Integrated control of blister blight disease in tea using the biocontrol agent Ochrobactrum anthropi strain BMO-111 with chemical fungicides

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 aimed to evaluate the integration of Ochrobactrum anthropi BMO-111 and chemical fungicides (copper oxychloride and hexaconazole) against blister blight disease of tea.

Methods and Results

Application of the liquid culture of O. anthropi BMO-111 (36-h-old culture broth) was found to be effective in combined sprays with individual chemical fungicides (copper oxychloride and hexaconazole). Spray application of O. anthropi BMO-111 to tea bushes improved the biochemical parameters such as the levels of chlorophyll, polyphenols, and catechins in the harvestable tea shoots. Moreover, in the microplot and large scale trials, the integrated treatment of every two O. anthropi BMO-111 sprays followed by a single fungicides spray was found to be more efficient than the stand alone O. anthropi BMO-111 or chemicals sprays. Further, pathogenicity study employing Swiss albino mice showed no mortality in the test animals when challenged with O. anthropi BMO-111 through oral, intravenous and intranasal routes.

Conclusions

The field trials clearly established that O. anthropi BMO-111 has capability to reduce incidence in integrated management of blister blight disease of tea and safe to use in the field.

Significance and Impact of the Study

The results indicate that O. anthropi BMO-111 can be used as an agricultural input in the integrated crop protection systems.

Introduction

Tea (Camellia sinensis (L.) O. Kuntze) is one of the most popular beverages in the world owing to its taste, the stimulative effect, and also for its health benefits. Perennial habit of the tea plant, peculiar cultural conditions and warm humid climate of the tea growing areas are highly conducive for disease development (Baby 2002). Leaf diseases are more important due to the obvious reason that tea plant is cultivated for its young succulent leaves (Muraleedharan and Chen 1997). Among the leaf diseases, blister blight caused by Exobasidium vexans is the most important one. The disease is known to occur in almost all tea growing areas of Asia. E. vexans is an obligate parasite with no alternate host (Fig. 1a–c). Hence, its life cycle has to be completed on tea plant itself. The entire life cycle is completed in 11 days under conducive weather conditions or else it could extend up to 28 days (Venkataram 1964). The disease affects directly the harvestable crop (tender leaves and stem) from which tea is manufactured. Hence, the crop loss is enormous and the quality deterioration is noticed even below the 35% disease threshold level. Comparisons between the crop harvested from tea fields protected by fungicide spray and those left unsprayed indicated a loss of 50% to blister blight disease in six months (Venkataram 1970).

Figure 1.

Blister blight disease of tea. (a) Abaxial surface view of mature blister. (b) Mature, sporulating blister lesions. (c) Necrotized lesions on leaves.

Fungicidal protection is the prime strategy in the control of plant diseases. In tea, the maximum fungicide usage was for the control of leaf diseases such as blister, grey blight and anthracnose (Muraleedharan and Chen 1997). Chemical fungicides have shown some promising results in control the blister blight disease. Overuse of chemical pesticides causes phytotoxicity, fungicide residues, and environmental pollution and thus, the use of microbial biological control agents has drawn attention and has been considered as a promising alternative to chemical pesticides (Goldman et al. 1994). However, it is difficult to assess 100% suppressibility of plant disease only by application of microorganisms. Therefore, the integrated use of chemical pesticides and microorganisms may be one of the practical methods applicable in the field. By this method, a reduction in the amount of chemical pesticides used in is anticipated (Mandeel 1996; Kondoh et al. 2001). Integrated approach of disease management using chemical and biological components is likely to be more attractive to growers than using them in isolation.

Integrated disease management is a long-term control strategy that combines biological, cultural, and chemical tactics. Integrated disease management has been shown experimentally to be more effective than classical methods (such as biological or chemical control only) (Tang and Cheke 2008). In our previous studies, Ochrobactrum anthropi BMO-111 was more efficient than chemical fungicides in controlling blister blight diseae of tea (Sowndhararajan et al. 2013). In support to our study, the biological control potential of the bacterium O. anthropi in controlling the fungal diseases of plants under field and in vitro conditions were evaluated by some authors (Chaiharn et al. 2009; Chakraborty et al. 2009). Therefore, an attempt was made to reducing the use of chemicals by combined and alternate with O. anthropi BMO-111 in integrated management practices against blister blight disease of tea. Further, the effect of spray treatments on the biochemical parmeters of tea leaves and pathogenicity study of the isolate using mice model were carried out.

Materials and methods

Study area

The field study was carried out in the tea plantations of Parry Agro Industries Ltd., Valparai, Coimbatore district, Tamil Nadu, India during June to December, 2007. Thirty-year-old tea bushes (Camellia sinensis (L.) O. Kuntz.) of Assamica variety were used to evaluate the efficacy of O.  anthropi BMO-111 against Exobasidium vexans causing blister blight disease. The plantations are located in the Western Ghats at an elevation of c. 2200 m mean sea level (MSL). 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.

Biocontrol agent used in this study

The isolate BMO-111 has been isolated from the phylloplane of tea, which showed higher antagonistic activity against Pestalotiopsis theae and E. vexans under in vitro. Further, the isolate effectively decreases the blister blight disease incidence under microplot field experiment (Sowndhararajan et al. 2013). Based on the results of physicochemical studies and 16S rDNA analysis, the isolate BMO-111 was identified as O.  anthropi and 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.

Formulation of BMO-111 for field application

The 36-h-old culture broth from the optimized medium was directly used for the spray experiments. The formulation of O. anthropi BMO-111 contained 1 × 108 colony forming unit (CFU) ml−1 of the bacterial cells and 0·5 ml l−1 of Triton E. Generally for spray treatments, 5 l of 36-h-old O. anthropi BMO-111 culture and 150 ml of Triton E were mixed with 300 l of water for every one hectare of tea plantation.

Optimized medium for the growth of O. anthropi BMO-111

Peptone5·0 g
Beef extract3·0 g
Sodium chloride5·0 g
Mannitol5·0 g
Distilled water1000·0 ml
pH7·0

Combined efficacy of O. anthropi BMO-111 with chemical fungicides in blister blight disease management under microplot trial

The treatments include O. anthropi BMO-111 and chemical fungicide [copper oxychloride (COC) + hexaconazole] sprays in isolation as well as in combined schedules. The BMO-111 formulation contains 1 × 108 CFU ml−1 of 36-h-old O. anthropi BMO-111 cultures in nutrient broth medium. The chemical fungicide used was a mixture of COC (0·3% w/v) + hexaconazole (0·3% v/v) in water. In the combined schedule, the following formulations of chemical fungicide and Oanthropi BMO-111 were mixed and used. Each treatment had 100 tea bushes and the spray schedule was maintained at 15-day-interval.

Treatment details

T1Untreated control (sterile nutrient broth medium)
T2O. anthropi BMO-111 (1 × 108 CFU ml−1)
T3O. anthropi BMO-111 (1 × 108 CFU ml−1) + COC (0·15% w/v)
T4O. anthropi BMO-111 (1 × 108 CFU ml−1) + hexaconazole (0·15% v/v)
T5O. anthropi BMO-111 (1 × 108 CFU ml−1) + COC (0·15% w/v) + hexaconazole (0·15% v/v)
T6Chemical fungicide (COC, 0·3% w/v + hexaconazole, 0·3% v/v)
T7Integrated trial: O. anthropi BMO-111 (1 × 108 CFU ml−1) and COC (0·3% w/v) + hexaconazole (0·3% v/v)
Every two rounds of O. anthropi BMO-111 sprays followed by one round of COC + hexaconazole spray.

The blister blight disease incidence was recorded for 90 days. Disease assessments were made during plucking rounds at 15-day-interval by picking ten samples of 50 harvestable shoots (three leaves and a bud) were randomly collected from individual plots and examined individually for the presence of lesions and expressed as percent disease incidence (Sowndhararajan et al. 2013).

Reisolation of O. anthropi BMO-111

In the experimental field, O. anthropi BMO-111 was reisolated during every plucking round by using leaf washing technique (Sowndhararajan et al. 2013). After the isolation, the bacterium was confirmed based on the zone of inhibition against the Pestalotiopsis theae.

Estimation of chlorophylls

The level total chlorophyll was estimated by the method described by Sadasivam and Manickam (1996). One gram of harvestable shoots was weighed, homogenized in 20 ml of 80% acetone with pestle and mortar, under diffused light and centrifuged at 3000 g for 15 min. The supernatant was kept aside and the residue was extracted again with 80% acetone till supernatant became colourless. Supernatant samples were pooled together and the total volume was made upto 100 ml with 80% acetone. The absorbance was measured at 645 nm and 663 nm against 80% acetone blank. Chlorophylls were calculated according to the equation given below:

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where, A, absorbance at specific wavelength; V, final volume (100 ml) of chlorophyll extract in 80% acetone; W, fresh weight of tissue extracted.

Estimation of total phenolics

The total phenolic content was determined according to the method of Bray and Thrope (1954). One ml of Folin – ciocalteu reagent (1 : 1 dilution) was added to 1 ml of ethanol extract followed by 2 ml of 20% Na2CO3. The mixture was heated in a boiling water bath for 1 min. The blue colour developed was diluted to 25 ml with distilled water and read at 725 nm. Ethanol (80%) served as the reagent blank and catechol as the standard.

Quantification of tea catechins and caffeine by HPLC

Tea catechins such as epicatechin (EC), epicatechin gallate (ECG), epigallocatechin (EGC), epigallocatechin gallate (EGCG) and catechins, gallic acid and caffeine in blister infected tea shoots and BMO-111 and chemical fungicide sprayed tea shoots were measured. The known quantity of sample was extracted with 25 ml methanol. The extract was filtered through membrane filter (0·45 μm) into HPLC vials. A 10 μl sample of filtrate was injected to a Shimadzu HPLC system (Kyoto, Japan) equipped with a diode array detector (SPD-M10Avp). Phenomenex Luna C-18 (2) column (4·6 mm ID × 25 cm, 5 μm) was used for the analysis. The mobile phase was a mixture of 0·1% orthophosphoric acid (A) and acetonitrile (B). The gradient used was: 0–12 min, 15% B; 12–22 min, 25% B; 22–30 min, 15% B. The flow rate and column temperature were maintained as 1·0 ml min−1 and 35°C respectively. Tea polyphenols were detected at 280 nm. Authentic standards of EC, ECG, EGC, EGCG, catechin, gallic acid and caffeine procured from Sigma-Aldrich, USA were used as standards and for spiking test. The resolution peaks were recorded on the HPLC chart according to the retention time of each compound. The concentrations of tea polyphenols were quantified from standard curves. Relative distribution of these constituents was expressed in percentage of individual component.

Integrated blister blight disease management under large scale field trial

A large scale field trial was designed to evaluate the efficacy of O. anthropi BMO-111 and chemical fungicides in blister blight disease management in an integrated spray schedule. Five hectare plots, each with 28 000–30 000 tea bushes were used in the trial. The O. anthropi BMO-111 formulation contained 1 × 108 CFU ml−1 of 36-h-old O. anthropi BMO-111 cultures in optimized nutrient broth medium and 0·5 ml l−1 of Triton E in water and was applied at the rate of 5 l culture in 300 l water hectare−1. The chemical fungicide used was a mixture of (COC) (210 g), hexaconazole (210 ml) and Triton E (150 ml) in 300 l water hectare−1. Spray schedule was conducted on every fifth day by using tank sprayer. For integrated treatment, every two O. anthropi BMO-111 sprays were followed by a single chemical fungicide spray. Spray schedule was conducted at 5-day-interval.

The blister blight disease incidence was observed for 75 days at 15-day-interval. Disease assessments were made at each plucking round by picking fifty samples of 100 harvestable shoots each (three leaves and a bud) at random as described in the previous field experiment.

Treatment details

T1O. anthropi BMO-111
T2Chemical fungicide (COC + hexaconazole)
T3Integrated trial (O. anthropi BMO-111 and COC + hexaconazole)
Every two rounds of O. anthropi BMO-111 sprays followed by one round of COC + hexaconazole spray.

Safety aspects of O. anthropi BMO-111

Bacterial strains and cultivation

The test bacterial isolate O. anthropi BMO-111 and the pathogenic clinical isolate Pseudomonas aeruginosa (positive control) were grown in nutrient broth at 30°C for 18 h over a rotary shaker at 150 rev min−1 and used for the inoculation of experimental animals.

Animals

The experiment was carried out using male Swiss albino mice (20–25 g) procured from the animal house, IRT Perundurai Medical College, Erode, India. On arrival, the animals were placed at random and allocated to treatment groups in polypropylene cages with paddy husk bedding. Animals were maintained at a temperature of 24 ± 2°C and relative humidity of 30–70%. A 12 : 12 light: day cycle was followed. All animals were allowed free access to water and fed with standard commercial rat chaw pellets (Hindustan Lever Ltd., Mumbai, India). All the experimental procedures and protocols in this study were reviewed by the Institutional Animal Ethics Committee, Nandha College of Pharmacy, Preundurai, Erode, Tamil Nadu (Regd. No. 688/2/C-CPCSEA) and were in accordance with guidelines of animal ethics.

Pathogenicity study

Rats were divided into nine groups (n = 6 animals). The nine groups were further divided into three treatment groups such as groups 1–3 were oral treatment [0·5 ml (of 107 CFU ml−1)], groups 4–6 were intrtavenous treatment [0·1 ml (of 107 CFU ml−1)] and groups 7–9 were intranasal treatment [0·5 ml (of 107 CFU ml−1)]. Groups 1, 4 and 7 were received plain sterile medium as control. Groups 2, 5 and 8 were received the culture of P. aeruginosa as positive control. Groups 3, 6 and 9 were administered with the culture of test organism (O. anthropi BMO-111). Oral dose was administered by using oral cavage tube. Intravenous administration was done through tail vein using a 26 gauge needle. Intranasal administration of the bacterial suspension into nasal cavities was carried out using an aerosol applicator (Porter et al. 1991; Landry et al. 2003). All the trated animals were monitored continuously from day 1 to day 15 for any adverse symptoms and mortality.

Statistical analysis

The data were subjected to a one-way analysis of variance (anova) [SPSS Software 9.0 (SPSS Inc., Chicago, IL, USA)]. 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 (DMRT). Values expressed are means of three replicate determinations (n = 3) ± standard deviation (SD).

Results

Effect of combined efficacy of O. anthropi BMO-111 with chemical fungicides

The efficacy of foliar application of O. anthropi BMO-111 was more effective than the chemical fungicide (COC + hexaconazole) treatment. However, when the biopesticide O. anthropi BMO-111 was combined with COC or hexaconazole or COC + hexaconazole, the blister blight control was still more effective. On the other hand, the integrated disease management (every two O. anthropi BMO-111 spray followed by a single chemical fungicide spray) was superior over all other treatments in the long run (90 days after first spray) (Table 1).

Table 1. Efficacy of combined (T3–T6) or alternate (T7) application of Ochrobactrum anthropi BMO-111 with chemical fungicides on the blister blight disease incidence on tea plants under microplot trial with 5-day-interval sprays
TreatmentDisease incidence (%)
Day 15Day 30Day 45Day 60Day 75Day 90
  1. T1, untreated control; T2, 1 × 108 CFU ml−1 of BMO-111; T3, BMO-111 (1 × 10CFU ml−1) + COC (0·15% w/v); T4, BMO-111 (1 × 10CFU ml−1) + hexaconazole 0·15% v/v); T5, BMO-111 (1 × 10CFU ml−1) + COC (0·15% w/v) + hexaconazole (0·15% v/v); T6, COC (0·3% w/v) + hexaconazole (0·3% v/v); T7, every two BMO-111 (1 × 108 CFU ml−1) sprays were followed by a single chemical fungicide (COC 0·3% w/v + hexaconazole 0·3% v/v) spray. Triton E was added to the spray formulation as surfactant at the rate of 0·5 ml l−1.

  2. Mean values followed by different superscript letters in a column are significantly different (< 0·05) according to Duncan's test.

T1 – Untreated control6·3d13·1d18·7c14·4f12·1d14·5e
T2 – BMO-1113·3a6·1b8·2b3·4a3·2a5·4c
T3 – BMO-111 + COC3·4a6·4b8·5b7·3e5·0c7·2d
T4 – BMO-111 + hexaconazole4·2b8·3c8·6b4·1b4·2b5·8c
T5 – BMO-111 + COC + hexaconazole4·1b5·3a6·7a3·2a3·3a4·2b
T6 – COC + hexaconazole5·2c6·3b8·7b6·1d4·5b7·1d
T7 – Alternate treatment6·5d6·2b7·2a5·2c4·3b3·1a

Reisolation of O. anthropi BMO-111

Population of O. anthropi BMO-111 was detected up to 10CFU ml−1 in BMO-111, BMO-111 + hexaconazole and alternate treatment plots. Whereas, O. anthropi BMO-111 combined with COC and COC + hexaconazole markedly decreases the population of O. anthropi BMO-111 (10CFU ml−1 and 10CFU ml−1, respectively).

Biochemical parameters of harvestable tea shoots as influenced by blister blight disease and spray protection

Spray treatment with O. anthropi BMO-111 significantly enhanced the total phenol content in tea shoots than the chemical fungicide treatment. On the other hand, in the blister blight infested shoots, the total phenolic content was 15·85% lower than the chemical fungicide treated harvestable shoots. In the blister infected shoots, the level of chlorophyll content also decreased.

Generally, O. anthropi BMO-111 treatment increased the catechin levels such as epigallocatechin (EGC), catechin (C), epicatechin (EC), epigallocatechin gallate (EGCG) and epicatechin gallate (ECG), gallic acid and caffeine than chemical fungicide treatment. However, in the blister infected tea shoots, all the said biochemical parameters were the lowest (Table 2).

Table 2. Effect of blister blight disease and spray protection on the biochemical parameters of harvestable tea shoots
Biochemical parameterBlister infectedBMO-111 treatedaChemical treatedb
  1. Values are means of three replicate determinations ± SD.

  2. a

    1 × 108 CFU ml−1 of O. anthropi BMO-111.

  3. b

    Copper oxychloride (0·3%) + hexaconazole (0·3%).

  4. Mean values followed by different superscript letters in a column are significantly different (< 0·05) according to Duncan's test.

Total chlorophyll (mg g−1)0·69 ± 0·03c0·95 ± 0·05a0·82 ± 0·02b
Total phenolics (%)21·90 ± 1·82c28·81 ± 1·02a27·58 ± 1·64b
Epigallocatechingallate (EGCG) (%)7·27 ± 0·59c11·14 ± 0·81a10·78 ± 0·74a
Epigallocatechin (EGC) (%)2·50 ± 0·19a2·81 ± 0·21a2·69 ± 0·24a
Epicatechingallate (ECG) (%)1·38 ± 0·15c2·21 ± 0·16a1·92 ± 0·11b
Epicatechin (EC) (%)0·60 ± 0·01a0·62 ± 0·02a0·62 ± 0·01a
Catechin (%)0·37 ± 0·04c0·81 ± 0·08a0·51 ± 0·07b
Gallic acid (%)0·10 ± 0·02b0·19 ± 0·02a0·19 ± 0·01a
Caffeine (%)1·86 ± 0·15b3·01 ± 0·09a2·94 ± 0·17a

Integrated blister blight disease management under large scale field trial

The results of the integrated blister blight disease management using O. anthropi BMO-111 and fungicides (COC + hexaconazole) as foliar sprays are presented in Fig. 2. It is evident from the data that the integrated treatment (every two O. anthropi BMO-111 sprays followed by a single fungicide spray) is more efficient than either O. anthropi BMO-111 spray or chemical fungicide spray over the observation period of 75 days. In the present study, however, O. anthropi BMO-111 achieved better performance when compared to the conventional field practice of chemical fungicides (COC + hexaconazole) in the field trial. The efficiency of disease control increased further when both the above said components were integrated, i.e. every two O. anthropi BMO-111 sprays were followed by a single chemical fungicide (COC + hexaconazole) spray.

Figure 2.

Efficacy of integrated treatment in blister blight disease management under large scale field trial. BMO-111 culture broth at the rate of 5 l for every 300 l of water to give 1 × 108 CFU ml−1 was applied per hectare. Chemical fungicide: COC (210 g) + hexaconazole (210 ml) in 300 l of water per hectare. Alternate treatment: every two BMO-111 sprays followed by a single chemical fungicide (COC + hexaconazole) spray. Triton E was added to the spray formulation as surfactant at the rate of 0·5 ml l−1 Bars having different letters are significantly different (< 0·05) according to Duncan's test. image BMO-111; image Chemical fungicides; image Alternate.

Pathogenicity of O. anthropi BMO-111

To ascertain the safety of the biopesticide O. anthropi BMO-111 to the handlers and other non-target organisms, a pathogenecity study was carried out in mice model with P. aeruginosa as positive control. The results of the animal study are presented in Table 3. There was no change in the general behavior of animals after treatment with O. anthropi BMO-111 or with plain medium. However in treatment with P. aeruginosa, the animals showed convulsion and muscle relaxation.

Table 3. Pathogenecity study with BMO-111 in the Swiss albino mice model
TreatmentDead animals after treatmentaSurvived animals (on day 15)
Oral routeIntravenal routeIntranasal route
  1. a

    Six animals/treatment.

  2. Control, sterile, plain medium; BMO-111 and P. aeruginosa; Oral treatment, 0·5 ml of 107 CFU ml−1 per animal; Intravenous treatment, 0·1 ml of 107 CFU ml−1 per animal; Intranasal treatment, 0·5 ml of 107 CFU ml−1 per animal.

Control00018
BMO-11100018
Pseudomonas aeruginosa (positive control)24012

Mortality was reported only in P. aeruginosa administered groups. In oral route administration of P. aeruginosa cells, one animal died on 3rd day and another on 9th day. In the intravenal treatment also, one animal died four hours after administration of P. aeruginosa and three animals after 24 h of treatment. There was no mortality either in control (plain medium) or O. anthropi BMO-111 treated groups (Table 3). There were no noticeable changes in body weight between treatment and control groups.

Discussion

The integrated microplot field experiment was conducted mainly for the assessment of compatibility between O. anthropi BMO-111 and chemical fungicides on blister blight disease management. This field experiment demonstrated that O. anthropi BMO-111 is suitable for integrated treatment where the biopesticide was combined with COC and hexaconazole. Generally, copper fungicides were successfully used to control blister blight in tea plantations for more than five decades. The efficacy was improved when triazoles were used in combination with copper oxychloride. Among the triazoles, mixture of copper oxychloride with hexaconazole provided superior control (Chandramouli and Agnihothrudu 1989; Premkumar et al. 1998). In several disease management strategies, the addition of fungicide at reduced rates with biocontrol agents has significantly enhanced disease control, compared to treatments with biocontrol agent alone (Frances et al. 2002; Buck 2004). Integrated use of biocontrol agent with reduced dose of fungicide was effective against Fusarium crown and root rot of tomato (Omar et al. 2006), Rhizoctonia root rot, take-all disease of spring wheat (Duffy 2000) and post harvest diseases of fruits (Chand-Goyal and Spotts 1996) compared with the individual components of disease management.

Synergistic action of fungicides and fungicide-tolerant biocontrol isolates was reported to be beneficial in management of other phytopathogenic fungi (Conway et al. 1997; Buck 2004; Omar et al. 2006). Kishore et al. (2005) found that the combination of chlorothalonil-tolerant Pseuomonas aeruginosa GSE 18 and chlorothalonil (500 μg ml−1) reduced the severity of late leaf spot in groundnut comparable to chlorothalonil (2000 μg ml−1) alone. Pseudomonas fluorescens EPS 288 and Bacillus subtilis Rb14-C, in combination with reduced dose of fungicides, were equally effective as the standard fungicides alone in the control of Penicillium expansum on pear fruits (Frances et al. 2002) and damping off in tomato plants (Kondoh et al. 2001) respectively. The present study also demonstrated that the integrated treatment where O. anthropi BMO-111 was combined with COC and hexaconazole gave maximum disease control. However in the long run, two sprays of O. anthropi BMO-111 alternated with a single chemical (COC + hexaconazole) spray was found to be most effective. The higher disease control in alternate treatment than combined treatment might be due to the incompatibility of BMO-111 and COC. In the reisolation of O. anthropi BMO-111 results confirmed that the combined application of BMO-111 with COC reduced the population when compared with BMO-111 and alternate treatment.

Tea polyphenols include groups of compounds of different chemical structure possessing variable biological properties. Green tea leaves contains six major catechins: epicatechin (EC), epicatechin gallate (ECG), epigallocatechin (EGC), epigallocatechin gallate (EGCG), catechin (C), and gallocatechin (GC) (Ninomiya et al. 1997). Catechins are flavanols, forming nearly 30% of the dry weight of green tea and their content is higher in young leaves (Balentine 1992). Chemically catechins are water soluble, colourless compounds and impart astringency to tea infusions. These compounds contribute to the bitterness, astringency and sweet aftertaste of tea beverage (Hara et al. 1995).

A marked depression in the levels of total phenols and catechins were observed in blister blight infected green-shoots (Table 1). This suggested how blister blight led to gross quality deterioration and lower market valuation of tea. It is well established that total phenols, especially catechins, and the activity of oxidative enzymes, particularly polyphenol oxidase(s) in tea shoots are directly associated with the quality of made tea (Howard 1978; Robertson 1983; Stephen Thanaraj and Ramachandran Seshadri 1990). Since the spray treatment with O. anthropi BMO-111 effectively controlled the blister blight disease, it also significantly increased the chlorophyll, polyphenol and catechins levels in tea shoots, which was comparable to chemical fungicide treatment.

The gram negative bacteria under the genus Ochrobactrum are widely distributed in the environment and may be part of normal flora of the large intestine (Holmes et al. 1988; Moller et al. 1999; Lebuhn et al. 2000). However, there is an element of suspicion that strains belonging to O. anthropi may emerge as opportunistic human pathogens because this is the most common species recovered from many clinical specimens. Hence, the pathogenicity of O. anthropi strain BMO-111, if any, was evaluated in mice model by introducing the bacterium through intravenous, intranasal and oral routes as stated in the published literatures (Porter et al. 1991; Landry et al. 2003).

The results of the pathogenicity study demonstrated that O. anthropi BMO-111 is non-pathogenic even when challenged in mice at concentrations as high as 0·5 ml of 1 × 107 CFU ml−1 per animal through oral inoculation, O.1 ml of 1 × 107 CFU ml−1 per animal as intravenous inoculation and 0·5 ml of 1 × 107 CFU ml−1 per animal as intranasal inoculation. The biological defense mechanism of the mice appears completely adequate to deal with any pathogenicity from BMO-111 even when challenged by a very large number of viable cells.

Conclusion

In the present study, the integrated approach of disease management involving chemical means of control with a biological component O. anthropi BMO-111 is likely to yield better results in long run than the fungicide or biological approach in isolation. Certain strains of O. anthropi are known to be pathogenic, but the present isolate, BMO-111, isolated from tea phylloplane, did not show any apparent pathogenic effect based on animal pathogenicity study. These results clearly suggested that O. anthropi BMO-111 has potential to be used as biopesticide for the effective management of blister blight disease on tea.

Ancillary