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.     

Variation in Heterobasidion annosum detected by Random Amplified Polymorphic DNAs

The production of Random Amplified Polymorphic DNAs (RAPDs) by Polymerase Chain Reaction (PCR) was used to detect variation in the isolates of Heterobasidion annosum with various geographical origins. Specific RAPD products were detected for each of the intersterility groups: the European S, F, P and the North American S group. There was considerably more polymorphism found among European S than P isolates. The analysis of RAPDs was shown to be a simple and a fast way to generate DNA markers specific to the previously established intersterility groups and thus useful for diagnostic purposes.     

Intersterility groups of Heterobasidion annosum and their host specificity in Bulgaria

The occurrence of 46 strains of Heterobasidion annosum from four forest areas in Bulgaria is reported. Three intersterility groups, P, S and F, were identified for the first time in populations of this pathogen in Bulgaria. A strict host specificity of different intersterility groups was observed even in mixed stands. The P group was isolated from Pinus sylvestris and Pinus nigra and occasionally from stumps of Abies alba. It was rare in artificial pine plantations on dry sites, whereas it was more common on more moist sites. The F group caused severe damage on Abies alba but was not found, even as a saprotroph, on admixed pines. This is the first report of the F group on Abies alba in the Balkans. The S group was found only on Picea abies.     

Phylogenetic analysis of oyster mushrooms (<Emphasis Type="Italic">Pleurotus</Emphasis> spp.) based on restriction fragment length polymorphisms of the 5′ portion of 26S rDNA

Polymorphism analysis of the 5 portion of 26S rDNA from 34 Pleurotus strains (12 intersterility groups) collected mainly from Asia was performed. By combining the restriction fragment length polymorphism (RFLP) patterns obtained from digestions with seven restriction enzymes, the 34 Pleurotus strains were assigned to 11 RFLP types. Ten RFLP patterns corresponded with biological species, but one pattern was found in intersterility groups I (P. ostreatus complex), II (P. pulmonarius complex), and VIII (P. eryngii). The phylogenetic tree suggests that Pleurotus species have evolved in two patterns based on 26S rDNA RFLP data. One major cluster comprising the P. ostreatus clade is separated by relatively short branches, suggesting that the P. ostreatus complex, the P. pulmonarius complex, P. eryngii, and P. nebrodensis (intersterility groups I, II, VIII, and IX, respectively), share a recent common ancestor. RFLP data did not distinguish the species in the intersterility groups I, II, and VIII. The other major cluster apparently divided into five sublevel clusters in early stages of evolution and these clades split into terminal nodes in late stages of evolution: the P. calyptratus-salmoneostramineus clade, the P. cornucopiae-ulmarius clade, the P. dryinus clade, the P. corticatus clade, and the P. cystidiosus-smithii clade.     

Intersterility group differentiation in Heterobasidion annosum using ribosomal IGS1 region polymorphism

Summary The intergenic spacer region 1 (IGS1) of the nuclear ribosomal RNA gene repeat was examined in three European intersterility groups of the wood‐rot fungus Heterobasidion annosum. The IGS1 elements were informative for restriction fragment length polymorphism (RFLP) fingerprinting the fungal intersterility groups following digestion with two different four‐cutter restriction endonucleases. The fingerprints of isolates from the same intersterility group from different localities shared some fragments in common, but also showed specific fragments suggesting that RFLP of the IGS1 region might be used in population studies. RFLP patterns between isolates from different intersterility groups differed more markedly. RFLP were also detected in the IGS1 region of North American strains of H. annosum and in the related species Heterobasidion araucariae. The results indicate that ribosomal IGS1 region fingerprinting could be a useful tool to identify the fungal intersterility group in Heterobasidion annosum.     

Hosts and distribution of the intersterility groups of Heterobasidion annosum in the highlands of Greece

The intersterility groups of 127 pure cultures of H, annosum collected from different host trees in 12 mountain areas in Greece were identified. The F group commonly caused butt rot on the fir species Abies cephalonica and Abies borisii regis. It was the only type of H. annosum found in pure fir forests. The P group caused serious root rot in pinus sylvestris stands in north-eastern Greece. In more southern mountain areas it often colonized stumps of pinus nigra but seldom killed this tree species. The S group was found in natural forests of Picea abies in northern Greece, causing butt rot of spruce. In mixed forests, the intersterility groups of H. annosum were found relatively often in stumps of tree species other than their main hosts, although some host preference seemed to occur also in stump colonization.     

Distribution of Heterobasidion annosum intersterility groups in Poland

Specimens of Heterobasidion annosum were collected in 104 different stands in 43 regions of Poland. Pure cultures originating from 439 collections were identified in mating tests. Three intersterility groups, P, S and F, of H. annosum were found. Their occurrence in Poland was connected with the natural distribution of the main hosts: Pinus sylvestris, Picea abies and Abies alba, respectively. P was the most common intersterility group of H. annosum in Poland, causing mortality in Scots pine plantations and root rot in older stands. It was also isolated from Betula pendula, P. abies, Larix decidua, Fagus sylvatica and Carpinus betulus. The S group was present in the southern and north‐eastern parts of the country, causing root and butt rot mostly in spruce stands. The F group occurred in the south of Poland, in the mountains, highlands and lowland up to the northern border of the distribution of fir. It was found only on stumps, old dead trees and logs. There was no evidence of damage caused by the F group on A. alba trees.     

Spore deposition of Heterobasidion annosum coll. in Picea abies stands of North Karelia,eastern Finland

Spore deposition of Heterobasidion annosum coll.was measured with the wood disc method in 20 Norway spruce stands in June, July and September 1999 in North Karelia, eastern Finland. The study area crossed the presently known northern boundary of the distribution area of H. annosum (62°45′N). The spore deposition rate decreased northwards and in the northernmost parts of the study area, deposition was detected only in September. On average 71% of the spore deposition consisted of intersterility group P and 29% of intersterility group S. The proportion of S‐type in the deposition increased northwards. Most of the isolated colonies were homokaryons, but the proportion of heterokaryons increased with increasing deposition rate.     

Presence of double‐stranded RNA in Heterobasidion annosum

A search for double‐stranded RNA (dsRNA) was conducted among 204 European isolates of the pathogenic fungus Heterobasidion annosum. Nucleic acids were extracted and purified by cellulose CF‐11 chromatography or lithium chloride precipitation. dsRNA was present in eight of the isolates and was confirmed by nuclease digestion. The dsRNA elements ranged between 1.8 and 2.4 kbp and were found in two H. annosum intersterility groups, S and P. Partial amino acid sequence information from one dsRNA element showed significant homology to RNA‐dependent RNA polymerases from several fungal partitiviruses. This is the first report of the presence of dsRNAs in H. annosum. Possible implications of dsRNA for the biology of the fungus and the potential for biological control are discussed.     

In vitro inhibition of Heterobasidion annosum by Phaeotheca dimorphospora

The antagonistic fungus Phaeotheca dimorphospora has been tested in vitro on agar plates and wood discs to evaluate its potential use against P (Eastern Canada) and S (Finland) intersterility groups strains of Heterobasidion annosum using two methods. Production of antifungal metabolites by P. dimorphospora was demonstrated on agar plates as well as on Pinus resinosa and Picea abies discs using the bi‐layer technique. Diffusible metabolites inhibited the growth of the pathogen on both substrates. Direct confrontation on wood discs showed that a 7‐day period of advanced growth by P. dimorphospora completely prevented the colonization of the tissue by H. annosum. Control was partial when the antagonist had only one day of advanced growth. Growth inhibition was superior against S group strains with less than 1% of the samples colonized by H. annosum compared with 22% for P group strains. Moreover, P. dimorphospora was isolated in more than 83% of the samples in the absence of the pathogen in both wood species. In vitro tests demonstrated that P. dimorphospora is able to colonize red pine as well as Norway spruce tissue and to use wood components for the production of antifungal metabolites.     

An intersterility group of Heterobasidion annosum specialized to Abies alba

A third intersterility group of H. annosum in Europe has been identified from Italy. It is specialized to Abies alba., and hence is called the F (fir) group. It is closely related to the European S group, but is sexually incompatible with the geographically adjacent S population occurring in the Alps.     

Abies sibirica in the Ural region is attacked by the S type of Heterobasidion annosum

Specimens of Heterobasidion annosum were collected in seven mixed stands of Abies sibirica and Picea abies in Yekaterinburg and Perm regions on the western side of the Ural Mountains, Russia. Pure cultures were isolated from 60 collections and identified in mating tests. Only the S intersterility group was found, attacking both fir and spruce. Based on the appearance of clamp connections in matings, the compatibility of 12 homokaryons from the Ural area with 14 S group homokaryons from western Europe was 100%, with 13 F group homokaryons from southern Europe 76% and 9 P group homokaryons from western Europe 3%. The results show that A. sibirica, in contrast to A. alba, is very susceptible to the S type H. annosum.     

Identification of Kenyan Armillaria isolates by cultural morphology,intersterility tests and analysis of isozyme profiles

Three groups of morphologically distinct isolates were observed among nine Kenyan Armillaria isolates based on their mycelium and rhizomorphs characteristics. Seven of the isolates were interfertile with testers of North American biological species III, VII and IX. However, tests with benomyl segregants BEN 433, BEN 157-10 and BEN AVK were intersterile with the same testers and also with the European A. mellea, A. lutea and A. ostoyae. The analysis of isozyme profiles showed that morphologically similar isolates had similar isozyme profiles of esterases. Their profiles however differed from those of the European A. mellea, A. lutea and A. ostoyae. On the basis of intersterility tests and analysis of isozyme profiles, the Kenyan isolates should be considered different.     

大别山不同龄级映山红种群遗传多样性的SSR分析

[目的]利用SSR标记比较大别山黄狮寨不同年龄级映山红(Rhododendron simsii Planch.)种群的遗传多样性及遗传结构,探究映山红不同世代间遗传多样性的变化规律,为大别山野生映山红资源的合理利用和高效保护提供科学依据。[方法]按照基径大小和丛枝多少将大别山黄狮寨典型映山红种群划分为老树、成树、小树、幼苗4个年龄级,筛选出12对多态性强的SSR引物用于PCR扩增,扩增产物经6%变性聚丙烯酰胺凝胶电泳检测并银染。构建"0/1"矩阵,利用POPGENE 32. 0软件分析种群遗传多样性。基于Nei's遗传距离,采用软件MEGA5. 0进行UPGMA聚类。[结果]不同龄级的映山红遗传多样性差别较大,幼苗和老树种群多样性最差,小树种群多样性最丰富。12个微卫星标记观测等位基因数为3～9个,平均5. 08;有效等位基因数为2. 254 9 6. 129 7,平均3. 460 5;观察杂合度HO和期望杂合度HE分别为0. 676 4～0. 881 2和0. 607 7～0. 690 7。Shannon信息指数(I)以小树群体最高,成树次之,幼苗最低。近交系数Fis为-0. 638 3～0. 174 4,平均为-0. 294 6;总近交系数Fit为-0. 615 1～0. 270 6,平均为-0. 162 1,表明各龄级种群内主要繁殖方式为杂交。分子方差分析(AMOVA)表明89. 76%的遗传变异存在于年龄级内,仅10. 24%存在于年龄级间。基因流水平高,仅1个位点Nm 1。遗传一致度最高的为小树和成树种群。[结论]大别山黄狮寨映山红种群遗传多样性丰富,种群间中度分化,遗传变异主要存在于年龄级内。     

Estimation of dispersal gradients of S- and P-type basidiospores of Heterobasidion annosum

The number of airborne deposits of the root pathogen Heterobasidion annosum was counted with a wood disc method at distances 5–1000 m from one S-type and one P-type infection centre in stands of Picea abies in June, July and August, 1995. The level of background deposition in the area was determined in four healthy control stands. Based on intersterility group determination of deposits, only measurements within 100 m of the infection centres could be used in estimations of dispersal gradients owing to confusion with background deposition. A power law gradient model adequately described the reduction of deposits with increasing distance from the infection centres. Gradients were fairly similar in four directions around the infection centres as the wind speeds were low (3.2m/s) during the collection periods. The gradients were steeper around the P-type centre, probably due to its lower spore production capacity. The background deposition level (2.8 colonies/m²× h) was reached at distances 1255 m and 98 m from the S-type and P-type centre, respectively.     

Interfertility between North American and European strains of Phlebiopsis gigantea

Thirteen homokaryotic strains of Phlebiopsis gigantea from Canada, six strains from the US and 10 strains from Europe were paired in all possible combinations in order to determine the degree of interfertility between them. The diagnosis of interfertility was based on the production of heterokaryotic fruit bodies in the pairings. Among the resulting 406 pairings, 253 (62%) fruited. Within the strains originating from Canada, USA and Europe, 64, 80 and 64% of the pairings fruited, respectively. The fruiting frequency in pairings between the Canadian and US strains was 65%, between the Canadian and European strains 55%, and between the US and European strains 67%. True hybridization between the European and North American P. gigantea was shown by analysing the single‐spore progeny using DNA fingerprinting. In spite of the relatively low interfertility in pairings within and between continents, no clear indication of the existence of intersterility groups was found. The low interfertility is probably due to the ageing of the pure cultures and to deficient fruiting ability of certain heterokaryons on agar medium. The results strongly suggest that although the North American and European strains of P. gigantea show some differentiation they can be regarded as belonging to the same biological species.     

Genetic diversity of the hyperparasite Sphaerellopsis filum on Melampsora willow rusts

The non‐specific rust hyperparasite Sphaerellopsis filum occurs naturally on Melampsora rusts of many species of the genus Salix as well as on a large range of other rust genera worldwide. To study the genetic diversity of the hyperparasitic fungus 77 S. filum isolates collected from rusts on willow clones from plantations, clone collections and natural habitats of different sites were investigated using polymerase chain reaction ‐ restriction fragment length polymorphism (PCR‐RFLP) analysis of the rDNA internal transcribed spacer regions including 5.8S rDNA and sequence analysis. Additionally, strains from Melampsora poplar rusts (4) and strains of Puccinia abrupta from Parthenium hysterophorus (5) and of P. obscura from Bellis perennis (1) were used for comparisons. Results of genetic analysis demonstrated distinct variation within the S. filum isolates. Two main groups with more than 32% difference between their nucleotide sequences were distinguished, indicating two taxa within S. filum. Within the first main group three profiles (I, II, III) were detected. The differences between these profiles were about 12%. The variation within each profile was very low (less than 2%). The second main group comprised two profiles (IV, V), which differed in 12 to 16% of their nucleotide positions. The isolates of group IV possessed a higher variation (up to 5%) within the group than those of the first main group (I, II, III). Group V was only represented by a single isolate. Neither interrelations between the S. filum profiles and the Melampsora genotypes nor a spatial distribution could be detected. It is remarkable that the six strains of S. filum from Puccinia rusts belong to one subgroup.     

Variation in growth of Heterobasidion annosum among clones of Picea abies incubated for different periods of time

Thirty‐seven 4‐year‐old clones of Picea abies were inoculated with one isolate of the S intersterility group of Heterobasidion annosum in a greenhouse. The dehardened cuttings were organized in three different groups with four to six ramets in each group. All groups were inoculated on the same day shortly after shoot elongation. The groups were sampled for H. annosum growth after 34 (group 1), 83 (group 2) and 182 days (group 3), respectively. Measured parameters were cutting height and diameter, vigour index of the cuttings, infection incidence, mortality rate and fungal growth in the stem. The height of the cuttings was almost constant during the 6 months of incubation, whereas the diameter increased by about 10% during the same period. The proportion of living cuttings containing H. annosum decreased with time (99.5, 93 and 67% infection in groups 1, 2 and 3, respectively) and differed significantly among clones in group 3. Mortality rate increased with time (0.5, 22 and 37% mortality in groups 1, 2 and 3, respectively) and differed significantly among clones in groups 2 and 3; the same clones being most susceptible for both times. Mean fungal growth into the wood was significantly different among groups and among clones within each group. The ranking position for fungal growth was similar in the three groups. The results indicate that frequency of infection success, mortality rate and fungal growth are clone‐dependent factors. Broad sense heritability varied between 0.08 and 0.25 for fungal growth and lesion length at the three incubation periods. Infection success frequency was initially not different among clones but in the longer incubation periods there were significant differences among clones, indicating differences in resistance. Fungal growth in wood differed among separate host clones irrespectively of the length of inoculation period. The vigour of the cuttings seem to influence the length of fungal extension only in the initial stage of the infection.     

用RAPD标记检测与杨树生长和物候期有关的QTLs

取美洲黑杨（母本）和青杨（父本）杂交获得的Ｆ２群体样本８０株,应用ＲＡＰＤ标记检测与Ｆ２群体３个数量性状（苗高、地径和封顶期）有关的ＱＴＬｓ。在Ｆ２九体中,一年生苗高、地径和封顶期性状表现显著分离,基本符合正态分布。单因子方差分析检测出与苗高、地径和封顶期性状相关的７个、６个和３个标记座位。并计算了各标记对相关性状变异的贡献率。与数量性状相关的标记有不同的亲本来源。ｔ检查结果各标记显带型和不显带型     

Genetic diversity and association analyses of fruit traits with microsatellite ISSRs in Sapindus

Sapindus mukorossi Gaertn. and S. delavayi Franchet are among the most valuable species in the genus Sapindus for their commercially exploitable plant oils and chemicals. However, few studies have addressed genetic variation and improvement for either species. We evaluated the genetic diversity of germplasm from selected plus trees within a wide region and established the relationship between fruit traits and molecular markers. An association analysis based on inter-simple sequence repeats(ISSRs)provided a genetic basis for studies of fruit traits. A total of 247 loci were detected by scanning 61 trees of S. mukorossi and S. delavayi using 16 ISSR markers. Genetic diversity parameters were estimated for selected superior trees(or germplasm) and S. mukorossi and S. delavayi were categorized into two main groups, as well as into four groups within S. mukorossi. An association analysis between the ISSR markers and 14 fruit traits used the TASSEL MLM model. A genetic structure analysis differentiated S.mukorossi and S. delavayi. Eighteen ISSR loci associated with 13 fruit traits(P<0.005) were identified, with 13, 1,and 4 loci associated with seed oil production, fruit saponin production, and fruit quality, respectively. Using this information, a core collection was selected with adequate genetic diversity and good seed oil characters. Our results demonstrate the feasibility of effectively estimating fruit trait associations in Sapindus using ISSR markers, and the method is applicable and valuable for select germplasm conservation. The markers obtained in this study are potentially useful for molecular-assisted breeding of Sapindus spp.