Acta agriculturae Slovenica, 117/4, 1–7, Ljubljana 2021 doi:10.14720/aas.2021.117.4.2345 Original research article / izvirni znanstveni članek In vitro antifungal potential of surfactin isolated from rhizospheric Bacil- lus thuringiensis Berliner 1915 against maize (Zea mays L.) fungal phy- topathogen Fusarium graminearum Schwabe Muddasir KHAN 1, 2 , Muhammad SALMAN 1 , Abdullah 1 , Syed Hussain SHAH 1 ; Muhammad ISRAR 3 Received September 16, 2021; accepted November 29, 2021. Delo je prispelo 16. septembra 2021, sprejeto 29. november 2021 1 Department of Health and Biological Sciences, Abasyn University Peshawar, Khyber Pakhtunkhwa, Pakistan 2 Corresponding author, e-mail: mk03025678947@gmail.com, muddasir.khan0302@gmail.com 3 Department of Microbiology, Abbottabad University of Science and Technology, Khyber Pakhtunkhwa, Pakistan In vitro antifungal potential of surfactin isolated from rhi- zospheric Bacillus thuringiensis Berliner 1915 against maize (Zea mays L.) fungal phytopathogen Fusarium graminearum Schwabe Abstract: Fusarium graminearum fungus cause signifi- cant loss in maize (Zea mays L.) and other cereal crops all over the world. The usage of chemical agents cause severe envi- ronmental problems. Bacillus species and other plant growth- promoting bacteria (PGPR) play key role in biopesticide devel- opment. A wide range of environmentally safe antimicrobial agents are already being manufactured. The current investiga- tion was focused on exploring the antifungal activity of Bacillus thuringiensis lipopeptide surfactin against fungal phytopatho- gen Fusarium graminearum. B. thuringensis was isolated from the rhizosphere of maize crop and cultivated to produce lipo- peptides. Surfactin was identified by high-performance liquid chromatography (HPLC) from the extract at 210 nm, retention time 3-5 minutes and the obtained peaks area was 3.990. The growth of F. graminearum was successfully inhibited by surfac- tin at different concentrations. Among these, 80 % concentra- tion showed the highest zone of inhibition in comparison to 60 %, 40 % and 20 % concentrations (p < 0.005), respectively. The current study concludes B. thuringensis lipopeptide surfac- tin has a high potential to inhibit the growth of F. graminearum. Key words: surfactin; Bacillus; biological control; HPLC; Fusarium graminearum In vitro protiglivni potencial surfaktina, izoliranega iz bak- terije Bacillus thuringiensis Berliner 1915 iz rizosfere koruze (Zea mays L.) proti patogeni glivi Fusarium graminearum Schwabe Izvleček: Gliva Fusarium graminearum povzroča znantne izgube v pridelku koruze in drugih žit širom po svetu. Uporaba kemičnih sredstev za zatiranje povzroča resne okoljske proble- me. Vrste iz rodu Bacillus in druge rast vzpodbujajoče bakterije (PGPR) igrajo ključno vlogo pri razvoju biopesticidov. Proizve- den je bil že širok spekter okolju prijaznih antimikrobnih agen- sov. Raziskava se osredotoča na uporabo protiglivne aktivnosti lipopeptidnih surfaktinov iz bakterije Bacillus thuringiensis proti patogeni glivi Fusarium graminearum. Bakterija B. thu- ringensis je bila izolirana iz rizosfere posevka koruze in gojena za proizvodno lipopeptidov. Surfaktin je bil določen s tekočin- sko kromatografijo visoke ločljivosti (HPLC) iz izvlečka pri 210 nm, retencijskim časom 3-5 minut, dobljeni višek je bil 3.990. Rast patogene glive je bila uspešno zavrta pri različnih koncen- tracijah surfaktina. 80 % koncentracija surfaktina je pokazala največjo sposobnost zaviranja v primerjavi s koncentracijami 60 %, 40 % in 20 % (p < 0,005). Na osnovi te raziskave lahko zaključimo, da ima lipopeptidni surfaktin iz bakterije B. thu- ringensis velik potencial za zaviranje rasti glive F. graminearum. Ključne besede: surfaktin; Bacillus; biološka kontrola; HPLC; Fusarium graminearum Acta agriculturae Slovenica, 117/4 – 2021 2 M. KHAN et al. 1 INTRODUCTION Globally Bacillus thurenginisis is considered to be the most predominant soil-dwelling bacterium found in the plants rhizosphere known for their antimicrobial properties. Aforementioned, Bacillus strains are known as plant growth promoting rhizobacteria (PGPR) that are associated with plants tolerance against biotic, and abiotic stresses caused by certain fungal phytopathogens (Saxena et al., 2019). In this context the worldwide major loss of maize and other cereal crops is due to fungal phy- topathogens. The repertoire of fungal phytopathogens in- cluding Acremonium alternatum Link (Pal and Gardener, 2006), Ustilago maydis (DC.) Corda [ (Kwon et al., 2021), Aspergillus niger van Tieghem, Aspergillus flavus Link, Puccinia sorghi Schwein., Fusarium species (Rehman et al., 2021), Helminthosporium, Alternaria, Rhizopus, Peni- cillium, Drechslera (Snetselaar and McCann, 2017), Mac- rophomina phaseolina (Tassi) Goid., and Colletotrichum graminicola D.J. Politis (Saleem et al., 2012), cause varie- ties of disease in maize. Maize (Zea mays L.) is the most important cereal crop in the world, covering 75 % of the food require- ments all over the world (Hussain et al., 2013). In Pa- kistan, among the cereal crops, maize is the third most important crop, after wheat and rice. Among these, glob- ally the most important and significant phytopathogen is Fusarium graminearum, which causes significant loss of grain crops (Rauwane et al., 2020). The wide range of dis- eases caused by this plant pathogen includes; fruit rots, Fusarium head blight (FHB), wilts, and root rots (Kant et al., 2011). Chemical compounds have been used to manage these fungal phytopathogens for many decades. They have a potential to generate major environmental prob- lems. Alternative and less environmentally detrimental measures are required to control these plant diseases. Bacillus species and other PGPR play a key role among biopesticides. They produce various antimicrobial com- pounds such as enzymes, lipopeptides, and antibiotics that stimulate plant development while inhibiting patho- genic microbes (Shafi et al., 2017). For B. thurenginisis cyclic peptides including, surfactin, mycobacillin, myco- subtilin, subtilin, bacilysin, fengycin, bacillomycin, and iturin are reported that exhibit both antibacterial, and antifungal properties (Khan et al., 2021; Ntushelo et al., 2019). Surfactin is a lipopeptide composed of cyclic dep- sipeptides of β-hydoxy hepta with possible amino acid combinations of alanine, valine, leucine, or isoleucine at positions 2, 4, and 7 in the cyclic depsipeptide moiety and β-hydoxy fatty acid chain variants of C 13 to C 16 in the cyclic depsipeptide moiety and β-hydoxy fatty acid chain variant (Hue et al., 2001). According to the investigations surfactin has natural antifungal properties produced by Bacillus spp. that could inhibit the growth of certain fun- gal species including, F. graminearum (Khan et al., 2021), Fusarium oxysporum Schlecht. emend. Snyder & Hansen (Kim et al., 2010), Colletotrichum gloeosporiodes (Penz.) Penz. & Sacc. (Snook et al., 2009), Fusarium verticillioides (Sacc.) Nirenberg (Dunlap et al., 2011), and Fusarium moniliforme (Sacc.) Nirenberg (Vitullo et al., 2012). Therefore, the current study was designed to iso- late and characterize B. thuringiensis lipopeptide from rhizospheric soil and also to assess its antifungal effica- cy against the fungal phytopathogen F. graminearum of maize. 2 MATERIALS AND METHODS 2.1 BACTERIAL AND FUNGAL ISOLATION A total of 20 maize rhizospheric soil samples were collected from various locations in Peshawar, Pakistan, for the isolation of B. thuringiensis (Figure 1). B. thur- ingiensis was identified using colony morphology, gram staining, and biochemical-tests such as citrate hydroly- sis, catalase, indole production, nitrate reduction, Vog- es-Proskauer (VP), motility, H 2 S production, and crys- tal formation (Amin et al., 2015). F. graminearum was isolated using a sample acquired from a diseased maize plant in Peshawar, Pakistan (Figure 1), and identified us- ing colony morphology and microscopic analysis (Uddin et al., 2019; John et al., 2006). 2.2 LIPOPEPTIDE EXTRACTION AND IDENTIFI- CATION In a shaking flask containing nutrient broth medi- um (Oxoid™), all morphological and biochemical based confirmed isolated colonies of B. thuringiensis were in- jected. The flask was incubated for 16 hours at 30 o C with shaking incubator at 200 rpm. Afterward the culture was transferred to an Erlenmeyer flask containing 99 ml of Tryptic Soy Broth (TSB) medium (Oxoid™) and incubat- ed overnight at 30 o C with shaking incubator at 200 rpm. The optical density (OD) of the B. thuringiensis growth curve was measured at 600 nm using a spectrophotom- eter (Shimadzu, UV-1800). After the decline phase of B. thuringiensis growth, the culture was removed and cen- trifuged at 6000 rpm for 30 minutes. The supernatant was filtered using a sterile 0.22 µm filter (Mater et al., 2009). The extract was then centrifuged for 10 minutes at 1000 rpm and 20 o C. The deposit was dissolved in a solution Acta agriculturae Slovenica, 117/4 – 2021 3 In vitro antifungal potential of surfactin ... against maize (Zea mays L.) fungal phytopathogen Fusarium graminearum Schwabe of methanol (Analytical grade, VWR Chemicals BDH®) and water (50:50, v/v) and filtered again using a 0.22 µm filter membrane. For purification, the sample was treated three times with 20 ml chloroform (VWR Chemicals BDH®). The bottom layer was collected and chloroform was evaporated at 50 o C temperature by using a hotplate stirrer. Methanol was used to dissolve the residue. Sur- factin from the extract were identified by introducing 50 µl of the extract into a Shimadzu 20A UV-Vis HPLC at a wavelength range of 200–250 nm. The isocratic HPLC method was employed, along with a 4.6 × 150 mm C-18 normal phase column (Mater et al., 2009). For the iden- tification of surfactin by HPLC experiment, acetonitrile was utilized as a mobile phase. Surfactin were discovered after comparing the observed peak to previously pub- lished data (Meena et al., 2014). 2.3 ANTIFUNGAL ACTIVITY OF LIPOPEPTIDE EXTRACT To test the antifungal activity of surfactin, four 5 mm wells were created on potato dextrose agar (PDA) (Oxoid™) using a sterilized cork borer. The methanol was used as a control and also used to create concentrations of the lipopeptide extract of 20 %, 40 %, 60 %, and 80 %, respectively. The wells were filled with 200 µl of methanol (control), 20 %, 40 %, 60 %, and 80 % concentrations of lipopeptide extract, respectively. A colony of active grow- ing F. graminearum was placed in the middle of media plates using sterile forceps and incubated at 30 o C for 3-7 days. Five repetitive antifungal analysis of the extracted lipopeptde was done by the same method described above. The inhibitory zones were measured and recorded (Mater et al., 2009). The obtained mean zone of inhibi- tions was analyzed using a one-way ANOVA test using the Statistical Packages for Social Sciences (SPSS) version 23.0 software and Microsoft Excel. 3 RESULTS AND DISCUSSION 3.1 BACTERIAL ISOLATE In 20 rhizospheric soil samples B. thuringiensis 12 isolates were confirmed by various criteria such as, col- ony morphology, gram staining, and biochemical assays (Table 1). Previous results revealed that Bacillus species are primarily found in rhizospheric soil and that their metabolites have antibiotic characteristics as they can inhibit or restrict the development of other microorgan- isms (Amin et al., 2015). 3.2 FUNGAL ISOLATE In context to this study, F. graminearum was isolated from infected maize plants and identified using colony morphology (white to pinkish), and microscopic assess- ment (Hyaline septate hyphae, two to multi-celled and Figure 1: (A) Sampling site of maize rhizospheric soil for the isolation of B. thuringiensis, (B) Diseased maize for the isolation of F. graminearum Acta agriculturae Slovenica, 117/4 – 2021 4 M. KHAN et al. sickle-shaped) in the current investigation. Fusarium head blight (FHB) disease is caused by F. graminearum in maize. This fungus exhibit certain sign of early bleaching during infection which could reduce grain production and quality (Ntushelo et al., 2019). 3.3 LIPOPEPTIDE IDENTIFICATION According to the current study findings, B. thuring- iensis was grown to produce lipopeptides, and the optical density (OD) of the growth curve was measured (Figure 2). Lipopeptides isolated from B. thuringiensis were ana- lyzed by HPLC using acetonitrile as the mobile phase. At 210 nm and retention period 3-5 minutes, the observed peak area was 3.990 (Figure 3), which is similar to the peaks found earlier in surfactin literature data (Mubarak et al., 2015). Previous studies are in agreement with our findings. According to the Deepak and Jayapradha (2015), they identified lipopeptide surfactin by HPLC which are produced by B. thuringiensis. In another study, the lipopeptde fengycin produced by B. thuringiensis was identified by HPLC techniques (Kim et al., 2004). 3.4 ANTIFUNGAL ACTIVITY OF LIPOPEPTIDE B. thuringiensis lipopeptide surfactin against the development of F. graminearum was investigated in this work. The surfactin lipopeptide efficiently suppressed the growth of F. graminearum (Figure 4). According to ear- lier research, isolated Bacillus spp. from the rhizosphere, particularly B. subtilis, reduced the growth of F. gramine- arum. Bacillus spp. is also effective in the prevention of Fusarium head blight (FHB) and root rot; they stimu- late plant development and inhibit the mycelial growth of fungal infections through antagonistic action (Herba et al., 2020; Madhi et al., 2020; Dukare et al., 2020). In this study, lipopeptide surfactin from B. thuringiensis was tested against F. graminearum at 20 %, 40 %, 60 %, and 80 % concentrations (Figure 4). The zone of inhibi- tion was the greatest at the 80 % concentration, followed by the 60 %, 40 %, and 20 % concentrations (p < 0.005), respectively. These findings are in agreement with previ- ous report, in which the surfactin action against F. ox- ysporum (Deepak and Jayapradha, 2015) was screened. According to a recent study, microorganisms were iso- lated from plant anthers and wheat kernels to test their antagonistic activity against F. graminearum, the causa- tive agent of Fusarium head blight (FHB). B. subtilis has a strong antifungal impact on F. graminearum mycelium, sporulation, and DON formation, with inhibition values of 87.9 %, 95.6 %, and 100 %, respectively (Zhao et al., 2014). 4 CONCLUSION Lipopeptides obtained from Bacillus species have less negative environmental effects as compared to chemical compounds. The current study concluded that B. thuringiensis isolated from the rhizosphere of maize crop may produce lipopeptide surfactin, which has a high potential to inhibit the growth of F. graminearum. The study is also emphasizing surfactin as potential bio- logical control agent with widespread usage. We are also encouraging other researchers to take advantage of newly invented techniques to explore mechanism of action of various Bacillus strains against phytopathogens. Tests Results Colony Morphology Circular, rough, opaque, fuzzy white or slightly yellow Gram Staining Gram Positive Shape Rod shaped Motility Positive Catalase Positive Indole production Negative Citrate utilization Positive H2S production Negative Crystals formation Positive Identified Strains B. thuringiensis Table 1: Morphological and Biochemical characteristics of B. thuringiensis Figure 2: Optical density (OD) of the growth curve of B. thur- ingiensis at 600 nm wavelength Acta agriculturae Slovenica, 117/4 – 2021 5 In vitro antifungal potential of surfactin ... against maize (Zea mays L.) fungal phytopathogen Fusarium graminearum Schwabe Figure 3: HPLC Chromatogram of B. thuringiensis lipopeptide surfactin obtained at 210nm, retention time between 3-5 minutes and peak area 3.990 Figure 4: B. thuringiensis lipopeptide surfactin zone of inhibition (mean) against F. graminearum at various concentrations (p < 0.005) Acta agriculturae Slovenica, 117/4 – 2021 6 M. 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