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- Molecular approach to characterize ectomycorrhizae fungi from Mediterranean pine stands in PortugalPublication . Ragonezi, Carla; Caldeira, A. Teresa; Martins, M. Rosário; Salvador, Cátia; Santos-Silva, Celeste; Ganhão, Elsa; Klimaszewska, Krystyna; Zavattieri, Maria AmelyStone pine (Pinus pinea L.), like other conifers, forms ectomycorrhizas (ECM), which have benefi cial impact on plant growth in natural environments and forest ecosystems. An in vitro co-culture of stone pine microshoots with pure mycelia of isolated ECM sporocarps was used to overcome the root growth cessation not only in vitro but also to improve root development during acclimation phase. Pisolithus arhizus (Scop.) Rauschert and Lactarius deliciosus (L. ex Fr.) S.F. Gray fungi, were col lected, pure cultured and used in in vitro co-culture with stone pine microshoots. Samples of P. arhizus and L. deliciosus for the in vitro co-cultures were collected from the pine stands southwest Portugal. The in situ characterization was based on their morphotypes. To confirm the identity of the collected material, ITS amplification was applied using the pure cultures derived from the sporo carps. Additionally, a molecular profile using PCR based genomic fingerprinting comparison was executed with other genera of Basidiomycetes and Ascomycetes. Our results showed the effective ness of the techniques used to amplify DNA polymorphic sequences, which enhances the characte rization of the genetic profile of ECM fungi and also provides an option to verify the fungus identity at any stage of plant mycorrhization.
- Histological studies of mycorrhized roots and mycorrhizal-like-structures in pine rootsPublication . Ragonezi, Carla; Zavattieri, MariaSeveral studies have shown the potential of using Ectomycorrhizal (ECM) fungi in conifer micropropagation to overcome the cessation of adventitious root development. In vitro inoculation promotes the re-growth of the root system induced previously by auxin treatments, facilitating acclimation and diminishing the losses of plants because of a weak root system that is incapable of water and nutrient absorption. During a series of mycorrhization experiments, cryostat and ultrafine cuts were used to study the morpho-histological transformation of the symbiotic roots. To obtain cryostat cuts from pine roots a method frequently used for animal tissue was adopted. Molecular methods allowed fungi identification in all the mycorrhization phases and in the acclimation of derived plants. Mycorrhizal-like-structures derived from in vitro culture and axenic liquid cultures of roots were microscopically analyzed and compare with mycorrhizal roots.
- A novelty system for biotization of plant microshoots and collection of natural compoundsPublication . Castro, Mário Rui da Costa Basílio e; Ragonezi, Carla; Oliveira, Paulo Guilherme Leandro de; Zavattieri, Maria AmelyAn in vitro plant microshoot culture system composed of two phases; a liquid phase overlaid by a floating solid phase, which is described in detail herein. This system is designed to enable the extraction of natural compounds released/disseminated into the liquid phase during root growth, thus facilitating their processing and biochemical characterization. The solid phase holds the plant afloat and enables the simultaneous culture of a microorganism, yet avoiding its penetration into the liquid phase, where the roots are submerged. Both phases can be independently formulated as required for growth optimization of both organisms. Considering the closed system and known variables described in this patent, applications of the described method include testing with pesticides, herbicides, and other similar products.
- Adventitious rooting of conifers: influence of biological factorsPublication . Zavattieri, Maria Amely; Ragonezi, Carla; Klimaszewska, KrystynaVegetative propagation of superior conifer trees can be achieved, e.g., through rooted cuttings or rooted microshoots, the latter predominantly through in vitro tis sue culture. Both techniques are used to achieve rapid multiplication of trees with favorable genetic combinations and to capture a large proportion of the genetic diversity in a single generation cycle. However, adventitious rooting of shoots (cuttings) is often not efficient due to various problems, such as scarcity of roots and cessation of their growth, both of which limit the application of vegetative propagation in some conifer species. Many factors are involved in the adventitious rooting of shoots, including physical and chemical ones, such as plant growth regula tors, carbohydrates, light quality, temperature and rooting substrates, or media [reviewed by Ragonezi et al. (Trees 24(6):975–992, 2010)]. The focus of this review is on biological factors, such as inoculations with Agrobacterium rhizogenes, plant-growth promoting rhizobacteria and other endophytes, and mycorrhizal fungi, which were found to stimulate adventitious rooting. These microor ganisms could contribute not only to adventitious root development but also to help in protecting conifer plants against pathogenic microorganisms, facilitate acclimation and transplanting, and contribute to more sustainable, chemical-free forests.