The ecology of fungal cankers on Cupressus macrocarpa in southern England

Bark isolations from cankers on Cupressus macrocarpa in southern England yielded Seiridium cardinale, Pestalotiopsis funerea and Pestalotiopsis monochaetioides. The recovery pattern of the three fungi indicated that only S. cardinale was a primary pathogen; the two Pestalotiopsis species being secondary. Pathogenicity data obtained following the inoculation of C. macrocarpa branches with mycelium of the three fungi confirmed this view. No evidence of vegetative incompatibility was found in S. cardinale, but five vegetative-compatibility (vc) groups were found in P. funerea and six in P. monochaetioides. Different vc groups of the two Pestalatiopsis species were often found in the same S. cardinale lesion, suggesting that a number of separate colonization events were involved. It is possible that this sometimes leads to complete replacement of the pathogen.     

Pathogenic variation of Seiridium spp. isolated from cankered Cuppressaceae hosts in New Zealand

Field and glasshouse inoculation tests with isolates of S. cardinale and S. unicorne (Cke & Ell.) Sutton on C. macrocarpa and C lusitanien showed symptoms caused by the two pathogens to be indistinguishable. Isolates of S. cardinale showed an overall higher pathogenicity and less variability than those of S. unicorne. Neither pathogen seemed capable of infecting hosts outside the Cupressaceae, An isolate of Boesewinkel originating from Crvptowcria and supposed to be a distinct species from the anamorph of Lepteutypa cupressi, failea to infect Cryptomeria; neither could L. cupressi and S. cardinale, though all of them readily infected C. macrocarpa and C. lusitanica. Isolates of S. unicoene showed vast differences in pathogenicity in any one test, but many of the Isolates also showed extremi pathogenicity variation between repeat tests, hence pathogenicity determination became uncertain or probalistic. There is no evidence that the decline of S cardinale in this country is due to reduced pathogenicity or to possible interaction with S. unicorne in host infection.     

Seiridium cardinale cankers in a tolerant Cupressus sempervirens clone under naturally CO2‐enriched conditions

Ramets of the Cupressus sempervirens clone Bolgheri were raised in three CO₂ concentrations in a naturally CO₂‐enriched environment [360 (control), 680 and 1670 μmol mol^–1 CO₂]. Bolgheri is patented as resistant to Seiridium cardinale. The ramets were artificially inoculated using a virulent isolate of S. cardinale. Healing of the resulting cankers was visually assessed over 6 years. Six months after inoculation, cankers on trees grown at 680 μmol mol^–1 were significantly larger than the controls. However, no significant trend was recorded at later dates. In conclusion, the Bolgheri clone maintained its resistance to S. cardinale and could be used to control cypress canker even in increasing atmospheric CO₂ concentrations.     

Sphaeropsis canker,a new disease of cypress in Greece

The fungus Sphaeropsis sp. is reported for first time in Greece to cause cankers on Cupressus sempervirens. The cultural characteristics on PDA, spore shape and size and canker morphology were identical to those of the fungus described as Diplodia pinea f. sp. cupressi in Israel. The cankers of Sphaeropsis sp. are characterized by resin exudation, with fissuring of the bark over a dry sector of the wood. The pathogenicity of the Sphaeropsis sp. was proved by artificial inoculations on selected cypress clones resistant and susceptible to Seiridium cardinale. The mean canker length of the susceptible clone was significantly more than that of the resistant clones. It appears that the defence reaction in cypress against both fungi is similar. In artificial inoculations on Pinus halepensis and Pinus pinea, Sphaeropsis sp. was nonpathogenic. During a 3‐year period, in an experimental plot in western Peloponnese, the fungus spread from one, initially, to 13 cypress clones. Drought stress during the summer appears to be the main factor predisposing the cypress plants to become susceptible to the fungus.     

Genetic differences between autochthonous and breeding populations of common cypress (Cupressus sempervirens L.) in Greece

Individuals were selected for resistance against the imperfect fungus Seiridium cardinale Wag in six common cypress populations (Cupressus sempervirens L.) in Greece. A total of 109 resistant clones and 140 trees from their base populations were surveyed at several allozyme loci. The comparison yielded information on changes in genetic variation due to artificial selection. The genetic structure of the breeding populations was similar to their base populations. Nevertheless, the number of rare alleles among the resistant clones had decreased, showing a similar trend at most loci and for most comparisons. Possible implications for breeding strategies are discussed.     

Pathogenic variability of various isolates of Seiridium cardinale,S. cupressi and S. unicorne inoculated on selected Cupressus clones and seedlings

Comparative studies of the pathogenicity of eight isolates of three Seiridium species were carried out by stem inoculations on Cupressus sempervirens and C. glabra seedlings and clones. Seiridium cardinale and S. cupressi caused bigger and more severe cankers than all strains of S. unicorne. During the first nine months after inoculation, the canker length of S. cardinale was larger than the cankers of S. cupressi. However, during the second year after inoculation, S. cupressi cankers were larger and more severe than those of 5. cardinale.     

Genetic variability of the ‘bark canker resistance’ character in several natural provenances of Cupressus sempervirens

Several Aegean (Greece) and Anatolian (Turkey) cypress provenances were studied for resistance variability to bark canker, a disease caused by the fungal pathogen Seiridium cardinale. The investigation also examined whether the low disease rate within the natural area of cypress was due to genetic or geographic‐climatic reasons. Results demonstrated strong variability for the ‘bark canker resistance’ character, in particular for trees within families. As trees from the provenances studied were not found to have genetic superiority for bark canker resistance, the above‐mentioned low disease rate could be due to geographic‐climatic barriers that inhibit the development of the fungus or its ability to infect the host. Several half‐sib progenies exhibited high resistance, suggesting that this character is totally inherited through the maternal line. Should this finding be confirmed by further research, it would facilitate the task of genetic improvement for resistance, allowing progenies of resistant trees to be obtained.     

Responses of Cupressus and Chamaecyparis callus tissues to inoculations with Seiridium cardinale

Inoculations of callus generated from micropropagated shoots of Cupressus sempervirens, Cupressus macrocarpa, Cupressus torulosa and Chamaecyparis lawsoniana showed that callus of species resistant or relatively resistant to Seiridium cardinale supported significantly less surface growth of the pathogen than callus of susceptible species. In dual cultures of Cupressus sempervirens or Cupressus torulosa callus with S. cardinale, inhibition of fungal growth towards the callus was correlated with known field resistance of these host species. Lignification, accumulation of polyphenolics and possible suberization of cell walls were detected in inoculated callus. This response was also correlated with known field resistance of the tested host species.     

Morphological and cultural variation of Seiridium spp. from cankered Cupressaceae hosts in New Zealand

Studies of Seiridium. spp. isolated from cankered cypresses and related hosts in New Zealand indicate that only 2 species are present, namely, 5. unicorne and 5. cardinale, following sdtton, and not 3 species as proposed by boesewinkel. Apart from a difference in the presence/absence of conidial appendages, the two pathogens are similar in many biological characteristics. Isolates of S. unicorne showed a continuum of variation in conidial size and cultural morphology making it difficult to justify splitting of the species.     

Responses in the bark of Cupressus sempervirens clones artificially inoculated with Seiridium cardinale under field conditions

By 70 days after inoculation, resistant and tolerant clones of field-grown Cupressus sempervirens restricted penetration of the bark by Seiridium cardinale, forming ligno-suberized boundary zones which included four to six layers of cells with suberized cell walls. This response contrasted with that in susceptible clones, where only two to four layers of suberized cells were formed in discontinuous bands around inoculation sites. Detection of multiple layers of thin suberized boundary zones in susceptible clones indicated repeated penetration of the wound/infection-induced barrier by S. cardinale during a single growing season. The methods described will be valuable in the development of rapid, early and accurate screening programmes for durable resistance to Seiridium canker in Cupressus species.     

‘Le Crete 1’ and ‘Le Crete 2’: two newly patented Seiridium cardinale canker‐resistant cultivars of Cupressus sempervirens

In the Mediterranean area, common cypress (Cupressus sempervirens) has traditionally been used as a multipurpose tree, for its symbolic and ornamental role, for its valuable timber, as well as for windbreaks and soil protection. The epidemic spread of the Seiridium cardinale canker has limited the use of this tree since the 1970s, inducing researchers to develop a breeding programme of cypress aimed at selecting canker‐resistant lines for different uses and to support a flourishing trade of cypress plants. ‘Le Crete 1’ and ‘Le Crete 2’ described here are two new canker‐resistant C. sempervirens varieties patented in 2010, selected through a 13‐year assessment of their response to artificial inoculations and growth traits. Both are characterized by a rapid growth and by a columnar and fastigiated habit that confers them a notable ornamental effect. Preliminary observations showed also that both tend to produce yearly few microsporophylls and little pollen. ‘Le Crete 2’ was also selected for the high growth rate it maintained on heavy, clayey soils.     

AFLP analyses of California and Mediterranean populations of Seiridium cardinale provide insights on its origin,biology and spread pathways

Seiridium cardinale is regarded as the most important agent responsible for the disease of cupressaceous hosts referred to as Cypress canker. The fungus was first described in California and is currently reported in all continents. A recent study based on seven SSR loci has suggested that California populations may represent the source of the epidemic in the Mediterranean. In this study, 185 AFLP markers were used on an expanded sample size of 125 isolates to determine whether the Mediterranean population may indeed be derived from the California one and to compare the reproductive modes of populations in the two regions of the world. Additionally, amova , NJ, STRUCTURE, principal component analysis and β‐tubulin sequence analyses were employed to infer the presence of genetic structure within and between populations. The distribution of pairwise AFLP similarity coefficients suggests Mediterranean populations are reproducing only clonally, while California populations are reproducing both clonally and sexually. amova indicates Mediterranean and California populations are currently genetically isolated, but NJ and STRUCTURE analyses both suggest ancestral Mediterranean genotypes belong to the California population. No alleles were private to either population, and the presence of identical or quasi‐identical genotypes at large distances supports the notion that movement of infected cypress plants is responsible for the global spread of the disease. Surprisingly, STRUCTURE identified a second cluster of Mediterranean genotypes distinct from the basal mixed California–Mediterranean cluster. This second cluster either may have originated from the first one under the selection pressure imposed on the pathogen in the new Mediterranean environments or may be the result of a further introduction from California or elsewhere. Cumulatively, the evidence presented here suggests that S. cardinale may be native or long naturalized in California and that two genetically distinct groups are present in the Mediterranean, with obvious implications for further studies on this disease.     

Genetic relationships among genetically distinct forms of Melampsora larici‐epitea and related species based on AFLP data

Amplified Fragment Length Polymorphism (AFLP) was used to study genetic relationship among leaf rust fungi (Melampsora spp.) occurring on Salix species in short‐rotation coppice. Special interest was paid to a newly identified rust fungus found on S. viminalis in southern Sweden, morphologically similar to M. larici‐epitea but with a distinct DNA profile. Genetic distances among 40 Melampsora isolates collected from S. viminalis, S. dasyclados, S. viminalis x dasyclados, S. daphnoides, S. acutifolia and M. larici‐populina were calculated based on 101 AFLP markers. Neighbour‐joining analysis revealed the presence of six clusters, which corresponded exactly to predefined groups, namely three formae speciales of M. larici‐epitea, a stem‐infecting form of Melampsora on S. viminalis, the newly identified Melampsora on S. viminalis and a group consisting of two isolates from the poplar rust M. larici‐populina. All six clusters were well supported by bootstrap analysis (84 to 100% support). The newly identified Melampsora on S. viminalis was indicated to be genetically separated from M. larici‐epitea as well as from the stem‐infecting form of Melampsora.     

Current status of the Dutch elm disease pathogen populations affecting Ulmus minor in Greece

One hundred and eight Ophiostoma ulmi sensu lato isolates were collected from field elm trees with symptoms in 14 Prefectures of Greece. The purpose of this study was to assign Greek isolates to species and afterwards subspecies of the DED fungi and to analyse the genetic variability within the Greek populations of these pathogens. Isolates were compared with six reference strains belonging to O. ulmi and the two subspecies of O. novo‐ulmi. The structure of the population has been analysed by means of morpho‐physiological features (growth rates, colony morphology and fertility responses) and by DNA sequencing and PCR‐RFLP amplification of the cerato‐ulmin (cu) and the colony type (col1) gene regions. According to fertility tests, both subspecies of O. novo‐ulmi were detected in Greece, but none of the isolates collected was identified as O. ulmi. O. novo‐ulmi ssp. novo‐ulmi occurred more frequently than ssp. americana (73 and 35 isolates, respectively) and their ranges overlapped. All isolates that behaved as ssp. novo‐ulmi in the fertility tests had the cu, as well as the col1 profile of ssp. novo‐ulmi. Surprisingly, all isolates that behaved as O. novo‐ulmi ssp. americana in the fertility test had the cu, as well as the col1 (with one exception) profile of O. novo‐ulmi ssp. novo‐ulmi. A possible explanation for this inconsistency could be the occurrence of hybridization between the two subspecies in Greece.     

An integrative study of Sakia sisanganensis n. sp. (Rhabditida: Tylenchidae) from Sisangan forest,Iran, and new morphological observations for the genus

Further data on the morphology (the lip region characters) and phylogeny of the genus Sakia are presented. The new observations were based on scanning electron microscopy (SEM) and light microscopy (LM). A new species, Sakia sisanganensis n. sp., was recovered from rotten wood of a dead beech tree (Fagus orientalis) in northern Iran, herein described and illustrated based on an integrative approach, that is morphological, morphometric and molecular characters. The new species is characterized by a combination of the following features: fine transverse striae and vestigial single band in the lateral field in SEM. Labial area dorso‐ventrally flattened. Oral region with two concentric hexagonal plates, the inner one apparently elevated. Amphidial openings short, slit‐like. Stylet delicate. Median bulb fusiform to spindle‐shaped with weak valvular apparatus. Spermatheca functional. Tail filiform with faintly pointed tip and males common. The new species was morphologically compared with four known species of the genus, viz., S. alii, S. arboris, S. castori and S. indica, all having indistinct lateral fields. Molecular phylogenetic analyses were performed based on the small subunit ribosomal RNA gene (SSU rDNA). In the Bayesian inference (BI), S. sisanganensis n. sp. with two isolates was strongly supported as a sister taxon of a clade harbouring S. arboris + Lelenchus species. However, in the maximum likelihood (ML) analysis, the new species formed a clade with S. arboris, thus supporting the reciprocal monophyly of the genera Sakia and Lelenchus. Accordingly, the test of monophyly was performed (using Bayes factor) and the results did not reject the monophyly of sakia (i.e., S. sisanganensis n. sp. and S. arboris as sister taxa) based on the currently available data.     

Distribution and ecology of Armillaria species in Greece

Five Armillaria species were identified in a nationwide survey in Greece. Armillaria mellea was present in coniferous and broad-leaved forests in most of the areas examined, except the high altitudes (above 1100 m) of the mountains of north Greece. It was found to cause significant damage in fir forests as well as in fruit orchards and vineyards. Armillaria gallica was common in coniferous and broad-leaved forests in the high altitudes of central and northern Greece, predominating in the beech forests. The fungus was a weak parasite or a saprophyte of forest trees and was occasionally found on cultivated plants. Armillaria ostoyae was not found in southern and central parts of the country, but it has a wide distribution in the mountain forests of northern Greece and causes significant damage on fir, black pine, Scots pine and spruce. Armillaria cepistipes was recorded at high altitudes (1400–1800 m) on two mountains of northern Greece, mostly as a saprophyte in coniferous and broad-leaved forests. Armillaria tabescens was rare in the forests of Greece; it was found to cause disease in almond tree orchards.     

Phylogeographic variation among isolates of the Sirococcus conigenus P group

In this study the phylogeographic variation among isolates of the Sirococcus conigenus P group and the phylogenetic relationships of S. conigenus with Sirococcus clavigignenti‐juglandacearum and other species previously placed in the genus Sirococcus were investigated. A collection of 33 isolates originating from Picea, Pinus and Larix in Europe, North America and Bhutan were characterized by sequence analyses of the internal transcribed spacer (ITS) region (including ITS1, 5.8S ribosomal DNA, ITS2) of the nuclear rDNA and a portion of the β‐tubulin gene. In phylogenetic analyses most isolates from pine, spruce and larch formed a distinct clade, representing the P group of S. conigenus, which was separated from the T group of this pathogen. Four isolates from Picea in Europe and Canada formed a third clade within S. conigenus and these isolates are referred to as the S group. The P group consisted of five distinct ITS haplotypes, which partly differed in their optimum growth temperature and their growth rates at 25°C on malt extract agar. Nested clade analysis resolved the five haplotypes into three distinct clades and revealed significant genetic/geographic associations for some of the haplotypes. Parsimony analysis of the small subunit (18S) ribosomal DNA sequences confirmed the phylogenetic affinities between S. conigenus and S. clavigignenti‐juglandacearum. In contrast, Godronia cassandrae and Hormococcus conorum, which formerly had been placed in the genus Sirococcus, were found to be only distantly related to S. conigenus and S. clavigignenti‐juglandacearum.     

Host specificity of co‐infecting Botryosphaeriaceae on ornamental and forest trees in the Western Balkans

The Botryosphaeriaceae is a diverse family of endophytes and fungal pathogens of mainly woody plants. We considered the host range and distribution of these fungi by sampling diseased ornamental and forest trees and shrubs in Serbia, Montenegro, Bosnia and Herzegovina, spanning a Mediterranean and a Continental climatic region. In total, ten Botryosphaeriaceae species were identified in the Western Balkans and with the exception of Sphaeropsis visci and Phaeobotryon cupressi, which occurred on one host, all the species had a broader host range. Phaeobotryon cupressi was found only in the Mediterranean region and S. visci, Dothiorella sp., Dothiorella sarmentorum and Diplodia seriata were present only in the Continental region. Pathogenicity tests were conducted on a variety of hosts from which the Botryosphaeriaceae species were isolated. These included leaves and/or stems of seedlings of 21 hosts, and cut leaves and/or branches of six hosts. Moreover, stems of seedlings of Chamaecyparis lawsoniana, Cedrus deodara, Picea omorika, Pinus patula and Eucalyptus grandis were inoculated as hosts from which some or all of the Botryosphaeriaceae species used for inoculation were not isolated. Inoculations showed that the majority of these fungi could also co‐infect hosts other than those from which they were isolated. The results suggest that most of the species have broad host ranges and can potentially cause disease on a broad range of tree species under certain conditions.     

Identification and geographic distribution of vegetative compatibility types of Cryphonectria parasitica and occurrence of hypovirulence in Greece

Castanea sativa, one of the most precious forest trees in Greece, is affected by the spread of chestnut blight all over the country. Identification and mapping of the vegetative compatibility (vc) types of the fungus Cryphonectria parasitica was carried out and the occurrence of hypovirulence was checked. In 11 chestnut populations distributed all over Greece, 627 isolates were obtained and their vc type was determined. Four vc types were identified by means of tester isolates: EU‐12 was found to be the dominant vc type reaching a percentage of 88%, with EU‐2 (6%), EU‐10 (3%) and EU‐1 (2%) following. EU‐12 was found in all 11 populations investigated whereas EU‐2 was isolated from five populations and EU‐10 and EU‐1 were found only in one population each. Natural hypovirulence was also found in five populations of 11 examined. Perithecia were not found in either population, a finding supported by the fact that all Greek isolates examined belonged to a single mating type, MAT‐1. The low diversity in vc types, the absence of the perithecial stage and the occurrence of natural hypovirulence in some areas support the feasibility of successful application of biological control on the national level in Greece.     

Characterization of North American Armillaria species: genetic relationships determined by ribosomal DNA sequences and AFLP markers

Phylogenetic and genetic relationships among 10 North American Armillaria species were analysed using sequence data from ribosomal DNA (rDNA), including intergenic spacer (IGS‐1), internal transcribed spacers with associated 5.8S (ITS + 5.8S), and nuclear large subunit rDNA (nLSU), and amplified fragment length polymorphism (AFLP) markers. Based on rDNA sequence data, the nLSU region is less variable among Armillaria species than the ITS + 5.8S and IGS‐1 regions (nLSU < ITS + 5.8S < IGS‐1). Phylogenetic analyses of the rDNA sequences suggested Armillaria mellea, A. tabescens and A. nabsnona are well separated from the remaining Armillaria species (A. ostoyae, A. gemina, A. calvescens, A. sinapina, A. gallica, NABS X and A. cepistipes). Several Armillaria species (A. calvescens, A. sinapina, A. gallica, NABS X and A. cepistipes) clustered together based on rDNA sequencing data. Based on the isolates used in this study, it appears that techniques based on IGS‐1, ITS + 5.8S, and/or D‐domain/3′ ends of nLSU are not reliable for distinguishing A. calvescens, A. sinapina, A. gallica and A. cepistipes. However, AFLP data provided delineation among these species, and AFLP analysis supported taxonomic classification established by conventional methods (morphology and interfertility tests). Our results indicate that AFLP genetic markers offer potential for distinguishing currently recognized North American Biological Species (NABS) of Armillaria in future biological, ecological and taxonomic studies.