The action of allicin against Armillaria spp. in vitro

The effect of allicin (a stabilized garlic extract product) at five different concentrations (0, 20, 30, 50 and 100 mg/l) was studied in vitro on the growth rate of 100 isolates of Armillaria gallica and A. mellea. Isolates were obtained from 41 host genera growing in gardens located in 39 counties in the United Kingdom. Agar plugs of the actively growing Armillaria isolates were added to the centre of malt agar plates infused with allicin, and radial mycelium growth was measured on days 7, 14 and 21. The total number of rhizomorphs and length of rhizomorphs were also measured. Relative growth rates were calculated as the growth rate relative to the controls (0 mg/ l). The relative growth of each isolate at each allicin concentration was used to estimate EC₅₀ values for A. mellea and A. gallica populations as well as individual isolates. EC₅₀ values for both Armillaria spp. increased over time. The mean EC₅₀ values for A. mellea of 16.0, 26.4 and 102 mg/l (days 7, 14 and 21, respectively) were higher than those for A. gallica (8.8, 7.9 and 11.0 mg/l) and probably relate to the more aggressive nature of A. mellea. Isolates with higher EC₅₀ values were also more likely to produce more rhizomorphs. At allicin concentrations of 20 and 30 mg/l, the production of rhizomorphs and the growth rates of A. mellea isolates were stimulated, when compared to the control treatments. From this study's findings, it appears that the field use potential of allicin is limited, due to better inhibition of the less virulent A. gallica, than the more aggressive A. mellea.     

Stimulation of Armillaria mellea growth by plant hormones in relation to the concentration and type of carbohydrate

Thalli of Armillaria mellea grew vigorously and produced abundant rhizomorph after 21 days of incubation on a glucose-L-asparagine medium supplemented with auxin, but grew poorly and failed to produce rhizomorphs on a non-supplemented medium or on one supplemented with either gibberellic acid or kinetin. Replacement of glucose in auxin-supplemented medium with arabinose, fructose, galactose, maltose, sucrose or starch resulted in poor thallus growth and a lack of rhizomorph development. The superiority of glucose as a carbon source for thallus growth appears to be related to its superiority as a substrate for metabolism by A. mellea.     

Penetration of Picea sitchensis root bark by Armillaria mellea,Armillaria ostoyae and Heterobasidion annosum

Penetration of root bark tissues of Picea sitchensis by Armillaria ostoyae, Armillaria mellea and Heterobasidion annosum was examined in the absence of wounds, in superficial wounds (rhytidome tissues removed to expose the secondary phloem) and in wounds to the depth of the vascular cambium (deep wounding). Both species of Armillaria penetrated bark without prior wounding, but neither species formed rhizomorphs in this treatment. Armillaria ostoyae penetrated to 39 cell layers in depth by 48 days after inoculation of unwounded bark, whereas A. mellea penetrated 25 cell layers in the same time. Armillaria mellea penetrated superficial wounds significantly more rapidly than did A. ostoyae. Both species produced rhizomorphs within wounded host tissues. Inoculation of deep wounds with Armillaria resulted in a greater depth of bark necrosis with A. mellea than with A. ostoyae. In the absence of wounding, H. annosum failed to penetrate root bark tissues, but in both superficial and deep wounds hyphae penetrated beyond the ligno–suberized boundary zone (LSZ) by 12 days after inoculation. Where no inoculations were made, superficial or deep wounding led within 25 days to the restoration of a structurally continuous LSZ, and by day 48 the wound periderm (WP) was fully differentiated. In inoculated wounds, however, formation of the LSZ and WP was delayed or inhibited in most trees, particularly following inoculation with A. ostoyae or A. mellea. Suberization in the LSZ and WP remained diffuse and discontinuous 48 days after inoculation. Moreover, the presence of WP did not prevent further penetration of the tissues by the pathogens. Variations between trees in the depth of pathogen penetration were noted, possibly indicating differing susceptibilities of individual host genotypes. The possible host factors involved in resistance to penetration of root bark tissues by Armillaria and Heterobasidion are discussed.     

Penetration and colonization of oak roots by Armillaria mellea in Wisconsin

The level of Armillaria mellea activity on root systems of living and herbicide-killed oâks was assessed. Epiphytic rhizomorphs were almost always present on living and dead oak roots. After an oak was killed or completely defoliated, A. mellea usually penetrated directly through the root-collar bark and spread rapidly in the cambium. Sapwood in the root collar and main lateral roots was colonized within 2 years. The production of thizomorphs from this region was assessed on trees that had been killed by herbicide sprays 2 to 14 years before sampling.     

The effect of Armillatox on the mycelial growth and rhizomorph production by Armillariella mellea in culture

Cultural studies have shown that unautoclaved Armillatox, a proprietary fungicide, when incorporated in an agar medium killed inocula of A. mellea mycelia at 550 ppm concentration and rhizomorphs at 50 000 ppm. Evidence on detoxification of Armillatox in agar culture and its probable role on new rhizomorph production is reported. Although Armillatox at 10000 ppm appeared to cause profuse production of new rhizomorphs in liquid culture, the difference in the dry weight between the treated and the control Samples has been found to be statistically insignificant.     

Influence de divers types de matière organique sur l'initiation et la croissance des rhizomorphes d'Armillariella mellea (Vahl) Karst. dans le sol

Influence of different organic materials on the initiation and growth of rhizomorphs of Armillariella mellea (Vahl) Karst. in the soil. The influence of different types of organic material on the initiation, growth, and branching of subterranean rhizomorphs of Armillariella mellea (Vahl) Karst. was studied under experimental conditions. Substances rich in lignin and cellulose stimulate rhizomorphic growth while reducing the number of rhizomorphs and their degree of branching. Substances rich in starch and organic nitrogen reduce both the growth and initiation of rhizomorphs, sometimes to a great extent.     

Recherche sur la nutrition azotée de plusieurs souches1 de ĽArmillaria mellea

Investigation of nitrogen nutrition in different strains of Armillaria mellea. I. Utilisation of different sources of nitrogen. The assimilation of different forms of nitrogen - NO₃-, NH₄+ and L-asparagine by isolates of Armillaria mellea from France and Poland was investigated. Both the growth aspect, in which mycelial biomass production was related to the utilisation of nitrogen sources, and the morphological aspect, involving aerial mycelium, sclerotia and rhizomorphs, were studied. Significantly difterent effects were recorded for the influence of nitrogen source, the period of growth and their interaction on the growth of various isolates. Different nitrogen sources significantly affected sclerotial and rhizomorph formation. Isolates of A. mellea, geographically and genetically different, were shown to be also physiologically specialised as was also C. tabescens.     

Examination of low-pathogenicity isolates of Armillaria mellea from natural stands of Picea abies in Middle-Europe

In certain indigenous Picea abies Karst. stands in the Thuringian Forest (Middle-Europe), the incidence of butt rot and die-back of saplings caused by Armillaria mellea (Vahl ex Fr.) Karst. is conspicuously low in spite of a general spread of this fungus on stumps of clear-fellings. The majority of 43 isolates of A. mellea recovered from 13 highland infection centres were unable to form the brown type of rhizo-morphs (BR) in soil but could form the white type rhizomorphs (WR) on sterile substrates. In contrast, 17 isolates from lowland hardwood stands of average disease incidence produced both types of rhizo-morphs on both substrates. Results of inoculation of excised stem and root sections of Picea abies and Fagus sylvatica L. and saplings of P. abies associated with soils of different fertility levels indicated that the low disease index of certain P. abies stands is caused by the dominance of low-pathogenicity strains of A. mellea which lack the ability to form BR and penetrate non-injured bark of roots. Moreover, the poor podzolic highland soil types may contribute more to increase the survival rate and resistance of transplanted P. abies saplings than to reduce the spread of A. mellea.     

Stump infection by Armillaria in first-rotation conifers

Information about the entry of Armillaria into first-rotation pine and spruce stands was obtained by searching for infected stumps, rhizomorph systems or trees that had been killed. In pines Armillaria foci were very rare. In pure Norway spruce Armillaria lutea and A. mellea were detected in stumps but rhizomorphs did not extend into the soil; in Norway spruce mixed with oak, by contrast, A. lutea sometimes produced extensive rhizomorph systems. In Sitka spruce small groups of trees had been killed by A. ostoyae. All foci investigated in conifers contained different genotypes of Armillaria and probably originated from spore infection of stumps created by thinning. Some implications of these findings are discussed.     

Ecology of Armillaria species in managed forests and plantations in Serbia

The distribution of Armillaria species was investigated in Serbian forest ecosystems, in relation to the main host species attacked, forest‐types, geography and altitude. In total, 388 isolates were identified from 36 host species in 47 sites. Armillaria gallica was the most commonly observed species with the widest distribution and with an altitudinal range of 70–1450 m, it was the dominating Armillaria species in lowland alluvial forests and in Quercus and Fagus forests at higher elevations. Armillaria mellea occurred in Quercus spp. – dominated forests in the north and central regions at 70–1050 m. Sixty‐eight per cent of the A. mellea isolates were collected from living hosts, most commonly in declining conifer plantations. Armillaria ostoyae was distributed in the cooler coniferous forest types and plantations in the Dinaric Alps in the south of Serbia, at 850–1820 m. Armillaria cepistipes was found in the eastern and southern hilly and mountainous regions of the country, at 600–1900 m. Most isolates were obtained from conifers and rhizomorphs in the soil around decaying stumps. Armillaria tabescens was found only on dead oak material in the northern and eastern regions of the country at altitudes lower than 600 m.     

Armillaria root rot: Artificial inoculation and development of the disease in greenhouse1

Inoculation of five softwood provenances have revealed that they vary considerably with regard to their percent infection, percent mortality, severity of disease symptoms and progress of infection. Sitka spruce was the most susceptible species and served as a better substrate for further development of mycelium and rhizomorphs as compared to Norway and black spruces and Scots pine. The fungus always entered through uninjured surface of root collar and/or roots, the latter with more than 2 mm diameter. There was a conspicuous reduction m the total and annual growth in height in A. mellea-infected seedlings.     

Rhizomorph production and stump colonization by co‐occurring Armillaria cepistipes and Armillaria ostoyae: an experimental study

In managed spruce forests, Armillaria cepistipes and A. ostoyae are efficient stump colonizers and may compete for these resources when they co‐occur at the same site. The aim of this experiment was to quantify the mutual competitive ability of the two Armillaria species in producing rhizomorphs and in colonizing Norway spruce (Picea abies) stumps. Five isolates of A. cepistipes and two isolates of A. ostoyae were simultaneously inoculated pair‐wise into pots containing a 4‐year‐old spruce seedling. For comparison, each isolate was also inoculated alone. One year after inoculation, stumps were created by cutting down the seedlings. Six months after creation of the stumps, rhizomorph production and stump colonization were assessed. Armillaria spp. were identified from 347 rhizomorphs and 48 colonized stumps. Armillaria cepistipes dominated both as rhizomorphs in the soil and on the stumps. Nevertheless, A. ostoyae was relatively more frequent on the stumps than in the soil and A. cepistipes was relatively more frequent in the soil than on the stumps. In both species, the ability to colonize the stumps in simultaneous inoculations was significantly reduced compared with single inoculations. In respect to rhizomorph production, simultaneous co‐inoculations had a slightly stimulatory effect on A. cepistipes and no significant effect on A. ostoyae. Our study suggests a rather neutralistic co‐existence of A. cepistipes and A. ostoyae as rhizomorphs in the soil. Concerning the ability to colonize stumps, the two species experience a mutual negative effect from the interaction, probably because of interspecific competition.     

A tree-ring study of Norway spruce infected with the wood-destroying fungus Armillaria mellea

A stand otPicea abies (L.) Karst. was studied using the method of tree-ring analysis. The stand involved non-infected trees and trees at different stages of infection by the wood-destroying fungus Armillaria mellea (Vahl. ex Fr.) Kummer [Armillariella mellea (Vahl.) Karst.].     

Phylogenetic analysis of Japanese Armillaria based on the intergenic spacer (IGS) sequences of their ribosomal DNA

To determine the phylogenetic positions of two new species, Armillaria jezoensis and Armillaria singula, and one new subspecies, Armillaria mellea suhsp. nipponica, the nucleotide sequences of the intergenic spacers (IGS) of their ribosomal DNA were investigated, and compared with those of tour other Armillaria species from Japan, and those of nine Armillaria species from Europe and North America. We conclude that Armillaria jezoensis, and Armillaria singula belong to the Armillaria gallica cluster as Armillaria cepistipes, Armillaria gallica and Armillaria sinapina from Japan. Two isolates of Armillaria ostoyae from Japan were placed within the Armillaria ostoyae cluster. Armillaria mellea subsp. nipponica had an IGS sequence as long as the IGS of Armillaria mellea from Europe and North America. However, the IGS sequences of Armillaria mellea subsp. nipponica, whose basidium base lacks a clamp connection could not be satisfactorily aligned with the IGS sequences of other species possessing this morphological feature.     

Comparison of the virulence of Armillaria cepistipes and Armillaria ostoyae on four Norway spruce provenances

The basidiomycetes Armillaria cepistipes and Armillaria ostoyae frequently occur in the same forest stand. In this study, we determined the virulence of 20 isolates of A. cepistipes and 16 isolates of A. ostoyae on four different provenances of 2‐year‐old Norway spruce (Picea abies). Within 30 months after inoculation, 1.1 and 19.1% of the seedlings inoculated with A. cepistipes and A. ostoyae, respectively, had died or were dying. The incidence of dead and dying seedlings varied between 3 and 49% among the A. ostoyae isolates. The virulence of an isolate was positively correlated to its ability to produce rhizomorphs. One Norway spruce provenance showed significantly lower susceptibility to A. ostoyae than the other three. Rhizomorphs of both Armillaria species were attached to the root surface. The attached rhizomorphs of A. ostoyae, however, were associated with significantly more lesions. The virulence of the isolates was not correlated with their wood‐degrading capability for either of the Armillaria species.     

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.     

Infection by Armillaria mellea and some Factors Affecting Host Resistance and the Severity of Disease

Infection by A. mellea in Britain occurs by means of rhizomorphs;the transfer of mycelium at root contacts is probably not importantas a means of spread. Following the initial phase of rhizomorphpenetration, established infection in the cambium is precededby ectotrophic spread of mycelium in the bark scales. Field investigation confirmed reports in the literature thatin susceptible conifers the greatest number of deaths generallyoccurs approximately 6–8 years after planting. However,attacks can also occur in much older crops and the reasons forthis are discussed. Inoculation experiments showed that suppressed trees and thoseexperiencing conditions of reduced light intensity are moresusceptible to infection than dominant trees and trees in fulllight. Species susceptibility in a variety of soils was studiedby inoculation of trees 20–30 cm tall. Of the coniferstested Pseudotsuga menziesii and Abies grandis were more resistantthan Picea abies, P. sitchensis, Larix kaempferi and Pinus sylvestris.Quercus robur and Fagus sylvatica were more resistant than allthe conifers except A. grandis. Soil type influenced diseasedevelopment and the proportion of trees infected was higherin an acidic sand soil than in an alkaline clay soil.     

Armillaria in an ancient broadleaved woodland

In an ancient broadleaved woodland in eastern England that had been coppiced regularly for over 700 years Armillaria mellea and A. gallica were common, A. tabescens was moderately frequent, whilst A ostoyae and A cepistipes were local. Fruit bodies were often abundant in areas coppiced the previous winter. The largest focus of A gallica occupied 9 ha and was probably over 500 years old. Large trees were rarely killed but A mellea caused some root decay in birches. The number of coppice shoots killed by Armillaria was small but became greater as the period since coppicing increased; A gallica was most often involved. Alder coppice was the most, and hazel coppice the least, affected. The productivity of coppiced woodland in general seemed little reduced by Armilaria.     

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.     

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.