Introduction

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 Núñez 2004}). In citrus, D. citri develops of its biological cycle exclusively in the vegetative young shoots under five centimeters long (^{Fernández and Miranda 2005}). It has been determined that D. citri locates its host through its volatile compounds (^{Wenninger et al. 2009}; ^{Patt and Sétamou 2010}). The hosts of D. citri release different numbers of volatile (^{Wenninger et al. 2009}; ^{Patt and Sétamou 2010}; ^{Robbins et al. 2012}). Attributed to the plant-insect and plant-pathogen interactions (^{Patt and Sétamou 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 Rutaceae´s 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

Results

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 β-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, β-elemene, α-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, β-myrcene compounds, D-limonene, β-ocimene, γ-elemene, β-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 β-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.

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. 

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. 

Discussion

Different authors have analyzed the volatile compounds released by the host plants of D. citri (^{Wenninger et al. 2009}; ^{Patt and Sétamou 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 Sétamou 2010}; ^{Hare 2011}). ^{Mann et al. (2012)} determined that C. Liberibacter asiaticus induces the release of β-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, β-myrcene, D-limonene, β-ocimene, γ-elemene, β-citral, geraniol, caryophyllene, and β-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}).

Conclusions

The young shoots with and without HLB symptoms released volatile compounds. The compounds D-limonene, β-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.