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Histological studies of mycorrhized roots and mycorrhizal-like-structures in pine roots
Publication . Ragonezi, Carla; Zavattieri, Maria
Several 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 compounds
Publication . Castro, Mário Rui da Costa Basílio e; Ragonezi, Carla; Oliveira, Paulo Guilherme Leandro de; Zavattieri, Maria Amely
An 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.
Mycorrhiza-like structures in rooted microshoots of Pinus pinea L.
Publication . Castro, M. R.; Ragonezi, C.; Klimaszewska, K.; Lima, M.; de Oliveira, P.; Zavattieri, M. A.
Pinus pinea L. (stone pine) is one of the major plantation species in Iberian
Peninsula, being Portugal the largest edible seed producer in the world. The
induction and improvement of in vitro rhizogenesis of microshoots of Pinus pinea
was developed in our laboratory using a co-culture system with ECM fungi. In the
acclimation phase in mixed substrates, or in rhizotrons, anatomical and
morphological studies were done to observe the evolution of the root system in
microshoots from the co-culture system vs. control plants. Extensive dichotomous
and coralloid branching of lateral roots occurred spontaneously in inoculated and
control plants as well. Moreover, similar branching occurred in liquid culture of
excised seedling roots without the presence of ECM fungi. The striking similarity of
these organs with pine ectomycorrhizas prompted their anatomical analysis;
however the presence of Hartig net was not confirmed. These results suggested that
the development of ECM-like structures might have occurred spontaneously.
Molecular approach to characterize ectomycorrhizae fungi from Mediterranean pine stands in Portugal
Publication . Ragonezi, Carla; Caldeira, A. Teresa; Martins, M. Rosário; Salvador, Cátia; Santos-Silva, Celeste; Ganhão, Elsa; Klimaszewska, Krystyna; Zavattieri, Maria Amely
Stone 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.
O-coumaric acid ester, a potential early signaling molecule in Pinus pinea and Pisolithus arhizus symbiosis established in vitro
Publication . Ragonezi, C.; Teixeira, D.; Caldeira, A. T.; Martins, M .R.; Santos-Silva, C.; Ganhão, E.; Klimaszewska, K.; Zavattieri, M. A.
During ectomycorrhizal (ECM) establishment, biochemical signals lead to the development of complex structures
in both the plant and the fungus that ultimately result in the formation of an ectomycorrhiza. The cross-talk
between partners begins before physical contact. Our objective was to investigate the chemical nature of the
signals during the first stages of in vitro mycorrhization of Pinus pinea with Pisolithus arhizus. For this purpose a
double-phase solid liquid medium was expressly developed for the co-culture in order to simplify the extraction
and further molecules analysis. O-coumaric acid ester was identified using HPLC UV and LC DAD MS on the
second day of co-culture and its presence was detected for up to 10 days. These results contribute to the
characterization of biochemical signals during pre-colonization involving conifer species and an ECM fungus,
and demonstrate the suitability of the double-phase medium developed for the growth of both organisms and for the analysis of released chemical mediators.
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Funding agency
Fundação para a Ciência e a Tecnologia
Funding programme
5876-PPCDTI
Funding Award Number
PTDC/AGR-CFL/71437/2006