Seasonal Variation of Plant Defense Inductor Ellagitannins in Strawberry Leaves under Field Conditions for Phytosanitary Technological Applications.

Many natural compounds can activate the plant immunity, and for this reason, they have attracted special interest in crop disease management. Previously, we isolated from strawberry leaves an ellagitannin (HeT), which elicits plant defense responses. In this research, we investigated bioactive compounds from field-collected strawberry leaves capable of inducing defense responses in Arabidopsis thaliana against a bacterial pathogen. Methanolic extracts of strawberry leaves sampled at different months were obtained and compared. The highest content of total soluble phenolic compounds was found in the methanolic extracts of leaves sampled in December (DME). The defense response induced in A. thaliana by DME was attributed to two ellagitannins, the HeT and galloyl-HHDP-glucose. Both compounds exhibited phytoprotective effects against Pseudomonas viridiflava and induced the expression of PDF1.2 and PR1 genes. These results provide an economic value to strawberry leaves, normally discarded at the end of the harvest stage of the crop, as a raw material for plant health enhancer bioinputs.

Soybean-Macrophomina phaseolina-Specific Interactions and Identification of a Novel Source of Resistance.

Charcoal rot, caused by the fungus Macrophomina phaseolina, is an economically important disease of soybean (Glycine max) worldwide. Objectives of the present research were to (i) study the genetic and pathogenic diversity in a collection of M. phaseolina isolates from Argentina and Paraguay and (ii) develop an improved in vitro phenotyping method to evaluate disease response of soybean genotypes to M. phaseolina isolates. Cluster analysis showed no clear association among simple sequence repeat profiles, year of collection, pathogenicity, and geographical origin of the isolates from Argentina and Paraguay. Subsequently, the response of four soybean genotypes against seven M. phaseolina isolates was evaluated in the field and the results were confirmed using the in vitro assay developed. This assay, which is based on root disease development on soybean seedlings, allowed the detection of a differential level of aggressiveness among the isolates on four soybean genotypes. The results suggest the existence of specific interactions among soybean genotypes and M. phaseolina isolates. In addition, cultivar Munasqa RR showed a superior response against M. phaseolina compared with DT 97-4290 (moderately resistant), thus becoming a novel source of resistance to charcoal rot.

The histone-like protein HupB influences biofilm formation and virulence in Xanthomonas citri ssp. citri through the regulation of flagellar biosynthesis.

Citrus canker is an important disease of citrus, whose causal agent is the bacterium Xanthomonas citri ssp. citri (Xcc). In previous studies, we found a group of Xcc mutants, generated by the insertion of the Tn5 transposon, which showed impaired ability to attach to an abiotic substrate. One of these mutants carries the Tn5 insertion in hupB, a gene encoding a bacterial histone-like protein, homologue to the ß-subunit of the Heat-Unstable (HU) nucleoid protein of Escherichia coli. These types of protein are necessary to maintain the bacterial nucleoid organization and the global regulation of gene expression. Here, we characterized the influence of the mutation in hupB regarding Xcc biofilm formation and virulence. The mutant strain hupB was incapable of swimming in soft agar, whereas its complemented strain partially recovered this phenotype. Electron microscope imaging revealed that impaired motility of hupB was a consequence of the absence of the flagellum. Comparison of the expression of flagellar genes between the wild-type strain and hupB showed that the mutant exhibited decreased expression of fliC (encoding flagellin). The hupB mutant also displayed reduced virulence compared with the wild-type strain when they were used to infect Citrus lemon plants using different infection methods. Our results therefore show that the histone-like protein HupB plays an essential role in the pathogenesis of Xcc through the regulation of biofilm formation and biosynthesis of the flagellum.

The Endophytic Strain Klebsiella michiganensis Kd70 Lacks Pathogenic Island-Like Regions in Its Genome and Is Incapable of Infecting the Urinary Tract in Mice.

Klebsiella spp. have been isolated from many different environmental habitats but have mainly been associated with nosocomial acquired diseases in humans. Although there are many recently published sequenced genomes of members of this genus, there are very few studies on whole genome comparisons between clinical and non-clinical isolates, and it is therefore still an open question if a strain found in nature is capable of infecting humans/animals. Klebsiella michiganensis Kd70 was isolated from the intestine of larvae of Diatraea saccharalis but genome analysis revealed multiple genes associated with colonization and growth promotion in plants suggesting an endophytic lifestyle. Kd70 cells labeled with gfp confirmed capability of root colonization and soil application of Kd70 promoted growth in greenhouse grown sugarcane. Further genomic analysis showed that the Kd70 genome harbored fewer mammalian virulence factors and no pathogen island-like regions when compared to clinical isolates of this species, suggesting attenuated animal/human pathogenicity. This postulation was corroborated by in vivo experiments in which it was demonstrated that Kd70 was unable to infect the mouse urinary tract. This is to the best of our knowledge the first experimental example of a member of a pathogenic Klebsiella spp. unable to infect a mammalian organism. A proteomic comparison deduced from the genomic sequence between Kd70 and several other K. michiganensis strains showed a high similarity with isolates from many different environments including clinical strains, and demonstrated the existence of conserved genetic lineages within this species harboring members from different ecological niches and geographical locations. Furthermore, most genetic differences were found to be associated with genomic islands of clinical isolates, suggesting that evolutionary adaptation of animal pathogenicity to a large extent has depended on horizontal gene transfer. In conclusion our results demonstrate the importance of conducting thorough in vivo pathogenicity studies before presupposing animal/human virulence of non-clinical bacterial isolates.

Development of PSP1, a Biostimulant Based on the Elicitor AsES for Disease Management in Monocot and Dicot Crops.

In this work, we present a novel biostimulant for sustainable crop disease management, PSP1, based on the plant defense-elicitor AsES, an extracellular protease produced by the strawberry fungal pathogen Acremonium strictum. Fungal fermentation conditions and downstream processing were determined to maximize extracellular protein production, product stability and a high plant defense-eliciting activity, as monitored by anthracnose resistance in supernatant-treated strawberry plants subsequently infected with a virulent strain of Colletotrichum acutatum. Fermentation batches were shown to reduce anthracnose development by 30-60% as compared to infected non-treated plants. Product formulation was shown to be stable for 6 months when stored at temperatures up to 45°C and toxicological tests showed that PSP1 was harmless to beneficial organisms and non-toxic to mammalian species at concentrations 50 times higher than those used in plant experiments. Furthermore, disease protection studies using dilutions of PSP1 indicated that there is a minimum threshold protease activity needed to induce pathogen defense in strawberry and that this induction effect is dose-independent. A significant characteristic of PSP1 is its broad-range protection against different diseases in various crop species. In soybean, PSP1 reduced the symptomatology by 70% of Corynespora cassiicola, etiological agent of the target spot. This protection effect was similar to the commercial inducer BION 500 WG based on BTH, and both products were shown to induce an oxidative burst and up-regulated PR1-gene expression in soybean. Furthermore, a double PSP1-treatment on greenhouse-grown sugarcane plants provided protection against bacterial red stripe disease caused by Acidovorax avenae and a double foliar application of PSP1 on field-grown wheat plants significantly increased resistance against Fusarium graminearum, causal agent of head blight disease, manifested mainly in an increased seed germination rate. In summary, these disease protection studies demonstrated an effective control against both bacterial and fungal pathogens in both monocot and dicot crop species, which together with its low production cost, effectiveness at low concentrations, long shelf-life, tolerance to high temperatures, harmlessness to non-target organisms and simple handling and application, make PSP1 a very promising candidate for effective and sustainable disease management in many crop species.

Elicitor-Based Biostimulant PSP1 Protects Soybean Against Late Season Diseases in Field Trials.

Currently, fungicide application in soybean production accounts for an important amount of global pesticide use, and it is therefore most desirable to find new healthier and more environmental friendly alternatives for the phytosanitary management in this crop. In this study, we present convincing evidence for effective induction of disease protection by the agricultural biostimulant PSP1, a formulation based on the plant-defense eliciting activity of the fungal protease AsES (Acremonium strictum elicitor subtilisin), in multiple field trials in Argentina. PSP1 was shown to combine well with commercial spray adjuvants, an insecticide, a herbicide and fungicides used in Argentinian soybean production without losing any defense-inducing activity, indicating an easy and efficient adaptability to conventional soybean production and disease management in the region. Results from multiple soybean field trials conducted with different elite genotypes at several locations during two consecutive growing seasons, showed that PSP1 is able to induce an enhanced pathogen defense which effectively reduced late season disease (LSD) development in field-grown soybean. This defense response seems to be broad-range as disease development was clearly reduced for at least three different fungi causing LSDs in soybean (Septoria glycines, Cercospora kikuchii and Cercospora sojina). It was noteworthy that application of PSP1 in soybean alone gave a similar protection against fungal diseases as compared to the commercial fungicides included in the field trials and that PSP1 applied together with a fungicide at reproductive stages enhanced disease protection and significantly increased grain yields. PSP1 is the first example of an elicitor-based strategy in order to efficiently control multiple fungal diseases under field conditions in the soybean crop. These results show the feasibility of using induced resistance products as complements or even full-good replacements to currently used chemical pesticides, fulfilling a role as important components of a more sustainable crop disease management system.

Ellagitannin HeT obtained from strawberry leaves is oxidized by bacterial membranes and inhibits the respiratory chain.

Plant secondary metabolism produces a variety of tannins that have a wide range of biological activities, including activation of plant defenses and antimicrobial, anti-inflammatory and antitumoral effects. The ellagitannin HeT (1-O-galloyl-2,3;4,6-bis-hexahydroxydiphenoyl-ß-d-glucopyranose) from strawberry leaves elicits a strong plant defense response, and exhibits antimicrobial activity associated to the inhibition of the oxygen consumption, but its mechanism of action is unknown. In this paper we investigate the influence of HeT on bacterial cell membrane integrity and its effect on respiration. A ß-galactosidase unmasking experiment showed that HeT does not disrupt membrane integrity. Raman spectroscopy analysis revealed that HeT strongly interacts with the cell membrane. Spectrochemical analysis indicated that HeT is oxidized in contact with bacterial cell membranes, and functional studies showed that HeT inhibits oxygen consumption, NADH and MTT reduction. These results provide evidence that HeT inhibits the respiratory chain.

Resistance to citrus canker induced by a variant of Xanthomonas citri ssp. citri is associated with a hypersensitive cell death response involving autophagy-associated vacuolar processes.

Xanthomonas citri ssp. citri (X. citri) is the causal agent of Asiatic citrus canker, a disease that seriously affects most commercially important Citrus species worldwide. We have identified previously a natural variant, X. citri AT , that triggers a host-specific defence response in Citrus limon. However, the mechanisms involved in this canker disease resistance are unknown. In this work, the defence response induced by X. citri AT was assessed by transcriptomic, physiological and ultrastructural analyses, and the effects on bacterial biofilm formation were monitored in parallel. We show that X. citri AT triggers a hypersensitive response associated with the interference of biofilm development and arrest of bacterial growth in C. limon. This plant response involves an extensive transcriptional reprogramming, setting in motion cell wall reinforcement, the oxidative burst and the accumulation of salicylic acid (SA) and phenolic compounds. Ultrastructural analyses revealed subcellular changes involving the activation of autophagy-associated vacuolar processes. Our findings show the activation of SA-dependent defence in response to X. citri AT and suggest a coordinated regulation between the SA and flavonoid pathways, which is associated with autophagy mechanisms that control pathogen invasion in C. limon. Furthermore, this defence response protects C. limon plants from disease on subsequent challenges by pathogenic X. citri. This knowledge will allow the rational exploitation of the plant immune system as a biotechnological approach for the management of the disease.

Isolation of cellulolytic bacteria from the intestine of Diatraea saccharalis larvae and evaluation of their capacity to degrade sugarcane biomass.

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.

XbmR, a new transcription factor involved in the regulation of chemotaxis, biofilm formation and virulence in Xanthomonas citri subsp. citri.

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 could be 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 knockout of xbmR led to a substantial downregulation of fliA that encodes a σ(28) transcription factor, as well as fliC and XAC0350 which are potential member of the σ(28) regulon. XAC0350 encodes an HD-GYP domain c-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.

Genome-wide annotation of the soybean WRKY family and functional characterization of genes involved in response to Phakopsora pachyrhizi infection.

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. RESULTS: As 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.

Rapid and sensitive detection of Candidatus Liberibacter asiaticus by loop mediated isothermal amplification combined with a lateral flow dipstick.

BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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.

Surface barriers of mandarin 'okitsu' leaves make a major contribution to canker disease resistance.

Field evaluations have shown that Satsuma mandarin (Citrus unshiu) '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; however, this period is shorter than that seen in the susceptible mandarin 'Clemenules' (C. clementina). Under controlled growth conditions, the resistance of 'Okitsu' 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 compared with '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.

Purification and characterization of AsES protein: a subtilisin secreted by Acremonium strictum is a novel plant defense elicitor.

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, are reported. The defense eliciting activity present in culture filtrates was recovered and purified by ultrafiltration (cutoff, 30 kDa), anionic exchange (Q-Sepharose, pH 7.5), and hydrophobic interaction (phenyl-Sepharose) chromatographies. Two-dimensional 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 by the expression of defense-related genes (i.e. PR1 and Chi2-1). Accumulation of reactive oxygen species (e.g. H2O2 and O2(˙)) and callose was also observed in Arabidopsis. By using degenerate primers designed from the partial amino acid sequences and rapid amplification reactions of cDNA ends, the complete AsES-coding cDNA of 1167 nucleotides was obtained. The deduced amino acid 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 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 eliciting activity in plants. This finding could contribute to develop disease biocontrol strategies in plants by activating its innate immunity.

Characterization of a variant of Xanthomonas citri subsp. citri that triggers a host-specific defense response.

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 subsp. citri (X. citri). To gain insight into the molecular epidemiology of CBC, 42 Xanthomonas isolates were collected from a range of Citrus spp. across 17 different orchards in Tucumán, Argentina and subjected to molecular, biochemical, and pathogenicity tests. Analysis of genome-specific X. citri markers and DNA polymorphisms based on repetitive elements-based polymerase chain reaction showed that all 42 isolates belonged to X. citri. Interestingly, pathogenicity tests showed that one isolate, which shares >90% genetic similarity to the reference strain X. citri T, has host range specificity. This new variant of X. citri subsp. citri, named X. citri A(T), which is deficient in xanthan production, induces an atypical, noncankerous chlorotic phenotype in Citrus limon and C. paradisi and weak cankerous lesions in C. aurantifolia and C. clementina leaves. In C. limon, suppression of canker development is concomitant with an oxidative burst; xanthan is not implicated in the phenotype induced by this interaction, suggesting that other bacterial factors would be involved in triggering the defense response.

HrpM is involved in glucan biosynthesis, biofilm formation and pathogenicity in Xanthomonas citri ssp. citri.

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 ß-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.

Host association of Spodoptera frugiperda (Lepidoptera: Noctuidae) corn and rice strains in Argentina, Brazil, and Paraguay.

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.

Temporal accumulation of salicylic acid activates the defense response against Colletotrichum in strawberry.

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.

Identification and characterisation of a novel class I endo-ß-1,3-glucanase regulated by salicylic acid, ethylene and fungal pathogens in strawberry.

The identification of a full length cDNA encoding an endo-ß-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 ß-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 ß-1,3-glucanase encoded by FaOGBG-5 may be implicated in plant defence against biotic and abiotic stress.

White-fruited Duchesnea indica (Rosaceae) is impaired in ANS gene expression.

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.