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Investigating the bioactivity of cells and cell-free extracts of Streptomyces griseus towards Fusarium oxysporum f. sp. cubense race 4
Biological Control 66 (2013) 204–208
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Investigating the bioactivity of cells and cell-free extracts of Streptomyces griseus towards Fusarium oxysporum f. sp. cubense race 4 Fathima Ameena Zacky, Adeline Su Yien Ting ⇑ School of Science, Monash University Sunway Campus, Jalan Lagoon Selatan, 46150 Bandar Sunway, Selangor Darul Ehsan, Malaysia
h i g h l i g h t s First documentation of antifungal
activities of Streptomyces griseus towards FOC race 4. Cells and cell-free extracts of S. griseus have comparable antifungal properties. Chitinases and b-1,3-glucanases are higher in cell-free extracts. Extracts have less bioactivity than cells of S. griseus when applied to soil.
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Article history: Received 30 September 2012 Accepted 3 June 2013 Available online 13 June 2013 Keywords: Antifungal activity Biological control agent Crude extracts Fusarium oxysporum f. sp. cubense race 4 Streptomyces griseus Wilt disease
g r a p h i c a l a b s t r a c t Both the cells and extracts of S. griseus have antifungal activity, inhibiting FOC race 4 via hyphal damage.
a b s t r a c t The antifungal activity of viable cells of Streptomyces griseus (St 4) and its cell-free extracts were investigated against the pathogenic Fusarium oxysporum f. sp. cubense race 4 (FOC race 4), causal agent of wilt disease in bananas. Results from in vitro and soil assays showed cells and cell-free extracts of S. griseus were able to inhibit FOC race 4 with varying degree of success. Antifungal activity was attributed to chitinase and b-1,3-glucanase, detected in both cells and cell-free extracts, which caused lysis of fungal cell wall and inhibited sporulation. Interestingly, b-1,3-glucanase and chitinase activities were significantly higher in cell-free extracts compared to cells, with 8.30 and 5.43 against 7.96 and 4.95 U mL 1, respectively. Application to soil however, showed inoculation using S. griseus cells were more effective in suppressing growth of FOC race 4 than crude extracts, with 6 log10 CFU of FOC race 4 g 1 soil enumerated compared to 7 log10 CFU of FOC race 4 g 1 soil after 20 days. To summarize, this study has shown that cell-free extracts of S. griseus have antifungal properties but may not be suitable for soil application in its current form (liquid suspension). Further investigations on bioformulation may address this limitation. Ó 2013 Elsevier Inc. All rights reserved.
1. Introduction Streptomyces spp. are established biocontrol agents of important soil-borne diseases (Behal, 2000; Doumbou et al., 2001). They have specifically been demonstrated to suppress wilt incidences in cotton, tomato and banana plants (Reddi and Rao, 1971; Getha and Vikineswary, 2001; Anitha and Rabeeth, 2009; Kim et al., 2011). ⇑ Corresponding author. Fax: +60 3 5514 6364. E-mail addresses: [email protected], [email protected] (A.S.Y. Ting). 1049-9644/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.biocontrol.2013.06.001
Streptomyces spp. demonstrate several modes of action with antibiosis and competition for nutrients and space being the most common. On rare occasions, hyphal parasitism has been identified as a mode of action (Tu, 1988; Gonzalez-Franco and Hernandez, 2009). Antibiosis by Streptomyces spp. is the most effective mechanism, where production of antibiotics inhibits the growth of the pathogen efficiently (Genilloud et al., 2011; Nakano et al., 2011; Roberts et al., 2011). In addition, production of extracellular cell-wall degrading enzymes (chitinase, b-1,3-glucanase, cellulase, b-glucosidase, amylase, xylanase and mannase) also inhibits pathogen growth significantly (Spear et al., 1993; Tweddell et al., 1994;
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Sepulveda and Crawford, 1998; Trejo-Estrada et al., 1998; Prapagdee et al., 2008; Gonzalez-Franco and Hernandez, 2009; Shafique et al., 2009). Additionally, some species such as Streptomyces pilosus, have ferrichrome siderophores that allows the sequesteration of iron as a growth limiting factor (Müller et al., 1984). All these contribute to effective biocontrol activity of the Streptomyces spp. Streptomyces spp. are commonly applied by introducing their cell biomass (viable inoculants) into the soil (Reddi and Rao, 1971; Anitha and Rabeeth, 2009). This has resulted in several cases of successful control, but also raises concerns on the possible detrimental effect of Streptomyces spp. due to prolonged purposeful introduction into the soil. One such concern from their purposeful introduction into soil is their inhibitory effect on the indigenous microflora community in the soil, especially beneficial microflora such as mycorrhizal fungi, as continuous bioaugmentation with cells of Streptomyces spp. may result in persistent antibiotic production thus implicating growth of microflora (Fitter and Garbaye, 1994). Whether these effects are transitory or not, is secondary as this will become a limiting factor in bioaugmentation or biofertilization approaches using beneficial microbes. Other concerns include possible transfer of antibiotic resistance (common trait in Streptomyces spp.) to other bacteria, especially with prolonged co-existence with residual cells of Streptomyces spp. (Courvalin, 1994). The other limitation to the use of viable inoculant is quality control and the economical aspect of having to re-culture, maintain and harvest large batches of Streptomyces spp. after every cycle (Sabaratnam and Traquair, 2002). The use of crude extracts produced by Streptomyces spp. present an interesting alternative to address limitations from using cell biomass. In this study, we investigated the antifungal activity of crude extracts from Streptomyces griseus (isolate St 4) against FOC race 4, subsequently comparing the biocontrol activities of both the viable cells and cell-free extracts. Our study is significant as currently, literatures on the potential of using cell-free extracts as a strategy in biocontrol, especially towards wilt disease caused by FOC race 4 is limited. Most research focused on the use of whole cells to render control. There is also novelty in our approach to evaluate the antifungal activity of S. griseus towards FOC race 4 as this far, S. griseus has only been known to inhibit other Fusarium pathogens such as Fusarium sp. causing crown rot (Ting et al., 2010), and tomato wilt (Anitha and Rabeeth, 2010). This paper therefore reports the results of the antifungal activity of cells and cell-free extracts of S. griseus (St 4) towards FOC race 4, demonstrated from in vitro and soil assays. 2. Materials and methods 2.1. Culture establishment The isolate St 4 was streaked on Actinomycetes Isolation Agar (AIA) (Difco) and incubated at room temperature (24 ± 2 °C). This isolate originated from soil and has shown antifungal activity towards Fusarium sp., responsible for crown rot disease of banana (Ting et al., 2010). Based on 16s RNA sequencing, isolate St 4 was identified as Streptomyces griseus (GenBank ID: NC_010572). The pathogenic Fusarium oxysporum f. sp. cubense race 4 (FOC race 4) was obtained from Professor Dr. Sariah Meon (Universiti Putra Malaysia) and established on Potato Dextrose Agar (PDA, Difco) at 24 ± 2 °C. Both isolates were cultured for 7 days prior to use in subsequent tests. 2.2. Extraction of crude extracts from S. griseus One milliliter of S. griseus (108 cfu mL 1) derived from 7-day-old cultures, was inoculated into 250 mL of ISP-2 broth (0.4% glucose
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(F.S. Chemicals), 0.4% yeast extract (Difco), 1.0% malt extract (Oxoid), pH 7). The broth culture was incubated on a rotary shaker (150 rpm, 24 ± 2 °C) for 5 days. Crude extracts were extracted by centrifuging broth culture (9000 rpm) at 0 °C for 20 min. The extracts were further filter-sterilized (0.45 lm filter, Sartorius) prior to use.
2.3. Evaluating antifungal activities of S. griseus and its cell-free extracts in vitro The antifungal activity of S. griseus (St 4) and its cell-free extracts was determined from plate assay, microscopic analysis of hyphal degradation, and soil assay.
2.3.1. Plate assay for detection of non-volatile antifungal properties Antifungal activity of S. griseus (St 4) was detected by streaking loopfuls of S. griseus cells perpendicularly on PDA (Difco) and incubated for 7 days at 24 ± 2 °C. After 7 days, a 0.5 cm-mycelial plug of FOC race 4 was placed 2.5 cm from the streak. The agar plate was then incubated for another 7 days at 24 ± 2 °C. Controls were prepared using sterile distilled water for streaking. The radial growth of FOC race 4 (R2) co-inoculated with S. griseus was calculated against the radial growth co-inoculated with sterile distilled water (R1), and the percentage of inhibition of radial growth (PIRG %) determined (Ting et al., 2010). This test had six replicates for each treatment, and was repeated twice. The antifungal potential of cell-free extracts of S. griseus was also detected using the plate assay. Filter-sterilized extracts (prepared as from Section 2.2) were mixed in double-strength molten PDA based on the following ratio; 1:1, 1:2, 1:3 and 1:4 (crude extracts: double-strength PDA) (Leelasuphakul et al., 2008). Once the agar has solidified, a 0.5 cm-mycelial plug of FOC race 4 was inoculated centrally on the agar. Six replicates was prepared, and plates were incubated for 7 days at 24 ± 2 °C. Control plates (0:1) were prepared and incubated similarly, substituting the extracts with sterile distilled water. The diameter of FOC race 4 (D2) inoculated on agar supplemented with extracts was calculated against the diameter of FOC race 4 on agar with sterile distilled water (D1), and the percentage of inhibition of diameter growth (PIDG %) determined (Ting et al., 2010). This test was also repeated twice.
2.3.2. Detection of hyphal degradation in FOC race 4 Riddell’s (1950) slide culture technique was employed to demonstrate the antifungal activity of S. griseus and its cell-free extracts on the hypha of FOC race 4. Briefly, a square block of PDA (1 1 cm) was placed on a sterile glass slide. This slide was placed on top of two glass rods in a Petri dish, with a 9-cm diameter filter paper (Whatman) positioned at the base of the Petri dish. The filter paper was moistened with 4–5 drops of sterile water. To determine possible hypha degradation upon exposure to cells of S. griseus, one side of the agar block was point-inoculated with loopfuls of S. griseus and the corresponding opposite side with teased mycelium of FOC race 4. A sterile coverslip was then placed on the inoculated agar block. The petri dish was sealed with parafilm and incubated for 5 days at 24 ± 2 °C. After 5 days, the coverslip was removed and placed onto a slide containing two drops of lactophenol cotton blue (Merck) and viewed under light microscope to determine the occurrence of hyphal degradation. Control was established by inoculating FOC race 4 and sterile distilled water (control) on two opposite sides of the block. The procedure was repeated for detection of hyphal degradation by cell-free extracts using agar blocks amended with crude extracts (agar blocks from 1:1, 1:2, 1:3 and 1:4 as in Section 2.3.1). Triplicates were prepared for all assessments. This assessment was repeated twice.
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2.3.3. In vitro testing of bioactivity of S. griseus and its cell-free extracts in the soil Soil mixture comprised of top soil, organic peat (coconut husk) and sand, was prepared according to the ratio of 3:2:1, respectively. The soil mixture was autoclaved twice (121 °C, 15 min) and allowed to cool overnight. The pH of the sterilized soil was measured and modified to pH 5.5 using 1 N HCl and 1 N NaOH. Approximately 200 g of the sterile soil was weighed, placed into an opened polybag and accounted as one replicate. To this soil, 5 mL (108 cfu mL 1) of S. griseus was added and incubated at 24 ± 2 °C for 5 days. After 5 days, 5 mL of FOC race 4 (106 cfu mL 1) was mixed into the soil. Four replicates were prepared. For the next 20 days, viable counts of FOC race 4 were enumerated at every 5day interval. At each sampling interval, 1 g of soil was randomly sampled, serial dilutions performed, and aliquots (0.1 mL) from each dilution factor (10 1 to 10 6) was spread-plated onto PDA supplemented with 50 lg lL 1 streptomycin (Sigma–Aldrich). Agar plates were incubated 3–5 days at 24 ± 2 °C, and FOC race 4 colonies were enumerated (CFU g 1soil) (Asha et al., 2011). Antifungal activities of cell-free extracts were determined using the same method, substituting cell suspension of S. griseus with 5 ml of crude extracts. Controls were also prepared using sterile distilled water as a replacement. This assessment was repeated twice. 2.4. Evaluating chitinase and b-1,3-glucanase activities from S. griseus and cell-free extracts The viable cells and cell-free crude extracts from S. griseus were assayed for chitinase and b-1,3-glucanase activities, to compare their activities and roles in inhibiting FOC race 4. The activity of extracellular chitinases was determined daily for 7 days, using methods described by Imoto and Yagishita (1971). Firstly, 1 ml of 108 cfu mL 1 of S. griseus was inoculated into 250 mL of ISP-2 broth, and incubated on a rotary shaker (150 rpm, at 24 ± 2 °C). To detect chitinase activity from cells of S. griseus, 1 ml of the culture suspension was transferred into 0.1% colloidal chitin. The reaction mixture was incubated in a water bath (40 °C) for 1 h. The reaction was subsequently stopped by adding 0.05% w/v potassium ferricyanide (F.S. Chemicals) (in 0.5 M sodium carbonate (F.S. Chemicals)) and re-boiling for 15 min. After cooling, the reaction mixture was centrifuged (6000 rpm, 10 min) and the absorbance read at 420 nm. Chitinase activity was derived from a standard curve established from N-acetylglucosamine (Sigma–Aldrich) (0– 30 lmol). One unit of chitinase activity is expressed as the amount of enzyme that catalyzes the release of 1 lmol of N-acetylglucosamine in 60 min at 40 °C. The procedure was repeated for cell-free extracts, substituting culture suspension with cell-free supernatant obtained after centrifuging (6000 rpm, 0 °C, 10 min). Six replicates were performed for both cell and cell-free assessments. This assessment was repeated twice. Extracellular b-1,3-glucanase activity was determined according to Singh et al. (1999). Cultures were established similarly as for chitinase assay. At daily intervals, 1 mL of cell suspension was mixed with 1 mL of reagent consisting of 0.9 mL of 0.2 M sodium acetate buffer (pH 5.3) and 0.1 mL of 2% w/v laminarin (Sigma). The reaction mixture (total volume of 2 mL) was incubated for 1 h in a water bath (40 °C). The reaction mixture was stopped by adding 3 mL of 3,5-dinitrosalicylic acid (DNS) (Fluka) (1% w/v 3,5-dinitrosalicylic acid, 30% potassium sodium tartrate (F.S. Chemicals) in 2 M sodium hydroxide (Bendosen)) and boiling (water bath at 100 °C) for 5 min. After cooling to room temperature, the absorbance of the mixture was read at 575 nm. b-1,3-Glucanase activity was derived from a standard curve established using various concentrations of glucose (F.S. Chemicals) as a standard (0–15 lmol) and expressed as the amount of enzyme that catalyzes the release of 1 lmol of glucose in 60 min at 40 °C. For assay
using cell-free extracts, cell-free supernatant used were obtained as described for chitinase assay. Six replicates were performed for both cell and cell-free assessments. This assessment was also repeated twice. 2.5. Statistical analysis The data obtained were analyzed with Analysis of Variance (ANOVA) (p < 0.05) and means compared with Tukey’s test (HSD(0.05)). All statistical analysis was performed using Statistical Analysis Software (SAS). 3. Results and discussion 3.1. Antifungal activities of cells and cell-free extracts of S. griseus The antifungal activities of S. griseus and its cell-free extracts were evident from the plate assay, microscopic analysis on hypha degradation, and soil assay. From these tests, we observed that both cells and cell-free extracts of S. griseus had antifungal activities, with mean inhibition values of 54.6% and 33%, respectively. Our results confirmed the antifungal potential of S. griseus, attributed to the inhibitory compounds produced extracellularly (El-Abyad et al., 1993; Prapagdee et al., 2008). The inferior inhibition values from filtrate-incorporated test compared to streak test is not a concern as it is a common occurrence attributed to the nature of the test (Prapagdee et al., 2008). We also observed that the concentrations of the cell-free extracts also influenced the antifungal activity with more diluted extracts (1:3, 1:4) having poorer inhibitory effect compared to less diluted extracts (1:1, 1:2) (Fig. 1). As such, extracts are used without dilution in all our investigations. Inhibition towards FOC race 4 by both cells and cell-free extracts of S. griseus was the result of the extracellular compounds produced, such as cell-wall degrading enzymes and antibiotics which damaged the hypha. This was evident in our microscopic analysis where hypha of FOC race 4, co-inoculated with cells of S. griseus and on extract-amended agar blocks, showed signs of hypha swelling and cytoplasm aggregation (Fig. 2A). Cell-wall degradation may have also occurred as hypha were not consistently stained blue with Lactophenol Cotton Blue (LCB) indicating fissures along the hyphal wall, unlike in control (Fig. 2B). Poor sporulation was also detected for FOC race 4 exposed to S. griseus or its extracts (Fig. 2A) compared to control (Fig. 2B). Induced hyphal abnormalities (distortion, stunting, hyphal protuberances) and the absence of spores observed are typical symptoms of consequences of lytic activities or antibiotics from biocontrol agents which destroys germ tube or induces lysis of the spore itself (Lockwood and
Fig. 1. Inhibition (%) of FOC race 4 by cell-free extracts incorporated into doublestrength molten PDA. Values are means of six replicates. Means with the same letters are not significantly different according to Tukey’s Studentized Range Test (HSD(0.05)).
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Fig. 2. Hyphal distortion and protuberances, and the absence of spores as result of co-inoculation with S. griseus cells and cell-free extracts (A). Normal hyphal structure and sporulation is detected in control (treated with sterile distilled water) (B). Hyphal structures are stained with Lactophenol Cotton Blue and viewed under light microscopy at 400 magnification. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 3. Effect of cells (cells) and cell-free extracts (extracts) of S. griseus on FOC race 4 in sterile soils. CFU count for FOC race 4 g 1 soil was enumerated for 20 days and compared to control (treatment with sterile distilled water). Values presented are means of four replicates. Vertical bars indicate standard errors. Means with the same letters within the same day of assessment are not significantly different according to Tukey’s Studentized Range Test (HSD(0.05)).
Lingappa, 1963; Debono and Gordee, 1994; Getha and Vikineswary, 2001). These qualitative observations were adequate to indicate that both cells and cell-free extracts from S. griseus have antifungal activities towards FOC race 4. They also strongly suggest the presence of extracellular cell-wall degrading enzymes such as chitinase and b-1,3-glucanases (Reynolds, 1954; Mahadevan and Crawford, 1997; Narayana, 2009), which was subsequently detected and reported in Section 3.2. In the soil assay, the bioactivity of viable cells of S. griseus towards FOC race 4 were detected by day 15–20, as the CFU count for FOC race 4 at day 20 (6 log10 CFU g 1 soil) was significantly lower than in soils from control (7 log10 CFU g 1 soil) (Fig. 3). In control, CFU count for FOC race 4 appeared to have increased in the absence of cells of S. griseus. We observed that treatment with cell-free extracts did not significantly reduce CFU count of FOC race 4 (Fig. 3). A study by Anitha and Rabeeth (2009) reported observations with similar trend where cells of their S. griseus were more effective in controlling Fusarium wilt in tomato than crude or partially purified chitinase enzymes. They attributed this to cells being able to multiply and produce a constant level of enzymes, whereas crude extracts have a finite concentration of antifungal compounds. Our study agreed with Anitha and Rabeeth (2009) as there was a significant decrease in CFU count of FOC race 4 from the use of viable cells compared to extracts.
Fig. 4. Enzymatic activity (U mL 1) of chitinases and b-1,3-glucanase from cell-free extracts and cells of S. griseus. Values for each column are means of 42 samples. Means with the same letters and captions assessed within the same enzyme, are not significantly different according to Tukey’s Studentized Range Test (HSD(0.05)).
3.2. Enzymatic activities of cells and cell-free extracts of S. griseus Higher b-1,3-glucanase activities were detected in both cells and cell-free extracts of S. griseus compared to chitinase activity (Fig. 4). Both these enzymes are signature active enzymes, responsible for the antifungal activities of S. griseus (Trejo-Estrada et al., 1998; Yedidia et al., 1999; Anitha and Rabeeth, 2010). Assays
Fig. 5. Chitinase activities (U mL 1) from cells and cell-free extracts of S. griseus in 7 days. Vertical bars indicate standard errors. Means with the same letters within the same day of assessment are not significantly different according to Tukey’s Studentized Range Test (HSD(0.05)).
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Fig. 6. b-1,3-Glucanase activities (U mL 1) from cells and cell-free extracts of S. griseus in 7 days. Vertical bars indicate standard errors. Means with the same letters within the same day of assessment are not significantly different according to Tukey’s Studentized Range Test (HSD(0.05)).
showed higher b-1,3-glucanase and chitinase activities in cell-free extracts compared to cells, with 8.30 and 5.43 compared to 7.96 and 4.95 U mL 1, respectively (Fig. 4). This suggests the potential for use of extracts as they have similar levels of enzyme activity to S. griseus cells. These enzymes are also responsible for the damage to the hypha and spores of FOC race 4 observed in the microscopic test conducted, by breaking down the main cell wall components; chitin and b-glucans (Mahadevan and Crawford, 1997). We observed that both chitinase and b-1,3-glucanase from extracts have higher activities in the first 2 days, but there were no difference in activities thereafter (Figs. 5 and 6). In fact, both cells and extracts showed declining trend of enzymatic activities from day 1 to day 7 (Figs. 5 and 6). This has also been observed by Prapagdee et al. (2008) and Nahed (2011), suggesting that enzymatic activities contribute to antifungal activities of S. griseus. 4. Conclusion This study has shown that cells and crude extracts of S. griseus have antifungal properties towards FOC race 4. While the S. griseus cells performed well in the in vitro test (plate assay, microscopic analysis, enzymatic assays), crude extracts were ineffective in soil assays despite the high levels of chitinases and b-1,3-glucanases. Hence the most effective biocontrol agent for FOC race 4 in a soil environment would be a pre-treatment of S. griseus cells. Further studies may be carried out to optimize the application of crude extracts such as with the use of bioformulation. Acknowledgments Authors extend their gratitude to Monash University Sunway Campus for the funding to conduct the project. The authors would also like to thank Professor Sariah Meon (Universiti Putra Malaysia) for the provision of pure cultures of FOC race 4. References Anitha, A., Rabeeth, M., 2009. Control of Fusarium wilt of tomato by bioformulation of Streptomyces griseus in green house condition. AJBAS 1, 9–14. Anitha, A., Rabeeth, M., 2010. Degradation of fungal cell walls of phytopathogenic fungi by lytic enzyme of Streptomyces griseus. Plant Sci. 4, 61–66. Asha, B.B., Chandra Nayaka, S., Udaya Shankar, A.C., Srinavas, C., Niranjana, S.R., 2011. Biological control of F. oxysporum f. sp. lycopersici causing wilt of tomato by Pseudomonas fluorescens. Int. J. Microbiol. Res. 3, 79–84. Behal, V., 2000. Bioactive products from streptomyces. Adv. Appl. Microbiol. 47, 113–156.
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Investigating the bioactivity of cells and cell-free extracts of Streptomyces griseus towards Fusarium oxysporum f. sp. cubense race 4 Fathima Ameena Zacky, Adeline Su Yien Ting ⇑ School of Science, Monash University Sunway Campus, Jalan Lagoon Selatan, 46150 Bandar Sunway, Selangor Darul Ehsan, Malaysia
h i g h l i g h t s First documentation of antifungal
activities of Streptomyces griseus towards FOC race 4. Cells and cell-free extracts of S. griseus have comparable antifungal properties. Chitinases and b-1,3-glucanases are higher in cell-free extracts. Extracts have less bioactivity than cells of S. griseus when applied to soil.
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Article history: Received 30 September 2012 Accepted 3 June 2013 Available online 13 June 2013 Keywords: Antifungal activity Biological control agent Crude extracts Fusarium oxysporum f. sp. cubense race 4 Streptomyces griseus Wilt disease
g r a p h i c a l a b s t r a c t Both the cells and extracts of S. griseus have antifungal activity, inhibiting FOC race 4 via hyphal damage.
a b s t r a c t The antifungal activity of viable cells of Streptomyces griseus (St 4) and its cell-free extracts were investigated against the pathogenic Fusarium oxysporum f. sp. cubense race 4 (FOC race 4), causal agent of wilt disease in bananas. Results from in vitro and soil assays showed cells and cell-free extracts of S. griseus were able to inhibit FOC race 4 with varying degree of success. Antifungal activity was attributed to chitinase and b-1,3-glucanase, detected in both cells and cell-free extracts, which caused lysis of fungal cell wall and inhibited sporulation. Interestingly, b-1,3-glucanase and chitinase activities were significantly higher in cell-free extracts compared to cells, with 8.30 and 5.43 against 7.96 and 4.95 U mL 1, respectively. Application to soil however, showed inoculation using S. griseus cells were more effective in suppressing growth of FOC race 4 than crude extracts, with 6 log10 CFU of FOC race 4 g 1 soil enumerated compared to 7 log10 CFU of FOC race 4 g 1 soil after 20 days. To summarize, this study has shown that cell-free extracts of S. griseus have antifungal properties but may not be suitable for soil application in its current form (liquid suspension). Further investigations on bioformulation may address this limitation. Ó 2013 Elsevier Inc. All rights reserved.
1. Introduction Streptomyces spp. are established biocontrol agents of important soil-borne diseases (Behal, 2000; Doumbou et al., 2001). They have specifically been demonstrated to suppress wilt incidences in cotton, tomato and banana plants (Reddi and Rao, 1971; Getha and Vikineswary, 2001; Anitha and Rabeeth, 2009; Kim et al., 2011). ⇑ Corresponding author. Fax: +60 3 5514 6364. E-mail addresses: [email protected], [email protected] (A.S.Y. Ting). 1049-9644/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.biocontrol.2013.06.001
Streptomyces spp. demonstrate several modes of action with antibiosis and competition for nutrients and space being the most common. On rare occasions, hyphal parasitism has been identified as a mode of action (Tu, 1988; Gonzalez-Franco and Hernandez, 2009). Antibiosis by Streptomyces spp. is the most effective mechanism, where production of antibiotics inhibits the growth of the pathogen efficiently (Genilloud et al., 2011; Nakano et al., 2011; Roberts et al., 2011). In addition, production of extracellular cell-wall degrading enzymes (chitinase, b-1,3-glucanase, cellulase, b-glucosidase, amylase, xylanase and mannase) also inhibits pathogen growth significantly (Spear et al., 1993; Tweddell et al., 1994;
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Sepulveda and Crawford, 1998; Trejo-Estrada et al., 1998; Prapagdee et al., 2008; Gonzalez-Franco and Hernandez, 2009; Shafique et al., 2009). Additionally, some species such as Streptomyces pilosus, have ferrichrome siderophores that allows the sequesteration of iron as a growth limiting factor (Müller et al., 1984). All these contribute to effective biocontrol activity of the Streptomyces spp. Streptomyces spp. are commonly applied by introducing their cell biomass (viable inoculants) into the soil (Reddi and Rao, 1971; Anitha and Rabeeth, 2009). This has resulted in several cases of successful control, but also raises concerns on the possible detrimental effect of Streptomyces spp. due to prolonged purposeful introduction into the soil. One such concern from their purposeful introduction into soil is their inhibitory effect on the indigenous microflora community in the soil, especially beneficial microflora such as mycorrhizal fungi, as continuous bioaugmentation with cells of Streptomyces spp. may result in persistent antibiotic production thus implicating growth of microflora (Fitter and Garbaye, 1994). Whether these effects are transitory or not, is secondary as this will become a limiting factor in bioaugmentation or biofertilization approaches using beneficial microbes. Other concerns include possible transfer of antibiotic resistance (common trait in Streptomyces spp.) to other bacteria, especially with prolonged co-existence with residual cells of Streptomyces spp. (Courvalin, 1994). The other limitation to the use of viable inoculant is quality control and the economical aspect of having to re-culture, maintain and harvest large batches of Streptomyces spp. after every cycle (Sabaratnam and Traquair, 2002). The use of crude extracts produced by Streptomyces spp. present an interesting alternative to address limitations from using cell biomass. In this study, we investigated the antifungal activity of crude extracts from Streptomyces griseus (isolate St 4) against FOC race 4, subsequently comparing the biocontrol activities of both the viable cells and cell-free extracts. Our study is significant as currently, literatures on the potential of using cell-free extracts as a strategy in biocontrol, especially towards wilt disease caused by FOC race 4 is limited. Most research focused on the use of whole cells to render control. There is also novelty in our approach to evaluate the antifungal activity of S. griseus towards FOC race 4 as this far, S. griseus has only been known to inhibit other Fusarium pathogens such as Fusarium sp. causing crown rot (Ting et al., 2010), and tomato wilt (Anitha and Rabeeth, 2010). This paper therefore reports the results of the antifungal activity of cells and cell-free extracts of S. griseus (St 4) towards FOC race 4, demonstrated from in vitro and soil assays. 2. Materials and methods 2.1. Culture establishment The isolate St 4 was streaked on Actinomycetes Isolation Agar (AIA) (Difco) and incubated at room temperature (24 ± 2 °C). This isolate originated from soil and has shown antifungal activity towards Fusarium sp., responsible for crown rot disease of banana (Ting et al., 2010). Based on 16s RNA sequencing, isolate St 4 was identified as Streptomyces griseus (GenBank ID: NC_010572). The pathogenic Fusarium oxysporum f. sp. cubense race 4 (FOC race 4) was obtained from Professor Dr. Sariah Meon (Universiti Putra Malaysia) and established on Potato Dextrose Agar (PDA, Difco) at 24 ± 2 °C. Both isolates were cultured for 7 days prior to use in subsequent tests. 2.2. Extraction of crude extracts from S. griseus One milliliter of S. griseus (108 cfu mL 1) derived from 7-day-old cultures, was inoculated into 250 mL of ISP-2 broth (0.4% glucose
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(F.S. Chemicals), 0.4% yeast extract (Difco), 1.0% malt extract (Oxoid), pH 7). The broth culture was incubated on a rotary shaker (150 rpm, 24 ± 2 °C) for 5 days. Crude extracts were extracted by centrifuging broth culture (9000 rpm) at 0 °C for 20 min. The extracts were further filter-sterilized (0.45 lm filter, Sartorius) prior to use.
2.3. Evaluating antifungal activities of S. griseus and its cell-free extracts in vitro The antifungal activity of S. griseus (St 4) and its cell-free extracts was determined from plate assay, microscopic analysis of hyphal degradation, and soil assay.
2.3.1. Plate assay for detection of non-volatile antifungal properties Antifungal activity of S. griseus (St 4) was detected by streaking loopfuls of S. griseus cells perpendicularly on PDA (Difco) and incubated for 7 days at 24 ± 2 °C. After 7 days, a 0.5 cm-mycelial plug of FOC race 4 was placed 2.5 cm from the streak. The agar plate was then incubated for another 7 days at 24 ± 2 °C. Controls were prepared using sterile distilled water for streaking. The radial growth of FOC race 4 (R2) co-inoculated with S. griseus was calculated against the radial growth co-inoculated with sterile distilled water (R1), and the percentage of inhibition of radial growth (PIRG %) determined (Ting et al., 2010). This test had six replicates for each treatment, and was repeated twice. The antifungal potential of cell-free extracts of S. griseus was also detected using the plate assay. Filter-sterilized extracts (prepared as from Section 2.2) were mixed in double-strength molten PDA based on the following ratio; 1:1, 1:2, 1:3 and 1:4 (crude extracts: double-strength PDA) (Leelasuphakul et al., 2008). Once the agar has solidified, a 0.5 cm-mycelial plug of FOC race 4 was inoculated centrally on the agar. Six replicates was prepared, and plates were incubated for 7 days at 24 ± 2 °C. Control plates (0:1) were prepared and incubated similarly, substituting the extracts with sterile distilled water. The diameter of FOC race 4 (D2) inoculated on agar supplemented with extracts was calculated against the diameter of FOC race 4 on agar with sterile distilled water (D1), and the percentage of inhibition of diameter growth (PIDG %) determined (Ting et al., 2010). This test was also repeated twice.
2.3.2. Detection of hyphal degradation in FOC race 4 Riddell’s (1950) slide culture technique was employed to demonstrate the antifungal activity of S. griseus and its cell-free extracts on the hypha of FOC race 4. Briefly, a square block of PDA (1 1 cm) was placed on a sterile glass slide. This slide was placed on top of two glass rods in a Petri dish, with a 9-cm diameter filter paper (Whatman) positioned at the base of the Petri dish. The filter paper was moistened with 4–5 drops of sterile water. To determine possible hypha degradation upon exposure to cells of S. griseus, one side of the agar block was point-inoculated with loopfuls of S. griseus and the corresponding opposite side with teased mycelium of FOC race 4. A sterile coverslip was then placed on the inoculated agar block. The petri dish was sealed with parafilm and incubated for 5 days at 24 ± 2 °C. After 5 days, the coverslip was removed and placed onto a slide containing two drops of lactophenol cotton blue (Merck) and viewed under light microscope to determine the occurrence of hyphal degradation. Control was established by inoculating FOC race 4 and sterile distilled water (control) on two opposite sides of the block. The procedure was repeated for detection of hyphal degradation by cell-free extracts using agar blocks amended with crude extracts (agar blocks from 1:1, 1:2, 1:3 and 1:4 as in Section 2.3.1). Triplicates were prepared for all assessments. This assessment was repeated twice.
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2.3.3. In vitro testing of bioactivity of S. griseus and its cell-free extracts in the soil Soil mixture comprised of top soil, organic peat (coconut husk) and sand, was prepared according to the ratio of 3:2:1, respectively. The soil mixture was autoclaved twice (121 °C, 15 min) and allowed to cool overnight. The pH of the sterilized soil was measured and modified to pH 5.5 using 1 N HCl and 1 N NaOH. Approximately 200 g of the sterile soil was weighed, placed into an opened polybag and accounted as one replicate. To this soil, 5 mL (108 cfu mL 1) of S. griseus was added and incubated at 24 ± 2 °C for 5 days. After 5 days, 5 mL of FOC race 4 (106 cfu mL 1) was mixed into the soil. Four replicates were prepared. For the next 20 days, viable counts of FOC race 4 were enumerated at every 5day interval. At each sampling interval, 1 g of soil was randomly sampled, serial dilutions performed, and aliquots (0.1 mL) from each dilution factor (10 1 to 10 6) was spread-plated onto PDA supplemented with 50 lg lL 1 streptomycin (Sigma–Aldrich). Agar plates were incubated 3–5 days at 24 ± 2 °C, and FOC race 4 colonies were enumerated (CFU g 1soil) (Asha et al., 2011). Antifungal activities of cell-free extracts were determined using the same method, substituting cell suspension of S. griseus with 5 ml of crude extracts. Controls were also prepared using sterile distilled water as a replacement. This assessment was repeated twice. 2.4. Evaluating chitinase and b-1,3-glucanase activities from S. griseus and cell-free extracts The viable cells and cell-free crude extracts from S. griseus were assayed for chitinase and b-1,3-glucanase activities, to compare their activities and roles in inhibiting FOC race 4. The activity of extracellular chitinases was determined daily for 7 days, using methods described by Imoto and Yagishita (1971). Firstly, 1 ml of 108 cfu mL 1 of S. griseus was inoculated into 250 mL of ISP-2 broth, and incubated on a rotary shaker (150 rpm, at 24 ± 2 °C). To detect chitinase activity from cells of S. griseus, 1 ml of the culture suspension was transferred into 0.1% colloidal chitin. The reaction mixture was incubated in a water bath (40 °C) for 1 h. The reaction was subsequently stopped by adding 0.05% w/v potassium ferricyanide (F.S. Chemicals) (in 0.5 M sodium carbonate (F.S. Chemicals)) and re-boiling for 15 min. After cooling, the reaction mixture was centrifuged (6000 rpm, 10 min) and the absorbance read at 420 nm. Chitinase activity was derived from a standard curve established from N-acetylglucosamine (Sigma–Aldrich) (0– 30 lmol). One unit of chitinase activity is expressed as the amount of enzyme that catalyzes the release of 1 lmol of N-acetylglucosamine in 60 min at 40 °C. The procedure was repeated for cell-free extracts, substituting culture suspension with cell-free supernatant obtained after centrifuging (6000 rpm, 0 °C, 10 min). Six replicates were performed for both cell and cell-free assessments. This assessment was repeated twice. Extracellular b-1,3-glucanase activity was determined according to Singh et al. (1999). Cultures were established similarly as for chitinase assay. At daily intervals, 1 mL of cell suspension was mixed with 1 mL of reagent consisting of 0.9 mL of 0.2 M sodium acetate buffer (pH 5.3) and 0.1 mL of 2% w/v laminarin (Sigma). The reaction mixture (total volume of 2 mL) was incubated for 1 h in a water bath (40 °C). The reaction mixture was stopped by adding 3 mL of 3,5-dinitrosalicylic acid (DNS) (Fluka) (1% w/v 3,5-dinitrosalicylic acid, 30% potassium sodium tartrate (F.S. Chemicals) in 2 M sodium hydroxide (Bendosen)) and boiling (water bath at 100 °C) for 5 min. After cooling to room temperature, the absorbance of the mixture was read at 575 nm. b-1,3-Glucanase activity was derived from a standard curve established using various concentrations of glucose (F.S. Chemicals) as a standard (0–15 lmol) and expressed as the amount of enzyme that catalyzes the release of 1 lmol of glucose in 60 min at 40 °C. For assay
using cell-free extracts, cell-free supernatant used were obtained as described for chitinase assay. Six replicates were performed for both cell and cell-free assessments. This assessment was also repeated twice. 2.5. Statistical analysis The data obtained were analyzed with Analysis of Variance (ANOVA) (p < 0.05) and means compared with Tukey’s test (HSD(0.05)). All statistical analysis was performed using Statistical Analysis Software (SAS). 3. Results and discussion 3.1. Antifungal activities of cells and cell-free extracts of S. griseus The antifungal activities of S. griseus and its cell-free extracts were evident from the plate assay, microscopic analysis on hypha degradation, and soil assay. From these tests, we observed that both cells and cell-free extracts of S. griseus had antifungal activities, with mean inhibition values of 54.6% and 33%, respectively. Our results confirmed the antifungal potential of S. griseus, attributed to the inhibitory compounds produced extracellularly (El-Abyad et al., 1993; Prapagdee et al., 2008). The inferior inhibition values from filtrate-incorporated test compared to streak test is not a concern as it is a common occurrence attributed to the nature of the test (Prapagdee et al., 2008). We also observed that the concentrations of the cell-free extracts also influenced the antifungal activity with more diluted extracts (1:3, 1:4) having poorer inhibitory effect compared to less diluted extracts (1:1, 1:2) (Fig. 1). As such, extracts are used without dilution in all our investigations. Inhibition towards FOC race 4 by both cells and cell-free extracts of S. griseus was the result of the extracellular compounds produced, such as cell-wall degrading enzymes and antibiotics which damaged the hypha. This was evident in our microscopic analysis where hypha of FOC race 4, co-inoculated with cells of S. griseus and on extract-amended agar blocks, showed signs of hypha swelling and cytoplasm aggregation (Fig. 2A). Cell-wall degradation may have also occurred as hypha were not consistently stained blue with Lactophenol Cotton Blue (LCB) indicating fissures along the hyphal wall, unlike in control (Fig. 2B). Poor sporulation was also detected for FOC race 4 exposed to S. griseus or its extracts (Fig. 2A) compared to control (Fig. 2B). Induced hyphal abnormalities (distortion, stunting, hyphal protuberances) and the absence of spores observed are typical symptoms of consequences of lytic activities or antibiotics from biocontrol agents which destroys germ tube or induces lysis of the spore itself (Lockwood and
Fig. 1. Inhibition (%) of FOC race 4 by cell-free extracts incorporated into doublestrength molten PDA. Values are means of six replicates. Means with the same letters are not significantly different according to Tukey’s Studentized Range Test (HSD(0.05)).
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Fig. 2. Hyphal distortion and protuberances, and the absence of spores as result of co-inoculation with S. griseus cells and cell-free extracts (A). Normal hyphal structure and sporulation is detected in control (treated with sterile distilled water) (B). Hyphal structures are stained with Lactophenol Cotton Blue and viewed under light microscopy at 400 magnification. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 3. Effect of cells (cells) and cell-free extracts (extracts) of S. griseus on FOC race 4 in sterile soils. CFU count for FOC race 4 g 1 soil was enumerated for 20 days and compared to control (treatment with sterile distilled water). Values presented are means of four replicates. Vertical bars indicate standard errors. Means with the same letters within the same day of assessment are not significantly different according to Tukey’s Studentized Range Test (HSD(0.05)).
Lingappa, 1963; Debono and Gordee, 1994; Getha and Vikineswary, 2001). These qualitative observations were adequate to indicate that both cells and cell-free extracts from S. griseus have antifungal activities towards FOC race 4. They also strongly suggest the presence of extracellular cell-wall degrading enzymes such as chitinase and b-1,3-glucanases (Reynolds, 1954; Mahadevan and Crawford, 1997; Narayana, 2009), which was subsequently detected and reported in Section 3.2. In the soil assay, the bioactivity of viable cells of S. griseus towards FOC race 4 were detected by day 15–20, as the CFU count for FOC race 4 at day 20 (6 log10 CFU g 1 soil) was significantly lower than in soils from control (7 log10 CFU g 1 soil) (Fig. 3). In control, CFU count for FOC race 4 appeared to have increased in the absence of cells of S. griseus. We observed that treatment with cell-free extracts did not significantly reduce CFU count of FOC race 4 (Fig. 3). A study by Anitha and Rabeeth (2009) reported observations with similar trend where cells of their S. griseus were more effective in controlling Fusarium wilt in tomato than crude or partially purified chitinase enzymes. They attributed this to cells being able to multiply and produce a constant level of enzymes, whereas crude extracts have a finite concentration of antifungal compounds. Our study agreed with Anitha and Rabeeth (2009) as there was a significant decrease in CFU count of FOC race 4 from the use of viable cells compared to extracts.
Fig. 4. Enzymatic activity (U mL 1) of chitinases and b-1,3-glucanase from cell-free extracts and cells of S. griseus. Values for each column are means of 42 samples. Means with the same letters and captions assessed within the same enzyme, are not significantly different according to Tukey’s Studentized Range Test (HSD(0.05)).
3.2. Enzymatic activities of cells and cell-free extracts of S. griseus Higher b-1,3-glucanase activities were detected in both cells and cell-free extracts of S. griseus compared to chitinase activity (Fig. 4). Both these enzymes are signature active enzymes, responsible for the antifungal activities of S. griseus (Trejo-Estrada et al., 1998; Yedidia et al., 1999; Anitha and Rabeeth, 2010). Assays
Fig. 5. Chitinase activities (U mL 1) from cells and cell-free extracts of S. griseus in 7 days. Vertical bars indicate standard errors. Means with the same letters within the same day of assessment are not significantly different according to Tukey’s Studentized Range Test (HSD(0.05)).
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Fig. 6. b-1,3-Glucanase activities (U mL 1) from cells and cell-free extracts of S. griseus in 7 days. Vertical bars indicate standard errors. Means with the same letters within the same day of assessment are not significantly different according to Tukey’s Studentized Range Test (HSD(0.05)).
showed higher b-1,3-glucanase and chitinase activities in cell-free extracts compared to cells, with 8.30 and 5.43 compared to 7.96 and 4.95 U mL 1, respectively (Fig. 4). This suggests the potential for use of extracts as they have similar levels of enzyme activity to S. griseus cells. These enzymes are also responsible for the damage to the hypha and spores of FOC race 4 observed in the microscopic test conducted, by breaking down the main cell wall components; chitin and b-glucans (Mahadevan and Crawford, 1997). We observed that both chitinase and b-1,3-glucanase from extracts have higher activities in the first 2 days, but there were no difference in activities thereafter (Figs. 5 and 6). In fact, both cells and extracts showed declining trend of enzymatic activities from day 1 to day 7 (Figs. 5 and 6). This has also been observed by Prapagdee et al. (2008) and Nahed (2011), suggesting that enzymatic activities contribute to antifungal activities of S. griseus. 4. Conclusion This study has shown that cells and crude extracts of S. griseus have antifungal properties towards FOC race 4. While the S. griseus cells performed well in the in vitro test (plate assay, microscopic analysis, enzymatic assays), crude extracts were ineffective in soil assays despite the high levels of chitinases and b-1,3-glucanases. Hence the most effective biocontrol agent for FOC race 4 in a soil environment would be a pre-treatment of S. griseus cells. Further studies may be carried out to optimize the application of crude extracts such as with the use of bioformulation. Acknowledgments Authors extend their gratitude to Monash University Sunway Campus for the funding to conduct the project. The authors would also like to thank Professor Sariah Meon (Universiti Putra Malaysia) for the provision of pure cultures of FOC race 4. References Anitha, A., Rabeeth, M., 2009. Control of Fusarium wilt of tomato by bioformulation of Streptomyces griseus in green house condition. AJBAS 1, 9–14. Anitha, A., Rabeeth, M., 2010. Degradation of fungal cell walls of phytopathogenic fungi by lytic enzyme of Streptomyces griseus. Plant Sci. 4, 61–66. Asha, B.B., Chandra Nayaka, S., Udaya Shankar, A.C., Srinavas, C., Niranjana, S.R., 2011. Biological control of F. oxysporum f. sp. lycopersici causing wilt of tomato by Pseudomonas fluorescens. Int. J. Microbiol. Res. 3, 79–84. Behal, V., 2000. Bioactive products from streptomyces. Adv. Appl. Microbiol. 47, 113–156.
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