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Keywords:

  • Trichoderma pseudokoningii SMF2;
  • serine protease;
  • nematicidal activity;
  • characterization;
  • gene clone;
  • Meloidogyne incognita

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Authors' contribution
  9. References
  10. Supporting Information

Trichoderma pseudokoningii SMF2 is a biocontrol fungus with inhibitory ability against phytopathogenic fungi. Here, a crude extract of strain SMF2 in a solid ferment exhibited strong nematicidal activity against Meloidogyne incognita, and a novel serine protease SprT with nematicidal activity was purified from the crude extract. Protease SprT has a molecular mass of 31 kDa, a pH optimum of 8.5, and a temperature optimum of 60–65 °C. It had good thermostability, and was stable in an alkaline environment. SprT could degrade bovine serum albumin, lysozyme, and gelatin, and its activity was enhanced by many metal ions. The cuticles of nematodes treated by protease SprT obviously crimpled. Purified protease SprT could kill juveniles of M. incognita and inhibit egg hatch, suggesting that it is involved in the nematicidal process of T. pseudokoningii SMF2. The full-length cDNA gene-encoding protease SprT was cloned by rapid amplification of cDNA ends. Sequence analysis showed that SprT is a monodomain subtilase containing 284 amino acid residues. It had higher identities and a closer relation to the nematicidal serine proteases (59–69%) from nematode parasitic fungi than to the serine proteases (<50%) from Trichoderma. Protease SprT represents the first well-characterized subtilase with nematicidal activity from Trichoderma.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Authors' contribution
  9. References
  10. Supporting Information

Plant-parasitic nematodes, such as Meloidogyne spp., cause extensive damage to crops every year (Casas-Flores & Herrera-Estrella, 2007). Because chemical control of nematodes leads to environmental and resistance problems, more and more attention is being paid towards developing fungal biocontrol agents. Trichoderma spp. are saprophytic fungi that are highly interactive in root, soil, and foliar environments, and have been widely described as biocontrol agents against phytopathogens (Harman, 2006). Trichoderma species, such as Trichoderma harzianum, Trichoderma lignorum, Trichoderma koningii, and Trichoderma viride, were reported to suppress Meloidogyne spp. populations (Windham et al., 1993; Khan & Saxena, 1997; Spiegel & Chet, 1998; Sharon et al., 2001). Culture filtrates from Trichoderma virens G1-3 can inhibit the egg hatch and second-stage juveniles (J2s) mobility of root-knot nematode Meloidogyne incognita (Meyer et al., 2000). Recently, Trichoderma asperellum and Trichoderma atroviride were reported to show an in vitro parasitic effect in Meloidogyne javanica eggs and larvae (Sharon et al., 2007). The demonstration of the nematicidal ability of Trichoderma species suggests their potential in nematode biocontrol.

Nematophagous fungi, including nematode-trapping fungi and parasitic fungi, are natural enemies of nematodes (Siddiqui & Mahmood, 1996). Extracellular hydrolytic enzymes, including serine protease, chitinase, lipase, and collagenase, are believed to play key roles in the infection process of parasitic fungi against plant-parasitic nematodes (Yang et al., 2007). Many serine proteases with nematicidal activity have been purified and cloned from parasitic fungi, such as pSP-3 from Paecilomyces lilacinus (Bonants et al., 1995), VCP1 from Pochonia chlamydosporia (Morton et al., 2003), Ver112 from Lecanicillium psalliotae (Yang et al., 2005), etc. These proteases are all subtilisin-like serine proteases, have similar molecular masses ranging from 32 to 39 kDa, and share a broad range of protein substrates including casein, gelatin, and eggshells. They can destroy the nematode cuticle and kill nematodes, and are important virulence factors.

Trichoderma species have a complex extracellular proteolytic system, in which serine proteases have long been attributed to their antagonistic and biocontrol activities (Kredics et al., 2005). However, serine proteases with nematicidal activity isolated from Trichoderma have hardly been reported. The subtilisin-like serine protease PRB1 from T. atroviride appeared to participate in virulence against M. javanica (Sharon et al., 2001). Another purified trypsin-like serine protease PRA1 from T. harzianum was reported to inhibit the egg hatch of M. incognita (Suarez et al., 2004). Trichoderma spp. SMF2 is a biocontrol fungus with strong inhibitory ability against phytopathogenic fungi (Song et al., 2006). Strain SMF2 was described as T. koningii in our previous report (Song et al., 2006), because it produced the same kinds of peptaibols with T. koningii (Huang et al., 1995). However, from analyses of its colony and conidiophore morphology (Supporting Information, Figs S1 and S2) and its 18S rRNA gene and internal transcribed spacer sequences (GenBank accession number FJ605099), strain SMF2 was recently identified as Trichoderma pseudokoningii (see Supporting Information). In this study, T. pseudokoningii SMF2 was shown to have strong nematicidal ability against M. incognita, and one important virulence factor, a novel nematicidal serine protease SprT, was identified from a crude extract of strain SMF2 in solid fermentation.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Authors' contribution
  9. References
  10. Supporting Information

Organisms and culture conditions

Trichoderma pseudokoningii SMF2 was a laboratory stock obtained from soil, and was routinely maintained on potato dextrose agar medium. For nematicidal activity assay and enzyme production, it was fermented in a solid medium containing 6 g wheat straw, 4 g bran, and 15 mL inorganic salt solution [% (w/v): KH2PO4, 1.25; (NH4)2SO4, 1.25; MgSO4, 0.3; CaCl2, 0.3%] at 28 °C for 5 days. After fermentation, the solid ferment was extracted with 10-fold sterile-distilled water at room temperature. The crude extract was used for nematicidal activity assay and protease purification.

Root samples of tomato, with a very typical root-knot symptom, were collected from a greenhouse in Weifang, China, and stored at 4 °C before use. Meloidogyne incognita eggs were extracted from egg masses in the root knot, and sterilized using 1% sodium hypochlorite as described by Sun et al. (2006). The J2s were hatched from eggs at 25 °C for 7 days, and then collected using a Baermann funnel. For nematicidal assay, both M. incognita eggs and J2s were washed with sterile M9 buffer (Na2HPO4 6 g L−1, KH2PO4 3 g L−1, NaCl 0.5 g L−1, NH4Cl 1 g L−1), and finally prepared in sterile-distilled water.

Purification of protease SprT

The crude extract of the SMF2 solid ferment was concentrated by ultrafiltration (10 000 Da cut-off membranes, Millipore), and then placed on a DEAE Sepharose Fast Flow (Amersham Bioscience) column (1.6 cm × 18 cm) pre-equilibrated with HAc buffer (20 mM, pH 5.6). It was eluted with a linear gradient of 0–0.2 M NaCl and monitored at 280 nm. The activity of every fraction toward casein was assayed, and the active fractions of the peak were collected. The purity and molecular mass of the purified protease was analyzed by 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as described by Laemmli (1970). The protein concentration was determined using the Bradford method (Bradford, 1976). The purified protease was named SprT and stored at −20 °C for further analysis.

Enzymatic assays and characterization of protease SprT

The activity of protease SprT toward casein was assayed at 55 °C for 15 min using the method described before (Chen et al., 2003). One unit of enzyme activity was defined as the amount of enzyme used for the production of 1 μmol tyrosine min−1. SprT activities to proteins bovine serum albumin (BSA), lysozyme, and gelatin were assayed using the same method as for casein. Proteolytic activities toward p-nitroanilide substrates (Sigma) were determined in Na2HPO4-NaH2PO4 [phosphate-buffered saline (PBS)] buffer (20 mM, pH 8.5) at 55 °C for 10 min using Peek's method (Peek et al., 1993). The release of p-nitroaniline (p-NA) was quantified by detecting A405 nm. One unit of enzyme activity was defined as the amount of enzyme used for the production of 1 μmol p-NA min−1. The effect of temperature (40–70 °C), pH (7.5–10.6) on the activity and stability of SprT, and the effect of inhibitors and metal ions on the activity of SprT were determined using casein as the substrate. Protease preparations were incubated with inhibitors and metal ions at room temperature for 30 min and the residual activities were measured at pH 8.5, 55 °C.

Cloning of the full-length sprt cDNA gene

Total RNA from T. pseudokoningii SMF2 was extracted according to the protocol of the RNeasy Plant Mini Kit (Qiagen). The N-terminal amino acid sequence of SprT was analyzed using an Applied Biosystem Procise 491 protein sequencer by automated Edman degradation, and was determined to be SYVSQSGAPWGLGRI. blast analysis at National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/) showed that this sequence has the highest identities to some subtilases from fungi (P85157, 93%; AAC99421, 85%; and AAR26028, 80%). Based on the N-terminal amino acid sequence of SprT and the conserved sequence of the catalytic domain of subtilases, a pair of degenerate primers, CFP and CRP (Table S4), was designed for cloning the conserved region of sprt gene. A 750-bp DNA fragment was amplified by RT-PCR, and the sequencing result indicated that it is a part of the sprt gene. Accordingly, several sense and antisense primers (Table S4) were designed for rapid amplification of cDNA ends (RACE) to amplify the whole sprt gene. RACE was performed according to the protocol of the SMART RACE cDNA Amplification Kit (Clontech). Consequently, a 547 bp upstream sequence and a 208 bp downstream sequence of the 750-bp fragment were amplified and sequenced. The full-length sequence of sprt cDNA was assembled and verified by PCR, and then deposited in GenBank under the number EF362571. Amino acid sequences of eight reported nematicidal serine proteases from nematophagous fungi and two mycoparasitism-related serine proteases (PRB1 and Tvsp1) from Trichoderma were aligned with SprT, and a neighbor-joining tree was constructed with mega 3.1 (Kumar et al., 2004).

Nematicidal activity assay

Enzyme solutions containing a crude extract of strain SMF2 solid ferment (50 U of protease activity), purified protease SprT (20 U), or boiled purified protease SprT in a total volume of 100 μL were prepared with PBS buffer (20 mM, pH 7.2). Fifty J2s or 100 early-stage eggs of M. incognita were separately added to the enzyme solutions and incubated at 28 °C for 24 h. The dead nematodes in the enzyme solutions containing J2s were counted under a light microscope, and the mortality was calculated. Eggs in enzyme solutions were collected and washed with sterile-distilled water three times, and incubated in sterile-distilled water at 28 °C. The numbers of J2s were periodically counted during 16 days of incubation, and the cumulative percentage hatch was calculated and analyzed as described (Suarez et al., 2004). Three parallels and three repeats were performed. The cuticle changes in protease SprT-treated J2s were observed using a scanning electron microscope (SEM) using Tikhonov's method (Tikhonov et al., 2002). Morphological changes of dead M. incognita eggs and J2s were observed under a light microscope, and photographs were taken using an inverted microscope.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Authors' contribution
  9. References
  10. Supporting Information

Nematicidal ability of T. pseudokoningii SMF2 against M. incognita

Trichoderma pseudokoningii SMF2 was fermented in a solid medium, and the crude extract of its solid ferment displayed strong nematicidal activity. The mortality of M. incognita J2s reached 100% after being treated with the crude extract for 24 h, and the eightfold diluted crude extract still retained moderate nematicidal ability, with a mortality of 44.47%. The dead nematode J2s appeared tetanic under a light microscope. There were several nutrient fat particles in live J2s (Fig. S3a), but most fat particles were decomposed, and vacuoles appeared in the dead J2s (Fig. S3b).

The effect of the crude extract of SMF2 solid ferment on M. incognita egg hatch was also studied with a water extract of a sterile blank solid ferment medium as the control. The ratio of egg hatch in the treated group was much lower than that in the control group. After being incubated for 6, 10, 12, and 16 days, the cumulative percentage of egg hatch in the control group reached 49.2%, 53.3%, 56.6%, and 78.3%, while that of eggs treated by the crude extract was only 0.5%, 1.9%, 2.1%, and 2.8%, respectively. Under a microscope, the eggs in the control group had a smooth surface and appeared brighter, but the eggs treated with the crude extract appeared darker, and the number of eggshell layers obviously reduced (Fig. 1).

image

Figure 1.  Morphological changes of Meloidogyne incognita eggs caused by a crude extract of a Trichoderma pseudokoningii SMF2 solid ferment. (a) control eggs (× 400); (b) eggs treated with a crude extract (50 U) at 28°C for 24 h (× 400). Arrows indicate the reduction of eggshell layers.

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Purification and characterization of protease SprT from T. pseudokoningii SMF2

Six peaks were observed in the whole anion-exchange chromatography process as shown in Fig. 2a. The third peak showed proteolytic activity, and its purity was identified by SDS-PAGE. As shown in Fig. 2b, only one band was observed, and the molecular mass was about 31 kDa. The purified enzyme was named as SprT, and its recovery was 14% (Table S5).

image

Figure 2.  Purification and characterization of protease SprT. (a) Anion-exchange chromatography elution profile of the concentrated crude extract. (b) SDS-PAGE (12.5%) analysis of purified protease SprT. The numbers on the left indicate molecular masses of protein standards. (c) Thermostability of SprT. The purified SprT was incubated at 50°C (-▪-) and 60°C (-•-) for 2 h and the residual activity was assayed every 20 min with casein as the substrate. (d) pH stability of SprT. The purified SprT was incubated in glycine-NaOH buffer with pH 8.0–10.6 for 1 h, and then the residual activity was assayed with casein as the substrate. The activity of SprT (1 mg mL−1) before incubation in glycine-NaOH buffer was defined as 100%.

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With casein as the substrate, protease SprT had the highest activity at 60–65 °C (Fig. S4a) and pH 8.5 (Fig. S4b). It exhibited good thermostability at temperatures <50 °C, retaining 70% activity after incubation at 50 °C for 2 h (Fig. 2c). Its long-term thermostability was also demonstrated at 40 °C for several days, with residual activities of 78%, 63.5%, 58%, and 54% after 1-, 2-, 3-, and 4-day incubations, respectively. SprT was stable in alkaline buffer, retaining 50% activity after incubation at pH 10.6 for 1 h (Fig. 2d). Phenylmethylsulfonyl fluoride inhibited 99.6% SprT activity, while o-phenanthroline had little inhibitory effect on its activity, suggesting that SprT is a serine protease rather than a metalloprotease. In addition, <1% of SprT activity was inhibited by EDTA. Metal ions Ca2+, Cu2+, Mg2+, Co2+, Li+, and K+ exerted an activation effect on SprT activity, while Fe2+ exerted an inhibitory effect (Table 1). Protease SprT could degrade proteins BSA, gelatin, and lysozyme. Among the synthetic substrates, SprT displayed the highest activity toward AAPF. In addition, SprT showed a relatively high activity toward FAAF and AAPL, but no activity toward AAPR and AAPK, suggesting that SprT prefers an aromatic or a bulky nonpolar amino acid at the P1 site (Table 2).

Table 1.   Effect of inhibitors and metal ions on SprT activity*
Chemicals (concentration)Residual activity (%)
  • *

    Purified SprT was incubated with different inhibitors and metal ions at room temperature for 30 min, and the residual activity of SprT was assayed at 55°C, pH 8.5, with casein as the substrate, which was presented as percentage of control. The activity of purified SprT without the listed chemicals was taken as control (100%).

  • PMSF, phenylmethylsulfonyl fluoride; o-P, o-phenanthroline.

Control100
PMSF (1 mM)0.4 ± 0.1
EDTA (3 mM)99.3 ± 4.7
o-P (1 mM)97.2 ± 0.5
Li+ (1 mM)126.2 ± 7.4
K+ (1 mM)112.7 ± 2.2
Ca2+ (1 mM)137.3 ± 6.0
Fe2+ (1 mM)59.6 ± 3.0
Co2+ (1 mM)163.5 ± 6.7
Cu2+ (1 mM)152.7 ± 3.5
Mg2+ (1 mM)119.9 ± 0.2
Table 2.   Substrate specificity of protease SprT*
SubstratesActivity (U mg−1)
  • *

    The activities of SprT (1 mg mL−1) toward protein substrates (20 mg mL−1) were measured as described in Materials and methods. One unit is defined as the amount of enzyme needed for the release of 1 μmol tyrosine min−1. The activities of SprT toward synthetic substrates (0.5 mg mL−1) were measured by Peek's method, and 1 U is defined as the amount of enzyme needed for the production of 1 μmol p-nitroaniline (p-NA) min−1.

Casein361 ± 2
BSA293 ± 2
Gelatin180 ± 1
Lysozyme387 ± 2
Suc-Ala-Ala-Pro-Phe-p-NA2410 ± 20
Suc-Phe-Ala-Ala-Phe-p-NA909 ± 17
Suc-Ala-Ala-Pro-Leu-p-NA415 ± 10
Suc-Ala-Ala-Leu-p-NA146 ± 9
Benzoyl-Phe-Val-Arg-p-NA25.0 ± 1.6
Suc-Ala-Ala-Pro-Arg-p-NA0
Suc-Ala-Ala-Pro-Lys-p-NA0

Nematicidal ability of purified protease SprT against M. incognita

Pure protease SprT showed a high nematicidal activity against M. incognita J2s. The mortality of J2s reached 37.3% after a 24-h treatment with SprT, higher than the mortality (1.4%) of J2s in PBS buffer, the mortality (30%) of J2s treated by boiled SprT, but lower than the mortality (57%) of J2s caused by the diluted crude extract, which had the same protease activity units as the purified SprT. The effect of SprT on J2s cuticle was observed by SEM. The untreated nematodes had a smooth surface with distinct striaes and lateral lines (Fig. 3a). In contrast, after a 24-h treatment with SprT, the cuticle of J2s obviously crimpled (Fig. 3b). These results indicated that the purified protease SprT has a cuticle-degrading ability. Moreover, the purified SprT could obviously inhibit egg hatch of M. incognita. After incubation for 3, 6, 12, and 15 days, the hatch rates of the eggs in the PBS buffer were 6.2%, 19.8%, 28.9%, and 51.7%, and the hatch rates of eggs treated by boiled SprT were 3%, 25.5%, 36%, and 47.6%, while the hatch rates of the eggs treated by purified SprT were only 0.98%, 8.92%, 8.97%, and 13.64%, respectively. These results suggested that SprT could destroy M. incognita eggs and thereby inhibited egg hatch.

image

Figure 3.  SEM observation of Meloidogyne incognita cuticle treated with protease SprT. (a) Control without protease; (b) treatment with purified protease SprT (20 U) at 28°C for 24 h.

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Molecular cloning and phylogenetic analysis of protease SprT

The whole cDNA cloned from strain SMF2 has a size of 1505 bp, containing an 1176 bp ORF of gene sprt, a 153 bp upstream sequence, and a 176 bp downstream sequence (Fig. S5). Gene sprt encodes a protein of 391 amino acid residues and shows a marked bias on the codon usage, with 60% of all codons ending in cytosine. Analysis of the predicted amino acid sequence showed that it is a subtilisin-like serine protease precursor. This precursor contains a signal peptide sequence of 18 amino acid residues (Met1-Ala18), which was predicted by signalp (Emanuelsson et al., 2007). Based on the N-terminal sequence of mature SprT determined, a propeptide of 89 amino acid residues (Thr19-Asn107) between the signal peptide and the mature enzyme could be determined. Mature SprT has 284 amino acid residues (Ser108-Gly391), and contains a peptidase_S8 catalytic domain. Analysis of the catalytic domain using the conserved domain database (http://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml) indicated that SprT is an S8 family (subtilase) serine protease (Marchler-Bauer et al., 2007). The predicted catalytic triad in SprT is Asp148, His179, and Ser334. The calculated molecular mass of mature SprT is 28.6 kDa, slightly lower than that (31 kDa) analyzed by SDS-PAGE, which might be due to some post-translational modifications (five glycosylation sites were predicted in the sprt gene sequence) (Fig. S5).

The complete amino acid sequence of SprT showed the highest identity (69%) with a predicted serine protease (ABO32256) from the parasitic fungus P. lilacinus. High identities were also found with cuticle-degrading serine protease PR1A (66%) from Metarhizium anisopliae (St Leger et al., 1992), and other nematicidal serine proteases (63% with VCP1, 60% with Ver112, and 59% with pSP-3) from parasitic fungi. In contrast, protease SprT showed low identities with serine proteases PRB1 (40%) from T. atroviride (Geremia et al., 1993) and Tvsp1 (40%) from T. virens (Pozo et al., 2004), and nearly no homology with the nematicidal serine protease PRA1 (CAC80694) from T. harzianum. Phylogenic analyses based on the amino acid sequences of SprT and representative serine proteases from Trichoderma and nematophagous fungi were performed, and these proteases appeared to be divided into three groups in the tree (Fig. 4). Nematicidal serine proteases (Mlx, PII, Aoz1) from nematode-trapping fungi formed one group, antifungal serine proteases (PRB1, Tvsp1) from Trichoderma formed another group, and nematicidal serine proteases (PR1B, pSP-3, Ver112, PR1A, and VCP1) from parasitic fungi and protease SprT from T. pseudokoningii SMF2 formed the third group. SprT was most closely related to PR1B, but was located far from the proteases from Trichoderma. The above analyses suggest that SprT is a novel serine protease with nematicidal activity from Trichoderma.

image

Figure 4.  Phylogenetic analysis of SprT and the serine proteases from nematophagous fungi. The phylogenetic tree was constructed by the neighbor-joining method using mega 3.1 software (Kumar et al., 2004). The PAM model and 1000 bootstrap replicates were used. The bootstrap percentages >50% were indicated on the branches. PR1B, Metarhizium anisopliae, AAC49831; PR1A, M. anisopliae, CAC95049; VCP1, Pochonia chlamydosporia, CAD20578; pSP-3, Paecilomyces lilacinus, AAA91584; Ver112, Lecanicillium psalliotae, Q68GV9; PRB1, Trichoderma atroviride, AAA34211; Tvsp1, Trichoderma virens, AAO63588; PII, Arthrobotrys oligospora, CAA63841; Aoz1, A. oligospora, AAM93666; Mlx, Monacrosporium microscaphoides, AAW21809.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Authors' contribution
  9. References
  10. Supporting Information

Trichoderma is a good candidate for the biological control of plant-parasitic nematodes. Extracellular proteases and also nonenzymatic virulence factors were reported to be associated with the nematicidal ability of Trichoderma (Meyer et al., 2000; Sharon et al., 2001; Suarez et al., 2004). However, information on the nematicidal virulence factors from Trichoderma is still insufficient. In this study, the crude extract of a solid-state ferment of T. pseudokoningii SMF2 was shown to display strong nematicidal ability against root-knot nematode M. incognita. It could kill J2s and inhibit hatching of early-stage eggs, but had no inhibitory effect on hatching of last-stage eggs (data not shown). The eggshell structures of early-stage eggs were changed on treatment with the crude extract. Similar results have been observed in the incubation of nematode eggs with serine protease VCP1 from Verticillium chlamydosporium, and also proteases from P. lilacinus (Segers et al., 1994; Khan et al., 2004). The proteolytic activities produced by T. harzianum were suggested to be responsible for the biocontrol activity against nematodes (Sharon et al., 2001), indicating that proteases might play a role in the nematicidal activity of Trichoderma. Correspondingly, a serine protease SprT with nematicidal activity was isolated from the crude extract of T. pseudokoningii SMF2.

Nematicidal serine proteases from nematode parasitic fungi have been extensively studied, and most of these proteases are subtilases with similar biochemical characteristics and similar sequences (Yang et al., 2007). In contrast, reports on proteases from Trichoderma with nematicidal activity are rather less. The cDNA gene cloning and sequence analysis showed that protease SprT from strain SMF2 is a novel subtilase. It shows higher identities and is more related to the serine proteases from parasitic fungi than to those from Trichoderma. In addition, SprT has biochemical characteristics that are similar to the serine proteases from nematode parasitic fungi. It was stable in an alkaline environment and possessed good long-term thermostability, remaining active for several days at 40 °C. Protease SprT also exhibited good proteolytic ability, and many metal ions commonly present in soil could enhance its activity. SEM observation showed that SprT has cuticle-degrading ability, because M. incognita treated with SprT showed structural changes in the nematode cuticle, and this is consistent with the report that serine proteases from nematode parasitic fungi are involved in the penetration of nematode cuticles (Yang et al., 2007).

When the crude extract and purified protease SprT with the same protease activity were used to treat M. incognita J2s, protease SprT showed lower nematicidal activity than the crude extract, which indicated that protease SprT was an important, but not the only virulence factor of T. pseudokoningii SMF2 against M. incognita. In recent years, several attempts have been made to use Trichoderma as biocontrol agents to control harmful nematodes (Rao et al., 1998; Spiegel et al., 2006). The strong nematicidal activity of the crude extract of strain SMF2 indicates that it has potential as a biological nematocide. Currently, its nematicidal effect in the greenhouse and field is being studied.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Authors' contribution
  9. References
  10. Supporting Information

The work was supported by the Hi-Tech Research and Development program of China (2007AA091504), the National Natural Science Foundation of China (30870047), and the Specialized Research Fund for the Doctoral Program of Higher Education (20060422053).

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Authors' contribution
  9. References
  10. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Authors' contribution
  9. References
  10. Supporting Information

Fig. S1. Colony of strain SMF2 on PDA plate after cultivation at 28°C for 1, 2, 3, 4 and 5 days.

Fig. S2. Conidia aggregation (a), conidia (b), conidiophore (c), and mycelium (d) morphology of Trichoderma spp.

Fig. S3. Morphological change of Meloidogyne incognita J2s caused by crude extract of Trichoderma pseudokoningii SMF2 solid ferment.

Fig. S4. The optimum temperature and pH of protease SprT.

Fig. S5. Nucleotide sequence of gene sprt and its deduced amino acid sequence.

Table S1. Primer sequences for cloning of 18S rRNA gene and ITSs gene of strain SMF2.

Table S2. Partial results of the 18S rRNA gene sequence of strain SMF2 aligned with that of a number of Trichoderma strains in the GenBank&sol;EMBL.

Table S3. Partial results of the ITS sequence of strain SMF2 aligned with that of a number of Trichoderma strains in the GenBank&sol;EMBL.

Table S4. Primer sequences for cloning of sprt gene.

Table S5. Purification of protease SprT from Trichoderma pseudokoningii SMF2.

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