INTRODUCTION
⌅Soybean (Glycine max L. Merr.) is one of the most cultivated legumes in the world, mainly because of its high oil and protein content (11. SoyStats. A Reference Guide to Important Soybean Facts and Figures. American Soybean Association. 2022. Available online: http://soystats.com/ (accessed on 28 July 2022) .). In Cuba, in order to replace the importation of grains, soybean production recently arose as an economic policy priority with a notable increase of the country area destined to soybean cultivation, for different uses (22. Roján-Herrera O, Maqueira-López LA, Santana-Ges I de los M, Miranda-Sierra CA, Núñez-Vázquez M. Productividad de cultivares de soya en dos épocas de siembra. CulTrop. 2022;43(1):e05. Disponible en: https://ediciones.inca.edu.cu/index.php/ediciones/article/view/1641).
A wide variety of pathogens can infect soybean and cause significant yield losses (33. Yu S-F, Wang C-L, Hu Y-F, Wen Y-C, Sun Z-B. Biocontrol of Three Severe Diseases in Soybean. Agriculture. 2022;12(9):1391. DOI: 10.3390/agriculture12091391). The expansion of soybean cultivation in the world has caused the number and severity of the diseases affecting this crop to increase, and, among these diseases, phytoplasmas have acquired significant importance in the last years.
Phytoplasma diseases have had great impact on cropswith significant losses in production yield and quality (44. Kumari S, Nagendran K, Rai AB, Singh B, Rao GP, Bertaccini A. Global Status of Phytoplasma Diseases in Vegetable Crops. Front Microbiol. 2019;10:1349. DOI: 10.3389/fmicb.2019.01349, 55. Rao GP, Bertaccini A, Fiore N, Liefting LW, editors. Phytoplasmas: Plant Pathogenic Bacteria - I: Characterisation and Epidemiology of Phytoplasma - Associated Diseases. Singapore: Springer Singapore; 2018. DOI: 10.1007/978-981-13-0119-3). According to Bertaccini and Lee (66. Bertaccini A, Lee I-M. Phytoplasmas: An Update. In: Rao GP, Bertaccini A, Fiore N, Liefting LW, editors. Phytoplasmas: Plant Pathogenic Bacteria - I. Singapore: Springer Singapore; 2018. DOI: 10.1007/978-981-13-0119-3_1), phytoplasmas infect more than 300 plant species worldwide, which limited the growth and photosynthesis of herbaceous and tree species (77. Bertaccini A. Plants and Phytoplasmas: When Bacteria Modify Plants. Plants. 2022;11(11):1425. DOI: 10.3390/plants11111425).
Several soybean diseases have been associated with phytoplasma infections around the world: (i) a disease caused by ‘Candidatus Phytoplasma asteris’ was detected in soybean plants in Wisconsin (88. Lee ME, Grau CR, Lukaesko LA, Lee I-M. Identification of aster yellows phytoplasmas in soybean in Wisconsin based on RFLP analysis of PCR-amplified products (16S rDNAs). Canadian Journal of Plant Pathology. 2002;24(2):125-30. DOI: 10.1080/07060660309506986); (ii) in Costa Rica, stunting, little leaf, shoot proliferation and aborted seed pods were associated with a putative new group related to group 16SrXII (99. Villalobos W, Moreira L, Rivera C, Lee I-M. First Report of New Phytoplasma Diseases Associated with Soybean, Sweet Pepper, and Passion Fruit in Costa Rica. Plant Disease. 2009;93(2):201-201. DOI: 10.1094/PDIS-93-2-0201C), which was later proposed as ‘Candidatus Phytoplasma costaricanum’ and designated as 16SrXXXI group (1010. Lee I-M, Bottner-Parker KD, Zhao Y, Villalobos W, Moreira L. ‘Candidatus Phytoplasma costaricanum’ a novel phytoplasma associated with an emerging disease in soybean (Glycine max). International Journal of Systematic and Evolutionary Microbiology. 2011;61(12):2822-6. DOI: 10.1099/ijs.0.029041-0); (iii) phytoplasma-infected soybean has been reported in Taiwan (1111. Wang C-J, Chien Y-Y, Liao P-Q, Chiu Y-C, Chen Y-K, Yang J-Y. First Report of 16SrII-V Phytoplasma Associated with Green Manure Soybean (Glycine max) in Taiwan. Plant Disease. 2021;105(7):2012. DOI: 10.1094/PDIS-12-20-2714-PDN); (iv) in Iran, phytoplasmas reported being transmitted by the seed (1313. Zamharir MG, Shameli S. Molecular evidence of seed transmission of soybean bud proliferation and seed pods abortion phytoplasma disease. Indian Phytopathology. 2022;75(3):889-93. DOI: 10.1007/s42360-022-00518-4) were found in soybean plants with symptoms of proliferation of sprouts and abortion of seeds and pods (1212. Zamharir MG, Aldaghi M. First report of a ‘Candidatus Phytoplasma trifolii‘‐related strain associated with soybean bud proliferation and seed pod abortion in Iran. New Disease Reports. 2018;37(1):15-15. DOI: 10.5197/j.2044-0588.2018.037.015).
In Cuba, the presence of Aster yellows group phytoplasma and the evidence of two new subgroups 16SrI associated with stunting, chlorosis, crinkles, and aborted seed pods were reported in soybean (1414. Acosta K, Silva FN, Zamora L, Quiñones M, Martínez R, Piñol B, et al. Detection and molecular characterization of an aster yellows group phytoplasma associated with soybean and evidence of two new 16SrI subgroups in Cuba. Journal of Plant Pathology. 2015;97(2):339-44. Available from: http://www.jstor.org/stable/24722474). However, new soybean symptoms characterized by blistering and severe leaf mosaics were observed in the country. The objective of this research was to determine the possible presence of other phytoplasma groups infecting soybean in the country.
MATERIALS AND METHODS
⌅Sample prospecting
⌅From January to August 2014, surveys were conducted in soybean fields of Cuban Incasoy-27 cultivar in the localities of Holguin province in the eastern region of the country. Samples were randomly collected from 57 symptomatic plants displaying symptoms of blistering and severe leaf mosaic (Fig. 1) and from 10 symptomless plants.
Phytoplasma detection by nested PCR/
⌅Total DNA was extracted from soybean plants according to Leiva et al. (1515. Leyva Martinez RM, Quiñones Pantoja ML, Piñol Perez B, Piloto Sardiñas E, Acosta Perez K. Detección de infección mixta de ‘Candidatus phytoplasma sp.’ y begomovirus afectando el cultivo de la soya en la región oriental de Cuba. Rev. Protección Veg. 2019;34(2). Disponible en: https://censa.edicionescervantes.com/index.php/RPV/article/view/1028). For phytoplasma detection by nPCR, the combination of universal primers that target the phytoplasma 16S rRNA gene was used to amplify a 1.45 kb fragment from the first PCR reaction (primer pair R16mF2/R1) and a 1.25 kb fragment from the nested PCR (nPCR) reaction (primer pair R16F2n/R16R2) (1515. Leyva Martinez RM, Quiñones Pantoja ML, Piñol Perez B, Piloto Sardiñas E, Acosta Perez K. Detección de infección mixta de ‘Candidatus phytoplasma sp.’ y begomovirus afectando el cultivo de la soya en la región oriental de Cuba. Rev. Protección Veg. 2019;34(2). Disponible en: https://censa.edicionescervantes.com/index.php/RPV/article/view/1028).
Phytoplasma identification by RFLP analysis
⌅Each nPCR product was digested separately with the restriction endonucleases BstUI, HhaI, HpaII and KpnI (Promega) following the manufacturer’s instructions. Digestion products were separated by electrophoresis on 3 % agarose gel and visualized by ethidium bromide staining in a transilluminator under UV light. RFLP patterns were compared with previously published patterns for different phytoplasmas (1616. Lee I-M, Gundersen-Rindal DE, Davis RE, Bartoszyk IM. Revised Classification Scheme of Phytoplasmas based on RFLP Analyses of 16S rRNA and Ribosomal Protein Gene Sequences. International Journal of Systematic Bacteriology. 1998;48(4):1153-69. DOI: 10.1099/00207713-48-4-1153).
Cloning and sequencing analysis of the 16SrDNAr region by BLASTn
⌅Positive fragments of 1250 bp were purified using the Gel Band Purification Kit (GE Healthcare) and ligated into pGEM T-easy vector (Promega) according to supplier’s instructions for subsequent transformation of competent cells of Escherichia coli DH5. Three inserts of each strain were sequenced in both directions using M13 primers (Macrogen, South Korea). The 16SrDNA sequences were compared with those available in GenBank at National Center of Biotechnology by BLASTn (1717. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. Journal of Molecular Biology. 1990;215(3):403-10. DOI: 10.1016/S0022-2836(05)80360-2).
Insilico RFLP analysis and phylogenetic construction
⌅Virtual RFLP analysis was performed for the 16S rDNA sequences (1250 bp) of the Cuban phytoplasma strains using the software program iPhyClassifier (1818. Zhao Y, Wei W, Lee I-M, Shao J, Suo X, Davis RE. Construction of an interactive online phytoplasma classification tool, iPhyClassifier, and its application in analysis of the peach X-disease phytoplasma group (16SrIII). International Journal of Systematic and Evolutionary Microbiology. 2009;59(10):2582-93. DOI: 10.1099/ijs.0.010249-0, 1919. Zhao Y, Wei W, Lee I-M, Shao J, Suo X, Davis RE. The iPhyClassifier, an Interactive Online Tool for Phytoplasma Classification and Taxonomic Assignment. In: Dickinson M, Hodgetts J, editors. Phytoplasma. Totowa, NJ: Humana Press; 2013. (Methods in Molecular Biology; vol. 938). DOI: 10.1007/978-1-62703-089-2_28). Each 16S rDNA sequences were digested in silico with AluI, BamHI, BfaI, BstUI, DraI, EcoRI, HaeIII, HhaI, HinfI, HpaI, HpaII, KpnI, Sau3AI, MseI, RsaI, SspI, and TaqI restriction enzymes. RFLP in silico patterns were compared with subgroup phytoplasmas reported to 16SrIII X-disease group (1818. Zhao Y, Wei W, Lee I-M, Shao J, Suo X, Davis RE. Construction of an interactive online phytoplasma classification tool, iPhyClassifier, and its application in analysis of the peach X-disease phytoplasma group (16SrIII). International Journal of Systematic and Evolutionary Microbiology. 2009;59(10):2582-93. DOI: 10.1099/ijs.0.010249-0, 2020. Lee I-M, Polashock J, Bottner-Parker KD, Bagadia PG, Rodriguez-Saona C, Zhao Y, et al. New subgroup 16SrIII-Y phytoplasmas associated with false-blossom diseased cranberry (Vaccinium macrocarpon) plants and with known and potential insect vectors in New Jersey. Eur J Plant Pathol. 2014;139(2):399-406. DOI: 10.1007/s10658-014-0396-7) and 16SrIX group ‘Ca. phytoplasma phoenicium’ (2121. Casati P, Quaglino F, Abou-Jawdah Y, Picciau L, Cominetti A, Tedeschi R, et al. Wild plants could play a role in the spread of diseases associated with phytoplasmas of Pigeon pea witches’-broom group (16SrIX). Journal of Plant Pathology. 2016;98(1):71-81. DOI: 10.4454/JPP.V98I1.026).
The 16S rDNA gene sequences were aligned with MUSCLE algorithm (2222. Edgar RC. MUSCLE: a multiple sequence alignment method with reduced time and space complexity. BMC Bioinformatics. 2004;5(1):113. DOI: 10.1186/1471-2105-5-113) implemented in MEGA v. 6.06 (2323. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. MEGA6: Molecular Evolutionary Genetics Analysis Version 6.0. Molecular Biology and Evolution. 2013;30(12):2725-9. DOI: 10.1093/molbev/mst197). A phylogenetic tree for phytoplasma sequences was constructed using the maximum likelihood (ML) method with Tamura-Nei nucleotide substitution model and gamma-distributed. Bootstrapping (3,000 replications) was used to estimate the stability and support of the branches.
RESULTS AND DISCUSSION
⌅Phytoplasma detection by nested PCR
⌅Nested PCR fragments of phytoplasmas (1250 bp) were detected in 18 out of 57 symptomatic soybean plants (Table 1). No amplification was obtained from total DNA extracted from asymptomatic plants. These results indicate the presence of phytoplasmas in the 31.58 % of samples displaying blistering and severe leaf mosaic symptoms. Phytoplasmas were detected by nested PCR in a low percentage (31.58 %) of symptomatic soybean plants. The remaining symptomatic samples may have resulted negative because of the low phytoplasma concentration or the occurrence of any other pathogen in these soybean plants . In this sense, soybean plants with symptoms such as mosaic, blistering, leaf yellowing, and floral abortion were found positive to Begomovirus in Cuba (2424. Leyva R, Quiñones M, Acosta K, Piñol B, Xavier C, Zerbini F. Primer informe de Tobacco Yellow Crinkle virus infectando el cultivo de la soya en Cuba. Rev. Protección Veg. 2019;34(2). Disponible en: https://censa.edicionescervantes.com/index.php/RPV/article/view/1038). Further studies will focus on whether mixed infection of begomoviruses and phytoplasmas could be occurring in soybean plants in the country.
The incidence of phytoplasma was higher in Holguín, Uñas and La Rosa (6, 5 and 5 positive plants, respectively, in the samples tested) than in La División (2 positive plants in 5 tested).
| Locality | # of samples collected | Phytoplasma Positive1 | 16SrIII2 | 16SrIX2 |
|---|---|---|---|---|
| Uñas | 21 | 5 | 3 | 2 |
| Holguín | 11 | 6 | 4 | 2 |
| La Rosa | 19 | 5 | 4 | 1 |
| La División | 5 | 2 | 2 | - |
| San Mateo | 1 | - | - | - |
| Total | 57 | 18 | 13 | 5 |
1Phytoplasma detected by nested-PCR followed by RFLP with HaeIII2Phytoplasma groups differentiation by RFLP analysis
Phytoplasma identification by RFLP analysis
⌅The presence of 16SrIII (13 samples) and 16SrIX (5 samples) groups were identified when they were compared with patterns previously reported by (1616. Lee I-M, Gundersen-Rindal DE, Davis RE, Bartoszyk IM. Revised Classification Scheme of Phytoplasmas based on RFLP Analyses of 16S rRNA and Ribosomal Protein Gene Sequences. International Journal of Systematic Bacteriology. 1998;48(4):1153-69. DOI: 10.1099/00207713-48-4-1153) (Table 1).
The thirteen samples of the 16SrIII-Cu177 strain showed unique profiles with HpaII endonuclease and a similar pattern with HhaI endonuclease (Fig. 2 A) with other 16SrIII phytoplasma subgroups (X-disease group) when it was compared with previously reported patterns (1616. Lee I-M, Gundersen-Rindal DE, Davis RE, Bartoszyk IM. Revised Classification Scheme of Phytoplasmas based on RFLP Analyses of 16S rRNA and Ribosomal Protein Gene Sequences. International Journal of Systematic Bacteriology. 1998;48(4):1153-69. DOI: 10.1099/00207713-48-4-1153).
However, the two 16SrIX-Cu205 and three 16SrIX-Cu185 strains showed profiles identical to the 16SrIX-A subgroup ‘Ca. Phytoplasma phoenicium’ (Fig. 2B). These results suggest that the Cuban soybean is infected by phytoplasmas within to two distinct subgroups belonging to the 16SrIII and 16SrIX groups, a novel one (16SrIII-Cu177 strain), and these two subgroups occurring in the field.
Analysis of the 16SrDNA region sequencing by BLASTn
⌅The sequence (1250 bp) of the 16SrIII-Cu177 strain (GenBank accession no KU749597) showed the highest nucleotide (nt) sequence identity (99 %) with several sequences of 16SrIII phytoplasma strains identified in different hosts. Also, this sequence showed high nt sequence identity (98.88 %, 1233 bp identical out of 1247 bp compared) with Manihot esculenta witches'-broom phytoplasma (GU193977). Additionally, the strain detected in soybean showed nt sequence identity of 98.79 % with a 16SrIII phytoplasma detected in clover in Lithuania (KC283217) and crotalaria in Brazil (KF941133), which are crop legumes.
The sequences of 16SrIX-Cu205 (KU749595) and 16SrIX-Cu185 (KU749596) showed nt sequence identity of 100 % with each other and the highest nt sequence identity (99.36 %, 1242 bp identical out of 1250 bp compared) with several sequences of 16SrIX phytoplasma strains including 'Crotalaria juncea' witches'-broom phytoplasma (KF941131) and Pigeon pea witches'-broom phytoplasma (KJ817871) detected in legumes.
In silico RFLP analysis and phylogenetic construction
⌅In silico RFLP analysis indicated genetic variability in the 16SrIII phytoplasma associated with soybean (Fig. 3), and it confirmed the results obtained by conventional RFLP (Fig. 2). Unique in silico RFLP patterns were observed with HpaII (Fig. 3A) for the 16SrIII-Cu177 strain. The 16SrIX-Cu205 and 16SrIX-Cu185 strains showed a profile similar to those from members of the 16SrIX-A subgroup for all enzymes analyzed (Fig. 3B). Together, the results of conventional and in silico RFLP indicate that the 16SrIII-Cu177 variant is a new subgroup within the 16SrIII group, and the 16SrIX-Cu205 and 16SrIX-Cu185 strains belong to the 16SrIX-A subgroup.
Phylogenetic analysis based on the 16S rDNA sequences of the 16SrIII soybean X-Disease phytoplasma and ‘Ca. Phytoplasma phoenicium’strains detected in this study and those of 53 other phytoplasmas yielded the consensus tree presented in Figure 4. The 16SrIII Soybean X-Disease phytoplasma strain grouped into a same branch of the ‘Ca. Phytoplasma pruni’ phytoplasma (16SrIII-A), which in turn clustered closely to other phytoplasmas of the 16SrIII group, including Soybean veinal necrosis phytoplasma (AF177383) detected in Lithuania (Fig. 4).
The 16SrIII-Cu177 strain grouped into distinct branches, closely to Cirsium white leaf phytoplasma (AF373106) showing a greater genetic distance to other phytoplasma sequences (Fig. 4), thus providing additional evidence that these phytoplasmas represent a new subgroup. The 16SrIX-Cu205 and 16SrIX-Cu185 strains grouped ito a same branch of the ‘Ca. Phytoplasma phoenicium’, which in turn clustered closely to other phytoplasmas of the 16SrIX group, including Pigeon pea witches-broom phytoplasma (AF248957), which is a member of the 16SrIX-A subgroup detected in the US. (Fig. 4)
Results presented here confirm the occurrence of phytoplasmas in soybean plants exhibiting blistering and severe leaf mosaic symptoms in Cuba. Based on RFLP patterns (HpaII), the strain 16SrIII-Cu177 is a member of a new ribosomal subgroup and is proposed to be named 16SrIII-Z, while the 16SrIX-Cu205 and 16SrIX-Cu185 strains are members of 16SrIX group ‘Ca. Phytoplasma phoenicium’, which were classified within the 16SrIX-A subgroup.
Soybean X-Disease phytoplasma and ‘Ca. Phytoplasma phoenicium strains were characterized as members of the 16SrIII and 16SrIX groups, respectively, based on RFLP analysis of PCR-amplified 16S rDNA using the restriction endonucleases BstUI, HhaI HpaII and KpnI.
Other soybean diseases have been associated with phytoplasmas from other groups, including a disease causing symptoms of stunting, chlorosis, crinkle and aborted seed pods that was associated with a 16SrI group phytoplasma in Cuba (1414. Acosta K, Silva FN, Zamora L, Quiñones M, Martínez R, Piñol B, et al. Detection and molecular characterization of an aster yellows group phytoplasma associated with soybean and evidence of two new 16SrI subgroups in Cuba. Journal of Plant Pathology. 2015;97(2):339-44. Available from: http://www.jstor.org/stable/24722474). In Costa Rica, the 16SrXXXI group was proposed as the new ‘Ca. P. costaricanum’ associated with soybean stunt symptoms (1010. Lee I-M, Bottner-Parker KD, Zhao Y, Villalobos W, Moreira L. ‘Candidatus Phytoplasma costaricanum’ a novel phytoplasma associated with an emerging disease in soybean (Glycine max). International Journal of Systematic and Evolutionary Microbiology. 2011;61(12):2822-6. DOI: 10.1099/ijs.0.029041-0). Additionally, a disease caused by a subgroup 16SrII-V phytoplasma was reported in Taiwan (1111. Wang C-J, Chien Y-Y, Liao P-Q, Chiu Y-C, Chen Y-K, Yang J-Y. First Report of 16SrII-V Phytoplasma Associated with Green Manure Soybean (Glycine max) in Taiwan. Plant Disease. 2021;105(7):2012. DOI: 10.1094/PDIS-12-20-2714-PDN), and another in Iran, showing symptoms of bud proliferation and aborted seed pods was associated with phytoplasmas of the 16SrVI group (1212. Zamharir MG, Aldaghi M. First report of a ‘Candidatus Phytoplasma trifolii‘‐related strain associated with soybean bud proliferation and seed pod abortion in Iran. New Disease Reports. 2018;37(1):15-15. DOI: 10.5197/j.2044-0588.2018.037.015).
These facts suggested that a high variability of phytoplasmas could be occurring in the soybean fields of Cuba. Accordingly, a high genetic variability of 16SrI phytoplasmas was reported in soybean in the same region by (1414. Acosta K, Silva FN, Zamora L, Quiñones M, Martínez R, Piñol B, et al. Detection and molecular characterization of an aster yellows group phytoplasma associated with soybean and evidence of two new 16SrI subgroups in Cuba. Journal of Plant Pathology. 2015;97(2):339-44. Available from: http://www.jstor.org/stable/24722474). Our results confirm this hypothesis, with the detection of phytoplasmas belonging to two groups, including one novel subgroup.
Phylogenetic analysis was consistent with RFLP analyseswhen showed that Soybean X-Disease phytoplasma classified as 16SrIII-Z subgroup cluster within the same clade with Ca. Phytoplasma pruni (16SrIII group). In addition, the 16SrIII-Z subgroup was the most frequently detected one (72.22% of the infected samples).
The results presented at this point indicate that phytoplasmas identified in soybean plants in the country are genetically diverse. The identification of two phytoplasma groups, including a new subgroup in soybean plants from production fields, reinforce the urgency of developing a more effective crop management strategy. Further studies are required to identify their potential vectors, as well as a more reliable molecular characterization tool to analyze phytoplasma diversity in soybean varieties, which may have important epidemiological implications for the disease control.