Isolation, characterization of glycolipid type biosurfactant from endophytic Acinetobacter sp. ACMS25 and evaluation of its biocontrol efficiency against Xanthomonas oryzae

Author’s Accepted Manuscript Title: Isolation, characterization of glycolipid type biosurfactant from endophytic Acinetobacter sp. ACMS25 and evaluation of its biocontrol efficiency against Xanthomonas oryzae Deivaraj Shalini, Abitha Benson, Ram Gomathi, Allen John Henry, S Jerrita, Manoharan Melvin Joe www.elsevier.com/locate/bab

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S1878-8181(16)30467-4 http://dx.doi.org/10.1016/j.bcab.2017.07.013 BCAB590

To appear in: Biocatalysis and Agricultural Biotechnology Received date: 5 December 2016 Revised date: 20 July 2017 Accepted date: 20 July 2017 Cite this article as: Deivaraj Shalini, Abitha Benson, Ram Gomathi, Allen John Henry, S Jerrita and Manoharan Melvin Joe, Title: Isolation, characterization of glycolipid type biosurfactant from endophytic Acinetobacter sp. ACMS25 and evaluation of its biocontrol efficiency against Xanthomonas oryzae, Biocatalysis and Agricultural Biotechnology, http://dx.doi.org/10.1016/j.bcab.2017.07.013 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Title: Isolation, characterization of glycolipid type biosurfactant from endophytic Acinetobacter sp. ACMS25 and evaluation of its biocontrol efficiency against Xanthomonas oryzae

Deivaraj Shalini1, Abitha Benson2, Ram Gomathi1, Allen John Henry1, Jerrita S, and Manoharan Melvin Joe1 1

Department of Microbiology, School of Life Sciences, VELS University, Velan Nagar, Pallavaram Chennai600117, Tamilnadu, India 2

Department of Biotechnology, School of Life Sciences, VELS University, Velan Nagar, Pallavaram Chennai-600117, Tamilnadu, India 3

Department of 1Department of Electronics and Communication Engineering, , VELS University, Pallavaram, Chennai -600117, India.

*Corresponding author Department of Microbiology, School of Life Sciences, VELS University, Velan Nagar, Pallavaram Chennai-600117, Tamilnadu, India. Phone: +917395841132. E-mail: [email protected]

ABSTRACT

1

Acinetobacter sp. ACMS25, which showed inhibitory effect on Xanthomonas oryzae pv. oryzae XAV24 was tested for biosurfactant production. Preliminary characterization showed that this strain was positive for hemolytic and oil spreading activity and was able to reduce the surface tension of water from 71.9 to 37.6 mN/m. The biosurfactant produced by this strain was characterized as a ‘glycolipid-type’ based on thin layer chromatography (TLC), fourier transform infrared spectroscopy (FTIR) and proton nuclear magnetic resonance (1HNMR) analysis. Inhibition studies showed that this biosurfactant was able to reduce the specific growth rate of X. oryzae by 38.4% and spermosphere population by 43.5%. Glycolipid biosurfactant treatment improved the germination and vigor index, when challenge inoculated with X. oryzae and this treatment was able to offer 76.9% disease protection efficiency against the rice blight disease in rice. Keywords: Endophytic bacteria; Acinetobacter sp.; glycolipid biosurfactant; Xanthomonas oryzae pv. oryzae

1. Introduction Endophytic bacteria after

successful survival in the spermosphere and

rhizosphere are known to colonize different plant parts such as stem, leaf and other reproductive organs (Rosenblueth and Martínez-Romero, 2006; Compant et al., 2011; Reinhold-Hurek and Hurek, 2011;). During this period of colonization they form metabolic association with the host plants (Compant et al., 2005) to enable them to selectively adapt to different ecological niches (Gray and Smith, 2005; Hallmann et al., 1997). These endophytic bacterial association with the host plant can play a major role in improving plant growth and offering protection against various pathogens (Bent and Chanway, 1998; Chanway, 1997).

2

To offer protection endophythic bacteria are known to synthesize several metabolites with antagonistic properties such as siderophores, antibiotics and biosurfactants (Brader et al., 2014; Joe et al., 2012). Among these antimicrobials, the use of biosurfactants against various plant pathogenic microorganisms as an alternative to the synthetic chemicals and antibiotics is gaining momentum (Nihorimbere et al., 2011; Sachdev and Cameotra, 2013). These biosurfactants, are low molecular weight surfaceactive amphiphilic biomolecules produced by a diverse group of microorganisms, that can reduce the surface tension either at the air/water interfaces or the interfacial tension at oil/water interfaces (Banat et al., 2010). Taking this into account, the present study was conducted to evaluate the biosurfactant production of Acinetobacter sp. ACMS25, which showed inhibitory effect against X. oryzae p.v. oryzae. This was followed by purification and characterization of biosurfactant. In vitro and greenhouse studies were carried out to evaluate the efficiency of biosurfactant against X. oryzae. X. oryzae is one of the most devastating diseases in rice, throughout the world and in particular in Asia (Ou, 1985). Being a seed borne pathogen and due to its high epidemic potential, it has been considered as a threat to all rice cultivars throughout the world. Though the use of pesticides and other antibiotics has been recommended against this pathogen, residual toxicity and development of antibiotic resistance has made researchers look for other viable alternatives. 2. Materials and Methods 2.1. Bacterial strains growth and maintainance Acinetobacter xylosoxidans used in the present study was isolated from a medicinal plant Catharanthus roseus (Karthikeyan et al. 2012), this strain was selected after preliminary screening for antagonistic activity from numerous strains of our lab collection. The bacterial strains were maintained at -20 °C as glycerol stocks. Prior to use 3

they were grown overnight in nutrient broth at 28± 2 °C, 120 rpm or in nutrient agar medium at 28± 2 °C for 24-36 h. Strain X. oryzae p.v. oryzae XAV24 was obtained from the Department of Microbiology, Faculty of Agriculture Annamalai University and this strain was maintained in Wakimoto’s agar (Mew and Misra, 1994). PCR based detection for asymptomatic leaves/seedlings was carried out using the primers TXT4F and TXT4R with the conditions as described earlier (Sakthivel et al., 2001) and the clip method devised by Kauffman et al. (1973) to test the virulence/pathogenicity of this strain. 1.2.Antagonistic activity screening against X. oryzae An agar disk technique described by Visser et al. ( 1986) was used for the initial screening of endophytic bacterial strains for antagonistic activity against X. oryzae. Briefly, pour plates were made using the selected bacterial strain by mixing one ml of a 48 h broth culture in 20 ml of Nutrient agar. After incubation at 30 °C for 48 h, disks with a diameter of 7 mm were stabbed from the agar. The disks were placed on MacConkey agar (Himedia, India) covered with suspensions of 48 h old X. oryzae cultures. After an incubation period of 36 h at 25 °C, the diameter of clear zones surrounding the disks was measured. 1.3.Hemolytic activity For hemolytic activity, Acinetobacter sp. ACMS25 was streaked on freshly prepared blood agar plates and incubated at 37 °C for 48-72h. Results were recorded based on the type of clear zone observed and classified as α-hemolysis (colony was surrounded by greenish zone), β-hemolysis (colony was surrounded by a clear white zone) and γ-hemolysis (no change in the medium surrounding the colony) according to the protocol of Carrillo and Mardaraz ( 1996). 1.4.Drop-collapse test

4

Screening for biosurfactant production was performed using qualitative dropcollapse test as described by Bodour and Maier (1998). Briefly, two µL of oil was applied to 96-well micro plates and left to equilibrate for 24 h. After 24 h, five µL of 48 h old cultural filtrate (after removing the cells by centrifugation at 10,000 × g) was transferred to oil-coated well regions and the drop size was observed after 1 min with the aid of magnifying glass. The result was considered positive for biosurfactant production, when the drop was flat and negative, when cultures produced rounded drops; indicative of the lack of biosurfactant production as described by Youssef et al. (2004). 1.5.Oil spreading technique Oil spreading technique was done according to the methods of Morikawa et al. (2000). Briefly, ten μl of oil was added to 40 ml of distilled water placed in a petridish to form a thin oil layer. To this ten μl of culture supernatant was gently placed on the center of the oil layer. If biosurfactant is present in the supernatant, the oil was displaced and a clearing zone was formed. 1.6.Emulsification index determination and biosurfactant quantification Biosurfactant production at different time periods was monitored in M9 mineral salt medium (MSM) (Sigma) (inoculated with 108 log Cfu mL-1 bacteria) supplemented with 2% glycerol. Emulsification index (EI) was determined by adding 2 ml of kerosene oil with 2 ml of culture broth described above, the contents were centrifuged for 2 min at 10,000 × g, and allowed to stand for 24 h. The %EI24 is given as percentage of height of emulsified layer divided by total height of the liquid column according to the methods of Ochsner et al., (1994). Biosurfactant production was quantified by phenol-sulphuric acid method and denoted as rhamnose equivalents with the standard curve prepared using rhamnose (Chandrasekharan and BeMiller, 1980). 1.7.Extraction of crude biosurfactant 5

Extraction of crude biosurfactant was done according to the methods of Yakimov et al. (1997) with required modifications. Twenty four h grown bacterial cells (cfu> 8 Log Cfu ml-1) were collected by centrifugation at 12,000 × g, 10 min and concentrated HCl was added to reduce the pH of supernatant to approximately 2.0 and maintained at 4 °C to precipitate the biosurfactant. After 24 h, the biosurfactant was precipitated by using one ml of solvent mixture of chloroform: methanol (3:1 v:v) and the solvent was evaporated to obtain the crude biosurfactant as described by Rooney et al. (2009). 1.8.Thin layer chromatography Ten µL of biosurfactant was placed in silica gel aluminium plates (Merck) at the point of origin near the bottom. Biosurfactant was eluted using a solution containing a mixture of chloroform-methanol-water (65:15:2), and the spots were visualized using iodine vapors for visualization of lipids and anthrone reagent dissolved in concentrated H2SO4 at a final concentration of 2% to visualize the glycolipid spots (Ramana and Karanth, 1989) and their Rf values were observed according to the methods of SoberonChavez and Maier (2011) . This fraction was scraped and eluted with chloroform: methanol (1:2, v/v) mixture. The solvent fraction was centrifuged at 5000 ×g for 10 min to remove the silica gel. The aliquots were micro-filtered and concentrated by air-drying and used for further analysis. 1.9. Fourier transform infrared (FT-IR) spectroscopy: The IR spectra of TLC-purified biosurfactant were recorded in a FT-IR spectrometer (Thermo Niocolet, AVATAR 330 FFT-IR system, Madison WI 53711-4495) in the 4000400 cm-1 spectral region using potassium bromide solid cells. The analysis was done in the Department of SAIF IIT Guindy, Chennai, India. The spectra recorded for the different pellets was analyzed using the standard methods described previously ( Lang and Wagner, 1987; Pornsunthorntawee et al., 2009; Yin et al., 2009). 6

1.10.

Purification of biosurfactant

TLC spots specifying the glycolipid were scraped (from 4-5 plates and, the contents were pooled) and dissolved in 1 ml methanol, centrifuged at 15,000 × g for 3 min at 4 °C to remove the silica gel. The supernatant was collected, air dried for 24 h and dissolved in 25 μl methanol and the contents were centrifuged and dried. The dried fraction was further purified in a silica gel (60–120 mesh) column eluted with a gradient of chloroform and methanol as described by Sharma et al. (2015). 1.11.

H NMR analysis and CMC determination

1

The biosurfactant sample purified as described above was analyzed by 1H NMR analysis in a solution of methanol. The sample was subjected to 1H NMR analysis at Indian Institute of science with an analytical 300 MHz solid state NMR. Critical micelle concentration (CMC) of purified biosurfactant was measured based on a plot of surface tension (Du Noüy ring tensiometer) vs surfactant concentration and CMC was expressed as mg ml-1. Effect on growth rate of X. oryzae The growth rate of X. oryzae as influenced by the addition of different antimicrobials was evaluated as described by Schoenknecht et al. (1985) with required modifications. Briefly, different antimicrobials at a concentration of 1 μg/mL were added to the test broth. The treatment groups were incubated for 48 h, with sampling done at different time periods and the bacterial growth at different time periods was recorded. The specific growth rate of X. oryzae as influenced by different antimicrobials was calculated using the formulae µ = (log10 N - log10 N0) 2.303) / (t - t0). The samplings were taken at an initial time period of 8h (t0), followed by another sampling done at 24h (t). N and N0 represent the number of bacteria at a time period of 8h and 24h, repectively. The experiment was repeated twice with a minimum of three

replications to confirm results. 1.12.

Seed surface survivability

For testing the seed surface survivability of X. oryzae against biosurfactant, surface sterilized seeds were coated with 1, 2 and 3% of biosurfactant. Positive control 7

was maintained with streptomycin and NaOCl @200 mg per 100g of seed. Four h after treatment the seeds were challenge inoculated with X. oryzae @ 8 Log Cfu g-1. Surviving bacterial population in the spermosphere was determined by using serial dilution and plating in modified Wakimoto's agar (Mew and Misra, 1994). 1.13.

Germination and vigour index

Germination test was carried out using the paper towel method. Seeds were placed in the germination paper presoaked in distilled water and incubated for 14 days at 24 ± 1 °C. After 14 days the germinated seeds were counted and expressed as percentage. The vigour index was calculated by using the formula as described by Abdul-Baki and Anderson (1973).

1.14.

Disease incidence under pot culture conditions

Seeds from the above mentioned treatments were transplanted in pots filled with sterilized soil, sand and manure in the ratio of 1:1:1. Plants were watered periodically with sterile distilled water and plants were maintained under water logged condition throughout the study period. Thirty days after transplantation, the disease incidence percentage was recorded by counting the number of infected plants. The percent protection offered was calculated by formula: (control -treated)/control *100. The experiment was repeated twice under similar conditions to confirm the results. 2. Results and Discussion 3.1. Screening for antagonistic activity against X. oryzae Among twenty four Plant growth-promoting Rhizobacteria (PGPR) strains from our lab collection tested for their in vitro growth inhibition activity against X. oryzae p.v. oryzae, seven isolates showed antagonistic effect and four isolates were also found to be positive for biosurfactant production (Data not shown). Among these four isolates evaluated Acinetobacter sp. ACMS 25, which showed the highest antagonist activity against X. oryzae was selected for further study. Though reports regarding the 8

biosurfactant production in endophytic bacteria and their use for the control of X. oryzae is scarce; Pathak and Keharia (2013) reported the isolation of Bacillus subtilis K1 isolated from aerial roots of Ficus benghalensis capable of secreting a mixture of three cyclic lipopeptides surfactins, iturins and fengycins with a high degree of heterogeneity and good emulsification activity. 2.2.Biosurfactant characterization Isolate Acinetobacter ACMS25 was found to be positive for hemolytic activity as evident by its ability to grow and to exhibit clear zone on blood agar (Fig 1a). Results on drop collapse tests show that the drop collapse activity of the crude biosurfactant was comparable to that of the surfactant SDS. Oil spreading technique also gave positive result with the crude biosurfactant (Fig 1b) and the crude biosurfactant was able to reduce the surface tension of water 71.9 to 37.6 mN/m (Data not shown). Though methods such as hemolytic activity, positive result for drop collapse test and oil spreading testing were considered as primary screening techniques for biosurfactant production, the real potential for microbial surfactant is determined by their capability to reduce the surface tension of production medium. An effective biosurfactant can decrease the surface tension of distilled water from 72.0 to 35.0 mN m-1 (Mulligan, 2005). In our present study biosurfactant was found to be positive for all the preliminary tests and was able to reduce the surface tension of water. In the mineral salt medium supplemented with glycerol (2%), at the end of the fermentation period of 72 h, an emulsification index (EI24%) of 64.6 was recorded (Fig 1c). Structural characterization of biosurfactant based on FTIR analysis is provided in Fig 2a. Based on FTIR analysis a band appearing on 3412 cm-1 confirmed the presence of OH stretching of hydroxyl group. The adsorption peak at 2296 shows the presence of C≡C bond. Characteristic peak observed at 1647 cm-1 showed the presence of C=C carbonyl group. The presence of bands at the regions of 1400-1600 cm-1 show the 9

presence of C=C stretching vibrations. The finger print region below 1200 cm-1 represents different kinds of C-H, C-O, and CH3 vibrations. The absorption at 1062 cm-1 confirmed the presence of C-O-C vibrations. The characteristic bands observed at 3412, 2296, 1647, and 1064 cm-1 confirmed the presence of a glycolipid type biosurfactant based on the earlier reports (Arutchelvi and Doble, 2010; Pornsunthorntawee et al., 2008 ; Nalini and Parthasarathi, 2014). TLC analysis visualized using iodine vapor revealed the presence of lipids at Rf value of 0.28-0.87 (Fig 2b).

Detection of

carbohydrates using anthrone reagent in concentrated H2SO4 revealed a peak at an Rf value of 0.83(Fig 2c). CMC of the biosurfactant was determined from the surface tensions of the biosurfactant at constant value of surface tension of 25.8 mN/m at 3.25 biosurfactant concentration (%) (Fig 2d). Kiran et al. (2010) who reported a Rf value of 0.81 for a glycolipid type biosurfactant from Brevibacterium casei MSA19 and Nalini and Parthasarathi (2014) reported an Rf value of 0.85 for a di-rhamnolipid type biosurfactant from Serratia rubidaea SNAU02. The column purified biosurfactant fraction exhibited a single band at an Rf value of 0.63 (Fig 3a). The structural characterization of biosurfactant studied by 1H NMR in provided in Fig 3b. The peaks at 1.13-1.22 correspond to CH3 on the sugar moiety in the biosurfactant. The peaks at 2.4 and 2.8-2.9 correspond to the CH2-COO on β-hydroxy fatty acids. The peak at 3.28 corresponds to –CH-OH on sugar moiety and the peak at 8.18 corresponds to CH2CH. From the 1HNMR results, TLC and FTIR analysis Based on positive anthrone test, characteristic bands observed at 3412, 2296, 1647, and 1064 cm-1 and a peak value of 3.28, which correspond to sugar moiety in NMR analysis and also in comparison with other studies (Lotfabad et al., 2010; Wei et al., 2005) it is confirmed that biosurfactant is a glycolipid type. 2.3.Effect of biosurfactant on specific growth rate of X. oryzae

10

The influence of biosurfacatant on the specific growth rate of X. oryzae was evaluated under in vitro conditions (Table 1). The biosurfactant was able to reduce the specific growth rate of X. oryzae by 38.4%. This inhibitory effect was similar to that of antibiotic and NaOCl treatments. Gomaa et al. (2013) reported that the addition of biosurfactant to the growth medium of Staphylococcus aureus resulted in the reduction of bacterial cell growth, total lipids, total proteins, RNA and DNA of the bacterial cell. These authors reported that this may be due to the reason that these antimicrobial agents affect the growth and multiplication of bacterial cells by interacting with receptors in the cell. Biosurfactant treatment significantly reduced the survivability of X. oryzae on the seed surface of rice. Biosurfactant treatment lead to reduction in the bacterial population by 43.5%, when compared to control treatment, 30.9% in NaOCl treatment, and 23.5% in streptomycin treatment was observed. To the best of our knowledge no study has been conducted on the influence of ‘glycolipid-type’ biosurfactant treatment on spermosphere survivability of X. oryzae. However, rhamnolipids have shown excellent antimicrobial activity against various pathogenic fungi,

‘Gram-positive’

bacteria

and

‘Gram-negative’

bacteria including

Salmonella typhimurium, Escherichia coli, Enterobacter aerogenes (Benincasa et al., 2004; Haba et al., 2003). This antibacterial mechanism of the biosurfactant is due to their amphipathic nature of the biosurfactant, which may interact with the phospholipids of the bacterial cell (Ortiz et al., 2006) and may cause bacterial cellular damage by increasing the permeability of the cell membrane (Sánchez et al., 2006). 2.4.Effect of biosurfactant on germination percentage, vigor index and disease incidence Significant reduction in the germination percentage, root and shoot length under in vitro and greenhouse conditions owing to X. oryzae inoculation was observed. However, this negative influence was reduced, when treated with biosurfactant. 11

Biosurfactant treatment improved germination (91.1%), root length (9.1 cm), shoot length (19.8 cm), and vigour index (2632.7) (Table 2). This treatment also showed 20.0% disease incidence and was able to offer 76.9% disease protection efficiency. Our previous report Joe et al. (2015) showed that ‘surfactant-based’ nanoemulsion treatment could be used effectively for the treatment of soft rot caused by Pectobacterium carotovorum sub sp. carotovorum in potato tubers. Numerous researchers (De Jonghe et al., 2005; Kim et al., 2000; Perneel et al., 2008; Sharma et al., 2007) reported that rhamnolipids could be used effectively against the plant pathogenic fungi such as Botrytis sp., Rhizoctonia sp., Phythium sp. and Phytophora sp. In the present study, we have isolated and characterized a ‘glycolipid-type’ biosurfactant from Acinetobacter sp. ACMS25 based on TLC, FTIR and NMR analysis. In vivo

and

greenhouse

study

results

on

using

biosurfactant

against

X. oryzae was encouraging and this may be extended against other pathogens. These ecofriendly biosurfactant may replace the chemical pesticides and chemically defined surfactants in the near future. Acknowledgements We are grateful to VELS University for their support. Authors thank DST-SERB for the Grant No. SB/YS/LS- 79/2013, “Development of endophytic bacterial consortium from selected medicinal plants of Western Ghats of India,” Reference Abdul-Baki, A.A., Anderson, J.D., 1973. Vigor determination in Soybean Seed by Multiple Criteria1. Crop Science 13, 630. Arutchelvi, J., Doble, M., 2010. Characterization of glycolipid biosurfactant from Pseudomonas aeruginosa CPCL isolated from petroleum-contaminated soil. Lett. Appled Microbiol. 51, 75–82.

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Figure Legends Fig 1. Bosurfactant characterization based on a) hemolytic activity of biosurfactant positive isolates in blood agar plates, and b) oil spreading technique. c) Growth and biosurfactant production as indicated by emulsification index %EI 24 in MSM with 2% glycerol.

Fig 2. Characterization of ‘glycolipid-type’ biosurfactant from Acinetobacter sp. ACMS25 a) FTIR analysis of biosurfactant, TLC showing the production of a ‘glycolipid-type’ biosurfactant showing the presence of b) lipids and c) sugars d) Graph drawn on surface tension vs concentration of biosurfactant.

Fig 3. Characterization of glycolipid type biosurfactant by a) TLC analysis b) NMR analysis

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a)

b)

1.6

c) c)

60

1.2

50

1.0 %EI24

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OD 600 nm

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30 0.6 20

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12h 18h 24h 30h 36h 42h 48h 54h 60h 66h 72h

Time in h

Fig 1.

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Emulsification index(%EI24)

Growth at OD 600 nm

1.4

70

100.0 95

BS11

90 85 80

a)

75 70 65 60

723

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)

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Fig 2. 20

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Fig 3.

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b)

Table 1. Effect of glycolipid seed treatment on the specific growth rate (k) h−1 and survival of X. oryzae cv. oryzae in the rice seed treatment Treatments Specific growth rate Seed surface Xanthomonas population (k) h−1 Log Cfu g-1 Crude extract 0.78b 6.2±0.4a,b BS (1%) 0.66c 5.7±0.5b BS (2%) 0.59c,d 4.3±0.3b,c BS (3%) 0.56d 3.9±0.5c Streptomycin 0.54d 5.6±0.4b NaOCl 0.52d 5.1±0.2b Pathogen only 0.91a 6.9±0.6a Control BS - Biosurfactant, Initial inoculation load used for seed challenge tests is >8 Log Cfu mL-1. Different lower case letters after values (a–d) indicate that there is a significant difference at a P value of 0.05 as determined by DMRT.

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Table 2. Effect of glycolipid seed treatment on the growth under in vitro condition and Percentage disease incidence (PDI) and Percentage disease protection against Xanthomonas oryzae cv. oryzae Treatment

BS (1%) BS (2%) BS (3%) Streptomycin NaOCl Pathogen Control

Germination Percentage (%) 80.0c 88.8b 91.1b 82.2c 77.7d 64.4e 97.7a

Root Shoot Length Length 7.6d 17.6b 8.4c 18.9a 9.1b 19.8a 8.2c 19.1a 6.4e 15.4c 4.8f 13.4d 10.1a 15.9c

Vigour Index 2016.0e 2424.2c 2632.7a 2244.0d 1693.8f 1172.1g 2540.2b

Dry wt. mg Plant-1. 372.6b 334.8c 401.9a 388.6b 342.6c 294.5d 412.6a

Disease incidence percentage 40.0b 26.6d 20.0e 26.6d 33.3c 86.6a 0.0

Disease Protection (%) 53.8d 69.2b 76.9a 69.2b 61.5c -

GP- germination percentage, RL-root length, SL-shoot length and VI- vigor index under in vitro conditions with observations made at 21 DAS. Disease incidence and disease protection under pot culture conditions. Different lower case letters after values (a–d) indicate that there is a significant difference at a P value of 0.05 as determined by DMRT. Percentages obtained are subjected to arcsine-transformations before statistical analysis.

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