Identification of the genus Armillaria by specific amplification of an rDNA-ITS fragment and evaluation of genetic variation within A. ostoyae by rDNA-RFLP and RAPD analysis

Genetic variation among Armillaria ostoyae isolates was studied by rDNA-restriction fragment length polymorphism (RFLP) and random amplified polymorphic DNA (RAPD) analysis. A total of 20 A. ostoyae isolates, mainly obtained from Picea spp. of different geographical origins, were examined. Southern hybridization of whole-cell DNAs digested with AvaII and probed with biotin-labelled cloned rDNA from Saccharomyces carlsbergensis allowed the differentiation of five RFLP groups. UPGMA cluster analysis of RAPD profiles (138 scorable bands) generated by 10 decamer primers (OPA 01-OPA 10) grouped the isolates in subclusters at similarity levels between 40% and 96%, indicating high intraspecific genetic variability. Some isolates of different geographical origins subgrouped together, suggesting that similar mutational events have occurred independently and that genetic exchange and recombination occurs among the DNAs in natural populations. The potential role of historical and current spread of spruce plants on the genetic variation of A. ostoyae isolates in Europe is discussed. Using the primer pair ARM-1 and ARM-2, an Armillaria-specific ITS-DNA fragment of about 660 bp was obtained. No intraspecific RFLP of this amplicon could be revealed, indicating low genetic variability of this region. The established informative RFLP and RAPD markers and also the Armillaria-specific ITS-DNA fragment may be powerful tools for further epidemiological, phylogenetic and host-pathogen interaction studies with A. ostoyae.     

Isozyme and RAPD polymorphisms in Heterobasidion annosum in Italy

Isozyme and random amplified polymorphic DNA (RAPD) polymorphisms were used to study variability in a group of 41 isolates from the Italian population of Heterobasidion annosum. The isolates belonged to the intersterility groups P and S, and particularly to the group that is most widely distributed in Italy, group F. Isozyme analysis was effective in identifying the three intersterility groups and revealed a high degree of genetic divergence within the P group isolates; the mannose phosphate isomerase (MPI-2) locus was diagnostic in the attribution of isolates to the more correlated F and S groups. RAPDs were detected following amplification by the polymerase chain reaction (PCR). 74 RAPD fragments, obtained through amplifications with eight primers, were scored. Isolates from the 3 intersterility groups were clearly divergent based on analysis of RAPD markers. However, a similarity index calculated for the isolates within the F population indicated a high uniformity of the isolates collected throughout the Italian peninsula.     

Adaptive genetic diversity and population structure of the “algarrobo” [<Emphasis Type="Italic">Prosopis chilensis</Emphasis> (Molina) Stuntz] analysed by RAPD and isozyme markers

The “algarrobo” [Prosopis chilensis (Molina) Stuntz] is a tree species that represents an important natural resource in arid and semi-arid regions of Argentina. In this paper, we analysed and compared the variability of 46 RAPD (Random Amplified Polymorphic DNA) loci with previous estimates obtained from 12 isozyme markers in nine Argentinean populations of P. chilensis representative of the whole range of this species in Argentina. We evaluated the population structure and the existence of genetic variants associated with environmental variables. Expected heterozygosity (H _e) estimated from RAPD varied significantly among populations and regions. Hierarchical analysis of genetic variability (AMOVA) showed that most (88.1%) of the total diversity occurs within populations, the component among populations within regions (9.3%) was intermediate, while the between-region component was the lowest (2.6%). All three variance components were highly significant. The MDS plot from pair-wise Φ _ST matrix was consistent with the highly significant among-region differentiation indicated by the AMOVA. All 12 variable isozyme loci and 26 out of 46 RAPD loci showed significant or highly significant association with at least one geographic/climatic variable according to the stepwise multiple regression analysis. These results imply that the genetic differentiation among populations is better explained by environmental or biogeographical grounds than by geographical distances, suggesting gametic disequilibrium with loci responsible for the adaptation to particular environmental conditions. The information from RAPD markers would provide a relevant criterion to preserve genetic diversity in programmes for conservation and rationale use of this species.     

Direct genotyping of the poplar leaf rust fungus,Melampsora medusae f. sp. deltoidae,using codominant PCR‐SSCP markers

Two anonymous DNA markers that are revealed by single‐strand conformational polymorphism (SSCP) analysis were developed for detection of polymorphisms in Melampsora medusae f. sp. deltoidae (Mmd). Mono‐uredinial isolates of Mmd were first obtained, DNA was extracted from urediniospores and random amplified polymorphic DNA (RAPD) products of eight mono‐uredinial isolates were separated on a SSCP gel to identify differences among them. Bands representing putative polymorphic loci among the eight isolates tested were excised from the SSCP gel and re‐amplified by polymerase chain reaction (PCR), and then cloned and sequenced. A primer pair was designed to amplify a DNA fragment of a size suitable for SSCP analysis (<600 bp) for two out of three DNA fragments sequenced. Each set of primers amplified a PCR product for all eight isolates that were initially used to generate them and the resulting PCR products were analysed by SSCP. Polymorphisms among isolates were identified for both putative loci. The two primer pairs amplified a PCR product of the expected size on an additional 32 mono‐uredinial isolates of Mmd tested. From the overall 40 mono‐uredinial isolates tested, 5 and 11 alleles were detected, and 12 and 34 isolates showed to be heterozygous, as indicated by the presence of more than two bands on the SSCP gel, at loci A and B, respectively. The primer pairs were tested for specificity against 106 fungal isolates belonging to various taxa, including other rusts, and against DNA extracted from greenhouse‐grown healthy poplar leaves. DNA amplification products of the expected size were obtained only when Mmd DNA was present. Optimization of PCR conditions with these two primer pairs allowed genotyping directly from single uredinia extracted from infected leaves, thus alleviating the need to culture the fungus to characterize individuals, hence making it possible to process large numbers of samples for population studies.     

RAPD variation in Gremmeniella abietina attacking Pinus sylvestris and Pinus contorta in northern Sweden

Genomic DNA from 81 isolates of Gremmeniella abietina collected from eleven plantations each of Pinus sylvestris and Pinus contorta in northern Sweden was studied using RAPD markers. The DNA variation between and within populations and the race and type distribution of G. abietina populations, causing symptoms similar to those of the North American race, were studied. The degree of genetic similarity was greater among G. abietina isolates from the same geographical areas than among isolates from different geographical areas, regardless of whether they were isolated from P. sylvestris or P. contorta. RAPD variation was greatest in the central parts of northern Sweden, suggesting that sexual reproduction has been somewhat more important there than further north or south. Only the RAPD fragments characteristic of the EU race of G. abietina were found in the material tested. The RAPD pattern described as characteristic of the northern type within the EU race was identified in 62% of the isolates. Divergence from the expected profile was due to differences in occurrence of fragments OPA12-1400 and 12-1500. This indicates that this part of the RAPD profile cannot be treated as diagnostic for the northern type. A conclusion of practical importance is that there is a considerable risk of G. abietina spreading from infected P. contorta plantations to adjacent areas with indigenous P. sylvestris regeneration, and vice versa, owing to the indicated lack of host-specificity of the pathogen. It is possible, however, that host-specific strains exist, but do not differ in their RAPD profiles.     

A molecular toolkit for population genetic investigations of the ash dieback pathogen Hymenoscyphus pseudoalbidus

The recently described ascomycete fungus Hymenoscyphus pseudoalbidus (anamorph: Chalara fraxinea) causes the current dieback of ash (Fraxinus excelsior) in large parts of Europe. The origin of this species and its relation to the native cryptic species Hymenoscyphus albidus are still enigmatic. The spatiotemporal pattern of the epidemic is typical for an introduced invasive species. However, the presence of two cryptic species indicates that hybridization or mutation might have been involved in driving speciation in this case. In this study, we present a set of 18 polymorphic microsatellite markers to study these processes in more detail on a population genetic level. Markers were designed such that they can be amplified in three individual multiplex PCRs and analysed in two fragment analysis runs. We thoroughly tested the marker set for pairwise linkage among loci, selective neutrality and Mendelian inheritance. Additionally, the markers were applied on two large collections of isolates derived from study sites in Germany. Population genetic calculations suggested a low yet significant level of differentiation, a large genotypic diversity and a limited genetic diversity within populations. Furthermore, we present additional data concerning the phylogenetic relation between H. albidus and H. pseudoalbidus, which seems to be more distantly related to each other than expected previously.     

First record of Chalara fraxinea in Finland and genetic variation among isolates sampled from Åland,mainland Finland,Estonia and Latvia

We have isolated and confirmed the identification of ash dieback fungus (Chalara fraxinea, teleomorph Hymenoscyphus albidus) for the first time in Finland. In a preliminary analysis, considerable amount of genetic variation was detected among 20 Finnish (Åland and mainland Finland), one Latvian and 11 Estonian isolates, analysed by random amplified microsatellite (RAMS) markers.     

RAPD Marker Diversity Within and Among Natural Populations of the Clonal Tree Cryptomeria japonica D. Don.

Abstract

The Japanese cedar, Cryptomeria japonica D. Don., is one of the most important endemic species in Japan. A long history of heavy logging has resulted in natural populations being discontinuously distributed and scattered among small, restricted areas. An understanding of the patterns of genetic variability among and within populations of C. japonica is important for conserving the genetic resources of this economically important species. We studied genetic variation by random amplified polymorphic DNA (RAPD) markers in C. japonica of Kyoto prefecture, western Japan. A total of 398 plants sampled from six natural populations were analyzed by ten arbitrarily chosen decamer primers, resulting in 50 highly reproducible RAPD bands. Analysis of molecular variance partitionated the RAPD variation into the among- and within population components. The within-population component accounted for 93.75% of the variation. The among-population component accounted for only 6.25%. Indirect estimates of gene flow indicated that the average number of migrants exchanged among six populations per generation was 3.72. A Mantel test for each population did not yield significant correlations between geographic and genetic distances. The extent and distribution of C. japonica diversity in the Kyoto prefecture is in agreement with the conclusion that long-lived, wind-pollinated, out-crossing species with wide ranges retain a considerable amount of genetic diversity within populations and exhibit little genetic differentiation among populations.     

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

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.     

Persistence of a biocontrol strain of Phlebiopsis gigantea in conifer stumps and its effects on within‐species genetic diversity

Fungal isolations and genetic fingerprinting were used to determine whether Phlebiopsis gigantea stump treatment against Heterobasidion annosum sl. using a single genotype (Rotstop) would affect the genetic diversity of P. gigantea populations. The survival time of P. gigantea was longer in Norway spruce (Picea abies) stumps compared to Scots pine (Pinus sylvestris) as no isolates were obtained from pine stumps 6 years after treatment, whereas in about half of the spruce stumps the fungus was still present. The usage of Rotstop did not seem to increase the occurrence of the fungus 5 years after the treatment in fresh (1‐year‐old) untreated stumps within the same forest stands. All the isolates from the 6‐year‐old treated spruce stumps were identical in genotype with the Rotstop‐strain, whereas all isolates from the fresh untreated spruce and pine stumps differed from it. Within the treated pine stand, the biocontrol usage seemed to have caused a slight reduction in genetic markers not related to Rotstop, but there were no statistically significant differences between the marker frequencies and the local natural population. Thus, Rotstop is not likely to cause any immediate threat to the genetic diversity of P. gigantea.     

Biscogniauxia nummularia infecting beech (Fagus sylvatica) trees and sympatric plants of the sedge Carex brevicollis

Biscogniauxia nummularia is known for its association with beech (Fagus sylvatica), on which it occurs as a saprotroph and a pathogen causing strip cankers following water stress. This fungus has also been reported as a dominant endophytic species in plants of the sedge Carex brevicollis growing in the understory of beech forests and adjacent grasslands in Sierra de Urbasa (Navarre, Spain). In this area, stromata of B. nummularia were observed in dead and living wood of beech trees at several locations where plants of C. brevicollis also contained the fungus as an endophyte. Pure cultures obtained from stromata of B. nummularia on F. sylvatica trees were compared to endophytic isolates from symptomless C. brevicollis. Culture morphology and micromorphology as well as rDNA sequences of the internal transcribed spacer (ITS) regions were identical, suggesting that B. nummularia from beech can also live endophytically in C. brevicollis. It is unknown whether the endophytic strains of Carex might have a role as an inoculum source for the infection of beech trees, or whether they represent a dead end in the life cycle of the fungus.     

Characterization of microsatellites in Fusicladium effusum,cause of pecan scab

Pecan scab, caused by the plant pathogenic fungus Fusicladium effusum, is the most destructive disease of pecan. Little is known of the population genetic diversity of this pathogen. In this study, microsatellites were mined from the F. effusum genome, and flanking primers were subsequently designed. A total of 275 microsatellites were screened, and 33 selected primers produced reliable, polymorphic markers against 46 isolates of F. effusum from 11 diverse locations in the southeastern USA. The number of alleles per microsatellite locus ranged from two to 17, and the polymorphic information content (PIC) from 0.475 to 0.911. A unique pattern informative combination (UPIC) analysis of three groups of 12 isolates each and 33 primers consistently showed a minimum number of markers required for the maximum discrimination of isolates equal to 3. The characteristics of the unique patterns (UP) and informative contents (IC) were very similar. However, the primers that were selected by UPIC were not necessarily the same for each of the three groups. Using all 46 isolates showed each locus was polymorphic, with a single‐population level Shannon's information index of 1.516, indicating substantial diversity. These markers show a range in polymorphic content and power of discrimination that will be valuable tools for studies of genetic diversity in F. effusum.     

Genetic diversity and population structure of Raffaelea quercus‐mongolicae,a fungus associated with oak mortality in South Korea

Raffaelea quercus‐mongolicae is a fungus associated with oak wilt and deemed to cause extensive oak mortality in South Korea. Since the discovery of this fungus on a dead Mongolian oak (Quercus mongolica) in 2004, the mortality continued to spread southwards in South Korea. Despite continued expansion of the disease and associated significant impacts on forest ecosystems, information is lacking about the origin and genetic diversity of R. quercus‐mongolicae. Restriction‐site‐associated DNA (RAD) sequencing was used to assess genetic diversity and population structure among five populations (provinces) of R. quercus‐mongolicae in South Korea. In total, 179 single nucleotide polymorphisms (SNPs) were identified among 2,639 RAD loci across the nuclear genome of the 54 R. quercus‐mongolicae isolates (0.0012 SNPs per bp), which displayed an overall low expected heterozygosity and no apparent population structure. The low genetic diversity and no apparent population structure among South Korean populations of this ambrosia beetle‐vectored fungus support the hypothesis that this fungus was introduced to South Korea.     

A comparison of UP‐PCR and RAPD markers to study genetic diversity of Fusicladium effusum (G. Winter), cause of pecan scab

Fusicladium effusum infects pecan causing yield loss, but no information is available on the genetic diversity of F. effusum. Randomly amplified polymorphic DNAs (RAPDs) and universally primed polymerase chain reaction (UP‐PCR) were compared to detect polymorphisms on a group of 20 isolates of F. effusum from 11 geographical locations in the southeastern USA. Two tests (run 1 and 2) of both the RAPD and UP‐PCRs were conducted to assess the repeatability of the methods, and the markers scored on agarose gels. In addition, the UP‐PCR markers from run 1 were scored using an automated capillary system. Both RAPDs and UP‐PCR markers detected a high level of polymorphism among the scored markers (92 and 91% of RAPD markers, and 86 and 87% of manually scored UP‐PCR markers in run 1 and 2 were polymorphic, respectively; 93% of UP‐PCR markers were polymorphic when scored using the automated system). Unweighted paired group method of arithmetic averages (UPGMA) analysis showed both RAPDs and UP‐PCR markers individually identified each isolate, producing three groupings, but only the groupings based on run 1 and 2 of the UP‐PCR contained the same isolates. Bootstrap analysis based on the Dice coefficient produced phenograms from the UP‐PCR data with weak to moderate node support (≥54) for the primary branch, but no support for the RAPDs data (≤34). A Mantel test of runs 1 and 2 using RAPDs or UP‐PCR showed good agreement (r = 0.8761 and 0.8289, p < 0.0001), but poor agreement between RAPDs and UP‐PCR. UP‐PCR results based on the interisolate Dice coefficients showed a weak to strong association with distance. Based on these results, both RAPDs and UP‐PCR markers were capable of demonstrating polymorphisms and identifying relationships among isolates of F. effusum; however, UP‐PCR markers appear to be more reliable.     

Genotypic diversity and distribution of Sphaeropsis sapinea within Pinus radiata trees from northern Spain

Sphaeropsis sapinea is an important latent pathogen of Pinus spp., outbreaks of which have a considerable impact on plantations. This study considers the population diversity and distribution of S. sapinea in northern Spain at different spatial scales from single plantations to a wide area covered by Pinus radiata trees. Estimation of genotypic diversity is an important component of the analysis of the genetic structure of plant pathogen populations. Ten simple sequence repeat (SSR) markers were used, together with vegetative compatibility tests, to study the genetic diversity among S. sapinea isolates. Polymorphism analysis at SSR loci is a simple and direct approach for estimating the genetic diversity of S. sapinea isolates. From a total of 86 isolates collected from four different areas, 14 microsatellite haplotypes and 13 vegetative compatibility groups (VCGs) were identified. The percentage of maximum genotypic diversity, based on Stoddart and Taylor's index, for microsatellites of the northern Spain population ranged from 14.6% to 38.1% and from 8.0% to 29.4% for VCGs. Analysis of these markers and vegetative compatibility groups confirmed that S. sapinea reproduces mainly asexually due to its reduced genotypic diversity in spatially close populations. Isolates of S. sapinea from northern Spain populations were predominantly monomorphic at the tested SSR loci. Vegetative compatibility groups also indicate a low level of genetic variability in these samples, which appear to be clonal.     

RAPD analysis of genetic relationships among Sphaeropsis sapinea isolates

Genetic relationships were studied among 23 isolates of Sphaeropsis sapinea collected from China, the United States, England, South Africa and Chile by using a random amplification of a polymorphic DNA (RAPD) analytical method. One hundred and 35 DNA fragments were amplified with 12 random primers by a polymerase chain reaction PCR technique and 96.3% were polymorphic. The genetic dendrogram based on RAPD analysis showed that the S. sapinea isolates could be divided into three types. Isolate CWS41 from Chile was separated genetically as the first type that was different from other isolates and isolates F2 and J2 from China comprised the second group. The third RAPD group accommodated other isolates including the B morphotype isolate CWS43 from the United States. __________ Translated from Journal of Nanjing Forestry University, 2006, 30(1): 13–16 [译自: 南京林业大学学报]     

Effect of Regeneration Method on RAPD-Based Genetic Variation of Cyclobalanopsis glauca (Fagaceae)

Cyclobalanopsis glauca is a dominant species of evergreen broad-leaved forests in mainland China. This study compares the genetic variation of an artificially regenerated population with its donor population and two other wild populations, by using RAPD markers. A total of 74 clear, reproducible bands were scored for 12 RAPD primers; 72 were polymorphic (P = 97.3%). AMOVA revealed that most genetic variation was within populations and only 10.35% was among populations. Various measures indicated that there is no difference in genetic diversity between the planted and the original populations. Φ_ST between the planted offspring population and the donor population was larger than those between the planted and other two natural populations, indicating that artificial regeneration might lead to biased genetic composition, given that temporal differentiation is usually lower than spatial differentiation. This divergence may be due to unequal seed production among the maternal individuals and viability differences among seeds.     

Genetic diversity in Nordic and Baltic populations of Chondrostereum purpureum: a potential herbicide biocontrol agent

We analysed genetic variation in the natural populations of a potential herbicide biocontrol agent, Chondrostereum purpureum, in Nordic and Baltic countries using random amplified microsatellite markers. The results showed high genetic diversity among the populations of this fungus, but almost a complete lack of local differentiation. The results implicate that any local strain from the area can be used as a biocontrol agent without a fear of introducing new genotypes to treatment areas.