YEASTS AS BIOCONTROLAGENTPresented by:ROUF AHMAD*Student of M.Sc MicrobiologyMajor Advisor: Dr.(Mrs.) UrmilaGupta Phutela
INTRODUCTION• The terms “biological control” and its abbreviated synonym“biocontrol” have been used in different fields of...
•In both fields, the organism that suppresses the pest or pathogen isreferred to as the biological control agent (BCA).•De...
Bio-control Agents• Parasitic Insects• Predatory Insects3
Bacteria (Bacillus ) fungus (Trichoderma)yeast (Tilletiopsis pallescens)4
Why yeasts?• Do not produce allergenic spores or mycotoxins, as many mycelialfungi do• Can grow at low oxygen levels and w...
Development of yeast biological controlagentsA program of a biocontrol agent developmentinvolves two main phases:• Discove...
Flow diagram of development of a biocontrol agent7
Isolation, selection and identification of a yeastbiological control agentCharacteristics of an ideal antagonist1. Genetic...
Prerequisite for the effectiveness of biocontrol agents:• they have to colonize,• Survive• multiply in the environment tha...
• Another important factor is the number of cells needed to effectivedisease control. To develop the antagonists at a comm...
Mechanisms of action 11
• Demonstrated by several studies for antagonists such as Aureobasidiumpullulans (Bencheqroun et al. 2007), Cryptococcus h...
• Iron competition was reported as the main mode of action of M.pulcherrima (Saravanakumar et al. 2008) to inhibit Botryti...
Parasitism and lytic enzymes production• Attachment of microorganisms to thepathogen hyphae is an important factor.• Facil...
(A) healthy hyphae; (B) heavy yeast colonization around the hyphal tips of the pathogen;(C,D) accumulation of extracellula...
(E) welling and beads in hyphae of the pathogen colnized by yeast cells; (F&G) pitting in the hyphal cell wall resulting i...
Production of antibiotic/antimicrobial compoundsby yeasts• The antibiotics produced by Pseudozyma flocculosa are a mixture...
Induction of host plant defences by yeast biocontrol agents19
EXAMPLES:-• P. guillermondii has been shown to stimulate the production ofethylene in grapefruit (Wisniewski et al 1991)• ...
Scanning electron microscope images of Penicillium digitatum spores and germ tubes water-treated (controls) orCandida oleo...
(E and F) Enlargement of Penicillium digitatum germ tubes after 48 h (G and H) Enlargement of Penicilliumdigitatum spores ...
Diagram of possible interactions between host, pathogen and antagonist23
Production of yeast biological control agent• One factor limiting commercial interest in biocontrol is the high cost ofpro...
Formulation of a biological control agent• Formulations of microbial biomass can be of 2 types:1. Dry2. Liquid.• Dry formu...
• Due to the loss of viability during the drying process and storage ofseveral microorganisms and the relatively high cost...
Improvement of yeast biological controlFollowing are some approaches:• Antagonistic mixtures• Manipulation of nutritional ...
Antagonistic mixture• Antagonistic mixtures improve their spectrum of activity andreduce the cost of treatments (by reduci...
Scanning electron micrographs of Botrytis cinerea conidia induced to germinate on strawberry leaves andinteracting with Pi...
Manipulation of nutritional environment• The nutrients should be chosen preferably by being metabolized by theantagonist a...
Pre-harvest application• It enhances the biocontrol system, by allowing the antagonist to havelonger interaction with the ...
Genetic manipulation of antagonists• Genes responsible for biocontrol activity, or for increasing the ecologicalcompetence...
Physiological improvements• Most common aim of this is to improve the ecological fitness ofthe microorganism.• Example:Eco...
Integration with other methods• Enhancement of biocontrol activity was achieved in pome fruitscombining ammonium molybdate...
Using yeasts to control fungal pathogens on cucumber. Blastospores of the yeast Tilletiopsis pallescens from a 72-h-old li...
The various stages of the development of a pathogen and resulting plant disease can be reduced by the application ofyeast ...
Applications of yeasts as biocontrol agents1. Reduction of soil borne fungal plant diseases using yeasts:• Candida valida,...
2.Reduction of diseases of aerial plant tissues in fieldand green house environments using yeastsYeast Disease Assessment ...
Effect of S. cerevisiae treatment on foliar disease incidence on sugar beetplants under field conditions (right) compared ...
Efficacy of biological treatments on powdery mildew and cercospora leaf spotTreatments Infection%Diseaseseverity%Efficienc...
3.Reduction of postharvest decays by field application of yeastantagonistsYeast Disease Assessment utilized ReferenceAureo...
Fruit rot of citrus showing the effectiveness of biocontrol with Pichiaguillermondii (U.S.-7).42
4.Marine yeasts as biocontrol agents in marine animals• Probiotics• Defined by Verschuere et al. (2000) as “a live microbi...
Cell wall (immuno-stimulants)• The β-1,3-glucans, mannoprotein, deacylated chitin or chitin in yeast cell wall,even the wh...
Siderophore• It has been confirmed that yeasts produce only hydroxamate-type compound(Riquelme 1996).• Over 300 yeast stra...
Chemical structure of fusigen and anguibactin46
Killer toxin• Studies have shown that some marine yeasts are also pathogenic to somemarine animals.• Some Candida spp., Me...
• It is generally regarded that the mechanisms of killer toxin system arebinding of killer toxin to cell wall, the formati...
• It was found Williopsis saturnus WC91-2, Pichia guilliermondii GZ1,Pichia anomala YF07b, D. Hansenii and Aureobasidium p...
Conserved domains of killer toxins (a) and alignment of amino acids of 11.0 kDakiller toxin and pfam09207 (b). The 11.0 kD...
CLUSTAL W program alignment of the deduced sequences of exo-β-1,3-glucanases of different yeasts .51
Genetic basis for killer phenotype expression in yeastYeast Genetic basis Toxin gene Ref.S. cerevisiae dsRNA virus M1-, M2...
Receptor-mediated mode of action of the yeast K1 and K28 viraltoxins.53
FUTURE ASPECTS• To carry out more research studies on the pathogen-biocontrol agentinteractions and host-biocontrol agent ...
Conclusion• The increasing interest in alternatives to fungicides hasproduced much research in biocontrol agents, but with...
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  1. 1. YEASTS AS BIOCONTROLAGENTPresented by:ROUF AHMAD*Student of M.Sc MicrobiologyMajor Advisor: Dr.(Mrs.) UrmilaGupta Phutela
  2. 2. INTRODUCTION• The terms “biological control” and its abbreviated synonym“biocontrol” have been used in different fields of biology, mostnotably entomology and plant pathology.• In entomology, it has been used to describe the use of live predatoryinsects, entomopathogenic nematodes, or microbial pathogens tosuppress populations of different pest insects.• In plant pathology, the term applies to the use of microbialantagonists to suppress diseases as well as the use of host-specificpathogens to control weed populations.1
  3. 3. •In both fields, the organism that suppresses the pest or pathogen isreferred to as the biological control agent (BCA).•Defined as the suppression of damaging activities of one organismby one or more other organisms, often referred to as natural enemies.• It refers to the purposeful utilization of introduced or resident livingorganisms, other than disease resistant host plants, to suppress theactivities and populations of one or more plant pathogens.2
  4. 4. Bio-control Agents• Parasitic Insects• Predatory Insects3
  5. 5. Bacteria (Bacillus ) fungus (Trichoderma)yeast (Tilletiopsis pallescens)4
  6. 6. Why yeasts?• Do not produce allergenic spores or mycotoxins, as many mycelialfungi do• Can grow at low oxygen levels and water activity(aw)• Can produce extracellular polysaccharides that enhance theirsurvival and restrict pathogen colonization sites• Can use nutrients rapidly and proliferate at a faster rate(Raspor et al 2010)5
  7. 7. Development of yeast biological controlagentsA program of a biocontrol agent developmentinvolves two main phases:• Discovery• Commercial development6
  8. 8. Flow diagram of development of a biocontrol agent7
  9. 9. Isolation, selection and identification of a yeastbiological control agentCharacteristics of an ideal antagonist1. Genetically stable2. Effective at low concentrations against a wide range of pathogens3. Ability to survive under adverse environmental conditions4. Simple and inexpensive nutritional requirements inexpensive toproduce and formulate with long shelf-life5. Easy to dispense6. Compatible with commercial processing practices7. Resistant to most common pesticides8. Non pathogenic for the human health and host commodityWisniewski and Wilson (1992)8
  10. 10. Prerequisite for the effectiveness of biocontrol agents:• they have to colonize,• Survive• multiply in the environment that normally is occupied by thepathogen . (Manso and Nunes 2011)• Once a potential antagonist is selected the next step is the secondaryscreening.• Nunes et al (2001a) tested in „Blanquilla‟ pears the activity of 247bacteria and yeasts against Penicillium expansum, and only 2%inhibited decay by 50% or more.9
  11. 11. • Another important factor is the number of cells needed to effectivedisease control. To develop the antagonists at a commercial scale,they must be effective at reasonable concentrations for commercialdevelopment.(Janisiewicz 1997)• Reported concentrations able to control postharvest diseasesvaried in yeasts from 2 107 cfu ml−1 of Candida sake (Viñas et al.1998) to 2 109 cfu ml−1 of Pichia guillermondii.(Droby et al 1997)10
  12. 12. Mechanisms of action 11
  13. 13. • Demonstrated by several studies for antagonists such as Aureobasidiumpullulans (Bencheqroun et al. 2007), Cryptococcus humicola (Filonow et al.1996), Debaryomyces hansenii (Chalutz et al. 1988), Metschnikowiapulcherrima (Saravanakumar et al. 2008), or Rhodotorula glutinis (Castoria etal. 1997)To compete successfully with the pathogen the biocontrol agent should :• grow rapidly• use low concentrations of nutrients• Be better adapted to the environment(Nunes et al 2001b)Competition for nutrients and/or space13
  14. 14. • Iron competition was reported as the main mode of action of M.pulcherrima (Saravanakumar et al. 2008) to inhibit Botrytis cinerea,Alternaria alternata and P. expansum development in apples stored at 1 Cfor 8 months under controlled atmosphere (2% O2 and 3% CO2).(Zhang et al 2007)14
  15. 15. Parasitism and lytic enzymes production• Attachment of microorganisms to thepathogen hyphae is an important factor.• Facilitates a more efficient depletion ofnutrients from the area subjacent to themycelium, or serves as a mechanicalbarrier to nutrient uptake by the fungi(Droby et al 1992)• P. guillermondii also shows a high activityof the enzyme β-1,3-glucanase that couldresult in the degradation of the fungal cellwalls. Chitinases also degrade fungal cellwalls.(Jijakli and Lepoivre 1998)15
  16. 16. (A) healthy hyphae; (B) heavy yeast colonization around the hyphal tips of the pathogen;(C,D) accumulation of extracellular matrix around the colonized hyphaeAntagonistic yeast cells Pichia anomala interacting with hyphae of B. theobromae16
  17. 17. (E) welling and beads in hyphae of the pathogen colnized by yeast cells; (F&G) pitting in the hyphal cell wall resulting in aconcave appearance of the hyphal surface under the attached yeast cell as well as disruption in the hyphae; (H) fungalhyphae of B. theobromae were totally killed and penetrated by cells of the antagonistic yeast.17
  18. 18. Production of antibiotic/antimicrobial compoundsby yeasts• The antibiotics produced by Pseudozyma flocculosa are a mixture of fatty acid-containing derivatives thataffect membrane permeability of the target organisms, thereby inhibiting their growth.Antimicrobial compounds (A. pullulans, M. Pulcherrima)Volatile alkaline compounds volatile organic compoundsAmmonia, HCN2-methyl-1-butanol isobutyric acid ethyl propionate phenylethylalcoholvolatiles(Mercierand Jiménez 2004)• Volatile compounds were shown to mediate the inter-colony signal but could have a possible antagonisticeffect and potential use in the biocontrol of pathogens18
  19. 19. Induction of host plant defences by yeast biocontrol agents19
  20. 20. EXAMPLES:-• P. guillermondii has been shown to stimulate the production ofethylene in grapefruit (Wisniewski et al 1991)• Candida famata (F35) stimulates the production of the phytoalexins,scoparone and scopoletin in the wound site of oranges. (Arras 1996)• Candida oleophila was found to induce resistance to P. digitatumwhen applied in the surface of both wounded and unwoundedgrapefruit. (Droby et al 2002)• Biocontrol agents C. oleophila and M. fructicola have the ability toinduce defence-related oxidative responses in apple and citrus fruit,either on intact fruit surfaces, or around wounds.( Macarisin et al 2010)22
  21. 21. Scanning electron microscope images of Penicillium digitatum spores and germ tubes water-treated (controls) orCandida oleophila-treated wound sites. A and B, Wound surfaces after 24 h C and D, Wound surfaces after 48 h20
  22. 22. (E and F) Enlargement of Penicillium digitatum germ tubes after 48 h (G and H) Enlargement of Penicilliumdigitatum spores after 48 h. Sp = spore; H = hyphae; AH = abnormal hyphae; Gt = germ tube; and SSp =swollen spore. 21
  23. 23. Diagram of possible interactions between host, pathogen and antagonist23
  24. 24. Production of yeast biological control agent• One factor limiting commercial interest in biocontrol is the high cost ofproduction. (Fravel et al 1999)• Both solid and liquid fermentation systems have been used for the massproduction of biocontrol agents (Lewis and Papavizas 1991), though ingeneral yeasts are produced by liquid fermentation.• For the yeast C. sake a medium based on cane molasses, a by-productfrom sugar industry, has been successfully used as a growing substrate(Abadias et al 2003)24
  25. 25. Formulation of a biological control agent• Formulations of microbial biomass can be of 2 types:1. Dry2. Liquid.• Dry formulation products include: wettable powders, dusts, andgranules. Dry formulation processes normally comprises freeze-drying, fluidized bed drying or spray drying.• Liquid formulation products consist of biomass suspensions inwater, oils, or combinations of both (emulsions).25
  26. 26. • Due to the loss of viability during the drying process and storage ofseveral microorganisms and the relatively high cost of the dryingtechnology, liquid formulation could be an alternative process.• Abadias et al. (2003) demonstrated that isotonic liquid formulation of C.sake could overcame the viability problems observed in the solidformulation process.(with 77% cell viability after 7 months at 4 Csorbitol-modified medium).• A similar approach was carried out in the liquid formulation ofRhodotorula minuta, using glycerol to reduce water activity and xanthangum as a viscosity-enhancing agent, however, loss of viability wasobserved after 6 months of storage at 4 C.(Patiño-Vera et al 2005)26
  27. 27. Improvement of yeast biological controlFollowing are some approaches:• Antagonistic mixtures• Manipulation of nutritional environment• Preharvest application• Genetic manipulation of antagonists• Physiological improvements• Integration with other methods 27
  28. 28. Antagonistic mixture• Antagonistic mixtures improve their spectrum of activity andreduce the cost of treatments (by reducing the biomass of antagonistrequired to achieve control).• The antagonistic action is actually, the action of a community ofmicroorganisms that suppresses the disease through differentmechanisms of action (Janisiewicz and Korsten 2002)• Mixtures of biocontrol agents are effective when:1. They have different modes of action2. Different ecological attributes28
  29. 29. Scanning electron micrographs of Botrytis cinerea conidia induced to germinate on strawberry leaves andinteracting with Pichia guilermondii and Bacillus mycoides.29
  30. 30. Manipulation of nutritional environment• The nutrients should be chosen preferably by being metabolized by theantagonist and not by the pathogen.(Janisiewicz 1997)• The application of sugar analog 2-deoxy-D-glucose, at 0.2%, showed toimprove Candida saitona biocontrol activity of green mould in lemon andorange, resulting in a control level similar to that of the fungicideimazalil.(El-Ghaouth et al 2000)• Similar effects were reported with C. saitona and C. sake on pome fruit.(Nunes et al 2001)• Nutritional composition can also influence the production of metabolites,such as cell wall-degrading enzymes.(Wisniewski et al 1991)30
  31. 31. Pre-harvest application• It enhances the biocontrol system, by allowing the antagonist to havelonger interaction with the pathogen and to colonise tissues before thearrival of the pathogen.• Field application of a combined treatment of the yeast C. sake andbacterium P. syringae showed an enhancement of biocontrol activityagainst P. expansum on apples and pears in comparison with control byantagonists applied separately(Teixidó et al 2010)• Similar results were obtained in postharvest treatments using a mixture ofC. sake and P. agglomerans in apples and pears(Nunes et al 2002)31
  32. 32. Genetic manipulation of antagonists• Genes responsible for biocontrol activity, or for increasing the ecologicalcompetence, could be introduced in biocontrol agents.• For example:• Insertion of genes or over-expression of endogenous genes responsible forantifungal activity (such as cell wall degrading enzymes)• Insertion of genes for better utilization of available nutrients• Genetic improvement can be achieved by chemical and physicalmutation, sexual hybrids, homokaryons and genetic manipulationse.g., directed mutagenesis, protoplast fusion, genetic analysis offusants, recombination transformation.32
  33. 33. Physiological improvements• Most common aim of this is to improve the ecological fitness ofthe microorganism.• Example:Ecological fittness and environmental stress tolerance of thebiocontrol yeast Candida sake was improved by manipulation ofintracellular sugar alcohol and sugar content.33
  34. 34. Integration with other methods• Enhancement of biocontrol activity was achieved in pome fruitscombining ammonium molybdate with C. sake (Nunes et al. 2002c), andin papaya using sodium bicarbonate and C. oleophila.(Gamagae et al 2004)• Other compounds such as sugar analogs, calcium salts and organic acidshave been combined with biological methods to manage postharvestdecay. (Ippolito et al 2005)• A result of this integrated approach of biocontrol systems is thedevelopment of a “second generation” of commercial products such“Biocoat” whose main components are Candida saitoana and chitosan or“Biocure” also with C. saitoana and lysozyme. Both products alsocontain other additives, such as sodium bicarbonate.(Wisniewski et al 2007)34
  35. 35. Using yeasts to control fungal pathogens on cucumber. Blastospores of the yeast Tilletiopsis pallescens from a 72-h-old liquid brothculture. Cucumber leaf heavily infected with powdery mildew [Podosphaera (Sect. Sphaerotheca) xanthi ] growth and sporulation35
  36. 36. The various stages of the development of a pathogen and resulting plant disease can be reduced by the application ofyeast biological control agents. Different agents can target single or multiple stages of pathogen development asindicated.36
  37. 37. Applications of yeasts as biocontrol agents1. Reduction of soil borne fungal plant diseases using yeasts:• Candida valida, Rhodotorula glutinis and Trichosporon asahii isolatedfrom the rhizosphere of sugar beet, individually were the most successfulthan other treatments in promoting plant growth and reducing root diseaseincited by Rhizoctonia solanii.( El-Tarabily and Sivasthamparam 2006)• El-mahalway (2004) reported that rhizosphere yeasts Saccharomycesunispora and Candida steatolytica reduced wilt of beans caused byFusarium oxysporum via the production of antifungal metabolites.37
  38. 38. 2.Reduction of diseases of aerial plant tissues in fieldand green house environments using yeastsYeast Disease Assessment utilized ReferenceAureobasidium pullulans Brown rot blossomBlight of cherriesFire blightForced blossom in mistchamberField test on applesduring floweringWittig et al.1997Kunz 2007Pseudozyma flocculosa Powdery mildew Cucumber, rose, wheat:greenhouseAvis and belanger 2002Cryptococcus andCandida spp.Late blight Tomato plants : greenhouseJunior et al. 2006Cryptococcus flavescens Fusarium head blight Wheat field Khan et al 2004Pichia membranifaciens Gray mold Grape vine plantlets Masih and Paul 2002Candida guillermondi Gray mold Tomato seedlings:growthchamberBuck and Jeffers 200438
  39. 39. Effect of S. cerevisiae treatment on foliar disease incidence on sugar beetplants under field conditions (right) compared with untreated ones (left)39
  40. 40. Efficacy of biological treatments on powdery mildew and cercospora leaf spotTreatments Infection%Diseaseseverity%Efficiency%Infection%Diseaseseverity%Efficiency%Control 27.9 67.10 o.o 85.7 39.2 0.0Topsin oo.o 00.0 100 14.3 3.33 91.61S.cerevisiae 6.33 16.00 76.15 78.6 29.o 26.06P. albicans 4.80 21.67 67.70 72.2 30.68 21.77C. sake 4.50 17.00 74.13 76.4 18.5 52.83Rhizo-N 20.0 14.0 79.13 78.9 17.70 54.87POWDERY MILDEW CERCOSPORA LEAF SPOT40
  41. 41. 3.Reduction of postharvest decays by field application of yeastantagonistsYeast Disease Assessment utilized ReferenceAureobasidiumpullulansVarious post harvestrotsCherry fruit Ippolito et al 2005Candida sake Blue mold Apple fruit Teixido et al 1999Rhodotorula minuta Anthracnose Mango fruit Patina-vera et al 2005Cryptococcus laurentii Side rot Pear fruit Sugar et al 2005Cryptococcus albidus Gray mold Straw berry fruit Helbig 200241
  42. 42. Fruit rot of citrus showing the effectiveness of biocontrol with Pichiaguillermondii (U.S.-7).42
  43. 43. 4.Marine yeasts as biocontrol agents in marine animals• Probiotics• Defined by Verschuere et al. (2000) as “a live microbial adjunct which has a beneficialeffect on the host by modifying the host-associated or ambient microbial community byensuring improved use of the feed or enhancing its nutritional value, by enhancing the hostimmunity response towards diseases, or by improving the quality of its environment”.• Using probiotics is a new method for inhibition of the pathogenic bacteria in rearinganimals in maricultural industries.• The ability of yeast genera Yarrowia, Metschnikowia, Candida, Debaryomyces,Kluyveromyces, Pichia, Saccharomyces, Hanseniaspora, Kloeckera, Exophiala,Leucosporidium, Cryptococcus, Sporobolomyces, Rhodotorula, and Trichosporon tocolonize the intestine of fish microbiota has been confirmed (Gatesoupe 2007).43
  44. 44. Cell wall (immuno-stimulants)• The β-1,3-glucans, mannoprotein, deacylated chitin or chitin in yeast cell wall,even the whole cell of S. Cerevisiae, Candida sake have been successfully usedas immuno-stimulants in fish and shellfish against bacterial and viral infection.(Gatesoupe 2007)• The efficacy of a marine yeast Candida sake as source of immuno-stimulant toIndian white shrimp Fenneropenaeus indicus was estimated. The results showthat marine yeast C. sake at 10% in diet (w/w) may be used as an effective sourceof immuno-stimulant in F. Indicus.(Sajeevan et al 2006)44
  45. 45. Siderophore• It has been confirmed that yeasts produce only hydroxamate-type compound(Riquelme 1996).• Over 300 yeast strains isolated from different marine environments werescreened for their ability to produce siderophore. Among them, only the yeaststrain HN6.2 which was identified to be A. pullulans, was found to produce thehighest level of the siderophore.• Under the optimal conditions, this produces 1.1 mg/ml of the siderophore. This isable to inhibit cell growth of Vibrio anguillarum and Vibrio parahaemolyticus,isolated from the diseased marine animals and it was found to be Fusigen.(Wang et al.2009b)45
  46. 46. Chemical structure of fusigen and anguibactin46
  47. 47. Killer toxin• Studies have shown that some marine yeasts are also pathogenic to somemarine animals.• Some Candida spp., Metschnikowia bicuspidata, Cryptococcus spp.,Sporobolomyces salmonicolor, and Trichosporon sp. are the pathogens ofamago (Oncorhynchus rhodurus), chinook salmon (Oncorhynchustshawytscha), the githead seabream (Sparus aurata), crab (Portunustrituberculatus), and teach (Tinca tinca), respectively.(Gatesoupe 2007)• Killer toxins produced by some yeast strains are low molecular massproteins or glycoprotein toxins which kill sensitive cells of the same orrelated yeast genera without direct cell– cell contact.(Magliani et al 1997)47
  48. 48. • It is generally regarded that the mechanisms of killer toxin system arebinding of killer toxin to cell wall, the formation of trans-membranechannels, ion leakage, arrest of cell division, interference with thesynthesis of glucan in the cell wall and cell death, induction of DNAdamage and apoptosis and a strong β- 1,3-glucanase activity.(Magliani et al. 2008)• Multiple yeast strains from seawater, sediments, mud of salterns, guts ofmarine fish, and marine algae for killer activity against the yeastM.bicuspidata (pathogenic to crab P. trituberculatus;) were screened48
  49. 49. • It was found Williopsis saturnus WC91-2, Pichia guilliermondii GZ1,Pichia anomala YF07b, D. Hansenii and Aureobasidium pullulansHN2.3 could secrete toxin into the medium and kill the pathogenic yeast.• Finally, it was observed that the marine-derived W. saturnus WC91-2 hasmuch higher killing activity and wider killing activity spectra than themarine-derived P. anomala YF07b.(Wang et al. 2008a)49
  50. 50. Conserved domains of killer toxins (a) and alignment of amino acids of 11.0 kDakiller toxin and pfam09207 (b). The 11.0 kDa killer toxin is produced by the marine-derived W. saturnus WC91-2; Pfam09207 is the known yeast killer toxin superfamily50
  51. 51. CLUSTAL W program alignment of the deduced sequences of exo-β-1,3-glucanases of different yeasts .51
  52. 52. Genetic basis for killer phenotype expression in yeastYeast Genetic basis Toxin gene Ref.S. cerevisiae dsRNA virus M1-, M2-, M28-dsRNADignard et al 1991H. uvarum dsRNA virus M-dsRNA Schmitt et al 1997Z. bailii dsRNA virus M-dsRNA Schmitt et al 1994U. maydis dsRNA virus M-dsRNA Park et al 1994K. lactis linear dsDNAplasmidpGKl1 Gunge et al 1981P. acaciae linear dsDNAplasmidpPac1 Hayman et al 1991Pichia inositovora linear dsDNAplasmidpPin1 Worsham et al 1990Pichia kluyveri chromosomal not identified Young et al 1978Pichia farinosa chromosomal SMK1 Suzuki et al 199452
  53. 53. Receptor-mediated mode of action of the yeast K1 and K28 viraltoxins.53
  54. 54. FUTURE ASPECTS• To carry out more research studies on the pathogen-biocontrol agentinteractions and host-biocontrol agent interactions.• Use of biotechnology and nanotechnology in improvement of biocontrolmechanisms and strategies.• Importantly, there is still a wealth of opportunity for the discovery of newantagonists because only a small fraction of the earth‟s microflora hasbeen identified and characterized.• Fundamental knowledge on thephysiology, genetic traits and molecularbasis of colonization, survival and differentiation of biocontrol agents onplant tissue is needed.54
  55. 55. Conclusion• The increasing interest in alternatives to fungicides hasproduced much research in biocontrol agents, but with only afew products in the market.• There is still a need for a deep research in many aspects of postharvest biocontrol, to make biological control more effective,offering more commercial products and spread, evengeneralize, to the use of postharvest biofungicides.55

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