ORIGINAL ARTICLE

A phytoplasma representative of a new subgroup 16SrI-Z associated with Bunchy Top Symptoms (BTS) on papaya in Cuba

Fitoplasma representante de un nuevo subgrupo, 16SrI-Z, asociado con síntomas de Cogollo Arrepollado del Papayo (PBT) en Cuba

Karel I. Acosta-Pérez¹*, Berta E. Piñol-Pérez², Loidy Zamora-Gutierrez², Madelaine L. Quiñones-Pantoja², Ileana Miranda-Cabrera², Norma E. Leyva-López³, Yaima Arocha-Rosete⁴

¹ Universidad de Las Tunas (ULT), Israel Santos, CP 75200, Las Tunas, Cuba

²Centro Nacional de Sanidad Agropecuaria (CENSA), Apdo. 10, San José de Las Lajas, CP 32700, La Habana, Cuba

³Centro Interdisciplinar de Investigación para el Desarrollo Integral Regional, Instituto Politécnico Nacional (CIIDIR-IPN), Unidad Sinaloa, México

⁴Sporometrics, Toronto, Ontario, Canada

ABSTRACT

Phytoplasmas are associated with two papaya diseases in Cuba, Papaya Bunchy Top (PBT) and Bunchy Top Symptoms (BTS), which decrease fruit production. Assessment of the 16S ribosomal DNA by nested PCR, conventional RFLP, sequencing, and in silico RFLP analyses allowed the identification of a new phytoplasma subgroup designated BTSp-16SrI-Z associated with BTS disease in the eastern region of the country. BTSp-16SrI-Z subgroup was detected in 60.42 % of the surveyed plants with crown leaf chlorosis and necrosis of the young leaves. Results confirmed that this phytoplasma strain was consistently associated with the observed BTS symptoms, implying new epidemiological constraints for the disease management.

Key words: papaya, nested PCR, phytoplasma, sequencing, in silico RFLP.

RESUMEN

Los fitoplasmas se asocian con dos enfermedades del papayo en Cuba, Cogollo Arrepollado del Papayo (PBT) y Síntomas de Cogollo Arrepollado (BTS), que disminuyen la producción de frutos de papaya. La detección del ADN ribosomal 16S por análisis de PCR anidada, RFLP convencional, secuenciación y RFLP in silico permitió la identificación de un nuevo subgrupo de fitoplasmas 16SrI-Z asociado a la enfermedad BTS en la región oriental del país. El fitoplasma BTSp-16SrI-Z se detectó en el 60,42 % de las plantas muestreadas con clorosis en las hojas de la región apical y necrosis en las hojas jóvenes. El resultado confirma que este aislado de fitoplasma está consistentemente asociado con síntomas de BTS, lo cual implica nuevas restricciones para el manejo de la enfermedad.

Palabras clave: papayo, PCR anidada, fitoplasma, secuenciación, RFLP in silico.    

INTRODUCTION

Phytoplasmas are wall-less, non-cultivable, phloem-limited prokaryotes that affect more than 1000 plant species worldwide (1). Molecular-based analyses have proven to be more accurate and reliable than biological criteria previously used for phytoplasma identification, characterization and diagnosis (2). Meanwhile, RFLP in silico analysis has been proposed for the identification of phytoplasma subgroups (3).

Several phytoplasma diseases have been reported worldwide affecting papaya crops, including dieback, yellow crinkle and mosaic in Australia (4) and Nivun Haamir dieback in Israel (5). Others papaya diseases associated with phytoplasmas are dieback in Ethiopia (6), yellow crinkling in Mexico (7), and an axillary shoot proliferation in India (8).

In Cuba, two diseases associated with wall-less prokaryote have been reported: Papaya Bunchy Top (PBT) and Bunchy Top Symptom (BTS), both causing severe losses in papaya crops. Besides, a rickettsia-related proteobacterium associated with PBT was also detected in papaya with PBT symptoms in 2003 (9). However, the causal agents have not been unequivocally identified. 

Initial surveys carried out in the provinces of Havana and Villa Clara detected phytoplasmasof the groups 16SrX (apple proliferation) (9) and 16SrXVII ('Candidatus Phytoplasma caricae') (10) in association with BTS. Later, BTS was associated with the group 16SrII ('Ca. P. aurantifolia') in the eastern region of the country (11). A group 16SrI phytoplasma has also been associated with papaya affected with BTS-like symptoms in Cuba (12). Finally, a mixed infection including a rickettsia and two new phytoplasma subgroups has been reported in BTS-affected plants (13), and a rickettsia-related proteobacterium is a generalized pathogen in papaya plants affected by bunchy top disease in Cuba (14).

The present research was aimed to determine the classification of a phytoplasma associated with the chlorosis of crown leaves and the necrosis of young leaves, symptoms observed on papaya plants with BTS in Cuba.

MATERIALS AND METHODS

Leaf samples were taken from forty-eight papaya plants with symptoms of chlorosis on the crown leaves and necrosis on the young leaves and from eight symptomless plants growing in papaya plantations in the eastern (Holguín, Las Tunas and Granma provinces) region of Cuba in 2009.

DNA of phytoplasma reference controls belonging to the group 16SrI (American aster yellows, AAY) were obtained from the collection at Rothamsted. In addition, DNA of pepper little leaf (PLL, subgroup 16SrI-S), tomato little leaf (ToLL, subgroup 16SrI-T) and potato purple top (PPTJAL8, subgroup 16SrI-U, and PPT-SON18, subgroup 16SrI-V) were used for the direct comparison of the RFLP patterns.

DNA was extracted from 300 mg of leaf tissue from 48 symptomatic papaya plants by the method of Doyle & Doyle (15). A nested PCR assay was performed using puReTaq Ready-To-Go PCR beads (Amersham Biosciences) with phytoplasma 16S rDNA primers R16mF1/R16mR1 for the first round and R16F2n/R16R2 (16) for the nested reaction. A programmable thermocycler (MJ Research) was used. PCR conditions were those previously described (13).

PCR products were digested with the restriction endonucleases AluI, BfaI, BstUI, HhaI, HinfI, HpaII, MseI, Sau3AI, RsaI and TaqI (New England Biolabs), according to the manufacturer's instructions. Digestion products were electrophoresed on 3% agarose gels and visualized under UV light after staining with ethidium bromide in a transilluminator. RFLP patterns were compared with previously published patterns of phytoplasma in papaya (13).

Phytoplasma R16F2n/R16R2 amplicon with diverse RFLP patterns, PCR-amplified from plants with BTS from each region of Cuba, were purified on spin columns (QIAquick gel extraction kit, Qiagen). PCR products were inserted in the pGEM-T-Easy vector (Promega) and cloned in Escherichia coli DH5a. The inserts were sequenced in both directions with M13 primers by the Sequencing Service of the School of Life Sciences, University of Dundee, UK (http://www.dnaseq.co.uk).

Virtual RFLP was performed for the 16S rDNA sequences (1246 bp) of the papaya phytoplasma strains by using virtual gel analysis in the iPhyClassifier program. Each 16S rDNA sequence was digested in silico with AluI, BamHI, BfaI, BstUI, DraI, EcoRI, HaeIII, HhaI, HinfI, HpaI, HpaII, KpnI, Sau3AI, MseI, RsaI, SspI, and TaqI restriction endonucleases (3).

The 16S rDNA sequences obtained were aligned using the MUSCLE algorithm implemented in MEGA v. 6.0 (17). A phylogenetic tree was constructed using the maximum likelihood (ML) method with the general time reversible nucleotide substitution model with gamma-distributed rate variation in invariant sites (G+I). Bootstrapping (3,000 replications) was performed to estimate the stability and support for the branches.

RESULTS

The detection of phytoplasma DNA in 85.42% of the 48 papaya plants displaying symptoms (Figure 1) by nPCR/HaeIII supported the presence of phytoplasma in the diseased papaya plants.

In vitro RFLP analysis of the R16F2n/R16R2 amplicon obtained from all the nPCR/HaeIII positive plants and the phytoplasma controls belonging to 16SrI subgroups (Figure 2), showed that BstUI profiles of the BTSp-16SrI-Z strain (twenty-nine samples) displayed a polymorphic pattern different from that displayed by the several subgroups used as controls (PPT-16SrI-S, PPT-16SrI-T, PPT-16SrI-U and PPT-16SrI-V), including 16SrI-B and 16SrI-X subgroups associated with BTS disease in a previous study (13).

When the same BstUI endonuclease was used, the BTSp-16SrI-Z strain also showed a pattern different from that of the rest of the 16SrI subgroups reported by Lee et al. (18).

Results of the in silico RFLP analysis confirmed  the previous  results obtained by the conventional RFLP with a restriction endonuclease. To the novel phytoplasma strain BTSp-16SrI-Z, polymorphism was observed with BstUI (Figure 3), which demonstrated the genetic divergence of this strain within the group 16SrI and established a new subgroup associated with BTS affected papaya plants.

The phylogenetic tree strongly supports the results obtained by the conventional RFLP analysis, the new strain, identified as BTSp-16SrI-Z (JF781311), clustered with the strain identified as PYC from papaya in Mexico (FJ602434) in a phylogenetic branch different from the strains BTSp-16SrI-X (JF781308) and BTSp-16SrI-B (JF781307), all in the group 16SrI (Figure 4). However, they clustered in different branches from phytoplasmas associated with PYC and papaya mosaic (PM) in Australia (Y10096 and Y10097).

DISCUSSION

Results from the present study confirmed the previously reported association of the 16SrI phytoplasma  group  with BTS in Cuba (12,13).

In addition, for the first time it is reported a new 16SrI phytoplasma subgroup identified in papaya plants exhibiting symptoms of chlorosis of the crown leaves and necrosis of the young leaves, named BTSp-16Sr-Z.

The conventional RFLP allowed identification and differentiation of a new 16SrI subgroup associated with BTS-affected papaya plants in Cuba. The novel strain BTSp-16SrI-Z was detected in 60.42% of the samples displaying chlorosis of the crown leaves and necrosis of the young leaf symptoms. This strain is different of BTSp-16SrI-X identified by Acosta et al. (13) in the same region. Similarly, this approach has been used to distinguish among phytoplasma subgroups associated with papaya diseases such as the 16SrI-C subgroup in Mexico (7) and a 16SrII group in India (8,19).  

In silico RFLP with sequences of the strain BTSp-16SrI-Z present in BTS-affected papaya plants confirmed the presence a new phytoplasma subgroup within the 16SrI group. This    technique    has    been    used    in    the identification of new phytoplasma subgroups within groups (3,20-22).

Phylogenetic analysis strongly supported the results obtained by both conventional and in silico RFLP, showing that the strain BTSp-16SrI-Z clustered within the same branch with the strains BTSp-16SrI-B and BTSp-16SrI-X. However, they clustered in different branches from the phytoplasmas associated with PYC and papaya mosaic (PM) in Australia, which were  classified  as subgroup  16SrII-D.  In this sense, Padovan and Gibb (23) indicated that yellow crinkle and dieback diseases were caused by two different groups of phytoplasma strains, and that the main factors increasing phytoplasma diversity in papaya were the hemipteran vector abundance and the presence of weed hosts growing in the same agroecosystem. However, in our case, ecological niches in unmanaged areas of the eastern region may favour strain diversity. Similarly,   Acosta   et   al.   (13)   found    that ecological niches in weedy areas favoured strains diversity of the phytoplasma group 16SrII in papaya. In the weedy ecosystems, papayas are apparently propagated through true seed, suggesting that, assuming the lack of seed transmission, insect vector(s) may carry diverse phytoplasma strains to or from neighbouring plant species, including weeds.

In summary, the papaya-infecting phytoplasmas from eastern Cuba exhibited a remarkable genetic diversity. The extensive biodiversity of 16SrI strains suggests an ongoing evolution of phytoplasmas in adaptation to their geo- and bioecological niches. The strain diversity observed in the present study could be explained by differences in vector species. In either case, co-existence of diverse phytoplasma strains in the same geographical location and in the same host species may favour phytoplasma-phytoplasma, phytoplasma-vector, and phytoplasma-plant interactions, providing increased opportunities for genetic recombinations and the emergence of new phytoplasma plant diseases.

ACKNOWLEDGEMENTS

We acknowledge the Rothamsted International Fellowship Program provided by Rothamsted Research, Harpenden, UK, the Mexican Foreign Affair Secretariat, and CIIDIR-IPN, Sinaloa, for the support of this research project.

REFERENCES

Recibido: 23/8/2016

Aceptado: 26/3/2017

* Autor para correspondencia: Karel I. Acosta-Pérez. E-mail: karel0978@gmail.com