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010_JPP430RP(Rojas)_331 20-07-2011 14:34 Pagina 331 Journal of Plant Pathology (2011), 93 (2), 331-335 Edizioni ETS Pisa, 2011 331 AETIOLOGY OF CHILI PEPPER VARIEGATION FROM YURÉCUARO, MÉXICO M. Camacho-Tapia1, R.I. Rojas-Martínez1, E. Zavaleta-Mejía1, M.G. Hernández-Deheza1, J.A. Carrillo-Salazar2, A. Rebollar-Alviter3 and D.L. Ochoa-Martínez1 1 Instituto de Fitosanidad, Colegio de Postgraduados, Montecillo 56230, México Genéticos y Productividad Genética, Colegio de Postgraduados, Montecillo 56230, México 3 Centro Regional Morelia, Universidad Autónoma de Chapingo, Morelia, Michoacán 58000, México 2 Recursos SUMMARY In summer 2008, symptoms resembling those of a virus disease, i.e. chlorotic veins, deformation of the leaves and fruits, albinism and browning, were observed in chile pepper (Capsicum annuum L.) fields at Yurécuaro (Michoacán, México). However, ELISA tests were negative for the viruses most commonly associated with this crop. Based on these results, and knowing that in the state of Sinaloa (México), Candidatus Liberibacter solanacearum (Ca.L.s.) was detected in chile plants showing shortened internodes, apical chlorosis and symptoms similar to those of “yellow psillid” in potato, it was thought that the symptoms of chile in Yurécuaro could be induced by this bacterium. In this study the etiology of the variegation of chile was determinated. Symptomatic plants were collected in the Yurécuaro region in order to detect in leaves and seeds the possible causal agent by PCR and transmission electron microscopy, and to transmit the pathogen by grafting, Bactericera cockerelli, and through seed. In leaf tissue and seed of affected plants, Ca L.s. was detected by PCR, using primers OA2 and OI2c. The bacterium was also detected in thin sectioned phloem cells of affected leaves. Disease symptoms were reproduced in plants grafted with infected tissue and in those exposed to adults of B. cockerelli collected from diseased plants. The evidence obtained indicates that the variegation of chile pepper in Yurécuaro is induced by Ca.L.s. Key words: Candidatus Liberibacter solanacearum, transmission electron microscopy, polymerase chain reaction, Bactericera cockerelli. INTRODUCTION The green chile pepper (chile, Capsicum annuum L.) is the most widely grown vegetable crop in México, with a cultivated area of 149,114 ha in 2007 (SIAP, 2007). The main producing Mexican states are: Sinaloa (694,634 tons), Chihuahua (564,256 tons), Zacatecas (209,331 tons), San Luís Potosí (133,402 tons), Tamaulipas (125,482 tons), and Michoacán (93,424 tons). The main Michoacán municipalities growing chile are Yurécuaro, Tanhuato, Coahuayana, Vista Hermosa and Ecuandureo (SIAP, 2009). During 2008, a new phytosanitary problem of chile arose in Yurécuaro, which resembled a viral infection because of the symptoms shown by affected plants, i.e. variegation, deformation of the leaves and albinism, that recalled those induced by Tobacco etch virus (TEV) (Plant Virus Online, 2008). However, ELISA tests using commercial kits (Agdia, USA) for the presence of TEV, Tobacco mosaic virus (TMV), Tobacco ringspot virus (TRSV), Cucumber mosaic virus (CMV), Alfalfa mosaic virus (AMV), Tomato spotted wilt virus (TSWV), and Tomato aspermy virus (TAV) were negative. Knowing that Candidatus Liberibacter solanacearum (Ca.L.s.) was found in New Zealand (MAF, 2008) in chile plants with mottling, yellowing, leaf deformation and sudden death and with chile plants with symptoms similar to those of “yellow psillid” of potato in Sinaloa, (Munyaneza et al., 2009), it was hypothesized that this bacterium could be involved in the aetiology of the disease occurring in Yurécuaro. Ca.L.s. is also the casual agent of Zebra chips of potato in the USA (Abad et al., 2009) and New Zealand (Liefting et al., 2008), affects other solanaceous crops, such as tomato (Solanum lycopersicum) (Liefting et al., 2008, 2009) and is transmitted by the psyllid Bactericera cockerelli (Hansen et al., 2008). As, mentioned, Munyaneza et al. (2009) have reported the presence of Ca.L.s. in symptomatic chile but did not determine whether this bacterium is the actual agent causal of the disease. This investigation was therefore carried out with the aim of establishing if there were an aetiological relationship between Ca.L.s. and the chile variegation disease present in Yurécuaro. MATERIAL AND METHODS Corresponding author: R.I. Rojas-Martinez Fax: 595.95.20200 ext.1623 E-mail: [email protected] Symptomatic chile plants of cvs Centella and Tajin were collected at Yurécuaro (northwest of the Mi- 010_JPP430RP(Rojas)_331 332 20-07-2011 14:34 Pagina 332 Etiology of chile pepper variegation from Mexico choacán state at 20º20’ north and 102º17’ west) including 10 fruits for seed extraction and taken to the laboratory for testing. Seed samples comprised also seeds harvested by the growers and from commercial lots of cvs Centella, Tajin, Big-Brother and Camino Real. DNA extraction. DNA was extracted from 0.3 g tissue from symptomatic and symptomless leaves according to Ahrens and Seemüller (1992) and from 0.2 g of seeds with the Qiagen extraction kit (Quiagen, USA). The quantity and quality of the extracted DNA was determined spectrophotometrically (Lambda Bio model 10, Perkin Elmer) at 260 nm and with electrophoresis in 0.8% agarose gel, respectively. PCR detection of Ca. Liberibacter solanacearum. PCR was conducted using Ca.L.s.-specific primers OA2 and OI2c, designed on the 16S rDNA region, that amplify a 1160 bp fragment (Liefting et al., 2008, 2009). Amplification took place in a BIO-RAD thermocycler, model i-Cycler, with an initial denaturation at 94ºC for 5 min, followed by 35 cycles at 95ºC for 30 sec for denaturation, 58ºC for 30 sec for annealing and 72ºC for 1 min for extension, and 10 min at 72ºC for final extension. Five µl of the PCR product were loaded onto a 1% agarose gel and run by electrophoresis at 90 V. Gels were stained with ethidium bromide (0.5 µg ml-1 for 10 min) and acquired with a BIO-RAD photodocumenter (Gel-Doc model 2000). For molecular weight reference, a 1 Kb marker (Gibco BRL, USA) was used. PCR products were cleaned with a Wizard kit (Promega, USA) then cloned in Escherichia coli using a TOPO-TA cloning kit (Invitrogen, USA) in accordance with manufacturer’s instructions. Sequencing of 16S rDNA clones was by the Automatic Sequencer 3700xl DNA Analyzer (Applied Biosystems, USA). Sequences were aligned with those available in GenBank, utilizing the BLAST tool of NCBI (http://www.ncbi.nlm.nih.gov/ BLAST/) Seeds in which Ca.L.s. was detected, were germinated and the seedlings transplanted into eight unicel containers with three seedlings each, that were placed in a cage with insect-proof netting to prevent contact with insect vectors. The seedlings were kept under observation for 2 months for symptom appearance. Transmission by grafting. Tissue from symptomatic chile plants were grafted onto 15 healthy plants. The cuts were made diagonally to ensure the greatest contact area between graft and host and the grafting area was covered with parafilm. The plants were covered with plastic bags for a week to maintain humidity and were kept in the greenhouse until symptoms appeared. Transmission by Bactericera cockerelli. Four symptomatic plants that carried a large number of B. cockerelli Journal of Plant Pathology (2011), 93 (2), 331-335 nymphs were collected from the field and taken to a glasshouse where they were placed inside entomology quality cages to avoid contact with other insects. Once two generations of the insects had been obtained, DNA was extracted from 10 samples, each consisting of three insects, and subjected to PCR analysis. Once the presence of Ca.L.s. in the colony of insects was confirmed, groups of 10 healthy chile plants held in the greenhouse inside cages with insect-proof netting were exposed to 50 adult insects to allow transmission. Phylogenetic relationships. A phylogenetic tree was constructed with the nucleotide sequence of Ca L.s. present in B. cockerelli amplified with primers OA2 and OI2c and comparable sequences retrieved from GenBank: Ca. L. solanacearum (FJ957897), Ca. L. solanacearum (EU834130), Ca. L. solanacearum (FJ957896), Ca. L. solanacearum (FJ395204), Ca. L. solanacearum (FJ395205), Ca. L. solanacearum (FJ395206), Ca. L. solanacearum (GU373049), Ca. L. solanacearum (EU935004), Ca. L. psyllaurous (EU812559), Ca. L. africanus (EU921620), Ca. L. asiaticus (EU921615), and Ca. L. americanus (EU921625). Seven distinct bacteria from this group were included as roots: Brucella melitensis (AY513507), Synorhizobium spp. (AY500255), Bradyrhizobium japonicum (EU481826), Rhodospirillum rubrum (NC_007643), Wolbachia spp. (DQ412085), Escherichia coli (NC_010473), and Helicobacter pylori (EU544200). The phylogenetic tree was produced using DNASTAR Lasergene with alignment by Clustal V. Electron microscopy. The main vein was excised from leaves of symptomatic chile plants and cut to small fragments that were fixed in 6% glutaraldehyde in 0.1 M phosphate buffer, pH 7.2. After washing in phosphate buffer, tissue fragments were post-fixed in osmium tetroxide for 1 h, dehydrated with graded alcohol dilutions and embedded in epoxy resin. Sections 120 nm thick were cut with an ultramicrotome, stained with 1% uranyl acetate, and were observed with a JOEL JEM-1200 EX electron microscope at 60 kV. RESULTS AND DISCUSSION All symptomatic but not the symptomless tissue samples from the field were PCR-positive for Ca.L.s. (Fig. 1). Positive amplification was also obtained from DNA extracted from seeds produced by symptomatic plants and from seeds produced by plants obtained from seeds of diseased plants (Fig. 2A). When nucleotide sequences of PCR amplicons were compared by BLAST with those deposited in GenBank, 100% identity was found with the nucleotide sequence of Ca.L.s. (FJ957897). Chile plants obtained from seed of diseased fruit 010_JPP430RP(Rojas)_331 20-07-2011 14:34 Pagina 333 Journal of Plant Pathology (2011), 93 (2), 331-335 Fig. 1. PCR products obtained with primers OA2 and OI2c in plants of Capsicum annuum L. Lanes 1 and 18: molecular weight marker 1 Kb (Invitrogen); lane 2, asymptomatic plant of Capsicum annuum L. (control); lane 3, leaf with albinism; lane 4, variegated leaf; lane 5, variegated leaf with yellow veins; lane 6, leaf showing browning; lane 7, leaf with blistering and deformation of the lamina; lane 13, negative control (injected sterile water). showed the typical variegation symptoms 2 months after transplanting (Fig. 2B). Comparable symptoms developed also in chile plants 90 days after grafting with tissue from diseased plants. The leaves showed initial chlorosis of the tips followed by necrosis. After 2 months, there was deformation of the leaves of new shoots and the variegation was more evident. After 90 days, PCR tests were positive for the presence of Ca.L.s. Fig. 2. A. PCR products obtained with primers OA2 and O12c. Lane 1, Molecular weight markers of 100 bp; lane 2, negative control; lanes 3-11, DNA from seed of the cultivars evaluated: 3-4 Centella, 5-6 Tajin, 7-8 seed collected by the grower, 9-10 Big-brother, 11 Camino Real. B. Chile pepper plant (Capsicum annuum L.) with symptoms of variegation obtained 2 months after germination of seed from plants with symptoms of variegation. Camacho-Tapia et al. 333 After six weeks exposure of chile plants to insects collected from the field, typical variegation symptoms were observed (Fig. 3B) and PCR detected the bacteria in the insects that had fed for 2 months on the plants (Fig. 3A). Nucleotide sequences of the amplified product and DNA from symptomatic plants and from insects that had been feeding on them (accession Nos HQ379735, HQ379737) were 98% homologous with the nucleotide sequence of Ca.L.s. genome deposited in GenBank (FJ957897). The phylogenetic tree comprising the sequence of the 1,160 kb amplicon from the 16Sr region of the bacterium from Yurécuaro and those from isolates deposited in GenBank showed a close relationship with the bacteria Ca. Liberibacter psyllaurous and Ca. Liberibacter solanacearum (Fig. 4). Bacilliform and circular structures 4 µm long and 0.1 µm in diameter, interpreted as longitudinal and cross sections, respectively, of walled bacterial cells were observed in the vascular cylinder of the main veins of symptomatic chile leaves (Fig. 5 A-B). These dimensions are within the range reported for Ca.L.s. by Tanaka et al. (2007), Bové (2006) and Liefting et al. (2009). The symptoms induced by Ca.L.s. in chile fields of Yurécuaro are different from those reported in New Zealand in the same crop, possibly because of the different cultivars involved, but recall those described from citrus, i.e. mottling, chlorotic veins, deformation of the fruits, and shrunken coffee-coloured seed (Garnier et al., 2000; Khairulmazmi et al., 2008; Da Graca et al., 2004). Fig. 3. A. PCR products obtained with the primers OA2 and O12c. Lane 1, Molecular weight markers of 100 bp; lane 2, negative control; lanes 3-6, DNA from insects. B. Chile pepper plant (Capsicum annum L.) with symptoms of variegation, yellowing and reduced leaf area obtained after 90 days exposure to the insects Bactericera cockerelli. 010_JPP430RP(Rojas)_331 334 20-07-2011 14:34 Pagina 334 Etiology of chile pepper variegation from Mexico Journal of Plant Pathology (2011), 93 (2), 331-335 Fig. 4. Phylogenetic tree of the 16S rRNA region of bacteria, made in MegAlign by the alignment method V of Clustal with the accession numbers obtained from GenBank. The scale bar indicates a substitution for each 100 nucleotides. The date of Ca.L.s. introduction into Mexico is uncertain. The disease has been reported in 2009, whilst the presence of B. cockerelli the vector responsible for its dissemination dates back to 1947 (Plesch, 1947, cited by Vega et al., 2008), long before this bacterium became a threat for solanacous plants. Fig. 5. Transmission electron photomicrographs of phloem tissue of chile pepper infected by Ca. Liberibacter solanacearum; the arrows indicate oblong bodies in the phloem of the central veins of symptomatic leaves. In practice, control of Ca.L.s. in potato crops has focused on vector management due to the phloem-restricted localisation of Liberibacter and dissemination operated primarily by insect vectors (Bové, 2006). Results of this investigation suggest that the most likely route through which the bacterium arrived to Yurécuaro is the seed, which is still contributing to its dissemination locally. Disease symptoms were observed before the insect vector was detected in chili fields (unpublished observations), thus supporting the likelihood that seeds play a relevant role in pathogen dissemination. According to Schuster and Coyne (cited by Orvin and Hinkle, 1976), bacteria may enter the seed via the vascular system, travel to the germination tube of pollen grains and the hilum of mature seed and invade their dorsal suture. Later, they may migrate to the funiculus through the raphe, pass to the seed coat, and rest there for a long periods of time. Since the presence of bacteria in seed lots represents the primary source of inoculation, one of the measures that may help reducing the phytosanitary problem, is the use of bacterium-free seeds. On the other hand, integrated management of B. cockerelli is necessary to avoid disease spread within a field. In the absence of chile crops, the insect vector has other host plants, and the bacterium can also survive as it was recorded from Solanum betaceum and Physalis peruviana (Liefting et al., 2009). 010_JPP430RP(Rojas)_331 20-07-2011 14:34 Pagina 335 Journal of Plant Pathology (2011), 93 (2), 331-335 REFERENCES Abad J.A., Bandla M., French-Monar R.D., Liefting L.W., Clover G.R.G., 2009. First report of the retection of ‘Candidatus Liberibacter’ species in Zebra chip disease-infected potato plants in the United States. Plant Disease 93: 108. Ahrens U., Seemueller E., 1992. Detection of DNA of plant pathogenic mycoplasma like organism by a polymerase chain reaction that amplifies a sequence of the 16S rRNA gene. Phytopathology 82: 828-832. Bové J.M., 2006. Huanglongbing: a destructive, newly-emerging, century-old disease of citrus. Journal of Plant Pathology 88: 7-37. Da Graca J.V., Korsten L., 2004. Citrus Huanglongbing: review, present status and future strategies. Diseases of Fruits and Vegetables 1: 229-245. Garnier M., Jagoueix-Eveillard S., Cronje P.R., Le Roux H.F., Bové J.M., 2000. Genomic characterization of a Liberibacter present in an ornamental rutaceous tree, Calodendrum capense, in the Western Cape Province of South Africa. Proposal of ‘Candidatus Liberibacter africanus subsp. capensis’. International Journal of Systematic and Evolutionary Microbiology 50: 2119-2125. Hansen A.K., Trumble J.T., Stouthamer R., Paine T.D., 2008. A New Huanglongbing Species, “Candidatus Liberibacter psyllaurous”, found to infect tomato and potato, is vectored by the psyllid Bactericera cockerelli (Sulc).Applied and Environmental Microbiology 74: 5862-5865. Khairulmazmi A., Kamaruzaman S., Habibuddin H., Jugah K., Syed O.S.R., 2008. Occurrence and spread of Candidatus Liberibacter asiaticus, the causal agent of Huanglongbing disease of citrus in Malaysia. Research Journal of Agriculture and Biological Sciences 4: 103-111. Liefting L.W., Perez-Egusquiza C.Z., Clover G.R.G., Anderson J.A.D., 2008. A new ‘Candidatus Liberibacter’ species in Solanum tuberosum in New Zealand. Plant Disease 92: 1474. Liefting L.W., Sutherland P.W., Ward L.I., Paice K.L., Weir Received September 6, 2010 Accepted January 3, 2011 Camacho-Tapia et al. 335 B.S., Clover G.R.G., 2009. A New ‘Candidatus Liberibacter’ species associated with diseases of solanaceous crops, Plant Disease 93: 208-214. MAF, 2008. Sección de Bioseguridad del Ministerio de Agricultura y Silvicultura de Nueva Zelanda. www.biosecurity. govt.nz (date accessed: August, 2010). Munyaneza J.E., Sengoda V.G., Crosslin J., Garzon Tiznado J.A., Cardenas Valenzuela O.G., 2009. First report of Candidatus Liberibacter solanacearum in pepper plants in México. Plant Disease 93: 1076. Orvin J., Hinkle N.F., 1976. Bacteria ovules and seeds. Applied and Enviroment Microbiology 32: 694-698. Plant Viruses Online, 2008. Descriptions and List from the VIDE Database. http://pvo.bio-mirror.cn/refs.htm (date accessed: August, 2008). Plesch D.J., 1947. The potato psyllid Paratrioza cockerelli (Sulc), its biology and control. Montana Agricultural Experimental Station Bulletin 446: 95. Schuster M.L., Coyne J.J., 1974. Survival mechanisms of phytopathogenic bacteria. Annual Review of Phytopahology 12: 199-221. SIAP (Servicio de Información Agroalimentaria y Pesquera), 2007. Agricultura, Producción anual por cultivo. www.siap.gob.mx (date accessed: August , 2010). SIAP (Servicio de Información Agroalimentaria y Pesquera), 2009. Agricultura, Producción anual por cultivo. www.siap.gob.mx (date accessed: August, 2010). Tanaka F.A.O., Colleta-Filho H.D., Alves K.C.S., Spinelli M.O., Machado M.A., Kitajima E.W., 2007. Detection of the “Candidatus liberibacter americanus” in phloem vessels of experimentally infected Cataranthus roseus by scanning electron microscopy. Fitopatologia Brasileira 32: 519. Vega G.M.T., Rodriguez M.J.C., Diaz G.O., Bujanos M.R., Mota S.D., Martinez C.J.L., Lagunes T.A., Garzón T.J.A., 2008. Susceptibilidad a insecticidas en dos poblaciones mexicanas del salerillo, Bactericera cockerelli (Sulc) (Hemiptera: Triozidae). Agrociencia 42: 463-471.