1. Biological control of plant pathogens

2. Rhizosphere microorganisms are found in the area surrounding and influenced by the plant roots.
Rhizoplane microorganisms are found on the surface of plant roots.

5. Exudates: carbohydrates and proteins secreted by roots;
Attracts bacteria, fungi, nematodes, protozoa
Bacteria and fungi are like little fertilizer bags
Nematodes and protozoa eat and excrete the fertilizer

6. Why use biological control?
Biological control agents are
Expensive
Host specific
Chemical pesticides are:
Cost-effective
Easy to apply
Broad spectrum

7. Why use biological control?
Chemical pesticides
Implicated in ecological, environmental, and human health problems
Require yearly treatments
Broad spectrum
Toxic to both beneficial and pathogenic species
Biological control agents
Non-toxic to human
Not a water contaminant concern
Host specific
Only effect one or few species

8. Mechanisms of biological control of plant pathogens
Antagonism
Are biological agents that reduce the numbers or disease producing activities of the pathogen through one or more of the following mechanisms:-

9. Mechanisms of biological control of plant pathogens
Antibiosis
– inhibition of one organism by another as a result of diffusion of an metabolic products such as antibiotic, lytic agents, enzymes, volatile compounds and other toxic substances.
Antibiotic production common in soil bacteria and fungi
Example: zwittermicin antibiotic production by Bacillus cereus against Phytophthora root rot in alfalfa

10. Mechanisms of biological control of plant pathogens
Nutrient competition
– competition between microorganisms for limited sources such as carbon, nitrogen, O2, iron, and other nutrients or spaces.
Most common way organisms limit growth of others

11. Mechanisms of biological control of plant pathogens
Destructive mycoparasitism
Occurs when the antagonist invades the pathogen by secreting enzymes such as chitinase, cellulase, glucanase, and other lytic enzymes.
The parasitism of one fungus by another
Direct contact
Cell wall degrading enzymes
Some produce antibiotics
Example
Trichoderma harzianum, used as seed treatment against pathogenic fungus

12. Requirements of successful biocontrol
Highly effective biocontrol strain must be obtained or produced
Be able to compete and persist
Be able to colonize and proliferate
Be non-pathogenic to host plant and environment

13. Requirements of successful biocontrol
Inexpensive production and formulation of agent must be developed
Production must result in biomass with excellent shelf live
To be successful as agricultural agent must be
Inexpensive
Able to produce in large quantities
Maintain viability

14. Requirements of successful biocontrol
Application must permit full expression of the agent
Must ensure agents will grow and achieve their purpose
Coiling of Trichoderma around a pathogen. (Plant Biocontrol by Trichoderma spp. Ilan Chet, Ada Viterbo and Yariv Brotman)

15. Plant pathogen control by Trichoderma spp.
Trichoderma spp. are present in nearly all agricultural soils
Antifungal abilities have been known since 1930s
Mycoparasitism
Nutrient competition
Agriculturally used as biocontrol agent and as a plant growth promoter
                                                                                                                                                                                                       

16. T22 strain
Uses antibiosis and predation against soil
borne pathogens such as Pythium,
Rhizoctonia, Fusarium and Sclerotina

17. Plant pathogen control by Trichoderma spp.
Action against pathogenic fungi
Attachment to the host hyphae by coiling
Lectin-carbohydrate interaction
(Hubbard et al., Phytopathology 73: ).

18. Plant pathogen control by Trichoderma spp.
Action against pathogenic fungi
2. Penetrate the host cell walls by secreting lytic enzymes
Chitinases
Proteases
Glucanases
(Ilan Chet, Hebrew University of Jerusalem).

19. Trichoderma spp. attach to the host hyphae via coiling, hooks and appressorium like bodies, and penetrate the host cell wall by secreting lytic enzymes.
Trichoderma recognizes signals from the host fungus, triggering coiling and host penetration.
A biomimetic system consisting of lectin-coated nylon fibers was used to study the role of lectins in mycoparasitism. Using this system we could also identify specific coiling-inducing molecules.

20. Plant pathogen control by Trichoderma spp.
Some strains colonize the root with mycoparasitic properties
Penetrate the root tissue
Induce metabolic changes which induce resistance
Accumulation of antimicrobial compounds

21. Plant pathogen control by Trichoderma spp.
Commercial availability
T-22
Seed coating
Protects roots from diseases caused by Pythium, Rhizoctonia and Fusarium
Interacts with the Rhizosphere, near the root hairs and increases the available form of nutrients needed by plants.

22. Plant pathogen control by Trichoderma spp.
Future developments
Transgenes
Biocontrol microbes contain a large number of genes which allow biocontrol to occur
Cloned several genes from Trichoderma as transgenes
Produce crops which are resistant to plant diseases
Currently not commercially available

23. The success of Trichoderma strains as BCA is due to:
High reproductive capacity
Ability to survive under very unfavorable conditions
Efficacy in the utilization of nutrients
Capacity to modify the rhizosphere ( colonization)
Strong aggression against phytopathogenic fungi
Efficacy in promoting plant growth and defense mechanisms.
Production of antibiotics.

24. Note
Trichoderma is more effective in acidic than alkaline soils.
Trichderma strains grow rapidlly when inoculated in the soil because they are naturally resistant to many toxic compounds including herbicides, fungicides and pesticides such DDT and phenolic compounds.
Resistance to toxic compounds

25. Plant root colonization
Trichoderma strains must:
Colonize plant roots prior to stimulation of plant growth and protection against infections.
Colonization implies the ability to adhere and recognize plant roots, penetrate the plant, and withstand toxic metabolites produced by the plants in response to invasion by a foreign organism.

26. Root colonization by Trichoderma strains frequently enhances:
Biofertilization
Root colonization by Trichoderma strains frequently enhances:
Root growth and development.
Crop productivity (increase up to 30%)
Resistance to a biotic stress
Uptake and use of nutrients.
Enhance solubilization of soil nutrients
Increase root hair formation.

27. Stimulation of plant resistance and plant defense mechanism
Strains of Trichoderma added to the Rhizosphere protect plants against numerous of pathogens. e.g those that produce aerial infections, including viral, bacterial and fungal pathogens, which points to the induction of resistance mechanisms similar to the hypersensitive response in plants.

28. Hypersensitive response –is a mechanism used by plants, to prevent spread of infection by microbial pathogens.
At a molecular level, resistance results in an increase in the concentration of metabolites and enzymes related to defense mechanisms, such as the enzyme phenyalanine ammonio lyase (PAL) degrades phenyalanine to harmless products ammonia and trans cinnamic acid. Chitinase, and glucanase –enzymes that break down glucans glucans are important structural compounds in the cell wall of plant and fungi.

29. Chitinase are digestive enzymes that break down glycosidic bonds in chitin because chitin composes the cell walls of fungi and exoskeleton elements of some animals (including arthropods=insects)

31. Antibiosis
Antibiosis occurs during interactions involving low molecular weight diffusible compounds or antibiotics produced by Trichoderma.
Most Trichoderma strains produce volatile and non volatile toxic metabolites that impede colonization by antagonized microorganisms among these metabolites the production of:

32. Harzianic acid---------antibacterial
Alamethicins which increase the permeability of membranes produced by T. viride
Tricholin is a potent of cell free protein synthesis through damage to large ribosomal subunits.
Gliovirin toxic to Pythium ultimum
Glisoprenin antifungal
Heptelidic acid antibacterial
Peptaibols active against fungi in late growth and insecticidal when fed to larvae

33. Mycoparasitism
Mycoparasitism the direct attack of one fungus on another, is a very complete process that involves sequential events, including recognition, attack and subsequent penetration and killing of the host.
Trichoderma spp parasitize fungal plant pathogens , the parasites extends hyphal branches toward the target host, coil around and attaches to within appressorium like bodies, and punctures it mycelium.

35. Mycoparasitism Cell wall degrading enzymes
1- glucanases ----it has been shown B-1,3-glucanases inhibit spore germination.
2- proteases inactivate hydrolytic enzymes produced by this pathogen on bean leaves.

36. Parasitism
Parasitism ---fungi that parasitize other fungi by the following steps
Detection of target.
Chemotactic growth.
Chemical and physical recognition of host.
Stimulation of extracellular enzymes..

37. After inoculation, the culture were filtered through 0.22 mm filters’
Determination of antifungal properties of culture filtrate of Trichoderma harzianum
Each T.harzianum isolate was separately inoculated into 100 ml PDB (potato dextrose broth) and inoculated at 20 c for 10 days.
After inoculation, the culture were filtered through 0.22 mm filters’
The a liquates 2 ml of these filtrates were placed in sterile petri dishes and 25ml of 25% strength PDA at 45c was added.

38. After agar solidified, mycelial discs of the pathogens (7 mm in diameter) obtained from activity growing colonies were placed gently in the center of the agar plates.
The petri dishes were incubated at 20c for 6 days.
Growth of the pathogens was recorded by measuring the diameter of the colonies each day. There were 3 replicates for each experiment.

39. Competition for infection sites. Competition for nutrients.
Fusarium spp
Certain non pathogenic races of Fusarium spp are able to protect plants from species pathogens.
The proposed mechanisms involved in Fusarium spp biological control include:
Competition for infection sites.
Competition for nutrients.
Local and systemic induced resistance.

40. Fusarium spp
Non pathogenic Fusarium oxysporum have received much attention and have been used in many field and green house trials conducted with watermelon, tomato, radish, and cucumber.
Combining the non pathogenic F. oxysporum with the bacterial strains Pseudomonas putida two different disease suppressive mechanisms act together to enhance suppresion of fusarium wilt.

41. Bacterial biocontrol
Many different bacterial strain can mediated biocontrol most important are Pseudomonas and Bacillus strains but several other bacteria are also known to mediate plant protection.
Pseudomonas are probably the most studied Rhizobacteria in biocontrol but Bacillus has one big advantage over Pseudomonas

42. Bacillus produce spores that are resistant to stress, it can survive high temperature, extreme PH, chemical and mechanical stress.
Bacillus are more convenient to use in the fields as it is easier to handle and apply providing commercial benefits.
The protection mechanism differs among strains several different methods can be used at the same time to combat the pathogen.
1. Alteration of the plant cell wall that causes an increased protection to pathogens has been found to occur with both Bacillus subtilis and Pseudomonas aeruginosa.

43. 2. Formation of biofilm on plant roots by the bacteria makes the plants less sensetive to infection.
3. Competition for growth space and nutrients is another important factor.
4. Production of antibiotics and other harmful compounds by the bacteria.

44. Formation of biofilm on plant roots

45. 5. Synthesis of salicylic acid by bacteria can make the plant more tolerant to pests and pathogens by stimulating systemic acquired resistance SAR, a common defense program induced in plants to combat pathogens.

46. 6. Induction of induced systemic resistance ISR in the plant is another way that bacteria can protect plants.
Such as Serratia marcescens induce systemic resistance ISR to fungal, bacterial and viral pathogens.
Selection of bacteria known to mediate biocontrol
Bacillus amyloliquefaciens
Bacillus subtilis
Bacillus polymyxa
Bacillus lichenformis

47. 5. Bacillus cereus
6. Bacillus pumilis
7. Pseudomonas fluorescens
8. Pseudomonas chlororaphis
9. Enterobacter cloacae

48. Antibiotics
Bacteria are known to produce a wide array of antibiotics.
Many bacteria are able to produce several different antibiotics that have a broad range and sometimes overlap in their function.
These antibiotics play a significant role in biocontrol.

49. Bacteria are also able to synthesize enzymes like chitinase, proteases, lipases and 1,3 glucanases that are all harmful for microorganisms and further improves the biocontrol efficacy.
It produces antibiotics such as difficidin, and oxydifficidin that have activity against a wide spectrum of aerobic and anaerobic bacteria.

50. It produces bacitracin, bacillin and bacillmycin B
Some bacteria are generally improved to produce more or new antibiotics to provide better protection Bacillus subtilis also produces iturin(lipopeptide antibiotic which make against phytopathogenic fungi )

51. Bacillus subtilis produces Zeatin is a plant hormone derived from the purine adenine, it is a member of the plant growth hormone family known as cytokinins
Zeatin (in vitro anti aging effect on human skin fibriblast.

53. Competition
One way that beneficial can protect plants from pathogens is through competition.
Bacteria also is made more efficient with the help of siderphore.
Siderophore are the extracellular low molecular weight Fe III specific ligands that are used for bacteria to scavenge iron from the environment.

54. Siderophore solubilises iron which then is transported into the bacterial cells using specific receptors.
This gives the bacteria the possibility to deplete the available iron source from other potentially harmful bacterial strains.
Siderophores have earlier been shown to be essential to some bacteria that protect plants.

56. PGPR plant growth promoting rhizobacteria
Can increase plant growth and vitality through production of phytohormones like auxins, gibberellins, abscisic acid, ethylene and cytokinins.
Gibberellins = promoting growth and elongation of cells + stimulates the cells of germinating seeds.
Auxins = increase the elongation of cells
Cytokinins= promote cell division

57. Auxin Indole-3-acetic acid (IAA) and its conjugates.
Synthesized from tryptophan
IAA Structure

58. Gibberellin Family of 125 compounds.
Gibberellin acid (GA3)is the most common.
Synthesized in young shoots and developing seed.

59. Gibberellin promotes Stem elongation
Cell division and elongation in stems
Germination if cold or light treatment is required.
Enzyme production, eg. α-amylase.
Fruit set

60. Cytokinins Adenine derivatives. Zeatin is the most common.
Synthesized in root tips and developing seed.
Transported in xylem.

61. Cytokinin promotes Cell division if auxin is present.
Lateral bud growth
Leaf expansion by cell enlargement
Stomatal opening
Chloroplast development

62. H2C=CH2 Ethylene Gas Synthesized from methionine
Most tissues can synthesize ethylene in response to stress.
Chemical Structure

63. Ethylene stimulates Defense response to wounding or disease
Release from dormancy
Shoot growth and differentiation
Root growth and differentiation
Adventitious root formation

64. Abscisic Acid
Synthesized from glyceraldehyde-3-phosphate through carotenoid pathway.
Synthesized in roots, mature leaves, and seeds.

65. Jasmonates
Jasmonic acid is most common
Methyl jasmonate

66. Jasmonate function Induce tuberization Important in plant defense
Inhibit growth
Inhibit germination
Promote pigmentation

67. Salicylic Acid Synthesized from phenylalanine.
Promotes production of pathogenesis related proteins.

68. Another way is to increase the amounts of available nutrients like fixed nitrogen, phosphorous and iron solubilised from soil.

69. Plant innate immunity
A major plant defense against pathogens has evolved as the innate immunity system. Using various pattern recognition receptor proteins plants can identify pathogen associated molecular patterns PAMPs of potential pathogens and show a basal defense response.
The innate immunity system is widespread in nature and seems to have evolved early explaining extensive similarities found between animals, insects and plants.

70. Biological fungicides
Gliocladium against Rhizoctonia
Trichoderma against Rhizoctonia
Penicillium against Rhizoctonia
Fusarium against Puccinia and verticillum

71. Most fungi produce inhibitory metabolites examples:
Gliocladium produces a diketopeprazine that kills Pythium because of coagulation of proteins in the cytoplasm.
Volatile pyronens produced by Trichoderma appear to reduce damping off caused by Rhizoctonia.

72. damping off ----------kill seeds or seedlings before or after they germinate.
Salicylic acid which produced by pathogens
Salicylic acid leads to the expression of pathogenesis related protein PRP
PRP lyse invading cells
----reinforce cell wall to resist infections

73. Growth inhibition of Pythium ultimum by the Trichoderma virens– produced antibiotic gliovirin: A, parent strain, and B, gliovirin-deficient mutant.

74. Growth inhibition of Rhizoctonia solani by the Trichoderma virens–produced antibiotic gliotoxin: A, gliotoxin-amended medium, and B, nonamended medium.

75. Mycoparasitism of Rhizoctonia solani by Trichoderma virens: A, parent strain coiling around host hyphae, and B, mycoparasitic-deficient mutant with no coiling or penetration of host hyphae.

76. • Control of root and foliar pathogens
Trichoderma
• Control of root and foliar pathogens
Induced resistance
Biological control of diseases by direct attack of plant pathogenic fungi
• Changes in the microfloral composition on roots
• Enhanced nutrient uptake
• Enhanced solubilization of soil nutrients
• Enhanced root development
• Increased root hair formation

77. Trichoderma
Mycoparasitism
Antibiosis
Competition for nutrients or space
Tolerance to stress through enhanced root and plant development
Solubilization of inorganic nutrients
Induced resistance
Inactivation of the pathogen’s enzymes

78. Trichoderma