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Different endophyte communities colonize buds of sprouts compared to

mature trees of mountain birch recovered from moth herbivory

Running title: Bud microbiome of mountain birch after herbivory

Pirjo Koivusaari 1, Johanna Pohjanen 1, Piippa R. Wäli1, Saija H.K. Ahonen 1, Karita

Saravesi1, Anna Mari Markkola, Kaisa Haapala 1, Marko Suokas 1, Janne J. Koskimäki

1, Mysore V. Tejesvi 1, 2, Anna Maria Pirttilä 1*

1 Ecology and Genetics, University of Oulu, PO Box 3000, FIN-90014 Oulu, Finland

2 Chain Antimicrobials, Teknologiantie 2, FIN-90590 Oulu, Finland

*Corresponding author.

Anna Maria Pirttilä

Ecology and Genetics

University of Oulu

POB 3000

90014 Oulu, Finland

Key words: arctic, bacteria, diversity, Betula, next-generation sequencing, shoot

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Abstract

Plant meristems were earlier thought to be sterile. Today meristem-associated shoot

endophytes are mainly reported as contaminants from plant tissue cultures, the number

of observed species being very low. However, the few strains characterized have the

capacity for infecting host cells and affecting plant growth and development. Here we

studied the communities of endophytic bacteria in the buds of mountain birch exposed

to winter moth herbivory, to identify differences between sprouts and branches of

mature birch trees. Mountain birch of the high subarctic is cyclically exposed to winter

moth and produces sprouts to generate new trees as a survival mechanism. The majority

(54 %) of operational taxonomic units (OTU) belonged to Xanthomonadaceae and

Pseudomonales of Proteobacteria. Most of the observed species were classified as

Xanthomonas (28 %). Sprout buds had the highest diversity, containing approximately

three times more species, and significantly more (43 %) Pseudomonas species than the

mature trees (14 %) (Figure 4). Our results demonstrate that endophytic communities

of buds are richer than previously thought. We suggest that the meristem-associated

endophytes should be studied further for a possible role in sprouting and aiding

regeneration of trees.

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Introduction

Endophytic bacteria that live in plant tissues have most often been studied in the roots

of crop plants where they colonize the apoplast or dead cells (Hardoim et al., 2015).

However, shoot-associated endophyte communities significantly differ from the root-

associated communities by colonization and diversity (Moore et al., 2006, Mano et

al., 2006, 2007, Izumi et al., 2008, Yrjälä et al., 2010, Compant et al., 2011).

Therefore, endophytic bacteria that colonize plant shoots can have different traits than

root endophytes considering host growth and development.

Plant shoot tips or buds were long thought to be sterile (Pierik, 1997). Even today

they are rarely studied for endophytes, apart from the occurrence of microbes in plant

tissue cultures (Reed et al., 1998, Kamoun et al., 1998, Laukkanen et al., 2000, Pirttilä

et al., 2000, Van Aken et al., 2004, Thomas et al., 2007, Ulrich et al., 2008,

Quambusch et al., 2014). The endosymbiont Methylobacterium extorquens

DSM13060 isolated from bud meristems of Scots pine is the best studied so far. This

bacterium can induce growth and development of host seedlings to the same level as

mycorrhizal fungi (Pohjanen et al., 2014). Unlike many bacterial root endophytes, the

meristem-dwelling endophytes of Scots pine do not induce plant growth through

hormones, but other compounds, such as adenine ribosides, may be responsible for

the growth effect (Pirttilä et al., 2004). Furthermore, the meristem-associated

endosymbionts of Scots pine, M. extorquens DSM13060 and Pseudomonas synxantha

DSM13080, colonize host cell interior where they aggregate around the nucleus

(Pirttilä et al., 2000, Koskimäki et al., 2015). The M. extorquens DSM13060 infection

is aided by methyl-esterified 3-hydroxybutyrate oligomers that have antioxidant

activity (Koskimäki et al., 2016). M. extorquens genome encodes several so-called

nucleomodulins, eukaryotic transcription factors that can interfere with host

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transcription and metabolism (Koskimäki et al., 2015). All this data indicate that

meristem-associated endophytes can have an overlooked role in the growth and

development of their hosts. Therefore, these endophytes deserve detailed studies on

their diversity and distribution in plants.

Mountain birch (Betula pubescens ssp. czerepanovii ((N. I. Orlova) Hämet-Ahti) is a

tree comprising the treeline forest as the single species at the subarctic of

Fennoscandia. Besides constant herbivory by e.g. reindeer, mountain birch forests are

regularly exposed to massive outbreaks of lepidopteran herbivores, autumnal moth

(Epirrita autumnata L.) and winter moth (Operophtera brumata L.) (Tenow et al.,

2007). During repeated outbreaks, the larvae of the autumnal moth that feed on

mountain birch leaves can permanently kill birch forest within large areas, whereas

winter moth damage has earlier been restricted to river valleys and seashores (Tenow

1972, Jepsen et al., 2008). However, in 2008, the winter moth spread to exceptionally

large areas of northernmost parts of Finland, destroying ~400 km2 of mountain birch

forest, likely due to climate warming (Jepsen et al., 2008, Kaukonen et al., 2013).

Some birch trees can survive the attacks by producing defense compounds

unfavorable for the moth larvae, by compensating the loss, and by producing new

leaves after the feeding (Tenow et al., 2005, Schoonhoven et al., 2006, Tømmervik et

al., 2005, Wielgolaski 2005). Due to high herbivory pressure and extreme growth

conditions, the main mechanism of propagation and survival of mountain birch after

moth herbivory, and in general, is sprouting (Karlsson et al., 2004). Whereas fungal

endophytes and epiphytes of mountain birch leaves and their role in herbivory

resistance have been extensively studied (e.g. Helander et al., 1993, Lappalainen and

Helander 1997, Ahlholm et al., 2002), bacterial endophytes have not been considered

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in relation to herbivory. To characterize the diversity and niche occupation of

meristem-associated endophytes, we analyzed by next-generation sequencing (NGS)

the bacterial communities of buds of mountain birch after winter moth herbivory.

Materials and Methods

Experimental setup

Material used in the study was mountain birch buds collected from subarctic

Kaldoaivi Wilderness Area located in Utsjoki, Finland (Mieraslompolo 7 724° 056’

N, 510° 527’ E; 255 m) in August 2012 (Figure 1). Samples were collected from an

area where altogether 400 km2 of mountain birch forest had suffered from severe

winter moth attack (Kaukonen et al., 2013, Saravesi et al., 2015). Samples were taken

from five tree groups (OP5, OP7, OP8, OP10, OP13), comprising susceptible

(partially recovered) and tolerant (totally recovered controls) mature mountain birches

and their sprouts (Figure 1, Table I). Three branch samples of 5-15 cm in length were

collected from each tree. In addition, sprout samples (max. three per tree) were

collected from all groups expressing sprouting and having live sprouts (Figure 1,

Table I). The initial sample size was 45 (one bud per branch or sprout, three branches

or sprouts per tree), and after collection the samples were transported at +4 °C to

laboratory, where buds were separated and stored frozen (-80 °C) until used for DNA

isolation. Each bud was treated individually in the analysis. During handling in the

laboratory, some buds were dismissed and the resulting final sample size in the

analyses was 33 buds (Table I).

DNA Extraction

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Before DNA extraction, the bud samples were surface sterilized to exclude epiphytes.

Samples were kept in 4 % calcium hypochlorite for 15 minutes and rinsed three times

with sterile water for ten minutes in a laminar flow hood. After the surface

sterilization, samples were ground in liquid nitrogen. DNA was extracted from 40 mg

of frozen plant material by using DNeasy Plant Mini Kit (Qiagen, USA). Before using

the kit, two additional steps were done. First, 300 µl of lysozyme in TE-buffer (50

mg/ml) was added, and the samples were incubated for 15 minutes at room

temperature. Then, 30 µl of 10 % SDS and 10 µl of proteinase K (20 mg/ml) were

added, and samples were incubated at 50 °C for 15 minutes. After this, the kit

protocol was followed according to manufacturer’s instructions from step 1 (addition

of AP1 buffer) onwards. The pure DNA was eluted with 50 µl of molecular grade

water and stored at -20 °C until used. NanoDrop 1000 (Thermo Scientific,

Wilmington, USA) was used to quantify the DNA.

Designing of primers specific for endophytic bacteria

For testing specificity of primers, DNA was isolated as described above from leaves

of downy birch (Betula pubescens Ehrh.) collected from the Biodiversity Unit, Oulu,

Finland (65° N; 25°30’ E; 5 m), which comprises botanical greenhouse gardens and

field collections (http://www.oulu.fi/biodiversityunit/), and DNA of

Methylobacterium extorquens DSM13060 was isolated as described by Koskimäki et

al., (2015).

The 16S rDNA genes of Methylobacterium extorquens DSM13060 (AF267912),

Amycolicicoccus subflavus DQS3-9A1 (NR_116057), Lactobacillus rhamnosus NT10

(JN813101), Olivibacter ginsengisoli Gsoil 060 (NR_041504), Pinus taeda

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chloroplast (KC427273), and Abies homolepis mitochondria (AB029360) were

aligned in ClustalW. The alignment was manually scanned for short (~20 nt)

sequences similar between the bacterial and different from chloroplast and

mitochondria sequences. The selected potential primers were tested for hairpin, self-

dimer and hetero-dimer formation in OligoAnalyzer Tool 3.1 (IDTDNA) and

analyzed by programs Probe Match (Cole et al., 2014) in RDP and BLASTN (Benson

et al., 2013) in Genbank for similarity with bacteria, chloroplast and mitochondrial

sequences. The primers selected for Ion Torrent sequencing were F22 (5-AGC AGC

CGC GGT AAT ACG W-3; Escherichia coli 16S gene positions 518-537, Genbank

accession no. J01859.1) and R21 (5-TAA TCC TGT TYG CTC CCC AC-3; E. coli

positions 769-789).

Gradient PCR

Optimal PCR conditions for microbial DNA amplification with minimal amplified

plant mitochondrial or chloroplast DNA were then tested by gradient PCR (Veriti®

96-Well Thermal Cycler, Thermo Scientific, Finland), i.e. that the primers would only

bind to microbial DNA, and not to chloroplast, mitochondrial or birch genome DNA.

DNA of downy birch and Methylobacterium extorquens DSM13060 were used as the

control template. Theoretical melting temperatures were 59.3 °C for F22 and 55.6 °C

for R21, and the temperature gradient tested was from 50.7 °C to 62.4 °C. The

following cycling conditions were used in gradient PCR: 4 min; 27 cycles of 95 °C,

15 sec; 55 °C or 57 °C (gradient ± 5 °C), 15 sec; 72 °C, 30 sec after an initial

denaturation of 95 °C. PCR reactions were performed in 50-µl reactions, each

containing 1x Phusion GC buffer, 10 µM of forward and reverse primers, 0.2 mM

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dNTPs, 50 ng of DNA template and 0.5 U of Phusion High-Fidelity DNA Polymerase

(Thermo Scientific, Espoo, Finland).

Amplification of bacterial rRNA genes by Ion Torrent

A fragment of the 16S small-subunit ribosomal gene was amplified with the primers

F22 and R21. The F22 primer contained an Ion Torrent sequencing adapter sequence

A (Lifescience Technologies, USA), 10-bp unique barcode sequence and one

nucleotide linker. The R21 primer contained an Ion Torrent adapter trP1 sequence.

The amplification was performed in 50-µl reactions, each containing 1x Phusion GC

buffer, 0.4 µM of forward and reverse primers, 0.2 mM dNTPs, 0.5 U of Phusion

High-Fidelity DNA Polymerase, 3 % dimethyl sulfoxide (DMSO), 0.2 mg/ml of

bovine serum albumin (BSA), and 20 ng of genomic community DNA of each

mountain birch bud sample as the template. For sequencing, each individual PCR

product was purified with Ampure XP reagent (Agencourt Bioscience, CA, USA),

concentration was measured with Quant-IT PicoGreen reagent (ThermoFisher

Scientific), and size and purity were determined with MultiNA capillary electrophosis

system (Shimadzu, Kyoto, Japan). Samples were then pooled in equivalent amounts,

the resulting pool was re-purified with AMPure XP reagent, and the final DNA

concentration was measured with Quant-IT PicoGreen reagent. Ion Torrent PGM

sequencing was performed at Biocenter Oulu Sequencing Center (Univ. Oulu, Oulu,

Finland) using 400 bp chemistry and 314 v2 kit.

Bioinformatics and statistical analyses

The Ion Torrent sequences were processed and analyzed using the state-of-the-art

procedures of QIIME (Caporaso et al., 2010). Sequences were quality filtered with the

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following adjustments: sequences shorter and longer than 200-600 bp length and

sequences with a quality score under 20 were removed. UCLUST (Edgar 2010) was

used for the de novo operational taxonomic unit (OTU) picking with a sequence

similarity value of 97%, and for taxonomic assignments. Chimeric sequences were

identified using blast fragments approach (Altschul et al., 1990) and removed from the

data. GreenGenes (McDonald et al., 2012, Werner et al., 2012) was used as a reference

database. Alpha diversity indices (Chao1, Shannon, Simpson) were calculated in

QIIME and statistical significances were tested with the Mann-Whitney U test in SPSS

program between tree groups, and between tolerant and susceptible trees, and between

mature trees and sprouts.

Isolation and identification of endophytic bacteria from buds of mature mountain

birch branches

An additional sampling of 42 buds was performed on randomly selected three

mountain birches from the same forest where the initial sampling took place, with the

aim of isolating endophytic Pseudomonas sp. for further testing. The samples were

collected as branches of 5-15 cm, as the selected trees had no sprouts. After

collection, the samples were transported at +4 °C to the laboratory, where buds were

separated from branches and immediately processed further. The buds were surface

sterilized with 4 % calcium hypochlorite for 15 min and rinsed three times with sterile

water. The buds were individually ground with mortar and pestle in 100 µl sterile

distilled water. The mixture of ground tissue and water was transferred with a pipette

onto LB-medium and grown for up to eight months. Emerging microbial growth was

isolated and tentatively classified under light microscope as filamentous fungi, yeasts,

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or bacteria. Bacterial isolates were transferred onto a new LB plate and further

identified by sequencing the internal transcribed spacer region (ITS) of ribosomal

RNA genes.

Briefly, a few bacterial cells were taken from a pure fresh culture in a PCR reaction

mixture of 0.4 µM primers (FGPS1490-72: TGCGGCTGGATCCCCTCCTT,

FGPL132-38: CCGGGTTTCCCCATTCGG) (Normand et al., 1996), 1x Phusion GC

buffer, 0.2 mM dNTPs, 0.5 U of Phusion High-Fidelity DNA Polymerase and water up

to 20 µl. The following cycling conditions were used in PCR: 30 cycles of 98 °C, 10

sec; 65 °C, 15 sec; 72 °C, 30 sec after an initial denaturation of 98 °C for 3 min. The

resulting PCR products were sequenced according to the manufacturer’s instructions

(Abi Prism BigDye Terminator Cycle Sequencing Kit and AbiPrism 377 DNA

Sequencer, PerkinElmer).

Results

Specificity of sequencing primers

The 16S ribosomal RNA gene was scanned thoroughly using alignments of species

from various bacterial classes and plant mitochondria and chloroplast, to find short

20-bp regions that would be most specific to bacteria and not mitochondria and

chloroplast. The primers were designed to amplify a 200-300-nucleotide long region

suitable for sequencing with the Ion Torrent Sequencer, and to contain a

hypervariable region of the ribosomal 16S RNA gene. As a result, the primers F22

and R21 were identified as the best primer candidates for Ion Torrent sequencing,

generating a 271-bp long PCR product from the hypervariable region v4 of bacterial

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16S rRNA gene. When the specificity of the primers F22 and R21 was tested by

gradient PCR, 2-7 bands were amplified from downy birch DNA at temperatures of

50.7-62.4 °C (Figure 2). In the temperature of 62.4 °C, only one band was amplified

of the same size as the PCR product from the genomic DNA of Methylobacterium

extorquens DSM13060 (Figure 2). Therefore, the most specific amplification was

obtained at the annealing temperature of 62.4 °C and used in the following PCR

reactions.

Total microbiome of mountain birch buds

From the original sample collection, 12 bud samples were dismissed for various

reasons, and therefore the final number of sequenced bud samples was 33 (Table I).

Specifically, 26 mature tree bud samples, of which 14 originated from susceptible

trees and 12 from tolerant trees, and 7 sprout bud samples were successfully analyzed

(Table I). Among all sequences analyzed, 16 % were mitochondrial and 6 %

chloroplast, which were removed from the dataset. A total of 182 operational

taxonomy units (OTUs) were identified from the complete dataset. The majority of

the OTUs (84 %) belonged to the phylum Proteobacteria. In addition, OTUs

belonging to phyla Firmicutes (9 %), Bacterioidetes (4 %) and Actinomycetales (2.5

%) were identified among the microbiome of mountain birch buds (Figure 3). At the

family level, the majority of the OTUs were identified into the families

Xanthomonadaceae (33 %) and Pseudomonales (21 %). The OTUs belonged mainly

to genera Pseudomonas (21 %), Xanthomonas (28 %) and Burkholderia (10 %),

Stenotrophomonas (5 %), Achromobacter (4 %), Lactococcus (4 %),

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Lachnospiraceae (4 %), Cloacibacterium (3 %), Sphingomonas (3 %), and

Curvibacter (3 %).

In the complete dataset, the Shannon and Simpson indices varied in the samples

between 0.65 - 4.49 and 0.27 - 0.94, respectively (Table II), indicating the overall

varying bacterial diversity in the community. We found no statistically significant

differences in the bacterial diversity measured by Shannon, Simpson and Chao-1

indices between susceptible and tolerant trees, or between mature trees and sprouts

(Table III).

The microbiome of mature tree buds

The majority of the OTUs in the bud microbiome of mature trees belonged to the

family Xanthomonadaceae (33 %). In addition, members of Burkholderiales (11 %),

Pseudomonadeacae (14 %), Firmicutes (9 %), Bacteroidetes (5 %) and

Actinomycetales (3 %) were present (Figure 4a). There were no statistically

significant differences in the microbiomes between the tree groups, or between

tolerant and susceptible trees. OTUs belonging to the genera Curvibacter (3 %),

Tepidimonas (3 %), Achromobacter (5 %), Lactococcus (5 %) and Cloacibacterium

(4 %) were more abundant, although not statistically significantly, in the buds of

mature trees than sprouts.

The microbiome of sprout buds

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The highest diversity of endophytes was observed in the buds collected from sprouts.

On average, three times more OTUs were observed in sprout buds than in mature tree

buds. The microbiome of sprout buds consisted mainly of OTUs belonging to

Pseudomonacaeae (43 %), and members of Xanthomonadaceae (10 %),

Enterobacteriales (8 %) and Burkholderiales (9 %) were the minority (Figure 4b).

Overall, the sprout bud microbiome had fewer OTUs belonging to phyla Firmicutes

(3 %), Bacteroidetes (3 %) and Actinomycetales (1 %), than that of mature trees.

Sprout buds had more members belonging to Erwinia (5 %), Ralstonia (3 %),

Sphingomonas (7 %), Rhizobium (3 %) and Methylobacterium (2 %) than mature tree

buds. However, these differences were not statistically significant. The only

statistically significant difference (p=0.011) was the relative abundance of

Pseudomonas species (43 %) in buds collected from sprouts than those from mature

trees (14 %).

Cultivable endophytic bacteria of mountain birch buds

Altogether 15 microbial strains were isolated from 12 mountain birch buds among the

total of 42 buds examined (28.6 % isolation frequency). Of the strains isolated, nine

were filamentous fungi, two yeasts, and four bacterial strains, but due to the objective

of isolating Pseudomonas spp., the filamentous fungi and yeasts were not studied

further. Two bacterial strains had identical sequences of internal transcribed spacer

(ITS) region of ribosomal RNA gene and were similar with Micrococcus luteus sp.

(Genbank No. CP007437.1). Two other strains also had identical ITS sequences and

were classified as an unknown strain with the closest match of 27 % for

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Corynebacterium deserti strain (CP009220.1) in BLAST database search. One M.

luteus strain and one Corynebacterium-like strain originated from the same bud.

Discussion

Overall, our knowledge on microbial diversity in plant buds is scarce. Earlier, it was

thought that the bud tissues are the most sterile tissues of the plant (Pierik 1997). The

first reports on endophytes in plant buds have demonstrated zero to few species

present, obtained by isolation from bud-derived tissue cultures (Reed et al., 1998,

Kamoun et al., 1998, Laukkanen et al., 2000, Pirttilä et al., 2000, Van Aken et al.,

2004). For example, a few strains of endophytic bacteria were isolated from the tissue

cultures of hazelnut (Reed et al., 1998) and a Pseudomonas sp. was identified from

micropropagated sour cherry plantlets (Kamoun et al., 1998). We have earlier isolated

Methylobacterium extorquens, Pseudomonas synxantha and Mycobacterium sp. from

buds or bud-derived tissue cultures of Scots pine (Laukkanen et al., 2000, Pirttilä et

al., 2000) and localized these bacteria as endophytes in the meristematic tissues of

pine buds (Pirttilä et al. 2000, 2005). An important discovery was made when an

AFLP-cDNA library was sequenced from buds of poplar (Rohde et al. 2007).

Bacterial sequences were identified in the library, indicating an active community

participating in dormancy of the poplar buds. However, the libraries were made from

non-sterilized samples (Rohde et al. 2007) and therefore could also represent

epiphytic activity. Recently, Miliute et al. (2016) isolated altogether 38 bacterial

strains from an undefined number of surface-sterilized apple buds, which suggested

that buds may harbor a richer community of endophytic bacteria than previously

thought.

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In the present study, the number of endophytes was at the same level in the buds of

mountain birch, as is found in other plant tissues by next-generation sequencing. For

example, root segments of Populus deltoides contained 83 ± 78 endophytic OTUs

identified by pyrosequencing (Gottel et al., 2011). Our study demonstrates that as

many as 182 OTUs can be identified in a bud endophytic microbiome. Although

surface sterilization protocols do not eliminate potential epiphytic DNA remains, this

is significantly more than expected, considering the 0-3 isolates typically obtained. In

our study, the isolation technique resulted in two bacterial strains obtained from 42

bud samples. In contrast, bacterial endophytes isolated from root, or stem, typically

reach higher numbers. For example, elm trees can have cultivable bacteria between

39-62 haplotypes in stems and roots (Mocali et al., 2003). This suggests that plants

harbor mainly unculturable endophytes in the bud tissues.

It is not possible to conclude, based on this study, whether high diversity of bacteria

in buds is beneficial, harmful, or neutral for the host plant. The fact that sprouts had

significantly higher diversity of microbiome in the buds than adult trees is regardless

interesting in the light of existing research data. For example, the meristem-associated

endophyte of Scots pine, M. extorquens DSM13060, can induce growth and health of

the host through production of adenine ribosides and methyl-esterified 3-

hydroxybutyrate oligomers, and by directly affecting host metabolism (Pirttilä et al.,

2004, Koskimäki et al., 2015, Pohjanen et al., 2014, Koskimäki et al., 2016).

However, the role of bud endophytes of mountain birch in sprouting and growth

remains to be studied in the future.

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The bacterial microbiome of mountain birch has not been earlier studied. Izumi et al.,

(2008) analyzed the endophytes of mature silver birch (Betula pendula Roth) trees by

culturing and identifying the isolated strains by sequencing. They discovered Bacillus

spp. and Acinetobacter spp. as the most common endophytes in silver birch stem and

leaves, but neither of these were identified in mountain birch buds in our study.

Similarly, Izumi et al., (2008) reported none of the species in silver birch that we

found abundant in mountain birch buds, Xanthomonas, Pseudomonas, and

Burkholderia. This is not surprising, considering the completely different habitats and

environmental growth conditions, as well as different tissues of these two species

studied. Mountain birch is the main tree species present in the ecosystem of the

subarctic treeline, where it forms monocultures.

In our study, Pseudomonas spp. were significantly more abundant in sprout buds than

in buds of mature trees. After herbivory, the mature trees of mountain birch produce

more sprouts as a recovery mechanism (Karlsson et al., 2004). In general,

Pseudomonas spp. are often isolated as endophytes, and some strains have been

reported as plant growth promoting, P. synxantha, from buds of Scots pine (Pirttilä et

al., 2004), four Pseudomonas sp. isolates (Oa_2, O_16, D_7, Ga_1) from apple buds

(Miliute et al., 2016), and P. putida W619 from Populus trichocarpa × deltoides

(Taghavi et al., 2009). Because Pseudomonas spp. are normally rather easy to

cultivate on laboratory media, we attempted to isolate these bacteria from buds of

mountain birch for testing their potential growth effect on birch in vitro. However, no

Pseudomonas strains grew out from the bud tissues, instead we obtained a

Micrococcus sp. and a Corynebacterium-like strain. This means that the

Pseudomonas spp. present in mountain birch buds are probably unculturable, and

might have more or less intimate relationship with their hosts. We suggest that the

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Pseudomonas spp. of mountain birch buds should be studied further for a possible

role in sprouting and aiding regeneration of trees.

Acknowledgements

We would like to thank the Nature Conservation Societies of Oulu and Kuopio,

Finland. Gerhardus Lansink, Nelli Mikkola, Ruwanthi Tissera and Visa Kamppari are

gratefully acknowledged for technical help. We declare no conflict of interest or

relationship, financial or otherwise, that might be perceived as influencing an author’s

objectivity is considered a potential source of conflict of interest.

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Table I. Experimental setup describing the samples obtained from each tree group (OP).

Tree Group Tree status Branch buds Sprout buds

Tolerant 1 1

OP5 Susceptible 3 *

Tolerant 3 *

OP7 Susceptible 3 1

Tolerant 3 1

OP8 Susceptible 3 2

Tolerant 3 *

OP10 Susceptible 3 2

Tolerant 2 *

OP13 Susceptible 2 *

* No living sprouts present

Table II. Alpha diversity indices (Chao1, Shannon, Simpson) of the bacterial

communities in mountain birch bud samples from mature trees (susceptible or tolerant)

and their sprouts.

Tree group Tree status Simpson Shannon Chao1

OP5 Tolerant 0.80 2.45 7

OP7 Tolerant 0.84-0.88 2.75-3.42 14.5-

21.25

OP8 Tolerant 0.5-0.94 1.0-4.33 3.0-

24.67

OP10 Tolerant 0.67 1.58-1.79 6.0-7.0

OP13 Tolerant 0.61-0.81 1.45-2.96 3.0-

31.0

OP5 Susceptible 0.28-0.8 0.65-2.32 2.0-

15.0

OP7 Susceptible 0.64-0.81 1.87-2.52 6.5-

11.0

26

OP8 Susceptible 0.75-0.89 2.0-3.53 10.0-

27.0

OP10 Susceptible 0.55-0.56 1.37-2.17 4.0-

34.17

OP13 Susceptible 0.79-0.92

3.17-3.93

30.0-

49.0

OP5 Tolerant sprout 0.89 4.49 72.21

OP8 Tolerant sprout 0.56 1.37 4.0

OP7 Susceptible

sprout

0.85 3.66 58.20

OP8 Susceptible

sprout

0.56-0.91 1.37-3.74 4-35.17

OP10 Susceptible

sprout

0.90-0.93

3.84-4.69

32.0-

57.05

27

Table III. Statistical significances of the Simpson, Shannon and Chao1 indices of the

bacterial communities in mountain birch bud samples between tolerant and susceptible

trees, and mature trees and sprouts, tested by the Mann-Whitney U test in SPSS.

Simpson’s index Shannon’s index Chao1

Tree status Mann-

Whitney U

p-value Mann-Whitney U p-value Mann-

Whitney U

p-value

Tolerant ×

Susceptible

79 0.325 65 0.107 59.5 0.059

Mature ×

Sprout

61.5 0.438 76.5 0.980 47 0.135

28

Figure Legends

Figure 1. Schematic diagram of the experimental setup and example trees. a) Bud

samples were collected from branches and sprouts of five mountain birch tree groups

suffered from severe moth attack. Each tree group contained a tolerant (totally

recovered) and a susceptible (partially recovered) tree, and their sprouts, if present. (b-

d) Examples of mountain birch trees sampled after winter moth herbivory. b) Tolerant

birch tree from group OP5 which had leaves in the majority (>70 %) of all branches, c)

Susceptible birch tree from group OP13 which had re-emerged leaves in some (30-70

%) branches, d) Sprout of susceptible birch from group OP8.

Figure 2. Testing the specificity of primers F22 and R21 on bacterial ribosomal 16S

rRNA gene. The sample DNAs were genomic DNA of Methylobacterium extorquens

DSM13060 (lanes 10-14, 15-21) and downy birch DNA (lanes 2-9, 22-27). Lanes 1,

15, Mass Ruler (Fermentas). The products were separated on 1 % agarose gel stained

with ethidium bromide. Products are shown from gradient PCR reactions with

annealing temperatures of 50.7 °C (lanes 2, 10), 51.6 °C (lanes 3, 11), 52.7 °C (lane 4),

54.0 °C (lanes 5, 12), 55.4 °C (lanes 6, 13), 56.8 °C (lanes 7, 14), 57.0 °C (lane 8), 57.4

°C (lanes 9, 16), 58.8 °C (lanes 17, 22), 59.4 °C (lane 23) 60.1 °C (lanes 18, 24), 61.2

°C (lanes 19, 25), 62.0 °C (lanes 20, 26), 62.4 °C (lanes 21, 27).

29

Figure 3. The relative abundance of total microbiome of mountain birch buds (N=

33). The most dominant genera found were Xanthomonas (28 %), Pseudomonas (21

%), Burkholderia (10 %) and Stenotrophomonas (5 %).

Figure 4. The microbiomes of buds of mature trees (a) and sprouts (b). a) The

majority of the OTUs in the bud microbiome of mature trees belonged to the family

Xanthomonadaceae (33 %). b) The microbiome of sprout buds consisted mainly of

OTUs belonging to Pseudomonacaeae (43 %).