Surveys for phytoplasmas and begomoviruses were conducted in common bean fields of cv. ´Bat-304´ from October to March in 2011 to 2013 in the provinces of Havana and Mayabeque in south-western Cuba. The leave samples were taken from eight farms of three municipalities: Cotorro (23°01′34″N 82°14′51″O) in Havana province, and San José de Las Lajas (22°58′04″N 82°09′21″O) and Güines (50°52′04″N 1°52′25″E) in Mayabeque province. The midribs were removed from the collected leaves with a sterile scalpel and subjected to total DNA extraction (2424. Doyle JJ, Doyle JL. Isolation of plant DNA from fresh tissue. Focus. 1990; 12:13-15.).

For both PCR assays, specific for phytoplasmas and begomoviruses, ´Mexican potato purple top phytoplasma´, belonging to ´Ca. Phytoplasma asteris´ (16Sr-I group) and TYLCV were used, respectively, as the positive controls. They were from the Interdisciplinary Centre of Research for Integral Regional Development of National Polytechnic Institute (CIIDIR-IPN), Unit Sinaloa, Mexico.

To detect phytoplasma DNA, it was performed a nested PCR assay using universal primers that amplify the 16S rRNA gene of phytoplasmas: R16mF2/R1 for the first round and R16R2/F2n for the nested reaction (2525. Gundersen DE, Lee IM. Ultrasensitive detection of phytoplasmas by nested-PCR assays using two universal primer pairs. Phytopathologia Mediterranea. 1996; 35:144-151.). A dilution to 1/30 was made from the first round to the second. A PCR assay including the Rep-DGSAR (XbaI)/ and a pCP70-BamH1 primer pair was used for the detection of begomoviruses (2626. Argüello-Astorga G, Ruiz-Medrano R. An iteron-related domain is associated to Motif 1 in the replication proteins of geminiviruses: identification of potential interacting amino acid-base pairs by a comparative approach. Archives of Virology. 2001; 146:1465-1485.). PCR products were electrophoresed in 1 % agarose gels, and visualized under UV transillumination, after staining with ethidium bromide as recommended by the manufacturer.

The obtained sequences of both phytoplasmas and begomoviruses were compared with those of reference at the GenBank by BLASTn (2727. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. Journal of Molecular Biology. 1990; 215:403-410.) and aligned using the MUSCLE algorithm implemented in MEGA v. 6.0 (2828. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. MEGA5: Molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony. Methods. Molecular Biology and Evolution. 2011; 28:2731-2739.). For 16S rDNA sequences a phylogenetic tree was constructed using the maximum likelihood (ML) method with the Hasegawa-Kishino-Yano (HKY) model and gamma distribution rate variation (+G). For the sequences of DNA-A and DNA- B begomoviruses, a phylogenetic tree was constructed using the Maximum Likelihood method with the Tamura-Nei model and gamma distribution rate variation (+G). Bootstrapping (3000 replications) was used to estimate the stability and support for the branches.

A total of 99 symptomatic common bean plants exhibited symptoms of mosaic, crinkle, leaf size reduction, leaf deformation, yellowing, chlorosis and dwarfism (Figure 1).

Phytoplasmas were detected by nested PCR in 55 out of 99 leaf samples, while begomoviruses were detected by PCR in 63 out of 99 symptomatic common bean plants from all localities of Havana and Mayabeque provinces (Table 1). No phytoplasma amplification was obtained from those symptomless plants collected.

Based on the molecular analysis, the province with the lowest infection incidence was Havana, from which with only two leaf samples were positive to phytoplasma and four samples to begomovirus (Table 1).

Mixed infections were detected in 44 leaf samples from common bean plants expressing phytoplasma and begomovirus disease-like symptoms (Table 1). These results confirmed that the common bean plants exhibiting the above mentioned symptoms were associated with begomoviruses and phytoplasmas.

The detection of both pathogens in 44 samples established an occurrence probability of mixed infections of 0.44 in the total plants evaluated. The occurrence probability of mixed infections, P (AB), was calculated. The probability level of begomoviruses (B) occurring in first instance was 0.72, and of phytoplasmas (A) was 0.80. These results suggest that the occurrence probability of a mixed infection will be 1.11 times higher if phytoplasmas are first detected in the field samples.

Using KpnI endonuclease, the fifty-five common bean samples positive to phytoplasma showed identical profiles to those of ‘Ca. Phytoplasma asteris’ (16SrI-B subgroup) (Figure 2). Similar results were obtained with MboI endonuclease (data not showed). These results indicate that common bean is infected in Cuba by group 16SrI of phytoplasmas.

The amplified genomic DNA from representative symptomatic common bean samples infected by a begomovirus exhibited a KpnI fragments of ~2,6 kbp. BLASTn analysis showed that the clones corresponded to begomoviral DNA-A (2644 nt) and DNA-B (2608 nt), respectively. DNA-A (KU160634) showed a nucleotide identity of 98 % with the begomovirus BGYMV isolates (AJ544531), (KX185517; KX185518), isolated previously in Cuba. In turn, DNA-B (KU145406) sequences obtained from 20 % of the clones showed a sequence identity of 95,71 %, 94,33 %, 93,84 %, and 90,75 % with the begomovirus strains BGYMV-US (DQ119825), BGYMV-MX (AF173556), BGYMV-DO (L01636), and BGYMV-GU (M91605).

The 16S rDNA sequences of three clones of Cuban Phaseolus phytoplasma (KR732321, KR732322, and KR732323) showed their highest sequence identity (99 %) with those of phytoplasma members of the group 16SrI, ‘Ca. Phytoplasma asteris’, including the Chinese periwinkle phyllody phytoplasma (GU113145) and ‘Canavalia ensiformis' yellowphytoplasma (KR232799).

The phylogenetic analysis based on the 16S rDNA sequences of the 16SrI phytoplasma strains detected in this study and of those of 14 other phytoplasmas yielded the consensus tree shown in Figure 3.

The 16SrI Cuban Phaseolus phytoplasma strains grouped into a same branch of the AY phytoplasma, which in turn clustered closely to other phytoplasma strains of the 16SrI group previously detected in several crops in Cuba (Fig.3).

The phylogenetic analysis of the DNA-A of BGYMV (KU160634) detected in this study was placed within the monophyletic cluster of the Mesoamerican begomovirus group (Fig.4), more closely related to the strain BGYMV-CU (KX185517; KX185518) reported in Cuba. The analyses carried out with the B component sequence (data not show) confirmed that obtained for the A component, because BGYMV CU (KU145406) was placed closely to BGYMV-US (DQ119825) in the monophyletic cluster with Mesoamerican isolates.

The symptoms observed on the common bean plants in the fields of the provinces Havana and Mayabeque examined in the present study slightly differed from those observed by Zamora et al. (66. Zamora L, Acosta K, Martínez Y. First report of 'Candidatus Phytoplasma asteris' (16SrI group) affecting common bean in Cuba. New Disease Reports. 2012; 25:4. [http://dx.doi.org/10.5197/j.2044-0588.2012.025.004]) in production areas of San Jose de Las Lajas, Mayabeque, which these authors described as short internodes, dwarfism, leaf yellowing, shoot proliferation, and chlorosis of crown leaves in approximately 10 % of the plants. They also differed from those described by Moreira et al. (2929. Moreira L, Villalobos W, Saborío RG, Garita L, Castro-Robleda S, Romero-Cano J, et al. A phytoplasma associated with ‘amachamiento’ disease of dry common bean in Costa Rica. Plant Pathol. New Disease Reports. 2009; 19:31.), associated with ''amachamiento'' in Costa Rica.

The presence of phytoplasmas and begomoviruses was confirmed by PCR in 72,72 % of the common bean plants collected. Single infection of phytoplasmas and begomoviruses was detected in 11,11 % and 17,17 %, respectively, of the total plants tested. A mixed infection by both phytoplasma and begomovirus pathogens was obtained in 44,44 % of the plants surveyed. This is the first work that describes the molecular characterization of phytoplasmas and begomoviruses coinfecting plants in the common bean crop. Leyva et al. (88. Leyva R, Quiñones M, Piñol B, Piloto E, Acosta K. Detection of mixed infection of ‘Candidatus phytoplasma sp.’ and begomoviruses sp affecting soybean crop in the eastern region of Cuba. Rev. Protección Veg. 2019; 34(2):10. E-ISSN: 2224-4697) detected a 15 % of coexistence of begomoviruses and phytoplasmas in the soybean crop in Cuba.

Based on the sequence of DNA-A component, the begomovirus was identified as BGYMV, confirming the previous results of Echemendía et al. (1212. Echemendía AL, Ramos PL, Villarreal N, Martínez AK, González G, Morales FJ. Caracterización del virus del mosaico amarillo dorado del frijol en Cuba. Fitosanidad. 2010; 14:11-17.) and Chang et al. (55. Chang-Sidorchuk L, González-Álvarez H, Martínez-Zubiaur Y. Begomoviruses infecting common beans (Phaseolus vulgaris L.) in production areas in Cuba. Spanish Journal of Agricultural Research. 2018; 16(2):1006.). BGYMV has been reported affecting common bean in several countries of Central America, South America and the Caribbean (55. Chang-Sidorchuk L, González-Álvarez H, Martínez-Zubiaur Y. Begomoviruses infecting common beans (Phaseolus vulgaris L.) in production areas in Cuba. Spanish Journal of Agricultural Research. 2018; 16(2):1006., 99. Morales FJ, Castaño M. Enfermedades virales del Frijol Común en América Latina. Centro Internacional de Agricultura Tropical. Unidad de Virología (CIAT); Palmira, Colombia. 2008. 86 págs.).

Other begomoviruses that have been reported in common bean in America are Bean calico mosaic virus (BCaMV); Tomato golden mosaic virus (TGMV), Tomato yellow leaf curl virus (TYLCV), Rhynchosia golden mosaic virus (RhGMV), Sida mottle virus (SiMoV), Tobacco leaf curl Cuba virus (TobLCCuV), Common bean mottle virus (CBMoV) and Common bean severe mosaic virus (CBSMV), and BGMV (55. Chang-Sidorchuk L, González-Álvarez H, Martínez-Zubiaur Y. Begomoviruses infecting common beans (Phaseolus vulgaris L.) in production areas in Cuba. Spanish Journal of Agricultural Research. 2018; 16(2):1006.,88. Leyva R, Quiñones M, Piñol B, Piloto E, Acosta K. Detection of mixed infection of ‘Candidatus phytoplasma sp.’ and begomoviruses sp affecting soybean crop in the eastern region of Cuba. Rev. Protección Veg. 2019; 34(2):10. E-ISSN: 2224-4697,3030. Rodríguez-Pardina PE, Zerbini FM, Ducasse DA. Genetic Diversity of Begomoviruses infecting soybean, bean and associated weeds in Northwestern Argentina. Fitopatol. Bras. 2006; 31:342-348.,3131. Fernandes FR, Cruz ARR, Faria JC, Zerbini FM, Aragao FJL. Three distinct begomoviruses associated with soybean in central Brazil. Arch. Virol. 2009; 154:1567-1570.,3232. Mendez-Lozano J, Perea-Araujo LL, Ruelas-Ayala RD, Leyva-Lopez NE, Mauricio-Castillo JA, Arguello-Astorga GR. A begomovirus isolated from stunted and chlorotic soybean plants in Mexico is a new strain of Rhynchosia golden mosaic virus. Plant Dis. 2006; 90:972.,3333. Martínez-Zubiaur Y, Quiñones M, Fonseca D, Potter J, Maxwell DP. First report of Tomato yellow leaf curl virus associated with beans, Phaseolus vulgaris, in Cuba. Plant Dis. 2002; 86;814.,3434. Morales FL, Anderson PK. The emergence and dissemination of whitefly-transmitted geminivirus in Latin America. Arch Virol. 2001; 146:415-441.). The latter virus caused losses in 100,000 ha in North Argentina.

The characterization of the full-length sequence of DNA-B component of BGYMV has a significant impact on BGYMV epidemiology in the country since DNA-B is associated with the virus cell-to-cell movement through the plant plasmodesmata and the plant host range and symptoms (3535. Hanley-Bowdoin L, Settlage SB, Orozco BM, Nagar S, Robertson D. Geminiviruses: Models for plant DNA replication, transcription and cell cycle regulation. Critical Review in Biochemical and Molecular Biology. 2000; 35:105-140.).

The confirmation of the detection of the 16SrI group in common bean, particularly the subgroup 16SrI-B, is of a great epidemiological impact for phytoplasma infection in common bean in Cuba. This is the phytoplasma group with the widest plant and vector host range and the most complex epidemiology (3636. Firrao G, Garcia-Chapa M, Marzachì C. Phytoplasmas: genetics, diagnosis and relationships with the plant and insect host. Frontiers in Bioscience. 2007; 12:1353-1375.). It is a noteworthy fact that the symptoms described in common bean in 2012 differ from those observed in the present study.

The high incidence of both BGYMV and the 16SrI phytoplasma in common bean fields indicates that it is required a more effective disease management strategy to target both pathogens and their vectors simultaneously.

BGYMV is prevalent in common bean cultivated areas in Havana and Mayabeque provinces, suggesting that surveillance of this virus, which has devastated common bean fields in South America, required being reinforced.

In view of the capability of BGYMV and the 16SrI phytoplasma to coexist on a same common bean host plant, the Cuban policy for management of common bean diseases is required to be revised more in detail. This is a potential threat for the the common bean crop in Cuba. Additional studies on the epidemiology of both plant pathogens are also needed to develop more effective strategies for crop management and disease control.