The effect of cell free supernatant of S. hydrogenans strain DH16 on mycelial growth of A. brassicicola was further determined in liquid culture according to Li et al. (2011). The 3 days old culture supernatant at concentrations of 0, 0.5, 1, 5, 7.5, 10, and 20% was supplemented into potato dextrose broth (PDB, 50 ml). Each flask was inoculated with single mycelial disk of 6 mm diameter of test fungus and uninoculated SCN broth served as control. After 7 days of incubation at 28◦C, mycelial dry weight was determined, and growth inhibition was calculated as follow:

```
[(Weight of untreated mycelium − Weight of treated mycelium)]
 Weight of untreated mycelium × 100
```
The experiment was repeated twice with three replicas.

### Effect of Culture Supernatant on Fungal Morphology of A. brassicicola

The effect of cell free culture supernatant of streptomycete on morphology of A. brassicicola was studied microscopically. Mycelium of A. brassicicola was taken from periphery of the inhibition zone around the well (containing culture supernatant of streptomycete) and from control plate and placed on glass slide in a drop of sterile water. The coverslip was placed on the film and then visualized under bright field microscope at 40× (Olympus). Microphotographs were taken using a digital camera.

### Effect of Streptomyces DH16 Culture Supernatant on Spore Germination

To study the effect of culture filtrate on spore germination of A. brassicicola, 100 µl of fungal spore suspension made in PDB (105 spores ml−1 ) were mixed with 100 µl culture filtrate of different concentrations (0, 0.5, 1, 5, 7.5, 10, and 20% v/v; prepared using uninoculated production medium) and incubated at 28◦C. In control, culture filtrate was replaced with 100 µl of SCN broth. After incubation of 8 h, 50 µl of each suspension were placed on sterile glass slide. After placing coverslip, slide was observed under microscope by counting about 50 spores.

### Extracellular Conductivity

The effect of antifungal metabolites on cellular leakage was studied by determining the extracellular conductivity of supernatants obtained from mycelial suspensions (A. brassicicola) treated with culture supernatant of DH16 (Lee et al., 1998). To obtain mycelial growth of fungus. Erlenmeyer flask (250 ml) containing 50 ml of PDB was inoculated with single mycelial disk of 6 mm diameter from 5-day-old PDA plate of test fungus. After 3 days of incubation at 28◦C in PDB, mycelial growth was collected and washed thoroughly with sterile double distilled water. Washed mycelium (3 mg) was then added to flask containing 20 ml of culture supernatant of DH16. Mycelial suspensions were centrifuged at 10,000 rpm for 10 min to obtain supernatants, first immediately after the addition of mycelium, second after 12 h and third after 24 h of treatment. The experiment was repeated three times. Electrical conductivity was measured using a conductivity meter.

### Direct Observations of Antagonistic Effects of Streptomyces DH16 on Spore Germination and Growth of A. brassicicola in Soil Environment

A buried slide technique was used to determine the effect of streptomycete on fungal pathogen, directly in soil environment (Stevenson, 1956). 50 g of soil was sieved, air dried and sterilized in 100 ml glass beaker by autoclaving for 30 min at 121◦C. A 7 ml spore suspension (1 × 108 cells) of streptomycete was prepared. Spores were then thoroughly washed with sterile distilled water and inoculated into the sterile soil and incubation of soil was done at 28◦C for 7 days. A 10 ml spore suspension of 5 days old A. brassicicola (1 × 105 spores/ml) was mixed with 100 ml of sterile molten agar (1.8%), and 1 ml of it was coated on sterile glass slides. After solidification of the agar layer, slides were carefully inserted vertically into the beakers containing sterile soil alone (control) or DH16 inoculated soil and incubated at 28◦C. At the end of every stated period, i.e., 2, 3, 4, and 6 days, slides were removed and examined immediately under a compound microscope at magnification of 400×. The experiment was conducted twice.

## Efficacy of Culture Supernatant and Cells of Streptomyces DH16 As Seed Treatment against A. brassicicola to Control Damping Off

### Seed Treatment

The biocontrol potential of cell free culture filtrate and cells of S. hydrogenans strain DH16 as seed treatment against A. brassicicola using radish seeds was determined. Seeds were surface sterilized by immersing in sodium hypochlorite (1%) for 10 min. and then washed repeatedly with sterilized distilled water. The sterilized seeds were first artificially infected with the pathogen prior to antagonist treatment. The seeds were immersed for 4 h in fungal spore suspension in presence of 1% carboxymethyl cellulose (CMC; 105–107 spores/ml). These pathogen infested seeds were further given second treatment viz. (i) soaked in different concentrations (5, 10, and 20% v/v) of culture supernatant of antagonist/(ii) soaked in cell suspension of antagonist prepared in 1% CMC (107–108 /ml).

In another treatment, uninoculated sterilized seeds were treated with (i) 1% CMC only (control), (ii) cells of antagonist only (107–108 ), and (iii) cell free culture supernatant only. After 1 h of second treatment, all seeds were dried in laminar flow on a sterile filter paper and used for further experiments.

### Blotter Test

The moistened blotters were first used to determine the effect of antagonist to reduce damping off on radish plants grown from artificially infected seeds. Thirty seeds per treatment were placed in Petri dishes (10 seeds per plate) already lined with moist filter paper and covered loosely with another filter paper. Number of germinated seeds, and healthy and diseased seedlings were recorded after incubation of 7 days at 28◦C in the dark. Seedling vigor (V) was determined by measuring root and shoot lengths and was calculated according to the equation:

$$\mathbf{V} = (\mathbf{L\_s} + \mathbf{L}\mathbf{r}) \times \mathbf{G}$$

Where Ls is average shoot length in mm and Lr is average root length in mm and G is % germination (Andresen et al., 2015). The experiment was repeated twice.

### In vivo Pot Experiment

Same treatments were given to seeds as described above, except in case of culture supernatant. For pot experiments, culture supernatant at a concentration of 10% was used as it showed significantly better results in blotter test. Seeds were sown in pots containing autoclaved soil with 10 seeds per pot. The pots were kept under natural conditions (month of February, 20 ± 2 ◦C, 14 h light/10 h dark) and were watered daily. Seed germination was recorded after 15 days of sowing on the basis of above ground hypocotyls. Emergence of healthy seedlings, and mean fresh and dry weights of emerged plants were recorded. Disease incidence on radish plants was determined after 28 days on the basis of percentage of diseased seedlings.

The ability of the strain DH16 for root and rhizosphere colonization (seeds treated with Streptomyces cells) was also determined. Roots (from 10 plants) were recovered from the

pots 28 days after planting and were cut into 1 cm pieces and suspended in 1 ml of autoclaved distilled water. Similarly, rhizosphere soil (10 soil samples) adhered to roots was carefully removed, weighed (1 g) and serially diluted. Then 100 µl from root aliquots and soil dilution (10−4 ) were spread on SCNA plates and cfu/ml were calculated after incubation for 7 days at 28◦C.

### Biocontrol Potential of Culture Supernatant and S. hydrogenans As Foliar Treatment against A. brassicicola to Control Black Leaf Spot

The biocontrol potential of cells of S. hydrogenans and its cell free culture supernatant against A. brassicicola was also studied as foliar treatments using whole plants. Surface sterilized seeds of radish were sown in pots containing autoclaved soil. After 15 days, the leaves were given different treatments (i) inoculation with 10 µl of fungal pathogen (105 spores/ml; inoculated control), (ii) co-inoculation of pathogen (10 µl) and culture supernatant of antagonist (10 µl of 10%), (iii) co-inoculation of pathogen and cells of antagonist (107– 108 cells/ml), and (iv) non-inoculated healthy control (water only).

In another treatment, cell suspension/culture supernatant of S. hydrogenans was also applied to soil containing uninoculated radish plants.

After 20 days of various treatments, the plants were uprooted, fresh and dry weights of plants were recorded. Disease incidence was determined on the basis of dry weight of plants compared to control plants. The experiment was repeated twice.

### Statistical Analysis

All the experiments were repeated twice and the data (expressed as the mean ± SD) obtained from these experiments were subjected to statistical analysis. Tukey's post hoc test was done with the help of ASSISTAT (7.7 β) to compare the means.

### RESULTS

### Suppression of A. brassicicola in Liquid Broth by Culture Supernatant

Present study demonstrated inhibition of mycelial growth of A. brassicicola grown in broth supplemented with culture supernatant of DH16. In comparison to control, the mycelial dry weight of pathogen in PDB was significantly lowered in the presence of culture supernatant and this suppression of mycelial growth was found to be depended on the concentration of bioactive metabolites present in the culture supernatant. More than 50% inhibition was achieved at concentration of 5% and complete inhibition of mycelial growth occurred at 20% antifungal metabolites (P = 0.05; **Table 1**). Furthermore, the change in pH of the spent medium varied between 6.5 and 7.15, which suggested that the resulted mycelial inhibition was not due to pH change.

TABLE 1 | Effect of filter sterile culture supernatant of Streptomyces hydrogenans DH16 on mycelial growth of Alternaria brassicicola when supplemented in potato dextrose broth at different concentrations.

aMeans ± SD of two independent experiments, followed by the different letters within a column are significantly different according to Tukeys Test with P ≤ 0.05. bFinal pH of the spent medium.

### Effect of Culture Supernatant on Fungal Morphology

The inhibition of fungal pathogen by bioactive metabolites in the culture supernatant of the Streptomyces DH16 prompted us to examine the effect of its metabolites on the spore and mycelial structures. Microscopic studies demonstrated severe morphological abnormalities such as hyphal swellings resulting in bulbous structures, granular cytoplasm, leakage of cellular materials, thinning of hyphae, discoloration of hyphae, caused by metabolites present in the culture supernatant. Extracellular metabolites completely inhibited the sporulation along with loss of pigmentation (**Figure 1**).

### Effect of Culture Supernatant on Spore Germination

The effect of culture supernatant on spore germination was studied by incubating spores of A. brassicicola with different concentrations of supernatant. In control, 65% of the spores germinated after 8 h. At lower concentrations of 0.5 and 1%, the germination was not greatly affected as compared to control. However, it was significantly reduced to 25 and 10% at concentrations of 5 and 7.5%, respectively (p ≤ 0.0001). Concentrations of 10 and 20% were found to be completely lethal (no spore germination), and resulted in loss of pigmentation and shrinkage of spores.

### Extracellular Conductivity

Exposure of A. brassicicola to the antifungal metabolites of S. hydrogenans strain DH16 for 12 and 24 h resulted in increased levels of extracellular conductivity as compared to control, which showed leakage of cellular electrolytes from test fungus due to loss of cell wall/cell membrane integrity (**Figure 2**).

### Buried Slide Technique

The results of buried slide technique demonstrated 100% spore germination in control soil after 2 days of incubation (**Figure 3**). However, germination was significantly inhibited (only 7.14%) in streptomycete inoculated soil. With further incubation, germ

FIGURE 1 | Effect of culture supernatant of Streptomyces hydrogenans strain DH16 on Alternaria brassicicola mycelial morphology. (A) Firm control mycelium (B,C) treated hyphae. Arrows indicate abnormalities like distorted mycelial structure, swellings, leakage of cellular material, loss of pigmentation in mycelium.

tubes developed to form long hyphal threads in control soil where as in treated soil, small germ tubes of half the length of spores were formed in germinated spores. After 6 days incubation, complete mycelial structure with new sporulation was seen in control soil whereas in treated soil, germ tubes with loss of pigmentation and high vacuolization were observed.

## Biocontrol Efficiency of Streptomyces DH16 Cells and Culture Supernatant As Seed Treatment

### In vitro Plate Assay

In vitro biocontrol potential of cells and culture supernatant of streptomycete against A. brassicicola was studied as seed treatment in radish. Seed germination, number of healthy seedlings and seedling vigor were found to differ significantly in treated and non-treated seeds (P ≤ 0.05; **Table 2**). In the seeds treated with pathogen alone, the percentage of seed germination and healthy seedlings, fresh and dry weights, and seedling vigor were significantly lower as compared to the uninoculated control seeds. On the other hand, treatment of pathogen infested seeds with culture supernatant at the highest concentration of 20% significantly improved seed germination and seedling vigor to 80% and 1538, respectively and were comparable to control. The percentage of healthy seedlings (90%) and their fresh and dry weights were also significantly higher in treated seeds. Similarly, treatment of pathogen infected seeds with antagonist also significantly improved all the parameters as compared to pathogen infested seeds. Additionally, seeds treated with streptomycete/culture supernatant only were found to be healthier than the uninoculated control seeds. Strain DH16 showed significant stimulatory effect and an increase of 21– 35% over control in various growth parameters of seedlings was observed.

### In vivo Pot Assay

The effect of seed treatment on germination of seeds and growth of emerged seedlings was observed for plants grown in soil. Culture supernatant at concentration of 10% (v/v) was selected for further experimentation, since extracellular metabolites at this concentration were found to be strongly inhibitory to the pathogen as shown in in vitro blotter test. Significant differences in percentages of seed germination, disease incidence, healthy seedlings, and in fresh and dry weights of emerged plants were found between the different treatments (**Figure 4**). The pathogen infected radish seeds when treated with Streptomyces DH16 cells/metabolites, germinated and emerged as healthy seedlings, at significantly (P ≤ 0.001) higher rates (70–95%)

as compared to seeds treated with pathogen only (5–25%). Similarly, disease severity was also significantly reduced in treated seeds in both the treatments. Although the fresh and dry weights of plants emerged from seeds treated with pathogen and antagonists were lower than the water treated control plants but the plants were higher and stronger than the plants emerged from seeds treated with pathogen only. However, the treatment of seeds with S. hydrogenans DH16 cells/ culture supernatant only, significantly enhanced the germination rate and growth of emerged plants with significantly (p ≤ 0.05) higher fresh and dry weights as compared to control plants (**Figure 5**). When serially diluted aliquots of root segments and rhizosphere soil of plants

TABLE 2 | Effect of S. hydrogenans strain DH16 and its metabolites on seed germination, growth of seedlings and seedling vigor in R. sativus during in vitro blotter assays carried out for 7 days

Data represents Means ± SD of two separate tests (total 60 seeds for each treatment) where seeds were artificially infested with pathogen and then given different treatments with streptomycete cells and culture filtrate, different letters with in the column are significantly different according to Tukeys test; p ≤ 0.01.

two experiments with five different treatments of artificially A. brassicicola inoculated seeds, each treatment consist of three plates per experiment with 10 seeds per plate, i.e., total 60 seeds for each different treatment. Error bars represent approximate 95% confidence limit. Same letters on the bar are not significantly different according to Tukeys test (p ≤ 0.01); C, Control (Water only); P, Pathogen only; PE, Pathogen + Extract; PC, Pathogen + Streptomyces cells; SC, Streptomyces cells only.

emerged from streptomycete treated seeds were plated, the cfu counts of 1 × 107 /cm and 1 × 108 /g soil, respectively, were observed.

### Biocontrol Potential of Culture Supernatant and Streptomyces DH16 As Foliar Treatment

**Figure 6** demonstrates the biocontrol ability of Streptomyces DH16 and its culture supernatant as effective foliar treatments to control black leaf spot disease on radish leaves. In control plants, the symptoms of disease caused by A. brassicicola were clearly visible with 66.81% disease severity and yellowing of leaves and therefore under developed roots. On the other side, symptoms of disease were rarely observed in plants treated with antagonist and its metabolites which significantly reduced the disease severity to 6.78 and 1.47%, respectively (p ≤ 0.01). The fresh and dry weights of treated plants were also significantly higher over the pathogen inoculated plants (p ≤ 0.05). Weight of tap roots (edible part) in treated plants was comparable to the control plants, in contrast to pathogen treated plants where they were not developed properly. As compared to uninoculated control plants, edible tap roots with high fresh weight were obtained in plants where soil was irrigated with spore suspension/culture supernatant of S. hydrogenans DH16 (**Figure 7**).

disease. (A) Percentage of disease incidence (B) fresh weight of plants (C) dry weight of plants (D) weight of root tuber. Error bars represent approximate 95% confidence limit. Same letters on the bar are not significantly different according to Tukeys test (p ≤ 0.01); C, Control (Water only); P, Pathogen only; PE, Pathogen + Extract; PC, Pathogen + Streptomyces cells; SC, Streptomyces cells only.

### DISCUSSION

Biological control of various plant diseases that reduce crop yields has gained the attention of researchers due to its safe and ecofriendly application and therefore, will play vital role in continuous supply of food to the increasing world population. Among various groups of microbial biocontrol agents, actinobacteria especially, number of Streptomyces spp. has been reported as potential biological agents to control various phytopathogenic fungi (Mukherjee and Sen, 2006; Prabavathy et al., 2006; Bressan and Figueiredo, 2008; Li et al., 2011).

This study demonstrated in vitro and in vivo potential of S. hydrogenans strain DH16 to control black leaf spot and damping off of R. sativus caused by A. brassicicola. In the present investigation, in vitro studies revealed inhibition of mycelial growth and spore germination of A. brassicicola in the presence of culture supernatant of S. hydrogenans. With increasing concentration of culture supernatant, suppression of both

FIGURE 7 | Biocontrol potential of S. hydrogenans DH16 and its culture filtrate as foliar application on A. brassicicola causal agent of leaf spot disease in pot experiments. Radish plants 20 days after inoculation with (A) pathogen (B) pathogen + Streptomyces cells (C) pathogen + Streptomyces extract (D) water only (E) Streptomyces DH16 only (F) Streptomyces extract.

mycelial growth and conidial germination increased and almost complete inhibition was observed at concentration of 20%. These results coincides with earlier reports of Prapagdee et al. (2008) and Li et al. (2011) who also studied the effect of culture filtrates of Streptomycesspp., S. hygroscopicus, and S. globisporus JK-1, on the mycelial growth of Colletotrichum gloeosporioides and Sclerotium

rolfsii, and Magnaporthe oryzae, respectively. In contrast, fistupyrone, a metabolite from endophytic Streptomyces sp. TP-A0569, showed inhibitory effects on spores of A. brassicicola but could not inhibit its mycelial growth on PDB and PDA even at concentration of 1000 ppm (Aremu et al., 2003).

Antibiotics from actinobacteria antagonize phytopathogenic fungi by inducing various morphological alterations such as stunting, distortion, swelling, hyphal protuberances in mycelial structure or the highly branched appearance of fungal germ tubes (Gunji et al., 1983). Getha and Vikineswary (2002) observed severe morphological changes such as swellings, distortions, and excessive branching in Fusarium oxysporum f.sp. cubense race 4 caused by extracellular metabolites from S. violaceusniger strain G10. Prapagdee et al. (2008) also reported the absence of C. gloeosporioides conidia as one of the malformations caused by S. hygroscopicus and its sterile culture filtrates.

Similarly, in the present study microscopic observations of fungal mycelia from the margins of the inhibition zones (resulted from culture broth of strain DH16) revealed severe structural alterations in vegetative cells and spores, which indicated that metabolites probably attack the cell wall/cell membrane. These findings further confirmed the antibiosis as the selective mechanism of antifungal activity of strain DH16. One of the antifungal compounds of S. hydrogenans strain DH16 has been purified and characterized to be 10-(2,2-dimethyl-cyclohexyl)- 6,9-dihydroxy-4,9-dimethyl dec-2-enoic acid methyl ester (SH2; Kaur et al., 2016b).

The mechanism of antibiosis is considered to be advantageous in biological control of plant diseases because antimicrobials can diffuse rapidly in nature, and thus, direct contact between the pathogen and antagonist is not indispensable (Hajlaou et al., 1994). In addition, loss of pigmentation (melanin) in hyphae as well as spores of A. brassicicola was also observed. Loss in integrity of cell wall/cell membrane was further confirmed by leakage of cellular materials (electrolytes) indicated by changes in extracellular conductivity.

Organic farming needs alternative seed treatments to eliminate or at least effectively reduce the seed-borne pathogens (Amein et al., 2011). Biological seed treatments with microbial antagonists are attractive alternatives to the chemical pesticides because the latter lead to changes in the metabolic profiles of rhizosphere biodiversity (Correa et al., 2009) whereas the bacterization of seeds with microorganisms does not alter the beneficial rhizosphere bacterial community. Moreover, the seed bacterization method has been proved to be effective as the biocontrol agent can rapidly and extensively grow and cover the surface of the seeds and can protect the plants from invading soil-borne pathogens (Kanini et al., 2013).

In recent years, the use of crude secondary metabolites produced by Streptomyces spp. are also gaining importance in crop protection and these metabolites may be a supplement or an alternative to chemical pesticides (Zacky and Ting, 2013; Harikrishnan et al., 2014). Therefore, in this study both the cells as well as extracellular antifungal metabolites of S. hydrogenans strain DH16 were evaluated for their in vivo biocontrol potential against A. brassicicola. The treatment of pathogen infested radish seeds with streptomycete cells/culture supernatant lead to statistically significant (p ≤ 0.05) improvement in seed germination, seedling vigor and plant weight, in both blotter assay as well as in in vivo pot experiments and thus reduced the frequency rate of damping off of seedlings.

In addition to disease control, cells as well as culture supernatant of Streptomyces DH16 significantly enhanced vigor index and other agronomic parameters (fresh and dry weights) over uninoculated control when applied as seed dressing. These results indicate the plant growth promoting prospect of S. hydrogenans DH16 in the absence of pathogen stress which is to its root colonizing ability as indicated by the adherence of bacterium to the roots and associated soil. The ability to colonize roots is an important trait of plant growth promoting microorganisms for their beneficiary effects (Bouizgarne, 2013) and is also related to the effectiveness of biocontrol activity against pathogens (Bull et al., 1991). To our best knowledge, only one report is available in literature where seed treatment is used to control A. brassicicola. In 1987, Tahvonen and Avikainen reported the biocontrol of seed borne A. brassicicola of cruciferous plants with a powdery preparation of a Streptomyces sp. However, till date no biological seed treatment method has been used commercially.

Furthermore, it is challenging to obtain pathogen free seeds of Brassica spp., due to the lack of foliar fungicides (Kohl et al., 2010). Alternaria species have also been reported to develop resistance against dicarboximides and phenylpyrroles (Iacomi-Vasilescu et al., 2004). Therefore, cells and metabolites of S. hydrogenans strain DH16 were also evaluated as foliar application to control black leaf spot by A. brassicicola. Both the cells as well as metabolites caused reduction of black leaf spot on R. sativus and significantly increased the size and weight of swollen stem as campared to negative control (fungal inoculated plant). In addition, plants irrigated with spore suspension/culture supernatant of streptomycete showed growth enhancement over the untreated control plants which further confirm the PGP potential of the S. hydrogenans DH16. Fistupyrone is the only metabolite reported earlier from Streptomyces sp. which displayed in vivo suppression of black leaf spot caused by A. brassicicola on the seedlings of Chinese cabbage (Igarashi et al., 2000).

The outcomes of the current study clearly indicate the great potential of S. hydrogenans DH16 (a less studied streptomycete) as another potent biocontrol agent which can be used to control both seed borne as well as foliar pathogen, A. brassicicola. The importance of the study lies in the fact that both spores/mycelium and extracellular metabolites of this streptomycete reduced the incidence of A. brassicicola, therefore, this strain might be used as a biofungicide in two forms, one having spores and other containing antifungal metabolites. Additionally, this strain is also having insecticidal (Kaur et al., 2014), nematicidal (Kaur et al., 2016a) and plant growth promoting activities (data communicated) which make it superior over already reported chemical and biofungicides (mycostop, fistupyrone, actinovate, and rhizovit) and thus can also be used as bioinsecticide and bio-fertilizer in addition to biofungicide.

### AUTHOR CONTRIBUTIONS

fpls-07-01869 December 14, 2016 Time: 16:19 # 12

TK was involved in the planning and execution of the research work; analysis and interpretation of the data; manuscript writing following the suggestions of the research supervisor. RM as research supervisor of TK was involved in the design and planning of research work; analysis and interpretation of data; drafting as well as critical editing of the manuscript for intellectual subject matter. Both the authors approved the final version of the manuscript for publication and agreed to be accountable for all aspects of the work in ensuring that questions

### REFERENCES

related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

### ACKNOWLEDGMENTS

We duly acknowledge University Grants Commission (UGC), New Delhi for providing funds to accomplish this work. TK acknowledges the grant of fellowship under UPE (University with Potential for Excellence) scheme of University Grants Commission, New Delhi, India.

aurantiogriseus VSMGT1014 against sheath blight of rice disease. World J. Microbiol. Biotechnol. 30, 3149–3161. doi: 10.1007/s11274-014-1742-9

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2016 Manhas and Kaur. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

fmicb-07-01004 June 29, 2016 Time: 11:19 # 1

\*

# Purification and Characterization of a New Antifungal Compound 10-(2,2-dimethyl-cyclohexyl)-6,9 dihydroxy-4,9-dimethyl-dec-2-enoic Acid Methyl Ester from Streptomyces hydrogenans Strain DH16

#### Talwinder Kaur1 , Amarjeet Kaur1 , Vishal Sharma2 and Rajesh K. Manhas1

1 Department of Microbiology, Guru Nanak Dev University, Amritsar, India, 2 Department of Pharmaceutical Sciences, Guru Nanak Dev University, Amritsar, India

#### Edited by:

Gero Benckiser, Retired from Justus-Liebig-Universität Gießen, Germany

### Reviewed by:

Wubei Dong, Huazhong Agricultural University, China Nan Yao, Sun Yat-sen University, China

> \*Correspondence: Rajesh K. Manhas<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Plant Biotic Interactions, a section of the journal Frontiers in Microbiology

Received: 22 April 2016 Accepted: 13 June 2016 Published: 29 June 2016

#### Citation:

Kaur T, Kaur A, Sharma V and Manhas RK (2016) Purification and Characterization of a New Antifungal Compound 10-(2,2-dimethyl-cyclohexyl)-6,9 dihydroxy-4,9-dimethyl-dec-2-enoic Acid Methyl Ester from Streptomyces hydrogenans Strain DH16. Front. Microbiol. 7:1004. doi: 10.3389/fmicb.2016.01004 In agriculture, biocontrol agents have been emerged as safe alternative to chemical pesticides where Streptomyces spp. and their metabolites constitute a great potential for their exploration as potent agents for controlling various fungal phytopathogens. The present study reports an antifungal compound purified from Streptomyces hydrogenans strain DH16, a soil isolate, using silica gel chromatography and semi preparative HPLC. The compound was characterized using various spectroscopic techniques (IR, 1H and 13C NMR) and named 10-(2,2-dimethyl-cyclohexyl)-6,9-dihydroxy-4,9-dimethyl-dec-2 enoic acid methyl ester (SH2). Compound (SH2) showed significant inhibitory activity against fungal phytopathogens and resulted in severe morphological aberrations in their structure. Minimal inhibitory and minimal fungicidal concentrations of the compound ranged from 6.25 to 25 µg/ml and 25 to 50 µg/ml, respectively. In vivo evaluation of the compound showed strong control efficacy against Alternaria brassicicola, a seed borne pathogen, on radish seeds. In comparison to mancozeb and carbendazim, the compound was more effective in controlling damping off disease. Additionally, it promoted plant growth with increased rate of seed germination, and displayed no phytotoxicity. The compound retained its antifungal activity after its exposure to temperature of 100◦C and sunlight for 1 h. Furthermore, the compound (SH2) when tested for its biosafety was found to be non-cytotoxic, and non-mutagenic against Salmonella typhimurium TA98 and TA100 strains. This compound from S. hydrogenans strain DH16 has not been reported earlier, so this new compound can be developed as an ideal safe and superior biofungicide for the control of various fungal plant diseases.

Keywords: Streptomyces hydrogenans DH16, antifungal compound, fungal phytopathogens, biofungicide, biocontrol

## INTRODUCTION

fmicb-07-01004 June 29, 2016 Time: 11:19 # 2

Plant pathogens, especially fungi are one of the major threats to agricultural productivity of economically important crops worldwide (Cornelissen and Melchers, 1993). Chemical fungicides are used in current agricultural practices to combat these phytopathogens. However, long term application of these chemicals has resulted in severe negative impacts on environment and human health. Furthermore, their indiscriminate and repeated use has triggered the emergence of resistance in phytopathogens, due to which several important chemical fungicides have lost their efficacy against the resistant pathogens in the field (Yang et al., 2008). These limitations of chemical fungicides and increased public concern for pesticide free food highlight the discovery and development of new safer fungicides (Coloretti et al., 2007).

In recent years, control of plant diseases using microorganisms and their bioactive metabolites has drawn greater attention as better alternative to chemical fungicides. The antifungal antibiotics of microbial origin are safe, broad spectrum, less toxic to host plants, easily biodegradable in the biosphere, thus low residue levels in environment and food. Cycloheximide and streptomycin from Streptomyces griseus were successfully used to control fungal and bacterial diseases in plants, respectively, for the first time (Leben and Keitt, 1954). Since then, many attempts were made to explore various antibiotics from microorganisms for control of plant diseases and some viz. blasticidin S, polyoxin, kasugamycin, validamycin, gopalamycin, dorrigocins, geldanamycin, nigericin, fistupyrone, jiggangmycin, phenyl acetic acid, azalomycin have been developed as fungicides for agricultural use (Hochlowski et al., 1996; Kim and Hwang, 2007).

Streptomyces spp. hold considerable importance in biocontrol of various plant diseases caused by diverse range of plant pests. These bacteria are the largest hub for the antimicrobial agents, and approximately two third of economically important antibiotics developed for agricultural use are from Streptomyces spp. Their use as potent biocontrol agents against phytopathogenic fungi has been reported by various workers (Yuan and Crawford, 1995; Xiao et al., 2002; Kanini et al., 2013; Faheem et al., 2015) and is mainly due to the production of various antifungal compounds (Doumbou et al., 2001; Xiong et al., 2012; Palaniyandi et al., 2013; Nguyen et al., 2015) and cell wall degrading enzymes such as chitinases and glucanases (Gupta et al., 1995; Taechowisan et al., 2003, 2005; Quecine et al., 2008). Streptomycetes have also been used as biofungicide formulations containing live mycelium or spores and their active metabolites. For example, mycostop (containing S. griseoviridis K61), Actinovate and actinoiron (containing Streptomyces lydicus WYEC 108) and RhizovitR (S. rimosus) are commercial biofungicides used to control plant diseases caused by Phytophthora spp., Fusarium spp., Pythium spp., Alternaria brassicicola, Botrytis sp., and Rhizoctonia solani (Tahvonen and Avikainen, 1987; Marten et al., 2000).

Although different antifungal compounds from Streptomyces spp. have been reported but it is just the tip of the ice berg that has been explored. Therefore, in continuous demand for new bioactive metabolites for plant protection, the present study reports the purification, characterization and biological evaluation of a new antifungal compound from S. hydrogenans, a strong antagonist against various fungal phytopathogens. The biosafety of the compound was also evaluated using Ames Mutagenicity and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide) cytotoxicity tests.

### MATERIALS AND METHODS

### Microorganisms and Maintenance

Streptomyces hydrogenans strain DH16 (GenBank accession no. JX123130) was isolated from soil procured from Dalhousie (32.53◦ N, 75.98◦ E), Himachal Pradesh, India (Kaur and Manhas, 2014) and maintained on starch casein nitrate agar slants at refrigeration temperature (4◦C). Twenty percent glycerol stocks were also prepared and stored at −20◦C during the study. The three test phytopathogenic fungi viz. Alternaria brassicicola (MTCC 2102), A. solani (MTCC 2101), and Colletotrichum acutatum (MTCC 1037) were obtained from Microbial Type Culture Collection (MTCC), Institute of Microbial Technology (IMTECH), Chandigarh, India. Fusarium moniliforme, Alternaria alternata were isolated in lab. All the fungal cultures were maintained on Potato dextrose agar (PDA) slants at 4◦C.

### Production of Antifungal Metabolites

Production of antifungal metabolites from S. hydrogenans strain DH16 was carried out according to Kaur and Manhas (2014). The actinobacterium was grown on starch casein nitrate agar medium at 28◦C for 7 days and then growth was scrapped and transferred aseptically into the SCN broth to develop seed culture. After 48 h of incubation, the seed culture was inoculated into 250 ml Erlenmeyer flasks containing 50 ml of production medium containing g/L: starch, 12; soyabean meal, 2.5; K2HPO4, 1.8; NaCl, 2; Casein, 0.3; MgSO4, 0.05; FeSO4, 0.01; and CaCO3, 0.02. The fermentation was carried out at 28◦C at 180 × g, and after 3 days of incubation, culture broth was centrifuged at 10000 × g for 20 min at 4◦C to obtain cell free culture supernatant.

### Extraction and Purification of Metabolites

For the recovery of active metabolites, culture supernatant (5 l) was extracted twice with equal volume of ethyl acetate (EA). The organic phase was separated, treated with Na2SO4 and then concentrated to dryness under vacuum at 45◦C using rotary evaporator (BUCHI Rota vapor R-200). For the purification of antifungal compounds, the resulting solids (1 g) re-dissolved in small volume of methanol were subjected to column chromatography using silica gel (60–120 mesh size; column, 1.0 cm × 35 cm) packed and pre-equilibrated with chloroform. The column was then eluted step-wise with linear gradients of: chloroform/methanol (100:0, 90:10, 80:20,70:30, 60:40, 50:50, 40:60, 30:70, 20:80, 10:90, and 0:100) at a flow fmicb-07-01004 June 29, 2016 Time: 11:19 # 3

rate of 2 ml/min. About 200 ml of each gradient was used for elution, and a total of 88 fractions of 25 ml each were collected and concentrated. Each fraction was then subjected to disk diffusion assay to determine their antifungal activity against A. brassicicola and fractions showing antifungal activity were pooled together and concentrated. Final purification of the active compounds was achieved by preparative RP-HPLC: Shimadzu Microsorb MV, 100 mm × 10 mm ID, 10 µm, flow rate 3 ml/min, gradient Acetonitrile:H2O 50% in 5 min, 50–70% in 15 min and 70–50% in 17 min, and UV detection at 210 nm. All the peaks of chromatogram were collected using a fraction collector coupled with the HPLC system, then concentrated and screened for antifungal activity. Three peaks which showed activity were rechromatographed with the same solvent system to check the purity to homogeneity level.

### Characterization of Active Peak 2

The peak **2** was characterized based on various physicochemical and spectroscopic properties. Appearance, color, odor, and solubility were determined according to the standard procedures. The UV-Visible spectrum was recorded qualitatively on UV-Visible Spectrophotometer (Shimadzu) in the range of 200– 400 nm using acetonitrile as reference solvent.1H NMR and 13C NMR spectra were recorded in chloroform-d [99.8 atom% D, containing 0.1% (v/v) tetramethylsilane (TMS)] at 25◦C on 500 MHz AVANCE III Bruker spectrometer equipped with a 5 mm double channel solution state probe. The chemical shifts are reported in parts per million (ppm) relative to TMS (δ0.0) used as internal standard. Mass spectrum (HR-MS) was recorded with Bruker MICROTOF II spectrometer. IR spectrum was recorded with Perkin–Elmer FTIR-C92035 Fourier-Transform spectrophotometer in the range 400–4000 cm−1 by using CHCl3 as the medium for the preparation of the samples.

## Antifungal Activity of Purified Compound

The antifungal activity of the purified compound SH2 was tested against A. brassicicola, A. solani and C. acutatum, F. moniliforme and A. alternata causing various diseases on diverse host plants. The activity was determined in terms of zone of inhibition by using Kirby–Bauer well diffusion assay (Bauer et al., 1996). Wells of 4 mm diameter were made on PDA plates seeded with the test fungal pathogens. Then aqueous solutions of purified compound, cycloheximide and chemical control agents (carbendazim and mancozeb) were made at concentration of 1 mg/ml and 100 µl of each were added into wells. The diameters of the resultant zones of inhibition were measured in mm after 48–72 h of incubation. Each experiment was performed in duplicates and repeated thrice.

## Effect of Purified Compound on Fungal Morphology

The effect of purified compound **(SH2)** on morphology of A. brassicicola and F. moniliformae was studied microscopically. Mycelia of A. brassicicola and F. moniliforme were taken from periphery of the inhibition zones around the well (containing **SH2**) and from control plate and placed on glass slide in a drop of sterile water. The coverslip was placed on the film and then visualized under bright field microscope at 40× (Olympus). Microphotographs were taken using a digital camera.

### Minimal Inhibitory Concentration (MIC) and Minimal Fungicidal Concentration (MFC)

Minimal inhibitory concentration of EA extract was worked out by 96 well microtitre plate method (Díaz-Dellavalle et al., 2011) using different concentrations (12.5, 25, 50, 100, 250, 500, and 1000 µg/ml) of extract in EA. The spore suspension of test fungus was prepared by scrapping the spores from 5-dayold PDA culture plate with fresh PDB. In 96 well microplates, 100 µl of fungal spore suspension (1 × 105 spores/ml) was mixed with 100 µl of extract of different concentrations. Control well contained 100 µl of fungal spore suspension and 100 µl of EA. Control blanks consisted of 100 µl of extract of different concentrations with 100 µl of PDB and other contained 100 µl of PDB and 100 of EA only. The microplates were incubated at 28◦C and readings were taken with microplate reader at 595 nm after 48 h. MIC values were calculated by comparing the growth in wells containing extract to the growth in control wells and is the lowest concentration that resulted in 80% inhibition in growth compared to the growth in control well. Further MFC was determined by plating 20 µl of the broth from each well on fresh PDA plates. The plates were then incubated at 28◦C until growth was visible in the control subculture. The MFC will be that lowest concentration where no visible growth will be observed on plate. The experiments were performed in triplicates. Similarly, the MIC and MFC values for purified compound were determined using different concentrations (6.25, 12.5, 25, 50, 100, and 200 µg/ml).

### Biocontrol of A. brassicicola on Raphanus sativus Seeds by Purified Compound

In vitro and in vivo experiments were used to evaluate the biocontrol potential of compound to control A. brassicicola on surface sterilized radish seeds. The sterilized seeds were first artificially infested with the pathogen by immersing them for 4 h in spore suspension, prepared from 5-day-old A. brassicicola, in presence of 1% carboxy methyl cellulose (CMC; 105–107 spores/ml). The pathogen infested seeds were then soaked in 1 mg/ml solution of compound and chemical control agents. After 1 h, all seeds were dried in laminar flow on a sterile filter paper and used for further experiments. Each treatment consisted of three replicates with 10 seeds each.

### In Vitro Blotter Test

The moistened blotters were first used to determine the effect of compound to reduce damping off due to seed-borne A. brassicicola on radish plants grown from artificially infected seeds. Three replicates of 10 seeds per treatment were placed in Petri dishes (10 seeds per plate) already lined with moist filter paper and covered loosely with another filter paper. The number of germinated seeds and healthy and diseased seedlings were recorded after 7 days of incubation at 28◦C in the dark. Seedling vigor (V) was determined by measuring root and shoot lengths of 10 seedlings (selected randomly) and was calculated according to the equation:

$$\mathbf{V} = (\mathbf{L\_s} + \mathbf{L\_t}) \times \mathbf{G}$$

Where Ls is average shoot length in mm and Lr is average root length in mm and G is % germination (Andresen et al., 2015).

### In Vivo Pot Experiment

fmicb-07-01004 June 29, 2016 Time: 11:19 # 4

Treated seeds were sown in pots containing sterilized soil with 10 seeds per pot. The pots were kept under natural conditions and were watered daily. Seed germination, emergence of healthy seedlings, mean fresh, and dry weights of emerged plants and seedling vigor were recorded after 15 days of sowing. To determine the dry weight of plants, harvested plants were placed separately on filter papers in oven at 60◦C for 48 h and then weighed using weighing balance.

### Stability of Compound SH2

To determine heat stability, compound **SH2** was heated at 37, 50, 70, and 100◦C for 1 h. Photostability was also tested by exposing the compound separately to sunlight for 1 h. All the treated samples were then checked for residual activity with respect to untreated control against C. acutatum.

### Safety Evaluation

Purified compound **(SH2)** was evaluated for toxicity testing viz. phytotoxicity, mutagenicity, and cytotoxicity.

### Phytotoxicity Testing

Phytotoxicity was checked by treating sterilized seeds with purified compound. In control, the compound was replaced with water. Treated seeds were then sown in sterilized soil and data for important agronomic parameters (seed germination, seedling vigor and weight of plants) were recorded after 10 days.

### Mutagenicity Studies

The mutagenicity of purified compound was determined using Salmonella histidine point mutation assay proposed by Maron and Ames (1983) with slight modifications suggested by Bala and Grover (1989). For toxicity testing, 0.1 ml of bacterial culture and 0.1 ml of compound **SH2** at different concentrations (50, 100, and 250 µg/plate) was added to 2 ml of top agar and poured onto the minimal agar plates followed by incubation at 37◦C for 48 h. To determine the spontaneous reversion which is characteristic of the tester strains (TA98 and TA100), negative control (0.1 ml bacterial culture + 0.1 ml DMSO per plate) was run while 4-Nitro-o-phenylenediamine (20 µg/0.1 ml/plate) and sodium azide (2.5 µg/0.1 ml/plate) were used as a positive control mutagens for strains TA98 and TA100, respectively. After incubation for 48 h, the number of revertant his+ bacteria colonies were scored. The mutagenic potential of the purified compound was determined by comparing the number of colonies with control plates where no test compound as well as mutagen was added.

### In Vitro Cytotoxicity

The MTT assay was used for determining in vitro cytotoxicity following the method given by Mosman (1983) using Chinese Hamster Ovary (CHO), a non-tumor or normal cell line obtained from National Centre for Cell Science (NCCS), Pune (India). Cells of CHO were grown in tissue culture flask in complete growth medium [Roswell Park Memorial Institute (RPMI)- 1640 medium with 2 mM glutamine, 100 units ml-1 streptomycin and supplemented with 10% Foetal Calf Serum (FCS) and 100 units/ml penicillin] at 37◦C in an atmosphere of 5% CO2 and 90% relative humidity. The cells at subconfluent stage were harvested from the flask by treatment with trypsin (0.05% in PBS containing 0.02% EDTA) for determination of cytotoxicity. Cells with viability of more than 98%, as determined by trypan blue exclusion, were used for assay. The cell suspension of 1 × 105 cells/ml was prepared in complete growth medium for determination of cytotoxicity. Stock solution of purified compound (100 µg/ml) was prepared in sterile filtered DMSO. The compound was serially diluted with complete growth medium containing 50 µg/ml of gentamicin to obtain working test solutions of different concentrations.

The 100 µl of cell suspension (1 × 105 cells/ml) were seeded in 96-well tissue culture plates (MicrotestTM, Falcon, USA), followed by addition of 100 µl of each concentration (30, 50, and 100 µg/ml) of **SH2**, after 24 h incubation. Respective control cells were treated with medium only. After 42 h of treatment the spent medium was discarded by inverting the plate on a tissue towel and 100 µl of MTT prepared in non-serum medium (0.5 mg/ml; without FCS) was added to each well. After 2 h of incubation, medium was discarded and blue formazan crystals formed by MTT reaction were dissolved in 100 µl of dimethyl sulfoxide (DMSO, Emplura R , Millipore) in each well. The color was read at 540 nm in the Elisa plate reader (Multiscan R EX by Labsystems, Finland). The proliferation of cells under treatment was assessed according to following formula:

$$\text{Percentage of refrigeration} = \frac{\text{Absorbance of test}}{\text{Absorbance of control}} \times 100$$

% Growth inhibition = 100 – % Cell growth

### Statistical Analysis

All the experiments were repeated twice and the data (expressed as the mean ± SD) obtained from these experiments were subjected to statistical analysis. Tukey's post hoc test was done with the help of ASSISTAT (7.7 beta) to compare the means.

### RESULTS

### Isolation and Purification of the Compound

The streptomycete DH16 was grown in SCN broth for the production of active antifungal metabolites. After third day of incubation, broth was centrifuged, extracted, and concentrated to yield dark brown residue which was subjected to silica gel column chromatography for isolation of active compounds. Three active fmicb-07-01004 June 29, 2016 Time: 11:19 # 5

fractions (no. 9–11) eluted with chloroform:methanol (90:10) in silica gel chromatography showed antifungal activity and then pooled together based on their similar TLC pattern and concentrated. The pooled fraction was then further fractionated on semi-preparative HPLC and individual peaks were collected. The peak with retention time of 9.61 min showed antifungal activity against the test pathogenic fungus (A. brassicicola). To check the homogeneity of active compound, collected peak was chromatographed under similar conditions and single peak was obtained which indicated the purity of the compound (**Figure 1**).

### Physical Properties of the Purified Compound

The compound was light yellowish in color and was odorless. It was soluble in most of the organic solvents but was sparingly soluble in water.

### Chemical Characteristics of the Purified Compound (SH2) from S. hydrogenans DH16

The compound responsible for antifungal activity was characterized by various spectroscopic techniques such as IR, 1H and 13C NMR spectra, and mass spectroscopy. **IR** (CHCl3): νmax (cm−1 ) = 3441, 3054, 2986, 2685, 2305, 1754, 1606, 1422, 1267, 1151, 908, 896, 752; 1**H NMR** (500 MHz, CDCl3): δ = 7.45 (d, 1H, J = 5.5 Hz, C2-H), 6.13 (distorted d, 1H, J = 4.2 Hz, C3-H), 5.06–5.03 (m, 1H, C6-H), 3.66 (dist. s, 3H, OCH3), 1.80–1.77 (m, 4H), 1.70–1.65 (m, 4H), 1.52–1.37 (m, 8H), 1.17–1.14 (m, 8H, 2x CH3, C7-H), 0.92–0.85 (m, 8H,); 13**C NMR** (150 MHz, CDCl3): δ = 173.1 (C = O), 156.2 (C-2), 121.5 (C-3), 83.3, 72.8, 71.7, 41.1, 39.9, 34.2, 33.1, 32.3, 29.8, 29.6, 26.3, 24.9, 23.5, 14.6, 8.2; **MS** (TOF, ESI): m/z:calculated for C21H38O4: 354.2; found: 377.1 [M+ Na]+ (Supplementary Figures 1–5).

In the proton spectrum proton of C-2 appeared as doublet at δ 7.45 with J = 5.5 Hz and proton of C-3 appeared as distorted doublet at δ 6.13 with J = 4.2 Hz. Proton of remaining carbons in the compound showed multiple resonances by two bond and three bond couplings. 13C NMR of the compound showed carbonyl resonance at δ 173.1 which is further revealed by IR spectrum showed band at 1754 cm−1 . Carbon NMR also showed resonances of two olefinic carbons at 156.2 (C-2) and 121.5 (C-3), respectively. All aliphatic carbon resonances of compound also appeared in 13C NMR below 100 ppm. The alcoholic function is confirmed by IR spectrum band at 3441 cm−1 and also reveals the other stretching and bending vibrations of functionalities in compound; its mass spectrum showed the molecular ion peak at m/z 377.1 (M+ Na)+, which corresponds to the molecular formula of compound. On the basis of these observations, the purified compound is proposed to be 10-(2,2-Dimethyl-cyclohexyl)-6,9-dihydroxy-4,9-dimethyldec-2-enoic acid methyl ester (**Figure 2**).

### Antifungal Activity of Compound (SH2)

**Table 1** depicts the antifungal spectrum of purified compound at concentration of 100 µg against various fungal pathogens. The results showed that it significantly inhibited the test fungi with inhibition zones in the range of 15–40 mm as against 15–32 mm zones resulted from carbendazim and mancozeb. In case of C. acutatum and Alternaria spp., the purified compound was more effective as compared to chemical control agents. In addition to antifungal activity, the purified compound also showed inhibitory activity against Candida albicans and Bacillus subtilis (data not shown).

### Antifungal Effects of Purified Compound

The effect of purified compound on spore germination and hyphal morphology was studied for A. brassicicola and F. moniliforme. Microscopic observations showed that purified compound significantly inhibited spore germination in both the fungal pathogens as compared to control (p < 0.05). Severe

fmicb-07-01004 June 29, 2016 Time: 11:19 # 6

#### TABLE 1 | Antifungal activity of purified compound SH2 and chemical control agents when used at a concentration of 100 µg.

Means followed by different letters with in a row are significantly different; Tukeys HSD, p ≤ 0.05).

morphological abnormalities such as hyphal swellings resulted in bulbous structures, thinning of hyphae, discoloration of hyphae were also observed. Additionally, purified compound also resulted in pigmentation loss in mycelial structure of A. brassicicola (**Figure 3**). MIC and MFC values of the purified compound and EA extract of streptomycete were determined by 96 well plate method and are shown in

#### TABLE 2 | MIC and MFC values of crude extract and purified compound SH2 against phytopathogenic fungi.

aND, not determined.

**Table 2**. The crude extract showed significant antifungal activity against all the tested fungi with MIC values of 50, 25, 100, 50, and 250 µg/ml for A. brassicicola, C. acutatum, A. solani, A. alternata, and F. moniliforme, respectively. The MIC and MFC values of purified compound were 25 and 50 µg/ml for A. brassicicola and were 6.25 and 25 µg/ml for C. acutatum.

### Biocontrol of A. brassicicola

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The results of in vitro as well as in vivo experiments showed ability of compound to control A. brassicicola (**Table 3**). In comparison to chemical control agents, the compound purified from S. hydrogenans strain DH16 was found to be more potent causing significant inhibition of pathogen on seeds and resulting in emergence of healthy seedlings. In in vivo experiments, no germination was observed in seeds, treated with pathogen only. Treatment of pathogen infested seeds with compound improved seed germination and seedling vigor to 71.4% and 1890, respectively, and are comparable to control. On the other hand delayed seed germination of 42 and 14.2% was observed in case of mancozeb and carbendazim treated seeds, respectively. The percentage of healthy seedlings and their fresh and dry weights were also significantly higher in case of seeds treated with compound (p ≤ 0.05).

### Stability of Compound

No loss in antifungal activity of compound was observed after its exposure to temperatures up to 70◦C. However, a decrease of 12.5 and 37.5% in the residual activity was observed after boiling (100◦C for 1 h) and autoclaving (121◦C for 30 min), respectively. The compound was also found to be photostable as only 5% loss was observed in activity against C. acutatum.

### Toxicity of Compound

To work out the biosafety of the purified compound, phytotoxicity, Ames mutagenicity and MTT cytotoxicity tests were carried out. The compound was found to be nonphytotoxic because the seedlings emerged from seeds treated with compound showed increase in all the growth traits (shoot length, root length, seedling vigor). Rather than showing any phytotoxicity, **SH2** triggered as well as enhanced seed germination as compared to water treated seeds. The emerged seedlings were also found to be healthier than the control plants as shown by higher seedling weights. Antifungal compound (**SH2**) was also non-mutagenic at all the concentrations used in the experiment. The numbers of revertant colonies in both the positive controls were numerous; whereas, the numbers colonies of bacteria in the presence of the purified compound were similar to that of spontaneous revertant colonies for TA98 and TA100 (**Table 4**). Further, the compound showed insignificant cytotoxicity, i.e., only 11.6% inhibition (or 88.8% viable cells) against CHO cell line at the highest tested concentration (**Figure 4**).

### DISCUSSION

Streptomyces species produce vast array of antifungal compounds which play important role in biocontrol of various fungal plant diseases. These bacteria constitute major portion of total antibiotics used in agricultural sector and are still great reservoirs of new antibiotics (Tanaka and Omura, 1993). This study further adds to the potential of Streptomyces spp. as unexhausted source of potent antifungal compounds which can be exploited as biofungicides for agricultural use.

TABLE 3 | Invitroandin vivoprotective effect of purified compound SH2 to control A. brassicicolaon seeds

 of R. sativus.

fmicb-07-01004 June 29, 2016 Time: 11:19 # 8

aValues are Means ± SD (n = 3) followed by different letters with in a column are significantly different; Tukeys HSD, p ≤ 0.05; bMean number of spontaneous revertants as determined in assays without SH2 and standard mutagens; cMean number of revertants induced by reference mutagens, sodium azide and 4-Nitroo-phenylenediamine (NPD); dMean number of revertants induced by DMSO in the absence of extract and mutagen; eMean number of revertants induced by SH2 at different concentrations.

Here in this work, purification of compound from the culture extracts of S. hydrogenans strain DH16 using various chromatographic techniques resulted in apparently new antifungal compound with broad spectrum activity against different phytopathogenic fungi. This compound is proposed to be 10-(2,2-Dimethyl-cyclohexyl)-6,9-dihydroxy-4,9-dimethyl-dec-2-enoic acid methyl ester on the basis of various spectroscopic techniques.

The importance of the study lies in the fact that it is the first report on production of new potential antifungal compound by this species. The purified compound (**SH2)** showed more activity against phytopathogenic fungi esp. Alternaria spp. and Colletotrichum spp. as compared to cycloheximide and chemical fungicides (carbendazim and mancozeb). Carbendazim exhibited no activity and mancozeb showed weak activity against all the tested Alternaria spp. Similarly, indole-3-carboxylic acid, another bioactive compound from Streptomyces sp. TK-VL\_333 showed better activity than that of mancozeb whereas less activity than carbendazim when tested against F. oxysporum (a wilt pathogen; Kavitha et al. (2010). However, compound **SH2** isolated in the present study was found to be superior to both the fungicides in terms of activity against Fusarium sp. also.

The low MIC and MFC values of purified compound which varied from 6.25 to 200 µg/ml depending upon the sensitivity of test fungi further demonstrated its effectiveness to control the fungal plant pathogens. It showed lowest MIC value (6.25 µg/ml) against C. acutatum and C. gloeosporioides and highest value (100 µg/ml) against F. moniliforme. In contrast, the MIC value of 3-methylcarbazole produced by Streptomyces LJK109 against C. gloeosporioides was found to be high (30 µg/ml; Taechowisan et al., 2012). Hwang et al., 2001 reported the MIC values in the range of 50 to >1000 µg/ml of compounds, phenylacetic acid, and sodium phenylacetate isolated from Streptomyces humidus

strain S5-55 which are again higher than the compound **SH2** of present study.

The biocontrol potential of purified compounds obtained from Streptomyces spp. in controlling different fungal phytopathogens and reducing their disease incidences in in planta experiments has been reported (Matsuda et al., 1998; Hwang et al., 2001; Bordoloi et al., 2002; Kavitha et al., 2010). In current study also, the in vitro and in vivo experiments showed that the compound effectively controlled the development of seed borne damping off of radish seedlings caused by A. brassicicola when used at the concentration of 1 mg/ml whereas the mancozeb showed less control efficacy against the disease at the same concentration. Carbendazim was found to be least effective. In the absence of pathogen, the seedlings emerged from compound treated seeds were found to be more healthier than the seedlings in control. Similarly, SPM5C-1 from Streptomyces sp. PM5 when applied at 500 and 250 µg/ml significantly suppressed the sheath blight disease in rice and also increased the growth parameters as compared to the control in the absence of the pathogen (Prabavathy et al., 2006).

For commercial application of biologically active compounds (to be used as biopesticides/plant growth promoting agent) in agriculture sector, it is important to determine their phytotoxicity. Cycloheximide (isolated from S. griseus), the first compound used to control fungal and bacterial diseases in plants, showed phytotoxicity. Therefore, use of cycloheximide as an agent for plant disease control is restrained because of its toxicity to the host plants (Ford et al., 1958). The application of carbendazim, a systemic fungicide showed phytotoxicity by negatively affecting the plant biomass in Nicotiana tabacum (García et al., 2003). Walia et al. (2014) demonstrated the deleterious impact of mancozeb on soil microflora, nitrification, ammonification, carbon mineralization, soil enzymes, and soil microbial biomass which in turn may result in harmful effects on nutrient uptake and plant growth. However, the antifungal compound purified in present work did not show any phytotoxicity in both in vitro and in vivo experiments. Rather, it enhanced the rate of seed germination and seedling vigor in fmicb-07-01004 June 29, 2016 Time: 11:19 # 9

radish seedlings compared to control plants and therefore can also be used for enhancing plant growth in addition to controlling the pathogens. This data further suggests the superiority of compound **SH2** over chemical fungicides both in terms of activity as well as phytotoxicity.

The ability to tolerate various factors (light, temperature, and pH) in natural environment is very crucial for any agro active compound. Therefore, for commercial application, compound should be thermostable, photostable, and pH stable. The compound obtained from S. hydrogenans strain DH16 was found to be highly thermo and photostable. Prapagdee et al. (2008) and Jayaprakashvel et al. (2010) also reported considerable thermo and photostability of antifungal compounds produced by S. hygroscopicus and Trichothecium roseum MML00l, respectively.

Further toxicity of the compound was tested by Ames mutagenicity test and in vitro cytotoxicity test. Ames test is useful in correlating in vitro bacterial mutagenesis with in vivo carcinogenicity in animals because positive Ames test indicates that the tested chemical is mutagenic and therefore may act as a carcinogen, because cancer is often linked to a mutation. The purified compound **SH2** obtained from S. hydrogenans strain DH16 was found to be potentially bio safe as it did not show any mutagenic response (at all tested concentrations) against S. typhimurium strains TA98 and TA100 in Ames mutagenicity test. The present study gets further credence as the results of MTT assay revealed **SH2** to be non-cytotoxic as 88.8% viable cells of CHO cell line were observed in its presence at the highest tested concentration.

### CONCLUSION

This study demonstrates the purification and characterization of a new heat and photo stable antifungal compound, with plant growth promoting potential, from S. hydrogenans strain DH16, showing more promising activity against a variety of fungal phytopathogens as compared to standard chemical fungicides. The non-phytotoxic, non-mutagenic, and non-cytotoxic nature of the compound suggests that it might serve as a new, safe, and broad spectrum biofungicide to combat serious plant diseases. Therefore, the compound **SH2** can be developed as a better replacement to chemical fungicides as effective plant chemotherapeutic agent.

### REFERENCES

### AUTHOR CONTRIBUTIONS

TK was involved in the planning and execution of the research work; analysis and interpretation of the data; manuscript writing following the suggestions of the research supervisor. VS analyzed, interpreted and characterized the compound on the basis of different spectroscopic techniques and drafted related content of the manuscript. AK provided fungal cultures Fusarium moniliforme, Alternaria alternata, and Alternaria mali; helped in analysing the data and editing of the manuscript. RM as research supervisor of TK was involved in the design and planning of research work; analysis and interpretation of data; drafting as well as critical editing of the manuscript for intellectual subject matter. All authors approved the final version of the manuscript for publication and agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

## FUNDING

We duly acknowledge University Grants Commission (UGC), New Delhi for providing funds to accomplish this work.

### ACKNOWLEDGMENTS

TK acknowledges the grant of fellowship under UPE (University with Potential for Excellence) scheme of University Grants Commission, New Delhi, India. We duly acknowledge Dr. Saroj Arora, Professor, Department of Botanical and Environmental Sciences for her help to determine the cytotoxicity of the compound and Shruti Chabba, Research Scholar, Department of Chemistry, Guru Nanak Dev University for help in operating NMR and MS softwares.

### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fmicb. 2016.01004

phytopathogenic fungus Alternaria spp. Chilean J. Agric. Res. 71, 231–239. doi: 10.4067/S0718-58392011000200008

griseus H7602 against Phytophthora capsici. J. Basic Microbiol. 55, 45–53. doi: 10.1002/jobm.201300820

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2016 Kaur, Kaur, Sharma and Manhas. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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# Induced Systemic Resistance against *Botrytis cinerea* by *Bacillus cereus* AR156 through a JA/ET- and *NPR1*-Dependent Signaling Pathway and Activates PAMP-Triggered Immunity in *Arabidopsis*

Pingping Nie1, 2, Xia Li 1, 2, Shune Wang1, 2, Jianhua Guo1, 2, Hongwei Zhao1, 2 \* and Dongdong Niu1, 2 \*

*1 Department of Plant Pathology, College of Plant Protection, Nanjing Agricultural University, Nanjing, China, 2 Key Laboratory of Integrated Management of Crop Diseases and Pests, Nanjing Agricultural University, Ministry of Education, Nanjing, China*

#### *Edited by:*

*Gero Benckiser, University of Giessen, Germany*

#### *Reviewed by:*

*Akanksha Singh, Central Institute of Medicinal and Aromatic Plants, India Prashant Singh, Lancaster University, UK*

*\*Correspondence:*

*Dongdong Niu<EMAIL_ADDRESS>Hongwei Zhao<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Plant Microbe Interactions, a section of the journal Frontiers in Plant Science*

*Received: 09 December 2016 Accepted: 08 February 2017 Published: 28 February 2017*

#### *Citation:*

*Nie P, Li X, Wang S, Guo J, Zhao H and Niu D (2017) Induced Systemic Resistance against Botrytis cinerea by Bacillus cereus AR156 through a JA/ET- and NPR1-Dependent Signaling Pathway and Activates PAMP-Triggered Immunity in Arabidopsis. Front. Plant Sci. 8:238. doi: 10.3389/fpls.2017.00238*

Induced resistance response is a potent and cost effective plant defense against pathogen attack. The effectiveness and underlying mechanisms of the suppressive ability by *Bacillus cereus* AR156 to *Pseudomonas syringae* pv. *tomato* DC3000 (*Pst* DC3000) in *Arabidopsis* has been investigated previously; however, the strength of induced systemic resistance (ISR) activity against *Botrytis cinerea* remains unknown. Here, we show that root-drench application of AR156 significantly reduces disease incidence through activation of ISR. This protection is accompanied with multilayered ISR defense response activated via enhanced accumulation of PR1 protein expression in a timely manner, hydrogen peroxide accumulation and callose deposition, which is significantly more intense in plants with both AR156 pretreatment and *B. cinerea* inoculation than that in plants with pathogen inoculation only. Moreover, AR156 can trigger ISR in *sid2-2* and *NahG* mutants, but not in *jar1*, *ein2* and *npr1* mutant plants. Our results indicate that AR156-induced ISR depends on JA/ET-signaling pathway and *NPR1*, but not SA. Also, AR156-treated plants are able to rapidly activate MAPK signaling and *FRK1*/*WRKY53* gene expression, both of which are involved in pathogen associated molecular pattern (PAMP)-triggered immunity (PTI). The results indicate that AR156 can induce ISR by the JA/ET-signaling pathways in an *NPR1*-dependent manner and involves multiple PTI components.

Keywords: *Bacillus cereus* AR156, induced systemic resistance, salicylic acid (SA), jasmonic acid (JA), ethylene (ET)

### INTRODUCTION

Plants are protected from pathogen attack through activation of innate immune system, which is a consequence of co-evolution between plants and their pathogens (Jones and Dangl, 2006). The emergence of pathogens is first detected by pattern recognition receptors (PRRs) that are localized on plant cell membrane. PRRs percept the conserved pathogen identification

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molecules known as pathogen-associated molecular patterns (PAMPs), which include flagellin, lipopolysaccharides, glycoproteins, or chitin (Jones and Dangl, 2006). The interaction between PRRs and PAMPs consequently triggers the socalled PAMP-triggered immunity (PTI), which initiates many immune responses, including oxidative burst, callose deposition, activation of the MAPK (mitogen-activated protein kinase) cascade, and defense-related gene expression (Altenbach and Robatzek, 2007; Schwessinger and Zipfel, 2008). During the course of evolution, some successful pathogens emerged that can successfully infect plants by suppressing PTI. This suppression is achieved by secreting virulent proteins generically termed effectors, which causes effector-triggered susceptibility (ETS) consequently (Speth et al., 2007). In response to effectors, some plants have evolved resistance (R) proteins, which can recognize effectors directly or indirectly, and elicit effectortriggered immunity (ETI). ETI usually is accompanied with a hypersensitive response (HR) at the infection site, which is thought to restrict biotrophic pathogen growth (Chisholm et al., 2006; Jones and Dangl, 2006).

Beyond defense response against an intermediate infection, resistance can be induced by a temporarily prior infection that is effective for a certain period of time, and to a broad spectrum of pathogens temporarily after (Fu and Dong, 2013). The induced defense responses can be activated by pathogen infection, microbial symbiosis, or other elicitation, such as wounding. There are two types of induced resistances that are phenotypically hard-to-distinguished: the induced systemic resistance (ISR) and systemic acquired resistance (SAR). ISR is a systemic resistance induced by some non-pathogenic rhizobacteria that can suppress disease in plants (van Loon et al., 1998). In contrast, SAR is an induced resistance that develops in whole plants in response to a temporally earlier local exposure to a pathogen. In both SAR and ISR, phytohormone signaling pathways, such as salicylic acid (SA), jasmonic acid (JA), and ethylene (ET) are found to play crucial regulatory roles (Glazebrook, 2001).

Induced systemic resistance (ISR) has been reported in many plant species, such as rice, bean, carnation, cucumber, radish, tobacco, tomato, and Arabidopsis, which is effective against a broad spectrum of plant pathogens, ranging from fungi, bacteria, to viruses, and even to insect herbivores (Van der Ent et al., 2008; Pieterse et al., 2014). ISR requires JA and ET signaling pathways and is associated with the expression of the gene encoding plant defensin 1.2 (PDF1.2) (Van Oosten et al., 2008). For example, the rhizobacterial strain Pseudomonas fluorescens WCS417r has been shown to trigger ISR in several plant species and in Arabidopsis, where it functions through JA/ET signaling pathways and in a NPR1-dependent manner (Pieterse et al., 2002). However, dependence on both SA- and JA/ET-signaling pathways is also observed. For example, we previously reported that the ISR mediated by the rhizobacterium Bacillus cereus strain AR156 requires both the SA and JA/ET signaling pathways and NPR1 (Niu et al., 2011). Also, colonization of Arabidopsisroots by Trichoderma atroviride IMI 206040 induces the expression of SA and JA/ET pathways simultaneously to confer resistance against hemibiotrophic and necrotrophic phytopathogens (Salas-Marina et al., 2011).

In most cases, ISR is associated with a potentiated defensive capacity, which is termed "priming". Priming does not cause a direct induction of resistance-related genes, or enhance the production of phytohormones or hormone-responsive genes in systemic tissues. Instead, ISR enhances the sensitivity to hormones rather than their synthesis. Therefore, priming is costeffective in increasing plant resistance and is more efficient in activating defense mechanisms upon pathogen attack (Conrath et al., 2006; Pastor et al., 2013). Beneficial rhizobacteria trigger ISR by priming the plant for potentiated activation of varieties of cellular defense responses, such as oxidative burst (Ahn et al., 2007), cell-wall reinforcement (Heil and Bostock, 2002), defense-related enzymes accumulation (Rahman et al., 2014), and secondary metabolites production (Yedidia et al., 2003).

In our previous study, the beneficial bacterium B. cereus AR156 was demonstrated to trigger ISR in Arabidopsis through both the SA- and JA/ET- signaling pathways, which lead to enhanced resistance to bacterial infection (Niu et al., 2011). As an effort to further dissect the mechanism of AR156-mediated ISR, we are prompted to explore more protective potential of this bacterium. Botrytis cinerea, a necrotrophic fungus causing gray mold disease, is considered an important pathogen around the world. In this study, we show that B. cereus AR156 treatment inhibits B. cinerea infection in Arabidopsis through activation of ISR. The potency of induced protection is lost in jar1, ein2 and npr1 mutant but unaffected in sid2-2 and NahG plants, implicating the JA/ET signaling pathway and NPR1 are required but the SA signaling pathway is dispensable for AR156-induced ISR to B. cinerea infection. We also show that the primed defense in AR156-treated plants is mediated by enhanced activation of multiple PTI defense responses.

## MATERIALS AND METHODS

### Plants and Growth Conditions

Arabidopsis thaliana plants were maintained at 22◦C with a 12-h light/12-h dark photoperiod. Arabidopsis thaliana ecotype Col-0 and mutants were cultivated in vermiculite. All plants were used for experiments when they were 4 weeks old. The mutants used in this study (sid2-2, NahG, jar1, ein2, npr1) were described elsewhere (Staswick et al., 1992; Bowling et al., 1994; Delaney, 1994; Alonso et al., 1999).

### AR156 Treatment, Pathogen Inoculation, and Disease Assays

Bacillus cereus AR156 was grown on Luria-Bertani (LB) agar plates at 28◦C for 24 h. Subsequently, bacterial cells were pelleted by centrifugation and were resuspended in sterile 0.85% NaCl with a final concentration of 5 × 108 CFU/ml. For a protection assay, AR156 or corresponding mock (0.85% NaCl) was root-drench applied 7 days prior infection. For fungal infection experiments, 4-weeks-old plants were used. Botrytis cinerea strain B1301 was cultivated on PSA agar medium for 7 days. Spores were collected in B. cinerea infection buffer to prepare the inoculum and adjusted to a final concentration of 1 × 106 spores/ml. Inoculation was carried out by depositing a 10 ul droplet on each side of the midvein. Ten inoculated

plants for each genotype were placed in plant growth room maintained at a high humidity. For each time point, at least three biological replicates were analyzed. In planta fungal growth was examined by analyzing the transcript levels of B. cinerea actin gene (BcActin) using primer BcActin-1F (5′ -TCC AAG CGT GGT ATT CTT ACC C-3′ ) and BcActin-1R (5′ - TGG TGC TAC ACG AAG TTC GTT G-3′ ). The Arabidopsis actin gene (AtActin2) amplified by primer AtActin2-1F (5′ -GGC GAT GAA GCT CAA TCC AAA CG-3′ ) and AtActin2-1R (5′ -GGT CAC GAC CAG CAA GAT CAA GAC G-3′ ) was used as an internal control.

### RNA Extraction and qRT-PCR Analysis of Gene Expression

Total RNA was extracted from Arabidopsis leaves with TRIzol Reagent (Invitrogen, San Diego, CA, U.S.A). In brief, 1 ug total RNA was used for cDNA synthesis by using a commercial reverse transcription system (TaKaRa Biotech, Dalian, China). After the cDNA was diluted 10 times, 2 ul diluted cDNA was used for realtime quantitative PCR with the following program: 40 cycles at 95◦C for 30 s, 55◦C for 30 s, and 72◦C for 34 s. Three replications were performed for each sample. The data were normalized with AtActin, and the means of three replications were presented. Primers used in qRT-PCR were listed in Table S1.

### Examination of Hydrogen Peroxide Accumulation and Callose Deposition

Hydrogen peroxide accumulation and callose deposition examination was performed according to previously described procedures (Niu et al., 2011). Briefly, for accumulation of ROS, Arabidopsis leaves from at least three different plants were stained with DAB solution (1 mg of diaminobenzidine per milliliter, pH 3.8) for 8 h dark at 25 to 28◦C. After being cleared with 96 % (vol/vol) ethanol and preserved in 50% (vol/vol) ethanol, hydrogen peroxide was visualized as dark-brown precipitate under the light microscope. For callose deposition, Arabidopsis leaves were immerged in 5 ml of destaining solution (Acetic acid/ethanol = 5:95) (vol/vol) and were infiltrated by applying a vacuum for 5 to 10 min. Leaves were incubated in a 60◦C water bath for 20 to 30 min to clear chlorophyll. The chlorophyll-free leaves were gently rinsed with water and were then soaked in 3 to 5 ml of 0.01% (wt/vol) aniline blue staining solution containing 150 mM K2HPO4 (pH 9.5) kept in dark for 2 to 4 h. After staining, Arabidopsis leaves were gently rinsed with water and were then mounted on microscope slides that were observed under an epifluorescence microscope with a UV excitation filter. Levels of callose deposition were quantified using Imge J software and expressed relative to total leaf area as described (Luna et al., 2011).

### Protein Extraction and Analysis

Plant tissue was ground in liquid nitrogen and total proteins were extracted using 2 × SDS loading buffer. The samples were resolved on SDS–PAGE gels and transferred onto nitrocellulose membranes. The blots were probed with appropriate antibodies: monoclonal mouse anti-α tubulin (1:4,000 dilution); polyclonal rabbit anti-PR1 (1:2,000 dilution). For MAPK activity assay, sample were analyzed by Western blotting using monoclonal rabbit phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) XP antibodies (Cell Signaling Technology, #4370S, 1:2,000 dilution). For these assays, α-tubulin was used as a loading control.

## RESULTS

### *B. cereus* AR156 Induces an Effective ISR against *Botrytis cinerea* Infection

We previously found in Arabidopsis that B. cereus AR156 could prime the whole plant for an induced resistance to Pst DC3000 infection. In this study, we designed experiments to test whether AR156 is able to induce ISR against B. cinerea as well. Arabidopsis was first pretreated with AR156 or mock (0.85% NaCl) for 7 days in a root-drench application manner, followed by B. cinerea infection. Effect of AR156 on ISR was examined by the plant performance against pathogen infection. Two days after pathogen inoculation, mock-treated plants showed typical symptoms of B. cinerea disease-severe necrosis around inoculating loci, leaves yellowing, and watersoaked spots surrounded by the spores (**Figure 1A**). In contrast, plants with AR156-pretreatment exhibited a significant (P < 0.01) reduction on disease symptoms, manifested by smaller necrosis size, less yellowing, compared with mock-treated plants (**Figure 1B**). As an indicator of the effectiveness of elimination of pathogen infection, fungal hyphae were measured 2 days after inoculation (dpi) by qRT-PCR. In agreement with the reduced disease symptom observed on leaf surface, significantly reduced fungal hyphae were detected inside leaf tissue from AR156 pretreated plants than that with control pre-treatment, indicating AR156 effectively protected Arabidopsis from B. cinerea infection (**Figure 1C**).

### AR156 Induces ISR by Potentiating PR1 Expression, Hydrogen Peroxide Accumulation, and Callose Deposition in Arabidopsis

Establishment of ISR is usually accompanied with potentiated activation of various cellular defense responses against pathogen infection, which was called priming (Conrath et al., 2001). To investigate whether AR156-induced ISR was accompanied with primed defense responses in systemic leaves, we measured the expression level of defense-related protein PR1. With AR156 or control pre-treatment alone, PR1 expression was almost negligible prior pathogen infection, indicating AR156 by itself in not capable of inducing PR1 expression (**Figure 2A**). At 12 hpi, elevated PR1 expression was detected in B. cinerea-infected leaves, indicating the Arabidopsis innate immunity system recognizes and responds rapidly against B. cinerea infection. Moreover, plants with AR156 pre-treatment accumulated much higher PR1 than the control treated plants did. These results indicate that in plants with AR156 pretreatment, the innate immunity system is at a potentiated status so that plants could launch a much accelerated defense response upon infection (**Figure 2A**).

was determined by ratios of *BcActin/AtActin*. A Student's *t*-test was used to determine significant differences between the AR156-treated sample and the control (\*\**P* < 0.01). The means values ± *SD* (*n* = 12) from one representative experiment among three independent repeats are shown.

To investigate the molecular mechanism of AR156-mediated ISR, H2O2 accumulation and callose deposition pattern were examined in plants inoculated with B. cinerea, with or without AR156 pretreatment. H2O2 accumulation and callose deposition are two rapid responses elicited by pathogen infection, intensity and rapidity of which are hallmarks of successful immune response. AR156 neither induce the production of H2O2 at 12 hpi, nor 24 hpi, indicating that AR156 itself is not an elicitor of plant defense (**Figure 2B**). At 12 hpi, B. cinerea infection alone did not induce measurable H2O2 accumulation, indicating at this stage of infection, plants were not able to deploy an effective defense response yet, in term of H2O2 accumulation. However, in plants pretreated with AR156, detectable H2O2 level was observed, indicating AR156 pretreatment potentiated plants for an expiated immune response (**Figure 2B**). At 24 hpi, detectable H2O2 level was observed in plants inoculated with B. cinerea, indicating an initiation of H2O2-mediated defense response later than 12 hpi but before 24 hpi. In accordance with observation made at 12 hpi, AR156 pretreatment increased the extent of H2O2 accumulation upon B. cinerea infection (**Figure 2B**). We also compared callose deposition between plants with and without AR156 pretreatment (**Figures 2C,D**). Similar to H2O2 accumulation, AR156 alone did not induced any detectable callose deposition. With B. cinerea infection, at 12 hpi, callose deposition was detectable, indicating plants were able to perceive the pathogen infection and response efficiently. Moreover, in plants pretreated with AR156, callose deposition was noticeably enhanced, indicating a potentiated defense response due to AR156 pretreatment. In agreement, callose deposition at 24 hpi exhibited similar pattern. Taken together, our results indicate that AR156 primes plants for accelerated and enhanced immune capacity, which is induced only upon pathogen attack and leads to rapidly activated cellular defense responses in systemic tissue.

### AR156-Mediated ISR Is Dependent of the JA/ET Signaling Pathway and *NPR1*

To further elucidate the molecular mechanisms responsible for AR156-triggered ISR, plants defective in different hormone signaling pathways were analyzed. As indicated in **Figure 3A**, 2 days after pathogen infection, B. cinerea caused visible disease symptom on both wild type (Col-0) and sid2-2, NahG, jar1, ein2, npr1 mutant plants, indicating none of the mutant plants exhibited an automatic and effective immune response against B. cinerea infection (**Figure 3A**, control set). Pretreatment with AR156 led to a reduction in disease symptom on Col-0, sid2- 2 and NahG plants, indicting protection mediated by AR156 is still functional in these mutants as effective as in corresponding

deposition were observed under light and epifluorescence microscopes with a UV excitation filter. Relative callose quantities in droplet inoculated leaves of 4-week-old plants. Callose was quantified from digital microscopy photographs (D). Shown are mean areas of callose per leaf relative to total leaf area ± *SD* (*n* = 24). A Student's *t*-test was used to determine significant differences between the AR156-treated sample and the control (\*\**P* < 0.01). Similar results were obtained in three independent repeats.

wild type plants. In contrast, no discernable difference could be observed between the control- and AR156-treated plants on the jar1, ein2 and npr1 mutant plants, indicating the AR156-mediated protection was jeopardized in these mutant plants (**Figure 3A**, AR156 set). In consistence, plants with AR156 pretreatment exhibited a significant (P < 0.01) reduction in necrosis size on Col-0, sid2-2 and NahG plants, but not the jar1, ein2 and npr1 mutant plants, when compared with controltreated plants (**Figure 3B**). Fungal growth in each signaling mutant plants were also examined at 2 dpi. In consistence with the disease symptom observed on leaf surface, dramatically reduced fungal growth was detected in Col-0, sid2-2 and NahG plants. In contrast, this protection was abolished in jar1, ein2 and npr1 mutant plants (**Figure 3C**). These results suggested that AR156-mediated ISR functions by activating the JA/ET signaling pathway and is dependent on NPR1.

It is known that plant innate immunity is modulated by phytohormone signaling networks. To investigate the signaling pathways employed by AR156-mediated ISR, we inspected the transcription of signaling pathway reporter genes, such as PR1, PR2, PR5, and PDF1.2 by RT-PCR. Transcriptions of PR1, PR2, PR5, and PDF1.2 were strongly induced at 48 hpi in Col-0, sid2-2 and NahG mutant plants pretreated with AR156 and inoculated with B. cinerea. Impressively, transcriptions of all these genes were not responsive to B. cinerea infection in the jar1, ein2 and npr1 mutant plants, perfectly conforming to the defective ISR mediated by AR156 (**Figure 4A**). Furthermore, cellular defense responses activated by AR156-mediated ISR was investigated in the above-mentioned five signaling mutant and their corresponding wild type Arabidopsis plants. H2O2 accumulation and callose deposition were detectable at 12 hpi in Col-0, sid2-2 and NahG plants with AR156 pre-treatment, but not detectable (or to a much lesser degree) in jar1, ein2 and npr1 mutant at the same time point. At 24 hpi, a combination of AR156 pretreatment and B. cinerea inoculation led to more H2O2 accumulation and callose deposition in the leaves of Col-0, sid2-2 and NahG plants, compared with the control-pretreatment group. At the same time point, H2O2 accumulation and callose deposition were very weak in jar1, ein2 and npr1 plants in both control- and AR156-treated plants (**Figures 4B–D**).

### AR156-Induced ISR Activates PTI Components

PAMP-triggered immunity (PTI) plays an important role for plant immunity against B. cinerea infection, in which activation of MAPKs is one of the earliest sign of PTI. Therefore, activation of MAPKs could be used as a good indicator for activated PTI, as well as defense responses (Eckardt, 2011; Meng and Zhang, 2013; Singh et al., 2013). To further investigate the relationship between AR156-induced ISR and PTI, antibodies specifically recognize MPK3 and MPK6 were used. In plants with control pretreatment, expression of MPK3 and MPK6 were detected 10 min after B. cinerea inoculation, which was then gradually decrease through 30 to 60 mpi. In contrast, in plants pretreated with AR156, we observed a sustained and gradually increased activation of MPK3 and MPK6 from 10 to 60 min after B. cinerea inoculation, which attained its maximums at 60 min (**Figure 5A**). Our results suggest that the AR156-mediated ISR is associated with an activated and enduring PTI response. MPK3 and MPK6 activation was also examined in sid2-2 and NahG mutant plants pretreated with AR156. Interestingly, sustained activation of MPK3 and MPK6 from 10 to 60 min was also detected in spite of the defect on SID2

(**Figure 5B**) or over-expression of NahG (**Figure 5C**). Our results indicate that MPK3 and MPK6 induction is SA-dependent. This may explain why AR156-mediated ISR is SA-independent.

To further support our hypothesis, we examined other PTI marker gene, such as the flg22-induced receptor-like kinase 1 (FRK1) and WRKY53 (Asai et al., 2002; Singh et al., 2012). qRT-PCR analysis showed that FRK1 and WRKY53 were activated and remained active by B. cinerea alone from 10 to 60 min. However, in AR156-pretreated plants, both the expression level was significantly increased after B. cinerea infection, when compared to the control-pretreated plants (**Figure 5D**). These results confirmed that there is a close association between AR156 mediated ISR and a rapid and sustained activation of PTI.

### DISCUSSION

Induced systemic resistance (ISR) has been recognized as an effective biological control agent that could induce plant defense against a broad range of pathogens. Our precious studies demonstrated that B. cereus AR156 is a plant growth–promoting rhizobacterium that can induce resistance against Pst DC3000 on Arabidopsis and tomato (Niu et al., 2011, 2012). In particular, our study in Arabidopsis showed that the AR156-mediated ISR against the biotrophic pathogen Pst DC3000 is dependent on SA and NPR1, but not the ET/JA signaling pathway. In the current study, we demonstrated that the AR156-mediated ISR is also effective against the necrotrophic pathogen B. cinerea, and this protection is mediated by the ET/JA-, but not the SA-signaling pathway. However, we did identified NPR1 as an indispensable component of this specific ISR, despite the fact that other SA signaling pathway components were not involved. Therefore, our results indicate that the AR156-mediated ISR is effective against both bio- and necro-trophic pathogens, which make it a good candidate for broad-spectrum biological control agent. Meanwhile, our results also indicate that NPR1 could potentially function independent of the SA signaling pathway.

In recent years, many studies have associated PGPRs with improving plant health by enhancing defense against a broad range of pathogens (Pieterse et al., 2014; Rahman et al., 2014; Ma et al., 2016). B. cereus AR156 is a plant growth–promoting rhizobacterium that induces resistance against Pst DC3000 on Arabidopsis and tomato (Niu et al., 2011, 2012). The aim in this study was to explore the potential role of AR156 in eliciting ISR in Arabidopsis against B. cinerea infection. Roots application

#### FIGURE 4 | Continued

value of *AtActin*, which is assigned to 1. Data are presented as the means ± *SD* from three independent experiments and different letters above the columns represent statistically significant differences (*p* < 0.01) between Col-0, *sid2-2*, *NahG*, *jar1*, *ein2* and *npr1* mutant plants. *In situ* detection of accumulation of H2O2 (B) and callose deposition (C, D) after inoculation with *B. cinerea*. Accumulation of H2O2 and callose deposition in leaves were detected by DAB staining and aniline blue staining, respectively. Relative callose quantities in droplet inoculated leaves of 4-week-old plants. Callose was quantified from digital microscopy photographs. Shown are mean areas of callose per leaf relative to total leaf area ± *SD* (*n* = 24). A Student's *t*-test was used to determine significant differences between the AR156-treated sample and the control (\*\**P* < 0.01).

Frontiers in Plant Science | www.frontiersin.org February 2017 | Volume 8 | Article 238

the AR156-treated sample and the control (\*\**P* < 0.01).

as an internal control. The means values ± *SD* from three independent repeats are shown. A Student's *t*-test was used to determine significant differences between

FIGURE 6 | A proposed model of AR156-mediated ISR against biotrophic and necrotrophic pathogens. When plants are pretreated with AR156, an ISR omnipotent to pathogens with both biotrophic and necrotrophic life styles is induced. When plants in a potentiated immune status is infected by a biotrophic pathogen, such as *Pst* DC3000, both SA and JA/ET signaling pathways are activated. Through a mechanism dependent on *NPR1*, downstream defense-related genes, such as *PR1*, *PR2*, *PR5*, and *PDF1.2* are expressed, and cellular defense responses, such as H2O2 accumulation, callose deposition are activated; when plants are challenged with necrotrophic pathogens, such as *B. cinerea*, only the JA/ET signaling pathways is activated. The necrotrophic-effective ISR is also dependent on *NPR1.* Dashed lings: protective function to other unidentified elicitors.

of AR156 significantly reduced necrosis diameter and inhibited fungal growth on the leaves of Arabidopsis plants (**Figure 1**). Since AR156 only colonized the roots but B. cinerea was a foliar applied, the lack of direct contact between these two parties indicates that it is ISR, instead of a direct limitation of pathogen, leads to the observed resistance to B. cinerea infection.

Under primed condition, the induction of defense-related PR genes following pathogen challenge has been reported in several plant-pathogen interaction (Ahn et al., 2007; Niu et al., 2011). This is also true in ISR against several hemibiotrophic and necrotrophic pathogens, such as the Harpophora oryzae-primed defense genes in the rice–Magnaporthe. oryzae interaction (Su et al., 2013) and Bacillus subtilis-induced PR genes in tomato challenged with Erwinia carotovora subsp. carotovora (Chandrasekaran and Chun, 2016). In this study, we found that PR1 protein expression was stronger in plants with AR156 pretreatment and B. cinerea infection than that in plants with pathogen infection only (**Figure 2A**). This is in consistence with reported results (Su et al., 2013; Chandrasekaran and Chun, 2016) and suggests that the induced PR expression level also contributes to defense against hemibiotrophic and necrotrophic pathogens. Rapid production of cellular defense responses in plant cells, such as quick H2O2 accumulation and callose deposition (Conrath et al., 2002), has been recognized as hallmark events induced by ISR-triggering bacteria (Ahn et al., 2007; Rahman et al., 2014). H2O2 and callose accumulation play important roles in plants response to B. cinerea infection (Mengiste, 2012; Schwessinger and Ronald, 2012). We found that AR156-pretreated plants accumulated higher H2O2 and callose levels than control-treated plants following B. cinerea infection (**Figures 2B–D**), suggesting that AR156 primes plants for accelerated and enhanced disease resistance capacity by activating cellular defense responses in systemic tissue.

Therefore, it interesting to seek how the defense signal is transmitted to remote tissues where the pathogen threaten has not reach yet. We investigated the involvement of SA and JA/ET signaling pathway components, which were previously demonstrated to be involved in the AR156-induced defense responses (Niu et al., 2011, 2012, 2016). AR156-induced ISR was abolished in jar1 and ein2 mutants, suggesting that the defense response is induced by AR156 through JA/ET-signaling pathways. This observation is in consistence with that the JA/ET-signaling pathways are more effective against infections by necrotrophic pathogens. Components involved in the SA signaling pathway were also examined. In our study, both sid2- 2 and NahG plants showed comparable disease symptoms to wild type plants, and similar necrosis diameter and fungal growth in plants pretreated with AR156. SID2 is one of the genes involved in SA synthesis, defect of which leads to reduced cellular SA accumulation. NahG plants are transgenic Arabidopsis expressing an SA hydroxylase (NahG) that degrades SA to catechol. Unaffected ISR in these two independent SA defective plants indicates that the AR156-mediated ISR against B. cinerea is independent of cellular SA level, and pretty much neither of the SA signaling pathway. ISR in NahG transgenic plants and defective of ISR by jar1, ein2 and npr1 have been reported in Arabidopsis previously (Pieterse et al., 1996; Ryu et al., 2003). However, there is a difference between the previous findings and ours that in their experiment, rhizobacterium treatment was not associated with induction of PR gene (van Wees et al., 1999; Verhagen et al., 2004), whereas augmented PR1 protein level was observed in Arabidopsis pretreated with AR156 and then infected with B. cinerea (**Figure 2A**).

Interestingly, we previously demonstrated that in AR156 mediated ISR against the biotrophic Pst DC3000, both the SAand JA/ET-signaling pathways were simultaneously activated (Niu et al., 2011). In contrast, when we analyze the AR156 mediated ISR against the necrotrophic B. cinerea in this study, we concluded that this type of ISR was accompanied by the activation of the JA/ET-signaling pathways, but not the SA signaling pathway. It is generally accepted that biotrophic pathogens, which acquire nutrient supply from live host cells, are more vulnerable to defense through SA-signaling pathway; whereas necrotrophic pathogens, which benefit from host cell death, are better restrained by a JA/ET-dependent defense (Grant and Lamb, 2006). So we speculated that the favorable signal transduction pathway promoted during ISR not only depends on the ISR-inducing strains and the host plants (Pieterse et al., 2002; Choudhary and Johri, 2009; Shoresh et al., 2010), but also on the pathogens the ISR apply to.

Intriguingly, AR156-induced ISR was noticeably jeopardized in the npr1 mutant, the gene of which encodes a redox-sensitive transcriptional regulator of SA-dependent responses. NPR1 also is a mediator of SA-JA cross talk, and a regulator of SAR and ISR (Pieterse et al., 2014). Upon activation by SA, NPR1 acts as a transcriptional coactivator of a large set of PR genes as we observed in our study. This clearly laid a discrepancy between the induction of SA-dependent PRs and the independency on SA synthesis and cellular content, which may suggest a SA-unrelated function of NPR1. Indeed, NPR1 was shown to be required for the SA-independent but JA/ET-dependent ISR triggered by P. fluorescens WCS417r (Pieterse et al., 1998). More and more evidence point to a cytosolic function of NPR1 in JA/ET signaling and ISR (Spoel et al., 2003; Ramirez et al., 2010; Pieterse et al., 2012). It is worthy to note that NPR1 are highly expressed in Arabidopsis roots (Iyer-Pascuzzi et al., 2011), which may imply a potential role in regulating root-associated immune responses including ISR.

Our results also showed that, in AR156-primed Arabidopsis, pathogen infection triggered expression of defense-related genes, and enhanced hydrogen peroxide accumulation and callose deposition (**Figure 2**). However, we found that induced expression of PR1, PR2 and PR5 was also observed in sid2-2 and NahG mutants, which are defective for SA accumulation. This is a surprise to us because PR1, PR2, and PR5 were generally recognized as markers of salicylic acid-dependent disease responses, which should be non-responsive in SAdeficient mutants (Vlot et al., 2009). Our results suggest that in the AR156-mediated ISR against B. cinerea, PR1, PR2 and PR5 were induced through a SA-independent signaling pathway. This is supported by a prior study in which constant MPK3 and/or MPK6 activation causes PR1 induction independent of SA (Tsuda et al., 2013). Taken together, PRs gene activation in sid2-2 and NahG by AR156 pretreatment and B. cinerea infection could be a consequence of activated JA/ET signal pathways and induced MAPKs cascade.

Previous studies already indicate the importance of MAPK signaling in plant defense against infections (Asai et al., 2002; Zipfel et al., 2004). In this study, MAPK activation was detected at 10 min and decreased at 60 min in the leaves of plants only inoculated with B. cinerea, but this induction initiated at about the same time but remained very strong at 60 min in those treated with AR156 and inoculated with B. cinerea (**Figure 4A**). This indicated that AR156-pretreatment induced stronger MAPK activation than plants without pretreatment. MPK3 and MPK6 are positive regulators of plant defense responses controlling ET (Tena et al., 2011; Meng and Zhang, 2013) and JA biosynthesis (Schweighofer and Meskiene, 2008). MPK3 and MPK6 are essential for plant defense against B. cinerea (Ren et al., 2008; Han et al., 2010; Galletti et al., 2011; Mendez-Bravo et al., 2011). This is consistent with our finding that AR156-induced ISR against B. cinerea is mediated by JA/ET-signaling pathways. qRT-PCR analysis of the MAMP-specific early-defense marker genes, such as FRK1 and WRKY53 showed that MAMP-mediated defense responses occur rapidly after treatment with AR156 and inoculation with pathogen (**Figure 5D**), implying that AR156 induces SAR through the activation PTI response.

In our previous study, the AR156-mediated ISR could efficiently protect plants against infections by biotrophic pathogen, such as Pst DC3000. This ISR simultaneously activate the SA- and the JA/ET-dependent signaling pathways, as evident by the induced expression of PR1, PR2, PR5, and PDF1.2 (Niu et al., 2011). In this study, we demonstrated that AR156 was also effective in protecting plant from infection by necrotrophic pathogens, such as B. cinerea. However, in this case, the JA/ET signaling pathways but not the SA signaling pathway is involved. In both cases the induced ISR is associated with similar defense responses, such as activated cellular defense responses, such as H2O2 accumulation, callose deposition, and expression of some defense related genes. Therefore, we propose a model that AR156-mediated ISR is effective against infection by pathogens with different life cycles. In this model, when plants are pretreated with AR156, ISR is activated by equipping plants with a potentiated immune status that is omnipotent to pathogens with both biotrophic and necrotrophic life styles. When plants in potentiated status are infected by a biotrophic pathogen, such as Pst DC3000, both SA and JA/ET signaling pathways are activated. Through a mechanism dependent on NPR1, downstream defense-related genes, such as PR1, PR2, PR5, and PDF1.2 are expressed, and cellular defense responses, such as H2O2 accumulation, callose deposition are activated; when plants are challenged with necrotrophic pathogens, such as B. cinerea, only the JA/ET signaling pathways is activated. Once again through a NPR1-dependent mechanism, downstream defense responses are activated, leading to increase resistance (**Figure 6**). However, whether the role of NPR1 is conserved between the SA and JA/ET signaling pathways is unclear to us. Further study is needed to clarify the versatile function of NPR1 in AR156-mediated ISR, which is of great significance in promoting the application of AR156 in crops protection.

### AUTHOR CONTRIBUTIONS

DN and HZ designed the study. PN, XL, and SW performed the experiments. All authors analyzed the data. DN and HZ wrote the manuscript. All authors contributed to the research and approved the final version of the manuscript.

### ACKNOWLEDGMENTS

We thank Profs. Zhou J.M. (National Institute of Biological Sciences, Beijing) and Qi Y.J. (Tsinghua University, Beijing) for kindly providing us with the seeds of Arabidopsis mutants, Prof. Dong H.S. (Nanjing Agricultural University, Nanjing) for Arabidopsis transgenic line NahG, Prof. Wei L.H (Jiangsu Academy of Agricultural Sciences, Nanjing) for Botrytis cinerea strain B1301. This work was supported by the National Natural Science Foundation of China (31501621) to Niu N. and a Fundamental Research Funds for the Central Universities (KYTZ201403) and a PhD Programs Foundation of Ministry of Education of China (B0201300664) to HZ.

### REFERENCES

### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fpls.2017. 00238/full#supplementary-material

septate endophyte Harpophora oryzae to rice blast disease. PLoS ONE 8:e61332. doi: 10.1371/journal.pone.0061332

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2017 Nie, Li, Wang, Guo, Zhao and Niu. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Thyme Oil Reduces Biofilm Formation and Impairs Virulence of Xanthomonas oryzae

Akanksha Singh1 , Rupali Gupta1 , Sudeep Tandon2 and Rakesh Pandey1 \*

1 Department of Microbial Technology and Nematology, Central Institute of Medicinal and Aromatic Plants, Council of Scientific and Industrial Research, Lucknow, India, 2 Chemical Processing Department, Central Institute of Medicinal and Aromatic Plants, Council of Scientific and Industrial Research, Lucknow, India

#### Edited by:

Gero Benckiser, Justus-Liebig-Universität Gießen, Germany

#### Reviewed by:

Adam Schikora, Julius Kühn-Institut, Germany Giovanni Di Bonaventura, Università degli Studi "G. d'Annunzio" Chieti-Pescara, Italy

> \*Correspondence: Rakesh Pandey<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Plant Microbe Interactions, a section of the journal Frontiers in Microbiology

Received: 15 November 2016 Accepted: 29 May 2017 Published: 13 June 2017

#### Citation:

Singh A, Gupta R, Tandon S and Pandey R (2017) Thyme Oil Reduces Biofilm Formation and Impairs Virulence of Xanthomonas oryzae. Front. Microbiol. 8:1074. doi: 10.3389/fmicb.2017.01074 Xanthomonas oryzae pv. oryzae (Xoo), a common bacterial plant pathogen regulates its virulence and biofilm formation attribute via a chemical method of communication. Disabling this mechanism offers a promising alternative to reduce the virulence and pathogencity of the microorganism. In this study, the effect of thyme (THY) oil on Quorum Sensing mediated synthesis of various virulence factors and biofilm formation was analyzed. Treatment of Xoo with 500 ppm THY oil displayed a significant diminution in swimming, swarming, exopolysaccharide and xanthomonadin secretion. However, no effect was observed on bacterial growth kinetics and metabolic activity of the cells. Results were further authenticated by RT-qPCR as significant reduction in motA, motB, and flgE genes was observed upon THY oil treatment. Similarly, the expression of some extracellular enzyme genes such as endoglucanase, xylanase, cellobiosidase, and polygalacturonase was also found to be significantly reduced. However, biochemical plate assays revealed insignificant effect of 500 ppm THY oil on secretion of protease, cellulase, and lipase enzymes. The rpfF gene known to play a crucial role in the virulence of the phytopathogenic bacteria was also significantly reduced in the THY oil treated Xoo cells. HPTLC analysis further revealed significant reduction in DSF and BDSF signaling molecules when Xoo cells were treated with 500 ppm THY oil. Disease reduction was observed in in vitro agar plate assay as lesion length was reduced in THY oil treated Xoo cells when compared with the alone treatment. GC–MS result revealed thymol as the active and major component of THY oil which showed potential binding with rpfF gene. Application of 75 µM thymol resulted in downregulation of gumC, motA, estA, virulence acvB and pglA along with rpfF. The other genes such as cheD, flgA, cheY, and pilA, were not found to be significantly affected. Overall, the results clearly indicated THY oil and its active component Thymol to be a potential candidate for the development of anti-virulence agent which in future when applied in combination with conventional bactericides might not only help in lowering the dose of bactericides but also be successful in curbing the disease progression in rice.

Keywords: biofilm, exopolysaccharide, thyme oil, virulence factors, quorum sensing

## INTRODUCTION

fmicb-08-01074 June 9, 2017 Time: 15:47 # 2

Xanthomonas oryzae pv. oryzae (Xoo), a Gram-negative bacterium, causes bacterial blight, one of the most disastrous diseases of rice know to cause an annual yield losses up to 60% (Niño-Liu et al., 2006). It enters in plant tissue either through wounds or hydathodes and travels to the xylem vessels where it actively multiplies resulting in blight disease on rice leaves. Alternatives for disease control and management strategies are very limited and mainly dependent on the resistant cultivars and usage of antibiotics and chemicals (Mansfield et al., 2012). Given that there are limited effective antibacterial compounds for controlling Xoo, search to develop cost effective and novel strategies that have minimal environmental impact is the need of hour. One such new wrinkle is the use of anti-virulence or anti-quorum sensing (QS) agents that do not kill the bacteria but restrains the production of disease triggering virulence factors (Tay and Yew, 2013).

Like other bacterial genera production of an array of virulence factors such as extracellular polysaccharides (EPS) and various enzymes is under the control of cell-to-cell conversation system (QS). Xoo is known to produce signaling molecules by an intricate regulatory procedure relying on the concentration of QS molecules (Karatan and Watnick, 2009). The process is customarily engaged in governing genes associated with biofilm maturation, motility, competence, and virulence factors production (Xu et al., 2006; Hegde et al., 2009). QS is reported to exist in different bacterial species, with an assortment of diverse signaling molecules like N-acylhomoserine lactones (AHLs), diffusible signal factors (DSF) family signals, oligopeptides and autoinducers-2 (AI-2) (Chen et al., 2002; Barrios et al., 2006; Boon et al., 2008; Deng et al., 2010; Monnet and Gardan, 2015).

Among the different signaling molecules, DSF family signals produced by an array of bacterial pathogens such as Xylella fastidiosa, X. campestris pv. campestris (Xcc), Stenotrophomonas maltophilia including Xoo (Deng et al., 2014; Ryan et al., 2015) permit sensing their fellow natives and thereby coordinate individual task to form resistant biofilm. Key players involved in Xoo regulating the QS pathway include DSF, an intermediate chain fatty acid 'cis-11-methyl-2-dodecenoic acid' and BDSF (cis-2-dodecenoic acid). Basically, coordination and control of QS in Xanthomonads is carried out by the rpf gene cluster (He et al., 2010; Ryan and Dow, 2011).

Among the various factors responsible for eliciting disease symptoms, biofilm and cell degrading enzymes play an important role. Multiple studies have reported that QS-deficient mutants formed thinner and more disorganized biofilms compared to the wild-types (Tomlin et al., 2005). Recently, a study by Li and Wang (2014) reported effective restrainement of citrus canker symptoms caused by X. citri subsp. citri on upon application of foliar-applied biofilm inhibitors D-leucine and 3-indolylacetonitrile (IAN). Similarly, several essential oils were also found to inhibit biofilm formation capacity of Pseudomonas aeruginosa and Staphylococcus aureus (Kavanaugh and Ribbeck, 2012). Keeping in view these findings, we hypothesized that Thymus vulgaris plant oil (THY oil) might provide effective control against blight disease by acting as QS antagonists. To test the hypothesis, the present investigation was thus carried out with the aim to identify the role of THY oil on virulence traits such as biofilm formation, extracellular enzymes production, signaling molecules and consequently its effect on disease development when inoculated on rice plants along with Xoo.

### MATERIALS AND METHODS

### Xanthomonas Strain and Growth Conditions

The X. oryzae pv. oryzae (Xoo) strain AS29 was isolated from susceptible rice cultivar BLB Pusa Basmati 1 (India) and further identified using 16S-rDNA gene sequence. The identified strain was submitted to GenBank database (Accession Number KX010418) and routinely grown on PME (Peptone Malt Extract) (HiMedia, India) plates (0.5% peptone, 3% malt extract, 1.5% bacto agar) at 26 ± 2 ◦C.

### Spectrophotometric Biofilm Assay

The protocol for measuring biofilm production was carried out following the method described by Dunger et al. (2014). Overnight grown XooAS29 culture was inoculated into 2 mL PME broth with 1:1000 dilution so that OD600 reached 0.6. To the 96-well U-plastic titre plate (SPL Life Sciences, Co., Ltd; South Korea), 1 µL culture was added to 99 µL PME broth in each well. For evaluating the consequence of THY oil, 100–1000 ppm were initially used to assess their effect on biofilm formation. The titre plate was incubated without shaking at 28◦C for 10 h. The incubated cells were then stained with crystal violet (CV) dye for 20 min. The unbound dye was removed by rinsing twice with sterile distilled water (SDW). Finally, the well-bound dye was solubilized in 200 µL dimethyl sulfoxide (100% DMSO) and quantified spectrophotometrically by recording absorbance at 595 nm.

### Microscopic Analysis of Biofilm

Since, 500 ppm oil was most efficient in the aforementioned method; the outcome of this concentration on biofilm (microscopic visualization) in contrast to XooAS29 alone (control) was measured using polyvinyl chloride (PVC) assay (Krzy´sciak et al., 2014). The XooAS29 culture (OD600 = 0.6) was supplemented into 1 mL of PME broth consisting of 500 ppm THY oil for 14 h at 28◦C. The PVC plate wells were washed twice with SDW to eliminate the buoyant planktonic cells after the incubation time. The bacterial cells adhering to the surface were stained with 0.5% CV and 0.2% methylene blue (MB) dyes, respectively and incubated for 5 min at room temperature. The surplus dye was eliminated by pipetting out and the plates were again kept for air drying at room temperature. The dried plates were further imaged at a magnification of 20X (Olympus BH2, Japan) under a light microscope. The XooAS29 culture was allowed to grow on the cover glass submerged in PME medium in the absence and presence of specific concentration of oil and incubated for 10–12 h. The biofilm formed were further stained with green fluorescent dye 20 mM SYTO-9 and observed under

fluorescent microscope (Leica DMR epifluorescence microscope, Germany).

### Metabolic Activity and Growth Kinetics

In the metabolic activity assay, a stock solution of 0.002% (w/v) resazurin reagent (Sigma, United Kingdom; filter sterilized) was prepared (Mariscal et al., 2009) and stored at −20◦C. XooAS29 culture was grown with and without 500 ppm THY oil in PME broth and incubated for 24 h. The culture was centrifuged and the cells were washed with saline. Individually XooAS29 (100 µL) was added to each well of 96 well titre plate followed by addition of 20 µL resazurin solution. The resazurin fluorescence activity (λex: 560 nm and λem: 590 nm) was measured after 60 min of incubation at 37◦C. To test the growth inhibitory effect of THY oil, XooAS29 was grown in PME broth with and without effective concentrations of THY oil (500 ppm) using a shaking incubator for 8–13 h at 25 ± 2 ◦C and the absorbance was recorded at 600 nm in triplicate samples collected hourly. Xoo cells treated with oil served as treatment while cells grown in the absence of oil was taken as control.

### Swimming, Swarming, and Wetness Assay

XooAS29 inoculum was grown as mentioned above and prior to carrying out the assay the final density was adjusted to 1 × 108 CFU mL−1 . For conducting the motility assays, XooAS29 cells mixed with 500 ppm THY oil were gently inoculated in the center of the solidified PME agar plates. 1 µL suspension droplets were carefully placed in the center of soft swimming plates (3% malt extract, 0.5% peptone, 0.3% agar), and (2) swarm plates (3% malt extract, 0.5% peptone, 0.5% agar), and kept for incubation at 25 ± 2 ◦C. The swimming and swarming traits were evaluated after 24 h by calculating the diameter of the bacterial zone. An equivalent quantity of only XooAS29 inoculum without oil in the respective media served as control.

The wetness assay of the colonies on swarm plates was calculated by the capillary-drop method as delineated by Wang et al. (2005). Capillary tubes of 0.5 mL capacity were mildly positioned on the treatment and control colonies for 30 s and the length of fluid entered was measured in millimeters. Xoo cells treated with oil served as treatment while cells grown in the absence of oil was taken as control. Five tubes were placed on different points on swarm plates for calculating the average and SD values.

### Quantification of Exopolysaccharides (EPS) and Xanthomonadin

For EPS, XooAS29 culture with and without 500 ppm THY oil was grown on PME media for 24 h at 28 ± 2 ◦C. To detect the production of EPS from bacterial cells, the cells were scraped off the plates and resuspended in 15 mL SDW. Centrifugation at 15,000 × g for 12 min was done and the supernatant was collected for further analysis. The collected supernatant was muddled up with 1% (w/v) potassium chloride and double volume of 100% ethanol and further incubated at −20◦C overnight. Again centrifugation was done and the precipitated EPS was left overnight for drying at 55◦C before determining the total dry weight. Expression of the results was done relative to the cell density.

Measurement of xanthomonadin pigment was done as mentioned in the method illustrated by Wang et al. (2015). The XooAS29 cells collected by centrifuging 4 mL broth suspension with and without 500 ppm THY oil was mixed with 1 mL 100% methanol. The concoctions were further incubated in darkness for 10 min kept on rotating shaker followed by centrifugation at 12,000 × g for 8 min to collect the supernatant. The xanthomonadin pigment was estimated by measuring the absorbance at OD445 and the result was denoted relative to the cell density measured before the assay (OD595).

### Extracellular Various Enzymatic Assays

The fresh colony of XooAS29 strain was grown in 10 mL of PME liquid medium in presence and absence of 500 ppm THY oil at a starting OD600 of 0.05. After incubation for 24 h, the XooAS29 culture at an OD600 of 1.8 was centrifuged at 12,000 rpm for 12 min and the supernatant obtained was used for enzymatic plate assays (Yang and Tseng, 1988). The extracellular protease activity was measured by a radial diffusion assay in agar plates containing skimmed milk as substrate with 0.5% skimmed milk and 2% (wt/vol) agar. The media was poured in the plates and wells of 6 mm diameter were cut out with the help of a cork borer. The cell suspension (with or without THY oil) was applied in the well of the plates. Clearing zones around the colony due to the utilization of the substrate were measured after 24 h of incubation at 28 ± 2 ◦C.

Extracellular cellulase activity was calculated as explained previously (Maki et al., 2011) using 2% CMC (Sigma-Aldrich) as substrate. Xylanase activity was also assayed on 1% agarose plates containing 0.5% RBB-Xylan and PME agar media (He et al., 2010). Positive xylanase activity was indicated by production of a white halo around the well in the plate.

For endoglucanase activity, XooAS29 culture with and without oil was pipetted into 6 mm diameter wells cut into CMC agar plates (1% agar, 0.125% CMC in 0.05 M potassium phosphate buffer, pH 6.0). The plates were incubated for 48 h at 28◦C and then developed with Congo red dye (Barber et al., 1997). Extracellular lipase/esterase activity was assayed on PME media containing 0.01% CaCl2 and 1% Tween 80 (Rajeshwari et al., 2005). The white crystals surrounding colonies was measured in medium containing CaCl2 and Tween 80 indicating positive lipase activity. All the assays were repeated three times, independently, in triplicates.

### Expression Analysis by Real-Time RT-qPCR

For RNA isolation, single celled colony of XooAS29 with and without 500 ppm THY oil was transferred to 10 mL PME medium at 28 ± 2 ◦C and sampled when the OD595 reached 2.0 (Xu et al., 2015). RNA was isolated by following the manufacturer's instructions of Trizol (Invitrogen, Carlsbad, CA, United States) method and RNA purity was evaluated

with a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, United States). Total RNA was used for synthesizing first-stranded cDNA using c-DNA verso reverse transcriptase (Thermo Scientific) following the instructions mentioned in the kit. The realtime RT-PCR was carried out in 96-well plates using a 7500 fast real-time PCR system (Applied Biosystems) with a Sybr Green master mix (Fermentas) following the manufacturer's instructions (Li and Wang, 2014). The gene expression was done in triplicate in a total volume of 25 µL including a passive reference dye (ROX) (Fermentas). The designing of gene specific primers (**Table 1**) was done on the basis of the available genome sequence of X. oryzae pv. oryzae strain KACC10331. The primers synthesized targeted 21 genes that were earlier recognized to be related to motility, chemotaxis, hydrolytic enzymes, virulence, and DSFs. As an endogenous control DNA gyrase subunit B encoding gene gyrB was used and the relative transcript level was calculated using the method 2−deltadelta CT described by Livak and Schmittgen, 2001.

### Quantification of QS Factors

XooAS29 culture with and without 500 ppm THY oil was individually allowed to grow as mentioned above and sampled when the OD595 reached 2.0. DSF (cis-11-methyl-2-dodecenoic acid) and BDSF (cis-2-dodecenoic acid) were quantified as illustrated by He et al. (2010) and Xu et al. (2015). Five liters of XooAS29 supernatant with and without 500 ppm THY oil was individually collected by centrifugation at 9,000 rpm for

TABLE 1 | Genes used in quantitative reverse transcription polymerase chain reaction.

40 min at 4◦C. Equal volume of ethyl acetate was used for extracting the obtained supernatant whose pH was adjusted to 4.0. The removal of organic phase was done by rotary evaporator at 40◦C, and the residue obtained was dissolved in 20 mL of methanol. The final dried extract obtained was dissolved in HPLC grade methanol. The signaling molecules present in ethyl acetate extracted samples were detected through prewashed aluminum HPTLC plates (10 cm × 10 cm), coated with silica gel 60- GF254 (Merck). 2 µl of each sample was applied in triplicate on preactivated TLC plate using the Camag Linomat 5 automated TLC applicator equipped with a 100-µl Hamilton syringe. Hexane: diethylether: acetic acid (8:2:0.1) was implemented as mobile phase to develop TLC plate in Camag twin glass chamber. After developing the TLC plate to 90 mm distance, the plate was removed and dried at room temperature for 1 h. Developed plate was documented under 254 and 366 nm, and then transferred into previously saturated iodine chamber to visualize the spot under day light. The plates were then scanned at 270 nm with a Camag TLC Scanner with winCATS 3 software, using the deuterium lamp. The densitograms was recorded and peak area of both samples was observed.

### Virulence Assay on Rice Plants