Commercialization of Postharvest Biocontrol: Barriers and Opportunities

Commercialization of Postharvest Biocontrol: Barriers and Opportunities

Food quality, safety and environment still a major challenge

Organic Produce: Increasing Demand

Consolidation of forces to reduce pesticide use current situation Food safety has become competitive among supermarket chains Each supermarket chain now have there own MRLs (maximum residual level) Increased pressure by media and NGOs demanding produce free of chemical residues Pathogen resistance to fungicides Key Fungicides have been withdrawn from the market

Naturally Suppressive Microorganisms on fruit Surfaces

Biocontrol agents of Postharvest Diseases Candida famata Candida sake Candida oleophila Candida guilliermondii Candida pulcherima Candida reukaufii Candida saitoana Candida musae Candida quercitrusa Pichia guilliermondii Pichia anomala Pichia membranefaciens Pichia fermentans Cryptococcus laurentii Cryptococcus albidus Cryptococcus infirmo-miniatus Metschnikowia fructicula Metschnikowia pulcherima Debaryomyces hansinii Debaryomyces roseus Aureobasidium pullulans Cleocera apiculata Issatchenkia orientalis Rodotorola glutinis Pantoea agglomerans Pseudomonas syringae Bacillus subtilis

Yeasts as biocontrol agents Indigenous to wide variety of fresh produce. Widely used in food and Beverage industries. Easy to culture and to mass produce. Genetically stable. Do not produce antibiotic substances. Colonize rapidly plant surfaces. Survive under adverse environmental and nutritional conditions. Easy to formulate, long shelf life.

Postharvest Biocontrol Products: the Long Way to the Market Toxicology testing Registration Commercial testing Up scale production Formulation The involvement of a commercial company in the development process is crucial Isolation & screening Laboratory testing Pilot & semi commercial testing Average time : 7-10 years Cost : ~ $ 10 million Time

The First Generation of Biocontrol Products for Postharvest Disease Control (Late 1990 s) Registered Products: US & IS US & IS US S.Africa

Limited to the US

Registered for: Strawberry Metschnikowia fructicola SHEMER Carrots Chalara Citrus Botrytis Rhizopus Table grapes Pepper Penicillium Sweet potato Botrytis Aspergillus Botrytis Rhizopus

Lleida Candifruit Candida sake CPA-1 Liquid formulation Commercially available - summer 2008 Application on pears and apples

InovaCure Neova Technologies Inc. Abbotsford BC, Canada inovapure (Lysozyme) Pancreatic Enzymes Ovoproducts Appalachian Fruit Research Station, Kearneysville, WV, USA

Products in advanced stages of Candida oleophila Leasafree International, Lille, France Pantoea agglomerrans

commercial use of these products was and remains limited and accounts for only a very small fraction of the potential market. The short comings: Inconsistency Variability Low levels of control compared to chemicals Formulation stability and activity Applicability Small size companies

Aspire for the Control of Postharvest Decay of Citrus - commercial citrus packinghouse trials

Decayed fruit/ 10 m Strawberry Botrytis control- commercial observation 3 days post spray 10 days post spray a a a a b b b b NTC Shemer NTC Shemer One acre plot

Variables Involved in Postharvest Biocontrol systems Inoculum load and previous infections. Fruit maturity and variability. Variability in treated commodity (previous chemical treatments, handling, latent infections). Formulation Application methods

Where we go? Is it time for new paradigm? If postharvest biocontrol is going to be commercially successful a broader concept of biological control would be needed

Breaking Out of the Entomology Paradigm Entomologists control an organism (insect) with an organism(antagonist, parasite, or predator) CONTROL OF AN ORGANISM Plant pathologists control a disease (process) not an organism CONTROL OF A PROCESS

The disease (process) can be interrupted at different levels: The pathogen - interfering with the contamination and infection process pre-harvest chemical or cultural practices physical treatments (heat / cold) biocontrol agents (pre- & postharvest) Micro-environment (infection site) ph Nutritional composition Surficants/adjuvants salts organic acids Host susceptibility Plant growth regulators MA/CA Ethylene inhibitord Induction of resistance

Multi-system approach Combination treatments 2 or more treatments with the same purpose (eg. combination of pre- and post-harvest treatments for mold control) Complementary treatments 2 or more treatments with different purposes when combined result in extra benefits Direct or indirect effect on the pathogen

Control of decay in cold stored Oranges (cv Tarocco) Commercial packing-house (SICILY) Decay (%) 100 % 100 80 60 Non Treated Control Shemer Imazalyl+wax 40 20 0 7 14 21 46 Storage at 10 C (days) (Treatments included a hot carbonate prewash)

DECAY (%) Integrated Control of Penicillium decay on Organic Ruby Red Grapefruit 16 14 12 10 8 6 4 2 a 5 days at 5 C + 10 days of shelf life a b 0 HW Control SBC+HW SBC+HW+Shemer Commercial test Packinghouse test March 2002

DECAY (%) Prevention of Rhizopus decay during storage Organic packing-house 100 90 80 70 60 50 40 30 20 10 0 NTC היקש g/l) Shemer (2 שמר 2 גרם g/l) g/l+ 2 )PBC ( 1 גרם 2 שמר ( +תוסף LSD 0.05 Shemer (2 g/l) + PBC (1g/L) 0 5 10 Days after טיפול application ימים אחרי

Rot (%) SHEMER with hot water pre-wash and additives for improved Botrytis control on Peppers 30 a 20 ab 10 bc bc 0 NTC Storage 7 O C RH 95: Shelf at 20C: 23 days 4 days HW HW+ SHEMER +Add 1 HW + SHEMER c HW+ SHEMER +Add 2 S23-06 Each treatment included 4 replicates of 5 kg peppers

Decay (%) Synergistic effect of steam and Shemer against Chalara elegans water 20 Shemer 15 10 5 steam 0 Steam-Shemer Eshel et al. Iprodione

The Current barriers: Lack of basic information on antagonist-host interactions Stability and efficacy of commercial formulations Ecology and physiology of biocontrol agents Mechanism/s of action biocontrol agents

Formulation is key factor for effective biocontrol product 10 Log cfu / g SHEMER 8 6 4 2 204-272-1 room temperature room temperature in package 4 C 0 100 200 300 400 500 600 days Shelf life of at least one year

Ecology of yeast biocontrol agents on fruit surfaces: Colonization and spatial distribution Biofilm formation and its relationship with activity Effect of fruit physiology

Lack of basic understanding of the physiology and mechanism/s of action of postharvest biocontrol agents Direct effects competition for nutrients and space mycoparasitism lytic enzymes Biocontrol agent Induction of Resistance PR proteins Phytoalexines Lignin ROS production Pathogen Host

Induction of Resistance by yeast antagonists Control Yeast

The time course production of superoxide anion by Candida oleophila cells applied to apple fruit wounds or on nutrition media, as detected by NBT staining. Hours after inoculation What 3h is the role 6h of ROS in the 24h biocontrol system? 48h Effect In vitroof fruit type, maturity, ripening? Effect of ROS on yeast physiology and resident natural microflora? Possible involvement of ROS in signal transduction 3h pathways 6 h leading to 48h resistance? MAPK cascade? Hours after inoculation Wounds

Production of H 2 O 2 in response to wounding and treatment of lemon fruit with Metschnikowia fructicola Time after inoculation 18h 42h 66h Control W W W W W Yeast W

Production of H 2 O 2 in response to wounding and treatment of apple fruit with Metschnikowia fructicola Water Control yeast 5 hpi W W 48hpi W W Granny Smith

Lack of basic understanding of the physiology and mechanism/s of action of postharvest biocontrol agents Direct effects competition for nutrients and space mycoparasitism lytic enzymes Pathogen Biocontrol agent?? Pathogenicity factors Induction of Resistance PR proteins Phytoalexines Lignin ROS production Host

Effect of the host on biocontrol efficacy Pichia fermentans Pathogenic pseudohyphae Antagonistic Vegetative growth Giobbe, et al, (2007). FEMS Yeast Research 7, 1389-1398.