STIMULATORY AND INHIBITORY EFFECTS OF UVA AND UVB RADIATION ON SOME PHYSIOLOGICAL AND PATHOGENIC CHARACTERISTICS OF FUNGAL BIOCONTROL AGENTS TO ENHANCE MYCOHERBISTAT EFFECTIVENESS
by
Feridon Ghasem Khan Ghajar B. Sc. University of Tehran, M. Sc. University of Western Sydney
A thesis submitted in fulfilment of requirements for the degree of Doctor of Philosophy
College of Science, Technology and Environment
University of Western Sydney
Penrith South DC NSW 1797
Australia
February 2004
To Mehri and Farideh, whose love and support have made the completion of this project possible.
i
ACKNOWLEDGEMENTS
I would like to gratefully acknowledge the supervisory panel, Associate Professor Paul Holford (UWS), Dr Eric Cother (Principal Research Scientist, NSW Agriculture, Orange Agricultural Institute, Orange) and Professor Andrew Beattie (UWS) for their supervision and encouragement throughout this study.
I also grateful to:
Dr Alison McInnes (School of Environment and Agriculture, UWS) for her assistance with the DNA analysis of D. avenacea isolates.
Dr S. D. Hetherington (NSW Agriculture, Orange Agricultural Institute, Orange) for providing the isolates of D. avenacea.
Mr Oleg Nicetic for UV measurements and providing meteorological data.
Technical officers, present and past, Mr Jim Sheehy, Ms Liz Darley, Dr Sumedha Dharmaratne, Mrs Kim Northcott and Mr Gary Morgan for their technical assistance throughout this study.
Mrs Kylie Kaszonyi and Mrs Robynne Warne for their assistance with propagating and maintaining the test plants.
ii
DECLARATION
The work presented in this thesis is, to the best of my knowledge and belief, original except as acknowledge in the text. I hereby declare that I have not submitted this material, either in full or in part, for a degree at this or any other institution.
Feridon Ghasem Khan Ghajar February 2004
iii TABLE OF CONTENTS
Dedication i Acknowledgements ii Declaration iii Table of contents iv Abbreviations xi Abstract xii
CHAPTER 1: GENERAL INTRODUCTION 1 1.1 WEEDS: DEFINITION AND IMPORTANCE 1 1.2 WEED MANAGEMENT 4 1.2.1 Cultural control method 5 1.2.2 Mechanical control methods 6 1.2.3 Chemical control methods 7 1.2.4 Biological control methods 9 1.2.4.1 Classical strategy 10 1.2.4.2 Inundative strategy 11 1.2.5 Integrated weed management (IWM) 14 1.3 BIOLOGICAL CONTROL OF WEEDS USING MYCOHERBICIDES 16 1.3.1 Creating mycoherbicides 16 1.3.1.1 Discovery phase 17 1.3.1.1.1 Selection of target weeds 17 1.3.1.1.2 Selection of fungal pathogens 18 1.3.1.2 Development phase 19 1.3.1.3 Deployment phase 20 1.3.2 Formulation of mycoherbicides 21 1.3.3 Products on the market 26 1.3.4 Benefits, constraints and prospects 28
iv 1.4 PLANT-PATHOGEN SYSTEMS STUDIED IN THIS PROJECT 32 1.4.1 Alisma lanceolatum and Damasonium minus- Rhynchosporium alismatis 32 1.4.1.1 Alisma lanceolatum and Damasonium minus 32 1.4.1.2 Rhynchosporium alismatis 35 1.4.2 Xanthium spinosum-Colletotrichum orbiculare 36 1.4.2.1 Xanthium spinosum 36 1.4.2.2 Colletotrichum orbiculare 40 1.4.3 Avena fatua-Drechslera avenacea 42 1.4.3.1 Avena fatua 42 1.4.3.2 Drechslera avenacea 46 1.5 EFFECTS OF ULTRAVIOLET (UV) RADIATION ON FUNGAL PATHOGENS 47
1.5.1 Ozone (O3), UV radiation and climate change 47 1.5.2 UVB and fungal pathogens 48 1.6 OBJECTIVES OF THIS THESIS 49
CHAPTER 2: OPTIMISING SPORULATION AND PATHOGENICITY IN DRECHSLERA AVENACEA 51 2.1 INTRODUCTION 51 2.2 MATERIALS AND METHODS 55 2.2.1 Fungal isolates 55 2.2.2 Data analysis 55 2.3 EXPERIMENTAL 56 2.3.1 Effect of agar media and temperature on conidium production 56 2.3.2 Effect of pH of the medium on conidium production 59 2.3.3 Effect of carbohydrate content on conidium production 60 2.3.4 Effect of NUV intensity on conidium production on ½OMA and CZA 61 2.3.5 Effect of agar media and light quality on conidium production 63
v 2.3.6 Effect of continuous dark and light, diurnal or combined light conditions and constant or alternating temperature on conidium production on ½OMA 64 2.3.7 Effect of photoperiodism on conidium production on ½OMA 66 2.3.8 Effect of media and light quality on subsequent virulence of conidia produced by D. avenacea 67 2.3.9 Applicability of culture conditions for other isolates 69 2.3.10 Effect of culture age on conidium production 70 2.4 DISCUSSION 72
CHAPTER 3: GENETIC VARIATION IN DRECHSLERA AVENACEA ISOLATES DETERMINED BY RAPD-PCR ANALYSIS 82 3.1 INTRODUCTION 82 3.2 MATERIALS AND METHODS 84 3.2.1 RAPD-PCR analysis 84 3.2.1.1 DNA extraction 84 3.2.1.2 Oligonucleotide primers, PCR amplification and electrophoresis 85 3.2.2 Fragment analysis 86 3.3 RESULTS 87 3.4 DISCUSSION 91
CHAPTER 4: EFFECTS OF ULTRA-VIOLET RADIATION, SIMULATED OR AS NATURAL SUNLIGHT, ON CONIDIUM GERMINATION AND APPRESSORIUM FORMATION OF FUNGI WITH POTENTIAL AS MYCOHERBISTATS 93 4.1 INTRODUCTION 93 4.2 MATERIALS AND METHODS 96 4.2.1 Fungal pathogens: maintenance and conidia description 96 4.2.2 Preparing conidium suspensions for exposure to UV radiation 97 4.2.3 Assessment of conidium germination and appressorium formation 97
vi 4.2.4 Suitability of different types of glassware for UV radiation studies 98 4.2.5 Exposure of suspensions of conidia to simulated UV radiation 98 4.2.6 Experiment 1. Determination of photomorphogenic and damaging wavelengths 99 4.2.7 Experiment 2. Effect of UVB radiation exposure at different periods of time (UVB doses) 100 4.2.8 Natural sunlight experiments 100 4.2.8.1 The effects of full-spectrum natural sunlight and sunlight without UVB and interactions with temperature 100 4.2.8.2 The effect of different levels of solar irradiation on germination of conidia 102 4.2.9 Meteorological measurements 103 4.2.10 Statistical analysis 104 4.3 RESULTS 105 4.3.1 Conidia description 105 4.3.2 Suitability of different types of filter for UV radiation studies 106 4.3.3 Experiment 1. Determination of photomorphogenic and damaging wavelengths 107 4.3.3.1 R. alismatis 107 4.3.3.2 C. orbiculare 108 4.3.3.3 D. avenacea 110 4.3.4 Experiment 2. Effect of UVB irradiation for different periods of time (UVB doses) 111 4.3.4.1 R. alismatis 111 4.3.4.2 C. orbiculare 112 4.3.5 Dose-response curves 113 4.3.5.1 R. alismatis 113 4.3.5.2 C. orbiculare 114
vii
4.3.6 Natural sunlight experiment 115 4.3.5.3 The effects of full-spectrum natural sunlight or sunlight without UVB and interactions with temperature 115 4.3.5.3.1 R. alismatis 115 4.3.5.3.2 C. orbiculare 120 4.3.5.3.3 D. avenacea 126 4.3.5.4 The effect of different levels of solar irradiation on germination of conidia 132 4.3.5.4.1 R. alismatis 132 4.3.5.4.2 Dose-response curves 136 4.3.5.4.3 C. orbiculare 139 4.3.5.4.4 Dose-response curves 141 4.4 DISCUSSION 144 4.4.1 Effects of simulated sunlight 144 4.4.2 Effects of natural sunlight 147 4.4.3 Practical implications 154
CHAPTER 5: PHOTOSTABILISATION OF FUNGI WITH POTENTIAL AS MYCOHERBITATS BY UV PROTECTANTS 155 5.1 INTRODUCTION 155 5.2 MATERIALS AND METHODS 158 5.2.1 Conidium suspensions 158 5.2.2 Mineral and plant oils 158 5.2.3 UV protectants 158 5.2.4 Solubility of UV protectants 158 5.2.5 Toxicity testing 159 5.2.5.1 R. alismatis 159 5.2.5.2 C. orbiculare 160 5.2.6 Selection of UV protectants 160 5.2.6.1 R. alismatis 160 5.2.6.2 C. orbiculare 161 5.2.7 Natural sunlight experiment 161
viii 5.2.8 Spectrophotometer studies 162 5.2.9 Statistical analysis 162 5.3 RESULTS 163 5.3.1 Solubility of UV protectants 163 5.3.2 Toxicity testing 163 5.3.2.1 R. alismatis 163 5.3.2.2 C. orbiculare 166 5.3.3 Selection of UV protectants 168 5.3.3.1 R. alismatis 168 5.3.3.2 C. orbiculare 173 5.3.4 Natural sunlight experiment 174 5.3.5 Spectrophotometer studies 176 5.4 DISCUSSION 179
CHAPTER 6: EFFECT OF UV PROTECTANTS AND UVB RADIATION, SIMULATED OR AS NATURAL SUNLIGHT ON PATHOGEN-HOST PLANT INTERACTIONS 187 6.1 INTRODUCTION 187 6.2 MATERIALS AND METHODS 190 6.2.1 Conidium suspensions 190 6.2.2 Water- and oil-compatible UV protectants 190 6.2.3 Leaf disc bioassays for anthracnose development 190 6.2.3.1 Exposure to simulated UVB radiation 190 6.2.3.2 Exposure to full-spectrum natural sunlight 192 6.2.4 Pot-in-field experiments 192 6.2.5 Statistical analysis 196 6.3 RESULTS 197 6.3.1 Leaf disc bioassays for anthracnose development 197 6.3.1.1 Exposure to simulated UVB radiation 197 6.3.1.2 Exposure to full-spectrum natural sunlight 199 6.3.2 Pot-in-field experiments 204 6.4 DISCUSSION 207
ix CHAPTER 7: GENERAL DISCUSSION AND CONCLUSIONS 210 7.1 R. alismatis 210 7.2 C. orbiculare 215 7.3 D. avenacea 219 7.4 Summary 225
REFERENCES 226
PUBLICATIONS 250
x Abbreviations
AFLP amplified fragment length polymorphism ANOVA analysis of variance APPs aryloxyphenoxypropionates CA carrot agar CHDs cyclohexanediones CIE Commission Internationale d’Eclairage CMA cornmeal agar CTAB cetyl-trimethyl ammonium bromide CZA Czapek Dox agar DAI days after inoculation dATP deoxyadenosine-5' -triphosphate dCTP deoxycytosine-5' -triphosphate dGTP deoxyguanine-5' -triphosphate DNA deoxyribonucleic acid dTTP deoxythymine-5' -triphosphate EDTA ethylenediaminetetraacetic acid, disodium salt FOP aryloxyphenoxypropionate GBA green bean agar IWM integrated weed management LBA lima bean agar L/D alternating 12 h white light and dark LSD least significant difference MA malt extract agar MCPA monochloro phenoxy acetic acid MPDA malt extract-peptone-dextrose agar MSMA monosodium methylarsonate MYA malt and yeast extract agar NUV near-ultraviolet NUV/D alternating 12 h near-ultraviolet radiation and dark OMA oatmeal agar pABA p-aminobenzoic acid PCA potato-carrot agar PCR polymerase chain reaction PDA potato-dextrose agar RAPD random amplified polymorphic DNA RFLP restriction fragment length polymorphism RNA ribonucleic acid SST selective spray-topping UV ultraviolet V8JA V-8 juice agar VPD vapour pressure deficit WA water agar WAI weeks after incubation YDA yeast extract-dextrose agar
xi
ABSTRACT
Many candidate mycoherbicides have shown promise in the laboratory or greenhouse, but most have been ineffective in the field. Factors limiting mycoherbicide efficiency include temperature and humidity. Results from this thesis indicate that solar radiation has both a damaging effect (reduction in germination) limiting efficacy and a photomorphogenic effect (appressorium induction) increasing efficacy. The study has also shown significant interactions between temperature and solar radiation on the survival of conidia of potential mycoherbistats. Therefore, solar radiation should be considered as third major component of the environment that should be considered when trying to produce mycoherbistats. Three pathosystems, Rhynchosporium alismatis-Alisma lanceolatum and Damasonium minus, Colletotrichum orbiculare-Xanthium spinosum and Drechslera avenacea- Avena fatua have been investigated in relation to the effects of solar radiation in an attempt to enhance disease development. The effect of different periods of exposure to UVB on conidium germination of R. alismatis indicated, firstly, low UVB doses only caused delays in the germination of conidia whereas higher doses killed conidia as well as causing delays in the germination of any survivors. Appressorium formation by R. alismatis conidia was induced by UVA radiation. About 50% of germinated conidia produced appressoria when exposed to a total solar dose of 2.80 MJ m-2 and a corresponding UVA dose of 44.6 kJ m-2. Conidium germination of R. alismatis 6 h incubation post- exposure was halved for doses of solar radiation, Dsolar, DUVA and DUVB, of about 4 MJ m-2, 60 and 12 kJ m-2, respectively. High level of protection against UVB radiation was provided with conidia treated with the water-compatible UV protectants, riboflavin, proline, melanin, folic acid and ascorbic acid. Microcycle conidiation following exposure to sunlight and treatment with 5% aqueous solutions of proline or pyridoxine was observed. Microcycle conidiation during periods of fluctuating RH or solar radiation may have epidemiological consequences when
xii primary conidia fail to infect and it may be possible to induce secondary infections through this mechanism. Conidium germination of C. orbiculare 16 h incubation post-exposure was -2 halved for doses of solar radiation, Dsolar, DUVA and DUVB, of about 1 MJ m , 20 and 4 kJ m-2, respectively. Appressorium formation by conidia of C. orbiculare was stimulated following exposure to a UVA dose of 4.24 kJ m-2. About 50% of germinated conidia produced appressoria 12 h after incubation. After exposure to natural sunlight at UVB irradiance of 0.63 W m-2, anthracnose development on leaf discs 3 DAI inoculated with conidia formulated in folic acid, proline, tyrosine, ascorbic acid, D-C-Tron at 1%, melanin at 0.01%, Codacide or D-C-Tron at 5% was increased compared with the control conidia formulated in water alone. The most suitable conditions for conidium production by D. avenacea were determined to be growth on half strength oat meal agar with an initial pH of 7 at
20°C under continuous UVA illumination for 2 weeks. Stimulatory effects of UVA radiation on sporulation, subsequent infectivity of conidia and appressorium formation by D. avenacea were found. Conidium germination of D. avenacea was not adversely affected by UVB radiation. Appressorium formation by D. avenacea appeared to be UVA dose dependent. High numbers of appressoria were formed after exposure to a UVA dose of 101.1 kJ m-2 with approximately 80% of germinated conidia forming appressoria. Considerable variation in sporulation and the virulence of conidia of D. avenacea was detected among isolates from different geographic areas. RAPD-PCR analysis also revealed variation at molecular level among these isolates. With the findings presented in this thesis and further research on disease development under different conditions, in combination with the formulation of conidia in suitable UV protectants, a computer simulation modelling the conditions leading to epidemics caused by C. orbiculare, D. avenacea and R. alismatis could be constructed. This may help to optimise time of application and development of disease by forecasting the optimum infection days and assessing the potential regions suitable for these mycoherbistats. It may be possible to manipulate fungal application time in order to expose conidia to doses of solar radiation that are not harmful to conidium germination and which stimulate appressorium formation. However, additional protection may be needed.
xiii
CHAPTER 1
GENERAL INTRODUCTION
1.1 WEEDS: DEFINITION AND IMPORTANCE
Invasive plants, commonly called harmful, noxious or weedy plants, are frequently defined as plants growing where they are not wanted or plants out of place. They are a serious problem almost everywhere; in parks, preserves, refuges, rangelands, forests, agricultural fields, urban green spaces, roadsides, railway lines, lakes and waterways. Weeds severely threaten the yield and quality of produce, biodiversity, habitat quality and ecosystem functions.
The unwanted plant or plant-out-of-place definitions imply that any plant species may become a weed in particular situations. These definitions do not distinguish plants that possess truly weedy characteristics from those that are only occasional nuisances. Ross and Lembi (1999) described certain definable characteristics that set a weed apart from other plant species. These characteristics include one or more of the following features: abundant seed production (and thus potentially large populations), rapid population establishment, seed dormancy, long- term survival of buried seeds, adaptations for spread, the presence of vegetative reproductive structures and the capacity to occupy sites disturbed by human activities. According to these characteristics, a very broad definition of the term
1 ‘weed’ is given by Navas (1991): ‘A plant that forms populations that are able to enter habitats cultivated, markedly disturbed or occupied by man, and potentially depress or displace the resident plant populations which are deliberately cultivated or are of ecological and/or aesthetic interest.’
Relatively few plants have the characteristics of true weeds. Of the total number of plants in the world (about 250,000 species), only a few thousand are thought to behave as weeds (Ross and Lembi, 1999). Of these, about 200 species or
0.08% of the total are recognised as major problems in world agriculture (Holm et al., 1997). Holm et al. (1997) suggest that these 200 species account for 90% of the loss in world food crops. Only about 25 species or 0.01% of the total cause the major weed problems in any one crop.
Weeds compete with crop plants for nutrients, soil moisture and sunlight. The intensity of weed competition depends upon: (a) the type of weed species; (b) the severity of weed infestation; (c) the duration of weed infestation; (d) the competing ability of crop plants; and (e) the climatic conditions which affect weed and crop growth (Rao, 2000).
Reduction in crop yield has a direct correlation with weed competition.
According to Aldrich and Kremer (1997) weeds may reduce crop yields in two ways:
(1) by reducing the amount of harvestable product (grain, forage and so on) produced by the crop; and (2) by reducing the amount of crop actually harvested. Weeds can cause sizable machine losses when grain crops are harvested. The losses can be a result of: (1) the additional bulk provided by weeds interfering with threshing and separating of grain; and (2) weeds interfering with actual cutting and movement of the grain into the harvester (Aldrich and Kremer, 1997).
2 Depending on the degree of competition, weeds reduce crop yields by 10% to
25% (Rao, 2000). Parker and Fryer (1975) estimated that the world was losing annually 11.5% of the total food production. That is, if all the weeds in food crops were controlled, current world food production would be higher by 11.5 % or 450 million tonnes. The economic impact of weeds on the United States economy in the early 1990s equalled or exceeded USD 20 billion, with the agricultural sector alone accounting for USD 15 billion (Bridges, 1994). Combellack (1989) estimated the total cost of Australian weeds to be AUD 2 billion in 1986, of which AUD 137 million was for the purchase of herbicides. Crop yield loss from weeds is highest in the tropics. For example, in rice, the mainstay of Asia’s economy, proper weed control increases gain yields by 20% to 75% (Rao, 2000). In extreme weed situations, weed management could triple the yield of rice.
Weeds can have a detrimental effect on crop quality as well as quantity, particularly in harvested crops in which the presence of weed contaminants can result in direct monetary loss due to dockage (Ross and Lembi, 1999). For example in
Australia, cleaning 1% of the national wheat crop harvested with wild oats as contaminants at AUD 5/t would cost approximately AUD 1 million/year (Medd,
1996a).
Some weeds actively eliminate competition by producing toxins that enter the soil and prevent the normal growth of other plants (Rice, 1984). This phenomenon, known as allelopathy, reduces crop development more than is normally expected from competition for water, light, and nutrients alone. Allelopathic potential has been suggested in about 90 weed species (Putnam, 1986).
Weeds can also increase production costs because they harbour other pests
(Zimdahl, 1999). Insects, nematodes, fungi, bacteria and viruses use weeds as
3 alternate hosts, thereby increasing opportunities for those organisms to persist in the environment and reinfest crops in succeeding years.
1.2 WEED MANAGEMENT
Rao (2000) states that in weed management, the primary objective is to maintain an environment that is as detrimental to weeds as possible by employing both preventive and post-infested control measures by using various methods either alone or in combination. Weed management is essentially a skilful combination of prevention, control and eradication techniques in a crop or environment. These techniques are defined by Ross and Lembi (1999) as follows:
• Prevention is keeping a weed from being introduced into an uninfested area. Successful implementation depends on sanitation, the prevention of seed production, and the prevention of seed and other propagule spread. Weed and seed certification laws and regulations are enacted to meet these objectives.
• Control is the suppression of a weed to the point that its economic (or harmful) impact is minimised. It is the practice most frequently conducted once a weed is established. Control methods do not prevent all plants in an area from reproducing. A reservoir of propagules usually remains so that control practices must be continued year after year.
• Eradication is the elimination of all plants and plant parts of a weed species from an area. Eradication includes the destruction of seeds as well as any vegetative propagules such as rhizomes, and tubers. Elimination of a weed usually can be achieved only in the case of new, small-scale infestations. Once a large area becomes infested, almost no practical methods exist to eliminate long-lived buried seeds. Weed control techniques have been adopted widely because control is easier to do than prevention or eradication. Control can be made to work well with short- term economic or cultural planning goals. In contrast, prevention and eradication require long-term thinking and planning. Factors to consider in selecting control
4 techniques include compatibility, effectiveness and environmental effects. Control techniques include biological, mechanical, chemical and cultural applications.
Because of the complexity of environmental, economic and cultural concerns associated with weed management, programs that are based on a combination of techniques tend to be most successful.
1.2.1 Cultural control methods
Cultural methods are well known to farmers and weed scientists; they are regularly used even in the absence of conscious efforts to control weeds. Cultural methods include crop competition, planting date and population, crop rotation, companion cropping and fertility manipulation (Zimdahl, 1999).
The success of a weed management program depends on the competitiveness of crops and crop cultivars with weeds. Crops and cultivars with high plant vigour, which grow more rapidly, have an advantage over slow-growing and late-emerging weeds. They compete better with weeds for plant nutrients, sunlight, soil moisture and carbon dioxide. For example, the most weed suppressive of 20 winter wheat cultivars reduced weed biomass 82% compared to the least suppressive cultivar
(Wicks et al., 1986).
Another aspect of crop management is crop rotation. Crop rotation is feasible for annual, biennial, or short-term perennial crops. It is not available for long-term, established perennial crops. When one crop is grown for many years (monoculture), some weeds, if they are present in the soil seed bank, will be favoured and their populations will increase. Weed-crop associations can be changed by changing crop, time of planting, or weed control methods (Zimdahl, 1999). Wild oat seed reserves
5 can be reduced by winter fallowing in association with a rotation of wheat and sorghum over summer (Martin and Felton, 1993).
1.2.2 Mechanical control methods
Mechanical or physical methods of weed control have been employed ever since man began to grow crops. Mechanical methods range in complexity from hand hoeing to tillage operations with multicomponent machines such as cultivators. The most commonly used mechanical methods are hand hoeing and pulling, tillage, mowing, mulching, flooding and burning (Ashton and Monaco, 1991).
Hand hoeing and pulling are the earliest and most primitive type of weed control. They are still the major methods of control in underdeveloped countries. Of the 350 million farmers in the world, approximately 250 million still rely on hoes and wooden ploughs to weed and cultivate their crops (Hill, 1982). Hand hoeing, however, is more time consuming and expensive than other methods of weed control when conducted on large areas with heavy infestations of weeds. It is more practical for small areas or for high value crops.
Tillage provides effective weed control efficiently and economically. Soil erosion and the need for timely operations are the two major limitations. Use of tillage equipment on wet soils can result in compacted soil layers. Mowing can provide control of tall growing weeds and at least prevent them from producing seed.
Species with growing points near the soil surface are not controlled effectively by mowing. Mowing after pollination has limited to no adverse effect on seed production (Ross and Lembi, 1999).
6 1.2.3 Chemical control methods
The major area of expansion in weed control technology since World War ΙΙ has been the development of herbicides. Chemical control is generally more economical, more convenient and more effective than mechanical control. Over 400 herbicides have been developed and registered in the world for weed control in agricultural and non-agricultural systems (Rao, 2000). Today, sales of herbicides have outstripped those of all other classes of pesticides. Currently, herbicides constitute 55% of the world pesticide market (Rao, 2000). In 1990, herbicides accounted for 65% of total pesticides used in the United States and 85% of pesticides used on cropland (Delvo, 1990). A report by the United States Environmental
Protection Agency (Donaldson et al., 2002) has shown that in the U.S. the amount of herbicides used over the period 1980 to 1999 has fallen slightly from 1,053,000 lb of active ingredient in 1980 to 956,000 lb in 1999. However, over the same period annual expenditure on herbicides has risen from USD 1,104 m in 1980 to USD 1,546 m in 1999. Hence, the value of the herbicide market has increased.
When a herbicide comes in contact with a plant, its action is influenced by the morphology and anatomy of the plant as well as by numerous physiological and biochemical processes that occur within the plant. According to Ashton and Monaco
(1991), these processes include: 1) absorption; 2) translocation; 3) molecular fate of the herbicide in the plant; and 4) effect of the herbicide on plant metabolism. The interaction of these plant factors with the herbicide determines the effect of a specific herbicide on a given plant species. When one plant species is more tolerant to the chemical than another plant species, the chemical is considered to be selective
(Ashton and Monaco, 1991). A herbicide that is selective at a low rate may become non-selective when applied at a higher rate. For every herbicide, there is an optimum
7 rate at which it maintains its selective characteristic; this rate varies from one weed or crop species to another (Rao, 2000).
One of the major distinguishing characteristics between different herbicide programs is the time the chemicals are applied. These timings are determined by the species of weed, the time of germination of weed and crop plant and the growth stage of the weeds (Rao, 2000). In the broadest sense, herbicides can be applied either directly to the soil (soil-applied) or directly to the foliage of weeds (foliar-applied).
More specific application timings are preplanting, preemergence and postemergence
(Ashton and Monaco, 1991). Foliar-applied herbicides can be either contact or translocated herbicides. The translocated herbicides move from the site of entry to the site of action via the phloem or xylem. But contact herbicides move very little or not at all from the point of entry (Rao, 2000). Soil-applied herbicides are translocated chemicals that persist for long periods and are absorbed by germinating seedlings and established weeds.
Although herbicide use is not without problems, the benefits have far exceeded the risks. The most common problems associated with herbicides are related to their inherent ability to kill plants. Problems include injury to non-target vegetation, crop injury and residues in the soil from the previous season.
Inconsistent weed control also is a frequent problem. The continued use of some herbicides has resulted in the occurrence of herbicide resistance in some weed species (Ross and Lembi, 1999).
In general, the public has a negative perception of any synthetic chemical applied to the environment, including herbicides. The inability of the public to distinguish among insecticides, fungicides and herbicides has led to herbicides being categorised with chemicals that are detrimental to the environment. These concerns
8 have resulted in severe restrictions and regulations on the use of herbicides and have greatly increased the cost of development of new herbicides.
1.2.4 Biological control methods
Although organic herbicides have played a major role in providing the food and fibre required by the world population, as outlined above, there has been increasing concern about their safety in food products, their adverse impact on environment and the development of resistance to herbicides. These factors, coupled with rising costs of developing, testing and registering herbicides, have provided the impetus to develop alternative weed management strategies. In this context, biological control as an alternative or supplemental weed management method has the potential to play a major role in agricultural production systems.
Biological control is defined as the action of parasites, predators or pathogens in maintaining another organism’s population at a lower average density than would occur in their absence (Zimdahl, 1999). The term was first used by H.S. Smith
(DeBach, 1964). Biological approaches to weed management involve the use of agents such as insects, nematodes, fungi, bacteria and viruses. In fact, insects and fungi have long been used in biological control of noxious weeds.
The term, biological control, is often applied to the use of plant and microbial-based phytotoxic compounds, although, in essence, there is no difference between the use of a biologically produced chemical and one manufactured by synthetic chemical methods. Many fungi produce natural products that are responsible for phytotoxicity. In these cases, symptoms produced by the fungus or by its phytotoxin alone are identical and weeds can be controlled by using either the fungus or the phytotoxin. An example of this has been the study of Alternaria
9 alternata (Fr.:Fr.) Keissl. and its phytotoxin, AAL-toxin, by Boyette and Abbas
(1995). A. alternata f. sp. lycopersici was initially described as a pathogen of susceptible tomato varieties. The toxin responsible for the pathogenesis was discovered to be AAL-toxin. A. alternata itself has not been found to be pathogenic to other weed and crop species. However, AAL-toxin has been shown to have a broad host range including jimsonweed (Datura stramonium L.), black nightshade
(Solanum nigrum L.) and prickly sida (Sida spinosa L.) (Boyette and Abbas, 1995).
Several important crops are not significantly affected by AAL-toxin including cotton
(Gossypium hirsutum L.), corn (Zea mays L.), and soybean [Glycine max (L.) Merr.].
AAL-toxin has been patented for use as a herbicide. Other potentially useful natural product herbicides from micro-organisms are reviewed by Cutler (1999).
Biological control methods in general are gaining in importance. Rodgers
(1993) has estimated that biological pesticide sales are increasing by 10–25% per year while agrochemical markets are either static or shrinking. There are two main biological control strategies involving the use of living organisms for biological control: the classical strategy and the inundative strategy. These are discussed below.
1.2.4.1 Classical strategy
The classical or inoculative approach involves the importation and release of one or more natural enemies that attack the target weed in its native range into areas where the weed is introduced and is troublesome and where its natural enemies are absent. The objective of classical biological weed control is generally not eradication of the weed species, but the self-perpetuating regulation of the weed population at acceptable low levels (Wapshere, 1982). The classical strategy differs
10 from the inundative strategy primarily in that it is an ecological rather than a technological response to a weed problem.
The ideal biological agent causes severe damage to the target weed and is not a risk to other plant species in the area of introduction. Thus, safety regulations must be followed before organisms can be released into a new geographical area. These regulations vary from country to country. Australia is the only country which has an act of parliament covering biological control, the Biological Control Act 1984 No.
139 of 1984 (Cullen and Delfose, 1985). In other countries, the biological control of weeds is covered under existing plant protection and safety legislation.
Prior to 1970, insects were the main biocontrol agent, but subsequently fungal pathogens, especially rust fungi, have become increasingly more prevalent as biocontrol agents of weeds. One of the finest examples of biological control of weeds using insects was the introduction of the pyralid moth (Cactoblastis cactorum
Berg.) into Australia in 1926 (Dodd, 1927) for control of Opuntia stricta Haw.
(prickly pear). By 1935, about 95% of the prickly pear infestation had been destroyed.
A dramatic example of a successful introduction of an exotic plant pathogen is the rust, Puccinia chondrillina Bub. & Syd., introduced into Australia for control of skeleton weed, Chondrilla juncea L. (Hasan, 1972). Skeleton weed was effectively controlled in wheat (up to 79% control), resulting in a saving of approximately AUD 20 million annually (Cullen, 1985).
1.2.4.2 Inundative strategy
The inundative or bioherbicide strategy employs the concerted application of large doses of inoculum to a target weed (Charudattan, 1988). A single application
11 of the bioherbicide may be sufficient or repeated applications may be required throughout a season. The control agent and target weeds are usually natives. The inundative strategy employs ecological knowledge but is essentially technological.
The best control agents must be amenable to large-scale mass production and have a reproductive method that allows rapid population increase. Organisms used for inundation have been pathogens or nematodes rather than arthropods, which do not satisfy the above-mentioned criteria.
The majority of candidate bioherbicides studied are fungal pathogens.
Nevertheless, recently it has been shown that it is possible to consider the use of bacteria as bioherbicides. Bacterial bioherbicide research is focused primarily on use of rhizobacteria. In contrast to fungal bioherbicides using foliar plant pathogens, which have been mostly directed towards dicotyledonous weeds, rhizobacteria have been directed towards grassy weeds (monocotyledons) in cereal crops. More than
1000 rhizobacteria isolated from prairie soils have been screened for their inhibitory effects against annual grassy weeds (Boyetchko, 1997). Host-range tests with several isolates suppressive to downy brome demonstrated that the effects of the rhizobacteria and their secondary metabolites are host-specific and the amount of reduction in root growth was found to be concentration-dependent (Boyetchko and
Holmstrom-Ruddick, 1996). These authors also showed that the rhizobacteria had a little or no detrimental effects on non-target hosts.
Johnson et al. (1996) reported that the spray application of Pseudomonas syringae pv. tagetis (Hellmers) Young, Dye & Wilkie in aqueous buffer with a surfactant produced severe disease in Canada thistle, common ragweed, sunflower and certain other members of the Compositae under field conditions. Spray application of the bacterium without surfactant was ineffective on all reported hosts.
12 Zhou and Neal (1995) demonstrated that two strains of Xanthomonas campesteris pv. poannua Egli & Schmidt (X-PO) were similarly virulent to annual bluegrass (Poa annua L.) in both growth chamber and field tests. Johnson et al (1996) also showed that the X-PO had an ability to control annual bluegrass when applied by spray during mowing as the bacteria entered through mowing injuries, causing a lethal, systemic wilt. Application of the bacterium to annual bluegrass in the absence of fresh mowing injuries failed to produce symptoms. One of the challenges confronting the use of plant pathogenic bacteria as a biocontrol agent is the requirement of free water for dispersal and the need for wounds or natural openings, such as stomata or lenticels, for entry of the bacteria into the plant (Johnson et al.,
1996).
The terminology associated with the inundative strategy varies depending on the type of agent used. The general term ‘biopesticide’ applies to any biological agent or biologically derived chemical used to control pests. The term ‘bioherbicide’ is used when the target is a weed. The terms ‘biopesticide’ or ‘bioherbicide’ imply that the target is killed. All biological agents do not necessarily kill, but may have different effects on the target. Crump et al. (1999) suggest a standardised usage to clarify existing terms through a hierarchical classification of existing terms and the introduction of new terms such as biopestistat, bioherbistat and mycoherbistat.
Because the fungal pathogens used in this study suppress plant growth rather than causing death (Cother and Gilbert, 1994a; Hetherington et al. 2002), these fungi are best considered in their biocontrol roles as mycoherbistats. A mycoherbistat is defined as an inundatively applied fungal pathogen that reduces the competitive ability of the target weed to below a desired threshold (Crump et al., 1999).
Therefore, later in this thesis, from Section 1.3 onwards, the term mycoherbistat will
13 be only used when referring to fungal pathogens under this study. The term mycoherbicide (a fungal pathogen that when applied inundatively kills a plant by causing a disease) will be used for the pathogens in other studies as this term is used by the authors of these studies.
1.2.5 Integrated weed management (IWM)
Successful and sustainable weed management systems are those that employ combinations of techniques rather than relying on one. Biological control is easy to combine with other methods because, once established, it can be self-perpetuating.
To be successful, an integrated system requires a thorough knowledge of ecology of the weed-crop system. Knowledge of a farmer’s production goals and farming system is necessary but not sufficient. When the goal is weed management, thorough ecological understanding is required (Wapshere et al., 1989). A successful, integrated, weed management system is one where the weed population is reduced and maintained below a level causing economic losses.
Although weed management integrates preventive, cultural, mechanical, chemical and biological practices, the use of herbicides is an important component of integrated weed management (IWM) strategies for most major crops produced in the
United States (Smith, 1991). This reliance on herbicides is due to the availability of more than 140 herbicides for use in programs that combine preplant, preemergence and postemergence treatments for control of important weeds in most crops. In contrast, only two bioherbicides are registered for use in crop sites. The use of plant pathogens as well as other biological agents to control weeds offers a comparatively untapped source of technology for selective weed control in crops (Quimby and
Walker, 1982). Although little research has been conducted on the discovery and
14 development of pathogens for weed control compared with chemical herbicides, additional scientific and financial resources should identify new pathogens with mycoherbicide potential and permit successful use of additional biological weed control agents in IWM systems (McWhorter, 1984).
In USA, intensive research was conducted on the integration of the mycoherbicide COLLEGO with chemical herbicides, fungicides and insecticides, each alone and combined. Many chemical pesticides applied in tank mixtures or sequentially with COLLEGO have inhibited COLLEGO infection and control of northern jointvetch (Aeschynomene virginica (L.) B.S.P.) in rice crops while others applied in specific management programs have had a synergistic effect or no effect on COLLEGO activity. Consequently, interactions between chemical pesticides and plant pathogens with microbial herbicide potential range from enhancement to suppression of disease incidence (Smith, 1991).
Synergism between plant pathogenic fungi and crop protection chemicals has long been recognised in relation to crop disease. Iatrogenic disease, that is disease resulting from man’s management of the crop, especially in relation to chemical inputs, have been reviewed by Griffiths (1981). Plants use various physical and biochemical mechanisms to defend against pathogen infectivity, including callose deposition, hydroxyproline-rich glycoprotein accumulation, pathogenesis-related proteins (PR-proteins), phytoalexin production, lignin and phenolic formation and free radical generation. Some herbicides, plant growth regulators, specific enzyme inhibitors and other chemicals can alter these defences (Hoagland, 1996). Research has shown some synergistic interactions of fungal pathogens and herbicides with relevance to weed control. An example of this has been studied by Sharon et al.
(1992) in biochemical interaction of Alternaria cassiae Jurair & Khan and sicklepod
15 (Cassia obtasifolia L.). The pathogen caused an elevated level of production of a flavonoid phytoalexin in sicklepod that was found to be fungitoxic. Treatment with glyphosate suppressed this defence response of the weed by lowering phytoalexin production. Thus, glyphosate acted synergistically with this pathogen, by suppressing weed defences. Twenty-fold less glyphosate than is normally phytotoxic was required to suppress the phytoalexin production and increased the intensity of infection. Compatibility of pathogens with herbicides suggests that they could be integrated into existing weed management strategies to increase efficacy of pathogen-herbicide combinations, reduction of herbicide and pathogen levels required for weed control and expanded pathogen host range.
1.3 BIOLOGICAL CONTROL OF WEEDS USING MYCOHERBICIDES
Daniel et al. (1973) introduced the concept of the mycoherbicide.
Mycoherbicides are usually indigenous plant pathogenic fungi that are applied in inundative doses of inoculum to control weeds using techniques and methodology similar to that used with chemical herbicides (TeBeest and Templeton, 1985). The application of a massive dose of inoculum and its proper timing would shorten the lag period for inoculum build-up and pathogen distribution, essential for natural plant disease epidemics. This high dose of inoculum makes it possible to use any natural or artificial conditions favourable for disease development (Daniel et al., 1973).
1.3.1 Creating mycoherbicides
There are three phases, discovery, development and deployment, before a fungal pathogen is successfully registered and commercially available as a mycoherbicide (Templeton, 1982).
16 1.3.1.1 Discovery phase
In this phase, the target weed is selected and a survey of potential fungal pathogens is carried out. The suitability of a fungal pathogen can be estimated by understanding the disease cycle and by deduction from our knowledge of similar pathogens of economic crops that have been studied in great detail.
1.3.1.1.1 Selection of target weeds
Several efficient mycoherbicides have not been developed for commercial use by industry because of their low market potential (Charudattan, 1991). The market for a mycoherbicide that targets only one weed is quite restricted unless the product is active on an economically important weed that escapes control with traditional methods. Thus, from a commercial standpoint, weed species that have developed resistance to chemical herbicides or those for which no other methods are available for their control are ideal targets for mycoherbicides.
Factors like the morphology of the target weed can influence the level of control achieved. Generally, grassy weeds (monocotyledons) are more difficult to control with foliar mycoherbicides because their growing points are well protected from infection by foliar pathogens (Greaves and McQueen, 1992). Therefore, the majority of mycoherbicide projects have been aimed at dicotyledonous weeds
(Charudattan, 1991). With most weeds, plant death is more likely to occur if pathogens infect stems at or below the cotyledonary node. Regeneration through regrowth from roots or rhizome buds is a major obstacle to achieving long-term control of perennial weeds with foliar mycoherbicides (Greaves and McQueen,
1992). Perhaps complete control of a weed is not necessary. For example, a non- specific fungal seed pathogen, Pyrenophora semeniperda (Brittlebank and Adam)
17 Shoem., reduces seed production and seedling emergence of several annual grassy weeds in arable land (Medd and Campbell, 1996). The effect of sublethal infections by a mycoherbicide can be greatly enhanced by crop competition (Ditommasso et al.,
1996).
1.3.1.1.2 Selection of fungal pathogens
Factors such as conidium production, conidium characteristics, type of parasitism, culture methods, pathogenicity or host specificity are the main criteria for the selection of fungal pathogens as potential mycoherbicides. Daniel et al. (1973) defined the essential characteristics a pathogen must have for selection as a mycoherbicide as: (a) it must be able to produce abundant and durable inoculum in artificial culture; (b) it must be genetically stable and specific for the target weed; and (c) it must be able to infect and kill the weed in environments of reasonably wide latitude. More than half of candidate mycoherbicides tested belong to
Hyphomycetes, a large and varied class of conidial and non-sporulating fungi. The
Coelomycetes, which include Colletotrichum species, come second, indicating these fungi satisfy most of above-mentioned requirements. To be able to assess strain stability, genetic profiles of a mycoherbicide must be developed and variation assessed. In initial study of the genetic variability of Drechslera avenacea was made in Chapter 3.
The type of parasitism exhibited by a fungus affects its ability to serve as a mycoherbicide (Templeton et al., 1979). Since obligate parasites are typically less damaging to their hosts than facultative parasites or facultative saprophytes, they have less potential for use as mycoherbicides in situations requiring rapid and complete control (Charudattan, 1991). The technical difficulties in producing
18 obligate parasites are also a barrier to their use, but advances in ex planta culturing of obligate parasites may create new opportunities in the future for mycoherbicidal use of obligate fungi.
1.3.1.2 Development phase
The first step in the development phase is the isolation of the collected pathogens in pure culture, re-establishment of disease and its re-isolation using basic plant pathology methods. After successful re-isolation of the pathogen, the infective units of the candidate mycoherbicide must be evaluated. From a standpoint of practicality and economics, the infective units of the candidate mycoherbicide must be produced in a timely and cost-effective manner (Boyette et al., 1991). Some fungi, such as Rhizoctonia spp., normally do not produce conidia; therefore, mycelial fragments are used as inoculum. Because many of these types of fungi are rather non-specific in host preference, they have not been extensively evaluated as mycoherbicides (Templeton et al., 1979). However, infection of the host directly by mycelia growing from the substrate has been observed for Sclerotinia sclerotiorum
(Lib.) de Bary (Brosten and Sands, 1986). With few exceptions, the most suitable infective units are fungal conidia (Boyette et al., 1991). Therefore, sporulation behaviour and ability to sporulate on a different range of solid or liquid media are evaluated for each candidate mycoherbicide. If spore production is sufficient, the optimal environmental conditions for infection and its environmental tolerance are determined. The most virulent fungi will be tested for their host specificity and for efficacy under field conditions. The determination of host range is an important component in the development phase. The safety of non-target economic and wild
19 plants must be considered before experimental release and commercial use
(Weideman and TeBeest, 1990).
Efficacy is defined by Charudattan (1989) as the ability to provide a satisfactory amount, speed, and ease of weed control. In practice, all of these three aspects must favour the mycoherbicide’s use. Thus, the level or amount of weed control should be satisfactorily high, the control should be rapid depending on the weed situation, and it should be easy to use over a fairly broad range of environmental conditions and with ongoing pesticide schedules to obtain satisfactory performance of the mycoherbicide.
1.3.1.3 Deployment phase
In the deployment phase, selected fungal pathogens that have shown the potential to become an efficacious, safe, economical and a high-quality product in the development phase are developed for practical use. At this stage, a patent is obtained either for the novel use of a pathogen or for the production or application process. The pathogen is mass-produced and formulated as product that must be stable, viable and effective for a reasonable length of time. A shelf life of 1–2 years
(Daigle and Connick, 1990) is desirable in order to allow for packaging, distribution and storage at the point of sale and on the farm. After successful performance of the formulated product, the pathogen is registered as a mycoherbicide and is sold commercially.
An important consideration during the deployment of any biological control agent is the possible of tracking its spread and its frequency within local populations of the pathogen. There is the possibility of gene flow, changes in gene frequency due to movement of genes from one population to another (Slatkin, 1987), resulting in
20 the introduction of rare virulence alleles from the released organism into a local population of the pathogen (Templeton et al., 1979). Gene flow is affected by factors such as the isolation of populations by distance or natural barriers to dispersal can allow evolutionary forces such as selection, mutation and genetic drift to affect gene flow in local populations (Hintz et al., 2001). Analysis of the genetic structure of a species using molecular methods such as RAPD-PCR can reveal information about its population structure and permit risk analysis. Initial genetic finger printing of isolates of D. avenacea was carried out in Chapter 3 to determine if this type of study is possible for this mycoherbistat.
1.3.2 Formulation of mycoherbicides
Formulation of mycoherbicides have been recently reviewed by Greaves et al. (1998); Green et al. (1998); Boyetcho et al. (1999); and Daigle and Connick
(2002). The most frequently cited reason for variable field performance of a mycoherbicide is the constraint imposed by climate after application. Fungal conidia generally require free water, or at least exposure to high humidity, to germinate and to infect the plant and frequently, moisture is not available for long enough after application. Similarly, the conidium may be inhibited by low or high temperatures, by UV radiation or lack of available nutrients. Most of these constraints can be reduced or eliminated by appropriate formulation (Greaves et al., 1998).
Formulation is the blending of an active ingredient, such as fungal conidia, with a carrier or solvent and often other adjuvants in order to alter physical characteristics of the mycoherbicide to a more desirable form. This includes diluting the final product to a common potency, enhancing stability and biological activity,
21 improving mixing and spraying and integrating the mycoherbicide into a pest management system (Boyette et al., 1996).
The development of formulations is essential for mycoherbicides to be an alternative option in weed management. Most formulations of biological control agents are largely based upon techniques developed for formulation of agrochemicals
(Rhodes, 1990); however, the use of organic solvents and surfactants can be detrimental to biological propagules (Connick et al., 1991a). Connick et al. (1991a) suggested that processes and ingredients employed by the food industry have promise for mycoherbicide formulations, such as alginate and invert emulsions made from lecithin. To date, most mycoherbicides formulations have concentrated on maintaining fungal agent viability in storage and reducing dew requirements.
The type of formulation used for a mycoherbicide depends upon the type and mode of action of the pathogen and available application technology. A granular formulation, based on alginate (Walker and Connick, 1983) or wheat gluten
(Connick et al., 1991a) for example, is best suited for soil-applied mycoherbicides.
Granular formulations may buffer environmental extremes, serve as a food base for the agent and make the mycoherbicide less likely to be washed away by rainfall so increasing its persistence (Boyette et al., 1991). Another advantage of granular formulations is that they allow controlled release or growth of the organism from the formulation. This means that the organism can replicate in the environment and grow to the site of action, provided a suitable substrate and environmental conditions are available (Rhodes et al., 1990).
In the case of foliar-applied mycoherbicides, the propagule remains on the leaf surface after application and is exposed to rainwash, abrasion, UV radiation and desiccation, all of which may reduce its viability (Greaves and Maqueen, 1990;
22 Rhodes, 1993). Many potential mycoherbicides require more than 12 h of free water
(dew period) for conidium germination and penetration of the host (Auld and Morin,
1995). Therefore, specific timing of applications is necessary to coincide with moist environmental conditions (Klein et al., 1995). Such problems may be overcome by developing formulations that protect fungal conidia until host penetration is complete
(Rhodes, 1990) and, in so doing, increase the window of timing of application.
The simplest mycoherbicide delivery system for foliar application contains fungal conidia formulated as a sprayable suspension in water (Connick et al., 1990).
This formulation is used most frequently in the early stages of evaluation of a potential mycoherbicide for efficacy (Daigle and Connick, 1990). However, various adjuvants and amendments have been used either to improve or modify conidium germination, pathogen stability and virulence, environmental requirements or host preference of a potential mycoherbicide (Boyette, et al., 1996). An adjuvant is defined by Foy (1989) as a compound that assists or modifies the action of a principal active ingredient. Adjuvants encompass a wide range of compounds including surfactants, sticking agents, inert carriers, antifreezing compounds, humectants, sunscreening agents, antievaporation agents and micronutrients
(Prassad, 1993; 1994).
The first formulations were surfactant-based formulations, which were already widely used with chemical herbicides. Surfactants perform two functions: they help to disperse the conidia in the tank mix and they serve as wetting agents to minimise runoff and the resulting loss of active ingredient from the target weed
(Daigle and Connick, 1990). In addition, surfactants may improve propagule adhesion and interfere with the structural integrity of the target surface waxes (Geyer and Schonherr, 1988) thereby assisting host penetration by the pathogen.
23 Surfactants, such as the Tween series, have been used widely in mycoherbicide formulations (Daigle and Cotty, 1991).
Since certain synthetic surfactants were found to be toxic to fungi (Prasad,
1993; 1994), there has been interest in the use of biosurfactants in mycoherbicide formulations. For example, the peptidolipid, viscosin, produced by Pseudomonas fluorescens (Flugge) Lehmann & Neumann has potential for practical application as a biosurfactant (Laycock et al., 1991). Schisler et al. (1991) demonstrated that strains of phylloplane bacteria could be employed to promote in vitro appressoria formation by conidia of Colletotrichum truncatum (Schwein.) Andrus & W.D.
Moore and, when used as co-inoculants, enhance the severity of disease symptoms incited on Sesbania exaltata (Raf.) Rydb. ex A.W. Hill exposed to only 6 h of dew.
These authors suggested the possible contribution of these bacteria to leaf wetability.
Nutrients, mainly carbohydrates, have been added to formulations for utilisation by the control agent to enhance mycoherbicide performance (Womack and
Burge 1993). For example, the addition of small quantities of sucrose and gum xanthan to aqueous conidium suspensions of C. truncatum increased conidium germination and the severity of anthracnose on Desmodium tortuosum (Sw.) DC.
(Cardina, 1986).
Recently, oil-in-water and water-in-oil emulsions have attracted much attention. The first emulsion used in mycoherbicidal formulation, an invert emulsion, was developed by Quimby et al. (1988) to improve the control of C. obtusifolia
(sicklepod) using A. cassiae. It contained paraffin wax, mineral paraffinic oil and soybean oil in its oil phase. Lecithin was used as the emulsifier and the water to oil ratio was 1:1. The formulation significantly reduced the dew period requirement of
A. cassiae as the emulsion retarded water evaporation and provided a water source
24 for conidium germination and infection of the target weed. The conidia were applied in an aqueous carrier followed by an overspray of an invert emulsion applied with specialised air-assist atomising nozzles. Later, Daigle et al. (1990) improved the emulsion so that only one spray was needed, incorporating the conidia into the emulsion itself. Despite their advantages, there are a number of disadvantages associated with invert emulsion formulations. Invert emulsions are complex to prepare and highly viscous so that conventional spray apparatus cannot be used for their application. Connick et al. (1991b) reported development of an invert emulsion that exhibited lower viscosities and greater water retention properties. Yang and
Jong (1995) found that an invert emulsion formulation of Myrothecium verrucaria
(Albertini & Schwein) Ditmar:Fr. could be easily applied using a garden sprayer at temperatures of 25°C and above, but problems associated with increasing viscosity occurred at 20°C. Womack et al. (1996) developed an invert emulsion to assess soybean oil as an alternative, polar, oil phase for the delivery of Ascochyta pteridis
Bres. conidia to Pteridum aquilinum (L.) Kuhn. The formulation was highly stable with low viscosity and showed no evidence of fungitoxicity. The formulation actually promoted germ tube extension and was able to retain water longer than the 9 h dew period required by A. pteridis for infection.
Oil suspension emulsions of mycoherbicides have been investigated as less expensive, easy to prepare alternatives to oil invert emulsion formulations, which can be applied with conventional spray equipment and effectively used at relatively reduced volumes. Egley and Boyette (1995) found that after a 24–72 h dew delay,
89–97% control of S. exaltata was achieved using conidia formulated in an unrefined corn oil emulsion. The emulsion enhanced mycoherbicide efficacy by stimulating
25 conidia germination by protecting the conidia during a dew-free period, hence, increasing weed infection when a dew occurred.
1.3.3 Products on the market
Mycoherbicide research programs are being undertaken throughout the world.
Three books (Charudattan and Walker, 1982; Hoagland, 1990; TeBeest, 1991) and several reviews have been published (Wilson, 1969; Templeton et al. 1979; TeBeest and Templeton, 1985; Charudattan, 1988; TeBeest et al. 1992; TeBeest, 1996;
Charudattan, 2000; Khetan, 2001). Charudattan (2000) in his latest review considers that for the small number of potential biocontrol pathogens explored so far, about
250 by some recent estimates, the return on the investment made in mycoherbicide research has been quite good.
A total of six fungal pathogens have been registered for commercial use as mycoherbicides since 1980 (Table 1.1). DeVine, the first mycoherbicide to be registered (Kenney, 1986), is based on chlamydospores of the fungus produced upon fermentation in a vegetable juice medium. Chlamydospores, the infective units, are not stable and the formulated material has a shelf-life of only 6 weeks and must be handled like fresh milk (Anonymous, 1981).
COLLEGO is a selective postemergence mycoherbicide. It is formulated as a two-compound product, with one component consisting of water-suspendible, dried conidia and the other containing a water-soluble conidium rehydrating agent
(Anonymous, 1982). Both components are packaged in an 18.9 L plastic mixing container. A mixture containing 0.95 L of rehydrating agent and one package of
10 conidia will treat 4 ha. The dried conidia are sold in unit packs of 75.7 × 10 viable conidia. Components of the two packages are added to the desired volume of water
26 just prior to application. Since 1997, when Encore Technologies began marketing
Collego, the market size for this mycoherbicide has increased steadily to 24,000 ha in
1999 (Charudattan, 2000).
TABLE 1.1. Registered mycoherbicides (adapted from Khetan, 2001). Pathogen Target Weed Crop Trade Name Manufacturer Alternaria cassiae Sicklepod Soybean Casst Mycogen Corp (Cassia San Diego, CA obtusifolia)
Colletotrichum Northern Rice and Collego Pharmacia & gloeosporioides f. jointvetch irrigated Upjohn sp. aeschynomene (Aeschynomene soybean Kalamazo, MI; virginica) Encore Technologies, Minnetonka, MN
Phytophthora Strangler vine Citrus DeVine Abbott Labs. Palmivora (Butl.) (Morrenia groves Chicago, IL, USA odorata)
Colletotrichum Round-leaved Vegetable BioMal Philom Bios gloeosporioides mallow (Malva crops and Saskatoon, Canada f. sp. malvae pusilla) straw- berries
Puccinia Yellow Sugarcane, Dr. Tifton Innovation canaliculata nutsedge maize, BioSedge Crop., Tifton, GA (Schw.) (Cyperus potato, esculentus) cotton and soybean
BioMal is formulated by using silica gel as a carrier. It provides over 90% control of the target weed. The wettable powder formulation disperses easily in water and is applied as a spray to the weed (Mortensen, 1988).
CASST causes a foliar blight disease on its host plant. It is applied at the rate of 1.1 kg/ha in 76.7 L water, is formulated an oil-based adjuvant and has been considered for commercial development as a wettable powder (Bannon, 1988).
27 Dr. Biosedge causes a rust disease on its host plant. It has been reported to be effective in controlling target weed using the inundative approach (Phatak et al.,
1987).
1.3.4 Benefits, constraints and prospects
Benefits
From society’s point of view, the use of mycoherbicides to control weeds provides an environmentally-friendly approach that is the main benefit of the mycoherbicide strategy. In general, most mycoherbicides do not damage non-target organisms, are not toxic to mammals and do not contaminate soil or groundwater
(Auld, 1991).
The development of resistance of a weed to mycoherbicide is lower than to a chemical herbicide, possibly due to limited field use and the multiple mechanisms involved in pathogenesis (Greaves and MacQueen, 1992). In addition, their use can retard the development of resistance to existing chemical herbicides by reducing the frequency of use.
The potential of mycoherbicides, in particular, is seen in areas that are served inadequately by chemical herbicides. These areas include: (1) control of parasitic weeds; (2) control of weeds closely related to crops, in which case a high degree of selectivity is necessary; (3) control of weeds resistant to chemical herbicides; and (4) control of weeds infecting small, specialised areas where development of chemical herbicides would be too costly (Templeton et al., 1986).
Mycoherbicides are cheap to produce. For example, COLLEGO cost approximately USD 2 million in research and development in the late 1970s and
28 early 1980s compared with USD 15 to 20 million to discover and develop a chemical herbicide at that time (Templeton, 1986).
Constraints
Auld and Morin (1995) itemised constraints to the development of commercial bioherbicides into four areas: (1) biological constraints; (2) environmental constraints; (3) technological constraints; and (4) commercial limitations. This section is briefly organised in the same manner.
Biological constraints. Biological constraints include host variability and host range (Gabriel, 1991; Leonard, 1982). Mycoherbicides are often too specific to one or a small group of plants. From the perspective of safety and registration this is desirable where a weed is closely related to the crop in which it is to be controlled.
Economically, this limits the potential use of candidate mycoherbicides because it is rare that only one weed species predominates in row crop situations (McWhorter and
Chandler, 1982). The simplest way to overcome this limitation is by applying mixtures of pathogens to mixed weed populations. For example, the rice weeds northern jointvetch and winged water primrose [Ludwigia decurrens (Walt.) D.C.] can be simultaneously controlled with a single application of Colletotrichum gloeosporioides (Penz.) Penz. & Sacc. f. sp. aeschynomene and C. gloeosporioides f.sp. jussiaea (Boyette et al., 1979). It also may be possible to alter the host selectivity of some mycoherbicides through formulation. Host selectivity of
Alternaria crassa (Sacc.) Rands., a mycoherbicide for jimsonweed (D. stramonium), can be altered either by the addition of water-soluble filtrates of jimsonweed or dilute fruit pectin to conidium suspensions. Several resistant plant species exhibited various elevated levels of susceptibility following these amendments, including
29 hemp sesbania, eastern black nightshade (Solanum ptycanthum Dunn.), cocklebur
(Xanthium strumarium L.) and showy crotalaria (Crotalaria spectabilis L.). Through proper timing and placement of inoculum, it is possible that this system could be used in a practical method to increase the host range of this pathogen (Boyette and
Abbas, 1994).
Many fungal pathogens are not sufficiently pathogenic to control a weed when used alone as mycoherbicides. To overcome this problem, attempts have been made to exploit synergistic interaction with chemical herbicides (Hoagland, 1996)
(see also Section 1.2.5).
Environmental constraints. The main limitation on mycoherbicides is the constraints on performance imposed by environmental variables (Greaves et al.,
1998). Extensive attempts have been made to overcome these constraints (see
Section 1.3.2). Compatibility of mycoherbicides with other pesticides is another major problem that has to be considered, as mycoherbicides are likely to be part of an integrated pest management system (Hoagland, 1996).
Technological constraints. Mass-production of viable, infective, and genetically stable propagules of a plant pathogen is a major requirement in the development of a mycoherbicide (Churchill, 1982). Current industrial preference favours submerged liquid fermentation over solid-substrate fermentation to produce mycoherbicide products (Churchill, 1982). Although successful, cost-effective and readily available, this technique is not suitable for fungi that do not sporulate in submerged culture. Sporulation in culture could be a difficult step, requiring special techniques. This, coupled with technological problems in mass culturing, formulation, shelf life (preferably 1–2 years) and delivery could add further constraints.
30 Commercial limitations. The small niche markets of most potential mycoherbicides to date have deterred industry from getting involved in mycoherbicide production due to limited opportunities to recover the cost of development, registration and large-scale production (Charudattan, 1990).
Moreover, mycoherbicides are intended to be used and regulated in a manner similar to the chemical herbicides (TeBeest and Templeton, 1985); therefore, the mycoherbicide technology is much like that of the chemical herbicides. Although it is generally assumed that mycoherbicides will be less costly to develop than conventional chemical herbicides, there will, nonetheless, be a cost associated with mycoherbicides that must be borne by the users. Because of this, mycoherbicides can be used only in situations such as crops where the expense of weed control is justified. Under this strongly economic demand, it is imperative that a candidate mycoherbicide for development be highly efficacious and cost-effective
(Charudattan, 1989). There are a limited number of cases where mycoherbicides have been
Prospects
From scientific and practical perspectives, inundative control of weeds with indigenous fungi is a successful and promising technology. In spite of the commercial limitations on the development of mycoherbicides, research in this field has been sustained by the public’s demand for non-chemical, weed control alternatives. However, mycoherbicides are considered as complementary adjuvants to current IWM systems rather than as alternatives to chemical herbicides.
Several research priorities need to be addressed in the near future in order to advance this field further (Charudattan, 1991). The most important ones are: (1)
31 development and registration of additional mycoherbicides; (2) integration of mycoherbicides with chemical pesticides; (3) development of suitable formulations to improve viability, efficacy and ease of application of mycoherbicides; (4) genetic improvement of pathotypes for altered host range, increased virulence, toxin production and resistance/tolerance to chemical pesticides; (5) increased public and private funding as well as administrative support for research and development of mycoherbicides; and (6) education of scientists unfamiliar with mycoherbicides and for technology transfer to the end user.
1.4 PLANT-PATHOGEN SYSTEMS STUDIED IN THIS PROJECT
Three plant pathogenic fungi, Rhynchosporium alismatis (Oudem) J.J. Davis, a pathogen of Alisma lanceolatum With. and Damasonium minus (R.Br) Buch.,
Colletotrichum orbiculare (Berck. et Mont.) v. Arx, a pathogen of Xanthium spinosum L., and Drechslera avenacea (M.A. Curtis ex Cooke) Shoem., a pathogen of Avena fatua L., were selected as suitable plant-pathogen systems on the basis of the findings of Cother and Gilbert (1994a,b), Hetherington and Auld (1996) and
McRae (1989), respectively. Detailed descriptions of above mentioned plant- pathogen systems are given below:
1.4 1 Alisma lanceolatum and Damasonium minus-Rhynchosporium alismatis
1.4.1.1 Alisma lanceolatum and Damasonium minus
A. lanceolatum (water plantain) and D. minus (starfruit) belong to the family of the Alismataceae which is a monocotyledonous family of erect, aquatic or marsh herbs mainly from the Northern Hemisphere, usually with perennial rootstocks.
These species are emergent, that is, their leaf blades and flowers are above water
32 level. Leaves on long petioles arise from the base of the stems (Auld and Medd,
1992).
A. lanceolatum is a perennial aquatic plant with lanceolate leaves on petioles up to 800 mm long (Figure 1.1). Each leaf has prominent parallel longitudinal vein and the flowers have pale pink petals in large open panicles. A. lanceolatum spreads from corms or seeds and can produce up to 25000 seeds/plant (Haymann, 1988).
This introduced species has become a significant weed in water-sown rice in NSW since it was first recorded in the early 1980s. Management options appear limited, while water sowing remains the most utilised sowing method (Pollock et al., 1994).
FIGURE 1.1. Alisma lanceolatum (from Anon a, undated)
D. minus, which is a native of Australia, is an annual or short-lived perennial aquatic plant. Its leaves have 3–5 longitudinal parallel veins. The species is
33 recognised by its characteristic small (about 10 mm diameter) star-like fruit that is composed of 6–10 (usually 9) united triangular sections (Figure 1.2). D. minus is widespread in NSW and occurs in all other States. It is a weed of rice and occasionally of channel systems (Auld and Medd, 1992).
FIGURE 1.2. Damasonium minus (from Auld and Medd, 1992)
Control strategies for these weeds have been reliant on the herbicide, Londax
(bensulfuron-methyl w.p.). A. lanceolatum has been a difficult weed to control with only variable results obtained using rates of Londax up to three times the recommended dosage (Pollock, 1992). Additionally, the emergence of D. minus populations resistant to Londax in Australia (Graham et al., 1996) makes these weeds a good targets for biological control with mycoherbicides.
34 1.4.1.2 Rhynchosporium alismatis
R. alismatis belongs to the order of Moniliales, the largest group of the
Deutromycetes and is often separated as the class Hyphomycetes. Spermosporina alismatis (Oudem) U. Braun has been reported as a synonym for R. alismatis
(Jahromi et al., 1998). No teleomorph has been reported.
R. alismatis has been recorded in Africa, Europe and North America on A. plantago-aquatica L., Sagittaria heterophyla Bert. ex Steud., S. rigida Pursh and S. subcordatum (Punithalingham, 1988; Far et al., 1989) and on A. plantago-aquatica var. orientale in China (Tai, 1979). Cother et al. (1994) reported the occurrence of
R. alismatis for the first time in Australia causing necrotic leaf spots on two important aquatic weeds of rice, A. lanceolatum and D. minus and also on the native species A. plantago-aquatica. This was the first record of R. alismatis in any country on A. lanceolatum and D. minus.
Cother and Gilbert (1994a) reported the effects of R. alismatis on the growth of A. lanceolatum and showed its pathogenicity to, and growth suppression of, five other species in the Alismataceae (Cother and Gilbert, 1994b). In addition to
Alismataceae weeds, R. alismatis infects Vallisneria (Hydrochardiaceae), Triglochin
(Juncaginaceae), Marsilea (Marsileaceae), barley, oats, triticale, lupin, soybean, lettuce and tomato, but in most instances, it produces only hypersensitive flecks or other minor symptoms. The use of this pathogen as a mycoherbistat for weeds in the
Alismataceae is considered to pose a negligible risk to crops grown adjacent to, or in rotation with, rice crops in southern Australia (Cother, 1999).
Cultures of R. alismatis are slow growing and compact, initially white but later pinkish/orange, sporulating after 3 days to produce hyaline, straight to slightly curved, 1-septate conidia from hyaline conidiogenous cells (Cother et al., 1994). R.
35 alismatis sporulates on a range of media and those based on lima beans produce the largest numbers of viable and infective conidia (Jahromi et al., 1998). Sporulation, germination and germ-tube elongation are greatest at 25ºC and 30ºC. The fungus also sporulates abundantly in liquid media (Jahromi et al., 1998). Cother and Van de
Ven (1999) reported a significant influence of nutrient composition of liquid-shake cultures on sporulation by R. alismatis and on their subsequent infectivity to A. lanceolatum.
1.4.2 Xanthium spinosum -Colletotrichum orbiculare
1.4.2.1 Xanthium spinosum
X. spinosum, commonly known as Bathurst burr, is a shrubby plant originating in South America. It is in the Composite or daisy family, Asteraceae, which includes many weeds, such as thistles, as well as useful plants. It has spread to become a major weed in many regions of the world including the Mediterranean,
North and South Africa, the USA and New Zealand. Bathurst burr is believed to have first arrived in Australia in the early 1800s as burrs tangled in the tails of horses imported from Chile (Maiden, 1920). It became established near Bathurst in NSW from where it takes its name (Martin and Carnahan, 1982). Bathurst burr has been recorded in all states and territories of Australia. It is prevalent in the warm temperate to semi-arid regions but does not occur or is uncommon in the cooler tablelands and the Australian Capital Territory (Hocking and Liddle, 1986).
Bathurst burr is an erect, much-branched, bushy plant. It usually grows to about 45 cm high but may reach 1.3 m (Parsons, 1976). It is best recognised by the presence of three-pronged, straw coloured spines set in the angle between leaf stalk and stem (Holm et al., 1977) (Figure 1.3). The leaves are arranged alternately on the
36 stem. They are thick in texture with three lobes; a large central lobe and two smaller side lobes. The upper surface is dark green with prominent veins, while the lower surface is pale with downy hairs. The stem is hairless (Parsons and Cuthbertson,
1992). Bathurst burr flowers are small and inconspicuous. They are greenish white and set in the angle of the leaves. Those near the top of the stem are mostly male while female flowers form further down. Egg-shaped burrs about 10 mm long form from the female flowers. These are covered with numerous hooked spines, perfectly adapted for attaching to wool and other fibres (Figure 1.4) (Parsons and Cuthbertson,
1992).
FIGURE 1.3. Bathurst burr has conspicuous spines and burrs (from DPIWE, 2004)
Bathurst burr is mainly a pasture weed but it is also a weed of summer growing crops such as cotton, sugarcane, grapes, soya beans, maize and some vegetables (Holm et al., 1977). The weed spreads entirely by seeds. Burrs are usually carried by people or animals, attached to clothes, fur or other fibres by their hooked spines (Parsons, 1976). Burrs may also be dispersed by water or as a
37 contaminant in the seed of summer crops or in weedy hay (Holm et al., 1977).
Although Bathurst burr is a summer growing annual plant, seeds can germinate at other times of the year if conditions are favourable. It reproduces only from seed.
Some mature plants (Figure 1.5) survive well into winter, with the result that burrs may be found for most of the year (Parsons and Cuthbertson, 1992).
FIGURE 1.4. Burrs showing hooked spines (from AWTA, 2004)
The plant flowers from January until autumn, producing up to 150 burrs per plant. Each burr has two cells, with each cell containing one brown, flattened seed about 9 mm long. Seeds are known to remain viable for at least three years (Parsons,
1976). Dormancy depends on the permeability of the seed coat. High temperatures break this down. Day length and daytime temperatures may also control germination. The result is a staggered germination, which makes seedlings difficult to control and allows the plant to exploit the variable environmental conditions under which it grows.
38
FIGURE 1.5. Mature plant of Bathurst burr (from Snowy River Shire Council, 2002)
Bathurst burr seedlings are poisonous, especially the cotyledons (seed leaves). Young plants are quite palatable to stock, but older plants are not eaten
(Everist, 1974). The main problem in Australia is burr contamination of wool
(Martin and Carnahan, 1982). The burrs are difficult to remove during processing, so buyers pay lower prices for contaminated fleeces. Burrs also make stock unpleasant to handle.
Control programs should aim to prevent seeding for 3–4 years. On arable land, cultivation is effective when carried out in the seedling stage but must be repeated after each germination. On non-arable areas, spraying with 2, 4-D or
MCPA is effective, particularly on young plants. To be sure of preventing seeding, plants must be treated before any burrs are formed. Ametryn kills seedlings in situations where 2, 4-D and MCPA might cause damage to nearby susceptible plants such as cotton, but is non-selective. Other herbicides that give selective control in a number of crops are available. They include metribuzin, MSMA, picloram, dicamba,
39 2, 4-DB, MCPA, fluometuron, atrazine and pendimethalin (Parsons and Cuthbertson,
1992).
Bathurst burr has been eliminated from many areas by hand hoeing but the mistake is often made of hoeing after the burrs have formed and leaving the plants to die where they are cut. Seeds in these burrs may have already developed or at least are capable of maturing on the cut plants. Thus, little is achieved by cutting when the burrs have formed unless the plants are gathered and destroyed, preferably by burning (Parsons and Cuthbertson, 1992).
1.4.2.2 Colletotrichum orbiculare
C. orbiculare belongs to a species complex within the anamorph genus of
Colletotrichum Cordal in the order Melanconiales of Coelomycetes which is a small, acervuli-forming group of the Deutromycotina. Glomerella lagenarium Stev. has been reported as the teleomorph (Sutton, 1992). Nine synonyms have been reported for C. orbiculare (Sutton, 1980). The most common synonym is C. lagenarium
(Pass.) Ell. & Halst.. The feasibility of using C. orbiculare as mycoherbicide to control X. spinosum has been extensively studied by McRae (1989).
In addition to Xanthium, C. orbiculare can infect certain members of the
Apiaceae, Asteraceae, Boraginaceae, Canellaceae, Cucurbitaceae, Fabaceae,
Malvaceae, Myrtaceae and Solanaceae families. However, the symptoms on these families may only consist of small patches of dead cells due to the induction of the hypersensitive response. Infection on X. spinosum by C. orbiculare may be expressed as leaf and seedling blight or as stem anthracnose, depending upon the age of the host plant, the initial inoculum and environmental conditions (McRae, 1989).
40 The C. orbiculare in Australia was originally called C. xanthii Halst. by
Butler (1951); however, no material of Butler's collection remains. Comparison of recent isolates from Xanthium spinosum with C. xanthii-type material demonstrates that the two are distinctly different morphologically (McRae, 1989). Current isolates fit the description of C. orbiculare as defined by Simmonds (1965) and Sutton
(1980).
Identification of C. orbiculare, as well as the whole concept of species in the genus Colletotrichum, is often uncertain, ambiguous and unreliable (Sutton, 1992).
Identification keys are mainly based on conidium shape, conidium size and host specificity (Sutton, 1980); however, the morphological characteristics can vary widely between isolates of the same species.
C. orbiculare is readily isolated and cultured on potato dextrose agar at 25°C in the dark. Conidia are produced abundantly on a number of artificial media under fluorescent lights. The optimum temperature for sporulation is 20–25°C and for conidium germination is 22–27°C. The conidium matrix contains enzymes such as cellulase, non-specific esterases, invertase, pectin esterase and pectic lyase that may facilitate fungal penetration (McRae and Stevens, 1990).
C. orbiculare demonstrated good efficacy in killing X. spinosum in controlled environments 14–16 days after inoculation when exposed to optimum dew temperatures of 20–25°C for periods of 8–48 h. A period of darkness during the dew period, after conidium germination, enhanced disease development (McRae and
Auld, 1988). Under field conditions the kill, varied from 81–100% after eight weeks and was highest when conidia were applied to younger plants at a concentration of
106 per mL in the late afternoon (McRae, 1989; Auld et al., 1990).
41 1.4 3 Avena fatua-Drechslera avenacea
1.4.3.1 Avena fatua
The genus Avena belongs to the family of the Poaceae (Gramineae) that contains the important crop plant, oats and several species referred to as ‘wild oats’.
Wild oats are among the more serious weeds of cereals throughout the world. The most important of these is Avena fatua, which is a weed of more than 20 crops in 55 countries (Auld and Medd, 1992).
A. fatua is an erect annual grass with extensive fibrous root system growing to a height of 60–100 cm (Figure 1.6). Its leaves are flat, with broad base and acute apex, 7–20 cm long and 5–15 mm wide. Culms are smooth, erect, stout and in small tufts whilst the sheaths are smooth or slightly hairy on the margins on younger plants. The panicle is loose and open, slender branches ascending and rough (Figure
1.7). The spikelets are made up of 2 to 3 florets enclosed by a pair of papery bracts
(Figure 1.8); each floret bears a long, abruptly bent bristle. The seeds of A. fatua are
6–10 mm long and the most characteristic distinction from cultivated oats is the presence of long silky hairs, especially around its base. The panicles of wild oats are looser and more widely spaced than those of cultivated oats. The seeds shatter and fall to the ground before wheat or a cereal is harvested and a small percentage may persist in the soil for many years depending on edaphic and other conditions. The seeds contaminate the crop seeds and get disseminated to new places. Freshly harvested seeds are dormant but germinate well after a prolonged storage at 20–25°C
(Rao, 2000).
42
FIGURE 1.6. Avena fatua (from ANON b, undated)
Wild oats are weeds of great economic importance and represent a serious form of degradation in winter cereals, which are the major crops in Australia (Medd,
1996a). Medd and Pandey (1990) estimated a national yield loss of 102 330 t (0.82% of total harvest) which represented a loss of approximately AUD 20 million/year for the 1987–88 cropping season. They also reported the value of herbicides used to control wild oats during the same period, together with their cost of application. On this basis, the annual cost of wild oats to the Australian wheat industry was conservatively estimated to be around AUD 42 million. This consisted of almost
AUD 30 million expenditure on herbicides and their application and over AUD 13 million due to lost wheat yield from the competitive effects of wild oats that survived in the crop.
43 Contrary to common belief, wild oat seeds are short-lived and under arable conditions only a small proportion of seeds survive for longer than three years. Seed bank (or the reservoir) flux rates are therefore high, with around 60–70% annual rates of loss (in addition to losses attributable to recruitment) (Medd, 1996b). Wild oats strongly depend on their seeds for survival, multiplication and invasion. The management implications are that if seed input is reduced, seed banks decline rapidly. Thus, the key to successfully managing wild oats is to minimise seed production and the number of seeds returned to the soil, since these add to the seed bank from which subsequent infestations are recruited. This can be achieved by cultural methods such as rotating to summer crops which involves winter fallowing, rotating to pastures, forage cropping, long fallowing or the use of selective spray- topping (Jones and Medd, 1997).
FIGURE 1.7. Fruiting branch of Avena fatua (from Stuber, 2002)
Selective spray-topping (SST) is the technique of using selective herbicides to control seed production of a weed within a crop prior to head emergence.
44 Significant decreases in wild oat seed production and seed banks were achieved as a result of either pre-emergence or post-emergence application of herbicides Avadex
RBW (triallate) and Puma RS (fenoxaprop and mefenpyr), respectively, applied in combination with SST (Edwards et al., 1998).
FIGURE 1.8. Spikelet of Avena fatua (from Wilson, 1998)
In Australia, aryloxyphenoxypropionates (APPs) and cyclohexanediones
(CHDs) herbicides are the most widely used for post-emergence applications, whilst triallate is the key pre-emergence herbicide for the control of wild oats in winter cereal crops. Widespread and persistence use of APPs and CHDs herbicides have resulted in appearance of resistant wild oat populations. Mansooji et al. (1992) reported the resistance to FOP (aryloxyphenoxypropionate) herbicides in wild oats.
In north-eastern Victoria, 6% of 1992 cropping paddocks contained diclofop-methyl resistant wild oats (Walsh, 1995), compared with 4% of 1993 cropping paddocks in the mid-north of South Australia (Nietschke et al., 1996). In southern New South
Wales, the level of diclofop-methyl resistant wild oats had increased from 3% in
1991 to 5% in 1994 (Nietschke and Medd, 1996).
45 The extent of its resistance to selected herbicides, its economic importance as a weed, widespread distribution and annual life cycle make A. fatua a good target weed for biological control with mycoherbicides. This indicates that mycoherbicides could be a potential component of any integrated management system for wild oats.
1.4.3.2 Drechslera avenacea
D. avenacea is included in the order Moniliales of the Hyphomycetes in the sub-division of Deuteromycotina. Pyrenophora chaetomioides (Speg) has been reported as its teleomorph (Shoemaker, 1962). Eight synonyms for D. avenacea and four synonyms for its teleomorph have been reported (Shoemaker, 1962).
Helminthosporium avenae (Eidam) and D. avenae (Eidam) Sharif are the name most frequently used for this fungus. A description of D. avenacea is given by Shoemaker
(1959, 1962).
D. avenacea causes a distinctive leaf spot on species of Avena and also infects Trisetum. The spots are small at first and often surrounded by a red margin.
The diseased leaves later become necrotic and the fungus sporulates on dead leaves and culms (Shoemaker, 1962). Hetherington and Auld (1996) reported the severe damage of D. avenacea to wild oats in pathogenicity tests. Optimisation of environmental conditions and inoculum density may produce even more damage to wild oats. Kastanias and Chrysayi-Tokousbalides (2000) reported a pathotype of D. avenae that exhibited host-specificity, being pathogenic to Avena sterilis L. but not to a number of related or unrelated species tested.
46 1.5 EFFECTS OF ULTRAVIOLET (UV) RADIATION ON FUNGAL
PATHOGENS
1.5.1 Ozone, UV radiation and climate change
UV radiation, the region of the electromagnetic spectrum between visible light and X-rays, is a natural component of sunlight and thus a part of the environment in which life evolved and to which it is adapted. Ultraviolet A (UVA) radiation is defined by the Commission Internationale d'Eclairage (CIE) as the wavelengths between 315–400 nm, while wavelengths between 280–315 nm are classed as UVB and shorter wavelengths between 200–280 nm as UVC (Webb,
2000). With a few exceptions, research into the biological effects of UVB radiation has been stimulated by concern over stratospheric ozone depletion. Attention has been focused on UVB since ozone depletion has far greater effects on this waveband than on UVA (Madronich et al., 1995). Ozone depletion will have no effect on UVC radiation as it does not penetrate to the ground due to strong absorption by gases, including oxygen (Madronich et al., 1995). Ozone loss between 1979 and 1991 averaged around 4–6% per decade at northern temperate latitudes (Hollandsworth et al., 1995) but depletion varied markedly with season. Stratospheric concentrations of ozone-depleting substances and the resulting ozone depletion will peak within the next decade (Madronich et al., 1995). However, ozone will not return to pre-1980 levels for several decades and increased UVB radiation may remain a concern until late into the 21st century, especially given recent evidence that global warming may exaggerate ozone depletion (Shindell et al., 1998).
47 1.5.2 UVB and fungal pathogens
The effects of UVB on phytopathogenic fungi have been the subject of recent reviews by Manning and Tiedemann, 1995; Ayres et al. (1996); Krupa et al. (1998); and Paul (1997; 2000). The effects of UVB on host plants appear to be largely a function of photomorphogenic responses, while effects on fungal pathogens may include both photomorphogenesis and damage (Paul, 2000). For many fungi, the main photomorphogenic effect is on sporulation, but this is generally considered primarily a UVA response (Manning and Tiedemann, 1995). Wavelengths that are effective at inducing sporulation at low doses may inhibit sporulation at higher doses
(Leach, 1971). The few action spectra for the inhibition of conidium germination or germ tube growth that have been defined appear to be broadly comparable with those for fungal photomorphogenesis (Maddison and Manners, 1973). Many studies using a wide range of pathogens show that UV can kill fungal spores and inhibit germination or germ tube growth of phytopathogenic fungi (Aylor and Sanogo,
1997; Caesar and Pearson, 1983; Maddison and Manners, 1973; Parnell et al. 1998;
Rotem and Aust, 1991; Rotem et al. 1985; Rasanayagam et al. 1995; Stevenson and
Pennypacker, 1988; Willocquet et al. 1996). Impaired conidial culturability and delayed germination have also been shown for entomopathogenic fungi (Braga et al.
2001a; 2002; Moore et al. 1993; 1996). However, not all pathogens are vulnerable to such damage (Rotem et al., 1985) and, even within fungal species, there is marked variation between genotypes in their response to UV light (Rasanayagam et al.,
1995).
In vitro fungal growth, conidium production, conidium germination and, where appropriate, appressorium formation can be used to predict the potential of selected plant pathogens as mycoherbicides (Daniel et al., 1973). The influence of
48 temperature, humidity and UV radiation on the behaviour of a potential mycoherbicide, both in relation to growth and conidium production as well as behaviour on plant-pathogen interactions, is of a particular importance, as is the time in which conidium germination and appressorium formation take place. Many candidate mycoherbicides have shown promise in the laboratory or greenhouse, but most have been ineffective in the field (Yang and TeBeest, 1993). Additionally, for some candidate mycoherbicides, control efficacy was not consistent from year to year nor from field to field. These contradictions indicate a lack of understanding of one or more important ecological factors or mechanisms contributing to the suppression of weeds by fungal pathogens in the field (Yang and TeBeest, 1993). A better understanding of the effects of UV radiation, simulated or as natural sunlight on C. orbiculare, D. avenacea and R. alismatis conidia would bridge an important gap in our knowledge. This knowledge would provide information for programs of improvement of radiation tolerance or time of application for these fungi with potential as mycoherbistats.
1.6 OBJECTIVES OF THIS THESIS
The main objective of the work reported in this thesis was to investigate the stimulatory and inhibitory effects of radiation, simulated or as natural sunlight on the development of fungi with potential as mycoherbistats with a view to enhancing mycoherbistat effectiveness under field conditions. An objective of the thesis was to determine were reaction of three potential mycoherbistats, R. alismatis C. orbiculare and D. avenacea, to UV light and then focus on the pathogen(s) showing the greatest sensitivity to this form of irradiation. The optimal conditions for sporulation and subsequent virulence of R. alismatis (Jahromi et al. 1998; Cother and Van de Ven,
49 1999) and C. orbiculare (McRae, 1989; McRae and Stevens, 1990) have already been determined. However, these parameters were unknown for D. avenacea and needed to be determined before further research could be conducted. Therefore, in
Chapter 2 the conditions affecting sporulation and virulence of D. avenacea with particular reference to stimulatory effect of UVA radiation were investigated.
Molecular characterisation of isolates of D. avenacea was performed in Chapter 3 followed to identify genetic variation in this pathogen. Identification of variation is important in relation to the identification of isolates of different virulence and would also be useful to assess the release of a single isolate in a natural population.
The specific objectives of this thesis are:
• To optimise sporulation and pathogenicity in D. avenacea
• To determine the genetic variation in D. avenacea isolates using RAPD-PCR
analyses
• To study the effects of UV radiation as simulated or natural sunlight on the
conidium germination and appressorium formation of fungi with potential as
mycoherbistats
• To evaluate the effects of oil- or water-compatible UV protectants on conidium
germination and appressorium formation of fungi with potential as mycoherbistat
in order to protect the spores from sunlight
• To examine the overall effect of UV radiation and various UV protectants on
host-pathogen interactions using the leaf disc bioassays and pot-in-field
experiments.
50
CHAPTER 2
OPTIMISING SPORULATION AND PATHOGENICITY
IN DRECHSLERA AVENACEA
2.1 INTRODUCTION
Drechslera avenacea has been evaluated as a mycoherbicide for weedy
Avena species by several authors (Wilson, 1987; Zonjian and Yangham, 1996;
Hetherington et al. 1998; Hetherington and Auld, 2001). One of the most important characteristics for any mycoherbicide to be successful is that the fungal pathogen must produce abundant and durable inoculum on artificial media (Daniel et al. 1973).
Mass production on agar media can provide sufficient inoculum for laboratory and field trials but, more importantly, these studies provide valuable knowledge for subsequent optimisation of scale-up production.
D. avenacea, as with most other Drechslera spp., is traditionally a poor sporulator in culture and much variation exists between isolates. Turner and Millard
(1931) made a detailed study of a wide range of media containing different carbohydrates and other nutrients to determine their capacity to induce sporulation.
Sporulation only occurred on media containing sterilised oat leaves and then only sparsely. Weston (1933) inoculated Petri dishes containing potato agar with D.
51 avenacea and after three days, the covers of the Petri dishes were replaced with discs of Sanalux glass, one-half of which was painted with Indian ink and then irradiated with a Hanovia quartz mercury-vapour lamp. One week later, the mycelia of the irradiated halves were strongly pigmented and abundant sporulation occurred whereas no conidia were produced on the non-irradiated halves. Weston (1936) reported that the fungus sporulated when the cultures were exposed for different periods to natural irradiation out-of-doors or to visible white light of high intensity.
Leach (1962) induced sporulation in D. avenacea by culturing the fungus on potato dextrose agar, malt agar and Czapek Dox agar exposed continuously to near- ultraviolet (NUV) radiation. Wilson (1987) reported that D. avenacea failed to sporulate after 4 weeks incubation on V8-juice agar when exposed to 12 h white light/12 h dark cycle at 20ºC: in contrast, sporulation was stimulated under 12 NUV light/12 h dark. She also reported that the fungus sporulated on potato carrot, oatmeal, half strength oatmeal, green bean, wild oat leaf extract and Sachs agar when exposed to 12 h NUV/12 h dark at 16ºC for 22 days. Thus, medium, light quality and photoperiod affect the sporulation of D. avenacea. However, the studies outlined above did not vary each of these factors for a range of isolates.
Kumagi (1984) divides the fungi into three groups according to the light-dark cycle necessary for conidiation: (1) light is required for the induction of conidiophores but conidium development is suppressed by light; (2) light is not required for the induction of conidiophores and conidium development is suppressed by light; (3) and light is required for the induction of conidiophores and conidium development is not suppressed by light. Based on whether or not light is needed to induce conidiophores, the first and third types can be called photo-induced sporulators whilst the second types are called non-photoinduced sporulators. Leach
52 (1967) also showed that for some Fungi Imperfecti, such as Alternaria dauci (Kuhn)
Groves & Skolko, NUV radiation will bring about the inductive phase of sporulation
(conidiophore development), but the terminal phase (conidium production) requires a period of darkness. Leach (1967) showed that the greatest number of conidia produced by A. dauci were obtained when a 12 h exposure to NUV radiation was given at a high temperature (30–35ºC) and the subsequent dark period was at a lower temperature (18 or 20ºC). For other species, such as Drechslera catenaria (Drechs.)
Ito, there were no distinct inductive or terminal phases, and Leach (1967) also concluded that if these two phases were indeed present, then it would appear that they must have similar or closely overlapping temperature optima. D. catenaria would sporulate satisfactorily under continuous NUV irradiation even more when exposure was followed by darkness. Leach classified the two groups of fungi as
‘diurnal sporulators’ and ‘constant temperature sporulators’, respectively.
Light and temperature requirements for conidiophore development and conidium production have not been fully studied for D. avenacea. The purpose of the work reported in this chapter was to find a placement for D. avenacea in the
Leach and Kumagi classifications and to devise a simple method using readily available material for sporulation of D. avenacea that would produce more conidia than traditional cultural methods whilst maintaining virulence. The steps taken were to: (1) determine the optimum agar medium, pH, carbohydrate content, temperature and light conditions for conidium production; (2) evaluate the intensity and quality of
NUV and white fluorescent light as continuous, diurnal or combined light conditions on conidium production; (3) determine the effects of agar medium and light quality on the subsequent virulence of D. avenacea conidia; and (4) evaluate the
53 applicability of the best conditions for a range of isolates showing variation in sporulation.
54 2.2 MATERIALS AND METHODS
2.2.1 Fungal isolates
The isolates used in this study were obtained from the New South Wales
Agriculture herbarium (Table 2.1). The isolates were maintained on potato dextrose agar incubated at 25°C in the dark. Mycelial plugs (5 mm diameter) taken from the periphery of 5-day-old cultures were used as inoculum throughout this study.
TABLE 2.1. Origin of Drechslera avenacea isolates used in this research Code Isolate Accession number Geographic origin A RS 1295124-2 IMI 375958 1 km W of Mathoura B RS 1295156 IMI 375692 17 km N of Leeton, NSW C RS 129561-1 IMI 375961 30 km S of Wagga Wagga, NSW D RS 099584-1 IMI 375959 10 km N of Narrabri, NSW E RS 0995209-1 IMI 374564 82 km S of Armidale, NSW F RS 0995151-3 IMI 374567 Kyogle, NSW G RS 109571-1 IMI 374568 40 km N of Cowra NSW H WA 109610 IMI 374565 20 km N of Moora WA
2.2.2 Data analysis
A completely randomised experimental design including three replicates for each treatment was used and each experiment was performed twice. Homogeneity of variances of data was determined using Bartlett’s test and data that were heteroscedastic subjected to square root or log transformation before further analysis.
The mean percentage values for each of the respective disease severity classes or percentage of necrotic leaf area were converted to arcsines before analysis. Analyses of variance were performed using STATISTICA software release 6.0 (StatSoft Inc.,
Tulsa OK, USA, 2001). Treatment means were compared by Fisher’s LSD tests at the 5% significance level.
55 2.3 EXPERIMENTAL
2.3.1 Effect of agar media and temperature on conidium production
Materials and Methods. Seventeen agar media were tested for their ability to induce sporulation: carrot agar (CA), cornmeal agar (CMA), Czapek Dox agar
(CZA), green bean agar (GBA), malt extract-peptone-dextrose agar (MPDA), malt and yeast extract agar (MYA), lima bean agar (LBA), 2% malt extract agar (MA), oatmeal agar, half-strength oatmeal agar (½OMA), potato-carrot agar (PCA), potato- dextrose agar (PDA), ½ PDA (Sigma), Alternaria sporulation medium (S-medium),
V-8 juice agar (V8JA), yeast extract-dextrose agar (YEDA) and 2% water agar
(WA). These media were prepared according to the methods described by Dhingara and Sinclair (1995) except for PDA, which was prepared following the manufacturer’s instructions (Sigma). The media (20 mL) were dispensed into plastic
Petri dishes (90 mm diameter) before inoculation with isolate A. The cultures were incubated at 25°C for 3 days in continuous darkness and then further incubated at 15,
20, 25 or 30°C under alternating 12 h periods of white light and darkness (L/D). The white light was obtained from a set of 3 cool-type fluorescent lamps (FL30CW/29, light emission wavelength 350–700 nm, China Electric MFG, Taiwan). The
-2 -1 -2 intensity of white light at a distance of 300 mm was 67 µE m s or 0.075 W m , as measured using a Lambda LI-189 light meter calibrated by quantum sensor (LI-
COR, Lincoln, USA) and UV sensor type UV2/AP (Delta-T Devices, Cambridge,
England), respectively. Four weeks after incubation (WAI) at the treatment temperatures, the agar surfaces were flooded with 10 mL of sterile water and the were scraped off with a flamed microscope slide. The resulting conidium suspensions were passed through a 150 µm sieve and a drop of lactophenol cotton
56 blue was added to the conidium suspension to kill and preserve the conidia before counting using a haemocytometer. Six readings for each replicate were recorded.
In a second experiment, conidium production by Isolate A was determined on a full-strength and half-strength preparation of a commercial (Sigma) formulation of
OMA. The plates were inoculated and incubated at 25ºC under continuous darkness for 3 days before being subjected to a 12L/12D photoperiod regime at 20ºC.
Conidium production was assessed 1 WAI as described above. The ½OMA was used in the experiments described below.
Results. No sporulation occurred on CA, WA, LBA, CMA, MA, MPDA,
MYA, PCA, S-Medium and V8JA at any temperature tested. On the various media,
Isolate A showed different degrees of sensitivity to temperature (Table 2.2). On
CZA and ½OMA, the isolate sporulated at all temperatures. On OMA, no sporulation occurred at 30°C, on PDA and GBA no conidia were produced at 25 and
30°C, whilst on YEDA and ½PDA sporulation did not occur at the lowest temperature (15°C). On all media, except CZA, maximum sporulation clearly occurred at 20°C. However, on CZA, there was no difference in sporulation between cultures incubated at 15 and 20°C. At 20°C, clear differences in the number of conidia produced on the different media were apparent. Maximum production occurred using OMA, with moderate conidium production on CZA, ½OMA, GBA and YEDA. Conidium production was poor on PDA. It should be noted that there is an apparent contradiction in the mean values by medium for conidium production and their ranking according to Fisher’s LSD test as conidium production on CZA and
OMA have the same means but are placed in different homogenous groups. The data were highly homoscedastic and to remove this departure from normality the data were subjected to log transformations: Fisher’s LSD test was performed on the
57 transformed data. The homogenous groups found using Fisher’s LSD test were the same as those determined by Tukey’s HSD test. It is assumed that this apparent anomaly is due to the different variations in conidium production found on CZA and
OMA at the different temperatures. On CZA similar numbers of conidia were found at three of the four temperatures used. In contrast, on OMA, a high number of conidia were found at one temperature and low numbers on the others.
On the commercial formulation of OMA (Sigma), 0.6 × 104 and 1.1 × 104 conidia mL-1 were produced on full-strength and half-strength OMA, respectively: these results were not statistically significant from each other.
TABLE 2.2. Effect of agar media and temperature on conidium production by D. avenacea under alternating 12 h light (67 µE m-2 s-1) and dark conditions for 4 weeks. Number of conidia mL-1 (× 104) Temperature (ºC) Average Agar by mediuma 15 20 25 30 medium CZA 0.46 (b) 0.41 (b) 0.30 (b) 0.06 (a) 0.31 (D) ½OMA 0.08 (a) 0.30 (b) 0.06 (a) 0.02 (a) 0.11 (C) OMA 0.08 (a) 1.13 (c) 0.04 (a) 0.00 (a) 0.31 (D) PDA 0.04 (a) 0.08 (a) 0.00 (a) 0.00 (a) 0.03 (B) GBA 0.02 (a) 0.46 (b) 0.00 (a) 0.00 (a) 0.12 (B) YEDA 0.00 (a) 0.41 (b) 0.00 (a) 0.00 (a) 0.10 (AB) ½PDA 0.00 (a) 0.02 (a) 0.00 (a) 0.00 (a) 0.00 (A)
Average by 0.09 (L) 0.40 (M) 0.05 (N) 0.01 (O) temperature Within the body of the table, the data were compared by 1-way ANOVA with means followed by the same letter (lower case) not being significantly different according to Fisher’s LSD test at P < 0.05. Differences between treatment means were determined using factorial ANOVA with means followed by the same letter (upper case) not being significantly different according to Fisher’s LSD test at P < 0.05. aYEDA = yeast extract dextrose agar, PDA = potato dextrose agar, ½ PDA = half- strength potato dextrose agar, GBA = green bean agar, OMA = oatmeal agar, ½OMA = half-strength oatmeal agar, CZA = Czapek Dox agar.
58 2.3.2 Effect of pH of the medium on conidium production
Materials and Methods. The pH of CZA was adjusted to 5, 5.5, 6, 6.5, 7,
7.5 or 8 with 1 M HCl or 1 M NaOH before autoclaving. The media were inoculated with isolate A and incubated at 25°C under continuous darkness for 3 days before being transferred to a L/D regime at 20°C. Conidium production was determined 4
WAI as described in Section 2.3.1.
In a second experiment, pH of ½OMA was adjusted to 7 or not adjusted (6.5).
The media were inoculated with isolate A and incubated as described above.
Results. The pH of CZA significantly (P < 0.001) affected the production of conidia (Figure 2.1). The maximum number of conidia was produced at pH 7 (0.2 ×
4 1 10 mL- ). On media where the pH was < 7, sporulation decreased as the pH became lower until, at pH 5.5, sporulation ceased. Sporulation also decreased markedly if the pH of the media was > 7. Significantly more conidia were also produced on
½OMA, when the pH was adjusted to 7.0 compared with the medium prepared according to the manufacturer’s instructions where pH (6.5) was not adjusted (data not presented).
59 25 ) 2 c 20 ( x 10 -1 15
10 b
5 ab ab a Number of conidiaNumber mL aa 0 55.566.577.588.5 pH of medium
FIGURE 2.1. Influence of the pH of Czapek Dox agar on conidium production by -2 D. avenacea when grown under an alternating 12 h light (67 µE m 1 s- ) and dark cycle at 20°C for 4 weeks. Means followed by the same letters are not significantly different at P < 0.05 according to Fisher’s LSD test.
2.3.3 Effect of carbohydrate content on conidium production
Materials and Methods. The CZA was prepared with sucrose concentrations of 10, 20, 30 or 60 g L-1 and the pH of the media were adjusted to 7.
The media were inoculated with isolate A and incubated at 25°C under continuous darkness for 3 days before being transferred to a L/D regime at 20°C. Conidium production was determined 4 WAI as described in Section 2.3.1.
Results. The sucrose content of CZA significantly (P < 0.0005) affected the production of conidia (Figure 2.2). The maximum number of conidia was produced
-1 4 -1 at sucrose concentration of 30 g L (0.1 × 10 mL ). Increasing the sucrose concentration from 30 to 60 or decreasing it to 10 or 20 g L-1 significantly reduced
60 conidium production. Conidium production was the lowest at the highest sucrose concentration (60 g L-1). ) 2 140 b 120 (x 10 -1 100 80 60 40 a a 20 c 0 Number of conidiaNumber mL 10 20 30 60 Sucrose content (gL-1)
FIGURE 2.2. Influence of the sucrose content of Czapek Dox agar on conidium production by D. avenacea when grown under an alternating 12 h -2 -1 light (67 µE m s ) and dark cycle at 20°C for 4 weeks. Means followed by the same letters are not significantly different at P < 0.05 according to Fisher’s LSD test.
2.3.4 Effect of NUV intensity on conidium production of D. avenacea grown on
½OMA and CZA
Materials and Methods. Conidium production by isolate A was determined on ½OMA and CZA at 20ºC under 3 conditions: 10 NUV lamps held at distance of
600, 300 or 150 mm from the cultures. Cultures on ½OMA were also incubated at
15°C at a distance of 150 mm. The experiment was conducted in a growth chamber
(Thermoline Ltd, Australia) maintained at 70% RH. The temperatures were regulated by circulating cool water through the platform supporting the samples.
NUV radiation was obtained from a set of 10 NARVA type BLB fluorescent lamps
(LT18W/073, light emission wavelength 315–400 nm, Crompton Lighting Ltd,
Sydney, Australia). Distances of 600, 300 and 150 mm corresponded to NUV (315–
400 nm) 6.66 W m-2, 0.01 W m-2; 14.56 W m-2, 0.02 W m-2, respectively, and to mid-
61 ultraviolet (UVB) (280–315 nm) intensities of 22.8 W m-2, 0.05 W m-2, respectively.
The intensities were measured using UV sensor types UV2/AP (peak sensitivity 373
± 2 nm) and UV2/BP (peak sensitivity 313 ± 2 nm) (Delta-T Devices, Cambridge,
England). Conidia were harvested 1 WAI and conidium production was determined as described in Section 2.3.1.
Results. There was a significant (P < 0.001) interaction between agar medium and NUV intensity on conidium production. A similar number of conidia were produced on CZA at the various intensities tested (Table 2.3). On ½OMA, on average, conidium production was approximately three times higher than on CZA; however, there was a significant interaction with the intensity of irradiation.
Conidium production was highest (6.0 × 104 mL-1) under moderate irradiation (14.56
W m-2). Increasing the amount of irradiation from 14.56 to 22.78 or decreasing it to
6.66 W m-2 significantly reduced conidium production. At 6.66 W m-2, sporulation was twice that which occurred on CZA. Increasing the intensity to 14.56 W m-2 significantly increased conidium production by three-fold compared with the lower intensity. However, if the intensity was increased further to 22.78 W m-2 the number of conidia produced was markedly reduced and was similar to that found on CZA.
On the ½OMA, 0.6 × 104 and 0.4 × 104 conidia mL-1 were produced at 20 ºC and
15ºC, respectively, at the high intensity: these results were not statistically significant from each other.
62 TABLE 2.3 Effect of NUV (315–400 nm) and UVB (280–315 nm) intensity on conidium production by D. avenacea grown on half-strength oatmeal agar (½OMA) and Czapek Dox agar (CZA) at 20ºC for 1 week. Distance of Number of conidia mL-1 (× 104) lamps from Intensity Intensity Agar medium cultures of NUV of UVB Average by (mm) (Wm-2) (Wm-2) ½OMA CZA intensity 600 6.66 0.01 2.0 ± 0.2 (b) 1.0 ± 0.2 (a) 1.5 ± 0.2 (A) 300 14.56 0.02 6.0 ± 0.4 (c) 1.1 ± 0.2 (a) 3.6 ± 1.1 (B) 150 22.78 0.05 0.6 ± 0.1 (a) 1.1 ± 0.2 (a) 0.8 ± 0.1 (C)
Average by 2.9 ± 0.8 (L) 1.1 ± 0.1 (M) medium Within the body of the table, the data were compared by 1-way ANOVA with means followed by the same letter (lower case) not being significantly different according to Fisher’s LSD test at P < 0.05. Differences between treatment means were determined using factorial ANOVA with means followed by the same letter (upper case) not being significantly different according to Fisher’s LSD test at P < 0.05.
2.3.5 Effect of agar media and light quality on conidium production
Materials and Methods. Conidium production by isolate A was determined on ½OMA and CZA. The pH of the media was adjusted to 7 before autoclaving. The plates were inoculated and incubated at 25°C under continuous darkness for 3 days before being subjected to a L/D photoperiod regime at 20ºC. Exposure to 12 h of alternating near ultraviolet radiation and dark (NUV/D) period was also included as a
-2 reference treatment. The intensity of NUV at a distance of 300 mm was 0.54 µE m s-1 (14.56 W m-2). Conidium production was assessed 1WAI.
Results. A significant interaction in terms of conidium production occurred between light quality and agar medium. The number of conidia produced by isolate
A on ½OMA and CZA exposed to white light was the same as the cultures grown on
CZA and exposed to NUV (Table 2.4). However, sporulation was ~6-fold higher on
½OMA plates exposed to NUV/D. The number of conidia mL-1 harvested from
OMA and ½OMA plates exposed to L/D condition at 20ºC was statistically similar and relatively more conidia were produced on ½OMA (data not presented).
63 TABLE 2 4. Effect of agar medium and light quality on conidium production by D. avenacea after incubation at 20ºC for 1 week on half-strength oatmeal agar (½OMA) and Czapek Dox agar (CZA). Number of conidia mL-1 (× 104) Light conditions Agar media L/Da NUV/D Average ½ OMA 1.1 ± 0.5 (a)b 6.0 ± 0.4 (b) 3.5 ± 1.1 (A) CZA 1.1 ± 0.2 (a) 1.1 ± 0.2 (a) 1.1 ± 0.1 (B)
Average 1.1 ± 0.2 (L) 3.6 ± 1.1 (M) aL/D = alternating 12 h white light (67 µE m-2 s-1) and dark, NUV/D = alternating 12 h near-ultraviolet radiation (14.56 W m-2) and dark. bWithin the body of the table, the data were compared by 1-way ANOVA with means followed by the same letter (lower case) not being significantly different according to Fisher’s LSD test at P < 0.05. Differences between treatment means were determined using factorial ANOVA with means followed by the same letter (upper case) not being significantly different according to Fisher’s LSD test at P < 0.05.
2.3.6 Effect of continuous dark and light, diurnal or combined light conditions and constant or alternating temperature on conidium production on ½OMA
Materials and Methods. Conidium production by isolate A was determined on ½OMA under 11 light and temperature conditions; (1) NUV/D25°C + L/D20°C;
(2) NUV/D20°C + L/D20°C; (3) L/D20ºC + NUV/D20°C; (4) NUV/D20°C; (5)
NUV20ºC; (6) L/D20°C + L/D15°C; (7) L20°C + L/D20°C; (8) L/D20°C + D20ºC;
(9) L/D20ºC; (10) L20°C; (11) D20°C. For combined light and temperature treatments (e.g., NUV/D25°C + L/D20°), after 3 days in the initial condition (i.e., 12 h NUV/12 h D at 25°C), cultures were transferred to the second treatment (i.e., 12 h
L/12 h D at 20°C) and maintained under these conditions for the remainder of the experiment. For all treatments, the intensity of white and NUV light at culture level
2 -1 -2 -2 -1 -2 was 67 µE m- s (0.075 W m ) and 0.54 µE m s (14.56 W m ), respectively.
Conidia were harvested and assessed 1 WAI.
Results. Conidium production was stimulated by either white light or NUV irradiation (Table 2.5) and cultures incubated in continual darkness (D20) had the
64 lowest number of conidia. Of these two light qualities, NUV stimulated conidium production more than white light. The two continuous irradiation treatments induced the greatest number of conidia with NUV producing twice the number of conidia as white light. Incubation under a L/D photoperiod can induce conidium production.
However, the number of conidia is ~6-fold less than under continuous illumination with white light, and the L/D cycle reduced conidium production. This effect of the
L/D photoperiod can be seen in the L20 + L/D20 treatment which produced 15-fold fewer conidia than were produced under continuous white light and in the NUV/D20
+ L/D20 treatment where conidium production was 4-fold less than under L/D20 +
NUV/D20. This adverse effect of exposure to L/D photoperiod on conidium production was overcome when cultures were subsequently exposed to continuous dark or NUV/D photoperiod. However, the positive effect of continuous dark exposure was not comparable to NUV/D photoperiod. Although many of the combined treatments successfully induced conidium production, none were as effective as continuous illumination. Temperature treatments that were found to inhibit conidium production in an earlier trial (Table 2.2) were also found to be inhibitory in this trial.
65 TABLE 2.5. Effect of continuous darkness, continuous light, diurnal or combined light and constant or alternating temperature treatments on conidium production by D. avenacea grown on half-strength oatmeal agar for 1 week. Light and temperature NUVa dose L dose Number of conidia conditions (kJ m-2) (J m-2) mL-1 (× 104) D20b 0.00 0.00 0.03 ± 0.03 (a) L/D20 + cL/D15 21.16 20.26 0.15 ± 0.09 (ab) L20 + L/D20 30.24 28.94 0.46 ± 0.29 (bc) L/D20 + D20 9.07 8.68 3.41 ± 0.20 (de) L/D20 21.16 20.26 1.10 ± 0.54 (c) L20 41.32 40.52 6.97 ± 1.82 (f) NUV/D25 + L/D20 1899.06 11.63 0.48 ± 0.04 (bc) NUV/D20 + L/D20 1899.06 11.63 2.64 ± 0.34 (d) L/D20 + NUV/D20 2525.03 8.77 4.81 ± 0.47 (ef) NUV/D20 4402.94 0.16 6.04 ± 0.44 (f) NUV20 8805.88 0.32 11.04 ± 0.98 (g) Means ± standard error followed by the same letter are not significantly different at P < 0.05 according to Fisher’s LSD test. aNUV doses = total energy absorbed by colonies during the exposure time at the NUV (315–400 nm) intensity of 14.56 W m-2 and 0.07 W m-2 for NUV and white light, respectively, L doses = total energy at the white light (350–700 nm) intensity of 67 µmol m-2 s-1 and 0.54 µmol m-2 s-1 for white and NUV light, respectively. bD = continuous dark, L = continuous white light, L/D = alternating 12 h white light and dark, NUV/D = alternating 12 h near-ultraviolet radiation and dark, light treatments followed by temperature (°C) treatments. cFirst treatment for 3 days, followed by second treatment for 4 days.
2.3.7 Effect of photoperiodism on conidium production on ½OMA
Materials and Methods. Conidium production was determined on D. avenacea (isolate A) grown on ½OMA under the following 7 photoperiods at 20ºC;
(1) 24 h L; (2) 20 h L/4 h D; (3) 16 h L/8 h D; (4) 12 h L/ 12 h D; (5) 8 h L/16h D;
(6) 4 h L/20 D; (7) 24 h D. Exposures to continuous NUV, 20 h NUV/4 h D and 12 h
NUV/12 h D were also included as reference treatments. The intensity of white and
NUV lights was the same as Section 2.3.6. Conidium production was determined 1
WAI.
Results. Conidium production was significantly (P < 0.001) affected by photoperiodic treatments (Table 2.6). In this experiment, conidium production was
66 again maximal under continuous irradiation, with NUV inducing more conidia than white light. Conidium production was not reduced if the irradiated period was interrupted with a 4 h dark period. However, if the dark period was greater than 4 h, conidium production declined with, in general, the decline being directly related to the length of the dark period. The exception to this trend was the 12 h L/12 h D treatment that was particularly inhibitory.
TABLE 2.6. Effect of photoperiod on conidium production by D. avenacea grown on half-strength oatmeal agar at 20ºC for 1 week. Photoperiodic NUVa dose L dose Number of conidia treatments (kJ m-2) (J m-2) mL-1 (× 104) 24 h L 41.32 40.52 7.7 ± 1.1 (f) 20 h L/4h D 35.28 33.76 7.3 ± 0.9 (f) 16 h L/8 h D 28.22 27.01 5.2 ± 0.8 (de) 12 h L/12 h D 21.16 20.26 0.8 ± 0.2 (b) 8 h L/16 h D 14.11 13.50 3.4 ± 0.1 (c) 4 h L/ 20 h D 7.05 6.75 4.1 ± 0.8 (cd) 24 h D 0.00 0.00 0.0 ± 0.0 (a) 24 h NUV 8805.88 0.32 11.0 ± 0.6 (g) 20 h NUV/4 h D 7338.24 0.27 11.6 ± 0.4 (g) 12 h NUV/ 12 h D 4402.94 0.16 6.0 ± 0.2 (ef) Means ± standard error followed by the same letter are not significantly different at P < 0.05 according to Fisher’s LSD test. aNUV doses = total energy absorbed by colonies during the exposure time at the NUV (315–400 nm) intensity of 14.56 W m-2 and 0.07 W m-2 for NUV and white light, respectively, L doses = total energy at the white light (350–700 nm) intensity of 67 µmol m-2 s-1 and 0.54 µmol m-2 s-1 for white and NUV light, respectively.
2.3.8 Effect of media and light quality on subsequent virulence of conidia produced by D. avenacea
Materials and Methods. Seeds of Avena byzantina L. (cv. Coolabah) were sown in a commercial potting mix (Debco Ltd, NSW, Australia) in seed trays.
Seedlings at the one-leaf stage were transplanted into 120-mm diameter plastic pots
(three per pot) containing the same potting mix. Osmocote, slow release fertiliser
(Scotts Ltd, NSW, Australia), was added to each pot. In addition, Aquasol (23:4:18,
67 N:P:K), a fast acting soluble fertiliser (Hortico Ltd, NSW, Australia) was applied weekly to seedlings. The plants were kept in a temperature controlled glasshouse 25
± 5°C fitted with an automatic watering system. The plants were inoculated when they had 3 fully expanded leaves.
Conidia for virulence tests were produced on ½OMA and CZA (Oxoid) incubated at 20°C for 2 weeks under continuous NUV or L conditions. Conidia were
4 harvested and the conidium concentration adjusted to 2×10 conidia/mL by dilution with sterile water. Conidium suspensions were amended with 0.1% Tween 20 and sprayed on the plants until runoff using an airbrush sprayer (Paasche Ltd, Illinois,
USA) at 125 kPa. Control plants were sprayed with water amended with 0.1%
Tween 20 only. All plants were covered with clear, rigid, plastic containers to maintain a high humidity and kept in darkness for 24 h at 25°C. The plants were then moved to a controlled temperature growth cabinet at 20°C, with a diurnal light cycle of 12 h.
Disease severity was assessed on all plants of each treatment within a pot by visual estimation of the percentage of necrotic leaves, using the disease rating scale developed by Horsfall and Barratt (1945). The disease rating scale consisted of 12 class values representing the percentage of disease severity as 0 = 0%, 1 = 0 to 3%, 2
= 3 to 6%, 3 = 6 to 12%, 4 = 12 to 25%, 5 = 25 to 50%, 6 = 50 to 75%, 7 = 75 to
88%, 8 = 88 to 94%, 9 = 94 to 97%, 10 = 97 to 99% and 11 = 100%. Disease severity was recorded at 9 days after inoculation (DAI).
Results. Light quality significantly (P < 0.001) affected the subsequent virulence of conidia with an interaction between agar medium and light quality during incubation. The virulence of conidia produced by isolate A on ½OMA and
CZA exposed to white light was the same as that from cultures grown on CZA and
68 exposed to NUV (Table 2.7). However, the conidia produced on ½OMA under NUV irradiation significantly increased the area of necrotic tissue by 9 DAI.
TABLE 2.7. Effect of agar medium and light quality on the subsequent virulence of conidia produced by D. avenacea grown on half-strength oatmeal agar (½OMA) and Czapek Dox agar (CZA) at 20ºC for 2 weeks. Necrotic leaf area (%) 9 DAIa Light conditions Average by Agar medium Lb NUV medium ½ OMA 58.1 ± 1.0 (a) 72.5 ± 2.6 (b) 65.5 ± 3.3 (A) CZA 58.5 ± 3.7 (a) 61.8 ± 3.5 (a) 60.1 ± 2.4 (A)
Average by light 58.3 ± 1.7 (L) 66.9 ± 3.0 (M) quality Within the body of the table, the data were compared by 1-way ANOVA with means followed by the same letter (lower case) not being significantly different according to Fisher’s LSD test at P < 0.05. Differences between treatment means were determined using factorial ANOVA with means followed by the same letter (upper case) not being significantly different according to Fisher’s LSD test at P < 0.05. aDAI = day after inoculation. bL = continuous white light (67 µE m-2 s-1), NUV = continuous near-ultraviolet radiation (14.56 W m-2).
2.3.9 Applicability of culture conditions for other isolates
Materials and Methods. To test the applicability of these treatments for a wider range of isolates, seven additional isolates (B–H) of D. avenacea were inoculated on ½OMA and then incubated under continuous NUV at 20°C. Details of the test isolates are given in Table 2.1. Conidium production was determined 1 WAI.
The virulence of conidia was determined as described in Section 2.3.8 with conidium suspensions containing 5 × 104 conidia mL-1. Disease severity was recorded 9 DAI.
Results. There were significant (P < 0.001) differences in sporulation among isolates from different geographic areas. Isolates A and B produced the highest number of conidia mL-1 followed by isolate D (Table 2.8). The number of conidia harvested from cultures of isolates C, E, F, G and H was not significantly different.
69 The degree of pathogenicity of six different isolates was also significantly (P <
0.001) different on common oat. Isolate D showed the highest degree of pathogenicity, and isolates A and B the least, although they produced the highest number of conidia (Table 2.8).
TABLE 2.8. Conidium production of D. avenacea isolates after incubation for 1 week at 20ºC on half-strength oatmeal agar under continuous NUV and their subsequent conidium virulence on A. byzantina. -1 4 a Isolate Number of conidia mL (× 10 ) Necrotic leaf area (%) 9 DAI A 11.6 ± 0.7 (bc) 14.3 ± 2.8 (a) B 15.0 ± 0.8 (c) 17.2 ± 5.8 (ab) C 3.3 ± 0.2 (a) 26.8 ± 2.6 (bc) D 10.4 ± 1.6 (b) 51.8 ± 2.6 (e) E 2.8 ± 0.7 (a) 34.5 ± 1.4 (cd) F 5.3 ± 1.3 (a) 40.5 ± 1.9 (d) G 3.4 ± 1.6 (a) Not assessedb H 2.4 ± 1.5 (a) Not assessed Means ± standard error followed by the same letters are not significantly different at P < 0.0 according to Fisher’s LSD test. aDAI = day after incubation bInsufficient numbers of conidia produced for inoculation.
2.3.10 Effect of culture age on conidium production
Materials and Methods. Isolate B, which produced highest number of conidia, was cultured on ½OMA under continuous NUV at 20°C. This experiment was conducted to determine the optimal time for harvesting the maximum number of conidia. Conidium production was assessed 1, 2, 3 and 4 WAI.
Results. Conidium production increased with culture age until cultures were
6 -1 three weeks old. At this age, 1.122 × 10 mL conidia were produced, which is
5 -1 statistically similar to conidium production at 2 WAI (9.89 × 10 mL ) (Figure 2.3).
70 120 c ) 4 100 b ( x 10
-1 80
60 b
40
20 a Number of conidia Number mL 0 1234 Week after incubation
FIGURE 2.3. Effect of culture age on conidium production by D. avenacea on half- strength oatmeal agar at 20ºC under continuous NUV irradiation (14.56 W m-2). Means are followed by the same letter are not significantly different at P < 0.05 according to Fisher’s LSD test.
71 2.4 DISCUSSION
The first part of this study focussed on optimising the conditions for conidium production by D. avenacea. It is evident that when formulating media for
D. avenacea, or other fungi, the effects of temperature, nutrition and irradiation cannot be assessed independently as significant interactions occur between them. For
D. avenacea, in general there was a temperature optimum of 20°C for conidium production. Leach (1962) induced sporulation of D. avenacea on PDA, MA and
CZA under continuous NUV radiation at 21ºC. Wilson (1987) also reported that D. avenacea sporulated on PCA, OMA, ½OMA V8JA and GBA when it was exposed to NUV/D at 16ºC. However, in these experiments, a range of temperatures was not included for comparison and to achieve maximum conidium production. The optimum temperature of 20ºC found in this study is comparable with that reported for conidium production by D. teres (Sacc.) Shoem. (21°C) on Gerber’s rice agar
(Onesirosan and Banttari, 1969) and for D. graminea (Rabenh. Ex Schecht) Shoem.
(20°C) reported by Grbavac (1981). However, the temperature range over which the conidia were produced was greatly influenced by nutrition. With the exception of
CZA, conidium production declined markedly when the temperature of incubation was varied by ±5°C from the optimum. In addition, the magnitude of the temperature range at which conidia were produced varied significantly with medium.
For example on CZA, high numbers of conidia were produced at the lower three temperatures tested and there was no significant decline in conidium production at 15 and 25°C. The cause of the interaction with medium is not known. The range does not seem to be related to the number of conidia produced at 20°C, as there is a narrower range for conidium production on YEDA than ½OMA despite more conidia being produced on YEDA.
72 The number of conidia produced at 20ºC appears to be related to the pH and nutritional environment of the growth medium. As shown by this study, the maximum number of conidia on CZA at 20°C was produced at pH 7 and a sucrose concentration of 30 g L-1. Increasing or decreasing pH and sucrose content from these optimal conditions significantly reduced the number of conidia. Media containing low concentrations of sugar tend to produce higher number of conidia
(Elliott, 1949). In this study, the lowest number of conidia was produced on CZA containing the highest level of sucrose (60 g L-1). Jackson and Bothast (1990) showed that in addition to the carbon concentration of a medium, the carbon: nitrogen (C:N) ratio, also significantly influenced the number of conidia produced by
C. truncatum. They also showed that inorganic nitrogen sources produced lower number of conidia. Sporulation could also be affected by carbohydrate quality.
Pande and Varma (1992) showed that Drechslera hawaiiensis Bugnicourt produced higher numbers of conidia on monosaccharides than di- or polysaccharides.
Therefore, variation in sporulation under L/D condition observed in this study could be attributable to pH, carbohydrate quality or concentration, C:N ratio and nitrogen source of growth medium.
In terms of conidium production, not only was there a general plasticity to temperature of cultures grown on CZA but also a general response to light intensity and quality on this medium. On CZA, conidium production by D. avenacea did not vary with respect to intensity of irradiation with NUV nor with light quality under both L/D and NUV/D conditions. However, D. avenacea on ½OMA exposed to the
NUV/D regime, not only produced more conidia than on CZA but production varied with intensity. These results show why CZA can be considered as a good general
73 medium for fungal growth and can be used to induce sporulation on many fungi
(Dhingara and Sinclair, 1995). However, it is far from optimal for D. avenacea.
Most light delivery systems also generate heat and closed culture vessels act like green houses, trapping heat, which causes increased temperature within the culture medium (Griffin, 1994). Therefore, when experimenting with different light intensities, it is important to separate the effect of intensity from that of temperature.
In this study, this was done by including an additional temperature treatment (15º) at highest intensity (distance of 150 mm). At this intensity, there was no significant difference in conidium production when the temperature was reduced from 20–15ºC indicating significant effect of intensity rather than heat effect on conidium production.
These results suggest that UV radiation has two effects on the sporulation of
D. avenacea: induction and inhibition of sporulation, both of which are intensity dependent. Since it has been reported that UV radiation induces not only sporulation but also DNA damage such as pyrimidine dimer formation (Kihara et al, 1999), it is possible that decrease of sporulation under high intensity of NUV radiation was caused by DNA damage. Thus, the number of NUV lamps and distance between lamps and cultures should be adjusted to achieve maximum conidium production and minimum DNA damage.
As shown by this study, ½OMA is the most favourable medium for sporulation of D. avenacea when exposed to NUV radiation. The highest sporulation on natural media prepared from host materials has also been reported on detached barley leaves and barley straw extract agar for D. teres (Deadman and Cook, 1985) and D. graminae (Babadoost and Johnston, 1998) respectively. It has been proposed by Trione (1981) that light-induced sporulation of fungi is associated with
74 endogenous compounds called P-310s, due to their maximum absorption at 310 nm.
This suggests that there might be such compounds in dead material from host plants that are essential for the maximum sporulation of these fungi or that compounds in these media in some way stimulate the production of P-310s. Further studies are needed to isolate and identify such compound(s), which may contribute to developing effective synthetic media for sporulation of these fungi. Trione (1981) further suggested that P-310s could replace the stimulus of light by inducing sporulation of certain fungi in the dark. Therefore, P-310s may be useful for the mass production of D. avenacea in submerged fermentation, which is more favourable than solid-substrate fermentation for the production of mycoherbicides
(Churchill, 1982). P-310s are not the only compounds that stimulate conidium production. For example, Stemphylium solani G.F Weber, which normally requires
NUV radiation for sporulation, can be induced to sporulate by treatment with ergosterol (Sproston and Setlow, 1968). Therefore, the exact nature of the compound(s) affecting sporulation in the media trialed in this study needs to be determined.
This study has shown that light is required to induce a high level of sporulation by D. avenacea. The highest sporulation occurred when the cultures were incubated under continuous irradiation and, of the two types of radiation used,
NUV induced 1.6-times the number of conidia as white light. Leach (1962) showed that on MA medium under continuous white when NUV and blue light were removed, conidia of D. avenacea were sparsely produced. In contrast, Weston
(1936) reported that under high intensity of white light when visible light including blue light was removed, no conidia were produced by D. avenacea. In this study, the cultures of D. avenacea grown under white light received low NUV doses and only
75 received NUV wavelengths between 350–400 nm whereas those given NUV light received a much higher NUV dose and received NUV wavelengths between 315–400 nm. Also, cultures grown under white light received high doses of radiation in the wavelength range between 400–700 nm whereas cultures under NUV did not receive any wavelengths > 400 nm. This suggests that NUV is better at stimulation of conidium production than white light. It is also possible that the stimulatory effect of continuous white light is coming from the low levels of NUV (350–400 nm), which also emitted from the light source. The inhibitory effect of NUV (315–400 nm) at high intensity has been shown in this study. Therefore, it is possible to increase conidium production under higher intensity of white light source emitting NUV
(350–400 nm) even more than produced under optimal NUV intensity (14.56 W m-2).
The optimal white light intensity for conidium production needs to be determined.
Higher numbers of conidia were produced from cultures given a L/D20 +
NUV/D20 treatment compared with those under a NUV/D20 + L/D20 or L20 +
L/D20 treatment. These results can be explained if, firstly, conidiophore development is stimulated by NUV wavelengths in the range of 315–400 nm and, secondly, conidiophore development is also stimulated by L/D conditions whereas conidium production is reduced by a L/D treatment. In support of this idea,
Onesirosan and Banttari (1969) also found that conidiophore formation of D. teres required NUV radiation in the region between 310–355 nm. According to Kumagi
(1984), the induction of conidiophore formation in photoinduced sporulators is promoted either by NUV and blue light shorter than 530 nm or by NUV alone. Data from this study suggest that this is the case for D. avenacea. However, morphological/biochemical studies are required to confirm the exact nature of the stimulation given by NUV.
76 The inclusion of a diurnal dark period during incubation reduced sporulation under both types of irradiation as did continuous white light followed by either a period of darkness or a L/D photoperiod. Surprisingly, it was the L/D treatment that gave the greatest reduction in conidium production suggesting that the dose of irradiation given was not the limiting factor. Wilson (1987) also reported that D. avenacea failed to sporulate when exposed to a L/D treatment whereas the fungus sporulated under NUV/D conditions. As shown by this study, the inhibitory effect of
L/D period on sporulation was overcome by subsequently exposing cultures to
NUV/D or continuous darkness. However, the number of conidia produced under
L/D + D treatment was not comparable to NUV/D, NUV or L treatments. The same manner of recovery from the inhibitory effect of a L/D period on sporulation was also reported by Masangkay et al., (2000) for A. alternata f.sp. sphenocleae. The results of this part of the study also suggest that after conidiophores of D. avenacea are developed due to stimulation of NUV, subsequent conidium production can occur in the darkness or under certain radiation exposures. Onesirosan and Banttari (1969) suggested that some wavelengths in the full fluorescent spectrum are inhibitory to conidium production by D. teres as, after conidiophore development, conidium production only occurred in darkness or in the absence of wavelengths between 355 and 495 nm. The inhibitory effect of blue light (360–530 nm) on conidium production by Alternaria cichorii Nattrass was also reported by Vakalounakis and
Christias (1981). Zimmer and McKeen (1969) showed that under continuous radiation in which wavelengths between 370–510 nm were present, only conidiophores of A. dauci were formed. Conidium production did not occur unless the irradiation exposure was followed by a sufficient period of darkness or the fungus was subjected to radiation of wavelengths 510 nm or above. Blue light does not
77 appear to have the same inhibitory effects with D. avenacea, as cultures exposed to continuous white light source that also emit blue light wavelengths. It is possible that this is due to interactions between the doses of inhibitory or stimulatory of wavelengths.
Stimulatory and inhibitory effects of L/D period on conidiophore development and conidium formation of D. avenacea, respectively, have been shown in this study. However, the results from this study suggested that, in terms of sporulation, D. avenacea is sensitive to photoperiod. To test this idea, cultures were subjected to photoperiod treatments with different periods of white light from continuous light through to continuous darkness. In general, sporulation gradually increased with an extension or reduction to a 12 h photoperiod. This was in contrast to findings of Sato and Takeda (1991) and Zimmer and McKeen, (1969) for D. teres and A. dauci, respectively, where sporulation decreased with an extension or reduction in the 12 h photoperiod. In contrast to inhibitory effect of a 12 h L/D period on sporulation of D. avenacea, L/D period has been reported as the optimal conditions for the sporulation of D. teres (Onesirosan and Banttari, 1969), D. graminae (Teviotdale and Hall, 1976) and P. semeniperda (Campbell et al., 2003).
However, the L/D treatment particularly suppressed conidium production of D. avenacea indicating that whatever the biochemical mechanism of sporulation for photoinduced sporulators may be, it cannot be identical for all Drechslera spp. The cause of this repeatable observation needs to be determined. Maximum sporulation was achieved under continuous irradiation and was not reduced until the dark period was greater than 4 hours duration. This was the same whether the irradiation was
NUV or white light. On the other hand, the inhibitory effect of L/D period on
78 sporulation of D. avenacea was overcome by subsequently exposing cultures to
NUV/D or continuous darkness.
A photoreceptor system called mycochrome has been proposed by Kumagai
(1978) for a blue and NUV reversible photoreaction controlling the sporulation of some fungi such as Bipolaris oryzea (Breda de Haan) Shoem. and Botrytis cinerea
Pers.:Fr.. The B form of this mycochrome upon absorption of blue light is changed into the NUV form, which absorbs strongly in the NUV. The absorption of NUV radiation changes the NUV form into B form, which is active for conidium formation. The results of this study present evidence for a transformation of the
NUV form of mycochrome to its B form under darkness in D. avenacea.
Mycochrome reversal under darkness was also reported in B. cinerea and A. cichorii by Tan (1974) and Vakalounakis and Christias (1986), respectively. However, it is likely that in D. avenacea the transformation of the NUV form of mycochrome to its
B form under NUV or white light is more active for conidium formation than under darkness.
The phenomenon of photoreactivation (the reduction of lethal and mutagenic effects of UV radiation by simultaneous or subsequent irradiation with blue/NUV light) in fungi has been observed mainly in relation to the survival of the conidia under UV radiation (Sametz-Baron et al., 1997). Kihara et al. (1999) proposed that the effects of UV (induction of sporulation and DNA damage) and those effects of blue light (inhibition of sporulation and photoreactivation) would interact in a complex manner in the process of light perception to gene expression for sporulation in B. oryzae. They also suggested that the photoreceptors might share homology with DNA photolyase. A DNA photolyase gene, phr, in fungi has been isolated from
Saccharomyces cerevisiae Hansen (Sancar, 1985) and Neurospora crassa Shear et
79 Dodge (Yajima et al., 1991). Therefore, cloning and analysis of D. avenacea photolyase gene could be a case for further investigations of sporulation controlled by the mycochrome system.
Higher numbers of conidia were produced under L/D20 and NUV/D20 +
L/D20 treatments compared with L/D20 + L/D15 and NUV/D25 + L/D20, respectively. These results indicate that higher temperatures are not required to induce conidiophore production nor that lower temperature is better for the production of conidia. A constant temperature of 20ºC appears to be optimal for both stages of conidium production. This is in contrast to findings of Grbavac (1981) who showed that the incubation of D. graminae cultures below 20°C following incubation at temperatures > 20°C stimulated the fungus to sporulate. Thus, D. avenacea can be considered to be a ‘constant temperature/continuous light sporulator’. Also, D. avenacea falls into Kumagi’s (1984) third group as light is required for the induction of conidiophores and conidium development is not suppressed by light. These results have not been previously shown for D. avenacea.
This study has shown that the most suitable condition for conidium production by D. avenacea was ½OMA with an initial pH of 7.0 at 20°C under
7 continuous NUV for 2 weeks. Under these conditions, on average 1.12 × 10 conidia per plate were produced, which is the highest sporulation for any Drechslera spp. yet reported. Fungi from this genus are traditionally regarded as poor sporulators.
However, for a method of conidium production to be useful, it must work for a large number of isolates. In this study, these conditions were tested using 8 isolates.
Although variation in conidium production was found, all isolates produced a reasonable number of conidia. It is, therefore, likely that these conditions would be generally successful for other isolates of this pathogen.
80 Virulence is an important feature for a fungal isolate to be a successful mycoherbicide. Virulence is affected by many factors including variability in conidium size and internal nutrients, conidium hydration rate, conidium adhesion to plant surfaces, the number of germ tubes per conidium, germ tube length, appressorium formation, penetration of plant tissue and toxin production (Van Dyke,
1989). In this study, exposure to NUV light resulted in a significant increase in the subsequent virulence of D. avenacea conidia when grown on ½OMA but not CZA.
The virulence of D. avenacea isolates is to some extent a function of toxin production (Nozoe et al. 1965; Kastanias and Chrysayi-Tokousbalides, 2000). It is possible the toxin produced by D. avenacea is activated by NUV radiation. A light- activated toxin has been found in several Cercospora spp. (Daub and Ehrenshaft,
2000).
Considerable variation in sporulation and virulence of D. avenacea was detected among isolates from different geographic areas. They can be grouped into three categories: (1) high sporulator-low virulence (A and B); (2) low sporulator- moderate virulence (C, E and F); and (3) high sporulator-high virulent (D). Isolate
D, therefore, potentially possesses two important features that should allow it to be developed into a successful mycoherbistat. However, since the virulence test was conducted on susceptible cultivated oat (A. byzantina), virulence testing of all isolates would have to be conducted on a wider range of plant hosts.
81
CHAPTER 3
GENETIC VARIATION IN DRECHSLERA AVENACEA
ISOLATES DETERMINED BY RAPD-PCR ANALYSIS
3.1 INTRODUCTION
Variation in virulence and sporulation among the isolates of D. avenacea from different geographical locations has been shown in Chapter 2. The genetic stability of a pathogen is an important requirement in mycoherbicide selection.
Genetic variation of the population must be examined to assess the release of a single isolate on the natural population (Hintz et al., 2001). Knowledge of the genetic structure of pathogen populations has, therefore, important implications for mycoherbicide development.
Attention has focused on molecular-based methods to determine the genetic variation among the isolates of fungi. These methods include: restriction fragment length polymorphism (RFLP) (Bruns et al., 1991); amplified fragment length polymorphism (AFLP) (Majer et al., 1996); ribosomal RNA analysis (White et al.,
1990) and methods utilising the polymerase chain reaction (PCR) (Mullis and
Faloona, 1987).
82 A PCR-based method, termed RAPD (random amplified polymorphic DNA), avoids the need for restriction enzymes, blotting, probing or cloning and produces fragment differences similar to RLFP analysis (Williams et al., 1990). Arbitrary primers are used to generate RAPD fragments (Williams et al., 1990) which, when analysed by gel electrophoresis, can provide a fingerprint profile for any particular target genome. Problems associated with RAPDs include the appearance and disappearance of minor bands with different runs and variability between thermocyclers from different manufacturers (Ellsworth et al., 1993). However, taking this into consideration, the appearance or absence of the major bands is a quick and easy method to identify a genet (Bidochka, 2001). For plant pathogenic fungi, RAPD analyses have been used to analyse genetic variation among isolates of
Leptosphaeria maculans (Desm.) Ces. et de Not (Goodwin and Annis, 1991),
Corynespora cassiicola (Berk. & Curt.) Wei (Silva, et al., 1995) and D. teres (Peever and Milgroom, 1994; Peltonen et al., 1996). Generating RAPD profiles with five arbitrary 10-mer primers revealed polymorphisms suitable for screening differentiation in D. teres isolates (Peltonen et al., 1996). The objective of this chapter was to determine the degree of detectable genetic variation among D. avenacea isolates, collected from different geographical locations. This was assessed by RAPD-PCR analyses using arbitrary primers previously used at UWS to detect variation in fungal species.
83 3.2 MATERIALS AND METHODS
3.2.1 RAPD-PCR analysis
3.2.1.1 DNA extraction
Each of 8 D. avenacea isolates (Table 2.1) was cultured at 25°C in darkness for two weeks on PDA plates (3 replications each) containing sterile filter papers on the surface. Genomic DNA was extracted using the method of Rogers and Bendich
(1988) with small modifications. The mycelium was scraped off and ground to a fine powder under liquid nitrogen with a mortar and pestle. DNA samples were stored at
–20°C before extraction. 50–100 mg of powdered mycelium was placed in a 1.5 mL
Eppendorf tubes together with 600-800 µL 2 × CTAB buffer (100 mM Tris-HCl pH
8-to-9, 1.4 M NaCl, 25 mM EDTA, 2% CTAB). The tubes were vortexed to suspend mycelial powder and incubated at 65°C for 30 min. One volume of chloroform was then added, the tubes were then vortexed briefly before being centrifuged for 10 min at maximum speed. The upper phase was transferred to new 1.5 mL Eppendorf tubes and the DNA precipitated with 600 µL cold (–20°C) isopropanol. The DNA was collected by centrifugation at maximum speed for 5 min. The supernatant was discarded and the pellets were gently washed with 70% EthOH. The DNA was dissolved in 100 µL TE (10 mM Tris-HCl pH, 1 mM EDTA pH 8). The concentration of DNA was determined by measuring the absorbance of the solutions
-1 at 280 nm. Appropriate dilutions were made to give 1 and 10 ng µL .
84 3.2.1.2 Oligonucleotide primers, PCR amplification and electrophoresis
RAPD-PCR fingerprints were generated using three oligonucleotide primers:
RPO1 (5'-AATTTTCAAGCGTCGTGCCA-3')
RPO4 (5'-GGAAGTCGCC-3')
RPO5 (5'-AGTCGTCCCC-3') as described by Richardson et al. (1995). PCR amplifications were performed in a total volume of 10 µL using a gradient thermal cycler (Model PC-960G Corbett
Research, Australia). Amplification reactions contained: 2 µL of 5 × PCR buffer
(335 mM Tris-HCl, pH 8.8; 83 mM (NH4)2SO4; 1 mM each of dATP, dCTP, dGTP
-1 and dTTP; 1 mg gelatine mL and 2.25% Triton X-100); 0.6 µL of 25 mM MgCl2; 2
µL of oligonucleotide primer (30 ng); 1 µL of DNA template; 0.1 µL of Taq
-1 polymerase (5 U µL , Boehringer Mannheim) and MilliQ water to a final volume of
10 µL. The temperature cycle for PCR with primers RPO4 and RPO5 was: 5 cycles of 30 s denaturation at 92°C, 2 min annealing at 40°C and 90 s extension at 72°C; followed by 35 cycles of 5 s at 92°C, 25 s at 45°C and 90 s at 72°C; followed by a final cycle of 10 s at 92°C, 20 s at 45°C and 5 min at 72°C. For RPO1, essentially the same cycles were used, however, the annealing temperature was increased to 50,
55 and 55°C for the 5 ×, 35 × and 1 × cycles, respectively. A cooling cycle of 30 min at 25°C was also added. A positive control (DNA extracted from
Mesorhizobium ciceri, strain CC1192) and a negative control (water) were included in each set of PCR amplifications.
An experiment was performed to determine the optimum concentration of
DNA for PCR. Dilutions of the stock template of samples A1–D1 were made to give
-1 1 and 10 ng µL DNA for each extract. One µL of these dilutions and 1 µL of
85 undiluted template were used for amplification together with the RPO5 primer and banding patterns compared.
Amplified PCR products were separated on 3% agarose gels using a combination of SPP-1 bacteriophage DNA restricted with EcoR1 (GeneWorksTM,
Adelaide, Australia) and Molecular Weight Marker VIIITM (Boehringer Mannheim) in the size marker lanes. Gels were run in 1 × TBE (89 mM Tris-base, 89 mM boric acid, 2 mM EDTA, pH 8.0) buffer at 60 V for 7 h using a Protean II gel electrophoresis apparatus (BioRad Corporation). Gels were then stained with ethidium bromide and imaged over a UV transilluminator using Polaroid TM 667 film and/or a BioRad GelDocTM system.
3.2.2 Fragment analysis
DNA bands in each amplification were identified using Molecular Analyst
Fingerprinting DST (Version 1.6 software, Bio-Rad) with the identity of each band being confirmed by eye. The presence or absence of each DNA band in each DNA profile generated by the different primers was assessed with a score of 1 being given if a band was present and 0 if the band was absent. Bands that were common to each of the three replicates were used to derive a data matrix from which the genetic distances between isolates were calculated using the method of Nei and Li (1979).
These distances were used for analysis by multidimensional scaling using
STATISTICA (Version 6, StatSoft, Inc.). For all samples, bands were compared with those occurring in the water (blank) controls. Where the bands in sample tracks corresponded to those in the control, they are removed from the analysis.
86 3.3 RESULTS
There was no appreciable difference between profiles from the stock and diluted templates of A1–D1 isolates (Figure 3.1). Therefore, 1 µL of stock templates was used for further PCR amplifications. The primers generated 33, 22 and 25 bands for RPO1, RPO4 and RPO5, respectively, that were consistent between the replicates of each isolate. Of these primers, RPO1 was most discriminatory (Figure 3.2). Using this primer, different fragment profiles were found for all isolates with the exception isolates G and F: the patterns for these latter two isolates were identical. For primers,
RPO4 and RPO5, there was no difference between the banding patterns of the isolates with the exception of isolate C whose banding pattern was different from the other isolates.
87
A B C D S PC 1 2 3 1 2 3 1 2 3 1 2 3 W S
2,810
1,510 1,390
720 692
489 404 320
242
FIGURE 3.1. To determine the optimum concentration of target DNA required for successful amplification using the primer RPO5, genomic DNA of D. avenacea isolates (A, B, C and D) was serially diluted and subjected to PCR. Target DNA concentrations were: 1, undiluted (stock template); 2, 10 ng µL-1 DNA; 3, 1 ng µL-1 DNA as indicated for each isolate. The size standards (lane S) are the combination of bacteriophage SPP-1 DNA digested with EcoRI and Molecular Weight Marker VIII TM indicated as number of base pairs. Lane PC is a positive control (DNA extracted from Mesorhizobium ciceri, strain CC1192) and lane W is a negative control (water with no template DNA).
88 S H A A B B B S E C C D D D W S
2,810
1,510 1,390
720 692
489 408
320 242
S C E E F F F S G G G A H H W S
A
FIGURE 3.2. DNA fingerprinting of D. avenacea isolates with 3 replicates each using RAPD-PCR with oligonucleotide primer RPO1. Lane letters correspond with the letters of individual isolates in Table 2.1. The size standards (lane S) are the combination of bacteriophage SPP-1 DNA digested with EcoRI and Molecular Weight Marker VIII TM indicated as number of base pairs and lane W is water control with no template DNA.
89 Multidimensional scaling separated the isolates into three groups (Figure 3.3).
The first group consisted of isolates A, B and D that were all genetically distinct but related to each other. A second group consisted of isolates E, F, G and H. Again, these isolates were closely related with the three primers being unable to distinguish isolates F and G from each other. The last group consisted of isolate C. This isolate was genetically very different from the other isolates and could clearly be separated from them of the profiles generated with all three primers.
FIGURE 3.3. Relationships between the 8 accessions of D. avenacea determined by multidimensional scaling using genetic distances derived using the method of Nei and Li (1979) from RAPD profiles.
90 3.4 DISCUSSION
Conclusions from these results should be considered preliminary in view of the limited number of isolates and markers tested. To examine the genome of N. crassa, 88 RAPD markers were employed (Williams et al., 1990): this study has used only three primers. Also, variation was found among the replicates used and only bands that occurred in all the replicates were used for analysis. This type of variation is common and can be ascribed to a number of reasons including variations in DNA template to primer ratios, inhibitory compounds from the DNA isolation procedure as well as genetic variation amongst the isolates themselves.
Despite the limitations, certain genetic relationships were apparent. The variation in sporulation observed between two groups A, B, D and E, F, G, H (Table
2.8) correlated with groups separated by multidimensional scaling analysis from genetic distances of isolates. The primers used in this study were not able to differentiate virulence of D. avenacea isolates (as determined in Chapter 2) and were chosen as they had identified genetic variation amongst isolates of Morchella sp. and
Alternaria alternata and A. cucumerina in laboratories at UWS. RAPD markers have been successfully used to differentiate pathotypes in L. maculans (Goodwin and
Annis, 1991). RAPD profiles generated by 14 decamer primers of arbitrary sequence did reveal significant differences between some of the C. cassiicola isolates and succeeded in differentiating all but two of the strains (Silva et al., 1995). The primers used by Peltonen et al. (1996) were not able to differentiate virulence of D. teres isolates. Peever and Migroom (1994) found no correlation between RAPD genetic diversity and genetic variation in resistance of D. teres isolates to sterol biosynthesis inhibiting fungicides. According to them RAPDs were best suitable for population genetic studies of fungi. However, population genetic studies based
91 solely on RAPD analysis should also be interpreted carefully due to the various sources of polymorphisms generated during RAPD assay (Clark and Lanigan, 1993;
Lynch and Milligan, 1994). Smedegard-Peterson (1976) found that conidia and hyphal cells of D. graminae and D. teres were multinucleate. Smedegard-Peterson
(1976) used symptom expression to define pathotypes of these pathogens. However; there was instability in the symptoms expressed by each pathotype and he considered that heterokaryosis might be a possible source of this instability. From the sectoring that occasionally occurred in culture, Wilson (1987) also concluded that D. avenacea might be a heterokaryon. Sectoring also occurred in this study. Analysis of the sectors may help shed light as to whether D. avenacea is multinucleate and a heterokaryon. Further analysis of this sort should reveal other primers that discriminate between the strains. Once DNA profiles of the pathogen have been determined, they can be used to assess strain stability.
It may be possible to use RAPDs or similar methods to develop markers for traits of interest. Such traits could include conidium production and virulence. The development of these markers could potentially reduce the need for time-consuming virulence testing. Genetic markers may also be used to identify genes associated with spore production or pathogenicity. Understanding of the genetic structure of this endemic fungal pathogen will allow tracking of the fate of any strain that is released. According to Hintz et al (2001) it is ideal that the strain chosen for development as a mycoherbicide should be genetically similar to local populations to reduce the risk of introducing novel alleles into a local population. As described by
Bidochka (2001), DNA profiles can be used in the identification of the introduced agent in a biological impact assessment and may provide valuable information about fungal epidemiology.
92
CHAPTER 4
EFFECTS OF ULTRA-VIOLET RADIATION,
SIMULATED OR AS NATURAL SUNLIGHT, ON
CONIDIUM GERMINATION AND APPRESSORIUM
FORMATION OF FUNGI WITH POTENTIAL AS
MYCOHERBISTATS
4.1 INTRODUCTION
In order to study the specific behaviour (photomorphogenic or damaging effect) of wavelengths in the UV region on fungal pathogens, it is best to use a narrow spectrum of UV radiation. Filters provide the simplest way to limit the spectrum of wavelengths (Jagger, 1967). Cut-off filters allow radiation above a critical wavelength to pass through. Band-pass filters transmit radiation in a certain region only, excluding wavelengths on both sides of two critical wavelengths.
The sharpness of cut-off of a glass filter is variable. A good filter will go from virtually zero transmittance (1%) to full transmittance (actually about 92% because of reflection losses) over a waveband of about 20 nm (Jagger, 1967). For some purposes, ordinary laboratory items make good filters. Window glass, Pyrex,
93 Corex, and Plexiglass form useful cut-off filters for various UV regions (Jagger,
1967). There are some problems associated with the use of these filters. They cannot be assumed to cut off all wavelengths below the value specified for them; the relationship between transmission and wavelength is a sigmoid curve (albeit steep) so they transmit a little radiation beyond the stated cut-off wavelength (Ayres et al.,
1996). To achieve precision in the damaging wavelength, experiments can be performed through construction of an action spectrum using filters with various cut- off wavelengths. These glass filters were used to separate the effects of UVA and
UVB radiation for subsequent experiments.
A photomorphogenic effect (sporulation) of UVA radiation has been shown for D. avenacea. Wavelengths that are effective at inducing sporulation at low doses may inhibit sporulation at higher doses (Chapter 2). However, there are no published data regarding other effects of UV radiation (UVA and UVB) on the three potential mycoherbistats used in this study nor on their host-pathogen interactions. Therefore, the objective of this chapter was to determine the photomorphogenic or damaging effects of UV radiation (UVA and UVB) provided from a source, simulated or as natural sunlight, on conidium germination and appressorium formation of these potential mycoherbistats.
The work described in this chapter was to (1) determine photomorphogenic and damaging wavelengths using different cut-off filters; (2) determine the effect of
UVB radiation exposure at different periods of time (UVB doses); (3) investigate the effects of full-spectrum natural sunlight and of sunlight from which UVB radiation was removed (designated sunlight without UVB) and any interactions with temperature; (4) investigate the effects of full sunlight at different times of a day.
The effect of UV radiation, especially UVB on host-pathogen interactions and
94 mycoherbistatic activity were studied under in vitro and in vivo conditions and are reported in Chapter 6.
95 4.2 MATERIALS AND METHODS
4.2.1 Fungal pathogens: maintenance and conidia description
The fungal pathogens used in this study were obtained from the New South
Wales Agriculture herbarium with reference numbers of DAR 73158, DAR 48942, and IMI 375958 for R. alismatis, C. orbiculare and D. avenacea, respectively. R. alismatis was maintained on lima bean agar (LBA). To make LBA, 50 g of lima beans, previously soaked in 500 mL for 10 h, were blended, 15 g of agar was then added and the suspension made up to 1 L prior to autoclaving. The fungus was stored and maintained in McCartney bottles in a refrigerator at 5ºC and transferred to fresh media at 3-monthly intervals. R. alismatis sporulated abundantly on LBA when incubated in the dark at 25°C (Jahromi et al., 1998). C. orbiculare was maintained on PDA at 25ºC in the dark. Cultures of C. orbiculare were regularly inoculated onto X. spinosum and reisolated to produce conidial matrix, which was required for a high rate of germination and also to ensure the pathogenicity of the isolate (McRae, 1989). This fungus sporulated abundantly when incubated in the dark at 25°C. D. avenacea was maintained on half-strength oatmeal agar (½ OMA) and incubated under 20 h UVA / 4 h dark at 20°C (Chapter 2). Conidium suspensions of R. alismatis, C. orbiculare and D. avenacea were prepared from agar plate cultures. Conidium length, conidium width and number of cells per conidium were measured on 25 conidia. Measurements were made using Image-Pro Plus software (Media Cybernetics, Maryland, USA).
96 4.2.2 Preparing conidium suspensions for exposure to UV radiation
Fungal cultures were maintained on the relevant media. Conidium suspensions to be tested were prepared by pouring 10 mL of sterile distilled water on to the agar plates and then scraping the surface of the cultures to liberate the conidia.
Suspensions were poured into McCartney bottles, Tween 20 was added to give a
0.01% (v/v) solution; the suspension was then sonicated for 2–3 min and sieved through a 90 µm mesh. The concentration of conidia was determined using a
6 -1 haemocytometer then adjusted to approximately 1×10 conidia mL for R. alismatis
5 -1 and C. orbiculare and 1×10 conidia mL for D. avenacea. For exposure to UV radiation, sterile pieces of cellophane membranes were laid on the 50 mm diameter
Petri dish bases. 0.2 mL of suspension was pipetted onto the cellophane and then spread across the whole area of the dish to give a depth of c. 100 µm. This corresponds to the diameter of a large droplet in ultra-low volume spraying (Moore et al., 1993). The plates were left for at least 30 min to ensure that all conidia had settled before exposure to irradiation. This method was used in all experiments reported in this chapter.
4.2.3 Assessment of conidium germination and appressorium formation in the dark
After irradiation, fungal conidia on pieces of cellophane membrane were placed on 90 mm agar plates. Membranes covered in conidium suspensions of R. alismatis and D. avenacea were placed on the surface of 2% WA whilst those covered in conidia of C. orbiculare were placed on PDA. The plates were incubated at 25°C in the dark before assessing germination at 40 × magnification for R. alismatis and C. orbiculare or 10 × magnification for D. avenacea. A total of 200–
97 300 conidia were examined in several fields of view for each assessment with a higher number of conidia counted when germination was low. Germination was defined as a visible germ tube at least as long as the width of the conidium. The number of germinated conidia that produced appressoria was also counted. At least
100 conidia were examined for each assessment.
4.2.4 Suitability of different types of glassware for UV radiation studies
To determine the transmission of UV radiation, pieces of Anumbra Petri dishes, Pyrex glass beakers and window glass were examined in a Cary spectrophotometer (Varian, NSW, Australia). The transmittance of glassware was measured between 250–500 nm.
4.2.5 Exposure of suspensions of conidia to simulated UV radiation
Irradiation tests were carried out in a controlled environment chamber
(Thermoline Pty Ltd, Australia) maintained at 25°C with a RH of 70%, using artificial UV radiation supplied by 10 UVB Philips TL 20 tubes (Davis Ultra Violet
Pty Ltd, Australia). Radiation from these UVB tubes ranged in wavelength from
265–400 nm, with a peak near 315 nm. The intensities of UVB and UVA radiation were measured using UV sensor types UV2/BP (peak sensitivity 313 ± 2 nm) and
UV2/AP (peak sensitivity 373 ± 2 nm) (Delta-T Devices, Cambridge, England). The average UVB and UVA fluxes measured at three locations in the chamber at a distance of 300 mm from source were 4.07 and 0.63 W m-2, respectively, along the length of the tubes.
98 4.2.6 Experiment 1. Determination of photomorphogenic and damaging wavelengths
R. alismatis, C. orbiculare and D. avenacea were cultured as described in
Section 4.2.1. Conidium suspensions were made from 7-day-old cultures as described in Section 4.2.2. Conidium suspensions in Petri dishes were exposed to
UVB and UVA irradiances of 4.07 and 0.63 W m-2 for 2 h corresponding to UVB and UVA doses of 29.30 and 4.53 kJ m-2, respectively. To determine damaging or photomorphogenic wavelengths, conidium suspensions on cellophane disks were placed under different types of glassware that filtered out wavelengths below specified levels. The treatments used were: unfiltered, Pyrex, Anumbra, 4-mm-thick window glass, or no exposure to light. The intensities of UVB and UVA and corresponding doses under this glassware are presented in Table 4.1. At the end of exposure, pieces of cellophanes containing fungal conidia were placed on relevant media and incubated at 25ºC in the dark. Conidium germination and appressorium formation were assessed after 6 h incubation for R. alismatis and D. avenacea and 12 h for C. orbiculare using methods described in Section 4.2.3. The experiments were performed twice with three replicates for each treatment.
TABLE 4.1. The intensities of UVA and UVB with corresponding doses transmitting through different types of glassware for 2 h exposure. a -2 -2 -2 -2 Types of glassware IUVA (Wm ) IUVB (Wm ) DUVA (kJ.m ) DUVB (kJm ) Pyrex 250 mL 0.57 2.32 4.10 16.70 Anumbra Petri-dish 0.59 1.11 4.24 7.99 4-mm-thickwindow 0.47 0.09 3.38 0.64 glass a IUVA = irradiance of incident UVA (315–400 nm), IUVB = irradiance of incident UVB (280–315 nm), DUVA = dose (irradiance × time) of UVA radiation, DUVB = dose of UVB radiation.
99 4.2.7 Experiment 2. Effect of UVB radiation exposure at different periods of time (UVB doses)
Conidium suspensions were made from 7-day-old cultures of R. alismatis and
C. orbiculare as described in Section 4.2.2. Conidium suspensions on Petri dishes were placed under 250 mL Pyrex beakers and then exposed at a UVB irradiance of
2.32 W m-2 for 15, 30, 60, 120 min or no exposure to light. These times correspond to UVB doses of 2.08, 4.17, 8.35 and 16.70 kJ m-2, respectively. This intensity of
UVB under Pyrex (2.32 W m-2) was comparable with average radiation from natural sunlight (2.65 W m-2) between 11:00 and 15:00 (Eastern standard day-light saving time) during summer in Australia on 21 January 2002, which was measured with the same UVB sensor. At the end of each exposure, pieces of cellophane containing fungal conidia were placed on relevant media and incubated at 25ºC in the dark.
Conidium germination was assessed after 3, 6 and 12 h incubation for R. alismatis and 8 and 16 h for C. orbiculare as described in Section 4.2.3. The experiments were performed twice with three replicates for each treatment.
4.2.8 Natural sunlight experiments
4.2.8.1 The Effects of full-spectrum natural sunlight and sunlight without UVB and interactions with temperature
Conidium suspensions of R. alismatis, C. orbiculare and D. avenacea in 50 mm Petri dishes were prepared from 7-day-old cultures as described in Section 4.2.2.
The suspensions were exposed to full sunlight between 10:00–14:00 or 11:00–15:00 h (Eastern standard day-light saving time) at Richmond, NSW with the exception of the experiment performed on 5 December where the conidia were exposed between
100 12:00–16:00 h. The latitude, longitude and elevation of Richmond are 33º 37' 6" south, 150º 44' 45" east and 19 m above mean sea level, respectively.
These experiments were conducted twice for each fungus during clear sunny days. The experiments were performed on 21 January and 18 March 2002; 5
December 2002 and 7 January 2003; 3 April 2002 and 5 December 2002 for R. alismatis, C. orbiculare and D. avenacea, respectively. The conidia were exposed under three different conditions: (1) exposure to full-spectrum sunlight (SUN); (2) exposure to full-spectrum sunlight from which UVB light was removed by double 3- mm thick window glass mounted above the irradiated Petri dishes [this treatment designated as sunlight without UVB (SUN-B)]; and (3) sunlight filtered by covering the Petri dishes with two pieces of citrus leaf (SHAD). The intensity of UVA radiation passing through double 3-mm thick window glass (SUN-B) was measured using UV sensors as described in Section 4.2.5.
In addition to the different conditions of exposure, conidium suspensions were also exposed under four different temperatures. The Petri dishes were floated on water in water baths whose temperatures were adjusted at 10, 20, 30 or 40ºC. For the lowest temperature, the water was kept cold by the addition of freezer blocks and ice. At end of the exposure, pieces of cellophane containing fungal conidia were placed on relevant media and incubated at 25°C in the dark. Conidium germination and appressorium formation were assessed after 6 h incubation for R. alismatis, after
3 h (first experiment) and 6 h (second experiment) for D. avenacea and after 16 h for
C. orbiculare as described in Section 4.2.3.
101 4.2.8.2 The effect of different levels of solar irradiation on germination of conidia
Conidium suspensions of R. alismatis and C. orbiculare in 50 mm Petri dishes were prepared from 7-day-old cultures as described in Section 4.2.2. Petri dishes were floated on water adjusted to 30ºC in a water bath and were then exposed to varying durations of sunlight at different times of day. Exposures to sunlight were performed twice for each fungus on different clear sunny days as follows:
R. alismatis
(1) 18 March 2002. Samples were exposed to sunlight for 1 h periods
between 11:00 and 17:00 h. Further aliquots of conidia were exposed
between 11:00 and 15:00 h and also between 15:00 and 17:00 h. Three
replicates were used for each treatment. At the end of exposure, the
pieces of cellophane containing fungal conidia were placed on 2% WA
and incubated at 25°C in the dark. Conidium germination and
appressorium formation were assessed after 6 h incubation as described
in Section 4.2.3.
(2) 11 April 2002. Samples were exposed to sunlight for 1 h periods between
11:00 and 15:00 h. Conidia were also exposed at 11:00 h and 3 Petri
dishes (replicates) were removed after 2, 3 and 4 h. Further aliquots of
conidia were exposed between 12:00–12:30 and 13:00–15:00 h.
Conidium germination and appressorium formation were assessed after 3
and 16 h as described in Section 4.2.3.
C. orbiculare
(1) 7 January 2003. The design of (2) above was used, but the exposure
between 12:00–12:30 was not included.
102 (2) 18 January 2003. Samples were exposed to sunlight for 15 min to 4 h.
Conidia were exposed for 15, 30, 45 or 60 min between 11:00–17:00 h.
Conidia were also exposed for 2 h between 11:00–13:00, 14:00–16:00 or
15:00–17:00 h and 4 h between 11:00–15:00 h. At end of the exposure,
pieces of cellophane containing fungal conidia were placed on PDA and
incubated at 25°C in the dark. Conidium germination was assessed after
16 h incubation as described in Section 4.2.3.
4.2.9 Meteorological measurements
Air temperature, relative humidity (RH) and global solar irradiance (watts per square metre) were provided by a meteorological station located about 500 m from the experimental site. Irradiance in the UVB and UVA wavelengths were measured using Delta UV sensors (Delta-T Devices, Cambridge, England). The notation used to represent the radiation measurements made by each of instruments is summarised in Table 4.2. The cumulative dose of radiation (irradiance × time) during each exposure period was obtained by the integration of the radiation signals over the appropriate time period.
TABLE 4.2. Nomenclature Symbol Explanation Unit -2 Dsolar Dose of total solar irradiance MJ m -2 DUVA Dose of UVA (315–400 nm) radiation kJ m -2 DUVB Dose of UVB (280–315 nm) radiation kJ m -2 Isolar Irradiance of incident solar radiation W m -2 IUVA Irradiance of incident UVA (315–400 nm) W m -2 IUVB Irradiance of incident UVB (280–315 nm) W m RH Relative humidity Percent t Exposure time h Tair Outdoor air temperature ºC
103 4.2.10 Statistical analysis
Both laboratory and sunlight experiments were performed twice with three replicates for each treatment. Laboratory experiments were performed in a completely randomised design and the results from the two trials were pooled.
Homogeneity of variances of data was determined using Bartlett’s test and data that were heteroscedastic were subjected to arcsine transformation before analysis.
Analyses of variance were performed using STATISTICA software release 6.0
(StatSoft Inc., Tulsa OK, USA, 2001). Treatment means were compared by Fisher’s
LSD tests at the 5% significance level. The results of the germination tests for conidia exposed or not exposed to simulated or natural sunlight were compared by fitting percent germination versus the dose of UVB, UVA and total solar radiation.
These comparisons were performed by regression analysis (curve estimation) and the model with the highest r2 was selected. All regression analyses were performed using SPSS software release 11.0 (SPSS Inc., Chicago, IL, USA, 2001) at the 5% significant level.
104 4.3 RESULTS
4.3.1 Conidia description
R. alismatis produced hyaline, straight to slightly curved 1-septate conidia
(Figure 4.1). Conidium length ranged from 12.41 µm to 18.76 µm with a mean value of 14.78 µm and conidium width ranged from 2.08 µm to 2.79 µm with an average of
2.46 µm.
FIGURE 4.1. Freshly harvested conidia of R. alismatis. Bar = 15 µm.
C. orbiculare produced hyaline, aseptate, straight or very slightly curved, clavate conidia with an obtuse apex and which tapered towards the base (Figure 4.2).
The length of the conidia ranged from 9.67 µm to 12 µm with a mean value of 10.85
µm and their width ranged from 3.91 µm to 5.53 µm with an average of 4.53 µm.
FIGURE 4.2. Fungal conidia of C. orbiculare. Bar = 10 µm.
105 These conidium sizes and other characteristics are similar to the most recent descriptions of C. orbiculare produced by Sutton (1992).
The conidia of D. avenacea were cylindrical with hemispheric ends (Figure
4.3). The length of the conidia ranged from 18.26 µm to 83.19 µm with an average of 53.19 µm. The width at the broadest part of each conidium ranged from 10.21 µm to 18.01 µm with an average of 13.47 µm. The conidia were pigmented with young conidia being light green whilst older conidia were yellowish brown and were divided by 1 to 6 septa. These conidium characteristics match the descriptions of D. avenacea produced by Shoemaker (1962).
FIGURE 4.3. Fungal conidia of D. avenacea. Bar = 20 µm
4.3.2 Suitability of different types of filter for UV radiation studies
A comparison of the spectral characteristics and the absorption cut-offs for
Pyrex glass, Anumbra Petri dish and window glass are presented in Figure 4.4 and
Table 4.3. Pyrex glass transmitted all wavelengths above 279 nm quite efficiently whereas Anumbra Petri dishes transmitted all wavelengths above 291 nm. 4-mm- thick window glass transmitted all wavelengths above 317 nm.
106 TABLE 4.3. Approximate cut-off wavelengths for different proportion of the UV spectrum. Approx. wavelength (nm) for transmittance of Type of filter 1% 5% 50% Pyrex 250 mL 279 284 306 Anumbra Petri-dish 291 297 319 4-mm-thick window glass 317 322 341
100 Pyrex 250 mL Anumbra Petri-dish 4-mm-thick window glass 80
60
40 Transmittance (%) Transmittance
20
0 260 280 300 320 340 360 380 400 Wavelengths (nm)
FIGURE 4.4. Spectral characteristics of various filters.
4.3.3 Experiment 1. Determination of photomorphogenic and damaging wavelengths
4.3.3.1 R. alismatis
There were significant differences between UV filters in conidium germination and appressorium formation for R. alismatis (P < 0.001) (Figure 4.5).
107 UV radiation below 290 nm greatly reduced conidium germination while radiation between 290 and 315 nm caused slight reduction in this parameter. Although the radiation passing through Anumbra Petri dish decreased conidium germination compared with that passing through window glass and the dark control, these wavelengths significantly increased appressorium formation compared with the dark control (Figure 4.5).
100 Conidium germination C C Appressorium formation 80 B
60 Percent 40 b
b 20 c A a A a 0 Unfiltered Anumbra (290 nm) No exposure Pyrex (280 nm) Window glass (315 nm) Type of filter
FIGURE 4.5. The effects of UV radiation (265–400 nm) transmitted through different filters on conidium germination and appressorium formation of R. alismatis. Conidium germination and appressorium formation were assessed after 6 h incubation following exposure. Intensities and doses corresponding to the different type of filters are given in Table 4.1. Bars labelled with the same letter are not significantly different at P < 0.05 according to Fisher’s LSD test.
4.3.3.2 C. orbiculare
There were significant differences between the UV filters on both conidium germination and appressorium formation (P < 0.001) (Figure 4.6). Radiation with
108 wavelengths below 290 nm had a marked deleterious effect on conidium germination and appressorium formation whereas radiation above 290 nm had no harmful effect.
Radiation below 280 nm killed conidia showing the germicidal effect of UVC (200–
280 nm), whilst radiation between 280–290 nm caused a significant reduction in conidium germination and appressorium formation compared with the dark control.
Radiation above 290 nm did not affect conidium germination but significantly increased appressorium formation compared with the dark control. About 50% of germinated conidia produced appressoria (Figure 4.6).
100 D Conidium germination CD Appressorium formation C 80
60 c c Percent 40 B
20 b
A a a 0 Unfiltered Anumbra (290 nm) No exposure Pyrex (280 nm) Window glass (315 nm)
Type of filter
FIGURE 4.6. The effects of UV radiation (265–400 nm) transmitted through different types of glassware on conidium germination and appressorium formation of C. orbiculare. Conidium germination and appressorium formation were assessed after 12 h incubation following exposure. Intensities and doses corresponding to the different types of filters are given in Table 4.1. Bars labelled with the same letter are not significantly different at P < 0.05 according to Fisher’s LSD test.
109 4.3.3.3 D. avenacea
There were significant differences between treatments (P < 0.001) in conidium germination of D. avenacea (Figure 4.7). The unfiltered treatment significantly decreased conidium germination compared with other treatments. No appressoria were formed under any treatments tested.
100 b b b b
80
60
40 Conidium germination (%) germination Conidium 20
0 a Unfiltered Anumbra (290 nm) No exposure Pyrex (280 nm) Window glass (315 nm) Type of filter
FIGURE 4.7. The effects of UV radiation (265–400 nm) transmitted through different filters on conidium germination of D. avenacea. Conidium germination was assessed after 6 h incubation following exposure. Intensities and doses corresponding to the different type of filters are given in Table 4.1. Bars labelled with the same letter are not significantly different at P < 0.05 according to Fisher’s LSD test.
110 4.3.4 Experiment 2. Effect of UVB irradiation for different periods of time
(UVB doses)
4.3.4.1 R. alismatis
There was significant effect of both the duration of exposure (P < 0.0001) to
UVB and incubation time post-exposure (P < 0.0001) as well as a significant interaction between exposure time and hours of incubation post-exposure (P <
0.001). After 6 or 12 h compared with 3 h incubation, no significant delays in germination were observed in the dark control (Figure 4.8). After 3 and 6 h incubation, conidium germination following 15, 30, 60 and 120 min exposure to
UVB radiation was significantly less than the dark control, whereas after 12 h incubation, only conidia given 60 and 120 min exposure to UVB had a lower conidium germination than the dark control.
100 i i hi hi 3 h 6 h 80 gh 12 h fg
60 ef e
40 de d d cd Conidium germination (%) 20 b bc a 0 0153060120
Minutes of exposure
FIGURE 4.8. Conidium germination of R. alismatis after exposure for 15, 30, 60 and 120 min to UVB radiation at an intensity of 2.32 W m-2. Conidium germination was assessed after 3, 6 and 12 h incubation following exposure. Bars labelled with the same letter are not significantly different at P < 0.05 according to Fisher’s LSD test.
111 4.3.4.2 C. orbiculare
There were significant differences in germination among conidia exposed to
UVB for different periods of time and also between the 8 and 16 h incubation periods post-exposure (P < 0.0001) (Figure 4.9). There was no significant interaction between exposure and incubation period. After 8 h incubation post-exposure for conidia exposed to UVB for 60 and 120 min, conidium germination was significantly less than the dark control. After 16 h incubation post-exposure, conidium germination was less than the dark control for conidia exposed to UVB for 30, 60 and 120 min.
100
f 8 h 16 h 80 ef de
60 cd
40 bc b b b Conidium germination (%) germination Conidium 20
a a 0 0 15 30 60 120 Minutes of exposure
FIGURE 4.9. Conidium germination of C. orbiculare after exposure for 15, 30, 60 and 120 min to UVB radiation at an intensity of 2.32 W m-2. Conidium germination was assessed after 8 and 16 h incubation following exposure. Bars labelled with the same letter are not significantly different at P < 0.05 according to Fisher’s LSD test.
112 4.3.5 Dose-response curves
The percentage germination of conidia in relation to UVB doses is shown in
Figures 4.10 and 4.11 and models to describe the relationships are given in Table 4.4.
4.3.5.1 R. alismatis
When the conidium germination of R. alismatis was expressed as a function of UVB dose, it could be described by cubic relationship for both 3 and 12 h incubation post-exposure (Figure 4.10). Conidium germination was halved after a
UVB dose of approximately 4 kJ m-2 after 3 h incubation whereas a 50% reduction after 12 h incubation occurred with a UVB dose of 10 kJ m-2.
100
80 3 h 12 h
60
40 Conidium germination (%) germination Conidium 20
0 0 2 4 6 8 1012141618
UVB dose (KJ.m-2)
FIGURE 4.10. Effect of UVB irradiation on conidium germination of R. alismatis after 3 and 12 h incubation post-exposure, expressed as a function of energy supplied during exposure at a UVB irradiance of 2.32 W m-2.
113 4.3.5.2 C. orbiculare
When the doses of UVB radiation applied to conidium suspensions for 15,
30, 60 and 120 min were plotted as a function of conidium germination, the dose response for C. orbiculare could be described by an exponential equation for both 8 and 16 h incubation post-exposure (Figure 4.11). The declines in germination were similar and a high level of reduction in conidium germination occurred after an energy input of 4 kJ m-2. Conidium germination was halved after a UVB dose of approximately 3 kJ m-2 after 8 h incubation whereas a 50% reduction after 16 h incubation occurred with a UVB dose of 8 KJ.m-2.
100
80 8 h 16 h 60
40 Conidium germination (%) 20
0 0 2 4 6 8 1012141618
-2 UVB dose (KJ.m )
FIGURE 4.11. Effect of UVB irradiation on conidium germination of C. orbiculare after 8 and 16 h incubation post-exposure, expressed as a function of energy supplied during exposure at a UVB irradiance of 2.32 W m-2.
114 TABLE 4.4. Models describing the relationships between conidium germination of potential mycoherbistats (R. alismatis and C. orbiculare) and energy input (total solar, UVA and UVB radiation) in the figures shown in this chapter. All models were significant at P < 0.05. Figure Details Modela r2b dfb Model type 4.10 3 h 65.2447-15.723x+1.3879x2-0.0408x3 0.903 11 Cubic 12 h 82.5348+9.6052x-1.9414x2+0.0688x3 0.949 11 Cubic
4.11 8 h 46.8940 e-0.2317x 0.859 13 Exponential 16 h 89.8814 e-0.0680x 0.908 13 Exponential
4.23 A 80.8843-12.062x+0.4976x2 0.376 24 Quadratic B 81.8530-0.7961x+0.0021x2 0.389 24 Quadratic C 63.7823-1.5053x+0.0006x2 0.273 24 Quadratic
4.24 3 h A 78.7974-13.702x-0.2633x2+0.0981x3 0.573 26 Cubic 3 h B 76.3104-0.9200x+0.0012x2+1.205x3 0.539 26 Cubic 3 h C 78.1948-4.7696x-0.0229x2+0.0038x3 0.565 26 Cubic 16 h A 100.670-15.589x+1.9315x2-0.1438x3 0.602 26 Cubic 16 h B 103.294-1.2473x+0.0118x2-5.05x3 0.627 26 Cubic 16 h C 100.631-5.5125x+0.2440x2-0.0064x3 0.601 26 Cubic
4.27 A 57.9287-29.553x+4.8612x2-0.2345x3 0.767 20 Cubic B 55.6911-1.9120x+0.0209x2-7.05x3 0.725 20 Cubic C 56.7508-9.5323x+0.5096x2-0.0079x3 0.746 20 Cubic
4.28 A 59.8754-27.523x+4.7656x2-0.2755x3 0.430 41 Cubic B 59.6896-1.8549x+0.0222x2-9.05x3 0.424 41 Cubic C 59.4272-8.6565x+0.4798x2-0.0090x3 0.430 41 Cubic a -2 -2 Where x = dose (kJ m or MJ m ) bCoefficient of determination and degrees of freedom of the regression.
4.3.6 Natural sunlight experiment
4.3.6.1 The effects of full-spectrum natural sunlight or sunlight without UVB and interactions with temperature
4.3.6.1.1 R. alismatis
The average of magnitude of environmental conditions encountered during the exposures is given in Table 4.5. In the first experiment, ANOVA showed significant differences within radiation (P < 0.001) and temperature treatments (P <
115 0.001). There were also significant interactions between radiation and temperature treatments (P < 0.04). Shaded (SHAD) and sunny minus UVB (SUN-B) conditions were always more favourable to conidium germination than full sunlight (SUN)
(Figure 4.12). Under full sunlight, conidium germination was the greatest at the coldest temperature and conidium germination decreased with increasing temperature. For conidia given the SUN-B treatment, the 30ºC and 40ºC treatments did not differ significantly but differed from the 10ºC and 20ºC treatments. The
UVA intensity and corresponding dose under SUN-B treatment were 8.57 W m-2 and
123. 40 kJ m-2, respectively. Under SHAD condition, conidium germination decreased significantly at 40ºC only compared with other temperature treatments
(Figure 4.12). No appressoria were formed after 6 h incubation post-exposure in this experiment.
TABLE 4.5. Average of magnitude of environmental conditions encountered during the exposures of R. alismatis conidia under natural sunlight. a t Isolar IUVA IUVB Dsolar DUVA DUVB Tair RH 21/01/02 11:00–15:00 827.18 13.18 2.65 11.91 189.79 38.16 31.90 55.26 18/03/02 11:00–15:00 805.33 13.14 2.61 11.60 189.22 37.58 27.43 61.97 11:00–12:00 766.26 12.62 2.44 2.76 45.43 8.78 25.56 66.21 12:00–13:00 835.73 13.84 2.76 3.01 49.82 9.94 26.85 64.28 13:00–14:00 841.65 13.74 2.77 3.03 49.46 9.97 28.00 59.88 14:00–15:00 778.18 12.38 2.46 2.80 44.57 8.86 29.28 57.51 15:00–16:00 653.75 9.94 1.91 2.35 35.78 6.88 30.26 54.45 16:00–17:00 482.14 7.09 1.25 1.74 25.52 4.50 30.56 51.58 15:00–17:00 567.17 8.51 1.58 4.08 61.27 1.38 30.42 53.14 11/04/02 11:00–15:00 596.58 9.22 1.70 8.59 132.77 24.53 25.60 67.57 11:00–14:00 677.64 10.49 1.92 7.32 113.29 20.84 25.22 68.38 11:00–13:00 699.11 10.94 2.01 5.03 78.77 14.49 24.77 68.81 11:00–12:00 698.45 11.05 2.01 2.51 39.78 7.26 24.46 70.42 12:00–12:30 709.25 11.16 2.06 1.28 20.09 3.70 24.94 66.61 12:00–13:00 700.10 10.83 2.00 2.52 38.99 7.23 25.09 67.16 13:00–14:00 635.41 9.62 1.76 2.29 23.83 6.37 26.11 67.55 14:00–15:00 353.08 5.46 1.03 1.27 19.66 3.47 26.76 65.15 13:00–15:00 494.72 7.53 1.40 3.56 54.22 10.09 26.43 66.35 aVariables are defined in Table 4.2.
116 1st experiment (21 January 2002) 120 10oC o 20 C a a a 100 o a 30 C a o 40 C 80 b bc bc 60 bc
cd 40 Conidium germination (%) germination Conidium d 20
e 0 SUN SU N-B SHAD Irradiation treatment
FIGURE 4.12. Effect of irradiation treatments on conidium germination of R. alismatis tested under 3 light conditions at 4 different temperatures. Germination was assessed after 6 h incubation following exposure. SUN: Full sunlight; SUN-B: Sun without UVB; SHAD: Shade. Bars labelled with same letter are not significantly different at P < 0.05 according to Fisher’s LSD test.
In the second experiment, ANOVA also showed significant (P < 0.0001) differences between radiation treatments, temperatures and the interaction between them on conidium germination. In addition, the trends with respect to irradiation and temperature were also the same (Figure 4.13A). For conidia given the SUN treatment, germination was again affected by temperature. However, at 10ºC conidium germination was greater than in the first experiment whilst at the other temperatures it was reduced. For the SUN-B treated conidia, there appears to be no
117 significant difference between the two experiments. Finally, for the SHAD treatment, germination at 30ºC was reduced compared with the conidia at 10ºC or
20ºC whilst in the first experiment conidia at 30ºC germinated as well as the conidia at lower temperatures.
There were significant (P < 0.001) differences between radiation treatments on appressorium formation whereas effect of temperature treatments was not significantly different. There was also a significant (P < 0.01) interaction between radiation treatments and temperatures on appressorium formation (Figure 4.13B).
Appressorium formation was the highest at 20ºC in the SUN-B treatment with approximately 40% of germinated conidia forming appressoria. Appressorium formation was reduced by increasing or decreasing temperature. However, it was comparable with appressorium formation at 30ºC. The UVA intensity and corresponding dose under SUN-B treatment were 8.55 W m-2 and 123.12 kJ m-2, respectively. In the SUN treatment, appressorium formation was reduced to below
20% and in the SHAD treatment to below 5% (Figure 4.13B).
118 2nd experiment (18 M arch 2002) 100 a a a a a A
80 10oC b b b 20oC 30oC 60 o 40 C b
40 c Conidium germination (%) germination Conidium 20 d d 0 SUN SUN-B SHAD 100 B
80
60
c 40
Appressorium formation(%) bc 20 ab
ab ab a a a a a a a 0 SUN SUN-B SHAD Irradiation treatment
FIGURE 4.13. Effect of irradiation treatments on conidium germination and appressorium formation of R. alismatis tested under 3 light conditions at 4 different temperatures. Conidium germination and appressorium formation were assessed after 6 h incubation following exposure. SUN: Full sunlight; SUN-B: Sun without UVB; SHAD: Shade. Bars labelled with same letter are not significantly different at P < 0.05 according to Fisher’s LSD test.
119 4.3.6.1.2 C. orbiculare
For C. orbiculare, the first experiment took place on 5 December 2002 and the second on 7 January 2003. The average of magnitudes of environmental conditions encountered during the exposures is given in Table 4.6. In the first experiment, conidium germination was significantly (P < 0.0001) affected by the radiation treatments. However, there were no significant differences between temperatures and no significant interaction. Conidium germination was the highest in the SUN-B treatment with ~80% of conidia germinating. In the SHAD treatment germination was reduced to ~40% and in the SUN treatment to ~10% (Figure
4.14A).
Appressorium formation was also significantly (P < 0.00001) affected by the radiation treatments. However, there were no significant differences between temperatures and no significant interaction. Appressorium formation was the highest in the SUN-B treatment followed by the SHAD and SUN treatments, respectively
(Figure 4.14B). The UVA intensity and corresponding dose under SUN-B treatment were 10.62 W m-2 and 152.92 kJ m-2, respectively. Most appressoria were melanised. Non-melanised appressoria were rarely formed (Figure 4.15).
120 TABLE 4.6. Average of magnitude of environmental conditions encountered during the exposures of C. orbiculare conidia under natural sunlight. a t Isolar IUVA IUVB Dsolar DUVA DUVB Tair RH 05/12/02 12:00–16:00 1002.02 16.33 3.13 14.42 235.15 45.07 30.49 55.86 07/01/03 11:00–15:00 979.61 15.00 3.06 14.10 216.00 44.06 34.60 11:00–14:00 1091.66 16.84 3.41 11.78 181.87 36.82 34.22 11:00–13:00 1075.65 16.72 3.36 7.74 120.38 24.19 33.77 11:00–12:00 1030.83 16.26 3.22 3.71 58.53 11.59 33.46 12:00–13:00 1117.27 17.18 3.49 4.02 61.84 12.56 34.09 13:00–14:00 1123.67 17.09 3.51 4.04 61.52 12.63 34.11 14:00–15:00 631.79 8.08 1.75 2.27 29.08 6.30 34.76 13:00–15:00 826.64 13.09 2.73 5.95 94.24 19.65 34.43 18/01/03 11:00–15:00 1008.42 15.12 3.15 14.52 217.72 45.36 34.00 11:00–13:00 1021.23 15.41 3.19 7.35 110.95 22.96 33.17 11:00–12:00 970.01 14.75 3.03 3.49 53.10 10.98 32.74 12:00–13:00 1075.65 16.08 3.36 3.87 57.88 12.09 33.09 13:00–14:00 1008.42 14.86 3.15 3.63 53.49 11.34 34.11 14:00–15:00 986.01 14.80 3.08 3.54 53.28 11.08 34.76 15:00–17:00 709.54 10.17 2.04 5.10 73.22 14.68 14:00–16:00 826.64 13.21 2.73 5.95 95.11 19.65 15:00–16:00 747.41 11.63 2.38 2.69 41.86 8.56 16:00–17:00 600.08 8.73 1.71 2.16 31.42 6.15 13:00–13:15 1094.86 16.32 3.42 0.98 14.68 3.07 34.16 13:00–13:30 1085.26 16.16 3.39 1.95 29.08 6.10 34.26 13:00–13:45 1008.42 14.80 3.15 2.72 39.96 8.50 34.22 14:00–14:30 1024.43 15.29 3.20 1.84 27.52 5.76 34.46 15:00–15:30 769.40 11.78 2.45 1.38 21.20 4.41 aVariables are defined in Table 4.2.
121 1st experiment (5 December 2002) 100 d A d d 80 10oC 20oC cd 30oC o 60 40 C bcd
abcd
40 abc abc abc
Conidium germination (%) germination Conidium abc 20 ab
0 a SUN SUN-B SHAD 100 B
80
60
40 d cd
Appressorium formation(%) bcd 20 abc abc ab ab ab a aaa 0 SUN SUN-B SHAD Irradiation treatment
FIGURE 4.14. Effect of irradiation treatments on conidium germination and appressorium formation of C. orbiculare tested under natural sunlight at 4 different temperatures. Conidium germination and appressorium formation were assessed after 16 h incubation following exposure. SUN: Full sunlight; SUN-B: Sun without UVB; SHAD: Shade. Bars labelled with same letter are not significantly different at P < 0.05 according to Fisher’s LSD test.
122 A
MA
MA
B
NA
C
MA
FIGURE 4.15 Melanised (MA) (A and C) and non-melanised appressoria (NA) (B) formed by C. orbiculare. Bar = 10 µm
123 In the second experiment, ANOVA showed significant differences in the effect of radiation treatments (P < 0.0001) and temperatures (P < 0.00001) on conidium germination. There were also significant interactions between radiation and temperature treatment (P < 0.006). For conidia given the SUN treatment, conidium germination was again the lowest. However, at 10ºC conidium germination was less than in the first experiment (Figure 4.16A). For SUN-B treatment, conidium germination at all temperatures was less than in the first experiment. However, a similar trend was observed for SUN-B treated conidia at 10,
20 and 30ºC whilst at 40ºC conidium germination was significantly reduced. Finally, for the SHAD treatment, germination at 30 and 40º were reduced compared with the conidia at 10 or 20ºC whilst in the first experiment conidium germination was similar at all temperatures.
Appressorium formation was significantly affected by temperature and radiation treatments (P < 0.00001). There were also significant interactions between radiation and temperature treatments (P < 0.00001) (Figure 4.16B). For conidia given the SUN treatment, no appressoria were formed at any temperature tested. For the SUN-B treated conidia, a similar level of appressorium formation was observed, but no appressoria were formed at 40ºC in this experiment. The UVA intensity and corresponding dose under SUN-B treatment were 9.76 W m-2 and 140.54 kJ m-2, respectively. For the SHAD treatment, no appressoria were formed at 30 and 40ºC.
However, at 10 and 20ºC, appressorium formation was greater than in the first experiment.
124 2nd experiment (7 January 2003) 100 A
o 80 10 C c 20oC 30oC c 40oC cc 60 c
40
b Conidium germination (%) germination Conidium 20 ab ab ab ab a a 0 SUN SUN-B SHAD
100 B
80
60
40
c bc bc Appressorium formation(%) bc 20 b
aaaa a aa 0 SUN SUN-B SHAD Irradiation treatment
FIGURE 4.16. Effect of irradiation treatments on conidium germination and appressorium formation of C. orbiculare tested under natural sunlight at 4 different temperatures. Conidium germination and appressorium formation were assessed after 16 h incubation following exposure. SUN: Full sunlight; SUN-B: Sun without UVB; SHAD: Shade. Bars labelled with same letter are not significantly different at P < 0.05 according to Fisher’s LSD test.
125 4.3.6.1.3 D. avenacea
The first experiment took place on 3 April 2002 and the second one on 5
December 2002. The average of magnitude of the environmental conditions encountered during the exposures is given in Table 4.7. In the first experiment, conidium germination was significantly (P < 0.0001) affected by the radiation and temperature treatments. However, there were no significant interactions between radiation and temperature treatments. Under the SUN treatment, conidium germination was the highest at the coldest temperature and significantly decreased with increasing temperature (Figure 4.17A). Under the SUN-B treatment, the 10, 20 and 30ºC did not differ significantly from each other but differed from the 40ºC treatment. Under SHAD treatment, conidium germination was high at 10 and 20ºC and low at 30 and 40ºC.
TABLE 4.7. Average of magnitude of environmental conditions encountered during the exposures of D. avenacea conidia under natural sunlight. a t Isolar IUVA IUVB Dsolar DUVA DUVB Tair RH 03/04/02 10:00–14:00 696.59 10.80 1.99 10.03 155.52 28.65 23.42 63.97 05/12/02 12:00–16:00 1002.02 16.33 3.13 14.42 235.15 45.07 30.49 55.86 aVariables are defined in Table 4.2.
There were significant differences among radiation (P < 0.001) and temperature (P < 0.0001) treatments on appressorium formation. There was also significant (P < 0.005) interaction between radiation and temperature treatments
(Figure 4.17B). For conidia given SUN treatment, at 10 and 20ºC about 60% of germinated conidia produced appressoria whereas no appressoria were produced at
30 and 40ºC. For the SUN-B treated conidia, at 20ºC about 80% of germinated conidia produced appressoria and numbers of appressoria were reduced by increasing
126 or decreasing temperature. However, appressorium formation at 20ºC was comparable with 30ºC. The UVA intensity and corresponding dose under SUN-B treatment were 7.02 W m-2 and 101.08 kJ m-2, respectively. For the SHAD treatment, appressorium formation was similar at all temperatures where between 5–
20% of germinated conidia produced appressoria (Figure 4.17B). Comparison of germinating conidia, with and without appressorium formation can be seen in Figure
4.18.
127 1st experiment (3 April 2002) 100
A d d 10oC d
80 cd 20oC d bcd
30oC c b bcd 40oC 60 bc
40 b Conidium germination (%) germination Conidium
20
a a
a 0 SUN SUN-B SHAD
100 B
f 80
f
e
60 def
e
d c bcde 40 abcd
Appressorium formation(%) 20
c
b
a
b
a a a a 0 a SUN SUN-B SHAD Irradiation treatment
FIGURE 4.17. Effect of irradiation treatments on conidium germination and appressorium formation of D. avenacea tested under natural sunlight at 4 different temperatures. Conidium germination and appressorium formation were assessed after 3 h incubation following exposure. SUN: Full sunlight; SUN-B: Sun without UVB; SHAD: Shade. Bars labelled with same letter are not significantly different at P < 0.05 according to Fisher’s LSD test.
128
A
AP
AP
B
FIGURE 4.18. Comparison of germinating conidia of D. avenacea, with (A) and without (B) appressorium (AP) formation. Bar = 20 µm
129 In the second experiment, conidium germination was significantly (P < 0.01) affected by temperature treatments. However, there were no significant differences between radiation treatments and also no interaction. Under SUN irradiation, the 10,
20 and 30ºC treatments did not differ significantly; however, the 20ºC differed from
40ºC treatment (Figure 4.19A). There was little significant difference in conidium germination at the different temperatures in the SUN-B and SHAD treatments.
Appressorium formation was significantly affected by radiation (P < 0.0001) and temperature treatments (P < 0.005). However, there was no significant interaction between radiation and temperature treatments (Figure 4.19B). For the
SUN treatment, appressorium formation was similar at 10, 20, 30ºC and differed significantly from 40ºC. For the SUN-B treated conidia, appressorium formation was the highest at 20ºC and reduced significantly by increasing or decreasing temperature. The UVA intensity and corresponding dose under SUN-B treatment were 10.62 W m-2 and 152.92 kJ m-2, respectively. Finally, for the SHAD treatment, appressorium formation was the lowest and similar at all temperatures tested (Figure
4.19B).
130 2nd experiment (5 December 2002) 100 A
d
d
d d c d
c c c
b
c
b b c
c
b
bcd a bcd
b b a
80 a
a ab a 60
40 Conidium germination (%) germination Conidium 20
0 SUN SUN-B SHAD
100 B 10oC 20oC o 80 30 C 40oC
60
40 c
c
c c
Appressorium formation Appressorium formation (%) b
20 b
b a
a
a
a a
a
a a 0 SUN SUN-B SHAD Irradiation treatments
FIGURE 4.19. Effect of irradiation treatments on conidium germination and appressorium formation of D. avenacea tested under natural sunlight at 4 different temperatures. Conidium germination and appressorium formation were assessed after 6 h incubation following exposure. SUN: Full sunlight; SUN-B: Sun without UVB; SHAD: Shade. Bars labelled with same letter are not significantly different at P < 0.05 according to Fisher’s LSD test.
131 4.3.6.2 The effect of different levels of solar irradiation on germination of conidia
4.3.6.2.1 R. alismatis
The first experiment took place on 18 March 2002 and the second one on 11
April 2002. The average of magnitude of environmental conditions encountered during exposure times are given in Table 4.5. In the first experiment, conidium germination (P < 0.00001) and appressorium formation (P < 0.002) were significantly affected by different exposures to full natural sunlight (Figure 4.20).
The highest reduction in conidium germination occurred after 1 and 4 h exposure to full sunlight between 13:00–14:00 and 11:00–15:00, respectively, followed by
12:00–13:00 and 15:00–17:00 treatments. Maximum appressorium formation occurred after 1 h exposures between 14:00–15:00 and 15:00–16:00 to full sunlight with ~35-50% of conidia producing appressoria. Appressorium formation in other treatments was either reduced (~1–10%) in comparison with these values or did not occur.
132 100 Conidium germination Appressorium formation C 80 C C C C 60 c Percent 40 B B bc
ab 20 a A A a a a a a 0 No exposure 12:00-13:00 14:00-15:00 16:00-17:00 11:00-15:00 11:00-12:00 13:00-14:00 15:00-16:00 15:00-17:00 Time of exposure
FIGURE 4.20. Mean percent conidium germination and appressorium formation of R. alismatis conidia after exposure to full-spectrum sunlight at different times of the day (18/03/02). Data were recorded 6 h incubation post-exposure. Corresponding solar, UVA and UVB doses for exposure times are given in Table 4.5. Bars labelled with the same letter are not significantly different at P < 0.05 according to Fisher’s LSD test.
In the second experiment, there were significant (P < 0.00001) differences between time of exposure and duration of incubation but no significant interaction.
Conidium germination after exposure to full sunlight between 13:00–14:00, 13:00–
15:00, 11:00–13:00, 11:00–14:00 and 11:00–15:00 were significantly less than in the dark control after 3 h incubation post-exposure whereas after 16 h incubation, exposure between 11:00–12:00, 12:00–13:00, 13:00–15:00, 11:00–13:00, 11:00–
14:00 and 11:00–15:00 were significantly less than in the dark control (Figure 4.21).
Assessment after 16 h incubation was not performed as microcycle conidiation
(Figure 4.22) interfered with the assessment process. Exposure to sunlight between
133 13:00–14:00, 13:00–15:00 and 11:00–15:00 delayed the germination of the surviving conidia of R. alismatis. No significant delays in germination were observed in the dark control (Figure 4.21). No appressoria had formed 3 h post-exposure. 16 h post- exposure appressoria had formed; however, there were no significant differences among treatments on (data not presented).
120 3 h
j 16 h j 100 i
ij h
j
j
i
i
h
h i ghij
g
g
h i
80 f
g h
f e g
h f
g
f
g f e 60 e
defg g
f
e
f
d
e e c
e
d d
c d c
c
40 b
b
d c
Conidium germination (%) germination Conidium
b a
c
b
20 a
b
b
a a
a 0 No esposure 12:00-12:30 13:00-14:00 13:00-15:00 11:00-14:00 11:00-12:00 12:00-13:00 14:00-15:00 11:00-13:00 11:00-15:00 Time of exposure
FIGURE 4.21. Mean percent germination of R. alismatis conidia after exposure to full-spectrum sunlight at different times of the day (11/04/02). Data were recorded after 3 and 16 h incubation post-exposure. Corresponding solar, UVA and UVB doses for exposure times are given in Table 4.5. Bars labelled with the same letter are not significantly different at P < 0.05 according to Fisher’s LSD test.
134 A
NC
OC
B
NC
C
NC
FIGURE 4.22. Microcycle conidiation under natural sunlight by R. alismatis demonstrating that germ tubes arise from the original conidium (OC) producing single (A and C) or multiple (B) new conidia (NC). Bar = 15 µm
135 4.3.6.2.2 Dose-response curves
When the effect of full-spectrum sunlight on conidium germination of R. alismatis was expressed as a function of dose of total solar, UVA and UVB irradiances, non-linear relationships were obtained that showed a significant decrease in germination for conidia exposed to sunlight. Models to describe the relationships are given in Table 4.4. In the first experiment, conidium germination versus total solar, UVA and UVB doses could be described by a quadratic model. Germination was halved 6 h after incubation post-exposure for doses of solar radiation, Dsolar,
-2 -2 DUVA and DUVB, of about 4 MJ m , 60 and 12 kJ m , respectively, based on the model (Figure 4.23).
In the second experiment, although the data obtained 3 and 16 h post- exposure showed a great deal of variability, they could be described by a significant cubic relationship (Figure 4.24). Germination after 3 h incubation post-exposure was halved for doses of solar radiation of 3 MJ m-2, 45 and 9 kJ m-2, respectively, for
Dsolar, DUVA and DUVB. Conidium germination 16 h after exposure was halved for
-2 -2 doses of solar radiation of 5 MJ m , 80 and 15 kJ m , respectively, for Dsolar, DUVA and DUVB.
136 100
80 A
60
40
20
Conidium germination (%) germination Conidium 0 024681012
D -2 solar (MJ.m ) 100 B 80
60
40
20
Conidium germination (%) germination Conidium 0 0 20 40 60 80 100 120 140 160 180 200
D -2 UVA (KJ.m ) 100 C 80
60
40
20
Conidium germination (%) germination Conidium 0 0 4 8 1216202428323640
D -2 UVB (KJ.m )
FIGURE 4.23. Percent germination of R. alismatis conidia exposed to various doses of full-spectrum sunlight on 18/03/02. Conidium germination was assessed after 6 h incubation post-exposure. Panel (A) shows the relationship between germination and the dose of total solar radiation; (B) with the dose of UVA; and (C) with the dose of UVB.
137 100 3 h A 80 16 h
60
40
20
Conidium germination (%) germination Conidium 0 0123456789
D -2 solar (MJ.m ) 100 3 h B 80 16 h
60
40
20
Conidium germination (%) germination Conidium 0 0 20406080100120140
D -2 UVA (KJ.m ) 100 3 h C 80 16 h
60
40
20
Conidium germination (%) 0 0 2 4 6 8 101214161820222426
D -2 UVB (KJ.m )
FIGURE 4.24. Percent germination of R. alismatis conidia exposed to various doses of full-spectrum sunlight on 11/04/02. Conidium germination was assessed after 3 and 16 h incubation post-exposure. Panel (A) shows the relationship between germination and the dose of total solar radiation; (B) with the dose of UVA; and (C) with the dose of UVB.
138 4.3.6.2.3 C. orbiculare
The first experiment took place on 7 January 2003 and the second on 18
January 2003. The average of magnitude of environmental conditions encountered during the exposure times are given in Table 4.6. In the first experiment, conidium germination was significantly (P < 0.00001) affected by time of exposure to natural full sunlight. Conidium germination was significantly decreased in all treatments compared with dark control (Figure 4.25).
100
80
a 60
40 Conidium germination (%) germination Conidium 20 b bc cd cd ddd cd 0 No exposure 12:00-13:00 14:00-15:00 11:00-13:00 11:00-15:00 11:00-12:00 13:00-14:00 13:00-15:00 11:00-14:00 Time of exposure
FIGURE 4.25. Mean percent germination of C. orbiculare conidia after exposure to full-spectrum sunlight at different times of the day (07/01/03). Data were recorded after 16 h incubation post-exposure. Corresponding solar, UVA and UVB doses for exposure times are given in Table 4.6. Bars labelled with the same letter are not significantly different at P < 0.05 according to Fisher’s LSD test.
139 In the second experiment, conidium germination was also significantly (P <
0.00001) affected by time of exposure. The dark control, exposure between 13:00–
13:15 and 15:00–15:30 did not differ significantly but they differed from all other treatments (Figure 4.26). In these treatments germination was reduced by at least
50% or did not occur at all.
100
80
d 60 d d
40
c c c
Conidium germination (%) germination Conidium 20 bc abc abc ab a a a aa a 0 11:00-15:00 11:00-13:00 11:00-12:00 12:00-13:00 13:00-13:15 13:00-13:30 13:00-13:45 13:00-14:00 14:00-14:30 14:00-15:00 15:00-15:30 15:00-16:00 16:00-17:00 14:00-16:00 15:00-17:00 No exposure No Time of exposure
FIGURE 4.26. Mean percent germination of C. orbiculare conidia after exposure to full-spectrum sunlight at different times of the day (18/01/03). Data were recorded after 16 h incubation post-exposure. Corresponding solar, UVA and UVB doses for exposure times are given in Table 4.6. Bars labelled with the same letter are not significantly different at P < 0.05 according to Fisher’s LSD test.
140 4.3.6.2.4 Dose-response curves
When the effect of full-spectrum sunlight on conidium germination of C. orbiculare was expressed as a function of dose of total solar, UVA and UVB irradiances, non-linear relationships were obtained that showed a significant decrease in germination for conidia exposed to sunlight. Models to describe the relationships are given in Table 4.4. In the first experiment, conidium germination versus total solar, UVA and UVB doses were well described by cubic relationship. Total loss of germination 16 h incubation post-exposure occurred for doses of solar radiation,
-2 -2 Dsolar, DUVA and DUVB, of just over 4 MJ m , 60 and 12 kJ m , respectively.
Germination was halved for doses of solar radiation, Dsolar, DUVA and DUVB, of about
1 MJ m-2, 20 and 4 kJ m-2 based on the model (Figure 4.27).
In the second experiment, conidium germination versus total solar, UVA and
UVB doses were also followed significant cubic relationships 16 h incubation post- exposure. Germination was almost halved for doses of solar radiation, Dsolar, DUVA
-2 -2 and DUVB, of about 1.5 MJ m , 20 and 5 kJ m , respectively (Figure 4.28).
141 100 A 80
60
40
20
Conidium germination (%) germination Conidium 0 024681012
D -2 solar (MJ.m ) 100 B 80
60
40
20
Conidium germination (%) germination Conidium 0 0 20 40 60 80 100 120 140 160 180 200
D -2 UVA (KJ.m ) 100 C 80
60
40
20
Conidium germination (%) germination Conidium 0 0 4 8 1216202428323640
D -2 UVB (KJ.m )
FIGURE 4.27. Percent germination of C. orbiculare conidia exposed to various doses of full-spectrum sunlight on 07/01/03. Conidium germination was assessed after 16 h incubation post-exposure. Panel (A) shows the relationship between germination and the dose of total solar radiation; (B) with the dose of UVA; and (C) with the dose of UVB.
142 100 A 80
60
40
20
Conidium germination (%) germination Conidium 0 012345678
D -2 solar (MJ.m ) 100 B 80
60
40
20
Conidium germination (%) germination Conidium 0 0 20406080100120
D -2 UVA (KJ.m ) 100 C 80
60
40
20
Conidium germination (%) germination Conidium 0 0 2 4 6 8 10 12 14 16 18 20 22 24
D -2 UVB (KJ.m )
FIGURE 4.28. Percent germination of C. orbiculare conidia exposed to various doses of full-spectrum sunlight on 18/01/03. Conidium germination was assessed after 16 h incubation post-exposure. Panel (A) shows the relationship between germination and the dose of total solar radiation; (B) with the dose of UVA; and (C) with the dose of UVB.
143 4.4 DISCUSSION
4.4.1 Effects of simulated sunlight
There have been few studies on conidium germination and appressorium production by R. alismatis and C. orbiculare. Jahromi et al. (1998) found that conidia of R. alismatis began to germinate on 2% WA after 2 h incubation at temperatures between 20 and 35ºC in the dark with maximum rate occurring after 6 h incubation at 25 and 30ºC. Appressorium formation did not occur or were not assessed in their experiments. McRae (1989) showed that germination of C. orbiculare conidia on X. spinosum leaf discs occurred 5 and 8 h after inoculation with maximum level (~75%) being reached by 24 h after inoculation at 25ºC.
Appressoria began to form somewhere between 5 and 8 hours after inoculation with a maximum level (~40%) being reached by about 36 h after inoculation. In contrast, conidium germination on nitrocellulose membranes commenced somewhere between
0-2 h after inoculation with maximum level (~93%) being reached by 12 h after inoculation and appressorium formation commenced between 2-4 h after inoculation with maximum level (~96%) being reached by 15.5 h after inoculation. Throughout the experiments described in the Results section above, for each pathogen, assessment times for conidium germination and appressorium formation were modified in light of the data gained from each experiment and also to fit the particular objectives of the experiment.
The inhibitory and stimulatory effects of UV radiation on conidium germination and appressorium formation on the potential mycoherbistats were shown clearly. Construction of an action spectrum in the UVB range (280–315 nm) indicated that wavelengths below 315 nm were the most damaging in terms of
144 conidium germination for both C. orbiculare and R. alismatis. However, for these species appressorium formation was increased significantly by wavelengths between
290 and 315 nm compared with dark control. This indicates that appressorium formation is stimulated by UV light between 290–400 nm. The promotion of appressorium formation by UV radiation (315–400 nm) has also been shown for C. lagenarium (Zahirul Islam and Honda, 1996).
There was no adverse effect on conidium germination of D. avenacea exposed to UVB range for 2 h at irradiance of 2.32 W m-2. Among other important functions, fungal conidium pigments are thought to provide protection from the detrimental effects of light (Leach, 1971). In this regard, it is not surprising that conidia of D. avenacea, the only pigmented species investigated, were the most resistant to UVB radiation. However, wavelengths below 280 nm (UVC) killed the conidia of D. avenacea but the UVC radiation is mainly screened out by atmosphere. Fungal conidia in the field are mainly exposed to UVB (280–315 nm) and UVA (315–400 nm) radiation (Caldwell, 1981).
The triggering of fungal conidium germination by light is rare; however, it does occur in some species (Sussman and Halvorson, 1966). The results of this study indicate that the germination capability of conidia in three species was not improved by exposure to UVA radiation (315–400 nm) compared with the dark control.
This study has shown that the most damaging wavelengths on conidium germination for R. alismatis and C. orbiculare were between 280–315 nm, which transmitted quite efficiently through Pyrex. Thus, Pyrex was selected to study the effect of UVB radiation exposure at different periods of time (UVB doses) on conidium germination of R. alismatis and C. orbiculare. UVB radiation caused
145 damage to conidia of R. alismatis and C. orbiculare as shown by both a delay and reduction in germination. Initial results from stimulated sunlight exposure showed that the germination of C. orbiculare conidia decreased exponentially as UVB dose increased. For R. alismatis, the reduction in germination could be described by a cubic relationship. Generally, germination figures for conidia in exposed and unexposed treatments were more similar after the longer post-exposure incubation times. This supports the view that germination can be delayed by exposure to UVB radiation. The results of this study indicate, firstly, that responses to UVB radiation are strongly dose dependent, and secondly, low UVB doses only caused delays in the germination of conidia whereas higher doses killed conidia or caused delays in the germination of survivors. A strong delay in the germination of surviving conidia of
Verticillium lecanii (Zimm.) Viegas and Aphanocladium album Gams. (Braga et al.,
2002), Metarhizium anisopliae (Metschnikofi) Sorokin (Braga, et al., 2001a) and
Metarhizium flavoviride Gams & Rozsypal (Moore et al., 1993) was also observed.
Since germination was delayed rather than abolished in a study of
Cladosporium cucumerinum Ellis & Arth. by Owens and Krizek (1980), these authors proposed that UVB affected protein synthesis by post transcriptional effects.
Several highly conserved checkpoints among eukaryotic and prokaryotic cells can interrupt the cell cycle as soon as damage to genetic material and other cell structures is detected (Petrocelli and Slingerland, 2000; Carr, 2001). This mechanism, by preventing the duplication of mutated DNA and the segregation of fractured chromosomes, increases the chances of survival of daughter cells and of the maintenance of genetic material integrity (Elledge, 1996). In the case of conidia exposed to radiation, the consequence of the interruption of the cell cycle is the temporary interruption of germination (Braga et al., 2001a).
146 4.4.2 Effects of natural sunlight
Various fungi differ in the length of their survival due to their intrinsic properties (Sussman, 1968). It has been repeatedly stated that two components of the environment that limit the utilisation and effectiveness of potential mycoherbicides are temperature and moisture as humidity or free water (Greaves et al. 1989; TeBeest and Templeton, 1985). Among the environmental influences, solar radiation in general, and its UV wavelength in particular, exert the main effect on survival
(Rotem et al., 1985). The impact of UVB radiation on fungal populations in natural habitats has not been extensively studied. The current study is the first to quantitatively assess the effect of solar radiation on the germination of R. alismatis,
C. orbiculare and D. avenacea conidia.
There has been a long-held belief, particularly among plant pathologists, that sunlight is detrimental to conidium germination. However, apart from the fact that the confounding effects of temperature have often been ignored, the role of UVB has seldom been distinguished from that of UVA, or control treatments have been based on the total and unrealistic exclusion of UVB (Ayres et al., 1996). An early exception was the work of Maddison and Manners (1973) in which, they clearly demonstrated by use of cut-off filters to modify both sun and lamp light, that UVB
(0.4 W cm-2 at 300 nm and 5.8 W cm-2 at 310 nm) inhibited by 90% the germination of conidia of Puccinia striiformis West. In the current study, four different temperatures and two controls (SHAD and SUN-B) were used to separate the effects of radiation and temperature. From the fact that (A) conidium germination among the identical temperature treatments for controls (SHAD and SUN-B) was generally more than SUN treatment and (B) although the temperature in samples under the window glass (SUN-B) was not measured but estimated to be higher than in samples
147 exposed directly to sunlight, it can be assumed that incubation temperature was not the major factor controlling germination in the present investigations. This reinforces the conclusion (e.g. Rotem and Aust, 1991) that UV radiation in general and its UVB portion in particular in sunlight, is the primary cause of conidium mortality. Using Mylar filters held in screens above bean plants (Phaseolus vulgaris
L.) in the field, Caesar and Pearson (1983) also demonstrated that the UVB component of sunlight reduced the viability of ascospores of S. sclerotiorum.
In both experiments with R. alismatis, increased germination losses under
SUN treatment resulted from higher temperatures. Similar results have been reported for the survival of conidia of Uncinula necator (Schweiniz) Burrill (Willocquet et al., 1996) and Metarhizium flavoviride Gams & Rozsypal (Moore et al., 1996).
Generally, germination figures for conidia were more similar under SUN-B and
SHAD conditions and they were always more favourable to conidium germination than SUN condition. In contrast to R. alismatis, conidium germination of C. orbiculare did not differ under the SUN treatment at all temperatures tested and always was generally lower than SUN-B and SHAD conditions. Therefore, for R. alismatis it may be possible to manipulate the fungal application time in order to reduce the adverse effect of sunlight, particularly at the beginning of the growing season of the target weeds when the temperature and radiation are moderate, but this seems not to be applicable for C. orbiculare.
In the first experiment, under SUN condition, conidium germination of D. avenacea was the highest under coldest temperature and significantly decreased with increasing temperature (Figure 4.17A); however, in the second experiment, germination percentages were similar for all treatments (Figure 4.19A). In the first experiment, conidium germination was assessed 3 h post-exposure whilst in the
148 second experiment it was assessed 6 h after exposure. This indicates that conidium germination of D. avenacea could be delayed at higher temperatures (30 and 40ºC).
For C. orbiculare, at some temperatures tested, conidium germination was higher under SUN-B than SHAD condition. This result indicates that conidium germination could be improved by sunlight without UVB radiation. This effect was not observed for R. alismatis and D. avenacea.
Temperatures in direct sunlight in the experimental site during December and
January (summer in Australia) were often 30–35ºC over the midday period (Table
4.5 and 4.6). Material sprayed on to dead vegetation could reach much higher temperatures than those reached on living vegetation (Moore et al., 1996). Actively transpiring leaves, on the other hand, can be 1 to 2ºC cooler than the air temperature depending on wind speed and vapour pressure deficit (VPD) of the air (Gates, 1980).
Thus, the conidia on Petri dishes floated on water at 30ºC, which were used in the full sunlight experiments, are environmentally realistic and should represent conidia naturally deposited on the upper surface of a leaf. It should be emphasised that the exposures were performed during the time of day and year of maximum irradiance and on cloudless or almost cloudless days. Thus, these conditions represent some of the harshest that would be experienced by conidia. Factors such as shading, cloudiness and seasonal variations in irradiance will enhance the chances of conidium survival, germination and infection of host plants in the field.
For C. orbiculare, initial results from the simulated experiments showed that germination decreased exponentially as UVB dose increased, i.e. the rate of decrease in germination was highest at low levels of UVB doses. These results were in contrast to those obtained from the natural sunlight experiments. Here, germination of conidia could be described by a significant cubic relationship. These can be
149 partially explained by the variation in intensity of UVB in full sunlight at different times of a day. These relationships (exponential and cubic models describing the effect of UV radiation on germination) are similar to those found by Jones et al.
(1993) for a nuclear polyhedrosis virus and by Moore et al. (1996) for M. flavoviride.
Conidia of R. alismatis exposed to natural sunlight showed similar cubic relationships to the simulated sunlight. The exponential relationships are similar to those in the single-hit target model, which are more typical of single-stranded DNA or RNA organisms than double-stranded ones (Harm, 1980). There was a variation in susceptibility to UV radiation between these two pathogens. Conidium germination of R. alismatis was halved 16 h incubation post-exposure for UVB dose of 15 kJ m-2 whereas conidium germination of C. orbiculare was halved for UVB dose of about 5 kJ m-2, respectively. Conidia of C. orbiculare are deactivated by much lower doses of radiation indicating C. orbiculare is more susceptible to UV radiation than R. alismatis.
The process of reciprocity implies that exposure for an extended period during a clear day in spring, when the solar elevation and flux are low, can be just as damaging as exposure for a short period of sunshine on a summer day, when solar elevation and flux are higher. This is because these two exposures deliver the same dose (Ayres et al., 1996). Owens and Krizek (1980) established that there was reciprocity between irradiance and period of exposure in their system (this was true at 325 nm, although not at 265 nm) for C. cucumerinum. In contrast, reciprocity was not observed when conidia of M. anisopliae were exposed to a total UVB dose of
17.3 kJ m-2 (Braga et al., 2001b). In this study, conidium germination of C. orbiculare was higher after a UVB dose of 6.1 kJ m-2 given by exposure between
16:00–17:00 at an irradiance of 1.7 W m-2 compared with a similar UVB dose
150 obtained by exposure between 13:00–13:30 at an irradiance of 3.39 W m-2, as shown in Figure 4.26. Conidium germination was also higher after a UVB dose of 8.5
KJ.m-2, obtained by exposure between 15:00–16:00 at an irradiance of 2.3 W m-2 compared with similar dose obtained by exposure between 13:00–13:45 at an irradiance of 3.1 W m-2, as shown in Figure 4.26. This indicates non-reciprocity when conidia of C. orbiculare exposed to these specific UVB doses. These results indicates that, although UVB damage is considered to be proportional to dose, for C. orbiculare a short period of high-intensity irradiance is be more damaging than an equal dose at a lower intensity given over longer period. However, for R. alismatis, conidium germination was not different after a UVB dose of 3.7 kJ m-2 obtained by exposure between 12:00–12:30 at an irradiance of 2.06 W m-2 from a similar dose obtained by exposure between 14:00–15:00 at an irradiance of 2.06 W m-2, as shown in Figure 4.21. This result indicates that conidium germination of R. alismatis shows reciprocity, at least when exposed to a total UVB dose of 3.7 kJ m-2. The finding that conidium germination may demonstrate reciprocity or non-reciprocity illustrates the importance of evaluating the impacts of irradiance in order to optimise the application time for these potential mycoherbistats.
Conidium germination was usually assessed after 3, 6, 12 or 16 h incubation for R. alismatis conidia exposed to UV or natural sunlight. Assessment after longer periods of incubation was not performed due to microcycle conidiation. These secondary conidia produced from germ tubes eventually become detached and could interfere with the assessment process (Figure 4.22). Microcycle conidiation is the production of spores following conidium germination without an intervening phase of vegetative growth (Hanlin, 1994; Smith et al., 1981) and has been described in more than 100 fungal species (Hanlin, 1994). Conidia of many fungal species may
151 use microcycle conidiation as a survival mechanism when conditions are unfavourable for vegetative growth. This method of conidiation of R. alismatis during exposure to sunlight may have epidemiological consequences and in particular subsequent mycoherbistatic activity when primary conidia fail to infect, but this has not been studied.
Generally, conidia of all three pathogens that received the SUN-B treatments produced many more appressoria than conidia in the SUN and SHAD treatments and appressorium formation was the highest at 20°C. In addition to the indirect effects on a host, light can directly influence appressorium formation (Emmett and Parbery,
1975). Requirements for specific light-temperature regimes for appressorium formation are well known for rust diseases. Several authors suggest that a two-phase regime is necessary to promote appressorium formation. Phase one is usually a 2 h period at about 20°C, while phase two involves a similar period at a temperature increased by approximately 5°C (Kim and Rohringer, 1974; Maheshwari et al., 1967;
Pavgi and Dickson, 1961). However, there is a controversy over the need for dark during phase one (Emmett and Parbery, 1975). In this study, samples were incubated at 25°C in the dark after exposure in order to avoid the effect of photoreactivation
(Jagger, 1958). This study indicates that sunlight from which UVB wavelengths
(280–315 nm) have been removed at initial temperature of 20°C stimulates appressorium initiation of these pathogens used in this current study. Mature appressoria of anthracnose fungi generally are pigmented with melanin (Staples and
Hoch, 1987). In this study, non-melanised appressoria were rarely formed by conidia of C. orbiculare. This indicates that sunlight without UVB radiation not only stimulates appressorium initiation but also stimulates appressorium melanisation.
Kubo et al. (1984) showed that C. lagenarium appressoria must be melanised in
152 order to form a successful penetration peg. This stimulation of melanised appressoria has practical implications for use of C. orbiculare as a mycoherbistat.
Conidia of C. orbiculare that received UVA treatment (window glass) in the laboratory produced more appressoria than conidia under window glass (SUN-B) in natural sunlight. About 50% of germinated conidia produced appressoria when exposed to a UVA dose of 4.24 kJ m-2 after 12 h incubation following exposure at
25°C whereas in the natural sunlight experiment about 15% of germinated conidia
-2 produced appressoria when exposed to a UVA dose of 140.54 kJ m at 20°C.
Appressorium formation has been shown to be optimal between 15–20°C on leaf discs with the maximum level (~70%) being reached 26 h after inoculation (McRae,
1989). Therefore, the data from the simulated and natural sunlight experiments shows that the amount of UV radiation had a greater effect on appressorium formation than did temperature. In contrast to C. orbiculare, conidia of R. alismatis that received UVA treatment (window glass) in the laboratory produced less appressoria than conidia under window glass (SUN-B) in natural sunlight. About
30% of germinated conidia produced appressoria when exposed to a UVA dose of
4.24 kJ m-2 in the laboratory whereas in the natural sunlight experiment about 50% of germinated conidia produced appressoria when exposed to a UVA dose of 44.57 kJ m-2 and no appressoria were formed by conidia exposed to a UVA dose of 123.40 kJ m-2. For D. avenacea no appressoria were formed by conidia exposed to the UVA dose of 4.24 kJ m-2 in the laboratory whereas in the natural sunlight experiments about 80 and 20% of germinated conidia produced appressoria when exposed to
UVA doses of 101.08 and 152.92 kJ m-2, respectively. These results indicate, firstly, appressorium initiation of these pathogens is stimulated by UVA radiation.
Secondly, appressorium initiation is UVA dose dependent and reduced or increased
153 with high doses. Reduction of appressorium formation in Puccinia helianthi Schw. by increasing light intensity has also been shown by Sood and Sackston (1972).
Lastly, there is a variation among these potential mycoherbistats in the UVA dose requirements for 50% appressorium formation with D. avenacea requiring highest dose followed by R. alismatis then C. orbiculare.
4.4.3 Practical implications
Variability in susceptibility to UV radiation has been shown among the three species of potential mycoherbistats. C. orbiculare is the most susceptible to UV radiation followed by R. alismatis then D. avenacea. The rapid inactivation of conidia and stimulation of appressorium formation of germinated conidia exposed to
UVB and UVA radiation, respectively, have serious implications for practical field control of target weeds, where a rapid and high germination rate is highly desirable in order to minimise the dew dependency and penetration period of these potential mycoherbistats. With the knowledge of how conidia of these potential mycoherbistats react to climate presented in this chapter, rapid conidium germination and appressorium formation could be achieved by manipulation of the time at which they are applied. Conidia could be applied earlier in the day so that they received sufficient UVA to stimulate appressorium formation. However, additional protection from UVB may be needed. Therefore, a suitable application time and any working formulation of these potential mycoherbistats should be designed to maximise both velocity (rate and extent) of germination and appressorium formation, possibly by inclusion of appropriate UV protectants with absorbance peak in the range between
280–315 nm. The use of UV protectants is examined in Chapters 5 and 6.
154
CHAPTER 5
PHOTOSTABILISATION OF FUNGI WITH
POTENTIAL AS MYCOHERBISTATS
5.1 INTRODUCTION
Studies of the effect of UV radiation (Chapter 4) and dew period requirements (Hetherington and Auld, 2001; McRae and Auld, 1988) on the potential mycoherbistats, R. alismatis, C. orbiculare and D. avenacea, suggest that specific formulations are required to achieve maximum efficacy on their host. Thus, the first objective of the studies in this chapter was to determine what compounds would act as UV protectants to significantly prolong the life of conidia of these fungi when exposed to natural sunlight. The second objective of these studies was to determine if compounds that stimulate conidium germination and appressorium formation can be found to increase virulence and consequently to reduce the dew dependency of potential mycoherbistats. UV protectants and stimulants may act synergistically and may provide the basis for the formulation of the mycoherbistats. R. alismatis and C. orbiculare, the two pathogens most sensitive to UVB radiation, were selected for this aspect of study.
155 Sunscreens act by physically reflecting and scattering light, by selectively absorbing radiation, or by converting short wavelengths to harmless, longer ones
(Jones and Burges, 1998). Reflectors include titanium oxide. Absorbents can be physical or chemical; the latter type contains derivatives of paramino benzoates, salicylates, cinnamates and benzophenones (Shaath, 1990). Natural absorbents that accompany the pathogens in microbial products confer variable UV protection
(Burges and Jones, 1998) and include amino acids (Ignoffo and Garica, 1995), B vitamins (Shapiro, 1985) and nitrogenous metabolic products (Ignoffo and Garica,
1994).
Relatively few studies have been conducted on the use of these types of protectants with fungal pathogens, as most studies have involved viruses or bacteria
(Killick, 1990; Martignoni and Iwai, 1985: Morris, 1983; Shapiro, 1992). With respect to fungi, most studies have concentrated on fungal pathogens of insects.
Exposure of conidia of M. flavoviride in water to simulated sunlight for 1 h resulted in 4.7% germination after 24 h incubation compared with 36.5% for conidia formulated in oil (Moore et al. 1993). Inglis et al. (1995) also reported that on glass surfaces, UVB radiation rapidly reduced the viability of conidia of Beauveria bassiana (Balsamo) Vuillemin in water whereas in oil there was 74% less reduction, indicating considerable protection by the oil. The addition of 1% oxybenzone resulted in 81.9% conidium germination in M. flavoviride after 3 h exposure and 48 h incubation compared with 28.1% in oil without the sunscreen (Moore et al. 1993).
The water-compatible fluorescent brightener, Tinopal LPW significantly increased survival of conidia of B. bassiana compared with the water control. However, conidium survival in the field was not enhanced by three oil-compatible adjuvants tested: oxybenzone, octyl-salicylate and ethyl-cinnamate (Inglis et al. 1995).
156 Similarly, conidium survival was not enhanced by adding 2% oxybenzone to a ULV oil formulation of M. flavoviride in Mali (Shah et al. 1998). Therefore, various formulations can enhance or reduce conidium viability.
The specific objectives of this chapter were to: (1) test and compare water formulations, and mineral and plant oil formulations of potential UV sunscreens for their toxicity towards conidia of C. orbiculare and R. alismatis; (2) determine the absorption spectra of the oils and sunscreens with different modes of action in order to select substances with a high degree of UVB (280-315 nm) absorbance; (3) use those oils and sunscreens with the greatest UVB absorption to test their ability to affect the survival of conidia of R. alismatis and C. orbiculare when exposed to UV radiation.
157 5.2 MATERIALS AND METHODS
5.2.1 Conidium suspensions
R. alismatis and C. orbiculare were cultured as described in Sections 4.2.1.
Conidium suspensions were made from 7-day-old cultures as described in Section
4.2.2.
5.2.2 Mineral and plant oils
Two mineral oils and three plant oils were examined in this study. The mineral oils were nC24 Ampol D-C-Tron (Ampol Ltd, Sydney, Australia) and nC24
Fuchs Spray Oil Universal (Fuchs, Ltd, Victoria, Australia). The plant oils were
Codacide (Microcide Ltd, Suffolk, UK), Synetrol (Cobbett Ltd, Hornsby, Australia) and Cottonseed oil (Sigma).
5.2.3 UV protectants
A range of UV protectants were obtained from various manufacturers (Table
5.1). Water-soluble UV protectants consisted of amino acids, B vitamins, optical brighteners, antioxidants and Anatase. All sunscreens tested were oil compatible.
5.2.4 Solubility of UV protectants
The solubilities of the UV protectants were determined in water and oil at room temperature (20 to 22°C). The production of a 5% solution of the UV protectants in both oil and water were attempted using a sonicator for 2 min. When
5% of a particular compound did not dissolve in either water or the oil, the highest concentration that dissolved was determined.
158 TABLE 5.1. Chemical names and suppliers of UV protectants used in this study. Group Chemical Concentration Company Amino acids Glutamic acid 5% ICN Biomedicals Proline 5% Sigma Tryptophan 5% Fluka Tyrosine 5% GIBCO
B Vitamins Folic acid 1% ICN Biomedicals Pyridoxine 5% Sigma Riboflavin 5% Sigma
Antioxidants Ascorbic acid 5% ICN Biomedicals Propyl gallate 0.1% Sigma
Sunscreens Benzyl cinnamate 5% Aldrich Ethyl cinnamate 5% Aldrich Eastern 5% ECCO p-aminobenzoic acid (pABA) 5% Sigma Tinuvin 171 (phenol, 2-(2H- benzotriazol-2-yl)-6-dodecyl- 4-Methyl) 5% Meury Ltd
Miscellaneous Anatase (titanium (IV) oxide) 5% Sigma Brightener 28 5% Sigma Melanin (synthetic) 0.1% Sigma
5.2.5 Toxicity testing
5.2.5.1 R. alismatis
The oils, 5% oil-in-water emulsions, were tested for toxicity. In addition, the compounds in Table 5.1 were formulated in water or D-C-Tron, at the concentrations given in Table 5.1, and tested for toxicity. To do this, suspensions of conidia of R.
6 -1 alismatis of 1×10 conidia mL were made in each solution. After 16 h at 25°C, 1 mL aliquots of the suspension were pipetted onto cellophane overlaying 2% water agar. Conidium germination was assessed after 6 h as described in Section 4.2.3.
159 5.2.5.2 C. orbiculare
Aqueous solutions of proline, tyrosine, ascorbic acid and folic acid at a concentration of 1%, melanin at 0.01%, 5% emulsions of Codacide and D-C-Tron were tested for toxicity. In addition, two oil-compatible sunscreens, Eastern and
Tinuvin (at 1%) were formulated in 5% emulsions of Codacide and D-C-Tron, respectively, and were also tested for toxicity. These oil emulsions or aqueous
6 -1 solutions of the UV protectants were prepared in a suspension of 1×10 conidia mL .
One mL aliquots were spread directly a layer of cellophane on PDA plates. A control plate of conidia and water only was included. The plates were incubated in the dark at 25°C. Conidium germination and appressorium formation were assessed
24 h after inoculation as described in Section 4.2.3.
5.2.6 Selection of UV protectants
5.2.6.1 R. alismatis
Conidia were suspended in each of the formulations of the water-soluble UV protectants at 1 or 5%. The 1% concentration was used to test the protective effect of compounds at lower concentrations and also for those that were toxic at 5%.
Melanin and propyl gallate were tested at a concentration of 0.1%. Oil-compatible sunscreens were only tested at 5%. Two hundred µL of each conidium suspension were pipetted onto cellophane overlaying 50 mm diameter Petri dishes and then exposed to UVB radiation for 2 h at irradiance of 2.32 W m-2 as described in Section
4.2.7. At the end of the exposure, the pieces of cellophane containing conidia were placed onto 2% water agar and incubated at 25°C in the dark. Conidium germination was assessed at 3 and 6 h intervals, as described in Section 4.2.3.
160 5.2.6.2 C. orbiculare
The UV protectants that were found to be most effective for R. alismatis were selected to test for their ability to protect conidia of C. orbiculare. Conidium
6 -1 suspensions containing 1×10 conidia mL were exposed to UVB radiation as described above. At the end of the exposure, the pieces of cellophane containing the conidia were placed on PDA and incubated at 25°C in the dark. Conidium germination was assessed after 16 h.
5.2.7 Natural sunlight experiment
Experiments were conducted to test the feasibility of using mineral or plant oils as possible carriers for R. alismatis conidia. Oil-in-water emulsions of D-C-Tron
6 -1 and Codacide at 10% (v/v) were prepared in suspensions of 1×10 conidia mL .
Two hundred µL of each aliquot were pipetted onto cellophane overlaying 50 mm diameter Petri dishes. The Petri dishes were floated on water adjusted to 30°C in a water bath and were then exposed to full-spectrum sunlight between 13:00–15:00 h on 11 April 2002. A control plate of conidia and water only was included. Two suspensions of conidia were exposed to full-spectrum sunlight from which UVB light was removed by two pieces of 3 mm thick window glass mounted above the irradiated Petri dishes and sunlight filtered by covering the Petri dishes with two pieces of citrus leaf were also included in the experiment. At the end of the exposure, the pieces of cellophane containing conidia were placed onto 2% water agar and incubated at 25°C in the dark. Conidium germination was assessed at 3 and
16 h intervals, as described in Section 4.2.3. 10% emulsion of Codacide was tested for toxicity as described in Section 5.2.5.1.
161 5.2.8 Spectrophotometer studies
The first series of experiments used the oils alone or oil-in-water emulsions.
The absorbance spectra of the following oils and emulsions were obtained using a
Cary spectrophotometer (Varian, NSW, Australia) with a scanning range between
250–500 nm: D-C-Tron, Codacide oil and oil-in-water emulsions of D-C-Tron and
Codacide at concentrations of 1 and 5%. In a second series of experiments, the absorbance spectra of the water-compatible UV protectants tyrosine, proline, folic acid, ascorbic acid and melanin in deionised distilled water, were measured. All chemicals were tested at a concentration of 1% except melanin, which was tested at
0.01%. In a third series of experiments, the absorbance spectra of the oil-compatible
UV protectants (all at 5%), Tinuvin, benzyl cinnamate, Eastern and tryptophane in
D-C-Tron oil, were obtained. In all experiments, deionised distilled water was used as a reference standard.
5.2.9 Statistical analysis
Laboratory experiments using R. alismatis were performed twice with three replicates for each treatment, and the results from two trials were pooled. Toxicity and sunscreen selection tests on C. orbiculare were not repeated due to agreement of the results with earlier findings for R. alismatis. Homogeneity of variances of data was determined using Bartlett’s test and data that were heteroscedastic were subjected to appropriate transformations before analysis. Analyses of variance were performed using STATISTICA software release 6.0 (StatSoft Inc., Tulsa OK, USA,
2001). Treatment means were compared by Fisher’s LSD tests at the 5% significance level.
162 5.3 RESULTS
5.3.1 Solubility of UV protectants
Of the four amino acids tested, tyrosine, proline and glutamic acid at concentrations of 5% were highly soluble in water. However, the solubility of tryptophane was low in water but it was highly soluble in oil. The B-group vitamins
(folic acid, pyridoxine and riboflavin) were highly soluble at concentrations of 5% in both water and oil. All cosmetic sunscreens, pABA, benzyl and ethyl cinnamate,
Tinuvin 171 and Eastern dissolved to produce 5% solutions in oil. Ascorbic acid, propyl gallate, anatase and fluorescent brightener were soluble in water. Melanin was marginally soluble in water and formulations of melanin (0.1 and 0.01%) in water were used for toxicity test and UV protection.
5.3.2 Toxicity testing
5.3.2.1 R. alismatis
In the first series of toxicity tests, the germination of conidia of R. alismatis was significantly (P < 0.001) affected by suspension in certain of the oils (Figure
5.1). The mineral oils (Fuchs and D-C-Tron) were not toxic to non-germinated conidia treated for 16 h at 25°C whereas all of the plant oils (Codacide, Synetrol and cottonseed) were toxic with Codacide and Synetrol being more toxic than cottonseed oil. None of the oil-in-water emulsions were toxic, including those made from the three plant oils. Appressorium formation was assessed after 6 h and was found to be low (1–6 % of conidia) and there were no differences amongst treatments.
In the second series of toxicity tests, all oil-compatible UV protectants were tested in D-C-Tron oil at concentration of 5%. Of the five UV protectants tested,
163 only ethyl cinnamate and pABA were toxic to non-germinated conidia of R. alismatis
(P < 0.001) after 16 h treatment at 25°C (Figure 5.2). The UV protectant, benzyl cinnamate increased conidium germination compared with the control.
g fg fg efg 100 efg def de d ) 80 c 60
40
20 Conidium germinationConidium (% b 0 a
Fuchs onseed Synetrol D-C-TronCodacide W+Fuchs Cott W+Synetrol W+D-C-TronW+Codacide Water control W+Cottonseed Oils and oil emulsions
FIGURE 5.1. Effects of oil and 5% oil-in-water emulsions (W+) on conidium germination of R. alismatis. Conidium germination was assessed 6 h incubation post-treatment. Bars labelled with same letter are not significantly different at P < 0.05 according to Fisher’s LSD test.
164 b b c 100 ab a
80
60
40
20
germinationConidium (%)
dd 0
te namate D-C-Tron cin l Water control y D-C-Tron+PABA D-C-Tron+TinuvinD-C-Tron+Eastern
D-C-Tron+Ethyl cinnama D-C-Tron+Benz Oil-compatible UV protectants
FIGURE 5.2. Effects of oil-compatible UV protectants on conidium germination of R. alismatis. Conidium germination was assessed 6 h post- treatment. All the UV protectants were tested at concentration of 5% in D-C-Tron. Bars labelled with same letter are not significantly different at P < 0.05 according to Fisher’s LSD test.
In the third series of toxicity tests, water-compatible UV protectants were tested in 5% aqueous solutions (Figure 5.3). Of the 11 UV protectants tested, only brightener 28, propyl gallate, folic acid and glutamic acid were toxic (P < 0.001).
Melanin and propyl gallate at a concentration of 0.1% and folic acid at 1% were not toxic (data not presented). Microcycle conidiation was initiated by conidia treated with proline and pyridoxine.
165 100 aaa aa aaa
80
60
d 40
20
Conidium germinationConidium (%) c b b 0
tase ProlineAna Folic acid Tyrosine PyridoxineRiboflavin Tryptophan Water control Propyl gallateAscorbic acid Glutamic acid
Fluorescent brightener Water-compatible UV protectants
FIGURE 5.3. Effects of water-compatible UV protectants on conidium germination of R. alismatis. Conidium germination was assessed 6 h post- treatment. All water-compatible UV protectants were tested at a concentration of 5%. Bars labelled with same letter are not significantly different at P < 0.05 according to Fisher’s LSD test.
5.3.2.2 C. orbiculare
Conidium germination and appressorium formation were significantly (P <
0.00001) affected by some of the UV protectants (Figure 5.4). After 24 h at 25°C, none of the water or oil-compatible UV protectants were toxic to non-germinated conidia of C. orbiculare with none of the treatments reducing germination.
However, conidium germination was enhanced by treatment with 1% Eastern in a
166 5% emulsion of Codacide, by a 5% emulsion of D-C-Tron and by a 1% Tinuvin in a
5% emulsion of D-C-Tron.
Appressorium formation was significantly increased by 1% aqueous solutions of proline and tyrosine (Figure 5.4). Appressorium formation was unaffected by treatment with melanin and folic acid but was reduced by the remaining treatments.
d d d
100 cd c bc abc ab a a Conidium germination 80 Appressorium formation
60 E E DE
Percent 40 CD
20 BC AB AB AB A A 0
1% 01%
astern 1% Proline 1% E Water controlTyrosine Folic acid 1%Codacide 5%D-C-Tron 5% Melanin 0. Ascorbic acid 1% ( 5%)+
Codacide emulsionD-C-Tron emulsion (5%)+Tinuvin 1% UV protectants
FIGURE 5.4. Effects of water and oil-compatible UV protectants on conidium germination and appressorium formation of C. orbiculare. Conidium germination and appressorium formation were assessed 24 h after treatment. Bars labelled with same letter are not significantly different at P < 0.05 according to Fisher’s LSD test.
167 5.3.3 Selection of UV protectants
5.3.3.1 R. alismatis
There were significant differences (P < 0.00001) between water-compatible
UV protectants in their ability to protect conidia from UV damage as assessed by conidium germination. There was also a significant difference in the proportion of conidia germinating at the two assessment times (P < 0.0002). However, there was no significant interaction between treatment with UV protectants and assessment time.
After 2 h exposure to UVB radiation, conidium germination in all formulations of water-compatible protectants was greater than that of control conidia treated with water alone (Figure 5.5). This affect was seen at both 3 and 6 h post- exposure assessment times. When assessed both 3 and 6 h post-exposure, four of the nine formulations provided a high level of protection with conidia treated with riboflavin, proline and melanin having germination levels similar to the dark control and with folic acid inducing a higher level of germination than the dark control.
168 100 l kl kl kl j jkl jkl 3 h
80 ijk ij hij
ghij 6 h ghi
60 ghi g fgh fg f efg
40 def de cd 20 bc Conidium germinationConidium (%) ab a 0
Water Proline Melanin Folic acid Tyrosine PyridoxineRiboflavin Tryptophan AscorbicPropyl acid gallate
Water (dark control) Water-compatible UV protectants
FIGURE 5.5. The influence of water-compatible UV protectants on the germination of conidia of R. alismatis subjected to a 2 h exposure to UVB radiation at an irradiance of 2.32 W m-2. Conidium germination was assessed 3 and 6 h post-exposure. All UV protectants were tested at a concentration of 1% except melanin and propyl gallate, which were tested at 0.1%. Bars labelled with same letter are not significantly different at P < 0.05 according to Fisher’s LSD test.
In the second experiment, there were also significant differences (P <
0.00001) in the germination of conidia treated by water-compatible protectants as well as between incubation times; however, there was no significant interaction between these factors. At the 3 h assessment period, conidium germination in 6 of the 8 water-compatible formulations was greater than that of conidia exposed to
UVB radiation in water alone (Figure 5.6). At the 6 h assessment time, conidium germination in all formulations was greater than the water treatment. At this time,
169 there was no difference between the germination of conidia in the dark control and those treated with ascorbic acid. In addition, there appeared to be no delay in germination as there was no significant difference between the two assessment times.
In addition to ascorbic acid, pyridoxine provided a level of protection resulting in levels of conidium germination similar to the dark control 6 h after incubation; however, germination was delayed as there was a significant difference between the percentages of conidia germinating at the two assessment times.
With the exception of cottonseed oil, all other treatments resulted in germination percentages of 40% or greater compared with 1% in the water control and 95% in the dark control: UV exposure also resulted in a delay in germination.
Although cottonseed oil gave a significant increase in protection, conidium germination was only 12%.
170 100 i hi hi 3 h ghi 80 ghi 6 h fgh
60 efg defg def def def 40 def cde bcd
20 bc bc abc Conidium germinationConidium (%) ab a 0 a
Fuchs n seed Water Synetrol o TyrosineD-C-Tron Codacide Pyridoxine Cott Ascorbic acid
Water (dark control) Water-compatible UV protectants
FIGURE 5.6. The influence of water-compatible UV protectants on the germination of conidia of R. alismatis following 2 h treatment with UVB radiation at an irradiance of 2.32 W m-2. Conidium germination was assessed 3 and 6 h post-exposure. All UV protectants were tested at a concentration of 5%. Bars labelled with same letter are not significantly different at P < 0.05 according to Fisher’s LSD test.
Results for the third experiment were similar to the previous two trials in that conidium germination was significantly (P < 0.00001) affected by treatment with oil- compatible protectants and that there was also a significant difference in germination between the two assessment times. Also, in this experiment there was no significant interaction between protectant treatment and assessment time. The five sunscreens significantly increased the survival of the conidia (Figure 5.7), but exposure caused a significant delay in germination. The main benefits of the sunscreens were seen when the conidia were incubated for 6 h. At this time, Tinuvin and Eastern provided
171 significantly more protection than the other sunscreens with germination percentages being similar to those of water and D-C-Tron-treated dark controls. The germination of conidia treated with the other compounds was 2 to 3.5 times greater than for irradiated conidia treated with D-C-Tron only.
j j 100 j j ij ij 3 h
80 hi 6 h h
60 gh fg efg
40 def cde cd
20 bc abc ab a Conidium germinationConidium (%) 0
han namate Water yptop cin D-C-Tron yl
D-C-Tron+Eastern Water (dark control)D-C-Tron+Tinuvin D-C-Tron+RiboflavinD-C-Tron+Tr D-C-Tron (dark control) D-C-Tron+Benz Oil-compatible UV protectants
FIGURE 5.7. The influence of oil-compatible UV protectants on the germination of conidia of R. alismatis following a 2 h exposure to UVB radiation at an irradiance of 2.32 W m-2. Conidium germination was assessed 3 and 6 h post-exposure. All UV protectants were tested at a concentration of 5% in D-C-Tron. Bars labelled with same letters are not significantly different at P < 0.05 according to Fisher’s LSD test.
172 5.3.3.2 C. orbiculare
There were highly significant (P < 0.000001) differences in the protection afforded to the conidia of C. orbiculare by the different protectants. Tinuvin formulated in an emulsion of D-C-Tron and aqueous solutions of proline or folic acid all resulted in conidium germination levels similar to the dark control (Figure 5.8).
With the exception of Codacide, all other treatments gave an approximate two-fold increase in germination over the irradiated water control. There was no significant difference between treatment with 5% emulsion of Codacide and the irradiated control.
173 100 d d cd 80 bcd bc b b b 60 b
40 a
20 a Conidium germinationConidium (%)
0
1% 01% Water astern 1% Proline 1% D-C-Tron 5% Codacide 5% TyrosineFolic acid 1% Melanin 0. (5%)+E Ascorbic acid 1% Water (dark control)
D-C-Tron emulsionCodacide (5%)+Tinuvin emulsion 1% UV protectants
FIGURE 5.8. The influence of oil or water-compatible UV protectants on the germination of conidia of C. orbiculare following a 2 h exposure to UVB radiation at an irradiance of 2.32 W m-2. Conidium germination was assessed 16 h post-exposure. Bars labelled with same letter are not significantly different at P < 0.05 according to Fisher’s LSD test.
5.3.4 Natural sunlight experiment
The experiment took place on 11 April 2002 between 13:00–15:00 h and the environmental conditions encountered during the treatment are given in Table 4.5.
Conidium germination of R. alismatis was significantly (P < 0.00001) affected by treatment with oil-emulsions (10%). There was also a significant difference in the
174 percentage of conidia germinating at the two assessment times: there was no significant interaction between treatment with oil and assessment time. At the 3 h assessment time, the oil emulsions did not increase the germination of the irradiated conidia. The effect of the oil emulsions was seen when the conidia were incubated for 16 h, by which time oil emulsions significantly increased the germination of the exposed conidia compared with the irradiated (SUN) conidia in water (Figure 5.9).
The oil emulsions provided protection similar to SUN-B treatment and were little different from the shaded (SHAD) treatment. Codacide emulsion at a concentration of 10% was not toxic to non-germinated conidia (data not presented).
f 100 3 h de de ef de 80 16 h cd 60 bc 40 ab ab 20 a 0 Conidium germinationConidium (%) ) ) ron) T (water
SUN (Water SUN-B SHAD (water)
SUN (water+10%D-C-SUN (water+10%Codacide) Radiation and formulation treatments
FIGURE 5.9. The influence of oil emulsions and radiation treatments on the germination of conidia of R. alismatis following a 2 h exposure to natural sunlight between 13:00–15:00 h at an average UVB irradiance of 1.40 W m- 2. Conidium germination was assessed 3 and 16 h post-exposure. SUN: full sunlight; SUN-B: sun without UVB; SHAD: shade. Bars labelled with same letter are not significantly different at P < 0.05 according to Fisher’s LSD test.
175 5.3.5 Spectrophotometer studies
In the first series of studies, the mineral oils (D-C-Tron and Codacide) alone had considerable UV absorption (4–5 units) between 310–350 nm without any distinct peaks (Figure 5.10). Oil-in-water emulsions at a concentration of 1% had similar absorptions to the oils from which they were made. Oil emulsions at a concentration of 5% had significant UV absorption (10 units) between 280–300 nm with apparent absorption peaks at 350 and 370 nm.
12 Deionised distilled water D-C-Tron emulsion 1% 10 300 nm 350 nm 370 nm Codacide emulsion 1% D-C-Tron emulsion 5% 8 Codacide emulsion 5% D-C-Tron oil 6 Codacide oil Absorbance 4
2
0
0 0 0 0 0 0 0 28 30 32 34 36 38 40
Wavelength (nm)
FIGURE 5.10. The absorbance spectra of oils and oil-in-water emulsions at concentrations of 1 and 5% (v/v).
In the second series of studies, folic acid showed a high degree of absorption
(10 units) between 280–300 nm and at wavelengths greater than 360 nm (Figure
5.11). Tyrosine and melanin showed similar absorbance spectra to the oils with
176 absorbances of (3–6 units) over the range of wavelength tested without any distinct absorption peaks. Ascorbic acid only absorbed UV light between 280–300 nm whilst proline had very little UV absorption.
12 Deionised distilled water Tyrosine 300 nm 360 nm 10 Proline Folic acid 8 Ascorbic acid Melanin
6 290 nm
Absorbance 4 300 nm 2
0
280 300 320 340 360 380 400 Wavelength (nm)
Figure 5.11. The absorbance spectra of water-compatible UV protectants at concentrations of 1 and 0.1% (melanin) (w/v).
In the third series of studies, oil-compatible UV protectants in D-C-Tron at concentrations of 5% were examined. All UV protectants had considerable UV absorption (10 units) between 280–300 nm (Figure 5.12). In general, the UV protectants also absorbed light strongly at wavelengths greater than 350 nm. There appeared to be little absorption between 300–350 nm with absorption in this region being due to the carrier oil.
177 12 Deionised distilled water 300 nm 350 nm D-C-Tron 10 D-C-Tron+Tinuvin D-C-Tron+Benzyl cinnamate 8 D-C-Tron+Eastern D-C-Tron+Tryptophan 6
Absorbance 4
2
0
280 300 320 340 360 380 400 Wavelength (nm)
Figure 5.12. The absorbance spectra of oil-compatible UV protectants at concentration of 5% (w/v), Tinuvin (v/v) in D-C-Tron.
178 5.4 DISCUSSION
Additives, in particular UV protectants to be used with a particular fungus, must be checked for intrinsic toxicity, both singly and in combination with any other additives that are to be included in the final product (Daigle and Cotty, 1991).
Because conidium germination and appressorium formation are of great significance to a mycoherbicide (and mycoherbistat) rather than germ tube growth (Greaves et al.,
1998), in this study conidium germination and appressorium formation were assessed to check for toxicity of oils and selected UV protectants on the potential mycoherbistats under study. In this study, potential fungistatic effects of the sunscreens and oils were not separated from fungicidal effects. The reason for this is that fungistatic compounds would delay conidium germination and practically, in the field, a delay in germination caused by them would be likely to kill the conidium due to adverse environmental conditions. This study found that all plant oils tested were toxic to non-germinated conidia of R. alismatis whereas the mineral oils were not.
However, none of oil-in-water emulsions (5%) were toxic, including those made from plant oils. Therefore, at this concentration, all oils could form a constituent of a formulation for the conidia of the two pathogens tested.
The toxicity of plant oils has also been shown by Potyka (1995). She examined ten plant oils in which the conidia of Colletotrichum dematium (Pers. ex
Fr.) Grove failed to germinate either on glass slides or on tissue culture plates, where as conidium germination was much greater in an emulsion of these oils in solutions of Tween 40. Greaves et al. (1998) states that adverse effects of oils on conidium germination may not indicate that the oils are intrinsically toxic, but may merely reflect the formation of an oil coat around the conidium, preventing hydration, oxygen uptake, or both. In this study, the two mineral oils were not toxic, indicating
179 that oils per se are not toxic to, nor have adverse effects on, the conidia of R. alismatis. Similarly, Calpouzos et al. (1959) showed that mineral oils did not prevent conidium germination or infection by Mycosphaerella musicola Leach ex
Mulder. However, this study on R. alismatis suggests the plant oils possess some sort of toxicity. As the mineral oils where shown not to be toxic, D-C-Tron mineral oil was selected as a carrier for oil-compatible UV protectants.
The result of this study indicates that both the water and oil-compatible UV protectants can exert a range of effects on conidia of R. alismatis. The response of R. alismatis conidia to amino acids, B vitamins and sunscreens was different. Of four amino acids tested at a concentration of 5%, only glutamic acid was toxic at this concentration. Of the five sunscreens, ethyl cinnamate, pABA; and the three B vitamins tested, only folic acid was toxic. However, folic acid tested at a concentration 1% was not toxic. The UV protectants found most effective for R. alismatis were selected to test for toxicity and for their ability to protect conidia of C. orbiculare against UVB radiation. Similarly, none of these selected UV protectants were toxic to non-germinated conidia of C. orbiculare. Although appressorium formation was reduced by some treatments, 1% aqueous solutions of tyrosine and proline significantly increased the appressorium formation by germ tubes of C. orbiculare. Stimulatory effects of amino acids on appressorium formation have also been shown for C. capsici (Syd.) Butler & Bisby (Muruganandam et al., 1987).
Conidium germination of C. orbiculare was enhanced by treatment with 5% emulsion of D-C-Tron compared with the control. However, appressorium formation of C. orbiculare was reduced by 5% emulsion of D-C-Tron. Improving conidium germination or inducing appressorium formation should be considered for
180 UV protectant selection, which could have dual effects, to protect conidia from sunlight and to reduce dew dependency of conidia.
R. alismatis conidia germinated and developed secondary conidia on conidiophores produced from germ tubes after 16 h incubation following exposure to
UV or natural sunlight (Chapter 4). This microcycle conidiation was also observed when conidia were treated with 5% aqueous solutions of proline and pyridoxine during toxicity tests. These results indicate that microcycle conidiation not only can be induced by stressful environmental conditions (Fernandez et al., 1991; Rossier et al., 1977; Rotem and Bashi, 1969) but also can be induced by some chemicals, which are not toxic to conidia. This alternative method of maintaining inoculum potential during periods of fluctuating RH or solar radiation may have epidemiological consequences when primary conidia fail to infect. This needs to be investigated in detail with a view to formulating the fungus in such compounds.
Most water-compatible UV protectants tested provided a degree of protection from artificial UVB radiation, but an exposure period of 2 h caused a significant delay in germination after 3 h incubation post-exposure. Of the water-compatible UV protectants, folic acid, proline at a concentration of 1%, melanin at 0.1% and ascorbic acid at 5% gave the best protection similar to water (dark control) without any delay in germination of conidia in R. alismatis. Proline and folic acid at a concentration of 1% also gave the best protection to C. orbiculare. These compounds have previously been shown to protect bacteria and viruses. A 1% aqueous solution of proline provided significant protection of baculovirus (Ignoffo and Garcia, 1995). Shapiro (1985) showed the protective effect of 1% aqueous solution of folic acid in nucleopolyhedrosis virus. Remarkably, the insect melanin was highly effective at only 0.0003% in its ability to protect Bacillus thuringiensis
181 Berliner var. israelensis (Liu et al., 1993). Ascorbic acid provided some level of protection of baculovirus at 0.1 and 1% (Ignoffo and Garcia, 1994). In this study, when the concentrations of ascorbic acid and melanin were decreased to 1 and
0.01%, respectively, the protective effect of these compounds significantly decreased. The effectiveness of a UV protectant covering the organisms depends on the concentration of both the protectant and the organisms in a suspension (Burges and Jones, 1998). Therefore, at the same concentration of organisms (bacteria or viruses) or propagules of an organism (conidia), it is likely that much higher concentrations of the same compounds are required to fully protect fungal conidia than those required for bacteria or viruses due to the size of fungal spores. On the other hand, UV protectants, which are effective at low concentration and are less costly, are economically feasible for use as a formulation adjuvant for field application (Ignoffo and Garcia, 1995). Burges and Jones (1998) reported that the use of UV protectants at 1–10% in low-or-high-volume tank-mixes is wasteful.
Therefore, future research should focus on the identification of more effective UV protectants at lower concentrations and other less costly substitutes might be found to use as spray-tank additives. Recently, photoprotection of conidia of B. bassiana provided by 0.02% aqueous solution of the alkaloid, berberine, was reported by
Cohen et al. (2001).
All oil-compatible UV protectants tested provided a degree of protection from artificial UVB radiation compared with water or oil alone when exposed to
UVB radiation (Figure 5.7), but exposure period of 2 h caused a significant delay in germination 3 h incubation post-exposure. Only two UV protectants, Tinuvin and
Eastern gave protection to the conidia resulting in gemination levels similar to the dark control after 6 h incubation; both of these compounds are UV absorbants. D-C-
182 Tron alone did not provide any protection but D-C-Tron-in-water emulsion provided significant protection. This could be attributed to the absorbance spectra of the oil and the oil-in-water emulsion. The oil alone had considerable UV absorption between 310–350 nm whereas the emulsion had considerable absorption between
280–300 nm (Figure 5.10).
In this study, oil-compatible UV protectants gave less protection than water compatible UV protectants although some of them such as folic acid (Figure 5.11) and benzyl cinnamate (Figure 5.12) had similar UV absorption (10 units) between
280–300 nm. This may be explained, at least, in part, by physical behaviour of the carriers. Since oil spreads more than water on a surface, less protectant would be sited over conidia to stop UV light in oil than in water. Burges (1998) also stated that, since water evaporates rapidly, it would drag the screen into an ever-decreasing volume and deposit it in concentric rings, as in the familiar rings around a drying puddle on a path. Finally the last remaining liquid would retreat around solid particles such as conidia, displacing most protectant around them. Because oil evaporates slowly, less protectant would be expected to aggregate around conidia in oil.
The absorption spectra of the potentially useful UV protectants showed a wide variety of absorbance capabilities. All UV protectants absorbed wavelengths between 280 and 400 nm but absorbance maxima differed between compounds. The pH and the solvent or carrier alters the spectra (Shaath, 1990; Moore et al., 1993).
The benzoates are polar compounds and when used as sunscreens may show shifts in their absorbance maxima, depending upon polarity of the solvent used. It has been noted for polar sunscreen compounds, a change from a polar to a less polar solvent increases the wavelength of their maximum absorbance (Shaath, 1990). In contrast,
183 relatively non-polar compounds, such as cinnamates, may show a shift to lower wavelengths under similar conditions (Shaath, 1990). In addition to polarity changes, UV protectants may also be affected by pH. Changes in absorption maxima due to the acidity of plant oils (e.g., pH 4.6 for cotton-seed oil) cannot be ruled out.
This is particularly important when comparing absorption spectra from UV protectants in mineral oils, where the pH is near neutrality (Moore et al., 1993). In this study, mineral oil D-C-Tron was used as a carrier for oil-compatible protectants.
Therefore, when changing the carrier, the UV protectants should be re-examined to check any changes in their absorption spectra.
Since the most damaging wavelengths which also caused delay in germination were in the UVB (280–315 nm) region and appressorium formation was stimulated by UVA (315–400 nm) (Chapter 4), a favourable UV protectant should have a peak in the UVB region that extends little into UVA region and should not cause any delay in germination of conidia when incorporated into formulations. UV protectants should also be cost-effective to use as spray-tank additives. Furthermore, from the environmental viewpoint, UV protectants are preferably either environmentally innocuous or easily biodegradable to prevent soil and water pollution. Oil emulsions even at 10% did not increase survival of the conidia exposed to natural sunlight after 3 h incubation; exposure caused a significant delay in germination (Figure 5.9). Results from this study indicate that folic acid, proline, ascorbic acid, tyrosine, melanin and propyl gallate are potentially useful UV protectants, which fulfil all or part of the above criteria. This study showed that melanin, at a low concentration did not delay conidium germination and of the compounds tested in this study appears to be the most efficient in terms of the protection given from UVB radiation. Furthermore, melanin is a natural product
184 easily biodegradable in the soil (Liu et al., 1993). Melanins are dark, pigmented polymers that often protect fungi and other organisms against UV radiation, radio waves, desiccation and temperature extremes (Bell and Wheeler, 1986). Caesar-
Tonthat et al. (1995) have recently shown that Gaeumannomyces graminis var. graminis, a filamentous soil ascomycete, exhibited enhanced cell wall melanin accumulation when exposed to as little as 0.01 mM CuSO4 in minimal broth culture.
Rowley and Pirt (1972) also noted that melanisation of Aspergillus nidulans greatly increased in a medium containing iron. These results show the prospect of growing the potential mycoherbicides on media conducive to the formation of melanised conidia by the fungi and should render them more resistant to UV radiation, desiccation and high temperatures.
Although proline and tyrosine are good protectants, an amino acid would not be economically feasible to use (Ignoffo and Garica, 1995). Of the B vitamins tested as natural absorbents, folic acid was the best at a concentration of 1%. Folic acid is a naturally occurring biochrome in insects and other animals (Burges and Jones, 1998) acts as a UV absorber. However, it needs to be determined if lower concentrations of folic acid than those used in this study give protection. Of the two antioxidants tested, 5% aqueous solution of ascorbic acid provided high level of protection resulting in conidium gemination levels similar to the dark control and when the concentration of ascorbic acid was decreased to 1%, the protective effect of this compound significantly decreased. Another antioxidant, propyl gallate showed some level of protection at a low concentration of 0.1% but conidium gemination was lower than observed in the dark control. Ignoffo and Garica (1994) reported that a low concentration of propyl gallate might be used in tank mixes because of low cost and common use as a food additive. Taking an overall view, the best protectants
185 could be listed in approximate order of merit as: melanin>folic acid>propyl gallate>ascorbic acid>proline>tyrosine. However, the utilisation of UV protectants to increase the efficacy of a mycoherbistat will depend on whether UV protectants prolong the survival of conidia sufficiently to enhance subsequent infectivity.
Several leaf bioassays and pot-in-field experiments are required to test the robustness of the protection afforded in these experiments and forms the basis for Chapter 6.
186
CHAPTER 6
EFFECT OF UV PROTECTANTS AND UVB
RADIATION, SIMULATED OR AS NATURAL
SUNLIGHT ON PATHOGEN-HOST PLANT
INTERACTIONS
6.1 INTRODUCTION
The effects of UV radiation on potential mycoherbistats may include photomorphogenic responses (mediated by UVA) and damage (mediated by UVB)
(Chapter 4). The effects of increased UVB on plant-pathogen interactions have been studied in only a few pathosystems and there are no published data regarding the direct effect of UV (UVA and UVB) irradiation on mycoherbistat-target weed interactions. According to Runeckels and Krupa (1994), available evidence clearly shows that the effects of UVB on the incidence and development of plant diseases on crop plants is dependent upon the crop cultivar and age, pathogen type (obligate versus facultative biotroph), pathogen inoculum level, type of plant organ infected and the timing and duration of the elevated UVB exposure.
The responses of plant-pathogen interactions to UVB appear to be variable.
Carns et al. (1978) studied several diseases caused by fungi and found that UVB
187 irradiation either reduced the viability of fungal spores and disease severity or had no effect. In contrast, Biggs and Webb (1986) found that infection of field-grown wheat by the leaf rust fungus, Puccinia recondita Rob. ex Desm., increased significantly in a susceptible cultivar (Red Hart) but not in the resistant cultivar (Florida 301) under enhanced UVB irradiation. Similarly, Orth et al. (1990) found that the severity of cucumber scab (caused by C. cucumerinum) was increased on a susceptible cultivar but not on a resistant cultivar. They also concluded that a high dose of UVB promoted disease if given before inoculation of cucumber leaves with the pathogen but reduced infection if given after inoculation due to inhibitory effects on fungal development. Similarly, Naito et al. (1996) determined that the disease incidence caused by damping-off fungus, Fusarium oxysporum Schlechtend.:Fr., on spinach
(Spinacia oleracea L.) over a 15 day period on plants treated with a UVB irradiance of 1.0 W m-2 or at two different visible light levels was 70 to 80% compared with about 40% for those not grown under UVB.
In the plant-pathogen interactions cited above, increased UVB after inoculation tends to reduce disease, perhaps due to direct damage to the pathogen, although responses vary markedly between and within pathogen species. Increased
UVB before inoculation causes a range of effects in different systems, but an increase in subsequent disease is a common response, perhaps due to changes in host surface properties or chemical composition (Paul, 2000).
Since C. orbiculare was the potential mycoherbistat most susceptible to UVB radiation (Chapter 4), the X. spinosum-C. orbiculare pathosystem was selected for this study. Experiments were conducted to determine whether post-inoculation UVB exposure and UV protectants had a direct effect on disease development and subsequent mycoherbistatic activity. Pre-inoculation UVB exposure studies are not
188 investigated because they are not of great significance to mycoherbistat development.
The work described in this chapter was to determine the effects of: (1) UV protectants and exposure to artificial UVB radiation on the host-pathogen interaction using a leaf disc bioassay; (2) UV protectants and exposure to natural sunlight on the host-pathogen interaction using a leaf disc bioassay; (3) UV protectants and exposure to natural sunlight on the host-pathogen interaction using whole plants grown in pots
(pot-in-field experiments).
189 6.2 MATERIALS AND METHODS
6.2.1 Conidium suspensions
C. orbiculare was cultured as described in Section 4.2.1. A suspension of 1 ×
106 conidia mL-1 was made from 7-day-old cultures as described in Section 4.2.2.
6.2.2 Water- and oil-compatible UV protectants
Conidia of C. orbiculare were prepared in 1% (w/v) solutions of the water- compatible UV protectants, ascorbic acid, folic acid, proline and tyrosine to give
6 -1 conidium concentrations of 1 × 10 conidia mL . The same concentration of conidia was also formulated in a 0.01% (w/v) solution of melanin. Conidia were also prepared in:
5% (v/v) oil-in-water emulsion of D-C-Tron;
5% (v/v) emulsion of D-C-Tron containing 1% Tinuvin;
5% (v/v) oil-in-water emulsion of Codacide; and
5% (v/v) emulsion of Codacide containing 1% Eastern.
A conidium suspension was also prepared in water only.
6.2.3 Leaf disc bioassays for anthracnose development
6.2.3.1 Exposure to simulated UVB radiation
Water agar (WA; 0.5% w/v), containing 1 ppm benzylaminopurine (ICN,
Biomedicals Inc, USA) to delay senescence, was autoclaved and dispensed aseptically into 50-mm diameter Petri dishes. X. spinosum leaves were cut from healthy, glass-house-grown plants. The leaves were surface-sterilised in a 1% chlorine solution for 1 min, rinsed in sterile distilled water and blotted dry on sterile
190 filter paper. Leaf discs (8 mm diameter) were cut with a cork borer and placed carefully (avoiding surface damage) on the surface of the agar (3 discs each plate).
The discs were inoculated in the centre or, if necessary, off-centre to avoid placing the inoculum on the mid vein. Three discs were inoculated with 4 µL aliquots of conidium suspensions, as described in Section 6.2.2. The plates were then exposed to UVB radiation (under Pyrex) for 2 or 3 h at irradiance of 2.32 W m-2 as described in Section 4.2.7, in a controlled temperature growth chamber at 25°C. Control treatments consisted of leaf discs inoculated with conidia applied in water only that were: (1) exposed to UV radiation; (2) covered with polyester film, Mylar 500 A
(DuPont Ltd, Victoria, Australia) and exposed to UV radiation (UVA treatment) for the same period; and (3) covered with aluminium foil and maintained in the dark for the duration of the exposure period. Following exposure, the plates were wrapped in plastic film and incubated in growth chamber at 25°C with a diurnal light cycle of 12 h.
Plates were examined after 3 and 9 days post-exposure incubation and scored using the following ordinal scale developed by Cother and Van de Ven (1999): 0–no symptoms; 1–mark left by inoculation droplet; 2–sparse necrotic flecking at inoculation site; 3–dense necrotic flecking at inoculation site; 4–dense or sparse necrotic flecking with chlorosis; 5–whole incubation point necrotic; 6–necrosis or chlorosis extending beyond inoculation site; 7–necrosis and chlorosis beyond inoculation site; 8–necrosis and chlorosis < half the disc; 9–extensive necrosis and chlorosis > half the disc; 10–entire disc chlorotic/necrotic.
191 6.2.3.2 Exposure to full-spectrum natural sunlight
Leaf discs were prepared and placed on the surface of 0.5% WA in 90-mm plastic Petri dishes (6 discs each plate) and were inoculated with 4 µL aliquots of conidium suspensions prepared from UV protectants as described in Section 6.2.3.1.
The plates were floated on water in plastic trays and exposed to natural sunlight on
15 February 2003 between 15:15–16:15; due to rainfall, the plates were covered and again exposed between 17:15–18:30 h. Meteorological measurements were provided as described in Section 4.2.9. Control treatments consisted of leaf discs inoculated with conidia applied in water only that were: (1) exposed to full-sunlight (SUN); (2) covered with polyester film, Mylar 500 A (DuPont Ltd, Victoria, Australia) and exposed to sunlight without UVB radiation (SUN-B) for the same period; and (3) covered with aluminium foil and maintained in the dark for the duration of the exposure period (SHAD). Following exposure, the plates were wrapped in plastic film and incubated in a growth chamber at 25°C, with a diurnal light cycle of 12 h.
Plates were examined after 3, 9 and 14 days post-exposure incubation and scored as described in Section 6.2.3.1.
6.2.4 Pot-in-field experiments
Fruits of X. spinosum were each cut at the distal end with a scalpel to expose the tips of the two seeds. This technique was used by McRae (1989) for more uniform and rapid germination. All cut fruit were sown in a commercial potting mix
(Debco Ltd, NSW, Australia) in seed trays. After 10 days, the seedlings were transplanted into 100-mm diameter plastic pots containing the same potting mix.
Osmocote, a slow release fertiliser (Scotts Ltd, NSW, Australia), was added to each pot. In addition, Aquasol (23:4:18, N:P:K), a fast acting soluble fertiliser (Hortico
192 Ltd, NSW, Australia) was regularly applied to seedlings. The plants were kept in a temperature-controlled glasshouse at 25 ± 5°C fitted with an automatic watering system. Six-week old plants were used for further work.
6 -1 Conidium suspensions of 1 × 10 conidia mL were prepared in 5% emulsions of D-C-Tron and Codacide and aqueous solutions of folic acid (1% w/v) and melanin (0.01% w/v), as described in Section 6.2.2. The suspensions were then sprayed on the 6-week-old plants until runoff using an airbrush sprayer (Paasche Ltd,
Illinois, USA) at 125 KPa. The inoculated plants in the pots were then exposed to the natural sunlight on 29 December 2002 between 12:30–16:30 and 15:30–18:30 h.
Meteorological measurements were provided, as described in Section 4.2.9. Control treatments consisted of plants inoculated with conidia applied in water only that were: (1) exposed to full-sunlight (SUN); (2) placed within a metal frame covered by polyester film, Mylar 500 A (DuPont Ltd, Victoria, Australia) and exposed to sunlight (SUN-B) for the same period; and (3) covered with aluminium foil and maintained in the dark for the duration of the exposure period (SHAD) (Figure 6.1).
Following exposure, all plants were covered with clear, rigid, plastic containers to maintain dew period and kept in the dark for 24 h at 25°C. The plants were then moved to a controlled temperature growth cabinet at 25°C with a diurnal light cycle of 12 h.
Disease severity was assessed daily for 26 days on all plants of each treatment within a pot, using the rating system developed by McRae et al. (1988): 1– no disease; 2–basal lesion (a stem lesion produced below the cotyledonary node) only; 3–leaf lesions only; 4–leaf and stem lesions; 5–basal lesion and leaf/stem lesions; 6–dead.
193 Aqueous suspensions of UV protectants and oil-in-water emulsions without fungal conidia were sprayed onto plants to observe phytotoxicity: three replications/treatment were included. The plants were assessed for any signs of phytotoxicity over a period of 2 weeks.
194
A
B
FIGURE 6.1. Pot-in-field trial showing Xanthium spinosum plants within metal frame covered with Mylar 500 A (SUN-B treatment) (A) or covered with aluminium foil (SHAD treatment) (B).
195 6.2.5 Statistical analysis
Leaf disc bioassays were performed with three replicates (Petri dishes) for each treatment. For experiments using artificial light, 3 leaf discs on each Petri dish were used whilst for experiments with natural sunlight 6 leaf discs on each plate were used. Data were subjected to logit transformation of original scores divided by
10. These transformed rating scales were used to normalise the error variance
(Cother and Van de Ven, 1999). Transformed data were analysed using two-way
ANOVA to identify main treatment effects and interactions. Pot-in-field experiments were performed with four replicates for each treatment. Data from the semi-field experiments were analysed by comparing the cumulative arithmetic means of daily disease ratings of each treatment up to the day when half the plants in the most effective treatment were dead (MR50). This has previously been found to be a very sensitive measure for comparing a range of different treatments (McRae et al.,
1988). In addition, the daily mean ratings until the experiment was concluded (26 days after inoculation) were also compared. Analyses of variance were performed using STATISTICA software release 6.0 (StatSoft Inc., Tulsa OK, USA, 2001).
Treatment means were compared by Fisher’s LSD tests at the 5% significance level.
196 6.3 RESULTS
6.3.1 Leaf disc bioassays for anthracnose development
6.3.1.1 Exposure to simulated UVB radiation
In the two trials comparing the protective effects of various sunscreens, there were significant differences in anthracnose development on leaf discs following exposure to UVB radiation for 2 h at irradiance of 2.32 W m-2, both among the treatments (P < 0.003, Trial 1; P < 0.0002, Trial 2) and between the two assessment times (P < 0.0001, Trial 1; P < 0.0001, Trial 2). There were no significant interactions between UV protectant and assessment time in either trial.
In Trial 1, exposure to UVA (Petri dishes covered in Mylar) increased disease severity 3 days after inoculation (DAI) compared with the dark control and with conidia formulated in water only and exposed to UVB (Figure 6.2). However, 9 DAI there was no significant difference amongst these three treatments. When assessed 3
DAI, all of the protectants had increased the disease rating above the levels of the water only control. However, there was a lot of variance in the data and only proline and the 5% emulsion of D-C-Tron gave statistically significant increases over control levels. The variation in disease rating 9 DAI appeared to be greater than at the first assessment time with again only conidia formulated in proline or the emulsion of D-
C-Tron causing significantly more disease than the control.
In Trial 2, 3 DAI, leaf discs inoculated with conidia formulated in water only showed less disease than all other treatments (Figure 6.3). Conidia formulated in water only and exposed to UVB caused less disease than those formulated in water and either exposed to UVA (dishes covered in Mylar) or those kept in the dark.
There was no significant difference in disease rating between these latter treatments.
197 Conidia formulated in UV protectants and exposed to UVB caused more disease than water control. All formulations resulted in similar disease ratings as the UVA treatment. When assessed 9 DAI, there was no significant difference in disease rating between leaf discs in the water control, those of the UVA treatment and those treated with conidia formulated in protectants with the exception of those in 5% D-C-
Tron. Conidia in this latter formulation caused more disease than those exposed to
UVB.
8 3 DAI h 7 9 DAI g
6 fgh efgh efgh
5 defgh cdef 4 cdefgh cdefg cdefg bcde 3 cdefg cde cdef bcd bcd Disease ratingDisease bcd bcd abcd bcd 2 bcd abc
1 a 0 a
1% 01% Water vin 1% Proline 1% inu Tyrosine T Water (Mylar) Folic acid 1% Codacide 5%D-C-Tron 5% Melanin 0. 5%+Eastern 1% Ascorbic acid 1% Water (dark control)
Codacide emulsionD-C-Tron emulsion 5%+ UV protectants
FIGURE 6.2. Effects of water and oil-compatible UV protectants on anthracnose development on leaf discs of Xanthium spinosum caused by Colletotrichum orbiculare following a 2 h exposure to UVB radiation at irradiance of 2.32 W m-2. Disease was assessed 3 and 6 days after inoculation (DAI). Bars labelled with same letter are not significantly different at P < 0.05 according to Fisher’s LSD test.
198 7 h 3 DAI gh 6 9 DAI fgh efgh efgh 5 cdefg defgh efgh cdefg defgh
4 cdef cdef
3 bcde bcd bc bc bc Disease ratingDisease
2 b b b b 1 a 0
) ) l r er % % % % ro a at 1% yl d 1 1% 1% 5% nt W i ne ne d .01 de on 1 on 5 M li si ci i co r ( ac o o in 0 ac k e ic ic a at l Pr b lan C-Tr C-Tr dar Tyr e Cod ( W Fo cor D- D- er M at As W Water-compatible UV protectants
FIGURE 6.3. Effects of water-compatible UV protectants on anthracnose development caused by Colletotrichum orbiculare on Xanthium spinosum leaf discs following 3 h exposure to UVB radiation at an irradiance of 2.32 W m-2. Disease was assessed 3 and 9 days after inoculation (DAI). Bars labelled with same letter are not significantly different at P < 0.05 according to Fisher’s LSD test.
6.3.1.2 Exposure to full-spectrum natural sunlight
The average of magnitude of environmental conditions encountered during the exposures of inoculated leaf discs under natural sunlight can be seen in Table 6.1.
There were highly significant (P < 0.00001) differences in anthracnose development on leaf discs following exposure to sunlight between both treatment and assessment times (Figs 6.4 and 6.5A-C). Disease scores assessed 3 DAI were significantly higher in all treatments than the water control where the conidia were exposed to full sunlight (SUN). Levels of disease in shade and SUN-B treatment were similar to those caused by conidia formulated in UV protectants with the exception of those in ascorbic acid and 5% D-C-Tron. Conidia in these latter two treatments caused more
199 disease than all other treatments (Figure 6.4). Similar results were observed 9 and 14
DAI except that only conidia formulated in 5% D-C-Tron produced disease scores higher than the SHAD and SUN-B treatments.
TABLE 6.1. Average of magnitude of environmental conditions encountered during the exposures of inoculated leaf discs or plants under natural sunlight. a t Isolar IUVA IUVB Dsolar DUVA DUVB Tair RH WS 29/12/02 8.5 12:30–16:30 995.6 15.6 3.1 14.3 225.9 44.7 30.1 50.9 15:30–18:30 567.1 8.4 1.58 6.12 96.55 17.0 30.4 50.9 15/02/03 1.0 15:15–16:15 215.9 3.1 0.6 0.7 11.3 2.2 34.8 43.3 17:15–18:30 133.6 2.4 0.3 0.6 11.1 1.7 a -1 Variables are defined in Table 4.2. WS shows average wind speed (m s ) for a day.
8
l 3 DAI 7 9 DAI
kl 14 DAI 6 jkl ijkl ijkl hijk
5 hijk hijk ghijk fghij fghij fghij
4 fghij fghi fgh fgh fgh efg cdef fgh cdef def
3 cdef Disease ratingDisease bcde bc bcd b 2 b b b b b 1 a 0
) D) B) % % % % % % - 1 1 1 1 5 5 .0 e n n e d 1% (SUN 0 ine 1% n id (SHA n acid l si aci (SUN er c o ac c er ani i Pr yro er rbi Wat el Fol T D-C-TroD-C-TroCod Wat Wat M co As Water-compatible UV protectants
FIGURE 6.4. Effects of water-compatible UV protectants on anthracnose development caused by Colletotrichum orbiculare on Xanthium spinosum leaf discs following exposure to natural sunlight between 15:15–16:15 and 17:15–18:33 on 15/02/03 at UVB irradiances of 0.63 and 0.39 W m-2, respectively. Disease was assessed 3, 9 and 14 days after inoculation (DAI). Bars labelled with same letter are not significantly different at P < 0.05 according to Fisher’s test.
200 Water only
Proline 1%
Melanin 0.01%
FIGURE 6.5A. Leaf discs of Xanthium spinosum 14 days after inoculation with Colletotrichum orbiculare conidia formulated in UV protectants following exposure to natural sunlight between 15:15–16:15 and 17:15–18:30 on 15/02/03 at UVB irradiances of 0.63 and 0.39 W 2 m- , respectively.
201 Water only
D-C-Tron 1%
Ascorbic acid 1%
FIGURE 6.5B. Leaf discs of Xanthium spinosum 14 days after inoculation with Colletotrichum orbiculare conidia formulated in UV protectants following exposure to natural sunlight between 15:15–16:15 and 17:15–18:30 on 15/02/03 at UVB irradiances of 0.63 and 0.39 W m-2, respectively.
202 Water (SUN)
Water (SHAD)
Water (SUN-B)
FIGURE 6.5C. Leaf discs of Xanthium spinosum 14 days after inoculation with Colletotrichum orbiculare conidia formulated in water only following exposure to natural sunlight between 15:15–16:15 and 17:15–18:30 on 15/02/03 at UVB irradiances of 0.63 and 0.39 W m-2, respectively.
203 6.3.2 Pot-in-field experiments
Solar radiation, temperature, RH and wind speed encountered within two trials on 29/12/02 are shown in Table 6.1. In the first trial (exposure between 12:30–
16:30), there were no significant differences between treatments at MR50 whereas at the end of the experiment (26 DAI) anthracnose development was the lowest in D-C-
Tron and Codacide treatments (Table 6.2). In the second trial (exposure between
15:30–18:30), there were no significant differences between treatment at MR50 or at end of experiment (26 DAI). However, the size of the dead plants in both experiments indicated that the application of conidia formulated in water alone and exposed to SUN-B was the first treatment in which all plants died (Figure 6.6 and
6.7). In both trials, formulation in UV protectants reduced disease and prolonged the life of the plants. There was no sign of the toxicity of UV protectants on X. spinosum in either of the two trials.
Table 6.2. Effect of UV protectants following exposure to natural sunlight between 12:30–16:30 on 29/12/02 on the anthracnose development of Xanthium spinosum 26 DAI. Radiation and formulation treatments Mean daily disease rating up to 26 DAIa b c Water (SUN) 5.11 ± 0.11a Water (SUN-B) 5.34 ± 0.05a Water (SHAD) 5.28 ± 0.14a Melanin 0.01% w/v (SUN) 5.15 ± 0.33a Codacide 5% v/v (SUN) 4.76 ± 0.36ab D-C-Tron 5% v/v (SUN) 4.10 ± 0.40b aDAI = days after inoculation bSUN = full sunlight, SUN-B = sun without UVB, SHAD = shade c Means ± standard errors followed by the same letters are not significantly different at P < 0.05 according to Fisher’s LSD test.
204
6 Water (SHAD) Water (SUN-B) 5 Water (SUN) Melanin 0.01% 4 Codecide 5% 3 D-C-Tron 5%
Disease ratingDisease 2
1
0 1 3 5 7 9 1113151719212325 Days after exposure
FIGURE 6.6. Effects of water-compatible UV protectants on anthracnose development caused by Colletotrichum orbiculare on Xanthium spinosum following exposure to natural sunlight between 12:30– 16:30 h on 29/12/02 at UVB irradiance of 3.11 W m-2. Disease was assessed on a 1–6 scale daily as described in Section 6.2.4 up to 26 days incubation post-exposure.
205 6 Water (SHAD) Water (SUN-B) 5 Water (SUN) 4 Melanin 0.01% Folic acid 1% 3 Codacide 5% D-C-Tron 5% 2 Disease ratingDisease
1
0 1 3 5 7 9 1113151719212325 Days after exposure A
D-C-Tron 5% Control Codacide 5% SUN Melanin SUN-B SHAD 0.01% Folic acid 1%
5
B
FIGURE 6.7. (A) Effects of water-compatible UV protectants on anthracnose development caused by Colletotrichum orbiculare on Xanthium spinosum following exposure to natural sunlight between 15:30– 18:30 h on 29/12/02 at UVB irradiance of 1.58 W m-2. Disease was assessed on a 1–6 scale daily as described in Section 6.2.4 up to 26 days incubation post-exposure. (B) A visual representation of experimental treatments affecting anthracnose disease development 16 days after exposure.
206 6.4 DISCUSSION
Post-inoculation, UVB (simulated or natural sunlight) exposure of X. spinosum leaf discs inoculated with conidia of C. orbiculare formulated in UV protectants led to increased anthracnose development. Although the effect of the various UV protectants on anthracnose development was similar to the dark control, it was clear that some UV protectants in combination with radiation exposure resulted in more severe disease than that developing in the dark control (Figure 6.2 and 6.4). However, this was not observed when the exposure time increased from 2 to 3 h. These results suggest that, firstly, appressorium formation is stimulated by certain UV wavelengths leading to higher levels of infection. Secondly, responses to these wavelengths are dose dependent. Since protectants such as ascorbic acid and
D-C-Tron in which conidia were formulated mostly absorbed UV light between 280–
300 nm (Chapter 5), the stimulation of appressorium formation could be attributable to UVA component of light source. Induction of appressorium formation by UVA radiation and their dose dependency have also been shown in Chapter 4.
Melanin (0.01% w/v), folic acid (1% w/v), D-C-Tron and Codacide (5% v/v) significantly increased anthracnose development on leaf discs following exposure to natural sunlight: of these treatments D-C-Tron was the most effective. However, when these treatments were applied to whole plants in pots in the pot-in-field experiments, there were no significant differences between treatments at MR50. This time has previously been found to be most suitable for comparing a range of different treatments (McRae et al., 1988).
Discrepancies in the anthracnose development caused by conidia in leaf disc bioassays and pot-in-field trials may be due to a proportion of the total fungal inoculum applied being protected from direct and/or extended exposure to UV
207 radiation by shading due to plant architecture and topography. This discrepancy could also be attributed to high wind speed on the day when experiments were conducted (Table 6.1) which caused the leaves not to be exposed directly to sunlight for most of the time during the experiments, particularly in SUN treatments. In addition, higher solar intensity and the extended periods of exposure in the pot-in- field experiments (Table 6.1) seemed to swamp any beneficial effects of the UV protectants. Other mitigating factors include the presence of overcast conditions and diurnal variations in solar radiation and temperature, which may allow for some degree of biochemical and molecular recovery in exposed conidia (Hunt et al., 1994;
Moore et al., 1996).
However, the proportion of the dead plants in both pot-in-field experiments indicated that plants under Mylar (SUN-B treatment) were killed more quickly than in other treatments, even the SHAD treatment (Figure 6.6 and 6.7). The RH close to the surface of the Mylar was not measured but it can be assumed that the higher temperature under the Mylar would reduce the RH. Although these changed environmental conditions may have affected either conidium germination and/or appressorium formation, it is more likely that UVA wavelengths stimulated conidium germination and appressorium formation, as was found in Chapter 4.
In the pot-in-field experiments, conidia in water-based formulations killed the plants more quickly than conidia in oil-based formulations (Figure 6.6 and 6.7), which is in agreement with the results in Chapter 5 indicating water-compatible UV protectants gave more protection than oil-compatible UV protectants for exposed conidia. Inglis et al. (1995) suggested that a possible reason why oil-soluble sunscreen gives poor protection against UV radiation is because the oil component is partly absorbed by leaf tissues, leaving conidia exposed. However, in this study D-
208 C-Tron was the most effective treatment in the leaf disc bioassays suggesting that absorption is not the cause of reduced protection. Since the solar dose in the bioassay experiment was much lower than that in the pot-in-field experiment (Table
6.1), D-C-Tron could be effective at lower solar doses. Inconsistent effects using mineral oils to reduce the dew dependency of C. orbiculare in field evaluations have also been reported by Klein et al. (1995).
It is evident from the leaf disc bioassays reported in this chapter that infectivity of conidia formulated with UV protectants or in treatments where UVB was removed was significantly increased compared to the dark control. These studies also show that the effects of UV protectants on anthracnose development following exposure to sunlight vary depending upon timing, duration of exposure,
UV protectant (water or oil compatible) and UV protectant concentration. A small number of UV protectants were tested in the pot-in-field experiments. If conidium persistence and their subsequent infectivity need to be increased, more effective UV protectants, particularly at higher solar doses in combination with different time of exposure need to be evaluated in a series of pot-in-field and field trials.
209
CHAPTER 7
GENERAL DISCUSSION AND CONCLUSION
7.1 R. alismatis
A summary of results arising from this study for R. alismatis was as follows:
1) UV radiation below 290 nm greatly reduced conidium germination of R. alismatis, while radiation between 290 and 315 nm caused slight reduction in this parameter after 6 h incubation following an exposure of 2 h.
2) The wavelengths between 290 and 400 nm at a dose of 4.24 kJ m-2 significantly increased appressorium formation compared with the dark control.
3) The effect of different periods of exposure to UVB on conidium germination of R. alismatis indicated, firstly, that the response to UVB radiation is strongly dose dependent. Secondly, UVB doses caused delays in the germination of conidia.
210 4) When the conidium germination of R. alismatis was expressed as a function of
UVB dose, it could be described by a cubic relationship. Conidium germination was halved after a UVB dose of approximately 4 kJ m-2 after 3 h incubation whereas a
50% reduction after 12 h incubation occurred with a UVB dose of 10 kJ m-2.
5) In the natural sunlight experiments, shaded and sunny minus UVB conditions were always more favourable to conidium germination of R. alismatis than full sunlight. In the SUN treatment under 4 different temperatures, conidium germination was the greatest under the coldest temperature (10°C) and there was an interaction between irradiation and temperature, leading to the enhancement of the lethal effects of UVB on conidium germination at high temperatures.
6) Appressorium formation was highest at a temperature of 30°C following exposure between 14:00–15:00 h with a total solar dose of 2.80 MJ m-2 and a corresponding
UVA dose of 44.57 kJ m-2. About 50% of germinated conidia produced appressoria.
7) In the first natural sunlight experiment, conidium germination 6 h incubation post- exposure was halved for doses of solar radiation, Dsolar, DUVA and DUVB, of about 4
MJ m-2, 60 and 12 kJ m-2, respectively. In the second experiment, conidium germination after 3 h incubation post-exposure was halved for doses of solar
-2 -2 radiation of 3 MJ m , 45 and 9 kJ m , respectively, for Dsolar, DUVA and DUVB.
Conidium germination 16 h after exposure was halved for doses of solar radiation of
-2 -2 5 MJ m , 80 and 15 kJ m , respectively, for Dsolar, DUVA and DUVB.
211 8) In this study, reciprocity was observed when R. alismatis conidia were exposed to total UVB dose of 3.7 kJ m-2.
9) R. alismatis conidia germinated and developed secondary conidia on conidiophores produced from germ tubes after 16 h incubation following exposure to
UV or natural sunlight. This microcycle conidiation was also observed when conidia were treated with 5% aqueous solutions of proline and pyridoxine during toxicity tests.
10) The mineral oils (Fuchs and D-C-Tron) were not toxic to non-germinated conidia whereas all of the plant oils (Codacide, Synetrol and cottonseed) were toxic. None of oil-in-water emulsions were toxic at the concentrations tested.
11) A high level of protection against UVB radiation was provided with conidia treated with riboflavin, proline, melanin, folic acid and ascorbic acid. All these water-compatible UV protectants were tested at a concentration of 1% except melanin and ascorbic acid, which were tested at concentrations of 0.1% or 5%, respectively.
12) Tinuvin and Eastern at a concentration of 5% provided significantly more protection than the other oil-compatible sunscreens. However, the protectants could not prevent a significant delay in germination of conidia.
212 13) Oil-in-water emulsions of D-C-Tron and Codacide at 10% (v/v) did not increase the germination of the irradiated conidia when assessed 3 h after treatment.
However, a protective effect of the emulsions was seen when conidia were incubated for 16 h, by which time oil emulsions significantly increased the germination of the exposed conidia compared with the irradiated conidia formulated in water.
The above-mentioned have practical implications for applying R. alismatis to
A. lanceolatum and D. minus infesting rice crops during summer. It is possible to manipulate the fungal application time in order to expose conidia to doses of solar radiation that are not harmful to conidium germination and which stimulate appressorium formation. However, additional protection may be needed. In vitro, germination of exposed conidia to UVB was enhanced by a range of UV protectants belonging to different functional groups, with water-compatible UV protectants being more effective. These protectants have not been studied in the leaf discs bioassays or field experiments, but provide a number of options for non-toxic adjuvants for formulating this mycoherbistat.
Natural sunlight experiments showed that lower germination resulted from higher temperatures. Therefore, it is possible to manipulate the fungal application time in order to reduce the adverse effect of sunlight, particularly at the beginning of the growing season of the target weeds when the temperature and radiation are moderate. Manipulating the fungal application time and incorporating UV protectants into formulations as the result of further research including in vivo experiments should lead to the creation of a product that will give effective epidemics.
213 This study used an isolate, DAR 73158, supplied by NSW Agriculture,
Orange. Variability in susceptibility to UVB radiation of conidia among isolates of entomopathogenic Hyphomycetes has been shown (Fargues et al. 1996; Braga et al.
2001c). Cother and Van de Ven (1999) showed significant variation in pathogenicity among isolates of R. alismatis. Strain selection in relation to radiation tolerance should be a further area of focus for the development of R. alismatis as a mycoherbistat.
Yang and TeBeest (1993) proposed a model to describe efficacy of mycoherbicide use. This model takes into account the level of primary and secondary weed host infection and rate of disease development to predict effectiveness of mycoherbicide use over time. Currently, most research with mycoherbicides, including R. alismatis, does not address the importance of secondary infection. This may be because, conceptually, mycoherbicides have been treated as chemical herbicides (Yang and TeBeest, 1993). This study suggests that control efficacy of a mycoherbicide is not similar to that of a chemical herbicides, which, in general, are not affected by environmental conditions. In addition to stimulation of microcycle conidiation following exposure to sunlight, this microcycle conidiation was also observed when conidia of R. alismatis were treated with 5% aqueous solutions of proline or pyridoxine during toxicity tests. This alternative method of maintaining inoculum potential during periods of fluctuating RH or solar radiation may have epidemiological consequences when primary conidia fail to infect. Therefore, it is possible to induce secondary infection to occur in a short period of time leading to higher levels of infection. This needs to be investigated in detail with a view to either formulating the fungus in compounds that stimulate
214 microcycle conidiation or exposing inoculum to specific solar doses that cause this effect.
7.2 C. orbiculare
A summary of results arising from this thesis for C. orbiculare was as follows:
1) Radiation with wavelengths below 290 nm had a marked deleterious effect on conidium germination of C. orbiculare whereas radiation above 290 nm had no harmful effect.
2) The wavelengths between 290 and 400 nm at a dose of 4.24 kJ m-2 significantly increased appressorium formation compared with the dark control. About 50% of germinated conidia produced appressoria 12 h after incubation following a 2 h exposure.
3) After 8 h incubation post-exposure, conidium germination was significantly less than the dark control for conidia exposed to UVB for 60 and 120 min.
4) When the conidium germination of C. orbiculare was expressed as a function of
UVB dose, the relationship could be described by an exponential equation for both 8 and 16 h incubation post-exposure. Conidium germination was halved after a UVB dose of approximately 3 kJ m-2 after 8 h incubation whereas a 50% reduction after 16 h incubation occurred with a UVB dose of 8 k m-2.
215 5) In the natural sunlight experiments, germination of conidia and appressorium formation by C. orbiculare conidia were always highest in the SUN-B treatment followed by the SHAD and SUN treatments. 16 h after exposure, about 30% of
-2 germinated conidia produced appressoria at 30°C with a UVA dose of 152.92 kJ m .
6) In the natural sunlight experiment, for conidium germination 16 h incubation post- exposure there was a cubic relationship between conidium germination and total solar, UVA and UVB irradiation. Germination was halved for doses of solar
-2 -2 radiation, Dsolar, DUVA and DUVB, of about 1 MJ m , 20 and 4 kJ m , respectively, based on the model.
7) In this study, non-reciprocity was observed when C. orbiculare conidia were exposed to a total UVB dose of either 6.1 or 8.5 kJ m-2. A short period of high intensity irradiance was more damaging than an equal dose given over a much longer period.
8) After 24 h treatment at 25°C, conidium germination on agar plates was enhanced by formulation in 1% Eastern in a Codacide emulsion, by a 5% emulsion of D-C-
Tron, and by 1% Tinuvin in a 5% emulsion of D-C-Tron. Appressorium formation was significantly increased by 1% aqueous solutions of proline or tyrosine.
Appressorium formation was unaffected by treatment with melanin and folic acid but was reduced by a 5% emulsion of D-C-Tron and Codacide.
216 9) High level of protection, resulting in levels of conidium germination similar to the dark control, against UVB radiation was provided for conidia treated by 1% Tinuvin in a 5% emulsion of D-C-Tron and 1% aqueous solutions of proline or folic acid.
10) After 2 h exposure to UVB radiation, C. orbiculare conidia formulated in proline
(1% v/v) or D-C-Tron (5% v/v) significantly increased the anthracnose development on leaf discs 3 and 9 DAI compared with control conidia treated with water alone, either exposed or not exposed.
11) After 3 h exposure to UVB radiation, anthracnose development after 3 days on leaf discs, inoculated with conidia formulated in ascorbic acid (1% w/v), melanin
(0.01% w/v), Codacide or D-C-Tron (5% v/v) was similar to conidia formulated in water alone where the discs were kept in the dark or exposed under Mylar which removed UVB wavelengths (UVA treatment).
12) After exposure to natural sunlight at UVB irradiances of 0.63 and 0.39 Wm-2, anthracnose development on leaf discs 3 DAI inoculated with conidia formulated in folic acid, proline, tyrosine, ascorbic acid, D-C-Tron at 1%, melanin at 0.01%,
Codacide or D-C-Tron at 5% was significantly higher than control conidia formulated in water alone and exposed to UVB. Also, anthracnose development in these treatments was similar to, or higher (ascorbic acid and D-C-Tron) than, from conidia formulated in water only and exposed under shaded conditions (SHAD) and from conidia exposed to the SUN-B treatment.
217 13) In the pot-in-field experiments, the application of conidia formulated in water alone and exposed to SUN-B was the first treatment in which all plants died.
Following this treatment, the order of treatments which caused plant death was: formulation in water and exposed to full sunlight; formulation in water and plants kept in the shade. In both trials, formulation in sunscreens reduced disease and prolonged the life of the plants.
This study used an isolate, DAR 48942, supplied by NSW Agriculture,
Orange. C. orbiculare was the potential mycoherbistat most susceptible to UVB radiation. Therefore, strain selection to irradiation tolerance should be included in further investigation. The feasibility of using this isolate as a mycoherbistat has been extensively studied by McRae (1989). Field application (Auld et al., 1990) and other studies (Auld, 1993; Klein et al., 1995; Chittick and Auld, 2001) have been conducted to reduce the dew dependency of this isolate. Inconsistent effects using this isolate in field evaluations have always been observed. With knowledge of how the conidia of C. orbiculare react to the climate as detailed above, rapid conidium germination and appressorium formation could be achieved by manipulation of the time at which conidia are applied. Currently, most spray applications are made late in the afternoon (McRae, 1989). However, the work in this study indicates that this may not be the best time to apply the conidia, as they need a period of exposure to solar radiation to stimulate conidium germination and appressorium formation. In the field, such rapid germination and appressorium formation would subsequently lead to rapid primary infection and minimise the dew dependency of C. orbiculare.
However, if conidia are to be applied earlier in the day then additional protection would be needed to significantly reduce the adverse effects of solar
218 radiation. It is evident from this study that subsequent infectivity of conidia when formulated with UV protectants could be significantly increased. To achieve optimal environmental conditions for primary infection, further research needs to be conducted including application of conidia formulated in water alone or in a wide range of effective UV protectants. Results from this study show the prospects for finding a suitable protectant; firstly, they should protect the conidia from extended periods of exposure; secondly, have a high level of absorbance in the range between
280–315 nm; and lastly, provide protection at low concentrations. A series of pot-in- field or field trials need to be performed at different times of day on days with different levels of irradiation and humidity. Consequently, it will be possible to optimise time of application for successful performance.
7.3 D. avenacea
The results arising from this study for D. avenacea can be summarised as follows:
1) Conidium production was affected by nutrition, temperature, luminosity regimes, pH and incubation period. The most suitable conditions for conidium production by
D. avenacea were ½OMA with an initial pH of 7.0 at 20°C under continuous NUV for 2 weeks.
2) Although variation in conidium production was found among isolates tested, all isolates produced a higher quantity of conidia than reported in other studies under the conditions cited in Point 1 above.
219 3) Conidium production was significantly affected by NUV intensity. Conidium production was highest under moderate irradiation (14.56 W m-2). Increasing the amount of irradiation from 14.56 to 22.78, or decreasing it to 6.66 W m-2 significantly reduced conidium production.
4) A constant temperature at 20°C appeared to be optimal for both conidiophore formation and conidium production.
5) Conidiophore development was stimulated by either white light or NUV light. Of these two light qualities, NUV stimulated conidiophore development more than white light. After conidiophore development, conidium production occurred either in the dark or in the light but conidium production was higher in the light.
6) Exposure to NUV light resulted in a significant increase in subsequent virulence of conidia of D. avenacea.
7) Considerable variation in virulence of D. avenacea was detected among isolates from different geographic areas.
8) RAPD-PCR analysis revealed genetic variation among D. avenacea isolates.
9) In the laboratory, conidium germination of D. avenacea was not adversely affected by a UVB dose of 16.70 kJ m-2 after 6 h incubation following exposure. No appressoria were formed under any treatments tested.
220 10) Under natural sunlight, conidium germination of D. avenacea could be delayed
-2 with a high UVB dose (28.65 kJ m ) at high temperatures (30 or 40°) whereas conidium germination was not adversely affected even with a UVB dose of 45.07 kJ
-2 m at 10 or 20°C.
11) Maximum appressorium formation occurred when stimulated by irradiation by
UVA.
12) Appressorium formation appeared to be UVA dose dependent. No appressoria were formed by conidia exposed to a UVA dose of 4.24 kJ m-2 in the laboratory whereas in the natural sunlight experiments high numbers of appressoria were formed after a UVA dose of 101.08 kJ m-2 with approximately 80% of germinated conidia forming appressoria. Increasing the amount of UVA dose from 101.08 to
152.92 kJ m-2 reduced appressorium formation.
One of the most important characteristics for a fungal pathogen to be successful as a mycoherbistat is that it must produce abundant and durable conidia on artificial media (Daniel et al., 1973). During this study, the efficiency of conidium production of D. avenacea was increased from inconsistent production of
5 approximately 2.35 × 10 conidia/plate (Wilson, 1987) to consistent production of
7 1.12 × 10 conidia/plate under the conditions described above. Krupinsky (1986) found isolates of Drechslera bromi (Died.) Shoem. that differed in their conidium production and he concluded that there were probably isolates of Drechslera spp. that would sporulate poorly at times regardless of the medium. In this study, the conditions that were found to be optimal were tested using 8 isolates from different
221 geographic areas. Although variation in conidium production was found, all isolates produced a reasonable quantity of conidia. It is, therefore, likely that those conditions would be generally successful for other isolates of the pathogen.
In addition to variation in conidium production, considerable variation in the virulence of D. avenacea was also detected among isolates from the different geographic areas. Some isolates showed two key features i.e., high sporulation and high virulence that should allow them to be developed into a successful mycoherbistat. However, to select a good mycoherbistat, the sporulation and pathogenicity of a large number of isolates would have to be examined.
For small-scale inoculum production where economics are not a primary concern, relatively expensive materials, such as agar can successfully be used to induce sporulation (Boyette et al., 1991). However, from the standpoint of practicality and economics, mass production in submerged fermentation is more favourable than solid-substrate fermentation for the production of mycoherbicides
(Churchill, 1982). Thus the sporulation of D. avenacea in liquid culture is desirable if this pathogen is to be commercialised as a mycoherbistat. Fungal conidia are more suitable than mycelial fragments as a source of inoculum (Boyette et al., 1991).
Turner and Millard (1931) showed that the rate of disease development caused by D. avenacea after mycelial inoculation was lower than that from inoculation with conidia. Additionally, the durability, longevity and viability of mycelium are generally much less than that of conidia (Churchill, 1982). However, Wilson (1987) showed that D. avenacea was only able to produce mycelium in liquid culture.
In this study, maximum sporulation was produced on ½OMA under NUV radiation. It has been proposed by Trione (1981) that light-induced sporulation by fungi is associated with endogenous compounds called P-310s, due to their
222 maximum absorption at 310 nm. This suggests that there might be such compounds in this medium or that the medium stimulate the production of P-310s. Trione (1981) further suggested that P-310s could replace the stimulus of light by inducing sporulation of certain fungi in the dark. Thus, further studies are needed to isolate and identify such compound(s) and to determine whether they may contribute to developing effective synthetic media for sporulation of D. avenacea in submerged fermentation.
It is evident from this study that sporulation, virulence and genetic variation testing of large numbers of naturally occurring strains from a wide geographic area could lead to strain improvement through increased virulence of the pathogen. One factor that may be considered during strain selection is the production of a fungal toxin. The virulence of D. avenacea isolates is, to some extent, a function of toxin production (Nozoe et al., 1965). An isolate of D. avenacea that is pathogenic to A. sterilis, but not to a number of related or unrelated species, was identified by
Kastainias and Chrysayi-Tokousbalides, 2000. These authors identified a metabolite, pyrenophorol that was toxic to A. sterilis and considerably less so to A. fatua. In this study, exposure to NUV light resulted in significant increase in the subsequent virulence of D. avenacea conidia. It is possible the toxin produced by D. avenacea is activated by NUV radiation. This has to be confirmed. A light-activated toxin has been found in several Cercospora spp. (Daub and Eherenshaft, 2000).
The practical possibilities of applying D. avenacea to wild oats infesting winter wheat crops are an important consideration. The results from this study showed that conidium germination of D. avenacea was not adversely affected at a range of temperatures under natural sunlight, whereas appressorium formation showed a narrow plasticity to temperature and solar radiation, in particular to UVA
223 doses. Large numbers of appressoria were formed at a temperature of 10 or 20°C with moderate solar and corresponding UVA doses of 10.03 MJ m-2 and 101.08 kJ m-2, respectively. Winter temperature and solar radiation dose may be conducive to conidium germination and, in particular, to appressorium formation as long as conidia are exposed to optimal solar radiation doses. Measurements of solar or UVA intensity could be used to select appropriate times for the application of this organism.
A rapid conidium germination and appressorium formation may reduce the dew dependency of D. avenacea, which has a relatively short dew period (8-12 h) for infection (Hetherington and Auld, 2001). Lindhout et al. (2001) showed that conidium germination of D. avenacea can be accelerated by iron chelators.
However, appressorium stimulation may be more important than stimulation of germination, because without appressorium formation, no infection will occur. Thus with this knowledge of complex environmental requirements for appressorium stimulation and incorporating humectants or germination stimulants such as iron chelators to formulations of D. avenacea, it may possible to create an effective epidemic and obtain substantial weed control.
224 7.4 Summary
Temperature and moisture, as humidity or free water, have always been considered two major components of the environment that limit the exploitation and effectiveness of mycoherbicides. Results from this thesis indicate that solar radiation had both a damaging effect (reduction in germination) limiting efficacy and a photomorphogenic effect (appressorium induction) increasing efficacy. The study has also shown significant interactions between temperature and solar radiation on the survival of conidia of potential mycoherbistats. Therefore, solar radiation should be considered as a third major component of environment that should be considered when trying to produce mycoherbistats. Yang and TeBeest (1993) state that studies that identify factors limiting the performance of mycoherbicides should share the same principles as those that determine the critical factors of epidemics for disease forecasting. With the findings presented in this thesis and further research on disease development under different conditions, in combination with formulating conidia in suitable UV protectants, a computer model of the epidemics caused by C. orbiculare,
D. avenacea and R. alismatis could be constructed. This may help to optimise time of application and development of disease by forecasting the optimum infection days and assessing the potential regions suitable for these mycoherbistats.
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PUBLICATIONS
Parts of the work in this dissertation have been submitted for publication.
Feridon Ghajar, Paul Holford, Eric Cother and Khalaf Alhussaen. (2004). Optimising sporulation and pathogenicity in Drechslera avenacea. (Submitted) Mycological Research.
Feridon Ghajar, Paul Holford, Eric Cother and Andrew Beattie. (2004). Effects of UV radiation, simulated or as natural sunlight, on conidium germination and appressorium formation of fungi with potential as mycoherbistats. (Submitted) Biocontrol Science and Technology.
Feridon Ghajar, Paul Holford, Eric Cother and Andrew Beattie. (2004). Photostabilisation of fungi with potential as mycoherbistats. (Submitted) Australasian Plant Pathology.
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