Bruno Mori

Coexistence of predominantly nonculturable rhizobia with diverse, endophytic bacterial taxa within nodules of wild legumes

A previous analysis showed that Gammaproteobacteria could be the sole recoverable bacteria from surface-sterilized nodules of three wild species of Hedysarum. In this study we extended the analysis to eight Mediterranean native, uninoculated legumes never previously investigated regarding their root-nodule microsymbionts. The structural organization of the nodules was studied by light and electron microscopy, and their bacterial occupants were assessed by combined cultural and molecular approaches. On examination of 100 field-collected nodules, culturable isolates of rhizobia were hardly ever found, whereas over 24 other bacterial taxa were isolated from nodules. None of these nonrhizobial isolates could nodulate the original host when reinoculated in gnotobiotic culture. Despite the inability to culture rhizobial endosymbionts from within the nodules using standard culture media, a direct 16S rRNA gene PCR analysis revealed that most of these nodules contained rhizobia as the predominant population. The presence of nodular endophytes colocalized with rhizobia was verified by immunofluorescence microscopy of nodule sections using an Enterobacter-specific antibody. Hypotheses to explain the nonculturability of rhizobia are presented, and pertinent literature on legume endophytes is discussed.

Phaeomoniella chlamydospora-Grapevine Interaction : Histochemical Reactions to Fungal Infection

Histochemical reactions of one-year-old potted micropropagated vines (rootstock 1103 Paulsen) to inoculation with Phaeomoniella chlamydospora were studied. Microscopic examination of the vine wood showed that the fungus spread through the wood tissue, albeit slowly. Starting from the roots it required nine months to colonise the first 20-25 cm of the grapevine stems. The slow spread of P. chlamydospora is thought to be due to the defence response initiated by the vines: production of tyloses, including accumulation of phenols in the vessels and adjacent tissue, and the deposition of unidentified defence-response substances, probably stilbene-like substances from the cell-wall surrounding the infection

Oak (Quercus robur L.) seedling responses to a realistic increase in UV-B radiation under open space conditions

Plants show different levels of sensitivity to ultraviolet-B (UV-B) radiation and acclimation to high UV-B irradiances had been developed in many species. A field experiment was performed during 1995 to study the adaptative responses of oak plants (Quercus robur L.) to artificial UV-B supplementation The most important effect of UV treatment was the increase in leaf thickness and the accumulation of phenolic compounds in the secondary wall of the adaxial epidennis of leaves in UV treated plants; the secondary wall of cells in the abaxial epidermis, that is not directly exposed to supplemental UV-B. did not present any alterations. No effects were observed on photosynthesis while plant morphology was sensible to UV treatment.

Differential Staining for Cellulosic and Modified Plant Cell Walls

A simple method to enhance the staining of cell wall components for fluorescence microscopy is described. In stems of Nicotiana tabacum and needles of Pinus eldarica lignin, the cuticle and unsaturated lipids are indicated by a purple-red fluorescence while pectocellulosic components fluoresce pale blue.

Insights into a hydration regulating system in Cupressus pollen grains

BACKGROUND AND AIMS Hydration, rupture and exine opening due to the sudden and large expansion of intine are typical of taxoid-type pollen grains. A hemispheric outgrowth external to the exine was observed on Cupressus and Juniperus pollen grains before the intine swelling and exine release. However, the actual existence of this permanent or temporary structure and its precise role in pollen hydration is still being debated. The aim of this paper is to collect information on the actual presence of this peculiar outgrowth on the surface of the Cupressus pollen grain, its structure, composition and function. METHODS Pollen grains of several Cupressus species were observed using various techniques and methodologies, under light and fluorescence microscopy, phase-contrast microscopy, confocal microscopy, scanning electron microscopy, and an environmental scanning electron microscope. Observations were also performed on other species with taxoid-type pollen grains. KEY RESULTS A temporary structure located just above the pore was observed on Cupressus pollen grains, as well as on other taxoid-type pollens. It is hemispheric, layered, and consists of polysaccharides and proteins. The latter are confined to its inner part. Its presence seems to regulate the entrance of water into the grains at the beginning of pollen hydration. CONCLUSIONS The presence of a temporary structure over the pore of taxoid-type pollen grains was confirmed and its structure was resolved using several stains and observation techniques. This structure plays a role in the first phases of pollen hydration.

Old World and New World Cupressus pollen: morphological and cytological remarks

The aim of this work was to collect new information about pollen morphology and pollen wall structure comparing Cupressus species from the Old World and New World. Cupressus is a nonmonophyletic genus that includes species that appear to be divided into Old World and New World clades. Observations in this study on cypress pollen indicate that grain size and composition of intine β-glucans are different between the considered Old World and New World species. Different from all the other American cypress species, pollen of C. macrocarpa reacted to dying in a similar manner to Old World species. Rehydrated pollen grains collected from 20 Asian, Afro-Mediterranean and American cypress species were measured under a light microscope. The size of the pollen grains and the percentage of intine in relation to the pollen grain diameter were significantly different between Old World and New World species. Pollen wall composition was tested after addition of different dyes to the hydration solution, and subsequent observations were carried out by light and fluorescence microscopy. Lugol and calcofluor staining showed differences in composition of the middle and inner intine layers between New World and Old World species.

Soft Dehydration in the Embedding Method for Transmission Electron Microscopy

SUMMARYA technique for the preparation of biological material for transmission electron microscopy has been successfully tested. After a partial dehydration with ethanol, GMA is used to complete dehydration and to favor the infiltration of the Spurrs resin used for embedding. This procedure enhances sectioning quality of problem materials and, at the same time, minimizes the loss of cell constituents.

Morphogenic effect of colchicine in Cichorium intybus L. root explants cultured in vitro

Abstract A simple and efficient protocol for in vitro shoot regeneration from storage root expiants of witloof chicory (Cichorium intybus L.) has been used to study the effect of colchicine at different concentrations, and colchicine in combination with kinetin or 2,4-D or with 2,4-D and kinetin, in comparison with kinetin and control (no treatment), upon this type of morphogenic event. The results demonstrated a strong stimulatory effect of colchicine at low concentration on shoot regeneration frequency when compared to the number of shoots formed in the control. This effect was comparable to that of kinetin, a growth regulator that induces shoot differentiation. By contrast, the presence of 2,4-D completely inhibited shoot regeneration. Colchicine at high concentration did not induce shoots, and caused the complete death of the expiants. A carefully histological analysis of the expiants during the in vitro culture period showed the formation of numerous meristematic zones (precursors of adventitious bud and shoot development) in the parenchyma mass; these meristems were present only in the expiants subjected to the colchicine and to the other treatments able to stimulate shoot differentiation. Nuclei of different sizes were observed in the explants treated with colchicine at low dose, the largest, probably, polyploids. Polyploid nuclei, in fact, were found in the regenerated plants.

IN VITRO ORGANOGENESIS OF CHESTNUT (CASTANEA SATIVA MILL.) COTYLEDON EXPLANTS: RESPONSES TO GROWTH REGULATORS AND DEVELOPMENTAL ASPECTS

SummaryAdventious root and shoot formation was obtained from cotyledon fragments of chestnut (Castanea sativa Mill.) and these processes followed two phases. In a first stage after detachment of the embryonic axis, the cotyledon fragments in culture formed a cotyledon petiole, which elongated for about 6d. Thereafter, root primordia arose at the tip of the cotyledon petioles, followed by normal root development. In some cases, the cotyledon, petioles showed adventitious shoot regeneration from a nodular structure previously formed at the end of the petioles. The presence or absence of growth regulators did not significantly influence root regeneration, whereas cytokinins stimulated shoot formtion. The processes of root and shoot differentiation were studied also at the histological level. Observation with a light microscope showed that the developing root apical meristems were connected with a vascular bundle of the cotyledon petiole. Similarly, shoot bud meristem connections were observed with vascular tissue inside the nodular structure.

Morpho-anatomical effects of pollutants on Pinus pinea L. needles

Italian coastal pinewoods (Pinus pinea L.) are frequently polluted by surfactants (contained in sea spray) and by ozone. This paper examines the morpho-anatomical features of needles collected from trees naturally exposed to these two pollutants. While sea spray causes acute damage rapidly leading to the death of the needle, ozone and other gaseous pollutants induce histochemical alterations causing an increase in the sclerophyllous conditions of the tissues. The paper examines the possible interaction of these two types of pollution in natural conditions and their effect on the entire needle.