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Keywords:

  • population dynamics;
  • inoculum density;
  • root colonization;
  • mycelia quantification;
  • Pinus radiata;
  • Trichoderma hamatum

Abstract

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

The plant health- and growth-promoting biological inoculant (bio-inoculant) Trichoderma hamatum LU592 was transformed with the constitutively expressed green fluorescent protein (gfp) and hygromycin B resistance (hph) genes to specifically monitor the isolate in the root system of Pinus radiata within a strong indigenous Trichoderma population. A modified dilution plating technique was developed to allow the determination of the mycelia proportion of total propagule levels. LU592 was shown to colonize the rhizosphere most effectively when 105 spores per pot were applied compared with inoculum concentrations of 103 and 107 spores per pot. LU592 extended its zone of activity beyond the rhizosphere to at least 1 cm away from the root surface. A positive relationship was shown between P. radiata root maturation and the spatial and temporal proliferation of LU592 in the root system. A steep increase in mycelia levels and proportion of penetrated root segments was observed after 12 weeks. This study reinforces the value of genetic markers for use in ecological studies of filamentous fungi. However, despite isolate-specific recovery of the introduced isolate, it was shown that total propagule counts do not always correlate with the amount of viable mycelium present in the root system. Therefore, it is proposed that the differentiation of mycelia from spores and root penetration is used as more accurate measures of fungal activity.


Introduction

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

One of the biggest constraints to successful integration of beneficial microorganisms in commercial agricultural systems is their inconsistent performance (Harman, 2000). Extensive work has been carried out to understand the interactions between biological control agents and various plant pathogens (Harman et al., 2004). However, there is still a need to better understand the interactions between the agent and the plant to successfully and more predictably use those beneficial microorganisms. Several studies have attempted to monitor introduced fungal isolates using reporter systems. For instance, strains of Fusarium oxysporum were monitored and quantified in the tomato root system by microscopic observations and activity measurements using constitutively expressed GUS (β-glucuronidase) or GFP (green fluorescent protein) as markers (Lagopodi et al., 2002; Papadopoulou et al., 2005). The colonization behaviour of Trichoderma spp. on tomato and cucumber roots was determined using GFP reporter systems (Lu et al., 2004; Chacón et al., 2007). However, all of the above studies worked with sterile, artificial systems or in the absence of a plant (Bae & Knudsen, 2000; Orr & Knudsen, 2004). Green et al. (2001) and De Souza et al. (2008) reported a reduced ability of Trichoderma spp. to colonize and penetrate roots in nonsterile conditions compared with sterile conditions. Further, previous work on bacterial bio-inoculants demonstrated that in vitro performance does not necessarily correlate with in vivo behaviour (Schottel et al., 2001; Gravel et al., 2005; Adesina et al., 2009). There are still major challenges when it comes to studying the population dynamics of bio-inoculants in nonsterile plant ecosystems. Only a few studies have attempted to quantify specific Trichoderma strains in nonsterile plant ecosystems using GUS activity or hygromycin B (hygB) resistance (Green & Jensen, 1995; Lo et al., 1998) or, prior to this, using benomyl-resistant mutants (Papavizas, 1981; Ahmad & Baker, 1987).

It is recognized that population size, survival period and distribution need to be related to the biological activity of the introduced isolate within the root system (Lo et al., 1998; Paulitz, 2000; Orr & Knudsen, 2004). Several studies focused on microscopic observations of hyphae to identify the potential of the agent to interact with the plant or pathogen (Lagopodi et al., 2002; Chacón et al., 2007; Watanabe et al., 2007). The presence of mycelia of an introduced isolate was often associated with biological activity (the ability of the strain to interact with its environment) (such as Maor et al., 1998; Lo et al., 1998; Green et al., 2001; Orr & Knudsen, 2004; Bae & Knudsen, 2005). However, qualitative microscopic observations, although valuable in themselves, need to be supplemented with quantitative assessments to allow conclusions about root colonization behaviour. When isolate-specific recovery was achieved (using strains resistant to inhibitors), its correlation with actual biological activity was not known. Bae & Knudsen (2000) demonstrated that a shift from hyphal growth to abundant spore production (high overall propagule levels) decreased the biocontrol efficacy of a gfp-tagged strain of Trichoderma harzianum.

This study focussed on Trichoderma hamatum LU592 that has been previously shown to improve growth and survival of commercially grown Pinus radiata seedlings (Hohmann et al., 2011). The health- and growth-promoting effects of LU592 were associated with its ability to colonize the P. radiata rhizosphere and penetrate the roots. However, two application methods (introducing LU592 at different inoculum densities) resulted in similar growth benefits, but gave differences in rhizosphere colonization and root penetration.

The first objective of this study was to transform LU592 with the gfp and hygB resistance (hph) genes to enable its monitoring and recovery in a nonsterile environment. The second objective was to develop a modified dilution plating technique to quantitatively differentiate mycelia from spores. Both root penetration ability and mycelia enumeration were incorporated into all assessments as indicators of fungal activity. These tools were used to investigate (1) the influence of inoculum density on the establishment of LU592 in the P. radiata root system, (2) the activity of LU592 as a function of distance from the root surface and (3) its population dynamics in the presence and absence of the plant.

Materials and methods

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

Plants and fungi

The isolate T. hamatum LU592 was used in this study, as described in Hohmann et al. (2011). The ITS and TEF1 regions were sequenced and compared with the T. hamatum genotype, which defined LU592 as T. hamatum. The ITS sequences were identical to the T. hamatum LU593 genotype (GenBank Accession ITS1-AY241456).

Seeds of the P. radiata seedline AO 880:692 × 268:539 were used in all plant-based experiments, as described in Hohmann et al. (2011).

Agrobacterium-mediated transformation and comparison of physiological traits

The construction of the binary vector for constitutive expression of GFP (p0300GFP) was carried out by M. Carpenter and is described in Carpenter et al. (2008). The p0300GFP vector was constructed by digesting pCT74 (Lorang et al., 2001) with EcoRI and Kpn1 to remove the gfp/hph cassette on a single fragment that was then cloned into the multi cloning site of p0300. The transformation procedure was based on Zeilinger's (2004) with the following modifications. Agrobacterium tumefaciens EHA105 containing p0300GFP was cultured in minimal medium (Carsolio et al., 1994) containing 10 mM glucose and amended with 50 μg mL−1 kanamycin and 10 μg mL−1 rifampicin (Sigma-Aldrich® Inc.). The culture was diluted to an optical density at 660 (OD660 nm) of 0.15 in induction medium (minimal medium, 40 mM 2-(N-morpholino)ethanesulphonic acid, pH 5.3, 0.5% glycerol (w/v), 200 μM acetosyringone) and cultured until an OD660 of 0.25–0.3 was obtained. Subsequently, 100 μL of 108 spores mL−1 of T. hamatum LU592 was mixed with 200 μL of A. tumefaciens culture. The mixture was plated on sterile cellophane filters placed on induction medium and incubated for 2 days at 22 °C. Filters were then transferred onto potato dextrose agar (PDA; Difco™ Laboratories) amended with 300 μg mL−1 hygromycin B (hygB; Total Lab Systems, New Zealand) and 200 μg mL−1 timentin (GlaxoSmithKline Plc., UK) and incubated at 22 °C. Emerging colonies were purified by subculturing three times onto PDA amended with 300 μg mL−1 hygB. The presence of gfp was verified by epifluorescence microscopy (fluorescence mirror unit U-MWIBA3: excitation wavelengths 460–495 nm, emission wavelengths 510–550 nm and a dichroic mirror peak wavelength of 505 nm; Olympus UIS2 fluorescent microscope; Olympus New Zealand Ltd, New Zealand) and PCR using the primers GFPIDfwd (5′-ACGTAAACGGCCACAAGTTC-3′) and GFPIDrev (5′-TGCTCAGGTAGTGGTTGTCG-3′). Purified transformants were tested for mitotic stabilization by subculturing actively growing hyphae 10 times onto nonselective PDA. Growth rates of the subcultured transformants were then compared with those of the initial transformants that had been stored in 25% glycerol at −80 °C on PDA amended with 300 μg mL−1 hygB. Subsequently, genomic DNA (3 μg) was digested with either EcoRI or Acc65I (New England Biolabs), size fractionated by gel electrophoresis and Southern blotted using standard procedures (Sambrook et al., 1989).

After mitotic stabilization of the transformants by single-spore isolation, three transformants were compared with the wild type by measurement of the following physiological characteristics: mycelial growth rates and spore production on different media, and spore viability. Three transformed isolates of T. hamatum LU592 (LU592/A, LU592/B and LU592/C) and its wild type were transferred onto either a nutrient-rich medium PDA or a nutrient-poor soil extract agar medium (SEA; 20% soil extract, 12 g L−1 bacteriological agar (Sigma-Aldrich®), 0.5 g L−1 K2HPO4·7H2O, pH 6.8). Three replicate plates for each treatment were incubated at 20 °C in 12-h light/12-h dark conditions, and the growth rate of the mycelium was measured on three consecutive days. After 14 days, the spores of each colony were harvested and quantified as described in Hohmann et al. (2011). To assess spore viability, an aliquot of each spore suspension was diluted to 106 spores mL−1 and then mixed with an equal volume of potato dextrose broth (PDB; Difco™ Laboratories) resulting in a final concentration of 5 × 105 spores mL−1 in ½-strength PDB and incubated at 20 °C in darkness. After 24 h, the percentage of germinated spores was determined. In addition, the soil-sandwich technique, as described by Wakelin et al. (1999), was used to compare the mycelial growth of transformants and their wild types across a filter membrane in a nonsterile environment. A deep (2.5 × 9.0 cm) Petri dish was half filled with nonsterile potting mix (50% composted pine bark, 25% peat and 25% pumice with the addition of 12.5 kg m−3 slow-release fertilizer [1.5 kg m−3 CaMg(CO3)2, 2 kg m−3 CaSO4·2H2O coarse, 2 kg m−3 CaSO4·2H2O fine, 1 kg m−3 hydraflo II G wetting agent, 6 kg m−3 osmocote (N : P : K : Mg 15 : 4 : 9 : 1.5)] adjusted to 80% water-holding capacity and a sterile nitrocellulose filter membrane (9 cm diameter, 0.45 μm pore size; Sartorius plc, Germany) placed on top. The transformed isolates of T. hamatum LU592 and its wild type were transferred to the sterile nitrocellulose filter membranes covered by another sheet of sterile nitrocellulose filter membrane, and the filter membranes were then covered with nonsterile potting mix adjusted to 80% water-holding capacity. Five replicates per isolate were incubated at 20 °C in darkness. After 6 days, fungal mycelia on each filter membrane was visualized by staining with lactophenol cotton blue (MicroAnalytix Pty Ltd, New Zealand) and the colony diameter determined.

Visualization and recovery in nonsterile potting mix

Stratified P. radiata seeds (Hohmann et al., 2011) were sown into 50-mL pots containing nonsterile potting mix. Five pots were inoculated with 105 spores per pot of LU592/C (by pipetting 1 mL of a 105 spores mL−1 suspension onto the surface) and incubated at 20 °C and 12-h/12-h light/dark conditions. For the untreated control, an equal volume of sterile distilled water (SDW) was applied to each pot. Seedlings were irrigated by application of 6 mL of SDW every 2 days. After 3 weeks, potting mix adhering to the root surface (rhizosphere) was taken and assessed for fluorescing hyphae and conidiophore structures using epifluorescence microscopy as described. Rhizosphere samples (one from each replicate) were also taken to determine Trichoderma colony-forming units (CFU) onto Trichoderma Selective Medium – Lincoln University (TSM-LU; McLean et al., 2005) and TSM-hyg (TSM-LU without Terrachlor (Pacific Growers Supplies Ltd, New Zealand) amended with 300 μg mL−1 hygB) using the dilution plating technique as described by Hohmann et al. (2011): 10 g of rhizosphere sample was mixed with 90 mL of sterile 0.01% distilled water agar and shaken for 10 min. Serial dilutions were made, and aliquots of each dilution was spread onto three replicate plates of TSM-LU and TSM-hyg. The plates were then incubated in the dark at 20 °C. Characteristic Trichoderma colonies were then recorded after 7 and 14 days and CFU g−1 of oven-dry potting mix determined. On TSM-LU, all Trichoderma colonies (indigenous + introduced Trichoderma) and fluorescent colonies (introduced Trichoderma only) were recorded after 7, 14 and 21 days. Simultaneously, Trichoderma CFU were recorded on TSM-hyg after 7, 14 and 21 days.

Differentiation of spores and mycelia by filtration dilution plating

A filtration dilution plating technique was developed to indirectly determine the proportion of the overall CFU as mycelia by subtracting the CFU levels arising from spores from the total CFU levels.

A pure mycelial suspension was obtained as follows: ½-strength PDB was inoculated with 1 μL of a 105 spores mL−1 suspension and incubated for 3 days at 20 °C in total darkness. Mycelia concentrations were determined by dilution plating. Three replicates of nonsterile potting mix (100 g each replicate) were inoculated with either a mycelia or spore suspension to achieve two different concentrations of 104 and 106 CFU g−1 fresh potting mix. Three samples of each replicate were taken to confirm the concentrations by dilution plating onto TSM-hyg. The first dilution of each sample was then taken and filtered through two, three or four layers of sterile miracloth to determine how many layers result in the maximum filtration of mycelia. The CFU levels of the filtered samples were then determined by dilution plating onto TSM-hyg and compared with the total CFU levels before filtration.

Effect of inoculum density on rhizosphere colonization (experiment 1)

Two-compartment rhizosphere study containers (RSC) described by Chen et al. (2002) were used in this experiment to determine (1) the rhizosphere competence of T. hamatum LU592/C when applied at three different inoculum concentrations and (2) the activity, as indicated by mycelium/spore ratio, of LU592/C as a function of distance from the root surface.

To create one RSC, two 50-mL compartments (PVC cylinder) were filled with potting mix, combined (separated by a 25-μm mesh nylon membrane) and sealed with duct tape. Stratified P. radiata seeds were sown in the upper compartment at 0.5–1 cm depth (one seed per RSC). One millilitre of T. hamatum LU592/C at 103, 105 and 107 spores mL−1 or a water control (SDW) was applied to the top of 10 RSC within two randomized blocks. Seedlings were grown at a 22°C/18°C day/night regime under 15-h/9-h light/dark conditions and irrigated every day by applying water (3 mL within the first 2 months and 6 mL after 2 months). Once a month, 0.6 g of a solid fertilizer (Osmoform®; Scotts, the Netherlands) was added to each RSC. After 6 months, the two compartments were separated, and the upper portion of the lower compartment was taken to sample rhizosphere discs of 0–2, 3–5 and 8–10 mm below the nylon membrane. Bulk potting mix was sampled in the lower portion of the lower compartment (25–30 mm below the nylon membrane). The filtration dilution plating technique was used to determine total and spore-only LU502/C CFU levels in each sample. The root system in the upper compartment was separated from the potting mix, divided into three sections, upper, middle and lower, and surface sterilized (1-min soak in 70% ethanol, 1-min soak in 2.5% sodium hypochlorite, three SDW rinses; Shishido et al., 1996). Then, each section was cut into 1-cm root segments and plated onto TSM-hyg and incubated at 20 °C. After 7 days, the proportion of root segments colonized by LU592/C was determined.

Spatio-temporal distribution of LU592/C in presence/absence of P. radiata roots (experiment 2)

This experiment aimed to determine the spatial and temporal distribution of T. hamatum LU592/C in the presence and absence of P. radiata seedlings.

Root containers described by Bourguignon (2008) were used with the following modifications: 50-mL conical centrifuge tubes (diameter: 30 mm; Sigma-Aldrich®) were cut vertically into two equal halves and the bottom tip removed to allow drainage of excess water. The two halves of each tube were combined and sealed with duct tape. The root containers were filled with potting mix, and one stratified P. radiata seed per container was sown at a depth of 0.5–1 cm. One millilitre of a 5 × 106 spores mL−1 spore suspension of LU592/C or a water control (SDW) was applied to the top of 40 root containers within two randomized blocks containing either potting mix and a seed or potting mix only. Seedlings were incubated as described for experiment 1. After 3 dpi (days postinoculation), 4, 8, 12 and 16 wpi (weeks postinoculation), eight root containers per treatment were harvested, split open and 2-cm3 discs of potting mix sampled at three different depths (2, 5 and 8 cm). Total and spore-only CFU levels of LU592/C were determined for each sample using the filtration dilution plating technique. Roots within each disc were separated from the potting mix, surface sterilized, as described in 'Experiment 1: effect of inoculum density on rhizosphere colonization', cut into ~1-cm pieces, plated onto TSM-hyg and incubated at 20 °C. After 7 days, the proportion of root segments colonized by LU592/C was determined. Root subsamples were randomly sampled and examined at each sampling time for fluorescent fungal structures using epifluorescence microscopy. Wherever GFP-fluorescent fungal structures were observed on the root surface, the root section was surface sterilized and plated onto TSM-hyg and incubated at 20 °C to determine potential root penetration at that site.

Statistical analyses

All data were analysed using standard analysis of variance (anova) with factorial treatment structure and interactions. Trichoderma population assessment data were log10-transformed to satisfy the assumption of normality for anova (Olsen, 2003). Mean separation between treatments was analysed using the unrestricted least significant difference (LSD; = 0.05) test according to Saville (2003). The least significant effect (LSE) test was used to determine whether mycelia proportion means were significantly different from zero. Data were analysed using the statistical software GenStat v. 9.0 (VSN Internation Ltd), and unless stated otherwise, all presented data are shown as the back-transformed mean.

Results

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

Agrobacterium-mediated transformation and comparison of physiological traits

Two rounds of transformation resulted in three putative transformants, these were labelled LU592/A, LU592/B and LU592/C. Transformants showed uniform colony growth of 2–3 mm day−1 on hygB-amended PDA, and mycelium showed fluorescence under excitation. Mycelium of LU592/C showed the strongest fluorescence. Conidia of all three transformants showed fluorescence with variations in intensity. After ten subcultures on nonselective PDA, all three transformants showed fluorescence of conidia and mycelium and were able to grow on hygB-amended PDA. There were no differences between the mean growth rates of colonies of the original transformants, which have been stored at −80 °C, and colonies after ten subcultures on nonselective PDA confirming the mitotic stability of the gfp/hph cassette insertion. Southern hybridization confirmed the successful insertion of the construct into the genome of all three transformants. Both digestion enzymes demonstrated a single-copy integration. All fragment sizes were of higher molecular weight than the 3-kb gfp/hph cassette indicating a successful integrative transformation for all transformants. There were no background signals for the wild-type genomic DNA.

A comparison of spore production, germination and growth rates on different media for the three transformants is shown in Table 1. LU592/A and LU592/C did not differ from the wild type in any of the physiological traits. The only significant difference in the wild type was detected for LU592/B on SEA where mycelial growth was reduced by 2.7%. Of the three transformants, LU592/C was the most characteristic of the wild type in terms of colony morphology, growth, spore production and germination and also showed strongest GFP fluorescence. Therefore, it was chosen for further studies.

Table 1. Comparisons of the three transformants with LU592 wild type for spore production, spore germination and growth rate
TreatmentSpore production (spores mL−1)% Spore germinationaGrowth rate (mm day−1)
PDASEAPDASEASSA
  1. Means (= 5 for SSA and = 3 for the other assessments) in each column followed by the same letter are not significantly different.

  2. PDA, potato dextrose agar; SEA, soil extract agar; SSA, soil-sandwich assay.

  3. a

    Averaged across spores harvested from both agar types.

LU592/A4.5 × 108 a1.6 × 106 a99.2 a30.7 a18.7 a9.8 a
LU592/B6.0 × 108 a1.6 × 106 a98.8 a30.2 a17.9 b10.6 a
LU592/C4.5 × 108 a2.4 × 106 a98.5 a30.0 a18.2 a10.7 a
Wild type7.4 × 108 a2.7 × 106 a98.5 a29.9 a18.4 a10.6 a

Recovery and visualization of T. hamatum LU592 transformants in nonsterile potting mix

Hyphal and conidiophore structures were visualized in nonsterile potting mix 2 weeks after inoculation. The conidiophores showed strong and uniform fluorescence, whereas hyphal structures had weaker and more variable fluorescence intensity. LU592/C was successfully recovered from the P. radiata rhizosphere by dilution plating onto TSM-hyg. No significant differences were observed between LU592/C CFU levels determined from the number of fluorescent colonies on TSM-LU (2.7 × 103 CFU g−1 dry potting mix) and the number of colonies on TSM-hyg (1.7 × 103 CFU g−1 dry potting mix). This indicates that selection pressure did not influence the recovery rate. Inoculation with LU592/C did not result in significantly higher Trichoderma CFU levels (1.2 × 104 CFU g−1 dry potting mix) compared with the un-inoculated control (9.8 × 103 CFU g−1 dry potting mix). The overall Trichoderma population determined on TSM-LU (LU592/C + indigenous Trichoderma population), 1.2 × 104 CFU g−1 dry potting mix, was significantly higher than the Trichoderma population determined on TSM-hyg (LU592/C only; 1.7 × 103 CFU g−1 dry potting mix).

Differentiation between spores and mycelia by filtration dilution plating

Mycelia of LU592/C were recovered from nonsterile potting mix onto TSM-hyg after being filtered through no, two, three or four layers of miracloth (Fig. 1). Two layers of miracloth significantly reduced mycelia levels by 89.4% and 98.6% when inoculated at 104 and 106 CFU g−1 potting mix, respectively. Three and four layers of miracloth significantly reduced mycelia numbers by 98.1–98.8% and 99.8–99.9% when inoculated at 104 and 106 CFU g−1 potting mix, respectively. The filtration efficiency increased with higher mycelia concentrations (10-fold between the 104 and 106 application rates). For conidial suspensions applied at 104 and 106 spores g−1 potting mix, there was no significant difference in CFU counts when filtered through no, two, three and four layers of miracloth. Based on these results, the filtration dilution plating technique with three or four layers of miracloth was used in subsequent experiments to determine proportions of the overall CFU as mycelia, as an underestimation of mycelia levels by < 2% is considered acceptable.

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Figure 1. Trichoderma hamatum LU592/C population recovered from potting mix immediately after inoculation with two different concentrations (conc.) of either mycelium or conidia of LU592/C after filtration through two, three or four layers of miracloth and their respective controls (no layers of miracloth). Error bars represent the standard error of the means (= 3). CFU, colony-forming units; LSD, least significant difference.

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Experiment 1: effect of inoculum density on rhizosphere colonization

Spore viability was determined for all applications of T. hamatum LU592/C in both experiments 1 and 2. Spore germination ranged between 98.2% and 98.9%. LU592/C was not recovered from roots or potting mix samples from the untreated controls in any of the experiments.

After 6 months, LU592/C established in all the subsystems sampled, the rhizosphere, endorhizosphere and bulk potting mix, at all three inoculum concentrations. Overall LU592/C CFU levels when inoculated with 105 spores per RSC (1.8 × 104 CFU g−1 dry potting mix) were significantly higher by 4.8 and 3.6 times compared with CFU levels when inoculated with 103 and 107 spores per RSC, respectively (Table 2). The proportions of the overall CFU as mycelia were 24.6% and 23.6% when inoculated with 105 and 107 spores per RSC, respectively. When inoculated with 103 spores per RSC, mycelia proportions were 0.7%, which was below the detection level according to the LSE test.

Table 2. Trichoderma hamatum LU592/C population recovered from the rhizosphere and surface-sterilized roots of Pinus radiata seedlings 6 months after inoculation with three different concentrations of LU592/C
TreatmentRhizosphere 
CFU g−1 dry potting mix% Total CFU as myceliaEndorhizosphere % Internally colonized root segments
  1. Means (= 10) in each column followed by the same letter are not significantly different.

  2. CFU, colony-forming units; RSC, rhizosphere study container.

103 conidia3.7 × 103 a0.7 a51.1 a
105 conidia1.8 × 104 b24.6 b80.6 c
107 conidia4.6 × 103 a23.6 b66.7 b

LU592/C was isolated from the upper, middle and lower parts of 6-month-old P. radiata roots. A trend similar to the overall CFU levels in the rhizosphere was observed where the highest proportion of internally colonized root segments (mean over all root parts) occurred when inoculated with 105 spores per RSC (80.6%), and this was significantly higher compared with levels when inoculated with 103 spores per RSC (51.1%) and 107 spores per RSC (66.7%) (Table 2). Internal colonization of the upper (older) part of the root system (mean over all treatments) was significantly higher with 80.0% colonized root segments compared with the recovery of LU592/C from the middle (62.8%) and lower (55.6%) parts of the root. However, this trend was only observed for the 105 and 107 applications. No significant differences between the three root sections were observed for the 103 application.

The rhizosphere potting mix closest to P. radiata roots (0–2 mm) contained the highest LU592/C CFU levels (mean over all treatments) at 1.3 × 104 CFU g−1 dry potting mix, and this was significantly higher than CFU levels at 4–6, 8–10 and 25–30 mm (bulk) distances by 2.4, 2.8 and 3.5 times, respectively (Table 3). This colonization pattern was observed for all three inoculum concentrations. The proportions of the total CFU as mycelia (mean over all treatments) were 25.1%, 20.9% and 19.4%, respectively, for the 0–2, 4–6 and 8–10 mm samples. Mycelia proportions of the bulk potting mix were below the detection level.

Table 3. Trichoderma hamatum LU592/C population recovered from the rhizosphere at four different distances from the root surface of Pinus radiata seedlings 6 months after inoculation with LU592/C
Distance from root surfaceCFU g−1 dry potting mix% Overall CFU as mycelia
  1. Means (= 10) in each column followed by the same letter are not significantly different.

  2. CFU, colony-forming units.

0–2 mm1.3 × 104 b25.1 b
4–6 mm5.2 × 103 a20.9 b
8–10 mm4.6 × 103 a19.4 b
25–30 mm (bulk)3.7 × 103 a0.5 a

Experiment 2: spatio-temporal distribution of LU592/C in the presence/absence of P. radiata roots

LU592/C established throughout the system in the presence of P. radiata seedlings with CFU levels (mean over all sampling depths) increasing from 1.5 × 101 CFU g−1 dry potting mix at 3 days postinoculation (dpi) to 4.2 × 104 CFU g−1 dry potting mix at 16 weeks postinoculation (wpi) (Fig. 2). This increase was significantly greater compared with the bulk potting mix (absence of the plant). The same trend was observed for each sampling depth. At 3 dpi, most of the P. radiata seeds had germinated (> 90%), and the emerging radicle had penetrated into the top sampling depth. LU592/C CFU levels for both treatments (plant present or absent) were below 103 and 102 CFU g−1 dry potting mix in the top and centre sections, respectively (data not shown). LU592/C was not recovered from the bottom section at that time. Between 3 dpi and 4 wpi, LU592/C CFU levels in the root zone increased significantly by 63 times compared with four times in the bulk potting mix (Fig. 2). The decrease in LU592/C population between 4 and 8 wpi relative to the overall increase (measured using the regression slope) was significantly greater in the root zone compared with the bulk potting mix. Between 4 and 16 wpi, the LU592/C population in the root zone was significantly higher at each assessment compared with the bulk potting mix. With regard to the spatial distribution, significantly higher colonization of LU592/C was seen in the centre and bottom sections of the pot when the plant was present. At the end of the experiment (16 wpi), the population of LU592/C in the bottom section was significantly higher in the presence of the plant with 1.4 × 104 CFU g−1 dry potting mix compared with 35 CFU g−1 dry potting mix in the absence of the plant (Fig. 3). The CFU levels in the top section in the presence of P. radiata roots were 1.7 × 105 CFU g−1 dry potting mix, which was 11.9 times higher than the levels in the corresponding bottom section, compared with a difference of 190 times between the top and bottom sections without the plant.

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Figure 2. Trichoderma hamatum LU592/C population recovered from potting mix over time in the presence or absence of Pinus radiata seedlings. Values are means (= 8) of all depths. The dotted line indicates a significant difference between the two treatments in the decrease in CFU between 4 and 8 wpi relative to the overall increase. Error bars represent the standard error of the means. CFU, colony-forming units; dpi/wpi, days/weeks postinoculation.

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Figure 3. Trichoderma hamatum LU592/C population recovered from potting mix 16 weeks postinoculation at three different depths (top: 2 cm, centre: 5 cm and bottom: 8 cm) in the presence (+) or absence (−) of Pinus radiata seedlings. Overall bars are means (= 8) across five sampling times. Bar areas show the proportions of conidia and mycelia of the overall CFU levels. Error bars represent the standard error of the means. CFU, colony-forming units.

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At 3 dpi, the proportion of the total CFU as mycelia was 43.6% in the top section of the root system (Fig. 4). Four weeks later, mycelia levels decreased in the top section to below the detection level and increased in the centre section to 29.0%. At 8 wpi, the overall mycelia proportions decreased below the detection level and increased rapidly at 12 wpi to 36.5%, 54.2% and 58.5% for the top, centre and bottom sections, respectively. At 16 wpi, the highest proportion of the total CFU as mycelia was recovered from the centre section of the root system with 89.0%. The proportions of CFU as mycelia in both the top and bottom section at that time were 23.1% and 76.4%, respectively. The proportion of CFU as mycelia in the plantless system remained below the detection level at all sampling times (data not shown). Similar to the trend in mycelia proportions, the proportion of LU592/C colonized root segments significantly increased from 19.4% to 74.3% between 12 and 16 wpi (Fig. 4). No internal root colonization was observed at 3 dpi. Similar to the trend observed in experiment 1, colonization of the top section of the root system (mean over all sampling times) was significantly greater (40.1% internally colonized root segments) compared with the centre and bottom sections (both with 20.8%).

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Figure 4. Trichoderma hamatum LU592/C mycelia proportions recovered from potting mix at three different depths (top: 2 cm, centre: 5 cm and bottom: 8 cm) and recovered from surface-sterilized roots (mean over all depths) of Pinus radiata seedlings over time. Filled values (mycelia proportions) indicate means above the detection level. Error bars represent the standard error of the means (= 8). Dpi/wpi, days/weeks postinoculation.

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Microscopic observations did not detect fluorescent fungal structures on the roots 3 dpi. At 8 dpi, fluorescent mycelia was seen colonizing the root surface, including root hairs and bark particles in proximity to roots (Fig. 5). At 12 and 16 wpi, LU592/C root colonization was more frequently detected than at 8 wpi (Fig. 6). LU592/C was seen to colonize all parts of the root system. Fluorescent mycelia were observed on healthy root tips (Fig. 6a1) and along lateral roots (Fig. 6b1). Fluorescent conidiophore structures were observed along lateral roots (Fig. 6c1, c2, d1). Conidiophores were easily detectable owing to their bright fluorescence and concentrated occurrence. Nonfluorescent fungal structures were often found in proximity to fluorescent fungal structures (Fig. 6c, c1 (arrows), d, d1). In addition, variation in fluorescence intensity was observed for hyphae of LU592/C at some locations. Roots not colonized by LU592/C were also frequently observed. Wherever GFP-fluorescent fungal structures were found on the root surface, LU592/C was recovered from within the roots. Microscopic examinations of root subsamples were laborious and time-consuming. The occurrence of fluorescent fungal structures was not frequently detected. Therefore, no conclusions could be drawn about preferred root colonization sites. No fluorescent fungal structures were observed for the untreated control.

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Figure 5. Representative photographs of Pinus radiata root colonization by fluorescent Trichoderma hamatum LU592/C 8 weeks postinoculation of experiment 2. (a) fluorescent mycelia colonizing root hairs, (b) fluorescent mycelia colonizing bark particle next to a lateral root (upper left corner) and (c) fluorescent mycelia colonizing the root surface.

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Figure 6. Representative photographs of Pinus radiata root colonization by fluorescent Trichoderma hamatum LU592/C 16 weeks postinoculation of experiment 2. (a, a1) fluorescent mycelia colonizing elongating root tip, (b, b1) fluorescent mycelia colonizing a lateral root, (c, c1, c2) fluorescing conidiophore structures attached to the root surface next to a nonfluorescent conidiophore structure (c and c1, arrow), (d, d1) fluorescent conidiophore structure next to a lateral root and surrounded by nonfluorescent mycelia. Photographs a, b, c and d were taken under daylight, photographs a1, b1, c1, c2 and d1 were taken under GFP-excitation.

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Discussion

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

The techniques used in this study enabled detailed ecological studies of an introduced isolate within a large indigenous Trichoderma population. Isolate-specific recovery was achieved by successful integration of marker genes into the genome of T. hamatum LU592. The transformant LU592/C was mitotically stable, showed phenotypic similarity to its wild type and could be visualized in and recovered from nonsterile potting mix. The exclusion of nontarget microorganisms allowed the detection of isolate concentrations as low as 35 CFU g−1 dry potting mix. This resulted in an increase in the sensitivity of the dilution plating technique by at least a factor of ten compared with detection levels achieved in the same system reported recently (Hohmann et al., 2011).

The rhizosphere and endorhizosphere competence of T. hamatum LU592, reported in a previous study (Hohmann et al., 2011), was confirmed in more detail. LU592 was shown to be both a rhizosphere colonizer and root endophyte colonizing the entire root system both externally and internally. More specifically, experiment 1 showed the greatest colonization of the P. radiata root system by LU592/C when pots were inoculated with 105 spores compared with 103 and 107 spores. Not only were total CFU levels highest in the rhizosphere, but the ability to internally colonize the roots was also best at the 105 inoculum rate. Similar mycelia proportions were found between the 105 and 107 treatments. As mycelia levels provide a better indication of biological activity than total CFU levels, this result probably explains why these two inoculation rates resulted in equivalent beneficial effects on P. radiata growth and health reported in a previous study (Hohmann et al., 2011). A possible explanation for the lower root colonization of LU592 at the highest inoculum concentration is the Allee effect. The Allee effect is known in the field of animal ecology and describes the density dependency of a population on its ability to establish in a new environment (Kramer et al., 2009; Tobin et al., 2009; A. Liebhold, pers. commun.). Etienne et al. (2002) reported for the insect Drosophila melanogaster that the Allee effect creates a lower and upper boundary to the population size. The initial population distribution and density determined whether a population was established, while resource availability was responsible for the subsequent population persistence. These findings were recently confirmed within microbial communities (Bonde et al., 2004; Aspray et al., 2006; Kadam & Velicer, 2006) and highlight the importance of identifying the optimum inoculum concentration, that is, to say, the optimum population density within the new environment to result in establishment. The Allee effect (a lower and upper boundary to the population size) would explain why higher inoculum concentrations do not necessarily result in higher population sizes and do not increase the beneficial effects to the plant. As a consequence, determining the optimum Trichoderma inoculum concentration is of vital importance for the success of a commercial bio-inoculant. Not only can overdosing result in detrimental side effects to the plant (Ousley et al., 1993; Rousseau et al., 1996; Brimner & Boland, 2003) but can also unnecessarily increase the inoculum production costs of the agent.

The population dynamics of T. hamatum LU592 were examined in experiment 2. Overall CFU levels of LU592 increased significantly over time in the P. radiata root system compared with the bulk potting mix (potting mix system without the plant). This establishment is supported by the mycelia and internal root colonization data. The mycelia enumeration indicated that a substantial proportion of the applied Trichoderma spores had germinated in the top section after 3 days. The nutrient-rich spermosphere probably initiated the germination of the spores.

A decrease in the number of propagules 8 weeks after seedling emergence was reported previously (Hohmann et al., 2011). At the time, seedlings were transferred from the glasshouse to the outside, and therefore, it was assumed that a change in environmental conditions caused a decrease in the Trichoderma population. However, a similar pattern was observed in this study where seedlings were grown under controlled conditions for the duration of the experiment. The decline in overall CFU and mycelia levels could have been caused by depletion in nutrients, assuming that the maturing root did not yet provide enough nutrients. It is known that Trichoderma spp. are very sensitive to various types of disturbances and stimuli (Lu et al., 2004). Maturing P. radiata roots experience physiological changes that can influence structure and function of soil microbial communities (Shi et al., 2011ab). Therefore, another possibility might be that changes in the composition of root exudates caused a decrease in overall Trichoderma populations. This assumption is supported by the fact that there was an increase in overall CFU and mycelia levels in the 4-week-old root system (centre section) indicating sufficient nutrient availability. Propagule numbers increased after 12 weeks when an abundance of roots was observed in the centre and bottom sections of the pot. An increase in mycelia levels and the number of internally colonized root segments also indicated high fungal activities at that time, both of which may have been caused by increased nutrients released by the roots.

The LU592 population in the bulk potting mix increased moderately over time and stabilized at around 103 CFU. Previous reports demonstrated long-term establishment of applied Trichoderma spp. at a similar level in natural soils and plant rhizosphere (Papavizas, 1981; Lewis & Papavizas, 1984; Leandro et al., 2007; Longa et al., 2009). However, mycelia ratios were below the detection level in the bulk potting mix after 16 weeks and 6 months in experiments 2 and 1, respectively. A weak ability to proliferate in the bulk potting mix was observed. These results are in accordance with Fahima & Henis (1990) who concluded that T. hamatum is a poor competitor for dead organic substrates, but uses root exudates as a food source. In contrast, composted bark and compost added to potting mix has recently been shown to stimulate T. hamatum activity, reducing disease incidence and enhancing plant vigour (Khan et al., 2004; Hoitink et al., 2006). There seems to be a synergistic effect on the activity of T. hamatum when both composted organic substrates and plant roots are present.

This study demonstrated that conclusions based on overall CFU numbers can lead to misinterpretations. Firstly, relatively high overall CFU levels do not necessarily indicate high fungal activity as shown in the bulk potting mix of experiments 1 and 2 or in the rhizosphere of the 103 application of experiment 1. This issue with the traditional dilution plating method has been reported extensively (Ahmad & Baker, 1987; Parkinson & Coleman, 1991; Paulitz, 2000; Green et al., 2001; Orr & Knudsen, 2004). Secondly, lower overall CFU levels do not necessarily indicate a low biological activity of Trichoderma. This was shown in experiment 2 when 43.6% of a total CFU level of < 103 was shown to be mycelia around the emerging radicle of 3-day-old seedlings. Also, the proportion of LU592 CFU as mycelia was maintained within a distance of at least 1 cm from the root surface, whereas total CFU counts suggested the zone of activity to be restricted to < 2 mm.

Chen et al. (2002) demonstrated the rhizosphere of P. radiata seedlings to be limited to within 5 mm of the root. LU592 clearly showed an affinity for P. radiata roots, but was also able to extend its zone of activity beyond the rhizosphere and, therefore, could have mobilized nutrients from the potting mix medium making them available to the plant. Previous reports support this assumption that demonstrated N and P mobilization by Trichoderma spp. in the root zone of pine trees (Blakeman, 1978; Koide & Kabir, 2001; Wu et al., 2003, 2005). Another possible mode of action by which LU592 elicits its benefits to the plant could be competition with minor root pathogens owing to the ability of LU592 to colonize and penetrate the entire root system including taproot, lateral roots, root tips and root hairs. In particular, this study showed qualitative evidence of the presence of T. hamatum LU592 on P. radiata root tips (external and internal), which are potential infection sites for root pathogens (Green et al., 2001; Lagopodi et al., 2002). Root tip colonization by Trichoderma spp. has been demonstrated in other systems including annual crops and Douglas-fir (Mousseaux et al., 1998; Yedidia et al., 2000; Green et al., 2001). Some of the microscopic observations suggest that mycelia of LU592 penetrated the outer root cortex colonizing between epidermal cells. This is a well-known feature reported for the genus Trichoderma in various plants (Waid, 1956; Harman et al., 2004; Vinale et al., 2008) and is supported by the fact that LU592 was recovered from every surface-sterilized root sample where fluorescent mycelia were visible beforehand. It has been suggested that the fungus occupies nutritional niches forming a symbiotic rather than parasitic relationship with the plant (Harman et al., 2004). Specific experiments are now required to address each of the potential modes of action.

This study answered key questions about the spatial and temporal distribution of T. hamatum LU592 in the root system of P. radiata. The importance of an optimum inoculum application was highlighted, which is essential to not only enhance root colonization and plant vitality, but also to minimize inoculum production costs. The total CFU as mycelia reached up to 89% in the 16-week-old rhizosphere. After 6 months, the mycelia data suggest that those proportions stabilize at 20–25%. Future studies could observe mycelia levels in relation to change in root exudate quantity and composition over longer periods (years). The results of this study lead to the hypothesis that Trichoderma experiences cyclic patterns of higher and lower fungal activity. Evidence was provided that the biological activity associated with the distribution of an introduced isolate is more critical than the distribution itself. Therefore, in future ecological studies of Trichoderma bio-inoculants, it is recommended to supplement traditional quantification techniques with root penetration and mycelia assessments to provide additional knowledge about fungal activity.

Acknowledgements

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

The authors thank Lincoln University Biotron Manager, Stuart Larsen, for technical support and Dr Leandro Lopes Loguercio for valuable discussions. This research was funded by the New Zealand Foundation for Research, Science and Technology.

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

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information
FilenameFormatSizeDescription
fem1340-sup-0001-FigS1.docWord document99KFig. S1. Southern hybridisation analyses showing the successful single copy integration of the gfp/hph cassette into the genome of the three transformants LU592/A (A), LU592/B (B) and LU592/C (C) digested with either EcoRI (left) or Acc65I (right).

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