[Show abstract][Hide abstract] ABSTRACT: Citrus Huanglongbing (HLB) is the most devastating bacterial citrus disease worldwide. Three Candidatus Liberibacter species are associated with different forms of the disease: Candidatus Liberibacter asiaticus, Candidatus Liberibacter americanus and Candidatus Liberibacter africanus. Amongst them, Candidatus Liberibacter asiaticus is the most widespread and economically important. These Gram-negative bacterial plant pathogens are phloem-limited and vectored by citrus psyllids. The current management strategy of HLB is based on early and accurate detection of Candidatus Liberibacter asiaticus in both citrus plants and vector insects. Nowadays, real time PCR is the method of choice for this task, mainly because of its sensitivity and reliability. However, this methodology has several drawbacks, namely high equipment costs, the need for highly trained personnel, the time required to conduct the whole process, and the difficulty in carrying out the detection reactions in field conditions.
A recent DNA amplification technique known as Loop Mediated Isothermal Amplification (LAMP) was adapted for the detection of Candidatus Liberibacter asiaticus. This methodology was combined with a Lateral Flow Dipstick (LFD) device for visual detection of the resulting amplicons, eliminating the need for gel electrophoresis. The assay was highly specific for the targeted bacterium. No cross-reaction was observed with DNA from any of the other phytopathogenic bacteria or fungi assayed. By serially diluting purified DNA from an infected plant, the sensitivity of the assay was found to be 10 picograms. This sensitivity level was proven to be similar to the values obtained running a real time PCR in parallel. This methodology was able to detect Candidatus Liberibacter asiaticus from different kinds of samples including infected citrus plants and psyllids.
Our results indicate that the methodology here reported constitutes a step forward in the development of new tools for the management, control and eradication of this destructive citrus disease. This system constitutes a potentially field-capable approach for the detection of the most relevant HLB-associated bacteria in plant material and psyllid vectors.
[Show abstract][Hide abstract] ABSTRACT: Field evaluations have shown that Satsuma mandarin (Citrus unshiu) cv. 'Okitsu' is one of the mandarin cultivars that shows substantial resistance to Xanthomonas citri subsp. citri (X. citri), the causal agent of citrus bacterial canker disease. However, the mechanisms underlying this resistance are not well understood. In this study we have shown that 'Okitsu' leaves are nevertheless susceptible to X. citri infection during a period of their development, but this period is shorter than that seen in the susceptible mandarin cv. 'Clemenules' (Citrus clementina). Under controlled growth conditions the resistance of 'Okitsu' cultivar to X. citri was associated with the age of the leaf and was evident in spray-inoculated plants but not in those inoculated by infiltration. Furthermore, X. citri showed reduced attachment and biofilm formation in 'Okitsu' leaves as compared to 'Clemenules'. Taken together, our data suggest that structural features of the 'Okitsu' leaf surface, such as the physical properties of the cuticle, are involved in the resistance to X. citri.
[Show abstract][Hide abstract] ABSTRACT: Xanthomonas citri spp. citri (Xcc)develops a biofilm structure both in vitro and in vivo. Despite all the progress achieved by studies regarding biofilm formation, many of its mechanisms remain poorly understood. This work focuses on the identification of new genes involved in biofilm formation and how they are related to motility, virulence and chemotaxis in Xcc. A Tn5 library of approximately 6,000 Xcc (strain 306) mutants was generated and screened to search for biofilm formation defective strains. We identified 23 genes whose association with the biofilm formation resulted in a novelty. The analysis of the 23 mutants revealed not only the involvement of new genes in biofilm formation but also reinforced the importance of exopolysaccharide production, motility and cell surface structures in this process. This collection of biofilm defective mutants underscores the multifactorial genetic program underlying the establishment of biofilm in Xcc.
[Show abstract][Hide abstract] ABSTRACT: In this work the purification and characterization of an extracellular elicitor protein, designated AsES, produced by an avirulent isolate of the strawberry pathogen Acremonium strictum, is reported. The defense-eliciting activity present in culture filtrates was recovered and purified by ultrafiltration (cut-off 30 kDa), anionic exchange (Q sepharose, pH 7.5) and hydrophobic interaction (Phenyl sepharose) chromatographies. 2D-SDS/PAGE of the purified active fraction revealed a single spot of 34 kDa and pI 8.8. HPLC (C2/C18) and MS/MS analysis confirmed purification to homogeneity. Foliar spray with AsES provided a total systemic protection against anthracnose disease in strawberry, accompanied with the expression of defense-related genes (i.e. PR1, Chi2-1). Accumulation of ROS (e.g. H2O2, O2(.-)) and callose was also observed in Arabidopsis. By using degenerate primers designed from the partial aminoacid sequences and RACE reactions, the complete AsES-coding cDNA of 1167 nucleotides was obtained. The deduced aminoacid sequence showed significant identity with fungal serine proteinases of the subtilisin family, indicating that AsES is synthesized as a larger precursor containing a 15-residue secretory signal peptide, and a 90-residue peptidase inhibitor I9 domain in addition to the 283-residue of mature protein. AsES exhibited proteolytic activity in vitro and its resistance-eliciting activity was eliminated when inhibited with PMSF, suggesting that its proteolytic activity is required to induce the defense response. This is, to our knowledge, the first report of a fungal subtilisin that shows defense-eliciting activity in plants. This finding could contribute to develop disease biocontrol strategies in plants, by activating its innate immunity.
Journal of Biological Chemistry 03/2013; · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Citrus is an economically important fruit crop that is severely afflicted by Asiatic citrus bacterial canker (CBC), a disease caused by the phytopathogen Xanthomonas citri ssp. citri (X. citri). To gain insight into the molecular epidemiology of CBC, a total of 42 Xanthomonas isolates were collected from a range of Citrus species across seventeen different orchards in Tucumán, Argentina; and subjected to molecular, biochemical and pathogenicity tests. Analysis of genome-specific X. citri markers and DNA polymorphism based on rep-PCRs showed that all 42 isolates belonged to X. citri. Interestingly, pathogenicity tests showed that one isolate, which shares more than 90% genetic similarity to the reference strain X. citri T, has host range specificity. This new variant of X. citri ssp. citri, named X. citri A(T), which is deficient in xanthan production, induces an atypical non-cankerous chlorotic phenotype in Citrus limon and C. paradisi and weak cankerous lesions in C. aurantifolia and C. clementinaleaves. In C. limon, suppression of canker development is concomitant with an oxidative burst; however, xanthan is not implicated in the phenotype induced by this interaction, suggesting that other bacterial factors would be involved in triggering the defense response.
[Show abstract][Hide abstract] ABSTRACT: In an incompatible interaction between Colletotrichum fragariae and strawberry plants, the accumulation of phenolic compounds in plant leaves was observed. A particularly abundant penta-esterified ellagitannin that accumulated in response to pathogen attack was identified as 1-0-galloyl-2,3;4,6-bis-hexahydroxydiphenoyl-β-d-glucopyranose (HeT) by mass spectroscopy and nuclear magnetic resonance. Foliar application of purified HeT prior to inoculation with a virulent pathogen was shown to increase resistance toward C. acutatum in strawberry plants and to Xanthomonas citri subsp. citri in lemon plants. The induced resistance in strawberry was associated with a rapid oxidative burst, callose deposition, a transient increase of salicylic acid in phloem, and induction of gene expression responsive to salicylic acid. Results obtained suggested that HeT could be a common plant defense response molecule capable of inducing pathogen resistance in different plant species.
[Show abstract][Hide abstract] ABSTRACT: Xanthomonas citri ssp. citri (Xcc) is the causal agent of citrus canker. This bacterium develops a characteristic biofilm on both biotic and abiotic surfaces. A biofilm-deficient mutant was identified in a screening of a transposon mutagenesis library of the Xcc 306 strain constructed using the commercial Tn5 transposon EZ-Tn5 Tnp Transposome (Epicentre). Sequence analysis of a mutant obtained in the screening revealed that a single copy of the EZ-Tn5 was inserted at position 446 of hrpM, a gene encoding a putative enzyme involved in glucan synthesis. We demonstrate for the first time that the product encoded by the hrpM gene is involved in β-1,2-glucan synthesis in Xcc. A mutation in hrpM resulted in no disease symptoms after 4 weeks of inoculation in lemon and grapefruit plants. The mutant also showed reduced ability to swim in soft agar and decreased resistance to H (2) O (2) in comparison with the wild-type strain. All defective phenotypes were restored to wild-type levels by complementation with the plasmid pBBR1-MCS containing an intact copy of the hrpM gene and its promoter. These results indicate that the hrpM gene contributes to Xcc growth and adaptation in its host plant.
[Show abstract][Hide abstract] ABSTRACT: The identification of a full length cDNA encoding an endo-β-1,3-glucanase (FaOGBG-5) from strawberry (Fragaria × ananassa Duch) is reported. The analysis of the deduced amino acid sequence of FaOGBG-5 showed that it shares typical structural features and a high degree of identity with other plant β-1,3-glucanases of the class I. The expression of FaOGBG-5 in plants infected with a virulent isolate of Colletotrichum acutatum and an avirulent isolate of Colletotrichum fragariae was examined. Induction of expression was observed with both pathogens but exhibited a delayed high expression with the virulent one. Additionally, the accumulation of FaOGBG-5 transcripts was also observed after treatments with the stress related hormones salicylic acid and ethylene. Results obtained suggest that the β-1,3-glucanase encoded by FaOGBG-5 may be implicated in plant defence against biotic and abiotic stress.
[Show abstract][Hide abstract] ABSTRACT: Spodoptera frugiperda (J.E. Smith) is composed of two genetically distinct strains, the so-called corn strain and the rice strain. Whether the two strains differ in their host use is unclear, because laboratory experiments have not been able to show consistent host performance or preference differences between them, and field studies showed high rates of hybridization, as well as some degree asymmetric host use. To determine the distribution of the two strains and their association with host plants, we collected fall armyworm larvae from different crops (corn, rice, alfalfa, and sorghum) and grasses in 15 different localities over 4 yr in Argentina, Brazil, and Paraguay. The strain identity was analyzed using two polymorphisms in the mitochondrial cytochrome oxidase subunit I gene. We identified the corn and rice haplotypes and three types of populations were characterized based on the frequencies of the individuals that belonged to any of these haplotypes: in 44% of populations the corn haplotype predominated, in 44% of populations the rice haplotype was the most frequent, and 11% of populations showed both haplotypes at similar proportions. In total, eight populations (47%) showed the expected pattern, two populations (12%) were polymorphic within the same field, and seven populations (41%) showed the inverse pattern. Taken together, there was no consistent pattern of host association between the two sympatric genotypes and their respective host plants. This investigation supports the need for additional studies to determine which other forces keep the genotypes separate, and what is the degree of genetic differentiation between these populations.
Journal of Economic Entomology 04/2012; 105(2):573-82. · 1.60 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Many authors have reported interactions between strawberry cultivars and pathogenic microorganisms, yet little is known about the mechanisms triggered in the plant. In this paper we examine the participation of the salicylic acid (SA) signaling pathway involved in the response of Fragaria x ananassa cv. Pájaro plants to pathogens. Strawberry plants were challenged with the virulent strain M11 of Colletotrichum acutatum, or with the avirulent strain M23 of Colletotrichum fragariae which confers resistance to the former. Our study showed that the isolate M23 induced a temporal SA accumulation that was accompanied with the induction of PR-1 gene expression in strawberry plants. Such events occured after the oxidative burst, evaluated as the accumulation of hydrogen peroxide and superoxide anion, and many hours before the protection could be detected. Similar results were obtained with exogenously applied SA. Results obtained supports the hypothesis that strawberry plants activate a SA mediated defense mechanisms that is effective against a causal agent of anthracnose. In contrast, plants inoculated with M11 did not show oxidative burst, SA accumulation or PR1 gene induction. This is the first report about a defense response signaling pathway studied in strawberry plants.
Plant Physiology and Biochemistry 02/2012; 54:10-6. · 2.78 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: • Premise of the study: Duchesnea indica is a wild strawberry-like species that has red fruits. In a recent survey in the highlands of Tucumán (Argentina), a plant of D. indica with white fruits was discovered. The aim of this study was to investigate whether the white-fruited character was due to a phenotypic or genotypic change. The stability and heritability of the character and the expression of genes involved in anthocyanins synthesis were studied and compared with red-fruited genotypes. This study contributes to understanding the molecular basis of some factors involved in fruit pigmentation, a horticulturally and taxonomically important trait. • Methods: Stability and heritability of the white-fruited character were evaluated in plants obtained by asexual propagation or by sexual crosses between the white- and red-fruited genotypes. Asexual multiplications were carried out by stolon rooting and sexual multiplications by germination of achenes obtained from crosses. The expression level of the genes involved in the synthesis and regulation of the anthocyanins pathway (CHS, F3H, DFR, ANS, and MYB10) were evaluated by RT-PCR using specific primers. • Key results: Plants with the white-fruited character always yielded white-fruited progeny when propagated asexually, whereas in sexually propagated plants fruit color depended on the mother. Red-fruited mothers yielded red-fruited progeny, and white-fruited mothers yielded fruits ranging from dark pink to white. Molecular analysis suggested that the white-fruited character was due to the low expression of the ANS gene. • Conclusions: Results obtained indicate that the white-fruited character was stable. Mother progenitors exert a strong influence on the expression of the white-fruited character. The white-fruited phenotype is due to the impairment or downregulation of the ANS gene.
American Journal of Botany 12/2011; 98(12):2077-83. · 2.59 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Actin depolymerizing factors (ADFs) have been recently implicated in plant defense against pathogenic fungi, associated with the cytoskeletal rearrangements that contribute to establish an effective barrier against fungal ingress. In this work, we identified a DNA fragment corresponding to a part of a gene predicted to encode an ADF-like protein in genotypes of Fragaria ananassa resistant to the fungus Colletotrichum acutatum. Bulked segregant analysis combined with AFLP was used to identify polymorphisms linked to resistance in hybrids derived from the cross between the resistant cultivar 'Sweet Charlie' and the susceptible cultivar 'Pájaro'. The sequence of one out of three polymorphic bands detected showed significant BLASTX hits to ADF proteins from other plants. Two possible exons were identified and bioinformatic analysis revealed the presence of the ADF homology domain with two actin-binding sites, an N-terminal phosphorylation site, and a nuclear localization signal. In addition to its possible application in strawberry breeding programs, these finding may contribute to investigate the role of ADFs in plant resistance against fungi.
[Show abstract][Hide abstract] ABSTRACT: In 1994, a group whose aims were both scientific research and technological development was set up. This team was inspired by the increasing demand of the agro-industrial sector in the North West of Argentina. Besides financial considerations inherent to the productive process, social and environmental issues were borne in mind when research had to be carried out. The conceptual premises of scientific work were the study, preservation and a judicious use of biodiversity; equally important was the relentless search for phytosanitary management alternatives which were friendly to both humans and the environment. Thus, in 1997, the frst National Program of Strawberry Genetic Breeding in Argentina started at INSIBIO (CONICET-UNT), Tucumán. This Institute coordinated this program on a national level. Thanks to an agreement between INSIBIO and the Estación Experimental Agroindustrial Obispo Colombres (EEAOC), in 2002, the EEAOC's Biotechnology Department, a Unit Associated to INSIBIO, was founded. One of the main goals of this department was both to develop and adapt technologies to support institutional ongoing programs which intended not only to increase productivity but also to confer more sustainability to those agro-industries related to sugar and alcohol, soybean and citrus. And most importantly, a new scientific and technological team constituted by professionals working with other groups from different research centers nationwide carne into existence. Their common goal has been the improvement of agro-industrial productive processes in order to help build a society which is fair and equitable for both the present and the future.
BAG. Journal of basic and applied genetics. 06/2011; 22(1).
[Show abstract][Hide abstract] ABSTRACT: Morphological, anatomical, and molecular techniques were used to characterize wild strawberry and wild strawberry-like species in northwest Argentina. Characteristics of leaves, flowers, runners, achenes, and genomic DNA polymorphisms were used to analyze similarities among Potentilla tucumanensis Castagnaro & Arias, Duchesnea indica (Andr.) Focke, and Fragaria vesca L. Comparison of phenograms obtained by using morphological and anatomical traits or genomic DNA characters revealed similar clustering of the species. Both phenograms suggest that D. indica is more closely related to P. tucumanensis than to F. vesca. Using the randomly amplified polymorphic DNA (RAPD) technique with specific primers, we detected polymorphic bands that permit the identification of P. tucumanensis, D. indica, and F. vesca. In addition, we report new morphological and anatomical characters that can be used as diagnostic traits for better identification of species in reproductive and vegetative states.Key words: Fragaria, Potentilla, Duchesnea, RAPD, DNA fingerprinting, morphological traits, anatomical traits.
Canadian Journal of Botany 02/2011; 78(4):547-556. · 1.40 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Molecular diagnosis of sistemic sugarcane diseases in Argentina: methodology adjustment and applicationsSystemic diseases represent one of the main factors affecting sugarcane production. The knowledge of crop sanitary conditions and the correct identification of phytopathogens are key factors to reduce losses caused by them. To diagnose diseases as early as possible is crucial, so techniques that are sensitive, fast, accurate and easy to use are essential. Since 2005, molecular diagnosis based on polymerase chain reaction has been applied at Estación Experimental Agroindustrial Obispo Colombres, for detecting four systemic sugarcane diseases: ratoon stunting (Leifsonia xyli sp xyli), leaf scald (Xanthomonas albilineans), Sugarcane mosaic virus (ScMV) and yellow leaf syndrome (Sugar cane yellow leaf virus, ScYLV). This paper presents the methodological optimization of molecular diagnosis and compares its efficiency with that of ELISA immunochemistry technique. The molecular approach showed greater sensitivity for the detection of both the bacterial and viral diseases evaluated. The implementation of molecular analysis constitutes a technological advance for regional sugar industry, which will not only help to reduce disease incidence, but also avoid the entrance of new pathogens whenever sugarcane germplasm is introduced from other regions.
Revista industrial y agrícola de Tucumán. 12/2010; 87(2):01-11.
[Show abstract][Hide abstract] ABSTRACT: Evaluation of somaclonal variation in in vitro produced sugarcane plants through molecular markers In vitro culture of plant tissue can produce somaclonal variation, which consists of genetic modifications in cultured cells and tissues. This may constrain the use of this technique in massive micropopagation, especially if such change causes an agronomically relevant phenotypical modification. In this work, a methodology based on the comparison of AFLP (Amplified Fragment Length Polymorphism) molecular marker profiles was developed for detecting somaclonal variation in in vitro propagated sugarcane plants. To optimize AFLP technique application to sugarcane plants, six conventionally propagated genotypes and two types of samples (tender leaves and meristems) were used. Somaclonal variation was determined in micropropagated lines of these genotypes after six months of micropropagation. Molecular profile differentiation of the selected genotypes was achieved with 19 primer combinations. Differential profiles were detected in LCP85-384 and TUCCP77-42 micropropagated lines with 3 of the 19 primer combinations. This result demonstrated that the technique can be used to detect somaclonal variants and that there are different susceptibility levels to in vitro culture among genotypes. Therefore, micropropagation methodology was adjusted to each multiplied genotype so as to ensure genetic purity of in vitro propagated plants.
Revista industrial y agrícola de Tucumán. 12/2010; 87(2):13-21.
[Show abstract][Hide abstract] ABSTRACT: The lack of naturally occurring resistance to Citrus psorosis virus (CPsV) has demanded exploitation of a transgenic approach for the development of CPsV-resistant sweet orange plants. Transgenic sweet orange plants producing intron-hairpin RNA transcripts (ihpRNA) corresponding to viral cp, 54K or 24K genes were generated and analyzed at the molecular and phenotypic levels. Two independent CPsV challenge assays demonstrated that expression of ihpRNA derived from the cp gene (ihpCP) provided a high level of virus resistance, while those derived from 54K and 24K genes (ihp54K and ihp24K) provided partial or no resistance. The presence of small interfering RNA molecules (siRNAs) in the ihpCP transgenic sweet orange plants prior to virus challenge, indicated that CPsV resistance was due to pre-activated RNA silencing, but siRNAs accumulation level was not directly correlated to the degree of the triggered virus resistance among the different lines. However, pre-activation of the RNA-silencing machinery and a certain minimum accumulation level of siRNA molecules targeting the viral genome are key factors for creating virus-resistant plants. This is the first report of resistance in citrus plants against a negative-strand RNA virus as CPsV.
Journal of Biotechnology 11/2010; 151(1):151-8. · 3.18 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Xanthomonas axonopodis pv. citri (Xac) is the causative agent of citrus canker. This bacterium develops a characteristic biofilm on both biotic and abiotic surfaces. To evaluate the participation of the single flagellum of Xac in biofilm formation, mutants in the fliC (flagellin) and the flgE (hook) genes were generated. Swimming motility, assessed on 0.25 % agar plates, was markedly reduced in fliC and flgE mutants. However, the fliC and flgE mutants exhibited a flagellar-independent surface translocation on 0.5 % agar plates. Mutation of either the rpfF or the rpfC gene, which both encode proteins involved in cell-cell signalling mediated by diffusible signal factor (DSF), led to a reduction in both flagellar-dependent and flagellar-independent surface translocation, indicating a regulatory role for DSF in both types of motility. Confocal laser scanning microscopy of biofilms produced in static culture demonstrated that the flagellum is also involved in the formation of mushroom-shaped structures and water channels, and in the dispersion of biofilms. The presence of the flagellum was required for mature biofilm development on lemon leaf surfaces. The absence of flagellin produced a slight reduction in Xac pathogenicity and this reduction was more severe when the complete flagellum structure was absent.
[Show abstract][Hide abstract] ABSTRACT: In this review, we summarise the current knowledge on three pathogens that exhibit distinct tissue specificity and modes of pathogenesis in citrus plants. Xanthomonas axonopodis pv. citri causes canker disease and invades the host leaf mesophyll tissue through natural openings and can also survive as an epiphyte. Xylella fastidiosa and Candidatus Liberibacter are vectored by insects and proliferate in the vascular system of the host, either in the phloem (Candidatus Liberibacter) or xylem (X. fastidiosa) causing variegated chlorosis and huanglongbing diseases, respectively. Candidatus Liberibacter can be found within host cells and is thus unique as an intracellular phytopathogenic bacterium. Genome sequence comparisons have identified groups of species-specific genes that may be associated with the particular lifestyle, mode of transmission or symptoms produced by each phytopathogen. In addition, components that are conserved amongst bacteria may have diverse regulatory actions underpinning the different bacterial lifestyles; one example is the divergent role of the Rpf/DSF cell-cell signalling system in X. citri and X. fastidiosa. Biofilm plays a key role in epiphytic fitness and canker development in X. citri and in the symptoms produced by X. fastidiosa. Bacterial aggregation may be associated with vascular occlusion of the xylem vessels and symptomatology of variegated chlorosis.
Applied Microbiology and Biotechnology 06/2010; 87(2):467-77. · 3.69 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Genetic diversity of sugarcane mosaic virus complex in Tucuman, ArgentinaSugarcane mosaic is one of the most important systemic diseases of sugarcane. Sugarcane mosaic virus (SCMV) and Sorghum mosaic virus (SrMV) are the causal agents of the disease. Sugarcane leaves with mosaic symptoms were analysed by RT-PCR-RFLP (reverse transcriptase-polymerase chain reaction- restriction fragment length polymorphism) and the nucleotide sequences of the coat protein (CP) genes amplified to assess the presence and genetic diversity of both viruses in the sugarcane producing area in Tucumán. Using the primers SCMVR3/F4, 93% of samples were SCMV positive and 33% of them had the E strain RFLP profile, while the rest produced nine different profiles that did not match those of any known strains. Sequence analyses showed that 20% of the samples with the unknown profiles were highly identical to the SCMV D strain, while the rest differed significantly from each other. The presence of the flexuous virions typical of potyviruses was confirmed by transmission electron microscopy. Also, using the specific primers, the presence of SrMV was detected in 90% of the samples, and co-existence of both viruses was found in 85% of the samples. RFLP analysis determined the presence of SrMV strains M and I in 68% and 14% of the samples, respectively, while in approximately 18% of the cases, both M and H strains were present. No RT-PCR product was produced by either SCMV or SrMV primer pairs in one symptomatic sample, suggesting the presence of another pathogen producing similar symptomatology.
Revista industrial y agrícola de Tucumán. 12/2009; 86(2):1-6.