Colonisation of Barley Roots by Endophytic Fusarium Equiseti and Pochonia Chlamydosporia: Effects on Plant Growth and Disease J.G

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. Macia-Vicente´ 1, 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) Ramon´ 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 Abstract Biological control; Fusarium equiseti; Gaeumannomyces graminis var. tritici;plant Colonisation of plant roots by endophytic fungi may confer beneﬁts to the host growth promotion; Pochonia chlamydosporia; such as protection against abiotic or biotic stresses or plant growth promotion. root endophytes; take-all. The exploitation of these properties is of great relevance at an applied level, either to increase yields of agricultural crops or in reforestation activities. Correspondence Fusarium equiseti is a naturally occurring endophyte in vegetation under J.G. Macia-Vicente,´ Laboratory of Plant Pathology, Department of Marine Sciences stress in Mediterranean ecosystems. Pochonia chlamydosporia is a nematode egg- and Applied Biology, Multidisciplinary Institute parasitic fungus with a worldwide distribution. Both fungi have the capacity to for Environmental Studies (MIES) Ramon´ colonise roots of non-host plants endophytically and to protect them against Margalef, University of Alicante, Apto 99, phytopathogenic fungi under laboratory conditions. The aim of this study 03080 Alicante, Spain. was to evaluate the root population dynamics of these fungi under non- Email: [email protected] axenic practical conditions. Both fungal species were inoculated into barley roots. Their presence in roots and effects on plant growth and incidence Received: 12 September 2008; revised version of disease caused by the pathogen Gaeumannomyces graminis var. tritici were accepted: 26 June 2009. monitored periodically. Both fungi colonised barley roots endophytically over doi:10.1111/j.1744-7348.2009.00352.x 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 such as wilt and take-all fungi was found (Maciá-Vicente et al., 2008b). Bona fide biocontrol fungi, which include Endophytism is a ubiquitous phenomenon that occurs important antagonists of plant pests and diseases (e.g. in all plants and environments. Its importance in plant nematophagous and entomopathogenic fungi), can also development and distribution is starting to be unrav- elled. Fungal endophytes may help their host plants behave endophytically (Lopez-Llorca et al., 2006). adapt to habitats, protect against biotic or abiotic stresses, Fusarium equiseti, a naturally occurring endophyte from promote plant growth or facilitate soil nutrient uptake vegetation under stress in Mediterranean ecosystems, (Sieber, 2002; Rodriguez et al., 2004; Schulz & Boyle, can also colonise non-host roots (Maciá-Vicente et al., 2005; Rodriguez & Redman, 2008). In studies of root 2008a,b). This species produces toxins antagonistic to endophytism in natural plant communities under stress fungal pathogens and plant parasitic nematodes (Nitao (Maciá-Vicente et al., 2008a), a large component of fungal et al., 2001; Horinouchi et al., 2007; Maciá-Vicente et al., root endophytes antagonistic to important root pathogens 2008b). Fusarium equiseti also controls parasitic plants

Ann Appl Biol 155 (2009) 391–401 © 2009 The Authors 391 Journal compilation © 2009 Association of Applied Biologists Root colonisation by endophytes and effects on host plant J.G. Macia-Vicente´ et al.

(Kirk, 1993). Pochonia chlamydosporia is a nematode egg- this purpose, we inoculated both fungal species in bar- parasitic fungus and occurs widely in cyst and root-knot ley roots to ensure their endophytic development and nematode-infested soils around the world (Kerry, 1993). grew the inoculated plants under greenhouse conditions. Pochonia chlamydosporia also parasitizes economically We monitored the presence of the two endophytes in important phytopathogenic fungi, including the take-all roots periodically, either in the rhizoplane or within the fungus Gaeumannomyces graminis var. tritici (Leinhos & roots. We also tested the effects of endophytic coloni- Buchenauer, 1992; Ehteshamul et al., 1994; Jacobs et al., sation on plant growth. In a second experiment, we 2003; Monfort et al., 2005). All these properties make included a fungal root pathogen and evaluated the influ- F. equiseti and P. chlamydosporia as potential biocontrol ence of the endophytic development of F. equiseti and agents for both phytopathogenic fungi and plant parasitic P. chlamydosporia on the presence of the pathogen in the nematodes. roots and root disease. For this experiment, we selected The plant benefits of endophytism can be exploited G. graminis var. tritici, the causal agent of take-all, a root in agriculture (e.g. crop protection/adaptation or yield disease of cereal crops worldwide. Gaeumannomyces grami- increase) or in ecosystem restoration (e.g. plant adap- nis var. tritici has been used extensively in root biology tation to degraded environments). Practical use of the studies and is a model pathogen for experimental systems endophytic capacities of selected fungi may come from the such as the one presented herein. use–in alternative hosts–of natural endophytes, which confer benefits to their original hosts (Rodriguez et al., Materials and methods 2008). Alternatively, beneficial fungi (e.g. biocontrol fungi) with endophytic capabilities can be inoculated Fungal material in roots (Lopez-Llorca et al., 2006) or other plant tissues Fusarium equiseti Corda (Saccardo) isolates, 10/3.3.1 (Gomez-Vidal ´ et al., 2006; Vega, 2008). The successful (Fe10331) and 45/1.2.1 (Fe45121), and P. chlamydosporia application of endophytes depends on our understanding (Goddard) Zare & Gams isolates, 123 (Pc123) and INEM- of their behaviour in the plant host and their interactions VC-21 (Pc21), were selected for inoculation experiments. with other rhizospheric microorganisms. This under- Fusarium equiseti Fe10331 was isolated from roots of standing includes the study of the dynamics of fungal Corynephorus canescens (Poaceae) growing in a sandy soil colonisation of plant tissues. and Fe45121 from roots of Lygeum spartum (Poaceae) Colonisation of barley roots by F. equiseti and growing in a salt marsh (Maciá-Vicente et al., 2008a). P. chlamydosporia has already been characterised under Pochonia chlamydosporia Pc123 was isolated from Heterodera axenic laboratory conditions (Bordallo et al., 2002; Lopez- avenae infected eggs in SW Spain (Olivares-Bernabeu & Llorca et al., 2002; Maciá-Vicente et al., 2008b, 2009). Lopez-Llorca, 2002) and Pc21 from soil and egg masses However, there are to date no reports on their long-term of Meloidogyne sp. infecting kiwifruit trees at Metaponto capacity to persist endophytically within roots of plants (Italy). Gaeumannomyces graminis (Saccardo) Arx & Olivier growing in non-axenic conditions. Pochonia chlamydospo- var. tritici Walker (Ggt) was kindly provided by Dr K. ria has previously been inoculated into rhizospheric soil of Sivasithamparam, University of Western Australia, and different crop plants, for control of both fungal pathogens has been used previously for co-inoculation of barley and nematode parasites (Bourne et al., 1996, 1998; Mon- plants with Fe10331, Fe45121 and Pc123 under axenic fort et al., 2005). The presence of the fungus in plant conditions (Monfort et al., 2005; Maciá-Vicente et al., roots under non-axenic conditions was not reported in 2008b). these studies. This may be an important feature because P. chlamydosporia seems to be a poor competitor when Inoculation of barley roots with fungal endophytes introduced into the soil (Monfort et al., 2006). We propose that root endophytism provides biocon- Barley (Hordeum vulgare L. var. distichum) roots were trol agents targeted to soil pathogens (e.g. F. equiseti or inoculated with four plugs (5 mm in diameter) of P. chlamydosporia) with an adaptative advantage by low- Fe10331, Fe45121, Pc123 or Pc21 growing on corn meal ering competition with soil microbiota. Fungi within the agar (CMA, BBL, Sparks, MD, USA) in tubes containing root can easily find nutrients and environmental condi- sterilised vermiculite, as described in Maciá-Vicente et al. tions suitable for multiplication. In this way, endophytism (2008b). Control treatments consisted of tubes with plugs could create a stable source of inoculum to sustain the of non-colonised CMA. Plants were placed in a growth populations of the microorganism in the rhizospheric chamber with a photoperiod of 16/8 h (light/dark) at ◦ soil. The aim of this study was to understand the effects 23 C for 7 days. Plants were then transferred to 1 L pots of endophytic colonisation of barley roots by F. equiseti with pasteurised sandy soil from Ragusa (Italy). Pots were and P. chlamydosporia on plant growth and disease. For placed in a greenhouse and irrigated periodically.

392 Ann Appl Biol 155 (2009) 391–401 © 2009 The Authors Journal compilation © 2009 Association of Applied Biologists J.G. Macia-Vicente´ et al. Root colonisation by endophytes and effects on host plant

Five plants from each treatment were sampled at inter- percentage of effective root length (ERL) per plant (Aberra vals of 7 (from tubes with sterilised vermiculite), 21, et al., 1998) were then estimated. 35, 49 and 63 (from pots with soil) days after inocula- Plant roots were either non-sterilised or surface tion (dai). Fresh shoot and root weight per plant were sterilised as described above and cut into approximately recorded upon sampling. Roots of each plant were then 1 cm long fragments. Ten root fragments per plant split into two halves. One was left untreated whereas the were plated onto CMA supplemented with 50 mg mL−1 other one was surface sterilised in 1% sodium hypochlo- streptomycin, 50 mg mL−1 penicillin G and 1% Triton rite for 1 min, washed three times in sterile distilled X-100. After 5–7 days, colonisation of the root pieces was water (1 min each) and blotted onto sterilised filter recorded, and developing fungal colonies were isolated paper. Non-sterilised and surface-sterilised roots were cut on fresh PDA for fungal identification. Percentage of root into approximately 1 cm fragments and six root pieces colonisation by endophytes, Ggt and other filamentous per plant were plated onto CMA supplemented with fungi was calculated. Remaining root material was stored ◦ 50 mg mL−1 streptomycin, 50 mg mL−1 penicillin G and at –76 C for further studies. 1% Triton X-100 (Sigma, St. Louis, MO, USA). Sterilised root pieces were imprinted onto plates with the same Polymerase chain reaction detection of Ggt medium before plating to evaluate the efficacy of the in barley roots surface sterilisation method (Hallmann et al., 2006). After 5–7 days, fungal colonisation of root pieces was recorded, Because adequate quantification of Ggt in roots could not and developing fungal colonies were isolated on potato be achieved by culturing techniques, its presence in roots dextrose agar (PDA, Oxoid Ltd, Hampshire, UK) for iden- was assessed by polymerase chain reaction (PCR)-based tification. Percentage of root colonisation by endophytes methods. Surface-sterilised root systems from plants sam- (F. equiseti and P. chlamydosporia) and other filamentous pled 63 dai were used for genomic DNA extraction. fungi was then calculated as: Nd/Nt·100, where Nd is the Roots of three plants per treatment were selected ran- number of root pieces from which the fungi were detected domly for this experiment. DNA was extracted from and Nt the total number of root pieces. surface-sterilised roots as follows: approximately 0.6–1 g of root tissue was ground in liquid nitrogen and DNA ◦ was extracted at 65 Cfor1hwith4mLofDNAextrac- Co-inoculation of barley roots with fungal root tion buffer containing 100 mM Tris–HCl pH 8.4, 1.4 M endophytes and Ggt NaCl, 25 mM EDTA (Sigma), 2% CTAB (Sigma) and 2% Co-inoculation of barley roots with either Fe10331, low weight polyvinylpyrrolidone (PVP, Sigma). Extracts Fe45121, Pc123 or Pc21 and Ggt was performed as were purified with one volume phenol: chloroform: described in Maciá-Vicente et al. (2008b). Control treat- isoamyl-alcohol (IAA)(25:24:1) and then with one vol- ments were inoculated with Ggt only and included four ume chloroform: IAA (24:1) and precipitated in one uncolonised plugs of CMA instead of the endophyte. volume isopropanol. DNA pellets were washed twice in Inoculated plantlets were kept in a growth chamber with 70% ethanol, air-hood dried and resuspended in 1 × TNE the same conditions previously mentioned for 7 days, and buffer. RNAse A (Sigma) was added to each treatment ◦ then transferred to 1 L pots with a non-sterilised sandy and tubes were incubated for 30 min at 37 C. Extracts soil from Arenal de Biar (SE, Spain; Monfort et al., 2006). were again purified in phenol: chloroform: IAA and chlo- Ten plants per treatment were sampled at intervals of roform: IAA and precipitated in two volumes absolute 7 (from tubes with sterilised vermiculite), 35 and 63 dai ethanol. Pellets were again washed twice in 70% ethanol (from pots with soil). Plants were rated for disease using and allowed to dry, and DNA was finally resuspended an arbitrary scale of 0–4 with intervals of 0.5 (Cotterill & in 1 × TE buffer. The extraction method used resulted in Sivasithamparam, 1987). Shoot and root fresh weight per DNA suspensions containing brown material, which could plant were recorded, and the root system of each plant be phenolic compounds such as lignin (Chen et al., 1996). was then carefully scanned with an EPSON Expression These compounds inhibited PCR amplification (data not 1680 Pro scanner (Seiko Epson, Nagano, Japan) for shown). Therefore, DNA extracts were further purified further analysis. Root images were digitised with the using the GENECLEAN SPIN Kit (Qbiogene Inc., Carlsbad, software Photoshop CS2 (Adobe); root length above CA, USA) following the manufacturer’s instructions. Puri- symptoms were coloured blue, symptoms red and root fied extracts were quantified using H6024 Hoechst stain length below symptoms green. Red–green–blue colours (Sigma) as described in Ausubel et al. (2002) and stored at ◦ of digitised images were detected separately using the 4 C until use. To perform positive controls for PCR reac- software analySIS (Soft Imaging System GmbH, Munster, ¨ tions, genomic DNA from Ggt pure cultures was obtained Germany). Total root length, mean lesion length and as in O’Donnell et al. (1998), with minor modifications.

Ann Appl Biol 155 (2009) 391–401 © 2009 The Authors 393 Journal compilation © 2009 Association of Applied Biologists Root colonisation by endophytes and effects on host plant J.G. Macia-Vicente´ et al.

Primers NS5 (5-AACTTAAAGGAATTGACGGAAG-3) remained relatively constant with time (Fig. 1). Gener- and GGT-RP (5-TGCAATGGCTTCGTGAA-3)wereused ally, the two isolates of each fungal species behaved for PCR detection of Ggt in roots as in Fouly & in a similar manner: Fe10331 and Fe45121 populations Wilkinson (2000), with modifications. Amplifications decreased gradually in the ectorhizosphere, whereas their were performed in a total volume of 50 μL, containing: endophytic populations were stable at around 7% and 1 × Flexi buffer, 2 mM MgCl2, 0.2 mM dNTP, 0.5 μM 20% colonisation. Colonisation at 63 dai reached higher each of the primers, 10 ng of DNA template and 2.5 units values than in non-sterilised roots at the same time of Taq DNA polymerase (Promega Corporation, Madison, (Fig 1A and Fig. 1B). Pc123 and Pc21 had completely WI, USA). A positive control included 1 ng of Ggt genomic colonised the outer root system 7 dai, as assessed by DNA. A negative control, in which genomic DNA was plating of non-sterilised roots. However, its incidence replaced by water, was used as a test for contamination. was reduced severely (from 100% to 0% for Pc123 Temperature cycling was carried out in a PTC-100 and 7% for Pc21) after plants were transferred to pots Thermal cycler (MJ Research, Waltham, MA, USA). An with soil (Fig. 1C and Fig. 1D). Isolation percentages of ◦ initial denaturation step at 93 C for 3 min was followed both P. chlamydosporia isolates from surface-sterilised roots ◦ by 35 cycles of denaturation at 93 C for 1 min, annealing were comparable with those of F. equiseti isolates 7 dai ◦ ◦ at 57 C for 1 min and extension at 72 C for 1 min. A final (13%–23%), but decreased in subsequent samplings of ◦ step at 72 C for 5 min was performed after the cycles. plants from pots (Fig. 1C and Fig. 1D). An aliquot of 10 μL amplification products were loaded The presence of both F. equiseti and P. chlamydosporia onto a 2% electrophoresis agarose gel, stained with SYBR as endophytes affected colonisation of barley roots by Green I (Sigma) and photographed under ultraviolet other filamentous fungi. In treatments with endophytes, light. Bands in the gel corresponding to 410-bp Ggt- the isolation of other fungi from surface-sterilised roots specific amplicon were excised, purified with Ultrafree decreased significantly (K–W, P < 0.05) when compared columns (Millipore Corp., Cork, Ireland) and sequenced with that of uninoculated controls, except at 49 dai (Macrogen Inc., Seoul, Korea). Sequences amplified from (Fig. 1). On the contrary, the percent colonisation root extracts were compared with the sequence from Ggt of filamentous fungi other than endophytes obtained pure cultures and with those in the GenBank database from non-sterilised roots was different between roots using BLAST analysis (Altschul et al., 1990). inoculated with F. equiseti or P. chlamydosporia. Although colonisation of the ectorhizosphere of roots inoculated with either Fe10331 or Fe45121 with other fungi Data analysis increased gradually with time following a reduction Data were checked for normality using the Shapiro–Wilk of F. equiseti populations, overall colonisation by other test, and Levene’s test was used to study homogeneity fungi was significantly lower than that in controls of variance across groups. Data following a normal distri- (K–W, P < 0.05; Fig. 1A, Fig. 1B and Fig. 1E). For roots bution were compared using two-way ANOVA for differ- inoculated with P. chlamydosporia, isolation of other fungi = ences between treatments and/or sampling times. Non- did not differ (K–W, P 1) from control treatments: after normal data were compared using the Kruskal–Wallis the transfer of the plants to pots with soil, colonisation (K–W) rank sum test. Either Student’s t-test or Wilcoxon of the root by soil contaminants reached values of test with corrections for multiple testing was used for 83%–100% (Fig. 1C, Fig. 1D and Fig. 1E). pair-wise comparisons. In all cases, significance level con- sidered was 95%. All analyses were performed using the Effects of root colonisation by F. equiseti R 2.5.1 software (R Development Core Team, 2007). and P. chlamydosporia on plant growth Colonisation of roots by F. equiseti isolates had virtu- Results ally no effect on plant growth (Fig. 2). On the contrary, P. chlamydosporia had a clear plant growth-promoting Rhizosphere dynamics of F. equiseti and P. chlamydosporia effect on barley (Fig. 2). Plants inoculated with either iso- Both F. equiseti and P. chlamydosporia occurred with a late Pc123 or Pc21 demonstrated a significant (ANOVA, high isolation frequency (from approximately 78% to P < 0.05) increase in shoot weight with respect to con- 100% colonisation) in non-sterilised roots 7 dai, from trols from 49 dai onwards (Fig. 2A). At 63 dai, the shoot plants growing in tubes with vermiculite. Their occur- weight of plants inoculated with either Pc123 or Pc21 rence decreased in roots after transplanting to pots with was 1.6- and 1.9-fold that of uninoculated control plants, soil (Fig. 1). Both fungal species were isolated at low fre- respectively. This growth promotion by P. chlamydosporia quency from surface-sterilised roots, but their presence was also observed in roots, with a significant (ANOVA,

394 Ann Appl Biol 155 (2009) 391–401 © 2009 The Authors Journal compilation © 2009 Association of Applied Biologists J.G. Macia-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 395 Journal compilation © 2009 Association of Applied Biologists Root colonisation by endophytes and effects on host plant J.G. Macia-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 Pc21 showed a significantly higher (K–W, P < 0.05) isola- with controls (Fig. 2B). At this time, the rate of weight tion percentage than Pc123 (65% and 9%, respectively) in increase with respect to the control plants was 1.9 for the ectorhizosphere 35 dai. Isolation rates of both Pc123 Pc123 and 1.8 for Pc21. Furthermore, plants inoculated and Pc21 at 63 dai did not differ significantly (K–W, with P. chlamydosporia presented an early development of P > 0.05) in non-sterilised and in surface-sterilised roots spikes at 63 dai, when compared with plants from the (9%–30%; Fig 3C and Fig 3D). other treatments (data not shown). Quantification determined by culturing Ggt from infected roots could only be achieved from plantlets growing in culture tubes with sterilised vermiculite 7 ± Rhizosphere dynamics of F. equiseti and P. chlamydosporia dai. In this case, isolation rates ranged between 2 0.6% ± in the presence of Ggt and 6 1.3% in non-sterilised roots, with no significant differences among treatments. In surface-sterilised roots, The patterns of endophyte colonisation of Ggt-infected values ranged between 0 ± 0% for Fe10331 and Pc21 and barley roots differed from those of plants inoculated with 4 ± 1.3% in control treatments, with no significant differ- the endophytes only (Fig. 3). In this experiment, root ences. At 35 and 63 dai, sterile dark-pigmented mycelia colonisation was scored at 7, 35 and 63 dai. Fe10331 were frequently recovered from roots of plants in all treat- displayed higher colonisation percentages, for both non- ments, but these could not be related to Ggt either in CMA sterilised and surface-sterilised roots than in the previous or PDA culture media, because of a lack of sporulation experiment, and significantly (K–W, P < 0.05) higher of the colonies. Therefore, the presence of Ggt in barley colonisation rates than Fe45121 at 7 and 35 dai (Fig. 3A). roots was assessed by PCR using primers specific to Ggt. All Fe45121 was isolated at lower levels from both non- PCR reactions including DNA from barley roots inoculated sterilised and sterilised roots after 7 days of growth in with Ggt only and those with both Ggt plus endophytes, sterilised vermiculite, and from non-sterilised roots from and with DNA extracted from Ggt pure cultures, yielded pots with soil at 35 dai (Fig. 3B), than in the experi- amplification products. All amplification products showed ment with the endophyte alone (Fig. 1B). Colonisation several unspecificities, but approximately 410-bp Ggt- by Fe10331 and Fe45121 of non-sterilised and surface- specific amplicon was found in all treatments (data not sterilised roots was not significantly different (K–W, shown). PCR products from seven independent amplifica- P > 0.05) at 63 dai (20%–44%). Pc123 followed the tion reactions were purified from the gels and sequenced. same pattern of colonisation of the root as in the previous In all cases, sequences matched those obtained from Ggt experiment, but with higher colonisation rates (Fig. 3C). pure cultures and from GenBank databases.

396 Ann Appl Biol 155 (2009) 391–401 © 2009 The Authors Journal compilation © 2009 Association of Applied Biologists J.G. Macia-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 397 Journal compilation © 2009 Association of Applied Biologists Root colonisation by endophytes and effects on host plant J.G. Macia-Vicente´ et al.

Isolation of fungal contaminants from surface-sterilised and two isolates of the nematode egg-parasitic fungus roots was significantly lower (K–W, P < 0.05) only in P. chlamydosporia (Pc123 and Pc21), were tested for ability plants treated with Fe10331 than in controls with Ggt to colonise barley roots endophytically and for their effects alone 63 dai (from 31% to 8%, respectively), whereas on plant growth and the take-all caused by Ggt, in pot both Fe10331 and Pc21 reduced the occurrence of other trials under greenhouse conditions. filamentous fungi in non-sterilised roots at 35 dai (from All isolates were able to colonise barley roots, where 89% to 52% and 54% respectively) and Fe10331 at 63 they remained endophytically for at least 2 months dai (from 31% to 8%; Fig. 3). Therefore, inhibition of under non-axenic conditions. Both F. equiseti and the presence of fungi other than the inoculated ones P. chlamydosporia have been found in previous studies to seemed to be correlated with the population size of the colonise barley roots under laboratory conditions using endophytes at each sampling time (Fig. 3). 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 long- Effects of root colonisation by F. equiseti and term study of the endophytic behaviour of both fungal P. chlamydosporia on take-all disease species under non-axenic conditions. Fusarium equiseti No effect was found on fresh shoot and root weight either appeared to be a more efficient rhizosphere coloniser than in plants inoculated with F. equiseti or P. chlamydosporia P. chlamydosporia, in terms of population size and compe- and Ggt with respect to control treatments with Ggt tition with other fungal root colonisers. Although both only. Both P. chlamydosporia isolates Pc123 and Pc21 species started with comparable levels of root colonisation significantly increased (ANOVA, P < 0.05) the total under axenic conditions, both outside and within the root root length 7 dai (0.584 ± 0.033 and 0.577 ± 0.039 m, cortex, transferring the plants to soil affected their capac- respectively) when compared with the other treatments ities to colonise roots in different manners. Fusarium equi- (0.430 ± 0.038 to 0.529 ± 0.023 m). This effect was lost seti persisted in the ectorhizosphere at high levels, which after the transfer of plants to soil, and from 35 dai onwards decreased gradually with time, whereas their endophytic root lengths did not differ significantly in all treatments. occurrence was constant with slight changes over the No significant differences were found among treat- duration of the experiment (approximately 2 months). ments and/or time in disease ratings, with values ranging The gradual decrease in the presence of F. equiseti in between 0.75 ± 0.22 and 1.7 ± 0.22. However, mean the ectorhizosphere was accompanied by a correspond- lesion length increased significantly (K–W, P < 0.05) ing increase in other soil-inhabiting fungi. These fungi with time in all treatments except for the treat- started colonising the ectorhizosphere to a lower extent ment with isolate Fe10331, in which a reduction in than in uninoculated controls. This effect could be related lesion length occurred from 35 (0.121 ± 0.027 m) to 63 to the reduction of Ggt growing axenically in culture (0.096 ± 0.036 m) dai. Larger differences in lesion length tubes observed in Maciá-Vicente et al. (2008b) and could were observed at 63 dai. At this time, plants inoculated be an indicator of the competitive ability of the fungus in with either Fe10331 or Fe45121 showed a significant the rhizosphere. On the contrary, P. chlamydosporia disap- reduction (K–W, P < 0.05) in the mean lesion length peared rapidly from the root surface after transfer of the (0.096 ± 0.036 and 0.097 ± 0.031 m, respectively) com- plants to soil, and its frequency in the ectorhizosphere pared with control plants (0.289 ± 0.122 m). remained low during the whole experiment. Endophytic Percent effective root length (ERL) did not vary among colonisation of P. chlamydosporia remained constant with treatments and sampling dates, except for Pc123 and Pc21, time, but was lower than that observed for F. equiseti.In which showed a reduction in ERL after transfer to pots treatments with P. chlamydosporia, complete colonisation with soil. At 63 dai, ERL of plants with Pc21 (71.1 ± 5.7%) of the outer root by other soil fungi occurred immediately was significantly lower (K–W, P < 0.05) than that of after transferring plants to soil, similar to uninoculated the same treatment at the first sampling (96.1 ± 1.4%). controls. As was observed with F. equiseti, the presence of It was, however, statistically similar (K–W, P > 0.05) P. chlamydosporia reduced the occurrence of other fungi to values found for other treatments at the same time in the root cortex. (77.7 ± 4.3to97.3 ± 1.7%). 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 experi- Discussion ment, the increase reached almost twice that observed Two F. equiseti isolates (Fe10331 and Fe45121) obtained for uninoculated controls. This effect has also been in Maciá-Vicente et al. (2008a) and screened in vitro for observed in wheat and tomato plants inoculated with antagonism against Ggt in Maciá-Vicente et al. (2008b), P. chlamydosporia (Monfort et al., 2005; Siddiqui & Akhtar,

398 Ann Appl Biol 155 (2009) 391–401 © 2009 The Authors Journal compilation © 2009 Association of Applied Biologists J.G. Macia-Vicente´ et al. Root colonisation by endophytes and effects on host plant

2008), but in these plants growth promotion was lower root regions, suggesting an evasion of the plant defences than that found in this study. This is probably because by the fungal endophyte. Both qPCR and confocal laser of our method of fungal inoculation, which was targeted scanning microscopy studies with GFP-tagged isolates are to plant roots rather than soil. Plant growth promotion currently being used under the same experimental sys- conferred by endophytic colonisation has been described tems used in the present work to reanalyse these results. previously, and this effect can include phenomena such Root behaviour of both F. equiseti and P. chlamydosporia as direct production of specific substances [i.e. phyto- isolates was also checked in the presence of the cereal hormones such as indole-3-acetic acid (IAA)] (Nassar fungal pathogen Ggt. Fe10331 and Pc123 followed et al., 2005), assistance in mineral supply and nutrient colonisation patterns similar to those observed in uptake, especially of phosphorus, from soil (Jumpponen the experiment with the endophytes alone. However, & Trappe, 1998; Sieber, 2002), increase in the availabil- Fe45121 displayed a reduction in root colonisation rate ity of carbohydrates and/or CO2 resulting from fungal and Pc21 showed a higher degree of root colonisation, metabolism (Jumpponen & Trappe, 1998) or stimulation than that observed in experiments with the endophytes of nitrate reduction (Sherameti et al., 2005). Some of alone. After the transfer of barley plants to soil, Ggt these effects, such as increased phosphorus availability, could not be isolated from roots. Ggt rarely produces are similar to that found for mycorrhizal associations. asexual structures in culture, and most isolates do not In fact, some mutualistic endophytes (i.e. Phialocephala produce sexual structures at all (Elliott, 2005). Therefore, fortinii) form colonisation patterns similar to the Hartig differentiation of the dark hyphae of the pathogen net and a thin patchy mantle, typical of ectomycorrhizae from other pigmented mycelia isolated from barley roots (Fernando & Currah, 1996). The plant growth promo- was difficult or unreliable by means of observation of tion observed in the present paper does not seem to be morphological characters. Application of a PCR detection related to formation of rhizospheric hyphal nets. This is procedure for Ggt (Fouly & Wilkinson, 2000) was connected with the low root colonisation rates found for therefore used as an alternative. This method allowed P. chlamydosporia. This points to alternative mechanisms, direct detection of Ggt in the roots of all plants analysed. A such as production by the fungus of specific compounds clear suppressive effect by F. equiseti and P. chlamydosporia (e.g. growth regulators), as the main effectors of plant on the take-all disease on barley plants could not be growth promotion driven by P. chlamydosporia.Further detected, in spite of previous positive results under studies are required to assess this hypothesis. Re-isolation laboratory screening conditions (Monfort et al., 2005; methods in culture media used in this study have limita- Maciá-Vicente et al., 2008b). Although such preliminary tions, which may include a false correlation between the screening procedures are commonly recommended as isolation rate and the real presence of the fungus (Schulz components of a sequence of assays in the search for & Boyle, 2005). It is difficult to discriminate whether biological control agents, quite often results between the low levels of P. chlamydosporia found in the roots are simple laboratory and greenhouse practical growing real, or they are an artefact of the methodology applied. conditions do not match, as a consequence of the increase The use of more accurate methods for the detection in the experimental variables (Knudsen et al., 1997). and quantification of fungi in the rhizosphere, such as In this study, a technique for inoculation and long-term real-time quantitative PCR (qPCR) techniques or genetic root endophytic colonisation by two fungal species was transformation with the GFP reporter gene in conjugation developed. Non-sterilised sandy soil used in this work with microscopic studies, will help to assess these results was non-receptive to Pc123 in previous studies (Monfort (Ciancio et al., 2005; Rosso et al., 2007; Maciá-Vicente et al., 2006). That is, fungal growth of P. chlamydosporia et al., 2009). In recent research, these methodologies was inhibited when applied to non-sterilised soil, proba- were developed for the characterisation of the endo- bly because of soil microbial activities. According to our phytic colonisation of barley roots by both F. equiseti results, root tissues of the host plant offer a ‘‘safe’’ envi- and P. chlamydosporia under laboratory growth conditions ronment for the fungus to escape soil competition (‘lack of (Maciá-Vicente et al., 2009). In these studies, first coloni- receptivity’). Colonisation of the endorhizosphere could sation events consisted of a massive fungal growth on the also act as an additional source of fungal inoculum to root periphery, with single hyphae colonising the cortical the soil. In this sense, previous work reported abundant tissues inter- and intracellularly. Subsequent colonisa- chlamydospore production by P. chlamydosporia on the tion events involved a reduction of the outer root fungal rhizoplane, and to a lesser extent in the cortex of bar- biomass and loss of viability of the endophytic mycelium ley and tomato roots colonised by the fungus (Bordallo in some root regions that could be a consequence of elic- et al., 2002). Independent of their initial root colonisa- itation of plant defence responses. In spite of the latter, tion rates, both types of root endophytes continued to well-established viable hyphae were detected in other colonise roots endophytically, although levels were low.

Ann Appl Biol 155 (2009) 391–401 © 2009 The Authors 399 Journal compilation © 2009 Association of Applied Biologists Root colonisation by endophytes and effects on host plant J.G. Macia-Vicente´ et al.

These results suggest that barley roots support certain Bourne J.M., Kerry B.R. (1998) Effect of the host plant on root colonisers. It seems that a single inoculation of the the efficacy of Verticillium chlamydosporium as a biological endophytes in culture tubes with barley plants growing control agent of root-knot nematodes at different axenically was not enough to keep high endophytic pop- nematode densities and fungal application rates. Soil ulations of the fungi inoculated. Therefore, their presence Biology and Biochemistry, 31, 75–84. gradually decreased when plants were transferred to soil Chen W., Gray L.E., Grau C.R. (1996) Molecular to reach a low and stable colonisation level with time. differenciation of fungi associated with brown stem rot Long-term endophytic colonisation may hence depend and detection of Phialophora gregata in resistant and susceptible soybean cultivars. 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