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.     

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. 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.     

Resistance and tolerance in juvenile interior Douglas‐fir trees Pseudotsuga menziesii var. glauca artificially inoculated with Armillaria ostoyae

Plants utilize two general strategies to cope with pathogen attack. They either limit or resist the pathogen (termed ‘resistance’) or they cope with the disease by surviving and growing (termed ‘tolerance’). Both strategies tend to increase plant fitness; however, there are possible costs, trade‐offs and interactions associated with each strategy. This study focused on five half‐sib interior Douglas‐fir families [Pseudotsuga menziesii var. glauca (Beissn.) Franco] that were putatively classified as either resistant or susceptible to Armillaria ostoyae in a previous greenhouse study of seedling families challenged with A. ostoyae. We compared their survival rates in the greenhouse study with results of juvenile trees from the same five families that were artificially inoculated in field conditions. Both resistance and tolerance appeared to be operating in the field test trees, and a possible trade‐off between resistance and tolerance was detected. Significant differences were detected among the five families for stem radial growth following infection at the tree root collar. Compared with the putatively susceptible families, resistant families had smaller lesions and lower proportional root collar girdling. Tolerant families generally had larger lesions but demonstrated better growth when diseased than resistant families. One tolerant family that was a good survivor in the greenhouse survival study presented vertically shaped lesions that were large in area but had greatly reduced proportional root collar girdling. While a second family showed tolerant traits in the field study, its poor survival in the greenhouse study agreed with the large horizontally spreading lesions associated with high root collar girdling in the field study trees. Survival rankings of the five families in the greenhouse study mostly agreed with results from the field study based on the proportion of collar girdling among families. These host responses are discussed with respect to stability, quantity and quality of stands and products.     

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.     

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.     

Detection of a chitinase-like protein in the roots of Douglas-fir trees infected with Armillaria ostoyae and Phellinus weirii

Protein was extracted from root bark of 11- and 25-year-old interior Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) trees that were naturally infected with Armillaria ostoyae (Romagnesi) Herink. The proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Root bark tissue adjacent to infected areas had a significantly higher protein concentration than healthy tissue (P < 0.05), whereas the protein concentration of infected tissue was consistently lower (P < 0.05) than that of healthy tissue. The SDS-PAGE profiles of healthy, infected, and adjacent-to-infected root bark tissues revealed significant differences in concentrations of a 29.3-kDa protein. The N-terminal amino acid sequence of the 29.3-kDa protein displayed significant homology (P = 0.013) to a basic endochitinase. Use of a polyclonal antibody raised against the 29.3-kDa putative endochitinase-like protein (ECP) indicated differences in the quantities of ECP in healthy roots compared with roots infected with A. ostoyae in 11- and 25-year-old interior Douglas-fir trees. The antibody was also used to screen for the presence of the 29.3-kDa protein in roots of 24-year-old coastal Douglas-fir (Pseudotsuga menziesii var. menziesii) trees that were artificially inoculated with and colonized by Phellinus weirii (Murr.) Gilbn. The amount of ECP was elevated in root bark of coastal Douglas-fir in response to P. weirii infection, although in lower quantities relative to those found in the A. ostoyae-interior Douglas-fir pathosystem. The sequence homology of the ECP with a basic chitinase, together with its increased synthesis in response to two fungal pathogens, indicate a possible role for this protein in the defense of Douglas-fir against fungal pathogens.     

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.     

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.     

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.     

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.     

Fungal endophytes in woody roots of Douglas‐fir (Pseudotsuga menziesii) and ponderosa pine (Pinus ponderosa)

The fungal community inhabiting large woody roots of healthy conifers has not been well documented. To provide more information about such communities, a survey was conducted using increment cores from the woody roots of symptomless Douglas‐fir (Pseudotsuga menziesii) and ponderosa pine (Pinus ponderosa) growing in dry forests on the eastern slope of the Cascade Mountains in Washington state, USA. Fungal isolates were cultured on standard media, and then were identified using a combination of molecular and morphological methods. Fungal genera and species identified in this study will provide baseline data for future surveys of fungal endophytes. Examination of internal transcribed spacer (ITS1 and ITS2) and 5.8S rDNA sequences and morphology of cultured fungi identified 27 fungal genera. Two groups predominated: Byssochlamys nivea Westling (20.4% of isolations) and Umbelopsis species (10.4% of isolations). This is the first report of B. nivea within large woody roots of conifers. Both taxa have been previously identified as potential biological control agents. Although some trends were noted, this study found no significant evidence of host species or plant association effects on total recovery of fungal endophytes or recovery of specific fungal taxa.     

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.     

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.     

The effect of Trifluralin on the formation of ectotrophic mycorrhizae in some pine species I. Toxicity to mycorrhiza forming fungi

Laboratory experiments were conducted to test the effect of various concentrations of Trifluralin on certain fungi. The ectotrophic mycorrhizal fungi tested were: Amanita citrina Schaefer, Tricholoma pessundatum (Fr.) Quel., and Tricholoma saponaceum (Fr.) Qtiel. All exhibited sensitivity to Trifluralin concentrations. The consequence of such fungitoxicity was discussed.     

Fungi associated with Ips typographus on Picea abies in southern Poland and their succession into the phloem and sapwood of beetle‐infested trees and logs

This study dealt with the species distribution and frequency of fungi associated with the bark beetle Ips typographus (Scolytidae) on spruce trees of various states of health in southern Poland. The spruce trees were assessed by their degree of defoliation and damage of their crowns and trunks after attack by I. typographus. The state of health of trees from which samples were obtained was related to varying stages of brood development of I. typographus. Fungi were isolated from phloem taken from and around insect galleries and the sapwood underneath brood systems. Samples were taken from ‘healthy‐looking’, weakened, wind‐fallen and wind‐broken trees as well as from trap trees. The mycobiota associated with I. typographus was quite diverse in respect of the number of detected species, 65 fungal taxa were obtained from the phloem of trees infested by I. typographus, and 36 taxa occurred in the sapwood underneath insect galleries. The spectrum of fungi mainly consisted of ascomycetes and anamorphic fungi. The ophiostomatoid fungi were represented by 14 species and were the most numerously represented group in all niches examined. The most frequent ophiostomatoid species were Ceratocystis polonica, Ophiostoma ainoae, O. bicolor, O. penicillatum, O. piceae and O. piceaperdum. The frequency of occurrence of ophiostomatoid fungi differed significantly between the phloem and sapwood as well as in relation to the varying states of health of the spruce trees. These quantitative differences in the mycobiota of I. typographus between spruce trees belonging to different health categories can be explained by successional patterns of fungal colonization of host tissues following attack by I. typographus. The pathogenic species C. polonica was the primary invader, occurring most frequently in the sapwood of ‘healthy‐looking’ trees. Ophiostoma bicolor, O. penicillatum and O. piceaperdum also occurred during the early stages of brood development of I. typographus on ‘healthy‐looking’ trees, but they mainly colonized the phloem. In contrast, O. ainoae, O. minuta and O. piceae likely follow the aforementioned species as secondary and tertiary invaders into the phloem and the sapwood of spruce trees.     

Dieback Disease of Pines with Special Reference to Corsican Pine, Pinus Nigra var. Calabrica schn.: I. The Nature of the Disease Symptoms and their Development in Relation to the Crown and to Aspect

In north Yorkshire the earliest symptoms of disease appear atthe base of the bud in January. A small area of tissue in theregion of the lowest leaf primordium becomes necrotic, the protoplastsbeing damaged and the cells filled with resin. This stage isrevealed externally by bleeding of fresh, clear resin on tothe outer bud scales. From the bud the necrosis extends intothe shoot and into the needle bases. Buds of the lower branches are the most susceptible, the diseaseproceeding upwards through the crown and usually killing onlycurrent year shoots. An analysis of distribution of disease reveals that north-facingslopes are heavily infected while trees on south aspects aregenerally vigorous and healthy. A distinction is made between dieback disease which occurs inpoorly ventilated, sheltered stands and debility resulting fromexposure, or from nutritional disorders.     

Dynamic changes of telomeric restriction fragment (TRF) lengths in cells during the developmental process from embryos to seedlings and a comparison with the embryonal calli in Ginkgo biloba L.

Telomeres are the structures that locate at the terminals of linear eukaryotic chromosomes. They can play essential roles in many cellular processes. The terminal location of Arabidopsis-type TTTAGGG tandem repeats were thought to be highly conserved. The terminal location of Ginkgo biloba L. consisting of TTTAGGG tandem repeats, were confirmed by Bal31 exonuclease degradation and Southern blotting. By comparing telomeric restriction fragment (TRF) lengths at different developmental stages from embryos to seedlings, a fluctuant tendency towards variation was found in these samples. The TRF length of embryos was also compared with that of embryonal calli and an upward trend was discovered in callus culture. The results suggest that there should be a telomerase mechanism or/and ALT mechanism for the maintenance of telomere length.