Phenotypic Variability and Genetic Diversity of the Pathogenic Fungus Macrophomina phaseolina from Several Hosts and Host Specialization in Strawberry.

Macrophomina phaseolina, is a pathogenic soil-borne fungus that affects more than 500 plant species, causing various types of disease to several crops, among which is the crown and root rot disease in strawberry. Its wide variability has been characterized reiteratively in the literature, but little is known about its virulence mechanisms. Morphological, physiological, genetic and phytopathogenic parameters were evaluated among 32 isolates of Macrophomina from different hosts occurring in Argentina and Spain. Colony characteristics, average size of microsclerotia, chlorate phenotype and mycelial growth at different temperatures (5º-40 °C), and pH (3.0-8.0) were recorded. The morphological and physiological traits were heterogeneous and did not show any association with the genetic structure nor with their pathogenicity. Most of the isolates (71.9%) exhibited chlorate-sensitive phenotype. The optimal growth temperature range was between 25 °C and 35 °C, and the optimal pH varied between 4.0 and 6.0. The genetic structure analyzed with four DNA markers (EF-1α, ITS, CAL and TUB) showed little diversity among isolates of M. phaseolina, with no clear association with the country of origin, but a significant association with the host. Based on their phylogenetic affinity, one isolate was reclassified as M. pseudophaseolina and another one as M. tecta. It is the first report of M. pseudophaseolina causing charcoal rot on beans, in Argentina, and the first report of M. tecta outside Australia. Pathogenicity tests on strawberry plants revealed marked host specialization, being the isolates obtained from strawberry more virulent than those from other hosts.

Effect of fertilisation with black table olive wastewater solutions on production and quality of tomatoes cultivated under open field conditions.

This study was aimed at making progress on the valorisation of table olive wastewater that currently represent a big environmental problem for factories. Concentrates from vacuum evaporation of the wastewater generated during processing of black ripe olives treated with KOH were tested as fertilisers of tomato plants in open field assays for three consecutive crops. Fertilisation was performed by drip irrigation every 15 days; the first treatment being 15 days after transplanting, and a total of five fertilisation treatments were carried out. A phytotoxic effect was not observed on plants or fruit in any case but higher yield (fruit/plant and g/plant) was obtained in comparison to irrigation with only tap water. Moreover, the combined use of the olive concentrate with inorganic nitrogen supply (NH4NO3), in order to comply with the nutrient needs of plants, was also tested. The results demonstrated that the olive concentrates could be a good substitute for inorganic potassium (as KNO3) during the cultivation of tomato plants without any negative effect on the tomato quality (pH, °Brix, sugars, organic acids) or content of bioactive substances (phenolic compounds and carotenoids).

Progress on green table olive processing with KOH and wastewaters reuse for agricultural purposes.

Table olive wastewaters are seriously polluting and a difficult to treat effluent worldwide, mainly due to their high content in sodium. An alternative approach could be the treatment of the olives with KOH instead of NaOH, in order to reuse the olive streams as biofertilizers. In this study, the debittering of olives with KOH was investigated at pilot plant scale in two olive seasons. The results indicated that a concentration between 1.7 and 2.0% of KOH (similar to that employed with NaOH) led to a fermented product with the same physicochemical and organoleptic characteristics than the traditional one. The spent lyes and washing waters from the KOH treatments were gathered and vacuum evaporated, giving rise to a concentrated solution rich in potassium (52 g/L) that was tested as biofertilizer in open tomato fields. Furthermore, the drip irrigation of the tomato plants with a combined olive solution and mineral fertilizer (NH4NO3) produced similar tomato yield and quality than the irrigation with only mineral fertilizer (NH4NO3 + KNO3). Overall, it has been demonstrated that Spanish-style green olives can be processed with KOH and the effluents valorized to be used as biofertilizer.

Partial Activation of SA- and JA-Defensive Pathways in Strawberry upon Colletotrichum acutatum Interaction.

Understanding the nature of pathogen host interaction may help improve strawberry (Fragaria × ananassa) cultivars. Plant resistance to pathogenic agents usually operates through a complex network of defense mechanisms mediated by a diverse array of signaling molecules. In strawberry, resistance to a variety of pathogens has been reported to be mostly polygenic and quantitatively inherited, making it difficult to associate molecular markers with disease resistance genes. Colletotrichum acutatum spp. is a major strawberry pathogen, and completely resistant cultivars have not been reported. Moreover, strawberry defense network components and mechanisms remain largely unknown and poorly understood. Assessment of the strawberry response to C. acutatum included a global transcript analysis, and acidic hormones SA and JA measurements were analyzed after challenge with the pathogen. Induction of transcripts corresponding to the SA and JA signaling pathways and key genes controlling major steps within these defense pathways was detected. Accordingly, SA and JA accumulated in strawberry after infection. Contrastingly, induction of several important SA, JA, and oxidative stress-responsive defense genes, including FaPR1-1, FaLOX2, FaJAR1, FaPDF1, and FaGST1, was not detected, which suggests that specific branches in these defense pathways (those leading to FaPR1-2, FaPR2-1, FaPR2-2, FaAOS, FaPR5, and FaPR10) were activated. Our results reveal that specific aspects in SA and JA dependent signaling pathways are activated in strawberry upon interaction with C. acutatum. Certain described defense-associated transcripts related to these two known signaling pathways do not increase in abundance following infection. This finding suggests new insight into a specific putative molecular strategy for defense against this pathogen.

Expression of the ß-1,3-glucanase gene bgn13.1 from Trichoderma harzianum in strawberry increases tolerance to crown rot diseases but interferes with plant growth.

The expression of antifungal genes from Trichoderma harzianum, mainly chitinases, has been used to confer plant resistance to fungal diseases. However, the biotechnological potential of glucanase genes from Trichoderma has been scarcely assessed. In this research, transgenic strawberry plants expressing the ß-1,3-glucanase gene bgn13.1 from T. harzianum, under the control of the CaMV35S promoter, have been generated. After acclimatization, five out of 12 independent lines analysed showed a stunted phenotype when growing in the greenhouse. Moreover, most of the lines displayed a reduced yield due to both a reduction in the number of fruit per plant and a lower fruit size. Several transgenic lines showing higher glucanase activity in leaves than control plants were selected for pathogenicity tests. When inoculated with Colletotrichum acutatum, one of the most important strawberry pathogens, transgenic lines showed lower anthracnose symptoms in leaf and crown than control. In the three lines selected, the percentage of plants showing anthracnose symptoms in crown decreased from 61 % to a mean value of 16.5 %, in control and transgenic lines, respectively. Some transgenic lines also showed an enhanced resistance to Rosellinia necatrix, a soil-borne pathogen causing root and crown rot in strawberry. These results indicate that bgn13.1 from T. harzianum can be used to increase strawberry tolerance to crown rot diseases, although its constitutive expression affects plant growth and fruit yield. Alternative strategies such as the use of tissue specific promoters might avoid the negative effects of bgn13.1 expression in plant performance.

Antimicrobial activity of olive solutions from stored Alpeorujo against plant pathogenic microorganisms.

The aim of this work was to assess the in vitro antimicrobial effects that wastewaters from alpeorujo oil extraction have against phytopathogenic bacteria and fungi. Alpeorujo was stored for 6 months and then processed to extract its oil, pomace, and a new liquid waste (OWSA), which was characterized by its content in phenolic compounds. OWSA at 20% decreased bu >4 log the population of Erwinia spp., Pseudomonas spp., and Clavibacter spp. viable cells in test tubes, whereas OWSA at 50% in agar medium was necessary to inhibit mycelial growth of most fungi. It was found that the bactericidal effect was due to the joint action of low molecular mass phenolic compounds, although neither hydroxytyrosol, its glucosides, hydroxytyrosol glycol, nor a glutaraldehyde-like compound individually explained this bioactivity. Hence, OWSA constitutes a promising natural solution to fight plant phytopathogenic bacteria and fungi.

Evidence for a positive regulatory role of strawberry (Fragaria x ananassa) Fa WRKY1 and Arabidopsis At WRKY75 proteins in resistance.

Knowledge of the molecular basis of plant resistance to pathogens in species other than Arabidopsis is limited. The function of Fa WRKY1, the first WRKY gene isolated from strawberry (Fragaria x ananassa), an important agronomical fruit crop, has been investigated here. Fa WRKY1 encodes a IIc WRKY transcription factor and is up-regulated in strawberry following Colletotrichum acutatum infection, treatments with elicitors, and wounding. Its Arabidopsis sequence homologue, At WRKY75, has been described as playing a role in regulating phosphate starvation responses. However, using T-DNA insertion mutants, a role for the At WRKY75 and Fa WRKY1 in the activation of basal and R-mediated resistance in Arabidopsis is demonstrated. At wrky75 mutants are more susceptible to virulent and avirulent isolates of Pseudomonas syringae. Overexpression of Fa WRKY1 in At wrky75 mutant and wild type reverts the enhanced susceptible phenotype of the mutant, and even increases resistance to avirulent strains of P. syringae. The resistance phenotype is uncoupled to PATHOGENESIS-RELATED (PR) gene expression, but it is associated with a strong oxidative burst and glutathione-S-transferase (GST) induction. Taken together, these results indicate that At WRKY75 and Fa WRKY1 act as positive regulators of defence during compatible and incompatible interactions in Arabidopsis and, very likely, Fa WRKY1 is an important element mediating defence responses to C. acutatum in strawberry. Moreover, these results provide evidence that Arabidopsis can be a useful model for functional studies in Rosacea species like strawberry.