[Show abstract][Hide abstract] ABSTRACT: As a strategy to find efficient lignocellulose degrading enzymes/microorganisms for sugarcane biomass pretreatment purposes, 118 culturable bacterial strains were isolated from intestines of sugarcane-fed larvae of the moth Diatraea saccharalis. All strains were tested for cellulolytic activity using soluble carboxymethyl cellulose (CMC) degrading assays or by growing bacteria on sugarcane biomass as sole carbon sources. Out of the 118 strains isolated thirty eight were found to possess cellulose degrading activity and phylogenetic studies of the 16S rDNA sequence revealed that all cellulolytic strains belonged to the phyla γ-Proteobacteria, Actinobacteria and Firmicutes. Within the three phyla, species belonging to five different genera were identified (Klebsiella, Stenotrophomonas, Microbacterium, Bacillus and Enterococcus). Bacterial growth on sugarcane biomass as well as extracellular endo-glucanase activity induced on soluble cellulose was found to be highest in species belonging to genera Bacillus and Klebsiella. Good cellulolytic activity correlated with high extracellular protein concentrations. In addition, scanning microscopy studies revealed attachment of cellulolytic strains to different sugarcane substrates. The results of this study indicate the possibility to find efficient cellulose degrading enzymes and microorganisms from intestines of insect larvae feeding on sugarcane and their possible application in industrial processing of sugarcane biomass such as second generation biofuel production.
AMB Express 12/2015; 5(1):15. DOI:10.1186/s13568-015-0101-z
[Show abstract][Hide abstract] ABSTRACT: Sugarcane commercial variety RA 87-3 was transformed with a genetic construct harboring the epsps gene from Agrobacterium strain CP4 conferring tolerance to glyphosate and nptII gene for kanamycin selection. Transformed lines were multiplied in greenhouse, and herbicide tolerance was evaluated using different concentrations (3, 4, 8 and 16 l/ha) of glyphosate (Helm 48 % p/v). All herbicide-tolerant (HT) lines were field tested to confirm glyphosate tolerance and perform preliminary evaluations of phenotypic resemblance to parental cultivar. All transformed lines maintained herbicide tolerance, but many showed phenotypic changes and/or growth aberrations. Ten HT lines, showing close growth resemblance to RA 87-3, were analyzed using nine compulsory morphologic markers proposed by the International Union for the Protection of New Varieties of Plants (UPOV) and 339 molecular markers. Out of the ten HT lines tested, six showed minor morphologic and genetic variations and were selected for field testing over two vegetative crop cycles (plant cane and first ratoon) at two production areas in Argentina. The six field-tested HT lines were found to be almost indistinguishable when comparing agronomic and industrial characteristics and chemical composition. Stable heritance of the CP4 epsps gene and glyphosate tolerance throughout different clonal generations were confirmed by RT-qPCR and Southern blot. Taking into account all results, two out of the six lines tested were selected for a possible commercial release. Our study confirms the utility of genetic transformation as a complementary tool to classical breeding procedures and highlights the usefulness of UPOV traits together with molecular markers for early selections of transgenic events that closely resemble their parental genotype.
[Show abstract][Hide abstract] ABSTRACT: Huanglongbing (HLB), a devastating citrus disease caused by the bacterium “Candidatus Liberibacter spp.”, is now responsible for significant economic losses worldwide. Yet, no effective disease control has been found, and the non-cultivability of the bacterium has severely hampered studies on the pathogen. The 16S rDNA gene is a well-characterized sequence, essential for cell survival, and is used for bacterial identification or assignment of close relationships at the genus and species levels. Quantitative Real-Time PCR (qPCR) assays based on 16S rDNA genes are widely used in the detection of “Ca. Liberibacter spp.” in multiplex reactions. We have developed for the first time a set of qPCR primers based on the conserved 16S rDNA gene, which specifically and simultaneously detects in a singleplex reaction, all three bacterial species associated with HLB, and can differentiateCa.Liberibacter asiaticus or africanus from americanus by their characteristic melting curves. The assay is very sensitive, and it was possible to amplify expected DNA fragments with an efficiency of 98 % using the Syber Green system and a Ct value lower than tested methods for HLB diagnosis. The application of this fast, simple and efficient detection methodology could also be important in the detection of all species of HLB-associated Liberibacters and could contribute to early pathogen detection, a crucial step in the development of preventive strategies aimed at avoiding the dissemination of this devastating disease in HLB-free areas.
Scientia Agricola 01/2015; 72(3):252-259. DOI:10.1590/0103-9016-2013-0417 · 0.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: This work identifies a compound able to inhibit Xanthomonas citri subsp. citri (citrus canker agent) growth in vitro and in vivo. First, we isolated bacteria from citrus leaf surfaces that were able to inhibit X. citri subsp. citri in vitro. Among the selected isolates, we focused on one with remarkable activity. The strain was characterized as Pseudomonas fluorescens after sequencing its 16S rDNA and analyzing the sequence with BLASTn. The purification and chemical analysis of the active compound allowed us to assign the inhibitory activity to enantio-pyochelin (E-Pch). Since this molecule is a siderophore, we wondered if the inhibition observed was a result of iron scavenging. Surprisingly, when we supplemented media with an excess of iron, we observed practically no change in the inhibition activity. In an attempt to identify the action mechanism of E-Pch, we evaluated the ability of E-Pch to generate reactive oxygen species (ROS) within a culture of X. citri subsp. citri and its correlation with the inhibitory activity. In fact, we observed increased ROS levels when E-Pch was added. In addition, the reducing agent, ascorbic acid, lowered ROS levels and the antibiotic activity, implying that inhibition is probably caused by oxidative stress. Finally, we studied the use of E-Pch in a model of canker disease. E-Pch reduced canker formation on leaves of ‘Eureka’ and ‘Lisbon’ lemon cultivars. These results show E-Pch as a promising compound for citrus canker biocontrol.
[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; 201(2). DOI:10.1111/jac.12106 · 2.44 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: Yellow leaf disease, caused by Sugarcane yellow leaf virus (SCYLV), is widespread around the world but very little information is available on this viral disease in Argentina. Therefore, the aims of the study were to assess the presence of SCYLV, analyze its distribution in the main sugarcane production areas of Argentina, characterize the virus, and determine histological alterations caused by its presence. For this purpose, 148 sugarcane samples with and without symptoms were collected in 2011 and 2012 from the province of Tucuman. One additional sample was collected in Salta, a different geographical, agroecological, and producing region. Results showed that SCYLV is widely distributed in commercial varieties of sugarcane throughout Tucuman in both symptomatic and asymptomatic leaves. A low but statistically significant positive correlation with virus detection and disease symptoms was found. BRA-PER was the only genotype detected by reverse-transcription polymerase chain reaction and sequence analysis of the SCYLV capsid protein gene. SCYLV-positive samples showed high starch levels in bundle sheath cells, whereas the asymptomatic ones, probably in an early stage of infection, were found to contain more chloroplasts. Symptomatic noninfected samples presented crystal formation probably associated with phytoplasma infection.
[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). DOI:10.1007/s10658-013-0279-3 · 1.49 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.
[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: Brown rust, caused by the fungus Puccinia melanocephala, is responsible for important yield losses in sugarcane production globally and it is therefore an important objective to introduce resistance to this disease in breeding programs. A major gene, Bru1, has been shown to confer resistance to P.
melanocephala strains from different parts of the world and two molecular markers, R12H16 and 9O20-F4, closely associated to this gene have been previously reported. The usefulness of these molecular diagnostic markers in order to predict a rust resistant phenotype under natural high pressure inoculums conditions was analyzed. A total of 129 sugarcane accessions were evaluated under field infection for resistance or susceptibility to brown rust and subsequently screened for presence or absence of the two Bru1 diagnostic markers. A total of 49 genotypes (38 %) were phenotyped as resistant to brown rust but only eight (16.3 %) of them were harboring the Bru1 gene. To determine overall frequency of the Bru1 in the local sugarcane germplasm collection, 190 additional genotypes were examined. Presence of Bru1, as determined by the diagnostic markers, was detected in only 7 % of the genotypes evaluated. In conclusion, Bru1 diagnostic markers enable positive selection for brown rust resistance in sugarcane and moreover allowed detecting at least one additional source(s) of resistance. Interestingly, whilst only little genetic variability of rust resistance independent of Bru1 has been reported previously, this alternative genetic resource(s) found in our local germplasm constitutes the predominant one and should be helpful in order to amplify the narrow genetic basis for brown rust resistance in sugarcane.
[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.
[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.