Powdery Mildew, Podosphaera Xanthii and Potato - Botrytis Cinerea

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Histological study of the interactions between Cucumber - powdery mildew, Podosphaera xanthii and Potato - Botrytis cinerea

Anindita Chakraborty Wageningen UR

Histological study of the interactions between Cucumber - powdery mildew, Podosphaera xanthii and Potato - Botrytis cinerea

Anindita Chakraborty Registration number: 811206156020

MSc Minor Thesis (PBR-80424) for the partial fulfilment of Master of Sciences in Plant Biotechnology Specialization of Molecular Plant Breeding and Pathology

Supervisors Dr. Yuling Bai

Daily Supervisor Dr. Michela Appiano Kaile Sun

Examiner Dr. Anne-Marie Wolters Dr. Evert Jacobsen

July, 2015

Laboratory of Plant Breeding, Wageningen University and Research Centre, Wageningen, The Netherlands.

Acknowledgements

I would like to take the opportunity to convey my heartfelt gratitude to my supervisor Dr. Yuling Bai for her constructive supervision, valuable suggestions and relentless support to initiate and accomplish this research work. Her patience, wisdom and critical guidance to the project approach allowed me the freedom to work in a spectacular way. I would also like to express my sincere appreciation to Dr. Michela Appiano and Kaile Sun, my daily supervisors, for their special instructions on operating instruments and other laboratory knowledge required to my lab-work, as well as continuous guidelines for writing a scientific report during this research, motivation and encouragement to reach my destination. I am very thankful to Dr. Evert Jacobsen and Dr. Ageeth van Tuinen for their valuable time to provide academic guidance, insightful comments and critical remarks in improving the manuscript. Importantly I should not forget to express my sincere thanks to Dr. Anne-Marie Wolters and Dr. Jan van Kan for their advice, cooperation and valuable suggestion to carry out these experiments. I am also indebted to many student colleagues and the technical staff of the laboratory of plant breeding, WUR for their consistent support throughout my project. Moreover, I could not but mention the name of Patrick Hendrickx, for his helps in microscopy techniques. In a nutshell, I got myself as a successful student of the project having support from the above mentioned respectful people; such accomplishment would not have been possible without their contribution.

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Thesis outline

This minor thesis is comprised of two experiments with the aim to provide insights into plant microbes interaction through histological analysis.

In the first experiment, histological study was carried out to elucidate the mechanism of interaction of plants of a F2 population of cucumber (F2-pm3) with cucumber powdery mildew, Podosphaera xanthii. This pathogen is an obligate biotrophic fungus. The staining for the histological analysis was performed using trypan blue solution. Development of pathogen structures (appressorium, haustorium, hyphae) and response of host tissue (Hypersensitive response, HR) were examined by light microscopy in susceptible and resistant plants.

In the second experiment, the infection progress of Botrytis cinerea, a necrotrophic fungus, was examined on potato plants. Leaf samples were stained in a medium using X-gluc. Histological study was performed to compare the development of the infection of Botrytis cinerea in susceptible control and DND1 silenced potato transformants.

Contents

1 Histological study of Podosphaera xanthii on cucumber ...... 1 1.1 Introduction ...... 1 1.1.1 Cucumber ...... 1 1.1.2 Cucumber Powdery Mildew ...... 1 1.1.3 Disease symptoms...... 2 1.1.4 Life cycle of Powdery mildew pathogen ...... 2 1.1.5 Host-pathogen interaction ...... 4 1.1.6 Objective of the present research ...... 6 1.2 Materials and methods ...... 7 1.2.1 Plant materials ...... 7 1.2.2 Sample collection ...... 7 1.2.3 Staining of sample ...... 7 1.2.4 Histological analysis ...... 7 1.3 Results ...... 9 1.3.1 Conidia germination and development ...... 9 1.3.2 Primary haustorium formation ...... 10 1.3.3 Host reaction ...... 11 1.3.4 Secondary structures and host reaction ...... 12 1.4 Discussion ...... 13 1.4.1 Infection structure development ...... 13 1.4.2 Host responses ...... 14 1.5 Summary ...... 17 1.6 References ...... 18 1.7 Appendices ...... 20 2 Histological study of Botrytis cinerea on potato ...... 21 2.1 Introduction ...... 21 2.1.1 Botrytis cinerea ...... 21 2.1.2 Infection cycle of Botrytis cinerea ...... 21 2.1.3 Resistance mechanism of the host plants against B. cinerea ...... 22 2.1.4 Susceptibility (S) genes ...... 24 2.1.5 DND1 ...... 25 2.1.6 Objective of present study ...... 25 2.2 Materials and methods ...... 26 2.2.1 Plant materials ...... 26 2.2.2 Pathogens description ...... 26 2.2.3 Inoculation and sample collection ...... 26 2.2.4 Staining of samples ...... 26 2.2.5 Histological analysis ...... 27

2.3 Results ...... 28 2.3.1 Observation at 0.5 hours post inoculation (hpi) ...... 28 2.3.2 Observation at 3 hours post inoculation (hpi) ...... 28 2.3.3 Observation at 6 hours post inoculation (hpi) ...... 29 2.3.4 Observation at 10 hours post inoculation (hpi) ...... 30 2.3.5 Observation at 24 hours post inoculation (hpi) ...... 31 2.3.6 Observation at 48 hours post inoculation (hpi) ...... 32 2.4 Discussion ...... 35 2.5 Summary ...... 38 2.6 References ...... 39 2.7 Appendices ...... 43

1 Histological study of Podosphaera xanthii on cucumber

1.1 Introduction

1.1.1 Cucumber Cucumber, Cucumis sativus L. (2n=2x=14) belongs to the genus Cucumis under the family Cucurbitaceae, is one of the widely consumed vegetables all over the world (Cavagnaro et al., 2010). The Cucurbitaceae or cucurbit family consists of diverse group of species including cucumber, melon, watermelon, squash, pumpkin etc. (Pérez-García et al., 2009). Cucumber is presumed to be from Indian origin and domesticated in different regions with diversified climatic conditions such as temperate and subtropical regions (Cavagnaro et al., 2010; Qi et al., 2013; Wóycicki et al., 2011). Cucumis sativus var. sativus L. is the scientific name of the cultivated variety of cucumber but its wild type Cucumis sativus var. hardwickii still exists (Qi et al., 2013). Qi et al., (2013) used some cucumber lines collected from a wide geographic distribution to characterize pattern of genetic variation of cucumber and divided them into four geographic groups (Figure 1). The Indian group consists of cucumber lines collected from India including some lines that are morphologically similar to the wild form Cucumis sativus var. hardwickii. The others three groups contain only cultivated form Cucumis sativus var. sativus. While the Eurasian group contains cucumber lines from central and western Asia, Europe and the United states, the East Asian group from China, Korea and Japan, and the Xishuangbanna group from south-western China (Qi et al., 2013). The yield and productivity of cucumber are influenced by different biotic and abiotic factors such as temperature, drought and pathogens (Wang et al., 2015). Powdery mildew is one of the common and widespread diseases of cucurbits among diverse range of diseases (Pérez-García et al., 2009).

Figure 1. Four groups of cucumber with their fruits morphology (Source: Qi et al., 2013)

1.1.2 Cucumber Powdery Mildew Powdery mildew fungi are obligate biotrophic plant pathogens that depend on their living host strictly for the completion of their life cycle. They establish a long-lasting host-pathogen interaction and cause numerous losses of crop production. Powdery mildew fungi cause greater yield losses of many important crops including cereals (e.g. barley, wheat), vegetables (e.g. gourd, melon, squash)

1 as well as ornamentals (e.g. lilac). Although many vegetable crops are infected by powdery mildew, perhaps the most severely affected group are cucurbits (Vela-Corcía et al. 2015). In cucurbits, two fungi species, Podosphaera xanthii (P. fusca) and Golovinomyces orontii (G. cichoracearum) are responsible for causing powdery mildew diseases (Martínez-cruz et al., 2014). However, Podosphaera xanthii is the main causal agent of powdery mildew in cucurbits in many countries of the world and considered the most important limiting factor for the production of cucurbits (Vela-Corcía et al., 2015).

1.1.3 Disease symptoms Podosphaera xanthii produces easily recognizable symptoms similar to that of other powdery mildews (Pérez-García et al., 2009). In cucurbits, disease can be recognized by the presence of a visual white powdery mass on the surface of leaves, petioles and stems, even sometimes on fruits (Figure 2). White, talcum-like, powdery mass is mainly composed of fungal mycelia and conidia (Martínez-cruz et al., 2014). Fungal colonies may join together and cover the entire top leaves surface in favourable environmental conditions. It reduces the photosynthetic capacity of the plant when the fungus feeds nutrients from the plant. Consequently, it causes yellowing of the leaf and can also lead to death of the leaf; even the whole plant may die through severe infection. The number, size and quality of the fruit of the infected plant can be hampered, which may severely affect the total yield. The infected cucumber fruits may be malformed, sunburned and sometimes may ripen prematurely, but the attack of powdery mildew on fruits of cucurbits is not so common (Pérez-García et al., 2009).

Figure 2. Symptoms of powdery mildew on the leaf of cucumber plant (F2-pm3-8) after 3 weeks of inoculation

1.1.4 Life cycle of Powdery mildew pathogen Pathogen powdery mildew is a true ascomycete fungus, belongs to the order Erysiphales that contains only one family, Erysiphaceae (Hückelhoven, 2005). The life cycle of Powdery mildew is divided in sexual and asexual stages (Pérez-García et al., 2009; Martínez-cruz et al., 2014).

Asexual cycle is mainly considered as responsible for most of the pathogenic damage. Conidium, the spore of Podosphaera xanthii, produces a short germ tube after landing on a host. The germ tube

2 swells at its tip and produces an essential penetration organ called primary appressorium to penetrate the host cuticle and cell wall. After breakdown of the cell wall barrier, the fungus produces primary haustorium inside a plant epidermal cell. It is noteworthy that, Podosphaera xanthii only can invade epidermal cells. Haustorium is a specialized fungal structure that makes direct relationship with the plant by facilitating nutrient uptake from it and releasing effectors (Martínez-cruz et al., 2014). A first or primary hypha arises from the primary appressorium or another pole of the conidium. Primary hypha forms secondary appressorium that produces secondary haustorium (Figure 3). Subsequently, primary hypha branches and forms secondary hyphae and conidiophores arise vertically from some of these hyphae. Five to ten ovoid shaped conidia are produced at the tip of each conidiophore in chains. The mat of secondary hyphae and conidia forms the white mycelium on the plant surface that looks like white powder, a typical symptom of powdery mildews (Figure 4) (Pérez-García et al., 2009).

Figure 3. Schematic representation of Podosphaera xanthii fungal structures during infection

Podosphaera xanthii is a heterothallic fungus. Chasmothecium (previous name was cleistothecium), a type of fruiting bodies is formed by sexual reproduction only after occurrence of two hyphae with opposite mating types. Chasmothecia are considered as sources of inoculum in overwintering. A chasmothecium contains one ascus with eight ascospores (sexual spore) (Figure 4). Chasmothecium of cucurbit powdery mildew is rarely or never observed in most of the important cucurbits growing areas, but it is assumed that ascospores have the ability to cause a disease outbreak similar to that of asexual conidia (Pérez-García et al., 2009).

Figure 4. The life cycle of Podosphaera xanthii (Source: Pérez-García et al., 2009)

1.1.5 Host-pathogen interaction Plants are infected by different types of pathogen that causes plant damage to a lesser or greater extent. But some plants can tolerate or resist pathogen so that they can survive and produce yield. So, pathogen needs to overcome all defense mechanisms of the immune system of the plant in order to establish an infection (Agrios, 1997).

Plant possesses multi-layered defence systems to cope with different types of stresses including microbial pathogen. The defense mechanisms of plant consists of two main lines which are called pre-existing or passive defense and induced or active defense. Pre-existing defense could be structural or chemical defense such as wax or cuticle layer, anti-microbial compounds, presence of inhibitors in plant cells, lack of essentials factors like recognition factors (specific molecules or structures) of pathogen or host, host receptors for toxins (Agrios, 1997). Induced or active defense mechanisms are divided into two groups: PAMP-triggered immunity (PTI) and effector-triggered immunity (ETI) and have been described using the zigzag model by Jones and Dangl, (2006).

The first layer of active defence is induced by pathogen derived elicitors (microbe- or pathogen- associated molecular patterns, PAMPs) or host derived elicitors (damaged-associated molecular patterns, DAMPs) caused by pathogen infection. PAMPs can be conserved pathogen structures such as bacterial flagella or fungal cell wall polysaccharides that are detected as non-self molecules by pattern recognition receptors (PRRs) of plants and can prevent further colonization of pathogen. This mode of plant immunity is called PAMP-triggered immunity (PTI) and the features of PTI are reactive oxygen species (ROS) production, callose deposition, stomatal closure, defense hormones induction such as salicylic acid (SA), jasmonic acid (JA) and ethylene (ET) etc. (Hückelhoven, 2005; Jones and Dangl, 2006; Nicaise et al, 2009).

Some pathogens produce effectors that are able to supress PTI resulting in effector-triggered susceptibility (ETS). When a particular effector is specifically recognised by one of the resistance (R) genes, an immune response called effector-triggered immunity (ETI) is initiated. ETI is a faster and stronger version of PTI that triggers the defence responses and activates a form of programmed cell death referred to as hypersensitive response (HR) at the infection site. Most of the R genes encode nucleotide binding site-leucine rich repeat (NB-LRR) proteins and the recognised effector is called avirulence (Avr) protein. In this situation, pathogens that fail to cause disease, are called avirulent pathogens and the host is called resistant. Thus the interaction is called incompatible. On the contrary, a compatible interaction occurs when the pathogen is virulent and host is susceptible (Glazebrook, 2005; Jones and Dangl, 2006).

The resistance mechanism to powdery mildew pathogen is roughly categorized into two types: pre- and post-haustorial resistance (Pérez-García et al., 2009). Powdery mildew pathogens need to penetrate cell walls to establish haustorial feeding structure in the host cell. Therefore, they must overcome the physical barrier given by the host cell walls. Deposition of cell wall appositions called papilla at the site of infection acts as a physical barrier that hampers pathogen penetration into the cell. Though the biochemical constitution of papilla may vary among plant species, the ß-1,3-glucan polymer callose is an abundant component of cell wall appositions (Underwood W, 2012). Pre- haustorial resistance is mainly mediated by papilla formation, not related with hypersensitive response (HR). Post-haustorial resistance is usually associated with hypersensitive response (HR) against powdery mildew (Figure 5) (Pérez-García et al., 2009). HR is a localized induce cell death at the infection site of host cell that restricts the growth and spread of pathogen and provides resistance of host against pathogen. HR can occur in one cell or very few cells and remain unnoticed in a resistant plant (Agrios, 1997).

Figure 5. Diagram representing the infection process of powdery mildew with plant responses

1.1.6 Objective of the present research

Previously, plants of a F2 population of cucumber, F2-pm3, were inoculated with a suspension of spores of the cucumber powdery mildew, Podosphaera xanthii, and phenotypically observed after 21 days. As a result, some plants resulted susceptible while others were found more resistant. Despite the fact that the study of powdery mildew is not new, little is known about the interaction between Podosphaera xanthii and cucumber. Therefore, the objective of this present study was to examine the spore development of Podosphaera xanthii and the response of susceptible and resistant cucumber plants through the histological analysis.

1.2 Materials and methods

1.2.1 Plant materials

Cucumber F2 population called F2-pm3 was used in the present experiment. Seed sowing, plant cultivation, inoculation of leaf with cucumber powdery mildew, phenotypic observation, collection and preservation of leaf samples were already done by our group. Nine plants (F2-pm3-1, F2-pm3-5,

F2-pm3-7, F2-pm3-8, F2-pm3-11, F2-pm3-31, F2-pm3-40, F2-pm3-61, F2-pm3-80) were used in this present experiment (Table 1).

Table 1. Plant materials used in the present study with their corresponding phenotypes Plant number Phenotype F2-pm3-1 Susceptible (S) F2-pm3-5 Resistant (R) F2-pm3-7 Resistant (R)

F2-pm3-8 Susceptible (S) F2-pm3-11 Susceptible (S)

F2-pm3-31 Resistant (R) F2-pm3-40 Resistant (R) F2-pm3-61 Susceptible (S) F2-pm3-80 Resistant (R)

1.2.2 Sample collection Initially all plants showed almost similar powdery mildew symptoms on the leaves. After 3 weeks, it was possible to distinguish resistant plants from susceptible ones. Then, a fresh true leaf per plant was inoculated with powdery mildew pathogen. Inoculated cucumber leaves were collected at 72 hours post inoculation (hpi). Leaves were cut in pieces of around 3 cm2 and four samples were taken from each inoculated cucumber leaf. Bleaching and preservation of samples was done by putting leaf samples in a 1:3 (V/V) acetic acid/ethanol solution immediately after cutting.

1.2.3 Staining of sample For staining, leaf samples were boiled for 5-10 minutes at 90°C in 1:2 (V/V) lactophenol/ethanol solution with 0.03% trypan blue. Immediately, leaf samples were transferred into the tubes of the saturated chloral hydrate (5kg:2L; W/V). Samples were kept in saturated chloral hydrate for 24-72 hours to discolour. Then each sample was mounted on glass slides with 1:1 (V/V) glycerol/water solution, a cover slip was placed over the glycerol/water solution and sealed tightly using transparent nail polish.

1.2.4 Histological analysis For histological analysis, Zeiss Axiophot light microscope was used. Proper adjustment of the microscope was done to get a good microscopic image. At the beginning, condenser was set to bright field and moved towards middle position using the condenser focusing knob and Köhler illumination method (Köhler A., 1894) was followed to generate even illumination of the sample. The focusing knob was used to make specimen focused that was kept during the whole alignment period. The luminous field diaphragm was made narrow and focused the image of the luminous field diaphragm

7 by changing the height of the condenser with the condenser focusing knob. When the image of the luminous field diaphragm was in sharp focus but not in centre, the condenser centering screws on the condenser was used to move it in centre. When the image was in almost centre, the luminous field diaphragm was opened until it fills the entire image field. Then the contrast was needed to be optimized as aperture diaphragm of the condenser was opened. The size of the aperture was corrected to maintain a suitable balance between contrast and resolution. An eyepiece was removed from the mount and looked into the tube directly. The diameter of the aperture diaphragm was adjusted in order to illuminate at least by 2/3 of the pupil diameter. Then eyepiece was inserted again and luminous filed diaphragm was set again when the objective was changed. The condenser phase stop was checked to make sure it matched to the chosen phase contrast objective. Then the sample was checked with 100X magnification and pictures were taken using Axiocam ERcs. A total of 25 germinated spores (Infection unit, IU) were counted for each plant to examine pathogen structures and plant responses.

1.3 Results Prepared slides of cucumber leaf samples, taken at 72 hpi, were analysed at microscope to understand the development of the powdery mildew infection and the reaction of the host cells. Germinated spores, infection structures (appressorium, haustorium), dense hyphal growth and host response (HR) were observed. For statistical analysis, two independent samples t-test were used to determine if there was a significant difference in the numbers and percentage of different structures and reaction of susceptible and resistant plants. Images of the interaction between Podosphaera xanthii and cucumber in both compatible and incompatible reaction are given in the Figure 6.

Figure 6. Interaction of Podosphaera xanthii with susceptible (A) and resistant (B) cucumber plant. A.1 shows a conidium with an appressorium; A.2 shows the mature haustorium inside of the epidermal cell of cucumber leaf; B.1 shows a conidium with an appressorium; B.2 shows epidermal cell with hypersensitive response (HR)

1.3.1 Conidia germination and development For the present study, 25 IUs (Infection unit) per plant were examined microscopically in order to study the infection structures. The number of non-germination conidia did not significantly differ between resistant and susceptible plants (P-value=0.268, α=0.05). The percentage of conidia forming primary appressorium was the same for susceptible and resistant plants and corresponded to 100%

(Appendix I). The size of the conidium was bigger than the epidermal cell and the structure of appressorium of Podosphaera xanthii was flattened. Germ tubes of Podosphaera xanthii was not supposed to be originated from the poles of the conidium (Figure 6-A.1 and 6-B.1). The number of primary hyphae was almost similar in both types of plant as no significant difference was found (P- value=0.199, α=0.05) (Table 2).

Table 2. Results of the histological analysis on susceptible and resistant plants

1.3.2 Primary haustorium formation Haustoria of Podosphaera xanthii were only found in epidermal leaf cell and two morphologically different haustoria were observed, a mature structured haustorium and an undeveloped haustorium. A mature haustorium consisted of vacuole in the central zone called haustorial body (hb), several haustorial lobes (hl) and it was separated by extrahaustorial membrane (em) (Figure 7). An undeveloped haustorium was found with small haustorial body without all recognizable features.

Figure 7. Pictures of the haustorium of Podosphaera xanthii; a mature haustorium in A and an undeveloped haustorium in B (hb- haustorial body, hl- haustorial lobe and em- extrahaustorial membrane)

The percentage of primary haustorium formation in susceptible and resistant plants varied significantly: 100% for susceptible plants and 87% for resistant plants (both in average) (Figure 8, Table 2). The p-value of t-test was 0.006 which was less than 0.05. Moreover, mature haustoria (Figure 7-A) was found in most of the susceptible plants whereas haustoria in a resistant plant looked like undeveloped without haustorial lobes (Figure 7-B) or with few lobes.

Figure 8. Incidence of infection units in % that initiated haustorium formation in resistant (R) and susceptible (S) plants

1.3.3 Host reaction Hypersensitive response (HR) was visible in all resistant plants and never present in susceptible plants (Figure 10, Table 2, Appendix I). In all resistant plants, epidermal cell with HR was found though the percentage of infection units (IU) that triggered HR was varied in different plants from 32% to 76% (Appendix I). We found recognizable number of IU initiated HR where haustorium was inside the epidermal cell. In such cases, haustorium seemed to be undeveloped without lobes or presenting few lobes (Figure 9). Any incidence of papillae formation was not observed in any plant infected by Podosphaera xanthii (Appendix I).

Figure 9. Pictures of epidermal cell with Hypersensitive response (HR) to Podosphaera xanthii (hb- haustorial body and em- extrahaustorial membrane)

Figure 10 Incidence of infected units in % that triggered hypersensitive response (HR) in resistant (R) and susceptible (S) plants

The number of infection units that triggered HR in epidermal cells of each resistant plant was counted. The number and percentage of IU that inducing HR comprised with haustorium inside the cells among the total number were also calculated. The percentages of IU that triggered HR after haustorium formation were 75%, 82%, 88%, 71% and 90% in resistant plants called F2-pm3-5, F2- pm3-7, F2-pm3-31, F2-pm3-40 and F2-pm3-80 respectively (Figure 11, Appendix II). The result indicated that the HR was initiated in the cell after the haustorium formation.

Figure 11 Diagram representing the incidence of infection units that triggered hypersensitive response (HR) in resistant plants. Coloured bars indicate the number of IU triggered HR with haustorium (orange coloured part, percentage is also mentioned) and without haustorium (green coloured part) inside the epidermal cell

1.3.4 Secondary structures and host reaction Development of secondary structures such as appressorium, haustorium as well as plant response (HR) were visible in both types of plants, but no significant difference was observed between susceptible and resistant plants (Table 2). The development of secondary hyphae were also seen in both types of plants, but it was not possible to count the number.

1.4 Discussion Conidial development was examined in susceptible and resistant plants in the present study. The germination of conidium, formation of appressorium, haustorium development and host response were observed histologically in both types of plants.

1.4.1 Infection structure development Histological examination showed that conidium germination, appressorium formation, primary hyphae development were almost similar without significant difference in both types of resistant and susceptible plants. It was found that conidium germination and appressorium development could be affected by host resistance. The surface characteristics of host plants like hirsuteness, cuticular wax and environment can also greatly influence conidial germination. The number of germ tube and appressorium formation can differ in two types of hosts because of their leave surface characteristics (Sedlářová et al., 2009). In the present study, we did not get any difference of conidium germination and appressorium formation in both types of plants.

In a biotrophic interaction, one of the most important organs is the haustorium (Martínez-cruz et al., 2014). In the present study, significant difference was visible for haustorium development between susceptible and resistant plants. All observed IUs of Podosphaera xanthii produced haustorium in epidermal cells of susceptible plants and the most of these were fully matured. But in resistant plants, fully mature haustoria were absent. The most of the haustoria in resistant plants were co- located with HR in the same epidermal cell, so that haustorium could be contracted due to the presence of HR. We also observed some undeveloped haustoria even in the cell where HR was absent. The more time could be required for the maturity of these haustoria whereas we only taken samples at 72 hpi.

Kuzuya et al., (2006) worked on the interaction of Podosphaera xanthii with different melon lines and found that well-developed haustoria formation consists of haustorial body, surrounded by several haustorial lobes that were separated from epidermal cells by extrahaustorial membrane in compatible interaction. They found that haustoria formation occurred within 24 hpi but mature haustoria development occurs within 120-240 hpi. In compatible interaction, the hyphae elongation, new conidia initiation and sporulation was found within 48 hpi, 120 hpi and 240 hpi respectively (Kuzuya et al., 2006). But in present study, we observed mature haustoria at 72 hpi of compatible interaction of Podosphaera xanthii with cucumber plants.

However, Kuzuya et al., (2006) also found two types of resistance in melon Podosphaera xanthii in incompatible interaction. They observed haustoria formation within 24 hpi with no or little germ tube branching. The haustoria were undeveloped without haustorial lobes around the haustorial body within 120 hpi (Type i resistance). Moreover, they also observed another pattern: conidia germination and haustorium formation occurred within 24 hpi, some secondary germ tubes and hyphal development were also observed within 48-120 hpi, but no sporulation occurred within 120 hpi. The haustoria seemed developing slowly despite of lacking lobes or having few lobes (Type ii resistance). In both types of resistance, the haustorium was not developed. Although they did not mention any specific reason of this, but they found callose accumulation around the haustorial neck of few haustoria. Moreover, they also found different responses in some melon lines depending on

13 the races of Podosphaera xanthii. In present study, we also observed undeveloped haustoria without lobes or few numbers of lobes in resistant plants at 72 hpi. Moreover, we still noticed development of secondary appressorium, haustoria and hyphae in both types of interactions without any significant difference.

Martínez-cruz et al., (2014) studied the haustorium of Podosphaera xanthii in the interaction with cucurbits cells. Initially they used confocal laser scanning microscopy (CLSM) and observed haustorium with three structural parts such as body, lobes and extrahaustorial membrane. For confirmation, they used transmission electron microscope (TEM) and observed some characteristics of the haustorium of Podosphaera xanthii. The haustorial lobes of Podosphaera xanthii cover the complete haustorial body whereas in Blumeria graminis f. sp. hordei, lobes grow at the poles and look as figure-like structures. The outline of extrahaustorial membrane was highly irregular like others powdery mildew pathogens. They also observed that a large vacuole was almost completely occupying in the haustorial body cytoplasm and such vacuole was surrounded by several vesicles. They divided the function of different parts of haustorium. The functions of haustorial body are mainly protein and effectors synthesis, as well as metabolism of nutrients taken from host plants. The functions of haustorial lobes are nutrients uptake, nutrients delivery and effectors exchange with plant cells. In the present study, we also observed haustorial lobes that were originated around the entire haustorial body, vacuole in haustorial body and irregular extrahaustorial membrane. But we could not see the vesicles around the vacuole of haustorial body. They conducted experiment using different staining methods such as toluidine blue, uranyl acetate with CLSM and TEM to examine the Podosphaera xanthii infected cucurbits leave with haustorial complex.

In case of secondary infection structures, we saw appressorium, haustorium as well as plant response (HR) in both types of plants without significant difference. But counting the number of secondary hyphae was not possible as the length of hyphae originated from different conidia was very long and overlapped each other producing net like structures. It was impossible to identify the conidium from which a particular hypha originated. We were not able to measure the colony size of IU for the same reason. In addition, the microscopy study of cucumber leaves was not so easy to perform. The surface of the leaf is irregular and not smooth, we observed epidermal cells and mesophyll cells in the same level sometimes, that make it difficult to focus.

1.4.2 Host responses Hypersensitivity response is a form of active defence mechanism and occurs during or after the first haustorium formation in powdery mildew infection that causes necrosis of the cell. Other defence mechanisms, such as phytoalexins production, pathogenesis-related proteins production, are also associated with HR. HR is usually of two types: a fast HR and a slow HR, depending on their stage of activation. Fast HR occurs during active resistance in early infection stage, while slow HR occurs when resistance is weaker and late. So in slow HR, pathogen may continue growing and reproducing at low level, thus, resistant plants show more necrosis and chlorosis around infection sites than susceptible plants (Niks et al., 2011). It was found that symptoms of powdery mildew were visible initially in both types of cucumber leaves used in the present study and leaves of susceptible plants were more green than leaves of the resistant ones (Figure 12). Moreover, the fungal growth was still continuing after the first HR production and formation of secondary hyphae was similar in both plants. The

14 production of slow HR perhaps was the reason behind showing the resistance of cucumber plants against Podosphaera xanthii.

Figure 12. Cucumber leaves of susceptible and resistant plants. A and B - leaves of susceptible plants and C and D - leaves of resistant plants (Plant number F2-pm3-59 was not included in the present histological study)

Kuzuya et al., (2006) found hypersensitive response, callose accumulation and lignification of epidermal cells were the cause of showing resistance of melon lines to Podosphaera xanthii. It was mentioned that the differences in timing of the HR were playing major role in showing differences in resistance in incompatible interactions. Type i resistance occurred due to the rapid production of HR within 24 hpi, whereas HR production was slower in type ii resistance between 48 to 120 hpi. In our present study we found some secondary structures after production of primary HR. The probable cause of this could be the production of slow HR. Again Martínez-cruz et al., (2014) stated that the haustorial lobes of Podosphaera xanthii increase the contact of surface area with the host cell that helps to exchange of materials as their lobes cover the entire haustorial body. Thus conidia easily get some nutrients because of their more contact with host cell and can continue growing if the HR production is occur in late.

In the present study, we did not find any pre-haustorial resistance such as papillae formation. But we found HR in the cucumber leaf epidermal cells within 72 hpi, especially localized in a single cell. HR was only found in resistance plants whereas susceptible plants did not show any HR. We found post- haustorial resistance as we observed most of the HR with haustorium in the same epidermal cell. We

15 also got some HR without haustorium, but it could be possible for staining problem. In some slides, staining was so high that makes whole epidermal cell dark blue and creates difficulty to distinguish HR and haustorium. Therefore IU of Podosphaera xanthii triggered HR in cucumber plants after the formation of haustorium in epidermal cells (post-haustorial resistance).

The present study aimed at the examination of the development of different structures of Podosphaera xanthii on susceptible and resistant cucumber leaf and the understanding of the underlined resistance mechanism. However, further modification could be suggested despite having significant differences in observation between susceptible and resistant plants. The use of the trypan blue solution was a critical stage during the staining process. Sometimes, it was difficult to distinguishing between plant cells and fungal structures, like haustorium, especially when the haustorium and HR were in the same epidermal cell. The double-staining method could be a possible solution to overcome such problem. Marques et al., (2013) performed a double staining method of cotton blue and safranin to distinguish fungal cell and plant cell, and found this method was very effective while studying Colletotrichum acutatum infected petals of Valencia sweet orange. As double-staining method has not been used in powdery mildew study yet, it could be a good alternative to get more precise result. In addition, in the present study, we used only one time point i.e. 72 hpi to investigate fungal development. It could be interesting to repeat the experiment using also more time points such as 12, 24, 48, 120 (and including 72 hpi) to understand if there are differences in the fungus growth at different stages and obtaining more accurate timing of HR cell death.

Overall, the findings of this study have improved our knowledge of the different structures of Podosphaera xanthii that are involved in the successful disease establishment on cucumber as well as the effects occurring in the infected cucumber cells. Understanding the interaction between Podosphaera xanthii and cucumber will provide the basis to set up future experiments and could lead to the development of new and effective strategies for disease control.

1.5 Summary Powdery mildew is one of the common and widespread disease of cucumber. Podosphaera xanthii is the main causal agent of powdery mildew in cucurbits throughout the world. This study investigated the spore development of Podosphaera xanthii and responses of the host in susceptible and resistant cucumber plants of a F2 segregating population (F2-pm3). A histological analysis was performed through trypan blue staining technique to visualise the interaction of Podosphaera xanthii on cucumber leaves at 72 hpi.

In both cases, it was possible to observe germinated spores, appressorium, haustorium and dense hyphal growth during microscopic examination. No considerable difference in conidial germination and primary hyphae formation was noticed between susceptible and resistant plants. However, the average percentage of primary haustorium formation in susceptible and resistant plants varied significantly, i.e. 100% and 87% respectively. Furthermore, two morphologically different haustoria of Podosphaera xanthii were visible during the infection process. A mature haustorium, consisting in haustorial body, haustorial lobes and extrahaustorial membrane, was observed in most of the susceptible plants, while in the resistant plants it seemed undeveloped without or with few haustorial lobes.

A significant difference was also found in the occurrence of HR in both types of plants. HR was not observed in any susceptible plant, but was visible in all resistant plants. In addition, in the resistant plants, most of the HRs co-localized with epidermal cells containing a haustorium which suggests a post-haustorial defence mechanism based on HR in resistant plants. No other host responses, like papillae formation, were observed in any of the plants infected by Podosphaera xanthii. Thus, the HR appeared to be an important underlying resistance mechanism and could also help to enrich our knowledge of resistance to Podosphaera xanthii.

1.6 References Agrios G. N. (1997). Plant Pathology, 4th edition. ISBN 0-12-044564-6. Academic Press Inc., 525 B Street, Suite 1900, San Diego, CA 92101-4495, USA. 93 -114 pp.

Cavagnaro P.F, Senalik D.A., Yang L., Simon P.W., Harkins T.T., Kodira C.D., Haung S., Weng Y. (2010). Genome-wide characterization of simple sequence repeats in cucumber (Cucumis sativus L.). BMC Genomics, 11:569. doi: 10.1186/1471-2164-11-569.

Glazebrook J. (2005). Contrasting mechanisms of defense against biotrophic and necrotrophic pathogens. The Annual Review Phytopathology, 43:205-227.

Hückelhoven R. (2005). Powdery mildew susceptibility and biotrophic infection strategies. FEMS Microbiology Letters, 245:9–17.

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

Köhler A. (1894). New Method of Illimination for Phomicrographical Purposes. Journal of the Royal Microscopical Society, 14:261–262.

Kuzuya M., Yashiro K., Tomita K., Ezura H. (2006). Powdery mildew (Podosphaera xanthii ) resistance in melon is categorized into two types based on inhibition of the infection processes. Journal of Experimental Botany, 57(9): 2093–2100.

Martínez-cruz J., Romero D., Dávila J.C., Pérez-García A. (2014). The Podosphaera xanthii haustorium, the fungal Trojan horse of cucurbit-powdery mildew interactions. Fungal Genetics and Biology, 71:21–31.

Marques J.P.R., Soares M.K.M., Appezzato-Da-Gloria B. (2013). New staining technique for fungal- infected plant tissues. Turkish Journal of Botany, 37:784-787.

Nicaise V., Milena Roux M., Zipfel C. (2009). Recent Advances in PAMP-Triggered Immunity against Bacteria: Pattern Recognition Receptors Watch over and Raise the Alarm. Plant physiology, 150: 1638-1647.

Niks, R.E., Parlevliet, J. E., Lindhout, P., & Bai, Y. (2011). Breeding crops with resistance to diseases and pests. Wageningen Academic Publishers, The Netherlands.

Pérez-García A., Romero D., Fernández-Ortuño D., López-Ruiz F., Vicente A.D., Torés J.A. (2009). The powdery mildew fungus Podosphaera fusca (synonym Podosphaera xanthii), a constant threat to cucurbits. Molecular Plant Pathology, 10 (2):153–160.

Qi J., Liu X., Shen D., Miao H., Xie B., et al. (2013). A genomic variation map provides insights into the genetic basis of cucumber domestication and diversity. Natural genetics, 45(12):1510-1515.

Sedlářová M., Lebeda A., Mikšíková P., Duchoslav M., Sedláková B., McCreight J.D. (2009). Histological aspects of Cucumis melo PI 313970 resistance to Podosphaera xanthii and Golovinomyces cichoracearum. Journal of Plant Diseases and Protection, 116 (4):169–176.

Underwood W. (2012). The plant cell wall: a dynamic barrier against pathogen invasion. Frontiers in plant science, 3(85):1-5.

Vela-Corcía D, Romero D., Torés J.A., Vicente A.D., Pérez-García A. (2015). Transient transformation of Podosphaera xanthii by electroporation of conidia. BMC Microbiology, 15:20.

Wang J., Pan C., Wang Y., Ye L., Wu J., Chen L., Zou T., Lu G. (2015). Genome-wide identification of MAPK, MAPKK, and MAPKKK gene families and transcriptional profiling analysis during development and stress response in cucumber. BMC Genomics, 16:386. DOI 10.1186/s12864-015-1621-2.

Wóycicki R., Witkowicz J., Gawroński P., Dabrowska J., Lomsadze A., et al. (2011). The Genome Sequence of the North-European Cucumber (Cucumis sativus L.) Unravels Evolutionary Adaptation Mechanisms in Plants. PLoS ONE, 6(7):e22728. doi:10.1371/journal.pone.0022728.

1.7 Appendices

Appendix I. Result from histological analysis

Plant Phenotype Observed Primary Seconday Non-germinated number IU no Appressorium No of hyphae Haustorium Papillae HR Appressorium Papillae HR Haustorium Spores no

F2-pm3-1 S 25 100% 79 100% - - 12% - - 12% 32

F2-pm3-5 R 25 100% 67 92% - 32% 8% - 16% 12% 19

F2-pm3-7 R 25 100% 82 88% - 68% 28% - 12% 16% 25

F2-pm3-8 S 25 100% 81 100% - - 48% - - 48% 40

F2-pm3-11 S 25 100% 81 100% - - 24% - - 24% 24

F2-pm3-31 R 25 100% 78 88% - 40% 12% - - 4% 9

F2-pm3-40 R 25 100% 70 76% - 56% 40% - 32% 12% 19

F2-pm3-61 S 25 100% 72 100% - - 16% - - 8% 16 F2-pm3-80 R 25 100% 60 92% - 76% 16% - - 16% 31

Appendix II. The HR reaction of resistance plants

Spores Spores producing HR with Observed Plant No Phenotype producing HR visualized Haustorium IU No (Total No) No Average F2-pm3-5 R 25 8 6 75% F2-pm3-7 R 25 17 14 82% F2-pm3-31 R 25 10 8 88% F2-pm3-40 R 25 14 10 71% F2-pm3-80 R 25 19 17 90%

2 Histological study of Botrytis cinerea on potato

2.1 Introduction

2.1.1 Botrytis cinerea Botrytis cinerea (B. cinerea) is a broad host range necrotrophic pathogen and a model for the study of necrotrophic fungi (Williamson et al., 2007). It is an ascomycete in the family of Sclerotiniaceae, which infects more than 200 host plants and causes gray mould disease worldwide (Van Kan, 2006; Smith et al., 2014). It is estimated that around 20% of the crop losses (pre- and post-harvest) in the world are caused by B. cinerea (Smith et al., 2014).

2.1.2 Infection cycle of Botrytis cinerea B. cinerea possesses multiple tools like enzymes, toxic compounds, phytotoxic metabolites to facilitate penetration, to kill host plant cells and to decompose plant tissue, and uses a variety of mechanisms to subdue host defences (van Kan, 2006). Conidia of B. cinerea form a germ tube within 2-4 hours post inoculation (hpi) and appressoria to penetrate the host cuticle within 8 hpi. An appressorium of B. cinerea does not always contain a septum that can separate it from the germ tube. The highly melanized wall that is essential to generate extreme high osmotic pressure for penetrating, is also absent in Botrytis appressoria (van Kan, 2006). Therefore, penetration of the cuticle and epidermis is most likely a biochemical process. After development of the germ tube, cuticle degrading activity was detected at the tip of the germ tube in the form of esterase activity (van Kan, 2006). The penetration peg of B. cinerea grows into the anticlinal cell wall of the epidermal cells at 16-24 hpi (Peters, 2015). The anticlinal cell wall is rich in pectin. B. cinerea needs enzymes to degrade pectin such as endopolygalacturonases (endo-PGs) and pectin methylesterases (PMEs) to be able to penetrate the cell wall (van Kan, 2006). At 24-48 hpi, mesophyll cell layers show high H2O2 concentrations as a result of both the pathogenic reactive oxygen species (ROS) accumulation and host defensive responses. High ROS concentration and secretion of cell wall degrading enzymes causes loss of cell wall integrity enabling B. cinerea to utilize the necrotic plant material for its own growth. Subsequently, hyphae expand further away from the original infection site and start producing hyphae in necrotic mesophyll layers (Peters, 2015) (Figure 1).

Figure 1. Different developmental stages of Botrytis cinerea upon penetration of the host. The image is not bound to a specific host, it is a general developmental pathway. Abbreviations: cu- cuticle; co- conidia; gt- germ tube; ap- appressorium; peg- penetration peg; epi- epidermis; mes- mesophyll; hpi- hours post inoculation; ROS- reactive oxygen species (Source: Peters, 2015)

2.1.3 Resistance mechanism of the host plants against B. cinerea Yet, no full resistance against B. cinerea has been found in plants. However, partial resistance has been found in accessions of Solanum habrochaites LYC4, a wild relative of the cultivated tomato (Finkers et al., 2007). Although, significant advances are made in understanding the interaction between plants and Botrytis cinerea, the resistance mechanism is still unclear. In recent years, the majority of resistance mechanisms to B. cinerea are identified where the cuticle and cell wall structure of the plant (Bourdenx et al., 2011; Buxdorf et al., 2014), the production of ROS (Asselbergh et al., 2007; Pietrowska et al., 2014) and the contribution of defence signalling pathways (Díaz et al., 2002; Beyers et al., 2014) were found to be involved.

2.1.3.1 Cuticle and cell wall structure The cuticle is a protective layer that covers all aerial parts of the plant and acts as a physical barrier against penetrating fungi and other pathogens. The main chemical component of the cuticle is cutin, a polyester polymer associated with various types of waxes. The cuticle greatly contributes to the defence of plants against different types of pathogens (Buxdorf et al., 2014).

The cuticle is produced by a complex pathway regulated by several enzymes. Buxdorf et al (2014) found that SlSHINE3, a transcription factor, affects the leaf cuticle as well as defence responses of tomato against Botrytis cinerea. Transgenic tomato plants overexpressing SlSHINE3 (SlSHN3-OE) were more resistant whereas silenced lines (Slshn3-RNAi) were more sensitive to Botrytis. Induction of pathogenesis related genes PR1a and Allene Oxide Synthase (AOS) in SlSHN3-OE was increased two- fold compared to wild type at 72 hpi. SlSHN3-OE plants produced more cutin monomers in contrast to the silenced plants (Slshn3-RNAi) which produced less cutin monomers as compared to wild type. This result suggested that the amount of cutin monomers and activation of the defense genes modulate the defense response (Buxdorf et al., 2014).

Cutin monomers are released from the cuticle in response to cutinase activity, which can act as elicitors and trigger defense responses. Therefore, the modification of cuticle permeability could facilitate less efficient perception of elicitors and cause delay of the defence activation. Bourdenx et al., (2011) stated that CER1 is a key gene in cuticle metabolism which is linked to wax formation. Modification of CER1 expression changes cuticle permeability. In Arabidopsis CER1-overexpression plants, the cuticle permeability was reduced and plants were more susceptible to the necrotroph Sclerotinia sclerotiorum (Bourdenx et al., 2011).

After breaching the cuticle the hyphae of B. cinerea grow within the outer periclinal cell wall of the epidermis, before the invasion of underlying tissue. Collapsing of the anticlinal cell walls could block the passage of B. cinerea into the underlying tissue resulting in decreased susceptibility. The epidermal anticlinal cell wall fortification was detected more abundantly in the abscisic acid deficient sitiens tomato mutant, whereas only few cells had limited cell wall modification in wild type. The modification was caused by H2O2 driven cross-linking of cell wall proteins and incorporation of phenolic compounds. Fungal growth was blocked by these cell wall modification that was involved in showing resistance of sitiens (Asselbergh et al., 2007).

2.1.3.2 Production of ROS An oxidative burst is a common early response of plant cells against pathogen. Reactive oxygen species (ROS) that includes superoxide ion (O2-), hydroxyl radical (OH*) and hydrogen peroxide

(H2O2) are produced during oxidative burst. ROS contribute to the activation of defense responses including direct antimicrobial action, cell wall modification, phytoalexin production, hypersensitive responses (HR) and triggering of systemic acquired resistance (SAR) (Asselbergh et al, 2007; El-Komy, 2014; Pietrowska et al., 2014).

The hypersensitive response (HR), a localized programmed cell death (PCD), is initiated by the rapid production of reactive oxygen species (ROS). HR is considered one of the successful defense strategies of plants against biotrophic pathogen attack to limit the spread of infection. In necrotrophic infection, enhanced ROS generation was also found but their role is still controversial. Since host cell death during HR is considered advantageous for the necrotrophs (Pietrowska et al., 2014; Patel et al., 2015), plant defense related ROS production facilitates the colonization by necrotrophic pathogens (Asselbergh et al., 2007). B. cinerea secretes superoxide dismutase (SOD) during the penetration of the cuticle that induces oxidative burst. Two genes encoding Nicotinamide adenine dinucleotide (NADPH) oxidase have been identified in B. cinerea, which is the key enzyme of

ROS production and has a great effect on virulence. NADPH-oxidase generates O2- that can be converted to H2O2 by SODs (Segmüller et al. 2008). Bcsod1 mutants produce lower levels of H2O2 resulting in retarded lesion formation and reduced virulence on hosts (van Kan, 2006). Research supports the assumption that B. cinerea triggers HR to accomplish pathogenicity in Arabidopsis, tobacco and other plants and thus host cell death acts as a determinant of host susceptibility in the infection of Botrytis species. However, ROS accumulation is not always a representant of susceptibility, HR may also be responsible host resistance to necrotrophs. Therefore, the timing and intensity of oxidative burst greatly influenced by the failure or success of B. cinerea infection in the host (Pietrowska et al., 2014).

In order to establish a successful infection, B. cinerea must imply protective mechanisms against the antimicrobial effects of ROS produced by the host as well as itself. Mannitol, an effective antioxidant, secreted by B. cinerea acts as a pathogenicity factor that supports the infection process. However, plants produce and secrete mannitol dehydrogenase (MTD), a catabolic enzyme, to counter the effects of mannitol producing fungi. It was found that transgenic tomato plants overexpressing MTD showed resistance against B. cinerea (Patel et al., 2015).

2.1.3.3 Effects of Hormones Resistance of plants against B. cinerea that is based on inducible defense strategies is regulated by various signalling pathways. Plant hormones such as salicylic acid (SA), jasmonic acid (JA), ethylene (ET) and abscisic acid (ABA) are considered as key elements of these pathways (Beyers et al., 2014). Generally, the SA-dependent defense pathway is active against biotrophic pathogens whereas the JA- dependent pathway is the main pathway for defense responses against necrotrophic pathogens. SA- and JA-dependent pathways can be either antagonistic or synergistic. A flexible signalling network can be created by crosstalk between SA and JA that can help the plant to tune defense responses against pathogens (Angulo et al., 2015; El Oirdi et al., 2011). The contribution of SA in plants resistance against B. cinerea is a complex process. It was found that B. cinerea activates the SA signalling pathway which antagonizes the JA pathway and so helps the fungus to develop disease in tomato plant (El Oirdi et al., 2011). However, SA was found to be involved in resistance of Arabidopsis against B. cinerea (Ferrari et al., 2003). Moreover, ET signalling was also found in contributing resistance of tomato against B. cinerea. Plants pretreated with ET resulted in decreased susceptibility to B. cinerea, and plants treated with an inhibitor of ET showed increased susceptibility (Díaz et al., 2002).

B. cinerea produces several metabolites and enzymes such as botrydial, botcinolides, oxalic acids and Nep1-like proteins to cause cellular plant death. The phytotoxin botrydial induces two pathogenesis- related proteins, PR1 and PDF1.2 that are involved in defence responses regulated by salicylic acid (SA) and jasmonic acid (JA) respectively. Botrydial manipulates host defence SA and JA signalling pathways to trigger the HR on the hosts and thus facilitates B. cinerea to feed on necrotic tissues (Rossi et al., 2011). Oxalic acid and Nep1-like proteins can trigger programmed plant cell death that is beneficial to the necrotrophic pathogens (Noda et al., 2010; van Kan, 2006).

The ABA-deficient sitiens mutant of tomato shows strong resistance to B. cinerea through the fast activation of ROS production and cell wall modification than the wild type (Asselbergh et al., 2007). Moreover, the hormone strigolactone is found to contribute to the plant resistance to B. cinerea. A strigolactone-deficient tomato mutant (Slccd8) was found to show the reduction of other plant defense hormones such as JA, SA and ABA and was susceptible to B. cinerea (Torres-Vera, 2014).

2.1.4 Susceptibility (S) genes In order to establish a compatible interaction, most of the pathogens need cooperation of the host along with overcoming plant immunity. Plant genes that facilitate infection of the pathogen and support a compatible relation can be considered susceptibility genes or S genes. According to van Schie & Takken, (2014) S genes facilitate infection process of a pathogen by allowing pre-penetration requirements (host recognition, penetration) or supressing host immune signalling or sustaining post-penetration requirements (metabolic or structural needs, pathogen proliferation). A loss of function, mutated S gene does not support the compatible interaction anymore and therefore limits

24 the pathogen to develop disease symptoms (van Schie & Takken, 2014). One of the most studied S genes is the barley Mlo gene, where a mutant allele confers broad spectrum resistance to Blumeria graminis f.sp. hordei (Bgh), a powdery mildew (PM) pathogen (Humphry et al., 2006; Zheng et al., 2013). Although, initially found in barley, this mlo based resistance was later also found for related mildews in other plant species such as Arabidopsis, tomato, pea, pepper and bread wheat (Zheng et al., 2013; Appiano et al., 2015).

Other genes that are involved in susceptibility against Botrytis cinerea are LePG and LeEXP1 (Cantu et al., 2008), RWA2 (Manabe et al., 2011), FDH (Voisin et al., 2009) which are involved in the pathogen activation and penetration, FDH (Voisin et al., 2009), CESA4 and CESA7 (Hernandez-Blanco et al., 2007), MYB46 (Kim et al., 2012), RST1 (Chen et al., 2005) are involved in defense suppression and pathogen sustenance and LePG and LeEXP1 (Cantu et al., 2008) are involved in replication.

2.1.5 DND1 In this study, we focused on the Arabidopsis Defense No Death 1 (AtDND1) gene (Yu et al, 1998) encoding a cyclic nucleotide-gated cation channel (CNGC; also known as AtCNGC2, Leng et al, 1999) that allows passage of transmembrane ion such as Ca2+, K+ and other cations (Clough et al., 2000). In Arabidopsis, dnd1 mutants were reported to show enhanced resistance against a variety of fungal, bacterial and viral pathogens (Yu et al., 2000; Jurkowski et al., 2004; Genger et al., 2008). Arabidopsis dnd1 mutants were found to show resistance to Pseudomonas syringae and strong restriction of pathogen growth without the presence of HR cell death (Yu et al., 2000). Studies suggested that dnd1 transformants show elevated levels of salicylic acid (SA) and an induction of pathogenesis-related (PR) genes (Clough et al., 2000).

2.1.6 Objective of present study In the previous study, the size of the lesion caused by B. cinerea on inoculated leaves of tomato and potato was examined. The smaller sized lesion was been found in more silenced DND1 plant leaves compared to leaves of susceptible control and less silenced DND1 plants. Then, a previous histological study was conducted to know the disease development of B. cinerea on tomato leaves. Inhibition of fungal growth was found in DND1 silenced tomato (Figure 2). Now, we also want to know the responses of potato. Therefore, the objective of this present study was to examine the B. cinerea infection progress in a susceptible control of potato and resistant StDND1 silenced potato transformants through histological analysis.

Figure 2. Results of histological study of B. cinerea infection on tomato (Source: Peters, 2015)

2.2 Materials and methods

2.2.1 Plant materials Potato cultivar Désirée and three DND1 silenced potato transformants (DND1A-6, DND1A-5, DND1A- 17) were used in the present experiment. RNA interference (RNAi) silenced transformants were made by our group in Désirée background (Table 1). Shoots were multiplied and grown in-vitro. After 3 weeks rooted in-vitro plants were transplanted into soil in greenhouse. Plants were grown in the greenhouse with 75% relative humidity in a 16h light/ 8h dark condition.

Table 1. Plant materials used: Potato cultivar Désirée and 3 more or less silenced potato transformants

Plant materials Characteristics Désirée Susceptible to B. cinerea DND1A-6 DND1 silenced transformants (Less silenced, 20%) DND1A-5 DND1 silenced transformants (More silenced, 80%) DND1A-17 DND1 silenced transformants (More silenced, 80%)

2.2.2 Pathogens description For the inoculation, the genetically modified (GM) Botrytis cinerea strain SAS56 which contains a pCutGUS was used (Van Kan et al., 1997). Spore suspension of B. cinerea in PDB (Potato Dextrose Broth, 12g/L) medium at a density of 3 x 105 spores/ml was provided by Jan van Kan of the laboratory of Phytopathology.

2.2.3 Inoculation and sample collection After 4-6 weeks in the greenhouse, when the plants were at the 9-10th leaf stage, 3th or 4th leaf below the first expanded leaf was selected for inoculation. A full composite leaf with 5 leaflets was harvested and stuck in flora foam placed in plastic trays covered with transparent plastic bags. Leaflets were inoculated on the upper side with 6-8 droplets of 2 µl of the GM B. cinerea spore suspension. After inoculation, the trays with leaves were covered with plastic bags to obtain a high humidity of around 100%. Then leaves were incubated in a climate chamber at 20℃, 14h light/10h darkness.

For GUS staining, inoculated leaves were punched out with a 1-cm-diameter cork bore at 6 time points, i.e. 0.5, 3, 6, 10, 24 and 48 hpi (hours post inoculation). Two technical and three biological replicates were used for each time point. In the meantime, two plants from each genotype were inoculated with only PDB medium and used as the mock in the experiment.

2.2.4 Staining of samples Collected leaf discs of potato were incubated in a medium containing X-gluc (5-bromo-4-chloro-3- indolyl β-D glucuronide, Biosynth AG) overnight at 37°C. The medium of 50 mM phosphate buffer was containing 0.5 mg/ml X-gluc, 0.1 mM KFeCN and 0.05% (v/v) triton-X100 with pH 7. In the next day, leaf discs were put in 96% ethanol, and repeated this step until plant tissue became clear by removing chlorophyll. Afterwards, leaf discs were transferred into new tubes with saturated chloral

26 hydrate (5kg:2L; W/V) and kept for a week to make samples clear and transparent. Then samples were mounted on glass slides with 1:1 (V/V) glycerol/water solution. A cover slip was placed over the glycerol/water solution and sealed tightly using transparent nail polish.

2.2.5 Histological analysis Prepared slides were observed with a Zeiss Axiophot bright field microscope (Zeiss, Germany). The samples were checked with 10X and 40X magnification and pictures were taken using a Axiocam ERcs.

2.3 Results Samples were collected at 6 different time points, 0.5, 3, 6, 10, 24 and 48 hpi to examine the disease development process of X-Gluc stained B. cinerea. Among all plant materials, Désirée is the wild type potato plant and DND1A-6 is the less silenced potato transformant (20% silenced), whereas DND1A-5 and DND1A-17 (80% silenced) both are much more silenced potato transformants. The microscopic image was taken by using same adjustment of light in order to get uniform images of all leaf samples.

2.3.1 Observation at 0.5 hours post inoculation (hpi) Blue coloured conidia were found on the leaf surface of potato cultivar Désirée and DND1 silenced transformants. The blue colour indicates that enzymatic activity in the pathogen had started earlier than 0.5 hpi (Figure 3).

Figure 3. Individual conidia on potato leaf samples of different genotypes after GUS staining at 0.5 hpi

2.3.2 Observation at 3 hours post inoculation (hpi) Germinated conidia were found on the surface of the leaf in wild type and silenced potato plants. But the number of the germinated conidia seemed less in the more silenced DND1 transformants, DND1A-5 and DND1A-17 than in Désirée and the less silenced DND1A-6 (Figure 4). Conidia produced a germ tube and few of them also produced an appressorium at 3 hpi (Figure 5).

Although, germinated conidia were growing on the surface of the leaf of all plants, some appressoria already started to penetrate the cuticle layer on Désirée and DND1A-6 leaves. It seemed that less conidia were attached on leaves of DND1A-5 and DND1A-17. Moreover, a more intensive blue coloured background was noticed on the leaves of Désirée and DND1A-6 (Figure 4 and 5).

Figure 4. Germinated conidia on leaves of Désirée and DND1 silenced plants leaf after GUS staining at 3 hpi

ap gt

Figure 5. Growth of germinated conidia on leaves of Désirée and DND1 silenced plants after GUS staining at 3 hpi, (co- Conidium, gt- Germ tube and ap- Appressorium)

2.3.3 Observation at 6 hours post inoculation (hpi) At 6 hpi, more attached conidia and a more intensive blue coloured background were also noticed on Désirée and DND1A-6 leaf (Figure 6). Hyphae of B. cinerea were developing between the cuticle and epidermal layer on the leaf of Désirée and less silenced DND1A-6 whereas less developing hyphae were developing on the cuticle layer in DND1A-5 and DND1A-17 plants (Figure 7).

Figure 6. GUS stained germinated conidia of B. cinerea on leaves of Désirée and DND1 silenced plants at 6 hpi

Figure 7. More detailed pictures (40X) representing the growth of B. cinerea hyphae on leaves of Désirée and DND1 silenced plants at 6 hpi, (co- Conidium, gt- Germ tube and ap- Appressorium)

2.3.4 Observation at 10 hours post inoculation (hpi) Hyphae of B. cinerea were long and developing between the cuticle and epidermal layer on the leaf of wild type and silenced plants at 10 hpi. The colour of conidia and hyphae were more dark blue at the inoculation sites on Désirée and less silenced DND1A-6 than on the leaf of DND1A-5 and DND1A- 17 (Figure 8). Again, some hyphae were found already trying to penetrate the epidermal layer on Désirée and less silenced DND1A-6 leaf whereas much less hyphae were developing on the cuticle

30 layer in DND1A-5 and DND1A-17 (Figure 9). In addition, the phenomenon of cell death on the epidermal layer of the leaf was observed in both wild type and silenced plants at 10 hpi (Appendix I).

Figure 8. Growth and colour differences of B. cinerea hyphae, after GUS staining, on leaves of Désirée and DND1 silenced plants at 10 hpi

Figure 9. GUS stained B. cinerea hyphae was entering inside the epidermal cell on the leaf of Désirée at 10 hpi

2.3.5 Observation at 24 hours post inoculation (hpi) At 24 hpi, the development of hyphae of B. cinerea was found in the mesophyll layer of the leaf of the wild type and the less silenced plant (Figure 10). But it seemed that B. cinerea was struggling to enter the mesophyll layer of leaves of more silenced DND1 plants and continuing to grow on the epidermal layer searching for the right place of penetration during the same time. Furthermore, DND1 silenced plants showed weak coloured hyphae and conidia on DND1A-5 and DND1A-17 leaves than on leaves of Désirée and less silenced DND1A-6 (Figure 11).

A B

Figure 10. The growth of B. cinerea hyphae, after GUS staining, on the epidermal layer (A) and inside the mesophyll layer (B) of a Désirée leaf at 24 hpi

Figure 11. Development of B. cinerea, after GUS staining, on inoculated leaves of Désirée and DND1 silenced plants at 24 hpi

2.3.6 Observation at 48 hours post inoculation (hpi) At 48 hpi, we observed the first symptom development of B. cinerea in Désirée and silenced plants by naked eye. The disease symptoms such as spreading of brown coloured necrotic spots surrounded by a water soaked lesion were found on inoculated leaves of Désirée and the less silenced plant, DND1A-6. However, brown necrotic spots with chlorotic response (yellowing cell death) were observed in more silenced plants indicating an unsuccessful infection attempt of B. cinerea on the inoculated leaves of DND1A-5 and DND1A-17 (Figure 12).

48 hpi 48 hpi

Figure 12. Disease symptoms of B. cinerea on leaves of Désirée and DND1A-5 silenced plant at 48 hpi

Again similar results were found in the GUS stained leaf samples where big blue coloured spots were indicating a dense network of growing hyphae of B. cinerea on leaved of Désirée and DND1A-6. On leaves of DND1A-5 and DND1A-17, a much lower amount of blue with yellow colour was observed indicating a very small fungus growth surrounded by the yellowing cell death. No fungus growth was observed on the leaf inoculated by only PDB (Figure 13).

Figure 13. GUS stained leaf samples of different genotypes at 48 hpi with B. cinerea (A) and PDB (B)

Therefore, the observations indicated that in the early stage, DND1 silencing may hamper the attachment of B. cinerea on the leaf surface as we found, after GUS staining, lower numbers of the conidia present on leaves of DND1 silenced plants at 3 and 6 hpi than on leaves of the control plants. During 6 to 24 hpi, the colour of the hyphae was light blue on leaves of more silenced DND1 plants compared to leaves of wild type and less silenced plants indicating that the expression of the GUS gene could be interrupted by DND1 silencing. Moreover, at 48 hpi, the infection of B. cinerea was

33 found to be restricted on leaves of a more silenced DND1 plants. In over all, it can be said that DND1 silencing is hampering the growth of hyphae of B. cinerea on an inoculated leaf (Figure 14).

Figure 14. Differences of the development of B. cinerea between Désirée and more silenced DND1 plant at different time points after inoculation (Scale bar = 100 μm)

2.4 Discussion Conidia of B. cinerea are serving as the primary source of inoculum. Conidial germination and adhesion on the surface of plant tissue are important events before penetration of the host tissue starts. When conidia landed on the plant leaf, after germination it has to penetrate the surface which is covered by the cuticle that is composed of cutin and wax. Under normal conditions, B cinerea conidia germinate, form germination tubes and produce infection structure called appressorium on the surface. The appressorium of B cinerea is not able to penetrate host tissue by physical pressure alone and it is thought to enter the plant surface by secreting enzymes that can breach the surface. B cinerea produces multiple tools that are capable to kill plant cells such as phytotoxic metabolites and proteins, oxalic acid and hydrogen peroxide. The ultimate goal of the pathogen is not to kill the plant, rather to decompose plant tissue and utilize the host derived nutrient by converting plant biomass to fungal mass for its own growth. B cinerea also produces cell wall degrading enzymes such as pectinases, cellulases and hemicellulases for colonizing plant tissue, releasing carbohydrates for consumption as well as some of these enzymes are also required for showing full virulence (Choquer et al., 2007; van Kan, 2006; Williamson et al., 2007).

At 0.5 hpi, we found the number of conidia was fluctuated among and between the different genotypes. We found the tendency of the conidia to move along the vein of the leaf (Appendix II-A). That is why counting the number of conidia could not be so accurate in several cases after droplet inoculation when two drops of spore suspension could mix together or some conidia of a drop run out along the vein. Another observation was that the disease infection of B cinerea was hampered when conidia was in a vein of the leaf (Appendix II-B).

In the present study, we found B cinerea conidia started the process of germination in wild type, Désirée and less silenced DND1A-6 leaf within 0.5 hpi as the blue coloured conidia indicated. The conidia showed a germ tube at 3 hpi, but some germ tubes produced already an appressorium in order to penetrate the cuticle layer at that moment. At 6 hpi, hyphae continued to grow between the cuticle and epidermal cell layer but some hyphae were found to start penetration in the epidermal layer within 10 hpi. Again, we found the hyphal growth in mesophyll layers within 24 hpi and subsequently, a disease symptom such as water socked spreading of necrotic area was found on the leaves of Désirée and DND1A-6 within 48 hpi (Figure 14).

However, we made some different observations in the more silenced DND1 transformants, DND1A-5 and DND1A-17. We found that more silenced DND1 plants inhibit the growth of B cinerea at each time point compared to Désirée and the less silenced DND1A-6. Much more conidia started to produce a germ tube and an appressorium at 3 hpi but they were still on the surface of the cuticle layer until 6 hpi with less numbers of conidia. In DND1 silenced plants the B. cinerea grew until the epidermal layer but seemed struggling to develop further into the mesophyll layer. In the later stage of 48 hpi, fungal growth was found to be restricted by lack of plant response. Moreover, another notable feature was the light blue coloured hyphae on DND1A-5 and DND1A-17 leaves after GUS staining (Figure 14).

We observed auto-necrotic cells on leaves of DND1A-5 and DND1A-17 which were the result of DND1 silencing. We checked some samples inoculated only by PDB without pathogen and found also auto- necrosis in all leaf samples of DND1A-5 and DND1A-17 after inoculating with B. cinerea or PDB (Figure 13). Again, we found the similar pattern of hyphae growth including light blue colour in

35 normal and auto-necrotic cells in all leaf samples (Appendix III). It is concluded that growth of hyphae of B. cinerea was not prevented by the auto-necrotic spots visible on the leaves of DND1 transformants.

In early stage (at 3 and 6 hpi) we found that the number of conidia was lower in DND1 silenced plants than in the control that indicating that DND1 silencing hampers the conidial attachment to the leaf surface. As we put the spore suspension as drop on the leaf and later we stained it in liquid, so that some conidia could be overflowed. Because of the above indicated technical problem, we were not able to count accurate numbers of conidia at 3 and 6 hpi. However, in later time points we did not find clear number differences of conidia between inoculated leaves of wild type and DND1 silenced plants. The conidia may need more time to attach to the leaf surface of DND1 silenced plants. At later times, the hyphae were already inside and stuck well to the plant, so that the staining procedure could not make problems anymore.

We also observed other colour differences in the present study. We found a clear blue colour background around the inoculation sites on leaves of Désirée and DND1A-6. At later stages, we also observed less coloured hyphae of B. cinerea in leaves of DND1A-5 and DND1A-17 than in those of the susceptible control plants. In the present study we used a GUS gene construct which was made by using a promoter of a B. cinerea cutinase gene encoding cutA and an uidA reporter gene to study fungal development during colonization of the host. The blue colour was visualized when the GUS gene was expressed under the control of the cutA promoter (van Kan et al., 1997). Blue colour background could be the result of over expression of the GUS gene in Désirée and DND1A-6. When such a GUS gene is over expressed, it could leak out the blue X-Gluc products during incubation at 37°C. On the contrary, in the DND1 silenced plants, DND1A-5 and DND1A-17, the GUS gene was expressed initially but later the gene expression could be interrupted. Therefore no leakage was observed during incubation and for the same reason, the colour of the hyphae was less intensive blue in leaves of DND1 silenced plants.

We found that the hyphae growth of B. cinerea was inhibited in DND1 silenced potato plants. Almost similar results of the lack of growth progression of B. cinerea was found in DND1 silenced tomato plants (Figure 2) (Peters, 2015). Moreover, in DND1 silenced Arabidopsis plants, constitutively increased expression of PR genes such as PR-1 and salicylic acid (SA) was found against Pseudomonas syringae. DND1 silenced plants showed strong restriction of pathogen growth in the absence of HR cell death (Yu et al., 1998). However, SA treated wild type plants did not inhibit lesion formation or growth of B. cinerea at all (Govrin and Levine, 2000). Although there are some contradictions, but it was stated that B. cinerea triggers hypersensitive response (HR) that facilitates its colonization in host plants. Therefore, it was reported that DND1 enhances resistance to B. cinerea because of its capability to restrict programmed cell death responses of the host (Govrin and Levine, 2000).

It was found that Arabidopsis plants, expressing fungal cutinase (CUTE plants), were complete resistant to B. cinerea. The germination of conidia was found normal but the hyphal growth of B. cinerea after the spore germination was inhibited on CUTE plant tissue compared to that of wild type plants (Appendix IV). It was found that the effects of cutinase to display immunity against B. cinerea was indirect by the modification of the cuticle. When cell wall-targeted fungal cutinase was expressed in plants, resistance was developed by releasing fungitoxic substances from the surface of the leave and by overexpressing genes coding for lipid transfer protein (LTP), peroxidase and the

36 proteinase inhibitor families. These responses were found independent of SA, JA and ET signalling pathways (Chassot et al., 2007). There could be a relation between DND1 silencing and the development of cuticle defects.

It was reported that the occurrence of a biotrophic phase was found in necrotrophic pathogen during the initial stages of disease establishment. Host responses, specially oxidative burst are occurring to supress the growth of the fungus in living tissue. When the fungus is established, it starts to continue necrotrophic life and inducing apoptotic cell death that provides nutrients to the fungus and facilitates the spreading of lesions. The plant limits the spread of this type of cell death by inducing autophagy contributing to resistance (Kabbage et al., 2013). In the present study we found that, in wild type potato, B. cinerea produced disease symptoms by spreading cell death (apoptosis). But in DND1 silenced potato plant, spreading of cell death was restricted by plant responses that could be classified to be autophagy. Sclerotinia sclerotiorum, a necrotrophic fungus found to secrete oxalic acid (OA) in Arabidopsis that induces apoptotic cell death and cause rapid spreading of disease. It is also presumed that OA can supress autophagy (Kabbage et al., 2013). In another study it was found that Botrytis elliptica, a specialized pathogen of lily, induces programmed cell death (PCD) or apoptosis by secreting toxins. In compatible interaction of Botrytis elliptica and lily, small water- soaked infection spots that were developed by spreading from the dark, dry necrotic regions were found. But during incompatible interaction, tiny dark spots consisting of a few or single dead cells appeared without spreading these lesions. It was found that the blocking of Ca2+ influx is one of the reasons that causes inhibition of cell death and the spreading of necrotic lesions by B. elliptica (Baarlen et al., 2004). Arabidopsis AtDND1 was also found to be involves in the passage of Ca2+, K+ and other cations (Clough et al., 2000). Therefore, silencing of DND1 in potato may block the Ca2+ diffusion that could be involved in autophagy and may affect the spreading of lesion growth by restricting apoptosis.

The result of the previous histological study of B. cinerea on tomato leaves (Peters, 2015) was almost similar to the present report. It was found that DND1 silencing inhibits the growth of the fungus on tomato leaves. Another observation was that the hyphal length were smaller on DND1 silencing tomato leaves compare to the leaves of wild type until 22 hpi (Figure 2). However, we did not observe any differences of the length of B. cinerea hyphae from 10 hpi to 24 hpi in the present study. It seemed that the hyphae was growing but struggling to enter into the mesophyll layer of the leaf at 24 hpi. We observed that the morphological differences in DND1 silenced tomato and potato plants. DND1 silencing tomato leaves were smaller and more curved than leaves of wild type tomato (Appendix V) where DND1 silencing potato leaves were almost similar sized compared to wild type (Figure 12). In addition, more auto-necrotic cells was present in DND1 silenced tomato leaves compare to potato leaves. These morphological differences of DND1 tomato and potato plants could influence results of both two studies.

In conclusion, it can say that DND1 silencing hampers the growth process of B. cinerea on leaves of potato. However, the mechanism behind the resistance is not so clear yet. Disruption of DND1 function may cause the activation of other plant responses. Therefore, further study of gene expression can be suggested to know the level of salicylic acid (SA), cutinase and autophagy in the DND1 silenced plants compare to susceptible control. These results will increase our knowledge on mechanisms of the disease resistance in plant species.

2.5 Summary Although the majority of breeding focuses on R-gene induced resistance, S-gene induced loss of susceptibility still remains less explored. In this study StDND1 silencing was used to mimic loss of function mutation in potato. Here we investigated the reaction of the necrotrophic fungus B. cinerea in wild type and DND1 silenced potatoes. A GUS expressing GM strain of B. cinerea was used in histological study to visualise the infection process on the leaf microscopically. Potato cultivar Desiree and the transformant (DND1A-6) with a low degree of DND1 silencing showed the expected infection process and disease symptoms. B cinerea conidia produced a germ tube and an appressorium at 3 hpi, breached the cuticle layer and grew further in the epidermal layer at 10 hpi. At 24 hpi, the hyphae were already in the mesophyll layer and the disease symptom was visualized at 48 hpi. Two transformants (DND1A-5, DND1A-17) with a high degree of DND1 silencing showed a much lower degree of susceptibility which was visible already from the moment that conidia were landed on the leaf surface. In the early stage, lower number of conidia was on leaves of DND1 silenced plants. At 24 hpi the hyphae were in the epidermal layer but seemed struggling to develop further to the mesophyll layer. Later an unsuccessful infection attempt of B. cinerea was found at 48 hpi on leave of DND1A-5 and DND1A-17. In over all, it can be concluded that DND1 silencing may limit the growth of B. cinerea hyphae from the beginning, leading to a low degree of susceptibility.

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2.7 Appendices

Appendix I. Cell death was observed in epidermal cells of B. cinerea inoculated leaf samples at 10 hpi after staining by 3,3ʹ-diaminobenzidine (DAB). Picture was taken from another experiment of our group.

Appendix II. B. cinerea conidia were found to move along the vein (A) and as a consequence the disease infection was hampered on the leaf (B). The disease symptom is marked as red arrow and infection failure in a vein is mark as a blue border.

Appendix III. Gus-stained B. cinerea development on the auto necrosis area in DND1A-5 plant.

Appendix IV. Trypan blue staining of B. cinerea inoculated wild type Arabidopsis and CUTE leave. (Source: Chassot et al., 2007).

Appendix V. Comparison of DND1 silenced plants (left side) with wild type tomato plant (right side). Pictures were taken when both plants were 5 weeks old. (Source: Peters, 2015).