Antagonistic Activity of Indigenous Rhizobacteria through Biosynthesis of Indole-3-Acetic Acid (IAA), Hydrogen Cyanide (HCN), and Siderophores

Aye Khaing; Theint Theint Win; ay Thi Oo; Pengcheng Fu

Research Article

Austin J Biotechnol Bioeng. 2021; 8(1): 1110.

Antagonistic Activity of Indigenous Rhizobacteria through Biosynthesis of Indole-3-Acetic Acid (IAA), Hydrogen Cyanide (HCN), and Siderophores

Aye Khaing^1,2, Theint Theint Win^1,2, Kay Thi Oo^1,2 and Pengcheng Fu¹*

¹State Key Laboratory of Marine Resource Utilization in South China Sea, Hainan University, Haikou, Hainan Province, China

²Department of Biotechnology Research, Ministry of Education, Kyaukse, 05151, Myanmar

*Corresponding author: Pengcheng Fu, State Key Laboratory of Marine Resource Utilization in South China Sea, Hainan University, 58 Renmin Avenue, Haikou, Hainan Province 570228, China

Received: May 31, 2021; Accepted: June 29, 2021; Published: July 06, 2021

Abstract

This study aimed to isolate indigenous Antagonistic Bacteria (AB) against common soil-borne phytopathogens, including Rhizoctonia solani, Phythium sp., Fusarium oxysporum. Biosynthesis of Indole-3-Acetic Acid (IAA), generation of Hydrogen Cyanide (HCN), and siderophores production were assessed for their involvement in the antagonistic activities. Rhizospheric soil of bean roots, sunflowers, wheat, rice, and humic and semi flooded soils were used to isolate twenty-one bacterial strains for phytopathogenic antagonism. It was found that nine isolates have potential antagonistic activity against three common soilborne pathogens. Antifungal productivity for IAA, HCN, and siderophores was screened on the isolates while nine ABs were identified. To choose statistically significant isolate for the formulation, the principal component analysis was performed with five variables (IAA production, siderophores production index, antagonistic activities against three phytopathogens). Among the nine isolates, the isolate Pseudomonas alicaligenes shew a positive correlation with all variables. In particular, the strain demonstrated to be an antagonistic strain against the fungal pathogens and a strong producer of siderophores, HCN and IAA, We prepared the biocontrol agents with rice flour, glutinous rice flour, Monosodium Glutamate and Chitosan that were found to maintain the up to three months for convenience of field applications.

Keywords: Antagonistic bacteria; Phytopathogen; Siderophore; Indole-3- acetic acid; Hydrogen cyanide; Soil-borne

Introduction

Agriculture is a critical sector in national economy for developing countries like Myanmar. In order to enhance crop productivity, chemical fertilizers and pesticides are applied to achieve higher yields for the income of farmers, although it contributes to the destruction of microbial species and ecosystems. It is also deposited pollutants in the environment, causing contamination, bioaccumulation, and biomagnifications in the farmlands. To be worse, fertilizer abuse can not only impact soil health, but also promote the growth of soil-borne pathogens which causes heavy loss in crop harvest [1-4]. The immense use of chemical fertilizers in agriculture has even become threats to consumer health [5]. Accordingly, the attention in environmentally sustainable farming techniques and organic farming has increased [6].

Capable of suppressing plant disease, plant growth Rhizobacteria are used in agriculture as a substitute to agrochemicals to increase crop quality and yield through different mechanisms, i.e. Production of siderophores, HCN, antibiotics, antifungal enzymes, vying for colonisation sites, nutrients, and induction of pervasive stress tolerance [6-9]. The Native microbial compositions are classes of innate microbial consortia, which inhabit in soil. They are capable of biodegradation, nitrogen fixation, and enhancement for soil fertility. In particular, Native microbial compositions play an important role in protection of crops from invasion of soil-borne plant pathogens by producing bacteriocins and other inhibitory substances. They contend with the plant pathogens for vital nutrients and host cell receptors, which will block the pathways for the soil-borne pathogens in the rhizosphere to enter higher plants through their roots [10,11].

Among the bacterial metaboilites, siderophores are a group of small biomolecules with high Fe³⁺ scavenging ability [12] which are capable for chelation and transportion of this element into bacterial cells. Siderophores have been effectively used as a biocontrol agent against several soil-borne phytopathogens. It would significantly lessen the amount of Fe accessible to microflora of the rhizosphere and hinder fungal growth [13]. In addition, down-regulation of IAA as signaling is attributed to plant defence mechanisms against a wide range of phytopathogenic bacteria [14,15]. Hydrogen Cyanide (HCN) is a broad-spectrum antimicrobial compound that is associated with the biological control of root diseases [16]. It has been implicated in the inhibition of various root diseases in tobacco, cucumber and tomato [17]. The importance of HCN in biocontrol has been studied by several researchers in the topics such as antifungal activity [18-20], nematocidal activity [21,22].

There are two objectives for this study: the first one at the mocribial level to isolate and screen indigenous antagonistic bacteria from different rhizospheric soils. The second one was at metabolic level to screen the antagonistic related compound biosynthesis (IAA, HCN, siderophores) and to formulate the most efficient preserving agents for selected antagonist.

Materials and Methods

Soil samples collection and isolation of bacterial strains

Rhizospheric soil samples have been collected from various crop lands, such as wheat, corn, bean root and sunflower lands, and flooded/humic soilx from various locations around the Kyaukse district of the Mandalay region of Myanmar. The pH of the soil samples was between 7.5 and 8.8, and the temperature between 28 and 35°C. Microbial species have been isolated by a serial dilution and steak plate method. One gram of dried soil was weighed and applied in a sterile test tube to 9 ml of double-distilled water (dd H₂O) and shaken well using a vortex mixer; this stock solution was then diluted serially up to 10^-7, and 0.1 mL of diluted sample dilution was inoculated on LB (Luria Bertani) media surface and incubated for 2 days at 35°C [23]. Following the Hans Gram staining method, the pure colonies were colonially and microscopically examined and preserved as glycerol stock for further uses [24].

Pathogenic fungal strains

The soil-borne pathogens Rhizoctonia solani, Phythium sp., Fusarium oxysporum were used in these research projects. The first two were collected from the Biotechnology Research Department in Myanmar and the second one supported by the state key laboratory of Marine Resource Utilization in South China Sea, Hainan University, China.

Screening of antagonistic activity

The antagonistic activities of all twenty-one isolates were tested in dual culture against common soil-borne plant pathogenic fungi such as Fusarium oxysporum, Phythium sp., and Rhizotonia solani. The pathogens were inoculated in the middle of the PDA-containing Petri plate, and bacteria were streaked 3 cm away from either side of the pathogen and incubated for 7 days at 28°C. The experiments were performed in triplicate and used a petri plate inoculated with a pathogen alone in the absence of an antagonist as a control. The inhibitory effect of control growth (absence of antagonists) was monitored by determining the radial growth of fungal mycelium on each plate and using the formula:

Inhibition of mycelia growth (%) = X-Y/ X × 100 [3].

Molecular identification of antagonistic isolates

The genomic DNA of nine antagonistic isolates was extracted using the TIANamp Bacteria DNA Kit of TIANGEN Company as instructed by the manufacturer. Genomic DNA was amplified by the amplification of universal primers; 27F (5-AGTTTGATC TTGGCTCAG-3) and 1492 R (5-GTTACCTTGTTACGACTT-3). The resulting amplicons were sequenced by 3730 XL DNA analyzers (Applied Biosystems, USA). The NCBI BLAST database (https://blast. ncbi.nlm.nih.gov/Blast.cgi?PAGE= Nucleotides) was used to perform sequence recognition.

Siderophore activity by plate screening method

Nine isolated strains were grown on modified azurol-S chromium agar plates as defined by [25]. In this experiment, calcium sulfate (60.5 mg) was dissolved in 50 mL of deionized water and the resulting solution was added to Fe (III) solution (10mL). This solution was slowly combined with Hexadecyltrimethylammonium Bromide (HDTMA) (72.9 mg) dissolved in 40 mL of water during stirring. The dark-blue solution that resulted was autoclaved, cooled to 50/60°C, and mixed with 15 g L^-1 agar containing 900 mL of sterile MM9 [26]. The bacterial strain was inoculated and incubated in the dark after solidification of the medium on Petri plates (28°C for 5 days). The emergance of the yellow-orange area from the dark blue around the colonies was positive. The zone index for siderophore activity has been determined using the formula;

Index = (colony + zone) diameter/colony diameter.

Characterization of siderophores

Fiss Minimal Medium (FMM) was prepared as given by R. Pal and K. Gokarn (2010), to test the production activity of bacterial siderophores; 5 g of KH₂PO₄ was dissolved in 924 ml ddH₂O, adjusted the pH at 6.8 with 6 M NaOH solution, 10 ml 0.001 gL^-1 MnSO4 solution, 10 ml 0.4 gL^-1 MgSO4 solution, 10 ml 0.005 gL^-1 solution ZnCl2. Sterilized, the medium cooled (60°C). The cooled medium was added with 10 ml of 50 gL^-1 sterile glucose solution, and mixed with the medium L-asparagine solution (5 gL^-1 asparagine dissolved in 30 ml ddH₂O). The isolates were incubated for 2 days in a water bath at 27°C. After incubation, the samples were centrifuged at a rate of 10000 rpm at 21°C for 10 minutes. Further experiments were donducted to identify specific siderophores [27]. The siderophore was classified as catechol or hydroxamate using the following procedure:

Catecolate type of siderophore was observed by using the Arnow test [28], which selectively detects the simple catechol-type siderophores. In this study, the catechol interacts in the presence of nitric acid by establishing a yellow color and becomes red in excess of sodium.

Hydroxamate type of siderophore was screened by ferric perchlorate (Atkin) assay in which Atkin reagent (0.0771 g of FeClO₄ in 100 ml of water with 1.43 ml of HClO₄) was used. In this experiment, 2.5 ml of Atkin reagent was added to 0.5 ml of supernatant after centrifugation and incubated at room temperature for 5 minutes [29]. Orange colour development indicates a positive reaction to hydroxate siderophore, while pale yellow is a negative reaction.

Screening of Hydrogen Cyanide (HCN) production by antagonestic isolates

Feigl-Anger paper method, based on Reddy et al. (2016), was used for the screening of HCN with isolates. The reaction was prepared by soaking a filter paper in a chloroform solution mixture containing equal volumes of 1% (w/v) copper ethylacetoacetate and 1% (w/v) 4, 4’-methylenebis (Sigma-Aldrich). In a 24-well microtiter plates which contains about 500 μl of prepared metabolite extract (prepared in section 2.2.2) was covered with the Fiegl-Anger paper and sealed airtight with a cover using binding clips. The plate was chilled at -20°C for 1 h and defrosted at room temperature for 30 min to develop clear blue spots on each reaction which is the positive reaction of HCN.

Screening for Indole Acetic Acid (IAA) production by antagonistic isolates

In order to assess IAA, the key auxin associated with physiological processes in many plants, pure nine antagonistic isolates were grown aseptically in LB broth comprising tryptophan (100 mgL^-1). These inoculated media were then incubated at 180-200 rpm at 30°C for 48 hours on the rotary shaker. One ml of filtrate was mixed with 2 ml of Salkowski’s reagent (20 ml of 35 per cent perchloric acid: 2 ml of 0.5 N FeCl₃) and incubated for 30-120 min in a room temperature under dark conditions. The Pink colour showed IAA production and an optical density of 530 nm was measured using the UV-VIS spectrophotometer [30]. The quantity of generated IAA was linked to a prepared standard curve.

Carrier formulation and shelf life of the selected isolate

Bio-formulations of selected isolates have been preserved by combining broth culture with previously sterilized (rice flour, monosodium glutamate, chitosan and glutinous rice flour) through a variety of combinations. These tablets have been made and kept at room temperature. Regular intervals 30 days to 3 months from the date of mixing were used to measure the shelf life of the formulations. A serial dilution of the agar plate method was used to count the colony-forming unit (cfu). Twelve types of carrier systems have been planned as follows:

T-1: 5 g of rice flour and 0.125 g of Monosodium Glutamate (MSG)

T-2: 5g of rice flour, 0.125g of MSG and 0.02 g of chitosan

T-3: 5g of glutinous rice flour and 0.125 g of MSG

T-4: 5 g of glutinous rice flour, 0.125 g of MSG and 0.02 g of Chitosan

T-5: 2.5 g each of rice flour and glutinous rice flour and 0.125 g of MSG

T-6: 2.5 g each of rice flour and glutinous rice flour, 0.125 g of MSG and 0.02 g of Chitosan

T-7: 5 g of rice flour

T-8: 5 g of glutinous rice flour

T-9: 2.5 g each of rice flour and glutinous rice flour

T-10: 2.5 g each of rice flour and skimmed milk

T-11: 2.5 g each of glutinous rice flour and glutinous rice flour

T-12: 5g of skimmed milk

Statistical analysis

All studies have been performed in at least three biological replications. Each sample was typically calculated in three technical repeats. Minitab 17 statistical software was used to interpret quantitative data (Minitab Ltd., Pennsylvania State University). Data were Analyzed using One-Way Variance Analysis (ANOVA). All results were viewed as means (SD), which suggests that not sharing the same letter is statistically different (p0.05). The Prcomp function in the R software stats package was used to perform multivariate Principal Component Analysis (PCA) (R Core Team, 2013).

Results and Discussion

Isolation, screening of antagonistic activity and identification of antagonistic isolates

The present study is focused on the exploration of possible biofertilizer strains from rhizospheric soil samples from the Kyaukse region of Myanmar. 21 bacterial strains have been isolated from 10 separate sample sites. Isolation was carried out based on various cultural morphologies to screen antagonistic activity using a dual culture system.

Dual plate inhibition experiments were performed using PDA, and significant differences in mean growth diameter were determined using the Turkey test (Table 1). Among the 21 strains analyzed, 9 of the strains listed in Table 1 showed an inhibitory effect on the soil-borne plant phytopathogens Rhizoctonia solani, Phythium sp., Fusarium oxysporum. The isolates were coded as BR1, BR2 (from Bean root rhizosphere), WM, WM2 (from wheat rhizosphere), FS1 and FS2 (from floated soil), HS1, HS2 (from humic soil) and SF (from sunflower rhizosphere). In the present analysis, growth inhibition against Fusarium oxysporum, isolates of FS3 possessed the highest activity (72.3±1.1 per cent). For Phythium sp., BR1 and FS3 had the largest reduction (51.6±1.7 and 56.7±1.3 per cent). WM2 had the highest inhibition against Rhizoctonia solani (66.7±1.2). The growth inhibition tests of WM isolates against three phytopathogens had yielded similar results. It is seen that the maximum growth inhibition of 62.7±0.7 per cent of Fusarium oxysporum, followed by 56.13±1.3 for Phythium sp. and 30.13±0.5 per cent for Rhizoctonia solani. Bacterial antagonism is a dynamic, well-known and well-established mechanism [31]. Antagonistic exploration has shown that the influent growth of bacteria had hindered the growth of fungal pathogens [6]. After additional 10-day incubation, pathogenic mycelia were no longer cover the surface of the inhibition ring, suggesting that the antagonism was very high [32]. Antagonistic testing revealed that confluent bacterial growth inhibited the development of fungal phytopathogens [31,6].

Table 1: The Phytopathogen inhibition activity of Isolates.




  
    
    Rhizoctonia solani
    Pythium sp.
    Fusarium Oxysporum
  
  
    BR1 
    38.3±0.6ab 
    51.6±1.7b 
    63.9±0.6bc 
  
  
    BR2 
    45.4±2.7ab 
    12.5±1.1g 
    58.3±0.9d 
  
  
    FS2 
    34.3±0.9ab 
    28.5±0.7f 
    67.1±0.9b 
  
  
    FS3 
    44.9±0.4ab 
    56.4±0.9a 
    72.3±1.1a 
  
  
    HS1 
    33.8±0.7ab 
    45. 3±0.8c 
    62.9±1.6c 
  
  
    HS2 
    32.3±1.8ab 
    45.1±0.6c 
    73.5±1.5a 
  
  
    SF 
    27.9±0.9b 
    38. 6±0.9d 
    61.1±0.9cd 
  
  
    WM 
    30.5±0.4b 
    56.6±1.3a 
    62.7±0.7c 
  
  
    WM2
    66.7±1.2a 
    34.3±0.9e 
    63.1±1.3c



Table 1: The Phytopathogen inhibition activity of Isolates.

Among the 21 isolates, nine isolates with antagonestics activity were identified through cultural, microscopic and 16S rRNA gene sequences. The microscopic morphology of nine antagonistic isolates was shown in Figure 1. Each strain’s 16S rRNA was sequenced and classified using BLAST on the NCBI database (www.ncbi.nlm.nih. gov/BLAST). As shown in Table 2, the 16S rRNA matches 6 different Pseudomonas spp., Leclercia sp., Citrobacter sp., Enterobacter sp.. Also, Table 2 summarized the isolation source, cultural, microscopic, and their accession number [23], we thus selected Pseudomonas sp. as the biocontrol agent to overcame the existing tailback for their commercial use.

Figure 1: Microscopic Morphology of antagonistic isolates (a) HS1 (b) HS2 (c) BR1 (d) BR2 (e) FS2 (f) FS3 (g) SF (h) WM (i) WM2.

    
    
    Figure 1: Microscopic Morphology of antagonistic isolates (a) HS1 (b) HS2 (c) BR1 (d) BR2 (e) FS2 (f) FS3 (g) SF (h) WM (i) WM2.

Table 2: Characteristics of antagonistic isolates.




  
    No. 
    Isolates 
    Strains 
    Accession No. 
    Isolation source 
    Colonial morphology 
    Microscopic morphology    (shape) 
  
  
    1 
    BR1 
    Pseudomonas    plecoglossicida
    MT349504 
    Bean    root 
    White,    mucoid 
    G-ive,    rod 
  
  
    2 
    BR2 
    Pseudomonas    furukawaii
    MT355447 
    Bean    root 
    Brown,    rhizoid 
    G-ive,    rod 
  
  
    3 
    FS3 
    Pseudomonas    alcaligenes
    MT355448 
    Flooded    soil 
    Light    green 
    G-ive,    rod 
  
  
    4 
    SF 
    Pseudomonas    oleovarans
    MT355450 
    Sunflower 
    Brown,    rhizoid 
    G-ive,    rod 
  
  
    5 
    WM 
    Pseudomonas    alicaligenes
    MT355451 
    Wheat    plant 
    Deep    Brown, rhizoid 
    G+ive,    rod 
  
  
    6 
    WM2 
    Pseudomonas    mendocina
    MT355493 
    Wheat    plant 
    Brown 
    G+ive,    rod 
  
  
    7 
    HS1 
    Leclercia    adecarboxylata
    MT378327 
    Humate    soil 
    light    green 
    G-ive,    rod 
  
  
    8 
    HS2 
    Citrobacter    youngae
    MT378331 
    Humate    soil 
    White,    dull 
    G-ive,    rod 
  
  
    9 
    FS2 
    Enterobacter    cloacae
    MT872006 
    Flooded    soil 
    Light    green 
    G-ive,    Curve rod



Table 2: Characteristics of antagonistic isolates.

Detection and characterization of siderophores

The development of a yellow to orange colored area on a blue CAS agar plate suggesting siderophore production was reported in 9 isolates. The color shift from blue to orange was caused by siderophores removing Fe from the dye (Figure 2), which was also representative of the hydroxymate form of siderophore [33]. Hydroxymate type of siderophore was also confirmed by the Atkin method. Basically, siderophores are categorized into three categories according to Messenger and Ratledge, 1985 [34]: hydroxamate produced by both bacteria and fungi, and catecholate produced only by bacteria. Table 3 showed the siderophore production index and the siderophore form produced by nine antagonist isolates. For the control of several fungal pathogens that cause plant diseases, siderophores producing bacteria as a biocontrol agent can play a crucial role. [6]. Our results confirm previous research suggesting that siderophore-mediated competition in disease incidence reduction has resulted in pathogen elimination in the rhizosphere [35,36].

Figure 2: Characteristics of antagonistic isolates.

    
    
    Figure 2: Characteristics of antagonistic isolates.

Table 3: Siderophore production index and types of siderophore.




  
    Isolates 
    Siderophores    production index 
    Types of Siderophore 
  
  
    BR1 
    2.89±0.73a 
    Hydroxymate,    Catecolate 
  
  
    BR2 
    1.64±0.11c 
    Hydroxymate,    Catecolate 
  
  
    FS2 
    1.24±0.08d 
    Hydroxymate,    Catecolate 
  
  
    FS3 
    1.27±0.06d 
    Hydroxymate,    Catecolate 
  
  
    HS1 
    1.23±0.08d 
    Hydroxymate,    Catecolate 
  
  
    HS2 
    1.19±0.04d 
    Hydroxymate,    Catecolate 
  
  
    SF 
    1.68±0.05bc 
    Hydroxymate,    Catecolate 
  
  
    WM 
    1.87±0.03c 
    Hydroxymate,    Catecolate 
  
  
    WM2 
    1.18±0.04d 
    Hydroxymate,    Catecolate



Table 3: Siderophore production index and types of siderophore.

HCN production

HCN productivity of the nine isolates are presented in Figure 3. HCN is a volatile compound which plays a critical role in biological control of some soil-borne diseases [37]. HCN is one of the known biocontrol agent based on its ascribed toxicity against plant pathogens [38,39]. All nine strains showed a positive result of hydrogen cyanide production (Figure 4). Apart from biocontrol properties, biogenic HCN could enhance other plant growth-promoting properties, as well as increase the nutrient availability [38].

Formulated tablets	Time (Day)
Formulated tablets	30	60	90
T-1	6x10 7	5x10 5	1x105
T-2	3x10 8	5x10 5	1x103
T-3	1.2x107	4.5x10 5	1x105
T-4	1.1x107	7.5x10 5	7x104
T-5	1.4x107	6.5x10 5	5x104
T-6	5x10 9	1x10 7	1.5x107
T-7	5x10 9	5.5x10 6	-
T-8	1x107	5x10 5	5x104
T-9	1.3x107	1x106	1x101
T-10	1.1x109	2x107	1x104
T-11	1x10 5	1.5x107	5x102
T-12	1x107	1x106	-

Antagonistic Activity of Indigenous Rhizobacteria through Biosynthesis of Indole-3-Acetic Acid (IAA), Hydrogen Cyanide (HCN), and Siderophores

Abstract

Introduction

Materials and Methods

Soil samples collection and isolation of bacterial strains

Pathogenic fungal strains

Screening of antagonistic activity

Molecular identification of antagonistic isolates

Siderophore activity by plate screening method

Characterization of siderophores

Screening of Hydrogen Cyanide (HCN) production by antagonestic isolates

Screening for Indole Acetic Acid (IAA) production by antagonistic isolates

Carrier formulation and shelf life of the selected isolate

Statistical analysis

Results and Discussion

Isolation, screening of antagonistic activity and identification of antagonistic isolates

Detection and characterization of siderophores

HCN production

IAA production

Principal Component Analysis (PCA)

Formulation for preserved selected antagonist and its viability

Conclusions

Conflicts of Interest

Funding Statement

References