Somatic Incompatibility Tests Research Articles

First Report of the Armillaria Root Disease Pathogen, Armillaria solidipes, on Black Oak (Quercus velutina) in North Carolina, USA.

Armillaria root disease is among the largest causes of mortality and lost productivity of widely ranging horticultural, urban, and forest trees/shrubs in diverse boreal, temperate, and tropical regions around the world (Kim et al. 2022). Damage from Armillaria root disease will likely increase in response to changing climate and extreme weather because environmental stress can predispose host trees to Armillaria root disease (Murray and Leslie 2021; Kim et al. 2021). On August 14, 2021, a mature black oak (Quercus velutina) ca. 29 m in height and 64 cm DBH experienced a complete structural failure originating at the root plate, falling onto a major highway within the Pisgah National Forest near Brevard, North Carolina (35°16'50.876"N, 82°42'51.785"W, elevation 680 m) during sunny, calm conditions. No above-ground indications of predisposing disturbance, injury, or insect/disease activity were observed. Advanced wood decay, however, was present in many structural roots of the exposed root plate; abundant rhizomorphs attached to the surfaces of most roots were noted (Supplemental Figure 1). One large primary root (ca. 23 cm in diameter) exhibited clear signs of Armillaria root disease at the point of failure as evidenced by an extensive decay column, abundant black rhizomorphs on the root surface, and white mycelial fans (a sign of pathogenicity) within the root itself (Supplemental Figure 1). Rhizomorph samples were established in culture, two Armillaria isolates (PNF#001R-1 and PNF#001R-2) were examined, and pairing tests showed that both isolates belonged to the same genet (PNF#001R-1 = PNF#001R-2). Based on translation elongation factor 1α (tef1) sequences, both isolates (one genet) were identified as A. solidipes (GenBank accession no. OP823701), showing 98% similarity with A. solidipes tef1 sequences (e.g., MH879015) in GenBank. Also, nine replications of somatic incompatibility tests of the isolate with five North American Armillaria spp. [A. solidipes, A. mellea, A. gallica, A. mexicana, and Desarmillaria caespitosa (= North American A. tabescens)] showed 67% compatibility with A. solidipes, compared with 0-22% for A. mellea, D. caespitosa, A. gallica, and A. mexicana. All test species but A. mexicana are reported to occur in the eastern and/or southern USA, while A. mexicana occurs in a similar environment in Mexico (Elías-Román et al. 2018). To our knowledge, this is the first confirmed report of A. solidipes in North Carolina, USA, where it was causing disease on black oak, and this is the most southerly confirmed case of A. solidipes in eastern North America. Although Armillaria inoculation tests are impractical, black oak was previously reported as a host of A. solidipes in Massachusetts, USA (Brazee and Wick 2009). Armillaria solidipes is considered as the most important Armillaria root disease pathogen of conifer forests in western USA (Lockman and Kearns 2016), but it has been suggested that A. solidipes can thrive in northern hardwood forests that reside near conifer forests (Brazee and Wick 2009). Climate change is predicted to increase damage caused by A. solidipes on conifers in the western USA (Kim et al. 2021); however, it is undetermined if the occurrence of A. solidipes-caused disease in North Carolina is related to climate change or how climate change could influence this disease across the region. More surveys are needed to assess the impact of Armillaria root disease on health of mixed forests in the eastern USA.

Successful cultivation of black morel, Morchella sp. in Japan.

True morels (Morchella spp.) are economically important edible fungi cultivated mainly in China. Japan is one of the top importers of morels, but there are no reports on the distribution of major cultivated species. To investigate the possibility of black morel cultivation in Japan, phylogenetic analysis, mating type analysis, and field cultivation tests were conducted using domestically collected strains. A total of 172 strains were isolated from the spores of wild ascomata collected from 15 locations. Mating type analysis for 118 strains revealed 28 strains had only MAT1-1-1, 40 strains had only MAT1-2-1, and 48 strains had both MAT genes. Seven strains were inoculated in March 2020 at field cultivation test site. Mycelial growth and conidial layer formation were observed within a month. Ascomata were observed on April 2021 for one of the tested strains. Phylogenetic analysis revealed that both the observed ascomata and fruited strains were Morchella sp. Mel-21, which is one of the cultivated species in China. Moreover, no antagonism was observed in the somatic incompatibility test between strains isolated from observed ascomata and spawn strain. These results suggest that the ascomata originated in the inoculated spawn, a finding that will contribute to commercial morel cultivation in Japan.

First report of Armillaria cepistipes causing root disease on Populus trichocarpa (black cottonwood) in Oregon, USA.

Populus trichocarpa Torr. and Gray (black cottonwood) is an economically and ecologically important tree species native to western North America. It serves as a model tree species in biology and genetics due to its relatively small genome size, rapid growth, and early reproductive maturity (Jansson and Douglas 2007). Black cottonwood is susceptible to root rot caused by at least one species of Armillaria (Raabe 1962), a globally distributed genus that exhibits diverse ecological behaviors (Klopfenstein et al. 2017) and infects numerous woody plant species (Raabe 1962). However, several Armillaria spp. have been isolated from Populus spp. in North America (Mallet 1990), and the most recent report of Armillaria on P. trichocarpa used the now ambiguated name A. mellea (Vahl.) Quel. (see Raabe 1962). In April 2016, mycelial fans and rhizomorphs of an unknown Armillaria species (isolate WV-ARR-3) were collected from P. trichocarpa in a riparian hardwood stand ca. 5.5 km east of Springfield, Oregon, USA (44°3'21.133"N, 122°49'39.935"W). The host was dominant in the canopy, large in diameter (ca. 90-cm dbh) relative to neighboring trees, and exhibited minimal crown dieback (ca. < 5%). A mycelial fan was observed destroying living cambium beneath the inner bark, indicating pathogenicity. The isolate was cultured on malt extract medium (3% malt extract, 3% dextrose, 1% peptone, and 1.5 % agar) and identified as A.cepistipes on the basis of somatic pairing tests and translation elongation factor 1α (tef1) sequences (GenBank Accession No. MK172784). DNA extraction, PCR, and tef1 sequencing followed protocols of Elías-Román et al. (2018). From nine replications of somatic incompatibility tests (18 tester isolates representing six North American Armillaria spp.), the isolate showed high intraspecific compatibility (colorless antagonism) with three A. cepistipes tester isolates (78%), but low compatibility with the other Armillaria spp. (0 - 33%) that occur in the region. Isolate WV-ARR-3 yielded tef1 sequences with a 99% identity to A. cepistipes (GenBank Accession Nos. JF313115 and JF313121). A second isolate (WV-ARR-1; GenBank Accession No. MK172783) with a nearly identical sequence was collected from a maturing P. trichocarpa in a riparian stand ca. 8 km northeast of Monroe, Oregon (44°21'47.57"N, 123°13'14.415"W) along the Willamette River, downstream from the McKenzie river tributary where WV-ARR-3 was collected. Armillaria cepistipes has been reported on Alnus rubra (red alder) in Washington, USA (Banik et al. 1996) and on broad-leaved trees in British Columbia, Canada (Allen et al. 1996). It is generally considered to be a weak pathogen on broad-leaved trees in the Pacific Northwest, but it is also associated with pathogenicity on both coniferous and deciduous trees in Europe (e.g., Lygis et al. 2005). However, a recent phylogenetic study suggested that North American A. cepistipes is phylogenetically distinct from Eurasian A. cepistipes (Klopfenstein et al. 2017), butadditional studies are needed to determine the formal taxonomic status of North American A. cepistipes. To our knowledge, A. cepistipes has not been previously confirmed on P. trichocarpa in the U.S.A. or formally reported as a pathogen of any Populus species in North America. Continued studies are needed to determine the distribution, host range, and ecological role of A. cepistipes in riparian forests of the Pacific Northwest, while monitoring its populations under changing climates.

ANALYSIS OF GENOTYPIC DIVERSITY PROVIDES A FIRST GLIMPSE ON THE PATTERNS OF SPREAD OF THE WOOD DECAY FUNGUS PERENNIPORIA FRAXINEA IN AN URBAN PARK IN NORTHERN ITALY

Perenniporia fraxinea is a fungal pathogen causing wood decay in roots and bole of a wide variety of broadleaf tree species. Despite its ecological importance, little is know about the infection biology of this fungus and in particular of its ability to infect trees through the mycelia growth through root contacts. To clarify its spreading mechanisms, a genetic analysis of 20 P. fraxinea isolates obtained from basidiomata collected from closely located Robinia pseudoacacia and Quercus robur trees in the Vernavola Urban Park (Pavia, Italy) and in surrounding areas was performed. Random Amplified Microsatellites (RAMs) fingerprinting was conducted allowing to distinguish 19 different haplotypes. High intrapopulation diversity was confirmed by somatic incompatibility tests (SITs), which were performed by dual- culturing isolates in vitro in all possible combinations, resulting in detection of 16 compatibility groups. These results, together with Non-metric MultiDimensional Scaling (NMDS) analysis on genetic data, suggest that spread through root contacts is unlikely for P. fraxinea. In addition, a significant negative correlation between spatial distribution and kinship coefficients was observed in isolates from the Vernavola Urban Park, suggesting a limited dispersal potential of P. fraxinea basidiospores. This report provides a first glimpse of the primary mechanisms of spread of P. fraxinea.

First Report of the Armillaria Root Disease Pathogen, Armillaria sinapina, on Subalpine Fir (Abies lasiocarpa) and Quaking Aspen (Populus tremuloides) in Colorado

In July 2014, mycelial fans (isolates CO104F, CO106F, and CO108F) of Armillaria sp. were collected from forest trees in Colorado. These isolates were all identified as A. sinapina based on a somatic pairing test against 18 tester isolates representing six North American Armillaria spp. and nucleotide sequences of the translation elongation factor 1alpha (tef-1alpha; GenBank Accession Nos. KT327065, KT327066, and KT327067). Based on nine replications of somatic incompatibility tests, all isolates showed high intraspecific compatibility (colorless antagonism) with three A. sinapina tester isolates (average 77%), but low compatibility with closely related Armillaria spp. (average 0 to 26%).

KARAKTERISTIK MORFOLOGI Ganoderma steyaertanum YANG MENYERANG KEBUN BENIH Acacia mangium DAN Acacia auriculiformis DI WONOGIRI, JAWA TENGAH

Acacia mangium and Acacia auriculiformis seed orchards on the island of Java are associated with a different species of Ganoderma. The importance of G. steyaertanum as a pathogen of forest trees has not been previously highlighted. The aim of this study is to look at the characteristics of the G. steyaertanum which attacks two Acacia seed orchads at Wonogiri Central Java. Activities undertaken are to identify the signs of the G. steyaertanum both in the field and in the laboratory. Characterization of fungi is conventionally done by observing the morphology of signs, such as the shape and color of the fruit body, shape and color of mycelium. Morphological characteristic applied in this study has proved to be effective to identify the pathogen. G. steyaertanum was isolated from fruitbodies of affected trees and pathogenicity tests confirmed Koch’s postulates. Somatic incompatibility tests demonstrated high genetic variability of the pathogen.

Ganoderma steyaertanum as a root‐rot pathogen of forest trees

SummaryTwo Acacia seed orchards, one each of A. mangium and A. auriculiformis, in Central Java have suffered high losses from root rot associated with a species of Ganoderma that is distinct from G. philippii, which causes extensive losses in A. mangium plantations in Sumatra and Kalimantan. Ganoderma steyaertanum was consistently isolated from roots of affected trees in the Javanese orchards and pathogenicity was confirmed by satisfying Koch's postulates. The mode of spread of the pathogen was examined by analysing the distribution of tree deaths in the A. mangium seed orchard and conducting somatic incompatibility tests among G. steyaertanum isolates from diseased trees. Spatial analyses indicate that, after an initially random disease distribution, the pattern of tree deaths was clustered. This is consistent with spore colonisation of thinned stumps followed by vegetative spread through root‐to‐root contact, as has been observed for other root pathogens such as Heterobasidion annosum. Somatic incompatibility tests demonstrated high genetic variability of the pathogen with root‐to‐root spread also confirmed.

UJI INKOMPATIBILITAS SOMATIK DAN PERTUMBUHAN JAMUR GANODERMA DARI KEBUN BENIH GENERASI PERTAMA Acacia auriculiformis DI WONOGIRI, JAWA TENGAH

Seed orchard A. auriculiformis F1 at Wonogiri, Central Java has attacked by ganoderma caused root rot. To determine the genetic variation, the pattern of spread of ganoderma necessary to somatic incompatibility test and measure the growth. This study used 8 isolates of the fungus ganoderma. Fungi were grown on PDA (Potato dextrose agar), made parental isolates and paired with each other. The results showed that all pairings indicated incompatible reactionsm except self pairings. All self-pairings showed compatible reactions that indicated by miycelia merged on PDA forming a single colony. Incompatible reaction zone is characterized by sparse zone, demarcation line and pigmentation. The results of somatic incompatibility test, ganoderma have different genotypes or it is not a single colony. The result indicates that the distribution of root rot in Seed orchard A. auriculiformis F1 at Wonogiri, Central Java not only occured by root to root contact.

UJI INKOMPATIBILITAS SOMATIK DAN PERTUMBUHAN JAMUR GANODERMA DARI KEBUN BENIH GENERASI PERTAMA Acacia auriculiformis DI WONOGIRI, JAWA TENGAH

Seed orchard A. auriculiformis F1 at Wonogiri, Central Java has attacked by ganoderma caused root rot. To determine the genetic variation, the pattern of spread of ganoderma necessary to somatic incompatibility test and measure the growth. This study used 8 isolates of the fungus ganoderma. Fungi were grown on PDA (Potato dextrose agar), made parental isolates and paired with each other. The results showed that all pairings indicated incompatible reactionsm except self pairings. All self-pairings showed compatible reactions that indicated by miycelia merged on PDA forming a single colony. Incompatible reaction zone is characterized by sparse zone, demarcation line and pigmentation. The results of somatic incompatibility test, ganoderma have different genotypes or it is not a single colony. The result indicates that the distribution of root rot in Seed orchard A. auriculiformis F1 at Wonogiri, Central Java not only occured by root to root contact.

Development of SCAR Markers to Determine the Mating Types of Lepista nuda Protoplast Monokaryons

Lepista nuda (Bull. ex Fr.) Cooke belongs to Tricholomataceae and is an edible fungus with both economic and medical value. Mycelia were isolated from the fruiting bodies of L. nuda and were used to prepare the protoplast monokaryons. One hundred and fifteen monokaryons were obtained and their mating types were determined using somatic incompatibility tests. Protoplast monokaryons segregated into either the A1B1 or the A2B2 mating types. Inter-simple sequence repeats and sequence-related amplified polymorphism fingerprinting were used to analyse the mating types of these protoplast monokaryons and 16 sequence-characterised amplified region primers were developed to efficiently differentiate between the monokaryon mating types. Multiplex PCR analyses were also established. The data presented here outline a method for the precise and rapid identification of protoplast monokaryon mating types, which has the promise to shorten the period required for conventional crossbreeding.

First Report of the Armillaria Root Disease Pathogen, Armillaria gallica, on Douglas-fir (Pseudotsuga menziesii) in Arizona

In August 2010, a mycelial fan (isolate AZ32F) of Armillaria sp. was collected from the root collar of a living Douglas-fir tree on the Mogollon Rim within the Coconino National Forest (approximate location 34°25'31.26″N, 111°20'41.04″W, elevation 2,293 m) in central Arizona. Mycelial fans under the bark of living trees are a sign of pathogenicity, and symptoms of the diseased tree included resinosis, sloughing bark, and thinning crown. The infected tree was located on a south-facing slope with approximately 30% tree cover, dominated by ponderosa pine (Pinus ponderosa), with lesser components of Douglas-fir and Gambel oak (Quercus gambelii). Based on three replications of somatic incompatibility tests against 24 tester isolates representing seven North American Armillaria spp., isolate AZ32F showed 100% intraspecific compatibility (colorless antagonism) with all four A. gallica isolates, 22% compatibility with A. calvescens, and 0% compatibility with the remaining Armillaria spp. Based on GenBank BLASTn of isolate AZ32F sequences, the partial LSU-IGS1 (GenBank Accession No. KF186682) showed 99 to 100% similarity to A. gallica and two other related Armillaria spp. with 99 to 100% coverage, and translation elongation factor-1 alpha (tef-1α) sequences (KC525954) showed 96% similarity to A. gallica (JF895844) with 100% coverage. Thus, isolate AZ32F was identified as A. gallica, based on somatic incompatibility tests and DNA sequences (partial LSU-IGS1 and tef-1α). Although the isolate is identified as A. gallica with similarities to other North American isolates, evidence is mounting that currently recognized A. gallica likely represents a species complex that comprises multiple phylogenetic species (4). Previous surveys in Arizona have noted A. mellea and A. solidipes (as A. ostoyae) (3), but A. gallica has never been previously confirmed in this state. Within North America, A. gallica is commonly reported east of the Rocky Mountains and in West Coast states of the United States, where it infects hardwoods and conifers including Douglas-fir (1,2). Its ecological behavior ranges from saprophyte to weak/aggressive pathogen (1,2). Because damage by A. gallica appears to increase on hosts predisposed by stress (1), further surveys are needed to document its distribution, frequency, and ecological behavior in the southwestern United States, where climate change will likely cause tree stress due to maladaptation. Continued surveys for Armillaria spp. will better determine their potential threat within the geologically and ecologically unique Mogollon Rim of Arizona.

Occurrence of Ganoderma adspersum on Pinus pinea

During a survey in a pine stand in Rome, six basidiomes of Ganoderma adspersum were collected from declining 50-60 year-old Pinus pinea . Species identification was accomplished through observation of macroscopic and microscopic morphological traits. Furthermore, ITS sequences of the collected specimens, 28 samples collected elsewhere, one isolate from the German collection of Microorganisms and Cell Cultures (DSMZ), and 21 sequences from GenBank were analyzed using MP and UPGMA. Both analyses showed that isolates fell into four well-delineated clades, namely G. adspersum , G. applanatum , G. resinaceum and G. lucidum . All the isolates collected from P. pinea were confirmed as G. adspersum . Previously, G. adspersum had been reported on hardwood trees and infrequently on a few conifers. Only sporocarps identified on the basis of morphological characters as G. applanatum have sporadically been recorded on P. pinea ; there is no previous record of G. adspersum on this host. Somatic incompatibility tests showed that infections, probably via basidiospores, and symptoms apparently associated with wood colonization by G. adspersum , worsened during the 3-year course of the study.

Comparative analysis on the diversity of Auricularia auricula-judae by physiological characteristics, somatic incompatibility and TRAP fingerprinting

Phenotypic traits (physiological characteristics and somatic incompatibility) and genotypic traits (target region amplification polymorphism TRAP) were used to study the diversity of 32 main cultivars of Auricularia auricula-judae in China. Twenty seven important and stable physiological indexes were evaluated; somatic incompatibility test (SIT) reaction was described from three aspects: type, pigment, and intensity; 16 pairs of TRAP primer combinations produced 535 unambiguous and reproducible DNA fragments, of these 524 (97.9%) were polymorphic. Dendrograms were constructed by Unweighted Pair-group Method with Arithmetic Averages (UPGMA) method, and the principal coordinate analysis (PCO) of the three methods (physiological characteristics, SIT intensity and TRAP) exhibited similar clustered patterns, revealing that all the tested strains could be divided into three distinct groups, each of which was correlated with different geographical regions. Most strains originated from the same area were with a narrow genetic basis and could possibly be domesticated from the local wild-type strains, some strains were suspected to be synonymous. The grouping information obtained in the present work provides significant information for further genetic improvement of A. auricula-judae.

The distribution of closely related large genets of Heterobasidion parviporum in a Todo fir (Abies sachalinensis) stand in Hokkaido, Japan

Summary The distribution of genets of a root and butt rot pathogen Heterobasidion parviporum was studied in a 68-year-old Abies sachalinensis plantation in Japan. A total of 33 pure cultures of H. parviporum were isolated from diseased Todo fir stumps after clear-felling. Individual genets of the fungus were identified by three different methods: somatic incompatibility test, random amplified polymorphic DNA, and DNA microsatellite analyses. The combined results of the three analyses identified at least eight genets within a 60 × 100 m plot. One genet, consisting of only one isolate, was genetically differentiated from the other genets by all three methods; the other 32 isolates, however, were grouped with minor discrepancies, into seven genetically close genets. The genetic differentiation was lower than that recorded previously in Europe. A single genet infected 1–15 trees in the plot. The longest distance of the two most isolated trees of a single genet was 51 m. The close genetic relationship between genets and their large sizes suggest that basidiospores from restricted sources (just a few fruiting bodies) infected the site before establishment of the present Todo fir stand and that the disease has spread mainly by vegetative growth of the mycelia through root contact. Absence of fruiting bodies of the fungus in the study plot also suggests the greater importance of vegetative growth than spore dispersal for the spread of this fungus.

UJI SOMATIK INKOMPATIBILITAS Ganoderma philippii UNTUK MENGETAHUI POLA SEBARAN PENYAKIT BUSUK AKAR PADA TANAMAN Acacia mangium

Ganoderma philipii is a fungi causing root rot disease that have spread widely in Acacia mangium plantation. Study on mode of disease spreading is necessary to develop ways of controlling the spread of the disease. Somatic incompatibility test was carried out using fungal cultures that has been identified as G. philippii from two sites of different rotation. Each location showing different type of incompatibility. Compatible reaction was observed at Logas of first rotation indicating that the isolates are not genotypically distinct and may come from closely related clones. At this site the spread of root rot occurred by root to root contact. Incompatible reaction was observed at Deras of second rotation indicating that G. philipii isolate are genotypically distinct individuals and of different clones, eventhough they share same morphological characters. Genetic diversity found in G. philippii suggested that sexual reproduction of basidiocarps in an important factor and is strongly implicated as one of the main modes of dispersal as well as root to root contact.

Distribution of genets of Cylindrobasidium argenteum in a river valley forest as determined by somatic incompatibility, and the significance of basidiospores for its dispersal

Cylindrobasidium argenteum, a causal pathogen of white stem blight of a variety of broadleaf trees and shrubs, is thought to be transmitted to healthy trees through contact with infected branches of neighboring trees. To clarify whether basidiospores of C. argenteum are involved in the dispersal and infection process, we investigated distribution of genets of the fungus in a river valley forest using a somatic incompatibility test. Thirty-eight isolates were collected from diseased trees along two census lines at the bottom of Kikuchi River valley in southern Japan. In addition, 50 isolates were collected from four experimental plots on the slope of the valley. When the isolates from the valley bottom were paired in all combinations, a narrow clear zone line appeared in 701 out of 703 pairings, indicating that the material included 36 different genotypes, and suggesting that airborne basidiospores play an important role for dispersal of C. argenteum. In contrast, vegetative spread proved to be more frequent on the slope of the valley. On lower parts of such slopes, opportunities for healthy trees to meet infectious agents may increase because infected broken branches frequently move downwards on the slope. Based on these results, we suggest that C. argenteum adopts following dissemination strategies: (1) it spreads chiefly by basidiospores and infects trees, and (2) after colonizing stems or branches and producing an extensive mycelial mat, secondary infections of adjacent trees occur by contact with infectious agents such as diseased branches.

Impact of forest fire on occurrence of Heterobasidion annosum s.s. root rot and other wood-inhabiting fungi in roots of Pinus mugo

Summary Forest fi res result in disturbance of wood- and soil-inhabiting fungal communities and also have an impact on the persistence of rot-causing fungi that are present in infected root systems. The aims of the present study were to (1) investigate the occurrence of the root pathogens Heterobasidion and Armillaria in root disease centres on burned and non-burned sites in Pinus mugo forest and (2) study the impact of forest fi re on the community structure of other wood-inhabiting fungi colonizing tree roots. A total of 18 root disease centres were investigated in 120-year-old plantations on sandy dunes in the Curonian Spit of western Lithuania, 14 of which were located on burned and 4 on non-burned sites. A total of 484 fungal isolates representing 16 taxa were obtained from 270 P. mugo root systems sampled at 10 – 30 cm in the soil. The two most frequently isolated fungi were Trichoderma viride and Penicillium sp. found in all disease centres and 96.7 per cent and 39.6 per cent of root systems, respectively. Heterobasidion annosum s.s. was the most commonly isolated basidiomycete found in 15 disease centres. Overall, it was isolated from 19.3 per cent of the root systems. The fungus gave growth from 42.5 per cent of root systems on non-burned and from 15.2 per cent of root systems on burned sites, and the difference was statistically signifi cant (chi-squared test; P < 0.001); thus forest fi re in disease centres seemingly had reduced occurrence of the pathogen. Nevertheless, H. annosum s.s. persisted on 11 burned sites, and on four of those fresh sporocarps were observed. Armillaria borealis was less commonly isolated (3.0 – 5.0 per cent of the root systems). Somatic incompatibility tests with 35 strains of H. annosum s.s., isolated from six (two from burned and four from non-burned) disease centres, showed genetically diverse populations of the fungus even within small and compact areas (0.01 – 0.07 ha). Fungal communities and species richness were moderately similar in burned and non-burned sites. Results of the study suggest that forest fi res on sandy soil can reduce the occurrence of H. annosum s.s. in disease centres in P. mugo plantations.

Distribution of Genets of Perenniporia subacida in Stands of Chamaecyparis obtusa (Japanese Cypress) Determined by AFLP Fingerprints and Somatic Incompatibility.

The clonal structure of Perenniporia subacida, a wood-rotting basidiomycete, was studied in a 30-year-old stand (site A; 1 ha) and a 43-year-old stand (site B; 0.5 ha) of Chamaecyparis obtusa in northern Kagawa Prefecture, Japan. A total of 110 isolates from decayed trees, stumps, wooden fences, logs, and mycelial mats were analyzed by amplified fragment length polymorphism (AFLP) and somatic incompatibility test (SIT). The results of AFLP, which agreed with those of SIT, revealed that the P. subacida population consisted of at least 17 genets at site A and 6 genets at site B. Individual genets were found in 1 to 9 trees at site A and in 1 to 7 trees at site B. One particular genet had spread over a distance of 70 m. Root contacts were observed among roots of decaying stumps and living trees at both sites. White mycelial mats were often found where roots made contact between stumps and neighboring trees. These results suggest that P. subacida spreads vegetatively over significant distances through root contacts.

Distinguishing homokaryons and heterokaryons in Phellinus sulphurascens using pairing tests and ITS polymorphisms

Phellinus sulphurascens Pilát causes laminated root rot of coniferous species in both western North America (WNA) and Asia. Accurate somatic incompatibility tests for mapping population structures have been difficult to conduct for P. sulphurascens because no single, unambiguous criterion has allowed differentiation of homokaryotic and heterokaryotic isolates. In a population study of P. sulphurascens in WNA, two types of ITS sequences were found in the single spore and vegetative isolates. All single spore isolates (SSIs) had either ITS type-1 or type-2 whereas some vegetative isolates had both ITS types. The segregation pattern for inheritance of ITS, which we observed in SSIs from eight basidiocarps, suggested that each ITS type occurred in a different nucleus and that each basidiospore inherited only one ITS type. In four SSIs from Russia and eight heterokaryotic isolates from Japan, nine different ITS types, referred to as type-3 to -11, were detected. A variety of pairing tests conducted between known Asian and WNA homokaryon and heterokaryon isolates did not always give consistent results with respect to fungal mat morphologies and formation of demarcation lines. However, the ITS types that occurred after pairing tests did follow consistent patterns. Thus, using ITS polymorphisms and pairing tests between Asian tester isolates and 49 vegetative isolates from WNA, we were able to accurately distinguish between homokaryotic and heterokaryotic isolates.

Genetic population structure of three Armillaria species at the landscape scale: a case study from Swiss Pinus mugo forests

Armillaria species are plant pathogens that cause Armillaria root rot and are known to cause mortality of mountain pines ( Pinus mugo) in the Swiss National Park in the Central Alps. The identity of isolates and the spatially explicit population structure of the Armillaria species were investigated in a 3.3 km 2 study area in the Swiss National Park. In total, 242 Armillaria isolates, 205 from wood samples and 37 from epiphytic rhizomorphs, were collected. Species were identified using haploid–diploid pairings and genets were determined using intraspecific somatic incompatibility tests. The population structure differed markedly among the Armillaria species. A. cepistipes and A. borealis mainly occurred as genets of small spatial extent (mean 0.2 ha, and 0.6 ha), whereas A. ostoyae formed significantly larger genets (mean 6.8 ha). The largest A. ostoyae genet extended over approx. 37 ha. Several disease centres associated with Heterobasidion annosum were found to be embedded within large Armillaria genets. The extension of large A. ostoyae genets suggests that forests that occupy the study area have developed in the presence of these Armillaria genets. The finding of large Armillaria genets supports the assumption that large genets occur in areas with cold climate and little precipitation.