Compuestos volatiles del limon persa y limon mexicano asociados con sintomas de HLB (Huanglongbing).
The Asian citrus psyllid, Diaphorina citri (Kuwayama, 1908) (Hemiptera: Psyllidae), is the vector of the bacteria Candidatus Liberibacter asiaticus (Jagoueix et al. 1994), which produce the disease known as Huanglongbing or HLB in the genus Citrus (Garnier et al. 2000; Halbert and Nunez 2004). In citrus, D. citri develops of its biological cycle exclusively in the vegetative young shoots under five centimeters long (Fernandez and Miranda 2005). It has been determined that D. citri locates its host through its volatile compounds (Wenninger et al. 2009; Patt and Setamou 2010). The hosts of D. citri release different numbers of volatile (Wenninger et al. 2009; Patt and Setamou 2010; Robbins et al. 2012). Attributed to the plant-insect and plant-pathogen interactions (Patt and Setamou 2010; Hare 2011). Mann et al. (2012) determined that C. Liberibacter asiaticus induces volatile compounds in C. aurantium and C. sinensis young shoots. Also, D. citri has a higher response to volatile produced by infested young shoots than those by asymptomatic young shoots of both Rutaceaes species. The detection and understanding of the role that volatiles play in the chemical ecology of D. citri can be used to formulate management strategies for this insect, as well as in the production of resistant or tolerant cultivars through traditional and genetic engineering (Robbins et al. 2012). The detection of trees infected with C. Liberibacter asiaticus is done by visual inspection of the symptoms, electron microscopy, polymerase chain reaction (PcR), and real-time PCR (Salcedo et al. 2010). However, the use of these techniques is limited because the bacteria is in low concentrations in the infected trees and symptoms of HLB do not appear on leaves for months to years after initial infection (Gottwald 2010). This increases the possibility of misdiagnosis and the need to seek new alternatives to detect C. Liberibacter asiaticus in citrus more efficiently (Pacheco et al. 2012). In this report, the aim was to quantify the abundance and concentration of the volatile compounds released from the young shoots of Persian lemon (C. latifolia Tanaka) and Mexican lemon [Citrus aurantifolia (Christm.) Swingle] with and without HLB symptoms.
Materials and methods
Plant material. Young shoots were cut from eight-yearold trees in the flowering stage (January to March in 2013). Samples of Persian lemon tree shoots were collected in Santiago Ixcuintla, Nayarit and samples of Mexican lemon tree shoots were collected in Tecoman, Colima. In both species of lemon, shoots of 1 to 3 cm were collected following the sampling methodology described by the National Agroalimentary Health and Safety Service (Servicio Nacional de Sanidad, Inocuidad y Calidad Agroalimentaria-SENASICA 2010). Shoots with symptoms were collected from trees with mature leaves with asymmetric mottling, green islands and marked positive for HLB by SENASICA (SENASICA 2010). The healthy shoots were collected in commercial plots with trees without report or HLB symptoms. The collected shoots were covered with bee wax in order to avoid water loss or aroma change (Flamini et al. 2007). Then, they were covered with a paper towel, deposited in a polyethylene bag with a sealing device and transported, in a cooler gel cooler, to the laboratory of the Autonomous University of Aguascalientes in Aguascalientes, Mexico (SENASICA 2010). In the laboratory, the wax layer was removed and the shoots without the presence of nymphs or eggs of D. citri were selected.
Collection of volatile compounds. The volatile compounds were sampled from: T1) asymptomatic Persian lime (C. latifolia) young shoots (BAA), T2) symptomatic Persian lime young shoots (AAS), T3 asymptomatic Mexican lime (C. aurantifolia) young shoots, T4) symptomatic Mexican lime young shoots, and T5) a vial without plant sample (control). Each treatment consisted of ten replicates and the experiment was repeated five times. The collection was done using the solid-phase microextraction (SPME) technique using a polydimethylsiloxane fiber (PDMS-DVB, Supelco, Bellefonte, PA). The wax was removed from the stem base and weighed one gram of plant samples (young shoots). The sample was maintained at room temperature for 20 seconds and placed in a 10 ml glass vial with a polyethylene cap (Sigma-Aldrich[R], Toluca, Mexico). Then, the fiber was introduced into the vial and the volatile compounds were captured for 60 minutes. The fiber was removed and placed in the injection port of a gas chromatograph (Agilent 6850 Series II; Agilent, Foster City, CA) linked to a mass selective detector (model 5975C, Agilent, Foster City, CA). The chromatography analyses were done with the instrumentation method described by Patt and Setamou (2010), using a 30 m DB-WAX column, 0.320 mm internal diameter and 0.25 [micro]m film thickness (Agilent[R], Folsom, CA, USA).
Identification and quantification of the volatile compounds. The compounds were identified based on the retention time and mass spectrum of the standard library compounds NIST 2002 version (National Institute of Standard and Technology). The abundance and concentration of each compound were determined by contrasting the area of the volatile compounds collected from the young shoots against the area of the pure standard of D-limonene, 6 % pure (Sigma-Aldrich) (Gonzalez-Palomares et al. 2009; Lin et al. 2010).
Data analysis. To determine statistical differences, we applied a Student's t-test for independent samples to the concentration value of the volatiles collected in both types of the two species of lemon young shoots. All statistical tests were considered as two-tailed, and the significance level was set at the value of P < 0.05. The continuous variable in this study showed a non-normal distribution by KolmogorovSmirnov (Chakravarti et al. 1967) and Shapiro-Wilk tests (Shapiro and Wilk 1965). Hence, statistical analysis was performed with the variable after logarithmic transformation of data. Analyses were performed in the 2004 version SAS statistical package (SAS 2004).
In asymptomatic and symptomatic Persian lime tree shoots 29 and 21 volatile compounds, respectively, were collected (Table 1). In both types of shoots, 16 compounds were identified in common. In contrast, in asymptomatic Mexican lime tree shoots, 13 compounds were identified. While in symptomatic shoots of the same lemon species, eight volatiles were collected. In both types of buds, five compounds were identified in common. In asymptomatic and symptomatic lemon tree shoots of both species the following compounds were collected: D-limonene, caryophyllene, and [beta]-ocimene in different amounts and concentrations (Table 2).
Furthermore, analyses of the amount and concentration of the volatiles collected showed statistically significant differences between the two lemon species. The citronellal, [beta]-elemene, [alpha]-terpineol, Isogeraniol and lavandulol compounds were more abundant and in higher concentration in asymptomatic Persian lime tree shoots (Fig. 1; P < 0.05 and P < 0.005 by Student's t-test). In contrast, [beta]-myrcene compounds, D-limonene, [beta]-ocimene, [gamma]-elemene, [beta]-citral and geraniol were collected in higher concentration in symptomatic than in asymptomatic shoots of the same lemon tree species. In the case of the volatiles collected in the Mexican lemon tree, the elemene compound was collected in higher concentration in asymptomatic tree shoots. While in this same lemon species, compounds: D-limonene, caryophyllene and [beta]-farnesene were collected in greater concentration in symptomatic than in asymptomatic tree shoots (Fig. 2; P < 0.05, P < 0.005 and P < 0.001 by Student's t-test). The volatile compound D-limonene was present in both species of lemon tree. However, this compound was collected in higher concentration (13.33 mg / mL) in symptomatic Mexican lemon tree shoots.
Different authors have analyzed the volatile compounds released by the host plants of D. citri (Wenninger et al. 2009; Patt and Setamou 2010; Robbins et al. 2012). In this report, volatiles were collected in Persian lime and Mexican lime tree shoots. In both lime, asymptomatic and symptomatic shoots released different numbers of volatile. Asymptomatic and symptomatic shoots released similar compounds but in different amounts and concentrations. This may be attributed to the plant-insect and plant-pathogen interactions (Patt and Setamou 2010; Hare 2011). Mann et al. (2012) determined that C. Liberibacter asiaticus induces the release of [beta]-ocimene and D-limonene in C. aurantium and C. sinensis young shoots. And that the higher percentage found in D. citri responds to these volatile compounds emitted from asymptomatic tree shoots of both species of Rutaceae. In this study, [beta]-myrcene, D-limonene, [beta]-ocimene, [gamma]-elemene, [beta]-citral, geraniol, caryophyllene, and [beta]-farnesene had higher concentrations in shoots from trees with symptoms of Huanglongbing. This is of biological significance and could be used for early detection of the disease in different species of lemon trees.
For example, when growing Capsicum annuum L. it has been determined that infected, healthy and mechanically damaged Coletotrichum spp. fruits emit different numbers of volatile compounds. Such information is used in rating their quality in the commercialization of these vegetables (In-Kyung et al. 2007).
The young shoots with and without HLB symptoms released volatile compounds. The compounds D-limonene, [beta]-ocimene and caryophyllene were collected in greater concentrations in the infected tree young shoots in both species of lemon tree. These results indicate the feasibility of early detection of trees infected with this bacterium.
We thank the Consejo Nacional de Ciencia y Tecnologia (CONACyT) (National Council for Science and Technology) for funding this project.
CHAKRAVARTI, I. M.; LAHA, R. G.; ROY, J. 1967. Handbook of Methods of Applied Statistics, John Wiley, 392 p.
FERNANDEZ, M.; MIRANDA, I. 2005. Comportamiento de Diaphorina citri Kuwayama (Hemiptera: Psyllidae). Parte III: Relacion entre el ciclo de vida y el brote vegetativo foliar. Revista de Proteccion Vegetal 20 (3): 161-164.
FLAMINI, G.; TEBANO, M.; CIONI, P L. 2007. Volatiles emission patterns of different plant organs and pollen of Citrus limon. Analytica Chimica Acta 589 (1): 120-124.
GARNIER, M. S.; JAGOUEIX, E.; CRONJE, R. P.; LE ROUX, F. G.; BOVE, M. J. 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 (1): 2119-2125.
GONZALEZ-PALOMARES, S.; ESTARRON-ESPINOSA, M.; GOMEZ-LEYVA. J. F.; ANDRADE-GONZALEZ, I. 2009. Effect of the temperature on the spray drying of roselle extracts (Hibiscus sabdariffa L.). Plant Foods for Human Nutrition 64 (1): 62-67.
GOTTWALD, T. R. 2010. Current epidemiological understanding of Citrus Huanglongbing. Annual Review of Phytopathology 48 (10): 119-139.
HALBERT, S. E.; NUNEZ, C. A. 2004. Distribution of the Asian citrus psyllid, Diaphorina citri Kuwayama (Rhynchota: Psyllidae) in the Caribbean basin. Florida Entomologist 87 (3): 401-402.
HARE, J. D. 2011. Ecological role of volatiles produced by plants in response to damage by herbivorous insects. Annual Review of Entomology 56: 161-180.
IN-KYUNG, K.; ABD EL-ATY, A. M.; HO-CHUL, S.; HYANG BURM, L.; IN-SEON, K.; JAE-HAN, S. 2007. Analysis of volatiles compounds in fresh healthy and diseased peppers (Capsicum annuum L.) using solvent free solid injection coupled with gas chromatography-flame ionization detector and confirmation with mass spectrometry. Journal of Pharmaceutical and Biomedical Analysis 45 (3): 487-494.
LIN, Y. S.; ROAN, F. S.; LEE, L. C.; CHEN, Z. I. 2010. Volatile organic components of fresh leaves as indicators of indigenous and cultivated citrus species in Taiwan. Bioscience, Biotechnology and Biochemistry 74 (4): 806-811.
MANN, R. S.; ALI, J. G.; HERMANN, J. G.; TIWARI, S. L.; PELZSTELINSKI, K. S.; ALBORN, H. T.; STELINSKI, H. T. 2012. Induced release of plant-defense volatiles "Deceptively" attracts insect vector to plants infected with a bacterial pathogen. PLoS Pathogens 8 (3): 1-13.
PACHECO, C. J.; SAMANIEGO, R. J.; FONTES, P. A. 2012. Tecnologia para el manejo integrado del psilido Diaphorina citri Kuwayama (Hemiptera: Psyllidae) en citricos en Sonora. INIFAP. Centro de Investigacion Regional del Noroeste. Campo Experimental Norman E. Borlaug. Cd. Obregon, Sonora, Mexico. Folleto Tecnico N. 88. ISBN: 978-607-425-817-2.
PATT, J. M.; SETAMOU, M. 2010. Responses of the Asian citrus psyllid to volatiles emitted by the flushing shoots of its Rutaceous host plants. Environmental Entomology 39 (2): 618-624.
ROBBINS, P. S.; ROCCO, T. A.; LUKASZ, L. S.; STEPHEN, L. S. 2012. Volatile profiles of young leaves of Rutaceae spp. varying in susceptibility to the Asian Citrus Psyllid (Hemiptera: Psyllidae). Florida Entomologist 95 (3): 774-776.
SALCEDO, B. D.; HINOJOSA, R. A.; MORA-AGUILERA, G.; COVARRUBIAS, G. L.; DEPAOLIS, F.; MORA, S.; CINTORA, G. C. 2010. Evaluacion del impacto economico de Huanglongbing (HLB) en la cadena citricola mexicana. Instituto Interamericano de Cooperacion para la Agricultura (IICA). Direccion General de Sanidad Vegetal del SENASICASAGARPA. Available at: http://argus.iica.ac.cr/Esp/regiones/ norte/mexico/Publicaciones%20de%20la%200ficina/B2009e. pdf. [Review date: 5 April 2016].
SAS-INSTITUTE. 2004. SAS/STAT[R] 9.1 User's Guide, 2nd Ed. SAS Institute, Inc., Cary, NC.
SENASICA. 2010. Protocolo de actuacion para la deteccion del Huanglongbing. Direccion General de Sanidad Vegetal. Direccion de Proteccion Fitosanitaria. Available at: http://www. siafeson.com/sitios/simdia/docs/protocolos/02Protocolodeactua cionantelaemergenciadelHLB. [Review date: 5 April 2016].
SHAPIRO, S. S.; WILK, M. B. 1965. "An analysis of variance test for normality (complete samples)". Biometrika 52 (3/4): 591-611.
WENNINGER, E. J.; STELINSKI, L. L.; HALL, D. G. 2009. Role of olfactory cues, visual cues, and mating status in orientation of Diaphorina citri Kuwayama (Hemiptera: Psyllidae) to four different host plants. Environmental Entomology 38 (1): 225-234.
Received: 9-Jun-2016 * Accepted: 20-Oct-2017
MENDOZA-PENA, E.; CIBRIAN-TOVAR, J.; VELAZQUEZGONZALEZ, J.; TAFOYA-RANGEL, F.; AZUARADOMINGUEZ, A. 2018. Volatile compounds of Persian and Mexican lime associated with HLB (Huanglongbing) symptoms. Revista Colombiana de Entomologia 44 (1): 19-24. Enero-Junio 2018.
ESTRELLA MENDOZA-PENA (1), JUAN CIBRIAN-TOVAR (2), JULIO VELAZQUEZ-GONZALEZ (3), FELIPE TAFOYA-RANGEL (4) and AUSENCIO AZUARA-DOMINGUEZ (5)
(1) Maestra en ciencias, Colegio de Postgraduados, Montecillo, Estado de Mexico, 56230, Mexico, email@example.com. (2) Doctor en ciencias, Colegio de Postgraduados, Montecillo, Estado de Mexico, 56230, Mexico, firstname.lastname@example.org. (3) Doctor en Ciencias, Instituto Nacional de Investigaciones Forestales, Agricolas y Pecuarias, Campo Experimental Delicias, Carretera Delicias-Rosales Km 2,5, Chihuahua, 56576, Mexico, email@example.com. (4) Doctor en Ciencias, Universidad Autonoma de Aguascalientes, Aguascalientes, Aguascalientes, 20131, Mexico, firstname.lastname@example.org. (5) Doctor en Ciencias, Tecnologico Nacional de Mexico/I.T. de Cd. Victoria, Ciudad Victoria, Tamaulipas, 87010, Mexico, email@example.com. Corresponding autor: Ausencio Azuara-Dominguez. Doctor en ciencias, Tecnologico Nacional de Mexico/I.T. de Cd. Victoria, Ciudad Victoria, Tamaulipas, 87010, Mexico, firstname.lastname@example.org.
Caption: Figure 1. Analysis of the concentration of volatile compounds collected in asymptomatic (SA) and symptomatic (SS) Citrus latifolia trees shoots. Significantly different from the corresponding control value: * P < 0.05, ** P < 0.005 and *** P < 0.001.
Caption: Figure 2. Analysis of the concentration of volatile compounds collected in asymptomatic (SA) and symptomatic (SS) Citrus aurantifolia trees shoots. Significantly different from the corresponding control value: * P < 0.05, ** P < 0.005 and *** P < 0.001.
Table 1. Abundance of volatile compounds emitted by young shoots asymptomatic and symptomatic of Citrus latifolia and Citrus aurantifolia. Compound Abundance (%) Tr (min) C. latifolia C. aurantifolia SA SS SA SS [alpha]-pineno 3.55 0.53 0.50 Sabinene 5.42 0.59 [beta]-thujene 5.42 3.44 [beta]-myrcene 6.38 0.52 4.88 [beta]-pinene 6.38 2.62 D-limonene 7.22 33.38 45.73 21.62 80.78 [beta]-phellandrene 7.44 0.89 0.63 Eucaliptol 7.50 1.58 2.10 [gamma]-terpinene 8.38 0.27 [beta]-ocimene 8.53 7.25 10.82 1.62 7.30 [sigma]-elemene 14.00 0.81 [delta]-elemene 14.02 1.09 5.51 Carene 14.06 0.78 Terpineol 14.07 0.48 Citronellal 14.30 1.58 0.34 [beta]-citronellal 14.31 1.01 Linalool 16.06 1.03 1.12 [alpha]-bergamotene 16.84 0.96 2.50 Elemene 16.94 6.35 1.32 Methylgeranate 16.94 0.35 [beta]-elemene 16.95 1.55 0.85 Caryophyllene 17.11 0.54 0.86 1.63 2.63 [gamma]-elemene 18.09 0.35 1.40 6.06 [alpha]-cariofileno 18.80 1.13 [beta]-citral 19.13 10.18 12.64 [alpha]-terpineol 19.58 1.39 0.26 D-germacrene 19.70 0.69 1.63 3.31 [alpha]-bisabolene 20.10 1.40 [beta]-bisabolene 20.10 2.61 1.64 Neryl acetate 20.14 1.24 Citral 20.27 8.20 46.37 [alpha]-citral 20.27 5.74 [alpha]-farnesene 20.56 2.45 [beta]-farnesene 20.58 1.38 2.90 Citronellol 21.12 0.64 [beta]-citronellol 21.12 1.06 Perillal 21.40 0.47 Isogeraniol 22.04 1.76 0.34 Geraniol 22.87 3.11 4.99 Lavandulol 22.87 10.53 3.81 Tr = Retention time. SA = Shoots asymptomatic. SS = Shoots symptomatic. Table 2. Concentration of volatile compounds emitted by young shoots asymptomatic and symptomatic of Citrus latifolia and Citrus aurantifolia. Concentration ([micro]g/mL) Compound C. latifolia C. aurantifolia SA SS SA SS [alpha]-pinene 0.08 0.08 Sabinene 0.08 [beta]-thujene 0.49 [beta]-myrcene 0.07 0.81 [beta]-pinene 0.07 D-limonene 4.76 7.55 0.91 13.33 [beta]-phellandrene 0.13 0.10 Eucaliptol 0.23 0.35 [gamma]-terpinene 0.04 [beta]-ocimene 0.96 1.51 0.07 0.21 [sigma]-elemene 0.02 [delta]-elemene 0.16 0.23 Carene 0.11 Terpineol 0.07 Citronellal 0.23 0.06 [beta]-citronellal 0.14 Linalool 0.15 0.05 [alpha]-bergamotene 0.14 0.11 Elemene 0.27 0.04 Methylgeranate 0.06 [beta]-elemene 0.22 0.14 Caryophyllene 0.08 0.14 0.00 0.17 [gamma]-elemene 0.05 0.23 0.26 [alpha]-caryophyllene 0.05 [beta]-citral 1.45 2.09 [alpha]-terpineol 0.20 0.04 D-germacrene 0.10 0.27 0.14 [alpha]-bisabolene 0.06 [beta]-bisabolene 0.37 0.05 Neryl acetate 0.20 Citral 1.17 1.96 [alpha]-citral 0.95 [alpha]-farnesene 0.35 [beta]-farnesene 0.00 0.18 Citronellol 0.11 [beta]-citronellol 0.15 Perillal 0.07 Isogeraniol 0.25 0.06 Geraniol 0.44 0.82 Lavandulol 1.50 0.63 SA = Shoots asymptomatic. SS = Shoots symptomatic.