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.     

A rapid and holistic approach to screen susceptibility of Prunus species to Armillaria root rot

We describe a holistic in vitro technique for inoculating roots of Prunus species with Armillaria solidipes, which is faster and more successful than previous methods. This method allows associated active and passive host defences to be assessed. Sterile root segments of three Prunus spp. were placed next to or on top of 14‐day‐old cultures of A. solidipes. At 21 day, the success of fungal penetration and length of the fungal colonization, and host responses were evaluated. Also, an anti‐Armillaria and anti‐Cladosporium activity detection assays were conducted by utilizing the root periderm, the first tissue that needs to be penetrated by fungus. These methods revealed the variation in the relative tolerance of three Prunus spp. to A. solidipes. The overall success of fungal colonization in the wounded and intact root, host defence, and antifungal activity significantly differed among three Prunus spp. Results indicate that this in vitro method can be used as a preliminary step in screening tree species to Armillaria spp.     

Distribution of Armillaria species in upland Ozark Mountain forests with respect to site,overstory species composition and oak decline

Armillaria root disease is a contributing factor to oak decline in the Ozark Mountains of central USA. We have identified Armillaria gallica, Armillaria mellea, and Armillaria tabescens in Quercus‐Carya‐Pinus forests of the region. Presence/absence patterns of each Armillaria species as well as all possible Armillaria species combinations were analysed by contingency tables and/or stepwise logistic multiple regressions with principal characteristics of the studied sites and forest stands, both quantitative and qualitative: geographic land‐type association, bedrock type, landform position, slope direction (aspect), soil type and soil surface stone cover, down woody debris, abundance and basal area of woody vegetation and decline mortality by species. Most decline mortality consisted of two red oak species (section Erythrobalanus, Quercus coccinea and Quercus velutina), which also were most sensitive to Armillaria infection. Site characteristics related to the distributions of Armillaria species and decline mortality were also related to the preponderance of Q. coccinea and Q. velutina, regional vegetation history (i.e. conversion of Pinus echinata stands to hardwoods), and the different strategies of territory acquisition and spread of the Armillaria species involved. The presence of A. gallica may reduce the activity of more virulent Armillaria species.     

Yield reduction in spruce infected with Armillaria solidipes in the southern interior of British Columbia

The effect of non‐lethal root infection on the yield of planted spruce (Picea engelmannii x glauca hybrid spruce) in southern British Columbia was determined in a 34‐year‐old, mixed‐species stand infected with Armillaria solidipes Peck. Yield reduction correlated with trees’ infection duration. Individual trees infected for 24 years had about 42% less volume than their healthy equivalents. Using the incidence of infection in stands in this area and the individual losses per tree, reduction at the stand level was approximated at 11% and increasing.     

Effects of wounding and fungal infection with Armillaria ostoyae in three conifer species. II. Host response to the pathogen

Structural responses in the bark and wood were described following penetration by Armillaria ostoyae in the roots of 20‐ to 30‐year‐old Douglas‐fir, western hemlock and western redcedar trees. Tissue necrosis presumably caused by fungal exudates was commonly observed at inoculum contact. In Douglas‐fir and western hemlock, A. ostoyae interfered with the initiation of active defence mechanisms involving the development of a lignified zone of impervious tissue (IT), necrophylactic periderm (NP) formation and compartmentalization of infected woody tissue. Breaching of IT and NP barriers was frequent, particularly around the clusters of sclereid cells in western hemlock. In western redcedar, the IT zone was inconspicuous. Induced rhytidome formation occurred in western redcedar either simultaneously with or after completion of NP development. The formation of this tissue facilitated en masse sloughing of infected tissue from the surface of roots. In western redcedar, traumatic phloem resin ducts formed in tangential bands surrounding the margin of expanded lesions. Effective compartmentalization in western redcedar was achieved by a barrier zone comprised of a higher‐than‐average number of axial parenchyma that accumulated polyphenolic deposits. A combination of host‐mediated defence mechanisms in western redcedar resulted in a significantly higher frequency of effective resistance reactions than in western hemlock or Douglas‐fir.     

Wood-inhabiting fungi in stems of Fraxinus excelsior in declining ash stands of northern Lithuania,with particular reference to Armillaria cepistipes

Abstract

Stem bases of 210 Fraxinus excelsior trees of three different health categories were sampled by the means of an increment borer in declining ash stands in northern Lithuania. From this number, 15 sound-looking, 132 declining and 63 dead trees from three discrete plots yielded 352 isolates, representing 75 fungal species. In addition, mycelial fans and rhizomorphs typical of Armillaria spp. from 205 and 20 trees, respectively, were sampled and subjected to fungal isolations. Species richness was similar in trees from each health category, but community structures differed, indicating that species composition of wood-inhabiting fungi in stems changes along with the changes in tree health condition. Armillaria cepistipes was the most common species (86 isolates from 210 wood samples, or 41.0%), isolated more frequently and consistently than any other potential tree pathogen. It also showed abundant occurrence on a majority of trees in the form of mycelial fans and rhizomorphs, from which 64 and 14 isolates of the fungus were obtained, respectively. The population structure of A. cepistipes revealed the presence of 53–93 genets per hectare, some of which extended up to 30–55?m.     

Identification of the Armillaria root rot pathogen in Ethiopian plantations

Armillaria root rot is a well‐known disease on a wide range of plants, world‐wide. In Ethiopia, the disease has previously been reported on Pinus spp., Coffea arabica and on various native hardwoods. The causal agent of the disease has been attributed to Armillaria mellea, a species now known to represent a complex of many different taxa. The aim of this study was to determine the extent of Armillaria root rot and the identity of the Armillaria sp. in Ethiopian plantations. As part of a plantation disease survey in 2000 and 2001, samples were collected in plantations at and around Munessa Shashemene, Wondo Genet, Jima, Mizan and Bedele, in south and south‐western Ethiopia. Basidiocarps were collected and their morphology studied. Morphological identification was confirmed by sequencing the intergenic spacer (IGS‐1) region of the ribosomal rRNA operon and comparing data with published sequences of Armillaria spp. Armillaria isolates were collected from Acacia abyssinica, Pinus patula, Cedrela odorata and Cordia alliodora trees. Sporocarps were found on stumps of native Juniperus excelsa. Basidiocarp morphology and sequence data suggested that the fungus in Ethiopia is similar to that causing disease of Pinus spp. in South Africa and previously identified as A. fuscipes. This identification was confirmed for all isolates, based on sequence data. Armillaria fuscipes is known to be common in southern Africa. Its widespread occurrence in Ethiopia suggests that it is also the major cause of Armillaria root rot in that country.     

Structural host responses of Acacia mangium and Eucalyptus pellita to artificial infection with the root rot pathogen,Ganoderma philippii

Structural host responses of young Acacia mangium and Eucalyptus pellita to infection with the root rot pathogen Ganoderma philippii were described. To our knowledge, this is the first report investigating the histology of root responses infected with G. philippii. The infected roots of A. mangium were characterized by the presence of a continuous multilayered mycelial sheath (~100–200 μm thick), fungal outgrowths and the production of a wound periderm. In contrast, roots of E. pellita were characterized by a mycelial sheath and fungal outgrowths and a wound periderm comprising of only one to four rows of cells.     

Calonectria metrosideri,a highly aggressive pathogen causing leaf blight,root rot,and wilt of Metrosideros spp. in Brazil

The genus Metrosideros includes several tree, shrub and vine species, native to the Pacific Islands. Seedlings from 25 seed lots of Metrosideros polymorpha and two seed lots of M. tremuloides with symptoms of root rot, stem girdling, wilting and round, purple leaf spots were observed in the Forestry Nursery at the Universidade Federal de Viçosa, Brazil. In the original disease site, seedling mortality reached up to 71% in M. polymorpha and 34% in M. tremuloides. Single conidial cultures obtained from infected leaf, root and stem samples of M. polymorpha were used to identify the fungal species. Morphological characters and DNA sequences of four loci, containing partial sequences of β‐tubulin (TUB2), histone H3 (HIS3), calmodulin (CAL) and the elongation factor (tef‐1α) genes of three isolates, indicated that they belong to a new species, described here as Calonectria metrosideri sp. nov. Potting medium infestation and inoculation of seedlings of M. polymorpha with an inoculum suspension at 1 × 10⁴ conidia ml^?1 induced typical symptoms of the disease (leaf spots, root rot and wilt), similar to those observed under natural conditions. Calonectria metrosideri was re‐isolated, which fulfilled Koch's postulates, and confirmed its status as a pathogen.     

Phytotoxins produced by the oak pathogen Discula quercina

Two phytotoxic metabolites, p‐hydroxybenzaldehyde and indol‐3‐aldehyde, were isolated and identified by spectroscopic methods from solid cultures of Discula quercina, an endophytic fungal pathogen frequently associated with oak decline in Italy. In addition, the fungus produced an unusual acyclic keto acid as a major metabolite, which was identified by spectroscopic and chemical methods as 5‐oxo‐6E,8E‐octadecadienoic acid. In leaf puncture assays on Quercus suber and Q. ilex leaves, indol‐3‐aldehyde proved to be more toxic than p‐hydroxybenzaldehyde, while 5‐oxo‐6E,8E‐octadecadienoic acid was inactive even at the highest concentration used (1 mg ml^?1). Although indol‐3‐aldehyde is a known microbial metabolite, its phytotoxic activity has hitherto not been reported. This report is the first to describe the production of phytotoxic compounds by D. quercina.     

Pathogenicity and virulence of Armillaria sinapina and host response to infection in Douglas‐fir,western hemlock and western redcedar in the southern Interior of British Columbia

The frequency of infection, lesion characteristics and anatomical changes in phloem and cambial tissues caused by Armillaria sinapina were studied on inoculated trees of Douglas‐fir, western hemlock and western redcedar and compared with results of A. ostoyae inoculations previously reported on the same host species. Similar percentages of inoculations resulting in infection of roots on the three hosts indicate that A. sinapina and A. ostoyae are equally pathogenic. Armillaria ostoyae was more virulent than A. sinapina as demonstrated by fungal exudates from A. ostoyae inoculum blocks, which appeared to cause lesions on roots; the higher frequency at which lignified impervious tissue (IT) and necrophylactic periderm (NP) developed in bark and following cambial invasion, compartmentalization; the large proportion of roots that showed no visible host response; the large zones of IT formed under continuous stimulation by A. ostoyae advancing in inner bark; and the high frequency of breaching of NP barriers. Spread of A. sinapina mycelium in host species appeared slower than that of A. ostoyae, particularly in Douglas‐fir and western hemlock. In western redcedar, A. ostoyae induced stronger host responses than those following invasion by A. sinapina, which included further expansion of the induced rhytidome response, traumatic phloem resin duct formation and higher numbers of polyphenolic parenchyma comprising its barrier zone. Where damage by A. sinapina ensued, it was always associated with high inoculum potential. The ecology of virulent and less virulent species of Armillaria in natural forests is discussed.     

Recent insights into the pandemic disease butternut canker caused by the invasive pathogen Ophiognomonia clavigignenti‐juglandacearum

In the 25 years following the initial 1967 report of the disease, butternut canker was able to quickly spread throughout the entire range of butternut (Juglans cinerea) in North America, from Minnesota in the upper Midwest to Tennessee in the south and Quebec in the north‐east. The speed of this dispersal is notable as butternut trees do not make up a significant proportion of any single forest type. Instead, they are usually found sparingly in most mixed hardwood forests. In this review, we synthesize the current knowledge of the invasion process of the butternut canker pathogen, Ophiognomonia clavigignenti‐juglandacearum, an invasive fungal pathogen, that as its emergence has spread across North America and is now found wherever butternut naturally occurs. Taxonomic studies have determined that the fungus belongs in the genus Ophiognomonia, which includes a number of saprophytes, endophytes and pathogens of members of the Fagales, rather than the genus Sirococcus, which includes several important pine pathogens. The ability of fungus to be dispersed by rain splash, transported on and in beetle vectors, transmitted by infected seed and successfully colonized several species of Juglans and Carya have all likely contributed to the rapid increase in abundance and severity of disease and tree mortality in the invaded forest ecosystems. Recent genomic and population genetic analyses have determined that there were at least three emergence events. A less virulent strain of the fungus likely has been present in the north eastern United States for over a century, but it was the emergence of a more virulent strain of the fungus in Minnesota and Wisconsin in the 1960s that resulted in range‐wide mortality and pushed butternut to be listed as an endangered species in Canada and a number of states in the United States.     

Antifungal properties of terpenoids in Picea abies against Heterobasidion parviporum

The antifungal properties of 14 major oleoresin‐constitutive terpenoids of Norway spruce (Picea abies) against Heterobasidion parviporum were evaluated in vitro at three gradient concentrations, 0.1, 0.2 and 0.4 μmol/cm², on potato dextrose agar medium. Eight monoterpene hydrocarbons (+)‐ and (?)‐α‐pinene, (?)‐β‐pinene, (+)‐3‐carene, myrcene, (+)‐ and (?)‐limonene, terpinolene; four oxygenated monoterpenes α‐terpineol, terpinen‐4‐ol, 1,8‐cineole, bornyl acetate; and two resin acids abietic acid and dehydroabietic acid were selected. Abietic and dehydroabietic acids showed the highest antifungal activities at all concentrations. Among oxygenated monoterpenes, bornyl acetate and α‐terpineol showed antifungal activity at the highest concentration. Among monoterpene hydrocarbons, (+)‐α‐pinene showed similar activity to terpinen‐4‐ol and 1,8‐cineole at the highest concentration; however, it was lower than α‐terpineol and bornyl acetate. Other monoterpene hydrocarbons inhibited mycelial growth by <10%. Re‐extraction of medium surfaces after the test period revealed that most α‐terpineol and terpinen‐4‐ol remained unchanged on the surface but monoterpene hydrocarbons completely disappeared. However, notable fungal transformed products were observed on surfaces applied with 1,8‐cineole and bornyl acetate. Thus, mycelial growth inhibition of monoterpenoids might be caused by complex functions such as biodegradation and/or detoxification by interaction between mycelium and compound. These results provide a basis for future studies considering the role of chemodiversity in the comprehensive chemical defence of P. abies.     

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.     

Development of a quantitative real‐time PCR assay for the detection of Phytophthora austrocedrae,an emerging pathogen in Britain

A TaqMan real‐time PCR assay was developed for Phytophthora austrocedrae, an emerging pathogen causing severe damage to juniper in Britain. The primers amplified DNA of the target pathogen down to 1 pg of extracted DNA, in both the presence and absence of host DNA, but did not amplify any of the non‐target Phytophthora and fungal species tested. The assay provides a useful tool for screening juniper populations for the disease.     

Mycelial fan formation of three sympatric Armillaria species on excised stem segments of Picea abies

Mycelial fan formation was studied in five Armillaria cepistipes, ten A. borealis and ten diploid and six haploid A. ostoyae strains on excised stem segments of Picea abies. Stem segments were either non‐autoclaved or autoclaved, representing dying and dead wood, respectively. To confirm the identity of mycelial fans on non‐autoclaved stem segments, re‐isolations were made and isolates characterized with microsatellite markers. Mycelial fan formation on autoclaved stem segments was fast and reliable for most of the tested Armillaria strains. On non‐autoclaved stem segments, mycelial fan formation was slower, more erratic and less predictable. Mycelial fan formation was fastest in A. cepistipes closely followed by A. borealis and was slowest in A. ostoyae. For two A. cepistipes and four A. ostoyae strains (all diploid), growth rates of mycelial fans were estimated in a time course experiment. They ranged between 5.1 and 8.7 mm/day for autoclaved and between 1.4 and 4.7 mm/day for non‐autoclaved stem segments. The haploid A. ostoyae strains also formed mycelial fans on autoclaved stem segments, but typically slower and less reliably than the diploid strains. Whether haploid strains are able to produce mycelial fans on non‐autoclaved stem segments remains unknown because of accidental diploidization of the original haploid strains which was likely caused by basidiospores introduced into the study system on the non‐autoclaved stems. Overall, the method developed in this study may be useful for further investigations into the genetic, physiological and biochemical nature of mycelial fan formation in the genus Armillaria.     

Direct quantitative real‐time PCR assay for detection of the emerging pathogen Neonectria neomacrospora

The epidemic outbreak in northern Europe of Neonectria neomacrospora, the causal agent of dieback in Abies spp., led the European and Mediterranean Plant Protection Organization (EPPO) to include the pathogen on its alert list in 2017. Effective monitoring of this pathogen calls for a rapid and sensitive method of identification and quantification. A probe‐based real‐time PCR (qPCR) assay based on the β‐tubulin gene was developed for the detection and quantification of N. neomacrospora in infected wood samples, and directly for ascospores. This study presents the first published species–specific molecular detection assay for N. neomacrospora. The analytical specificity was validated on taxonomically closely related fungal species as well as on 18 fungal species associated with the host (Abies sp.). The analytical sensitivity was tested on naturally infected wood, on purified pathogen DNA in a matrix of host DNA and on N. neomacrospora ascospores for detection of airborne inoculum. The latter was tested both with a DNA extraction step prior to qPCR and without DNA extraction by direct qPCR on collected ascospores. The assay was specific to N. neomacrospora, with a sensitivity of 130 fg purified DNA, or 10 ascospores by direct qPCR. Omitting DNA extraction and amplifying directly on unpurified ascospores improved assay sensitivity significantly.     

A major facilitator superfamily transporter in Colletotrichum fructicola (CfMfs1) is required for sugar transport,appressorial turgor pressure,conidiation and pathogenicity

The major facilitator superfamily (MFS) is one of the largest membrane‐protein families. To investigate the role of MFS proteins in the fungal plant anthracnose pathogen Colletotrichum fructicola, the CfMFS1 gene was deleted. This resulted in reduced mycelial growth, conidial yield and decreased virulence on tea oil camellia leaves. In addition, ?Cfmfs1 showed increased sensitivity to osmotic stress and to a cell‐wall stressor. Further analysis revealed that CfMfs1 is required for conidial penetration and appressorial turgor pressure, both important for fungal pathogen invasion. Confocal fluorescence microscopy showed that CfMfs1 is localized to membranes of both hyphae and conidia, suggesting that it may be a membrane transporter. Our study provides evidence that CfMfs1 has a role in conidiation, sugar transport, stress response, conidial penetration, appressorial turgor pressure and virulence against tea oil camellia.