Institute of Plant Nutrition Faculty of Natural Sciences

Effect of sulfur nutrition on the resistance of tomato (Solanum lycopersicum L.)

against Pseudocercospora fuligena

A Thesis Submitted in Partial Fulfillment of the Requirements for the Degree Master of Science in International Horticulture

(Major in Plant Nutrition)

By

San Shwe Myint

Supervisors:

Prof. Dr. Walter J. Horst Prof. Dr. Bernhard Hau

Prof. Dr. Thomas Debener

September 2007

DEDICATION

In the memory of my father

ii

ACKNOWLEDGEMENTS

I wish to express my sincere gratitude to Prof. Dr. Walter J. Horst for supervising this work. I

am grateful indeed for his invaluable advice, facilitated supports and resourceful discussion in

the course of this study.

I am also thankful to Prof. Dr. Bernhard Hau and Prof. Dr. Thomas Debener for being my

second and third supervisors and for their critical advice.

My particular thanks go to Dr. Gregor Heine for his guidance and assistance towards the

realization of this manuscript. His much patience, constructive criticisms and total

involvement in this work will never be forgotten.

I am grateful to Prof. Dr. Jutta Papenbrock and Ms. Julia Volker from Institute of Botany,

Hanover for collaborative support in laboratory analysis and fruitful discussion.

I am indebted to Dr. Angelika Stass, Dr. Schulte auf’m Erley Gunda, Dr. Dejene Eticha,

André Specht, Andres F. Rangel, Benjamin Klug and Hendrik Fuehrs for their enthusiastic

discussion and helpful comments. Many thanks to Ingrid Dusy, Tanja Edler, Anne Herwig,

Gerlinde Geisler, Hartmut Wieland, H. Geyer and Alexander Klein for their helpful technical

assistance. I do appreciate the academic competency and team-work of staff members from

our Institute of Plant Nutrition.

My grateful thanks go to DAAD and Leibniz University Hannover for the two years financial

support. To my honesty, I am very much impressed by Prof. Dr. H. Stuetzel and Prof. Dr.

D.M. Hoermann for keeping this program, “Master of Science in International Horticulture”

academically competitive and socially dynamic.

Best regards to friends Dr. Ni Ni Tun and Igor, Ms. Kyin Than and Mie Mie, and Ms.

Thandar Nyi for making me feel at home and encouraging and supporting me during my time

in Hanover.

Last, not least, I have to thank my family for their never-ending love, complete understanding

and sustained endurances.

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TABLE OF CONTENTS

LIST OF FIGURES .................................................................................................................. v

LIST OF TABLES.................................................................................................................... vi

ABBREVIATIONS................................................................................................................. viii

ABSTRACT ............................................................................................................................... x

1. INTRODUCTION ............................................................................................................ 1

2. LITERATURE REVIEW................................................................................................. 3

2.1. Tomato Black Leaf Mold (BLM)............................................................................... 4

2.1.1. Outbreaks of BLM ............................................................................................. 4

2.1.2. Disease symptoms and morphology of P. fuligena............................................ 5

2.2. Plant Resistance.......................................................................................................... 6

2.3. Importance of Sulfur Nutrition on the Plant Resistance ............................................ 6

2.4. Sulfur Assimilation and Metabolism in Plants........................................................... 7

2.5. Role of Sulfur Containing Defense Compounds (SDCs) in Plant Resistance ........... 9

2.5.1. Glutathione (GSH) ........................................................................................... 10

2.5.2. Glucosinolates (GSLs) ..................................................................................... 11

2.5.3. Phytoalexins ..................................................................................................... 11

2.5.4. Elemental sulfur (S0) ........................................................................................ 11

2.5.5. Sulfur-rich proteins (SRP)................................................................................ 12

2.5.6. Hydrogen sulfide (H2S).................................................................................... 13

2.6. Sulfur-induced Resistance (SIR) or Sulfur-enhanced Defense (SED)..................... 13

3. OBJECTIVES AND HYPOTHESES............................................................................ 15

3.1. Objectives................................................................................................................. 15

3.2. Hypotheses ............................................................................................................... 15

4. MATERIALS AND METHODS.................................................................................... 16

4.1. Plant Cultivation....................................................................................................... 16

4.2. Treatments and Experimental Design ...................................................................... 17

4.3. Cultivation of P. fuligena ......................................................................................... 18

4.4. Inoculation................................................................................................................ 18

4.4.1. Inoculation of whole leaves.............................................................................. 18

4.4.2. Localized inoculation for microscopic examinations....................................... 18

iv

4.5. SPAD Measurement and Fresh Weight ................................................................... 19

4.6. Visual Estimation of Disease Severity ..................................................................... 20

4.7. Microscopic Examinations....................................................................................... 21

4.7.1. Experimental setup........................................................................................... 21

4.7.2. Staining and microscopy .................................................................................. 22

4.8. Chemical Analysis.................................................................................................... 23

4.8.1. Determination of total sulfur ............................................................................ 23

4.8.2. Determination of water soluble compounds..................................................... 23

4.9. Statistical Analysis ................................................................................................... 26

5. RESULTS ....................................................................................................................... 27

5.1. Effects of Sulfur Nutrition on the Growth and Sulfur Status of Tomato Plants ...... 27

5.1.1. Chlorophyll content (SPAD value) .................................................................. 27

5.1.2. Shoot fresh weight............................................................................................ 29

5.1.3. Total sulfur and sulfur fractions ....................................................................... 29

5.1.4. Relationship between leaf sulfur status and SPAD values of plants................ 34

5.2. Effect of Sulfur Nutrition on P. fuligena infection .................................................. 34

5.2.1. Areas under disease development .................................................................... 34

5.2.2. Microscopic examinations................................................................................ 38

5.2.3. Relationship between fungal growth and disease severity............................... 42

5.3. Metabolites in Relation to Sulfur Supply and P.fuligena Infection ......................... 42

5.3.1. Total soluble proteins, total amino acids and carbohydrates ........................... 42

5.3.2. Sulfur-containing metabolites: Cysteine and GSH .......................................... 45

5.4. Relationship between Disease Severity and Sulfur Status ....................................... 47

5.5. Correlations between Different Sulfur Fractions ..................................................... 49

6. DISCUSSION................................................................................................................. 52

6.1 Role of Sulfur Nutrition on Sulfur Metabolism of Tomato Plants........................... 52

6.2 Interaction between Sulfur Nutrition and P. fuligena Infection............................... 60

7. REFERENCES .............................................................................................................. 65

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LIST OF FIGURES

Figure 1: Biotic, abiotic and man-related factors that can induce stress-related reactions of terrestrial plants (Adapted from Lichtenthaler, 1996; Reigosa et al., 1999). .......................................... 3

Figure 2: Symptoms of tomato black leaf mold (BLM); (A) visible lesions on upper leaf surface in the early stages of infection; (B) leaves roll, dry and develop sooty appearance in the advanced stage. 5

Figure 3: Sulfur assimilation as a platform for the biosynthesis of sulfur-containing defense compounds (SDC) (Adapted from Rausch and Wachter, 2005). ............................................................ 8

Figure 4: Sulfur-containing metabolites that are involved in the constitutive and induced protection of plants against pests and diseases (Bloem et al., 2005). ....................................................................... 9

Figure 5: Inoculation of P.fuligena on localized areas of the lower leaf surface, (A) agar ring (0.13 cm2) made from 3% agar solution; (B) mycelium block (0.2 cm2) scratched off the cultured plate..... 19

Figure 6: Correlation coefficients (r) for the relationship between percentage and scale values of disease severity (leaf area infected) at the two different time points (15 and 21 dpi). [Note: Experiment four, 2007]…………….. ....................................................................................................................... 21

Figure 7: Detailed structures of P. fuligena examined under light microscope. (A) cylindric- obclavate conidia and conidiophores from infected leaf; (B) fascicle of divergent conidiophores growing out of cuticle of infected leaf. [scale bars = 50 µm]................................................................ 22

Figure 8: Different degrees of leaf greenness in relation to differential sulfur supply, (A) small pale leaf with severe sulfur deficiency; (B) light green leaf with moderate deficiency; (C) well supplied leaf……………….. ............................................................................................................................... 28

Figure 9: SPAD readings of three leaves respectively of experiments (A) one (winter 2006), (B) two (summer 2006) and (C) three (winter 2007). The plants were grown in nutrient solution at three sulfur supply levels (0/50, 500, 5000 µM). Means (n=6) ± SD with different letters for each leaf are significantly different at p < 0.05 (Tukey test). *, ** and *** indicate significant difference at P < 0.05, 0.01 and 0.001 respectively (F test); ns, not significant. .............................................................. 28

Figure 10: Shoot fresh weights of 13-week-old tomato plants from experiment one (winter 2006) and two (summer 2006) treated with three sulfur supplies 0, 500 and 5000 µM. The plants were pre-cultured in a complete nutrient solution for five weeks (experiment one) or two weeks (experiment two) before the start of sulfur treatments. Means (n=12) ± SD followed by the different letters are significantly different at p < 0.05 (Tukey test); ***, significant at p < 0.001 (F test). ......................... 29

Figure 11: Relationship between total sulfur contents and SPAD readings of different leaves from three experiments conducted in different environmental conditions. R2 values are shown with levels of significance as ** and *** for p < 0.01 and 0.001, respectively........................................................... 34

Figure 12: Disease development of leaf 4 and 5 as affected by sulfur supply and P. fuligena infection (Experiment one, winter 2006). The plants were pre-cultured for five weeks in a complete nutrient solution before the start of sulfur treatment and inoculation was done three weeks later. Dots are means ± SD of six replicates. Different letters indicate significant difference at p < 0.05 (F test). *, ** and *** denote levels of significance at P < 0.05, 0.01 and 0.001; ns, not significant. ................... 35

Figure 13: Differences in lesions development of P. fuligena infected tomato leaves treated with 50 µM (S-) and 5000 µM (S+) sulfur. The developments of lesions were restricted in leaves under 5000 µM sulfur supply level (Experiment four, summer 2007)..................................................................... 36

Figure 14: Disease development of leaf 8 as affected by sulfur supply and P. fuligena infection (Experiment two, summer 2006). The plants were pre-cultured for two weeks in a complete nutrient solution before the start of sulfur treatments and inoculation was done five weeks later. Dots are means ± SD of six replicates. Different letters indicate significant difference at p < 0.05 (F test). ** and *** indicate levels of significance at p < 0.01 and 0.001 respectively; ns, not significant. .......... 36

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Figure 15: Disease development of leaf 7, 8 and 9 under three sulfur levels (µM: 50, 500, 5000) and P. fuligena infection (Experiment three, winter 2007). Sulfur treatment was started on five-week-old plants and inoculation was done three weeks later. Dots are the means (n=6) ± SD. Lines with similar letters are not significantly different at p < 0.05 (F test). *** indicates level of significance at p< 0.001; ns, not significant. ...................................................................................................................... 37

Figure 16: P. fuligena related parameters observed under microscopic examinations, (A) infected stomata stained with Coomassie brilliant blue and surrounding areas with yellow coloration in the incubation phase; (B) conidiophores and hyphae growing out of stomata in the infection phase; (C) structures found on abaxial surface of an infected leaf sample; (a) conidia, (b) hyphae, (c) conidiophores, (d) penetrated stomata. [scale bars = 50 µm]................................................................ 39

Figure 17: Effect of sulfur supply on the number of penetrated stomata and fungal structures examined at 7, 14 and 21 dpi (days post-inoculation). Bars represent the means ± SD of six replicates. Values with different letters are significantly different at p < 0.05 (Tukey test). *** indicates level of significance at p < 0.001; ns, not significant. ........................................................................................ 41

Figure 18: Effect of sulfur supply on the number of stomata penetrated by P. fuligena at 1, 4, 8 and 12 dpi (days post-inoculation). Bars represent the means ± SD of six replicates. * and *** indicate levels of significance at p< 0.05 and 0.001, respectively; ns, not significant. ...................................... 41

Figure 19: Relationship between number of penetrated stomata per mm2 and disease severity of leaf 12 at 21 dpi after six weeks treatment of three sulfur levels (50, 500, 5000 µM; experiment 3). R2 is given with significance level as (+) for P < 0.1..................................................................................... 42

Figure 20: Cysteine (Cys) and Glutathione (GSH) contents of (A) leaf 4 at 31 dpi after 8 weeks of sulfur treatment (Experiment one) and (B) leaf 7 at 33 dpi after 10 weeks of sulfur treatment (Experiment two). Sulfur treatment was started on five-week- and two-week-old plants in each experiment. Bars are means ± SD of six replicates. *, ** and ***indicate significance at P < 0.05, 0.01 and 0.001, respectively (F test); ns, not significant. Small and capital letters stand for comparison of sulfur at fungal inoculation – and + respectively (Tukey test). ............................................................. 46

Figure 21: Cysteine (Cys) and Glutathione (GSH) contents of leaf 6, 8 and 9 harvested at 18, 24 and 27 dpi respectively. Sulfur treatment was started on five-week-old plants and inoculation was done three weeks later. Bars represent the means (n=6) ±SD. *, ** and *** denote levels of significance at P < 0.05, 0.01 and 0.001 respectively (F test); ns, not significant; F-, without inoculation; F+, with inoculation; dpi, days post-inoculation. ................................................................................................ 47

Figure 22: Relationships between (A) total sulfur and severity (%), and (B) GSH contents and severity (%) of leaf 4 at 31 dpi as affected by different sulfur supplies (Experiment one). .................. 48

Figure 23: Relationships between disease severity and (A) total sulfur and (B) GSH at different harvests (Experiment three). In the scale, the value “7” indicates the highest disease severity and “0” the lowest…..................................................................................................................................... ......48

LIST OF TABLES

Table 1: Mineral compositions of tomato leaves as affected by sulfur treatment (µM: 0, 500, 5000). [Note: Experiment two conducted in summer, 2006] ........................................................................... 17

Table 2: Layout of greenhouse experiments (3 x 2 factorial; CRD). ................................................. 17

Table 3: The rating scale used in estimation of disease severity according to (Godoy et al.) ........... 20

Table 4: Total sulfur, SO4-S and organic sulfur contents of (A) leaf 4 and at 31 dpi after seven weeks of sulfur treatment and (B) leaf 5 at 37 dpi after eight weeks of sulfur treatments [Experiment one,

vii

winter 2006] with or without inoculation with P. fuligena. Plants were pre-cultured in a complete nutrient solution for five weeks before the start of sulfur treatment. The values are means ± SD of six replicates. (P) / (N), positive or negative effect of a factor; * and *** indicate levels of significance at p < 0.05 and 0.001 respectively; ns, not significant; nd, not determined. ............................................. 31

Table 5: Total sulfur, SO4-S and organic sulfur contents of leaf 7 after 10 weeks of sulfur treatment [Experiment two, summer 2006] in the case of without inoculation. Plants were pre-cultured in a complete nutrient solution for two weeks before the start of sulfur treatment. Means (n=6) ± SD followed by different letters are significantly different at p < 0.05; ***, level of significance at p < 0.001 (Tukey test). ................................................................................................................................ 32

Table 6: Total sulfur, SO4-S and organic sulfur contents of leaf 6 (18 dpi), leaf 8 (24 dpi) and leaf 9 (27 dpi) [Experiment three, winter 2007]. Plants were pre-cultured in a complete nutrient solution for five weeks before the start of sulfur treatment. P. fuligena inoculation was done three weeks later. Means (n=6) ± SD followed by different letters are significantly different at p < 0.05 (Tukey-test). * and *** indicate levels of significance at P< 0.05 and 0.001; ns, not significant (F test); (P) / (N), positive / negative effect of a factor. ..................................................................................................... 32

Table 7: Number of penetrated stomata and fungal structures per mm2 at 4 and 11 dpi as affected by sulfur supply and P. fuligena for inoculation methods. The values are means ± SD of six replicates. * and ** denote levels of significance at P < 0.05 and 0.01, respectively (F test); ns, not significant; dpi, days post-inoculation; (P) / (N), positive / negative effect of a factor. ................................................. 40

Table 8: Metabolites concentrations in the leaf 4 (31 dpi) of 12-week-old tomato plants as affected by P. fuligena infection and seven weeks of sulfur treatment (Experiment one). Mean values (n=6) ± SD followed by different letters are significantly different at p < 0.05 (Tukey test). Small and capital letters are for the comparison of fungus and sulfur levels, respectively. F-, without inoculation; F+, with inoculation; nd, not determined..................................................................................................... 43

Table 9: Metabolites concentration in the leaf 7 of 12-week-old plants as affected by 10 weeks of sulfur treatment without inoculation (Experiment two). Means (n = 6) ± SD followed by different letters are significantly different at p < 0.05 (Tukey test). .................................................................... 43

Table 10: Metabolites concentrations in the leaf 9 (27 dpi) of 12-week-old tomato plants as affected by P. fuligena infection and seven weeks sulfur treatment (Experiment three). Mean values (n = 6) ± SD followed by different letters are significantly different at p < 0.05 (Tukey-test). Small and capital letters are for comparison of fungus and of sulfur levels, respectively. F-, without inoculation; F+, with inoculation. ............................................................................................................................. 44

Table 11: Correlation coefficients (r) for the relationships between sulfur fractions in leaf 4 of tomato plants differentially supplied with sulfur at 31 dpi (Experiment one) without and with inoculation by P. fuligena. .................................................................................................................... 49

Table 12: Correlation coefficients (r) for the relationships between sulfur fractions in leaf 7 of tomato plants (Experiment two) after seven weeks of differential sulfur treatments without infection……......................................................................................................................................... 50

Table 13: Correlation coefficients (r) for the relationships between sulfur compounds in the leaf 6 (18 dpi), leaf 8 (24 dpi) and leaf 9 (27 dpi) of tomato plants differentially supplied with sulfur without and with P. fuligena inoculation, (A) without inoculation; (B) with inoculation; +, *, ** and *** denote significant levels at P < 0.1, 0.05, 0.01 and 0.001 respectively; ns, not significant. ................. 50

viii

ABBREVIATIONS

ADP Adenosine diphosphate

ANOVA Analysis of variance

APR APS reductase

APS Adenosine- 5`phosphosulfate

ATP Adenosine triphosphate

AVRDC Asian vegetable research and development center

BLM Black leaf mold

Cys Cysteine

dd H2O Double-distilled water

dpi Days post-inoculation

DTT Dithiothreitol

DW Dry weight

Exp. Experiment

Fed ox Oxidized ferredoxin

Fed red Reduced ferredoxin

Fe-EDTA Ferric ethylenediamine-tetraacetic acid

F- No-inoculation with P. fuligena

F+ Inoculation with P. fuligena

FW Fresh weight

GR Glutathione reductase

GS Glutathione synthetase

GSH Reduced glutathione

GSSG Oxidized glutathione/glutathione disulfide

GSL(s) Glucosinolates

H Harvest

h hours

HPLC High performance liquid chromatography

L Leaf

LCD L-cysteine desulfhydrase

min. Minutes

ml mililiter

ix

mg GluE g -1DW Miligram glucose equivalent per gram dry weight

mmol GluE g -1 DW Milimole glutamate equivalent per gram dry weight

mS cm -1 Mili-siemens per centimeter

µM Micromolar

µmol Micromole

n Number

nm Nanometer

nmol Nanomole

NiR Nitrite reductase

NR Nitrate reductase

OAS O-acetylserine

OAS- TL O-acetylserine (thiol)lyase

P. fuligena Pseudocercospora fuligena

PAPS 3`-phophoadenosine- 5`- phosphosulfate

PRP(s) Pathogenesis-related protein (s)

r Coefficient of correlation

R2 Coefficient of determination

ROS Reactive oxygen species

SAR Systemic-acquired resistance

SAT Serine acetyl-transferase

SD Standard deviation

SDC(s) Sulfur containing defense compound(s)

SED Sulfur-enhanced defense

SIR Sulfur-induced resistance

SPAD Single photon avalande diode

SRP(s) Sulfur-rich protein(s)

TOA Tomato oatmeal agar

Tris Tris (hydroxymethyl)- aminomethane

x

ABSTRACT

Black leaf mold caused by Pseudocercospora fuligena, is a major foliar disease of tomato in

the tropics and subtropics. Though the fungicidal properties of leaf applied elemental sulfur

(S0) have been long known, it was recently discovered that enhancing the plant S nutritional

status through sulfate applications to the roots also has the potential to improve the disease

resistance of plants (S-enhanced defense, SED).

In the current study, the role of S nutrition on the resistance of tomato against black leaf mold

was investigated in a hydroponics system under controlled conditions. Three levels of S [µM:

0/50, 500, 5000] were applied with or without inoculation with P. fuligena. The progress of

the disease was monitored on the leaf level and plant metabolites suspected to be involved in

the plant - pathogen interaction were determined.

Sulfur deficient plants were characterized by decreased contents of chlorophyll (SPAD value)

mainly in younger leaves and leaf S-fractions. Protein and total sugars contents also tended to

decline in the leaves of S-deficient plants whereas amino acids and starch accumulated under

severe S-deficiency. The total S, SO4-S and glutathione (GSH) contents were more responsive

to sulfur supply than organic S and cysteine (Cys). The proportion of SO4-S in the leaves was

higher in S-enriched than in S-deficient plants.

Tomato plants under moderately S-deficient conditions were more susceptible to P. fuligena

infection than plants well supplied with S. Microscopic examinations also revealed that the

number of penetrated stomata was lower in the leaves of S-supplied plants. However, under

severe S deficiency fungal infection was greatly inhibited. No consistent differences in either

disease rating or microscopic studies were found between 500 or 5000 µM S treatments. In

well S-supplied plants GSH contents slightly increased in the early phases of infection, but

substantially decreased in the course of the disease in all treatments. Significant negative

relationships were found between disease severity and total S and GSH respectively. It is

concluded that S nutrition plays a role in enhancing the constitutive resistance of tomato

against black leaf mold. Underlying biochemical and molecular mechanisms remain to be

elucidated.

1

1. INTRODUCTION

Tomato (Solanum lycopersicum L.) is one of the most widely grown vegetables for fresh

markets and processing in the world (Hanson et al., 2001). Although generally adapted to a

wide range of environmental conditions, the crop is attacked by many pests and diseases

(Jones et al., 1991). Tomato black leaf mold (BLM) caused by the fungus Pseudocercospora

fuligena (Roldan) Deighton, is a widespread foliar disease in the tropics and subtropics (Wang

et al., 1995) and the most severe fungal disease under conditions of protected cultivation in

Thailand. Despite that both, disease and pathogen were described as early as 1938 (Roldan,

1938), information about the pathogen and especially about plant-pathogen interaction in

relation to the mineral element nutrition of the plant is limited.

Being in tune with the ideas of sustainable agriculture, it is inevitably necessary to minimize

the use of fungicides in plant protection. A promising option is to optimize the mineral

nutrition of plants because mineral fertilizers are among the environmental factors influencing

both, the tolerance (i.e. the ability to endure a disease without yield reduction) and the

resistance (i.e. the ability to withstand the infection) of plants against pathogens. In general,

plants well-equilibratedly supplied with all nutrients have a higher fitness and thus can

tolerate a pathogen infection to a higher degree than plants suffering from nutrient deficiency

(Sieling, 1990).

Sulfur (S) is an essential macroelement for plant life and has numerous biological functions

(Marschner, 1995; Leustek et al., 2000). Though the fungicidal effect of foliar applied

elemental S (S0) is known for a long time it is a relatively new discovery that soil-applied S in

the form of sulfate can also have a positive effect on plant health (Haneklaus et al., 2002).

In the second part of the 20th century, air pollution with S dioxide (SO2) became a major

global concern but the S demands of many crops might have been partially covered by the

resulting sulfur depositions. However, in response to drastic reduction of SO2 emissions

started by the governments of many industrialized countries (Helsinki Protocol, 1979),

decreasing inputs of S to agro-ecosystems led to S deficiencies since the early 1980s which

have been regularly observed under field conditions mainly in the northern part of Europe

(Schnug et al., 1995). As a consequence, reduction of yield and product quality and decrease

in plant resistance against certain diseases became obvious for many crops (Brokenshire et al.,

2

1984) under greenhouse (Wang et al., 2003) and field conditions (Schnug et al., 1995;

Bourbos et al., 2000; Klikocka et al., 2004; Bloem et al., 2004; Salac et al., 2004).

Sulfur is taken up by plants in its inorganic sulfate form from the soil or as SO2 and hydrogen

sulfide (H2S) from the atmosphere. Plants assimilate and reduce sulfate to sulfide which is

incorporated into cysteine and further converted to methionine (Leuctek and Saito, 1999;

Dubuis et al., 2005). Cysteine is the precursor of most S-containing defense compounds

which are involved in resistance reactions of plants against pathogens (Rausch and Wachter,

2005; Salac et al., 2005). Due to an improved dynamic of pathogen- induced adaptations of S

assimilation and formation of S-containing defense compounds, S-enhanced defense reactions

may be possible (Hell and Leustek, 2005).

Therefore, this study aims at investigating the significance of sulfate nutrition on the

resistance of tomato against P. fuligena infection and underlying physiological mechanisms

involved in S-enhanced defense (SED) of the plants. It is expected that the ample application

of inorganic sulfate will improve the resistance of tomato against the fungal diseases.

3

2. LITERATURE REVIEW

Plants are exposed to diverse forms of external stress factors in different stages of growth and

development. Although there are several adaptive mechanisms that the plants may have

developed to cope with these inducers, plant response to a particular stress depends on the

nutritional status of the plant. In general, the resistance against diseases is improved when the

nutrient supply is increased from deficiency to the optimum range (Graham, 1983).

Lichtenthaler (1996) and Reigosa et al. (1999) suggested the stress factors that can be

summarized into abiotic, biotic and man-related factors as shown in Fig.1. Out of many stress

factors, fungal diseases are among the most serious biotic factors threatening the plant’s life

and interacting with other factors like mineral nutrient deficiency or toxicity.

Biotic factors- Pathogens

(virus, fungus, bacteria)- Herbivores

(phytophagia, animals, insects)- Other plants

(parasitism, allelopathy, competition)

Abiotic factors- Temperature- Water- Radiation- Chemicals- Others

(like wounding, magnetic and electric fields)

Man-related factors- Pollution

(e.g. agrochemicals, smog, acid rain, heavy metals)

- Nutrient deficiency- Excess of nutrients and salts- Climatic change- Soil compaction- Radiation

Biotic factors- Pathogens

(virus, fungus, bacteria)- Herbivores

(phytophagia, animals, insects)- Other plants

(parasitism, allelopathy, competition)

Abiotic factors- Temperature- Water- Radiation- Chemicals- Others

(like wounding, magnetic and electric fields)

Man-related factors- Pollution

(e.g. agrochemicals, smog, acid rain, heavy metals)

- Nutrient deficiency- Excess of nutrients and salts- Climatic change- Soil compaction- Radiation

Figure 1: Biotic, abiotic and man-related factors that can induce stress-related reactions of terrestrial plants (Adapted from Lichtenthaler, 1996; Reigosa et al., 1999).

4

2.1. Tomato Black Leaf Mold (BLM)

Tomato (Solanum lycopersicum L. former Lycopersicon esculentum Mill.) is a member of the

Solanaceae or nightshade family. Originating in South and Central America, tomato is now

grown world-wide for its edible fruits. Besides the major limiting factors like nutrients, water

and climate, biotic pressure from insects and pathogens can drastically reduce tomato yield

(Beri, 2005) and product quality. Tomato black leaf mold (BLM) is a foliar fungal disease of

tomato caused by Pseudocercospora fuligena (Roldan) Deighton (= Cercospora fuligena

Roldan). BLM, formerly known as Cercospora leaf mold, was first observed and reported in

the Philippines (Roldan, 1983) and was subsequently reported to be widespread in tropical

and subtropical Asia (Wang et al., 1995). It is also known to occur in Nigeria and southern

U.S.A (Hsieh and Goh, 1990).

2.1.1. Outbreaks of BLM

A study in the Asian Vegetable Research and Development Center (AVRDC) in Taiwan

indicated a high degree of susceptibility in fresh-market hybrid tomato (Hartman, et al.,

1991). In 1989-90, black leaf mold caused extensive damage on tomatoes with a yield loss of

32% in Taiwan (Hartman and Wang, 1992). The same study indicated that fruit marketability

is not affected because P. fuligena does not cause direct damage to fruits but to the leaves.

However, both fruit weight and number of fruits were reduced. In Japan, the disease was

found to be widely distributed and caused severe reductions in yield (Yamada, 1951).

Recently in 2005, a severe outbreak of BLM in tomato cv. Santa Clara was observed in

protected cultivation conditions in Brazil (Halfeld-Vieira et al., 2006). Particularly, under

conditions of protected cultivation in South Thailand, black leaf mold turned out to be the

most severe fungal tomato disease (Heine, Moran-Puente and Horst, 2006; Hau and Pongarm,

2005). Cultivars tolerant of BLM were reported to be identified and have been used to

minimize disease losses in Florida (Jones. et al., 1991). But it appears to be not widely known

to many growers and little information about resistant lines is available to date.

Outbreak of BLM is mainly influenced by day and night temperature, relative humidity, and

leaf wetness. High relative humidity, moderate to warm day and cool night temperatures favor

disease development (Jones et al., 1991). Prolonged periods of leaf wetness also correlate

5

with high disease severity. During the rainy season the disease may become more serious

when warm temperatures prevail. In Thailand, BLM naturally occurred under the conditions

of protected cultivation in the greenhouses. Hau and Pongarm (2005) highlighted that the

disease can spread not only by spores disseminated by splashing rain, running water and

machinery (Hsieh and Goh, 1990) but also by wind-borne spores, and suggested that older

leaves seemed to be more susceptible to BLM.

2.1.2. Disease symptoms and morphology of P. fuligena

Fig. 2 shows the symptoms of BLM. Disease symptoms begin with the development of

irregularly shaped chlorotic spots on the leaves, with sporulation evident mainly on the lower

surface. In advanced stages, the lesions enlarge and coalesce, with abundant dark sporulation

on both surfaces. Moreover, the leaves roll upward, die prematurely, and generally remain

hanging on the plant with a soot-covered appearance (Hartman, Wang, et al., 1995). In

inoculated plants, the disease progresses slowly on younger plants but increases rapidly as

plants aged (Hartman and Wang, 1992). According to Cerkauskas (2004), major symptoms

occur on the foliage but they may also occur on petioles, stems, and fruit peduncles (but not

on the fruit itself).

(B)(A)

Figure 2: Symptoms of tomato black leaf mold (BLM); (A) visible lesions on upper leaf surface in the early stages of infection; (B) leaves roll, dry and develop sooty appearance in the advanced stage.

6

Hsieh and Goh (1990) described the morphology of P. fuligena. Conidiophores of the

pathogens are loosely fasciculate, 2-7 septate or very dense, pale olivaceous to very pale

brown, uniform in color, straight to sinuous, tip round or truncate, not branched, 0-3 septate

with constriction and 15-60 x 3-5 µm. The conidia were described as subhyaline to pale

olivaceous, cylindric to cylindro-obclavate, and straight to mildly curved, tip rounded, base

long obconic to long obconically truncate, 2-9 septate and 20-90 x 2.5-4 µm.

2.2. Plant Resistance

Resistance mechanisms of plants can be distinguished into two major groups, constitutive

resistance and induced resistance. The first one is non-specific and not restricted to certain

host-pathogen combinations. On the contrary, induced resistance is often restricted to certain

combinations of plants and pathogens. This includes the synthesis of phytoalexins and the

hypersensitive response.

Defense metabolites produced by plants can also be classified in two groups: constitutive

substances (prohibitins or phytoanticipins) which are involved in initial plant defense (Mohr

and Schopfer, 1994) and induced metabolites formed in response to an infection involving de

novo enzyme synthesis, known as phytoalexins (Van Etten et al., 1994; Grayer and Harborne,

1994).

When the constitutive plant defense is overcome by pathogens, different mechanisms are

mobilized within the plant tissues including signal recognition and transduction,

hypersensitive response (programmed cell death), reinforcement of mechanical barriers,

development of systemic acquired resistance (SAR), induction of pathogenesis-related

proteins, release of toxic aglycones, and accumulation of newly produced phytoalexins

(Kombrink and Schmelzer, 2001; Grayer and Kokubun, 2001).

2.3. Importance of Sulfur Nutrition on the Plant Resistance

Sulfur is a macronutrient necessary for growth and physiological functioning of plants and the

S content in plants ranges from 0.1 to 6% of dry weight (0.03 to 2 mmol g-1 DW) (De Kok et

7

al., 2002). The fungicidal impact of foliar elemental S applications has been known for long

time. A new role of sulfur (S) in the resistance of crops against diseases became obvious in

central Europe at the end of the 1980s when atmospheric S depositions caused by traffic and

industrial production were greatly reduced due to clean air laws and S deficiency became a

widespread problem in European agriculture (Booth et al., 1991; Kjellquist and Gruvaeus,

1995; Knudsen and Pedersen, 1993; Pedersen, 1992; Richards, 1990). At the same time,

increasing infestations of crops with certain diseases became evident. One example is the

infestation of oilseed rape with Pyrenopeziza brassicae which was observed in regions that

were never infested before (Paul, 1992; Schnug and Ceynowa, 1990; Schnug et al., 1995).

With the application of soil-applied S, the resistance of different crops was found to increase

against various fungal diseases under greenhouse (Wang et al., 2003) as well as field

conditions (Schnug et al., 1995; Bourbos et al., 2000; Klikocka et al., 2004; Bloem etal.,

2004; Salac et al., 2004). A case study from Scotland demonstrated that S-fertilization

increases oilseed rape resistance to Pyrenopeziza brassicae (Schnug et al., 1995) and Bourbos

et al. (2000) found a repressive effect of sulfate application on grapes infected with Uncinula

necator. In a recent field study with potato, a significant decrease in potato tuber infection

with Rhizoctonia solani was observed when plants were supplemented with potassium sulfate

or elemental sulfur (Klikocka et al., 2005). However, so far no investigation of the effect of

sulfur application has been done concerning BLM caused by the fungus Pseudocercospora

fuligena.

2.4. Sulfur Assimilation and Metabolism in Plants

Sulfur assimilation and the production of low-molecular-weight S-containing defense

compounds (SDCs) are shown in Fig. 3. Sulfur is taken up by the roots in the form of sulfate

and the subsequent reduction of sulfate to sulfide and its incorporation into cysteine (Cys)

takes place in the chloroplast. As a first step of S-assimilation, sulfate is activated by ATP via

ATP sulfurylase in the leaves. The product, adenosine-5`- phosphosulfate (APS) is reduced to

sulfite by APS reductase, with the tripeptide glutathione (GSH) acting as an electron donor.

Alternatively, APS is further activated by APS kinase to form 3`-phophoadenosine-5`-

phosphosulfate (PAPS), which is required for various reactions, including the biosynthesis of

glucosinolates. Sulfite is reduced by sulfite reductase (SiR) to H2S, which is incorporated into

8

O-acetylserine (OAS) via O-acetylserine (thiol)lyase (OAS-TL) to form cysteine. The amino

acid cysteine is the primary product of S-assimilation and functions as precursor of most other

organic sulfur compounds in plants like S-rich proteins (SRPs) and glutathione (GSH).

Furthermore, cysteine is the donor of reduced S for glucosinolate biosynthesis and for the

synthesis of phytoalexins. Finally, H2S can be released from cysteine via the action of L-

cysteine desulfhydrases (LCD). In contrast, S0 is probably released from GSH. Excess sulfite

can be converted to sulfate by the activity of sulfite oxidase, a reaction that uses O2 as

electron acceptor and leads to the formation of H2O2, which could act as signal for defense

reactions.

H2O2H2O2

Figure 3: Sulfur assimilation as a platform for the biosynthesis of sulfur-containing defense

compounds (SDC) (Adapted from Rausch and Wachter, 2005).

9

2.5. Role of Sulfur Containing Defense Compounds (SDCs) in Plant

Resistance

Sulfur containing defense compounds (SDCs) are proposed to have a strong impact on the

potential for defense reactions of plants. Bloem et al. (2005) pointed out that the formation of

SDCs saturates at a higher S supply than the demand for plant growth, suggesting that supply

of supra-optimal S for growth can still increase pathogen resistance. The formation of various

forms of SDCs, including glutathione (GSH) and secondary metabolites can be stimulated

with increasing sulfate supply. Hirai, et al (2003) proposed that an increase in the level of the

cysteine precursor O-acetylserine (OAS) is dependent on both, pathogen infection and S

nutrition. Rausch and Wachter (2005) mentioned that during pathogen infection, additional

sinks for reduced cysteine S arise from secondary metabolite synthesis, release of H2S or the

formation of S-rich proteins (SRPs), antimicrobial polypeptides like defensins and thionins.

Fig. 4 shows the involvement of S-containing metabolites in constitutive and induced plant

defense mechanisms.

S-containing metabolites

GlucosinolatesThiols (GSH)Defensins, thionins (S-rich proteins)

H2S-release (and other reduced gaseous S-compounds)

Accumulation of elemental SPathogen related molecules,defensins, thionins, phytoalexins

Constitutive Defense

Induced Defense

Alkaloids, TerpenoidsPhenolicsCyanogenic glucosidesSaponins….

Enzymatic reactions..

AlkaloidsTerpenoidsPhenolics…

S-containing metabolites

GlucosinolatesThiols (GSH)Defensins, thionins (S-rich proteins)

H2S-release (and other reduced gaseous S-compounds)

Accumulation of elemental SPathogen related molecules,defensins, thionins, phytoalexins

Constitutive Defense

Induced Defense

Alkaloids, TerpenoidsPhenolicsCyanogenic glucosidesSaponins….

Enzymatic reactions..

AlkaloidsTerpenoidsPhenolics…

Figure 4: Sulfur-containing metabolites that are involved in the constitutive and induced protection of plants against pests and diseases (Bloem et al., 2005).

10

2.5.1. Glutathione (GSH)

Glutathione (L-γ-glutamyl-L-cysteinyl glycine; GSH) is an S-containing compound of the

primary metabolism and is the most abundant non-protein thiol in the plant ranging from 0.1 -

3 mM. GSH is synthesized in two steps. First γ-glutamyl-cysteine is synthesized from

cysteine and glutamate catalyzed by γ-glutamyl-cysteine synthetase. Second gluthathione is

synthesized from γ-glutamyl- cysteine and glycine catalyzed by glutathione synthetase (GS).

Both steps of the synthesis of glutathione are ATP dependent reactions (De Kok et al., 2005).

Glutathione is predominantly present in its reduced stage and represents a storage and

transport form of reduced S in plants. At the same time, GSH controls sulfate influx into the

plant at the level of sulfate uptake and xylem loading in the roots (Rennenberg and

Herschbach, 1995). Glutathione is part of the anti-oxidative system of plant cells preventing

damage by active oxygen species, which are synthesized as a reaction to biotic and abiotic

stresses. It rapidly accumulates after fungal attack and may act as systemic messenger

carrying information concerning the attack to non-infested tissues (Foyer and Rennenberg,

2000; Edwards et al., 1991). Foyer and Noctor (2001) mentioned the important molecular and

biochemical aspects of glutathione in plants. These include (i) storage and long distance

transport of reduced S, (ii) regulation of enzyme activity (iii) antioxidant defense through

peroxide metabolism (GSH peroxidase and ascorbate peroxidase), (iv) signal molecule, (v)

synthesis of pytochelatins, (vi) detoxification of xenobiotics and (vii) redox regulation and

buffering.

The GSH content is relatively more sensitive to S nutrition than most other organic S

compounds in the soluble fraction (Anderson, 1990). Sulfur-deficient plants have very low

GSH concentrations and S fertilization strongly increases the free thiol content (De Kok et al.,

1981; Schnug et al., 1995). However, when the cells subsequently encounter S deficiency, the

produced GSH is taken up and used as S source. This implies that the production of GSH is

determined by the availability of sulfate and the production of GSH declines when the S

supply is restricted (Anderson, 1990). These findings indicated that S-deficient plants are

more vulnerable to stress factors, which are usually compensated for by the GSH system.

Accordingly, increasing the S supply from the deficient to the optimum range should have a

positive effect on resistance mechanisms provided by the GSH pathways.

11

2.5.2. Glucosinolates (GSLs)

Glucosinolates (GSLs) are low-molecular-mass nitrogen (N)- and S-containing secondary

compounds, that are produced by Brassicaceae and the glucosinolate content in vegetative

and generative plant tissues significantly depends on S fertilization (Schnug, 1997).

Glucosinolate can increase the resistance of plants against infection of pests and pathogens

(Mithen, 1992). The effect is mediated by the formation of bio-active isothiocyanates via

enzymatic hydrolysis of glucosinolates when the plant tissue is damaged as a result of

pathogen attack. Theses compounds and other products of GSL hydrolysis have been shown

to be toxic or inhibitory to many species of fungi and bacteria (Greenhalgh and Mitchell,

1976). However, the involvement of glucosinolates in plant defense against pathogen is still

controversial.

2.5.3. Phytoalexins

Pathogen attack and abiotic elicitors induce the synthesis of S-containing phytoalexins such as

brassinin and camalexin (Kliebenstein, 2004). Pedras et al. (1997) described 23 different

phytoalexins from Brassicaceae and other crucifers, and showed that plants can accumulate

different phytoalexins depending on the stress factor. The capability to synthesize

phytoalexins is widespread but plant families are specialized on the production of certain

phytoalexins, e.g., sesquiterpenoide structure in Solanaceae and indole-related ring system in

Brassicaceae (Gross, 1993; Pedras et al., 1998). Their synthesis is triggered by elicitors either

formed by the pathogen (e.g., substances on the cell wall of the fungus) or by the host (e.g.,

breakdown products of the plant cell wall) after infestation (Mohr and Schopfer, 1994). Since

the formation of precursors of phytoalexin depends on the S status, it can be speculated that S

nutrition can influence the formation of phytoalexins.

2.5.4. Elemental sulfur (S0)

The finding that elemental S (S0) may have a function as phytoalexin is relatively new (Beffa,

1993; Cooper et al., 1996; Resende et al., 1996). Williams et al. (2002) demonstrated the

12

accumulation of S0 in the vascular tissue of tomato plants in response to infestation with V.

dahliae and the degree of S0 formation correlated with disease resistance. The formation of

elemental S is concentrated in hypersensitive cells and it was shown that in these cells

elemental S can reach levels required for pathogen inhibition in vitro (Cooper et al., 1996;

Mansfield, 2000).

The exact mode of action of S0 is still elusive. It is thought that after S0 enters the fungal cells,

it is reduced to hydrogen sulfide or acts as an electron acceptor at the level of the respiratory

chain (Tweedy, 1981). In contrast, Beffa (1993) found that increasing concentrations of S0 to

spores of P. viticola resulted in a dramatic increase in oxidized glutathione (GSSG), and

concluded that the fungicidal action of S0 is more likely related to the oxidation of important

sulfydryl groups than to the competitive interaction between S0 and oxygen at the level of the

respiratory chain.

In line with the association of S0 with the hypersensitive response the expression of many

other genes for disease resistance were also reported as a result of S0 accumulation (Jabs and

Slusarenko, 2000).

2.5.5. Sulfur-rich proteins (SRP)

Sulfur-rich non-storage plant proteins are the major group of low molecular weight

antimicrobial peptides, accumulating after pathogen attack. They can be divided into plant

defensins and thionins which act as enzyme inhibitors (Bohlmann, 1993) and lipid transfer

proteins (Van Loon et al., 1994). It was observed that thionins isolated from barley leaves

inhibited the growth of phytopathogenic fungi in vitro experiment (Bohlmann et al., 1988)

and thionins have already been used to create transgenic plants resistant to phytopathogenic

fungi (Carmona et al., 1993). Defensins are regarded as a new family of thionins (Collila,

1990) and also known as super antimicrobial peptide. Although the mode of action is not yet

clear, many plant defensins display a strong antifungal capacity, indicating that they can act as

defense proteins (Thevissen, 1999). Many studies proposed that defensins are involved in

both constitutive and induced resistance (Chiang and Hadwiger, 1991; Gu et al., 1992; Terras

et al., 1995; Parashina et al., 2000).

13

2.5.6. Hydrogen sulfide (H2S)

The release of gaseous S containing compounds can be a fast, stress-induced reaction (Bloem

et al., 2005). Hydrogen sulfide (H2S), which is the predominant form of volatile S

compounds, has been characterized as fungi-toxic and the resistance of crops against certain

pest and diseases could be related to increased H2S emissions of plants (Sekiya et al., 1982;

Beauchamp et al., 1984; Schroeder, 1993).

According to Rennenberg (1983, 1984), the release of H2S regulates the size of the cysteine

pool, maintaining it at a low level because cysteine is cytotoxic. When cysteine-consuming

processes are inhibited and the concentration of cysteine increases, sulfide is emitted as H2S

(Rennenberg and Filner, 1982). The release of H2S may take place before or after cysteine

synthesis and the same compounds that inhibit the incorporation of cysteine into GSH

promote the evolution of H2S.

Studies in plants infected with Verticillium dahliae showed a negative correlation between S

contents in plants and infection rates (Burandt et al., 2001). An increasing total S content of

the plants was associated with decreasing L-cysteine desulfhydrase (LCD) and increasing O-

acetyl-L-serine-(thiol)lyase (OAS-TL) activity. This indicates that the evolution of volatile S

compounds is directly linked to the S nutritional status of the crops and that release of sulfide

has an important role in the defense system of plants against fungal infection.

Nevertheless measurements of H2S evolution from living plants are difficult to record and

there are only a few measurements available for the release of gaseous S compounds.

Moreover, measurements from detached leaves or plant parts result in an overestimation of

H2S emission by the crop (Bloem et al., 2005). Research on the direct relationship between

the gaseous release of H2S and the S nutrition and fungal infestation of plants is still scarce.

2.6. Sulfur-induced Resistance (SIR) or Sulfur-enhanced Defense (SED)

During the present decade, much progress has been achieved on sulfur research using the -

omics and molecular genetic tools. However, studies on the plant-pathogen interaction at the

metabolite level are still scare. In 1995, the term ‘Sulfur-Induced Resistance’ (SIR) was

proposed by Ewald Schnug and colleagues at the 9th International Rapeseed Congress. They

14

reported on the observation that supplementary S-fertilization reduced the incidence of fungal

pathogens on Brassica crops. However, this term was challenged in a recent review on S

metabolism by Rausch and Wachter (2005) who stated, “….Unfortunately, the term ‘SIR’ is a

misnomer because S nutrition does not ‘induce’ resistance but rather enhances defense

operations and might even, in the case of GSH, act indirectly by improving general plant

performance under abiotic stress. Also, the term ‘induced resistance’ has a different

connotation in plant pathology. Therefore we propose that ‘SIR’ should be replaced with the

term S-enhanced defense.”

Regarding to the leaf pathogen P. fuligena, the effect of S nutrition on the plant resistance was

not yet investigated in any crop. It is therefore unknown if external S supply can improve the

resistance of tomato against P. fuligena. Providing a positive effect of S supply on disease

resistance, a detailed study of the changes in the level of plant metabolites would bring an

insight into the underlying physiological mechanisms.

15

3. OBJECTIVES AND HYPOTHESES

3.1. Objectives

The objectives of this study are:

To investigate the role of Sulfur nutrition on the resistance of tomato against

Pseudocercospora fuligena infection

To characterize the changes in metabolites as affected by Sulfur nutrition and

Pseudocercospora fuligena infection

To analyze the relationships between these changes and resistance mechanism of

tomato

3.2. Hypotheses

Supra-optimal Sulfur nutrition can enhance the defense of tomato against

Pseudosercospora fuligena.

Interaction between Sulfur nutrition and infection with Pseudocercospora fuligena

can induce changes in plant Sulfur metabolism.

16

4. MATERIALS AND METHODS

4.1. Plant Cultivation

Tomato (Solanum lycopersicum L.) seeds of the variety King Kong II (Known-You Seed Co.,

Ltd, Taiwan) were germinated on water-soaked filter paper at 25°C. After three days, the

germinated seeds were transferred to a “sandwich system” which consisted of two layers of

foam over-layered with moistened filter paper supported by PVC plates. The sandwich system

was placed in a plastic container provided with water and kept in a growth chamber. One

week later, the seedlings were carefully transferred from the sandwich system to 10-liter-pots

containing a complete nutrient solution with the following composition: (µM): Ca(NO3)2 x

H2O 800; NH4NO3 20; K2SO4 350; MgSO4 x 7H2O 325; KH2PO4 100; H3BO3 8; CuSO4 x

5H2O 0.2; ZnSO4 x 7H2O 0.2; MnSO4 x H2O 2; Na2MoO4 x 2H2O 0.1 and Fe-EDTA 40. The

nutrient solution was continuously aerated and the pH adjusted to 6.

After three to four weeks, the plants were transferred into a mist chamber with an average

temperature of more than 25°C day/night and humidity of more than 90% under natural light.

Individual plants were set into 10-liter-pots and supported by plastic strings. Except negligible

concentration (i.e., 2.4 µM) the basic nutrient solution was free of S and composed of (µM):

Mg(NO3)2 x 6H2O 325; KNO3 500; Ca(NO3)2 x 4H2O 400; KH2PO4 200; H3BO3 8; CuSO4 x

5H2O 0.2; ZnSO4 x 7H2O 0.2; MnSO4 x H2O 2; Na2MoO4 x 2H2O 0.1 and Fe-EDTA 40.

Electrical conductivity (~ 1 mS cm-1), pH (6.0 - 6.5) and nitrate concentration (120 mg L-1)

were checked three times a week. When the nitrate concentration dropped down to the half,

additional nutrient solution was applied to meet the original concentration. In any case the

whole solution was renewed and S treatment was given weekly (see below). In experiment 1

and 2, plants analysis was performed at harvest (Tab. 1).

17

Table 1: Mineral compositions of tomato leaves as affected by sulfur treatment (µM: 0, 500, 5000). [Note: Experiment two conducted in summer, 2006]

Sulfur supply 0 µM 500 µM 5000µM Macro elements [mg g-1DW] N 35 ± 4.4 a 26 ± 2.7 b 25 ± 2.4 b Ca 70.9 ± 3.9 c 90.6 ± 5.3 a 79.7 ± 4.6 b Mg 6.8 ± 0.9 ab 8.2 ± 1.4 a 5.7 ± 0.8 b K 7.8 ± 2.2 b 18.8 ± 5.1 a 21.5 ± 4.5 a P 10.7 ± 1.4 a 8.3 ± 1.6 b 6.6 ± 1.2 b S 1.7 ± 0.6 c 10.0 ± 0.3 b 20.0 ± 1.1 a Micro elements [µg g-1DW] Fe 30.4 ± 13.6 b 62.8 ± 4.3 a 70.0 ± 4.2 a Mn 58.5 ± 5.1 a 97.3 ± 35.8 a 93.7 ± 26.6 a Zn 56.9 ± 9.9 a 60.4 ± 10.5 a 54.8 ± 5.9 a Cu 33.5 ± 5.4 b 51.5 ± 9.9 a 41.0 ± 8.3 ab Mo 12.7 ± 4.1 a 8.1 ± 2.4 b 2.7 ± 1.3 c

4.2. Treatments and Experimental Design

The experiments were conducted in a factorial, completely randomized design (CRD) with six

replications. Three levels of sulfur were used with two fungal treatments [i.e., without (-) and

with (+) inoculation] as shown in Tab. 2. Sulfur was given as calcium sulfate (CaSO4)

whereas Ca was balanced by CaCl2 application. To avoid chloride toxicity in the lowest and

the middle sulfur supply level, Ca was balanced only up to the half of the concentration of

sulfur of the highest supply level. The lowest S level of 0 µM used in experiment 1 and 2 was

replaced by 50 µM in experiment 3 and 4 to avoid severe S deficiency (Tab. 2). In experiment

1, 3 and 4, the plants were pre-cultured with complete nutrient solution for five weeks, but in

experiment 2, two weeks.

Table 2: Layout of greenhouse experiments (3 x 2 factorial; CRD).

Treatment CaSO4

[µM] CaCl2 [µM]

P. fuligena inoculation

1 - 2

0 / 50 2500 / 2450 +

3 - 4

500 2000 +

5 - 6

5000 0 +

18

4.3. Cultivation of P. fuligena

The isolate of P. fuligena was received from greenhouse grown tomatoes of the experimental

site of the AIT, Thailand. Tomato oatmeal agar (TOA) based on Hartman et al. (1991) was

used as growing media for the fungus. Fifty grams of tomato leaves and 15 g of oat meal were

boiled separately in 500 ml water, and mixed with 25 g of agar. The pH was adjusted to 6.5

by using 0.1 M HCl and 0.1 M NaOH. P. fuligena was cultured and maintained at 25ºC and

18/6 h light.

4.4. Inoculation

Inoculation was done when plants were at least seven weeks old. Petri dishes with fungal

colonies were filled with 10 ml of distilled water and the surfaces of the plates were carefully

scraped with a glass slide to dislodge fungal structures (Wang et al., 1995). The solution was

then filtered through a 40-µm-mesh to remove agar fragments. A few drops of non-ionic

detergent (Tween® 20, AppliChem) were added to the suspension before inoculation.

4.4.1. Inoculation of whole leaves

The inoculum suspension was sprayed with a hand sprayer on both surfaces of selected leaves

(leaf 4 and 5 in experiment 1; leaf 6, 7 and leaf 8 in experiment 2; leaf 6, 7, 8 and 9 in

experiment 3 and leaf 9 and the whole plant in experiment 4 until runoff. Depending on the

sizes and number of leaflets, 10 - 20 ml of suspension was used for each leaf (node). Leaves

of control plants were sprayed with distilled water.

4.4.2. Localized inoculation for microscopic examinations

To optimize the inoculation process with regard to a more detailed examination, local

inoculation on the specified area was done on the lower surface of leaf 10 (Exp. 3) using the

following methods.

19

4.4.2.1. Agar ring method

Agar (3%) rings with a diameter of 0.2 cm were attached to the lower leaf surface and the

outer margin of the agar ring was immediately sealed by 1% agar solution (50°C). There were

three agar rings per leaflet. Subsequently, about 0.5 ml of inoculum suspension was sprayed

to the inner area (0.13 cm2) of the agar ring using a 3 ml syringe (Fig. 5A).

4.4.2.2. Mycelium block

Mycelia blocks (0.2 cm2) were carefully stamped out off the TOA plates using a sterilized

forceps and put on the lower leaf surface with the side containing the fungus next to the leaf

(Fig. 5B). The blocks were then sealed as described above. Three mycelium blocks were used

for each leaflet.

(A) (B)(A) (B)

Figure 5: Inoculation of P.fuligena on localized areas of the lower leaf surface, (A) agar ring (0.13 cm2) made from 3% agar solution; (B) mycelium block (0.2 cm2) scratched off the cultured plate.

4.5. SPAD Measurement and Fresh Weight

In order to detect the effect of S treatments on the chlorophyll contents, SPAD values of non-

inoculated plants were measured using chlorophyll meter (SPAD-502, Minolta, Japan).

20

Leaves at the same ages of inoculated leaves were selected for SPAD measurement. At

harvest, shoot fresh weights were recorded in experiment 1 and 2.

4.6. Visual Estimation of Disease Severity

Assessment of disease progress was started 2 - 3 days after the appearance of first visible

symptoms. Disease severity was estimated using a scored chart and carried on at two-day-

interval until the time of harvest. Disease severity estimated as percent leaf area infected was

transformed into an appropriate rating scale for data analysis as described in Tab. 3. The

rating scale employed was based on Godoy et al. (1997) and modified by Hau (personal

communication). In order to find out the relationship between percentage and scale values of

disease estimations, correlation coefficient (r) was computed using the data from experiment

4 taken at early (15 dpi) and middle stage of infection (21 dpi) as shown in Fig. 6. There was

a strong and highly significant correlation between the two ways of expression.

Table 3: The rating scale used in estimation of disease severity according to (Godoy et al.)

Percent leaf area

infected

Scale value

0 0

> 0 - 1 1

> 1 - 2 2

> 2 - 5 3

> 5 - 10 4

> 10 - 25 5

> 25 - 50 6

> 50 7

21

Severity [%]0 5 10 15 20 25

Seve

rity

[Sca

le]

0

1

2

3

4

5

621 dpi15 dpi

y = 0.7305 x + 0.338r = 0.96***n = 18

y = 2.0946 x 0.2778

r = 0.83***n = 18

Figure 6: Correlation coefficients (r) for the relationship between percentage and scale values of disease severity (leaf area infected) at the two different time points (15 and 21 dpi). [Note: Experiment four, 2007]

4.7. Microscopic Examinations

4.7.1. Experimental setup

Parallel to the main experiments, three trials of microscopic studies were conducted in the

mist chamber. In the first trial, the inoculum suspension was sprayed on both surfaces of leaf

7, using a hand sprayer as for normal inoculation. Leaf discs (1 cm diameter) from inoculated

leaf were sequentially harvested at 7, 14 and 21 dpi for microscopic examinations. In the

second trial, aiming at optimizing the infection process, localized inoculation was performed

using the two approaches described above (No. 4.4) with three replications each. For check,

normal spraying was also employed on the other two leaflets from the same node. Samples

were taken at 4 and 11 dpi. In both experiments, one replication consisted of 3 leaf discs

randomly taken from the selected leaflet. Leaves of the same node from non-inoculated plants

served as control. In the third trial, normal spray was applied to leaf 9 (L9) with six

22

replications. Microscopic examinations were done at four different time points (i.e., 1, 4, 8

and 12 dpi). Each replicate consisted of six leaf discs (1 cm diameter).

4.7.2. Staining and microscopy

The harvested leaf discs were immediately treated with 70% ethanol (5 ml) and kept

overnight in order to decolorize the leave tissues. Staining was done according to Wolf and

Fric (1981) using three different solutions. For solution one (S1), trichloroacetic acid (0.15%)

was added to a mixture of ethanol and chloroform (75:25 (v/v)). To prepare solution two

(S2), trichloroacetic acid (15% in H2O) and Coomassie Brilliant Blue R-250 (0.6% in 99%

methanol) were mixed by 50:50 (v/v) for one day. Deviating from original protocol, the

solution was then diluted 1/10 with dd H2O in order to avoid over-staining of the tissues.

Solution three (S3) was prepared from 5% acetic acid, 20% Glycerin and 75% H2O.

(A) (B)(A)(A) (B)(B)

Figure 7: Detailed structures of P. fuligena examined under light microscope. (A) cylindric- obclavate conidia and conidiophores from infected leaf; (B) fascicle of divergent conidiophores growing out of cuticle of infected leaf. [scale bars = 50 µm]

The samples were treated with 2 ml of S1 for one day in order to fix the fungal structures and

remove chlorophyll, and then transferred to new glasses. For staining, the leaf discs were

23

treated by 3 ml of S2 for 30 min. The dyes in the samples were washed out by treating with

S3 before mounting on microscopic slides. Fungal growth and related changes in cell

structure were examined under a light microscope (x 200). The number of stained stomata

supposed to be penetrated and all detected structures attributed to the fungus were counted

together for evaluation of fungal growth.

4.8. Chemical Analysis

4.8.1. Determination of total sulfur

Total S was determined by using a CNS analyzer (Elementar, Vario EL III, Germany). Leaf

samples were dried at 65°C for three days and ground to fine particle sizes using a plant mill.

Around 20 mg of leaf material was used for analysis.

4.8.2. Determination of water soluble compounds

4.8.2.1. Sample extraction

For SO42- determination, 100 g of fresh tissue was mixed with 2 ml of dd H2O and

homogenized at a speed of 30-1 for 3 min. using a ball mill (MM200, Retsch, Germany). The

homogenates were incubated on a dump-roller for 2 h and the extracts were transferred to test

tubes. After addition of 6 ml of dd H2O, the extracts were filtered through cellulose

membrane filters (0.45 µm pore size, Micro science, Germany). For the sample extraction for

proteins, amino acids and carbohydrates, 0.2 g of fresh leaf material was mixed with 9.9 ml of

dd H2O and 100 µl of ethanol (70%). After 30 min. incubation, the extracts were filtered

through blue band filter paper (Macherey-Nagel, Germany). For cysteine and GSH, about 50

mg of fresh leaf materials homogenized in liquid nitrogen were mixed with 250 µl of HCl (0.1

M) and incubated for 15 min. at 4°C. Samples were centrifuged twice for 5 min. at 14000 rpm

and 4°C.

24

4.8.2.2. Determination of SO42-

The measurement of leaf sulfate (SO42-) was performed using 2- aminoperimidine as color

reagent. Tissue extracts (1.5 ml) were mixed with 2 ml acetate buffer (1% sodium acetate +

0.5 M acetic acid) and 1 ml color reagent (0.16% of 2- aminoperimidine hydrobromide). After

addition of 2 ml HCl (0.05 M), the solution were filtered through cellulose membrane filters

(0.45 µm pore size, Micro science). After mixing 150 µl of the resulting solution with 150 µl

of NaNO2 (1%), SO42- was determined photometrically at 420 nm. Samples of experiment 3

were prepared by extracting 20 mg dried sample with 10 ml of water and incubated for 2 h.

After filtration through blue band filter paper (Macherey-Nagel, Germany) the determination

of SO42- was performed at 420 nm wavelength by using a continuous flow analyzer (SANplus

System, SKALAR, Germany).

4.8.2.3. Determination of total sugars

Total sugars were measured with anthrone according to Umbreit et al. (1972). The extract (50

µl) of sample was added to 900 µl dd H2O and 2 ml anthrone solution (0.2% anthrone in

H2SO4 conc.). Samples were mixed thoroughly and boiled in a water-bath for 10 min. After

cooling down to room temperature, total sugars were photometrically determined at 620 nm.

4.8.2.4. Determination of starch

Frozen leaf materials (0.2 g) were homogenized in liquid nitrogen. Homogenates were mixed

with 2 ml of K2PO4 buffer (25 mM; pH 7.5) and incubated for 30 min. at 4°C. After

centrifugation (12000 rpm, 10 min.), the pellet was mixed with 1 ml K2PO4 (25 mM; pH 7.5)

and agitated using a vortex mixer. After centrifugation at 12000 rpm for 10 min, the pellet

was incubated in 1 ml of perchloric acid (17.5%) for 30 min. followed by centrifugation at

12000 rpm for 10 min. The supernatant was taken and diluted with dd H2O (1:4 (v/v)). 900 µl

of dd H2O and 2 ml of anthrone solution (0.2%) were added to 0.1 ml of the extract and the

solution was thoroughly mixed by vortexing. Extraction was done for 10 min. at 100°C in a

water bath. After cooling down to room temperature, starch contents (Glucose equivalent)

were photometrically quantified at 620 nm.

25

4.8.2.5. Determination of total soluble proteins

The total soluble protein was determined according to Bradford (1976). The sample extract

(25 µl) was mixed with 275 µl of Bradford reagent [0.1g Coomassie brilliant blue G-250

(Sigma) in 50 ml of ethanol (95%), added to 100 ml phosphoric acid (85%), diluted to 1000

ml H2O]. After 15 min., soluble protein was measured at 595 nm against a bovine serum

albumin (BSA) standard.

4.8.2.6. Determination of total amino acids

Total amino acids were measured by using the ninhydrin method according to Yemm and

Cocking (1955) was used. Solution (A) was prepared by mixing 1 ml of KCN stock solution

(0.066%) with 49 ml of Ethylenglycol-monoethylether (99%). Citric acid (0.2 M) was

separately prepared at pH 5 (Solution (B)). 100 µl of leaf extract was added to 1 ml of citrate

buffer (solution (A) + (B)) and 1 ml of ninhydrin reagent (0.2%). The solution was thoroughly

mixed and boiled in a water bath for 15 min. After cooling down to room temperature, the

total amino acids (Glutamate equivalent) were photometrically determined at 570 nm.

4.8.2.7. Determination of cysteine (Cys) and glutathione (GSH)

Free cysteine and GSH were measured by HPLC according to Hell and Bergmann (1990). For

reduction, aliquots (100 µl) of the extract was mixed with 130 µl ultra-pure H2O, 20 µl Tris (1

M, pH 8, filtered), 10 µl DTT (10 mM, fresh) and 10 µl NaOH (0.08 M, filtered) and

incubated in the dark for 1 h. For derivatization of sulfhydryl groups, the sample extract was

mixed with 20 µl of 10 mM bromobimane in acetonitrile (Sigma- Taufkirchen, Germany) and

incubated at room temperature. After 15 min., 710 µl of acetic acid (5%) was added for

stabilization. At each step of reduction, derivatization and stablization, vortexing and a short

centrifugation followed. Separation, detection and quantification of fluorescent was achieved

by reversed phase column (Waters Nova- Pak C18, 4.6 x 250 mm) and a Hitachi HPLC

System running with a gradient of 100% methanol and 0.1 M potassium acetate buffer as

eluents. Color eluent was monitored by fluorescence detector (380/480 nm, Sense x 16 and

Gain High).

26

4.9. Statistical Analysis

Results were analysed by using the GLM option of SAS Version 9.1.3 (SAS 2004, Institute

Cary, NC). One or two factorial ANOVA was employed in order to analyze the effect of each

treatments and interaction among the treatments. The Tukey test was used for mean

separation at 5% significance level (P < 0.05). Pearson correlation coefficients (r) and R2

values were computed using PROC CORR and PROC REG procedures.

27

5. RESULTS

5.1. Effects of Sulfur Nutrition on the Growth and Sulfur Status of Tomato

Plants

In order assess the role of S nutrition on the response of tomato plants, leaf greenness, shoot

fresh weight, total S and SO4-S contents in the leaves were determined.

5.1.1. Chlorophyll content (SPAD value)

In tomato, S deficiency can be recognized by pale leaves due to the reduction of leaf

chlorophyll contents.

Fig. 8 shows tomato leaves supplied with different levels of S. In the case of severe S

deficiency, leaves were yellow, small and the leaf edges curled upwards whereas the

moderately S-deficient plants exhibited only a slight decrease of leaf greenness compared to

well S-supplied plants.

Fig. 9A, 9B and 9C show the SPAD values of three experiments conducted in winter 2006

(Exp. 1), summer 2006 (Exp. 2), and winter 2007 (Exp. 3). In experiment 1 and 2, first

symptoms of S deficiency were observed two weeks after the beginning of S deprivation.

SPAD values of the 0 µM treatment were lower than 500 and 5000 µM treatments. This

difference was more pronounced in younger than in older leaves. No significant difference

was found between 500 and 5000 µM supply except in leaf 10 (L10) of experiment 1. The

interactions between leaf and S observed in experiment 1 and 2 indicate that the effect of S

supply altered depending on the position of leaves.

In experiment 3 where the 0 µM was replaced by 50 µM S supply, no significant treatment

effect or interaction was observed (Fig. 9C). Under the conditions of 500 and 5000 µM S

supply, SPAD values of the plants from experiment 2 (summer) ranged by 50 – 60 whereas

that in experiments 1 and 3 (winter) showed below 40, indicating that there was an effect of

climatic condition on chlorophyll biosynthesis of plants.

28

(A) (B) (C)

Figure 8: Different degrees of leaf greenness in relation to differential sulfur supply, (A) small pale leaf with severe sulfur deficiency; (B) light green leaf with moderate deficiency; (C) well supplied leaf.

Leaf 6 Leaf 8 Leaf 90

10

20

30

40

50

60

ANOVA (A) (B) (C)

Leaf *** ** nsSulfur *** ** nsL x S * *** ns

Leaf 4 Leaf 5 Leaf 100

10

20

30

40

50

60

Leaf 6 Leaf 7 Leaf 8

(B)aa aa

b

b

b

aa

bc

a

0 µM 500 µM 5000 µM (A)

ab

(C)50 µM 500 µM 5000 µM

ba a

a a

Chl

orop

hyll

cont

ents

[SPA

D v

alue

]

a aa aa a a aa

Figure 9: SPAD readings of three leaves respectively of experiments (A) one (winter 2006), (B) two (summer 2006) and (C) three (winter 2007). The plants were grown in nutrient solution at three sulfur supply levels (0/50, 500, 5000 µM). Means (n=6) ± SD with different letters for each leaf are significantly different at p < 0.05 (Tukey test). *, ** and *** indicate significant difference at P < 0.05, 0.01 and 0.001 respectively (F test); ns, not significant.

29

5.1.2. Shoot fresh weight

Tab. 4 shows shoot fresh weights of tomato plants taken from experiment 1 (winter 06) and 2

(summer 06). In experiment 1, no effect of S treatments was observed. But in experiment 2,

the fresh weight of S-starved plants were significantly lower than S-supplied plants,

indicating a tremendous reduction of growth. No difference was found between 500 µM and

5000 µM S treatments in experiment 1 (winter). Comparing two experiments, shoot fresh

weights of the plants from experiment 1 (winter) revealed 40% as compared to experiment 2

(summer).

Sulfur supply [µM]0 500 5000

Shoo

t fre

sh w

eigh

t [g

plan

t -1]

0

200

400

600

800

1000

1200Exp. 1 (winter)Exp. 2 (summer)

a aC

a

ABA ANOVA

Sulfur ***Exp. ***Sulfur x Exp. ***

Figure 10: Shoot fresh weights of 13-week-old tomato plants from experiment one (winter 2006) and two (summer 2006) treated with three sulfur supplies 0, 500 and 5000 µM. The plants were pre-cultured in a complete nutrient solution for five weeks (experiment one) or two weeks (experiment two) before the start of sulfur treatments. Means (n=12) ± SD followed by the different letters are significantly different at p < 0.05 (Tukey test); ***, significant at p < 0.001 (F test).

5.1.3. Total sulfur and sulfur fractions

S status of the plants was investigated by measuring total S and SO4-S. Organic S was

calculated as the difference between total S and SO4-S. Both fractions of total S and SO4-S

30

readily increased with increasing S supply (Tab. 4, 5 & 6). Organic S was also found to

increase with increasing S supply in experiment 1 and 2 (Tab. 4 and 5) but not in experiment

3 (Tab. 6) where 0 µM S was replaced with 50 µM S.

Tab. 4 and 5 show the contents of S fractions for experiment 1 and 2, respectively. In

respective of fungal inoculation, the average total S contents in the leaves (leaf 4 + 5) of

experiment 1 were 10.1 (8.4 – 11.7), 15.8 (14.7 – 16.9) and 28 (27.1 – 28.9) mg g-1 DW

whereas that in the leaf 7 of experiment 2 were 1.7, 10.2 and 19.9 mg g-1 DW for the S supply

levels of 0, 500, and 5000 µM, respectively. For all S supplies, total S content was visibly

lower in the experiment 2 (summer) (Tab. 5) than in the experiment 1 (winter) (Tab. 4).

For both of experiments 1 and 2 (Tab. 4 & 5), SO4-S and organic S fractions followed the

trend of total S in response to S treatment. It was observed that the contents of both fractions

readily increased with increasing S supply. Additionally, the proportion of SO4-S (% of total

S) was lower in S-starved (0 µM S) than in S-supplied plants, which was reflected by 34%

(19.4 – 54.6), 39% (27.7 – 58.9) and 52% (34.0 – 65.1) for the three of S supplies 0, 500 and

5000 µM, respectively. It clearly shows that SO4-S accumulated faster than total S in response

to increasing S supply. No effect of fungus was found on all S fractions in experiment 1 (Tab.

4). SO4-S tended to increase with fungal inoculation mainly in the S-supplied plants but it was

not significant.

Tab. 6 shows the leaf S-fractions of the plants from experiment 3 separately described for

three harvests. In agreement to experiment 1 and 2, a highly significant positive effect of S

supply was observed on all S fractions (i.e. total S, SO4-S and organic S). Moreover, there

was a highly significant negative effect of harvests (leaves) on all S fractions. The average

contents of all S fractions were the highest at harvest 1 (L6) and the lowest at harvest 3 (L9),

implying that there was a negative relationship between leaf position and S status. No effect

of P. fuligena was observed on all S fractions. Again, a tendency to increase the contents of

total S and SO4-S was found in S-enriched plants in relation to fungal infection. For all S

fractions, no interaction was found between the factors (Harvest (H) x Sulfur (S) x Fungus

(F)).

Similar to the results of experiment 1 and 2, the proportion of SO4-S (% of total S) in the

leaves increased with increasing S supply, which was ranged by 45%, 75% and 80% for the

three S levels of 50, 500 and 5000 µM, respectively (Tab. 6). Again, it clearly showed the

31

faster accumulation of SO4-S than total S in response to increasing S supply. Interestingly, the

higher the leaf position the lower the contents but the higher the proportion of SO4-S. The

proportion of SO4-S exhibited 58%, 66% and 76% for three leaves L6 (H1), L8 (H2) and L9

(H3), respectively. Differences of SO4-S proportion between leaf positions were big in S

supply of 50 µM [31 vs. 61%] and 500 µM [65 vs. 85%] but small in 5000 µM [77 vs. 80%].

Table 4: Total sulfur, SO4-S and organic sulfur contents of (A) leaf 4 and at 31 dpi after seven weeks of sulfur treatment and (B) leaf 5 at 37 dpi after eight weeks of sulfur treatments [Experiment one, winter 2006] with or without inoculation with P. fuligena. Plants were pre-cultured in a complete nutrient solution for five weeks before the start of sulfur treatment. The values are means ± SD of six replicates. (P) / (N), positive or negative effect of a factor; * and *** indicate levels of significance at p < 0.05 and 0.001 respectively; ns, not significant; nd, not determined.

(A) Leaf 4 (31 dpi)

Treatments Leaf sulfur fractions Sulfur Fungus Total S SO4-S Organic S [µM] [mg g -1DW] [mg g -1 DW] (%) [mg g -1 DW] 0 - 8.41 ± 0.97 4.59 ± 1.03 (54.6) 3.82 ± 1.40 500 - 14.69 ± 2.22 8.65 ± 0.88 (58.9) 6.04 ± 1.49 5000 - 27.12± 2.08 15.21 ± 1.79 (56.1) 11.91 ± 3.37 0 + 8.27 ± 1.06 5.13 ± 1.33 (62.0) 3.01 ± 0.77 500 + 14.33 ± 1.43 9.50 ± 1.31 (66.3) 4.83 ± 2.66 5000 + 26.39 ± 2.10 16.42 ± 1.96 (62.2) 9.97 ± 3.02 Sulfur *** *** *** Fungus ns ns ns Sulfur x Fungus ns ns ns

(B) Leaf 5 (37 dpi)

Treatments Leaf sulfur fractions

Sulfur Fungus Total S SO4-S Organic S [µM] [mg g -1 DW] [mg g -1 DW] (%) [mg g -1 DW] 0 - 11.67 ± 0.82 2.26 ± 1.53 C (19.4) 9.41 ± 2.32 C 500 - 16.99 ± 0.88 4.70 ± 0.99 B (27.7) 12.29 ± 1.68 B 5000 - 28.88 ± 2.77 9.80 ± 1.49 A (34.0) 19.07 ± 2.01 A 0 + 11.01 ± 1.19 nd nd 500 + 18.37 ± 1.74 nd nd 5000 + 30.53 ± 2.40 nd nd Sulfur *** *** *** Fungus ns Sulfur x Fungus ns

32

Table 5: Total sulfur, SO4-S and organic sulfur contents of leaf 7 after 10 weeks of sulfur treatment [Experiment two, summer 2006] in the case of without inoculation. Plants were pre-cultured in a complete nutrient solution for two weeks before the start of sulfur treatment. Means (n=6) ± SD followed by different letters are significantly different at p < 0.05; ***, level of significance at p < 0.001 (Tukey test).

Leaf sulfur fractions Sulfur supply Total S SO4-S Organic S [µM] [mg g -1 DW] [mg g -1 DW] (%) [mg g -1 DW] 0 1.70 ± 0.67 C 0.46 ± 0.28 C (27.2) 1.24 ± 0.57 B 500 10.24 ± 0.19 B 3.18 ± 1.24 B (31.1) 7.06 ± 1.05 A 5000 19.93 ± 1.33 A 12.97 ± 0.90 A (65.1) 6.96 ± 1.40 A *** *** ***

Also in organic S content, it was found to decrease with higher leaf position, which accounted

for 6.36, 4.17 and 2.5 mg g-1 DW for L6 (H1), L8 (H2) and L9 (H3), respectively (Tab. 6). In

contrary to experiment 1 and 2, organic S contents did not show a positive response to

increasing S supply.

For total S, there was an interaction between S and fungus (F). It showed that total S in 50 µM

treatment was not different from 500 µM without inoculation. Moreover, total S in 50 µM S

was depleted in response to inoculation.

In both of SO4-S and organic S, an interaction between harvest date (H) and S was found. In

the leaves supplied with sub-optimal (50 µM) S level, no effect of harvest (leaves) was found

on SO4-S contents. For organic S, H2 (L8) and H3 (L9) showed no difference in the leaves

supplied with 5000 µM S. At H1 (18 dpi), there was a negative effect of inoculation on

organic S.

33

Table 6: Total sulfur, SO4-S and organic sulfur contents of leaf 6 (18 dpi), leaf 8 (24 dpi) and leaf 9

(27 dpi) [Experiment three, winter 2007]. Plants were pre-cultured in a complete nutrient solution for

five weeks before the start of sulfur treatment. P. fuligena inoculation was done three weeks later.

Means (n=6) ± SD followed by different letters are significantly different at p < 0.05 (Tukey-test). *

and *** indicate levels of significance at P< 0.05 and 0.001; ns, not significant (F test); (P) / (N),

positive / negative effect of a factor.

Factors Leaf sulfur fractions Harvest Sulfur

[µM] Fungus Total S

[mg g -1 DW] SO4-S [mg g -1 DW]

(%) Organic S [mg g -1 DW]

H1 50 - 12.50 ± 2.57 3.90 ± 0.90 (31.2) 8.59 ± 1.75 a L6 (18 dpi) 500 - 13.74 ± 1.01 8.94 ± 0.89 (65.1) 4.80 ± 1.01 b 5000 - 25.32 ± 1.72 19.57 ± 1.24 (77.3) 5.75 ± 0.63 b 50 + 11.19 ± 0.68 3.38 ± 0.51 (30.2) 7.81 ± 0.45 A 500 + 15.43 ± 1.11 9.94 ± 0.71 (64.4) 5.49 ± 0.61 B 5000 + 26.47 ± 3.21 20.78 ± 2.37 (78.5) 5.70 ± 1.55 B mean 17.44 11.09 (57.8) 6.36 H2 50 - 10.12 ± 1.04 4.28 ± 0.77 (42.3) 5.84 ± 0.32 a L8 (24 dpi) 500 - 10.76 ± 1.40 8.13 ± 1.11 (75.6) 2.63 ± 0.57 c 5000 - 21.82 ± 1.28 17.79 ± 1.30 (81.5) 4.03 ± 0.58 b 50 + 8.67 ± 1.39 3.73 ± 0.96 (43.0) 4.94 ± 0.55 A 500 + 10.43 ± 1.43 8.25 ± 1.93 (79.1) 2.18 ± 0.72 B 5000 + 22.11 ± 1.83 16.74 ± 1.47 (75.7) 5.37 ± 0.81 A mean 13.99 9.82 (66.2) 4.17 H3 50 - 6.74 ± 1.97 4.08 ± 0.74 (60.5) 2.66 ± 0.40 b L9 (27dpi) 500 - 8.78 ± 1.32 7.45 ± 1.08 (84.9) 1.33 ± 0.43 c 5000 - 20.49 ± 1.49 16.39 ± 1.68 (80.0) 4.10 ± 0.74 a 50 + 5.48 ± 0.60 3.54 ± 0.72 (64.6) 1.94 ± 0.69 AB 500 + 7.82 ± 1.26 6.46 ± 1.55 (82.6) 1.36 ± 0.60 B 5000 + 20.12 ± 1.57 16.50 ± 2.34 (82.0) 3.62 ± 1.47 A mean 11.57 9.07 (75.8) 2.50 Harvest (N)*** (N)*** (N)*** Sulfur *** *** *** Fungus ns ns ns Harvest x Sulfur ns * *** Harvest x Fungus ns ns ns Sulfur x Fungus * ns ns Harvest x Sulfur x Fungus ns ns ns

34

5.1.4. Relationship between leaf sulfur status and SPAD values of plants

To assess the relationship between total S and SPAD values, coefficient of determination (R2)

was computed (Fig. 11). For all experiments, a highly significant relationship was found

between total S and SPAD values. The relation was stronger in summer than in winter.

Total Sulfur [mg g -1 DW]

0 5 10 15 20 25 30 35

Chl

orop

hyll

cont

ent

[SPA

D u

nit]

0

10

20

30

40

50

60

70

Exp. 2 (summer 06)Exp. 3 (winter 07)

Exp. 1 (winter 06)

y = 13.742 Ln (x) + 17.35R2 = 0.85 ***n = 18

y = 0.189 x + 33.96R2 = 0.37 ***n = 36

y = 0.155 x + 31.88R2 = 0.16 **n = 54

Figure 11: Relationship between total sulfur contents and SPAD readings of different leaves from three experiments conducted in different environmental conditions. R2 values are shown with levels of significance as ** and *** for p < 0.01 and 0.001, respectively.

5.2. Effect of Sulfur Nutrition on P. fuligena infection

5.2.1. Areas under disease development

In the current experiments, the first visible symptoms of tomato black leaf mould (BLM)

developed between 14 - 20 days after inoculation. Since the first symptoms were not clear

enough for a good estimation, disease ratings always started 2 - 3 days later.

In experiment 1 (Fig. 13) the first symptoms occurred 20 days after inoculation. It was

observed that disease development was slower in plants supplied with 5000 µM S than in the

35

other treatments and the difference was significant at 25 and 27 dpi for leaf 4 and at 25, 27

and 29 dpi for leaf 5. Analyses of variances showed a significant difference in disease severity

between the 500 µM S treatment and the 5000 µM S treatment for leaf 5 but not for leaf 4.

Days post- inoculation23 25 27 29 31

Dis

ease

sev

erity

0

1

2

3

4

5

6

70

1

2

3

4

5

6

70 µM S500 µM S5000 µM S

ANOVALeaf nsTime ***Sulfur ***L x T nsL x S nsT x S nsL x T x S ns

Leaf 4

ns

*

ns

*

AB

Leaf 5

ABC

ns

ns

ns

B

***

*

Figure 12: Disease development of leaf 4 and 5 as affected by sulfur supply and P. fuligena infection (Experiment one, winter 2006). The plants were pre-cultured for five weeks in a complete nutrient solution before the start of sulfur treatment and inoculation was done three weeks later. Dots are means ± SD of six replicates. Different letters indicate significant difference at p < 0.05 (F test). *, ** and *** denote levels of significance at P < 0.05, 0.01 and 0.001; ns, not significant.

36

Figure 13: Differences in lesions development of P. fuligena infected tomato leaves treated with 50 µM (S-) and 5000 µM (S+) sulfur. The developments of lesions were restricted in leaves under 5000 µM sulfur supply level (Experiment four, summer 2007).

ANOVATime ***Sulfur ***T x S ns

Days post- inoculation18 20 22 24 26 28 30 33

Dis

ease

sev

erity

0

1

2

3

4

5

6

70 µM S500 µM S5000 µM S

A

BB

Figure 14: Disease development of leaf 8 as affected by sulfur supply and P. fuligena infection (Experiment two, summer 2006). The plants were pre-cultured for two weeks in a complete nutrient solution before the start of sulfur treatments and inoculation was done five weeks later. Dots are means ± SD of six replicates. Different letters indicate significant difference at p < 0.05 (F test). ** and *** indicate levels of significance at p < 0.01 and 0.001 respectively; ns, not significant.

37

Days post- inoculation15 17 19 21 23 25 27

0

1

2

3

4

5

6

Dis

ease

sev

erity

0

1

2

3

4

5

60

1

2

3

4

5

6

7

50 µM S500 µM S5000 µM S

ANOVALeaf nsTime ***Sulfur ***L x T ***L x S nsT x S nsL x T x S ns

Leaf 7

AA

B

Leaf 8

AA

B

Leaf 9AA

B

Time *** Sulfur * Time x Sulfur ns

Time *** Sulfur ** Time x Sulfur ns

Time *** Sulfur ** Time x Sulfur ns

ns

ns

ns

*ns

ns

*

*

**

*

ns

ns nsns *

ns*

ns

Figure 15: Disease development of leaf 7, 8 and 9 under three sulfur levels (µM: 50, 500, 5000) and P. fuligena infection (Experiment three, winter 2007). Sulfur treatment was started on five-week-old plants and inoculation was done three weeks later. Dots are the means (n=6) ± SD. Lines with similar letters are not significantly different at p < 0.05 (F test). *** indicates level of significance at p< 0.001; ns, not significant.

38

Fig. 13 shows the difference of disease levels between S-starved and S-enriched leaves. The

lesions development was visibly inhibited in well S-supplied plants mainly in the later stages

of infection. Moreover, it was observed that the leaves of extremely S-deficient plants had a

larger chlorotic area surrounding the disease lesions. In the leaves of the supraoptimal S-

treatment, the colony-free areas looked green and healthy

Contrary to the expectation, extreme S-deficiency observed in experiment 2 led to a strong

inhibition of fungal infection (Fig.14). As a consequence, disease development in S- starved

plants became delayed resulting in a lower severity than in plants supplied with S. This lower

disease severity found in extremely S-deficient condition was linked to the changes in leaf

phenotype (e.g., starchy surface and anthocyanin formation; Fig. 8).

In order to avoid the severe deficiency, the 0 µM S treatment was replaced by 50 µM S in

experiment 3 (Fig. 15). In agreement to experiment 1, repressive effect of S nutrition on the

disease levels was also observed showing higher severity in sup-optimal S (50 µM) as

compared to well S-supplied plants. The development of disease symptoms was significantly

retarded for 2 - 3 days when plants were supplied with 500 or 5000 µM S. No difference was

found between the middle and the high S supply level. Analysis of variances on each leaf

showed that there was a significant negative effect of S on disease severity and no interaction

between time and S. It indicates that the effect of S was consistent for all leaves analyzed (i.e.,

L7, L8 and L9).

5.2.2. Microscopic examinations

Since the stomata are the main site of penetration for P. fuligena microscopic examination

focused on changes in stomata pores.

At 7 dpi, a number of colorized stomata were found after staining with Coomassie blue and

fungal structures were observed in the proximity of the stained stomata mainly beneath the

cuticle (Fig. 16A). Stained stomata were considered to be penetrated and the structures

observed beneath the cuticle were taken into account for examination of fungal growth. No

treatment difference in infected stomata was found at 7 and 14 dpi, although the number of

penetrated stomata slightly increased by time (Fig. 17). At 21 dpi, after the occurrence of

39

visible symptoms on the leaf, fruiting bodies and mycelia could be clearly seen under the

microscope (Fig. 16B and 16C), but conidia were hardly found. At this stage, the number of

infected stomata and of fungal structures was significantly lower in the leaves of plants

supplied with 500 and 5000 µM S compared to 50 µM S as shown in Fig. 17.

(C)

a

d

b

c

(A) (B)

(C)

a

d

b

c

(C)(C)

a

d

b

c

(A) (B)(A) (B)(A)(A) (B)(B)

Figure 16: P. fuligena related parameters observed under microscopic examinations, (A) infected stomata stained with Coomassie brilliant blue and surrounding areas with yellow coloration in the incubation phase; (B) conidiophores and hyphae growing out of stomata in the infection phase; (C) structures found on abaxial surface of an infected leaf sample; (a) conidia, (b) hyphae, (c) conidiophores, (d) penetrated stomata. [scale bars = 50 µm]

In order to optimize the conditions for the evaluation of the infection process a localized

inoculation was performed on a defined area using two approaches (mycelium block and ring)

as can be seen in Fig. 5. For comparison, normal spraying was also carried out on the other

leaflets from the same node of localized inoculation.

40

At 4 dpi, no significant difference was found between the S treatments. At 11 dpi, a lower

number of fungal colonies, which were localized in stomata, were observed in 5000 µM S

treatment compared to the 50 and the 500 µM S treatments. When comparing the inoculation

methods, the number of fungal related parameters was the highest in the ring methods and the

lowest in the normal spray for both measured points (i.e. 4 dpi and 11 dpi). There was no

interaction between the methods demonstrating that the effect of S on fungal growth was not

affected by the inoculation method.

In the last trial employed in summer 2007, more intensive study was carried out in the early

stages of infection process at four different time points (i.e. 1, 4, 8 and 12 dpi). Although there

was a consistent trend of less infected stomata for the 500 and the 5000 µM supply than for

the 50 µM supply, no significant effect was observed for any sampling date. However,

analysis of variance clearly indicated that there was a significant difference between low and

high S treatments (Fig. 18) but no difference between the 500 and the 5000 µM supply level.

Table 7: Number of penetrated stomata and fungal structures per mm2 at 4 and 11 dpi as affected by sulfur supply and P. fuligena for inoculation methods. The values are means ± SD of six replicates. * and ** denote levels of significance at P < 0.05 and 0.01, respectively (F test); ns, not significant; dpi, days post-inoculation; (P) / (N), positive / negative effect of a factor.

Number of penetrated stomata and fungal structures per mm 2 4 dpi 11 dpi Sulfur supply [µM]

Normal spray

Mycelium block

Ring spray

Normal spray

Mycelium block

Ring spray

50 8.39 ± 2.0 10.51 ± 4.6 8.86 ± 4.7 11.17 ± 3.0 13.79 ± 2.5 20.69 ± 2.8 500 7.40 ± 0.9 12.59 ± 3.0 14.28 ± 1.4 11.08 ±1.3 13.15 ± 6.5 21.12 ± 9.3 5000 5.66 ± 1.1 9.24 ± 5.6 9.33 ± 0.9 6.60 ± 2.9 9.88 ± 2.0 11.64 ± 3.00 Method * ** Sulfur ns (N)* M x S ns ns

41

ANOVATime ***Sulfur ***T x S ***

Days post- inoculation7 14 21N

o. o

f pen

etra

ted

stom

ata

and

fung

al

stru

ctur

es m

m -2

0

5

10

15

20

25

30

3550 µM S 500 µM S 5000 µM S

aa

aa a a

b

a a

Figure 17: Effect of sulfur supply on the number of penetrated stomata and fungal structures examined at 7, 14 and 21 dpi (days post-inoculation). Bars represent the means ± SD of six replicates. Values with different letters are significantly different at p < 0.05 (Tukey test). *** indicates level of significance at p < 0.001; ns, not significant.

Days post-inoculation1 4 8 12

No.

of p

enet

rate

d st

omat

a m

m-2

0

5

10

15

20

25

30

3550 µM S500 µM S5000 µM S

ANOVATime ***Sulfur **T x S ns

aa a

aa a

a

a a

a

aa

Figure 18: Effect of sulfur supply on the number of stomata penetrated by P. fuligena at 1, 4, 8 and 12 dpi (days post-inoculation). Bars represent the means ± SD of six replicates. * and *** indicate levels of significance at p< 0.05 and 0.001, respectively; ns, not significant.

42

5.2.3. Relationship between fungal growth and disease severity

In order to determine the relationship between fungal growth and areas under disease

infection, coefficient of determination (R2) was evaluated. Fig. 19 shows the relationship

between number of penetrated stomata per mm2 and disease severity of leaf 12 at 21 dpi.

There was a relation with confidence level of 90% (p < 0.1).

No. of penetrated stomata per mm2 of leaf 12 at 21 dpi

0 5 10 15 20 25 30 35

Seve

rity

of le

af 1

2 at

21

dpi

0.0

3.5

4.0

4.5

5.050 µM S500 µM S5000 µM S

y = 0.0187 x + 3.7623R2 = 0.29+

Figure 19: Relationship between number of penetrated stomata per mm2 and disease severity of leaf 12 at 21 dpi after six weeks treatment of three sulfur levels (50, 500, 5000 µM; experiment 3). R2 is given with significance level as (+) for P < 0.1.

5.3. Metabolites in Relation to Sulfur Supply and P.fuligena Infection

5.3.1. Total soluble proteins, total amino acids and carbohydrates

Protein tended to decrease with decreasing S supply. In experiments 1 (Tab. 8) and 3 (Tab.

10), protein content was induced by P. fuligena infection mainly in S supplied plants, which

was significant in 5000 µM S supply. In experiment 2, a drastic decrease of protein in the

leaves was found in the condition of extreme S deficiency (Tab. 9). The data of proteins under

fungal infection was not available for experiment 2.

43

Table 8: Metabolites concentrations in the leaf 4 (31 dpi) of 12-week-old tomato plants as affected by P. fuligena infection and seven weeks of sulfur treatment (Experiment one). Mean values (n=6) ± SD followed by different letters are significantly different at p < 0.05 (Tukey test). Small and capital letters are for the comparison of fungus and sulfur levels, respectively. F-, without inoculation; F+, with inoculation; nd, not determined.

Sulfur supply 0 µM 500 µM 5000 µM Water soluble proteins [mg g -1 DW] F - 12.9 ± 2.4 aA 17.3 ± 4.7 aA 15.5 ± 5.1 bA F + 16.4 ± 4.4 aB 20.2 ± 1.8 aAB 24.3 ± 5.2 aA Total amino acids [mmol GluE g -1 DW] F - 0.066 ± 0.010 aA 0.072 ± 0.007 aA 0.063 ± 0.003 bA F + 0.072 ± 0.013 aA 0.079 ± 0.014 aA 0.080 ± 0.013 aA Total sugars [mg GluE g -1 DW] F - 33.0 ± 2.9 aA 38.3 ± 6.5 aA 33.3 ± 4.1 aA F + 36.3 ± 7.8 aA 41.9 ± 3.0 aA 35.0 ± 3.7 aA Starch [mg GluE g -1 DW] F - 59.1 ± 10.1A 33.3 ± 8.0 B 34.6 ± 8.5 B F + nd nd nd

Table 9: Metabolites concentration in the leaf 7 of 12-week-old plants as affected by 10 weeks of sulfur treatment without inoculation (Experiment two). Means (n = 6) ± SD followed by different letters are significantly different at p < 0.05 (Tukey test).

Sulfur supply 0 µM 500 µM 5000 µM Water soluble proteins [mg g -1 DW]

5.96 ± 2.65 C 16.55 ± 5.15 B 24.47 ± 2.8 A

Total amino acids [mmol GluE g -1 DW]

0.24 ± 0.10 A 0.10 ± 0.02 B 0.08 ± 0.02 B

Total sugars [mg GluE g -1 DW]

96.0 ± 14.6 B 68.0 ± 12.3 B 138.0 ± 28.8 A

Starch [mg GluE g -1 DW]

123.6 ± 49.6 A 27.4 ± 15.6 B 40.5 ± 17.0 B

44

Table 10: Metabolites concentrations in the leaf 9 (27 dpi) of 12-week-old tomato plants as affected by P. fuligena infection and seven weeks of sulfur treatment (Experiment three). Mean values (n = 6) ± SD followed by different letters are significantly different at p < 0.05 (Tukey-test). Small and capital letters are for comparison of fungus and sulfur levels, respectively. F-, without inoculation; F+, with inoculation.

Sulfur supply 50 µM 500 µM 5000 µM Water soluble proteins [mg g -1 DW] F - 14.1 ± 3.8 aA 12.6 ± 2.2 aA 13.2 ± 3.8 bA F + 12.2 ± 4.2 aB 12.9 ± 2.1 aAB 17.6 ± 2.5 aA Total amino acids [mmol GluE g -1 DW] F - 0.093 ± 0.03 aA 0.106 ± 0.02 aA 0.107 ± 0.04 aA F + 0.133 ± 0.04 aA 0.119 ± 0.01 aA 0.129 ± 0.03 aA Total sugars [mg GluE g -1 DW] F - 26.8 ± 6.8 aA 39.8 ± 14.9 aA 42.9 ± 12.6 aA F + 37.3 ± 6.4 aA 37.6 ± 8.7 aA 46.9 ± 10.2 aA Starch [mg GluE g -1 DW] F - 26.3 ± 10.2 aA 17.1 ± 2.0 aAB 16.1 ± 3.3 aB F + 19.1 ± 5.1 aA 19.5 ± 7.0 aA 17.0 ± 3.2 aA

Concentration of total amino acids was not affected by S treatment in experiment 1 and 3. But

in experiment 2, the extremely S-starved plants exhibited 2-folded increase in comparison to

S-supplied plants. With fungal infection, amino acids tended to increase in all S supply levels,

but a significant increase was found only in 5000 µM S treatment of experiment 1 (Tab. 8).

In experiment 1 and 3, contents of total sugars was not affected by S treatment, however there

was a tendency to increase with 5000 µM S supply. With fungal inoculation, a slight increase

of sugars was found in all S supply levels but it was not significant. In experiment 2, sugar

content in 5000 µM S treatment was significantly higher than other treatments (Tab. 9).

In all experiments, a significant accumulation of starch was found in the S-deficient plants,

getting more distinctive under the conditions of severe S-deficiency observed in experiment 2.

Starch content under inoculation was available only for experiment 3 and it was not affected

by fungus.

45

5.3.2. Sulfur-containing metabolites: Cysteine and GSH

Fig. 20 shows the Cys and GSH contents of experiment 1 and 2 conducted in summer and

winter 2006, respectively. In both experiments Cys concentration was significantly affected

by S supply. In experiment 1, fungal inoculation led to a decline of Cys contents under

conditions of low S supply (0 µM) but to an increase in plants supplied with S, which

explains the significant interaction between S and fungus analyzed. However, no difference of

Cys concentration was found between 500 µM and 5000 µM S treatments. In experiment 2,

no effect of fungus on Cys content and no interaction were found (Fig. 20B).

A highly significant accumulation of GSH contents was found in the plants supplied with S in

both experiments 1 and 2. Extremely low content of GSH was found in the 0 µM S supply in

experiment 2, representing less than 1% of either 500 or 5000 µM supply levels. In both

experiments highly significant negative effect of fungus was found on GSH contents. In

inoculated plants, GSH contents increased with increasing S supply, which was significant in

experiment 2 (Fig. 20B).

Fig. 21 shows the Cys and GSH concentrations in the leaves (leaf 6, 8 and 9) from experiment

3 harvested at 18 dpi (H1), 24dpi (H2) and 27 dpi (H3), respectively. There were highly

significant effects of all factors (i.e. harvest (H), S and fungus (F)) on both of Cys and GSH

concentrations. Cys decreased from H1 to H3 while GSH increased. For all leaves sampled,

the pools of Cys and GSH were induced by S supply but depleted by P. fuligena infection.

Interaction of H x S was found on both pools of Cys and GSH, which indicated that the effect

of S supply was affected by harvest. For Cys, there was a highly significant positive effect of

S at H1 and H2 but not at H3. GSH concentration increased significantly at H1 and H3 but

not at H2 (Fig. 21).

An interaction of H x F found in GSH indicated that the increase of GSH with increasing

harvest (from 18 dpi to 27 dpi) was highly significant without inoculation but not significant

when plants were inoculated.

The interaction of H x S x F was observed in the analysis of Cys. It showed that there was no

effect of S at H3 when the plants were inoculated. Evaluating the effect of fungus, highly

46

significant negative effects of fungal inoculation were found at H2 for the 5000 µM S level

and at H3 for 50 µM S.

P. fuligena infectionF- F+

Cys

tein

e an

d gl

utat

hion

e co

nten

ts [n

mol

. g

-1 F

W]

0

2

4

6

8

10

12

ANOVA : CysSulfur **Fungus *S x F ***

ANOVA : GSHSulfur ***Fungus ***S x F **

ANOVA : CysSulfur ***Fungus nsS x F ns

ANOVA : GSHSulfur ***Fungus ***S x F ***

0

2

4

620

40

60

80GSHa

b

A

ab

AA

(B)

0

2

4

6

8

10

12

0 µM S500 µM S5000 µM S

Cys

a ab

AA

B

(A)

CysA

b

a a

B

B

F- F+0

2

4

620

40

60

80 GSHa

BB

a

b

A

Figure 20: Cysteine (Cys) and Glutathione (GSH) contents of (A) leaf 4 at 31 dpi after 8 weeks of sulfur treatment (Experiment one) and (B) leaf 7 at 33 dpi after 10 weeks of sulfur treatment (Experiment two). Sulfur treatment was started on five-week- and two-week-old plants in each experiment. Bars are means ± SD of six replicates. *, ** and ***indicate significance at P < 0.05, 0.01 and 0.001, respectively (F test); ns, not significant. Small and capital letters stand for comparison of sulfur at fungal inoculation – and + respectively (Tukey test).

47

18 dpi

Glu

tath

ione

[nm

ol .

g-1 F

W]

0

5

10

15

20

25

Cys

tein

e [n

mol

. g-1

FW]

0.0

0.5

1.0

1.5

2.0

2.550 µM S500 µM S5000 µM S

ANOVA: GSHHarvest ***Sulfur ***Fungus ***H x S *H x F ***S x F nsH x S x F ns

ANOVA: CysHarvest ***Sulfur ***Fungus ***H x S ***H x F nsS x F nsH x S x F **

CysS *** F ns S x F ns

S ns F * S x F ns

S *** F *** S x F ns

F- F-F- F+ F+F+

GSH

24 dpi 27 dpi

S ns F * S x F ns

S *** F ** S x F **

S* F ns S x F ns

Figure 21: Cysteine (Cys) and Glutathione (GSH) contents of leaf 6, 8 and 9 harvested at 18, 24 and 27 dpi respectively. Sulfur treatment was started on five-week-old plants and inoculation was done three weeks later. Bars represent the means (n=6) ±SD. *, ** and *** denote levels of significance at P < 0.05, 0.01 and 0.001 respectively (F test); ns, not significant; F-, no inoculation; F+, inoculation; dpi, days post-inoculation.

5.4. Relationship between Disease Severity and Sulfur Status

Relationships between severity and total S and GSH, respectively were evaluated in

experiment 1 (winter 2006) and 3 (winter 2007). In experiment 1 (Fig. 22), a significant non-

48

linear relation was observed between disease severity and total S (R2 = 0.25*), and between

disease severity and GSH (R2 = 0.53*). In experiment 3 (Fig. 23) a highly significant negative

relationships was found between total S and disease severity at 18 and 24 dpi. The correlation

between severity and GSH was significant (R2 = 30*) only at 27 dpi (leaf 9).

Total S [mg/g DW]0 5 10 15 20 25 30 35

Dis

ease

sev

erity

[%

leaf

are

a of

lesi

ons]

0

10

20

30

40

50

60

GSH [nmol/g FW]0 1 2 3 4 5 6 7

50 µM S500 µM S5000 µM S

(A)y = -14.059Ln(x) + 71.076R2 =0.25*

(B)y = -20.68Ln(x) + 50.099R2 = 0.53*

Figure 22: Relationships between (A) total sulfur and severity (%), and (B) GSH contents and severity (%) of leaf 4 at 31 dpi as affected by different sulfur supplies (Experiment one).

GSH [nmol g -1 FW]

0 5 10 15 20 25

Total S [mg g -1 DW]

0 5 10 15 20 25 30 35

Seve

rity

0

1

2

3

4

5

6

7(A) (B) y = - 0.0413 x + 5.6147

R2 = 0.30 *

y = 0.0253 x + 4.1147R2 = 0.07 ns

y = - 0.0545 x + 3.0473R2 = 0.09 ns

y = 5.8252 x-0.0569

R2 = 0.17 ns

y = - 0.0218 x + 4.7234R2 = 0.30 *

y = - 0.0422 x + 3.2533R2 = 0.34 *

18 dpi (leaf 6) 24 dpi (leaf 8) 27 dpi (leaf 9)

Figure 23: Relationships between disease severity and (A) total sulfur and (B) GSH at different harvests (Experiment three). In the scale, the value “7” indicates the highest disease severity and “0” the lowest.

49

5.5. Correlations between Different Sulfur Fractions

Correlations between the different S-containing compounds are shown in Tab. 11

(Experiment 1), Tab. 12 (Experiment 2) and Tab. 13 (Experiment 3) separately for inoculated

and non-inoculated plants.

For non-inoculated plants, all experiments showed a strong, positive correlation between total

S and SO4-S, organic S and GSH, respectively. In experiment 2 and leaf 9 of experiment 3,

total S also had a relation with Cys at p < 0.1. Correlations between SO4-S and organic S was

highly significant in experiment 1 and 2 but not in experiment 3. SO4-S contents was not

related to Cys contents in all experiments but closely related to GSH in all experiments except

leaf 6 and 8 of experiment 3. In all experiments, organic S was positively related with GSH

but not always with Cys. A positive relation was observed between Cys and GSH in

experiment 2 and leaf 9 of experiment 3, but the relation was negative in leaf 6 of experiment

3.

For the case of P. fuligena inoculation, correlations between S and S-containing compounds

from experiment 1 and 3 are described in Tab. 11 and 13. In experiment 1, there were highly

significant positive relations between all S-fractions, except for GSH. A significant negative

relation between Cys and GSH was found in experiment 1.

Table 11: Correlation coefficients (r) for the relationships between sulfur fractions in leaf 4 of tomato plants differentially supplied with sulfur at 31 dpi (Experiment one) without and with inoculation by P. fuligena.

P. fuligena inoculation

Sulfur fraction Total S (mg g-1 DW)

SO4-S (mg g-1 DW)

Organic S (mg g -1 DW)

Cys (nmol g -1 FW)

SO4-S 0.93 *** Organic S 0.92 *** 0.72 ** Cys - 0.41 ns - 0.32 ns - 0.44 +

F -

GSH 0.71** 0.68 ** 0.64 ** -0.38 ns

SO4-S 0.97 *** Organic S 0.97 *** 0.92 *** Cys 0.75 ** 0.79 ** 0.79 **

F +

GSH - 0.30 ns - 0.38 ns - 0.41 ns - 0.67 *

50

Table 12: Correlation coefficients (r) for the relationships between sulfur fractions in leaf 7 of tomato plants (Experiment two) after seven weeks of differential sulfur treatments without infection.

Sulfur fraction Total S (mg g-1 DW)

SO4-S (mg g-1 DW)

Organic S (mg g -1 DW)

Cys (nmol g -1 FW)

SO4-S 0.96 *** Organic S 0.82 ** 0.62 * Cys 0.52 + 0.44 ns 0.74 * GSH 0.65 * 0.77 * 0.72 * 0.80 ***

In experiment 3, total S and SO4-S showed a strong and highly significant relation in all

leaves. Close relationships of total S and SO4-S to Cys and GSH respectively were also

observed mainly in the advanced stages of infection (i.e. 27 dpi (L9)). At 18 dpi, organic S

was negatively correlated with GSH. Both Cys and GSH showed a negative relationship with

total S and organic S at 24 dpi (L8) but positive relationship at 27 dpi (L9). It shows that GSH

had a significant relationship with total and SO4-S, but not with organic S. As expected, a

positive relationship between Cys and GSH was found at 27 dpi (L9).

Table 13: Correlation coefficients (r) for the relationships between sulfur compounds in the leaf 6 (18 dpi), leaf 8 (24 dpi) and leaf 9 (27 dpi) of tomato plants differentially supplied with sulfur without and with P. fuligena inoculation, (A) without inoculation; (B) with inoculation; +, *, ** and *** denote significant levels at P < 0.1, 0.05, 0.01 and 0.001 respectively; ns, not significant.

(A) Without inoculation

Harvest Sulfur fraction Total S (mg g-1 DW)

SO4-S (mg g-1 DW)

Organic S (mg g -1 DW)

Cys (nmol g -1 FW)

SO4-S 0.95 *** Organic S - 0.14 ns - 0.44 + Cys 0.02 ns 0.16 ns - 0.52 *

18 dpi (Leaf 6)

GSH 0.56 * 0.36 ns 0.55 * - 0.62 **

SO4-S 0.96 *** Organic S - 0.12 ns - 0.37 ns Cys 0.014 ns 0.21 ns - 0.75 ***

24dpi (Leaf 8)

GSH 0.27 ns 0.25 ns 0.03 ns 0.13 ns

SO4-S 0.97 *** Organic S 0.57 * 0.36 ns Cys 0.48 + 0.39 ns 0.66 **

27 dpi (Leaf 9)

GSH 0.67 ** 0.65 ** 0.71 ** 0.56 *

51

(B) With inoculation

Harvest Sulfur fraction Total S (mg g-1 DW)

SO4-S (mg g-1 DW)

Organic S (mg g -1 DW)

Cys (nmol g -1 FW)

SO4-S 0.98 *** Organic S - 0.38 ns - 0.54 * Cys 0.32 ns 0.07 ns - 0.37 ns

18 dpi (Leaf 6)

GSH 0.37 ns 0.66 * - 0.60 * 0.03 ns

SO4-S 0.97 *** Organic S 0.47 + 0.25 ns Cys - 0.59* -0.49+ - 0.84***

24dpi (Leaf 8)

GSH - 0.19 ns - 0.06 ns - 0.31ns 0.18 ns

SO4-S 0.98 *** Organic S 0.64 ** 0.49 * Cys 0.15 ns 0.15 ns 0.17 ns

27 dpi (Leaf 9)

GSH 0.59 ** 0.63 ** 0.34 ns 0.64 **

52

6. DISCUSSION

6.1 Role of Sulfur Nutrition on Sulfur Metabolism of Tomato Plants

Sulfur (S) in plant metabolism is an essential component for the synthesis of a wide range of

S-containing metabolites including the important amino acids Cys and Met (Hesse and

Hoefgen, 2003; Saito, 2004). A useful approach to study the regulation of S metabolism is to

expose plants to S starvation, followed by the analysis of changes in metabolite

concentrations (Nikiforova et al., 2005).

It is well known that under conditions of S starvation, plants mechanisms for increased

acquisition are activated. In contrast, the mechanisms of how plants sense and signal changes

in the availability of (S) nutrients at the cellular or whole-plant level are not yet fully

understood (Schachtman and Schin, 2007).

In general, a lack of sulfate availability for the plant leads to a reduced assimilation activity

(Hirai et al., 2003; Hirai and Saito, 2004) and decreased tissue contents of total S, reduced

amounts of chlorophyll and total protein, accumulation of starch, free amino acids and

phenolic compounds, decreased concentration of glutathione (GSH) and cysteine (Cys) and

reduced plant growth (Marschner, 1995; Blake-Kalff et al., 1998; Kutz et al., 2002; Lencioni

et al., 1997; Migge et al., 2000; Nikiforova et al., 2003; Prosser et al., 2001; Nikiforova et al.,

2003, 2005).

Accordingly, determination of the S status in the plant became a prerequisite in the current

study. The S status of the plants was characterized based on the chlorophyll content (SPAD

value), total S and SO4-S. Moreover changes in concentrations of thiols cysteine (Cys) and

glutathione (GSH), and other products of primary metabolism in response to S nutrition were

analyzed.

Chlorophyll contents (SPAD value)

The visible phenotype of S starvation typically consists of yellow-green young leaves while

mature leaves remain dark green. This phenomenon is different from the symptoms of

53

nitrogen deficiency where young leaves remain green due to supply of nitrogen remobilized

from degrading mature leaves (Hell, 1997). This is supported by the current experiments

where decreases in the SPAD readings of the low S treatment were mainly found in younger

leaves, meaning that younger leaves were more susceptible to S deficiency than older leaves

(Fig. 9A & 9B). The same pattern was found in oilseed rape grown in hydroponics system

(Blake-Kalff et al., 1988). In green leaves a high proportion of protein (at least 25 % of total

protein) is located in the chloroplasts. Under S deficiency shortage of the S-containing amino

acids cysteine (Cys) and methionine (Met) inhibits protein synthesis which leads to decreased

chlorophyll content in leaves (Marschner, 1995). A drastic decrease in the S-containing

metabolite SAM (S-adenosyl methionine), which is required in a methylation step during

chlorophyll biosynthesis accounts for the inhibition of chlorophyll formation in S-deficient

plants (Nikiforova et al., 2005). The extremely low SPAD values in the 0 µM S treatment

(accompanied with retarded plants growth; Fig. 10) found in experiment two (Fig. 9B) may be

attributed to a short period of pre-culture, followed by a long period of S withdrawal (9

weeks) under high light intensity.

Only small differences in SPAD readings were found between the 500 and the 5000 µM S

treatment, suggesting that 500 µM S is the optimum concentration for chlorophyll

biosynthesis. However, SPAD readings alone are not sufficient to characterize the internal S

status of the plant.

Plants well supplied with S revealed higher SPAD values in the summer (Fig. 9B) than in the

winter (Fig. 9A and 9C). This result is in contrary to results from rice (Resurreccion et al.,

2002), where chlorophyll contents were always higher in low-light-grown than in high-light-

grown plants. The discrepancy may be due to differences in plant specific light responses

between tomato and rice. In the summer, tomato plants have adapted to high light intensity by

increasing chlorophyll formation in order to maximize the photosynthesis. This adaptation can

be reflected by the observation of bigger plants and higher fresh weight (Fig. 10) of the plants

grown in summer than in winter. In line with the work of Resurreccion et al. (2002), the data

from current study suggests that the negative impact of S deficiency on chlorophyll contents

was relatively small under low light- (winter) than under high light-intensity (summer) as can

be seen in Fig. 9B. In experiment three (winter 07) where 0 µM was replaced by 50 µM S

(Fig. 9C), no deficiency symptoms and no difference in SPAD readings were observed

54

between treatments. This could be explained by high sulfate reserves from the pre-treatment

phase in the plants and upregulation of sulfate uptake under limited supply.

Fresh weight

Shoot fresh weights of plants in experiment 2 (summer) was higher than that in experiment

1(winter) mainly in S-enriched conditions (Fig. 10). In winter, the plants have to adapt with

low light intensity which resulted in decreased formation of chlorophyll and low shoot fresh

weight. Since S deficiency was not extreme, the growth was not effected resulting in no

treatment differences. However, in summer, there was more chlorophyll biosnythesis and high

nutrient demand. In response to a lack of S supply, plant growth became severely retarded

leading to lower biomass yield. With balanced nutrients supply, light induced nutrient demand

was fulfilled for high photosynthesis, and it led to bigger plant growth and higher shoot fresh

weight.

Sulfur status of the leaves

The efficacy of S fertilization can be verified best by determining total S contents. Studies

regarding the effect of S deprivation on total S content have been performed for oilseed rape

(Lencioni et al., 1997), sugar beet (Thomas et al., 2000), rice (Resurreccion et al., 2002),

onion (McCallum et al., 2002) and Arabidopsis (Nikiforova et al., 2003). However up to date,

little precise information is available about the effect of S supply on total S and SO4-S status

of tomato.

As expected, in the current study increased total S contents were found in plants well supplied

with sulfate. In experiment 1, average total S in the leaves (4+5) represented 10.1, 15.8 and 28

mg g-1 DW whereas in experiment 2, 1.7, 10.2 and 19.9 mg g-1 DW for the S supply levels of

0, 500 and 5000 µ, respectively. The marked differences in total S between the treatments

clearly indicate that S was readily taken up with increased supply. However, there was a

variation in S contents of experiments mainly in the 0 µM S treatment which can be explained

by a number of influencing factors like pre-culture period, treatment period, leaf age/position

55

and climatic conditions (temperature & light). In experiment 2 (summer, 06), the severe S

deficiency of S-deprived plants was well characterized by very low S status (1.7 mg g-1 DW).

This indicates that the depletion of total S under S deprivation was more drastic in the

summer than in the winter, which can be attributed to the differences in plants’ nutrient

demands between summer and winter. In general, nutrient demands of the plants are higher in

summer than in winter. Accordingly, a prolonged S- deprivation in summer resulted in a

drastic reduction of total S and reduced growth (Fig. 10). In experiment 3, the reason for the

lower depletion of S can be the replacement of the 0 µM treatment with 50 µM. Supra-

optimal S supply (i.e. 5000 µM) resulted in two-folded increase of total S compared to the

500 µM S treatment among all experiments.

The ranges of 20-30 mg S g-1 DW seemed to be the maximum for tomato because with five

weeks of 5000 µM supply, total S could have accumulated to 26 mg g-1 DW which was found

in experiment 3 (Tab. 6), and further increase became limited in spite of prolonged period (i.e.

10 weeks) supply with 5000 µM. This can be due to the fact that the uptake of sulfate by the

roots and its transport to the shoot is strictly controlled (De Kok et al., 2002), and there should

have been a down-regulation of uptake under S enriched conditions (Clarkson and Saker,

1989; Hawkesford et al., 1993)

Sulfur status depends also on leaf ages being lower in younger and higher in older because of

the low export of S from mature leaves. This is because the soluble S pool (mainly SO42-) of

leaves approaching maturity is too small to support significant redistribution to younger

leaves (Sunarpi and Anderson, 1997).

In experiment 3 (Tab. 6), average S status of three harvests were 17.44, 13.99 and 11.57 for

leaf 6, 8 and 9, respectively, clearly showing a negative relation of S status with leaf

positions. Moreover the average contents of SO4-S (11.1, 9.8 and 9.1 mg g-1 DW) and organic

S (6.4, 4.2 and 2.5 mg g-1 DW) also followed the same pattern as total S. It leads to an

assumption that the higher the leaf position, the lower the S status regardless of leaf age.

Interestingly, in spite of decreased actual contents, the proportion of SO4-S (% of total S) was

found to increase with increasing leaf positions. This pattern was more pronounced under 50

µM S. It has been reported in soybean that sulfate in the transpiration stream is predominantly

delivered to developing leaves despite of similar transpiration in mature and young leaves

(Smith and Lang, 1988). However in the current study, tomato does not seem to agree with

56

this statement may be because of inefficient activation of cellular sulfate pool (Hell, 1996),

which might be particularly more specific to tomato plants.

Freney et al. (1978) mentioned that sulfate contents of plants are a more sensitive marker for

the current S nutritional status than total S contents and the proportion of sulfate S to total S

content is the best indicator. This statement is fairly supported by the data from present study

since SO4 -S was found to be more sensitive to S supply than total S. However, the proportion

of SO4-S in leaf 4 of experiment 1 (Tab. 4) failed to reflect the actual S status mainly in S-

sufficient plants.

It is known that despite of high concentrations, SO42-can hardly be redistributed to S deficient

tissues (Hell, 1996) but results from wheat (Gilbert et al., 1997), oilseed rape (Blake-Kalff et

al., 1998) and onion (McCallum et al., 2002) showed a drastic decrease of SO42- under S

starvation. This is however supported by current results (Tab. 4B and 5), where it was

depleted to 27-19.4% of total S, indicating that SO42- pool may contribute the most (Blake-

Kalff et al., 1998) to the developing sinks under S starvation. Experiment 1 and 2 show the

decrease of organic S under S-starvation. This decrease was linked to the decline of protein

contents in S-deficient plants, showing the probable remobilization of S from the insoluble

fractions (mainly protein S). However, S part from insoluble pool will be available for

recycling only when proteins are hydrolyzed (proteolysis), which can be proved by the

observation of the reduction of chlorophyll contents and proteins found in the leaves under

extreme S starvation (Fig. 8). Since little is known about the regulatory aspects of the

remobilization of S from mature leaves (Blake-Kalff et al., 1998), further studies on this

aspect need to be done.

In all experiments, total S and SO4-S readily accumulated more under supra-optimal (5000

µM) than optimum (500 µM) S supply. But the additional contribution of supra-optimal S

supply was less in SO4-S proportion and organic S contents particularly when the plants were

growing in the S-sufficient conditions.

57

Sulfur containing metabolites: Cysteine and Glutathione

The S-containing metabolites cysteine (Cys) and glutathione (GSH) showed positive

correlations with total S and SO4-S status (Tab. 12 & 13). Also in Arabidopsis, a decrease in

total thiols was found after S deprivation (Kutz et al., 2002). Lencioni et al. (1997) and

Nikiforova et al. (2003) demonstrated a decrease in the cysteine pool of S-starved oilseed rape

and Arabidopsis.

In leaves, more than 50% of GSH is localized in the chloroplasts where it may reach

milimolar concentrations (Rennenberg and Lan Moureux, 1990). About 2% of the organic

reduced S in the plant is present in the water soluble thiol (-SH) fraction, and under normal

conditions the tripeptide glutathione accounts for more than 90% of this fraction (De Kok and

Stulen, 1993).

GSH has been proposed to act as a regulatory signal to decrease the SO42- uptake in the roots

(Rennenberg et al., 1989; Lappartient and Touraine, 1996), but it is unknown whether it also

acts as a signal for the leaf-to-leaf translocation of S. It appears that having constitutively

formed GSH, the plant uptake for external supply will be decreased accordingly. In

experiment 3 (Fig. 21) comparable level of GSH pool found in 50 µM S supply level, might

be attributed to the constitutively formed GSH pool.

In principle, after the withdrawal of the external sulfur supply, the youngest leaves will be

able to convert SO42-, GSH and GSLs to insoluble S (protein S) (Blake-Kalff et al., 1998).

According to Rennenberg (1984), GSH can be exported from younger leaves and the GSH

pool in these leaves is then replenished to the original level by utilization of the SO42-pool

(Blake-Kalff et al., 1998).

Cooper and Williams (2004) observed that SO42- and GSH levels in leaves increased under

high SO42- supply. In experiment 2 and 3, Cys and GSH contents tended to increase with S

supply in non-inoculated plants (Fig. 20 & 21). However, with some exceptions (e.g. GSH in

leaf 7 of experiment 2) the difference was rather small and plants of the suboptimal S supply

level (50 µM) were able to sustain thiols in a similar concentration range as plants well-

supplied with S. GSH was found to be more responsive to S supply than cysteine content.

This can be explained by the toxic properties of cysteine. Plant cells are able to regulate the

cysteine pool. When the cysteine concentrations reach a critical level, it is degraded in order

58

to keep concentrations within a tolerable range (Schmidt and Jäger, 1992). In experiment 3,

thiol concentrations in the leaves were much lower compared to experiment 1 and 2. The

reason for this difference is not clear.

Proteins, amino acid and carbohydrates

Amino acids and proteins are the major organic compounds resulting from NO3- assimilation

(Barneix and Causin, 1996). The reduction in protein concentration under conditions of S

deprivation was observed in experiment 2 (Tab. 9), where a lack of S supply for a prolonged

period (10 weeks) led to the drastic reduction of protein and chlorophyll contents even in the

matured leaves. This can be explained by the finding that half of the internal S is allocated to

the protein fraction (Nikiforova et al., 2005). This effect seems to be restricted to conditions

of severe S deficiency and no difference in protein concentrations was found in experiment 1

or 3 (Tab. 8 & 10). This can be explained by protein homeostasis suggested by Nikiforova et

al. (2005).

Accumulation of nitrogenous amino acids is well known to be one of the characteristics of S

deficiency. According to Brunold (1993) a high accumulation of amino acid in the S-deficient

condition could be readily explained by increased proteolysis and reduction in the rate of

protein synthesis due to the absence of the key S containing amino acids, cysteine and

methionine. Under optimum S nutrition and under mild deficiency conditions the amino acid

content in plants is usually balanced in a delicate way (Höfgen et al., 1995). This can be

reflected by the results of experiment 1 and 3, where no difference was found between S

treatments. But, under prolonged and severe S deficiency, amino acid homeostasis tended to

be impaired resulting in the net accumulation of total amino acids. Ruiz et al. (2005) found in

bean that the amino acids reached their highest concentrations under zero S supply and

proteins lowest concentration, while the plants at high S supply resulted in an opposite effect

namely high protein contents and low amino acids concentration. In agreement to this, the

current study showed that amino acids and protein in severly S deficient plants were very low

compared to S supplied plants as found in experiment 2 conducted in summer (Tab. 9).

Eaton (1951) described that proteolysis accounted for the accumulation of amino acids. This

explanation appears reasonable because in the present experiment, lack of S supply for a

59

prolonged period (10 weeks) led to the drastic reduction of protein and chlorophyll contents

even in the mature leaves. This leads to the conclusion that amino acids may accumulate as a

consequence either of impaired nitrogen metabolism or of the hydrolysis of proteins

(proteolysis). It also suggests on the basis of this result that remobilization of insoluble S

(mainly protein S) will be a new source not only for growing sinks but also for the

maintenance of physiological functions under a prolonged S deprivation. Proteolysis in well

functioning old leaves is in fact unlikely to occur unless the severe S deficiency is combined

with high nutrient demand.

For sugar, the highest levels were found at the 5000 µM S compared to 500 µM and 0 µM

supply levels in the experiment conducted in the summer (Tab. 9). Even though proteolysis

was reported to cause a decrease in total sugar content (Eaton, 1951) this can not explain why

the sugar content in S-enriched plants (500 µM) was as low as in S-starved plants found in

experiment 2 (Tab. 9). In the winter experiments (i.e. experiment 1 and 3), sugar contents

were hardly affected by varying S supplies. It implies that the plant possesses an adaptation

mechanism for sugar causing homeostasis under different S supply levels. These findings are

supported by a study in alfalfa and tomato showing that S deficiency did not result in

alteration of sugar levels except under conditions of severe S deficiency (Rendig and

McComb, 1961).

Starch was reported by Marschner (1995) to accumulate in leaves of S deficient plants as a

consequence either of impaired carbohydrate metabolism at the sites of production (the

source) or of low demand at the sink sites (growth inhibition). In line, in current study starch

accumulation in leaves of the S-starved plants was consistently observed under different

environmental conditions (Tab. 8, 9 & 10). This clearly indicates that starch accumulation is a

more distinctive indicator of S deficiency than sugars depletion. However, it is unlikely to

observe these changes upon short periods of S deprivation.

60

6.2 Interaction between Sulfur Nutrition and P. fuligena Infection

P. fuligena infection

Sulfur nutritional status of plants was reported to have a strong impact on the natural

resistance against pathogens (Schnug et al., 1995). A significant repressive effect of soil-

applied S on the on the infection of oilseed rape with Pyrenopeziza brassicae, grapes with

Uncinula necator, and potato tubers with Rhizoctonia solani was found (Bourbos et al., 2000;

Klikocka et al., 2004; Schnug et al., 1995).

In current study, disease levels of tomato black leaf mold were more severe in S-starved as

compared to S-enriched plants. In experiment 3, S supply had no influence on the disease

severity of P. fuligena (Fig. 14) during the early stage of infection indicating that the disease

was not affected by S treatment before the appearance of visible symptoms in spite of the high

S status of S-treated plants. This leads to an assumption that the internal S pool is not a

decisive factor for resistance reactions against the fungus and implies that there was a

temporal discrepancy between the S status of the plant and resistance reactions. However,

when maintaining the S supply the disease development was delayed as compared to plants

not supplied with S. The positive effect of S nutrition on the plant resistance was consistent in

two out of four experiments (Fig. 11 & 14). This finding is in agreement with the results from

oilseed rape (Dubuis, 2004) infected by Brassicca napus where disease resistance was

negatively affected by S deficiency.

There are two main arguments to explain why S deficient plants become more susceptible to

pathogens (Dubuis, 2004). First, the increased susceptibility could be caused by a specific

effect of S deficiency on the accumulation of S-containing defense compounds such as

phytoalexins, GSL and cysteine-rich antifungal polypeptides which have important roles in

disease resistance. A lack of cysteine could lead to reduced glutathione (GSH) levels and a

disturbance of the central cellular active oxygen scavenging system, the GSH-ascorbate cycle.

Nikiforora et al. (2002) suggested that a loss of antimicrobial activity in S deficient plants

might contribute to their higher susceptibility. Second, S deficiency could lead to a general

reduction of plant fitness that causes an enhanced susceptibility to stress.

Experiment 2 (summer 2006) where severe S deficiency was observed, was an exception. It

showed that disease development was highly retarded on plants suffering from severe S

61

deficiency. Fungal infection might have failed to establish already in the penetration phase,

probably because of physical barriers due to starch accumulation and stomata closure. Even

more probable, an accumulation of phenolic compounds (Nikiforova et al., 2005), which may

have accumulated in severely S-deficient leaves characterized by changes of leaf coloration

e.g., anthocyanin formation (Fig. 8A), may have prevented the challenges of fungal infection.

This could be the main reason why plants in extremely S-starved conditions did not respond

to fungal infection since the phenolic compounds are highly toxic to pathogens as found in in-

vitro (Chérif et al., 1994) and in- situ as indicated by destroyed fungal hyphae (Chérif et al.,

1992). Although fungal infection was repressed in this way, it is unrealistic to impose the

plants to a severe S deficiency as a strategy.

The microscopic examinations revealed that S nutrition has the potential to delay the infection

stages of P.fuligena. Since P. fuligena penetration is known to be either cuticular or stomatal,

stained stomata are assumed to be a marker for fungal penetration/invasions. Stained stomata

seemed to be penetrated by fungal chitin which got stained with Coomassie dye and they were

quantitatively related to infection phase. Unfortunately the stained stomata were not

exclusively found in infected leaves but also in leaves of control plants. The reasons could be

contamination when the leaves were inoculated or interference of other fungi like Botrytis that

was also sporadically occurred in the mist chamber. However, in the third trial (Fig. 18)

employed on the plants from experiment 4 (summer 2007), the plants were encountered no

contamination problems. Moreover stained/penetrated stomata were exclusively found in

inoculated leaf samples. Noticeably changes in tissue coloration (Fig. 16A) around the

infected stomata were also found. It suggests that stained stomata can be used as a marker to

detect the fungal penetration and/or early establishment of infection. Whether S has a direct

impact on mycelium growth or sporulation is not clear. Detailed studies on infection

processes of P. fuligena need to be performed. Plant S status appeared to induce some

changes in relation to P. fuligena infection in the early incubation phase. The question of

which form of S is responsible for this can not be answered by the current data. In an in-vitro

test, the mycelium growth of P. fuligena was inhibited by high rates of elemental S (Heine,

personal communication) and elemental S was found to be involved in the resistance of

tomato against vascular pathogens (Williams et al., 2002). For this purpose, further tests

should be done using detached leaves from plants differently supplied with sulfur.

62

Changes in metabolites in relation to P. fuligena infection

Free Cys and GSH are S-containing compounds of primary metabolism. These metabolites

are found to be involved in plant resistance against fungal pathogens (Vidhyasekaran, 2000;

Gullner and Kömives, 2001). In greenhouse and field experiments, De Kok et al. (1981),

Schnug et al. (1995) and Bloem et al. (2004) found a significant relationship between S status

and the cysteine and GSH content.

Current study agrees fairly to the others’ findings. Without inoculation, all S fractions (i.e.,

total S, SO4-S and organic S) were significantly correlated with GSH, but not always with

Cys concentration in all experiments (Tab. 11, 12 & 13A). It clearly indicated GSH can be

used as a suitable indicator of plant S status. A close relation between Cys and GSH was

found only in younger leaf (Tab. 12 & 13A). With fungal infection, the relationships between

S fractions became unclear not because of S fractions but of the dramatic fluxes of thiols.

In the advanced stages of infection, when the disease severity reached maximum, the

depletion of GSH was found to very high while Cys pool was less affected. This response of

thiols is in contrary to other studies which normally reported that fungal infections generally

yields an increase in the GSH content (Vanacker et al., 2000; Gullner and Kömives, 2001;

Williams et al., 2002).

In the present study, in spite of a heavy decline induced by P. fuligena, S supply could have

maintained the positive effect or replenished the GSH pool to a certain functional level after

being challenged with P. fuligena especially in the late stage of infection (Fig. 20 & 21). This

was related to a lower disease severity (Fig. 12 & 15). Salac et al. (2005) suggested the

possible reason why GSH and Cys declined faster and to a higher extend in S deficient plants.

In the infected plant tissues these metabolites were consumed during metabolic processes

involved in disease reactions but could be replenished only in plants provided with external S

(Fig. 20 & 21). They added another possibility as that the plant tissue was severely damaged

by the pathogen resulting in a shift of anabolic in the favor of catabolic processes. Necrotic

leaf areas are composed of dead cells and a complete degradation or translocation from Cys

and GSH in/from necrotic plant tissue is assumed. Accordingly, a slight decrease in the Cys

and GSH content might be expected in visually infected leaf discs if the necrosis is not so

severe, but in the severe cases, drastic reduction in GSH and Cys may be attributed to

metabolic changes in the decaying leaf tissue (Fig. 20 & 21). Comparing experiment 1 (33

63

dpi) and 3 (27 dpi), GSH decline was more drastic in experiment 1 than in experiment 3

possibly because of differences in disease levels. This implies that the more severe the disease

severity the more depletion of GSH in tomato (particularly cv. King Kong II) which can

clearly be seen in experiment 3 where three harvests (18, 24 and 27 dpi) were taken. At 18 dpi

GSH levels in S supplied plants even showed a tendency of a slight increase, followed by

depletion at 24 dpi and 27 dpi.

Interestingly, at 27 dpi, highly significant positive effect of S on GSH occurred concomitantly

with highly significant negative effect of fungus. This represents the dynamic flux of GSH

pool being imposed by interaction of P. fuligena (depletion) and S nutrition (accumulation).

Under the attack of fungus, Cys can be rapidly degraded to H2S or metabolized to other

compounds that are putatively involved in pathogenesis (Salac et al., 1005). Moreover it was

reported (Bloem et al., 2004) that the activity of LCD (Cys consuming (degrading) enzyme)

was up-regulated under fungal infections. Sulfide is a byproduct of this degradation. Since

H2S is highly fungi-toxic the improved resistance of plants with high S supply may be linked

to fungal induced H2S formation concomitantly with the probable formation other S-

containing defense compounds like PRPs and phytoalexins. However, H2S determination and

activities of the enzymes involved were not determined in this experiment.

May et al. (1996) suggested that GSH concentrations were not related to the predisposition of

plants to infection. A decrease in GSH pool has been reported in oat and tomato leaves

infected with necrotrophic fungal pathogens (Gönner and Schlösser, 1993). The GSH pool in

the current experiments was depleted after P.fuligena infection in a similar way (Fig. 20 &

21). In tobacco mosaic virus infected tobacco plants, increased levels of GSH were related to

a reduced necrotization of infected tissues (Gullner et al., 1999). This result is in agreement

with present study where disease severity and GSH contents showed a significant negative

relationship (Fig. 22 & 23). In barley and oat infected with a biotrophic fungal pathogen

Blumeria graminis, GSH concentration increased in resistant but not in susceptible lines

(Vanacker et al., 1998, 2000). But in Arabidopsis mutants, the depletion of GSH levels by

70% was found in response to fungal and bacterial pathogens, but this did not change the

susceptibility (May et al., 1996). Accordingly, in the present study, it is not surprising that the

depletion of GSH levels was observed since tomato cultivar (King Kong II) is susceptible to

P. fuligena,. If not continuously supplied with S, susceptibility of KK2 will not be changed

too in spite of drastic GSH depletion.

64

Soluble protein content was found to increase in response to P. fuligena inoculationn mainly

in the 5000 µM S treatment (Tab. 5 & 6). Increase in soluble protein of leave tissues under

fungal infection could be directly linked to the formation of pathogenesis proteins released

either by the pathogen during infection process (Segarra et al., 2003) or by the host plant to

counteract the pathogen attack (Zareie, et al., 2002). The questions of if this increase of

soluble protein has a direct relation to apoplastic proteins or if the tomato plant can release PR

proteins under high S supply can not be answered by the present study. Further analysis of

proteomes and metabolomes are necessary to clarify this. Amino acids accumulation was also

observed significantly in 5000 µM S of (experiment 1) in relation to infection which may be

attributed to pathogen induced oxidative reactions.

In conclusion, total S, SO4-S, the proportion of SO4-S (as % of total S) and GSH all seem to

be the suitable indicators of S nutritional status in tomato plants. Extreme S deficiency leads

to the decreases of proteins and chlorophyll contents whereas increases of starch and total

amino acids contents. The optimized nutrition of S in the form of sulfate plays an important

role in enhancing the constitutive resistance of tomato against black leaf mold caused by P.

fuligena. Sulfur nutrition in combination with P. fuligena infection causes the changes in

plant S metabolism. Underlying biochemical and molecular mechanisms of these changes

remain to be elucidated.

65

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