Canadian Journal of Plant Pathology

ISSN: 0706-0661 (Print) 1715-2992 (Online) Journal homepage: http://www.tandfonline.com/loi/tcjp20

Detection and identification of the coconut lethal yellowing phytoplasma in weeds growing in coconut farms in Côte d’Ivoire

Y. Arocha Rosete, H. ATTA Diallo, J.L. Konan Konan, A.E.P. Kouamé, K. Séka, K.D. Kra, M.N. Toualy, K.E. Kwadjo, W.A.M.P. Daramcoum, N.I. Beugré, B.W.M. Ouattara, C.G. Kouadjo Zaka, K. Allou, N.D. Fursy-Rodelec, O.N. Doudjo- Ouattara, N. Yankey, S. Dery, A. Maharaj, M. Saleh, R. Summerbell, N. Contaldo, S. Paltrinieri, A. Bertaccini & J. Scott

To cite this article: Y. Arocha Rosete, H. ATTA Diallo, J.L. Konan Konan, A.E.P. Kouamé, K. Séka, K.D. Kra, M.N. Toualy, K.E. Kwadjo, W.A.M.P. Daramcoum, N.I. Beugré, B.W.M. Ouattara, C.G. Kouadjo Zaka, K. Allou, N.D. Fursy-Rodelec, O.N. Doudjo-Ouattara, N. Yankey, S. Dery, A. Maharaj, M. Saleh, R. Summerbell, N. Contaldo, S. Paltrinieri, A. Bertaccini & J. Scott (2016) Detection and identification of the coconut lethal yellowing phytoplasma in weeds growing in coconut farms in Côte d’Ivoire, Canadian Journal of Plant Pathology, 38:2, 164-173, DOI: 10.1080/07060661.2016.1191044 To link to this article: http://dx.doi.org/10.1080/07060661.2016.1191044

Accepted author version posted online: 19 Submit your article to this journal May 2016. Published online: 20 Jun 2016.

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Download by: [University of Toronto Libraries] Date: 14 February 2017, At: 08:27 Can. J. Plant Pathol., 2016 Vol. 38, No. 2, 164–173, http://dx.doi.org/10.1080/07060661.2016.1191044

Bacteria and phytoplasmas/Bactèries et phytoplasmes

Detection and identification of the coconut lethal yellowing phytoplasma in weeds growing in coconut farms in Côte d’Ivoire

Y. AROCHA ROSETE1, H. ATTA DIALLO2, J.L. KONAN KONAN3, A.E.P. KOUAMÉ2, K. SÉKA2, K.D. KRA2, M.N. TOUALY2, K.E. KWADJO2, W.A.M.P. DARAMCOUM3, N.I. BEUGRÉ3, B.W.M. OUATTARA2, C.G. KOUADJO ZAKA3, K. ALLOU3, N.D. FURSY-RODELEC2, O.N. DOUDJO-OUATTARA4, N. YANKEY5, S. DERY5, A. MAHARAJ1, M. SALEH1, R. SUMMERBELL1, N. CONTALDO6, S. PALTRINIERI6, A. BERTACCINI6 AND J. SCOTT1,7

1Sporometrics, 219 Dufferin Street, Suite 20C, Toronto, ON M6K 3J1, Canada 2Université Nangui Abrogoua, 02 BP 801, Abidjan 02, Abidjan, Côte d’Ivoire 3Station de Recherche ‘Marc Delorme’, Centre National de Recherche Agronomique (CNRA), Abidjan, 07 BP 13, Côte d’Ivoire 4Swiss Center of Scientific Research, Abidjan, Côte d’Ivoire 5Council for Scientific Research Program–Oil Palm Research Institute–Coconut Research Program (CSIR-OPRI), Off Well Road Box 254, Sekondi, Western Region, Ghana 6Department of Agricultural Sciences, Alma Mater Studiorum, University of Bologna viale Fanin 42, Bologna, 40127, Italy 7Department of Agricultural Sciences, Plant Pathology, Alma Mater Studiorum, University of Bologna, viale G. Fanin 42, 40127 Bologna, Italy

(Accepted 15 May 2016)

Abstract: Coconut farms located in the southern coast of Grand-Lahou in Côte d’Ivoire are severely affected by a lethal yellowing disease (CILY) associated with the group 16SrXXII-B, ‘Candidatus Phytoplasma palmicola’-related strains. Given the high prevalence of weed species on most of the farms, plants growing within and in the periphery of five selected coconut farms were assessed for the presence of the CILY phytoplasma to identify potential alternative hosts. A total of 396 plant samples belonging to 84 plant species and 35 botanical families were collected. Total DNA was extracted and tested by nested PCR with primers targeting the 16S rRNA and the translocation protein (secA) phytoplasma genes, and sequenced. Twenty samples from six plant species and five botanical families yielded PCR amplicons of the expected size, and both the secA and 16S rDNA sequences showed over 99% similarity with that of the Côte d’Ivoire lethal yellowing phytoplasma previously identified from coconut palms grown in Grand-Lahou coconut farms. Plant species from the families Poaceae (Paspalum vaginatum, Pennisetum pedicillatum), Verbenaceae (Stachytarpheta indica), Plantaginaceae (Scoparia dulcis), Phyllanthaceae (Phyllantus muellerianus) and Cyperacea (Diplacrum capitatum) were positive for the presence of the CILY phytoplasma, suggesting they may have epidemiological implications for disease spread in coconut farms in Grand-Lahou.

Keywords: 16S rRNA, 16SrXXII phytoplasma, alternative hosts, ‘Candidatus Phytoplasma palmicola’-related strains, coconut, Côte d’Ivoire lethal yellowing, secA

Résumé: Les exploitations agricoles de la côte sud de Grand-Lahou, en Côte d’Ivoire, où l’on cultive la noix de coco, sont gravement touchées par le jaunissement mortel (JM) associé au groupe 16SrXXII-B des souches apparentées à ‘Candidatus Phytoplasma palmicola’. Étant donnée la forte prévalence des mauvaises herbes sur la plupart des fermes, des plantes poussant dans cinq exploitations ciblées ou en périphérie de celles-ci ont été évaluées au regard du phytoplasme du JM afind’identifier de possibles hôtes alternatifs. En tout, 396 échantillons de plantes appartenant à 84 espèces et 35 familles botaniques ont été collectés. L’ADN total a été extrait et testé par PCR par amorces incluses ciblant l’ARNr 16S et les gènes du phytoplasme de la protéine de translocation (secA), puis séquencé. En tout, 20 échantillons provenant de 6 espèces

Correspondence to: Y. Arocha Rosete. E-mail: [email protected]

© 2016 The Canadian Phytopathological Society Detection and identification of a CILY phytoplasma 165 de plantes et de 5 familles botaniques ont fourni des amplicons de taille attendue découlant de la PCR, et les séquences de la secA ainsi que de l’ARNr 16S ont affiché une similitude de 99% avec celles du phytoplasme du jaunissement mortel de la Côte d’Ivoire préalablement identifié chez les cocotiers cultivés à Grand-Lahou. Des espèces de plantes de la famille des Poaceae (Paspalum vaginatum, Pennisetum pedicillatum), des Verbenaceae (Stachytarpheta indica), des Plantaginaceae (Scoparia dulcis), des Phyllanthaceae (Phyllantus muellerianus) et des Cyperacea (Diplacrum capitatum) se sont révélées positives quant à l’occurrence du phytoplasme du JM, ce qui suggère qu’elles peuvent avoir des implications épidémiologiques quant à la propagation de la maladie dans les exploitations de Grand-Lahou.

Mots clés: ARNr 16S, cocotier, hôtes alternatifs, jaunissement mortel de la Côte d’, Ivoire, phytoplasme 16SrXXII, secA, souches apparentées à ‘Candidatus Phytoplasma palmicola’

fi Introduction conserved nature of the 16S rRNA gene makes it dif cult to discriminate among closely related strains (Duduk & Phytoplasmas are cell wall-less prokaryotic intracellular Bertaccini 2011; Marcone 2014). Therefore, assays based plant pathogens belonging to the class Mollicutes (Marcone on non-ribosomal target genes such as the secAgeneare 2014) which originated from Gram-positive bacteria. Lethal beingused(Dickinson&Hodgetts2013;Yankeyetal.2014; yellowing (LY)-like or Lethal Decline (LD)-like phyto- Valiunas et al. 2015) as a support to the 16S rRNA gene- plasma diseases have decimated millions of coconut groves based identification and classification system, and to distin- and affected livelihoods of thousands of smallholder farm guish among closely related phytoplasmas. communities that depend on coconut palm trees in Africa, Phytoplasmas possess a double life cycle that involves Central America and the Caribbean (Danyo 2011; Sullivan replication in phloem-feeding Hemipteran insects and plant &Harrison2013). Côte d’Ivoire lethal yellowing (CILY) hosts. The latter involve over 1000 plant species, including disease is rapidly expanding into new coconut-growing vil- crops of economic importance worldwide (Duduk & lages of Grand-Lahou, a coastal commune in southern Côte Bertaccini 2011). Phytoplasma vectors and alternative host d’Ivoire, and sub-prefecture of the Grand-Lahou Department plants play an active role in the development of epidemics in the Lagunes District. The disease has already destroyed (Lee et al. 2003; Weintraub & Beanland 2006). Alternative over 400 hectares of coconut groves and continues to expand host plants of phytoplasma-associated diseases have been from the most CILY-affected areas (Arocha-Rosete et al. identified worldwide, and include either cultivated species 2014). Moreover, the destruction of the coconut palms or weeds. Examples of phytoplasma diseases whose sec- makes lands previously covered more exposed and therefore ondary plant hosts have been identified include the alfalfa more prone to degradation, which adds serious environmen- witches’ broom phytoplasma in Iran (Esmailzadeh-Hosseini tal repercussions (Yankey et al. 2009). et al. 2011), Napier grass stunt phytoplasma in East Africa CILY symptoms resemble those of the Cape St. Paul Wilt (Arocha & Jones 2010), and 'bois noir' phytoplasma in Disease (CSPWD) that wreaked great havoc to the coconut grapevine in Italy (Mori et al. 2015). industry in Ghana during the last 30 years (Eziashi & Plant species found within surrounding crop fields Omamor 2010;Danyo2011). The CILY phytoplasma has have been shown to play a role in spreading phytoplasma become a phytosanitary risk for the Ivorian coconut industry diseases, as they may act as alternative reservoirs for the (Arocha-Rosete et al. 2014). Based on the analysis of the 16S phytoplasmas (Weintraub & Beanland 2006) or their rRNA gene, it was identified as a member of the subgroup insect vectors (Mori et al. 2015). This makes the design 16SrXXII-B (Arocha-Rosete et al. 2014), officially and implementation of effective disease control strategies described as ‘Candidatus Phytoplasma palmicola’-related very difficult. So far, no alternative plant reservoirs have strains (Harrison et al. 2014) along with the CSPWD strain been identified for the CSPWD or the CILY phytoplasma from Ghana. Both CILY and CSPWD phytoplasmas have strains. This paper reports on the detection and identifica- been shown to be closely related to the phytoplasma strains tion of the CILY phytoplasma in plant species growing in associated with ‘Awka Wilt’ in Nigeria and LY in CILY-affected coconut farms of Grand-Lahou. Mozambique (LYM), which form the subgroup 16SrXXII- A, officially described as ‘Candidatus Phytoplasma palmi- cola’ (Harrison et al. 2014). DNA-based technology is the Materials and methods best approach for the detection, identification and character- Plant sampling ization of phytoplasmas, and relies on the analyses of the 16S rRNA sequences, and their actual and/or virtual RFLP Non-coconut plant species growing within and adjacent to (restriction fragment length polymorphism) profiles (Lee five coconut farms were randomly sampled during 2015. et al. 1998; IRPCM 2004; Wei et al. 2007). However, the Five individual plants were collected per species in each of Y. A. Rosete et al. 166 the CILY-affected villages: Braffedon, Palmindustrie V1, (Thompson et al. 1994) and phylogenetic trees were con- Adjadon, Yaokro and Badadon, at distances of 18 km, structed using the neighbour-joining method with the pro- 26 km, 34 km, 41 km and 48 km, respectively, from gram MEGA version 6.0 (Tamura et al. 2013) with default Grand-Lahou. Plant samples were placed in labelled plastic values and 1000 replicates for bootstrap analysis. bags and transported to the laboratory in a cooler with ice packs for further testing. Plant species collected were bota- nically identified at the University of Nangui Abrogoua Results (Abidjan) and the Swiss Center of Scientific Research in Plant sampling Côte d’Ivoire (Hutchinson & Dalziel 1972;Poilecot1995; fi Aké 2002; Hawthorne & Jongkind 2006). A total of 396 individual plants were collected from the ve coconut-growing villages of Grand-Lahou (Table 1): Braffedon (84); Badadon (163); Palmindustrie V1 (109); Polymerase chain reaction (PCR) Yaokro (25); and Adjadon (15), corresponding to 84 plant species and 35 botanical families, from which species from Leaves of the plant species collected were disinfected around 15 families have been previously identified as reser- with 70% ethanol, and midribs were separated from voirs for diverse phytoplasmas worldwide (Arocha & Jones leaves using a sterile scalpel. Total DNA was extracted 2010). using a CTAB small-scale protocol from 1 g of finely chopped midribs (Doyle & Doyle 1990), and used as a template for nested PCR assays with universal primers Detection of CILY phytoplasma that target the phytoplasma 16S rRNA gene, the inter- Phytoplasma DNA was amplified from 20 samples corre- genic spacer and the beginning of the 23S rRNA. Primers sponding to six plant species and five botanical families included P1/P7 (Deng & Hiruki 1991; Schneider et al. with G813/AwkaSR primers (yielding amplicons of 1995) for the direct PCR reaction and G813/AwkaSR approximately 875 bp), from which 19 produced secA (Tymon et al. 1998) for the nested reaction. amplicons (of approximately 600 bp). CILY phytoplasma For all PCR reactions, 50 ng of DNA template were added detection was consistent and reproducible from all samples

to a 25 µL PCR reaction (GE Healthcare, UK). For the nested that yielded both G813/AwkaSR and secA amplicons. Only reaction, 1 µL of the 40-fold diluted direct PCR product was three out of the 20 samples that gave positive results with used. Primers SecAfor1⁄SecArev3 (direct PCR) and the other assays also yielded P1/P7 amplifications. Cox SecAfor5⁄SecArev2 (nested reaction) were used to amplify primers yielded amplification from 387 out of the 396 plants the secA gene that encodes the protein translocase subunit collected, including all samples giving positive results. Nine (Hodgetts et al. 2008). One microlitre of the direct PCR samples did not produce any amplicons with both primer product was diluted 30-fold and used as a DNA template combinations G813/AwkaSR and CoxF3/B3 corresponding for the nested PCR (Dickinson & Hodgetts 2013). Samples to Newbouldia laevis, Ixora sp. and Paullinia pinnata were also tested with primers CoxF3 and CoxB3 that amplify (Braffedon), N. laevis, Albertisia cordifolia and Cleome the plant cytochrome oxidase (cox) gene to assess the pre- ciliate (Badadon), Dichapetalum heudelotii and Dissotis sence of PCR inhibitors (Dickinson & Hodgetts 2013). Five rotundifolia (Palmindustrie V1). This suggested the pre- microlitres of the PCR products were separated in a 1.5% sence of possible PCR inhibitors in those samples, so they agarose gel, and visualized with SYBR Safe (Invitrogen, were not considered for further analyses. USA), in a UV gel documenter (Alpha Innotech, USA). Samples that gave positive results with the G813/AwkaSR assay included the following: (i) from Yaokro village: Stachytarpheta indica L. Vahl (family Verbenaceae) Sequence analysis (Fig. 1A); Scoparia dulcis L. (family Plantaginaceae) Representative G813/AwkaSR PCR amplicons were puri- (Fig. 1B); Diplacrum capitatum (Willd.) Boeckeler (family fied on spin columns (Omega Bio-Tek, USA), cloned using Cyperacea) (Fig. 1F); (ii) from Adjadon village: Paspalum p-GEMT Easy Vector (Promega, USA), and sequenced bi- vaginatum Sw. (family Poaceae) (Fig. 1C); and (iii) from directionally. The consensus G813/AwkaSR sequences Badadon, Braffedon and Palmindustrie V1 villages: were blasted (Altschul et al. 1990)atNCBI(http://www. Pennisetum pedicillatum Trin (family Poaceae) (Fig. 1D), ncbi.nlm.nih.gov) for phytoplasma sequence homology. and Phyllanthus muellerianus L. (family Phyllanthaceae) Sequences obtained were aligned using Clustal W (Fig. 1E). Detection and identification of a CILY phytoplasma 167

Table 1. List of plant species sampled from CILY-affected coconut farms in Grand-Lahou. Location Botanical ID Botanical Family CILY phytoplasma positive/No. plants collected

Braffedon Newbouldia laevis Bignoniaceae 0/3 Nelsonia canescens Acanthaceae 0/5 Paspalum sp. Poaceae 0/5 Pennisetum pedicellatum Poaceae 2/5 Rottboellia cochinchinensis Poaceae 0/5 Eragrostis tenella Poaceae 0/5 Panicum sp. Poaceae 0/5 Ageratum conyzoides Asteraceae 0/5 Chromolaena odorata Asteraceae 0/5 Vernonia cinerea Asteraceae 0/5 Schwenckia americana Solanaceae 0/5 Flagellaria paniculata Flagellariaceae 0/3 Desmodium adscendens Fabaceae 0/5 Dialium guineense Fabaceae 0/5 Sida acuta Malvaceae 0/5 Triumphetta romboidea Malvaceae 0/5 Kyllinga africana Cyperaceae 0/5 Paullinia pinnata Sapindacea 0/3 Zehneria capillacea Cucurbitaceae 0/5 Achyranthes aspera Amanranthaceae 0/5 Phyllanthus muellerianus Phyllanthaceae 3/5 Adenia lobata Passifloraceae 0/5 Adenia cissampeloides Passifloraceae 0/5 Nauclea latifolia Rubiaceae 0/5 Morinda lucida Rubiaceae 0/5 Ixora sp. Rubiaceae 0/5 Badadon Cassia occidentalis Fabaceae 0/5 Dalbergia saxatilis Fabaceae 0/5 Millettia zechiana Fabaceae 0/5 Albizia adianthifolia Fabaceae 0/5 Baphia nitida Fabaceae 0/5 Phyllanthus reticulatus Phyllanthaceae 0/5 Phyllanthus muellerianus Phyllanthaceae 2/5 Phyllanthus amarus Phyllanthaceae 0/5 Triumfetta rhomboidea Malvaceae 0/5 Hibiscus tiliaceus Malvaceae 0/5 Catharanthus roseus Apocynaceae 0/5 Rauvolfia vomitoria Apocynaceae 0/5 Momordica charantia Cucurbitaceae 0/5 Croton lobatus Euphorbiaceae 0/5 Ricinus communis Euphorbiaceae 0/5 Passiflora foetida Passifloraceae 0/5 Cleome ciliate Cleomaceae 0/3 Ipomoea sp. Convolvulaceae 0/5 Celosia trigyna Amaranthaceae 0/5 Chromolaena odorata Asteraceae 0/5 Vernonia cinerea Asteraceae 0/5 Cyperus sp. Cyperaceae 0/5 Ocimum gratissimum Lamiaceae 0/3 Blighia welwitschii Sapindaceae 0/3 Albertisia cordifolia Menispermaceae 0/3 Waltheria indica Malvaceae 0/5 Mitracarpus scaber Rubiaceae 0/5 Cnestis ferruginea Connaraceae 0/3 Morinda morindoides Rubiaceae 0/5 Lantana camara Verbenaceae 0/5 Sterculia tragacantha Sterculiaceae 0/3 Newbouldia laevis Bignoniaceae 0/3 Anchomanes difformis Araceae 0/5 Kohautia senegalensis Rubiaceae 0/5 Oplismenus burmannii Poaceae 0/5 Pennisetum pedicellatum Poaceae 3/5

(Continued) Y. A. Rosete et al. 168

Table 1. (Continued.)

Location Botanical ID Botanical Family CILY phytoplasma positive/No. plants collected

Palmindustrie V1 Dissotis rotundifolia Melastomataceae 0/3 Pennisetum pedicellatum Poaceae 3/5 Digitaria horizontale Poaceae 0/5 Rauvolfia vomitoria Apocynaceae 0/5 Landolphia hirsuta Apocynaceae 0/5 Chromolaena odorata Asteraceae 0/5 Leptoderris miegei Fabaceae 0/5 Puearia phaseoloides Fabaceae 0/5 Centrosema pubescens Fabaceae 0/5 Cyclosorus oppositifolius Thelypteridaceae 0/3 Scleria sp. Cyperaceae 0/5 Chrysophyllum welwitschii Sapotaceae 0/3 Alchornea cordifolia Euphorbiaceae 0/5 Diodia rubricosa Rubiaceae 0/5 Dissotis rotundifolia Melastomataceae 0/3 Crossostemma laurifolium Passifloraceae 0/5 Chrysobalanus icaco Chrysobalanaceae 0/3 Nephrolepis bisserata Lomariopsidaceae 0/3 Clerodendrum splendens Lamiaceae 0/3 Ipomoea involucrata Convolvulaceae 0/5 Dichapetalum heudelotii Dichapetalaceae 0/3 Scleria boivnii Cyperaceae 0/5 Althernanthera brasiliensis Amanranthaceae 0/5 Parquetina nigrescens Asclepiadaceae 0/5 Glyphaea brevis Malvaceae 0/5 Yaokro Lantana camara Verbenaceae 0/5 Stachytarpheta indica Verbenaceae 3/5 Pterididium aquilinium Dennstaedtiaceae 0/5 Scoparia dulcis Plantaginaceae 2/5 Diplacrum capitatum Cyperaceae 1/5 Adjadon Panicum maximum Poaceae 0/5 Paspalum vaginatum Poaceae 1/5 Catharanthus roseus Apocynaceae 0/5 Total 20/396

Text in bold corresponds to plant species that host the CILY phytoplasma.

Sequence and phylogenetic analysis The phylogenetic trees based on the 16S rDNA (Fig. 2) and secA(Fig. 3) sequences showed that the CILY phyto- The BLAST analysis showed that the respective G813/ plasma strains detected in samples from the five botanical AwkaSR and secA sequences of the CILY phytoplasmas families grouped within the same subgroup 16SrXX-B, identified from the weeds S. indica (KU644563; enclosing the CILYphytoplasma strains previously reported KU644569), S. dulcis (KU644564; KU644570), D. capita- in the CILY-affected Grand-Lahou villages, and the tum (KU644565; KU644571), P. vaginatum (KU644566; CSPWD phytoplasma from Ghana, which form the ‘Ca.P. KU644572), P. pedicillatum (KU644567; KU644573) and palmicola’-related strains lineage, closely related to P. muellerianus (KU644568; KU644574) shared 100% 16SrXXII-A ‘Ca.P.palmicola’ (Harrison et al. 2014). sequence identity to each other and over 99% with that of the CSPWD phytoplasma strain from Ghana (Harrison et al. 2014) and CILY phytoplasma strains previously identified in Discussion the Grand-Lahou villages of Adjadon (KU216443; KU304343), Amanikro (KU216447), Badadon Among the 35 botanical families identified in the natural (KU216451; KU304348), Braffedon (KU216444; vegetation present between rows and beneath the coconut KU304346), Palmindustrie V1 (KU216456; KU304353) tree canopies in the farms of the five villages of Grand-Lahou, and Yaokro (KU216441; KU304337). the highest number of plant species collected belonged to the Detection and identification of a CILY phytoplasma 169

Fig. 1 (Colour online) Plant species growing in CILY-affected coconut farms of Grand-Lahou identified as potential alternative hosts for the CILY phytoplasma.

Fig. 2 Phylogenetic tree based on the G813/AwkaSR sequences of the CILY phytoplasma and reference 16Sr phytoplasma groups. ‘Ca.P. sp’: ‘Candidatus Phytoplasma sp.’; CILY: Côte d’Ivoire lethal yellowing; CSPWD: Cape St. Paul Wil Disease; H. crudus: Haplaxius crudus; V1: Palmindustrie V1; LYM: Lethal Yellowing – Mozambique; LD: Lethal Decline. Ribosomal group/subgroup when available are reported before the GenBank accession numbers in parentheses. Y. A. Rosete et al. 170

Fig. 3 Phylogenetic tree based on the secA sequences of the CILY phytoplasma and selected 16Sr phytoplasma groups. CILY: Côte d’Ivoire lethal yellowing; V1: Palmindustrie V1; CSPWD: Cape St. Paul Wilt; LYM: Lethal Yellowing – Mozambique; LD: Lethal Decline. Ribosomal group/subgroup when available are reported before the GenBank accession numbers in parentheses. families Poaceae (45) and Fabaceae (45), followed by the Adjadon village, and P. pe di ci ll at um from the villages of families Rubiaceae (35), Asteraceae (30), Malvaceace (25) Badadon, Braffedon and Palmindustrie V1. A very well- and Apocynaceae (20). known phytoplasma associated with Pennisetum species Few plant families have been reported as hosts of (from the Poaceae family) is the Napier grass stunt phyto-

coconut lethal yellowing phytoplasmas. Asteraceae spe- plasma, which causes a severe stunt disease on Napier cies such as Emelia forsbegii and Synedrella nodiflora grass (P. purpureum Schumach) in Ethiopia (group have been identified as secondary hosts for the provi- 16SrIII, ‘Ca.P.pruni’) (Arocha et al. 2009), and in sional candidate species ‘Ca. P. palmae’ (group 16SrIV) Uganda and Kenya (group 16SrXI, ‘Ca. P. oryzae’) in Jamaica (Brown et al. 2008). Palm species, such as the (Arocha & Jones 2010). Poaceae conjugatum is another African fan palm (Borassus aethiopum) and oil palm member of the Poaceae family that harbours a phytoplasma (Elaeis guineensis) from the family Arecaceae, to which of group 16SrXIV, ‘Ca. P. cynodontis’ in Singapore (Koh coconut palms belong, have been recently reported as et al. 2008). In addition, plant species of the Poaceae alternative hosts of the group 16SrXX-A ‘Ca. P. palmi- family, such as Panicum maximum, Sorghum arundina- cola’ in Mozambique (Bila et al. 2015). ceum, Andropogon gayanus and Oryza barthii have been Plant species from five botanical families, namely recorded as weed species commonly found in coconut Poaceae, Verbenaceae, Plantaginaceae, Phyllanthaceae areas of Nigeria affected by the ‘Awka Wilt’ phytoplasma and Cyperacea tested positive for the CILY phytoplasma (Eziashi et al. 2013). with primers G813/AwkaSR and secA. With primers P1/ From the Verbenaceae family, a 16SrI (‘Ca.P.asteris’) P7, only three out of 20 samples positive for the CILY phytoplasma was found in Verbena x hybrida plants in phytoplasma yielded amplification. This may be attribu- Turkey (Přibylová et al. 2014). The lethal yellowing (LY) ted to the well-known low titre and irregular distribution phytoplasma (16SrIV) was identified from Stachytarpheta of phytoplasmas in the phloem of the infected plants (Lee jamaicensis, a weed member of the Verbenaceae, which has et al. 1998). imposed new challenges for the vector epidemiology and The family Poaceae (Gramineae) has the largest reported management of LY in Jamaica (Brown & McLaughlin number of plant species associated with phytoplasmas 2011). worldwide, and includes plants such as rice, wheat, barley, Plantago lanceolata L., from the family Plantaginaceae, oats, bamboo, sorghum, sugarcane, maize, most millet, and was identified as a host of ‘Ca.P.asteris’ in the Czech a large range of grasses like Bermuda grass (Arocha & Republic (Fránová & Simková 2009), and as one of the Jones 2010). Poaceae species that gave positive results for preferred hosts for Hyalesthes obsoletus, insect vector of the CILY phytoplasma included P. vaginatum from the grapevine ‘bois noir’ disease in Switzerland (Kessler et al. Detection and identification of a CILY phytoplasma 171

2011). Plantago major is another species within the vector has been identified for any of the West African phyto- Plantago genus that hosts the ‘stolbur’ phytoplasma in plasmas affecting coconut palms, and associated with a lethal Serbia (Josic et al. 2012). yellowing-like disease, including CILY. No conclusive evi- Phytoplasmas from groups 16SrVI, ‘Ca.P.trifoli’ and dence is presented in our study that supports an epidemiolo- 16SrI, ‘Ca.P.asteris’ have been identified in plant species gical role of S. indica, S. dulcis, D. capitatum, P.vaginatum, P. from the Phyllanthaceae family, including species such as pedicillatum or P. muellerianus for CILY. However, these P. am aru s L. (Samad et al. 2004)andP. ni ruri L. in India plant species were growing within the coconut plantations (Chaube et al. 2015). Cyperus rotundus L. is a member of confirmed as infected by the CILY phytoplasma, which was the Cyperaceae family that has been reported as a host for also detected from the six plant species. Therefore, these the phytoplasma group 16SrII, ‘Ca. P. aurantifolia’ in Cuba species, although not confirmed as alternative hosts for the (Zamora et al. 2015). Other species within that family such CILY phytoplasma, may pose a threat for the spread of the as C. difformis, Mariscus sp. and Kyllingia sp. have been CILY phytoplasma in the coconut groves of Grand-Lahou. identified as common plants growing in coconut fields Although many of the plant species found in coconut planta- affected by CSPWD in Ghana (Danyo 2011). tions from the East, Central and Western regions in Ghana Although Pennisetum was found in Badadon, coincide with those surveyed from coconut farms in Grand- Braffedon and Palmindustrie V1, it was not among the Lahou, attempts to identify alternative hosts for the CSPWD plant species surveyed from the villages of Yaokro and phytoplasma in Ghana have not yet been successful (Yankey Adjadon. On the other hand, S. indica, S. dulcis and et al. 2009). D. capitatum were limited to Yaokro, and P. vaginatum Phylogenetic analysis also supported the utilization of limited to Adjadon. It is not clear if there is any environ- the nested PCR system based on both 16Sr RNA and mental factor driving the differential distribution or occur- secA genes as a diagnostic tool for the detection of the rence of S. indica, S. dulcis and D. capitatum in Yaokro, CILY phytoplasma. In particular, the 16Sr DNA/secA or P. vaginatum in Adjadon, or P. pedicillatum in either nested PCR was shown to be a robust approach for Badadon, Braffedon or Palmindustrie V1. It is possible detection of the CILY phytoplasma in weed species grow- that these specific plant species were not available at the ing within and in the periphery of coconut farms in time of sampling, or that the sampling itself was too Grand-Lahou. It is noteworthy that the CILY-affected limited. A larger sampling size would help to include coconut farms surveyed were very poorly weeded; in more representative plant species, and assess them for particular, the villages of Badadon, Braffedon and the presence of the CILY phytoplasma, and further unveil Palmindustrie V1 exhibited a highly diverse and vigorous any possible role in disease epidemiology. vegetation. Weeds are well known sources of inoculum This study provides first world reports of S. indica, for phytoplasma diseases (Weintraub & Beanland 2006; S. dulcis, D. capitatum, P. vaginatum, P. pedicillatum Mirzaie et al. 2007; Arocha et al. 2009); therefore their and P. muellerianus as phytoplasma hosts. These plant control may greatly reduce disease pressure and prevent species are also being reported as secondary reservoirs outbreaks. of the CILY phytoplasma. The confirmations were The successful uses of secA PCR in support of the based on more than 99% sequence identity in 16Sr 16Sr RNA-based phytoplasma identification and classifi- RNA and secA genes when compared with CILY phy- cation (Hodgetts et al. 2008; Valiunas et al. 2015), and for toplasma strains identified from the same CILY- detecting a number of phytoplasmas, including the affected coconut farms surveyed in Grand-Lahou. No CSPWD strain in Ghana, have been reported (Yankey characteristic symptoms such as yellowing, witches’ et al. 2014). The secA phylogeny of the CILY phyto- broom, phyllody, or stunting, commonly associated plasma identified from the alternative host plants was in with phytoplasmas (Lee et al. 1998) were observed in good agreement with the phylogenetic grouping based on any of these plant species. This suggests that asympto- the 16S rRNA gene. Both phylogenies confirmed the matic reservoirs are present naturally for CILY phyto- distinction of three ‘Ca. Phytoplasma’ taxa based on plasma infections, as is the case of phytoplasmas their geographic origins: Americas (LY), East Africa identified in sugarcane and other grasses in northern (Tanzanian LD) and West Africa (Nigerian LD, CSPWD Australia (Tran-Nguyen et al. 2000). in Ghana, and CILY in Côte d’Ivoire). Phytoplasma epidemics are greatly influenced by the num- The use of the 16Sr DNA/secA PCR system to confirm ber and abundance of insect vectors and also by alternative/ CILY phytoplasma-presence, and the removal of S. indica, reservoir plants (Sharon et al. 2005). Alternative phytoplasma S. dulcis, D. capitatum, P. vaginatum, P. pedicillatum and plant hosts may harbour additional insect vectors capable of P. muellerianus, should be recommended for the CILY spreading the disease (Mori et al. 2015). So far, no insect management strategy in Grand-Lahou. Although just Y. A. Rosete et al. 172 identified as putative alternative hosts, the surveillance of Arocha Y, Jones P. 2010. Phytoplasma diseases of the Gramineae. In: Weintraub P, Jones P, editors. Phytoplasmas: genomes, plant hosts and these plant species in both CILY-affected and CILY-free – fi vectors. Oxfordshire (UK): CABI publishing; p. 170 187. coconut farms will signi cantly contribute to better man- Arocha Y, Zerfy T, Abebe G, Proud J, Hanson J, Wilson M, Jones P, agement of CILY, and improve the livelihood of small- Lucas J. 2009. Identification of potential vectors and alternative plant holder coconut farmers of Grand-Lahou. hosts for the phytoplasma associated with Napier grass stunt disease in The removal of alternative hosts for phytoplasmas com- Ethiopia. J Phytopathol. 157:126–132. Arocha-Rosete Y, Konan Konan JL, Diallo AH, Allou K, Scott JA. bined with the use of resistant plant material has proven to be 2014. Identification and molecular characterization of the phytoplasma highly effective for the management of lethal yellowing-like associated with a lethal yellowing-type disease of coconut in Côte diseases. In Jamaica, a coconut rehabilitation programme d’Ivoire. Can J Plant Pathol. 36:141–150. ‘Black Approach’ has been successfully employed to control Bila J, Högberg N, Mondjana A, Samils B. 2015. African fan palm (Borassus aethiopum) and oil palm (Elaeis guineensis) are alternate hosts LY by combining the replacement of infected trees using a of coconut lethal yellowing phytoplasma in Mozambique. Afr J Biotech. locally developed resistant variety and the removal of alter- 14:3359–3367. native reservoirs of the LY phytoplasma (Coconut Industry Brown S, Been BO, McLaughlin WA. 2008. First report of the presence of Board 2012). the lethal yellowing group (16Sr IV) of phytoplasmas in the weeds Emilia fosbergii and Synedrella nodiflora in Jamaica. Plant Pathol. 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