Identification of Armillaria species in Japan using PCR-RFLP analysis of rDNA intergenic spacer region and comparisons of Armillaria species in the world

Armillaria species from Japan were characterized using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of the intergenic spacer region-1 (IGS-1) of ribosomal DNA (rDNA). Eleven different digestion patterns by restriction endonuclease Alu I were found among 70 isolates of seven Armillaria species in Japan. Isolates within Armillaria nabsnona, A. ostoyae, A. cepistipes, and Japanese biological species E showed the same Alu I digestion patterns. Five Alu I patterns were detected for A. gallica, three patterns for A. mellea, and two patterns for A. tabescens. Seven Armillaria species in Japan were clearly distinguished by using the profiles obtained when PCR products were digested with Alu I, Msp I, and Hae III restriction enzymes. There was considerable variability of Alu I restriction sites within the IGS-1 between the isolates of five Armillaria species, A. gallica, A. nabsnona, A. cepistipes, A. mellea, and A. tabescens, in Japan and those of their European and North American counterparts.     

Identification of European Armillaria species by analysis of isozyme profiles

Studies were carried out to test the possibility of identifying European Armillaria species by using isozyme patterns. Twenty-two different enzymes were used to analyse the haploid and diploid mycelium extract of Armillaria borealis, Armillaria cepistipes, Armillaria gallica, Armillaria mellea, Armillaria ostoyae and Armillaria tahescens. Tests for fumarase (E.C. 4.2.1.1.), aconitase (E.C. 4.2.1.3.), leucine-amino peptidase (E.C. 3.4.11.1.), isocitrate dehydrogenase (E.C. 1.1.1.42.), shikimic dehydrogenase (E.C. 1.1.1.25), glucose-6-P-dehydrogenase (E.C. 1.1.1.49.), malic enzyme (E.C. 1.1.1.40.), 6-P-gluconic dehydrogenase (E.C. 1.1.4.4.), pectin esterase (E.C. 3.1.1.11.), and pectic lyase (E.C. 4.2.99.3.) did not reveal enzyme activity. Isozyme profiles of acid phosphatase (E.C. 3.1.3.2.), phospho-gluco-isomerase (E.C. 5.3.1.9.), peroxidase (E.C. 1.11.1.7.), polyphenoloxidase (E.C. 1.14.18.), malic dehydrogenase (E.C. 1.1.1.37.), glutamic dehydrogenase (E.C. 1.4.1.3.) and superoxide dismutase (E.C. 1.15.1.1.) were ineffective for species identification. In contrast, esterase (E.C. 3.1.1.1.), glutamic-oxalacetic transaminase (2.6.1.1.), phospho-gluco-mutase (E.C. 2.7.5.1.), alcohol dehydrogenase (E.C. 1.1.1.1.), and polygalacturonase (E.C. 3.2.1.15.) isoenzyme patterns showed enough polymorphism to allow the identification of the different Armillaria species. However, it is necessary to compare several enzyme profiles for a conclusive identification. Intraspecific crosses of A tabescens were confirmed by the presence of a heteromeric isozyme pattern of alcohol dehydrogenase and phospho-gluco-mutase.     

Identification of European Armillaria species by restriction-fragment-length polymorphisms of ribosomal DNA

Identification of European Armillaria species using ribosomal DNA (rDNA) restriction-fragment-length polymorphisms (RFLPs) was studied. A total of 44 Armillaria isolates representing five European biological species were examined. Whole-cell DNAs were digested with one or two (double digest) restriction endonucleases and probed with a cloned plasmid carrying one complete rDNA repeat copy of Saccharomyces carlsbergensis. Applying the restriction endonuclease Bgl II in combination with either Eco RI, Bam Hl or Hin dIII, it was possible to detect rDNA RFLPs allowing differentiation between all five European Armillaria species investigated in the study. The most conclusive results were obtained in the rDNA/Ava II RFLP pattern. All biological species showed unique Ava II banding patterns. Generally speaking, the interspecific similarities were around 42% and lower, indicating a distinct species separation. The analysis of rDNA RFLP patterns by a restriction-enzyme-rDNA-probe combination (for instance, Ava II or Bgl II/Hin dIII; probe pMY 60) is a practical means of identifying European Armillaria species for further taxonomic, phylogenetic and host-pathogen interaction studies.     

Distribution of Armillaria species in upland Ozark Mountain forests with respect to site,overstory species composition and oak decline

Armillaria root disease is a contributing factor to oak decline in the Ozark Mountains of central USA. We have identified Armillaria gallica, Armillaria mellea, and Armillaria tabescens in Quercus‐Carya‐Pinus forests of the region. Presence/absence patterns of each Armillaria species as well as all possible Armillaria species combinations were analysed by contingency tables and/or stepwise logistic multiple regressions with principal characteristics of the studied sites and forest stands, both quantitative and qualitative: geographic land‐type association, bedrock type, landform position, slope direction (aspect), soil type and soil surface stone cover, down woody debris, abundance and basal area of woody vegetation and decline mortality by species. Most decline mortality consisted of two red oak species (section Erythrobalanus, Quercus coccinea and Quercus velutina), which also were most sensitive to Armillaria infection. Site characteristics related to the distributions of Armillaria species and decline mortality were also related to the preponderance of Q. coccinea and Q. velutina, regional vegetation history (i.e. conversion of Pinus echinata stands to hardwoods), and the different strategies of territory acquisition and spread of the Armillaria species involved. The presence of A. gallica may reduce the activity of more virulent Armillaria species.     

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.     

Armillaria species distribution on symptomatic hosts in northern hardwood and mixed oak forests in western Massachusetts

Armillaria spp. are some of the most important forest pathogens in mixed hardwood forests of southern New England, yet their role as prominent disturbance agents is still not fully appreciated. We investigated the distribution of Armillaria species across eight separate stands of northern hardwood and mixed oak forests in western Massachusetts. We were specifically interested in the Armillaria species distribution from live, symptomatic hosts and not in determining overall incidence in the forest. From 32 plots (16 within each forest type), 320 isolates were collected. Armillaria was routinely encountered causing disease of live trees. In total, 89% (286/320) of all isolations came from live hosts exhibiting symptoms of root and butt rot. Overall, A. gallica was the dominant species in each forest type, making up 88/160 (55%) isolates from northern hardwood and 153/160 (96%) of all isolations from mixed oak stands. However, northern hardwood forests showed much greater species diversity, as A. calvescens, A. gemina, A. ostoyae, and A. sinapina were all found. At one site, a northern hardwood forest surrounding a high elevation spruce-fir forest, A. ostoyae was the most abundant species encountered. All five Armillaria species were found causing disease of live hosts, including A. gemina, a species considered by some as weakly virulent. Armillaria gallica was found on 22/23 tree species’ sampled, and was found most often causing butt rot.     

Armillaria trapping in aPinus koraiensis plantation in northeast China

Trap bags were used to assess the presence of anArmillaria species in aPinus koraiensis plantation in Heilongjiang, China. 216 trap bags containing bark were placed in three plots at 2 × 2m spacing. After 10 weeks, the presence ofArmillaria was determined from the growth of rhizomorphs in the trap bags. The trap bag method proved successful in determining the presence of viableArmillaria rhizomorphs where only a few dead trees showed signs of the disease. The distribution of positive traps corresponded generally with the presence of rhizomorphs in the soil taken from the trap holes. However, the trap bags were superior to soil samples for evaluating the presence of viable rhizomorphs. The fresh, abundant rhizomorphs were easily identified by persons inexperienced in the assessmentof Armillaria root rot. Several practical problems need to be solved before the method is applicable in general forest management in China.     

Study on isozyme electrophoresis ofMartes zibellina L.

Four isozymes, such as Malate dehydrogenase (MDH), Alchol dehydrogenase (ADH), Peroxidae (POD) and Esterase (EST) in six tissues (heart, liver, kidney, muscle, eye, gonad) ofMartes zibellina L., were analyzed by means of vertical polyacrylamide gel electrophoresis (PAGE). The results indicated that the zymograms of these four isozymes in different tissues were different from each other, i.e. there existed apparent tissue-specificitity in these isozymes inMartes zibellina L.. Characteristic enzyme band was found both in POD zymogram and in EST zymogram. Moreover, the characteristic enzyme band in POD isozyme would be of some value to sexual identification ofMartes zibellina L. (Responsible Editor: Zhu Hong)     

Characterization of North American Armillaria species: genetic relationships determined by ribosomal DNA sequences and AFLP markers

Phylogenetic and genetic relationships among 10 North American Armillaria species were analysed using sequence data from ribosomal DNA (rDNA), including intergenic spacer (IGS‐1), internal transcribed spacers with associated 5.8S (ITS + 5.8S), and nuclear large subunit rDNA (nLSU), and amplified fragment length polymorphism (AFLP) markers. Based on rDNA sequence data, the nLSU region is less variable among Armillaria species than the ITS + 5.8S and IGS‐1 regions (nLSU < ITS + 5.8S < IGS‐1). Phylogenetic analyses of the rDNA sequences suggested Armillaria mellea, A. tabescens and A. nabsnona are well separated from the remaining Armillaria species (A. ostoyae, A. gemina, A. calvescens, A. sinapina, A. gallica, NABS X and A. cepistipes). Several Armillaria species (A. calvescens, A. sinapina, A. gallica, NABS X and A. cepistipes) clustered together based on rDNA sequencing data. Based on the isolates used in this study, it appears that techniques based on IGS‐1, ITS + 5.8S, and/or D‐domain/3′ ends of nLSU are not reliable for distinguishing A. calvescens, A. sinapina, A. gallica and A. cepistipes. However, AFLP data provided delineation among these species, and AFLP analysis supported taxonomic classification established by conventional methods (morphology and interfertility tests). Our results indicate that AFLP genetic markers offer potential for distinguishing currently recognized North American Biological Species (NABS) of Armillaria in future biological, ecological and taxonomic studies.     

Molecular‐based identification and phylogeny of Armillaria species from Serbia and Montenegro

Armillaria causes problems of root rot, kill trees and decay wood in the forests of Serbia and Montenegro, but the species involved have not hitherto been identified. The aim of this study was to identify field isolates collected on 25 localities. Identification was based on restriction fragment length polymorphism (RFLP) analysis of intergenic spacer 1 (IGS1) region and comparisons of IGS1 sequence with those available on NCBI database. Phylogenetic analysis was performed on sequence information from selected isolates to determine possible interrelationships between isolates with different banding patterns and previously identified tester isolates of five European Armillaria species. Five Armillaria species were identified in 90 isolates obtained from forests in Serbia and Montenegro. Armillaria gallica was most frequently isolated, followed by A. cepistipes, A. mellea, A. ostoyae and A. tabescens; two isolates remained unidentified. Restriction digestion of IGS1 amplification products with AluI produced 10 RFLP patterns. Patterns G4 (400, 250, 180) for A. gallica and pattern X (400, 180, 140) for isolates 74 and 79 are reported for the first time in European isolates. Eight RFLP patterns were observed after restriction with TaqI. Two patterns each were observed for A. ostoyae and A. gallica, and one each for A. cepistipes, A. mellea, A. tabescens and isolates 74 and 79. Parsimony analyses based on the IGS1 region placed the isolates into four clades: one including A. mellea, the second containing A. gallica–A. cepistipes isolates, while isolates of A. ostoyae and A. borealis were in the third clade. Armillaria tabescens differed from all annulate species. Phylogenetic analysis supported the conclusion that European Armillaria species are closely related and separated from a common ancestor in the near past. According to this survey five European Armillaria species are present in the forests of Serbia and Montenegro, while A. borealis is not present in the studied ecosystems.     

Polymorphism analysis of Fagaceae and DNA-based identification ofFagus species grown in Japan based on therbcL gene

Fagaceae species in Japan were identified by restriction fragment length polymorphism (RFLP) and sequence comparison of a region ofrbcL. Of nine restriction endonucleases used for digestion, three (MspI,RsaI,HaeIII) produced different restriction patterns in Fagaceae. Digestion byMspI yielded four patterns: Fagus species,Castanea crenata, Pasania glabra, and others. Digestion byRsaI andHaeIII afforded two patterns:Fagus species and others. These facts indicate thatCastanea crenata andPasania glabra can be identified byMspI restriction patterns ofrbcL. Sequence comparison of a region of therbcL gene among 20 species of Fagaceae showed that: (1) they could be divided into seven groups; (2) there is a site mutation betweenFagus crenata andF. japonica. The latter indicates that the wood of both Fagus species are identifiable at the species level, which is not the case using conventional methods. This result indicates the possibility of wood identification based on DNA polymorphism in Fagaceae at the intrageneric level.Part of this paper was presented at the 46th annual meeting of the Japan Wood Research Society, Kumamoto, April 3–5, 1996 and the 47th annual meeting of the Japan Wood Research Society, Kochi, April 3–5, 1997     

Rapid identification of polymorphic sequences in non‐model fungal species: the PHYLORPH method tested in Armillaria species

Development of molecular markers for phylogenetic, population genetics and phylogeographic studies remains arduous in non‐model species with low or no genomic resources. Sequencing the whole or a large part of the genome of the target species using next‐generation sequencing technologies is considered a promising method, although it still needs a large investment in bioinformatics. To quickly find polymorphic markers in fungal species, we tested an alternative method, named PHYLORPH. This method allows users to quickly target polymorphic regions of single copy genes in fungi using public databases. We applied this method to Armillaria species, which are important fungal pathogens and saprophytes playing a central role in the dynamics of forest ecosystems worldwide. We isolated 32 single copy genes with numerous single nucleotide polymorphism (SNP) sites. A genetic analysis of two French populations validated the polymorphism of 80 among 92 SNPs tested, and seven of these sequences exactly reconstructed the known phylogenetic tree of four tested Armillaria species. These results confirmed that the PHYLORPH method is efficient to identify various markers at both the intra‐ and interspecific levels for fungal species with no or few previous genetic markers.     

Influence of wood species on the sawdust-based cultivation ofPleurotus abalonus andPleurotus eryngii

Mycelial growth and fruit body formation ofPleurotus abalonus andP. eryngii cultured on various sawdust-based substrates from different wood species were investigated. Growth onCryptomeria japonica substrate resulted in good mycelial growth and a high yield of fruit bodies.Larix kaempferi substrate was unsuitable for the cultivation of these mushrooms. The fruit body formation rate correlated with mycelial growth from all the wood species tested. Although differences were found for mycelial growth and fruit body formation on various wood species, there were no wood species that were completely unsuitable exceptL. kaempferi. These results show that a wide range of wood species can be used for the cultivation ofP. abalonus andP. eryngii.     

Karyotype analysis of interspecific fusants of basidiomycetes by pulsed-field gel electrophoresis

The purpose of this research was to analyze the karyotype of the interspecific fusants of twoPleurotus species. Auxotrophic mutants derived from the cultivated strain ofP. ostreatus andP. cornucopiae were used. Protoplasts were fused electrically, and the fusants were selected under auxotrophic complementation. Esterase isozyme analysis showed that several fusants had isozyme bands originating from both parental strains, and others had unilateral isozyme bands. The fusant that had expressed isozyme bands of both parental strains showed chromosomal DNA bands of both of the parental strains in pulsedfield gel electrophoresis analysis. Despite the above results, the chromosomal composition of the fusants obtained by the pulsed-field gel electrophoresis did not exhibit all of the bands of both fusion parents.     

Characterization of Canadian isolates of Chondrostereum purpureum by protein content,API ZYM and isozyme analyses

Seventeen isolates of Chondrostereum purpureum, collected from New Brunswick, Vancouver Island, and south-eastern British Columbia, Canada, were compared to determine the degree of genetic variation among isolates from different host species and different biogeographic locations. Several characteristics, including mycelial protein content, fungal biomass, API ZYM profile (semi-quantitative assay to detect the presence of 19 different enzymatic activities in an organism), and isozyme patterns were measured in these isolates. All 17 isolates shared similar API ZYM profiles, with only minor variation observed in the level of expression of specific enzymes. Of the 13 isozyme systems examined, eight (acid phosphatase, alkaline phosphatase, β-esterase, β-glucosidase, malate dehydrogenase, peroxidase, phosphoglucomutase, and polyphenoloxidase) produced measurable activity. No qualitative differences were observed between the isolates for all of these enzymes. There appeared to be limited variation between the isolates collected from across Canada and there was no correlation between specific API ZYM, and isozyme patterns and either host plant or geographical source of the isolates.     

Influence of host plant species of Bemisia tabaci (Genn.) (Hom., Aleyrodidae) on some of the biological and ecological characteristics of the entomophagous Serangium parcesetosum Sicard (Col., Coccinellidae)

The entomophagous Serangium parcesetosum Sicard (Col., Coccinellidae) is an effective predator of some whitefly species. However, information on the influence of the preys host plant species on its biological and ecological characteristics is still lacking in the literature. Therefore, the current study focuses on the possible influence of three greenhouse and two field host plant species of Bemisia tabaci (Genn.) (Hom., Aleyrodidae) on the number of eggs laid by S. parcesetosum. In addition, because of the economic importance and widespread planting of cucumber in greenhouses and cotton in the field, these plants were selected for further investigation into the development, mortality, longevity and reproduction of S. parcesetosum at a high temperature in the laboratory. Results showed that S. parcesetosum was able to lay eggs on all five host plant species of B. tabaci, whether greenhouse or field plants. However, among the three greenhouse plant species studied, S. parcesetosum females laid the highest number of eggs on cucumber followed by tomato and then sweet pepper. Of the two field plant species, significantly higher numbers of eggs were laid on tobacco than on cotton. S. parcesetosum could develop either on cucumber or on cotton as preferable host plant species for B. tabaci. There were significant differences in mean developmental duration of larval instars of the same sex between both host plant species; the duration was significantly shorter on cucumber than on cotton. There were no significant differences for mean total developmental duration from egg to adult emergence between both host plant species within the same sex; females showed a mean of 15.9 days and males of 15.1 days on cucumber, while on cotton the means were 17.2 days for females and 16.2 days for males. Total mortality percentage of S. parcesetosum during development from egg to adult stage was lower on cucumber than on cotton, 20.6 and 23.8%, respectively. Longevity of S. parcesetosum varied according to host plant species and sex with a mean of 63.4 days for females and 50.3 days for males on cucumber, and 92.4 days for females and 52.5 days for males on cotton. On cucumber, mean period of oviposition of S. parcesetosum was significantly longer than on cotton. Mean total fecundity was significantly higher on cucumber than on cotton, with means of 97.7 and 31.0 eggs/female, respectively.     

Identification and distribution of Armillaria species associated with an oak decline event in the Arkansas Ozarks

Forests in the Ozark Mountains of northern Arkansas recently experienced a widespread oak decline event. Armillaria, a root rot fungus, has been associated with other oak decline events and may have been an important contributing factor to tree mortality in this event. Although Armillaria has been identified from the Ozark Mountains in Missouri, it has never been investigated in the Arkansas Ozarks. Molecular diagnostic techniques were used in this study to identify species of Armillaria present on roots removed from dead trees of two common oak species, northern red oak, Quercus rubra L., and white oak, Q. alba L., from three geographic areas and on three topographic positions – ridges, south‐ and west‐facing benches. Armillaria(A. mellea, A. gallica or A. tabescens) was identified from 31% of root samples taken from 102 trees in seven of nine sample plots. Armillaria mellea, occurred most often (20 samples, both oak species on seven plots) followed by A. gallica (10 samples, northern red oak only on four plots), and A. tabescens occurred twice (on northern red oak in a single plot). Thus, all three Armillaria species occurred on northern red oaks while A. mellea was the only species recovered from white oaks. Results varied by topographic position with samples from tree roots on ridges having the fewest positive identifications, one of 29. West‐facing benches had the highest positive samples with 20 of 41 testing positive and trees on south‐facing benches were intermediate with 11 of 32 samples from infected trees. This study documents the occurrence of three species of Armillaria in the Arkansas Ozarks and their association with oak mortality resulting from an oak decline event coupled with a red oak borer, Enaphalodes rufulus, outbreak. Further, it documents some potential variation in host/pathogen combinations and forest site conditions.     

Laccase isoenzyme patterns of European Armillaria species from culture filtrates and infected woody plant tissues

Polyacrylamide isoelectric focusing with specific staining for laccase activity was used to characterize laccase from European Armillaria species (Armillaria ostoyae, Armillaria mellea, Armillaria gallica, Armillaria cepistipes). The enzyme was extracted from culture media either supplemented, or not, with pine sawdust, and also from Pinus pinaster naturally infected by A. ostoyae, or artificially inoculated with A. mellea and A. ostoyae. Some differences in banding patterns were found for Armillana isolates according to the species and the culture media, but a common band at pI = 3.4 was found in all the extracts tested, independently of their origin (culture filtrate or wood).     

Development and verification of a diagnostic assay based on EF‐1 α for the identification of Armillaria species in Northern Europe

The overall aim of this study was to develop a new, reliable and rapid diagnostic assay for differentiating six European Armillaria species based on variation in their elongation factor‐1 alpha (EF‐1 α) gene sequences and to verify a set of species‐specific primers on 61 Armillaria isolates from Europe. Partial sequences of the EF‐1 α gene obtained in Armillaria borealis, Armillaria cepistipes, Armillaria gallica, Armillaria mellea, Armillaria ostoyae and Armillaria tabescens revealed sufficient interspecific variation to distinguish among species using nested primers. These primers gave unambiguous bands when tested on representative isolates of five of these species. However, the EF‐1 α sequences of European A. borealis isolates clustered into two distinct clades, termed here AbX and AbY. Specific primers were subsequently designed and tested successfully on both AbX‐type and AbY‐type A. borealis isolates. The taxonomy of A. borealis needs to be elucidated to determine whether a new, as yet unnamed Armillaria taxon exists in Europe. Three A. borealis isolates were also found to have heterozygous sites in their EF‐1 α sequences, which suggests that the gene could exist in more than one copy or that these isolates contain hybrid sequences. A pyrosequencing method was also developed, targeting a small region of EF‐1 α intron 4, which was able to differentiate European Armillaria isolates to the species level and additionally could distinguish AbX‐type and AbY‐type A. borealis isolates.     

Ecology of Armillaria species on conifers in Japan

Distribution, host preference and pathogenicity of Japanese Armillaria species on conifers were investigated on the basis of field collections of 65 isolates. We identified seven Armillaria species from 19 conifer species including six major Japanese plantation conifers using mating tests and sequences of the translation elongation‐1 α gene. Armillaria mellea, Armillaria ostoyae, Armillaria cepistipes and Armillaria sinapina were frequently collected, whereas Armillaria nabsnona, Armillaria tabescens and a biological species Nagasawa’s E were rare. On the basis of host condition when the isolates were collected, A. mellea, A. ostoyae, A. cepistipes and A. tabescens are considered as moderate to aggressive pathogens of conifers in Japan.