Effects of wounding and fungal infection with Armillaria ostoyae in three conifer species. II. Host response to the pathogen

Structural responses in the bark and wood were described following penetration by Armillaria ostoyae in the roots of 20‐ to 30‐year‐old Douglas‐fir, western hemlock and western redcedar trees. Tissue necrosis presumably caused by fungal exudates was commonly observed at inoculum contact. In Douglas‐fir and western hemlock, A. ostoyae interfered with the initiation of active defence mechanisms involving the development of a lignified zone of impervious tissue (IT), necrophylactic periderm (NP) formation and compartmentalization of infected woody tissue. Breaching of IT and NP barriers was frequent, particularly around the clusters of sclereid cells in western hemlock. In western redcedar, the IT zone was inconspicuous. Induced rhytidome formation occurred in western redcedar either simultaneously with or after completion of NP development. The formation of this tissue facilitated en masse sloughing of infected tissue from the surface of roots. In western redcedar, traumatic phloem resin ducts formed in tangential bands surrounding the margin of expanded lesions. Effective compartmentalization in western redcedar was achieved by a barrier zone comprised of a higher‐than‐average number of axial parenchyma that accumulated polyphenolic deposits. A combination of host‐mediated defence mechanisms in western redcedar resulted in a significantly higher frequency of effective resistance reactions than in western hemlock or Douglas‐fir.     

Effects of wounding and fungal infection with Armillaria ostoyae in three conifer species. I. Host response to abiotic wounding in non‐infected roots

A parallel investigation to characterize host reactions involved in wound repair following abiotic injury and challenge with Armillaria ostoyae in three conifer species (Douglas‐fir, western hemlock and western redcedar) was conducted in the southern Interior of British Columbia. In this study, we characterize wound healing in roots following freezing injuries to the bark. Five weeks following wounding, all conifers developed lignified impervious tissue (IT) around killed tissue, and a necrophylactic periderm (NP) formed internally abutting IT, although IT was difficult to discern in western redcedar. Phellogen restoration in the secondary phloem of some western hemlock roots was retarded around large clusters of sclereids. Displacement of phloem fibre cells occurred in western redcedar but did not appear to delay NP formation. In roots exhibiting expansion of necrosis to the vascular cambium, callus tissue was evident at the margin of the wound. Novel host reactions in western redcedar involving induced rhytidome formation and the formation of traumatic phloem resin ducts are described.     

Lesion formation and host response to infection by Armillaria ostoyae in the roots of western larch and Douglas‐fir

The process of lesion formation and host response to natural infection by Armillaria ostoyae were studied in the roots of western larch (Larix occidentalis) and Douglas‐fir (Pseudotsuga menziesii ssp. glauca) trees in the three age classes, 6–8, 18–19 and 85–95 years. The characteristics of lesions on infected roots were recorded and bark samples were dissected from infection points and lesion margins in the field and stored in liquid nitrogen for macroscopic study in the laboratory. Infection in the roots of 6‐ to 8‐year‐old trees advanced freely, overcoming any host resistance, quickly girdling the root collar and killing the trees. In 18‐ and 19‐year‐old trees, however, 43% of infections on western larch and 27% of the infections on Douglas‐fir roots were confined to lesions bounded by necrophylactic periderms with multiple bands of phellem. Host response was similar in 85‐ to 95‐year‐old trees, but the percentage of confined lesions was higher than in younger trees. The results suggest that larch shows resistance to A. ostoyae at a younger age and with greater frequency than Douglas‐fir.     

Necrophylactic periderm formation in the roots of western larch and Douglas‐fir trees infected with Armillaria ostoyae. II. The response to the pathogen

Periderm formation was studied in bark samples collected from the roots of western larch (Larix occidentalis) and Douglas‐fir (Pseudotsuga menziesii) trees infected with Armillaria ostoyae. Necrophylactic periderms were formed in advance of infection and successfully restricted continued fungal spread in 68 and 45% of the samples collected from 10‐ and 27‐year‐old western larch, respectively. However, all periderms formed in 11‐ and 25‐year‐old Douglas‐fir had been breached by the advancing fungus. In both species, necrophylactic periderms were commonly breached at the junction of the periderm with the vascular cambium. In western larch, stone phellem often comprised the external phellem layer of necrophylactic periderms with multiple bands of phellem. In 27‐year‐old western larch, infection was often confined to discrete lesions bounded by multiple periderms with multiple bands of phellem. In both tree species, phellem production was greater in response to infection than in response to abiotic wounding.     

Epidemiology of Armillaria root disease in Douglas‐fir plantations in the cedar‐hemlock zone of the southern interior of British Columbia

Results are presented from several studies on the epidemiology of Armillaria ostoyae in Douglas‐fir plantations in the interior cedar‐hemlock (ICH) biogeoclimatic zone of British Columbia. Two plantations were monitored for mortality by A. ostoyae and other agents for 35 years after establishment. In these and other plantations ranging in age from 7 to 32 years, one or more of the following factors were determined: source of inoculum, mode of spread and characteristics of lesions on roots of excavated trees; symptom expression in relation to tree age and damage to the root system and years from initial infection to death on trees killed by the fungus. Mortality from A. ostoyae began in both plantations about 5 years after planting, reaching 30% in one and 11% in the other after 35 years. The spatial pattern of mortality was similar to that reported from New Zealand, France and South Africa; however, the temporal pattern differed, beginning later and, instead of declining, continuing at a nearly constant rate to the present. To age 10, nearly all infections were initiated by rhizomorphs; as plantations aged, the proportion of infections occurring at root–root contacts increased. In seven plantations, in moist and wet subzones of the ICH, from 23 to 52% of Douglas‐firs had root lesions, with the higher incidences occurring on moist sites. The occurrence of aboveground symptoms, reduced leader growth and basal resinosis, was related to the percentage of root length colonized by A. ostoyae on trees with more than 30% of root length killed. Average time from infection to death increased from 1 to 2 years at age 6 to 22 years at age 33. The outlook for timber yield from Douglas‐fir plantations in the cedar‐hemlock zone is discussed. Management alternatives for reducing damage from A. ostoyae when regenerating sites are reviewed.     

Structural responses in bark to mechanical wounding and Armillaria ostoyae infection in seedlings of Pinus sylvestris

The sequence of events leading to the restoration of an intact periderm surface in the stem bark of Pinus sylvestris following wounding and challenge with Armillaria ostoyae was determined. A sub-erized impervious tissue (SIT) was produced outside the necrophylactic periderm (NP). Al-though A. ostoyae initially infected wounded bark with intracelfularly growing hyphae, these died before penetrating deeply into the bark. Later, intercellularly growing rhizomorphs, which had also developed from the inoculum, penetrated the NP and infected the functional phloem/ cambial zone. Compared to the reactions in uninfected wounded bark, the A. ostoyae infection delayed the formation of NP and reduced the frequency of cells involved in lignification and in SIT, but increased the number of lignified cell layers. The rhizomorph infection reached deeper and lignification was more pronounced in defoliated seedlings than in non-defoliated ones. The importance of structural responses in defence against A. ostoyae is discussed.     

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.     

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.     

Necrophylactic periderm formation in the roots of western larch and Douglas‐fir trees infected with Armillaria ostoyae. I. The response to abiotic wounding in non‐infected roots

The sequence of events leading to necrophylactic periderm formation was studied throughout the year following the abiotic wounding of the non‐infected roots of 10‐ and 27‐year‐old western larch (Larix occidentalis) and 11‐ and 25‐year‐old Douglas‐fir (Pseudotsuga menziesii) trees that were infected with Armillaria ostoyae. The sequence was the same for both ages and species of trees. Wound repair was more rapid in the summer compared with the spring and autumn. Following cell hypertrophy, a zone of lignified impervious tissue was in the initial stages of formation within 10 days of wounding in the summer and 14 days in the spring or autumn. The new phellogen produced a layer of phellem three to four rows of cells thick after 20 days in the summer or 40 days in the spring. Modified cells abutting the inner boundary of the impervious zone frequently developed thick lignified abaxial walls and thin suberized adaxial walls. A typical exophylactic periderm in healthy root bark tissue of both western larch and Douglas‐fir consisted of stone phellem one to four rows of cells thick and a layer of thin‐walled phellem three to six rows of cells thick in western larch and two to three rows thick in Douglas‐fir, a single row of phellogen cells and one to three rows of phelloderm cells. Mature thin‐walled phellem cells had pigmented contents, red in western larch and light brown in Douglas‐fir. In response to wounding, 27‐year‐old western larch and 25‐year‐old Douglas‐fir developed necrophylactic periderms with annual bands of phellem. The bands included a layer of phellem that was six to 12 and nine to 15 rows of cells thicker than the layer of phellem observed in the respective naturally developed exophylactic periderms. Fifty days following wounding in the summer, stone phellem, one to three rows of cells thick, was observed in the necrophylactic periderm of 10‐year‐old trees. When fully developed, the necrophylactic periderm in 27‐year‐old western larch also had a layer of stone phellem three to five rows of cells thick in each band. Stone phellem development was only sporadic in 25‐year‐old Douglas‐fir. Wounds in the winter showed no signs of activity associated with repair until dormancy broke in the spring.     

Traumatic resin canals as markers of infection events in Douglas‐fir roots infected with Armillaria root disease

In response to an infection, traumatic resin canals (TRCs) are formed in the roots of many conifers, which may be used to determine the timing and sequence of infection events essential for epidemiological studies of root diseases. Juvenile Douglas‐fir (Pseudotsuga menziesii) tree roots at coastal and interior sites in British Columbia were wounded at various times of the year or were inoculated with an isolate of Armillaria ostoyae, and root sections were taken to determine the timing and extent of TRC formation. Naturally infected Douglas‐fir were also examined to determine the extent of the TRCs in infected and uninfected roots on infected trees and in the lower stem. Wounds made in March and October had poor or no TRC formation while the summer wounds responded strongly and were associated with resin soaking. Roots wounded in October did not respond until the following year in all trees except one. Trees produced TRCs and resin soaked tracheids at all times in response to the fungal inoculations. The most striking difference between wounding or fungal inoculation was the multiple bands of TRCs produced in response to the fungus. TRCs at natural A. ostoyae infections were found 92% of time in roots at the stem junction and 74% of the time in the stem at soil line. TRCs were produced in uninfected roots on infected trees but disappeared with increasing distance from the initiating lesion. TRCs can be used to time yearly and seasonal root infections when they can be traced from an identified lesion.     

Tree vigour and the susceptibility of Douglas fir to Armillaria root disease

In this study the effects of thinning, fertilization and pruning on the vigour of Douglas fir (Pseudotsuga menziesii) and its susceptibility to Armillaria root disease were explored. Tree vigour was defined as the relative capacity for tree growth, expressed as the above-ground biomass increment per unit of photosynthetic tissue, or growth efficiency (GE). It has been hypothesized that trees with higher GE can better resist pathogen attack, and that GE can be used as a predictor of tree susceptibility to disease. In a previous study, four Douglas fir plantations were thinned, fertilized and pruned in all combinations, and the effects of these treatments on tree vigour were measured after 10 years. Root disease was not a factor in the initial study design, and mortality was ignored until 8 years after the treatments were applied. The results of an earlier study were utilized and the correlation between Armillaria root disease incidence and the effects of earlier stand treatments on tree growth was tested. Armillaria ostoyae was the primary mortality agent in the study area. Disease incidence on infested subplots ranged from 2 to 20%. Armillaria ostoyae incidence was the highest at medium tree density (6.1%), slightly lower on the low density (5.6%), and lowest on the unthinned plots (3.8%). There were no significant correlations between disease incidence and previous tree growth. The vigour of trees that became symptomatic or died by 1993 was not significantly different in 1983–85 from the vigour of trees that remained asymptomatic. On these sites, in areas of infestation, A. ostoyae is killing the largest, fastest growing trees, as well as less vigorous trees. Armillaria continues to cause mortality, regardless of the growth efficiency or growth rate of the host.     

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.     

Phylogeographic patterns of Armillariaostoyae in the western United States

Nuclear ribosomal DNA regions (i.e. large subunit, internal transcribed spacer, 5.8S and intergenic spacer) were sequenced using a direct‐polymerase chain reaction method from Armillaria ostoyae genets collected from the western USA. Many of the A. ostoyae genets contained heterogeneity among rDNA repeats, indicating intragenomic variation and likely intraspecific hybridization. Intragenomic variation was verified by visually editing base‐sequence offsets in regions with insertions/deletions, and using sequence‐specific internal primers to resequence heterogeneous regions. Phylogenetic analyses with Bayesian Inference methods were used to define groups within A. ostoyae. Analysis of A. ostoyae from outside the western USA indicated the presence of a Circumboreal group of A. ostoyae that also occurs in Utah; two other phylogeographic groups were associated with the Rocky Mountain and Pacific Northwest regions of the USA. Mixed sequence types, an indication of intraspecific hybrids, were common in some geographic regions. Hybridization events may have influenced species evolution, contributing to variation in pathogenicity and virulence. The occurrence of these groups and intraspecific hybrids also indicates that paleogeography and paleoclimate may have influenced the phylogeography of A. ostoyae. In addition, other Armillaria species were examined for evolutionary relationships with the groups of A. ostoyae. These findings will provide a basis for future research relating ecological function to genetic diversity within A. ostoyae.     

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.     

Evidence of resistance to hemlock dwarf mistletoe (Arceuthobium tsugense) in western hemlock (Tsuga heterophylla) clones

Records from western hemlock (Tsuga heterophylla) trees naturally exposed to infection by hemlock dwarf mistletoe (Arceuthobium tsugense) and controlled inoculations of grafted branch scions from a selection of these trees indicated a repeatability of the levels of resistance to the disease in the clonal material. Evidence pointed to a resistance mechanism operating within, rather than outside, the host branch.     

Midcanopy growth following thinning in young-growth conifer forests on the Olympic Peninsula western Washington

Midcanopy layers are essential structures in “old-growth” forests on the Olympic Peninsula. Little is known about which stand and tree factors influence the ability of midcanopy trees in young-growth forests to respond to release; however, this information is important to managers interested in accelerating development of late-successional structural characteristics. We examined basal area growth response of midcanopy trees following variable-density thinning in an effort to determine the effect of thinning and local environment on the release of western hemlock (Tsuga heterophylla (Raf.) Sarg.) and western redcedar (Thuja plicata ex. D. Don) on the Olympic Peninsula in western Washington. Release was measured as the difference between average annual basal area growth over the 5-year prior to thinning and the 3-to-6 year period following thinning. Results indicate that while growth rates were similar prior to thinning (5.4 cm² year⁻¹in both thinned and unthinned patches) midcanopy trees retained in a uniformly thinned matrix grew significantly more (8.0 cm² year⁻¹) than those in unthinned patches (5.4 cm² year⁻¹) for western hemlock and for western redcedar. Crown fullness and crown crowding affected the release of western hemlock in the thinned matrix. Initial tree size, relative age, local crowding and measures of crown size and vigor affected the release of western redcedar in the thinned matrix. Our results indicate that midcanopy western hemlock and western redcedar retain the ability to respond rapidly with increased growth when overstory competition is reduced and the magnitude of response is related to neighborhood variables (intracohort competition, overstory competition, and tree vigor), thus suggest that variable-density thinning can be an effective tool to create variability in the growth of midcanopy trees in young-growth stands. We expect that this rapid response will produce even greater variability over time.     

Parasitic association of the mycetophagous wood nematode,Bursaphelenchus fraudulentus with the honey fungus Armillaria ostoyae

Honey fungi, Armillaria spp. are common and economically important pathogens of a wide range of tree species grown both in the forest and orchard cultures worldwide. Our field research in 73 forest stands across Poland has shown a common association of the mycetophagous wood nematode, Bursaphelenchus fraudulentus with Armillaria‐infected trees. The data reported here provide the first insight into the parasitic interaction of a nematode and Armillaria ostoyae. In laboratory conditions, B. fraudulentus reproduced on A. ostoyae, caused substantial damage to its mycelium and, finally, killed the whole colony within a short time. In ageing cultures, the nematode produced resting (dauer) juveniles. After artificial inoculation to Armillaria‐infected trees, the nematode population persisted under the bark, continued feeding and mass reproduction on the mycelium, and dispersed over the mycelial fans. The ability of B. fraudulentus to develop and reproduce on the surface and inside the fungal rhizomorphs suggests that these structures could facilitate the nematode dispersion in the environment. Since B. fraudulentus is not pathogenic to the host tree, its parasitic association with A. ostoyae may contribute to natural regulation of this fungus in the environment. The observed characteristics of this nematode suggest that it could potentially be used as a biocontrol agent of honey fungi in forest and orchard trees.     

Feeding responses of the western dry-wood termite <Emphasis Type="Italic">Incisitermes minor</Emphasis> (Hagen) (Isoptera: Kalotermitidae) against ten commercial timbers

Five Japanese timbers, four timbers from the USA, and one Malaysian timber were evaluated for their resistance to the invasive dry-wood termite Incisitermes minor (Hagen) using laboratory choice and no-choice feeding tests with holed specimens. The highest survival rates of I. minor in both the heartwood and sapwood no-choice feeding tests were more than 70% after 3 months. When offered sapwood and heartwood choice feeding tests and the combined choice feeding tests, the highest survival rates of I. minor were more than 75% after 3 months. With regards to the percentage of wood mass losses in the no-choice and choice feeding tests, karamatsu (Larix leptolepsis), buna (Fagus crenata), and Douglas fir (Pseudotsuga menziesii) were classified as “resistant” species among the ten sapwood specimens. In the heartwood no-choice and choice feeding tests, the resistant species were buna, karamatsu, Douglas fir, sugi (Cryptomeria japonica), akamatsu (Pinus densiflora), and western red cedar (Thuja plicata). The ranking of the resistance of the ten commercial timbers against I. minor was buna > karamatsu > sugi > western red cedar > Douglas fir > rubber > western hemlock > hinoki > spruce.     

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.