R. Danti

Sequence and Simple-Sequence Repeat Analyses of the Fungal Pathogen Seiridium cardinale Indicate California Is the Most Likely Source of the Cypress Canker Epidemic for the Mediterranean Region

Seiridium cardinale is the pathogenic fungus of unknown origin responsible for a world pandemic known as cypress canker affecting several species of Cupressaceae in both the Northern and Southern Hemisphere. In this study, a comparative genetic analysis of worldwide populations was performed using sequence analysis of a portion of the ?-tubulin locus and seven polymorphic simple-sequence repeat (SSR) loci on 96 isolates. Sequence analysis identified two distinct ?-tubulin alleles, both present in California. Only one of the two alleles was detected in the Mediterranean basin, while two isolates from the Southern Hemisphere were characterized by the presence of the allele absent from the Mediterranean. SSRs identified a total of 46 multilocus genotypes (MGs): genotypic diversity was always higher in the California population, and calculations of the index of association (I(A)) determined the presence of linkage disequilibrium associated with the absence of sexual reproduction only in the Mediterranean population but not in California. In 50 instances, the same MG was found at great geographic distances, implying a role played by humans in spreading the disease. Network analysis performed on SSR data identified three clusters of MGs: California, Morocco, and the rest of the Mediterranean. Both the Morocco and the Mediterranean clusters were linked to the California cluster. Coalescent analysis identified insignificant migration between California and Italy, as expected in the presence of a single introduction event, and very high migration from Italy into Greece, as expected of an outbreak still in exponential growth phase and starting from an Italian source.

An increase in transmission-related traits and in phenotypic plasticity is documented during a fungal invasion

The adaptive rapid evolution of phenotypic traits is potentially a key contributor to invasiveness, but has been relatively little studied for the fungi, even though these organisms are responsible for devastating losses in agriculture and natural resources. In this study, we compare biologically relevant phenotypic characters of spore-generated individuals from two native and two invasive populations of the fungal pathogen Seiridium cardinale to infer which traits may be adaptive and rapidly evolving during an ongoing biological invasion. Results show that: (1) lower growth rate and smaller spore size are selected for in invasive populations, independent of the stage of invasion; (2) there is no selection evident towards increased rapid sporulation, but overall reproductive potential increases in later stages of the invasions; and (3) demographic plasticity of most traits increases during the initial stages of invasion, but decreases in a later phase. Comparisons against levels of neutral genetic variation (Qst-Fst comparisons) showed that the decrease in spore size is strongly adaptive, despite the trade-off of reduced viability. Lesion size of isolates inoculated on the naive Italian cypress host was not correlated with their growth rate, and was significantly lower in invasive than in native populations. This last result indicates that rate of host colonization is a complex trait affected both by host and pathogen, which may not be necessarily adaptive and/or which may not easily evolve. In summary, the success of S. cardinale as an invasive in the Mediterranean basin is associated with reduced spore size and increased plasticity of almost all traits in initial phases, followed by further decreased spore size, increased overall sporulation, and decreased plasticity in a second phase of the invasion. Interestingly, growth rate by population results show that invasive populations are well adapted only to moderate temperatures, while native populations fare well also when exposed to relative extremes in temperature. This different adaptation suggests a “master-of-some” specialization scenario for the invasion by S. cardinale in the Mediterranean.

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.

Ecology of invasive forest pathogens

Invasive forest pathogens are a major threat to forests worldwide, causing increasing damage. The knowledge of both the specific traits underlying the capacity of a pathogen to become invasive, and the attributes predisposing an environment to invasion are to be thoroughly understood in order to deal with forest invasions. This paper summarizes the historical knowledge on this subject. Many aspects of the ecological processes underlying alien forest pathogens invasions are still unknown, which raises several scientific issues that need further study. The introduction of invasive forest pathogens to areas where naïve hosts are found, is mainly due to global plant trade. Rapid transportation and reduced delivery times increase the chances of survival of pathogen propagules and of their successful establishment in new environments. In forest pathogens, the reproduction mode seems not to be a crucial determinant of invasiveness, as highly destructive pathogens have a variety of reproductive strategies. The most important drivers of forest pathogen invasions appear to be (a) great adaptability to new environmental conditions; (b) efficient dispersal over long and short distances, possibly assisted by the capacity to form novel associations with endemic and/or alien insect vectors; (c) the ability to exchange genetic material or hybridize with resident or alien species. Moreover, these features interact with some key traits of the invaded environment, e.g. environmental variability and biodiversity richness. Host resistance and natural enemies may occur as a result of rapid selection/adaptation after the epidemic phase of invasion.

The Epidemic Spread of Seiridium cardinale on Leyland Cypress Severely Limits Its Use in the Mediterranean

Leyland cypress (× Hesperotropsis leylandii) is a fast-growing conifer used in most temperate regions as an ornamental tree for hedges and screens, and is one of the most commercially important trees in Europe. In recent years, severe diebacks and mortality due to cypress canker have been observed on Leyland cypress plantations in Southern Europe. This study was conducted to evaluate (i) the spread and impact of cypress canker caused by Seiridium cardinale in plantations of a sample area of 1,250 km2 in central Italy, (ii) the response of the most commonly grown Leyland cypress varieties to artificial inoculation with to S. cardinal, and (iii) the pathogenicity of S. cardinale isolates obtained from Leyland cypress. Of the 1,411 surveyed trees, 11.4% had been killed by cypress canker and 43.9% of the living trees were affected by the disease. The number of diseased or dead trees and the percentage of cankered trunks was significantly correlated with the mean trunk diameter of the plantations. Six months after inoculation, the size of developed cankers was significantly different among the inoculated Leyland cypress cultivars but all of them showed markedly larger cankers than the C. sempervirens canker-resistant control clone. All of the tested S. cardinale isolates obtained from Leyland cypress also caused cankers on Cupressus sempervirens when inoculated as conidial suspensions or mycelia. Leyland cypress is highly prone to contract cypress canker in the Mediterranean due to its high susceptibility to S. cardinale infections, low genetic variability among the grown cultivars, and cracks which form on fast-growing trunks, favoring entry of the fungus into the inner bark and the occurrence of infections.

Somatic plant regeneration from selected common cypress ( Cupressus sempervirens L.) clones resistant to the bark canker disease

The effectiveness of a protocol for somatic embryogenesis in conifers requires both the proliferation of embryonal masses and their conversion into somatic plants. Despite several successful protocols developed for Pinaceae, species belonging to Cuperessaceae family are often characterized by a problematic and unsatisfactory maturation of somatic embryos. Hence, the main goal of this study was to overcome the problem of embryo maturation and plant regeneration in Cupressus sempervirens, a Mediterranean species widely used for its ornamental value, timber production and interest in reforestation programmes. Embryogenic lines were produced from selected canker-resistant genotypes of common cypress; the effect of polyethylene glycol (PEG), desiccation period, medium composition and culturing period on the somatic embryo maturation and conversion, were evaluated. Despite significant variations observed among genotypes, the PEG based medium was the most effective for somatic embryo maturation. Germination and conversion of mature somatic embryos took place after three months of culture in a low-sucrose LP medium with activated charcoal. A short desiccation period failed to improve the germination rate of the mature somatic embryos. To our knowledge this is the first protocol reporting on somatic plant regeneration from somatic embryos of C. sempervirens.

Terpene arms race in the Seiridium cardinale – Cupressus sempervirens pathosystem

The canker-causing fungus Seiridium cardinale is the major threat to Cupressus sempervirens worldwide. We investigated the production of terpenes by canker-resistant and susceptible cypresses inoculated with S. cardinale, the effect of these terpenes on fungal growth, and the defensive biotransformation of the terpenes conducted by the fungus. All infected trees produced de novo terpenes and strongly induced terpenic responses, but the responses were stronger in the canker-resistant than the susceptible trees. In vitro tests for the inhibition of fungal growth indicated that the terpene concentrations of resistant trees were more inhibitory than those of susceptible trees. The highly induced and de novo terpenes exhibited substantial inhibition (more than a fungicide reference) and had a high concentration-dependent inhibition, whereas the most abundant terpenes had a low concentration-dependent inhibition. S. cardinale biotransformed three terpenes and was capable of detoxifying them even outside the fungal mycelium, in its immediate surrounding environment. Our results thus indicated that terpenes were key defences efficiently used by C. sempervirens, but also that S. cardinale is ready for the battle.

Flammability of Two Mediterranean Mixed Forests: Study of the Non-additive Effect of Fuel Mixtures in Laboratory

In the Mediterranean region, wildfires are a major disturbance, determined by ecosystem and forest species characteristics. Both the flammability and resistance to fire of a mixed forest may vary from those of the individual species. Two mixed Mediterranean woodlands, a Cupressus sempervirens and Quercus ilex stand in Italy; and a Juniperus thurifera and Quercus faginea stand in Spain were investigated. Laboratory flammability tests were conducted on live foliage, litter samples and on litter beds from individual and mixed species to evaluate: (i) the flammability traits of the mixtures of live foliage and litter samples; (ii) whether the flammability of the two-species mixtures are non-additive, i.e., differ from expected flammability based on arithmetic sum of the single effects of each components species in monospecific fuel; (iii) the ignition success and initial fire propagation in litter beds. Flammability tests were also conducted on bark samples to estimate the resistance of the tree species to fire. The ignitibility of live foliage was lower and the combustibility was higher in Cupressaceae than in Quercus. Non-additive effects were observed in some flammability components of live foliage and litter, especially in the mixtures of C. sempervirens and Q. ilex. Ignitability and combustibility were higher and lower than expected, respectively, and tended to be driven by Quercus), while the consumability was lowered more than expected by both Cupressaceae. The ignition success in the litter beds was low, especially for the presence of Cupressaceae that increase the bulk density of the mixtures. Cupressaceae, which have a thinner bark, suffered more damage to the cambium after shorter exposure to the heat source than Quercus species. In all the species studied, time to reach lethal temperatures in the cambium was dependent on thickness rather than on flammability of the bark. The study findings revealed that tree species may influence flammability of mixed fuels disproportionately to their load. The studied species showed to exert a contrasted effect on flammability of the mixtures, increasing ignitability and decreasing combustibility and consumability well out of their proportion in the mixture. This may potentially influence fire dynamics in mixed forests.

Clonal consistency of wood technological properties in canker-resistant Cupressus sempervirens clones at two contrasting sites

Cupressus sempervirens L. (Mediterranean cypress) has been traditionally used as a multipurpose tree. In the past, its wood was extensively used as a highly durable raw material in the Mediterranean, but nowadays, production of cypress wood is constrained by the lack of exploitable woods and plantations and by the spread of bark canker. In this study, the wood properties of canker-resistant clones specifically meant for timber production were assessed in two different locations. The aim was to evaluate the effect of genotype and environment on physical and mechanical properties of wood and its bonding quality. Four ramets of each of 10 clones were sampled in both sites, wood density, shrinkage, hardness, and the bonding strengths when glued together with two different adhesives were determined, and clonal repeatabilities and genetic correlations were also estimated. Clonal consistency for wood traits was moderate to high within and across environments, far higher than for growth- and morphological traits. Indications are that selection based exclusively on tree height would result in a lower wood density and hardness. Bonding strength reflected the combination of the material properties and the selected adhesive: For adhesive M (polyvinyl acetate dispersion), it was negatively correlated with density, whilst for adhesive E (emulsion polymerization isocyanate), it was not as influenced. Thus, even favouring higher-density wood (and therefore not only hardness but also higher shrinkages), adhesive E would give excellent bonding.