Composition of volatiles of citrus varieties and their effects on the behavior of Diaphorina citri

Abstract

Huanglongbing (HLB) is the most devastating citrus disease worldwide, with no resistant varieties currently available. Effective management of HLB relies heavily on controlling its primary vector, the Asian citrus psyllid (<italic>Diaphorina citri</italic>, ACP). This study aimed to evaluate the attractiveness of different citrus varieties to ACP using a four-arm olfactometer and to characterize the volatile organic compounds (VOCs) extracted from these plants. The varieties tested included three sweet orange scions (‘Valencia’, ‘Westin’, and ‘Pera’) and three rootstocks (‘Rangpur’ lime, ‘Sunki Tropical’, and ‘BRS Bravo’). VOCs were extracted using HS-SPME and identified via GC-MS. Results indicated that ‘Sunki Tropical’, ‘Rangpur’ lime, and ‘Valencia’ were significantly more attractive to ACP compared to other varieties. The reduced attractiveness of rootstock genotypes, such as ‘BRS Bravo’ and the control ‘Flying Dragon’, was correlated with higher levels of germacrene D and γ-elemene. Conversely, the increased attractiveness of ‘Rangpur’ lime and ‘Sunki Tropical’ was associated with abundant D-limonene. In grafted scions on ‘Rangpur’ lime, linalool was identified as a key compound linked to the attractiveness of ‘Westin’ and ‘Valencia’. These findings suggest that the differential attractiveness of citrus varieties to ACP is influenced by specific VOC profiles.

Key words
Citrus greening; Volatile organic compounds; D citri; Citriculture; Four-Arm Olfactometer

INTRODUCTION

Huanglongbing (HLB) is currently the most serious citrus disease worldwide. It is associated with three species of phloem-restricted α-proteobacteria, Candidatus Liberibacter asiaticus, Ca. L. africanus and Ca. L. americanus (Teixeira et al. 2005a, b, Bové 2006, Teixeira et al. 2007). Recently, the occurrence of a new species in northern Colombia, Ca. L. caribbeanus was reported. The bacteria were detected in sweet orange (Citrus sinensis Osbeck) and in Diaphorina citri Kuwayama, 1908 (Hemiptera: Psyllidae). It is still unknown whether this species causes the same damage reported for HLB (Keremane et al. 2015).

The Asian and American species are transmitted by the psyllid D. citri and the African species by the psyllid Trioza erytreae Del Guercio, 1918. Insect vectors are primarily responsible for spreading HLB in orchards, although grafting contaminated material and producing seedlings in unprotected areas also contribute to its spread (Lopes & Frare 2008, Lopes et al. 2009, Guo et al. 2024).

As for the hosts of D. citri, despite the preference for Murraya paniculata (L.) Jack, known as orange jasmine (Halbert & Manjunath 2004, Borgoni et al. 2014), all citrus varieties can host the insect (Westbrook et al. 2011). However, some species such as ‘Sunki’ mandarin, Citrus sunki (Nava et al. 2007) and ‘Cleopatra’ mandarin, Citrus reshni (Tsagkarakis & Rogers 2010) may negatively affect oviposition, development, and survival of the insect. The negative effect caused by trifoliate genotypes, Poncirus trifoliata species, and Casimiroa edulis, on insect selection and colonization is also mentioned (Westbrook et al. 2011), as well as different reactions of citrus genotypes to HLB infection (Ramadugu et al. 2016).

Despite this knowledge, there is still no HLB-resistant variety of Citrus. Thus, disease control is based on inoculum reduction (removal of symptomatic plants) and the use of greenhouse produced HLB-free seedlings (Bové 2006). Population reduction of D. citri is also crucial in the management of HLB (Bové 2006, Belasque Júnior et al. 2010, Bassanezi et al. 2020). Among the promising pest control methods, behavioral control stands out, either using pheromones or allelochemicals (Fancelli et al. 2018, Amorós et al. 2019, Zanardi et al. 2018, 2019, Volpe et al. 2020, Yan et al. 2020, Silva et al. 2023).

D. citri responds to odors emitted by plants in olfactory bioassays, as demonstrated in both Y-tube and 4-way olfactometers bioassay (Onagbola et al. 2011, Signoretti 2014, Wu et al. 2015, Fancelli et al. 2018, Silva et al. 2023). Attractiveness can vary between infected and healthy plants. The psyllid is initially attracted to infected plants, probably because they are induced to release methyl salicylate, a compound that is associated with host attractiveness to the vector. However, over time, in the absence of new branches, the psyllid changes its choice to healthy plants (Mann et al. 2012, Wu et al. 2015, Silva et al. 2023), because the release of MeSA is not stable; over the course of the infection, it tends to decrease (Martini et al. 2018).

Studies to identify volatiles have been carried out in parallel with olfactometer bioassays to associate VOCs emitted by citrus plants with attractiveness to D. citri (Mann et al. 2012, Silva et al. 2023). The compound D-limonene, abundantly present in citrus, was associated with attractiveness to the insect (Mann et al. 2012). It was observed that most genotypes of Citrus sp., and other genotypes, that were less preferred by the psyllid had the VOCs phytol, (Z)-β-ocimene, (E)-caryophyllene, β-elemene and α-humulene (Andrade et al. 2016). The attractiveness of citrus varieties to D. citri seems to be associated with the synergistic effect of more than one class of the main VOCs released by plants (Patt & Sétamou 2010, da Silva et al. 2024).

Understanding the behavior of D. citri towards VOCs of different genotypes of cultivated citrus, constitutes an important tool in the control of the psyllid and, consequently, in the management of the HLB (Sanches et al. 2016). In Brazil, the production of sweet oranges is very relevant, with the ‘Pera’ variety grafted onto the ‘Rangpur’ lime standing out as the most cultivated in the country (Fundecitrus 2024, Silva et al. 2024). However, there is currently a need to diversify varieties in the orchard due to phytosanitary issues associated with the trend of expanding citrus farming to non-conventional areas due to HLB (Napoleão et al. 2023). Citrus rootstock selection can significantly contribute to crop resilience under climate change scenarios (Costa et al. 2025), in addition to providing the canopy with resistance to HLB (Bowman et al. 2016, Stover et al. 2016, 2018, Kunwar et al. 2021, Bodaghi et al. 2022, Gill et al. 2024).

In this sense, to contribute to the management and prevention of HLB, the objective of this study was to characterize citrus varieties regarding their attractiveness to D. citri through olfactometry bioassays and evaluation of the profile of VOCs emitted by them.

MATERIALS AND METHODS

Plant selection – Scion and rootstock varieties

Three sweet orange scion varieties [Citrus sinensis (L.) Osbeck] (‘Valencia’, ‘Westin’ and ‘Pera’) established on ‘Rangpur’ lime rootstock were used, as well as three rootstock varieties: ‘Rangpur’ lime (Citrus limonia Osbeck), ‘Sunki Tropical’ [C. sunki (Hayata) Hort. ex Tanaka] and ‘BRS Bravo’ hybrid (C. sunki x (C. limonia x P. trifoliata). ‘Flying Dragon’ [Poncirus trifoliata (L.) Raf.] and orange jasmine (Murraya paniculata L. Jack) seedlings were used as the least preferred (George & Lapointe 2018) and highly preferred control to D. citri (Patt & Sétamou 2010), respectively.

Three plants of each variety (measuring between 50 and 70 cm) were used for the olfactometry bioassays and three plants for VOC extraction, according to the approach used in other studies (Qian et al. 2024, Fauziah et al. 2022). The sweet orange seedlings were produced at Fazenda Gavião, Entre Rios – Bahia [11°54’07”S 38°16’00”W] and the rootstock and orange jasmine plants at Embrapa Cassava and Fruits, in Cruz das Almas, BA [12°40’51”S 39°05’11”W]. In both cases, coconut fiber was used as a substrate. Plants were kept in screen cages until they reached the age of eight months for bioassays and volatile extraction.

Rearing and selection of Diaphorina citri

Branches containing D. citri nymphs in final instars (fourth and fifth) were collected from orange jasmine plants. The nymphs were transferred to the laboratory and kept in PVC frame cages (40 cm wide x 40 cm deep x 40 cm high) covered with an anti-aphid screen and with a zippered opening on one side, made of transparent and resistant plastic. As adults emerged, they were captured and separated by sex with the aid of a stereomicroscope at 6x magnification. The distinction between males and females was made based on the final portion of the abdomen of psyllids in ventral view (Aubert 1987).

Females were separated for this study and kept in cages containing two orange jasmine plants for feeding, and males were discarded, as they do not respond to plant odors like females (Wenninger et al. 2009, Moghbeli Gharaei et al. 2014). The rearing room temperature was maintained at 26 ± 2°C, relative air humidity at 70 ± 10% and a 14h photophase (Nava et al. 2007).

Four-Arm Olfactometer Bioassay - Series I

Olfactometry bioassays consisted of tests to verify the attractiveness of selected scion and rootstock varieties to D. citri, HLB vector psyllid. For this purpose, the seedlings of the three scion varieties and the three rootstocks were compared to the M. paniculata and ‘Flying Dragon’ controls, also the controls were compared to each other. The bioassays were carried out in laboratory, at a temperature of 25 ± 1°C and relative humidity at 70 ± 10%. The tests were carried out from 9:00 am to 3:00 pm, period of greatest insect activity (Sétamou et al. 2012).

The airflow was maintained at 0.5 L min-1. Polyester bags were used as aeration chambers to isolate the aerial part of the plants from each of the treatments. The upper part of each bag had two outlets at the ends, one of them was connected to the flow meter regulating the air inlet, and the other to the olfactometer. Connections were made using polytetrafluoroethylene hose (Teflon®).

A four-arm olfactometer (Pettersson 1970, Fancelli et al. 2018, Alquézar et al. 2021) measuring 16.5 cm × 16.5 cm and thickness of 2 cm was used. The insect was introduced through the circular air drainage hole (0.8 cm in diameter) located in the center of the olfactometer. The odors of each treatment were evaluated in two arms of the olfactometer (2×2 configuration), alternately, forming a configuration of an x. The central area (where there is a mixture of odors) was considered the non-response area.

Females aged between four and seven days remained without food for a period of one hour to one and a half hour. For each treatment, 22 repetitions were evaluated, and the psyllids were exposed for 10 min to odors in each repetition. Each individual insect was considered a repetition. The plants of each variety served as treatments, and the insects’ responses to these treatments were analyzed accordingly. Psyllids that remained motionless in the non-response area for more than two minutes were considered non-responsive. Non-response rate was around 20%.

Four-Arm Olfactometer Bioassay - Series II

In series II, considering the results obtained in series I, new tests were performed to confirm repellency of ‘Flying Dragon’ and the attractiveness of ‘BRS Bravo’ and ‘Westin’ genotypes (Vet et al. 1983). The bioassays were performed as described in the previous section, except for the number of repetitions, which was 20. In the attractive configuration (1×3), volatiles of the genotype being tested were supplied in only one arm of the olfactometer, the other entries were supplied with clean air. In the repellent configuration (3×1), three arms received the odor of the test plant against 1 entry with clean air.

In both series of bioassays, the choices made by D. citri were recorded in the System for data acquisition in olfactometry bioassays – SOLF, using a numerical keyboard (Fancelli et al. 2017). White light was used, emitted by LED lamps positioned above the olfactometer to simulate daylight when the insect feeds on citrus plants. To eliminate environmental influences of light and odor, the arena was rotated 90° at each repetition in all treatments.

VOC extraction procedure

The extraction of VOCs was performed using headspace solid-phase microextraction (HS-SPME) and manual sampling (Mesquita et al. 2017). Three plants of each variety were selected, and each plant was sampled three times. The average value was then calculated for each plant, resulting in a total of three samples per plant variety. Leaf selection was standardized by harvesting the three youngest leaves with the blade completely open. For the ‘Flying Dragon’ control, which has trifoliate leaves and smaller leaves than the other varieties, the leaves just below the third youngest leaf were also collected. 0.2 g of fresh mass were used per replicate, which were macerated with a glass rod in a 20 mL vial. The vials were sealed with a Teflon®-coated silicone septum, which were placed in an aluminum block on a heating plate set at 62°C, where they remained for 11 min. Then, the cap seal was punctured by the sampler and the fiber (DVB/CAR/PDMS, 50/30 μm) was exposed for 47 min for adsorption of volatiles. Subsequently, the fiber was removed from the vial and inserted into the GC injector for thermal desorption of the analytes at 250 ºC for 3 min.

Gas Chromatography-Mass Spectrometry (GC-MS)

GC-MS analysis was performed using a Shimadzu GCMS-QP2010 Plus (Kyoto, Japan), operated in electron impact ionization mode at 70 eV. Thermal desorption injection was performed with an injector in splitless mode. Chromatographic separation was performed on a DB–5 MS column ((5%-phenyl) dimethylpolysiloxane; 30 m × 0.25 mm i.d., film thickness 0.25 μm, Restek, Bellefonte, USA). The GC oven operated with the following heating program: initial temperature of 35 °C for 5 min, increasing from 0.7 °C min-1 to 70 °C, remaining for 1 min; then 3.0 °C min-1 to 90 °C, remaining for 1 min; then 1.0 °C min-1 to 120 °C, remaining for 1 min; and, finally, increasing at a rate of 15 °C min-1 to 280 °C. The helium carrier gas was maintained at a constant flow of 0.60 mL min-1 (14.2 Kpa) and a constant linear velocity of 27.9 cm s-1. The temperature of the transfer line and ionization source was 220 °C, with a total analysis time of 105 min.

Analytes identification was performed by calculation of their retention indices, injections of a homologous series of n-alkanes (C₈ to C₂₂), commercial standards, and by comparison of the mass spectra obtained in the analyzes with the spectra from the NIST reference database (NIST – 147,198 compounds).

Statistical Analysis

In both series of bioassays, the evaluated variables, residence time and number of entries to the arena, were tabulated in electronic spreadsheets (Microsoft Excel 2016). These variables were submitted to the Shapiro-Wilk test to verify the normality of the treatments. Means were compared using the Wilcoxon test or paired t-test (p ≤ 0.05).

In the 3×1 or 1×3 configuration, the mean of the variables for the three arms was determined and compared with that of the single entry (Hegde et al. 2011, Sobhy et al. 2017). The analyses were performed using the statistical program R (R Core Team 2018).

Relative peak areas of the compounds detected and identified in the samples were autoscaled and submitted to the multivariate analysis of Sparse Partial Least Squares Discriminant Analysis (sPLS-DA) and hierarchical cluster analysis (HCA) using Metaboanalyst 5.0 software (Pang et al. 2021) to verify the formation of groups between the varieties based on the similarity and on the relative values of the VOCs areas (Hotelling 1936).

RESULTS

Four-Arm Olfactometer Bioassay - Series I

For the residence time of D. citri in the arms containing the odors of the orange jasmine and the odors of the different treatments, the only one that presented significant difference was the ‘BRS Bravo’ hybrid, with less time than in the orange jasmine (t = 2.2004, df = 21, p = 0.0196). There was no significant difference between orange jasmine and the other treatments (‘Sunki Tropical’, W = 128, df = 21, p = 0.4806; ‘Rangpur’ lime, t = 0.5247, df = 21, p = 0.3027; ‘Valência’, t = 0.37031, df = 21, p = 0.3574; ‘Westin’, t = 0.7342, df = 21, p = 0.2355; ‘Pera’, t = 0.1088, df = 21, p = 0.4572) (Fig. 1a).

Figure 1
Residence time of Diaphorina citri females in response to odors emitted by scion varieties grafted on ‘Rangpur lime’ (RL) and citrus rootstocks, compared to the orange jasmine control (Murraya paniculata) (a) and Flying Dragon control (Poncirus trifoliata) (b). Number of entries of D. citri females in response to odors emitted by scion varieties grafted on ‘Rangpur lime’ (RL) and citrus rootstocks, compared to the orange jasmine control (M. paniculata) (c) and Flying Dragon control (P. trifoliata) (d), in a 4-way arena. The mean or median values for * and for ** are different based on the t test or Wilcoxon test, respectively (P < 0.05 and P < 0.01); n.s. denotes non-significant differences.

Regarding the ‘Flying Dragon’ control, the treatment ‘Pera’ (t = 1.4310, df = 21, p = 0.1007) was the only one that did not differ in terms of time spent by females of D. citri. For the other combinations, the psyllid spent more time in the treatments than in the control (‘BRS Bravo’, t = 1.9200, df = 21, p = 0.0343; ‘Sunki Tropical’, t = 2.1212, df = 21, p = 0.0230; ‘Rangpur’ lime, t = 3.1588, df = 21; p = 0.0024; ‘Valência’, t = 2.3391, df = 21, p = 0.0147; ‘Westin’, t = 1.8680, df = 21, p = 0.0379) (Fig. 1b). Significant difference was also found between controls (‘Flying Dragon’ x orange jasmine) (t = 1.7694, df = 21, p = 0.0457).

Considering the number of entries in the olfactometer arms, there was no significant difference in any of the treatments compared to orange jasmine (‘BRS Bravo’, W = 100,5, df = 21, p = 0.1232; ‘Sunki Tropical’, W = 72, df = 21, p = 0.2435; ‘Rangpur’ lime, W = 36.5, df = 21, p = 0.3738; ‘Valencia’, W = 57, df = 21, p = 0.3877; ‘Westin’, W = 57, df = 21, p = 0.0741; ‘Pera’, W = 78, df = 21, p = 0.4807) (Fig. 1c). However, when treatments were compared to the ‘Flying Dragon’ control, there were significantly more entries into arms containing odors of the ‘Rangpur’ lime treatment (W = 18, df = 21, p = 0.0088) and ‘Westin’ (t = 2.1585, df = 21, p = 0.0213). For treatments orange jasmine (W = 62.5, df = 21, p = 0.2622), ‘BRS Bravo’ (W = 73, df = 21, p = 0.0948), ‘Sunki Tropical’ (W = 55.5, df = 21, p = 0.4209), ‘Valencia’ (W = 118.5, df = 21, p = 0.0723), and ‘Pera’ (W = 71, df = 21, p = 0.2053), there was no difference (Fig. 1d).

In this study, the ‘BRS Bravo’ hybrid rootstock was less attractive to the insect in terms of residence time in comparison to the orange jasmine attractive pattern (Fig. 1a), although it did not differ from this genotype in terms of the number of entries (Fig. 1c). It also did not differ from the least preferred pattern (‘Flying Dragon’) in terms of number of entries (Fig 1d), despite the attractiveness revealed by residence time (Fig. 1b). Considering that C. sunki and C. limonia, attractive genotypes to D. citri (Fig. 1b and 1d), are part of the cross that originated ‘BRS Bravo’ [C. sunki x (C. limonia x P. trifoliata), this genotype was further evaluated in the attractive configuration, while ‘Flying Dragon’, a natural mutation clone of P. trifoliata, was tested in the repellent configuration.

In relation to the other rootstock genotypes, differences were only found in comparisons with the least preferred pattern, with greater residence time and number of entries for ‘Rangpur’ lime (Fig. 1b and 1d). For ‘Sunki Tropical’, a higher value was found only for residence time (Fig. 1b). Among scion varieties, ‘Westin’ was the most attractive one in terms of both residence time and number of entries (Fig. 1b and 1d). Thus, it was subsequently tested in the attractive configuration. ‘Valencia’ promoted a longer residence time (Fig. 1b). The variety ‘Pera’, in turn, was the least attractive, not differing from ‘Flying Dragon’ control, despite having a longer residence time, not supported by statistical evidence, though (Fig. 1b and 1d).

Four-Arm Olfactometer Bioassay - Series II

The ‘Flying Dragon’ rootstock genotype was tested for repellency. ‘BRS Bravo’ seedlings and ‘Westin’ plants were tested in the attractive configuration, to confirm the previously detected patterns. The repellency of the ‘Flying Dragon’ genotype was confirmed, and the insects preferred the arms containing clean air, remaining longer in these areas (t = 2.5094, df = 19, p = 0.0107) and with a greater number of entries (W = 149, df = 19, p = 0.0148) (Fig. 3).

Figure 3
(a) Score plot of sPLS-DA obtained from the VOC profile of different treatments; (b) sPLS-DA loading vectors highlighting the 10 compounds that most discriminate between sample groups.

The results show that in comparison to ‘BRS Bravo’ hybrid, the insect remained for a similar time in the arm containing air (t = 1.1312, df = 19, p = 0.1026), despite having a greater number of entries than in the arm containing air (W = 158, df = 19, p = 0.0237) (Fig. 2). These results partially corroborate with the pattern of a greater attractiveness of the volatiles of this genotype compared to ‘Flying Dragon’ (series I results), probably due to the repellency exerted by the volatiles of the latter genotype. Interestingly, the highest number of entries was verified only when the volatiles of ‘BRS Bravo’ were compared with air (Fig. 2) and not when compared with those of ‘Flying Dragon’. For the ‘Westin’ scion grafted onto ‘Rangpur’ lime, no attractive effect on the insect was observed, both in terms of residence time (W = 125, df = 19, p = 0.2276) and in the number of entries (W = 95, df = 19, p = 0.1897). Therefore, the previously observed result is due to the repellency exerted by the volatiles of ‘Flying Dragon’ on D. citri (Fig. 1b and 1d).

Figure 2
Residence time (a) and number of entries of female of Diaphorina citri (b) in response to odors emitted by citrus genotypes in a 4-way arena, compared to clean air. ‘Flying Dragon’ rootstock was tested in a repellent configuration. For ‘BRS Bravo’ rootstock and ‘Westin’ variety, an attractive configuration was used. * Mean values are different based on the Wilcoxon test (P < 0.05) or t test (P < 0.05), n.s. not significant.

Detection and Identification of citrus volatile compounds through Gas Chromatography-Mass Spectrometry

Forty-three volatile organic compounds (VOCs) were identified in the genotypes included in this research (Table I), 21 in M. paniculata, 27 in ‘Flying Dragon’, 40 in the ‘BRS Bravo’ hybrid, 42 in ‘Rangpur’ lime, 39 in ‘Sunki Tropical’ and for the scion genotypes, 40 in ‘Westin’ and ‘Valencia’, and 39 in ‘Pera’, being these three grafted on ‘Rangpur’ lime tree. Most of these compounds, detected by using the HS-SPME-GC-MS technique, belong to the class of monoterpene hydrocarbons (32.56%), followed by the class of sesquiterpene hydrocarbons (30.23%). Other compounds such as oxygenated monoterpenes (9.31%), alcohols (9.31%), aldehydes (6.97%) and others (11.62%) are among the identified VOCs.

Table I
Relative composition (%) of VOCs identified in controls and in scion and rootstock varieties (n=9 replicates).

No.	Compound	LRIbEXP	LRIcLIT	Murraya paniculata	‘Flying Dragon’	‘BRS Bravo’	‘Rangpur lime’	‘Sunki Tropical’	‘Westin’	‘Pera’	‘Valência’
1	hexanala	803	803	0.14	0.10	0.07	0.05	0.01	0.08	0.08	0.05
2	2-hexanal	855	855	1.57	1.19	0.86	0.86	0.49	0.96	1.18	0.74
3	4-hexen-1-ol	859	866	0.59	0.60	0.51	0.21	0.52	0.43	0.71	0.66
4	2-hexen-1-ol	872	872	0.10	0.08	0.05	0.09	0.02	0.03	0.05	0.03
5	hexanol	874	874	0.02	0.23	0.13	0.04	0.01	0.03	0.06	0.04
6	2,4-hexadienal	911	910	0.02	0.05	0.10	0.04	0.02	0.09	0.10	0.06
7	α-thujene	927	923	-	-	0.14	0.18	1.38	0.88	1.22	1.28
8	α-pinenea	931	931	-	-	0.79	0.85	3.72	0.69	0.81	0.84
9	camphene	945	945	-	-	0.07	0.07	0.38	0.02	0.01	0.02
10	β-pinene	975	975	-	-	7.44	7.38	18.17	9.13	11.93	13.31
11	β-myrcenea	994	994	0.01	3.51	0.59	1.13	2.57	2.47	2.56	2.62
12	α-phellandrenea	1002	1002	-	0.01	0.12	0.08	0.48	0.96	0.89	0.97
13	3-carene	1006	1006	-	-	0.04	0.19	0.03	6.29	5.39	5.35
14	D-limonenea	1029	1031	-	0.03	3.33	24.87	4.34	3.56	3.27	3.13
15	cis-β-ocimenea	1044	1044	-	0.05	0.72	0.60	0.56	0.42	0.35	0.51
16	trans-β-ocimene	1059	1058	-	-	0.37	0.15	1.18	0.70	1.08	1.20
17	α-terpinolene	1085	1084	-	-	0.06	0.09	0.86	2.76	2.39	2.63
18	linaloola	1099	1099	-	-	0.36	0.99	2.85	3.95	10.67	11.72
19	citronellal	1165	1165	-	-	0.11	-	0.03	1.39	0.93	0.65
20	terpinen-4-ol	1179	1179	-	-	-	0.06	0.26	0.21	0.23	0.32
21	α-terpineol	1190	1190	0.06	-	0.14	0.81	0.73	1.33	0.98	1.65
22	decanala	1205	1205	-	0.27	0.42	0.43	0.10	0.01	0.05	0.01
23	octyl acetate	1216	1216	-	0.04	0.02	0.02	-	-	-	0.02
24	citronellol	1231	1231	-	-	0.15	1.10	0.06	0.93	0.61	1.33
25	β-citral	1242	1242	-	-	-	3.26	-	7.40	2.85	3.64
26	decanol	1275	1274	-	0.05	0.10	5.09	0.02	12.35	4.92	6.16
27	tridecane	1301	1300	-	0.05	0.38	0.24	-	-	-	-
28	methyl geranoate	1325	1323	-	-	-	0.01	-	0.59	0.63	0.27
29	α-cubebene	1344	1343	1.66	0.33	0.34	0.18	0.36	0.05	0.04	0.05
30	citronellol acetate	1357	1357	-	-	0.30	0.56	0.01	0.01	-	-
31	copaene	1368	1368	1.57	0.28	0.75	1.04	0.67	0.42	0.24	0.30
32	β-elemene	1379	1375	0.04	0.70	0.69	0.02	0.52	1.49	1.72	1.00
33	cis-α-bergamotene	1404	1404	2.95	0.11	0.12	0.39	0.02	0.23	0.39	0.38
34	β-caryophyllenea	1412	1412	34.22	14.53	15.54	12.68	10.78	5.68	6.68	5.41
35	γ-elemene	1436	1435	0.52	11.07	10.67	3.10	7.03	-	-	-
36	α- caryophyllene	1450	1450	6.78	2.68	3.10	1.84	2.83	2.37	2.79	1.77
37	β-farnesene	1466	1458	-	-	3.42	0.48	0.03	1.46	2.45	3.06
38	germacrene D	1482	1482	5.14	25.47	9.54	0.11	0.38	0.74	1.07	0.54
39	β-iononea	1489	1489	0.75	0.54	0.40	0.07	0.22	0.16	0.17	0.08
40	elixene	1495	1492	26.25	3.52	5.44	1.53	3.58	0.36	0.38	0.40
41	α-muurolene	1497	1497	0.40	0.65	0.86	0.23	0.59	0.37	0.43	0.25
42	α-farnesene	1515	1514	0.28	1.31	0.59	2.81	0.29	0.09	0.09	0.08
43	δ-cadinene	1528	1528	5.66	1.29	1.53	0.70	1.08	0.40	0.41	0.43

• aIdentification confirmed by comparison with mass spectra and retention times of analytical standards. _LRIbEXP = linear retention index relative to n-alka_nes (_C8 a C22) obtained using a Rxi-5 MS capillary column. _LRIcLIT = linear retention index published in the literature.

The major compounds in orange jasmine were β-caryophyllene and elixene, which together added up to 60.47%. Germacrene D, β-caryophyllene and γ-elemene were the most abundant compounds in ‘Flying Dragon’ (51.07%). For the ‘BRS Bravo’ hybrid, the compounds β-caryophyllene, γ-elemene and germacrene D added up to 35.75% of abundance. D-limonene and β-caryophyllene, added up to 37.55%, were the main compounds in ‘Rangpur’ lime tree. β-pinene and β-caryophyllene represented 28.95% of the compounds found in the ‘Sunki Tropical’ rootstock. The main compounds found in scion varieties were similar, however for the ‘Westin’ variety, decanol and β-pinene accounted for 21.48% of the volatile profile; for ‘Pera’ and ‘Valencia’, β-pinene and linalool added up to 22.60% and 25.03%, respectively.

From the Sparse Partial Least Squares Discriminant Analysis (sPLS-DA) it was possible to visualize a profile difference between the different groups of samples (Fig. 3a). The score chart shows that, in general, there is a separation between the sample groups based on their VOC profile. Distinct groups are observed for the samples of the control groups M. paniculata and P. trifoliata, as well as observed for the ‘Sunki Tropical’ rootstock variety, which clustered closer to the ‘Rangpur’ lime and ‘BRS Bravo’ varieties. In addition, a single group was formed by the samples of sweet orange scion varieties ‘Valencia’, ‘Westin’ and ‘Pera’. This indicates that there is a great similarity in the VOC profile of these three varieties.

The loading plot (Fig. 3b), generated from the sPLS-DA analysis, highlights the compounds that contributed the most to differentiating the various sample groups. It is possible to observe that these ten highlighted compounds presented the lowest concentration, or even absence of them, in the samples of P. trifoliata and M. paniculata, with the exception of β-caryophyllene, which was found at the highest concentration in M. paniculata. Volatiles of the attractive rootstocks (‘Rangpur’ lime and ‘Sunki Tropical’) showed intermediate to low concentrations of these compounds (Fig. 3b).

Using the data matrix containing the identified compounds in the different varieties, hierarchical cluster analysis (HCA) was performed in combination with a heat map, enabling the clustering of different groups based on compound similarity (Fig. 4). The rootstock varieties ‘Rangpur’ lime and ‘Sunki Tropical’ formed a distinct cluster, which was more closely related to the group comprising ‘Flying Dragon,’ ‘BRS Bravo,’ and M. paniculata than to the group formed by the scion varieties (‘Westin,’ ‘Pera,’ and ‘Valencia’). A greater variability was observed in the profiles of the scion variety samples, leading to the formation of subgroups within this cluster.

Figure 4
Dendrogram associated with the heat map of the volatile compounds identified in the genotypes that were used in this study. At the top: dendrogram of the samples; on the left side: dendrogram of the compounds detected. The color of the cells refers to relative abundances, red represents high abundance and blue low abundance.

DISCUSSION

Our results show variation in insect behavior associated with VOCs emitted by different citrus genotypes. Additionally, it made it possible to infer the effect of VOCs from different rootstocks and rootstock/scion combinations on insect response when confronted with VOCs from a suitable host, orange jasmine, and another unsuitable to the insect, Poncirus trifoliata (‘Flying Dragon’). Such results are very important for understanding host selection mechanisms of D. citri and for identifying and indicating citrus genotypes that are less attractive to the insect.

The analysis of the VOCs profile made it possible to determine groups of genotypes and evaluate them in terms of their attractiveness in the insect. Two main classes of volatiles (monoterpene and sesquiterpene hydrocarbons) were identified, which are mainly involved in plant defense against pests and diseases (Alquézar et al. 2017, Ninkuu et al. 2021, Li et al. 2023).

In the heat map, the influence of each compound for the groups of samples is evidenced. The group formed by rootstocks ‘Rangpur’ lime, ‘Sunki Tropical’, and ‘BRS Bravo’ showed a high concentration of compounds such as β-caryophyllene and β-pinene. However, for ‘BRS Bravo’, the compound germacrene D stands out among predominant VOCs of the genotype. This VOC was associated with the control of herbivorous insects (Li et al. 2019).

In the olfactometry bioassays, ‘BRS Bravo’ proved to be less attractive than orange jasmine, which can be attributed to the presence of germacrene D in this rootstock. The presence of the ‘BRS Bravo’ genotype in the same cluster of ‘Rangpur’ lime and ‘Sunki Tropical’ can be explained by its genealogy. However, it is interesting to observe that the bioassays results reflect a different pattern, closer to the P. trifoliata genotype, which is also one of the parental genotypes. As previously mentioned, ‘BRS Bravo’ was less attractive to the psyllid than orange jasmine; however, it was not as repellent as ‘Flying Dragon’. This could be a consequence not only of the concentration of germacrene D but also due to the higher content of γ-elemene in the latter, compared to ‘BRS Bravo’. These two compounds were present in higher proportions in non-attractive samples of pummelo (C. maxima) to D. citri (Killiny et al. 2020). In the present study, they occurred in low quantities, or they were absent in attractive genotypes.

The rootstock ‘BRS Bravo’ has already been identified as a good substitute for ‘Rangpur’ lime tree (Carvalho et al. 2016). The use of this rootstock confers reduction in scion size, greater efficiency in the production of good quality fruits, high tolerance to drought and precocity in production (Ramos et al. 2015). The combination of this rootstock with the ‘Pera’ scion showed a reduction in water loss during evapotranspiration and great efficiency in the use of water for CO₂ assimilation (Carvalho et al. 2016).

The attractiveness of volatiles from ‘Rangpur’ lime and ‘Sunki Tropical’ compared to ‘Flying Dragon’ can be explained by the abundance of D-limonene. This response is more evident for ‘Rangpur’ lime due to the higher concentration of this compound. D-limonene has been identified as attractive to D. citri in a dose-dependent manner (Mann et al. 2012), as well as methyl salicylate, linalool and citral (Liu et al. 2021). It also increases insect trapping when presented as a component of a synthetic host-plant blend (Amorós et al. 2019).

If, on one hand, D-limonene is considered a common volatile in citrus, playing an important role in host selection (Mann et al. 2012), on the other hand, orange jasmine lacks the presence of D-limonene in its volatile profile (Table I). Probably because of this, in our study, orange jasmine was less attractive than ‘Rangpur’ lime when compared to ‘Flying Dragon’. Orange jasmine is recognized as the preferential host for D. citri (Halbert & Manjunath 2004, Borgoni et al. 2014) and recommended as a trap plant for the insect (Tomaseto et al. 2019).

Conversely, ‘Flying Dragon’ rootstock is mostly indicated as a less suitable host for the insect (Westbrook et al. 2011, Richardson & Hall 2013, Fancelli et al. 2018). Trifoliate genotypes induce low oviposition and inadequate nymphal development (Halbert & Manjunath 2004, George & Lapointe 2018) and are associated with HLB tolerance (Folimonova et al. 2009, Ramadugu et al. 2016). The repellent effect of ‘Flying Dragon’ on D. citri was confirmed in olfactometry bioassays (Fig. 1-3), which can be attributed to higher concentration of germacrene D and γ-elemene, as previoulsy discussed.

However, the great abundance of β-caryophyllene stands out in its volatile profile (Table I). This compound, in certain doses, has been reported as a psyllid repellent, an effect verified for Arabidopsis thaliana (L.) Heynh plants, a non-host plant for D. citri (Alquézar et al. 2017) and for ‘Valencia’ plants genetically modified aiming at the constitutive emission of β-caryophyllene (Alquézar et al. 2021). This discussion becomes complex because orange jasmine showed an even higher β-caryophyllene content than that found for ‘Flying Dragon’ and was attractive to the insects. Several researchers reported an attractive effect of β-caryophyllene on the psyllid (Patt & Sétamou 2010, Patt et al. 2011, Wang et al. 2020). There is little information about the bioactivity of volatiles regarding their effects on ACP behavior (Silva et al. 2016, 2023). Additionally, VOCs’ concentration and proportion are key factors to be considered aiming to manipulate insect behavior (Patt et al. 2011, 2014, 2018, Aksenov et al. 2014, Silva et al. 2023).

Regardless of the evaluated variety, the composition of the odor emitted and its discrimination by the insect can change according to the properties of the mixture of VOCs. Compounds that are attractant or repellent when isolated may have the opposite effect when combined (Webster et al. 2010, Amorós et al. 2019, Signoretti 2014). Indeed, examining volatile compound profiles is crucial for understanding insect chemical ecology. However, it is important that such analysis be complemented by behavioral and electroantennographical studies (Volpe et al. 2024). This holistic approach ensures a deeper insight into the potential impact of these compounds on insect behavior.

Furthermore, it is important to highlight that in this study, the VOC profile was obtained using the HS-SPME technique, a method that may release compounds from disrupted cells that are not naturally emitted under field conditions, potentially altering the volatile profile perceived by the Asian citrus psyllid (ACP). While this approach does not fully replicate natural conditions (Tholl et al. 2006), it was selected for its practicality, sensitivity, and widespread application in similar studies. For example, the SPME technique has been successfully employed in recent research to analyze plant volatiles and their interactions with insects (Zhuo et al. 2022, Qian et al. 2024, Kanjana et al. 2025, Fauziah et al. 2022). These studies underscore the value of SPME in providing meaningful insights into VOC profiles and their ecological roles, even if the method does not entirely mimic natural emission patterns.

Finally, in the group formed by the scion varieties ‘Westin’, ‘Pera’, and ‘Valencia’, a higher concentration of β-pinene, decanol, and linalool was observed. Similar compounds had already been reported for the ‘Valencia’ scion (Beloti et al. 2017). Linalool is an important indicator of the presence of shoots in plants for D. citri (Patt & Sétamou 2010). This compound triggers an attraction response in the insect, more intense at low concentrations (Signoretti 2014) and it is present in the profile of VOCs emitted by CaLas-infected plants, which are even more attractive to the insect than those from healthy plants, so they can be useful to manipulate the insect behavior (Mann et al. 2012, Aksenov et al. 2014, Martini et al. 2020).

However, it should be considered that, in this study, linalool, individually may not be responsible for the attractiveness to D. citri, since it is present in abundance in VOCs of the ‘Pera’ genotype, which was not attractive to the insect. This low attractiveness to the psyllid for ‘Pera’ scion grafted on ‘Rangpur’ lime may be associated with the relative higher emission rates of β-caryophyllene and germacrene D compared to the other two scion-rootstock combinations.

The non-attractiveness of volatiles from ‘Pera’ orange plants to the psyllid was also verified in olfactometry bioassays (Tomaseto et al. 2019, Volpe et al. 2020). Despite this behavior, this genotype is reported to be highly susceptible to the insect in the field (Bergmann et al. 1994). It is known that field results do not only reflect the effect of plant volatiles, considering that host selection is a very complex process (Patt & Sétamou 2010). Multimodal cues are involved in this process, including visual, olfactory, and gustatory stimuli (Wenninger et al. 2009, Rohde et al. 2013, Aksenov et al. 2014). Additionally, physical characteristics, the proportion of nutrients present in the shoots, and the composition of the phloem sap also determine the success or failure of the insect in colonizing a host (Sétamou et al. 2016).

The observed results corroborate partially with Alves et al. (2014), that pointed out that ‘Valencia’ was more attractive than ‘Westin’ and ‘Pera’ and it was the most suitable for the development of D. citri. Alves et al. (2018) also indicated that D. citri showed greater preference and higher food consumption of this variety when compared to ‘Sicilian’ lemon tree and ‘Hamlin’ sweet orange. However, in a Y-tube olfactometer bioassay, volatiles from ‘Valencia’ seedlings attracted D. citri females only when the plants had been previously infested by the insect (Moghbeli Gharaei et al. 2014). ‘Rangpur’ lime proved to be a better host than orange jasmine and ‘Sunki’ mandarin orange concerning oviposition and nymphal viability (Nava et al. 2007), but in scion/rootstock interaction study, the results can be very different depending on the genotypes evaluated (Alves et al. 2018). This demonstrates that the interaction between the scion and the rootstock also influences the relationship between the psyllid and the plant, so it should be considered in studies of attractiveness between D. citri and citrus, as well as the evaluation of the behavior of these combinations in relation to the HLB. Disease-tolerant rootstocks can be very useful in dealing with HLB, as they allow plants to survive with minimal effects on crop production or productivity (Bowman et al. 2016, Stover et al. 2016, 2018, Kunwar et al. 2021, Bodaghi et al. 2022).

As noted in recent studies (Bowman et al. 2016, Stover et al. 2016, 2018, Kunwar et al. 2021, Bodaghi et al. 2022, Gill et al. 2024, Costa et al. 2025), selecting appropriate citrus rootstocks can significantly enhance crop resilience under climate change scenarios and improve resistance to Huanglongbing (HLB). Our findings contribute to this body of knowledge by providing insights into how the Asian citrus psyllid (ACP) responds to volatiles from different citrus varieties, which could inform the development of more effective pest management strategies and HLB-resistant citrus cultivars.

Further tests should be conducted to evaluate the behavior and biology of the insect with different canopy combinations using the rootstock recommended in this study, ‘BRS Bravo’. Additionally, the potential of these combinations to reduce the spread of HLB-associated bacteria should be investigated. A promising research direction is using highly attractive bait plants for trap crops alongside repellent or less suitable plants for the insect. The attractive potential of ‘Valencia’ and ‘Westin’ scions should be further studied in field conditions. If confirmed, enhanced insect and disease management strategies will be necessary to prevent significant production losses.

Acknowledgements

The authors would like to thank Embrapa Mandioca e Fruticultura and the Secretaria da Agricultura, Pecuária, Irrigação, Pesca e Aquicultura do Estado da Bahia (SEAGRI) for the infrastructure made available to carry out this work; to the Fundação de Amparo à Pesquisa do Estado da Bahia (FAPESB), for financial support to the research project T.O. RED0040/2014; to the Coordenação de Aperfeiçoamento de Pessoal de Ensino Superior (CAPES) - Financing Code 001 for granting the master’s scholarship to L. C. Oliveira.

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Publication Dates

• Publication in this collection
  22 Sept 2025
• Date of issue
  2025

History

• Received
  6 Sept 2024
• Accepted
  27 May 2025