YEASTS AS BIOCONTROL AGENT

Presented by:ROUF AHMAD

*Student of M.Sc MicrobiologyMajor Advisor: Dr.(Mrs.) Urmila

Gupta Phutela

INTRODUCTION

• The terms “biological control” and its abbreviated synonym “biocontrol” have been used in different fields of biology, most notably entomology and plant pathology.

• In entomology, it has been used to describe the use of live predatory insects, entomopathogenic nematodes, or microbial pathogens to suppress populations of different pest insects.

• In plant pathology, the term applies to the use of microbial antagonists to suppress diseases as well as the use of host-specific pathogens to control weed populations.

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•In both fields, the organism that suppresses the pest or pathogen is referred to as the biological control agent (BCA).

•Defined as the suppression of damaging activities of one organism by one or more other organisms, often referred to as natural enemies.

• It refers to the purposeful utilization of introduced or resident living organisms, other than disease resistant host plants, to suppress the activities and populations of one or more plant pathogens.

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Bio-control Agents• Parasitic Insects

• Predatory Insects

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Bacteria (Bacillus ) fungus (Trichoderma)

yeast (Tilletiopsis pallescens)

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Why yeasts?

• Do not produce allergenic spores or mycotoxins, as many mycelial fungi do

• Can grow at low oxygen levels and water activity(aw)

• Can produce extracellular polysaccharides that enhance their survival and restrict pathogen colonization sites

• Can use nutrients rapidly and proliferate at a faster rate

(Raspor et al 2010)

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Development of yeast biological control agents

A program of a biocontrol agent development involves two main phases:

• Discovery

• Commercial development

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Flow diagram of development of a biocontrol agent

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Isolation, selection and identification of a yeast biological control agent

Characteristics of an ideal antagonist

1. 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 to produce 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 commodity

Wisniewski and Wilson (1992)

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Prerequisite for the effectiveness of biocontrol agents:

• they have to colonize, • Survive• multiply in the environment that normally is occupied by the

pathogen . (Manso and Nunes 2011)

• Once a potential antagonist is selected the next step is the secondary screening.

• Nunes et al (2001a) tested in ‘Blanquilla’ pears the activity of 247 bacteria and yeasts against Penicillium expansum, and only 2% inhibited decay by 50% or more.

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• Another important factor is the number of cells needed to effective disease control. To develop the antagonists at a commercial scale, they must be effective at reasonable concentrations for commercial development.

(Janisiewicz 1997)

• Reported concentrations able to control postharvest diseases varied 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)

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Mechanisms of action 11

• Demonstrated by several studies for antagonists such as Aureobasidium pullulans (Bencheqroun et al. 2007), Cryptococcus humicola (Filonow et al. 1996), Debaryomyces hansenii (Chalutz et al. 1988), Metschnikowia pulcherrima (Saravanakumar et al. 2008), or Rhodotorula glutinis (Castoria et al. 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 space

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• 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°C for 8 months under controlled atmosphere (2% O2 and 3% CO2).

(Zhang et al 2007)

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Parasitism and lytic enzymes production

• Attachment of microorganisms to the pathogen hyphae is an important factor.

• Facilitates a more efficient depletion of nutrients from the area subjacent to the mycelium, or serves as a mechanical barrier to nutrient uptake by the fungi (Droby et al 1992)

• P. guillermondii also shows a high activity of

the enzyme β-1,3-glucanase that could result in the degradation of the fungal cell walls. Chitinases also degrade fungal cell walls. (Jijakli and Lepoivre 1998)

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(A) healthy hyphae; (B) heavy yeast colonization around the hyphal tips of the pathogen;

(C,D) accumulation of extracellular matrix around the colonized hyphae

Antagonistic yeast cells Pichia anomala interacting with hyphae of B. theobromae16

(E) welling and beads in hyphae of the pathogen colnized by yeast cells; (F&G) pitting in the hyphal cell wall resulting in a concave appearance of the hyphal surface under the attached yeast cell as well as disruption in the hyphae; (H) fungal hyphae of B. theobromae were totally killed and penetrated by cells of the antagonistic yeast.

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Production of antibiotic/antimicrobial compounds by yeasts

• The antibiotics produced by Pseudozyma flocculosa are a mixture of fatty acid-containing derivatives that affect membrane permeability of the target organisms, thereby inhibiting their growth.

Antimicrobial compounds (A. pullulans, M. Pulcherrima)

Volatile alkaline compounds volatile organic compounds

Ammonia, HCN

2-methyl-1-butanol isobutyric acid ethyl propionate phenylethyl alcohol volatiles

(Mercierand Jiménez 2004)

• Volatile compounds were shown to mediate the inter-colony signal but could have a possible antagonistic effect and potential use in the biocontrol of pathogens

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Induction of host plant defences by yeast biocontrol agents

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EXAMPLES:-

• P. guillermondii has been shown to stimulate the production of ethylene 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. digitatum

when applied in the surface of both wounded and unwounded grapefruit. (Droby et al 2002)

• Biocontrol agents C. oleophila and M. fructicola have the ability to induce defence-related oxidative responses in apple and citrus fruit, either on intact fruit surfaces, or around wounds.

( Macarisin et al 2010)

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Scanning electron microscope images of Penicillium digitatum spores and germ tubes water-treated (controls) or Candida oleophila-treated wound sites. A and B, Wound surfaces after 24 h C and D, Wound surfaces after 48 h

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(E and F) Enlargement of Penicillium digitatum germ tubes after 48 h (G and H) Enlargement of Penicillium digitatum spores after 48 h. Sp = spore; H = hyphae; AH = abnormal hyphae; Gt = germ tube; and SSp =

swollen spore. 21

Diagram of possible interactions between host, pathogen and antagonist

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Production of yeast biological control agent

• One factor limiting commercial interest in biocontrol is the high cost of production. (Fravel et al 1999)

• Both solid and liquid fermentation systems have been used for the mass production of biocontrol agents (Lewis and Papavizas 1991), though in general yeasts are produced by liquid fermentation.

• For the yeast C. sake a medium based on cane molasses, a by-product from sugar industry, has been successfully used as a growing substrate (Abadias et al 2003)

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Formulation of a biological control agent

• Formulations of microbial biomass can be of 2 types:

1. Dry

2. Liquid.

• Dry formulation products include: wettable powders, dusts, and granules. Dry formulation processes normally comprises freeze-drying, fluidized bed drying or spray drying.

• Liquid formulation products consist of biomass suspensions in water, oils, or combinations of both (emulsions).

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• Due to the loss of viability during the drying process and storage of several microorganisms and the relatively high cost of the drying technology, 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 solid formulation process.(with 77% cell viability after 7 months at 4°C sorbitol-modified medium).

• A similar approach was carried out in the liquid formulation of Rhodotorula minuta, using glycerol to reduce water activity and xanthan gum as a viscosity-enhancing agent, however, loss of viability was observed after 6 months of storage at 4°C.

(Patiño-Vera et al 2005)

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Improvement of yeast biological control

Following are some approaches:

• Antagonistic mixtures

• Manipulation of nutritional environment

• Preharvest application

• Genetic manipulation of antagonists

• Physiological improvements

• Integration with other methods27

Antagonistic mixture

• Antagonistic mixtures improve their spectrum of activity and reduce the cost of treatments (by reducing the biomass of antagonist required to achieve control).

• The antagonistic action is actually, the action of a community of microorganisms that suppresses the disease through different mechanisms of action (Janisiewicz and Korsten 2002)

• Mixtures of biocontrol agents are effective when:

1. They have different modes of action

2. Different ecological attributes

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Scanning electron micrographs of Botrytis cinerea conidia induced to germinate on strawberry leaves and interacting with Pichia guilermondii and Bacillus mycoides.

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Manipulation of nutritional environment

• The nutrients should be chosen preferably by being metabolized by the antagonist and not by the pathogen.

(Janisiewicz 1997)

• The application of sugar analog 2-deoxy-D-glucose, at 0.2%, showed to improve Candida saitona biocontrol activity of green mould in lemon and orange, resulting in a control level similar to that of the fungicide imazalil.

(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)

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Pre-harvest application

• It enhances the biocontrol system, by allowing the antagonist to have longer interaction with the pathogen and to colonise tissues before the arrival of the pathogen.

• Field application of a combined treatment of the yeast C. sake and bacterium P. syringae showed an enhancement of biocontrol activity against P. expansum on apples and pears in comparison with control by antagonists applied separately

(Teixidó et al 2010)

• Similar results were obtained in postharvest treatments using a mixture of C. sake and P. agglomerans in apples and pears

(Nunes et al 2002)

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Genetic manipulation of antagonists

• Genes responsible for biocontrol activity, or for increasing the ecological competence, could be introduced in biocontrol agents.

• For example:

• Insertion of genes or over-expression of endogenous genes responsible for antifungal activity (such as cell wall degrading enzymes)

• Insertion of genes for better utilization of available nutrients

• Genetic improvement can be achieved by chemical and physical mutation, sexual hybrids, homokaryons and genetic manipulations e.g., directed mutagenesis, protoplast fusion, genetic analysis of fusants, recombination transformation.

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Physiological improvements

• Most common aim of this is to improve the ecological fitness of the microorganism.

• Example:

Ecological fittness and environmental stress tolerance of the biocontrol yeast Candida sake was improved by manipulation of intracellular sugar alcohol and sugar content.

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Integration with other methods

• Enhancement of biocontrol activity was achieved in pome fruits combining ammonium molybdate with C. sake (Nunes et al. 2002c), and in papaya using sodium bicarbonate and C. oleophila.

(Gamagae et al 2004)

• Other compounds such as sugar analogs, calcium salts and organic acids have been combined with biological methods to manage postharvest decay. (Ippolito et al 2005)

• A result of this integrated approach of biocontrol systems is the development 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 also contain other additives, such as sodium bicarbonate.

(Wisniewski et al 2007)

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Using yeasts to control fungal pathogens on cucumber. Blastospores of the yeast Tilletiopsis pallescens from a 72-h-old liquid broth culture. Cucumber leaf heavily infected with powdery mildew [Podosphaera (Sect. Sphaerotheca) xanthi ] growth and sporulation

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The various stages of the development of a pathogen and resulting plant disease can be reduced by the application of yeast biological control agents. Different agents can target single or multiple stages of pathogen development as indicated.

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Applications of yeasts as biocontrol agents

1. Reduction of soil borne fungal plant diseases using yeasts:

• Candida valida, Rhodotorula glutinis and Trichosporon asahii isolated from the rhizosphere of sugar beet, individually were the most successful than other treatments in promoting plant growth and reducing root disease incited by Rhizoctonia solanii.

( El-Tarabily and Sivasthamparam 2006)

• El-mahalway (2004) reported that rhizosphere yeasts Saccharomyces unispora and Candida steatolytica reduced wilt of beans caused by Fusarium oxysporum via the production of antifungal metabolites.

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2.Reduction of diseases of aerial plant tissues in field and green house environments using yeasts

Yeast Disease Assessment utilized Reference

Aureobasidium pullulans Brown rot blossomBlight of cherriesFire blight

Forced blossom in mist chamber

Field test on apples during flowering

Wittig et al.1997

Kunz 2007

Pseudozyma flocculosa Powdery mildew Cucumber, rose, wheat: greenhouse

Avis and belanger 2002

Cryptococcus and Candida spp.

Late blight Tomato plants : green house

Junior et al. 2006

Cryptococcus flavescens Fusarium head blight Wheat field Khan et al 2004

Pichia membranifaciens Gray mold Grape vine plantlets Masih and Paul 2002

Candida guillermondi Gray mold Tomato seedlings:growth chamber

Buck and Jeffers 2004

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Effect of S. cerevisiae treatment on foliar disease incidence on sugar beet plants under field conditions (right) compared with untreated ones (left)

AA

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Efficacy of biological treatments on powdery mildew and cercospora leaf spot

Treatments Infection %

Disease severity%

Efficiency%

Infection%

Disease severity%

Efficiency%

Control 27.9 67.10 o.o 85.7 39.2 0.0

Topsin oo.o 00.0 100 14.3 3.33 91.61

S.cerevisiae 6.33 16.00 76.15 78.6 29.o 26.06

P. albicans 4.80 21.67 67.70 72.2 30.68 21.77

C. sake 4.50 17.00 74.13 76.4 18.5 52.83

Rhizo-N 20.0 14.0 79.13 78.9 17.70 54.87

POWDERY MILDEW CERCOSPORA LEAF SPOT

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3.Reduction of postharvest decays by field application of yeast antagonists

Yeast Disease Assessment utilized Reference

Aureobasidium pullulans

Various post harvest rots Cherry fruit Ippolito et al 2005

Candida sake Blue mold Apple fruit Teixido et al 1999

Rhodotorula minuta Anthracnose Mango fruit Patina-vera et al 2005

Cryptococcus laurentii Side rot Pear fruit Sugar et al 2005

Cryptococcus albidus Gray mold Straw berry fruit Helbig 2002

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Fruit rot of citrus showing the effectiveness of biocontrol with Pichia guillermondii (U.S.-7).

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4.Marine yeasts as biocontrol agents in marine animals

• Probiotics

• Defined by Verschuere et al. (2000) as “a live microbial adjunct which has a beneficial effect on the host by modifying the host-associated or ambient microbial community by ensuring improved use of the feed or enhancing its nutritional value, by enhancing the host immunity response towards diseases, or by improving the quality of its environment”.

• Using probiotics is a new method for inhibition of the pathogenic bacteria in rearing animals in maricultural industries.

• The ability of yeast genera Yarrowia, Metschnikowia, Candida, Debaryomyces, Kluyveromyces, Pichia, Saccharomyces, Hanseniaspora, Kloeckera, Exophiala, Leucosporidium, Cryptococcus, Sporobolomyces, Rhodotorula, and Trichosporon to colonize the intestine of fish microbiota has been confirmed (Gatesoupe 2007).

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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 used as 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 to Indian white shrimp Fenneropenaeus indicus was estimated. The results show that marine yeast C. sake at 10% in diet (w/w) may be used as an effective source of immuno-stimulant in F. Indicus.

(Sajeevan et al 2006)

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Siderophore

• It has been confirmed that yeasts produce only hydroxamate-type compound (Riquelme 1996).

• Over 300 yeast strains isolated from different marine environments were screened for their ability to produce siderophore. Among them, only the yeast strain HN6.2 which was identified to be A. pullulans, was found to produce the highest level of the siderophore.

• Under the optimal conditions, this produces 1.1 mg/ml of the siderophore. This is able 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)

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Chemical structure of fusigen and anguibactin46

Killer toxin

• Studies have shown that some marine yeasts are also pathogenic to some marine animals.

• Some Candida spp., Metschnikowia bicuspidata, Cryptococcus spp., Sporobolomyces salmonicolor, and Trichosporon sp. are the pathogens of amago (Oncorhynchus rhodurus), chinook salmon (Oncorhynchus tshawytscha), the githead seabream (Sparus aurata), crab (Portunus trituberculatus), and teach (Tinca tinca), respectively.

(Gatesoupe 2007)

• Killer toxins produced by some yeast strains are low molecular mass proteins or glycoprotein toxins which kill sensitive cells of the same or related yeast genera without direct cell– cell contact.

(Magliani et al 1997)

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• It is generally regarded that the mechanisms of killer toxin system are binding of killer toxin to cell wall, the formation of trans-membrane channels, ion leakage, arrest of cell division, interference with the synthesis of glucan in the cell wall and cell death, induction of DNA damage and apoptosis and a strong β- 1,3-glucanase activity. (Magliani et al. 2008)

• Multiple yeast strains from seawater, sediments, mud of salterns, guts of marine fish, and marine algae for killer activity against the yeast M.bicuspidata (pathogenic to crab P. trituberculatus;) were screened

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• It was found Williopsis saturnus WC91-2, Pichia guilliermondii GZ1, Pichia anomala YF07b, D. Hansenii and Aureobasidium pullulans HN2.3 could secrete toxin into the medium and kill the pathogenic yeast.

• Finally, it was observed that the marine-derived W. saturnus WC91-2 has much higher killing activity and wider killing activity spectra than the marine-derived P. anomala YF07b.

(Wang et al. 2008a)

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Conserved domains of killer toxins (a) and alignment of amino acids of 11.0 kDa killer 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 superfamily

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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 yeast

Yeast Genetic basis Toxin gene Ref.

S. cerevisiae dsRNA virus M1-, M2-, M28-dsRNA

Dignard et al 1991

H. uvarum dsRNA virus M-dsRNA Schmitt et al 1997

Z. bailii dsRNA virus M-dsRNA Schmitt et al 1994

U. maydis dsRNA virus M-dsRNA Park et al 1994

K. lactis linear dsDNA plasmid

pGKl1 Gunge et al 1981

P. acaciae linear dsDNA plasmid

pPac1 Hayman et al 1991

Pichia inositovora linear dsDNA plasmid

pPin1 Worsham et al 1990

Pichia kluyveri chromosomal not identified Young et al 1978

Pichia farinosa chromosomal SMK1 Suzuki et al 1994

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Receptor-mediated mode of action of the yeast K1 and K28 viral toxins.

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FUTURE ASPECTS

• To carry out more research studies on the pathogen-biocontrol agent interactions and host-biocontrol agent interactions.

• Use of biotechnology and nanotechnology in improvement of biocontrol mechanisms and strategies.

• Importantly, there is still a wealth of opportunity for the discovery of new antagonists because only a small fraction of the earth’s microflora has been identified and characterized.

• Fundamental knowledge on thephysiology, genetic traits and molecular basis of colonization, survival and differentiation of biocontrol agents on plant tissue is needed.

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Conclusion

• The increasing interest in alternatives to fungicides has produced much research in biocontrol agents, but with only a few products in the market.

• There is still a need for a deep research in many aspects of post harvest biocontrol, to make biological control more effective, offering more commercial products and spread, even generalize, to the use of postharvest biofungicides.

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