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.

Strawberry fatty acyl glycosides enhance disease protection, have antibiotic activity and stimulate plant growth.

An increasing interest in the development of products of natural origin for crop disease and pest control has emerged in the last decade. Here we introduce a new family of strawberry acyl glycosides (SAGs) formed by a trisaccharide (GalNAc-GalNAc-Glc) and a monounsaturated fatty acid of 6 to 12 carbon atoms linked to the glucose unit. Application of SAGs to Arabidopsis thaliana (hereafter Arabidopsis) plants triggered a transient oxidative burst, callose deposition and defense gene expression, accompanied by increased protection against two phytopathogens, Pseudomonas viridiflava and Botrytis cinerea. SAGs-induced disease protection was also demonstrated in soybean infected with the causal agent of target spot, Corynespora cassiicola. SAGs were shown to exhibit important antimicrobial activity against a wide-range of bacterial and fungal phytopathogens, most probably through membrane destabilization, and the potential use of SAGs as a biofungicide for postharvest disease protection was demonstrated on lemon fruits infected with Penicillium digitatum. Plant growth promotion by application of SAGs was shown by augmented primary root elongation, secondary roots development and increased siliques formation in Arabidopsis, whereas a significant increment in number of seed pods was demonstrated in soybean. Stimulation of radicle development and the induction of an auxin-responsive reporter system (DR5::GUS) in transgenic Arabidopsis plants, suggested that SAGs-stimulated growth at least partly acts through the auxin response pathway. These results indicate that strawberry fatty acid glycosides are promising candidates for the development of environmental-friendly products for disease management in soybean and lemon.

Colletotrichum acutatum M11 can suppress the defence response in strawberry plants.

MAIN CONCLUSION: Colletotrichum acutatum M11 produces a diffusible compound that suppresses the biochemical, physiological, molecular and anatomical events associated with the defence response induced by the plant defence elicitor AsES. The fungal pathogen Colletotrichum acutatum, the causal agent of anthracnose disease, causes important economical losses in strawberry crop worldwide and synthetic agrochemicals are used to control it. In this context, the control of the disease using bioproducts is gaining reputation as an alternative of those toxic and pollutant agrochemicals. However, the success of the strategies using bioproducts can be seriously jeopardized in the presence of biological agents exerting a defence suppression effect. In this report, we show that the response defence induced in plant by the elicitor AsES from the fungus Acremonium strictum can be suppressed by a diffusible compound produced by isolate M11 of C. acutatum. Results revealed that strawberry plants treated with conidia of the isolated M11 or the culture supernatant of the isolate M11 suppress: ROS accumulation (e.g., H2O2, O2·- and NO), cell wall reinforcement (e.g., lignin and callose), and the up-regulation of defence-related genes (e.g., FaPR1, FaCHI23, FaPDF1.2, FaCAT, FaCDPK, FaCML39) induced by the elicitor AsES. Additionally, we show that the defence suppressing effect causes a systemic sensitization of plants. Results presented here highlights the necessity to make an integral study of the microbiome present in soils and plant biosphere before applying defence activation bioproducts to control crop diseases.

Inducible expression of Bs2 R gene from Capsicum chacoense in sweet orange (Citrus sinensis L. Osbeck) confers enhanced resistance to citrus canker disease.

Transgenic expression of the pepper Bs2 gene confers resistance to Xanthomonas campestris pv. vesicatoria (Xcv) pathogenic strains which contain the avrBs2 avirulence gene in susceptible pepper and tomato varieties. The avrBs2 gene is highly conserved among members of the Xanthomonas genus, and the avrBs2 of Xcv shares 96% homology with the avrBs2 of Xanthomonas citri subsp. citri (Xcc), the causal agent of citrus canker disease. A previous study showed that the transient expression of pepper Bs2 in lemon leaves reduced canker formation and induced plant defence mechanisms. In this work, the effect of the stable expression of Bs2 gene on citrus canker resistance was evaluated in transgenic plants of Citrus sinensis cv. Pineapple. Interestingly, Agrobacterium-mediated transformation of epicotyls was unsuccessful when a constitutive promoter (2× CaMV 35S) was used in the plasmid construction, but seven transgenic lines were obtained with a genetic construction harbouring Bs2 under the control of a pathogen-inducible promoter, from glutathione S-transferase gene from potato. A reduction of disease symptoms of up to 70% was observed in transgenic lines expressing Bs2 with respect to non-transformed control plants. This reduction was directly dependent on the Xcc avrBs2 gene since no effect was observed when a mutant strain of Xcc with a disruption in avrBs2 gene was used for inoculations. Additionally, a canker symptom reduction was correlated with levels of the Bs2 expression in transgenic plants, as assessed by real-time qPCR, and accompanied by the production of reactive oxygen species. These results indicate that the pepper Bs2 resistance gene is also functional in a family other than the Solanaceae, and could be considered for canker control.

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.

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.

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.