draft - · pdf filedraft 2 abstract: fluorapatite-solubilizing bacteria were isolated from...

Draft

Phosphate-solubilizing bacteria isolated from

ectomycorrhizal mycelium of Picea glauca are highly efficient at fluorapatite weathering

Journal: Botany

Manuscript ID cjb-2016-0089.R1

Manuscript Type: Article

Date Submitted by the Author: 08-Jun-2016

Complete List of Authors: Fontaine, Laurent; Université Laval, Département des sciences du bois et de la forêt Thiffault, Nelson; Ministère des Forêts, de la Faune et des Parcs, Direction de la recherche forestière Paré, David; Natural Resources Canada, Canadian Forest Service Fortin, J.-André; Université Laval, Département des sciences du bois et de la forêt Piché, Yves; Université Laval, Département des sciences du bois et de la forêt

Keyword: Ectomycorrhizal fungi, Mineral weathering, Phosphate solubilizing bacteria, Apatite, Burkholderia

https://mc06.manuscriptcentral.com/botany-pubs

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Phosphate-solubilizing bacteria isolated from ectomycorrhizal mycelium of Picea

glauca are highly efficient at fluorapatite weathering

Laurent Fontainea,*, Nelson Thiffaultb, David Paréc, J.-André Fortina, and Yves Pichéa

a Département des sciences du bois et de la forêt, Faculté de foresterie, de géographie et

de géomatique, Université Laval, 2405 de la Terrasse, Québec, QC G1V 0A6, Canada

b Direction de la recherche forestière, Ministère des Forêts, de la Faune et des Parcs, 2700

Einstein, Québec, QC G1P 3W8, Canada

c Natural Ressources Canada, Canadian Forest Service, Laurentian Forestry Centre, 1055

du PEPS, P.O. Box 10380 Stn. Sainte-Foy, Québec, QC G1V 4C7, Canada

* Corresponding author: Laurent Fontaine (email: [email protected])

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Abstract: Fluorapatite-solubilizing bacteria were isolated from the hyphosphere of the

ectomycorrhizal (ECM) fungus Wilcoxina sp., a dominant species in the Picea glauca

rhizosphere. Hundreds of strains isolated from the ascomycete Wilcoxina sp. could

dissolve tricalcium phosphate, while only 27 of them could produce clarification halos on

fluorapatite-amended solid medium. Most of the fluorapatite-solubilizing strains

belonged to the Burkholderia genus. Scanning electron microscopy observations have

shown that these efficient phosphate-solubilizing bacteria (PSB) were able to completely

solubilize fluorapatite crystals within 22 h. The efficient PSB Burkholderia sp. strain 205

and Curtobacterium sp. strain 168 were tested for their ability to associate with a

genetically distant fungal host while fulfilling their phosphate solubilizing function.

Burkholderia sp. strain 205 successfully associated with the Basidiomycete Laccaria

bicolor when hydroxyapatite was the only phosphorus source available to the fungus,

while there was no bacterial development when L. bicolor could access soluble

phosphorus as well. Optical microscopic observation of L. bicolor associated with

Burkholderia sp. revealed extensive colonization of fungal hyphae by the bacterium.

These results suggest an important role of bacteria-ECM fungi associations in white

spruce phosphate nutrition.

Key words: Ectomycorrhizal fungi, apatite, mineral weathering, phosphate solubilizing

bacteria, hyphosphere, Burkholderia.

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Résumé: Des bactéries solubilisatrices de fluorapatite ont été isolées à partir de

l’hyphosphère du champignon ectomycorhizien (ECM) Wilcoxina sp., une espèce

dominant la rhizosphère de Picea glauca. Des centaines de souches isolées de

l’ascomycète Wilcoxina sp. ont dissout le phosphate tricalcique, alors que seulement 27

d’entre elles ont clarifié le milieu amendé en fluorapatite. La majorité des souches

dissolvant la fluorapatite appartiennent au genre Burkholderia. Des observations réalisées

en microscopie électronique à balayage ont montré que ces bactéries solubilisatrices de

phosphates (BSP) efficaces pouvaient dissoudre complètement des cristaux de

fluorapatite en 22 h. Les souches de BSP efficaces Burkholderia sp. 205 et

Curtobacterium sp. 168 ont été testées quant à leur capacité de s’associer avec un hôte

génétiquement distant tout en remplissant leur fonction de BSP. La souche 205 de

Burkholderia sp. s’est associée avec succès au basidiomycète Laccaria bicolor lorsque

l’hydroxyapatite était la seule source de phosphore accessible au champignon, alors

qu’une source de phosphore soluble inhibait la croissance bactérienne. Des observations

en microscopie à fond clair de L. bicolor en association avec Burkholderia sp. ont révélé

une importante colonisation bactérienne des hyphes. Ces résultats suggèrent une

importance des associations bactéries-champignons ECM dans la nutrition phosphatée de

l’épinette blanche.

Mots-clés: Champignons ectomycorhiziens, apatite, alteration minérale, bactérie

solubilisatrice de phosphate, hyphosphère, Burkholderia.

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Introduction

In boreal forests, soil properties shape the composition of plant communities,

which in turn enable the development of diverse communities of ectomycorrhizal fungi

(ECM) and their associated bacteria. The availability of nutrients in the soil as well as the

composition of soil minerals influence the development of ECM fungi (Rosling et al.

2004) and bacterial diversity (Uroz et al. 2012). For example, carbon allocation to ECM

mycelium formed by Pinus sylvestris L. and Paxillus involutus S.L. (Batsch) Fries was

significantly higher in soils amended with calcium phosphate compared with a control

treatment amended with quartz (Smits et al. 2012). Moreover, P. involutus releases large

amounts of organic acids when host growth is limited by a phosphorus deficiency (van

Schöll et al. 2006). Exudation of these organic acids by the ECM mycelium should

provide associated bacteria with a significant source of carbon (Olsson and Wallander

1998). This probably explains why mineral weathering bacteria are more abundant in the

ECM hyphosphere than in bulk soil (e.g. Scleroderma citrinum; Uroz et al. 2007).

Therefore, these bacteria closely associated with ECM hyphae are more effective in

mineral weathering than those isolated from the rest of the soil (Calvaruso et al. 2007).

This suggests a tripartite relationship, in which the ECM fungus receives photosynthates

that are then released as organic acids, which are substrates that can be assimilated by

certain soil bacteria. In exchange, these bacteria would mobilize ions from minerals

(Sokolova 2011), which are immediately absorbed by the ECM fungus and then

transmitted to the host (Calvaruso et al. 2010). According to this model, soil fertility in

boreal forest would be influenced by the content and weatherability of soil minerals as

well as by the mineral weathering capacities of fungal and bacterial communities

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associated with forest stands. The existence and role of this tripartite relationship in the

nutrition of boreal forest tree species has been observed for Pinus resinosa Sol. ex. Aiton

in microcosm experiments (Balogh-Brunstad et al. 2008).

Among the nutrients essential for tree development, the abundance and forms of

phosphate minerals found in the soil define the fertility of a site (Hahm et al. 2014). The

crystalline properties of phosphate minerals determine their reactivity (Elliott 1994) and

thus their availability as phosphorus sources for soil microorganisms. In turn, apatite

crystalline structures are determined by calcium, phosphate and fluoride substitutions

(McDonnell 1973). An amorphous mineral such as tricalcium phosphate (TCP) is easily

weathered by microorganisms, while crystalline phosphate minerals are much more

resistant to corrosion (Bashan et al. 2012). Igneous rocks contain apatites that can be

classified into the three subgroups of hydroxyapatite, chlorapatite and fluorapatite, the

last one being the most recalcitrant to chemical weathering (Dorozhkin 2011). Access to

the nutrients contained in soil minerals by plants and their associated microorganisms is

of particular interest in boreal and mountainous regions because of the relatively early

stage of soil development and, hence, the availability of unweathered minerals due to the

recent glacial retreat (Milner et al. 2007). We chose to study the microbial flora of white

spruce (Picea glauca (Moench) Voss), a species typical of the boreal zone, known for its

sensitivity to phosphorus availability (Quesnel and Côté 2009).

Based on our assumption of the tripartite relationship between the host, ECM

fungi and phosphate solubilizing bacteria (PSB), we predicted that the most abundant

ECM fungi in the rhizosphere of white spruce would be associated with potentially

cultivable PSB. Our hypothesis also suggests that PSB receive organic carbon from ECM

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mycelium when the only source of phosphorus is insoluble. Finally, it predicts that the in

vitro weathering of fluorapatite by PSB could be observed in less than a day when

crystals are incubated in a bacterial suspension.

Therefore, our overall objective was to verify the role of the bacterial microbiota

associated with ECM fungi in fluorapatite weathering, an important source of phosphorus

in mineral soils of igneous origin. To do so, our specific objectives were to i) isolate and

identify PSB from the mycelium of an ubiquitous ECM fungus associated with P. glauca,

ii) demonstrate in vitro fluorapatite solubilization by PSB and iii) validate in vitro the

close association between PSB and ECM fungi.

Materials and methods

PSB source

Potential fluorapatite-solubilizing bacteria were isolated from the rhizosphere of

four P. glauca seedlings. The host plants were aged between 5 and 15 years and located

in plantations. The collection site is located approximately 75 km north of Québec City

(Canada) (lat. 46°57'55.3'' N; long 71°30 55.9'' W) within the balsam fir (Abies balsamea

(L.) Mill.) – white birch (Betula papyrifera Marsh.) bioclimatic domain described by

Saucier et al. (2009). The seedlings were taken along with a layer of MOR organic

material (approx. 3 cm thick). The B-horizon from the surrounding stands (Humo-Ferric

podzol; Soil Classification Working Group 1998) was collected to serve as the mineral

soil substrate for 8 L transplantation pots.

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Maintenance of host plants

After transplantation, seedlings were maintained in a greenhouse for 18 months

(September 2012 – April 2014). Seedlings were submitted to a 16 h photoperiod at

temperatures ranging from 18–23 °C. The plants were watered once a week with tap

water. No weeding was made during this period; thus, herbaceous species (e.g.

Chamaerion angustifolium (L.) Holub, Poa pratensis L., Pilosella aurantiaca (L.)

F.W.Schultz & Schultz-Bip.) proliferated in the pots. After the first trimester in the

greenhouse, the seedlings became dormant and were lifted from that state by being kept

for 4 months at 4 °C in complete darkness.

Characterization of PSB hosting ECM morphotypes

ECM morphotypes were collected in order to use them as a PSB inoculum source

to be grown on solid and liquid media. Mycorrhizal morphotypes were collected in June

2013 for liquid cultures and in February 2014 for solid cultures. While growing in the

greenhouse, the seedlings produced large amounts of long roots running along the

perimeter of the pots, which made it possible to harvest short roots without disturbing the

substrate. The most abundant ECM were identified visually, then collected and screened

under a dissecting microscope to ensure only one species was present in the sample.

Molecular identification of the main ectomycorrhizal morphotype

Five ectomycorrhizas of the dominant morphotype were randomly selected from

three seedlings to proceed with the ECM fungal DNA extraction. They were ground with

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mortar and pestle in 100 µl of TTE buffer (0.002 M EDTA, 0.02 M Tris, 0.0008 M Triton

X-100) and heated at 95 °C for 10 min. The resulting product was centrifuged and the

supernatant retained as total DNA.

A polymerase chain reaction (PCR) (Saiki et al. 1988) was performed on the

internal transcribed spacers (ITS) of the ribosomal genes in a reaction volume of 25 µl

containing 0.2 mM dNTP mix, 0.5 µM primer ITS-1F and ITS-4, 2.5 µl PCR buffer

(Qiagen, Hilden, Germany), 1.5 mM MgCl2, BSA 0.2 mg·ml-1, 3 U Taq (Qiagen) and

2 µl of total DNA. Amplification was performed on a PTC-225 Thermal Cycler (MJ

Research, Waltham, Massachusetts) under the following conditions: 94 °C for 4 min,

followed by 34 cycles at 94 °C, 55 °C and 72 °C for 1 min, and finally at 72 °C for 10

min.

Amplification success was confirmed by migration of the PCR product on agarose

gel before proceeding to sequencing. The sequencing was performed on a 16 capillary

genetic analyzer (3130XL model, Applied Biosystems, Thermo Fisher Scientific Inc.,

Burlington, ON).

Isolation of PSB on solid selective medium

We isolated BSP closely associated with the identified ECM morphotype. In order

to obtain a diversity of strains, we used the spread plate method on agar media containing

only insoluble phosphorus sources. A subsample of ectomycorrhizas subjected to

molecular analysis was selected to form the bacterial inoculum. Ectomycorrhizas were

washed vigorously under running tap water to remove particles and bacteria not firmly

bound to the fungal sheath. The washing was performed on a 1 mm mesh sieve. The

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freshly cleaned ectomycorrhizal samples were then transferred to a sterile aqueous

solution of NaCl 8.5 g·L-1 (9x their fresh mass). In this saline solution, ectomycorrhizas

were finely ground with a sterile set of mortar and pestle. The resulting suspension was

considered as a 10-1 dilution. Serial dilutions were carried out in the same saline solution

to obtain a bacterial suspension factor of 10-4. That 10-4 dilution was used as inoculum for

spread plating on selective medium at 300 µl per plate. The medium was double layered

to facilitate the observation of clarification halos. The bottom layer, based on the NBRIP

medium (Nautiyal 1999), contained 10 g glucose, 5 g MgCl2·6H2O, 250 mg

MgSO4·7H2O, 200 mg KCl, 200 mg KH2PO4, 100 mg (NH4)2SO4, 200 mg yeast extract

(Difco Laboratories, Detroit, Michigan), 50 mg cycloheximide (Sigma Chemical Co., St.

Louis, Missouri) and 10 g agarose (Amresco, Solon, Ohio) per litre. The upper layer

contained, per litre of medium, 2.5 g Ca3PO4 (TCP) (Fisher Science Education, Hannover

Park, Illinois) and 10 g of agarose. The media were poured into 80 mm Petri dishes at a

rate of 20 ml for the bottom layer and 5 ml for the upper layer. The initial isolation of

phosphate-solubilizing strains was carried out on a medium containing TCP as an

insoluble phosphorus source. The stability of the weathering phenotype was verified with

three subcultures on TCP. Strains exhibiting stable expression of the desired phenotype

were subcultured on an amended layer containing 2.5 g·L-1 hydroxyapatite (Sigma-

Aldrich, St. Louis, Missouri). The strains having successfully dissolved hydroxyapatite

were finally tested on a medium amended with 2.5 g·L-1 carbonated fluorapatite from the

Grenville geological province (Arianne Phosphate, Saguenay, Québec) containing 1%

fluorine.

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PSB isolation by enrichment in selective liquid medium

PSB were obtained by enrichment in selective liquid medium using the same

inoculum as for isolation on solid media. The enrichment was initiated with 200 µl of 10-2

dilution of ground ectomycorrhizas. Again, the NBRIP selective medium (Nautiyal 1999)

was used, but this time with fluorapatite ore rather than TCP as an amendment in order to

immediately select bacterial strains able to grow on igneous apatite. The bacteria were

grown in 125 ml flasks containing 50 ml of medium, sealed with foam plugs, and

incubated at 26 °C with agitation at 100 rpm. The medium was sterilized in an autoclave

with a liquid cycle for 20 min. A benomyl solution was added to each experimental unit

after autoclaving to obtain a medium concentration of 3 mg·L-1. After adding the

inoculum, the flasks were incubated for one week and subcultured in fresh media.

Transplanting was done with an inoculation loop so that subculture would favour the

most abundant strains in the selection of colony forming units inoculating the fresh

medium. In turn, this should promote the selection of the most competitive fluorapatite

weathering bacteria. After seven subcultures, the bacterial communities were transferred

to solid medium to allow the isolation of strains. The strains unable to weather TCP on

solid medium were rejected.

Ultrastructural observations of fluorapatite weathering by an efficient PSB

Fluorapatite weathering by an efficient bacterial strain was observed using

scanning electron microscopy (SEM). The elemental composition of the fluorapatite ore

was determined by X-ray photoelectron spectroscopy. The bacterial strain PSB 205,

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selected because of its effectiveness in fluorapatite dissolution, was propagated in flasks

containing the liquid medium Tryptic Soy Broth (Difco Laboratories, Detroit, Michigan)

maintained at 26 °C under agitation at 180 rpm. The microbial cultures were centrifuged,

the supernatant removed, followed by washing of the bacteria by resuspension in a

solution of NaCl 0.15 M. This suspension was centrifuged again to remove the

supernatant. The bacterial pellet was resuspended in phosphorus-free NBRIP medium to

produce a bacterial concentration of 1×106 CFU per ml. Two fluorapatite ores were used,

with fluorine concentrations of 1% (Arianne Phosphate, Saguenay, Québec) and 4%

(Mine Arnaud, Sept-Îles, Québec), respectively. The fluorapatite ore was washed with

distilled water, open air dried, and then autoclaved for 20 min. Ferromagnetic particles

were removed from the ore using a magnet. Carbon tape adhered to a stainless steel SEM

stub was used as a specimen holder for the ores (Goldstein 2003). Fluorapatite was

deposited on the carbon tape and loose particles were removed with an air jet.

Two methods were used to incubate the bacterial strain PSB 205 in the presence

of fluorapatite. The first was limited to covering the ore attached to the SEM stub with a

drop of bacterial suspension. The second method consisted in placing the fluorapatite-

bearing stub in a cavity of a sterile multiwell plate (24 Well Multiwell, Becton Dickinson,

Franklin Lakes, NJ) and then filling the well with the bacterial suspension to cover the

fluorapatite and incubating under agitation at 60 rpm. The first and second methods were

used after 6 and 22 h of incubation, respectively. Controls involving incubation in

bacteria-free growth medium were used to confirm that mineral weathering did not occur

spontaneously.

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Observations were made using a JEOL JSM-7600F SEM model equipped with an

energy-dispersive X-ray spectrometer (EDS) with a resolution of 1 nm at 15 kV and

1.4 nm at 1 kV. The conventional secondary electron detector (LEI) was used. No

conductive coatings (Au, C, Pt) were applied during sample preparation. Initial

observations were conducted on virgin fluorapatite crystals. The most conspicuous

crystals were identified in order to be easily located after incubation with PSB strain 205.

Micrographs of the whole crystals and their edges were taken. After incubation,

fluorapatite was washed under a stream of distilled water for 1 min. The sample was

dried under vacuum before being inserted back into the SEM column. The crystals

observed in their virgin state were found, photographed again and subjected to qualitative

observations. The mineralogical composition was also determined by X-ray

photoelectron spectroscopy.

Resynthesis of close associations between three PSB strains and an ECM fungus

The ECM fungus Laccaria bicolor (Maire) P.D.Orton strain B3-25#2 was

selected for its symbiotic properties, especially its inability to solubilize apatites. As for

bacteria, PSB strains (168, 205 and 260) were chosen for their ability to efficiently

weather fluorapatite. The reconstitution of associations between PSB strains and L.

bicolor was carried out in a bicompartmented Petri dish culture system. Initially, the

phosphate-free proximal compartment was inoculated with L. bicolor to promote

exploration of the distal compartment containing a phosphorus source when the proximal

compartment contained all essential nutrients, with the exception of phosphorus (Fig. 1).

Meeting of the ECM fungus with each PSB strain occurred in the distal compartment,

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comprising one of three sources of phosphorus: i) soluble phosphorus (soluble P), ii)

hydroxyapatite (HA), or iii) soluble phosphorus + hydroxyapatite (soluble P + HA). Each

treatment was inoculated or not with three bacterial strains able to solubilize fluorapatite.

The experiment also included two controls with bacteria growing without the fungus on a

medium of soluble phosphorus or hydroxyapatite. Each treatment was repeated three

times.

The proximal compartment contained a modified MNM medium (Marx 1969)

without phosphorus. This medium consisted of 0.15 g MgSO4 · 7H2O, 0.025 g NaCl, 5 g

glucose, 0.26 g KCl, 0.05 g CaCl2, 1 ml trace elements solution, 0.5 ml FeEDTA , 100 ug

thiamine HCl, 0.340 g l-alanine, and 10 g agar per litre of medium. Distal compartments

contained 10 g agar, 0.5 g KH2PO4 (soluble P), 2.5 g hydroxyapatite (HA) or 0.5 g

KH2PO4 (soluble P), and 2.5 g hydroxyapatite (HA) per litre. The media were sterilized

in an autoclave with a liquid cycle for 20 min.

The proximal compartments were each inoculated with two 5 mm diameter fungal

plugs taken at the edge of a 1-month-old colony of L. bicolor grown on GYME medium.

The plugs were formed using a cork borer and placed at the centre of the proximal

compartment. The morphologically distinct PSB strains 168, 205 and 260 were deposited

in the distal compartment under a laminar flow hood. The bacteria were deposited once L.

bicolor colonies had grown 1.5 cm past the septum. The bacteria were then deposited in

the proximal compartment using a micropipette as three 2 µl drops of an aqueous

suspension containing 1×106 CFU per ml and set 1.5 cm apart, a few mm from the edge

of the fungal colonies.

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The diameters of the bacterial colonies were used as a proxy for growth and

analyzed according to a complete block experimental design using an analysis of variance

(ANOVA). In the presence of a significant F value, the means were compared using a

priori orthogonal contrasts to discern meaningful differences in growth for the bacterial

strains 168, 205 and 260 in terms of i) the soluble or insoluble nature of the phosphorus

source available to L. bicolor in the distal compartment, and ii) the presence or absence

of a soluble phosphorus source when an insoluble phosphorus source was always

available to L. bicolor in the distal compartment.

The data were examined to verify normality and homogeneity of variance with

standard graphical methods. All analyses were performed using the GLM procedure of

SAS v.9.32 software (SAS Institute Inc., Cary, North Carolina). Effects were considered

significant at the α = 0.05 threshold.

Molecular identification of PSB strains

PSB strains selected by culture on solid medium and in liquid medium were

identified based on 16S rRNA genes. Molecular identification was performed on 27

strains of PSB having produced clarification halos on fluorapatite ore on solid media and

on the nine strains isolated by enrichment in liquid media displaying a stable weathering

phenotype. The strains were grown on Tryptic Soy Broth (Difco Laboratories, Detroit,

Michigan) for a period of 7 days at 26 °C, with agitation at 180 rpm. We collected and

centrifuged 100 µl of culture medium containing the bacteria. The supernatant was

removed and the pellet resuspended in 100 µl of TTE buffer. This suspension was then

heated at 95 °C for 10 min. The resulting product was centrifuged and the supernatant

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retained as total DNA. A polymerase chain reaction (PCR) was performed in the same

way as for the ectomycorrhizal morphotype, except for the primers (358E and 907R,

Weidner et al. 1996) and the use of three U Taq instead of one. After validation of the

amplification success, the sequences were edited using the Bioedit software v.7.1.3 (Ibis

Biosciences, Carlsbad, California). Identity of the taxa was determined by comparing the

sequences obtained with those deposited in GenBank

(http://www.ncbi.nlm.nih.gov/genbank/). A phylogenic tree was created with the Mega5

software (Tamura et al. 2011) using the UPGMA method with 100 bootstrap replicates.

Results

Isolation of PSB closely associated with the dominant ECM morphotype in the P. glauca

rhizosphere

The most abundant ECM morphotype found in association with P. glauca, used as

our source of PSB, matched the fungus Wilcoxina sp. with 95-98% similarity to ITS of

the ribosomal genes with sequences returned by GenBank.

The spread plating of crushed Wilcoxina sp. mycorrhizae produced 455 colonies

displaying clarification halos on TCP-amended solid medium. Only 140 strains were

retained based on their capacity to produce clarification halos after being subcultured

three times (Table 1). Of these 140 phenotypically stable strains, 98 clarified the

hydroxyapatite amended medium. Finally, 27 strains produced clarification halos on the

fluorapatite ore amendment. Clarification halos typically became visible on TCP within

24-48 h, while those on fluorapatite required a minimum of 1 week to be discernible.

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Enrichment in liquid medium produced 39 strains that were later grown on solid

media. Of these 39 strains, nine showed a stable weathering phenotype on three

subcultures on TCP-amended medium. The weathering phenotype remained after the

strains were cultured on rich media.

Identification of PSB taxa

The strains isolated on solid media and whose DNA was successfully amplified

mostly belong to the genus Burkholderia (Fig. 2). Burkholderia spp. are divided into two

taxa with a support of 1 (Fig. 2). The other bacterial strains obtained on solid media

belong to the genus Curtobacterium, while those obtained by enrichment in liquid media

were identified as Leifsonia sp. The phylogenic tree is rooted by Pseudozyma aphidis, an

environmental yeast isolated by enrichment in liquid media.

Ultrastructural observations of fluorapatite weathering by Burkholderia sp. 205

Fluorapatite weathering by the PSB Burkholderia sp. 205 was successfully

observed with both incubation methods. With the drop method, the edges of fluorapatite

crystals containing 1% fluorine showed signs of corrosion after only 6 h of incubation

(Fig. 3b), compared to intact fluorapatite (Fig. 3a). Complete dissolution of fluorapatite

crystals containing 1 % fluorine was observed after 22 hours of incubation with the

immersion method (Figs. 3c and 3d). There were no signs of weathering for the controls

incubated in bacteria-free growth medium. The surface study of a fluorapatite crystal

shows an elemental composition consisting mainly of phosphorus, calcium and oxygen

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while also showing traces of fluorine, silica and iron1. These fluorapatite crystals are

commingled with dust and layers of complex oxides resistant to weathering. Furthermore,

the crystals of fluorapatite containing 4% fluorine (Figs. 3e and 3f) are more resistant to

weathering and have different patterns of etching than those containing 1% fluorine (Fig.

3e).

Close association between PSB and ECM fungi

The association between L. bicolor and PSB strains in the distal compartment of

bicompartmented plates demonstrated that the growth of Burkholderia sp. 205 depended

on the nature of the phosphorus source made available to the fungus. Indeed, when L.

bicolor could acquire phosphorus on its own, (Fig. 4a), the growth of Burkholderia sp.

205 could not be observed on the hyphae of the ECM fungus located in the distal portion

of the plate (Fig. 4a inset). In contrast, when L. bicolor was deprived of a soluble source

of phosphorus (Fig. 4b), Burkholderia sp. 205 displayed growth on the hyphae of the

symbiotic fungus (Fig. 4b inset). Thus, the presence of hydroxyapatite in the absence of

soluble phosphorus produced a significantly increased growth for Burkholderia sp. 205,

for the unknown strain 260 and, to a lesser, non-significant level, for Curtobacterium sp.

168 (Fig.5; Table 2).

Discussion

The PSB we isolated from ECM fungi were able to rapidly dissolve fluorapatite.

The enrichment in liquid medium yielded the most competitive strains for growth in

defined medium whose sole source of phosphorus was fluorapatite ore. In contrast, 1 See supplementary figure 1.

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enrichment on solid medium better expressed the diversity of species able to grow in

these conditions. However, the low diversity of bacterial strains obtained either by

isolation on solid media or by enrichment in liquid medium does not match the

complexity of bacterial communities typically found in soils (Sharma et al. 2005). The

PSB community potentially sensitive to culture-dependent isolation is also limited

because it comes from the immediate environment of the hyphae of one ECM fungus,

Wilcoxina sp. (Scheublin et al. 2010). The use of a selective medium and the removal of

bacterial strains that did not produce clarification halos also limited the isolation of

strains able to solubilize phosphates. We further limited our estimation of the

taxonomical diversity of isolated PSB strains by restricting 16S rRNA-based

identification to the strains that were successful in dissolving fluorapatite under in vitro

conditions only.

Our results confirm the presence of PSB in the hyphosphere of Wilcoxina sp., a

dominant and abundant ectomycorrhizal fungus in P. glauca stands (Sokolski et al.,

unpublished results). Obviously, the role of phosphate solubilization in boreal forests is

not restricted to the three genera isolated in this study (Uroz and Frey-Klett 2011).

Moreover, the strains found to be the most effective for in vitro phosphate solubilization

might display a lessened weathering activity when grown in hyphospheric communities

maintained in microcosms (Calvaruso et al. 2006). A semi-quantitative estimation of

biomass under natural conditions of the Burkholderia sp. isolated in this study could be

established by quantifying the fraction of bacterial DNA they represent within

hyphospheric communities.

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We observed that the bacterial strains capable of dissolving fluorapatite were

fewer than those that were capable of dissolving TCP. This variation in the PSB

community appears to be modulated by the lower reactivity of apatites characterized by

the presence of a crystal lattice that is more resistant to bacterial chemical attack

compared with precipitated amorphous phosphates such as TCP (Zhao et al. 2011). Out

of 203 TCP-solubilizing strains, 98 dissolved hydroxyapatite and 27 succeeded in

dissolving fluorapatite ore. Fluorapatite is the most resistant type of apatite to weathering

(Bashan et al. 2012).

Ultrastructural observations of fluroapatite weathering by Burkholderia sp. 205

support that biotic factors contributing to mineral weathering could achieve impressive

rates; Burkholderia sp. 205 growing in a static drop of NBRIP medium succeeded in

corroding the edges of fluorapatite crystals within six hours. When this same fluorapatite

was incubated under agitation in a Burkholderia sp. 205 suspension of NBRIP medium,

less than 1 day was sufficient to observe the dissolution of whole crystals. However,

complete dissolution in 22 h was observed on fluorapatite with a fluorine content of 1%

per mass. When the experiment was repeated for 23 h with fluorapatite containing 4%

fluorine, we only observed partial dissolution. The fluorine content therefore appears to

be a determining factor in the availability of phosphorus from fluorapatite crystals when

the time scale remains substantially the same. The first signs of weathering were

observed on crystal edges. This is due to the energy of lattice edges being greater than

that of flat surfaces; edges are thus the most reactive regions of particles (G. L'Espérance

(personal communication, 2015)).

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Whereas some ECM fungi were ineffective at mineral weathering when grown

axenically in vitro, they are still abundant in nature (Calvaruso et al. 2010). This apparent

contradiction could be explained by an ecological role that is possibly largely limited to

the absorption and transport of nutrients. Those ECM fungi receive carbon from host

photosynthesis, which they exude in the immediate vicinity of minerals containing useful

elements for plant growth (Paris et al. 1996). This release supports the activity of bacteria

possessing mineral weathering capabilities. In return, the fungus absorbs minerals and

transfers them to its host. The model ECM fungus L. bicolor alone is ineffective in

apatite weathering but, in nature, this ECM fungus is associated with effective apatite

weathering bacteria (Frey-Klett 2005; Uroz et al. 2007) and has a tendency to increase

plants’ Mg and K budgets in microcosm experiments when co-inoculated with efficient

biotite weathering bacteria (Koele et al. 2009). Among the Glomeromycota, several

bacterial species preferentially attach to active fungal hyphae (Toljander et al. 2005;

Taktek et al. 2015). This situation also occurs in Basidiomycetes. For example, the

apatite weathering bacterium Burkholderia terrae migrates on the hyphae of Lyophyllum

sp. and carries other bacterial species (Warmink et al. 2010). This ability to assist the

comigration of strains unable to achieve it on their own is widespread among the genus

Burkholderia (Nazir et al. 2012). In light of our isolation of strains from the hyphosphere

of Wilcoxina sp., the ubiquity of the Burkholderia genus in forest soils (Lepleux et al.

2012) has been confirmed as encompassing the boreal forest.

Our study innovates by demonstrating the non-specificity of the fungal host for

Burkholderia sp. 205, which hails from the hyphosphere of an Ascomycete (Wilcoxina

sp.) and then associates with a Basidiomycete (L. bicolor). The fungal host exuded

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substances necessary for the growth of bacteria and their hydroxyapatite solubilizing

activity, providing a source of phosphorus available to L. bicolor. Bacteria developed and

completed their alleged ecological function as they migrated on the hyphae of their

fungal host. This comigration undoubtedly facilitates the colonization of large amounts of

substrates by Burkholderia sp. 205 due to the exploration capacity of the fungus host

which greatly exceeds that of the bacterium.

In conclusion, efficient PSB were isolated from the ECM ascomycete Wilcoxina

sp., hosted by P. glauca, and identified as belonging to the Burkholderia, Curtobacterium

and Leifsonia genera. Our study was essentially observational and qualitative in nature

though. For example, our observations would have been strengthened by assessing the

number of bacteria in the distal portion of the plate by extracting the contents,

homogenizing and preparing dilution series for plate counts, measuring P contents within

the fungal hyphae, the supernatant of the well or the microbial biomass. Nevertheless, our

ultrastructural observations demonstrated rapid in vitro fluorapatite solubilization by

Burkholderia sp. 205, and the close association between PSB and ECM fungi was

validated in monoxenic conditions. Although our results have neither been reproduced

with L. bicolor in symbiosis nor under field conditions, and are thus not to be

generalized, they suggest that the efficiency of ECM symbiosis in acquiring P from soil

minerals relies on hyphospheric PSB.

Acknowledgements

We thank Philippe Plamondon (Centre for Characterization and Microscopy of Materials,

(CM)²), Serge Sokolski and Franck Stefani for the quality of their technical work as well

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as Isabelle Lamarre for text editing. We are thankful for the advice provided by Salma

Taktek and Jean-Guy Catford, and grateful for the help of interns Gwenola Plougoulen

and Mathieu Paradis. Special thanks also extended to Armand Séguin, two anonymous

reviewers and the Associate Editor, whose relevant comments served to improve a

preliminary version of this manuscript. This study was funded by the Fonds de recherche

du Québec – Nature et technologies.

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Tables

Table 1. Number of bacterial strains grown on amended solid media successful at

dissolving tricalcium phosphate (TCP), hydroxyapatite and fluorapatite.

Amendment Subculture

Nb.

Colonies

TCP - 455

TCP 1 428

TCP 2 203

TCP 3 140

Hydroxyapatite - 98

Fluorapatite (< 45µm) - 27

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Table 2. A priori orthogonal contrasts comparing colony diameters of Burkholderia sp.

strain 205, unknown species strain 260, and Curtobacterium sp. strain 168 growing in the

presence of L. bicolor in the distal compartment containing various sources of

phosphorus (soluble, insoluble, or both).

Phosphorus source Strain P

Soluble P vs. Hydroxyapatite

205 < 0.0001

260 < 0.0001

168 0.0691

Soluble P + hydroxyapatite vs.

Hydroxyapatite

205 < 0.0001

260 < 0.0001

168 0.0143

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Figure captions

Fig. 1. Experimental set-up of dual compartment culture media driving L. bicolor to

explore both compartments in order to obtain all the essential nutrients for its growth.

Proximal compartment: agar medium without phosphates (P), inoculum L. bicolor

represented by grey circles. Distal compartment: agar with either i) soluble phosphorus

(soluble P), ii) hydroxyapatite (HA), or iii) soluble phosphorus + hydroxyapatite (soluble

P + HA). Bacterial inocula represented by three black dots: strains 168, 205 and 260.

Fig. 2. Phylogenic tree illustrating the relationship between the PSB strains associated

with Wilcoxina sp. that were isolated on selective solid and liquid media and that are

capable of solubilizing fluorapatite.

Fig. 3. SEM images of fluorapatite crystals. (a) Virgin fluorapatite crystal with sharp

edges. (b) Edge of a fluorapatite crystal after 6 h of incubation in the presence of

Burkholderia sp. strain 205. Edges are corroded and the etching pattern does not follow

crystalline axes. (c) and (d) Fluorapatite crystal before (c) and after (d) incubation by

immersion for 22 h in the presence of Burkholderia sp. strain 205. In image d, the

remnants of the particle still present on the carbon tape are complex oxides. (e) and (f)

Fluorapatite crystals after immersion for 23 h in the presence of Burkholderia sp. strain

205. Those crystals were part of an ore containing 4% fluorine. The crystals are partially

weathered, and weathering patterns follow crystalline axes.

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Fig. 4. Reconstitution of the association between L. bicolor and Burkholderia sp. strain

205. Arrows indicate where the bacterial inoculum was deposited. (a) Laccaria bicolor in

the presence of Burkholderia sp. strain 205 in the distal compartment with the soluble

P+hydroxyapatite treatment. Inset: the hyphae of L. bicolor devoid of PSB. (b) Laccaria

bicolor in the presence of Burkholderia sp. strain 205 on the distal compartment of the

hydroxyapatite treatment. Inset: the hyphae of L. bicolor covered with PSB.

Fig. 5. Mean diameter of colonies of Burkholderia sp. strain 205, unknown species strain

260 and Curtobacterium sp. strain 168 growing in the presence of L. bicolor in the distal

compartment containing sources of phosphorus, either soluble (KH2PO4), insoluble

(hydroxyapatite), or both simultaneously. Error bars represent standard deviation.

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Figure 1 Experimental set-up of dual compartment culture media driving L. bicolor to explore both compartments in order to obtain all the essential nutrients for its growth. Proximal compartment: agar

medium without phosphates (P), inoculum L. bicolor represented by grey circles. Distal compartment: agar with either i) soluble phosphorus (soluble P), ii) hydroxyapatite (HA), or iii) soluble phosphorus +

hydroxyapatite (soluble P + HA). Bacterial inocula represented by three black dots: strains 168, 205 and 260. (Fig. 1)

123x129mm (300 x 300 DPI)

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Figure 2 Phylogenic tree illustrating the relationship between the PSB strains associated with Wilcoxina sp. that were isolated on selective solid and liquid media and that are capable of solubilizing fluorapatite.

(Fig. 2) 247x158mm (300 x 300 DPI)

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Figure 3 SEM images of fluorapatite crystals. (a) Virgin fluorapatite crystal with sharp edges. (b) Edge of a fluorapatite crystal after 6 h of incubation in the presence of Burkholderia sp. strain 205. Edges are corroded and the etching pattern does not follow crystalline axes. (c) and (d) Fluorapatite crystal before (c) and after

(d) incubation by immersion for 22 h in the presence of Burkholderia sp. strain 205. In image d, the remnants of the particle still present on the carbon tape are complex oxides. (e) and (f) Fluorapatite crystals after immersion for 23 h in the presence of Burkholderia sp. strain 205. Those crystals were part of an ore

containing 4% fluorine. The crystals are partially weathered, and weathering patterns follow crystalline axes. Fig. 3

338x406mm (300 x 300 DPI)

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Figure 4 Reconstitution of the association between L. bicolor and Burkholderia sp. strain 205. Arrows indicate where the bacterial inoculum was deposited. (a) Laccaria bicolor in the presence of Burkholderia sp. strain 205 in the distal compartment with the soluble P+hydroxyapatite treatment. Inset: the hyphae of L.

bicolor devoid of PSB. (b) Laccaria bicolor in the presence of Burkholderia sp. strain 205 on the distal compartment of the hydroxyapatite treatment. Inset: the hyphae of L. bicolor covered with PSB.

Fig. 4 398x203mm (300 x 300 DPI)

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Figure 5 Mean diameter of colonies of Burkholderia sp. strain 205, unknown species strain 260 and Curtobacterium sp. strain 168 growing in the presence of L. bicolor in the distal compartment containing sources of phosphorus, either soluble (KH2PO4), insoluble (hydroxyapatite), or both simultaneously. Error

bars represent standard deviation. Fig. 5

287x153mm (300 x 300 DPI)

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