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The effect of Bacillus subtilis Vru1 encapsulated in alginate – bentonite coat-

ing enriched with titanium nanoparticles against Rhizoctonia solani on bean

Roohallah Saberi-Rise, Mojde Moradi Pou

PII: S0141-8130(19)36022-2
DOI: https://doi.org/10.1016/j.ijbiomac.2019.10.197
Reference: BIOMAC 13698

To appear in: International Journal of Biological Macromole-

cules

Received Date: 31 July 2019

Revised Date: 23 October 2019
Accepted Date: 23 October 2019

Please cite this article as: R. Saberi-Rise, M.M. Pou, The effect of Bacillus subtilis Vru1 encapsulated in alginate
– bentonite coating enriched with titanium nanoparticles against Rhizoctonia solani on bean, International
Journal of Biological Macromolecules (2019), doi: https://doi.org/10.1016/j.ijbiomac.2019.10.197

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 The effect of Bacillus subtilis Vru1 encapsulated in alginate – bentonite

coating enriched with titanium nanoparticles against Rhizoctonia solani on bean

Roohallah Saberi-Riseh*a, Mojde moradi pourb

a,bDepartment of Plant Protection, Faculty of Agriculture, Vali-e-Asr University of Rafsanjan,

Iran.

*
Corresponding author: Roohallah Saberi-Riseh

Associate professor, Department of Plant Protection, Faculty of Agriculture, Vali-E-Asr University of Rafsanjan, Iran

Fax: 0098-3431312041

E-mail address: r.saberi@vru.ac.ir

Tel: 0098-9131932624

1
Abstract

This research was investigated the colonization efficiency and survival rate of nanoencapsulated

Bacillus subtilis Vru1 prepared with sodium alginate (NaAlg), starch and bentonite and, this

efficiency for controlling Rhizoctonia solani. The XRD and FTIR analysis in this research

indicated the absence of chemical reactions and correct mixing efficiency were funded with

alginate, bentonite and starch. The highest release of B.subtilis Vru1 was 4×109 in 45th days of

storage. The quantity of colonization by B.subtilis Vru1 nanocapsules was lower than that of free

B.subtilis Vru1 at days 5-20th and was significantly higher than that of free B.subtilis VRU1 and

encapsulated B.subtilis VRU1 without Titanium dioxide nanoparticle, after day 35th. This level

was maintained for up to 45th day. The results of this experiments indicated Vru1 nanocapsules

with 90% had the highest and free Vru1 with 60% had the lowest inhibition of R.solani on bean

and Vru1 capsule without titanium nanoparticles (TNs) decreased the disease by 75%. The

nanoencapsulated B.subtilis Vru1 strain significantly increased the bean vegetative growth

parameters. This finding probably attributed to the enhancement in the number of the bacterium

and the high level of metabolites production such as indole-3-acetic acid. Thus, nanocapsule

formulation is potential alternatives for sustainable agriculture.

Keywords: Nanoencapsulation, PGPR, Starch, Titanium nanoparticles

2
1. Introduction

Beans (Phaseolus vulgaris L.) have the highest cultivation area than other legumes in Iran. In Iran,

about 23 million tons of dry beans and 20 million tons of green beans were harvested from 30

million hectares in 2012. Bean in addition to direct human use, its product residues can be used as

animal feed and organic manure. Rhizoctonia solani is one of the most destructive pathogens in

agriculture and that infects a wide range of crops such as bean, potato and, etc. [1]. This pathogen

has the parasitic ability and it can survive as a saprotroph in the soil [2,3]. This very destructive

fungi can attack main parts of the plants such as roots, stems and etc. to cause seed decay, damping-

off of seedlings, wire stem and sore shin, root rot, hypocotyl and stem canker, bottom rot or head

rot, and storage rots a variety of crops, including agronomical, forestry and ornamental species

worldwide [1]. Microbial synergy in the rhizosphere is an important factor in the maintenance of

plant and soil productivity. Soil bacteria stimulate plant growth by interfering in the soil activities,

which improves the reserve of available nutrients to the plant [4]. Several experimental indicated

the plant growth promoting rhizobacteria to soil improves the plant growth by some mechanism

such as HCN, siderophore, auxin (indole-3-acetic acid) and, etc. [5,6]. Plant growth-promoting

rhizobacteria are promising biocontrol agents for use in plant protection but their efficacy is

variable in field conditions. Therefore, bacterial formulations that can protect, help and increase

biocontrol will be of great advantage to agriculture. Microbial survival amount in the soils depends

on biotic and abiotic factors. A main role of the formulation is to supply a more suitable

environment for the prolonged survival in the soil. It is opined that the encapsulation method

assistances to enhancement the survival rate and easy delivery of bacteria cultures. It also supports

the bacterial cells from the harmful environment and thereby decreasing cell loss. In recent years

many experimental formulations based on polymers have been done [7] and showed to be potential

3
bacterial carriers [8]. Capsules were protecting the microorganisms against biotic and abiotic

stresses and releasing bacterium to the soil slowly when the polymers are degraded by soil

microorganisms [7]. The encapsulation of bacterium can raise cell survival during storage, and

encapsulated cells could be released into the purpose medium in a slow and controllable method,

which long-range enhancement effectiveness. Also, after a long period of storage, bacteria do not

lose their ability to increase plant growth. Titanium dioxide nanoparticles (TNs) have one of the

important materials in nanotechnology. Recently ideas have emerged to use TNs in agricultural.

TNs have been indicated to have adhesive effects on the bacterium. Park et al, [9] showed that

titanium dioxide nanoparticle enhancement adhesion of bacterium on to surfaces. These properties

of TNs could be applied to guide bacterium to a place where they are required. A suitable

formulation, encapsulating Bacillus subtilis with sodium-alginate, starch, and bentonite, enrich

with TNs to control Rhizoctonia solani infection of bean plants will be important and effective.

This research aims are to develop a novel microencapsulation system using alginate, bentonite,

starch and TNs to protect the biocontrol bacteria against harmful environmental conditions, and to

enhancement their survival rates and controlling of Rhizoctonia solani infection in bean plants.

2. Materials and methods

Rhizoctonia solani was retrieved from the Fungus collection maintained at the University of

Tehran and Bacillus subtilis Vru1 was selected from biological control collection of the Vali-e-

Asr University of Rafsanjan.

2.1. Laboratory tests for plant growth-promoting (PGP) activities of Vru1 strain

In vitro indole-3-acetic acid production by the strain was studied on the method described by Patten

and Glick [10]. Detection of siderophore production was done in chrome azurol agar plates [11].

4
Phosphate solubilization activity with isolates was measured on tricalcium phosphate agar [12].

Protease and cellulase were characterized on solid agar plate method as outlined in Maurhofer et

al. [13] and Kasana et al. [14], respectively. The lipopeptide production was done according to

Sarwar et al. [15], and the Vru1 bacterium ability to chitinase production was assayed with the

method described by Saima et al. [16] with some modification.

2.2. Fungal inhibition zone

The growth inhibition of R. solani by Vru1 strain was measured on PDA (potato dextrose agar).

Overnight cultures of Vru1 strain was spotted in PDA by 1 cm from the edge of plates and, 0.5 cm

mycelia disc from the fresh culture of R.solani placed in the center of the plate. Plates were

incubated at 27 °C. After five days, the diameter of the inhibition zone was measured [17].

2.3. Preparation of microcapsules

The bentonite was purified by soaking dried grounded bentonite into 5 volume of water for 24h;

then, the suspension was stirred for 50 min and was allowed to stand for a period of 5h. Finally,

the suspension was centrifuged and then precipitate dried at 70˚C and was used in formulation.

The encapsulation of Bacillus subtilis Vru1 cells with the composites of sodium alginate (NaAlg),

bentonite and starch was carried out by according to the method described by He et al. [18].

Suspension having different compositions were prepared using sodium alginate (1.5%), bentonite

(4%), starch (3%), TiO2 nanoparticle (10 ppm) contents, and the composite suspension were mixed

evenly with bacterium strain broth at a 2:1 ratio. The mixtures, including bacterium strain cells,

were added drop-wise into the 150 ml of CaCl2 (2%) crosslinking solution. After about two h, the

capsules obtained were cleaned two or three times with sterile water and dried in ovens at 40˚C.

2.3.1. SEM investigation of the microcapsule

5
The morphology of the microcapsules produced and its size was determined by scanning electron

microscopy (EM 320).

2.3.2. FT-IR Spectroscopy

The FTIR of the sodium-alginate, bentonite, starch and microcapsules formulation was captured

using KBr pellet technique (Nicolt. IS10, 60625-1, American). The spectra were prepared over the

range from 4000 to 400cm−1.

2.3.3. XRD (X-ray diffraction)

For investigation the morphology of the sodium-alginate, starch, bentonite and microcapsules

formulation was used A D8 Advance (Bruker) X-ray diffractometer. Diffractograms were

recorded from 10° to 70° (2θ) at an angular speed of 1° (2θ) min−1.

2.3.4. Evaluation of release of the encapsulated bacterium in the phosphate buffer

In vitro release of the bacterium from the nanocapsules was determined using a dialysis method,

the method with some modification [19]. Approximately 1 g of nanocapsules was placed in the

dialysis cellulosic membrane bag, which was sealed and added into 400 ml of phosphate buffer,

(pH = 7.4). The system was thermostated at 28 ± 2˚C and magnetically stirred at 200 rpm. Every

day, all of the viable bacteria were counted for colony counting after serial dilution [20].

2.3.5. Determination of B. subtilis Vru1 in the root of plants

To characterized the colonization quantity of Vru1 nanocapsules, Vru1 capsules without TNs and

free B.subtilis Vru1 on the bean roots were taken out at 5, 10, 15, 20, 25, 30, 35, 40 and 45 days

of inoculation. Then, 1 gram of roots in each treatment was measured, washed, and dissolved in

phosphate-buffer (137 mM NaCl, 2mM KH2PO4, 10 mM Na2HPO4, 2.7 mM KCl). After 2h,

6
bacteria were detached from the root and the viable bacteria in phosphate buffer were counted

using the NA agar plates and incubated at 28˚C for 24h [18].

2.4. Preparation of inoculant

300 g barley seeds were poured into the 1-liter flask and autoclaved three times at 121°C, 1.5 atm

pressure for 20 minutes. Six mycelia discs of 7-day-old R.solani in PDA were added into the flasks

and stored at 27ºC for 21days.

To use the bacterium strain during the research, cell suspension of Vru1 strain was prepared in

sterile distilled water and the concentration adjusted to 1010 CFU mL-1 (OD 0.5 at 530nm =1010)

using a spectrophotometer (U-2000, Hitachi Instruments, Tokyo, Japan). Then, 10 ml of bacterium

suspension (1010 CFU/ml) was used for each kilogram of potting soil.

2.5. Greenhouse experiments

Bean seeds were sterilized by soaking in 2.5% sodium hypochlorite for 3 min and washed several

times with sterile water and allowed to natural drying. The germination was performed by sowing

seeds on 1% water agar in a petri dish. After three days, four germinated seeds were placed in pots

that contained 2 kg of sterilized soil. To determine the potential of the B. subtilis Vru1 applications

for bean, experiments were conducted using either bacterium nanocapsules or free .The treatments

were as follows: T-0 with 20 ml free B.subtilis Vru1; T-1 with 20 ml free B.subtilis Vru1 and 4 g

of barley seeds included R.solani; T-2 with 4 g encapsulated B.subtilis Vru1 bacterium and no

pathogen; T-3 with 4 g encapsulated B.subtilis Vru1 bacterium and 4 g of barely seeds included

R.solani, T-4 with 4 g of barely seeds included R.solani, T-5 with nanocapsule without bacterium,

T-6 control, (B.subtilis Vru1 content in each treatment: 1010 CFU/treatment). Five Bean seeds

were sown in each pot, and each treatment included 10 replicates. Moisture was held by irrigating

7
sterile water three times a week. Forty-five days after planting, three plants from each treatment

were randomly harvested, and the data on plant growth factors, such as dry weight, fresh weight,

and plant height, were prepared and disease percentage was recorded.

2.6. Statistical analysis:

The data of evaluation of bacterium viability, the colonization amount and growth factor

measurement were analyzed in one-way ANOVA. Significant the SAS 9.1 (SAS Institute, Inc,

Cary, NC, USA) were used to analyzed the data and to compere mains.

3. Results

3.1. Siderophore production

The results of the siderophore experiment showed that Vru1 strain was able to produce siderophore

and changed the color of the chrome azurol S (CAS) agar medium from blue to orange. The

diameters of the orange halo around colony approximately was 20 mm in 2nd days after incubation

(Fig. 1).

3.2. indole-3-acetic acid production

According to the result of indole-3-acetic acid producing with B.subtilis Vru1 strain indicated that

this strain has the high ability to IAA producing. So that, this strain produced 19.3µg IAA ml-1

(Fig. 1).

3.3. Cellulase and protease enzymes production

The results of cellulase and protease test revealed that Vru1 produced both enzymes. Diameters of

the halo zone of Vru1 colonies on Skim Milk Agar (SMA) medium was 2.17 cm after 48 h. The

8
cellulose production was determined by clear halo zone around of bacterial colony after staining

with congored (Fig. 1).

3.4. Phosphate solubilizing activity

The presence of colorless halos (2.14cm) around the Vru1 colony after 7 days suggested that

phosphate solubilizing activity was positive in Vru1 bacterium (Fig. 1).

Lipopeptide and chitinase enzymes production

Lipopeptide production with B.subtilis Vru1 indicated significant antifungal activity (76.6%)

against R.solani. On the basis of colloidal chitin degradation and zone of clearance around the

bacterium colony showed this strain had ability of chitinase production (Fig. 1).

3.5. Fungal inhibition zone

Zone of inhibition produced by the Vru1 strains was measured in millimeters and it was observed

that Vru1 strain was caused inhibition zone more than 20mm (Fig. 2).

3.6. SEM of capsules

SEM photographs of nanocapsules shown in Fig. 3. The nanocapsules were an almost globular

form with irregular surfaces and its size was approximately 150 micrometers.

3.7. FTIR

Fourier-transform infrared spectroscopy (FTIR) was used to determine the NaAlg-B-S

nanocapsule formulations. The spectra of sodium-alginate indicated characteristic peaks in the area

of 3300-3600 cm-1 because of the O-H stretching of H2O. C-H stretching band has appeared at

2921 cm-1. Two bands were shown at 1619 and 1421 cm-1, were determined to the O-H bending

of the H2O band and COO- stretching, respectively. NaAlg indicated a peak at 1320 cm-1 for C-H

9
bending stretching and a particular peak at 1126 cm-1 related to the C-O stretching vibration of

CH2-OH. The peak at 614 cm-1 associated with the vibration of the Na-O bond. The analysis of

starch indicated the absorption bands at 3420 cm-1 related to the O-H stretching vibration, and the

band at 2931 cm-1 was due to C-H stretching in glucose. The sharp absorption band at 1654 cm-1

maybe originated from stoutly bound water available in the starch molecules. The peaks at 1366

corresponded to O-H bending and the peaks at 1082 cm-1 related to C-O stretching. The band

absorption at 765 cm-1 was due to the vibrational the polysaccharides. In the infrared spectroscopy

of bentonite, the wide band around 3434 cm-1 was related with the OH- stretching mode of water,

while the sharp band at 3630 cm-1 is associated the octahedral sheet in the bentonite. The band at

1638 cm-1 was determined to the -OH bending vibrations of water in the bentonite, and the band

at 1038 cm-1 showed the Si-O stretching vibrations. The band at 520 cm-1 and 462 cm-1 related to

Si-O-Al deformation, and Si-O-Si deformation, respectively. By contrasting the FTIR spectra of

nanocapsule with those of NaAlg, bentonite and starch, according to the results of Chougala et al.

(2017), the appeared peaks at 464 cm-1 and 3422 cm-1 related to TiO2 nanoparticles and presented

all of the special bands of NaAlg, starch and bentonite, which showed that no chemical reaction

happened between NaAlg, bentonite and starch (Figs. 4-7).

3.8. XRD

X-ray diffractograms of NaAlg, bentonite, starch and their formulated with TNs showed in Fig 8.

NaAlg exhibited no crystal peak in Fig. 7. Starch showed three peaks in 2θ =15.12°, 2θ =17.90°and

2θ =23.70°. Bentonite had nine reflection peaks between 2θ of 19˚ and 62˚, which as follows: 2θ

=19.83˚, 2θ =27.84˚, 2θ =29.50˚, 2θ =35.04˚, 2θ =61.95˚, 2θ =26.67˚, 2θ =20.89˚, 2θ =45.83˚and

2θ =31.5˚. The diffraction patterns of nanocapsule formulation did exhibit new peaks of

crystallinity in 2θ =29.53˚.

10
3.9. Evaluation of release and bacterium viability in the phosphate buffer

The effects of storage at 27˚ C on the release of B.subtilis Vru1 from nanocapsules are presented

in Fig. 5. The quantitative assessment of bacterium growth upon storage at 27˚C showed that

bacterium growth reduced with time. The number of released bacterium from nanocapsules

decreased after 40 days of storage at 27˚C (Fig. 9). The nanocapsules significantly improved

(P<0.01) the survival rate of B.subtilis Vru1 compared with the free B.subtilis Vru1. He et al. [16]

indicated that CaAg-B-S microcapsules of P. putida Rs-198 are less susceptible to salinity stress

compared to P. putida Rs-198 without coating.

3.10. Changes of root colonization amount of B.subtilis Vru1

Root colonization is the main factor in the survival ability of bacterium [28]. Therefore, the

colonization amount of the free B.subtilis Vru1, Vru1 capsules without TNs and Vru1

nanocapsules (Vru1 capsules + TNs) to bean root were analyzed. Fig. 9 Indications that the

quantity of colonization by B.subtilis Vru1 formulation was less than that by free B.subtilis Vru1

from 5 to 20 days (Fig. 10). The use of encapsulated Vru1 led to a population concentration in the

bean root that was similar to that obtained with cells freely added to the soil on days 25th. Anyway,

the quantity of bacterium colonization in the rhizosphere under the nanoencapsulated B.subtilis

Vru1 treatments was significantly higher than that the plants treatments by the B.subtilis Vru1

without coating and Vru1 capsules without TNs in 30th days and was retained up to 45 days. Our

research indicated that bean root colonization by B.subtilis Vru1 nanocapsules could occur at high

levels (log CFU = 9-9.1 per g fresh root) at 45 days. The B.subtilis Vru1 bacterium trapped in

nanocapsules was continually and slowly released into the soil, leading to the colonization of the

bean root.

11
3.11. Greenhouse experiments

The result of these experiments indicated that efficiency bacterium strain and its encapsulation was

different in case of R.solani suppression. Two treatments, including Vru1 bacterium and Vru1

nanocapsules were able to control the disease more than 60%. Plants mortality was significantly

decreased (P < 0.01) by 90 to 60% in bean plants treated with nanoformulated Vru1 and free B.

subtilis Vru1. All bean plants inoculated with R.solani had died after 45 days after inoculation

(Fig. 11). The results indicated that use of nanoformulations prepared with Vru1 strain in bean

plants in greenhouse conditions significantly (P<0.01) reduced the R.solani growth, better than

Vru1 capsule without TNs. So that, the Vru1 nanocapsule and Vru1 capsule without TNs decreased

the disease by 90% and 75% respectively.

Plant growth as measured through shoot fresh weight, root fresh weight, shoot and root dry weight

and plant height were significantly affected by nanoformulated of bacterium strain. The formulated

plants with B.subtilis Vru1 nanocapsules recorded higher amount in all the parameters measured

compared to control (Fig. 12). Vru1 nanocapsules resulted in an enhancement in the assessed

growth parameters in R.solani inoculated and non-inoculated plants compared to plants that were

not inoculated with the bacteria strains. There were significant (P<0.01) differences in shoot

length, root fresh weight, root dry weight, shoot fresh weight and shoot dry weight (Table 1).

4. Discussion

Rhizoctonia solani is considered the important soil borne pathogenic fungus able of causing intense

damage to agricultural products such as bean, cotton and potato [21, 22, 23].

Several B.subtilis strains had demonstrated their effectiveness on the control of plant diseases, due

to their ability to produce a wide variety of antifungal compounds, such as volatiles, enzymes and

12
lipopeptides. Vru1 strain applied in this study also showed several enzyme activities such as IAA,

protease, cellulase production and some metabolites production. The ability of this strain to

produce some metabolites suggested that it can act on R.solani growth by antibiosis. Proteases also

play the main role in antifungal activity by degrading the protein link in the external fungal layers

and the use of the fungal proteins for nutrition [24,25]. Protease enzyme can contribute to the

activity of bacterium to control fungal diseases. The exoprotease was enhancing the biocontrol

activity against Pythium [26]. There are a many strains with the efficiency to induce resistance in

several plants by siderophore production [27]. Some research showed that production of

pyoverdines increasing the biocontrol ability of the Pseudomonads fluorescent [28-29].

Siderophore competition for iron is one of the mechanisms of biocontrol inhibiting soil-borne

bacteria [29]. Zone of inhibition indicating susceptibility to antimicrobial compound secreted by

the bacteria. It has been demonstrated that B. subtilis can produce some antifungal metabolites

such as bacitracin, subtiline, bacillomycin and bacillin [30]. Lipopeptides help bacteria to survive

and grow by allowing them to instate of multicellular communities supporting increased

colonization and motility abilities [31]. Chitinase plays an important role in the decomposition of

chitin and since chitin is a main composition of the cell walls of R. solani, also chitinases hydrolyze

cell wall chitin eventually leading to cell death [16].

Peaks observed in starch can be due to regular crystallization and its close molecular packing.

According to the results of Brindley et al. [31], it can be seen that the peak of montmorillonite

indicated at 2θ =19.85˚, 2θ =27.79˚, 2θ =29.81˚, 2θ =35.1˚, 2θ =61.86˚ corresponding to the results

of this study. Also, reflections pattern bagcellulose membrane of the quartz was observed in 2θ

=20.89˚, 2θ =26.85˚, 2θ =31.77˚, 2θ =45.50˚, which was similar to the results of Salem et al. [32].

Which, according to the results of Lee et al. [34] can be due to the presence of Titanium dioxide

13
nanoparticles (TNs) during the formulation process. The arrangement of the crystal structure

probably imputed to the egg box area along the orientation of the alginate chain. The XRD results

indicated well mixing efficiency between alginate, bentonite and starch.

We select Vru1 strain for formulation and greenhouse experiments because Vru1 colonies the root

of important plants and produces different enzymes and several antibiotic compounds, which are

main biocontrol factors for suppression of plant diseases [35].

The successful PGPR by the free cell inoculum usually are restricted to in vitro studies but, in

several examples, this fails to efficiency under field conditions. This is mainly due to limitations

in retaining a threshold level of the primary bacteria inoculum in the adverse soil conditions as to

stimulate plant growth significantly [36].

A key limitation to successfully commercializing useful microorganisms is overcoming difficulties

in formulating a viable, user-friendly final product and cost-effective. The extension of new

microbial formulations is a challenging function and needs greater endeavor in terms of funding

and research than making significant development in this field [37].

Encapsulation causes gradual and controlled bacteria release from the matrix of the alginate-

bentonite gel bead on inoculation into the soil and simplifies in establishing the stable PGPB

population and the probabilities of reduction in population over time can be minimized.

The essential advantages of alginate inoculants are their degradation in the soil, their nontoxic

nature and their slow release of the entrapped bacterium into the soil [7]. The final test for each

PGPB is its ability to colonize its aim plant roots and to produce growth factors [38,39]. Bacteria

inoculation should be applied for the farmer and simple to use. They are should stay viable in the

dry soil for a long time, until it rains, and should proliferate rapidly and colonize the root system

14
of the seedling when the seed germinates. Over the last years, the major development in

nanotechnology has been achieved. The advances of nanotechnology have significantly expanded

the application domain of nanomaterials in different fields such as agriculture, including plant

growth and germination, plant protection, production and pathogen detection [40]. Hong et al. [41]

demonstrated that TNs could decrease production rates of free radicals and raise the superoxide

dismutase and chloramphenicol acetyltransferase in vivo to sustain membrane structure stability

in spinach. Our aim for use TNs in encapsulated was to investigate if TNs could be used to support

useful rhizobacteria in the colonization of plant roots. There was a substantial increase in the root

colonization when the B. subtilis microcapsule with TNs was inoculated. These high colonization

amounts could not be seen with free B. subtilis Vru1 and microcapsule of the bacterium without

TNs. This means that there is an interaction between bacterium and nanoparticles. The results

showed the nanoparticles help the colonization of the roots. When the plants had been treated with

nanocapsule of B.subtilis Vru1 the roots were covered to a higher extent with the bacterium. We

suggest that first the TNs helped bacterium colonize the plants root causing higher biomass of

bacterium. This character was the primary criteria for selecting TNs as an additional supplement

particularly with alginate in our study. B.subtilis Vru1 and its nanocapsule indicated significant

inhibitory effects on the growth of R.solani in vitro and in vivo. This result is in a match with those

of Yang et al. [42] and Ben Khedher et al. [43], who reported the efficacy of B.subtilis in

controlling of R.solani. The in vivo investigation showed the biocontrol ability of the

nanoencapsulation of Vru1 against R.solani. Indeed, beans plant treatment with Vru1 nanocapsule

significantly suppressed R.solani growth and increased plant growth, when compared to the

untreated plants. Statistical analysis of data on the root canker caused by R.solani on bean showed

that there are significant differences between treatments. The Vru1 nanocapsule was able to control

15
root canker disease (90% biocontrol efficacy) more efficiently than the commercial free Vru1

strain. The result of Russo et al. [41] indicated that a higher enhancement in colonization was

observed using a liquid formulation. Root colonization is a main factor in plant growth promotion

and the survival ratio of the bacterium. Therefore, the colonization ability and survival of the B.

subtilis Vru1 strain to bean root was analyzed. Fig. 9 shows that the amount of colonization by B.

subtilis Vru1 nanocapsule was less than that by B. subtilis Vru1 without coating from 5 to 20 days.

The use of nanocapsule of bacterium led to a population concentration in the bean root that was

similar to obtained with free bacterium added to the soil from 20 to 30 days. The content of

bacterium colonization in the bean root under the B. subtilis Vru1 nanocapsule treatments was

significantly higher than that under the free B. subtilis Vru1 treatments on 35 days and was retained

up to 45 days. Our research showed that bean root colonization by encapsulated B. subtilis Vru1

could occur at high levels (log CFU = 9-9.1 per g fresh root) at 45 days. This study indicates the

applied technology of inoculum formulation for soil applications.

Acknowledgments

The authors acknowledge Vali-e-Asr University of Rafsanjan for providing the research materials

and funds

16
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 Table1: Effect of B.subtilis VRU1 encapsulation on fresh weight, dry weight and plant height of bean

Treatment Shoot Shoot fresh root fresh Shoot dry root dry
Length weight weight weight weight
(cm) (g) (g) (g) (g)
Nanoformulation 17a 3.42
a 2.18a 1.43a 0.84a
(VRU1)
Nanoformulation 14.33b 3.13ab 2.17a 1.13ab 0.68a
(VRU1) + R.solani
Bacteriuml strain 13.33bc 2.94bc 1.97b 0.94bc 0.46b
(VRU1)
Bacteriuml strain 11.66cd 2.81bcd 1.9bc 0.80bcd 0.4bc
(VRU1) + R.solani
Nanocapsule without 11.33d 2.65cde 1.81bcd 0.64cde 0.31bc
bacterium
R.solani 9.33e 2.53de 1.75cd 0.53de 0.25c
Control 8.33e 2.37e 1.71d 0.39e 0.21c
Notes: Mean ± standard errors. Significant differences are according to student’s test with p≤0.05. (Each value is an
average of three replication)

Fig. 1. A: Production of indole-3-acetic acid. B: Production of cellulase. C: Production of protease enzyme. D:

phosphate solubilization. E; siderophore production. F:. Production of chitinase enzyme

23
 Fig. 2. Antifungal Activity of B.subtilis Vru1

Fig. 3. SEM images of NaAlg-B-S nanocapsules

24
Fig.4. FTIR spectra of NaAlg

Fig. 5. FTIR spectra of bentonite.

25
Fig.6. FTIR spectra of starch

Fig. 7. FTIR spectra of NaAlg-B-S nanocapsules.

26
Fig.8. X-ray diffraction patterns of pure N: NaAlg, S: starch, B: bentonite and Ca: NaAlg-B-S
nanocapsules
.

27
 12

release bacterium (log CFU/g)

10 b b c
d d
e
8 f
g
i h
6
j

k
4

0
T0 T5 T10 T15 T20 T25 T30 T35 T40 T45 T50 T55 T60
Time (d)

Fig. 9. Release of B.subtilis Vru1 from nanocapsules in phosphate buffer during two month.

h
12
a
b
viable bacterium (log CFU/g)

10 c b a
c
d d b a
8 c
e e d
g f f e
f f
6 h h g g
i h h
4

0
T5 T10 T15 T20 T25 T30 T35 T40 T45
Time (days)

Free capsules without TNs nanocapsules

Fig. 10. Changes in the viable bacterium amount of B.subtilis VRU1 colonizing on the bean root

28
 120
a
b
100 c

Disease inhibition
80 d

60

40

20 e

Treatments

Fig.11. Effect of bean plants inoculated with B.subtilis Vru1 strain and its formulation on the disease inhibition
under greenhouse conditions

Fig. 12. The effect of B.subtilis Vru1 strain and its nanoformulation on growth parameters in bean plants.

29