Phylogeography and host range of Armillaria gallica in riparian forests of the northern Great Plains,USA

Root disease pathogens, including Armillaria, are a leading cause of growth loss and tree mortality in forest ecosystems of North America. Armillaria spp. have a wide host range and can cause significant reductions in tree growth that may lead to mortality. DNA sequence comparisons and phylogenetic studies have allowed a better understanding of Armillaria spp. taxonomic diversity. Genetic sequencing has facilitated the mapping of species distributions and host associations, providing insights into Armillaria ecology. These studies can help to inform forest management and are essential in the development of disease risk maps, leading to more effective management strategies for Armillaria root disease. Armillaria surveys were conducted on publicly owned lands in North Dakota, South Dakota, and Nebraska, U.S.A. Surveyed stands consisted of riparian forests ≥0.4 hectares in area. Armillaria was found at 78 of 101 sites. A total of 57 Armillaria isolates—associated with 12 host tree species—were used for DNA sequencing of the translation elongation factor‐1 alpha (tef1) gene. Armillaria gallica was the only species identified within the study sites. Results suggest that A. gallica is a common root pathogen of hardwood trees in riparian forests of the northern Great Plains with a wider host range and geographic distribution than previously recognized.     

Fungal lesion length and expansion rate for the root pathogen Armillaria ostoyae in Douglas‐fir affecting root colonization and damage

Plant lesions affect disease impact, progression and host resistance. Root lesions caused by Armillaria ostoyae (Romagn.) Herink were inspected on 24‐ to 34‐year‐old planted Douglas‐fir (Pseudotsuga menziesii (Mirb.) Franco) pulled from the soil in five locations. Four hundred seventy‐nine lesions were measured for length (mm), expansion rate (mm/year) and lesion type. Lesion types were patch lesions on a root side, girdled lesions covering the root circumference and root collar lesions on the stem below ground. Non‐linear mixed modelling revealed that fungal lesion length at girdled and collar lesions increased but the expansion rate slowed as a power function of time, indicating increasing secondary inoculum and host resistance. Average fungal lesion expansion rate in girdled root and collar lesions was 127 mm the first year which then declined non‐linearly to 26 mm/year after 10 years. Fungal lesion length in patch lesions showed little change after the first year of infection. For girdled and collar lesions, fungal lesion length increased with tree size at the time of infection. Sites with a longer frost‐free period had shorter girdled root and stem lesions, probably because of more active host defence. Results indicate that mortality would be rare from one infection event. Furthermore, patch lesions pose a lower risk of mortality than other lesions because of a lower fungal lesion expansion rate and secondary inoculum. Tree size, lesion position, lesion type and fungal lesion expansion rate need to be considered when assessing the risk of lesions to individual tree survival. Techniques to minimize the disease impact are discussed.     

Parasitic association of the mycetophagous wood nematode,Bursaphelenchus fraudulentus with the honey fungus Armillaria ostoyae

Honey fungi, Armillaria spp. are common and economically important pathogens of a wide range of tree species grown both in the forest and orchard cultures worldwide. Our field research in 73 forest stands across Poland has shown a common association of the mycetophagous wood nematode, Bursaphelenchus fraudulentus with Armillaria‐infected trees. The data reported here provide the first insight into the parasitic interaction of a nematode and Armillaria ostoyae. In laboratory conditions, B. fraudulentus reproduced on A. ostoyae, caused substantial damage to its mycelium and, finally, killed the whole colony within a short time. In ageing cultures, the nematode produced resting (dauer) juveniles. After artificial inoculation to Armillaria‐infected trees, the nematode population persisted under the bark, continued feeding and mass reproduction on the mycelium, and dispersed over the mycelial fans. The ability of B. fraudulentus to develop and reproduce on the surface and inside the fungal rhizomorphs suggests that these structures could facilitate the nematode dispersion in the environment. Since B. fraudulentus is not pathogenic to the host tree, its parasitic association with A. ostoyae may contribute to natural regulation of this fungus in the environment. The observed characteristics of this nematode suggest that it could potentially be used as a biocontrol agent of honey fungi in forest and orchard trees.     

An agglutination test for fast and specific detection of Erwinia psidii

Dieback and wilt, caused by Erwinia psidii (Ep), is one of the most important emergent diseases of Eucalyptus spp. in Brazil. Currently, pathogen detection relies on isolation of bacteria from infected plant tissue and either identification based on morphological, physiological and biochemical tests or DNA amplification using the polymerase chain reaction (PCR), which in many cases is laborious and cumbersome. Considering the need for a simpler and more rapid, yet reliable, method for detecting the pathogen, we obtained a polyclonal antibody (anti‐Ep) and developed an agglutination test for specific detection of E. psidii. The antiserum was produced against the E. psidii strain LPF534 and tested against 101 E. psidii isolates from Eucalyptus spp.; three E. psidii isolates from Psidium guajava; 23 Ralstonia solanacearum and 18 Xanthomonas axonopodis isolates pathogenic to Eucalyptus spp.; and seven endophytic isolates from Eucalyptus spp., three of which are phylogenetically related to the genus Erwinia. Results of direct ELISA indicated that a concentration as low as 3.5 µg/ml of the anti‐Ep antibody was able to detect the E. psidii antigen and that the antibody did not cross‐reacted with other bacteria pathogenic and non‐pathogenic to Eucalyptus spp. In the agglutination test, the anti‐Ep antibody showed positive reaction with all strains of E. psidii tested whereas cross‐reaction with none of the strains that belong to other taxonomic groups was observed. The agglutination test showed a detection limit of 10⁵ colony‐forming units (CFU)/ml, and its specificity was the same as that obtained by PCR amplification using E. psidii‐specific primers. These results demonstrate that the agglutination test developed here is a useful tool for specific, fast and inexpensive detection of E. psidii although only operational on pure bacterial suspensions and not yet directly from infected tissues.     

Individual resistance of Fraxinus angustifolia clones to ash dieback

The presumed resistance of individual ash trees to ash dieback caused by invasive pathogen Hymenoscyphus fraxineus is an important issue for the maintenance of ash in European forests. All known studies regarding the resistance of ash trees to ash dieback were conducted in plantations and stands of F. excelsior; however, no such data exist for F. angustifolia. Crown damage assessments were performed over four consecutive years between 2009 and 2012 at a F. angustifolia clonal plantation in Hra??ica, Slovenia. Inoculation of H. fraxineus into the branches of the most and least damaged clones of F. angustifolia and leaf phenology assessments was performed to verify the presence of defence mechanisms that limit fungal growth or promote disease escape. Additionally, root collars of selected clones were inspected for fungal infections. The crown damage assessments showed considerable differences among F. angustifolia clones, indicating genetic variability in susceptibility to ash dieback. Crown dieback progressed significantly over the 4‐year time period; the mean crown damage of individual clones in 2012 varied between 16.7% and 83.8%. Significant differences among F. angustifolia clones were found in the inoculation trials and leaf phenology assessments. However, defence mechanisms such as early leaf flushing, early leaf shedding and the ability to inhibit pathogen growth in host tissues were not confirmed. High frequency of Armillaria spp. and H. fraxineus root collar infection demonstrated the need for whole tree inspection to determine causal agent of damages on individual ash trees. Armillaria spp. may be highly associated with ash decline epidemiology.     

Fungal root endophytes of Quercus robur subjected to flooding

In August 2011, fungal endophytes were isolated from fine (0.1–0.5 cm diameter) and structural (0.6–2.0 cm) roots from eighteen Q. robur trees at six sites in a Q. robur‐dominated forest displaying symptoms of oak decline. Five sites were subjected to periodic flooding for 3 months in 1997 and 1 month in 2010. Fungal colonization was detected in 94% of 1296 root fragments examined. Fungi representing 126 taxa from Zygomycota (four species), Ascomycota (117 species) and Basidiomycota (five species) were isolated using culture‐based methods and identified by morphotyping and sequencing of the ITS 1 and 2 rDNA. The most frequent eudominants (with colonization frequencies ≥10%) were Aspergillus spp., Cosmospora sp., Ilyonectria radicicola, Pezicula radicicola, Pyrenochaeta cava and six species of Trichoderma. Species composition did not vary greatly between sites, but only 3% of species occurred in all sites. Number of species was higher in roots subjected to floods. Eudominants and dominants present only on flooded sites included Aspergillus spp., Chaetomium globosum, Cosmospora sp., I. radicicola, Sporothrix inflata, Trichoderma harzianum, T. pubescens and T. virens. Structural roots were usually colonized by a higher number of species than fine roots. The majority of species detected belong to Class 2 NC‐endophytes. Pezicula radicicola and Phialocephala fortinii belong to Class 4 NC‐endophytes.     

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.     

Pinus taeda L. response to differential inoculum density of Leptographium terebrantis colonized toothpicks

Bark beetle‐vectored ophiostomatoid fungi, Leptographium terebrantis, is inoculated on the roots and lower stems of stressed Pinus species during the feeding activity of bark beetle. To determine the exact host response following inoculation, it is critical to challenge the host with a realistic amount of fungal inoculum. Thus, we designed a series of stepwise experiments using L. terebrantis colonized toothpicks which focused on the inoculum transfer from the toothpicks to excised Pinus taeda stem segments and living saplings, respectively, at different inoculum densities. The toothpicks served as a substrate for fungal growth and sporulation and the inoculation showed their utility in eliciting host's response to the pathogen. The inoculated fungus caused blue‐stain and sapwood occlusions in P. taeda stems and saplings, respectively. The volume of occluded, visually damaged sapwood increased by 1.96 cm³ per radial inoculation point on average. Fungal colonized toothpicks can be used as a suitable alternative to agar discs for studying bark beetles vectored fungi and their host interactions.     

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.     

Temporal evolution of collar lesions associated with ash dieback and the occurrence of Armillaria in Belgian forests

Ash dieback, caused by the fungus Hymenoscyphus fraxineus, has been observed in Europe for several years. In Belgium, the disease was first reported in 2010. Besides crown defoliation and dieback, collar lesions have sometimes been reported. To evaluate the prevalence and the progression of collar lesions and crown defoliation in ash dieback‐affected stands of various ages, a survey was conducted in 2013 and 2014 on 268 ash trees (Fraxinus excelsior) originating from 17 Walloon forest stands. The results showed that the proportion of trees with collar lesions greatly increased between June 2013 and September 2014 and that there appeared to be no significant link between a tree's diameter‐at‐breast height (DBH) and collar lesion occurrence. The mean percentage of defoliation increased in each forest stand across time, with observations conducted in September 2013 and 2014 showing a positive correlation with the mean percentage of trees with collar lesions. Molecular tests were carried out on 103 additional trees originating from 12 of the 17 stands to evaluate the occurrence of H. fraxineus and Armillaria spp. at the collar level. Most of the trees (98%) were infected by H. fraxineus. In contrast, only 41% of the samples were infected with Armillaria spp., most commonly A. gallica and A. cepistipes. This study discusses the role of Armillaria spp. and the rapid increase in the number of trees with collar lesions within the context of the evolution of ash dieback in Europe.     

Dothistroma spp. in Western Ukraine and Georgia

Dothistroma needle blight (DNB) is among the most serious foliar diseases affecting Pinus spp. globally. Infected needles were collected from potential host species in four locations in western Ukraine and in four locations in eastern Georgia during spring–summer 2015 to update the knowledge on pathogen distribution in these countries. Dothistroma spp. were detected using isolation, sequencing and species‐specific priming (SSPP) PCR. Two new hosts for Dothistroma spp. were recorded in western Ukraine: D. septosporum on Pinus nigra var. australica and D. pini on P. nigra var. mollet. D. septosporum was found on 15‐year‐old P. strobus in western Ukraine. New hosts for D. septosporum were recorded in Georgia on 5‐ to 10‐year‐old naturally regenerated P. sylvestris var. hamata and on 40‐ to 50‐year‐old P. ponderosa trees. D. pini was found for the first time in Georgia on 30‐ to 40‐year‐old P. nigra trees. The work confirmed the presence of both D. septosporum and D. pini in western Ukraine and Georgia, and demonstrated new hosts for both Dothistroma species.     

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.     

Ophiostomatoid fungi associated with root‐feeding bark beetles on Scots pine in Poland

Ophiostomatoid fungi are known to be associated with various species of bark beetles. However, information about fungal associates of root‐feeding bark beetles in Europe is still fragmentary. For this reason, the fungal associates of Hylastes ater, H. opacus and Hylurgus ligniperda on Pinus sylvestris were isolated and identified. A total of 743 fungal isolates were collected and separated into 10 morphological groups. Analyses of ITS rDNA and partial β‐tubulin gene sequences confirmed that these groups represented distinct species. The 10 species included a total of 13 associations between fungi and bark beetles that had not been recorded previously. All of the bark beetles examined were frequently associated with ophiostomatoid fungi. The fungal diversity and relative abundance of species were very similar in the three species of root‐feeding bark beetles. The most commonly encountered associates of these beetles were Grosmannia radiaticola, Leptographium lundbergii, L. procerum and L. truncatum. Insect infestation data furthermore suggest that Hylastes spp. and Hg. ligniperda are also important vectors of the fungal pathogen Sphaeropsis sapinea.     

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.     

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.     

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.     

Impact of Botryosphaeria,Diplodia and Neofusicoccum species on two Eucalyptus species and a hybrid: From pathogenicity to physiological performance

Species of Botryosphaeriaceae are associated with canker and dieback of Eucalyptus spp. worldwide, but little is known about their effect on the host physiology. The aim of this study was to evaluate the impact of Botryosphaeriaceae isolates from nine species in three genera (Botryosphaeria, Diplodia and Neofusicoccum), previously isolated from eucalypts, on three different Eucalyptus hosts (seedlings of E. nitens, cuttings of E. globulusand of E. globulus× E. cypellocarpa). An approach combining standard pathogenicity trials with evaluation of plant morpho‐physiological parameters was used. The size of the lesions produced revealed differences in fungal aggressiveness and host susceptibility. Isolates of Neofusicoccum kwambonambienseand Diplodia corticolawere the most aggressive, while Botryosphaeria dothidea and Diplodia seriataisolates were the least aggressive. In general, hybrid E. globulus× E. cypellocarpa plants developed smaller lesions, followed by E. nitens and E. globulus. Eucalyptus nitensplants showed minimal modifications on the morpho‐physiological profile when infected, although more severe symptoms and mortality were observed. This is probably due to a more variable genetic background of the plants. However, in general, no direct association between putative fungal aggressiveness and plant physiological disorders could be found. Results suggested that under optimal growth conditions plants manage to cope with pathogen attack and maintain their physiological performance.     

Pathogenicity of Thekopsora areolata from seed orchards in Finland on Prunus spp. and Picea abies

Pathogenicity of cherry‐spruce rust, Thekopsora areolata, was investigated by inoculations with aeciospores from seven Norway spruce, Picea abies, seed orchards that had suffered from successive severe rust epidemics in the 2000s in Finland. Detached leaves of Prunus spp. were inoculated in the laboratory using aeciospores from cones of various ages. In the greenhouse, live Prunus padus plants were inoculated, and possible autoecism of the rust was tested by inoculations of Picea abies seedlings. Thirty‐five spore sources from the seed orchards formed uredinia on Prunus spp. in the laboratory 2 weeks after incubation, but no telia developed. In the greenhouse, uredinia developed on live P. padus, but no rust symptoms, cankers or sporulation were detected on Picea abies. Thus, no evidence of autoecism was observed among the T. areolata populations and therefore all populations, suggesting all those tested from the Finnish seed orchards were heteroecious.     

Wood‐rotting basidiomycetes are a minor component of fungal communities associated with Acacia hybrid trees grown for sawlogs in South Vietnam

Acacia hybrid (Acacia mangium × Acacia auriculiformis) clones are widely planted in Vietnam with a total of approximately 400,000 ha to meet the demand for pulpwood, sawn timber and wood chip exports. Silvicultural techniques such as pruning and thinning have been applied to improve productivity and sawlog quality of Acacia hybrid plantations. However, those techniques may also create opportunities for wood decay fungi to enter the Acacia hybrid stems through wounds and cause stem defects that reduce sawlog quality and the value of the plantation. The presence of fungal decay agents in Acacia hybrid trees was examined in two Vietnamese plantations. In July 2011, just prior to a second thinning, discoloured wood samples were taken from a three‐year‐old Acacia hybrid plantation at Phan Truong Hai for the isolation of fungi. In July 2012, approximately 18 months after pruning and thinning treatments, discoloured wood samples were taken from a three‐year‐old Acacia hybrid plantation at Nghia Trung for the isolation of fungi. DNA sequencing of the rDNA ITS identified the isolates. In May 2015, approximately 4 years after thinning and fertilizer treatments, discoloured and decayed wood samples were taken from the above (7‐year‐old) Acacia hybrid plantation at Phan Truong Hai for fungal identification. DNA was extracted directly from discoloured and decayed wood samples and fungal rDNA ITS amplicons sequenced on a Roche 454 sequencer. The results showed that silvicultural treatments did not affect the fungal communities associated with discoloured and decayed wood of Acacia hybrid plantation at Phan Truong Hai. A total of 135 fungal species or OTUs (operational taxonomic units) were identified, including 82 members of Ascomycota and 52 Basidiomycota.