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Home , Cassava, Jatropha curcas

Università degli Studi di Padova Facoltà di Agraria Dipartimento di Biotecnologie Agrarie

Laurea Magistrale in Scienze e Tecnologie Agrarie

Jatropha curcas L., a potential . On field research in .

Relatore: Prof. Mario Malagoli, Università degli Studi di Padova Correlatore: Dr. ir. Raymond Jongschaap, Wageningen University and Research centre, Research International

Laureando: Berardo da Schio Matricola n° 588712

Anno Accademico 2009/2010 If you use information from this M.Sc. dissertation document, please cite and refer to: da Schio, B., 2010. curcas L., a potential bioenergy crop. On field research in Belize. M.Sc. dissertation. Padua University, Italy and Wageningen University and Research centre, Plant Research International, the Netherlands. “Sub umbra floreo”.

National motto of Belize.

Table of Contents

List of Abbreviations...... 7 Abstract...... 11 1.Introduction...... 13 1.1 Energy crisis and global climate...... 13 1.2 L., a potential bioenergy crop...... 18 1.2.1 Knowledge gaps in Jatropha curcas L. research...... 28 1.2.2 Selection of knowledge gaps and justification...... 31 1.3 Aim and objectives of the thesis...... 33 2.Materials and Methods...... 35 2.1 Research at PRI Wageningen, the Netherlands...... 35 2.2 On field research in Belize, ...... 36 2.2.1 Climate data...... 39 2.2.2 Maya Ranch trial...... 40 2.2.3 Warrie Head trial...... 44 2.2.4 Central Farm trial...... 45 2.3 Statistical analysis...... 49 3.Results and discussions...... 51 3.1 Important drivers for Jatropha curcas L. growth and development and how are these for Belize...... 51 3.1.1 Radiation and light interception...... 51 3.1.2 Temperature...... 52 3.1.3 Water...... 53 3.1.4 Vapour pressure and wind speed...... 53 3.2 Response of genetically different accessions to available resources in Belize.....55 3.2.1 dimension and weight...... 55 3.2.2 Seed rate...... 58 3.2.3 development at nursery stage: LA, fresh weight, taproot length....60 Table of Contents

3.3 Resources use efficiency and optimization for jatropha crop conditions in Belize...... 63 3.3.1 Plant density...... 63 3.3.2 Plant spacing...... 64 3.3.3 Crop management...... 71 3.4 Discussions...... 80 4.Conclusions...... 85 5.Acknowledgements...... 89 Annex 1. Growth parameters and sustainability indicators tables...... 91 Annex 2. Experimental designs...... 99 References...... 107

6 List of Abbreviations AET...... Actual Evapo-Transpiration aft. …...... after (in Annex 1) agr. …...... agricultural (in Annex 1) BNMS...... Belize National Meteorological Service BT...... Bullet CBD...... Convention on Biological Diversity CDM...... Clean Development Mechanism CF...... Central Farm COCyTECH.. Consejo de Ciencia y Tecnología comm. …...... communication DM...... Dry matter DMA...... Dry matter assignment EEP...... Energy and Environment Partnership EM...... Effective Micro-organisms ERA-ARD..... Agricultural Research for Development; Dimension of the European Research Area EU...... European Union FACT...... Fuel on Agricultural Common Technology FAO...... Food and Organization of the GHG...... Greenhouse gases GUARD...... Galen University – Applied Research and Development for Sustainability Institute HI...... Harvest index IEA...... International Energy Agency IPCC...... International Panel on Climate Change JC...... Jatropha curcas L. (in Annex 1) LA...... Area LAI...... Leaf Area Index LCA...... Life Cycle Analysis MR...... Maya Ranch NBS...... Nucleotide Binding Site NGO...... Non Governmental Organization List of Abbreviations

OAS...... Organization of the American States OM...... Organic Matter PET...... Potential Evapo-Transpiration pm...... parameter (in Annex 1) PRI...... Plant Research International PS...... Production system (in Annex 1) T,M,B...... Top, Middle, Bottom branches (in Annex 1) TSDF...... Tropical Studies and Development Foundation UB...... University of Belize UNEP...... United Nations Environment Programme UNDP...... United Nations Development Programme UNFCCC...... United Nations Framework Convention on Climate Change USD...... United States Dollar WH...... Warrie Head WUR...... Wageningen University and Research centre

8 Ai miei genitori,

e ai miei nonni.

Abstract

Potentials of bioenergy crop Jatropha curcas L. are investigated through field trials in Cayo District, Belize, Central America. Crop growth and development are monitored in two jatropha plantations of one and six years, respectively in Warrie Head and Maya Ranch. A third plantation is set up in Central Farm, in the framework of the '1 st Coordinated Call for a Transnational Research Activity under the ERA-ARD Net: Bioenergy – an opportunity or threat for the rural poor', in the project 'Bioenergy in and Central America – Opportunities and Risks of Jatropha and Related '.

Biomass development is assessed through measurements of dry yield, LAI development, length of effective branch, seed dimensions and weight, fruit to seed ratio and seed to kernel ratio and seed germination test in relation to different crop variables, according to the different trials: management, such as pruning and cropping system (monoculture, and living fence), genotype, plant spacing and plant density. Outputs on biomass development are linked with weather variables data, kindly provided by the National Meteorological and Hydrological service of Belize.

Results indicate that Jatropha curcas L. has promising potentials to play a decisive role in bioenergy scenario in Belize and the Region, for the favourable pedoclimatic situation and the availability of genetic resources.

Key words: Jatropha curcas L. – – LAI development – Belize – .

Riassunto Il potenziale della coltura energetica Jatropha curcas L. viene investigato in prove di campo nel Distretto di Cayo, Belize, America Centrale. La crescita e lo sviluppo della coltura vengono monitorati in due piantagioni di uno e sei anni, rispettivamente a Warrie Head e a Maya Ranch. Una terza piantagione viene messa a dimora a Central Farm, nel contesto della '1 st Coordinated Call for a Transnational Research Activity under the ERA-ARD Net: Bioenergy – an opportunity or threat for the rural poor', nel progetto 'Bioenergy in Africa and Central America – Opportunities and Risks of Abstract

Jatropha and Related Crops'.

Lo sviluppo della biomassa è stimato attraverso misurazioni di: produzione di frutta, sviluppo del LAI, lunghezza effettiva dei rami, dimensioni e peso dei semi, rapporto in peso di frutto e seme, prove di germinazione, in relazione a variabili differenti, secondo le diverse prove: gestione colturale, come potatura e sistema colturale (monocoltura, consociazione e recinto verde) genotipo, spaziatura e densità. Lo sviluppo della biomassa viene confrontato in relazione ai dati sulle variabili climatiche, gentilmente messi a disposizione del servizio Meteorologico ed Idrologico Nazionale del Belize.

I risultati indicano che Jatropha curcas L. mostra promettenti potenziali nello scenario bioenergetico del Belize e della Regione, in ragione della situazione pedoclimatica favorevole e della disponibilità di risorse genetiche.

Parole chiave: Jatropha curcas L. – Biocombustibili – indice di area fogliare LAI – Belize – Agricoltura tropicale.

12 1. Introduction

Energy availability and energy use are issues of global concern and have been under research worldwide for a long time. Attention is mostly given to reduce energy consumption and to detect new and resources, in order to cope with the world energy crisis. Two main processes are responsible for this situation: first, the increasing population and development rates are rapidly multiplying the global energy demand in many countries; secondly, the great share that traditional fossil fuels occupy on the global energy consumption can hardly be sustained. In fact, the ongoing fossil fuels depletion has reached a stage where the current reserves seem not enough for future needs. Furthermore the role of fossil fuels on climate change and global warming can no longer be neglected (IPCC, 2007).

1.1 Energy crisis and global climate

High prices, the risks of fossil fuel dependence and the increasing greenhouse gas (GHG) emissions derived from this kind of fuels are the main reasons to find new and renewable energy sources for the coming years (FAO, 2008). In fact the current energy crisis is mainly due to the high dependence on fossil fuels. It is now evident that oil consumption is drastically increasing, while the reserves are rapidly diminishing (Dowlatabadi, 2006), so it would be more relevant to talk about oil crisis than energy crisis. Current global trends in energy supply and consumption are patently unsustainable: environmentally, economically and socially. That indicates a global need to secure the supply of reliable and affordable energy and to effect a rapid transformation to a low-carbon efficient and environmentally benign system of energy supply. It is also clear that current energy fossil fuel consumption trend will have severe consequences on natural ecosystems and social communities. Switching to renewable energy will therefore reduce global warming and curb actual trends (IEA, 2006). These are the reasons why a search for alternatives to fossil fuels, such as renewable energy from solar, wind, water, biomass and nuclear, has been provoked.

All renewable energy options have pros and cons and a global analysis should be undertaken for each one of them. Various aspects of renewable energies will be 1.Introduction discussed, but nuclear energy will not be taken into consideration.

The matter of renewable energy involves different forms and sources of energy. According to the Unified Bio-Energy Terminology definition (FAO, 2004), renewable energy consists of energy produced and/or derived from sources infinitely renovated (hydro, solar, wind) or generated by renewable combustibles (sustainably produced biomass). Renewable energy offers an interesting option to reduce fossil fuels dependence and to reach climate change mitigation and if well consciously and sustainably managed, it may help to preserve biodiversity, water and reserves, as well as human livelihoods.

Due to its importance, international actors have expressed themselves on the subject. The United Nations Framework Convention on Climate Change (UNFCCC, 1992) supports bioenergy as one of the “precautionary measures to anticipate, prevent or minimize the causes of climate change”. The 1997 Kyoto Protocol to the UNFCCC recognizes the importance of renewable energy as a contributor to mitigating climate change, with a view to assisting developing countries in achieving sustainable development and enabling industrialized countries to comply with their quantitative emission targets thanks to the Clean Development Mechanism. The Convention on Biological Diversity (UNEP, 1992) is relevant to sustainable bioenergy development as it commits parties to biodiversity conservation, the sustainable use of its components and the fair and equitable sharing of benefits arising from the use of genetic resources.

Many different solutions to face worldwide changes on climate and on energy demand are available. Alternatives to fossil fuels seem in fact numerous. The appropriate solution to alleviate fossil fuel crisis and related climatic problems may vary according to the local situation and taking into account social, economic and environmental spheres. An interesting option that should be seriously taken into account in relation to the local factors is represented by biofuels produced directly or indirectly from biomass. They have been subject of many claims and researches during the past decades, and they are now classified by the FAO as “First-generation” and “Second-generation” biofuels. First generation biofuels are the ones mainly derived from food-crops,

14 1.Introduction including - and -based bioethanol and oilseed based ; while Second generation biofuels are the ones derived from non-food crop agricultural and forestry products, making use of the lignin, cellulose and hemicellulose components of (FAO, 2008). Both first and second generation biofuels present good opportunities but also have negative sides. In fact, on the one hand they are a remarkable option to combat climate change and to reduce fossil fuel dependency, while on the other hand they can directly or indirectly affect and do not play a relevant role in GHG emissions mitigation. It is important to bear in mind that biofuels often compete for land, water and resources and they can provoke an increase of food price. However, in an optimized setting, (local) energy supply may contribute to productivity increase and to the prosperity of livelihoods. A way to check the effectiveness of climate change mitigation through production and use should be found, which means identifying tools that allow comparing energy and GHG emission balances (inputs and outputs) and indicating if a better action is effectively accomplished. Ultimately, the impact of biofuels on livelihood should not be disregarded.

To date, energy and GHG emission balances are of high concern as researches on this issue provoke great discussions. To loose this worry, life cycle assessments (LCA) are used as tools that take into account different parameters and variables to define the best improvement on an environmental scale: impacts are evaluated after different decisions and management actions over the whole life cycle of a specific product. Indeed, the benefits coming from renewable energies' strategy and management implementation are linked to many variables, as the good opportunity of favourably impacting the environment and the farmers' livelihoods. With regard to all these concerns, a further broad question is attracting people’s attention and animating the worldwide debate on energy: the struggle between local and global energy. In fact, energy consumption is an issue present everywhere the humanity stands, and people are arguing on who is the actor, on how and why the energy (or oil) production and distribution are controlled. Globally, there is an increasing energy demand and a global concern on occurring climate change, as well as a worldwide spread awareness of the necessity of finding

15 1.Introduction alternatives. With regard to that, an emergent question is showing up: what consequence would bring the development of a global energy based community and what if it is local energy based. According to the previous discussion, it is important to remember that global energy production can involve high energy losses due to the transportation itself and risks of energy market monopoly or oligopoly. Local energy options seem to be socially sustainable and economically viable, if proper infrastructures and services are provided. Local energy development starts at a local scale, identifying the potential alternatives to put into effect: in this regard, biofuels from agricultural crops often represent a valuable opportunity. In relation to the local environmental and social situations and to the pedoclimatic characteristics, many could be the suitable crops: sugar crops (sugar , , sweet sorghum), starchy crops (, , , , potatoes, ) and cellulosic material (switchgrass, Mischantus , willow, poplar, crop ) for the production of ; oil crops (, oil palm, , sunflower, peanut, Jatropha ) for the production of biodiesel; biomass coming from different crops but also from agro-industrial by-products and municipal wastes for the production of (FAO, 2008).

However, despite some possible negative impacts, many are the potential benefits of bioenergy development. Indeed, it is clear the strong call for implementation strategies that will act on the development of this sector. Often, well balanced policies and accurate actions can strongly operate to maximize positive effects and reduce the negative ones. As positive effects there are: diversification of agricultural output, stimulation of rural development and contribution to poverty reduction, increase in food prices and higher income for farmers, development of infrastructure and employment in rural areas, lower greenhouse gas emissions, increased investment in land rehabilitation, new revenues generated from the use of wood and agricultural residues, and from carbon credits, reduction in energy dependence and diversification of domestic energy supply, especially in rural areas, access to affordable and clean energy for small and medium-sized rural enterprises. On the other hand, potential negative effects are addressed to as reduced local food availability if plantations replace

16 1.Introduction subsistence farmland and increased food prices for consumers. Demand for land for energy crops may increase deforestation, reduce biodiversity and increase GHG emissions. Increased number of pollutants, modification to requirements for vehicles and fuel infrastructure, higher fuel production costs, increased wood removals leading to degradation of forest ecosystems, displacement of small farmers and concentration of land tenure and incomes, reduced soil quality and fertility from intensive cultivation of bioenergy crops, distortion of subsidies on other sectors and creation of inequities across countries represent some other alarming impacts (FAO, 2008).

A strong focus on this issue is necessary and more importance should be given to the problems of biofuels’ production, by looking at them from an agricultural point of view. To what concerns the fuels coming from food crops, the main problems seem to be the competition for their destination as human food or animal feedstock and from the competition for agricultural land resources, water, energy and . The fear coming from the cultivation of food crops for energy production is also due to the mismanagement of these crops. In fact, while climate change mitigation can be partly achieved (if it is truly proved for some produce, for others should be cautiously explored), mismanagement can lead to a loss of biodiversity, especially in the case of repeated monoculture over the years, and in a reduced efficiency of water and soil resources that are of vital importance. That is especially the case at the moment in non- industrialized countries, where the demand for large area to place bioenergy plantations is increasing (Wood, 2005; von Braun and Meinzen-Dick, 2009; Daey Ouwens et al. , 2007). The other option, represented by the use of “second-generation” biofuels, seems to be of difficult and slow spreading in most countries at the present day, due to the lack in technology knowledge and resources for processing the lignin component that is still under development (FAO, 2008). What should not be forgotten is that second generation biofuels might not affect the availability of food but they do compete for land, water, nutrients and energy.

Thence, biofuels represent a valuable solution to mitigate global warming and to reduce oil dependence. Nevertheless, significant objections are still heard against their use, as

17 1.Introduction their consequences on communities could be positive as well as negative. Major concerns lie in the selection of the fuel crop and its management in the cultivation and processing steps: solutions may vary according to the regional context. Moreover, the use of some fuel crops is controversial, as they have been proven to be not enough efficient or useful. For some others, more research is required and their use as an energy feedstock will heavily depend on how this research is carried out and how the results are presented. Among these last mentioned, a relatively new energy crop is Jatropha curcas L. (Figures 1.1 and 1.2).

Figure 1.1. Jatropha curcas L. plantation at Figure 1.2. Jatropha curcas L. , in Maya fruiting stage, in Maya Ranch, Belize, July Ranch, Belize, July 22 nd , 2009. 22 nd , 2009.

1.2 Jatropha curcas L., a potential bioenergy crop

Jatropha curcas L. is a deciduous monoecious perennial or small tree belonging to the botanical family of , to the tribe Jatropheae of the subfamily Crotonoideae. Common name varies according to the region: in English it is called 'physic nut', while in Italian it is known as 'ricino d'inferno'. J. curcas probably originated in southern or neighbouring parts of Central America, which are the only areas where it has often been collected from undisturbed vegetations. It was then distributed all over the world by Portuguese seafarers in the XVII century and is now

18 1.Introduction naturalized throughout the and (Figure 1.3). Different parts of J. curcas are used for a range of medicinal purposes; moreover it is a source of oil used for production and as a source of energy, as mentioned before; it is also an important hedge plant (Baldrati, 1950; Henning, 2007). Hereunder, a review of jatropha plants, fruits, and (Figures 1.4, 1.5, 1.6 and 1.7).

Figure 1.3. 'Global indication of the most suitable climate conditions for the growth of Jatropha ( J. curcas L.) (30°N, 35°S) and Oil palm ( Elaeis guineensis Jacq.) (4°N, 8°S)'. Source: Claims and Facts on Jatropha curcas L., Jongschaap et al. , 2007.

Figure 1.4. Row of six years old Jatropha Figure 1.5. Jatropha curcas L. dry fruit coats curcas L. plants pruned at four years, in and seeds. In the background, Arachis Maya Ranch, Belize, July 22 nd , 2009. pintoii growing as intercrop with jatropha, in Maya Ranch, Belize, July 22 nd , 2009.

19 1.Introduction

Figure 1.6. One year old Jatropha curcas L. Figure 1.7. Six years old fruiting Jatropha plant, in Warrie Head, Belize, July 22 nd , curcas L. plants, in Maya Ranch, Belize, July 2009. 22 nd , 2009.

The plant ecophysiology and the botanical features have been investigated. The plant develops a deep taproot and initially four shallow lateral (Figure 1.8). The stem, arising from a thick, perennial rootstock, with watery to whitish latex, has a bark smooth, grey or reddish, shiny, peeling off in papery scales. Leaves are alternate, simple, petioled and glabrous, with a blade broadly ovate in outline, usually shallowly 5-lobed and margins usually entire. Terminal contain unisexual . The fruit is a broadly ellipsoid capsule, smooth-skinned containing three ellipsoid seeds, 1-2 cm long, mottled black and coarsely pitted. Growth in J. curcas is intermittent and sympodial, dormancy is induced by fluctuations in rainfall, temperature and light but not all plants respond simultaneously. Pollination seems to be carried out by honeybees and beetles (Bhattacharaya et al. , 2005) and moths (Henning, 2007). In flowering, the female flowers open one or two days before the males one; male flowers last only one day. Seed never sets in indoor cultivation unless the flowers are pollinated by hand. J. curcas occurs in semi-arid tropical and warm subtropical climates with mean pedoclimatic surviving requirements as the followings: daily temperatures of 20-30°C, annual rainfall of 300-600mm (but resistant to periods of of up to seven months), absence of frost (Figure 1.9).

20 1.Introduction

Figure 1.8. Particular of Jatropha curcas L. Figure 1.9. Six years old Jatropha curcas L. system. Central taproot and four lateral plantation after an extraordinary dry month, roots are evident, in Belmopan, Belize, in Maya Ranch, October 28 th , 2009. November 27 th , 2009.

The main inputs for the production of oil-bearing fruits of J. curcas are land area including the prevalent site characteristics, plantation establishment practices and plantation management practices. The outputs are the seeds and other biomass elements (Achten et al. , 2008). J. curcas can grow in a wide range of : on degraded, sandy or gravelly and even saline soil with low nutrient content. Nevertheless, clay soils are unsuitable for the plant if water logging or saturation occurs due to the climatic conditions. It is clear that J. curcas responds highly when growing on well aerated soils. Sandy to loamy soils seem to be a best fit. Optimal pH reaction is considered between 6 and 8.5. The plant is well adapted to marginal soils with low nutrient content but in order to support a high biomass production the crop shows a high demand for nitrogen and fertilization (Henning, 2007; Daey Ouwens et al. , 2007). Among soil proprieties, pH, EC, CaCO 3, organic C and clay significantly affect the availability of nutrients, thus soil conditions reflect the effect of jatropha cultivation practices on a degraded soil. From the perspective of both soil structure and carbon and nitrogen sequestration, jatropha cultivation under minimal soil disturbance can serve ‘environmental functions’. In fact, jatropha cultivation improves soil resistance to wind and enhanced macro-aggregate stability to water erosion. Under jatropha, increased potential carbon sequestration rates are possible as stable micro-aggregates

21 1.Introduction can offer protection to organic carbon. Therefore jatropha cultivation programmes will not only serve as a source of income-generation to resource-poor farmers but will also improve the quality of their soils in the long run (Ogunwole et al. , 2008).

Propagation is done by seeds or cuttings. Plants raised from seed are more resistant to drought than those raised from cuttings, because of the taproot they develop. The development of root system is then different, according to the originating part of the plant (Achten et al. , 2007). The taproot enables a straighter and deeper root system growth so to extract moisture from deeper layers of the soil. This root structure is also preferable in intercropping systems to minimize the competition for water and nutrients between the different crops. Thereafter, nursery-grown seedlings have a higher survival rate than direct-seeded ones and produce seeds earlier (Figure 1.10). Seeds in nursery or direct seeding with seed treatment is recommended (Daey Ouwens et al. , 2007). Seed soaking in cold water for 24 hours is suggested for better and quick germination (Kaushik et al. , 2007), although it might influence more the germination celerity than its rate (Sengfelder, personal comm.). At the onset of the rains the seedlings can be planted in the field (Heller, 1996). It was noted that spacing of plants is a trade off between biomass and fruit production. Thus, optimum spacing is differently achieved depending on weather situation, site characteristic and intended objective (Achten et al. , 2008).

Irrigation will depend on the climatic conditions of the location. Although J. curcas can survive precipitation as low as 300mm by shedding its leaves, it does not produce well under such conditions. Minimum and optimal rainfalls to produce fruits are assessed on values of 600mm ha -1 y-1 and 1000-1500mm ha -1 y -1 . Water and rains after periods of drought will induce blossoming. Hence, too much rain and humidity will provoke fungus, thus high rainfall might require other spacing (Daey Ouwens et al. , 2007). Indeed, an economic sustainable oil production is achieved with higher minimum requirements of water of at least 750 mm annual rainfall or supplementary (Henning, 2007). Plantations aiming at oil production might also need artificial or organic fertilization. Fertilizers at least compensate the nutrient removal due to harvest or management practice (e.g. pruning). Simultaneous reclamation of barren lands and

22 1.Introduction will inevitably imply use of fertilizer and irrigation (Achten et al. , 2008).

Pruning is a very important issue as it determines to a large extent, although not completely, seed yield in each site and it can facilitate manual and mechanical harvesting of fruits (Figure 1.11). Canopy size determines the maximum number of flowering branches. Large on a low planting density or smaller plants on high densities can apparently both result in sufficient flowering branches (Daey Ouwens, 2007). The pruning should be done when the tree sheds leaves and enters a period of dormancy (Kaushik et al. , 2007), that is usually coinciding with the dry season. Cultural practices in new plantations, thence, include regular weeding, pruning and fertilization. Standard management count on a plant density field design of 1350-2500 plants ha -1 (Henning, 2007).

Figure 1.10. Jatropha curcas L. seedlings a week after sowing, in Bullet Tree, Belize, Figure 1.11. Six years old Jatropha curcas L. September 30 th , 2009. plant pruned two years before, in Maya Ranch, Belize, July 22 nd , 2009.

Diseases and pests attacks should not be underestimated. Opinions on this issue are contrasting. In fact, Henning (2007) says that intervention against pests and diseases occurs rarely and just in the case of powdery mildew ( Uncinula necator ), Alternaria spp., and caterpillars of Spodoptera litura and several species of phytophagous beetles, and particular attention must be put on intercrops grown together with J. curcas , as it

23 1.Introduction can be an alternative host (e.g. ). While Daey Ouwens et al. (2007) say that the plant is vulnerable to most common pests and diseases found in food crops, adding that most of these pests and diseases can be treated fairly easily and, if required, biologically.

Basic agricultural operations are done manually, and so harvesting and separation of seeds from fruits. Handling after harvesting foresees a careful exsiccation in the shade until 6-9% moisture content. Subsequently the extraction of the oil can be done following different techniques (Henning, 2007). The reported yields range from extremely low to high; Jongschaap et al. (2007) conclude to a potential yield range of 1.5-7.8 dry seed ha -1 y -1 . As mentioned above, yield depends on site characteristics (temperature, radiation, rainfall, soil type and soil fertility), (a strict selection of seeds or cuttings leads to more uniformity in offspring and higher yields per plant), plant age and management (propagation method, spacing, pruning, fertilizing, irrigation, etc.) (Daey Ouwens et al. , 2007; Achten et al. , 2008).

The oil contained in the seeds, around 35% by weight (Baldrati, 1950; Kandpal and Madan, 1995; Ginwal et al. , 2004; Jongschaap et al. , 2007), has to be expelled or extracted. For extraction of the jatropha oil two main methods have been identified: mechanical extraction (with manual ram press or engine driven screw press) and chemical extraction (aqueous enzymatic or solvent oil extraction). Finally the oil may be refined in a continuous reactor to produce biofuel or diesel oil and as a valuable by-product. The oil quality is dependent on the interaction of environment and genetics (Jongschaap and van Loo, 2009; Achten et al. , 2008). Thus, the cake attributes change in relation to the oil characteristics and the oil extraction method used. Anyway, the cake contains high-quality and various toxins. The presence of biodegradable toxins makes the fertilizing cake simultaneously serving as biopesticide/insecticide and molluscicide. Anyway, it is advisable to check the absence of in the crops grown on jatropha cake fertilized land, certainly in crops for human consumption. Digesting the cake and bringing the effluent back to the field is thought to be the best practice at present from an environmental point of view. Due to

24 1.Introduction the toxicity of the seeds and oils, some attention should be paid to the human health and work environment impact categories (Achten et al. , 2008).

Available genetic resources show several types of J. curcas . Low-toxic type from Mexico, type with larger leaves and larger but fewer fruits and seeds from , male sterile type which produce more fruits than normal types, just to quote some examples. A study on 200 J. curcas accessions from different regions around the world highlighted, among other characteristics, the differences in oil composition that could be regionally identified (Jongschaap and van Loo, 2009). In order to start breeding the genetic variation needs to be assessed. Most plant material used so far is derived from simple selection within semi-wild populations or landraces. Between-plant variation of vigour and seed yield are tremendous and great genetic improvement in seed yields and other important characteristics may, therefore, be expected from systematic breeding (Figure 1.12). Obviously, oil yield per hectare will dominate breeding objectives for J. curcas for biofuel production. Cultivars with compact growth would facilitate harvesting (Henning, 2007). Literature reports lack of genetic variation. To date, it is assessed a high phenotypic variation (e.g. plant architecture) in material from (Figure 1.13). Genetic variability was found low in African and Indian J. curcas accessions but high in Guatemalan and other Latin American ones. Diversity in J. curcas should be found in wild species, in their centre of origin in Mexico, Central and (Jongschaap and van Loo, 2009; Montes et al. , 2008). programmes should be carried out after a more through analysis of the existing genetic resources and variability, that would allow getting the characteristics that would be required. Among the most important and urgent features to be investigated, there are: toxicity of seeds, oil-seed and seedcake (source of that could be suitable for animal feedstock); drought resistance and water requirements under different pedoclimatic situations; plant susceptibility to pests and diseases.

25 1.Introduction

Figure 1.12. Seed coats and kernels of three Figure 1.13. Particular of stem and lateral accessions of Jatropha curcas L., in branches of a year old Jatropha curcas L. Belmopan, Belize, November 2 nd , 2009. plant, in Warrie Head, July 22 nd , 2009.

Concerning environmental impacts of J. curcas production system, the main issues are the energy balance, impact on global warming potential and land use impact. Energy balance of J. curcas biodiesel is reported to be positive and the total energy inputs into the crop to the energy output ratio is estimated at 1:4-5 (Henning, 2007). The available information shows that energy balance improvement options lay in the cultivation, where irrigation and fertilization are the most energy intensive practices, and transesterification steps. How positive the balance is in reality, will mainly depend on how efficiently the by-products of the system are used. Impact on global warming potential showed a reduction of GHG emissions for the production of biodiesel from J. curcas in comparison to fossil fuels. However, the removal of (semi-) natural forest for the introduction of J. curcas is expected to have a significant negative effect on the GHG balance of the whole life cycle. Ultimately, it is expected that land occupation impact of J. curcas on the soil will be positive, as the plant is observed to improve soil structure, is strongly believed to control and prevent soil erosion and sequestrates carbon. Nonetheless, being an exotic species in most actual growing areas, the impact of land use change towards J. curcas on biodiversity is expected to be negative, although this will largely depend on the mix of land use which is replaced by J. curcas and on how the plant is cultivated (Achten et al. , 2008).

J. curcas ’ considerable potential as an oil crop for biofuel purposes at relatively low

26 1.Introduction costs and modest demands on the local agro-ecosystem has received much attention in recent years. It is foreseen that within the next decade or so, J. curcas will become a major source of renewable energy in the drier rural areas of (sub) tropical Asia, Africa and America (Henning, 2007). The promise of J. curcas as a species to produce high quantity and quality feedstock for bioenergy is considerable. First, yields are expected to increase over the years as seed improvement takes effect; they are expected to reach 6 to 7 ton dry seeds ha -1 y -1 within few years, but only under optimal climate conditions, using high yielding strains, and optimal soil fertility conditions. Looking at such promise, it is concluded that jatropha might be an alternative for other oil producing plants such as oil palm, especially for less humid areas (Daey Ouwens, 2007).

The role of J. curcas in bioenergy generation looks like to be of great interest; in fact, biodiesel production from jatropha seeds give an optimistic impression of the capability to combine the low-technology inputs required for oil production with other agricultural interesting claims on the plant. In fact, many are the claims regarding J. curcas, and its development as an agricultural crop appears to have many positive effects. The present- day hype for this plant comes from the theoretical combination of all the good features that characterize J. curcas cultivation, however they are not always scientifically proven and barely come out altogether simultaneously in the same site. In fact, many good characteristics of this plant appear to exist because of its rusticity and an intensive exploitation it is not like to be supported by scientifically sound agronomic data. Indeed, often good resistance characteristics are not linked with high productivity values (Jongschaap et al. , 2007). To understand better the limits and the potentials that J. curcas crop growth may result from a more intensive and focused farming, the main agronomic characteristics, the physiological behaviours, the reactions to different sites and the relevance of genotype or environmental influences should be known, and if not, they should be meticulously detected and, afterwards, explained to farmers. Keeping this assessment in mind and looking at an objective of J. curcas cultivation techniques improvement, knowledge gaps on botanical features, potential utilizations, claims and facts regarding J. curcas and its production system will be hereunder briefly described

27 1.Introduction and explained. In any case, a clear statement can be extrapolated: the plant cannot perform all its functions together at the best level.

1.2.1 Knowledge gaps in Jatropha curcas L. research

Despite the huge interest that J. curcas production system has attracted by now, a lack of knowledge and available data is evident. The main gaps concern some basic agronomic characteristics, the application of the good agricultural practices, the development of a complete J. curcas production system (including oil yield characteristics and oil processing) and the input/output balances at all these stages. Moreover, information is still required to assess the nutrient requirements and the dry matter assignment in different agricultural settings and pedoclimatic conditions. There is lack of data also in reported genetic variability that would allow beginning plant breeding programmes. Further research towards the discovery of the potential of utilization of other J. curcas products and by-products would be very welcome.

Accurate data on yield and on its characteristics are missing. In order to respond to the J. curcas oil production want of a list of countries, in which incredible large areas, that are drastically increasing, are planned to be grown with this plant, reliable information on crop requirements and climate/soil characteristics are still very much required (Daey Ouwens et al. , 2007). Much agronomic and breeding work needs still to be done to maximize oil production potential per ha and thus improve the economic sustainability of jatropha oil production. To this, rapid multiplication techniques and facilities have to be developed to make improved planting material available in adequate amounts. This is especially urgent as planting of unimproved material not only leads to low returns on investments but may also lead to a loss of interest in this crop (Henning, 2007).

Concerning the plant cultivation, substantial efforts should be made to streamline observations in current jatropha planting sites, to implement specific experiments for unravelling the impact of different production factors on crop performance and to exchange knowledge and information, in order to prevent unjustified investments. It is recognized that the main knowledge gaps are situated in the cultivation step, where a

28 1.Introduction description of the best practice and the potential environmental risks or benefits are needed. In fact, basic agronomic proprieties are not exhaustively understood and the environmental effects have not been investigated yet (Achten et al. , 2008). Correct spacing should be identified depending on different intended objectives and much has still to be learned from plant manipulation, from more or less intensive pruning or curving of branches, at different moments. As a new technology, the micropropagation is being developed by Manurung (Daey Ouwens et al. , 2007; Achten et al. , 2008). Taproot potential has not been investigated scientifically. The susceptibility of J. curcas to pests and diseases is a source of discussion and is believed to depend on the management intensity. More experiments are needed where the growth effect on different kinds of crops are monitored in intercropping systems. Impacts on soil structure, water-holding capacity, soil decomposition, organic matter content and soil biological activity should be brought under detailed investigation as well. Dominant role of environment over genetics in seed size, seed weight and oil content (Achten et al. , 2008) should be more deeply investigated. Much research is still necessary to improve yield, to exactly understand the energy efficiency of the plant under different conditions, to allow use of bioproducts such as oil cake as animal fodder (Daey Ouwens et al. , 2007).

Nutrient requirements for maximum oil production are not well-defined for J. curcas (Henning, 2007). No information is available on nutrient cycles and the impact on soil biological life (Achten et al. , 2008). The relation between plant nutrients, organic matter content of the soil and and yields is not fully understood (Daey Ouwens et al. , 2007). Jatropha has not been domesticated yet and basic knowledge of its soil- plant relations is required for the development of appropriate agricultural techniques. Very little is known about foliar nutrient content of jatropha and soil-plant relationship, which is essential to domesticate the plant and establish the nutrients requirements (Chaudhary et al. , 2008).

The input levels to optimize the harvest index (HI) in given conditions are yet to be quantified. Very limited information is available regarding acidification, eutrophication,

29 1.Introduction and other LCA impact categories of the J. curcas production cycle. Increased investigation of the cultivation step in the production of jatropha biodiesel will enable researchers to assess the specific contribution of the plant in these impact categories as well. As the reduction of global warming potential is one of the main aims of the J. curcas biodiesel system, this confirms the research need on input-responsiveness of the J. curcas cultivation step.

Good documented yield data are still scarce. Seed yield and biomass production in different environmental and abiotic settings, varying provenances or accessions and applying different levels of different inputs should be monitored in order to discover the input-responsiveness of the plant in different settings as well as the interactions between the different inputs and the interaction between the environmental and genetic set-ups and the inputs. Notwithstanding, there is still insufficient information to account the nutrient and water needs for specific environmental and genetic set-ups. The actual potential of J. curcas cultivation should be explored, as it is not clear if the plant is able to produce ecologically and socio-economically viable amounts of energy in barren situation (Achten et al. , 2008). From selection of basic plant material up to yield, there are many options, with a lot of variation in available data and not enough information for optimization. More research should also be initiated on medicinal proprieties of different parts of the plant, e.g. wound healing, antimalarial and anti-HIV effects, and investigation of the agronomic and medicinal potential of other Jatropha species would be valuable as well (Henning, 2007).

J. curcas is still a wild plant with a wide variation in growth, production and quality characteristics. In order to start breeding towards high yielding biodiesel plantations, the best suitable germplasm has to be identified for different cultivation situations (Henning, 2007; Daey Ouwens et al. , 2007; Achten et al. , 2008). For this reason, research makes progresses thank to new patent free molecular marker technology : conserved sequence based on NBS-gene family. A starting point in this sense could reasonably be intercrossing ‘elite’ J. curcas accessions (e.g. ‘Cabo verde’) with low toxic and toxic accessions as starting point for breeding, now that genetic analysis of

30 1.Introduction segregating population is possible (Montes et al. , 2008). More research is necessary on oil content, oil quality/acidic composition and the influence of environment and genetics on it. oil can be used as base for liquid engine fuel in various ways; choice of extraction method is clearly dependent on the intended scale of activity; crucial research and development options lay in the maximization of the transesterification efficiency at minimal cost. About this, an important issue is the improvement in the catalytic process, certainly the recovery and the reuse of the catalyst. As part of the option of decentralized processing units, low-cost, robust and versatile small-scale oil transesterification designs should be developed (Achten et al. , 2008).

1.2.2 Selection of knowledge gaps and justification The above mentioned statements and knowledge gaps lead the interest and the requirement of further research that is now the case. A broad investigation appears to be necessarily addressed to all the mentioned topics; however the evident risk of being imprecise would necessary bring to a selection. As, to date, some major knowledge gaps of the whole J. curcas production system are in the cultivation step, a deep analysis into that will put in evidence the need of looking for growth variables in monoculture, intercropping and hedges, as well as looking for sustainability indicators, also in these three production systems.

Growth Parameters are of major concern to understand net primary production of the plant, its potential and actual energy and water use efficiencies, its nutrient requirements. In general, growth parameters are needed to better understand the ecophysiology of the plant so to allow a full implementation of its potentials, adding the required inputs. Concerning J. curcas growth parameters, a general overview will be given. Firstly, a description is presented of growth parameters by plant parts: seed, root, stem/wood, leaf, , fruit and whole plant (Annex 1a). In this section some of the reported data are followed by annotations that would explain whether there were exceptional conditions when the data were recorded. That is the case of seed germination, oil quality, apical dominance and fitness. In fact, seed germination varies

31 1.Introduction apparently depending on different pre-treatments; oil quality, that is its physical- chemical proprieties and its constituent composition, varies under environmental conditions and genotype (Jongschaap and van Loo, 2009; Achten et al. , 2008). To what concern apical dominance and plant fitness, it must be said that plant breeding is still in its infancy. Variability in plant architecture between different accessions is reported (Montes, 2008). Then, growth parameters are intended under different farming condition. Three cropping systems are taken into account: monoculture, intercropping and living fence. Same growth parameters in the three cases are reported and differences between values will highlight differences between farming systems (Annex 1b).

The potential of jatropha oil production at a small scale and its implementation as a tool for rural development lead to a necessary investigation of sustainability indicators . A sustainability indicator is a parameter that allows understanding the impact of an action at different levels, mainly environmental, economic and social. This indicator should be clear enough to describe accurately the input/output ratio of each step of an entire process and tell whether it is more or less sustainable. In the case of the J. curcas production system, sustainability indicators will be identified and, firstly, divided into agronomic, environmental, economic and social spheres (Annex 1c). These categories were chosen to better explain the interactions between J. curcas production system internal and external factors and actors. Then, an overview of sustainability indicators will be given for different cropping systems: monoculture, intercropping and living fence (Annex 1d), as above considered. A deeper investigation in this sense will allow to define which one of the production systems is expected to be more sustainable. Here a right and proper specification: often the results depend on local or regional variables that are unable to be chosen or modified.

A relief of basic parameters coming out from the analysis of growth variables and sustainability indicators and an attempt to link them together should eventually give a general idea of growing patterns and impacts of the different J. curcas production systems.

32 1.Introduction

1.3 Aim and objectives of the thesis

J. curcas production system is attracting worldwide Institutions, Companies and Organizations attention, both for its oil yield suitable for biodiesel uses and for its capability to sustainably interact with rural tropical and sub-tropical world. A lot of projects are now taking place in Countries all over the world to assess the feasibility of the set-up of J. curcas production systems and their implementation at different scales and in relation with different realities and conditions.

The present work is a part of a wider research promoted by the 1 st Coordinated Call for a Transnational Research Activity under the ERA ARD net (the Agricultural Research for Development; Dimension of the European Research Area): “Bioenergy – an opportunity or threat to the rural poor”. Specifically, a joint consortium of academic, governmental and private institutions has proposed the interdisciplinary research and capacity development project “Bioenergy in Africa and Central America – Opportunities and Threats of Jatropha and Related Crops”, thus selecting two main foci: a crop, the Jatropha curcas L., and the regions, Central America and East Africa. The objective of ERA-ARD is to follow the growth and development of Jatropha curcas by monitoring important dynamic crop variables in different production systems and densities, and relate them to the environmental circumstances. The opportunity that I have to join this ERA ARD proposal has included a research on crop growth and processing at Tropical Studies and Development Foundation, in Belmopan, Belize, under the scientific guidance of Plant Research International, Wageningen, the Netherlands.

The main research activity was held in Belize and consisted on gathering crop growth and development data from existing plantations (LAI, yield) and to set up a field trial, in view of establishing the growth and development of Jatropha curcas L., and searching the links between these data to the environmental variables. The aim to share knowledge and to set up discussions on biofuels at national and regional levels is also pursued. In Belize, the goodness of jatropha as an oil feedstock and as a tool for rural development at small and medium farm size will be evidenced by further research, looking at the results in the coming years.

33

2. Materials and Methods

The work was structured and developed in two periods. The first period, from 14 th April to 14 th May 2009, was spent at Plant Research International (PRI) of the Wageningen University and Research centre, the Netherlands, and was directed to the bibliographic research and study of the state of the art on the jatropha plant and the whole jatropha system, with a focus on the cultivation. Research activities have also been undertaken in the greenhouse, such as for leaf area index (LAI) and light interception measurements, and in the laboratory, such as for oil extraction. The second period, from 15 th July to 15 th December, was dedicated to the field research in Belmopan, Belize, at the NGO Tropical Studies and Development Foundation (TSDF), with the collaboration of Galen University – Applied Research and Development for Sustainability Institute (GUARD) and the University of Belize (UB).

2.1 Research at PRI Wageningen, the Netherlands

During the research period in Wageningen twenty-six jatropha plants from four different accessions from Central America coded as 160 (7 plants), 176 (9 plants), 177 (7 plants) and 184 (4 plants) were grown in a greenhouse experiment for leaf area (LA) measurements and for assessing the interception capacity of photosynthetically active radiation (PAR). The plants were five months old, they were daily watered in the morning and evening and exposed to 12 hours of light, from 7.00 to 19.00 hours. For each plant, cotyledons and leaves were counted, and length (L) and width (W) of each leaf was measured; then leaf area was calculated according to the following formula:

A=0.84 ∗L∗W 0.99

(Liv Soares et al. , 2007). Jatropha light interception was also measured through a beam ('SunScan Canopy Analysis System, type p.SS1'), which could measure the fraction of photosynthetically active radiation (PAR) intercepted by the plants at different levels in the canopy (top, middle and bottom) and in fifteen different plant densities (Table 2.1). This research phase allowed testing a methodology for LA estimation on field and light interception calculations that were later used for the research activities in Belize.

Part of the research activity was carried out in the chemical laboratory, where oil was 2.Materials and Methods extracted from jatropha seeds and weighed. Kernels were separated from shells, smashed and mixed with the solvent: 7.5ml of hexane per round of extraction (3 times) with about 0.4g to 1.0g of kernel (van Loo, 2009, personal comm.).

Table 2.1. Fifteen J. curcas plant set-ups for LAI and light interception measurements (Jongschaap et al. , unpublished data)

Row Plant TreatmentDistance Distance Length Width Area (m) (m) (m) (m) (m2) 1 1,00 0,90 3,00 4,50 13,50 2 1,00 0,60 3,00 3,00 9,00 3 1,00 0,45 3,00 2,25 6,75 4 1,00 0,30 3,00 1,50 4,50 5 0,90 0,90 2,70 4,50 12,15 6 0,90 0,60 2,70 3,00 8,10 7 0,90 0,45 2,70 2,25 6,08 8 0,90 0,30 2,70 1,50 4,05 9a 0,60 0,90 1,80 4,50 8,10 9b 0,60 0,60 1,80 3,00 5,40 10 0,60 0,45 1,80 2,25 4,05 11 0,60 0,30 1,80 1,50 2,70 12 0,45 0,45 1,35 2,25 3,04 13 0,45 0,30 1,35 1,50 2,03 14 0,30 0,30 0,90 1,50 1,35

2.2 On field research in Belize, Central America

Belize, Central America, has a total land area of 22.966 km 2. The Country is located at 17°15' N, 88°45' W, with climate characterized by a dry and a rainy (June to November) season and mean annual rainfall from 1524mm in the north to 4064mm in the south (Figures 2.1 and 2.2) (CIA World Fact Book, 2009; Belize National Meteorological Service, 2009). Central Region, where the trials of the present research are located, shows a primary and secondary rainfall maxima occurring in June and September; in this region, main soil type is cambisol (FAO et al. , 2009). In the Country, jatropha is known as physic nut and used for curative purposes; just recently it received more attention by the Ministry of Agriculture and some private investors, although policies on bioenergy and market for jatropha products are lacking.

36 2.Materials and Methods

Field trials were performed in three areas in Belize: at Maya Ranch, a six year old jatropha plantation; at Warrie Head, a one year old jatropha plantation; at Central Farm, a new plantation of jatropha, whose design was set up at Wageningen. These three trials were treated and monitored trough the study of different variables: crop growth and development was monitored through the Leaf Area Index (LAI) measurement (a non- destructive method useful in yearly biomass production assessment) in all the three trials, while yield was measured only at Maya Ranch. Measurements on seed dimension and a germination test, other biomass production and crop development indicators, were performed at Bullet tree, the nursery for the jatropha seedlings destined to Central Farm trial. Environmental variables taken into consideration were: climatic data (the same for the three trials) and soil samples (at Central Farm trial, only). Locations of meteorological stations and experimental sites are shown in figure 2.3.

Figure 2.2. Map of Belize and its Districts (source: http://www.watersidebelize.co m/images/belize_map.jpg ).

37 2.Materials and Methods

Figure 2.3. Map of Belize and location of meteorological stations (Philip Goldson International Airport and Central Farm) and experimental sites (Maya Ranch, Warrie Head, Bullet Tree and Central Farm). (Source: http://www.hydromet.gov.bz/AgroClimat_Stations.htm ).

38 2.Materials and Methods

2.2.1 Climate data

Climate data collection was a kind concession from the Belize National Meteorological service (www.hydromet.gov.bz). Weather variables were recorded on a daily basis (Table 2.2), through two climate stations: one station was settled at Philip Goldson International Airport (17°32' N, 88°18' W) and recorded radiation (kJ m -2 d -1 ), vapour pressure (kPa) and wind speed (m s -1 ); the second one, in Central Farm (17°11' N, 89°00' W), recorded Min and Max temperatures (°C) and precipitation (mm d -1 ). Both stations were selected as the closest available to trials and able to record the required data on a daily basis. However, actual weather variables in the experimental sites might slightly differ. In fact, trials occurred in different locations at around 100km to 140km from Philip Goldson International Airport and 1km to 25km apart from Central Farm weather stations (Figure 2.3).

Table 2.2. Weather variables required on a daily basis.

Abbreviation Weather variable Unit Rad Radiation (kJ m -2 d -1 ) Tmax Maximum temperature (°C) Tmin Minimum temperature (°C) VP Vapour pressure (kPa) WS Wind speed (m s -1 ) P Precipitation (mm)

39 2.Materials and Methods

2.2.2 Maya Ranch trial

Maya Ranch field trial, with an area of almost 0.5ha, is located at the 4 th mile on the Caracol Road, in Cayo District, at an altitude of 150 meters above the sea level (Figures 2.4 and 2.5), in a location out of the main routes. The area is surrounded by tropical rainforest and was formerly used as pasture for sheep, then abandoned. In June 2003, TSDF transplanted around five hundred jatropha seedlings, from seeds harvested from wild spontaneous and backyard grown isolated trees. Jatropha was planted in the context of an Organization of American States (OAS) funded agro-forestry project, that involved the planting of a teak plot ( Tectona grandis ) that still exist beside the jatropha field, and the sowing of Habanero peppers ( Capsicum sp.) and Arachis pintoii as suitable inter-crops for jatropha. To date, A. pintoii is still growing on the south-east side of the jatropha plantation and is giving positive feedback since it is a leguminous able to control weed and its cultivation does not require much labour. A. pintoii , known in Belize as Pinto peanut (English) and Maní forrajero (Spanish), is a perennial herb that develops a strong taproot and forms a dense mat of stolons and rhizomes up to 20 cm deep; there are low and highland species (up to 1400m) that show high tolerance to shade and drought. It has been used as a forage legume in intensively managed grass/legume pastures and tree plantations, or as a ground cover in tree plantations (Cook, 1992). Its key benefits are weed control, nitrogen fixation and ability to lower surface temperature for better soil health and moisture.

The project was temporarily suspended, in 2006/2007, and the whole area was left abandoned, until July 2009, when the jatropha field became a trial to the purpose of the current research for which an inventory was done, on 22 nd July. The inventory lead to the following results: 458 plants were counted, organized in 8 rows. The distances are 4m between rows and 2m between the plants within the row (1250 plants ha -1 ). The field is oriented on a south-north axis and a slight slope gradient is present in this direction, from the south side where the upper part is, to the north, at the bottom of the field (lower part). In the field, three sets of jatropha plants were recognized: i) the plants of the two west side rows and the last six or seven plants of each row at the bottom of the

40 2.Materials and Methods field appeared smaller and less vigorous than the others, and they were barely bearing fruits, as a result probably of less deep soils on this side and water logging at the bottom of the field; ii) about one third of the plants towards the northern part had been pruned in 2006/2007, they presented long and straight upward branches and already some fruits; iii) the remaining two thirds consisted on the largest trees, with many branches that closed completely the rows and were bearing a lot of fruits in groups of three to eight racemes, A. pintoii was found growing below these plants. Two-meter high wild herbaceous vegetation was growing along the field and in the surroundings, which have been cut by machete twice during the growing season, in July and October 2009.

Beginning a research activity on a field at that stage required some quick decisions, therefore two trials were initiated: they have been developed in separate times during the growing season but both of them have been set up in the same field and considered the same jatropha plantation. The first trial A (27th July – 10 th August) focused on the harvest, while the second trial B (1 st September – 1 st December) on LAI measurements, as described below.

Figure 2.4. Jatropha curcas L. row in Figure 2.5. Jatropha curcas L. plantation, in intercropping with Arachis pintoii, in Maya Maya Ranch, Belize, August 11 th , 2009. Ranch, Belize, August 11 th , 2009.

For the first trial A, 'pruning' and 'intercropping' were identified as already existing treatments, and two plots were selected within the field: one with pruned plants growing in monoculture (named 'pruned'), and, the other one with not-pruned plants under which A. pintoii was vigorously growing (named 'not pruned')(see annex 2, table a). Fruits

41 2.Materials and Methods were harvested and dried separately by plot. The harvest was done on 7 th July and 10 th August, 2009, and fruits were left drying under a roof during four to six weeks. After this period, the fruits have been weighed on a 100g precise scale separately as follows: 7th July-pruned, 7 th July-not pruned, 10 th August-pruned and 10 th August-not pruned. From each of the four groups, a 2% fruit sample (between 140g and 740g) was picked and coats and seeds were separated and weighed on a 1g precise scale. From each of the four groups, a 100g seed sample was chosen and shells and kernels separated and weighed on a 1g precise scale. Moreover, one thousands of randomly selected fruits were opened: the number of seeds per fruit was counted and the average coat to seed weight ratio was measured on a 1g precise scale. A 100 seed sample was crushed and shell to kernel ratio was measured on a 1g precise scale.

For the second trial B, two already existing treatments were identified: pruning and intercropping, and three plots were selected according to their cultural management, as follows: 'intercrop/not pruned', 'monoculture/pruned' and 'monoculture/not pruned' (see annex 2, table b). 'Pruning' referred to those plants that had been pruned two years before at an height of 60cm, and, as intercrop, A. pintoii was maintained because its easy-to-cultivate characteristics and its reciprocal benefits with J. curcas had been noted. To record jatropha growth and development, 90 monitoring plants were selected for LAI measurements: 36 plants from the 'intercrop/not pruned' plot, 18 from the 'monoculture/pruned' plot, 36 from the 'monoculture/not pruned' plot. LAI measurements and estimation have been performed on 1 st September, 1 st November, 1 st December, according to the methodology described in table 2.3. The choice of different amount of monitoring plants in the plots was necessarily taken as a trade off between the existing situation and the new research objectives. Furthermore, on the 1 st November and the 1 st December, the length and the effective length of representative branches of the monitoring plants were measured to report the leaf production and fall off trends. Effective branch is expressed as the segment of the branch in which green leaves are still growing. This methodology allowed to monitor two aspects of plant growth and development: first, to record the branch part actually involved in solar radiation

42 2.Materials and Methods interception and to monitor how its length changes during the season; secondly, to observe the plant dry matter production potential and its nutrients cycle, for what concern leaf production and decay, from the soil deeper layers to the leaves back to the soil, on the superficial layers.

Further calculations were made to find out intercepted solar radiation by the leaves of the monitoring plants, applying the following formula:

1−e−k∗LAI  from 's Law formula for light interception, first described for plants by Monsi and Saeki (1953), where k=0.68, in the case of J. curcas L. (Jongschaap, unpublished data).

Table 2.3. Estimation method of the Leaf area per tree. 1Estimation method per leaf presented at Expert seminar on J. curca s L. (March 2007) (Jongschaap et al. , 2007).

(example unit data) Measure the length of a representative branch a 0.75 (m) Count the number of leaves on this branch b 17 (#) From the mid-section of the representative branch, select c 21.5 (cm) a representative leaf. Measure the length from the point where the leaf sheath is attached to the petiole to the opposite point of the leaf Measure the maximum width of the leaf, perpendicular d 18.5 (cm) on axis of the previous measurement Estimate how often this representative branch ‘fits’ in e 5.5 (#) the monitoring tree (e.g.5.5 similar branches) Leaf Area 1 = 0.0001 m 2 cm -2 * b * 0.84 * (c * d) 0.99 * e 3.50 (m 2 tree -1 )

43 2.Materials and Methods

2.2.3 Warrie Head trial

Warrie Head field trial, about 0.8ha large, is located just off the 9 th mile of the Western Highway, from Belmopan to San Antonio, in Cayo District, at an altitude of 45 meters above the sea level (Figures 2.6 and 2.7). The area is a private property, surrounded by forest, and at the border of the Belize River. In 2008 the land was cleared from the prevalent graminaceae wild vegetation and ploughed. In July of the same year, in the framework of an OAS-EEP research project, TSDF planted around five thousands jatropha seedlings of two accessions (from Guatemala and Cuba, the Cuban being called 'Cabo Verde'), at two different spacings (3*1.7m alternated with 1.7*1.7m and 4*1m) but resulting in the same plant density of 2500 plants ha -1 . Water soluble polyacrylamide (ultra-fine water-soluble polymer/acrylamide providing benefits as soil conditioning agents) in one half and effective micro-organisms (a biological product that contains a mixture of beneficial organisms, such as lactic acid bacteria, yeast, and phototrophic bacteria) on the other half of the field were sprayed (Baumgart and Sengfelder, personal comm.). On the short sides of the trial, additional jatropha seedlings were transplanted and not sprayed with micro-organisms. An irrigation system was installed. In October 2008, almost two thirds of the jatropha plants were uprooted and died, as a consequence of an extraordinary flood from the Belize River. The field was left abandoned until its rehabilitation as a trial for the current research, in July 2009.

Figure 2.6. Jatropha curcas L. plantation and Figure 2.7. Jatropha curcas L. plantation particular of irrigation system (not in use), in (distance 1.7m between the two central Warrie Head, Belize, August 11 th , 2009. rows), in Warrie Head, Belize, August 11 th , 2009. 44 2.Materials and Methods

For this trial, the preparatory inventory lead to the following results and assumptions: 1476 plants were counted, the majority of them being the additional plants in the sides of the EEP-OAS trial; these plants survived the flood because they were growing in a slight hilly area; the treatments still existing after the flood were genotype and plant spacing; at this point on time the micro-organisms previously sprayed are most likely lost and not able to interfere in the plantation; concerning the plant size, a great phenotype variability was evident (individual height from 0.3m to 3m), but plant architecture appeared quite similar and suggested a strong competition for light with wild herbaceous vegetation; apparently, soil is deep. During the trial, the field was cleared by machete and bush-hogger twice, in July and in October 2009. As shown in annex 2, table c, the field is organized in four groups, divided by genotype and plant spacing in: Guatemala-(3*1.7)*1.7m 2 (328 plants), Cuba-(3*1.7)*1.7m 2 (370 plants), Cuba-4*1m 2 (387 plants) and Guatemala- 4*1m 2 (391 plants), where (3*1.7)*1.7m 2 spacing consists in the 'double row' and 4*1m 2 is 'single row'. Both spatial design result in 2500 plants ha -1 . The research activity, then, started from this situation and focused on LAI measurements. Each group was divided in three sub-plots and six monitoring plants have been chosen from each sub-plots. In total, the same 72 plants (18 per group) were selected for LAI measurements four times in the growing season on 2 nd September, 2 nd October, 2 nd November and 2 nd December. The methodologies used for LAI measurements and estimation and for effective branch length measurements are similar to the ones applied for Maya Ranch trial, as well as the formula used to calculate intercepted solar radiation.

2.2.4 Central Farm trial

The trial in Central Farm was established in the framework of the project 'ERA-ARD Biofuels in Africa and Central America 2009-2013'. The preparatory work consisted on the experimental design finalization and on the seed retrieval. The on field activities have been carried out in two places: the sowing was done in a nursery at Bullet tree and, two months after, the seedlings were transplanted on field at Central Farm.

45 2.Materials and Methods

The nursery was set up at Bullet tree, three miles off the west end of the Western Highway, in Cayo District, at an altitude of 80 meters above the sea level (Figures 2.8 and 2.9). At the nursery stage data on seed dimension were taken, a germination test, LA measurements and destructive biomass assessment were performed, from the seed to the two month old seedlings. The nursery was located in a vegetable organic farm, and teak seedlings were present, too. Once a week all the plants, including the jatropha, were sprayed with a micro-organisms mixture that granted the sanity of the seedlings during this raising period. For the purpose of the ERA-ARD project, three seed accessions have been used: the Belize local one, collected in Maya Ranch, the Guatemalan G17 provided by the company 'Biocombustibles de Guatemala' and a low-toxic Mexican provided by the 'Universidad Autónoma Chapingo'. The average 100-seed weight was established weighing 937 Mexican seeds, 822 Guatemalan and 1000 Belizean on a 1g precise scale. The length and the width of hundred seeds per accessions were measured, and the length to width ratio was calculated. Hundred seeds per accession were crushed, and shell and kernel were weighed separately. After these measurements, on the 23 rd September, 1144 seeds per accessions were sown in black cylindrical perforated plastic bags, 20cm high and 10cm in diameter, filled with local soil and husks (Figure 2.8); they were watered twice daily and sprayed with an EM mixture once a week. More Belizean seeds were sown: 288 on the same day and 1088 on the 9 th October, in the context of the EEP project. Surveys at the nursery were done on the 30 th September, 2 nd and 7 th October, and on the 21 st October for the last sown seeds: germinated seeds and standing plants were counted. At the nursery stage, ten plants per accessions were randomly selected and LA measurements were performed one month after the germination (3 rd November), and at the moment of transplanting (26 th November,), using the same methodology explained for Maya Ranch trial. On the 26 th November, the same ten plants per accessions were uprooted and dried under a roof for two weeks: on the 11 th December dicotyledons, leaves, petioles, stems and roots were counted and weighed on a 1g precise scale. All stems and some leaves of Guatemalan (11 leaves) and Belizean (1 leaf) accessions were still green and fresh. Tap root lengths were measured.

46 2.Materials and Methods

Figure 2.8. Sowing Jatropha curcas L. Figure 2.9. Jatropha curcas L. nursery, in seeds, in Bullet Tree, Belize, September Bullet tree, November 2 nd , 2009. 23 rd , 2009.

On the 26 th November, 1494 jatropha plants were transplanted on a 0.6ha field at UB Campus, according to the design in annex 2, tables d and e. Additionally, 504 plants were transplanted on the north side of the experimental plot (264 plants, 0.15ha) and on the south (240 plants, 0.14ha) (see annex 2, table f). The field selected is located on the top of a hilly area at Central Farm, in Cayo District, at an altitude of 60 meters above the sea level (Figure 2.10). The field is east to west oriented and has a pig house on the east side, a vegetable garden and the additional jatropha on the south, trees on the west and the natural forest on the north, beyond the additional jatropha strip. There is a slight slope gradient going downwards from south to north. Formerly, there was an orange plantation, then, nearly half of the field was covered by meadow and half cultivated with corn (on the east side). The field has been bush-hogged, cleared and prior the transplanting. The transplanting followed the trial set up: a randomized blocks design with three factors and three repetitions (see annex 2, table d). The factors were three genotypes: Mexican, Guatemalan and Belizean; three agricultural production systems: monoculture, intercropping with A. pintoii and living fence; two plant densities of about 1250 and 2500 plants ha -1 , organized in spacing of 3.7*2.2m 2 and 3.7*1.1m 2 in the plot (monoculture and intercropping) and 0.25m and 0.5m in the living fence. There were twelve treatments per block, repeated in three blocks, resulting in a total of 36 sub- plots in the field. Moreover, six treatments repeated four times for a total of 24 segments

47 2.Materials and Methods in the living fence. In each of the thirty-six block, six monitoring plants (which had at least 1 border row in the sub-plot) were selected and LAI measurement was performed, on the 2 nd December, using the same methodology explained for Maya Ranch trial (Figure 2.11). For the statistical analysis, although, the design was reinterpreted, as, at the time of the first LAI measurement, intercropping was not available and genotype and plant density were the only factors. The trial was treated as a complete randomized block design for the analysis of the variance, though not two repetitions per three blocks have been considered but totally six repetitions for each treatment.

Figure 2.10. Transplanted Jatropha curcas Figure 2.11. LAI measurements of Jatropha L. seedlings at 60 days, in Central Farm, curcas L. with technicians from the Ministry Belize, November 26 th , 2009. of Agriculture and the University of Belize, an example of knowledge sharing and institutional strengthening, in Central Farm, Belize, December 2 nd , 2009.

On the 10 th December, after the transplanting of jatropha but before A. pintoii was in the ground, representative soil samples were taken from each of the three blocks at 0-20cm, 20-50cm and 50-100cm depths. For the size of the experiment, six random pits per block were prepared and samples from the three depths extracted with an auger. In total, nine soil samples were collected for different soil characteristics (Table 2.4).

48 2.Materials and Methods

Table 2.4. Soil variables required for 1 st soil characterization.

Abbreviation Soil variable Unit % Sand (%) Silt% Silt (%) Clay% Clay (%) BD Bulk density (g cm -3 ) OM% Organic Matter content (%) C% Carbon content (%) Norg% Organic Nitrogen content (%) N nitrogen (ppm) P Available Phosphorous (ppm) K Potassium (ppm)

2.3 Statistical analysis

The collected data were analysed and organized in factorial spreadsheets, using OpenOffice.org Calc. Average, standard deviation and standard error of the treatments of each trial were calculated. Thereafter, descriptive methods have been used for climate data, fruit yield at Maya Ranch, jatropha seed dimensions and germination rate in Bullet Tree; while, on data collected at Maya Ranch (LAI and effective branch length), at Warrie Head (LAI and effective branch length) and at Central Farm (LAI) the software Anova97 ver. 3.12 (Onofri, 1997) was used to perform the analysis of the variance. In each trial, monitoring plants were grouped and averaged by treatment and block (or repetition), and the average of these groups were compared, to the purpose of the ANOVA. To differentiate the means, the Duncan test with P=0.01 was applied in all the cases, except where otherwise specified.

49

3. Results and discussions

Outcomes of the preparatory research in Wageningen and of the on field research in Belize, for the period July – December 2009, are presented in this chapter.

3.1 Important drivers for Jatropha curcas L. growth and development and how are these for Belize

Weather variables, soil composition and nutrients availability are main drivers that contribute to characterize the pedoclimatic condition of an agro-ecological zone and directly influence crop growth and development. In the current research it was possible to gather climate data, provided by the Belize National and Meteorological Service. To this purpose, the closest available climate stations were used: in Central Farm (1km to 25km apart from the experimental sites), to record temperature (Min and Max) and precipitation, and at Philip Goldson International Airport (100km to 140km apart from the experimental sites), to record radiation, vapour pressure and wind speed. With regard to this, it should be reminded that this situation may have influenced some findings of this research: in fact, it is possible that radiation, vapour pressure and wind speed values recorded in a station by the coast of the sea and close to a large urban area (Belize City, 70000 inhabitants) may differ from actual values occurred more than 100km in the inland in less populated areas. Hereunder, climatic situation in Belize for the year 2009 is summarized on a daily basis.

3.1.1 Radiation and light interception

Over the whole year 2009, radiation showed the highest mean value in May, where it reached more than 18kJ m -2 d-1 ; since then, mean monthly values were constantly and gradually declining to 12kJ m -2 d -1 recorded in December (Figure 3.1).

To analyse resource use efficiency, solar radiation interception was measured. Light interception formula for jatropha has been calculated in a trial at PRI Wageningen, where measurements and calculations on jatropha at different plant densities indicate that value of coefficient 'k' is 0.68. However, it changes according to different LAI: in fact, for LAI>7, k=0.55 and for LAI<1-1.5, k=0.75 (Jongschaap, unpublished data). 3.Results and discussions

Weather variables, Belize, 2009 Mean monthly values - Philip Goldson International Airport station 20

15

Radiation [kJ m -2 d-1] 10

5

0 JFMAMJJASOND Figure 3.1. Mean monthly values of radiation recorded at Philip Goldson International Airport meteorological station, in Belize, 2009. Source: Belize National Meteorological Service , personal communication.

3.1.2 Temperature

Mean monthly temperatures have been recorded to be between 15 and 35°C, with average Maximum temperature around 31°C and minimum around 21°C. Both Max and Min temperature values were above the mean from April to October (Figure 3.2).

Weather variables, Belize, 2009 Mean monthly values - Central Farm station 40

35

30

25 Temp. Max. [°C] Temp. Min. [°C] 20

15

10

5

0 JFMAMJJASOND Figure 3.2. Mean monthly values of minima (Temp. Min.) and maxima (Temp. Max.) temperatures recorded at Central farm meteorological station, in Belize, 2009. Source: Belize National Meteorological Service , personal comm.

52 3.Results and discussions

3.1.3 Water

A total yearly precipitation of 1476mm was recorded in Central Farm, where the driest period occurred from February to May, with about only one sixth of the yearly precipitation (245.1mm); while the wettest months have been June, July, August and November, totalling 792.2mm, equivalent to more than a half of total yearly precipitation. Oddly, October was recorded to be exceptionally dry, being the driest month with only 31.4mm of rainfall (Figure 3.3). Given this information, the beginning of the growing season of jatropha could have been most likely established in June, when important precipitations occurred on the 5 th (72mm) and between 16 th and 20 th (86.4mm).

Rainfall distribution, Belize, 2009 Total monthly values - Central Farm station 300

250

200 Tot. Precip. [mm] Days of rain [#] 150 Mean Precip. [mm]

100

50

0 JFMAMJJASOND Figure 3.3. Total monthly values of precipitation (Tot. Precip.), number of days of rain and mean monthly precipitation (Mean Precip.) recorded at Central Farm meteorological station, in Belize, 2009. Source: Belize National Meteorological Service , personal comm.

3.1.4 Vapour pressure and wind speed

Vapour Pressure was at lowest levels of around 25kPa in the beginning of the year, then increased from April, reaching maximum values between June and September when it was recorded at around 32kPa; it finally came down to values close to 27-28kPa in the last two months of the year (Figure 3.4).

53 3.Results and discussions

Weather variables, Belize, 2009 Mean monthly values - Philip Goldson International Airport station 35

30

25

20 Vap.Press. [kPa]

15

10

5

0 JFMAMJJASOND Figure 3.4. Mean monthly values of vapour pressure (Vap.Press.) recorded at Philip Goldson International Airport meteorological station, in Belize, 2009. Source: Belize National Meteorological Service , personal communication.

Wind speed mean values over the year were recorded to be around 3m s -1 , with slightly above-the-mean values from January to July and slightly below-the-mean in the rest of the year (Figure 3.5).

Weather variables, Belize, 2009 Mean monthly values - Philip Goldson International Airport station 5

4

3 Wind Speed [m s-1]

2

1

0 JFMAMJJASOND Figure 3.5. Mean monthly values of wind speed recorded at Philip Goldson International Airport meteorological station, in Belize, 2009. Source: Belize National Meteorological Service , personal communication.

54 3.Results and discussions

3.2 Response of genetically different accessions to available resources in Belize

Different accessions have been used in the three trials: local Belizean, in Maya Ranch, and Cuban and Guatemalan, in Warrie Head, where plants were already in the ground; while in Central Farm, seeds from Mexico, Guatemala and Belize were analysed and sown. In this section, firstly, seed morphological characteristics, and, secondly, responses to agro-ecological condition in Belize are reported. Results achieved in Central Farm trial are treated in the frame of work package 1 'Crop growth and processing' of the ERA-ARD project 'Bioenergy in Africa and Central America – Opportunities and Threats of Jatropha and Related Crops'. Results obtained refer to seed dimension, germination, LA, fresh seedling weight and taproot length, during the nursery stage in Bullet Tree, and to LAI measurements during transplanting in Central Farm.

3.2.1 Seed dimension and weight

Measurements of length, width and hundred seed weight of J. curcas L. were determined for three genotypes: a low-toxic Mexican, a Guatemalan (G17), and a local Belizean accession. Guatemalan seeds were recorded among the largest for dimensions, (Figures 3.6, 3.7 and 3.8) and weight (Figure 3.9). Guatemalan average seed length was above 1.8cm and had 100-seed weight of 73g. Mexican and Belizean seeds had an average length slightly under 1.8cm and a 100-seed weight, well below Guatemalan values, at 58g for Mexican seeds and 55g for Belizean seeds (Figure 3.10).

55 3.Results and discussions

Length of jatropha seeds of three accessions, Belize, 2009 Bullet Tree, nursery 50

40

30 Mexico Guatemala 20 Belize

10 Frequency [# of seeds] of Frequency [# 0 1.35 1.45 1.55 1.65 1.75 1.85 1.95 2.05 2.15 Class central value [cm] Figure 3.6. Length of jatropha seeds as average of a hundred seed sample per accession. Seed accessions are from Mexico (low-toxic), Guatemala (G17) and Belize (local). 2009.

Width of jatropha seeds of three accessions, Belize, 2009 Bullet Tree, nursery 50

40

30 Mexican Guatemalan 20 Belizean

10 Frequency [# of seeds] of Frequency [# 0 0.82 0.87 0.92 0.97 1.02 1.07 1.12 1.17 1.22 Class central value [cm] Figure 3.7. Width of jatropha seeds as average of a hundred seed sample per accession. Seed accessions are from Mexico (low-toxic), Guatemala (G17) and Belize (local). 2009.

56 3.Results and discussions

Length:width ratio of jatropha seeds of three accessions, Belize, 2009 - Bullet Tree, nursery 50

40

30 Mexico Guatemala 20 Belize

10 Frequency [# of seeds] of Frequency [# 0 1.46 1.54 1.62 1.7 1.78 1.86 1.94 2.02 2.1 Class central value Figure 3.8. Length to width ratio of jatropha seeds as average of a hundred seed sample per accession. Seed accessions are from Mexico (low-toxic), Guatemala (G17) and Belize (local). 2009.

Weight of 100 seeds of jatropha of three accessions, Belize, 2009 Bullet Tree, nursery 80 70 60 50 Shell [g] 40 Kernel [g] 30 20 10

Weight100 of seeds [g] 0 Mexico Guatemala Belize Accessions Figure 3.9. Weight of 100 seeds of J. curcas L. of accessions from Mexico (low- toxic), Guatemala (G17) and Belize (local). 2009.

57 3.Results and discussions

Average dimensions of jatropha seed of three accessions, Bullet Tree, nursery 2 1.8 1.6

1.4 Mexico 1.2 Guatemala 1 Belize 0.8 0.6 0.4 0.2 0 Length [cm] Width [cm] L:W ratio Weight [g] Figure 3.10. Length, width, length to width ratio (L:W ratio) and weight of jatropha seeds as average of a hundred seed sample per accession. Seed accessions are from Mexico (low-toxic), Guatemala (G17) and Belize (local). 2009.

3.2.2 Seed germination rate

After measuring the seed dimensions, 1144 seeds per accession were sown and germination rate and standing plants were recorded three times. One week after sowing, the majority of seeds already germinated, however differences among accessions were noticed; more than 85% of Mexican and Guatemalan seeds germinated, while only 70% of Belizean seeds germinated after one week. Two weeks after sowing, Mexican seeds recorded the highest germination rate (96%), followed by Guatemalan (91%) and Belizean (80%) seeds (Figure 3.11); after that moment, germination was not surveyed any longer. Standing plants, that are upraised plants with open cotyledons, were counted at the same time intervals and results were calculated as percentage over sown and germinated seeds. Significantly different results were obtained for the three genotypes. At the time of the first survey, less than 3% of jatropha plants from Mexican seeds were standing, while Guatemalan and Belizean showed standing rates of 37% and 22% over sown seeds (Figure 3.12) and 44% and 32% over germinated seeds (Figure 3.Error: Reference source not found). However, two weeks after sowing, more than 99% of

58 3.Results and discussions plants from germinated seeds of all the accessions were standing (Figure 3.13).

Germination rate [%] of 1144 jatropha seeds of three accessions, Belize, 2009 - Bullet Tree, nursery 100 90 80

70 Mexico 60 Guatemala 50 Belize 40 30 20 10 [%] of germinatedof [%] seeds 0 2009.09.23 2009.09.30 2009.10.02 2009.10.07 Figure 3.11. Germination rate of J. curcas L. seeds of three accessions, on 1144 seeds per accession monitored in intervals of 7, 9 and 14 days after sowing. Seeds from Mexico (low-toxic), Guatemala (G17) and Belize (local). 2009.

Standing plants as [%] of sown jatropha seeds of three accessions, Belize, 2009 - Bullet Tree, nursery 100 90 80

70 Mexico 60 Guatemala 50 Belize 40 30 20

[%] of standingof [%] plants 10 0 2009.09.23 2009.09.30 2009.10.02 2009.10.07 Figure 3.12. Standing plants as percentage of sown seeds of J. curcas L. of three accessions. 1144 seeds per accession monitored in intervals of 7, 9 and 14 days after sowing. Seeds from Mexico (low-toxic), Guatemala (G17) and Belize (local). 2009.

59 3.Results and discussions

Standing plants as [%] of germinated seeds of jatropha of three accessions, Belize, 2009 - Bullet Tree, nursery 100 90 80 70 Mexico 60 Guatemala 50 Belize 40 30 20 [%] of standingof [%] plants 10 0 2009.09.23 2009.09.30 2009.10.02 2009.10.07

Figure 3.13. Standing plants as percentage of germinated seeds of J. curcas L. of three accessions. 1144 seeds per accession monitored in intervals of 7, 9 and 14 days after sowing. Seeds from Mexico (low-toxic), Guatemala (G17) and Belize (local). 2009.

In general, Mexican low-toxic seeds showed a slower but more effective germination characteristic, Guatemalan accession G17 were the quickest plants to stand with a satisfying germination rate, while Belizean local seeds depicted the lowest, but acceptable, germination rate and an average standing plant percentage compared to the other accessions.

3.2.3 Biomass development at nursery stage: LA, fresh weight, taproot length

During two months at the nursery stage, leaf area (LA) measurements on jatropha seedlings of the three accessions were performed twice, one month after germination and at the time of transplanting. The analysis of the variance revealed high significant differences for the factor 'date' and the interaction 'genotype x date'. In fact, at the first survey in the beginning of November, no significant differences were found among the three genotypes. Instead, in the second survey, after a month, significant differences were found between Mexican and Belizean genotypes, while LA of Guatemalan

60 3.Results and discussions seedlings were not significantly different from the other two accessions. Considering the effect of date separately per accessions, it was found that LA of Belizean seedlings did not differ significantly between dates, while LA of both Mexican and Guatemalan seedlings were greater and significantly different at the time of the second survey than of the first (Figure 3.14).

LA measurements on jatropha of three accessions, Belize, 2009 Bullet Tree, nursery 0.040

0.035 a

0.030 ab Mexico 0.025 bc c bc Guatemala 0.020 Belize c 0.015

0.010 LA [m2*plant-1] LA 0.005

0.000 2009.11.03 2009.11.26 Figure 3.14. Mean LA of ten jatropha seedlings of three accessions, from Mexico (low-toxic), Guatemala (G17) and Belize (local), measured about one and two months after germination. 2009. The same letters above the histograms indicate that between the treatments significant differences were not found at the Duncan test, with P=0.01.

Additional information about early biomass development of jatropha seedlings came out from the measurements of taproot length and weight of ten individuals per accession at the time of transplanting. Results on taproot length analysis indicated that there is a significant difference, for P=0.05, between Guatemalan and Mexican accessions, while Belizean accession did not significantly differ from the two other accessions (Figure 3.15). Fresh seedlings of Guatemalan origin seedlings produced more biomass than others in the first two months of life (Figure 3.16), which can be related to the seed dimensions, as an indication for the growth reserves available in the seeds. Belizean accessions with more or less same seed weight but with lower kernel weight (Figure 3.9) grow slower than the other accessions, but invest relatively more dry matter in

61 3.Results and discussions roots.

Taproot length of jatropha seedlings of three accessions Belize, 2009 - Bullet Tree, nursery 30

a 25 ab

20 b Mexico Guatemala 15 Belize [cm] 10

5

0 Taproot [cm] Figure 3.15. Taproot length of ten jatropha seedlings of three accessions, from Mexico (low-toxic), Guatemala (G17) and Belize (local). 2009. The same letters above the histograms indicate that between the treatments significant differences were not found at the Duncan test, with P=0.05.

Fresh weight of jatropha seedlings of three accessions Belize, 2009 - Bullet Tree, nursery 25

20

Mexico 15 Guatemala Belize [g] 10

5

0 Fresh w eight [g] Figure 3.16. Fresh weight of ten jatropha seedlings of three accessions, from Mexico (low-toxic), Guatemala (G17) and Belize (local). 2009.

Jatropha seedlings dry weight could not be properly measured, since a stove was not

62 3.Results and discussions available. As a result, two weeks after uprooting and air drying, the young plants' stems were still green. However, some indicative data were acquired: in two weeks, uprooted seedlings lost from one quarter to one third by weight; stems were still green and accounted for the three quarters of the weight; the remaining weight (one quarter) was divided over roots, petioles and leaves according to the ratio 4:1:4, approximately, for all three accessions.

3.3 Resources use efficiency and optimization for jatropha crop conditions in Belize

In the three trials, different agricultural features were considered (plant density in Central Farm trial, plant spacing in Warrie Head trial and crop management in Maya Ranch trial). LAI development and effective branch length were recorded in order to measure part of the plant biomass development and to reveal differences between different treatments, in view to eventually evaluate resources use efficiency and optimization for the crop.

3.3.1 Plant density

One week after transplanting jatropha seedlings in Central Farm, LAI measurements were performed, on December 2 nd . Data showed the plant density as the factor that influenced the LAI values in the different combinations (genotype x plant density). At the same plant density, no significant differences were found between genotypes (Figure 3.17). Although not significantly different, LAI of Belizean plants were the lowest among genotypes at high density (circa 2500 plants ha -1 ) and LAI of Guatemalan plants were the highest among genotypes at low density (circa 1250 plants ha -1 ).

63 3.Results and discussions

LAI measurements on jatropha of three accessions and two plant densities, Belize, 2009 - Central Farm trial

0.005 a

a ab 0.004

0.003 Mexico bc Guatemala c c 0.002 Belize

LAI [m2*m-2] LAI 0.001

0.000 ca 1250 plants*ha-1 ca 2500 plants*ha-1 Plant density Figure 3.17. LAI measurements of J. curcas L. two month old seedlings of three accessions, from Mexico (low-toxic), Guatemala (G17) and Belize (local), at two plant densities, Central Farm, Belize, 2009.

LAI measurements performed after transplanting in the fields of Central Farm resulted in smaller values than the ones in the nursery in Bullet Tree, even if occurred one week after. The reason might be searched in the loss of leaves by the plants during the transportation from the nursery to the field ('transplanting shock').

3.3.2 Plant spacing

Results from Warrie Head trial deal with LAI and length of effective branch, measured in four treatments, as a result of the combination of two genotypes (Guatemalan and Cuban jatropha accession), and two plant spacing designs, single row (4*1m 2) and double (3*1.7)*1.7m 2. To compare the mean values of the four treatments, LAI values are first presented by date and in a summary graph for all dates. On September 2 nd , the only statistically significant difference was found between the 'Guatemala-double row' plot and the 'Cuba-single row', with LAI values greater than 0.08 in the first one and below 0.06 in the second. The other two plots, 'Cuba-double row' and 'Guatemala-single row', did not show statistically significant differences with any of the treatments (Figure 3.18). At the

64 3.Results and discussions time of the second survey, on October 2 nd , no differences were noticed between the LAI values for the four treatments (Figure 3.19). Later on in the season, statistically significant differences were observed between LAI values in 'Cuba-double row' treatment, with more than 0.06m 2 of leaf per m 2 of soil, and the two accessions in 'single row', both of them having LAI between 0.03 and 0.04, but these were not significantly different. LAI of jatropha in 'Guatemala-double row' plot was not significantly different from the other three treatment (Figure 3.20). Again, towards the end of the growing season, on December 2 nd , no significantly differences were observed between the LAI of jatropha growing under the four treatments, being at values around 0.025 (Figure 3.21).

LAI measurements on jatropha, Belize, 2009 September 2nd - Warrie Head trial 0.09 a

0.08 ab 0.07 b ab 0.06 0.05 0.04 0.03 0.02 LAI [m2*m-2] LAI 0.01 0 Cuba, (3*1.7)*1.7m2 Guatemala, 4*1m2 Guatemala, (3*1.7)*1.7m2 Cuba, 4*1m2 Treatment (Genotype, Spacing) Figure 3.18. LAI measurements on J. curcas L. on September, 2 nd , in Warrie Head, Belize, 2009. The same letters above the histograms indicate that between the treatments significant differences were not found at the Duncan test, with P=0.01.

65 3.Results and discussions

LAI measurements on jatropha, Belize, 2009 October 2nd - Warrie Head trial 0.09 0.08 a 0.07 a a 0.06 a 0.05 0.04 0.03 0.02 LAI [m2*m-2] LAI 0.01 0 Cuba, (3*1.7)*1.7m2 Guatemala, 4*1m2 Guatemala, (3*1.7)*1.7m2 Cuba, 4*1m2 Treatment (Genotype, Spacing) Figure 3.19. LAI measurements on J. curcas L. on October, 2 nd , in Warrie Head, Belize, 2009. The same letters above the histograms indicate that between the treatments significant differences were not found at the Duncan test, with P=0.01.

LAI measurements on jatropha, Belize, 2009 November 2nd - Warrie Head trial 0.09 0.08 0.07 a 0.06 0.05 ab 0.04 b b 0.03 0.02 LAI [m2*m-2] LAI 0.01 0 Cuba, (3*1.7)*1.7m2 Guatemala, 4*1m2 Guatemala, (3*1.7)*1.7m2 Cuba, 4*1m2 Treatment (Genotype, Spacing) Figure 3.20. LAI measurements on J. curcas L. on November, 2 nd , in Warrie Head, Belize, 2009. The same letters above the histograms indicate that between the treatments significant differences were not found at the Duncan test, with P=0.01.

66 3.Results and discussions

LAI measurements on jatropha, Belize, 2009 December 2nd - Warrie Head trial 0.09 0.08 0.07 0.06 0.05 0.04 0.03 a a a a 0.02 LAI [m2*m-2] LAI 0.01 0 Cuba, (3*1.7)*1.7m2 Guatemala, 4*1m2 Guatemala, (3*1.7)*1.7m2 Cuba, 4*1m2 Treatment (Genotype, Spacing) Figure 3.21. LAI measurements on J. curcas L. on December, 2 nd , in Warrie Head, Belize, 2009. The same letters above the histograms indicate that between the treatments significant differences were not found at the Duncan test, with P=0.01.

An overview of the LAI measurements resulting from monitoring of jatropha in the Warrie Head plantation from September to December is hereunder provided. Spacing is influencing LAI of jatropha in the first part of the monitoring period, while effects caused by genotype are not statistically significant. This behaviour suggests a greater biomass development capacity in plants in a double row than the ones, more closed, in the single row, at the same plant density of 2500 trees ha -1 . However, the jatropha plants were relatively young (only fifteen months) and have never been pruned, and they did not have many lateral branches. Factor 'date' showed its effects especially reducing differences towards the end of the season. LAI values in 'Guatemala-double row' had a more gradual reduction towards the end of the growing season than LAI in the other three treatments. 'Cuba-double row' LAI showed an abrupt difference between November and December, while LAI of jatropha in 'single row', between October and December (Figure 3.22). The design of the Warrie Head experiments were blocked perpendicular on the slope gradient; however, it might be that the location of the 'Guatemala-double row' treatment, closer to the margins of the forest and to the river, could have attenuated the effects of the weather variables along the season, especially in

67 3.Results and discussions the drought of October. Moreover, the analysis of the variance highlighted differences among the blocks, with the block in the middle having generally higher values than the ones on the sides.

Additional information on jatropha growth potentials is obtained through the calculation of intercepted radiation by the plants in the growing season. As they are mainly depending on the LAI, values of intercepted radiation are following the same LAI trends discussed above. Solar radiation was slightly decreasing from September to December, however, due to limited interception capacity of the low LAI, plants were only able to use a small amount of the available radiation (Figure 3.23; note the log scale). This situation might suggest a potential improvement of solar radiation use by jatropha, in all the situations. Basically, to improve radiation interception, LAI should be increased by management actions that, on the one hand, induce more and larger branches and leaves that stay green longer in the season, such as pruning (strongly recommended as the plantation is still young), and, on the other hand, increase plant density and plant capability to completely close the rows, such as spacing at transplanting and curbing

68 3.Results and discussions branches, thus to optimize the use of available space.

Solar radiation, jatropha LAI and intercepted radiation, Belize, 2009 Warrie Head trial 100

10

Radiation [kJ m -2 d-1] 1 LAI [m2 m-2] Intercepted radiation [kJ m -2 d-1] 0.1

0.01 SONDSONDSONDSOND Treatment (from left to right: Guatemala- and Cuba-double row, Cuba- and Guatemala-single row ) and date (September, October, November, December)

Figure 3.23. Solar radiation, LAI of jatropha under different treatments (combinations of two genotypes and two spacings: Guatemala-double row, Cuba-double row, Cuba-single row and Guatemala-single row) and intercepted radiation are represented from September to December, in Warrie Head, Belize, 2009.

The 'effective branch' length measurements supported the results obtained for LAI, although not completely, highlighting statistically significant differences between dates, October 2 nd and December 2 nd (Figure 3.24) and blocks. No significant differences were found between genotypes and spatial arrangement of the rows. The lengths of effective branch in Warrie Head trial were similar to the ones in Maya Ranch, during the same dates of survey, mainly around 10-12cm in November and 4-5cm in December. However, in relative terms, length of effective branch was almost double in Maya Ranch than in Warrie Head, being around 20% and 10% of the whole branch length in the first site and around 10% and 5% in the second, in the dates considered (Figure 3.25).

69 3.Results and discussions

Jatropha 'effective branch' length, Belize, 2009 Time series - Warrie Head trial

20 a a a 15 ab abc abc abc abc 2009.10.02 10 2009.11.02 bc 2009.12.02 bc 5 c c

Effective branch [cm] Effective 0 Cuba, (3*1.7)*1.7m2 Guatemala, 4*1m2 Guatemala, (3*1.7)*1.7m2 Cuba, 4*1m2 Treatment (Genotype, Spacing) Figure 3.24. Length of the 'effective branch' (part of the branch with green leaves on), in J. curcas L., Warrie Head, Belize, for October 2 nd , November 2 nd and December 2 nd , 2009. The same letters above the histograms indicate that between the treatments significant differences were not found at the Duncan test, with P=0.01.

Jatropha 'effective branch' relative lenght, Belize, 2009 Time series - Warrie Head trial 25

20

15 2009.10.02 2009.11.02 10 2009.12.02

5

0 Cuba, (3*1.7)*1.7m2 Guatemala, 4*1m2 Guatemala, (3*1.7)*1.7m2 Cuba, 4*1m2

Effective branch totalbranch asEffective of [%] Treatment (Genotype, Spacing) Figure 3.25. Relative length of the 'effective branch' (part of the branch with green leaves on) as percentage of the whole branch, in J. curcas L., Warrie Head, Belize, for October 2 nd , November 2 nd and December 2 nd , 2009.

70 3.Results and discussions

3.3.3 Crop management

As in the case of Warrie Head trial, assessing jatropha biomass development was the objective of the research in the Maya Ranch plantation, as well. Results from Maya Ranch trial refer to yield, LAI and length of the effective branch.

Firstly, results from fruit and seed production are given. It must be underlined that yield values are referred to as dry weight production. Separately, for the two plots considered and for the two dates of harvesting, yield values are given under three forms: reported as harvested per plot, as an average per plant and extrapolated to a unit surface basis (kg ha -1 ). Data are indicating different production potentials in the plots considered (Table 3.1). Over the season, in the treatment 'Not pruned' , where plants have never been pruned and were growing in intercropping with A. pintoii , jatropha fruit production was double than the one in the treatment 'Pruned' , where, instead, jatropha plants were growing in monoculture and have been pruned two years before (Figure 3.26). Specifically, fruit production from 'Not pruned' treatment was almost triple at the moment of the first harvest and one tenth higher at the second harvest. However, fruit production trends revealed important differences between the time of the two harvests; in fact, during the season, production from 'Not pruned' treatment decreased almost by a half, while production from 'Pruned' plot increased by 70% (Figures 3.27 and3.28). Cautiously, it has to be reminded here that production losses seemed to be significant: in fact, by the time of the first harvest, fruits were already on the ground and have not been collected; moreover, after the second and last harvest, fruits have been seen on the plants and on the ground up till mid November, and some plants were still flowering in October. These considerations suggest that potential production is bigger than measured, although it is difficult to determine how bigger. An estimation of the production that could not be harvested may be possible, considering the fruiting period (mid July to end October), the harvested yield in the period from July 27 th to August 10 th and its trend for the different treatments, the average dry fruit production per plant (0.26kg for ' Not pruned' and 0.14kg for 'Pruned' treatment), the number of branches per plant (23 for 'Not pruned' and 16 for 'Pruned' treatment), the fruit ripening period (40-80 days), the

71 3.Results and discussions

LAI development and the weather variables trends. It may be concluded that dry fruit potential yields would have been around 400-450kg in 'Not pruned' treatment and around 250-300kg in 'Pruned' treatment.

Table 3.1. Dry fruit production of J. curcas L. with two treatments: i)never pruned and in intercropping with A. pintoii (Not pruned), and ii) pruned two years before and in monoculture (Pruned), in Maya Ranch, Belize, 2009.

Fruit Production Not pruned Pruned Area (m2) 1728 960 # of plants 216 120 Yield 2009.07.27 (kg) 37 7 kg*plant-1 0.17 0.06 kg*ha-1 214 73 Yield 2009.08.10 (kg) 20 10 kg*plant-1 0.09 0.08 kg*ha-1 116 104 Total yield (kg) 57 17 kg*plant-1 0.26 0.14 kg*ha-1 330 177

Total jatropha yield, Belize, 2009 Maya Ranch trial 350

300

250 Fruit [kg*ha-1] 200 Seed [kg*ha-1] Kernel [kg*ha-1] 150

[kg*ha-1] 100

50

0 Not pruned Pruned Figure 3.26. J. curcas L. dry fruit, dry seed and kernel total productions for 'Not pruned' and 'Pruned' treatments, in Maya Ranch, Belize, 2009. Jatropha plants were growing in intercropping with A. pintoii and have never been pruned ('Not pruned' treatment) or were growing in monoculture and have been pruned two years before ('Pruned' treatment).

72 3.Results and discussions

Jatropha yield from 'Not pruned' plot, Belize, 2009 Maya Ranch trial 250

200

Fruit [kg*ha-1] 150 Seed [kg*ha-1] Kernel [kg*ha-1] 100 [kg*ha-1]

50

0 2009.07.27 2009.08.10 Figure 3.27. J. curcas L. dry fruit, dry seed and kernel productions from 'Not pruned' treatment, represented by harvests on the 27 th July and 10 th August, in Maya Ranch, Belize, 2009. Jatropha plants were growing in intercropping with A. pintoii .

Jatropha yield from 'Pruned' plot, Belize, 2009 Maya Ranch trial 250

200

Fruit [kg*ha-1] 150 Seed [kg*ha-1] Kernel [kg*ha-1] 100 [kg*ha-1]

50

0 2009.07.27 2009.08.10 Figure 3.28. J. curcas L. dry fruit, dry seed and kernel productions from 'Pruned' treatment, presented by harvests on the 27 th July and 10 th August, in Maya Ranch, Belize, 2009. Jatropha plants have been pruned two years before and were growing in monoculture without intercropping.

Further measurements and calculations on the harvested yield have been undertaken to establish dry matter assignment in the fruit. Seed to fruit ratio and kernel to seed ratio

73 3.Results and discussions were calculated, separately per date and per treatment. On a dry matter basis, seed weights were 70.5% to 74.3% of the dry fruit weight; while kernel weight was between 63% and 65% of the dry seed weight (Table 3.2).

Table 3.2. Dry fruit weight, seed to fruit ratio and kernel to seed ratio of J. curcas L. in two treatments: i)never pruned and in intercropping with A. pintoii ('Not pruned'), and ii) pruned two years before and in monoculture ('Pruned'), in Maya Ranch, Belize, 2009. 2009.07.27 2009.10.08 Total 2009 Not pruned Pruned Not pruned Pruned Not pruned Pruned Fruit (kg) 37 7 20 10 57 17 Fruit sample (g) 740 140 400 200 1140 340 of which seed (g) 530 104 282 141 812 245 Seed:Fruit (%) 71.62 74.29 70.5 70.5 71.23 72.06 Seed sample (g) 100 100 100 100 200 200 of which kernel (g) 64 65 63 65 127 130 Kernel:Seed (%) 64 65 63 65 63,5 65 Fruit (kg*ha-1) 214 73 116 104 330 177 Seed (kg*ha-1) 153 54 82 73 235 128

A thousand dry fruit sample was randomly collected from the whole production of the trial: seed dry matter was about 71% of dry fruit weight, which agreed with previous measurements (Table 3.3). An average value 2.65 seeds per fruit was recorded (Table 3.4).

Table 3.3. J. curcas L. fruit coat to seed ratio, on a dry weight basis, from one thousand dry fruit samples randomly collected in Maya Ranch, Belize, 2009. DRY ITEM QUANTITY (#) WEIGHT (g) (%) Fruits 1000 1681 100.00 Coats 1000 485 28.85 Seeds 2648 1196 71.15

Table 3.4. Number of seeds per fruit of J. curcas L., from one thousand dry fruit samples randomly collected in Maya Ranch, Belize, 2009. Number of fruits with: (#) 4 seeds 2 3 or 2 seeds 962 1 seed 36 Average # of seeds*fruit-1 2.65

74 3.Results and discussions

At Maya Ranch trial, further research was carried out to determine biomass development by monitoring LAI and length of the effective branch. At the time of the first survey, on September 1 st , monitored plants were grouped according to their management (Not pruned/Intercropping, Pruned/Monoculture or Not pruned/Monoculture), showed LAI values between 0.7 and 0.8 for the 'Pruned/Monoculture' and the 'Not pruned/Intercropping' treatments, which were significantly larger than the values of 'Not pruned/Monoculture' treatment (Figure 3.29); although, this difference was not significant later on the season. LAI values were much smaller (around 0.2; Figures 3.30, and 3.31) for the Not pruned/Monoculture treatment. LAI values reported in Maya Ranch are altogether presented in Figure 3.32, where management and date effect can be visually compared.

LAI measurements on jatropha, Belize, 2009 September 1st - Maya Ranch trial 0.9 a 0.8 a 0.7 0.6 0.5 0.4 0.3 b

LAI [m2*m-2] LAI 0.2 0.1 0 Not pruned/Intercropping Pruned/Monoculture Not pruned/Monoculture Management Figure 3.29. LAI measurements on J. curcas L. on September, 1 st , in Maya Ranch, Belize, 2009. The same letters above the histograms indicate that between the treatments significant differences were not found at the Duncan test, with P=0.01.

75 3.Results and discussions

LAI measurements on jatropha, Belize, 2009 November 1st - Maya Ranch trial 0.9 0.8 0.7 0.6 0.5 0.4 0.3 a a LAI [m2*m-2] LAI 0.2 0.1 a 0 Not pruned/Intercropping Pruned/Monoculture Not pruned/Monoculture Management Figure 3.30. LAI measurements on J. curcas L. on November, 1 st , in Maya Ranch, Belize, 2009. The same letters above the histograms indicate that between the treatments significant differences were not found at the Duncan test, with P=0.01.

LAI measurements on jatropha, Belize, 2009 December 1st - Maya Ranch trial 0.9 0.8 0.7 0.6 0.5 0.4 0.3 a

LAI [m2*m-2] LAI a 0.2 a 0.1 0 Not pruned/Intercropping Pruned/Monoculture Not pruned/Monoculture Management Figure 3.31. LAI measurements on J. curcas L. on December, 1 st , in Maya Ranch, Belize, 2009. The same letters above the histograms indicate that between the treatments significant differences were not found at the Duncan test, with P=0.01.

76 3.Results and discussions

LAI measurements on jatropha, Belize, 2009 Time series - Maya Ranch trial 0.9 a 0.8 a 0.7 0.6 September 0.5 November 0.4 December b 0.3 b b b

LAI [m2*m-2] LAI b 0.2 b 0.1 b 0 Not pruned/Intercropping Pruned/Monoculture Not pruned/Monoculture Management Figure 3.32. LAI measurements on J. curcas L. in Maya Ranch, Belize, 2009. Data are presented in time series, according to the surveys over the season, occurred in September 1 st , November 1 st and December 1 st . The same letters above the histograms indicate that between the treatments significant differences were not found at the Duncan test, with P=0.01.

Values of intercepted radiation by jatropha plants in Maya Ranch trial followed the same trends discussed for the LAI. Solar radiation values are the same as for Warrie Head, since they have been recorded in the same meteorological station. It is clear that there is a considerable loss of solar radiation by jatropha in all the situations, over the season (Figure 3.33; note the log scale). Similar management actions as discussed for plantation at Warrie Head should be undertaken.

77 3.Results and discussions

Solar radiation, jatropha LAI and Intercepted Radiation, Belize, 2009 Maya Ranch trial 100

10

Radiation [kJ m -2 d-1] 1 LAI [m2 m-2] Intercepted radiation [kJ m -2 d-1] 0.1

0.01 SONDSONDSOND Treatment (from left to right: Not Pruned/Intercropping, Pruned/Monoculture, Not Pruned/Monoculture) and month (September, October, November, December)

Figure 3.33. Solar radiation, LAI of jatropha under different treatments (managements: Not pruned/Intercropping, Pruned/Monoculture and Not pruned/Monoculture) and intercepted radiation are represented from September to December, in Maya Ranch, Belize, 2009.

The lengths of the effective branch have been recorded twice during the season and results showed values of 12cm to 18cm on November 1st and around 4cm on December 1st . However, treatments did not evidence statistically significant differences on effective branch length, while differences appeared considering the date of survey (except in the case of 'Pruned/Monoculture' plot).Overall, the longest effective branch of 'Not pruned/Monoculture' plot in November and the shortest of 'Not pruned/Intercropping' plot in December have been pinpointed (Figure 3.34). In figure 3.35 the length of the effective branch in relation to the total length of the representative branch is presented.

78 3.Results and discussions

Jatropha 'effective branch' length, Belize, 2009 Time series - Maya Ranch trial 20 a

16

ab abc 12 2009.11.01 8 2009.12.01 bc c bc 4

Effective branch [cm] Effective 0 Pruned/Monoculture Not pruned/Intercropping Not pruned/Monoculture Management Figure 3.34. Length of the 'effective branch' (part of the branch with green leaves on), in J. curcas L., Maya Ranch, Belize, for November 1 st and December 1 st , 2009. The same letters above the histograms indicate that between the treatments significant differences were not found at the Duncan test, with P=0.01.

Jatropha 'effective branch' relative length, Belize, 2009 Maya Ranch trial 40 35 30 25 20 2009.11.01 2009.12.01 15 10 5 0 Pruned/Monoculture Not pruned/Intercropping Not pruned/Monoculture

Effective branch totalbranch asEffective of [%] Management Figure 3.35. Relative length of the 'effective branch' (part of the branch with green leaves on) as percentage of the whole branch, in J. curcas L., Maya Ranch, Belize, for November 1 st and December 1 st , 2009.

Regrettably, the survey could not be completed over the whole growing season, so the growth curve for LAI and length of the effective branch can not be presented.

79 3.Results and discussions

It is noted that there is a sudden lowering of LAI values in the period September – November, especially in 'Not pruned/Intercropping' and 'Pruned/Monoculture' treatments; while in the period November – December LAI measured in the same treatments is decreasing much slower, and even is increasing in 'Not pruned/Monoculture' treatment, although differences are not significant. This fact can be linked with the relatively low precipitation in October and a remarkable restart of the rainfall in November and December. The additional information, provided by the length of effective branch, suggest that between November and December the branch length in which green leaves were growing was reduced but the leaf area did not vary, since effective branch length decreased while LAI values kept being similar. Indeed, from November to December, some leaves died thus reducing the space occupied on the branch and some others leaves grew or in area or in number. Due to the favourable precipitation events in November and December, jatropha might have developed and increased the area of the already existing leaves or a new significant sprouting might have occurred.

Observations in the two experimental sites (6 year old Maya Ranch and 1.25 year old Warrie Head) lead to the following considerations. Results on biomass development (LAI and effective branch) suggest that, even if the length of representative branch was recorded to be greater in Warrie Head trial, jatropha plants in Maya Ranch had a significantly larger LA and longer segments of branch with green leaves and more branches. The reason for this is the different age, management (pruning and plant density), genotype and degree of adaptation to the location; therefore, reasons of different plant development should be cautiously studied.

3.4 Discussions

Field research in Belize allowed to monitor Jatropha curcas L. growth and development under a wide range of conditions. Many different environmental variables have influenced jatropha growth in the surveyed trials. Trials in Maya Ranch, Warrie Head,

80 3.Results and discussions

Central Farm (and Bullet Tree) represent a wide range of different environmental factors and differences in plant genotype, age, crop management (treatments: pruning, intercropping, spacing), that unravelling the effects among these factors in jatropha growth and development is challenging. At this point in time, after a relatively short period of on field research, it is difficult to weigh the outputs and quantify the outcomes of all the experiments considered. Last but not least, there may exist a difference in weather variables that could not be specified per location, since the same meteorological stations were used in the experimental sites. Some constraints were encountered and scientific logic had to be developed in the process. Observations for the three trials with possible comparisons are highlighted and specific actions on further research activity are suggested.

As far as it is known, Belizean oldest jatropha plantation is located in Maya Ranch and it has to be considered a valuable source of information. The present investigation provides additional information to research previously undertaken in Maya Ranch. So far, it looked like jatropha in Maya Ranch received a benefit from the intercropping with Arachis . Not pruned plants might have larger biomass development, while pruned plants keep both leaf and fruit production slightly delayed in the season: the reasons could be that the plants still invest more biomass in new branches and have a more compact but intense growth. Drought in October might have negatively affected leaves development, but re-occurring rains in November restored the plants. Management actions, such as weeding, could have influenced jatropha growth more, by eliminating those plants that compete for resources and by leaving the vegetable material in the field, as organic fertilizer. In general, the jatropha plantation at Maya Ranch is looked after with interest and this potential could be valued even more, since a selection within the local genetic resources could start from here. To follow this aim, production per single plant in a defined production system need to be assessed, in order to start a selection that would eventually lead to a breeding program. For this purpose, harvested seed should be isolated per tree and number of fruits per should be monitored. The seed production reached in 2009 does not justify a commercial implementation of the crop.

81 3.Results and discussions

Measures have to be undertaken in order to increase the production. An action to improve crop production is making better use of the available space, that is to occupy the inter- an intra-row space in order to give the plant a better chance of developing lateral branches, bringing along the benefit of reducing weed growth; bending long branches downward without breaking them could have a beneficial impact in order to achieve the mentioned objective; also in this sense, knowledge in jatropha cultivation is increasing and suggestions are made about yearly pruning in the dry season, setting principal branches at about 50cm from the ground, leaving about 20cm of the original branches and about 7-8 branches per plant (Stanningen, personal comm.). Moreover, it has to be understood why, within the plot, some plants are weaker, smaller and were not yielding; with regard to this, soil analysis are strongly recommended. After all, Maya Ranch is an ideal place where to test different management actions on local genetic material, so to analyse the responses and elaborate a jatropha cultivation decision- support system.

The Warrie Head trial is set up with some of the considerations mentioned for Maya Ranch, with a few exceptions. At Warrie Head, the plantation is five years younger and three times bigger, and moreover it involves different genotypes and management treatments. In general, LAI values measured in Warrie Head trial were, even at a double plant density, about ten times smaller than the ones measured in Maya Ranch. Plant spacing, at Warrie Head plantation, played a significant role in LAI development, with the 'double-row' system being more efficient, even if plant architecture (single stem) of both systems and genotypes did not appear the most efficient for optimized seed production. Firm actions should be undertaken, i.e. start with pruning to induce branching (Stanningen, personal comm.). The crop in Warrie Head might have suffered from the drought in October, as well, and the response to the unexpected rains in November was not so evident. For further investigations, consequences of intercropping or application of effective microorganism should be analysed. Furthermore, the Cuban accession (referred to as 'Cabo-Verde') should be surveyed, as 'Cabo Verde' is the most productive accession to date in many areas. Further research could be implemented

82 3.Results and discussions since an irrigation system is available, where river water can be used to overcome water shortage. At this point and time, TSDF together with the Energy and Environment Partnership (EEP) is aiming to continue the research and the set up of a breeding program should bring along beneficial consequences also in this situation. Further investigations both in Warrie Head and Maya Ranch would necessarily have to deal with the fact that plants were already in the ground and some factors would have to be considered fixed; in any case the surrounding environment is promising and expectations are high.

Adding knowledge and experience to current research is the jatropha plantation in Central Farm, one of the latest pilot project set up in Belize, thanks to the ERA-ARD transnational call. Results of the first three months of surveying, since its original implementation, brought to considerations that should be discussed with similar trials within the net. According to the data analysis, it seemed that the largest seeds, from Guatemalan accession G17, resulting in a considerable germination rate, gave also the largest amount of biomass in seedlings. The low-toxic accession from Mexico and the local accession from Belize showed different germination patterns, but eventual biomass development did not differ significantly. Mexican low-toxic accession seemed to have a superior germination power resulting in the highest germination rate and a discrete degree of adaptation for its greater increase in LA between the two dates of survey. Belizean local accession, instead, showed satisfying patterns of germination, growth and development, although considered average when compared to the other two accessions. A selection of plant material should be done as the next step in developing Belize jatropha scenario. Hopefully, the project outputs will contribute to a beneficial application of jatropha system in Belize and worldwide. A summary of results of this work is reported in the column 'Research in Belize', in tables a,b,c and d of annex 1.

83

4. Conclusions

Energy demand and environmental awareness are pushing world community to search for alternatives to fossil fuels, both in developing and developed countries. With regard to this search for alternatives, a significant question is emerging about new energy source, what choice should at the same time mitigate global warming, reduce oil dependence, and, possibly, improve quality of life globally. Thence, numerous solutions to face energy hunger worldwide, without compromising the health of the planet, seem available nowadays and may vary according to regional peculiarities. In fact, issues, such as food security, land property and division, technology availability, market competition, social disparity and natural constraints among others, might affect local and regional decision-making processes towards selection and definition of a sustainable energy system. In this sense, comprehensive policies are strongly recommended to initiate such sustainable energy process, together with the implementation of research and development projects on new suitable energy sources.

The present research investigated the cultivation of Jatropha curcas L., as one of the potential candidate crops as renewable energy source in Belize is, indeed, jatropha . This crop appears to be a suitable and valuable option to meet the needs of the country because of its remarkable features. Findings and challenges that the start up of a jatropha based bioenergy system would bring are hereunder discussed.

The implementation of jatropha cultivation in Belize is an opportunity because of the following good reasons. Jatropha is native to Belize, and researches suggested genetic resources availability and a proper pedoclimatic situation for its growth and development in the country. Moreover, land is available and the integration of jatropha in existing farming systems (especially intercropping and agro-forestry systems) or its use as hedge seem feasible and helpful, as its cultivation is easy to implement at a small-farm scale and low technology inputs are required. In fact, being a perennial drought tolerant energy crop brings to a series of positive patterns, even at low seed production levels: there are no needs for fulfilment of annual agricultural practices that often require appropriate machinery and structures (e.g. tillage, sowing); it withstand the possibility of one or more years of interruption of cultivation for climatic or human reasons; its cultivation provides valuable co-products such as leaves,latex, fruits coats, 4.Conclusions seed cake and shells for medicinal uses, fertilizers, insecticides, soap production or combustibles, as well, according to researchers' judgements and findings in similar agro-ecological zones (Baldrati, 1950; Heller, 1996; Rijssenbeek and Togola, 2007; Kumar and Sharma, 2008; Jongschaap et al. , 2007; Achten et al. , 2008; Ogunwole et al. , 2008). Low technology inputs are required on the cultivation step ,where most of the management actions are done manually (pruning and harvesting, mainly), and on oil processing. In fact, BD20 blends (20% of vegetable oil and 80% of diesel) are possible and, after all, would greatly reduce Country costs for energy.

In Belize, interests on jatropha have been shown at local national and regional levels. In the Country, the Ministry of Agriculture is going to start up a five acre jatropha pilot plantation to optimize the agro-technology and bring it to farmers (Martinez, personal comm.); private investors are increasing since few years, both for research purposes and commercial activity set up; the NGO Tropical Studies and Development Foundation Belize is conducting research projects together with the Energy and Environment Partnership (EEP), the Organization of the American State (OAS), the ERA-ARD net, and its role as pioneer on jatropha research throughout the Country is remarkable. At regional level, Central America and Caribbean Countries are cooperating and pushing for a concrete development of bioenergy regulations and for a deep technology research. Particularly, Belize, represented by the Ministry of Agriculture, the University of Belize and TSDF, signed a Memorandum of Understanding, planning further collaboration and research together with , Costa Rica, , Guatemala, , Mexico, Nicaragua, and at the 'First Reunion of the Biofuels Research and Development Network' of the Mesoamerican Project (Primera Reunión de la Red Mesoamericana de Investigación y desarrollo en Biocombustibles, 23-27 de Agosto, 2009, Tuxtla-Gutierrez, , México). With regard to this, institutional strengthening at national and regional scale is recommended, and, in Belize, the creation of a platform to host highly scientific researches and knowledge exchange would be very welcome.

Next steps that would allow to unravel jatropha potentials in Belize include: the

86 4.Conclusions selection of high-yielding individuals and the set up of a breeding programme; feasibility studies of jatropha cultivation in different Districts; regulation of bioenergy sector, to promote the birth of an equitable market for jatropha and other bioenergy products. What is more, the existing knowledge should be integrate with next findings and the wide range of possibilities linked to a jatropha production system should be shared with farmers and growers associations. Ultimately, as motivating the first actors (the farmers) in this value chain is essential, possibilities should be exhaustively investigated both on developing agro-forestry applications suitable in Clean Development Mechanism projects or other rural development and bioenergy projects and, what is more, oil extraction and processing technology should be brought and developed in Belize.

87

5. Acknowledgements

All around the world, many friends and tutors I met along the road and I thank them all for helping and talking and walking with me to the slopes of the mountain. My sincere gratitude goes to Sylvia and Alex Laasner and TSDF Belize, who welcomed me from the very beginning until the last day, providing the basic equipment to carry out the research activity in the field and in the office and indicating me the borderline between the 'endless research' and the implementation of research findings into concrete actions; to Michael Rosberg, Marion Cayetano and Sylvia Carillo from Galen University, who stimulate a debate on jatropha (and helped me getting the visa); to Dr. Holder, Dr. Mendez and Maynor Hernandez from University of Belize, for their essential collaboration; to Ann Gordon and Michelle Smith of the National Meteorological and Hydrological service for their most valuable contribution in providing all the climatic data; to Eva, Toby, Alan and all TSDF team, who could translate theory to practice, implementing the experimental design so efficiently; to the Mexican Secretary of Foreign Affairs and to COCyTECH and the State of Chiapas, Mexico, who permitted to bring the debate on bioenergy and jatropha at international level, fostering cooperation and knowledge exchange between Central American States, during the First Reunion of the Mesoamerican Network on Biofuels Research and Development in Tuxtla-Gutierrez, Chiapas; to Pio Saki from the University of Belize and to Clifford Martinez from Belizean Ministry of Agriculture, who joined me, as TSDF representative, and represented Belize in that reunion; to Hans Stanningen, for sharing his knowledge on jatropha cultivation; to John and Richard, to the friends of 6, Bladen street, especially Mrs and her familiy and, the best chess player, nurse Robert John Ilao, to take care of me and make my experience in Belize more sustainable. Some friends, in Belize, are pictured in figures 5.1, 5.2, 5.4 and 5.3.

I thank professor Berti, for the valuable contribution on structuring the statistical analysis of this thesis.

My gratitude also goes to my friends and my family in Italy, to Papà, who stimulates a critic dialogue on agriculture, together with Diego and my friends of studies Mariano, Lorenzo, Marco, Alvise, Alessandro, Giulio and Linda; to Mamma, Clara, Nicola, 5.Acknowledgements

Maria, Girolamo and all the members of my family to support (and 'sopport') me until here, especially Zia Agnese, who taught me the first steps in the world of Research, and Zia Lavinia, who constantly stimulate my research and interests forwarding me agricultural information and news on jatropha. I thank my grandparents Nonna Paola and Nonno Berardo, Nonna Clara and Nonno Beppi, for their best lessons of life, being the most precious model I have ever known. Last but not least, I thank Valentina, for patiently reviewing drafts of this M.Sc. thesis, finding better expressions, being an outsider, and for showing me the power of love.

I apologize if I missed someone but I guarantee: I did not forget anyone.

Figure 5.1. Harvesting with Rubelio and his Figure 5.2. Transplanting jatropha with Toby, two brothers, in Maya Ranch, Belize, July Alan and TSDF team, in Central Farm, 2009. Belize, November 2009.

Figure 5.4. Surveying jatropha plantations, with Hans Stanningen and Toby Sengfelder, Cayo District, Belize, October, 2009. Figure 5.3. Surveying jatropha plantations, with Raymond Jongschaap, Cayo District, Belize, October, 2009 90 Annex 1. Growth parameters and sustainability indicators tables

Annex 1a. Growth parameters by plant part

Growth Parameter Unit or evaluating pm Measurement Literature Research SEED in Belize Seed germinability % 89 (a) Kaushik et al., 2007 80-96 Seed germination energy days to germination 10 Henning, 2007 7-14 (a) Seed yield (dry seed) t*ha-1*yr-1 1.5-7.8 Jongschaap et al., 2007 0.13-0.24 (b) Seed size length (cm) 1-2 Henning, 2007 1.8-1.9 width (cm) 1-1.1 HI-seed kg*kg-1 0,35 Jongschaap et al., 2007 100-seed weight g 63 Singh et al., 2008 54-73 1000-seed weight g 400-730 Henning, 2007 Shell:Kernel ratio by DM % 34.3:65.7 Openshaw, 2000; Singh et al., 2008 33-37:63-67 Oil content in seed % 33.6-37.0 Rivera Lorca and Ku Vera, 1997 Oil content in kernel % 21-74 Shah et al., 2005 Oil yield l*ha-1 439-2.217 Jongschaap et al., 2007 HI-oil l*kg-1 0,1 Jongschaap et al., 2007 Oil quality Different compounds and composition Henning, 2007; Jongschaap and van Loo, 2009 DMA-seed % 32 Jongschaap (personal comm.) DMA-shell % 11 Jongschaap (personal comm.) DMA-kernel % 21 Jongschaap (personal comm.) Nutrient composition (NPK, …) % existing data Jongschaap et al., 2007 ROOT # roots*plant-1 n 8-16 (j) Kaushik et al., 2007 Length (aft. 90 days) cm 10-17 Kaushik et al., 2007 19-25 (c) Root hairs yes/no Explored area by roots cm³ Water uptake mm*ha-1 Nutrient uptake (NPK) kg*ha-1*yr-1 existing data Jongschaap (personal comm.) Mychorrization (after inoculation) yes/no yes Jongschaap et al., 2007 DMA % 8 Jongschaap (personal comm.) Nutrient composition (NPK, …) % existing data Jongschaap et al., 2007 STEM/WOOD Cuttings survival rate (T,M,B) % 42-72-88 Achten et al., 2008 Seedling survival rate % 95-100 Kaushik et al., 2007 Total height (aft. 90 days) cm 29-47 Kaushik et al., 2007 17-20 (c) Total height m 1.15-1.34 Kaushik et al., 2007 0.5-2.5 (d) ; 2-4 (e) Collar diameter (aft. 90 days) cm 8-11 Kaushik et al., 2007 Crown size m 1.25-1.52 Kaushik et al., 2007 # branches *plant-1 (optimum) n 25 Henning, 2007 1-4 (d) ; 11-40 (e) Apical dominance low/high Montes et al., 2008 (f) DMA % 23 Jongschaap (personal comm.) Nutrient composition (NPK, …) % existing data Jongschaap et al., 2007 LEAF # leaves*plant-1 (aft. 90 days) n 13-20 Kaushik et al., 2007 1-5 (c) Leaf size length (cm) 7-18 Henning, 2007 width (cm) 5.5-18 Henning, 2007 LA m²leaf*plant-1 0.02-0.9 (k) Light interception k 0.55-0.68-0.75 Jongschaap (unpublished data) (g) Clorophyll content SPAD values existing data Jongschaap (unpublished data) PET, AET mm*day-1 Photosyntetic activity existing data Jongschaap (unpublished data) DMA % 23 Jongschaap (personal comm.) Nutrient composition (NPK, …) % existing data Jongschaap et al., 2007 FLOWER # male flowers*plant-1 n # female flowers*plant-1 n Male:female ratio n 13:1, 29:1 Achten et al., 2008 Location of flowers Female major axis, male lateral, branch age Henning, 2007 Cross:self-pollinated Abdelgadir et al., 2008 Pollination actors Moths yes(/no) Henning, 2007 Honeybees yes(/no) Abdelgadir et al., 2008 Small flower and abortion % up to 60 Jongschaap et al., 2007 FRUIT # fruits*plant-1 n 24-240 # fruits*branch-1 n 3-8 Fruit size length (cm) 2.5 Singh et al., 2008 Fruit setting % Ripening days 90 Coat:Seed ratio by DM n 30:70 Openshaw, 2000 29:71 HI-fruit kg*kg-1 0.50 Jongschaap et al., 2007 DMA % 46 Jongschaap (personal comm.) DMA-coat % 14 Jongschaap (personal comm.) Nutrient composition (NPK, …) % existing data Jongschaap et al., 2007 (continues in the next page) Annex 1. Growth parameters and sustainability indicators tables

PLANT Growth Parameter Unit or evaluating pm Measurement Literature in Belize Plant energy efficiency Nutrient cycles (macro/micro) Chaudary et al., 2008 Plant potential lifespan years 30-50 Henning, 2007 Genotype Fitness (h) Hormons Quality Quantity Location in plant organs Stress tolerance Abiotic Temperature (optimum) °C 20-28 Achten et al., 2 008; 20-33 (i) Rainfall (optimum) mm*ha-1*yr-1 1000-1500 Daey Ouwens et al., 2007 1476 R.U. % Radiation kJ*m-2* d-1 12-17 (i) Vapour pressure kPa 28-32 (i) Wind speed m*s-1 4-6 (i) Biotic Pest (types) Phytophagous, powdery mildew; more Henning, 2007; Daey Ouwens et al., 2007 Disease (types and plant organ) Cassava mosaic virus; more Henning, 2007; Daey Ouwens et al., 2007 Nutrient competition Water competition Energy competition

(a) The value may vary according to different pre-treatments. (b) Data not fully reliable for Belize (see section 3.2). (c) 60 days after sowing. (d) 1 year old plantation. (e) 6 years old plantation. (f) It is reported variability in plant architecture among different accessions. (g) 'k' varies according to LAI values, respectively >7; 7>LAI>1,5; <1,5. (h) Plant breeding still in its infancy. (i) Calculated on Minimum and Maximum monthly values in the growing season (June – December 2009). (j) At different planting times. (k) According to plant age and period during the growing season.

94 Annex 1b. Growth parameters by crop system

CROP SYSTEM Growth Parameter Unit or evaluating pm Measurement Literature Research Monoculture in Belize Plant density (a) plants*ha-1 1100-2500 Heller, 1996; Henning, 2007 1250-2500 Light interception radiation intercepted*m-2 Energy use efficiency LAI Canopy size m³ Total aerial biomass kg*ha-1 Root system development kg*ha-1 m³*plant Seed yield (dry seed) kg*ha-1 HI-seed Water uptake mm*ha-1 PET, AET mm*ha-1*day-1 Water use efficiency Nutrient uptake (NPK) kg*ha-1*yr-1 existing data Jongschaap (personal comm.) Plant potential lifespan years Interactions with neighbour plants Effects Intercropping Plant density (a) plants*ha-1 Light interception Energy use efficiency LAI Canopy size m³ Total aerial biomass kg*ha-1 Root system development kg*ha-1 m³*plant Seed yield (dry seed) kg*ha-1 HI-seed Water uptake mm*ha-1 PET, AET mm*ha-1*day-1 Water use efficiency Nutrient uptake (NPK) kg*ha-1*yr-1 existing data Jongschaap (personal comm.) Plant potential lifespan years Interactions with neighbour plants Effects Living fence Plant density (a) plants*ha-1 Light interception Energy use efficiency LAI Canopy size m³ Total aerial biomass kg*ha-1 Root system development kg*ha-1 m³*plant Seed yield (dry seed) kg*ha-1 HI-seed Water uptake mm*ha-1 PET, AET mm*ha-1*day-1 Water use efficiency Nutrient uptake (NPK) kg*ha-1*yr-1 existing data Jongschaap (personal comm.) Plant potential lifespan years Interactions with neighbour plants Effects

(a) It should always be specified for a complete understanding of following data. Annex 1c. Sustainability indicators by sphere of interest

Sustainability indicators Unit or evaluating pm Measurement Literature Research Crop energy balance (input:output) 1:4-5 Henning, 2007 in Belize AGRONOMIC Soil Structure macro-aggr stab. + 6-30% Chaudhary et al., 2007 soil bulk density - 20% Ogunwole et al., 2007 OM content Microbial activity Microbial diversity Soil moisture retention increased Kumar and Sharma, 2008 Erosion control On farm use of by-products yes/no yes Nutrient cycles (NPK) kg*ha-1*yr-1 Jongschaap (personal comm.) Water Plant water use efficiency (seeds) kg*m-3 0.615-1.314 Abdrabbo and Atta, 2008 Plant water use efficiency (oil) kg*m-3 0.154-0.393 Abdrabbo and Atta, 2008 Actual water use no data Jongschaap et al., 2007 Water pollution Nutrient cycles Air Nutrient cycles ENVIRONMENTAL Soil Soil recovery Spaan et al., 2004; Kumar et al., 2008) Soil preservation Spaan et al., 2004 Biodiversity (macro/micro-flora/fauna) # species*m² Acidification g SO4²--eq*ha-1*yr-1 no data IFEU Institute, 2008 Water Water consumption l*week-1*plant-1 Abdrabbo and Atta, 2008 Groundwater leaching g NO3-*ha-1*yr-1 Eutrophication g P2O5-eq*ha-1*yr-1 no data IFEU Institute, 2008 Air GHG emission kg CO2-eq 56,7 Prueksakorn and Gheewala, 2006 GHG emission balance neutral Carbon sequestration g CO2*ha-1*yr-1

ECONOMIC Labour cost $*man-1*d-1 10-15USD Income generation $*kg-1 By-products $*kg-1 Fossil fuel independence $ Renewable energy production $*J-1 SOCIAL Diversify agr. activity # agr. activities Labour generation man*day-1*ha-1 Annex 1d. Sustainability indicators by crop system

CROP SYSTEM Sustainability indicators Unit or evaluating pm Measurement Literature Research Monoculture in Belize Energy Input l of petrol*ha-1 Output eq-l of petrol*ha-1 Water Input (irrigation) mm*ha-1*yr-1 Consumption mm*ha-1*yr-1 Nutrient Nutrient input (NPK, …) kg*ha-1*yr-1 Nutrient Removed kg*ha-1*yr-1 existing data Jongschaap (personal comm.) Others Foreign molecules input (ie:) kg*ha-1*yr-1 Amendments input kg*ha-1*yr-1 On farm use of by-products from JC PS kg*ha-1*yr-1 Labour input man*day-1*ha-1 Interactions with neighbour plants Effects

Intercropping Energy Input l of petrol*ha-1 Output eq-l of petrol*ha-1 Water Input (irrigation) mm*ha-1*yr-1 Consumption mm*ha-1*yr-1 Nutrient Nutrient input (NPK, …) kg*ha-1*yr-1 Nutrient Removed kg*ha-1*yr-1 existing data Jongschaap (personal comm.) Others Foreign molecules input (ie:pesticide) kg*ha-1*yr-1 Amendments input kg*ha-1*yr-1 On farm use of by-products from JC PS kg*ha-1*yr-1 Labour input man*day-1*ha-1 Interactions with neighbour plants Effects

Living fence Energy Input l of petrol*ha-1 Output eq-l of petrol*ha-1 Water Input (irrigation) mm*ha-1*yr-1 Consumption mm*ha-1*yr-1 Nutrient Nutrient input (NPK, …) kg*ha-1*yr-1 Nutrient Removed kg*ha-1*yr-1 existing data Jongschaap (personal comm.) Others Foreign molecules input (ie:pesticide) kg*ha-1*yr-1 Amendments input kg*ha-1*yr-1 On farm use of by-products from JC PS kg*ha-1*yr-1 Labour input man*day-1*ha-1 Interactions with neighbour plants Effects

Annex 2. Experimental designs

ABCDEFGH Not pruned o o o o o o o o 1 o o o o o o o o 2 o o o o o o o o 3 o o o o o o o o 4 o o o o o o o o 5 o o o o o o o o 6 o o o o o o o o 7 o o o o o o o o 8 o o o o o o o o 9 o o o o o o o o 10 o o o o o o o o 11 o o o o o o o o 12 o o o o o o o o 13 o o o o o o o o 14 o o o o o o o o 15 o o o o o o o o 16 o o o o o o o o 17 o o o o o o o o 18 o o o o o o o o 19 o o o o o o o o 20 o o o o o o o o 21 o o o o o o o o 22 o o o o o o o o 23 o o o o o o o o 24 o o o o o o o o 25 o o o o o o o o 26 o o o o o o o 27 o o o o o o o 28 o o o o o o o 29 o o o o o o o 30 o o o o o o o 31 o o o o o o o 32 o o o o o o o 33 o o o o o o 34 o o o o o o 35 o o o o o 36 o o o 37 o o o 38 Pruned o o o o o o o o 39 o o o o o o o o 40 o o o o o o o o 41 o o o o o o o o 42 o o o o o o o o 43 o o o o o o o o 44 o o o o o o o o 45 o o o o o o o o 46 o o o o o o o o 47 o o o o o o o o 48 o o o o o o o o 49 o o o o o o o o 50 o o o o o o o o 51 o o o o o o o o 52 o o o o o o o o 53 o o o o o o o o 54 o o o o o o o o 55 o o o o o o o o 56 o o o o o o o o 57 o o o o o o o o 58 o o o o o 59 Not yielding o o o o o 60 o o o 61 o o o 62 o o o 63 o o o 64 o o o 65 o o 66 Table a. Maya Ranch trial yield design. ABCDEFGH o o o o o o o o 1 Block 1 o o o o o o o o 2 Not pruned, o o o o o o o o 3 Monoculture Not pruned, o o o o o o o o 4 Intercropping o o o o o o o o 5 o o o o o o o o 6 o o o o o o o o 7 o o o o o o o o 8 o o o o o o o o 9 o o o o o o o o 10 o o o o o o o o 11 o o o o o o o o 12 o o o o o o o o 13 Block 2 o o o o o o o o 14 o o o o o o o o 15 o o o o o o o o 16 o o o o o o o o 17 o o o o o o o o 18 o o o o o o o o 19 o o o o o o o o 20 o o o o o o o o 21 o o o o o o o o 22 o o o o o o o o 23 o o o o o o o o 24 o o o o o o o o 25 Block 3 o o o o o o o o 26 o o o o o o o 27 o o o o o o o 28 o o o o o o o 29 o o o o o o o 30 o o o o o o o 31 o o o o o o o 32 o o o o o o o 33 o o o o o o 34 o o o o o o 35 o o o o o 36 o o o 37 o o o 38 o o o o o o o o 39 Block 1 o o o o o o o o 40 o o o o o o o o 41 Pruned, o o o o o o o o 42 Intercropping o o o o o o o o 43 o o o o o o o o 44 o o o o o o o o 45 o o o o o o o o 46 Block 2 o o o o o o o o 47 o o o o o o o o 48 o o o o o o o o 49 o o o o o o o o 50 o o o o o o o o 51 o o o o o o o o 52 o o o o o o o o 53 Block 3 o o o o o o o o 54 o o o o o o o o 55 o o o o o o o o 56 o o o o o o o o 57 o o o o o o o o 58 o o o o o 59 o o o o o 60 o o o 61 o o o 62 o o o 63 o o o 64 o o o 65 o o 66 Table b. Maya Ranch trial LAI design 4 3 2 GB 4 3 2 CB 7 6 5 4 3 2CA7 6 5 4 3 2GA 95 98 99 99 94 98 100 95 GB: Guatemala,4*1m 53 57 55 52 51 52 50 51 49 51 46 48 45 38 CB: Cuba, 4*1m o o o CA: Cuba, (3*1,7)*1,7m GA: Guatemala, ( 3*1,7)*1,7

o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o oo o oo oo oo oo o o o o o o o o o oo oo oo oo oo o o o o o o o oo o oo oo oo oo o o o o o o o o o o o o o o o o o o oo oo oo oo oo oo o o o o o o o o o oo oo oo oo oo oo o o o o o o o o o oo oo oo oo oo oo o o o o o o o o o o o o o o o o o oo oo oo oo oo oo o o o o o o o o o o oo oo oo oo oo oo o o o o o o o o o oo oo oo oo oo oo o o o o o o o o o o o o o o o o oo oo oo oo oo oo o o o o o o o o o oo o oo oo oo oo o o o o o o o o o o oo oo oo oo oo oo o o o o o o o o o o o o o o o o o o oo oo oo oo oo oo o o o o o o o o o o oo oo oo oo oo oo o o o o o o o o o o o o o o o o o o oo oo oo oo oo oo o o o o o o o o o o oo oo oo oo oo oo o o o o o o o o o o oo oo oo oo oo oo o o o o o o o o o o o o o o o o o o oo oo oo oo oo oo o o o o o o o o o o oo oo oo oo oo oo o o o o o o o o o o oo oo oo oo oo oo o o o o o o o o o o o o o o o o o o oo oo oo oo oo oo o o o o o o o o o o oo oo oo oo oo oo o o o o o o o o o o oo oo oo oo oo oo o o o o o o o o o o o o o o o o o o oo oo oo oo oo oo o o o o o o o o o oo oo oo oo oo oo o o o o o o o o o oo oo oo oo oo oo o o o o o o o o o o o o o o o o o o oo oo oo oo oo oo o o o o o o o o o o oo oo oo oo oo oo o o o o o o o o o o oo oo oo oo oo oo o o o o o o o o o o o o o o o o o oo oo oo oo oo oo o o o o o o o o o oo oo oo oo oo oo o o o o o o o o o oo oo oo oo oo oo o o o o o o o o o o o o o o o o o oo oo oo oo oo oo o o o o o o o o o oo oo oo oo oo oo o o o o o o o o o oo oo oo oo oo o o o o o o o o o o o o o o o o o o oo oo oo oo oo o o o o o o o o o oo oo oo oo oo o o o o o o o o o o o o o o o o o oo oo oo oo oo o o o o o o o o o oo oo oo oo oo o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o Table c. Warrie Head trial design ERA-ARD Biofuels in Africa and Central America 2009-2013 – Planting Plan 26 November 2009 Gradient >>> Block I Block II Block III

01oooooo 02oooooo 13oooooo 14 o o o 25oooooo 26 o o o 01

ooaoboo ooaoboo ooaoboo o a b o ooaoboo o a b o

oocodoo oocodoo oocodoo o c d o oocodoo o c d o

ooeofoo ooeofoo ooeofoo o e f o ooeofoo o e f o 02

ooooooo ooooooo ooooooo o o o o ooooooo o o o o

03oooooo 04 o o o 15oooooo 16 o o o 27oooooo 28oooooo 03

ooaoboo o a b o ooaoboo o a b o ooaoboo ooaoboo

oocodoo o c d o oocodoo o c d o oocodoo oocodoo

ooeofoo o e f o ooeofoo o e f o ooeofoo ooeofoo 04

ooooooo o o o o ooooooo o o o o ooooooo ooooooo

05 o o o 06oooooo 17 o o o 18 o o o 29oooooo 30 o o o 05

o a b o ooaoboo o a b o o a b o ooaoboo o a b o

o c d o oocodoo o c d o o c d o oocodoo o c d o

o e f o ooeofoo o e f o o e f o ooeofoo o e f o 06

o o o o ooooooo o o o o o o o o ooooooo o o o o

07oooooo 08 o o o 19oooooo 20oooooo 31oooooo 32 o o o 07

ooaoboo o a b o ooaoboo ooaoboo ooaoboo o a b o 111m

oocodoo o c d o oocodoo oocodoo oocodoo o c d o

ooeofoo o e f o ooeofoo ooeofoo ooeofoo o e f o 08

ooooooo o o o o ooooooo ooooooo ooooooo o o o o

09oooooo 10 o o o 21 o o o 22 o o o 33 o o o 34 o o o 09

ooaoboooabooabooabooabooabo

oocodooocdoocdoocdoocdoocdo

ooeofoooef ooef ooef ooef ooef o 10

ooooooooooooooooooooooooooo

11 o o o 12 o o o 23oooooo 24oooooo 35oooooo 36 o o o 11

o a b o o a b o ooaoboo ooaoboo ooaoboo o a b o

o c d o o c d o oocodoo oocodoo oocodoo o c d o

o e f o o e f o ooeofoo ooeofoo ooeofoo o e f o 12

o o o o o o o o ooooooo ooooooo ooooooo o o o o

24 23 22 21 20 19 18 17 16 15 14 13

52m

Table d. Central Farm trial design (1/2) Row D1 D2

G1 Mexico accession 3,7 2,2 1,1 m I1 No Intercropping Intercrop: Arachis pintoii

G2 Belize accession 3,7 2,2 1,1 m Jatropha curcas O Border row plant

G3 Guatemala accession 3,7 2,2 1,1 m I2 Intercropping X Monitoring plant

1250 2500 trees ha-1

Treatments G D I Treatment randomization plots

1 1 1 1 11 3 12 5 8 2

2 1 1 2 7 5 11 1 12 4

3 1 2 1 2 8 9 6 7 10

4 1 2 2 12 1 3 4 11 6

5 2 1 1 4 6 2 10 5 1

6 2 1 2 9 10 8 7 3 9

7 2 2 1

8 2 2 2 Treatment randomization horizontal hedge (left to right)

9 3 1 1 1 8 12 6 4 7 3 9 11 5 2 10

10 3 1 2

11 3 2 1 Treatment randomization horizontal hedge (top to bottom)

12 3 2 2 1 6108 4 7112125 3 9

Plots row plants plants plot-1 Plants for plots Field size (m2) 6440 m2

Plants per plot D1 5 4 20 18 plots x 20 plants 360

D2 5 7 35 18 plots x 35 plants 630 990 plants

Living fence D1 D2 Plants per fence Plants per treatment unit in fence

Field length 111 m 0,25 0,50 m 55,5 m / 0.25 + 55,5 m / 0.5 = 333 D1 37

Field width 52 m 0,25 0,50 m 26 m / 0.25 + 26 m / 0.5 = 156 489 plants D2 19

Distance fence from other plots Total plants 1479 plants

At least 3,7 m

Table e. Central Farm trial design (2/2) Additional plants: 240 Additional plants: 255

19m ooooooooooo o o o o o ooooooooooo 103,6m ooooooooooooooo ooooooooooo o o o o o ooooooooooo ooooooooooooooo ooooooooooo o o o o o ooooooooooo ooooooooooooooo ooooooooooo o o o o o ooooooooooo 38m oooooooooooooo ooooooooooo o o o o o ooooooooooo ooooooooooooooo o o o o o ooooooooooo o o o o o oooooooooooooo

o o o o o ooooooooooo o o o o o oooooooooooooo

o o o o o ooooooooooo o o o o o oooooooooooooo

o o o o o ooooooooooo o o o o o o o o o o o o

o o o o o ooooooooooo o o o o o o o o o o o o

38 m o o o o o o o

Table f. Extra land (above and right) for additional plants at o o o o o o o Central Farm o o o o o o

o o o o o o

o o o o o o

o o o o o o

o o o o o o

ooooooooooo

o o o o o o o o o o

o o o o o o o o o o

o o o o o o o o o

o o o o o o o o o

o o o o o o o o o

o o o o o

o o o o o

o o o o o

o o o o

103,6m o o o o 9m References

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111 If you use information from this M.Sc. dissertation document, please cite and refer to: da Schio, B., 2010. Jatropha curcas L., a potential bioenergy crop. On field research in Belize. M.Sc. dissertation. Padua University, Italy and Wageningen University and Research centre, Plant Research International, the Netherlands.