Elisabeth wood defense seminar final

Elisabeth M. Wood

Aberdeen Research and Extension CenterDepartment of Plant, Soil and Entomological Sciences

University of Idaho

Major Professor: Dr. Phillip Wharton

Agenda Introduction

Potato Storage Diseases Volatile Compounds

Objectives Will plant derived volatile organic compounds work to control

postharvest potato pathogens?

Results in vitro studies Mode of action studies in vivo studies

Conclusions

Introduction

Potatoes are a food source for millions of people

Can be processed and utilized in a variety of ways making them a highly desirable crop

Able to be stored for many months after harvest

Time spent in storage can lead to loss from disease

Controlled by carefully monitoring storage temperature, humidity, and sanitation

Spread easily and quickly through storage, especially if potatoes are damaged during harvest

In storage, one of the only defense mechanisms available to potatoes is their impermeable skin

Only a few fungicides approved for direct use on edible product

Expensive control measures and losses from disease can be costly to producers

There is a need for better disease control methods

Potato Storage Diseases

Blemish diseases of potatoes caused by 3

main pathogens:

P. atrosepticum is the causal agent of potato soft rot and pit rot

Infected in wet soils (anaerobic conditions), high humidity and warm temperatures in field and storage

The bacterium contains pectolytic enzymes

• Pectobacterium atrosepticum

• Colletotrichum coccodes• Helminthosporium solani

Blemish diseases of potatoes caused by 3

main pathogens:

C. coccodes is the causal agent of blackdot

Symptoms on stems and roots, and senesce prematurely (early die off)

Infected tubers have sooty lesions (microsclerotia)

Does not spread in storage

• Pectobacterium atrosepticum

• Colletotrichum coccodes

• Helminthosporium solani

Blemish diseases of potatoes caused by 3

main pathogens:

H. solani is the causal agent of silver scurf

Infected by soil-borne inoculum

Storage spread via air-borne conidia

Infected potatoes show silvery raised lesions and rough skin

• Pectobacterium atrosepticum

• Colletotrichum coccodes

• Helminthosporium solani

Storage rots of potatoes caused by 3

main pathogens:

P. erythroseptica is the causal agent of pink rot

Stolons infected in water-saturated soils. In storage, infection spreads in warm and humid conditions via zoospores.

Softened tuber tissue turns pink when exposed to air

• Phytophthora erythroseptica

• Pythium ultimum• Phytophthora infestans

Storage rots of potatoes caused by 3

main pathogens:

P. ultimum is the causal agent of pythium leak

Infected during harvest through wounds when humidity and temperatures are high

Infected potatoes have soft, discolored tuber tissue and watery discharge

• Phytophthora erythroseptica

• Pythium ultimum• Phytophthora infestans

Storage rots of potatoes caused by 3

main pathogens:

P. infestans is the causal agent of late blight

Infected by plant material or soil, high humidity and warm temperatures in field and storage. Infection can spread quickly in storage

Infected potatoes show rust colored tissue and darkened lenticels

• Phytophthora erythroseptica• Pythium ultimum

• Phytophthora infestans

Other potato diseases, 3 more pathogens:

F. sambucinum is a causal agent of dry rot

Infected by soil-borne inoculum, in storage spreads quickly and is able to infiltrate wounded skin

Infected potatoes have visible mycelia in tuber wounds

Dry rot infection is often followed by soft rot infection (P. atrosepticum)

• Fusarium sambucinum

• Alternaria solani• Sclerotinia sclerotiorum

A. solani is the causal agent of potato early blight

Infected when tuber skin is wounded during harvest

Typically a foliar pathogen, but infected potatoes have sunken, corky, lesions that extend into tuber tissue

• Fusarium sambucinum

• Alternaria solani• Sclerotinia sclerotiorum

Other potato diseases, 3 more pathogens:

S. sclerotiorum is the causal agent of white mold

Potato stems and leaves infected in the field at flowering, kills stems and can cause early senesce

Not typically a tuber pathogen in the USA, but has been shown to infect tubers under specific conditions resulting in tuber tissue decay

• Fusarium sambucinum• Alternaria solani

• Sclerotinia sclerotiorum

Other potato diseases, 3 more pathogens:

Infected potato images courtesy of Queensland Government Department of Agriculture, Fisheries, and Forestry

Agenda Introduction

Potato Storage Diseases Volatile Compounds

Objectives Will plant derived volatile organic compounds work to control

postharvest potato diseases?

Results in vitro studies Mode of action studies in vivo studies

Conclusions

2E-hexenal is a naturally produced volatile compound (lipoxygenase pathway)

Previous studies show anti-fungal and anti-bacterial properties

Approved by the FDA as a food/flavor additive

Volatile nature allows for highly effective control methods such as fumigation or headspace treatment

2E-hexenal2E-hexenal

Volatile compounds to control disease

H 3C

Acetaldehyde is a naturally produced volatile compound (pyruvic acid and pyruvate decarboxylase)

Previous studies show slowing of the ripening process of fruit

Previous studies show anti-fungal properties

Volatile nature allows for highly effective control methods such as fumigation or headspace treatment

Volatile compounds to control disease

Acetaldehyde

Agenda Introduction

Potato Storage Diseases Volatile Compounds

Objectives Will plant derived volatile organic compounds work to control

postharvest potato diseases?

Results in vitro studies Mode of action studies in vivo studies

Conclusions

Objectives

Which volatile compound is the most effective and at what concentration in vitro?

Can this volatile compound control other potato pathogens in vitro?

What is the mode of action of this volatile compound?

Can this volatile compound control these pathogens in vivo?

In vitro methods

Results: Acetaldehyde least effective

Acetaldehyde Did not inhibit the growth of

any of the blemish pathogens

None of the treatment volumes of acetaldehyde were significantly different from the untreated control

Results: P. atrosepticum2.5 µL/L of 2E-hexenal was capable of inhibiting growth of P. atrosepticum completely in vitro.

() UTC () IS Check () 2.5 µL/L () 5 µL/L () 7.5 µL/L () 10 µL/L

2.5 µL/L

Untreated Control

Results: C. coccodes2.5 µL/L of 2E-hexenal was capable of slowing C. coccodes growth, but 5 µL/L completely inhibited growth in vitro.

Untreated Control

() UTC () IS Check () 2.5 µL/L () 5 µL/L () 7.5 µL/L () 10 µL/L

2.5 µL/L5 µL/L

Results: H. solani2.5 µL/L of 2E-hexenal was able to inhibit the growth of H. solani completely in vitro.

2.5 µL/L

Untreated Control

() UTC () IS Check () 2.5 µL/L () 5 µL/L () 7.5 µL/L () 10 µL/L

Objectives

Which volatile compound is the most effective and at what concentration in vitro? 2E-hexenal at 5 µL/L for potato blemish

pathogens

Which volatile compound is the most effective and at what concentration in vitro? 2E-hexenal at 5 µL/L for potato blemish pathogens

Can this volatile compound control other potato pathogens in vitro?

What is the mode of action of this volatile compound?

Can this volatile compound control these pathogens in vivo?

Results: P. erythroseptica2.5 µL/L was capable of slowing P. erythroseptica growth, 5 µL/L capable of inhibiting growth completely in vitro.

Untreated Control

() UTC () IS Check () 2.5 µL/L () 5 µL/L () 7.5 µL/L () 10 µL/LP. erythroseptica in vivo

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Results: P. ultimum2.5 µL/L was capable of capable of inhibiting P. ultimum growth completely in vitro.

2.5 µL/L

Untreated Control

P. ultimum in vitro

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Results: P. infestans2.5 µL/L was capable of capable of inhibiting P. infestans growth completely in vitro.

2.5 µL/L

Untreated Control

P. infestans in vitro

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Results: F. sambucinum7.5 µL/L was capable of inhibiting F. sambucinum growth completely in vitro, with 2.5 and 5 µL/L able to slow the growth of the pathogen in vitro.

F. sambucinum in vitro

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() UTC () IS Check () 2.5 µL/L () 5 µL/L () 7.5 µL/L () 10 µL/L

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2.5 µL/L5 µL/L7.5 µL/L

Results: A. solani5 µL/L was capable of capable of inhibiting A. solani growth completely in vitro, although 2.5 µL/L did slow the growth of the pathogen in vitro.

Untreated Control

() UTC () IS Check () 2.5 µL/L () 5 µL/L () 7.5 µL/L () 10 µL/LA. solani in vitro

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Results: S. sclerotiorum2.5 µL/L was capable of inhibiting S. sclerotiorum growth completely in vitro.

2.5 µL/L

Untreated Control

() UTC () IS Check () 2.5 µL/L () 5 µL/L () 7.5 µL/L () 10 µL/LS. sclerotiorum in vitro

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Objectives

Can this volatile compound control other potato pathogens in vitro? 7.5 µL/L 2E-hexenal toxic to all tested

pathogens in vitro

Which volatile compound is the most effective and at what concentration in vitro? 2E-hexenal at 5 µL/L for potato blemish pathogens

Can this volatile compound control other potato pathogens in vitro? 7.5 µL/L 2E-hexenal toxic to all tested pathogens in

vitro

What is the mode of action of this volatile compound?

Can this volatile compound control these pathogens in vivo?

Blemish pathogen C. coccodes and H. solani Microscopy experiments were

completed to understand the relationship between 2E-hexenal and the two fungal blemish pathogens Germination rate Hyphal elongation

C. coccodes germination in vitro2.5 µL/L capable of completely inhibiting C. coccodes conidial germination in vitro.

2.5 µL/L

Untreated Control

C. coccodes hyphal elongation in vitro2.5 µL/L 2E-hexenal capable of significantly decreasing hyphal elongation of C. coccodes in vitro.

2.5 µL/L

Untreated Control

H. Solani germination in vitro2.5 µL/L capable of strongly inhibiting conidial germination in vitro. H. solani has a notably slower rate of germination than C. coccodes.

2.5 µL/L

Untreated Control

H. Solani hyphal elongation in vitro2.5 µL/L 2E-hexenal capable of somewhat decreasing hyphal elongation of H. solani in vitro. However, with low germination rates and very slow rates of growth the results are more modest.

2.5 µL/L

Untreated Control

Results: Mode of Action

C. coccodes Blackdot

H. solani Silver Scurf

C. acutatum untreated cell

C. acutatum : 2E-hexenal

Arroyo, F. T., Moreno, J., Daza, P., Boianova, L., and Romero, F., 2007. Antifungal activity of strawberry fruit volatile compounds against Colletotrichum acutatum. Journal of Agricultural and Food Chemistry. 55:5701–5707

2E-hexenal inhibits C. coccodes and H. solani conidial germination and significantly reduces hyphal elongation

Other research has shown that 2E-hexenal scrambles cellular membranes disrupting organelles and important cellular functions

C. acutatum untreated cell

C. acutatum : 2E-hexenal

Objectives

What is the mode of action of this volatile compound? 2E-hexenal inhibited conidial

germination as well as hyphal elongation in the blemish pathogens C. coccodes and H. solani

Which volatile compound is the most effective and at what concentration in vitro? 2E-hexenal at 5 µL/L for potato blemish pathogens

Can this volatile compound control other potato pathogens in vitro? 7.5 µL/L 2E-hexenal toxic to all tested pathogens in vitro

What is the mode of action of this volatile compound? 2E-hexenal inhibits conidial germination as well as hyphal

elongation in the blemish pathogens C. coccodes and H. solani

Can this volatile compound control these pathogens in vivo?

In vivo methodsLarge Scale Trials(C. Coccodes)

Small Scale Trials

Molecular and Visual Quantification

Results: C. coccodes large-scale trialUnder large-scale experimental conditions, 2E-hexenal did not inhibit the growth of C. coccodes over 5 months in storage.The amount of C. coccodes present on naturally infected potatoes increased over time in storage on the untreated control.

Results: in vivo large-scale trial

Results indicate that 2E-hexenal was not effective in controlling C. coccodes in vivo, why?

Small-scale trials designed to over come these possible issues

Poor circulation of 2E-hexenal

Respiring potatoes may interact with 2E-hexenal and decrease concentration

Broad sample collection times

C. coccodes does not spread in storage, symptoms worsen

Improvements

In vivo methodsSmall Scale Trials

Potatoes treated with 2E-hexenal absorbed the volatile compound for up to 5 days after treatment

Molecular and Visual Quantification

Both the tuber peel (2 mm) and tuber tissue absorbed 2E-hexenal, absorption was independent of treatment volume and tuber surface area

Un-inoculated Tubers

Results: C. coccodes small scaleUnder small-scale experimental conditions, 2E-hexenal did not inhibit the growth of C. coccodes In vivo.

Untreated Control

50 µL/L

Results: P. atrosepticum small-scale trialUnder small-scale experimental conditions, 2E-hexenal did not inhibit the growth of P. atrosepticum in vivo, and in fact may have increased disease severity due to anaerobic conditions.

All replications

Bacterial Soft Rot SymptomsBacterial Pit Rot Symptoms

50 µL/L

Untreated Control

Results: P. erythrosepticaUnder small-scale experimental conditions, 2E-hexenal did not inhibit the growth of P. erythroseptica in vivo.

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Results: P. ultimumUnder experimental conditions, 2E-hexenal did not inhibit the growth of P. ultimum in vivo.

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50 µL/L

Results: H. solaniUnder experimental conditions, 2E-hexenal inhibited the growth of H. solani in vivo at the minimum treatment volume of 5 µL/L.

50 µL/L

Untreated Control

Results: in vivo small-scale trial

Results indicate that 2E-hexenal controlled H. solani in vivo but nothing else, why?

Decrease treatment volumes

Decrease treatment time

Increase oxygen presence and volatile circulation

Treat prior to inoculation

Low oxygen conditions (P. atrosepticum) due to increased treatment concentrations

Anoxic conditions lower potato defense mechanisms

Rot pathogens penetrate tuber tissue deeper than volatile

C. coccodes microsclerotia

H. solani rapid sporulation on tuber surface

Improvements

Objectives

Can this volatile compound control these pathogens in vivo? 2E-hexenal inhibited H. solani in vivo at 5

µL/L

Which volatile compound is the most effective and at what concentration in vitro? 2E-hexenal at 5 µL/L for potato blemish pathogens

Can this volatile compound control other potato pathogens in vitro? 7.5 µL/L 2E-hexenal toxic to all tested pathogens in vitro

What is the mode of action of this volatile compound? 2E-hexenal inhibits conidial germination as well as hyphal

elongation in the blemish pathogens C. coccodes and H. solani

Can this volatile compound control these pathogens in vivo? 2E-hexenal inhibited H. solani in vivo at 5 µL/L

Agenda Introduction

Potato Storage Diseases Volatile Compounds

Objectives Will plant derived volatile organic compounds work to control

postharvest potato diseases?

Results in vitro studies Mode of action studies in vivo studies

Conclusions

Overall Conclusions: Acetaldehyde was not

effective

2E-hexenal was effective

7.5 µL/L of 2E-hexenal inhibited growth of all tested pathogens in vitro

Mode of action: inhibition of conidial germination and prevention of hyphal elongation in fungal blemish pathogens at 2.5 µL/L

This shows promise for in vivo control, but more to work is needed for commercialization

Applications:

Low treatment volumes

Fumigation

Active packaging

Further research Improved In vivo studies Inclusion complexes and

polymeric plastic films (packaging)

With further research, 2E-hexenal could be used to

control the growth of postharvest potato pathogens,

and an alternative to postharvest fungicides.

Almenar, E., Auras, R., Wharton, P., Rubino, M., & Harte, B. (2007). Release of acetaldehyde from β-cyclodextrins inhibits postharvest decay fungi in vitro. Journal of Agricultural and Food Chemistry, 55, 7205–7212.

Thank you for your time.

Committee:Phillip WhartonNora OlsenJoe KuhlRafael Auras

Potato Pathology Crew:Tim MilesLaura MilesKatie FairchildDarrah Ricard

Equipment Support:Joe KuhlPamela HutchinsonLouise-Marie Dandurand

Thank you for your time