BIOCONTROL AGENT – Bacillus subtilis

BIOCONTROL AGENT – Bacillus subtilis

General Characteristics of Bacillus subtilis

• Gram’s classification – Gram positive
• Shape – Rod shape
• Arrangement - Single, pair or chains
• Size – Measures 4 –10 μm long and 0.25 – 1.0 μm in diameter
• Family – Bacillaceae
• Motility – Motile (Swarming motility)
• Capsule – Absent
• Endospores – Present (Central spores)
• Respiration – Facultative anaerobic.
• Optimum Temperature – Grow in the mesophilic temperature range. The optimal temperature is 25 °C to 35 °C.
• Optimum pH – 8.0
• Bacillus subtilis is also called as Hay Bacillus or Grass Bacillus.
• The word Bacillus refers to the shape of the bacteria (rod-shaped) and subtilis means slim, slender.
• Bacillus subtilis bacteria secrete enzymes such as Amylase, Protease, Pullulanase, Chitinase, Xylanase and Lipase. These enzymes are produced commercially and this enzyme production represents about 60 % of the commercially produced industrial enzymes.  

History of Bacillus subtilis

• Previously, Bacillus subtilis was known as Vibrio subtilis, this bacterium was discovered by Christian Gottfried Ehrenberg in 1835.
• Vibrio subtilis was renamed as Bacillus subtilis in 1872 by Ferdinand Cohn.

Habitat of Bacillus subtilis

• The main habitat of endospore forming Bacillus organisms is the soil. Likewise, Bacillus subtilis is most commonly found in soil environments and on plant undergrowth. 

Bacillus subtilis as Plant disease control agent

• Numerous species of Bacillus have been identified as plant-growth promoting bacteria (PGPB) and Biocontrol agents (BCA). The most commonly studied PGPB are Bacillus amyloliquefaciens, Bacillus licheniformis, and Bacillus subtilis.
• The most common direct mechanisms of PGPB are phytohormone production, the acquisition of nutrients such as phosphorous and nitrogen, and the control of pathogens through various means, for example, through the synthesis of hydrolytic enzymes, antifungal compounds, lipopeptides, or antibiotics.
• The indirect mechanisms of PGPB include protection from abiotic stress brought about by drought, salinity, etc., the triggering of specific defense-related pathways, particularly the induction of systemic resistance (ISR) against pathogens and pests and the release of volatile organic compounds.
• Bacillus subtilis are also used on plants as a Fungicide.
• The bacteria Bacillus subtilis, colonized on root systems, compete with disease causing fungal organisms.
• Bacillus subtilis use as a fungicide fortunately does not affect humans.
• Bacillus subtilis bacteria secrete enzymes such as Amylase, Protease, Chitinase, Xylanase and Lipase. The enzyme Chitinase is highly involved in the biocontrol of plant diseases.
• Bacillus subtilis have become attractive biological control agents due to their ability to produce hard, resistant Endospores and antibiotics which control a broad range of plant pathogens.
• Bacillus subtilis competes with other microorganisms by producing antibiotics (Surfactin, Iturin, and Fengycin) that either kill competition or reduce their growth rate.
• Bacillus subtilis was reported to induce SAR (Systemic Acquired Resistance) against bacterial pathogens, whereby the plant's defenses are triggered prior to pest incursion.
• In addition, Bacillus subtilis hinders spore germination in plant pathogens and prevents pathogens from attaching to the plant. 

Crop Recommendation

• Potato, Cucumbers, Eggplant, Tomatoes, Chilli, Cut flowers, Orchards, Vineyards Ornamentals in greenhouses, lawns and nurseries.

Target Diseases

• Phytophtora sp., Aspergillus sp., Pythium sp., Rhizoctonia solani, Fusarium sp., Botrytis cinerea, Sclerotium rolfsii, Sclerotinia sp. and Ustilogo sp. 

Isolation of Bacillus subtilis

• Bacillus subtilis are isolated from the soil sample by Serial Dilution Method (Spread Plate Method or Pour Plate Method).
• The common culture medium used for the isolation of Bacillus subtilis is Bacillus Differentiation Agar.

Microscopic characteristics of Bacillus subtilis

• Gram staining – Bacillus subtilis was observed as Violet coloured, rod shaped Gram positive bacteria arranged as single, pair or chains.
• Motility test (Hanging Drop Method) – Motile rods (Swarming motility).

Cultural characteristics of Bacillus subtilis

• Bacillus Differentiation Agar is recommended for differentiation between Bacillus cereus and Bacillus subtilis based on Mannitol fermentation. Yeast autolysate provide necessary nitrogenous source for growth of Bacillus subtilis. Magnesium sulphate and Potassium chloride supports sporulation. Ammonium phosphate maintains buffering action. Bromocresol purple act as a pH indicator to detect Mannitol fermentation.
• In Bacillus Differentiation Agar, Bacillus subtilis shows Yellow coloured colonies.

Figure – 1: Growth of Bacillus subtilis in Bacillus Differentiation Broth

Figure – 2: Colony morphology of Bacillus subtilis in Bacillus Differentiation Agar

Biochemical characteristics of Bacillus subtilis

a)     Catalase test – Positive

b)     Oxidase test – Negative

c)     Urease test – Negative

d)     Indole test – Negative

e)     Methyl Red (MR) test – Negative

f)      Voges Proskauer (VP) test – Positive

g)     Citrate utilization test – Positive

h)    Starch hydrolysis test – Positive

i)      Gelatin hydrolysis test – Positive

j)      Esculin hydrolysis – Negative

k)     O-F test - Positive

Formulation of Bacillus subtilis

• Commercial preparations of Bt comes in various forms such as solids, liquids, powders, tablets, granules, ball agents, liquid suspensions, and oil emulsions.