Implementation of pre-harvest techniques in emerging agroforestry systems to increase the yield of cocoa tree (Theobroma cacao L.).

Cocoa is one of the most important tropical fruits worldwide, its importance lies in its use in the food, cosmetic and pharmaceutical industries. Cocoa yield has been affected by different environmental, cultural and phytosanitary aspects. The emergence of new growing areas allows exploring the possibility of generating new economic and ecological systems that comply with current trends in organic farming. For them, pre-harvest practices such as pruning and soil fertilization are two necessary tools to control the productivity of cocoa agroecosystems. Therefore, the objective of this research was to analyses the implementation of pre-harvest techniques and the quality soil to increase the yield in a cocoa agroecosystem in an emerging zone in the Huasteca Potosina of Mexico. The work was carried out in an emerging zone in the cultivation of cocoa in three different zones delimited in 30 × 30 m. Thinning and pruning practices were carried out to keep the space clear and observe the influence on fruit yield. In addition, the quality of the soil was measured in terms of physical conditions and nutrient content. 25 kg/ha of nitrogen, 22 kg/ha of P2O5, 24 kg/ha of K2O and 4 kg/ha of magnesium were added following the recommendation of the fertilization laboratory. The physical properties of the pod were also analyzed, such as size, weight, number of grains and color. And some of the cocoa bean such as size, weight and hardness, all these parameters to measure the average yield of cocoa pods. The results show a clear influence of the soil quality and pre-harvest practices on the physical properties of the fruit and the total yield from 472.36 ± 52.01 to 520.06 ± 104.91 kg. However, other aspects are also modified, such as the increase in the size of the pod and the cocoa bean. Other aspects such as the color of the pod and the hardness of the grain do not present statistical difference. In conclusion, pre-harvest practices together with the application of fertilizers are factors that positively influence the yield of cocoa fruit. Some of the limitations of this research were the age of the plants and the local plant species.

Activating stress memory: eustressors as potential tools for plant breeding.

Plants are continuously exposed to stress conditions, such that they have developed sophisticated and elegant survival strategies, which are reflected in their phenotypic plasticity, priming capacity, and memory acquisition. Epigenetic mechanisms play a critical role in modulating gene expression and stress responses, allowing malleability, reversibility, stability, and heritability of favourable phenotypes to enhance plant performance. Considering the urgency to improve our agricultural system because of going impacting climate change, potential and sustainable strategies rely on the controlled use of eustressors, enhancing desired characteristics and yield and shaping stress tolerance in crops. However, for plant breeding purposes is necessary to focus on the use of eustressors capable of establishing stable epigenetic marks to generate a transgenerational memory to stimulate a priming state in plants to face the changing environment.

Analysis of the Population Dynamics of Whitefly (Bemisia tabaci [Hemiptera: Aleyrodidae]) Under Greenhouse Conditions.

Whiteflies (Bemisia tabaci) represent an insect pest in horticulture. It serves as a vector for transmitting phytopathogens that inhibit the correct development of plants, affecting crop performance. In this research, whitefly population model was proposed to provide a tool that predicts the pest spread within a crop under greenhouse conditions. The analysis, calibration, and validation of the models, based on logistic functions, were implemented for the three stages (egg, nymph, and adult) of the life cycle of this organism. Temperature (°C), relative humidity (%), initial population (number/cm2), and Growing Degree-Day (GDD) were considered as input variables to describe each development stage. The statistical analysis for the model validation included the coefficient of determination (R2), the percentage standard error of prediction (%SEP), the average relative variance (AVR), and the efficiency coefficient (E). The first period for calibration consisted of 43 d (204.3 GDD), and the second period for validation consisted of 36 d (171.1 GDD). The model efficiently predicts the population growth for the egg, nymph, and adult stages since the values of R2 were 0.9856, 0.9918, and 0.9436, and the values of %SEP were 12.4, 11.9, and 75.1% for the egg, nymph, and adult stages, respectively. Moreover, the validation model obtained an R2 of 0.9287 for the egg stage, 0.9645 for the nymph stage, and 0.9884 for the adult stage. Meanwhile, the values of %SEP were 10.38, 16.89, and 32.59% for the egg, nymph, and adult stages, respectively. In both cases, the values suggest an adequate fit for the model.

Exogenous fragmented DNA acts as a damage-associated molecular pattern (DAMP) inducing changes in CpG DNA methylation and defence-related responses in Lactuca sativa.

Damage-associated molecular patterns (DAMPs) have been studied recently to understand plant self-nonself recognition in response to attack by biotic and abiotic stresses. Extracellular DNA has emerged as a possible DAMP. As a DAMP DNA seems to function depending on the phylogenetic scale and has been tested in a few plant species. This study aimed to evaluate the possible role of self DNA (sDNA) as a DAMP by analysing changes in CpG DNA methylation and defence-related responses in lettuce (Lactuca sativa L.) as a model plant. sDNA and nonself DNA (nsDNA) from Capsicum chinense Murray (both species belong to the same clade, Asterids) stimulated aberrant seed germination and root growth in lettuce seedlings. Similar resultswere obtained with nsDNA obtained from Acaciella angustissima (Mill.) Britton & Rose plants (belonging to the clade Rosids I), although at significantly higher concentrations. Moreover, in most cases, this behaviour was correlated with hypomethylation of CpG DNA as well as defence responses measured as altered gene expression associated with oxidative burst and production of secondary metabolites (phenylpropanoids) related to coping with stress conditions. Our results suggested that extracellular and fragmented DNA has a role as a DAMP depending on phylogenetic closeness in plants as lettuce, inducing epigenetic, genetic and biochemical changes within the plant. The importance of our results is that, for the first time, they demonstrate that sDNA acts as a DAMP in plants, changing CpG DNA methylation levels as well as increasing the production of secondary metabolites associated with defence responses to stress.

Effect of foliar application of salicylic acid, hydrogen peroxide and a xyloglucan oligosaccharide on capsiate content and gene expression associatedwith capsinoids synthesis in Capsicum annuum L.

Capsinoids are non-pungent analogues of capsaicinoids in pepper (Capsicum spp). The absence of pungency, in addition to their biological activities similar to that of capsaicinoids such as anti-inflammatory, antimicrobial, and antioxidant properties, makes capsinoids an excellent option for increasing use in human and animal nutrition, as well as health and pharmaceutical industries. There are only few sources of pepper producing capsinoids, and one of them (accession 509-45-1), Capsicum annuum L., is a potential source for increasing capsinoids content using strategies as controlled elicitation during plant production in the greenhouse. In this research we evaluated the effect of weekly and one-day-before-harvest foliar applications of hydrogen peroxide, salicylic acid and a xyloglucan oligosaccharide on the concentration of capsiate in fruits of this pepper accession, as well as the gene expression of phenylalanine ammonia-lyase (pal), putative aminotransferase (pamt), capsaicin synthase (at3) and ß-keto acyl synthase (kas). Results showed that the two tested concentrations of H2O2 significantly increased capsiate content and gene expression associated with capsaicinoids (pamt, at3 and kas) and the phenylpropanoids (pal) pathways. Plant yield was not affected using this induction strategy. Our results indicated that the pre-harvest and weekly application of hydrogen peroxide and xyloglucan oligosaccharide improved production of capsiate in C. annuum L.

The fermented non-digestible fraction of common bean (Phaseolus vulgaris L.) triggers cell cycle arrest and apoptosis in human colon adenocarcinoma cells.

Cancer is a leading cause of death worldwide with colorectal cancer (CRC) ranking as the third contributing to overall cancer mortality. Non-digestible compounds such as dietary fiber have been inversely associated with CRC in epidemiological in vivo and in vitro studies. In order to investigate the effect of fermentation products from a whole non-digestible fraction of common bean versus the short-chain fatty acid (SCFAs) on colon cancer cells, we evaluated the human gut microbiota fermented non-digestible fraction (hgm-FNDF) of cooked common bean (Phaseolus vulgaris L.) cultivar Negro 8025 and a synthetic mixture SCFAs, mimicking their concentration in the lethal concentration 50 (SCFA-LC50) of FNDF (hgm-FNDF-LC50), on the molecular changes in human colon adenocarcinoma cells (HT-29). Total mRNA from hgm-FNDF-LC50 and SCFA-LC50 treated HT-29 cells were used to perform qPCR arrays to determine the effect of the treatments on the transcriptional expression of 84 genes related to the p53-pathway. This study showed that both treatments inhibited cell proliferation in accordance with modulating RB1, CDC2, CDC25A, NFKB and E2F genes. Furthermore, we found an association between the induction of apoptosis and the modulation of APAF1, BID, CASP9, FASLG, TNFR10B and BCL2A genes. The results suggest a mechanism of action by which the fermentation of non-digestible compounds of common bean exert a beneficial effect better than the SCFA mixture by modulating the expression of antiproliferative and pro-apoptotic genes in HT-29 cells to a greater extent, supporting previous results on cell behavior, probably due to the participation of other compounds, such as phenolic fatty acids derivatives and biopetides.

A new strain of tomato severe leaf curl virus and a unique variant of tomato yellow leaf curl virus from Mexico.

The complete genome sequence of a distinct variant of tomato yellow leaf curl virus-Israel (TYLCV-IL) and the DNA-A sequence of a new strain of tomato severe leaf curl virus (ToSLCV) isolated in San Luis Potosi, Mexico, are described and analyzed. The TYLCV-IL[MX:SLP:11] variant differs from all TYLCV-IL isolates described so far by a unique 42-nt duplicated sequence comprising a part of the conserved stem-loop element of the virion-strand replication origin and adjacent regulatory sequences. TYLCV-IL[MX:SLP:11] was associated with tomato chino La Paz virus (ToChLPV-B[MX:SLP:11]) in a Solanum pimpinellifolium plant, and with pepper huasteco yellow vein virus (PHYVV-[MX:SLP:11]) and ToSLCV-GT[MX:SLP:11] in a Solanum lycopersicum plant. In addition, a distinct ToSLCV exhibiting low sequence identity (<89 %) to other ToSLCV isolates from Mexico was found in a tomato plant collected in the same field. Sequence analysis of this new ToSLCV strain indicates that it is a recombinant of close relatives of ToSLCV-GT[MX:SLP:11] and ToChLPV-B[MX:SLP:11] found in mixed infections with TYLCV-IL[MX:SLP:11].

Homologue expression of a ß-xylosidase from native Aspergillus niger.

Xylan constitutes the second most abundant source of renewable organic carbon on earth and is located in the cell walls of hardwood and softwood plants in the form of hemicellulose. Based on its availability, there is a growing interest in production of xylanolytic enzymes for industrial applications. ß-1,4-xylan xylosidase (EC 3.2.1.37) hydrolyses from the nonreducing end of xylooligosaccharides arising from endo-1,4-ß-xylanase activity. This work reports the partial characterization of a purified ß-xylosidase from the native strain Aspergillus niger GS1 expressed by means of a fungal system. A gene encoding ß-xylosidase, xlnD, was successfully cloned from a native A. niger GS1 strain. The recombinant enzyme was expressed in A. niger AB4.1 under control of A. nidulans gpdA promoter and trpC terminator. ß-xylosidase was purified by affinity chromatography, with an apparent molecular weight of 90 kDa, and showed a maximum activity of 4,280 U mg protein(-1) at 70°C, pH 3.6. Half-life was 74 min at 70°C, activation energy was 58.9 kJ mol(-1), and at 50°C optimum stability was shown at pH 4.0-5.0. ß-xylosidase kept residual activity >83% in the presence of dithiothreitol (DTT), ß-mercaptoethanol, sodium dodecyl sulfate (SDS), ethylenediaminetetraacetate (EDTA), and Zn(2+). Production of a hemicellulolytic free xylosidase showed some advantages in applications, such as animal feed, enzymatic synthesis, and the fruit-juice industry where the presence of certain compounds, high temperatures, and acid media is unavoidable in the juice-making process.

Identification of the minimal sequence required for vascular-specific activity of Tomato mottle Taino virus Replication-associated protein promoter in transgenic plants.

A 597 nt fragment from Tomato mottle Taino virus (ToMoTV) DNA-A, with 459 nt located upstream of the Replication-associated protein translation start codon, was tested for promoter activity in solanaceous plants. The promoter activity of this fragment (pRep(459::Rep)) was demonstrated when it was introduced upstream the uidA reporter gene into tobacco, potato and tomato plants by genetic transformation. It became active in 7-day-old transgenic tobacco seedlings as revealed by a vascular-specific pattern of gene expression which was maintained during the continued growth of the plant. Transformed potato and tomato plants also showed a vascular-specific pattern of expression. In comparative assays, pRep(459::Rep) showed an expression activity 10-40-fold less than the 35S promoter from Cauliflower mosaic virus. To delimit the minimal cis-acting elements necessary for vascular specificity of this promoter, a set of PCR deletion mutants of pRep(459::Rep) (pRep(459), pRep(324), pRep(203), pRep(145), pRep(132) and pRep(115)), were generated and used to transform tobacco plants. Transgenic tobacco plants belonging to all the pRep versions were blue stained in the vascular system except those from the pRep(115) version. The results described in this report demonstrate that the minimal sequences necessary for the pRep promoter activity are confined in a segment of 132 nts (located between the nts 2454 and 2585 of the ToMoTV DNA A) and that this promoter harbors those elements sufficient for vascular-specific expression.

Tomato mottle Taino virus pseudorecombines with PYMV but not with ToMoV: implications for the delimitation of cis- and trans-acting replication specificity determinants.

Over the last decade, the tomato production in Cuba has been affected by new whitefly-associated diseases. In addition to the well-documented presence of Tomato yellow leaf curl virus (TYLCV) along the island, the occurrence of bipartite begomoviruses has also been reported. One of them, tentatively named Tomato mottle Taino virus (ToMoTV), has now been cloned and characterized at the molecular level. Its genomic organization is similar to other bipartite geminiviruses. Phylogenetic analyses placed ToMoTV in a subcluster with other geminiviruses isolated in the Caribbean Basin: Tomato mottle virus (ToMoV), Bean dwarf mosaic virus, Abutilon mosaic virus, Sida golden mosaic virus and Potato yellow mosaic virus (PYMV). Biolistic inoculation of tobacco and tomato plants with cloned viral DNA showed that ToMoTV pseudorecombines with PYMV-GP as predicted by the identity of their iterative elements, whereas it does not show the same ability with ToMoV, even when their replication-associated proteins (Rep and REn) show the highest percentage of similarity. A comparative analysis of Rep proteins from begomoviruses that are able to produce viable reassortants suggests that some key elements for virus replication specificity are located in the first ten amino acids of this protein.

Identification of a sequence element involved in AC2-mediated transactivation of the pepper huasteco virus coat protein gene.

The geminivirus AC2 gene product transactivates the expression of the coat and movement protein (CP and BV1) genes, and this effect seems to be mediated by specific although hitherto unknown cis-acting elements. In this work we examined regions from the CP and BV1 gene promoters of pepper huasteco virus (PHV) to define the sequence elements involved in regulation by AC2. Results from transient gene expression and transgenic plant assays suggest that a truncated 115-nt CP promoter is still responsive to the viral transactivator. This promoter contains three elements similar to a sequence motif termed conserved late element (CLE), which is found in the regulatory regions of many geminiviruses and that was previously suggested, on a theoretical basis, to be a potential functional target for AC2 (Argüello-Astorga et al. (1994), Virology 203, 90-100). To confirm these results, an oligonucleotide containing two CLE motifs was synthesized and characterized in gain-of-function experiments. Transient expression assays showed that this 29-nt sequence is able to confer AC2 responsiveness to heterologous promoters. A smaller oligonucleotide (16 nt) containing a single CLE also conferred this activity. In addition, when the CLE motifs were mutated in their original context (truncated 115-nt promoter), this modified promoter lost its ability to be transactivated by AC2. All these results support the involvement, at least in the case of PHV, of CLE sequences in the process of transactivation.

Complementation of coat protein mutants of pepper huasteco geminivirus in transgenic tobacco plants.

ABSTRACT The role of the pepper huasteco virus (PHV) coat protein (CP) gene during the infection was investigated in three different hosts by using mutations that produced truncated proteins and by complementation assays in transgenic plants. The infectivity analysis revealed that mutants that express truncated CP (CP7 and CP191) behave like the wild-type virus when inoculated onto pepper and Nicotiana benthamiana plants in terms of symptom expression and viral DNA movement. On the contrary, the CP7 mutant was unable to systemically infect tobacco plants, whereas only 10% of the plants inoculated with the CP191 mutant became infected. The CP7 mutant was complemented by coinoculating it with another geminivirus (taino tomato mottle virus). No complementation was observed in plants from nine transgenic tobacco lines expressing CP under the control of the cauliflower mosaic virus (CaMV) 35S promoter. However, 3 out of 10 lines expressing CP under the control of its own promoter (693 nucleotides) were able to complement the CP7 mutant. Interestingly, upon infection, the levels of CP mRNA in 693CP plants increased dramatically, probably due to transactivation of the CP promoter by the viral protein AC2.

Analysis of the infectivity of monomeric clones of pepper huasteco virus.

The infectivity of several monomeric clones of pepper huasteco virus was investigated. All clones were infectious when inoculated excised from the plasmid DNA. However, only certain clones were infectious when inoculated in the non-excised form. Constructs in which the cloning site lies inside regions or genes involved in replication (e.g. Rep-binding site, rep and AC2-AC3 genes) were not infectious, whereas constructs in which the site was located inside the CP or BC1 genes were infectious. A clone that interrupts the BV1 gene was not infectious suggesting an early of BV1 during the establishment of the infection. Linear viral clones containing different DNA fragments at both extremes were also infectious although with a lower efficiency. Analysis of the progeny suggested a precise excision mechanism since in most cases only wild type virus was recovered. The results suggest that excision could be linked to replication through a very specific recombination process.

Geminivirus replication origins have a group-specific organization of iterative elements: a model for replication.

A phylogenetic and structural analysis of the intergenic region of 22 dicot-infecting and 8 monocot-infecting geminiviruses was carried out. The analysis allowed the identification of iterative sequence motifs 8-12 nucleotides in length, whose organization (number, orientation, and spacing) is highly conserved within each of the three major lineages of dicot-geminiviruses, according to the phylogeny derived from the amino acid sequences of the replication-associated protein (AL1). The iterated elements differ in sequence even between closely related viruses, and are found in the vicinity of the putative TATA box of the AL1 gene in all dicot-infecting geminiviruses. Analogous elements were identified also in monocot-infecting geminiviruses, but the arrangement was different, since one of the iterative sequences is part of the conserved hairpin structure essential for replication of all the members of this viral family. We propose here that the iterated sequences are the specific binding sites of the geminiviral replication-associated proteins and show that the hypothesis is in agreement with the experimental data available to date. Additionally, a model of geminivirus replication that involves the participation of host transcription factors in the process is presented.