Different Polyphenolic Parenchyma Cell and Phloem Axial Resin Duct-Like Structure Formation Rates in Cupressus sempervirens Clones Infected with Seiridium cardinale.

The aim of this study was the characterization of constitutive and induced defense mechanisms in the bark tissues of Cupressus sempervirens before and after infection with the bark fungus Seiridium cardinale, which is responsible for cypress canker disease. The time-course development of polyphenolic parenchyma (PP) cells and phloem axial resin duct (PARD)-like structures in the phloem was investigated in two C. sempervirens clones, one resistant and one susceptible to the disease, through anatomical and histological observations carried out by light microscope during a 19-day trial. PP cells were constitutively more abundant in the canker-resistant clone (R clone) compared with the susceptible clone (S clone), whereas PARD-like structures were not present in the bark of untreated plants of both clones. PP cells increased in both clones as a response to infection, but in the R clone, they were more abundant 5 and 12 days after inoculation. After inoculation, PARD-like structures appeared in the phloem after 5 days in the R clone and only after 12 days in the S clone. Even the number of cells surrounding the PARD-like structures was higher in the R clone 5 and 12 days after inoculation compared with the S clone. These observations demonstrate a faster phloem response of the R clone in the early phase of the infection. This may slow down initial growth of the fungus, contributing to the resistance mechanism.

Different allergenicity of pollen extracts of three Mediterranean cypress species accounted for cytological observations.

BACKGROUND: Cypresses play an important role in the urban landscape of the Mediterranean region, releasing a huge amount of allergenic airborne pollen which causes a specific pollinosis in exposed people. OBJECTIVE: The aim of this work is to evaluate, in vivo and in vitro, the potential allergenicity of Cupressus macrocarpa pollen, and to compare it with the allergenicity observed for C. arizonica and C. sempervirens. METHODS: Pollen extracts of the three species were prepared, to determine their protein profile through SDS PAGE analysis and to evaluate their allergenic potential through EAST inhibition assays and SPT. Pollen grain composition was evaluated using a cytochemical approach with optical microscopy. RESULTS: SDS PAGE, EAST inhibition and SPT indicated the higher allergenic potential of C. arizonica compared to C. sempervirens and C. macrocarpa. No significant differences in allergenic potential were found between the latter two species. Cytochemical observations reveal higher ß-glucans and protein content in the intine of C. arizonica during hydration. CONCLUSION: The higher protein content found in C. arizonica pollen grains extract may be due to higher enzyme activity leading to the movement of ß-glucans and pectins from the intine to the partially developed pollen cell wall during hydration. This could explain the higher potential allergenicity of C.arizonica in respect to C. macrocarpa and C. sempervirens.

Possible land management uses of common cypress to reduce wildfire initiation risk: a laboratory study.

Accurate determination of flammability is required in order to improve knowledge about vegetation fire risk. Study of the flammability of different plant species is essential for the Mediterranean area, where most ecosystems are adapted to natural fire but vulnerable to recurrent human-induced fires, which are the main cause of forest degradation. However, the methods used to evaluate vegetation flammability have not yet been standardized. Cupressus sempervirens is a native or naturalized forest tree species in the Mediterranean area that is able to tolerate prolonged drought and high temperatures. The aim of this study was to characterize the flammability of C. sempervirens var. horizontalis at particle level by using different bench-scale calorimetry techniques (mass loss calorimeter, epiradiator and oxygen bomb) to determine the main flammability descriptors (ignitability, sustainability, combustibility and consumability) in live crown and litter samples. Our findings indicate that this variety of cypress is relatively resistant to ignition because of the high ash content, the high critical heat flux, the high time to ignition displayed by both crown and litter samples and the ability of the leaves to maintain a high water content during the summer. We also discuss the possibility of exploiting some morphological, functional and ecological traits of the species to construct a barrier system (with selected varieties of cypress) as a promising complementary land management tool to reduce the fire spread and intensity in a Mediterranean context.

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.

First Report of Botryosphaeria iberica Associated with Dieback and Tree Mortality of Monterey Cypress (Cupressus macrocarpa) in Algeria.

Stem cankers and branches showing bark discoloration, fissuring, resin exudation leading to dieback, crown wilting, and tree mortality have been observed since late spring 2008 on 40-year-old Cupressus macrocarpa (Hartw.) trees planted in forests mixed with Juniperus oxycedrus L. and Acer monspessulanum L. in Taffet, near Ain Abbessa, in the district of Bougaa, Algeria (36°18'57″N; 05°06'33″E; 1,400 m elevation). In 2010, approximately 60% of the C. macrocarpa trees were diseased. For fungal isolations, cankered branches were surface sterilized with ethanol. After removal of the outer bark, fragments of necrotic inner bark taken from the margin of cankers were plated on potato dextrose agar (PDA). Most of the colonies were identified as Botryosphaeria iberica (Phillips, Luque & Alves) based on comparison of morphological traits and DNA sequences with known isolates of the fungus (1). Pestalotiopsis funerea colonies were also obtained, although with less frequency. B. iberica colonies on PDA were dark green with aerial mycelium and optimum growth at 25°C. Pycnidia were produced after 3 weeks of incubation at 20°C under a 12-h near UV light photoperiod on water agar amended with autoclaved cypress seeds. Conidia were brown, one-septate, oval to oblong, and 24.2 (20.1 to 27.4) × 11.2 µm (8.8 to 14.1) (n= 50). An isolate was deposited at the Centralbureau voor Schimmelculture as CBS 130984. DNA was extracted from freeze-dried mycelium and amplified using primers ITS1 and ITS4. The amplified DNA sequence of B. iberica isolate CBS 130984 from Algeria (GenBank Accession No. JN836991) showed 100% homology with sequences of B. iberica isolates obtained from dead and cankered bark of oaks from Spain and Italy (GenBank Accession Nos. AY573216, AY573214, AY573213, AY573210, AY573202, and AY573201). Stem inoculations were performed in the greenhouse on 10 4-year-old, grafted plants of C. macrocarpa growing in 5-liter pots using isolate CBS 130984. A 3-mm plug taken from the margin of a colony grown on PDA for 1 week was inserted in a circular wound of the same size made in the bark with a cork borer where the stem diameter was approximately 1 cm. Inoculations were repeated in June 2010 and June 2011. Five months after inoculations, small rounded to elongated lesions (1.0 to 2.5 cm long), sometimes with resin exuding cracks, were visible on all inoculated stems. Control trees, inoculated with sterile PDA plugs, showed no canker development. B. iberica was successfully reisolated from the necrotic bark surrounding the inoculation sites. No significant differences in canker size were observed between the two replicated experiments. Some Botryosphaeria species that are found on a variety of hosts are also known to cause cankers and dieback of cypress; among these are B. stewensii, B. obtusa, B. dothidea, and B. ribis, often acting as weak pathogens (2,3). Considered weakly virulent in causing dieback of grapevine (4) and, to our knowledge, reported here for the first time on Cupressaceae, B. iberica caused cankers and dieback of C. macrocarpa trees that had probably been weakened by repeated drought events occurring in Algeria during the last 10 years. References: (1) A. Phillips et al. Mycologia 97:513, 2005. (2) E. Punithalingam and J. M. Waller. IMI Descriptions of Fungi and Bacteria 40, Sheet 394, 1973; (3) E. Punithalingam and P. Holliday. IMI Descriptions of Fungi and Bacteria. 40, Sheet 395, 1973; (4) R. Úrbez-Torres et al. Plant Dis. 93:584, 2009.

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.

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.

First Report of Seiridium unicorne Causing Bark Cankers on a Monterey Cypress in California.

In June 2009, dieback of distal branches and resin exudation associated with bark lesions were observed in an adult Cupressus macrocarpa tree in Sonoma County, California (Glenn Ellen; 38°21'N, 122°31'W, elevation 233 m). The fungal pathogen, Seiridium unicorne (Cooke and Ellis) Sutton, was obtained by plating fragments of necrotic bark from the margins of branch cankers on potato dextrose agar (PDA). Identification was based on cultural, morphological, and molecular traits (2,3). Colonies on PDA were dense, cottony, off-white at first and then turning pale gray-green, and 2.3 and 4.3 cm in diameter after 1 and 2 weeks of growth at 20°C, respectively. Colonies of the fungus showed a faster radial growth at 20°C than at 25°C. Acervuli were abundantly produced on water agar amended with autoclaved cypress seeds after 2 to 3 weeks at 18°C under a mixture of fluorescent and near UV light. Conidia were six celled (five euseptate), fusiform, 20.9 to 35.2 × 7.11 to 10.57 µm, straight or slightly curved, with four, brown median cells, and with end cells bearing unbranched appendages 2 to 5 µm long. The DNA sequence of a portion of the ß-tubulin locus (GenBank Accession No. HQ678171) revealed a 100% homology with sequences of S. unicorne isolates from Portugal and South Africa, while being clearly distinct from sequences of S. cupressi and S. cardinale isolates (2). Greenhouse stem inoculations were performed by underbark placement of a 3-mm plug taken from the margins of a colony of the fungus grown on PDA. Inoculations were repeated twice in the spring and fall of 2010 on 10 C. macrocarpa saplings grown in pots for 3 years. Three months postinoculation, the pathogen could be successfully reisolated from the edges of 15 to 30 mm long elliptical lesions, present on each one of the inoculated saplings. The observed S. unicorne isolate is atypical because of its shorter appendages compared with the form reported in the literature (2,3). Because of its shorter conidial appendages and in vitro temperature optimum of 18 to 20°C, the fungus described here is similar to an unnamed Coryneum sp. observed by Wagener on C. macrocarpa (4). S. unicorne is a pathogen of many Cupressaceae in Africa, New Zealand, Japan, and some U.S. states (Georgia, South Carolina, Kansas, and Texas) (3), and although it was mentioned in a USDA Plant Quarantine Division report from 1963 as found on cypress in San Francisco (1), it has never been officially reported from California. Since similar disease symptoms were observed on many Cupressaceae in the course of an extensive survey performed in 2009 in California, it may be important to evaluate the relative incidence of S. unicorne compared with that of S. cardinale, a pathogen more commonly reported in association with the disease (4). References: (1) D. F. Farr and A. Y. Rossman. Fungal Databases. Systematic Mycology and Microbiology Laboratory, ARS, USDA. Retrieved from http://nt.ars-grin.gov/fungaldatabases/fungushost/fungushost.cfm , 1/19/2011. (2) P. Krokene et al. Mycologia 96:1352, 2004. (3) N. A. Tisserat. Plant Dis. 75:138, 1991. (4) W. W. Wagener. J. Agric. Res. 58:1, 1939.

Permanent Genetic Resources added to Molecular Ecology Resources database 1 January 2009-30 April 2009.

This article documents the addition of 283 microsatellite marker loci to the Molecular Ecology Resources Database. Loci were developed for the following species: Agalinis acuta; Ambrosia artemisiifolia; Berula erecta; Casuarius casuarius; Cercospora zeae-maydis; Chorthippus parallelus; Conyza canadensis; Cotesia sesamiae; Epinephelus acanthistius; Ficedula hypoleuca; Grindelia hirsutula; Guadua angustifolia; Leucadendron rubrum; Maritrema novaezealandensis; Meretrix meretrix; Nilaparvata lugens; Oxyeleotris marmoratus; Phoxinus neogaeus; Pristomyrmex punctatus; Pseudobagrus brevicorpus; Seiridium cardinale; Stenopsyche marmorata; Tetranychus evansi and Xerus inauris. These loci were cross-tested on the following species: Agalinis decemloba; Agalinis tenella; Agalinis obtusifolia; Agalinis setacea; Agalinis skinneriana; Cercospora zeina; Cercospora kikuchii; Cercospora sorghi; Mycosphaerella graminicola; Setosphaeria turcica; Magnaporthe oryzae; Cotesia flavipes; Cotesia marginiventris; Grindelia Xpaludosa; Grindelia chiloensis; Grindelia fastigiata; Grindelia lanceolata; Grindelia squarrosa; Leucadendron coniferum; Leucadendron salicifolium; Leucadendron tinctum; Leucadendron meridianum; Laodelphax striatellus; Sogatella furcifera; Phoxinus eos; Phoxinus rigidus; Phoxinus brevispinosus; Phoxinus bicolor; Tetranychus urticae; Tetranychus turkestani; Tetranychus ludeni; Tetranychus neocaledonicus; Tetranychus amicus; Amphitetranychus viennensis; Eotetranychus rubiphilus; Eotetranychus tiliarium; Oligonychus perseae; Panonychus citri; Bryobia rubrioculus; Schizonobia bundi; Petrobia harti; Xerus princeps; Spermophilus tridecemlineatus and Sciurus carolinensis.