Arabidopsis thaliana Relationship between classification of Rhizoctonia solani species complex and pathogenicity on

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Relationship between classification of Rhizoctonia solani species complex and pathogenicity on Arabidopsis thaliana

A Thesis

Submitted to the Graduate school of Environmental and Life Science in partial fulfilment of the requirements for the degree doctor of philosophy in Agriculture

Division of Agricultural and Life Science Graduate School of Environmental and Life Sciense

OKAYAMA UNIVERSITY

Mai Mohsen Ahmed Abdelghany Ahmed March 2022

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I

Contents list

Contents Page

Acknowledgements II

Abbreviations III

List of figures IV

List of tables IV

Abstract of the thesis V

Chapter 1: General introduction 1

1.1. Rhizoctonia solani Khün 1

1.2. Infection process of R. solani 1

1.3. Pathogenicity and host range of R. solani 2

1.4. Grouping of R. solani isolates 3

1.5. Plant defense mechanisms against pathogens 4

1.6. Role of phytohormones in Arabidopsis defense 5

1.7. Aim of the study 7

1.8. References 8

Chapter II:

The relationship between the AGs and subgroups of R. solani and its host specificity. 12

2.1. Introduction 12

2.2. Materials and methods 13

2.3. Results 16

2.4. Discussion 23

2.5. References 25

Chapter III

Investigation of the infection mechanism of R. solani through the infection behavior on Arabidopsis plants with exogenous application of phytohormones

27

3.3. Results 28

3.4. Discussion 31

3.5. References 34

Chapter IV

Investigation of the infection mechanism of R. solani through the infection behavior on Arabidopsis plants with mutations in defense pathways

36

4.3. Results 41

4.4. Discussion 52

4.5. References 54

Final conclusion and future prospects 59

Appendices 61

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ACKNOWLEDGEMENTS

I would like to express my deepest gratitude to my unique supervisor, professor NOUTOSHI Yoshiteru. This project would not have been possible without your thoughts, guidance, concern, your kind help through all the work, and that you always had time to discuss.

Your door was never closed.

I would like to extend my gratitude to my co-supervisors Professor Kazuhiro TOYODA, Professor Yuki ICHINOSE, for their suggestions through this work are much appreciated and acknowledged. Also, I want to express my gratitude to Professor Hidenori MATSUI and Professor Mikihiro YAMAMOTO for their suggestions and comments during this work.

I would like to acknowledge my colleagues, the currents and the formers from the Laboratory of Plant Pathology and the Genetic Engineering, and also Egyptian friends for their helping to cope with lab work and daily activities in Okayama.

Also, I want to express my very profound gratitude to my wonderful family whose love, guidance, and prayers are with me in whatever I pursue. Finally, I cannot express my full gratitude to my dear husband who sacrificed a lot of his rest and his time just to push me forward, thank you from all my heart.

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Abbreviations

AGs: anastomosis groups

cerk1-2: chitin elicitor receptor kinase1-2 mutant coi1-1: coronatine insensitive1-1 mutant

DMSO: dimethyl sulfoxide dpi: day post inoculation

eds1-2: enhanced disease susceptibility1 mutant ein2-1: ethylene insensitive2-1 mutant

ET: ethylene

ETI: effector-triggered immunity etr1-1: ethylene resistant1-1 mutant HR: hypersensitive response ICS1: isochorismate synthase1 JA: jasmonic acid

jin1-1: jasmonic acid insensitive1-1 mutant LAR: local acquired resistant

LRRs: leucine-rich repeats MS: Murashige and Skoog

nahG: salicylic acid-deficient transgenic line

npr1-5: non-expressor of pathogenesis-related genes1-5 mutant pad4-1: phytoalexin deficient4-1 mutant

PAMP: pathogen-associated molecular pattern PDA: potato dextrose agar

PR: pathogenesis-related proteins PTI: pattern-triggered immunity SA: salicylic acid

SAR: systemic acquired resistance

sid2-2: salicylic acid induction deficient2-2 mutant

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List of figures

Figure Page

Figure 1: Cultural morphology of Rhizoctonia solani isolates used in this study

on PDA medium 17

Figure 2: The growth rates of Rhizoctonia solani isolates used in this study 18 Figure 3: Infectivity of Rhizoctonia solani Japanese isolates in detached leaves

of Arabidopsis thaliana 20

Figure 4: Infectivity of Rhizoctonia solani Japanese isolates in soil-grown

Arabidopsis thaliana seedling 22

Figure 5: Effects of pretreatment of the defense-related phytohormones, salicylic acid (SA), jasmonic acid (JA), and ethylene (ET) on disease resistance of A. thaliana leaves against Rhizoctonia solani Japanese isolates

30 Figure 6: Effects of pretreatment of the defense-related phytohormones, salicylic

acid (SA), jasmonic acid (JA), and ethylene (ET) on disease resistance of Arabidopsis thaliana plants to Rhizoctonia solani Japanese isolates inoculated into soil

31 Figure 7: Infectivity of the Rhizoctonia solani Japanese isolates in detached

leaves of sid2-2 mutant 43

Figure 8: Infectivity of the Rhizoctonia solani Japanese isolates in detached

leaves of npr1-5 mutant 44

leaves of eds1-2 mutant 45

leaves of pad4-1 mutant 46

leaves of jin1-1 mutant 47

leaves of coi1-1 mutant 48

leaves of ein2-1 mutant 49

leaves of etr1-1 mutant 50

leaves of cerk1-2 mutant 51

Figure 16: Infectivity of the soil-inoculated Rhizoctonia solani isolates on

different Arabidopsis mutants 52

List of tables

Table 1: Rhizoctonia solani isolates used in this study and their pathogenicity to

upper- and underground tissues of Arabidopsis thaliana Col-0. 15

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Abstract of the thesis

Rhizoctonia solani is a necrotrophic destructive plant pathogen with a wide host range and causes various crop diseases worldwide. R. solani is a species complex and it consists of 13 anastomosis groups (AGs). R. solani in the same AGs can fuse their hyphae to expand their genetic diversity. Some AGs are divided into subgroups according to their pathogenicity, host specificity, cultural morphology, nutritional requirements, optimum temperature, and hyphal anastomosis frequency. Although this pathogen causes serious agricultural damage in the world, its virulence mechanism remains unclear.

I. Relationship between AGs and subgroups of R. solani and its host specificity

R. solani AG-I IA isolates were empirically isolated as causal agents of rice sheath blight and AG-2-1 was frequently isolated from Brassicaceae around the world. Therefore, it has been believed that there is a certain relationship between the pathogenicity of R. solani and AGs and subgroups. However, many exceptions have been reported. For example, AG-1 IA isolates were identified as pathogens of soybean and some brassica species. Also, AG-2-1 isolates were reported to infect pea, wheat, and barley. Thus, this study aims to clarify if the classifications of R. solani have relatedness to its host and tissue specificity of the virulence.

For this purpose, surveillance of the infectivity of various isolates of R. solani to particular plant species is useful.

In this study, seventeen R. solani isolates collected in Japan were used. They include 4 AG-1 isolates, 8 AG-2 isolates, and single isolates for each of AG-3, AG-4, AG-5, AG-6, and AG-7, sampled from different diseased crops and soils. Their pathogenicity towards the dicot model plant Arabidopsis thaliana was evaluated using leaf and soil inoculation methods. For leaf inoculation assay, the infection aggressiveness of the R. solani isolates was categorized into 4 levels, highly-virulent, moderately-virulent, weak-virulent, and non-virulent. In addition

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to R. solani AG-2, which is frequently isolated from Brassicaceae (same family of Arabidopsis), AG-1, AG-4, AG-5, and AG-7 were also found to be pathogenic to Arabidopsis leaves. The virulence levels differed even within the same AGs and subgroups. Among 3 AG-1 IA isolates (2 isolates from rice and 1 isolate from sudangrass), only a single isolate exhibited severe necrotic lesion. Among 3 AG-2-2 IIIB isolates sampled from rice, broccoli, and welch onion, only a single isolate was found to be highly pathogenic.

For root inoculation assay, the aggressiveness of the isolates was categorized into 3 levels, highly-virulent, moderately-virulent, and non-virulent. In this case, the R. solani isolates AG-1, AG-2, and AG-4 were strongly virulent. On the other hand, AG-3, AG-5, AG-6, and AG-7 did not exhibit obvious symptoms. Among 3 AG-1 IA isolates, the same isolate which is virulent on the leaf was also pathogenic in this assay system. Unlike leaf inoculation assay, all the 3 AG-2-2 IIIB isolates were pathogenic. Comparing the results of the two different inoculation methods, 2 AG-2 isolates, AG-5, and AG-7 exhibited the leaf-specific virulence, while 2 AG-2 isolates were only pathogenic to roots. These results indicate that the AGs and their subgroups are not enough to determine virulence, host specificity, or tissue specificity of R. solani.

II. Investigation of infection mechanism of R. solani using host defense response

To understand the infection mechanism of the plant pathogen, plant defense responses that effectively counteract the pathogen provide useful information. Arabidopsis leaves or seedlings were treated with defense-related phytohormones, salicylic acid (SA), jasmonic acid (JA), or ethylene (ET) and the infection behaviors of the R. solani isolates were compared with the control treatment. As a result, these treatments had no obvious effects on the symptoms formations by the highly-virulent isolates in both inoculation systems. The treatment with SA or ET slightly enhanced the susceptibility to the weak- and non-virulent isolates on Arabidopsis leaves. These results indicate that induction of a single defense pathway does not confer disease

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resistance to R. solani. This pathogen should be able to overcome the defense induced by every single defense-related phytohormone.

The infection behavior of the R. solani isolates was evaluated using various Arabidopsis single mutants that are impaired in salicylic acid biosynthesis and signalling (sid2, npr1, eds1, and pad4), jasmonic acid signalling (jin1, coi1), and ethylene signalling (ein2, etr1). In addition, the mutants for the receptor of chitin elicitor (cerk1-2) were used. All these mutants displayed similar levels of resistance/susceptibility to the tested R. solani isolates compared with wild- type in both inoculation assays, except for the npr1 and cerk1-2 mutants in response to the AG- 1 IA (MAFF305232). Although this AG-1 IA isolate was non-pathogenic to wild-type Arabidopsis plants, the symptoms were produced on the leaves of these mutants. The major component of the nonhost resistance of A. thaliana to this R. solani isolate would rely on pattern-triggered immunity induced by chitin perception. Generally, for the virulent isolates, they would fully suppress the host defense so that the effect of the impairment of a single defense-related gene used in this study would not be observed at a detectable level. For the non-virulent isolates, at least the loss of a single of these genes did not induce susceptibility.

Thus, the results suggest that the infection mechanism of R. solani is found to vary greatly among isolates. Also, the nonhost resistance of A. thaliana to R. solani may consist of various layers of defense pathways.

In conclusion, this study revealed that AGs and subgroups of R. solani would be unrelated to their host specificity and tissue specificity. In A. thaliana, the disease resistance to R. solani was not induced by any defense-related phytohormones tested. Also, the Arabidopsis nonhost resistance to various R. solani isolates, except for a single AG-1 IA, was not impaired by the loss of defense genes. This is contrasting to the previous results that the pre-treatment of SA conferred resistance to R. solani AG-I IA in Brachypodium distachyon and rice. In rice, the SA deficiency by NahG transgene increased susceptibility to R. solani. These results

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suggest the difference in infection behavior of R. solani among host plant species. On monocotyledonous plants, it would pass through a biotrophic interaction, however, it might be almost nothing or very short in A. thaliana.

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Chapter I

General introduction

1.1. Rhizoctonia solani Khün

Genus Rhizoctonia is a highly heterogeneous group of filamentous fungi that shares a lot of similarities. The group contains several plant pathogens which provide economically serious damage in agriculture. One of the important species is Rhizoctonia solani Kühn (teleomorph Thanatephorus cucumeris). The fungus binomial name is derived from ancient Greek: Rhiza- root, ktonos- murder. R. solani was first identified on potato in 1858 [Ceresini, 1999] and it now can be found worldwide with an almost unlimited host range [Adams, 1988].

Rhizoctonia solani goes through two life cycles; anamorph (asexual state) and teleomorph (sexual state) [Singleton et al.,1992]. R. solani rarely forms basidiospores (sexual spores) and does not produce asexual spores [Bienkowski, 2012]. Because of the scarcity of the sexual spores, R. solani exists as vegetative hyphae and sclerotia in nature. Sclerotia are an encapsulated, tight hard hyphal clump that protects and preserves the fungus during unfavorable conditions. The fungus is dispersed mainly via sclerotia contaminated in residual diseased plant materials or soil [Ceresini, 1999; Keijer, 1996]. R. solani can stay in the soil as a saprophyte for long periods. They survive on nutrients leaked from growing plants or decaying plant materials [Doornik, 1980].

1.2. Infection process of R. solani

Passive entry is not the usual infection mechanism for R. solani [Keijer, 1996; Weinhold

& Sinclair, 1996]. R. solani initiates germination of hyphae from sclerotia when they recognize particular chemicals (ex: amino acids, sugars, organic acids, and phenols) in the host plant root exudates [Keijer, 1996]. The entry frequently occurs at weak spots where the protecting layer

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is relatively easily broken down [Weinhold & Sinclair, 1996]. After the first contact, the loose and unattached hyphae start to grow over the plant. Within a few hours, the hyphae flatten and directionally grow over the epidermal cells. Then, the T-shaped hyphal branches are formed to make an infection machinery so-called infection cushion that attaches strongly to the host epidermis [Keijer, 1996]. From the infection cushion, many infection hyphae elongate and swollen hyphal tips concurrently form infection pegs that penetrate the cuticle and epidermal cell walls into the host epidermal tissue and outer layer of the cortex [Demirci & Döken, 1998].

Penetration is established by using hydrostatic pressure and degrading enzymes such as cutinases [Baker & Bateman, 1978], pectinases [Bertagnolli et al., 1996; Jayasinghe et al., 2004], and xylanases [Peltonen, 1995]. When it invades the host, the fungus starts to grow inter- and intra-cellularly by degrading the tissue, which can be seen as necrotic lesions on epidermal tissue of shoots, roots, and stolons, or as damping-off of the young seedlings [Demirci & Döken, 1998].

1.3. Pathogenicity and host range of R. solani

R. solani infects various plant species. It attacks members of the Poaceae (e.g. maize, rice, wheat, barley, and oat), Fabaceae (e.g. soybean, peanut, dry bean, alfalfa, chickpea, lentil, and field pea), Solanaceae (e.g. tobacco, potato, tomato), Amaranthaceae (e.g. sugar beet), Brassicaceae (e.g. canola, radish, cauliflower, cabbage, and broccoli), Rubiaceae (e.g. coffee), Malvaceae (e.g. cotton), Asteraceae (e.g. lettuce), Araceae (e.g. pothos), Moraceae (e.g. ficus), and Linaceae (e.g. flax) family, as well as ornamental plants and forest trees [Ogoshi, 1996;

Ajayi-Oyetunde & Bradley, 2018]. On such plant species, it produces diverse symptoms including pod rots, stem canker, black scurf, seedling blight, and pre- and post-emergence damping-off, in addition to foliarand sheath blights [Baker, 1970; Ogoshi, 1996].

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Since the teleomorphic (sexual) state of R. solani is rare, the identification of this fungus is based on the characteristics of vegetative hyphae. R. solani is identified from the color of the mycelium on agar media which can vary from nearly white to dark brown or almost black, with irregularly shaped, undifferentiated, light brown to dark brown sclerotia. The diameter of vegetative hyphae is more than 7 μm and the hyphal branches occur at 90˚ angles. There are more than two nuclei in apical compartments of hyphae [Parmeter & Whitney 1970; Ogoshi, 1987]. Grouping of R. solani isolates in the species complex is accomplished by evaluation of the anastomosis reactions. Anastomosis is defined as the compatibility of hyphal fusion reaction between different but closely related isolates [Anderson et al., 1972]. The earliest reports about anastomosis reactions in R. solani were done by Matsumoto et al. date to the early 20th century and it has since been modified by several authors [Carling, 1996] to produce the present-day concept of anastomosis groups (AGs). Anastomosis grouping revealed that R.

solani is a species complex consisting of non-interbreeding [Anderson et al., 1972] or reproductively isolated populations [Anderson, 1982]. Using this classification system, genetically similar isolates that readily undergo hyphal fusion and cytoplasmic and nuclei exchange were placed in the same AG, while isolates that fail to achieve hyphal contact, hyphal fusion, and nuclear exchange were considered members of different AGs [Ogoshi, 1987;

Matthew & Brooker, 1991]. This Anastomosis or fusion between isolates leads to expansion of the genetic diversity of the fungus [Webster & Weber, 2007]. To date, R. solani is divided into 13 AGs designatedas AG-1 through AG-13, as the AG-BI group has been integrated into AG-2 [Carling, et al., 2002]. Some of theanastomosis groups (AG-1, AG-2, AG-3, AG-4, AG- 6, AG-8, and AG-9) have been further divided into multiple subgroups according to their pathogenicity, host specificity, cultural morphology, nutritional requirements, optimum temperature, and hyphal anastomosis frequency [Ogoshi, 1987; Sneh et al., 1991].Currently,

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there are 28 subgroups, AG-1 (IA, IB, IC, ID, IE, IF), AG-2 (1, t, Nt, 2IIIB, 2IV, 2LP, 3, 4), AG-3 (TB, PT, TM), AG-4 (HG-I, HG-II, HG-III), AG-6 (HG-I, GV), AG-8 (1,2,3,4), and AG-9 (TP, TX) [Ajayi-Oyetunde & Bradley, 2018].

Regarding the AGs and subgroups classification and their pathogenicity, it has been believed that host specificity and virulence are related to the AGs and subgroups. For instance, the isolates belonging to AG-1 IA were frequently isolated from infected rice with sheath blight disease and it has been recognized as a rice pathogen. However, it also has been reported as pathogens of soybean and some Brassica species [Hua, et al., 2014; Jones & Belmer, 1989].

Similarly, the AG-2-1 isolates were frequently isolated from Brassicaceae [Ogoshi, 1987;

Carling & Leiner, 1986; Paulitz et al., 2006], but it was also found to infect pea, wheat, and barley [Stodart et al., 2007; Ozer et al., 2021]. Thus, the relationship between such classifications and virulence of the isolates towards specific host plants remains elusive.

In addition to host specificity, tissue specificity can also provide important aspects for the virulence mechanism of R. solani. The R. solani AG-2-1 was found to be virulent to Arabidopsis detached leaves and induced severe necrotrophic symptoms, while its colonization was restricted in roots when it was inoculated into the soil [Kidd et al., 2021]. The relationship between R. solani AGs and subgroups along with tissue-specific virulence is also unclear.

1.5. Plant defense responses against pathogens

Plants have assembled many strategies to protect them from pathogen attacks and invasions. Structural barriers on the plant surfaces are the first host protection system that pathogens have to overcome in order to start the infection. It includes hairs, spikes, resins, waxes on the cuticle, and the strongly built cell wall [Agrios, 1997; Nürnberger et al., 2004].

A wide array of secondary chemical compounds in plant cells and cell walls also protect plants against pathogens. They are constitutively expressed in healthy plants, either in their active

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forms or as inactive precursors ready to react when the integrity of the tissue is damaged [Bennett & Wallsgrove, 1994; Morrissey & Osbourn, 1999].

Once microbial enemies break such preexisting defense layers and reach the cell membrane, plants recognize pathogens. This is a key feature of the onset of the induced plant defense. A broad spectrum of actions is brought into the induced defense system [Nürnberger

& Lipka, 2005]. It includes the formation of new cell walls designated papillae at the pathogen entry site with callose, an abnormal cell wall component. It also includes the synthesis of antimicrobial chemical molecules and proteins. By the receptor molecules deployed at the cell surface, plants recognize microbe-derived molecules such as chitin, peptidoglycan, lipopolysaccharides, flagellin-derived peptide, and so on that exists in the broad range of microorganisms. After that, plants induce an immunity response named pattern-triggered immunity (PTI) [Jones & Dangl, 2006; Chisholm, et al., 2006]. It activates transcription of a set of defense-related genes that confer basal resistance against a broad range of microorganisms. Plant pathogens can suppress PTI by secreting effector molecules that target and prevent the machinery of PTI. Plants counteract pathogens by inducing alternative defense systems so-called effector-triggered immunity (ETI). Effector proteins or their actions are recognized by plant immune sensor proteins encoded by disease resistance (R) genes. ETI confers stronger and more durable resistance compared with PTI [Jones & Dangl, 2006;

Chisholm et al., 2006].

1.6. Role of phytohormones in plant defense

Plant hormones are a class of chemical compounds that are required in small quantities to regulate the growth and development of plants. Plant hormones also play a critical role in regulating defense responses against abiotic and biotic stresses. Due to the sessile nature of plants, plant hormones have a central role in connecting the signaling networks managing an

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array of processes. Their precise signals are required for healthy plant growth, successful completion of its life cycle with the production of seeds. External stimuli such as abiotic and biotic factors, including environmental factors, microbes, pests, and herbivores affect not only the hormone signaling networks but also the cellular hormone levels. In addition to the classical plant hormones such as ethylene (ET), auxin (AUX), cytokinin (CTK), gibberellic acid (GA), and abscisic acid (ABA), research in the past two decades has led to the discovery of new hormones such as salicylic acid (SA), jasmonic acid (JA), brassinosteroid (BR), nitric oxide (NO), and others including peptide hormones and polyamines [Shigenaga & Argueso, 2016;

Berens et al., 2017].

PTI and ETI as well as systemic acquired resistance (SAR) are regulated by defense- associated phytohormones such as salicylic acid (SA), jasmonic acid (JA), and ethylene (ET) [Bostock, 2005]. The SA-induced signalling pathway is associated with defense against biotrophic pathogens that evade PTI by effectors to steal nutrients from alive plant cells. SA induces the production of plenty of antimicrobial molecules, such as pathogenesis-related (PR) proteins and phytoalexins, and restricts pathogen colonization. On the other hand, the JA/ET- induced signalling pathway(s) are typically associated with necrotrophic pathogens that kill plant cells to obtain nutrients [Glazebrook, 2005; Bari & Jones, 2009; Muthamilarasan &

Prasad, 2013; Li, et al., 2019].

Interestingly, many of these hormones are produced by plant pathogenic microbes as secondary metabolites. Moreover, some pathogens may overcome host defense responses with the interference with host hormone signalling. Also, some effectors produced by plant pathogens directly influence hormone levels or the hormone signalling network components to manipulate host resources to their advantage [Shigenaga & Argueso, 2016; Berens et al., 2017].

The result of these hormonal interferences between plants and pathogens determines the

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outcome of host-pathogen interactions, and it also affects the hormone signaling pathways responsible for nutrient acquisition, growth, and defense of the plant.

Plant-microbe interactions can be manipulated by the application of plant hormones directly or by interference in hormone signalling processes to assess responses that affect plant defense [Delaney et al., 1994; Tjamos et al., 2005].

1.7. Aim of the study

In this study, we aim to clarify if the AGs and subgroups classifications of R. solani have relatedness to its virulence against the hosts and tissues or not. This was done by evaluating the pathogenicity of the different R. solani isolates belonging to different AGs against Arabidopsis thaliana in two inoculation systems targeting the leaves and the roots (Chapter II).

The second aim of our study is trying to investigate the infection mechanism of R. solani towards the dicot model plant Arabidopsis. This was done by investigating the Infection behavior of R. solani towards different Arabidopsis mutants (Chapter III). In addition to evaluating the effects of phytohormone on induction of resistance or susceptibility of Arabidopsis thaliana towards R. solani (Chapter IV).

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Nürnberger, T., Lipka, V. (2005) Non-host resistance in plants: new insights into an old phenomenon. Mol. Plant Pathol. 6, 335–345.

Ogoshi, A. (1987) Ecology and pathogenicity of anastomosis and intraspecific groups of Rhizoctonia solani Kuhn. Annu. Rev. Phytopathol. 25:125–43.

Ogoshi, A. (1996) Introduction – the genus Rhizoctonia. In. Rhizoctonia species: taxonomy, molecular biology, ecology, pathology and disease control. Eds. Sneh B, Jabaji-Hare S, Neate S, Dijst G. Kluwer Academic Publishers, The Netherlands. pp. 1–9.

Ozer, G., Imren, M., Bozoglu, T., Dababat, A.A. (2021) First report of Rhizoctonia solani AG2- 1 on roots of wheat in Kazakhstan. Plant Dis.

Parmeter, J.R., Whitney, H.S. (1970) Taxonomy and nomenclature of the imperfect state. In.

Rhizoctonia solani Biology and pathology. Ed. Parmeter J.R. University of California Press, USA. pp. 7–19.

Paulitz, T.C., Okubara, P.A., Schillinger, W.F. (2006) First report of damping-off of canola caused by Rhizoctonia solani AG 2-1 in Washington State. Plant Dis. 90, 829

Peltonen, S. (1995) Comparison of xylanases production by fungal pathogens of barley with special preference to Bipolaris sorokiniana. Mycol. Res. 99, 717–723.

Shigenaga, A.M., Argueso, C.T. (2016) No hormone to rule them all: interactions of plant hormones during the responses of plants to pathogens. Semin. Cell Dev. Biol. 56, 174–189.

Singleton, L.L., Mihail, J.D., Rush, C.M. (1992) Methods for research on soil-borne phytopathogenic fungi. The Amer Phytopathol Soc. 157–165.

Sneh, B., Burpee, L. Ogoshi, A. (1991). Identification of Rhizoctonia Species, The Amer.

Phytopathol. Soc. Press, St. Paul, Minn, USA.

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Stodart, B.J., Harvey, P.R., Neate, S.M., Melanson, D.L., Scott, E.S. (2007) Genetic variation and pathogenicity of anastomosis group 2 isolates of Rhizoctonia solani in Australia. Mycol.

Res. 111, 891–900.

Tjamos, S.E., Flemetakis, E., Paplomatas, E.J., and Katinakis, P. (2005) Induction of resistance to Verticillium dahliae in Arabidopsis thaliana by the biocontrol agent K-165 and pathogenesis-related proteins gene expression. Mol. Plant-Microbe Interact. 18, 555–561.

Webster, J., Weber, R.W.S. (2007) Introduction to Fungi, 3rd edition.; Cambridge University Press

Weinhold, A.R., Sinclair, J.B. (1996) Rhizoctonia solani: penetration, colonization and host response. In. Rhizoctonia species: taxonomy, molecular biology, ecology, pathology and disease control. Eds. Sneh B, Jabaji-Hare S, Neate S, Dijst G. Kluwer Academic Publishers, The Netherlands. pp. 163–174.

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Chapter II

Relationship between AGs and subgroups of R. solani and its virulence and host specificity

2.1. Introduction

As mentioned in Chapter I, R. solani species are classified into 13 AGs. The hyphae of the same AG can fuse or anastomose with each other to expand their genetic diversity [Webster

& Weber, 2007; Carling, 1996]. Some anastomosis groups can be divided into multiple subgroups according to their pathogenicity, host specificity, cultural morphology, nutritional requirements, optimum temperature, and hyphal anastomosis frequency [Ogoshi, 1987; Sneh et al., 1991].

The relationship between the pathogenicity of R. solani and AG and subgroup classification remains elusive. For example, AG-1 IA isolates are usually known as a causal pathogen of rice sheath blight, but it also has been reported as pathogens of soybean and some Brassica species [Hua et al., 2014; Jones & Belmar, 1989]. Also, AG-2-1 isolates were frequently isolated from Brassicaceae species [Ogoshi, 1987; Carling & Leiner, 1986; Paulitz et al., 2006]. However, it was also found to infect pea, wheat, and barley [Stodart et al., 2007;

Ozer et al., 2021]. This fact does not necessarily prescribe the host range or preferential host species of a particular AG and subgroup.

To clarify if there is a relationship between host specificity and AGs and subgroups, it would be useful to survey the infectivity of R. solani various isolates to a single plant species.

In the previous studies, the pathogenicity of 35 R. solani isolates, belonging to AG-1, AG-2, AG-3, and AG-4, was tested on kidney beans, soybeans, red pepper, radish, sugar beet, and cabbage, and the virulence of the same isolate was varied on the different hosts [Bolkan &

Ribeiro, 1985]. Keijer et al. surveyed the host range of 32 R. solani isolates (ranging from AG-

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1 to 6) using two Brassicaceae (cauliflower and A. thaliana) and three Solanaceae (eggplant, tomato, and potato) plant species, and detected a certain degree of pathogenicity to plant species in the same family as the originally isolated plant species [Keijer et al., 1997]. R. solani isolates belonging to AG-8 and AG-2-1 isolated from wheat, barley, lentil, fallow, onion, canola, and chickpea from Washington State in the US were evaluated for their infectivity to wheat and canola [Jaaffar et al., 2016]. All the AG-2-1 isolates, but not the AG-8, except a single isolate, showed symptoms in canola. Moreover, AG-2-2 IIIB isolated from maize was found to be very aggressive against cauliflower [Pannecoucque & Hofte, 2009].

In the same context, the relationship between R. solani AGs and subgroups along with tissue-specific virulence is also unclear. Tissue specificity can also provide important aspects for the virulence mechanism of R. solani. The R. solani AG-2-1 was found to be virulent to Arabidopsis detached leaves and induced severe necrotrophic symptoms, while its colonization was restricted in roots when it was inoculated into the soil [Kidd et al., 2021].

To further investigate the relationship between AG groups and subgroups and host and tissue specificity, in this study, the infectivity of R. solani isolates collected in Japan from diseased crop species and soils were surveyed using a model plant A. thaliana.

2.2. Materials and methods

2.2.1. Fungal isolates and plant materials

Seventeen Rhizoctonia solani isolates were obtained from the Genebank of the National Agricultural Research Organization (NARO) in Japan. The isolates were classified by the researchers into different AGs by applying hyphal anastomosis assay (face-to-face culture assay). The sources of these isolates are summarized in Table 1. The isolates were maintained on potato-dextrose-agar (PDA) medium (24 g/L Difco™ potato dextrose broth, and 2% Agar).

Arabidopsis seeds were surface sterilized using sodium hypochlorite for five minutes, then

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suspended in sterile water and washed five times. The seeds were stored for three days at 4 ˚C in dark condition before sowing. Arabidopsis seeds were plated on ½ strength Murashige and Skoog (MS) medium plates (2.23 g/L MS salt (Nihon Pharmaceutical, Tokyo, Japan) enriched with 0.1% (v/v) Gamborg’s vitamin solution (Sigma-Aldrich, St. Louis, MO, USA), 1% (w/v) sucrose, 0.05% MES, and 0.8% Agar, pH 5.7 with KOH). Two-week-old Arabidopsis seedlings were transplanted into the soil (Supermix-A; Sakata Seed, Kanagawa, Japan) and grown for 2 weeks in a long-day growth chamber with LED lights (Nippon Medical & Chemical Instruments, Osaka, Japan) under a 16 h light/ 8 h dark photoperiod at 23 ˚C.

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Table 1: Rhizoctonia solani isolates used in this study and their pathogenicity to upper- and underground tissues of Arabidopsis thaliana Col-0.

AGs MAFF

number¹ Name Growth

(days)² Source

Infection severity³ Leaves Roots

AG-1 IA 305230 C-325 3 Rice +++ ++

AG-1 IA 305219 C-54 6 Rice - -

AG-1 IA 305232 C-501 3 Sudangrass - -

AG-1 IC 243448 RD1 3 Carrot +++ +++

AG-2-1 II 305203 6 7 Six-rowed barley ++ -

AG-2-2 IIIB 305244 C-329 7 Rice - ++

AG-2-2 IIIB 726525 RS-B 3 Broccoli +++ +++

AG-2-2 IIIB 242301 WLS81 4 Welch onion + +++

AG-2-2 IV 241951 SBF1 4 Broccoli ++ +++

AG-2-2 IV 242303 SD1 4 Soybean ++ +++

AG-2-3 237259 H5-210 7 Wheat ++ -

AG-2 BI 305228 SH-1-2 7 Soil - -

AG-3 IV 305250 C564 14 Potato - -

AG-4 IIIA 305225 BO-3 3 Cauliflower +++ +++

AG-5 305256 SH-30 7 Soil + -

AG-6 305262 UB-7-1-A 4 Soil - -

AG-7 305551 1529 3 Radish field soil + -

aMAFF numbers are descriptors for the microorganism genetic resources assigned by NARO (National agriculture and food research organization) Genebank in Japan.

bDays that were taken by the fungus to fill the 90-mm Petri plate.

cInfection severity visually assessed, (+++) severe symptoms, (++) moderate symptoms, (+) weak symptoms, (-) no symptoms.

2.2.2. Inoculation tests

2.2.2.1. Detached leaf inoculation assay

This assay was performed by detaching rosette leaves from 4-week-old Arabidopsis plantlets and were placed onto moistened Whatman filter paper in a petri dish. The cut sites were wrapped with a moist paper wiper (KimWipes; Nippon Paper Crecia, Tokyo, Japan).

Mycelial plugs (6 mm diameter) were prepared with a biopsy punch (BP-30F; Kai Corporation,

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Tokyo, Japan) from R. solani-grown PDA medium when the mycelial growth reaches the plate edges and they were placed on the middle of the detached Arabidopsis leaf [Mukherjee et al., 2010]. The plates were covered and placed in a tray with a clear plastic lid lined with moist paper towels to keep high humidity. Plates were incubated at 23 ˚C in a long-day growth chamber. The developed symptoms were observed and photographed daily until the fifth-day post-inoculation (dpi). Non-colonized PDA plugs were used as a control. The assay was performed at least three times with three biological replicates each to confirm the reproducibility.

2.2.2.2. Soil inoculation assay

Soil inoculation was carried out following the method of Bowyer, et al., (1995]. Briefly, 70-mL capacity pots were filled with soil and four mycelial PDA plugs (6 mm diameter) were inoculated. The pots were kept in a container with a clear plastic lid at 23 ˚C for 6 days. Two- week-old A. thaliana seedlings grown on MS agar were transplanted into the inoculated soil.

We planted two plants in one pot. Photographs were taken at 3, 5, 7, 10, and 14 dpi, and seedling survival rates at 7 and 14 dpi were scored. Soil containing four non-colonized PDA plugs was used as a control. The assay was carried out three times with three biological replicates each to confirm the reproducibility of the results.

2.3. Results

2.3.1. Phenotypic characterization and growth rate of R. solani isolates

In our study, we used 17 different R. solani isolates belonging to AG-1 to AG-7 obtained from different diseased plants and soils. Six isolates were isolated from Graminaceae plants, three isolates were obtained from Brassicaceae plants, four isolates were obtained from different dicot diseased plants, and four isolates were recovered from infected soils. To

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compare the isolates with the cultural morphologies, the photographs of the R. solani-grown PDA media for the isolates were taken after growing for 21 days at 23 ˚C and they were summarized in Figure 1. In this study, three isolates for each AG-1 IA and AG-2-2 IIIB were used, but their culture morphologies were varied even within the same AG and subgroup.

Figure 1: Cultural morphology of Rhizoctonia solani isolates used in this study on PDA medium The isolates were grown on PDA medium for 21 days at 25 ˚C. 1: AG-1 IA (MAFF305230), 2: AG-1 IA (MAFF305219), 3: AG-1 IA (MAFF305232), 4: AG-1 IC, 5: AG- 2-1 II (MAFF305203), 6: AG-2-2 IIIB (MAFF305244), 7: AG-2-2 IIIB (MAFF726525), 8:

AG-2-2 IIIB (MAFF242301), 9: AG-2-2 IV (MAFF241951), 10: AG-2-2 IV (MAFF242303), 11: AG-2-3 (MAFF237259), 12: AG-2 BI (MAFF305228), 13: AG-3 IV (MAFF305250), 14:AG-4 IIIA (MAFF305225), 15: AG-5 (MAFF305256), 16: AG-6 (MAFF305262), 17: AG- 7 (MAFF305551).

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Next, the time taken for the mycelium to reach the edge of the medium was measured and summarized in Table 1. Also, the hyphal growth rates were calculated (Figure 2). The growth rates of the 3 AG-1 IA isolates, as well as AG-2-2 IIIB were also not similar.

Figure 2: The growth rates of Rhizoctonia solani isolates used in this study The length (cm) of mycelium elongated over the course of a day of each isolate on PDA medium were measured and the growth speed was calculated. The red columns represent the isolates belonging to the same AG and the same subgroup.

2.3.2. Pathogenic behavior of the R. solani isolates in Arabidopsis leaves

To investigate the relationship between pathogenicity of R. solani and AGs and host specificity, we evaluated the virulence of the collected Japanese R. solani isolates (Table.1) on Arabidopsis thaliana, a model dicotyledonous plant. In this study, we firstly evaluated their infectivity to detached leaves of A. thaliana Col-0. The mycelial agar plugs of each R. solani isolate were inoculated on the Arabidopsis detached leaves placed in moist Petri dishes, and the photographs of the inoculated leaves were taken at 0, 3, and 5 dpi (Figure 3). In this study, the virulence aggressiveness of the isolates was classified into 4 levels; highly-virulent (+++), moderately-virulent (++), weak-virulent (+), and non-virulent (-), and the results were summarized in Table 1. In this assay system, it is possible that the apparent virulence intensity

0 0.5 1 1.5 2 2.5 3 3.5

AG-1 IA AG-1 IA AG-1 IA AG-1 IC AG-2-1 II AG-2-2 IIIB AG-2-2 IIIB AG-2-2 IIIB AG-2-2 IV AG-2-2 IV AG-2-3 AG-2 BI AG-3 AG-4 AG-5 AG-6 AG-7

Growth rate (cm/day)

R. solaniisolates

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depends on the growth speed of each isolate. To confirm this point, the virulence of each isolate was compared with their growth rates measured (Figure 2 and Table 1). As a result, it seems that the growth speed seems not to be correlated with the virulence level of the isolates.

The pathogenic symptoms were observed in the R. solani isolates AG-1, AG-2, AG-4, AG-5, and AG-7 but not AG-3 and AG-6. Note that the virulence level of AG-5 and AG-7 were categorized as weak virulent and their symptoms were only detected after 5 dpi. While, AG-1, AG-2, and AG-4 showed obvious symptoms in Arabidopsis leaves. However, the virulence level differed within the same AGs and subgroups (Figure 3). In this study, there are three AG-1 IA (two isolates were isolated from rice (MAFF305230 and 305219) and one isolate was isolated from Sudangrass (MAFF305232)), and, among them, only the single isolate (MAFF305230) exhibited severe necrotic lesion on leaves. As for AG-2-2 IIIB, three different isolates (MAFF305244 from rice, MAFF726525 from broccoli, and MAFF242301 from Welch onion) were tested. In this case, just a single isolate (MAFF726525) was found to be pathogenic against Arabidopsis leaves (Figure 3). These results indicate that the AGs and subgroups are unrelated to the virulence of R. solani to the particular host.

The R. solani isolates used in this study were sampled from various host sources but some of them could make severe infections on Arabidopsis leaves. For example, the AG-1 IA (MAFF305230) isolated from an infected rice plant could infect Arabidopsis leaves. Also, AG- 1 IC, AG-2-1 II, AG-2-3, and AG-2-2 IV (MAFF242303) isolated from diseased carrot, barley, wheat, and soybean, respectively, presented virulence on Arabidopsis leaves (Figure 3). These results indicated that these isolates have a certain virulence that can overcome the immunity mechanism of at least two different plant species. The virulence of these isolates is not limited to the phylogenetically-related plant species. Based on these results, the classifications of R.

solani with AGs and subgroups seem to be unrelated to the host specificity of the isolates.

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Figure 3. Infectivity of Rhizoctonia solani Japanese isolates in detached leaves of Arabidopsis thaliana Leaves were inoculated with 6-mm mycelial PDA plugs and incubated in a humid condition at 23 ˚C for 5 days. Photographs were taken at 0-, 3-, and 5-days post inoculation (dpi). Plain PDA plugs without fungus were used as a control. The assays were performed three times with three biological replicates. Bars, 1 cm.

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2.3.3. Pathogenic behavior of the R. solani isolates in Arabidopsis roots

Next, the virulence of the R. solani isolates in Arabidopsis roots was tested. The agar plugs prepared from the R. solani-grown PDA media were inoculated into the soil and incubated for 6 days. Then, Arabidopsis young seedlings were transplanted and the photographs of the pots were taken at 0, 7, and 14 dpi (Figure 4). In this case, the virulence of the isolates was categorized into 3 levels; highly-virulent (+++), moderately-virulent (++), and non-virulent (-) as summarized in Table.1. The R. solani isolates AG-1, AG-2, and AG-4 were strongly virulent in this assay system. On the other hand, AG-3, AG-5, AG-6, and AG-7 did not exhibit obvious symptoms. The AG-5 and AG-7 represented weak virulence on leaf tissue, but their pathogenicity was not detected in the root inoculation assay, although we cannot exclude the possibility of their weak virulence to root tissue that can be detected at a microscopic level. The AG-1 IA (MAFF305230), AG-1 IC (MAFF243448), and AG-4 IIIA (MAFF305225), which were virulent in leaf inoculation, were also pathogenic to underground plant parts. The AG-1 IA isolates (MAFF305219 and MAFF305232), which were nonpathogenic isolates on leaves, also did not induce symptoms in the soil inoculation. The AG- 2-1 II (MAFF305203) and AG-2-3 (MAFF237259) were pathogenic to leaves but they were nonpathogenic to roots. While the AG-2-2 IIIB (MAFF305244 and MAFF242301) were nonpathogenic to leaves but they were pathogenic to roots (Figure 4). In this assay, it can be concluded that there is no correlation between AGs and subgroups classifications and the virulence and host specificity of the isolates. This is the same conclusion obtained from the leaf inoculation assay.

When comparing the pathogenicity of each isolate on the two different inoculation systems (leaves and soil inoculations), we found that some isolates seem to have different pathogenic behavior towards different tissue. For example, AG-2-1 II and AG-2-3 were virulent isolates against Arabidopsis leaves (Figure 3), but they did not produce any symptoms

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when inoculated into the soil (Figure 4). In contrast, two isolates belonging to AG-2-2 IIIB (MAFF 305244 and MAFF242301) were non-virulent to the Arabidopsis leaves but caused severe infection when inoculated into the soil (Figures 3 and 4). Also, AG-5 and AG-7 shared this behavior with AG-2, as both could cause infection on the leaves while could not produce any symptoms in the soil-inoculation assay (Figures 3 and 4). From these results, we conclude that these isolates may have tissue-specific virulence against Arabidopsis leaves and roots.

Figure 4: Infectivity of Rhizoctonia solani Japanese isolates in soil-grown Arabidopsis thaliana seedlings PDA plugs prepared from R. solani-grown medium were inoculated to soil and incubated for 6 days. Then, 2-week-old Arabidopsis seedlings grown on MS agar plate medium were transplanted. Photographs were taken at 0-, 7-, and 14-days post inoculation (dpi).

Plain PDA plugs without fungus were used in the control treatment. The assays were performed three times with three biological replicates.

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In this study, we evaluated the pathogenicity of seventeen R. solani isolates. It includes three different AG-1 IA isolates that were recovered from rice and Sudangrass. We previously tested the virulence of these isolates on detached leaves of Brachypodium distachyon, and found that all of them were pathogenic [Kouzai et al., 2018]. These results suggest that they have a virulence mechanism for monocotyledonous plants. Among them, in this study, only the AG-1 IA (MAFF305230) isolated from rice showed pathogenicity to A. thaliana. This means that it would have virulence mechanism(s) adapted to both monocots and dicots. This might make this isolate hypervirulence on B. distachyon compared with the other two isolates as demonstrated in Kouzai, et al., (2018). According to the records of NARO, MAFF305230 was isolated at Fukuoka, Japan in 1975, MAFF305219 was also obtained at Fukuoka in 1966, and MAFF243956 was from Kagoshima in 1999. The comparative genomics approach would be able to reveal the molecular basis underlying this difference virulence regarding host specificity.

The eight AG-2 isolates were tested this time, and seven of them except AG-2 BI were pathogenic to Arabidopsis. This is basically consistent with the previous consensus that AG-2 is relatively compatible with Brassicaceae. The source of these isolates is varied and it includes Poaceae, Brassicaceae, Amaryllidaceae, and Fabaceae. The AG-2-2 IIIB and AG-2-2 IV from Broccoli and AG-2-2 IV from Soybean exhibited pathogenicity to both upper-and underground tissues. The defense mechanisms of these plant species might be relatively similar, otherwise, these isolates acquire alternative pathogenicity genes for different host species in the past. The AG-2-1 II from six-rowed barley and AG-2-3 from rice have virulence to A. thaliana leaves but not roots. The AG-2-2 IIIB isolates from rice and welch onion showed preferable virulence to roots. These results suggest that the virulence mechanisms in the AG-2 group are not common and differed in each isolate. These isolates could be useful materials to study tissue-

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specific immunity mechanisms in plants and the virulence mechanism of R. solani for its breaking down.

The AG-4 IIIA used in this study was found to be pathogenic to both upper- and underground parts of A. thaliana. Because the source of this isolate is Cauliflower, its virulence mechanism would be adaptable to the same family A. thaliana. At this moment, it is unclear if the virulence mechanisms of AG-4 isolates are varied or not. The AG-2 BI, AG-5, AG-6, and AG-7 used in this study are less or nonpathogenic to A. thaliana. This seems to be consistent with the fact that their sources are soil. However, it does not exclude the possibility that these AG groups contain virulent isolates to A. thaliana or other plant species in nature. Surveillance of virulence using at least several different isolates is needed to understand this point.

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Bowyer, P., Clarke, B.R., Lunness, P., Daniels, M.J., Osbourn, A.E. (1995) Host range of a plant pathogenic fungus determined by a saponin detoxifying enzyme. Science 267, 371–374.

Bolkan, H.A., Ribeiro, W.R.C. (1985) Anastomosis groups and pathogenicity of Rhizoctonia solani isolates from Brazil. Plant Dis. 69, 599–601.

Carling, D.E. (1996) Grouping in Rhizoctonia solani by hyphal anastomosis. In Rhizoctonia Species: Taxonomy, Molecular Biology, Ecology, Pathology and disease control; Sneh, B., Jabaji-Hare, S., Neate, S., Dijst, G., Eds.; Kluwer Academic: Dordrecht, The Netherlands, pp.

37–47.

Carling, D.E., Leiner, R.H. (1986) Isolation and characterization of Rhizoctonia solani and binucleate R. solani-like fungi from aerial stems and subterranean organs of potato plants.

Phytopathology 76, 725–729.

Hua, G.K.H., Bertier, L., Soltaninejad, S., Höfte, M. (2014) Cropping systems and cultural practices determine the Rhizoctonia anastomosis groups associated with Brassica spp. in Vietnam. PLoS ONE 9, e111750.

Jaaffar, A.K.M., Paulitz, T.C., Schroeder, K.L., Thomashow, L.S., Weller, D.M. (2016) Molecular characterization, morphological characteristics, virulence, and geographic distribution of Rhizoctonia spp. in Washington State. Phytopathol. 106, 459–473.

Jones, R.K., Belmar, S.B. (1989) Characterization and pathogenicity of Rhizoctonia spp.

isolated from rice, soybean, and other crops grown in rotation with rice in Texas. Plant Dis.

73, 1004–1010.

Keijer, J., Korsman, M.G., Dullemans, A.M., Houterman, P.M., De Bree, J., Van Silfhout, C.H.

(1997) In vitro analysis of host plant specificity in Rhizoctonia solani. Plant Pathol. 46, 659–

669.

Kidd, B.N. Foley, R. Singh, K.B., Anderson, J.P. (2021) Foliar resistance to Rhizoctonia solani in Arabidopsis is compromised by simultaneous loss of ethylene, jasmonate and PEN2 mediated defense pathways. Sci. Rep. 11, 2546.

Kouzai, Y., Kimura, M., Watanabe, M., Kusunoki, K., Osaka, D., Suzuki, T., Matsui, H., Yamamoto, M., Ichinose, Y., Toyoda, K., et al. (2018) Salicylic acid-dependent immunity contributes to resistance against Rhizoctonia solani, a necrotrophic fungal agent of sheath blight, in rice and Brachypodium distachyon. New Phytol. 217, 771–783.

Mukherjee, A.K., Carp, M.J., Zuchman, R., Ziv, T., Horwitz, B.A., Gepstein, S. (2010) Proteomics of the response of Arabidopsis thaliana to infection with Alternaria brassicicola.

J. Proteom. 73, 709–720.

Ogoshi, A. (1987) Ecology and pathogenicity of anastomosis and intraspecific groups of Rhizoctonia solani Kuhn. Annu. Rev. Phytopathol. 25:125-43.

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Ozer, G., Imren, M., Bozoglu, T., Dababat, A.A. (2021) First report of Rhizoctonia solani AG2- 1 on roots of wheat in Kazakhstan. Plant Dis. 105, 3744.

Pannecoucque, J., Hofte, M. (2009) Interactions between cauliflower and Rhizoctonia anastomosis groups with different levels of pathogenicity. BMC Plant Biol. 9, 95.

Paulitz, T.C., Okubara, P.A., Schillinger, W.F. (2006) First report of damping-off of canola caused by Rhizoctonia solani AG 2-1 in Washington State. Plant Dis. 90, 829.

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Res. 111, 891–900.

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Chapter III

Investigation of the infection mechanism of R. solani through the infection behaviour on Arabidopsis plants with exogenous application of phytohormones

3.1. Introduction

Gene knockout approach is useful to understand the virulence mechanism of the pathogens at a molecular level. But currently, it is impossible for R. solani due to its multinucleate nature and unavailable of transformation. As an alternative way, plant defense responses against the pathogen can provide information indirectly for the virulence mechanism of R. solani.

Plants have a complex immune system to defend themselves against biotic and abiotic stresses. In plant immunity, defense-related-phytohormones such as salicylic acid (SA), jasmonic acid (JA), and ethylene (ET) play pivotal regulatory roles [Berens et al., 2017]. The phytohormone SA is a phenolic compound involved in various plant processes including growth, flowering, senescence, and responses against abiotic and biotic stress [Raskin, 1992, Vlot et al., 2009, Dempsey et al., 2011]. SA has been extensively studied for its role in local and systemic acquired resistance (LAR and SAR) against biotrophic and hemibiotrophic pathogens [Glazebrook, 2005; Vlot et al., 2009; Dempsey et al., 2011]. The JA pathway has long been thought to allow plants to cope with various environmental stresses including attacks by necrotrophic pathogens and herbivores [Thomma et al., 1998; Glazebrook, 2005]. More recently, it has also been shown that JA-mediated defenses contribute to resistance against some biotrophic or hemibiotrophic pathogens [Thaler et al., 2004; Riemann et al., 2013;

Lemarie et al., 2015]. The gaseous phytohormone ET is well known for its role in fruit ripening and plant senescence [Burg & Burg, 1965; Grbić & Bleecker, 1995; Bleecker & Kende, 2000].

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Activation of ET signaling confers plant resistance upon pathogen attack, suggesting that ET signaling also plays a role in plant defense [Hoffman et al., 1999; Thomma et al., 1999;

Berrocal-Lobo et al., 2002; Yang et al., 2017].

In this chapter, the effect of the pre-application of the different phytohormones SA, JA, and ET in the induction of resistance or susceptibility in A. thaliana plants against the different isolates of R. solani was studied.

3.2. Materials and methods 3.2.1. Phytohormones treatments

Sodium salicylate (Wako, Osaka, Japan), methyl jasmonate (Wako), and ethephon (Sigma-Aldrich), an ethylene generator, were used as phytohormones. They were dissolved in dimethyl sulfoxide (DMSO) then diluted with distilled water to prepare 100 µM solutions (final DMSO concentration is 0.1%) supplemented with 0.04% (v/v) tween-20. Four-week-old A.

thaliana plants grown on soil were sprayed with the phytohormone solutions and incubated for 48 h at the same growth condition. Then, the detached rosette leaves were prepared and inoculated with R. solani PDA plugs as described in Chapter II [Kouzai et al., 2018a]. For the soil inoculation method, 10-day-old A. thaliana seedlings grown on ½MS were sprayed with the phytohormones solutions and kept in the same growth condition for 48 h. Then, seedlings were transplanted in the R. solani-inoculated soil as described before.

3.3. Results

3.3.1. The effects of exogenously applied SA, JA, and ET on the resistance in Arabidopsis leaves to R. solani

Arabidopsis Col-0 plants were treated with the defense-related phytohormones, SA, JA, and ET (1 mM each). Forty-eight hours after exogenously applied phytohormones the leaves