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.     

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.     

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.     

Response of balsam fir and hemlock roots to injuries

Drill wounds in balsam fir and hemlock roots activated the nonspecific resistance mechanisms of compartmentalization in wood and necrophylactic periderm in bark. Tangential bands of resin ducts localized around the wounds constituted the barrier zones in the secondary xylem of conifer roots. Barrier zones were more extensive in roots which showed symptoms characteristic of invasion by fungi and bacteria after wounding. This observation supports an expanded definition of barrier zones; barrier zones may form not only in response to mechanical wounds but also in response to xylem injury caused by pathogens. Multiple bands of resin ducts were common in young xylem when bark lesions developed around wounds. Necrophylactic periderms isolared dead bark from living bark. Development of phellem cells with dark contents and thick suberized walls, typical of exophylactic periderm, followed initiation of necrophylactic periderm. The wound responses were similar in both balsam fir and hemlock.     

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.     

The periderms of three North American conifers

Summary The anatomy of periderms in three North American conifers are described and compared. The phellems of balsam fir and eastern hemlock consist of tangential bands of thin-walled phellem (TnP) separated by one, or sometimes more, cell layers of thick-walled phellem (TkP). The phellem of white spruce contains tangential bands of TnP and TkP, as well as one to several cell layers of crystalliferous phellem (CP) abaxially adjacent to every TkP zone. The TkP, and to a lesser extent the TnP, are distinctive for each of the three conifers.The terminations of phellem growth cycles in fir and hemlock are delineated by thickened adaxial suberinic walls in the last-formed layer of TnP cells. In spruce, the adaxial suberinic walls of the last-formed layer of CP cells are thickened. TkP marks the geginning of phellem growth cycles in all three conifers.TkP cells are true suberized phellem cells, not phelloids.     

Pathogenicity and virulence of Armillaria sinapina and host response to infection in Douglas‐fir,western hemlock and western redcedar in the southern Interior of British Columbia

The frequency of infection, lesion characteristics and anatomical changes in phloem and cambial tissues caused by Armillaria sinapina were studied on inoculated trees of Douglas‐fir, western hemlock and western redcedar and compared with results of A. ostoyae inoculations previously reported on the same host species. Similar percentages of inoculations resulting in infection of roots on the three hosts indicate that A. sinapina and A. ostoyae are equally pathogenic. Armillaria ostoyae was more virulent than A. sinapina as demonstrated by fungal exudates from A. ostoyae inoculum blocks, which appeared to cause lesions on roots; the higher frequency at which lignified impervious tissue (IT) and necrophylactic periderm (NP) developed in bark and following cambial invasion, compartmentalization; the large proportion of roots that showed no visible host response; the large zones of IT formed under continuous stimulation by A. ostoyae advancing in inner bark; and the high frequency of breaching of NP barriers. Spread of A. sinapina mycelium in host species appeared slower than that of A. ostoyae, particularly in Douglas‐fir and western hemlock. In western redcedar, A. ostoyae induced stronger host responses than those following invasion by A. sinapina, which included further expansion of the induced rhytidome response, traumatic phloem resin duct formation and higher numbers of polyphenolic parenchyma comprising its barrier zone. Where damage by A. sinapina ensued, it was always associated with high inoculum potential. The ecology of virulent and less virulent species of Armillaria in natural forests is discussed.     

The relationship between Swiss needle cast symptom severity and level of Phaeocryptopus gaeumannii colonization in coastal Douglas‐fir (Pseudotsuga menziesii var. menziesii)*

This study examined 108 15‐year‐old Douglas‐fir (Pseudotsuga menziesii var. menziesii) trees to investigate whether trees exhibiting less severe Swiss needle cast (SNC) symptoms were more resistant (had less fungal colonization) or more tolerant (maintained healthy foliage under similar infection levels). Trees were sampled from six open pollinated families that were categorized into three disease severity groups (two families for each group; mild, moderate and severe disease symptoms). The amount of retained foliage and level of discoloration were visually assessed on trees in the field. Fungal colonization (as determined by proportion of stomata occluded with pseudothecia and by amount of Phaeocryptopus gaeumannii DNA in sampled needles) was measured on 1‐ and 2‐year‐old needles in the laboratory. Trees in the different disease severity groups were similar with respect to amount of fungus in their needles, yet the trees in the mild symptom group retained higher proportions of needles and maintained greener foliage. The relationship between amount of P. gaeumannii in needles and SNC symptom severity was statistically significant (p < 0.05) for amount of fungal DNA in 1‐year‐old needles and average needle retention (NR) over the last four growing seasons. Average NR decreased with increased amount of pathogen DNA in the mild disease symptom families. This relationship was reversed in the severe disease symptom group and there was no relationship in the moderate disease symptom group. Because the amount of P. gaeumannii DNA in foliage did not differ significantly among the groups, differences in symptom severity were attributed to tolerance, not resistance. Visual scoring of individual trees for average NR over the past four growing seasons could be used to effectively assess for SNC tolerance in Douglas‐fir.     

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.     

Stamm- und Wurzelfäulen in Douglasien,Pseudotsuga menziesii (Mirb.) Franco1

Stem and root rot of Douglas fir, Pseudotsuga menziesii (Mirb.) Franco. In 20 year old Douglas fir most of the butt rot was caused by Fomes annosus. Stem decay was central as well as eccentric reaching the sap wood in either case. The side roots of 20 year old Douglas fir were compared with those of a 40 year old stand with butt rot. In individual trees with decay there were less roots with Fomes annosus decay in the younger stand. Calocera viscosa was more abundant in the older stand. Soil conditions which might have favoured root rot in the young stand are discussed.     

Role of periderm in resistance of Eucalyptus marginata roots against Phytophthora cinnamomi

Phytophthora cinnamomi Rands invaded phloem tissue preferentially in Eucalyptus marginata Sm. (Jarrah) roots of both primary and secondary structure. Rate of fungal growth in the “primary” roots was less than in roots with secondary thickening. In the main field study jarrah roots between 1.5–2.5 mm in diameter, were inoculated with P. cinnamomi at regular intervals, over 12 months and in most cases invasion of the phloem was halted within the first 8 weeks of infection. In the majority of roots which exhibited secondary structure, lesion shape reflected the pattern of periderm development. Some lesions were peripheral in the outer phloem as cylindrical periderms had formed close to the vascular cambium. Other lesions were limited to a sector of the roots; periderm had formed radially through the secondary phloem and a zone of suberization was often continuous through the cambial zone and xylem. Formation of periderm at the edge of lesions was a multi-step process with various changes occurring, including lignification and suberization of walls and accumulation of autofluorescent compounds in cell walls and vacuoles. The layers of suberized and lignified cells formed near lesion fronts, were necrophylactic periderms. Necrophylactic periderm formation generally preceded the establishment of periderm of the exophylactic type.     

Fungal lesion length and expansion rate for the root pathogen Armillaria ostoyae in Douglas‐fir affecting root colonization and damage

Plant lesions affect disease impact, progression and host resistance. Root lesions caused by Armillaria ostoyae (Romagn.) Herink were inspected on 24‐ to 34‐year‐old planted Douglas‐fir (Pseudotsuga menziesii (Mirb.) Franco) pulled from the soil in five locations. Four hundred seventy‐nine lesions were measured for length (mm), expansion rate (mm/year) and lesion type. Lesion types were patch lesions on a root side, girdled lesions covering the root circumference and root collar lesions on the stem below ground. Non‐linear mixed modelling revealed that fungal lesion length at girdled and collar lesions increased but the expansion rate slowed as a power function of time, indicating increasing secondary inoculum and host resistance. Average fungal lesion expansion rate in girdled root and collar lesions was 127 mm the first year which then declined non‐linearly to 26 mm/year after 10 years. Fungal lesion length in patch lesions showed little change after the first year of infection. For girdled and collar lesions, fungal lesion length increased with tree size at the time of infection. Sites with a longer frost‐free period had shorter girdled root and stem lesions, probably because of more active host defence. Results indicate that mortality would be rare from one infection event. Furthermore, patch lesions pose a lower risk of mortality than other lesions because of a lower fungal lesion expansion rate and secondary inoculum. Tree size, lesion position, lesion type and fungal lesion expansion rate need to be considered when assessing the risk of lesions to individual tree survival. Techniques to minimize the disease impact are discussed.     

Endochitinase activity in the apoplastic fluid of Phellinus weirii‐infected Douglas‐fir and its association with over wintering and antifreeze activity

Extracellular proteins were extracted from Phellinus weirii infected Douglas‐fir (Pseudotsuga menziesii var. menziesii) roots and needles to examine endochitinase activity. Chitinases have been associated with the plant's defence response against fungal attack because they hydrolyse chitin, a structural component of fungal cell walls. Protein separation using sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE) followed by Western immunoblot analysis using a polyclonal antibody specific to an endochitinase‐like protein (ECP) resulted in the detection of up to three polypeptides between 27 and 30 kDa in size. Two‐dimensional gel electrophoresis (2‐D PAGE) followed by Western immunoblot analysis revealed that the apoplastic fluid contained multiple ECP isoforms with isoelectric points (pIs) ranging from 5.3 to 5.8 and molecular masses of 27–30 kDa. Chitinase activity in needle and root tissues was measured spectrophotometrically using a colorimetric assay. A gel overlay technique using glycol chitin as a substrate for endochitinase was applied to confirm that the ECP antibody detected an enzymatically active protein. The apoplastic fluid collected from P. weirii‐infected winter Douglas‐fir needles showed anti‐freeze activity and seasonal analysis of needle tissue showed some evidence of ECP accumulation in winter months. ECP was distributed systemically throughout the tree. Increased levels of endochitinase activity in the region of P. weirii infection supports a physiological role for ECP in the plant defence response.     

Virulence of Fusarium oxysporum and F. commune to Douglas‐fir (Pseudotsuga menziesii) seedlings

Fusarium species can cause damping‐off and root rot of young conifer seedlings, resulting in severe crop and economic losses in forest nurseries. Disease control within tree nurseries is difficult because of the inability to characterize and quantify Fusarium spp. populations with regard to disease potential because of high variability in isolate virulence. Fusarium isolates were collected from healthy and diseased seedlings of Douglas‐fir (Pseudotsuga menziesii) and western white pine (Pinus monticola) from a nursery in Idaho, USA. Molecular markers such as DNA sequences (mitochondrial small subunit and nuclear translation elongation factor 1‐alpha) and amplified fragment length polymorphism were used to identify isolates as either F. oxysporum or F. commune. In addition, diagnostic primers were developed to detect and distinguish F. commune from F. oxysporum. In vitro and greenhouse virulence tests were completed on Douglas‐fir germinants and seedlings. For Douglas‐fir germinants and seedlings, F. oxysporum isolates generally caused less severe symptoms, whereas most F. commune isolates caused mortality through damping‐off. This is the first report of direct evidence that F. commune can cause damping‐off disease on Douglas‐fir seedlings under greenhouse conditions.     

Growth of Phlebiopsis gigantea in wood of seven conifer species

Heterobasidion parviporum and Heterobasidion annosum are widely distributed root‐rot fungi that infect conifers throughout Europe. Infection of conifer stumps by spores of these pathogens can be controlled by treating fresh stumps with a competing non‐pathogenic fungus, Phlebiopsis gigantea. In this study, growth of three Latvian strains of P. gigantea and the biological control agent ‘Rotstop’ strain was evaluated in stem pieces of Norway spruce, Scots pine, lodgepole pine, Douglas‐fir, Weymouth pine, Siberian larch and Sitka spruce. The growth rates of one H. parviporum and one H. annosum isolate were also measured in the same stem pieces. The growth rate of P. gigantea varied greatly in wood of different conifer species. It was higher in the three pine species, lower in Norway spruce and lowest in Sitka spruce and Siberian larch, and in Douglas‐fir, this fungus did not grow. The largest area of wood occupied by P. gigantea was in lodgepole pine. Growth of Latvian isolates of P. gigantea in the wood of Pinus and Picea species was comparable to that of the Rotstop isolate. Consequently, stump treatment with local P. gigantea isolates should be recommended. However, our results suggest that Douglas‐fir stump treatment against Heterobasidion by P. gigantea may be ineffective and other stump treatment methods should be considered.     

Quantification of Phaeocryptopus gaeumannii colonization in Douglas‐fir needles by ergosterol analysis

Current assessments of infection levels of Phaeocryptopus gaeumannii, the incitant pathogen of Swiss needle cast disease on Pseudotsuga menziesii, typically rely on surveys of abundance of fruit bodies on diseased needles. The relationship between this measure and internal fungal colonization is unknown. In this article, a series of experiments to determine whether ergosterol can be used to quantify P. gaeumannii internal colonization within Douglas‐fir needles is reported. It was found that ergosterol content in seven commonly occurring Douglas‐fir foliar fungi is proportionally related to biomass, and in P. gaeumannii this relationship is not affected by age of the culture. Furthermore, at four sites tested, P. gaeumannii was the most common fungus species isolated from Douglas‐fir needles, accounting for approximately 50% of the isolations. Ergosterol content in these needles was best related to P. gaeumannii despite the presence of other fungi. The strong relationship between ergosterol and P. gaeumannii is attributed to its greater contribution to total fungal biomass compared with all other fungi present within Douglas‐fir needles.     

Butt rot in conifers caused by Serpula himantioides (Fr.) Karst.

Serpula himantioides was found to be a causal agent of butt rot in Douglas fir and Japanese larch in northern Germany. The decay started predominantly from tap roots and main side roots and extended few meters into the stem. In final stages of rot the basal heartwood was completely destroyed and the residual width of healthy sapwood was reduced. Laboratory tests revealed a tolerance by the fungus to extremely acid substrates.     

Dothistroma spp. in Western Ukraine and Georgia

Dothistroma needle blight (DNB) is among the most serious foliar diseases affecting Pinus spp. globally. Infected needles were collected from potential host species in four locations in western Ukraine and in four locations in eastern Georgia during spring–summer 2015 to update the knowledge on pathogen distribution in these countries. Dothistroma spp. were detected using isolation, sequencing and species‐specific priming (SSPP) PCR. Two new hosts for Dothistroma spp. were recorded in western Ukraine: D. septosporum on Pinus nigra var. australica and D. pini on P. nigra var. mollet. D. septosporum was found on 15‐year‐old P. strobus in western Ukraine. New hosts for D. septosporum were recorded in Georgia on 5‐ to 10‐year‐old naturally regenerated P. sylvestris var. hamata and on 40‐ to 50‐year‐old P. ponderosa trees. D. pini was found for the first time in Georgia on 30‐ to 40‐year‐old P. nigra trees. The work confirmed the presence of both D. septosporum and D. pini in western Ukraine and Georgia, and demonstrated new hosts for both Dothistroma species.     

Ein Beitrag zur Regeneration und zur Infektion von Douglasienwurzeln (Pseudotsuga menziesii [Mirb.] Franco) durch Pilze aus dem Boden1

On the regeneration and infection of roots of Douglas fir (Pseudotsuga menziesii [Mirb.] Franco) by fungi present in the soil. 167 roots in 4 stands were severed 40–60 cm from the base of the tree. At the proximal cut ends of the thick roots of the 40 years old trees on loamy sand, either replacement roots or callous tissue were formed (only a small percentage of those roots was invaded by rot causing Basidiomycetes), or no new roots and no callus were formed and root rot fungi (Fomes annosus, Coniophora puteana, Resinicium bicolor) had invaded the root sections from the soil. On the root sections of the 35 and 55 years old Douglas firs on sand, with the exception of 2 roots, neither replacement roots nor callus were formed; Fomes and Coniophora had invaded the thicker roots of the older trees from the soil.     

Larval performances and life cycle completion of the Siberian moth, <Emphasis Type="Italic">Dendrolimus sibiricus</Emphasis> (Lepidoptera: Lasiocampidae), on potential host plants in Europe: a laboratory study on potted trees

The Siberian moth, Dendrolimus sibiricus, Tschtv. is the most harmful defoliator of coniferous forests in North Asia. The pest has already spread over the Urals and continues moving westwards. Recently, it has been recommended for quarantine in member countries by European and Mediterranean Plant Protection Organization (EPPO). The performances of the pest on coniferous species planted in Europe were assessed on a range of potted trees corresponding to the spectrum of economically important conifers in the EU: European larch Larix decidua, Norway spruce Picea abies, Scots pine Pinus sylvestris, European black pine Pinus nigra, and the North American species: Douglas fir Pseudotsuga menziesii and grand fir Abies grandis. Larvae showed a potential to survive and complete the development on all these host tree species. Favorable hosts were grand fir, European larch, and Douglas fir that allowed higher survival, better larval development, and as a result, yielded heavier pupae and adult moths with higher longevity. Black pine was a poor host but, however, could still support larval and pupal development. Norway spruce and Scots pine had an intermediate behavior. If accidentally introduced to Europe, the Siberian moth may become especially damaging in forest stands predominated by European larch and by the North American firs. Norway spruce and especially the two-needle pines will be less prone to intensive defoliation by this species. The fact that the pest may damage the range of economically important coniferous species should be taken into account in the pest risk assessment for Europe and also for North America where the Siberian moth occurrence is considered likely.