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.     

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.     

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.     

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.     

Reaction of Armillaria ostoyae to forest soil microfungi

Fungi isolated from the oak (Quercus robur) rhizosphere were tested for their effects on rhizomorph formation and growth of 16 isolates of Armillaria ostoyae sampled in three localities in western Poland. The number of rhizomorphs, number of rhizomorph apices, and rhizomorph length and weight increased most in the presence of Penicillium lanosum, Penicillium notatum, Cylindrocarpon destructans, Penicillium spinulosum and Mycelium radicis atrovirens α and, to a lesser extent, in the presence of Nectria grammicospora. Inhibition of rhizomorph formation was caused by Trichoderma hamatum and Trichoderma viride in two A. ostoyae isolates and by M. radicis atrovirens α and P. spinulosum in one A. ostoyae isolate. It is suggested that variation in sensitivity to microbial stimulation within A. ostoyae is associated with the environmental and nutritional conditions of its original habitat. Isolates from nutrition‐rich localities, with 20% of the land area covered by deciduous trees, were particularly susceptible to stimulation by rhizosphere fungi.     

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.     

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.     

Plant recovery from seedling-derived shoot tips ofFaidherbia albida grown in vitro

In vitro cultures were initiated from shoots taken from seedlings ofFaidherbia albida on Murashige and Skoog based medium supplemented with different combinations of 6-benzylaminopurine (BA) and -naphthaleneacetic acid (NAA). The shoots grew and rooted on all media. Rooting and vigorous growth were most successful on medium supplemented with 10^–7M NAA alone on which 87% of the shoots formed roots. Seventy-one percent of plantlets which were transferred to soil were successfully established and nodulated with the nativeRhizobium. The procedures provide a basis for the development of in vitro techniques for rapid multiplication and physiological studies of the species.     

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.     

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).     

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.     

Stimulation of Armillaria ostoyae vegetative growth by tryptophol and rhizomorph produced by Zygorhynchus moelleri

Tryptophol (an indole-3-ethanol analogue which is a major secondary metabolite produced by Zygorhynchus moelleri) stimulated the growth of 13 out of 18 isolates of Armillaria ostoyae in culture. Rhizomorph production of 16 out of 18 isolates of A. ostoyae was enhanced by the presence of Z moelleri in oak branch segments. Tryptophol can be considered as a growth-promoting substance stimulating the vegetative growth of A. ostoyae in culture, and Z. moelleri as a fungus stimulating the rhizomorph formation on oak wood.     

Effect of different nitrogen and carbon sources and concentrations on the mycelial growth of ectomycorrhizal fungi under in-vitro conditions

Effect of different nitrogen and carbon sources and their concentrations in liquid media on the mycelial growth of six different ectomycorrhizal (ECM) fungal species was studied. Differences were found in the utilization of the different nitrogen and carbon sources between the fungal species. All the species showed better mycelial growth in the medium containing ammonium as nitrogen source. Growth was low in all species in medium in which nitrogen was supplied in nitrate form. All the ECM isolates investigated showed reduced growth in the medium containing maleic acid as the carbon source. The effect of glucose and di-ammonium hydrogen orthophosphate concentrations on mycelia growth of all the six fungal species was studied with ranges for glucose of 2–40 g/l, and for di-ammonium hydrogen orthophosphate of 2–20 g/l. Cortinarius fulvoconicus and Cortinarius flexipes showed maximal mycelial growth at a glucose concentration greater than 20 g/l. Suillus luteus, Scleroderma citrinum, Laccaria laccata, and Tricholoma aurantium showed maximal growth at a glucose concentration of 20 g/l. All six species showed maximal mycelial growth at 5–10 g/l of di-ammonium hydrogen orthophosphate concentration and with increased concentration mycelial growth in all species decreased.     

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.     

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.     

Regeneration and multiplication of Albizia procera Benth. through organogenesis

Vegetative propagation techniques are recognized as indispensable tools for mass multiplication of important multipurpose trees adopted in different agroforestry systems. Albizia procera, one among important species, is difficult to propagate commercially either by stem / root cuttings or layering. A study was undertaken to develop procedure for its in vitro regeneration through organogenesis. Explants collected from 15±2 yr-old mature plus trees and from 15 days old juvenile seedlings were regenerated with exogenous application of different hormones. Epicotyl and hypocotyl explants excised from juvenile seedlings showed higher callusing than axillary bud and shoot tip explants derived from mature trees. Benzylaminopurine (BA) at 3 μg/l was most effective, which induced hundred percent callusing in epicotyl and hypocotyl explants in 1/2 Murashige and Skoog (MS) medium. Callus originated from axillary buds and apical shoot tips of mature trees failed to form organs, however callus derived from epicotyl and hypocotyl explants proliferated and formed de novo shoots and leaflets. A concentration of 3 μg/l of BA was found effective for shoot proliferation. Shoots grew vigorously in 2 μg/l gibberellic acid (GA₃) treatment and rooted in 1/2 MS medium supplemented with indole-3-butyric acid (IBA) and indole-3-acetic acid (IAA). Rooting was most successful on medium supplemented with 6 μg/l IBA alone on which 93.3% of the shoots formed roots. Sand or vermiculite supplemented with 4 ml of yoshida solution proved as best hardening media, which recorded 70-80% survival of plantlets. One year old tissue culture raised plants had comparatively more height, collar diameter, biomass, and root shoot ratio than plants raised from cuttings and seeds of the same age. The procedures enumerated provide a basis for the development of in vitro techniques for rapid multiplication of A. procera. This revised version was published online in June 2006 with corrections to the Cover Date.     

Establishment of an in vitro plant regeneration protocol for Casuarina cunninghamiana Miq. via indirect organogenesis

An in vitro plant regeneration protocol via indirect organogenesis from morphogenetic callus was established for Casuarina cunninghamiana Miq. Effects of plant growth regulator NAA (naphthaleneacetic acid) and BAP (6-benzylaminopurine), sucrose and AgNO₃ on callus induction, adventitious bud differentiation and shoot development were examined. Explants used were epicotyl fragments from 45-day-old seedlings. The largest callus (4.29 mm in diameter) was obtained after 1 month on a basic culture medium consisting of Murashige and Skoog ? macro- and full strength micro- elements, Nitsch and Nitsch vitamins, supplemented with 0.54 μM NAA, 3.30 μM BAP, and 30 g L⁻¹ sucrose. The calli were subcultured in the same medium above for 2 months. They were then cultured for another 2 months for adventitious bud differentiation and shoot development. The highest mean adventitious bud differentiation, number of shoots formed per callus and number of shoots ≥2 cm long per callus (47.50%, 27.38 and 4.75, respectively) were achieved on the above medium modified with NAA at 0.27 μM and supplemented with AgNO₃ 1 mg L⁻¹. Shoots were successfully rooted without plant growth regulator and the rooted plantlets survived and grew normally. This protocol for in vitro plant regeneration provides a tool not only for vegetative propagation but also for plant genetic transformation and gene function studies of C. cunninghamiana.     

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.].     

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.     

Establishment of cell suspension line of <Emphasis Type="Italic">Populus tomentosa</Emphasis> Carr.

Leaves of fine Populus tomentosa genotype TC152 were used as explants to establish cell suspension lines. The effects of plant growth regulators on callus induction and establishment of cell suspension lines were studied. The callus induction rate was the highest on a MS solid medium supplemented with 1.0 mg·L^-1 2,4-D. A cell suspension line could be obtained by inoculating calli which were not subcultured into a MS liquid medium supplemented with 1.5 mg·L^-1 2,4-D. The best subculture medium was MS ＋ 0.8 mg＇L-1 2,4-D ＋ 30 g·L^-1 sucrose with a subculture cycle of seven days.