Diversity of Bacteria Associated with Bursaphelenchus xylophilus and Other Nematodes Isolated from Pinus pinaster Trees with Pine Wilt Disease

The pinewood nematode (PWN), Bursaphelenchus xylophilus, has been thought to be the only causal agent of pine wilt disease (PWD), however, since bacteria have been suggested to play a role in PWD, it is important to know the diversity of the microbial community associated to it. This study aimed to assess the microbial community associated with B. xylophilus and with other nematodes isolated from pine trees, Pinus pinaster, with PWD from three different affected forest areas in Portugal. One hundred and twenty three bacteria strains were isolated from PWN and other nematodes collected from 14 P. pinaster. The bacteria strains were identified by comparative analysis of the 16S rRNA gene partial sequence. All except one Gram-positive strain (Actinobacteria) belonged to the Gram-negative Beta and Gammaproteobacteria. Most isolates belonged to the genus Pseudomonas, Burkholderia and to the family Enterobacteriaceae. Species isolated in higher percentage were Pseudomonas lutea, Yersinia intermedia and Burkholderia tuberum. The major bacterial population associated to the nematodes differed according to the forest area and none of the isolated bacterial species was found in all different forest areas. For each of the sampled areas, 60 to 100% of the isolates produced siderophores and at least 40% produced lipases. The ability to produce siderophores and lipases by most isolates enables these bacteria to have a role in plant physiological response. This research showed a high diversity of the microbial community associated with B. xylophilus and other nematodes isolated from P. pinaster with PWD.


Introduction
The importance and potentially devastating impact of tree diseases was recognized early in the 20th century with severe epidemics associated with the introduction of new pathogens to native forest ecosystems. The pinewood nematode (PWN), Bursaphelenchus xylophilus [1] is the causal agent of the Pine Wilt Disease (PWD). Native to North-America, it was introduced to Japan and has spread into China, Korea and into Europe (Portugal and Spain) [2]. The etiology of the disease has not been well understood although PWN was confirmed to be the causative agent [3]. In general, browning of the tissues is caused by oxidation of phenols which occurs as a result of cellular disorganization [4,5]. Since the physiological and histological changes in the diseased trees occur before a rapid increase in the number of nematodes, it is alleged that other participants might be involved in the pathological process [6]. Bacteria in association with B. xylophilus have been proposed to be needed for PWD development in Pinus thunbergii [7], but although studies were performed on isolates, none of the studies [7][8][9][10][11][12] included the molecular analysis of the microbial community associated with the nematode. Furthermore, the presence of bacteria in plant tissues (endophytes) is usually recognized as positive for the plant. The term endophyte is defined as 'an organism inhabiting plant organs that at some time in its life can colonize internal plant tissue without causing apparent harm to the host' [13].
The objective of this study was to evaluate the diversity and stability of the bacterial community associated with B. xylophylus and other nematodes isolated from P. pinaster with PWD.

Sampling areas
Pinus pinaster from three different areas affected by PWD in Portugal were sampled: one located between Alcácer do Sal and Grândola (Z), south Portugal (Setúbal District) and two other areas located in Coimbra District, Central Portugal, Malhada Velha, Arganil (M) and Avô, Oliveira do Hospital (A) (Fig. 1). The area Z is affected with PWD since 1999 and includes mainly P. pinaster and P. pinea trees with more than 30-35 years old. In this area, 4 symptomatic P. pinaster trees were sampled together with 4 asymptomatic P. pinaster trees. The areas M and A are affected with PWD since 2008 and include mainly P. pinaster, with a sparse number of P. radiata, Quercus and Eucalyptus trees. The area M included 109 trees: 11 Quercus, 7 Eucalyptus, 85 P. pinaster and 6 P. radiata. In this area, 12 P. pinaster were symptomatic/dead and 2 P. radiata also showed symptoms. The sampling area A included a total of 116 P. pinaster and 15 Quercus trees where 18 P. pinaster were symptomatic. The presence of nematodes was screened in all symptomatic trees.

Plant material
Sampling was performed in Spring-Summer. Each sample consisted of pinewood cross-sections from cut trees or wood obtained by drilling a 5 mm diameter hole to a depth of 10 to 15 cm with a sterilized hand brace drill (Haglof, Mora, Sweden). Fourteen P. pinaster trees were sampled at the trunk at breast height and at the top of the tree. The wood samples were placed in labelled and sealed individual plastic bags and divided in subsamples for microbiological analysis and nematodes screening. All samples were kept at 4uC and analyzed within 24 h. The diameter of sampled trees was measured at breast height (DBH) and the trees classified into 6 symptom classes based on the symptoms they expressed: 0 -tree without symptoms, I -,10% brown leaves, II -10-50% brown leaves, III -50-80% brown leaves, IV -.80% brown leaves, V-dead tree without leaves.

Nematode screening and identification
Nematodes were extracted, from triplicates of 20 g of each cross-section, using modified Baermann funnels [14]. After 48 h, the suspensions were collected and observed using an inverted stereomicroscope. The identification of PWN and other nematodes was based on the diagnostic morphological characters. B. xylophilus identification was confirmed molecularly by a satellite-DNA species-specific based technique [15].

Microbial community associated with PWN and with other nematodes
The bark and sapwood of each sub-sample were removed under sterile conditions and the wood cut in ca. 2 cm chips. The wood pieces were placed in Petri dishes with R2A medium and incubated at 25uC, for three days. All bacterial colonies were isolated from the trails made by the nematodes on the medium. In order to isolate bacteria associated only with PWN, wood chips from infected wood samples from area Z were sterilized and processed as previously described and the bacterial colonies were selected only from the trails made by PWN on the medium. Molecular identification of PWN was performed using the satellite-DNA species-specific based technique [15].
Bacterial isolates were grouped by RAPD typing. RAPD fragments were amplified by PCR, using primer OPA-03 (59 -   AGT CAG CCA C -39) (Operon Technologies, Inc. Alameda, California, USA) together with crude cell lysates. DNA profiles for 123 isolates were grouped on basis of visual similarities of the fragments analyzed by electrophoresis in a 2% agarose gel stained with ethidium bromide. Reproducibility of the patterns was tested. Nematodes were also extracted from infected wood samples from area Z, identified as B. xylophilus on the basis of morphological characters and sterilized with 0.1% sodium hypochlorite and washed with sterilized distilled water. The nematodes were then centrifuged and the resulting nematode pellet was homogenized with sterilized distilled water. DNA was extracted according to Nielsen et al. [16], the 16S rRNA gene from bacteria in the homogenate was amplified by PCR, cloned and sequenced as described below.
16S rRNA gene sequence of the bacterial isolates from nematode trails and nematode homogenates Amplification of a nearly full-length 16S rRNA gene sequence from bacterial isolates and from nematodes homogenates was performed by PCR with primers 27F (59 -GAG TTT GAT CCT GGC TCA G -39) and 1525R (59 -AGA AAG GAG GTG ATC CAG CC -39) [17]. The PCR reaction mix (50 ml) contained: reaction buffer (1.5 mM MgCl 2 , 50 mM KCl and 10 mM Tris-HCl, pH 8.3), 100 mM (each) deoxynucleoside triphosphates (Promega, Madison, Wisconsin, USA), 0.2 mM (each) primer and 1.5 U Taq polymerase (Sigma, St. Louis, Missouri, USA). The PCR was performed with 30 cycles: 1 min at 94uC, 1 min at 55uC, and 1 min at 72uC. PCR products with 1500 bp obtained from isolates were purified using the JET Quick PCR Purification Spin Kit (Genomed GmbH, Löhne, Germany) according to the manufacturer's instructions, and sequenced as described below. PCR products from nematode homogenates were cloned into pGEM-T Easy (Promega), transformed into E. coli XL1-Blue, extracted, amplified and purified according to standard procedures, and sequenced as described below.

DNA sequence analyses
The 16S rRNA genes from 61 strains associated with nematodes trails and representing all RAPD groups and PCR products from nematode homogenates were subjected to amplification (or reamplification in the case of the clones) for sequencing. Automated sequencing of the purified PCR products was performed using dRodamina terminator cycle-sequencing kit and ABI 310 DNA Sequencer (Applied Biosystems, Foster City, California, USA) according to the manufacturer's instructions.

Phylogenetic analyses
All sequences were compared with sequences available in the EMBL/GenBank database using BLAST network services and with sequences in the Ribosomal Database Project II (RDP) [18]. Sequences were initially aligned with the CLUSTAL X program [19], visually examined, and relocated to allow maximal alignment. To obtain a more accurate phylogenetic assignment of the OTUs, the aligned sequences were divided into phyla and, in the case of Proteobacteria, into classes. Sequences were also checked for chimeric properties by using CHIMERA_CHECK program of RDP [18]. The method of Jukes and Cantor [20] was used to calculate evolutionary distances and phylogenetic dendrograms were constructed by the neighbor-joining method using the MEGA4 package [21].

Siderophore production and proteolytic activity
All isolates were screened for their ability to produce siderophores when cultivated in CAS medium at 25uC during 48 h [22]. Strains developing an orange halo were considered as positive. The ability to degrade Tween 20, 40, 60 and 80 at concentration of 1.0% in R2A medium was tested after 3 and 5 days incubation at 25uC. Inoculated medium showing an opaque halo around the zone of growth was considered positive. Skim Milk Agar (R2A:skim milk, 1:1, w/w) was used to detect proteolytic activity. Strains showing a transparent halo around the zone of growth were considered positive.

Nucleotide sequence accession numbers
The 16S rRNA gene sequences of the isolates reported in this study have been deposited in EMBL database under the accession numbers from HQ538775 to HQ538819, from FJ784694 to FJ984701, FJ784703 from FJ784705 to FJ784710 and the cloned 16S rRNA genes sequences under the accession numbers FJ784711 and from FJ784713 to FJ784716.

Nematode screening and identification
Bursaphelenchus xylophilus and other nematodes (Families Rhabditidae and Aphelenchoididae) were detected in 14 symptomatic trees (class III to V) from the 3 different affected areas, 4 from area Z, 5 from area M and 5 from area A ( Fig. 1 and Table 1). Nematodes of family Rhabditidae and Aphelenchoididae were found in both symptomatic P. radiata. Microbial community associated with PWN and with other nematodes Nematode-associated bacteria were isolated from samples of 11 P. pinaster with PWN (Table 1). One hundred and twenty three strains were isolated from nematode trails (Fig. 2). The number of isolated bacteria and the species isolated were not related with the symptomatology or with the DBH of trees. These strains were grouped into 61 RAPD-types on basis of visual similarities. These strains belonged to two different phylogenetic groups: Betaproteobacteria and Gammaproteobacteria and one strain belonged to the Actinobacteria (family Microbacteriaceae) (Fig. 3). The strains of the Betaproteobacteria belonged to the genus Burkholderia (19.7%) and one strain was identified as Janthinobacterium agaricidamnosum, of the family Oxalobacteriaceae (ZR1-2). The isolates belonging to the Gammaproteobacteria were identified as strains of the family Enterobacteriaceae (41.0%), family Pseudomonadaceae (34.4%) and one strain was identified as Luteibacter rhizovicinus (Xanthomonadaceae) (M24-cE1).
The family Enterobacteriaceae was represented by 10 different species (Fig. 4A). The most abundant species were Yersinia intermedia (20%) and S. marcescens (16%). The genera Pantoea, Cronobacter, Erwinia and Ewingella each included 12% of the isolates. The family Burkholderiaceae was represented by six species but the majority of the strains belonged to the species B. tuberum (38% of the Betaproteobacteria) (Fig. 4B). The second most abundant species was B. glathei (23%). The family Pseudomonadaceae was represented by 6 species with the most abundant species being Pseudomonas lutea (57%) (Fig. 4C).
The bacterial isolates associated only to B. xylophilus isolated from Grândola (area Z) were identified as belonging to the genus Burkholderia (50%) and to the Enterobacteriaceae (37.5%) and one strain was identified as P. lutea. One strain from the genus Janthinobacterium sp. (Oxalobacteriaceae) was isolated associated with PWN (Fig. 3).
Nematodes from Arganil trees (area M) carried bacteria from the genus Pseudomonas (59.4%), and Burkholderia (12.5%) and from the family Enterobacteriaceae (21.9%). The only Luteibacter and Actinobacteria strains were isolated in this area. In Oliveira do  Hospital (area A), 92.3% of the strains associated with the nematodes belonged to the Enterobacteriaceae and only 7.7% to the genus Pseudomonas.
No single isolated bacterial species associated with B. xylophilus and other nematodes was common to all of the different forest areas samples. The Enterobacteriaceae associated with nematodes present in two different areas (M and A) were strains belonging to the species Ewingella americana, Cronobacter dublinensis, and Pantoea cypripedii. Strains from Burkholderia glathei and P. lutea were, respectively, the only Burkholderiaceae and Pseudomonadaceae, isolated from two areas (Z and M).

Biochemical characterization
All isolates were tested for lipases, proteases and siderophore production (Fig. 5). Most strains produced siderophores, from 60% of the isolates from Grândola (area Z) to 100% of the isolates from Oliveira do Hospital (area A). Proteases were produced only by the isolates from Oliveira do Hospital (30%) and by one isolate from Arganil (area M), but lipases were produced by 35 strains (63.6%) from the different forest areas. Lipase activity was best when using Tween 40 as substrate.

Discussion
PWD has been detected in Portugal affecting P. pinaster. Therefore, it is important to understand first if the PWN isolated from infected trees carries bacteria and, second, what is the diversity of the bacteria associated with PWN and with other nematodes.
Previous works studied bacteria associated with PWN in the perspective that bacteria produce toxins that play an important role in PWD [23]. Bacteria producing toxins to both callus and the seedlings of Japanese black pine (P. thunbergii) were described. Zhao and Lin [11] described bacteria attached to the surface of PWN and Guo et al. [8] reported 209 as the average number of bacteria attached to the surface of PWN. Furthermore, authors claimed that healthy P. thunbergii did not possess bacteria in the tissues. This was against what was reported by Pirttilä et al. [24] which described the presence of bacteria in P. sylvestris tissues.  According to literature the isolates carried by PWN in different countries are mainly species of Pseudomonas (in China) and Bacillus (in Japan), with both genera in Korea [23]. Furthermore, the number of reported isolated bacterial strains associated with PWN and identified in the literature is low [25] and no information exists about differences in diversity of the bacteria carried by PWN or by other nematodes isolated from different forest zones.
In the present study, bacteria strains were isolated from B. xylophilus and other nematodes (Families Rhabditidae and Aphelenchoididae) isolated from P. pinaster with different symptom classes and different DBH. The number of isolated bacteria and the species isolated were not related with the symptomatology or with the DBH. The major phylogenetic groups of bacteria isolated associated with PWN and other nematodes present in trees with PWD were Enterobacteriaceae and strains from the genera Burkholderia and Pseudomonas. Burkholderia are reported for the first time associated with B. xylophilus, although strains from this genus are usually reported as endophytes. Additionally, Gram-positive bacteria associated with PWN were detected by molecular methods and one strain was isolated from nematode trails but none belonged to the genus Bacillus. Furthermore, the dominant populations were different according to the different areas. These differences could be explained by differences in the endophyte community of different tree species (from China, Japan, Korea and Portugal) and differences in the soil community. In this work, Enterobacteriaceae isolates belonged to different species but the most abundant was the species Y. intermedia (with one fifth of the strains), and species S. marcescens, Pantoea cypripedii, Cronobacter dublinensis, Ewingella americana and species belonging to the genus Erwinia. This phylogenetic group was reported to belong to the endophytic community of citrus, cocoa, eucalypti, soybean and sugar cane [26]. In fact, species Erwinia tasmaniensis and Cronobacter dublinensis were already reported as beneficial endophytes, while Ewingella americana, Erwinia psidii and Pantoea cypripedii are reported phytopathogens [27]. It is yet unclear if these Enterobacteriaceae are connected with the normal flora of P. pinaster or with PWD. Within the Burkholderiaceae, the species B. tuberum was the most common isolate but only carried by nematodes isolated from P. pinaster from Grândola (area Z). The presence of this species can most probably be related with its presence in the endophytic community of pine trees since Burkholderia species and its diversity were related with plant species and land use management [28]. The species of the genus Pseudomonas have been considered to be in mutualistic symbiosis with PWN and co-responsible for PWD [29].
Although species of the genus Pseudomonas were isolated from nematodes of all sites, the results of this study do not point to the possibility of a Pseudomonas species existing associated with PWN in Portugal, as none of the isolated Pseudomonas species was found in all 3 sampling areas. In fact, with the exception of P. lutea, which was isolated from nematodes of two different sampling areas, all the other isolated Pseudomonas species were different from area to area. Moreover, molecular techniques did not allow the detection of Pseudomonas in surface-disinfected nematodes. All species identified were described already associated with plants or from soil or even carried by insects. The type strain of P. lutea is able to solubilize phosphate [30] but P. constantinii is described as plant pathogenic bacteria [27]. Therefore additional research is needed in order to find out which is the role of these bacteria when associated to PWN and/or inside the plant.
The microbial community associated with Portuguese PWN and with other nematodes, produced siderophores and different lipases and did not produce proteases (casein hydrolysis). Plant lipases have been described as playing a role in plant defense [31]. Furthermore, the product of Tween 20 and 80 hydrolysis have been reported to be of essential importance in the regulation of a plant defense response [32]. Therefore, the lipase ability of the bacterial community could have a role, to be explained, in activation of plant defense.
This study contributes to the characterization of the diversity of the microbial community associated with Bursaphelenchus xylophilus and other nematodes present in Pinus pinaster with PWD. The majority of the strains isolated belonged to phylogenetic groups usually isolated as endophytic bacteria. Further work will be needed to understand the role of these bacteria in PWD.