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. 2024 Apr 14;10(8):e29692. doi: 10.1016/j.heliyon.2024.e29692

Impact of bacterial volatiles on the plant growth attributes and defense mechanism of rice seedling

Tushar Goyal a, Arpan Mukherjee a, Gowardhan Kumar Chouhan a, Anand Kumar Gaurav a, Deepak Kumar a, Saman Abeysinghe b, Jay Prakash Verma a,⁎
PMCID: PMC11040113  PMID: 38660266

Abstract

Rice is a major dietary element for about two billion people worldwide and it faces numerous biotic and abiotic stress for its cultivation. Rice blast disease caused by Magnaporthe oryzae reduce up to 30 % rice yield. Overuse of synthetic chemicals raises concerns about health and environment; so, there is an urgent need to explore innovative sustainable strategies for crop productivity. The main aim of this study is to explore the impact of bacterial volatiles (BVCs) on seedling growth and defense mechanisms of rice under in-vitro condition. On the basis of plant growth promoting properties, six bacterial strains were selected out of ninety-one isolated strains for this study; Pantoea dispersa BHUJPVR01, Enterobacter cloacae BHUJPVR02, Enterobacter sp. BHUJPVR12, Priestia aryabhattai BHUJPVR13, Pseudomonas sp. BHUJPVWRO5 and Staphylococcus sp. BHUJPVWLE7. Through the emission of bacterial volatiles compounds (BVCs), Enterobacter sp., P. dispersa and P. aryabhattai significantly reduces the growth of rice blast fungus Magnaporthe oryzae by 69.20 %, 66.15 % and 62.31 % respectively. Treatment of rice seedlings with BVCs exhibited significant enhancement in defence enzyme levels, including guaiacol peroxidase, polyphenol oxidase, total polyphenols, and total flavonoids by a maximum of up to 24 %, 48 %, 116 % and 80 %, respectively. Furthermore, BVCs effectively promote shoot height, root height, and root counts of rice. All BVCs treated plant showed a significant increase in shoot height. P. dispersa treated plants showed the highest increase of 60 % shoot and 110 % root length, respectively. Root counts increased up to 30% in plants treated with E. cloacae and Staphylococcus sp. The BVCs can be used as a sustainable approach for enhancing plant growth attributes, productivity and defence mechanism of rice plant under biotic and abiotic stresses.

Keywords: Plant growth promoting microorganisms (PGPM), Bacteria volatiles compounds (BVCs), Rice, Seedling growth and defense elevation

Highlights

  • •

    Six bacteria selected as PGPM for biocontrol ability of its volatiles.

  • •

    Enterobacter sp. BHUJPVR12 showed significant reduction 69.20 % of M. oryzae.

  • •

    BVCs showed significant increase plant growth & defense elevation of rice seedling.

  • •

    BVCs treated plant synthesized more guaiacol peroxidase, polyphenol oxidase enzyme.

  • •

    BVCs have ability to improve plant sustainability under biotic & abiotic stresses.

1. Introduction

Exponential increase in the human population over the decades elevates the demand for agricultural products globally. Rice (Oryza sativa L.) is a major dietary element for approximately two billion people worldwide. Consequently, over 100 nations cultivate rice across the world, however the top ten rice producers accounts for about 85 % of global production [1]. Among this India and China accounts approximately 50 % of total rice production globally [2]. In addition, the current food demands rise 20 % in rice production for avoiding food crisis. To meet the global food demand, the synthetic chemicals and biofertilizers have been extensively used [3]. However, as health and environmental concerns are increasing, there is a shift towards the application of biofertilizers, including endophytic bacteria and their byproducts.

Innumerable microbial species interact with plants in both mutualistic and parasitic way. Out of these, plant endophytes are residents of the inside plant parts in mutualistic way and emits a complex mixture of volatiles, secondary metabolites, plant and microbial hormone [4]. This relationship helps to host plant for taking up essential nutrients and generate tolerance against various biotic and abiotic stresses [5]. From seed germination onwards, plants are in contact with bacterial volatiles compounds (BVCs). Microbial volatiles promote improve seed germination and plant biomass, and to protect plants from abiotic and biotic stress BVCs plays a pivotal role for inducing plant systemic tolerance.

Global rice production is severely impacted by rice blast disease, which is caused by a filamentous fungus namely Magnaporthe oryzae (synonym of Pyricularia oryzae) [6]. Up to 30 % of yield losses of rice possibly due to the rice blast disease [7]. Multiple strategies have been employed to reduce the severity of this condition, among them application of endophytes and their byproducts in different bioformulation having inhibitory effects on the blast fungus and plant growth promotion as well. From literature approximately 2000 VOCs have been identified which were emitted by a bacteria and fungi and were discovered on a limited number of microorganisms. BVCs are categorized in alcohols, alkenes, ketones, tepenoids, and other chemical classes [[8], [9], [10]].

BVCs possess the ability to stimulate plant defense mechanisms by elevating the activity of defense related enzymes such as polyphenol oxidase, guaiacol peroxidase, total flavonoids, and total polyphenols. To fufill the gaps on BVCs and their impact on rice plant growth and elevating the defense response mechanisms, this study is (a) to identify the rice endophytes having properties of reducing blast severity through their volatiles (b) to promote plant growth by the action of volatiles and (c) to enhance synthesis of defense related enzymes in plant by the action of BVCs. We hypothesised that BVCs have ability to show positive impact on plant growth, development and biocontrol under biotic and abiotic stresses by elevating plant defense systems.

2. Material and methods

2.1. Isolation of endophytic strains from rice plant

Isolation of endophytic bacteria from rice were done by serial dilution methods. Different plants part were washed under the tap water and deionized water to remove dirt particles. Different parts (seeds, leaf, root and, stem) cut into sections 2, 3 cm and surface sterilised by using 0.1 % mercury chloride (HgCl2) and 70 % ethanol [11]. The bacterial endophytes were then isolated from the sterilised rice plant parts via crushing and serial dilution in agar and broth containing nutrient agar (NA) and tryptone soya agar or trypto-casein soy agar (TSA). Then the agar plates and broth inoculated with rice plant parts were incubated at 28 ± 2 °C for 2–5 days, and the broth was incubated in an incubator with shaking at 130 rpm. Following incubation, many microbial colonies were found on the particular plates. Based on the colony morphology, shapes, sizes, colours, we picked the microbial colony and prepared slant and glycerol stock and stored in −20 °C and −80 °C.

2.2. Biochemical characterization of isolated bacterial strains

Gram staining and biochemical properties were used to characterize isolated endophytic isolates [12,13].The plant growth promoting biochemical properties of isolated rice plant endophytes were explored by investigating the indole-3-acetic acid (IAA) [14], NH3 (ammonia) production [15], phosphate solubilization [16,17], siderophore production [18], zinc solubilization [19], Silicate solubilization [20,21], Potassium solubilization [22], and antagonistic effect of BVCs were observed against the plant pathogen Magnaporthe oryzae [23,24].

2.3. Molecular characterisation of isolates

DNA of endophytes were isolated by using CTAB (cetyltrimethylammonium bromide) methods [25], and the identification of isolates were done by PCR, which were conducted by using the universal 16s rRNA gene (Forward primer 27F 5′-AGAGTTTGATCCTGGCTCAG-3′, and Reverse primer 939R 5′-TACGGTTACCTTGTTACGACTT-3′) [26]. The PCR products were resolved in 1.2 % (w/v) agarose gel, alongside a 100 bp DNA ladder (Thermo Scientific, Lithuania). Predominantly 1000 bp PCR products were observed. For the sequencing of amplified 16s rRNA genes, purification of the PCR products was carried out by PCR purification kit (Invitrogen, PCR purification kit, USA).

2.4. Effects of the endophytic volatile components on rice seedling growth

Rice seeds surface sterilisation was done by using of 0.1 % mercury chloride and 70 % ethanol [11]. We took two opened small (4 & 8 cm) petri plates containing microbial inoculum and other contain sterilised rice seed and placed both petri plate inside a bigger plate (15 cm). After that bigger petri plate covered with lid and sealed properly to evaluate the effect of BVCs on seed germination of rice and defense elevation. Then the whole plate placed in the plant growth chamber (12 h and 12 h duration of light and dark respectively, moisture was ranging from 75 to 80 %, and the temp. was ranged from 28 to 30 °C). The whole plant growth promoting experiments was measured for 15 days. The height of root, shoot and numbers of root and leaves were recorded.

2.5. Plant biochemical assay

After the experiments, plants were collected and measured the activity of different plant defence enzymes including; guaiacol peroxidase [[27], [28], [29]], polyphenol Oxidase [[30], [31], [32]], total phenols [28,33] and flavonoids content [34].

2.6. Statistical analysis

This study was conducted with seven treatments having three replicates. The research data were represented as mean ± SD. Data analysis was done by use of ANOVA with Duncan comparison tests using SPSS software. The significant values were taken at p ≤ 0.05.

3. Results

3.1. Isolation and culture collection

A total of 91 morphologically diverse endophytes were isolated from different parts of the rice plant samples. On the basis of their plant growth promoting biochemical properties (IAA production, phosphate solubilization, potassium solubilization, ammonium production, siderophore production, Silicate solubilization, Zinc solubilization) only 4 endophytes were selected for the study of their volatiles on plant growth promotion and defense activation. Another 2 microorganisms were taken from lab that were previously characterized in Chouhan et al. (2023) [35] namely BHUJPVWRO5 (Pseudomonas sp.), and BHUJPVWLE7 (Staphylococcus sp.).

3.2. Characterization of plant growth promoting properties of rice endophytes

Biochemical characteristics (IAA production, phosphate solubilization, potassium solubilization, ammonium production, siderophore production, silicate solubilization, zinc solubilization) were used to characterize the isolates. Upon 48h of incubation, out of 91 only 47 isolated strains from rice plants and rhizosphere soil produced IAA where Enterobacter sp. strain BHUJPVR12 (56.54 μg/ml), Staphylococcus sp. strain BHUJPVWLE7 (56.07 μg/ml) Pseudomonas sp. strain BHUJPVWRO5 (49.62 μg/ml) and Enterobacter cloacae strain BHUJPVR02 (42.29 μg/ml) produced highest amount of IAA. 27 isolated endophytes showing positive result for ammonia production in the range from 9.16 to 21.30 μg/ml and the highest activity was showing in Priestia aryabhattai strain BHUJPVR13 (21.30 μg/ml). All endophytes solubilized phosphate, but the most significant strain was Enterobacter cloacae strain BHUJPVR02 (943.51 μg/ml) and Pantoea dispersa strain BHUJPVR01 (823.51 μg/ml). Enterobacter sp. strain BHUJPVR12 (38.6 μg/ml) and Pseudomonas sp. strain BHUJPVWRO5 (14.45 μg/ml) significantly produced siderophore in comparison to other isolates. All selected strains solubilize zinc, but Pantoea dispersa strain BHUJPVR01 and Pseudomonas sp. strain BHUJPVWRO5 being the most effective solubilizers. Pseudomonas sp. strain BHUJPVWRO5, Staphylococcus sp. strain BHUJPVWLE7, and Pantoea dispersa strain BHUJPVR01 solubilize more silicate than the others. Pseudomonas sp. strain BHUJPVWRO5 having the highest potassium solubilizing activity, followed by Pantoea dispersa strain BHUJPVR01, Enterobacter cloacae strain BHUJPVR02, Enterobacter sp. strain BHUJPVR12, and Staphylococcus sp. strain BHUJPVWLE7 (Table 1).

Table 1.

Plant growth promoting properties of selected endophytic strains.

Isolate IAA production (ug/ml) (with tryptophan) Ammonia production PSB Siderophore production Zinc Silica Potassium
BHUJPVR01 14.90 ± 1.02a 11.01 ± 1.26ab 823.51 ± 8.48c 6.62 ± 0.81ab +++ ++ +
BHUJPVR02 42.29 ± 0.11c 11.46 ± 1.55ab 943.51 ± 14.12d 4.69 ± 0.79a ++ + +
BHUJPVR12 56.54 ± 2.21d 9.16 ± 1.47a 741.94 ± 11.12b 38.6 ± 5.76e + + +
BHUJPVR13 25.62 ± 0.43b 21.30 ± 1.64c 578.81 ± 34.82a 3.02 ± 1.14a ++ – –
BHUJPVWRO5 49.62 ± 0.11c 18.51 ± 1.64bc 607.82 ± 34.82ab 14.45 ± 1.55c +++ +++ +++
BHUJPVWLE7 56.07 ± 2.21d 14.45 ± 1.55b 609.00 ± 34.82ab 21.30 ± 1.64d + +++ +
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Note: Here BHUJPVR01= Pantoea dispersa; BHUJPVR02 = Enterobacter cloacae; BHUJPVR12 = Enterobacter sp.; BHUJPVR13= Priestia aryabhattai; BHUJPVWRO5= Pseudomonas sp.; BHUJPVWLE7 = Staphylococcus sp. Data are shown as mean ± SD (n = 3), and in each column the superscript letters are used to show the statistical relationship between isolates based on ANOVA and Duncan multiple post-hoc test (P ≤ 0.05).

3.3. Identification of culturable rice endophytes by 16s rDNA sequencing

On the basis on their PGP properties 4 isolates were selected for 16s rDNA sequencing. From the analysis of their sequence, we found out that BHUJPVR01 is Pantoea dispersa, BHUJPVR02 is Enterobacter cloacae, BHUJPVR12 is Enterobacter sp. and BHUJPVR13 is Priestia aryabhattai (Table 2, Supplementary Figs. 1A and B).

Table 2.

Molecular identification of isolates through 16srDNA sequencing.

Strain Bacterial Name Accession number Percent identity match Reference organism Reference organism Accession no.
BHUJPVR01 Pantoea dispersa OQ892233 100 % Pantoea dispersa strain AA9 MT275631
BHUJPVR02 Enterobacter cloacae OQ892234 100 % Enterobacter cloacae strain MBB8 MT138639
BHUJPVR12 Enterobacter sp. OR603106 97.83 % Enterobacter sp. strain HSTU-ASn40 MN559048
BHUJPVR13 Priestia aryabhattai OR603107 100 % Priestia aryabhattai strain AYG1023 OQ569480
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3.4. Effect of bacterial volatile compounds (BVCs) on the growth of rice blast fungus Magnaporthe oryzae

Bacterial volatile compounds from selected microorganisms were evaluated for the potential of antifungal activity on rice pathogenic fungi M. oryzae. All the microorganisms significantly reduce the mycelial growth of fungi but T4 (Enterobacter sp. BHUJPVR12), T2 (P. dispersa BHUJPVR01) and T5 (P. aryabhattai BHUJPVR13) up to 69.20 %, 66.15 % and 62.31 %, respectively (Table 3, Fig. 1A and B).

Table 3.

Impact of bacterial volatiles compounds (bVOCs) for growth inhibition of plant pathogenic fungus.

Treatment Average diameter (cm) Percent growth inhibition (PGI)
T1 4.33 ± 0.05a –
T2 1.47 ± 0.05e 66.15
T3 1.73 ± 0.05d 59.99
T4 1.33 ± 0.11e 69.20
T5 1.63 ± 0.11d 62.31
T6 1.93 ± 0.11c 55.35
T7 2.10 ± 0.10b 51.53
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Note: Treatment T1 = Control; T2 = Pantoea dispersa BHUJPVR01; T3 = Enterobacter cloacae BHUJPVR02; T4 = Enterobacter sp. BHUJPVR12; T5 = Priestia aryabhattai BHUJPVR13; T6 = Pseudomonas sp. BHUJPVWRO5; T7 = Staphylococcus sp. BHUJPVWLE7. Data are shown as mean ± SD (n = 3), and in each column the superscript letters are used to show the statistical relationship between isolates based on ANOVA and Duncan multiple post-hoc test (P ≤ 0.05).

Fig. 1.

Fig. 1

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A: Antifungal activity of bacterial volatiles against Magnaporthe oryzae for showing growth inhibition as compare to control; B: Diameter of fungal growth inhibition by bacterial volatiles compunds. Note: Treatment T1 = Control; T2 = Pantoea dispersa BHUJPVR01; T3 = Enterobacter cloacae BHUJPVR02; T4 = Enterobacter sp. BHUJPVR12; T5 = Priestia aryabhattai BHUJPVR13; T6 = Pseudomonas sp. BHUJPVWRO5; T7 = Staphylococcus sp. BHUJPVWLE7. Data are shown as mean ± SD (n = 3), and in each column the superscript letters are used to show the statistical relationship between isolates based on ANOVA and Duncan multiple post-hoc test (P ≤ 0.05).

3.5. Effects of bacterial volatile compounds (BVCs) on rice plant growth promotion

After 15 days of BVCs treatment shoot length, root length, leaf number and root number were recorded, and a significant difference were recorded when compared to the control (T1). All BVCs treated plants showed a significant increase in height, with T2 (P. dispersa BHUJPVR01) and T5 (P. aryabhattai BHUJPVR13) treated plants have longer shoot length of 14.17 and 12.03 cm, respectively, as compared to control T1 (8.83 cm). Apart from T3 (E. cloacae BHUJPVR02), all BVCs treated plants showed higher root length of up to 10.70 cm when compared to control (T1) 5.10 cm. In treatments T3 (E. cloacae BHUJPVR02), T4 (Enterobacter sp. BHUJPVR12) and T7 (Staphylococcus sp. BHUJPVWLE7) root counts were also increased to 14.33, 14.00 and 14.33 than control (T1), respectively (Fig. 2 & Table 4).

Fig. 2.

Fig. 2

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Figure represents the effect of bacterial volatiles on growth of rice seedlings, Note: Treatment T1 = Control; T2 = Pantoea dispersa BHUJPVR01; T3 = Enterobacter cloacae BHUJPVR02; T4 = Enterobacter sp. BHUJPVR12; T5 = Priestia aryabhattai BHUJPVR13; T6 = Pseudomonas sp. BHUJPVWRO5; T7 = Staphylococcus sp. BHUJPVWLE7.

Table 4.

Bacterial volatiles compounds mediated plant growth promotion of rice seedlings at 15 days.

Treatment Shoot length Root length Leaf numbers Root numbers
T1 08.83 ± 2.63a 05.10 ± 1.15a 2.67 ± 0.57a 11.00 ± 1.00a
T2 14.17 ± 1.25c 10.70 ± 2.98b 3.00 ± 0.00a 11.67 ± 0.57a
T3 11.13 ± 1.06ab 05.27 ± 0.66a 3.00 ± 0.00a 14.33 ± 0.57b
T4 10.90 ± 0.17ab 10.00 ± 1.48b 3.00 ± 0.00a 14.00 ± 1.00b
T5 12.03 ± 0.84bc 10.00 ± 0.79b 3.00 ± 0.00a 11.33 ± 2.51a
T6 11.10 ± 0.87ab 10.00 ± 1.00b 3.00 ± 0.00a 12.67 ± 1.15ab
T7 11.37 ± 0.72b 09.53 ± 2.66b 3.00 ± 0.00a 14.33 ± 0.57b
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Note: Treatment T1 = Control; T2 = Pantoea dispersa BHUJPVR01; T3 = Enterobacter cloacae BHUJPVR02; T4 = Enterobacter sp. BHUJPVR12; T5 = Priestia aryabhattai BHUJPVR13; T6 = Pseudomonas sp. BHUJPVWRO5; T7 = Staphylococcus sp. BHUJPVWLE7. Data are shown as mean ± SD (n = 3), and in each column the superscript letters are used to show the statistical relationship between isolates based on ANOVA and Duncan multiple post-hoc test (P ≤ 0.05).

3.6. Effect of BVCs on the defense related enzymes

BVCs treated 15 days old rice seedlings were assayed for their defense enzymes such as guaiacol peroxidase and polyphenol oxidase. All treatments showed enhanced activity of POX enzymes, with T4 (Enterobacter sp.) and T6 (Pseudomonas sp.) having the maximum activity of 0.59 and 0.57 Unit/L, respectively, as compared to the T1 control 0.47 unit/L. PPO activity was also enhanced in all treatments where T3 (E. cloacae) and T6 (Pseudomonas sp.) showing the maximum activity up to 0.37 Unit/L. Total polyphenols and total flavonoids were found more in BVCs treated rice seedlings (Fig. 3 & Table 5).

Fig. 3.

Fig. 3

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Defense elevation of bacterial volatiles treated rice plant. Treatment T1 = Control; T2 = Pantoea dispersa BHUJPVR01; T3 = Enterobacter cloacae BHUJPVR02; T4 = Enterobacter sp. BHUJPVR12; T5 = Priestia aryabhattai BHUJPVR13; T6 = Pseudomonas sp. BHUJPVWRO5; T7 = Staphylococcus sp. BHUJPVWLE7. Data are shown as mean ± SD (n = 3), and in each column the superscript letters are used to show the statistical relationship between isolates based on ANOVA and Duncan multiple post-hoc test (P ≤ 0.05).

Table 5.

Enzymatic activity of rice plants treated with bVOCs.

Treatment Guaiacol peroxidase (Unit/L) Polyphenol oxidase (Unit/L) Total polyphenols (GAE/gFW) Total flavonoids (μg RE/gFW)
T1 0.47 ± 0.02a 0.25 ± 0.03a 18.02 ± 0.49a 30.30 ± 0.42a
T2 0.56 ± 0.01bc 0.33 ± 0.04b 18.84 ± 0.45a 44.03 ± 0.94b
T3 0.54 ± 0.09bc 0.37 ± 0.01c 25.76 ± 0.92c 36.98 ± 3.94ab
T4 0.59 ± 0.01c 0.35 ± 0.01bc 21.79 ± 0.10b 32.73 ± 0.64a
T5 0.50 ± 0.02ab 0.37 ± 0.05c 21.99 ± 0.20b 54.48 ± 4.84c
T6 0.57 ± 0.04c 0.34 ± 0.04bc 31.27 ± 0.12d 29.45 ± 0.52a
T7 0.56 ± 0.01bc 0.36 ± 0.05c 38.97 ± 0.24e 44.27 ± 0.99b
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Note: GAE-Gallic acid; FW- Fresh weight & RE-Rutin equivalent. Treatment T1 = Control; T2 = Pantoea dispersa BHUJPVR01; T3 = Enterobacter cloacae BHUJPVR02; T4 = Enterobacter sp. BHUJPVR12; T5 = Priestia aryabhattai BHUJPVR13; T6 = Pseudomonas sp. BHUJPVWRO5; T7 = Staphylococcus sp. BHUJPVWLE7. Data are shown as mean ± SD (n = 3), and in each column the superscript letters are used to show the statistical relationship between isolates based on ANOVA and Duncan multiple post-hoc test (P ≤ 0.05).

4. Discussion

In this study, we explored the impact of bacterial volatiles on the growth of rice plant and their impact on defense related enzymes. Based on the PGP properties (IAA production, phosphate solubilization, potassium solubilization, ammonium production, siderophore production, silicate solubilization and zinc solubilization), only 6 strains were selected for the study. In here, we demonstrated the effect of bacterial volatiles on plant growth promotion, elevation of defense related enzymes and inhibition of rice blast fungus M. oryzae in plate assay.

In our study, we were targeting fungal pathogen M. oryzae, the causative agent of rice blast disease. Blast disease can cause significant yield loss of up to 30 % and in some instance up to 100 % yield loss was observed [7,36]. It was previously reported, bacterial volatiles can inhibit the growth of plant pathogen of up to 69 % [37]. For instance, several microbial cultures are known to produce volatile compounds making plant more resistant to phytopathogen attack by elevating their systemic resistance [[38], [39], [40]]. In this study, we demonstrated that bacterial volatiles reduce the mycelial growth of rice blast fungus M. oryzae up to 69.20 % in plate assay. All selected strains effectively reduce the growth of plant pathogenic fungus by the action of their volatiles, with Enterobacter sp. strain BHUJPVR12 reduces maximum growth followed by P. dispersa strain BHUJPVR01 and P. aryabhattai BHUJPVR13.

Ryu et al. (2003) [41], first reported the positive impact caused by bacterial volatiles promoting the growth of Arabidopsis, since then very few research was conducted on the growth promotion of rice by the action of bacterial volatiles [10]. In our experimental setup, six isolates were used to see if their volatiles could promote rice growth when grown on two petri plates with only airborne signals being transferred between the bacteria and the plants. Our findings showed that bacterial volatiles promote rice plant growth in the form of shoot height, root height, root numbers and leaf numbers. When rice seedlings were exposed to bacterial volatiles for 15 days, all treated plants showed a significant increase in shoot height, with P. dispersa BHUJPVR01 treated plants showed the highest increase of 60.48 % as compared to control. Similarly, more than 85 % increment was observed in root length of plants treated with bacterial volatiles of P. dispersa BHUJPVR01, Enterobacter sp. BHUJPVR12, P. aryabhattai BHUJPVR13, Pseudomonas sp. BHUJPVWRO5 and Staphylococcus sp. BHUJPVWLE7. Leaf counts were practically identical to control, whereas root counts increased a maximum of up to 30 % in plants treated with E. cloacae BHUJPVR02 and Staphylococcus sp. BHUJPVWLE7.Additional study was conducted for estimation of elevated defense response in plants treated with bacterial volatiles by assaying defense related enzymes such as guaiacol peroxidase, polyphenol oxidase, total polyphenols, and total flavonoids. Bacterial volatiles treated plants exhibited enhanced activity of guaiacol peroxidases, polyphenol oxidases, and increased levels of total phenols and flavonoids. Guaiacol peroxidases activity was observed highest in Enterobacter sp. strain BHUJPVR12 and Pseudomonas sp. strain BHUJPVWRO5 treated rice plants. Polyphenol oxidase activity was found highest in E. cloacae strain BHUJPVR02 and Pseudomonas sp. strain BHUJPVWRO5 treated rice plants. According to a recent study, tomato leaves exposed to bacterial volatiles exhibited increased level of phenolics, which improves plant resistance to F. oxysporum f.sp. lycopersici and effectively promoted plant growth as compared to untreated pathogen challenged control [42]. Increased phenol content in plant was due to the exposure of bacterial volatiles and were responsible for induced systemic resistance (ISR) and systemic acquired resistance [43]. Few studies reported that, 3-pentanol and 6-pentyl-α-pyron volatiles produced by Bacillus amyloliquefaciens strain IN937a and Trichoderma, respectively, induce systemic resistance via modulation of salicylic acid and jasmonic acid pathways resulting in reduction of phytopathogenic attack in pepper and Arabidopsis thaliana [44,45]. Many phenols such as gallic acid were reported to protect plant from fungal pathogen by degrading fugal chitin by enhancing the activities of chitinases and peroxidases [46,47]. From aforementioned reports, it was clear that our finding of increased level of phenols in volatiles treated plants was indication of elevated defense response against fungal pathogens. Furthermore, Tang et al. (2023) [48] reported that flavonoids treatment can contribute to plant resistance to whitefly (Bemisia tabaci) by the callose accumulation and jasmonic acid expression. In corelation with above results, our findings of increased level of flavonoids in volatiles treated rice plants may show more resilience towards insect pest attack. Our findings align with several studies demonstrating that increased levels of defence enzymes in plants leads to the breakdown of reactive oxygen species (ROS), effectively protecting plants from pathogenic attack [[49], [50], [51], [52]]. Considering the outcomes of the above study, we may hypothesize that bacterial volatiles exposure could promote plant growth and defense response in rice plants from a variety of biotic and abiotic stresses.

5. Conclusion

Bacterial volatile compounds (BVC) from plant growth promoting bacterial strains could be an ideal strategy to increase plant growth by improving plant physiological property and suppressing the disease. The volatiles compounds of rice endophytes Enterobacter sp. BHUJPVR12, Pantoea dispersa BHUJPVR01 and Priestia aryabhattai BHUJPVR13 directly reduce growth of the rice pathogens Magnaporthe oryzae by 69.20 %, 66.15 %, and 62.31 %, respectively under in vitro condition experiments. A significant increase of plant growth attributes is observed in the rice plants treated with volatiles of Pantoea dispersa BHUJPVR01, Enterobacter cloacae BHUJPVR02 and Staphylococcus sp. BHUJPVWLE7 as compared to untreated plants. Beside the plant growth BVCs also elevated the defense activity of host plants by activation of key defense related enzymes such as guaiacol peroxidase, polyphenol oxidase, total phenol and total flavonoids content which are able to improve plant sustainability under biotic and abiotic stresses. The bacterial volatiles compounds will be an effective technology for biocontrol against different phytopathogen. This technology will be environment friendly, cost effective and socially acceptable for agricultural production.

CRediT authorship contribution statement

Tushar Goyal: Writing – original draft, Validation, Methodology, Formal analysis, Data curation, Conceptualization. Arpan Mukherjee: Writing – review & editing, Methodology, Formal analysis. Gowardhan Kumar Chouhan: Software, Formal analysis, Conceptualization. Anand Kumar Gaurav: Software, Methodology, Formal analysis, Data curation. Deepak Kumar: Software, Methodology, Formal analysis, Data curation. Saman Abeysinghe: Writing – review & editing, Project administration. Jay Prakash Verma: Writing – review & editing, Supervision, Project administration, Investigation, Funding acquisition, Conceptualization.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgement

Authors JPV thankful to Science & Engineering Research Board (SERB), (EEQ/2021/001083) and DST (DST/INT/SL/P-31/2021), Banaras Hindu Univeristy-IoE (6031) and “Promotion Trans-Disciplinary Research grant of IoE” for financial assistance for research work related to research on phytomicrobiome, plant-microbe interaction and developing plant growth promoting microbial metabolites. AM thanks to PMRF fellowship scheme for financial support to research work.

Footnotes

Appendix A

Supplementary data to this article can be found online at https://doi.org/10.1016/j.heliyon.2024.e29692.

Appendix A. Supplementary data

The following is/are the supplementary data to this article:

Supplementary Fig. 1A

Gel electrophoresis image representing DNA isolated from rice plant associated endophytes. Note: 1 = Marker (100 bp); 2 = BHUJPVR01; 3 = BHUJPVR02; 4 = BHUJPVR12; 5 = BHUJPVR13; Supplementary Fig. 1B: Gel electrophoresis image of the purified PCR amplification product, Note: 1 = Marker (100 bp); 2 = BHUJPVR01; 3 = BHUJPVR02; 4 = BHUJPVR12; 5 = BHUJPVR13

mmc1.docx (1.1MB, docx)

References

  • 1.Rice | FAO | Food and Agriculture Organization of the United Nations. https://www.fao.org/markets-and-trade/commodities/rice/en/ (accessed December 12, 2023) Vol XXI ISSUE No. 1, April, 2018.
  • 2.Bandumula N. Rice production in asia: key to global food security, proceedings of the national academy of sciences India section B. Biol. Sci. 2018;88:1323–1328. doi: 10.1007/S40011-017-0867-7. [DOI] [Google Scholar]
  • 3.Mukherjee A., Chouhan G.K., Gaurav A.K., Jaiswal D.K., Verma J.P. New and Future Developments in Microbial Biotechnology and Bioengineering. Elsevier; 2021. Development of indigenous microbial consortium for biocontrol management; pp. 91–104. [DOI] [Google Scholar]
  • 4.Sharifi R., Jeon J.S., Ryu C.M. Belowground plant-microbe communications via volatile compounds. J. Exp. Bot. 2022;73:463–486. doi: 10.1093/JXB/ERAB465. [DOI] [PubMed] [Google Scholar]
  • 5.Ravelo-Ortega G., Raya-González J., López-Bucio J. Compounds from rhizosphere microbes that promote plant growth. Curr. Opin. Plant Biol. 2023;73 doi: 10.1016/J.PBI.2023.102336. [DOI] [PubMed] [Google Scholar]
  • 6.Mageshwaran V., Paulraj S., Nagaraju Y. Millet Rhizosphere. Springer Nature Singapore; Singapore: 2023. Current insights into the role of rhizosphere bacteria in disease suppression in millets; pp. 121–147. 2023. [DOI] [Google Scholar]
  • 7.Yan X., Tang B., Ryder L.S., Maclean D., Were V.M., Eseola A.B., Cruz-Mireles N., Ma W., Foster A.J., Osés-Ruiz M., Talbot N.J. The transcriptional landscape of plant infection by the rice blast fungus Magnaporthe oryzae reveals distinct families of temporally co-regulated and structurally conserved effectors. Plant Cell. 2023;35:1360–1385. doi: 10.1093/PLCELL/KOAD036. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Schulz S., Dickschat J.S. Bacterial volatiles: the smell of small organisms. Nat. Prod. Rep. 2007;24:814–842. doi: 10.1039/B507392H. [DOI] [PubMed] [Google Scholar]
  • 9.Schmidt R., Etalo D.W., de Jager V., Gerards S., Zweers H., de Boer W., Garbeva P. Microbial small talk: volatiles in fungal-bacterial interactions. Front. Microbiol. 2016;6 doi: 10.3389/FMICB.2015.01495/FULL. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Almeida O.A.C., de Araujo N.O., Mulato A.T.N., Persinoti G.F., Sforça M.L., Calderan-Rodrigues M.J., Oliveira J.V. de C. Bacterial volatile organic compounds (VOCs) promote growth and induce metabolic changes in rice. Front. Plant Sci. 2023;13 doi: 10.3389/FPLS.2022.1056082/FULL. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Mukherjee A., Singh S., Gaurav A.K., Chouhan G.K., Jaiswal D.K., de Araujo Pereira A.P., Passari A.K., Abdel-Azeem A.M., Verma J.P. Harnessing of phytomicrobiome for developing potential biostimulant consortium for enhancing the productivity of chickpea and soil health under sustainable agriculture. Sci. Total Environ. 2022;836 doi: 10.1016/J.SCITOTENV.2022.155550. [DOI] [PubMed] [Google Scholar]
  • 12.Aneja K. 2003. Experiments in Microbiology, Plant Pathology and biotechnology. [Google Scholar]
  • 13.Mukherjee A., Singh B.K., Verma J.P. Harnessing chickpea (Cicer arietinum L.) seed endophytes for enhancing plant growth attributes and bio-controlling against Fusarium sp. Microbiol. Res. 2020;237 doi: 10.1016/J.MICRES.2020.126469. [DOI] [PubMed] [Google Scholar]
  • 14.Bric J.M., Bostock R.M., Silverstone S.E. Rapid in situ assay for indoleacetic acid production by bacteria immobilized on a nitrocellulose membrane. Appl. Environ. Microbiol. 1991;57:535–538. doi: 10.1128/AEM.57.2.535-538.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Cappuccino J.G., Sherman N. third ed. Benjamin/Cummings Publication Co; New York, NY: 1992. Microbiology, a Laboratory Manual; pp. 125–179. [Google Scholar]
  • 16.Pikovskaya R.I. Mobilization of phosphorus in soil in connection with the vital activity of some microbial species. Mikrobiologiya. 1948;17:362–370. doi: 10.1016/S0048-9697(03)00262-6. [DOI] [Google Scholar]
  • 17.Watanabe F.S., Olsen S.R. Test of an ascorbic acid method for determining phosphorus in water and NaHCO3 extracts from soil. Soil Sci. Soc. A. J. 1965;29:677–678. doi: 10.2136/sssaj1965.03615995002900060025x. [DOI] [Google Scholar]
  • 18.Alexander D.B., Zuberer D.A. Use of chrome azurol S reagents to evaluate siderophore production by rhizosphere bacteria. Biol. Fertil. Soils. 1991;12:39–45. doi: 10.1007/BF00369386. [DOI] [Google Scholar]
  • 19.Bunt J.S., Rovira A.D. Microbiological studies of some subantarctic soils. J. Soil Sci. 1955;6:119–128. doi: 10.1111/J.1365-2389.1955.TB00836.X. [DOI] [Google Scholar]
  • 20.Nautiyal C.S. An efficient microbiological growth medium for screening phosphate solubilizing microorganisms. FEMS Microbiol. Lett. 1999;170:265–270. doi: 10.1111/J.1574-6968.1999.TB13383.X. [DOI] [PubMed] [Google Scholar]
  • 21.Kang S.M., Waqas M., Shahzad R., You Y.H., Asaf S., Khan M.A., Lee K.E., Joo G.J., Kim S.J., Lee I.J. Isolation and characterization of a novel silicate-solubilizing bacterial strain Burkholderia eburnea CS4-2 that promotes growth of japonica rice (Oryza sativa L. cv. Dongjin) Soil Sci. Plant Nutr. 2017;63:233–241. doi: 10.1080/00380768.2017.1314829. [DOI] [Google Scholar]
  • 22.Prajapati K., Sharma M.C., Modi H.A. Isolation of two potassium solubilizing fungi from ceramic industry soils. Life Sci. Leaf. 2012;5:71–75. [Google Scholar]
  • 23.Velmurugan S., Ashajyothi M., Charishma K., Kumar S., Balamurugan A., Javed M., Karwa S., Prakash G., Subramanian S., Gogoi R., Eke P., Kumar A. Enhancing defense against rice blast disease: unveiling the role of leaf endophytic firmicutes in antifungal antibiosis and induced systemic resistance. Microb. Pathog. 2023;184 doi: 10.1016/J.MICPATH.2023.106326. [DOI] [PubMed] [Google Scholar]
  • 24.Patel A., Sahu K.P., Mehta S., Balamurugan A., Kumar M., Sheoran N., Kumar S., Krishnappa C., Ashajyothi M., Kundu A., Goyal T., Narayanasamy P., Kumar A. Rice leaf endophytic Microbacterium testaceum: antifungal actinobacterium confers immunocompetence against rice blast disease. Front. Microbiol. 2022;13 doi: 10.3389/fmicb.2022.1035602. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Minas K., Mcewan N.R., Newbold C.J., Scott K.P. Optimization of a high-throughput CTAB-based protocol for the extraction of qPCR-grade DNA from rumen fluid, plant and bacterial pure cultures. FEMS Microbiol. Lett. 2011;325:162–169. doi: 10.1111/J.1574-6968.2011.02424.X. [DOI] [PubMed] [Google Scholar]
  • 26.Mukherjee A., Gaurav A.K., Patel A.K., Singh S., Chouhan G.K., Lepcha A., Pereira A.P. de A., Verma J.P. Unlocking the potential plant growth-promoting properties of chickpea (Cicer arietinum L.) seed endophytes bio-inoculants for improving soil health and crop production. Land Degrad. Dev. 2021;32:4362–4374. doi: 10.1002/LDR.4042. [DOI] [Google Scholar]
  • 27.Castillo F.J., Penel C., Greppin H. Peroxidase release induced by ozone in Sedum album leaves. Involvement of Ca2+ Plant Physiol. 1984;74:846–851. doi: 10.1104/PP.74.4.846. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Sarker U., Oba S. Augmentation of leaf color parameters, pigments, vitamins, phenolic acids, flavonoids and antioxidant activity in selected Amaranthus tricolor under salinity stress. Sci. Rep. 2018;8 doi: 10.1038/S41598-018-30897-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Eke P., Kumar A., Sahu K.P., Wakam L.N., Sheoran N., Ashajyothi M., Patel A., Fekam F.B. Endophytic bacteria of desert cactus (Euphorbia trigonas Mill) confer drought tolerance and induce growth promotion in tomato (Solanum lycopersicum L.) Microbiol. Res. 2019;228 doi: 10.1016/J.MICRES.2019.126302. [DOI] [PubMed] [Google Scholar]
  • 30.Sikora M., Swieca M., Franczyk M., Jakubczyk A., Bochnak J., Zlotek U. Biochemical properties of polyphenol oxidases from ready-to-eat lentil (Lens culinaris medik.) sprouts and factors affecting their activities: a search for potent tools limiting enzymatic browning. Foods. 2019;8 doi: 10.3390/FOODS8050154. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Ashajyothi M., Kumar A., Sheoran N., Ganesan P., Gogoi R., Subbaiyan G.K., Bhattacharya R., pepper Black, Piper nigrum L. Associated endophytic Pseudomonas putida BP25 alters root phenotype and induces defense in rice (Oryza sativa L.) against blast disease incited by Magnaporthe oryzae. Biol. Control. 2020;143 doi: 10.1016/J.BIOCONTROL.2019.104181. [DOI] [Google Scholar]
  • 32.Alam A., Kataria P., N. V. Assessment of superoxide dismutase, catalase, and peroxidase activities in aspergillus sp. and cladosporium sp. J. Adv. Sci. Res. 2021;12:249–254. doi: 10.55218/JASR.202112334. [DOI] [Google Scholar]
  • 33.Ainsworth E.A., Gillespie K.M. Estimation of total phenolic content and other oxidation substrates in plant tissues using Folin-Ciocalteu reagent. Nat. Protoc. 2007;2:875–877. doi: 10.1038/NPROT.2007.102. [DOI] [PubMed] [Google Scholar]
  • 34.Sarker U., Oba S. Drought stress effects on growth, ROS markers, compatible solutes, phenolics, flavonoids, and antioxidant activity in Amaranthus tricolor. Appl. Biochem. Biotechnol. 2018;186:999–1016. doi: 10.1007/s12010-018-2784-5. [DOI] [PubMed] [Google Scholar]
  • 35.Chouhan G.K., Mukherjee A., Singh S., Gaurav A.K., Kumari B., Verma J.P. Developing eco-friendly endophytic bioinoculants for enhancing productivity and soil fertility in wheat. Agron. J. 2023;115:1640–1652. doi: 10.1002/AGJ2.21349. [DOI] [Google Scholar]
  • 36.Devanna B.N., Jain P., Solanke A.U., Das A., Thakur S., Singh P.K., Kumari M., Dubey H., Jaswal R., Pawar D., Kapoor R., Singh J., Arora K., Saklani B.K., Anilkumar C., Maganti S.M., Sonah H., Deshmukh R., Rathour R., Sharma T.R. Understanding the dynamics of blast resistance in rice-Magnaporthe oryzae interactions. J. Fungi. 2022;8 doi: 10.3390/JOF8060584. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Thangaraj P., Subbiah K.A., Sevugapperumal N., Uthandi S., Damodarasamy A., Shanmugam H. Activity of volatiles induced by microbes and natural plants stifled the growth of Pythium aphanidermatum - the damping off in Tomato. BMC Plant Biol. 2023;23 doi: 10.1186/S12870-023-04351-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Garbeva P., Weisskopf L. Airborne medicine: bacterial volatiles and their influence on plant health. New Phytol. 2020;226:32–43. doi: 10.1111/nph.16282. [DOI] [PubMed] [Google Scholar]
  • 39.Patel A., Kumar A., Sheoran N., Kumar M., Sahu K.P., Ganeshan P., Ashajyothi M., Gopalakrishnan S., Gogoi R. Antifungal and defense elicitor activities of pyrazines identified in endophytic Pseudomonas putida BP25 against fungal blast incited by Magnaporthe oryzae in rice. J. Plant Dis. Prot. 2021;128:261–272. doi: 10.1007/S41348-020-00373-3. [DOI] [Google Scholar]
  • 40.De la Cruz-López N., Cruz-López L., Holguín-Meléndez F., Guillén-Navarro G.K., Huerta-Palacios G. Volatile organic compounds produced by cacao endophytic bacteria and their inhibitory activity on moniliophthora roreri. Curr. Microbiol. 2022;79 doi: 10.1007/S00284-021-02696-2. [DOI] [PubMed] [Google Scholar]
  • 41.Ryu C.M., Faragt M.A., Hu C.H., Reddy M.S., Wei H.X., Paré P.W., Kloepper J.W. Bacterial volatiles promote growth in Arabidopsis. Proc. Natl. Acad. Sci. U.S.A. 2003;100:4927–4932. doi: 10.1073/PNAS.0730845100. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Singh P., Singh J., Ray S., Vaishnav A., Jha P., Singh R.K., Singh H.B. Microbial volatiles (mVOCs) induce tomato plant growth and disease resistance against wilt pathogen Fusarium oxysporum f.sp. lycopersici. J. Plant Growth Regul. 2023 doi: 10.1007/S00344-023-11060-6. [DOI] [Google Scholar]
  • 43.Fincheira P., Quiroz A. Microbial volatiles as plant growth inducers. Microbiol. Res. 2018;208:63–75. doi: 10.1016/J.MICRES.2018.01.002. [DOI] [PubMed] [Google Scholar]
  • 44.Choi H.K., Song G.C., Yi H.S., Ryu C.M. Field evaluation of the bacterial volatile derivative 3-pentanol in priming for induced resistance in pepper. J. Chem. Ecol. 2014;40:882–892. doi: 10.1007/S10886-014-0488-Z. [DOI] [PubMed] [Google Scholar]
  • 45.Kottb M., Gigolashvili T., Großkinsky D.K., Piechulla B. Trichoderma volatiles effecting Arabidopsis: from inhibition to protection against phytopathogenic fungi. Front. Microbiol. 2015;6 doi: 10.3389/FMICB.2015.00995/FULL. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46.Nguyen D.M.C., Seo D.J., Lee H.B., Kim I.S., Kim K.Y., Park R.D., Jung W.J. Antifungal activity of gallic acid purified from Terminalia nigrovenulosa bark against Fusarium solani. Microb. Pathog. 2013;56:8–15. doi: 10.1016/J.MICPATH.2013.01.001. [DOI] [PubMed] [Google Scholar]
  • 47.Akhtar N., Mannan M.A.U., Pandey D., Sarkar A., Sharma H., Kumar M., Ghosh A. Potent antifungal properties of gallic acid in Sarcochlamys pulcherrima against Candida auris. Biotechnologia. 2023;104:105–119. doi: 10.5114/BTA.2023.127202. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 48.Tang J., Shen H., Zhang R., Yang F., Hu J., Che J., Dai H., Tong H., Wu Q., Zhang Y., Su Q. Seed priming with rutin enhances tomato resistance against the whitefly Bemisia tabaci. Pestic. Biochem. Physiol. 2023;194 doi: 10.1016/J.PESTBP.2023.105470. [DOI] [PubMed] [Google Scholar]
  • 49.Bhattacharyya C., Banerjee S., Acharya U., Mitra A., Mallick I., Haldar A., Haldar S., Ghosh A., Ghosh A. Evaluation of plant growth promotion properties and induction of antioxidative defense mechanism by tea rhizobacteria of Darjeeling, India. Sci. Rep. 2020;10 doi: 10.1038/s41598-020-72439-z. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 50.Awan Z.A., Shoaib A., Khan K.A. Variations in total phenolics and antioxidant enzymes cause phenotypic variability and differential resistant response in tomato genotypes against early blight disease. Sci. Hortic. 2018;239:216–223. doi: 10.1016/j.scienta.2018.05.044. [DOI] [Google Scholar]
  • 51.Kumar S., Chandra R., Behera L., Sudhir I., Meena M., Singh S., Keswani C. Microbial consortium mediated acceleration of the defense response in potato against Alternaria solani through prodigious inflation in phenylpropanoid derivatives and redox homeostasis. Heliyon. 2023;9 doi: 10.1016/j.heliyon.2023.e22148. 11. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 52.Zhu F., Wang J., Jia Y., Tian C., Zhao D., Wu X., Liu Y., Wang D., Qi S., Liu X., Li L., Jiang Z., Li Y. Bacillus subtilis GB519 promotes rice growth and reduces the damages caused by rice blast fungus Magnaporthe oryzae. PhytoFront. 2021;1:330–338. doi: 10.1094/PHYTOFR-12-20-0041-R. [DOI] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Fig. 1A

Gel electrophoresis image representing DNA isolated from rice plant associated endophytes. Note: 1 = Marker (100 bp); 2 = BHUJPVR01; 3 = BHUJPVR02; 4 = BHUJPVR12; 5 = BHUJPVR13; Supplementary Fig. 1B: Gel electrophoresis image of the purified PCR amplification product, Note: 1 = Marker (100 bp); 2 = BHUJPVR01; 3 = BHUJPVR02; 4 = BHUJPVR12; 5 = BHUJPVR13

mmc1.docx (1.1MB, docx)

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