• TABLE OF CONTENTS
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 Title Page
 Acknowledgement
 Table of Contents
 Abstract
 Introduction
 Literature review
 The photosynthetic competence of...
 The photosynthetic competence of...
 The photosynthetic competence of...
 General conclusions
 Appendices
 References
 Biographical sketch














Title: Photosynthetic competence of bean leaves with rust and anthracnose
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 Material Information
Title: Photosynthetic competence of bean leaves with rust and anthracnose
Physical Description: Book
Language: English
Creator: Lopes, Daniela Biaggioni, 1969-
Publisher: State University System of Florida
Place of Publication: <Florida>
<Florida>
Publication Date: 1999
Copyright Date: 1999
 Subjects
Subject: Plant Pathology thesis, Ph. D   ( lcsh )
Dissertations, Academic -- Plant Pathology -- UF   ( lcsh )
Genre: government publication (state, provincial, terriorial, dependent)   ( marcgt )
bibliography   ( marcgt )
theses   ( marcgt )
non-fiction   ( marcgt )
 Notes
Summary: ABSTRACT: The effects of two important diseases of common bean (Phaseolus vulgaris) on leaf photosynthesis were studied under controlled conditions. Uromyces appendiculatus and Colletotrichum lindemuthianum, the causal agents of rust and anthracnose, respectively, were inoculated separately or together in the same leaf, and several levels of disease intensity were considered in the study. The photosynthetic rate of leaves with rust was reduced mainly within the area of the lesion, and thus there was little effect of the disease in the remaining green area of the leaf. Increased rates of respiration and loss of chlorophyll from the leaf tissue apparently were the major factors responsible for the reduction of photosynthetic rates on diseased leaves. The optimal quantum yield and the electron transport rate, which are parameters of chlorophyll fluorescence related to the efficiency of the photosynthetic apparatus, were reduced in leaves with high rust severity after the appearance of the fleck symptoms. Both chlorophyll content and color of the leaves were well correlated to relative photosynthetic rates on healthy and diseased leaves with different nutritional status. Leaf color and chlorophyll content could, thus, be used as potential predictors of leaf photosynthetic rate. The reduction in the photosynthetic rates of bean leaves with anthracnose was greater than that caused by rust. The photosynthesis in the green area beyond the necrotic symptoms of anthracnose was severely impaired shortly after the appearance of the symptoms. Factors associated with the reduction in the rate of photosynthesis were decreased stomatal conductance and increased rates of respiration. No obvious interaction was observed between the rust and anthracnose pathogens, when both were simultaneously inoculated in the same leaf.
Summary: ABSTRACT (cont.): The photosynthetic rate and the electron transport rate of leaves with both diseases were determined by the proportion of leaf tissue with anthracnose. The impact of rust and anthracnose on bean leaf photosynthesis should be considered in assessments of the proportion of healthy tissue in diseased leaves. The accurate assessment of the healthy portion of the leaf could improve the use of concepts such as healthy leaf area duration and healthy leaf area absorption, which are valuable predictors of crop yield.
Summary: KEYWORDS: photosynthesis, Phaseolus vulgaris, bean, rust, Uromyces appendiculatus, anthracnose, Colletotrichum lindemuthianum
Thesis: Thesis (Ph. D.)--University of Florida, 1999.
Bibliography: Includes bibliographical references (p. 141-155).
System Details: System requirements: World Wide Web browser and PDF reader.
System Details: Mode of access: World Wide Web.
Statement of Responsibility: by Daniela Biaggioni Lopes.
General Note: Title from first page of PDF file.
General Note: Document formatted into pages; contains ix, 157 p.; also includes graphics.
General Note: Vita.
 Record Information
Bibliographic ID: UF00100667
Volume ID: VID00001
Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
Resource Identifier: oclc - 47680071
alephbibnum - 002456827
notis - AMG2158

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Table of Contents
    Title Page
        Page i
        Page ii
    Acknowledgement
        Page iii
        Page iv
        Page v
    Table of Contents
        Page vi
        Page vii
    Abstract
        Page viii
        Page ix
    Introduction
        Page 1
        Page 2
        Page 3
        Page 4
        Page 5
    Literature review
        Page 6
        Page 7
        Page 8
        Page 9
        Page 10
        Page 11
        Page 12
        Page 13
        Page 14
        Page 15
        Page 16
        Page 17
        Page 18
        Page 19
        Page 20
    The photosynthetic competence of bean leaves with rust
        Page 21
        Page 22
        Page 23
        Page 24
        Page 25
        Page 26
        Page 27
        Page 28
        Page 29
        Page 30
        Page 31
        Page 32
        Page 33
        Page 34
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        Page 53
        Page 54
        Page 55
        Page 56
        Page 57
        Page 58
        Page 59
        Page 60
    The photosynthetic competence of bean leaves with anthracnose
        Page 61
        Page 62
        Page 63
        Page 64
        Page 65
        Page 66
        Page 67
        Page 68
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        Page 89
        Page 90
        Page 91
        Page 92
        Page 93
        Page 94
        Page 95
    The photosynthetic competence of bean leaves with rust and anthracnose
        Page 96
        Page 97
        Page 98
        Page 99
        Page 100
        Page 101
        Page 102
        Page 103
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        Page 106
        Page 107
        Page 108
        Page 109
        Page 110
        Page 111
        Page 112
    General conclusions
        Page 113
        Page 114
        Page 115
        Page 116
        Page 117
    Appendices
        Page 118
        Page 119
        Page 120
        Page 121
        Page 122
        Page 123
        Page 124
        Page 125
        Page 126
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        Page 128
        Page 129
        Page 130
        Page 131
        Page 132
        Page 133
        Page 134
        Page 135
        Page 136
        Page 137
        Page 138
        Page 139
        Page 140
    References
        Page 141
        Page 142
        Page 143
        Page 144
        Page 145
        Page 146
        Page 147
        Page 148
        Page 149
        Page 150
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        Page 152
        Page 153
        Page 154
        Page 155
    Biographical sketch
        Page 156
        Page 157
Full Text











PHOTOSYNTHETIC COMPETENCE OF BEAN LEAVES
WITH RUST AND ANTHRACNOSE















By

DANIELA BIAGGIONI LOPES


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA


1999
































To Ronaldo and Maria Jose















ACKNOWLEDGMENTS


I would like to thank all members of my Supervisory Committee for their

continuous intellectual support and for their belief in my ability to conduct my research

project. I am very grateful to Dr. Richard Berger for accepting me in his program and for

his trust, which allowed me to develop an intellectual independence that will certainly be

important for the rest of my career. I would like to thank Dr. David Mitchell for being a

true mentor and friend, and for making me realize, through his example, that I really want

to teach plant pathology. I thank Dr. James Kimbrough for his support and for sharing his

excitement about fungi. I thank Dr. Kenneth Boote for his insightful comments about my

research and for allowing me to work with his Walz fluorometer. I would like to thank

Terry Davoli for her friendship and for her sincere disposition in helping me with my

work. The data collection would not have been possible without her help and patience. I

thank Eldon Philman and his assistants for being always available and for answering

promptly all of my needs in the greenhouse. I would like to thank all of the department

staff for their efficiency and kindness.

I am grateful to Scott Kowalski, Galin Jones, Jay Harrison, and Jack Bishko, from

the IFAS Statistics Department, for their assistance with my statistical analysis. I would

like to thank Rea Leonard and Dr. Terry Nell, from the Department of Environmental

Horticulture, for allowing me to use their LI-6200 Portable Photosynthesis System and

their gas tank.









I would also like to thank Dr. Armando Bergamin Filho, from ESALQ/USP, for

the support he gave to my plans to come to the University of Florida and for his

encouragement and friendship throughout my stay here. I extend my thanks to Renato

Bassanezi for sharing his thoughts and questions about the common subject of our

research projects. I am very grateful to Dr. William Zettler for giving me the unique

opportunity of working with him in his undergraduate course. I extend my thanks to

Mark Elliott for being such a wonderful partner in preparing and conducting the labs. It

was a real pleasure to work with both of them. I would like to thank the Department of

Plant Pathology, the Student Government, and IFAS for the travel grants that allowed me

to present my work in two different scientific meetings.

I feel lucky to have met such a diverse and interesting group of graduate students

in this department. I thank Simone Tudor, Gustavo Astua-Monge, and S. Chandramohan

for the experiences we shared during these four and a half years. Special thanks to Carlos

Forcelini, Bob Kemerait, and Erin Rosskopf for giving me emotional and intellectual

support to endure my Ph.D. program. They have all my respect and admiration as friends

and colleagues. I would like to thank all the friends I made while helping to build the

Brazilian Student Association at UF (BRASA), for sharing their dreams and 'saudades.'

I hope I can pay back, in service, the public money that my country invested on

me. I will be always grateful to the Conselho Nacional de Desenvolvimento Cientifico e

Tecnol6gico (CNPq), Brazil, for the financial support that allowed me to come to the

University of Florida. Special thanks to Jessy Alves Pinheiro for her efficiency in

handling any problems and questions I had during these years.









Most thanks go to Ronaldo Lopes and Maria Jose Biaggioni Lopes for their

unconditional love and their support to anything I ever decided to do. All of my

accomplishments in life are also theirs. I could never have gone through this experience

without the support of my husband, Fabiano Toni. He is a true and loving friend, always

reminding me of what is really important in life. I also thank my baby for his quiet being,

and for giving me such a wonderful perspective for the future.















TABLE OF CONTENTS


page


A C K N O W L E D G M E N T S .............................................................................. ...............iii

A B S T R A C T .........................................................................................v iii

CHAPTERS

1 IN TR OD U CTION ............................. ........................................ ......... 1

2 L ITE R A TU R E R E V IE W .................................................................. .... ................... 6

Photosynthesis and Stress Physiology................................. .......................... 6
Disease as a Stress Factor.................. ... ........... .............. 7
Measuring Photosynthesis in vivo ..... ............................................................. 13

3 THE PHOTOSYNTHETIC COMPETENCE OF BEAN LEAVES WITH
R U S T ............................................................................... 2 1

Introduction .................................................................................... ............ ........ 21
M material and M ethods............................................................ .......................... 23
Results ........... ... ... ............ ...... ..................... .............. 30
D discussion ......................................... 48

4 THE PHOTOSYNTHETIC COMPETENCE OF BEAN LEAVES WITH
A N T H R A C N O SE ..................................................................... .. ...... ...... .. 6 1

Introduction ................................... ............................... ......... 61
M materials and M ethods ............................................................. ................ 63
R results .................................... .................................. ......... 69
Discussion ............................................ 86

5 THE PHOTOSYNTHETIC COMPETENCE OF BEAN LEAVES WITH
RU ST AND ANTHRACNOSE ......................................................... ......... ..... 96

Introduction .................................... ............................... ........ 96
M material and M ethods............................................................ .......................... 97
R results ................................................................ ....... ......... 101









D discussion ......................................... 109

6 GENERAL CON CLU SION S .............. .......................................................... 113


APPENDICES

A EFFECT OF THE REMOVAL OF THE GROWING POINT OF BEAN
PLANTS ON THE PHOTOSYNTHESIS OF DISEASED LEAVES...................... 118

B SA S P R O G R A M ..................................................................... .. .... .................. 12 1

C COMPARISON OF TWO METHODS TO ESTIMATE LEAF
CHLOROPHYLL CONTENT ............ ....................................................... 122

D RESULTS FROM EXPERIMENT 3-6, CHAPTER 3 ............................................ 125

E ASSESSMENT OF ANTHRACNOSE SEVERITY WITH A
DIAGRAMMATIC SCALE OF SYMPTOMS ............................................ .. 127

F EFFECTS OF DUAL INFECTIONS OF Uromyces appendiculatus AND
Colletotrichum lindemuthianum ON THE MONOCYCLIC PARAMETERS
OF BEAN RUST AND BEAN ANTHRACNOSE .................................................. 129

R E F E R E N C E S ............................................................... .. .. ........ .... . ........... 14 1

BIOGRAPHICAL SKETCH................. ... ........... ............. 156















Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

PHOTOSYNTHETIC COMPETENCE OF BEAN LEAVES
WITH RUST AND ANTHRACNOSE

By

Daniela Biaggioni Lopes

May 1999

Chairman: Richard D. Berger
Major Department: Plant Pathology

The effects of two important diseases of common bean (Phaseolus vulgaris) on

leaf photosynthesis were studied under controlled conditions. Uromyces appendiculatus

and Colletotrichum lindemuthianum, the causal agents of rust and anthracnose,

respectively, were inoculated separately or together in the same leaf, and several levels of

disease intensity were considered in the study.

The photosynthetic rate of leaves with rust was reduced mainly within the area of

the lesion, and thus there was little effect of the disease in the remaining green area of the

leaf. Increased rates of respiration and loss of chlorophyll from the leaf tissue apparently

were the major factors responsible for the reduction of photosynthetic rates on diseased

leaves. The optimal quantum yield and the electron transport rate, which are parameters

of chlorophyll fluorescence related to the efficiency of the photosynthetic apparatus, were

reduced in leaves with high rust severity after the appearance of the fleck symptoms. Both









chlorophyll content and color of the leaves were well correlated to relative photosynthetic

rates on healthy and diseased leaves with different nutritional status. Leaf color and

chlorophyll content could, thus, be used as potential predictors of leaf photosynthetic rate.

The reduction in the photosynthetic rates of bean leaves with anthracnose was

greater than that caused by rust. The photosynthesis in the green area beyond the necrotic

symptoms of anthracnose was severely impaired shortly after the appearance of the

symptoms. Factors associated with the reduction in the rate of photosynthesis were

decreased stomatal conductance and increased rates of respiration.

No obvious interaction was observed between the rust and anthracnose pathogens,

when both were simultaneously inoculated in the same leaf. The photosynthetic rate and

the electron transport rate of leaves with both diseases were determined by the proportion

of leaf tissue with anthracnose.

The impact of rust and anthracnose on bean leaf photosynthesis should be

considered in assessments of the proportion of healthy tissue in diseased leaves. The

accurate assessment of the healthy portion of the leaf could improve the use of concepts

such as healthy leaf area duration and healthy leaf area absorption, which are valuable

predictors of crop yield.















CHAPTER 1
INTRODUCTION

The fact that plant diseases cause crop losses was the very reason for the birth of

plant pathology as a science, in the last century. Nevertheless, after 150 years, it is still

difficult to make accurate predictions about the magnitude of loss that a specific disease

(or diseases) is capable of causing in a particular season. Reliable estimates of the impact

of a disease on yield are a prerequisite to the establishment of any crop protection strategy

(Zadoks and Schein, 1979).

Empirical loss models are used commonly to search for a relationship between

disease intensity and yield loss, at one specific time or at several times, during a growing

season. However, this relationship is often inconsistent, primarily because the highest

possible yield is different for each field, locale, and season due to differences in

environmental and edaphic factors. Also, the relationship between crop yield and

intensity of disease can be disappointing, particularly if factors related to the host

(developmental stage, defoliation, leaf area) are not considered (Rouse, 1988; Waggoner

and Berger, 1987).

Alternatively, a mechanistic approach to loss models would be to describe the

various physiological processes of plant growth and to incorporate the effects of diseases

on these processes, usually as a crop simulation model. The use of mechanistic models is

of paramount importance for a fuller understanding of yield response to disease (Gaunt,

1987; Loomis et al., 1979; Loomis and Adams, 1983; Pinnschmidt et al., 1994). Effect of






2


pests and pathogens on crop carbon flow processes can be classified into seven groups:

tissue consumers, leaf senescence accelerators, stand reducers, light stealers,

photosynthetic rate reducers, assimilate sappers, and turgor reducers (Boote et al., 1983).

The first four categories would represent major effects of pests and pathogens on

radiation interception, and the last three represent major effects on radiation use

efficiency (Johnson, 1987). The quantification of the damaging effects of pathogens on

crop growth would make it possible to couple these effects to crop growth simulators to

predict reductions in yield.

In several publications on crop growth physiology, crop production has been

shown to be closely related to the amount of leaf area available (Watson, 1947), the

duration of this leaf area (Watson, 1952), and the amount of insolation the plant is able to

use during the season (Charles-Edwards, 1982; Gallagher and Biscoe, 1978; Monteith,

1972; Monteith, 1981). Monteith (1977) defined crop productivity as the product of

radiation interception and radiation use efficiency. As early as 1955, plant pathologists

began to notice the importance of leaf area to disease assessments (Last, 1955). Diseases

reduced the leaf area duration and the green leaf area (Lim and Gaunt, 1981; Lim and

Gaunt, 1986a; Lim and Gaunt, 1986b). Waggoner and Berger (1987) subtracted the

diseased leaf area from the leaf area integrated over time and came up with the concept of

healthy leaf area duration (HAD). Since yield is determined by the energy absorbed

during the season, the concept of healthy leaf area absorption (HAA) was also introduced

(Waggoner and Berger, 1987).

Waggoner and Berger (1987) proposed that HAD and HAA were much better

predictors of yield compared to disease intensity as a predictor of yield loss, since HAD









and HAA add biological realism and flexibility to the empirical approaches. These

concepts proved valid for many different pathosystems, such as Phytophthora infestans

on potato (Haverkort and Bicamumpaka, 1986; Rotem et al., 1983a; Rotem et al., 1983b;

van Oijen 1990), Alternaria solani on potato (Johnson and Teng, 1990), Aschochyta

fabae on Viciafaba (Madeira et al., 1988), Pyricularia oryzae on rice (Pinnschmidt and

Teng, 1993), Erysiphe graminis on wheat (Daamen and Jorritsma, 1990), and

Phaeoisariopsis griseola on common bean (Bergamin et al., 1997).

The concepts of HAD and HAA should also apply to crops with multiple

pathogens and even with insect pests (Berger, 1988). Under field conditions, the attack of

more than one plant pathogen at the same time is very common, especially in tropical

areas (Savary and Zadoks, 1991). Nevertheless, the list of published studies on multiple

pathogens, based on the synecological approach (Kranz and Jorg, 1989), in relation to

crop loss, is not very extensive (Johnson, 1992; Johnson et al., 1986; Savary et al., 1988;

Savary and Zadoks, 1991; Savary and Zadoks, 1992a; Savary and Zadoks, 1992b; Savary

and Zadoks, 1991; Simkin and Wheeler, 1974; van de Wal and Cowan 1974; van der Wal

et al., 1975}. Disease assessment becomes more complicated with more than one disease

in the same plant. Several authors agree that to assess the effects of a complex of

diseases, the healthy leaf area of the plant should be the focus of attention (Berger, 1988;

Johnson and Teng, 1990; Kranz and Jorg, 1989). The duration and absorption of the

healthy leaf area can provide a valuable measure to integrate all components in the

complex (Berger, 1988).

Johnson (1987) raised several interesting questions concerning the relation of

HAA and yield. First, is it enough to know the amount of intercepted radiation by the









green portion of a canopy to predict yield? For many pests and diseases this was

demonstrated to be so (Waggoner and Berger, 1987), but for some other pathosystems it

may be different (Johnson, 1987). Second, how do different pathogens affect radiation

interception and radiation use efficiency, the factors that determine productivity? In fact,

there are examples of pathogens that cause a greater reduction in photosynthesis than that

expected by the severity alone (Bastiaans, 1991; Bastiaans et al., 1994; Boote et al., 1980;

Bourgeois and Boote, 1992).

The determination of radiation use efficiency has been done under field

conditions, by estimating the slope of the line that relates yield to HAA, in plots with

different disease intensities. If the slopes are constant for the different situations, the

radiation use efficiency is considered not to be affected by the disease (Aquino et al.,

1992; Waggoner and Berger, 1987). However, some researchers believe that only direct

measurements of photosynthetic rates in healthy and diseased plants can show whether

the photosynthetic activity of the green leaf area is being affected by disease. Examples

of crop loss studies that were based on the effects of the harmful agent on physiological

processes are given by Rabbinge and Rijsdijk (1981), Boote et al. (1983), Rabbinge et al.

(1985), van Roermund and Spitters (1990), and Bastiaans (1991).

The concept of a virtual lesion, introduced by Bastiaans (1991), can help in the

classification of pathogens according to their effect on the radiation use efficiency of their

hosts. According to Bastiaans (1991), the virtual lesion is the proportion of leaf tissue,

equal to or larger than the visual lesion (proportion of leaf tissue with visible symptoms),

in which photosynthesis is severely reduced. The ratio between virtual and visual lesion

size is defined as the parameter 3, which characterizes the effect of the pathogen on leaf









photosynthesis for an entire range of disease severities. A 3 value of 1.0 would be

interpreted as no detrimental effect on photosynthesis beyond the lesioned area.

Thus, to extend the use of HAD and HAA to different pathosystems and to study

the effect of multiple pathogens on the same plant, there is a need to determine, for

specific and multiple plant-pathogen interactions, how the green tissue of the diseased

plant is being affected. To address this question, the pathosystems Phaseolus vulgaris-

Uromyces appendiculatus and P. vulgaris-Colletotrichum lindemuthianum were chosen

as model systems for the work presented here. The effects of each disease on the

photosynthetic competence of bean leaf and also the effects of the interaction of these two

fungi on the same leaf were quantified. Photosynthetic competence of a diseased leaf is

defined here as the ability of that leaf to perform photosynthesis when compared to a

similar healthy leaf.















CHAPTER 2
LITERATURE REVIEW


Photosynthesis and Stress Physiology


Dry matter production, and consequently crop yield, is largely determined by the

amount of solar radiation intercepted by the green leaf area and by radiation use

efficiency (Monteith, 1977; Monteith, 1981). Thus, photosynthesis is the key

physiological process to understand crop yield potential and how this potential can be

affected by various stresses. Most stress factors impact photosynthesis, even if they do

not affect the composition of the photosynthetic apparatus directly (Lichtenthaler, 1996).

Plant stress is a very broad concept and may be defined as "any unfavorable

condition or substance that affects or blocks a plant's metabolism, growth or

development" (Lichtenthaler, 1996, p.4). It includes natural stress factors, such as low or

high temperatures, shortage or excess of water, attacks by pests and diseases; and

anthropogenic factors, such as herbicides, excess of nutrients and air pollutants. To

understand this concept, it is important to differentiate between the effects of short-term

and long-term stress, as well as between low stress events, which could be overcome, and

chronic stress events, which could lead to irreparable damage.

Photosynthetic rate is generally reduced in plants subjected to stress, but the

mechanisms underlying this reduction are dependent on the stress factor involved. For

example, water stress may cause an increase in the carbohydrate status of the plant, which









indirectly may induce the photosynthetic rate to fall. Also, stomatal closure is believed to

be influential in this case (Chaves, 1991). High temperatures will directly damage the

photosynthetic apparatus, which causes a loss of thylakoid membrane function and the

inactivation of photosystem II (PSII) (Paulsen, 1994). Similarly, low temperatures will

alter the function of membranes and enzymes in chilling-sensitive plants, which can cause

damage to PS II reaction centers (Guy, 1994).

Disease as a Stress Factor


Very few generalizations can be made regarding the ways pathogens affect

photosynthesis. In most host/pathogen interactions, both net and gross photosynthetic rate

decline, respiration rate increases and chlorophyll is lost from the tissue as infection

progresses (Scholes, 1992).

Due to the scope of the work, only the reported effects of plant pathogenic fungi

will be reviewed here. Plant pathogenic fungi can be divided in two groups, based largely

on their nutritional behavior: biotrophic and necrotrophic fungi. Obligate fungal

pathogens, such as rusts and powdery mildews, do not kill their host plants immediately;

these biotrophic fungi are dependent upon viable host tissue to complete their

development. Necrotrophic fungi, such as the blight and rotting fungi, usually invade

only structural tissues killed before the spread of the pathogen, and they can survive just

as well on decaying or dead host tissue (Agrios, 1997). As the pattern of response and the

actual reduction in photosynthesis are related to the type of trophic relationships

(Shtienberg, 1992), biotrophic and necrotrophic fungi are treated separately here.









Necrotrophic Fungi

The major effect of disease in depressing crop yields is through the reduction on

light interception (Haverkort and Bicamumpaka, 1986; Madeira et al., 1988; Waggoner

and Berger, 1987), which clearly happens when leaves turn completely brown and

necrotic as a result of infection by a necrotrophic fungus. Necrotic lesions are

photosynthetically useless. However, there may be an additional effect on the

photosynthesis of non-infected areas of a diseased leaf, on symptomless leaves of

diseased plants, and on infected areas before cell death and necrosis occur.

For Rhynchosporium secalis on barley leaves, Martin (1986) observed larger

reductions in maximum net photosynthesis than would be expected simply from the

reduction in green leaf area. He suggested that reduced photosynthetic rates occurred in

the green parts of the leaf, as well as in lesioned tissue. Also, for higher severities, there

was a decrease in photorespiration; however, there was an increase in the carbon dioxide

compensation point, the resistance of the mesophyll, and the resistance of the stomata to

the diffusion of CO2. Bourgeois and Boote (1992) also found a reduction in the

photosynthesis of peanut leaflets infected with Cercosporidium personatum that was

greater than expected, when the area with visible symptoms was considered. They

observed that a disease severity of 15% caused a 65% reduction in photosynthetic rate.

Leaf blast, caused by Pyricularia oryzae, also reduced net photosynthesis and

transpiration of infected rice leaves beyond what would be explained by severity alone

(Bastiaans, 1991; Bastiaans, 1993). Conversely, van Oijen (1990) reported that

photosynthesis was not impaired in healthy tissue of potato plants infected with

Phytophthora infestans. In this latter study, differently from the previously mentioned









studies, the CO2 assimilation was measured on the symptomless leaves of diseased plants

and not on the green area of partially diseased leaves.

Some necrotrophic fungi reduce photosynthesis by inducing defoliation (leaf

senescence accelerators) and thereby reducing light interception. Boote et al. (1980)

demonstrated that this happens with Cercospora leafspot on peanut. Photosynthesis of

diseased canopies was reduced by loss of leaves, which abscised as a result of the

infection; also, diseased leaves that remained on the plants were less efficient in fixing

C02.

Vascular wilt fungi are necrotrophic fungi that systemically infect plants,

occupying the xylem vessels and causing wilt symptoms. There is some evidence that,

although stomatal conductance was reduced in plants infected with these fungi, the

reduction in photosynthesis is not always due to fungal-induced water stress. From

analysis of CO2 assimilation (A) versus intercellular CO2 concentration (Ci) response

curves, it has been shown that some wilts directly affect carboxylation efficiency

(Hampton et al., 1990; Pennypacker et al., 1990). Hampton et al. (1990) suggested that

Verticillium dahliae reduced photosynthesis in cotton initially (before visible symptoms)

through nonstomatal processes, which were not directly mediated by water-deficit stress.

In contrast, leaves with symptoms exhibited decreased photosynthesis due to combined

chlorosis, water stress, and stomatal closure. They concluded that the resultant decrease

in photosynthetic capacity was the product of two distinct mechanisms of pathogenicity.

Pennypacker et al. (1990), working with Verticillium albo-atrum on alfalfa, indicated that

the reduction in the amount and activity of carboxylase enzyme (Rubisco) was the factor









responsible for reduced net photosynthesis of infected plants, rather than the reduced

internal CO2 concentrations caused by reduced stomatal conductance.

Biotrophic Fungi

It can not be assumed that all biotrophic agents act in a similar manner concerning

the mechanisms of infection. Most powdery mildews form haustoria only in epidermal

cells. Thus, carbohydrates and other nutrients must pass through these cells prior to

uptake by the fungus. In contrast, rust and downy mildews parasitize epidermal,

mesophyll and bundle sheath cells; therefore, they have a more direct access to pools of

carbohydrates. Such differences in growth within the leaf may have profound

consequences on the mechanism of inhibition of photosynthesis (Scholes, 1992).

Many different mechanisms have been proposed to explain reductions in the rate

of photosynthesis in infections by obligate fungi. Reduced net photosynthesis associated

with large increases in mesophyll resistance were reported in barley with brown rust

(Owera et al., 1981) and in sugar beet with powdery mildew (Gordon and Duniway,

1982). This increase in mesophyll resistance to CO2 diffusion was suggested to be caused

by metabolic alterations within chloroplasts or changed amounts of photosynthetic

machinery.

Rusts and powdery mildews were reported to affect photosynthetic electron

transfer in two ways: inhibiting non-cyclic photophosphorylation and inducing loss of

components of the photosynthetic electron transfer chain. Powdery mildew on sugar beet

and rust on broad bean induced an inhibition of non-cyclic photophosphorylation in

isolated chloroplasts (Magyarosy et al., 1976; Montalbini and Buchanan, 1974). In both

of these pathosystems, inhibition resulted from a diminution of electron flow from water









to NADP. Magyarosy and Malkin (1978) observed that the cytochrome content of the

electron transport chain was decreased by about one-third in chloroplasts from powdery

mildew-infected sugar beet plants. However, from measurements of chlorophyll

fluorescence in leaves of barley with powdery mildew, caused by Blumeria graminis f.sp.

hordei, there was no direct effect of the pathogen on the capacity for electron transfer in

this pathosystem (Scholes et al., 1990).

Loss of chlorophyll from the leaf tissue is commonly reported for plants infected

with biotrophic pathogens; it is usually closely correlated with decreases in

photosynthetic rate (McGrath and Pennypacker, 1990; Tang et al., 1996). For chloroplast

measurements made within individual pustules of Uromyces muscari on bluebell leaves,

the chloroplast volume, the chlorophyll concentration, and the ratio of chlorophyll a:b

declined, but there was little reduction in chloroplast number per unit area. This was

interpreted as a loss of chlorophyll from individual chloroplasts (Scholes and Farrar,

1985). In contrast, Ahmad et al. (1983) reported that the reduction in the rate of net

photosynthesis in barley leaves infected with brown rust was not due to reduced CO2

fixation per chloroplast, but was ascribed to a decrease in the number of functional

chloroplasts. In isolated chloroplasts from diseased and healthy plants, Ahmad et al.

(1983) found that surviving chloroplasts in diseased leaves functioned at least as well as

those from healthy leaves.

RuBP carboxylase/oxygenase (Rubisco) and other enzymes of the Calvin cycle

also can be affected. The infection of barley with B. graminis f.sp. hordei decreased

RuBP carboxylase activity per unit area, which was attributed to a decrease in the amount

of enzyme and in enzyme activity (Scholes et al., 1994; Walters and Ayres, 1984). The









activity of other enzymes of the pentose phosphate pathway (3-phosphoglycerate-kinase

and glyceraldehyde-3-phosphate dehydrogenase) was also reduced after inoculation with

mildew. The net result of this reduction in enzyme activity would be a decrease in the

regeneration of RuBP and possibly a reduction in the storage of various carbohydrates

(Walters and Ayres, 1984). Roberts and Walters (1988) found that the activity of RuBP

carboxylase was reduced in rust pustule areas of leek leaves infected with Puccinia allii .

Losses in total soluble protein and chlorophyll were observed within the pustule area but

not in the region between pustules.

More recently, feedback inhibition of photosynthesis following carbohydrate

accumulation has been investigated as a possible mechanism of reduction in

photosynthesis (Scholes, 1992; Scholes et al., 1994). The pathosystems studied in these

cases were B. graminis on barley and on wheat. Based on measurements of the rate of

photosynthesis, chlorophyll-a fluorescence, and freeze clamping of leaves throughout the

infection cycle, an increase in the activity of acid invertase and, consequently, an

accumulation of glucose, fructose, and sucrose, was observed. These events resulted in

the inhibition of the rate of photosynthesis in infected leaves due to loss of activity and

amount of photosynthetic enzymes of the Calvin cycle. Scholes et al. (1994) suggested

that the down-regulation of the Calvin cycle occurred either as a result of end-product

inhibition or, more probably, as a direct effect of carbohydrates on the expression of

genes encoding photosynthetic enzymes. In the sequence, electron transfer would be

down-regulated in response to a decreased demand for ATP and NADPH, and

photoinhibition and accelerated loss of chlorophyll from the leaf would occur. In

conclusion, this pathogen was thought to be altering the source/sink relations in the leaf









(Scholes, 1992). Increased activity of acid invertase and accumulation of carbohydrate

were also detected in leaves ofArabidopsis thaliana infected with Albugo candida, and

these were closely correlated with the decrease in photosynthesis, chlorophyll content,

and Rubisco activity in those leaves (Chou et al., 1995; Tang et al., 1996)

Measuring Photosynthesis in vivo


Measurements of photosynthesis in vivo, under the environmental conditions in

which the plant is growing, is an important step to determine the status of radiation-use

efficiency. The most commonly used methods to measure photosynthesis in vivo in the

last decade were carbon dioxide exchange measurements and chlorophyll fluorescence

emission. The principles and applications of these techniques are discussed here.

CO2 Exchange Measurements

Measurement of CO2 exchange with infrared gas analyzers is an instantaneous and

non-destructive technique that provides an unambiguous and direct measure of the net

rate of photosynthetic carbon assimilation (Long and Hallgren, 1993). Leaves, individual

plants, or a stand of plants are usually enclosed in a transparent chamber and the rate of

CO2 assimilation by the plant material is determined by measuring the change in the CO2

concentration of the air flowing across the chamber. Portable infrared gas analyzer

systems have made this technique very popular for measurements of individual leaves in

field conditions.

The principle of infrared gas analysis is based on the absorption of infrared

radiation, at specific wavelengths, by hetero-atomic gas molecules, such as CO2, H20,

and NH3 (Hall and Rao, 1994; Long and Hallgren, 1993). Gas molecules consisting of









two identical atoms, such as 02 or N2, do not absorb infrared radiation. The main

absorption peak for CO2 is at 4.26 pim, with secondary peaks at 2.66, 2.77, and 14.99 1Pm.

The only other hetero-atomic gas present in air with an absorption spectrum overlapping

that of CO2 is water vapor, which could interfere significantly with the determination of

CO2 concentration. This interference can be overcome simply by drying the air or by the

use of interference filters that preferentially isolate the 4.3-pim peak for detection. An

infrared gas analyzer has three basic parts: an infrared source; two parallel cells, the

analysis and the reference cell; and an infrared radiation detector. Equal amounts of

radiation pass into the two cells. There is a continuous flow of the sample gas through

the analysis cell and C02-free air is used in the reference cell. The detector is divided

into two chambers, linked to the cells, but separated by a thin metal diaphragm.

Radiation passing through the reference cell enters one chamber and radiation passing

through the analysis cell enters the other. Both chambers, also filled with C02, will

absorb radiation, the amount available for absorption being proportional to the amounts

absorbed within the cells. Pressure changes within the chambers will vibrate the

diaphragm and produce a voltage reading. The CO2 assimilation rate is expressed as the

amount of CO2 assimilated per unit leaf area and time (pimol CO2 m-2 S-1).

The environment of the enclosed leaf or plant is influenced by the design of the

chamber; thus, it is important to monitor and, if possible control, the environmental

variables that are associated with the determination of CO2 assimilation rates (Cheeseman

and Lexa, 1996; Long and Hallgren, 1993). Commonly, there is a system of air-

circulation within the chamber, and sensors are included to determine leaf and air









temperature, vapor pressure and relative humidity, and light intensity (photosynthetically

active radiation).

The parameters that commonly can be obtained by gas exchange systems are

photosynthetic rate, transpiration rate, intercellular CO2 concentration, and stomatal

conductance or resistance. The calculations of these parameters in commercial

equipment are done by pre-programmed models that link the disappearance of CO2 from

the chamber to the activity of the photosynthetic apparatus (Cheeseman and Lexa, 1996).

Other models are required to interpret the measurements and the

interdependencies among the variables measured. The models developed by Farquhar

and his group (Farquhar et al. 1980; Farquhar and von Caemmerer, 1982) summarized

more than 10 years of research in this area and are still the basis for interpretation of gas-

exchange measurements. The relationships of CO2 assimilation (A) to external limiting

factors, such as quantity of incident light, ambient temperature, and CO2 concentration,

allow quantitative assessments of the effects of environmental variables on different steps

in the diffusion pathway and are widely used in ecophysiological studies. For example,

the initial slope of the assimilation versus intercellular CO2 concentration (A:Ci) curve

reflects the activity status of Rubisco; CO2 assimilation at saturating light and saturating

CO2 is assumed to be limited by the potential rate of regeneration of the substrate of

carboxylation (ribulose-l,5-bisphosphate, RuBP). Further, the initial slope of the

relationship A vs. incident light is an estimate of the maximum light-use efficiency (or

quantum efficiency) of the light reaction of photosynthesis. The light-saturated

assimilation rate is considered a measurement of the photosynthetic capacity of the leaf,









as it varies with almost all environmental variables that influence photosynthesis

(Beyschlag and Ryel, 1998; Long and Hallgren, 1993).

Chlorophyll Fluorescence

Chlorophyll fluorescence, obtained in vivo with a fluorometer, has been called, in

the last decade, the "plant physiologist's stethoscope" because it measures the efficiency

of the photosynthetic activity and thus can provide approximate estimates of the vitality

and vigor of a plant in its environment (Bolhar-Nordenkampf and Oquist, 1993; Foyer,

1993; Hall and Rao, 1994). Measurements of chlorophyll fluorescence have been used

widely, not only in routine studies on photosynthesis, but also in other related areas, such

as studies on environmental stresses, screening for stress tolerance in plant breeding, and

studies on herbicide and air pollution (Foyer, 1993). The non-invasiveness, speed of data

acquisition, and high sensitivity are often cited as outstanding advantages of this

technique (Foyer, 1993; Schreiber et al., 1998).

When a molecule of chlorophyll absorbs light, it becomes excited to a higher

energy state. An excited molecule is not stable and the electrons return rapidly to their

ground energy level, releasing the absorbed photon energy basically in three ways: (1) the

electronic excitation energy is transferred by resonance to another acceptor molecule,

which results in photosynthetic electron transport; (2) it can release part of the excitation

as heat; and (3) it can emit the rest of the energy as a photon of lower energy content and

higher wavelength, a phenomenon called fluorescence (Hall and Rao, 1994). Since these

decay processes are competitive, changes in the photosynthetic activity and dissipative

heat emission will cause complementary changes in the intensity of the emitted

fluorescence (Bolhar-Nordenkampf and Oquist, 1993). In limiting low light and









maximum quantum yield, about 80 to 90% of the absorbed light energy will be dissipated

via photosynthetic quantum conversion, 5 to 15% by heat emission and only about 0.5 to

2% by fluorescence (Lichtenthaler, 1996). Most of the fluorescence emitted at room

temperature emanates from photosystem II (PSII), and thus fluorescence yield is related to

the efficiency of electron transport through PSII.

Chlorophyll fluorescence displays characteristic changes in intensity, termed the

Kautsky effect (Kautsky and Franck, 1943), that follow the induction of photosynthesis in

previously dark-adapted plants. Upon illumination, the fluorescence yield rises quickly in

the first second and then it may take several minutes to decline to a terminal level. The

fast rise in fluorescence yield is related to primary processes of PSII, whereas the decline

in yield is related to interactions between processes in the thylakoid membranes and

metabolic processes in the stroma, primarily carbon metabolism (Bolhar-Nordenkampf

and Oquist, 1993).

The parameters used in the quantification of fluorescence emission are derived

from the Kautsky curve of induction (Bolhar-Nordenkampf and Oquist, 1993; Schreiber

et al., 1998; van Kooten and Snel, 1990). Minimal fluorescence, or Fo, is an immediate

rise in fluorescence following weak illumination of dark-adapted photosynthetic tissues;

minimal fluorescence is measured when all reaction centers are open for primary

photochemistry, and it provides a reference for all other fluorescence parameters of the

induction curve. When a sufficiently strong light is applied, fluorescence increases from

Fo to a peak level, which is the maximum fluorescence, Fm. This rise reflects a gradual

increase in the yield of chlorophyll fluorescence, as the reaction centers become

increasingly reduced and the rate of photochemistry concurrently declines. Maximum









fluorescence is the maximum level of fluorescence, when all the reaction centers are

closed and no photochemistry is taking place. Variable fluorescence, Fv, equals the

fluorescence increase from Fo to Fm. The ratio Fv/Fm is a measure of the efficiency of

excitation energy capture by open PSI reaction centers, and it is usually called maximal

quantum yield of PSH. This ratio has a typical range of 0.75-0.85 in non-stressed healthy

plants. Quantum yield also can be assessed during illumination, and in this case it is

called effective quantum yield (Genty et al., 1989). There is a ubiquitous relationship

between the effective quantum yield and the quantum efficiency of photosynthetic carbon

assimilation, which is the photosynthetic efficiency at any light intensity, mol C02/mol of

photon (Genty et al., 1989; Seaton and Walker, 1990). The introduction of the effective

quantum yield parameter also made it possible to estimate electron transport rate through

PSH. Electron transport rate can be obtained by multiplying the effective quantum yield

by the fraction of incident irradiance absorbed by PSII, which is considered to be about

84% of the incident irradiance.

The fluorescence yield is lowered, or quenched, by two fundamentally different

mechanisms. The first type of reduction in fluorescence yield is essentially linked to

photochemistry, as it is controlled by the rate of re-oxidation of the first stable electron

acceptor to PSH. This is called photochemical quenching (qP). The second type of

quenching mechanism is called non-photochemical quenching (qN), and this is mainly

determined by de-excitation through heat generation. The largest contributor to non-

photochemical quenching is the energy-dependent quenching created by the hydrogen ion

concentration gradient across the thylakoids. This is induced in response to light

saturation and it is indicative of photo-protective regulations (Bolhar-Nordenkampf and









Oquist, 1993; Scholes and Horton, 1993). The quenching parameters are also derived

from the photosynthetic induction curve (Scholes and Horton, 1993).

It is well documented that PSII antenna and reaction centers are particular

sensitive to a number of stress factors, such as high temperatures, chilling, freezing,

drought, and excessive radiation (Bolhar-Nordenkampf and Lechner, 1988; Comic and

Briantais, 1991; Comic, 1994; Oquist and Ogren, 1985; Scheuermann et al., 1991). Few

reports, in this last decade, have dealt with the effects of biotic stress factors, such as

plant pathogens, on the activity ofPSII (Balachandran et al., 1994b; Moll et al., 1995;

Raggi, 1995). Since all these stresses affect the function of PSII, directly or indirectly,

fluorescence can be used as a tool to quantify the stress response and to understand stress

response mechanisms (Bolhar-Nordenkampf and Oquist, 1993).

The recent progress in chlorophyll fluorescence research is closely linked to the

development of modulated fluorometers, which allow detection of fluorescence yield

under normal daylight, whereas the previous techniques had to be applied only in dark

environments. Numerous reviews of the principles and details of the modulated light

fluorescence and the equipment developed to measure it are available in the literature

(Bolhar-Nordenkampf and Oquist, 1993; Foyer, 1993; Schreiber et al., 1986; Schreiber et

al., 1993; Schreiber and Bilger, 1993).

Although commercial devices for measurement of chlorophyll fluorescence

provide valuable information on the state of the photosynthetic apparatus in healthy and

stressed plants, they have an intrinsic disadvantage. Fluorescence data can be collected

only from one leaf point per measurement, and the researcher needs to determine the

fluorescence signals of various leaf points to obtain an approximate realistic figure of the









photosynthetic metabolism (Kramer and Crofts, 1996; Lichtenthaler, 1996). Fluorescence

video imaging has been used recently in situations where spatially resolved fluorescence

information is desirable. The ability to take video images during saturation pulses

superimposed onto continuous illumination has allowed the mapping of spatial variations

in the efficiency of PSII or in qN over the leaf surface (Daley et al., 1989; Genty and

Meyer, 1994; Lichtenthaler, 1996). This approach seems to be particularly suitable to

study changes in the photosynthetic metabolism of diseased leaves (Balachandran et al.,

1992; Balachandran et al., 1994a; Daley et al., 1989; Peterson and Aylor, 1995; Rolfe and

Scholes, 1995; Scholes and Rolfe, 1995; Scholes and Rolfe, 1996). Infected leaves are

often heterogeneous, consisting both of cells directly invaded by the pathogen and cells

that are not invaded but that can be modified by the pathogen's presence. The changes in

the photosynthetic metabolism in the different regions of an infected leaf can be

quantified with fluorescence imaging systems.















CHAPTER 3
THE PHOTOSYNTHETIC COMPETENCE OF BEAN LEAVES WITH RUST


Introduction


Bean rust, caused by Uromyces appendiculatus (Pers.: Pers.) Unger, inflicts major

production problems worldwide (Stavely and Pastor-Corrales, 1989). It is especially

damaging in humid areas, where periodic severe epidemics are common. Reported yield

losses range from 18-100% and these losses are directly related to the time and severity of

disease (Stavely, 1984; Stavely, 1991; Townsend, 1939; Venette and Jones, 1982). Under

greenhouse conditions, severe yield reduction occurred when plants were inoculated in

the pre-flowering, flowering, and pod-filling stages (Almeida et al., 1977). Reliable

estimates of future losses depend on the understanding of the epidemiological variables

that intensify the progress of the disease (Amorim et al., 1995; Berger et al., 1995) and on

accurate assessment of the impact of rust on the performance of the bean crop (Bergamin

and Amorim, 1996; lamauti, 1995).

Waggoner and Berger (1987) proposed that healthy leaf area duration (HAD) and

healthy leaf area absorption (HAA) were much better predictors of yield compared to

disease intensity as a predictor of yield loss, since HAD and HAA add biological realism

and flexibility to the empirical approaches. The concepts of HAD and HAA proved valid

for the yield loss assessment of many different pathosystems such as Phytophthora

infestans on potato (Haverkort and Bicamumpaka, 1986; Rotem et al., 1983a; Rotem et









al., 1983b; van Oijen 1990), Alternaria solani on potato (Johnson and Teng, 1990),

Aschochytafabae on Viciafaba (Madeira et al., 1988), Pyricularia oryzae on rice

(Pinnschmidt and Teng, 1993), Blumeria graminis on wheat (Daamen and Jorritsma,

1990), and Phaeoisariopsis griseola on common bean (Bergamin et al., 1997). The yield

of bean plants affected by rust was related to HAD and HAA, but yield had no significant

relationship with the area under the disease progress curve (AUDPC) (lamauti, 1995).

The AUDPC has been considered as the best measure of the intensity of an epidemic

(Campbell and Madden, 1990).

In addition to affecting the amount of intercepted radiation, some foliar pathogens

can affect the efficiency of radiation use by the plant (Bastiaans, 1991; Bastiaans et al.,

1994; Boote et al., 1980; Bourgeois and Boote, 1992; Johnson, 1987). In such reported

cases, the assessment of HAD and HAA can be less than accurate if the effects of the

disease on the healthy area are not considered (Johnson, 1987). The determination of

radiation use efficiency of diseased plants, from the slope of the line that relates yield to

HAA, has been done under field conditions in plots with different disease intensities. If

the slopes are constant for the different situations, the radiation use efficiency is

considered to be unaffected by the disease (Aquino et al., 1992; Waggoner and Berger,

1987). However, some researchers believe that only direct measurements of

photosynthetic rates in healthy and diseased plants can show whether the photosynthetic

activity of the green leaf area is being affected by disease (Bastiaans, 1991; Boote et al.,

1983; Rabbinge and Rijsdijk, 1981; Rabbinge et al., 1985; van Oijen 1990; van

Roermund and Spitters 1990). Thus, the present study focused on the quantification of

the effects of rust on the photosynthesis of leaves of common bean (Phaseolus vulgaris)









to verify whether assessments of HAA and HAD for bean plants with rust should take

into account any effects of the disease on radiation use efficiency.

Material and Methods


Plant Material and Inoculum

Bean plants (Phaseolus vulgaris) of the susceptible cultivar Rosinha were used in

all experiments. The plants were grown, one plant per pot, in 4-liter pots filled with

Metromix, and watered daily to field capacity. The plants were fertilized every other day

with Peter's fertilizer (20-20-20, 1 g/1 of water), unless stated otherwise. The growing

point of each plant was removed above the fourth or fifth leaf, to restrict the

indeterminate growth of the cultivar and to facilitate the handling of the plants. This

procedure did not affect the relative photosynthetic rate of diseased plants (Appendix A).

Urediniospores of race 86 of Uromyces appendiculatus were collected

periodically from pustules formed on 'Rosinha' plants, allowed to dry for 24 hours in a

closed silica gel container, and stored under -4C in glass vials submitted to vacuum. The

viability of the sample was tested before each inoculation. A diluted suspension of

urediniospores was plated on water-agar, and the percentage of germinated spores after a

12-hour period was determined. To prepare the inoculum, urediniospores were

suspended in sterile distilled water with 0.01% Tween-20, and the suspension was

agitated for 30 minutes. The concentration of spores was determined with a

haemocytometer, and the suspension was then adjusted to the desired concentration by

dilution.









Determination of Virtual Lesion Size

Bean plants were grown outdoors for Experiments 3-1 (November) and 3-2

(October), or under greenhouse conditions, for Experiment 3-3 (March). The plants were

fertilized weekly with Peter's fertilizer (20-20-20, 2 g/1 of water), and watered daily to

field capacity. Temperatures ranged from 14 to 300C for the outdoor experiments, and

from 18 to 300C for the experiment in the greenhouse. For the experiments conducted

outdoors, both sides of the expanding third trifoliate leaf were sprayed with a suspension

of urediniospores, amended with 0.01% of Tween-20 and 0.05% of the humectant

Metamucil. For the experiment in the greenhouse, the second leaf was inoculated and

each plant was enclosed for 15 hours in a clear plastic bag, which served as a dew

chamber. Suspensions of urediniospores of different concentrations (0, 2x102, 2x103, or

2x104 viable spores/ml) were used to obtain leaves with a wide range of rust severity.

The net photosynthetic rate at light saturation of healthy and diseased leaves was

determined by gas-exchange measurements using the LI-6200 Portable Photosynthesis

System (LI-COR, Inc.). Measurements were taken at a range of light intensities from

1000 to 1500 jpmol photon m-2s-1 of photosynthetically active radiation (400-700 nm) for

the experiments outdoors, and 800 to 1000 jpmol photon m-2s-1 for the experiment in the

greenhouse. The measurements were taken 10-12 days after the inoculation, when

symptoms were well developed. A portion of a bean leaf, still attached to the plant, was

placed in a 1-liter leaf chamber for 20 seconds, while the infra-red gas analyzer measured

the rate of assimilation of CO2. Simultaneously, sensors in the chamber measured CO2

concentration, air temperature, photosynthetically active radiation, relative humidity, and

vapor pressure, which were used to calculate the net photosynthetic rate, expressed in









Pjmol CO2 m -2s and the stomatal conductance, expressed in mol H20 m2 s -. The leaf

was then detached from the plant and, later, the rust severity was determined by the

number of lesions (pustule + halo) and an estimate of the average lesion size obtained

with an ocular micrometer. Chlorophyll pigments of the leaves were then extracted in

80% acetone and quantified with a spectrophotometer, according to the methodology

described by Amon (Arnon, 1949).

The equation P/Po=(1-x)P was used to relate relative photosynthetic rate (Po/Po) to

disease severity (x), where 3 represents the ratio between virtual and visual lesion sizes

(Bastiaans, 1991). Relative photosynthetic rate is the ratio of the photosynthetic rate of a

diseased leaf to the average photosynthetic rate of the healthy control leaves. The

parameter 3 was obtained by non-linear regression, using the procedure PROC NLIN

(method DUD) of SAS (SAS Institute, Cary, NC; release 6.12 for personal computers).

Analysis of covariance, using time as a covariate, was used with a linearized form of the

Bastiaans' model (see Appendix B) to verify if the results of the three experiments could

be pooled.

Effects of Fertilization on the Photosynthetic Competence of Bean Leaves with Rust

Bean plants growing under greenhouse conditions received the following

fertilization treatments, applied every other day: (a) no fertilizer, (b) half of the

recommended dosage of Peter's fertilizer for daily fertilization (0.5 g/1 of water), (c) full

recommended dosage of the fertilizer (1 g/1 of water). The experiment was conducted

twice. Temperatures inside the greenhouse ranged from 25 to 350C during the day, and

from 22 to 25 C during the night, for Experiment 3-4 (June). For Experiment 3-5









(March), the range of temperature was from 18 to 300C during the day, and around 18C

during the night.

The expanded first trifoliate leaf of all plants was inoculated with suspensions of

urediniospores. Spore concentrations of 0, 2x103 or 2x104 viable spores/ml were used

to obtain several levels of rust severity. Control leaves of each treatment received only

water with 0.01% Tween-20. Gas-exchange and fluorescence parameters were measured

10-12 days after inoculation, when symptoms were fully developed. The range of light

intensity during gas-exchange and fluorescence measurements was 800-1000 jPmol

photon m-2s1

The chlorophyll content and average color of control and diseased leaves were

determined. The objective of the quantification of chlorophyll and color was to verify if

these variables were related to photosynthetic rate or fluorescence parameters. If there

was such a relationship, it would be possible to use chlorophyll or color, which are easier

to assess, to make inferences about the photosynthetic status of a specific leaf. The

estimated chlorophyll content of the leaves was determined using a SPAD-502

chlorophyll meter (Minolta Co., Ltd.). The SPAD-502 measures peak chlorophyll

absorbance at 650 nm and non-chlorophyll absorbance at 940 nm. A microprocessor

calculates a SPAD value, which is proportional to the relative optical density, based on

the ratio of absorbancies of the two wavelengths. The SPAD values correspond to actual

amounts of chlorophyll present in the tissue (Appendix C); lower values of SPAD

represent less chlorophyll in the tissue and more yellowing. The area measured by the

equipment is very small (6 mm2), thus a minimum often readings were taken for each

leaf and then averaged.









The average color of the same leaves was determined using a Color Reader CR-10

(Minolta). The color reader has a built-in light source, which ensures uniform

illumination of the object. The CR-10 expresses colors numerically in the L*a*b color

space, also referred to as CIELAB. In this color space, L indicates lightness, and a and b

are chromaticity coordinates, representing the red-green direction and the yellow-blue

direction, respectively (Minolta, 1989). Six readings were taken and averaged for each

leaf. The average reading of each leaf was then compared to the average color of the

control leaves, which in this case were the healthy leaves of plants fertilized with 100%

of the recommended rate. These latter leaves had the darkest green color and the most

chlorophyll. The color difference (AEab) between any specific leaf and the average color

of the controls was calculated by using the equation AEab = (AL)2 + (a)2 + (Ab)2,

where AL, Aa, and Ab are, respectively, the differences in L, a, and b values between the

leaf color and the average color of the control leaves.

The equation Px=Po(l-x)1 was applied to relate relative photosynthetic rate (Px/Po)

to disease severity (x), where Px is the photosynthetic rate of a diseased leaf, and Po is, in

this case, the average photosynthetic rate of the healthy control leaves for each fertilizer

treatment. The 3 parameter for each fertilizer treatment was obtained by non-linear

regression, using SAS. The equation Rx=Ro(1-x)+cRox, introduced by Bastiaans and

Kropff in 1993, was used to relate dark respiration Rx to disease severity (x) at each

fertilizer level, where Ro is the rate of dark respiration of healthy leaves and a expresses

the ratio between the respiration of a lesioned area and that of an identical area of healthy









tissue. This function assumes that an increase in respiration is restricted to the visible

lesion area (Bastiaans and Kropff, 1993).

Analysis of covariance was used with a linearized form of the Bastiaans' model to

verify the significant differences among the levels of fertilizer, and also to determine if

the results of the experiments could be pooled. The non-linear estimation module of the

software package STATISTICA (release 5.1 for Windows, StatSoft, Inc.) was used to

obtain the best fitting model to describe the relationships between color or chlorophyll to

the photosynthesis variables. The evaluation of model fitness was based on the values of

the coefficients of determination and the homogeneity of distribution of the residuals.

Measurement of Chlorophyll Fluorescence during Disease Development

Bean plants were grown under 300-400 pjmol photon m-2s-1 and a photoperiod of

12 hours in a growth room. The temperature in the room ranged from 13 to 180C during

the night, and from 24 to 320C during the day. To obtain plants with different levels of

severity, suspensions of urediniospores of either of two concentrations (103 or 104 viable

spores/ml) were sprayed onto both surfaces of the first trifoliate leaf. One of the three

leaflets was protected from inoculation and was considered as a non-inoculated area of

inoculated leaves. Control plants were sprayed with water with 0.01% Tween-20.

Inoculated leaves were enclosed in clear plastic bags for 18 hours in the dark.

This experiment was conducted three times, under similar environmental

conditions. In Experiments 3-6 and 3-7, three circular areas of 2 cm in diameter, on

inoculated and on non-inoculated areas, were randomly marked on each leaf after

inoculation. These areas contained lesioned tissue, as well as green asymptomatic tissue.

Severity assessments and fluorescence measurements were performed on these marked









areas. Rust severity was determined 12-14 days after inoculation by the number of

pustules and the average lesion area. The lesion area was considered to be the area of the

sporulating pustule and the adjacent chlorotic halo. Leaves with 18-30% symptomatic

area were considered as low severity, and leaves with 65-85% symptomatic area were

considered as high severity. In Experiment 3-8, chlorophyll fluorescence was measured

in smaller areas delimited by an aluminum foil frame that exposed a circular area of 1

mm in diameter. The use of this frame made it possible to measure fluorescence

specifically on the lesioned area, as well as in areas between lesions, in leaves with low

severities. In leaves with high severity, only measurements on lesioned areas were taken,

since there were few isolated green areas in the affected tissues. In Experiment 3-8,

leaves with 30-38% symptomatic area were considered as low severity, and leaves with

65-80% were high severity.

Minimal fluorescence (Fo), maximal fluorescence (Fm), optimal quantum yield

(Fv/Fm), effective quantum yield, and electron transport rate were the parameters of

chlorophyll fluorescence measured in attached leaves, using the PAM-2000 modulated

light fluorometer (Walz, Germany). The PAM-2000 is a portable instrument that can be

used to measure in vivo fluorescence of photosynthesizing plant tissue. Modulated

chlorophyll fluorescence techniques use the repetitive application of brief saturated light

pulses in addition to the continuous actinic illumination used to drive photosynthesis

(Foyer, 1993). Minimal fluorescence, maximal fluorescence, and optimal quantum yield

were determined on leaves that were dark-adapted for 20 minutes. A modulated light

beam of very low intensity was applied to the dark-adapted leaf, which induced a weak

initial rise in fluorescence to a low level (Fo). This response is considered to be the









emission of fluorescence which occurs when all PSII reaction centers are open. A brief

strong light pulse was then added to the modulated beam to cause light saturation and to

close all reaction centers at once (Fm). Optimal quantum yield was calculated from Fo

and Fm, and it is considered as an indication of the potential photosynthetic efficiency of

a leaf. Measurements similar to the ones taken on dark-adapted leaves were then

obtained when the leaves were re-adapted to 300-400 pjmol photon m- s1, to calculate the

effective quantum yield and the electron transport rate.

The fluorescence parameters were determined on all plants before inoculation and

then at frequent intervals during a period of 14 days. For Experiment 3-7, the chlorophyll

of leaves of similar age and severity as those of the measured leaves was extracted in 80%

acetone and quantified on days 7, 10, and 14 after inoculation, according to the procedure

described by Amon (1949). The response of each fluorescence parameter over time, for

healthy plants and plants with low or high severity, was analyzed with the SAS repeated

measures procedure.


Results


Net Photosynthetic Rate, Chlorophyll Content, and Stomatal Conductance

In all three experiments, the net photosynthetic rates were reduced in leaves with

more than 5% rust severity (Table 3.1). Reductions of photosynthetic rate were

proportional to the area affected by the disease. In Experiment 3-3, leaves that had












Table 3.1. Net photosynthetic rate and total chlorophyll content for bean leaves with different levels of rust severity.

Experiment 3-1 Experiment 3-2 Experiment 3-3
Rust No. Photosynthetic No. Photosynthetic Total No. Photosynthetic Total
severity of rate of rate chlorophyll of rate chlorophyll
leaves pmol CO2 m-2S-1 leaves pmol C02 m-2-1 mg/cm2 leaves pmol C02 m-2s mg/cm2
1

0.00 5 18.490.48b 10 17.550.40 0.0230.0015d 9 13.460.55 0.0240.0012
<0.01 5 20.041.59 0 -- 2 14.070.65 0.0250.0027
0.01-0.05 6 18.450.86 7 16.991.13 0.0210.0016 4 12.060.39 0.0220.0005
0.05-0.10 0 7 14.780.97 0.0190.0024 0
0.11-0.20 4 11.693.23 9 13.181.16 0.0220.0011 8 11.910.45 0.0240.0012
0.21-0.30 7 12.540.81 8 11.50.91 0.0210.0007 3 10.460.50 0.0210.0002
0.31-0.40 0 5 9.370.65 0.0150.0017 0
0.41-0.50 2 8.930.96 3 7.260.73 0.0150.0039 0
0.51-0.70 0 0 -- 5 5.291.01 0.0110.0024
0.71-0.90 0 0 -- 5 2.370.44 0.0070.0010
a Proportion of leaf area with rust.
b Mean net photosynthetic rate standard error of the mean.
0 Chlorophyll was extracted from individual leaves and quantified using methodology described by Arnon (1949).
d Mean chlorophyll content standard error of the mean.









between 70 and 90% severity had net photosynthetic rates close to zero. The chlorophyll

content markedly decreased only in leaves with rust severity higher than 30%.

Stomatal conductance, which represents the degree of stomatal opening and is

directly proportional to the transpiration rate, also decreased with increases in disease

severity (Figure 3.1a). The ratio of intercellular CO2 to ambient CO2 (Ci/Ca), which

describes the diffusion of CO2 from the atmosphere to intercellular spaces and is

responsive to mesophyll photosynthetic capacity, slightly increased at higher severities in

all three experiments (Figure 3.1b).

Virtual Lesion Size

The values of 3, for the relationship of relative photosynthetic rate to rust severity,

ranged from 0.88 to 1.54 (Figure 3.2). These values were either not significantly different

from one or were very close to one, which is an indication that the virtual lesions and the

visual lesions were of similar sizes for this combination of rust isolate and bean cultivar.

The values of the coefficient of determination obtained with the model Px=Po(l-x)P for

the three experiments were similar or higher than the values reported in the literature for

this type of experiment (Bastiaans, 1991; Goodwin, 1992). The higher coefficient of

determination for the experiment conducted in the greenhouse (Experiment 3-3) than in

the experiments conducted outside is probably due to the better control of environmental

conditions.

Effects of Fertilization on the Photosynthetic Competence of Bean Leaves with Rust

The nutritional condition of the leaf determined its level of photosynthetic

activity. The absolute average values of net photosynthetic rate, chlorophyll content, and












































0.0 0.2 0.4 0.6 0.8 1.0
Rust severity









Figure 3.1. Effects of rust severity on bean on (a) stomatal conductance and on (b) ratio
of intercellular to ambient CO2 concentration (Ci/Ca). The regression lines for
(a) are y=0.69-0.50x (r2=0.77) for Exp. 3-1, y=0.59-0.4x (r2=0.10) for Exp. 3-2,
and 1.75-1.97x (r2=0.16) for Exp. 3-3; for (b) the equations are
y=0.86+0.06x(r2=0.54) for Exp. 3-1, y=0.79+0.16x (r2=0.33) for Exp. 3-2, and
y=0.85+0. lx (r2=0.14) for Exp. 3-3.





















1.0



0.5



0.0
S 1.5 b beta=1.54 (SE =0.14)
r2= 0.66




0

r *
o 0 .





1.5 C beta=0.88 (SE=0.07)
r2= 0.91


1.0

** S
O^ **
0.5



0.0 ...
0.0 0.2 0.4 0.6 0.8 1.0

Rust severity








Figure 3.2. Effects of rust severity (x) on the relative photosynthetic rate (Px/Po) of bean
leaves. The values of beta (3) were obtained with the model P/Po =(1-x) P;
(a) Experiment 3-1, (b) Experiment 3-2, (c) Experiment 3-3.









electron transport rate for the healthy control leaves increased with increasing rates of

fertilizer (Table 3.2). Dark respiration was higher in diseased plants than in the healthy

controls in all fertilizer treatments (Figure 3.3) in Experiment 3-5. According to the

equation proposed by Bastiaans (1993), Rx=Ro(l-x)+cRox, respiration rates in the lesions

were 4.8, 6.3, and 5.1 times higher than respiration of green leaf tissue, respectively, for

plants with 0%, 50%, and 100% of the recommended rate of fertilizer (Figure 3.3a).

Also, almost twice as much chlorophyll was lost in non-fertilized plants than in fertilized

ones in both Experiments 2-4 and 2-5, as rust severity increased (Figure 3.3b).

In both experiments, the values of 3, the parameter that describes the relationship

between relative photosynthetic rate and disease severity, were significantly lower for the

group of plants that received no fertilizer (1.66 for Exp. 3-4 and 1.14 for Experiment 3-5)

than the values obtained for the plants that received 100% of the recommended rate of

fertilizer (2.42 for Experiment 3-4 and 1.82 for Experiment 3-5) (Figure 3.4). For

Experiment 3-4, 3 values were 1.66, 2.15, and 2.42 for 0, 50%, and 100% fertilizer rates,

respectively. These values were all significantly different from one. For Experiment 3-5,

the 3 values of 1.14 for the treatment with no fertilizer and 1.13 with 50% of the fertilizer

rate were not significantly greater than one, but the 3 value of 1.82 for the treatment with

100% of the fertilizer rate was greater than one. The values of the 3 parameter different

from one were interpreted as a virtual lesion area slightly larger than the visual lesion in

these experiments.

For the combined data of Experiments 3-4 and 3-5, the apparent quantum yield of

CO2 assimilation (mol CO2 assimilated/mol of quanta absorbed) of healthy and diseased












Table 3.2. Absolute average values of net photosynthetic rate, chlorophyll content, and electron transport rate for healthy control
leaves of bean.

Experiment 3-4 Experiment 3-5

Fertilizer Photosynthetic Chlorophyll Electron Photosynthetic Chlorophyll Electron
treatment rate content transport rate rate content transport rate

Pmol CO2 m-2s- SPAD valuesb Pmol CO2 m-2' Pmol C2 m-2s- SPAD values Pmol CO2 m2s-1
1 1 1

No fertilizer 9.610.7a 31.021.7 62.265.5 8.850.3 25.920.6 72.973.5

50% fertilizer rate 13.100.4 34.130.6 72.505.2 16.340.8 31.170.7 105.344.9

100% fertilizer rate 16.731.2 38.522.2 108.255.7 18.000.8 38.000.9 133.558.7

a Mean net photosynthetic rate standard error of the mean.
b Values obtained with the Minolta Chlorophyll Meter SPAD-502; 15 readings on non-necrotic areas were taken for each leaf.

















a






0
Sv


O No fertilizer
sigma 4.79 (SE=0.43; r2 0.76)
50% fertilizer rate
sigma 6.36 (SE 0.53; r20.85)
v 100% fertilizer rate
-- sigma-5.1 (SE 0.77; r20.52)

0.0 0.2 0.4 0.6 0.8 1


b 0 No fertilizer
-- y=27.9-21.9x (r2=0.84)
50% fertilizer rate
,v v . y=32 10.9x (r2=0.28)
v v 100% fertilizer rate
SV -- y37.7 -13.4x (r2=0.39)
o 0 o .......... .... -V -


S ""



OO 0

8 o


0.0 0.2 0.4 0.6 0.8 1.0

Rust severity









Figure 3.3. Effects of rust severity on (a) dark respiration (Rx) (Experiment 3-5) and (b)
chlorophyll content of bean leaves (combined data from Experiments 3-4 and
3-5) with different nutritional status. Sigma (o) values were obtained with the
equation Rx=Ro(1-x)+oRox, where Ro is the average dark respiration of the
healthy leaves, and x is the rust severity, expressed as a proportion of the leaf
area. SPAD values are the values given by the chlorophyll meter (SPAD-
502).




















































0.0 0.2 0.4 0.6 0.8 0.0 0.2 0.4 0.6 0.8 1.0


Rust severity


Figure 3.4. Effects of rust severity (x) on the relative photosynthetic rate (Px/Po) of
bean leaves with different nutritional status; The values of beta (3) were
obtained with the model Px/Po =(l-x) P; (A) Experiment 3-4; (B)
Experiment 3-5.









leaves with the three levels of nutrition correlated well with the effective quantum yield

obtained through fluorescence measurements (Figure 3.5). Also, color differences and

estimated chlorophyll content had good relationships with both relative photosynthetic

rate and relative electron transport rate, best described by a negative exponential model

(Figures 3.6 and 3.7). The greater the difference between the color of a diseased leaf and

the average color of healthy, well fertilized leaves, the lower the photosynthetic and

electron transport rates. Higher contents of chlorophyll corresponded to higher

photosynthetic and electron transport rates.

Measurement of Chlorophyll Fluorescence during Disease Development

Experiments 3-6 and 3-7 had similar results; thus, only the results of Experiment

3-7 are presented here. The data from Experiment 3-6 are presented in Appendix D.

Relative changes in the parameters of chlorophyll fluorescence were first observed during

the fleck stage, 5 to 7 days after inoculation, for the group of leaves that later had 65-85%

of rust severity (Figures 3.8 and 3.9). All the fluorescence parameters were reduced in

the presence of high rust severity. Fourteen days after inoculation, when disease

symptoms were very evident, the values of minimal fluorescence (Fo), maximal

fluorescence (Fm), optimal quantum yield (F,/Fm), effective quantum yield, and electron

transport rate were, respectively, 69%, 39%, 74%, 57%, and 56% of the average values of

these parameters in healthy control leaves.

The average chlorophyll content of the leaves with high severity (65-85%

severity) was 54% lower than the content of healthy control leaves at the fleck stage

(Figure 3.10). At 14 days after inoculation, when chlorotic halos surrounding the






























0.025


0.020


0.015


0.010


0.005


0.000


-0.005


0.1 0.2 0.3 0.4

Effective quantum yield (relative units)


Figure 3.5. Relationship between effective quantum yield of photosystem II and quantum
yield of CO2 assimilation in bean plants with different levels of rust severity
and nutritional status.


v
O nofertilizer
* 50% fertilizer rate
v 100% fertilizer rate V
Sy= -0.0032+0.047x (r2=0.6) V

.




0 0 v




0
0



















10 1.2 v 50% fertilizer rate
v v 100% fertilizer rate
V
0 -- y= 1.065exp(-0.059x)


c *


0.6 0 "0, o o
Sv vo o .
.v o
0.3 % o_ '0 o

a o o
0 o

0.0 -0
0 nofertilizer
1.2 -7 50% fertilizer rate
S v ,v 100% fertilizer rate
0 -- y=0.989exp(-0.038x)
D v r2=0.68
0.9 -


_0.0- v o Co
0. a- v0 0


0,3 o o


ai b o
o.o ----, ,-
0 10 20 30 40

Units of color difference












Figure 3.6. Responses of (a) relative photosynthetic rate and (b) relative electron
transport rate to units of color difference, at the different levels of fertilizer.
Both rates are expressed as fractions of the average value of all healthy bean
leaves at the recommended fertilizer rate of 100%. Data of Experiments 3-4
and 3-5 were combined in these graphs.


























































0.6


0.9


Relative chlorophyll content


Figure 3.7.


Responses of (a) relative photosynthetic rate and (b) relative electron
transport rate to relative chlorophyll content (SPAD values), at the different
levels of fertilizer. Both rates and the values of SPAD are expressed as
fractions of the average value of all healthy bean leaves at the recommended
fertilizer rate of 100%. Data of Experiments 3-4 and 3-5 were combined in
these graphs.


S 1.2


o
r-
. 0.9


0.6
0
r--



0.3
| 0.-0

7T 0.0


0.0 -
0.0


o nofertilizer
* 50% fertilizer rate
v 100% fertilizer rate
Sy=0.082*exp(2.404x)



0
O5'


o oI
**
. .w


000
r 0 o


a



o nofertilizer
50% fertilizer rate
v 100% fertilizer rate
- y=0.163*exp(1.717x) v v/
v 'v /v



0. VVV

*
0



go
0 b

cb 0


-LaD r








43









0.25 0.25


0.20 0.20

0.15 0.15

0.10 0.10

-- control
0.05 lowseverity 0.05
high severity
0.00 0.00
c
1.0 1.0

0.8 0.8


E 0.6 0.6

0.4 0.4

0.2 0.2

0.0 0.0
0.9 e 0.9


0.8 0.8


L 0.7 0.7


0.6 0.6


0.5 0.5
0 2 4 6 8 10 12 14 16 0 2 4 6 8 10 12 14 16

Days after inoculation Days after inoculation










Figure 3.8. Minimal fluorescence (Fo), maximal fluorescence (Fm), and optimal quantum
yield (Fv/Fm) during rust development, in inoculated (a, c, e) and non-
inoculated (b, d, f) areas of bean leaves with different rust severities. The
fluorescence parameters are expressed in relative units. The percentage of area
affected by rust was 18-30% in leaves with low severity, and 65-85% in leaves
with high severity.






























a 0.7

S 0.6


-- control
- low severity
-- high severity


0 2 4 6 8 10 12 14 16

Days after inoculation


d


0 2 4 6 8 10 12 14 16

Days after inoculation


Effective quantum yield, in relative units, and electron transport rate

(jmol m2s-1) during rust development, in inoculated (a, c) and non-
inoculated (b, d) areas of bean leaves with different rust severities. The
percentage of area affected by rust was 18-30% in leaves with low severity,
and 65-85% in leaves with high severity.


0.7

O) 0.6

E 0.5


CT
c~ 0.4

oD 0.3

t 0.2
1,
w 0.1

0.0
120
(D
o100

O 80

0 60
4-'
O 40



0


Figure 3.9.

























0.05
healthy control
i low s ever ity
0.04 l high severity
E


0.03

07
0 0.02


O 0.01 -



0.00 -
day 7 dayl0 dayl4
Days after inoculation













Figure 3.10. Chlorophyll content of bean leaves with different rust severities during
disease development. The percentage of area affected by rust was 18-30% in
leaves with low severity and 65-85% in leaves with high severity.









pustules were well developed, the chlorophyll content of those leaves was around 25% of

the control.

Leaves with 18-30% severity did not differ significantly from the healthy control

leaves in any of the measured parameters during disease development. Rust development

in symptomatic areas of inoculated leaflets did not interfere with the fluorescence

parameters of the non-inoculated leaflet of the same leaf (Figures 3.8 and 3.9). The

noticeable peaks in the graphs for minimal and maximal fluorescence, on day 8 after

inoculation, occurred one day after the apical portion of all plants was removed to avoid

shading of the leaves of interest. Whatever the reason for this sudden rise in fluorescence,

it probably did not interfere with the effects of rust on leaf photosynthesis, as it occurred

for all three groups of plants, on both inoculated and non-inoculated areas. On the

following day, fluorescence emission returned to the levels observed prior to the removal

of the apical portion.

For Experiment 3-8, where fluorescence was measured on the lesioned area and

areas between lesions in leaves with low severity, maximal fluorescence and optimal

quantum yield were reduced inside the lesions, beginning at 10-11 days after inoculation

(Figure 3.11). Effective quantum yield and electron transport in the chlorotic areas of

leaves with low severity were reduced on day 11, but were not significantly different from

the other treatments on day 12. Fluorescence in the green area between lesions on leaves

with low severity remained unaffected when compared to the healthy control leaves. All

fluorescence parameters were reduced on leaves with high severity when compared to the

healthy control group. Minimal fluorescence on leaves with high


















0.04



0.03



LL 0.02



0.01



0.00
5 6 7 8 9 10 11 12


0.8

E
'> 0.7
LU-


0.12


0.04


0.00
5 6 7 8 9 10 11 12 13


S0.8
(D

E 0.6


DT 0.4
(D

(D 0.2
wL


5 6 7 8 9 10 11 12 13

125


5 6 7 8 9 10 11 12 13


loo
100

0
75


c 50
o -*- control
t lowseverity/green
- 25 lowseverity/chlorotic
high severity
0
5 6 7 8 9 10 11 12 13

Days after inoculation


Figure 3.11. Minimal fluorescence (Fo), maximal fluorescence (Fm), optimal quantum
yield (Fv/Fm), effective quantum yield, and electron transport rate during rust

development in healthy control and inoculated bean leaves with different rust
severities. In leaves with low severity (30-38%), fluorescence was measured
in green areas (between lesions) and chlorotic areas (inside the lesions). The

percentage of area affected by rust was 65-80% in leaves with high severity.









severity was significantly lower than the control leaves on days 10 to 12. Maximal

fluorescence and optimal quantum yield were significantly reduced from the onset of the

fleck stage, 6 days after inoculation. Effective quantum yield and electron transport rate

on the same leaves were significantly reduced on days 8 to 12.

Discussion


The effects of rust on the photosynthetic competence of common bean have been

described by various researchers. Decreased photosynthetic rates (Livne, 1964; Raggi,

1980), increased respiration (Daly et al., 1961; Livne, 1964; Raggi, 1980), changes in the

patterns of translocation of photosynthates (Livne and Daly, 1966), and increase in

invertase activity (Wagner and Boyle, 1995) were the main effects observed during

infection by the rust pathogen. Also, Moll et al. (1995) reported loss of chlorophyll in the

pustule area and diminished chlorophyll fluorescence during rust development, which

indicated reduced photosynthetic activity. In most of these studies, the effects of rust on

the physiology of the bean plant were investigated using only heavily infected leaves.

Rates of photosynthesis, respiration, and loss of chlorophyll are dependent upon infection

density (Scholes, 1992). Thus, the quantification of disease effects on plant physiology

must take the density of infections into consideration.

A virtual lesion consists of a visual lesion and an adjacent area, in which the

photosynthetic activity is zero. This concept was introduced by Bastiaans (1991), as a

basis for a model that quantifies photosynthetic competence of diseased leaves. The

relation between disease severity and photosynthesis is described with a single parameter,

3, the ratio of virtual and visual lesions. Values of 3 greater than one are interpreted as









an indication that the disease not only reduced the leaf area capable of photosynthetic

activity, but also affected the photosynthesis in the remaining green leaf tissue. For some

pathosystems involving necrotrophic organisms, the virtual lesion could result from the

production and diffusion of toxins in the surrounding area of the symptomatic tissue

(Bastiaans, 1991). However, diffusible toxins have not been associated with infections by

biotrophic pathogens (Gay, 1984), and thus, biochemical studies complementary to

photosynthetic measurements are needed to clarify the phenomenon.


The values of 3 reported for diseases caused by obligate parasites are 1.26 0.17

for Puccinia recondita on winter wheat (Bastiaans, 1991, based on data from Spitters et

al., 1990), and 8.74 1.70 for Blumeria graminis on winter wheat (Bastiaans, 1991,

based on data from Rabbinge et al., 1985). The reported 3 values for facultative parasites

are 3.04 0.18 and 3.74 0.19 for Pyricularia oryzae on rice (Bastiaans, 1991); 2.68

0.22 for Xanthomonas campestris pv. phaseoli on common bean (Goodwin, 1992); 7.24 +

0.75 for Colletotrichum lindemuthianum on common bean (Bassanezi et al., 1997); 11.0

+ 3.5 for Cercospora leafspot of peanut (van der Werf et al., 1990, based on data from

Boote et al., 1980); 2.1 0.61 for Rhynchosporium secalis on barley (van der Werf, 1990,

based on data from (Martin, 1986); and 1.66 0.34 for Septoria nodorum on wheat (van

der Werf, 1990, based on data from Rooney, 1989). In the present study, most of the 3

values obtained were not different from one; the remaining values were between one and

two (Figures 3.2 and 3.4). These values of 3 are similar to the value of 1.26 0.16

obtained for brown rust of wheat (Spitters et al., 1990), the only other reported value for a

pathosystem involving a rust pathogen. The conclusion, in the present study, was that









there was little effect of rust infection on the remaining green area of leaves of 'Rosinha'

bean, but some environmental factors may cause variation in this response. Light and

temperature conditions during plant growth and symptom development, and the

nutritional status of the plant are factors that may have contributed to the observed

variation between experimental outcomes.

The determination of the 3 value, using the equation Px/Po = (1-severity)P,

depends on (i) the reliability of the estimates of the average photosynthetic rate of healthy

control plants (Po); (ii) the photosynthetic rates of diseased plants (Px) with a wide range

of severities; and (iii) the proportion of leaf area affected by the disease. Some aspects

related to the physiology of the plant that should be taken into consideration when

determining 3 values include: (a) photosynthetic rates should be determined at light

saturation to ensure that the maximum rate (Pmax) is obtained (Bastiaans, 1991); (b) if

possible, leaves of similar physiological age or position in the canopy should be selected

for the measurements (Goodwin, 1992; Shtienberg, 1992); and (c) the environmental

conditions in which the measurements are taken should not be very different from the

conditions under which the plants were grown (Daly, 1976). The aspects listed above are

suggested to reduce the variation in the values of photosynthetic rate obtained, as an

intrinsic variation among leaves is expected.

The accurate assessment of the proportion of leaf area affected and the time of this

assessment in relation to symptom development can also influence the 3 values obtained.

For diseases that do not have clearly confined symptoms, it is certainly more difficult to

assess severity accurately. The accuracy of disease assessment should be taken into

consideration when interpreting the 3 values.









Assessments of severity at early stages of symptom development may result in a

different 3 value than the one obtained when symptoms are fully advanced. In a

preliminary experiment (results not shown), the 3 value, determined for bean leaves

inoculated with rust showing fleck symptoms, was 4.7 0.77 (r2=0.49). The maximum

severity estimated at that stage was 5%. Four days later, when the symptoms were well

developed, with chlorotic halos surrounding the sporulating pustules, the maximum

severity was estimated at 63%, and the 3 value was 1.27 0.07 (r2=0.95).

Chlorophyll fluorescence, obtained in vivo with a fluorometer, has been called, in

the last decade, the "plant physiologist's stethoscope" because it measures the efficiency

of the photosynthetic activity, and thus can provide approximate estimates of the vitality

and vigor of a plant in its environment (Bolhar-Nordenkampf and Oquist, 1993; Foyer,

1993; Hall and Rao, 1994). Measurements of chlorophyll fluorescence have been used

widely, not only in routine studies on photosynthesis, but also in other related areas, such

as studies on environmental stresses, screening for stress tolerance in plant breeding, and

studies on herbicide and air pollution (Foyer, 1993). The non-invasiveness, speed of data

acquisition, and high sensitivity are often cited as outstanding advantages of this

technique (Foyer, 1993; Schreiber et al., 1998). Plant diseases certainly can be

considered stressful to the physiology of the plant, and, thus, chlorophyll fluorescence

may provide valuable information on the photosynthetic competence of diseased leaves.

Chlorophyll fluorescence reflects the efficiency of light utilization on photosystem

II (PSII). It was shown that there is an ubiquitous curvilinear or biphasic relation between

the effective quantum yield of fluorescence and the apparent quantum yield of CO2

assimilation (mol C02/mol quanta) (Comic and Briantais, 1991; Genty et al., 1989;









Seaton and Walker, 1990). This relationship may allow good estimates of photosynthetic

rate through measurements of fluorescence, without recourse to analysis of gaseous

exchange between leaf and environment. Inhibition of primary photochemistry, electron

transport, or carbon metabolism caused by environmental stresses will affect the function

of PSII and will be expressed as changes in fluorescence yield. These changes in

fluorescence yield can provide quantitative information about plant responses to the

duration and intensity of the stress (Bolhar-Nordenkampf and Oquist, 1993).

Minimal fluorescence (Fo) represents the emission from molecules of chlorophyll

a on the light harvesting antenna associated with PSII, prior to the excitation energy being

used for electron transport (Krause and Weis, 1984). It is therefore independent of

photochemical events, but dependent upon the chlorophyll content of the tissue and on

the ultrastructure of the thylakoid membrane. There was a slight increase in Fo on the

bean leaves infected by the rust pathogen during the fleck stage, but it later decreased

during sporulation. Similar results were reported by (Scholes and Farrar, 1985), for

measurements taken on bluebell leaves infected with rust, caused by Uromyces muscari.

These authors proposed two hypotheses to explain the temporary increase in Fo before

sporulation. It could be due to an inactivation of some PSII reaction centers, caused by a

reducing compound produced transiently by the pathogen; or it could be caused by a

disorientation of a proportion of the chlorophyll a molecules within the thylakoid

membrane during colonization of the tissue by the pathogen. The subsequent decrease in

Fo may be explained by the loss of chlorophyll from the tissue, which was very

pronounced at the onset of sporulation (Figure 3.10).









Maximal fluorescence (Fm) is a measure of the oxidation-reduction status of the

electron acceptors between PSII and PSI, and is a direct indicator of PSI activity (Scholes

and Farrar, 1985). Environmental stresses that cause thylakoid damage, such as heat and

freezing stresses, usually lower Fm. The Fm values of leaves with high rust severity were

reduced beginning at fleck stage. After sporulation, these values were less than 40% of

the control values (Figure 2.8c). In leaves with lower rust severity, Fm was reduced only

in the chlorotic areas, but not in areas between lesions (Figure 3.1 lb). Maximal

fluorescence was also reduced in plants infected by other pathogens, such as Uromyces

muscari on bluebell (Scholes and Farrar, 1985), Taphrina deformans on peach (Raggi,

1995), and tobacco mosaic virus on tobacco (Balachandran and Osmond, 1994).

The optimal quantum yield (Fv/Fm) provides information on the potential

photosynthetic efficiency of PSII, and it is often discussed as a vitality index (Moll et al.,

1995). It has a range of 0.75 to 0.85 for healthy leaves of many plant species and

ecotypes, and decreases under stress (Bolhar-Nordenkampf and Oquist, 1993). In this

study, there was a reduction in Fv/Fm on bean leaves with high rust severity (Figure 3.8e)

and on the lesioned areas of leaves with low severity (Figure 3.11 c), largely due to the

decrease in Fm values. These results are in accordance to the findings of (Moll et al.,

1995), with the same pathosystem.

Effective quantum yield is the quantum yield of the non-cyclic electron flow

(Foyer, 1993; Genty et al., 1989). It is proportional to the concentration of the open PSII

reaction centers and to the efficiency with which these centers capture and use excitation

energy. It is the basis to calculate the electron transport rate. Both effective quantum

yield and electron transport rate suffered similar reductions on highly diseased leaves, but









these variables were not affected outside the area of the pustules in leaves with lower

severities (Figures 3.8 and 3.11d and 3.11e).

Moll et al. (1995) suggested that the diminished efficiency of use of the excitation

energy may be due to ultrastructural changes within the chloroplasts. Another hypothesis

to explain the depression in photochemical efficiency is the down-regulation of PSII

photochemistry, as a consequence of a reduced demand for NADPH and ATP in the

chloroplasts, resulting from inhibition of the Calvin cycle (Krupa et al., 1993).

On bean leaves with high rust severity, reductions in the fluorescence parameters

were first noticeable at the fleck stage (5 to 7 days after inoculation). Similarly,

alterations in photosynthetic rate and dark respiration were not noticed before the fleck

stage for this same pathosystem (Daly et al., 1961; Livne, 1964; Raggi, 1980). However,

(Peterson and Aylor, 1995), through chlorophyll fluorescence imaging, were able to

observe enhanced fluorescence emission 3 days after inoculation of bean leaves with rust,

when symptoms were not visible. This observation was interpreted as an early sign of

alteration in light utilization for photosynthesis.

A reduction in the Fv/Fm ratio is considered symptomatic of a phenomenon called

photoinhibition, which is a light-dependent inhibition of photosynthesis (Baker, 1993;

Foyer, 1993; Krause, 1988). When a healthy, non-stressed plant is exposed to light levels

considerably higher than the light conditions experienced during growth, this plant will

face an excess of absorbed excitation energy. The thylakoids have a mechanism to

increase the rate of dissipation of excitation energy as heat, when the rate of electron

transport cannot meet the rate of excitation of the reaction centers. The end-result of this









process is an inhibition of photosynthesis, as a consequence of a decreased efficiency of

light utilization with the increase in light intensity.

Stressed plants will face the same problems described above, but at lower light

intensities (Baker, 1993; Bolhar-Nordenkampf and Oquist, 1993; Osmond, 1994).

Photoinhibition is thus considered in the study of stress physiology, as a secondary stress

response, usually triggered by other stress factors that reduce the ability of the plant to

assimilate CO2. The accumulation of the end-products of the light-reactions of

photosynthesis (ATP and reductants) will result in an inhibition of electron transport.

Under such conditions, damage to the reaction-center proteins can occur if the excess of

excitation energy in the pigment antenna is not successfully dissipated. Osmond (1994)

called this condition chronic photoinhibition, which can have two possible outcomes:

photon protection or photon damage. Photon protection will be the result of a decrease in

the efficiency of photosynthesis by means of dissipating the excess of photons as heat,

before these photons can reach the reaction centers. A decline in dark-adapted Fv/Fm

values, accompanied by a decline in Fo is considered diagnostic of mechanisms that

engage photon protection, and result in little photon damage. Photon damage occurs

when the photoprotective capacity is exceeded, and the excess of photons irreversibly

degrades certain components of the PSII reaction centers. In this case, Fv/Fm declines, but

Fo increases.

Balachandran and Osmond (1994) reported that chlorotic tissue of expanding

leaves of tobacco infected with TMV were chronically photoinhibited (low Fv/Fm, high

Fo). These authors concluded that, in tissue infected with viral particles, photoinhibitory

damage was occurring as a result of illumination of chloroplasts having impaired PSII.









The photon damage then led to photooxidation of chlorophyll and, thus, to patchy

chlorosis. From the results with bean rust in the present study, it is concluded that the

reduced carbon assimilation resulting from the infection induced chronic photoinhibition

on bean leaves. The F,/Fm ratio was clearly reduced in severely diseased leaves, and in

chlorotic areas of leaves with low severity, in comparison to the control leaves (Figures

3.8e and 3.1 Ic). The transient increase in Fo during the fleck stage and its subsequent

decrease during sporulation (Figure 3.8a) was interpreted as both photon damage and

photon protection may occur in this system under moderate light conditions (400 Pjmol

photon m-2 S-1).

Low availability of essential mineral elements for growth impairs the

photosynthetic capacity of a plant. The question in the present study was whether bean

rust would have a greater impact on the photosynthesis of non-fertilized plants than on

well-fertilized plants. The answer was that increases in disease severity did not cause

greater reductions on the photosynthetic rates of plants that received no fertilizer, when

compared to the reductions on plants with 100% of fertilizer. In fact, the opposite was

true in the present experiments. The photosynthetic rates of healthy control plants that

received no fertilizer were 43-51% lower than the rates of well-fertilized control plants.

Although the healthy control plants of the three fertilizer treatments initially had different

photosynthetic rates, all plants with 90-100% rust severity, independently of their

nutritional status, would have rates of photosynthesis close to zero or negative values,

according to the regressions performed. Consequently, leaves of well fertilized plants

lost more efficiency per unit area than leaves of non-fertilized plants, when both had the

same area affected by disease. The impairment of photosynthesis in areas with higher









relative efficiency was the cause for the larger proportional reductions of photosynthetic

rates on plants with 100% fertilizer, and their slightly higher 3 values (Figure 3.4).

Physiological processes other than photosynthesis, but closely related to it, were

also affected by rust infection. In the present study, decreased stomatal conductance,

increased respiration, and losses of chlorophyll from leaf tissue were observed in

response to increases in rust severity.

Stomatal functioning is believed to be integrated with photosynthesis in such a

way that it optimizes the use of water while only marginally limiting the photosynthetic

process (Farquhar and Sharkey, 1982). The stomatal conductance of bean leaves

decreased with their reduced photosynthetic capacity, as rust severities increased (Figure

3.2a). This positive linear relationship between conductance and carbon assimilation was

also observed for leaves with various nutritional and water status, age, and levels of viral

infection (Hall and Loomis, 1972; Schulze and Hall, 1982; Wong et al., 1979). The

simultaneous reduction of carbon assimilation and transpiration rate is generally caused

by two different mechanisms, one based on an increase in carboxylation resistance, and a

second based on an increase in stomatal resistance (Farquhar and Sharkey, 1982;

Rabbinge et al., 1985). Carboxylation resistance is the resistance of the mesophyll to CO2

diffusion. Stomatal resistance, which is the inverse of stomatal conductance, represents

the degree of stomatal closure. If stomatal closure were the cause of the reduced

assimilation, a reduction in the intercellular CO2 would be observed. The slight increase

in the Ci/Ca ratio is an indication that the flow of CO2 from the stomatal cavities to the

carboxylation sites was not affected (Figure 3.2b). Consequently, it is possible to

conclude that mesophyll resistance to carboxylation increased in diseased leaves.









Increased rates of dark respiration in bean leaves with rust are in accordance with

previous reports on respiratory changes for this pathosystem (Daly et al., 1961; Raggi,

1980). The higher rates of respiration reported were observed during sporulation, but

were believed to be related mostly to host respiration (Daly et al., 1961). The

contribution of fungal respiration to the increases in the rate of respiration was considered

negligible.

The rate of respiration in the light is comparable to the rate of respiration in the

dark (Azc6n-Bieto and Osmond, 1983). Respiration rate contributes significantly to the

total CO2 exchange in illuminated leaves. Increased respiration was partially responsible

for the reduced photosynthetic rates observed in bean leaves in the present study, because

the rate obtained by gas-exchange measurements was a net photosynthetic rate, which

included CO2 effluxes and refixation, besides the CO2 assimilation by the leaf tissue.

Rates of dark respiration inside the lesions were determined to be five to six times higher

than in non-infected green tissue (Figure 3.3a), according to Bastiaans' equation

(Bastiaans and Kropff, 1993). Possible mechanisms of increased respiration in tissues

infected by biotrophic pathogens include: (a) wound respiration due to regenerative

activity of tissue physically damaged by the invading pathogen; (b) enhanced

consumption of ATP and reductants through increased biosynthesis; and (c) increased

levels of starch and soluble sugars that accumulate in the cells as a result of blocked

translocation (Hutcheson and Buchanan, 1983; Smedegaard-Petersen, 1984).

The percentage of losses of chlorophyll from diseased leaves in relation to control

leaves had similar values to the proportion of the tissue visually chlorotic, which was the

basis for assessments of severity. This was true for all experiments (Table 3.1, Figure









3.3b, Figure 3.10), independent of the methodology used to assess chlorophyll content of

the tissue. However, Moll et al. (1995) reported that chlorotic areas in bean leaves with

rust still retained a certain amount of chlorophyll, and that the pigment was also reduced

in green areas between lesions, in comparison to non-infected control leaves. Leaf

chlorophyll content had a good relationship with net photosynthetic rates and electron

transport rates in bean leaves with different nutritional status and rust severities (Figure

3.7). This is in accordance with reports in which the leaf chlorophyll content is often well

correlated with leaf nitrogen status, Rubisco activity, and photosynthetic activity (Evans,

1983; Seeman et al., 1987; Thompson et al., 1996). Also, chlorophyll is believed to be a

sensitive indicator of many types of plant stress. Leaf color, which is largely dependent

upon chlorophyll content, was also a good indicator of the photosynthetic status of bean

leaves. When expressed as the difference in color from a healthy control standard,

relative leaf color was strongly related to photosynthetic rates and electron transport rates

of healthy and diseased bean leaves (Figure 3.6).

The relationships obtained between leaf color, chlorophyll content, and the

photosynthetic parameters were obtained for healthy and rust-infected leaves, with

different levels of nutrition. Thus, if the relationships found here could be validated in

field experiments, estimates of photosynthetic activity of plants with rust could be done

on larger scales. A few healthy, well-fertilized plants, growing in the same conditions as

the plants of interest, could be used as healthy control standards. The instruments used to

determine leaf color and chlorophyll content of the leaves, respectively the Color Reader

CR-10 and the SPAD-502 Chlorophyll Meter, are portable, easy to operate, and relatively









inexpensive when compared to the equipment necessary to measure gas-exchange or

fluorescence.

The photosynthetic competence of bean leaves infected with rust was reduced,

beginning at fleck stage, when assessed under controlled conditions. Loss of chlorophyll

and higher respiration rates in the diseased tissue are believed to be the main factors

determining this reduction. The reduced rates of photosynthesis observed were

proportional to the proportion of leaf tissue with visual symptoms, for most of the

experiments in the present study. However, in a few cases, the reduction was slightly

higher than what could be explained by rust severity. It was concluded that there is

probably very little interference of bean rust in the efficiency with which the intercepted

radiation is utilized by the plant. Thus, it would not be necessary to correct severity

assessments based on visual symptoms to have accurate assessments of HAD or HAA. It

should be noticed, though, that these conclusions need to be verified for other

combinations of bean cultivar and pathogen race.

The quantitative determination of the effects of a disease on the physiology of

individual leaves is the first step towards a broader understanding of crop losses

(Bastiaans et al., 1994). The future steps would be to determine the effects of disease on

the physiology of whole plants and, then, to integrate this information on plant

performance over an entire season.















CHAPTER 4
THE PHOTOSYNTHETIC COMPETENCE OF BEAN LEAVES WITH
ANTHRACNOSE


Introduction


Severe epidemics of bean anthracnose, caused by Colletotrichum lindemuthianum

(Sacc. and Magnus) Briosi. and Cav., usually occur when contaminated seeds are planted

in locations with cool to moderate temperatures, frequent rains, and high humidity, as in

temperate and subtropical zones and high altitudes in the tropics (Tu, 1992). Reported

yield losses due to bean anthracnose were up to 100% for highly susceptible cultivars and

between 27 and 88% for moderately susceptible cultivars (Guzman et al., 1979; Shao and

Teri, 1985). Serious losses occurred when bean plants became infected in the first 5

weeks of development (Guzman et al., 1979).

In the past, the control of bean anthracnose relied heavily on race-specific

resistance(Tu, 1992). However, C. lindemuthianum has high pathogenic variability and

new races of the pathogen are reported frequently (Menezes and Dianese, 1988). Thus,

integrated disease management is considered the most effective approach to minimize the

yield losses to anthracnose. The control measures in an integrated disease management

program include use of non-infected seeds, race non-specific resistance, and chemical

treatment. Reliable estimates of yield losses are essential to assess the success of any

disease management program. The reliability of estimates of yield loss due to a disease

depends on the understanding of the epidemiological variables that intensify the progress









of disease and on accurate assessment of the impact of the disease on crop performance

(Bergamin and Amorim, 1996; Berger et al., 1995).

Waggoner and Berger (1987) proposed that healthy leaf area duration (HAD) and

healthy leaf area absorption (HAA) were much better predictors of yield compared to

disease intensity as a predictor of yield loss, since HAD and HAA add biological realism

and flexibility to the empirical approaches. The concepts of HAD and HAA proved valid

for the yield loss assessment of many different pathosystems, such as Phytophthora

infestans on potato (Haverkort and Bicamumpaka, 1986); Rotem et al., 1983a and 1983b;

van Oijen, 1990), Alternaria solani on potato (Johnson and Teng, 1990), Aschochyta

fabae on Viciafaba (Madeira et al., 1988), Pyricularia oryzae on rice (Pinnschmidt and

Teng, 1993), Blumeria graminis on wheat (Daamen and Jorritsma, 1990), and

Phaeoisariopsis griseola on common bean (Bergamin et al., 1997). The yield of bean

plants affected by anthracnose, expressed in number of pods per plant or grams of seeds

per plant, was positively correlated to HAD and HAA, but yield had no significant

relationship with the area under the disease progress curve (AUDPC) (Nunes and

Bergamin, 1996). The AUDPC has been considered as the best measure of the intensity

of an epidemic (Campbell and Madden, 1990).

In addition to affecting the amount of intercepted radiation, some foliar pathogens

can affect the efficiency of radiation use by the plant (Bastiaans, 1991; Bastiaans et al.,

1994; Boote et al., 1980; Bourgeois and Boote, 1992; Johnson, 1987). In the reported

cases, the assessment of HAD and HAA can be less than accurate if the effects of the

disease on the healthy area are not considered (Johnson, 1987). The determination of

radiation use efficiency of diseased plants has been done under field conditions, from the









slope of the line that relates yield to HAA, in plots with different disease intensities. If

the slopes are constant for the different situations, the radiation use efficiency is

considered to be unaffected by the disease (Aquino et al., 1992; Waggoner and Berger,

1987). However, some researchers believe that only direct measurements of

photosynthetic rates in healthy and diseased plants can show whether the photosynthetic

activity of the green leaf area is being affected by disease (Bastiaans, 1991; Boote et al.,

1983; Rabbinge and Rijsdijk, 1981; Rabbinge et al., 1985; van Oijen 1990; van

Roermund and Spitters 1990). Thus, the present study focused on the quantification of

the effects of anthracnose on the photosynthesis of leaves of common bean (Phaseolus

vulgaris) to verify whether assessments of HAA and HAD for bean plants with

anthracnose should take into account any effects of the disease on radiation use

efficiency.

Materials and Methods


Plant Material and Inoculum

Bean plants (Phaseolus vulgaris) of the susceptible cultivar Rosinha were used in

all experiments. The plants were grown, one plant per pot, in 4-liter pots filled with

Metromix, and watered daily to field capacity. The plants were fertilized every other day

with Peter's fertilizer (20-20-20, 1 g/1 of water). The growing point of each plant was

removed above the fourth or fifth leaf, to restrict the indeterminate growth of the cultivar

and to facilitate the handling of the plants.

Conidia of C. lindemuthianum, race kappa, were produced in Mathur's C

modified medium (Balardin et al., 1997). A suspension of conidia was spread on the









surface of the medium to obtain maximum sporulation. Ten- to fourteen-day-old cultures

were rinsed with sterile distilled water to prepare the concentrated spore suspension,

which was then diluted to obtain the desired concentrations. The viability of the conidia

was determined prior to the preparation of the suspensions. A diluted suspension of

conidia was spread on the surface of water-agar medium, and the percentage of

germinated conidia was determined after 12 hours of incubation.

Measurement of Gas-Exchange on Bean Leaves with various Levels of Anthracnose
Severity

The experiment was conducted twice (Experiments 4-1 and 4-2) with bean plants

grown under greenhouse conditions. The temperature ranges in the greenhouse, 18-21C

during the night and 24-35C during the day, were similar for both experiments. In

Experiment 4-1, the conidial suspensions used in the inoculation had 104, 5 x 104, 105, or

5 x 105 viable spores/ml. These suspensions were sprayed onto both surfaces of first

trifoliate leaves. Control plants were sprayed with sterile distilled water. The plants were

then enclosed in plastic bags and transferred to a growth room at 210C, in which they

were maintained for 30 hours, and then returned the greenhouse. For Experiment 4-2, the

third trifoliate leaf was inoculated and the plants were maintained in the greenhouse,

enclosed in plastic bags for 16 hours during the night and early morning.

Gas-exchange measurements were taken 6 and 7 days after inoculation, when

symptoms were well developed. The net photosynthetic rate at light saturation and the

stomatal conductance of healthy and infected leaves were determined using the LI-6200

Portable Photosynthesis System (LI-COR Inc.). Measurements were taken at a range of

light intensity of 700 to 1000 Cmol photon m-2 s-1 with the method described in Chapter
light intensity of 700 to 1000 jamol photon m- s1 with the method described in Chapter









3. For Experiment 4-1, the severity of the measured leaves was estimated using a

diagrammatic scale of anthracnose symptoms (Godoy et al., 1997). For Experiment 4-2,

the necrotic lesions of each leaf were traced onto transparent plastic; the plastic, as well

as the actual leaf from where the lesions were traced, were then run through an area meter

(LI-3000 Portable Area Meter, LI-COR). The severity was calculated as the proportion of

leaf area with lesions.

In Experiment 4-2, other variables were also determined in healthy and diseased

leaves. The electron transport rate and effective quantum yield were determined under

the same conditions as the photosynthetic rate with the PAM-2000 fluorometer (Walz,

Germany). After the plants were adapted for 30 minutes in the dark, the rate of dark

respiration was determined with the LI-6200.

After the determination of severity on the detached leaves was concluded, the

chlorophyll content of the leaves was assessed with the SPAD-502 Chlorophyll Meter

(Minolta Co., Ltd.). Fifteen readings were taken and then averaged for each leaf. The

readings were taken on non-necrotic areas of the diseased leaves. The average leaf color

was also determined on non-necrotic areas, with the CR-10 Color Reader (Minolta), and

then expressed as a value of color difference in relation to the average color of the healthy

leaves. The objective of the quantification of chlorophyll and color was to verify if these

variables were related to photosynthetic rate or fluorescence parameters. If there is such a

relationship, it may be possible to use chlorophyll or color to make inferences about the

photosynthetic status of healthy and diseased leaves.

The equation P/Po=(1-x)1 was used to relate relative photosynthetic rate (Po/Po) to

disease severity (x), where 3 represents the ratio between virtual and visual lesion sizes









(Bastiaans, 1991). Relative photosynthetic rate was the ratio of the photosynthetic rate of

a leaf to the average rate of the healthy control leaves. The parameter 3 was obtained by

non-linear regression, using the procedure PROC NLIN (method DUD) of SAS (SAS

Institute, Cary, NC; release 6.12 for personal computers). The equation R,-Ro(1-x) +

oRox, introduced by Bastiaans and Kropff in 1993, was used to relate dark respiration Rx

to disease severity (x), where Ro is the rate of dark respiration of healthy leaves and a

expresses the ratio between the respiration of a lesion and that of an identical area of

healthy tissue. This function assumes that an increase in respiration is restricted to the

visible lesion area (Bastiaans and Kropff, 1993).

Effects of Fertilization on the Photosynthetic Competence of Bean Leaves with
Anthracnose

Bean plants growing under greenhouse conditions received the following

fertilization treatments, every other day: (a) no fertilizer, (b) half of the recommended

dosage of Peter's fertilizer for daily fertilization (0.5 g/1 of water), or (c) full

recommended dosage of the fertilizer (1 g/1). The experiment was conducted twice

(Experiments 4-3 and 4-4). Temperatures inside the greenhouse ranged from 25 to 300C

during the day and from 18 to 20 C, during the night, for both experiments.

The expanded third trifoliate leaf in all plants was inoculated with suspensions of

conidia, sprayed onto both leaf surfaces. Two suspensions with different concentrations

(5 x 103 or 5 x 104 viable conidia/ml) were used to obtain levels of anthracnose severity.

Control leaves of each treatment were sprayed with sterile distilled water. All plants were

enclosed in plastic bags for 16 hours during the night and early morning. Measurements

were taken 6 and 7 days after inoculation, when symptoms were fully developed. The









range of light intensity during the measurements was 700-900 jPmol photon m- s -. Net

photosynthetic rate and stomatal conductance were determined by gas-exchange

measurements using the LI-6200 Portable Photosynthesis System. Electron transport rate

and effective quantum yield were measured with the PAM-2000 fluorometer, under the

same environmental conditions. Estimates of chlorophyll content and average color of

control and diseased leaves were obtained as described above.

The equation P,-Po(1-x)P was applied to relate relative photosynthetic rate (Px/Po)

to disease severity (x), where Px is the photosynthetic rate of a diseased leaf, and Po is, in

this case, the average photosynthetic rate of the healthy control leaves for each fertilizer

treatment. The 3 parameter for each fertilizer treatment was obtained by non-linear

regression, using SAS. Analysis of covariance was used with a linearized form of the

Bastiaans' model to verify the significant differences among the levels of fertilizer and

also to determine if the experiments could be pooled. The non-linear estimation module

of the software package STATISTICA (release 5.1 for Windows, StatSoft, Inc.) was used

to obtain the best fitted model to describe the relationships between color or chlorophyll

to the photosynthesis variables. Evaluation of model fitness was based on the values of

the coefficients of determination and the homogeneity of the distribution of the residuals.

Measurement of Chlorophyll Fluorescence during Disease Development

Two independent experiments (Experiments 4-5 and 4-6) were conducted to

determine the changes in fluorescence parameters during the development of anthracnose

symptoms. Bean plants were grown under 300-400 jpmol photon m-2 s-1 and a

photoperiod of 12 hours in a growth room. The temperature in the room ranged from 13

to 180C during the night, and from 24 to 32C during the day. To obtain plants with









different levels of severity, suspensions of conidia of different concentrations (5 x 104 or

5 x 103 viable spores/ml, for Experiment 4-5; and 7.5 x 105, 7.5 x 104, or 7.5 x 103 viable

spores/ml, for Experiment 4-6) were sprayed onto both surfaces of the first trifoliate leaf,

in different groups of plants. One of the three leaflets was protected from inoculation by

a plastic bag, and was then considered as a non-inoculated area of inoculated leaves.

Control plants were sprayed with sterile distilled water. Inoculated plants were enclosed

in clear plastic bags for 18 hours in the dark.

In Experiment 4-5, leaves with low severity had a range of 0.1 to 0.4% of disease

and leaves with high severity had from 1 to 7% of disease. For Experiment 4-6, leaves

with low, medium, and high severity had, respectively, 0.8-2%, 3.6-10%, and 16-25% of

their area with symptoms of anthracnose. Disease severity was estimated at the end of the

experiments using a diagrammatic scale of anthracnose symptoms (Godoy et al. 1997).

Minimal fluorescence (Fo), maximal fluorescence (Fm), optimal quantum yield

(Fv/Fm), effective quantum yield, and electron transport rate were the parameters of

chlorophyll fluorescence measured in attached leaves, using the PAM-2000 modulated

light fluorometer. The PAM-2000 is a portable instrument that can be used to measure in

vivo fluorescence of photosynthesizing plant tissue. Modulated chlorophyll fluorescence

techniques use the repetitive application of brief saturated light pulses in addition to the

continuous actinic illumination used to drive photosynthesis (Foyer, 1993). Minimal

fluorescence, maximal fluorescence, and optimal quantum yield were determined on

leaves that were dark-adapted for 20 minutes. A modulated light beam of very low

intensity was applied to the dark-adapted leaf, which induced a weak initial rise in

fluorescence to a low level (Fo). This response is considered to be the emission of









fluorescence which occurs when all PSII reaction centers are open. A brief strong light

pulse was then added to the modulated beam to cause light saturation and to close all

reaction centers at once (Fm). Optimal quantum yield was calculated from Fo and Fm, and

this is considered an indication of the potential photosynthetic efficiency of a leaf.

Measurements similar to the ones taken on dark-adapted leaves were then obtained when

the leaves were re-adapted to 300-400 pjmol photon m-2 s-1 to calculate the effective

quantum yield and the electron transport rate.

The first readings were taken before inoculation (day 0), and then at frequent

intervals during 12 days for Experiment 4-5. In Experiment 4-6, the same fluorescence

parameters were measured from day 0 to day 6 after inoculation. After day 6 the leaves

with high severities of anthracnose collapsed due to disease development. In both

experiments the parameters of chlorophyll fluorescence were measured at random

locations on the leaves before the appearance of symptoms. The necrotic areas of the leaf

were avoided when symptoms were visible. The responses of each fluorescence

parameter over time for healthy leaves and diseased leaves were analyzed with the SAS

repeated measures procedure.


Results


Net Photosynthetic Rate and Related Variables

In both experiments, the reduction in the net photosynthetic rate of leaves with

anthracnose was greater than the increase of severity (Table 4.1). The average net

photosynthetic rate of leaves with 20% severity was reduced in both experiments to about

30% of the rate for the healthy control leaves. For the same leaves, the electron transport









rate in Experiment 4-2 was reduced to 74% of the rate for the healthy control leaves,.

The estimated chlorophyll content of non-necrotic areas in Experiment 3-2 did not

decrease significantly in leaves with up to 20% severity. The dark respiration rate in the

diseased areas increased 26 fold in negative values when compared to healthy tissue

(Figure 4.1).

Stomatal conductance, which represents the degree of stomatal opening and is

directly proportional to the transpiration rate, decreased with increases in disease severity

(Figure 4.2). This reduction in stomatal conductance was described by a negative

exponential model (Figure 4.2a). The ratio of intercellular CO2 to ambient CO2 (Ci/Ca),

which describes the diffusion of CO2 from the atmosphere to intercellular spaces, also

decreased at higher severities, in both experiments, according to a power function (Figure

4.2b).

The apparent quantum yield of CO2 assimilation, expressed as mol CO2/ mol of

quanta, was obtained by dividing the absolute values of photosynthetic rate by the light

intensity at the specific moment of the measurement. The apparent quantum yield of CO2

assimilation was positively correlated to the effective quantum yield, which is a

fluorescence parameter related to the efficiency of the photosystem II (Figure 4.3).

Virtual Lesion Size

The value of 3, for the relationship of relative photosynthetic rate to anthracnose

severity, was 8.46 for Experiment 4-1, and 12.18 for Experiment 4-2 (Figure 4.4). The 3

values were greater than one, which was an indication that the virtual lesion was much

larger than the visual lesion, for this combination of C. lindemuthianum isolate and bean












Table 4.1. Net photosynthetic rate, electron transport rate, and estimated chlorophyll content for bean leaves with different levels of
anthracnose severity.

Experiment 4-1 Experiment 4-2


Anthracnose No. Photosynthetic Anthracnose No. Photosynthetic Electron Chlorophyll
severity of rate severity of rate transport rate content
leaves pmol CO2 m-2s-1 leaves pmol CO2 m-2s S mol CO2 m-2s-1 SPAD
1 values


0.00 13 19.270.52b 0.00a 9 16.430.55 121.42.45 36.5310.63d

<0.01 6 14.781.14 <0.01 5 15.060.87 124.45.07 36.520.63

0.01-0.05 6 10.290.89 0.01-0.02 9 14.250.62 118.83.56 36.410.41

0.05-0.10 7 12.370.88 0.02-0.03 6 12.090.34 109.84.60 35.330.96

0.11-0.20 8 5.991.42 0.03-0.05 7 10.390.77 107.07.26 35.240.58

0.21-0.35 3 2.751.30 0.05-0.2 4 5.0651.68 90.47.89 35.701.48
a Proportion of leaf area with symptoms.
b Mean photosynthetic rate standard error.
0 Chlorophyll was estimated with the Minolta Chlorophyll Meter SPAD-502; 15 readings on non-necrotic areas were taken for each
leaf.
d Mean amount of chlorophyll standard error.


























E r2=0.41
0(


o










Anthr acnos e sever ity




















Figure 4.1. Effects of anthracnose severity on dark respiration rate (R,) of bean leaves.
Sigma (a) values were obtained with the equation Rx=Ro(1-x) + aRox, where
Ro is the average dark respiration rate of the healthy leaves, and x is
anthracnose severity, expressed as a proportion of the leaf area.







73








1.2 8a Experiment 4-1
E 0 Experiment 4-2
S-- y=0.65*exp(-32.6*x)+0.14
Sr2=0.72

0.8



O

0
S0.4

*


n 0.0
Experiment 4-1
1.0 O Experiment 4-2
y= -0.22(xosi)+0.84
r2=0.44


0.8 t **0*




0.6




0.4
0.0 0.1 0.2 0.3 0.4

Anthr acnos e s ever ity
















Figure 4.2. Effects of anthracnose severity in bean leaves on (a) stomatal conductance
and on (b) ratio of intercellular to ambient CO2 concentration (Ci/Ca).





























0 y=-0.014+0.08x -
0.03 r2= 0.62 ^



S 0.02
2 00 6
O 0


-co'


I 0.00 -


0.0 0.1 0.2 0.3 0.4 0.5 0.6
Effective quantum yield (relative units)


















Figure 4.3. Relationship between effective quantum yield of the photosystem II and
apparent quantum yield of CO2 assimilation in bean plants with different
levels of anthracnose severity.


















1.4 a
beta=8.46 (S E =1.09)
1.2 r2=0.70

1.0

0.8 0
0,6
t 0.6 -

S 0.4 a

0.2

0.0

14 b beta=12.18 (SE=1.02)
S 1.2 r2=0.82

1.0

0.8

0.6

0.4

0.2

0.0 ,
0.0 0.1 0.2 0.3 0.4

Anthr acnos e s ever ity












Figure 4.4. Effects of anthracnose severity (x) on the relative photosynthetic rate (Px/Po)
of bean leaves. The values of beta (3) were obtained with the model Px/Po
=(l-x) P; (a) Experiment 4-1, (b) Experiment 4-2.









cultivar. The values of the coefficient of determination for the three experiments were

higher than the values reported in the literature for this type of experiment (Bastiaans,

1991; Goodwin, 1992). The higher coefficient of determination for the model in

Experiment 4-2 compared to that for Experiment 4-1 was probably due to greater

precision in the assessment of anthracnose severity in the second experiment, which may

have reduced the variability of the data.

Effects of Fertilization on the Photosynthetic Competence of Bean Leaves with
Anthracnose

The nutritional condition of the leaf determined its level of photosynthetic

activity. The absolute average values of net photosynthetic rate, chlorophyll content, and

electron transport rate for the healthy control leaves in Experiments 4-3 and 4-4 were

significantly lower in plants that did not receive any fertilizer than in plants that received

50 or 100% of the recommended rate of fertilizer (Table 4.2). The results of the two

experiments for the several variables analyzed were combined, based on lack of

significant differences in the initial statistical analysis. At the levels of severity obtained

in these experiments, there was no reduction on the chlorophyll content of diseased

leaves, when compared to the healthy control leaves at all levels of fertilizer (Figure

4.5a). Leaf color also was not significantly affected by the levels of anthracnose severity

observed (Figure 4.5b). Despite the absence of a significant relationship between

anthracnose severity and leaf color or chlorophyll content, the two latter variables were

correlated with relative photosynthetic rate and relative electron transport, when plants at

all levels of fertilizer were considered (Figures 4.6 and 4.7).












Table 4.2. Absolute average values of net photosynthetic rate, chlorophyll content, and electron transport rate for healthy control bean
leaves.

Experiment 4-3 Experiment 4-4

Fertilizer Photosynthetic Chlorophyll Electron Photosynthetic Chlorophyll Electron
treatment rate content transport rate rate content transport rate
Pimol CO2 m-2s-1 SPAD values"b Pmol m-2S-1 Pmol CO2 m-2s- SPAD values Pimol m-2s-1
1

No fertilizer 9.670.44a 22.200.85 69.404.08 11.220.81 27.240.94 88.423.91

50% fertilizer rate 15.440.44 31.810.70 106.574.0 15.20.78 32.481.17 112.564.3

100% fertilizer rate 14.100.65 31.541.33 95.463.23 17.660.58 35.250.69 126.522.9

a Means standard errors.
b Values obtained with the Minolta Chlorophyll Meter (SPAD-502); 15 readings on non-necrotic areas were taken for each leaf.














































0 00
0.00


0.01 0.02 0.03 0.04 0.05 0.06


Anthr acnos e s ever ity










Figure 4.5. Effects of anthracnose severity on (a) chlorophyll content and (b) color of
bean leaves with different nutritional status. SPAD values are the values
given by the chlorophyll meter (SPAD-502, Minolta). Color is expressed as
units of difference between the color of a given leaf and the average color of
the well fertilized, healthy control leaves.

















(U
" 1.2






y
0,
| 0.8



(U
_0



0.0


0 5 10 15 20

Units of color difference


25 30


Figure 4.6. Responses of (a) relative photosynthetic rate and (b) relative electron
transport rate to units of color difference at the different levels of fertilizer.
Both rates are expressed as fractions of the average value of all healthy bean
leaves at the recommended fertilizer rate of 100%. Data from Experiments
3-3 and 4-4 were combined in these graphs.


















1.2

.5
O
_c-

S0.8


0--
0.4


ry

0.0


6 0.8 1.0

Relative chlorophyll content


Figure 4.7.


Responses of (a) relative photosynthetic rate and (b) relative electron
transport rate to relative chlorophyll content (SPAD values), at the different
levels of fertilizer. Both rates and the values of SPAD are expressed as
fractions of the average value of all healthy bean leaves at the recommended
fertilizer rate of 100%. Data from Experiments 4-3 and 4-4 were combined
in these graphs.


No fertilizer
o 50% fertilizer rate
A 100% fertilizer rate
-- y= -0.46+1.356x AE
r2=0.56 A



**

*0 F

0

a


No fertilizer
D 50% fertilizer rate
A 100% fertilizer rate A A
-- y= -0.17+1.12x
r2=0.62 D D





*




b









The values of 3, the ratio of virtual lesion to visual lesion size, for all three levels

of fertilization were greater than one (Figure 4.8). The value of 3 for the group of plants

that received no fertilizer (11.51) was significantly lower than the value obtained for the

plants that received 50% of the recommended rate of fertilizer (18.56), but it did not

differ significantly from the value for the treatment with 100% fertilizer (11.85). The

treatments that received 50% and 100% of fertilizer rate did not have significantly

different values of P. The variability observed in the data was high, and only a small part

of this variability could be explained by the model that was used.

Measurement of Chlorophyll Fluorescence during Disease Development

For Experiment 4-5, there was a significant reduction over time in maximal fluorescence,

electron transport rate, and effective quantum yield, in non-necrotic areas of inoculated

leaves that had 1 to 7% severity (Figure 4.9b, 4.10a, 4.10b). These parameters were

reduced in comparison to healthy tissue after the appearance of symptoms of anthracnose,

6 days after inoculation. Minimal fluorescence and optimal quantum yield were not

reduced in leaves with 1 to 7% severity in comparison to healthy control leaves (Figure

4.9a, 4.9c). Leaves with 0.1-0.4% severity did not differ significantly from the healthy

control leaves in any of the measured parameters during disease development.

Anthracnose development in symptomatic areas of inoculated leaflets did not interfere

with the fluorescence parameters in the non-inoculated leaflet of the same leaf (Figures

4.9d, e, fand 4.10c and d).

For Experiment 4-6, maximal fluorescence, optimal quantum yield, electron

transport rate, and effective quantum yield were reduced in the non-necrotic areas of



















1.2

0

0.8 69

*

0.4


a
0.0

1.2
SDD


0.8 E



0.4

b
0.0
1.2 A
A


0.8



0.4


C
0.0
0.00 0.01


0.02 0.03 0.04 0.05 0.06

Anthr acnos e s ever ity


Figure 4.8. Effects of anthracnose severity (x) on the relative photosynthetic rate (Px/Po)
of bean leaves with (a) no fertilizer, (b) 50% of the recommended fertilizer
rate, and c) 100% fertilizer rate. The values of beta (3) were obtained with the
model Px/Po = (1-x) ; the data from Experiments 4-3 and 3-4 were combined
in these graphs.































-*- healthy control
- 0.1 -0.4% severity
- 1-7% severity
b


0 2 4 6 8 10 12 14

Days after inoculation


0.5
0


2 4 6 8 10 12 14

Days after inoculation


Figure 4.9. Minimal fluorescence (Fo), maximal fluorescence (Fm), and optimal quantum
yield (Fv/Fm) during anthracnose development in non-necrotic areas of
inoculated (a, b, c) and non-inoculated (d, e, f) areas of bean leaves with
different anthracnose severities.


0.08


0.08

0.7

-

0.6
LL

0.5



0.4

0.8


0.7


















100


-*- healthy control
- 0.1 -0.4% severity
- 1-7% severity


0 2 4 6 8 10

Days after inoculation


60

a
-40

0.7



0.6



0.5


b
0.4
12 14 0


2 4 6 8 10 12

Days after inoculation


Figure 4.10. Electron transport rate (pmol m-2s-) and effective quantum yield, in relative
units, during anthracnose development in non-necrotic areas of inoculated
(a, b) and non-inoculated (c, d) areas of bean leaves with different
anthracnose severities.


-0
0) 0.7

E

S0.6





w
0.4

0.4




















0.6

E
LL 0.4


0.2


0.0


0 1 2 3 4 5 6 0 1 2 3 4 5 6


1)
C oo
2100
t


60

r-
0 40


LU
G) 20
w


- healthy control
- 0.8-2% severity
--- 3.6-10% severity
- 16-25% severity


0 1 2 3 4 5 6 0 1 2 3 4 5 6


healthy control
0.8-2% severity
-- 3.6-10% severity
16-25% severity

0 1 2 3 4

Days after inoculation


5 6


Figure 4.11. The effect of initial development of anthracnose in bean leaves on (a)
minimal fluorescence (Fo), (b) maximal fluorescence (Fm), (c) optimal
quantum yield (Fv/Fm), (d) effective quantum yield (all in relative units), and

(e) electron transport rate (pmol m-21).


0.2




" 0.1
LL. 0.1


E
,^ 0.7
I,









leaves with 16-25% severity, as soon as the first symptoms of anthracnose were visible (5

days after inoculation) (Figures 4.1 Ib, c, d, and e). Minimal fluorescence was not

reduced for any of the levels of severity during disease development. Leaves with 0.8 to

2% or 3.6 to 10% severity did not differ from the healthy control leaves 5 days after

inoculation. The fluorescence parameters of non-inoculated areas of inoculated leaves

were not affected by the disease (data not shown).

Discussion


According to Luttrell's (1974) definition, Colletotrichum lindemuthianum is a

hemibiotrophic fungus. It exhibits a two-phase infection process involving an initial

biotrophic phase, during which the pathogen establishes itself in living host cells,

followed by a visibly destructive phase (Bailey et al., 1992; O'Connell and Bailey, 1991).

The symptoms of the disease become visible when the pathogen switches to necrotrophic

nutrition, and they are characterized by tissue maceration and water soaking. An

increased activity of pectin lyases observed in infected tissues is associated with the

switch from the transient biotrophic phase to the necrotrophic phase, in which extensive

dissolution of cell walls occurs in advance of the pathogen (Wijesundera et al., 1989).

The impact of C. lindemuthianum on photosynthesis is associated with the

necrotrophic phase of the infection. In plants of Vigna sesquipedalis, the photosynthetic

rate and the chlorophyll content of the leaves were reduced, and the respiration rate

increased when necrotic symptoms appeared (Wong and Thrower, 1978a). Reduced

photosynthetic and transpiration rates were reported for plants ofPhaseolus vulgaris with

different levels of anthracnose severity (Bassanezi et al., 1997). Colletotrichum









lindemuthianum had no significant effect on the translocation of current photosynthate

from diseased leaves of V. sesquipedalis (Wong and Thrower, 1978b). The pathogen

induced only a slight accumulation of photosynthates during the first days of

pathogenesis.

The quantification of the impact of C. lindemuthianum on the photosynthesis of

bean leaves is the first step to characterize the potential yield losses caused by

anthracnose. The concept of virtual lesion, introduced by Bastiaans (1991), is the basis

for a model that quantifies photosynthetic competence of diseased leaves. A virtual

lesion consists of a visual lesion and an adjacent area, in which the photosynthetic activity

is zero. The relation between disease severity and photosynthesis is described by a single

parameter, 3, the ratio of virtual and visual lesions. Values of 3 greater than one are

interpreted as an indication that the disease not only reduced the leaf area capable of

photosynthetic activity, but also affected photosynthesis in the remaining green leaf

tissue. In the present study, the values of 3 obtained (8.46 and 12.18) are an indication

that the virtual lesion induced by this pathogen is much larger than the visual lesion,

which means that there was a great impact of anthracnose on the photosynthesis of the

remaining green tissue of bean leaves of 'Rosinha'. These results are similar to the high

value of 3 (7.24 + 0.75) reported by Bassanezi et al. (1997) for a different cultivar of

common bean, 'Carioca Comum'. Since the same race of the pathogen, race kappa, was

used in both studies, the difference in the 3 values could be due to a difference in the

cultivar reaction to infection. However, this hypothesis should be investigated carefully,

since differences in 3 values among cultivars may not imply genotypic differences in

tolerance. Bastiaans and Roumen (1993) concluded that the 3 parameter was not a









suitable selection criterion in breeding rice for tolerance to leaf blast, as the 3 values were

similar for three susceptible cultivars with different relative infection efficiencies.

The method of severity assessment also may have influenced the determination of

the 3 values. The same diagrammatic scale of anthracnose symptoms (from Godoy et al.,

1997) was used by Bassanezi et al. (1997) and in the present study (Experiment 4-1) to

obtain the values of 3 of 7.24 and 8.46, respectively. For the experiment in the present

study in which the value of 12.18 was obtained (Experiment 4-2), the actual severity was

assessed by determining the ratio between the area of the lesions and the area of the leaf.

In a preliminary experiment (Appendix E), conducted before Experiment 4-2, the ratings

anthracnose severity present in the leaves obtained with the diagrammatic scale were

overestimated, when compared to the actual values. If anthracnose severity were

overestimated by the use of the scale, the resultant 3 value would tend to be lower than it

would be with values of actual severity.

Besides the fact that the lesions of anthracnose on the leaves are irregularly

shaped, which makes the assessment of severity difficult, there are other characteristics of

the symptomatology of this disease that further complicate the scenario. The necrotic

symptoms of anthracnose are localized on the leaf veins, which most likely interferes with

the transport of water and assimilates in the leaf. Moreover, in very susceptible cultivars,

such as the one used in the present study, the leaf tissue adjacent to the lesions usually

becomes water-soaked. On leaves with high disease severity, the water-soaked regions

rapidly coalesce and large portions of the leaf can collapse in less than one day after

necrotic symptoms are first observed. Also, lesions of the same size but in different

positions on the leaf may have a different impact on the leaf physiology. Bastiaans and









Roumen (1993) observed that blast lesions on the central vein of rice leaves caused a

marked reduction in the photosynthetic rate of the distal part of the leaf, while lesions on

either side of the central vein caused only localized effects.

While the net photosynthetic rate of bean leaves, determined by gas-exchange

measurements, was drastically reduced with increases in anthracnose severity, the effects

on the chlorophyll fluorescence parameters were less severe. Similar to the results with

bean rust reported in Chapter 3, there were no relative changes in the fluorescence

parameter in non-inoculated areas of inoculated leaves in any of the levels of severity

investigated.

Minimal fluorescence (Fo) represents the emission from molecules of chlorophyll

a of the light harvesting antenna associated with PSH prior to the excitation energy being

used for electron transport (Krause and Weis, 1984). It is therefore independent of

photochemical events, but dependent upon the chlorophyll content of the tissue and on

the ultrastructure of the thylakoid membrane. In this study, Fo was not altered by

anthracnose development, even at high severity levels (Figures 4.9a and 4.1 la), which

may be due to the lack of change in chlorophyll content in the early development of this

disease (Experiment 4-2, Table 4.1).

Maximal fluorescence (Fm) is a measure of the oxidation-reduction status of the

electron acceptors between PSI and PSI and a direct indicator of PSH activity (Scholes

and Farrar, 1985). Environmental stresses that cause thylakoid damage, such as heat and

freezing stresses, usually lower Fm. The Fm values of leaves with high anthracnose

severity were reduced by 28%, in relation to the control leaves, as soon as the necrotic

symptoms were visible (Figure 4.1 Ib). In leaves with lower anthracnose severity, Fm was









reduced only a few days after the appearance of symptoms (Figure 4.9b). Maximal

fluorescence was also reduced in plants infected by other pathogens, such as

Phaeoisariopsis griseola on common bean (Bassanezi, 1995), Uromyces muscari on

bluebell (Scholes and Farrar, 1985), Taphrina deformans on peach (Raggi, 1995), and

tobacco mosaic virus on tobacco (Balachandran and Osmond, 1994).

The optimal quantum yield (Fv/Fm) provides information on the potential

photosynthetic efficiency of PSII, and it is often discussed as a vitality index (Moll et al.,

1995). It has a range of 0.75 to 0.85 for healthy leaves of many plant species and

ecotypes, decreasing under stress (Bolhar-Nordenkampf and Oquist, 1993). The

calculation of this parameter depends on the values of Fo and Fm. In the present study,

Fv/Fm was unchanged in leaves with low severity (Figures 4.9c and 4.1 Ic). The reduction

in Fm for plants with 16-25% anthracnose severity induced a reduction of 13% in Fv/Fm,

compared to the control leaves (Figure 4.1 1c). This reduction in Fv/Fm may be an

indication that mechanisms of photon protection are being activated in response to the

stress caused by the disease development. Photon protection is the result of a decrease in

the efficiency of photosynthesis by means of dissipating the excess of photons as heat,

before these photons can reach the reaction centers (Osmond, 1994).

Effective quantum yield is the quantum yield of the non-cyclic electron flow

(Foyer, 1993; Genty et al., 1989). It is proportional to the concentration of the open PSII

reaction centers, and to the efficiency with which these centers capture and use excitation

energy. It is the basis for the calculation of the electron transport rate. Both effective

quantum yield and electron transport rate suffered reductions, compared to the control

leaves, on diseased leaves with 1-7% severity (Figure 4.10a and b) and 16-25% severity









(Figure 4.1 d and e). The reduction in these parameters coincided with the appearance of

necrotic symptoms, which occurred 5 to 6 days after the inoculation.

Chlorophyll fluorescence is a measure of the efficiency of light utilization on PSII.

There is a ubiquitous curvilinear or biphasic relationship between the effective quantum

yield of fluorescence and the apparent quantum yield of CO2 assimilation (mol CO2/mol

quanta) (Comic and Briantais, 1991; Genty et al., 1989; Seaton and Walker, 1990). This

relationship may allow good estimates of photosynthetic rate through measurements of

fluorescence, without recourse to analysis of gaseous exchange between leaf and

environment. Inhibition of primary photochemistry, electron transport, or carbon

metabolism caused by environmental stresses will affect the function of PSI and will be

expressed as changes in fluorescence yield. These changes in fluorescence yield can

provide quantitative information about plant responses to the duration and intensity of the

stress (Bolhar-Nordenkampf and Oquist, 1993). In the present study, there was a linear

relationship between effective quantum yield and apparent quantum yield of CO2

assimilation (Figure 4.3). However, the linear regression did not pass through the origin,

which means that for bean leaves with anthracnose, the effective quantum yield of PSII is

not zero when the efficiency of CO2 assimilation is zero, as would be theoretically

expected (Genty et al., 1989; Seaton and Walker, 1990).

There was a clear imbalance between the drastic inhibition in net photosynthetic

rate and the moderate decreases in the photochemical reactions (Fv/Fm and electron

transport rate) in bean leaves with anthracnose. Raggi (1995) observed a similar trend for

peach leaves infected with Taphrina deformans. He suggested that the refixation of the

CO2 released by stimulated respiration could partially explain the disparity between gas-




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