Evaluación del efecto del filtrado crudo del hongo Rosellinia necatrix en el crecimiento de brotes y cultivos embriogénicos de olivo

Rosellinia necatrix es un patógeno muy extendido en plantaciones de aguacate del sur de la Península Ibérica, afectando también, ocasionalmente, a plantaciones de olivo. Nuestro grupo está abordando la obtención de variantes somaclonales de olivo y aguacate, que muestren mayor tolerancia a este patógeno. En este trabajo se presentan los resultados en olivo, tras la exposición de cultivos embriogénicos a dosis crecientes de filtrado crudo del hongo. Se utilizó callo embriogénico, obtenido a partir de radícula, cultivado en medio líquido ECO (Pérez-Barranco et al. 2009 PCTOC 97: 243-251) durante 3 semanas en agitación, y posteriormente filtrado (malla de 2 mm) para separar la fracción fina. Esta fracción fue cultivada durante otras 3 semanas en medio ECO líquido conteniendo un 20, 40, 60 u 80% de filtrado crudo de Rosellinia necatrix, tras su esterilización en frio. Finalmente, el callo embriogénico fue cultivado en placa en medio ECO sólido para su recuperación. El filtrado del hongo al 20% no afectó al crecimiento del callo embriogénico; sin embargo, a partir del 40% el crecimiento fue inhibido, salvo en algunas placas, donde se observó proliferación de callo tras 2 meses en medio de cultivo sin filtrado. Se han obtenido varias líneas que han sobrevivido tras la exposición al 40, 60 y 80% de filtrado del hongo. Estas líneas están siendo cultivadas de nuevo en presencia de la misma concentración de filtrado del hongo para evaluar su comportamiento. Posteriormente, se abordará la recuperación de plantas. Paralelamente, brotes de olivo regenerados a partir de la línea embriogénica P1 han sido cultivados en medio de multiplicación (Vidoy-Mercado et al. 2012, Acta Hort. 949: 27-30) suplementado con filtrado crudo del hongo (60%), durante varios subcultivos, para evaluar el efecto del filtrado en el crecimiento de los brotes.Universidad de Málaga, Campus de Exelencia Internac ional Andalucía Tech. Proyecto AGR-7992 (P11-Junta de Andalucía

Agrobacterium-mediated transformation of olive (Olea europaea L.) with an antifungal protein from Aspergillus giganteus.

Broad-spectrum resistance to pests and diseases is difficult to obtain through classical breeding programs, hence, this is a targeted trait for accelerating the development of major olive cultivars using plant trans- formation technologies. Olive Verticillium wilt, caused by Verticillium dahliae, is considered to be an important constraint for cultivation of olive trees (López-Escudero and Mercado-Blanco 2010). Different transgenic approaches have been proposed to engineer plants for resistance to fungal diseases, including production of antifungal proteins (Gurr and Rushton 2005). Regarding this approach, among different anti- fungal compounds, the antifungal protein (AFP) from Aspergillus giganteus can be considered a promising candidate for practical applications in crop protection (Meyer 2007). AFP is a defensin-like protein that belongs to a group of small-sized secretory proteins rich in cysteine residues. The protein possesses in vitro antifungal activity inhibiting the growth of several fungal pathogens. Previous work has already shown that afp gene can be expressed in transgenic rice plants inducing resistance to the fungus Magnaporthe grisea and indicating the usefulness of such approach for protection against rice blast. (Coca et al. 2004). In this work, transgenic olive plants were generated by Agrobacterium-mediated transformation as des- cribed by Torreblanca et al. (2010). The AGL-1 strain containing the pBIN61-afp binary vector was used. This plasmid contains the nptII gene for paromomycin selection and a chemically synthesized codon-op- timized afp gene under the control of the 35S CaMV promoter. Globular somatic embryos derived from a mature seed of cultivar `Picual ́ were transformed obtaining an average success rate around 2%. Plants were regenerated from six independent lines and transgenic nature was confirmed by PCR studying nptII and afp insertion. With the aim of studying whether the afp gene can be used to induce resistance against fungal diseases in olive, susceptibility to the fungal pathogens Rosellinia necatrix and Verticilium dahliae will be evaluated. In addition, the inhibitory effect of proteins extracts from transgenic leaves on the in vitro growth of these fungal pathogens will also be examined.Universidad de Málaga. Campus de Excelencia Internacional Andalucia Tec

Contribution to improvement of the traditional extraction of olive oil by pressure from whole and stoned olives by addition of a co-adjuvant (talc)

The olive growing is one of the strategic sectors of the Algerian economy. Traditional olive culture located in the mountains of Kabylia offers typical oil widely preferred by a large part of the Algerian consumers. However, this ancestral culture risks not only abandonment but suffers much more from uncontrolled of the good practices surrounding this local product requiring a valorization accompanied by improvement. Indeed, the difficulty to extract the total oil contained in the fruit is one of the main obstacles of the extraction method particularly by pressure. Nevertheless, some actions as the addition of co-adjuvant during the malaxing process allow improving efficiency of the extraction process. Our results indicate that the addition of 2.5% of talc as a co-adjuvant to a moistless paste obtained from whole olives significantly improves the oil yield by about 4.4% without altering the acidity compared to the control. Similarly, stoning improves the moisture of the pressed mass although without improving yield of the oil characterized by a slight decrease in acidity compared to other extracted oils. Therefore, our results confirm the beneficial effect of talc on the extraction of olive oil and contribute to the improvement of the traditional extraction by pressure to enhance the value of this local product

Usage of the Heterologous Expression of the Antimicrobial Gene afp From Aspergillus giganteus for Increasing Fungal Resistance in Olive

The antifungal protein (AFP) produced by Aspergillus giganteus, encoded by the afp gene, has been used to confer resistance against a broad range of fungal pathogens in several crops. In this research, transgenic olive plants expressing the afp gene under the control of the constitutive promoter CaMV35S were generated and their disease response against two root infecting fungal pathogens, Verticillium dahliae and Rosellinia necatrix, was evaluated. Embryogenic cultures derived from a mature zygotic embryo of cv. ‘Picual’ were used for A. tumefaciens transformation. Five independent transgenic lines were obtained, showing a variable level of afp expression in leaves and roots. None of these transgenic lines showed enhanced resistance to Verticillium wilt. However, some of the lines displayed a degree of incomplete resistance to white root rot caused by R. necatrix compared with disease reaction of non-transformed plants or transgenic plants expressing only the GUS gene. The level of resistance to this pathogen correlated with that of the afp expression in root and leaves. Our results indicate that the afp gene can be useful for enhanced partial resistance to R. necatrix in olive, but this gene does not protect against V. dahliae