Mode of action of Bacillus subtilius as plant pathogen biocontrol agent it includes mode of action and introduction ..

1. University of Horticultural science, Bagalkot
College of Horticulture, Bagalkot, 587104
Presentation on : Bacillus species as versatile weapons for plant pathogens
Presented By: Nagesh
Ist Ph.D Plant Pathology
UHS,Bagalkot Submitted to: Dr. Kiran Kumar K. C.
Asst. Prof. Plant Pathology
UHS, Bagalkot

2. Introduction
1. Bacillus subtilis, also known as the hay bacillus or grass bacillus.
2. It is a Gram-positive, catalase-positive bacterium.
3. Its found in soil and the gastrointestinal tract of ruminants and humans.
4. A member of the genus Bacillus,
5. These are endospores producing bacteria.
6. These endospores protects the bacteria from tolerate extreme environmental conditions.
7. B. subtilis has historically been classified as an obligate aerobe, though evidence exists that it is a facultative
aerobe.
8. B. subtilis is considered the best studied Gram-positive bacterium.
9. B. subtilis is model organism to study bacterial chromosome replication and cell differentiation.
10. It is one of the bacterial champions in secreted enzyme production and used on an industrial scale by
biotechnology

3. 1. There was a long-held belief that the gram-positive soil bacterium Bacillus subtilisis a strict aerobe. But recent
studies have shown that B. subtilis will grow anaerobically, either by using nitrate or nitrite as a terminal electron
acceptor, or by fermentation.
2. In terms of popularity as a laboratory model organism, B. subtilisis often used as the Gram positive equivalent of
Escherichia coli, an extensively studied Gram-negative bacterium.
3. Bacillus subtilis are rod-shaped bacteria that are Gram-positive (Perez 2000). The cell wall is a rigid structure
outside the cell. It is composed of peptidoglycan, which is a polymer of sugars and amino acids. The
peptidoglycan that is found in bacteria is known as murein.
4. Bacillus subtilis is a Gram-positive bacterium, rod-shaped and catalase-positive. It was originally named Vibrio
subtilis by Christian Gottfried Ehrenberg, and renamed Bacillus subtilis by Ferdinand Cohn in 1872.
5. B. subtilis cells are typically rod-shaped, and are about 4-10 micrometers (μm) long and 0.25–1.0 μm in diameter.
As other members of the genus Bacillus, it can form an endospore, to survive extreme environmental conditions
of temperature and desiccation.

4. Reproduction:
1. B. subtilis can divide symmetrically to make two daughter cells (binary fission), or asymmetrically,
2. producing a single endospore that can remain viable for decades and is resistant to unfavorable environmental.
drought, salinity, extreme pH, radiation, and solvents.
3. Prior to the process of sporulation the cells might become motile by producing flagella, take up DNA from the
environment, or produce antibiotics.
4. These responses are viewed as attempts to seek out nutrients by seeking a more favourable environment, enabling
the cell to make use of new beneficial genetic material or simply by killing of competition.
Transformation:
Natural bacterial transformation involves the transfer of DNA from one bacterium to another through the surrounding
medium.
In B. subtilis, length of transferred DNA is greater than 1271 kb (more than 1 million bases).
The transferred DNA is likely double-stranded DNA and is often more than a third of the total chromosome length of
4215 kb.
It appears that about 7-9% of the recipient cells take up an entire chromosome.

5. 1. In order for a recipient bacterium to bind, take up exogenous DNA from another bacterium of the same species
and recombine it into its chromosome, it must enter a special physiological state called competence.
2. Competence in B. subtilisis induced toward the end of logarithmic growth.
3. The antibiotic bacitracin was determined to be affective on Gram-positive bacteria only (Jamil 2007).
4. Other antibiotics that Bacillus subtilis form are polymyxin, difficidin, subtilin, and mycobacillin.
5. Polymyxin is affective against Gram-negative bacteria, whereas difficidin has a broader spectrum (Todar).
6. Bacillus subtilis bacteria secrete enzymes, "such as amylase, protease, pullulanase, chitinase, xylanase, lipase,
among others.
7. These enzymes are produced commercially and this enzyme production represents about 60% of the
commercially produced industrial enzymes

7. Plant growth promotion as an indirect weapon
Higher level of plant growth-promoting hormones (GA3 and IAA) and defence-related enzymes (peroxidase (PO),
polyphenol oxidase (PPO) and superoxide dismutase).
IAA has an important role in origination and emergence of adventitious roots.
It also enhances shoot development by influencing cell expression, division and differentiation.
The GA3 plays it role in combination with auxin for elongation of plant and leaf bud formation.

8. Lipopeptides- and antibiotics:
756 of such peptides have various degrees of antifungal properties.
The mechanisms by which microorganisms lead fungi to death include blockage, distraction and holes formation in the
cell wall and cell membranes of the fungi.
Table1.Summary of Bacillus-produced lipopeptides and antibiotics, their mode of action and target pathogens

10. Systemically induced disease resistance
The bacterial strain EXTN-1 was proved to be effective and they also
showed that reduction of disease was not due to direct antagonism
but as a result of elicitation of host plant resistance genes.
The main components of systemic-induced resistance are phenolic
compounds, genetic and structural modifications, plant resistance
activators, and activation of enzymatic weapons.
Triggering of phenolic compounds:
B. subtilus 174 against Fusarium wilt of tomato.
(phenyl ammonia lyase (PAL), PPO and PO)
B. subtilis strain FZB-G has the ability to trigger phytohormones
precursor which plays an important role in signal transduction.
AUBS1 for systemic resistance induction against sheath blight of rice.
(They also found that the increased levels of thaumatin and b-1-3-
glucanases with 17 and 30 kDa).

11. Fig. 7: Transgenic tobacco plants with the prp1/PAT gene fusion for identification of resistance inducers. Plants 120 h
after treatment with FZB24® (left) or with blank formulation (right). Test plants were treated with 15l of Basta/ha.
Photograph taken 9 days after herbicide treatment.
The activation of the prp1 promoter by FZB24® is clearly demonstrated by the reduction of herbicide damage.
prp1 – promoter of the
proteins that accompany
pathogenesis development
from potatoes
• chit2a – promoter of a
chitinase gene from peanuts
• Vst1 – promoter of the
stilbene synthase gene from
vines
All three promoters were activated by the treatment with FZB24
prp1
Kilia et al., 2000

12. Bacillus species are also capable of producing enzymes like chitinase and b-1,3-glucanase having a very strong lytic activity.
(oxidative enzymes such as PPO, PO and PAL)
Table 2. Summary of Bacillus-induced systemic resistance principle eliciting factors and target pathogens

13. Colonization
Interspecies competition causes reduction in growth, productiveness and other activities of the competing organisms.
Table 3. Summary of important Bacillus strains in relation to colonization, their target pathogens and host plants

14. Fig. 3: Tomato roots (cv ‘Minibell’) on gelrite
medium colonized by FZB24®-Bacillus subtilis.
Application of the bacteria was done by seed
dressing
Fig. 2: Scanning electron micrograph of a pea
root with adhering FZB24® Bacillus subtilis cells

15. In vitro spore germination evaluation
Korsten et al., 1995

16. Korsten et al., 1995

17. Spores can survive treatments that rapidly and efficiently kill other bacterial forms, including high temperatures
(even 100°C), ionizing radiation, chemical solvents, detergents and hydrolytic enzymes.
Figure 1| The sporulation cycle of Bacillus subtilis.

18. cytosolic factor
nucleoid occlusion
Figure 2| Cell division during growth (a) and sporulation (b) of B. subtilis

20. Table 1 Effect of soil drench treatments immediately after sowing with the endophytic Bacillus subtilis strain
E1R-j on seedling growth and take-all disease of wheat in greenhouse at 25C.
Lt, length; FW, fresh weight; DW, dry weight; Ht, height; DI, disease index; DR, disease reduction (%)
Liu et al., 2009

21. Fig. 1. The morphology of the endophytic bacterial strain
E1R-j observed by electron microscopy.
Fig. 2. Phylogenetic tree based on 16S rDNA sequence
homology of strain E1R-j

22. Table 2: Influence of endophytic Bacillus subtilis strain E1R-jon plant height and yield parameters (seeds per head and
thousand kernel weight) in field experiments (2006/2007 and 2007/ 2008 at harvest. In 2007/2008 the
fungicide Triadimefon was included in the experiment.

23. Fig. 2. Scanning electron micrographs showing the effects of Bacillus subtilis E1R-j on the morphology of the hyphae of
Gaeumannomyces graminis var. tritici (Ggt).

24. Fig. 3. Transmission electron micrographs of colonization of roots of wheat seedlings by Gaeumannomyces graminis var.
tritici (Ggt) in absence and presence of the endophytic strain E1R-jof Bacillus subtilis,3days after inoculation (dai).
Root Cortex of Host
cell wallcavity oftheroot

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