Antioxidant activity of nine Fabaceae species growing in Serbia and Montenegro

The aim of this study was to investigate antioxidant capacity of nine Fabaceae species collected on the mountains of Serbia and Montenegro. Antioxidant assays with various reaction mechanisms were used, including total phenolic content by Folin-Ciocalteu, DPPH radical scavenging capacity, Trolox equivalent antioxidant capacity (TEAC) values by ABTS radical cation and inhibition of liposome peroxidation. The investigated plants exhibited strong antioxidant capacity in all the tested methods, and among them, Lathyrus binatus, Trifolium pannonicum, and Anthyllis aurea were found to be the most active.     

Ecology of Armillaria species in managed forests and plantations in Serbia

The distribution of Armillaria species was investigated in Serbian forest ecosystems, in relation to the main host species attacked, forest‐types, geography and altitude. In total, 388 isolates were identified from 36 host species in 47 sites. Armillaria gallica was the most commonly observed species with the widest distribution and with an altitudinal range of 70–1450 m, it was the dominating Armillaria species in lowland alluvial forests and in Quercus and Fagus forests at higher elevations. Armillaria mellea occurred in Quercus spp. – dominated forests in the north and central regions at 70–1050 m. Sixty‐eight per cent of the A. mellea isolates were collected from living hosts, most commonly in declining conifer plantations. Armillaria ostoyae was distributed in the cooler coniferous forest types and plantations in the Dinaric Alps in the south of Serbia, at 850–1820 m. Armillaria cepistipes was found in the eastern and southern hilly and mountainous regions of the country, at 600–1900 m. Most isolates were obtained from conifers and rhizomorphs in the soil around decaying stumps. Armillaria tabescens was found only on dead oak material in the northern and eastern regions of the country at altitudes lower than 600 m.     

Rapid identification of polymorphic sequences in non‐model fungal species: the PHYLORPH method tested in Armillaria species

Development of molecular markers for phylogenetic, population genetics and phylogeographic studies remains arduous in non‐model species with low or no genomic resources. Sequencing the whole or a large part of the genome of the target species using next‐generation sequencing technologies is considered a promising method, although it still needs a large investment in bioinformatics. To quickly find polymorphic markers in fungal species, we tested an alternative method, named PHYLORPH. This method allows users to quickly target polymorphic regions of single copy genes in fungi using public databases. We applied this method to Armillaria species, which are important fungal pathogens and saprophytes playing a central role in the dynamics of forest ecosystems worldwide. We isolated 32 single copy genes with numerous single nucleotide polymorphism (SNP) sites. A genetic analysis of two French populations validated the polymorphism of 80 among 92 SNPs tested, and seven of these sequences exactly reconstructed the known phylogenetic tree of four tested Armillaria species. These results confirmed that the PHYLORPH method is efficient to identify various markers at both the intra‐ and interspecific levels for fungal species with no or few previous genetic markers.     

Development of species‐specific PCR primers on rDNA for the identification of European Armillaria species

Attempts to design species‐specific PCR primers from six European Armillaria species in the ribosomal RNA genes are reported. Primers were developed on the basis of the nucleotide sequence variability of the internal transcribed spacers (ITS) and the intergenic spacer (IGS1) of the ribosomal DNA. Four sets of primers gave specific PCR products for Armillaria tabescens, Armillaria mellea and Armillaria ostoyae. However, due to the high sequence similarities between Armillaria borealis and Armillaria ostoyae and between Armillaria cepistipes and Armillaria gallica no species specific amplification was obtained for these taxa.     

Initial characterization of an unidentified Armillaria isolate from Serbia using LSU‐IGS1 and TEF‐1‐α genes

Armillaria species have a global distribution and play variable ecological roles, including causing root disease of diverse forest, ornamental and horticultural trees. Accurate identification of Armillaria species is critical to understand their distribution and ecological roles. This work focused on characterizing an unidentified Armillaria isolate from a Serbian forest using pairing, sequencing of the partial large subunit and intergenic spacer‐1 regions of rDNA (LSU‐IGS1) and the translation elongation factor‐1 alpha gene (tef‐1α) genes, and phylogenetic analyses. Despite previously obtained LSU‐IGS1 RFLP patterns that matched the newly described North American Armillaria altimontana, pairing tests and phylogenetic analyses of LSU‐IGS1 and tef‐1α sequences clearly demonstrate that the unidentified isolate is not A. altimontana. Based on LSU‐IGS1, Armillaria gallica isolates were polyphyletic, and the Serbian isolate clustered with a subset of European A. gallica isolates within a well‐supported clade (99%). Based on tef‐1α, the Serbian isolate appeared as a separate, well‐supported clade (97%) that was basal to other poorly resolved, polyphyletic clades containing European A. gallica isolates. It is speculated that the unidentified Armillaria isolate from Serbia could represent an evolutionary ancestral state because of its separate, basal position compared with other clades comprising polyphyletic European A. gallica isolates. Alternatively, this unidentified Serbian isolate could represent an unusual hybrid because of its high‐level sequence heterogeneity, represented by multiple two‐nucleotide codes, within tef‐1α. Further characterization is needed to confirm the taxonomic status and ecological/evolutionary significance of this unique, unknown Armillaria isolate from Serbia.     

Development and verification of a diagnostic assay based on EF‐1 α for the identification of Armillaria species in Northern Europe

The overall aim of this study was to develop a new, reliable and rapid diagnostic assay for differentiating six European Armillaria species based on variation in their elongation factor‐1 alpha (EF‐1 α) gene sequences and to verify a set of species‐specific primers on 61 Armillaria isolates from Europe. Partial sequences of the EF‐1 α gene obtained in Armillaria borealis, Armillaria cepistipes, Armillaria gallica, Armillaria mellea, Armillaria ostoyae and Armillaria tabescens revealed sufficient interspecific variation to distinguish among species using nested primers. These primers gave unambiguous bands when tested on representative isolates of five of these species. However, the EF‐1 α sequences of European A. borealis isolates clustered into two distinct clades, termed here AbX and AbY. Specific primers were subsequently designed and tested successfully on both AbX‐type and AbY‐type A. borealis isolates. The taxonomy of A. borealis needs to be elucidated to determine whether a new, as yet unnamed Armillaria taxon exists in Europe. Three A. borealis isolates were also found to have heterozygous sites in their EF‐1 α sequences, which suggests that the gene could exist in more than one copy or that these isolates contain hybrid sequences. A pyrosequencing method was also developed, targeting a small region of EF‐1 α intron 4, which was able to differentiate European Armillaria isolates to the species level and additionally could distinguish AbX‐type and AbY‐type A. borealis isolates.     

Ecology and distribution of Armillaria species in Norway

The occurence of Armillaria species was assessed in Norway, enabling the northern‐most distribution of this genus to be determined in Europe. Four Armillaria species were found in Norway. Armillaria borealis was the most common species occurring on woody vegetation to the permafrost zone (ca. 69°N). Armillaria cepistipes was present in southern and central Norway, but was not found further than 66°N. Armillaria solidipes and Armillaria gallica were rare, found at only one locality each; 59°40′ and 59°32′, respectively. Armillaria species were found on 14 hosts, but there was no significant difference between occurrence of A. borealis and A. cepistipes on declining and dead trees. Phylogenetic analyses separated each species into separate clades. All isolates of A. borealis, except one, and most isolates of A. solidipes were in separate clades. However, a subclade within the A. borealis clade was formed of two A. ostoyae and one A. borealis isolates. Two small A. cepistipes genets were found in a declining oak stand.     

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.     

Rhizomorph production and stump colonization by co‐occurring Armillaria cepistipes and Armillaria ostoyae: an experimental study

In managed spruce forests, Armillaria cepistipes and A. ostoyae are efficient stump colonizers and may compete for these resources when they co‐occur at the same site. The aim of this experiment was to quantify the mutual competitive ability of the two Armillaria species in producing rhizomorphs and in colonizing Norway spruce (Picea abies) stumps. Five isolates of A. cepistipes and two isolates of A. ostoyae were simultaneously inoculated pair‐wise into pots containing a 4‐year‐old spruce seedling. For comparison, each isolate was also inoculated alone. One year after inoculation, stumps were created by cutting down the seedlings. Six months after creation of the stumps, rhizomorph production and stump colonization were assessed. Armillaria spp. were identified from 347 rhizomorphs and 48 colonized stumps. Armillaria cepistipes dominated both as rhizomorphs in the soil and on the stumps. Nevertheless, A. ostoyae was relatively more frequent on the stumps than in the soil and A. cepistipes was relatively more frequent in the soil than on the stumps. In both species, the ability to colonize the stumps in simultaneous inoculations was significantly reduced compared with single inoculations. In respect to rhizomorph production, simultaneous co‐inoculations had a slightly stimulatory effect on A. cepistipes and no significant effect on A. ostoyae. Our study suggests a rather neutralistic co‐existence of A. cepistipes and A. ostoyae as rhizomorphs in the soil. Concerning the ability to colonize stumps, the two species experience a mutual negative effect from the interaction, probably because of interspecific competition.     

PCR‐based identification of Erysiphe pulchra and Phyllactinia guttata from Cornus florida using ITS‐specific primers

The internal transcribed spacer (ITS) regions of rDNA and the intervening 5.8S rRNA gene for the powdery mildew fungi Erysiphe (sect. Microsphaera) pulchra and Phyllactinia guttata were amplified using standard polymerase chain reaction (PCR) protocols and the universal primer pairs, ITS₁ and ITS₄. PCR products for ITS were analysed by electrophoresis in a 1.5% agarose gel and sequenced. The size of the amplified ITS products (approximately 650 bp) were not sufficiently different to allow reliable differentiation of E. pulchra and P. guttata; however, their sequences were distinct. Specific primers for E. pulchra and P. guttata were developed and evaluated for use as diagnostic tools. The diagnostic band size from E. pulchra‐specific primer pair was 568 bp while the P. guttata band was 597 bp; the two primer pairs were highly specific to E. pulchra and P. guttata. Comparison of ITS sequences with information in the GenBank showed a very close similarity between sequences of E. pulchra isolates from Cornus florida in the USA and isolates collected on Cornus kousa in Japan. BLAST analysis of the sequence of the 650‐bp band from P. guttata revealed a close alignment with sequences of P. moricola (92%), P. kakicola (94%), and P. fraxini (92%). The sequence of P. guttata in C. florida also had a 98% identity with P. guttata in Calycanthus occidentalis and 94% identity with P. guttata in Corylus cornuta.     

Ecology of Armillaria species on conifers in Japan

Distribution, host preference and pathogenicity of Japanese Armillaria species on conifers were investigated on the basis of field collections of 65 isolates. We identified seven Armillaria species from 19 conifer species including six major Japanese plantation conifers using mating tests and sequences of the translation elongation‐1 α gene. Armillaria mellea, Armillaria ostoyae, Armillaria cepistipes and Armillaria sinapina were frequently collected, whereas Armillaria nabsnona, Armillaria tabescens and a biological species Nagasawa’s E were rare. On the basis of host condition when the isolates were collected, A. mellea, A. ostoyae, A. cepistipes and A. tabescens are considered as moderate to aggressive pathogens of conifers in Japan.     

Identification of Armillaria species in Japan using PCR-RFLP analysis of rDNA intergenic spacer region and comparisons of Armillaria species in the world

Armillaria species from Japan were characterized using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of the intergenic spacer region-1 (IGS-1) of ribosomal DNA (rDNA). Eleven different digestion patterns by restriction endonuclease Alu I were found among 70 isolates of seven Armillaria species in Japan. Isolates within Armillaria nabsnona, A. ostoyae, A. cepistipes, and Japanese biological species E showed the same Alu I digestion patterns. Five Alu I patterns were detected for A. gallica, three patterns for A. mellea, and two patterns for A. tabescens. Seven Armillaria species in Japan were clearly distinguished by using the profiles obtained when PCR products were digested with Alu I, Msp I, and Hae III restriction enzymes. There was considerable variability of Alu I restriction sites within the IGS-1 between the isolates of five Armillaria species, A. gallica, A. nabsnona, A. cepistipes, A. mellea, and A. tabescens, in Japan and those of their European and North American counterparts.     

Laccase isoenzyme patterns of European Armillaria species from culture filtrates and infected woody plant tissues

Polyacrylamide isoelectric focusing with specific staining for laccase activity was used to characterize laccase from European Armillaria species (Armillaria ostoyae, Armillaria mellea, Armillaria gallica, Armillaria cepistipes). The enzyme was extracted from culture media either supplemented, or not, with pine sawdust, and also from Pinus pinaster naturally infected by A. ostoyae, or artificially inoculated with A. mellea and A. ostoyae. Some differences in banding patterns were found for Armillana isolates according to the species and the culture media, but a common band at pI = 3.4 was found in all the extracts tested, independently of their origin (culture filtrate or wood).     

Susceptibility to Armillaria novae‐zelandiae among clones of Pinus radiata

Options are currently limited for the management of armillaria root disease in plantations of Pinus radiata in New Zealand. One possibility may be to plant genetically resistant clones on infested sites. Studies were undertaken over four consecutive years to examine variation in inherent resistance in P. radiata. Rooted cuttings in pots were treated with wood segment cultures of Armillaria novae‐zelandiae and disease symptoms were monitored during the following summer. Disease was severe among inoculated cuttings in all studies, with an overall mean of 54% plants infected. There were significant differences in both infection and mortality between studies and between four pathogen isolates, but not among 25 radiata pine clones. The rate at which symptoms first appeared also did not vary significantly between clones. Results suggest that while genetic resistance cannot be ruled out as an option in P. radiata, potential gains may be limited in extent and costly to achieve.     

Distribution,ecology and host range of Armillaria species in Albania

Species of Armillaria were identified from 645 isolates obtained in a nation‐wide survey in Albania. The material was collected from ca. 250 permanent plots, established for monitoring forest health, and from forests and orchards attacked by Armillaria. Armillaria mellea s.s. occurred on several coniferous and broadleaved trees in most areas examined, although it was absent above 1100–1200 m in northern Albania. This species damaged Abies and Quercus spp. and, to a lesser extent, other forest trees. Armillaria mellea was also commonly recorded causing damage in orchards and vineyards. Armillaria gallica was a common saprophyte or weak pathogen in coniferous and deciduous forests at altitudes from 600 to 1600 m, and less commonly on oaks at lower altitudes. Armillaria ostoyae was rare in central and southern Albania, but common in northern Albania, causing significant damage to pine and other conifers, mostly at altitudes from 600 to 1800 m. Armillaria cepistipes was recorded at altitudes from 800 to 1800 m as a saprophyte or weak pathogen on conifers and deciduous trees, mostly in beech and silver fir forests. Armillaria tabescens was found in oak forests at altitudes from sea level to 900 m. In orchards, A. tabescens occasionally attacked almond and pear trees. Armillaria borealis was found in a few locations in northern Albania, at altitudes from 800 to 1800 m.     

Pathogenicity of fungi associated with ash dieback symptoms of one‐year‐old Fraxinus excelsior in Montenegro

In addition to Hymenoscyphus fraxineus, two fungi identified as Diaporthe eres aff. and Fusarium sambucinum aff. were also isolated from necrotic bark lesions on declining one‐year‐old Fraxinus excelsior in a forest stand in Montenegro. To examine their involvement in ash decline, a pathogenicity test was performed using under bark inoculations on one‐year‐old Fraxinus excelsior. Hymenoscyphus fraxineus was included as comparison. All three fungal species proved highly pathogenic towards one‐year‐old seedlings although lesion sizes differed significantly between the different species. Hymenoscyphus fraxineus was most aggressive, followed by F. sambucinum aff., while D. eres aff. caused the smallest lesions. This study demonstrates for the first time the ability of isolates in the D. eres and F. sambucinum species complexes to cause decline on one‐year‐old common ash seedlings.     

Identification of European Armillaria species by analysis of isozyme profiles

Studies were carried out to test the possibility of identifying European Armillaria species by using isozyme patterns. Twenty-two different enzymes were used to analyse the haploid and diploid mycelium extract of Armillaria borealis, Armillaria cepistipes, Armillaria gallica, Armillaria mellea, Armillaria ostoyae and Armillaria tahescens. Tests for fumarase (E.C. 4.2.1.1.), aconitase (E.C. 4.2.1.3.), leucine-amino peptidase (E.C. 3.4.11.1.), isocitrate dehydrogenase (E.C. 1.1.1.42.), shikimic dehydrogenase (E.C. 1.1.1.25), glucose-6-P-dehydrogenase (E.C. 1.1.1.49.), malic enzyme (E.C. 1.1.1.40.), 6-P-gluconic dehydrogenase (E.C. 1.1.4.4.), pectin esterase (E.C. 3.1.1.11.), and pectic lyase (E.C. 4.2.99.3.) did not reveal enzyme activity. Isozyme profiles of acid phosphatase (E.C. 3.1.3.2.), phospho-gluco-isomerase (E.C. 5.3.1.9.), peroxidase (E.C. 1.11.1.7.), polyphenoloxidase (E.C. 1.14.18.), malic dehydrogenase (E.C. 1.1.1.37.), glutamic dehydrogenase (E.C. 1.4.1.3.) and superoxide dismutase (E.C. 1.15.1.1.) were ineffective for species identification. In contrast, esterase (E.C. 3.1.1.1.), glutamic-oxalacetic transaminase (2.6.1.1.), phospho-gluco-mutase (E.C. 2.7.5.1.), alcohol dehydrogenase (E.C. 1.1.1.1.), and polygalacturonase (E.C. 3.2.1.15.) isoenzyme patterns showed enough polymorphism to allow the identification of the different Armillaria species. However, it is necessary to compare several enzyme profiles for a conclusive identification. Intraspecific crosses of A tabescens were confirmed by the presence of a heteromeric isozyme pattern of alcohol dehydrogenase and phospho-gluco-mutase.     

Field survey,isolation, identification and pathogenicity of Phytophthora species associated with a Mediterranean‐type tree species

Corymbia calophylla (marri), a keystone tree species in the global biodiversity hot spot of southwestern Australia, is suffering decline and mortality associated with a canker disease caused by the endemic fungus Quambalaria coyrecup. Phytophthora species are frequently isolated from the rhizosphere of C. calophylla, and a hypothesis is that Phytophthora root infection is predisposing C. calophylla to this endemic canker pathogen. Field surveys were conducted in both anthropogenically disturbed and undisturbed C. calophylla stands, from where a total of 100 rhizosphere soil samples, from both healthy and cankered trees, were collected. Phytophthora species were isolated from 26% of the samples collected, with Phytophthora incidence significantly higher on disturbed stands than in natural forests (73% and 27%, respectively). Five Phytophthora species were recovered, including P. cinnamomi, P. elongata, P. multivora, P. pseudocryptogea and P. versiformis. Under‐bark inoculations with the Phytophthora isolates caused significant lesion lengths in excised C. calophylla stems. Corymbia calophylla response to pot infestation trials in the glasshouse varied between Phytophthora species and isolates, with isolates of P. cinnamomi and P. multivora causing a significant reduction in seedling root volume and often leading to seedling death. This study demonstrates that root disease caused by Phytophthora species, especially P. cinnamomi and P. multivora, has the ability to adversely affect C. calophylla health. This study leads the way to do a dual inoculation trial with the canker pathogen Q. coyrecup, and different Phytophthora species to investigate if Phytophthora root infection predisposes C. calophylla to this canker disease.     

Armillaria ostoyae associated with dying 60‐year‐old Scots pines in northern Turkey

We investigated an Armillaria root disease centre in a 60‐year‐old, naturally regenerated Pinus sylvestris stand located at 1250 m altitude in Sinop province in northern Turkey. Based on sequence analysis of the internal transcribed spacer region of the rDNA, the mycelial fans sampled from the infected trees were Armillaria ostoyae. Multilocus genotyping indicated that the fans belonged to a single genet, at least 0.2 ha in size. Drought as a predisposing factor is discussed. The study area received 50–70 and 25–50% of the normal precipitation in May, June and August in 2009 and in August and September in 2010, respectively. Moreover, earlier excavation work to prepare a forest road had probably disrupted the water table in the affected stand.     

Rhizomorph growth habit,saprophytic ability and virulence of 15 Armillaria species

The rhizomorph branching habit in soil, competitive saprophytic ability and virulence were determined for 15 species of Armillaria from Europe, North America and Australia and New Zealand. In soil, rhizomorphs of northern hemisphere species branched either monopodially or dichotomously, whereas all five species from Australia and New Zealand branched dichotomously. The dry weight of rhizomorphs produced in soil by isolates of a species and by species was very variable. Species with monopodially branched rhizomorphs had significantly higher saprophytic colonization scores than dichotomously branched species and scores were significantly higher in Garry oak than Douglas‐fir segments and in fresh than autoclaved segments. The damage to Douglas‐fir seedlings caused by isolates of most dichotomously branched species was significantly greater than that caused by monopodially branched species. Species producing dichotomously branched rhizomorphs were more aggressive than monopodially branched species, killing 80% (vs. 17%) of seedlings that died during the first year of the 2‐year experiment.