SCREENING FOR RESISTANCE TO ROOT ROT IN LUPIN

Gayathri Udayangika Beligala

A Thesis

Submitted to the Graduate College of Bowling Green State University in partial fulfillment of the requirements for the degree of

MASTER OF SCIENCE

August 2016

Committee:

Vipaporn Phuntumart, Advisor

Helen Michaels

Paul Morris

© 2016

Gayathri Beligala

All Rights Reserved iii ABSTRACT

Vipaporn Phuntumart, Advisor

Phytophthora sojae is a destructive pathogen belonging to the class Oomycota. It has a very narrow host range and infects only and lupin. Infection by P.sojae overcomes most soybean resistance genes. Therefore, lupins can be perceived as a potential reservoir of novel resistance genes that can be engineered into soybean lines. produce motile zoospores that swim towards plant roots and subsequently cause root rot diseases. This study focuses on determining the chemotactic behaviors of zoospores and pathogenicity of P. sojae 2 (P6497) towards roots of ten annual lupin ( spp.) lines and a perennial line (L. perennis). The latter was further divided into three subgroups based on the degree of speckling on the seed coat; darkly speckled, lightly speckled and seeds with no speckles. Root chemotaxis assay showed no statistically significant differences between all the lupin lines tested. For pathogenicity assay, I measured disease incidence and disease severity. Interestingly, two annual lupin lines exhibited resistance to the infection of P. sojae. There was no correlation between chemotaxis of zoospores and disease severity of P. sojae on lupin. This suggests that the attack of zoospores on its host roots does not necessarily lead to root rot symptoms as there is a risk for the pathogen to be recognized by the host receptors which restrict the pathogenic invasion. When chemotactic behaviors of zoospores were tested against the metabolites extracted from lupin seeds exhibiting different seed coat phenotypes, the zoospore attachment was significantly higher towards the metabolites extracted from seed coats of all three seed phenotypes compared to the control with a preferential response to the seed coat extract of dark seeds. It is evident that zoospore attractants possibly , are present at higher amount in the seed coats of darkly speckled lupin iv seeds compared to seeds with no speckles. v

This thesis is dedicated to my dearest father A.T. Wijesooriya, my mother R.B.M. Chandrani and

my beloved husband Dilshan Beligala for their endless support. vi ACKNOWLEDGMENTS

Although it is just my name appears on the cover of this thesis, a great many people contributed to make this attempt a success.

Foremost, I would like to express my gratitude to my advisor Dr. Vipaporn Phuntumart for her guidance and continuous support in completing this project. I have been amazingly fortunate to have her as my guide.

I’m deeply grateful to my committee members Dr. Paul Morris and Dr. Helen Michaels who shared their valuable ideas with me and guided me throughout my research.

I thank Satyaki Ghosh and all the other fellow lab mates from Phuntumart lab, for helping me in different situations.

I’m also grateful to Jacob Sublet and Paige Marie for their various forms of support during my graduate study especially in the statistical analyses. Many thanks and appreciations also go to

Frank Schemenauer who helped me in growing and taking care of my lupin plants.

With much happiness I thank my dear friends, especially Menaka Ariyaratne and Cedirc Jackson for supporting me in numerous ways throughout my research work and Dayal Wijayarathna for helping me in compiling this thesis.

Most importantly, none of this would have been impossible without the love and strength of my husband Dilshan Beligala and my parents to whom this thesis is dedicated to. I have to mention my immediate family Renuka Ranaweera, Samarasinghe Beligala and Lakshan Beligala for their love and encouragement.

Without the continued efforts and support of all these people, I would have not been able to bring my work to a successful completion. vii

TABLE OF CONTENTS

Page

CHAPTER I.………………………………………………………………...... 1

1.1 Lupins…………………………………………...... 1

1.2 Lupin pathogens ...... 2

CHAPTER II…….………………………………………………………………………..... 4

2.1 Oomycetes...... 4

2.2 Molecular mechanisms of pathogenesis ...... 7

2.3 Sources of resistance to ...... 9

CHAPTER III…………………………………………………………………………….... 14

3.1 Zoospore chemotaxis to host plant metabolites ...... 14

3.2 Secondary metabolites as defense molecules ...... 15

3.3 Objectives ...... 18

3.4 Hypotheses ...... 18

CHAPTER IV. METHOD ...... 19

4.1 Evaluation of zoospore-chemotactic responses of P. sojae towards lupin roots . 19

4.1.1 Plant materials ...... 19

4.1.2 Preparation of zoospores ...... 22

4.1.3 Chemotaxis assays ...... 23

4.2 Determination of chemotactic response towards metabolites present in different

seed coat phenotypes of lupin…..………………………………………………. 24

4.3 Pathogenicity assays ...... 25

4.3.1 Pathogen inoculation ...... 25 viii

4.3.2 Evaluation of disease severity ...... 25

4.4 Data analysis…...... 27

CHAPTER V. RESULTS ...... 28

5.1 Chemotaxis assays ...... 28

5.1.1 Zoospore-root attraction assays ...... 28

5.2 Evaluation of disease severity ...... 30

5.3 Evaluation of disease incidence ...... 34

5.4 Zoospore chemotaxis towards seed coat extracts ...... 37

CHAPTER VI. DISCUSSION...... 40

6.1 Zoospore-root attraction assays ...... 40

6.2 Screening of lupin accessions for root rot resistance under laboratory condition 42

6.3 Zoospore chemotaxis towards seed coat extracts ...... 45

CHAPTER VII. CONCLUSIONS AND FUTURE DIRECTIONS ...... 48

REFERENCES……………………………………………………………………………… 50 ix

LIST OF FIGURES

Figure Page

1 Schematic representation of the phenylpropanoid pathway ...... 16

2 Seeds of annual lupin lines used for pathogenicity screening ...... 21

3 Three phenotypes of perennial lupin seeds categorized based on the degree of

Speckling……………………………………………………………………………. 22

4 Incubation of lupin roots with a suspension of zoospores (8×103spores/ml) in an

assay chamber ...... 24

5 Root rot severity of infected lupin seedlings rated according to a visual scale of 0-5

compared to resistant soybean (SB-W82) and susceptible soybean (SB-W) ...... 26

6 Abundance of zoospore attachment along a lupin root ...... 29

7 A) Zoospores encysting and germinating on the root surface of elongation zone B)

Zoospores encysting and germinating on root hairs ...... 29

8 Chemoattraction of zoospores to the roots of lupin lines tested ...... 30

9 Disease severity of lupin lines tested ...... 34

10 Disease incidence (percentage plant death) of the lupin lines tested ...... 35

11 Comparison of zoospore attachment and disease severity ...... 36

12 Average number of zoospores attached to a unit area of the agar plug (1.2 mm2)

containing the metabolites extracted from three seed phenotypes of perennial lupin 38

13 Representation of microscopic images of zoospore accumulations on the agar plugs

containing metabolites extracted from three seed phenotypes of perennial lupin ..... 39

x

LIST OF TABLES

Table Page

1 Annual and perennial lupin lines used for chemotaxis and pathogenicity screening

of P. sojae ...... 20

2 Nonparametric comparison with the resistant control using Steel Method ...... 33

1

CHAPTER I

1.1 Lupins

Lupinus sp. commonly known as either lupin or lupine is a genus of legumes grown in South and

North America, Africa and Mediterranean regions. Lupin is becoming extensively recognized as a human and an animal food additive due to its high nitrogen and high phenolic content.

Although lupin seeds are rich in proteins, its use as a nutrient supplement is restricted due to their high fiber and high alkaloid content and therefore, low-alkaloid cultivars are being introduced.

Although lupin could not achieve the broad recognition of soybean in the food industry, lupins were used by human as a high protein diet since ancient times (Gladstones et al., 1998). The actual number of species belonging to the genus Lupinus L. is not clearly documented but it is suggested to be over two hundred (Drummond et al., 2012). Most commonly cultivated Lupinus species include Lupinus albus L. (white lupin), L. luteus L. (yellow lupin), and L. angustifolius

L. (blue or narrow-leafed lupin), and L. mutabilis (Sweet or Andean lupin).

Lupin seeds contain certain secondary metabolites including alkaloids and isoflavones (Katagiri et al., 2002). Phenolic compounds such as isoflavones are known to be one of the key signaling compounds that play a significant role in both positive and negative plant-microbial interactions.

It has been reported that isoflavones exuded by the roots of certain lupin species act as signaling molecules in symbiosis between nitrogen fixing rhizobacteria and legumes (Gagnon, 1998).

Lupins are nodulated mostly by the rhizobial species belonging to the genus Bradyrhizobium

(Jarabo-Lorenzo et al., 2003). High potential of lupin to fix nitrogen by these symbionts has promoted the use of lupin as a green manure as well as their felicity to use in rotation with other 2

cash crops to increase the yield of subsequent crops.

1.2 Lupin pathogens

Not many pathogens have been reported to cause diseases in lupin. Diseases that damage lupin

most frequently are anthracnose and Fusarium wilt. Lupin anthracnose caused by Colletotrichum lupinei has been detected in L. albus in Western Australia in 1996 (Nirenberg et al., 2002; Shea et al., 2008). This disease is highly destructive in susceptible varieties and is found to be a seed borne disease (pathogen survives on old lupin trash and in or on infected seed). Fusarium wilt is a disease caused by Fusarium oxysporum f. sp. lupinei which resulted in a huge loss in lupin production in Europe in the 1970s. Fusarium diseases of lupin are soil borne and could be either vascular wilts or root diseases including root rot, hypocotyl rot, pod rot and damping off.

Pleiochaeta setosa is another phytophathogenic fungus causing brown-leaf spot which is prevalent mostly in autumn-sawn crops (Huyghe, 1997). Uromyces lupineicolus causes rust disease during warm and dry periods leading to early defoliation. In 1960s, Phomopsis leptostromiformis was recognized to cause stem blight in young lupins especially in L. luteus.

This fungus produces mycotoxins termed phomopsins which cause the animal liver disease known as lupineosis (Culvenor et al., 1977; Williamson et al., 1994).

Phytopathogenic bacteria that infect lupin include Pseudomonas spp. and Xanthomonas spp.

(Hill, 1979; Wilkie et al., 1973). Phytopathogenic viruses such as Cucumber mosaic virus are also widely spread among lupin growing regions (Jones & McLean, 1989). Plenty of resistant lines have been bred against above diseases as well as for other beneficial traits.

There are few records published on lupin attacking soil borne pathogens belonging to class 3

Oomycota (in the kingdom Straminopiles). Members of the class Oomycota that are called

oomycetes include destructive root pathogens including Phytophthora spp. Among Phytophthora

pathogens, Phytophthora erythroseptica has the first records of its pathogenicity on white lupins

(L. albus) (Nikandrow et al., 2001; Trapero-Casas et al., 2000). In certain regions of the world,

P. megasperma and P. cinnamomi also reported to infect lupins (Jones & Johnson, 1969; Serrano et al., 2010). Jones & Johnson (1969) isolated Phytopththora species similar to P. sojae from L.

albus, L. luteus and L. angustifolius growing in naturally infested soil from Mississippi. It has

been suggested that lupins are tolerant to these pathogens due to the presence of isopentenyl

termed ‘’ that acts as a pre-infection antifungal agent (Harborne et al., 1976).

Bantignies et al. (2000) and Pinto & Ricardo (1995) have reported that L. luteus pocesses proteins homologous to PR family (pathogenicity related protein family) in healthy roots which may have a role in pre-infection defense mechanisms against pathogen attack. Similarly, Sikorski

et al. (1999) demonstrated the presence of two proteins belonging to PR10 class in yellow lupin

(L. luteus L. cv. Ventus). L. albus has also displayed elevated levels of phytoalexin in root

tissues in response to elicitors of Phytophthora pathogens (Cosio et al., 1996). The presence of surface receptors in L. luteus that bind β-glucan elicitors and subsequently leading to activation of defense responses is another evidence for existence of resistance in certain lupin species to plant pathogens (Ebel et al., 1995).

4

CHAPTER II

2.1 Oomycetes

Oomycetes are a distinct group of fungus-like filamentous microorganisms belonging to

Kingdom Straminopila. These organisms are taxonomically distinct from fungi and more closely

related to algae. The general characteristics of Class Oomycota include biflagellate zoospores, a

consisting of beta-glucans and cellulose, oogamous reproduction by gametangial contact and filamentous growth habit (Baldauf et al., 2000; Beakes et al., 2012; Dick, 2013; Fry

& Grünwald, 2010; Harshberger, 1917).

Class Oomycetes is divided into four subclasses; Saprolegniales, Leptomitales, Lagenidales, and

Peronosporales. Taxonomic classification of oomycetes is still undergoing debate and some

authors group the plant pathogenic genera Phytophthora and Pythium together in to a separate

class named Pythales (Schaechter, 2011). is a well-known

pathogen that caused an immense epidemic in fields in most of Europe in the 1940’s

(Bourke, 1993; Bourke, 1968). Members of the genus Phytophthora are known to cause various

types of root rot, stem rot and mildew in many legume species. More than 80 devastating species

of Phytophthora have been reported worldwide. Amongst them, Phytophthora sojae, which was

previously known as f. sp. glycinea, has been reported to cause the

highest damage to soybean fields (Hart et al., 1981). In 2006, the annual soybean yield was

reduced by 1.46 million metric tons in the United States because of Phytophthora root and stem

rot (Wrather et al., 2010). Certain members of the genus Phytophthora such as P. ramorum, P.

cinnamomi and P. parasitica have very broad host ranges. In contrast, P. sojae has a narrow host 5

range where their infection is limited primarily to soybean and may be lupins.

Pathogenic oomycetes are difficult to control with the majority of fungicides because they

genetically adapt to the chemicals applied (Erwin & Ribeiro, 1996; Tyler, 2007). Control of

oomycete pathogens becomes a challenge because not only the fungicide is ineffective but also

the affected underground parts are not visible (Tyler, 2007). To date, genetic resistance has become an effective method employed to combat oomycete pathogens (Tyler, 2007). In this

perspective, screening for disease resistance is an important prerequisite for breeding strategies.

When resistant genes within a species are limited, screening for resistant genes from closely related species and then incorporating them into susceptible host plants could provide protection against certain pathogens.

Phytophthora species are successfully adapted to diverse environments producing specialized

structures for survival, dispersal and infection. They produce sexual reproductive spores termed

‘oospores’ from fertilization of the female organ ‘’ by the male organ ‘antheridium’.

Oospores are thick-walled hard structures adapted for survival in soil or in plant tissue. Under

favorable conditions (when water is available) these oospores will be germinated to produce

mycelia which then produce spore bearing structures termed sporangia. When the soil is

saturated, these sporangia start releasing motile spores called zoospores. Zoospores can swim

towards plant roots which subsequently attack and cause root-rot diseases. Certain chemical

compounds exuded by plant roots such as flavones and isoflavones are known to attract some

soil pathogens and they are also vital for the host-specific soil pathogens to recognize and move

towards the host signals (Morris & Ward, 1992; Tyler, 2002). Once zoospores come into contact 6

with the surface of a root, they become encysted losing their flagella and forming a surrounding

cell wall. Sometimes encystment occurs prematurely and the cyst will either release a secondary zoospore or will survive in the soil (Tyler, 2002). It has been suggested that release, encystment and subsequent of zoospores depend on a series of Ca2+ signaling mechanisms

(Connolly et al., 1999). After the germ tube of a germinating cyst penetrates between root

epidermal cells, it forms a flattened hyphal organ termed ‘appressorium’. An infection peg grows

from the appressorium in order to feed on living cytoplasm of the host cell.

In early stages, the pathogen proliferates inside the host tissues exhibiting an asymptomatic biotrophic phase and later kills the host tissues by entering in to a destructive necrotrophic stage

characterized by disease symptoms. P. sojae infects the fine roots causing ‘root rot’. This root

necrosis causes the upper canopy to wilt. Root rot extends to the major roots and the main stem

resulting in death of the canopy.

In order to infect and to establish the infection in the host, an oomycete pathogen undergoes

several developmental stages in zoospore taxis, encysment, cyst germination and hyphal tropism.

All of these processes increase the chance of recognizing a potential host. Especially, most signal

molecules exuded by roots such as isoflavones have a crucial role in host recognition by

zoospores. To what extent this host recognition is necessary for establishing an infection still

remains in question. A number of studies have documented that zoospore taxis and pathogenicity are correlated (Chi & Sabo, 1978; Milholland, 1972) while some showed no correlation (Halsall,

1978; Raftoyannis & Dick, 2006; Tippett et al., 1976). Also, it is still unclear whether the 7

infective stages respond only to individual chemical metabolites or instead to a combination of chemicals such as sugars, amino acids, phenolic compounds, isoflavones, Ca2+ ions and soil pH.

2.2 Molecular mechanisms of pathogenesis

Principally, host plants possess two layers of immunity. The first layer of immunity is triggered

upon recognition of microbial- or pathogen-associated molecular patterns (MAMPs or PAMPs)

by the transmembrane pattern recognition receptors (PRRs). PAMP triggered immunity (PTI)

activates primary defense responses such as callose deposition, cell wall remodeling and

phytoalexin accumulation (Jones & Dangl, 2006). To establish a successful infection, all true

fungi and oomycete pathogens including Phytophthora species either overcome PTI or

manipulate cellular functions, especially defense circuitry of the host cells through an array of disease effector proteins. By secreting and utilizing a multitude of effectors, these pathogens establish effector-triggered susceptibility (ETS) to allow successful infection and tissue colonization (Huitema et al., 2004; Kamoun, 2006). To defend the action of pathogens’ effector proteins, plants evolved receptors (R proteins) that recognize these effectors to activate effector- triggered immunity (ETI), the second layer of immunity in plants. Phytophthora species are typically hemibiotrophic pathogens where they require living host cells to feed on during initial infection followed by necrotrophic colonization of the host tissue where the host cells are being killed. Typically these pathogens initially grow intercellularly and invade the host cells developing nutrient-absorbing structures termed haustoria. Surrounding the haustorium, an extrahaustorial matrix is formed between the plasma membranes of the pathogen and the host where all the pathogen effectors are secreted from the haustoria (Dodds et al., 2009; Kamoun, 8

2007). These effectors are defined as molecules that manipulate the structure and function of

host cells by which the pathogen infection is facilitated and host defense responses are triggered

(Huitema et al., 2004; Kjemtrup et al., 2000). Effectors can be categorized in to two classes based on their target site of translocation. Firstly, apoplastic or extra cellular effectors are

secreted into the host-pathogen interface and interact with surface receptors of the host. These

apoplastic effectors include cell wall degrading enzymes, inhibitors of plant enzymes, elicitins

and toxins (Hardham & Cahill, 2010). GIP1 and GIP2 are two examples for apoplastic effectors

that P. sojae produces to inhibit the endoglucanase activity of their soybean host (Rose et al.,

2002). The second class, cytoplasmic or intracellular effectors are translocated inside the host cells via infection vesicles and haustoria that develop inside host cells (Kamoun, 2006). These cytoplasmic effectors have a signal peptide for the role of secretion followed by a host-targeting

(HT) leader sequence at the N-terminal region which is required for the translocation of effector proteins inside host cells. There are two large classes of cytoplasmic effectors grouped based on

the nature of HT sequences. Most Phytophthora pathogens have the motif RXLR (where X is an

amino acid) followed by the sequence EER as their HT signal (Birch et al., 2008; Whisson et al.,

2007). The second class of cytoplasmic effectors is termed Crinkler effectors (CRN) and they

have a distinct LXLFLAK motif instead of RxLR at the N terminus (Haas et al., 2009;

Schornack et al., 2010). Amongst these two major classes of cytoplasmic effectors, CRNs occur

ubiquitously in oomycete plant pathogens whereas RxLR effectors are limited to Phytophthora

spp. and some members (Bozkurt et al., 2012; Schornack et al., 2010). Avr1b-1

is an example for cytoplasmic effector belonging to RxLR family and is secreted by P. sojae.

CRN1 and CRN2 are two examples for CRN family effectors and they are secreted by P. 9

infestans (Kamoun, 2006; Shan et al., 2004; Torto et al., 2003). As a result of co-evolution,

effectors secreted by pathogens are recognized by resistant proteins/receptor proteins that are

encoded by disease resistance genes or R genes activating effector-triggered immunity (ETI)

(Hein et al., 2009).

2.3 Sources of resistance to Phytophthora sojae

Disease resistance has become a critical research topic of interest at the present time in the field

of agriculture and science. Single gene resistance (also termed qualitative resistance) which is

activated in a gene-for-gene system has been utilized extensively to manage plant diseases where

resistance or susceptibility is controlled by pairs of interactive genes (Dorrance et al., 2007). Rps

(Resistance to P. sojae) gene resistance is quantitatively inherited, race specific and activates

defense responses only after infection by P. sojae. According to this model, there are two

possible alleles that carry avirulence and virulence in the pathogen as well as two alleles carrying

resistance and susceptibility in host. Hosts that carry a susceptible allele can be easily infected by

pathogens carrying either allele. If the host possesses a resistant allele, only a pathogen with a

virulent allele can infect that particular host. In a resistant host, growth of the mycelium does not

proceed beyond a hypersensitive lesion. The host resistance gene (Rps) is responsible for the recognition of pathogens in a race-specific manner whereas the pathogen’s avirulence (Avr) gene is involved in producing a race-specific effector protein leading to the activation of a series of biochemical events ending in a hypersensitive reaction (HR) (Dodds & Rathjen, 2010;

Fliegmann et al., 2003; Jiang & Tyler, 2012; Tyler, 2007). 10

Rps-induced defense signaling is very complex and the regulation of defense responses triggered

by Rps gene products is mediated through a network of phytohormone signals such as salicylic

acid, jasmonic acid and ethylene (Lin et al., 2014). To date, 21 Rps genes have been identified and 8 of these (Rps1a, Rps1b, Rps1c, Rps1k, Rps2, Rps3a, Rps6 and Rps7) have already been

utilized in breeding commercial soybean cultivars (Agrawal & Lively, 2002; Dorrance &

Schmitthenner, 2000; Flor, 1971; Lin et al., 2013; Schmitthenner, 1999; Sun et al., 2014; Sun et

al., 2011). This Rps family is predicted to encode proteins that contain nucleotide binding and

leucine rich repeat domains (Bhattacharyya et al., 2005; Gao & Bhattacharyya, 2008). The

structure of a protein coded by Rps1-k locus has been predicted by Bhattacharyya et al., (2005).

Six structural domains of the Rps1-k proteins were identified which consist of a coiled-coil motif

(CC), a nucleotide-binding adaptor conserved in apoptosis protease activating factor-1 (NB-

ARC), a leucine rich repeat domain (LRR) and a domain containing R gene products and a CED-

4 domain. A leucine-zipper like motif has been identified in the LRR domain and Rps1-k

proteins are divided in to two classes based on the absence of one LRR repeat in the C-terminal

LRR region. There are three proposed models that describe the recognition of pathogen effectors

by host receptor proteins that contain NB and LRR domains. The first model is direct recognition

where direct physical binding of effector molecule to the NB-LRR receptor triggers immune

signaling (Valent et al., 2000). The second model is an indirect effector recognition named

‘guard and decoy’ model. In this model, the effector induces a modification of an accessory

protein and this accessory protein is then recognized by the host’s NB-LRR receptor. This

accessory protein could be the pathogen’s virulence target (guard model) or a structural mimic of

it (decoy) (van der Hoorn & Kamoun, 2008). Lastly, the ‘bait and switch’ model is a system 11

where the effector molecule first interacts with an accessory protein (bait) that is associated with

NB-LRR receptor and this interaction event facilitates NB-LRR receptor to recognize the pathogen’s effector directly (Collier & Moffett, 2009). Recognition of effectors leads to the activation of NB-LRR proteins which subsequently translates signals to trigger the general immune responses. Activation mechanisms of NB-LRR proteins are complex and still needs to be explored. Downstream responses of NB-LRR compose of large number of regulatory events.

These include rapid calcium influx, oxidative burst, activation of mitogen-activated protein kinases (MAPKs), expression of defense genes and hypersensitive reaction (HR) (Dodds &

Rathjen, 2010). Expression of defense genes is regulated by salicylic acid, jasmonic acid and ethylene hormone pathways. As an example, expression of Rps1-k genes in soybean requires ethylene mediated Signaling pathway (Hoffman et al., 1999). The R gene mediated defense response often results in HR which is characterized by localized cell and tissue death at the site of infection within a few hours of pathogen contact (Hammond-Kosack & Jones, 1996). This local response often induces a nonspecific resistance throughout the plant, which is termed systemic acquired resistance (SAR) and this provides protection against a wide range of subsequent pathogens (Baker et al., 1997; Ryals et al., 1996). During HR, accumulation of a group of major proteins termed pathogenesis-related (PR) proteins takes place throughout the plant. These PR proteins are classified in to 17 families based on the basis of their serological properties, biological activity and structural differences (Loon et al., 2006; Sels et al., 2008; Van

Loon et al., 1994). PR1 to PR4, PR9 and PR11 proteins have been characterized as β-1,3- glucanases, chitinases and peroxidases whereas PR6, PR12, PR13, PR14 and PR 15 were characterized as proteinase-inhibitors, defensins, thionins, lipid-transfer proteins and oxalate 12

oxidases (Edreva, 2005; Sels et al., 2008). In recent years, a member in PR10 family that has

RNase activity has been identified in a resistant soybean variety and it has been found that this protein plays an important role against P.sojae infection (Fan et al., 2015; Xu et al., 2014). Some of these PR proteins manifest antimicrobial activities but the direct functional role of them in the

R gene-mediated defense responses has not yet been elucidated for all.

A resistant plant that contains a single R gene (Rps) is only effective for 8-15 years because genetic variability and high reproduction rate of pathogen populations make the plant vulnerable to new pathotypes (Dorrance et al., 2003; Lee et al., 2013; Ruberson, 1999; Tooley & Grau,

1984). Thus, additional sources of resistance must be sought.

Another type of host resistance is partial resistance or tolerance which is also known as rate- reducing, field or race-nonspecific resistance. Race-specific resistance is superior to partial resistance in terms of the capacity to restrict pathogen invasion and also plant breeders prefer race-specific resistance due to the convenience of incorporation. Unlike Rps gene mediated resistance, partial resistance delays the development of disease by reducing the rate of the pathogen’s colonization in host tissues. However, partial resistance is polygenic, quantitatively inherited and has the advantage of efficacy against all pathotypes although its genetic nature is complex (Dorrance et al., 2003; Tooley & Grau, 1984). There are a couple of studies in which quantitative trait loci (QTL) for partial resistance to P. sojae have been mapped. Some of these

QTLs map to regions different from known locations of Rps genes and some QTLs are associated with Rps gene loci (Burnham et al., 2003). Moreover, based on the fact that the epidermis of plant roots is the site of initial invasion by soil-borne pathogens, genes that confer 13

preformed suberin in the roots is found to be a QTL that could be a part of overall partial resistance of the soybean host to P. sojae (Thomas et al., 2007). The extent to which all these types of resistance occur in the tissues of soybean host has yet to be discovered. However, exploring new sources of partial resistance and novel Rps genes would benefit future soybean breeding programs.

14

CHAPTER III

3.1 Zoospore chemotaxis to host plant metabolites

Recognition of host plants by the soil borne pathogens is affected by physical properties as well as chemical properties of the host tissues. They include chemical metabolites diffusing from the surface of the host tissues in contact with soil environment. Zoospores produced by

Peronosporales members (Phytophthora and Pythium) respond chemotactically to varying concentrations of some amino acids such as aspartate, asparagine, glutamate and glutamine

(Carlile, 1983; Deacon & Donaldson, 1993; Morris & Ward, 1992; Suo et al., 2016; Tyler,

2002). A variety of compounds including the above amino acids are exuded to the soil from plant roots (Rovira, 1969). Exudation from roots varies with the environmental conditions and therefore host recognition by pathogens is also influenced by the environment. For instance, zoospores are attracted to ethanol at a concentration of 25 mM (Morris & Ward, 1992). Ethanol is exuded by the flooded roots and this may promote the infection of flood-stressed plants by zoosporic pathogens. Exudation of some amino acids by roots varies with alternate wetting and drying of the soil, light intensity, temperature fluctuations and the age of the plant (Toussoun et al., 1970). This may lead to alteration of disease incidence in plants by these changing environments. Compounds such as amino acids attract zoospores nonspecifically but mixtures of multiple compounds might have either synergistic or antagonistic effect on host-specific taxis

(Deacon & Donaldson, 1993).

Host specific taxis had been observed in Phytophthora and Pythium species that have narrow host range. For example, graminicolous pathogens Pythium graminicola and Pythium 15

arrhenomanes are attracted specifically to grass hosts (Mitchell & Deacon, 1986). This

specificity is suggested to be achieved through signal molecules that are exuded by plant roots.

Isoflavones is one of the flavanoid classes derived from the phenylpropanoid pathway, which

acts as a signaling molecule for the chemotactic attraction of plant pathogenic microbes. Studies

have reported that zoospores produced by Aphanomyces euteiches, a legume pathogen, exhibit

specific attraction towards , an isoflavone exuded by pea roots, at a concentration down

to 10 nM (Sekizaki & Yokosawa, 1988). Zoospores of A. cochlioides are attracted to isoflavone

cochliophilin, an isoflavone exuded by the roots of its host, spinach, at a concentration down to 1

nM (Horio et al., 1992). and are also host-specific isoflavones exuded from

soybean roots to which P. sojae zoospores attract at concentrations as low as 0.25 nM (Morris &

Ward, 1992; Tyler et al., 1996).

3.2 Secondary metabolites as defense molecules

Phenylpropanoids produced by most legumes are not only acting as signaling molecules to

attract pathogens but also are involved in stress and disease resistance. Isoflavones are

synthesized via phenylproponoid pathway that also produces various other flavanoid compounds

in plants. The enzyme phenylalanine ammonia lyase (PAL) initiates the phenylproponoid

pathway by deaminating L-phenylalanine to generate a series of phenylpropanoid compounds

including isoflavones, , tannins and anthocyanins (fig. 1). 16

Figure 1. Schematic representation of the phenylpropanoid pathway (Adapted from Gonzalez et

al., 2010). PAL; Phenylalanine ammonia lyase, C4H; cinnamic acid 4-hydroxylase, 4CL; 4-

coumarate-CoA ligase, CHS; chalcone synthase, CHR; chalcone reductase, CHI; chalcone

isomerase, IFS; isoflavone synthase, F3H; flavanone 3-hydroxylase, DFR; dihydroflavonol

reductase and IFR; isoflavone reductase.

These phenylpropanoid derivatives are ubiquitously found in plants and are involved in various

biological activities. pigments such as anthocyanins in flowers produce colors that

attract pollinators. present in leaves are important for physiological survival of the

plants (Harborne & Williams, 2000). However, all these classes of phenylpropanoid compounds are not found in all higher plants. For example, and stilbenes are two classes of phenylpropanoids that are mostly found in legumes. 17

Natural metabolites that are involved in plant defense can be divided in to three groups namely phytoanticipins, phytoalexins and signaling molecules (Dixon et al., 2002). Phytoanticipins are antimicrobial compounds that are always present in the plant tissues. One example for a phytoanticipin is prenylated isoflavones which are being synthesized during seedling development of yellow lupin (Tahara et al., 1994). Phytoalexins are antimicrobial compounds that are synthesized in response to a pathogenic invasion (VanEtten et al., 1994). Phytoalexins in legumes include simple isoflavones such as genistein and daidzein, and complex isoflavones such as wightenone (Jeandet et al., 2013). The synthesis of phytoalexins could be induced by biotic elicitors such as carbohydrates from fungal cell walls, lipids and microbial enzymes and abiotic elicitors such as mechanical injury (Soylu et al., 2002).

Moreover, phenylpropanoid pathway could give rise to signal molecules in plant defense and in growth regulation. Salicylic acid is an example of a signal molecule synthesized from cinnamate produced by PAL. Salicyclic acid regulates activation of local and systemic pathogen-induced defense genes and pathogen-induced cell death. In addition, salicylic acid is involved in plants responses to abiotic stresses such as temperature fluctuation, salt and oxidative stresses (Gunes et al., 2007).

Tannin is another phenylpropanoid-derived polymer involved in the expression of color in seeds.

Although tannin is an anti-nutritional factor in legumes, they are beneficial in terms of resistance to pathogens and insect pests. Tannins present in leaves increase the oxidative activities giving rise to reactive oxygen species which in turn cause damage to herbivores (Barbehenn et al.,

2006; Peters & Constabel, 2002). Studies have shown that legume seeds that have a high content 18

of phenolic compounds contribute to resistance towards root rot diseases (Clauss, 1961; Statler,

1970). It has been reported that bean varieties having black seeds and purple hypocotyls were more resistant to Fusarium root rot compared to normal varieties (Statler, 1970). Islam et al.,

(2003) reported that higher content of phenolic compounds including tannin in black beans is associated with resistance to anthracnose caused by Colletotrichum lindemuthianum. Seeds of faba bean containing high tannin content were found to be protected from soil borne pathogens

Fusarium and Pythium compared to zero-tannin faba beans. The chemical and physical nature of tannin was impeding infection by fungal spores, while zero-tannin seeds are more susceptible due to frequent cracking of seeds (Kantar et al., 1996).

3.3 Objectives

This study focuses on evaluating chemotaxis of zoospores and pathogenicity of P. sojae race 2

(P6497) towards different annual lupins and a perennial lupin (L. perennis).

3.4 Hypotheses

I. The degree of zoospore attraction varies for different lines of lupin and there is a

correlation between pathogenicity of P. sojae and zoospore chemotaxis.

II. Different lines of lupin show a differential response to infection by P. sojae race 2.

III. Different seed phenotypes of perennial lupin deliver different levels of chemical

signals for zoospore attraction.

19

CHAPTER IV. METHODS

4.1 Evaluation of zoospore-chemotactic responses of P.sojae towards lupin roots

4.1.1 Plant materials

Thirteen germplasm accessions (referred as ‘lines’ in this study) of annual lupins and a perennial lupin (L. perennis) with different seed phenotypes were used in this study (table 1). All germplasm was received as seeds (fig. 2). Annual lupins were obtained from USDA-ARS grain legume collection held at Pullman, WA, USA and these annuals include 5 species namely L. albus, L. mutabilis, L. luteus, L. angustifolius and L. succulentus.

20

Table 1. Annual and perennial lupin lines used for chemotaxis and pathogenicity screening of

P.sojae.

Species Reference PI numbera Origin Year Germinationb number collected

Lupinus albus LAB18 543018 NA 2010 G

L. succulentus LS28 284728 United States, 1989 G

California

L. succulentus LS90 577290 United States 1991 G

L. mutabilis LM86 457986 Peru, Cuzco 2010 NG

L. mutabilis LM02 458002 Peru, Cuzco 2009 NG

L. mutabilis LM25 478525 Peru 2010 G

L. luteus LL50 240750 Germany 2008 G

L. luteus LL66 384566 Portugal 2009 G

L. luteus LL35 516635 Morocco 2008 G

L. luteus LL19 660719 Morocco 2010 G

L. angustifolius LAN05 385105 Spain 2009 G

L. angustifolius LAN86 385086 Spain 2008 G

L. angustifolius LAN39 383239 Spain 2009 G

L. perennis LP - United States 2014 G aPlant Introduction number assigned by USDA. bGermination status: G; at least 1 seed germinated out of a random sample of 5 and NG; No seeds germinated out of a random sample of 5. NA; Not available

The perennial species (L. perennis) was provided by Dr. Helen J Michaels, Department of 21

Biological Sciences at BGSU. These perennial seeds were separated into three phenotypes based on their degree of speckling; white/no speckles, lightly speckled & darkly speckled (fig. 3).

Figure 2. Seeds of annual lupin lines used for pathogenicity screening. Details of the species and corresponding abbreviations are listed in table 1.

Although seeds of annual lines also display different seed coat colors, due to the limitation of annual seeds, only perennial lupin was studied for the effect of seed coat coloring on pathogenicity and chemotaxis of P. sojae.

22

Figure 3. Three phenotypes of perennial lupin seeds categorized based on the degree of speckling; white/no speckles, lightly speckled and darkly speckled.

Seeds were surface sterilized by submerging first in 2.5% sodium hypochlorite solution and then

in 80% ethanol each for 10 mins followed by rinsing three times with sterile deionized water.

Then to initiate germination, seeds were scarified mechanically by nicking the seed coats with a sterile blade. Scarified aseptic seeds were transferred to perforated plastic containers that were

filled with sterile distilled water, and the seeds were covered with sterile, wet paper towels until

roots emerged. Seeds were germinated for 4-6 days at 21-25 °C in dark.

4.1.2 Preparation of zoospores

Phytophthora sojae race 2 (P6497) was used to produce zoospores for both pathogenicity and

chemotaxis assays. P. sojae cultures were grown and maintained on V8 juice agar (15% V8) at

25 °C. Zoospores were prepared from 5-7 day-old P. sojae cultures. These cultures were flooded with sterile distilled water for 18 hours to induce formation of sporangia. Then, cultures were washed repeatedly with sterile distilled water at 15 minute intervals until they started releasing spores. The number of zoospores reached 105/ml after approximately 13 washes, which was 23

followed by incubation of washed cultures in water for 1-2 hours (Eye, 1978). Zoospore density was determined using a hemocytometer and the zoospore suspensions were adjusted to the desired concentration with sterile distilled water.

4.1.3 Chemotaxis Assays

Chemotaxis assays with zoospores produced from P. sojae were carried out using 5 days old seedlings produced from Lupinus spp. listed in table 1. The assays were performed as described by Morris et al. (1998) using an assay chamber that can hold up to 600 µl of liquid. Distal ends of the lupin roots (~1 cm from the tip) were incubated in a zoospore suspension at a concentration of 8×103 spores/ml in assay chambers (fig. 4). After 20-30 minutes of incubation, roots were rinsed with sterile distilled water. Then the root sections were photographed under a microscope and the number of zoospores attached along a fixed distance (0.25 mm) of the root starting 1-1.2 mm from the root tip were counted and calculated. Zoospore encystment on roots of seedlings derived from each L. perennis seed type was also compared.

24

Figure 4. Incubation of lupin roots with a suspension of zoospores (8×103spores/ml) in an assay chamber.

4.2 Determination of chemotactic response towards metabolites present in different seed coat phenotypes of lupin

As described previously, I studied L. perennis seeds for any effect of their seed coat coloration

(degree of speckling) on chemotactic behavior of zoospores.

Chemical metabolites, possibly complexes of flavonoids, were extracted in 80% methanol

according to the method described by Kelly and Opiyo (personal communication, May 30,

2014). The extract was resuspended in sterile distilled water (30 µl per 1 mg of seed coat) and

filter sterilized using 0.22 µm syringe filters.

Agarose plugs (0.5%) containing seed coat extracts at a concentration of 1 mg seed coat per 1 ml

of agarose solution were prepared. These plugs were placed in chemotaxis chambers filled with

600 µl of zoospore suspension (1x103/ml). After 15 minutes, chemoattraction of the plugs was 25

observed under microscope and photographed. Agarose plugs containing only sterile distilled

water was used as control.

For positive control, synthetic isoflavone genistein (30 μM) was incorporated in the agar plugs to observe chemotaxis. Genistein was obtained from Sigma Aldrich and stock solutions of 10 mM were prepared by dissolving genistein powder in DMSO (Dimethyl sulfoxide) and stored at -

20°C until further use.

4.3 Pathogenicity assays

4.3.1 Pathogen inoculation

The same annual lupin and L perrennis lines (table 1) were used for pathogenicity assays. Four to six day-old seedlings were produced as described in 4.1.1. Plants were inoculated by incubating the root of each plant (up to1 cm distance from the root tip) in a zoospore suspension

(8×103 spores/ml) for 20 mins. After incubation, roots were washed briefly with sterile distilled

water and root tips of each plant were photographed under microscope and numbers of zoospores

adhered to the root surface were counted. These plants were then transferred to a plastic tray

filled with distilled water and covered with polythene to prevent them from drying. The plants were kept for 4-6 days at room temperature to observe symptoms of the infection.

4.3.2 Evaluation of disease severity

Pathogenicity of P. sojae was evaluated by rating the disease symptoms and by calculating disease incidence at 5-6 days post inoculation. Symptoms on the diseased seedlings were observed and recorded using a visual scale from 1 to 5 (fig. 5). Susceptible (Williams) and 26

resistant (Williams 82) soybean seedlings were used as controls. Susceptible plants develop soft

and water-soaked lesions on the site of infection within the first 5 days after inoculation and die.

Plants that exhibit resistance develop a hypersensitive reaction (HR) which appears as brown pigmentation at the site of infection during the first 12 hours after infection.

Disease severity increases 0

Figure 5. Root rot severity of infected lupin seedlings rated according to a visual scale of 0-5 compared to resistant soybean (SB-W82) and susceptible soybean (SB-W). 0 = resistant to infection (the plant shows a hypersensitive response); 1 = brown lesions on 0-30% of tap root; 2

= lesions extended up to 30-70% of tap root and hypocotyl; 3 = water soaked lesions on 70-90% of the plant; 4 = tap root and hypocotyl extensively girdled; 5 = plant dead.

Disease incidence was rated according to the scale described by Dorrance et al. (2008): 25 plants died = resistant; 26 to 75% plants died = intermediate and more than 75% plants died

=susceptible. 27

4.4 Data analysis

Differences in zoospore-root chemotaxis were tested using Kruskal-Wallis nonparametric

ANOVA test (Wallis & Kruskal, 1952). Differences in disease severity scores were also tested using Kruskal-Wallis nonparametric ANOVA test and average disease severity scores of each line were compared with the control using Steel’s test for multiple comparison (Steel, 1959).

Chemotaxis data from the agarose plug assay were tested using ANOVA followed by Tukey’s

HSD to compare the means. P<0.05 was considered as statistically significant for all the tests.

All statistical analyses were performed using JMP 7.0 (SAS Institute, Inc, Cary, NC) software.

28

CHAPTER V. RESULTS

5.1 Chemotaxis Assays

5.1.1 Zoospore-root attraction assays

In this study, intact plant roots were used for the chemotaxis assays where ~1 cm from the root tips were immersed in a zoospore suspension. Zoospore attraction was abundant in the elongation zone right above the root cap starting from about 1-2 mm from the root tip (fig. 6).

Very few zoospores were adhered to the root cap and the area of cell differentiation/maturation.

Majority of the zoospores attached to the elongation zone appeared to encyst and continued forming the germ tube. Most zoospores attached and encysted on the elongation zone (fig. 6 and fig. 7-A) and a few zoospores were found to attract to the immediate zone of root hairs.

Zoospores can become trapped between the root hairs, and also seem to be encysting on the root hairs (fig. 7-B). Only those adhered to the root surface along a fixed distance (0.25 mm) were counted in the chemotaxis assays.

29

Elongation zone Root cap

Figure 6. Abundance of zoospore attachment along a lupin root.

Figure 7. A) Zoospores encysting and germinating on the root surface of elongation zone. B)

Zoospores encysting and germinating on root hairs.

The average density of the attached zoospores was not noticeably different on each lupin line

(fig. 8). After analyzing the chemotaxis data using Kruskal-Wallis ANOVA test, there was no significant difference (p value> 0.05) between the lines with respect to average number of zoospores attached along a fixed root length. 30

along tip root 0.25mm the along of Average number zoospores attached of Average

Line

Figure 8. Chemoattraction of zoospores to the roots of lupin lines tested. LAB18; L. albus

543018 (n=9), LS 28; L. succulentus 284728 (n=2); LM 25; L. mutabilis 478525 (n=3), LL 50; L. luteus 240750 (n=5), LL 66; L. luteus 384566 (n=6), LL 35; L. luteus 516635 (n=6), LL 19; L. luteus 660719 (n=9), LAN 05; L. angustifolius 385105 (n=12), LAN 86; L. angustifolius 385086

(n=11), LAN 39; L. angustifolius 383239 (n=6), LP-D; L. perennis darkly speckled seed (n=8),

LP-L; L. perennis lightly speckled seed (n=8), LP-N; L. perennis seed with no speckles (n=10),

SB-W; soybean (Glycine max) variety Williams (n=7), SB-W82; soybean variety Williams 82

(n=7).

5.2 Evaluation of disease severity

According to the pathogenicity assays performed, majority of the lupins were susceptible at varying degrees and many of them exhibited the typical symptoms of the root and hypocotyl rot.

The symptoms were first visible as a light brown soft rot in the roots. The infection spread as 31

water soaked lesions towards the hypocotyl leading to rots of roots and hypocotyls during the first 5 days post inoculation. A few lines (LAB 18, LL 35, LL 50, LL66 and LAN 86) survived from the disease compared to 100% rot of susceptible soybean variety Williams. Among the lines tested, many L. albus (LAB 18) and L. luteus (LL 35, LL 50 and LL66) seedlings showed a restricted dark brown lesion at the site of infection known as hypersensitive reaction (HR). HR is a form of localized programmed cell death that is visible as a localized tissue necrosis. It is a defense response resulting from the activation of resistance genes to restrict or even to kill the pathogen without further spreading of the pathogen in the surrounding host tissues (Hammond-

Kosack & Jones, 1996).

Germination of LM 25 and LS 28 lines was poor therefore it was not possible to include them in the second experiment. LS 90 did not germinate at all. Since the number of seeds obtained from

USDA was limited, the experiments could not be repeated for a third time with large replicates

(more than 15 replicates).

I observed variations in the tissue responses after inoculation in individual lines. Possible reasons for this variation of symptoms within lines can be due to environment-induced susceptibility such as temperature (Gijzen et al., 1996).

The root rot symptoms were scored according to a visual scale modified from the method described by Loyd et al. (2014). Average disease severity scores of each line were compared with resistant Williams 82 using Steel’s method for nonparametric comparison. LAB 18 and LL

35 showed the highest resistance according to the graph (fig 9). LAN 86 also may harbor resistance but further testing is required with higher sample numbers because of the high error bar. Resistance level was weaker in LL 66 and therefore it shows intermediate resistance 32

compared to Williams 82. The resistance of these four lines was comparable to the resistant

Williams 82 at a significance level of 0.05 (table 2). LS 28 also shows a comparable p value but it was opted out due to very low replicate number.

33

Table 2. Nonparametric comparison with the resistant control using Steel Method.

aScore Mean Line Control Difference bStd Err Dif P-Value

LL35 SB-W82 1.51 2.55 0.9997

LAB18 SB-W82 0.69 2.85 1.0000

LAN86 SB-W82 -0.97 2.78 1.0000

LL66 SB-W82 -7.71 2.86 0.0655

LS28 SB-W82 -6.79 2.45 0.0537

LM25 SB-W82 -7.85 2.39 0.0113

LL50 SB-W82 -8.63 2.67 0.0132

LL19 SB-W82 -10.24 2.89 0.0046

P-L SB-W82 -10.71 2.90 0.0026

LAN05 SB-W82 -10.74 2.80 0.0015

SB-W SB-W82 -12.26 2.89 0.0003

P-D SB-W82 -12.50 3.03 0.0005

LAN39 SB-W82 -13.27 3.04 0.0002

P-N SB-W82 -13.42 3.03 0.0001 aDifference between the mean severity score of each line and the resistant control SB-W82. bStandard error of the difference between score means. Boxed numbers show no significant difference from the control (p>0.05).

34

6 * 5 * * * * * 4 * * * 3

2

1

Average disease severity Average score 0

Line

Figure 9. Disease severity of lupin lines tested. LAB18; L. albus 543018 (n=20), LS 28; L.

succulentus 284728 (n=3); LM 25; L. mutabilis 478525 (n=5), LL 50; L. luteus 240750 (n=11),

LL 66; L. luteus 384566 (n=16), LL 35; L. luteus 516635 (n=12), LL 19; L. luteus 660719

(n=14), LAN 05; L. angustifolius 385105 (n=12), LAN 86; L. angustifolius 385086 (n=14), LAN

39; L. angustifolius 383239 (n=15), LP-D; L. perennis darkly speckled seed (n=15), LP-L; L.

perennis lightly speckled seed (n=13), LP-N; L. perennis seed with no speckles (n=15), SB-W;

soybean (Glycine max) variety Williams as susceptible control (n=13), SB-W82; soybean variety

Williams 82 as resistant control (n=10). Asterisks on a column indicate a statistically significant difference from the resistant soybean variety Williams 82 with a p-value below 0.05.

5.3 Evaluation of disease incidence

In addition to rating disease severity, disease incidence/percentage of dead plants was also

observed to determine root rot susceptibility/resistance. The scale described by Dorrance et al. 35

(2008) was used to evaluate disease incidence. According to disease incidence values, only two

lupin lines showed resistance to root rot with disease incidences less than 25% (fig. 10).

100% 90% 80% 70% 60% 50% 40% 30% 19% 20% 12% 10% 10% 0% Percentage plant death 6 days post 6 days inoculation Percentage plant death

Line

Figure 10. Disease incidence (percentage plant death) of the lupin lines tested. LAB18; L. albus

543018, LS 28; L. succulentus 284728; LM 25; L. mutabilis 478525, LL 50; L. luteus 240750,

LL 66; L. luteus 384566, LL 35; L. luteus 516635, LL 19; L. luteus 660719, LAN 05; L. angustifolius 385105, LAN 86; L. angustifolius 385086, LAN 39; L. angustifolius 383239, LP-L;

L. perennis lightly speckled seed, LP-D; L. perennis darkly speckled seed, LP-N; L. perennis no

speckles on seed, SB-W; soybean (Glycine max) variety Williams susceptible to P. sojae race 2

infection, SB-W82; soybean variety Williams 82 resistant to P. sojae race 2 infection.

Although the resistant and susceptible properties vary along lupin lines, such a variation in terms of zoospore attraction cannot be observed (fig. 11). 36

Figure 11. Comparison of zoospore attachment and disease severity. SB-W82; soybean variety

Williams 82, SB-W; soybean variety Williams, LS 28; L. succulentus 284728, LL 35; L. luteus

516635. 37

Correlation between zoospore chemotaxis and pathogenicity was tested using Spearman’s rho test and the results showed that the correlation between the two variables was low and not significant (p>0.05).

5.4 Zoospore chemotaxis towards seed coat extracts

According to the results of ANOVA, the amount of zoospores attached to seed coat extracts of the three seed phenotypes were significantly higher than the control with p values greater than

0.05 (fig. 12). The chemoattraction to darkly speckled seeds was significantly higher than lightly speckled seeds and the seeds with no speckles. But the zoospore count was not significantly different between lightly speckled and non-speckled seed phenotypes (fig. 12). This suggests that metabolites present in lupin seed coats were extracted into methanol and the zoospores exhibit a preferential response to these metabolites from all three seed phenotypes compared to the control

(water) (fig. 13). Also, it is evident that P. sojae zoospores exhibit a preferential response to seed coat extracts from dark seeds compared to the other two seed phenotypes possibly due to the high abundance of isoflavones in darkly speckled seed coat.

38

Figure 12. Average number of zoospores attracted to a unit area of the agar plug (1.2 mm2) containing the metabolites extracted from three seed phenotypes of perennial lupin; darkly speckled (n=5), lightly speckled (n=5) and seeds with no speckles (n=3). 3-5 zoospore counts per replicate. Means with different letters are significantly different (p<0.05). 39

Figure 13. Representation of microscopic images of zoospore accumulations on the agar plugs containing metabolites extracted from three seed phenotypes of perennial lupin; darkly speckled, lightly speckled and seeds with no speckles.

40

CHAPTER VI. DISCUSSION

6.1 Zoospore-root attraction assays

In previous studies, cut root segments of approximately 1-2 cm were used for the convenience of

inoculation and microscopic observation (Beagle-Ristaino & Rissler, 1983; Jones et al., 1991;

Messenger et al., 2000). However, using cut root segments causes alterations to the root

physiology and exudation properties that could subsequently affect the zoospore attraction in the

long term (Raftoyannis & Dick 2006). Previous reports that used cut root segments for zoospore

chemotaxis have found inconsistent results (Ho & Hickman, 1967). For this reason, intact plant

roots were chosen for zoospore-root chemotaxis assay in this study. Milholland (1972) and Chi

& Sabo (1978) reported that there is a positive correlation between zoospore chemotaxis and

susceptible plants while Tippett et al.(1976) and Halsall (1978) showed that there was no

relationship of zoospore chemotaxis with disease severity on susceptible plants.

Chi & Sabo, (1978) reported that susceptible roots showed increased accumulation of P.

megasperma zoospores compared to a resistant cultivar. They also observed that several non-

host legumes showed very weak or no chemotaxis of zoospores towards their roots. Another

study by Milholland (1972) also reported that a blueberry species susceptible to P. cinnamomi is positively related to higher zoospore accumulation on the roots compared to a resistant species.

In contrast, Halsall (1978) and Tippett et al. (1976) showed that there is no relationship of zoospore taxis or encystment with the pathogenicity of P. cinnamomi on root rot susceptible and resistant species. Raftoyannis & Dick (2006) examined the encystment density and pathogenicity of 12 isolates of Pythium and Phytopthora species on seven plant species (lucerne, 41

wheat, maize, sorghum, oat, sugar beet and tomato) that have been reported as hosts of these pathogens and they found no correlation between the zoospore encystment density and disease severity of the plant species tested.

In this study, the correlation between zoospore chemotaxis and the root rot susceptibility was low and not significant (p>0.05).

The zoospore attraction was more abundant in the elongation zone of the root compared to the root cap and the area of cell differentiation. Similar observations were documented in the literature (Gow, 1993; Hosseini et al., 2014; Wester et al., 2012) and this preferential accumulation of zoospores at the root elongation zone is suggested to be due to electrochemical and physical properties of plant roots (Gow et al., 1992; Miller et al., 1988).

Zoosporic pathogens exploit the host specific chemicals to locate and encyst on the host tissues.

For instance, zoospores of P. sojae are attracted to host-specific isoflavones (Morris & Ward,

1992) through a specific receptor based recognition system (Yang et al., 2013). After encystment, zoospores penetrate and invade the host cells in order to establish a successful infection. It has been suggested that the zoospore stage of the pathogens is non-specific unless the host selection takes place, and the specificity arises with the attempted penetration and invasion of the pathogen in the plant (van West et al., 2003).

In the current study using intact roots, I did not see a differential attachment of zoospores between resistant soybean variety Williams 82 and soybean variety Williams which is susceptible to P. sojae (race 2). A detailed examination of the sequential events that occur during infection is required to further investigate the effect of race-specific resistance on progression of 42

zoosporic pathogens. Enkerli et al. (1997) examined the progression of two P. sojae races (race 2 and race 8) on two soybean lines (Rps1b and Rps1a) that show resistance and susceptibility to each race using light and electron microscopes. In the virulent interaction, P. sojae showed a short biotrophic phase establishing many haustoria without inducing necrosis in the susceptible host. In the avirulent/incompatible interaction, the pathogen has triggered rapid cell necrosis with less numbers of haustoria and the pathogen rarely penetrated the endodermis towards the vascular tissue in the resistant host. These observations confirmed the results of previous histopathological studies of P. sojae on resistant and susceptible soybean roots (Beagle-Ristaino

& Rissler, 1983; Ward et al., 1981).

At the present time, the consensus opinion is that the specificity and the pathogenicity of zoospores are not prominent in the zoospore stage, but they become pathogenic at the subsequent stages of pathogenic invasion. Further examination on the invading hyphae would be necessary to determine what type of resistance the plant is equipped with.

6.2 Screening of lupin accessions for root rot resistance under laboratory condition

According to the gene-for-gene model, there is an interaction of corresponding pathogen Avr gene of P. sojae for each Rps gene of the host, resulting in activation of defense responses (Jiang

& Tyler, 2012; Tyler, 2007). Some of these Avr genes from P. sojae have been characterized and they are predicted to encode RxLR-type effectors. A single P. sojae isolate has one or more Avr genes that correspond to noted Rps genes (Dong et al., 2011). These isolates of P. sojae are categorized into races or pathotypes based on their virulence on soybean lines that contain different Rps genes (Robertson et al., 2009). Currently, over 200 pathotypes or races of P. sojae have been recognized (Abney et al., 1997; Dorrance et al., 2003; Dorrance et al., 2016; Kaitany 43

et al., 2001; Leitz et al., 2000). Since the complexity of the pathotypes continue to increase

through evolution (Dorrance et al., 2003; Dorrance et al., 2016; Robertson et al., 2009), the

emerging P. sojae populations develop resistance to Rps genes that are commonly deployed in

commercial cultivars (Kaitany et al., 2001). Thus, many soybean germplasm accessions are screened using different pathotypes of P. sojae to identify novel Rps genes (Burnham et al.,

2003; Kyle et al., 1998).

Phytophthora root rot was first observed in the midwestern United States (in Indiana in 1948 and

in Ohio in 1951), and the respective pathogen, P. sojae is believed to be native to the United

States (Schmitthenner, 1999). P. sojae has been reported to be a pathogen of lupin (Erwin &

Ribeiro, 1996; Jones & Johnson, 1969). Therefore, lupin might be a natural host of this pathogen

and there is a potential for lupin germplasm to develop resistance to one or more races of P.

sojae through co-evolution as there are no recent reports of lupin infecting P. sojae isolates.

Based on this fact, this study used 10 annual lupin lines (LM 02, LM 86 and LAN 05 lines did

not germinate) and selected seed phenotypes of a perennial lupin line to screen for resistance

against Phytophthora root rot using P. sojae race 2. The roots of 4-6 days-old seedlings were

inoculated with a zoospore suspension (8x103/ml) for 20 mins and then transferred to a

perforated tray filled with water, after a brief wash. Many studies screened soybean germplasm

for potential Rps genes by injecting an agar/mycelia slurry onto the wounded hypocotyls of the

seedlings using a syringe (Dorrance et al., 2008; Ping et al., 2015; Yang et al., 1996). While

many studies used a mycelial slurry for the screening of different soybean accessions to P. sojae

races, Kyle et al. (1998) inoculated soybean hypocotyls by injecting a zoospore suspension. Gao

et al., (2015) applied zoospore suspensions onto etiolated soybean seedlings to examine the P. 44

sojae pathogenicity. Inoculation of plant roots by zoospores was also performed for histopathological studies (Beagle-Ristaino & Rissler, 1983; Enkerli et al., 1997; Hahn et al.,

1985). In this study, I used a zoospore-root inoculation method to mimic the natural infection process in the field because zoospore is the primary infectious propagule of P. sojae and root tissues are the most frequent sites of infection in this pathogen’s natural environment (Dorrance et al., 2007).

In general, Rps gene resistance is active in the plants from germination onwards and partial resistance is activated typically after the appearance of first true leaves (Dorrance et al., 2007).

To avoid the confusion that might be created by the partial resistance, seedlings at the age of 4-6 days were used for the pathogenicity screening in this study. Soybean cultivar Williams 82 has the gene Rps1k against P. sojae race 2 that contains the avirulence gene Avr1k (Jing et al., 2015;

Yang et al., 1996). Therefore, Williams 82 was used as the resistant control and the soybean variety Williams that has no known Rps genes was used as the susceptible control (Lin et al.,

2013; Yang et al., 1996).

According to this study, only LL 35 and LAB 18 exhibited resistance against P. sojae race 2 with regard to both disease incidence data and disease severity data. Therefore, these two lines can be suggested as resistant against P. sojae (race 2) under laboratory conditions. Further molecular analysis is required to investigate whether this is due to R-mediated resistance or another source of resistance. Further screening of these lines for resistant genes can be optimized with the advent of newly developed screening systems.

Many phytopathogens including oomycetes deploy effector proteins to overcome the immune

reactions in host plants. The functions of bacterial effectors in pathogenesis have been 45

extensively studied and these effector proteins are secreted directly into the cytoplasm of plant

cells. Pseudomonas syringae is a bacterial pathogen of Arabidopsis, tobacco and tomato that

utilizes the type III secretion system (TTSS) to translocate their effectors to the host cell

cytoplasm where the N termini of their TTSS effectors carry the translocation signal (Grant et

al., 2006). Several groups have developed efficient methods to deliver oomycete RxLR effectors

into plant cells using bacterial TTSS (Rentel et al., 2008; Sohn et al., 2007). Sohn et al. (2007)

reported that fusion of effector genes from oomycete pathogen Hyaloperonospora parasitica to

the N terminus of TTSS effector of P. syringae (DC3000 mutant) induced HR in Arabidopsis

plants containing the corresponding R gene. By inoculation of the genetically engineered P.

syringae bacteria using leaf infiltration assays, the oomycete effector is expressed and processed

in the plant cell that will be recognized by the plant cell to induce HR. These findings led to in

planta high-throughput screening of germplasms to identify potential R genes.

6.3 Zoospore chemotaxis towards seed coat extracts

Germinating seeds imbibe water and release biologically active molecules such as amino acids,

peptides, alkaloids, flavonoids, anthocyanins, termpenoids and that are important for the

establishment of the seeds (Ndakidemi & Dakora, 2003). Amongst these exuded chemicals,

isoflavones are a group of flavonoids derived from phenylproponoid pathway (Winkel-Shirley,

2001). Isoflavones are of profound importance as nodulation signals for rhizobial species as well

as chemoattractants for oomycete pathogens (Steinkellner et al., 2007). These chemical/phenolic compounds present in the seed coat are released into the soil and some of them are transported to the leaves in developing seedlings. Ndakidemi & Dakora (2003) reported that seeds of some 46

legumes (cowpea, some beans and groundnut species) release significant amounts of flavonoids and anthocyanins when they are soaked in a solvent such as water or aqueous methanol.

Kim & Kim (1999) and Kim et al. (2007) extracted isoflavones from whole seeds of soybean and they reported that black soybean seeds have a higher amount of total isoflavones compared to yellow seeds on average. In general, distribution of isoflavones varies in different parts of soybean seeds, the embryo being the highest, and the seed coat, the lowest (Kim et al., 2007).

Ciabotti et al. (2016) extracted isoflavones (genistein, daidzein and ) from whole seeds of soybean using a methanol based extraction protocol followed by HPLC. They used four genotypes of soybean seed coats (dark yellow, light yellow, matte brown and glossy black) for their study and they found that, the dark-yellow seeded cultivar exhibited the highest isoflavone content, followed by black seeded cultivar having the next highest as compared to the genotypes studied. Similarly, Lee et al. (2010) reported that the yellow seeded soybean cultivars have a higher amount of total isoflavones compared to green and black seeded cultivars. In contrast,

Cho et al. (2013) reported that with a green seed coat have higher total isoflavones content compared to yellow and black seeds. However, previous studies suggest that the isoflavone content of the seeds varies significantly with a number of other factors including plant height, yield, and environmental factors such as crop year, location, rainfall, and temperature.

To examine whether there is any effect of the seed coat color (degree of speckling) on chemotactic properties of zoospores, I extracted metabolites (possibly isoflavones) from three seed phenotypes of perennial lupin using a method described by Kelly and Opiyo (personal communication, May 30, 2014). There are host-specific isoflavones such as genistein and 47

daidzein exuded by the plant roots that chemotactically attract P. sojae zoospores to soybean roots (Morris & Ward, 1992).

I incorporated these metabolite-extracts into agarose plugs for chemotaxis studies to observe the zoospore attraction and I found that the zoospore attraction was higher towards the seed coat extracts from darkly speckled compared to seeds with no speckles. However, there is no preferential accumulation of zoospores between the metabolites extracted from lightly speckled and non-speckled seeds. Possible reasons for this observation could be due to the seed coat metabolites (possibly isoflavones) were present in similar amounts in lightly speckled seeds and the seeds with no speckles and/or the extraction method is not sensitive enough to isolate all metabolites from seed coats. Since the amount of isoflavones present in one agarose plug was not quantified either as how many seed coats were present in one agarose plug or how much isoflavones were present in one agarose plug, I find it inconceivable to conclude that seed coat colors do not play roles in zoospore chemotaxis. Therefore, this experiment requires further testing.

48

CHAPTER VII. CONCLUSIONS AND FUTURE DIRECTIONS

In the chemotaxis assay, P. sojae zoospores did not show differential attachment on the roots of the tested lupin lines although the disease symptoms varied between them. The zoospore attraction seems to be non-host specific suggesting that zoospore attraction does not necessarily

lead to successful infection.

In the pathogenicity assay, the two lupin lines LAB 18 ( L. albus 543018) and LL 35 (L. luteus

516635) are found to be resistant against infection by P. sojae race 2 while LAN 86 (L. angustifolius 385086) and LL 66 (L. luteus 384566) show intermediate resistance.

When using seed coat extracts from lupin exhibiting different degrees of speckles, the zoospore attachment was significantly higher towards the metabolites extracted from seed coats of all

three seed phenotypes compared to the control. Furthermore, zoospores exhibited a preferential response to the seed coat extract of dark seeds compared to the non-speckled seeds.

To further confirm the resistance of LAB 18 and LL 35 against P. sojae, screening using multiple isolates of P. sojae is necessary. For this, a high throughput screening method developed by Sohn et al. (2007) and Anderson et al. (2012) can be used. This approach uses bacterial type III secretion system (TTSS) to deliver genes into plant cells. To perform the high- throughput screening, candidate P. sojae effectors that are expressed during infection can be delivered into plant cells via Pseudomonas syringae DC3000 by constructing surrogate bacterial

TTSS effector (possibly AvrRpm1 of P. syringae). These selected P. sojae effectors can be cloned with the promoter including the secretion-translocation signal of a TTSS effector into a vector that can replicate in P. syringae cells. The effector expression can then be confirmed by 49

tagging it with an HA epitope using immunoblot detection. Inoculation of P. syringae containing the effectors into the plant cells using leaf infiltration assays and the development of HR would indicate the resistance against certain effectors.

50

REFERENCES

Abney, T., Melgar, J., Richards, T., Scott, D., Grogan, J., & Young, J. (1997). New races of

Phytophthora sojae with rps 1-d virulence. Plant Disease, 81(6), 653-655.

Agrawal, A., & Lively, C. M. (2002). Infection genetics: Gene-for-gene versus matching-alleles

models and all points in between. Evolutionary Ecology Research, 4(1), 91-107.

Anderson, R. G., Casady, M. S., Fee, R. A., Vaughan, M. M., Deb, D., Fedkenheuer, K., . . .

McDowell, J. M. (2012). Homologous RXLR effectors from Hyaloperonospora

arabidopsidis and Phytophthora sojae suppress immunity in distantly related plants. The

Plant Journal, 72(6), 882-893.

Baker, B., Zambryski, P., Staskawicz, B., & Dinesh-Kumar, S. P. (1997). Signaling in plant-

microbe interactions. Science (New York, N.Y.), 276(5313), 726-733.

Baldauf, S. L., Roger, A. J., Wenk-Siefert, I., & Doolittle, W. F. (2000). A kingdom-level

phylogeny of eukaryotes based on combined protein data. Science, 290(5493), 972-977.

doi:10.1126/science.290.5493.972

Bantignies, B., Séguin, J., Muzac, I., Dédaldéchamp, F., Gulick, P., & Ibrahim, R. (2000). Direct

evidence for ribonucleolytic activity of a PR-10-like protein from white lupin roots. Plant

Molecular Biology, 42(6), 871-881. doi:10.1023/A:1006475303115

Barbehenn, R. V., Jones, C. P., Karonen, M., & Salminen, J. (2006). Tannin composition affects

the oxidative activities of tree leaves. Journal of Chemical Ecology, 32(10), 2235-2251. 51

Beagle-Ristaino, J. E., & Rissler, J. F. (1983). Histopathology of susceptible and resistant

soybean roots inoculated with zoospores of Phytophthora megasperma f. sp. glycinea.

Phytopathology, 73(4), 590-595.

Beakes, G. W., Glockling, S. L., & Sekimoto, S. (2012). The evolutionary phylogeny of the

oomycete “fungi”. Protoplasma, 249(1), 3-19.

Bhattacharyya, M. K., Narayanan, N. N., Gao, H., Santra, D. K., Salimath, S. S., Kasuga, T., . . .

Buzzell, R. I. (2005). Identification of a large cluster of coiled coil-nucleotide binding site–

leucine rich repeat-type genes from the Rps1 region containing Phytophthora resistance

genes in soybean. Theoretical and Applied Genetics, 111(1), 75-86. doi:10.1007/s00122-

005-1993-9

Birch, P. R., Boevink, P. C., Gilroy, E. M., Hein, I., Pritchard, L., & Whisson, S. C. (2008).

Oomycete RXLR effectors: Delivery, functional redundancy and durable disease resistance.

Current Opinion in Plant Biology, 11(4), 373-379.

Bourke, A. (1993). 'The visitation of god?'the potato and the great irish famine. Lilliput Press

Ltd.

Bourke, P. A. (1968). The use of the potato crop in pre-famine Ireland. Journal of the Statistical

and Social Inquiry Society of Ireland, 12(6), 72-96.

Bozkurt, T. O., Schornack, S., Banfield, M. J., & Kamoun, S. (2012). Oomycetes, effectors, and

all that jazz. Current Opinion in Plant Biology, 15(4), 483-492.

doi:http://dx.doi.org.ezproxy.bgsu.edu:8080/10.1016/j.pbi.2012.03.008 52

Burnham, K., Dorrance, A., Francis, D., Fioritto, R., & St Martin, S. (2003). 8, A new locus in

soybean for resistance to. Crop Science, 43(1), 101-105.

Carlile, M. J. (1983). Motility, taxis, and tropism in phytophthora. Phytophthora, it's Biology

Taxonomy, Ecology and Pathology, , 95-107.

Chi, C., & Sabo, F. (1978). Chemotaxis of zoospores of phytophthora megasperma to primary

roots of alfalfa seedlings. Canadian Journal of Botany, 56(7), 795-800.

Cho, K. M., Ha, T. J., Lee, Y. B., Seo, W. D., Kim, J. Y., Ryu, H. W., . . . Lee, J. H. (2013).

Soluble phenolics and antioxidant properties of soybean (glycine max L.) cultivars with

varying seed coat colours. Journal of Functional Foods, 5(3), 1065-1076.

doi:http://dx.doi.org/10.1016/j.jff.2013.03.002

Ciabotti, S., Silva, A., Juhasz, A., Mendonça, C., Tavano, O., Mandarino, J., & Gonçalves, C.

(2016). Chemical composition, protein profile, and isoflavones content in soybean

genotypes with different seed coat colors. International Food Research Journal, 23(2)

Clauss, E. (1961). The phenolic constituents of the testas of peas and their importance for

resistance to the agents of root rot. Naturwissenschaften, 61, 106.

Collier, S. M., & Moffett, P. (2009). NB-LRRs work a “bait and switch” on pathogens. Trends in

Plant Science, 14(10), 521-529. doi:10.1016/j.tplants.2009.08.001

Connolly, M. S., Williams, N., Heckman, C. A., & Morris, P. F. (1999). Soybean isoflavones

trigger a calcium influx in Phytophthora sojae. Fungal Genetics and Biology, 28(1), 6-11. 53

Cosio, E. G., Feger, M., Miller, C. J., Antelo, L., & Ebel, J. (1996). High-affinity binding of

fungal β-glucan elicitors to cell membranes of species of the plant family

Fabaceae. Planta, 200(1), 92-99.

Culvenor, C., Beck, A., Clarke, M., Cockrum, P., Edgar, J., Frahn, J., . . . Peterson, J. (1977).

Isolation of toxic metabolites of Phomopsis leptostromiformis responsible for lupinosis.

Australian Journal of Biological Sciences, 30(4), 269-278.

Deacon, J. W., & Donaldson, S. P. (1993). Molecular recognition in the homing responses of

zoosporic fungi, with special reference to Pythium and Phytophthora. Mycological Research,

97(10), 1153-1171. doi:10.1016/S0953-7562(09)81278-1

Dick, M. W. (2013). Straminipilous fungi: Systematics of the peronosporomycetes including

accounts of the marine straminipilous protists, the plasmodiophorids and similar organisms

Springer Science & Business Media.

Dixon, R. A., Achnine, L., Kota, P., Liu, C., Reddy, M., & Wang, L. (2002). The

phenylpropanoid pathway and plant defence—a genomics perspective. Molecular Plant

Pathology, 3(5), 371-390.

Dodds, P. N., Rafiqi, M., Gan, P. H. P., Hardham, A. R., Jones, D. A., & Ellis, J. G. (2009).

Effectors of biotrophic fungi and oomycetes: Pathogenicity factors and triggers of host

resistance. New Phytologist, 183(4), 993-999. doi:10.1111/j.1469-8137.2009.02922.x

Dodds, P. N., & Rathjen, J. P. (2010). Plant immunity: Towards an integrated view of plant–

pathogen interactions. Nature Reviews Genetics, 11(8), 539-548. doi:10.1038/nrg2812 54

Dong, S., Yu, D., Cui, L., Qutob, D., Tedman-Jones, J., Kale, S. D., . . . Gijzen, M. (2011).

Sequence variants of the phytophthora sojae RXLR effector Avr3a/5 are differentially

recognized by rps 3a and rps 5 in soybean. PLoS One, 6(7), e20172.

Dorrance, A., Kurle, J., Robertson, A., Bradley, C., Giesler, L., Wise, K., & Concibido, V.

(2016). Pathotype diversity of Phytophthora sojae in eleven states in the United States. Plant

Disease, , PDIS-08-15-0879-RE.

Dorrance, A., McClure, S., & DeSilva, A. (2003). Pathogenic diversity of Phytophthora sojae in

Ohio soybean fields. Plant Disease, 87(2), 139-146.

Dorrance, A., & Schmitthenner, A. (2000). New sources of resistance to Phytophthora sojae in

the soybean plant introductions. Plant Disease, 84(12), 1303-1308.

Dorrance, A. E., Berry, S. A., Anderson, T. R., & Meharg, C. (2008). Isolation, storage,

pathotype characterization, and evaluation of resistance for Phytophthora sojae in soybean.

Plant Health Progress, 10, 1094.

Dorrance, A. E., Mills, D., Robertson, A. E., Draper, M. A., Giesler, L., & Tenuta, A. (2007).

Phytophthora root and stem rot of soybean. The Plant Health Instructor, , 1.

Drummond, C. S., Eastwood, R. J., Miotto, S. T. S., & Hughes, C. E. (2012). Multiple

continental radiations and correlates of diversification in Lupinus (leguminosae): Testing for

key innovation with incomplete taxon sampling. Systematic Biology, 61(3), 443-460.

doi:10.1093/sysbio/syr126 55

Ebel, J., Feger, M., Kissel, U., Mithöfer, A., Waldmüller, T., Bhagwat, A. A., & Cosio, E. G.

(1995). Elicitor-binding proteins and signal transduction in the activation of a phytoalexin

defense response. Canadian journal of botany,73(S1), 506-510.

Edreva, A. (2005). Pathogenesis-related proteins: Research progress in the last 15 years. Gen

Appl Plant Physiol, 31(1-2), 105-124.

Enkerli, K., Mims, C., & Hahn, M. (1997). Ultrastructure of compatible and incompatible

interactions of soybean roots infected with the plant pathogenic oomycete phytophthora

sojae. Canadian Journal of Botany, 75(9), 1493-1508.

Erwin, D. C., & Ribeiro, O. K. (1996). Phytophthora diseases worldwide. American

Phytopathological Society (APS Press).

Eye, L. L. (1978). Factors affecting zoospore production by Phytophthora megasperma var.

sojae. Phytopathology, 68(12), 1766. doi:10.1094/Phyto-68-1766

Fan, S., Jiang, L., Wu, J., Dong, L., Cheng, Q., Xu, P., & Zhang, S. (2015). A novel

pathogenesis-related class 10 protein gly m 4l, increases resistance upon Phytophthora sojae

infection in soybean (Glycine max [L.] merr.). PloS One, 10(10), e0140364.

Fliegmann, J., SCHüLER, G., Boland, W., Ebel, J., & Mithöfer, A. (2003). The role of

octadecanoids and functional mimics in soybean defense responses. Biological Chemistry,

384(3), 437-446. 56

Flor, H. H. (1971). Current status of the gene-for-gene concept. Annual Review of

Phytopathology, 9(1), 275-296.

Fry, W. E., & Grünwald, N. J. (2010). Introduction to Oomycetes. The Plant Health Instructor.

DOI: 10.1094. The Plant Health Instructor.

Gagnon, H. (1997). Molecular signalling in Lupinus albus-Rhizobium lupini symbiosis (Doctoral

dissertation, Concordia University).

Gao, J., Cao, M., Ye, W., Li, H., Kong, L., Zheng, X., & Wang, Y. (2015). PsMPK7, a

stress‐associated mitogen‐activated protein kinase (MAPK) in Phytophthora sojae, is

required for stress tolerance, reactive oxygenated species detoxification, cyst germination,

sexual reproduction and infection of soybean. Molecular , 16(1), 61-70.

Gao, H., & Bhattacharyya, M. K. (2008). The soybean-phytophthora resistance locus Rps1-k

encompasses coiled coil-nucleotide binding-leucine rich repeat-like genes and repetitive

sequences. BMC Plant Biology, 8(1), 29-29. doi:10.1186/1471-2229-8-29

Gijzen, M., Mac Gregor, T., Bhattacharyya, M., & Buzzell, R. (1996). Temperature induced

susceptibility to Phytophthora sojae in soybean isolines carrying different Rps genes.

Physiological and Molecular Plant Pathology, 48(3), 209-215.

doi:http://dx.doi.org.ezproxy.bgsu.edu:8080/10.1006/pmpp.1996.0018

Gladstones, J. S., Atkins, C., & Hamblin, J. (1998). Lupins as crop plants: Biology, production

and utilization. CAB international. 57

Gow, N., Morris, B., & Reid, B. (1992). The electrophysiology of root-zoospore interactions.

Paper presented at the Perspectives in Plant Cell Recognition, Society for Experimental

Biology Seminar Series, , 48 173-192.

Gow, N. A. R. (1993). Nonchemical signals used for host location and invasion by fungal

pathogens. Trends in Microbiology, 1(2), 45-50.

doi:http://dx.doi.org.ezproxy.bgsu.edu:8080/10.1016/0966-842X(93)90031-L

Grant, S., Fisher, E., Chang, J., Mole, B., & Dangl, J. (2006). Subterfuge and manipulation: Type

III effector proteins of phytopathogenic bacteria. Annual Review of Microbiology, 60(1),

425-449. doi:10.1146/annurev.micro.60.080805.142251

Gunes, A., Inal, A., Alpaslan, M., Eraslan, F., Bagci, E. G., & Cicek, N. (2007). Salicylic acid

induced changes on some physiological parameters symptomatic for oxidative stress and

mineral nutrition in maize (Zea mays L.) grown under salinity. Journal of Plant Physiology,

164(6), 728-736.

Gutierrez-Gonzalez, J. J., Guttikonda, S. K., Tran, L. P., Aldrich, D. L., Zhong, R., Yu, O., . . .

Sleper, D. A. (2010). Differential expression of isoflavone biosynthetic genes in soybean

during water deficits. Plant & Cell Physiology, 51(6), 936-948. doi:10.1093/pcp/pcq065

Haas, B. J., Kamoun, S., Zody, M. C., Jiang, R. H., Handsaker, R. E., Cano, L. M., . . . Torto-

Alalibo, T. (2009). Genome sequence and analysis of the irish potato famine pathogen

Phytophthora infestans. Nature, 461(7262), 393-398. 58

Hahn, M. G., Bonhoff, A., & Grisebach, H. (1985). Quantitative localization of the phytoalexin

I in relation to fungal hyphae in soybean roots infected with Phytophthora

megasperma f. sp. glycinea. Plant Physiology, 77(3), 591-601.

Halsall, D. (1978). A comparison of phytophthora cinnamomi infection in Eucalyptus sieberi, a

susceptible species, and Eucalyptus maculata a field resistant species. Australian Journal of

Botany, 26(5), 643-655.

Hammond-Kosack, K. E., & Jones, J. D. (1996). Resistance gene-dependent plant defense

responses. The Plant Cell, 8(10), 1773-1791.

Harborne, J. B., & Williams, C. A. (2000). Advances in flavonoid research since 1992.

Phytochemistry, 55(6), 481-504.

Harborne, J. B., Ingham, J. L., King, L., & Payne, M. (1976). The isopentenyl isoflavone luteone

as a pre-infectional antifungal agent in the genus Lupinus. Phytochemistry, 15(10), 1485-

1487. doi:http://dx.doi.org/10.1016/S0031-9422(00)88921-9

Hardham, A. R., & Cahill, D. M. (2010). The role of oomycete effectors in plant–pathogen

interactions. Functional Plant Biology, 37(10), 919-925.

Harshberger, J. W. (1917). A text-book of mycology and plant pathology P. Blakiston's Son &

Company.

Hart, P., Lockwood, J. L., & Helsel, Z. R. (1981). Phytophthora root and stem rot of soybeans

Cooperative Extension Service, Michigan State University. 59

Hein, I., Gilroy, E. M., Armstrong, M. R., & Birch, P. R. J. (2009). The zig-zag-zig in oomycete-

plant interactions. Molecular Plant Pathology, 10(4), 547-562. doi:10.1111/j.1364-

3703.2009.00547.x

Hill, C. (1979). New plant disease records in New Zealand. New Zealand Journal of Agricultural

Research, 22(4), 641-645.

Hoffman, T., Schmidt, J. S., Zheng, X., & Bent, A. F. (1999). Isolation of ethylene-insensitive

soybean mutants that are altered in pathogen susceptibility and gene-for-gene disease

resistance. Plant Physiology, 119(3), 935-950.

Horio, T., Kawabata, Y., Takayama, T., Tahara, S., Kawabata, J., Fukushi, Y., . . . Mizutani, J.

(1992). A potent attractant of zoospores ofAphanomyces cochlioides isolated from its host,

spinacia oleracea. Experientia, 48(4), 410-414.

Ho, H., & Hickman, C. (1967). Factors governing zoospore responses of phytophthora

megasperma var. sojae to plant roots. Canadian Journal of Botany, 45(11), 1983-1994.

Hosseini, S., Heyman, F., Olsson, U., Broberg, A., Funck Jensen, D., & Karlsson, M. (2014).

Zoospore chemotaxis of closely related legume‐root infecting phytophthora species towards

host isoflavones. Plant Pathology, 63(3), 708-714.

Huitema, E., Bos, J. I. B., Tian, M., Win, J., Waugh, M. E., & Kamoun, S. (2004). Linking

sequence to phenotype in phytophthora–plant interactions. Trends in Microbiology, 12(4),

193-200. doi:http://dx.doi.org.ezproxy.bgsu.edu:8080/10.1016/j.tim.2004.02.008 60

Huyghe, C. (1997). White lupin (Lupinus albus L.). Field Crops Research, 53(1–3), 147-160.

doi:http://ezproxy.bgsu.edu:2068/10.1016/S0378-4290(97)00028-2

Islam, M. T., Ito, T., & Tahara, S. (2003). Host-specific plant signal and G-protein activator,

mastoparan, trigger differentiation of zoospores of the phytopathogenic oomycete

Aphanomyces cochlioides. Roots: The dynamic interface between plants and the earth (pp.

131-142) Springer

Jarabo-Lorenzo, A., Pérez-Galdona, R., Donate-Correa, J., Rivas, R., Velázquez, E., Hernández,

M., . . . León-Barrios, M. (2003). Genetic diversity of bradyrhizobial populations from

diverse geographic origins that nodulate lupinus spp. and ornithopus spp. Systematic and

Applied Microbiology, 26(4), 611-623.

doi:http://ezproxy.bgsu.edu:2068/10.1078/072320203770865927

Jeandet, P., Clément, C., Courot, E., & Cordelier, S. (2013). Modulation of phytoalexin

biosynthesis in engineered plants for disease resistance. International Journal of Molecular

Sciences, 14(7), 14136-14170. doi:10.3390/ijms140714136

Jiang, R. H., & Tyler, B. M. (2012). Mechanisms and evolution of virulence in oomycetes.

Annual Review of Phytopathology, 50, 295-318.

Jing, M., Ma, H., Li, H., Guo, B., Zhang, X., Ye, W., . . . Wang, Y. (2015). Differential

regulation of defense-related proteins in soybean during compatible and incompatible

interactions between Phytophthora sojae and soybean by comparative proteomic analysis.

Plant Cell Reports, 34(7), 1263-1280. 61

Jones, J. D., & Dangl, J. L. (2006). The plant immune system. Nature, 444(7117), 323-329.

Jones, J., & Johnson, H. (1969). Lupine a new host for Phytophthora megasperma var sojae.

Phytopathology, 59(4), 504-&.

Jones, R., & McLean, G. (1989). Virus diseases of lupins. Annals of Applied Biology, 114(3),

609-637.

Jones, S., Donaldson, S., & Deacon, J. (1991). Behaviour of zoospores and zoospore cysts in

relation to root infection by Pythium aphanidermatum. New Phytologist, 117(2), 289-301.

Kaitany, R., Hart, L., & Safir, G. (2001). Virulence composition of phytophthora sojae in

michigan. Plant Disease, 85(10), 1103-1106.

Kamoun, S. (2006). A catalogue of the effector secretome of plant pathogenic oomycetes.

Phytopathology, 44(1), 41.

Kamoun, S. (2007). Groovy times: Filamentous pathogen effectors revealed. Current Opinion in

Plant Biology, 10(4), 358-365. doi:10.1016/j.pbi.2007.04.017

Kantar, F., Hebblethwaite, P., & Pilbeam, C. (1996). Factors influencing disease resistance in

high and low tannin Vicia faba. The Journal of Agricultural Science, 127(01), 83-88.

Katagiri, Y., Hashidoko, Y., & Tahara, S. (2002). Localization of flavonoids in the yellow lupin

seedlings and their UV-B-absorbing potential. Zeitschrift Fur Naturforschung C-Journal of

Biosciences, 57(9-10), 811-816. 62

Kim, S., Hong, H., & Kim, S. (1999). Some properties and contents of isoflavone in soybean and

soybean foods. Korea Soybean Digest, 16(2), 35-46.

Kim, J., Hong, S., Jung, W., Yu, C., Ma, K., Gwag, J., & Chung, I. (2007). Comparison of

isoflavones composition in seed, embryo, cotyledon and seed coat of cooked-with-rice and

vegetable soybean (Glycine max L.) varieties. Food Chemistry, 102(3), 738-744.

doi:http://dx.doi.org/10.1016/j.foodchem.2006.06.061

Kjemtrup, S., Nimchuk, Z., & Dangl, J. L. (2000). Effector proteins of phytopathogenic bacteria:

Bifunctional signals in virulence and host recognition. ENGLAND: Elsevier Ltd.

doi:10.1016/S1369-5274(99)00054-5

Kyle, D., Nickell, C., Nelson, R., & Pedersen, W. (1998). Response of soybean accessions from

provinces in southern china to Phytophthora sojae. Plant Disease, 82(5), 555-559.

Lee, S., Mian, M. R., McHale, L. K., Wang, H., Wijeratne, A. J., Sneller, C. H., & Dorrance, A.

E. (2013). Novel quantitative trait loci for partial resistance to Phytophthora sojae in

soybean PI 398841. Theoretical and Applied Genetics, 126(4), 1121-1132.

Lee, S., Seguin, P., Kim, J., Moon, H., Ro, H., Kim, E., . . . Chung, I. (2010). Isoflavones in

korean soybeans differing in seed coat and cotyledon color. Journal of Food Composition

and Analysis, 23(2), 160-165. doi:http://dx.doi.org/10.1016/j.jfca.2009.08.005

Leitz, R., Hartman, G., Pedersen, W., & Nickell, C. (2000). Races of Phytophthora sojae on

soybean in Illinois. Plant Disease, 84(4), 487-487. 63

Lin, F., Zhao, M., Baumann, D. D., Ping, J., Sun, L., Liu, Y., . . . Doerge, R. W. (2014).

Molecular response to the pathogen Phytophthora sojae among ten soybean near isogenic

lines revealed by comparative transcriptomics. BMC Genomics, 15(1), 1.

Lin, F., Zhao, M., Ping, J., Johnson, A., Zhang, B., Abney, T. S., . . . Ma, J. (2013). Molecular

mapping of two genes conferring resistance to Phytophthora sojae in a soybean landrace PI

567139B. Theoretical and Applied Genetics, 126(8), 2177-2185.

Loon, L., Rep, M., & Pieterse, C. (2006). Significance of inducible defense-related proteins in

infected plants. Annual Review of Phytopathology, 44, 7.1-7.28.

Loyd, A., Benson, D. M., & Ivors, K. (2014). Phytophthora populations in nursery irrigation

water in relationship to pathogenicity and infection frequency of Rhododendron and Pieris.

Plant Disease, 98(9), 1213-1220.

Messenger, B., Menge, J., & Pond, E. (2000). Effects of gypsum on zoospores and sporangia of

Phytophthora cinnamomi in field soil. Plant Disease, 84(6), 617-621.

Milholland, R. (1972). Histopathology and pathogenicity of Botryosphaeria dothidea on

blueberry stems. Phytopathology, 62(6), 654-660.

Miller, A., Shand, E., & Gow, N. (1988). Ion currents associated with root tips, emerging laterals

and induced wound sites in Nicotians tabacum: Spatial relationship proposed between

resulting electrical fields and phytophthoran zoospore infection. Plant, Cell & Environment,

11(1), 21-25. 64

Mitchell, R., & Deacon, J. (1986). Differential (host-specific) accumulation of zoospores of

Pythium on roots of graminaceous and non-graminaceous plants. New Phytologist, 102(1),

113-122.

Morris, P. F., & Ward, E. W. B. (1992). Chemoattraction of zoospores of the soybean pathogen,

Phytophthora sojae, by isoflavones. Physiological and Molecular Plant Pathology, 40(1),

17-22. doi:10.1016/0885-5765(92)90067-6

Ndakidemi, P. A., & Dakora, F. D. (2003). Legume seed flavonoids and nitrogenous metabolites

as signals and protectants in early seedling development. Functional Plant Biology, 30(7),

729-745.

Nikandrow, A., Gilbert, R., Gunning, D., Lawler, M., Lindbeck, K., & Murray, G. (2001). First

report of Phytophthora sp. causing a root and basal stem rot of narrowleaf lupin in Australia.

Plant Pathology, 50(6), 811-811.

Nirenberg, H. I., Feiler, U., & Hagedorn, G. (2002). Description of Colletotrichum lupini comb.

nov. in modern terms. Mycologia, 94(2), 307-320.

Peters, D. J., & Constabel, C. P. (2002). Molecular analysis of herbivore-induced condensed

tannin synthesis: Cloning and expression of dihydroflavonol reductase from trembling aspen

(Populus tremuloides). The Plant Journal, 32(5), 701-712.

Ping, J., Fitzgerald, J. C., Zhang, C., Lin, F., Bai, Y., Wang, D., . . . Ma, J. (2015). Identification

and molecular mapping of Rps11, a novel gene conferring resistance to Phytophthora sojae

in soybean. Theoretical and Applied Genetics, , 1-7. 65

Pinto, P. M., & Ricardo, C. P. (1995). Lupinus albus L. pathogenesis-related proteins that show

similarity to PR-10 proteins. Plant physiology, 109(4), 1345-1351.

Raftoyannis, Y., & Dick, M. (2006). Effect of oomycete and plant variation on zoospore cover

and disease severity. Journal of Plant Pathology, , 95-101.

Rentel, M. C., Leonelli, L., Dahlbeck, D., Zhao, B., & Staskawicz, B. J. (2008). Recognition of

the Hyaloperonospora parasitica effector ATR13 triggers resistance against oomycete,

bacterial, and viral pathogens. Proceedings of the National Academy of Sciences of the

United States of America, 105(3), 1091-1096. doi:10.1073/pnas.0711215105

Robertson, A. E., Cianzio, S. R., Cerra, S. M., & Pope, R. O. (2009). Within-field pathogenic

diversity of Phytophthora sojae in commercial soybean fields in Iowa. Plant Health

Progress Doi, 10

Rose, J. K., Ham, K. S., Darvill, A. G., & Albersheim, P. (2002). Molecular cloning and

characterization of glucanase inhibitor proteins: Coevolution of a counter-defense

mechanism by plant pathogens. The Plant Cell, 14(6), 1329-1345.

Rovira, A. D. (1969). Plant root exudates. Botanical Review, 35(1), 35-57.

doi:10.1007/BF02859887

Ruberson, J. R. (1999). Handbook of pest management CRC Press. 66

Ryals, J. A., Neuenschwander, U. H., Willits, M. G., Molina, A., Steiner, H. Y., & Hunt, M. D.

(1996). Systemic acquired resistance. The Plant Cell, 8(10), 1809-1819.

doi:10.1105/tpc.8.10.1809 [doi]

Schaechter, M. (2011). Eukaryotic microbes Academic Press.

Schmitthenner, A. (1999). Phytophthora rot of soybean. Compendium of Soybean Diseases, 4th

Edn.the American Phytopathological Society Press, St Paul, , 39-42.

Schornack, S., van Damme, M., Bozkurt, T. O., Cano, L. M., Smoker, M., Thines, M., . . .

Huitema, E. (2010). Ancient class of translocated oomycete effectors targets the host

nucleus. Proceedings of the National Academy of Sciences of the United States of America,

107(40), 17421-17426. doi:10.1073/pnas.1008491107 [doi]

Sekizaki, H., & Yokosawa, R. (1988). Studies on zoospore-attracting activity. I. synthesis of

isoflavones and their attracting activity to Aphanomyces euteiches zoospore. Chemical and

Pharmaceutical Bulletin, 36(12), 4876-4880.

Sels, J., Mathys, J., De Coninck, B. M. A., Cammue, B. P. A., & De Bolle, M. F. C. (2008).

Plant pathogenesis-related (PR) proteins: A focus on PR peptides. Plant Physiology and

Biochemistry, 46(11), 941-950.

doi:http://dx.doi.org.ezproxy.bgsu.edu:8080/10.1016/j.plaphy.2008.06.011

Serrano, M. S., Fernández-Rebollo, P., De Vita, P., Carbonero, M. D., Trapero, A., & Sánchez,

M. E. (2010). Lupinus luteus, a new host of Phytophthora cinnamomi in spanish oak-

rangeland ecosystems. European Journal of Plant Pathology, 128(2), 149-152. 67

Shan, W., Cao, M., Leung, D., & Tyler, B. M. (2004). The Avr1b locus of Phytophthora sojae

encodes an elicitor and a regulator required for avirulence on soybean plants carrying

resistance gene Rps 1b. Molecular Plant-Microbe Interactions, 17(4), 394-403.

Shea, G., Thomas, G., Buirchell, B., Salam, M., McKirdy, S., Sweetingham, M., . . . Berger, J.

(2008). Case study: Industry response to the lupin anthracnose incursion in Western

Australia. Paper presented at the Proceedings of the 12th International Lupin Conference—

Lupins for Health and Wealth, 425-431.

Sikorski, M. M., Biesiadka, J., Kasperska, A. E., Kopcińska, J., Łotocka, B., Golinowski, W., &

Legocki, A. B. (1999). Expression of genes encoding PR10 class pathogenesis-related

proteins is inhibited in yellow lupine root nodules. Plant Science, 149(2), 125-137.

doi:http://dx.doi.org/10.1016/S0168-9452(99)00148-X

Sohn, K. H., Lei, R., Nemri, A., & Jones, J. D. G. (2007). The downy mildew effector proteins

ATR1 and ATR13 promote disease susceptibility in Arabidopsis thaliana. The Plant Cell,

19(12), 4077-4090. doi:10.1105/tpc.107.054262

Soylu, S., Bennett, M. H., & Mansfield, J. W. (2002). Induction of phytoalexin accumulation in

broad bean (Vicia faba L.) cotyledons following treatments with biotic and abiotic elicitors.

Turkish Journal of Agriculture and Forestry, 26(6), 343-348.

Statler, G. (1970). Resistance of bean plants to Fusarium solani f. phaseoli. Plant Disease

Reporter, 54, 698-699. 68

Steel, R. G. (1959). A multiple comparison sign test: treatments versus control. Journal of the

American Statistical Association, 54(288), 767-775.

Steinkellner, S., Lendzemo, V., Langer, I., Schweiger, P., Khaosaad, T., Toussaint, J., &

Vierheilig, H. (2007). Flavonoids and strigolactones in root exudates as signals in symbiotic

and pathogenic plant-fungus interactions. Molecules, 12(7), 1290-1306.

Sun, J., Li, L., Zhao, J., Huang, J., Yan, Q., Xing, H., & Guo, N. (2014). Genetic analysis and

fine mapping of RpsJS, a novel resistance gene to Phytophthora sojae in soybean [Glycine

max (L.) merr.]. Theoretical and Applied Genetics, 127(4), 913-919. doi:10.1007/s00122-

014-2266-2

Sun, S., Wu, X. L., Zhao, J. M., Wang, Y. C., Tang, Q. H., Yu, D. Y., . . . Xing, H. (2011).

Characterization and mapping of RpsYu25, a novel resistance gene to Phytophthora sojae.

Plant Breeding, 130(2), 139-143. doi:10.1111/j.1439-0523.2010.01794.x

Suo, B., Chen, Q., Wu, W., Wu, D., Tian, M., Jie, Y., . . . Wen, J. (2016). Chemotactic responses

of Phytophthora sojae zoospores to amino acids and sugars in root exudates. Journal of

General Plant Pathology, , 1-7.

Święcicki, W., Kroc, M., & Kamel, M Sc Katarzyna Anna. (2015). Lupins. Grain legumes (pp.

179-218) Springer.

Tahara, S., Katagiri, Y., Ingham, J. L., & Mizutani, J. (1994). Prenylated flavonoids in the roots

of yellow lupin. Phytochemistry, 36(5), 1261-1271. doi:10.1016/S0031-9422(00)89648-X 69

Thomas, R., Fang, X., Ranathunge, K., Anderson, T. R., Peterson, C. A., & Bernards, M. A.

(2007). Soybean root suberin: Anatomical distribution, chemical composition, and

relationship to partial resistance to Phytophthora sojae. Plant Physiology, 144(1), 299-311.

doi:pp.106.091090 [pii]

Tippett, J. T., Holland, A., Marks, G., & O'brien, T. (1976). Penetration of Phytophthora

cinnamomi into disease tolerant and susceptible eucalypts. Archives of Microbiology, 108(3),

231-242.

Tooley, P., & Grau, C. (1984). Field characterization of rate-reducing resistance to Phytophthora

megasperma f. sp. glycinea in soybean. Phytopathology, 74(10), 1201-1208.

Torto, T. A., Li, S., Styer, A., Huitema, E., Testa, A., Gow, N. A. R., . . . Kamoun, S. (2003).

EST mining and functional expression assays identify extracellular effector proteins from the

plant pathogen Phytophthora. Genome Research, 13(7), 1675-1685. doi:10.1101/gr.910003

Toussoun, T. A., Bega, R. V., & Nelson, P. E. (1970). Root diseases and soil-borne pathogens

Univ of California Press.

Trapero-Casas, A., Rodriguez-Tello, A., & Kaiser, W. (2000). Lupins, a new host of

Phytophthora erythroseptica. Plant Disease, 84(4), 488-488.

Tyler, B. M., Wu, M., Wang, J., Cheung, W., & Morris, P. F. (1996). Chemotactic preferences

and strain variation in the response of Phytophthora sojae zoospores to host isoflavones.

Applied and Environmental Microbiology, 62(8), 2811-2817. 70

Tyler, B. (2002). Molecular basis of recognition between Phytophthora pathogens and their

hosts. Annual Review of Phytopathology, 40, 137-167.

doi:10.1146/annurev.phyto.40.120601.125310

Tyler, B. M. (2007). Phytophthora sojae: Root rot pathogen of soybean and model oomycete.

Molecular Plant Pathology, 8(1), 1-8. doi:10.1111/j.1364-3703.2006.00373.x

Valent, B., McAdams, S. A., Hershey, H. P., Jia, Y., & Bryan, G. T. (2000). Direct interaction of

resistance gene and avirulence gene products confers rice blast resistance. The EMBO

Journal, 19(15), 4004-4014. doi:10.1093/emboj/19.15.4004 van der Hoorn, R. A. L., & Kamoun, S. (2008). From guard to decoy: A new model for

perception of plant pathogen effectors. The Plant Cell, 20(8), 2009-2017.

doi:10.1105/tpc.108.060194

Van Loon, L., Pierpoint, W., Boller, T., & Conejero, V. (1994). Recommendations for naming

plant pathogenesis-related proteins. Plant Molecular Biology Reporter, 12(3), 245-264. van West, P., Appiah, A. A., & Gow, N. A. (2003). Advances in research on oomycete root

pathogens. Physiological and Molecular Plant Pathology, 62(2), 99-113.

VanEtten, H. D., Mansfield, J. W., Bailey, J. A., & Farmer, E. E. (1994). Two classes of plant

antibiotics: Phytoalexins versus "phytoanticipins". The Plant Cell, 6(9), 1191-1192.

doi:10.1105/tpc.6.9.1191 [doi] 71

Kruskal, W. H., & Wallis, W. A. (1952). Use of ranks in one-criterion variance analysis. Journal

of the American statistical Association, 47(260), 583-621.

Ward, E., Stössel, P., & Lazarovits, G. (1981). Similarities between age-related and race-specific

resistance of soybean hypocotyls to Phytophthora megasperma var. sojae. Phytopathology,

71, 504-508.

Wester, E., Jensen, D. F., & Moller, J. D. (2012). Anatomical studies of cucumber roots

inoculated with Pythium zoospores. Biotic Interactions and Soil-borne Diseases, , 401.

Whisson, S. C., Boevink, P. C., Moleleki, L., Avrova, A. O., Morales, J. G., Gilroy, E. M., . . .

Chapman, S. (2007). A translocation signal for delivery of oomycete effector proteins into

host plant cells. Nature, 450(7166), 115-118.

Wilkie, J. P., Dye, D., & Watson, D. (1973). Further hosts of Pseudomonas viridiflava. New

Zealand Journal of Agricultural Research, 16(3), 315-323.

Williamson, P. M., Highet, A. S., Gams, W., Sivasithamparam, K., & Cowling, W. A. (1994).

Diaporthetoxica sp. nov., the cause of lupinosis in sheep. Mycological Research, 98(12),

1364-1368. doi:http://ezproxy.bgsu.edu:2068/10.1016/S0953-7562(09)81064-2

Winkel-Shirley, B. (2001). Flavonoid biosynthesis. A colorful model for genetics, biochemistry,

cell biology, and biotechnology. Plant Physiology, 126(2), 485-493. 72

Wrather, A., Shannon, G., Balardin, R., Carregal, L., Escobar, R., Gupta, G., . . . Tenuta, A.

(2010). Effect of diseases on soybean yield in the top eight producing countries in 2006.

Plant Health Progress Doi, 10, 2008-2013.

Xu, P., Jiang, L., Wu, J., Li, W., Fan, S., & Zhang, S. (2014). Isolation and characterization of a

pathogenesis-related protein 10 gene (GmPR10) with induced expression in soybean

(Glycine max) during infection with Phytophthora sojae. Molecular Biology Reports, 41(8),

4899-4909.

Yang, X., Zhao, W., Hua, C., Zheng, X., Jing, M., Li, D., . . . Wang, Y. (2013). Chemotaxis and

oospore formation in Phytophthora sojae are controlled by G-protein-coupled receptors with

a phosphatidylinositol phosphate kinase domain. Molecular Microbiology, 88(2), 382-394.

Yang, X., Ruff, R., Meng, X., & Workneh, F. (1996). Races of Phytophthora sojae in iowa

soybean fields. Plant Disease, 80(12), 1418-1420.