The damage caused by Cd toxicity to photosynthesis, cellular ultrastructure, antioxidant metabolism, and gene expression in young cacao plants are mitigated by high Mn doses in soil.

Manganese (Mn) is one of the essential mineral micronutrients most demanded by cacao. Cadmium (Cd) is highly toxic to plants and other living beings. There are indications that Mn can interact with Cd and mitigate its toxicity. The objective of this study was to evaluate the action of Mn on the toxic effect of Cd in young plants of the CCN 51 cacao genotype, subjected to different doses of Mn, Cd, and Mn+Cd in soil, through physiological, biochemical, molecular, and micromorphological and ultrastructural changes. High soil Mn doses favored the maintenance and performance of adequate photosynthetic processes in cacao. However, high doses of Cd and Mn+Cd in soil promoted damage to photosynthesis, alterations in oxidative metabolism, and the uptake, transport, and accumulation of Cd in roots and leaves. In addition, high Cd concentrations in roots and leaf tissues caused irreversible damage to the cell ultrastructure, compromising cell function and leading to programmed cell death. However, there was a mitigation of Cd toxicity when cacao was grown in soils with low Cd doses and in the presence of Mn. Thus, damage to the root and leaf tissues of cacao caused by Cd uptake from contaminated soils can be attenuated or mitigated by the presence of high Mn doses in soil.

Transcriptomic, osmoregulatory and translocation changes modulates Ni toxicity in Theobroma cacao.

Nickel is one of the most released trace elements in the environment and in the case of bioaccumulation in foods and beverages derived from cocoa beans can cause risk to human health. It is very important to understand how plants respond to toxic metals and which are the defense strategies they adopt to mitigate their effects. In the present study we used young plants of T. cacao, submitted to increasing Ni doses (0, 100, 200, 300, 400 and 500 mg Ni kg-1 soil) and evaluated them for a period of 30 days. Doses of Ni, from 300 mg of Ni kg-1 onwards in the soil, promoted changes in photosynthetic, antioxidant, osmoregulatory, transcriptomic and translocation levels, evidenced by the increase in the activity of antioxidant enzymes, proline, glycine betaine, upregulation of the metallothionein 2B gene (Mt2b), and lipid peroxidation of the cell membranes. Foliar gas exchange was severely affected at higher doses of Ni. In addition, reduced levels of stomatal conductivity and transpiration rate were observed from 300 mg Ni kg-1 dose onwards in the soil, which consequently affected CO2 assimilation. Phytostabilization and exclusion mechanisms control the translocation of Ni from the root to the shoot and reduce harmful effects on plant metabolism. Our results highlighted the toxicity of Ni, a trace element often underestimated in T. cacao. In particular, it was noted that doses of 100 and 200 Ni kg-1 soil, although high, do not induce toxicity in T. cacao plants. But Ni toxicity is observed from 300 mg Ni kg-1 soil onwards. This study contributed to the understanding of the harmful effects of higher doses of Ni in cacao plants and the biochemical processes the plant uses to mitigate the effects of this metal.

Mitigation of Pb toxicity by Mn in seedling of the cacao clonal CCN 51 genotype grown in soil: physiological, biochemical, nutritional and molecular responses.

Lead (Pb) is a highly toxic metal for humans, animals and plants even at low concentrations in the soil. The ingestion of chocolate produced from contaminated beans can contribute to consumer exposure to Pb. While, Mn is an element essential for plants and participates as enzymatic cofactors in several metabolic pathways. The objective of this study was to evaluate the influence of Mn on mitigation of Pb toxicity in seedling of the cacao clonal CCN 51 genotype grown in soils with different doses of Pb, Mn and Mn+Pb, through physiological, biochemical, molecular and nutritional responses. It was found that the seedling of the cacao clonal CCN 51 genotype grown in soils with high Pb, Mn and Mn+Pb contents accumulated these heavy metals in the roots and leaves. Mn doses reduced the Pb uptake by root system and prevented that the Pb accumulated at toxic levels in the roots and leaves of the plants. High doses of Pb applied in soil were highly toxic to the plants, leading, in some cases, them to death. However, no Mn toxicity was observed in cocoa plants, even at high doses in the soil. Uptake of Pb and Mn by the roots and its transport into the aerial part of the plant promoted changes in photosynthesis, leaf gas exchange, respiration, carboxylation and in the instantaneous efficiency of carboxylation, reducing in the treatments with the highest concentrations of Pb, and the emission of chlorophyll fluorescence, affecting the efficiency of photosystem 2 and the production of photoassimilates. Besides that, Pb, Mn and Mn+Pb toxicities activated defense mechanisms in plants that alter the gene expression of met, psbA and psbO, increasing in plants subjected to high concentrations of Pb and the activity of the enzymes involved in the cellular detoxification of excess ROS at the leaf level. In addition, high uptake of Mn by root system was found to reduced Pb uptake in plants grown with Mn+Pb in the soil. Therefore, application of Mn in the soil can be used to mitigate the Pb toxicity in seedling of the cacao clonal CCN 51 genotype grown in contaminated soils.

Impact of Ambient and Elevated [CO2] in Low Light Levels on Growth, Physiology and Nutrient Uptake of Tropical Perennial Legume Cover Crops.

At early stages of establishment of tropical plantation crops, inclusion of legume cover crops could reduce soil degradation due to erosion and nutrient leaching. As understory plants these cover crops receive limited irradiance and can be subjected to elevated CO2 at ground level. A glasshouse experiment was undertaken to assess the effects of ambient (450 µmol mol-1) and elevated (700 µmol mol-1) levels of [CO2] on growth, physiological changes and nutrient uptake of six perennial legume cover crops (Perennial Peanut, Ea-Ea, Mucuna, Pigeon pea, Lab lab, Cowpea) under low levels of photosynthetic photon flux density (PPFD; 100, 200, and 400 µmol m-2 s-1). Overall, total and root dry biomass, total root length, specific leaf area, and relative growth rates were significantly influenced by levels of [CO2] and PPFD and cover crop species. With few exceptions, all the cover crops showed significant effects of [CO2], PPFD, and species on net photosynthesis (PN ) and its components, such as stomatal conductance (gs) internal CO2 conc. (Ci), and transpiration (E). Increasing [CO2], from 450 to 700 µmol mol-1 and increasing PPFD from 100 to 400 µmol Ö¼m-2 Ö¼s-1 increased PN . Overall, the levels of [CO2], PPFD and species significantly affected total water use efficiency (WUETOTAL ), instantaneous water use efficiency (WUEINST ) and intrinsic water use efficiency (WUEINTR ). With some exceptions, increasing levels of [CO2] and PPFD increased all the WUE parameters. Interspecific differences were observed with respect to macro-micro nutrient uptake and use efficiency. With a few exceptions, increasing levels of [CO2] from 450 to 700 µmol mol-1 and PPFD from 100 to 400 µmol m-2 s-1 increased nutrient use efficiency (NUE) of all nutrients by cover crop species.

Growth and nutritional responses of wild and domesticated cacao genotypes to soil Cd stress.

Cadmium (Cd), a toxic non-essential metal, is easily accumulated in cacao tissues. This represents a risk for cacao exportation, and consequently it affects the economic well-being of the resource-poor-small-producers in Latin America. A greenhouse experiment was conducted with 53 wild and domesticated cacao genotypes to determine their response to Cd in terms of growth and Cd and essential nutrients accumulation. Cacao seedlings were grown for 6 months in an acidic soil with or without added Cd. The total concentration of macro (Ca, K, Mg, N and P) and micronutrients (B, Cu, Fe, Mn and Zn) as well as Cd were measured in shoots along with growth (biometric) parameters after harvest. The results revealed that even if there was a wide range of Cd concentrations among genotypes, there was a reduction in the concentration of essential nutrients in genotypes grown in Cd spiked soils, however these concentrations were not significantly different from the control. In the case of growth parameters, the effects of Cd were diverse across all genotypes some of them being more tolerant to Cd stress than others. Thus, different growth responses to Cd stress are related to a genotype effect. Based on their lower Cd concentration, a total of 11 cacao genotypes (AYP-22, PAS-105, UGU-126, ICT-1026, ICT-1087, ICT-1189, ICT-1292, PH-17, CCN-51, ICS-39 and TSH-565) are proposed here as low Cd-accumulating genotypes. Therefore, these genotypes are potentially useful as rootstock to reduce uptake and transport of Cd, especially in economically important cacao cultivars.

Rootstock-Mediated Genetic Variance in Cadmium Uptake by Juvenile Cacao (Theobroma cacao L.) Genotypes, and Its Effect on Growth and Physiology.

Grafting typically offers a shortcut to breed tree orchards throughout a multidimensional space of traits. Despite an overwhelming spectrum of rootstock-mediated effects on scion traits observed across several species, the exact nature and mechanisms underlying the rootstock-mediated effects on scion traits in cacao (Theobroma cacao L.) plants often remain overlooked. Therefore, we aimed to explicitly quantify rootstock-mediated genetic contributions in recombinant juvenile cacao plants across target traits, specifically cadmium (Cd) uptake, and its correlation with growth and physiological traits. Content of chloroplast pigments, fluorescence of chlorophyll a, leaf gas exchange, nutrient uptake, and plant biomass were examined across ungrafted saplings and target rootstock × scion combinations in soils with contrasting levels of Cd. This panel considered a total of 320 progenies from open-pollinated half-sib families and reciprocal full-sib progenies (derived from controlled crosses between the reference genotypes IMC67 and PA121). Both family types were used as rootstocks in grafts with two commercial clones (ICS95 and CCN51) commonly grown in Colombia. A pedigree-based best linear unbiased prediction (A-BLUP) mixed model was implemented to quantify rootstock-mediated narrow-sense heritability (h 2) for target traits. A Cd effect measured on rootstocks before grafting was observed in plant biomass, nutrient uptake, and content of chloroplast pigments. After grafting, damage to the Photosystem II (PSII) was also evident in some rootstock × scion combinations. Differences in the specific combining ability for Cd uptake were mostly detected in ungrafted rootstocks, or 2 months after grafting with the clonal CCN51 scion. Moderate rootstock effects (h 2> 0.1) were detected before grafting for five growth traits, four nutrient uptake properties, and chlorophylls and carotenoids content (h 2 = 0.19, 95% CI 0.05-0.61, r = 0.7). Such rootstock effects faded (h 2< 0.1) when rootstock genotypes were examined in soils without Cd, or 4 months after grafting. These results suggest a pervasive genetic conflict between the rootstock and the scion genotypes, involving the triple rootstock × scion × soil interaction when it refers to Cd and nutrient uptake, early growth, and photosynthetic process in juvenile cacao plants. Overall, deepening on these findings will harness early breeding schemes of cacao rootstock genotypes compatible with commercial clonal scions and adapted to soils enriched with toxic levels of Cd.

Comparative effectiveness of activated dolomite phosphate rock and biochar for immobilizing cadmium and lead in soils.

Sandy soils in Florida are vulnerable to toxic metal pollution, and it is necessary to identify desirable amendments for the remediation of metal contaminated soils. Sorption and incubation experiments were conducted to compare the effectiveness of dolomite phosphate rock (DPR), humic acid activated dolomite phosphate rock (ADPR) and biochar (BC) in immobilizing Cd2+ and Pb2+ in two representative agricultural soils in south Florida (Alfisol-Riviera and Spodosol -Ankona series). The results showed that the soils had a low sorption capacity for metals with maximum sorption of 0.767-3.30 mg/g. Application of amendments increased the maximum sorption by 4.2-4.8 times for Pb2+ and 1.5-2.2 times for Cd2+ in Alfisol soil, and 7.1-7.9 times for Pb2+ and 1.7-3.1 times for Cd2+ in Spodosol soil. ADPR was the most effective amendment for increasing the soil's sorption capacity for Cd2+ and Pb2+. 0.01 M CaCl2 extractable metals in the contaminated soils were significantly decreased by all the amendments, especially ADPR, which reduced extractable Cd2+ and Pb2+by 87.2 and 76.0% in Alfisol and 91.3 and 76.3% in Spodosol soil as compared to control. The amounts of extractable Cd2+ and Pb2+ were negatively correlated with soil pH and available P, indicating that the change of soil characteristics by amendments was the dominant mechanism for enhanced immobilization of metals in the contaminated soils. These results indicate that ADPR has great potential for remediating toxic levels of Cd2+ and Pb2+ in contaminated soils.

Physiological, ultrastructural, biochemical and molecular responses of young cocoa plants to the toxicity of Cr (III) in soil.

The objective of this study was to evaluate Cr toxicity in young plants of the CCN 51 Theobroma cacao genotype at different concentrations of Cr3+ in the soil (0, 100, 200, 400 and 600 mg kg-1) through physiological, ultrastructural, antioxidant and molecular changes. Doses of 400 and 600 mg Cr3+ kg-1 soil severely affected foliar gas exchange, promoted by damages in photosynthetic machinery evidenced by the decrease in CO2 fixation. Decreased expression of psbA and psbO genes, changes in enzymatic activity and lipid peroxidation also affected leaf gas exchange. A hormesis effect was observed at 100 mg Cr3+ kg-1 soil for the photosynthetic activity. As a metal exclusion response, the roots of the cocoa plants immobilized, on average, 75% of the total Cr absorbed. Ultrastructural changes in leaf mesophyll and roots, with destruction of mitochondria, plasmolysis and formation of vesicles, were related to the oxidative stress promoted by excess ROS. The activity of the antioxidant enzymes SOD, APX, GPX and CAT and the amino acid proline coincided with the greater expression of the sod cyt gene demonstrating synchronicity in the elimination of ROS. It was concluded, therefore, that the tolerance of the cocoa plants to the toxicity of Cr3+ depends on the concentration and time of exposure to the metal. Higher doses of Cr3+ in the soil promoted irreversible damage to the photosynthetic machinery and the cellular ultrastructure, interfering in the enzymatic and non-enzymatic systems related to oxidative stress and gene expression. However, the low mobility of the metal to the leaf is presented as a strategy of tolerance to Cr3+.

Path analysis of phenotypic traits in young cacao plants under drought conditions.

Drought is worldwide considered one of the most limiting factors of Theobroma cacao production, which can be intensified by global climate changes. In this study, we aimed to investigate the phenotypic correlation among morphological characteristics of cacao progenies submitted to irrigation and drought conditions and their partitions into direct and indirect effects. Path analysis with phenotypic plasticity index was used as criteria for estimation of basic and explanatory variables. The experiment was conducted in a greenhouse at the Cacao Research Center (CEPEC), Ilhéus, Bahia, Brazil, in a randomized block 21 x 2 factorial arrangement [21 cacao progenies obtained from complete diallel crosses and two water regimes (control and drought)] and six replications. In general, drought conditions influenced biomass production in most progenies, causing significant reductions in total leaf area, leaf number, leaf biomass, fine-roots length (diameter <1 mm), root volume and root area for considered drought intolerant. All progenies showed alterations in growth due to drought. Phenotypic plasticity was most strongly pronounced in root volume. Stem and root diameters, as well as stem dry biomass were the growth variables with the greatest direct effects on root volume under drought conditions, these characters being indicated in screening of cacao progenies drought tolerant.

Heavy metal accumulation in leaves and beans of cacao (Theobroma cacao L.) in major cacao growing regions in Peru.

Peru is one of the leading exporters of organic cacao beans in the world. However, the accumulation of heavy metals in cacao beans represents a problem for cocoa bean export and chocolate quality. The aim of this study was to investigate the distribution and accumulation of heavy metals in cacao leaves and cocoa beans in three major cacao growing regions of Peru. The study was conducted in cacao plantations of 10 to 15years old in three regions of Peru: North (Regions of Tumbes, Piura, Cajamarca, and Amazonas); Center (Regions of Huánuco and San Martin) and South (Junin and Cuzco). Samples of leaf and cacao beans were collected from 70 cacao plantations, and the nature of cacao clone or genotype sampled was recorded. The concentrations of heavy metals such as Cd, Cr, Cu, Fe, Mn, Ni, Pb and Zn in leaves and beans were determined using atomic absorption spectrophotometer. Overall, concentrations of heavy metals were below the critical limits; however, the presence of high levels of Cd in cacao grown in Amazonas, Piura, and Tumbes regions is of primary concern. Plantations of cacao with different cacao clones show differences in Cd accumulation both in leaves and cocoa beans. Therefore, it is promising to screen low Cd accumulator cacao genotypes for safe production of cacao on lightly to moderately Cd contaminated soils. Also, synergism between Zn and Cd present both in plant and soil suggests that Zn has a direct effect on Cd accumulation in cacao.

Photosynthetic, antioxidative, molecular and ultrastructural responses of young cacao plants to Cd toxicity in the soil.

Cadmium (Cd) is a highly toxic metal for plants, even at low concentrations in the soil. The annual production of world cocoa beans is approximately 4 million tons. Most of these fermented and dried beans are used in the manufacture of chocolate. Recent work has shown that the concentration of Cd in these beans has exceeded the critical level (0.6mgkg-1 DM). The objective of this study was to evaluate the toxicity of Cd in young plants of CCN 51 cacao genotype grown in soil with different concentrations of Cd (0, 0.05 and 0.1gkg-1 soil) through photosynthetic, antioxidative, molecular and ultrastructural changes. The increase of Cd concentration in the soil altered mineral nutrient absorption by competition or synergism, changed photosynthetic activity caused by reduction in chloroplastidic pigment content and damage to the photosynthetic machinery evidenced by the Fv/Fm ratio and expression of the psbA gene and increased GPX activity in the root and SOD in leaves. Additionally, ultrastructural alterations in roots and leaves were also evidenced with the increase of the concentration of Cd in the soil, whose toxicity caused rupture of biomembranes in root and leaf cells, reduction of the number of starch grains in foliar cells, increase of plastoglobules in chloroplasts and presence of multivesiculated bodies in root cells. It was concluded, therefore, that soil Cd toxicity caused damage to the photosynthetic machinery, antioxidative metabolism, gene expression and irreversible damage to root cells ultrastructure of CCN 51 cocoa plants, whose damage intensity depended on the exposure time to the metal.

Diallel Analysis and Growth Parameters as Selection Tools for Drought Tolerance in Young Theobroma cacao Plants.

This study aimed to estimate the combining ability, of T. cacao genotypes preselected for drought tolerance through diallel crosses. The experiment was conducted under greenhouse conditions at the Cacao Research Center (CEPEC), Ilhéus, Bahia, Brazil, in a completely randomized block design, in an experimental arrangement 21 x 2 [21 complete diallel crosses and two water regimes (control and stressed)]. In the control, soil moisture was kept close to field capacity, with predawn leaf water potential (ΨWL) ranging from -0.1 to -0.5 MPa. In the drought regime, the soil moisture was reduced gradually by decreasing the amount of water application until ΨWL reached -2.0 to -2.5 MPa. Significant differences (p < 0.05) were observed for most morphological attributes analyzed regarding progenies, water regime and their interactions. The results of the joint diallel analysis revealed significant effects between general combining ability (GCA) x water regimes and between specific combining ability (SCA) x water regimes. The SCA 6 genetic material showed high general combining ability for growth variables regardless of the water regime. In general, the water deficit influenced the production of biomass in most of the evaluated T. cacao crosses, except for SCA-6 x IMC-67, Catongo x SCA, MOC-01 x Catongo, Catongo x IMC-67 and RB-40 x Catongo. Multivariate analysis showed that stem diameter (CD), total leaf area (TLA), leaf dry biomass (LDB), stem dry biomass (SDB), root dry biomass (RDB), total dry biomass (TDB), root length (RL), root volume (RV), root diameter (RD) <1 mm and 1 <(RD) <2 mm were the most important growth parameters in the separation of T. cacao genotypes in to tolerant and intolerant to soil water deficit.

Effects of CO2 enrichment and drought pretreatment on metabolite responses to water stress and subsequent rehydration using potato tubers from plants grown in sunlit chambers.

Experiments were performed using naturally sunlit Soil-Plant-Atmosphere Research chambers that provided ambient or twice ambient CO2. Potato plants were grown in pots that were water sufficient (W), water insufficient for 12-18 days during both vegetative and tuber development stages (VR), or water insufficient solely during tuber development (R). In the ambient CO2 treatment, a total of 17 and 20 out of 31 tuber metabolites differed when comparing the W to the R and VR treatments, respectively. Hexoses, raffinose, mannitol, branched chain amino acids, phenylalanine and proline increased, although most organic acids remained unchanged or decreased in response to drought. Osmolytes, including glucose, branched chain amino acids and proline, remained elevated following 2 weeks of rehydration in both the ambient and elevated CO2 treatments, whereas fructose, raffinose, mannitol and some organic acids reverted to control levels. Failure of desiccated plant tissues to mobilize specific osmolytes after rehydration was unexpected and was likely because tubers function as terminal sinks. Tuber metabolite responses to single or double drought treatments were similar under the same CO2 levels but important differences were noted when CO2 level was varied. We also found that metabolite changes to water insufficiency and/or CO2 enrichment were very distinct between sink and source tissues, and total metabolite changes to stress were generally greater in leaflets than tubers.

Molecular, physiological and biochemical responses of Theobroma cacao L. genotypes to soil water deficit.

Six months-old seminal plants of 36 cacao genotypes grown under greenhouse conditions were subjected to two soil water regimes (control and drought) to assess, the effects of water deficit on growth, chemical composition and oxidative stress. In the control, soil moisture was maintained near field capacity with leaf water potentials (ΨWL) ranging from -0.1 to -0.5 MPa. In the drought treatment, the soil moisture was reduced gradually by withholding additional water until ΨWL reached values of between -2.0 to -2.5 MPa. The tolerant genotypes PS-1319, MO-20 and MA-15 recorded significant increases in guaiacol peroxidase activity reflecting a more efficient antioxidant metabolism. In relation to drought tolerance, the most important variables in the distinguishing contrasting groups were: total leaf area per plant; leaf, stem and total dry biomass; relative growth rate; plant shoot biomass and leaf content of N, Ca, and Mg. From the results of these analyses, six genotypes were selected with contrasting characteristics for tolerance to soil water deficit [CC-40, C. SUL-4 and SIC-2 (non-tolerant) and MA-15, MO-20, and PA-13 (tolerant)] for further assessment of the expression of genes NCED5, PP2C, psbA and psbO to water deficit. Increased expression of NCED5, PP2C, psbA and psbO genes were found for non-tolerant genotypes, while in the majority of tolerant genotypes there was repression of these genes, with the exception of PA-13 that showed an increased expression of psbA. Mutivariate analysis showed that growth variables, leaf and total dry biomass, relative growth rate as well as Mg content of the leaves were the most important factor in the classification of the genotypes as tolerant, moderately tolerant and sensitive to water deficit. Therefore these variables are reliable plant traits in the selection of plants tolerant to drought.

Altered physiology, cell structure, and gene expression of Theobroma cacao seedlings subjected to Cu toxicity.

Seedlings of Theobroma cacao CCN 51 genotype were grown under greenhouse conditions and exposed to increasing concentrations of Cu (0.005, 1, 2, 4, 8, 16, and 32 mg Cu L(-1)) in nutrient solution. When doses were equal or higher than 8 mg Cu L(-1), after 24 h of treatment application, leaf gas exchange was highly affected and changes in chloroplasts thylakoids of leaf mesophyll cells and plasmolysis of cells from the root cortical region were observed. In addition, cell membranes of roots and leaves were damaged. In leaves, 96 h after treatments started, increases in the percentage of electrolyte leakage through membranes were observed with increases of Cu in the nutrient solution. Moreover, there was an increase in the concentration of thiobarbituric acid-reactive substances in roots due to lipid peroxidation of membranes. Chemical analysis showed that increases in Cu concentrations in vegetative organs of T. cacao increased with the increase of the metal in the nutrient solution, but there was a greater accumulation of Cu in roots than in shoots. The excess of Cu interfered in the levels of Mn, Zn, Fe, Mg, K, and Ca in different organs of T. cacao. Analysis of gene expression via RTq-PCR showed increased levels of MT2b, SODCyt, and PER-1 expression in roots and of MT2b, PSBA, PSBO, SODCyt, and SODChI in leaves. Hence, it was concluded that Cu in nutrient solution at doses equal or above 8 mg L(-1) significantly affected leaf gas exchange, cell ultrastructure, and transport of mineral nutrients in seedlings of this T. cacao genotype.

Nutrients and nonessential elements in soil after 11 years of wastewater irrigation.

Irrigation of citrus (Citrus aurantium L. × Citrus paradise Macf.) with urban reclaimed wastewater (RWW) can be economical and conserve fresh water. However, concerns remain regarding its deleterious effects on soil quality. We investigated the ionic speciation (ISP) of RWW and potential impacts of 11 yr of irrigation with RWW on soil quality, compared with well-water (WW) irrigation. Most of nutrients (∼53-99%) in RWW are free ionic species and readily available for plant uptake, such as: NH(4+), NO(3-), K(+), Ca(2+), Mg(2+), SO(4)(2-), H(3)BO(3), Cl(-), Fe(2+), Mn(2+), Zn(2+), Co(2+), and Ni(2+), whereas more than about 80% of Cu, Cr, Pb, and Al are complexed with CO(3-), OH(-), and/or organic matter. The RWW irrigation increased the availability and total concentrations of nutrients and nonessential elements, and soil salinity and sodicity by two to three times compared with WW-irrigated soils. Although RWW irrigation changed many soil parameters, no difference in citrus yield was observed. The risk of negative impacts from RWW irrigation on soil quality appears to be minimal because of: (i) adequate quality of RWW, according to USEPA limits; (ii) low concentrations of metals in soil after 11 yr of irrigation with RWW; and (iii) rapid leaching of salts in RWW-irrigated soil during the rainy season.

C and N content in density fractions of whole soil and soil size fraction under cacao agroforestry systems and natural forest in Bahia, Brazil.

Agroforestry systems (AFSs) have an important role in capturing above and below ground soil carbon and play a dominant role in mitigation of atmospheric CO(2). Attempts has been made here to identify soil organic matter fractions in the cacao-AFSs that have different susceptibility to microbial decomposition and further represent the basis of understanding soil C dynamics. The objective of this study was to characterize the organic matter density fractions and soil size fractions in soils of two types of cacao agroforestry systems and to compare with an adjacent natural forest in Bahia, Brazil. The land-use systems studied were: (1) a 30-year-old stand of natural forest with cacao (cacao cabruca), (2) a 30-year-old stand of cacao with Erythrina glauca as shade trees (cacao + erythrina), and (3) an adjacent natural forest without cacao. Soil samples were collected from 0-10 cm depth layer in reddish-yellow Oxisols. Soil samples was separated by wet sieving into five fraction-size classes (>2000 µm, 1000-2000 µm, 250-1000 µm, 53-250 µm, and <53 µm). C and N accumulated in to the light (free- and intra-aggregate density fractions) and heavy fractions of whole soil and soil size fraction were determined. Soil size fraction obtained in cacao AFS soils consisted mainly (65 %) of mega-aggregates (>2000 µm) mixed with macroaggregates (32-34%), and microaggregates (1-1.3%). Soil organic carbon (SOC) and total N content increased with increasing soil size fraction in all land-use systems. Organic C-to-total N ratio was higher in the macroaggregate than in the microaggregate. In general, in natural forest and cacao cabruca the contribution of C and N in the light and heavy fractions was similar. However, in cacao + erythrina the heavy fraction was the most common and contributed 67% of C and 63% of N. Finding of this study shows that the majority of C and N in all three systems studied are found in macroaggregates, particularly in the 250-1000 µm size aggregate class. The heavy fraction was the most common organic matter fraction in these soils. Thus, in mature cacao AFS on highly weathered soils the main mechanisms of C stabilization could be the physical protection within macroaggregate structures thereby minimizing the impact of conversion of forest to cacao AFS.

Carbon storage in soil size fractions under two cacao agroforestry systems in Bahia, Brazil.

Shaded perennial agroforestry systems contain relatively high quantities of soil carbon (C) resulting from continuous deposition of plant residues; however, the extent to which the C is sequestered in soil will depend on the extent of physical protection of soil organic C (SOC). The main objective of this study was to characterize SOC storage in relation to soil fraction-size classes in cacao (Theobroma cacao L.) agroforestry systems (AFSs). Two shaded cacao systems and an adjacent natural forest in reddish-yellow Oxisols in Bahia, Brazil were selected. Soil samples were collected from four depth classes to 1 m depth and separated by wet-sieving into three fraction-size classes (>250 microm, 250-53 microm, and <53 microm)-corresponding to macroaggregate, microaggregate, and silt-and-clay size fractions-and analyzed for C content. The total SOC stock did not vary among systems (mean: 302 Mg/ha). On average, 72% of SOC was in macroaggregate-size, 20% in microaggregate-size, and 8% in silt-and-clay size fractions in soil. Sonication of aggregates showed that occlusion of C in soil aggregates could be a major mechanism of C protection in these soils. Considering the low level of soil disturbances in cacao AFSs, the C contained in the macroaggregate fraction might become stabilized in the soil. The study shows the role of cacao AFSs in mitigating greenhouse gas (GHG) emission through accumulation and retention of high amounts of organic C in the soils and suggests the potential benefit of this environmental service to the nearly 6 million cacao farmers worldwide.

Molecular, physiological and morphological analysis of waterlogging tolerance in clonal genotypes of Theobroma cacao L.

In soil, anoxia conditions generated by waterlogging induce changes in genetic, morphological and physiological processes, altering the growth and development of plants. Mass propagation of cacao (Theobroma cacao L.) plantlets (clones) is affected by waterlogging caused by heavy rains and irrigation methods used to induce rooting. An experiment was undertaken to assess the effects of a 45-day flooding (anoxia) on physiological and morphological traits of 35 elite cacao genotypes, aiming at potentially identifying those with greater tolerance to flooding of the growth substrate. Eighteen fluorochrome-labeled microsatellite (SSR) primer pairs were used to assess genetic variability among clones, with 248 alleles being amplified and used to calculate similarity coefficients. The resulting dendrogram indicated the presence of four major groups, in which two represented 60% and 31% of the genotypes tested. A general trend toward high levels of heterozygosity was also found for physiological and morphological traits. The survival index (IS) for flood tolerance observed varied from 30 to 96%. Clones TSA-654, TSA-656, TSA-792, CA-1.4, CEPEC-2009 and PH-17 showed an IS value above 94%, whereas CEPEC-2010, CEPEC-2002, CA-7.1 and VB-903 clones were those mostly affected by waterlogging, with IS value below 56%. All genotypes displayed lenticel and adventitious root formation in response to waterlogging, although with different intensities. To determine whether patterns of physiological response could be associated with tolerance to anoxia, a similarity-grouping analysis was performed using the ratio between waterlogged and control values obtained for a series of physiological variables assessed. No specific pattern of physiological and morphological responses to waterlogging was strictly associated with survival of plantlets. However, results revealed by the dendrogram suggest that absence of leaf chlorosis may be a proper trait to indicate cacao clones with higher survival rates under flooding conditions. Consequences of these findings are discussed in the context of developing improved strategies for mass production of clones from elite cacao genotypes.