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Indian Journal of Biotechnology Vol 13, April 2014, pp 247-255

Isolation and characterization of endophytic bacteria associated with chilli (Capsicum annuum) grown in coastal agricultural ecosystem N Amaresan1,2, V Jayakumar3 and N Thajuddin1* 1

Department of Microbiology, Bharathidasan University, Tiruchirapalli 620 024, India Division of Field Crops, Central Agricultural Research Institute (ICAR), Port Blair 744 105, India 3 Crop Protection Division, Sugarcane Breeding Institute, Coimbatore 641 007, India

2

Received 22 December 2012; revised 10 April 2013; accepted 7 May 2013 The antagonistic potentials of endophytic bacteria isolated from chilli plants were determined in vitro against four pathogens, viz., Sclerotium rolfsii, Fusarium oxysporum, Colletotrichum capsici and Pythium sp. The effect of endophytic bacteria towards these fungi revealed that most of the isolates showed antagonistic activity against Pythium sp. (37.8%), followed by 35.1% isolates against F. oxysporum and C. capsici, and 21.6% to S. rolfsii. The identification of potential bacterial isolates through Microbial Identification System (Biolog) and 16S rDNA sequencing of the isolates revealed the presence of 8 genera. Among them Bacillus species were the dominant antagonists. Characterized by BOX-PCR fingerprints, the 23 antagonistic endophytic bacterial (AEB) isolates represented 19 different cluster types. To explore the antagonistic mechanisms, the agar diffusion method was used to detect cell-wall degrading enzyme activity and siderophore secretion. The isolates BECS7, BECS4 and BECL5 showed clearly the growth promoting activity, reduction of disease incidence and high yield under field conditions. Hence, these isolates are promising plant growth promoting isolates showing multiple attributes that can significantly influence the chilli growth. The results of present study provide a strong basis for further development of these strain as bio-inoculants to attain the desired plant growth promoting activity in chilli growing fields. Keywords: Bacillus spp., Biolog, BOX-PCR, Capsicum annuum, microbial identification system, 16S rDNA

Introduction Endophytic bacteria are defined as bacteria that can be isolated from surface-disinfected plant tissues or extracted from within plants and are not observed to harm the host plants1. Endophytic bacteria have been isolated from the flowers, fruits, leaves, stems, roots and seeds of various plant species2 and have demonstrated a tremendous amount of diversity in plant hosts and in bacterial taxa as well3. Many factors, such as, plant genotype4,5, plant development and other biotic and abiotic environments5, are considered to influence the population structures of endophytic bacteria. Endophytic bacteria may play many important beneficial roles in the metabolism and physiology of the host plant6,7, including fixing atmospheric nitrogen, sequestering iron from the soil, solubilizing phosphates, synthesizing plant-growth hormones and suppression of ethylene production by 1-aminocyclopropane-1-carboxylate (ACC) deaminase, —————— *Author for correspondence: Tel: +91-431-2407082; Fax: +91-431-2407045 E-mail: [email protected]

degrading toxic compounds, inhibiting strong fungal activity and antagonizing bacterial pathogens. The internal plant tissues provide a protective environment for endophytic bacteria, which colonize an ecological niche similar to plant pathogens, especially vascular wilt pathogens8. Antagonistic endophytic bacteria (AEB) are promising agents for biological control agents. Berg and Hallmann6 confirmed that a significant portion of the indigenous endophytic bacteria in plant roots have antagonistic potential toward fungal pathogens, but little is known about the fluctuation and structure of the AEB population. In Andaman and Nicobar Islands of India, out of total geographical area of 8249 sq. km land, around 9% land is used for vegetable cultivation. Several diseases have been reported in Andaman and Nicobar Islands and some of them cause severe damage to the vegetable crops9. The objectives of the present study was to examine the diversity of AEB isolated from chilli and evaluate their antagonistic effects against some pathogenic fungi, such as, Sclerotium rolfsii, Fusarium oxysporum, Capsicum capsici and Pythium sp.

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Materials and Methods Sample Collection

Four localities of chilli cultivated areas (situated in different Islands of Andaman and Nicobar, India) endemic to wilt and root rot disease were selected for sample collection. Individual healthy and vigorous chilli plants with intact roots were collected in the pre-flowering stage and brought to the laboratory aseptically in an ice box and stored at 4°C. The samples were utilized for isolation of endophytes with in 48 h after collection. Isolation of Endophytic Bacteria

From the plant samples collected in each location, 5 g leaves were separated by excising the leaf parts proportionately from tip, middle and basal region. Similarly 5 g each of stem and root were collected to represent the sample from whole plant. Putative endophytic bacterial strains were isolated from leaf, root and stem of each chilli plant following the method of Zinniel et al10. The plant parts were surface sterilized and macerated individually with sterile mortar and pestle. The tissue extracts were diluted in 12.5 mM potassium phosphate buffer (pH 7.1) and plated on Nutrient agar for isolation of endophytic bacteria. The plates were incubated at 28±2ºC for 72 h, and total colonies development were enumerated, purified and maintained on slants. Antagonistic Activities against Plant Pathogenic Fungi

The antagonistic effects of endophytic bacterial isolates were tested in vitro against S. rolfsii, F. oxysporum, C. capsici and Pythium sp. The bacterial isolates were streaked at a distance of 3.5 cm from brink of individual Petri plate containing potato dextrose agar (PDA) medium. Mycelial disc (6 mm) from a 7-d-old PDA culture of the fungal pathogens was then placed on the other side of Petri dish and the plates were incubated at 28ºC for 4 d. The plates without endophytic bacteria served as control. The per cent inhibition was calculated by using the formula: I=(C–T)/C ×100, where I is per cent inhibition of mycelial growth over the control, C is mycelial growth of fungal pathogen in control and T is mycelial growth of fungus in endophytic bacteria inoculated plate. The experiment was carried out in three independent replicates. Production of Secondary Metabolites

The ability to produce indole-3-acetic acid (IAA) was quantified according to the method developed by

Sawar and Kremer11. The siderophore production was determined following the method of Schwyn and Neilands12. The phosphate solubilization ability was determined using Pikovaskayas agar amended with tri-calcium phosphate13, whereas HCN production was inferred by the qualitative method of Bakker and Schipper14. Identification with Biolog System

Selected isolates were tested for 73 carbohydrates and 23 chemical sensitivity patterns and identified using Biolog system kits. Bacterial isolates were raised on Biolog Universal Growth (BUG) medium and 24 h grown cultures were then suspended in inoculation fluid A (IFA) and inoculation fluid B (IFB), adjusted to required optical density and inoculated to 96 well plates. Plates were incubated at 30ºC and observed for colour development at intervals of 12 h. Colour development pattern was compared to the database and isolates were identified at species level. DNA Extraction and Amplification

The AEB were characterized by 16S rRNA gene partial sequence analysis. Genomic DNA was extracted using the method described elsewhere. The extracted DNA was dissolved in 20 µL TE buffer and used as the template for the PCR reactions. PCR amplifications were performed in a total volume of 50 µL by mixing 20 ng of the template DNA with 2.5 mM concentration of each deoxynucleotide triphosphate, 1 µM concentration of each primer of pA (5′-AGA GTT TGA TCC TGG CTC AG-3′) and pH (5′-AAG GAG GTG ATC CAG CCG CA-3′) as described by Edwards et al15 and 3 U of Taq DNA polymerase in 10× Taq buffer A (Genei, Bengaluru). These reactions were subjected to initial denaturation of 92ºC for 2 min and 10 sec, followed by 35 cycles of 92ºC for 1 min, 48ºC for 30 sec and 72ºC for 2 min and 10 sec, and a final extension step of 72ºC for 6 min and 10 sec using GeneAmp® PCR system 9700 (Applied Biosystems). The PCR products were resolved using 1% agarose gel. Sequencing and Phylogenetic Analysis

The partial sequencing of purified PCR product was undertaken at Oscimium Biosciences, Bangalore, India, using the above mentioned forward primer. 16S rRNA gene sequence of the isolate was compared with 16S rRNA gene sequences available by the BLASTN search in the NCBI, GenBank database (http://www.ncbi.nlm.nih.gov). Multiple sequence

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alignment was performed using Clustal X. The method of Jukes and Cantor16 was used to calculate evolutionary distances. Phylogenetic dendrogram was constructed by the neighbour-joining method and tree topologies were evaluated by performing bootstrap analysis of 1000 data sets using MEGA 4.1 (Molecular Evolutionary Genetic Analysis). The sequences were submitted to GenBank (acc. no. GU569854-67 & GU569878-79).

agar methods, where nutrient agar medium was supplemented with 0.01% CaCl2.H2O. Tween 80 sterilized for 20 min at 120ºC was added to the molten agar medium at 45ºC to give a final concentration of 1%. The medium was shaken until the Tween 80 had dissolved completely and then was poured onto Petri plates. An opaque halo formed around the colonies was considered as positive test.

BOX-PCR

Biocontrol and Yield under Field Conditions

To reveal the genetic diversity of endophytic bacteria, DNA extracted from the endophytic bacteria was amplified. Using the BOXA1R primer 5′-CTA CGG CAA GGC GAC GCT GAC G-3′, BOX-PCR was performed according to the procedure of Nick et al17. PCR products were separated by gel electrophoresis in 2% agarose in 0.5× Tris-borateEDTA buffer for 16 h and stained with ethidium bromide. The banding patterns were converted to a two-dimensional binary matrix (1 denotes presence of a band; 0 denotes absence of a band). Similarity matrices were calculated with the Dice coefficient and a cluster analysis of similarity matrices was performed using the unweighted pair group method with arithmetic averages (UPGMA) using NTSYSpcversion 2.10e18 . Analysis of Extra Cellular Enzyme Activity

The isolates were analyzed for production of 4 enzymes, viz., protease, amylase, cellulase and lipase, by plate method. Proteolytic activities of the cultures were screened qualitatively in a medium containing skimmed milk (HiMedia, Mumbai). Zones of precipitation of paracasein around the colonies appearing over the next 48 h were taken as evidence of proteolytic activity. The presence of amylolytic activity on plates was determined routinely using starch agar medium (HiMedia, Mumbai). After incubation at 28ºC for 5 d, the plates were flooded with Gram’s Iodine solution, a clear zone around the growth indicated hydrolysis of starch. For cellulase activity, mineral-salt agar plate containing 0.4% (NH4)2SO4, 0.6% NaCl, 0.1% K2HPO4, 0.01% MgSO4, 0.01% CaCl2 with 0.5% carboxymethylcellulose (CMC) and 2% agar (HiMedia, Mumbai) were surface inoculated. Iodine solution was used to detect cellulase activity as described by Kasana et al19. The clear zone formation around the growing colony was considered as positive. The lipase activity of bacterial isolates was determined by the diffusion

The talc-based bioformulation of the 8 individual strains (selected based on antagonistic, plant growth promoting and hydrolytic enzyme properties) were tested under field conditions to study their efficacy for large-scale adoption. Seed treatment, seedling root dipping, soil application and foliar spray of endophytic strains were conducted as described previously20 . Chilli seeds were soaked for 24 h in water containing talc-based formulation (4 g/kg of seeds) of individual strain. The seeds soaked in distilled water alone served as control. Treated seeds were allowed to sprout and sown in nursery bed. The seedlings were pulled out 30 d after sowing and planted in the main field; soil texture-sandy loam, pH-6.5, organic carbon-0.52%, electrical conducutivity0.2 dS/m, low in available N (215 kg/ha), P (10.5 kg/ha) and K (112 kg/ha. Recommended dose of fertilizer (150:100:50 kg of NPK/ha) was applied through urea, super phosphate and muriate of potash. The roots of chilli seedlings in bundles were dipped in water containing talc powder formulation (20 g/L) for 2 h and then transplanted. After draining the water from the field, the talc-based powder product (2.5 kg/ha) was mixed with 50 kg of farm yard manure (FYM) and broadcast 30 d after planting as soil application. For foliar spray, the talc-based product was dissolved in water (0.1%) and allowed to settle for 1 h, filtered through muslin cloth and the filtrate was sprayed 30 d after planting. The efficacy of the strains was assessed by comparing with the plants raised from the seeds treated with thiram (2.5 g/kg seeds) and foliar spray of fytolan (COC) (2.5 g/L) at 30 d after planting. Untreated control was also maintained. Natural incidence of damping off, root rot, leaf spot, fruit and leaf anthracnose and wilt were recorded in each plot from 40 d after planting. The yield was recorded at the time of harvest for all treatments. Two field trials were conducted with three replications in factorial randomized complete block design during 2009 and 2010 at the farmer’s field, Burmanallah,

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South Andaman. Standard plot size of 2×1 m2 was maintained for all treatments. Development of disease symptoms associated with chilli infection was observed and assessed based on the appearance of the symptoms. Disease development was expressed as disease incidence percent (DI, %) according to the formula: DI (%) =

No. of infected seedlings × 100 Total no. of seedlings assessed

Statistical Analysis

Statistical method data from the completely randomized design experiment on in vivo biocontrol activity were arc sine transformed prior to statistical analysis. Results were analyzed using one-way ANOVA (AGRES ANOVA package version 7.01, Tamil Nadu Agricultural University, India). The field experiment was repeated twice. Since the variances between the experimental trials were homogeneous, data were pooled and analyzed using one-way ANOVA21. Results

respectively (Table 2). Ten isolates (27.0%) showed significant antagonism towards all pathogens. Biolog Identification

Differentiation and identification of 23 bacterial strains showing antagonistic property were carried out using Biolog Micro Station System, an automated identification system. Test yielded a characteristic pattern of substrate utilization for each isolate, which was compared to a current GENIII database. Table 3 gives identity of the 23 isolates. Isolates BECS4, BECS5, BECR12, BECL10, BECL11, BECR13, BECR14, BECS7 and BECL5 gave similarity index less than 0.3. Based on the Biolog identification, biocontrol endophytic bacteria belonged to Bacillus spp. (12), Serratia spp. (4), Microbacterium spp. (2), Achromobacter sp. (1), Providencia sp. (1) and Enterobacter sp. (1). 16S rDNA Sequence and Phylogenetic Analysis

Sixteen bacterial strains, which showed less similarity index in Biolog identification, were identified on the Table 2—Antagonistic activity of chilli endophytic bacterial isolates against the phytopathogens No. Isolate name

Antagonistic Activity of Endophytes against Plant Pathogens

A total of 37 endophytic bacteria were isolated (11 from leaf and stem and 15 from root) from chilli plant samples (Table 1). The endophytic isolates were tested in vitro for antagonistic activity against 4 common fungal plant pathogens, viz., S. rolfsii (colar rot), Pythium sp. (damping off), F. oxysporum (wilt) and C. capsici (anthracnose). Among the isolates 13, 14, 13 and 8 strains were antagonistic to F. oxysporum, Pythium sp., C. capsici and S. rolfsii, Table 1—Endophytes isolated from chilli plants cultivated in Andaman and Nicoba Island No.

Place of collection

1

Mangulton

N: 11º36’30” E: 092º40’42”

2

Sadasnagar

N: 12º39’29.5” E: 092º53’28.5”

3

Neil Island

N: 11º49’30” E: 093º1’0”

5

R K Gram

N: 13º14’18.1” E: 092º58’34.0”

GPS data

Total

Plant parts Stem Leaves Root Stem Leaves Root Stem Leaves Root Stem Leaves Root

No. of phenotypes isolated 3 3 3 3 2 3 2 2 5 3 4 4 37

*Growth inhibition of pathogen over control (%)

S. rolfsii Pythium sp. C. capsici F. oxysporum 1 BECL1 23.0 32.6 30.0 21.9 2 BECS1 45.9 11.1 40.0 42.9 9.6 37.8 22.2 20.0 3 BECR1 4 BECR2 28.9 33.3 33.3 39.0 32.6 18.5 30.0 27.6 5 BECL4 6 BECS4 43.0 8.9 36.7 37.1 7 BECS5 33.3 33.3 36.7 39.0 46.7 33.3 34.4 48.6 8 BECR6 9 BECS8 33.3 41.5 31.1 37.1 10 BECS6 36.3 45.2 41.1 42.9 31.1 33.3 30.0 30.5 11 BECR12 12 BECL8 37.8 14.1 36.7 42.9 13 BECL9 37.8 25.9 46.7 33.3 11.1 20.0 31.1 42.9 14 BECL10 15 BECL11 38.5 19.3 43.3 37.1 16 BECR13 8.9 37.8 12.2 52.4 13.3 18.5 32.2 50.5 17 BECR14 18 BECR15 25.9 37.8 21.1 35.2 19 BECR16 15.6 28.9 38.9 42.9 20 BECS9 20.0 17.0 27.8 47.6 21 BECS7 37.8 45.9 35.6 42.9 22 BECS3 35.6 35.6 36.7 37.1 29.6 33.3 31.1 48.6 23 BECL5 CD (0.05) 0.37 0.40 0.33 0.43 SEd 0.19 0.20 0.17 0.22 *Values are mean of three replications

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basis of 16S rDNA partial sequences. Accordingly BECR2, BECR12, BECL10, BECL11, BECR14, BECS7, BECL5 and BECR13 were identified as Bacillus spp., BECS4, BECS5 and BECR16 were Achromobacter spp., BECL4 and BECR15 Providencia spp., BECR6 and BECS9 were Microbacterium spp., and BECS1 was Arthrobacter sp. (Table 4). Phylogenetic analyses of the strains based on the neighbor joining method resulted into 8 major clusters (Fig. 1). Cluster I formed with Bacillus sp. and B. megaterium, cluster II formed with Bacillus sp., cluster III formed with B. licheniformis, cluster IV with B. circulans, cluster V with Arthrobacter sp., cluster VI with Microbacterium spp. and cluster VII and VIII formed with P. rettgeri and Achromobacter spp., respectively. Table 3—Identification of bacterial isolates using Biolog system No.

Isolate name

Microbial Identification System (Biolog) Organisms identified

1

BECL1

2 3 4 5 6

BECS1 BECR1 BECR2 BECL4 BECS4

7 8

BECS5 BECR6

9

BECS8

10

BECS6

11 12

BECR12 BECL8

13 14 15 16 17 18 19 20 21 22 23

BECL9 BECL10 BECL11 BECR13 BECR14 BECR15 BECR16 BECS9 BECS7 BECS3 BECL5

Microbacterium imperiale Bacillus licheniformis B. circulans B. amyloliquefaciens Providencia rettgeri Achromobacter piechaudi A. piechaudi Enterobacter cloacae ss dissolvens Serratia marcescens ss marcescens S. marcescens ss marcescens B. cereus S. marcescens ss marcescens B. megaterium B. megaterium B. subtilis ss subtilis B. megaterium B. flexus P. rettgeri S. entomophila M. imperiale B. megaterium B. weihenstephanensis B. licheniformis

Probability Similarity 0.99

0.80

0.86 0.99 0.70 1.00 -

0.56 0.80 0.54 0.65 0.25

0.99

0.29 0.83

0.99

0.68

0.93

0.67

0.86 0.81 1.00 0.87 0.98 0.95 -

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Clustering of BOX Fingerprints of AEB

BOX-PCR analysis resulted in 7-18 specific PCR bands with fragments sized from 0.3 to 3.5 kb. The PCR generated patterns examined by cluster analysis showed a high genotypic diversity (Fig. 2). Based on a cut-off level of 0.50 genetic similarities, the 23 AEB isolates were placed in total of 21 clusters; 2 clusters with 2 isolates each and 19 clusters with a single isolate. The largest cluster containing BECL5 and BECR12 (Bacillus spp.) originated from Sadasnagar and R K Gram. The second largest cluster of BECS14 and BECS3 originated from R K Gram and Mangulton, corresponding to Bacillus spp.. AEB Characterization Based on Production of Hydrolytic Enzymes and Secondary Metabolites

The ability of the AEB isolates to produce cell-wall hydrolyzing secondary metabolites were investigated (Table 5). In total, 15 out of 23 AEB isolates had shown the protease activity: Bacillus spp. (9 isolates), Serratia spp. (3 isolates), Providencia spp. (2 isolate) and Enterobacter spp. (1 isolate). Further, cellulase activity was detected in 15 isolates: Bacillus spp. (9 isolates) and Achromobacter spp., Providencia spp. and Serratia spp. (2 isolates each). Twelve isolates Table 4—Identification of bacterial isolates based on 16S rDNA partial sequences and comparisons with system Biolog No. Isolate name

1 2 3 4

BECS1 BECR2 BECL4 BECS4

0.14 0.56

5 6

BECS5 BECR6

0.52 0.21 0.21 0.38 0.29 0.84 0.57 0.74 0.23 0.69 0.12

7 8 9 10 11 12 13

BECR12 BECL10 BECL11 BECR13 BECR14 BECR15 BECR16

Bacterial species identified based on Biolog

16S rDNA partial sequences

Bacillus licheniformis B. amyloliquefaciens Providencia rettgeri Achromobacter piechaudi A. piechaudi Enterobacter cloacae ss dissolvens B. cereus B. megaterium B. subtilis ss subtilis B. megaterium B. flexus P. rettgeri Serratia entomophila

Arthrobacter sp. Bacillus sp. P. rettgeri Achromobacter sp. A. xylosoxidens Microbacterium sp. Bacillus sp B. circulans B. megaterium B. megaterium Bacillus sp. P. rettgeri Achromobacter sp. Microbacterium sp. Bacillus sp. B. licheniformis

14 BECS9

Microbacterium sp.

15 BECS7 16 BECL5

B. megaterium B. licheniformis

% similarity in BLAST match 98 99 99 98 98 96 98 96 99 95 98 99 98 98 99 98

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Fig. 1—Neighbour joining phylogenetic tree based on 16S rRNA gene sequences and their closest phylogenetic neighbours. Bootstrap values are indicated at nodes. Scale bar represents observed number of changes per nucleotide position.

showed amylase activity: Bacillus spp. (8 isolates), Serratia spp. (2 isolates), Providencia sp. and Achromobacter sp (1 isolate each). Lipase activity was detected in only two isolate, while HCN production was undetected in AEB isolates. Furthermore, siderophore were found to be produced by 15 isolates: Bacillus spp. (9 isolates), Serratia spp. (4 isolates), Providencia sp. and Microbacterium sp. (1 isolate each). Majority of the isolates produced IAA in the range of 9.5 to 27.5 µg/mL, whereas isolate BECL5 (Bacillus sp.) produced 44.0 µg/mL and BECL4 (Providencia sp.) 84.5 µg/mL IAA. Only 7 isolates showed inorganic phosphate solubilization, in which Serratia spp. (3 isolates), Bacillus sp., Providencia sp., Enterobacter sp. and Microbacterium sp. had one isolate each. The mechanisms of AEB antagonism towards S. rolfsii, F. oxysporum, C. capsici and Pythium sp. are complex, but the excretion of siderophores and other enzyme production may play a crucial role. Biocontrol, Plant Growth and Yield under Field Conditions

The isolates from chilli tested for their influence on growth parameters showed considerable influence on

Fig. 2—Dendrogram showing the relationship of antagonistic endophytic isolates based on BOX-PCR fingerprints, genetic similarities (DICE) and UPGMA cluster analysis were calculated using NTSYSpc-version 2.10e program.

the chilli crops. They showed increased shoot and root length and also increased root and shoot biomass at the nursery stage. Results from the two field trail revealed that inoculation of talc based formulations with BECS7, BECS4 and BECL5 were effective in controlling almost all the diseases to the tune of chemical control. The magnitude of disease reduction varied between the individual strains; however, the same trend of effects for each treatment was observed on disease control in two field experiments. The yield was the highest in BECS4 (9.43 t/ha) treatment, followed by BECS7 (9.13 t/ha) and BECR6 (6.53 t/ha) in comparison to untreated control, which recorded the lowest yield (5.15 t/ha) (Table 6). Discussion A comprehensive study was conducted on chilli endophytic bacteria, which showed in vitro antagonistic response to S. rolfsii, C. capsici, F. oxysporum and Pythium sp., using a cultivation-based approach to characterize their behaviour. Previously, several studies on endophytic bacteria have also been performed in ginseng, cotton, sweet corn, canola and balloon flower. Similarly, Germida et al22 have also isolated 18 different endophytic bacterial genera from 220 isolates in canola roots. Thus, it seems clear that endophytic communities are diverse and the

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Table 5—Plant growth promoting and hydrolytic enzymes properties of antagonistic endophytic isolates from chilli No.

Isolate name IAA production (µg/mL)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

BECL1 BECS1 BECR1 BECR2 BECL4 BECS4 BECS5 BECR6 BECS8 BECS6 BECR12 BECL8 BECL9 BECL10 BECL11 BECR13 BECR14 BECR15 BECR16 BECS9 BECS7 BECS3 BECL5

Siderophore production

HCN production

PO4 solubilization

Amylase

Protease

Cellulase

Lipase

+ ++ + + + ++ + + + + + + + + ++

-

+ + + + + + +

+ + + + + + + + + + +++ ++ -

+ + +++ ++ ++ + + ++ ++ + + + + ++ + -

+++ + +++ + + + ++ ++ + ++ ++ + + ++ + -

+ + -

9.5 21.2 14.2 16.0 84.5 16.5 18.7 16.5 18.7 16.2 27.5 15.0 14.5 14.5 10.2 13.7 18.5 16.2 11.5 13.0 15.2 15.0 44.0

+ Weak reaction; ++ Moderate reaction; +++ Strong reaction Table 6—Evaluation of endophytic bacteria on disease incidence and yield of chilli under field conditions Diseases Treatment

Damping off (%)

Root rot (%)

Wilt (%)

Leaf spot (PDI)

Anthracnose on leaf (PDI)

Anthracnose on fruit (PDI)

BECS1 16.67 (24.98)d 20.37 (26.75)de 18.52 (25.39)bc 36.00 (36.83)bc 30.67 (33.62)cd 13.33 (21.33)cd BECS7 2.00 (6.69)a 0.00 (0.48)a 24.07 (29.28)c 33.33 (35.15)abc 22.00 (27.91)bc 4.00 (11.28)a cd c d abc bcd 31.33 (33.99) 8.00 (16.08)abc BECL11 11.33 (19.34) 17.59 (24.59) 23.15 (28.72) 32.67 (34.83) bc cd e abc ab BECR6 8.00 (16.08) 40.00 (39.22) 25.93 (30.56) 16.67 (24.05) 16.67 (23.94) 18.67 (25.55)de BECS5 9.33 (17.32)bcd 0.00 (0.48)a 22.22 (28.10)c 32.67 (34.80)abc 18.00 (25.02)ab 24.67 (29.70)e ab bc bc a ab BECS4 7.33 (15.33) 11.11 (19.38) 14.81 (22.35) 24.67 (29.70) 18.00 (24.96) 6.00 (14.05)ab BECR2 10.00 (18.06)bcd 12.04 (20.11)bc 24.07 (29.28)c 30.00 (33.16)ab 28.00 (31.94)cd 13.33 (21.33)cd bcd a ab bc b BECL5 9.33 (17.63) 0.00 (0.48) 13.89 (21.82) 34.67 (36.04) 21.33 (27.47) 8.67 (16.96)abc bc b a bc a Chemical 8.00 (16.21) 10.19 (18.17) 11.11 (19.38) 37.33 (37.61) 14.67 (22.48) 12.00 (19.85)bcd Control 14.67 (22.45)cd 25.93 (30.48)e 18.52 (25.39)bc 49.33 (44.62)d 28.67 (32.32)cd 24.00 (29.22)e CD (.05) 7.09 5.71 5.58 5.84 4.94 6.26 SEd 3.38 2.72 2.66 2.78 2.35 2.98 PDI=Plants disease index Values in parenthesis are arc-sine transformed values Means followed by same letter are not significantly different (p = 0.05) by DMRT on arcsine-transformed values

Yield (t/ha) 7.12 (11.73)bcd 9.13 (8.08)abc 7.72 (11.24)bcd 6.53 (10.25)cd 6.92 (9.09)bcd 9.43 (10.09)ab 7.05 (11.24)bcd 8.76 (10.35)abc 10.76 (9.38)a 5.15 (8.32)d 2.64 1.26

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extent of diversity may vary significantly between plant species. In the present study, among the six different bacterial genera isolated, Bacillus species were dominant in chilli plants. Many factors, such as, plant rotation, soil condition and phytopathogen population are known to influence the population structures of endophytic bacteria. Besides, endophytic bacteria in a single plant host are not restricted to a single species but comprise of several genera and species. To exploit endophytic bacteria for biological control, we further investigated the 23 AEB isolates. Based on Biolog identification 23 AEB consisted of 6 genera and the genera of Bacillus spp. (12 out of 23) were predominant among the AEB. The isolates which showed less similarity index in Biolog identification were further confirmed with 16S rDNA sequence analysis. Except 3 isolates, BECS1 (B. licheniformis) identified as Arthrobacter sp., BECR6 (E. cloacae) identified as Microbacterium sp. and BECR16 (S. entomophila) identified as Achromobacter sp., the remaining isolates identity was the same as in Biolog identification. Further, 3 AEB isolates (BECR6, BECL10 & BECR13) demonstrated ≤97% sequence similarities (identities ranged from 95-96%) with the most similar sequences of strains in the Ribosomal Database Project. Using BOX-PCR fingerprinting data, the 23 AEB isolates were clustered in 21 different groups (Fig. 2). Therefore, we can presume that, in the chilli plants, not only exist a high diversity of endophytic bacteria but also a large variety of AEB. This was not unexpected, as it has been previously reported that some genera harbour more antagonistic potential, for example, presence of AEB against the fungal and the bacterial pathogens in field-grown potato23. AEB from chilli samples showed the activity of different hydrolytic enzyme. Hydrolytic enzymes help the endophytic bacteria to enter into the plant root. Plant cell wall hydrolytic enzymes play important roles in plant-microbe interactions and intercellular colonization of microorganisms in plant roots. It has been suggested that hydrolytic enzymes might only be produced by endophytes during the early invasion phase and not after residing in the plant tissue24. In the present study, maximum hydrolytic enzyme activity was shown by Bacillus spp. (BECS1, BECR12, BECL9, BECL10, BECL11, BECR14, BECS7 & BECS3), Serratia spp. (BECL8 & BECR16) and Providencia sp. (BECR15). Further, lipase activity was shown by only 2 isolates (BECL10

& BECL11), while none of the isolates showed the production HCN. Almost all the AEB isolates showed the production of one or more enzymes detected in the present study. However, isolates BECL5, BECS9, BECS8, BECR2 and BECL1 did not show any enzyme production, suggesting that these isolates could have entered inside the plant by stomata, lenticels, wounds including broken trichomes, areas of emerging lateral roots and the germinating radical25. In the present study, the bacterial strains that produced different hydrolytic enzymes, such as, protease, cellulase, amylase and lipase, had also inhibited the growth of pathogenic fungi S. rolfsii, C. capsici, Pythium sp. and F. oxysporum. Nielson and Sorensen26 also demonstrated that lytic enzyme production acted as an antagonistic mechanism against R. solani, P. ultimum, F. culmorum and F. oxysporum. Further, the proportion of endophytes that were not only able to suppress pathogenic fungi but could also improve seed germination and plant growth have been found. Nejad and Johnson27 described the isolates of endophytic bacteria that significantly improved the seed germination and plant growth of oilseed rape and tomato. This phenomenon can be attributed to the ability of the isolates to produce IAA, as IAA positively influences root growth and development, thereby enhancing nutrient uptake28. The nature of endophytic relationship of bacteria with plants is least understood at present29. The intimate relationships between the endophytic bacteria and their hosts, however, make them natural candidates for selection as bio-control agents30. This further highlights the need for selecting bacterial types with high levels of plant habitat competence because it is often considered necessary for successful seed or root bacterization treatments before planting. In conclusion, in the present study, BECS7 (Bacillus sp.), BECL5 (B. licheniformis) and BECS4 (Achromobacter sp.) stands out as a possible candidate for use as bio-control agent with plant growth promoting (PGP) characteristics and these were isolated from plants grown under coastal agricultural ecosystems. Hence, it is proposed that potential strains observed in the present study can be deployed as bio-inoculants to increase chilli growth and yield, and to control the incidence of disease. Acknowledgment The authors are thankful to the Director, Central Agricultural Research Institute, Port Blair for

AMARESAN et al: ENDOPHYTIC BACTERIA ASSOCIATED WITH CHILLI

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