Helminthosporium solani: Biology, Occurrence, and Control

By

Chakradhar Mattupalli

A dissertation submitted in partial fulfillment of

the requirements for the degree of

Doctor of Philosophy

(Plant Pathology)

at the

UNIVERSITY OF WISCONSIN-MADISON

2013

Date of final oral examination: 04/29/2013

The dissertation is approved by the following members of the Final Oral Committee:

Amy O. Charkowski, Professor, Plant Pathology

Glen R. Stanosz, Professor, Plant Pathology

Douglas I. Rouse, Professor, Plant Pathology

James P. Kerns, Assistant Professor, Plant Pathology

Shelley H. Jansky, Associate Professor, Horticulture

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ABSTRACT

Potatoes are one of the top five organic vegetables purchased for home consumption in the

United States. Many biotic and abiotic factors limit organic potato production and to meet the

increasing consumer demand, especially from the fresh market, there is a need to produce good

quality blemish-free potatoes. This research shows that silver scurf(caused by Helminthosporium

solani) and black dot (caused by Colletotrichum coccodes) are two prevalent diseases in organic

potato production in Wisconsin, with high incidences across locations and cultivars. Seventy-five

percent and 94% of asymptomatic tubers assessed were positive for H. solani and C. coccodes

respectively. Because no cultivar has yet been identified that is completely resistant to silver

scurf, we performed minituber inoculation assays and identified a diploid interspecific hybrid

C287 that consistently supported little sporulation of H. solani, suggesting it has partial

resistance to silver scurf. Although important, H. solani is an understudied pathogen with poor

genomic information, and herein we report the draft genome of the fungus. The estimated

genome size of H. solani is ~ 35 megabases. In silico genomic analysis suggests that the fungus

possesses a large suite of genes encoding putative cell wall degrading enzymes.

We also explored alternate methods to control fungal pathogens of potato. We performed in

vitro tests with tobacco phylloplanins (Nt-phylloplanins) using an inexpensive microfluidic

approach to test for its inhibitory activity against plant and human fungal pathogens. Spores of

Colletotrichum coccodes and Aspergillus fumigatus treated with Nt-phylloplanins did not

germinate at the concentrations tested at 48 and 30 hours post treatment respectively. Inhibition

of spore germination of the human pathogen A. fumigatus by Nt-phylloplanins also reveals a new

venue for medicinal antifungal drug discovery that has not been explored previously.

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ACKNOWLEDGEMENTS

I am especially thankful to Dr. Amy Charkowski, my advisor, for accepting me into her

laboratory and instilling in me the confidence that led to the completion of this work. Her

enthusiasm, willingness to give her time, and constructive suggestions during the planning and

development of this research work were extraordinary. I am fortunate to work in her lab, which

provided me with various opportunities for conducting research, mentoring undergrads, writing

grant proposals and I am confident that these experiences will help shape my future career. I

would also like to thank my committee, Drs. Glen Stanosz, Doug Rouse, Jim Kerns and Shelley

Jansky for having provided me with valuable insights at critical stages of my research.

I would like to express my great appreciation to Dr. Ryan Shepherd, for introducing me

to phylloplanins and teaching protein-related work. I would like to offer special thanks to Dr.

Nancy Keller for her advice with phylloplanins and genome projects, and her lab group members

Joe Spraker, Erwin Berthier and Jon Palmer for their assistance with Aspergillus assays,

microfluidics and fungal transformations. Jeremy Glasner provided me with very valuable

assistance in sequencing the genome of Helminthosporium solani. I thank my lab group and

many undergrads for creating a friendly environment in the lab. I wish to acknowledge the care

and concern shown by my friends Nancy Koval, Angie Peltier, Bob Rand, Lenice Covert, Ana

Cristina Fulladolsa Palma and Gregorio Sanchez Perez. Karen Lackermann has been the most

encouraging and helpful friend I ever had and I also thank her for proofreading various chapters

of this dissertation.

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I thank my wife Radhika for her steady encouragement and patience, my son Neerajaksh

for his countless and precious smiles. Finally, I am indebted to my dear friend Vambarelli

Laxmikanth Varma for his unconditional support and motivation that enabled me to come to the

United States and pursue a PhD degree in Plant Pathology.

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TABLE OF CONTENTS

Abstract ……………………………………………………………………………….. i

Acknowledgements …………………………………………………………………… ii

List of tables ………………………………………………………………………....... v

List of figures ………………………………………………………………………….. vii

INTRODUCTION ……………………………………………………………………. 1

CHAPTER 1 ………………………………………………………………………….. 33

Evaluating incidence of Helminthosporium solani and Colletotrichum coccodes

on asymptomatic organic potatoes and screening potato lines for resistance to

silver scurf

CHAPTER 2 …………………………………………………………………………… 61

A microfluidic assay for identifying differential responses of plant and human fungal

pathogens to tobacco phylloplanins

CHAPTER 3 ……………………………………………………………………………. 83

A draft genome sequence reveals Helminthosporium solani’s arsenal for cell wall

degradation

SYNTHESIS ……………………………………………………………………………. 102

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LIST OF TABLES

INTRODUCTION

Table 1: Bacterial, fungal, and oomycete pathogens affecting potato tubers ………………. 31

Table 2: Comparison of four potato tuber surface blemish diseases that are common in

Wisconsin …………………………………………………………………………………… 32

CHAPTER 1:

Table 1. Soil type, previous crop, and planting and harvest dates for each sampling

location ……………………………………………………………………………………… 53

Table 2. Percentage of potato tubers asymptomatic for both silver scurf and black dot

after harvest from three locations in Wisconsin …………………………………………….. 54

Table 3. Percentage of asymptomatic tubers positive for Helminthosporium solani

and Colletotrichum coccodes after performing PCR and tuber incubation assays …………. 55

Table 4. Spore production on fourteen potato minituber lines inoculated with

Helminthosporium solani …………………………………………………………………… 56

Table 5. Kappa coefficient values showing agreement between tuber incubation

and PCR assays for detecting Helminthosporium solani and Colletotrichum coccodes

from asymptomatic tubers ……………………………………………………………………57

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CHAPTER 2

Table 1: Comparison of the effects of Nt-phylloplanins on fungal spore germination ……… 77

CHAPTER 3

Table 1: Comparison of the number of cellulose degrading genes among

ascomycete fungi …………………………………………………………………………….. 100

Table 2: Comparison of the number of hemicelluloses degrading genes among

ascomycete fungi …………………………………………………………………………….. 101

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LIST OF FIGURES

CHAPTER 1:

Figure 1. Percentage of asymptomatic tubers positive or negative for Helminthosporium

solani and Colletotrichum coccodes by tuber incubation (T) and PCR (P) assays ………….. 58

Figure 2. Percentage of tubers positive or negative for Helminthosporium solani (Hs) and

Colletotrichum coccodes (Cc) by either tuber incubation or PCR assay for asymptomatic

tubers and by tuber incubation assay for symptomatic tubers ……………………………….. 59

Figure 3. Conidia and conidiophores of Helminthosporium solani (left side) and sclerotia

of Colletotrichum coccodes (right side) observed in close proximity on a potato tuber

periderm under a dissecting microscope …………………………………………………….. 60

CHAPTER 2:

Figure 1: Schematic representation of microfluidic device fabrication process ……………. 78

Figure 2. Inhibition of Colletotrichum coccodes spore germination after treatment

with Nt-phylloplanins in tobacco leaf wash ………………………………………………… 79

Figure 3: Inhibition curve of Colletotrichum coccodes spore germination by

Nt-phylloplanins in tobacco leaf wash ……………………………………………………… 80

Figure 4. Inhibition of Aspergillus fumigatus spore germination after treatment

with Nt-phylloplanins in tobacco leaf wash ………………………………………………… 81

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Figure 5. Transient inhibition of Fusarium sambucinum spore germination by

Nt-phylloplanins in tobacco leaf wash …………………………………………………….... 82

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INTRODUCTION

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Potato (Solanum tuberosum L.) is an annual herbaceous plant belonging to the family

Solanaceae. The crop is well known to plant pathologists in particular due to the Irish potato

famine caused by the late blight disease in the mid 1840’s. In terms of human consumption,

potatoes are the third most important food crop in the world after rice and wheat. Potatoes are an

excellent source of low fat carbohydrates, vitamins B and C, as well as iron, potassium and zinc.

(CIP, 2013). Potatoes are consumed fresh, frozen, dehydrated, canned, or processed into forms

such as potato chips and French fries. In developed countries, up to 60% of potatoes are

consumed in a processed form (Kirkman, 2007). Potatoes are a critical crop in terms of food

security; as an example China, which is the world’s largest consumer of potatoes, intends to meet

50% of its increased food demand in the next 20 years through increased potato production (CIP,

2013).

Potatoes are propagated commercially using “seed” tubers. However, individual cells,

meristems, sprouts, true seeds, and stem cuttings can also be used as propagules (Struik and

Wiersema, 1999). A seed tuber produces sprouts in its eyes, which develop into shoots, and

produce roots from primordial on the sprouts. A potato plant is in fact a cluster of stems,

originating from one seed tuber or seed piece (Struik, 2007). Potatoes have modified under-

ground shoots called stolons, with elongated internodes, rudimentary leaves and hooked tips.

Tubers are the swollen parts of stolons bearing axillary buds and leaves, evident as eyes on the

tuber skin. The life cycle of a potato plant can be described through five growth stages: 1) sprout

development (sprouts develop from tubers and emerge from the soil) 2) vegetative growth

(leaves and stems develop from above-ground nodes, roots and stolons develop from below-

ground nodes) 3) tuber initiation (tubers start forming at stolon tips) 4) tuber bulking (tubers

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enlarge and develop as a strong sink organ), and 5) maturation (tuber growth ceases and aerial

parts start to senesce) (Miller and Hopkins, 2008).

Tubers are the economically significant portion of the potato plant and because they

develop in the soil, they are attacked by myriad pathogens that affect quality and yield. These

pathogens may cause symptoms on the tuber that are either external, internal or both (Table 1).

Fiers et al. (2012) recently reviewed various biotic, abiotic and management factors that

influence the occurrence and development of soil-borne potato pathogens. Understanding how

these pathogens attack the tuber requires some knowledge about the tuber periderm. In the first

part of this review, a general overview of how tubers naturally protect themselves from soil

micro-biota is provided. The second part of the review focuses on four pathogens that cause

potato tuber-surface blemish diseases that are commonly observed in Wisconsin.

Native and Wound Periderm:

The potato tuber surface is protected by a specialized suberin-rich layer called the native

periderm. This layer provides protection against water loss, mechanical damage, and pathogens

(Lulai, 2001). Epidermis exists on the developing tubers for a very short period which is

replaced by the native periderm and lenticels are formed below each stoma (Peterson and Barker,

1979). The native periderm is composed of three layers: phellem, phellogen, and phelloderm.

The phellem is the outermost layer consisting of suberized cells and is often referred to as the

skin of the potato tuber. The phellogen or the cork cambium is the middle layer and gives rise to

phellem and phelloderm. The phelloderm is the innermost layer of the periderm consisting of

parenchyma-like cells. Tubers with immature periderm are fragile and are susceptible to skinning

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and mechanical damage during harvest and handling operations. The reason for this fragile state

is because phellogen cells are meristematic and have thin cell walls, so they loosely hold the

phellem to the phelloderm, allowing easy scuffing from the tuber (Lulai and Freeman, 2001).

This wounding damage is overcome by killing the potato vines about 3 weeks before harvest

which promotes good skin-set (Lulai and Orr, 1993). Histological and immunolabeling studies

by Sabba and Lulai (2004) indicated that the phellogen walls are reinforced by pectin depositions

during native periderm maturation providing resistance to skinning injuries.

Tubers that are cut or wounded lack protection from native periderm and as the wounded

tuber begins to heal, it forms a wound periderm which is very similar in organization to the

native periderm (Sabba and Lulai, 2002). Before the formation of wound periderm, a suberized

closing layer may form. Suberin is a biopolymer composed of polyphenolic and polyaliphatic

domains. The two domains are spatially separate. The polyphenolic domain which is embedded

in the primary cell wall is cross-linked by glycerol to the polyaliphatic domain which is located

between the primary cell wall and the plasma membrane (Bernards, 2002). During wound

healing, differential deposition of these two domains play separate roles in conferring resistance

to bacterial and fungal pathogens. Lulai and Corsini (1998) observed that the polyphenolic

domain deposited within 2 to 3 days of wound healing provided resistance to the bacterial

pathogen Erwinia carotovora subsp. carotovora (causal organism of bacterial soft rot) but not

Fusarium sambucinum (causal organism of fungal dry rot). Resistance to F. sambucinum

developed after 5 to 7 days of wound healing process which corresponded with the completion of

deposition of the polyaliphatic domain. These results suggest that only complete suberin matrix

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(suberin polyphenolic and aliphatic domains) can provide resistance to both fungal and bacterial

infections.

The relative importance of potato diseases that cause external defects has been increasing

as the fresh market becomes a more significant source of potato sales. Fresh market consumers

demand washed potatoes with a good quality and healthy looking external appearance. Here we

will focus on the following four pathogens that cause external tuber defects and are common in

the state of Wisconsin: Rhizoctonia solani, Streptomyces scabies, Helminthosporium solani, and

Colletotrichum coccodes.

Rhizoctonia solani:

The Rhizoctonia disease complex of potatoes consists of two phases: Rhizoctonia canker

(infection of growing plants) and black scurf (infestation of tubers by sclerotia). This disease

complex is present wherever potatoes are grown (Banville and Carling 2001) and is caused by

the fungus, Rhizoctonia solani Kuhn (teleomorph: Thanatephorus cucumeris (Frank) Donk).

Isolates of R. solani are categorized into Anastomosis Groups (AGs) based on hyphal

incompatibility; AG-3 is the most prevalent AG found on potatoes (Tsror, 2010). Symptoms of

Rhizoctonia canker appear as reddish brown to black lesions leading to girdling of sprouts,

stolons and roots. Poor crop stands and delayed emergence occur as the fungus kills the growing

tip of the sprouts. Above ground symptoms may manifest in the form of aerial tubers in the axils

of branches and petioles, leaf rolling, stunting, rosetting and purple pigmentation of leaves

(Banville and Carling, 2001). Hymenia of the sexual stage of the fungus may be observed as a

white to gray powdery mass on the lower stems near the soil surface (Carling et al. 1989). The

other phase of the disease complex, black scurf, occurs on the tubers. The most obvious sign of

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this disease is the presence of flat or raised, irregularly shaped brown to black sclerotia. These

sclerotia are often referred as “the dirt that won’t wash off” as they adhere tightly to the tuber

skin (Banville et al. 1996). On tubers that are harvested before skin set, the pathogen can also

cause dark spots on tubers during storage (Buskila et al. 2011). Yield losses due to R. solani can

be as high as 20 to 30 % ( Banville 1989; Carling et al. 1989).

Disease cycle:

The primary sources of inoculum for this disease complex are mycelia and sclerotia

infested seed tubers and soil. Roots and stolons are infected at any time during the growing

season (Banville and Carling, 2001). After vine-killing and during tuber maturation, volatile

tuber exudates increase the formation of sclerotia on the tubers (Dijst, 1990). Chand and Logan

(1984) found an increase in the number of sclerotia per tuber on tubers harvested 4 weeks after

vine-killing as compared to those that were harvested one week after vine-killing. Incidence and

severity of black scurf have been shown to increase during storage (Chand and Logan, 1984;

Spencer and Fox, 1979).

Host-pathogen interactions:

Histopathological studies revealed that R. solani mycelia invade the tuber periderm

tissues inter-cellularly causing tissues surrounding the hyphae to collapse, arresting further

growth of the pathogen (Schaal, 1939; Chand and Logan, 1984). Hyphae did not penetrate

beyond the phellogen but in some cases extended to the cortical tissues (Chand and Logan, 1984;

Schaal, 1939; Spencer and Fox, 1979). However, when native periderm on tubers was removed

and the exposed areas of the tuber were artificially inoculated with R. solani, a thicker and less

organized closing layer with over suberized cell walls was formed. An increase in the expression

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of suberin biosynthesis potato genes such as StKCS6, CYP86A33, and POP_A was also observed

as a response to R. solani inoculation (Buskila et al. 2011).

Aoki et al. (1963) and Nishimura and Sabaki (1963) identified phytotoxic compounds such as

phenylacetic acid and its hydroxyl derivatives (meta-hydroxyphenylacetic acid and para-

hydroxyphenylacetic acid) produced from the culture filtrates of R. solani. Rioux et al. (2011)

compared putative pathogenesis related genes expressed by R. solani AG1 and AG3 in infected

rice leaves and potato sprouts respectively. They identified 12 genes similar between the two

pathosystems which play key roles in the early processes of infection such as appresorium

formation, cell wall degradation and toxin secretion. However, at present similar information

related to the genes involved in the tuber infection process is lacking.

Streptomyces spp.:

Common scab of potatoes is caused by Gram-positive, filamentous bacteria belonging to

the genus Streptomyces. Most of the 900 known species of Streptomyces are soil-inhabiting

saprophytes, except a few species which are pathogens on plants (Bignell et al. 2010a). Loria et

al. (2006) described various pathogenic species of Streptomyces that cause typical scab

symptoms. However, the most common pathogenic species of Streptomyces that cause common

scab of potato are: Streptomyces scabies (synonym S. scabiei), Streptomyces turgidiscabies and

Streptomyces acidiscabies. S. scabies is the oldest characterized scab pathogen with a worldwide

distribution while the other two Streptomyces species are newly emergent pathogens.

S. acidiscabies causes acid scab whose symptoms are indistinguishable from common scab, but

the pathogen occurs in soils with pH below 5.2 (Lambert and Loria, 1989). S. turgidiscabies

causes distinctly erumpent lesions on potato tubers in Japan (Miyajima et al. 1998). All three

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species are neither tissue nor host specific, causing scab lesions on various root and tuber crops.

They also cause root and shoot stunting and root tissue necrosis of model plants such as tobacco

and Arabidopsis (Bignell et al. 2010a). However, DNA homology studies suggest that these

species are not closely related (Miyajima et al. 1998; Healy and Lambert, 1991). Common scab

symptoms are mostly limited to the tubers, but the pathogen can also infect stolons (Loria,

2001). Initially, the lesions are circular or irregular and tan or brown-colored and enlarge as the

tuber swells. Commercially, infection of the first four to five formed internodes is critical for

scab infection as these early internodes expand more rapidly than the later-formed internodes,

causing more scabby lesions (Lapwood et al. 1970). As the lesions mature, they become rough in

texture, coalesce, grow up to 5 to 10 mm in diameter, and in severe cases cover most of the tuber

surface. When scab lesions are superficial, the disease is called russet scab, and when they are

sunken, it is called pitted scab. Likewise, if the lesions are raised, the disease is called erumpent

scab. Lesion types are influenced by several factors such as the pathogen strain, cultivar, age of

the tuber at the time of infection, and environmental factors (Loria, 2001). Common scab does

not affect tuber yield but unsightly corky lesions impact the marketability of the tubers (van der

Wolf and Boer, 2007).

Disease cycle:

Infected tubers and infested soil are the primary sources of inoculum for common scab

(Loria, 2001). The pathogen gains entry into the tuber through recently formed unsuberized

lenticels (Fellows, 1926). The early tuber development period (2 or 3 weeks after tuberization) is

the critical stage for common scab infection and symptoms develop within seven days after

exposure of the tuber to the pathogen (Lapwood and Hering, 1970; Khatri et al. 2011). Scab

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symptoms are more severe on the first formed internodes of the tuber as compared to the later-

formed internodes, because enlarging tubers are more susceptible to infection (Fellows, 1926;

Lapwood and Hering, 1970). Initially the pathogen grows vegetatively, producing a branched,

tangled network of filamentous cells, but as the tissues affected by scab mature the pathogen

produces reproductive hyphae that transform into spore chains (Jones, 1931).

Host-pathogen interactions:

After S. scabies gains entry into the tuber through immature lenticels, the meristem is

stimulated to form closely packed elongated cells which later collapse and turn brown. Cell

proliferation, expansion, and subsequent browning are attributable to thaxtomin, the toxin

produced by the pathogen (Lawrence et al. 1990). Gradually the meristematic activity ceases and

the last-formed cells suberize to form a wound cork which separates infected cells from the

healthy tissue below. The pathogen can further infect the healthy cells by growing through

incompletely suberized cells, leading to the formation of a second barrier of wound cork. These

barriers are formed as long as the tuber grows actively resulting in severe scab lesions (Jones,

1931). Khatri et al. (2011) studied pathogen-induced changes in the tuber periderm at 10 days

after tuber initiation in a hydroponic or a non-destructive pot culture system. They observed a

significant increase in phellem thickness, number of phellem cell layers and phellem suberin

content, and these changes were greater in the scab-resistant cultivar Russet Burbank than in the

susceptible cultivar Desiree. These results suggest that potato cultivars respond differentially to

scab infection by reinforcing their periderm structure.

Lawrence et al. (1990) were the first to establish the role of a toxin in S. scabies infection.

By artificially inoculating aspetically grown minitubers with either S. scabies or purified extracts

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obtained from scab lesions, they were able to reproduce common scab symptoms. They later

identified the phytotoxic agents as thaxtomin (in honor of Roland Thaxter who first described the

causal agent of common scab) A and B which are produced only by pathogenic Streptomyces

species. Thaxtomins are cyclic dipeptides (2, 5-diketopiperazines) derived from 4-nitro-

tryptophan and phenylalanine. The presence of a 4-nitroindole moiety plays a key role in

thaxtomin phytotoxicity (King and Calhoun, 2009). Inhibition of cellulose biosynthesis has been

identified as the primary mode of action of thaxtomins (Bignell et al. 2010a). So far, 11

members of the thaxtomin family have been characterized, 10 of which are produced by S.

scabies (King and Calhoun, 2009). Similarly, another class of phytotoxins called concanamycins

A and B were shown to be produced by S. scabies, but not by S. acidiscabies. Their role as a

virulence factor is yet to be determined (Natsume et al. 1998).

Another virulence factor that was accidently discovered while screening a cosmid library

is nec1 (Bukhalid and Loria 1997). When this gene was expressed in the non-pathogen

Streptomyces lividans, it caused necrosis of potato tuber disks and produced scab-like symptoms

on immature potato tubers. The nec1 gene is structurally conserved among S. scabies, S.

acidiscabies, and S. turgidiscabies, indicating that this gene may have been horizontally

transferred among these species (Bukhalid et al. 1998; Bukhalid et al. 2002). Although nec1 is

not directly involved in the biosynthesis of thaxtomin A, it is physically linked to thaxtomin A

biosynthesis genes (Bukhalid et al. 1998). Kers et al. (2005) described a large pathogenicity

island (discrete cluster of pathogenicity and virulence genes acquired by horizontal gene transfer)

of 325-660 kb in size conserved among the three pathogenic species of Streptomyces described

earlier. One of the genes identified in this pathogenicity island tomA, encodes a homologue of

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tomatinase enzyme, which can detoxify anti-microbial saponins secreted by plants. Although

Seipke and Loria (2008) showed that tomA is not important for pathogenicity in potatoes,

conservation of this gene among S. scabies, S. acidiscabies, S. turgidiscabies suggests a role in

plant-microbe interactions.

Coronofacic acid is a component of the phytotoxin coronatine, produced by plant

pathogens such as Pseudomonas syringae and Pectobacterium atrosepticum. Bignell et al.

(2010b) performed mutational studies and identified a secondary metabolite cluster that is

predicted to synthesize a compound similar to coronafacic acid. It is interesting to note that this

biosynthetic cluster is present only in S. scabies but not S. acidiscabies or S. turgidiscabies.

Genomic analysis of S. scabies 87-22 revealed the presence of other virulence factors such as

expansin-like proteins, cutinases and auxin biosynthetic genes. These results warrant further

investigations for their role and mechanism of action during host-pathogen interactions (Bignell

et al. 2010a).

Helminthosporium solani:

Silver scurf, caused by the imperfect fungus, Helminthosporium solani Durieu & Mont. is

a potato disease that affects tuber appearance. The disease is known to occur in various parts of

the world (Errampalli et al. 2001; Loria and Secor 2001). Symptoms are observed only on the

tubers and are localized to the periderm. At harvest, diseased tubers have grey lesions on the

periderm that appear silvery when moistened. The silvery appearance of the tubers is attributed

to air pockets that develop due to cellulolytic activity of the pathogen (Heiny and McIntyre,

1983). The outer cell layers of severely diseased tubers may slough off and H. solani infection

increases the permeability of the periderm to water vapor, resulting in tuber shrinkage and

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weight loss (Burke, 1938; Jellis and Taylor, 1974). In severely diseased tubers loss of

pigmentation occurs, especially on red-skinned cultivars reducing their fresh market value (Jellis

and Taylor, 1977). The disease affects the processing market as chips made from severely

diseased tubers have unacceptable black burnt edges (Holley and Kawchuck, 1996). Field studies

indicate that H. solani does not affect emergence and growth of the potato plants or tuber yields

(Mooi, 1968; Read and Hide, 1984; Cunha and Rizzo, 2004). Silver scurf has been gaining

economic importance in the past three decades due to the quality standards demanded by both

processing and fresh market consumers as well as the emergence of H. solani isolates resistant to

the broad-spectrum thiabendazole fungicides that are applied at postharvest by conventional

potato growers (Errampalli et al. 2001).

Disease cycle:

H. solani has a very narrow host range comprising only tuber-bearing Solanum species

and the weed species Solanum elaeagnifolium (Burke, 1938; Kamara and Huguelet, 1972;

Sethuraman et al. 1997; Rodriguez et al. 1995). The disease spreads both in the field and during

storage. Diseased seed tubers (Jellis and Taylor, 1977) and infested soil (Merida and Loria,

1994) act as the primary source of inoculum in the field while diseased seed tubers act as the

initial source of inoculum in storage (Rodriguez et al. 1996). After planting diseased seed tubers,

expansion of lesions occurs rapidly within 4 to 5 weeks so that the entire tuber surface is affected

(Jellis and Taylor, 1977). The mechanism of pathogen transmission from a diseased seed tuber to

its progeny tubers is unknown as the pathogen is not known to infect either stems or stolons

(Burke, 1938). Jellis and Taylor (1977) recovered viable conidia of H. solani from soil

surrounding progeny tubers at 14 weeks after planting and observed that the first infection sites

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on progeny tubers occur at the stolon end of the tubers. They attributed the increase in disease in

the field and during storage to the expansion of lesions on the diseased tubers as well as to the

spread of the pathogen from diseased tubers to healthy tubers. In studying H. solani conidial

production in storage, Rodriguez et al. (1996) found that conidia were produced throughout the

storage season, with production levels ranging from 0 to 12,000 conidia per day at 4oC and from

0 to 24,000 conidia per day at 10oC. H. solani colonized and sporulated in-vitro on senescent leaf

tissue of alfalfa, sorghum, rye, oats, corn and wheat suggesting the pathogen may be able to

survive saprophytically in soil (Merida and Loria, 1994). The pathogen can also survive in dry

soil of potato stores for a period of five months (Carnegie et al. 1996) and conidia may serve as

over-wintering structures for the pathogen (Kamara and Huguelet, 1972).

Host-pathogen interactions:

Histological studies by Hunger and McIntyre (1979) showed that the hyphae of H. solani

are localized to the periderm and that conidiophores arise from beneath the periderm. Fewer cell

layers were observed in infected (4 to 6 layers) compared to healthy (6 to 8 layers) periderm

(Hunger and McIntyre, 1979). Conidial germination occurred at 6 and 16 hours after inoculation

on tubers of Dark Red Norland (Martinez et al. 2004) and Norchip (Heiny and McIntyre, 1983),

respectively. Likewise, penetration by germ tubes into the periderm also differed between these

cultivars. For Dark Red Norland, germ tube penetration occurred 6 to 9 hours after inoculation,

whereas in Norchip germ tube penetration did not occur until five days after inoculation.

Appresoria-like structures and suberin deposits in cortical cell layers were noted by Heiny and

McIntyre (1983), but not by Martinez et al. (2004). Hyphae grow intra- or inter-cellularly and

move from one cell to another by direct cell wall penetration (Martinez et al. 2004). Martinez et

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al. (2004) observed necrosis in both colonized and neighboring cells and suggested that H. solani

may be a necrotroph.

Colletotrichum coccodes:

Colletotrichum coccodes (Wallr.) causes a tuber blemish disease called black dot of

potato, which has a world-wide distribution (Lees and Hilton, 2003). The presence of small black

sclerotia on tubers, roots, and above- and below-ground stems gives the disease its common

name. Diseased tubers may have gray-brown blemishes on the periderm, or less clearly delimited

discolored areas on which sclerotia may or may not be present (Jellis and Taylor, 1974). The

fungus has been shown to cause dark brown, deep sunken lesions following artificial inoculation

of mini-tubers stored at 5-15oC for 5 months (Glais and Andrivon, 2004). Diseased roots have a

stringy appearance when pulled out of the soil due to cortical tissue desiccation. The pathogen

infects the cortical tissues of the stem; after drying, amethyst discoloration on the inside of the

vascular cylinder is observed. Stolons may be attacked at any stage of plant growth and the

lesions cause severing, thus leaving a part of the stolon adhering to the stem end of the tuber

(Dickson, 1926). Diseased foliage bears dark brown to black lesions similar to those caused by

Alternaria solani, but the lesions of C. coccodes lack concentric rings (Johnson and Miliczky,

1993). The disease also causes a reduction in yield, in some cases even with very little symptom

expression (Johnson, 1994; Mohan et al. 1992; Barkdoll and Davis, 1992). Also, C. coccodes is

known to interact with other pathogens like Verticillum dahliae, causing increased disease

symptoms and more extensive fungal colonization in some potato cultivars (Tsror and

Hazanovsky, 2001). Fresh weight losses of 5 to 10 % were observed by Hunger and McIntyre

(1979) when tubers naturally infected with C. coccodes were stored for 18 weeks.

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Disease cycle:

Hosts of C. coccodes include vegetables such as tomato, pepper and squash in the

Solanaceae and Cucurbitaceae families (Dillard, 1992). Isolates of the pathogen collected from

potato were capable of producing typical anthracnose lesions on ripe tomato fruit and vice-versa

(Illman et al. 1959, Chesters and Hornby, 1965). The fungus can also colonize roots of winter

cherry, marrow and symptomless hosts such as chrysanthemum, white mustard, cress, cabbage

and lettuce (Chesters and Hornby, 1965). Also, C. coccodes colonizes the stems of rotation crops

such as yellow mustard, soybean, spring canola, alfalfa, oats, as well as the stems of a number of

weeds, including eastern black night shade, velvetleaf, giant foxtail and Timothy grass (Nitzan et

al. 2006a).

Black dot is a soil-borne and tuber-borne disease. Sclerotia are the over-wintering

structures. Greenhouse studies by Blakeman and Hornby (1966) found 53% of C. coccodes

sclerotia remained viable and no conidia remained viable after burying them in soil for 83 and 3

weeks respectively. Tuber-borne inoculum may be present as sclerotia on the tuber surface or as

hyphae in the vascular bundles (Tsror et al. 1999). Contaminated seed act as an important source

in introducing the disease to non-infested soils (Barkdoll and Davis, 1992). However, once

infested, soil-borne inoculum has a higher disease causing potential than tuber-borne inoculum

(Nitzan et al. 2008). Nitzan et al. (2008) conducted greenhouse studies and observed a nonlinear

trend in disease severity (sclerotial density on the roots and the length of the stem with visible

sclerotia) with increasing soilborne inoculum concentration. Aerial infection of potato foliage

can occur in certain regions where sandstorms and sprinkler irrigation are common. Johnson and

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Miliczky (1993) obtained infections by sandblasting potato foliage followed by inoculation with

a conidial suspension of C. coccodes. In this type of dispersal, blowing sand creates wounds on

the foliage and sprinkler irrigation aids in the dissemination of conidia and sclerotia.

Infection of below and above ground parts occurs early in the growing season (Davis and

Johnson, 2001). But, the pathogen has a latent period evident by restricted colonization in the

plant stems. As the plant enters the tuber bulking phase and starts to senesce, C. coccodes

colonization is activated and sclerotia develop on above and below-ground plant parts (Nitzan et

al. 2006b). However, Ingram and Johnson (2010) did not observe such a colonization pattern on

potato roots. Glais-Varlet et al. (2004) observed an increase in the development of black dot

symptoms upon artificial inoculation of minitubers stored at 5 to 15oC. These results suggest that

the disease may be able to spread on tubers during the storage period.

Host-pathogen interactions:

Not much literature is available for C. coccodes interactions with potato tubers. However,

considerable work was done in tomato on which the fungus causes anthracnose disease. The

extent to which these results can be extrapolated to potato is debatable owing to the differences

in the environment and the plant part affected. Reasons for slow progress on this subject may be

due to the under-estimation of the disease as the symptoms appear late in the cropping season,

typically coinciding with natural senescence. In addition, symptoms on stems and stolons

resemble those of Rhizoctonia canker, symptoms on leaves are similar to Verticillum wilt or

early blight and symptoms on tubers are similar to silver scurf (Davis and Johnson, 2001).

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Conclusion:

Potato growers must manage tuber surface defect diseases, but have few non-pesticide

based methods for this problem. All four diseases discussed in this review are soil- or tuber-

borne and are monocyclic, so control involves reducing the amount of initial inoculum. One way

to achieve this goal is by the use of certified seed potatoes. One challenge is to determine the

permissible level of surface defects in a certified seed tuber, which is especially difficult when

considering latent pathogens like C. coccodes (Glais-Varlet et al. 2004). The percentage of

surface defects that are permissible on certified seed tubers varies among states, with 5 % surface

defects permitted in some states like Wisconsin. However, in chapter 1, we show visual

assessment of tubers at harvest for silver scurf and black dot may be misleading because a very

high percentage of asymptomatic tubers are already infected with H. solani and C. coccodes.

As an alternative, growers could adopt new methods of producing seed tubers such as

seed potato sprout technology. This technology facilitates production of seed tubers by planting

detached sprouts, thus minimizing the risk of soil-borne pathogen movement (Souza-Dias et al.

2008). Reducing soil inoculum using fumigants is an option, but one that is limited due to

environmental concerns. Future efforts should focus on breeding for new cultivars resistant to

multiple diseases, a solution that is eco-friendly and has long-term viability. Although cultivated

potatoes are tetraploid, which complicates breeding efforts, the use of wild Solanum species in

potato breeding programs may be valuable as the progeny tubers following crosses with wild

relatives have long dormancy with good tuber size and set (Jansky and Peloquin 2006). For

instance, by crossing [H551 × S. chacoense] with [US-730 × (S. berthaultii × S. tarijense)], a

diploid interspecific clone C545 was obtained, which exhibited improved resistance to a number

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of important diseases including soft rot, common scab, pitted scab, early dying disease and early

blight (Jansky and Rouse 2003). Further in chapter 1, we show that these diploid inter-specific

clones exhibit partial resistance to other tuber diseases such as silver scurf.

Several cultural and chemical control methods are available for the management of the

tuber blemish diseases discussed here, although their effectiveness is limited (Errampalli et al.

2001; Lees and Hilton, 2003). As an addition to the existing disease management options, we

explored the possibility of utilizing leaf surface proteins that are naturally produced by tobacco

by testing for their inhibitory effects on fungal pathogens such as C. coccodes. The results of this

project are discussed in chapter 2.

This review demonstrates the dearth of information about the molecular basis of host-

pathogen interactions for the fungal pathogens discussed here (Table 2). With the advent of high

throughput sequencing techniques, the cost of obtaining genome data has dropped considerably.

This has led to the description of several fungal genomes and provided insights into the pathogen

biology, survival and infection strategies (Ellwood et al. 2010; de Wit et al. 2012; Islam et al.

2012; Manning et al. 2013). A next step to advance our understanding of fungal diseases of

potato is to sequence the genomes of fungi that infect tuber surfaces and to perform comparative

genomics to study factors involved in host-pathogen interactions. Based on what we know about

fungal pathogenicity, plant cell wall degrading enzymes and fungal toxins are likely candidates

for virulence factors important for infection of potato tubers. Because numerous fungi are present

in soil, but only these three species (H. solani, C. coccodes, and R. solani) commonly infect

potato tuber surfaces in Wisconsin, we anticipate that evidence of convergent evolution will be

found once the molecular determinants of pathogenicity have been identified for these fungi. In

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chapter 3, we address questions related to H. solani’s biology by generating the draft genome of

the fungus.

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D.K.L., Taylor, M.A., and Ross, H.A. Potato biology and biotechnology: Advances and

perspectives. p.219-233. Elsevier publications, Oxford, GB, UK.

88. Struik, P. C., and Wiersema, S. G. 1999. Seed Potato Technology. p. 383. Wageningen Pers,

Wageningen, the Netherlands.

89. Tsror, L., Aharon, M., and Erlich. O. 1999. Survey of bacterial and fungal seedborne diseases

in imported and domestic potato seed tubers. Phytoparasitica 27: 215-226.

90. Tsror, L., and Hazanovsky. M. 2001. Effect of coinoculation by Verticillium dahliae and

Colletotrichum coccodes on disease symptoms and fungal colonization in four potato

cultivars. Plant Pathol. 50: 483-488.

91. Tsror, L. 2010. Biology, epidemiology and management of Rhizoctonia solani on potato. J.

Phytopathol. 158: 649-658.

92. Van der Wolf, J. M., and De Boer, S. H. 2007. In: Vreugdenhil, D., Bradshaw, J.E., Gebhardt,

C., Govers, F., MacKerron, D.K.L., Taylor, M.A., and Ross, H.A. Potato biology and

biotechnology: Advances and perspectives. p.605. Elsevier publications, Oxford, GB, UK.

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Table 1: Bacterial, fungal and oomycete pathogens affecting potato tubers

Disease Causal agent Diagnosis

Bacterial ring rot Clavibacter michiganensis subsp. sepedonicus External and internal

Brown rot Ralstonia solanacearum External and internal

Soft rot Pectobacterium spp., Clostridium spp. External and internal

Common scab Streptomyces spp. External

Black dot Colletotrichum coccodes External

Black pit Alternaria alternata External and internal

Charcoal rot Macrophomina phaseolina External and internal

Early blight Alternaria solani External and internal

Fusarium dry rot Fusarium spp. External and internal

Gangrene Phoma foveata External and internal

Gray mold Botrytis cinerea External and internal

Late blight Phytophthora infestans External and internal

Leak Pythium spp. External and internal

Pink rot Phytophthora erythroseptica External and internal

Powdery scab Spongospora subterranea f. sp. subterranea External

Black scurf Rhizoctonia solani External

Rosellinia black rot Rosellinia spp. External and internal

Silver scurf Helminthosporium solani External

Skin spot Polyscytalum pustulans External

Stem rot Sclerotium rolfsii External and internal

Thecaphora smut Angiosorus solani External and internal

Verticillium wilt Verticillium spp. Internal

Wart Synchytrium endobioticum External

White mold Sclerotinia sclerotiorum External and internal

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Table 2: Comparison of four potato tuber surface blemish diseases that are common in

Wisconsin

Common Scab Black Scurf Black Dot Silver Scurf

Causal agent Streptomyces spp. Rhizoctonia

solani

Colletotrichum

coccodes

Helminthosporium

solani

Source of inoculum Infected tubers,

Infested soil

Infected tubers,

Infested soil

Infected tubers,

Infested soil

Infected tubers,

Infested soil

Primary inoculum Spores Mycelia and

sclerotia

Conidia Conidia

Invasion Filamentous cell

growth restricted

by formation of

wound cork

Hyphae grow

intercellularly

and localized to

the periderm, but

may sometimes

extend to cortex

Systemic and

hyphae colonize

cortical and

vascular bundle

cells

Hyphae grow

intra- and inter-

cellularly and

localized to the

periderm

Symptom/sign

development

Tuber Tuber Tuber, stolons,

roots, above-

ground stems

Tuber

Tuber

symptoms/signs

Corky lesions that

may be raised or

pitted or

superficial

Irregularly

shaped brown to

black sclerotia

adhering to the

periderm

Grey-brown

blemishes that

are less clearly

delimited and

with/without

sclerotia

Grey lesions on

the periderm that

appear silvery

when moistened

Overwintering

structures

Spores Mycelia and

sclerotia

Sclerotia Conidia

Active disease

spread in storage

No No No Yes

Pathogenicity and

virulence factors

during tuber-

pathogen

interactions

Thaxtomin, nec1,

concanamycins

Not known Not known Not known

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CHAPTER 1

Evaluating incidence of Helminthosporium solani and Colletotrichum coccodes on

asymptomatic organic potatoes and screening potato lines for resistance to silver scurf

This chapter has been published by the American Journal of Potato Research:

Mattupalli, C., Genger, R. K., and Charkowski, A. O. 2013. Evaluating incidence of

Helminthosporium solani and Colletotrichum coccodes on asymptomatic organic potatoes and

screening potato lines for resistance to silver scurf. American Journal of Potato Research doi:

10.1007/s12230-013-9314-3.

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Abstract

Silver scurf, caused by Helminthosporium solani, and black dot, caused by Colletotrichum

coccodes, cause tuber blemishes on potato (Solanum tuberosum L.) which affect processing and

fresh market trade. Tubers from ten cultivars were collected at harvest from three organic farms

in Wisconsin and categorized as symptomatic or asymptomatic based on visual symptoms of

silver scurf and black dot and/or signs of H. solani and C. coccodes. Tuber incubation and PCR

assays were performed on asymptomatic tubers to detect H. solani and C. coccodes. Tuber

incubation and PCR assays were in slight to fair agreement (kappa coefficient < 0.4) for

detecting both pathogens. Most asymptomatic tubers tested were positive by one or both assays

for H. solani (75%) or C. coccodes (94%). Minituber inoculation assays were also performed to

screen potato lines for resistance to silver scurf. Of the 14 lines tested, a diploid interspecific

hybrid, C287, had consistently low sporulation, suggesting it has partial resistance to silver scurf.

Since the majority of tubers harvested are already infected with one or both pathogens further

research should focus on organically acceptable management practices that may inhibit disease

development in field and in storage.

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Introduction

Potato (Solanum tuberosum L.) is the third most important food crop in the world for

human consumption (CIP 2012) and one of the top five organic vegetables purchased for home

consumption in the U.S. (Stevens-Garmon et al. 2007). Although organic potatoes comprise less

than one percent of total potato sales, Greenway et al. (2011) forecast a 19% annual growth rate

in U.S. per capita organic potato consumption from 2008 to 2013. Many biotic and abiotic

factors limit organic potato production. In order to meet the increasing consumer demand,

especially from the fresh market, there is a need to produce good quality blemish-free organic

potatoes.

Silver scurf, caused by the fungus Helminthosporium solani Durieu & Mont. is a little-

studied disease, particularly in organic potato production. At harvest, diseased tubers have grey

lesions on the periderm that appear silvery when moist. Under favorable storage conditions,

fungal sporulation makes tubers appear black and sooty. At later stages, the outer cell layers of

tubers slough off leading to tuber shrinkage and weight loss (Burke 1938; Jellis and Taylor

1974). Silver scurf has been gaining economic importance in the past three decades due to the

quality standards demanded by processing and fresh market consumers as well as the emergence

of H. solani isolates resistant to the broad-spectrum thiabendazole and thiophanate-methyl

fungicides (Errampalli et al. 2001; Geary et al. 2007). Initial infection of tubers by H. solani

occurs in the field either from the seed tuber (Jellis and Taylor 1977) or soil (Merida and Loria

1994). Disease spread occurs during storage, as air flow in storages aids in the dissemination of

fungal spores (Rodriguez et al. 1996).

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Colletotrichum coccodes (Wallr.) causes black dot, and the presence of small black

sclerotia on tubers, roots, and above- and below-ground stems gives the disease its common

name. Infected tubers may have gray-brown blemishes on the periderm, or less clearly delimited

discolored areas on which sclerotia may or may not be present (Jellis and Taylor 1974). The

pathogen can cause dark brown, deep sunken lesions on minitubers stored at 5 to 15 C for five

months (Glais and Andrivon 2004). Fresh weight losses of 5 to 10% occurred when tubers

naturally infected with C. coccodes were stored for 18 weeks (Hunger and McIntyre 1979). The

pathogen also infects roots, making them appear stringy when pulled out of the soil. Stolons

may also be attacked at any stage of tuber growth and the lesions can sever the stolon, leaving a

part of the stolon adhering to the tuber at harvest (Davis and Johnson, 2001). Foliar infections

manifest as dark brown to black lesions similar to those caused by Alternaria solani, but lacking

concentric rings (Johnson and Miliczky, 1993). Soil, infected seed tubers and air-borne inoculum

serve as sources of inoculum for this disease (Lees and Hilton 2003). The disease may be

overlooked as the symptoms appear late in the cropping season, coinciding with natural

senescence. In addition, symptoms on stems and stolons are similar to those of Rhizoctonia

canker, and on leaves similar to those of Verticillum wilt or early blight (Davis and Johnson,

2001).

Tuber symptoms of silver scurf and black dot are frequently confused owing to the

similarity of blemishes caused on the periderm, and because they may be present on the same

tuber (Jellis and Taylor, 1974). Depending on environmental conditions, both diseases can

increase during storage. Rodriguez et al. (1996) collected air-borne conidia of H. solani ranging

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from 0 to 12,000 conidia per day in storages at 4 C, and from 0 to 24,000 conidia per day in

storages at 10 C during one year of the study. They detected H. solani conidial production

throughout the storage season and also showed that greenhouse grown minitubers placed in the

storages could be infected by H. solani conidia. Although such extensive work has not been done

with black dot spread during storage, Mawson (1999) showed that preventing water

condensation during storage led to increased levels of black dot and decreased levels of silver

scurf on tubers. Latent infections by C. coccodes and H. solani have been documented (Glais-

Varlet et al. 2004; Tsror et al. 1999; Jellis and Taylor 1974) and may contribute to disease

increase during storage. To avoid continued underestimation of these diseases based on visual

symptoms alone, the frequency of latent infections on asymptomatic tubers, and the potential for

disease spread from such tubers, should be better understood. This information will help to

determine whether future efforts to manage these diseases in organic farming should focus on

reduction of inoculum spread or disease development in storage.

Organic potato production is unique from conventional production both in terms of

agronomic and plant protection practices. Fludioxonil, fludioxonil plus quintozene, and

azoxystrobin can reduce severity of silver scurf (Geary et al. 2007). However, these chemicals

cannot be used in organic potato production. Seed tuber treatments with salts acceptable in

organic production such as sodium carbonate and sorbic acid were not effective in controlling

silver scurf (Geary et al. 2007). In addition, to date, no potato cultivars with complete resistance

to silver scurf have been identified (Merida et al. 1994; Secor 1994; Thomas et al. 2005). Since

organic potato growers have few control strategies for silver scurf, we hypothesize that a high

percentage of organically grown potatoes, even if asymptomatic, are infected with H. solani

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and/or C. coccodes. With the availability of interspecific Solanum hybrids that have resistance to

multiple potato diseases (Jansky and Rouse 2003), resistance to silver scurf may be present in

potato germplasm suitable for breeding. Our objectives were to investigate the abundance of

asymptomatic tubers that harbor H. solani or C. coccodes in tubers harvested from organic potato

farms, and to identify potato germplasm that has resistance to silver scurf.

Materials and Methods

Tuber samples

Field trials were conducted during the 2011 growing season in conjunction with the on-farm

organic variety trials at three locations in Wisconsin: Antigo (A), Rosholt (B), and Cottage

Grove (C). At locations A and B, the experimental design was a randomized complete block

design with four replications at each location. The plots were hand planted and consisted of two

rows with 20 plants per row. Plots measured 1.8 m wide by 6.1 m long with 0.9 m row spacing.

At location C, a single replicate was planted, and cultivar location within the block was

randomized. The plots were hand planted, and consisted of single rows with 20 plants per row.

Plots measured 1.8 m wide by 6.1 m long with 0.9 m row spacing. Soil type, previous crop

history, and planting and harvest dates for each location are presented in Table 1. There were 31

cultivars planted at each location, including yellow, red, russet, and specialty cultivars. Of these

cultivars, 10 were selected that included multiple market classes and maturity groups, and are

commonly grown by conventional and/or organic farmers in Wisconsin (Table 2). Potatoes

were grown at these locations by farmers following organic management practices. The cultivars

planted had four different seed sources (Table 2) and all seed was grown under conventional

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management. Yield samples were harvested from measured lengths of each plot: 6.1 m lengths

for each plot row at location A, 3.05 m lengths of each plot row at location B, and 6.1 m lengths

of the single plot row at location C (data not shown). After harvest, the tubers from each plot

were bulked and thirty tubers of each cultivar were arbitrarily selected from one replicate plot at

each location. Sampled tubers were processed within a period of one week from the sampling

date, during which time the tubers were stored at 4 C. Each tuber was handled individually and

washed thoroughly under running tap water to remove soil deposits and then grouped as either

symptomatic or asymptomatic based on visual scoring. Tubers were considered to be

asymptomatic if they showed no signs of either H. solani or C. coccodes or symptoms of black

dot or silver scurf. Ten asymptomatic tubers were selected per cultivar per location for PCR and

tuber incubation assays, although in some cases there were fewer than ten asymptomatic tubers

available (Table 3). At location C there were fewer than ten asymptomatic tubers for some

cultivars. Hence, tubers that showed only signs of C. coccodes (sclerotia) but that lacked

symptoms of either disease and signs of H. solani were grouped as asymptomatic for silver scurf

but symptomatic for black dot (Table 3).

Tuber incubation assay

A humid chamber was made by placing a sterile paper towel in a 450 g deli container and

dispensing 10 ml of sterile distilled water onto the towel. A small rectangular portion on the side

of the container was cut out and the hole was sealed with Micropore 3M tape to facilitate

aeration. Each symptomatic (up to 15 tubers per cultivar per location) or asymptomatic tuber was

then placed in an individual deli container, and incubated in the dark at 24 C for a period of three

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40

weeks. Asymptomatic tubers were number coded so that the PCR and tuber incubation assays

were always conducted on the same tuber. At the end of the incubation period, the entire tuber

surface was examined for the presence of conidiophores and conidia of H. solani and sclerotia of

C. coccodes.

PCR assay

A one centimeter diameter circle of periderm was arbitrarily collected from each asymptomatic

tuber using a cork borer, excised and chopped into small pieces with a sterile blade and used for

DNA extraction. DNA was extracted from this tissue using the FastDNA SPIN Kit for Soil (MP

Biomedicals, Solon, OH) following the manufacturer’s protocol except that the homogenization

of the sample was carried out in a mini beadbeater (Biospec Products, Bartlesville, OK) for two

minutes. To detect H. solani, two sets of primer pairs (First-round: Hs1F1/Hs2R1; Nested:

Hs1NF1/Hs2NR1) developed by Cullen et al. (2001) were used to amplify ITS regions.

Sequences designed by Cullen et al. (2002) were used to amplify C. coccodes (First-round:

Cc1F1/Cc2R1; Nested: Cc1NF1/Cc2NR1). Each PCR reaction consisted of one µl of undiluted

potato DNA extract, 0.3 µM of each primer, and 1x PCR Master Mix (Promega Corporation,

Madison, WI) in 25 µl of total volume. One µl of the first-round PCR product was used for

nested PCR. Amplification of H. solani was carried out with cycling parameters slightly

modified from Cullen et al. (2001): 95 C for 150 s, followed by 30 cycles of 95

C for 45 s,

annealing at 66 C (first-round) and 60

C (nested) for 60 s, extension at 72

C for 90 s, and a final

elongation at 72 C for 5 min. All reactions were run in a Techne TC-512 thermocycler. H. solani

or C. coccodes DNA was used as the positive control and the negative control was nuclease free

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water (Promega Corporation, Madison, WI). PCR products were analyzed by electrophoresis on

2% agarose gel in 1X TAE buffer, stained with SYBR Safe DNA gel stain (Life Technologies,

Carlsbad, CA) and visualized under a UV transilluminator (Universal Hood II: Bio-Rad,

Hercules, CA). An asymptomatic tuber was considered positive for H. solani or C. coccodes if

the pathogen was detected by either PCR or the tuber incubation assay.

Minituber inoculation assay

Fourteen lines of minitubers were screened in two separate experiments (Table 4). Minitubers

grown in a nutrient film technique system were washed, surface disinfested with calcium

hypochlorite (0.5% active chlorine) for five minutes and rinsed twice in sterile water. B-AC-

16A isolate of H. solani recovered from an infected potato was grown on V8 medium containing

177 ml V8 juice, 20 g agar and 3 g CaCO3 per liter (Goth and Webb 1983). Cultures were

incubated in the dark at 24 C and a conidial suspension was prepared by scraping a four-week old

colony in a small volume of sterile water. The conidial suspension concentration was determined

with a hemacytometer and adjusted to the required concentration (105 spores per milliliter) by

dilution with 0.1% water agar. Lines C287, Dark Red Norland, Early Epicure and Epicure

Banana were screened in both experiments. A minimum of ten minitubers per line were

arbitrarily selected and inoculated with H. solani conidial suspension until wet using an

aspirator. Up to three inoculated minitubers of the same line were placed together in individual

humid chambers, which were designed as described earlier for the tuber incubation assay. Three

minitubers per line included as negative controls were sprayed with 0.1% water agar. After one

month, each minituber was placed in a tube containing 15ml of deionized water plus 10 µl of

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10% Tween20 and washed for five minutes on a laboratory shaker to extract conidia. The

minitubers were then removed from the suspension and weighed. To standardize across the

samples, the volume in each tube was adjusted to 20 ml with deionized water. A 10 µl portion of

this suspension was filtered through a membrane filter (Munck and Stanosz 2009) and conidia of

H. solani were counted with the aid of a dissecting microscope. The mean number of conidia

obtained from three such readings was adjusted for the total volume of water in which the

minituber was washed and divided by tuber weight to estimate the number of conidia extracted

per gram of minituber.

Statistical analyses

Tubers asymptomatic for silver scurf and black dot at harvest (Table 2), and the asymptomatic

tubers positive for H. solani and C. coccodes after performing tuber incubation and PCR assays

(Table 3) were binary responses. Hence, Fisher’s exact tests were performed to compare the

proportion of asymptomatic tubers among cultivars within a location and also to compare among

locations within a cultivar. PCR and tuber incubation assays were considered as two ratings

assessing an asymptomatic tuber, hence the inter-rating agreement between the assays was

measured using the kappa coefficient. The kappa coefficient lies between -1 and 1, where values

less than zero indicate less agreement than expected by chance and values greater than zero

indicate more agreement than expected by chance (Cohen 1960). Interpretation of the kappa

coefficient was made using an arbitrary benchmark scale (Landis and Koch 1977). For minituber

inoculation assays, significant differences were noted between the two experiments. Hence, a

linear model was fit treating the lines that were common in both experiments as separate lines.

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Comparisons among lines were made using ANOVA, with means separated when necessary

using Fisher’s LSD. All statistical analyses were performed with R software version 2.14.1.

Statistical significance was set at P < 0.05.

Results

Disease incidence at harvest

Ten potato cultivars were assayed for the incidence of black dot and/or silver scurf after harvest.

At each location, significant differences were observed among cultivars for the number of

asymptomatic tubers for silver scurf and/or black dot at harvest (P < 0.05). However, none of the

cultivars assayed were resistant to these diseases (Table 2). Barring three cultivars (Purple

Majesty, Freedom Russet and Dark Red Norland) from location A, and three cultivars (Freedom

Russet, Keuka Gold and Satina) from location B, more than 50% of the sampled tubers for each

cultivar were symptomatic for silver scurf and/or black dot. Significant differences (P < 0.05) for

the number of asymptomatic tubers at harvest were found among locations except for

Adirondack Blue and Adirondack Red (Table 2). The cultivar Freedom Russet was an extreme

example of this variability. All assayed Freedom Russet tubers from location B were

asymptomatic, while only 30% from location C appeared healthy.

Tuber incubation and PCR assays

Asymptomatic tubers were assayed for H. solani and C. coccodes by tuber incubation and PCR

assays to determine if these tubers carried either pathogen (Table 3). Tubers were considered

infected if they were positive for the pathogen by either assay. The majority of asymptomatic

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tubers assayed from these fields were infected with one or both of the pathogens: 75% of the

asymptomatic tubers assayed were positive for H. solani and 94% were positive for C. coccodes

(Fig. 1). The proportion of asymptomatic tubers that were positive for H. solani following tuber

incubation and PCR assays (Table 3), differed significantly among cultivars at each of the three

locations (P < 0.05). Only Adirondack Blue, Superior and Yukon Gold showed significant

differences among locations for incidence of H. solani on asymptomatic tubers. It is interesting

to note that tubers of Freedom Russet from location B showed no lesions at harvest (Table 1), yet

all tubers were infected with H. solani (Table 3). No significant differences were found among

locations or cultivars for C. coccodes (Table 3).

For both pathogens, among most of the cultivars and locations tested, there was slight to

fair agreement between the tuber incubation and PCR assays (Table 5). A moderate agreement

for H. solani was observed with Dark Red Norland and Yukon Gold. Location B had a

substantial agreement between the two H. solani assays. Tuber incubation and PCR assays did

not agree in 37% and 26% of the asymptomatic tubers assessed for H. solani and C. coccodes

respectively (Fig. 1). In samples where the two assays did not match, the tuber incubation assay

resulted in more positives for H. solani (29%) than C. coccodes (8%), while the PCR assay

detected more positives for C. coccodes (18%) than H. solani (8%).

The majority of the tubers that were assayed were infected with both H. solani and C.

coccodes. Both pathogens occurred on the same tuber in 69% of symptomatic and 65% of

asymptomatic tubers assessed (Fig. 2). In many cases, conidiophores of H. solani and sclerotia of

C. coccodes were observed in close proximity when the tuber periderm was viewed with the aid

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of a dissecting microscope (Fig. 3). Co-occurrence of these two fungi was reported earlier

(Nitzan et al. 2005), but there is essentially no information available on how these fungi may

interact on tuber surfaces.

Minituber inoculation assays

In the minituber inoculation assays, significant differences were observed among lines for H.

solani sporulation (Table 4). C287 consistently had the lowest conidial count per gram of

minituber, and Peruvian Blue had the highest level of sporulation. Epicure Banana from one

experiment had low sporulation that was not significantly different from C287, but it produced

higher number of conidia (24806 ± 3764) per gram of minituber in the other experiment,

suggesting variability either within the line or the experiments. However, since C287 supported a

low level of sporulation in both experiments, it appeared to have partial resistance to silver scurf.

Discussion

A high incidence of silver scurf and black dot was found across locations and cultivars

with 75% and 94% of asymptomatic tubers assessed positive for H. solani and C. coccodes

respectively. These results, coupled with the difficulty of distinguishing between the symptoms

of silver scurf and black dot, especially on blue-skinned cultivars, indicate that visual assessment

of the tubers at harvest for silver scurf and black dot symptoms is not predictive of pathogen

incidence. Since high incidences of both fungi were observed on asymptomatic tubers, the tuber

infections might have occurred shortly before harvest, or, alternatively infections may remain

latent in developing tubers. Consistent with these results, others have reported tuber latent

infections by C. coccodes (Glais-Varlet et al. 2004 and Tsror et al. 1999). Likewise, Jellis and

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Taylor (1974) did not observe symptoms of silver scurf until three to five weeks after infection.

Our results have direct implications for seed potato production. Black dot (Johnson et al. 1997)

and silver scurf (Geary and Johnson 2006) incidence increases with each year that potatoes are

replanted. Thus, organic farmers would likely face a very high incidence of both silver scurf and

black dot if they replant the tubers they harvested.

Since initial information about the presence of either pathogen on asymptomatic tubers

prior to our assays is unobtainable, this limited our ability to judge whether the tuber incubation

or PCR assay is more accurate. The fast, but relatively expensive PCR assay was in slight to fair

agreement with the slower, but inexpensive tuber incubation assay. The periderm samples for the

PCR assay were taken arbitrarily from each tuber and it is unlikely that these pathogens are

distributed evenly over the tuber surface at the time of harvest. Indeed, previous work showed

that C. coccodes is isolated at greater frequency from tuber stem ends than from either lateral

sections or bud ends (Johnson et al. 1997). These pathogens may also be present on the tuber but

lack sclerotia or fruiting bodies after incubation, which would result in low agreement between

the assays. Both scenarios likely occurred in this study since H. solani was detected more

frequently by the tuber incubation assay, suggesting that infected regions of some tubers were

not assayed, and C. coccodes was detected more frequently by PCR, suggesting that sclerotia did

not form consistently in our tuber incubation assay.

No cultivar has yet been identified that is completely resistant to silver scurf (Merida et

al. 1994; Secor 1994; Thomas et al. 2005). However, partial resistance to silver scurf was

reported in wild Solanum species such as S. demissum, S. chacoense, S. acaule, S. stoloniferum,

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S. oxycarpum and S. hondelmannii (Rodriguez et al. 1995). Previous studies indicate that tuber

assays are likely to give a more accurate assessment of cultivar resistance to silver scurf than

field tests, since disease severity increases with the length of time that tubers remain exposed to

the pathogen in the field and this time period varies between early- and late-maturing cultivars

(Merida et al. 1994). In this study, minitubers were used instead of field-grown potatoes, because

when surface disinfested field-grown tubers were inoculated and incubated, the majority of the

samples were lost due to tuber decay pathogens. Minitubers grown by the nutrient film technique

remain nearly pathogen free, which minimized tuber decay.

Screening for resistance was performed based on the ability of H. solani to sporulate on

the tuber surface, a measure of resistance for silver scurf used previously by Merida et al. (1994)

and Rodriguez et al. (1995). Of the 14 lines examined, only C287 had consistently low spore

counts in both minituber inoculation experiments. C287, a diploid (2n = 2x = 24) interspecific

hybrid line has wild Solanum species in its parentage [Female parent: (US-W730 × (S.

berthaultii × S. tarijense) and male parent: (US-W730 × S. tarijense)]. C287 is resistant to

Verticillium wilt and other surface blemish diseases such as common scab and black scurf

(Jansky and Rouse, 2003). The results suggest that C287 may also have partial resistance to

silver scurf, which may be usefully transferred to commercial cultivars as has been done for

common scab (Murphy et al. 1995) and Verticillium wilt (Frost et al. 2006) resistance.

The high prevalence of H. solani and C. coccodes on organic potatoes and the lack of

tuber resistance in currently available cultivars suggest that future research should also focus on

preventing disease development during the growing season and in storage. Field data indicate a

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complex relationship between pathogen incidence, disease expression, cultivar, and location.

Although the percentage of asymptomatic tubers (Table 2) and the frequency of H. solani

infections (Table 3) in asymptomatic tubers differed significantly between some cultivars and

locations, no clear trends were seen when comparing cultivars and locations. Further research

into the influence of soil characteristics and management choices on disease development during

the growing season is needed. For organic growers with operational flexibility and a ready

market, harvesting and marketing immediately at plant maturity may be an adequate control for

silver scurf, since disease severity increases if tubers remain in the field (Merida et al 1994).

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CIP (International Potato Center). Potatoes: facts and figures. http://cipotato.org/ potato/facts.

Accessed 14 December 2012.

Cohen, J. 1960. A coefficient of agreement for nominal scales. Educational and Psychological

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Cullen, D. W., A. K. Lees, I. K. Toth, and J. M. Duncan. 2001. Conventional PCR and real-time

quantitative PCR detection of Helminthosporium solani in soil and on potato tubers.

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Table 1. Soil type, previous crop, and planting and harvest dates for each sampling locationz

Aspect Location A Location B Location C

Soil type Langlade silt loam Sandy loam Dodge silt loam

Planting date 30 May 2011 20 May 2011 16 May 2011

Harvest date 11 October 2011 20 September 2011 17 October 2011

Previous crop Oats and Clover Alfalfa and Rye Popcorn

z Sampling locations A, B and C were farms located in Antigo, Rosholt, and Cottage Grove, WI

respectively.

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Table 2. Percentage of potato tubers asymptomatic for both silver scurf and black dot after harvest from three locationsx in Wisconsin

% Asymptomatic tubers

Seed Seed __________________________________________

Cultivar sourcey certification Maturity group Location A Location B Location C P-value

z

Market class: Blue

Adirondack Blue D Certified Medium 33.3 (30) 50.0 (30) 26.7 (30) 0.155

Purple Majesty R Certified Medium 82.1 (28) 31.0 (29) 10.0 (30) <0.001

Market class: Red

Adirondack Red D Certified Early to Medium 26.7 (30) 50.0 (30) 26.7 (30) 0.089

Chieftain C Certified Medium 20.7 (29) 40.0 (30) 0.0 (30) <0.001

Dark Red Norland L Foundation Early 60.0 (30) 0.0 (30) 10.0 (30) <0.001

Market class: Russet

Freedom Russet L Foundation Medium to late 65.4 (26) 100.0 (30) 30.0 (30) <0.001

Market class: Yellow

Keuka Gold D Certified Medium to late 46.7 (30) 63.3 (30) 10.0 (30) <0.001

Satina C Certified Medium early 30.4 (24) 53.3 (30) 23.3 (30) 0.047

Superior L Foundation Early 21.4 (28) 46.7 (30) 0.0 (30) <0.001

Yukon Gold L Foundation Early 26.7 (30) 36.7 (30) 10.0 (30) 0.045

P-valuez

<0.001 <0.001 0.002

x Sampling locations A, B and C were organic farms located in Antigo, Rosholt, and Cottage Grove, WI respectively. Numbers in

parentheses indicate sample size.

y Seed source: D= David Gallenberg farm, WI; C = Childstock farm, NY; L= Lelah Starks farm, WI; R= Rockey Farms, CO.

z Statistical analysis was carried out in R (Version 2.14.1) using Fisher’s exact test at 5% significance level.

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Table 3. Percentage of asymptomatic tubers positive for Helminthosporium solani and Colletotrichum coccodes after performing PCR

and tuber incubation assays

H. solani C. coccodes

_____________________________________________ _______________________________________________

Cultivarx Location

y

_______________________________________________________________________________________________

A B C P-valuez A B C P-value

z

____________________________________________________________________________________________________________

ADB 80.0 (10) 10.0 (10) 50.0 (10) 0.009 90.0 (10) 90.0 (10) 90.0 (8) 1.000

PMJ 60.0 (10) 33.3(9) 85.7 (7) 0.115 100.0 (10) 100.0(9) 71.4 (3) 1.000

ADR 75.0 (8) 100.0 (10) 100.0 (8) 0.172 87.5 (8) 90.0 (10) 62.5 (8) 0.458

CFT 83.3 (6) 100.0 (10) 100.0 (9) 0.240 100.0 (6) 100.0 (10) NA 1.000

DRN 30.0 (10) NA 66.7 (9) 0.179 100.0 (10) NA 100.0 (3) 1.000

FDR 100.0 (10) 100.0 (10) 100.0 (9) 1.000 100.0 (10) 80.0 (10) 100.0 (9) 0.310

KKG 90.0 (10) 80.0 (10) 100.0 (10) 0.754 80.0 (10) 100.0 (10) 100.0 (2) 0.567

STN 100.0 (7) 60.0 (10) 90.0 (10) 0.143 100.0 (7) 100.0 (10) 90.0 (4) 0.190

SUP 83.3 (6) 0.0 (10) 90.0 (10) <0.001 100.0 (6) 100.0 (10) NA 1.000

YKG 100.0 (8) 30.0 (10) 100.0 (10) <0.001 100.0 (8) 100.0 (10) 100.0 (3) 1.000

P-valuez 0.004 <0.001 0.004 0.483 0.507 0.410

x Cultivar: ADB= Adirondack Blue, PMJ = Purple Majesty, ADR = Adirondack Red, CFT = Chieftain, DRN = Dark Red Norland,

FDR= Freedom Russet, KKG = Keuka Gold, STN = Satina, SUP = Superior and YKG = Yukon Gold.

y Sampling locations A, B and C were organic farms located in Antigo, Rosholt, and Cottage Grove, WI respectively. Numbers in

parentheses indicate sample size.

z Statistical analysis was carried out using Fisher’s exact test at 5% significance level.

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Table 4. Spore production on fourteen potato minituber lines inoculated with Helminthosporium

solani

Line Number of conidia extracted per gram of minituber

Peruvian Blue 49283x ± 6437.7 a

Epicure Banana-Ay 24806 ± 3763.9 b

Peanut 23846 ± 6363.6 bc

Early Epicure-A 24307 ± 5815.1 bcd

Dark Red Norland-A 17793 ± 3845.4 bcd

Nosebag 12744 ± 2950.9 bcde

Superior 10957 ± 1854.3 cdef

Early Epicure-Bz 11717 ± 1882.1 def

Dark Red Norland-B 11892 ± 2456.1 def

White Cobbler 9920 ± 2633.9 ef

Scotia Blue 9728 ± 2278.9 ef

Trina 8514 ± 1596.4 ef

Pike 7021 ± 1372.3 ef

Round Blue Andean 10387 ± 6066.8 f

Tundra 3775 ± 1235.0 g

Epicure Banana-B 2360 ± 410.9 gh

C287-B 2031 ± 403.7 hi

C287-A 1216 ± 292.5 i

x Means (±SE) followed by the same lowercase letters are not significantly different within a

column (α = 0.05).

y Lines followed by the letter A are sporulation data from experiment 1

z Lines followed by the letter B are sporulation data from experiment 2

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Table 5. Kappa coefficient values showing agreement between tuber incubation and PCR assays

for detecting Helminthosporium solani and Colletotrichum coccodes from asymptomatic tubers

Kappa coefficienty

_______________________________________________

Cultivar / Locationz H. solani C. coccodes

Cultivar:

Adirondack Blue 0.11 0.24

Purple Majesty 0.21 0.0

Adirondack Red 0.16 0.19

Chieftain 0.04 0.0

Dark Red Norland 0.55 0.0

Freedom Russet 0.0 0.23

Keuka Gold 0.09 0.30

Satina 0.17 0.01

Superior 0.27 0.0

Yukon Gold 0.41 -0.07

Location:

A 0.24 0.25

B 0.62 0.09

C 0.03 0.30

y Kappa coefficients (Landis and Koch 1977): 0.81 to 1.00 = almost perfect agreement, 0.61 to

0.80 = substantial agreement, 0.41 to 0.60 = moderate agreement, 0.21 to 0.40 = fair agreement,

0.00 to 0.20 = slight agreement, <0.00 = poor agreement.

z Sampling locations A, B and C were organic farms located in Antigo, Rosholt, and Cottage

Grove, WI respectively.

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Figure 1. Percentage of asymptomatic tubers positive or negative for Helminthosporium solani

and Colletotrichum coccodes by tuber incubation (T) and PCR (P) assays. (T0P0) Negative for

both tuber incubation and PCR assays. (T1P1) Positive for both tuber incubation and PCR

assays. (T1P0) Positive for tuber incubation and negative for PCR assay. (T0P1) Negative for

tuber incubation and positive for PCR assay.

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Figure 2. Percentage of tubers positive or negative for Helminthosporium solani (Hs) and

Colletotrichum coccodes (Cc) by either tuber incubation or PCR assay for asymptomatic tubers

and by tuber incubation assay for symptomatic tubers. (Hs1Cc0) Positive for H. solani and

negative for C. coccodes. (Hs0Cc1) Negative for H. solani and positive for C. coccodes.

(Hs0Cc0) Negative for both H. solani and C. coccodes. (Hs1Cc1) Positive for both H. solani and

C. coccodes.

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Figure 3. Conidia and conidiophores of Helminthosporium solani (left side) and sclerotia of

Colletotrichum coccodes (right side) observed in close proximity on a potato tuber periderm

under a dissecting microscope.

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CHAPTER 2

A microfluidic assay for identifying differential responses of plant and human fungal

pathogens to tobacco phylloplanins

The structure of this chapter has been formatted with submission to PeerJ journal in mind:

Mattupalli C, Spraker JE, Berthier E, Charkowski AO, Keller NP, Shepherd RW. A microfluidic

assay for identifying differential responses of plant and human fungal pathogens to tobacco

phylloplanins. PeerJ.

Some of the data published in this chapter was done in Dr. Keller and Dr. Shepherd lab.

Berthier E fabricated the microfluidic plates and Spraker JE was responsible for the Aspergillus

assays.

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Abstract

Phylloplanins are defensive glycoproteins secreted onto leaf surfaces by trichome bearing plants

such as tobacco. They are of interest because of their anti-microbial properties, but like other

natural product bioactives, the assessment and screening of phylloplanin biological activity is

impeded by limited availabilities of active compounds. Here we report an inexpensive

microfluidic approach that requires only a few microliters of Nicotiana tabacum (tobacco)

phylloplanins (Nt-phylloplanins) to assess spore germination inhibition of plant and human

fungal pathogens. Spores of Colletotrichum coccodes and Aspergillus fumigatus treated with Nt-

phylloplanins did not germinate at 48 and 30 hours post treatment respectively. Nt-phylloplanins

transiently inhibited spore germination of Fusarium sambucinum, but had no detectable activity

against Alternaria alternata or Verticillium albo-atrum, demonstrating differential sensitivity of

fungi to Nt-phylloplanins. Inhibition of spore germination of the human pathogen A. fumigatus

by Nt-phylloplanins also reveals a new venue for medicinal antifungal drug discovery that has

not been explored previously.

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Introduction

The phyllosphere or microorganismal habitat of leaf surfaces is a complex and inhospitable

habitat exposed to continuous fluctuations in physical environment (Hirano and Upper, 2000). It

is inhabited by diverse genera of microbes including potential pathogens, that must also endure

the array of preformed and induced plant defences of the leaf surface or phylloplane (Lindow

and Brandl, 2003; Shepherd and Wagner, 2012). While many well-studied plant defenses and

defensive processes are triggered by pathogens as they access the plant interior or penetrate the

epidermal cell wall (Chisholm et al. 2006; Bent and Mackey 2007; Underwood and Somerville

2008), it is evident that sizable fortifications are also present at the outermost layers of the air-

plant interface, and consist mainly of secreted plant-produced biochemicals that act directly

against pathogens to hinder or obstruct their progress (Kennedy et al. 1992; Karamanoli and

Lindow 2006; Shepherd and Wagner 2007).

The leaf surface is composed of a pavement of epidermal cells interspersed with stomata,

trichomes and other leaf surface structures (Hirano and Upper, 2000). Trichomes are epidermal

protuberances developing outwards on the surface of various plant organs (Werker, 2000) and

can be broadly classified as simple/non-glandular and glandular secreting types. Glandular

secreting trichomes are present in about 30% of vascular plants and secrete secondary

metabolites such as terpenoids and phenylpropanoids that can provide disease and pest resistance

(Kelsey et al. 1984; Wagner et al. 2004). Shepherd et al. (2005) discovered that plants produce

and secrete to their leaf surfaces antifungal plant proteins termed phylloplanins. Native

phylloplanins of Nicotiana tabacum (Nt-phylloplanin) can be collected by gently agitating

undamaged leaves in water, lyophilizing the resulting solution and resuspending the lyophilized

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product in sterile water. With SDS-PAGE, Nt-phylloplanin is visible as four bands with

molecular masses of 16 kDa, 19 kDa, 21 kDa, and 25 kDa. The four bands all have the same N-

terminal amino acid sequence and are believed to arise from glycosyl additions to a single 13

kDa core polypeptide encoded by the phylloplanin gene (Accession AY705384). Homologues of

this gene have been reported to exist and be expressed only in N. tabacum trichome glands

(Harada et al. 2010).

Analyses of the phylloplanin promoter using reporter genes indicate that it directs expression

solely in short glandular secreting trichomes, thus revealing a highly specialized mechanism for

localization and delivery of proteins to leaf surfaces (Shepherd et al., 2005). Short glandular

trichomes are believed to continually secrete phylloplanins onto the leaf surface, although factors

such as leaf age might influence secretion. After leaf surface proteins are removed, their levels

are renewed within one week (Kroumova et al. 2007). Native Nt-phylloplanins have inhibitory

activity against spore germination and hyphal growth of the basidiomycete Rhizoctonia solani,

and the ascomycete Pyricularia oryzae (King et al. 2011). A recombinant 13 kDa Nt-

phylloplanin polypeptide produced in Escherichia coli inhibited spore germination of

Peronospora tabacina, causal agent of blue mold disease in tobacco (Shepherd et al. 2005).

Mechanized methods for harvesting phylloplane proteins on a large scale are not yet available.

This impeded our ability to screen for their antimicrobial properties and led us to explore for

techniques that are compatible with low volume of reagents. Microfluidic platforms are reputed

for temporal and spatial control of microenvironments concurrently allowing assays to be

performed in micro-channels using smaller volumes of samples (Paguirigan and Beebe, 2008).

To date, these microsystems have been successfully used for applications in drug discovery, cell

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culturing and characterization of fungal secondary metabolites (Dittrich and Manz, 2006; Gao et

al. 2011; Berthier et al. 2013). Here, we developed a simple microfluidic channel array that

enabled the in vitro study of antifungal effects of Nt-phylloplanin on plant and, for the first time,

human pathogens. We verified the hypothesis that Nt-phylloplanins have inhibitory effects on a

subset of fungi that have not been studied earlier and suggest that these molecules be considered

for development in medicinal venues.

Materials and Methods:

Nt-phylloplanin collection: Tobacco (Nicotiana tabacum TI 1068) plants were grown in 15.2-

centimeter diameter plastic pots in a greenhouse. MetroMix (Sun Gro Horticulture, BC, Canada)

was used as potting soil and a slow releasing fertilizer (Osmocote) was applied at the rate of 7.5

g per each pot. Nt- phylloplanins were collected by washing fully expanded leaves (undetached

from the plant) in 200 ml of distilled water with gentle agitation for 15 seconds. Leaves with

visible damage were not washed to avoid contamination from leaf sap. The wash solution was

then passed through a Miracloth (EMDMillipore, MA, USA) to filter debris and lyophilized to

dryness. The lyophilized powder was resuspended in 1 ml of sterile Milli-Q water, and

centrifuged at 12,000 × g for 5 minutes at 24 °C. The supernatant was filtered using a PVDF

membrane syringe filter (13 mm / 0.22 µm; Fisher Scientific, PA, USA) and stored at 4oC until

further use. Proteins were separated from the lyophilized product by sodium dodecyl sulfate –

polyacrylamide gel electrophoresis (SDS-PAGE) using a Mini-PROTEAN Tetra cell

electrophoresis system (Bio-Rad, Hercules, CA) and visualized with Coomassie Brilliant Blue.

Nt-phylloplanin concentration of 1 mg mL-1

was used in this study and the protein concentration

was estimated using BCA protein assay kit (Thermo Scientific Pierce, Rockford, IL).

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Proteinase treatment: Proteinase K immobilized on Eupergit C beads (200mg, Sigma-Aldrich,

MO, USA) was placed into mini-spin filters and 500 µL of leaf water wash samples were added

to these filters. The filters were then placed in a 1.5 mL microcentrifuge tube and incubated at

37oC overnight. Flow-through was collected by centrifuging the tubes at 5000 g for 10 minutes

which was again incubated at 95oC for 30 minutes, followed by a second centrifugation at 5000g

for 5 minutes to pellet any precipitate.

Fungal cultures: Alternaria solani (Asol11), Verticillium albo-atrum (AC186), Fusarium

sambucinum (PD1), Colletotrichum coccodes (DRN-MK), Aspergillus fumigatus (Af293) were

the fungal pathogens used for in vitro testing of phylloplanin activity. A. fumigatus was grown on

glucose minimal media (GMM) agar at 37ºC for 3 days. A. fumigatus spore suspensions were

made by applying 10 ml of sterile 0.01% Tween-20 solution to the plate and subsequently

agitating conidiophores with a cell spreader. A. solani was grown on clarified V8 media, F.

sambucinum on quarter strength PDA, C. coccodes and V. albo-atrum on full strength PDA.

Spore suspensions were prepared by scraping 10 to 15 day old colonies into a small volume of

sterile water. Spore suspensions were quantified using a haemocytometer and appropriate

dilutions were made with sterile ddH2O.

Microfluidic channel fabrication: Microfluidic channel assays were fabricated using a soft-

lithography method (Fig. 1). The mold was fabricated from a 15 cm diameter silicon wafer upon

which was spun 2 layers of SU8 epoxy photoresist, the first was set to a thickness of 150 µm and

the second to 500 µm. Masks for patterning the designs in the photoresist were drawn on Adobe

Illustrator (Adobe, USA) and printed at Imagesetter Inc (Madison, WI, USA). The soft-

lithography process was performed by mixing polydimethylsiloxane (PDMS) in a ratio of 1:10,

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degassing the mix in a vacuum desiccator, and pouring onto the mold. Following the application

of PDMS, a transparent sheet, a rectangle of silicone rubber, a rectangle of rigid polyester, and 4

kg of weights were placed on the mold. The molds were baked at 80˚C for 4 hours after which

the PDMS was removed from the mold and bonded via to a glass microscope slide following an

oxygen plasma surface activation treatment (FEMTO, Diener, Germany).

Spore Germination Assays: The spore germination assay was conducted in microfluidic channels

prepared according to the described method (Fig. 1). Each micro-channel was loaded with 20 µl

or 15 µl (A. fumigatus) total fluid volume, containing 10 µl or 13.5 µl (A. fumigatus) of

phylloplanin solution and 10 µl or 1.5 µl (A. fumigatus) spore suspension, premixed in

microcentrifuge tubes. Final spore concentration in each micro-channel was 4 × 102 for A. solani,

1.5 × 103 for A. fumigatus, 2 × 10

3 for C. coccodes and F. sambucinum, and 2 × 10

4 for V. albo-

atrum. Controls of proteinase treated phylloplanin solution were used and loaded in equal

volume to the treatment channels. Channels were incubated at room temperature in a moist

chamber to prevent evaporation from the channels. Germination rates were quantified

microscopically at 6, 12, 24, 30 or 48 hours post inoculation by counting 200 spores per

treatment. A spore was considered germinated if the length of the germ tube exceeds half the

length of the spore or multiple germ tubes developed. Each treatment had three biological

replicates.

Results and Discussion:

Due to the large volumes of water used for collecting phylloplanins from leaf aerial surfaces, the

samples first needed to be lyophilized to yield a concentrated product highly enriched in protein

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but very limited in volume and quantities. This aspect of sample recoverability was a significant

bottleneck in our ability to perform a large number of assays. Here, we developed a microfluidic

channel array enabling the efficient study of phylloplanins requiring only microliters of reagents

(Fig. 1). The accessible microscale assay leveraged simple pipette-based pumping methods

(Berthier and Beebe, 2007) allowing simple fabrication and use of the channels. The channels

positioned on a microscope slide allowed straightforward use and imaging at known locations

and devoid of typical meniscus effects that occur in microwell plates. Using this microscale

array, we performed tens of assays simultaneously with low volumes of Nt-phylloplanins, and

demonstrated that this assay will be broadly useful in further large-scale screening of natural

product bioactives against microorganisms.

Earlier studies indicated the antimicrobial effects of Nt-phylloplanins (Shepherd et al. 2005;

Kroumova et al. 2007; King et al. 2011) against an oomycete and two fungal pathogens. Here,

we show that the antimicrobial activity of Nt-phylloplanins is specific to some fungal species,

suggesting that fungal resistance mechanisms exist. Differential responses to antimicrobial

proteins by pathogens, which are likely driven by host-pathogen co-evolution, is not uncommon

(Segura et al. 1998; Segura et al. 1999; Van Damme et al. 1999; Vigers et al. 1992). For instance,

a 30 kDa protein isolated from leaves of Engelmannia pinnatifida strongly inhibited the growth

of plant pathogens such as Fusarium oxysporum, Alternaria solani, Gaeumannomyces graminis,

but not the human pathogen, Candida albicans (Huynh et al. 1996).

The inhibition of spore germination by tobacco leaf wash samples was tested in vitro against four

fungal plant pathogens (Alternaria solani, Verticillium albo-atrum, Fusarium sambucinum,

Colletotrichum coccodes), and a human pathogen (Aspergillus fumigatus). Nt-phylloplanins

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strongly inhibited C. coccodes and A. fumigatus, transiently inhibited Fusarium sambucinum,

and were inactive against A. solani and V. albo-atrum, thereby indicating a spectrum of pathogen

sensitivity (Table 1). C. coccodes spores treated with tobacco leaf wash sample did not germinate

at 48 hours post inoculation when treated with 1 mg mL-1

Nt-phylloplanins (Fig. 2). The LD50 for

Nt-phylloplanins against C. coccodes spores was approximately 0.1 mg mL-1

(Fig. 3) C.

coccodes spores failed to germinate when they were treated with tobacco leaf wash sample

overnight and washed twice with sterile water, thus the effect of Nt-phylloplanins was

irreversible (Fig. 2).

Few of the A. fumigatus spores germinated when incubated with tobacco leaf wash samples at

the Nt-phylloplanin concentrations tested, and this effect persisted for at least 30 hours post

inoculation (Fig. 4). The inhibition of A. fumigatus (Fig. 4) and C. coccodes (Fig. 2) spore

germination was eliminated when proteins were digested with proteinase-K, suggesting that Nt-

phylloplanins were necessary for inhibition.

Unlike C. coccodes and A. fumigatus, F. sambucinum spore germination was only transiently

inhibited by Nt-phylloplanins. F. sambucinum spores germinated in negative controls at 12 hours

post inoculation (hpi), but not when treated with Nt-phylloplanins. However, this inhibitory

effect started to degrade at 16 hpi and by 24 hpi no inhibitory effect was evident and extensive

hyphal growth occurred (Fig. 5).

The mechanism of action of Nt-phylloplanins remains unknown. Since, Nt-phylloplanins inhibit

C. coccodes and A. fumigatus (this work), Rhizoctonia solani and Pyricularia oryzae (King et al.

2011), but only transiently inhibit F. sambucinum and are ineffective against Alternaria solani

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and, Verticillium albo-atrum, this suggests that host-pathogen co-evolution may select for Nt-

phylloplanin resistance in some fungi. Knowledge of fungal resistance mechanisms and the ease

of acquisition of resistance to phylloplanins would aid in decisions on whether to develop these

antimicrobial proteins as a natural product for use in plant / human protection.

Aspergillus fumigatus is the most common Aspergillus species to cause human infections

(Dagenais and Keller, 2009) and it causes a spectrum of diseases such as allergic

bronchopulmonary aspergillosis, aspergilloma and invasive aspergillosis, depending on the

host’s immune status (Latge, 1999; Dagenais and Keller, 2009; Zaas and Alexander, 2009).

Currently there are four classes of antifungal agents with activity against Aspergillus: the

polyenes such as amphotericin B and nystatin; the triazoles such as itraconzole, voriconazole and

posaconazole; the echinocandins such as caspofungin and micafungin; and the allylamines such

as terbinafine (Chamilos and Kontoyiannis, 2005). However, treatments involving amphotericin

B and triazoles may cause nephric and hepatic toxicity (Wingard et al., 1999; Tan et al., 2006).

Lipid-based formulations of amphotericin B have reduced the incidence of nephrotoxicity, but

they do so at a significantly increased drug acquisition cost (Pound et al., 2011). Also, in some

countries such as UK, up to 15% of A. fumigatus strains have been found to be resistant to

various azoles (Mayr and Lass-Florl, 2011). This highlights the ongoing requirement for new

anti-fungal agents for this pathogen (Denning and Hope, 2010) and antimicrobial proteins are

now being explored as potential therapeutics. Over 1000 antimicrobial peptides are now known

to be produced by different classes of organisms, of which about 80 are plant defensins or plant

defense peptides produced by 50 plant species (AMSDatabase). To date, a majority of the

antimicrobial peptides utilized in drug development are of animal origin (Gordon et al., 2005).

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Our results suggest that examination of plant antimicrobial proteins, such as Nt-phylloplanins,

may prove fruitful for development of novel treatments for A. fumigatus.

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Table 1: Comparison of the effects of Nt-phylloplanins on fungal spore germination

Fungus Fungal family Disease Host Inhibition of

spore

germination

by Nt-

phylloplanins

Alternaria solani Pleosporaceae Early blight Plants No

Verticillium albo-atrum Plectosphaerellaceae Wilt Plants No

Fusarium sambucinum Nectriaceae Dry rot Plants Transient

Colletotrichum coccodes Glomerellaceae Black dot Plants Yes

Aspergillus fumigatus Trichocomaceae Invasive

aspergillosis

Humans Yes

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Figure 1: Schematic representation of microfluidic device fabrication process.

(1) A multilayered mold is fabricated using photolithography methods from a light-sensitive

negative photoresist SU8. Each layer of SU8 is spun on top of the previous one and patterned by

exposure to UV light. (2) Using soft-lithography, PDMS polymer is cured on the mold to create a

replicate of the channel array. (3) The PDMS layer is bonded to glass using an oxygen-plasma

surface activation method. (4) The result is a platform containing an array of microscale channels

on a microscope slide.

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Figure 2. Inhibition of Colletotrichum coccodes spore germination after treatment with Nt-

phylloplanins in tobacco leaf wash

C. coccodes spores were subjected to various treatments: water control, proteinase-K treated Nt-

phylloplanins, Nt-phylloplanins (1 mg mL-1

), treatment with Nt-phylloplanins overnight

followed by washing twice in sterile distilled water, and incubated in microfluidic channels (2 ×

103

spores per channel). Each point represents an average of three biological replicates with 200

spores assessed per each treatment. The data was arcsine transformed prior to being analyzed

using ANOVA, with means separated using Fisher’s LSD in R 2.15.3. Treatments with the same

lowercase letters are not significantly different at P < 0.05. Error bars represent standard error of

the mean.

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Figure 3: Inhibition curve of Colletotrichum coccodes spore germination by Nt-

phylloplanins in tobacco leaf wash

C. coccodes spores were treated with tobacco leaf wash containing various concentrations of Nt-

phylloplanins and incubated for a period of 12 hours. Each data point represents an average value

from three biological replicates.

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Figure 4. Inhibition of Aspergillus fumigatus spore germination after treatment with Nt-

phylloplanins in tobacco leaf wash

Spores of A. fumigatus were subjected to various treatments: GMM control, proteinase-K treated

Nt-phylloplanins, Nt-phylloplanins (1 mg mL-1

), and incubated in microfluidic channels (1.5 ×

103

spores per channel). Each point represents an average of three biological replicates with 200

spores assessed per each treatment. The data was arcsine transformed prior to being analyzed

using ANOVA, with means separated using Fisher’s LSD in R 2.15.3. Treatments with the same

lowercase letters are not significantly different at P < 0.05. Error bars represent standard error of

the mean.

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Figure 5. Transient inhibition of Fusarium sambucinum spore germination by Nt-

phylloplanins in tobacco leaf wash

A) F. sambucinum spores germinated in water controls at 12 hours post inoculation (hpi). B)

Non-germination of Nt-phylloplanin treated F. sambucinum spores at 12 hpi. C) Extensive

hyphal growth observed after treating F. sambucinum spores with Nt-phylloplanins at 24 hpi.

Bar = 20 µm.

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CHAPTER 3

A draft genome sequence reveals Helminthosporium solani’s arsenal for cell wall

degradation

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ABSTRACT

Helminthosporium solani, is a fungal pathogen belonging to the family Massarinaceae. It causes

blemishes on potato tubers, affecting processing and fresh market trade. Despite its world-wide

distribution, little is known about the biology of H. solani. Here we report the generation of a

draft genome sequence of H. solani with an estimated genome size of ~35 megabases, which is

also the first reference genome within the family Massarinaceae. We identified a large suite of

genes in the H. solani genome that encode putative cell wall degrading enzymes. Based on

comparison with other known genomes, we speculate that H. solani is a hemi-biotroph or

necrotroph. The presence of a large number of genes in the glycoside hydrolase (GH) 10 and 43

families, which aid in the hydrolysis of glucoronoarabinoxylan, also suggests that H. solani may

be able to survive on grass hosts or debris and indicates the need to re-examine the life cycle and

host range of this pathogen. The H. solani draft genome will be a valuable resource for

phylogenetic and pathogenicity studies on this pathogen.

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INTRODUCTION

The fungus Helminthosporium solani causes silver scurf disease of potatoes and has a worldwide

distribution (Errampalli et al. 2001). Potato tubers with silver scurf have grey lesions which are

localized to the periderm and appear silvery when moistened. Under favorable storage

conditions, fungal sporulation makes the tubers appear black and sooty. Loss of pigmentation

occurs in severely diseased tubers, especially on red-skinned cultivars (Jellis and Taylor, 1977),

reducing their fresh market value. Silver scurf also increases the permeability of the tuber skin to

water vapor, resulting in tuber shrinkage and weight loss (Burke, 1938; Jellis and Taylor, 1974).

Silver scurf negatively affects the processing market as chips made from severely infected tubers

have unacceptable black burnt edges (Holley and Kawchuck, 1996). Although silver scurf does

not affect tuber yields (Read and Hide, 1984; Cunha and Rizzo, 2004), the disease has been

gaining economic importance in the past three decades due to the quality standards demanded by

processing and fresh market consumers as well as the emergence of H. solani isolates resistant to

the broad-spectrum thiabendazole fungicides and thiophanate-methyl fungicides used to control

this disease (Errampalli et al. 2001; Geary et al. 2007).

Silver scurf spreads both in the field and during storage. Diseased seed tubers (Jellis and Taylor,

1977) and infested soil (Merida and Loria, 1994) act as the primary source of inoculum. H.

solani has a very narrow host range comprised of tuber-bearing Solanum species and the weed

species Solanum elaeagnifolium (Burke, 1938; Kamara and Huguelet, 1972; Sethuraman et al.

1997; Rodriguez et al. 1995). H. solani is an anamorphic fungus that can colonize senescent leaf

tissues of alfalfa, sorghum, rye, oats, corn, and wheat in vitro, and can also survive in dry soil of

potato stores for a period of five months (Merida and Loria, 1994; Carnegie et al. 1996). There

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are no known specialized over-wintering structures, however conidia may serve this purpose

(Kamara and Huguelet, 1972). Other aspects of the disease cycle such as pathogen spread from

mother tuber to daughter tubers and its overwintering phases are poorly understood.

Furthermore, genomic information for H. solani is scarce, as evidenced by the presence of only

36 nucleotide sequences of beta-tubulin and ribosomal genes in the National Center for

Biotechnology Information (NCBI). There is no information available regarding H. solani genes

required for virulence or pathogenesis. In this sense, H. solani is an understudied pathogen.

Helminthosporium solani is an ascomycete belonging to the order Pleosporales, the largest order

in the class Dothideomycetes (Kirk et al. 2008). Analyses of ribosomal DNA and ITS sequences

reveal that the closest relatives of H. solani is the wood saprophyte Helminthosporium velutinum

and Saccharicola bicolor, causal agent of leaf scorch disease of sugarcane (Olivier et al. 2000).

Currently H. solani is placed in the family Massarinaceae (Eriksson and Hawksworth, 2003).

With the exception of H. solani and S. bicolor, the majority of the members of Massarinaceae are

saprophytes (Liew et al. 2002; Zhang et al. 2009). Wood saprophytes possess multiple cell wall

degrading enzymes capable of hydrolyzing cellulose and hemicelluloses (Bucher et al. 2004;

Simonis et al. 2008; Sin et al. 2002). In a recent study, a large scale screening for hydrolytic

activity of pathogenic and non-pathogenic fungi has also revealed that plant pathogens are

promising sources for cell wall degrading enzymes (King et al. 2011). With the advent of high

throughput sequencing techniques, the cost of sequencing has dropped considerably. This has led

to the description of several fungal genomes with genes predicted to encode a number of

carbohydrate-active enzymes (Hane et al. 2007; Ellwood et al. 2010; de Wit et al. 2012; Islam et

al. 2012; Manning et al. 2013). Based on H.solani’s phylogeny and its growth in association with

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plants, we hypothesized that H. solani encodes genes involved in cell wall degradation. The

purpose of this study was to generate a draft genome of H. solani to gain insights into its biology

and identify candidate virulence factors that are important for colonization of potato tubers.

Materials and Methods:

Helminthosporium solani genomic DNA isolation

The H. solani isolate B-AC-16A was recovered from a diseased potato tuber. Pure culture of H.

solani was black in color when grown on V8 medium, conidiophores were septate and

unbranched, and conidia were dark brown obclavate (7-8 × 18-64 µm) with up to 8 septa (Goth

and Webb, 1983; Loria and Secor, 2001, Rivera-Varas et al. 2007). The single spore isolate of H.

solani was grown in minimal medium (Olivier and Loria, 1998) for 2 weeks in the dark at 24oC

on a rotary shaker. The fungal mycelia were then separated from the medium and placed

between paper towels to squeeze out the remaining medium. The mycelia were then lyophilized

overnight and DNA was extracted using phenol-chloroform-isoamyl alcohol (Bok and Keller

2012).

Sequencing of the H. solani genome and sequence assembly

H. solani genomic DNA was used for paired-end pyrosequencing on a full plate Roche (454)

Genome Sequencer FLX+ with Titanium chemistry. H. solani genomic DNA was also sequenced

in one lane of a 1X 100 bp flow cell on an Illumina HiSeq2000. We obtained approximately 1

million reads from the 454 sequencer and approximately 175 million reads from the Hiseq2000.

The genome was assembled from a combination of 454 and Illumina data using the Roche GS

De Novo Assembler (Newbler) Version 2.6 (Marqulies et al. 2005) The following parameters

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were used in the assembly- seed step 12, seed length 16, seed count 1, min overlap length 40,

minimum overlap identity 90, alignment identity score 2, alignment difference score –3. We used

all of the 454 data in the assembly. The first 25 million reads from the Illumina data were used in

the assembly to represent 50 X coverage of the genome assuming a genome size of 50 Mb.

Retrieval and gene modeling for cell wall degrading enzymes

Following genome assembly we retained all contigs larger than 100 bp and predicted genes using

the self-training gene prediction program GenMark-ES Version 2.3c (Borodovsky and Lomsadze

2011). The search for cell wall degradation-associated genes in the H. solani genome was also

performed with complete protein sequences from other ascomycetes such as Leptosphaeria

maculans and Cochliobolus heterostrophus. The sequences were used as query in a stand-alone

tblastn search with H. solani genome as the database (Tao, 2010). Preliminary annotations of

proteins sequences obtained from translation of the genes predicted by GeneMark were obtained

using InterProScan Version 4.8 (Zdobnov and Apweiler 2001) including membership in protein

families and Gene Ontology (Ashburner et al. 2000) categories.

Phylogenetic analyses:

Phylogenetic analyses of 16 putative cell wall degrading genes were conducted using MEGA

version 5.05 (Tamura et al. 2011). The amino acid sequences were aligned using Clustal W with

default parameters (Thompson et al. 1994). A neighbor-joining tree was constructed for each

sequence group and a bootstrap support for internal branches was estimated from 1000 pseudo-

replicates.

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RESULTS AND DISCUSSION

Helminthosporium solani is an understudied pathogen with few genomic resources. Here, we

report a draft genome sequence of H. solani, which is also the first reference genome within the

family Massarinaceae. The H. solani genome assembly consisted of 35.0 Mb in 9,248 contigs,

with about 34 Mb in contigs bigger than 500 bp (2939 contigs) that are contained in 486

scaffolds. The N50 scaffold size is 168,899 bp with an N50 contig size of 34,146 bp. The

assembly contained 874,415 Roche 454 reads and 19,976,474 Illumina reads.

We searched the H. solani contig sequences with the complete complement of predicted protein

sequences from the genomes of two closely related Ascomycete species Leptoshaeria maculans

and Cochiobolus heterostrophus C5 using tblastn (Altschul et al. 1990) with a threshold evalue

of 0.00001. The 36.36 Mb C. heterostrophus genome encodes13,336 predicted proteins and has

10,159 putative homologs in the H. solani genome. The 44.89 Mb L. maculans genome encodes

12,469 predicted proteins and has 8,459 putative homologs in the H. solani genome. The average

percentage identity between H. solani proteins and their homologs in either genome is about

60%. De novo gene prediction using GeneMark-ES revealed 14,862 predicted protein-coding

genes from the H. solani contig sequences. Approximately two-thirds of these predicted proteins

have some matching entry from the InterProScan searches.

H. solani genome sequence provided insight into several putative virulence genes, including

genes homologous to plant cell wall degrading enzymes, non-ribosomal peptide synthetases, and

polyketide synthetases. However, we focused our analysis on the plant cell wall degrading

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enzymes because of the large base of knowledge about the enzyme function and enzyme content

of plant-associated microbes.

H. solani encodes numerous plant cell wall degrading enzymes:

The complex polysaccharides of plant cell walls are hydrolyzed by a large variety of

carbohydrate-active enzymes (CAZymes) which are classified into families such as glycoside

hydrolases (GH), polysaccharide lyases, carbohydrate esterases and carbohydrate-binding

modules (Cantarel et al. 2008). Because the lifestyle of a fungus is generally well correlated with

CAZyme content, we compared the number of genes predicted to encode celluloytic and

hemicellulolytic enzymes in the genome of H. solani to a subset of ascomycetes that included a

saprotroph (Trichoderma reesei), necrotrophs (Fusarium graminearum, Sclerotinia sclerotiorum,

Botrytis cinerea), a hemibiotroph (Magnaporthe grisea) and a biotroph (Blumeria graminis). We

observed that H. solani has at least 71 putative genes encoding GH enzymes, which was

comparable to genomes of hemibiotrophs and necrotrophs (Table 1 and 2). For instance,

biotrophs generally possess fewer cell wall degrading enzymes than necrotrophs and

hemibiotrophs to evade plant defenses (deWit et al. 2012; Amselem et al. 2011; Goodwin et al.

2011). Based on GH enzyme content, H. solani likely has a hemibiotrophic or necrotrophic

lifestyle, which is in accordance with earlier histological studies where necrosis was observed in

both H. solani colonized and neighboring potato tuber cells (Martinez et al. 2004).

No evidence of horizontal gene transfer among putative cell wall degrading genes:

H. solani belongs to a family that consists mainly of saprophytes, suggesting that acquisition of

novel genes or gene duplication resulted in H. solani gaining the ability to colonize potato tuber

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surfaces. Because it is a tuber- and soil-borne pathogen and because soil is a complex

environment hosting diverse organisms, there are likely to be ample sources for horizontal gene

transfer for H. solani (Wenzl et al. 2005; Gorfer et al. 2011). Hence, we constructed phylogenetic

trees, a primary method for identification of horizontal gene transfer event, with a subset of 16

H. solani plant cell wall degrading gene homologs (Richards et al. 2011). However, all of the GH

enzyme gene sequence trees were congruent with the H. solani phylogeny (data not shown).

Hemicellulase enzyme content suggests that H. solani can degrade monocot plant cell walls:

Hemicelluloses are the second most abundant group of polysaccharides in the plant cell wall (van

den Brink and de Vries 2011). Xyloglucans and arabinoxylans constitute two major families of

hemicelluloses whose composition varies among plants. For instance, xyloglucans are

predominant in dicots and nongraminaceous monocots whereas arabinoxylans are primarily

present in monocot grasses in the family Poaceae (Albersheim et al. 2011). H. solani is a

pathogen on potato, which is a dicot plant. For this reason, we were surprised by the presence of

a single gene likely involved in xyloglucan degradation (family GH16) which was in contrast to

other fungi compared in this study and another eighteen fungal species in the class

Dothideomycetes (Ohm et al. 2012). This suggests that H. solani does not extensively digest

dicot hemicelluloses, which may explain, in part, its narrow host range. But the fact that H.

solani is well adapted to potato suggests that the pathogen may possess a different strategy. A

caveat to this analysis is that the gene number is not predictive of enzyme expression or activity,

as evidenced with T. reesei which produces highly efficient enzymes with a small number of

genes (Martinez et al. 2008).

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Glucuronoarabinoxylan, composed of a xylose backbone with arabinose and glucuronic acid side

chains is the major hemicellulose component of grass cell walls (van den Brink and de Vries

2011; Vogel 2008). Degradation of glucuronoarabionxylan is aided by endoxylanases (GH10)

and arabinofuranosidases (GH43). Fusarium graminearum and Magnaporthe grisea, which are

predominantly pathogens on grass hosts possesses 15 and 16 genes respectively that encode

GH10 and GH43 enzymes. It is interesting to note that H. solani possesses 13 GH10 and GH43

homologs, which is comparable with grass host pathogens. Although earlier in vitro studies

showed H. solani’s ability to colonize senescent grass leaf tissues (Merida and Loria 1994), our

genomic data suggest that H. solani may have a greater ability to survive on monocots than

dicots, which indicates the need to re-examine the life cycle and host range of H. solani. Further

studies should also focus on developing a transformation method for H. solani which would

allow functional analyses of candidate genes associated with cell wall degradation and elucidate

their role during pathogenesis.

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Table 1: Comparison of the number of cellulose degrading genes among ascomycete fungi

Fungal species

Lifestyle

Glycoside Hydrolase (GH) family

GH 1 GH 3 GH 5 GH 6 GH 7 GH12 GH45 GH61 GH74 Total

Trichoderma reesei a Saprotroph 2 8 3 1 2 1 1 3 1 22

Botrytis cinerea a Necrotroph 5 14 5 1 3 1 2 10 0 41

Sclerotinia sclerotiorum a Necrotroph 1 12 5 1 3 2 2 9 0 35

Fusarium graminearum a Necrotroph 3 17 3 1 2 2 1 13 1 43

Magnaporthe grisea a Hemi-biotroph 2 15 6 3 7 3 1 22 1 60

Helminthosporium solani b ? 3 15 7 2 5 1 1 17 0 51

Blumeria graminis f.sp. hordei a Biotroph 0 0 0 0 0 0 0 2 0 2

a Gibson et al. (2011)

b Data from this study

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Table 2: Comparison of the number of hemicelluloses degrading genes among ascomycete fungi

Fungal species

Lifestyle

Glycoside Hydrolase (GH) family

GH 10 GH 11 GH 16 GH 43 GH 51 GH 54 GH 62 GH 81 Total

Trichoderma reesei a Saprotroph 1 3 12 2 0 2 1 2 23

Botrytis cinerea a Necrotroph 2 2 11 4 3 1 1 2 26

Sclerotinia sclerotiorum a Necrotroph 2 3 10 3 2 1 0 1 22

Fusarium graminearum a Necrotroph 4 3 14 11 2 1 1 1 37

Magnaporthe grisea a Hemi-biotroph 6 5 10 10 3 1 4 2 41

Helminthosporium solani b ? 4 4 1 9 0 0 1 1 20

Blumeria graminis f.sp. hordei a Biotroph 0 0 3 0 0 0 0 1 4

a Gibson et al. (2011)

b Data from this study

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SYNTHESIS

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Silver scurf (causal agent: Helminthosporium solani) and black dot (causal agent: Colletotrichum

coccodes) are two potato surface blemish diseases that are frequently misidentified by potato

growers and whose importance in organic farming is poorly understood. Organic potato

production is unique from conventional production in terms of both agronomic and plant

protection practices. Synthetic pesticides are not permitted in organic farming which leaves the

crop vulnerable to various diseases. Very little on-farm research has been done to learn about the

importance of these diseases in organic potato farming. This dissertation is the first to

demonstrate that both H. solani and C. coccodes are highly prevalent in organic potato farms in

Wisconsin. This research also suggests that the visual assessment of tubers at harvest for silver

scurf and black dot may be misleading. Possible future work should focus on organic control

measures to prevent disease development during the growing season and in storage. With the

expanding fresh market for organic potatoes demanding blemish free potatoes, we believe there

is an immediate need for research on tuber surface defects.

In our experiments, we also observed H. solani and C. coccodes together on about 65% of the

asymptomatic and asymptomatic tubers assessed. Despite having different lifestyles, co-

occurrence of both pathogens in such a high frequency raises interesting ecological questions. At

one extreme, H. solani has a known host range limited to tuber-bearing Solanum species, some

saprophytic ability, and no known special over-wintering structures. At the other extreme, C.

coccodes has a broad host range, the ability to survive in crop debris, produce sclerotia, and

colonize the same host tissue as H. solani. Generally speaking, two species sharing a single niche

cannot co-exist for a long period of time, which raises questions about the nutrients that the

pathogens sequester from the tuber, the time of infection and the growth patterns affecting their

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reproductive potential. We have conducted preliminary experiments looking at carbon and

nitrogen utilization patterns of these pathogens, but more experiments need to be performed to

dissect this association and explore plausible interactions between these two pathogens.

As all current potato cultivars are susceptible to silver scurf, we screened potato cultivars and

lines for resistance to silver scurf. Mature tuber incubation assays performed to screen for

resistance were unsuccessful because 90% of the tubers were lost before the end of the assay due

to tuber decay pathogens. As an alternative to mature tubers, we explored the utilization of

hydroponically grown minitubers, which minimized tuber losses to decay pathogens. This work

demonstrated that minitubers can be successfully employed to screen for resistance to silver

scurf. This dissertation has identified a diploid inter-specific breeding line (C287) with

consistently low H. solani sporulation. C287 is known to have resistance to other significant

potato diseases such as verticillium wilt, common scab, and black scurf and findings from this

study suggest that C287 possesses partial resistance to silver scurf as well. Further work should

be focused on field and storage trials to evaluate silver scurf resistance of C287 at multiple

locations.

H. solani is a little studied pathogen with many unanswered questions regarding its biology and

lifecycle. For instance, little information is available regarding the genes involved in the

infection process, overwintering phase of the pathogen, and virulence factors. In order to address

some of these questions, we generated a draft genome sequence of H. solani, which is the first

reference genome in the family Massarinaceae and will be made available for public research.

This research led to the identification of a large suite of genes in the H. solani genome that

encode putative cell wall degrading enzymes. Based on comparison with other known genomes,

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we speculate that H. solani is a hemi-biotroph or necrotroph. Also, the presence of a large

number of genes in Glycoside Hydrolase (GH) 10 and 43 families, which aid in the hydrolysis of

glucoronoarabinoxylan, a major component of grass hemicelluloses, suggests that H. solani may

be able to survive on grass hosts and indicates the need to re-examine the host range of this

pathogen.

We believe draft genomic data will shed light on previously unknown aspects of the biology of

H. solani. In silico genomic analysis indicates H. solani may be a promising target for

identifying novel cell wall degrading enzymes for use in the biofuel industry. To this end, future

research should focus on performing enzymatic assays to obtain more detailed characterization

of the secretome of H. solani. It would also be useful to develop a new primer set for detecting

H. solani because the current nested primer set is time and cost prohibitive for use in diagnostics.

Analysis of the genome of H. solani also revealed several genes involved in secondary

metabolite production. Although our attempts to transform H. solani by biolistic transfection and

Agrobacterium-mediated transformation were unsuccessful, we strongly believe further work on

the development of a transformation procedure that would aid in performing functional

genomics.

Finally, we explored the antifungal properties of tobacco phylloplane (leaf surface) proteins to

elucidate their potential effects on pathogen development, and determine if they can be utilized

in post-harvest disease control. Due to the large volumes of water used for collecting

phylloplanins from leaf aerial surfaces, the samples first needed to be lyophilized to yield a

concentrated product highly enriched in protein. This aspect of sample recovery limited the

volume of phylloplanins and was a significant bottleneck in our ability to perform large numbers

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of assays. For this reason, we decided to use a microfluidic approach that has been successfully

used earlier for applications in drug discovery, cell culturing, and characterization of fungal

secondary metabolites. Using microfluidic channels, we were able to perform tens of assays

simultaneously with low volumes of phylloplanins, and demonstrated that this assay will be

broadly useful in further large-scale screening of natural product bioactives against

microorganisms.

Tobacco phylloplanins strongly inhibited spore germination of Colletotrichum coccodes and

Aspergillus fumigatus, transiently inhibited Fusarium sambucinum spore germination, and were

inactive against Alternaria solani and Verticillium albo-atrum, indicating a spectrum of pathogen

sensitivity that was not previously known. Most importantly, inhibition of spore germination of

the human pathogen A. fumigatus by tobacco phylloplanins reveals a new venue for medicinal

antifungal drug discovery that has not been explored previously. Future work should focus on

mass scale production of tobacco phylloplanins by expressing them in a heterologus system and

testing for inhibitory effects on additional species of Aspergillus that are human pathogens.

Lastly because the mechanism of action of phylloplanins remains unknown, further research

efforts should be directed at exploring the targets of phylloplanins.