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Screening and characterization of antimicrobial substances originated from entomopathogenic fungi
Journal of Asia-Pacific Entomology 19 (2016) 1053–1059
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Screening and characterization of antimicrobial substances originated from entomopathogenic fungi Tae Young Shin, Sung Min Bae, Soo Dong Woo ⁎ Department of Agricultural Biology, College of Agriculture Life & Environment Sciences, Chungbuk National University, Cheongju 28644, Republic of Korea
a r t i c l e
i n f o
Article history: Received 21 June 2016 Accepted 6 September 2016 Available online 28 September 2016 Keywords: Entomopathogenic fungi Antimicrobial activity Antimicrobial substance Culture filtrate
a b s t r a c t To illustrate the presence of different types of antimicrobial substances derived from entomopathogenic fungi, the antimicrobial activities of 342 fungi were evaluated, and their antimicrobial substances were analyzed. The antimicrobial activities of 342 fungi isolated under various conditions from different regions of Korea were evaluated against the bacterium Bacillus cereus and the fungus Botrytis cinerea using a dual culture technique on agar plates. The results showed that 170 isolates (49.7%) and 186 isolates (54.4%) inhibited the growth of B. cereus and B. cinerea, respectively. The culture filtrates of selected fungi completely suppressed the growth of the microorganisms, indicating the presence of antimicrobial substances. Characteristics of the antimicrobial substances such as thermostability, polarity, susceptibility to proteases and molecular weight varied not only within a culture filtrate but also with the fungal isolate. Different types of antimicrobial substances with different characteristics were present in the fungal culture filtrates. These substances may be useful not only as biocontrol agents against plant diseases but also in other industrial fields. This is the first study to demonstrate various kinds of antimicrobial substances from a large number of entomopathogenic fungi. This study suggests that novel antimicrobial substances could be isolated from entomopathogenic fungi. © 2016 Published by Elsevier B.V. on behalf of Korean Society of Applied Entomology, Taiwan Entomological Society and Malaysian Plant Protection Society.
Introduction Entomopathogenic fungi are natural enemies of insects and contribute to the regulation of their host populations (Vega, 2008). These fungi are widely distributed such as aquatic, forest, and agricultural habitats, and are often used as active ingredients of microbial pesticide (Shah and Pell, 2003; de Faria and Wraight, 2007). Their mode of action against insects involves attachment of conidia to insect cuticle, followed by germination, cuticle penetration, and internal dissemination throughout the insect body (Vega et al., 2012). During this process, fungal secreted enzymes, protein toxins, and secondary metabolites can overcome the host immune system, modify host behavior, and defend host resources against competing pathogens and saprophytes (Isaka et al., 2005; Molnar et al., 2010). Recently, several species of entomopathogenic fungi have been shown to have multiple roles in nature as endophytes, antagonists against plant pathogens and plant growth promoters (Vega et al., 2009; Ownley et al., 2010). In addition, the common entomopathogenic fungus Metarhizium robertsii could give a beneficial effect to plant by endophytic association with plant roots (Sasan and Bidochka, 2012). Potential antimicrobial effects of entomopathogenic fungi against
⁎ Corresponding author. E-mail address: [email protected] (S.D. Woo).
microorganisms have also been recently reported (Lee et al., 2005; Goettel et al., 2008; Lozano-Tovar et al., 2013). Dual action against insect pests and plant pathogens were studied in Beauveria spp., Metarhizium spp., Lecanicillium spp. and Clonostachys rosea with good results (Kim et al., 2007, 2010; Lozano-Tovar et al., 2013; Keyser et al., 2015). Therefore, such fungi are not only entomopathogenic but also useful microorganisms in various fields and many researchers are suggesting new paradigms for the use of such organisms in various fields (Vega et al., 2009). Although studies concerning bioactive substances from entomopathogenic fungi have been performed worldwide (Schmidt et al., 2003; Madla et al., 2005), they have been limited to a few fungal species and substances. In previous studies, we have isolated and identified 342 entomopathogenic fungal isolates including 28 species belonging to 20 genera from various habitats in Korean soils, and some of them isolates showed antibacterial, antioxidant and anticancer activities (Shin et al., 2013a,b, 2014). In this study, we tried to suggest the presence of different types of the antimicrobial substances from these entomopathogenic fungi using the bacterium Bacillus cereus and the fungus Botrytis cinerea. Both microorganisms are used for the antimicrobial screening test in many studies (Wilson et al., 1997; Lee et al., 2005). Especially, B. cinerea is a serious fungal pathogen causing gray mold diseases in many plant species and is difficult to control because of its various modes of attack (Dean et al., 2012). This study will serve as a guide for future entomopathogenic fungal resource programs.
http://dx.doi.org/10.1016/j.aspen.2016.09.008 1226-8615/© 2016 Published by Elsevier B.V. on behalf of Korean Society of Applied Entomology, Taiwan Entomological Society and Malaysian Plant Protection Society.
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Materials and methods Microorganisms The bacterium Bacillus cereus used for the antibacterial assays in this study was isolated from soil and cultured using BG media (1 g casamino acids, 10 g peptone, and 5 g dextrose in 1000 ml distilled water, pH 7.4). One ml of an overnight bacterial culture was inoculated into 100 ml of media and grown to mid-exponential phase (O.D.650 nm = ~ 0.5) at 25 °C with shaking at 150 rpm. The fungal isolate Botrytis cinerea GBYP-8 was obtained from the plant fungal disease laboratory of Chungbuk National University, Korea and used for the antifungal assay. To investigate their antimicrobial activities, 342 entomopathogenic fungal isolates from 20 different genera were used (Shin et al., 2013b). The entomopathogenic fungi were maintained on Potato dextrose agar (PDA) media at 25 °C in the dark. Fungal culture filtrates Fungal conidia were obtained by scraping a 2 week-cultured PDA plate and suspending the material in a 0.05% Tween-80 solution. The conidial suspension was vigorously agitated and filtered through cotton to remove the mycelial debris. After counting the number of conidia, 50 ul of the conidial suspension (2 × 106 conidia/ml) inoculated in 20 ml Sabouroud dextrose yeast broth (SDYB) (10 g Bacto-peptone, 40 g dextrose, and 10 g yeast extract in 1000 ml distilled water, pH 6.0) in a 100 ml flask. The samples were cultured at 25 °C in the dark and shaken at 150 rpm for 10 days. The percentage of viable conidia was determined prior to inoculation by conidia germination assay; in all cases, more than 90% of the conidia were determined to be viable. After 10 days, the samples were centrifuged at 10,000 ×g for 20 min at 4 °C, and the supernatants were filtered using a 5 um membrane filter (Advantec No. 2, Japan) to separate the crude extract from the mycelial and spore mass. Subsequently, the culture filtrates were adjusted to pH 5.1 and pH 7.4 with HCl and NaOH for the antibacterial and antifungal assays, respectively, and re-filtered through a 0.45 um membrane filter (Advantec Dismic-25cs, Japan). All cell-free fungal culture filtrates were stored at −70 °C until used.
plug that contained B. cinerea. The plate was incubated at 22 °C in the dark for 4 days, and the clear inhibition zone was measured. For the antifungal assay of the fungal culture filtrates, conidial suspensions of B. cinerea were diluted with Potato dextrose broth (PDB, pH 5.1) to 2 × 104 conidia/ml. One hundred ul of the conidial suspension was plated on each well of 96-well plates with 100 ul of fungal culture filtrate at different concentrations (1%, 10%, 20%, 40%, 60%, 80%, and 100%) and incubated at 22 °C for 48 h. The absorbance of each well was measured at 595 nm using a microplate reader. For the control, fungal conidial suspensions were cultured in SDYB medium with different concentrations of 20 mM citrate phosphate buffer in place of the fungal culture filtrate. Characterization of the antimicrobial substances To determine its heat stability, the culture filtrate was subjected to various temperatures (50 °C, 80 °C, 100 °C, and 121 °C) for 15 min. After the heat shock, the culture filtrate was quickly cooled to 25 °C, and its antimicrobial activity was evaluated. The hydrophilic/hydrophobic characteristics of the antimicrobial substances were determined by an acetone precipitation method. Four volumes of cold (−20 °C) acetone (Sigma, USA) was added to one volume of sample. The mixture was vortexed and incubated at −20 °C for 1 h. After incubation, the sample was centrifuged at 13,000 × g for 10 min at room temperature, and the pellet and supernatant were separated. The pellet was air dried for 30 min and the supernatant was evaporated by nitrogen. Both samples were adjusted to the original volume with distilled water, and then used in the antimicrobial activity assays. To test their protease stability, the culture filtrate was treated with proteinase K (Sigma, USA) or pronase E (Sigma, USA) at a final concentration of 1 mg/ml and 300 μg/ml at 37 °C for 2 h and 24 h, respectively. Then, the sample was treated at 121 °C for 15 min or 80 °C for 20 min to inactivate proteinase K and pronase E, respectively, before the antimicrobial activity assay was carried out. The molecular weight of an antimicrobial substance was estimated using an Amicon ultra centrifugal filter device (Molecular wt. cut off: 30 kDa, 3 kDa) (Millipore Corporation, Ireland). After ultrafiltration, the samples were adjusted to the original volume with distilled water, and then used in the antimicrobial activity assay.
Antibacterial activity assay
Results
To detect antibacterial activity, two Ø6 mm agar plugs containing fungi from 5 day-old PDA were placed 3 cm away from the center of a 90 mm petri dish containing SDYA and incubated at 25 °C in the dark for 2 days. After incubation, a B. cereus bacterial suspension (1 × 108 CFU/ml) was inoculated in the center of each dish using a spreader. The plate was incubated at 25 °C in the dark for 24 h, and the clear inhibition zone was measured. For the antibacterial assay of fungal culture filtrates, the B. cereus was diluted with medium to 5 × 105 CFU/ml. One hundred ul of bacterial suspensions was plated on each well of 96-well plates with 100 ul of fungal culture filtrate at different concentrations (1%, 10%, 20%, 40%, 60%, 80%, and 100%) diluted with SDYB and incubated at 25 °C for 16 h. The absorbance of each well was measured at 650 nm using a microplate reader (Molecular Devices, UK). For the control, bacterial suspensions were cultured in SDYB with different amounts of Dulbecco's PBS in place of the fungal culture filtrate.
Screening of fungi for antimicrobial activity
Antifungal activity assay A dual culture method was used to detect the antifungal activity against B. cinerea on SDYA. A Ø6 mm agar plug containing 1 week-old B. cinerea was placed at the center of an SDYA plate and two agar plugs containing different test fungi were placed 3 cm away from center
The antimicrobial activity of 342 entomopathogenic fungal isolates against B. cereus and B. cinerea was investigated on agar plates. The results indicated that the antimicrobial activities varied (Figs. 1, 2). Of the 342 isolates tested, antibacterial activity was presented in 49.7% (170) of the isolates. The antibacterial activity was divided into three categories; high activity with a clear zone larger than 10 mm (16 isolates, 4.7% frequency), medium activity with a clear zone of 5 to 9.9 mm (108 isolates, 31.6% frequency) and low activity with a clear zone less than 5 mm (46 isolates, 13.5% frequency). Antifungal activity to B. cinerea was present in 54.4% (186 isolates) of the isolates. The antifungal activity was also divided into three groups; high-activity with a clear zone greater than 5 mm (14 isolates, 4.1% frequency), medium activity with a clear zone of 2 to 4.9 mm (90 isolates, 26.3% frequency) and low activity with a clear zone less than 2 mm (82 isolates, 24% frequency). The antimicrobial activities observed in the various fungal isolates varied with genus and species (Tables 1, 2). The degree of antibacterial and antifungal activity in the various fungal isolates was different from each other. Antibacterial activity was more common in B. brongniartii, I. farinose, I. fumosorosea and M. anisopliae (Table 1), but antifungal activity was more common in B. bassiana and Isaria spp. (Table 2). The fungal isolates which showed the antifungal activity (186 isolates, 54.4% of
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Table 1 Antibacterial activity of fungi against B. cereus according to species (genus). Frequency (%) of antibacterial activity Isolates
Fig. 1. Representative inhibition by fungi to B. cereus on SDYA after 2 days. 1, I. javanica CB6T2W1 (no-activity); 2, M. anisopliae var. anisopliae CB7T3W1 (high-activity); 3, I. fumosorosea JB3F1W1 (low-activity); 4, B. bassiana JB7R3W1 (medium-activity); 5, M. anisopliae var. anisopliae KW3G8W1 (high-activity); 6, I. farinosa KB13T2W1 (highactivity); 7, M. anisopliae var. anisopliae KB20G2W1 (high-activity); 8, M. anisopliae var. anisopliae KB20G2G1 (high-activity). Scale bars: 10 mm.
total isolates) were more common than which showed antibacterial activity (170 isolates, 49.7% of total isolates). A total of 254 fungal isolates (74.3% of the total) had at least one of the antimicrobial activities (Table 3). Although a fungal isolate that had both high antibacterial and antifungal activity was not found, 102 isolates (29.8% of the total) had antimicrobial activity against both B. cereus and B. cinerea (Table 3).
Acremonium spp. Aspergillus spp. Beauveria bassiana B. brongniartii B. pseudobassiana Bionectria spp. Chaunopycnis spp. Clonostachys rosea Fusarium spp. Isaria farinosa I. fumosorosea Isaria spp. Lecanicillium spp. Metarhizium anisopliae var. anisopliae M. anisopliae var. lepidiotum M. flavoviride var. pemphigum Mucoromycotina spp. Myrothecium spp. Paecilomyces carneus P. marquandii Paraconiothyrium spp. Phialocephala spp. Pochonia spp. Purpureocillium lilacinum Simplicillium spp. Tolypocladium spp. Verticillium spp.
No. of isolates
HighLowNoactivity Medium-activity activity activity
1 8 110 8 8 7 23 1 2 12 6 22 8 64
0 0 0 25 0 0 0 0 50 8.3 0 0 0 14.1
0 25 42.7 62.5 25 0 8.7 0 50 66.7 33.3 13.6 0 45.3
0 25 10 0 0 14.3 26.1 0 0 8.3 33.3 18.2 25 20.3
100 50 47.3 12.5 75 85.7 65.2 100 0 16.7 33.3 68.2 75 20.3
1
0
0
100
0
16
0
6.3
6.3
87.5
1 5 3 5 2 1 17 4
0 0 0 0 0 0 5.9 0
0 20 66.7 0 0 0 11.8 0
0 20 33.3 0 0 0 0 0
100 60 0 100 100 100 82.4 100
1 3 3
100 0 33.3
0 33.3 0
0 0 0
0 66.7 66.7
* Percentage frequency % = number of isolates from each activity/number of isolates from each species.
Antimicrobial activity of culture filtrates Several fungal isolates that showed the highest antimicrobial activities (12 isolates with antibacterial activity and 7 isolates with antifungal activity) were used for the further study of the production of antimicrobial substances in the culture medium. The final pH after 10 days cultivation ranged between 3.4 and 5.7 and 4.76–7.78 for the antibacterial isolates and antifungal isolates, respectively (data not shown). Most of the selected fungal culture filtrates showed similar antimicrobial activity as indicated by the results of the dual culture assay, except for M. anisopliae var. anisopliae JB11F1W1 for antibacterial activity (Figs. 3, 4). The antimicrobial activities were concentration dependent (Figs. 3, 4). The culture filtrates of B. brongniartii CB12M5W1; I. farinosa KB13T2W1; M. anisopliae var. anisopliae KB20G2W1, KB20G2G1, KG22G3W1, KB14R1W1 had the highest antibacterial activity (Fig. 3). The culture filtrates of B. bassiana CB7S2W1 and I. farinosa KB7R1W1 showed the highest antifungal activity at a concentration as low as 1% (Fig. 4).
Characteristics of the active antimicrobial substances
Fig. 2. Representative inhibition by fungi to B. cinerea on SDYA after 4 days. 1, B. bassiana DK1R1W1 (medium-activity); 2, B. bassiana KW10G7W1 (high-activity); 3, B. bassiana JN13S1W1 (high-activity); 4, Chaunopycnis sp. JN17G2W1 (no-activity); 5, Myrothecium sp. JB11S2W2 (high-activity); 6, Aspergillus sp. CN1G5W1 (medium-activity); 7, M. anisopliae var. anisopliae KG21G2W1 (low-activity); 8, B. bassiana JB9S1W1 (mediumactivity). Scale bars: 10 mm.
The substances from six fungal culture filtrates that had the highest antibacterial activity were analyzed for their physico-chemical properties. The stability of culture filtrate to heat and protease treatment was evaluated. Heat or proteinase K treatment did not affect the antibacterial activity in any fungal culture filtrate, except for M. anisopliae var. anisopliae KB20G2W1 and KB20G2G1 (Table 4, Fig. S1A). To reduce the effect of high temperature on the inactivation of proteinase K, culture filtrates of M. anisopliae var. anisopliae KB20G1and KB20G2G1 were treated with pronase E to determine whether the active substance
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Discussion
Table 2 Antifungal activity of fungi against B. cinerea according to species (genus). Frequency (%) of antifungal activity Isolates Acremonium spp. Aspergillus spp. Beauveria bassiana B. brongniartii B. pseudobassiana Bionectria spp. Chaunopycnis spp. Clonostachys rosea Fusarium spp. Isaria farinosa I. fumosorosea Isaria spp. Lecanicillium spp. Metarhizium anisopliae var. anisopliae M. anisopliae var. lepidiotum M. flavoviride var. pemphigum Mucoromycotina spp. Myrothecium spp. Paecilomyces carneus P. marquandii Paraconiothyrium spp. Phialocephala spp. Pochonia spp. Purpureocillium lilacinum Simplicillium spp. Tolypocladium spp. Verticillium spp.
No. of isolates
HighLowNoactivity Medium-activity activity activity
1 8 110 8 8 7 23 1 2 12 6 22 8 64
0 12.5 4.5 0 0 0 0 0 0 8.3 0 9.1 0 0
0 25 60 0 0 0 4.3 0 0 16.7 0 36.4 12.5 6.3
0 25 28.2 25 50 0 26.1 0 0 25 33.3 22.7 25 28.1
100 37.5 7.3 75 50 100 69.6 100 100 50 66.7 31.8 62.5 65.6
1
0
0
100
0
16
0
6.3
18.8
75
1 5 3 5 2 1 17 4
0 40 0 0 0 0 5.9 0
0 20 33.3 0 0 0 11.8 0
0 20 0 0 50 0 0 25
100 20 66.7 100 50 100 82.4 75
1 3 3
0 66.7 0
100 0 0
0 0 0
0 33.3 100
* Percentage frequency % = number of isolates from each activity/number of isolates from each species.
was proteinaceous. Pronase E had no effect on the antibacterial activity of either strain (Fig. S1B), which indicated that the active substance was not a protein. The hydrophobic and hydrophilic properties of the antibacterial substances were evaluated by an acetone precipitation method. Antibacterial activity in the pellet was considered as hydrophilic, except for the active substance produced by I. farinosa KB1 3 T2 W1 (Table 4, Fig. S1C). The molecular weight of the antibacterial substance was estimated using the acetone precipitation samples. Substances with antibacterial activity had various molecular weights and different activities (Table 4, Fig. S1D). Four fungal culture filtrates with the highest antifungal activity were also analyzed. The substances had high thermal stability and retained their activity after proteinase K treatment (Table 4, Fig. S2A). The antifungal activities of two fungal culture filtrates from B. bassiana KW10G7W1 and I. farinosa KB7R1W1 were hydrophilic, and from B. bassiana JN13S1W1 and CB7S2W1, were observed as both hydrophobic and hydrophilic (Table 4, Fig. S2B). The molecular weights of the active substances varied from below 3 kDa to above 30 kDa as for the antifungal substances (Table 4, Fig. S2C).
Although entomopathogenic fungi have been studied solely as biocontrol agents against a variety of pests in various countries, their additional roles in nature are now under scrutiny, i.e., as endophytes, or antagonists of other microorganisms (Vega et al., 2009). Such research not only results in biocontrol agents against pests but also provides resources for the development of antimicrobial substances. Studies have been concerned with screening of limited number of entomopathogenic fungi species and fungal substances which have antimicrobial activities (Schmidt et al., 2003; Lee et al., 2005; Lozano-Tovar et al., 2013). In this study, we screened and analyzed various antimicrobial substances from a variety of entomopathogenic fungi to suggest the presence of various kinds of substances. Our fungal isolates showed antimicrobial activity against the bacterium B. cereus and the fungus B. cinerea, respectively. Lee et al. (2005) also reported anti-Bacillus or anti-Staphylococcus activity in 47 fungal isolates. Previous studies and our results suggest that many fungi may have antimicrobial activity against a wide variety of microorganisms. Beauveria spp., Metarhizium spp., Isaria spp. and Lecanicillium spp. are important entomopathogenic fungi as biocontrol agents against a variety of pests in many countries (Shah and Pell, 2003; de Faria and Wraight, 2007). Recently, these genera have been investigated for dual activity against pests and plant diseases (Goettel et al., 2008; Lozano-Tovar et al., 2013). In our study, these genera more frequently exhibited antimicrobial activity against B. cereus and B. cinerea compared to the rest of the isolates (Tables 1, 2). Although we did not confirm the pathogenicity of the studied fungi for specific pests, they may still be used for the development of novel dual-control agents against both pests and plant pathogens because many of the fungal isolates used in this study are pathogenic to insects (Shin et al., 2013b). It has been previously shown that the antimicrobial activity in entomopathogenic fungi was primarily due to secondary metabolites (Isaka et al., 2005; Lee et al., 2005; Molnar et al., 2010; Lozano-Tovar et al., 2013). This was also demonstrated in our study because most of the antimicrobial activities were present in the fungal culture filtrates (Figs. 3, 4). Various metabolites secreted by entomopathogenic fungi could overcome the host immune system, modify host behavior, and defend host resources against competing pathogens and saprophytes during the pathogenicity process (Isaka et al., 2005; Molnar et al., 2010). Such metabolites as beauvericin, bassianin, oosporein, and destruxins exhibit a wide variety of antimicrobial activities including insecticidal, anticancer, antioxidant, and antiviral, and therefore have been suggested as potential candidates for the development of new bioactive agents (Molnar et al., 2010; de Souza Santos et al., 2013). Especially, destruxins have been implicated as feasible, multifunctional anticancer drug candidates for preclinical development (Dornetshuber-Fleiss et al., 2013). However, their production is limited to a few fungal species. In addition, most entomopathogenic fungal bioactive substances have a low molecular weight (below 1000 g/mol) and have been reported to be peptides, but fungal bioactive substances of high molecular weight are rare (Quesada-Moraga and Vey, 2004). Although more diverse biological activities should be evaluated, the remarkable thermostability of the antimicrobial substances identified makes them good candidates not only for the development of novel biocontrol agents in plants but also
Table 3 Comparison of antimicrobial activities of fungi against B. cereus and B. cinerea. No. of fungi having antimicrobial activity against microorganisms (n = 254) Level of activitya Frequency a b
b
Highhigh 0 (0%)
Highmedium 8 (3.1%)
Highlow 5 (2%)
Mediummedium 35 (13.8%)
Mediumlow 45 (17.7%)
Lowlow 9 (3.5%)
Any antimicrobial activity against both B. cereus and B. cinerea. Percentage frequency( %) = number of each level activity/number of total any antimicrobial activity of isolates.
Highno 17 (6.7%)
Mediumno 75 (29.5%)
Lowno 60 (23.6%)
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Fig. 3. Antibacterial activity of entomopathogenic fungal culture filtrates at different concentrations (1%, 10%, 20%, 40%, 60%, 80%, and 100%) against B. cereus for 16 h. The vertical bars correspond to the standard error.
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Fig. 4. Antifungal activity of entomopathogenic fungal culture filtrates at different concentrations (1%, 10%, 20%, 40%, 60%, 80%, and 100%) against B. cinerea for 48 h. The vertical bars correspond to the standard error.
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Table 4 Physico-chemical properties of antimicrobial culture filtrates from entomopathogenic fungi. Properties Isolates Anti- B. cereus activity B. brongniartii CB12M5W1 I. farinosa KB13T2W1 M. anisopliae var. anisopliae KB20G2W1 KB20G2G1 KG22G3W1 KB14R1W1 Anti- B. cinerea activity B. bassiana KW10G7W1 JN13S1W1 CB7S2W1 I. farinosa KB7R1W1
Protein
Limited thermostability
Hydrophobicity/ hydrophilicity
Molecular weight
No
121 °C
hydrophilicity
3 kDa (lower)
No
121 °C
hydrophobicity
3 kDa (lower)
No No No No
100 °C 80 °C 121 °C 121 °C
hydrophilicity hydrophilicity hydrophilicity hydrophilicity
30 kDa (upper), 3 ~ 30 kDa,3 kDa (lower) 30 kDa (upper), 3 ~ 30 kDa 3 ~ 30 kDa 3 ~ 30 kDa
No No No
100 °C 121 °C 121 °C
hydrophilicity hydrophilicity hydrophilicity
30 kDa (upper), 3 ~ 30 kDa, 30 kDa (upper), 3 ~ 30 kDa, 30 kDa (upper), 3 kDa (lower)
No
121 °C
hydrophilicity
30 kDa (upper), 3 ~ 30 kDa, 3 kDa (lower)
for various industrial applications, such as in the food and livestock industries. In conclusion, this study proved the usefulness of the entomopathogenic fungi as a resource for bioactive substances for various applications by an analysis of a large number of such fungi. Further investigation will identify more novel bioactive substances from entomopathogenic fungi. Conflict of interest No conflict of interest declared. Acknowledgements This work was supported by a grant from the Next-Generation BioGreen 21 Program (Project No. PJ01102902), Rural Development Administration, Republic of Korea and Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2014R1A6A3A01009867). Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.aspen.2016.09.008. References de Faria, M.R., Wraight, S.P., 2007. Mycoinsecticides and Mycoacaricides: A comprehensive list with worldwide coverage and international classification of formulation types. Biol. Control 43, 237–256. de Souza Santos, M., Andrioli, W.J., de Morais Del, M.P.F., Bastos, J.K., Nanayakkara, N.D., Naal, R.M.Z.G., 2013. In vitro anti-allergic activity of the fungal metabolite pyridovericin. Int. Immunopharmacol. 15, 532–538. Dean, R., Van Kan, J.A., Pretorius, Z.A., Hammond-Kosack, K.E., Di Pietro, A., Spanu, P.D., Rudd, J.J., Dickman, M., Kahmann, R., Ellis, J., Foster, G.D., 2012. The Top 10 fungal pathogens in molecular plant pathology. Mol. Plant Pathol. 13, 414–430. Dornetshuber-Fleiss, R., Heffeter, P., Mohr, T., Hazemi, P., Kryeziu, K., Seger, C., Berger, W., Lemmens-Gruber, R., 2013. Destruxins: fungal-derived cyclohexadepsipeptides with multifaceted anticancer and antiangiogenic activities. Biochem. Pharmacol. 86, 361–377. Goettel, M.S., Koike, M., Kim, J.J., Aiuchi, D., Shinya, R., Brodeur, J., 2008. Potential of Lecanicillium spp. for management of insects, nematodes and plant diseases. J. Invertebr. Pathol. 98, 256–261. Isaka, M., Kittakoop, P., Kirtikara, K., Hywel-Jones, N.L., Thebtaranonth, Y., 2005. Bioactive substances from insect pathogenic fungi. Acc. Chem. Res. 38, 813–823.
Keyser, C.A., Jensen, B., Meyling, N.V., 2015. Dual effects of Metarhizium spp. and Clonostachys rosea against an insect and a seed-borne pathogen in wheat. Pest Manag. Sci. 72, 517–526. Kim, J.J., Goettel, M.S., Gillespie, D.R., 2007. Potential of Lecanicillium species for dual microbial control of aphids and the cucumber powdery mildew fungus, Sphaerotheca fuliginea. Biol. Control 40, 327–332. Kim, J.J., Goettel, M.S., Gillespie, D.R., 2010. Evaluation of Lecanicillium longisporum, Vertalec® against the cotton aphid, Aphis gossypii, and cucumber powdery mildew, Sphaerotheca fuliginea in a greenhouse environment. Crop. Prot. 29, 540–544. Lee, S.Y., Nakajima, I., Ihara, F., Kinoshita, H., Nihira, T., 2005. Cultivation of entomopathogenic fungi for the search of antibacterial compounds. Mycopathologia 160, 321–325. Lozano-Tovar, M., Ortiz-Urquiza, A., Garrido-Jurado, I., Trapero-Casas, A., QuesadaMoraga, E., 2013. Assessment of entomopathogenic fungi and their extracts against a soil-dwelling pest and soil-borne pathogens of olive. Biol. Control 67, 409–420. Madla, S., Methacanon, P., Prasitsil, M., Kirtikara, K., 2005. Characterization of biocompatible fungi-derived polymers that induce IL-8 production. Carbohydr. Polym. 59, 275–280. Molnar, I., Gibson, D.M., Krasnoff, S.B., 2010. Secondary metabolites from entomopathogenic Hypocrealean fungi. Nat. Prod. Rep. 27, 1241–1275. Ownley, B.H., Gwinn, K.D., Vega, F.E., 2010. Endophytic fungal entomopathogens with activity against plant pathogens: ecology and evolution. BioControl 55, 113–128. Quesada-Moraga, E., Vey, A., 2004. Bassiacridin, a protein toxic for locusts secreted by the entomopathogenic fungus Beauveria bassiana. Mycol. Res. 108, 441–452. Sasan, R.K., Bidochka, M.J., 2012. The insect-pathogenic fungus Metarhizium robertsii (Clavicipitaceae) is also an endophyte that stimulates plant root development. Am. J. Bot. 99, 101–107. Schmidt, K., Li, Z., Schubert, B., Huang, B., Stoyanova, S., Hamburger, M., 2003. Screening of entomopathogenic Deuteromycetes for activities on targets involved in degenerative diseases of the central nervous system. J. Ethnopharmacol. 89, 251–260. Shah, P.A., Pell, J.K., 2003. Entomopathogenic fungi as biological control agents. Appl. Microbiol. Biotechnol. 61, 413–423. Shin, T.Y., Ko, S.H., Lee, W.W., Bae, S.M., Choi, J.B., Woo, S.D., 2013a. Screening and evaluation of antibacterial metabolites from entomopathogenic fungi. Int. J. Ind. Entomol. 26, 89–94. Shin, T.Y., Lee, W.W., Ko, S.H., Choi, J.B., Bae, S.M., Choi, J.Y., Lee, K.S., Je, Y.H., Jin, B.R., Woo, S.D., 2013b. Distribution and characterisation of entomopathogenic fungi from Korean soils. Biocontrol Sci. Tech. 23, 288–304. Shin, T.Y., Bae, S.M., Choi, J.B., Woo, S.D., 2014. Simple and rapid screening of entomopathogenic fungi having radical-scavenging and anticancer activities. J. Asia Pac. Entomol. 17, 543–547. Vega, F.E., 2008. Insect pathology and fungal endophytes. J. Invertebr. Pathol. 98, 277–279. Vega, F.E., Goettel, M.S., Blackwell, M., Chandler, D., Jackson, M.A., Keller, S., Koike, M., Maniania, N.K., Monzon, A., Ownley, B.H., 2009. Fungal entomopathogens: new insights on their ecology. Fungal Ecol. 2, 149–159. Vega, F.E., Meyling, N.V., Luangsa-ard, J.J., Blackwell, M., 2012. Fungal Entomopathogens. In: Vega, F.E., Kaya, H.K. (Eds.), Insect Pathology. Academic Press Inc., New York, pp. 171–220. Wilson, C.L., Solar, J.M., El Ghaouth, A., Wisniewski, M.E., 1997. Rapid evaluation of plant extracts and essential oils for antifungal activity against Botrytis cinerea. Plant Dis. 81, 204–210.
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Journal of Asia-Pacific Entomology journal homepage: www.elsevier.com/locate/jape
Screening and characterization of antimicrobial substances originated from entomopathogenic fungi Tae Young Shin, Sung Min Bae, Soo Dong Woo ⁎ Department of Agricultural Biology, College of Agriculture Life & Environment Sciences, Chungbuk National University, Cheongju 28644, Republic of Korea
a r t i c l e
i n f o
Article history: Received 21 June 2016 Accepted 6 September 2016 Available online 28 September 2016 Keywords: Entomopathogenic fungi Antimicrobial activity Antimicrobial substance Culture filtrate
a b s t r a c t To illustrate the presence of different types of antimicrobial substances derived from entomopathogenic fungi, the antimicrobial activities of 342 fungi were evaluated, and their antimicrobial substances were analyzed. The antimicrobial activities of 342 fungi isolated under various conditions from different regions of Korea were evaluated against the bacterium Bacillus cereus and the fungus Botrytis cinerea using a dual culture technique on agar plates. The results showed that 170 isolates (49.7%) and 186 isolates (54.4%) inhibited the growth of B. cereus and B. cinerea, respectively. The culture filtrates of selected fungi completely suppressed the growth of the microorganisms, indicating the presence of antimicrobial substances. Characteristics of the antimicrobial substances such as thermostability, polarity, susceptibility to proteases and molecular weight varied not only within a culture filtrate but also with the fungal isolate. Different types of antimicrobial substances with different characteristics were present in the fungal culture filtrates. These substances may be useful not only as biocontrol agents against plant diseases but also in other industrial fields. This is the first study to demonstrate various kinds of antimicrobial substances from a large number of entomopathogenic fungi. This study suggests that novel antimicrobial substances could be isolated from entomopathogenic fungi. © 2016 Published by Elsevier B.V. on behalf of Korean Society of Applied Entomology, Taiwan Entomological Society and Malaysian Plant Protection Society.
Introduction Entomopathogenic fungi are natural enemies of insects and contribute to the regulation of their host populations (Vega, 2008). These fungi are widely distributed such as aquatic, forest, and agricultural habitats, and are often used as active ingredients of microbial pesticide (Shah and Pell, 2003; de Faria and Wraight, 2007). Their mode of action against insects involves attachment of conidia to insect cuticle, followed by germination, cuticle penetration, and internal dissemination throughout the insect body (Vega et al., 2012). During this process, fungal secreted enzymes, protein toxins, and secondary metabolites can overcome the host immune system, modify host behavior, and defend host resources against competing pathogens and saprophytes (Isaka et al., 2005; Molnar et al., 2010). Recently, several species of entomopathogenic fungi have been shown to have multiple roles in nature as endophytes, antagonists against plant pathogens and plant growth promoters (Vega et al., 2009; Ownley et al., 2010). In addition, the common entomopathogenic fungus Metarhizium robertsii could give a beneficial effect to plant by endophytic association with plant roots (Sasan and Bidochka, 2012). Potential antimicrobial effects of entomopathogenic fungi against
⁎ Corresponding author. E-mail address: [email protected] (S.D. Woo).
microorganisms have also been recently reported (Lee et al., 2005; Goettel et al., 2008; Lozano-Tovar et al., 2013). Dual action against insect pests and plant pathogens were studied in Beauveria spp., Metarhizium spp., Lecanicillium spp. and Clonostachys rosea with good results (Kim et al., 2007, 2010; Lozano-Tovar et al., 2013; Keyser et al., 2015). Therefore, such fungi are not only entomopathogenic but also useful microorganisms in various fields and many researchers are suggesting new paradigms for the use of such organisms in various fields (Vega et al., 2009). Although studies concerning bioactive substances from entomopathogenic fungi have been performed worldwide (Schmidt et al., 2003; Madla et al., 2005), they have been limited to a few fungal species and substances. In previous studies, we have isolated and identified 342 entomopathogenic fungal isolates including 28 species belonging to 20 genera from various habitats in Korean soils, and some of them isolates showed antibacterial, antioxidant and anticancer activities (Shin et al., 2013a,b, 2014). In this study, we tried to suggest the presence of different types of the antimicrobial substances from these entomopathogenic fungi using the bacterium Bacillus cereus and the fungus Botrytis cinerea. Both microorganisms are used for the antimicrobial screening test in many studies (Wilson et al., 1997; Lee et al., 2005). Especially, B. cinerea is a serious fungal pathogen causing gray mold diseases in many plant species and is difficult to control because of its various modes of attack (Dean et al., 2012). This study will serve as a guide for future entomopathogenic fungal resource programs.
http://dx.doi.org/10.1016/j.aspen.2016.09.008 1226-8615/© 2016 Published by Elsevier B.V. on behalf of Korean Society of Applied Entomology, Taiwan Entomological Society and Malaysian Plant Protection Society.
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Materials and methods Microorganisms The bacterium Bacillus cereus used for the antibacterial assays in this study was isolated from soil and cultured using BG media (1 g casamino acids, 10 g peptone, and 5 g dextrose in 1000 ml distilled water, pH 7.4). One ml of an overnight bacterial culture was inoculated into 100 ml of media and grown to mid-exponential phase (O.D.650 nm = ~ 0.5) at 25 °C with shaking at 150 rpm. The fungal isolate Botrytis cinerea GBYP-8 was obtained from the plant fungal disease laboratory of Chungbuk National University, Korea and used for the antifungal assay. To investigate their antimicrobial activities, 342 entomopathogenic fungal isolates from 20 different genera were used (Shin et al., 2013b). The entomopathogenic fungi were maintained on Potato dextrose agar (PDA) media at 25 °C in the dark. Fungal culture filtrates Fungal conidia were obtained by scraping a 2 week-cultured PDA plate and suspending the material in a 0.05% Tween-80 solution. The conidial suspension was vigorously agitated and filtered through cotton to remove the mycelial debris. After counting the number of conidia, 50 ul of the conidial suspension (2 × 106 conidia/ml) inoculated in 20 ml Sabouroud dextrose yeast broth (SDYB) (10 g Bacto-peptone, 40 g dextrose, and 10 g yeast extract in 1000 ml distilled water, pH 6.0) in a 100 ml flask. The samples were cultured at 25 °C in the dark and shaken at 150 rpm for 10 days. The percentage of viable conidia was determined prior to inoculation by conidia germination assay; in all cases, more than 90% of the conidia were determined to be viable. After 10 days, the samples were centrifuged at 10,000 ×g for 20 min at 4 °C, and the supernatants were filtered using a 5 um membrane filter (Advantec No. 2, Japan) to separate the crude extract from the mycelial and spore mass. Subsequently, the culture filtrates were adjusted to pH 5.1 and pH 7.4 with HCl and NaOH for the antibacterial and antifungal assays, respectively, and re-filtered through a 0.45 um membrane filter (Advantec Dismic-25cs, Japan). All cell-free fungal culture filtrates were stored at −70 °C until used.
plug that contained B. cinerea. The plate was incubated at 22 °C in the dark for 4 days, and the clear inhibition zone was measured. For the antifungal assay of the fungal culture filtrates, conidial suspensions of B. cinerea were diluted with Potato dextrose broth (PDB, pH 5.1) to 2 × 104 conidia/ml. One hundred ul of the conidial suspension was plated on each well of 96-well plates with 100 ul of fungal culture filtrate at different concentrations (1%, 10%, 20%, 40%, 60%, 80%, and 100%) and incubated at 22 °C for 48 h. The absorbance of each well was measured at 595 nm using a microplate reader. For the control, fungal conidial suspensions were cultured in SDYB medium with different concentrations of 20 mM citrate phosphate buffer in place of the fungal culture filtrate. Characterization of the antimicrobial substances To determine its heat stability, the culture filtrate was subjected to various temperatures (50 °C, 80 °C, 100 °C, and 121 °C) for 15 min. After the heat shock, the culture filtrate was quickly cooled to 25 °C, and its antimicrobial activity was evaluated. The hydrophilic/hydrophobic characteristics of the antimicrobial substances were determined by an acetone precipitation method. Four volumes of cold (−20 °C) acetone (Sigma, USA) was added to one volume of sample. The mixture was vortexed and incubated at −20 °C for 1 h. After incubation, the sample was centrifuged at 13,000 × g for 10 min at room temperature, and the pellet and supernatant were separated. The pellet was air dried for 30 min and the supernatant was evaporated by nitrogen. Both samples were adjusted to the original volume with distilled water, and then used in the antimicrobial activity assays. To test their protease stability, the culture filtrate was treated with proteinase K (Sigma, USA) or pronase E (Sigma, USA) at a final concentration of 1 mg/ml and 300 μg/ml at 37 °C for 2 h and 24 h, respectively. Then, the sample was treated at 121 °C for 15 min or 80 °C for 20 min to inactivate proteinase K and pronase E, respectively, before the antimicrobial activity assay was carried out. The molecular weight of an antimicrobial substance was estimated using an Amicon ultra centrifugal filter device (Molecular wt. cut off: 30 kDa, 3 kDa) (Millipore Corporation, Ireland). After ultrafiltration, the samples were adjusted to the original volume with distilled water, and then used in the antimicrobial activity assay.
Antibacterial activity assay
Results
To detect antibacterial activity, two Ø6 mm agar plugs containing fungi from 5 day-old PDA were placed 3 cm away from the center of a 90 mm petri dish containing SDYA and incubated at 25 °C in the dark for 2 days. After incubation, a B. cereus bacterial suspension (1 × 108 CFU/ml) was inoculated in the center of each dish using a spreader. The plate was incubated at 25 °C in the dark for 24 h, and the clear inhibition zone was measured. For the antibacterial assay of fungal culture filtrates, the B. cereus was diluted with medium to 5 × 105 CFU/ml. One hundred ul of bacterial suspensions was plated on each well of 96-well plates with 100 ul of fungal culture filtrate at different concentrations (1%, 10%, 20%, 40%, 60%, 80%, and 100%) diluted with SDYB and incubated at 25 °C for 16 h. The absorbance of each well was measured at 650 nm using a microplate reader (Molecular Devices, UK). For the control, bacterial suspensions were cultured in SDYB with different amounts of Dulbecco's PBS in place of the fungal culture filtrate.
Screening of fungi for antimicrobial activity
Antifungal activity assay A dual culture method was used to detect the antifungal activity against B. cinerea on SDYA. A Ø6 mm agar plug containing 1 week-old B. cinerea was placed at the center of an SDYA plate and two agar plugs containing different test fungi were placed 3 cm away from center
The antimicrobial activity of 342 entomopathogenic fungal isolates against B. cereus and B. cinerea was investigated on agar plates. The results indicated that the antimicrobial activities varied (Figs. 1, 2). Of the 342 isolates tested, antibacterial activity was presented in 49.7% (170) of the isolates. The antibacterial activity was divided into three categories; high activity with a clear zone larger than 10 mm (16 isolates, 4.7% frequency), medium activity with a clear zone of 5 to 9.9 mm (108 isolates, 31.6% frequency) and low activity with a clear zone less than 5 mm (46 isolates, 13.5% frequency). Antifungal activity to B. cinerea was present in 54.4% (186 isolates) of the isolates. The antifungal activity was also divided into three groups; high-activity with a clear zone greater than 5 mm (14 isolates, 4.1% frequency), medium activity with a clear zone of 2 to 4.9 mm (90 isolates, 26.3% frequency) and low activity with a clear zone less than 2 mm (82 isolates, 24% frequency). The antimicrobial activities observed in the various fungal isolates varied with genus and species (Tables 1, 2). The degree of antibacterial and antifungal activity in the various fungal isolates was different from each other. Antibacterial activity was more common in B. brongniartii, I. farinose, I. fumosorosea and M. anisopliae (Table 1), but antifungal activity was more common in B. bassiana and Isaria spp. (Table 2). The fungal isolates which showed the antifungal activity (186 isolates, 54.4% of
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Table 1 Antibacterial activity of fungi against B. cereus according to species (genus). Frequency (%) of antibacterial activity Isolates
Fig. 1. Representative inhibition by fungi to B. cereus on SDYA after 2 days. 1, I. javanica CB6T2W1 (no-activity); 2, M. anisopliae var. anisopliae CB7T3W1 (high-activity); 3, I. fumosorosea JB3F1W1 (low-activity); 4, B. bassiana JB7R3W1 (medium-activity); 5, M. anisopliae var. anisopliae KW3G8W1 (high-activity); 6, I. farinosa KB13T2W1 (highactivity); 7, M. anisopliae var. anisopliae KB20G2W1 (high-activity); 8, M. anisopliae var. anisopliae KB20G2G1 (high-activity). Scale bars: 10 mm.
total isolates) were more common than which showed antibacterial activity (170 isolates, 49.7% of total isolates). A total of 254 fungal isolates (74.3% of the total) had at least one of the antimicrobial activities (Table 3). Although a fungal isolate that had both high antibacterial and antifungal activity was not found, 102 isolates (29.8% of the total) had antimicrobial activity against both B. cereus and B. cinerea (Table 3).
Acremonium spp. Aspergillus spp. Beauveria bassiana B. brongniartii B. pseudobassiana Bionectria spp. Chaunopycnis spp. Clonostachys rosea Fusarium spp. Isaria farinosa I. fumosorosea Isaria spp. Lecanicillium spp. Metarhizium anisopliae var. anisopliae M. anisopliae var. lepidiotum M. flavoviride var. pemphigum Mucoromycotina spp. Myrothecium spp. Paecilomyces carneus P. marquandii Paraconiothyrium spp. Phialocephala spp. Pochonia spp. Purpureocillium lilacinum Simplicillium spp. Tolypocladium spp. Verticillium spp.
No. of isolates
HighLowNoactivity Medium-activity activity activity
1 8 110 8 8 7 23 1 2 12 6 22 8 64
0 0 0 25 0 0 0 0 50 8.3 0 0 0 14.1
0 25 42.7 62.5 25 0 8.7 0 50 66.7 33.3 13.6 0 45.3
0 25 10 0 0 14.3 26.1 0 0 8.3 33.3 18.2 25 20.3
100 50 47.3 12.5 75 85.7 65.2 100 0 16.7 33.3 68.2 75 20.3
1
0
0
100
0
16
0
6.3
6.3
87.5
1 5 3 5 2 1 17 4
0 0 0 0 0 0 5.9 0
0 20 66.7 0 0 0 11.8 0
0 20 33.3 0 0 0 0 0
100 60 0 100 100 100 82.4 100
1 3 3
100 0 33.3
0 33.3 0
0 0 0
0 66.7 66.7
* Percentage frequency % = number of isolates from each activity/number of isolates from each species.
Antimicrobial activity of culture filtrates Several fungal isolates that showed the highest antimicrobial activities (12 isolates with antibacterial activity and 7 isolates with antifungal activity) were used for the further study of the production of antimicrobial substances in the culture medium. The final pH after 10 days cultivation ranged between 3.4 and 5.7 and 4.76–7.78 for the antibacterial isolates and antifungal isolates, respectively (data not shown). Most of the selected fungal culture filtrates showed similar antimicrobial activity as indicated by the results of the dual culture assay, except for M. anisopliae var. anisopliae JB11F1W1 for antibacterial activity (Figs. 3, 4). The antimicrobial activities were concentration dependent (Figs. 3, 4). The culture filtrates of B. brongniartii CB12M5W1; I. farinosa KB13T2W1; M. anisopliae var. anisopliae KB20G2W1, KB20G2G1, KG22G3W1, KB14R1W1 had the highest antibacterial activity (Fig. 3). The culture filtrates of B. bassiana CB7S2W1 and I. farinosa KB7R1W1 showed the highest antifungal activity at a concentration as low as 1% (Fig. 4).
Characteristics of the active antimicrobial substances
Fig. 2. Representative inhibition by fungi to B. cinerea on SDYA after 4 days. 1, B. bassiana DK1R1W1 (medium-activity); 2, B. bassiana KW10G7W1 (high-activity); 3, B. bassiana JN13S1W1 (high-activity); 4, Chaunopycnis sp. JN17G2W1 (no-activity); 5, Myrothecium sp. JB11S2W2 (high-activity); 6, Aspergillus sp. CN1G5W1 (medium-activity); 7, M. anisopliae var. anisopliae KG21G2W1 (low-activity); 8, B. bassiana JB9S1W1 (mediumactivity). Scale bars: 10 mm.
The substances from six fungal culture filtrates that had the highest antibacterial activity were analyzed for their physico-chemical properties. The stability of culture filtrate to heat and protease treatment was evaluated. Heat or proteinase K treatment did not affect the antibacterial activity in any fungal culture filtrate, except for M. anisopliae var. anisopliae KB20G2W1 and KB20G2G1 (Table 4, Fig. S1A). To reduce the effect of high temperature on the inactivation of proteinase K, culture filtrates of M. anisopliae var. anisopliae KB20G1and KB20G2G1 were treated with pronase E to determine whether the active substance
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Discussion
Table 2 Antifungal activity of fungi against B. cinerea according to species (genus). Frequency (%) of antifungal activity Isolates Acremonium spp. Aspergillus spp. Beauveria bassiana B. brongniartii B. pseudobassiana Bionectria spp. Chaunopycnis spp. Clonostachys rosea Fusarium spp. Isaria farinosa I. fumosorosea Isaria spp. Lecanicillium spp. Metarhizium anisopliae var. anisopliae M. anisopliae var. lepidiotum M. flavoviride var. pemphigum Mucoromycotina spp. Myrothecium spp. Paecilomyces carneus P. marquandii Paraconiothyrium spp. Phialocephala spp. Pochonia spp. Purpureocillium lilacinum Simplicillium spp. Tolypocladium spp. Verticillium spp.
No. of isolates
HighLowNoactivity Medium-activity activity activity
1 8 110 8 8 7 23 1 2 12 6 22 8 64
0 12.5 4.5 0 0 0 0 0 0 8.3 0 9.1 0 0
0 25 60 0 0 0 4.3 0 0 16.7 0 36.4 12.5 6.3
0 25 28.2 25 50 0 26.1 0 0 25 33.3 22.7 25 28.1
100 37.5 7.3 75 50 100 69.6 100 100 50 66.7 31.8 62.5 65.6
1
0
0
100
0
16
0
6.3
18.8
75
1 5 3 5 2 1 17 4
0 40 0 0 0 0 5.9 0
0 20 33.3 0 0 0 11.8 0
0 20 0 0 50 0 0 25
100 20 66.7 100 50 100 82.4 75
1 3 3
0 66.7 0
100 0 0
0 0 0
0 33.3 100
* Percentage frequency % = number of isolates from each activity/number of isolates from each species.
was proteinaceous. Pronase E had no effect on the antibacterial activity of either strain (Fig. S1B), which indicated that the active substance was not a protein. The hydrophobic and hydrophilic properties of the antibacterial substances were evaluated by an acetone precipitation method. Antibacterial activity in the pellet was considered as hydrophilic, except for the active substance produced by I. farinosa KB1 3 T2 W1 (Table 4, Fig. S1C). The molecular weight of the antibacterial substance was estimated using the acetone precipitation samples. Substances with antibacterial activity had various molecular weights and different activities (Table 4, Fig. S1D). Four fungal culture filtrates with the highest antifungal activity were also analyzed. The substances had high thermal stability and retained their activity after proteinase K treatment (Table 4, Fig. S2A). The antifungal activities of two fungal culture filtrates from B. bassiana KW10G7W1 and I. farinosa KB7R1W1 were hydrophilic, and from B. bassiana JN13S1W1 and CB7S2W1, were observed as both hydrophobic and hydrophilic (Table 4, Fig. S2B). The molecular weights of the active substances varied from below 3 kDa to above 30 kDa as for the antifungal substances (Table 4, Fig. S2C).
Although entomopathogenic fungi have been studied solely as biocontrol agents against a variety of pests in various countries, their additional roles in nature are now under scrutiny, i.e., as endophytes, or antagonists of other microorganisms (Vega et al., 2009). Such research not only results in biocontrol agents against pests but also provides resources for the development of antimicrobial substances. Studies have been concerned with screening of limited number of entomopathogenic fungi species and fungal substances which have antimicrobial activities (Schmidt et al., 2003; Lee et al., 2005; Lozano-Tovar et al., 2013). In this study, we screened and analyzed various antimicrobial substances from a variety of entomopathogenic fungi to suggest the presence of various kinds of substances. Our fungal isolates showed antimicrobial activity against the bacterium B. cereus and the fungus B. cinerea, respectively. Lee et al. (2005) also reported anti-Bacillus or anti-Staphylococcus activity in 47 fungal isolates. Previous studies and our results suggest that many fungi may have antimicrobial activity against a wide variety of microorganisms. Beauveria spp., Metarhizium spp., Isaria spp. and Lecanicillium spp. are important entomopathogenic fungi as biocontrol agents against a variety of pests in many countries (Shah and Pell, 2003; de Faria and Wraight, 2007). Recently, these genera have been investigated for dual activity against pests and plant diseases (Goettel et al., 2008; Lozano-Tovar et al., 2013). In our study, these genera more frequently exhibited antimicrobial activity against B. cereus and B. cinerea compared to the rest of the isolates (Tables 1, 2). Although we did not confirm the pathogenicity of the studied fungi for specific pests, they may still be used for the development of novel dual-control agents against both pests and plant pathogens because many of the fungal isolates used in this study are pathogenic to insects (Shin et al., 2013b). It has been previously shown that the antimicrobial activity in entomopathogenic fungi was primarily due to secondary metabolites (Isaka et al., 2005; Lee et al., 2005; Molnar et al., 2010; Lozano-Tovar et al., 2013). This was also demonstrated in our study because most of the antimicrobial activities were present in the fungal culture filtrates (Figs. 3, 4). Various metabolites secreted by entomopathogenic fungi could overcome the host immune system, modify host behavior, and defend host resources against competing pathogens and saprophytes during the pathogenicity process (Isaka et al., 2005; Molnar et al., 2010). Such metabolites as beauvericin, bassianin, oosporein, and destruxins exhibit a wide variety of antimicrobial activities including insecticidal, anticancer, antioxidant, and antiviral, and therefore have been suggested as potential candidates for the development of new bioactive agents (Molnar et al., 2010; de Souza Santos et al., 2013). Especially, destruxins have been implicated as feasible, multifunctional anticancer drug candidates for preclinical development (Dornetshuber-Fleiss et al., 2013). However, their production is limited to a few fungal species. In addition, most entomopathogenic fungal bioactive substances have a low molecular weight (below 1000 g/mol) and have been reported to be peptides, but fungal bioactive substances of high molecular weight are rare (Quesada-Moraga and Vey, 2004). Although more diverse biological activities should be evaluated, the remarkable thermostability of the antimicrobial substances identified makes them good candidates not only for the development of novel biocontrol agents in plants but also
Table 3 Comparison of antimicrobial activities of fungi against B. cereus and B. cinerea. No. of fungi having antimicrobial activity against microorganisms (n = 254) Level of activitya Frequency a b
b
Highhigh 0 (0%)
Highmedium 8 (3.1%)
Highlow 5 (2%)
Mediummedium 35 (13.8%)
Mediumlow 45 (17.7%)
Lowlow 9 (3.5%)
Any antimicrobial activity against both B. cereus and B. cinerea. Percentage frequency( %) = number of each level activity/number of total any antimicrobial activity of isolates.
Highno 17 (6.7%)
Mediumno 75 (29.5%)
Lowno 60 (23.6%)
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Fig. 3. Antibacterial activity of entomopathogenic fungal culture filtrates at different concentrations (1%, 10%, 20%, 40%, 60%, 80%, and 100%) against B. cereus for 16 h. The vertical bars correspond to the standard error.
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Fig. 4. Antifungal activity of entomopathogenic fungal culture filtrates at different concentrations (1%, 10%, 20%, 40%, 60%, 80%, and 100%) against B. cinerea for 48 h. The vertical bars correspond to the standard error.
T.Y. Shin et al. / Journal of Asia-Pacific Entomology 19 (2016) 1053–1059
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Table 4 Physico-chemical properties of antimicrobial culture filtrates from entomopathogenic fungi. Properties Isolates Anti- B. cereus activity B. brongniartii CB12M5W1 I. farinosa KB13T2W1 M. anisopliae var. anisopliae KB20G2W1 KB20G2G1 KG22G3W1 KB14R1W1 Anti- B. cinerea activity B. bassiana KW10G7W1 JN13S1W1 CB7S2W1 I. farinosa KB7R1W1
Protein
Limited thermostability
Hydrophobicity/ hydrophilicity
Molecular weight
No
121 °C
hydrophilicity
3 kDa (lower)
No
121 °C
hydrophobicity
3 kDa (lower)
No No No No
100 °C 80 °C 121 °C 121 °C
hydrophilicity hydrophilicity hydrophilicity hydrophilicity
30 kDa (upper), 3 ~ 30 kDa,3 kDa (lower) 30 kDa (upper), 3 ~ 30 kDa 3 ~ 30 kDa 3 ~ 30 kDa
No No No
100 °C 121 °C 121 °C
hydrophilicity hydrophilicity hydrophilicity
30 kDa (upper), 3 ~ 30 kDa, 30 kDa (upper), 3 ~ 30 kDa, 30 kDa (upper), 3 kDa (lower)
No
121 °C
hydrophilicity
30 kDa (upper), 3 ~ 30 kDa, 3 kDa (lower)
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