rmfiRevista mexicana de fitopatologíaRev. mex. fitopatol0185-33092007-8080Sociedad Mexicana de Fitopatología A.C.10.18781/R.MEX.FIT.2403-2Nota FitopatológicaIdentificación de fitoplasmas asociados al Bunchy Top de la papaya en Colima, MéxicoValadez-RamírezPedroOchoa-MartínezDaniel Leobardo*1Valdovinos-PonceGuadalupe1Blanco-RodríguezEdith1Aranda-OcampoSergio1Ortega-AcostaCandelario1Buenrostro-NavaMarco Tulio2Rodríguez-BarajasJetzajary Ayerim2Torre-VelázquezLuis Rafael De la2Leopardi-VerdeCarlos Luis2Postgrado en Fitosanidad-Fitopatología, Colegio de Postgraduados, km 36.5 Carretera México-Texcoco, Montecillo, Texcoco, Estado de México, CP 56264. MéxicoColegio de PostgraduadosColegio de PostgraduadosTexcocoEstado de México56264MexicoUniversidad de Colima, Facultad de Ciencias Biológicas y Agropecuarias, Autopista Colima-Manzanillo km 40, Tecomán, Colima, México, CP 28930.Universidad de ColimaUniversidad de ColimaColima28930MexicoAutor de correspondencia: Daniel Leobardo Ochoa-Martínez ldaniel@colpos.mx16022025Sep-Dec2024423RMEXFIT240320503202419052024Este es un artículo publicado en acceso abierto bajo una licencia Creative CommonsResumenAntecedentes/Objetivo:
Fitoplasmas, rickettsias y virus se han detectado en plantas de papaya con la enfermedad Bunchy Top (BT). En agroecosistemas de papaya ubicados en Colima, México, durante 2019 se observaron plantas con síntomas similares al BT. Con la finalidad de determinar la presencia de fitoplasmas e identificar las especies y/o subgrupos asociados a esta enfermedad, se recolectaron plantas asintomáticas y con síntomas en cuatro municipios productores del estado, así como malezas e insectos asociados al cultivo.
Materiales y Métodos:
La detección e identificación de fitoplasmas se hizo a través de PCR, secuenciación y análisis filogenéticos de la subunidad SecA de la translocasa (secA) y 16S RNA ribosomal (16Sr), y PCR-RFLPs in vitro e in silico del gen 16Sr.
Resultados:
En papaya se identificaron fitoplasmas de los grupos 16SrI (subgrupo AF), 16SrX y 16SrXIII, en el 2.08% (4 de 192) de las muestras con síntomas. Los resultados del análisis RFLPs in silico del gen 16Sr mostraron la presencia de los (sub)grupos 16SrX y 16SrXIII. En malezas e insectos, se identificaron fitoplasmas del grupo 16SrI (subgrupos AF y B) en el 1.7% (3 de 174) y 1.1% (2 de 185) de las muestras analizadas, respectivamente. Las malezas portadoras de fitoplasmas fueron Amaranthus palmeri y Echinochloa colona, mientras que los insectos fueron Micrutalis calva y Balclutha mexicana.
Conclusión:
Se reporta por primera vez la presencia de fitoplasmas 16SrI-AF, 16SrX y 16SrXIII en plantas de papaya con síntomas de Bunchy Top en agroecosistemas del estado de Colima, México. Los fitoplasmas de los grupos 16SrX y 16SrXIII se registran por primera vez a nivel mundial y en México en papaya, respectivamente. Las malezas e insectos portadores de fitoplasmas constituyen nuevos registros como reservorios naturales y potenciales vectores de las bacterias.
Introducción
México es el tercer productor de papaya (Carica papaya) en el mundo y principal proveedor de fruta fresca a Estados Unidos de América y Canadá, donde las ventas anuales superan las 350 000 toneladas, con un valor de 335 millones de dólares (SIAP, 2020). En este país la producción de la caricácea está localizada en las regiones Sur-Sureste y Centro Occidente, siendo Oaxaca, Colima y Chiapas los principales estados productores (SIAP, 2020). El estado de Colima, ubicado en la región Occidente por la costa sur del Océano Pacífico, posee un registro de producción de 192 417 toneladas y es el principal exportador nacional (Consejo de Productores de Papaya del estado de Colima [COEPAPAYA A.C.], 2017; SIAP, 2020). En 2019, en 64 agroecosistemas de papaya visitados en cuatro municipios del estado de Colima, se observaron plantas con acortamiento de entrenudos que originaban el arrepollamiento de las hojas apicales. Estas plantas también presentaron hojas con clorosis, amarillamiento, deformación y/o necrosis marginal. Estos síntomas son típicos de la enfermedad Bunchy Top (BT) (Cook, 1931; Story y Halliwell, 1969), y semejantes a los descritos en Cuba como síndromes “papaya bunchy top” (PBT) y “bunchy top symptom” (BTS) (Acosta et al., 2013).
El BT es inducido por fitoplasmas, rickettsias y virus, a veces en infecciones mezcladas (fitoplasmas + rickettsias o fitoplasmas + virus), y se ha reportado en República Dominicana, Cuba, Haití, Jamaica, Puerto Rico, Costa Rica, Perú (Martorell y Adsuar, 1952; Story y Halliwell, 1969; Davis et al., 1998; Arocha et al., 2003; Acosta et al., 2013; Luis-Pantoja et al., 2015; Wei et al., 2017) y más recientemente en Nigeria (Kazeem et al., 2021).
Considerando que en México se han detectado fitoplasmas en plantas de papaya con síntomas parecidos al BT (Pogoshyan et al., 2004; Navarrete-Yabur et al., 2005; Rojas-Martínez et al., 2011), y que en Colima, esta enfermedad ha adquirido mayor importancia económica y su estudio se ha abordado escasamente (productores de papaya del estado de Colima, comunicación personal); se han planteado los siguientes objetivos: a) determinar la presencia de fitoplasmas en plantas de papaya con síntomas de BT, así como en malezas e insectos asociados al cultivo, y b) establecer la identidad a nivel grupo y/o subgrupo 16Sr.
En noviembre y diciembre de 2019, se recolectaron 256 muestras de papaya en 64 agroecosistemas comerciales localizados en los municipios de Tecomán, Colima, Ixtlahuacán y Armería. Del total de muestras, 64 fueron asintomáticas y 192 presentaron síntomas relacionados con BT. De cada planta se obtuvo una muestra compuesta constituida por tres hojas (lámina y pecíolo), cada una de las cuales se recolectó de los estratos superior, medio e inferior de la planta. De cada planta de papaya (sintomática o asintomática muestreada) se recolectaron (de acuerdo con su disponibilidad y mayor abundancia) hasta cinco especies de malezas presentes en un diámetro de 2 m. Asimismo, se colectaron insectos con una red entomológica de golpeo con tres barridos, de arriba y abajo, sobre la planta de papaya y malezas adyacentes. Los insectos se conservaron en etanol al 96 % para su posterior clasificación en morfoespecies. Las malezas e insectos que fueron positivos para fitoplasmas se identificaron a nivel especie.
A partir de nervaduras centrales y de pecíolos de hojas de papaya y malezas, así como ejemplares completos de insectos (uno a cinco, según tamaño y disponibilidad), se extrajo el ADN total con un protocolo de enriquecimiento con ADN de fitoplasma basado en CTAB (Ahrens y Seemuller, 1992) y con ayuda de un homogeneizador (Tissue Lyser, Qiagen, Alemania) (Stillson y Szendrei, 2020). El ADN se evaluó por espectrofotometría de luz UV (NadoDrop 2000, Fisher Scientific, EUA) y por amplificación por PCR del gen de la proteína ribosomal S16 (rps16) de plantas (Oxelman et al., 1997) o citocromo oxidasa subunidad I (COI) de insectos (Folmer et al., 1994), como controles internos en volúmenes de reacción de 25 μL con 50 ng de ADN.
La detección de fitoplasmas se realizó en 200 ng de ADN por PCR punto final anidada de los genes 16Sr (primera ronda: iniciadores P1/P7 y anidada: iniciadores R16F2n/R16R2 y R16mF2/R16mR1) (Deng y Hiruki, 1991; Schneider et al., 1995; Gundersen y Lee, 1996; Lee et al., 1993) y secA (primera ronda: iniciadores secAFor1/secARev3 y anidada: iniciadores SecAFor5/SecARev2) (Dickinson y Hodgetts, 2013).
La identificación se hizo por secuenciación, PCR-RFLPs in vitro e in silico del gen 16Sr y análisis filogenéticos de secA y 16Sr. Para la secuenciación, los amplicones de la PCR anidada para 16Sr (iniciadores R16mF2/R16mR1) y secA de las muestras positivas se purificaron con el kit Wizard® SV Gel and PCR Clean-Up System (Promega, EUA) y se secuenciaron en ambos sentidos con los iniciadores R16mF2/R16mR1 y SecAFor5/SecARev2, con el método de dideoxi o cadena terminal en Macrogen Inc. (Seúl, Corea del Sur). Las secuencias resultantes se analizaron con el programa MEGAX (Kumar et al., 2018) y la función BLASTn del Centro Nacional de Información Biotecnológica (NCBI) (https://blast.ncbi.nlm. nih.gov/Blast.cgi), para su depósito en el GenBank (https://submit.ncbi.nlm.nih. gov/).
Para las PCR-RFLPs in vitro, aproximadamente 1 μg de cada producto purificado de la PCR anidada para el gen 16Sr (iniciadores R16F2n/R16R2) de las muestras positivas, cuantificado por espectrofotometría de luz UV (NanoDrop 2000, Fisher Scientific, EUA) se digirió, por separado, con 10 U de las enzimas de restricción Taq I, Hae III, Sau 3AI, Kpn I, Tru I1 (Mse I), Alu I y Rsa I (Thermo Scientific, EUA), siguiendo las instrucciones del fabricante, para confirmar que el fragmento amplificado correspondiera a ADN de fitoplasmas (Lee et al., 1998). Los productos se separaron por electroforesis en geles de agarosa 3 % (p/v) en amortiguador TAE 1× y se tiñeron con bromuro de etidio (Sambrook y Russell, 2001).
Para los RFLPs in silico, las secuencias de los amplicones de la PCR anidada (iniciadores R16mF2/R16mR1), editadas manualmente con MEGAX (Kumar et al., 2018), se analizaron con el software iPhyClassifier (https://acortar.link/AikrtQ) para la asignación y/o confirmación de especies, grupos y subgrupos 16Sr.
Los análisis filogenéticos se hicieron independientemente con los genes secA y 16Sr. Para ello, secuencias de cada uno de los genes analizados de diferentes grupos de fitoplasmas reportados a nivel mundial (49 para secA y 51 para 16Sr) y de Bacillus subtilis (GenBank: X62035 para secA) o de Acholeplasma laidlawii (GenBank: U14905 para 16Sr) como grupos externos, se descargaron del GenBank y se alinearon con Clustal W. La construcción de las filogenias se realizó con el método de neighbor-joining con 1000 repeticiones de bootstrap. Todos los análisis se hicieron en MEGA X (Kumar et al., 2018).
Los fitoplasmas se detectaron en el 2.08 % (4 de 192) de plantas de papaya con síntomas asociados al BT (Cuadro 1). No se detectaron fitoplasmas en plantas asintomáticas. Las plantas positivas se recolectaron en los municipios de Colima e Ixtlahuacán (Cuadro 1) y presentaron tallos con acortamientos de entrenudos y hojas amarillas y cloróticas (Figura 1A). Las muestras positivas se confirmaron
Detección de fitoplasmas en papaya (<italic>Carica papaya</italic>), malezas e insectos asociados a su cultivo en Colima, México, durante noviembre y diciembre de 2019.
Municipio
Número de muestras positivas a fitoplasmas / Número de muestras analizadas (%)
Papaya
Malezas
Insectos
Armería
0/64 (0.0)
1/74 (1.4)
1/60 (1.7)
Colima
2/64 (3.1)
0/31 (0.0)
0/32 (0.0)
Ixtlahuacán
2/64 (3.1)
1/35 (2.9)
0/46 (0.0)
Tecomán
0/64 (0.0)
1/34 (2.9)
1/47 (2.1)
Total
4/256 (1.6)
3/174 (1.7)
2/185 (1.1)
Detección de fitoplasmas en agroecosistemas de papaya con Bunchy Top en Colima, México. <bold>A)</bold> Plantas de papaya con tallos con acortamiento de entrenudos y hojas amarillas y cloróticas, positivas a fitoplasmas. <bold>B)</bold> Perfiles RFLPs <italic>in vitro</italic> del gen <italic>16Sr</italic> (iniciadores R16F2n/R16R2) de los fitoplasmas en estudio. Carriles 1-4: muestras 1S-4S-papaya, Carriles 5-6: muestras 5S-6S-<italic>Amaranthus palmeri</italic>; Carril 7: muestra 7S-<italic>Echinochloa colona</italic>; Carril 8: muestra 8S<italic>Micrutalis calva</italic>; y Carril 9: muestra 9S-<italic>Balclutha mexicana</italic>. El ADN se digirió con 10 U de cada enzima y los productos se separaron por electroforesis en geles de agarosa 3% (p/v) en TAE 1×. Carril M: marcador de masa molecular 1 kb plus (Thermo Scientific, EUA).
por secuenciación de los genes secA (GenBank: ON303285-ON303288) y 16Sr (GenBank: PP348058-PP348061). Los análisis de secuencias de los genes secA y 16Sr con BLASTn, indican que, en dos de las cuatro muestras positivas, los fitoplasmas pertenecen al grupo 16SrI (GenBank secA: ON303286 y ON303287; 16Sr: PP348059 y PP348060), mientras que las dos restantes a los grupos 16SrX (GenBank secA: ON303288; 16Sr: PP348061) y 16SrXIII (GenBank secA: ON303285;16S: PP348058), respectivamente.
El perfil de los RFLPs in vitro del gen 16Sr (Figura 1B) mostró concordancia con los perfiles reportados por Lee et al. (1998) y con los obtenidos con iPhyClassifer (Figura 2), para las enzimas de restricción usadas, lo cual permitió autenticar el
Perfiles RFLPs <italic>in silico</italic> del gen <italic>16Sr</italic> de los fitoplasmas en estudio, generados con iPhyClassifier con 17 enzimas de restricción. A) muestra 1S-papaya; B) muestra 2S-papaya; C) muestra 3S-papaya; D) muestra 4S-papaya, E) muestra 5S-<italic>Amaranthus palmeri</italic>; F) muestra 6S-<italic>Amaranthus palmeri</italic>; G) muestra 7S-<italic>Echinochloa colona</italic>; H) muestra 8S<italic>Micrutalis calva</italic>; I) muestra 9S-<italic>Balclutha mexicana</italic>. Carriles MW: ADN de <italic>φ</italic>X174 digerido con <italic>Hae</italic> III.
ADN de los fitoplasmas detectados en este estudio. El análisis RFLPs in silico del gen 16Sr confirmó la presencia de las especies ‘Candidatus Phytoplasma asteris’ [grupo 16SrI (porcentajes de identidad: 99.44 y 99.60) y subgrupo 16SrI-AF (coeficiente se similitud: 1.00)], ‘Ca. Phytoplasma rhamni’ [grupo 16SrX (porcentaje de identidad: 93.31), subgrupo indeterminado] y ‘Ca. Phytoplasma hispanicum’ [grupo 16SrXIII (porcentaje de identidad: 99.37), subgrupo 16SrXIII-D (coeficiente se similitud: 0.95)] (Cuadro 2). Con respecto a los porcentajes de identidad y coeficiente de similitud obtenidos para los fitoplasmas de los grupos 16SrX y 16SrXIII, deberá analizarse con otros estudios, si se trata de nuevos (sub)grupos.
Fitoplasmas identificados en papaya, malezas e insectos asociados al cultivo en Colima, México, durante noviembre-diciembre de 2019.
Muestra (GenBank 16Sr)
‘Candidatus Phytoplasma sp.’
Grupo 16Sr (% identidad)
Subgrupo (coeficiente de similitud)
1S-papaya (PP348058)
‘Ca. Phytoplasma hispanicum’
16SrXIII (99.37)
D (0.95)y
2S-papaya (PP348059)
‘Ca. Phytoplasma asteris’
16SrI (99.44)
AF (1.00)
3S-papaya (PP348060)
‘Ca. Phytoplasma asteris’
16SrI (99.60)
AF (1.00)
4S-papaya (PP348061)
‘Ca. Phytoplasma rhamni’
16SrX (93.31)
NAz
5S-Amaranthus palmeri (PP348062)
‘Ca. Phytoplasma asteris’
16SrI (99.44)
AF (1.00)
6S-Amaranthus palmeri (PP348063)
‘Ca. Phytoplasma asteris’
16SrI (99.68)
B (0.98)
7S-Echinochloa colona (PP348064)
‘Ca. Phytoplasma asteris’
16SrI (99.60)
AF (1.00)
8S-Micrutalis calva (PP348065)
‘Ca. Phytoplasma asteris’
16SrI (99.52)
AF (1.00)
9S-Balclutha mexicana (PP348066)
‘Ca. Phytoplasma asteris’
16SrI (99.68)
B (0.98)
yPotencial nuevo subgrupo.
zNA: no aplica en función del bajo porcentaje de identidad del grupo asignado en iPhyClassifier.
La identificación de los grupos de fitoplasmas aquí obtenidos también se corroboró por inferencias filogenéticas de los genes secA y 16Sr, puesto que en las filogenias obtenidas para ambos genes de los fitoplasmas detectados en este estudio, se agruparon con secuencias correspondientes a los mismos grupos 16Sr de fitoplasmas previamente identificados en México (GenBank 16S: MN807429) o en otros países (Figura 3).
Para nuestro conocimiento, este estudio documenta por primera vez la presencia de fitoplasmas 16SrI-AF, 16SrX y 16SrXIII en plantas de papaya con BT en México y Colima. Además, a nivel mundial, es la primera vez que se reporta la presencia del grupo 16SrX en papaya. En México, la primera detección de fitoplasmas en papaya se hizo por microscopía electrónica de barrido en Baja California Sur (Poghosyan et al., 2004). Posteriormente, mediante biología molecular, en Yucatán,
Árboles filogenéticos de los genes <italic>secA</italic> (A) y <italic>16Sr</italic> (B) obtenidos con el método de neighbor-joining de grupos 16Sr de fitoplasmas identificados en agroecosistemas de papaya en Colima, México (círculos negros) y de grupos de fitoplasmas de otras partes del mundo. Los números de acceso se muestran en paréntesis. Muestras 1S-4S: papaya, muestras 5S-6S: <italic>Amaranthus palmeri</italic>, muestra 7S: <italic>Echinochloa colona</italic>, muestra 8S: <italic>Micrutalis calva</italic>, muestra 9S: <italic>Balclutha mexicana</italic>. La secuencia del gene <italic>secA</italic> de <italic>Bacillus subtilis</italic> y la del <italic>16Sr</italic> de <italic>Acholeplasma laidlawii</italic> se usaron como grupos externos. En cada filogenia, los valores de bootstrap (por 1000 réplicas, mayores a 70 %) se muestran en las ramas. La barra indica el número de sustituciones nucleotídicas por sitio.
Campeche, Quintana Roo (Navarrete-Yabur et al., 2005), Michoacán y Veracruz, donde las muestras positivas se asignaron al subgrupo 16SrI-C (Rojas-Martínez et al., 2011).
Además de su detección en México, el grupo 16SrI se ha reportado en papaya en Cuba (Acosta et al., 2011; 2017; Acosta-Pérez et al., 2017), Perú (Hodgetts et al., 2009), Sri-Lanka (Abeysinghe et al., 2014) y China (Yu et al., 2023), mientras que el 16SrXIII solamente en Brasil (Melo et al., 2013). Otros grupos de importancia económica en papaya son 16SrII (por ej. White et al., 1998; Panda et al., 2022), 16SrXII (por ej. Gibb et al., 1996; Barbieri et al., 2023), 16SrXV (Wei et al., 2017) y 16SrXVII (Arocha et al., 2005).
En México, nunca se ha reportado la presencia del grupo 16SrX (PogoshyanMelkonyan et al., 2019), mientras que el 16SrXIII sólo se ha asociado a plantas de papa y jitomate en Sinaloa (Santos-Cervantes et al., 2010) y Baja California Sur (Holguín-Peña et al., 2007). Próximamente, deberá determinarse si el bajo porcentaje de detección de fitoplasmas se debe a la presencia de otros organismos, como rickettsias o virus, que estén induciendo los síntomas estudiados (Davis et al., 1998; Arocha et al., 2003; Acosta et al., 2013; Luis-Pantoja et al., 2015).
En el caso de malezas e insectos, la detección de fitoplasmas fue positiva en tres de 174 (1.7%) y dos de 185 (1.1%) muestras, respectivamente, y con excepción de Ixtlahuacán, los ejemplares positivos se obtuvieron a pesar de no haber registrado fitoplasmas en plantas de papaya (Cuadro 1). Colima fue el único municipio donde no hubo malezas ni insectos positivos a fitoplasmas (Cuadro 1).
Las malezas en donde se detectaron los fitoplasmas fueron asintomáticas, dos de ellas se identificaron como Amaranthus palmeri (Amaranthaceae) (GenBank 16S: PP348062-PP348063, secA: ON303289-ON303290) y la otra como Echinochloa colona (Poaceae) (GenBank 16S: PP348064, secA; ON303291. Los insectos se identificaron como Micrutalis calva (Hemiptera: Membracidae) (GenBank 16S: PP348065, secA: ON303292) y Balclutha mexicana (Hemiptera: Cicadellidae) (GenBank 16S: PP348066, secA: ON303292) (Cuadro 2).
Los análisis RFLPs in silico del gen 16Sr permitieron asignar los fitoplasmas detectados a la especie ‘Ca. Phytoplasma asteris’ (grupo 16SrI, porcentaje de identidad: 99.44-99.68), subgrupos 16SrI-AF (coeficiente de similitud: 1.00) en A. palmeri (muestra 5S), E. colona (muestra 7S) y M. calva (muestra 8S), y 16SrI-B (coeficiente de similitud: 0.98) en A. palmeri (muestra 6S) y B. mexicana (muestra 9S) Cuadro 2).
La detección de fitoplasmas en insectos y malezas asociadas a los agroecosistemas de papaya, sugiere que los artrópodos diseminan las bacterias entre plantas y que las malezas pueden jugar un papel importante como fuente de inóculo (Duduk et al., 2018). Para México, la presente investigación es pionera en indagar si las malezas e insectos asociados a la papaya son portadores/fuente de inóculo de fitoplasmas, siendo la primera vez que se reportan como especies portadoras de fitoplasmas del grupo 16SrI en agroecosistemas de papaya. Las malezas se suman a los registros de las que se han asociado al cultivo de papaya, y que se han reportado como hospedantes de fitoplasmas 16SrII en Cuba y Etiopía, tales como Anoda acerifolia (Malvaceae), Euphorbia heterophylla (Euphorbiaceae), Malvastrum coromandelianum (Malvaceae) y Rhynchosia minima (Fabaceae) (Arocha et al., 2007; Bekele et al., 2011). Aun cuando A. palmeri es una maleza común en diferentes agroecosistemas mexicanos, solo se ha reportado como hospedante de fitoplasmas del grupo 16SrIII como maleza asociada al cultivo de lufa (Luffa acutangula) (Cucurbitaceae) (Santos-Cervantes et al., 2021).
Respecto a los insectos B. mexicana y M. calva, es la primera vez que se registran como portadores de fitoplasmas en agroecosistemas de papaya. Previamen te sólo se había documentado la detección positiva de fitoplasmas en ejemplares del género Orosius en Australia (Padovan y Gibb, 2001), de E. papayae en Cuba (Acosta-Pérez et al., 2010; Acosta et al., 2017) y E. stevensi en Trinidad (Haque y Parasram, 1973). Sin embargo, hasta la fecha, sólo se ha demostrado experimentalmente la capacidad vectorial de E. stevensi en Trinidad (Haque y Parasram, 1973) y de E. papayae en Puerto Rico (Adsuar, 1946) y Cuba (Acosta-Pérez et al., 2010; Acosta et al., 2017).
Sobre Balclutha, en México este género forma parte de la entomofauna en agroecosistemas de Vaccinium corymbosum (Pérez-Mejía et al., 2020), Vitis vinifera (Almendra-Paxtlan et al., 2021), Capsicum annuum (Velásquez-Valle et al., 2018) y Zea mays (Pinedo-Escatel y Moya-Raygoza, 2018), pero ni el género ni alguna especie se han reportado como portadores de fitoplasmas. En otros países, sólo en ejemplares de la especie B. hebe se ha detectado fitoplasmas del grupo 16SrXIII en Brassica oleracea (Canale y Bedendo, 2020), 16SrI en Solanum tuberosum (Girsova et al., 2016), 16SrII en Trigonella foenum-graecum (Malik et al., 2020) y 16SrIX en Prunus dulcis (Dakhil et al., 2011).
Con respecto a Micrutalis, su presencia en México se ha registrado en agroecosistemas de amaranto (Amaranthus hypocondriacus) y Prosopis (Salas-Araiza y Boradonenko 2006), y la especie M. calva en Z. mays (Pinedo-Escatel, 2014). Micrutalis calva posee capacidad vectorial del Tomato pseudo-curly top virus (TPCTV) en jitomate en Florida (Simons y Coe, 1958).
Con base en la importancia que tiene la papaya en México y en otros países donde se cultiva, se determinará próximamente si B. mexicana y M. calva tienen la capacidad de transmitir fitoplasmas en papaya y en las malezas que conforman su agroecosistema.
En este estudio se reporta por primera vez la presencia de fitoplasmas 16SrI-AF, 16SrX y 16SrXIII en plantas de papaya con síntomas de Bunchy Top en agroecosistemas del estado de Colima, México. Los fitoplasmas de los grupos 16SrX y 16Sr-XIII se reportan por primera vez a nivel mundial y en México en papaya, respectivamente. Las malezas Amaranthus palmeri y Echinochloa colona, y los insectos Micrutalis calva y Balclutha mexicana, asociados a los agroecosistemas de papaya, son portadores de fitoplasmas subgrupos 16SrI-AF y 16SrI-B, lo que los constituye nuevos registros como reservorios naturales y potenciales vectores de las bacterias.
Agradecimientos
Al Servicio Nacional de Sanidad, Inocuidad y Calidad Agroalimentaria (SENASICA México) por el financiamiento de este proyecto. A los productores de papaya del estado de Colima para la recolección de las muestras. A los revisores anónimos de este manuscrito, por sus valiosos comentarios. Pedro Valadez-Ramírez agradece al Colegio de Postgraduados por el apoyo otorgado para la realización de sus estudios de doctorado.
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International Journal of Systematic Bacteriology, 48, 941−951. 10.1099/00207713-48-3-941.WhiteDTBlackallLLScottPTWalshKB1998Phylogenetic positions of phytoplasmas associated with dieback, yellow crinkle and mosaic diseases of papaya, and their proposed inclusion in ‘Candidatus Phytoplasma australiense’ and a new taxon, ‘Candidatus Phytoplasma australasia’International Journal of Systematic Bacteriology48941−951941−95110.1099/00207713-48-3-941Yu, SS, Zhu, AN, Song, WW (2023). Carica papaya represents a new host of 16SrI-B subgroup phytoplasma associated with yellow symptoms in China. Plant Disease, 107, 211. 10.1094/PDIS-03-22-0692-PDN.YuSSZhuANSongWW2023Carica papaya represents a new host of 16SrI-B subgroup phytoplasma associated with yellow symptoms in ChinaPlant Disease10721121110.1094/PDIS-03-22-0692-PDNPhytopathological NoteIdentification of phytoplasmas associated with Bunchy Top disease of papaya in Colima, MexicoAbstractBackground/Objective:
Phytoplasmas, rickettsiae and viruses have been detected in papaya plants with Bunchy Top disease (BT). In 2019, papaya plants with BTlike 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.
Mexico is the third largest producer of papaya (Carica papaya) in the world and the main supplier of fresh fruit for the United States of America and Canada, where annual sales are greater than 350,000 tons, with a value of 335 million dollars (SIAP, 2020). In this country, the production of the caricaceae is located in the South-Southeast and Center-West regions, with the states of Oaxaca, Colima and Chiapas being the main producer states (SIAP, 2020). The state of Colima, located in the West on the coast of the Pacific Ocean, has a record of papaya production of 192,417 tons and is the main national exporter (Colima State Papaya Farmers Council [COEPAPAYA A.C.], 2017; SIAP, 2020).
In 2019, in 64 papaya agroecosystems visited in four municipalities of the state of Colima, plants showed stems with shortened internodes that caused the bunching of apical leaves. These plants also displayed leaves with chlorosis, yellowing, deformation and/or marginal necrosis. These symptoms are typical of the Bunchy Top (BT) disease (Cook, 1931; Story and Halliwell, 1969), and similar to those described in Cuba as “papaya bunchy top” (PBT) and “bunchy top symptom” (BTS) syndromes (Acosta et al., 2013).
BT is induced by phytoplasmas, rickettsiae and viruses, occasionally in mixed infections (phytoplasmas + rickettsiae or phytoplasmas + viruses), and it has been reported in the Dominican Republic, Cuba, Haiti, Jamaica, Puerto Rico, Costa Rica, Peru (Martorell and Adsuar, 1952; Story and Halliwell, 1969; Davis et al., 1998; Arocha et al., 2003; Acosta et al., 2013; Luis-Pantoja et al., 2015; Wei et al., 2017), and more recently, in Nigeria (Kazeem et al., 2021).
Considering that in Mexico phytoplasmas have been found in papaya plants with symptoms similar to BT (Pogoshyan et al., 2004; Navarrete-Yabur et al., 2005; Rojas-Martínez et al., 2011), and that in Colima this disease has become more economically important and its study has been scarcely covered (Colima papaya farmers, personal communication), the following goals have been set: a) to determine the presence of phytoplasmas in papaya plants with BT symptoms, as well as in weeds and insects related with the crop, and b) to establish their identity at the 16Sr group and/or subgroup level.
In November and December of 2019, 256 papaya samples were gathered from 64 commercial agroecosystems located in the municipalities of Tecomán, Colima, Ixtlahuacán and Armería. Out of all the samples, 64 were asymptomatic and 192 displayed the symptoms described earlier, related to BT. A compound sample was taken from every plant, consisting of three leaves (blade and petiole), each one collected from the upper, middle and lower strata of the plant. From every papaya plant (symptomatic or asymptomatic sampled) up to five species of weeds present within a 2-meter diameter were gathered (depending on their availability and greater abundance). Insects were also gathered with a beating entomological net using three downward sweeps on the papaya plant and adjacent weeds. The insects were kept in 96% ethanol for later classification into morphospecies. The weeds and insects that tested positive for phytoplasmas were identified at the species level. From the central veins and petioles of papaya leaves and weeds, as well as from the entire specimens of insects (one to five, depending on size and availability), the total DNA was extracted using a phytoplasma DNA enrichment protocol based on CTAB (Ahrens and Seemuller, 1992) and with the help of a homogenizer (Tissue Lyser, Qiagen, Germany) (Stillson and Szendrei, 2020). The DNA was evaluated by UV spectrophotometry (NadoDrop 2000, Fisher Scientific, USA) and by PCR amplification of the ribosomal S16 (rps16) gene of plants (Oxelman et al., 1997) or cytochrome oxidase subunit I (COI) of insects (Folmer et al., 1994), as internal controls in reaction volumes of 25 μL with 50 ng of DNA.
The detection of phytoplasmas was performed on 200 ng of DNA by nested endpoint PCR of the 16Sr genes (first round: primers P1/P7 and nested: primers R16F2n/R16R2 and R16mF2/R16mR1) (Deng and Hiruki, 1991; Schneider et al., 1995; Gundersen and Lee, 1996; Lee et al., 1993) and secA (first round: primers secAFor1/secARev3 and nested: primers SecAFor5/SecARev2) (Dickinson and Hodgetts, 2013).
Identification was performed by sequencing, PCR-RFLPs in vitro and in silico of the gene 16Sr and phylogenetic analysis of secA and 16Sr. For sequencing, the nested PCR amplicons for 16Sr (primers R16mF2/R16mR1) and secA of the positive samples were purified with the Wizard® SV Gel and PCR Clean-Up System kit (Promega, USA) and sequenced in both directions with the primers R16mF2/ R16mR1 and SecAFor5/SecARev2, using the dideoxy or chain termination method at Macrogen Inc. (Seoul, South Korea). The resulting sequences were analyzed with the MEGAX program (Kumar et al., 2018) and the BLASTn function of the National Center for Biotechnology Information (NCBI) (https://blast.ncbi.nlm.nih. gov/Blast.cgi), for deposition in the GenBank (https://submit.ncbi.nlm.nih.gov/).
For the in vitro PCR-RFLPs, approximately 1 μg of each purified product of the nested PCR for the 16Sr gene (primers R16F2n/R16R2) of the positive samples, quantified by UV spectrophotometry (NanoDrop 2000, Fisher Scientific, USA) was conducted separately with 10 U of the restriction enzymes Taq I, Hae III, Sau 3AI, Kpn I, Tru I1 (Mse I), Alu I and Rsa I (Thermo Scientific, USA), following the instructions by the manufacturer to confirm that the amplified fragment corresponded to the DNA of phytoplasmas (Lee et al., 1998). The products were separated by electrophoresis in 3% agarose gels (w/v) in a 1×TAE buffer and dyed with ethidium bromide (Sambrook and Russell, 2001).
For the in silico RFLPs, the sequences of the amplicons of the nested PCR (primers R16mF2/R16mR1), edited manually with MEGAX (Kumar et al., 2018) were analyzed using the software iPhyClassifier (https://acortar.link/AikrtQ) to assign and/or confirm species, 16Sr groups and subgroups.
The phylogenetic analyses were carried out independently with the secA and 16Sr genes. To do this, the sequences of each one of the analyzed genes from different groups of phytoplasmas reported worldwide (49 for secA and 51 for 16Sr) and from Bacillus subtilis (GenBank: X62035 for secA) or from Acholeplasma laidlawii (GenBank: U14905 for 16Sr) as outgroups were downloaded from the GenBank and aligned with Clustal W. Phylogenies were built with the neighborjoining method with 1000 bootstrap repetitions. All analyses were carried out in MEGA X (Kumar et al., 2018).
Phytoplasmas were detected in 2.08% (4 out of 192) papaya plants with symptoms related to BT (Table 1). No phytoplasmas were found in asymptomatic plants. The positive plants were gathered in the municipalities of Colima and Ixtlahuacán (Table 1) and they displayed stems with shortened internodes and yellow chlorotic leaves (Figure 1A). Positive samples were confirmed by sequencing the secA (GenBank: ON303285-ON303288) and 16Sr (GenBank: PP348058-PP348061) genes. Sequence analysis of the secA and 16Sr genes with BLASTn indicate that, in two of the four positive samples, the phytoplasmas belong to the 16SrI group (GenBank secA: ON303286 and ON303287; 16Sr: PP348059 and PP348060), whereas the remaining two belong to the 16SrX (GenBank secA: ON303288; 16Sr: PP348061) and 16SrXIII (GenBank secA: ON303285; 16Sr: PP348058) groups, respectively.
Detection of phytoplasmas in papaya (<italic>Carica papaya</italic>), weeds and insects associated with the crop in Colima, Mexico, in November and December of 2019.
Municipality
Number of positive samples to phytoplasmas / Number of analyzed samples (%)
Papaya
Weeds
Insects
Armería
0/64 (0.0)
1/74 (1.4)
1/60 (1.7)
Colima
2/64 (3.1)
0/31 (0.0)
0/32 (0.0)
Ixtlahuacán
2/64 (3.1)
1/35 (2.9)
0/46 (0.0)
Tecomán
0/64 (0.0)
1/34 (2.9)
1/47 (2.1)
Total
4/256 (1.6)
3/174 (1.7)
2/185 (1.1)
Detection of phytoplasmas in agroecosystems papaya with Bunchy Top disease in Colima, Mexico. <bold>A)</bold> Papaya plants showing stems with shortened internodes and yellow, chlorotic leaves, positive to phytoplasmas. <bold>B)</bold>
<italic>In vitro</italic> RFLPs profiles of gene <italic>16Sr</italic> (primers R16F2n/R16R2) of the phytoplasmas under study. Lanes 1-4: samples 1S-4Spapaya, Lanes 5-6: samples 5S-6S-<italic>Amaranthus palmeri</italic>; Lane 7: sample 7S-<italic>Echinochloa colona</italic>; Lane 8: sample 8S-<italic>Micrutalis calva</italic>; and Lane 9: sample 9S-<italic>Balclutha mexicana</italic>. 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).
The profiles of the in vitro RFLPs of the 16Sr gene (Figure 1B) were consistent with those reported by Lee et al. (1998) and with those obtained using iPhyClassifer (Figure 2) for the restriction enzymes used, which helped authenticate the DNA of the phytoplasmas found in this study. The in silico RFLPs analysis of the 16Sr gene confirmed the presence of the ‘Candidatus Phytoplasma asteris’ [group 16SrI (percentages of identity: 99.44 and 99.60) species and subgroup 16SrI AF (coefficient of similarity: 1.00)], ‘Ca. Phytoplasma rhamni’ [group 16SrX (percentage of identity: 93.31), undetermined subgroup] and ‘Ca. Phytoplasma hispanicum’ [group 16SrXIII (percentage of identity: 99.37), subgroup 16SrXIII-D (coefficient of similarity: 0.95)] (Table 2). Regarding the percentages of identity
<italic>In silico</italic> RFLPs profiles of gene <italic>16Sr</italic> of the phytoplasmas under study, generated with <italic>i</italic>PhyClassifier with 17 restriction enzymes. A) sample 1S-papaya; B) sample 2S-papaya; C) sample 3S-papaya; D) sample 4S-papaya, E) sample 5S-<italic>Amaranthus palmeri</italic>; F) sample 6S-<italic>Amaranthus palmeri</italic>; G) sample 7S-<italic>Echinochloa colona</italic>; H) sample 8S-<italic>Micrutalis calva</italic>; I) sample 9S-<italic>Balclutha mexicana</italic>. Lanes MW: <italic>φ</italic>X174 DNA digested with <italic>Hae</italic> III.
Phytoplasmas identified in papaya, weeds and insects associated with the crop in Colima, Mexico, in NovemberDecember of 2019.
Sample (GenBank 16Sr)
‘Candidatus Phytoplasma sp.’
Group 16Sr (% similarity)
Subgroup (coefficient of similarity)
1S-papaya (PP348058)
‘Ca. Phytoplasma hispanicum’
16SrXIII (99.37)
D (0.95)y
2S-papaya (PP348059)
‘Ca. Phytoplasma asteris’
16SrI (99.44)
AF (1.00)
3S-papaya (PP348060)
‘Ca. Phytoplasma asteris’
16SrI (99.60)
AF (1.00)
4S-papaya (PP348061)
‘Ca. Phytoplasma rhamni’
16SrX (93.31)
NAz
5S-Amaranthus palmeri (PP348062)
‘Ca. Phytoplasma asteris’
16SrI (99.44)
AF (1.00)
6S-Amaranthus palmeri (PP348063)
‘Ca. Phytoplasma asteris’
16SrI (99.68)
B (0.98)
7S-Echinochloa colona (PP348064)
‘Ca. Phytoplasma asteris’
16SrI (99.60)
AF (1.00)
8S-Micrutalis calva (PP348065)
‘Ca. Phytoplasma asteris’
16SrI (99.52)
AF (1.00)
9S-Balclutha mexicana (PP348066)
‘Ca. Phytoplasma asteris’
16SrI (99.68)
B (0.98)
yPotential new subgroup.
zNA: does not apply due to the low percentage of identity of the group assigned in iPhyClassifier.
and coefficients of similarity obtained for the phytoplasmas of groups 16SrX and 16SrXIII, this will have to be analyzed with other studies, if we are dealing with new (sub)groups.
The identification of the groups of phytoplasmas obtained here was also corroborated by phylogenetic inferences of the secA and 16Sr genes, as the phylogenies obtained for both genes of the phytoplasmas detected in this study grouped with sequences corresponding to the same 16Sr groups of phytoplasmas previously identified in Mexico (GenBank 16Sr: MN807429) or in other countries (Figure 3).
To our knowledge, this study is the first to document the presence of 16SrIAF, 16SrX and 16SrXIII phytoplasmas in papaya plants with BT in Mexico and Colima. In addition, globally, it is the first time that the presence of the 16SrX has been reported in papaya. In Mexico, the first detection of phytoplasmas in papaya was made by scanning electron microscopy in Baja California Sur (Poghosyan et al., 2004). Subsequently, via molecular biology in Yucatan, Campeche, Quintana Roo (Navarrete-Yabur et al., 2005), Michoacan and Veracruz, where the positive samples were assigned to the subgroup 16SrI-C (Rojas-Martínez et al., 2011).
Along with its detection in Mexico, the group 16SrI has been reported in papaya in Cuba (Acosta et al., 2011; 2017; Acosta-Pérez et al., 2017), Peru (Hodgetts et al., 2009), Sri Lanka (Abeysinghe et al., 2014) and China (Yu et al., 2023), whereas 16SrXIII, only in Brazil (Melo et al., 2013). Other groups of economic importance in papaya are 16SrII (White et al., 1998; Panda et al., 2022), 16SrXII (Gibb et al., 1996; Barbieri et al., 2023), 16SrXV (Wei et al., 2017) and 16SrXVII (Arocha et al., 2005).
Phylogenetic trees of the <italic>secA</italic> (A) and <italic>16Sr</italic> (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: <italic>Amaranthus palmeri</italic>, sample 7S: <italic>Echinochloa colona</italic>, sample 8S: <italic>Micrutalis calva</italic>, sample 9S: <italic>Balclutha mexicana</italic>. The gene sequence of <italic>secA</italic> of <italic>Bacillus subtilis</italic> and the <italic>16Sr</italic> gene sequence of <italic>Acholeplasma laidlawii</italic> 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.
In Mexico, the presence of group 16SrX has never been reported (PogoshyanMelkonyan et al., 2019), whereas 16SrXIII has only been associated with potato and tomato plants in Sinaloa (Santos-Cervantes et al., 2010) and Baja California Sur (Holguín-Peña et al., 2007). In the future, whether the low percentage of the detection of phytoplasmas is due to the presence of other organisms such as rickettsiae or viruses that may be inducing the studied symptoms is something that must be determined (Davis et al., 1998; Arocha et al., 2003; Acosta et al., 2013; Luis-Pantoja et al., 2015).
In the case of weeds and insects, the detection of phytoplasmas was positive in three out of 174 (1.7%) and two out of 185 (1.1%) samples, respectively, and with the exception of Ixtlahuacán, the positive samples were obtained regardless of not having recorded phytoplasmas in papaya plants (Table 1). Colima was the only municipality in which there were no weeds nor insects that were positive to phytoplasmas (Table 1).
The weeds in which phytoplasmas were found were asymptomatic, two of which were identified as Amaranthus palmeri (Amaranthaceae) (GenBank 16Sr: PP348062-PP348063, secA: ON303289-ON303290), and the other one, as Echinochloa colona (Poaceae) (GenBank 16Sr: PP348064, secA; ON303291. The insects were identified as Micrutalis calva (Hemiptera: Membracidae) (GenBank 16Sr: PP348065, secA: ON303292) and Balclutha mexicana (Hemiptera: Cicadellidae) (GenBank 16Sr: PP348066, secA: ON303292) (Table 2).
The in silico RFLPs analyses of the 16Sr gene helped assign the phytoplasmas detected to the species ‘Ca. Phytoplasma asteris’ (group 16SrI, percentage of identity: 99.44-99.68), subgroups 16SrI-AF (coefficient of similarity: 1.00) in A. palmeri (sample 5S), E. colona (sample 7S) and M. calva (sample 8S), and 16SrI-B (coefficient of similarity: 0.98) in A. palmeri (sample 6S) and B. mexicana (sample 9S) (Table 2).
The detection of phytoplasmas in insects and weeds related with the papaya agroecosystems suggests that the arthropods spread bacteria between plants and that weeds can play an important part as a source of inoculum (Duduk et al., 2018). For Mexico, this investigation pioneers in questioning whether weeds and insects related to papaya are carriers/sources of phytoplasma inoculum, since it is the first time they are reported as carrying species of the group 16SrI phytoplasmas in papaya agroecosystems. Weeds add to the records of those that have been associated with papaya crops and have been reported as hosts of 16SrII phytoplasmas in Cuba and Ethiopia, such as Anoda acerifolia (Malvaceae), Euphorbia heterophylla (Euphorbiaceae), Malvastrum coromandelianum (Malvaceae) and Rhynchosia minima (Fabaceae) (Arocha et al., 2007; Bekele et al., 2011). Although A. palmeri is a common weed in different Mexican agroecosystems, it has only been reported as a host of phytoplasmas of 16SrIII group phytoplasmas as a weed associated with the cultivation of luffa (Luffa acutangula) (Cucurbitaceae) (Santos-Cervantes et al., 2021).
Regarding insects B. mexicana and M. calva, it is the first time these are recorded as carriers of phytoplasmas in papaya agroecosystems. Previously, the positive detection of phytoplasmas was reported in specimens of the genus Orosius in Australia (Padovan and Gibb, 2001), E. papayae in Cuba (Acosta-Pérez et al., 2010; Acosta et al., 2017) and E. stevensi in Trinidad (Haque and Parasram, 1973). However, to date, only the vectorial capacity of E. stevensi in Trinidad (Haque and Parasram, 1973) and E. papayae in Puerto Rico (Adsuar, 1946) and Cuba (AcostaPérez et al., 2010; Acosta et al., 2017) has been proven.
Regarding Balclutha, in Mexico this genus is part of the entomofauna in Vaccinium corymbosum (Pérez-Mejía et al., 2020), Vitis vinifera (AlmendraPaxtlan et al., 2021), Capsicum annuum (Velásquez-Valle et al., 2018) and Zea mays (Pinedo-Escatel and Moya-Raygoza, 2018) agroecosystems, but neither the genus nor any species have been reported as carriers of phytoplasmas. In other countries, only in specimens of the B. hebe species have phytoplasmas of group 16SrXIII been found in Brassica oleracea (Canale and Bedendo, 2020), 16SrI in Solanum tuberosum (Girsova et al., 2016), 16SrII in Trigonella foenum-graecum (Malik et al., 2020) and 16SrIX in Prunus dulcis (Dakhil et al., 2011).
Regarding Micrutalis, its presence in Mexico has been recorded in amaranth (Amaranthus hypocondriacus) and Prosopis agroecosystems (Salas-Araiza and Boradonenko 2006), and the species M. calva in Z. mays (Pinedo-Escatel, 2014). Micrutalis calva has the vectorial capacity for the Tomato pseudo-curly top virus (TPCTV) in tomato in Florida (Simons and Coe, 1958).
Based on the importance of papaya in Mexico and other countries in which it is planted, it will soon be determined if B. mexicana and M. calva have the capacity to transmit phytoplasmas in papaya and in the weeds that make up its agroecosystem. This study reports for the first time the presence of phytoplasmas 16SrI-AF, 16SrX and 16SrXIII in papaya plants with symptoms of Bunchy Top disease in agroecosystems in the state of Colima, Mexico. The phytoplasmas of groups 16SrX and 16SrXIII are reported for the first time worldwide and in Mexico in papaya, respectively. The weeds Amaranthus palmeri and Echinochloa colona, and the insects Micrutalis calva and Balclutha mexicana, associated with papaya agroecosystems, are carriers of phytoplasma subgroups 16SrI-AF and 16SrI-B, making them new records as natural reservoirs and potential vectors of the bacteria.
Acknowledgments
To the National Agrifood Health, Safety and Quality Service (SENASICA Mexico) for funding this project. To the papaya farmers of the state of Colima for the collection of the samples. To the anonymous reviewers of this manuscript for their valuable comments. Pedro Valadez-Ramírez thanks the Colegio de Postgraduados for the support provided to carry out his doctoral studies.