Roberto Calamassi

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.

Morphological and ultrastructural aspects of dehydration and rehydration in leaves of Sporobolus stapfianus

The resurrection species Sporobolus stapfianus Gandoger has been studied by LM, TEM and SEM in order to define the leaf morphology and fine structure and to analyse the cellular changes occurring during the processes of dehydration and rehydration of the plant. Some characteristics of the fully hydrated leaf and some ultrastructural and physiological events which take place during leaf wilting are discussed in relation to their possible role in plant desiccation-tolerance.The leaves of S. stapfianus show several characteristics common among xerophytic species. In the resurrection leaf they could play a role in slowing down the drying rate, thus leaving time to activate the mechanisms protecting the cell structures against drought damage. Actually, the S. stapfianus leaves do not undergo important cellular alterations during dehydration. The chloroplasts, in particular, retain part of their photosynthetic pigments and thylakoid membranes. Upon rewatering leaf recovery is rather fast and the tissue structure and cell organization of the fully hydrated state are already regained after two days.

Soil and plant changing after invasion: the case of Acacia dealbata in a Mediterranean ecosystem.

Acacia dealbata Link (Fabaceae) is one of the most invasive species in the Mediterranean ecosystems of Europe, Africa and America, where it has been proved to exert strong effects on soil and plant communities. In Italy A. dealbata has been largely used for ornamental and forestry purpose and is nowadays spreading in several areas. The present study was addressed to evaluate the impacts on soil chemical properties, soil microbial communities and understory plant communities and to assess the relationships among these compartments after the invasion of A. dealbata in a typical Mediterranean shrubland. Towards these aims, a soil and vegetation sampling was performed in Elba Island where A. dealbata is invading the sclerophyllous native vegetation. Three levels of invasion status were differentiated according to the gradient from invaded, to transitional and non-invaded vegetation. Quantitative and qualitative alterations of soil chemical properties and microbial communities (i.e. bacterial and fungal communities) and above-ground understory plant communities were found. In particular, the invaded soils had lower pH values than both the non-invaded and transitional ones. High differences were detected for both the total N and the inorganic fraction (NH4(+) and NO3(-)) contents, which showed the ranking: invaded>transitional>non-invaded soils. TOC and C/N ratio showed respectively higher and lower values in invaded than in non-invaded soils. Total plant covers, species richness and diversity in both the non-invaded and transitional subplots were higher than those in the invaded ones. The contribution of the nitrophilous species was significantly different among the three invasion statuses, with a strong increase going from native to transitional and invaded subplots. All these data confirm that A. dealbata modifies several compartments of the invaded ecosystems, from soil chemical properties to soil and plant microbial communities determining strong changes in the local ecosystem processes.

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

Structure and ultrastructure of Pinus halepensis primary needles

Summary This study using light, transmission and scanning electron microscopy, describes the structure of Pinus halepensis primary needles collected from seedlings 22 and 24 weeks after emergence. P. halepensis is a circum-mediterranean pine of high importance for ecosystem maintenance and reforestation, in which primary needles are formed three to four years before being replaced by secondary needles. The needle surface shows spine-like structures and deeply sunken stomata, covered by wax, which however does not occlude the epistomatal chamber. Cross sections of the rhomboidal-shaped leaves show a uniseriate epidermis composed by living cells with a thin cuticle. Hypodermal cells do not occur under the epidermal layer. The plicate mesophyll, with large intercellular spaces and resin ducts, is also described. The vascular bundles are surrounded by transfusion tissue, bounded by a uniseriate endodermis. The endodermal cells have unmodified thin walls which lack Casparian strips, but plasmodesmata between adjacent endodermal cells occasionally occur. Moreover, plasmodesmata between mesophyll and endodermal, and endodermal and transfusion parenchyma cells interconnect these tissues. Transfusion tracheids are numerous with abundant bordered pits with a torus. The xylem has the usual structure, and the mature sieve elements have very thick fibrous walls. The fine structure of primary needles of the P. halepensis population in North Eubea (Greece) indicates that the typical morpho-anatomic characteristics of the pine leaf, which are generally correlated with resistance to aridity and are described for secondary needles, are not present. Rather, the features of non-vascular tissues may enable a high photosynthesis and are coherent with an (indefinite) growth model for the juvenile phase of P. halepensis .

Relations between apical structure and growth patterns in Pinus halepensis Mill, seedlings

Abstract The process of primary growth in 2-year-old seedlings of 11 populations of Pinus halepensis Mill. is described. At the end of the first growing season one type of apical structure was observed: type-1, a tuft of primary needles placed close together, surrounding and protecting a meristematic apex. At the end of the 2nd growing season, three types of apical structure were observed: type-1; type-2, a terminal winter bud; and type-3; a «bud» with characteristics of both type-1 and type-2. Morphological observation along with an anatomical examination of the winter bud led to the conclusion that the definitive growth pattern in juvenile P. halepensis is monocyclic with a variable number of summer shoots. This growth pattern is reached by some P. halepensis populations in 3–4 years, by contrast, in other pine species two years are usually needed. The populations studied differed both in growth potential (differences in number of cycles, ratio of first cycle to total growth, growth rates) and in the de...

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.

Dormancy of Fagus sylvatica L. buds III. Temperature and hormones in the evolution of dormancy in one-node cuttings

ABSTRACT The evolution of the growth potential of Fagus sylvatica L. buds in the autumn-winter period (September-March) was analysed. One-node cuttings (of apical, median and basal buds, and small buds at the base of the branch) were taken from one-year old shoots removed from adult beech plants (at Vallombrosa in the Tuscan Apennines) and placed in climatic chambers with a 12-h photoperiod and temperatures of 15° and 25°C. They were treated either with water or gibberellin and kinetin solutions. The trend in sprouting was followed, for all types of buds, for 60 days. The months of September and October constituted the period of entry into dormancy, which then reached a peak at the beginning of November. In December, the phase of rapid dormancy removal was already present. Restriction and subsequent widening of the thermic interval compatible with growth, establishment and later removal of the basitonal habitus were the characterising elements in the autumn-winter evolution. Kinetin had null or slightly inhibiting effects in all the examined period. Gibberellin did not modify the behaviour of buds approaching dormancy, whereas they strongly stimulated growth in the period of rapid dormancy removal, and exerted moderate effects in the subsequent period of slow residual dormancy removal.

Frost hardening and resistance in three Aleppo pine (Pinus halepensis Mill.) provenances

The paper investigates frost hardening and resistance in two-year-old seedlings of three Aleppo pine provenances (Litorale tarantino, Eubea, Kassandra). Starting in early autumn, field-raised seedlings were sampled fortnightly and subjected to temperatures of +4, 6, 12, and 17 C. Electrolyte release was measured before and after boiling of the samples, separated according to organ type. Conductivity values were converted into damage index It. The findings were then correlated to mean climate data (maximum, minimum, and mean temperature, theoretical sunshine duration) recorded over the 212 days prior to sampling. Acquired frost resistance increased gradually from the first sampling till January, as the day length and air temperature decreased. Subsequent hardening-dehardening fluctuations indicated that frost resistance in Aleppo pine is strongly dependent on microclimate conditions. Provenance Kassandra displayed the greatest frost resistance. The differences between the 3 provenances were not marked. Ale...