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.     

Initial reactions in sapwood of Norway spruce and Scots pine after wounding and infection by Heterobasidion parviporum and H. annosum

The xylem surface of seedlings, stem material and roots of Norway spruce (Picea abies) and Scots pine (Pinus sylvestris) were inoculated with strains of Heterobasidion annosum s. str. and H. parviporum s. str. The depth of necrosis in wounded spruce increased at a linear rate for at least seven weeks of incubation, but the rate of necrotic spread was significantly faster in infected wounds. In wounded pine the necrosis was maintained at a more superficial level for several weeks. Both spruce and pine sapwood were initially infected by hyphae of both species. In spruce, the hyphae advanced at a constant rate behind the necrotic front. On the contrary, after 1–2 weeks living H. parviporum hyphae were rare in pine rays. Heterobasidion annosum hyphae survived in pine rays, phloem and tracheids, despite a heavy accumulation of phenolics and resins and were able to penetrate into the sapwood at a linear rate although slower than infections in spruce. Histochemistry and quantitative estimates demonstrated that peroxidase activity was initially higher in spruce sapwood than in pine. Within three days of incubation, the activity in spruce sapwood disappeared concurrently with deepening necrosis. However, in pine, in both control and infected samples, there was a significant increase in peroxidase activity in the area surrounding the superficial necrosis, up to the wound surface and in the cambium and phloem around the wound. After wounding and infection, the content of soluble protein increased significantly in wood of older trees but not in seedlings. Infection resulted in an increased formation of lipophilic extractives in both spruce and pine but to a significantly greater degree in the latter, whereas the amount of hydrophilic compounds decreased in both. High‐performance liquid chromatography (HPLC) analyses of lipophilic extracts showed that inoculation of pine with the two species of Heterobasidion increased the amounts of pinosylvin, its monomethylether and several other phenolics as also resinous compounds. The results obtained may be relevant in explaining the known higher resistance of Scots pine to H. parviporum.     

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 role of Spulerina simploniella in the spread of chestnut blight

The bark miner Spulerina simploniella (Lepidoptera: Gracilariidae) was found in coppice chestnut (Castanea sativa) forests in Greece but was not found in chestnut orchards. Its larvae mine under the thin periderm of young trees, 4–10 years old, while the stem bark is still smooth. Under normal conditions it does not cause any damage to the trees. However, when chestnut blight caused by Cryphonectria parasitica is present in the area, the insect may be an agent of disease spread. Experiments revealed that spraying of 23 pupation sites with a C. parasitica conidiospore suspension caused canker formation at a rate of 100% in the coppice chestnut forests of Mount Athos, North Greece. It is believed that rain during the pupation period (approximately May 23 to June 15) may deposit conidiospores on the freshly exposed phloem and cause cankers. This bark miner has been detected in several parts of Greece, however, always in intensively managed chestnut coppice forests.     

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.     

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.     

Methyl jasmonate induces changes mimicking anatomical defenses in diverse members of the Pinaceae

Conifers have defenses such as the production of phenolic compounds and resins that can be induced by bark beetles and other invading organisms, but the signaling agents involved are unknown. The anatomical effects of methyl jasmonate (MJ), a potent inducer of certain plant defenses, were compared with wounding of the bark of 12-15-year-old trees of five conifer species. Wounding in all species resulted in tissue necrosis and wound periderm development immediately around the wound site. One cm from the wound, swelling of phloem polyphenolic parenchyma cells and phenolic accumulation were observed in Pseudotsuga menziesii (Mirb.) Franco, Picea pungens Engelman, Larix occidentalis Nutt. and Pinus monticola Douglas ex D. Don, but not in Taxus brevifolia Nutt. Traumatic resin ducts were formed in response to wounding in three species of Pinaceae, but not in P. monticola, which formed irregular clusters of cells rather than ducts. Taxus brevifolia did not form resin ducts in response to either wounding or MJ treatment. In the Pinaceae species studied, surface application of 100 mM MJ caused similar anatomical changes to those observed in response to wounding, including phenolic accumulation, cell swelling and traumatic resin duct formation, but it did not induce a wound periderm. Traumatic resin ducts differed in size among the study species, ranging from small in L. occidentalis to very large in P. menziesii. In P. menziesii, P. pungens and L. occidentalis, traumatic resin ducts were more abundant after MJ treatment than after wounding. We conclude that the octadecanoid pathway is likely involved in defense responses in stems of the Pinaceae, but not necessarily in other taxa.     

Lesion development in stems of rough‐ and smooth‐barked Eucalyptus nitens following artificial inoculations with canker fungi

A study of lesion development in stems of Eucalyptus nitens following artificial inoculations with canker fungi was carried out on 16‐year‐old plantation trees. In a first trial cambium bark wounds on smooth‐ and rough‐barked trees were inoculated with the mycelium of nine species of canker fungi, including Endothia gyrosa. In a second trial spores or mycelium of E. gyrosa were applied directly onto undamaged or superficially wounded bark surfaces. Infection subsequent to artificial inoculation via wounding (whatever the wounding technique or type of inoculum) resulted in significantly larger external lesions (mean lesion area up to 35.6 cm² 20 months after inoculation) on smooth bark compared with those on rough bark (up to 19.0 cm²). Microscopic studies of infected rough and smooth bark suggest that, once smooth bark is compromised by wounding and artificial inoculation, the particular anatomical structure of smooth bark may offer less mechanical resistance to post‐penetration hyphal spread in comparison with rough bark. It is suggested that at a pre‐penetration stage under natural conditions spores of E. gyrosa more easily infect rough bark via cracks associated with this type of bark but not present in smooth bark.     

The effect of periderm formation in the cortex of Pinus thunbergii on early invasion by the pinewood nematode

The distribution of cortical resin canals and periderm formation in the cortex of Pinus thunbergii was studied in relation to early invasion of the pinewood nematode, Bursaphelenchus xylophilus. Nematode invasion was restricted in stem cuttings of P. thunbergii in which periderm closed cortical resin canals. Early invasion of the nematodes was also restricted in stem cuttings where wound periderm had formed in response to prior nematode inoculation. It was concluded that early invasion of pinewood nematodes in living bark tissue is restricted by periderm in mature stems and by wound periderm that had formed as the result of previous nematode infection.     

Structural host responses of Acacia mangium and Eucalyptus pellita to artificial infection with the root rot pathogen,Ganoderma philippii

Structural host responses of young Acacia mangium and Eucalyptus pellita to infection with the root rot pathogen Ganoderma philippii were described. To our knowledge, this is the first report investigating the histology of root responses infected with G. philippii. The infected roots of A. mangium were characterized by the presence of a continuous multilayered mycelial sheath (~100–200 μm thick), fungal outgrowths and the production of a wound periderm. In contrast, roots of E. pellita were characterized by a mycelial sheath and fungal outgrowths and a wound periderm comprising of only one to four rows of cells.     

Variation of cell length in bark and wood of tropical trees

Summary The variation in length of cell elements both in bark and wood of a few tropical trees (Mangifera altissima, Mansonia altissima, Pentacme contorta, Pterocarpus marsupium, Shorea megrosensis, Shorea squamata, Triplochtion scleroxylon) is described. The phloem fibres and sieve tube members show a tendency for increase in length from periderm to cambium, just as the wood fibres and vessel members increase from pith to cambium. The phloem fibres are generally longer than wood fibres (40–70%). In woods with storeyed cambia the sieve tube members exhibit no variation, retaining a constant length throughout the width of the bark; the phloem fibres possess the normal trend of a length increase from periderm to cambium. The length variation of sieve tube members and phloem fibres with the height of the tree generally exhibits a decreasing trend from butt to top, although a slight increase in length of phloem fibres at the top was noticed.Presented at the IUFRO Division V Meeting, September 1973, Cape Town, South AfricaThanks are due to Mrs. K. Puchstein for help with the measurements. The financial support by the Deutsche Forschungsgemeinschaft and the Gesellschaft der Freunde und Förderer der Bundesforschungsanstalt für Forst- und Holzwirtschaft, Reinbek, is also gratefully acknowledged.     

Histopathologie du chancre cortical du cypres à Seiridium cardinale

Histopathology of cankers on Cupressus caused by Seiridium cardinale . The purpose of this work was to understand host defense reactions of cypress trees against S. cardinale canker to improve selection of clones resistant to the disease. Following infection, bark recovery depends on the neophellogenic efficiency to build a necrophylactic periderm (NP) that compartmentalizes the diseased tissue. The thicker the NP the more resistant the bark. Neophellogenic activity can be estimated by measuring the thickness of constitutive phelloderm of the bark of trees from natural plantings or seedlings. Potentially resistant clones having phelloderms thicker than 100μm will be selected for further experiments.     

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.     

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.     

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.     

Genetic variability of the ‘bark canker resistance’ character in several natural provenances of Cupressus sempervirens

Several Aegean (Greece) and Anatolian (Turkey) cypress provenances were studied for resistance variability to bark canker, a disease caused by the fungal pathogen Seiridium cardinale. The investigation also examined whether the low disease rate within the natural area of cypress was due to genetic or geographic‐climatic reasons. Results demonstrated strong variability for the ‘bark canker resistance’ character, in particular for trees within families. As trees from the provenances studied were not found to have genetic superiority for bark canker resistance, the above‐mentioned low disease rate could be due to geographic‐climatic barriers that inhibit the development of the fungus or its ability to infect the host. Several half‐sib progenies exhibited high resistance, suggesting that this character is totally inherited through the maternal line. Should this finding be confirmed by further research, it would facilitate the task of genetic improvement for resistance, allowing progenies of resistant trees to be obtained.     

Resinous stem canker development during the growing season of Chamaecyparis obtusa (Hinoki cypress) inoculated with pathogenic fungus Cistella japonica

Seasonal development of resinous stem canker was determined in Chamaecyparis obtusa trees wound‐inoculated with Cistella japonica in January 1996. Samples for anatomical analysis were harvested on 20 May, 9 July and 6 August in the second year of inoculation, and the sections were observed under light microscope. Resin exudation was most abundant in the second year, decreasing in the third. All new resin exudation in the second year was recognized at the May observation. New traumatic resin canal formation was observed in the August samples, and an expansion of necrotic lesions was observed mainly in summer samples in the inoculated trees and in naturally affected trees. Cistella japonica was re‐isolated from all bark lesions or inoculated wounds examined. These results suggest that the activity of Ci. japonica in the tree affects the seasonal development of resinous stem canker during the growing season of the trees.     

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.     

Role of Eucalyptus globulus wound wood extractives: evidence of superoxide dismutase‐like activity

Superoxide dismutase‐like activity of crude Eucalyptus globulus extracts obtained from new wound wood was measured using the water‐soluble tetrazolium salt (WST)‐1 assay. This in vitro assay determined the activity of superoxide radicals scavenging in a xanthine oxidase system. The 50% inhibition (IC₅₀) of formazan formation was estimated for fractionated samples and purified compounds isolated from the crude wound wood extract, i.e. engelitin and pedunculagin. Standards of gallic acid and pentagalloyl glucose were also measured. Fraction II as well as the compounds pentagalloyl glucose and pedunculagin showed the highest levels of antioxidant activity. Subsequent analysis of fraction II by high‐performance liquid chromatography (HPLC) coupled with negative ion electrospray mass spectrometry revealed the fraction to be dominated by hydrolysable tannins including pedunculagin, di‐, tri‐ and tetragalloylglucoses. These preliminary results provide some evidence for an antioxidant role of secondary metabolites in tree wound repair and defence.     

A new method to detect Lonsdalea quercina in infected plant tissues by real‐time PCR

Presymptomatic and accurate diagnoses of pathogens are essential for disease prediction and the timely application of bactericide. The bacterium Lonsdalea quercina (=Brenneria quercina) has been reported as the causal agent of drippy nut and bark canker disease on oak in California (US) and Europe. In recent years, it is also found on Populus × euramericana trees in Henan province of China. This bacterium causes longitudinal cankers of a few centimetres in size on the bark surface of the upper trunk. In this study, we developed two species‐specific PCR assays using primer pairs LqfF/LqfR and LqgF/LqgR for the rapid and accurate detection of the pathogenic bacteria in diseased plant tissues. The results show that the LqfF/LqfR primers amplified only a single PCR band of approximately 382 bp and the LqgF/LqgR primers yielded a PCR product of approximately 286 bp. The two primers were successfully adapted to real‐time PCR based on SYBR Green I used with the ABI 7500 system. The detection limit of the reaction was 0.1 pg genomic DNA per 20 μl PCR reaction volumes. The pathogen was mainly detected in the phloem of cankers as well as in the exudates of diseased trees, but was not found in the xylem or leaves. The size of pathogen in distribution was larger than the lesion. The results demonstrate that real‐time PCR assays can be used to detect the pathogen by extracting DNA directly from infected plant tissues. This method is a rapid, reliable method for the presymptomatic and accurate detection of L. quercina, providing a useful insight into epidemiological studies.