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Identification of phytoplasmas associated with Bunchy Top disease of papaya in Colima, Mexico
By Pedro Valadez Ramírez, Daniel Leobardo Ochoa Martínez*, Guadalupe Valdovinos Ponce, Edith Blanco Rodríguez, Sergio Aranda Ocampo, Candelario Ortega Acosta, Marco Tulio Buenrostro Nava, Jetzajary Ayerim Rodríguez Barajas, Luis Rafael De la Torre Velázquez, Carlos Luis Leopardi Verde
*Corresponding Author.
Received: 05/March/2024 – Published:
18/June/2024
DOI:
https://doi.org/10.18781/R.MEX.FIT.2403-2
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Abstract Background/Objective. Phytoplasmas, rickettsiae and viruses have been detected in papaya plants with Bunchy Top disease (BT). In 2019, papaya plants with BT-like symptoms were observed in agroecosystems of Colima, Mexico. In order to determine the BT-associated phytoplasmas species or subgroups, asymptomatic and symptomatic plants were collected from papaya agroecosystems in four papaya producer municipalities, as well as papaya-associated weeds and insects.
Materials and Methods. Phytoplasma detection and identification was conducted by PCR, sequencing and phylogenetics of translocase subunit SecA (secA) and 16S ribosomal RNA (16Sr) genes, and PCR-RFLPs in vitro and in silico for 16Sr gene.
Results. In papaya, phytoplasma groups 16SrI (subgroup AF), 16SrX, and 16SrXIII were identified in 2.08% (4 out of 192) symptomatic samples. The results of RFLPs in silico analysis showing the presence of 16SrX and 16SrXIII (sub)groups. In papaya-associated weeds and insects, phytoplasmas of group 16SrI (subgroups AF and B) were identified in 1.7% (3 out of 174) and 1.1% (2 out of 185) evaluated samples, respectively. Phytoplasma-carrying weeds were Amaranthus palmeri and Echinochloa colona; positive insects were Micrutalis calva and Balclutha mexicana.
Conclusion. It is the first time that phytoplasmas 16SrI-AF, 16SrX y 16SrXIII are associated with Bunchy Top disease of papaya in agroecosystems from Colima, Mexico. Phytoplasmas 16SrX y 16SrXIII are first reported in papaya plants at the world level and in Mexico, respectively. Phytoplasma-carrying weeds and insects are new records as natural reservoirs and potential vectors.
Keywords: Carica papaya, Candidatus Phytoplasma, weeds, insects
Figure 1. Detection of phytoplasmas in agroecosystems papaya with Bunchy Top disease in Colima, Mexico. A) Papaya plants showing stems with shortened internodes and yellow, chlorotic leaves, positive to phytoplasmas. B) in vitro RFLPs profiles of gene 16Sr (primers R16F2n/R16R2) of the phytoplasmas under study. Lanes 1-4: samples 1S-4S- papaya, Lanes 5-6: samples 5S-6S-Amaranthus palmeri; Lane 7: sample 7S-Echinochloa colona; Lane 8: sample 8S-Micrutalis calva; and Lane 9: sample 9S-Balclutha mexicana. The DNA was digested with 10 U of every enzyme and the products were separated by electrophoresis in 3% (w/v) agarose gels in 1×TAE buffer. Lane M: 1 kb plus molecular weight marker (Thermo Scientific, EUA).
Figure 2. in silico RFLPs profiles of gene 16Sr of the phytoplasmas under study, generated with iPhyClassifier with 17 restriction enzymes. A) sample 1S-papaya; B) sample 2S-papaya; C) sample 3S-papaya; D) sample 4S-papaya, E) sample 5S-Amaranthus palmeri; F) sample 6S-Amaranthus palmeri; G) sample 7S-Echinochloa colona; H) sample 8S-Micrutalis calva; I) sample 9S-Balclutha mexicana. Lanes MW: φX174 DNA digested with Hae III
Figure 3. Phylogenetic trees of the secA (A) and 16Sr (B) trees obtained using the neighbor-joining method of 16Sr groups of phytoplasmas identified in papaya agroecosystems in Colima, Mexico (black circles) and from phytoplasma groups from other parts of the world. Accession number are shown in parentheses. Samples 1S-4S: papaya, samples 5S-6S: Amaranthus palmeri, sample 7S: Echinochloa colona, sample 8S: Micrutalis calva, sample 9S: Balclutha mexicana. The gene sequence of secA of Bacillus subtilis and the 16Sr gene sequence of Acholeplasma laidlawii were used as outgroups. In each phylogeny, bootstrap values (from 1000 replicates, greater than 70%) are shown on the branches. The scale bar indicates the number of nucleotide substitutions per site
Table 1. Detection of phytoplasmas in papaya (Carica papaya), weeds and insects associated with the crop in Colima, Mexico, in November and December of 2019.
Table 2. Phytoplasmas identified in papaya, weeds and insects associated with the crop in Colima, Mexico, in November-December of 2019.
