Systemic infestation of Serratia entomophila AB2 through plant tissue inferred protection against insect pest and fungal pathogens

Serratia entomophila, a Gram negative non spore forming bacteria, is commercially exploited as biocontrol agent of pasture pest, grass grub (Costelytra zealandica, Coleoptera) in New Zealand as soil inoculants. As an exception, the strain S. entomophila AB2 used in this study is having insecticidal property against pod borer (Heliothis armigera, Lepidoptera), a phyllospheric pest. Expermental results showed high rate of mortality when the pests were fed with shoot portion of ground nut developed from bacteria treated seeds. Antifungal property of S. entomophila AB2 was recorded when tested in vivo using different parts of plant developed from bacteria treated and untreated seeds. In the present study, the systemic infestation was understood by isolation and enumeration of the inoculant from different plant tissues in specific medium and an increment in number within the tissues was also observed. This is the first report of systemic spreading of S. entomophila in plant parts to provide protection against insect and fungal pests.

Rhizosphere is a natural repository of diversified microorganisms.The pathogenic strains of pasture pests make them effective for pest control by rhizospheric application.However, S. entomophila AB2 is known to attack Lepidopteron pest in the phyllosphere (Chattopadhyay et al., 2012).It is important to study whether, the rhizospheric application of the strain AB2 could provide an effective protection against Lepidopteron pest.
Therefore, the main objective of the present study was to find out the efficacy of rhizospheric application of S. entomophila AB2 to control pest and fungal attack at phyllosphere in laboratory condition and the extent of bacterial dispersal inside the plant parts.

Experimental organism used
Bacterial strain S. entomophila AB2, was previously isolated, characterized and identified by our team and currently obtained from Prof. Sukanta K. Sen, Microbiology Division, Department of Botany, Visva-Bharati University, India.The 16S rRNA gene sequence was registered to Gene Bank (Gene Bank accession no.GU370899).The isolate was reported earlier for its pesticidal property against lepidopteran pest (Chattopadhyay et al., 2011(Chattopadhyay et al., , 2012)).
Bacterial culture was maintained as 50% glycerol stock at -20°C in brain heart infusion agar (BHI-agar, Hi-media, India).The working strain was grown in shake flask containing 100 ml of broth (4 g sugar, 1 g yeast extract, 0.2 g urea and 0.2 g NPK; pH 7.1).Fermentation was carried at 28°C for 30 h in an orbital shaker (Scigenics Biotech, India) at 240 rpm.

Fungal phytopathogens used
The phytopathogens, Aspergillus flavus MTCC 1973, Candida albicans MTCC 183 and Fusarium oxysporum MTCC 1753 were maintained in recommended media (http://mtcc.imtech.res.in/catalogue) and cultured using Potato Dextrose Agar (PDA, HiMedia, India) for in vitro and in vivo assessment of antifungal activity.For in vivo assessment of antifungal activity 8 mm discs were prepared from 5 day old cultures.

Rearing of bollworm
Heliothis armigera larvae were obtained from the field and maintained on a commercial diet (IM002, Hi-Media, India) in plastic vials (Tarsons, India) at constant ambient temperature (Chattopadhyay et al., 2011).In order to obtain a homogeneous mass of the test insects, the larvae were reared till emergence of adults.After rearing for two generations, 5-day-old larvae were used for bioassay (Chattopadhyay et al., 2011).

Bacterial treatment of seeds
Surface sterilized seeds of Arachis hypogaea var.Koushal G201 were treated with S. entomophila AB2 (2 ml of 10 9 cfu ml -1 seed-1) and air dried for 1 h in a laminar cabinet at RT (28±2°C).After treatment, the seeds were taken with a sterile forcep and placed on moistened blotter paper in Petri dishes (10 seeds/plate).The seeds were incubated at RT (28±2°C) for 10 days, maintaining the standard moisture condition (40%).

Efficacy against test insect
For bioefficacy test against Lepidopteron pest, the roots and shoots of germinated seeds were cut into bits of 1 cm maintaining sterility.To record the mortality rate after 72 h, experimental larvae (5-day old) were distributed into 5 separate batches, each containing 30 larvae but in separate vials with 3 g of feed at constant ambient temperature (28±2°C).Batch No. 1 was fed with commercial diet (IM002, Hi-Media, India).Batch No. 2 and No. 3 were fed with shoot bits and root bits (respectively) derived from 10 days old seedling of bacteria untreated seed.Similarly, Batch No. 4 and No. 5 were fed with shoot bits and root bits (respectively) derived from bacteria treated seed.

Efficacy against fungal phytopathogens
In vitro assessment of antifungal efficacy of S. entomophila AB2 was carried out with test fungi through bore well method using PDA.The inocula, S. entomophila AB2 (48 h old) and test fungi (168 h old) were put oppositely and 25 mm away from the periphery of the Petri plates, in three replicates (Dennis and Webster, 1971).Petri plates inoculated with the fungal phytopathogen alone served as the control.The plates were incubated at 28±2°C.After 72 h of incubation, the inhibition of mycelial growth (%) was calculated according to the study of Vincent (1927).
In vivo assessment of antifungal efficacy was carried out with seedling parts (10 days old) derived from bacteria untreated seed and bacteria untreated seeds.Roots/shoots were separated and cut in to bits as described at specific distance (2, 4, 6 and 8 cm away) from seed coat.The bits of root/shoot from bacteria treated/untreated seeds were surface sterilized and placed on Nutrient agar medium and incubated at 28°C for overnight.After incubation, 8 mm discs of test fungi were placed just opposite the plant parts, incubated at room temperature for five days and the inhibition zone, if any, was measured.

Assessment of systemic infestation into plant parts
Roots/shoots/leaves were separated at regular interval (day 4, 6, 8 and 10) from seedlings (derived from bacteria treated and untreated seeds).The roots and shoots were cut as stated and ground (1 g) individually in a sterile mortar-pestle and suspended in 9.9 ml sterile distilled water in tubes, maintaining asepsin.The tube containing sample was vortexed for 30 s and placed in slanting position in an orbital shaker (10 rpm) for 1 h.For enumeration of S. entomophila AB2, serially diluted suspensions were plated onto caprylate thallous agar (CTA) medium (O'Callaghan et al., 2002) supplemented with antibiotic Ampicillin (A).The colony count was taken against the UV lamp ( Kishore et al., 2005).

Statistical analysis
Each experiment was performed at least for 3 times unless stated otherwise.Standard deviation for each treatment was determined.The experimental data were statistically analyzed using box plot analysis.Pesticidal activity evaluated, through pest scouting and evaluation of mortality rate, on the basis of severity of infestations (Amer et al., 1999).

Efficacy of S. entomophila AB2 against pod borer
In vivo pesticidal activity was measured between 3.33-90.0%against the test pest.High mortality rate was recorded in Batch No. 4 (90%) and No. 5 (87%) of experimental larvae, fed with roots/shoots of bacteria treated seeds in comparison to Batch No.2 (7%) and No. 3 (7%) where experimental larvae were fed with roots/shoots of bacteria untreated seeds.While the lowest mortality rate was recorded in Batch No. 1 of experimental larvae fed with commercial diet.
These results clearly indicate that the shoot bits and root bits derived from bacteria treated seeds are with sufficient inoculants (S. entomophila AB2 population) to impart pesticidal activity to test larvae (Table 1).Availability of S. entomophila from phyllosphere region of tomato and banana are reported (Akutsu et al., 1993;Riveros et al., 2002).

Efficacy of S. entomophila AB2 against phytopathogenic fungi
In in vitro experiment, S. entomophila AB2 exhibited high spectrum of antifungal activity in a concentration dependent manner (Table 2) but found more antagonistic to C. albicans.
In in vivo experiment also, the plant parts from bacteria treated seeds showed positive activity against the test pathogens.Results (Table 3) clearly showed that S. entomophila AB2 was more antagonistic to C. albicans, as found from the in vitro study (Table 2).

Systemic movement of S. entomophila AB2 through plant parts
When ground nut seeds were treated with S. entomophila AB2 and allowed to germinate, the bacterial strain was re-isolated from emerging shoots, roots as well as leaves, after surface sterilization.In box plot on the 10 th day, the highest and lowest population of the inoculum were recorded from root (11 × 10 4 cfu g -1 ) and leaf (1.5 × 10 4 cfu g -1 ) respectively.The direct cell count from in vitro experiment shows a slow but steady increase of inoculant population in all plant parts from the 4 th to 10 th day of the experiment but the count was more pronounced in leaf tissue (Figure 1).

Conclusion
S. entomophila AB2, as a single biological agent for pest and disease management seems to be a lucractive alternative to chemical pesticides and fungicides in integrated crop management system.In the present study, it is understood that the seed application of S. entomophila AB2 could provide effective control against lepidopteran pest and fungal pathogens at the phyllosphere.It further assumes that the rhizospheric application would be reciprocated positively as an alternative to phyllospheric application because of systemic infestation of S. entomophila AB2 and colonization at different plant parts.This is the first report of systemic infestation of S. entomophila through plant tissue to impart protection against insect and fungal pests.

Table 3 .
In vivo efficacy of S. entomophila AB2 against fungal phytopathogens