PH12017000229A1

PH12017000229A1 - Yeast as biocontrol agent for pathogens

Info

Publication number: PH12017000229A1
Application number: PH12017000229A
Authority: PH
Inventors: B Exconde Severina; Jean G Batin Cynthia
Original assignee: Univ Of The Philippines Los Banos
Priority date: 2017-08-14
Filing date: 2017-08-14
Publication date: 2019-02-18

Abstract

The application of yeast as biocontrol agent for postharvest diseases that reduces dependence to chemical pesticides. Specifically, a number of yeasts were selected, tested and proven to be effective against post-harvest pathogens of some fruits and vegetables such as mango, banana, calamansi, peanuts, onion, garlic and tomato. A number of yeast antagonist that exhibited biocontrol efficacy against the pathogens of the said commodities were identified and processed as biocontrol agent product.

Description

The formulation is a mixture of active ingredients such as yeast cells with the. inert material.

A final formulation is easy to handle, stable over a range temperature preferably, -5 to 35°C and have a minimum shelf-life of two years at —_— temperature.

Pichia kudriavzevii, Saccharomyces cerevisiae, Debaryomyces hansenii, =

Torulopsis candida and Kodamaea ohmerii are formulated as wettable powder using - talc as carrier. = : y

YEAST APPLICATION FOR BIOCONTROL OF POSTHARVEST ~~ “ PATHOGENS OF FRUITS AND VEGETABLES =

FIELD OF THE INVENTION

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The present invention relates to the biological control of plant pathogens (e.g. ~ postharvest pathogens) in agricultural commodities such as fruits and vegetables.

More particularly, this invention relates to: (1) method for biologically controlling *~ plant diseases, such as anthracnose on horticultural commodities using antagonistic = yeast isolated from the surface of fruits and vegetables; (2) compositions useful in the said methods, and; (3) products produced in such methods.

BACKGROUND OF THE INVENTION :

Postharvest decays of fruits and vegetables account for significant levels of postharvest losses. It is estimated that about 20-25% of the harvested fruits and vegetables are decayed by pathogens during postharvest handling even in developed countries. Postharvest diseases caused by microbial pathogens account for millions of dollars in losses of both durable and perishable products every year. These diseases consist of various fungal infections that rot fruits and vegetables after harvesting.

Farmers often rely heavily on the use of synthetic fungicides to control these plant diseases. However, the environmental problems caused by excessive use and misuse of synthetic fungicides have led to changes in people’s attitudes towards the use of chemicals in agriculture.

The use of synthetic fungicides has long been the most effective disease control method (Eckert and Ogawa, 1988; Snowdon, 1992). However, due to the increasing concern about the potential harmful effects of fungicides on the environment and human health and the development of fungicide resistance in pathogens, researchers are trying to find alternative means to control postharvest diseases (Ragsdale and Sisler, 1994; Conway et al, 2004). Fungicide residues in the fruit and their possible effects on human health and the environment, and the }

development of fungicide resistance by these pathogens have stimulated the. evaluation of alternative control methods that are effective and safe to use (Eckert, - 1990). -

The past two decades has seen a steady increase in the interest of finding - alternatives to the use of synthetic fungicides for postharvest disease control. In® particular this has led to considerable research on the use of microbial antagonists as protective agents. Recently, biological control has been developed as an alternative to synthetic fungicide treatments and considerable success has been achieved using” antagonistic microorganisms to control both preharvest and postharvest diseases (Janisiewicz and Korsten, 2002).

The use of naturally occurring yeasts has attracted special interest because of the special attributes yeasts have over filamentous fungi and bacteria. The advantage of using yeasts in biocontrol of foliar and postharvest diseases is caused by the fact that they are major component of the epiphytic microbial community on the surface of leaves, fruits and vegetables (Wilson and Wisniewski, 1989). They are effective as biocontrol agents because they are phenotypically adapted to these niches and are able to effectively colonize and compete for nutrients and space on fruit and leaf surfaces (McLaughlin et al, 1990; Filonow, 1998).

Yeasts generally do not produce allergenic spores or mycotoxins as many mycelial fungi do, or antibiotic metabolites likely to be produced by bacterial antagonists (Droby and Chalutz, 1994). Yeasts have simple nutritional requirements and are able to colonize dry surfaces for long periods of time, as well as withstand many pesticides used in the postharvest environment (El-Tarabily and

Sivasithamparam, 2006). They can grow rapidly on inexpensive substrates in fermenters and are therefore easy to produce in large quantities (Druvefors, 2004). -

Past studies suggested modes of action with regard to biocontrol yeasts indicated a lower likelihood of any hazards to consumers (Arras et al, 1999).

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Competition for nutrients and space is believed to be the major mode of action. of antagonistic yeasts. Indeed, for these biocontrol agents, different mechanisms or combinations of mechanisms may be involved in the suppression of different plant diseases such as inhibition of the pathogen by antimicrobial compounds (antibiosis); = induction of plant resistant mechanisms; inactivation of pathogen germination factors - present in seed and root exudates and degradation of pathogenicity factors of the “pathogen such as toxins; and parasitism that may involve production of extracellular cell-wall degrading enzymes that can lyse pathogen cell walls. =

Antagonist-pathogen interaction has been shown to play a major role in biocontrol activities. Wisniewski et al. (2003) reported this kind of interaction between the yeast Pichia guilliermondii being intactly attached to the pathogen

Botrytis cinerea that even extensive washing with distilled water wouldn’t dislodge the attachment. Similar antagonist-pathogen interaction was exhibited by Pichia anomala which heavily colonized the hyphae of Botrydiplodia theobromae that caused swelling and beading until totally penetrated and destroyed (Mohammed and

Saad, 2009). In the presence of pathogen, the yeast cells produce lytic enzymes that can enhance the attachment ability of yeast to the hyphae of the pathogen (Wisniewski et al, 1991). The yeast-pathogen interaction is believed to be dependent on protein signal recognition between yeast cells and the hyphae of pathogens and attachment efficacy might be dependent on the secretion abilities of lytic enzymes such as B-1, 3-glucanase, exo-chitinase, and endo-chitinase (Chan and Tian, 2005).

There are various types of interactions between organisms from which arise the mechanism of biological control. Researchers thus, focused on characterization of the mechanism operating in different experimental set-ups. Yeast antagonism occurs in a spectrum of directionality and is associated with the amount of interspecies contact and specificity of the interactions (Nega, 2014). The narrow range of host - specification of antagonistic yeast limits its application as biocontrol agent in a wide array of plant diseases. This study addresses this problem by isolating and identifying yeast with antagonistic activity against some common postharvest pathogens specifically, Colletotrichum sp., Erwinia sp., Fusarium spp., Aspergillus sp,

Penicillium sp, Ralstonia sp., in the Philippine setting. -.

SUMMARY OF THE INVENTION t=

The present invention relates to a process of controlling plant pathogens by * application of antagonistic microorganisms comprising: the postharvest application = onto the agricultural commodities of at least one microorganism which is antagonistic = against plant pathogen. Wherein, the said microorganism is applied in an amount ol effective to control the diseases of agricultural commodities caused by pathogens. ~~

The second embodiment of the present invention relates to a process of application wherein the said antagonistic microorganisms against plant pathogen is applied as early as possible during postharvest.

The third embodiment of the invention relates to compositions which is effective in the execution of the said processes. Such composition include:

The composition comprising a mixture of, (1) at least one microorganism which is an antagonist against plant pathogen and, (2) a carrier for the said microorganism selected from the group consisting of a starch, talc, gum or mixtures therefore.

The composition wherein the microorganism is 10° to 108 colony forming unit per 20 grams of a carrier.

DETAILED DESCRIPTION OF THE IVENTION

Biological control, especially using antagonists (mainly yeasts and bacteria) of the plant pathogens, has gained considerable attention and appears to be promising as a viable supplement or alternative to chemical control.

Isolation and identification of yeasts with potential antagonistic activity

Epiphytic yeasts are isolated from surfaces of fruits and vegetable. Sources of yeast isolates are eight species of tropical fruits and vegetable namely, apple (Malus y domestica), banana (Musa sapientum), grape (Vitis vinifera), orange (Citrus sinensis), o pepper (Capsicum annuum), papaya (Carica papaya), mango (Mangifera indica) and & carrot (Daucus carota) sourced from a public market. _

Initially, yeast isolation from fresh fruits is done by skin washing. The fruit - peelings are suspended for enrichment in 50 mL sterile phosphate buffer solution © (PBS) in sterile flasks and shaken vigorously for few minutes. Grape fruits are = suspended for enrichment in 50 mL sterile phosphate buffered saline (PBS) contained ® in a sterile plastic bag and aseptically scrubbed to dislodge yeast from the surface. ©

Serial ten-fold dilutions of the washings from each source are prepared in sterile PBS.

Screening for yeast antagonists )

The yeast-pathogen direct interaction in vitro is examined using a light microscope. A 6mm agar bloc of the fungus is placed on the center of a 9cm diameter plate and a 2cm streak of the yeast is place 3cm away from the agar bloc. The dual cultures are incubated until margins of fungal colony and yeast are in contact. The mold-yeast interaction is observed under a light microscope, (Leitz) at 400x magnification. The affinities of the yeast to colonize the fungal mycelia are recorded according to Hashem and Alamri (2009) rating system: - negative (no attachment), + sparse attachment (less than 10 cells per hypha), ++ loose attachment (10-50 cells per hypha), +++ heavy attachment (more than 50 cells per hypha).

Mass production of effective yeast antagonists

Yeast isolates that exhibit antagonism against a number of pathogens are selected for mass production. Pilot plant biomass production processes of the yeast antagonists are done using sugar cane molasses as substrate. The cells are grown aerobically in the shake flasks for 48 hrs and then harvested by centrifugation at ) 5000rpm for 10mins, wash twice in sterile distilled water and resuspended in PBS with 10% glycerol.

Claims

ee —————— OR LT CLAIMS -

1. A composition for biocontrol of pathogens in plants comprising - a yeast species; and - a carrier. >»

2. A composition for biocontrol of pathogens in plants according to Claim 1 wherein the plants are banana, tomato, mango, calamansi, garlic, onion, or _ peanut.

3. A composition for biocontrol of pathogens in plants according to Claim 1 wherein the pathogen species are Colletotrichum musae, Colletotrichum gloeosporioides, Ralstonia solanacearum, Penicillium digitatum and Aspergillus flavus or a combination thereof.

4. A composition for biocontrol of pathogens in plants according to Claims 1 wherein the yeast species are Pichia kudriavzevii, Saccharomyces cerevisiae, Debaryomyces hansenii, -Turolopsis candida and Kodamaea ohmerii.or a ; 15 combination thereof.

5. A composition for biocontrol of pathogens in plants according to Claims 1, 2, 4, and 6 wherein, Pichia kudriavzevii is antagonistic against Colletotrichum musae in banana; Ralstonia solanacearum in tomato, Penicillium digitatum in calamansi and Aspergillus flavus in garlic.

6. A composition for biocontrol of pathogens in plants according to Claims 1, 2, 4, and 6 wherein Saccharomyces cerevisiae is antagonistic against * Colletotrichum gloeosporioides in mango, Penicillium digitatum in calamansi and Aspergillus flavus in garlic.

7. A composition for biocontrol of pathogens in plants according to Claims 1, 2, | 4, and 6 wherein Debaryomyces hansenii is antagonistic against Colletotrichum musae in banana, Aspergillus flavus in garlic and nuts.

8. A composition for biocontrol of pathogens in plants according to Claims 1,2, 4, and 6 wherein Turolopsis candida is antagonistic against Colletotrichum gloeosporioides in mango and Aspergillus flavus in garlic and nuts.

9. A composition for biocontrol of pathogens in plants according to Claims 1, 2, 4, and 6 wherein Kodamaea ohmerii is antagonistic against Colletotrichum i T iat gloeosporioides in mango, Penicillium digitatum in calamansi, Aspergillus. flavus in garlic and onion. on

10. The composition according to Claim 1 in which, the concentration of the yeast. strain is 10° to 108 CFU per 20 g carrier. @

11. The composition according to Claim 1 wherein the carrier is talc. : :