**3. Biocompetitive bacteria from agricultural soil**

Regard to biocompetitive bacteria, *Bacillus subtilis* was first introduced as an inhibitor of growth and AF production of aflatoxigenic fungi by Kimura and Hirano (1988) and the ef‐ fective compound, iturin A, had been patented for the control of AF in nuts and cereals (Kimura & Ono, 1988). Nowadays, ubiquitous inhabitants of agricultural soils i.e. the genera *Bacillus* and *Pseudomonas* are widely recognized as effective biocontrol agents of aflatoxigen‐ ic fungi. The broad host range, ability to form endospores and produce different biologically active compounds with a broad spectrum of activity made these bacteria as potentially use‐ ful biocontrol agents (Saharan & Nehra, 2011).

**3.2. Screening for antifungal activity by visual agar plate assay**

ed inhibition of AF production by the bacterium (Fig. 3).

For selecting bacteria that inhibit either fungal growth or AF production, a visual agar plate assay was used as described by Hua et al. (1999) with some modifications. A 5 µl aliquot of a conidial suspension (200 conidia/µl) of a norsolorinic acid (NA)-accumulating mutant of *As‐ pergillus parasiticus* NRRL 2999 was streaked on the center of a Potato dextrose agar (PDA) plate. A single streak of 10 µl aliquots of isolated bacteria grown overnight in 0.5X Tryptic soy agar (TSA; Difco, Becton Dickinson, Franklin Lakes, NJ) at 28°C was inoculated in pe‐ ripheral lines in distance of 1.5 cm from central line by tooth pick. Screen plates were incu‐ bated for 3-5 days at 28°C and assessed visually for antifungal phenotypes (Fig. 3). Antifungal activity was assessed by comparing the zone of fungal growth inhibition in fun‐ gus co-cultured with bacteria as tests, in comparison with control plates which were inocu‐ lated only with the fungus. The effect of bacteria on AF production was assessed from the underside of the fungus where a decrease in the red pigment (NA) in the mycelium indicat‐

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**Figure 3.** Visual agar plate assay shows screen identifying antagonistic bacteria with inhibitory activity against fungal (NA-accumulating mutant of *A. parasiticus* NRRL 2999) growth and/or NA accumulation (AF production):A) Control fungal culture against distilled water on both sides of GY agar.B) Control fungal culture against distilled water (left) and an antagonistic bacterium for fungal growth (right).C) Antagonistic bacteria for fungal growth with very weak inhibitory activity on NA accumulation on both sides.D) Antagonistic bacteria for both fungal growth and NA accumu‐

lation (left) and for only NA accumulation without affecting fungal growth (right).

#### **3.1. Soil sampling and bacterial isolation**

One-hundred fifty soil samples were collected from pistachio, maize and peanut fields locat‐ ed in different regions of Damghan, Sari and Astaneh cities during June-July 2009. Sam‐ pling was done according to the latitude of each field. Each soil comprised from ten subsamples each of approximately 1000 mm3 which were obtained using a sterile trowel at 10 m intervals. The subsamples were collected from the 50 mm top of the surface soil and then mixed thoroughly in a Nylon bag. The samples were air-dried in sterile Petri-dishes and stored at 4°C before use.

For bacteria isolation, 3 g of each soil sample was added to 10 ml of sterile normal saline solution (0.8 M), mixed vigorously by vortex for 2 min and centrifuge at 2500 rpm for 10 min. The amount of 10 µl aliquots of each sample supernatant was spread on to GY (Glucose 2%, Yeast extract 0.5%) agar and KB (King's B) agar plates and incubated for 3 days at 28°C. Discrete bacterial colonies were selected every 12 h and their purity was insured after trans‐ ferring to master GY plate by tooth pick spot technique as shown in Fig. 2.

**Figure 2.** Various bacterial colonies appeared on GY agar after 3 days cultivation of soil suspensions (A). Separation and purification of colonies by using pick spot technique on GY agar master plates (B).

#### **3.2. Screening for antifungal activity by visual agar plate assay**

**3. Biocompetitive bacteria from agricultural soil**

ful biocontrol agents (Saharan & Nehra, 2011).

**3.1. Soil sampling and bacterial isolation**

26 Aflatoxins - Recent Advances and Future Prospects

stored at 4°C before use.

Regard to biocompetitive bacteria, *Bacillus subtilis* was first introduced as an inhibitor of growth and AF production of aflatoxigenic fungi by Kimura and Hirano (1988) and the ef‐ fective compound, iturin A, had been patented for the control of AF in nuts and cereals (Kimura & Ono, 1988). Nowadays, ubiquitous inhabitants of agricultural soils i.e. the genera *Bacillus* and *Pseudomonas* are widely recognized as effective biocontrol agents of aflatoxigen‐ ic fungi. The broad host range, ability to form endospores and produce different biologically active compounds with a broad spectrum of activity made these bacteria as potentially use‐

One-hundred fifty soil samples were collected from pistachio, maize and peanut fields locat‐ ed in different regions of Damghan, Sari and Astaneh cities during June-July 2009. Sam‐ pling was done according to the latitude of each field. Each soil comprised from ten subsamples each of approximately 1000 mm3 which were obtained using a sterile trowel at 10 m intervals. The subsamples were collected from the 50 mm top of the surface soil and then mixed thoroughly in a Nylon bag. The samples were air-dried in sterile Petri-dishes and

For bacteria isolation, 3 g of each soil sample was added to 10 ml of sterile normal saline solution (0.8 M), mixed vigorously by vortex for 2 min and centrifuge at 2500 rpm for 10 min. The amount of 10 µl aliquots of each sample supernatant was spread on to GY (Glucose 2%, Yeast extract 0.5%) agar and KB (King's B) agar plates and incubated for 3 days at 28°C. Discrete bacterial colonies were selected every 12 h and their purity was insured after trans‐

**Figure 2.** Various bacterial colonies appeared on GY agar after 3 days cultivation of soil suspensions (A). Separation

and purification of colonies by using pick spot technique on GY agar master plates (B).

ferring to master GY plate by tooth pick spot technique as shown in Fig. 2.

For selecting bacteria that inhibit either fungal growth or AF production, a visual agar plate assay was used as described by Hua et al. (1999) with some modifications. A 5 µl aliquot of a conidial suspension (200 conidia/µl) of a norsolorinic acid (NA)-accumulating mutant of *As‐ pergillus parasiticus* NRRL 2999 was streaked on the center of a Potato dextrose agar (PDA) plate. A single streak of 10 µl aliquots of isolated bacteria grown overnight in 0.5X Tryptic soy agar (TSA; Difco, Becton Dickinson, Franklin Lakes, NJ) at 28°C was inoculated in pe‐ ripheral lines in distance of 1.5 cm from central line by tooth pick. Screen plates were incu‐ bated for 3-5 days at 28°C and assessed visually for antifungal phenotypes (Fig. 3). Antifungal activity was assessed by comparing the zone of fungal growth inhibition in fun‐ gus co-cultured with bacteria as tests, in comparison with control plates which were inocu‐ lated only with the fungus. The effect of bacteria on AF production was assessed from the underside of the fungus where a decrease in the red pigment (NA) in the mycelium indicat‐ ed inhibition of AF production by the bacterium (Fig. 3).

**Figure 3.** Visual agar plate assay shows screen identifying antagonistic bacteria with inhibitory activity against fungal (NA-accumulating mutant of *A. parasiticus* NRRL 2999) growth and/or NA accumulation (AF production):A) Control fungal culture against distilled water on both sides of GY agar.B) Control fungal culture against distilled water (left) and an antagonistic bacterium for fungal growth (right).C) Antagonistic bacteria for fungal growth with very weak inhibitory activity on NA accumulation on both sides.D) Antagonistic bacteria for both fungal growth and NA accumu‐ lation (left) and for only NA accumulation without affecting fungal growth (right).

Table 1 represents the results of antifungal phenotypes among soil bacteria isolated from pis‐ tachio, peanuts and maize fields. Different phenotypes were identified in all soils including NA and fungal growth inhibitors (type I), NA inhibitors (type II), growth inhibitors (type III) and finally non-inhibitors of NA and growth (type IV). The only exception were bacteria type II which was not isolated from peanuts field soils. In all fields, a pattern of type IV > type I > type III > type II were obtained regard to the number of antagonistic bacteria isolated. The phenotypes I and III are suitable candidates for biocontrol of AF-producing fungi in the field, while bacteria from type II are useful for elucidate AF biosynthesis pathway.

*3.3.2. Molecular identification*

Fig. 4 illustrates all the steps for molecular identification of antagonistic bacteria. Overnight bacterial cultures on LB medium at 30°C were streaked on TSA plates. Single colonies from cultures grown on 0.5X TSA at 28°C were suspended in 2.0 ml sterile distilled water. Bacteri‐ al cells were pelleted by centrifugation at 12,000 × g for 10 min. and resuspended in 0.1 ml sterile distilled water. Total DNA from bacteria was prepared from single colonies grown on TSA according to the QIAGEN instruction. The 16s rRNA gene fragment was amplified in PCR using 1 to 5 µl of each cell suspension as template and universal primers 27F (5´- AGAGTTTGATCMTGGCTCAG-3´) and 1525R (5´AAGGAGGTGWTCCARCC-3´) (Lane, 1991). The PCRs were carried out using approximately 500 ng of total bacterial DNA, 10 µl of 10x PCR buffer, 8 µl of MgCl2 (25 mM), 10 µl of deoxynucleoside triphosphates (dNTPs) (2 mM each), 3.3 µl of each primer (20 µM), 0.5 µl of *Taq* polymerase (5 U/µl), and enough

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**Figure 4.** Molecular identification of antagonistic bacteria using PCR and DNA sequencing:A) PCR reaction tempera‐ ture cycling; denaturing at 94°C, annealing at 55°C and extension at 72°C. Every cycle, DNA between primers is dupli‐ cated.B) An agarose gel stained with ethidium bromide shows PCR amplified bacterial DNAs (lines 2 to 13 from left). DNA molecular marker (100 bp DNA ladder) is shown in line 1 from left.C) Electroherogram data of purified DNA frag‐ ments of *Pseudomonas fluorescens* 82 which originated from sequence analysis by an ABI Prism Big Dye® Terminator

v3.1 Cycle Sequencing Kit (Applied Biosystems).

Milli Q water so that the final volume of the mixture was 100 µl.


**Table 1.** Visual agar plate assay of antifungal phenotypes among soil bacteria isolated from pistachio, maize and peanuts field of Iran on PDA plates using a norsolorinic acid (NA) mutant of *A. parasiticus* NRRL 2999.

#### **3.3. Identification of biocompetitive bacteria**

The strongest antagonistic bacteria recognized from initial screening on PDA by visual agar plate assay were selected for identifying at genus and species level.

#### *3.3.1. Biochemical identification*

Selected bacteria were first determined to be either Gram-positive or Gram-negative using potassium hydroxide (Gregersen, 1978). Catalase and oxidase enzymatic activities were also determined (Barrow & Feltham, 1993). Gram-positive isolates were identified using GP2 Mi‐ croPlates (Biolog), whereas Gram-negative isolates were identified using GN2 MicroPlates (Biolog), according to the instructions of the manufacturer. Identification was based on the similarity index of carbon source utilization by each isolate relative to that of identified ref‐ erence strains in the Biolog GP and GN databases.

#### *3.3.2. Molecular identification*

Table 1 represents the results of antifungal phenotypes among soil bacteria isolated from pis‐ tachio, peanuts and maize fields. Different phenotypes were identified in all soils including NA and fungal growth inhibitors (type I), NA inhibitors (type II), growth inhibitors (type III) and finally non-inhibitors of NA and growth (type IV). The only exception were bacteria type II which was not isolated from peanuts field soils. In all fields, a pattern of type IV > type I > type III > type II were obtained regard to the number of antagonistic bacteria isolated. The phenotypes I and III are suitable candidates for biocontrol of AF-producing fungi in the field,

while bacteria from type II are useful for elucidate AF biosynthesis pathway.

**Inhibitory bacteria**

Pistachio 290 37 + +

Maize 227 49 + +

Peanuts 87 19 + +

peanuts field of Iran on PDA plates using a norsolorinic acid (NA) mutant of *A. parasiticus* NRRL 2999.

plate assay were selected for identifying at genus and species level.

**Table 1.** Visual agar plate assay of antifungal phenotypes among soil bacteria isolated from pistachio, maize and

The strongest antagonistic bacteria recognized from initial screening on PDA by visual agar

Selected bacteria were first determined to be either Gram-positive or Gram-negative using potassium hydroxide (Gregersen, 1978). Catalase and oxidase enzymatic activities were also determined (Barrow & Feltham, 1993). Gram-positive isolates were identified using GP2 Mi‐ croPlates (Biolog), whereas Gram-negative isolates were identified using GN2 MicroPlates (Biolog), according to the instructions of the manufacturer. Identification was based on the similarity index of carbon source utilization by each isolate relative to that of identified ref‐

**Inhibition of**

9 + − 22 − + 222 − −

6 + − 13 − + 159 − −

0 + − 16 − + 62 − −

**NA Fungal growth**

**Fields of soil sampling**

28 Aflatoxins - Recent Advances and Future Prospects

**Total bacteria**

**3.3. Identification of biocompetitive bacteria**

erence strains in the Biolog GP and GN databases.

*3.3.1. Biochemical identification*

Fig. 4 illustrates all the steps for molecular identification of antagonistic bacteria. Overnight bacterial cultures on LB medium at 30°C were streaked on TSA plates. Single colonies from cultures grown on 0.5X TSA at 28°C were suspended in 2.0 ml sterile distilled water. Bacteri‐ al cells were pelleted by centrifugation at 12,000 × g for 10 min. and resuspended in 0.1 ml sterile distilled water. Total DNA from bacteria was prepared from single colonies grown on TSA according to the QIAGEN instruction. The 16s rRNA gene fragment was amplified in PCR using 1 to 5 µl of each cell suspension as template and universal primers 27F (5´- AGAGTTTGATCMTGGCTCAG-3´) and 1525R (5´AAGGAGGTGWTCCARCC-3´) (Lane, 1991). The PCRs were carried out using approximately 500 ng of total bacterial DNA, 10 µl of 10x PCR buffer, 8 µl of MgCl2 (25 mM), 10 µl of deoxynucleoside triphosphates (dNTPs) (2 mM each), 3.3 µl of each primer (20 µM), 0.5 µl of *Taq* polymerase (5 U/µl), and enough Milli Q water so that the final volume of the mixture was 100 µl.

**Figure 4.** Molecular identification of antagonistic bacteria using PCR and DNA sequencing:A) PCR reaction tempera‐ ture cycling; denaturing at 94°C, annealing at 55°C and extension at 72°C. Every cycle, DNA between primers is dupli‐ cated.B) An agarose gel stained with ethidium bromide shows PCR amplified bacterial DNAs (lines 2 to 13 from left). DNA molecular marker (100 bp DNA ladder) is shown in line 1 from left.C) Electroherogram data of purified DNA frag‐ ments of *Pseudomonas fluorescens* 82 which originated from sequence analysis by an ABI Prism Big Dye® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems).

The PCR mixtures were denatured at 95°C for 5 min, which was followed by 35 cycles of 94°C for 30s, 55°C for 30s, and 72°C for 90s and then a final extension at 72°C for 5 min. Am‐ plification was checked for purity by electrophoresis on a 1.0% agarose gel. The bands of in‐ terest were excised from the gel, and the DNA was purified using QIAquick PCR purification columns (Qiagen, Inc., Valencia, CA). Purified DNA fragments were sequenced using the same sets of primers that were used for amplification by an ABI Prism Big Dye® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems). Bacteria were identified based on sequence similarities to homologous 16S rRNA gene fragments in the Ribosomal Data‐ base Project database (Cole et al., 2005) (accessed at http://rdp.cme.msu.edu/index.jsp).

Identified bacteria (0.1 ml of bacterial inoculums containing Ca. 107

sion of *A. parasiticus* NRRL 2999 containing Ca 107

**4.1. Culture conditions for metabolite production**

**approach**

ed on 20 ml of PDB prepared in 100 ml capacity flasks and incubated for 48 h at 28°C in shak‐ ing condition (100 rpm). Cell free supernatant fluids were prepared by centrifuging the cultures at 23990×g for 15 min. The supernatant was supplemented with PDB to compensate for the consumption of nutrient by bacterial growth the pH of supernatant fluid was adjust‐ ed to that of the original medium. Supernatant fluids were sterilized by filtration through a 0.45 µm pore size nylon membrane. Five ml aliquots of sterilized bacterial supernatant were aseptically dispensed in 25 ml Erlenmeyer flasks and inoculated with 0.1 ml of a spore suspen‐

Terrestrial Bacteria from Agricultural Soils: Versatile Weapons against Aflatoxigenic Fungi

h at 28°C and analyzed for fungal growth and AF production. At the end of incubation peri‐ od, fungal mycelia were separated from culture medium using filter paper. Mycelia dry weight was determined as an index of fungal growth by incubating a known weight of fun‐ gal biomass at 80°C for 3 h and then until a constant weight was obtained. AF was extracted from the culture medium using chloroform. The chloroformic extracts were concentrated by a rotary evaporator (EYELA N-1000, Japan) to dryness. Quantitation of AFB1 was carried out us‐ ing HPLC (KNAUER D-14163 UV-VIS system, Germany) (Razzaghi-Abyaneh et al., 2007). Fif‐ ty ml of each sample (chloroformic extract) were injected into the HPLC column (TSKgel ODS-80TS; 4.6 mm ID × 150 mm, TOSOH BIOSCIENCE, Japan) and eluted at a flow rate of 1 ml/min. by water-acetonitrile-methanol (60:25:15, v/v/v) as mobile phase. AFB1 was meas‐ ured at wavelength of 365 nm. The elution time of the samples was compared with AFB1 stand‐ ards and quantified on the basis of the ratio of the peak area of samples to those of the standards. As shown in Table 2, secretory metabolites of all tested antagonistic bacteria includ‐ ing *Pseudomonas aeruginosa* (12 isolates), *Bacillus subtilis* (3 isolates), and one isolate of each *Pseudomonas chlororaphis*, *P. fluorescens* and *Bacillus amyloliquefaciens* inhibited both *A. parasiti‐ cus* growth and AFB1 production by different extents. Fungal growth was inhibited in the range of 15.3 to 72.7%, while AFB1 synthesis was suppressed by 18.7 to 96.9%. The highest in‐ hibition of fungal growth and AFB1 production was related to *P. fluorescens* 82 and *P. aerugino‐ sa* 168, respectively. In contrast to *Pseudomonas*, *Bacillus* species strongly inhibited fungal growth with a weak suppressive effect on AF production. All antagonistic bacteria except *P. aeruginosa* 155 from maize, *P. chlororaphis* 236 from peanuts and *P. fluorescens* 82 from pista‐ chio were capable of producing surfactants as a part of their pathogenesis system (Table 2).

**4. Purification of antifungal metabolites from soil bacteria: A practical**

As the first step for production of bioactive antifungals, different culture conditions includ‐ ing medium, incubation time and aeration should be optimized. In order to initial purifica‐ tion of inhibitory metabolites, the selected bacterium with strongest antifungal activity in initial screening was cultured on suitable liquid media such as GY (2% glucose, 0.5% yeast extract), SCD (2% bacto dextrose, 20% potato infusion), PDB (potato dextrose broth) or even KB (King´s B). The cultures were checked for optimal conditions of aeration (stationary cul‐ tures to shaking at different rpm from 100 to 250), incubation times (for at least 1 to maxi‐

CFU/ml) were inoculat‐

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31

conidia/ml. Cultures were incubated for 96

#### **3.4. Antagonistic activity against aflatoxigenic** *A. parasiticus* **NRRL 2999**

Cell free culture supernatants of inhibitory bacteria were used in an antagonistic assay sys‐ tem. Table 2 represents the strongest antagonistic bacteria which were identified by a combi‐ nation of biochemical and molecular methods in relation to their source of isolation.


**Table 2.** Inhibitory effects of the strongest antagonistic bacteria selected from screening plates of visual agar plate assay on *A. parasiticus* NRRL 2999 growth and AF production in Potato dextrose broth. Control fungal culture had a growth rate of 51.17 mg and an AFB1 amount of 697.78 ng/mg fungal dry weight.

Identified bacteria (0.1 ml of bacterial inoculums containing Ca. 107 CFU/ml) were inoculat‐ ed on 20 ml of PDB prepared in 100 ml capacity flasks and incubated for 48 h at 28°C in shak‐ ing condition (100 rpm). Cell free supernatant fluids were prepared by centrifuging the cultures at 23990×g for 15 min. The supernatant was supplemented with PDB to compensate for the consumption of nutrient by bacterial growth the pH of supernatant fluid was adjust‐ ed to that of the original medium. Supernatant fluids were sterilized by filtration through a 0.45 µm pore size nylon membrane. Five ml aliquots of sterilized bacterial supernatant were aseptically dispensed in 25 ml Erlenmeyer flasks and inoculated with 0.1 ml of a spore suspen‐ sion of *A. parasiticus* NRRL 2999 containing Ca 107 conidia/ml. Cultures were incubated for 96 h at 28°C and analyzed for fungal growth and AF production. At the end of incubation peri‐ od, fungal mycelia were separated from culture medium using filter paper. Mycelia dry weight was determined as an index of fungal growth by incubating a known weight of fun‐ gal biomass at 80°C for 3 h and then until a constant weight was obtained. AF was extracted from the culture medium using chloroform. The chloroformic extracts were concentrated by a rotary evaporator (EYELA N-1000, Japan) to dryness. Quantitation of AFB1 was carried out us‐ ing HPLC (KNAUER D-14163 UV-VIS system, Germany) (Razzaghi-Abyaneh et al., 2007). Fif‐ ty ml of each sample (chloroformic extract) were injected into the HPLC column (TSKgel ODS-80TS; 4.6 mm ID × 150 mm, TOSOH BIOSCIENCE, Japan) and eluted at a flow rate of 1 ml/min. by water-acetonitrile-methanol (60:25:15, v/v/v) as mobile phase. AFB1 was meas‐ ured at wavelength of 365 nm. The elution time of the samples was compared with AFB1 stand‐ ards and quantified on the basis of the ratio of the peak area of samples to those of the standards. As shown in Table 2, secretory metabolites of all tested antagonistic bacteria includ‐ ing *Pseudomonas aeruginosa* (12 isolates), *Bacillus subtilis* (3 isolates), and one isolate of each *Pseudomonas chlororaphis*, *P. fluorescens* and *Bacillus amyloliquefaciens* inhibited both *A. parasiti‐ cus* growth and AFB1 production by different extents. Fungal growth was inhibited in the range of 15.3 to 72.7%, while AFB1 synthesis was suppressed by 18.7 to 96.9%. The highest in‐ hibition of fungal growth and AFB1 production was related to *P. fluorescens* 82 and *P. aerugino‐ sa* 168, respectively. In contrast to *Pseudomonas*, *Bacillus* species strongly inhibited fungal growth with a weak suppressive effect on AF production. All antagonistic bacteria except *P. aeruginosa* 155 from maize, *P. chlororaphis* 236 from peanuts and *P. fluorescens* 82 from pista‐ chio were capable of producing surfactants as a part of their pathogenesis system (Table 2).
