Somatic Incompatibility Tests Research Articles

Incidence, host relations and population structure of Armillaria ostoyae in Colorado campgrounds

Armillaria root disease is common and widely distributed in campgrounds of southwestern Colorado. Armillaria ostoyae spreads clonally underground and kills and decays tree roots, causing mortality or predisposing the trees to windthrow. We intensively surveyed and mapped genets (clones) of the pathogen in two campgrounds on the San Juan National Forest and one on Grand Mesa National Forest (GMNF). Three additional campgrounds on the GMNF were also surveyed. Infection (based on mycelium under the bark on or near the root collar) of all sampled live trees was 10.5% (range 7.5–15.0) inside campgrounds and 12.7% (3.3–25.9) immediately outside campgrounds, suggesting that campground construction and management practices have not exacerbated the disease. Dominant trees had significantly greater incidence of infection than trees in other crown classes. Isolates of A. ostoyae were obtained from 379 trees, and genets were identified by somatic incompatibility tests and variation in DNA microsatellite markers. The pathogen occurred as one large genet in the spruce-fir campground and several large genets in each of the mixed conifer campgrounds. Based on the size and distribution of the genets, the campgrounds appeared to be almost completely colonized by mosaics of centuries old genets, and little expansion of genet territories (or disease centers) appeared possible without loss of territory by another genet. The diseased trees in the campgrounds were randomly distributed rather than being organized into discrete disease centers. Live subalpine fir had significantly lower incidence of infection (7.0%) than Engelmann spruce, blue spruce and Douglas-fir (12.0, 12.3 and 15.7%, respectively), but evidence suggests a higher rate of mortality in subalpine fir. Crown thinning and dieback were useful symptoms for detection, but basal resinosis was the most efficient symptom indicating infection. Use of a combination of aboveground symptoms to select trees for more intensive examination is the most efficient approach to detection of infected trees. The disease poses difficult obstacles to long-term management of safe vegetation in developed sites.

Planting Betula pendula on pine sites infested by Heterobasidion annosum: disease transfer, silvicultural evaluation, and community of wood-inhabiting fungi

Persistence of the root rot pathogen Heterobasidion annosum (Fr.) Bref. s.s. on infested areas and its transfer to a forest regeneration was studied in three forest sites in eastern Lithuania. The sites represented H. annosum disease centres in Pinus sylvestris L. stands, which were clear-felled and replanted with Betula pendula Roth 25 years prior to our study. Fungal isolation from trees and stumps on each site was performed on both replanted B. pendula and surrounding P. sylvestris from the previous generation. Low productivity of B. pendula stands (45.0–76.1 m3·ha–1), high mortality rates, and comparatively low vigor of trees (measured as crown densities) indicated a strong impact of root rot. Based on somatic incompatibility tests, we detected large spreading areas of clones of H. annosum (up to 48 m across) and old (35- to 40-year-old) clonal individuals. Territorial clones covered areas that encompassed both previous stands of P. sylvestris and current stands of B. pendula. Our results showed that H. annosum is able to persist in root systems of diseased trees for decades and readily attack birch replanted on infested sites. In addition, a total of 83 fungal species (out of 398 isolates) was found as a result of sampling 508 B. pendula, 49 P. sylvestris, 21 Juniperus communis L., and 1 Salix cinerea L. trees.

Analysis of genetic variation in Ganoderma Lucidum after space flight

A modified CTAB method was used in the extraction of total cellular DNA of Ganoderma lucidum. Four strains Cx, Ch, C3 and C4, and their counterparts, four space flown strains Sx, Xh, S3 and S4, were analysed by amplified fragment length polymorphism (AFLP) with several primer combinations. Polymorphic bands were detected between Sx and Cx, S3 and C3, respectively. Somatic incompatibility tests further confirmed their heterogeneity. However, no disparity between Sh and Ch, S4 and C4 was detectable. The results suggest that spaceflight may be used to accelerate breeding of Ganoderma lucidum strains for commercial cultivation.

European Pines May Be Simultaneously Infected by More Than One Species of Heterobasidion.

Heterobasidion annosum (Fr.:Fr.) Bref. sensu lato, one of the most damaging root and butt rot agents on conifers, was recently segregated into three species in Europe based on morphology, intersterility grouping (ISGs), and host preferences (3). These species include: H. annosum (Fr.) Bref. sensu stricto (ISG P) on Pinus, other conifers and some hardwoods; H. parviporum Niemelä & Korhonen (ISG S), primarily on Picea; and H. abietinum Niemelä & Korhonen (ISG F) on Abies. In the summer of 1998, a Swiss stone pine (Pinus cembra L.), growing at 1,900 m in a mixed spruce (Picea) and larch (Larix) forest in the Aosta Valley (northwest Italian Alps), was found infected by H. parviporum and H. annosum sensu stricto. The pine (approximately 14 m tall and at least 75 years old) was without crown symptoms, but the stem, stump, and all the main roots showed internal decay. Disks, 3 to 4 cm thick, were cut consecutively from the roots, stump, and stem, incubated, and examined for conidiophore production. After 8 days, 63 isolates were obtained from all disks taken from the stump and roots, and from disks taken up to 4 m above the collar in the stem. Isolates were identified by polymerase chain reaction (PCR) of mitochondrial and nuclear markers (2) and by sexual compatibility with testers of each European Heterobasidion spp. The stem and one root were colonized by H. parviporum while the other roots and most of the stump was colonized by H. annosum sensu stricto. Somatic incompatibility tests among conspecific isolates suggested that there was only one genet of each species. The coexistence of different Heterobasidion spp. (ISGs) in the same tree has been reported only in Pinus ponderosa Dougl. ex Laws. in California (1) and in Picea abies (L.) Karst. in Europe (4). To our knowledge, this is the first report of H. annosum sensu stricto on P. cembra and of a European pine to be simultaneously infected by more than one species of Heterobasidion.

Population structure of the endangered wood decay fungus Phellinus nigrolimitatus (Basidiomycota)

The population structure of five Fennoscandian geographic populations of the endangered wood-decay fungus Phellinus nigrolimitatus (Romell) Bourdot et Galzin was examined by analyses of nuclear ribosomal DNA (nrDNA) spacer sequences (ITS and IGS1) and a partial sequence of the elongation factor 1α gene (efa). A high level of sequence variation was observed in ITS and IGS1, suggesting restrictions in nrDNA homogenization in this taxon. Six polymerase chain reaction – restriction fragment length polymorphism (PCR-RFLP) markers, five located in nrDNA and one in efa, suggest that the geographic populations are genetically very similar, presumably owing to recent gene flow. However, linkage disequilibria were obtained in 50% of the cases in tests between the five nrDNA PCR-RFLP markers. The calculated FST values from the linked nrDNA markers and the unlinked efa marker were congruent, ranging from 0.006 to 0.042. In one geographic population, the efa locus showed significant deviation from Hardy– Weinberg expectations. Somatic incompatibility tests demonstrated that isolates derived from different basidiocarps and different logs belonged to different genets. In a microscale study including three logs, the independent assays of PCR-RFLP analysis and somatic incompatibility tests distinguished 10 genets. Life history traits and conservation status of P. nigrolimitatus are discussed in light of the results.Key words: Phellinus nigrolimitatus, population structure, somatic incompatibility, PCR-RFLP, nrDNA.

The population structure ofArmillaria ostoyaeandArmillaria sinapinain the central interior of British Columbia

The population structures of Armillaria ostoyae (Romagn.) Herink and Armillaria sinapina Bérubé & Dessureault were investigated at a study site near Williams Lake in the central interior of British Columbia. One hundred and twenty eight fungal isolates were examined from nine infection centers and individual genets were delineated using random amplified polymorphic DNA (RAPD) analysis and somatic incompatibility tests. Six A. ostoyae genets ranging in size from 0.70 to >15 ha were detected. The population structure of A. ostoyae was consistent with a clonal reproductive strategy, and infection centers were occupied by single A. ostoyae genets or ramets thereof. Eighteen relatively small A. sinapina genets were detected, with infection centers being occupied by multiple genets. Armillaria sinapina appears to be more pathogenic to coniferous hosts than previously reported in the region. Armillaria ostoyae appears to initiate new infections of available substrate via airborne basidiospores at a lower frequency than A. sinapina. However once established, A. ostoyae can spread quite aggressively and capture significant amounts of secondary resources, while A. sinapina is unable to do so. The results of somatic incompatibility tests used to differentiate genets corresponded with the results of RAPD analysis, with only one minor discrepancy.Key words: Armillaria, RAPD, population structure, genet, ramet, clone.

Impact of liming on the abundance and vigor of Armillaria rhizomorphs in Allegheny hardwoods stands

Abundance of rhizomorphs of Armillaria was characterized in 1995-1996 in 32 plots located in sugar maple (Acer saccharum Marsh.) stands in the Susquehannock State Forest (Pennsylvania, U.S.A.). All of the plots were thinned, and half of the plots were limed in 1985 when the plots were established. Frequency and abundance of Armillaria rhizomorphs in soil samples, on dead wood food bases (stumps, snags, fallen logs), and on the root collar of living sugar maples were determined in each plot. Rhizomorph vigor was evaluated by measuring their ability to colonize fresh striped maple (Acer pennsylvanicum L.) stem sections in the soil, or potato tubers in the laboratory. Isolates of Armillaria were obtained from rhizomorphs in the soil samples and species were determined by somatic incompatibility tests. Armillaria calvescens Bérubé & Dessureault was the major species present, representing about 66% of the isolates. Armillaria gemina Bérubé & Dessureault and Armillaria mellea (Vahl:Fr.) Kummer were also identified in the plots. Frequency of rhizomorphs in the soil, on food bases, abundance of rhizomorphs on root collars, as well as the proportion of rhizomorphs per plot that regenerated and (or) colonized fresh substrates were all correlated. However, abundance of ectotrophic rhizomorphs on the root collar was only weakly correlated with the other components of rhizomorph abundance and vigor. Frequency of the rhizomorphs as well as their ability to colonize fresh substrates were greater in plots either limed or with a high proportion of the basal area in sugar maple prior to thinning. By contrast, abundance of ectotrophic rhizomorphs on root collars was not affected by these factors.

Genetic characterization of heterothallic and non-heterothallic Armillaria mellea sensu stricto

The genetic variation of Armillaria mellea sensu stricto was studied within and among non-heterothallic populations from Japan and Africa, and heterothallic populations from Europe and North America, using somatic incompatibility tests and RAPD analysis. Non-heterothallic isolates from Japan and Africa were divided into four somatic compatibility (SI) groups (A, B, C and D). SI group A contained half of all Japanese isolates and all African isolates. SI groups B and C each contained four Japanese isolates and D contained one isolate. All European and North American isolates were clearly separated from each other by somatic incompatibility tests. From the RAPD analysis, three distinct groups were separated (non-heterothallic, European and North American groups). The non-heterothallic group was divided into three subgroups (Ia, Ib and Ic). Subgroup Ia and Ib corresponded to SI groups A and B, however, Ic contained SI groups C and D. Subgroup Ia, in which 12 of 14 isolates had the same haplotype, showed a little variation. Non-heterothallic populations were less variable than heterothallic populations. The distribution of the isolates belonging to each SI group overlapped within Japan.

Impact of liming on the abundance and vigor of <i>Armillaria</i> rhizomorphs in Allegheny hardwoods stands

Abundance of rhizomorphs of Armillaria was characterized in 1995-1996 in 32 plots located in sugar maple (Acer saccharum Marsh.) stands in the Susquehannock State Forest (Pennsylvania, U.S.A.). All of the plots were thinned, and half of the plots were limed in 1985 when the plots were established. Frequency and abundance of Armillaria rhizomorphs in soil samples, on dead wood food bases (stumps, snags, fallen logs), and on the root collar of living sugar maples were determined in each plot. Rhizomorph vigor was evaluated by measuring their ability to colonize fresh striped maple (Acer pennsylvanicum L.) stem sections in the soil, or potato tubers in the laboratory. Isolates of Armillaria were obtained from rhizomorphs in the soil samples and species were determined by somatic incompatibility tests. Armillaria calvescens Berube & Dessureault was the major species present, representing about 66% of the isolates. Armillaria gemina Berube & Dessureault and Armillaria mellea (Vahl:Fr.) Kummer were also identif...

Transfer of the P‐type of Heterobasidion annosum from old‐growth stumps of Picea abies to Picea abies and Larix×eurolepis

SummaryThe infection of Picea abies and Larix x eurolepis by Heterobasidion annosum was studied in felled trees in Sweden. Thinnings were carried out in two stands of L. x eurolepis, 15 and 20 years old, and in a 25‐year‐old stand of P. abies, established on a site heavily infected by H. annosum. The 15‐ and 20‐year‐old L. x eurolepis stands exhibited the greatest incidence of butt rot with infections amounting to 38 and 57%, respectively, of removed trees. The incidence of butt rot in the P. abies stand was only 5%. Heterobasidion annosum was the main butt rot causing fungus. All isolates of H. annosum were of the P‐intersterility group. Transfer of H. annosum from old‐growth P. abies stumps to trees felled in the thinning of the next generation was demonstrated by somatic incompatibility tests. However, the majority of infections could not be connected with decay already established in the previous generation. In L. c eurolepis, rot columns were frequent, including also several genotypes in the same stem, and typically positioned in the inner sapwood. The few infections of P. abies were situated in the heartwood.

Isoenzymes of Ganoderma species from southern South America

One hundred and thirty four dikaryotic isolates of Ganoderma, morphologically determined as G. adspersum, G. annulare, G. applanatum, G. brownii, G. lobatoideum, G. lobatum, G. lucidum, G. oerstedii, G. resinaceum, and G. tornatum, were examined by horizontal PAGE for eight enzymatic activities. A total of 94 bands were analysed. The objective was to evaluate the relatedness among the specific taxa by analysing numerically the isoenzymic patterns. To discern fungal individuals somatic incompatibility tests were used. Within each enzyme system several allozyme phenotypes could be observed. The enzymic systems analysed failed to yield diagnostic bands for each morphologically defined group, except the AKP system for G. resinaceum. Some bands were shared by all the isolates studied. On the other hand, diagnostic bands at the species complex level were encountered.

Spread of Stereum sanguinolentum vegetative compatibility groups within a stand and within stems of Picea abies

A total of 57 naturally established Stereum sanguinolentum isolates was obtained from artificially wounded Picea abies stems in a forest area of 2 ha in Lithuania. Somatic incompatibility tests revealed 27 vegetative compatibility groups (VCGs) that contained 1–10 isolates. There was no spatial clustering of S. sanguinolentum VCGs within the forest area. The extent of S. sanguinolentum decay was analysed in 48 P. abies stems, 9–26 cm in diameter at breast height. Within 7 years of wounding, the length of S. sanguinolentum decay column in stems was 107–415 cm (291.5 ± 77.3 cm on average), lateral spread of the fungus at the butt was 38–307 cm2 (142.3 ± 66.8 cm2) and decayed proportion of the stem cross-section at the wound site (the butt) was 3–84% (36.8 ± 19.7%). In average, S. sanguinolentum VCG that infected 10 trees exhibited more slow growth inside the stem than VCGs that infected only one tree, and vertical growth varied to a greater extent within this VCG than among different VCGs. Correlation between stem diameter and vertical spread of S. sanguinolentum was not significant (r = –0.103). Despite uniformity of debarked area on all stems 7 years ago (300 cm2), open wound sizes on individual trees at the time of study were between 97–355 cm2 (215.1 ± 59.2 cm2) indicating large differences in wound healing capacity.

Comparison of RAPD technique and somatic incompatibility tests for the identification ofPhlebiopsis giganteastrains

Somatic incompatibility assays and random amplified polymorphic DNA (RAPD) analysis were assessed and compared for use in monitoring the survival of introduced strains of Phlebiopsis gigantea, a fungal biocontrol agent used against Heterobasidion annosum root rot. Overall there was concordance between the two methods. All incompatible strains exhibited different RAPD profiles whilst compatible ones, with two exceptions, had the same RAPD profile. Somatic incompatibility tests and RAPD profiles generated by three primers, from which 11 markers were retained, could distinguish field-tested strains from among 60 strains from a population collected in Quebec and Ontario. Furthermore, both methods allowed us to clearly demonstrate the presence of inoculated strains 1 year after treatment. The combined use of these techniques provides a valuable tool for discriminating between different P. gigantea strains and conducting epidemiological studies. Key words: Heterobasidion annosum, vegetative incompatibility, random amplified polymorphic DNA, biological control, Pinus resinosa.

Population structure and genetic variation in Nectria fuckeliana

Population structure and genetic variation in Nectria fuckeliana Booth isolated from Picea abies (L.) Karst. in Sweden and Lithuania was studied using somatic incompatibility tests and DNA fingerprinting. All incompatibility pairings between different isolates of N. fuckeliana resulted in demarcation zones; thus, no vegetative compatibility groups were detected. Each isolate was distinguishable from all other isolates on the basis of banding patterns produced by amplification of DNA using the M13 primer. No country-specific markers were observed. Principal component analysis of amplified banding patterns separated the isolates from Sweden and Lithuania into two clusters, showing genetic differentiation between the geographical populations across the Baltic sea. An analysis of similarity matrix, calculated by the program SIMQUAL from the numerical taxonomy package NTSYS-pc, confirmed the separation of the isolates into the two groups. Low genetic differentiation was revealed within both the Swedish and Lithuanian geographical populations of the fungus. Local distances in the forest stand (100 m) had no influence on the genetic similarity of the N. fuckeliana isolates (R2 = 0.003). Key words: Nectria fuckeliana, DNA fingerprinting, genetic variation, somatic incompatibility, population structure.

Genetic variation within and among populations of Fomitopsis pinicola (Basidiomycetes)

RAPD polymorphisms were used to reveal the genetic population structure of Fomitopsis pinicola from 5 populations in southwestern Sweden with outliers in Finland and Norway. Eleven primers were used on 35 isolates. Using the Analysis of Molecular Variance (AMOVA), the total variance was divided into 3 hierarchical components: 84% within populations, 11% among regions, and 5% among populations within regions. The 3 large Swedish populations contained 95% of the variation within them. The statistical significance of these patterns was supported by permutation tests. The similarity index between all genets ranged from 0.10 to 0.72, with an average of 0.45. Genets from the same population could form more than one cluster in the neighbor‐joining analysis. Some of these clusters were also supported by parsimony jack‐knifing. This pattern is tentatively explained by establishment of spores from different basidiomata. The result of somatic incompatibility tests and RAPD markers were compared and this comparison indicated that compatible reactions do not necessarily imply genetic identity. Sampling from cut sections of infected trees revealed that multiple infections were present in a single tree and that the fungus probably infects host‐trees by basidiospores, arriving via the air. Each somatically incompatible genet characteristically monopolized only part of a resource unit. Spore trapping showed no evidence of long distance spore dispersal, but this is probably due to the limited experiment, since the genetic analysis suggested a high rate of gene flow.

Biology and Control ofConiophora eremophilaon Lemon Trees in Arizona

A survey of mature lemon trees showed an average of 30% of trees with symptoms of brown heartrot caused by Coniophora eremophila. Growth of C. eremophila inoculated into branches of Valencia orange, Marsh grapefruit, Orlando tangelo, or Lisbon lemon on rough lemon root-stock was significantly higher in lemon than in other types of citrus. C. eremophila inoculated into Lisbon lemon branches on trees established on rough lemon, volkameriana, macrophylla, Cleopatra mandarin, sour orange, or Troyer citrange rootstocks showed no significant differences in growth. Somatic incompatibility tests of isolates from one mature orchard demonstrated that isolates from different trees were incompatible. In vitro fungicide trials showed that NECTEC P paste and NECTEC blank paste effectively reduced decay on lemon blocks 15 weeks after inoculation with C. eremophila. Field fungicide trials showed that NECTEC P paste with fungicides, as well as the blank paste without fungicides, significantly inhibited the advance of the fungus 7 months after inoculation. Also, propiconazole at 10,000 μg/ml, imazalil at 20,000 μg/ml, or propiconazole at 10,000 μg/ml in combination with imazalil at 20,000 μg/ml significantly inhibited the advance of the fungus.

Spatiotemporal patterns in ectomycorrhizal populations

To understand the functioning and ecological roles of ectomycorrhizal fungi in natural ecosystems, it is necessary to have adequate knowledge of the spatial distribution of individual mycelial systems in populations and communities and how this distribution may persist or vary with time. However, this issue has attracted relatively little attention until recently. Moreover, the limited information available is mostly based on the distribution of sporocarps, which is at best an unreliable indicator of the location and activity of mycelia. More useful information can be obtained using somatic and sexual incompatibility tests, as well as molecular markers to trace the distribution of individual genets over a range of spatial and temporal scales. For example, it has been possible using this approach to demonstrate that while young populations tend to consist of numerous small mycelia, individuals in older populations tend to be fewer and larger but heterogeneous in scale. It has also been possible to verify the persistence over several years of mycorrhizal mycelial individuals. Such findings represent only the first step in the study of the spatiotemporal dynamics of ectomycorrhizal fungi, which promises to be a rich and important field for future research. Concepts concerning the process and mechanisms likely to affect distribution patterns are discussed. Key words: ectomycorrhiza, population structure, population dynamics, spatiotemporal patterns.

Variability among isolates of Xylaria cubensis as determined by isozyme analysis and somatic incompatibility tests

Thirty strains of Xylaria cubensis isolated from leaves of the Brazilian rainforest palm Euterpe oleracea were compared by means of somatic incompatibility tests and gel electrophoretic analysis in order to gain further insights into their population dynamics. Five enzyme systems were used to assess genetic variability. Isozyme results showed that somatically compatible isolates do not necessarily have the same phenotype. Comparison of isozyme banding patterns revealed a relatively high degree of genetic diversity within pop? ulations of X cubensis, suggesting that E. oleracea leaves are subjected to a constant immigration of fungal in? oculum. This genetic diversity is presumably the result of sexual reproduction and consequently development of new genotypes of X. cubensis in the forest surround? ing the palms.

Variability among Isolates of Xylaria cubensis as Determined by Isozyme Analysis and Somatic Incompatibility Tests

Variability among Isolates of Xylaria cubensis as Determined by Isozyme Analysis and Somatic Incompatibility Tests

Size, distribution and biomass of genets in populations of Suillus bovinus (L.: Fr.) Roussel revealed by somatic incompatibility.

The spatial distribution of genets in the ectomycorrhizal fungus Suillus bovinus (L.: Fr.) Roussel were studied in somatic incompatibility pairings of isolates from five Scots pine (Pinus sylvestris L.) stands differing in forest history and age, With increasing forest age, the size of genets increased while the number of genets and production of sporocarps per unit area decreased. There was an estimated 700-5700 genets ha-1 in younger forests and 30-120 ha-1 in older ones. The maximum size of genets was 1.7-5.3 m in the younger forest and to 17.5 m in the older ones. The production of sporocarps per unit area decreased with increasing forest age. Furthermore, production of sporocarps increased nonlinearly with the size of the genet, suggesting that genets become fragmented. Ergosterol measurements indicated that the fungal biomass of one genet consisted of 20-45% sporocarps and 55-80% mycorrhiza, not including extramatrical mycelia. Mycorrhizal aggregations in soil were mapped in two 5 m2 areas and, based on somatic incompatibility tests, all were round CO belong to the same genet as sporocarps present above ground. Production of spores per sporocarp was estimated to be 1.1-12.8 × 108 . The observations are discussed in terms of population ecology.