Antonie van Leeuwenhoek (2012) 102:677–687 DOI 10.1007/s10482-012-9766-3
ORIGINAL PAPER
Endophytic bacterial community living in roots of healthy and ‘Candidatus Phytoplasma mali’-infected apple (Malus domestica, Borkh.) trees Daniela Bulgari • Adem I. Bozkurt Paola Casati • Kadriye C ¸ ag˘layan • • Fabio Quaglino Piero A. Bianco
•
Received: 5 April 2012 / Accepted: 14 June 2012 / Published online: 30 June 2012 Ó Springer Science+Business Media B.V. 2012
Abstract ‘Candidatus Phytoplasma mali’, the causal agent of apple proliferation (AP) disease, is a quarantine pathogen controlled by chemical treatments against insect vectors and eradication of diseased plants. In accordance with the European Community guidelines, novel strategies should be developed for sustainable management of plant diseases by using resistance inducers (e.g. endophytes). A basic point for the success of this approach is the study of endophytic bacteria associated with plants. In the present work, endophytic bacteria living in healthy and ‘Ca. Phytoplasma mali’-infected apple trees were described by cultivation-dependent and independent methods. 16S rDNA sequence analysis showed the presence of the groups Proteobacteria, Acidobacteria, Bacteroidetes, Actinobacteria, Chlamydiae, and Firmicutes. In detail, library analyses underscored 24 and 17 operational taxonomic units (OTUs) in healthy and infected roots, respectively, with a dominance of Betaproteobacteria. Electronic supplementary material The online version of this article (doi:10.1007/s10482-012-9766-3) contains supplementary material, which is available to authorized users. D. Bulgari P. Casati F. Quaglino P. A. Bianco (&) Dipartimento di Scienze Agrarie e Ambientali – Produzione, Territorio, Agroenergia, Universita` degli Studi, via Celoria 2, 20133 Milan, Italy e-mail:
[email protected] A. I. Bozkurt K. C¸ag˘layan Plant Protection Department, Mustafa Kemal University, 31034 Antakya, Hatay, Turkey
Moreover, differences in OTUs number and in CFU/g suggested that phytoplasmas could modify the composition of endophytic bacterial communities associated with infected plants. Intriguingly, the combination of culturing methods and cloning analysis allowed the identification of endophytic bacteria (e.g. Bacillus, Pseudomonas, and Burkholderia) that have been reported as biocontrol agents. Future research will investigate the capability of these bacteria to control ‘Ca. Phytoplasma mali’ in order to develop sustainable approaches for managing AP. Keywords 16S rDNA Plant endophytes Apple proliferation Operational taxonomic units
Introduction Phytoplasmas are obligate parasitic symbionts of plants and insects belonging to the class Mollicutes that are transmitted by phloem-feeding insects (IRPCM Phytoplasma/Spiroplasma Working Team— Phytoplasma Taxonomy Group 2004). Apple proliferation (AP) is one of the most important phytoplasma diseases in Europe and it is associated with ‘Candidatus Phytoplasma mali’, a quarantine pathogen. AP induces a broad range of symptoms such as witches’brooms, rosettes, enlarged stipules, foliar reddening, growth suppression, and undersized fruits (Seemu¨ller and Schneider 2004). Because of its economic importance, scientific efforts are focused on the study of
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‘Ca. Phytoplasma mali’ characterization, molecular mechanisms of disease induction, transmission by insect vectors, and plant defense response induced by AP phytoplasma (Tedeschi and Alma 2006; Jarausch et al. 2007; Kube et al. 2008; Casati et al. 2011; Seemu¨ller et al. 2011). So far, due to the absence of AP resistant varieties, the management of AP mainly consists in insecticide treatment against the insect vectors and in the eradication of diseased plants. These treatments have a strong economic and environmental effect, representing a risk for both operators and final customers. Moreover, the new guidelines for the Common Agricultural Policy of the European Community (Dir 128/2009) required the use of sustainable agricultural practices with an eye on environmental safeguard. One of the most innovative solutions aims to substitute the insecticide treatment with biotic (e.g. endophytes) and abiotic resistance inducers able to trigger the natural plant defense responses. Recently, endophytes community associated with healthy and phytoplasma infected plants has been characterized (Bulgari et al. 2009; Martini et al. 2009) to find putative biocontrol agents. In nature, endophytic bacteria can promote plant growth by reducing the deleterious effects of plant pathogens through direct or indirect mechanisms. Bacteria can directly antagonize pathogens by competition for nutrients and production of allelochemicals and indirectly through the induction of systemic resistance (ISR) (Lugtenberg and Kamilova 2009). Endophytic bacteria have been isolated from a wide range of monocotyledonous and dicotyledonous plants (Ulrich et al. 2008; Brooks et al. 1994; Ferreira et al. 2008; Sun et al. 2008). The structure and composition of endophytic bacterial community can be influenced by various factors such as plant genotype, growth stage, management practices, and interaction with other organisms (Araujo et al. 2002; van Overbeek and van Elsas 2008; Sagaram et al. 2009; Hardoim et al. 2011). Recently, it was described that plant pathogens such as ‘Candidatus Liberibacter asiaticus’ and Flavescence dore´e associated phytoplasma can influence endophytic bacterial community composition (Trivedi et al. 2010; Bulgari et al. 2011). Interestingly, some bacterial strain isolated from healthy and ‘Candidatus Liberibacter asiaticus’ infected citrus roots showed the potential to suppress diseases (Trivedi et al. 2011). The employment of plant associated bacteria in plant protection is related to the understanding of bacteria-
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host interactions and the ability to formulate and spread the bacteria under field conditions (Hallmann et al. 1998). An important and basic point for the success of this approach is the study of endophytic bacteria associated with plants. In this work, we describe the endophytic bacterial community associated with healthy and AP phytoplasma-infected apple trees in order to obtain basic information for future studies focused on developing sustainable control strategies.
Materials and methods Plant material, DNA extraction and ‘Ca. Phytoplasma mali’ detection Ten apple (Malus domestica, Borkh.) plants were selected during field survey conducted in 2010 in Minoprio Foundation, Como, Italy; five of them were asymptomatic and five showed typical symptoms of AP disease. In April 2011, apple roots were collected from each of those plants. Apple roots were washed with tap water and sterilized by treating with ethanol 70 % for 3 min, sodium hypochlorite 2 % for 5 min, and ethanol 70 % for 30 s, and fivefold washing with sterile water. Total DNA was extracted from 5 g of roots with the method describe by Doyle and Doyle (1990), with some modifications. Extracted DNA was quantified by spectrophotometer (NanoDrop, Thermo Scientific, USA) and used as template for ‘Ca. Phytoplasma mali’ identification by polymerase chain reaction (PCR)-based amplification of ribosomal RNA genes. In detail, ‘Ca. Phytoplasma mali’ was detected by the use of primer pairs fAT/rAS specific for 16SrX phytoplasmal group (Smart et al. 1996). PCR conditions were as previously described (Smart et al. 1996). PCR products were separated on 1 % agarose gel and visualized by UV transilluminator. Isolation of endophytic bacteria from roots by cultivation Endophytic bacteria were identified by cultivation on different culture media. Apple roots (2 g) from 10 plants (Table 1) were sterilized as described above, ground in a pre-cooled mortar by pestle, and homogenized in a ‘ringer solution’ (Oxoid, Italy). Partial volume of homogenates (100 ll), serially diluted, was
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Table 1 Phytoplasma detection in roots of asymptomatic and symptomatic apple trees and the correspondent CFU (g) of endophytic bacteria isolated ID
Sanitary status
PCRa
CFU (g) LB agar
1
Asymptomatic
TSA
-
1.8 9 105
2.4 9 105
4
2
Asymptomatic
?
2.8 9 10
3.6 9 104
3
Asymptomatic
?
8.3 9 103
1.5 9 103
-
5
1.6 9 105
4
1.1 9 104
3
4 5
Asymptomatic Asymptomatic
?
1.1 9 10 7 9 10
6 7
Symptomatic Symptomatic
? ?
3 9 10 6 9 103
7.5 9 103 7 9 103
8
Symptomatic
?
9 9 103
1.1 9 104
9
Symptomatic
?
1.2 9 104
1.3 9 104
?
4
8.6 9 103
10
Symptomatic
1 9 10
a Specific detection of phytoplasma group 16SrX by PCRbased amplification of rRNA gene regions carried out by means of primer pair fAT/rAS
spread on Tryptic Soy Agar (TSA; Sigma, Italy) and Luria–Bertani (LB; Sigma, Italy) agar medium. The samples were incubated at 30 °C for 5 days. After growth, bacterial colonies were selected on the basis of phenotypic characters (shape, color, etc.). Bacterial density in the roots was calculated as CFU/g. Total DNA from bacterial colonies of root samples no. 1 and 4 (healthy plants), and no. 8 and 10 (infected plants) was extracted by microLYSIS (Microzone, Italy) according to the manufacturer’s instructions. 16S rRNA genes were amplified with the primers 27F and 1495R (Lane 1991) following PCR conditions previously described (Bulgari et al. 2009), and were sequenced with an ABI 3730 sequencer (Primm). Sequences were identified by comparison with the NCBI GenBank sequence database with the BLAST software. Nucleotide sequences of representative isolated bacteria were deposed at NCBI GenBank database at accession numbers from JQ291734 to JQ291749. 16S rRNA gene sequences were clustered in a neighbour-joining phylogenetic dendrogram bootstrapped 1,000 times with the software MEGA4 (Tamura et al. 2007). Endophytic bacteria identification by cultivationindependent methods Endophytic bacterial community associated with healthy and ‘Ca. Phytoplasma mali’-infected apple
roots was described by 16S rRNA gene library analyses. The bacterial 16S rDNA was amplified from apple root total DNAs with bacterial universal primer pairs 799f/1492r (Chelius and Triplett 2001). PCR reaction mixture (25 ll) contained 19 PCR buffer, 1.5 mM MgCl2, 0.2 mM dNTPs, 0.4 lM of each primer, 1.25 U AccuPrimeTM Taq polymerase High Fidelity (Invitrogen, Italy). The samples were amplified in thermocycler (Applied Biosystem, Italy); PCR reactions consisted of an initial denaturation at 94 °C for 5 min followed by 35 cycles of 1 min at 94 °C, 45 s at 52 °C, 1 min at 72 °C and finally 8 min at 72 °C. PCR products were separated on 1 % agarose gel and visualized by UV transilluminator. The 16S rDNA bacterial specific bands were excised and purified from the gel with the QIAquick Gel Extraction Kit (QIAGEN, Italy) according to manufacturer’s instructions. Purified products were cloned in the plasmid vector pCRII-TOPO (Invitrogen) and propagated in Escherichia coli as described (Shuman 1994). The plasmid DNA of each clone was extracted from E. coli colonies with the QIAGEN Plasmid Mini kit (QIAGEN), and sequenced with an ABI 3730 sequencer (Primm, Italy). Clone sequences were identified by comparison with National Center of Biotechnology Information (NCBI) GenBank database with the BLAST software (http://www.ncbi.nim.nih.gov/BLAST/). Taxonomic assignment and OTU determination Hierarchical taxa assignment in each library was carried out by using Classifier program of Ribosomal Database project (Wang et al. 2007) with a confidence level of 80 % (http://rdp.cme.msu.edu/classifier). Clone libraries were compared using Libcompare program of Ribosomal Database project (http://rdp. cme.msu.edu/comparison/comp.jsp). Comparison was performed on class and order level with a confidence level of 80 %. The diversity of the clone libraries was also investigated by rarefaction analyses. Rarefaction curve was calculated using PAST software (http:// folk.uio.no/ohammer/past/index.html). All the 16S rRNA gene sequences from clone libraries were clustered in operational taxonomic units (OTUs) with the software Bioedit 7.0.9 (http://www.mbio.ncs u.edu/BioEdit/bioedit.html). Nucleotide sequences sharing more than 97 % identity were clustered in the same OTU. Nucleotide sequences of one representative clone of each OTU were deposed at NCBI
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GenBank at accession numbers from JQ291750 to JQ291790.
Results and discussion Detection of ‘Ca. Phytoplasma mali’ in apple roots ‘Ca. Phytoplasma mali’ detection in root samples was carried out using primers fAT/rAS which target 16S rDNA and 16S–23S intergenic region of 16SrX phytoplasma group. Electrophoresis analysis showed a band of approximately 500 bp in all roots collected from symptomatic apple trees. PCR products were also found in root samples no. 2, 3 and 5 that are from asymptomatic plants (Table 1; Fig. 1). Some studies reported that asymptomatic apple trees can host phytoplasmas in the roots (Musetti et al. 2004). This phenomenon was also observed in apricot plants infected by ‘Ca. Phytoplasma prunorum’ that are asymptomatic instead the presence of phytoplasmas (Osler et al. 2000). Endophytic bacterial community described by cultivation-dependent methods The diversity of microbiota associated with healthy and phytoplasma-infected apple roots was investigated both with cultivation-dependent and -independent methods in order to increase the range of diversity explored in a sample. Before ‘Ca. Phytoplasma mali’ detection in root samples, endophytic bacteria were isolated on TSA and LB from all samples collected to
M
1
2
3
4
5
6
7
8
9
10
AP
AT
W
Fig. 1 Agarose gel electrophoresis of PCR products obtained by the use of fAT/rAS primers specific for ‘Candidatus Phytoplasma mali’. Lane M molecular weight marker 1 kb plus (Invitrogen); lanes 1–5 roots from asymptomatic apple trees; lanes 6–10 roots from symptomatic apple trees; AP: Catharanthus roseus infected by ‘Candidatus Phytoplasma mali’ strain AP, positive control; AT: Catharanthus roseus infected by ‘Candidatus Phytoplasma mali’ strain AT, positive control; W: water, PCR mix without DNA, negative control
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calculate CFU/g. In healthy plants bacteria CFU/g were 105, while in diseased plants were 103–104 (Table 1). Interestingly, the asymptomatic plants positive to phytoplasma detection were characterized by a CFU comparable to symptomatic plants. These findings are in agreement with the data reported in other studies (Trivedi et al. 2010; Bulgari et al. 2011). Endophytic bacteria isolation was performed on healthy apple roots (sample no. 1 and no. 4) and on infected roots (sample no. 8 and no. 10). Twelve colonies with different morphology were isolated from healthy roots and six colonies from infected roots. Sequences of the 16S rRNA gene identified Firmicutes of the genus Bacillus, Lysinibacillus and Paenibacillus; Gammaproteobacteria of the genus Pseudomonas (Fig. 2; Table S3). In detail, Lysinibacillus and Paenibacillus were isolated respectively only in healthy and AP-infected roots, while the other genera identified were shared by all samples analyzed. Six different Bacillus species were isolated from healthy apple trees and, among these, Bacillus amyloliquefaciens and Bacillus gibsonii were found also in infected plants; Bacillus aquimaris was identified exclusively in phytoplasma-infected roots. Interestingly, bacterial strains, here isolated, belong to genera widely studied for developing biocontrol strategies to contain plant pathogens (Choudhary and Johri 2009; Trivedi et al. 2011). Analysis of endophytic bacterial community by 16S rRNA gene libraries Endophytic bacterial community had been characterized in a wide range of woody and herbaceous plants (Lodewyckx et al. 2002), but no researches have described the microbial diversity associated with apple trees. In this study, 16S rRNA gene libraries from infected and uninfected apple roots were analyzed to describe the endophytic bacterial community. Primers 799f/1492r were employed because they discriminate bacterial 16S rDNA from chloroplast and mitochondrial DNAs (Chelius and Triplett 2001; Sun et al. 2008). A total of 120 clones were sequenced, 76 clones from the healthy roots and 44 from the infected ones. The coverage of libraries from uninfected and infected trees was 95 and 89 % respectively. Furthermore, the rarefaction curves also tended to plateau (Fig. 3), indicating that these libraries were sufficient to describe the endophytic bacterial
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Alphaproteobacteria (19 %). In the infected roots the majority of the clones clustered in Betaproteobacteria (31 %) and Gammaproteobacteria (27 %), while Alphaproteobacteria were found in lower percentage (Fig. 4a). This result differs from the data reported by Trivedi et al. (2010, 2011) that identified Alphaproteobacteria as the dominant bacterial class in ‘Ca. Liberibacter asiaticus’-infected citrus roots. As reported in other studies, Proteobacteria is the most abundant bacterial phylum identified in plants (Rosenblueth and Martinez-Romero 2006; Trivedi et al. 2010; Li et al. 2011), but the proportion of the Proteobacteria classes is influenced by plant genotype and physiology. In fact, plants can specifically attract
diversity. Clone sequences of the two libraries were organized in a taxonomic hierarchy using RDP classifier. Confidence level was positioned at 80 % in order to provide taxonomic affiliations and to perform library comparison using RDP tools, as reported by Trivedi et al. (2010). This confidence level allowed to resolve the bacterial diversity associated with the samples analyzed. Seven different bacterial classes were found in both libraries. Class of Actinobacteria was present only in infected roots while the class of Chlamydiae was detected only in healthy roots. The dominant bacterial classes in the library from healthy roots were Betaproteobacteria (34.2 %), Gammaprotebacteria (25 %), and
Fig. 2 Phylogenetic relationships based on partial 16S rRNA gene sequences obtained from the endophytic bacteria associated with healthy and phytoplasma-infected apple roots (this work) and closely related sequences, retrieved from GenBank. Bootstrap values higher than 50 % are displayed at tree nodes. GenBank accession numbers of nucleotide sequences are shown along with the name of the bacterial species. Bacterial strains isolated in this work are reported in bold character
Bacillus subtilis 1H2 (JQ291734) Bacillus amyloliquefaciens (JF899282) Bacillus amyloliquefaciens 4H7 (JQ291735) Bacillus methylotrophicus (JF899259) 95 56
Bacillus methylotrophicus 4H3 (JQ291736) Bacillus amyloliquefaciens 10D2 (JQ291737) Bacillus subtilis (JF907697) Bacillus gibsonii 4H6 (JQ291738) 100
Bacillus gibsonii (NR026143) Bacillus gibsonii 10D1 (JQ291739)
Bacillus sp. 4H10 (JQ291740) Bacillus aquimaris 10D4 (JQ291741) Bacillus aquimaris (JN182695) Bacillus altitudinis 4H9 (JQ291742) 100 Bacillus altitudinis (NR042337) 63
Bacillis cereus 4H5 (JQ291743) 100
Bacillus cereus (FJ937875) Bacillus megaterium 4H2 (JQ291744)
97
100 Bacillus megaterium (JN004170) Lysinibacillus sp. (HQ396802) Lysinibacillus fusiformis (NR042072)
99 99
Lysinibacillus fusiformis 4H11 (JQ291745) Lysinibacillus sphaericus (JN377789) Paenibacillus uliginis (JN638057)
99 59
Paenibacillus sp. 10D8 (JQ291746) Paenibacillus sp. (JF892726) 99
Pseudomonas poae (HQ876461) Pseudomonas sp. (AB626117)
55 Pseudomonas fluorescens 1H3 (JQ291747) 100 75 Uncultured Pseudomonas sp. (JF905983) Pseudomonas poae 1H7 (JQ291748) 51
Pseudomonas fluorescens (JF423119) Pseudomonas sp. 8D5 (JQ291749)
65 88
Pseudomonas putida (JN222977)
0.05
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Fig. 3 Rarefaction curves of 16S rRNA gene libraries of endophytic bacteria associated with healthy and phytoplasmainfected apple roots
Fig. 4 Composition of 16S rRNA gene libraries at a class level and b order level, determined by the use of RDP classifier tool
bacteria for their own ecological and evolutionary benefit (Bais et al. 2006). At the order level, libraries from uninfected and infected apple roots shared five orders with a different number of clones. On the other hand, five bacterial orders were identified exclusively in the library from healthy roots and three orders in the library from infected roots (Fig. 4b). For example, Pseudomonandales were present only in the library from healthy roots and represented the 15.7 % of the total clones. On the basis of sequence similarity, clone sequences of the two libraries were organized in OTUs. These analyses allowed to estimate bacterial richness associated with healthy and phytoplasma-
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infected apple roots. Most of the clones showed C98 % sequence similarity to the relative strains of GenBank. Twenty-four and 17 OTUs were identified in the libraries from healthy and infected roots, respectively (Tables S1, S2). These data showed that bacterial diversity in healthy roots is higher than that observed in infected ones. Also in previous work, clone library analysis of plant-associated bacteria in ‘Ca. Liberibacter asiaticus’-infected and uninfected citrus roots evidenced differences in the composition of their bacterial community (Trivedi et al. 2010). The influence of pathogen infection on endophytic bacterial community was also reported in healthy and phytoplasma-infected grapevine leaves (Bulgari et al. 2011). Such evidences, along with data from the present study, suggest that uncultivable bacterial pathogens can modify the composition of endophytic bacterial communities associated with infected plants. OTUs distribution and prevalence in uninfected and phytoplasma-infected apple roots were reported in Table 2. The sequences related to Proteobacteria represented the largest fraction of clone libraries. In detail, 7 and 5 OTUs of Betaproteobacteria group were identified in healthy and infected roots libraries, respectively. In healthy roots, the major number of clones (19.6 %) yielded best matches with bacteria of the family Oxalobacteriaceae; in infected roots, 10.8 % of clones showed the highest similarity with bacteria of the family Burkholderiaceae, genus Burkholderia. This bacterium has been reported as endophyte of pathogen-infected plants (Nowak et al. 1997; Bulgari et al. 2011), and in various uninfected host plants such as potato (Bensalim et al. 1998), tomato (Onofre-Lemus et al. 2009), and grapevine (Compant et al. 2008a). Several strains are known to enhance disease resistance in plants, contribute to better water management, and improve nitrogen fixation and overall host adaptation to environmental stresses (Ait Barka et al. 2000; Compant et al. 2008b). The dominant OTU in healthy roots yielded best matches with Gammaproteobacteria group, Pseudomonandaceae family, Pseudomonas fluorescens (15.7 % of clones). Interestingly, the genus Pseudomonas was the sole bacterium found by cultivationdependent and independent methods in association with healthy and infected apple trees. Pseudomonas is one of the most frequently occurring genera in plants (Lodewyckx et al. 2002). Previous studies emphasized the potential of Pseudomonas fluorescens
Gammaproteobacteria
Betaproteobacteria
Alphaproteobacteria
Group
99
Uncultured bacterium (GQ128327)
Legionellaceae
Burkholderiaceae
Burkholderia incertae sedis
Uncultured Legionella (HQ003528)
96
99
99
Uncultured bacterium (AY917770) Burkholderia sp. (AB531407)
99
Methylibium sp. (AB609313)
99
3
19
2
1
9
99
Collimonas fungivorans (AY593480)
1
99 99
5
6
2
26
2
1
1
3
1
2
4
14
Herbaspirillum hiltneri (DQ150565) Oxalobacteraceae bacterium (DQ337591)
Ramilibacter sp. (EU423304)
7
7 5.5
2.6
1.3
11.8
1.3
6.5
7.8
2.6
2.6
1.3
1.3
3.9
1.3
2.6
4
5
2
2
12
5
2
3
2
1
13
1
2
3
No. of clones
No. of OTUs
% of total clones
No. of OTUs
No. of clones
Diseased roots
Healthy roots
Massilia sp. (FR682709)
98
Variovorax boronicumulans (AB300597)
Comamonadaceae
Oxalobacteraceae
99
Methylovorus glucosetrophus (FR733702)
Methylophilaceae
98
97
98
Uncultured bacterium (GQ451197) Uncultured Devosia (JN679184)
98
Uncultured soil bacterium (DQ297946)
Hyphomicrobiaceae
98
Sphingomonas sp. (AB196253)
Sphingomonandaeae
99 96
Bradirhizobium sp. (FR753118) Uncultured Phenylobacterium (HM438573)
Uncultured Bradyrhizobium sp. (HQ674794)
Caulobacteraceae
99
Rhizobium giardinii (EU488750)
99
% Identity
Bradirhizobiaceae
GeneBank closest relative
Rhizobiaceae
Family
Table 2 Distribution of 16S rRNA gene clones of endophytic bacteria from healthy and AP phytoplasma-infected apple roots
4.4
10.8
4.3
6.5
4.3
2.1
2.1
4.4
% of total clones
Antonie van Leeuwenhoek (2012) 102:677–687 683
123
123 99
Dyella sp. (EF471222) Uncultured Dokdonella (FJ889343)
Xanthomonadaceae
Uncultured soil bacterium (HQ404595)
Actinobacteridae
Uncultured bacterium clone (FJ625364) Uncultured alpha proteobacterium (FR749752)
Unclassified Rhizobialesa
Uncultured Chlamydiales bacterium (EU852559)
Uncultured Actinobacterium (JN409141)
97
98
94
98
98
98
99
Uncultured Niastella (FJ984473) Mucilaginobacter sp. (HM204914)
98
Uncultured bacterium (HQ323235)
99
98
Uncultured Myxococcales (EU440666) Uncultured Acidobacteria (HM061771)
99
99
Uncultured bacterium (GU391672) Uncultured bacterium (EF492897)
99
99
Uncultured gammaproteobacterium (JF733397)
Acidimicrobidae
Sphingobacteriaceae
Chitinophagaceae
Acidobacteriaceae
Haliangiaceae
Unclassified Gammaproteobacteria
99
Steroidobacter sp. (AB548216)
Sinobacteraceae
99
Pseudomonas fluorescens (GU198113)
Pseudomonandaceae
% Identity
GeneBank closest relative
Family
Unclassified Proteobacteriaa
Chlamydiae
Actinobacteria
Bacteroidetes
Acidobacteria
Deltaproteobacteria
Group
Table 2 continued
1
1
3
1
2
1
1
1
1
4
3
2
9
3
3
1
1
2
4
3
12
1.3
1.3
5.5
3.9
2.6
3.9
1.3
1.3
5.5
3.9
15.7
2
1
1
2
2
3 1
5
5
2
2
2
2
4
7
1
2
No. of clones
No. of OTUs
% of total clones
No. of OTUs
No. of clones
Diseased roots
Healthy roots
4.4
2.1
10.8
4.4
4.4
4.4
15.2
2.1
4.4
% of total clones
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2.1 1
2.1 1
No. of OTUs No. of OTUs
These clones were not included in OTUs clustering due to the impossibility of classification
Uncultured bacterium clone (JF708360) Unclassified bacteriuma
a
99 Uncultured bacterium clone (EF492930) Unclassified Betaproteobacteriaa
Group
Table 2 continued
Family
98
% Identity GeneBank closest relative
No. of clones
% of total clones
Diseased roots Healthy roots
No. of clones
% of total clones
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as biocontrol agents of several plant diseases through the production of antibiotics (Duffy and De´fago 1999; Schouten et al. 2004), and the activation of induced systemic resistance (ISR) (Kavino et al. 2007; Verhagen et al. 2010). Moreover, the dominant OTU (15.2 % of clones) of infected apple roots shared best sequence similarity score with an uncultured Gammaproteobacterium. Alphaproteobacteria group has been characterized by 7 OTUs in healthy roots library and 2 OTUs in infected roots library. The families of Rhizobiaceae (5.5 % of library clones), Sphingomonandaceae, and Caulobacteriaceae were found only in healthy roots library, while the family Hyphomicrobiaceae only in phytoplasma-infected roots library. The level of diversity among other bacterial groups identified in apple roots was lower (Table 2); in particular, Acidobacteria were represented only by one OTU. Acidobacteria is a common group isolated from soil, but it has diminished or no representation in bacterial community associated with plants (Chelius and Triplett 2001). In the present study, cultivation as well as cultureindependent methods revealed differences among bacterial endophytes in terms of diversity and abundance. In fact, bacteria belonging to Firmicutes were identified only with cultivation dependent methods. However, the selectivity of cultivation as well as a preferential amplification of certain bacterial groups with universal primers could also cause the different abundance. A disparity in the representation of different bacterial classes, genera and species between isolate collection and clone library had also been observed in several other studies (Dunbar et al. 1999; Hengstmann et al. 1999; Chelius and Triplett 2001; Idris et al. 2004; Bulgari et al. 2009). Therefore, the combination of culturing methods and cloning analysis is needed for the study of the endophytic community associated with plants. Up to now, effective strategies for phytoplasma disease containment are mainly based on chemical treatment against the insect vectors (Weintraub and Wilson 2009). However, the use of insecticides causes adverse effects, such as residual toxicity and environmental pollution, and is incompatible with organic farming. Thus, other sustainable approaches to control phytoplasmas are desirable. Intriguingly, some of the endophytic bacteria identified in the present study in healthy and phytoplasma-infected apple trees have
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been reported as biocontrol agents against different plant pathogens. This finding opens the possibility to in-depth investigate the action of those bacteria against ‘Ca. Phytoplasma mali’ with the aim to project novel strategies for AP disease containment.
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