Productivity and Oil Content in Relation to Jatropha Fruit Ripening Under Tropical Dry-Forest Conditions

Article Productivity and Oil Content in Relation to Jatropha Fruit Ripening under Tropical Dry-Forest Conditions

Álvaro Cañadas-López 1,2,* , Diana Yasbhet Rade-Loor 3, Marianna Siegmund-Schultze 4, Marys Iriarte-Vera 3, Juan Manuel Domínguez-Andrade 5, Jesús Vargas-Hernández 6 and Christian Wehenkel 7

1 Universidad Laica Eloy Alfaro de Manabí, Facultad de Ingeniería Agropecuaria, Carrera Ingeniería Agropecuaria, Campus ULEAM-Extensión Chone, Av. Eloy Alfaro, Chone C.P. 130301, Provincia de Manabí, Ecuador 2 Instituto Nacional de Investigaciones Agropecuarias (INIAP), Estación Experimental Tropical Pichilingue, Programa de Forestaría, Km 5 vía Quevedo—El Empalme, Cantón Mocache C.P. 120501, Provincia Los Ríos, Ecuador 3 Escuela Superior Politécnica de Manabí (ESPAM-MFL), Centro de Investigación de las Carreras de la ESPAM-MFL (CICEM), Campus Politécnico Calceta, Sitio El Limón, Calceta, Cantón Bolívar C.P. 130250, Provincia de Manabí, Ecuador; [email protected] (D.Y.R.-L.); [email protected] (M.I.-V.) 4 Technische Universität Berlin, Environmental Assessment and Planning Research Group, Straße des 17, Juni 145, 10623 Berlin, Germany; [email protected] 5 Escuela Superior Politécnica del Litoral (ESPOL), ESPAE Graduate School of Management, Campus Las Peñas C.P. 090903, Provincia de Guayas, Ecuador; [email protected] 6 Colegio de Postgraduados, Posgrado en Ciencias Forestales, Montecillo, Texcoco, Mexico; [email protected] 7 Instituto de Silvicultura e Industria de la Madera, Universidad Juárez del Estado de Durango, Boulevard Guadiana #501, Ciudad Universitaria, Torre de Investigación, Durango C.P. 34120, Mexico; [email protected] * Correspondence: [email protected]; Tel.: +593-93-906-8644

Received: 27 August 2018; Accepted: 1 October 2018; Published: 4 October 2018

Abstract: Jatropha is promoted as a pro-poor bioenergy plant, while basic information about its productivity, age of maximum production, and oil content are missing. This study aims to determine the seed yield (dry weight) for three INIAP elite jatropha accessions, and to evaluate the changes in physical and chemical seed traits at the different fruit ripening stage in a split-plot design. Maximum seed production occurred four years after planting for the accessions CP041 and CP052, while for accession CP054, it occurred after the first year. CP041 was the most productive, with a mean of 316.46 g tree−1 year−1 (±76.50) over the 8-year study period. No significant differences in oil content were found among accessions, fruit ripening stage, and their respective interactions. Seed moisture content decreased drastically as the fruit ripening stage increased, from 40.5% ± 1.0% at fruit ripening stage 1 (greenish-yellow) down to 13.8% ± 0.4% at fruit ripening stage 4 (black-brown). No significant differences in seed weight were found among accessions, but it decreased as maturation progressed. Yellow fruits (stage 2) were the heaviest (62.4 g ± 1.5 g) and the black-brown fruits the lightest (44.3 g ± 1.9 g). The oil content (%) increased with seed weight up to the point of 58.3 g, but then decreased for heavier seeds.

Keywords: harvesting point; jatropha accessions; seed productivity; seed humidity; seed oil content

Forests 2018, 9, 611; doi:10.3390/f9100611 www.mdpi.com/journal/forests Forests 2018, 9, 611 2 of 13

1. Introduction Jatropha curcas L. (in the following: jatropha) is a shrub or small tree belonging to the tribe Joannesieae of the family Euphorbiaceae, adapted to arid zones, with potential use for biofuel production. Jatropha is commonly used in the Manabí province, Ecuador, as living fence for pasture division [1]. Jatropha had often been classified as an ideal “pro-poor” crop, due to its potential value for seed oil production in marginal and degraded areas. Consequently, substantial public and private investment has been made available for jatropha plantations in marginal lands [2]. Nonetheless, it has become clear that the requirements to obtain economically viable seed yields have been underestimated, and therefore, many investments have been withdrawn [3–5]. Despite this reversal, production derived from jatropha under favorable growing conditions continues to be a concept receiving considerable political and commercial interest [6,7], and efforts are being made to estimate the biomass production so that small jatropha producers could sell the carbon sequestration in the trees under the emissions trading scheme of the Clean Development Mechanism (CDM) of the Kyoto Protocol [8]. For example, in Ecuador, the Ministry of Electricity and Renewable Energy (Ministerio de Electricidad y Energía Renovable, MEER), with the support of private investors, implemented the “Jatropha for Galápagos” Project in 2011. The main objective of this project is to replace petro diesel by jatropha oil through the agro industrial development of jatropha in the continent [1]. The selected province is Manabí, which belongs to the tropical, dry forest zone [9], and focusing on smallholders. Determination of seed productivity using long-term field data is crucial for the management of jatropha plantations [10]. When regional or local data on seed productivity is missing, information from the literature is commonly used to evaluate the economic and financial feasibility of jatropha plantations [10]. However, estimates of jatropha production used in several economic studies range between 3000–7000 kg ha−1 year−1 [10]. In Ecuador, Rade et al. [2] observed a huge seed production variation during seven years’ data collection of the INIAP accession CP041 planted in jatropha live fences in Manabí, Ecuador (average of 243.32 g tree−1 year−1). In a jatropha pure plantation under tropical dry forest conditions, Cañadas et al. [1] reported an average jatropha dry seed productivity of 283.20 g tree−1 year−1 with 1677 trees per hectare for the INIAP accession CP041, but also with a broad variation in productivity from one year to the next. In addition to year-to-year variation in environmental factors, plantation age influences seed production. According to GTZ [11], jatropha plantations reach maturity at about eight years of age. Van Eijck et al. [10] highlighted that the jatropha production horizon is too short (10 years or less) to be able to reliably assess medium and long-term economic viability of jatropha plantations. However, a perspective on jatropha productivity in Ecuador was presented by Rade et al. [2], who found a negative covariance between jatropha dry seed production and time in a live fence for the INIAP accession CP041 (−991.35) and for traditional jatropha (−715.00) during seven years’ field observations. Moreover, jatropha oil content in seeds is known to vary between 40% to 60%, with protein content varying from 10% to 30% (although it is not usable due to the presence of phrobol ester and curcin contents) [12]. Jatropha fruits show different maturity degrees within the same tree as a result of a continuous fructification process [13]. Since physiological changes occur during the maturation stages [14], harvesting the fruits at an adequate maturation stage is an important factor determining seed quality and oil content. This information is essential for planning harvesting and processing, with the aim of optimizing oil extraction [15]. In addition, genetic variation of the lipid content of jatropha seeds has been found among genotypes [16]. Oil content and moisture change with time, and affect the seed quality and behavior in response to environmental changes, especially relative humidity [17]. Thus, it is important to evaluate changes in physical and chemical properties, including oil and moisture content, and dry weight of jatropha seed along the maturation process. These data are not only essential for harvest timing and equipment, but also for seed processing and storage [18]. The productivity of mature jatropha stands is poorly described under tropical dry forest conditions. Moreover, determination of optimum seed quality in relation to oil content during the jatropha fruit ripening process is of utmost importance to establish the optimum time for fruit harvesting in jatropha. Forests 2018, 9, x FOR PEER REVIEW 3 of 13 Forests 2018, 9, 611 3 of 13 harvesting in jatropha. The objective of this study was to evaluate the productivity and physical changes at four fruit ripening stages for three INIAP elite jatropha accessions. The objectiveForests 2018 of, 9, thisx FOR study PEER REVIEW was to evaluate the productivity and physical changes at four fruit3 of ripening 13 stages for three INIAP elite jatropha accessions. 2. Materialsharvesting and in Methods jatropha. The objective of this study was to evaluate the productivity and physical changes at four fruit ripening stages for three INIAP elite jatropha accessions. 2. Materials and Methods 2.1. Study Area 2. Materials and Methods 2.1. StudyThe st Areaudy was conducted at the Portoviejo Experimental Station (EEP) of the National Institute of AgriculturalThe2.1. Study study Area Research was conducted (INIAP) at during the Portoviejo the period Experimental from August Station 2009 to (EEP) December of the 2017. National The Institutegeographical ofThe Agricultural stcoordinatesudy was conducted Research are 0°0 at1′ (INIAP) theS andPortoviejo 80°23′ during Experimental W, the sector period Lodana, Station from (EEP) August canton of the 2009 Port Nationaloviejo, to December Instituteprovince 2017. of TheManabí. geographicalof Agricultural The EEP coordinatesis Researchlocated at (INIAP) are 47 0m.a.s.l,◦01 during0 S and with the 80 an ◦ period23 annual0 W, sectorfrom mean August Lodana, temperature 2009 canton to December of Portoviejo, 26.4 °C 2017. and province Theaverage of Manabannualgeographical í.rainfall The EEP of coordinates is798 located mm, at arewith 47 0°0 m.a.s.l, 1a′ Slarge and with 80°23′year an- to W, annual-year sector meanrainfall Lodana, temperature variability, canton Port ofoviejo,78% 26.4 average ◦ provinceC and averagerelative of annualhumidity,Manabí. rainfall and The a of totalEEP 798 mm,issun located-light with atsum a 47 large m.a.s.l,of year-to-year1159 with h year an −1annual rainfall. Figure mean variability, 1 shows temperature the 78% monthly of average 26.4 °Cprecipitation relative and average humidity, along annual rainfall of 798 mm, with a large year-to-year rainfall variability, 78% average relative andwith apotential total sun-light evapotranspiration, sum of 1159 h averaged year−1. Figure over 1the shows experiment’s the monthly period. precipitation A surplus along of water with humidity, and a total sun-light sum of 1159 h year−1. Figure 1 shows the monthly precipitation along potentialgenerally evapotranspiration,occurs in the course averagedof January over to March, the experiment’s while there period.is a water A surplusdeficit the of rest water of generallythe year. with potential evapotranspiration, averaged over the experiment’s period. A surplus of water occurs in the course of January to March, while there is a water deficit the rest of the year. generally occurs250 in the course of January to March, while there is a water deficit250 the rest of the year. 1 Precipitation - 1

- 250 250 1

200 PrecipitationEvapotranspiration 200-

1 - 200 Evapotranspiration 200 150 150 150 150 100 100 100 100

Precipitatio n50 month mm 50

Precipitation Precipitation mm month 50 50

Evapotranspiration mm month Evapotranspiration Evapotranspiration mm month 0 0 0 0 E E F F MM AA MM JJ Jl AA SS OO NN D D MonthsMonths FigureFigure 1. Water 1. Water balancebalance balance inin the thein the INIAP-EEP,INIAP INIAP-EEP,-EEP, 2009–2017. 2009 2009––2017.2017. The TheThe solid solid solid lineline line represents represents the the themonthly monthly monthly average average average of precipitationof precipitationof precipitation and and the and the dashed thedashed dashed line line theline the potentialthe potential potential evapotranspiration. evapotranspiration.

2.2. Soil 2.2. Conditions Soil Conditions A soil analysis for the study area was carried out in the INIAP-EEP (Table 1). The soil has a A soil analysis for the study area was carriedcarried out inin thethe INIAP-EEPINIAP-EEP (Table1 1).). TheThe soilsoil hashas aa neutral pH, with low levels of Nitrogen, Zinc, and Iron, medium levels of Boron, and high levels of neutral pH, with low levels of Nitrogen, Zinc, and Iron, medium levels of Boron, and high levels of Phosphorus, Potassium, Calcium, Magnesium, Copper, and Manganese. Phosphorus, Potassium, Calcium, Magnesium, Copper,Copper, andand Manganese.Manganese. Table 1. Soil chemical properties in the study area, INIAP-EEP, 2009. TableTable 1. Soil chemical properties in the study area, INIAP-EEP,INIAP-EEP, 2009.2009. NH4 P Zn Cu Fe Mn Zn B K Ca Mg pH NH4 P Zn Cu(in ppm)Fe Mn Zn B (in meqK 100Ca mL −1) Mg pH 7.3 17.12 23.01 1.82 (in7.14 ppm) 12.11 23.75 1.82 0.92 2.07(in meq20.90 1004.02 mL −1) 7.3 17.12 23.01 1.82 7.14 12.11 23.75 1.82 0.92 2.07 20.90 4.02 2.3. Preparation of Experimental Site

2.3. Preparation The land of Experimentalwas prepared Site by mechanized clearing, ploughing, and harrowing. Jatropha curcas L. cuttings were collected from the mother trees. Three INIAP jatropha accessions (CP041, CP052 and TheCP054) land were was used prepared in the present by mechanizedmechanized study. Diammonium clearing, phosphate ploughing, fertilizer and harrowing. (18-46-0) was JatrophaJatropha applied curcas curcasat a L.L. cuttingsdose were of 50 collected g per planting from the hole mothermother before cutttrees.trees.ings Three were INIAP transplanted. jatrophajatropha The accessions plantlets were (CP041, planted CP052 in and CP054)August were 2009 used as in 70 the-day present-old bare study. root transplants. Diammonium Potassium phosphate chloride fertilizer was subsequently (18-46-0)(18-46-0) wasapplied, applied at a at a dose ofdoseof 5050 of g g4 per gper per planting plantingtree, 30, hole 90, hole and before 120before cuttingsdays cutt afterings were transplanting. were transplanted. transplanted. Weeds The were plantlets The cut manually,plantlets were planted oncewere a monthplanted in August in ® 2009August aswith 70-day-old2009 a machete, as 70-daybare and- oldIgran root bare transplants. liquid root herbicide transplants. Potassium (Nufarm Potassium chloride Australia chloride was Limited: subsequently was Me lboumesubsequently applied,, VIC, Australia) applied, at a dose at of a was applied by spraying every 4 months, at a dose of 200 mL per 20 L of water. After jatropha 4dose g per of tree,4 g per 30, tree, 90, and30, 90, 120 and days 120 after days transplanting. after transplanting. Weeds Weeds were cutwere manually, cut manually, once aonce month a month with establishment, no fertilization, or pest or mite controls were provided. aw machete,ith a machete, and Igran and Igran® liquid® liquid herbicide herbicide (Nufarm (Nufarm Australia Australia Limited: Limited: Melboume, Melboume VIC,, VIC, Australia) Australia) was appliedwas applied by spraying by spraying every 4every months, 4 months, at a dose at of a 200 dose mL of per 200 20 Lm ofL water.per 20 After L of jatropha water. establishment, After jatropha noestablishment, fertilization, no or fertilization, pest or mite controlsor pest or were mite provided. controls were provided.

Forests 2018, 9, 611 4 of 13

2.4. Plot Sizes and Determination of Jatropha Productivity A split-plot design with three replications was used. The experiment covered a total of 2304 m2 (48 m × 48 m). The three INIAP jatropha accessions (INIAP CP041, INIAP CP052, INIAP CP054) were assigned to the main plots (768 m2), and the fruit ripening stage was considered as four sub-plots (64 m2) which were located in each of the main plots. The experimental unit (each sub-plot) included a total of 16 trees at a spacing of 2 × 2 m. From October 2009 to December 2017, jatropha fruits were harvested monthly from four trees in each sub-plot. Seed weight was measured with a precision balance after removing the pulp from the yellow fruits, extracting the seeds and drying them in a convection oven at 60 ◦C until constant weight.

2.5. Determination of Seed Weight, Moisture, and Oil Content at Different Fruit Ripening Stages In April 2012, when jatropha stands were three years old, fruits at several fruit ripening stages were harvested in order to determine the relationship between fruit ripening and seed characteristics, such as seed weight, moisture, and oil content. Once collected from the research plots, fruits were taken to the laboratory, where they were separated visually according to their ripening stage. Four fruit ripening stages were distinguished in the analysis: (a) early maturity (greenish-yellow fruits); (b) physiological maturity (yellow fruits); (c) over maturity (mottled-yellow fruits); and (d) senescent (black-brown fruits). After fruit separation, the fresh weight of 100 seeds per fruit ripening stage and replication were weighed with a precision balance. To determine seed moisture, a sample of 100 seeds from each replication was sun-dried for 30 days and then dehydrated by the standard hot air oven method at 105 ◦C ± 10 ◦C for 24 h to obtain the dry weight [19]. The moisture content was estimated as the weight loss (fresh weight - dry weight) of the sample, divided by the dry weight, expressed in percent. The oil content was determined for each seed sample by the solvent extraction method. The seeds were ground and placed in an extraction thimble. The oil was extracted using a Soxhlet apparatus with hexane for 6 h. The solvent material was evaporated with a rotary vacuum evaporator, and the remaining jatropha oil was weighed. The oil content of the seed was expressed as percent of dry matter mass. All the analyses were done in the seed laboratory of INIAP-EEP.

2.6. Statistical Analyses Analysis of variance (ANOVA) for seed dry weight, seed moisture, and oil content was performed with the MIXED procedure, using SAS software (V 9.4, SAS Institute Inc., Cary, NC, USA), and mean values were compared by a post-hoc Tukey’s multiple comparison test. A signiﬁcance level of 0.05 was assumed. The following linear model for the split-plot design was used:

Yijk = µ + Bi + Aj + Bi × Aj + Rk + Aj × Rk + εijk where Yijk is the value of the seed sample in the kth sub-plot of the jth main-plot in the ith block; µ is the population mean; Bi is the random effect of the ith block; Aj is the fixed effect of the jth accession; Bi × Aj is the random effect of the main-plot error; Rk is the fixed effect of the kth ripening stage; Aj × Rk is the fixed effect of the interaction between the jth accession and the kth ripening stage; and εijk is the experimental error. To determine the relationship between oil content (%) and seed weight for the seed samples harvested in April 2012 (n = 36), linear, quadratic and two-segment piecewise linear regression models were evaluated with the GLM procedure of SAS software, considering their respective coefficient of determination (R2) and mean square error (MSE). For piecewise linear regression, the optimal break point model (with the lowest MSE) was found before comparisons were made with the linear and quadratic regression models. Forests 2018, 9, 611 5 of 13

Forests 2018, 9, x FOR PEER REVIEW 5 of 13 3. Results Forests 2018, 9, x FOR PEER REVIEW 5 of 13 3. Results 3.1. Jatropha3. Results Seed Productivity The3.1. Jatropha dry seed Seed production Productivity per tree from October 2009 to December 2017 is shown in Figure2. 3.1. Jatropha Seed Productivity Three monthsThe dry after seed the productionjatropha plantation, per tree from the October fruit production 2009 to December began. 2017 The maximumis shown in jatrophaFigure 2.dried seedsThree productionThe months dry seed occurredafter production the jatropha at four-year-old per plantation, tree from plantations the October fruit production 2009 for theto December accessions began. The 2017 CP041maximum is shown and jatropha CP052,in Figure dried while 2. for accessionThreeseeds CP054 productionmonths it after occurred occurred the jatropha after at four theplantation,- ﬁrstyear- year.old the plantations fruit production for the began.accessions The CP041maximum and jatrophaCP052, whiledried Jatrophaseedsfor accession productionaccession CP054 occurred CP041 it occurred wasat four after the-yearmost the- oldfirst productive, plantations year. with for the 316.46 accessions± 76.50 CP041 g tree and−1 CP052,year−1 while(here and for accessionJatropha accessionCP054 it occurred CP041 was after the the mos firstt productive, year. with 316.46 ± 76.50 g tree−1 year−1 (here and in the following: mean followed by its standard error), taking into account all harvests from the early in theJatropha following: accession mean followedCP041 was by the its standardmost productive, error), taking with into316.46 account ± 76.50 all g harvests tree−1 year from−1 (here the early and fruits in the ﬁrst year up to the harvest at 8 years of age. The jatropha accession CP052 obtained an infruits the infollowing: the first meanyear upfollowed to the harvestby its standard at 8 years error), of age. taking The into jatropha account accession all harvests CP052 from obtained the early an −1 −1 −1 −1 averagefruitsaverage of in 314.02 ofthe 314.02 first± 63.19 year± 63.19 up g tree gto tree the−1 harvestyearyear−1 and atand 8CP054 years CP054 308.74of 308.74age. ± The 59.07± jatropha59.07 g tree g−1 accession treeyear−1. year CP052. obtained an average of 314.02 ± 63.19 g tree−1 year−1 and CP054 308.74 ± 59.07 g tree−1 year−1. 1000

1000900 CP041 1

- 900800 CP041CP052

1 - 800700 CP052CP054 700600 CP054 600500 500400 400300

300200 Dry Seed Seed Dry in Productiong tree

200100 Dry Seed Seed Dry in Productiong tree 1000 0 2009 2010 2011 2012 2013 2014 2015 2016 2017 2009 2010 2011 2012 2013Year 2014 2015 2016 2017 Year FigureFigure 2. Dry 2. Dry seed seed production production per per tree tree for for three three jatrophajatropha INIAPINIAP accessions accessions during during the theyears years 2009 2009–2017–2017 at INIAPFigureat INIAP Portoviejo 2. DryPortoviejo seed Research production Research Station, Station, per tree Ecuador. Ecuador. for three Vertical Verticaljatropha INIAP bars indicate indicate accessions the the standard during standard the error. years error. 2009–2017 at INIAP Portoviejo Research Station, Ecuador. Vertical bars indicate the standard error. 3.2. Seed3.2. DrySeed Dry Weight Weight and and Fruit Fruit Ripening Ripening Stage Stage 3.2. Seed Dry Weight and Fruit Ripening Stage ThereThere were were no nosignificant significant differences differences in in seed seed weight among among jatrophajatropha accessiaccessionsons or for or the for the interactioninteractionThere accessions accessions were no× significant fruit× fruit ripening ripening differences stage, stage, but in but seedseed seed weight weight was among was significantly significantlyjatropha accessiaffected affectedons (p < or 0.001) for (p < theby 0.001) interactionfruit ripening accessions stage (Table × fruit 2). ripening Tukey’s stage, (alpha but value seed weight of 0.05) was multiple significantly test showed affected two (p < distinctive 0.001) by by fruit ripening stage (Table2). Tukey’s (alpha value of 0.05) multiple test showed two distinctive fruitgroup ripening ranges of stage seed (Table ripening. 2). Tukey’sThe first (alphawas the value yellow of fruits, 0.05) multiplebeing the test heaviest, showed with two 62.4 distinctive g ± 1.5 g group ranges of seed ripening. The first was the yellow fruits, being the heaviest, with 62.4 g ± 1.5 g group(for 100 ranges seeds), of and seed the ripening. black-brown The fi fruitsrst was the the lightest, yellow with fruits, 44.3 being g ± 1.9the g heaviest, (Figure 3). with 62.4 g ± 1.5 g (for 100(for seeds), 100 seeds), and and the the black-brown black-brown fruits fruits the the lightest, lightest, with 44.3 44.3 g g ± ±1.91.9 g (Figure g (Figure 3). 3 ). 70 a a a 70 a 60 a a 60 50 b 50 b 40 40 30

Seed(g) weight 30

20 Seed(g) weight 20 10 10 0 0 Greenish-yellow Yellow Mottled-yellow Black-brown Greenish-yellow Yellow Mottled-yellow Black-brown Figure 3. Dry seed weight (100 seeds) at four jatropha fruit ripening stages. Vertical bars indicate the FigureFigurestandard 3. Dry 3. error.Dry seed seed weightDifferent weight (100 letters (100 seeds) seeds)indicate at at four afour significantjatropha jatropha difference fruit ripening ripening. stages. stages. Vertical Vertical bars bars indi indicatecate the the standard error. Different letters indicate a significant difference. standard error. Different letters indicate a signiﬁcant difference.

Forests 2018, 9, 611 6 of 13 Forests 2018, 9, x FOR PEER REVIEW 6 of 13

Table 2.2. SigniﬁcanceSignificance ((pp-value)-value) ofof effectseffects fromfrom jatrophajatropha accessionsaccessions andand fruitfruit ripeningripening stage on seed dry weight, moisturemoisture content,content, andand oiloil contentcontent inin aa jatrophajatrophaplantation plantation atat Portoviejo,Portoviejo, Ecuador.Ecuador.

SourceSource of of Variation Variation ( p(p-Value)-Value) EffectsEffects SeedSeed Weight Weight Seed MoistureMoisture Oil Oil Content Content AccessionAccession 0.96880.9688 <0.0001<0.0001 0.1497 0.1497 FruitFruit ripening ripening stage stage 0.00020.0002 <0.0001<0.0001 0.3239 0.3239 AccessionAccession× fruit × fruit ripening ripening stage stage 0.92060.9206 <0.0001<0.0001 0.2575 0.2575

3.3. Seed Moisture Content and Fruit Ripening Stage Highly signiﬁcantsignificant differences (p < 0.001) in seed moisture content were found among jatrophajatropha accessions, fruit ripening stage, and the interaction of these factors (Table2 2).). A A Tukey’s Tukey’s multiple multiple comparisoncomparison test showed three ranges of fruitfruit moisturemoisture contentcontent amongamong accessions.accessions. Accession CP052 had the highesthighest averageaverage moisturemoisture contentcontent withwith 37.2%37.2%± ± 4.1%, followed by CP041 withwith 31.3%31.3% ±± 3.3%,3.3%, and CP054 with 25.8% ± 3.0%.3.0%. As expected, fruit moisture content decreased as fruit ripening advanced, from an average value of 40.53% ±± 1.0%1.0% at at the the initial stage (greenish (greenish-yellow-yellow fruits), downdown toto 13.81%13.81% ±± 0.4% at the senescent stage (black-brown(black-brown fruits). Physiologically mature and over-matureover-mature fruits still have moisture contents over 30%, on average (Figure4 4).).

50 CP041 a a 45 CP052 a b 40 a CP054 a 35 b 30 a b

25 Seed(%)moisture 20

15 c 10 Greenish-yellow Yellow Mottled-yellow Black-brown Figure 4. Jatropha seed moisture variation between jatrophajatropha INIAPINIAP accessions along thethe fruit ripeningripening stages. INIAP INIAP Portoviejo Portoviejo Research Research Station, Station, Ecuador. Ecuador. Vertical Vertical bars bars indicate indicate the the standard standard error. error. Different lettersletters indicateindicate aa signiﬁcantsignificant difference.difference.

3.4. Oil Content and Fruit Ripening Stage No significantsignificant differencesdifferences in in oil oil content content were were found found among among accessions, accessions, fruit fruit ripening ripening stage, stage, or the or interactionthe interaction of these of these factors factors (Table (Table2). Mean 2). oilMean content oil content varied from varied 36.54% from 36.54%± 1.45% ± (accession1.45% (accession CP041) toCP041) 33.48% to ±33.48%1.08% ± (accession1.08% (accession CP052). CP052). Among Among fruit ripening fruit ripening stage, itstage, varied it varied from 36.01% from 36.01%± 1.60% ± (yellow1.60% (yellow stage) tostage) 33.31% to 33.31%± 1.10% ± 1.10% (black (black brown brown stage). stage). The coefficients The coefficients of variation of variation (a measure (a measure of the experiment’sof the experiment’s validity) validity) for main for plots main (9.92%) plots and (9.92%) subplots and (8.64%) subplots are (8.64%) acceptable are for acceptable field experiments. for field Oneexperiments. component One that component explains that the explains variation the in seedvariation oil content in seed inoiljatropha contentis in seed jatropha weight is seed (Figure weight5). However,(Figure 5). the However, relationship the relationship was not as simple was not as expected; as simple the as expected;linear regression the linear model regression based on mod seedel weightbased on explains seed weight only about explains 13.5% only of the about variation 13.5% in of seed the oilvariation content in expressed seed oil ascontent percentage expressed of seed as 2 masspercentage (Table of3). seed The mass quadratic (Table regression 3). The quadratic model showed regression a somewhat model showed better fit,a somewhat with an R better= 0.275, fit, indicatingwith an R2 that = 0.275, seed oilindicating content (%)that hasseed a maximumoil content value (%) has around a maximum 58 g of seed value mass, around decreasing 58 g of at bothseed sidesmass, ofdecreasing this point. at The both optimal sides two-segmentof this point. piecewise The optimal linear two regression-segment model, piecewise however, linear showed regression the 2 bestmodel, fit, however, with an R showed= 0.317 the and best the fit, lowest with MSE an R (2.72).2 = 0.317 and the lowest MSE (2.72).

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44 42

40 R2=0.951

36 34 R² = 0.1352 Oildcontent (%) 32

30 R2 = 0.317

28 R² = 0.275 26 30 35 40 45 50 55 60 65 70

Seed weight (g) Figure 5. Linear, quadratic, and two-segment piecewise linear regression models for oil content on Figure 5. Linear, quadratic, and two-segment piecewise linear regression models for oil content on seed weight of jatropha seed from three elite accessions. INIAP Portoviejo Research Station, Ecuador. seed weight of jatropha seed from three elite accessions. INIAP Portoviejo Research Station, Ecuador.

TableTable 3. Parameter 3. Parameter estimates estimates for for linear, linear, quadratic, quadratic, and and two-segmenttwo-segment piecewise piecewise linear linear regression regression equationsequations of oil of content oil content (y) (y) on on seed seed weight weight (x) (x) of of jatrophajatropha seed samples samples from from three three elite elite accessions. accessions. INIAP Portoviejo Research Station, Ecuador. INIAP Portoviejo Research Station, Ecuador. 2 Model Equation R CV (%) MSE p-Value b0 ± (se) b1 ± (se) b2 ± (se) 2 p ± ± ± ModelLinear Equationy = b0 + b1x R 0.135CV 8.695 (%) 3.012 MSE 0.02-Value 27.441 b0 (3.164)(se) 0.129 b(0.056)1 (se) n.a. b 2 (se) 2 LinearQuadratic y =y b 0= +b0 b +1 xb1x + b2x 0.1350.275 8.6958.082 2.8003.012 0.010.02 −8.218 27.441 (14.444) (3.164) 1.4990.129 (0.546) (0.056) −0.013 (0.005)n.a. § 2 QuadraticPiecewisey =y b =0 +b0b +1 xb1 +x b+2 bx2(x − t)(x0.2752) 0.317 8.0827.845 2.7182.800 0.0010.01 18.797−8.218 (4.082) (14.444) 0.3091.499 (0.079) (0.546) −0.617− (0.208)0.013 (0.005) Piecewise § y = b + b x + b (x − t)(x ) 0.317 7.845 2.718 0.001 18.797 (4.082) 0.309 (0.079) −0.617 (0.208) § For piecewise0 1 regression2 2model, t = 58.8, is the optimal break point and x2 is an indicator variable § For piecewise(x2 = 0 when regression x ≤ t; x2 model,= 1 when t = x 58.8, > t). is the optimal break point and x2 is an indicator variable (x2 = 0 when x ≤ t; x2 = 1 when x > t). The optimal piecewise linear regression model had a break point at 58.8 g of seed mass. Below Thethis optimalpoint, seed piecewise oil content linear is regression positively model related had to seed a break mass, point but at beyond 58.8 g of the seed break mass. point, Below the this point,relationship seed oil content becomes ispositively negative, so related seed oil to content seed mass, decreases but beyondas seed mass the breakincreases point, (Figure the 5). relationship becomes negative, so seed oil content decreases as seed mass increases (Figure5). 4. Discussion 4. Discussion 4.1. Jatropha Maturity and Productivity 4.1. JatrophaThe Maturity early stud andy undertaken Productivity by Jongschaap et al. [20] established that the maturity of jatropha as a crop depends on its geographical location, i.e., radiation and temperature levels, resulting in higher The early study undertaken by Jongschaap et al. [20] established that the maturity of jatropha as a net primary production potential in some in comparison to other latitudes. In general, these crop dependsplantations on its can geographical reach their maturity location, and i.e., radiation maximum and production temperature 3–4 yearslevels, after resulting planting. in higher This net primaryinformation production on jatropha potential stand in somematurity in comparisonis comparable to to other our results latitudes. in this In study. general, Under these dry plantations forest can reachconditions their in maturity the province and of maximum Manabí-Ec productionuador, seed production 3–4 years afterof jatropha planting. declined This after information four years on jatrophaforstand INIAP maturity jatropha accessions is comparable CP041 to and our CP052. results Nevertheless, in this study. INIAP Under jatropha dry forestaccession conditions CP054 in the provincereached maximum of Manab productioní-Ecuador, 1.4 seed years production after planting. of jatrophaSuch jatrophadeclined behaviour after was four observed years forin rain INIAP jatrophafedaccessions marginal lands CP041 of and Uttar CP052. Pradesch Nevertheless, (India), where INIAP the bestjatropha dry accessionseeds production CP054 reachedwas in the maximum first −1 productionyear, with 1.4 years a yield after between planting. 3.2 to Such4.1 t seedsjatropha ha behaviour [21]. was observed in rain fed marginal lands of These results do not agree with data provided by Euler and Gorriz [22], and Achten et al. [23], Uttar Pradesch (India), where the best dry seeds production was in the ﬁrst year, with a yield between who observed −that1 jatropha plantations show considerably lower dry seeds production at younger 3.2 toages 4.1 t due seeds to hathe inefficient[21]. interception of radiation, and therefore, the distribution of dry matter to Thesepermanent results biomass do not instead agree of with harvestable data provided parts such by as Euler fruits and seeds. Gorriz Our [22 results], and also Achten differ et from al. [23], who observed that jatropha plantations show considerably lower dry seeds production at younger ages due to the inefﬁcient interception of radiation, and therefore, the distribution of dry matter to permanent biomass instead of harvestable parts such as fruits and seeds. Our results also differ from the eight years required by jatropha to reach maturity in Kenya, as observed by GTZ [11]. Forests 2018, 9, 611 8 of 13

Jongschaap et al. [20] emphasized that a decrease in jatropha seed productivity has been reported for aging plantations. What is still unclear is whether or not this is a general phenomenon. The study conducted by Cañadas et al. [1] and Rade et al. [2] showed a decreasing production of seed for INIAP jatropha mono-plantation and a local jatropha material in live fences, Manabí, Ecuador. The seed production estimated in this study for jatropha is similar to that obtained for jatropha stands under 6 years of age in other regions of the world (Table4). Cañadas et al. [ 1] showed that, after having reached a maximum, jatropha production decreases over time. These results were accompanied by a high inter-annual variation in dry seed production, which was not related to the mean annual precipitation. Rade et al. [2] calculated a 7-year average of 0.24 kg tree−1 year−1 (±0.06) for jatropha INIAP CP041 accession in life fences, while the local jatropha varieties reached an average production of 0.18 kg tree−1 year−1 (±0.03) with comparatively little variation in dry seed production. Site-speciﬁc knowledge about the development of seed production of jatropha trees is fundamental for estimating their economic viability. Projections of seed production in the literature for more mature jatropha stands often lack sound scientiﬁc bases, or contain wrong assumptions [2].

Table 4. Published data for seed productivity of jatropha plantations from different countries.

Age of Jatropha Productivity Country, Region References Plantation in Years (in kg tree−1) Brazil 1 0.4 Saturnino et al. [24] Guatemala 1 0.8 Ouwens et al. [25] Indonesia 1 1.6–2.0 Ouwens et al. [25] Indonesia 1 1.8 Manurung [26] India, Pradesh 1 2.4 Lal et al. [21] India 1.25 1.7 Achten et al. [27] India 2 1.5 Ouwens et al. [25] Mali, Digini 2 0.3 Achten et al. [27] India 2.5 0.8 Ghosh et al. [28] India, Rajasthan 2.5 0.1–0.5 Achten et al. [27] Kenya 2–3 <1.0 Iiyama et al. [29] Tanzania, Arusha 3 0.5–2.0 Messemaker [30] Nicaragua 5 4.5 Heller [31] Ecuador, CP052, 041, 054 6 0.2–0.3 Cañadas et al. [1] Ecuador, CP041 7 0.2 Rade et al. [2] Ecuador, CP041, 052, 054 8 0.1–0.3 Present study

Jatropha stands in the present study had received minimal care in relation to plantation maintenance. This could signiﬁcantly affect the productivity potential and sustainability of the jatropha plantation. However, most small farmers do not have alternatives, and apply sub-optimal management practices [29], so the productivity shown in this research would be a real scenario for jatropha, when used in a pro-poor crop program.

4.2. Seed Dry Weight No statistical significance was detected in relation to seed weight of the three INIAP jatropha accessions investigated in the present study. This result contrasted with those reported by Ginwal et al. [32], who found statistical differences (p < 0.05) in relation to seed weight and seed size of diverse jatropha provenances of Central India. Similar results were obtained by Kaushik [33] in Andhra Pradesh, India. In the present study, however, a high statistical difference in seed weight was found across the various fruit ripening stages. This corresponds with the results obtained by Kaushik [33]. Rondanini et al. [34] emphasized the need to determine how temperature during seed filling could affect several characteristics of jatropha fruit. Not only the fruit ripening stage influences seed weight; the environmental conditions throughout the various fruit ripening stages also do Forests 2018, 9, 611 9 of 13 so, and for this reason, a broad variation in seed weight (from 32.6 g to 75.2 g) was found in Argentina [13]. Seed weights found in our study are similar to those obtained in the province of Andhra Pradesh, India by Rao et al. [16] in accessions CRDJ1 (77.4 g), CRDJ20 (78.3 g) and CRDJ6 (79.1 g). While Subramanyam et al. [35] reported a seed weight between 49.3 g and 74.2 g and Rathbauer et al. [36] between 49.3 g and 74.2 g and this broad variation was attributed to the diverse agro-ecological growth conditions and genetics. Wani et al. [37] found that seed weight for 100 seeds in Indian jatropha accessions varied between 44 g and 77 g. In Ecuador, Cañadas et al. [1] recorded an average weight of 72.6 g ± 0.77 g for 100 seeds in seven jatropha elite accessions of INIAP in Portoviejo-Manabí.

4.3. Seed Moisture Content According to Silva et al. [14] seed moisture content is not a good indicator of physiological ripening, since this parameter can vary among genotypes. In fact, seed moisture content varied widely among the jatropha accessions tested, from 25.8% in INIAP CP054 to 37.2% in INIAP CP052. Statistical differences were also identiﬁed in seed moisture contents among the fruit ripening stage, which was negatively and signiﬁcantly correlated. A clear gradient of moisture content as ripening advanced was established. A similar seed moisture content decline was described by Dias et al. [38] and Dias et al. [39]. The rapid humidity reduction is proportional to the reserve deposit compounds, which to a large extent replace the space occupied by water. Finally, water reduction occurs by the search for hygroscopic equilibrium between the seed water content and the environmental relative humidity [40]. The moisture content varies in relation to the maturation and ripening of the jatropha fruit with high water content in the physiological maturity stage and low moisture content in the senescent stage [40].

4.4. Oil Content The best jatropha INIAP accessions were collected from Manabí province (Ecuador) for further testing [1]. Jatropha local accessions had adapted to a wide range of edaphic and ecological conditions, suggesting that a considerable amount of genetic variability exists to be exploited for potential oil production. Osorio et al. [41] found a higher genetic variability of jatropha in Central American accessions compared to Asian, African, and South American accessions. Nevertheless, a variation of oil seed concentration was reported by Rao et al. [16] ranging between 29.8%–37.1%, by Subramanyam et al. [35] between 17.1%–38.8%, and by Kaushik et al. [42] 28.0%–38.8% from different genotypes evaluated under the same environmental conditions. Among INIAP’s jatropha accessions, no statistical differences were found in seed oil content; mean values varied by only 3% among the evaluated accessions. The small variation in jatropha oil content found in the present investigation is not sufficient as a viable selection option at a very early stage (germplasm collection) form seed base material. Singer et al. [43] mentioned that seed oil concentration does not only depend on the genotype, but is also affected by the environmental conditions during grain filling, i.e., mainly temperature, that modifies seed oil concentration and the fatty acid composition through changes in grain filling dynamics and biosynthetic activity. Jatropha oil is accumulated during the last third of the seed filling period [44], so a shortening of grain filling duration should lead to lower oil concentrations. Furthermore, changes in seed weight generated by different temperatures can affect seed oil concentration through changes in the seed coat-kernel relationship. Such seed weight variation was detected, but only through the fruit ripening stage. Fruit color has been associated as a visual indicator of the fruit ripening stage. In the current research, no statistical differences were found in oil content between fruit ripening stages. This fact has a practical application. The difference in price between a quintal (45 kg) jatropha fruit for US $10 and jatropha seed for US $12, as paid in Manabí province, is currently not a sufficient incentive to invest more labor which is necessary for the sale of seeds, because this would increase production Forests 2018, 9, 611 10 of 13 costs excessively [2]. The harvest timing of small producers within the project “Jatropha for Galápagos” would be the yellow fruit ripening stage, due to the greater weight. The oil content fluctuated from 33.3% in the dry fruits (fruit ripening stage 4) to 36% in yellow fruits (fruit ripening stage 2). Nevertheless, Silva et al. [14] had associated the jatropha fruit color with the oil content and germination power of seeds. The oil contents from jatropha INIAP accessions is comparable with the values reported by Wassner et al. [13] in Argentina, under subtropical conditions and precipitation of 1005 mm year−1, with a maximum seed oil content of 38.7% ± 0.6% and a minimum of 19.6% ± 1.8%. Wani et al. [37] for Jammu and Kashmir State in Northwest India obtained an oil concentration from 27.8% to 37.9%. Kaushik et al. [41] found an oil content range from 28% to 39% in the Haryana-India accession. Rao et al. [16] established between 30% to 37% of oil contained in Andhra Pradesh, India accessions. Other referential data on oil content is reported by Srivastava et al. [45], who observed values between 17.1% for accession IC5660864 to 34.5% for the accession IC558231 in New Delhi and China, and between 34.1% for accession S1 and 55.5% for accession S2 [46]. Higher oil content (61%–64%) was found in jatropha seeds coming from Malaysia, Indonesia, and Thailand [47]. In the Latin-American context, the jatropha oil content reported in Colombia with jatropha variety Brazil tested in two areas, varied just slightly from 37% in Vichada to 38% in Santander [48].

5. Conclusions Our study revealed that there are no differences in oil content among the three compared jatropha accessions, nor do oil content changes occur during the fruit ripening process. This means that, for oil content in the Ecuadorian jatropha accessions, the genotype and collection date of fruits under tropical dry forest conditions are not relevant. Under subtropical conditions, the harvest timing is as relevant as the jatropha genotypic selection [13]. Studies conducted by Rao et al. [16] and Karaj and Müller [49] pointed out that the significant statistical correlation between seed weight and oil content can be considered as an important trait for early selection of seed sources. A bilinear relationship for oil content and seed weight was observed in the present study, even though the coefficient of determination was not high. Better adjustment of the bilinear relationship between these variables was reported by Wassner et al. [13], where the harvest time was at the mottled-yellow color stage, characteristic of ripe fruit. In both studies the bilinear relationship showed similar trends, but the break point, where the linear relationship between oil content and seed weight changes in slope, was 58.8 g in our study, while in the subtropical zones of Argentina, the break point was at 62.5 g [13]. Additionally, the particular relationship between seed weight and oil content limits the usefulness of a rapid selection for genotypes with high oil concentration based on seed weight. Finally, it is safe to state that site-specific data on jatropha is necessary when aiming at promoting it, since reported variation is high. Basing management plans on literature data bears the risk of overestimating potential returns to farmers. Some authors reported rather high yields, which might be due to studies being undertaken under “optimal” conditions, which are different from usual farm management, as simulated in our study, where caring for jatropha trees is just one duty among many others. Our results provide important building blocks for a comprehensive cost-benefit analysis essential for realistic planning of jatropha plantations at farm, regional energy, and national levels.

Author Contributions: Á.C.-L., D.Y.R.-L. and M.I.-V. conceived, collected de data, designed the experiments and wrote the first draft; J.V.-H. and M.S.-S. expanded the statistical analyses; M.S.-S., J.M.D.-A. and C.W. carried out a detailed revision of the paper. Funding: This research received no external funding. Acknowledgments: We are thankful to the Director of the Portoviejo Experimental Research Station (EEP) of the National Institute of Agricultural Research (INIAP) period 2015–2016 and INIAP General Director for the jatropha data release and the updating of the information. Thanks to two anonymous reviewers for their very valuable comments. Conflicts of Interest: The authors declare no conflict of interest. Forests 2018, 9, 611 11 of 13

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