Extracellular proteases from Streptomyces phaeopurpureus ExPro138 inhibit spore adhesion, germination and appressorium formation in Colletotrichum coccodes

Authors

  • S.A. Palaniyandi,

    1. Center for Nutraceutical and Pharmaceutical Materials, Myongji University, Yongin, Gyeonggi-Do,, Korea
    2. Division of Bioscience and Bioinformatics, College of Natural Science, Myongji University, Yongin, Gyeonggi-Do,, Korea
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  • S.H. Yang,

    1. Center for Nutraceutical and Pharmaceutical Materials, Myongji University, Yongin, Gyeonggi-Do,, Korea
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  • J.-W. Suh

    Corresponding author
    1. Division of Bioscience and Bioinformatics, College of Natural Science, Myongji University, Yongin, Gyeonggi-Do,, Korea
    • Center for Nutraceutical and Pharmaceutical Materials, Myongji University, Yongin, Gyeonggi-Do,, Korea
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Correspondence

Joo-Won Suh, Division of Bioscience and Bioinformatics, College of Natural Science, Myongji University, Cheoin-gu, Yongin, Gyeonggi-Do 449-728, Korea.

E-mail: jwsuh@mju.ac.kr

Abstract

Aim

To study the antifungal mechanism of proteases from Streptomyces phaeopurpureus strain ExPro138 towards Colletotrichum coccodes and to evaluate its utilization as biofungicide.

Methods and Results

We screened proteolytic Streptomyces strains from the yam rhizosphere with antifungal activity. Forty proteolytic Streptomyces were isolated, among which eleven isolates showed gelatinolytic activity and antagonistic activity on C. coccodes. Of the 11 isolates, protease preparation from an isolate designated ExPro138 showed antifungal activity. 16S rDNA sequence analysis of the strain showed 99% similarity with Streptomyces phaeopurepureus (EU841588.1). Zymography analysis of the ExPro138 culture filtrate revealed that the strain produced several extracellular proteases. The protease preparation inhibited spore germination, spore adhesion to polystyrene surface and appressorium formation. Microscopic study of the interaction between ExPro138 and C. coccodes revealed that ExPro138 was mycoparasitic on C. coccodes. The protease preparation also reduced anthracnose incidence on tomato fruits compared with untreated control.

Conclusion

This study demonstrates possibility of utilizing antifungal proteases derived from antagonistic microbes as biofungicide.

Significance and Impact of the Study

Microbial proteases having the ability to inhibit spore adhesion and appressorium formation could be used to suppress infection establishment by foliar fungal pathogens at the initial stages of the infection process.

Introduction

Colletotrichum is one of the most economically damaging groups of phytopathogenic fungi that cause anthracnose on a number of crop plants (Kleemann et al. 2008). The conidia of Colletotrichum species are dispersed by raindrop splashes and adhere to the aerial parts of the host plant to initiate infection (Hughes et al. 1999). Colletotrichum spores have extracellular matrix (ECM) on their spore coat (Hutchison et al. 2002), which is essential for conidial attachment to host surface. The attached conidia form appressorium to establish infection (Sanogo et al. 2003). Colletotrichum coccodes causes anthracnose in tomato and induces disease symptoms in ripe fruits and mortality in seedlings (Redman and Rodriguez 2002). The disease symptoms can be seen as dark, sunken and circular lesions; as the disease progresses, black dots may be visible on the inner side of the lesions in tomato fruits (Chapin et al. 2006). These symptoms cause rejection of the tomato by the consumers (Chapin et al. 2006). Temperature, wetness, inoculum density and relative humidity are important factors that lead to infection by C. coccodes and disease development (Dillard 1989). When the disease is not properly managed, the yield loss can be up to 70% (Chapin et al. 2006).

The pathogenicity factor responsible for anthracnose symptoms on tomato fruits is a 78-kDa serine protease secreted by C. coccodes (Redman and Rodriguez 2002). C. coccodes establishes infection by local modulation of pH by ammonia secretion (Alkan et al. 2008), which in turn activates host NADPH oxidase activity and reactive oxygen species, enhancing host tissue decay (Alkan et al. 2009). Ammonium production by C. coccodes begins with the secretion of fungal protease that degrades host proteins and subsequent deamination of the amino acids (Prusky and Yakoby 2003).

Control of C. coccodes has been achieved through chemical fungicides, such as azoxystrobin, chlorothalonil, fixed copper and mancozeb (Chapin et al. 2006). However, C. coccodes has been reported to be unresponsive to chlorothalonil and mancozeb (Ingram et al. 2011), indicating development of fungicide resistance. An alternative to the use of chemical fungicides has been the use of biofungicides. Control of tomato anthracnose caused by C. coccodes has previously been reported with methyl jasmonate (Tzortzakis 2007), ozone (Tzortzakis et al. 2008) and Origanum vulgare oil vapour (Tzortzakis 2010). However, to the best of our knowledge, there are no reports on biofungicides of anthracnose disease in tomato.

Proteases produced by antagonistic microbes that degrade the extracellular matrix and block the infection processes of C. coccodes could be used as biofungicides of tomato anthracnose. Keeping these points in mind, we randomly screened for proteolytic Streptomyces from the yam rhizosphere. Streptomyces are among the important group of bacteria that are as such or their derivatives developed in to biofungicides. In this study, we report a C. coccodes-mycoparasitic Streptomyces phaeopurpureus strain designated ExPro138 that produced a battery of extracellular proteases that inhibit spore germination, spore adhesion, appressorium formation and infection establishment by C. coccodes on tomato fruits.

Materials and methods

Fungal pathogen

The fungal pathogen C. coccodes KACC40802 was obtained from RDA, Suwon, South Korea. C. coccodes was grown on Mathur's medium (2·5 g of MgSO4 · 7H2O, 2·7 g of KH2PO4, 1·0 g of Bacto peptone, 1·0 g of yeast extract, 10 g of sucrose per liter) containing chloramphenicol (250 mg l−1) at 28°C. Ten-day-old plate cultures were flooded with sterile distilled water containing 0·2% (v/v) Tween 80, conidia were scraped gently and the suspension was collected in a tube. Spore density was adjusted to 106 spores ml−1.

Screening and isolation of antifungal proteolytic actinobacteria from yam rhizosphere

Actinobacteria were isolated from soil adhering to yam roots collected from the Yeoju region, South Korea, using humic acid vitamin agar (HVA), following the method described by Hayakawa and Nonomura (1989). Screening for proteolytic activity of the colonies obtained in HVA plates was performed by transferring the colonies to skim milk agar. The plates were incubated at 28°C for 7 days. The cultures showing zones of clearance were screened for gelatinolytic activity by transferring the cultures to gelatinase screening medium containing 1% (w/v) gelatin, 0·5% (w/v) glucose, 0·1% (w/v) yeast extract, 0·35% (w/v) K2HPO4, 0·1% (w/v) KH2PO4 and 0·05% (w/v) MgSO4·7H2O. The petri dishes were incubated for 5 days. The gelatin plates were stained with Coomassie Brilliant Blue (CBB) R-250 for 30 min. The plates were subsequently destained with solution containing methanol, water and acetic acid in the ratio 5 : 4 : 1. The gelatinolytic colonies showing a zone of clearance were isolated and studied further for their antagonistic activity on C. coccodes.

In vitro antagonistic activity of selected proteolytic bacteria on Colletotrichum coccodes

The selected proteolytic isolates were evaluated for their antagonistic activity on C. coccodes by dual-culture in vitro assay on PDA plates. The actinobacteria were cultured on Bennett's agar medium (glucose 10 g l−1, yeast extract 1 g l−1, peptone 2 g l−1, beef extract 1 g l−1 and agar 15 g l−1) for 4 to 7 days and further inoculated by streaking on PDA at 1 cm from the edge of each petri dish. The inoculated petri dishes were incubated at 28°C for 3 days, and an agar block (0·5 cm in diameter) containing C. coccodes mycelia was placed at 5 cm from the actinobacteria. C. coccodes was also inoculated in a separate plate without actinobacteria as negative control. The dishes were incubated at 28°C for 7 days and examined for inhibition zone between the fungus and the actinobacteria. The level of inhibition was determined as reported previously (Palaniyandi et al. 2011).

Production and extraction of protease

The proteolytic actinobacterial isolates were cultured in liquid medium containing 0·5% (w/v) gelatin, 0·5% casein, 1% (w/v) glucose, 0·1% (w/v) yeast extract, 0·35% (w/v) K2HPO4, 0·1% (w/v) KH2PO4 and 0·05% (w/v) MgSO4•7H2O for 4 days at 28°C under shaking conditions (170 rev m−1). After 4 days, the culture was centrifuged at 13 000 g for 10 min to remove the actinobacterial mycelium. The supernatant was collected, and solid ammonium sulfate was added to 90% saturation and placed overnight at 4°C to salt out the proteins. The precipitated proteins were collected by centrifugation at 13 000 g for 30 min and reconstituted with 50 mmol l−1 Tris-HCl (pH 7·4) buffer. The protein solution was dialysed against a large volume of 50 mmol l−1 Tris-HCl buffer at 4°C for 18 h using a cellulose acetate dialysis membrane of 10-kDa cut-off. The dialysed crude protease preparation was used for further study.

Antifungal activity of protease preparation on Colletotrichum coccodes

Antifungal activity of the protease preparation was studied by agar diffusion assay. A well was cut at the centre of a petri dish containing PDA medium and was loaded with 200 μl of protease preparation from the actinobacterial strains. Three agar blocks of the same size containing C. coccodes were cut out from a profusely grown fungal plate, placed at equal distance from the central well, allowed to diffuse for 30 min at 4°C and incubated at 28°C for 5 days. Zone of inhibition was considered as a measure of antifungal activity (Singh and Chhatpar 2011).

Determination of protease activity

Collagenase/Gelatinase assay using azocollagen

Azocoll (azo dye-impregnated collagen) (Sigma-Aldrich, St Louis, MO, USA) degradation was performed as previously described by (Pruteanu et al. 2011).

Endoprotease activity assay using azocasein

Endoprotease activity was performed using azocasein following the procedure described by Megazyme (manufacturer of azocasein).

Detection of proteolytic activity by gelatin and casein zymography

Gelatin and casein zymography was performed as described by (Brown et al. 1990). Electrophoresis was carried out on a Mini-PROTEAN Tetra System (Bio-Rad, Hercules, CA, USA) and 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) containing 1 mg ml−1 gelatin (Sigma-Aldrich) or casein 1 mg ml−1. Aliquots of culture supernatant from the actinobacterial culture were mixed with sample buffer and applied to the polyacrylamide gel. Electrophoresis was run at 125 V constant voltages. Following electrophoresis, zymogram gels were washed in 2·5% Triton X-100 for 1 h to remove SDS and renaturation of the proteins. Subsequently, the gels were incubated in a developing buffer (50 mmol l−1 Tris-HCl, pH 7·6, containing 0·2 mol l−1 NaCl, 5 mmol l−1 CaCl2 and 0·02% (w/v) Brij-35) at 37°C for 4 h. Gels were stained with Coomassie brilliant blue R-250 and destained with distilled water. The resulting zymograms displayed clear zones on a blue (dark) background of intact substrate where proteolytic activity had occurred, and the substrate in the gel was degraded. Estimation of the molecular weight of the proteolytic enzymes by SDS-PAGE was performed using the Precision Plus protein standards (Bio-Rad).

Identification of the antagonistic strain

Genomic DNA was isolated from strain 138 using a bacterial genomic DNA isolation kit (CoreBio, Seoul, South Korea), following the manufacturer's protocol. The 16S rRNA gene was amplified by PCR using the following primers: 27F (forward), 5′-AGAGTTTG ATCATGGCTCAG-3′ and 1492R (reverse), 5′-AAGGAGGTGATCCARCCGCA-3′ (Coombs and Franco 2003). The PCR products were electrophoresed on a 1·2% agarose gel, purified with a gel elution kit and sequenced directly using the primers 27F and 785F (GGATTAGATACCCCGGTA) on an ABI 3730XL Capillary DNA Sequencer (Applied Biosystems, Foster City, CA, USA). The 16S rDNA sequence was searched for similarities to known sequences in the GenBank database (National Center for Biotechnology Information, National Library of Medicine) using the BLAST search program. The sequence was aligned with those of the reference strains using ClustalW (Thompson et al. 1994). A phylogenetic tree was constructed by the neighbour-joining method (Saitou and Nei 1987) using MEGA 5 software (Tamura et al. 2011).

Effect of strain 138 protease preparation on Colletotrichum coccodes spore germination, spore adhesion to hydrophobic surface and appressorium formation

The effect of protease preparation on C. coccodes spore germination was studied as described in Palaniyandi et al. (2011) with modifications. Twenty microlitres of spore suspension (105 spores ml−1) was mixed with 20 μl of a medium containing 0·4% (w⁄v) yeast extract and sucrose, with 25 μl of the protease preparation on a sterile microscope glass slide, and placed under sterile plastic petri dishes. The petri dishes were incubated at 28°C in a plastic tray containing sterile filter paper soaked with water to maintain humidity. After 24 h of incubation, total number of spores and number of germinated spores were counted using a hemocytometer, and the percentage of germinated spores was calculated. Spores untreated with protease preparation or treated with protease preparation inhibited with EDTA or Phenylmethanesulfonyl fluoride (PMSF) served as control.

The effect of protease produced by strain 138 on C. coccodes spore adhesion to hydrophobic surfaces was studied using the procedure described by Shimoi et al. (2010) with modifications. Fifteen microlitre of C. coccodes spore suspension (approx. 200 spores μl−1) was placed on a polystyrene slide together with 15 μl of protease preparation. Spore suspension mixed with distilled water or with boiled protease preparation and placed on polystyrene slide served as control. The set-up was incubated at 28°C in a humidified chamber for 4 h. After incubation, the number of spores that adhered to the polystyrene slide was counted under a phase-contrast microscope. The slides were then washed in distilled water with shaking for 100 rotations. After washing, the conidia on the polystyrene slides were counted again. The percentage of adhered conidia was calculated by the following formula:

% adhered conidia = conidial count after washing/conidial count before washing × 100. The experiment was performed three times to get confirmatory results. Appressorium formation was observed after 12 h of incubation.

Microscopic examination of interaction between Colletotrichum coccodes and strain 138

The interaction between strain 138 and C. coccodes was studied using a phase-contrast microscope (Olympus, Tokyo, Japan) and scanning electron microscope (SEM) (Hitachi S-3500N, Tokyo, Japan). A 10-μl spore suspension (104 spores ml−1) of C. coccodes was mixed with 20 μl of half-strength potato dextrose broth (PDB) and 10 μl of strain 138 spore suspension (104 spores ml−1), kept on a sterile microscope glass slide and placed under sterile plastic petri dishes. The petri dishes were incubated at 28°C in a humidified chamber. The slides were observed every 12 h for 3 days. Images were taken at regular intervals.

For SEM analysis, spore suspensions of C. coccodes and strain 138 were mixed and place on a 1-cm filter paper disc loaded with 50 μl of PDB. The filter paper discs were kept in sterile petri dishes and incubated at 28°C in a humidified chamber for 3 days. After 3 days, the filter discs were treated with osmium tetraoxide to fix the fungal and strain 138 mycelium and freeze-dried overnight. Photomicrographs of the samples were taken with a SEM at a voltage of 20 kV.

Effect of strain 138 protease preparation on tomato fruit infection by Colletotrichum coccodes

Fully ripe mature cherry tomato fruits were used for the study of anthracnose suppression by the protease preparation. The tomato fruits were washed in running tap water and 0·1% detergent and surface-sterilized to remove epiphytic microbes. The surface sterilization procedure included submerging the fruits in 0·05% sodium hypochlorite solution for 5 min with occasional shaking, followed by 70% ethanol for 1 min and washing in sterile distilled water for 5 times, 5 min each. The surface-sterilized fruits were treated with strain 138 protease preparation by spraying. The treated fruits were injured by piercing the skin with a sterile toothpick. The fruits were then inoculated with spores of C. coccodes by spraying with an atomizer, kept in a tray with sufficient humidity to avoid drying of the fruits and incubated at 28°C under dark condition. The control was inoculated with the pathogen the same way as described above and received no other treatment. The tomato fruits were also treated with protease preparation inhibited with EDTA or PMSF and benomyl 100 μg ml−1. The effect of the protease preparation on reduction in disease incidence on tomato fruits was assessed after 7 days of challenge inoculation with the pathogen. Incidence of infection was assessed as the percentage of infected tomato fruits among the total number of tomato fruits in each treatment group.

Statistical analysis

The experiments were repeated thrice to obtain confirmable results. Data were log10-transformed and subjected to one-way-anova. Comparison of means was made using the Tukey test using SPSS version 19 (IBM SPSS statistics).

Results

Proteolytic and antagonistic activity of Streptomyces isolates

A number of colonies showed proteolytic activity, which appeared to be common among the root-inhabiting Streptomyces. Forty proteolytic Streptomycete isolates were collected (Fig. S1). All of the 40 isolates were screened for gelatinolytic activity and antagonistic activity against C. coccodes. Among the 40 isolates, 11 isolates (3, 14, 36, 64, 100, 126, 132, 136, 138, 140 and 142) showed both gelatinolytic activity (Fig. S2a) as well as antagonistic activity (Fig. S2b) and were chosen for further study.

Antifungal activity of protease preparation on Colletotrichum coccodes

Among the 11 actinobacteria tested, the protease preparation from strain 138 showed significant inhibition of fungal growth in the well diffusion assay. The effect of the strain 138 protease preparation on the growth of advancing mycelium was observed to be not so strong (Fig. S3).

Protease activity of strain 138

The proteolytic activity of the culture filtrates from strain 138 was analysed using azocasein and azocollagen. Strain 138 showed both caseinolytic and collagenolytic activity (Fig. 1). The caseinolytic activity was inhibited by EDTA and PMSF. Inhibition by PMSF was higher than by EDTA (Fig. 1a), indicating the presence of serine proteases in the proteolytic preparation. In case of collagenolytic activity, EDTA caused higher inhibition than PMSF (Fig. 1b). The proteolytic activity of strain 138 culture supernatant is due to a mix of metalloproteases and serine proteases, as evidenced by the inhibition of proteolytic activity by PMSF (a serine protease inhibitor) and EDTA (a metalloprotease inhibitor) on azocasein (Fig. 1a) and azocollagen (Fig. 1b) degradation.

Figure 1.

Proteolytic activity of actinobacterial culture filtrates and the inhibitory effect of EDTA and PMSF. (a) Degradation of azocasein and (b) degradation of azocollagen. Data shown are average from three independent replicates. Error bars indicate standard deviation. An asterisk, double asterisk and no asterisk indicate significantly different from each other at < 0·05. (■) No inhibitor; (image_n/jam12212-gra-0001.png) EDTA and (□) PMSF.

Detection of proteolytic activity by zymography

Determination of proteolytic activity of the strain 138 revealed that the strain produced several extracellular proteases (Fig. 2). The zymogram patterns were different for gelatin zymography and casein zymography, indicating several enzymes in the protease preparation. Several proteolytic bands were not detected in SDS-PAGE (lane 1) stained with CBB (Fig. 2). Two prominent bands were observed in the CBB-stained SDS-PAGE, corresponding to the protease bands observed in the gelatin gel between 50 and 75 kD (Fig. 2). A larger band at the start of the resolving gel was observed, and a corresponding proteolytic band was observed in the gelatin and casein gel, indicating a multienzyme complex (Fig. 2).

Figure 2.

Electrophoretic analysis of proteases from ExPro138 culture filtrate precipitated with ammonium sulphate. Electrophoretic analysis of the protease preparation was carried out in a 10% (w/v) polyacrylamide gel. Gels were stained with Coomassie Brilliant Blue (CBB) R-250 and destained with distilled water. (M) Molecular weight marker, (A) protease preparation separated and stained with CBB R-250 in SDS-PAGE, (B) gelatin zymography of 138 protease preparation and (C) casein zymography of ExPro138 protease preparation. Estimated molecular weights of protease bands (1) 261 kD; (2) 68 kD; (3) 55 kD; (4) 176 kDa; and (5) 46 kD.

Identification of the antagonistic strains

The 16S rDNA sequence of strain 138 was searched for similarities with known sequences in GenBank using BLAST. Strain 138 (GenBank accession no. KC466283) shared 99% similarity to Streptomyces 16S rRNA gene sequences. Furthermore, nucleotide sequence alignment and phylogenetic analysis showed that the strain was closely related to S. phaeopurpureus HBUM174561 (EU841588.1) (Fig. 3).

Figure 3.

16S rDNA phylogenetic tree, showing the relationship between isolate ExPro138, and other Streptomyces sp. The tree was constructed using neighbour-joining method using the MEGA 5 program. Kimura-2 parameter was used as the nucleotide substitution model. The bootstrap values (%) presented at the branches were calculated from 1000 replications.

Inhibition of Colletotrichum coccodes spore germination, adhesion to hydrophobic surfaces and appressorium formation by strain 138 protease preparation

The protease preparation showed inhibition of spore germination of C. coccodes (Fig. 4). Treatment of C. coccodes spores with protease preparation from strain 138 showed inhibition of germination (Fig. 4b). When the protease was treated with EDTA or PMSF (Fig. 4c, d), spore germination was observed to the level of germination in the control (Fig. 4a).

Figure 4.

Effect of ExPro138 protease preparation on germination of Colletotrichum coccodes spores. (a) Control, (b) C. coccodes spores treated with the protease preparation, (c) C. coccodes spores treated with protease preparation inhibited with EDTA and (d) C. coccodes spores treated with protease preparation inhibited with PMSF.

Treatment of the spores with protease preparation from strain 138 significantly reduced the percentage of C. coccodes spore adhesion to polystyrene slides (Fig. 5b) compared with untreated control (Fig. 5a). The effect of the protease on spore adhesion was reversed when the preparation was boiled for 5 min before mixing with the spores (Fig. 5c). Treatment of the spores with the protease preparation also significantly affected appressorium formation, as evidenced by the reduced number of appressorium in the protease-treated spores (Fig. 5e) compared with untreated (Fig. 5d) and spores treated with heat-inactivated protease preparation (Fig. 5f).

Figure 5.

Effect of ExPro138 protease on spore adhesion to hydrophobic surface and appressorium formation. Spore adhesion to polystyrene surface in (a) untreated control, (b) spores treated with protease preparation and (c) spores treated with boiled protease preparation. Appressorium formation in (d) untreated control, (e) spores treated with protease preparation and (f) spores treated with boiled protease preparation. Black arrows indicate nongerminated spores and white arrows indicate appressorium. Error bars indicate standard deviation of the value obtained for three independent replicates. An asterisk, double asterisk and no asterisk in bar charts indicate significantly different from each other at < 0·05. Scale bar indicates 10 μm.

Interaction between strain 138 and Colletotrichum coccodes

The microscopic study of strain 138 and C. coccodes showed that 138 was able to parasitize the C. coccodes mycelium (Fig. 6). After 24 h, the C. coccodes mycelium was well established, and parasitization by strain 138 was not observed (Fig. 6a). However, after 2 days, the strain 138 mycelium showed profuse growth and colonization of the fungal mycelium (Fig. 6b). After 3 days, strain 138 could completely parasitize the C. coccodes mycelium, and lysis of the fungal mycelium was observed (Fig. 6c). The parasitized fungal mycelium had lost its cellular integrity compared with the nonparasitized mycelium (Fig. 6c). Strain 138 also showed penetration into fungal mycelium and intramycelial growth (Fig. 6d). SEM observation of the strain 138 and C. coccodes interaction at 72 h also showed coiling of the strain 138 mycelium around the fungal mycelium (Fig 6e), lysis and distortion of the fungal mycelium (Fig. 6f).

Figure 6.

Mycoparasitism of Colletotrichum coccodes by ExPro138 observed by phase-contrast and scanning electron microscopy: a, C. coccodes before parasitization (24 h); b, colonization of C. coccodes mycelia by strain 138 mycelia (48 h); c, parasitization of C. coccodes by strain 138 (72 h) showing coiling (thick arrow) of fungal mycelium by strain 138 mycelium and haustorium-like structure (small white arrows) formed by strain 138; d, penetration of fungal mycelium by strain 138 mycelium (white arrow) (72 h); (e) SEM picture showing coiling (72 h) of C. coccodes mycelium by strain 138 mycelium and lysis; (f) Lysis of C. coccodes mycelium (72 h). a – b, white arrows indicate strain 138 mycelium, and black arrows indicate C. coccodes mycelium; e – f, discontinuous arrows indicate lysis or disruption of C. coccodes mycelium. Scale bar indicates 10 μm.

Effect of strain 138 protease preparation on anthracnose development on tomato fruits

Protease preparation from strain 138 culture filtrate lowered the incidence of anthracnose development on tomato fruits after 5 days of challenge inoculation with C. coccodes (Fig. 7c). When the protease preparation was treated with EDTA or PMSF, the incidence of C. coccodes infection was observed to be equal to that of the pathogen-challenged control fruits (Fig. 7b, d, and e), indicating the involvement of protease in the antifungal activity. The disease incidence of protease-treated group was higher compared with benomyl (Fig. 7f).

Figure 7.

Effect of protease preparation on tomato fruits infection by Colletotrichum coccodes. (a) Noninoculated tomato fruits, (b) C. coccodes inoculated, (c) C. coccodes + protease preparation-treated, (d) C. coccodes + protease + EDTA-treated, (e) C. coccodes + protease + PMSF-treated, (f) C. coccodes + benomyl-treated. An asterisk indicates significantly different from C. coccodes inoculated group at < 0·05.

Discussion

The fungal cell wall consists of chitin, glucan, lipids and proteins. Hydrolytic enzymes that cleave these molecules have been reported to have antifungal activity (Haran et al. 1996). Proteases produced by antagonistic microbes play a significant role in their biocontrol activity (Flores et al. 1997; Pozo et al. 2004). In this article, we present the results of the study on the antifungal activity of the extracellular proteases from a S. phaeopurpureus strain designated ExPro138. The observation of low antifungal activity of the protease preparation (Fig. S3) compared with the antagonistic activity of strain 138 on C. coccodes (Fig. S2b) indicates that mechanisms other than protease activity may also be involved in the antagonism of the strain. However, the strain 138 was not observed to produce chitinase or glucanase (data not shown). Butanol extract of the culture filtrate did not show antifungal activity, but the strain produced siderophores (Data S1). Synergistic action of hydrolytic enzymes and siderophores can enhance the biocontrol efficacy of the antagonistic strain. Trichoderma harzianum cell wall hydrolytic enzymes were reported to act synergistically with fungitoxic substances and inhibit Botrytis cinerea (Lorito et al. 1994).

A study of the effect of the protease preparation on spore germination, spore adhesion and appressorium formation showed that the protease preparation significantly reduced spore germination (Fig. 4b), spore adhesion to hydrophobic surfaces (Fig. 5b) and appressorium formation (Fig. 5e). In addition, inhibition of this activity by EDTA or PMSF or heat inactivation further confirms the involvement of protease activity in reducing spore germination, adhesion and appressorium formation. Colletotrichum spores have been reported to possess preformed extracellular matrix (ECM) on the spore coat, which is necessary for adhesion to host surfaces (Hutchison et al. 2002). Spore adhesion to the host surface is the first step in the infection process and the adhered spores germinate and form appressorium (Shimoi et al. 2010). Disruption of the ECM will affect spore adhesion, which has been demonstrated by Hughes et al. (1999) with monoclonal antibodies generated against the spore surface glycoprotein of Colletotrichum lindemuthianum, and poor spore adhesion will affect appressorium formation (Bae et al. 2007). Inhibition of spore adhesion, appressorium formation and spore germination by protease preparation from strain 138 could be due to disruption of ECM proteins.

The mycoparasitic activity of strain 138 demonstrated in this study could be due to the synergistic activity of proteases. Protease production by antagonistic microbes has been implicated in mycoparasitism (Geremia et al. 1993). Streptomyces sp., such as Streptomyces lydicus WYEC108 (Yuan and Crawford 1995), Streptomyces griseus (Tu 1988) and Streptomyces griseoviridis (Tapio and Pohto-Lahdenperä 1991), have previously been reported to cause hyphal lysis and mycoparasitism upon direct interaction with pathogenic fungi. Mechanisms other than protease production were reported to play roles in mycoparasitism in all three afore-mentioned cases. Despite several reports available on antifungal activity of chitinases and glucanases from Streptomyces sp. (Trejo-Estrada et al. 1998a,b; Yang et al. 2005), the antifungal activity of a 20-kD protease isolated from Streptomyces sp. A6 has been the only available report on antifungal activity of a protease from a Streptomyces strain (Singh and Chhatpar 2011).

The strain 138 exhibited mycoparasitism by the formation of specialized haustorium-like structures, coiling of the fungal mycelium, penetration into the fungal mycelium and lysis (Fig. 6). A recent report showed that Streptomyces strains showing mycoparasitic activity against Verticillium dahliae exhibited mechanisms such as coiling around the fungal mycelium and lysis (Xue et al. 2013).

A study of the effect of the protease preparation on anthracnose development in tomato fruits by C. coccodes showed the effectiveness of the protease preparation in suppressing anthracnose. The protease preparation reduced the incidence of anthracnose on tomato fruits. Inhibition of this activity by EDTA or PMSF confirms the involvement of proteases in the reduction in disease incidence. The mechanism of action of the proteases could be at the level of initiation of infection by C. coccodes. Inhibition of the spore adhesion leads to less number of spore germination and appressorium formation on the plant surface that will result in low disease incidence. Appressorium formation by C. coccodes is an essential step in the infection of tomato fruits and development of anthracnose. Inhibition of appressorium formation by higher temperature (35°C) reduced the anthracnose symptoms on tomato fruits (Sanogo et al. 2003). A previous report by Shimoi et al. (2010) demonstrates a similar mechanism in that detachment of spore adhesion by proteolytic strains resulted in biological control of rice blast disease caused by Magnaporthe oryzae. Involvement of proteases in biological control has been well documented in T. harzianum (Elad and Kapat 1999) and Stenotrophomonas maltophilia (Dunne et al. 1997). The T. harzianum T39 protease imparted a 55% reduction in spore germination and 80% reduction in germ tube length in B. cinerea and reduced disease symptoms on bean leaves (Elad and Kapat 1999). S. maltophilia W81 inhibited the growth of Pythium ultimum by the production of extracellular protease and protected sugar beet from damping-off caused by P. ultimum. In the present study, we observed an average 50% reduction in anthracnose incidence on tomato fruits upon treatment with S. phaeopurpureus ExPro138 protease. Benomyl treatment at 100 μg ml−1 on average showed higher disease protection; however, statistical analysis showed no significant difference (at < 0·05) between protease treatment and benomyl treatment.

In conclusion, a reduction in anthracnose symptoms on tomato fruits was achieved following treatment with protease from S. phaeopurepureus ExPro138, which exhibits antagonism towards C. coccodes. The protease preparation was shown to be involved in the detachment of spores to hydrophobic surfaces and reduction in germination and appressorium formation by the spores. This mechanism is novel, and the detachment effect of proteases can be utilized to block the pathogen at the initial stages, or it can be used in combination with other hydrolytic enzymes or antibiotics to enhance its efficacy. Further work will be carried out to evaluate the possibility of developing a combination of S. phaeopurpureus ExPro138 protease with other antifungal secondary metabolites to enhance the biofungicidal activity.

Acknowledgements

This work was supported by a grant from the Next-Generation BioGreen 21 Program (No. PJ009007), Rural Development Administration, Republic of Korea. Sasikumar Arunachalam Palaniyandi is supported by the second stage of the BK21 (Brain Korea 21) Project.

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