Signalling responses in the bark and foliage of canker-susceptible and -resistant cypress clones inoculated with Seiridium cardinale.

The necrotrophic fungus Seiridium cardinale is the main responsible for Cypress Canker Disease (CCD), a pandemic affecting many Cupressaceae worldwide. The present study aims to elucidate the signalling of the early responses in the bark and foliage of CCD-susceptible and -resistant C. sempervirens clones to S. cardinale inoculation (SI and RI, respectively). In the bark of SI, a peaking production of ethylene (Et) and jasmonic acid (JA) occurred at 3 and 4 days post inoculation (dpi), respectively, suggesting an attempted plant response to the pathogen. A response that, however, was ineffective, as confirmed by the severe accumulation of malondialdehyde by-products at 13 dpi (i.e., lipid peroxidation). Differently, Et emission peaked in RI bark at 3 and 13 dpi, whereas abscisic acid (ABA) accumulated at 1, 4 and 13 dpi, resulting in a lower MDA accumulation (and unchanged levels of antioxidant capacity). In the foliage of SI, Et was produced at 1 and 9 dpi, whereas JA and salicylic acid (SA) accumulated at 1 and 3 dpi. Conversely, an increase of ABA and SA occurred at 1 dpi in the RI foliage. This outcome indicates that some of the observed metabolic alterations, mainly occurring as local defence mechanisms, might be able to gradually shift to a systemic resistance, although an accumulation of MDA was observed in both SI and RI foliage (but with an increased antioxidant capacity reported only in the resistant clone). We believe that the results reported here will be useful for the selection of clones able to limit the spread and damage of CCD.

Strong Induction of Minor Terpenes in Italian Cypress, Cupressus sempervirens, in Response to Infection by the Fungus Seiridium cardinale.

Seiridium cardinale, the main fungal pathogen responsible for cypress bark canker, is the largest threat to cypresses worldwide. The terpene response of canker-resistant clones of Italian cypress, Cupressus sempervirens, to two differently aggressive isolates of S. cardinale was studied. Phloem terpene concentrations, foliar terpene concentrations, as well as foliar terpene emission rates were analyzed 1, 10, 30, and 90 days after artificial inoculation with fungal isolates. The phloem surrounding the inoculation point exhibited de novo production of four oxygenated monoterpenes and two unidentified terpenes. The concentrations of several constitutive mono- and diterpenes increased strongly (especially α-thujene, sabinene, terpinolene, terpinen-4-ol, oxygenated monoterpenes, manool, and two unidentified diterpenes) as the infection progressed. The proportion of minor terpenes in the infected cypresses increased markedly from the first day after inoculation (from 10% in the control to 30-50% in the infected treatments). Foliar concentrations showed no clear trend, but emission rates peaked at day 10 in infected trees, with higher δ-3-carene (15-fold) and total monoterpene (10-fold) emissions than the control. No substantial differences were found among cypresses infected by the two fungal isolates. These results suggest that cypresses activate several direct and indirect chemical defense mechanisms after infection by S. cardinale.

Long-Term Conservation for the Safeguard of Abies nebrodensis: An Endemic and Endangered Species of Sicily.

The combined approaches between ex situ and in situ conservation are of great importance for threatened species in urgent need of protection. This study aims to develop concrete actions to preserve the relic of 30 adult trees of the Sicilian fir (Abies nebrodensis) from extinction using long-term germplasm conservation in liquid nitrogen (LN, -196 °C). Pollen grains were collected, and their moisture content (MC) was measured. Then, viability (2,3,5-tryphenyl tetrazolium chloride, TTC), in vitro germinability, and enzymatic antioxidant activity (ascorbate peroxidase, APX; catalase, CAT) were evaluated before and after cryopreservation. Seeds collected from mature cones underwent X-ray analysis, and only full seeds were used to excise the zygotic embryos (ZEs) for cryopreservation. The MC percentage of ZEs was determined, and then they were plunged in LN with (+PVS2) or without (-PVS2) Plant Vitrification Solution 2; untreated ZEs were used as a control. Viability (TTC test) and in vitro germination were assessed for all ZEs (+PVS2, -PVS2, and control). Embryogenic callus (EC) lines obtained from mature ZEs were cryopreserved applying the 'encapsulation-dehydration' technique. This study has allowed, after optimizing cryopreservation protocols for pollen, ZEs, and EC of A. nebrodensis, to establish the first cryobank of this endangered species in Polizzi Generosa (Palermo, Italy), inside the 'Madonie Regional Park'. The strategy developed for Sicilian fir conservation will pave the way for similar initiatives for other critically endangered conifer species.

Responses of a soil fungal community to severe windstorm damages in an old silver fir stand.

Forests are increasingly threatened by climate change and the Anthropocene seems to have favored the emergence and adaptation of pathogens. Robust monitoring methods are required to prevent biodiversity and ecosystems losses, and this imposes the choice of bioindicators of habitat health. Fungal communities are increasingly recognized as fundamental components in nearly all natural and artificial environments, and their ecosystem services have a huge impact in maintaining and restoring the functionality of ecosystems. We coupled metabarcoding and soil analyses to infer the dynamics of a fungal community inhabiting the old silver fir stand in Vallombrosa (Italy), which is known to be afflicted by both Armillaria and Annosum root rot. The forest was affected in 2015, by a windstorm which caused a partial falling and uprooting of trees. The remaining stand, not affected by the windstorm, was used as a comparison to infer the consequences of the ecosystem disturbance. We demonstrated that the abundance of pathogens alone is not able to explain the soil fungal differences shown by the two areas. The fungal community as a whole was equally rich in the two areas, even if a reduction of the core ectomycorrhizal mycobiome was observed in the wind-damaged area, accompanied by the increase of wood saprotrophs and arbuscular mycorrhizas. We hypothesize a reshaping of the fungal community and a potentially ongoing re-generation of its functionalities. Our hypothesis is driven by the evidence that key symbiotic, endophytic, and saprotrophic guilds are still present and diversified in the wind-damaged area, and that dominance of single taxa or biodiversity loss was not observed from a mycological point of view. With the present study, we aim at providing evidence that fungal communities are fundamental for the monitoring and the conservation of threatened forest ecosystems.

Characterization of Trichoderma species from forest ecosystems by high-throughput phenotypic microarray.

The use of beneficial organisms for the biocontrol of soil-borne pathogens in forestry is still poor explored. In this work, the nutritional demands of 10 previously selected isolates of Trichoderma for the biocontrol of forest soil-borne pathogens have been tested by Phenotype Microarray technology, to investigate about their C-source utilization and exploring the possibility to obtain a microbial consortia (SynCom), an innovative strategy for the biocontrol of plant disease. All Trichoderma isolates tested in this study showed a high spore germination percentage within 3 d and evidenced nutritional preference regardless of the species they belong to, and unrelated to their soil of origin. Results of growth curve analysis and MANOVA test revealed that all isolates assimilate a broad range of substrates, generally preferring complex compounds such as monosaccharides related compounds, nitrogen compounds, carboxylic acids and esters. No evidence of competition for nutritional resources have been observed among isolates of this study. As a result, a combination of different isolates could be proposed to obtain a SynCom useful for the practice of phytopathogen biocontrol in forestry. The addition of i-erythritol, adenosine and turanose to a growth substrate could be suggested as stimulating compounds for the growth of the selected Trichoderma isolates.

Genotypic and environmental effect on male flower production in Cupressus sempervirens clones and selection of genotypes with reduced pollen emission.

Common cypress (Cupressus sempervirens L.) is widespread in the Mediterranean area and is frequently planted as ornamental tree in parks and gardens. Like other species of Cupressus, common cypress releases a significant amount of the total annual airborne pollen in most regions and is known as responsible for winter pollinosis. Although variation in the production and release of pollen has been observed among C. sempervirens trees growing in urban areas, no information is available on effects due to genotype × environment interaction on this trait. In this study more than 150 C. sempervirens clones were analyzed for two to four consecutive years in clonal orchards situated in central Italy to evaluate variations in the production of male cones. Variance component ANOVA underscored an important genetic control of male flowering, with high repeatability (from 0.80 to 0.95) found in single environments. Analysis for combined sites or years (in a single site) showed significant effect of environment and genotype × environment interaction on the total variance. Intra-trait genetic correlations between environments were moderate to high (from 0.40 to 0.92), which indicates that male cone production of clones is fairly consistent across years and sites. Of the 10 clones characterized by the lowest mean male cone production, three showed good stability across environments based on the linear regression coefficient and Wricke's ecovalence. The mean cone production of these 10 clones was 5 to 10 times lower than the mean production observed in the same environment. These clones have both ornamental and hypoallergenic traits and hold promise for designing green spaces with low allergy impact.

Ultrastructure of Terpene and Polyphenol Synthesis in the Bark of Cupressus sempervirens After Seiridium cardinale Infection.

Cypress Canker Disease (CCD) pandemic caused by Seiridium cardinale is the major constraint of many Cupressaceae worldwide. One of the main symptoms of the disease is the flow of resin from the cankered barks. While inducible phloem axial resin duct-like structures (PARDs) have recently been characterized from an anatomical point of view, their actual resin production is still being debated and has never been demonstrated. Although the involvement of polyphenolic parenchyma cells (PP cells) in the bark of Cupressus sempervirens after S. cardinale infection was revealed in one of our previous studies using light microscopy, their evolution from the phloem parenchyma cells is yet to be clarified. This study investigated functional and ultrastructural aspects of both PARD-like structures and PP cells by means of more in-depth light (LM) and fluorescence microscopy (FM) combined with histochemical staining (using Sudan red, Fluorol Yellow, NADI Aniline blue black, and Toluidine blue staining), in addition to Transmission Electron Microscope (TEM). Two-year-old stem sections of a C. sempervirens canker-resistant clone (var. "Bolgheri"), artificially inoculated with S. cardinale, were sampled 5, 7, 14, 21, and 45 days after inoculation, for time-course observations. FM observation using Fluorol yellow dye clearly showed the presence of lipid material in PARD-like structures lining cells of the cavity and during their secretion into the duct space/cavity. The same tissues were also positive for NADI staining, revealing the presence of terpenoids. The cytoplasm of the ducts' lining cells was also positive for Sudan red. TEM observation highlighted the involvement of plastids and endoplasmic reticulum in the production of terpenoids and the consequent secretion of terpenoids directly through the plasma membrane, without exhibiting vesicle formation. The presence of a high number of mitochondria around the area of terpenoid production suggests that this process is active and consumes ATP. The LM observations showed that PP cells originated from the phloem parenchyma cells (and possibly albuminous cells) through the accumulation of phenolic substances in the vacuole. Here, plastids were again involved in their production. Thus, the findings of this work suggest that the PARD-like structures can actually be considered PARDs or even bark traumatic resin ducts (BTRD).

Localized reshaping of the fungal community in response to a forest fungal pathogen reveals resilience of Mediterranean mycobiota.

Mediterranean forests are facing the impact of pests such as the soilborne Phytophthora cambivora, the causal agent of Ink disease, and this impact is made more severe by global changes. The status and resilience of the soil microbial ecosystem in areas with such a disturbance are little known; however, the assessment of the microbial community is fundamental to preserve the ecosystem functioning under emerging challenges. We profile soil fungal communities in a chestnut stand affected by ink disease in Italy using metabarcoding, and couple high-throughput sequencing with physico-chemical parameters and dendrometric measurements. Since the site also includes an area where the disease symptoms seem to be suppressed, we performed several analyses to search for determinants that may contribute to such difference. We demonstrate that neither pathogen presence nor trees decline associate with the reduction of the residing community diversity and functions, but rather with microbial network reshaping through substitutions and new interactions, despite a conservation of core taxa. We predict interactions between taxa and parameters such as soil pH and C/N ratio, and suggest that disease incidence may also relate with disappearance of pathogen antagonists, including ericoid- and ectomycorrhizal (ECM) fungi. By combining metabarcoding and field studies, we infer the resilient status of the fungal community towards a biotic stressor, and provide a benchmark for the study of other threatened ecosystems.

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.

Involvement of phenazine-1-carboxylic acid in the interaction between Pseudomonas chlororaphis subsp. aureofaciens strain M71 and Seiridium cardinale in vivo.

Pseudomonas chlororaphis subsp. aureofaciens encompasses bacterial strains that effectively control phytopathogenic fungi through the production of the natural antibiotics named phenazines. In this work, the involvement of phenazine production in the interaction between the biological control agent P. chlororaphis subsp. aureofaciens M71 and the fungus Seiridium cardinale, a serious cypress pathogen, was investigated. Field trials were carried out to assess the role of phenazines in the control of S. cardinale in vivo. Results showed that P. chlororaphis subsp. aureofaciens M71 and 30-84, both able to produce phenazine-1-carboxylic acid (PCA), drastically reduced the canker development incited by S. cardinale. Conversely, strain M71b, a natural gacA mutant of P. chlororaphis subsp. aureofaciens M71, showed a decrease in PCA production and a reduction in controlling S. cardinale. These results were enforced by the reduction of canker size higher than 94% registered when 6µg of pure PCA was directly applied on each cypress wound. Furthermore, PCA was detected in cypress plant tissues only when P. chlororaphis subsp. aureofaciens M71 was interacting with S. cardinale for 30 days. All these data support that the biological control of S. cardinale achieved by the application of P. chlororaphis subsp. aureofaciens M71 relies mainly on the ability of the bacterial strain to produce PCA in planta.

Occurrence and transmission of mycovirus Cryphonectria hypovirus 1 from dejecta of Thyreophagus corticalis (Acari, Acaridae).

The natural spread of virus-induced hypovirulence is highly involved in the recovery of blighted chestnut stands and orchards in Italy and in Europe. The potential role of corticolous mites as vectors of hypovirulence in blighted chestnut Castanea sativa (Mill.) stands was pointed out in previous reports. Here, by using RT-PCR, mycovirus Cryphonectria hypovirus (CHV1) was detected in Thyreophagus corticalis mites reared on a hypovirulent strain in monoxenic cultures and in their faecal pellets. Cryphonectria parasitica mycelium derived from mites' dejecta was able to transmit CHV1 to the virulent strain determining its conversion to hypovirulent one. This converted strain induced healing cankers on excised stems, differently from the un-converted virulent strain. Our findings prove the spread of CHV1 by corticolous mites that feed on virus-infected fungus and emphasize their potential role as vectors.

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.

Decline in diversity and abundance of endophytic fungi in twigs of Fagus sylvatica L. after experimental long-term exposure to sodium dodecylbenzene sulphonate (SDBS) aerosol.

Sodium dodecylbenzene sulphonate (SDBS) is an anionic synthetic detergent found in polluted sea aerosol and is known for its harmful effects on leaf surface ultrastructure on conifers and broadleaved trees. Four-year-old saplings of European beech were sprayed weekly for three consecutive growing seasons with either a 50 mg l(-1) solution of SDBS in deionized water or with pure deionized water (control). Two- to three- year-old twigs were collected from SDBS-treated and control plants during the growing season one year after the last treatment to isolate endophytic fungi. The frequency of colonization by endophytic fungi was significantly lower on SDBS-treated plants (63.8%) than on control plants (85.4%). Multiple colonization of twigs occurred more frequently and diversity of endophyte species was higher in control plants than in SDBS-treated plants. Thirty-six fungal species were isolated from 360 twigs. Cladosporium cladosporioides, Coryneum compactum, Phialocephala dimorphospora, and a species each of Mycosphaerella and Phomopsis were the most abundant endophytes with frequencies of colonization of more than 5%. The abundance of the Phomopsis species proved to be significantly reduced by the SDBS treatment. Within the limits of the indoor experimental conditions, the obtained results suggest that long-term exposure of aerial parts of beech to SDBS can affect the amount and composition of endophytic fungal communities of lignified twigs. Degradation of the leaf epicuticular wax layer and changes of the assimilation capacity and leaf water content (transpiration) of the crowns are presumed to be responsible for the reduction of endophytic fungi detected in twigs of SDBS-treated plants.