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Characterization and Pathogenicity of Fusicladium eriobotryae, the Fungal Pathogen Responsible for Loquat Scab
No full textTo characterize the fungal pathogen responsible for loquat scab and establish differences in pathogenicity in loquat, eight strains identified as Fusicladium eriobotryae were isolated from either loquat leaves or fruit showing scab symptoms in Spain. Loquat plants belonging to the cv. Peluche were infected via a newly developed infection system that was based on spraying susceptible loquat plants with fungal spore suspensions, keeping the plants for I week in 100% humidity, and then transferring the plants to ambient relative humidity in greenhouses. Scab symptoms were analyzed and pathogenic characterization of all F eriobotryae strains revealed different degrees of aggressiveness. Based on infection progression and severity of scab symptoms, strain ST1 was confirmed as the most aggressive in cultivars in the Mediterranean region. Strain STI, which even caused chlorotic spots in loquat stems, is so aggressive it can be used to identify highly resistant cultivars using this in vivo system. Molecular characterization of internal transcribed spacer ribosomal DNA and, particularly, the glyceraldehyde 3-phosphate dehydrogenase gene, clearly distinguished loquat strains from Venturia inaequalis. Moreover, random amplified polymorphic DNA (RAPD) and microsatellite-primed polymerase chain reaction techniques were used to qualitatively discriminate between species and report the variations within fungal populations. Molecular variability was checked by comparing all the different strains and enabled the specific identification of F eriobotryae. Although no association was observed between any pattern and phenotypic traits, such as aggressiveness, RAPD provided a specific profile that allowed fungal identification
Development of a machine for the automatic sorting of pomegranate (Punica granatum) arils based on computer vision
No full textThe pomegranate is a fruit with excellent organoleptic and nutritional properties, but the fact that it is difficult to peel affects its commercialisation and decreases its potential consumption. One solution is to market the arils of pomegranate in a ready-to-eat form. However, after the peeling process, unwanted material, such as internal membranes and defective arils, is extracted together with good arils and must be removed on the packing line because the presence of such material shortens the shelf life of the product or deteriorates its appearance. For different reasons, the commercial sorting machines that are currently available for similar commodities (cherries, nuts, rice, etc.) are not capable of handling and sorting pomegranate arils, thus making it necessary to build specific equipment. This work describes the development of a computer vision-based machine to inspect the raw material coming from the extraction process and classify it in four categories. The machine is capable of detecting and removing unwanted material and sorting the arils by colour. The prototype is composed of three units, which are designed to singulate the objects to allow them be inspected individually and sorted. The inspection unit relies on a computer vision system. Two image segmentation methods were tested: one uses a threshold on the R/G ratio and the other is a more complex approach based on Bayesian Linear Discriminant Analysis (LDA) in the RGB space. Both methods offered an average success rate of 90% on a validation set, the former being more intuitive for the operators, as well as faster and easier to implement, and for these reasons it was included in the prototype. Subsequently, the complete machine was tested in industry by working in real conditions throughout a whole pomegranate season, in which it automatically sorted more than nine tons of arils
Membrane transporters and carbon metabolism implicated in chloride homeostasis differentiate salt stress responses in tolerant and sensitive Citrus rootstocks
No full textSalinity tolerance in Citrus is strongly related to leaf chloride accumulation. Both chloride homeostasis and specific genetic responses to Cl(-) toxicity are issues scarcely investigated in plants. To discriminate the transcriptomic network related to Cl(-) toxicity and salinity tolerance, we have used two Cl(-) salt treatments (NaCl and KCl) to perform a comparative microarray approach on two Citrus genotypes, the salt-sensitive Carrizo citrange, a poor Cl(-) excluder, and the tolerant Cleopatra mandarin, an efficient Cl(-) excluder. The data indicated that Cl(-) toxicity, rather than Na(+) toxicity and/or the concomitant osmotic perturbation, is the primary factor involved in the molecular responses of citrus plant leaves to salinity. A number of uncharacterized membrane transporter genes, like NRT1-2, were differentially regulated in the tolerant and the sensitive genotypes, suggesting its potential implication in Cl(-) homeostasis. Analyses of enriched functional categories showed that the tolerant rootstock induced wider stress responses in gene expression while repressing central metabolic processes such as photosynthesis and carbon utilization. These features were in agreement with phenotypic changes in the patterns of photosynthesis, transpiration, and stomatal conductance and support the concept that regulation of transpiration and its associated metabolic adjustments configure an adaptive response to salinity that reduces Cl(-) accumulation in the tolerant genotype
Gene stacking in 1-year-cycling APETALA1 citrus plants for a rapid evaluation of transgenic traits in reproductive tissues
No full textRapid flowering is crucial to perform functional genomic studies to investigate reproductive biology characteristics and fruit quality-related traits in fruit trees. However, long generation cycles of woody plants considerably delay this evaluation. Through genetic transformation, juvenile periods can be significantly shortened by overexpression of flower meristem-identity genes. Transgenic APETALA1 (AP1) citrus plants behave as rapid-cycling trees, since 1-year-old seedlings promptly show precocious flowering and fruiting. By transgene stacking into these short-generation AP1 and nptll/GUS-positive plants. expression of novel transgenes could theoretically be examined as quickly as 1 year after retransformation. Establishment of the selection and regeneration conditions for the production of retransformed individuals with marker genes is detailed in this communication. Hpt and bar genes were used as the second selectable marker genes. PCR and Southern blot analyses confirmed the recovery of retransformed shoots. AP1 transcript accumulation and GUS and GFP expression were assessed in leaves, and flowers and fruit organs of rapid-cycling retransformed lines, respectively, as early as I year after plant generation and during three consecutive years, demonstrating that the principle of stable transgene stacking on early-fruiting transgenic trees is feasible. (C) 2009 Elsevier B.V. All rights reserved
Comparative transcriptional survey between laser-microdissected cells from laminar abscission zone and petiolar cortical tissue during ethylene-promoted abscission in citrus leaves
Get PDFBackground: Abscission is the cell separation process by which plants are able to shed organs. It has a great impact on the yield of most crop plants. At the same time, the process itself also constitutes an excellent model to study cell separation processes, since it occurs in concrete areas known as abscission zones (AZs) which are composed of a specific cell type. However, molecular approaches are generally hampered by the limited area and cell number constituting the AZ. Therefore, detailed studies at the resolution of cell type are of great relevance in order to accurately describe the process and to identify potential candidate genes for biotechnological applications. Results: Efficient protocols for the isolation of specific citrus cell types, namely laminar abscission zone (LAZ) and petiolar cortical (Pet) cells based on laser capture microdissection (LCM) and for RNA microextraction and amplification have been developed. A comparative transcriptome analysis between LAZ and Pet from citrus leaf explants subjected to an in-vitro 24 h ethylene treatment was performed utilising microarray hybridization and analysis. Our analyses of gene functional classes differentially represented in ethylene-treated LAZ revealed an activation program dominated by the expression of genes associated with protein synthesis, protein fate, cell type differentiation, development and transcription. The extensive repertoire of genes associated with cell wall biosynthesis and metabolism strongly suggests that LAZ layers activate both catabolic and anabolic wall modification pathways during the abscission program. In addition, over-representation of particular members of different transcription factor families suggests important roles for these genes in the differentiation of the effective cell separation layer within the many layers contained in the citrus LAZ. Preferential expression of stress-related and defensive genes in Pet reveals that this tissue is likely to be reprogrammed to prevent pathogen attacks and general abiotic stresses after organ shedding. Conclusion: The LCM-based data generated in this survey represent the most accurate description of the main biological processes and genes involved in organ abscission in citrus. This study provides novel molecular insight into ethylene-promoted leaf abscission and identifies new putative target genes for characterization and manipulation of organ abscission in citrus
Activity-density of Pardosa cribata in Spanish citrus orchards and its predatory capacity on Ceratitis capitata and Myzus persicae
No full textThe wolf spider Pardosa cribata Simon is the most abundant ground-dwelling spider inhabiting citrus orchards in eastern Spain. However, little is known about its activity-density and its predatory role in the citrus agrosystem. Here we report on the activity-density of P. cribata monitored by pitfall traps, and on its capacity to prey on two citrus pests that appear both in the citrus canopy and the ground cover, Ceratitis capitata (Wiedemman) and Myzus persicae (Sulzer), respectively. Pardosa cribata was present in citrus orchards throughout the year, with a peak in spring and a higher peak in summer. Pardosa cribata preyed on adults and third-instar larvae but not on pupae of C. capitata. A type II functional response was obtained for teneral-like adults, with an estimated attack rate (a') of 0.771 +/- A 0.213 days(-1) and a handling time (T (h)) of 0.051 +/- A 0.013 days. Pardosa cribata also preyed efficiently on M. persicae, giving a type II functional response with an estimated attack rate and handling time of 2.833 +/- A 0.578 days(-1) and 0.031 +/- A 0.001 days, respectively. The data reported here indicate that this wolf spider could play an important role in regulating both these pests, and therefore might contribute to developing conservation biological control strategies for citrus pests
Identification of fruit yield loci controlling the salt tolerance conferred by solanum rootstocks
Get PDFThe rootstock effect on the fruit yield of a grafted tomato variety was genetically analyzed under salinity using as rootstock two populations of F(9) lines developed from a salt sensitive genotype of Solanum lycopersicum var. cerasiforme, as female parent, and two salt tolerant lines, as male parents, from S. pimpinellifolium, the P population ( 123 lines), and S. cheesmaniae, the C population ( 100 lines). There were rootstock lines from the two populations ( up to 65% in the P population) that raised the fruit yield of the commercial hybrid under saline conditions. It is shown that this salt tolerance rootstock effect is a heritable trait ( h(2) near 0.3), governed by at least eight QTLs. The most relevant component was the number of fruits. Thus most detected QTLs correspond to this component. In general, QTL gene effects are medium-sized, with contributions from 8.5 up to 15.9% at most, and the advantageous allele comes from the wild, salt tolerant species. Only two fruit yield QTLs on chromosomes P9 and C11 might correspond to fruit yield QTLs of the non-grafted lines indicating their root system dependence. A fruit yield QTL on chromosome 3 is acting epistatically in both populations. The epistatic interactions found were dominant and they were unveiled using the associated marker as cofactor in the composite interval mapping methodology. Therefore, an efficient and profitable utilization of wild germplasm can be carried out through the improvement of rootstocks that confer salt tolerance in terms of fruit yield to the grafted variety
Recovery and characterization of a Citrus clementina Hort. ex Tan. Clemenules haploid plant selected to establish the reference whole Citrus genome sequence
Get PDFBackground: In recent years, the development of structural genomics has generated a growing interest in obtaining haploid plants. The use of homozygous lines presents a significant advantage for the accomplishment of sequencing projects. Commercial citrus species are characterized by high heterozygosity, making it difficult to assemble large genome sequences. Thus, the International Citrus Genomic Consortium (ICGC) decided to establish a reference whole citrus genome sequence from a homozygous plant. Due to the existence of important molecular resources and previous success in obtaining haploid clementine plants, haploid clementine was selected as the target for the implementation of the reference whole genome citrus sequence. Results: To obtain haploid clementine lines we used the technique of in situ gynogenesis induced by irradiated pollen. Flow cytometry, chromosome counts and SSR marker (Simple Sequence Repeats) analysis facilitated the identification of six different haploid lines (2n = x = 9), one aneuploid line (2n = 2x+4 = 22) and one doubled haploid plant ( 2n = 2x = 18) of 'Clemenules' clementine. One of the haploids, obtained directly from an original haploid embryo, grew vigorously and produced flowers after four years. This is the first haploid plant of clementine that has bloomed and we have, for the first time, characterized the histology of haploid and diploid flowers of clementine. Additionally a double haploid plant was obtained spontaneously from this haploid line. Conclusion: The first haploid plant of 'Clemenules' clementine produced directly by germination of a haploid embryo, which grew vigorously and produced flowers, has been obtained in this work. This haploid line has been selected and it is being used by the ICGC to establish the reference sequence of the nuclear genome of citrus
Mejora genética de especies vegetales
Get PDFUna parte importantede la sociedad y de los medios de comunicación están convencidos de que la
mejora genética o transformación genética de las plantas cultivadas es un proceso reciente que serealiza
mediante la utilización de la biotecnología, particularmente mediante técnicas de ingeniería genética. Sin
embargo, está idea está totalmente alejada de la realidad ya que este proceso se ha producido de forma
ininterrumpida desde el inicio de la agricultura hace unos 10.000 años. Lo que es cierto es que durante este
periodo la mejora ha tenido distintas fases y los actores encargados de la misma han ido cambiando. Los
primeros “mejoradores” fueron los recolectores-cazadores que realizaron la domesticación de especies
silvestres e iniciaron la agricultura. Posteriormente los agricultores sometieron a las plantas a un proceso
de selección empírica durante varios milenios creando las variedades locales. Los descubrimientos sobre
la hibridación, la herencia y la genética hacen que los científicos tomen el relevo en la mejora de las plantas,
tímidamente durante el siglo XIX y de forma generalizada durante el siglo XX creando las variedades de
alto rendimiento más resistentes a plagas y enfermedades. Los desarrollos biotecnológicos de la segunda
mitad del siglo XX incorporaron una nueva pléyade de científicos en diversas disciplinas al proceso de mejora
genética de las plantas cultivadas con nuevas estrategias que abren horizontes insospechados en el
desarrollo de la agricultura, especialmente con la irrupción de las plantas transgénicas. La domesticación
de las especies y su mejora a través de las distintas fases constituyen un proceso evolutivo totalmente
artificial desde sus orígenes que nunca hubiese sucedido de forma espontánea en la naturaleza (García
Olmedo, 1998)
The S-Adenosyl-L-Homocysteine Hydrolase Gene ahcY of Agrobacterium radiobacter K84 Is Required for Optimal Growth, Antibiotic Production, and Biocontrol of Crown Gall Disease
No full textAgrobacterium radiobacter K84 is a commercial agent used worldwide to control crown gall disease caused by pathogenic isolates of A. tumefaciens. More than 2,000 transposon insertion derivatives of strain K84 were screened by a standardized greenhouse bioassay to identify mutants defective in biocontrol. Three mutants affected in biocontrol properties were identified. All three mutants displayed normal levels of attachment to tomato seed and root colonization. One of these mutants, M19-164, exhibited partial biocontrol and did not produce detectable levels of agrocin 84. In this mutant, the transposon is located in the agn locus of pAgK84, which codes for agrocin 84 biosynthesis. The second mutant, M19-158, also exhibited partial biocontrol and produced reduced amounts of agrocin 84 as a result of a mutation in a chromosomal gene of unknown function. The third mutant, M9-22, failed to biocontrol, was impaired in both growth in minimal medium and siderophore production, and failed to produce detectable levels of agrocin 84. The chromosomal gene ahcY, which encodes S adenosyl-L-homocysteine hydrolase, was disrupted in this mutant. Expression of a functional copy of ahcY in M9-22 restored all of the altered phenotypes. The fact that all identified biocontrol mutants exhibited a partial or total defect in production of agrocin 84 indicates that this antibiotic is required for optimum biocontrol. This study also identified two chromosomally encoded genes required for agrocin 84 production. That a mutation in ahcY abolishes biocontrol suggests that the intracellular ratio of S-adenosyl-L-methionine to S-adenosyl-L-homocysteine is an important factor for agrocin 84 biosynthesis. Finally, we demonstrate that the ahcY gene in strain K84 is also required for optimal growth as well as for antibiotic production and biocontrol of crown gall disease