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Keywords:

  • arbuscular mycorrhizal (AM) fungi;
  • hyphal healing mechanism (HHM);
  • monoxenic culture;
  • physical injury;
  • independent growth;
  • hyphal recovery

Abstract

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

The hyphal healing mechanism (HHM) has been shown to differ between Gigasporaceae and Glomeraceae. However, this process has not been considered under (severe) physical stress conditions. Scutellospora reticulata and Glomus clarum strains were cultured monoxenically. The impact of long distance separating cut extremities of hyphae and of multiple injuries within hyphae on the HHM was monitored. For long distances (>5000 μm) separating cut extremities, hyphae healing was observed in half the cases in S. reticulata and was absent in G. clarum. For multiple-injured hyphae, the HHM was always oriented towards the complete recovery of hyphae integrity in S. reticulata, while in G. clarum, the growing hyphal tips (GHTs) could indifferently reconnect cut sections, by-pass cut sections or develop into the environment. Hyphae behaviour under severe physical stress clearly differentiated S. reticulata from G. clarum, suggesting that both fungi have developed different strategies for colony growth to survive under adverse conditions.


Introduction

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

Hyphal fusion (i.e. anastomoses) is a mechanism by which different branches of the same or different hyphae fuse (Kirk et al., 2001). It indistinctly concerns intact and damaged hyphae, the latter being described as a hyphal healing mechanism (HHM).

The HHM has been largely observed in Ascomycetes and Basidiomycetes (Buller, 1933; Calonge, 1968; States, 1975). In arbuscular mycorrhizal (AM) fungi, the first tangible report of HHM was made by Gerdemann (1955). He observed ‘wound healing’ in germinating hyphae arising from Gigaspora sp. spores. Wound healing was observed in Gigaspora gigantea and rarely in Glomus intraradices (Mosse, 1988). Bago et al. (1999) also reported the existence of HHM in AM fungi and suggested this mechanism as a way to restrict damage induced by ageing and lytic events.

Recently, the process of HHM was scrutinized in detail by de la Providencia et al. (2005) in Glomeraceae and Gigasporaceae species grown in monoxenic culture. In Glomus, this mechanism was oriented towards the reconnection of the affected area by linking several hyphae in relative small vicinity or by recolonization of roots and substrate, while in Gigaspora and Scutellospora, the HHM led to the recovery of hyphal integrity. These data were obtained for short distances separating injured extremities (i.e. <100 μm), while the impact of long distances (several mm) as well as of multiples injuries within hyphae on the HHM remains unexplored. It is obvious that these events occur in any ecosystem and may influence the capacity of AM fungi of rapid colony increase, exploration and exploitation of new environments and substrates.

The objective of the present study was to compare the HHM in a member of Gigasporaceae and Glomeraceae, following severe physical damages, on the hyphae integrity and AM fungal colony development. We monitored and compared hyphal fusion in Scutellospora reticulata and Glomus clarum combining the monoxenic culture system and the video-camera imaging.

Materials and methods

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

Biological material

Scutellospora reticulata Koske (Koske, Miller and Walker) Walker and Sanders EMBRAPA CNPAB11 and G. clarum MUCL 46238 Nicolson and Schenck were used for the experiments. Monoxenic cultures of the two strains were established in association with Ri T-DNA transformed carrot (Daucus carota L.) roots on the modified Strullu–Romand (MSR) medium (Declerck et al., 1998) solidified with 3 g L−1 Gel Gro (ICN, Biomedicals, Inc., Irvine, CA), following the methodologies detailed in Cranenbrouck et al. (2005).

Experiment 1: Effect of long distance (>5000 μm) on the healing mechanism

Two-month old Petri dishes containing active growing cultures of S. reticulata or G. clarum were used. In each Petri dish, five hyphae (emerging from a root and ending by an apex), showing dense cytoplasmic/protoplasmic flow under a compound bright-field light microscope (magnification × 100), were selected. These hyphae were apart from each other by 30–50 mm to prevent interferences and were traced carefully under a dissecting microscope to guarantee the absence of a direct connection between them.

The selected hyphae were carefully cut with a scalpel in two positions separated by 5000 μm. The middle-isolated fragments were removed with a cork borer and the holes were filled by the addition of sterilized (121°C for 15 min) MSR medium cooled to 40°C in a water bath. The Petri dishes were then sealed and incubated in an inverted position in the dark at 27°C. The time-course of hyphal healing was measured under a dissecting microscope (magnification × 6.7 to × 40) and a compound bright-field light microscope (magnification × 50 to × 250). Images of growing hyphal tips (GHTs) were captured with a digital camera (model Leica DFC320; Leica Microsystems Ltd) coupled to a compound bright-field light microscope Olympus BH-2 and displayed on a 15-inch of hp pavilion ze4500 screen to reconstitute the sequence of the HHM using the image manager software: leica im50, version 4.0 Leica Microsystems Imaging solutions Ltd, Cambridge, UK. A total of 25 hyphae per strain were analysed (i.e. five per Petri dish). One replicate consisted in an Ri T-DNA transformed carrot root inoculated with one of the two strains.

Experiment 2: Effect of multiple injuries on the healing mechanism

Two-month-old Petri dishes containing active growing cultures of S. reticulata or G. clarum were used. In each Petri dish, five hyphae were selected following the same criteria as in experiment 1. Each selected hypha was carefully cut in three places, distant from each other by 350–700 μm, thus forming four sections: section I (the section attached to the root), sections II and III (the sections isolated at both sides of the hyphae) and section IV (the section corresponding to the apex side of the hypha) (Fig. 1a and b). The distance between each cut extremity was 40–100 μm. The Petri dishes were then sealed and the time-course reaction of hyphal healing was measured under a dissecting microscope (magnification × 6.7 to × 40) and a compound bright-field light microscope (magnification × 50 to × 250). Images of GHTs were captured using the same methodology as in experiment 1. A total of 25 hyphae per strain were analysed (i.e. five per Petri dish). One replicate consisted of an Ri T-DNA transformed carrot root inoculated with one of the two strains.

image

Figure 1.  Diagram showing the HHM in multiples-injured hyphae of Scutellospora reticulata and Glomus clarum. (a) In S. reticulata, (A) septa produced at the extremities of each cut section. (B) The first GHT appeared in section I. (C) Other GHTs appeared at the extremities of the other sections. In section IV, multiple septa were formed and a GHT appeared at the apex side, behind the last septum. The GHTs of sections I and II always fused previous to the other sections. (D) The healing proceeded further by connecting all the sections, without ordered pattern. After total reconnection of the sections, the GHT at the tip of the apex section became a main apex and started its elongation and exploration of the substrate. (b) In G. clarum, (E) Septa produced at the cut-extremities of each section. (F) The first GHT initiated its elongation from the cut-extremities but did not always correspond to section I. (G) Several GHTs produced within the hyphal sections and also multiples septa formed in the apex side of section IV but no GHT was noted at this side. (H) Reconnection of all the sections.

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Data collection and statistical analysis

For both experiments, the total hyphal length, number of spores and auxiliary cells were evaluated (data not shown) to select the adequate Petri dishes for the experiments. Only the cultures showing a development comparable to the general description of species in the literature referring to monoxenic culture (see ref. in Declerck et al., 2005) were considered.

After selection of the cultures and following injury (see Experiment 1 and 2), the time for septum formation, GHT initiation, contact and fusion between the GHTs were measured (de la Providencia et al., 2005). The total duration for hyphae to heal was measured and compared between the two strains in each experiment.

The data were statistically analysed using the software package statistica for Windows (Stat Soft, 2001). The data were subjected to an anova, and the mean values were ranked by the Student–Newman–Keuls test at P<0.05.

Results

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

Experiment 1: Effect of long distance (>5000 μm) on the healing mechanism

In S. reticulata, a septum was formed at variable distances (50–300 μm) behind each cut extremity within 11–17 min. The first GHT appeared after 294±71 min just behind the septum, and always corresponded to section I attached to the root. Subsequently, two to three additional GHTs developed from this section, 20–40 μm apart from the first GHT and also behind the septum (Fig. 2b). Each GHT ramified into secondary and lower order branches of decreasing diameter, sometimes ending in a profuse branching pattern (Fig. 2a). The section corresponding to the apex section (Fig. 2c) also developed two or three GHTs but generated a lower number of ramifications. The total number of new branches (i.e. ramifications) was 62±15. The GHTs produced at both cut extremities elongated in an apparent disorganized pattern and spread into the medium. Once two GHTs were apart about 100 μm, their growth pattern changed from tortuous to a more organized growth oriented towards each other (Fig. 2d). The total time for complete healing was 1405±185 min after hyphal injury. Fifty six percent of the injured hyphae were able to reconnect cut extremities and to re-establish the cytoplasmic/protoplasmic flow. Growth arrest was observed in all the GHTs that did not fuse and within the ramifications of lower order. This was evidenced by septa formation and cytoplasm/protoplasm retraction. Hyphal fusions between GHT's of the same section or with neighbouring hyphae belonging to the fungal colony were never observed.

image

Figure 2.  Effect of long distance on the hyphae healing mechanism in Scutellospora reticulata. (a) GHTs were produced just behind the septum (arrows) in the section attached to the root (discontinued arrow) and the section corresponding to the main apex (double arrowhead) ramifying in new branches growing in different directions. Note the contact point between two GHTs. (arrowhead). Scale bar: 550 μm. Whites arrows indicate where pictures in part b, c and d were taken. (b) and (c) Closed view of the cut extremities and GHTs emission (arrows) just behind the septum at both sections. Scale bar: 80 μm. (d) Closed view of the fusion between the two GHTs (arrowhead). Scale bar: 100 μm.

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In G. clarum, a septum was formed at the end of each cut extremity within 9–17 min. The first GHT was observed after 411±94 min, most often in the section attached to the root. This initiation was significantly slower (P<0.05) as compared with S. reticulata. In the same section, several other GHTs developed from the cut extremities and spread into the medium. In some cases, no main GHT was formed, but several GHTs of the same size emerged. They grew in straight lines, becoming new runner hyphae with several ramifications of secondary and lower order. The section corresponding to the apex section produced slender GHTs. Their growth was tortuous and poorly ramified. The total number of new branches (i.e. ramifications) was 25±8 and was significantly lower (P<0.05) than S. reticulata. No fusion between GHTs of the two sections was recorded, while fusions between branches emerging from the same cut extremity were often observed.

Experiment 2: Effect of multiples injuries on the healing mechanism

In S. reticulata, a septum was formed at the cut extremities of each section (Fig. 1a) within 9–16 min following injury. In addition, the apex zone of section IV produced several septa (Fig. 1aB and C). In this section, retraction of protoplasm was observed starting from the hyphal tip, with successive formation of several retraction septa separated from each other by about 30–50 μm isolating empty hyphal segments (Fig. 1aC). In all sections I, bidirectional cytoplasmic/protoplasmic flow was observed, while in sections II, III and IV, this flow corresponded to slight circular movements from one extremity to another of the section lasting in some cases, during the entire process of healing. Whatever the section, the GHTs always appeared behind the septa. The first GHT appeared after 321±80 min and always corresponded to section I (Fig. 1aB). Shortly after its initiation, other GHTs appeared at both extremities of the three other sections (Figs 1aC and 3a–c). Particularly, in section IV, new GHTs appeared at the cut extremity side and at the apex side, this last one just behind the last septum (Fig. 1aC). The number of new branches was 1.5±0.5 within hyphal sections. The reconnection between GHTs was first noted between section I and section II (Fig. 1aC). The healing proceeded further by connecting the other sections, without an ordered pattern (Fig. 1aD). In every case (100%), all the sections were reconnected and cytoplasmic/protoplasmic flow was re-established from section I to section IV. Once section IV was reconnected, the GHT at the apex section became a main apex in the hypha and started its elongation and exploration of the substrate (Fig. 1aD). The total duration for the multiple-injured hyphae to reconnect all sections and re-establish cytoplasmic/protoplasmic flow was 537±78 min.

image

Figure 3.  Hyphal re-growth in an isolated section of a Scutellospora reticulata hypha. (a) Isolated section of a multiple-injured hyphae of S. reticulata showing GHT emission at both extremities just behind the septum (arrowheads). Scale bar: 50 μm. (b, c) Closed view of the emission of GHT (arrowheads). Scale bar: 10 μm.

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In G. clarum, a septum was formed at the cut extremities of each section (Fig. 1bE) within 10–18 min following injury and also multiple septa were observed in the apex side of section IV (Fig. 1bF–H). Whatever the section, the GHTs always appeared at the cut extremities. The first GHT appeared 449±96 min after damage but did not always correspond to section I (Fig. 1bF). This initiation was significantly slower (P<0.05) as compared with S. reticulata. Bi-directional cytoplasmic/protoplasmic flow was detected within section I, while the slight movement of cytoplasmic–protoplasmic material was rarely observed in sections II, III and IV. The total duration for the multiple-injured hyphae to reconnect all sections and re-establish cytoplasmic/protoplasmic flow was 658±70 min. This duration was significantly slower (P<0.05) as compared with S. reticulata. However, this process only concerned 32% of the observed hyphae. Most often, the multiple-injured hypha repaired partially, reconnecting either (1) sections I and II, without fusion of the two other sections (2) section I with III or IV, therefore by-passing sections II and/or III and (3) no sections while several GHTs were produced in section I, spreading into the substrate colonizing new zones. The number of branches produced in G. clarum varied from two GHTs (total reparation of hypha) to 34 (partial reparation of hypha). This mean number (12±10) further significantly differed (P<0.05) from S. reticulata hyphae.

Discussion

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

In the present study, we demonstrated for the first time the ability of hyphae belonging to Glomeraceae and Gigasporaceae to heal and recover their integrity after severe physical damage. This mechanism was supported by the capacity of isolated short-length (350–700 μm) hyphal sections to shelter from the surrounding environment by rapid septa formation. Despite the absence of a true septa in the coenocytic mycelium of AM fungi, the members of Glomeromycota (de la Providencia et al., 2005) and filamentous fungi (Trinci & collinge, 1974; Collinge & Markham, 1985, 1987; Markham, 1994; Levina et al., 2000) share in common the isolation of damaged sections. This mechanism prevents massive cytoplasmic/protoplasmic leakage into the surrounding environment.

The short-length hyphal sections preserved their vitality for several hours in the absence of any connection with a main hypha or a host. These sections contained sufficient resources to activate the emergence of the GHT at each side of the injured extremities for both strains and just behind the last septum of the apex section in S. reticulata. In contrast to the dynamic protoplasmic/cytoplasmic movements observed in the cut section attached to the root, slow circular movements were observed from one to the other extremity in these sections that persisted for at least 7–9 h. The slight movements, combined with the emergence of GHTs suggested a capacity of these sections of short-term independent growth (for a review, see Azcón-Aguilar et al., 1999). Triacylglicerides and glycogen, the main carbon storage compounds of the AM fungi, could be mobilized to support the development of the new GHTs including the de novo synthesis of the chitinous cell wall, which surrounds the fungal hypha as well as other structures (Bago et al., 2000). Despite the emergence of GHTs at each extremity of the isolated sections, it was clear from our data that the activation of the first GHT always took place in the section attached to the root in S. reticulata and in almost all cases in G. clarum. This was probably due to the continuous and direct transfer of metabolites from the intraradical mycelium to the extraradical hyphae. GHT activation was subsequently observed in the other isolated sections and in the apex section, without an ordered pattern. Hyphal bridges (i.e. anastomoses) were observed between the sections of multiple-injured hyphae for both strains. These hyphal bridges were formed following a tip-to-tip or tip-to-peg fusion between two GHTs. However, in S. reticulata the HHM was oriented towards the complete recovery of hyphal integrity, while in G. clarum this mechanism was oriented towards hyphal recovery, regrowth into the environment or root recolonization. Such contrasting behaviour may reflect differences in the molecular and cytological events that govern the elongation and hyphal growth direction (Riquelme et al., 1998) and the cellular machinery required for hyphal fusion (Glass et al., 2004).

We further demonstrated for the first time that for distances exceeding 5000 μm between cut sections (Experiment 1), the growth was often disorganized and in the majority of cases, numerous branches were produced at the lateral side of the main GHT. This was particularly evident with S. reticulata. With this AM fungus, long distance separating cut sections resulted in the profuse emission of GHTs (62±15) that contrasted with the restricted number of GHT produced (1.5±1) when the distance was low (<100 μm – Experiment 2). When the distance between two GHTs belonging to the two cut sections decreased to a few micrometres, they started to grow towards each other. until contact and complete fusion. This suggested that the growth of GHTs towards each other are due to the elicitation of specific diffusible substances in a sequence of signal-response (de la Providencia et al., 2005). Previous results showed the same phenomenon in Rhizoctonia solani (McCabe et al., 1999) and Neurospora crassa (Hickey et al., 2002). It is well known that AM fungal hyphae are reactive to various molecules even at very low concentrations (e.g. sesquiterpenes –Akiyama et al., 2005) and some specific molecules are probably involved in the HHM. It is obvious that in a given environment where the signals may be diluted due to the long distance separating the two cut extremities, the production of multiple GHTs increases both the concentration of specific signals emitted and the possibility for signals to be perceived. Such a mechanism was not observed with G. clarum. A significantly lower number of GHTs were produced as compared with S. reticulata, and never were long distance separating cut sections observed to reconnect but rather were always replaced by regrowth of multiple GHTs issued from the section attached to the root into the medium. This could again be related to the more plastic behaviour of these GHTs, which could indifferently reconnect or regrow into the environment and possibly colonize roots.

In the present study, we demonstrated that the hyphal behaviour under physical stress clearly differentiated S. reticulata from G. clarum, suggesting that Glomeraceae and Gigasporaceae have developed different strategies for colony to survive under adverse conditions. Studying the molecular basis that trigger hyphal regrowth as well as processes like hyphal homing, fusion and interconnectedness will provide a better understanding of the dissimilar life history strategies of these genetically different groups in AM fungi, and help understand the mechanisms by which the AM fungal species composition is regulated in ecosystems.

Acknowledgements

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

This work was supported by a grant of La Commission pour la Coopération au Développement from the Université catholique de Louvain. SD gratefully acknowledges the financial support from the Belgian Federal Science Policy Office (contract BCCM C3/10/003). IdelaP and FF thanks Dr José R. Martin Triana director of INCA for supporting AM fungal research.

Statement

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

Mycothéque de I'Université catholique de Louvain (MUCL) is part of the Belgian coordinated collections of Microorganisms (BCCM).

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