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Keywords:

  • alginate entrapment;
  • arbuscular mycorrhizal fungi;
  • in vitro culture;
  • Malthusian fitness;
  • succinate dehydrogenase;
  • Trichoderma harzianum

Abstract

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

Aims:  This study was performed to explore the compatibility and applicability of plant beneficial micro-organisms (i.e. Trichoderma harzianum MUCL 29707 and Glomus sp. MUCL 41833) co-entrapped in alginate beads.

Methods and Results:  Spores of Glomus sp. and conidia of T. harzianum were immobilized in alginate beads and the impacts of the saprotrophic fungi on the presymbiotic and symbiotic phase of the arbuscular mycorrhizal (AM) fungi evaluated under strict in vitro culture conditions. Our results demonstrated the capacity of both micro-organisms in combination to regrowth outside the calcium alginate coating. The presence of T. harzianum did not hinder the AM fungal development but rather stimulated its spore production and fitness.

Conclusions:  The combination of T. harzianum MUCL 29707 with Glomus sp. MUCL 41833 in alginate beads may represent a reliable alternative inoculum formulation for application in sustainable agriculture.

Significance and Impact of the Study:  The entrapment in the alginate beads of two fungi (i.e. a saprotroph and a symbiont) having beneficial effects on plants represents a promising formulation for the development of inoculants adapted to field application.


Introduction

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

Microbes represent the unseen majority in soil and influence directly and indirectly the biodiversity, productivity, composition of plant communities and ecosystem functioning (van der Heijden et al. 2008). In the last decades, soil micro-organisms have been described, characterized and tested for their use as plant growth promoters and biocontrol agents (Kaewchai et al. 2009). Among these micro-organisms, an abundant literature has reported the beneficial effects of arbuscular mycorrhizal (AM) fungi (van der Heijden et al. 2008) and saprotrophic fungi from the genus Trichoderma (Harman et al. 2004) on plant growth and protection against biotic stresses. AM fungi that colonize the roots of nearly 80% of terrestrial plants (Smith and Read 2008) have been shown to increase plant performance and yield (van der Heijden et al. 2008), to improve soil quality (Rillig and Mummey 2006) and to reduce the damages caused by soilborne pathogens (Whipps 2004). Identically, Trichoderma spp. have been reported to control a broad spectrum of fungal pathogens via the combination of several mechanisms including mycoparasitism and antibiosis (Harman et al. 2004). Both micro-organisms in combination may thus represent a reliable alternative to reduce the application of pesticides in agro-environments. However, their broad-scale field application necessitates the development of adequate carriers allowing their advantageous combination (Bashan 1998).

Among the formulations frequently reported, the immobilization of cells within beads has been frequently mentioned for agricultural and environmental applications (see review by Bashan 1998). Immobilization (encapsulation/entrapment) within beads offers an excellent protection of cells against many biotic and abiotic environmental constraints (Bashan 1998) and increases the microbial survival and thus shelf life following inoculation into the field (Vassilev et al. 2001).

One of the most common materials used for the immobilization of micro-organisms is alginate, a natural, biodegradable polymer (Bashan 1998). Alginate entrapment methods have been used for the immobilization of AM fungi (Declerck et al. 1996) and Trichoderma sp. (Fravel et al. 1985) alone, but no studies reported yet the co-entrapment of both micro-organisms together.

In the last years, several studies have mentioned the beneficial effects of mixed nonentrapped inocula of both micro-organisms on plant growth (Calvet et al. 1993) as well as on the control of various plant diseases (Chandanie et al. 2009). Interestingly, De Jaeger et al. (2010a) also reported mycoparasitism of Trichoderma harzianum over AM fungi. As a consequence, the compatibility of AM fungi with Trichoderma spp. within beads should be first evaluated to guarantee the successful application of the co-entrapment technology.

Within this context, in vitro culture systems provide useful and powerful experimental tools to allow direct and nonperturbed microscopic observations of the interactions between micro-organisms (De Jaeger et al. 2010a). Using in vitro culture systems, the interaction between Glomus sp. MUCL 41833 and T. harzianum MUCL 29707 co-entrapped in alginate beads was investigated to determine whether (i) the co-entrapment process impacted the presymbiotic phase of the AM fungus and (ii) T. harzianum influences the symbiotic AM fungal development.

Materials and methods

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

Potato plantlets

In vitro propagated potato plantlets (Solanum tuberosum L., var. Bintje) were supplied by the Station de Haute Belgique in Libramont, Belgium. Plantlets were micropropagated every 5–6 weeks by placing nodal cuttings in sterile culture boxes (90 × 60 × 50 mm, 20 cuttings per box) containing 50 ml of Murashige and Skoog medium following the method described by Voets et al. (2005). The plantlets were kept in a growth chamber set at 21°C with a photoperiod of 16 h day−1 and a photosynthetic photon flux (PPF) of 300 μmol m−2 s−1.

Arbuscular mycorrhizal fungi

The AM fungus Glomus sp. MUCL 41833 was supplied by the Glomeromycota in vitro collection (GINCO –http://www.mycorrhiza.be/ginco-bel/index.php) on root organ cultures (ROC) of carrot (Daucus carota L.). The cultures were provided in Petri plates (90 mm diameter) on the modified Strullu–Romand (MSR) medium supplemented with 10 g l−1 sucrose, adjusted to pH 5·5 before sterilization (121°C for 15 min) and solidified with 3 g l−1 Phytagel™ (Sigma-Aldrich, Inc., St Louis, MI, USA) (Cranenbrouck et al. 2005). The Petri plates were incubated during 3 months in the dark in an inverted position at 27°C.

Trichoderma harzianum

A culture of T. harzianum Rifai MUCL 29707 was supplied by the Mycothèque de l’Université catholique de Louvain (MUCL –http://bccm.belspo.be/about/mucl.php) on potato dextrose agar (PDA) (Scharlau Chemie S.A, Barcelona, Spain). The identity of this strain was confirmed using the ITS1, 5·8S rRNA gene, ITS2 sequence and EF1 partial sequence (sequence submitted to Embl: Hx2000012494, Hx2000012335). The comparison using Trichblast database (International sub-commission on Trichoderma and Hypocrea taxonomy: http://www.isth.info/tools/blast/blast.php) confirmed the identity at 98% for the EF1 sequence with T. harzianum strain GJS92-61 and 100% for the ITS sequence with T. harzianum strain DAOM229907. A plug of gel containing several conidia and mycelium was placed into a sterile glass tube (1·5 ml) filled with 0·4 ml of 1% sterile distilled water-peptone (SDWP) (Duchefa, Haarlem, the Netherlands). The plug was swirled in the SDWP with a vortex mixer for 15 s, and 50 μl of the suspension was spread on PDA. The Petri plates were incubated for 4 days in the dark at 25°C and subsequently maintained at 4°C until needed.

Entrapment process of the AM fungus and Trichoderma harzianum in alginate beads

A solution of sodium alginate 20 g l−1 (Sigma-Aldrich, Inc.) was prepared by dissolution at 60°C before autoclaving for 15 min at 121°C.

Spores of the AM fungus were isolated from the ROC by solubilization of the MSR medium (Doner and Bécard 1991). The spores were subsequently maintained in deionized sterilized water (121°C for 15 min). Cluster of spores were separated with sterile needles under a dissecting microscope (Olympus SZ40; Olympus Optical GmbH, Hamburg, Germany) at 6·7–40× magnification. The isolated spores were then resuspended in the sodium alginate solution. The number of spores in the suspension was quantified under a bright-field light microscope (Olympus BH-2) at 50–250× magnification, and their concentration was adjusted to 300 individuals per ml of sodium alginate solution.

Conidia of T. harzianum were extracted from the PDA medium by adding 10 ml of deionized sterilized water on the surface of the fungal culture. The Petri plates were gently shaken for 5 min, and a suspension was sampled with a micropipette. Conidial suspension was quantified in a Thoma chamber (Fisher Scientific, Pittsburg, USA) under a bright-field light microscope at 50–250× magnification. A fraction of the conidial suspension was added to the sodium alginate solution under sterile conditions to obtain a final concentration of 150 conidia per ml of the sodium alginate solution.

For the co-entrapment process, spores of the AM fungus and conidia of T. harzianum were added to the same solution of sodium alginate following the method described earlier to have 300 spores of the AM fungus and 150 conidia of T. harzianum per ml of sodium alginate solution in final concentration.

The sodium alginate solutions (10 ml) containing spores of the AM fungus, conidia of T. harzianum or a combination of both micro-organisms were maintained under agitation in flasks. Droplets of 33 μl were sampled with a micropipette (Gilson, Middleton, WI, USA) and subsequently added into a 0·1 mol l−1 solution of sterilized (121°C for 15 min) CaCl2 under constant agitation for polymerization. The beads were maintained under agitation for 30 min (Declerck et al. 1996). The alginate beads were thereafter collected on a 100-μm-sterilized nylon mesh, washed with deionized sterile water and maintained at 4°C in a Petri plate. Each alginate bead contained an approximate of ten spores of the AM fungus (AMF treatment), five conidia of T. harzianum (T treatment) or a mixture of ten spores and five conidia of both fungi (AMF-T treatment).

Experiment 1: Evaluation of the percentage of potentially infective beads following co-entrapment of Glomus sp. MUCL 41833 with Trichoderma harzianum MUCL 29707

MSR medium lacking sucrose and vitamins (Voets et al. 2005) was sterilized at 121°C for 15 min and subsequently cooled in a water bath to approximately 35°C. The MSR medium was poured in the Petri plate (50 ml per plate), and five beads belonging either to the AMF treatment, T treatment or AMF-T treatment were embedded into the MSR medium before solidification. Petri plates were then incubated at 27°C in the dark in a completely randomized block design. Fifty replicates (i.e. beads) were considered per treatment.

Beads containing either the two fungi (AMF-T treatment) or one fungus (AMF treatment and T treatment) were examined under a bright-field light microscope at 50–250× magnifications to assess the percentage of potentially infective beads (%PIB). The %PIB was originally developed by Declerck et al. (1996) to evaluate the percentage of beads containing at least one germinated spore of AM fungi. This parameter was adapted in our study by evaluating the percentage of beads in which, at least, one germinated spore (%PIB-AMF) or conidia (%PIB-T) crossed the calcium alginate coating. In the case of bead containing both fungi, the %PIB was calculated as described earlier for each organism individually (%PIB-AMF′ and %PIB-T′) as well as for both organisms in combination (%PIB-AMF-T, in this case the calcium alginate coating should be crossed by both organisms). The %PIB-AMF-T, %PIB-AMF, %PIB-AMF′, %PIB-T and %PIB-T′ were plotted against time at 2, 3, 6 and 14 days.

Experiment 2: Impact of Trichoderma harzianum MUCL 29707 on the life cycle of Glomus sp. MUCL 41833

Similar to experiment 1, five beads belonging either to the AMF treatment, T treatment or AMF-T treatment were embedded into the MSR medium on a Petri plate. A 10-day-old in vitro produced potato plantlet was subsequently inserted in the Petri plate following the protocol described by Voets et al. (2005). Petri plates were covered with an opaque plastic bag and incubated horizontally in a growth chamber set at 20/16°C (day/night) with 70% relative humidity, a 16-h photoperiod and a PPF of 300 μmol m−2 s−1 in a completely randomized block design. Ten microlitres of MSR medium lacking sucrose and vitamins was added every 3 weeks in the Petri plates to maintain an adequate level of the MSR medium and to supply the plantlets with nutrients. Thirty-five replicates (i.e. Petri plates containing each five beads) were considered per treatment (i.e. AMF, AMF-T and T).

Evaluation of the %PIB in presence of potato plantlet

Beads containing either the two fungi (AMF-T treatment) or one fungus (AMF treatment and T treatment) were associated with potato plantlets and examined for the percentage of PIB (%PIB-T, %PIB-T′, %PIB-AMF, %PIB-AMF′ and %PIB-AMF-T) at 14 days, following the method described in experiment 1. Ten replicates per treatment were selected randomly among the 35 described earlier. One experimental unit, i.e. one replicate, consisted of a 10-day-old potato plantlet inoculated with five beads containing both fungi (AMF-T treatment) or a single fungus (AMF treatment or T treatment).

Malthusian fitness

To measure the survival success and reproductive effort of the AM fungus in presence of T. harzianum, the Malthusian fitness (MF) was calculated (Pringle and Taylor 2002). The MF is a measure of the instantaneous change in individual numbers over time in relation to the starting number of individuals. It is measured by comparing the number of spores at an initial time (N0) to the number of spores at a future time (Nt) as follows:

  • image

The N0 is the number of spores contained in each Petri plate at the inoculation time (e.g. five alginate beads containing each 10 spores = 50 spores) and the Nt corresponded to the number of spores measured at 4, 8, 12, 16 and 20 weeks after alginate beads association with a potato plantlet.

The number of newly produced spores of the AM fungus was estimated under a dissecting microscope at 6·7–40× magnifications following the protocol described in Voets et al. (2005). A grid of lines was marked on the bottom of each Petri plate. Spores were counted in each cell formed by the grid and summed over the entire Petri plate. The evaluation of the MF was conducted on the same Petri plates than those used in section ‘Evaluation of the %PIB in presence of potato plantlet’.

AM fungi root colonization and mycelium viability assessment

In the AMF-T and AMF treatments, the 25 remaining Petri plates were randomly divided into five groups of five replicates. Roots and extraradical mycelium were harvested at 4, 8, 12, 16 and 20 weeks after alginate beads’ inoculation.

Root colonization was assessed by staining according to the protocol described by Phillips and Hayman (1970), and colonization was estimated under the bright-field light microscope, following the method of McGonigle et al. (1990).

For the assessment of the extraradical mycelium viability, hyphae of both fungi were carefully extracted following solubilization of the MSR medium. The extraradical mycelium (ERM) viability was estimated using the succinate dehydrogenase (SDH) staining technique (Saito 1995). Samples were incubated overnight at room temperature in the staining solution, washed with deionized water during 15 min and immersed in 0·1% (w/v) acid fuchsin/lactic acid during 15 min. Hyphae were mounted on slides in 100% glycerol. The SDH activity was observed under a bright-field light microscope using the magnified intersect method (McGonigle et al. 1990). The hyphae were classified into SDH active and SDH inactive hyphae, corresponding to dark granular staining or without staining, respectively. This measure has been used previously to evaluate the antagonistic effect of T. harzianum on the AM fungi (De Jaeger et al. 2010a).

Spore germination and continuous culture

After 20 weeks, spores of the AM fungus isolated from five Petri plates of the AMF-T and AMF treatments used in sections ‘Malthusian fitness’ were tested for their capacity to germinate and their ability to reproduce the fungal life cycle following their association with potato plantlets under in vitro culture conditions.

Fifty spores were extracted from the AMF-T and AMF treatments following solubilization of the MSR medium and placed in Petri plates (i.e. ten isolated spores per Petri plate) containing the MSR medium lacking sucrose and vitamins and subsequently associated with a 10-day-old potato plantlet following the protocol described by De Jaeger et al. (2010b).

In the AMF-T treatment, both AM fungal spores and conidia of T. harzianum were present in the solubilized medium. As it was not possible to separate both fungi and isolate the AM fungal spores from the conidia of T. harzianum, a third treatment [AMF (+T) treatment] was included to discriminate the effect of the origin of the spore from the influence of T. harzianum. This treatment associated extracted spores sampled from the AMF treatment and conidial suspension of T. harzianum. During the MSR medium solubilization of AMF treatment, a fraction of conidial suspension extracted from the fungal culture on PDA medium was added to obtain the same concentration than measured with Thoma in the solubilization solution of the AMF-T treatment. Fifty spores were associated with potato plantlet as described previously.

Petri plates were incubated under the same conditions as described above. Spores were examined under a bright-field light microscope at 50–250× magnifications for germination at days 3, 7 and 11. Germination was considered when a hyphal tube emerging through the lumen of the subtending hyphae was observed.

For each treatment, the life cycle of the AM fungus was followed during 6 weeks. At that time, root colonization was assessed according to the method described elsewhere.

One experimental unit, i.e. one replicate, consisted of a 10-day-old potato plantlet associated with each treatment [i.e. AMF, AMF-T and AMF (+T) treatments]. Five experimental units were considered for each treatment.

Microscopic analysis

Images of the presymbiotic and symbiotic phases of the AM fungus were captured with a digital camera (Leica DFC320; Leica Microsystems Ltd, Cambridge, UK) coupled to a compound bright-field light microscope and displayed on a computer using an image manager software (Leica IM50, ver. 4·0; Leica Microsystems Imaging solutions Ltd).

Statistical analysis

Data analysis was performed with the statistical software sas Enterprise Guide (SAS Inc., Cary, NC). Data that were normally distributed and had homogeneous variances were subjected to an analysis of variance (anova). The Tukey’s honest significant difference test was used to identify the significant differences ( 0·05).

Results

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

Experiment 1: Evaluation of the percentage of PIB following co-entrapment of Glomus sp. MUCL 41833 with Trichoderma harzianum MUCL 29707

Spores of Glomus sp. MUCL 41833 and conidia of T. harzianum MUCL 29707 entrapped in alginate beads were able to germinate and to regrow outside the calcium alginate coating either when grown alone or in combination (Table 1). The first beads showing hyphal regrowth outside the alginate coat were observed at day 3 with the AM fungus (Fig. 1a) (in presence as well as in absence of the saprotroph) and at day 2 with T. harzianum (Fig. 1b) (in presence as well as in absence of the AM fungus). In the co-entrapment treatment, overlapping hyphae of T. harzianum and Glomus sp. were observed, but mycoparasitism by the saprotrophic fungus on the AM fungus was never noted neither on the spores nor on the regrowing hyphae.

Table 1.   Effect of entrapment on the percentage of potentially infective beads (%PIB) containing at least one germinated spore (%PIB-AMF) or conidia (%PIB-T) crossing the calcium alginate coating. In the case of bead containing both fungi, the %PIB was calculated as above for each organism individually (%PIB-AMF′ and %PIB-T′) as well as for both organisms in combination (%PIB-AMF-T). The %PIB-AMF-T, %PIB-AMF, %PIB-AMF′, %PIB-T and %PIB-T′ were plotted against time at 2, 3, 6 and 14 days
Time (days)23614
  1. Data represent means of 50 replicates (i.e. beads) (mean ± standard deviations).

  2. For each time of observation (i.e. 2, 3, 6 and 14 days following entrapment), values in the same column followed by identical letter did not differ significantly (P ≤ 0·05, Tukey’s test).

%PIB-T72·0 ± 4·5a86·0 ± 3·5a96·0 ± 1·9a96·0 ± 1·9a
%PIB-AMF0·0 ± 0·0b10·0 ± 3·0b68·0 ± 4·7b100·0 ± 0·0a
%PIB-T′80·0 ± 4·0a82·0 ± 2·5a94·0 ± 2·4a94·0 ± 2·4a
%PIB-AMF′0·0 ± 0·0b14·0 ± 3·5b84·0± 3·7c98·0 ± 1·4a
%PIB-AMF-T0·0 ± 0·0b12·0 ± 3·5b86·0 ± 3·7c96·0 ± 1·4a
image

Figure 1.  (a) Regrowth of hyphae of Trichoderma harzianum (MUCL 29707) (arrow) and germination of spore of Glomus sp. (MUCL 41833) (double arrowhead) extending out of the alginate polymer coating of the bead (empty arrowhead). Scale bar = 70 μm. (b) Focus on regrowth of hyphae of T. harzianum (arrow) through the alginate polymer coating of the bead (empty arrowhead). Scale bar = 70 μm.

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The %PIB estimated on the entrapped conidia of T. harzianum was already high at day 2, reached a maximum at day 6 and remained unchanged thereafter (i.e. %PIB-T and %PIB-T′–Table 1). In contrast, the %PIB estimated on the entrapped spores of Glomus sp. remained low until day 3, increased markedly at day 6 and reached a maximum at day 14 (i.e. %PIB-AMF and %PIB-AMF′–Table 1). A similar trend was observed for the %PIB estimated on both fungi in the co-entrapment treatment (%PIB-AMF-T –Table 1). Considering each fungus individually, no significant differences in the hyphal regrowth of T. harzianum were noted in absence (%PIB-T) or in presence of the AM fungus (%PIB-T′) during the 14 days of observation. Similarly, the regrowth of hyphae of the AM fungus did not differ in absence (%PIB-AMF) or in presence (%PIB-AMF-T) of T. harzianum, with the exception of day 6, where the hyphal regrowth of the AM fungus was significantly higher in the co-entrapped treatment (%PIB-AMF′) as compared to the treatment where the AM fungus was entrapped alone (%PIB-AMF). Considering concomitant regrowth of both micro-organisms (%PIB-AMF-T), we noted a significant difference with the %PIB-T at days 2, 3 and 6. At day 6, the %PIB-AMF was significantly lower as compared to the %PIB-AMF-T. At day 14, no significant differences between the treatments (%PIB-AMF, %PIB-AMF-T and %PIB-T) were observed (Table 1).

Experiment 2: Impact of Trichoderma harzianum MUCL 29707 on the life cycle of Glomus sp. MUCL 41833

Following association with potato plantlets, the conidia of T. harzianum entrapped in beads (i.e. AMF-T and T treatments) started to germinate and regrow outside the calcium alginate coating at day 1, while the spores of the AM fungus (i.e. AMF and AMF-T treatments) started to germinate within 2 days and crossed the alginate coating after 3 days. No significant differences in %PIB were observed at day 14 between the treatments. The values ranged from 98·0 ± 2·4% in the AMF-T treatment to 100·0% in the AMF treatment. The %PIB-T also reached 96·0 ± 1·9% and did not differ from the %PIB in the AMF-T treatment (98·0 ± 2·4%). The %PIB-T′ and %PIB-AMF′ in the co-entrapment treatment reached 98·0 ± 2·4% and 98·0 ± 1·6%, respectively.

In the AMF and AMF-T treatments, the first contacts between the AM fungal hyphae and the potato roots were observed within 6–14 days in absence as well as in presence of T. harzianum. After 4 weeks of culture, some AM hyphae developed in the MSR medium. At week 8, a network of AM hyphae covered the whole surface of the MSR medium. Runner hyphae ramifying into secondary and lower-order hyphae bearing hundreds of spores and branched absorbing structures were observed.

In the T and AMF-T treatments, T. harzianum developed a network of highly branched and septated hyaline hyphae bearing hundreds of conidiophores covering the whole surface of the MSR medium within 7 days. The presence of the AM fungus did not impact the development of the saprotroph.

In the AMF-T treatment, intermingled and overlapped mycelia of both fungi as well as mycoparasitism (i.e. coiling and penetration of the extraradical mycelium) of the AM fungal mycelium were observed. T. harzianum was detected in the intraradical mycelium of the AM fungus (i.e. hyphae and vesicles) and formed peloton of saprotrophic hyphae in the roots (data not shown).

The percentage of SDH activity measured in the hyphae of the AM fungus decreased with time but did not differ significantly in the presence or in the absence of T. harzianum (Fig. 2). In week 20, values ranged from 55·7 ± 4·6% in the AMF-T treatment to 58·7 ± 5·0% in the AMF treatment.

image

Figure 2.  Influence of Trichoderma harzianum MUCL 29707 on the viability of Glomus sp. MUCL 41833 measured by the percentage of succinate dehydrogenase (SDH) active extraradical hyphae of the arbuscular mycorrhizal (AM) fungus. AMF represents the AM fungus alone, and AMF-T represents the AM fungus associated with T. harzianum. Data represent means of five replicates. The bars indicate standard deviations. (inline image) AMF and (inline image) AMF-T.

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Whatever the treatment (AMF and AMF-T), the AM fungal spore production increased over time and the sporulation dynamics followed a sigmoidal curve (Fig. 3). No significant difference in the spore production between the treatments was observed during the first 8 weeks. Thereafter, a marked stimulation of AM fungal spore production was observed in the presence of T. harzianum. Values ranged from 2767 ± 938 spores in the AMF treatment to 11702 ± 3086 spores in the AMF-T treatment at week 12 and from 6867 ± 1594 spores in the AMF treatment to 24461 ± 3306 spores in the AMF-T treatment at week 20.

image

Figure 3.  Dynamics of spore production of Glomus sp. MUCL 41833 associated with an autotrophic potato plantlet (Solanum tuberosum cv. Bintje) in the presence (AMF-T treatment) or in the absence (AMF treatment) of Trichoderma harzianum MUCL 29707. Data represent means of ten replicates (mean ± standard deviations). Values followed by * differed significantly between treatments at P ≤ 0.05 (Tukey’s test) for a same time. (inline image) AMF and (inline image) AMF-T.

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The total root colonization increased in both treatments (i.e. AMF and AMF-T) over time (Fig. 4). A significant increase in AM fungal potato root colonization was observed in the AMF-T treatment as compared with the AMF treatment at weeks 12 and 16, with values ranging from 14·5 ± 5·1% in the AMF treatment to 18·9 ± 5·4% in the AMF-T treatment at week 12 and from 22·7 ± 4·9% in the AMF treatment to 30·1 ± 5·1% in the AMF-T treatment at week 16. At week 20, no significant difference was observed between the AMF and AMF-T treatment. No differences in arbuscules and vesicles colonization were observed between both treatments (Fig. 4).

image

Figure 4.  Influence of Trichoderma harzianum MUCL 29707 on the percentage of root colonization (hyphal, arbuscules and vesicles) by Glomus sp. MUCL 41833 of autotrophic potato plantlets (Solanum tuberosum). AMF represents the arbuscular mycorrhizal (AM) fungus alone, and AMF-T represents the AM fungus associated with T. harzianum. Data represent means of five replicates. The bars indicate standard deviations of total root colonization. Column followed by * differed significantly between treatments at P ≤ 0.05 (Tukey’s test). (inline image) vesicular; (inline image) arbuscular and (inline image) hyphal.

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For both treatments (i.e. AMF and AMF-T), the evolution of the MF followed the same pattern (Fig. 5). The values increased during the first 12 weeks (0·32 ± 0·03 in the AMF treatment and 0·45 ± 0·02 in the AMF-T treatment at week 12) and slowed down from week 12 to week 20 (0·24 ± 0·01 in the AMF treatment and 0·30 ± 0·06 in the AMF-T treatment at week 20).

image

Figure 5.  Malthusian fitness measured on the sporulation dynamics of Glomus sp. MUCL 41833 in the presence (AMF-T treatment) or in the absence (AMF treatment) of Trichoderma harzianum MUCL 29707. AMF represents the arbuscular mycorrhizal (AM) fungus alone, AMF-T represents the AM fungus associated with T. harzianum. Data represent means of ten replicates (mean ± standard deviations). Values followed by * differed significantly between treatments at P ≤ 0.05 (Tukey’s test). (inline image) AMF and (inline image) AMF-T.

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Spores produced in the presence/absence of T. harzianum were viable (Table 2). Germination of the spores was observed 2 days after plating on the MSR medium. The dynamics of spore germination did not differ between the spores sampled from the AMF or AMF-T treatments or in the AMF (+T) treatment (Table 2). Moreover, whatever the treatment considered [i.e. AMF, AMF-T and AMF (+T) treatments], the spores had the same colonization potential. The first contact between the AM fungal hyphae and the roots was observed within 15 days. After 6 weeks, typical AM extraradical structure was observed in both treatments and an average of 158 ± 69 spores were produced in the AMF treatment and ranged from 284 ± 85 spores in the AMF-T treatment to 234 ± 93 spores in the AMF (+T) treatment. At that time, the total root colonization ranged from 3·8 ± 2·6% in the AMF treatment to 5·2 ± 1·9% in AMF-T treatments and 5·1 ± 2·3% in AMF (+T) treatment.

Table 2.   Percentage of spore germination of Glomus sp. MUCL 41833 in presence (AMF-T and AMF (+T) treatments) or in absence (AMF treatment) of Trichoderma harzianum MUCL 29707, measured 3, 7 and 11 days after platting on the modified Strullu–Romand medium
Time (days)3711
  1. Data represent means of 50 replicates (mean ± standard deviations).

  2. For each time of observation (i.e. 3, 7 and 11 days following entrapment), values in the same column followed by identical letter did not differ significantly ( 0·05, Tukey’s test).

AMF78·0 ± 15·7a90·0 ± 9·9a94·0 ± 7·1a
AMF-T78·0 ± 6·4a86·0 ± 7·9a90·0 ± 5·8a
AMF (+T)86·0 ± 9·3a92·0 ± 4·3a96·0 ± 3·6a

Discussion

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

Nowadays, the application of beneficial micro-organisms is encouraged in agriculture because of their potential to improve plant disease resistance and to increase crop production in an environmentally friendly way. Inoculum of AM fungi and Trichoderma sp. is available commercially in a variety of forms ranging from wettable powders and granules to potting media (reviewed in Kaewchai et al. 2009). Among the carriers, alginate beads have been extensively used in laboratory studies for inoculum formulation and are compatible with industrial scaling-up (Bashan 1998; Kaewchai et al. 2009). One important advantage for immobilized microbes is the good storage capacity for practical applications (Plenchette and Strullu 2003; Sanyal et al. 2003). A number of studies have reported the germination and regrowth of spores of Glomus sp. and conidia of T. harzianum entrapped individually in alginate beads (Fravel et al. 1985; Declerck et al. 1996), but none, to our knowledge, investigated the co-entrapment of both fungi. Here, we demonstrated that the co-entrapment of both micro-organisms did not hinder germination and regrowth of Glomus sp. or T. harzianum. The saprotroph did not affect the AM symbiosis establishment but rather stimulate the extra- and intraradical AM fungal development and fitness.

In our study, the use of alginate beads as a carrier for the entrapment of in vitro produced spores of AM fungi and conidia of T. harzianum resulted in a high infective potential of both fungi entrapped individually as well as in combination. However, a significantly higher %PIB was noted with T. harzianum as compared to Glomus sp. in the early days following entrapment (i.e. days 2–6). This could be attributed to the higher velocity of germination and mycelium growth rate of T. harzianum. Indeed, from earlier studies conducted by De Jaeger et al. (2010a,b) with T. harzianum and by Giovannetti (2002) with various species of AM fungi, it was shown that during the early phase of growth (i.e. 24 h after conidia or spore incubation), T. harzianum grew between 1·64 and 10 times faster than the AM fungi germ tubes. As a result, T. harzianum crossed first the alginate coating.

For the establishment of the AM symbiosis, the first key events in the life cycle of AM fungi are propagule (i.e. spore in our case) germination, germ tube elongation and contact with the root surface of a mycotrophic plant. During these first events (grouped in the presymbiotic phase of AM fungal development), AM fungi are particularly sensitive to the presence of other micro-organisms (Jansa and Gryndler 2010). Synergistic as well as antagonistic interactions have been reported to impact the establishment of the AM symbiosis (McAllister et al. 1994a,b; Fracchia et al. 1998; Martinez et al. 2004). In the last decade, several studies have demonstrated the role of soluble exudates and volatile substances produced by Trichoderma species to stimulate or inhibit the development of the AM fungal presymbiotic phase (McAllister et al. 1994a; Fracchia et al. 1998, 2004; Martinez et al. 2004). Lee and Koske (1994) and De Jaeger et al. (2010a) further suggested that the mycoparasitic nature of Trichoderma sp. may represent an important component of the decline in AM fungal spore viability. Interestingly, in our experiment, no mycoparasitism of T. harzianum was noted on the spores of Glomus sp. in the alginate beads, suggesting their compatibility to co-exist in a close confined environment. As synergistic, antagonistic or neutral relationships between both fungi have all been reported (Gryndler 2000), it was suggested that variation in the interaction between AM and Trichoderma sp. during the AM presymbiotic phase may be attributed to differences between fungal species involved.

At present, little is known on the impact of T. harzianum on the AM fungal effectiveness, measured as the extent of fungal development and metabolic activity of the intra- or extraradical fungal structures (Tisserant et al. 1993; Guillemin et al. 1995). This might be because most studies were conducted under greenhouse and field conditions and have addressed the plant performance (Datnoff et al. 1995; Chandanie et al. 2009) rather than the interaction between both fungi. In our study, the presence of T. harzianum did not suppress, inhibit or impede the establishment of the mycorrhizal symbiosis. The potato roots were colonized within 4 weeks. This contradicts earlier studies reporting that Trichoderma sp. was able to decrease the AM root colonization when the saprotroph was inoculated before or at the same time to the AM fungi (McAllister et al. 1994b). The decrease in AM root colonization was not attributed to a direct effect of T. harzianum on the intraradical mycelium but to a negative effect on the AM presymbiotic development of the AM fungus (McAllister et al. 1994b), a mechanism that was not observed in our experiment.

Our results demonstrated a significant increase in AM intra- and extraradical mycelium length, in the presence of T. harzianum. The mechanisms behind this observation remain unclear but could potentially be attributed to exudates produced by the saprotroph as demonstrated by Fracchia et al. (2004). These authors reported an increase in the AM extra- and/or intraradical fungal development in the presence of exudates produced by Trichoderma sp. The development of the intraradical mycelium was modulated by the effects of the saprotrophic fungi on the extraradical phase of the AM fungi. Similar observations was made by Green et al. (1999) who demonstrated that the presence of T. harzianum, restricted to a root-free compartment, was able to modulate the development of the intraradical phase of Glomus sp. in cucumber roots. According to these studies, communication between the different parts of the AM mycelium was hypothesized (Hodge et al. 2010). Interestingly, Duffy et al. (2003) suggested that spore production was a stress response against parasitism. Similar results were obtained in the presence of Collembola, grazing on the AM mycelium (Hanlon 1981). This author demonstrated that low level of predation could stimulate the production of mycelium and spores as a stress reaction. In our study, visual evidence of coiling of T. harzianum around AM host hyphae and subsequent penetration of the extraradical mycelium supported this hypothesis.

The use of SDH has been proposed as a good method to evaluate the effects of biotic or abiotic factors on the AM fungal viability (Tisserant et al. 1993; Guillemin et al. 1995). The limited decrease in AM fungal viability observed in our study contrasted with earlier results reported by De Jaeger et al. (2010a). These authors demonstrated, under in vitro culture conditions and with the same isolates, a significant decrease in the AM fungal viability because of the presence of T. harzianum when the saprotrophic fungus was inoculated on an established AM fungal colony. As previously demonstrated, competition for nutrients (i.e. carbon and nitrogen) and space is an antagonistic mechanism of T. harzianum (Harman et al. 2004). Moreover, mechanisms related to mycoparasitism are strongly induced by the sensing of the host and/or when a poorly assimilable carbon or nitrogen source is available (Rincon et al. 2009). Consequently, it was suggested that the mycoparasitism process was expressed when T. harzianum was inoculated on an established AM network. In contrast, in our experimental study, the inoculation at the same time coupled with the fast mycelium development of T. harzianum allowed the saprotrophic fungus to access the resources and space before the AM fungus. As T. harzianum colonized the substrate prior to the invasion by the extraradical mycelium of Glomus sp., it was proposed that the absence of the host derepressed genes encoding for the parasitism. According to these hypotheses, it was suggested that the antagonistic effect of T. harzianum on the AM mycelium viability (i.e. SDH staining) may be decreased when the saprotrophic fungus was co-entrapped and inoculated at the same time as Glomus sp.

Interestingly, our results suggested that T. harzianum increased the reproductive activity of Glomus sp. as demonstrated by the MF measure. The newly produced spores were able to germinate, colonize a new host and reproduce the fungal life cycle. Consequently, as spores represent a main source of inoculum in soil (Smith and Read 2008), the presence of T. harzianum in mixed inoculum may represent an adequate mean to increase the inoculum potential of AM fungi on field.

In this study, we demonstrated that the co-entrapment represents a promising formulation for the development of inoculants combining AM fungi with T. harzianum. Our results clearly confirmed the capacity of both fungi in combination to regrowth outside the calcium alginate coating and to colonize a susceptible plant. This corroborates earlier findings combining AM fungi with cells of the P-solubilizing yeast Yarrowia lipolytica or Bradyrhizobium sp., a plant growth–promoting rhizobacterium (Vassilev et al. 2001; Weber et al. 2005). Our study demonstrated a weak mycoparasitism effect of T. harzianum that did not impact the AM fungal development but rather stimulated spore production. This study is the first that clearly demonstrates that the presence of T. harzianum modulates the fitness of an AM fungus. Even though the in vitro system is useful to investigate AM fungal–saprotrophic fungal interactions, the extrapolation of these results to field conditions should be carried out to test the reliability of this formulation under field conditions and to understand how beneficial micro-organisms interact with their plant host or respond to their environment.

Acknowledgements

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

This research was sponsored by the Direction générale opérationnelle de l’Agriculture, des Ressources naturelles et de l’Environnement du service public de Wallonie under contract number D31-1193, entitled ‘Valorisation de la microflore bénéfique des sols pour le contrôle de la flore pathogène des productions de pomme de terre comme alternative à l’utilisation des pesticides’.

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  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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