Journal of Plant Pathology (2003), 85 (2), 81-86 Edizioni ETS Pisa, 2003 81

DETECTION AND CHARACTERIZATION OF THE PHYTOPLASMA ASSOCIATED WITH MARIGOLD PHYLLODY IN MEXICO

R.I. Rojas-Martínez1, E. Zavaleta-Mejía1 I.M. Lee2, M. Martini2 and H.S. Aspiros3

1Instituto de Fitosanidad. Colegio de Postgraduados, Montecillo, México 56230 2Molecular Plant Pathology. USDA, ARS, Beltsville, Maryland 20705, USA 3Laboratorio de Biotecnología, INIFAP, Chapingo, Mexico 56230

SUMMARY disease is characterized by a variety of symptoms resem- bling those caused by phytoplasmal infections, namely Cempazuchil (marigold, Tagetes erecta L.) plants with shoot proliferation (witches’ broom), green petal flow- symptoms of phyllody, virescence, witches’ broom ers (virescence), leafy floral structures (phyllody), (shoot proliferation), apical dwarfing and yellowing dwarfing or yellowing. Diseased plants produce few were collected from fields in the States of Puebla, Mi- flowers with normal pigments. choacan, Guanajuato, and Estado de Mexico. They In Mexico, marigold phyllody was first reported by were examined for the presence of phytoplasmas by Zavaleta-Mejía et al. (1993), who found a 5% incidence nested polymerase chain reaction (PCR) using the uni- in experimental plots at Montecillo, Mexico, and a 50% versal primer pair R16mF2/R16mR1 followed by the incidence in commercial plots at Tecamachalco, Puebla. primer pair R16F2n/R16R2. Restriction fragment length Since then, the disease has spread into several states of polymorphism (RFLP) analysis of R16F2n/R16R2-PCR the central part of the country. products and the sequencing of the 16S rDNA indicat- The causal agent of marigold phyllody has never ed that the phytoplasma which induces the disease been identified. However, since electron microscopy known as “filodia del cempazuchil” (marigold phyllody) observations had shown that the disease could be belongs to the aster yellows group (16SrI), subgroup B. caused by a phytoplasma (Salamanca, 1995), a study Our results also indicated that the variety of symptoms was carried out for detecting and indentifying, by mo- shown by diseased Tagetes plants from different States lecular methods, which phytoplasma(s) was associated in Mexico were induced by the same phytoplasma. with marigold plants exhibiting different symptoms.

Key words: Apical darwfing, virescence, yellowing, marigold, phytoplasma, diagnosis, nested PCR. MATERIALS AND METHODS

Sources of infected marigold plants and reference INTRODUCTION phytoplasmas. Shoots from marigold plants with and without symptoms of phyllody, virescence, witches’ Tagetes species are economically important crops. broom, apical dwarfing and yellowing were collected They are used as ornamental (Wright, 1979) and phar- from commercial plantations in the States of Puebla, maceutical plants (Tostle, 1968), and for the protection Michoacan, Guanajuato, and State of Mexico, and of crops because of its fungicidal (Kourany and Arna- frozen in liquid nitrogen. son, 1988), insecticidal (Morallo and Decena, 1987), DNA (total nucleic acid) extracts were prepared and nematocidal properties (Ruelo and Davide, 1979; from plants showing each of the above symptoms as de- Rhoades, 1990). scribed by Ahrens and Seemüller (1992). Reference In Mexico, marigold (Tagetes erecta L), whose flow- phytoplasma DNA extracted from periwinkle plants in- ers contain high amount of xanthophylls, has become a fected with an Oklahoma aster yellows (OKAY) phyto- cash-crop because of high demand for natural pigments plasma, a member of the aster yellows group (16SrI), from the international food industry (Gunthener et al., subgroup B (16SrI-B), was provided by I.M. Lee. To 1973), thus its average annual production has increased avoid false negatives due to the possible presence of to 1500 ton ha-1 since 1980 (SAGAR, 1999). PCR inhibitors in marigold extracts, nested PCR was The production of useful marigold flowers for pig- performed. DNA samples were diluted in sterile dis- ments is now being threatened by marigold phyllody tilled water to a final concentration of 20 ng/ml. The disease which occurs in several Mexican states. This two universal primer pairs R16mF2/R16mR1 and R16F2n/R16R2 designed for amplification of phytoplas- ma 16S rDNA (Lee and Gundersen, 1993; Gundersen Corresponding author: R.I. Rojas-Martínez and Lee, 1996) were used to detect phytoplasma DNA Fax: 595.95.20200 ext. 1602 E-mail: [email protected] in samples prepared from marigold tissues. 82 Marigold phyllody phytoplasma Journal of Plant Pathology (2003), 85 (2), 81-86

PCR (35 cycles) was done with an automatic thermo- sity of Illinois), on a Power Mac G4. Uninformative cycler (Perkins-Elmer Cetus, Norwark, USA) using the characters were excluded from analyses. A phylogenetic primer pair R16mF2/R16mR1 for the first amplification tree was constructed by a heuristic search via random and the primer pair R16F2n/R16R2 (Gundersen and stepwise addition implementing the tree bisection and Lee, 1996) for the second amplification (nested PCR). reconnection branch-swapping algorithm to find the op- Conditions of the assay were: denaturation at 94ºC for 1 timal tree(s). Acholeplasma laidlawii was selected as the min (2 min for the first cycle), annealing for 2 min at out-group to root the tree. The analysis was replicated 55ºC, primer extension for 3 min with a final extension 1000 times. Bootstrapping was performed to estimate for 10 min at 72ºC. OKAY phytoplasma DNA was used the stability and support for the inferred clades. as positive control. PCR products were separated by electrophoresis in 1% agarose gel, stained with ethidium bromide, and visualized with an U.V. trans-illuminator. RESULTS RFLP analysis of PCR products from 16S rDNA Detection of phytoplasmas associated with marigold phytoplasma sequences amplified by nested PCR using phyllody. Twenty-five samples from plants showing each the primer pair R16F2n/R16R2 was by digestion with symptom type and 25 samples from asymptomatic the restriction enzymes AluI, HaeIII, HhaI, HpaII, MseI, plants were collected in each marigold growing region RsaI, KpnI, HinfI, ThaI and TaqI (GIBCO BRL, Bever- and tested for the presence of phytoplasmas. ly, MA, USA) according to the manufacturer instruc- Single-step PCR amplification did not detect phyto- tions. The restriction products were separated by plasmas in all symptomatic samples, whereas nested PCR electrophoresis in a 5% polyacrylamide gel and stained was successful with symptomatic and some asympto- with ethidium bromide. The RFLP patterns were com- matic samples (Table 1). For example, single-step PCR pared with electrophoretic patterns of known phyto- using the primer pair R16mF2/R16mR1 detected phyto- plasmas described by Lee et al. (1998). plasma 16S rDNA in only three out of seven marigold samples with witches’ broom symptoms (data no shown), Sequencing and analysis of 16S rDNA. The PCR- but in none of the samples with other types of symptom. amplified product (1.2 kbp 16Sr DNA fragment) was By contrast, nested PCR using the second primer pair cloned in Escherichia coli using TOPO-TA Cloning Kit R16F2n/R16R2, detected phytoplasmal 16Sr DNA in all (Invitrogen, Carsbad, CA, USA) according to manufac- samples from marigold plants showing phyllody, vires- turer’s instruction. The nucleotide sequences of selected cence, witches’ broom, apical dwarfing, and yellowing cloned 16S rDNA were determined by automated DNA (Fig. 1). Phytoplasmal 16S rDNA was also detected in a sequencing. BLAST comparison was performed using DNA sample from a symptoless plant (Fig. 1, lane 9). the 1.2 kb 16Sr DNA fragment. Phytoplasma identification in marigold. RFLP analy- Phylogenetic analysis. Phylogenetic interrelationships ses of nested PCR products with Alu, HpaII, HaeIII, among marigold phyllody (MgPh) strains and strains in HhaI, KpnI, HinfI, ThaI and Taq I gave identical collec- other phytoplasma groups were determined based on tive restriction patterns from all marigold phytoplasmas 16S rRNA gene sequences. Partial sequences of 16S rD- present in samples with various symptoms. These pat- NA (1.5 kb) from MgPh strains and representative phy- terns were identical to that of the reference phytopla- toplasma strains available in GenBank were aligned by sma OKAY-B (Fig. 2) and to those of members of aster using CLUSTAL 5, and DNASTAR’s Laser Gene soft- yellows group (16SrI), subgroup B (Lee et al., 1998). ware (DNASTAR, Madison, WI, USA). Cladistic analy- No discrimination was possible from RFLP patterns ob- ses were performed with PAUP (phylogenetic analysis tained with two additional restriction enzymes (MseI using parsimony), version 4.0 by D.L. Swofford (Univer- and RsaI) (data not shown).

Table 1. Source of marigold plants showing phyllody, virescence, witches’ broom, apical darwfing and yellowing, and average percentage of phytoplasma detection by single-step and nested PCR.

Symptoms Samples from each Location Single-step Nested locality (No.) PCR (%) PCR (%)

Phyllody 25 Puebla, Guanajuato, Michoacan, and Estado de 93 100 México Witches' broom 25 Puebla, Guanajuato, Michoacán, and Estado de 90 100 México Apical darwfing 25 Puebla, Guanajuato, Michoacán, and Estado de 85 100 México Yellowing 25 Puebla, Guanajuato, Michoacán, and Estado de 73 100 México Journal of Plant Pathology (2003), 85 (2), 81-86 Rojas-Martínez et al. 83

The results indicate that a single type of phytoplasma is associated with symptoms of phyllody, virescence, witches’ broom, apical dwarfing, and yellowing shown by marigolds from the States of Guanajuato, Michoa- can, Puebla and Estado de Mexico, as shown by RFLP profiles (Lee et al., 1998). Confirmation of this taxo- nomic affiliation was obtained by comparative comput- er-assisted analysis of the sequence of the 1.2 kb 16S rDNA fragment amplified from symptomatic marigold samples (GenBank accession numbers AY249247, AY249248 and AY249249) and phylogenetic analysis showed close relationship to one another and most closely related to subgroup 16SrI-B phytoplasmas in the Fig. 1. Nested PCR amplification of phytoplasmal 16S rDNA aster yellows phytoplasma group (Fig. 3). with primers R16mF2/R16mR1 followed by R16F2n/R16R2 from marigold plants showing phyllody (lanes 1 and 2), vire- scence (lanes 3 and 4), witches’ broom (lane 5), apical dwarf- ing (lane 6), yellowing (lane 7), and no symptoms (lane 8). Water control is in lane 9, OKAY-B positive control in lane 10, and DNA ladder in lane kb.

Fig. 2. RFLP analyses of PCR-amplified phytoplasmal 16S rDNA with primers R16F2n/R16R2 from marigold plants showing phyllody (lanes 1 and 2), virescence (lanes 3 and 4), witches’ broom (lanes 5 and 6), yellowing (lane 7), apical dwarfing (lane 8). OKAY-B positive control is in lane 9, DNA ladders are in the first and last lane of each panel. The DNA products were digested with restriction enzymes: Alu (panel A), Hpall (panel B), Kpnl (panel C), Hinfl (panel D), Hhaelll (panel E), Hhal (panel F), Thal (panel G), TaqI (panel H). 84 Marigold phyllody phytoplasma Journal of Plant Pathology (2003), 85 (2), 81-86

Fig. 3. Phylogenetic tree constructed by parsimony (PAUP version 4.0b, D. Swofford) analysis of near full length 16S rDNA se- quences from representative phytoplasma strains in the aster yellows group (16SrI) and other 16Sr phytoplasma groups. Se- quences were aligned with Clustal version 5 (DNAStar Lasergene software, Madison, WI). Acholeplasma laidlawii (M23932) was employed as the out-group to root the tree. Bar length represents 10 inferred character state changes. Branch lengths are propor- tional to the number of inferred character state transformations. Bootstrap values (measures of support for the inferred subclades) are shown on branches. Phytoplasmas or ‘Candidatus’ phytoplasma species and GenBank accession numbers are as follows: marigold phyllody phytoplasma strains associated with infected marigold with various symptoms: MgPh1(AY249249), MgPh2 (AY249248), and MgPh3 (AY249247); CLY, Cherry lethal yellows (AY197659); JWB, Jujube witches’-broom (AY197661); PY-In1, Indian peach yellows (AY197660); EY1, Elm yellows (AY197655); BLL, Brinjal little leaf (AF228052); CP, Clover prolif- eration (L33761); AshY, Ash yellows (AF189215) (‘Candidatus Phytoplasma fraxini’); LfWB, Loofah witches’-broom (AF248956); BGWL, Bermuda grass white leaf (Y16388); SCWL, Sugarcane white leaf (X76432); LY, Palm lethal yellowing (AF498309); TLD, Tanzanian lethal decline of coconut (X80117); PPWB, Pigeon pea witches’-broom (AF248957); CYE, Clover yellow edge (AF175304); WX, Western X (L04682); FBP, Faba bean phyllody (X83432); WBDL, Witches’-brrom disease of lime (‘Candidatus Phytoplasma aurantifolia’) (U15442); PnWB, Peanut witches’-broom (L33765); PpYC, Papaya yellow crinkle (‘Candidatus Phyto- plasma australasia’) (Y10097); PEP, Picris echioides phyllody (Y16393); HibWB26, Hibiscus witches’-broom (‘Candidatus Phyto- plasma brasiliense’) (AF147708); AT, Apple proliferation (‘Candidatus Phytoplasma mali’) (X68375); ESFYC, European stone fruit yellows (X68374); SpaWB, Spartium witches’-broom (X92869); BWB, Buckthorn witches’-broom (X76431); OAY, Oenothera virescence (M30790); PaWB, Paulownia witches’-broom (AY265206); ACLR-AY, apricot chlorotic leaf roll (AY265211); BBS3, Blueberry stunt (AY265213); STRAWB2, Strawberry multiplier (U96616); BB, Tomato big bud/Arkansas (L33760); CPh, Clover phyllody (L33762); AUGY, Australian grapevine yellows (‘Candidatus’ Phytoplasma australiense) (X95706); STOL, Stolbur of pepper (X76427). Journal of Plant Pathology (2003), 85 (2), 81-86 Rojas-Martínez et al. 85

DISCUSSION REFERENCES

In the present study we have employed molecularly- Ahrens U., Seemüller E., 1992. Detection of DNA of plant based tools for detection and identification of the puta- pathogenic mycoplasmalike organisms by a polymerase tive causal agent of marigold phyllody, and report for chain reaction that amplifies a sequence of the 16S rRNA the first time the presence of aster yellows phytoplasma gene. Phytopathology 82: 828-832. subgroup B (16SrI-B) in Mexico. Gunthener E., Carlson C.W., Olgon O.E., Kohler G.O., Liv- Biological properties, symptomatology in particular, ington A. L., 1973. Pigmentation of egg yolks by xantho- have been used in the past to classify phytoplasmas. phylls from corn, marigold, alfalfa and synthetic sources. They are still important tools for the preliminary identi- Poultry Science 52:1787-1790. fication of putative phytoplasmal diseases. However, Gundersen D.E., Lee I.M., 1996. Ultrasensitive detection of this approach is frequently inaccurate, as discussed by phytoplasmas by nested-PCR assay using two universal Lee and Davis (1992) and confirmed by the present primer pairs. Phytopathologia Mediterranea 35: 144-151. study, which has shown that all phytoplasma isolates de- Gundersen D.E., Lee I.M., Rehner S.A., Davis R.E., Kings- tected in Mexican marigolds belong to a single type, re- bury D.T., 1994. Phylogeny of mycoplasmalike organisms gardless of the symptoms shown in the field. (phytoplasmas): a basis for their classification. Journal of RFLP analysis of PCR-amplified 16S rDNA has been Bacteriology 176: 5244-5254. widely used to identify and classify phytoplasmas (Lee Kourany E., Arnason J.T., 1988. Accumulation of phototoxic et al., 1993; Gundersen et al., 1994; Lee et al., 1998), us- thiophenes in Tagetes erecta (Asteraceae) elicited by Fusa- ing several key restriction enzymes that differentiate rium oxysporum. Physiological and Molecular Plant Patholo- phytoplasma groups and subgroups (Lee et al., 1998). gy 26: 809-820 In the present study, the RFLP patterns obtained using Lee I.M., Davis R.E., 1992. Mycoplasmas which infect plants 10 restriction enzymes showed that all the phytoplasma and insects. In: Maniloff J., McElhaney R.N., Finch L.R., isolates associated with different symptoms in marigold Baseman J.B (eds.). Mycoplasmas: molecular biology and plants collected in several Mexican States were closely pathogenesis, pp. 379-390. American Society for Microbi- related to one another and to the phytoplasma strains ology, Washington D.C., USA. belonging to aster yellows group (16SrI), subgroup B. Lee I.M., Hammond R.W., Davis R.E., Gundersen D.E., The accuracy of this classification system has been vali- 1993. Universal amplification and analysis of pathogen 16S dated with previous studies (Schneider et al., 1993; Lee rDNA for classification and identification of mycoplasma- et al., 1998) and again with the present study by parallel like organisms. Phytopathology 83: 834-842. phylogenetic analysis with partial (1.2 kb) or near full Lee I.M., Dawn E., Gundersen-Rindal E., Davis R.E., 1998. length 16S rDNA sequences. Revised classification scheme of phytoplasmas based on Identification of the causal agent of marigold phyl- RFLP analyses of 16S rRNA and ribosomal protein gene lody should facilitate studies on the ecology of this dis- sequences. International Journal of Systematic Bacteriology ease, and the development of effective control meas- 48: 1153-1169. ures. The primary sources of marigold phytoplasma are Morallo R.B., Decena A., 1987. The activity, isolation and pu- unknown. Attempts to detect moleculary phytoplasmas rification of the insecticidal principles from Tagetes. Biolo- in seeds from symptomatic marigold plants have failed, gical Abstract 7: 81-87. confirming that they may not be seed-borne in this host (Rojas-Martínez et al., 2001). Vegetables, ornamental McCoy R.E., Caudwell A., Chang C.J., Chen T.A., Chiykows- plants, and weeds that frequently surround marigold ki L.M., Cousin M.T., Dale J.L., De Leeuw G.T.N., Golino D.A., Hackett K.J., Kirkpatrick B.C., Marwitz R., Petzold plots, can be infected by phytoplasma strains belonging H., Sinha R.H., Sugiura M., Whitcomb R.F., Yang I.L., to the same subgroup (16SrI-B) as that found in Zhu B.M., Seemüller E., 1989. Plant diseases associated marigold (Rojas-Martínez et al., 2002), and may serve with mycoplasmas-like organisms. In: Whitcom R.F., Tully as alternative hosts for both the pathogen and the vec- J.G.(eds.). The Mycoplasmas, 5, pp. 545-560. Academic tor (McCoy, et al., 1989). In Mexico, several insects Press, San Diego, USA. may be involved in the spread of marigold phyllody in Rhoades H.L., 1990. Relative susceptibility of Tagetes patula the field, possibly leafhoppers of the genera Scaphy- and Aeschynomene americana to plant nematodes in Flori- topius, Neokolla, and Idiodonus which were experimen- da. Nematropica 10: 116-120. tally shown to transmit the phyllody agent to periwin- kle and marigold (Salamanca, 1995). A survey is now Rojas-Martínez R.I., Zavaleta-Mejía E., Lee I.M., Aspiros H.S., 2001. El fitoplasma que induce la filodia del cem- underway in various Mexican areas for identifying with pazuchil (Tagetes erecta L.) no se transmite por semilla. Re- molecular methods the potential vectors of the phyl- vista Mexicana de Fitopatología 19: 309-311. lody phytoplasma and their role in the epidemics of marigold phyllody disease. Rojas-Martínez R.I., Zavaleta-Mejía E., Martinez-Soriano J.P., Lee I.M., 2002. Identification of phytoplasma associated with Cosmos (Cosmos bippinatus Cav.) phyllody and classi- fication by RFLP analysis of 16S rDNA. Revista Mexicana de Fitopatología 21: 83-86. 86 Marigold phyllody phytoplasma Journal of Plant Pathology (2003), 85 (2), 81-86

Ruelo S.J., Davide R.G., 1979. Studies on the control of organisms using restriction-site analysis of PCR-amplified Meloidogyne incognita. III. Integration of biological and 16S rDNA. Journal of General Microbiology 139: 519-527. chemical control. Philippine Agriculture 62:159-165. Tostle N.R., 1968. The etnobotany of Tagetes. Economic Salamanca C.M., 1995. Transmisión y epidemiología de la filo- Botany 22: 317-325. dia del cempoalxochitl (Tagetes erecta L.) en Tepanco de Wright M., 1979. Guia Practica Ilustrada para el Jardin. I, II Lopéz, Puebla. M. Sc. Thesis. Colegio de Postgraduados, Ed. Blume, Barcelona, Spain. Montecillo, México. Zavaleta-Mejía E., Cárdenas S.E., Gómez R.O., 1993. Organ- SAGAR (Secretaría de Agricultura, Ganadería y Desarrollo ismos tipo micoplasma asociados con la filodia del cem- Rural), 1999. Anuario Estadistico de la Producción Agrícola pazúchil. XX Congreso Nacional de Fitopatologia, Zacatecas de los Estados Unidos Mexicanos 1: 466-471. 1993, 15. Schneider B., Ahrens U., Kirkpatrick B.C., Seemüller E., 1993. Classification of plant-pathogenic mycoplasma-like

Received 19 March 2003 Accepted 11 June 2003