Ethics Statement

No specific permissions were required for sampling, since the study was supported by "Fundo Florestal Permanente" (Permanent Forest Fund), through a national project. The field studies did not involve endangered or protected species.

Bacterial isolation

Bacteria strains used in this work were isolated in Proença et al. [9]. Briefly, P. pinaster trees were sampled in three different Portuguese areas affected by PWD: one located in southern Portugal, Setúbal District, between Alcácer do Sal and Grândola (Z) and two other areas in Central Portugal, Coimbra District, located in Malhada Velha, Arganil (M) and Avô, Oliveira do Hospital (A). The area between Alcácer do Sal and Grândola (Z) is affected with PWD since 1999 and the areas M and A are affected with PWD since 2008 [20]. The presence of nematodes was screened in all symptomatic trees. The bark and sapwood of infected trees 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 25°C, for three days. All bacterial colonies were isolated from the trails made by the nematodes on the medium. Isolated strains were preserved in LB medium with 15% glycerol at -80°C.

Screening and identification of bacterial strains with nematicidal activity

The bacterial strains, isolated under the conditions described above, were grown in Casamino Acids liquid medium (CAA) [21] at 180 rpm, during 24h until the end of the exponential phase, at 26°C. The suspensions obtained were centrifuged (20 min, 4°C, 4000 rpm), in order to drastically reduce the number of cells, without removing any growth product (filtration with 0.22 µm filter showed to affect nematicidal activity in some strains). To test the nematicidal activity, 500 μl of each bacteria supernatant, with less than 0.06 O.D._600nm, were incubated with 75 (3 times 25) disinfected nematodes, for 48 hours at 26 °C. Nematodes were disinfected by sequential washes in sodium hypochlorite 0.1 % (one wash, 1 min at 4°C) and in 1 ml sterilized water (two washes, 3 min at 4°C), followed by centrifugation. Last water (100 μl) was inoculated on R2A for control of the disinfection efficiency.

The number of dead nematodes was assessed under the stereoscopic microscope. Nematodes were considered dead when linearized and not able to recover after being transferred to water. All tests were performed in triplicate and repeated at least two separate times. The controls - nematodes in CAA and nematodes in water - were incubated under the same conditions.

The bacterial strains with a significant nematicidal activity in the above screening were selected and identified by 16S rRNA gene sequencing and comparison with sequences available in the EMBL/GenBank database, using BLAST network services, and with sequences in the EzTaxon identification service [22].

Screening for extracellular proteases

To screen for the presence of proteases, all strains were grown in 50 ml of CAA at 26°C during 24h, centrifuged (20 min, 4°C, 4000 rpm) and the resulting supernatants were evaluated for the presence of proteins by SDS-PAGE using slab gels (1.5mm thick, 10% polyacrylamide) [23]. The proteins were stained with Coomassie Blue G-250. Additionally, the proteolytic activity of the supernatants was evaluated by zymography using 12.5% polyacrilamide gels containing 0.2% copolymerized gelatin. In parallel for comparison, a native gel without gelatin was also performed. Each supernatant (25 μl) was mixed, without boiling, with sample buffer (dilution 1:1 with 125 mM Tris-HCl, pH 6.8, containing 4% SDS and 20% glycerol) and applied to the gel. After electrophoresis, zymogram gels were renatured twice with 0.25% Triton-X-100, washed in distilled water and incubated overnight in activation buffer (Tris HCl 50 mM pH 8.0). Gels were stained with Coomassie Blue and proteolysis in zymograms was detected as clear zones in a blue background. The weight of the proteins was estimated with an appropriate marker (Precision Plus Protein Standard) for native gel analysis (Bio-Rad).

Extracellular proteases of the strain A88copa13 were assessed from supernatants as described above. To purify the extracellular proteases with proteolytic activity, the purification process was initiated with a step of concentration of the collected supernatant trough Millipore Centricon® Centrifugal Filter Unit with a cut-off for 30,000 MW. The protein content was determined in the elute by Bradford assay (Bio-Rad Protein Assay, Bio-Rad). The proteins were purified by anionic exchange in FPLC separation. The crude protein extract was applied to a Hitrap Q column (1 ml) (Amersham, Switzerland) equilibrated in (buffer A) 50 mM Tris HCl (pH 8.0) buffer. The column elution was performed by a linear gradient 0-50% of (buffer B) 50 mM Tris HCl (pH 8.0) with 1 M NaCl for 20 minutes at 1 ml/ min flow rate. The eluted proteins were detected at UV 280 nm. Fractions were collected and assayed for protease and nematicidal activities.

To assess the protease activity, 200 μl of each fraction and 2 μl of each of the 12 different substrates indicated in Table 1 were added to 50 mM Tris HCl, pH 8.0. All substrates were at 10 mM concentration. The release of AMC was measured for 20 minutes at 37°C, using a microplate fluorometer SpectraMAX-GeminiEM (Molecular Devices, Sunnyvale, USA), at an excitation wavelength of 380 nm and an emission wavelength of 460 nm. The fractions with the higher proteolytic activity were tested for nematicidal activity as described below. The proteolytic inhibition profile using Ala-AMC as substrate was determined by adding to the protease activity assay different inhibitors (Table 1).

MALDI-TOF identification of the proteases and gene amplification in strain A88copa13

The bands in native acrylamide gel, corresponding to proteins with proteolytic activity, were excised and placed into 1.5 mL microtubes, covered with water and sent to be sequenced by MALDI-TOF/TOF (MS / MS) at IPATIMUP, Porto, Portugal. Briefly, the samples were digested with trypsin, followed by the search of proteins from the mass spectrum of the peptides obtained (PMF). Peptide fragmentation spectra was obtained by peptide sequencing of MS / MS peaks with higher signal-noise. Peptides resulting from autolysis of trypsin were used as internal calibrants. Searches in databases of proteins and in the NCBInr Swiss-Prot/UniProtKB selections for taxonomic Bacteria and all entries were carried out.

The detection of the genes coding for the proteases in strain A88copa13 was done by PCR amplification with PCR reaction mix (50 μl) containing: reaction buffer (1.5 mM MgCl₂, 50 mM KCl and 10 mM Tris-HCl, pH 8.3), 100 μM (each) deoxynucleoside triphosphates (Promega, Madison, Wisconsin, USA), 0.2 μM (each) primer and 1.5 U Taq polymerase (Sigma, St. Louis, Missouri, USA). For the Serralysin gene amplification the primers were 50KRT (5'-TTACACGATAAAGTCCGTGGCG-3') and 50KFT (5'-TGAGTGGAATCGAACCAATGCA-3'). Amplification of Serine protease gene was performed by three sets of primers 70KF1 (5'-GCATGYGTTATCYGTCGTTTC-3') and 70KR1 (5'-CAGTTCGCCCTTATTATAMGCT-3'), 70KF2 (5'-AGTTAAGCGTTTCCTCRAC-3') and 70KR2 (5'-GATGCTCGCCTCTTCGTTCAC-3'), 70KF3 (5'-GAAACGGCCAAACGCAGCGTG-3') and 70KR3 (5'-ATCGTYKCGATGRGGGCTGA-3'). The PCR was performed with 30 cycles: 1 min at 94°C, 1 min at 53°C, and 1 min 30 sec at 72°C. PCR products obtained were purified using the JET Quick PCR Purification Spin Kit (Genomed GmbH, Löhne, Germany) according to the manufacturer’s instructions and sequenced.

Nematicidal activity of the proteases

The FPLC fractions, presenting higher protease activity, were tested for nematicidal activity against B. xylophilus (Bx-Portugal11AS) and other species of the genus Bursaphelenchus. The nematodes from the species B. tusciae (Bt-Italy), B. mucronatus (Bm-Portugal2) and B. conicaudatus (Bc-Japan) were used to evaluate the specificity of the proteases against the different species of nematodes, using the methodology described above. Inhibitors for the different classes of proteins were added to the FPLC fractions with protease activity and incubated during 20 min. Each mixture was then incubated with 25 nematodes, in triplicate, under the conditions described above, and the nematodes mortality was evaluated. Controls were performed by incubating nematodes with the protease inhibitors used, in order to evaluate their toxicity under the same conditions: 1 mM Pefabloc SC (Sigma), 10 mM EDTA (Sigma) and 10 mM 1, 10-Phenantrolin (Sigma Aldrish).

Scanning Electron Microscopy observations

PWNs subjected and not subjected to nematicidal tests were prefixed in 2% formaldehyde plus 2% glutaraldehyde in 0.1 M phosphate buffer (pH 7.2) for 12 h at 4°C. The nematodes were post-fixed with 2% osmium tetroxide for 30 min, and then dehydrated with serial ethanol treatments (30, 50, 70, 90, and 95%, and twice in 100% ethanol) for 30 min at each concentration. Then, the nematodes were dried by a critical point dryer (JCPD-5; JEOL, Tokyo) using liquid carbon dioxide, and sputter-coated with Au-Pd using a magnetron sputter (MSP-1S; Vacuum Device, Ibaraki, Japan). Scanning electron microscope (SEM) images were acquired using a VE-8800 (Keyence, Osaka, Japan).

Statistical Analysis

The significance of the differences, observed between the different treatments with Serratia sp. A88copa13, was evaluated with analysis of variance (ANOVA), using Tukey's Multiple Comparison Test, considering: 1) the amount of NaCl in the supernatant, 2) the different fractions obtained after FPLC of the supernatant, and 3) different species of nematodes. The statistical analysis was performed with GraphPadPrism v5.0 for windows, GraphPad software, San Diego, California, USA, www.graphpad.com. All differences were considered to be statistically significant for P < 0.05.

Screening of the bacterial nematicidal activity

Forty-seven bacterial strains (Table 2) were evaluated for their ability to kill B. xylophilus nematodes. From these, 21 strains shown to have nematicidal activity (100% of nematodes dead after the incubation period). Five belonged to the species Serratia plymuthica, five to Pseudomonas lutea, three to Serratia marcescens, two to Rhanella aquatilis and one strain to each of the species P. koreensis, P. constantinii, P. putida, P. moorei, Pantoea cypripedii and Curtobacterium pusillum (Table 2). None of the Burkholderia strains had nematicidal activity (less than 30% of the nematodes killed during the assay). The lack of nematicidal activity was also observed in one strain of the genus Yersinia and in two Erwinia strains, and in one P. putida strain.

All strains showed ability to produce siderophores except 4 strains from three different genera (Burkholderia, Pseudomonas and Serratia), all with different nematicidal activities. Proteolitic activity in skim milk was detected in the supernatant of P. constantinii, L. rizhinovicinus, and of the 4 strains of the species S. marcescens, all able to kill more than 70% the nematodes during the assays. Only strains from R. aquatillis (one strain), S. marcescens (four strains) and P. constantinii (one strain) were able to degrade all the different lipids tested including Tween 80 (Table 2). All these strains were able to kill more than 70% of the nematodes in the nematicidal assay.

The observation by SEM of the result of bacterial activity on the nematodes showed bacteria cells accumulated on the nematode cuticle (Figure 1). The incubation of the nematodes with bacteria lead to the degradation of the nematodes, and only bacteria-covered empty cuticles were visible after 48h incubation.

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Figure 1. SEM images of deposits of Serratia strain A88copa13 on the cuticle of the nematode Bursaphelenchus xylophilus.

Incubation after 24 h (A) and 48 h (B).

https://doi.org/10.1371/journal.pone.0079705.g001

Proteases in the supernatants of nematicidal bacteria

The strains with higher nematicidal activity were selected to be screened for the presence of extracellular substances and proteins with proteolytic capacity. The strains A25tronco1, A37tronco1, A88copa5, A88copa7 and A88copa13 produced soluble extracellular proteins with proteolytic activity when evaluated by zymography with gelatin. Strain S. marcescens A88copa13 was selected for further work, because it produced the proteins with highest activity in the zymogram gel (Figure 2).

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Figure 2. Proteins in the supernatants of nematicidal bacteria.

(A) SDS-PAGE of extracellular proteins from Serratia strains A88copa13 (2 and 3) and A88copa5 (4–6), molecular weight marker (1). Arrows indicate the band of 70 kDa (A-serine protease) and the band of 50 kDa (B-metalloproteinase serralysin) removed for sequencing by MALDI-TOF/TOF (MS / MS). (B) Zymogram using 12.5% polyacrilamide gel containing 0.2% copolymerized gelatin of extracellular proteins of Serratia strains A88copa13 (1 and 2) and A88copa5 (3 and 4) showing differences in protease activity.

https://doi.org/10.1371/journal.pone.0079705.g002

Identification of extracellular proteins with proteolytic activity

The production of extracellular proteins by strain A88copa13, under the growth conditions used, was independent of the presence of nematodes (added dead to the growth medium) during strain growth (data not show). During growth of strain A88copa13, aliquots of the supernatant were removed at different times and the nematicidal activity was evaluated. The nematicidal activity of the strain's supernatant increased during the exponential phase of the strain's growth and was maximal when the strain achieved 2.4 O.D._610nm (late exponential). Therefore, all assays were performed with aliquots removed from strain growth with 2.4 O.D._610nm. The amount used to purify the protein fractions was standardized to 50 ml of supernatant. The protein content of the supernatant was 1.23±0.08 mg/ml, when quantified after centrifugation, and shown to be stable.

The toxicity assays were incubated for three days, and during this period the number of dead nematodes was counted each 24h. No increase in the number of dead nematodes was observed after 48h incubation. After this time, a brown color developed in the supernatant and the proteolytic activity ceased.

The presence and the number of proteins in the supernatant of the different nematicidal strains was evaluated by SDS-PAGE. The band from the supernatant of strain A88copa13 with 50 kDa (Figure 2) was identified by MS / MS, with 100% statistical significance (SwissProt database), as the extracellular metalloproteinase serralysin. The band with 70 kDa was identified, with 100% statistical significance (SwissProt database), as an extracellular serine protease. In strain A88copa13, the gene sequence obtained for the 70 kDa protease (JX667979) was 95% similar to the serine protease gene sequence of S. marcescens IFO-3046 (M13469). On the other hand, the 50 kDa band was coded by a gene (JX667978) which had 96% similarity with the S. marcescens strain ZhenJiang insecticidal protein gene (EU999787).

The comparative analysis of the amino acid sequence obtained from nucleotide translation of the 70 kDa protease using BlastX showed an high homology (96%, 87%, 50% and 49%, respectively) with the serine proteases of the subtilisin-type serine proteases family from two strains of S. marcescens, (P09489, P29805) and with a smaller serine protease (60 kDa) from Xenorhabdus bovienii (YP_003468240) and X. nematophila (YP_003712486). Similarly, the comparative analysis of the amino acid sequence of the 50 kDa protease showed 99% homology with the zinc-dependent metalloproteinase, serralysin-like proteins from different strains of S. marcescens and S. nematophila (ACH90152, ABK55613, CAA39138 and ABZ81090).

Effect of protease inhibitors and metal ions on the enzyme activity

To confirm biochemically to which group the proteases in Serratia sp. A88copa13 belong, their activity was measured testing the supernatant in the presence of different protease inhibitors and protein substrates (Tables 1 and 3).

The digestion of the fluorogenic substrates at pH 7.0 showed that Alanine-AMC and Boc-Val-Pro-Arg were the substrates that induced more activity (Table 3). These peptides were typical identified as metalloprotease (aminopeptidase) substrate and serine protease substrate, respectively.

To further characterize the extracellular proteases, enzymatic assays were performed with Alanine-AMC in the presence of class-specific protease inhibitors and divalent ions. In the presence of the substrate Alanine-AMC, the inhibitor Pefabloc SC (4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride; AEBSF), when added in concentrations of 1 mmol/L, almost completely abolished the proteolytic activity of the supernatant, revealing a serine protease as a major active proteolytic enzymatic group (Table 1). The inhibitor 1,10-Phenanthroline, a chelating agent with high affinity to zinc, when added in concentrations of 10 mmol/L in the presence of Alanine-AMC as substrate, almost completely abolished the activity of the supernatant, also revealing an active metalloproteinase as a major proteolytic group, also.

In addition, with Alanine-AMC as substrate, Aprotinin showed to have an effect on the proteolytic activity of the supernatant and inhibited the proteolytic activity in 41%. EDTA is a metal ions chelator, which can chelate Ca²⁺ ions, and is recognized as an inhibitor of metalloproteinases. The addition of EDTA to the supernatant partially inhibited (26%) its proteolytic activity. Aspartic protease inhibitor, Pepstatin A, showed a moderate effect in the supernatant, with 32% of inhibition. Alkali metal ions showed a moderate effect on the enzymatic activity of the supernatant. However, Zn²⁺ inhibited the supernatant activity (66%) while Mn²⁺ increased it (152%).

The nematicidal activity of Pefabloc SC inhibitor was negative but EDTA and Phenantroline had a high nematicidal activity (P<0.05) (Figure 3). The high toxicity of the EDTA to the nematodes may suggest its action as a membrane permeabilizing agent, through the chelation of cations. When added to the supernatant, the amount of cations available is higher than in sterilized distilled water, and the EDTA free to chelate cations from membranes will be less, lowering its toxicity. On the other hand, the possibility of the strain to produce EDTA degrading proteins to the supernatant cannot be overruled [24].

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Figure 3. Bursaphelenchus xylophilus dead (stripes) and alive (grey) after the different in vitro treatments with Serratia strain A88copa13 (growth supernatant) and with proteases (FPLC fractions), after 24h incubation.

The fractions with proteolytic activity contained 0.1M NaCl. A- H₂O; B- Serratia strain A88copa13; C- Serratia strain A88copa13 + Pefabloc SC; D- H₂O + Pefabloc SC; E- Serratia strain A88copa13 + EDTA; F- EDTA + H₂O; G- FPLC protein fraction 9; H- FPLC protein fraction 19. Different symbols (*, †, &) above the bars indicate significantly different percentages of mortality (P < 0.05) between treatments.

https://doi.org/10.1371/journal.pone.0079705.g003

Nematicidal activity of FPLC fractions

Fractions of the supernatant separated by FPLC were tested for proteolytic activity and for their nematicidal activity (Figure 4A). A complete purification of the 2 proteases evaluated by SDS-PAGE and zymography was not achieved (Figure 4B). The FPLC fractions with proteolytic activity contained 0.1 M NaCl. Controls were performed in order to check the salinity effect on nematodes mortality. A control with the same salinity as the fractions tested (0.1 M NaCl), caused mortality similar to that observed with water (Figure 5). Statistical analysis showed that treatments with water (A, L, N, P), with proteins that did not bound to the FPLC column (B) and with buffer containing 0.1 M NaCl (G) did not induced nematodes mortality (P>0.05). The toxicity assays revealed that only two peaks showed nematicidal activity statistically comparable to the whole extract (other treatments with P<0.05). The FPLC fractions 9 and 19 showed the highest toxicity, although with significant statistical differences (P<0.05) in consequence, most probably, of the presence of an additional serine protease in fraction 19 when compared with fraction 9. The activity measured by the hydrolysis of substrates and by the inhibition of the activity, determined in the presence of the specific inhibitors of each protein, demonstrated that the serine protease had the highest nematicidal activity. After 24 h incubation, 73% mortality was observed when nematodes were incubated with fraction 19 (data not shown). Extending incubation increased B. xylophilus mortality to 83% (Figure 5). Dilution of these fractions lead to a decrease in toxicity. The nematicidal activity of fraction 19 after 24 h incubation was also observed against nematodes isolates of the species B. tusciae (Bt-Italy) (81%), B. conicaudatus (Bc-Japan) (13%) and also B. mucronatus (Bm-Portugal2) (10%). After 48h incubation, the nematicidal activity of this fraction increased significantly against Bm-Portugal2 and Bt-Italy (P<0.05) (Figure 5). Fraction 19 maintained the nematicidal activity against Bx-Portugal11AS until 72h of incubation but lost the activity when the incubation was prolonged (data not shown).

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Figure 4. Purification and characterization of the proteases in the growth supernatant of strain Serratia A88copa13.

(A) Anionic exchange chromatography on Hitrap Q column (1ml). Fractions 9 and 19 were eluted by using a linear gradient 0-50% of 50 mM Tris HCl (pH 8.0) with 1 M NaCl for 20 minutes at 1ml/ min flow rate. FPLC protein fraction 9 rich in metalloproteinase (1), FPLC protein fraction 19 rich in metalloproteinase and serine protease (2), A88copa13 supernatant (3), and molecular weight (MW) evaluated by SDS-PAGE silver stained (B) and by zymography using 12.5% polyacrilamide gel containing 0.2% copolymerized gelatin (C).

https://doi.org/10.1371/journal.pone.0079705.g004

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Figure 5. Bursaphelenchus xylophilus dead (stripes) and alive (grey) after the different in vitro treatments with Serratia strain A88copa13 (growth supernatant) and with proteases (FPLC fractions), after 48 h incubation.

The fractions with proteolytic activity contained 0.1M NaCl. (A) H₂O + Bx-Portugal11AS; (B) FPLC fraction containing the proteins that did not bound to the column + Bx-Portugal11AS; (C) Tris-HCl + (1M) NaCl + Bx-Portugal11AS; (D) Tris-HCl + (0.7M) NaCl + Bx-Portugal11AS; (E) Tris-HCl + (0.5M) NaCl + Bx-Portugal11AS; (F) Tris-HCl + (0.3M) NaCl + Bx-Portugal11AS; (G) Tris-HCl + (0.1M) NaCl + Bx-Portugal11AS; (H) FPLC fraction 19 + Bx-Portugal11AS; (I) Serratia strain A88copa13 (growth supernatant); (J) FPLC fraction 19 (75%) diluted with H₂O; (K) FPLC fraction 19 (50%) diluted with H₂O; (L) H₂O + Bt-Italy; (M) FPLC fraction 19 + Bt-Italy; (N) H₂O + Bc-Japan; (O) FPLC fraction 19 + Bc-Japan; (P) H₂O + Bm-Portugal2; (Q) FPLC fraction 19 + Bm-Portugal2. Results from triplicate assays. Different symbols (*, †, &) above the bars indicate significantly different percentages of mortality (P < 0.05) between treatments.

https://doi.org/10.1371/journal.pone.0079705.g005