Assessment of fruit aroma for twenty-seven guava (Psidium guajava) accessions through three fruit developmental stages

Scientia Horticulturae 238 (2018) 375–383

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Assessment of fruit aroma for twenty-seven guava (Psidium guajava) accessions through three fruit developmental stages Pamela Moona, Yuqing Fua, Jinhe Baib, Anne Plottob, Jonathan Cranea, Alan Chambersa, a b

T ⁎

Tropical Research and Education Center, University of Florida, 18905 SW 280th St, Homestead, FL, 33031, United States Horticultural Research Laboratory, USDA-ARS, 2001 South Rock Road, Ft Pierce, FL, 34945, United States

A R T I C LE I N FO

A B S T R A C T

Keywords: Aroma Fruit quality Ripening Titratable acidity

Guava (Psidium guajava) fruit are valued for their rich, tropical aroma, but fruit quality data are lacking for many important cultivars. Guava is native to warm-climate areas of the Americas and is grown commercially in southern Florida, USA. Varieties relevant to south Florida display a high level of morphological diversity, including fruit color and shape. Fruit are commercially available at either a mature green stage which are harvested prior to full aroma development, or as mature yellow fruit which possess a full aroma profile. We sampled 27 guava accessions growing at the University of Florida’s Tropical Research and Education Center for aroma at immature, mature green, and mature yellow fruit developmental stages. Variability in pulp pH (3–4.6) was not indicative of fruit maturity, but titratable acidity (0.17–3.6%) did decrease throughout fruit development. Aroma profiles showed significant differences among accessions. Some volatiles including hexanal, (E)-caryophyllene, and (E)-2-hexenal were common across guava accessions and were present at all developmental stages. Other compounds including (Z)-3-hexenyl acetate, ethyl butanoate, and ethyl octanoate had the greatest abundance only at the mature yellow stage of fruit development. Limonene, (E)-cadina-1,4-diene, and β-ionone were abundant in some accessions and not detected in others (21, 10, and 14 out of 27 accessions, respectively). This work describes the comparative diversity of guava fruit quality for a large number of accessions through fruit development for the first time. Furthermore, these fruit quality data could support future breeding work towards the genetic improvement of guava flavor.

1. Introduction Guava (Psidium guajava) is a high-value tropical fruit suitable for niche markets, but accession-level fruit quality data are lacking. Guava is native to tropical and warm subtropical areas from Mexico to Peru and was expanded throughout the cool subtropical to tropical regions of the world during the past 400 years (Cobley, 1976; Morton, 1987; Samson, 1984). Today guava is grown commercially throughout Latin America, the Caribbean, Asia, Africa, the Middle East, Australia, and the United States. Guava was reportedly introduced into Florida sometime between 1847 and 1887 (Popenoe, 1920; Reasoner, 1888). However, Native Americans and/or Caribbean islanders may have introduced guava earlier than this date. By 1886, guava plants were common across the southern half of the state. The first commercial planting was established at Palma Sola, Manatee County in 1912 and by 1948 there were about 400 acres (Smith, 1950). Today, the commercial guava industry in southern Florida includes production of both white and red pulp types estimated at ∼600 acres with a gross value of ∼$15 ⁎

million annually (Garcia et al., 2016). Guava fruit is primarily consumed fresh, but it can also be processed for juice, candy, puree, preserves, dried fruit, and ice cream. Fresh guava fruit is harvested commercially in Florida at two developmental stages for distinct markets. Fully expanded, white-fleshed fruit are harvested before full flavor development, chilled, and consumed crisp, like an apple. This product is favored by Asian-Americans. Fully mature, red fleshed, and full-flavored guava comprise the alternative commercial market in Florida, and are favored by Latin-American and Caribbean cultures. Having multiple fresh fruit markets is advantageous to growers seeking to maximize market opportunities. Fruit quality drives guava acceptance at the consumer level, but is significantly impacted by the plant accession, growing environment, and cultural practices. The diversity of guava fruit quality for multiple accessions or cultivars has been investigated by groups around the world including Pakistan (Mehmood et al., 2014), Malaysia (Yusof, 1989), Taiwan (Thaipong and Boonprakob, 2005), Mexico (Mondragon-Jacobo et al., 2008), and the U.S (Wilson, 1980). These

Corresponding author. E-mail addresses: pamoon@ufl.edu (P. Moon), yuqingf@ufl.edu (Y. Fu), [email protected] (J. Bai), [email protected] (A. Plotto), jhcr@ufl.edu (J. Crane), ac@ufl.edu (A. Chambers). https://doi.org/10.1016/j.scienta.2018.04.067 Received 2 February 2018; Received in revised form 27 April 2018; Accepted 28 April 2018 0304-4238/ © 2018 Elsevier B.V. All rights reserved.

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Table 1 Brief description of guava accessions used in this study. GRIN – Germplasm Resources Information Network. Genotype

Ripe Fruit Color

Planting Date

Source

Alhabari White Asian White Barbie Pink Blanca Blitch Giant Bangkok Hawaiian White Homestead Hong Kong Pink Indonesian Red Indonesian White Mexican Cream Patillo Queen Ruby x Supreme TREC 53-6550 Unknown-1 Unknown-2 Unknown-3 Unknown-4 Unknown-5 Unknown-6 Unknown-7 Unknown-8 Unknown-9 Webber x Supreme White Indonesian Seedless

white white pink pink pink white white pink pink pink white white pink pink pink pink pink pink pink pink white pink pink pink pink white white

1997 1997 2007 1996 1996 1997 1996 1997 1996 1997 1997 1997 1996 1997 1997 1997 1996 1996 1996 1996 1996 1996 1996 1996 1996 1997 1996

California Tropical Fruit Tree Nursery, Vista, CA. GRIN Acc. No. MIA-37659. Local selection, Homestead, FL. GRIN Acc. No. MIA-35034. Selected by Hopkins Rare Fruit Nursery, Immokalee, FL. Unknown origin. GRIN Acc. No. MIA-35030. Unknown origin. GRIN Acc. No. MIA-36760. Origin Thailand. Sourced California Tropical Fruit Tree Nursery, Vista, CA. Sourced from Hilo, HI. Seedling of Ruby x Supreme hybrid. GRIN Acc. No. MIA-36762. Origin Hong Kong. GRIN Acc. No. HSPI-15. Origin unknown. GRIN Acc. No. MIA-35033. Origin unknown. California Tropical Fruit Tree Nursery, Vista, CA. GRIN Acc. No. MIA-36763. Unknown origin. GRIN Acc. No. MIA-36764. Unknown origin. GRIN Acc. No. MIA-36765. Seedling selection. GRIN Acc. No. MIA-35024. Seedling selection. GRIN Acc. No. MIA-35031. Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Seedling selection. Origin Malasia. Sourced from Hilo, HI. GRIN Acc. No. MIA-35028.

Fig. 1. Average pH (n = 4) of 27 guava accessions at immature, mature green, and mature yellow stages. Bars represent standard errors.

important volatiles from a sensory perspective. While numerous studies on guava fruit quality have been published, quantifying fruit quality metrics for a large number of guava accessions is necessary to support local growers and their dual markets. This information can then be used to make cultivar recommendations and for selecting breeding parents to create the next generation of superior cultivars.

studies often include assessment of soluble solids, pH, titratable acidity, fruit color, and other fruit metrics. Guava aroma has also been deeply characterized usually for one or a few fully ripe and aromatic varieties (Bassols and Demole, 1994; Chen et al., 2006; Clery and Hammond, 2008; Jordán et al., 2003; Mahattanatawee et al., 2005; Pino and Bent, 2013; Pino et al., 2002, 1999; Porat et al., 2011; Soares et al., 2007; Steinhaus et al., 2008, 2009; Wilson, 1980). Many of these studies have cataloged aroma compounds in guava, or sought to identify the most

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Fig. 2. Average titratable acidity (n = 4) reported as % citric acid for 27 guava accessions at immature, mature green, and mature yellow stages. Bars represent standard error.

2. Materials and methods

unknown genotypes were also included in this study and named Unknown-1 through Unknown-9. All unknown samples were red fleshed except for the white fleshed Unknown-5 (Table 1).

2.1. Plant growth conditions The guava trees in this study are maintained at the University of Florida Tropical Research and Education Center in Homestead, Florida, USA. Most accessions were planted during 1996 and 1997. TREC 53–6550 and TREC 51-4967 were planted in 1950 and 1951, respectively. Tree spacing was 6.1 m within row by 6.1 m between rows. Up to five replicates of each guava accession was randomly assigned a field location. Inputs were optimized for guava production on the calcareous, high pH, restricted root depth soils of Miami-Dade County (Noble et al., 1996; Obreza et al., 1993). Trees were maintained with 1.8 kg/tree of granular 6-6-6 N-P-K each February, April, June and August. Granular potassium sulfate-magnesia (1.3 kg/tree) was applied after fruit set in May and again during fruit development each July. Secondary (Mg) and minor (Mn, Zn, Fe, Mo, and B) elements were applied in a mix (KeyPlex®350) to the foliage during March, May, July and September. Phosphorus acid (e.g., AgPhite®57) was applied to the foliage along with the secondary and minor element applications. 45 g chelated iron (Sequestrene®136–EDDHA) (45 g per tree) was mixed with water and applied as a soil drench in May and July. In general, trees were not sprayed to control insects and diseases. However, on an as-needed basis (e.g., high quality fruit are required) trees were sprayed with insecticide to control the Caribbean fruit fly (Anastrepha suspensa), scales, and mealybugs and/or fungicide to control red alga and anthracnose (Colletotrichum gloeosporioides). Plant mortality and tropical storms had caused some replicates to be lost or misidentified leading to accessions of unknown origin.

2.3. Fruit harvest Guava fruit to be analyzed for pH, titratable acidity, and aroma were harvested on September 7, 2016 in the morning. At least four representative clean and blemish-free fruit were selected for each guava sample at each developmental stage. Samples were bulked by tree and fruit developmental stage with biological replicates coming from each of two independent trees where available. Border row trees were avoided when possible. Three developmental stages were selected for analysis. The first developmental stage, immature green, included immature fruit that were approximately 3 cm in diameter, hard, and dark green in color. The second stage, mature green, included physiologically mature guava fruit that were fully expanded, firm, light green, and suitable for the early guava market. The third and final stage, mature yellow, included mature fruit with peel color-break at the early stages of fruit softening and were externally aromatic. 2.4. Sample processing Fruit samples were sliced, weighed, and blended with an equal weight of saturated sodium chloride solution. Two technical replicates of 6 mls for each sample were aliquoted into 20 ml Gerstel vials, flushed with nitrogen, and sealed with magnetic crimp caps for volatile analysis. Two replicates of 45 ml samples were aliquoted per sample for titratable acidity analysis. All samples were stored at −80 °C until analysis.

2.2. Guava accessions 2.5. pH and titratable acidity Several guava accession types were available for study including white fleshed (‘Alhabari White’, ‘Asian White’, ‘Indonesian White’, ‘Mexican Cream’, ‘Hawaiian White’, ‘Giant Bankok’, ‘Webber x Supreme’, ‘White Indonesian Seedless’), and red fleshed (‘Barbie Pink’, ‘Blanca’, ‘Blitch’, ‘Homestead’ (Campbell, 1990), ‘Hong Kong Pink’, ‘Indonesian Red’, ‘Patillo’, ‘Queen’, ‘Ruby x Supreme’, TREC 53-6550) types. ‘Homestead’ and ‘Ruby x Supreme’ are independent selections from a ‘Ruby’ by ‘Supreme’ cross. Additionally, ‘Webber x Supreme’ is a selection resulting from a cross between ‘Webber’ and ‘Supreme’. Nine

Fruit homogenate was thawed, centrifuged at 12,100 × g for 15 min, and the supernatant was analyzed for titratable acidity (TA) and pH. For TA, 6 g of the supernatant was diluted with 50 mL DI-water and titrated with 0.1 N sodium hydroxide to a pH 8.1 endpoint using a Metrohm 808 Titrando equipped with a Metrohm 730 sample changer and operated by software Tiamo 2.4 Light (Metrohm USA Inc., Westbury, NY). Whenever the titratable acidity was too low (no inflexion point in titration curve), the analysis was repeated with 10–15 g 377

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Table 2 47 volatiles putatively identified and quantified for clustering analysis. Retention time, Linear Retention Index (Kovats), and putative identification for each compound is shown. Averaged Genotype Fold Range and Averaged Stage Fold Range are the averaged max value divided by averaged min value for each compound by genotype and fruit developmental stage, respectively. Generalized aroma classifications among accessions were qualitatively assigned including Common (present in the majority of accessions), +/− (detected and not detected across accessions), and Ripe Only (only detected in ripe samples). Sensory aroma support is from published literature as follows: (a) Pino et al. (1999), (b) Pino and Bent (2013), (c) Porat et al. (2011), and (d) Steinhaus et al. (2008). Retention Time

LRI

Putative Identification

Averaged Genotype Fold Range

Averaged Stage Fold Range

4.38 7.49 11.41 14.74 14.90 17.01 17.43 20.34 21.31 23.22 23.39 23.61 24.04 24.20 24.95 25.35 25.53 25.80 25.88 29.34 29.96 31.83 32.32 36.82 38.71 39.36 40.36 40.57 41.13 41.33 41.72 42.00 42.32 42.40 42.49 42.63 42.89 43.00 43.35 43.44 43.59 43.94 44.18 44.30 44.53 46.38 46.58

501 599 710 796 800 851 861 929 951 995 998 1003 1013 1017 1034 1043 1047 1053 1055 1134 1149 1194 1206 1322 1375 1394 1423 1429 1447 1453 1465 1474 1484 1487 1490 1494 1503 1506 1518 1521 1526 1538 1546 1550 1558 1624 1631

Ethyl alcohol Ethyl acetate Methyl butanoate Ethyl butanoate Hexanal Unknown 2 (E)-2-Hexenal Methyl hexanoate α-Pinene 6-Methyl-5-hepten-2-one Myrcene Ethyl hexanoate (Z)-3-Hexenyl acetate Hexyl acetate Unknown 3 (Z)-β-Ocimene Limonene 1,8-Cineole (E)-β-Ocimene allo-Ocimene allo-neo-Ocimene Ethyl octanoate Octyl acetate β-Ionone Phenylpropyl isobutyrate α-Copaene (E)-α-Bergamotene α-Gurjunene (E)-Caryophyllene (E)-Cinnamyl acetate Unknown 1 Unknown 4 α-Humulene γ-Curcumene allo-Aromadendrene γ-Muurolene α-Zingiberene (Z)-α-bisabolene β-Bisabolene β-Selinene (Z)-γ-Bisabolene Δ-Cadinene (E)-Calamenene 7-epi-α-Selinene (E)-Cadina-1,4-diene Caryophyllenyl alcohol Unknown 5

435 1248 42209 71 2 5 4 29684 1043 130 40 867 6 20 105 140 62 136 25 98 174 99510 8402 8639 2655 21 104 35 4 214 345 7 13 33 64 213 104 49 18 37 80 21 131 59 341 47 689

27 11 13498 57 2 2 1 305 1 2 2 514 332 110 2 1 1 2 2 2 2 40710 246 1125 2237 3 3 4 2 7 3 3 3 3 2 4 3 3 2 2 3 3 2 3 5 4 2

Common

+/-

Yes

Ripe Only

Odor Description

Support

Yes Yes Yes Yes

Alcohol Pineapple, fruity Fruity Fruity Grassy – Green Fruity, sweet Herbal Citrus Spicy Fruity, wine Green, banana Sweet, fruity – Floral, herbal Citrus Herbal Sweet, herbal Green, fresh Floral Floral, fruity Fruity Floral Floral, fruity Woody, spicy Woody Woody Woody, spicy Sweet, cinnamon – – Woody Earthy Woody Herbal, woody Spicy Balsamic Balsamic Herbal Balsamic Herbal – Amber – Spicy –

b) c), b)

Yes Yes Yes Yes

Yes

Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes

Yes

Yes Yes Yes

Yes Yes Yes Yes

Yes Yes Yes Yes

Yes Yes

Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes

d), b) d), b) a), b) c) a) a) a) a), b) c) c), a), b) c) c), a), b) a) c), a) c) a), b) c), a), b) a) c)

c) c)

c) a) c), a) c), a) a) c) a) a) a)

a)

detector (5973 N; Agilent). The column oven was programmed to increase at 4 °C min−1 from the initial 40 °C to 230 °C, then ramped at 100 °C min−1 to 260 °C and held for 11.7 min for a total run time of 60 min. Helium was used as carrier gas at a flow rate of 1.5 mL min−1. Inlet, ionizing source, and transfer lines were kept at 250, 230, and 280 °C, respectively. Mass units were monitored from 30 to 250 m/z and ionized at 70 eV. Data were collected using a data system (ChemStation G1701 AA; Hewlett-Packard, Palo Alto, CA). A mixture of C-5–C-18 nalkanes was run at the beginning of each day to calculate retention indices (RIs). Compound identification was performed by comparison of mass spectra with library entries (NIST/EPA/NIH Mass Spectral Library, v. 2.0d, National Institute of Standards and Technology, Gaithersburg, MA), as well as by comparing RIs with published RIs on the same column (Adams and Brown, 2007; Kondjoyan and Berdagué, 1996).

of sample. 2.6. Aroma analysis Headspace sampling and GC–MS analysis were performed as previously reported (Bai et al., 2014). Frozen sample vials were thawed under tap water and loaded into the autosampler (Model MPS2; Gerstel Inc.) equipped with a cooled tray holder [a cooling plate (Laird Tech, Göteborg, Sweden) controlled by a Peltier Thermostat (CTC Analytics AG, Zwingen, Switzerland)]. Samples were held 0 to 16 h at 4 °C in the cooled tray until analyzed. For analysis, juice samples were incubated for 30 min at 40 °C. A 2-cm solid phase microextraction (SPME) fiber (50/30 μm DVB/Carboxen/PDMS; Supelco, Bellefonte, PA) was then exposed to the headspace for 30 min at 40 °C. After exposure, the SPME fiber was inserted into the injector of a GC–MS (Model 6890; Agilent, Santa Clara, CA) to desorb the extract for 15 min at 250 °C. The GC–MS equipment and settings were: DB-5 (60 m length, 0.25 mm i.d., 1.00 μm film thickness; J&W Scientific, Folsom, CA) columns, coupled with a MS 378

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Fig. 3. a–c. Volatile metabolites that are common among all guava accessions tested. Representative volatiles hexanal (a), (E)-caryophyllene (b), and (E)-2-hexenal (c) are shown (putative identification only). Bars represent standard errors (n = 4). d–f. Volatile metabolites detected primarily in mature yellow guava fruit. Representative metabolites (Z)-3-hexenyl acetate (d), ethyl butanoate (e), and ethyl octanoate (f) are shown (putative identification only). Bars represent standard errors (n = 4). g–i. Volatile metabolites that show presence/absence phenotypes are represented by limonene (g), (E)-cadina-1,4-diene (h), and β-ionone (i) (putative identification only). Error bars represent standard error.

response to ripening, but TA significantly decreased for most accessions from immature to mature yellow developmental stages.

2.7. Statistical analysis Tukey’s HSD (alpha = 0.05) was used for all multiple comparisons where statistical significance is reported. Titratable acidity values were multiplied by two to correct for dilution during sample preparation. All data were analyzed using SAS JMP Pro v13. Total peak area was used for analysis of volatile data. 47 non-coeluting compounds detected in at least two accessions were selected for Hierarchical Clustering and visualized using the Ward distance option. Metabolites that were consistent between sample replicates and common among many accessions were selected for relative quantification using peak area. Co-eluting metabolites were excluded from analysis.

3.2. Patterns of volatile metabolites Volatile metabolites can be common across many genotypes, vary with fruit development, or vary with accession due to genetic differences. Volatiles that were common among all accessions include two common aldehydes present in all plant tissue, hexanal and (E)-2-hexenal, as well as many of the terpene compounds (mono- and sesquiterpenes) (Table 2). Fig. 3a–c shows representative examples of volatiles that were found in all guava accessions tested. These include putatively identified hexanal, (E)-caryophyllene, and (E)-2-hexenal. The abundance of these volatiles can vary significantly, but were detectable in each cultivar at each stage of fruit development tested. Hexanal and (E)-2-hexenal showed a general trend of increasing from immature to mature yellow fruit. Conversely, (E)-caryophyllene decreased from immature to mature yellow for most accessions. Certain volatile metabolite patterns exemplify the impact of fruit development on the development of aroma. Fig. 3d–f shows three representative volatile metabolites that were abundant primarily in the yellow stage of fruit development. The selected metabolites were putatively identified as (Z)-3-hexenyl acetate, ethyl butanoate, and ethyl octanoate. (Z)-3-Hexenyl acetate was the most common across accessions for the ripening-associated volatile metabolites. The relative

3. Results 3.1. pH and titratable acidity We measured pH and titratable acidity to test if these traits could be used to classify guava fruit maturity. Fig. 1 shows the results for pH by accession and developmental stage. Internal pH ranged from 2.93 to 4.59. Titratable acidity ranged from 0.17 to 3.62, and these results are shown in Fig. 2. Large differences in pH and titratable acidity were observed among guava accessions particularly for ‘Blanca’, ‘Blitch’, ‘Patillo’, ‘Unknown 5′, and ‘Unknown 9′ that generally had lower pH and higher TA than other accessions. pH did not consistently change in 379

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Fig. 3. (continued)

than limonene, and decreased over developmental time in general. β–Ionone showed both strong presence/absence and ripening induction. Overall, these results show the value of testing multiple guava accessions in order to capture greater aroma diversity that could then be leveraged to enhance aroma profiles through breeding.

abundance of ethyl butanoate and ethyl octanoate varied greatly among the guava accessions, with some lacking detectable levels of one or both of these metabolites. Interestingly, volatiles that were detected at the ripe stage all belonged to the ester family, known to have fruity or “sweet” characters (Table 2). Some volatiles might be considered as “signature” compounds for certain accessions. Fig. 3g–i shows three representative volatile metabolites that follow a presence/absence pattern of detection among the tested guava accessions. Limonene, (E)-cadina-1,4-diene, and β-ionone were selected as representative volatiles. Limonene was present at all three developmental stages for those accessions that produced this metabolite. (Z)-Cadina-1,4-diene was less common across genotypes

3.3. Global aroma Forty-seven volatile components were selected to investigate aroma diversity among guava accessions (Table 2). Fig. 4 shows a scatterplot of these components for all guava accessions and developmental stages. Non-coeluting volatiles that were present in both replicates for at least 380

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Fig. 4. Scatterplot of 47 selected guava volatile metabolites identified by retention time as listed in Table 2 for all accessions. Immature, mature green, and mature yellow developmental stages are shown as dark green, light green, and yellow points, respectively. For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.

wider range of values from guava accessions grown in our conditions could be attributed to greater diversity being sampled, or the impacts from sampling fruit at different stages of fruit development. A few accessions had significantly higher TA than the others. TA for Unknown 5 was greater than 2.0% citric acid, which makes it extremely sour, but interestingly, its volatile composition at the yellow mature stage clustered with the mature samples. ‘Patillo’ and Unknown 9 were also in that category, with TA between 0.89 and 1.95 at the mature yellow stage. On the other hand, ‘Blitch’ also had high TA and its volatile composition clustered the mature yellow fruit together with the mature green and immature, suggesting that samples from this specific accession was not fully mature when harvested, in spite of external color. One replication of each TREC 53–6550 and ‘Blanca’, both also with high TA, fell into that category. It could be speculated that the samples of these biological replicates had not reached physiological maturity. Future studies should include the combination of all parameters, including external and internal color, soluble solids contents, and sensory evaluation to further pinpoint optimum maturity at the accession level. As expected, volatile metabolites varied by fruit developmental stage and among cultivars with a few exceptions. There were significant differences in metabolite abundance among cultivars, but these can also vary significantly with environment and should therefore be only be used to identify general trends. A general trend with increasing maturity was a decrease in terpenes over time, with esters only being produced at full maturity. This would greatly affect sensory quality between mature green and mature yellow fruit. There were examples of volatile metabolites that were similar across all cultivars and each fruit developmental stage. These metabolites could reasonably be expected to be detected in a number studies investigating guava volatiles. The abundance of many volatile metabolites depended on the developmental stage of the fruit. This could impact detection and should guide sampling strategies for future studies. Additionally, this information could be useful for targeting cultivars for different markets if the volatile metabolites have sensory impacts that influence consumer choice. Finally, there were a number of volatile metabolites with presence/absence phenotypes. These results could be useful for parental selection for future genetic studies if any of these compounds have the potential to be a desirable breeding target. The data generated in this study were not sufficient to confidently identify the unknown accessions. Future work would most likely require genetic markers to confidently identify these accessio. Otherwise, the unknowns could still be used as parents in a breeding program because of their unique fruit quality profiles.

two accessions were selected for analysis. The selected compounds were putatively identified, and inclusion of more metabolites was favored to explore aroma profile relationships among accessions. Fig. 4 shows that most volatiles increase in abundance at the mature yellow stage. Fewer volatiles decrease with maturity including α-pinene (RT = 21.311), 6methyl-5-hepten-2-one (RT = 23.219), myrcene (RT = 23.387), limonene (RT = 25.533), α-copaene (RT 39.359), and most of higher boiling point sesquiterpenes. 3.4. Clustering on aroma Data from the 47 volatile metabolites (Table 2) were used for hierarchical clustering of each guava accession and developmental stage. The results are shown in Fig. 5. The mature yellow samples tended to cluster more tightly than immature or mature green suggesting greater aroma changes on average between mature green and mature yellow developmental stages. There was no obvious pattern that would allow separation of guava accessions by flesh color. Comparing biological replicates from independent trees enabled additional accession-level insights. All ‘Blitch’ samples at each developmental stage clustered in the clade that mostly included immature or mature green samples suggesting that this cultivar exhibits fewer changes upon ripening than average. ‘Mexican Cream’ immature and mature yellow samples from both biological replicates clustered more closely than the mature green developmental stage suggesting metabolite fluctuations throughout ripening. ‘Patillo’, ‘Queen’, and ‘Webber x Supreme’ are examples where mature yellow samples were clearly separated from immature and mature green stages, and might therefore be expected to have the greatest changes in aroma at the mature yellow stage. None of the ‘Ruby x Supreme’ replicates clustered with the mature yellow samples of other accessions suggesting a different aroma profile at the mature yellow fruit stage. 4. Discussion Guava fruit are valued for their tropical sensory qualities, but quality traits vary by genotype, growing environment, and developmental stage. These traits must therefore be quantified in each production area to identify the best genotypes for specific markets and to allow indenficiation of superior genetic donors in the creation of new, superior guava varieties. Aroma analysis can also be greatly impacted by fruit developmental stage. Many fruits decrease in acidity as the fruit matures, and this can serve as an indicator of maturity. Although pH did not reliably increase over fruit development, TA did decrease, in general, over developmental time for most accessions. TA and pH results were similar to those other studies with fresh, ripe guava fruit (Mehmood et al., 2014; Mondragon-Jacobo et al., 2008; Soares et al., 2007; Thaipong and Boonprakob, 2005; Yusof, 1989). A

5. Conclusions This work demonstrates the wide variability in quality traits among a large number of guava accessions, and begins to relate that variability 381

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Fig. 5. Clustering guava accessions and developmental stages using 47 volatile metabolites. Developmental stages are highlighted as dark green (immature), light green (mature green), or yellow (mature yellow). For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.

Competing interests

to fruit development. It allowed the identification of those accessions that fail to develop the characteristic aroma profiles of mature yellow fruit, which would therefore render them to be primarily suitable for the green fruit market. This information would be useful for both growers and those interested in guava plant breeding and genetics.

The authors declare that they have no competing financial interests.

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Funding

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