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Inhibition of Phytophthora species by secondary metabolites produced by the dark septate endophyte Phialocephala europaea
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Inhibition of Phytophthora species by secondary metabolites produced by the dark septate endophyte Phialocephala europaea a,e € Christoph TELLENBACHa,b, Mark W. SUMARAHc,d, Christoph R. GRUNIG , d, J. David MILLER * a
Forest Pathology and Dendrology, Institute of Integrative Biology (IBZ), ETH Zurich, 8092 Zurich, Switzerland € € bendorf, Switzerland Eawag, Aquatic Ecology, Uberlandstrasse 133, 8600 Du c Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada, 1391 Sandford Street, London, ON, Canada N5V 4T3 d Department of Chemistry, Carleton University, 1125 Colonel By Drive, Ottawa, Ontario, Canada K1S 5B6 e Microsynth AG, Schuetzenstrasse 15, 9436 Balgach, Switzerland b
article info
abstract
Article history:
Dark septate fungal root endophytes of the Phialocephala fortinii s.l.eAcephala applanata
Received 5 July 2012
species complex (PAC) are widely distributed throughout the temperate and subtropical
Revision received 21 September 2012
regions of the Northern Hemisphere. Previous studies have shown that some PAC
Accepted 27 September 2012
members are pathogenic, others suppress oomycete root pathogens and some have no
Available online 2 December 2012
obvious effect on their Norway spruce (Picea abies) host. The activity of 85 PAC isolates
Corresponding editor:
against Phytophthora citricola s.l. was investigated by co-culture on plates. We identified
James White
a strain of Phialocephala europaea that significantly reduced the growth of P. citricola in vitro. Characterization of its extracellular metabolites resulted in the identification of four major
Keywords:
compounds, sclerin, sclerolide, sclerotinin A, and sclerotinin B. These compounds are
Dark septate endophytes
known for their positive as well as negative effects on plant growth. We found that sclerin
Disease suppression
and sclerotinin inhibited the growth of P. citricola in vitro at 150 mg ml1 (w1 mM). This is the
Norway spruce
first report of their production by Phialocephala and of activity of these compounds against
Phialocephala
an oomycete. Therefore, our data suggest that some PAC might reduce disease resulting
Phytophthora
from P. citricola by the production of antibiotics and plant growth promoting metabolites.
Sclerin
ª 2012 Elsevier Ltd and The British Mycological Society. All rights reserved.
Sclerotinin A
Introduction Dark septate endophytes (DSE) are abundant fungal root colonizers of a wide range of mycorrhizal and nonmycorrhizal plant species, particularly in woody plants.
They form a polyphyletic group of fungi that are characterized by melanized, septate hyphae (Stoyke et al. 1992). They can be found in all parts of the root system, from the tips to coarse € nig et al. 2008a). In conifers and roots at the stem bases (Gru ericaceous shrubs, the most prevalent members of the DSE are
* Corresponding author. Department of Chemistry, Carleton University, 1125 Colonel By Drive, Ottawa, Ontario, Canada K1S 5B6. Tel.: þ1 613 520 2600x1053; fax: þ1 613 520 3749. E-mail address: [email protected] (J.D. Miller). 1754-5048/$ e see front matter ª 2012 Elsevier Ltd and The British Mycological Society. All rights reserved. http://dx.doi.org/10.1016/j.funeco.2012.10.003
Inhibition of Phytophthora species by secondary metabolites
anamorphic Helotialean ascomycetes of the Phialocephala fortinii s.l.eAcephala applanata species complex (PAC) (Wang & € nig et al. 2008a). PAC Wilcox 1985; Ahlich et al. 1998; Gru members dominate the endophytic assemblages of conifer and Ericaceae roots in the Northern Hemisphere from polar to subtropical regions (Addy et al. 2000; Zhang et al. 2009; Queloz et al. 2011) and form communities of up to 10 sympatrically cooccurring species that remain stable for several years (Queloz et al. 2005). Apart from A. applanata, which shows a clear preference for trees of the pine family in forest ecosystems € nig et al. 2006), host with ericaceous-plant understory (Gru € nig et al. specialization seems to be low for PAC species (Gru 2008a). Moreover, PAC community composition is neither correlated with stand composition nor climate (Queloz et al. 2011). In laboratory studies, PACeNorway spruce interactions have been shown to be mainly strain-specific, ranging from highly virulent strains causing pronounced seedling mortality to avirulent strains causing no obvious disease symptoms. Nonetheless, even colonization by avirulent strains was associated with net costs for Norway spruce seedlings as biomass accumulation of all PAC-inoculated seedlings was reduced compared to PAC-free control seedlings in growth-cabinet studies (Tellenbach et al. 2011; Reininger et al. 2012). Despite increasing knowledge of PAC community ecology and plantePAC interactions, the ecological role of PAC in forest ecosystems still remains unresolved. Various authors have speculated that DSE and PAC induce resistance to abiotic stress, accelerate root turnover and mineralization, and suppress root pathogens (Mandyam & Jumpponen 2005; € nig et al. 2008a; Tellenbach et al. 2011). Schulz 2006; Gru Endophytes of grasses are well-known to protect their hosts against insect pests and fungal infections (Clay 1988), which appears also to be the case for endophytes of some conifer species as well as a number of tropical plants (Clay 1988; Arnold et al. 2003; Kageyama et al. 2008; Sumarah & Miller 2009). Moreover, endophytes of grasses and some tropical plants also improve drought tolerance (Arnold & Engelbrecht 2007; Kuldau & Bacon 2008; Rodriguez et al. 2009). In a recent study, Phialocephala subalpina isolates protected Norway spruce seedlings against two oomycete root pathogens, Phytophthora plurivora and Elongisporangium undulatum (Tellenbach & Sieber 2012), but the mechanism could not be determined from these experiments. However, the possible mechanisms could include mycoparasitism, improved root health and nutrient uptake (Schulz 2006), exploitation competition (Lockwood 1992) and/or interference competition, i.e., antibiosis (Wicklow 1992). Therefore, the aim of the present study was to test the hypothesis that some PAC strains produce metabolites that are toxic to Phytophthora.
Methods Screening for antagonistic PAC strains Pairwise plate assays were used to screen 85 PAC strains belonging to eight PAC species (Table 1) for possible suppressive effects against P. plurivora (syn. Phytophthora citricola s.l.) (Table 2 and Supplementary Fig 1), which is frequently
13
associated with root dieback of broadleaved trees, but which has also been found to cause mortality and fine root loss in Norway spruce (Nechwatal & Osswald 2001; Jung & Burgess 2009; Tellenbach & Sieber 2012). PAC strains were previously genotyped based on single-copy RFLP markers and sequence € nig & Sieber 2005; Gru € nig et al. 2007, 2008b). Most markers (Gru of the PAC strains tested had distinct multilocus haplotypes, i.e., strains represent different individuals (Table 1). Cultures of strains used during this study are preserved in the culture € rich and are accessible for all researchers. collection of ETH Zu All PAC strains were grown on 2 % malt extract agar (MEA, 2 % malt extract, Hefe Schweiz AG, Stettfurt, Switzerland; 2 % agar [w/v]) for 26 d prior to inoculation on 2 % carrot agar plates (2 % commercially available carrot juice; 2 % agar [w/v]) overlaid with sterilized cellophane sheets. Since the PAC growth rate € nig et al. 2008b) is slower (1.25 0.13 mm d1 20 C on MEA) (Gru than P. plurivora growth rate (6.3 0.1 mm d1 at 20 C on V8 agar) (Jung & Burgess 2009), PAC were inoculated 23 d prior to P. plurivora. One agar plug was excised from the growing margin of the PAC colony with a 4 mm diameter cork borer, inoculated 25 mm from the center of the plate, and incubated at 20 C in darkness. P. plurivora (syn. P. citricola s.l.) strains PLU 55, PLU 278 and PLU 279 (Table 1, Supplementary Fig 1) were grown on V8 agar (V8A; 350 ml V8, 5 g CaCO3 mixed and centrifuged for 20 min at 1000 g, supernatant 1:4-diluted with deionized water and 20 g agar added prior to autoclaving) for 5 d prior to inoculation. Similarly, inocula from the margin of the actively growing P. plurivora colonies were excised as above and inoculated 25 mm from the center of the plate on the opposite site of the PAC colony. There were 85 PAC strains times three P. plurivora isolates equaling 255 experimental units. The diameter of both the PAC and P. plurivora colonies were measured 3 and 15 d post inoculation of the latter and extension rates calculated. In addition, colony morphologies for both PAC and P. plurivora were characterized according to Wicklow et al. (1980).
Growth inhibitory effects of PAC culture exudates Based on the results of the dual-culture plate assay, eight PAC strains with different characteristics were selected for the analysis of potential antagonistic activity (Table 1). As the effect on all three P. plurivora isolates was similar in the plate assays, only strain Bu 14218 (PLU 279) was used in this screening. PAC strains were inoculated aseptically in triplicate into 100-ml Erlenmeyer flasks containing 50 ml of autoclaved 5 %-carrot juice. They were shaken at 100 rpm for 30 d at 20 C. Liquid cultures for each PAC strain were combined and filtered to remove the mycelial fraction. The filtrate was then sterilized by filtration using 0.45 mm filters (Sartorius, Tagelswangen, Switzerland) and mixed 1:1 [v/v] with autoclaved 5 % double-strength CA (5 %-carrot juice; 3 % agar [w/v]) that was cooled to 50 C. As a control, three sterile uninoculated 50 ml portions with 5 %-carrot juice were maintained under the same conditions as the PAC colonies and processed as described above. P. plurivora strains were grown on 5 %-CA agar plates for 2 weeks prior to inoculation. P. plurivora was inoculated in the center of the agar plate and growth radii were recorded 3 d and 8 d post inoculation to calculate mean colony growth. Per substrate there were nine to 12 replicates.
14
C. Tellenbach et al.
Table 1 e PAC strains included in the present study to assess suppressive effects of PAC against Phytophthora plurivora Taxon Phialocephala turiciensis
Phialocephala letzii
Phialocephala europaea
c
Phialocephala helvetica
Phialocephala uotolensis
Phialocephala subalpina
Strain
MLHa
CBS no
1_117_3 1_120_2 1_124_1 1_124_2 1_130_3 1_133_3 1_154_1 1_176_1 1_190_4
MLH_01 MLH_02 MLH_03 MLH_04 MLH_05 MLH_06 MLH_07 MLH_08 MLH_09
2_120_3 2_126_3 2_137_4 2_146_1 2_146_2 2_147_3 2_149_5 2_152_2 2_168_4 2_194_1 2_194_4
MLH_10 MLH_11 MLH_12 MLH_13 MLH_14 MLH_15 MLH_16 MLH_17 MLH_18 MLH_19 MLH_10
3_117_2 3_122_3 3_129_1 3_136_1 3_136_3 3_137_3 3_139_3 3_158_4 3_169_2 3_171_5 3_173_2 3_177_3
MLH_19 MLH_20 MLH_21 MLH_22 MLH_23 MLH_24 MLH_25 MLH_26 MLH_27 MLH_28 MLH_29 MLH_30
CBS 119269 CBS 119270
4_123_4 4_135_1 4_136_4 4_138_5 4_140_4 4_144_4 4_145_2 4_153_2 4_160_1 4_195_2
MLH_31 MLH_32 MLH_33 MLH_34 MLH_35 MLH_36 MLH_37 MLH_38 MLH_39 MLH_40
CBS 119272
5_134_3 5_220_1 5_234_5 5_252_7r 5_264_1r 5_265_1 5_265_3 5_265_4
MLH_41 MLH_42 MLH_43 MLH_44 MLH_45 MLH_46 MLH_46 MLH_47
6_16_1 6_2_7v 6_22_7v 6_30_4 6_35_6v 6_37_6v 6_53_6v 6_70_1 6_70_4 6_78_2 6_8_7v
MLH_48 MLH_49 MLH_50 MLH_51 MLH_52 MLH_53 MLH_54 MLH_55 MLH_56 MLH_57 MLH_58
CBS 119234
CBS 119264
CBS 119266
CBS 119268
CBS 119267
UAMH 11659
CBS 119271
CBS 119273
CBS 119274
CBS 119276 CBS 119277 CBS 119275
CBS 119279 CBS 119278
CBS 119280
Geographic origin
Plateb assay
Picea abies P. abies P. abies P. abies P. abies P. abies P. abies P. abies P. abies
€ richberg; Switzerland Zu € richberg; Switzerland Zu € richberg; Switzerland Zu € richberg; Switzerland Zu € richberg; Switzerland Zu € richberg; Switzerland Zu € richberg; Switzerland Zu € richberg; Switzerland Zu € richberg; Switzerland Zu
B C A/B A/B A/B B B B A
P. abies P. abies P. abies P. abies P. abies P. abies P. abies P. abies P. abies P. abies P. abies
€ richberg; Switzerland Zu € richberg; Switzerland Zu € richberg; Switzerland Zu € richberg; Switzerland Zu € richberg; Switzerland Zu € richberg; Switzerland Zu € richberg; Switzerland Zu € richberg; Switzerland Zu € richberg; Switzerland Zu € richberg; Switzerland Zu € richberg; Switzerland Zu
A A A A/B A/B B A/B A B B A
P. abies P. abies P. abies P. abies P. abies P. abies P. abies P. abies P. abies P. abies P. abies P. abies
€ richberg; Switzerland Zu € richberg; Switzerland Zu € richberg; Switzerland Zu € richberg; Switzerland Zu € richberg; Switzerland Zu € richberg; Switzerland Zu € richberg; Switzerland Zu € richberg; Switzerland Zu € richberg; Switzerland Zu € richberg; Switzerland Zu € richberg; Switzerland Zu € richberg; Switzerland Zu
B B B A/C B/C A B B A/B B B A
P. abies P. abies P. abies P. abies P. abies P. abies P. abies P. abies P. abies P. abies
€ richberg; Switzerland Zu € richberg; Switzerland Zu € richberg; Switzerland Zu € richberg; Switzerland Zu € richberg; Switzerland Zu € richberg; Switzerland Zu € richberg; Switzerland Zu € richberg; Switzerland Zu € richberg; Switzerland Zu € richberg; Switzerland Zu
A A/B A/C B B B B B A/B B
P. abies P. abies P. abies P. abies P. abies P. abies P. abies P. abies
€ richberg; Switzerland Zu Uetliberg; Switzerland Uetliberg; Switzerland Uetliberg; Switzerland Uetliberg; Switzerland Uetliberg; Switzerland Uetliberg; Switzerland Uetliberg; Switzerland
B B B B A/B B B B
P. abies Vaccinium myrtillus V. myrtillus P. abies V. myrtillus V. myrtillus V. myrtillus P. abies P. abies P. abies V. myrtillus
€ dmeren; Bo € dmeren; Bo € dmeren; Bo € dmeren; Bo € dmeren; Bo € dmeren; Bo € dmeren; Bo € dmeren; Bo € dmeren; Bo € dmeren; Bo € dmeren; Bo
B B B A A B A/B A B A A/B
Host
Switzerland Switzerland Switzerland Switzerland Switzerland Switzerland Switzerland Switzerland Switzerland Switzerland Switzerland
Culture assay x
x
x
x
x
Inhibition of Phytophthora species by secondary metabolites
15
Table 1 e (continued ) Taxon
Phialocephala fortunes s.s.
Acephala applanata
Strain
MLHa
CBS no
Host
Geographic origin
Plateb assay
6_9_6v
MLH_59
V. myrtillus
€ dmeren; Switzerland Bo
A
7_10_6v
MLH_60
V. myrtillus
€ dmeren; Switzerland Bo
A/B
7_39_7v 7_45_5 7_54_7v 7_6_7v 7_62_6v 7_62_7v 7_63_4 7_64_7v 7_K92_049 7_K93_395 7_K93_444
MLH_61 MLH_62 MLH_63 MLH_64 MLH_65 MLH_65 MLH_66 MLH_67 MLH_68 MLH_69 MLH_70
V. myrtillus P. abies V. myrtillus V. myrtillus V. myrtillus V. myrtillus P. abies V. myrtillus P. abies Pinus sylvestris P. sylvestris
€ dmeren; Switzerland Bo € dmeren; Switzerland Bo € dmeren; Switzerland Bo € dmeren; Switzerland Bo € dmeren; Switzerland Bo € dmeren; Switzerland Bo € dmeren; Switzerland Bo € dmeren; Switzerland Bo Odenwald; Germany Kevo; Finland Suonenjoki; Finland
A A B A A A A A/B A A A
T1_38_2 T1_39_2 T1_50_2 T1_51_3 T1_52_2 T1_55_1 T1_58_1 T1_75_3 T1_77_1 T1_78_1 T1_K92_079
MLH_71 MLH_72 MLH_73 MLH_74 MLH_75 MLH_76 MLH_77 MLH_78 MLH_79 MLH_80 MLH_73
P. abies P. abies P. abies P. abies P. abies P. abies P. abies P. abies P. abies P. abies P. abies
€ dmeren; Switzerland Bo € dmeren; Switzerland Bo € dmeren; Switzerland Bo € dmeren; Switzerland Bo € dmeren; Switzerland Bo € dmeren; Switzerland Bo € dmeren; Switzerland Bo € dmeren; Switzerland Bo € dmeren; Switzerland Bo € dmeren; Switzerland Bo € ntis; Switzerland Sa
A/B B A/B B A A/B A A A A A
CBS 119281
CBS 119282
CBS 114608 CBS 443.86
Culture assay
x
x
x
€ nig & Sieber 2005; Gru € nig et al. 2007; Gru € nig et al. 2008b). a Multilocus haplotype based on 11 single-copy RLFP probes and sequence markers (Gru b Categories of antagonism after Wicklow et al. (1980). A ¼ mutual intermingling of the two organisms, B ¼ inhibition of P. plurivora on contact; PAC continues to grow unchanged or at a reduced rate, through P. plurivora, C ¼ inhibition of P. plurivora at distance. c Strain grown for metabolite characterization and toxicity testing.
Characterization of secondary metabolites The strongest growth inhibition of P. plurivora in culture was observed with Phialocephala europaea strain 3_136_1 (UAMH 11659), which was then chosen for the secondary metabolite screening. A 2 % MEA slant of 3_136_1 was macerated in sterile water under aseptic conditions. The resulting suspension was used to inoculate five Glaxo bottles, each containing 1l of 2 % ME medium. The cultures were grown for 2 months at 25 C and then harvested and filtered. The filtrate was extracted with 2 equal volumes of ethyl acetate and then dried by rotary evaporation. The extract was re-suspended in acetonitrile and dried under nitrogen. The extract, 183 mg, was filtered and re-dissolved in acetonitrile and screened by LCeMS using electrospray ionization in both positive and negative ion mode as previously described (Sumarah et al.
2011). The extract yielded four major peaks by UV and MS that were isolated by semi-preparative HPLC using an Agilent 1100 HPLC with diode array detector and 250 10 mm Luna C18 column (Phemonomex, Torrence, California). The four peaks (compounds 1e4) were isolated and further analyzed by HRLCeMS and characterized by NMR.
Bioassay of the characterized compounds against P. citricola Sufficient quantities of compounds 1 and 3 (Fig 1) were isolated for testing against P. citricola s.l. strain BR519 (National Mycological Herbarium, Ottawa, ON, Canada) in sterile 96 well microplates at concentrations of 30 and 150 mg ml1. Unfortunately, it was not possible to use the P. plurivora strains for the screening because the transfer was not possible due to
Table 2 e Details of Phytophthora citricola s.l. strains used in the present study Straina PLU 55 (137/7a) PLU 278 PLU 279 BR519
Host
Species
Collector
Reference
Used for
Fagus sylvatica Quercus sp. Fagus sylvatica Citrus sinensis
P. plurivora P. plurivora P. plurivora P. citricola s.s.
J. Nechwatal T. Jung T. Jung G.A. Zentmyer
(Nechwatal & Osswald 2001) None None (Robideau et al. 2011)
Screening Screening Screening In vitro testing of metabolites
a PLU numbers refer to the collection of T. Jung (Jung & Burgess 2009).
16
C. Tellenbach et al.
5.0 ag def
4.5
b
bd
afg bdef
bde
4.0
3.5 c
Fig 2 e Structures of the secondary metabolites isolated from P. europaea strain 3_136_1; sclerin [1], sclerolide [2], sclerotinin A [3] and sclerotinin B [4]. T1 38_2
7 63_4
7 54_7v
6 2_7v
5 265_1
3 171_5
3 136_1
1 120_2
3.0
control
mean growth rate [mm d−1]
a
Fig 1 e Inhibitory effect of PAC culture filtrate against P. plurivora isolate PLU 279. Different letters indicate significant differences among extracts in a Tukey’s HSD test following ANOVA (P < 0.05). regulatory issues. The pure compounds were dissolved in DMSO and 5 ml of this solution were added to 200 ml of a macerated 24 hr old culture of P. citricola in V8 media. Individual tests included six replicates per plate and tests were performed in triplicate on different days; the plates were incubated at room temperature with constant shaking and were measured at 600 nm at time zero, 24 and 48 hr with a plate reader (Power Wave XS microplate reader (BioTek, Vermont, USA)). The antibiotic chloramphenicol (Sigma, St. Louis, MO) was used as the positive control at the same concentration (w1 mM) for all experiments. ANOVA followed by Tukey’s honestly significant difference (HSD) test was used to evaluate the in vitro bioassays using SYSTAT v. 13. The remaining statistics were performed using the R statistical package (R Development Core Team 2009).
Results and discussion Growth inhibition of P. plurivora (syn. P. citricola s.l.) by PAC strains In general, the effect of PAC strains was similar on growth of the three P. plurivora strains. P. plurivora cultures were overgrown by mycelium of PAC (Table 1), but no evidence of mycoparasitism was found for either PAC or P. plurivora after microscopic examination. The strains tested represented a variety of PAC species, some of which inhibited Phytophthora growth. Based on these results, eight strains with varying degrees of inhibition were selected, and the effect on the growth of P. plurivora after incorporating PAC culture filtrate into growth medium was tested (Table 1). There were significant differences among the culture filtrates as indicated by an ANOVA (F8,76 ¼ 231.6, P < 0.001). The culture filtrate of strain 3_136_1 (UAMH 11659) significantly reduced P. plurivora growth compared to the control and all other PAC strains (Fig 1; Tukey’s HSD test). In contrast, the filtrates of other strains showed less pronounced, but still significant, to no inhibitory effects. Four compounds were isolated and characterized from the culture filtrate of 3_136_1. The four biosynthetically-related
compounds were determined to be the known fungal metabolites: sclerin [1] HRMS m/z 235.0990 [M þ 1]þ, sclerolide [2] HRMS m/z 223.0984 [M þ 1]þ, sclerotinin A [3] HRMS m/z 253.1078 [M þ 1]þ and sclerotinin B [4] HRMS m/z 239.0929 [M þ 1]þ (Fig 2; Supplementary Fig 3) previously isolated from Sclerotinia sclerotiorum, a known plant pathogen. The spectroscopic data for these compounds were in agreement with published literature (Supplementary Figs 4 and 5). Sclerin (4.5 mg) was the major compound produced by strain 3_136_1. This compound was originally isolated by Satomura & Sato (1965), where it was described as a plant growth hormone. Sclerin was also shown to be phytotoxic to a number of plant species (Pedras & Ahiahonu 2004). Sclerotinin A (3.5 mg) was initially isolated by Sassa et al. (1968) along with sclerotinin B, where they were described as growth promoters of rice seedlings. Antibiotic assays of sclerin and sclerotinin A to P. citricola s.s. (syn. P. citricola s.l.) were performed in 96 well plates containing liquid V8 medium. DMSO had no effect on the strain tested. In vitro, 30 mg ml1 sclerin and sclerotinin A resulted in significant reduction in the growth of P. citricola vs. controls at 24 hr (F6,72 ¼ 200.2, P ¼ 0.04 and P ¼ 0.001 respectively; Supplementary Fig 2) but both were less toxic than chloramphenicol. After 48 hr, growth had recovered to the control levels. When the higher concentration (150 mg ml1) of sclerin and sclerotinin A were tested, significant reductions in the growth of P. citricola s.l. were observed at 24 hr (F6,72 ¼ 56.3, P < 0.001 and P < 0.001, respectively; Supplementary Fig 2) and at 48 hr for both test compounds. The potencies were comparable to a similar molar concentration of chloramphenicol. The results suggest that sclerin and sclerotinin A, the major compounds isolated from P. europaea strain 3_136_1, are either individually or synergistically responsible for some of the original growth inhibition of P. citricola s.l.
Conclusions After screening 85 PAC strains for inhibitory effect against P. plurivora and performing subsequent plate assays using the culture filtrates of eight strains, one strain of interest was identified. This strain, P. europaea 3_136_1, produced at least four compounds, which are reported for the first time from this genus and species. Two of these, sclerin and sclerotinin A, inhibited P. citricola s.s. in the low mM range. These compounds have not been reported as antimicrobial despite many studies on their biological effects. Previous researchers have shown
Inhibition of Phytophthora species by secondary metabolites
that sclerin and sclerotinin A and related compounds are phytotoxic in vivo (Pedras & Ahiahonu 2004). As noted above, one hypothesis for the mode of action for DSE was improvement in root health (Schulz 2006). Our data show that they are toxic to oomycetes of the P. citricola s.l. species complex. This study provides a mechanistic basis to support the idea that some PAC isolates protect their host against some oomycete root pathogens (Tellenbach & Sieber 2012). The selection scheme used to detect and characterize these metabolites was first to detect the variety of interaction types between PAC and P. plurivora, quantifying the strength of interaction afterward, and finally, targeting metabolites and assaying their antagonistic potential. Therefore, these results justify the need for further testing using PAC isolates to investigate the variability in the production of these metabolites and to inoculate conifer trees with isolate P. europaea 3_136_1 and other PAC strains to potentially reduce the damage resulting from Phytophthora in a more natural environment.
Acknowledgments We would like to thank Thomas Jung for providing the Phytophthora plurivora strains, and Ottmar Holdenrieder for helpful comments on a previous version of this manuscript. This work was partially supported by SNF Grant 3100A0-113977 from the Swiss National Science Foundation, Bern, Switzerland. This work was associated with the GEDIHAP project of the Competence Center Environment and Sustain€ rich, Switzerland. ability (CCES) of the ETH Domain, Zu
Supplementary data Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.funeco.2012.10.003.
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nov., formerly known as dark-septate endophyte Type 1. Mycologia 97: 628e640. € nig CR, Duo A, Sieber TN, 2006. Population genetic analysis of Gru Phialocephala fortinii s.l. and Acephala applanata in two undisturbed forests in Switzerland and evidence for new cryptic species. Fungal Genetics and Biology 43: 410e421. A, Sieber TN, 2007. Suitability of € nig CR, Brunner PC, Duo Gru methods for species recognition in the Phialocephala fortiniieAcephala applanata species complex using DNA analysis. Fungal Genetics and Biology 44: 773e788. € nig CR, Queloz V, Sieber TN, Holdenrieder O, 2008a. Dark Gru septate endophytes (DSE) of the Phialocephala fortinii s.l. e Acephala applanata species complex in tree roots: classification, population biology, and ecology. Botany 86: 1355e1369. A, Sieber TN, Holdenrieder O, 2008b. Assignment € nig CR, Duo Gru of species rank to six reproductively isolated cryptic species of the Phialocephala fortinii s.l.eAcephala applanata species complex. Mycologia 100: 47e67. Jung T, Burgess TI, 2009. Re-evaluation of Phytophthora citricola isolates from multiple woody hosts in Europe and North America reveals a new species, Phytophthora plurivora sp. nov. Persoonia 22: 95e110. Kageyama SA, Mandyam KG, Jumpponen A, 2008. Diversity, function and potential applications of the root-associated endophytes. In: Varma A (ed), Mycorrhiza. Springer, Berlin Heidelberg, pp. 29e57. Kuldau G, Bacon C, 2008. Clavicipitaceous endophytes: their ability to enhance resistance of grasses to multiple stresses. Biological Control 46: 57e71. Lockwood JL, 1992. Exploitation competition. In: Carroll GC, Wicklow DT (eds), The Fungal Community Its Organization and Role in the Ecosystem, vol. XXIV. Dekker, New York, NY. 976 S. Mandyam K, Jumpponen A, 2005. Seeking the elusive function of the root-colonising dark septate endophytic fungi. Studies in Mycology 53: 173e189. Nechwatal J, Osswald W, 2001. Comparative studies on the fine root status of healthy and declining spruce and beech trees in the Bavarian Alps and occurrence of Phytophthora and Pythium species. Forest Pathology 31: 257e273. Pedras MSC, Ahiahonu PWK, 2004. Phytotoxin production and phytoalexin elicitation by the phytopathogenic fungus Sclerotinia sclerotiorum. Journal of Chemical Ecology 30: 2163e2179. € nig CR, Sieber TN, Holdenrieder O, 2005. Monitoring Queloz V, Gru the spatial and temporal dynamics of a community of the tree-root endophyte Phialocephala fortinii s.l. New Phytologist 168: 651e660. € nig CR, Queloz V, Sieber TN, Holdenrieder O, McDonald BA, Gru 2011. No biogeographical pattern for a root-associated fungal species complex. Global Ecology and Biogeography 20: 160e169. R Development Core Team, 2009. R: a language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. Reininger V, Grunig CR, Sieber TN, 2012. Host species and strain combination determine growth reduction of spruce and birch seedlings colonized by root-associated dark septate endophytes. Environmental Microbiology 14: 1064e1076. Robideau GP, de Cock AWAM, Coffey MD, Voglmayr H, Brouwer H, saulniers NL, Eggertson QA, Bala K, Chitty DW, De € pper FC, Rintoul TL, Sarhan E, Gachon CMM, Hu C-H, Ku Verstappen ECP, Zhang Y, Bonants PJM, Ristaino JB, vesque CA, 2011. DNA barcoding of oomycetes with Le cytochrome c oxidase subunit I and internal transcribed spacer. Molecular Ecology Resources 11: 1002e1011. Rodriguez RJ, White Jr JF, Arnold AE, Redman RS, 2009. Fungal endophytes: diversity and functional roles. New Phytologist 182: 314e330.
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Sassa T, Aoki H, Namiki M, Munakata K, 1968. Plant growthpromoting metabolites of Sclerotinia sclerotiorum. I. Isolation and structures of sclerotinins A and B. Agricultural and Biological Chemistry 22: 1432e1439. Satomura Y, Sato A, 1965. Isolation and physiological activity of sclerin a metabolite of Sclerotinia fungus. Agricultural and Biological Chemistry 29: 337e344. Schulz B, 2006. Mutualistic interactions with fungal root endophytes. In: Schulz B, Boyle C, Sieber TN (eds), Microbial Root Endophytes, vol. 9. Springer, Berlin, Germany, pp. 261e279. Stoyke G, Egger KN, Currah RS, 1992. Characterization of sterile endophytic fungi from the mycorrhizae of subalpine plants. Canadian Journal of Botany 70: 2009e2016. Sumarah MW, Miller JD, 2009. Anti-insect secondary metabolites from fungal endophytes of conifer trees. Natural Product Communications 4: 1497e1504. Sumarah MW, Kesting JR, Sørensen D, Miller JD, 2011. Antifungal metabolites from fungal endophytes of Pinus strobus. Phytochemistry 72: 1833e1837.
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Tellenbach C, Sieber TN, 2012. Do colonization by dark septate endophytes and elevated temperature affect pathogenicity of oomycetes? FEMS Microbiology Ecology 82: 157e168. € nig CR, Sieber TN, 2011. Negative effects on Tellenbach C, Gru survival and performance of Norway spruce seedlings colonized by dark septate root endophytes are primarily isolate-dependent. Environmental Microbiology 13: 2508e2517. Wang CJK, Wilcox HE, 1985. New species of ectendomycorrhizal and pseudomycorrhizal fungi e Phialophora finlandia, Chloridium paucisporum, and Phialocephala fortinii. Mycologia 77: 951e958. Wicklow DT, 1992. Interference competition. In: Carroll GC, Wicklow DT (eds), The Fungal Community Its Organization and Role in the Ecosystem, vol. XXIV. Dekker, New York, NY. 976 S. Wicklow DT, Hesseltine CW, Shotwell OL, Adams GL, 1980. Interference competition and aflatoxin levels in corn. Phytopathology 70: 761e764. Zhang C, Yin L, Dai S, 2009. Diversity of root-associated fungal endophytes in Rhododendron fortunei in subtropical forests of China. Mycorrhiza 19: 417e423.
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Inhibition of Phytophthora species by secondary metabolites produced by the dark septate endophyte Phialocephala europaea a,e € Christoph TELLENBACHa,b, Mark W. SUMARAHc,d, Christoph R. GRUNIG , d, J. David MILLER * a
Forest Pathology and Dendrology, Institute of Integrative Biology (IBZ), ETH Zurich, 8092 Zurich, Switzerland € € bendorf, Switzerland Eawag, Aquatic Ecology, Uberlandstrasse 133, 8600 Du c Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada, 1391 Sandford Street, London, ON, Canada N5V 4T3 d Department of Chemistry, Carleton University, 1125 Colonel By Drive, Ottawa, Ontario, Canada K1S 5B6 e Microsynth AG, Schuetzenstrasse 15, 9436 Balgach, Switzerland b
article info
abstract
Article history:
Dark septate fungal root endophytes of the Phialocephala fortinii s.l.eAcephala applanata
Received 5 July 2012
species complex (PAC) are widely distributed throughout the temperate and subtropical
Revision received 21 September 2012
regions of the Northern Hemisphere. Previous studies have shown that some PAC
Accepted 27 September 2012
members are pathogenic, others suppress oomycete root pathogens and some have no
Available online 2 December 2012
obvious effect on their Norway spruce (Picea abies) host. The activity of 85 PAC isolates
Corresponding editor:
against Phytophthora citricola s.l. was investigated by co-culture on plates. We identified
James White
a strain of Phialocephala europaea that significantly reduced the growth of P. citricola in vitro. Characterization of its extracellular metabolites resulted in the identification of four major
Keywords:
compounds, sclerin, sclerolide, sclerotinin A, and sclerotinin B. These compounds are
Dark septate endophytes
known for their positive as well as negative effects on plant growth. We found that sclerin
Disease suppression
and sclerotinin inhibited the growth of P. citricola in vitro at 150 mg ml1 (w1 mM). This is the
Norway spruce
first report of their production by Phialocephala and of activity of these compounds against
Phialocephala
an oomycete. Therefore, our data suggest that some PAC might reduce disease resulting
Phytophthora
from P. citricola by the production of antibiotics and plant growth promoting metabolites.
Sclerin
ª 2012 Elsevier Ltd and The British Mycological Society. All rights reserved.
Sclerotinin A
Introduction Dark septate endophytes (DSE) are abundant fungal root colonizers of a wide range of mycorrhizal and nonmycorrhizal plant species, particularly in woody plants.
They form a polyphyletic group of fungi that are characterized by melanized, septate hyphae (Stoyke et al. 1992). They can be found in all parts of the root system, from the tips to coarse € nig et al. 2008a). In conifers and roots at the stem bases (Gru ericaceous shrubs, the most prevalent members of the DSE are
* Corresponding author. Department of Chemistry, Carleton University, 1125 Colonel By Drive, Ottawa, Ontario, Canada K1S 5B6. Tel.: þ1 613 520 2600x1053; fax: þ1 613 520 3749. E-mail address: [email protected] (J.D. Miller). 1754-5048/$ e see front matter ª 2012 Elsevier Ltd and The British Mycological Society. All rights reserved. http://dx.doi.org/10.1016/j.funeco.2012.10.003
Inhibition of Phytophthora species by secondary metabolites
anamorphic Helotialean ascomycetes of the Phialocephala fortinii s.l.eAcephala applanata species complex (PAC) (Wang & € nig et al. 2008a). PAC Wilcox 1985; Ahlich et al. 1998; Gru members dominate the endophytic assemblages of conifer and Ericaceae roots in the Northern Hemisphere from polar to subtropical regions (Addy et al. 2000; Zhang et al. 2009; Queloz et al. 2011) and form communities of up to 10 sympatrically cooccurring species that remain stable for several years (Queloz et al. 2005). Apart from A. applanata, which shows a clear preference for trees of the pine family in forest ecosystems € nig et al. 2006), host with ericaceous-plant understory (Gru € nig et al. specialization seems to be low for PAC species (Gru 2008a). Moreover, PAC community composition is neither correlated with stand composition nor climate (Queloz et al. 2011). In laboratory studies, PACeNorway spruce interactions have been shown to be mainly strain-specific, ranging from highly virulent strains causing pronounced seedling mortality to avirulent strains causing no obvious disease symptoms. Nonetheless, even colonization by avirulent strains was associated with net costs for Norway spruce seedlings as biomass accumulation of all PAC-inoculated seedlings was reduced compared to PAC-free control seedlings in growth-cabinet studies (Tellenbach et al. 2011; Reininger et al. 2012). Despite increasing knowledge of PAC community ecology and plantePAC interactions, the ecological role of PAC in forest ecosystems still remains unresolved. Various authors have speculated that DSE and PAC induce resistance to abiotic stress, accelerate root turnover and mineralization, and suppress root pathogens (Mandyam & Jumpponen 2005; € nig et al. 2008a; Tellenbach et al. 2011). Schulz 2006; Gru Endophytes of grasses are well-known to protect their hosts against insect pests and fungal infections (Clay 1988), which appears also to be the case for endophytes of some conifer species as well as a number of tropical plants (Clay 1988; Arnold et al. 2003; Kageyama et al. 2008; Sumarah & Miller 2009). Moreover, endophytes of grasses and some tropical plants also improve drought tolerance (Arnold & Engelbrecht 2007; Kuldau & Bacon 2008; Rodriguez et al. 2009). In a recent study, Phialocephala subalpina isolates protected Norway spruce seedlings against two oomycete root pathogens, Phytophthora plurivora and Elongisporangium undulatum (Tellenbach & Sieber 2012), but the mechanism could not be determined from these experiments. However, the possible mechanisms could include mycoparasitism, improved root health and nutrient uptake (Schulz 2006), exploitation competition (Lockwood 1992) and/or interference competition, i.e., antibiosis (Wicklow 1992). Therefore, the aim of the present study was to test the hypothesis that some PAC strains produce metabolites that are toxic to Phytophthora.
Methods Screening for antagonistic PAC strains Pairwise plate assays were used to screen 85 PAC strains belonging to eight PAC species (Table 1) for possible suppressive effects against P. plurivora (syn. Phytophthora citricola s.l.) (Table 2 and Supplementary Fig 1), which is frequently
13
associated with root dieback of broadleaved trees, but which has also been found to cause mortality and fine root loss in Norway spruce (Nechwatal & Osswald 2001; Jung & Burgess 2009; Tellenbach & Sieber 2012). PAC strains were previously genotyped based on single-copy RFLP markers and sequence € nig & Sieber 2005; Gru € nig et al. 2007, 2008b). Most markers (Gru of the PAC strains tested had distinct multilocus haplotypes, i.e., strains represent different individuals (Table 1). Cultures of strains used during this study are preserved in the culture € rich and are accessible for all researchers. collection of ETH Zu All PAC strains were grown on 2 % malt extract agar (MEA, 2 % malt extract, Hefe Schweiz AG, Stettfurt, Switzerland; 2 % agar [w/v]) for 26 d prior to inoculation on 2 % carrot agar plates (2 % commercially available carrot juice; 2 % agar [w/v]) overlaid with sterilized cellophane sheets. Since the PAC growth rate € nig et al. 2008b) is slower (1.25 0.13 mm d1 20 C on MEA) (Gru than P. plurivora growth rate (6.3 0.1 mm d1 at 20 C on V8 agar) (Jung & Burgess 2009), PAC were inoculated 23 d prior to P. plurivora. One agar plug was excised from the growing margin of the PAC colony with a 4 mm diameter cork borer, inoculated 25 mm from the center of the plate, and incubated at 20 C in darkness. P. plurivora (syn. P. citricola s.l.) strains PLU 55, PLU 278 and PLU 279 (Table 1, Supplementary Fig 1) were grown on V8 agar (V8A; 350 ml V8, 5 g CaCO3 mixed and centrifuged for 20 min at 1000 g, supernatant 1:4-diluted with deionized water and 20 g agar added prior to autoclaving) for 5 d prior to inoculation. Similarly, inocula from the margin of the actively growing P. plurivora colonies were excised as above and inoculated 25 mm from the center of the plate on the opposite site of the PAC colony. There were 85 PAC strains times three P. plurivora isolates equaling 255 experimental units. The diameter of both the PAC and P. plurivora colonies were measured 3 and 15 d post inoculation of the latter and extension rates calculated. In addition, colony morphologies for both PAC and P. plurivora were characterized according to Wicklow et al. (1980).
Growth inhibitory effects of PAC culture exudates Based on the results of the dual-culture plate assay, eight PAC strains with different characteristics were selected for the analysis of potential antagonistic activity (Table 1). As the effect on all three P. plurivora isolates was similar in the plate assays, only strain Bu 14218 (PLU 279) was used in this screening. PAC strains were inoculated aseptically in triplicate into 100-ml Erlenmeyer flasks containing 50 ml of autoclaved 5 %-carrot juice. They were shaken at 100 rpm for 30 d at 20 C. Liquid cultures for each PAC strain were combined and filtered to remove the mycelial fraction. The filtrate was then sterilized by filtration using 0.45 mm filters (Sartorius, Tagelswangen, Switzerland) and mixed 1:1 [v/v] with autoclaved 5 % double-strength CA (5 %-carrot juice; 3 % agar [w/v]) that was cooled to 50 C. As a control, three sterile uninoculated 50 ml portions with 5 %-carrot juice were maintained under the same conditions as the PAC colonies and processed as described above. P. plurivora strains were grown on 5 %-CA agar plates for 2 weeks prior to inoculation. P. plurivora was inoculated in the center of the agar plate and growth radii were recorded 3 d and 8 d post inoculation to calculate mean colony growth. Per substrate there were nine to 12 replicates.
14
C. Tellenbach et al.
Table 1 e PAC strains included in the present study to assess suppressive effects of PAC against Phytophthora plurivora Taxon Phialocephala turiciensis
Phialocephala letzii
Phialocephala europaea
c
Phialocephala helvetica
Phialocephala uotolensis
Phialocephala subalpina
Strain
MLHa
CBS no
1_117_3 1_120_2 1_124_1 1_124_2 1_130_3 1_133_3 1_154_1 1_176_1 1_190_4
MLH_01 MLH_02 MLH_03 MLH_04 MLH_05 MLH_06 MLH_07 MLH_08 MLH_09
2_120_3 2_126_3 2_137_4 2_146_1 2_146_2 2_147_3 2_149_5 2_152_2 2_168_4 2_194_1 2_194_4
MLH_10 MLH_11 MLH_12 MLH_13 MLH_14 MLH_15 MLH_16 MLH_17 MLH_18 MLH_19 MLH_10
3_117_2 3_122_3 3_129_1 3_136_1 3_136_3 3_137_3 3_139_3 3_158_4 3_169_2 3_171_5 3_173_2 3_177_3
MLH_19 MLH_20 MLH_21 MLH_22 MLH_23 MLH_24 MLH_25 MLH_26 MLH_27 MLH_28 MLH_29 MLH_30
CBS 119269 CBS 119270
4_123_4 4_135_1 4_136_4 4_138_5 4_140_4 4_144_4 4_145_2 4_153_2 4_160_1 4_195_2
MLH_31 MLH_32 MLH_33 MLH_34 MLH_35 MLH_36 MLH_37 MLH_38 MLH_39 MLH_40
CBS 119272
5_134_3 5_220_1 5_234_5 5_252_7r 5_264_1r 5_265_1 5_265_3 5_265_4
MLH_41 MLH_42 MLH_43 MLH_44 MLH_45 MLH_46 MLH_46 MLH_47
6_16_1 6_2_7v 6_22_7v 6_30_4 6_35_6v 6_37_6v 6_53_6v 6_70_1 6_70_4 6_78_2 6_8_7v
MLH_48 MLH_49 MLH_50 MLH_51 MLH_52 MLH_53 MLH_54 MLH_55 MLH_56 MLH_57 MLH_58
CBS 119234
CBS 119264
CBS 119266
CBS 119268
CBS 119267
UAMH 11659
CBS 119271
CBS 119273
CBS 119274
CBS 119276 CBS 119277 CBS 119275
CBS 119279 CBS 119278
CBS 119280
Geographic origin
Plateb assay
Picea abies P. abies P. abies P. abies P. abies P. abies P. abies P. abies P. abies
€ richberg; Switzerland Zu € richberg; Switzerland Zu € richberg; Switzerland Zu € richberg; Switzerland Zu € richberg; Switzerland Zu € richberg; Switzerland Zu € richberg; Switzerland Zu € richberg; Switzerland Zu € richberg; Switzerland Zu
B C A/B A/B A/B B B B A
P. abies P. abies P. abies P. abies P. abies P. abies P. abies P. abies P. abies P. abies P. abies
€ richberg; Switzerland Zu € richberg; Switzerland Zu € richberg; Switzerland Zu € richberg; Switzerland Zu € richberg; Switzerland Zu € richberg; Switzerland Zu € richberg; Switzerland Zu € richberg; Switzerland Zu € richberg; Switzerland Zu € richberg; Switzerland Zu € richberg; Switzerland Zu
A A A A/B A/B B A/B A B B A
P. abies P. abies P. abies P. abies P. abies P. abies P. abies P. abies P. abies P. abies P. abies P. abies
€ richberg; Switzerland Zu € richberg; Switzerland Zu € richberg; Switzerland Zu € richberg; Switzerland Zu € richberg; Switzerland Zu € richberg; Switzerland Zu € richberg; Switzerland Zu € richberg; Switzerland Zu € richberg; Switzerland Zu € richberg; Switzerland Zu € richberg; Switzerland Zu € richberg; Switzerland Zu
B B B A/C B/C A B B A/B B B A
P. abies P. abies P. abies P. abies P. abies P. abies P. abies P. abies P. abies P. abies
€ richberg; Switzerland Zu € richberg; Switzerland Zu € richberg; Switzerland Zu € richberg; Switzerland Zu € richberg; Switzerland Zu € richberg; Switzerland Zu € richberg; Switzerland Zu € richberg; Switzerland Zu € richberg; Switzerland Zu € richberg; Switzerland Zu
A A/B A/C B B B B B A/B B
P. abies P. abies P. abies P. abies P. abies P. abies P. abies P. abies
€ richberg; Switzerland Zu Uetliberg; Switzerland Uetliberg; Switzerland Uetliberg; Switzerland Uetliberg; Switzerland Uetliberg; Switzerland Uetliberg; Switzerland Uetliberg; Switzerland
B B B B A/B B B B
P. abies Vaccinium myrtillus V. myrtillus P. abies V. myrtillus V. myrtillus V. myrtillus P. abies P. abies P. abies V. myrtillus
€ dmeren; Bo € dmeren; Bo € dmeren; Bo € dmeren; Bo € dmeren; Bo € dmeren; Bo € dmeren; Bo € dmeren; Bo € dmeren; Bo € dmeren; Bo € dmeren; Bo
B B B A A B A/B A B A A/B
Host
Switzerland Switzerland Switzerland Switzerland Switzerland Switzerland Switzerland Switzerland Switzerland Switzerland Switzerland
Culture assay x
x
x
x
x
Inhibition of Phytophthora species by secondary metabolites
15
Table 1 e (continued ) Taxon
Phialocephala fortunes s.s.
Acephala applanata
Strain
MLHa
CBS no
Host
Geographic origin
Plateb assay
6_9_6v
MLH_59
V. myrtillus
€ dmeren; Switzerland Bo
A
7_10_6v
MLH_60
V. myrtillus
€ dmeren; Switzerland Bo
A/B
7_39_7v 7_45_5 7_54_7v 7_6_7v 7_62_6v 7_62_7v 7_63_4 7_64_7v 7_K92_049 7_K93_395 7_K93_444
MLH_61 MLH_62 MLH_63 MLH_64 MLH_65 MLH_65 MLH_66 MLH_67 MLH_68 MLH_69 MLH_70
V. myrtillus P. abies V. myrtillus V. myrtillus V. myrtillus V. myrtillus P. abies V. myrtillus P. abies Pinus sylvestris P. sylvestris
€ dmeren; Switzerland Bo € dmeren; Switzerland Bo € dmeren; Switzerland Bo € dmeren; Switzerland Bo € dmeren; Switzerland Bo € dmeren; Switzerland Bo € dmeren; Switzerland Bo € dmeren; Switzerland Bo Odenwald; Germany Kevo; Finland Suonenjoki; Finland
A A B A A A A A/B A A A
T1_38_2 T1_39_2 T1_50_2 T1_51_3 T1_52_2 T1_55_1 T1_58_1 T1_75_3 T1_77_1 T1_78_1 T1_K92_079
MLH_71 MLH_72 MLH_73 MLH_74 MLH_75 MLH_76 MLH_77 MLH_78 MLH_79 MLH_80 MLH_73
P. abies P. abies P. abies P. abies P. abies P. abies P. abies P. abies P. abies P. abies P. abies
€ dmeren; Switzerland Bo € dmeren; Switzerland Bo € dmeren; Switzerland Bo € dmeren; Switzerland Bo € dmeren; Switzerland Bo € dmeren; Switzerland Bo € dmeren; Switzerland Bo € dmeren; Switzerland Bo € dmeren; Switzerland Bo € dmeren; Switzerland Bo € ntis; Switzerland Sa
A/B B A/B B A A/B A A A A A
CBS 119281
CBS 119282
CBS 114608 CBS 443.86
Culture assay
x
x
x
€ nig & Sieber 2005; Gru € nig et al. 2007; Gru € nig et al. 2008b). a Multilocus haplotype based on 11 single-copy RLFP probes and sequence markers (Gru b Categories of antagonism after Wicklow et al. (1980). A ¼ mutual intermingling of the two organisms, B ¼ inhibition of P. plurivora on contact; PAC continues to grow unchanged or at a reduced rate, through P. plurivora, C ¼ inhibition of P. plurivora at distance. c Strain grown for metabolite characterization and toxicity testing.
Characterization of secondary metabolites The strongest growth inhibition of P. plurivora in culture was observed with Phialocephala europaea strain 3_136_1 (UAMH 11659), which was then chosen for the secondary metabolite screening. A 2 % MEA slant of 3_136_1 was macerated in sterile water under aseptic conditions. The resulting suspension was used to inoculate five Glaxo bottles, each containing 1l of 2 % ME medium. The cultures were grown for 2 months at 25 C and then harvested and filtered. The filtrate was extracted with 2 equal volumes of ethyl acetate and then dried by rotary evaporation. The extract was re-suspended in acetonitrile and dried under nitrogen. The extract, 183 mg, was filtered and re-dissolved in acetonitrile and screened by LCeMS using electrospray ionization in both positive and negative ion mode as previously described (Sumarah et al.
2011). The extract yielded four major peaks by UV and MS that were isolated by semi-preparative HPLC using an Agilent 1100 HPLC with diode array detector and 250 10 mm Luna C18 column (Phemonomex, Torrence, California). The four peaks (compounds 1e4) were isolated and further analyzed by HRLCeMS and characterized by NMR.
Bioassay of the characterized compounds against P. citricola Sufficient quantities of compounds 1 and 3 (Fig 1) were isolated for testing against P. citricola s.l. strain BR519 (National Mycological Herbarium, Ottawa, ON, Canada) in sterile 96 well microplates at concentrations of 30 and 150 mg ml1. Unfortunately, it was not possible to use the P. plurivora strains for the screening because the transfer was not possible due to
Table 2 e Details of Phytophthora citricola s.l. strains used in the present study Straina PLU 55 (137/7a) PLU 278 PLU 279 BR519
Host
Species
Collector
Reference
Used for
Fagus sylvatica Quercus sp. Fagus sylvatica Citrus sinensis
P. plurivora P. plurivora P. plurivora P. citricola s.s.
J. Nechwatal T. Jung T. Jung G.A. Zentmyer
(Nechwatal & Osswald 2001) None None (Robideau et al. 2011)
Screening Screening Screening In vitro testing of metabolites
a PLU numbers refer to the collection of T. Jung (Jung & Burgess 2009).
16
C. Tellenbach et al.
5.0 ag def
4.5
b
bd
afg bdef
bde
4.0
3.5 c
Fig 2 e Structures of the secondary metabolites isolated from P. europaea strain 3_136_1; sclerin [1], sclerolide [2], sclerotinin A [3] and sclerotinin B [4]. T1 38_2
7 63_4
7 54_7v
6 2_7v
5 265_1
3 171_5
3 136_1
1 120_2
3.0
control
mean growth rate [mm d−1]
a
Fig 1 e Inhibitory effect of PAC culture filtrate against P. plurivora isolate PLU 279. Different letters indicate significant differences among extracts in a Tukey’s HSD test following ANOVA (P < 0.05). regulatory issues. The pure compounds were dissolved in DMSO and 5 ml of this solution were added to 200 ml of a macerated 24 hr old culture of P. citricola in V8 media. Individual tests included six replicates per plate and tests were performed in triplicate on different days; the plates were incubated at room temperature with constant shaking and were measured at 600 nm at time zero, 24 and 48 hr with a plate reader (Power Wave XS microplate reader (BioTek, Vermont, USA)). The antibiotic chloramphenicol (Sigma, St. Louis, MO) was used as the positive control at the same concentration (w1 mM) for all experiments. ANOVA followed by Tukey’s honestly significant difference (HSD) test was used to evaluate the in vitro bioassays using SYSTAT v. 13. The remaining statistics were performed using the R statistical package (R Development Core Team 2009).
Results and discussion Growth inhibition of P. plurivora (syn. P. citricola s.l.) by PAC strains In general, the effect of PAC strains was similar on growth of the three P. plurivora strains. P. plurivora cultures were overgrown by mycelium of PAC (Table 1), but no evidence of mycoparasitism was found for either PAC or P. plurivora after microscopic examination. The strains tested represented a variety of PAC species, some of which inhibited Phytophthora growth. Based on these results, eight strains with varying degrees of inhibition were selected, and the effect on the growth of P. plurivora after incorporating PAC culture filtrate into growth medium was tested (Table 1). There were significant differences among the culture filtrates as indicated by an ANOVA (F8,76 ¼ 231.6, P < 0.001). The culture filtrate of strain 3_136_1 (UAMH 11659) significantly reduced P. plurivora growth compared to the control and all other PAC strains (Fig 1; Tukey’s HSD test). In contrast, the filtrates of other strains showed less pronounced, but still significant, to no inhibitory effects. Four compounds were isolated and characterized from the culture filtrate of 3_136_1. The four biosynthetically-related
compounds were determined to be the known fungal metabolites: sclerin [1] HRMS m/z 235.0990 [M þ 1]þ, sclerolide [2] HRMS m/z 223.0984 [M þ 1]þ, sclerotinin A [3] HRMS m/z 253.1078 [M þ 1]þ and sclerotinin B [4] HRMS m/z 239.0929 [M þ 1]þ (Fig 2; Supplementary Fig 3) previously isolated from Sclerotinia sclerotiorum, a known plant pathogen. The spectroscopic data for these compounds were in agreement with published literature (Supplementary Figs 4 and 5). Sclerin (4.5 mg) was the major compound produced by strain 3_136_1. This compound was originally isolated by Satomura & Sato (1965), where it was described as a plant growth hormone. Sclerin was also shown to be phytotoxic to a number of plant species (Pedras & Ahiahonu 2004). Sclerotinin A (3.5 mg) was initially isolated by Sassa et al. (1968) along with sclerotinin B, where they were described as growth promoters of rice seedlings. Antibiotic assays of sclerin and sclerotinin A to P. citricola s.s. (syn. P. citricola s.l.) were performed in 96 well plates containing liquid V8 medium. DMSO had no effect on the strain tested. In vitro, 30 mg ml1 sclerin and sclerotinin A resulted in significant reduction in the growth of P. citricola vs. controls at 24 hr (F6,72 ¼ 200.2, P ¼ 0.04 and P ¼ 0.001 respectively; Supplementary Fig 2) but both were less toxic than chloramphenicol. After 48 hr, growth had recovered to the control levels. When the higher concentration (150 mg ml1) of sclerin and sclerotinin A were tested, significant reductions in the growth of P. citricola s.l. were observed at 24 hr (F6,72 ¼ 56.3, P < 0.001 and P < 0.001, respectively; Supplementary Fig 2) and at 48 hr for both test compounds. The potencies were comparable to a similar molar concentration of chloramphenicol. The results suggest that sclerin and sclerotinin A, the major compounds isolated from P. europaea strain 3_136_1, are either individually or synergistically responsible for some of the original growth inhibition of P. citricola s.l.
Conclusions After screening 85 PAC strains for inhibitory effect against P. plurivora and performing subsequent plate assays using the culture filtrates of eight strains, one strain of interest was identified. This strain, P. europaea 3_136_1, produced at least four compounds, which are reported for the first time from this genus and species. Two of these, sclerin and sclerotinin A, inhibited P. citricola s.s. in the low mM range. These compounds have not been reported as antimicrobial despite many studies on their biological effects. Previous researchers have shown
Inhibition of Phytophthora species by secondary metabolites
that sclerin and sclerotinin A and related compounds are phytotoxic in vivo (Pedras & Ahiahonu 2004). As noted above, one hypothesis for the mode of action for DSE was improvement in root health (Schulz 2006). Our data show that they are toxic to oomycetes of the P. citricola s.l. species complex. This study provides a mechanistic basis to support the idea that some PAC isolates protect their host against some oomycete root pathogens (Tellenbach & Sieber 2012). The selection scheme used to detect and characterize these metabolites was first to detect the variety of interaction types between PAC and P. plurivora, quantifying the strength of interaction afterward, and finally, targeting metabolites and assaying their antagonistic potential. Therefore, these results justify the need for further testing using PAC isolates to investigate the variability in the production of these metabolites and to inoculate conifer trees with isolate P. europaea 3_136_1 and other PAC strains to potentially reduce the damage resulting from Phytophthora in a more natural environment.
Acknowledgments We would like to thank Thomas Jung for providing the Phytophthora plurivora strains, and Ottmar Holdenrieder for helpful comments on a previous version of this manuscript. This work was partially supported by SNF Grant 3100A0-113977 from the Swiss National Science Foundation, Bern, Switzerland. This work was associated with the GEDIHAP project of the Competence Center Environment and Sustain€ rich, Switzerland. ability (CCES) of the ETH Domain, Zu
Supplementary data Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.funeco.2012.10.003.
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