Novel Metabolites Identied From Bacillus Safensis and Their Antifungal Property Against Alternaria Alternata

Plant growth promoting rhizobacteria offer an effective and eco-sustainable solution to protect crops against phytopathogens. In the present study, Bacillus safensis STJP (NAIMCC-B-02323) from the rhizospheric soil of Stevia rebaudiana showed strong biocontrol activity against phytopathogen, Alternaria alternata. B. safensis STJP produced antifungal volatile organic compounds (AVOC). In the presence of AVOC, there was no conidia germination, mycelium growth was inhibited, and hyphae ruptured as observed by scanning electron microscopy (SEM). When mycelium of the fungus from bacterial treated plate was transferred into fresh potato dextrose agar plate, A. alternata could not grow. Extracted AVOC from B. safensis STJP were identied by thin-layer chromatography (TLC), fourier-transform-infrared spectroscopy (FT-IR) and gas-chromatography-mass spectrometry (GC-MS). In total 25 antifungal metabolites were identied by GC-MS analysis having alcohol, alkane, phenol, alkyl halide and aromatic compounds. Five compounds (phenol, 2,4-bis (1,1-dimethylethyl)-, 3-hexadecanol, pyrrolo(1,2-a)pyrazine-1,4-dione, 5,10-diethoxy-2,3,7,8-tetrahydro-1H,6H-dipyrrolo(1,2-a:1',2'-d)pyrazine and hexadecanoic acid) completely inhibited the mycelium growth, controlling spore formation and conidia germination of A. alternata. This study concluded that AVOC producing B. safensis can be used as a green-fungicide against A. alternata. Bacterial metabolites could pave the way for the development of next generation biopesticide. This can be a reliable technology to enhance the quality and reliability of biopesticides.


Introduction
According to estimates, the world population is expected to reach 9 billion by 2050 (FAO 2012). Hence, the demand for food is rising continuously so as to feed the ever growing population. Currently, the total cost of annual food production is USD 1300 billion, out of which 500 billion is lost every year due to biotic stresses such as weeds, insects, fungus, virus and bacterial diseases (Watts et al. 2018). Among all, phytopathogens (including bacteria and fungi) account for yearly loss of USD 120 billion during crop production and after harvesting (Shuping et al. 2017). Decrease in the crop yields and impact on economy due to phytopathogens are often more severe in developing and poor countries, particularly where economy largely depends on agriculture (Danial et al. 2020).
Deadly fungal phytopathogen Alternaria spp. is commonly responsible for the reduction of crop productivity all around the globe (Chakraborty and Newton 2011;Müller, et al. 2019). It is one of the common fungal genus, reported around the globe with endophytic, saprophytic and pathogenic properties and also responsible for reduction in crop productivity (Müller, et al. 2019). Alternaria spp. are opportunistic pathogens, causing widespread damage to a number of crops and results in up to 80% reduction of yield in affected elds (Nowicki et al. 2012). A. alternata is known to cause leaf spot disease in several crops including Stevia rebaudiana (Meena et al. 2017). The leaf spot disease is identi ed by the presence of dark brown color or necrotic lesions on the leaf (Danial et al. 2020). The symptoms of this disease appear after infection and during pathogenesis, when fungal metabolites impact the plant during active phase of growth (Thomma 2003). The spores of A. alternata are dispersed ubiquitously and can spoil cereals, grains, fruits, foods and damage vegetative parts of the plants and ultimately decrease their nutritive value by secretion of several toxic metabolites (Meena et al. 2017).
Crop rotation, cultural practices, or use of disease-resistant varieties are commonly employed for the management of soil borne pathogens (Roy et al. 2018), but these methods are not satisfactory for control of phytopathogens. Although, chemical pesticides are e cient way to control fungal pathogens and enhance crop productivity, but their application is being increasingly limited or banned due to negative impact on the soil fertility, microbial diversity, and ecosystem and causes toxicity to animals and humans (Alori and Babalola 2018; Prakash, and Arora, 2019). Beside these, green house emission and environmental pollution are also caused due to production and application of pesticides (Harman and Uphoff 2019). Hence, this situation calls for management strategies. Biopesticides are being used to control fungal diseases along with enhancing crop yields in an eco-friendly manner. Microorganisms with the abilities of biocontrol of phytopathogens are being used as biopesticides to realize the target of sustainable agriculture ). Low input biotechnology utilizing biocontrol agents for suppression of diseases and increased plant growth has now become a needful sector for research so as to obtain quality products which are reliable and equal or even more effective than chemical pesticides (Shumaila and Khan 2016).
Gram-positive Bacillus spp. are one of the front runners for use as biopesticides because of their ability to produce a number of secondary metabolites with very different structure and nature and also broad spectrum antifungal potential (Kapetanakou and Skandamis 2016; Kai 2020). Rhizospheric Bacillus spp. are widely known to produce secondary metabolites such as bacitracin, polymyxins, lipopeptide surfactin, subtilosin, lantibiotic, iturin and gramicidin (Stoica et al. 2019;Kaspar et al. 2019). There is a continuous need to extract and identify biocontrol metabolites to develop biopesticides with effective potential against fungal pathogens such as A. alternata. The objective of the present study was to investigate the biocontrol potential of B. safensis strain against A. alternata. Extracellular antifungal metabolites produced by B. safensis were identi ed and checked for inhibition of A. alternata so as to check the mechanism of biocontrol agent (BCA) and determine the effective anti-fungal biochemicals.

Microorganisms
B. safensis STJP NAIMCC-B-02323 taken for the study has already been reported as an effective plant growth promoting rhizobacteria (PGPR) (Prakash and Arora 2019; Prakash and Arora 2020). B. safensis STJP showed plant growth promoting (PGP) characters viz. solubilization of phosphate, zinc, potassium, production of siderophore, and indole acetic acid (IAA) as reported in earlier studies by the authors (Prakash and Arora 2019; Prakash and Arora 2020). A. alternata isolate P1 (accession number: KX494864) was procured from Laboratory for Rhizospheric Microbiology and Sustainable Agriculture, School for Environmental Science, Babasaheb Bhimrao Ambedkar University, Lucknow and grown (at 28 o C) and maintained on potato dextrose agar (PDA) and Czapex dox agar (CDA) at 4 o C for further use and experiments. The fungal isolate has also been already reported as a phytopathogen in an earlier study (Prakash and Arora 2019).

Rapid screening technique
A rapid screening test is a simple process, done in the laboratory on the Petri plate for screening of antagonistic microorganisms. For rapid screening test, fungal mycelia and hyphae were cut with the help of cork borer (from an already cultured PDA plate and kept in the centre of prepared PDA plate and STJP was spotted and streaked (on separate plates) around the fungal pathogen (Schoeman et al. 1994). Subsequently, Petri plate were incubated for 120 h at 28°C and calculated for fungal growth inhibition rate as per Schoeman et al. (1994).

Dual culture method
Dual culture test was performed on PDA (Himedia, Mumbai) for screening of biocontrol potential as per Arora et al. (2001). Brie y, the fungal mycelium was cut (5 mm) by cork borer (from a PDA plate grown with A. alternata after 5 days incubation period) and placed on the center of PDA plate. Afterwards, the fresh log phase culture of B. safensis STJP (on the basis of rapid screening) was streaked on two edges of Petri plate (Arora et al. 2001). Further, Petri plates were sealed with para lm and then incubated at 28°C for 120 h. After incubation period, the inhibition of fungal growth was observed and inhibition zone was calculated by using the formula: 100 x (C-B)/C; where; C is the diameter of fungal colony in control plate and B diameter of fungus in the dual culture plate.
2.4. Screening of bacteria for antifungal volatile compound production by seal plate method Bacterial strain B. safensis STJP was checked for the production of antifungal volatile compounds as per Fernando et al. (2005). Actively growing mycelia (disc) of A. alternata (5 mm) were kept at the center of Petri plate containing PDA. The overnight grown bacterial culture was spread on another Petri plate containing nutrient agar (NA). Petri dish containing fungal mycelia was inverted over the NA and both plates were immediately wrapped with para lm. The plates were further incubated for 120 h at 28 o C. Every 24 h, growth inhibition of A. alternata by bacterial isolate was observed in comparison to control plate (contained only mycelia) as per Fernando et al. (Kersten and Kirk 1987). After 120 h, fungal mycelia were detached from the plate and further con rmed for viability in a fresh Petri plate containing PDA. The experiments were repeated three times with ve replicates taken each time.

Growth inhibition of A. alternata by extracellular or intracellular metabolites
The overnight grown bacterial culture was inoculated in nutrient broth and incubated (28°C, 48 h). After incubation, broth was centrifuged at 5,000 rpm for 15 min to get the supernatant and pellet. Subsequently, two wells were made on the prepared PDA plate with the help of sterilized cork borer and the obtained supernatant and pellet were transferred in the well of Petri plate (separately), with A. alternata at centre point to check whether the biocontrol activity is by extracellular or intracellular metabolites (Kersten and Kirk 1987). Besides, control plate of A. alternata was also incubated simultaneously.
All the plates were incubated for 120 h at 28 o C.
2.6. Growth inhibition of A. alternata checked by scanning electron microscopy For sample preparation of scanning electron microscopy (SEM), the fungal mycelia were picked up from dual culture and control plates and xed with modi ed Karnovsky's xative (2% glutaraldehyde and 2% paraformaldehyde in sodium cacodylate buffer (0.05 M; pH 7.2) and incubated for 12 h at 4 o C (Kaminskyj and Dahms 2008). Further, the xed specimen sample was washed three times with sodium cacodylate buffer (0.05 M) for 12 min followed by washing with distilled water. Subsequently, dehydration was done of the specimen with an ethanol solution of concentration of 30%, 50%, 70%, 80%, and 90% (5 min), and lastly, absolute (100%) ethanol was used (three times) for dehydration of cell for 2 min (Hennequin et al. 2006). Afterwards, the specimen was further dried by using hexamethyldisilazane and with the help of a sputter coater, and specimen was nally coated with gold (MSC-101, JOEL). The prepared sample of the specimen was observed under SEM (Model: JSM-7610F, JEOL, Japan).

Extraction and puri cation of metabolites
For extraction of antifungal metabolites, 24 h culture of bacterium STJP (selected on the basis of PGP traits) was inoculated in NB and inoculated at 120rpm, 28 o C, 7 days (Model: Labtech LS1-3016R). After the incubation period, the broth was centrifuged (rpm: 10,000rpm, 15 min, 4 o C) (Centrifuge Model: Sigma 3-18KS) to obtain supernatant. The pH of the obtained supernatant was adjusted at 7.0 and subsequently it was dissolved in the solvent; chloroform + ethyl acetate + dichloromethane (DCM) in a ratio of 1:2:1 as per Hennequin et al. (Hennequin et al. 2006). Further, 50 ml of solventsupernatant mixture was lled in a separating funnel and mixed vigorously for 15 min. The funnel was then hanged on a stand and this process was done for 24 h at 28 o C. The transparent layer of the reaction mixture containing metabolites was removed from the separating funnel and the sample was ltered via Whatman number 42 containing 5 gm of sodium sulphate (anhydrous compound, HiMedia, Mumbai) as per Hennequin et al. (2006). The obtained sample containing antifungal metabolites was kept in a sterilized beaker, and nally, solvent was evaporated by using a rotatory vacuum evaporator (Model: UTS: 1.53, Thermo-scienti c).

Thin Layer Chromatography (TLC)
Thin layer chromatography (TLC) was done for the separation of different compounds present in the crude metabolite sample as per Reddy et al. (Reddy et al. 2009). Brie y, TLC was done on freshly prepared silica plate (20 cm long x 10 cm width) by using mobile phase [chloroform: methanol (80: 20)]. The crude metabolite (10 µg) was spotted on the silica plate and the solvent was allowed to run from bottom to top. The retardation factor (RF) value was calculated as per Reddy et al. (2009). The running lane was then dried thoroughly and the fraction of the compound was analyzed under UV light (254nm). Different bands of metabolites were scraped separately into microcentrifuge tubes and further centrifuged with ethyl acetate as per Gocan (2002). After centrifugation, the solvent was evaporated through a rotatory vacuum evaporator (Model: UTS: 1.53, Thermo-scienti c) and nally supernatant was taken for further experiment.

Fourier Transform Infrared Spectroscopy (FT-IR)
To analyze the functional groups of puri ed metabolites of bacterium STJP (obtained after TLC), the sample was dried in a hot air oven at 40 o C and subsequently analyzed as per Reddy et al. (2009). Brie y, the dried sample of metabolites was mixed with analytical grade potassium bromide (KBr) (IR Grade; purity ≥ 99%) (1:30 ratio) and the reaction mixture was thinly ground and further fused into a thin pellet (13mm X 1mm) by PCl hydraulic pressure with a 15 tons capacity under vacuum pressure. Afterwards, spectra absorbance was observed by FT-IR (Model: Nicolet™ 6700, Thermo Fisher Scienti c, USA). Further, the result was analyzed in the mid-infrared region from 4000 to 450 cm − 1 with a resolution of 4 cm − 1 . For spectrum peak, scanning was done and the pure KBr as a background spectrum was kept to measure in the ambient air. The processing of data was done using OMNIC™ (v7.4) software.

Gas Chromatography-Mass Spectrophotometry (GC-MS)
GC-MS was performed using a ame ionization detector (Model: Varian Star 3400 CX, OH, USA). Brie y, 15 m DB-1 megabore column of 100% dimethylpolysiloxane was used for the separation of the crude metabolite sample (Gocan 2002). The sample was injected into a fused silica column [(25m x 0.22mm x 0.25µm (length x width x thickness)] at 22 o C which was focused cryogenically with chilled acetone (time: 2 min). This column was previously joined with a Hewlett-Packard 5890 gas chromatograph, linked with a selective Hewlett-Packard mass detector. For the carrier gas, helium was used and the ow rate was adjusted at 1 ml/min. The temperature of GC-column was from 35-200 o C at 4 o C /min ramp rate. The result obtained in the form of unknown compounds were compared with mass spectra (MS) using NIST/EPA/NIH mass spectroscopy library (Version 17).

Examination of antifungal activity of volatile organic compounds
The metabolites produced by B. safensis STJP and detected through GC-MS analysis were purchased (mentioned in Table 3) from Sigma Aldrich, USA (synthetic chemicals) to antifungal activities against phytopathogen (A. alternata) and individually evaluated using seal plate method (Fernando et al. 2005). Brie y, a pure individual compound (purchased) with volume of 100 µl was prepared and spread onto sealed Petri plate in one compartment and a 5 mm mycelium disk of A. alternata was placed in another compartment. Afterwards, plates were incubated for 5 days at 28 o C for observation of result. For control, dimethyl sulfoxide (DMSO) was used as per Fernando et al. (2005).

Data analysis
Each treatment had ve replications and all the experiments were performed in triplicates. Data were analyzed using means and using standard deviation as per Gomez and Gomez (1984

Biocontrol activity
B. safensis STJP showed biocontrol activity against A. alternata in-vitro conditions as con rmed by rapid screening and dual culture methods and the zone of inhibition was 14.8 ± 1.34 mm (Fig. 1)

Extracellular or intracellular metabolites production
B. safensis STJP inhibited mycelia growth of A. alternata as con rmed by inhibition zone when supernatant was poured, whereas no inhibition zone was observed around the well in which pellet was added. This con rmed that the isolate STJP produced extracellular metabolites which were involved in inhibition of phytopathogen. Further, these extracellular metabolites were found to be volatile in nature as con rmed by seal plate method (

Scanning electron microscope (SEM) analysis
For SEM analysis, fungal mycelium was taken from dual culture plate in which B. safensis STJP inhibited the growth of A. alternata. Fungal mycelia of A. alternata were also taken from control plate. From the control plate, straight large catenate conidia chains with transverse septa of A. alternata were observed (Fig. 3a), whereas from bacterial treated (dual) plate, the destruction, and ruptured hyphae of fungus could be seen (Fig. 3b). Through SEM images it was con rmed that STJP showed strong antagonistic activity against A. alternata. On the basis of SEM analysis, it was proved that volatile nature of antifungal compounds was able to prevent conidia germination of A. alternata. Volatile compounds produced by B. safensis STJP as soil amendments, foliar, or root applications can be used for the control of A. alternata. SEM observations suggested that the mycelia of A. alternata was deformed, ruptured and disintegrated. In the presence of AVOC, reduced apical growth, cell disruption and irregular distortions in the phytopathogen Aspergillus avus with reduction in the thickness of mycelium and conidia were observed by Horak

Fourier transform-infrared spectroscopy (FT-IR)
FT-IR spectrum was obtained of the metabolites (dried extract of the supernatant) from B. safensis STJP (Fig. 4). The absorption peaks of the FT-IR spectrum were examined as per Gri th (1996). The absorption peak at 3610.0-3355.4 cm − 1 indicated the -OH group (-OH stretching) with strong intensity corresponded to the existence of alcohol, phenol, and carboxylic acid present in the metabolite sample (Fig. 4). Absorbance peak at 3406.5 cm − 1 is attributed to -O-H stretching corresponded to the presence of carboxylic and phenolic compounds. The absorbance peak 1620.8-1434 cm − 1 indicated the C-C stretching resembling the presence of aromatic compounds such as benzene, phenol, and toluene, and absorbance peak 1115.6 cm − 1 indicated the alkyl halide and peak 800.7-662.2 cm − 1 showed the presence of aliphatic compounds such as alkane or alkene. The absorbance peak at 601.1 cm − 1 attributed to alkyl halide and another peak 465.2 cm − 1 showed the alkane group. Overall, the absorbance spectrum of bacterial metabolite from B. safensis STJP showed the presence of alcohol, alkane, phenol, alkyl halide, and aromatic ring. Puškárová et al. (2017) reported that aliphatic and aromatic compounds produced by Bacillus spp. were more effective in growth inhibition of A. alternate. However, Wang et al. (2018) suggested that alcohol and phenolic compounds were also found to be e cient in early and post-harvest leaf spot disease control, by A. alternata.

Conclusion
It can be concluded that B. safensis can be an eco-friendly tool for promoting plant growth by using as a biocontrol agent against fungal phytopathogens. B. safensis produced a bouquet of antifungal volatile compounds against A. alternata. The antifungal compounds were mainly volatile in nature. The exploitation of B. safensis as a biopesticide would be a good option in for production of healthy food, reducing health problems related with chemical pesticides, and sustainable crop and agro-industry. The variety of extracellular metabolites produced by B. safensis may be involved in several other roles related to plant-microbe interactions which need to be explored further.