[Show abstract][Hide abstract] ABSTRACT: Xanthomonas citri subsp. citri (Xcc) is the causal agent of citrus canker. Biofilm formation on citrus leaves plays an important role in epiphytic survival of Xcc. Biofilm formation is affected by transposon insertion in XAC3733, which encodes a transcriptional activator of the NtrC family, not linked to a gene encoding a sensor protein, thus be could considered as an ‘orphan’ regulator whose function is poorly understood in Xanthomonas spp. Here we show that mutation of XAC3733 (named xbmR) resulted in impaired structural development of the Xcc biofilm, loss of chemotaxis and reduced virulence in grapefruit plants. All defective phenotypes were restored to wild-type levels by the introduction of PA2567 from Pseudomonas aeruginosa, which encodes a phosphodiesterase active in the degradation of cyclic diguanosine monophosphate (c-di-GMP). A knock-out of xbmR led to a substantial down regulation of fliA, which encodes a σ28 transcription factor, as well as fliC and XAC0350 which are potential member of the σ28 regulon. XAC0350 encodes an HD-GYP domain cyclic di-GMP phosphodiesterase. These findings suggest that XbmR is a key regulator of flagellar-dependent motility and chemotaxis exerting its action through a regulatory pathway that involves FliA and c-di-GMP.
[Show abstract][Hide abstract] ABSTRACT: Drought is a major limitation to crop yields worldwide. Screening for soybean yield under water deficit is often a bottleneck in breeding programmes. We assessed the validity of a standardized drought tolerance screening method to predict water-limited field performance of soybean in NW Argentina. First, to determine the phenological period when yield of glasshouse-grown plants was more sensitive to water deficit, we applied treatments during 21 days in V7, R3 or R5 stages, being the period from R5 to R6 the most critical for yield. Afterwards, two glasshouse experiments were carried out to quantify the tolerance of either eight or four genotypes, respectively, by applying a controlled water deficit of constant intensity during the critical period. Finally, yield data obtained in field trials in Argentina across several locations and seasons classified according to rainfall were analysed. Drought Susceptibility Index was calculated for each experiment and for field data, and rankings of tolerance were similar in all cases. This standardized method, which can be automated for high-throughput phenotyping, could represent a useful tool in breeding programmes for identifying soybean cultivars with improved performance under drought conditions.
Journal of Agronomy and Crop Science 10/2014; · 2.62 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background
Many previous studies have shown that soybean WRKY transcription factors are involved in the plant response to biotic and abiotic stresses. Phakopsora pachyrhizi is the causal agent of Asian Soybean Rust, one of the most important soybean diseases. There are evidences that WRKYs are involved in the resistance of some soybean genotypes against that fungus. The number of WRKY genes already annotated in soybean genome was underrepresented. In the present study, a genome-wide annotation of the soybean WRKY family was carried out and members involved in the response to P. pachyrhizi were identified.ResultsAs a result of a soybean genomic databases search, 182 WRKY-encoding genes were annotated and 33 putative pseudogenes identified. Genes involved in the response to P. pachyrhizi infection were identified using superSAGE, RNA-Seq of microdissected lesions and microarray experiments. Seventy-five genes were differentially expressed during fungal infection. The expression of eight WRKY genes was validated by RT-qPCR. The expression of these genes in a resistant genotype was earlier and/or stronger compared with a susceptible genotype in response to P. pachyrhizi infection. Soybean somatic embryos were transformed in order to overexpress or silence WRKY genes. Embryos overexpressing a WRKY gene were obtained, but they were unable to convert into plants. When infected with P. pachyrhizi, the leaves of the silenced transgenic line showed a higher number of lesions than the wild-type plants. Conclusions
The present study reports a genome-wide annotation of soybean WRKY family. The participation of some members in response to P. pachyrhizi infection was demonstrated. The results contribute to the elucidation of gene function and suggest the manipulation of WRKYs as a strategy to increase fungal resistance in soybean plants.
[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: We report Acremonium strictum as the causal agent of a new disease in strawberry plants (Fragaria x ananassa Duch.) in the Northwest of Argentina. Both the structure of conidiophores and the sequence spanning the internal transcribed spacers 1 and 2 (ITS1 and ITS2) of the nuclear ribosomal DNA (rDNA) allowed confirming the affiliation of the isolate, corresponding to A. strictum. An analysis of symptoms and lesions caused by the strain of A. strictum in susceptible cultivars showed that the typical symptoms are as follows: in an early stage, small necrotic light-brown spots in leaves and petioles increase in number and size as the disease progresses; in a more advanced stage, dark necrotic areas expand over petioles and leaves causing strangulation of petioles and the plant wilt. Crown rot was not observed even at a very advanced stage of the disease.
European Journal of Plant Pathology 12/2013; 137(4). · 1.71 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Abstract
SINCE 2001, THE ‘Estación Experimental Agroindustrial Obispo Colombres’ (EEAOC)
has been working on the ‘Vitroplantas Project’. On average 70 000 sugarcane plantlets
of commercial varieties are produced through in vitro meristem culture in order to
eliminate systemic diseases caused by bacteria and viruses. The sanitation of plant
material is achieved through in vitro culture of apical meristems from donor plants
previously hot-water-treated and grown for 3 years under greenhouse conditions with
anti-aphid screens. Systemic diseases are detected in both meristem donor plants and
micropropagated plantlets using different molecular diagnostic techniques. It is well
known that in vitro plant tissue culture can produce somaclonal variations, which
consist of genetic changes in cultured cells and tissues. In order to guarantee that
seedlings propagated in vitro are identical to parental varieties, molecular markers to
quantify and detect somaclonal variation are routinely applied. Thus, the aim of this
project is to guarantee healthy and genetically pure plantlets before transfer to the field
for seed cane propagation. After propagation and testing, in vitro plantlets undergo an
acclimatisation process in a specially adapted greenhouse at the EEAOC. In order to
avoid dehydration of plantlets, the process takes place in a greenhouse with very high
relative humidity (RH=80–100%) and low light intensity. After acclimatisation, two
more stages of conventional field propagation (Basic and Registered), are carried out
before the seed cane is finally distributed among sugarcane growers. The
implementation of the project ‘Vitroplantas’ has greatly improved the health and
biomass yields of sugarcane production in Tucuman, Argentina.
Proc. Int. Soc. Sugar Cane Technol.,. 10/2013; 28.
[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; · 4.60 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: The pepper Bs2 gene confers resistance to Xanthomonas campestris pv. vesicatoria (Xcv) pathogenic strains containing the avrBs2 avirulence gene in susceptible pepper and tomato. The avrBs2 gene is highly conserved in the Xanthomonas genus and when bacteria lack this gene their growth in a susceptible host is diminished, indicating that the avrBs2 gene product could confer an adaptive advantage to the pathogen. The avrBs2 of Xanthomonas citri subsp. citri (Xcc), cause of citrus canker, shares 96% homology with avrBs2 of Xcv. To evaluate if Bs2 could recognize avrBs2 of Xcc in citrus plants and thereby activate plant defence mechanisms to increase resistance to canker, transient expression experiments were conducted using Agrobacterium tumefaciens in lemon plants subsequently challenged with wildtype Xcc. The results showed that transient expression of Bs2 reduced canker formation in lemon and induced plant defence mechanisms, as shown by callose deposition and PR‐1 expression. Moreover, when an avrBs2 mutant of Xcc was used, no decrease in disease symptoms was observed. This work shows that the Bs2 gene from Solanaceae is functional in lemon, a member of the Rutaceae family. Therefore, Bs2 is a potential candidate gene for stable expression in transgenic citrus plants in order to improve resistance to canker disease.
[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: Sugarcane is known for its highly complex genetics and more knowledge is needed for better use and conservation of genetic materials. In order to identify genotypes and to assess genetic diversity, diverse data sets such as morphological and molecular markers are used as a general approach. To evaluate the usefulness of different markers, important sugarcane genotypes in Argentina were characterized by AFLP, SSR and morphological traits. All genotypes characterized were grouped in one main cluster in dendrograms using two independent softwares. Interestingly, local genotypes grouped together with USA varieties and no clear genetic differentiation could be found probably due to intensive germplasm exchange between these breeding programs. The molecular markers tested were useful for genetic diversity assessment as well as for genotype identification. These markers should be included in the internationally established characters for sugarcane variety protection as they give a better view on whole genome complexity. Additionally, genetic similarities obtained from molecular markers will provide more accurate information to breeders than the pedigree method, especially when considering the asymmetric genetic inheritance of sugarcane. Morphological traits are valuable tools to identify genotypes since they reflect external resemblance more than genetic relatedness. When they were combined with molecular markers the dendogram obtained revealed genetic relationships and the genetic diversity was better estimated. In summary, both methods appear to be useful, complementing each other and should be used together to assist sugarcane breeders in estimating genetic diversity, electing parents for crossings, identifying superior lines and to protect intellectual property rights.
[Show abstract][Hide abstract] ABSTRACT: Strawberry plants exposed to an avirulent isolate of Colletotrichum fragariae acquired strong resistance against a virulent strain of C. acutatum. Biochemical, morphological and molecular markers indicated that the strong defence response was associated with an oxidative burst and a transient accumulation of salicylic acid (SA). A maximum accumulation of H2O2 and O2− was observed 8 h after inoculation (hai), callose was detected 48 hai, and a peak of SA was observed 48 hai. Biochemical and phytopathogenic analyses carried out in non-treated tissues revealed that the defence response was systemic and remained fully active 60 days after the first inoculation. Experiments also showed that the resistance acquired by mother plants after the inoculation with the avirulent isolate could be passed to daughter plants through runners. Further characterization of the induced systemic resistance showed that the resistance was not only effective against a virulent strain of C. acutatum but also against Botrytis cinerea.
European Journal of Plant Pathology 04/2012; 135(4). · 1.71 Impact Factor
[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. · 1.61 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. · 2.35 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. · 2.46 Impact Factor