Biological control of plant pathogens

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 http://www.ars.usda.gov/is/pr/2002/021231.trichoderma.jpg

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., 1983. Phytopathology 73:655-659).

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. 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. 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. 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 togetherto 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 )