection of the hybridization signal with CPD-Star, according to the manufacturer's instructions (AlkPhos, Amersham LIFE SCIENCE).
RESULTS AND DISCUSSION
Very well defined and strong hybridization signals were yielded by 172/177 symptom papaya samples, 37/53 asymptomatic papaya samples, and 63/67 E. papayae samples (Figure 1).
The development of NAH signals by 97.1% of symptomatic samples suggests the robustness of the nrNAH assay as previously described (4) and corroborates the association of BTD with phytoplasmas extending the information of previous reports (3, 5). The development of NAH signals indicates that 40°C is the optimal hybridization temperature for the detection of the BTD phytoplasma in both papaya plant and E. papayae. Reference controls yielded similar NAH signals extending the use of this ribosomal probe for the detection of phytoplasmas of 16SrIII group, which is the most related phylogenetically to 16SrII phytoplasma group (5) where BTD phytoplasma belongs to. This points the need to identify highly variable genomic regions to achieve the specific detection of the BTD phytoplasma.
The 16SrII group has representatives in South-East Asia, South Pacific, Africa, Arabian Peninsula, Europe (Italy), Australia, and America (12) in many different plant species including citrus, peanut, potato, sweet potato, alfalfa, cacti, apple, faba bean, soybean, weeds (15, 23, 25, 26, 27, 34), and also includes two important diseases of papaya: papaya yellow crinkle (PYC) and papaya mosaic (PM) (16). However, as far as we know, it is the first record of 16SrII phytoplasma group in papaya in America and the Caribbean.
Phytoplasmas seem to have a limited distribution correlated to the geographic region (24). The 16SrII group along with members of 16SrXI, Candidatus Phytoplasma oryzae do appear to be restricted to the South-East Asia region (18, 19, 20, 22, 24, 30).
The phytoplasma associated with an original plant host can become dispersed and re-distributed throughout a wide area by the exchange of germplasm in the form of seeds and plants, which along with the potential of exotic insects to vector local phytoplasma pathogens can cause changes in the balance between disease and epidemic (24). The presence of phytoplasmas in coconut embryos (11), in vitro grown seedlings of alfalfa with witches' broom (23), symptomatic seedlings of wild carnation (31), as well as tomato seedlings and lime plantlets (8), leads to hypothesise that the BTD phytoplasma could have originally been imported through seed exchange, a route which is currently under investigation.
Latent infections of phytoplasma infections habitually occur for perennial and long cycle crops (2, 32). Phytoplasmas multiply and move throughout the plant during a period of incubation, where they can be detected in symptomless plants (10). Symptom expression may occur when the infected plant is under unfavourable growth conditions or other stress factors (32). ]]>
The study of BTD in the eastern Cuba yielded a 69.8% of BTD latent infection. Therefore, asymptomatic papayas are an escape route for the 16SrII phytoplasma, and may play an important epidemiological role in spreading the disease in the field. It is an important element for the future design of practices for BTD management. Further studies will be required to understand the genetic mechanisms of the BTD phytoplasma in symptomless papaya and the environmental factors involved. The fact that the nrNAH assay detects phytoplasma DNA at early stages makes this assay feasible to introduce in the seed certification and breeding programs throughout the country.O. argentatus has been identified as a putative candidate for transmission trials in Australia (28), although the identification of leafhopper vectors of papaya phytoplasmas is still under investigations. The BTD phytoplasma was detected in 94% of E. papayae collected from BTD affected papaya fields of eastern Cuba. These findings point to E. papayae as the potential vector of BTD disease in Cuba remaining as a target for future transmission studies. It also means a high constraint for the national papaya production as the apparently healthy papayas might be latent phytoplasma reservoirs from where vectors can acquire and spread it. Therefore, it is urgent to consider the control of E. papayae populations as part of the management of BTD (21, 24).
As far as we know, this is the firs report of using a ribosomal phytoplasma probe through an nrNAH assay for the detection of a phytoplasma associated with a papaya disease in America and the Caribbean. The ribosomal probe allows detecting the phytoplasma associated with BTD in both papaya plants and E. papayae, providing a valuable tool to support diagnosis purposes for the national quarantine, phytosanitary surveillance, seed certification and breeding programs. In addition, it is a start point to improve the nrNAH assay for the specific detection of such phytoplasma using highly variable genomic regions. On the other hand, the identification of alternative BTD hosts and E. papayae as the potential vector is a crucial element for the improvement of the BTD management in Cuba.
REFERENCES
1. Angelini E, Squizzato F, Lucchetta G, Borgo M. Detection of a phytoplasma associated with grapevine flavescence dorée in Clematis vitalba. Eur. J Plant Pathol. 2004;110:193-201.
2. Aljanabi S, Parmessur Y, Moutia Y, Saumtally S,Dookun A. Further evidence of the association of a phytoplasma and a virus with yellow leaf syndrome in sugarcane. Plant Pathol. 2001;50:628-636.
3. Arocha Y, Horta D, Peralta EL, Jones P. First report on molecular detection of phytoplasmas in papaya in Cuba. Plant Dis. 2003;87:1148.
4. Arocha Y, Horta D, Peralta EL, Jones P. Development of a non radioactive methodology for the generic diagnostic of phytoplasmas in Cuba. Rev. Protección Veg. 2004;19(2):118-122.
5. Arocha Y, López M, Piñol B, Fernández M, Picornell B, Almeida R, et al. `Candidatus Phytoplasma graminis' and `Candidatus Phytoplasma caricae', two novel phytoplasmas associated with diseases of sugarcane, weeds and papaya in Cuba. Int. J Syst. Evol. Microbiol. 2005;55:2451-63.
6. Arocha Y, Piñol B, Picornell B, Almeida R, Jones P. First report of the 16SrII (Candidatus Phytoplasma aurantifolia) group associated with a bunchy-top disease of papaya in Cuba. Plant Pathol. 2006;55:821.
7. Bai X, Zhang J, Ewing A, Miller S, Radeck A, Shevchenko D, et al. Living with genome instability: the adaptation of phytoplasmas to diverse environments of their insect and plant hosts. J Bacteriol. 2006;188(10):3682-3696.
8. Botti S, Calari A, Bertaccini A.Phytoplasma infection through seed transmission: further observations. In: proceedings of 16th International Organization of Mycoplasmology Congress, St. John's College, Cambridge, Jul 9-14; 2006.
9. Christensen N, Nicolaisen M, Hansen M, Schulz A. Distribution of Phytoplasmas in Infected Plants as Revealed by Real-Time PCR and Bioimaging. Mol. Plant-Microbe Interactions. 2004;17(11):1175-84.
10.Constable F, Gibb K, Symons R. Seasonal distribution of phytoplasmas in Australian grapevines. Plant Pathol. 2003;52:267-76.
11.Córdova I, Jones P, Harrison N, Oropeza C. In situ detection of phytoplasma DNA in embryos from coconut palms with lethal yellowing disease. Mol. Plant Pathol. 2003;4(2):99-108.
12.Davis M, Jacobson S, De La Rue S, Tran-Nguyen L, Gunua T, Rahamma S. Phytoplasma disease surveys in the extreme north of Queensland, Australia, and the island of New Guinea. Aust. Plant Pathol. 2003;32:269-77.
13.Davis M, Ying Z, Brunner B, Pantoja A, Fewerda F. Rickettsial relative associated with Papaya Bunchy Top disease.Current Microbiol. 1998;36:80-84.
14.Deng S, Hiruki C. Amplification of 16S rDNA genes from culturable and non culturable mollicutes. J. Microbiol. Methods. 1991;14:58-61.
15.Gibb K, Padovan A, Mogen B. Studies on sweet potato little leaf phytoplasma detected in sweet potato and other plant species in northern Australia. Phytopathology. 1995;85:169-74.
16.Gibb K, Persley D, Schneider B, Thomas J. Phytoplasmas associated with papaya diseases in Australia. Plant Dis. 1996;80:174-178.
17.Gibb K, Schneider B, Padovan A. Differential detection and generic relatedness of phytoplasmas in papaya. Plant Pathol. 1998;47:323-332.
18.Griffiths H, Wayne A, Smart C, Davis R. The phytoplasma associated with ash yellows and lilac witches' broom: `Candidatus Phytoplasma fraxinii'. Int. J Syst. Bacteriol. 1999;49:1605-1614.
19.Hiruki D, Wang K. Clover proliferation phytoplasma: `Candidatus Phytoplasma trifolii'. Int. J Syst. Bacteriol. 2004; 54:1349-1353.
20.IRPCM Phytoplasma/Spiroplasma Working Team-Phytoplasma Taxonomy Group. `Candida-tus Phytoplasma', a taxon for the wall-less, non helical prokaryotes that colonize plant phloem and insects. Int. J Syst. Evol. Microbiol. 2004; 54:1257-1269.
21.Jones P. Phytoplasma plant pathogens. Waller M, Lenné JM, Waller SJ, editor. Plant Pathologists Pocketbook, Part 12. CAB International, Oxford University Press, USA; 2002.
22.Jung H, Sawayanagi T, Wongkaew P, Kakizawa S, Nishigawa H, Wei W, et al. `Candidatus Phytoplasma oryzae', a novel phytoplasma taxon associated with rice yellow dwarf disease. Int. J Syst. Evol. Microbiol. 2003;53:1925-1929.
23.Khan AJ, Botti S, Paltrinieru S, Al-Subhi AM, Bertaccini A. Phytoplasmas in alfalfa seeds: infected or contaminated? In: Proceedings of 14th Congress International Organization for Micoplasmology. IOM Abstracts 205. 2002; p.148.
24.Lee IM, Davis R, Gundersen-Rindal D. Phytoplasma: phytopathogenic mollicutes. Ann. Rev. Microbiol. 2000;54:21-25.
25.Lee IM, Gundersen-Rindal , Davis R, Bartoszyk M. Revised classification of phytoplasmas based on RFLP analyses of 16s rRNA and ribosomal protein gene sequences. Int. J Syst. Evol. Microbiol. 1998;48:1153-1169.
26.Leyva-López NE, Aguilar-Rojas O, Leal-Klevezas DS, Martínez-Soriano JP. Presence of phytoplasmas in Mexican cacti. Phytopathology. 1999;89:545.
27.Leyva-López NE, Ochoa-Sánchez JC, Leal-Klevezas DS, Martínez-Soriano JP. Multiple phytoplasmas associated with potato diseases in Mexico. Can. J Microbiol. 2002,48:1062-1068.
28.Padovan A, Gibb K. Epidemiology of phytoplasma diseases in papaya in Northern Australia.J Phytopathol. 2001;149: 649-658.
29.Schenider B, Cousin M, Klinkong S, Seemüller E. Taxonomic relatedness and phylogenetic positions of phytoplasmas associated with diseases of faba bean, sunhemp, sesame, soybean and eggplant. Plant Dis.1995;102:225-232.
30.Seemüller E, Schneider B.`Candidatus Phytoplasma pyri' and `Candidatus Phytoplasma prunorum', the causal agents of apple proliferation, pear decline and European stone fruit yellows, respectively. Int. J Syst. Evol. Microbiol. 2004;54:1217-1226.
31.Seruga M, Vrek I, Koriae D. Dianthus croaticus Borb _a new host for phytoplasma from ribosomal groups 16SrI and 16SrIII. In: Proceedings of 15th International Congress for Micoplasmology.IOM Abstracts. 2004; p.122-123.
32.Tran-Nguyen L, Blanche K, Egan B, Gibb K. Diversity of phytoplasmas in northern Australian sugarcane and other grasses. Plant Pathol. 2000;49:666-679.
33.Tymon A, Jones P, Harrison N. Phylogenetic relationships of coconut phytoplasmas and the development of specific oligonucleotide PCR primers. Ann. Appl. Biol. 1998;132:437-452.
34.Zreik L, Carle P, Bove J, Garnier M. (1995): Characterization of the mycoplasma-like organism associated with witches' broom disease of lime and proposition of a Candidatus taxon for the organism "Candidatus Phytoplasma aurantifolia". Int. J Syst. Bacteriol. 1995;45: 449-453.
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(Recibido 24-1-2007; Aceptado 2-2-2007)
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