Biocontrol potential of phylloplane bacterium Ochrobactrum anthropi BMO-111 against blister blight disease of tea

Authors


Correspondence

Sellamuthu Manian, Department of Botany, School of Life Sciences, Bharathiar University, Coimbatore – 641046, Tamil Nadu, India. E-mail: sellamuthumanian@yahoo.com

Abstract

Aims

The present study was carried out to screen the phylloplane bacteria from tea for antagonism against grey blight caused by Pestalotiopsis theae and blister bight caused by Exobasidium vexans and to further evaluate the efficient isolates for disease control potential under field condition.

Methods and Results

A total of 316 morphologically different phylloplane bacteria were isolated. Among the antagonists, the isolates designated as BMO-075, BMO-111 and BMO-147 exhibited maximum inhibitory activity against both the pathogens under in vitro conditions and hence were selected for further evaluation under microplot field trial. Foliar application of 36-h-old culture of BMO-111 (1 × 108 colony-forming units ml−1) significantly reduced the blister blight disease incidence than the other isolates. The culture of BMO-111 as well as its culture filtrate effectively inhibited the mycelial growth of various fungal plant pathogens. The isolate BMO-111 was identified as Ochrobactrum anthropi based on the morphological and 16S rDNA sequence analyses.

Conclusions

It could be concluded that the biocontrol agent O. anthropi BMO-111 was effective against blister blight disease of tea.

Significance and Impact of the Study

Further study is required to demonstrate the mechanism of its action and formulation for the biocontrol potential against blister blight disease of tea.

Introduction

Tea [Camellia sinensis (L.) O. Kuntze], the oldest known beverage, is an intensively managed perennial monoculture crop, cultivated in large- and small-scale plantations. Its popularity is attributed to its sensory properties, relatively low retail price, stimulating effects and potential health benefits. India is the largest producer of tea, which accounts for 20% of global area and constitutes 28% of world production. Tea is manufactured from young shoots of the tea plant; hence, the leaf diseases are of great concern.

The grey blight disease caused by the fungus Pestalotiopsis/Pestalotia spp. is the second important foliar disease in tea. It was the first tea leaf disease to be reported from tea and created great panic in earlier days. It generally affects mature foliage, bare stalks and young shoots (Sanjay and Baby 2005). The blister blight incited by the fungus Exobasidium vexans Massee is the most serious leaf disease in all tea-growing areas of Asia. The pathogen is a biotroph with no alternate host, and the life cycle is completed on tea itself. The pathogen attacks harvestable tender shoots, inflicting enormous yield losses up to 40%, and the quality deterioration is noticed even below the 35% disease threshold level (Gulati et al. 1993). Spread of the disease is highly dependent upon weather conditions. The disease is favoured by the relative humidity in the range of 60–100%. The fungus produces extracellular enzymes that degrade the polysaccharides of the cell wall and thereby gains entry into the cells (Albersheim et al. 1969).

A variety of fungicides with different modes of action are used in tea for the control of various leaf diseases (Muraleedharan and Chen 1997). Although fungicides have shown some promising results in controlling the foliar pathogen, phytotoxicity and fungicide residues are the major problems besides causing environmental pollution and human health hazards. Furthermore, the repeated use of such chemicals encourages the development of resistance in the target organisms (Goldman et al. 1994). Under this scenario, the search continues for environmentally more friendly methods to control diseases that will contribute to the goal of sustainability in agriculture. There is a pressing need in tea industry either for exclusively utilizing biological products in disease management or for reducing the use of chemicals by supplementing with biological products in integrated management practices.

In line with this, biological control of foliar fungal diseases of tea by utilizing antagonistic micro-organism is also rarely practiced, but the results of preliminary research hold considerable promise for the biological management of tea diseases (Baby et al. 2004; Saravanakumar et al. 2007; Sanjay et al. 2008). Therefore, an attempt was made to screen micro-organisms isolated from phylloplane of tea and identify new biocontrol agents for the effective control of blister blight diseases of tea in the field.

Materials and methods

Study area

All the field studies were carried out in the tea plantations of Parry Agro Industries Ltd, Valparai, Coimbatore district, Tamil Nadu, India, during June 2006 to December 2007. Thirty-year-old tea bushes [C. sinensis (L.) O. Kuntz] of Assamica variety were used to evaluate the efficacy of phylloplane isolates against E. vexans causing blister blight disease. The plantations are located in the Western Ghats at an elevation of c. 2200 m MSL (mean sea level). There was drastic fluctuation in the weather pattern with a temperature range of 12–30°C, relative humidity of 85–95% and mean rain fall of c. 1500 mm per annum.

Isolation of phylloplane bacteria

Samples of tea plant (harvestable shoots containing three leaves and a bud) were collected from the different tea plantations (Murugalli, Sheikalmudi, Pudhukadu, Kalyanapandal and Iyerpadi) of Parry Agro Industries Ltd, Valparai, Coimbatore (District), Tamil Nadu, India, during June 2006.

Leaf washing technique was followed to isolate the phylloplane bacterial strains. The shoot samples (10 g) were suspended in 100 ml of sterile distilled water and kept in a rotary shaker at 150 rev min−1 for 15 min. The washing thus obtained was serially diluted up to 10−8 dilutions, and 0·1 ml from 10−5 to 10−8 dilutions was spread-plated on nutrient agar (NA) medium. After incubation at 20°C for 2 days, morphologically different colonies that appeared on the media were selected, subcultured and purified. The purified isolates were maintained in NA slants as well as in 20% glycerol vials.

Screening of the phylloplane bacteria for antagonistic activity

All the isolated phylloplane bacteria were subjected to primary screening against the test pathogen Pestalotiopsis theae on potato dextrose agar (PDA) medium. Pestalotiopsis theae, the foliar pathogen of tea, was isolated from the naturally infected tea leaves. The isolate was identified up to species level by referring the study by Guba (1961). The primary screening was carried out by dual-culture plate method and incubated at 30°C for 5 days. At the end of incubation period, the zone of inhibition was recorded by measuring the distance between the edges of the fungal mycelium and the antagonistic bacterium.

The phylloplane bacterial isolates showing antagonistic activity against P. theae were further screened for their potential to inhibit the spore germination of E. vexans. The basidiospores harvested in the beakers were suspended in sterile distilled water. The spore suspension was diluted (1 × 108 spores per ml) and used in spore germination study. One millilitre of culture filtrate of the bacterial isolate (36-h-old cultures grown on nutrient broth (NB) and centrifuged at 12 000 rev min−1 for 10 min to remove the cells) that showed antagonism against P. theae was mixed with 1 ml of E. vexans basidiospore suspension (1 × 108 spores per ml). In the control, 1 ml of sterile water was used in the place of culture filtrate. After 24 h of incubation, the spores of E. vexans were examined under microscope for germination, and the percentage of inhibition of spore germination was calculated. Based on the in vitro screening, the most effective bacterial strains were subjected to field experiment.

Evaluation of the efficacy of the antagonists under microplot trial

Selected phylloplane bacterial isolates (BMO-075, BMO-111 and BMO-147), based on their effectiveness under in vitro tests, were further evaluated in microplot trial for their potential in controlling blister blight disease under field condition. Broth cultures of these antagonistic bacterial isolates (36-h-old) containing 1 × 108 colony-forming units (CFU) ml−1 in water was applied as foliar spray. The biocontrol agent Pseudomonas fluorescens Pf1 (1 × 108 CFU ml−1) obtained from Tamil Nadu Agricultural University, Coimbatore, India, was included in the field study for comparison. In the blister blight disease trial plots, the field practice of chemical fungicide spray with copper oxychloride (COC) (0·3% w/v) + hexaconazole (0·3% v/v) was included as positive control. All the formulations were added with 0·5 ml l−1 of Triton E as surfactant.

The microplot experiment was conducted using complete randomized block design. There were totally nine treatments for each disease trial as given below, with three replications in each treatment. Each treatment replicate (microplot) had ten bushes, and the spray schedule was conducted on every fifth day.

Treatment details

  • Treatment 1: Untreated control (Sterile NB medium)
  • Treatment 2: BMO-075
  • Treatment 3: BMO-111
  • Treatment 4: BMO-147
  • Treatment 5: BMO-075 + BMO-111 (1 : 1 concentration)
  • Treatment 6: BMO-075 + BMO-147 (1 : 1 concentration)
  • Treatment 7: BMO-111 + BMO-147 (1 : 1 concentration)
  • Treatment 8: Ps. fluorescens Pf1
  • Treatment 9: Chemical fungicide – COC + hexaconazole

The blister blight incidence was recorded during plucking rounds at 15-day interval from 0 to 120 days. During each plucking round, five samples each containing 25 tea shoots were randomly collected from individual plots and examined individually for the presence of lesions and expressed as percentage of disease incidence.

Based on the performance of the antagonistic bacterial isolates in the microplot field experiment, the isolate BMO-111 was selected for further laboratory studies.

Effect of BMO-111 on spore germination

Conidia of P. theae from 7-day-old culture plates were harvested in sterile distilled water using a sterile brush, and the spore concentration was adjusted to 106 conidia per ml using distilled water. A 50-μl aliquot of the conidial suspension was mixed separately with equal volumes of 36-h-old BMO-111 culture broth, culture filtrate or the chemical fungicide (dithane M45, 0·3% w/v) in cavity slides. Conidial suspension with sterile medium served as the control. The slides were then placed in Petri plates lined with moist filter paper and incubated for 12 h at room temperature under dark condition. The slides were then observed for conidial germination under a light microscope. Five replications were maintained for each treatments, and the percentage of inhibition of conidial germination was calculated. In the case of E. vexans, the basidiospore suspension (108 spores per ml) was used for the study of inhibitory effect of BMO-111 on the basidiospore. The basidiospore germination was assessed as for the conidiospores of P. theae. Each experiment was repeated three times and the mean of inhibitory percentage recorded.

Antagonistic activity of BMO-111 against different phytopathogens

The isolate BMO-111 was tested for antagonistic activity against a wide range of phytopathogens viz. P. theae, Rhizoctonia solani, Phytophthora infestans, Fusarium oxysporum, Sclerotium rolfsii, Helminthosporium oryzae, Botryodiplodia theobromae and Curvularia lunata on PDA medium employing dual-culture technique. All the said phytopathogenic isolates were obtained from Centre for Advanced Studies in Botany, University of Madras, Chennai, India except P. theae that was isolated in this laboratory. Further, the culture filtrate of BMO-111 was bioassayed for antifungal effects by agar diffusion method. The isolate BMO-111 was grown on NB medium at 28·0°C in a rotary shaker (150 rev min−1) for 36 h. To obtain the cell-free supernatant, the culture broth was centrifuged at 12 000 rev min−1 for 10 min. The wells (9 mm diameter) were made into the PDA medium containing Petri plate using a sterile cork borer, and 100 μl of clear supernatant was loaded into each well for the assay of antagonistic activity against different pathogens. The dishes were preincubated at 4·0°C for 2 h to allow uniform diffusion into the agar. After preincubation, the plates were incubated at 30°C for 5 days. The antifungal activity of the culture filtrate was evaluated by measuring the diameter of inhibition zones observed. Each experiment was repeated three times and the mean of inhibition zones recorded.

Experiments to assess the possible mechanism(s) of biocontrol by BMO-111

The production of siderophores by BMO-111 was determined by the Chrome Azurol S (CAS) assay (Schwyn and Neilands 1987). Lytic enzymes production such as cellulase, amylase, protease, chitinase and lipase by substrate (Cellulose, Starch, Casein, Chitin and Tween 80, respectively) amended water agar medium. The production of hydrogen cyanide (HCN) was determined according to the method of Lorck (1948).

Extraction of antifungal metabolites

BMO-111 was grown in NB medium for 36 h at 37°C. The culture was centrifuged at 12 000 rev min−1 for 15 min, and the cell-free supernatant was used for further studies. The culture supernatant of BMO-111 was extracted with two volumes of various organic solvents (petroleum ether, diethyl ether, chloroform and ethyl acetate) using separating funnels. Further, the crude extracts were tested for their antifungal activity against the pathogen P. theae. The crude extract (100 μl) was placed in a 9-mm well made using a sterile cork borer in PDA medium. The antifungal activity was determined by measuring the inhibition zone of mycelial growth of the test pathogen caused by the crude extract.

Identification of the isolate BMO-111

Various physiological and biochemical tests were carried out for the identification of the isolate BMO-111 according to the methods outlined in the Bergey's Manual of Determinative Bacteriology.

The 16S rDNA sequencing was carried out with the help of the Institute of Microbial Technology (IMTECH), Chandigarh, India. The chromosomal DNA of BMO-111 was isolated according to the procedure described by Sharma and Singh (2005). The 16S rDNA was amplified using the primers 8-27f (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1500 r (5′-AGAAAGGAGGTGATCCAGGC-3′). Then the DNA fragments were eluted from the gel and purified using QIAquick gel extraction kit (Qiagen, Hilden, Germany). The rDNA sequence was determined by the dideoxy chain-termination method using the Big-Dye terminator kit using ABI 310 Genetic analyzer (Applied Biosystems, Foster City, CA, USA). The 16S rDNA sequence of the isolate BMO-111 generated in this work with 1338 bases was deposited in the GenBank (MTCC accession number 9026). The sequence was aligned with the other 16S rDNA sequences retrieved from the GenBank database. A sequence similarity search was performed using GenBank BlastN (Altschul et al. 1997). Sequences of closely related taxa were retrieved, aligned using Clustal X program, and the alignment was manually corrected. For the neighbour-joining analysis, the distances between the sequences was calculated using Kimura's two-parameter model. Bootstrap analysis was performed to assess the confidence limits of the branching (Felsenstein 1985).

Statistical analysis

The data were subjected to a one-way analysis of variance (anova) (spss 9.0; SPSS Inc., Chicago, IL). The significance of effects of treatments was determined by the magnitude of F value (< 0·05), and the treatment means were separated by Duncan's multiple range test. Values expressed are means of three replicate determinations (n = 3) ± standard deviation (SD).

Results

Isolation and screening of phylloplane bacteria

A total number of 57 phylloplane samples (tender leaves with mature blister blight lesions) were collected from the moderately to highly blister blight infested fields in different tea plantations of Parry Agro Industries Ltd, Valparai, Coimbatore (District), Tamil Nadu, India. From the samples, 316 phylloplane bacterial strains were isolated by leaf washing technique using NA medium.

The bacterial isolates from phylloplane of tender tea leaves showing mature blisters were screened for antagonism against P. theae by dual-plate assay. Among the 316 isolates tested, 262 failed to show any antagonism. The remaining 54 phylloplane bacterial isolates suppressed the growth of the test fungus P. theae with inhibition zones ranging from 5 to 21 mm. Among them, 29 isolates showed zone of inhibition of 5 mm, 22 showed 5–10 mm and three isolates showed zone of inhibition of >15 mm.

Of the culture filtrates of 54 effective isolates antagonistic against P. theae, only 26 inhibited spore germination of E. vexans. The same three isolates also showed higher mycelial inhibition against P. theae. These highly antagonistic bacterial isolates were designated as BMO-075, BMO-111 and BMO-147. They were selected for further evaluation of their biocontrol potential against blister blight disease under microplot field condition.

Efficacy of antagonists against blister blight disease of tea under microplot trial

The effective antagonists (BMO-75, BMO-111 and BMO-147) were evaluated for their efficacy to control the blister blight disease under microplot field trial. The blister blight disease incidence was observed for 120 days after the first spray, at 15 days interval. Among the various treatments, BMO-111 sprayed plots recorded significantly lower incidence of disease than the other treatments, followed by chemical fungicide treatment. Interestingly, the combination of any two isolates resulted in significantly higher disease incidence than the treatment with individual bacterial isolates. Compared with untreated control, BMO-111 and chemical treatment gave respectively 73·4% and 64·7% protection against blister blight disease incidence on day 120 after the initial spray (Table 1).

Table 1. Effect of antagonistic phylloplane bacteria on blister blight disease incidence in tea plants under microplot trial
TreatmentDisease incidence (%)*
0 days15 days30 days45 days60 days75 days90 days105 days120 days
  1. Values are means of three replicate determinations. Mean values followed by different superscripts in a column are significantly different (< 0·05).

  2. T2–T8, bacterial culture (1 × 108 colony-forming unit ml−1); T5–T7, combination (1 : 1); T9, copper oxychloride 0·3% (w/v) + hexoconazole 0·3% (v/v). Triton E was added to the spray formulation as surfactant at the rate of 0·5 ml l−1.

  3. *Scored at 15-day interval after initiation of 5-day spray cycles with antagonistic bacteria and fungicides.

T1 – Untreated control28·2a61·3f60·1e65·3g59·3f24·0f32·0d34·0d27·6e
T2 – BMO-07528·4a36·3c37·1b43·3d41·6c13·7c22·9b23·3b15·6d
T3 – BMO-11128·1a28·0a32·1a30·5b32·6b8·0a19·0a18·7a7·3a
T4 – EMO-14727·5a41·6d45·9c46·0e54·6e16·4d25·7c23·1b19·2e
T5 – BMO-075 + BMO-11128·7a59·0f54·9d54·0g53·3e19·3e26·3c24·9c21·2e
T6 – BMO-075 + BMO-14727·6a57·0f56·3d49·2f49·8d19·7e27·6c26·1c23·1f
T7 – BMO-111 + BMO-14728·2a47·6e51·1d51·3e50·6d19·0e28·0c23·4b19·5e
T8 – Pseudomonas fluorescens Pf127·3a36·5c36·9b34·4c35·7c10·2b22·3b22·7b12·4c
T9 – Chemical fungicides27·9a30·6b32·0a27·0a25·3a7·2a18·9a19·6a9·7b

Effect of BMO-111 on conidial germination of Pestalotiopsis theae and basidiospore germination of Exobasidium vexans

The conidial germination of P. theae was 85·3% in the sterile NB. However, treatment with BMO-111 culture (62·8%), BMO-111 culture filtrate (71·8%) or dithane M45 (73·4%) resulted in significant decreases in conidial germination (Table 2). The basidiospore germination of E. vexans was as high as 92·3% in sterile NB medium. However, BMO-111 culture/culture filtrate and the chemical fungicides remarkably inhibited the spore germination of E. vexans when compared with control (Table 2).

Table 2. Inhibitory effect of BMO-111 on conidial germination of Pestalotiopsis theae and spore germination of Exobasidium vexans
TreatmentConidial germination (%)Basidiospore germination (%)
  1. Values are means of three replicate determinations ± standard deviation.

  2. Values in parenthesis indicate percentage of inhibition over control.

  3. Mean values followed by different superscripts in a column are significantly different (< 0·05).

  4. COC, copper oxychloride.

BMO-111 culture31·7 ± 1·4 (62·8)b16·7 ± 1·4 (81·9)b
BMO-111 culture filtrate24·1 ± 0·8 (71·8)a13·1 ± 1·2 (85·8)a
Dithane M45 (0·3% w/v)22·7 ± 1·1 (73·4)a 
COC (0·3% w/v) + hexaconazole (0·3% v/v) 12·2 ± 0·7 (86·8)a
Sterile nutrient broth (control)85·3 ± 2·3c92·3 ± 3·1c

Screening of BMO-111 for antifungal activity against different fungal pathogens

Screening of BMO-111 against a wide range of plant pathogens revealed that the bacterial isolate effectively inhibited the mycelial growth of P. theae (Fig. 1), R. solani, P. infestans, F. oxysporum, S. rolfsii, H. oryzae, B. theobromae and C. lunata, and the inhibition zone ranged from 9 to 23 mm (Table 3). These pathogenic species demonstrated differences in their sensitivity to the antagonistic effect of the isolate BMO-111. The culture filtrate of the isolate BMO-111 also remarkably inhibited the mycelial growth of plant pathogens at the concentration of 100 μl per well. The inhibition zones produced by the culture filtrate ranged from 9 to 21 mm against the pathogenic fungi.

Figure 1.

Antagonistic activity of BMO-111 against Pestalotiopsis theae.

Table 3. Antifungal activity of the isolate BMO-111 against different plant pathogens
Plant pathogenInhibition zone (mm)
CultureCulture filtrate
  1. Values are means of three replicate determinations ± standard deviation.

Pestalotiopsis theae 23 ± 221 ± 3
Rhizoctonia solani 19 ± 415 ± 2
Phytophthora infestans 09 ± 112 ± 1
Fusarium oxysporum 11 ± 214 ± 1
Sclerotium rolfsii 13 ± 212 ± 2
Helminthosporium oryzae 14 ± 309 ± 3
Botryodiplodia theobromae 18 ± 220 ± 2
Curvularia lunata 11 ± 119 ± 3

Mechanism of biocontrol by Ochrobactrum anthropi BMO-111

Ochrobactrum anthropi BMO-111 was initially subjected to various in vitro tests to ascertain the mechanism of its biocontrol activity. It neither produced siderophores nor extracellular lytic enzymes nor HCN. However, antifungal activity was noticed only in the nonprotein fractions of its culture filtrate. Among the various solvents (petroleum ether, diethyl ether, chloroform and ethyl acetate) used for the extraction of antifungal metabolites from the culture filtrate, ethyl acetate extract exhibited maximum (23 mm) antifungal activity against P. theae in well diffusion method (Fig. 2).

Figure 2.

Antifungal activity of ethyl acetate extract of BMO-111 culture filtrate against Pestalotiopsis theae.

Identification of BMO-111

Morphology and physicochemical characteristics

The isolate BMO-111 showed good growth on NA. Light microscopic observation showed that it is a rod-shaped bacterium. Further, BMO-111 is Gram-negative, obligatory aerobic and grows under 5–40°C and pH 3–9. No colony pigment development was observed.

16S rDNA analysis

The 16S rDNA sequence of BMO-111 was generated for a total of 1338 nucleotide base pairs (Fig. 3). The sequence was compared with those of different reference species of Ochrobactrum, Brucella and Mycoplana available in the GenBank database. The sequence analysis showed the closest match of 100% similarity with O. anthropi. The high bootstrap support of the tree derived from the 16s rDNA analysis demonstrated that strain BMO-111 is a typical member of the genus Ochrobactrum (Fig. 4).

Figure 3.

16S rDNA sequences of BMO-111.

Figure 4.

Phylogenetic tree showing the position of Ochrobactrum anthropi BMO-111. Neighbour-Joining tree showing the position of strain BMO-111 compared with related organisms in a 16S rRNA gene tree. Numbers at nodes indicate levels of bootstrap support (%) based on 1000 resampled data sets. The scale bar indicates the number of nucleotide substitutions per site.

The results of the physicochemical studies further support the 16S rDNA analysis. Based on the above, the isolate BMO-111 is designated as O. anthropi BMO-111. The strain has been deposited in MTCC with accession number – 9026 (MTCC-9026). The gene sequence was also submitted to GenBank under accession number JX455164.

Discussion

Interest in biological control of plant pathogens has increased in recent years, partly due to public concern to the indiscriminate use of hazardous chemical pesticide. In biological control, the potential of antagonistic micro-organisms and the significance of antibiotics in biocontrol and more generally in microbial interactions have been highlighted (Raaijmakers et al. 2002). The present study was aimed at investigating the biological control of E. vexans, the causal organism of blister blight disease, using the antagonistic bacterial isolates from phylloplane of tea.

Leaf surfaces provide physical environments suitable for growth and reproduction of certain micro-organism. These phylloplane micro-organisms are influenced by external factors such as microclimate, anatomical features, physical variations, environmental changes and by the use of agrochemicals (de Jager et al. 2001). The occurrence of leaf inhabiting micro-organisms on leaf surface is reported in several plant species in both temperate and tropical countries (Kenerley and Andrews 1990; Fahmy and Ouf 1999). The ecological significance of microbes on plant surfaces has been reviewed by Pandey (1999). In the present study, a total of 316 bacterial strains were isolated from 57 phylloplane tea samples obtained from different tea estates in Valparai, Coimbatore district, Tamil Nadu, India. These phylloplane isolates exhibited different colony types, shape and colour.

Among the antagonists, three isolates designated as BMO-075, BMO-111 and BMO-147 exhibited maximum antagonistic activity against both the pathogens under in vitro conditions. Baby et al. (2004) also reported 82 antagonistic bacterial strains against P. theae among 889 phylloplane isolates. Reports are available that the phylloplane micro-organisms can be antagonistic to various foliar pathogens. Some examples are Sporobolomyces roseus antagonistic to Septoria nodorum (Fokkema and Van der Meulan 1976), Ps. fluorescens to Sartorial terypticae (Levy et al. 1992) and Bacillus spp. and Pseudomonas spp. to Phytophthora of rubber.

BMO-111 was effective in controlling blister blight disease of tea and was even better than the chemical fungicides (COC + hexaconazole) (Table 2). Similar study was conducted by Baby et al. (2004) who reported that the strains APB 78, CPB 77, MPB 138, WPB 104, WPB 109 and NLB 12 were promising biocontrol agents in controlling blister blight disease of tea. The phylloplane isolates BMO-111, BMO-075 and BMO-147 gave better disease control individually (Table 1). However, in combination they gave only poor disease control. This may be attributed to their incompatibility to coexist.

Ji et al. (2006) also reported that combined use of foliar biological control agent Pseudomonas syringae Cit7 and PGPR strain Ps. fluorescens 89B-61 provided significant control over bacterial speck and spot of tomato in each trial. However, combined use of these two strains did not enhance suppression of bacterial spot in 1998; bacterial speck in 1997, indicating that combined use of these two biological control agents is not always synergistic.

Several workers have attempted to control foliar fungal disease of tea using antagonistic phylloplane micro-organisms. Chakraborty et al. (1995) reported that the phylloplane micro-organisms isolated from tea were effective antagonists against the foliar pathogen Glomerella cingulata. Particularly, the bacterium Micrococcus luteus showed good antagonism to G. cingulata and P. theae (Chakraborty et al. 1998). Effectiveness of the phylloplane isolates of Bacillus subtilis in controlling black rot disease of tea has also been reported.

In the present study, we observed the variations in blister blight disease incidence during the experiment period. The disease incidence was mainly depends on the weather parameters. Generally, the pathogen infects tender harvestable tea shoots, and the pathogen completes its life cycle in a short span of 11–28 days. The entire life cycle is completed in 11 days under conducive weather conditions or else it could extend up to 28 days (Baby 2002). Hence, the blister blight disease incidence was varied in every plucking round (harvesting of three leaves and a bud). Average weekly sunshine hours ranging from 2 to 10 h and temperature range of 10–23°C favoured disease development. A mean daily rainfall ranging from 20 to 100 mm favoured the disease; however, with the rainfall above 120 mm, the relationship was weak. The disease was favoured by relative humidity in the range of 60–100% and the availability of tender shoots (Premkumar et al. 2002). The climatic factors in southern India and Sri Lanka favour blister blight epidemic over a period of 6 months in a year. The disease incidence and spore load in the atmosphere was high during the months of June to December. On the contrary, the disease incidence was comparatively low during dry months of January to March and completely absent in April and May.

Based on the data obtained on the control of blister blight disease under microplot field condition, the antagonistic isolate BMO-111 was chosen for further studies. Apart from its antagonistic activity against P. theae and E. vexans, the isolate BMO-111 also exhibited different levels of antagonistic activity against other fungal plant pathogens as indicated by strong inhibition zones. The microscopic studies revealed mycelial deformities in these plant pathogens in the zone of inhibition. The culture filtrate of BMO-111 also remarkably inhibited the growth of all the tested pant pathogens. This clearly indicates that the inhibitory principle is extracellular in nature and is being released in the culture during the growth of the antagonistic bacterium. Production of antifungal metabolites by antagonistic bacteria has been reported earlier by many authors (Kaur et al. 2006; Prabavathy et al. 2006). In the present study, BM0-111 did not produce siderophore, extracellular enzymes or HCN. Hence, some of the secondary metabolites present in the culture might be responsible for its biocontrol potential.

Encouraged by the antagonistic potential of the selected isolate BMO-111 in both in vitro and in vivo studies, an attempt was made to characterize and identify it. The taxonomic identity of the strain BMO-111 was ascertained by various physicochemical parameters and 16S rDNA sequencing as O. anthropi, and hence, the isolate was designated as O. anthropi BMO-111. Species of Ochrobactrum have been described as free-living α-proteobacteria. Ochrobactrum strains occur in different habitats including soil, rhizosphere, plant, water, animals and humans. The environmental isolates of Ochrobactrum spp. are frequently associated with plants, because they have mostly been isolated from the rhizosphere or the rhizoplane (Lebuhn et al. 2000).

The potential of the bacterium O. anthropi as a biofungicide in controlling the fungal diseases of plants under field and in vitro conditions was noted by few other workers. Chakraborty et al. (2009) reported that the tea rhizosphere isolate of O. anthropi, either in aqueous suspension or in talc formulation, induced growth of tea plants and suppressed brown root rot disease caused by Phellinus noxius. It induced defence responses in tea plants. Chaiharn et al. (2009) found that O. anthropi D 5.2 isolated from rice rhizosphere in northern Thailand exhibited a good antagonistic effect against F. oxysporum. An attempt was made to use O. anthropi and/or its cultured product for controlling soil blight of solanaceous plants caused by bacterial pathogen Pseudomonas solanacearum (Kazuharu and Hidenori 1997). In the present study, O. anthropi BMO-111 achieved better performance when compared with the conventional field practice of chemical fungicides (COC + hexaconazole).

Conclusion

It may be stated that the present study has successfully developed a new biocontrol agent, O. anthropi BMO-111. In vitro and field experiments have shown that the bacterium was effective in controlling blister bight disease of tea incited by E. vexans. As such, exploitation of this phylloplane bacterium for the control of the fungal plant pathogens is less expensive, safer and could serve as a good alternative to synthetic fungicides.

Ancillary

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