Growth Promotion and Bi-Control Approaches of Brown Root Rot Disease of Tea by Pseudomonas Aeruginosa ( PM 105 )

Tea (Camellia sinensis (L) O. Kuntze) belongs to the family Theaceae. It is a dicotyledonous perennial crop. Tea is the second largest consumed beverage in the world, after water. Total tea production of the world during 2007 was 3.527 million tones, and the total area under cultivation is around 5 million hectares [1]. Tea cultivation was initiated in India, around 1886, with the opening of few gardens under two tea companies of Assam. Barak Valley region of Assam have 35,000 hectares (approx.) area under tea cultivation, which annually produces 46,000 thousands Kg of tea (approx.) [2]. As it is a long duration plantation crop, it has become largely prone to be attacked by several pathogens [3]. Disease problems are found to be an integral part of the tea plant, which is under monoculture for over 150 years in North Eastern (N.E) region of India, including Barak Valley of Assam [4]. Petch describes Brown root rot disease, Fomes lamoensis, as the earliest known root disease of tea. Symptoms of brown root rot disease are: slow plant growth, yellowing and wilting of leaves, defoliation, branch dieback, and plant death. Soil represents a highly heterogenous environment for the microbiota inhabiting. Some are beneficial and some detrimental, while others may have no direct effect. Pseudomonas aeruginosea is one of the free living microorganisms, which can be considered as Plant Growth Promoting Rhizobacteria (PGPR), having the ability to colonize plants roots and stimulate growth of plants, and contributes to disease control [5-10]. Use of biological control agents such as Plant Growth Promoting Rhizobacteria (PGPR) can be a suitable approach for the biocontrol of disease [11,12]. This would also be an alternative to decrease the input chemical fertilizer, which is having detrimental effect on the environment [13]. Several mechanism have been involved to explain how the PGPRs stimulate plant growth, which may be either direct, e.g. production of growth hormones, phosphate solubilization, nitrogen fixation etc., or, indirect, viz., suppression of deleterious microorganisms by siderophore production, or secretion of antifungal metabolites [13]. In the present work, some observations were made on the potential of Pseudomonas aeruginosa in the growth promotion of young tea plants, and the control of tea root pathogen Fomes lamoensis under nursery condition. Materials and Methods


Introduction
Tea (Camellia sinensis (L) O. Kuntze) belongs to the family Theaceae. It is a dicotyledonous perennial crop. Tea is the second largest consumed beverage in the world, after water. Total tea production of the world during 2007 was 3.527 million tones, and the total area under cultivation is around 5 million hectares [1]. Tea cultivation was initiated in India, around 1886, with the opening of few gardens under two tea companies of Assam. Barak Valley region of Assam have 35,000 hectares (approx.) area under tea cultivation, which annually produces 46,000 thousands Kg of tea (approx.) [2]. As it is a long duration plantation crop, it has become largely prone to be attacked by several pathogens [3]. Disease problems are found to be an integral part of the tea plant, which is under monoculture for over 150 years in North Eastern (N.E) region of India, including Barak Valley of Assam [4]. Petch describes Brown root rot disease, Fomes lamoensis, as the earliest known root disease of tea. Symptoms of brown root rot disease are: slow plant growth, yellowing and wilting of leaves, defoliation, branch dieback, and plant death. Soil represents a highly heterogenous environment for the microbiota inhabiting. Some are beneficial and some detrimental, while others may have no direct effect. Pseudomonas aeruginosea is one of the free living microorganisms, which can be considered as Plant Growth Promoting Rhizobacteria (PGPR), having the ability to colonize plants roots and stimulate growth of plants, and contributes to disease control [5][6][7][8][9][10]. Use of biological control agents such as Plant Growth Promoting Rhizobacteria (PGPR) can be a suitable approach for the biocontrol of disease [11,12]. This would also be an alternative to decrease the input chemical fertilizer, which is having detrimental effect on the environment [13]. Several mechanism have been involved to explain how the PGPRs stimulate plant growth, which may be either direct, e.g. production of growth hormones, phosphate solubilization, nitrogen fixation etc., or, indirect, viz., suppression of deleterious microorganisms by siderophore production, or secretion of antifungal metabolites [13]. In the present work, some observations were made on the potential of Pseudomonas aeruginosa in the growth promotion of young tea plants, and the control of tea root pathogen Fomes lamoensis under nursery condition.

Study area
Barak valley is located in southern part of Assam, which falls under 24°80∕ and 25°80∕ N latitude and 92°15/ and 93°15/ E longitude. Rainfall of this region ranges from 2500 mm to 4000 mm, and temperature between 9.2°C to 36°C.

Collection of soil samples
Soil samples were collected from multi location areas of Barak valley. The sites of collection selected are mainly tea plantations. The soil samples were collected in plastic bags and brought to the laboratory without sealing, and then these samples were air dried, homogenized and sieved (200 mesh) to get uniform samples.

Isolation of pseudomonas aeruginosa
Pseudomonas aeruginosa (PM 105) isolation has been done according to Cappucino and Sherman [14].
(ITCC) of Indian Agricultural Research Institute (IARI), New Delhi, India. The pathogen was also isolated locally from the diseased root samples of tea, collected from Roskandy Tea Estate. This isolate was verified and confirmed, comparing the isolate received from ITCC.

In vitro antibiosis test with live organism
In vitro antagonism test was done by two methods, as recommended by Dileep Kumar [15]: Spot inoculation and Line inoculation.
Spot inoculation: For this, an actively growing mycelial disc of the pathogen (approx. 4 mm 2 ) was placed in the centre of the petri plate, and a loopful of the rhizobacterial strain was spot-inoculated in the periphery of the Petri plate, 2.0 cm inside at two opposite equidistant places and incubated at 28 ± 2°C. Inhibition zones were measured as distance (in cm) between the respective test bacteria PM 43 (antagonist) and the fungal pathogen F. lamoensis, after 7 days growth under incubation.

Line inoculation:
King's medium B (KB), Nutrient Agar (NA) and Potato Dextrose Agar (PDA), were used for examining the antagonism. For this, an actively growing mycelial disc (approx. 6mm 2 ) was placed at one side of the Petri plate, 2 cm inside of the periphery and a loopful of the rhizobacterial strain was streaked in a line, on the opposite side at a distance of 5 cm from the mycelial disc. The plates were incubated at 28 ± 2°C and inhibition zone was measured, as distance (in cm) between the respective rhizobacterial strain PM 43 (antagonist) and F. lamoensis (fungal tea pathogen), after 7 days of growth under incubation.

Growth promotion and disease suppression study under nursery condition
This was done according to Dileep Kumar and Bezbaruah [16]. The experiment was laid out in Completely Randomized Design (CRD), which was three set of each replication and repeated thrice. Under each replication, 10 numbers of tea saplings were treated as per treatment. The tea plants were planted in the polythene bags (21×15 cm), filled with a mixture of sterilized soil and Farm Yard Manure (FYM) in 3:1 ratio. Tea saplings with the treatment of F. lamoensis alone served as control, F. lamoensis+PM 105 and PM 105 alone, were challenge inoculated with the homogenized broth culture of the pathogen, F. lamoensis (10 days old) @50 ml/clonal plant, simultaneously bacteria grown on Nutrient broth (NB) medium (for 48 h) culture were applied on the tea clonal plants, with the same quantity. Hygienic condition was maintained by weeding, light forking and irrigation, as and when required to maintain the healthy condition of the tea plants and to minimize the interference of external factors, other than desired. After a month of planting, plants were again treated twice with respective pathogen and PGPR strains, as per different treatment specifications at 15 days interval. After 60 days following treatments, five numbers of representative tea plants were selected randomly and uprooted from the polythene bags with utmost care to keep the roots intact and then, washed gently under running tap water to remove the adhering soil particles. Data on growth promotion in terms of increase in shoot height, root length, chlorophyll content of leaves, fresh and dry weight of shoot, root and number of newly emerged leaves were recorded. Disease incidence was recorded upto 90 days of treatment. Per-cent Disease Incidence (PDI) and Percent Disease Control (PDC) were calculated, based on the disease incidence. The percentage of survival of the tea plants was also recorded up to 90 days of growth, following the treatment.

Assessment of the disease incidence
Occurrence of disease symptom was observed by recording the wilting of leaf, leading to drying of the plant. Randomly 15 plants were selected from different treatments in the nursery and the number of plants that wilted were counted, and the mean wilt incidence was expressed in percentage. Disease incidence was recorded upto 90 days of treatment. The percent disease incidence was calculated, by using the following formula. Percent disease control was calculated, based on the per-cent disease incidence.
Number of plants infected Percent disease incidence (PDI) = ×100 Total number of plants observed

Statistical analysis
All the data obtained were subjected to perform statistical tests of analysis of variance with least significant difference (LSD), with the help of SPSS (portable_PASW_statistics_18) software. (P ≤ 0.05).

Results
In in vitro antibiosis test, isolated bacterial strain of Pseudomonas aeruginosa was found to show remarkable significant results against the pathogen, which was able to make a zone of inhibition in all media such as PDA, NA and King's B, both in spot and line inoculations ( Figure 1). Highest zone of inhibition was observed in King's B medium with line inoculation (Table 1).

Growth promotion and disease suppression study under nursery condition
In this study, disease suppression was encountered by wilting of leaves to drying of tea plants. Hence, it is found that plants treated with only pathogen, by infesting the soil in the nursery (control) condition, showed drying of leaves (wilting), which occurred after 32 nd day onwards, whereas drying of tea plants occurred on the 78th day ( Table 2). On the other hand, the treatment in which the pathogen and the bacteria were inoculated to the same tea plants, (F. lamoensis+PM 105) drying of leaf was observed after 58 th day. There was no record of plants drying, and no symptoms were found in tea plants treated with bacterium only (PM 105), which remained healthy upto the last day (90 th day) of observation. It was also found that Pseudomonas aeruginosa treatment has shown significant result on the different growth promotion parameters on tea, in nursery condition. The treated tea plants were observed with the increased in all growth promotion parameters, over the non treated control (F. lamoensis alone). Among the parameters, highest number of new leaves, lateral branches, shoot height, root length, fresh weight of shoot, fresh weight of root, dry weight of shoot were observed in only bacteria treated plants (PM105), followed by the treatment of pathogen in the inoculated bacterized tea plants (F. lamoensis+PM 105) ( Table  3). The treatment also resulted in green leaves with high chlorophyll content, over non-treated plants (control).

Spot Inoculation Line inoculation
Highest percent increase over control in number of lateral branches (214.28%), shoot height (75.29%), root length (63.34%), fresh weight (190.12%) and dry weight of shoot (171.51%) were recorded in PM 105 treatments, whereas in the treatment where pathogen and PGPR both were applied, highest per cent increase over control in the number of new leaves (163.63%), fresh weight of root (97.18%) and dry weight of root (131.25%) were recorded in the treatment with F. lamoensis+PM 105 (Table 3).

Percent disease incidence under nursery conditions
Under nursery condition, tea plants which were grown in soil infested with F. lamoensis (control) have shown 73.00% disease incidence, whereas plants treated with the pathogen and inoculated with the bacteria (F. lamoensis+PM 105), the disease incidence was recorded 33.33% only, or 67.00% disease control vice-versa (Table 4).

Discussion
In the present study, the rhizobacterial strain Pseudomonas aeruginosa (PM 105) isolated from tea soil showed significant inhibition zone against the pathogen (Figure 1), both in spot and line inoculation method in vitro. Some PGPR's synthesize antifungal antibiotics, which inhibits growth of phytopathogenic fungi [15,17]. PGPR's are indigenous to soil and plant rhizosphere, and play a major role in the biocontrol of plant pathogens [13,18]. The bacterial strains have been reported to control many plant pathogens, through in vitro antibiosis study done by several workers [17][18][19][20][21][22]. Although the in vitro antibiosis test does not always correlate with the suppression of soil-borne plant disease, but because of the magnitude of the rhizosphere population and the lack of a more reliable method, in vitro screening of organism is a valuable tool to select the potential strains [23,24]. Exploitation of beneficial rhizobacteria for crop enhancement and disease control is relatively, a new and emerging area in agricultural biotechnology. Use of these rhizobacteria can result to physiological and chemical stimulation of the plant roots, resulting more rapid emergence, higher chlorophyll levels, and enhanced plant health [10,25]. Growth promotion can also depend on the suppression of either deleterious microorganisms of the soil, and rhizosphere that reduce plant growth and development of soil borne pathogens that cause diseases such as damping-off, rots and wilts [2]. Pseudomonas sp. are widespread bacteria in agricultural soil, used as PGPR [17]. Pseudomonas aeruginosa has been reported to be potential PGPR, that inhibited the growth of the pathogens on several crops [26,27]. Complex reciprocal interactions between soil, plant and microorganisms that occur, could also account for the population dynamics of the biocontrol agent [28]. Application of bacterial strains to the rhizosphere of tea plants for enhanced growth and productivity in terms of biomass, have been reported by some workers [18,28]. In the present nursery studies, it has been observed that the treatment with the bacterial strain PM 105 showed survival of the clonal tea plants for a longer period, whereas the fungus (pathogen) treated plants without bacterial treatment, died after 78 th day onwards after inoculation. It was also assumed that the bacteria treated tea plants grown in pathogen infested soil survived till the duration of observation, where number of new leaves, lateral branches, shoot height, root length, fresh weight of shoot and root, dry weight of shoot, dry weight of root and chlorophyll content increased. The highest disease control with reduced disease incidence was also found in F. lamoensis+PM 105 treated nursery plants.
Bacterial strain PM 105 was applied, either alone, or in combination with the pathogen under nursery condition showed remarkable performance in disease control, and in various growth and yield parameters. The consistent performance of PM 105 indicate its potential to be used as biocontrol agent and as a biofertilizer, in the case of brown root rot disease infested tea fields. This will also help in the enhancement of the growth and yield of tea in Barak valley, Assam, for the benefit of the tea industry of N.E. India in general, and of Barak Valley, in particular.    Table 4: Effect of different treatments on the disease incidence and percent disease control of tea plants (after 90 days).