105Proc. Fla. State Hort. Soc. 123: 2010.

Proc. Fla. State Hort. Soc. 123:105–108. 2010.

*Corresponding author; phone: (863) 956-1157, ext. 1477; email: rrouseff@ufl.edu

RefeReed ManuscRipt

A Comparison of Common and Different Volatiles in “White” Guava and ‘Valencia’ Orange Leaves

EbEnEzEr OnagbOla, JOhn SmOOt, lukaSz StElinSki, and ruSSEll rOuSEff*University of Florida, IFAS, Citrus Research and Education Center, 700 Experiment Station Road,

Lake Alfred, FL 33850

additional index woRds. static headspace, SPME, GC-MS

Static head-space volatile extraction was conducted to concentrate ‘Valencia’ orange and guava leaf volatiles, which were subsequently analyzed using gas chromatography–mass spectrometry (GC-MS). Forty-seven volatiles were identified in both guava and ‘Valencia’ leaves. There were 35 volatiles unique to each leaf type and 12 volatiles in com-mon. Guava leaf volatiles consisted primarily of esters whereas ‘Valencia’ leaf volatiles consisted almost exclusively of terpenes. For guava, there were 20 esters, 13 terpenes/sesquiterpenes, 7 aldehydes, 2, ketones, 2 alcohols, 2 furans, and one acid. ‘Valencia’ leaf volatiles consisted of 33 terpenes/sesquiterpenes, 7 alcohols, 4 aldehydes, and 3 esters. Of the 12 volatiles in common, 8 were terpenes, 3 were aldehydes, and one alcohol. Identification was based on matching sample MS fragmentation patterns with those in the NIST library as well as matching literature standardized reten-tion index values. Identifications were confirmed by matching observed fragmentation patterns and retention values with those of known standards run under identical conditions. The volatile profiles from guava and ‘Valencia’ orange leaves were profoundly different.

The Asian citrus psyllid [Diaphorina citri Kuwayama (He-miptera: Psyllidae)] vectors three Liberibacter species, which are the causal pathogens of greening/huanglongbing (Su and Huang, 1990; Tsai et al., 1988). Huanglongbing (HLB) is a phloem-limiting bacteria which produces rapid tree decline, fruit loss and eventual tree death (Bove, 2006; Capoor, 1963; Roistacher, 1996). Since first identified in south Florida in 1998 (Halbert et al., 1998), D. citri has spread to all citrus-producing regions in the state. It has become the major threat to U.S. citrus production (Michaud, 2001; Michaud and Olsen, 2004).

Interplanting guava (Psidium guajava L.) with citrus in Viet-nam has been reported to reduce populations of D.citri and the incidence of HLB (Beattie et al., 2006; Hall et al., 2007, 2008). This has been attributed to a repellent effect of guava volatiles. Because of the apparent protective effect of guava, a comparison study of ‘Valencia’ orange and guava volatiles was undertaken. Although dimethyl disulfide has been identified as a key biologi-cally active volatile produced by guava but not citrus (Rouseff and others 2008), there could be other guava volatiles which might contribute to this protective effect. In order to be protective, these additional volatiles had to be found only in guava or primarily in guava and not ‘Valencia’ orange.

Materials and Methods

leaf saMples. Husbandry methods for the “white” guava and ‘Valencia’ orange citrus plants used in these investigations have been described previously (Rouseff et al., 2008). ‘Valencia’ orange citrus was selected for analysis because it is one of the most highly cultivated citrus varieties in Florida (FDACS, 2003).

Secondary plant metabolites are typically not evenly distributed within plants (Loomis and Croteau, 1980). In order to maximize the amount of static volatile metabolites for analyses, we used fresh leaf flush [immature leaves at the growing shoots (Hall and Albrigo, 2007)], which are known to contain a higher proportion of plant metabolites (Hrutfior et al., 1974) compared with older leaves or other plant parts.

Gas chRoMatoGRaphy–Mass spectRoMetRy (Gc-Ms) analyses. Leaf volatiles were collected from ~3.5-g samples of guava and citrus flush for GC-MS analyses using the SPME static headspace technique following methods described previously by Rouseff et al. (Rouseff et al., 2008). At least three replicates of each volatile sample were analyzed.

The collected volatiles were analyzed and identified with a PerkinElmer® Clarus 500 quadrupole gas chromatograph coupled to a mass spectrometer (GC-MS). The GC-MS was equipped with TurboMass software (PerkinElmer, Shelton, CT) and a 60 m × 0.25 mm i.d. × 0.50 µm Restek (Stabilwax) capillary column. Helium was used as the carrier gas in the constant flow mode of 2 mL/min. The source was kept at 180 °C, and the transfer line and injector were maintained at 240 °C. The oven was programmed from 40 to 240 °C at 7 °C/min. We matched mass spectra with NIST 2005 version 2.0 standard spectra (NIST, Gaithersburg, MD) and identifications with spectral fit values equal to or greater than 800 and appropriate LRI values were considered positive. Mass spectral fragmentation patterns and retention index values of authentic standards were used to confirm identifications when available.

Results and Discussion

Valencia leaf Volatiles. Citrus volatiles are known to be attractive to D. citri (Wenninger et al., 2009). Most of the citrus-specific compounds reported in these studies were terpenes known to serve as attractants of other insect herbivores (Kasperbauer and

106 Proc. Fla. State Hort. Soc. 123: 2010.

Loughrin, 2004; Wallin and Raffa, 2004). In a recent study, Patt and Setamou (2010) reported that D. citri were attracted to the volatiles in C. limon and C. sinensis, but not C. paradisi. A synthetic mixture of terpenes, sesquiterpenes, and terpene alcohols (ß-ocimene, ß-caryoph-yllene, linalool, α-cubebene, D-limonene, and myrcene) was also found to be attractive to D. citri. It should be noted that the major leaf volatiles in ‘Valencia’ oranges were ocimene, sabinene, ß-caryophyllene, limonene, and ß-myrcene. This is similar to the synthetic mixture identified as being a psyllid attractant except all eight volatiles found in highest relative con-centration were all terpenes or sesquiterpenes (Table 1). Z-3-hexenyl acetate and linalool (a terpene alcohol) round out the top 10 ‘Valencia’ leaf headspace volatiles.As shown in Figure 1, about 90% of the total ‘Valencia’ leaf volatiles were terpenes or sesquiterpenes, with 34 of the 48 ‘Valencia’ leaf volatiles terpenes or sesqui-terpenes. These hydrocarbon terpenes included acylic terpenes such as ocimene and myrcene, cyclic terpenes such as limonene and sabinene, as well as bicyclic terpenes such as α-pinene and δ-3-carene. The few oxygen-containing volatiles included seven alcohols, four alde-hydes and three esters, which comprised only about 10% of the total leaf volatiles.

GuaVa leaf Volatiles. In contrast to the overwhelming hydrocarbon terpene content of ‘Valencia’ leaf volatiles, guava leaf volatiles consisted of almost 80% oxygenated volatiles consisting primarily of esters with smaller amounts of aldehydes (Fig. 2). Six of the top 10 volatiles were esters, consisting of: isoamyl 3-methylbutyrate, isoamyl 2-methylbutyrate, 2-methylbutyl 2-methylbutyrate, 3-methyl-3-butenyl-3-methylbutanoate, geranyl acetate and geranyl propionate. The other four major volatiles consisted of two aldehydes: Z-3-hexenal and (E,E)-2,4-hexadienal as well as two terpenes: (Z,E)-α-farnesene and cis-ocimene. As shown in Figures 1 and 2, the volatile profiles of ‘Valencia’ and guava leaves are profoundly different.

coMMon Volatiles. As shown by the lack of overlapping peaks in Figure 1, guava and ‘Valencia’ leaves have few volatiles in com-mon. Therefore, it is not surprising that D. citri exhibit completely different responses to volatiles from these two sources. A careful comparison of the overlapping chromatograms in Figure 1 as well as Table 1 data, indicate that only 12 of the roughly 50 volatiles were found in both ‘Valencia’ orange and guava. Nine of the 12 common volatiles were terpenes/ sesqui-terpenes and consisted of: α-pinene, limonene, neoalloocimen, α-copaene, ß-caryophyllene, α-farnesene, ß-farnesene, and an unidentified sesquiterpene (LRI=1755).

Table 1. Comparison of guava and ‘Valencia’ orange leaf volatiles separated on a polar (wax) column. Background color code: green = terpenes, blue = esters, red = aldehydes, pink = ketones, yellow = alcohols, orange = furans, black = acids, * = common volatiles.

Guava leaf volatiles Valencia orange leaf volatiles

Obs Area Obs AreaNo. RT LRI % Identification No. RT LRI % Identification

107Proc. Fla. State Hort. Soc. 123: 2010.

Fig. 2. Comparison of relative amounts of guava and ‘Valencia’ leaf volatiles by chemical group.

Fig. 1. Chromatograms from ‘Valencia’ and guava leaf volatiles. Blue = ‘Valencia’ leaf volatiles; Red = guava leaf volatiles. Numbers and letters correspond to volatiles listed in Table 1.

Conclusions

It is apparent that the volatile composition of guava and ‘Va-lencia’ orange leaves are profoundly different. Therefore, it is not surprising that D. citri are attracted to ‘Valencia’ leaf volatiles and repelled by guava volatiles. The 12 volatiles found to be in common are unlikely responsible for either the attractive or re-pulsive influence on ACP behavior. Compounds responsible for the differences in ACP behavior should be due to the remaining non common volatiles identified in Table 1.

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