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Keywords:

  • Spore germination;
  • Sugar residue;
  • Fatty acid;
  • Pinus sylvestris;
  • Picea abies

Abstract

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

The objective of this study was to correlate the occurrence of particular root and woody stump surface components with the ability of spores of the root rot fungus (Heterobasidion annosum) to adhere, germinate and establish on conifer tissues. With the aid of high performance liquid chromatography, several sugars (pinitol, xylitol, dulcitol, mannitol, D-glucose, mannose, fructose) were detected on both stump and fine root surfaces of Scots pine and Norway spruce. Of all the sugars observed, xylose and arabinose were poorly utilized for initiation of germ tube growth whereas spore germination was enhanced in the presence of D-glucose, mannose or fructose. Oxidation of these sugars by pretreatment of wood discs or roots with periodic acid abolished the ability of the spores to germinate. Non-sugar components such as long chain fatty acids on spores and root surfaces as detected with nuclear magnetic resonance were found to have a significant influence on adhesion and initiation of germ tube development. Removal of these aliphatic compounds from the root surface increased spore germination by 2-fold, whereas similar treatment on spores led to a 5-fold decrease in adhesiveness to root material. In vitro studies revealed that the di-ethyl ether extract from the roots had no long term adverse effect on spore germination which suggests that the fungus may possess the capability to detoxify this substance. Similarly, adhesion of spores was affected by low and freezing temperatures. The role of significant levels of mannitol and trehalose accumulated in spores and hyphae of the fungi on viability, survival and tolerance to adverse conditions such as oxidative stress, freezing and desiccation are discussed.


1Introduction

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

Heterobasidion annosum (Fr.) Bref. is a major pathogenic fungus of both young seedlings and mature trees, where it produces characteristic root and butt rot [1,2]. The fungus is spread by basidiospores to stumps and wounds and by mycelia via root contact from tree to tree [3]. Nothing is known however of factors likely to influence the establishment of the pathogen following initial contact with its conifer host. Earlier authors have studied the roots and woody tissues of coniferous trees mostly from the point of view of their role as a substrate for parasitic, endophytic and saprophytic fungi [4,5] and their physiological functions in forest ecosystems [6]. Less attention however has been focused on the biochemistry of the mucilage/surface constituents and their role during interaction with parasitic fungi and their survival in the ecosystem. Many authors have hypothesized that susceptibility of plant species to virulence factors in a pathogen may result from specific binding of homologous bacterial/fungal material to the corresponding cell surface molecules of host plants [7–9]. Little is known of the nature of similar cell surface interacting molecules in the H. annosum conifer pathosystem.

Furthermore, the mucilaginous sheath on spores or hyphae of several endophytic and pathogenic fungi/bacteria has been implicated in host–parasite interactions [10,11]. Bonfante-Fasolo [12] noted that extracellular sheaths on hyphae of mycorrhizal fungi are responsible for host recognition and attachment of the fungus to the roots. Jones [9] reported that contact between fungal mycelia and its host can be achieved by either formation of appressoria or through localized secretion of extracellular material. In addition, Jones [9] also noted that the survival of a species could also depend upon the successful attachment of dispersive propagules to substrates. Despite the importance of such adhesion in survival and pathogenicity, studies on the mechanisms and factors influencing the establishment of fungal pathogens following attachment to their host are still in their infancy in gymnosperm systems. Fungal parasites of angiosperm plants, unlike gymnosperms, have been well studied in this respect. In gymnosperms, particularly conifers, the root systems consist of morphologically and functionally diverse root types (i.e. fine and coarse roots). As a pre-requisite for establishment of root diseases in conifers [13,14], the host tissues and fungal material must come in contact at their cell surfaces. There is evidence that mucilage or polysaccharides may function in adhesion of fungal materials to host surfaces [15,16]. Other authors have also suggested that host surface components other than polysaccharides, e.g. proteins and lipids, may act as adhesives [17].

Reports from studies on agricultural crop plants have shown that physiochemical factors influencing adhesion competence and spore germination are likely to play a significant role in the competitiveness, dispersal and establishment of a pathogen in a particular ecosystem [9]. This study therefore reports on the characteristics of spore, root and stump surface molecules that may be involved in influencing the establishment and ecophysiological adaptation of the root rot fungi H. annosum to hosts.

2Materials and methods

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

2.1Host material

Scots pine (Pinus sylvestris L.) seeds (provenance Eksjo, 319-1-1) were purchased from a plant nursery. Fresh wooden discs from stumps of 20 year old Picea abies (L.) Karst (Norway spruce) and Scots pine were obtained from Lilla Ålbo, Sweden (courtesy of Prof. Martin Johansson).

2.2Fungal strains

H. annosum (Fr.) Bref., P-type (FP5), which attacks spruce, pine and broad-leaf trees, and S-type (FS6) which attacks mainly conifer seedlings and older spruce, were gifts from Dr. Kari Korhonen, Finnish Forest Research Institute, Helsinki, Finland. All fungal strains were maintained on Hagem agar [3].

2.3Seedling preparation

Scots pine seeds were surface-sterilized with 30% H2O2 for 15 min, washed in several changes of sterile water, sown on sterile 1% w/v water agar and left to germinate in the dark. Sprouting seedlings (10–15 days old) were used for spore attachment experiments.

2.4Recovery of the spores

Conidiospores from H. annosum were recovered from Petri-dishes containing Hagem agar medium [3] by filtering through glasswool and washing thoroughly with sterile water before being recovered by centrifugation (5000×g, 10 min at 4°C). Spore concentration for attachment studies was adjusted by dilution with sterile distilled water to give a final concentration of 106 spores ml−1 except where otherwise stated.

2.5Adhesion on detached live roots

For spore attachment experiments, primary roots (<2-mm diameter) were excised 10 mm from the tip and treated as indicated below.

2.5.1Effect of proteases, di-ethyl ether, periodic acid and KOH on adhesion to detached live roots

About 10 excised uninfected pine roots and pelleted fungal spores (106 ml−1) were incubated for 1 h in either pronase E (5 mg ml−1), 4.5% KOH, 2.5% periodic acid, di-ethyl ether or sterile water (as control). After 1 h, the roots and spores were thoroughly washed with three changes of sterile distilled water. Thereafter, the roots were placed in 200 μl of either fresh or treated spore suspension (106 ml−1). After 2–24 h, the roots were washed (4×) by gentle shaking in sterile water to dislodge unadhered spores. The number of adherent and germinated spores associated with the roots was then counted with the aid of a Leitz phase contrast microscope.

2.5.2Effect of temperature on adhesion

The effect of temperature on adhesion was assessed by incubation of roots in spore suspensions at −20, 4, 20, 35 and 70°C. The number of adherent spores associated with the roots was then counted with the aid of a Leitz phase contrast microscope as described above.

2.6Effect of pretreatment with di-ethyl ether, periodic acid and KOH on germ tube development on wood discs

Sapwood discs (7-mm diameter, 3-mm thick) from a 20 year old spruce stem were pretreated for 1 h in either di-ethyl ether, KOH (4.5%), periodic acid (2.5%) or sterile water as control. Thereafter, they were washed thoroughly with several changes of sterile water to remove traces of the chemicals followed by immersion in a solution containing 108 spores per ml. One batch (A) of the immersed wood discs was washed after 1 h to remove unadhered spores and then placed in 1% water agar, another batch (B) was placed directly on the 1% water agar without washing. The third batch (C) was left in the spore solution for 72 h, and then examined immediately by light microscopy whereas batch A and B were examined after 10 days for presence of germ tubes.

2.7Extraction of long chain fatty acids or wax like compounds with di-ethyl ether from spores and root material

Ether soluble extracts were extracted by immersing concentrated pellet of spores (ca. 300 mg), and excised 10-mm region of unsuberized fine roots of 12–15 day old seedlings and suberized roots of 5 year old plants in di-ethyl ether for 3 h. The supernatant was transferred into a preweighed clean container, air-dried in a fume hood and weighed. The weight of the ether soluble extract was noted.

2.7.1Effect of di-ethyl ether extract rich in long chain fatty acids and steroids on spore germination

Aliquots (50 μl) of varying concentration of the di-ethyl ether extract (100, 200, 300, 400, 500 μg) from suberized roots of 5 year old Scots pine seedlings were applied to the marked surface of 1% water agar, and allowed to dry. The marked areas were then inoculated with 20 μl of spore suspension (106 per ml). For the control experiment, 100–500 μl di-ethyl ether solvent alone was applied to the agar and treated as above. Samples were incubated for 1–4 days. The germinated spores were counted by examination under the microscope.

2.8Effect of sugars on germ tube development

The effects of sugars (mannose, arabinose, xylose, glucose, galactose, fructose) on spore germination was assessed by mixing 50 μl of spore suspension (106 per ml) and 50 μl sugar solution (0.5% w/v) and incubating at 20°C in a humid chamber. After overnight incubation, samples were examined using a microscope equipped with a calibrated ocular objective lens. The length of germ tube for each germinated spore per field of view using a 10× objective aperture was calculated. The average from a minimum of 15 spores in triplicates was taken. Sterile deionized water served as control.

2.9High performance liquid chromatography (HPLC) analyses of surface constituents of hyphae, spores and woody stumps

2.9.1Hyphae and spores

The fungi (H. annosum, S- and P-types) were pregrown in Hagem agar for 6 weeks. To each Petri-dish fully covered with mycelia and spores of the test fungus, 4 ml acetate buffer was added and shaken gently by hand for 20 s. Thereafter, 3 ml was removed, from the 12 Petri-dishes (for each fungus), pooled together, filtered through 0.20-μm filter and freeze dried. After freeze drying, the samples were reconstituted with 5 ml of deionized water. The uninoculated control plates were similarly treated.

2.9.2Woody stumps

Wooden discs (20-mm thick) from stumps of Norway spruce and Scots pine were used. Both the upper and lower cut surfaces from four pieces of 70-mm diameter stump cut from the tree species were rinsed five times with 5 ml of acetate buffer. Thereafter, 2.5 ml was recollected from each tree stump extract and the samples filtered (0.20-μm filter) and pooled. Samples were freeze dried and reconstituted with 3 ml of water.

Extracts were further filtered with a Cameo 13 N nylon syringe filter, 0.45 μm (MSI, Westboro, MA, USA) before analysis. Soluble sugars were analyzed by ion exchange chromatography on a Dionex DX300 HPAE, with a pulsed electrochemical detector using a column designed for carbohydrate analysis (CarboPac PA1, Dionex Corp., Sunnyvale, CA, USA). At least two replicate samples of each extract were analyzed and the retention times for peaks were compared with those of authentic standards for carbohydrates. Results were calculated as μg ml−1 of sugars. Analyses of soluble sugars for root samples were the same as previously described [14].

2.10Nuclear magnetic resonance (NMR) analyses

1H-NMR was run in CDCl3 at 30°C on di-ethyl ether extract of roots and spores. A Bruker DRX400 (400 MHz) instrument was used. Spectral width was 6410 Hz and the number of scans was 32 with repetitive time of 7.1 s.

2.11Preparation of samples for scanning electron microscopy (SEM)

Five root samples were harvested 16–48 h post inoculation. The excised root regions (first 10 mm) were pre-fixed in 3% (v/v) glutaraldehyde, washed in phosphate buffer (3×10 min) and post-fixed for 3 h in 1% w/v osmium tetroxide. After washing in distilled water (4×15 min), roots were dehydrated using a 10-step ethanol series (i.e. 10%, 20%, 30% until 100%), then an ethanol–acetone series (3:1, 2:2, 1:3, pure acetone, 10 min each), and dried using a Polaron critical point apparatus. Samples were mounted on stubs using double-sided cellotape and coated with gold using a Polaron E5000 sputter coater. Roots were observed using a Cambridge 150 SEM operated at 20 kV.

2.12Experimental design

All experiments were repeated on at least 3–4 separate occasions. For each treatment, 10 intact or excised roots were used except where otherwise indicated. Experiments for visual observations were performed several times. Statistical tests using multiple range comparisons were applied where necessary.

3Results

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

3.1Constituents of conifer root exudates

The surface of fine roots of conifer seedlings (Scots pine and Norway spruce) is covered with sheath like structures or sloughing cells [18] which are probably linked together by mucilage. The mucilage and sloughing cells were not uniformly distributed in all roots examined. In inoculated, detached live roots, spores or hyphal materials were preferentially associated with root areas covered with copious amounts of slime and mucilage. Analyses of the exudates within the slime and mucilage with aid of HPLC revealed that the major soluble sugars on the root surface were pinitol, xylitol, glucose and fructose. Low concentrations of galactose and mannose were detected in some samples but not in others. In addition, low levels of quinic and shikimic acid were also recorded.

3.2Carbohydrate constituents of Scots pine and Norway spruce stump surface

The carbohydrate constituents of a typical wood stump surface were also examined with HPLC using freshly cut stumps of a 20 year old Scots pine and Norway spruce. In woody stumps of both species, there were significantly greater accumulations of pinitol than other sugar alcohols (i.e. dulcitol, sorbitol and mannitol). Detectable levels of dulcitol were observed in Scots pine tissues but not in Norway spruce. Comparatively, mannitol and sorbitol were about 3-fold higher in Norway spruce than in Scots pine. Among soluble sugars, trehalose, fucose, salicin, arabinose, galactose, glucose, mannose, fructose, sucrose, raffinose, maltose were commonly observed (Fig. 1a,b). As expected, glucose and fructose were the major soluble sugars accumulated on both woody stumps. Among disaccharides, sucrose was the major sugar accumulated. As in root slime and mucilage, quinic and shikimic acid were also recorded.

image

Figure 1. HPLC chromatogram of soluble carbohydrate fractions from Scots pine and Norway spruce woody tissues. a: Peak identities for Scots pine μg ml−1: (3) pinitol, 50.0; (5) dulcitol, 1.3; (6) sorbitol, 1.3; (7) mannitol, 2.5; (8) trehalose, 2.5; (9) fucose, 1.3; (10) arabinose, 1.3; (11) galactose, 1.3; (12) glucose, 91.3; (13) mannose, 6.3; (14) fructose, 111.3; (15) sucrose, 277.5; (16) qinic acid, 2.5; (18) raffinose, 23.8; (19) shikimic acid, 5.0. b: Peak identities for Norway spruce μg ml−1: (4) pinitol, 30.0; (6) sorbitol, 3.8; (7) mannitol, 7.5; (8) trehalose, 2.5; (12) salicin, 1.3; (14) arabinose, 3.8; (15) galactose, 5.0; (16) glucose, 225.0; (17) fructose, 216.3; (18) sucrose, 158.8; (19) quinic acid, 11.3; (21) raffinose, 51.3; (22) maltose, 6.3; (23) shikimic acid, 13.8.

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3.3Carbohydrate exudates of spores and hyphae

The carbohydrate exhudates from spores of H. annosum (S- and P-type) were also analyzed with aid of HPLC. Relative to control values, the result revealed significant levels of accumulated mannitol and trehalose compared to other sugars. Accumulation of trehalose was about 20-fold higher in P-type than in S-type. Similarly, mannitol accumulation was 5-fold higher in P-type than S-type (Fig. 2a,b).

image

Figure 2. HPLC chromatogram of soluble carbohydrate fractions from H. annosum spores and mycelia. a: Peak identities for S-type of H. annosumμg ml−1: (3) pinitol, 1.1; (5) mannitol, 2.9; (6) trehalose, 1.7; (7) glucose, 0.6; (8) sucrose, 0.6. b: Peak identities for P-type of H. annosumμg ml−1: (4) mannitol, 12.6; (5) trehalose, 35.4; (6) glucose, 45.1; (8) fructose, 1.1; (10) sucrose, 0.6.

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3.4Chemical composition of di-ethyl ether extract from 12–15 day old and 5 year old Scots pine seedling roots and fungal spores

Di-ethyl ether extracts from Scots pine roots and fungal spores were analyzed with the aid of NMR. Analyses of extract from juvenile 12–15 day old roots revealed trace amounts of phenolics as shown in signals noted at frequency axis 6–8 (Fig. 3a). Double bond signals suggesting the presence of olephilic carbon protons were observed within frequency axis 4.5–6. Signals for aliphatic substances suggesting the presence of fatty acids, steroids, terpines and mixtures of CH2 chains were recorded within frequency axis 0.5–1.5. However, fewer aromatic compounds were extracted from juvenile roots compared to results from 5 year old Scots pine roots (Fig. 3b). With the 5 year old Scots pine roots, low amounts of phenolics were recorded in the extract as characterized by signals recorded at frequency axis 6–8. In addition, signals for olephilic protons at frequency axis 4.5–6 and anomeric signals suggesting the presence of sugars were picked up at frequency axis 3–4.5. Higher levels of aliphatic substances mainly fatty acids, terpines, and steroids characterized by aromatic rings and several hydroxyl groups were observed at frequency axis 0.5–2.5.

image

Figure 3. 1H-NMR analyses of di-ethyl ether extracts of non-suberized and suberized Scots pine roots and H. annosum spores. a: Signals observed for non-suberized Scots pine root at frequency axis (6–8) gave indications for trace amounts of phenolics, (4.5–6) olephilic carbon protons, (0.5–1.5) aliphatic substances. b: Signals observed for suberized Scots pine root at frequency axis (6–8) showed low amounts of phenolics, (4.5–6) olephilic carbon protons, (3.0–4.5) anomeric signals, (0.5–2.5) high amounts of aliphatic protons (fatty acids, steroids, terpines). c: Signals observed for H. annosum at frequency axis (0.7–1.7) aliphatic protons.

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With H. annosum (P-type) spores, almost no phenolic compounds or sugars were observed. The extract was found to contain mainly aliphatic substances or chains with terminal methyl groups and long chain fatty acids (Fig. 3c).

3.5Adhesion on detached live roots and woody tissues

Without treatment, H. annosum (P-type) spores adhere easily to fine roots (Fig. 4A). H. annosum spores formed germ tubes (Fig. 4B) on the root surface after 15–24 h. Using SEM, some of the adhered spores appeared clumped together. Clumping of the spores was partly due to the surface mucilage on spores which functions not only for attachment but also enables the spores to stick together (Fig. 4A). Following attachment and spore germination, direct contact with host cell tissues beneath the slime and mucilaginous covering on root material was established (Fig. 4C) as evidenced by degradation of the wax layer of the root surface possibly by pathogenic enzymes. In some roots, appressoria was formed within ridges on the root surfaces (Fig. 4D).

image

Figure 4. A–D: Scanning electron micrographs showing stages in spore adhesion and development on conifer roots. A: Firmly adherent conidiospores (arrowheads) of H. annosum on the root surface. Note that some of the adherent spores are clumped together. B: Following attachment and under suitable conditions, spores germinate (GC) on the root surface (R). C: Active growing germ tube of H. annosum (arrowheads) on root surface. Note distinct degradation (star) of mucilaginous sheath on root arreas surrounding hyphae (arrowheads). D: Appressorial formation (arrowheads) from actively growing hyphae within ridges of the root surface. Bars: A, 1.0 μm; B, C and D, 0.25 μm.

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In a separate experiment, attempts were made to find explanations for the reduced number of spores that germinated following attachment after pretreatment of roots with a number of chemicals (Table 1). Results revealed that only the pretreatment of Scots pine roots with di-ethyl ether increased significantly the number of spores that formed germ tubes on the root surface (Table 1). A similar observation was made with the S-type of H. annosum (data not shown). Further investigations on the impact of the di-ethyl ether extracts from suberized roots of 5 year old Scots pine seedlings showed over 80–100% repression of germ tube development in H. annosum (S-type) spores after 24 h incubation. However, the repressive action of the extract was short-lived as the fungi were able to produce germ tubes after incubation of 96 h (data not shown). In addition, selective removal of the root surface slime and mucilage by pretreatment with potassium hydroxide significantly reduced the number of H. annosum spores that were recorded germinating on root material. Removal of proteinaceous substances on root material by pretreatment with pronase E led to a significant reduction in the number of spores able to germinate on the root. Furthermore, oxidation of carbohydrate constituents on the roots by pretreatment with periodic acid abolished almost completely the germination of spores on the root surface (Table 1).

Table 1.  Numbers of fungal spores germinating to form infection structures on Scots pine roots (P. sylvestris) pretreated with various chemicals
  1. aValues represent the approximate number of germinated spores (per root) associated with sloughing cells and mucilage 12–20 h post inoculation. Results are average of three separate determinations. Ten root tips were examined for each replicate. Values followed by different letters are significant at P<0.05 (analyzed by multiple range comparisons).

H. annosuma (FP5)
Water20.0±12.0c
Periodic acid (2.5%)1.0±1.0b
Potassium hydroxide (4.5%)1.0±1.0b
Di-ethyl ether44.0±19.0a
Pronase E (5 mg ml−1)7.0±5.0d

To test if spore surface factors also influenced adhesion and spore germination, H. annosum spores were pretreated with pronase E, di-ethyl ether or periodic acid. Removal of proteins from spore surfaces by pretreatment with pronase E led to 2-fold decrease in the number of adhered spores and a 7-fold decrease in the number of germinated spores on root surfaces (Table 2). Similarly, pretreatment of spores with either di-ethyl ether or periodic acid led to a 5–6-fold decrease in spore adhesion and almost no germinated spores were recorded on root surfaces following these pretreatments. Repeating this experiment by pretreating spores of the S-type of the root rot fungus with di-ethyl ether gave the same result (data not shown). A series of further experiments were conducted to investigate the possible influence of temperature on spore adhesion. No difference was recorded in the number of adhered spores at −20°C and 4°C (data not shown). However, above 4°C there was a general trend that adhesion competence increased with temperature. The increase in adhesion following changes in temperature was not accompanied by increased germinability of the spores on root surfaces.

Table 2.  Effect of pretreatment of H. annosum (P-type) spores with various chemicals on attachment and development of germ tube on Scots pine roots
  1. aResults are mean (±S.D.) of three separate determinations. Samples were examined 2 h post inoculation. Observations were made 18 h post inoculation. For each replication, 10 root tips were examined. nd=not determined.

ParametersUngerminated spores/rootaGerminated spores/roota
Water47.0±17.014.0±7.0
KOHnd0.0±0.0
Pronase E21.0±13.02.0±1.0
Di-ethyl ether9.0±4.00.0±0.0
Periodic acid7.0±3.01.0±1.0

Results from earlier experiments [2,13] showed that H. annosum is capable of infecting conifer roots of all ages, therefore a parallel experiment with wood discs from stumps which represents the primary source of H. annosum infection of trees was conducted. By contrast to observations with fine roots, pretreatment of wood discs with di-ethyl ether had no effect on spore germination as the results were comparable to control values with or without treatment. Results further confirmed the observations noted for fine roots that removal of sugars and other carbohydrates by pretreatment with periodic acid or KOH considerably reduced the ability of spores to germinate and become established on host material (Table 3). Since sugars appear to have a strong influence on germ tube development, the impact of some selected sugars which are components of both root mucilage and stumps was investigated on spore germination (Table 4). Of all the sugars assessed, H. annosum showed increased germ tube growth in the presence of mannose, D-glucose and fructose while xylose and arabinose had no beneficial effect.

Table 3.  Effect of pretreating Norway spruce sapwood discs with chemicals on germination of H. annosum (S-type) sporesa
  1. aThe extent of spore germination and colonization was rated after microscopic examination as follows: −, no growth; +, traces of germinated spores; ++, few germinated spores; +++, moderate number; ++++, many germinated spores and extensive growth; +++++, very many germinated spores and extensive growth.

  2. bA: Wood discs were washed after 1 h incubation in spore suspension prior to transfer to 1% water agar. B: Wood discs were transferred directly to 1% water agar without washing. C: Wood discs were left in spore solution for 72 h before microscopic examination.

 Treatmentsb
 AbBbCb
Water+++++++++++++
Di-ethyl ether+++++++++++++
KOH+/−+
Periodic acid+/−
Table 4.  Effect of selected soluble sugars on spore germination and development in vitro
 Length of germ tube (μm), H. annosum (P-type)
Water (control)4.0±4.0
D-Glucose21.0±10.0
Fructose19.0±10.0
Xylose5.0±4.0
Mannose24.0±8.0
D-Galactose9.0±5.0
Arabinose6.0±3.0
Results are mean (±S.D.) of three separate determinations. Samples were examined after overnight incubation. For each replication, 15 spores were examined.

4Discussion

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

A major requirement for the dispersal of fungal propagules or a pathogen is contact with its host. Following contact, the survival of the pathogen will ultimately depend on its viability and ability to establish on the new host. Viability and development of the propagule are in turn influenced by nutrient conditions and requirements for both host and pathogen. As shown in this study, H. annosum, the most important pathogen of conifer trees, is no exception regarding its ecophysiological needs for adaptation to its host. The results clearly showed that the spores of H. annosum could easily adhere to fine roots. However, subsequent establishment and development of penetration structures seem to require some form of metabolic activity. This assumption is supported by evidence for degradation of the waxy layer on the roots possibly with the aid of esterase enzymes [19,20] secreted by preformed germ tubes and appressoria structures. Other investigators have noted that such phenomena may be related to the process of spore adhesion [20].

The long period of time required for germ tube development among H. annosum spores suggests that there is a limited supply of nutrients required to facilitate establishment of growth on host material. It is also possible that certain components of the host material are inhibitory to spore germination. Furthermore, the variations in the adhesiveness of individual H. annosum spores also suggests possible differences in their surface properties. Similarly, the low number of germinated spores relative to the total number of adhered spores could indicate that not all spores were viable or that the storage nutrients on individual spores necessary to initiate germ tube growth were insufficient. The tendency for spores to clump together during adhesion could be a strategy to overcome this deficiency as nutrients can be tapped from more viable spores to initiate germination. In contrast, the variations recorded in the number of germinated spores per individual root could also relate to possible differences in host surface constituents. This could imply that roots and stumps which lack the right nutritional constituents may escape infection or be poorly colonized.

Both woody tissues and the surface of fine roots contain almost the same types of carbohydrate nutrients as revealed by the HPLC analyses. The presence of pinitol, glucose, mannose, fructose and other sugars on the root surface indicates that these sugars might be secreted from the cytoplasm as a result of exocytic events. Understanding the role of each individual sugar in spore germination in vivo is more complicated (Asiegbu, F.O., unpublished) but in vitro studies showed that the spores had preference for particular sugars (i.e. glucose, fructose, mannose) than others (i.e. arabinose, xylose) tested. Interestingly, the two major sugars in spore mucilage were neither glucose nor fructose but rather mannitol and trehalose. The significant accumulation of these sugars relative to other soluble sugars could be a survival strategy for the fungus during adverse conditions such as desiccation and freezing following dispersal.

Studies [21,22] with other fungi (Mucor rouxii) and another basidiomycete (Agaricus bisporus) showed that trehalose is rapidly broken down to yield glucose and glucose-1-phosphate thus serving as carbon and energy source for further growth of emerging hyphae and for synthesis of the osmolyte mannitol. This could possibly explain the spore germination and hyphal growth observed in control cultures containing only 1% water agar (Asiegbu, unpublished). Furthermore, experimental results with tropical yeast strains (Saccharomyces cerevisiae) suggest that the disaccharide trehalose may be involved in stress tolerance by acting as a membrane protectant or reserve carbohydrate which may be mobilized during stress [23,24]. Trehalose has also been described to protect several enzymes including membrane enzymes (H+-ATPases) against destabilizing conditions [25–27]. Trehalose is known to prevent proteins from denaturing at high temperatures in vitro [28]. Further studies are needed to evaluate the ecological importance of these polyols to various H. annosum strains under field conditions.

Another carbohydrate accumulated in significant amount was mannitol. Studies with encapsulated yeast (Cryptococcus neoformans) have shown that glucose, fructose and mannose are good substrates for synthesis of mannitol [29]. This could also be the case for H. annosum strains. However, the observations of differential levels of mannitol accumulation within these strains suggest that fructose and mannose metabolism in S- and P-types of the root rot fungus may be different. Although the exact function and ecological significance of mannitol in these fungal strains are not known, studies with other fungal pathogens (C. neoformans; Alternaria alternata) suggest that mannitol may help to protect against oxidative death [30,31] by scavenging reactive oxygen intermediates (ROS).

Apart from carbohydrates, long chain fatty acids were major components of both root and spore surfaces. Although the precise ecological significance of the individual fatty acids was not analyzed, preferential removal of these compounds by di-ethyl ether treatment provided evidence that they might be involved in adhesion of the spores further facilitating dispersal. Other authors have also suggested that spore surface components other than polysaccharides (e.g. lipids) may act as adhesives [32]. By contrast, Clement et al. [33] demonstrated that solvent extraction of lipids from the surface of uredinospores of Uromyces viciae-fabae increased attachment of the spores to hydrophilic substrates. Since selective removal of fatty acids and other aliphatic substances by pretreatment with di-ethyl ether significantly blocked the level of spore adhesion, it was concluded that one of the major adhesive components of the spores is either a lipid, or bound to lipid. On the other hand, the inhibitory effects of the di-ethyl ether extract from root material to spore germination may be due to complexes formed with phenolics which were extracted along with the aliphatic compounds. The release from the repressive action of the extracts after prolonged incubation suggests that the fungus may be secreting extracellular enzymes such as esterase and cutinases and that this may also be related to adhesion [19,20].

Evidence that proteins on spore surfaces and mucilage influenced adhesion and spore germination was obtained by digestion of the conidia with proteases (pronase E). This implies that some of the adhesive substance is either a protein or bound to a protein. Moreover, the removal of proteinaceous components of the mucilage by pronase E appeared to delay germination of H. annosum spores. This suggests that the proteinaceous adhesive materials coating spores is either hydrolyzed or used for growth initiation during spore development. Furthermore, Clement et al. [33,34] suggested that in addition to lipids, proteins and polysaccharides, it is also possible that other factors such as positive/negatively charged molecules on host and spore surfaces may be involved in determining adhesion competence.

The results also demonstrated that adhesion of H. annosum was enhanced at higher temperatures of 30–70°C. Such an increase in adhesiveness at high temperatures was not accompanied by germinability of the spores. Clement et al. [33] made a similar observation with uredinospores of U. viciae-fabae. It is possible that the adhesive matrix on H. annosum spores is insensitive to heat treatment or that the adhesion process does not require metabolic energy in line with the conclusions of Mercure et al. [35] from their experiments with dead spores of Colletotrichum graminicola. In contrast, other authors have demonstrated that spore adhesion by Nectria haemotococca and Colletotrichum lindemuthianum depends on metabolism as adhesion was affected by temperature and sodium azide (respiratory inhibitor) treatment [36,37].

Finally, under natural conditions, basidiospores constitute an overwhelming source of spore inoculum than conidiospores (used in this study), but the two spore types seem to be equally capable of infecting stumps [38]. Similarly, in natural situations [38], wide variations have been noted in the viability of dispersed spores but no information is currently available on which factors are important. The differences in viability among adherent spores on the root surfaces observed in this study may be attributed to possible variations in accumulation and metabolism of storage sugars (trehalose and mannitol). Although the influence of abiotic factors on spore viability and survival has not been properly investigated, it is likely that their influence on surface constituents of spores may also have an impact on their viability. In a review article, Redfern and Stenlid [38] also reported variations in survival of airborne spores within susceptible stumps. In terms of survival within host tissues, it is possible that H. annosum strains secreting higher levels of ROS scavengers (e.g. mannitol) are probably well equipped to overcome residual host resistance and the hostile physical and chemical environment represented by fresh conifer stumps. Among fresh stumps, variations in the period of susceptibility ranging from 1 to 4 weeks has been recorded [38]. Little is known however on the reasons for the observed variations in the period of susceptibility during stump infections. Other authors have implicated photochemical oxidant injury [38] as possible reason for such variations. From the results of this study, it seems reasonable to presume that photochemical oxidation may have a direct effect on major essential nutrients (e.g. fructose, glucose, mannose) required for initial spore development.

These results provide experimental evidence on factors likely to influence the adhesion, survival and establishment of H. annosum spores on a new host. As also noted under natural conditions [38], adhesion and high spore density are not enough to influence the outcome of stump infection, spore viability and presence of essential nutrients are also important determinant factors. It is hoped that a better understanding of these adhesive mechanisms will be essential for the control of this very important pathogen of coniferous trees. This new insight may well open up other avenues of research in the study of diseases of forest trees.

Acknowledgements

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

This work was supported by grants from Swedish Council for Forestry and Agricultural Research (SJFR) and Vidfelts fond. Prof. Geoffery Daniel is acknowledged for critically reading the manuscript. I thank Ludmila Skoglund and Rolf Anderson for technical assistance.

References

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References
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