World Applied Sciences Journal 28 (12): 2229-2241, 2013ISSN 1818-4952© IDOSI Publications, 2013DOI: 10.5829/idosi.wasj.2013.28.12.2009

Corresponding Author: Krishnan Nagendran, Department of Plant Pathology, Centre for Plant Protection Studies,Tamil Nadu Agricultural University, Coimbatore, India.

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Management of Bacterial Leaf Blight Disease in Rice with Endophytic Bacteria

Krishnan Nagendran, Gandhi Karthikeyan, Mohammed Faisal Peeran, Muthuraj Raveendran, Kuppusamy Prabakar and Thiruvengadam Raguchander

Department of Plant Pathology, Centre for Plant Protection Studies,Tamil Nadu Agricultural University, Coimbatore, India

Abstract: Forty bacterial endophytes were isolated from different plant sources and tested for their efficacyagainst Xanthomonas oryza pv. oryzae inciting bacterial leaf blight disease in rice. Out of these, isolates viz.,Bacillus subtilis var. amyloliquefaciens (FZB 24), EPB 9, EPB10, EPCO 29 and EPCO 78 recorded a significantlyhigher inhibition of X. oryzae pv. oryzae over control in vitro. Among these efficient endophytes EPB 18, EPB11, EPCO 74, FZB24 and EPB 10 were promote the plant growth of rice seedlings significantly over the otherisolates and control. In the present study, rice plants (cv. ADT39) applied with FZB 24 through seed treatment@ 4g/kg + seedling dip @ 4g/l + soil application @ 500g/ha + foliar application @ 500g/ha recorded the lowestseverity of bacterial leaf blight (31.36 %) with a per cent reduction of 40 over control under glasshouseconditions. In addition, the B. subtilis (FZB 24) treated rice plants registred higher induction of defence relatedenzymes viz., peroxidase, polyhenol oxidase and phenylalanine ammonia lyase and resulted in higheraccumulation of total phenols compared to untreated control plants. The endophytes treated rice plotsregistered a significantly lower intensity of bacterial leaf blight (2.80%) compared to untreated control plots(19.82%), which also recorded a higher grain and straw yield.

Abbreviation: Xoo - Xanthomonas oryzae pv. oryzae; Xam - Xanthomonas axonopodis pv. mavacearum;PSA – Potato sucrose agar; ACC - 1-aminocyclopropane-1-carboxylic acid; DAPG – 2,4- di-acetylphloroglucinol

Key words: Endophytes Bacterial leaf blight of rice Bacillus Induced systemic resistanceXanthomonas oryza pv. oryzae

INTRODUCTION pathogens by the use of antagonistic microorganisms or

Rice is the most important staple food crop of more feasible disease control technology (Han et al. [6]).than half of the world population. Bacterial leaf blight Plants are constantly involved in interactions with acaused by Xanthomonas oryzae pv. oryzae Ish. is found wide range of bacteria. These plant-associated bacteriaworldwide and particularly destructive in Asia. The colonize the rhizosphere (rhizobacteria), the phyllospheredisease was endemic in Bihar (Srivastava and Rao [1]) and (epiphytes) and inside the plant tissues (endophytes).Tamil Nadu (Rajagopalan et al. [2]). Reduction in rice yield Endophytes are sheltered from environmental stressesmay be as high as 50 per cent was also recorded, when the and microbial competition by the host plant and theycrop was severely infected (Mew et al. [3]). It became a seem to be ubiquitous in plant tissues, having beendestructive disease of rice in Punjab and appeared in isolated from flowers, fruits, leaves, stems, roots andepiphytotic form in Ludhiana, Jalandhar, Patiala and seeds of various plant species (Kobayashi andSangur districts and caused about 30 per cent yield loss Palumbo [7]). Several bacterial endophytes have been(Chahal, [4]). Scientists have focused on biological shown to support plant growth and increase nutrientmethods to protect crops from invasion and infection uptake by providing phytohormones (Kang et al. [8])by the pathogen (Liu et al. [5]). The management of and biologically fixing nitrogen (Jha and Kumar [9]).

their secondary metabolites is now considered to be a

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Endophytic bacteria can not only promote plant growth Isolation of Endophytes: Source plants were manuallyand act as biocontrol agents, but also produce nature uprooted and brought to the laboratory. Root, stem andproducts to control plant diseases (Guan et al. [10]) and leaf sections (2-3cm long) were made using a sterilereducing disease severity (Senthilkumar et al. [11]). scalpel. The root samples were taken just below the soil

Bacillus species are among the most common line for younger plants and 5-10cm below the soil line forbacteria found to colonize plants endophytically older plants. Stem samples were first weighed and surface(Lilley et al. [12], Mahaffee and Kloepper, [13]) and it is sterilized with hydrogen peroxide (20%) for 10 min. andlikely that their endophytic ability could play a role in the rinsed four times with 0.02 M potassium phosphate bufferbiocontrol of vascular plant pathogens. Endophytic (pH 7.0). Root samples were surface disinfected withbacteria from potato tissue was found to be antagonistic sodium hypochlorite (1.05%) and washed in four changestowards Clavibacter michiganensis ssp. sepidonicus, the of 0.02 M phosphate buffer solution. Measured quantitycasual agent of bacterial ring rot of potato (Van Buren et of 0.1 ml aliquot from the final buffer wash was removedal. [14]) and against Erwinia carotovora var. atroseptica and transferred in 9.9 ml tryptic soya broth to serve ascausing potato soft rot (Sturz and Matheson, [15]). sterile check. Samples were discarded, if growth wasSome endophytic bacteria such as P. fluorescens 89B-61 detected in the sterile check within 48 h. Selected samplesinduces systemic resistance against P. syringae pv. were triturated in 9.9 ml of buffer in sterile pestle andlachrymans (Liu et al. [16]). Reiter et al. [17] screened the mortar. The triturate was serially diluted in potassiumendophytic bacteria against E. carotovora ssp. phosphate buffer solution and plated on Tryptic Soyaatroseptica revealed that 38 per cent of the endophytes Agar (TSA). Representatives of colony morphology wereprotected tissue culture potato plants from black leg transferred to fresh TSA plated as pure cultures (McInroydisease. With this background, the present study was and Kloepper [23]).carried out to isolate, screen and field evaluation of theeffective endophytic bacterial strains for the management Antagonism of Endophytic Bacterial Strains Againstof bacterial leaf blight disease of rice. Xoo: Cell suspension of Xoo was prepared in the sterile

MATERIALS AND METHODS the bacterial cell suspension (Xoo) was mixed with 19 ml

Bacterial Pathogen: Rice leaves showing typical bacterial Petri dishes. After solidification, sterile paper discsleaf blight symptoms were collected for isolation of (6mm diameter) were placed on the surface of the mediumXanthomonas oryzae pv. oryzae (Xoo) from wetlands of at 1 cm away from the side of the Petri dish and 5µl of theTamil Nadu Agricultural University, Coimbatore. The endophytic bacterial culture in NA broth of 4h old wascollected diseased leaves were ground with autoclaved applied to each disc. The plates were incubated at 37±2°Cpestle and mortar, suspended in 200 ml of sterile saline for and the inhibition of bacterial growth was measured 48h2 h. The suspensions were serially diluted 4 x 1:10 in test after the treatment (Salah et al. [24]).tubes. Aliquots of 0.05 ml were spread on to mXOS(Modified XOS agar; Di et al. [18]; Gnanamanickam et al. Plant Growth Promotion by Endophytes[19]) medium and incubated at 28 ± 2°C for 3-5 days. Preparation of Bacterial Inoculum: Endophytic bacteriaThe pathogenicity of the isolated Xoo was confirmed by were grown on KB broth with constant shaking at 100 Xgclip inoculation. Thirty-day-old rice plants grown in for 48 h at room temperature (28±2°C). Bacterial cells weregreenhouse with fully expanded leaves were clip harvested by centrifugation at 12,000 Xg for 15 min andinoculated with the sterile scissors dipped in bacterial bacterial cells were resuspended in Phosphate Buffersuspension (10 cfu ml ) (Kauffman et al. [20]). For (0.01 M, pH 7.0). The concentration was adjusted to8 1

inoculum preparation, the 36 h old bacterial cultures were approximately 10 cfu ml (OD595=0.3) with amultiplied on PSA for 48 h, centrifuged at 5000 x g for spectrophotometer and used as bacterial inoculum10 min and the bacterial pellets were washed with sterile (Thompson [21]).saline. Cell suspensions were adjusted to 10 cfu ml8 1

(Thompson [21]). Xoo was maintained on rice cultivar Seed Bacterization: Rice seeds (cv. ADT 39) were surfaceADT 39 in a greenhouse and on PSA (Sakthivel et al. [22]) sterilized with 2% sodium hypochlorite for 30 sec, rinsedand nutrient agar under laboratory conditions. in sterile distilled water and dried overnight under a sterile

distilled water to a concentration of 10 cfu / ml. One ml of7

of nutrient agar (NA) medium and poured onto the sterile

8 1

Sum of all numerical ratings 100Total number of leaves graded Maximum grade

PDI = ×

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air stream. Endophytic bacterial strains, inoculated into conditions. The effectiveness of the treatments on thetheir respective broths and bacterial suspension was intensity of bacterial leaf blight disease was observedprepared as mentioned above. The required quantity of fifteen days after pathogen inoculation, with a 0-9 scale ofseeds was soaked in bacterial suspension containing the Standard Evaluation System for rice (IRRI [28]) and3X10 bacteria ml for 2 h and dried under shade. the per cent disease indices were calculated using the8 1

Plant-Growth Promotion: The plant-growth promotingactivity of the bacterial endophytic strains was assessedon the basis of seedling vigour index as determined by thestandard roll towel method (ISTA [25]). Twenty five seedswere kept on presoaked germination paper. The seeds In addition, growth parameters like plant height andwere held in position with another presoaked germination number of tillers were also recorded at different timepaper strip on top of them and gently pressed. The intervals. Plant samples were also collected at differentpolythene sheet along with the seeds was then rolled and time intervals from different treatments to study theincubated in a growth chamber for 14 days. Three induced systemic resistance from the followingreplications were carried out for each treatment. The root treatments.and shoot length of individual seedlings was measuredand seed germination percentage calculated. T - ST - Seed treat with B. s. var. amyloliquefaciens

The vigour index was calculated using the formula of (FZB24) @4g/kgBaki and Anderson [26]: T - SD - Seedling dip with B. s. var. amyloliquefaciens

Vigour index = % germination X seedling length T - SA + FA - Soil + Foliar application with B. s. var.(shoot length + root length) amyloliquefaciens (FZB24) @500g each /ha

Glasshouse Study Seedling dip with FZB24@4g/l + Soil applicationApplication of Bioformulations: Bioformulation @500g/ha + Foliar spray @ 500g each /ha with FZB24@30containing B. subtilis var. amyloliquefaciens was used to Days After Transplantingtreat the rice plants. Rice seeds (ADT 39) were soaked in T - Chemical - Foliar spray with Streptomycin sulphate +double the volume of sterile distilled water containing the Tetracycline @ 600ppm + COC @ 0.25% as chemicalformulation. After 24 h, the suspension was drained off checkand the seeds were dried under shade for 30 min and T - IC - Inoculated controlsown in plastic pots along with untreated control seeds. T - HC - Healthy controlFor seedling dip, seedlings were removed 15 days aftersowing, tied in bundles and their roots were dipped in Induced Systemic Resistance: Plants were carefullywater containing bioformulation of B. subtilis (FZB 24) uprooted without causing any damage to root and leaffor 30 min. The seedlings were transplanted in plastic pots tissues at different time intervals viz., BI (Beforecontaining 0.014 m soil at the rate of two seedlings per Inoculation), 0, 12, 24, 48, 72, 96 and 120 h after challenge3

hill and four hills per pot (Nandakumar et al. [27]) and inoculation. Plant samples were homogenized withwere watered regularly to maintain a 1-cm water level. liquid nitrogen in a pre-chilled mortar and pestle.Soil application of bioformulation was applied at the time The homogenized plant tissues were used immediately orof planting under glasshouse conditions. The foliar spray were stored in deep freezer (-70°C) until used forwas given with bioformulation dissolved in water and the biochemical analysis. Variation in activity of defensesuspension was sprayed. Foliar spray with Streptomycin related enzymes, phenylalanine ammonia lyase (PAL),sulphate + Tetracycline @ 600ppm + Copper oxy chloride peroxidase (PO) and polyphenol oxidase (PPO) was@ 0.25% served as a chemical check. determined by enzyme assays according to Ross and

Bacterial Leaf Blight Disease Assessment: Three [32] respectively. The enzyme activity was expressedreplications were maintained for each treatment in as nmoles of cinnamic acid min g protein andcompletely a randomised design under glasshouse change in absorbance min mg of protein respectively.

formulae of McKinney [29]:

1

2

(FZB24) @4g/l3

T - ST + SD + SA + FA - Seed treat with FZB24@4g/kg +4

5

6

7

Sederoff [30], Hammerschmidt et al. [31] and Mayer et al

1 1

1 1

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Accumulation of total phenols was also estimated Statistical Analysis: The statistical analysis of the dataaccording to Zieslin and Ben Zaken [33] and it is were made using package IRRISTAT version 92 ofexpressed in mg of catechol/g of fresh tissue. Each of the International Rice Research Institute Biometrics unit,enzyme assays were atleast repeated three times to obtain Philippines.consistent results.

Native Polyacrylamide Gel Electrophoresis Analysis:The isoform profiles of PO and PPO were studied by Isolation and Pathogenicity: Xanthomonas oryzae pv.discontinuous native polyacrylamide gel electrophoresis oryzae (Xoo) were isolated on the mXOS agar medium(PAGE) (Laemmli [34]). The protein extract was prepared from the infected portion of leaf showing characteristicby homogenising 1 g of leaf sample in 2 ml of 0.1 M symptoms of water soaked yellowish lesions with wavysodium phosphate buffer pH 7.0 and centrifuged at margins on leaf blades. Xoo was artificially inoculated on16,000g for 20 min at 4°C. The samples were loaded into the leaves of susceptible rice variety ADT 39 by leaf clip8% polyacrylamide gels (Sigma, USA). After method under glass house conditions. Symptoms of waterelectrophoresis, PO isoforms were visualised by soaked lesions with wavy margin were observed atsoaking the gels in staining solution containing 10 days after inoculation of the pathogen.0.05% benzidine (Sigma Aldrich, Mumbai, India) and0.03% H O in acetate buffer (20 mM, pH 4.2) Isolation of Endophytes: Twenty six endophytic bacterial2 2

(Nadolny and Sequeira [35]). For assessing the strains were isolated from different field crop plants viz.,PPO isoform profiles, the gels were equilibrated for rice, greengram, cotton, redgram and ragi, medicinal plants30 min in 0.1% p-phenylene diamine, followed by the such as neem and noni, weed plants viz., Trianthema,addition of 10 mM catechol in the same buffer Acalypha, Tribulus, Cactus, Opuntia, Aloe and Agave.(Jayaraman et al. [36]). FZB24 strain of Bacillus subtilis var. amyloliquefaciens

Testing the Efficacy of Endophytes Against Bacterial Another thirteen strains of endophytes were obtainedLeaf Blight of Rice under Field Conditions: Two different from the culture collection centre of Department of Plantfield trials with rice cultivar ADT 39 were laid out in two Pathology, Tamil Nadu Agricultural University,different seasons under randomized block design with Coimbatore.three replications with a plot size of 4X3 m. The treatmentsdetails are as follows. In vitro Screening: Forty endophytic bacterial isolates

T. No. Treatment DetailsT Seed treat with FZB24@2g/kg1

T Seed treat with FZB24@4g/kg2

T Seedling dip with FZB24@2g/l3

T Seedling dip with FZB24@4g/l4

T Soil + Foliar app with FZB24@250g each /ha5

T Soil + Foliar app with FZB24@500g each /ha6

T T3 + Foliar app with FZB24@250g/ha7

T T4 + Foliar app with FZB24@500g/ha8

T T1 + T3 + Soil application @250g/ha + Foliar spray @9

250g each /ha with FZB24@30 Days After TransplantingT T2 + T4 + Soil application @500g/ha + Foliar spray @10

500g each /ha with FZB24@30 Days After TransplantingT Streptomycin sulphate + Tetracycline @ 600ppm + COC @11

0.25%T Control12

The observations on per cent incidence ofbacterial leaf blight were recorded on 90 days aftertransplanting. The growth parameters such as plantheight and number of tillers were recorded at differentintervals and the grain and straw yield was also recordedat the end of the crop.

RESULTS

was obtained from Novozymes South Asia Pvt. Ltd.

were screened against Xoo to test their efficacy ofinhibition. Among the isolates, FZB24, EPB 9, EPB 10,EPCO 29 and EPCO 78 showed the maximum inhibitionhalo of 20mm diameter followed by EPB 6, EPB 7, EPB 14and EPCO 16 with an inhibition halo of 19 mm diameter(Table 1).

Endophytic Bacterial Strains on Plant Growth Promotionof Rice: Rice seeds were treated with 40 endophyticbacterial strains separately and their effect on plantgrowth was studied by roll towel method in vitro.Endophytic bacterial isolates viz., EPB 18, EPB 11, EPCO74, FZB24 and EPB 10 were found to increase the vigourindex of the rice seedling significantly compared tountreated control. A maximum vigour index of 3343 wasrecorded by the EPB 18 isolate which was followed by theisolates viz., EPB 11, EPCO 74, FZB24 and EPB 10 withvigour index of 3225, 3127, 3035 and 3023 respectively.The untreated control has a very less vigour index of 1168(Table 1).

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Table 1: Effect of bacterial endophytic isolates against Xanthomonas oryzae pv. oryzae and its growth promoting activity on rice in vitroGrowth promotion activity-------------------------------------------------------------------------------------------------

S.No Isolates Inhibition zone of Antagonistic activity (mm) Root length (cm) Shoot length (cm) Germination (%) Vigour index*1 EPB 1 15.0 20.81 7.95 96 2761efg mn

2 EPB 2 15.0 19.58 7.93 94 2586efg o

3 EPB 3 14.0 20.90 7.20 100 2810fg jkl

4 EPB 4 12.0 20.20 7.70 100 2790hi lm

5 EPB 5 16.2 21.70 7.50 100 2920cde .h

6 EPB 6 19.0 21.30 9.33 98 3002ab ef

7 EPB 7 19.0 21.70 8.00 100 2970ab fg

8 EPB 8 15.6 18.54 8.21 98 2621def o

9 EPB 9 20.0 19.51 8.52 100 2803a klm

10 EPB 10 20.0 22.06 8.17 100 3023a de

11 EPB 11 15.0 23.54 8.71 100 3225efg b

12 EPB 12 10.6 19.09 8.49 84 2317ijk s

13 EPB 13 17.0 20.20 8.39 100 2859.cd i

14 EPB 14 19.0 19.11 8.12 100 2723ab n

15 FZB 24 20.0 21.90 8.45 100 3035a de

16 EPB 15 0.0 20.95 8.06 82 2379p r

17 EPB 16 15.0 15.99 8.31 100 2430efg q

18 EPB 17 17.0 20.44 8.00 100 2844cd ijk

19 EPB 18 14.0 23.88 9.55 100 3343 fg a

20 EPB 19 14.2 19.50 8.60 100 2810fg jkl

21 EPC 5 14.0 19.35 8.66 100 2801fg klm

22 EPC 8 17.8 19.39 7.90 100 2729bc n

23 EPCO 16 19.0 20.15 8.39 100 2854ab ij

24 EPCO 26 11.0 j 18.87 8.24 96 2603i o

25 EPCO 29 20.0 15.60 6.41 100 2201a t

26 EPCO 30 4.0 12.91 6.87 86 1701n v

27 EPCO 74 11.0 22.63 8.64 100 3127ij c

28 EPCO 78 20.0 16.63 8.64 94 2375a r

29 EPCO 81 2.0 20.99 8.48 100 2947o gh

30 EPCO 95 14.6 19.51 9.74 100 2925efg gh

31 EPCO 96 7.8 12.74 8.13 98 2045m u

32 EPCO 43 8.0 11.40 6.81 84 1529m w

33 EPCO 60 3.5 17.60 8.30 86 2227gh t

34 EPB 20 2.2 19.91 6.83 90 2407hi qr

35 EPB 21 8.3 19.90 8.55 92 2617lm o

36 EPB 22 12.3 21.60 8.10 100 2970hi fg

37 EPB 23 10.0 21.00 7.70 100 2870jkl i

38 EPB 24 9.2 20.44 8.00 88 2503klm p

39 EPB 25 9.0 13.00 6.01 88 1673klm v

40 EPB 26 8.0 20.7 8.06 90 2588m o

41 Control 0.0 8.70 5.90 80 1168 p x

* Mean of three replications. Means in a column followed by same superscript letters are not significantly different according to DMRT.

Efficacy of FZB24 under Glasshouse Conditions: 500g/ha with B. subtilis (FZB24) recorded a lowerBioformulation with endophytic Bacillus (FZB24) was intensity of 31.36 per cent BLB, which was 39.81 per centapplied to rice with different methods of application as reduction in intensity over control (Table 2).described earlier to determine its efficiency againstbacterial leaf blight pathogen, Xoo under glasshouse Effect of Endophytic Bioformulations on Plant Growthconditions along with chemical check of Streptomycin Parameters of Rice under Glass House Conditions:sulphate @ 600ppm + Copper oxy chloride @ 0.25%. Application of bioformulations enhanced the plantAmong the treatments, seed treatment @ 4g/kg + seedling growth in rice under glasshouse conditions compareddip @ 4g/l + soil application @ 500g/ha + foliar spray @ to untreated control and chemical treatment. The growth

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Table 2: Effect of B. subtilis (FZB24) treatments on the plant growth parameters and bacterial leaf blight intensity of rice (ADT39) under glasshouseconditions on 75 DAT

Bacterial leaf blight*---------------------------------------------------

T. No. Treatment Plant height* (cm) No. of Tillers* (PDI) % reduction over controlT Seed treat with FZB24@2g/kg 56.99 7.07 46.33 (42.89) 11.091

abc bcd c

T Seed treat with FZB24@4g/kg 57.37 7.84 36.49 (37.15) 29.982abc a f

T Seedling dip with FZB24@2g/l 56.80 6.89 50.33 (45.18) 3.423abc cd b

T Seedling dip with FZB24@4g/l 59.10 6.87 44.29 (41.71) 15.014ab cd d

T Soil + Foliar app with FZB24@250g each /ha 59.25 7.14 41.39 (40.03) 20.575ab bc e

T Soil + Foliar app with FZB24@500g each /ha 57.75 6.71 33.96 (35.64) 34.836abc de I

T T3 + Foliar app with FZB24@250g/ha 55.45 7.15 36.11 (36.93) 30.707cd bc g

T T4 + Foliar app with FZB24@500g/ha 57.86 7.42 33.16 (35.15) 36.378abc b j

T T1 + T3 + Soil application @250g/ha + Foliar spray @ 56.15 7.19 34.93 (36.22) 32.979bc bc h

250g each /ha with FZB24@30 DATT T2 + T4 + Soil application @500g/ha + Foliar spray @ 59.84 8.16 31.36 (34.05) 39.8210

a a k

500g each /ha with FZB24@30 DATT Streptomycin sulphate @600ppm + COC 0.25% 57.23 6.93 12.71 (20.88) 75.6111

abc cd l

T Control 53.10 6.36 52.11 (46.21) 0.0012d e a

* Mean of three replications. Means in a column followed by same superscript letters are not significantly different according to DMRT. Values in parentheses are arcsine-transformed values

paramaters viz., number of active tillers per hill and plant application @ 500g/ha + foliar spray @ 500g/ha withheight, were significantly higher in the treatment B. subtilis (FZB24) inoculated with Xoo compared tocombination of seed treatment @ 4g/kg + seedling dip @ untreated control plants. The activity was found to4g/l + soil application @ 500g/ha + foliar spray @ 500g/ha increase at 4 days after inoculation and, thereafter, it waswith the endophytic Bacillus FZB24 than other treatment declined. In contrast, the increased activity of PO andcombinations (Table 2). PPO was observed only up to the third day of Xoo

The treatment combination with seed treatment @ inoculation in untreated control plants and, thereafter,4g/kg + seedling dip @ 4g/l + soil application @ 500g/ha a drastic reduction in enzyme activity was documented+ foliar spray @ 500g/ha with B. subtilis (FZB24) recorded (Figure 1 and 2). Similarly, the assay of PAL from thehigher plant height of 59.84cm against 53.10cm on 75 days B. subtilis (FZB24) treated plants inoculated with Xooafter transplanting in untreated control. Similarly in case showed an enhanced activity compared to untreatedof number of productive tillers, the combination treatment control plants (Figure 5).of seed treatment @ 4g/kg + seedling dip @ 4g/l + soilapplication @ 500g/ha + foliar spray @ 500g/ha with Accumulation of Total Phenols: The application ofB. subtilis (FZB24) recorded higher number of tillers than B. subtilis (FZB24), as seed treatment @ 4g/kg + seedlingother treatments. Least number of tillers was observed in dip @ 4g/l + soil application @ 500g/ha + foliar spray @the untreated control plants (Table 2). 500g/ha resulted in higher accumulation of total phenols

Enhanced Activity of Defence Enzymes: The study of the inoculated and uninoculted healthy control plantsdisease resistance in rice plants treated with B. subtilis showed lower amount of accumulation of phenols than(FZB24) bioformulation revealed the higher activity and bacterized plants. A higher phenol accumulation wasexpression of defence-related proteins against Bacterial observed on 4th day of inoculation in above treatment.leaf blight pathogen. The activity of PO and PPO weremeasured in leaves from Xoo inoculated and B. subtilis Native PAGE Analysis of Defence Enzymes: The native(FZB24) bioformulations pretreated rice plants. B. subtilis PAGE analysis of enzyme extract from B. subtilis (FZB24)(FZB24) bacteria in different method of application treated as seed treatment @ 4g/kg + seedling dip @ 4g/ldiffered in their ability to stimulate PO and PPO in rice + soil application @ 500g/ha + foliar spray @ 500g/ha riceplants inoculated with Xoo. Increased PO and PPO plants inoculated with Xoo expressed four isoforms PO1,activity was observed in the combination treatment of PO2, PO3 and PO4, whereas in non-bacterised plants, onlyseed treatment @ 4g/kg + seedling dip @ 4g/l + soil two isoforms PO1 and PO4 were observed. (Figure 3).

in rice leaves challenged with Xoo (Figure 6). In contrast,

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Fig. 1: Induction of peroxidase activity in rice plants upon treated with endophytic B. subtilis (FZB24) challengaed withX. oryzae pv. oryzae under glasshouse cindition

Fig. 2: Induction of Polyphenol exidase activity in rice plants upon treated with emlophytic B. subtilis (FZB24)challenged with X. oryzae pv. oryzae under glasshouse condition

Fig. 3: Expression of PO isoforms in rice plants upon treatment with FZB24 and untreated control challenged withX. Oryzae pv. Oryzae under glasshouse conditions

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Fig. 4: Expression of PPO isoforms in rice plants upon treatment with FZB24 and untreated control challenged withX. Oryzae pv. Oryzae under glasshouse conditions

Fig. 5: Induction of Phenylalanine Ammonia Lyase in rice plants upon treated with B. Subtilis (FZB24) challenged withX. Aryzae pv. Oryzae under glasshouse condition

Fig. 6: Accumulation of total phenols activity in rice plants upon treated withendophytic B.subtilis (FZB24) challengedwith X. Oryzae pv. Oryzae under glasshouse condition

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Table 3: Effect of B. subtilis (FZB24) treatments on the plant growth parameters and bacterial leaf blight intensity of rice (ADT39) under Field conditions

Growth parametrs* Disease intensity* Yield*

------------------------------------------------------- ---------------------------------------------- ---------------------------------

S.No Treatment Plant Height 90 DAT (cm) No. of Tillers 60 DAT PDI % reduction over control Grain (kg/ha) Straw (kg/ha)

T Seed treat with FZB24@2g/kg 81.36 16.89 10.110 (18.53) 48.99 4078 63581j e c j i

T Seed treat with FZB24@4g/kg 82.58 17.52 7.580 (15.97) 61.76 4570 68132b b g b c

T Seedling dip with FZB24@2g/l 80.29 15.79 14.210 (22.13) 28.30 4042 61923k i b k j

T Seedling dip with FZB24@4g/l 81.93 15.78 10.110 (18.53) 48.99 4208 64384e j c h h

T Soil + Foliar app with FZB24@250g each /ha 81.85 16.11 9.670 (18.11) 51.21 4276 65615g h d f g

T Soil + Foliar app with FZB24@500g each /ha 81.48 15.51 9.220 (17.66) 53.48 4438 67736h k f e d

T T3 + Foliar app with FZB24@250g/ha 81.46 16.61 9.460 (17.9) 52.27 4220 66057i f e g e

T T4 + Foliar app with FZB24@500g/ha 81.93 17.16 5.780 (13.9) 70.84 4485 68418f c h c b

T T1 + T3 + Soil application @250g/ha + Foliar spray 82.20 16.93 5.720 (13.83) 71.14 4456 66029d d i d f

@ 250g each /ha with FZB24@30 DAT

T T2 + T4 + Soil application @500g/ha + Foliar spray 83.14 18.48 2.800 (9.62) 85.87 5025 705210a a j a a

@ 500g each /ha with FZB24@30 DAT

T Streptomycin sulphate @600ppm + COC 0.25% 82.51 16.30 2.070 (8.26) 89.56 4081 597711c g k i k

T Control 73.67 14.35 19.820 (26.42) 0.00 3720 544512l l a l l

* Mean of three replications.

Means in a column followed by same superscript letters are not significantly different according to DMRT.

Values in parentheses are arcsine-transformed values

Similarly, four isoforms of PPO1, PPO2, PPO3 and PPO4 DISCUSSIONwere observed in B. subtilis (FZB24) treated as seedtreatment @ 4g/kg + seedling dip @ 4g/l + soil application Endophytic bacteria colonize an ecological niche@ 500g/ha + foliar spray @ 500g/ha rice plants inoculated similar to that of plant pathogens, especially vascular wiltwith Xoo while in the controls, only one isoform was pathogens. Exploiting an additional microbial habitat fornoticed (Figure 4). biocontrol purposes might enhance overall biocontrol

Efficacy of B. subtilis var. Amyloliquefaciens (FZB 24) the endophytic agent could avoid unfavourableAgainst BLB under Field Conditions: Two differentfield trials conducted at different locations andseasons revealed that, among different treatments,application of B. subtilis (FZB24) bio-formulation asseed treatment @ 4g/kg + seedling dip @ 4g/l + soilapplication @ 500g/ha + foliar spray @ 500g/ha hasrecorded a lowest PDI of 2.80% with a per centreduction of 85.87% over control plots. The yieldattributing parameters viz., plant height and number ofactive tillers were recorded in both the field trials. Theplots treated with the treatment combination of seedtreatment @ 4g/kg + seedling dip @ 4g/l + soil application@ 500g/ha + foliar spray @ 500g/ha with B. subtilis(FZB24) recorded higher plant height of 83.14 cm on90 days after transplanting with maximum number oftillers as much as 18.48 on 60 days after transplantingwhere as untreated control plants recorded the plantheight of 73.67 cm with an average minimum number oftillers of 14.35. In addition to disease control, abovetreatment recorded a 35% increase in yield over untreatedcontrols, whereas in the chemical treatment, an increase ofonly 9.7% were recorded in both the trials (Table 3)through the severity is less.

efficacy and increase consistency in performance, since

conditions prevailing in the soil environment by enteringand localizing in the intercellular spaces of the epidermalcells of root tissues. Endophytic bacteria have shownsignificant control of diseases such as Fusariumvasinfectum in cotton (Chen et al. [37]; Van Buren et al.[38]), Verticillium albo-atrum, Rhizoctonia solani andClavibacter michiganesis subsp, sepedonicum inpotato (Nowak et al. [39]), Sclerotium rolfsii in bean(Pleban et al. [40]), Rhizoctonia solani, Pythiummyriotylum, Gauemannomyces graminis andHelerobasidium annosum in rice (Mukhopadhyay et al.[41]) and Fusarium moniliformae in maize (Hinton andBacon, [42]).

The development of biocontrol strategies usingendophytes is an emerging area in crop protection toreduce the damage caused by plant pathogens ineconomically important crops. Forty endophytes wereisolated from different plant sources. Similarly, endophyticbacterial isolates of Bacillus (39%), Pseudomonas(27.6%), Corynebacterium (16.7%), Actinomyces (11.1%)and Staphylococcus (5.6%) with enzymatic activity insolid media isolated from Jacaranda decurrens (amedicinal plant) were reported (Carrim et al. [43]).

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In the present study, endophytic bacterium, B. subtilis + seedling dip @4g/l + soil application @500g/ha + foliar(FZB24) was found to increase the vigour index of rice application @500g/ha both under glasshouse and fieldseedlings significantly and effectively inhibited the conditions. This treatment recorded more plant heightgrowth of Xoo in vitro. Similar report was given by with more number of tillers compared to untreated controlRajendran et al. [44] that endophytic Bacillus (EPC 5) plants. Upon artificial inoculation of X. oryzae pv. oryzae,inhibited the growth of Ganoderma lucidum in coconut. the endophytes treated (seed treatment @ 4g/kg +The endophytic Bacillus spp. CY22 isolated from balloon seedling dip @4g/l + soil application @ 500g/ha + foliarflower produced iturin A with antifungal activity against application @500g/ha on 30 days after transplanting)Rhizoctonia solani, Pythium ultimum and Fusarium plants recorded a significantly lower disease severity ofoxysporum (Cho et al. [45]). bacterial leaf blight compared to untreated control plants.

In addition to biological control, endophytic bacteria This finding is on par with findings of (Melnick et al.improved plant growth in different crops like potato [52]), that significant reductions of disease severity on(Sturz, [46]) and rice (Hurek et al. [47]). Hallmann et al. cacao leaf disks challenged with Phytophthora capsici[48] speculated that the observed plant growth promotion were recorded following colonization with endophyticin different crops might have been caused by enhanced Bacillus, BT8. Also Salah et al. [53] has found thatplant mineral uptake and improved plant water B. subtilis B49 found to be the most effective in inhibitingrelationships associated with the colonization of the growth of Xanthomonas axonopodis pv.endophytic strains. Some strains of Pseudomonas, malvacearum in vitro among 93 isolates of rhizobacteriaEnterobacter, Staphylococcus, Azotobacter and and also found to effectively control the bacterial blightAzospirillum produce plant growth regulators such as of cotton both under greenhouse and field conditions.ethylene, auxins or cytokinins and have, therefore, been Kloepper et al. [54] found that five of sixconsidered as causal agents for altering plant growth and rhizobacteria, which induced systemic resistance indevelopment. In addition to a direct mechanism for growth cucumber, exhibited both external and internalpromotion, plant growth promotion is also thought to be colonization. In addition, Pseudomonas fluorescens 89B-due to the suppression of deleterious microflora by 27 and Serratia marcescens 90-166 induced resistance inintroduced endophyte (Kloepper et al. [49]; Leifert et al. cucumber to Pseudomonas syringae pv. lachrymans as[50]). The beneficial effects of bacterial endophytes, well as to the fungal pathogens, F. oxysporum f. sp.however, vary and appear to operate through similar cucumerinum and Colletotrichum orbiculare (Liu et al.mechanisms as described for PGPR (Kloepper et al. [49]; [54]). In the present study, rice plants treated withHoflich et al. [51]). However, because of the different the bioformulation containing endophytic bacteriahabitats colonized, endophytes offer another tool for Bacillus subtilis (FZB24) with the treatment combinationdeveloping biological control strategies. By integrating of seed treatment @ 4g/kg + seedling dip @ 4g/l + soilthe use of bacterial endophytes with rhizosphere application @ 500g/ha + foliar spray @ 500g/ha andantagonists, a holistic biological control system could be challenged with Xoo showed higher induction of PO, PPOdeveloped that works against the pathogens. and PAL. The timing and expression patterns of the

The selection of bacterial endophytes for the defense mechanisms are important for the suppression ofmanagement of bacterial leaf blight of rice was done based pathogen. Higher level expression of defense relatedon the consideration of criteria such as growth promotion, proteins and timely accumulation of chemicals at theantagonistic activity against X. oryzae pv. oryzae, infection site certainly prevent the colonization ofantibiotics and ACC deaminase production. Considering pathogen in rice seedlings. The maximum accumulation ofall the above criteria Bacillus subtilis strain FZB24 was PO, PPO and PAL was observed on 4 days of challengeselected to assess its performance for the management of inoculation with Xoo after it starts declining.bacterial leaf blight of rice both under glasshouse and Similarly, the activities of PO, PPO and PAL werefield conditions. It was applied to the rice plants by found to be higher after two days of challenge inoculationdifferent methods of application such as seed treatment, after that it was declined in the cotton plants treated withseedling dip, soil application and foliar application at two endophytic Bacillus EPCO 16 and EPCO 102 upondifferent concentrations. It was also tried in combination challenge inoculation with X. axonopodis pv. malvacerumof different methods of application. (Rajendran et al. [55]). Thus, enhanced induction of

It was found that B. subtilis (FZB24) has performed defence related enzymes in endophyte treated plantswell in treatment combination viz., seed treatment @ 4g/kg might have been a part of ISR which eventually reduced

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the pathogen infection caused by X. oryzae pv. oryzae 6. Han, J.S., J.H. Cheng, T.M. Yoon, J. Song,upon artificial inoculation under glasshouse conditions.In the present study, higher levels of phenolics occurredin rice plants treated with endophytes against Xoo.Benhamou et al. [56] reported that the endophyticbacterium Serratia plymuthica raised levels of phenolicsin cucumber roots, affording protection against Pythiumultimum. Also Rajendran et al. [55] reported thatendophytic bacillus EPCO 16 and EPCO 102 increased theaccumulation of phenolics in cotton plant uponchallenged inoculation with Xam. Further, biocontrolstrains stimulate the activities of defence enzymes PO,PPO and PAL in plants that could be involved in thesynthesis of phytoalexins (Chen et al. [57]; van Loon andBakker [58]).

In the present study, incidence of bacterial leaf blightunder natural condition is found to be less in Bacillus(FZB 24) treated plots than the untreated plots. Incidencewas much lower in the treatment combination of seedtreatment @ 4g/kg + seedling dip @4g/l + soil application@500g/ha + foliar application @500g/ha on 30 days aftertransplanting. Also treated plots showed increased plantheight with maximum number of tillers and recorded highergrain and straw yield compared to untreated control plots.Similar result was observed in the work of Dunne et al.[59], that strain mixtures of DAPG producer P. fluorescensF113 and a proteolytic rhizobacterium enhanced the plantgrowth in terms of increased seedling vigour and grainyield.

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