Formulation of a novel multidimensional biopesticide for increasing plant productivity and reducing disease susceptibility. Som Banerjee*1, Shreya Baroi*2, Sohitri Mukherjee*, Devlina Ganguly*, Shreya Das*, Bikram Dhara*3, Arup Kumar Mitra*4. All authors have contributed to this poster equally. * Department of Microbiology, St. Xavier’s College (Autonomous), Kolkata, West Bengal 1 This author will be presenting the poster. 2This author prepared the poster. 3Project mentor. 4Pofessor-In-Charge.
ABSTRACT
Isolation and characterisation of microbes
Background: The increasing population pressure and a need for a second green revolution has impressed upon the scientific community the importance to exploit various beneficial plant-fungi-bacterial tripartite interactions and thereof develop a novel, multi-dimensional biopesticide to increase crop productivity and popularize the practice of organic farming. Rationale: The soil harbours several microorganisms like, Plant GrowthPromoting Rhizobacteria (Bacillus spp., Azotobacter spp.), Phosphate solubilizing bacteria (PSB), Potassium solubilizing bacteria (KSB) and entomopathogenic fungi (Metarhizium spp., Beauveria spp., Paecilomyces spp. and Trichoderma spp.) which increase nutrient uptake by plants and confer immunity against phytopathogens; the rationale of this project was to exploit the soil microbiota to act as biocontrol agents and further the goals of organic farming. Strategy: Several micobes were purified through serial dilution, pure colonies were isolated on different specific media, the colony characteristics and spore morphology were studied and furthermore inter-specific interaction studies were carried out. Results: Based on the results of the interaction studies a novel biopesticide comprising of bacterial as well as fungal species and organic supplements was formulated and applied on Okra plant (Abelmoschus esculentus). Pathogenesisrelated enzymes were isolated from the treated as well as control plants and assayed to check for the efficacy of the newly-formulated product in terms of inducing resistance in the plant host against phytopathogen (Alternaria spp.). It is often debated that the major disadvantage of bio-control include its variable efficacy and delayed response and this study aims to challenge this notion.
1. 2.
3.
4. 5. 6.
Isolation and characterization of bacterial and fungal species. Interaction studies between: A) Bacterial-bacterial species, B) Fungalfungal species and C) Bacterialfungal species. Formulation and application of novel biopesticide (for rhizospheric application) on Abelmoschus esculentus.
Name of the Organism
No. of colonies
Size
Shape
Elevation
Margin
Transparency
Gram nature
PSB
1890
Pin point
Boat
No elevation
Smooth
Opaque
Gram negative
Bacillus thuringiensis
3.0 mm
Circular
No elevation
Smooth
Opaque
Based on studies of the interaction studies, the following microorganisms were included in the final formulation: Trichoderma spp. Metarhizium spp. B. thuringiensis B. subtilis Azotobacter spp. PSB KSB
Gram positive
67
4.0 mm
Circular
No elevation
Serrated
Opaque
Gram positive
Bacillus subtilis
59
0.5 mm
Irregular
No elevation
Serrated
Opaque
Gram positive
Azotobacter spp.
1792
Pin point
Circular
No elevation
Smooth
Opaque
Gram negative
Azospirillum spp.
460
Pin point
Circular
No elevation
Smooth
Opaque
Gram negative
KSB
356
Pin point
Circular
No elevation
Smooth
Opaque
Gram negative
Figure 10: The novel biopesticide ready for application.
Organic supplement, carrier and microbial inoculants were mixed and the mixture was incubated for 10 days at 37°C.
pH
(b)
(a)
(c)
Colony character
Species 2
Trichoderma spp.
Granular, an irregular yellow zone without conidia present around the inoculum, white pustules found growing on the green mat of conidia.
Green
Metarhizium spp.
Olive green mycelium with milky white, cottony and round edges.
Olive green
Paecilomyces spp.
Colonies are floccose and white, the texture is wooly to powdery.
White
Azotobacter spp.
PSB
Azotobacter spp.
No inhibitory action was observed.
Moderately antagonistic growth was observed.
Azospirillum spp.
Azotobacter spp.
Species 1
Species 2
Bacillus megaterium
Bacillus subtilis
Pseudomonas fluorescens
Bacillus thuringiensis
Pseudomonas fluorescens
Bacillus thuringiensis
Bacillus subtilis
Bacillus megaterium
Bacillus thuringiensis
PSB
Pseudomonas fluorescens
Bacillus thuringiensis
KSB
KSB
Table 3: Interaction study between bacterial species on YMA plates.
8.4
Figure 11: The model organism used for testing the efficacy of the biopesticide is Abelmoschus esculentus (Okra plant).
Figure 12: The pathogenic effect was evaluated by application of a suspension of Alternaria spp. 7 (concentration: 7.8 × 10 cfu of spores per ml; corroborated with haemocytometer and microscope) to the Okra plant (both control and experimental set-up).
Result: PO activity was considerably higher in the experimental leaf as compared to the control leaf.
Pathogenicity Test β-1,3-glucanase Assay
To check for the efficiency of the biopesticide a pathogenicity was carried out where two major aspects were analysed post exposure to the phytopathogen, Alternaria spp.: (1) The number of spots appearing on the leaf and (2) area of infection of the leaf.
β-1,3-glucanase is a PR-2 family of pathogenesis-related protein which helps in defending the plant against fungal phytopathogens either alone or in association with chitinases or other antifungal proteins. They also play a key role in cell division, trafficking of material through the plasmodesmata, in withstanding abiotic stresses and governs physiological processes such as seed maturation and flower formation. The assay was carried out by the Laminarin-Dinitrosalicylate method.
Slight negative interaction was observed. No inhibitory action was observed.
Result: β-1,3-glucanase activity was considerably higher in the experimental leaf as compared to the control leaf.
No inhibitory action was observed.
Bacillus subtilis
Experimental set-up 2 (Soil + Biopesticide)
Peroxidase enzyme plays a key role in plants in response to biotic and abiotic stresses. During pathogen attack, peroxidases: (1) help in formation of lignin which contributes to the rigidity of plant and prevents pathogen entry, (2) are involved in scavenging of ROS which otherwise cause oxidative damage to the cells. Therefore, peroxidase is considered to be one of the enzymes which is associated with induction of resistance in plants. The assay was carried out by the Guaiacol-H2O2 method.
Figure 13: 48 hrs post application of biopesticide to experimental setup, suspension of Alternaria spp. was applied to the leaves of both (a) Experimental set-up and (b) Control set-up, and the leaves were covered with plastic bags.
Interaction No inhibitory action was observed. No inhibitory action was observed. No inhibitory action was observed. No inhibitory action was observed.
Negative interaction was observed..
5.0
Peroxidase Assay
Table 2: Colony characteristics of the fungal species.
Interaction
Experimental set-up 1 (Soil + Chemical pesticide)
These enzyme assays were performed 48 hrs after application of biopesticide to the Experimental Set-up but before conducting pathogenicity test.
Color of spore
Interaction Study Species 1
8.0
Enzyme Assay
Name of the organism
Figure 2: (a) Paecilomyces spp. plated on PDA plate. (b) Metarhizium spp. plated on PDA plates. (c) Metarhizium spp. observed under 400X magnification.
Control set-up (Soil only)
Table 7: Soil pH evaluation using a pH meter.
Table 1: Colony characteristics of the bacterial species.
Table 4: Interaction study between bacterial species on NA plates.
(d)
(c)
(b)
Figure 3: Interaction study between bacterial species on YMA plates, after 24 hours of incubation: (a) Azotobacter and PSB, (b) Azotobacter spp. and Azospirillum spp., (c) Azotobacter spp. and Pseudomonas fluorescens, (d) after 48 hours of incubation between Azotobacter spp. and Pseudomonas fluorescens. Species 1
Species 2
Interaction
Paecilomyces spp.
Trichoderma spp.
Negative interaction was observed.
Metarhizium spp.
Trichoderma spp.
No inhibitory action was observed.
(a)
(e)
(b)
(c)
(d)
Figure 5: Interaction study between bacterial species on NA plates, after 24 hours of incubation: (a) B. subtilis and B. megaterium, (b) P. fluorescens and B. thuringiensis, (c) PSB and P. fluorescens, (d) ) B. thuringiensis and B. megaterium, , (e) B. thuringiensis and B. subtilis, (f) KSB, B. subtilis and B. thuringiensis.
(f)
Table 5: Interaction study between fungal species on PDA plates.
Pathogenesis-related enzyme assays (PO and β-1,3-glucanase).
Figure 7: Graphical representation of growth of Trichoderma spp. in presence of different bacterial species.
Figure 6: Interaction study between Metarhizium spp. and Trichoderma spp. on PDA. The growth potential of the two species is variable. Growth of Trichoderma spp. was more vigorous than Metarhizium spp..
Pathogenicity test using Alterneria spp. Formulation of a liquid biopesticide for phyllospheric application.
(a)
(b)
(c)
(d)
(e)
(f)
Figure 17: Spores of Alternaria spp. were observed surrounding the necrotic epidermal tissues of the control leaf when observed under 400X magnification.
120
100
80
60
40
Figure 14: (a) Control leaf after defoliation, (b) Crosssectional view of epidermis of control leaf, (c) Crosssectional view of vascular bundle of control leaf, (d) Experimental leaf after defoliation, (e) Cross-sectional view of epidermis of experimental leaf, (f) Crosssectional view of vascular bundle of experimental leaf.
Length
Weight
Experimental fruit
10.4 cm
9.4 gm
Control fruit
6.2 cm (curling was observed)
5.1 gm
20
Fungal species
Bacterial species
Interaction
Azotobacter spp.
No inhibitory action was observed.
Future prospects:
Bacillus thuringeinsis
Negative interaction was observed.
1. Enzyme assay of other pathogenesis related enzymes such as chitinase, phenylalanine ammonia-lyase and polyphenol oxidase would be carried out.
Bacillus subtilis
No inhibitory action was observed.
Bacillus megaterium
No inhibitory action was observed.
Figure 8: Experimental set-up for bacterial-fungal interaction study (both the fungal and bacterial species were grown together).
0
PSB
Bacillus subtilis
Bacillus megaterium Bacillus thuringiensis
Control
Azotobacter
KSB
Figure 15: (a) Appearance of spots on control leaf after 3 days of exposure to phytopathogen, (b) No such spot appeared on experimental leaf.
Experimental
Trichoderma spp. PSB
No inhibitory action was observed.
KSB
No inhibitory action was observed.
Figure 8: Graphical representation of growth of Metarhizium spp. in presence of different bacterial species. 90
2. Pathogenicity test would be carried out using (a) a model organism other than Okra plant, (b) using fungal phytopathogen other than Alternaria spp. 3. To carry out elaborate scientific test to determine applicability of this novel biopesticide in the natural environment and to test for the shelf-life of the product.
45
Bacillus megaterium
Figure 1: Pure culturing of the bacterial species using quadrant streaking.
(a)
Materials and Methods:
Formulation and application of biopesticide
(a)
(a)
80
(b)
Figure 21: (a) Unnatural curling was observed in the fruits obtained from the control set-up. (b) Normal healthy growth observed in fruits from experimental set-up.
Table 8: Tabulation of different parameters of fruits obtained from the Okra plants.
(b)
70
Azotobacter spp.
No inhibitory action was observed.
Bacillus thuringiensis
No inhibitory action was observed.
Bacillus subtilis
No inhibitory action was observed.
Metarhizium spp. Bacillus megaterium
No inhibitory action was observed.
PSB
No inhibitory action was observed.
KSB
No inhibitory action was observed.
60 50
(a)
(b)
Figure 9: Control set-up (a) for bacterial-fungal interaction study (only the bacterial species were grown), (b) for bacterial-fungal interaction study (only the fungal species were grown).
40 30
Figure 16: Post exposure to phytopathogen, growth of new leaves was observed only in the experimental set-up but not in the control.
20 10 0 PSB
Bacillus subtilis
Bacillus megaterium Control
Bacillus thuringiensis
Azotobacter
KSB
Experimental
(a) Table 6: Interaction study between bacterial and fungal species on NA plates.
(b)
Figure 23: Spore suspension of Trichoderma spp. and Metarhizium spp. were separately mixed with diluted Neem oil and kept for 24 hours. The viability of spores was checked by plating them in PDA plates
Figure 24: A liquid formulation containing the fungal species, neem oil and Tween 20 was prepared. This would be sprayed on the phyllosphere of infected plants