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Chilean journal of agricultural research

versión On-line ISSN 0718-5839

Chil. j. agric. res. vol.79 no.2 Chillán jun. 2019 


Rootstock affects the blend of biogenic volatile organic compounds emitted by ‘Hass’ avocado

Ricardo Ceballos1 

Tommy Rioja2  3 

1Instituto de Investigaciones Agropecuarias, INIA Quilamapu, Av. Vicente Méndez 515, Chillán, Chile.

2Universidad Arturo Prat, Facultad de Recursos Naturales Renovables, Campus Huayquique Av. Arturo Prat s/n, Iquique, Chile.

3Universidad de Tarapacá, Facultad de Ciencias Agronómicas, Campus Azapa km 12, Arica, Chile.


Grafting, using tolerant rootstocks, has been necessary to increase avocado (Persea americana Mill.) production in drought, salinity, pest, and soil disease conditions. The avocado rootstock has shown an influence on scion vigor, nutrient absorption, fruit quality, and disease tolerance. Nevertheless, the avocado rootstock influence on the biogenic volatile organic compounds (BVOCs) emitted from ‘Hass’ shoots has not been reported. Our objective was to study the effect of two avocado rootstocks of the Mexican race on BVOC emitted by avocado ‘Hass’ shoots. We collected BVOCs emitted by ‘Hass’ avocado shoots grafted on ‘Mexicola’ and ‘Zutano’ rootstocks. All volatile collections were made from living plants for 24 h through a dynamic headspace technique. The chemical profiles were analyzed by gas chromatography coupled to a mass spectrometry (GC-MS). BVOC emission rates were highly variable in amount and composition. The monoterpene α-pinene was emitted at 5.06 ± 0.74, 0.73 ± 0.14, and 1.43 ± 0.61 μg mL-1 by graft ‘Hass’/‘Mexicola’, ‘Hass’/‘Zutano’ and ungrafted ‘Mexicola’, respectively. Grafted ‘Hass’ on ‘Mexicola’ emitted a wide variety of monoterpenes as β-pinene, cumene, 3-carene, R-limonene and (Z)-β-ocimene, whereas grafted plants on ‘Zutano’ only released α-pinene and cumene. Estragole was only detected on ungrafted ‘Mexicola’. We found that the chemical profile of volatile compounds released by ‘Hass’ grafted avocado plants was qualitatively and quantitatively influenced by rootstocks

Key words: Avocado; headspace collection; Persea americana; plant volatiles; rootstock


Modern agricultural practice of grafting vegetal material and rootstock use has allowed cultivating in unfavorable conditions such as extreme temperatures, drought, salinity and flooding (Colla et al., 2010; Schwarz et al., 2010; Reeksting et al., 2014). Resistant and/or tolerant rootstocks to insect pests and pathogens increase the production, scion vigor, and organoleptic fruit quality (Warschefsky et al., 2016).

Structural, biochemical and genetic factors influence the grafting process (Martínez-Ballesta et al., 2010). The scion- rootstock union and callus development produce molecular changes that affect scions’ defense mechanisms (Cookson et al., 2013; Trinchera et al., 2013; Lordan et al., 2017). On the other hand, the transport of RNA, DNA, phyto-hormones and proteins between scion and rootstock elicit phenotypic changes on grafted plants (Wang et al., 2016).

Secondary metabolites such as alkaloids, non-protein amino acids, amines, cyanogenic glycosides, glucosinolates, terpenoids, and phenolics are constitutively produced by plants (Jamwal et al., 2018; Moreno-Medina et al., 2018). Biogenic volatile organic compounds (BVOC) are emitted through intercellular spaces, whereas stomas release them into the environment (Kegge and Pierik, 2010). As a result, terpenoids, aldehydes, green leaf volatiles (GLV), indole, and aromatic compounds are released (Loreto et al., 2014). These compounds have important physiological functions, and direct and indirect defensive roles (Sabelis et al., 2011; Mithöfer and Boland, 2012). BVOC are chemical cues for the attraction or repellence of phytophagous insects and plant pathogens, providing key information on host-plant status (Ueda et al., 2012; Holopainen and Blande, 2013; Lu et al., 2015). Herbivore-induced plant volatiles (HIPVs) are emitted after herbivory (Arimura et al., 2011; Gols, 2014; Heil, 2014); thus, specific odor cues are exploited by predators and parasitoids. In addition, HIPVs are modified by arthropod species, herbivores density, genotypes and plant cultivars, plant ontogeny, and abiotic factors (Hare, 2010; Hare and Sun, 2011; Proffit et al., 2011; Becker et al., 2015; Clavijo, 2016; Rioja et al., 2016).

Avocado ‘Hass’ emits mainly GLV, alkane and monoterpene compounds (Bravo-Monzón and Espinosa-García, 2008; Rioja et al., 2016; 2018). Rincón-Hernández et al. (2011) described differences in the chemical profile of volatiles compounds on Guatemalan, Mexican and West Indian race. García-Rodríguez et al. (2016) found variability in the emitted compounds by ‘Hass’ trees propagated clonally. Most investigations have focused on mineral transportation rootstock effect, defense-gene expression, hormone production and abiotic stress tolerance. We studied the effect of two avocado rootstocks of the Mexican race on BVOC emitted by avocado ‘Hass’ -shoots.


Plants and BVOC collections

‘Hass’ avocado scions were grafted on two Mexican race rootstocks, ‘Mexicola’ and ‘Zutano’, and cultivated in 7 L containers filled with organic soil and peat. The plants were fertilized (Ultrasol 18-18-18, Soquimich, Santiago, Chile) and irrigated suitably. We included a set of ‘Mexicola’ ungrafted plants, cultivated as described above. The volatile collections were carried out during summer season, under semi-field conditions using a greenhouse (3 m × 2.5 m × 4 m), in Instituto de Investigaciones Agropecuarias INIA La Cruz (32°49’ S; 71°17’ W), Quillota, Región de Valparaíso, Chile.

For all in situ collections, we used 2-yr-old plants and a dynamic headspace technique with a positive/negative air system. We carefully enclosed a healthy and undamaged branch in a polyethylene terephthalate (PET) bottle (1.5 L) divided into halves along. The parts were secured with Teflon tape to avoid mechanical damage to leaves. A purified airstream (charcoal 8-20 mesh, Sigma-Aldrich, St. Louis, Missouri, USA) was pumped into the PET bottle at 1000 mL min-1 and extracted at 900 mL min-1 by a vacuum pump (BOECO, Hamburg, Germany). Plant volatiles were collected into chemical traps (13 cm in length and 5 mm in internal diameter) filled with 100 mg Porapak Q (80-100 mesh, Supelco, Bellefonte, Pennsylvania, USA) inserted into an upper outlet of the bottle. Traps were previously cleaned with 1 mL diethyl ether and conditioned for 2 h at 220 ºC under a constant stream of 40 mL min-1 of nitrogen (Ceballos et al., 2015). After 24 h of collection, we eluted the volatiles from Porapak using 1 mL hexane (95%, Sigma-Aldrich).

Chemical analysis of BVOC by GC-MS

An aliquot of 1 μL of the eluted samples was injected into a gas chromatographer coupled to a mass spectrometer (GCMS; QP2010 Ultra, Shimadzu, Kyoto, Japan), equipped with an RTx5 capillary column (30 m, 0.25 mm ID, 0.25 μm film thickness; Restek, Bellefonte, Pennsylvania, USA). The injection mode was split-less, and helium was used as the carrier gas with a constant flow at 1.0 mL min-1. The GC oven was programmed at 40 °C for 2 min and then increased at a rate of 5 °C min-1 until 225 °C. The acquisition was carried out in the mass range from 50 to 500 m/z, whereas the ionization was performed by an electron impact at 70 eV with an ion source at 230 °C. The BVOC identity was verified by comparing their retention times while mass spectrum was corroborated with a library database (NIST version 2.0, Standard Reference Data; National Institute of Standards and Technology [NIST], Gaithersburg, Maryland, USA). To quantify, a calibration curve was made with commercial standard of α-pinene (Dr. Ehrenstorfer GmbH, Augsburg, Germany) under the same chromatographic conditions described above.

Statistical analysis

Six replicates were performed for volatile’s entrainments and data from quantitative analysis of the emitted compounds were expressed as proportion of α-pinene concentration in the corresponding plant condition. Data were analyzed by Student’s t test or ANOVA (P < 0.05), according to its presence in two or three plants conditions, respectively.


The monoterpene α-pinene was emitted at 5.06 ± 0.74, 0.73 ± 0.14, and 1.43 ± 0.61 μg mL-1 (F = 21.232; p = 0.001) by graft ‘Hass’/‘Mexicola’, ‘Hass’/‘Zutano’ and ungrafted ‘Mexicola’, respectively. These concentrations were employed to standardize the emissions of the other compounds on each plant condition. The graft ‘Hass’/‘Mexicola’ emitted other monoterpenes as β-pinene, cumene, 3-carene, R-limonene and (Z)-β-ocimene, and ‘Hass’/‘Zutano’ emitted cumene (Table 1). Rioja et al. (2016) collected BVOC from intact avocado ‘Hass’ grafted on ‘Mexicola’ registering the same compounds although in different concentrations. Bravo-Monzón and Espinosa-García (2008) found that ‘Hass’ avocado grafted on ‘Criollo’ rootstock released high abundance of α-pinene, β-phellandrene, β-pinene, β-myrcene, α-cubebene, α-copaene, and β-caryophyllene. This may indicate that ‘Hass’ shoots are physiologically modified by the rootstock influence.

The graft ‘Hass’/‘Zutano’ released high amounts of alkanes as 2,4 dimethyl-heptane (t = 2.358; p = 0.040), 3,7 dimethyl- decane (t = 2.536; p = 0.029) and undecane, which were not detected in the volatile profiles emitted by ‘Hass’/‘Mexicola’. Graft ‘Hass’/‘Zutano’ released a profile of BVOC conformed by 87.5% alkanes, 1.9% GLVs and 1.1% monoterpenes, unlike the graft ‘Hass’/‘Mexicola’. Rioja et al. (2016) registered low quantities of BVOCs emitted from ‘Hass’-shoots grafted on ‘Mexicola’. Furthermore, Bravo-Monzón and Espinosa-García (2008) did not register emissions of BVOCs from ‘Hass’/‘Criollo’ combination. Seemingly, ‘Zutano’ increased volatile emissions from ‘Hass’ leaves.

An abundant emission of estragole was found only on ungrafted ‘Mexicola’ (Table 1). Bravo-Monzón and Espinosa- García (2008) registered that ‘Criollo’ plants, from the Mexican race just like ‘Mexicola’, emitted estragole at 54.75% of abundance. Contrary to this, ‘Hass’ did not emit estragole because of its predominantly Guatemalan race. Moreover, Torres-Gurrola et al. (2011) collected high amounts of estragole from intact ‘Criollo’ leaves. Yet, Rioja et al. (2016) did not detect estragole emissions from ‘Hass’ shoots. Therefore, the estragole compound would be a characteristic of the Mexican race. Higher amounts of aromatic aldehydes were released by ‘Mexicola’ shoots. The graft ‘Hass’/‘Mexicola’ did not emit 3-ethyl-benzaldehyde although ‘Hass’-shoots, grafted on ‘Zutano’, emitted significantly low amounts of 3-ethyl-benzaldehyde (t = 2.386; p < 0.044) in comparison to ‘Mexicola’ plants (Table 1). Therefore, the chemical profiles are different within avocado species as it in the monoterpenes, alkanes, phenylpropanoids and aldehydes biosynthesis, affected by the genotype rootstock in avocado plants.

Table 1 Concentration of biogenic volatile organic compounds (mean ± SE) collected from grafted and ungrafted Persea americana ‘Hass’ for 24 h. 

†Data are expressed as proportion of α-pinene concentration in the corresponding plant condition; α-pinene was emitted at 5.06 ± 0.74, 0.73 ± 0.14, and 1.43 ± 0.61 μg mL-1 by graft ‘Hass’/‘Mexicola’, ‘Hass’/‘Zutano’ and ungrafted ‘Mexicola’, respectively. Means sharing a letter, for each compound, do not differ significantly according to Tukey’s test (P < 0.05).

*Significant differences according Student’s t-test (P < 0.05).

Although, morphological and physiological parameters were not measured, ‘Hass’ shoots were vigorous and the emission of alkanes was stimulated when were grafted on ‘Zutano’ rootstock. Alkanes are major constituents of epicuticular waxes promoting drought tolerance, UV-radiation protection and defenses against insects and pathogens (Bernard et al., 2012; Bush and McInerney, 2013). Rootstocks can enhance tolerance to abiotic and biotic stressors, and its benefits has been studied several species as kiwifruit (Actinidia deliciosa), apple (Malus domestica), and pomegranate (Punica granatum) (Warschefsky et al., 2016). According to Warschefsky et al. (2016) many aspects of rootstock biology are at initial stages, including long-distance molecular signaling and the capacity of rootstocks to modulate interaction between plant and soil microbiomes.


Volatile profile released by grafted ‘Hass’ avocado plants was affected by both ‘Mexicola’ and ‘Zutano’ rootstocks. Grafted ‘Hass’ on ‘Mexicola’ emitted a wide variety of more monoterpenes than plants grafted on ‘Zutano’. Estragole was only detected on ungrafted ‘Mexicola’ plants, 3-carene, (Z)-β-ocimene, 2-propyl-1-pentanol, R-limonene and (2E, 6E)-4,5- dimethyl-2,6-octadiene were identified only in the volatile profile of ‘Hass’/‘Mexicola’ plants. Undecane, 2,3-dimethyl- heptane and 3,4,5-trimethyl-heptane were collected from ‘Hass’/‘Zutano’plants.


We extend our gratitude to Instituto de Investigaciones Agropecuarias staff and Centro Regional de Investigación La Cruz, Región de Valparaíso, Chile.


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Received: November 05, 2018; Accepted: February 12, 2019

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