Influence of Soil Nutrients, Tree Age, and Sandalwood Provenances on Sandalwood Oil Yield and Quality

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Introduction
Sandalwoods are a commercially and culturally important group of plant species belonging to the family Santalaceae [1]. Sandalwoods provide high-value essential oils used as fragrances and cosmetic ingredients. In Kenya, Osyris lanceolata (East African Sandalwood) is not region-specifc and can adapt to diferent environmental conditions [2]. Osyris lanceolata (Hochst. and Steud.) in the family Santalaceae is a hemiparasite dioecious tree species that requires a host plant to grow to its full potential [3]. Te species grows in transitional wet and dry zones and is found in most of the agroclimatic zones of Kenya [4]. In Kenya, sizeable populations have been recorded in Taita Taveta, Makueni, Kitui, Embu, Machakos, Kiambu, Laikipia, Pokot, Uasin Gishu, Narok, Samburu, Murang'a, Kajiando, Migori, Nyandarua, Marsabit, and Baringo counties. In its natural range, the species grows at an altitude of 900-2250 m above sea level (asl) in areas with a mean annual rainfall of 600-1600 mm. Naturally, it can grow in areas whose natural vegetation is dry woodland in ecological zone III and moist to dry forest in zone II. Osyris lanceolata requires well-drained soil and does not tolerate waterlogging [3,4].
Wild-harvested Osyris lanceolata from Kenya is illegally poached and smuggled mostly to India, where its oil is used as a substitute for Asian sandalwood, the oil of which is an ingredient in the multimillion dollar cosmetic, perfumery, and favor industries [5]. In Kenya, the global market per kilogram of O. lanceolata heartwood is estimated to be about 3.0 US dollars [6] compared to Santalum album at 100 US dollars [7]. Sandalwood oil is composed of natural compounds referred to as sesquiterpenoids, which give the essential oil its distinctive aroma. Osyris lanceolata has biochemical composition of 55 constituents in oil such as (E)-cis-epi-b-santalol, (Z)-a-Farnesene, (z)-cis-a-santalol, and Alpha-caryophyllene among others as compared to S. album with between 250 and 300 such as (Z) α santalol, (Z) β-santalol, and (Z)-epi-β-santalol [8]. In pharmaceutical industries, sesquiterpenoids are used as a composition of medicines for treating bronchitis, dry persistent coughs, laryngitis, diarrhea, dermatitis arthritis, and insomnia among other diseases [9]. In perfumery industries, sesquiterpenoids are efective in aromatherapy treatment because of the sweet powerful long-lasting aroma. Te sandalwood sesquiterpenoids make an excellent composition for scenting soaps and all kinds of perfumes [10]. Both O. lanceolata and S. album contain α and β santalols which are responsible for fragrance in sandalwood oil [8].
Domestication of Osyris is being encouraged to reduce overexploitation of the natural populations in Kenya by providing sandalwood markets with sustainable stocks grown in plantations [7]. High oil yields would make such enterprises more proftable and attractive to investors. Studies conducted in Tanzania revealed diferent provenances had contrasting oil yield that varied in quality [11]. Tis study sought to establish how age, soil, and agroecological factors infuenced oil yield and quality to add to the body of knowledge that would guide in selection of provenances and prescription of appropriate on-farm husbandry for the species. Te objective of this study was to determine the efect of age, soil, and agro-ecological factors on oil yield and quality of Osyris growing in natural stands.
Te study sites are in diferent Agro-Ecological Zones (AEZ) ranging from high and medium potential to semiarid and arid climatic zones. Table 1 shows a summary of agroclimatic characteristics of the study sites. II.

Soil Sampling and Analysis.
At each site, soil samples were collected under O. lanceolata trees to a depth of 0-25 cm using a soil auger. Soil was taken at three random sampling points around the Osyris tree and mixed to make a composite sample. Te soil samples were analysed for texture, pH, electrical-conductivity (EC), total nitrogen, total carbon, extractable phosphorus, calcium, magnesium, potassium, zinc, copper, manganese, and iron properties using standard laboratory procedures as described by Okalebo et al. [12].

Sampling of Osyris lanceolata Trees.
As the harvesting of sandalwood in its natural stands is legally proscribed, authorization to harvest O. lanceolata trees was obtained from relevant authorities. Four trees per site were permitted to be used for this research, which is adequate for research in the extraction and quality analysis of oil [13]. Mature trees were harvested at the base using a pruning saw, labeled, and packed in gunny bags for transportation in line with procedures described in Howes et al. and Brand et al. [14,15].

Age Determination of O. lanceolata Trees.
To determine the age of Osyris trees, a dendrochronological method was used as described by Kankare et al. [16]. Dried Osyris wood blocks were cut horizontally at the base to a uniform size of 2 cm thickness using an electric saw. Te rings present in the wood samples were counted using a magnifying glass, and the number of rings counted was assumed to correspond to the age in years.

Extraction of O. lanceolata Oil and Yield Determination.
Wood samples were air-dried at room temperature (22°C) for 20 days to avoid loss of volatile essential oils [17]. Smaller pieces of wood, about 1 cm 3 , were cut using an electric saw so they could be accommodated in the grinder. Tey were then ground into fne powder using a Dietz model D-73265 grinder. Te fne powder was stored in airtight plastic containers to prevent further moisture loss. One hundred grams of fne powder were weighed using an analytical weighing balance and placed into a separating funnel, where 300 ml of hexane (analar grade) was added and left to stand for 72 hours to give a yellow-coloured extract. Te extract was removed from the separating funnel and concentrated with a rotary evaporator (Yamato model: CF 301) under vacuum. Te oil obtained was weighed, and the yield was calculated as per Equation (1). Te oil was then transferred into 20 ml brown sample bottles to avoid light and stored in the freezer at −10°C awaiting further analysis.
where W1-weight of empty bottle, W2-weight of the bottle plus oil, and W3-weight of the ground sample used in extraction.

Determination of Oil Quality.
To determine the quality of oil, 10 μl of the distilled oil was pipetted into a 1.5 ml sample bottle and diluted with 1 ml of hexane. Additionally, 200 µl of the diluted sample was transferred into an insertion for gas chromatography-mass spectra (Agilent Technologies, model 7890 A) for the identifcation of compounds. Te method used for compound analysis was hexane volatiles at 35°C-280°C for 50 minutes per sample. Helium gas was used as a carrier gas, and GC-MS facilitated the identifcation of diferent compounds in the oil extracts at diferent time points of detection [18]. Areas were recorded for all detectable peaks, and percent concentration was calculated by taking the area of the peak divided by the total chromatogram area multiplied by 100. provenance, and oil data, where negative loadings indicated negative correlation and positive loadings indicated positive correlation. Te infuence of tree age in selected provenances on oil yield was ftted as a covariate.

Variation of Soil Nutrients in Diferent Study Sites.
Soil pH levels signifcantly varied in all study sites (p � 0.001). In Baringo, Maralal, Narosura, Marsabit, Namanga, and Ol Donyo Sabuk soils were acidic, while Mbooni, Murang'a, Kitui, Loita, and Chyulu were neutral. Only Homabay had alkaline soil. EC levels signifcantly difered amongst all study sites (p � 0.009) as shown in Table 2. Carbon levels difered signifcantly across all study sites (p � 0.001). Te highest carbon levels were recorded in Chyulu and Homabay, whereas the lowest levels of carbon were recorded in Maralal as shown in Table 2.
Nitrogen levels also difered signifcantly (p � 0.009) with the highest levels recorded in Chyulu and the lowest levels in Kitui. Phosphorus levels varied signifcantly (p � 0.001), with the highest levels in Homabay and Chyulu; all other sites had phosphorus levels below 15 ppm. Similarly, potassium levels varied signifcantly (p � 0.001), with Homabay having the highest levels while Namanga had the lowest levels ( Table 2).
Tese results show that Osyris naturally occurs in contrasting soil types and diferent agro-ecological zones with varying soil nutrient levels as demonstrated by the presence of the species at sites ranging from high-to medium-potential agricultural sites to semiarid and arid regions [19]. Tese fndings are consistent with previous studies of Osyris cutting across diferent AEZ with varying soil nutrient levels [11].

Infuence of Soil Nutrients on Oil Yield.
Tere was no signifcant diference on oil yield as infuenced by soil nutrient levels in all sites as indicated by nonsignifcant regressions and Pearson correlations (Table 3). Tis study contrasts with a previous study by Golubkina et al. [20], where increased soil nutrients showed benefcial efects on plant growth, development, and oil content in aromatic plants. A similar study done by Argyropoulou et al. [21] concurred with Golubkina's results, where growth performance and oil yield among aromatic plants such as spearmint and rosemary increased with the addition of soil nutrients. Te current study also contradicts the study by Argyropoulou et al. [21] on the cultivation of aromatic plants and the addition of nitrogen and potassium, which established a signifcant increase of oil quantity and quality in the aromatic plants. However, the signifcant diference in soil nutrients across all study sites could contribute to diferences in the total woody biomass of individual trees.

Infuence of Age on Oil
Yield. Te age of sampled trees ranged from 8 to 40 years. In this study, there is a very weak but positive signifcant infuence of age on oil yield in Osyris trees (R � 0.31, p � 0.04). Te strength of the relationship is shown by R 2 � 0.096, where 9.6% of observation made in oil yield can be attributed to Osyris's age while over 90.4% is explained by other factors. Similar studies in Western Australia by Brand and Prank [22] on Santalum spicatum plantations indicated signifcant diference in oil yield as infuenced by age. Increased oil yields in older trees could be contributed by the well-developed mature heartwood. Tese fndings concur with the current study which showed signifcant increase in oil yield with increasing age of Osyris trees ( Figure 2).
Bush and Pronk [23] revealed that S. album and S. yasi oil yield was also signifcantly positively infuenced by the age of the tree from which the oil was extracted. Teir study further revealed that Santalum spp. trees less than 16 years old growing in the Pacifc under much wetter conditions than the current study were not mature enough to produce essential oil due to the low formation of heartwood at that age. Mashra et al. [24] also reported a signifcant increase in oil yield with age; however, they concluded that there was no uniform pattern in heartwood formation and oil content with the increasing age of the tree which may be attributed to varying edaphoclimatic conditions.

Efect of Provenance on Oil Yield.
Te results of this study indicate that Osyris oil yield was not signifcantly diferent among the various provenances sampled (p � 0.19). Loita provenance, however, recorded the highest oil yield amongst the provenances (3.56%); this was followed by Baringo, which recorded an oil yield of 3.49% as shown in Table 4 below. Narosura provenance recorded the lowest oil yield of 1.50%, which was 57.9% lower than the Loita provenance. Te provenances which recorded higher oil yield were all found in agro-ecological zone III, except Mbooni and Ol Donyo Sabuk which fall under agro-ecological zone IV. Te diferences in oil yield among the diferent provenances can partially be attributed to the age of the trees selected for oil quantifcation. Age of trees selected in the various provenances signifcantly infuenced the oil yield (f (1,41) � 2.97, p � 0.008).
In Kenya, no previous studies have evaluated the efect of provenances on Osyris oil yield. A study by Mwang'ingo [25] reported that Osyris populations in Tanzania varied signifcantly in the amount of oil produced. A similar study [11], which assessed oil yield and quality variation between sexes in Osyris study, concluded that there were no significant diferences in oil yield between the diferent sexes. However, it reported that oil yield was signifcantly diferent (p � 0.001) among populations with the highest oil yield being 9.32%. Te study concluded that the reasons for observed trends in the quantity and quality of oil are still uncertain. Te observed variations could have resulted from environmental and genetic factors. Moniodis et al. [26] similarly reported a signifcant diference in oil yield in diferent provenances and soil types. Figure 3 explores the interaction of diferent variables under the current study. Te study revealed that the frst and second principal components accounted for 48.3% of the overall variation. Te study further revealed that there was a positive correlation amongst soil variables such as carbon, nitrogen, pH, EC, manganese, magnesium, and potassium. However, there was no correlation between oil yield and most soil variables. Tere was a negative correlation between International Journal of Forestry Research oil yield and soil EC. Oil yield is positively correlated to tree age and clay content in the soil. Ecosystems are complex and consist of interacting biotic and abiotic components [4].

Oil Quality Potential in Diferent Provenances in Kenya.
Te phytochemical composition of genus Osyris includes volatile constituents that are important in essential oils and are known as sesquiterpenes. Te superiority of sandalwood oil is determined by the level of alpha (α) and beta (β) santalol relative to the international oil quality standards for these compounds [27]. Te GC-MS analysis in this study recognized compounds belonging to groups of hydrocarbons, fatty acids, esters, alcohols, amines, sesquiterpenes, among others, in Osyris oil. Te GC-MS analysis on oil extracts from 12 sampled Osyris populations showed numerous compounds occurring at various peak intervals. However, the most common and abundant compounds from the sampled populations were Z-alpha-trans-bergamotol, alpha bisabolol, lanceol  Note. Signifcance codes: 0 " * * * ," 0.001 " * * ," 0.01 " * ," 0.05 ".," and n.s. not signifcant. 6 International Journal of Forestry Research cis, beta bisabolene, alpha santalol, cis-alpha-copaene-8ol, isopropyl myristate, and isopropyl palmitate (Table 5 and 6).

Beta Bisabolene.
Te highest concentration of this compound was 0.37% found in Baringo provenance, followed by Narosura 0.36%. Te compound was absent in oil extracted from Loita and Maralal provenances. However, there was no signifcant diference (p � 0.34) among oil extracted from the sampled provenances.

Cis-Alpha-Copaene-8-Ol.
Te compound was present in all sampled provenances except Murang'a and Namanga. Te highest concentration was 5.51% in Marsabit, followed by 2.44% in Loita provenances. Tere was no signifcant diference (p � 0.67) in all oil extracts from sampled provenances.

Isopropyl Myristate.
Te compound was present in all oil extracts from sampled provenances. Te highest concentration was 1.76% in Homabay and the lowest 0.21% in Narosura. Tere was no signifcant diference (p � 0.15) in all oil extracts from sampled provenances.
Tere was no signifcant diference in oil extracts from all sampled provenances.

Beta Santalol.
Tis compound was not among the common and abundant compounds, it was only detected in Narosura (1.78%) and Chyulu (0.14%) provenances. All sesquiterpenes in sandalwood oil are valueable. According to Arn and Acree [28], β bisabolene belongs to organics known as sesquiterpenoides which are terpenes with 3 consecutive isoprenes units used in fragrance Table 5: Te relative presence of common compounds in oil extracts from 12 Sandalwood provenances in Kenya.

Name of compounds
Baringo Chyulu Homabay Kitui Loita Maralal Marsabit Mbooni Muranga Namanga Narosura Ol Donyo Sabuk Z-alpha-transbergamotol   [29]. Jirovetz [30] revealed that α and β santalol have antimicrobial activities against klebsiella pneumonia and α santalol induces apoptosis in human prostate cancer cells [31]. Copaene is an attractant synergistic with quercetin, a favor used as an ingredient in dietary supplements contained in beverages and foods [32]. According to Christian [33], isopropopyl myristate is a pollar emollient used in cosmetics and topical pharmaceutical preparations for dry skin moisturizers. Isopropyll palmitate is a fatty acid ester used as a treatment for head lice, in fea, and tick killing products for pets [34]. Isopropopyl myristate is also used as a mouthwash disinfectant to remove bacteria from oral cavity [34]. Tis fatty acid ester is also used in the perfumery and pharmaceutical industries as an emollient, emulsifer, and plasticizer physical stabilizer in preparations of products such as creams, lotions, and eye makeups, [32]. According to Sharif [35], Bergamotol, cis lanceol, and α-bisabolol are sesquiterpene alcohols and they inhibit antifungal activity against Onychomycosis, Candida albicas, and Cryptococuccus neoformans. Tese sesquiterpenoids compounds have antiinfamatory, tissue-remodelling efects, and aromatherapy treatment, hence their wide use in pharmaceutical and perfumery industries. Tables 5 and 6 below show the presence/absence and relative abundance of the most common compounds for oil extracted from 12 Osyris provenances in Kenya.
Te objective of the current study was to support a programme on breeding, domestication, and conservation of Osyris lanceolata from its natural habitat to a farm in Kenya. Similar work done by Page [37] on Santalum austrocaledonicum, identifed superior germplasm which was used in domestication work in Vanuatu. Te oil quality results shows that Baringo, Mbooni, and Ol Donyo Sabuk populations can be selected for domestication as unique populations with Alpha santalol, while Narosura and Chyulu provenances can be targeted due to Beta santalol. However, the results of this study are based on samples collected in natural stands, and studies on planted Osyris should be done to confrm that these unique characteristics are maintained in established plantations. In addition, studies on younger Osyris woody samples ranging from18 years and below should be done for comparison of oil yield and quality to guide on the optimal age of rotation. Te studies should also focus on how ecological and soil characteristics infuence the volume of the heartwood to provide additional information to support the domestication of the species. Te current study was not able to sample the population on a farm as domestication is on the infancy stage in Kenya. Research on domestication of Osyris populations in Kenya should be conscious of challenges of genetic fragmentation [37] of Osyris across varying AEZs. Te study shows that the natural populations in Kenya have a lower oil yield than those of Tanzania (11) and are of inferior quality compared to Santalum album and Santalum austrocaledonicum [37,38]. It is therefore recommended that the domestication process would beneft from the introduction of Osyris populations from Tanzania and germplasm from populations of genus Santalum from the Asian continent to Kenya in order to shift focus from the wild Osyris populations to high-value sandalwood grown on farms.

Conclusion
In this study, soil nutrients varied signifcantly across the study sites, though there was no correlation between Osyris trees oil yield and most soil variables. Tese results of soil analysis show that Osyris can be planted in a wide range of soil types across diferent agro-ecological zones. Te age of Osyris trees infuenced the oil yield weakly but signifcantly, though the yield did not vary signifcantly amongst provenances. Loita provenance recorded the highest oil yield (3.56%), followed by Baringo (3.49%), whereas Narosura recorded the lowest yield (1.5%). Osyris trees which recorded high oil yield fall under agro-ecological zones (AEZs) III and IV. Te GC-MS oil quality results recorded nine common and most abundant compounds across the study sites which includes Z-alpha-trans-bergamotol, alpha bisabolol, lanceol cis, beta bisabolene, alpha santalol, beta satalol, cis-alphacopaene-8-ol, isopropyl myristate, and isopropyl palmitate. Tese compounds are used as a phytochemical composition of commercial essential oils which are responsible for favors and fragrances industries. However, the superiority of sandalwood oil is determined by the level of Alpha (α) and Beta (β) santalol assayed in sandalwood extracts, which is compared to the international oil quality standards. In this study, alpha santalol was detected in oil extracts from Baringo, Mbooni, and Ol Donyo Sabuk provenances and International Journal of Forestry Research 9 concentration varied signifcantly. Beta santalol was only detected in Narosura and Chyulu provenances. Based on the foregoing Baringo, Mbooni, Ol Donyo Sabuk, Narosura, and Chyulu provenances could ofer the best germplasm for onfarm propagation and domestication.

Data Availability
Data supporting the development of this manuscript can be availed by the corresponding author upon reasonable request.

Conflicts of Interest
Te authors declare no conficts of interest.