Presence of phytoplasma in Chaya an ancient Maya edible plant

Presença de fitoplasma em Chaya, uma planta comestível dos antigos Maias

Abstract

Cnidoscolus aconitifolius ssp. aconitifolius and C. souzae are shrubs that are widely distributed in the Yucatan Peninsula, Mexico. In recent years, they have shown symptoms characteristic of phytoplasma infection. Therefore, they were screened for the presence of phytoplasma using nested PCR with P1 and P7 primers, followed by R16F2n/R16R2 primers for universal phytoplasma detection. Through this protocol, phytoplasma was detected in both species that exhibited symptoms of infection. Sequence analysis, virtual RFLPs, and a phylogenetic tree confirmed that the Cnidoscolus species analyzed and sequenced are infected by phytoplasmas similar to those that infect cassava, belonging to subgroups 16SrIII-L. This study is the first to report the presence of phytoplasmas in chaya species.

Keywords:
Cnidoscolus; witches’s broom symptoms; Euphorbiaceae

Resumo

Cnidoscolus aconitifolius ssp. aconitifolius e C. souzae são arbustos amplamente distribuídos na Península de Yucatán, México. Nos últimos anos, eles apresentaram sintomas característicos de infecção por fitoplasma. Por isso, foram examinados para a presença de fitoplasma utilizando PCR aninhado com os primers P1 e P7, seguidos pelos primers R16F2n/R16R2 para detecção universal de fitoplasma. Por meio desse protocolo, o fitoplasma foi detectado em ambas as espécies que apresentavam sintomas de infecção. A análise de sequências, RFLPs virtuais e uma árvore filogenética confirmaram que as espécies de Cnidoscolus analisadas e sequenciadas estão infectadas por fitoplasmas semelhantes aos que infectam a mandioca, pertencentes aos subgrupos 16SrIII-L. Este estudo é o primeiro a relatar a presença de fitoplasmas em espécies de chaya.

Palavras-chave:
Cnidoscolus; sintomas da vassoura-de-bruxa; Euphorbiaceae

1. Introduction

The genus Cnidoscolus is distributed throughout the Americas, with Mexico having the greatest diversity, including 25 endemic species (Coelho et al., 2012). In the Yucatan Peninsula, this genus has three species: Cnidoscolus aconitifolius (Mill) I.M. Johnst, C. souzae McVaugh, and C. urens (L.) Arthur (Chin-Chan et al., 2021). Cnidoscolus aconitifolius and C. souzae are distributed in the Yucatan Peninsula, Guatemala, Belize and Honduras while C. urens is distributed from Mexico to Argentina (Chin-Chan et al., 2021).

Chaya was classified by Breckon (1975) as Cnidoscolus aconitifolius (Mill.) I.M. Johnst ssp. aconitifolius. This taxon includes a wide range of plant species, from wild to completely domesticated. According to Ross-Ibarra and Molina-Cruz (2002), there are four cultivated varieties: chayamasa, redonda, estrella, and picuda. 'Chayamansa' is the most clearly domesticated and is found in the greatest abundance on the peninsula, primarily used for feeding purposes (Bautista-Cruz et al., 2011). C. souzae, on the other hand, is not edible (Zapata-Estrella et al., 2014).

These plant species are robust evergreen shrubs with lobulated leaves from the Euphorbiaceae family (Ross-Ibarra and Molina-Cruz, 2002). They are widespread in the backyards of residences across the rural landscape of the Yucatan Peninsula. Cnidoscolus aconitifolius ssp. aconitifolius has been used for centuries by the Mayan people to treat various ailments and diseases. Nowadays, numerous medicinal properties are attributed to them, aiding in the improvement of conditions such as high blood pressure, poor blood circulation, high cholesterol, uric acid, and diabetes (Pérez-González et al., 2016), as well as for the preparation of ancestral foods (Pérez-González et al., 2016).

Phytoplasmas (genus 'Candidatus Phytoplasma') are intracellular plant pathogenic bacteria belonging to the class Mollicutes that affect a great number of cultivated species. They are transmitted by insect vectors when they feed on the sap of infected plants (Fernández-Barrera et al., 2024). The classical symptomatology of phytoplasmas includes virescence (greening of sepals and petals), phyllody (retrograde development of floral organs to leaves), witches’ broom (multiple shoots), yellowing of leaves, and stunting (Hogenhout et al., 2008). They are classified into groups according to the 16S rRNA gene sequence and restriction fragment length polymorphism (RFLP) analysis (Lee et al., 2000).

In recent years, plants of Cnidoscolus aconitifolius ssp. aconitifolius and C. souzae have shown characteristic symptoms of phytoplasma infection such as yellowing of leaves and witches’ broom. The presence of phytoplasmas in chaya species across the Yucatán Peninsula and other regions of Mexico has not been previously reported. Within the Euphorbiaceae family, phytoplasmas primarily affect cassava species (Manihot esculenta Crantz). The presence of phytoplasma subgroups 16SrIII-L and 16SrIII-H were identified in association with Cassava Frogskin Disease in Brazil (Souza et al., 2014) and Colombia (Alvarez et al., 2009) respectively.

Therefore, this study aimed to detect and molecularly characterize phytoplasmas present in C. aconitifolius ssp. aconitifolius and C. souzae in the Yucatan Peninsula, Mexico, in order to potentially identify the etiological agent.

2. Material and Methods

2.1. Sample collection

Cnidoscolus aconitifolius ssp. aconitifolius and C. souzae plants were surveyed from August 2022 to September 2023 within the provinces of Tixkokob and Dzitbalché, Mexico (20°59'45”N 89°23'57”W and 20°19'30”N 90°02'54”W). In the case of C. aconitifolius ssp. aconitifolius, we sampled the variety ‘chayamansa’ because it was the most predominant. These plants were located in the backyard of the houses. Leaves from ten ‘chayamansa’ plants showing typical symptoms of phytoplasma infection were collected and leaves from five plants showed no symptoms. Leaves from five C. souzae plants showing similar symptoms to those observed in the edible chaya plants were also collected. These plants were located on the side of the road and in the nearby bush or forest, not far from the edible chaya plants.

2.2. DNA extraction

To confirm the presence of phytoplasmas in C. aconitifolius ssp. aconitifolius and C. souzae, genomic DNA was extracted from 0.5 g of leaf tissue from symptomatic and asymptomatic plants. The nucleic acids were extracted using the CTAB extraction procedure described by Córdova et al. (2014) with minor modifications. In brief, each sample was pulverized in liquid nitrogen with a mortar and pestle, and 1 ml of CTAB buffer was added and incubated at 60°C for 30 minutes. After centrifugation for 5 minutes at 14,000 rpm, the supernatant was transferred to a new tube and added with 1 ml of phenol: chloroform:isoamyl alcohol (25:24:1) and centrifuged for 5 minutes at 14,000 rpm. The supernatant was transferred to a new tube and 0.1 volume of sodium acetate (pH 5.3) and 0.6 volumes of cold isopropanol were added. The sample was then incubated at -20°C for 1 hour, and centrifuged for 10 minutes at 14,000 rpm. The pellet was washed with 70% ethanol, dried, re-suspended in 100 µL of sterile water. Then, 3 µl of RNase (RNAse concentration of 1 mg/ml) was added and incubated at 37°C for 30 minutes. DNA was visualized by electrophoresis on a 1% agarose gel in TAE buffer for 30 minutes. The gel was then stained with ethidium bromide and observed with a UV transilluminator Gel-Doc (Bio-Rad). The concentration of all the extracted DNA was adjusted to 50 ng/μl and stored at 4°C until it was used for PCR amplifications.

2.3. Detection of phytoplasma by nested PCR using general primers

The presence of phytoplasmas in samples was determined using a PCR procedure with the universal phytoplasma primer pair P1 (Deng and Hiruki, 1991) and P7 (Schneider et al., 1995) in direct PCR, followed by R16F2n/R16R2 nested PCR primers (Lee et al., 1994). The PCR reaction was performed with final concentrations of 1X PCR buffer, 2.0 mM MgCl2, 50 ng genomic DNA, 200 µM of dNTPs, 1.5 U of Dream Taq DNA Polymerase (Thermo Scientific, USA), and 1 µM of each primer. For the nested PCR reaction, a 40-fold diluted template generated by P1/P7 primers was used. All amplifications were carried out in a PCR machine T100 Thermo Cycler (Bio-Rad) following the conditions reported by Deng and Hiruki (1991).

A phytoplasma previously detected in a coconut palm in our laboratory was used as a positive control, and one DNA sample from a palm showing no symptoms of LY was used as a negative control. The PCR products were analyzed by electrophoresis on 1% agarose gels, stained with GelRed nucleic acid stain (Biotium, USA), and visualized using a UV transilluminator Molecular Image Gel-Doc (Bio-Rad).

2.4. Sequencing, blast, virtual RFLP and phylogenetic analysis

Seven amplified 16S rRNA fragments from nested PCR were purified using the Zymoclean Gel DNA Recovery Kit (Zymo Research) following the instructions provided. These purified products were sequenced directly using phytoplasma universal primers R16F2n/R16R2 on an ABI® 3730XL DNA analyzer at the Laboratorio Nacional de Biotecnología Agrícola, Médica y Ambiental (LANBAMA) in Mexico.

The amplicon sequences of the R16F2n/R16R2-primed PCR products obtained from both types of diseased chaya species were assembled, curated manually, and analyzed using BLASTN software database to find the closest match in the GenBank (accessed on October 05, 2024) to obtain the percentage of sequence identity.

The phylogenetic analysis of the 16S rRNA gene tree was constructed using the sequences of 38 'Candidatus Phytoplasma' species available in GenBank (Supplementary material. Table 1). Evolutionary distances were computed using the maximum composite likelihood method, and the neighbor-joining method was used to construct the phylogenetic trees. The evolutionary history was inferred using the neighbor-joining method (Saitou and Nei, 1987) analysis employing MEGA version 11.0.11 software (Tamura et al., 2021), using a bootstrap with 1000 replicates. The DNA sequences of Acholeplasma laidlawii (M23932) were used as the out-group taxa to root the phylogenetic tree.

Computer-simulated RFLP analysis was conducted using the iPhyClassifier tool (Zhao et al., 2009) for the DNA sequences of each 16S rRNA gene from C. aconitifolius ssp. aconitifolius and C. souzae samples. Consensus sequences obtained were subjected to in silico digestion with 17 restriction endonucleases (AluI, BamHI, BfaI, BstUI, DraI, EcoRI, HaeIII, HhaI, HinfI, HpaI, HpaII, KpnI, Sau3AI, MseI, RsaI, SspI, and TaqI). The generated virtual RFLP profiles were compared with available representatives of 16S rRNA phytoplasma groups/subgroups.

3. Results

Cnidoscolus aconitifolius ssp. aconitifolius and C. souzae were sampled from two sites on the Yucatan Peninsula. Healthy chaya plants were shrubs, reaching heights of 1-2 meters, highly branching with large, three-lobed green leaves (Figure 1A). The flowers of healthy Cnidoscolus aconitifolius ssp. aconitifolius were small and usually white (Figure 1B), while infected plants showed yellowing and a reduction in the number of leaves (Figure 1C and D). The infected plants exhibited an excessive proliferation of shoots, indicative of witches' broom disease symptoms (WB) (Figure 1E and 1G). Healthy C. souzae plants were shorter than the edible variety, with smaller leaves (Figure 1F). The infected C. souzae plants displayed similar symptoms to the edible plants, primarily witches' broom (Figure 1G).

Figure 1
Cnidoscolus aconitifolius ssp. aconitifolius healthy plant (A), inflorescence of a healthy palm (B), chaya infected with phytoplasma showing yellowing of leaves (C), infected plant losing its leaves (D), witches’ broom symptom (E), healthy C. souzae (F), witches’ broom symptom of C. souzae (G).

3.1. Phytoplasma detection by PCR assays

Nested PCR assays using R16F2n/R16R2 primers successfully amplified 1.2 kb fragments of the 16S rRNA region from all symptomatic chaya plants, as well as the phytoplasma positive control (Figure 2). However, no DNA amplification products were detected in direct and nested PCR assays using the same primers when DNA from asymptomatic C. aconitifolius ssp. aconitifolius and C. souzae were tested.

Figure 2
Detection of phytoplasmas in Cnidoscolus aconitifolius ssp. aconitifolius and C. souzae plants using universal primers P1/P7 and R16F2n/R16R2n by nested amplification. Line M (molecular marker, 1 Kb), lane LY (Coconut palm infected by LY phytoplasma as positive control; lane 1, healthy palm as negative control; lanes 2-10 chaya plants with symptoms; lanes 11-13 chaya plants without symptoms.

3.2. Sequence analysis

Seven fragments of approximately 1.2 kb were sequenced from diseased plants. The sequences were deposited in the National Center for Biotechnology (NCBI) database, three for C. aconitifolius ssp. aconitifolius under the accession number: OR854627, OR878455, OR878456 and four for C. souzae: OR854628, OR878457, OR878458, OR878459. Five sequences, the four from C. souzae and one from C. aconitifolius ssp. aconitifolius (OR854627) presented 100% sequence identity with Cassava Frogskin Disease Phytoplasma belonging to 16SrIII-L subgroup, while two sequences from C. aconitifolius ssp. aconitifolius (OR878455, OR878456) presented 99.68% identity to 16SrIII-L subgroup (Supplementary material, Table 2).

Phylogenetic analysis of the 16S rDNA sequences of the seven strains in this study, together with18 phytoplasma sequences from the 16SrIII group and 20 from other groups, show that the amplicons cluster with phytoplasmas of the 16SrIII-L subgroup (Figure 3).

Figure 3
Phylogenetic tree based on 16S rRNA constructed using the Neighbor-Joining method in MEGA 11.0.11, displaying the relationships among Cnidoscolus species' phytoplasma strains and reference phytoplasma strains. Accession numbers are indicated in the tree. Acholeplasma laidlawii was utilized as an outgroup for tree construction. The number on branches corresponds to bootstrap values obtained from 1000 replicates.

3.3. In silico RFLP analysis

The sequences underwent in silico RFLP analysis using the iPhyclassifier software, which utilizes 17 restriction enzymes for phytoplasma characterization. The virtual RFLP patterns from the C. aconitifolius ssp. aconitifolius and C. souzae isolates showed that the profiles of five isolates (OR854627, OR854628, OR878457, OR878458, OR878459) matched the Cassava Frogskin Disease Phytoplasma classified in subgroup 16SrIII-L (EU346761) (Figure 4). However, two isolates (OR878455, OR878456) were identical to the reference pattern of phytoplasma Eggplant Giant Calyx Phytoplasma (HM589213) in subgroup 16SrIII-U. The enzyme that differentiate the subgroups was BstUI.

Figure 4
Virtual RFLP patterns resulting from the in silico analysis of the nucleotide sequences of phytoplasmas found in Cnidoscolus aconitifolius ssp. aconitifolius and C. souzae plants displaying symptoms of witches' broom (WBP) (OR878455, OR878456, OR854627, OR854628, OR878457, OR878458, and OR878459) (Figures 4B, C, E, F, H, I, J respectively). These patterns were compared to the sequence profiles of Eggplant Giant Calyx Phytoplasma (16SrIII-U) (HM589213) (Figure 4A), Cassava frogskin disease (16SrIII-L) (EU346761) (Figure 4D), and Clover yellow edge phytoplasma (16SrIII-B) (AF189288) (Figure 4G). The red box highlights the restriction enzyme that generates a distinct pattern. Molecular weight markers: 72, 118, 194, 234, 271, 281, 310, 603, 872, 1078, 1353bp.

4. Discussion

Cnidoscolus aconitifolius ssp. aconitifolius and C. souzae are commonly grown in the backyards of homes in the countryside of the Yucatan Peninsula. Recently, many plants of both chaya species have displayed symptoms similar to those caused by phytoplasma infections. The most frequent symptoms seen in infected chaya plants are witches' broom and yellowing of leaves, which are noticeable year-round but more prominent during the rainy season, eventually leading to the death of the plants.

The DNA amplicons obtained from infected chaya species were sequenced and analyzed. The BLAST sequence analysis revealed a high percentage of identity ranging from 100-99.68 with the Cassava Frogskin Disease Phytoplasma classified in the 16SrIII-L subgroup. Phylogenetic tree analysis of the phytoplasma 16Sr sequence also confirmed that the sequences isolated from both chaya species were clustered within the clade of the Cassava Frogskin Disease Phytoplasma in the 16SrIII-L subgroup. In silico RFLP pattern digestion showed an identical pattern to the subgroup 16SrIII-L for most sequences. However, only two isolates (OR878455, OR878456) from C. aconitifolius ssp. aconitifolius exhibited a digestion pattern identical to that of the Eggplant Giant Calyx Phytoplasma (HM589213) classified in the 16SrIII-U phytoplasma subgroup (Amaral-Mello et al., 2011). Despite this, the percentage of identity of these sequences to the 16SrIII-U phytoplasma subgroup was 99.19 which was lower than with subgroup 16SrIII-L (Supplementary material, Table 2). Overall, the majority of the results supported that phytoplasmas infecting chaya species belong to the 16SrIII-L subgroup.

The phytoplasma from the 16SrIII-L subgroup is known to infect cassava (Manihot esculenta Crantz), a staple food in developing countries (Alvarez et al., 2009; Souza et al., 2014). The Manihot and Cnidoscolus genera are closely related, belonging to the tribe Manihoteae in the Euphorbiaceae family. Symptoms reported in cassava include tuber aberrations, reduced diameter, and a woody appearance with injuries. While witches' broom symptoms have been reported in cassava in Argentina, Brazil, and Colombia (Flôres et al., 2013; Fernández et al., 2018; Alvarez et al., 2009, respectively), these symptoms appear to be different from the classical witches' broom symptoms caused by phytoplasma in cassava in Southeast Asia, where other groups of phytoplasma are present (Pardo et al., 2023).

This study demonstrated the presence of phytoplasma subgroup 16SrIII-L in C. souzae and C. aconitifolius ssp. Aconitifolius. This pathogen has a significant impact on edible chaya, affecting the availability of essential leaves for preparing various dishes and juices. Despite not being extensively cultivated in the Mesoamerican region, these species are valuable for their high nutritional content and have been proposed as potential crops in the region, underscoring the importance of characterizing their pathosystem.

Supplementary Material

Supplementary material accompanies this paper.

Supplementary material. Table 1.

List of phytoplasma 16Sr sequences used for phylogenetic analysis, including their classification and GenBank accession number (http://www.ncbi.nlm.nih.gov).

Supplementary material. Table 2.

The percentage sequence identity of phytoplasma sequences isolated from Cnidoscolus aconitifolius ssp. aconitifolius and C. souzae plants was compared with the specific sequences of each phytoplasma subgroup within group 16SrIII phytoplasma. The black box indicates the highest percentages of sequence identity observed among the CaWBP and CsWBP sequences of Chaya species with the different subgroups of phytoplasma characterized within group 16SrIII.

This material is available as part of the online article from https://doi.org/10.1590/1519-6984.282086

Acknowledgements

This work was supported partially by CONAHCYT Project number 320993.

References

• ALVAREZ, E., MEJÍA, J.F., LLANO, G.A., LOKE, J.B., CALARI, A., DUDUK, B. and BERTACCINI, A., 2009. Characterization of a phytoplasma associated with frogskin disease in Cassava. Plant Disease, vol. 93, no. 11, pp. 1139-1145. http://doi.org/10.1094/PDIS-93-11-1139 PMid:30754574.
  » http://doi.org/10.1094/PDIS-93-11-1139
• AMARAL-MELLO, A.P.O., ECKSTEIN, B., FLORES, D., KREYCI, P.F. and BEDENDO, I.P., 2011. Identification by computer-simulated RFLP of phytoplasmas associated with eggplant giant calyx representative of two subgroups, a lineage of 16SrIII-J and the new subgroup 16SrIII-U. International Journal of Systematic and Evolutionary Microbiology, vol. 61, no. Pt 6, pp. 1454-1461. http://doi.org/10.1099/ijs.0.019141-0 PMid:20622054.
  » http://doi.org/10.1099/ijs.0.019141-0
• BAUTISTA-CRUZ, A., ARNAUD-VIÑAS, M.R., MARTÍNEZ-GUTIÉRREZ, G.A., SÁNCHEZ-MEDINA, P.S. and PÉREZ-PACHECO, R., 2011. The traditional medicinal and food uses of four plants in Oaxaca, México. Journal of Medicinal Plants Research, vol. 5, no. 15, pp. 3404-3411.
• BRECKON, G.J., 1975. Cnidoscolus, section calyptosolen (Euphorbiaceae) in Mexico and Central America Davis: University of California, 926 p. Ph.D. Thesis.
• CHIN-CHAN, T., ORTIZ-GARCÍA, M.M., RUIZ-GIL, P.J. and MARTÍNEZ-CASTILLO, J., 2021. Genetic Diversity of Chaya (Cnidoscolus aconitifolius (Mill.) I. M. Johnst. ssp. aconitifolius) In Yucatan, Mexico, Its Putative Domestication Center. Polibotánica, vol. 51, pp. 185-201.
• COELHO, M.S., BELMIRO, M.S., SANTOS, J.C. and FERNANDES, G.W., 2012. Herbivory among habitats on the Neotropical tree Cnidoscolus quercifolius Pohl. in a seasonally deciduous forest. Brazilian Journal of Biology = Revista Brasileira de Biologia, vol. 72, no. 3, pp. 453-457. http://doi.org/10.1590/S1519-69842012000300006 PMid:22990814.
  » http://doi.org/10.1590/S1519-69842012000300006
• CÓRDOVA, I., OROPEZA, C., PUCH-HAU, C., HARRISON, N., COLLÍ-RODRÍGUEZ, A., NARVÁEZ, M. and SÁENZ, L., 2014. A real-time PCR assay for detection of coconut lethal yellowing phytoplasmas of group 16SrIV subgroups A, D and E found in the Americas. Journal of Plant Pathology, vol. 96, pp. 343-352.
• DENG, S. and HIRUKI, C., 1991. Amplification of 16S rRNA genes from culturable and nonculturable Mollicutes. Journal of Microbiological Methods, vol. 14, no. 1, pp. 53-61. http://doi.org/10.1016/0167-7012(91)90007-D
  » http://doi.org/10.1016/0167-7012(91)90007-D
• FERNÁNDEZ, F., USET, A., BAUMGRATZ, G. and CONCI, L., 2018. Detection and identification of a 16SrIII-J phytoplasma affecting cassava (Manihot esculenta Crantz) in Argentina. Australasian Plant Disease Notes, Australasian Plant Pathology Society, vol. 13, no. 1, pp. 1-5. http://doi.org/10.1007/s13314-018-0309-2
  » http://doi.org/10.1007/s13314-018-0309-2
• FERNÁNDEZ-BARRERA, M., CÓRDOVA-LARA, I., CHAN-RODRÍGUEZ, J.L., CASTILLO-VERA, A., BLANCO-RODRÍGUEZ, E., NIC-MATOS, G., OROPEZA-SALÍN, C. and SÁENZ-CARBONELL, L., 2024. Detection of 16SrIV-A phytoplasma DNA in Colpoptera sp. (Hemiptera: Nogodinidae) insects in Yucatan Peninsula, Mexico. Brazilian Journal of Biology = Revista Brasileira de Biologia, vol. 84, e257470. http://doi.org/10.1590/1519-6984.257470 PMid:35416853.
  » http://doi.org/10.1590/1519-6984.257470
• FLÔRES, D., HAAS, I.C., CANALE, M.C. and BEDENDO, I.P., 2013. Molecular identification of a 16SrIII-B phytoplasma associated with cassava witches’ broom disease. European Journal of Plant Pathology, vol. 137, no. 2, pp. 237-242. http://doi.org/10.1007/s10658-013-0250-3
  » http://doi.org/10.1007/s10658-013-0250-3
• HOGENHOUT, S.A., OSHIMA, K., AMMAR, E.D., KAKIZAWA, S., KINGDOM, H.N. and NAMBA, S., 2008. Phytoplasmas: bacteria that manipulate plants and insects. Molecular Plant Pathology, vol. 9, no. 4, pp. 403-423. http://doi.org/10.1111/j.1364-3703.2008.00472.x PMid:18705857.
  » http://doi.org/10.1111/j.1364-3703.2008.00472.x
• LEE, I.M., DAVIS, R. and GUNDERSEN-RINDAL, D., 2000. Phytoplasma: phytopathogenic Mollicutes. Annual Review of Microbiology, vol. 54, no. 1, pp. 221-255. http://doi.org/10.1146/annurev.micro.54.1.221 PMid:11018129.
  » http://doi.org/10.1146/annurev.micro.54.1.221
• LEE, I.M., GUNDERSEN-RINDAL, D.E., HAMMOND, R.W. and DAVIS, R.E., 1994. Use of micoplasmalike organisms (MLO) group-specific oligonucleotide primers for nested PCR assay to detect mixed MLO infections in a single host plant. Phytopathology, vol. 84, no. 6, pp. 559-566. http://doi.org/10.1094/Phyto-84-559
  » http://doi.org/10.1094/Phyto-84-559
• PARDO, J.M., CHITTARATH, K., VONGPHACHANH, P., HANG, L.T., OEURN, S., ARINAITWE, W., RODRIGUEZ, R., SOPHEARITH, S., MALIK, A.I. and CUELLAR, W.J., 2023. Cassava Witches’ Broom disease in southeast asia: a review of its distribution and associated symptoms. Plants, vol. 12, no. 11, pp. 2217. http://doi.org/10.3390/plants12112217 PMid:37299196.
  » http://doi.org/10.3390/plants12112217
• PÉREZ-GONZÁLEZ, M., GUTIÉRREZ, G. and JIMÉNEZ, M., 2016. Importancia nutricional, farmacológica y química de la chaya (Cnidoscolus chayamansa): revisión bibliográfica. Temas de Ciencia y Tecnología, vol. 20, no. 60, pp. 43-56.
• ROSS-IBARRA, J. and MOLINA-CRUZ, A., 2002. The ethnobotany of chaya (Cnidoscolus aconitifolius ssp. aconitifolius Breckon): a nutritious Maya vegetable. Economic Botany, vol. 56, no. 4, pp. 350-365. http://doi.org/10.1663/0013-0001(2002)056[0350:TEOCCA]2.0.CO;2
  » http://doi.org/10.1663/0013-0001(2002)056[0350:TEOCCA]2.0.CO;2
• SAITOU, N. and NEI, M., 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Molecular Biology and Evolution, vol. 4, no. 4, pp. 406-425. PMid:3447015.
• SCHNEIDER, B., SEEMÜLLER, E., SMART, D.C. and KIRKPATRICK, B.C., 1995. Phylogenetic classification of plant pathogenic mycoplasma-like organisms or phytoplasma. In: S. RAZIN and J.G. TULLY, eds. Molecular and diagnostic procedures in mycoplasmolgy Amsterdam: Elsevier, pp. 369-380. http://doi.org/10.1016/B978-012583805-4/50040-6
  » http://doi.org/10.1016/B978-012583805-4/50040-6
• SOUZA, A.N., DA SILVA, F.N., BEDENDO, I.P. and CARVALHO, C.M., 2014. A Phytoplasma Belonging to a 16SrIII-A Subgroup and dsRNA Virus Associated with Cassava Frogskin Disease in Brazil. Plant Disease, vol. 98, no. 6, pp. 771-779. http://doi.org/10.1094/PDIS-04-13-0440-RE PMid:30708630.
  » http://doi.org/10.1094/PDIS-04-13-0440-RE
• TAMURA, K., STECHER, G. and KUMAR, S., 2021. MEGA11: molecular evolutionary genetics analysis version 11. Molecular Biology and Evolution, vol. 38, no. 7, pp. 3022-3027. http://doi.org/10.1093/molbev/msab120 PMid:33892491.
  » http://doi.org/10.1093/molbev/msab120
• ZAPATA-ESTRELLA, H.E., SÁNCHEZ-PARDENILLA, A.D., GARCÍA-SOSA, K., ESCALANTE-EROSA, F., DE CAMPOS-BUZZI, F., MEIRA-QUINTÃO, N.L., CECHINEL-FILHO, V. and PEÑA-RODRÍGUEZ, L.M., 2014. Bioactive metabolites from Cnidoscolus souzae and Acmella pilosa. Natural Product Communications, vol. 9, no. 9, pp. 1319-1321. http://doi.org/10.1177/1934578X1400900925 PMid:25918802.
  » http://doi.org/10.1177/1934578X1400900925
• ZHAO, Y., WEI, W., LEE, I.M., SHAO, J., SUO, X. and DAVIES, R.E., 2009. 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, vol. 59, no. Pt 10, pp. 2582-2593. http://doi.org/10.1099/ijs.0.010249-0 PMid:19622670.
  » http://doi.org/10.1099/ijs.0.010249-0

Publication Dates

• Publication in this collection
  07 Feb 2025
• Date of issue
  2024

History

• Received
  08 Jan 2024
• Accepted
  28 Nov 2024