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Annals of Applied Biology ISSN 0003-4746 RESEARCH ARTICLE Colonisation of barley roots by endophytic Fusarium equiseti and Pochonia chlamydosporia: Effects on plant growth and disease J.G. Maciá-Vicente1 , L.C. Rosso2 , A. Ciancio2 , H.-B. Jansson1 & L.V. Lopez-Llorca1 1 Laboratory of Plant Pathology, Department of Marine Sciences and Applied Biology, Multidisciplinary Institute for Environmental Studies (MIES) Ramón Margalef, University of Alicante, Apto 99, 03080 Alicante, Spain 2 Istituto per la Protezione delle Piante, CNR, Via Amendola 165/A, 70126, Bari, Italy Keywords Biological control; Fusarium equiseti; Gaeumannomyces graminis var. tritici; plant growth promotion; Pochonia chlamydosporia; root endophytes; take-all. Correspondence J.G. Maciá-Vicente, Laboratory of Plant Pathology, Department of Marine Sciences and Applied Biology, Multidisciplinary Institute for Environmental Studies (MIES) Ramón Margalef, University of Alicante, Apto 99, 03080 Alicante, Spain. Email: [email protected] Received: 12 September 2008; revised version accepted: 26 June 2009. doi:10.1111/j.1744-7348.2009.00352.x Abstract Colonisation of plant roots by endophytic fungi may confer benefits to the host such as protection against abiotic or biotic stresses or plant growth promotion. The exploitation of these properties is of great relevance at an applied level, either to increase yields of agricultural crops or in reforestation activities. Fusarium equiseti is a naturally occurring endophyte in vegetation under stress in Mediterranean ecosystems. Pochonia chlamydosporia is a nematode eggparasitic fungus with a worldwide distribution. Both fungi have the capacity to colonise roots of non-host plants endophytically and to protect them against phytopathogenic fungi under laboratory conditions. The aim of this study was to evaluate the root population dynamics of these fungi under nonaxenic practical conditions. Both fungal species were inoculated into barley roots. Their presence in roots and effects on plant growth and incidence of disease caused by the pathogen Gaeumannomyces graminis var. tritici were monitored periodically. Both fungi colonised barley roots endophytically over the duration of the experiment and competed with other existing fungal root colonisers. Furthermore, colonisation of roots by P. chlamydosporia promoted plant growth. Although a clear suppressive effect on disease could not be detected, F. equiseti isolates reduced the mean root lesion length caused by the pathogen. Results of this work suggest that both F. equiseti and P. chlamydosporia are long-term root endophytes that confer beneficial effects to the host plant. Introduction Endophytism is a ubiquitous phenomenon that occurs in all plants and environments. Its importance in plant development and distribution is starting to be unravelled. Fungal endophytes may help their host plants adapt to habitats, protect against biotic or abiotic stresses, promote plant growth or facilitate soil nutrient uptake (Sieber, 2002; Rodriguez et al., 2004; Schulz & Boyle, 2005; Rodriguez & Redman, 2008). In studies of root endophytism in natural plant communities under stress (Maciá-Vicente et al., 2008a), a large component of fungal root endophytes antagonistic to important root pathogens Ann Appl Biol 155 (2009) 391–401 © 2009 The Authors Journal compilation © 2009 Association of Applied Biologists such as wilt and take-all fungi was found (Maciá-Vicente et al., 2008b). Bona fide biocontrol fungi, which include important antagonists of plant pests and diseases (e.g. nematophagous and entomopathogenic fungi), can also behave endophytically (Lopez-Llorca et al., 2006). Fusarium equiseti, a naturally occurring endophyte from vegetation under stress in Mediterranean ecosystems, can also colonise non-host roots (Maciá-Vicente et al., 2008a,b). This species produces toxins antagonistic to fungal pathogens and plant parasitic nematodes (Nitao et al., 2001; Horinouchi et al., 2007; Maciá-Vicente et al., 2008b). Fusarium equiseti also controls parasitic plants 391 Root colonisation by endophytes and effects on host plant (Kirk, 1993). Pochonia chlamydosporia is a nematode eggparasitic fungus and occurs widely in cyst and root-knot nematode-infested soils around the world (Kerry, 1993). Pochonia chlamydosporia also parasitizes economically important phytopathogenic fungi, including the take-all fungus Gaeumannomyces graminis var. tritici (Leinhos & Buchenauer, 1992; Ehteshamul et al., 1994; Jacobs et al., 2003; Monfort et al., 2005). All these properties make F. equiseti and P. chlamydosporia as potential biocontrol agents for both phytopathogenic fungi and plant parasitic nematodes. The plant benefits of endophytism can be exploited in agriculture (e.g. crop protection/adaptation or yield increase) or in ecosystem restoration (e.g. plant adaptation to degraded environments). Practical use of the endophytic capacities of selected fungi may come from the use–in alternative hosts–of natural endophytes, which confer benefits to their original hosts (Rodriguez et al., 2008). Alternatively, beneficial fungi (e.g. biocontrol fungi) with endophytic capabilities can be inoculated in roots (Lopez-Llorca et al., 2006) or other plant tissues (Gómez-Vidal et al., 2006; Vega, 2008). The successful application of endophytes depends on our understanding of their behaviour in the plant host and their interactions with other rhizospheric microorganisms. This understanding includes the study of the dynamics of fungal colonisation of plant tissues. Colonisation of barley roots by F. equiseti and P. chlamydosporia has already been characterised under axenic laboratory conditions (Bordallo et al., 2002; LopezLlorca et al., 2002; Maciá-Vicente et al., 2008b, 2009). However, there are to date no reports on their long-term capacity to persist endophytically within roots of plants growing in non-axenic conditions. Pochonia chlamydosporia has previously been inoculated into rhizospheric soil of different crop plants, for control of both fungal pathogens and nematode parasites (Bourne et al., 1996, 1998; Monfort et al., 2005). The presence of the fungus in plant roots under non-axenic conditions was not reported in these studies. This may be an important feature because P. chlamydosporia seems to be a poor competitor when introduced into the soil (Monfort et al., 2006). We propose that root endophytism provides biocontrol agents targeted to soil pathogens (e.g. F. equiseti or P. chlamydosporia) with an adaptative advantage by lowering competition with soil microbiota. Fungi within the root can easily find nutrients and environmental conditions suitable for multiplication. In this way, endophytism could create a stable source of inoculum to sustain the populations of the microorganism in the rhizospheric soil. The aim of this study was to understand the effects of endophytic colonisation of barley roots by F. equiseti and P. chlamydosporia on plant growth and disease. For 392 J.G. Maciá-Vicente et al. this purpose, we inoculated both fungal species in barley roots to ensure their endophytic development and grew the inoculated plants under greenhouse conditions. We monitored the presence of the two endophytes in roots periodically, either in the rhizoplane or within the roots. We also tested the effects of endophytic colonisation on plant growth. In a second experiment, we included a fungal root pathogen and evaluated the influence of the endophytic development of F. equiseti and P. chlamydosporia on the presence of the pathogen in the roots and root disease. For this experiment, we selected G. graminis var. tritici, the causal agent of take-all, a root disease of cereal crops worldwide. Gaeumannomyces graminis var. tritici has been used extensively in root biology studies and is a model pathogen for experimental systems such as the one presented herein. Materials and methods Fungal material Fusarium equiseti Corda (Saccardo) isolates, 10/3.3.1 (Fe10331) and 45/1.2.1 (Fe45121), and P. chlamydosporia (Goddard) Zare & Gams isolates, 123 (Pc123) and INEMVC-21 (Pc21), were selected for inoculation experiments. Fusarium equiseti Fe10331 was isolated from roots of Corynephorus canescens (Poaceae) growing in a sandy soil and Fe45121 from roots of Lygeum spartum (Poaceae) growing in a salt marsh (Maciá-Vicente et al., 2008a). Pochonia chlamydosporia Pc123 was isolated from Heterodera avenae infected eggs in SW Spain (Olivares-Bernabeu & Lopez-Llorca, 2002) and Pc21 from soil and egg masses of Meloidogyne sp. infecting kiwifruit trees at Metaponto (Italy). Gaeumannomyces graminis (Saccardo) Arx & Olivier var. tritici Walker (Ggt) was kindly provided by Dr K. Sivasithamparam, University of Western Australia, and has been used previously for co-inoculation of barley plants with Fe10331, Fe45121 and Pc123 under axenic conditions (Monfort et al., 2005; Maciá-Vicente et al., 2008b). Inoculation of barley roots with fungal endophytes Barley (Hordeum vulgare L. var. distichum) roots were inoculated with four plugs (5 mm in diameter) of Fe10331, Fe45121, Pc123 or Pc21 growing on corn meal agar (CMA, BBL, Sparks, MD, USA) in tubes containing sterilised vermiculite, as described in Maciá-Vicente et al. (2008b). Control treatments consisted of tubes with plugs of non-colonised CMA. Plants were placed in a growth chamber with a photoperiod of 16/8 h (light/dark) at 23◦ C for 7 days. Plants were then transferred to 1 L pots with pasteurised sandy soil from Ragusa (Italy). Pots were placed in a greenhouse and irrigated periodically. Ann Appl Biol 155 (2009) 391–401 © 2009 The Authors Journal compilation © 2009 Association of Applied Biologists J.G. Maciá-Vicente et al. Five plants from each treatment were sampled at intervals of 7 (from tubes with sterilised vermiculite), 21, 35, 49 and 63 (from pots with soil) days after inoculation (dai). Fresh shoot and root weight per plant were recorded upon sampling. Roots of each plant were then split into two halves. One was left untreated whereas the other one was surface sterilised in 1% sodium hypochlorite for 1 min, washed three times in sterile distilled water (1 min each) and blotted onto sterilised filter paper. Non-sterilised and surface-sterilised roots were cut into approximately 1 cm fragments and six root pieces per plant were plated onto CMA supplemented with 50 mg mL−1 streptomycin, 50 mg mL−1 penicillin G and 1% Triton X-100 (Sigma, St. Louis, MO, USA). Sterilised root pieces were imprinted onto plates with the same medium before plating to evaluate the efficacy of the surface sterilisation method (Hallmann et al., 2006). After 5–7 days, fungal colonisation of root pieces was recorded, and developing fungal colonies were isolated on potato dextrose agar (PDA, Oxoid Ltd, Hampshire, UK) for identification. Percentage of root colonisation by endophytes (F. equiseti and P. chlamydosporia) and other filamentous fungi was then calculated as: Nd /Nt ·100, where Nd is the number of root pieces from which the fungi were detected and Nt the total number of root pieces. Co-inoculation of barley roots with fungal root endophytes and Ggt Co-inoculation of barley roots with either Fe10331, Fe45121, Pc123 or Pc21 and Ggt was performed as described in Maciá-Vicente et al. (2008b). Control treatments were inoculated with Ggt only and included four uncolonised plugs of CMA instead of the endophyte. Inoculated plantlets were kept in a growth chamber with the same conditions previously mentioned for 7 days, and then transferred to 1 L pots with a non-sterilised sandy soil from Arenal de Biar (SE, Spain; Monfort et al., 2006). Ten plants per treatment were sampled at intervals of 7 (from tubes with sterilised vermiculite), 35 and 63 dai (from pots with soil). Plants were rated for disease using an arbitrary scale of 0–4 with intervals of 0.5 (Cotterill & Sivasithamparam, 1987). Shoot and root fresh weight per plant were recorded, and the root system of each plant was then carefully scanned with an EPSON Expression 1680 Pro scanner (Seiko Epson, Nagano, Japan) for further analysis. Root images were digitised with the software Photoshop CS2 (Adobe); root length above symptoms were coloured blue, symptoms red and root length below symptoms green. Red–green–blue colours of digitised images were detected separately using the software analySIS (Soft Imaging System GmbH, Münster, Germany). Total root length, mean lesion length and Ann Appl Biol 155 (2009) 391–401 © 2009 The Authors Journal compilation © 2009 Association of Applied Biologists Root colonisation by endophytes and effects on host plant percentage of effective root length (ERL) per plant (Aberra et al., 1998) were then estimated. Plant roots were either non-sterilised or surface sterilised as described above and cut into approximately 1 cm long fragments. Ten root fragments per plant were plated onto CMA supplemented with 50 mg mL−1 streptomycin, 50 mg mL−1 penicillin G and 1% Triton X-100. After 5–7 days, colonisation of the root pieces was recorded, and developing fungal colonies were isolated on fresh PDA for fungal identification. Percentage of root colonisation by endophytes, Ggt and other filamentous fungi was calculated. Remaining root material was stored at –76◦ C for further studies. Polymerase chain reaction detection of Ggt in barley roots Because adequate quantification of Ggt in roots could not be achieved by culturing techniques, its presence in roots was assessed by polymerase chain reaction (PCR)-based methods. Surface-sterilised root systems from plants sampled 63 dai were used for genomic DNA extraction. Roots of three plants per treatment were selected randomly for this experiment. DNA was extracted from surface-sterilised roots as follows: approximately 0.6–1 g of root tissue was ground in liquid nitrogen and DNA was extracted at 65◦ C for 1 h with 4 mL of DNA extraction buffer containing 100 mM Tris–HCl pH 8.4, 1.4 M NaCl, 25 mM EDTA (Sigma), 2% CTAB (Sigma) and 2% low weight polyvinylpyrrolidone (PVP, Sigma). Extracts were purified with one volume phenol: chloroform: isoamyl-alcohol (IAA)(25:24:1) and then with one volume chloroform: IAA (24:1) and precipitated in one volume isopropanol. DNA pellets were washed twice in 70% ethanol, air-hood dried and resuspended in 1 × TNE buffer. RNAse A (Sigma) was added to each treatment and tubes were incubated for 30 min at 37◦ C. Extracts were again purified in phenol: chloroform: IAA and chloroform: IAA and precipitated in two volumes absolute ethanol. Pellets were again washed twice in 70% ethanol and allowed to dry, and DNA was finally resuspended in 1 × TE buffer. The extraction method used resulted in DNA suspensions containing brown material, which could be phenolic compounds such as lignin (Chen et al., 1996). These compounds inhibited PCR amplification (data not shown). Therefore, DNA extracts were further purified using the GENECLEAN SPIN Kit (Qbiogene Inc., Carlsbad, CA, USA) following the manufacturer’s instructions. Purified extracts were quantified using H6024 Hoechst stain (Sigma) as described in Ausubel et al. (2002) and stored at 4◦ C until use. To perform positive controls for PCR reactions, genomic DNA from Ggt pure cultures was obtained as in O’Donnell et al. (1998), with minor modifications. 393 Root colonisation by endophytes and effects on host plant Primers NS5 (5 -AACTTAAAGGAATTGACGGAAG-3 ) and GGT-RP (5 -TGCAATGGCTTCGTGAA-3 ) were used for PCR detection of Ggt in roots as in Fouly & Wilkinson (2000), with modifications. Amplifications were performed in a total volume of 50 μL, containing: 1 × Flexi buffer, 2 mM MgCl2 , 0.2 mM dNTP, 0.5 μM each of the primers, 10 ng of DNA template and 2.5 units of Taq DNA polymerase (Promega Corporation, Madison, WI, USA). A positive control included 1 ng of Ggt genomic DNA. A negative control, in which genomic DNA was replaced by water, was used as a test for contamination. Temperature cycling was carried out in a PTC-100 Thermal cycler (MJ Research, Waltham, MA, USA). An initial denaturation step at 93◦ C for 3 min was followed by 35 cycles of denaturation at 93◦ C for 1 min, annealing at 57◦ C for 1 min and extension at 72◦ C for 1 min. A final step at 72◦ C for 5 min was performed after the cycles. An aliquot of 10 μL amplification products were loaded onto a 2% electrophoresis agarose gel, stained with SYBR Green I (Sigma) and photographed under ultraviolet light. Bands in the gel corresponding to 410-bp Ggtspecific amplicon were excised, purified with Ultrafree columns (Millipore Corp., Cork, Ireland) and sequenced (Macrogen Inc., Seoul, Korea). Sequences amplified from root extracts were compared with the sequence from Ggt pure cultures and with those in the GenBank database using BLAST analysis (Altschul et al., 1990). Data analysis Data were checked for normality using the Shapiro–Wilk test, and Levene’s test was used to study homogeneity of variance across groups. Data following a normal distribution were compared using two-way ANOVA for differences between treatments and/or sampling times. Nonnormal data were compared using the Kruskal–Wallis (K–W) rank sum test. Either Student’s t-test or Wilcoxon test with corrections for multiple testing was used for pair-wise comparisons. In all cases, significance level considered was 95%. All analyses were performed using the R 2.5.1 software (R Development Core Team, 2007). Results Rhizosphere dynamics of F. equiseti and P. chlamydosporia Both F. equiseti and P. chlamydosporia occurred with a high isolation frequency (from approximately 78% to 100% colonisation) in non-sterilised roots 7 dai, from plants growing in tubes with vermiculite. Their occurrence decreased in roots after transplanting to pots with soil (Fig. 1). Both fungal species were isolated at low frequency from surface-sterilised roots, but their presence 394 J.G. Maciá-Vicente et al. remained relatively constant with time (Fig. 1). Generally, the two isolates of each fungal species behaved in a similar manner: Fe10331 and Fe45121 populations decreased gradually in the ectorhizosphere, whereas their endophytic populations were stable at around 7% and 20% colonisation. Colonisation at 63 dai reached higher values than in non-sterilised roots at the same time (Fig 1A and Fig. 1B). Pc123 and Pc21 had completely colonised the outer root system 7 dai, as assessed by plating of non-sterilised roots. However, its incidence was reduced severely (from 100% to 0% for Pc123 and 7% for Pc21) after plants were transferred to pots with soil (Fig. 1C and Fig. 1D). Isolation percentages of both P. chlamydosporia isolates from surface-sterilised roots were comparable with those of F. equiseti isolates 7 dai (13%–23%), but decreased in subsequent samplings of plants from pots (Fig. 1C and Fig. 1D). The presence of both F. equiseti and P. chlamydosporia as endophytes affected colonisation of barley roots by other filamentous fungi. In treatments with endophytes, the isolation of other fungi from surface-sterilised roots decreased significantly (K–W, P < 0.05) when compared with that of uninoculated controls, except at 49 dai (Fig. 1). On the contrary, the percent colonisation of filamentous fungi other than endophytes obtained from non-sterilised roots was different between roots inoculated with F. equiseti or P. chlamydosporia. Although colonisation of the ectorhizosphere of roots inoculated with either Fe10331 or Fe45121 with other fungi increased gradually with time following a reduction of F. equiseti populations, overall colonisation by other fungi was significantly lower than that in controls (K–W, P < 0.05; Fig. 1A, Fig. 1B and Fig. 1E). For roots inoculated with P. chlamydosporia, isolation of other fungi did not differ (K–W, P = 1) from control treatments: after the transfer of the plants to pots with soil, colonisation of the root by soil contaminants reached values of 83%–100% (Fig. 1C, Fig. 1D and Fig. 1E). Effects of root colonisation by F. equiseti and P. chlamydosporia on plant growth Colonisation of roots by F. equiseti isolates had virtually no effect on plant growth (Fig. 2). On the contrary, P. chlamydosporia had a clear plant growth-promoting effect on barley (Fig. 2). Plants inoculated with either isolate Pc123 or Pc21 demonstrated a significant (ANOVA, P < 0.05) increase in shoot weight with respect to controls from 49 dai onwards (Fig. 2A). At 63 dai, the shoot weight of plants inoculated with either Pc123 or Pc21 was 1.6- and 1.9-fold that of uninoculated control plants, respectively. This growth promotion by P. chlamydosporia was also observed in roots, with a significant (ANOVA, Ann Appl Biol 155 (2009) 391–401 © 2009 The Authors Journal compilation © 2009 Association of Applied Biologists J.G. Maciá-Vicente et al. Root colonisation by endophytes and effects on host plant Figure 1 Dynamics of colonisation of barley roots inoculated with the endophytes (open circles) Fe10331 (A), Fe45121 (B), Pc123 (C) and Pc21 (D), and by other indigenous soil filamentous fungi (solid circles), assessed in both non-sterilised (solid lines) and surface-sterilised (dashed lines) roots. Values of colonisation by filamentous fungi other than Fusarium equiseti and Pochonia chlamydosporia in control (uninoculated) treatments are provided in plot E. Bars show standard errors. Ann Appl Biol 155 (2009) 391–401 © 2009 The Authors Journal compilation © 2009 Association of Applied Biologists 395 Root colonisation by endophytes and effects on host plant J.G. Maciá-Vicente et al. Figure 2 Effect of root colonisation by Fusarium equiseti and Pochonia chlamydosporia on barley growth: shoot fresh weight (A) and root fresh weight (B). Bars show standard errors. P < 0.05) root weight increase at 63 dai when compared with controls (Fig. 2B). At this time, the rate of weight increase with respect to the control plants was 1.9 for Pc123 and 1.8 for Pc21. Furthermore, plants inoculated with P. chlamydosporia presented an early development of spikes at 63 dai, when compared with plants from the other treatments (data not shown). Rhizosphere dynamics of F. equiseti and P. chlamydosporia in the presence of Ggt The patterns of endophyte colonisation of Ggt-infected barley roots differed from those of plants inoculated with the endophytes only (Fig. 3). In this experiment, root colonisation was scored at 7, 35 and 63 dai. Fe10331 displayed higher colonisation percentages, for both nonsterilised and surface-sterilised roots than in the previous experiment, and significantly (K–W, P < 0.05) higher colonisation rates than Fe45121 at 7 and 35 dai (Fig. 3A). Fe45121 was isolated at lower levels from both nonsterilised and sterilised roots after 7 days of growth in sterilised vermiculite, and from non-sterilised roots from pots with soil at 35 dai (Fig. 3B), than in the experiment with the endophyte alone (Fig. 1B). Colonisation by Fe10331 and Fe45121 of non-sterilised and surfacesterilised roots was not significantly different (K–W, P > 0.05) at 63 dai (20%–44%). Pc123 followed the same pattern of colonisation of the root as in the previous experiment, but with higher colonisation rates (Fig. 3C). 396 Pc21 showed a significantly higher (K–W, P < 0.05) isolation percentage than Pc123 (65% and 9%, respectively) in the ectorhizosphere 35 dai. Isolation rates of both Pc123 and Pc21 at 63 dai did not differ significantly (K–W, P > 0.05) in non-sterilised and in surface-sterilised roots (9%–30%; Fig 3C and Fig 3D). Quantification determined by culturing Ggt from infected roots could only be achieved from plantlets growing in culture tubes with sterilised vermiculite 7 dai. In this case, isolation rates ranged between 2 ± 0.6% and 6 ± 1.3% in non-sterilised roots, with no significant differences among treatments. In surface-sterilised roots, values ranged between 0 ± 0% for Fe10331 and Pc21 and 4 ± 1.3% in control treatments, with no significant differences. At 35 and 63 dai, sterile dark-pigmented mycelia were frequently recovered from roots of plants in all treatments, but these could not be related to Ggt either in CMA or PDA culture media, because of a lack of sporulation of the colonies. Therefore, the presence of Ggt in barley roots was assessed by PCR using primers specific to Ggt. All PCR reactions including DNA from barley roots inoculated with Ggt only and those with both Ggt plus endophytes, and with DNA extracted from Ggt pure cultures, yielded amplification products. All amplification products showed several unspecificities, but approximately 410-bp Ggtspecific amplicon was found in all treatments (data not shown). PCR products from seven independent amplification reactions were purified from the gels and sequenced. In all cases, sequences matched those obtained from Ggt pure cultures and from GenBank databases. Ann Appl Biol 155 (2009) 391–401 © 2009 The Authors Journal compilation © 2009 Association of Applied Biologists J.G. Maciá-Vicente et al. Root colonisation by endophytes and effects on host plant Figure 3 Dynamics of colonisation of barley roots inoculated with both Ggt and Fe10331 (A), Fe45121 (B), Pc123 (C) and Pc21 (D) endophytes (open circles), and by other indigenous soil filamentous fungi (solid circles), assessed in both non-sterilised (solid lines) and surface-sterilised (dashed lines) roots. Values of colonisation by filamentous fungi other than Fusarium equiseti and Pochonia chlamydosporia in control (Ggt only) treatments are provided in plot E. Bars show standard errors. Ann Appl Biol 155 (2009) 391–401 © 2009 The Authors Journal compilation © 2009 Association of Applied Biologists 397 Root colonisation by endophytes and effects on host plant Isolation of fungal contaminants from surface-sterilised roots was significantly lower (K–W, P < 0.05) only in plants treated with Fe10331 than in controls with Ggt alone 63 dai (from 31% to 8%, respectively), whereas both Fe10331 and Pc21 reduced the occurrence of other filamentous fungi in non-sterilised roots at 35 dai (from 89% to 52% and 54% respectively) and Fe10331 at 63 dai (from 31% to 8%; Fig. 3). Therefore, inhibition of the presence of fungi other than the inoculated ones seemed to be correlated with the population size of the endophytes at each sampling time (Fig. 3). Effects of root colonisation by F. equiseti and P. chlamydosporia on take-all disease No effect was found on fresh shoot and root weight either in plants inoculated with F. equiseti or P. chlamydosporia and Ggt with respect to control treatments with Ggt only. Both P. chlamydosporia isolates Pc123 and Pc21 significantly increased (ANOVA, P < 0.05) the total root length 7 dai (0.584 ± 0.033 and 0.577 ± 0.039 m, respectively) when compared with the other treatments (0.430 ± 0.038 to 0.529 ± 0.023 m). This effect was lost after the transfer of plants to soil, and from 35 dai onwards root lengths did not differ significantly in all treatments. No significant differences were found among treatments and/or time in disease ratings, with values ranging between 0.75 ± 0.22 and 1.7 ± 0.22. However, mean lesion length increased significantly (K–W, P < 0.05) with time in all treatments except for the treatment with isolate Fe10331, in which a reduction in lesion length occurred from 35 (0.121 ± 0.027 m) to 63 (0.096 ± 0.036 m) dai. Larger differences in lesion length were observed at 63 dai. At this time, plants inoculated with either Fe10331 or Fe45121 showed a significant reduction (K–W, P < 0.05) in the mean lesion length (0.096 ± 0.036 and 0.097 ± 0.031 m, respectively) compared with control plants (0.289 ± 0.122 m). Percent effective root length (ERL) did not vary among treatments and sampling dates, except for Pc123 and Pc21, which showed a reduction in ERL after transfer to pots with soil. At 63 dai, ERL of plants with Pc21 (71.1 ± 5.7%) was significantly lower (K–W, P < 0.05) than that of the same treatment at the first sampling (96.1 ± 1.4%). It was, however, statistically similar (K–W, P > 0.05) to values found for other treatments at the same time (77.7 ± 4.3 to 97.3 ± 1.7%). Discussion Two F. equiseti isolates (Fe10331 and Fe45121) obtained in Maciá-Vicente et al. (2008a) and screened in vitro for antagonism against Ggt in Maciá-Vicente et al. (2008b), 398 J.G. Maciá-Vicente et al. and two isolates of the nematode egg-parasitic fungus P. chlamydosporia (Pc123 and Pc21), were tested for ability to colonise barley roots endophytically and for their effects on plant growth and the take-all caused by Ggt, in pot trials under greenhouse conditions. All isolates were able to colonise barley roots, where they remained endophytically for at least 2 months under non-axenic conditions. Both F. equiseti and P. chlamydosporia have been found in previous studies to colonise barley roots under laboratory conditions using the same experimental system applied in this work (Bordallo et al., 2002; Maciá-Vicente et al., 2008b, 2009). To the best of our knowledge, this is the first longterm study of the endophytic behaviour of both fungal species under non-axenic conditions. Fusarium equiseti appeared to be a more efficient rhizosphere coloniser than P. chlamydosporia, in terms of population size and competition with other fungal root colonisers. Although both species started with comparable levels of root colonisation under axenic conditions, both outside and within the root cortex, transferring the plants to soil affected their capacities to colonise roots in different manners. Fusarium equiseti persisted in the ectorhizosphere at high levels, which decreased gradually with time, whereas their endophytic occurrence was constant with slight changes over the duration of the experiment (approximately 2 months). The gradual decrease in the presence of F. equiseti in the ectorhizosphere was accompanied by a corresponding increase in other soil-inhabiting fungi. These fungi started colonising the ectorhizosphere to a lower extent than in uninoculated controls. This effect could be related to the reduction of Ggt growing axenically in culture tubes observed in Maciá-Vicente et al. (2008b) and could be an indicator of the competitive ability of the fungus in the rhizosphere. On the contrary, P. chlamydosporia disappeared rapidly from the root surface after transfer of the plants to soil, and its frequency in the ectorhizosphere remained low during the whole experiment. Endophytic colonisation of P. chlamydosporia remained constant with time, but was lower than that observed for F. equiseti. In treatments with P. chlamydosporia, complete colonisation of the outer root by other soil fungi occurred immediately after transferring plants to soil, similar to uninoculated controls. As was observed with F. equiseti, the presence of P. chlamydosporia reduced the occurrence of other fungi in the root cortex. Pochonia chlamydosporia had an early growth-promoting effect in barley. Both Pc123 and Pc21 increased fresh shoot and root weight and, at the end of the experiment, the increase reached almost twice that observed for uninoculated controls. This effect has also been observed in wheat and tomato plants inoculated with P. chlamydosporia (Monfort et al., 2005; Siddiqui & Akhtar, Ann Appl Biol 155 (2009) 391–401 © 2009 The Authors Journal compilation © 2009 Association of Applied Biologists J.G. Maciá-Vicente et al. 2008), but in these plants growth promotion was lower than that found in this study. This is probably because of our method of fungal inoculation, which was targeted to plant roots rather than soil. Plant growth promotion conferred by endophytic colonisation has been described previously, and this effect can include phenomena such as direct production of specific substances [i.e. phytohormones such as indole-3-acetic acid (IAA)] (Nassar et al., 2005), assistance in mineral supply and nutrient uptake, especially of phosphorus, from soil (Jumpponen & Trappe, 1998; Sieber, 2002), increase in the availability of carbohydrates and/or CO2 resulting from fungal metabolism (Jumpponen & Trappe, 1998) or stimulation of nitrate reduction (Sherameti et al., 2005). Some of these effects, such as increased phosphorus availability, are similar to that found for mycorrhizal associations. In fact, some mutualistic endophytes (i.e. Phialocephala fortinii) form colonisation patterns similar to the Hartig net and a thin patchy mantle, typical of ectomycorrhizae (Fernando & Currah, 1996). The plant growth promotion observed in the present paper does not seem to be related to formation of rhizospheric hyphal nets. This is connected with the low root colonisation rates found for P. chlamydosporia. This points to alternative mechanisms, such as production by the fungus of specific compounds (e.g. growth regulators), as the main effectors of plant growth promotion driven by P. chlamydosporia. Further studies are required to assess this hypothesis. Re-isolation methods in culture media used in this study have limitations, which may include a false correlation between the isolation rate and the real presence of the fungus (Schulz & Boyle, 2005). It is difficult to discriminate whether the low levels of P. chlamydosporia found in the roots are real, or they are an artefact of the methodology applied. The use of more accurate methods for the detection and quantification of fungi in the rhizosphere, such as real-time quantitative PCR (qPCR) techniques or genetic transformation with the GFP reporter gene in conjugation with microscopic studies, will help to assess these results (Ciancio et al., 2005; Rosso et al., 2007; Maciá-Vicente et al., 2009). In recent research, these methodologies were developed for the characterisation of the endophytic colonisation of barley roots by both F. equiseti and P. chlamydosporia under laboratory growth conditions (Maciá-Vicente et al., 2009). In these studies, first colonisation events consisted of a massive fungal growth on the root periphery, with single hyphae colonising the cortical tissues inter- and intracellularly. Subsequent colonisation events involved a reduction of the outer root fungal biomass and loss of viability of the endophytic mycelium in some root regions that could be a consequence of elicitation of plant defence responses. In spite of the latter, well-established viable hyphae were detected in other Ann Appl Biol 155 (2009) 391–401 © 2009 The Authors Journal compilation © 2009 Association of Applied Biologists Root colonisation by endophytes and effects on host plant root regions, suggesting an evasion of the plant defences by the fungal endophyte. Both qPCR and confocal laser scanning microscopy studies with GFP-tagged isolates are currently being used under the same experimental systems used in the present work to reanalyse these results. Root behaviour of both F. equiseti and P. chlamydosporia isolates was also checked in the presence of the cereal fungal pathogen Ggt. Fe10331 and Pc123 followed colonisation patterns similar to those observed in the experiment with the endophytes alone. However, Fe45121 displayed a reduction in root colonisation rate and Pc21 showed a higher degree of root colonisation, than that observed in experiments with the endophytes alone. After the transfer of barley plants to soil, Ggt could not be isolated from roots. Ggt rarely produces asexual structures in culture, and most isolates do not produce sexual structures at all (Elliott, 2005). Therefore, differentiation of the dark hyphae of the pathogen from other pigmented mycelia isolated from barley roots was difficult or unreliable by means of observation of morphological characters. Application of a PCR detection procedure for Ggt (Fouly & Wilkinson, 2000) was therefore used as an alternative. This method allowed direct detection of Ggt in the roots of all plants analysed. A clear suppressive effect by F. equiseti and P. chlamydosporia on the take-all disease on barley plants could not be detected, in spite of previous positive results under laboratory screening conditions (Monfort et al., 2005; Maciá-Vicente et al., 2008b). Although such preliminary screening procedures are commonly recommended as components of a sequence of assays in the search for biological control agents, quite often results between simple laboratory and greenhouse practical growing conditions do not match, as a consequence of the increase in the experimental variables (Knudsen et al., 1997). In this study, a technique for inoculation and long-term root endophytic colonisation by two fungal species was developed. Non-sterilised sandy soil used in this work was non-receptive to Pc123 in previous studies (Monfort et al., 2006). That is, fungal growth of P. chlamydosporia was inhibited when applied to non-sterilised soil, probably because of soil microbial activities. According to our results, root tissues of the host plant offer a ‘‘safe’’ environment for the fungus to escape soil competition (‘lack of receptivity’). Colonisation of the endorhizosphere could also act as an additional source of fungal inoculum to the soil. In this sense, previous work reported abundant chlamydospore production by P. chlamydosporia on the rhizoplane, and to a lesser extent in the cortex of barley and tomato roots colonised by the fungus (Bordallo et al., 2002). Independent of their initial root colonisation rates, both types of root endophytes continued to colonise roots endophytically, although levels were low. 399 Root colonisation by endophytes and effects on host plant These results suggest that barley roots support certain root colonisers. It seems that a single inoculation of the endophytes in culture tubes with barley plants growing axenically was not enough to keep high endophytic populations of the fungi inoculated. Therefore, their presence gradually decreased when plants were transferred to soil to reach a low and stable colonisation level with time. Long-term endophytic colonisation may hence depend on an equilibrium between plant defences and the fungal rhizosphere competence, but could also be influenced by the methodology used for root inoculation. Even with the sub-optimal inoculation procedure of this study, F. equiseti and P. chlamydosporia isolates persisted as endophytes under non-axenic conditions. In the case of the putative endophyte P. chlamydosporia, this resulted in beneficial effects on the host plant growth. As suggested before, we are currently investigating practical and theoretical aspects of the root inoculation of fungi with endophytic capabilities and antagonistic potential against root pathogens (fungi and nematodes). The final aim is to optimise their biocontrol performance and crop growth promotion under agricultural conditions. Acknowledgements The authors want to thank Mathieu Gueroult for technical assistance. 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