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.     

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.     

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.     

The production of rhizomorphs by Armillaria mellea from stumps

The production of rhizomorphs by Armillaria mellea from stumps samples collected in East Anglia was studied: those of broad-leaved trees produced greater yields than those of pines and, in the absence of poisoning, over a much longer period after felling. The ability of A. mellea to form rhizomorphs is often reduced after prolonged growth in pine tissues. The interaction of A. mellea and other fungi is briefly considered. Some implications of these observations for forestry practice are discussed.     

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.     

Liquid culture and transformation of hinoki cypress (Chamaecyparis obtusa Sieb. et Zucc.)

Hinoki cypress (Chamaecyparis obtusa) is one of the most important timber resource forest trees in Japan. Because seed production from a seed orchard of hinoki cypress is not constant every year, micropropagation from a limited amount of material is useful. Up to now, the conventional tissue culture method using solid medium has been used. Here a new method using liquid culture in tubes rotated vertically is described. Shoot primordium of hinoki cypress was inoculated in Campbell and Durzan’s (CD) liquid medium containing different cytokinins (6-benzylaminopurine (BAP), Zeatin, thidiazurone (TDZ)), and the container tubes were rotated vertically around the axis at 2 times / min. Culture room temperature was 25°C and light condition was 16 h photoperiod per day of fluorescent lamps. Zeatin at 1μM concentration was the best for maintaining the shoot primordium production and TDZ induced callus on the surface of the shoot primordia. After shoot primordium multiplication in the liquid culture, they were transplanted to agar medium for shoot elongation. A high concentration of agar (up to 16 g/L) or AVF (anti vitrification factor from Dr. Nairn, 1995) was effective to prevent vitrification of the shoots. Transformation of shoot primordium was done using particle bombardment with vectors containingβ-glucuronidase (GUS) gene or herbicide resistance gene (bar). Positive result for transient transformation was observed with the histo-chemical study for transformation with GUS. Integration of a useful herbicidebar gene into the shoot primordium culture system was also tried and stably transformed plants were obtained. This is the first report of stable transformation of Japanese conifer using practically useful gene. The generous supply of AVF-B from Dr. B.J. Nairn, Tasman Forestry, NZ is also appreciated.     

Rhizomorph growth habit,saprophytic ability and virulence of 15 Armillaria species

The rhizomorph branching habit in soil, competitive saprophytic ability and virulence were determined for 15 species of Armillaria from Europe, North America and Australia and New Zealand. In soil, rhizomorphs of northern hemisphere species branched either monopodially or dichotomously, whereas all five species from Australia and New Zealand branched dichotomously. The dry weight of rhizomorphs produced in soil by isolates of a species and by species was very variable. Species with monopodially branched rhizomorphs had significantly higher saprophytic colonization scores than dichotomously branched species and scores were significantly higher in Garry oak than Douglas‐fir segments and in fresh than autoclaved segments. The damage to Douglas‐fir seedlings caused by isolates of most dichotomously branched species was significantly greater than that caused by monopodially branched species. Species producing dichotomously branched rhizomorphs were more aggressive than monopodially branched species, killing 80% (vs. 17%) of seedlings that died during the first year of the 2‐year experiment.     

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.     

Effects of metals and pH on in vitro growth of Armillaria ostoyae and other root and butt rot fungi of red spruce

Armillaria ostoyae, Perenniporia subacida, Resinicium bicolor and Scytinostroma galactinum, root and butt rot fungi found on red spruce, Picea rubens, were tested, in vitro, for their sensitivity to metals typically found in high elevation forest soils where red spruce grows. Rhizomorph production by A. ostoyae from woody inocula in soils from red spruce stands at three elevations at each of five mountainous sites in the eastern United States was inhibited completely in the mineral soil from all elevations at all sites, and was also reduced significantly in the organic horizon from the upper two elevations at three of the sites. Inhibition was correlated with concentrations of metal ions in the soil. Growth of rhizomorphs into an agar medium containing lead and other heavy metals was inhibited for isolates of A. ostoyae from red spruce, but not for an isolate of Armillaria gallica from sugar maple; aluminium inhibited rhizomorph growth of isolates of both species. Mycelial growth of all four root and butt rot fungi was inhibited by lead, aluminium and other heavy metals depending on the solubility and concentration of metal and pH of the medium; growth inhibition was usually greater at an initial pH of 3.5 than at pH 4.5. Metal ions inhibited radial growth of Armillaria species more than that of the other three fungi. Rhizomorph growth of Armillaria was inhibited more than radial growth. Because local spread of A. ostoyae occurs frequently by means of rhizomorph growth between near roots, increases in lead, aluminium and other metals in the forest floor may contribute to this fungus' scarcity in high elevation soils and reduced incidence of infection at these sites in the eastern United States.     

Spatial pattern of the density of Armillaria epiphytic rhizomorphs on tree collar in an oak stand

The density of Armillaria gallica epiphytic rhizomorphs on tree collar was mapped in a young stand of pedunculate oaks (Quercus robur). A quick method to estimate the rhizomorph density was developed, investigating 19 trees, and was used in the subsequent study. The method consists of measuring the frequency of rhizomorphs present on a grid applied to an exposed small area of the tree collar. The pattern in tree collar rhizomorph density was closely related to the pattern of stump colonization by Armillaria and stumps were therefore important as inoculum sources. Rhizomorph density on tree collar showed a strong aggregated spatial pattern which could not be explained by variability in the soil characteristics, in particular waterlogging, or by a spatial pattern in tree vigour. Moreover, rhizomorph density on tree collar did not depend on their dominance status.     

Quantification of Armillaria rhizomorphs in Wisconsin aspen sucker stands

Weights of rhizomorphs of undetermined Armillaria sp. obtained using ring-trench and core soil sampling methods around aspen stumps in stands at different intervals after harvest were correlated. The core method was used to sample a second series of stands twice (at an interval of two growing seasons) at radial distances of 0.5, 1.0, and 1.5 meters from stump centers. Stands were uncut (at both samplings) or 3, 6, 9, 12, or 15 years after harvest (at the time of the initial sampling). Rhizomorphs were obtained from less than half the cores from the uncut stand, and the quantities (weights and lengths) were relatively small from both samplings. Mean numbers of soil cores yielding rhizomorphs and the mean quantities obtained generally increased as a function of the interval after harvest (and from the first to the second sampling), except for the oldest stand.     

Mycorrhizal dependency and growth responses of Acacia nilotica and Albizzia lebbeck to inoculation by indigenous AM fungi as influenced by available soil P levels in a semi-arid Alfisol wasteland

A series of available phosphorus (Olsen) levels ranging from 10 to 40 ppm were achieved in a semi-arid soil. The influence of the levels of phosphorus on the symbiotic interaction between two subtropical tree species, Acacia nilotica and Albizzia lebbeck, and a mixed inoculum of indigenous arbuscular mycorrhizal (AM) fungi was evaluated in a greenhouse study. The extent to which the plant species depended on AM fungi for dry matter production decreased as the levels of soil P increased, but the degree of this decrease differed in the two species tested. Acacia nilotica colonized by AM fungi showed a significant increase in shoot P and dry matter at a soil P level of 10 ppm whereas in Albizzia lebbeck, such increase occurred at 20 ppm. Mycorrhizal inoculation response disappeared beyond soil P levels of 25 ppm in Acacia nilotica and 30 ppm in Albizzia lebbeck. Levels of soil P greater than 25 ppm suppressed AM fungus colonization in both species. Soil P levels of 30 and 40 ppm and 40 ppm caused negative mycorrhizal dependencies (MD) in Acacia nilotica and Albizzia lebbeck respectively. Values of MD for both species were negatively correlated with soil P levels. Based on the MD values, regression equations were developed to predict MD for given levels of available P.     

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.     

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.     

Epitypification,characterization and phylogenetic positioning of Pseudobeltrania cedrelae,the causal agent of pseudobeltrania spot on Cedrela fissilis

The fungus Pseudobeltrania cedrelae, the type species of the genus, is the causal agent of an important leaf spot in seedlings and adult plant of cedar (Cedrela fissilis). Due to the contradictory phylogenetic position of the genus Pseudobeltrania, epitypification of P. cedrelae was carried out based on a culture obtained from the same locality and host of the original type. Samples were collected, and 10 isolates of P. cedrelae associated with lesions on cedar leaflets were obtained. For morphological characterization, conidia, conidiophores, conidiogeneous cells, conidiogeneous loci and basal cells were taken both from the fungus obtained from leaf lesions and from that obtained in slide culture. Mycelial growth rates and sporulation were evaluated in six different culture media. For molecular phylogeny, maximum parsimony analyses were performed from the ITS and 28S sequences of the isolates. Both the morphological characteristics of the fungal structures obtained from symptomatic leaves and the slide culture technique presented variations. In foliar lesions, isolates presented the same morphological characteristics as the type material. Mycelial growth rate and sporulation of P. cedrelae were greatest on malt extract agar and V8 juice agar. Pseudobeltrania cedrelae was pathogenic when inoculated into healthy cedar plants. According to the phylogenetic tree, isolates grouped in the same clade, but in a distinct clade of Pseudobeltrania ocoteae. The results suggested that P. ocoteae belongs to the genus Hemibeltrania. This paper presents new information on P. cedrelae that contributes to clarifying the phylogeny of the Beltraniaceae.     

A quick method for the production of Dothistroma pini spores in culture

A new method for the production of Dothistroma pini spores in agar culture is described. Temperature, malt concentration, pH and inoculation density of spores were the main influencing factors.     

Hymenoscyphus albidus is not associated with an anamorphic stage and displays slower growth than Hymenoscyphus pseudoalbidus on agar media

Examination of isolates of Hymenoscyphus albidus from France revealed that this fungus does not form an anamorphic stage in culture. The lack of an asexual stage in this fungus is a conspicuous morphological difference to the ash dieback pathogen Hymenoscyphus pseudoalbidus, which is associated with its Chalara fraxinea anamorphic state. In growth studies on malt extract agar (MEA) and MEA amended with ash leaflets (ash leaf malt extract agar, AMEA) at 20°C, isolates of H. albidus grew slower than those of H. pseudoalbidus. On AMEA, the growth of cultures of both species was greatly enhanced.     

Wood-inhabiting fungi in stems of Fraxinus excelsior in declining ash stands of northern Lithuania,with particular reference to Armillaria cepistipes

Abstract

Stem bases of 210 Fraxinus excelsior trees of three different health categories were sampled by the means of an increment borer in declining ash stands in northern Lithuania. From this number, 15 sound-looking, 132 declining and 63 dead trees from three discrete plots yielded 352 isolates, representing 75 fungal species. In addition, mycelial fans and rhizomorphs typical of Armillaria spp. from 205 and 20 trees, respectively, were sampled and subjected to fungal isolations. Species richness was similar in trees from each health category, but community structures differed, indicating that species composition of wood-inhabiting fungi in stems changes along with the changes in tree health condition. Armillaria cepistipes was the most common species (86 isolates from 210 wood samples, or 41.0%), isolated more frequently and consistently than any other potential tree pathogen. It also showed abundant occurrence on a majority of trees in the form of mycelial fans and rhizomorphs, from which 64 and 14 isolates of the fungus were obtained, respectively. The population structure of A. cepistipes revealed the presence of 53–93 genets per hectare, some of which extended up to 30–55?m.     

Inhibition of Armillaria by bacteria isolated from soils of the Boreal Mixedwood Forest of Ontario

Pseudomonas fluorescens, Pseudomonas spp., Bacillus spp., Enterobacter spp., Serratia spp. and Agrobacterium radiobacter were isolated from root-free soils of the Boreal Mixedwood Forest of Ontario a, nd found to be capable of inhibiting the linear growth of Armillaria ostoyae in vitro. Isolates of P. fluorescens, Bacillus spp. and A. radiobacter were the most effective inhibitors of mycelial growth. To test the ability of the bacteria to suppress rhizomorph formation, A. gallica, which produces rhizomorphs in culture more consistently than does A. ostoyae, was used; only a small fraction of P. fluorescens and Bacillus spp. isolates were capable of preventing in vitro rhizomorph formation by A. gallica.     

Efficacy of spinosad and methoprene,applied alone or in combination,against six stored-product insect species

Efficacy of the insecticides spinosad and methoprene, applied alone or in combination to wheat, was evaluated against six stored-product insect species, Rhyzopertha dominica, Sitophilus oryzae, S. granarius, Cryptolestes ferrugineus, Oryzaephilus surinamensis, and Liposcelis bostrychophila. The concentrations of the insecticides were 0.1 and 0.5 ppm for spinosad and 1 and 5 ppm for methoprene. Parental mortality of R. dominica was 100% on wheat treated with either rate of spinosad, with no progeny production in any combination treatment. Parental mortality of S. oryzae did not exceed 62%, and progeny production was not reduced by any insecticide treatment. All S. granarius adults exposed on wheat treated with spinosad alone at 0.5 ppm were killed, but parental mortality was reduced when methoprene was added. Progeny production was reduced in treatments containing spinosad. Mortality of adult C. ferrugineus was 97% or greater in treatments containing spinosad, but adult mortality of O. surinamensis and L. bostrychophila was not reduced by insecticide treatments. The specific combinations of spinosad and methoprene evaluated in our study would have no benefit over spinosad used alone for control of any of the six species tested.