Modelling of spore release and alternative methods of control for stem rot (Sclerotinia sclerotiorum) in beans

Transcription

1 Modelling of spore release and alternative methods of control for stem rot (Sclerotinia sclerotiorum) in beans Chris Archer TAS Institute of Agricultural Research Project Number: VG2

2 VG2 This report is published by Horticulture Australia Ltd to pass on information concerning horticultural research and development undertaken for the vegetable industry. The research contained in this report was funded by Horticulture Australia Ltd with the financial support of the vegetable industry. All expressions of opinion are not to be regarded as expressing the opinion of Horticulture Australia Ltd or any authority of the Australian Government. The Company and the Australian Government accept no responsibility for any of the opinions or the accuracy of the information contained in this report and readers should rely upon their own enquiries in making decisions concerning their own interests. ISBN Published and distributed by: Horticultural Australia Ltd Level 1 5 Carrington Street Sydney NSW 2 Telephone: (2) Fax: (2) horticulture@horticulture.com.au Copyright 24

3 Modelling of spore release and alternative methods of control for Sclerotinia sclerotiorum in Beans Final report for Horticulture Australia Project Number: VG2 Prepared by Zi-Qing Yuan Tasmania Institute of Agricultural Research, University of Tasmania New Town, Tasmania November 23 Tasmanian Institute of Agricultural Research

4 Modelling of spore release and alternative methods of control for Sclerotinia sclerotiorum in Beans Final Report for Horticulture Australia Project Number: VG2 Project leader Chris Archer Tasmanian Institute of Agriculture Research University of Tasmania Report prepared by Dr. Zi-Qing Yuan Tasmanian Institute of Agriculture Research University of Tasmania 13 St. Johns Av. New Town, Tas 78 Ph: (3) Research Team Paul Schupp (Tasmanian Institute of Agriculture Research) Changyou Pan (Tasmanian Institute of Agriculture Research) Ziqing Yuan (Tasmanian Institute of Agriculture Research) Frank Hay (Tasmanian Institute of Agriculture Research) Shane Dullahide (Queensland Department of Primary Industry) Bob Davis (Queensland Department of Primary Industry) Scope of report This is the final report of the above project. It covers the conduct and results of the project in detail, and also includes media and technical summaries. This report presents the research results of a corporative effort from all the team members led by Chris Archer. Ziqing Yuan joined the research team in 22. During the final year of the project, Ziqing Yuan was involved in the establishment of the spore trapping trails including trial setting up, spore collecting, counting and data analyses. He prepared this report with the resignation of the project leader. Funded by This project was funded by the Horticultural Australia Limited (HAL). Acknowledgment Ziqing Yuan wishes to express thanks to TIAR and special appreciation to Chris Archer for this research and for his most valuable contribution as project leader. In addition thanks are due to all other team members, particularly to Chang-you Pan for her skilful technical assistance in spore counting. 1

5 Hoong Pung of Serve-Ag and Ian Porter of Agriculture Victoria are thanked for their assistance in discussing problems during the project. The provision of the bean fields for the experimental sites and the information on bean development by Simplot Australia, personally David Stirling and J. Rockliffe is greatly appreciated. Deep appreciation must also be extended to Horticulture Australia Limited whose funding made this research project possible. Disclaimer Any recommendations contained in this publication do not necessarily represent current Horticulture Australia policy. No person should act on the basis of the contents of this publication, whether as to matters of fact or opinion or other content, without first obtaining specific, independent professional advice in respect of the matters set out in this publication. 2

6 TABLE OF CONTENTS 1. MEDIA SUMMARY TECHNICAL SUMMARY INTRODUCTION INVESTIGATION OF ASCOSPORE RELEASE PROFILES OF SCLEROTINIA SCLEROTIORUM IN BEAN FIELDS INTRODUCTION METHODS RESULTS Spore release patterns of the 2-21 growth season Spore release patterns of the growth season Spore release patterns of the growth season Preliminary statistical modelling of seasonal patterns of ascospore dispersal DISCUSSION Seasonal and diurnal patterns of ascospore dispersal Seasonal ascospore dispersal and meteorological factors Diurnal ascospore dispersal and meteorological factors Modelling of seasonal patterns of ascospore dispersal Ascospore dispersal and disease incidence CONCLUSION DETERMINATION OF ALTERNATIVE CONTROL STRATGIES FOR SCLEROTINIA SCLEROTIORUM IN BEAN FIELDS INTRODUCTION METHODS RESULTS Spray trials for screening potential alternative chemical agents for management of S. sclerotiorum Pot trials for examining effect of cover crops on survival of overwintering sclerotia of S. sclerotiorum DISCUSSION TECHNOLOGY TRANSFER RECOMMENDATIONS AND FUTURE WORK REFRENCES APPENDIX: A milestone report on Queensland Trials in season

7 1. MEDIA SUMMARY A project was undertaken to investigate the environmental requirements for spore discharge in the pathogen by investigating S. sclerotiorum spore release patterns within beans using spore traps. The project also aimed to determine alternative control strategies for the pathogen by using non-host cover crops as a mechanism for inoculum reduction and by using systemic acquired resistance (SAR) elicitors to accentuate the natural plant resistance to infection. 1. The spore trapping studies revealed the seasonal and diurnal spore release patterns of S. sclerotiorum, a preliminary predictive model was formulated using the air temperature as an independent variable to forecast spore dispersal; the best time during the day or within a growth season for fungicide applications with reduced fungicide usage; the multi-peaked seasonal pattern of S. sclerotiorum may not be triggered by a single factor, but a combined influence of several factors including environmental and host conditions; measurement of soil and air temperatures (combined with soil moisture) at late development stage of beans (1-2 weeks before flowering) would be useful in helping farmers decide when to start their preventative fungicide program; using early flowering varieties of beans or adjusting the sowing date are optional practices to prevent infections of S. sclerotiorum. 2. The spray trials demonstrated that of the seven chemicals tested against infection of S. sclerotiorum, the systematic fungicides Benlate (benomyl) and Sumisclex (procymidone) were more effective than the contact fungicides Rovral (iprodione) Ronilan (vinclozolin) and Folicur (tebuconazole). No efficacy was found for the plant activator Salicylic acid and Amistar. However, the ability of Salicylic acid to induce the SAR response was likely concealed due to the evaluation method used in this study. 3. Pot trials in laboratories provided positive information on the effect of non-host crops as a cover crop or rotation crop of beans to manage S. sclerotiorum disease. Barley, spinach and faba beans are potential cover crops. Soil with moisture retaining capacity should be considered in recommending these cover crops. It is recommended that (a) measurement of soil and air temperatures (and soil moisture) 1-2 weeks before flowering would help farmers decide when to start their preventative fungicide program, and (b) early in the morning during the peak period of spore dispersal is the best time for fungicide applications during the day. It is also recommended that using early flowering varieties of beans or adjusting the sowing date (or early sowing) are optional practices to prevent infections of S. sclerotiorum. There is a need to provide more accurate information on spore release and disease forecast if more comprehensive mathematical models incorporating temperature, soil moisture, rainfall and humidity and host conditions are built. Extensive field trials would be helpful to better evaluate the significance of SAR elicitors and cover crops as alternative methods in the control S. sclerotiorum problems. 4

8 2. TECHNICAL SUMMARY Background Sclerotinia sclerotiorum is a major pathogen of many vegetable crops including beans, tomatoes, lettuce and cabbage. It can cause yield losses of both green and navy beans in Tasmania by plant death and severe infection of stems and pods. Control of S. sclerotiorum is currently achieved mainly by way of fungicide applications. As the primary initiator of disease is the release of spores discharged from overwintering sclerotia of the pathogen, any prediction of when spores are released will enable a narrowing of the timing window over which fungicide sprays need to be applied. There are other methods, such as using non-host cover crops for reducing the overwintering disease inoculum levels to reduce the incidence of disease within a crop, or using plant activators to induce immunity against diseases following infections by pathogen, known as systemic acquired resistance (SAR). Objective The primary aim of this project was to investigate the environmental requirements for spore discharge in the pathogen Sclerotinia sclerotiorum by investigating S. sclerotiorum spore release within beans using spore traps. The second aim of the project was to determine alternative control strategies for the pathogen by (a) using non-host cover crops as a mechanism for inoculum reduction and (b) using SAR elicitors to accentuate the natural plant resistance to infection. Project findings 1. Using spore traps to examine spore release, this project has provided a profile of conditions under which ascospores of S. sclerotiorum are released to be ascertained. This profile can now be used to construct a predictive model for the disease based on quantifiable environmental conditions such as rainfall, soil moisture, temperature etc which can be easily and economically measured on farm. More specifically, the spore trapping studies revealed Seasonal spore release of S. sclerotiorum is of multi-peaked seasonal patterns; Ascospores are produced continually through the growth seasons; S. sclerotiorum is a slow build up pathogen. Spore release of S. sclerotiorum was favoured by low temperatures (5-19ºC for air temperature, 13-21ºC for soil temperature) and high relative humidity; The multi-peaked seasonal pattern of S. sclerotiorum may not be triggered by a single factor, but a combined influence of several factors including environmental (eg. temperature, soil moisture, rainfall and humidity) and host conditions (eg. canopy development of beans), which are essential parameters for building a mathematical model of ascospore release forecast; The best predictive model was formulated through the utilisation of the air temperature as an independent variable to forecast spore dispersal; Although the diurnal spore dispersal patterns follow a night pattern, spore releases were favoured on the days with large contrast between minimum and maximum relative humidity; 5

9 The marked diurnal pattern gives an indication of the best time for fungicide applications during a day. 2. The spray trials demonstrated that of the seven chemicals tested against infection of S. sclerotiorum, the systematic fungicides Benlate (benomyl) and Sumisclex (procymidone) were more effective than the contact fungicides Rovral (iprodione) Ronilan (vinclozolin) and Folicur (tebuconazole). No efficacy was found for the plant activator Salicylic acid and Amistar. However, the ability of Salicylic acid to induce the SAR response was likely concealed due to the evaluation method used in this study. 3. Pot trials in laboratories provided positive information on the effect of non-host crops as a cover crop or rotation crop of beans for management of S. sclerotiorum disease. Four non- susceptible hosts, barley, oats, faba bean and spinach were tested for their effect on survival (germination) of overwintering sclerotia. Barley was the most effective cover crop, spinach the second followed by faba bean. No effect was found for oats. Barley, spinach and faba beans are potential cover crops. The significant high rates of sclerotial germination associated with the silty loam soil from northwestern Tasmania were attributed to the character of soil structures which is of higher water holding capacity than the sandy loam soil from Housten, southern Tasmania. Soil features relating to its moisture holding capacity, therefore, should be considered in recommending these cover crops. Recommendations and future work It is recommended that (a) measurement of soil and air temperatures (combined with soil moisture) 1-2 weeks before flowering would help farmers forecast spore release and decide when to start their preventative fungicide program, and (b) early in the morning during the peak period of spore dispersal is the best time for fungicide applications during the day. It is also recommended that using early flowering varieties of beans or adjusting the sowing date (or early sowing) are optional practices to prevent infections of S. sclerotiorum. Comprehensive mathematical models incorporating environmental variables (eg. temperature, soil moisture, rainfall and humidity) and host conditions (eg. canopy development of beans) are needed for spore release and disease forecast. Extensive field trials would be helpful to better evaluate the significance of SAR elicitors and cover crops as alternative methods in the control S. sclerotiorum problems. 6

10 3. INTRODUCTION Sclerotinia sclerotiorum (Lib.) de Bary is a common nectrotrophic pathogen with worldwide distribution causing various rots of stem, foliar and fruit on over 4 species of plants (Boland and Hall 1994). Important crops affected include oilseed rape, sunflower, tobacco and a wide range of vegetables such as beans, lettuce, cabbage, cauliflower, carrot, potato and many other flower crops. The stem and pod rots caused by this pathogen on beans, commonly called white mould are often highly destructive resulting in crop losses up to 1% under favourable conditions (Hall and Steadman 1991). In Tasmania, it can cause yield losses of both green and navy beans by plant death and severe infection of stems and pods. Contemporary control of Sclerotinia sclerotiorum is achieved predominantly by way of fungicide applications (Oliveira et al. 1999, Mueller et al. 1999, 22). Knowledge of the most appropriate time for fungicide application is based on what is essentially a trial and error process to determine a spray schedule for disease control. As the primary initiator of disease is the release of ascospores from apothecia of S. sclerotiorum, any prediction of when spores are released will enable a narrowing of the timing window over which fungicide sprays need to be applied to achieve control of the disease. The use of spore traps to examine spore release will enable a profile of conditions under which they are released to be ascertained. This profile can then be used to construct a predictive model for the disease based on quantifiable environmental conditions such as rainfall, soil moisture, temperature etc which can be easily and economically measured on farm. Although the primary method of control of Sclerotinia diseases in vegetables is primarily by way of fungicide application, there are other methods for reducing the overwintering disease inoculum levels or for reducing the incidence of disease within a crop. The use of non-host cover crops as a mechanism for inoculum reduction has been proved of great potential for disease control (Johnson et la. 1997, Reis and Santos 1985). The ability of plants to develop immunity against diseases following infections by pathogen, known as systemic acquired resistance (SAR) induced by SAR activators has also been applied in green beans (Phaseolus vulgaris cv. Dufrix) for control of several different diseases caused by fungi and bacteria (Siegrist et al. 1997). The primary aim of this project was to investigate the environmental requirements for spore discharge in the pathogen Sclerotinia sclerotiorum. This was achieved by investigating S. sclerotiorum spore release within beans using spore traps. A second aim of the project was to determine alternative control strategies for the pathogen. This was achieved by firstly using non-host cover crops as a mechanism for inoculum reduction and secondly using systemic acquired resistance (SAR) elicitors to accentuate the natural plant resistance to infection. 7

11 4. INVESTIGATION OF ASCOSPORE RELEASE PROFILES OF SCLEROTINIA SCLEROTIORUM IN BEAN FIELDS 4.1. INTRODUCTION Fungi reproduce by way of spores which are similar in function to plant seeds (Carlisle and Watkinson 1994). That is, they germinate to form a new colony thus permitting dispersal of the parent organism. Fungal spores are produced from specialised reproductive structures and occur in a highly diverse array of sizes and shapes. Despite this diversity fungal spores are very constant in shape, size, colour and form for any given species making them invaluable for species identification. There are two methods by which spores can be initiated in fungi, either sexually or asexually. Sexually initiated spores result from a mating between two different individuals or hyphae, whereas asexual spores result from a simple internal division or external modification of an external hyphae. Sclerotinia sclerotiorum is a homothallic fungus and does not produce asexual spores, ie. conidia, although small, globose, hyaline microconidia may be produced (Hall and Steadman 1991). The main method of spore formation for this pathogenic fungus takes place sexually within apothecia. Apothecia are small, tan coloured, funnel shaped cups on thin stalks which occur at ground level and arise from overwintering sclerotia that were formed in infected plants the previous season. Sclerotia are capable of surviving in the soil for five years or more. Development of the apothecia from sclerotia requires suitable environmental conditions with high moisture and warm weather favoured (usually in spring). Hyaline, ellipsoid, one-celled ascospores of S. sclerotiorum (Fig ) are released from apothecia when the small sacs called asci (Fig ) holding the spores absorb sufficient water to expel them forcibly. The spores released from the apothecia are carried to the plants on air currents and ejected spores are capable of travelling at least 2 metres when blown by the wind. Dependent on conditions, apothecia are capable of releasing spores for up to 8 days. Microconidia in S. sclerotiorum do not seem to be important in the dissemination of infections. Equally, although fungal mycelium can grow directly out of sclerotia to invade plants, it is uncommon and not a major form of disease spread. The disease caused by S. sclerotiorum is usually initiated when ascospores discharged from apothecia land on senescing flower petals. The spores germinate and colonise the petals. Within 3 days, senescent flowers will be completely colonised. Using the petals as a food source, the fungus then is able to infect other parts of the plant including leaves, branches, and pods. The characteristic white fluffy mycelium develops on the surface of the infected tissues within a few days, and hard, black, irregular shaped sclerotia are produced in ca days for overwintering (Abawi and Hunter 1979). Ascospore release and survival are therefore key stages in the lifecycle of S. sclerotiorum as illustrated in Fig The spore producing apothecia usually do not form until a dense crop canopy is present to provide the cool, moist conditions. Soil temperatures of 1-21ºC and high soil moisture levels for one to several weeks cause the coverwintering sclerotia to germinate and form the apothecia. Fungicide application is the major method of disease control. The goal of these applications is to protect not only the leaves and stems from infection, but also to prevent colonisation of the flower petals. Other control procedures for white mould include 8

12 measures such as lower plant densities or using varieties with upright growth habits, thus allowing the drying of the canopy and soil. The use of spore trapping for the purpose of developing predictive models has a long history in disease management research. Recent investigations using spore trapping include; Scherm et al. (1995) where the hypothesis that spore release and infection of lettuces by the pathogen Bremia lactucae occurred concurrently during mornings with prolonged leaf wetness was tested and validated. Reis and Santos (1985) have conducted spore trapping of Helminthosporium sativum above a trial investigating the effect of cover crops on inoculum reduction as a mechanism for determining trial treatment efficacy. Cooperman et al. (1986) monitoring spore release of the asparagus pathogen Cercospora aspargi from April to November determined that spore release for the pathogen occurred primarily over a six day period in late August in the North Carolina region in the United States. MacHardy and Gadoury (1986) have demonstrated the potential for a prediction model for the apple pathogen, Venturia inaequalis, based on spore trapping conducted in the USA, Australia and Italy and linked to monitoring of environmental parameters necessary for spore release. Latorre et al. (1985) has shown that the peak period for spore release of the pear scab pathogen (Venturia pirina) occurs over a two week period in September in Chile. The identification of this period allowed growers to concentrate fungicide applications at this time and achieve increased disease control. Patel et al. (1997) have investigated the effects of weather on groundnut crop growth and the development of groundnut late leaf spot disease in India, caused by the pathogen Phaeoisariopsis personata. Using spore traps, observations by these researchers allowed early detection of pathogen inoculum load and increased disease forecasting ability. 9

13 A B Fig : Sclerotinia sclerotiorum. A. ascospores, B. longitudinal section of the apothecial hymenium, C. an ascus containing eight ascospores. C 1

14 Ascospores infect wet blossoms and spread from them to other parts of the plant Sclerotia are produced among the white, fluffy mycelium on diseased plants Ascospores are carried by air and some of them land on bean plants DISEASE CYCLE OF WHITE MOULD OF BEANS Sclerotia overwinter on or in the soil Ascospores are produced by apothecia when they mature and Ascospores are produced by apothecia and ejected into the air as a cloud of spores Apothecia develop from sclerotia when the soil is wet for about 1 days Fig : Disease cycle of white mould caused by Sclerotinia sclerotiorum on beans (after Abawi and Hunter 1979) 11

15 4.2 METHODS Sampling Site Three fields at Kindred, Mersey Lea and Railton of north-western Tasmania near Devonport, were selected for spore trapping during the and growth seasons. Due to a failure of spore trap equipment, sampling at Railton site was not completed. The fields were sown to green beans (Phaseolus vulgaris) and managed by Simplot. Details on size and shape of each field, as well as development of beans were recorded (Table 4.2-1). All the fields were overhead watered periodically. No chemical sprays were applied during the growth seasons for any of the trapping sites. Table 4.2-1: Site description and the main development stages of beans at Mersey Lean and Kindred Date of development stage Growth season Location Size (ha.) Shape Sowing Flowering Harvest Mersey Lea 5.1 Rectangular Kindred 8.1 Square Mersey Lea 5.9 Rectangular Kindred 8.9 Triangular Spore trappings Spore release was monitored using two types of spore traps. A Burkard volumetric spore trap (Burkard Scientific Lit, Agronomics Division, Uxbridge, UK) was used for the site at Kindred. This spore trap is a Hirst-type, 7-day recording (battery-operated) spore sampler (Fig. 3.1). The trap collects airborne particles onto a transparent Melinex tape wrapped round a slowly rotating drum. Air is sucked at 1 l/min through an orifice of 14 x 2mm. The tape coated with 1% Gelvatol and an adhesive mixture (Vaseline + 1% paraffin wax in toluene) was moved through the orifice at a constant rate of 2mm/hour. After exposure for one week the tape was removed and cut into 48 mm (24 h) sections and mounted on microscope slides. For accuracy, the ascospores trapped on the entire tape surface were counted under a microscope (x4). All ascospores deposited on 14 mm wide tape which traverses every 2 mm along the slide (corresponding to 1 h intervals) were enumerated, and counts were converted into a concentration of spore numbers per cubic metre (/m 3 ) per hour (or per day) of air sampled following the manufacturer s instruction. Since the counting method was extremely time-consuming, to save time, the tape was then examined only by scanning (sweeping) four equidistant transects across the long axis of all the 48 mm tape at 2mm (1 h) intervals at a magnification of 4 x. The number of ascospores observed in each 1 h transect was corrected for the proportion of the tape examined and the volume of air sampled according to Kapyla and Penttinen (1981). 12

16 The trap was operated following the manufacturer s instructions and positioned approximately 4 cm above the ground at the centre of the field to collect spores in the atmosphere and within crop canopies. Spores were collected for about 2-3 months to cover the whole growth season from sowing to or/and after harvesting of beans. The collection period spanned 96 days from 28 Nov. 21 to 4 March 22 for the trial in 22, and in 23, 69 days from 1 Jan. to 2 March with one week break due to harvesting (2/2/3-27/2/3). A Quest volumetric spore trap (Melpat International Pty Ltd, Canningvale, Western Australia) was used for the site at Mersey Lea. This spore trap is also a battery-operated, 8- day recording device as is the Burkard spore trap. Instead of using a Melinex tape, a disc was used to collect spores with a Quest spore trap. The disc is divided into eight segments, with each segment being roughly the size of a normal microscopic disc, and is further subdivided into 24 hours. Spores collected on the surface of the discs were counted directly under the microscope. Air was sucked through an orifice of 14 x 2mm at 1 l/min. Calculation of spore concentration is the same as the Burkard trap. The trap was operated following the instructions provided by the manufacturer and positioned approximately 4 cm above the ground at the edge of the field to collect spores in the atmosphere. Spores were collected for 87 days from 7 Dec. 21 to 4 March 22 for the trial in 22, and in 23 from 1 Jan. to 2 March with one week break due to harvesting (2/2/3-27/2/3) Climatic data collection Rainfall, air temperature, humidity, soil moisture and soil temperature in both sites were recorded using automated weather stations (Gemini Data Loggers). Relative humidity and Leaf wetness were recorded quarterly using Tinytag Plus loggers (TGP-34 and TGP-93). Probes for measuring soil temperatures and soil moisture were inserted to a depth of 15 cm into the soil Disease incidence surveys Eight transects at each site were conducted randomly over the field 1-2 weeks before harvest. All plants within transects (ca. 5 to 1 depending on site and location) were assessed for evidence of S. sclerotiorum infection along a 5 metre tape. Plants showing any symptoms of infection on stems, leaves or on pods were counted as infected plants. Percentage infection incidence was then calculated Statistical analysis All raw spore counts were entered into Excel spread-sheets and converted to concentrations of spores/m 3. 13

17 Correlations between ascospore concentrations and meteorological factors including air and soil temperature, relative humidity and leaf wetness were calculated using Spearman s Correlation Test with GenStat (version 6.1) Analytic Software. Multiple linear and polynomial regression analyses were carried out also using GenState version 6.1. To examine the relationship between seasonal discharge patterns of ascospores and weather parameters, both hourly (a total count of each hour in all trapping days at each site) and daily average (a mean count of each day over 24 hours) spore concentrations were used. For diurnal discharge patterns, hourly average concentrations (a mean of each hour from : to 23: h over the trapping days where daily average spore concentration exceeded 5 spore/m 3 ) were tested. The spore data were not transformed because Spearman s test is valid for non-normal distributions. In all cases, meteorological parameters including maximum, minimum and average temperature, humidity and leaf wetness were considered as independent variables and spore counts as dependent variables. Concentration differences were analysed by way of analysis of variance (ANOVA) Fig : A Burkard volumetric spore sampler 14

18 4.3. RESULTS Ascospore dispersal of S. sclerotiorum in bean fields was monitored for three consecutive growth seasons at North Western Tasmania from 2 to 23. The airborne ascospores discharged from apothecia during the seasons were caught in spore traps at all trial sites and are illustrated in Figs Fig : One-celled, hyaline, ellipsoid to slightly fusiform ascospores of Sclerotinia sclerotiorum trapped in a green bean (Phaseolus vulgaris) field at Mersey Lea, Northern Tasmania in December 21 (Bar = 15 µm) The spore dispersal patterns and their correlations with weather parameters are presented separately in seasons. 15

19 Spore release patterns of the 2-21 growth season Intermittent mechanical failure of the principal Burkard spore trap resulted in a fragmented spore release profile for the 2-21 season and provided an inadequate data set for both sites. The limited data were analysed by way of analysis variance (ANOVA) to explore the significance of spore dispersal between days over a period of time and between hours during a day. Such ANOVA analyses were not used for the spore dispersal patterns from the following two seasons. The results presented here were the daily spore counts over a period of eight days from January 23 to 31, 21 at Mersey Lea site. Seasonal pattern of ascospore dispersal A significant difference (P<.1) with mean numbers of ascospores releasing was observed between days. A clear distribution pattern is shown in Fig A significant peak period of spore release was found starting to increase on day 23, peaking on day 25 and declining on day Mean no. of spores /1 24/1 25/1 26/1 27/1 28/1 29/1 3/1 Date Fig : Distribution of mean numbers (n=24) of Sclerotinia sclerotium ascospores releasing daily over a period of eight days from 23/1/1 to 31/1/1. (Bar represents standard error) Diurnal pattern of ascospore dispersal There were no significant differences (P=.51) between hours counted over 8 days of discharge, although a pattern of spores discharge was observed (Fig ). However, when an individual day is divided into four equal 6 hour periods (5: to 11: (approx. sun rise to noon), 11: pm to 17: (approx. noon to dusk), 17: to 23: (approx. sunset to midnight) and 23: to 5: (approx. midnight to sun rise)), a significant difference is demonstrated (P =.27) between time sections in mean spore numbers captured. The 16

20 spore discharge reached the maximum mean numbers during the night (from sunset to sunrise) and the lowest during the daylight hours. (Fig ) Mean no. of spores Time Fig : Distribution of mean numbers (n=8) of Sclerotinia sclerotium ascospores released during 24 hours over a period of eight consecutive days (Bar represents standard error). 2 Mean no. of spores h to 17h 17h to 23h 23h to 5h 5h to 11h Time sections Fig : Distribution of mean numbers (n=48) of Sclerotinia sclerotium ascospores releasing at different time sections over a period of eight days (Bar represents standard error) 17

21 Spore release patterns of the growth season Seasonal pattern of ascospore dispersal At Mersey Lea, ascospores were caught in spore traps in the growth seasons continually from pre-sowing through to harvest. Dispersal of spores, expressed as a concentration of spore numbers per cubic metre (/m 3 ) air, was small and fluctuated periodically. Daily average spore concentrations in 22 rarely (only 24 days out of a total 81 trapping days) exceeded 5 spores/m 3. There were two peak periods found for this site (Fig ). The first peak period started on January 1 till January 5, 22, having spore concentrations 243, 283, 396, 232 and 122 spores/m 3 respectively. For the second peak period, concentration values fluctuated from day to day, but tended to increase gradually from January 22, reaching the highest concentration of 578 spores/m 3 on January 31. After that day, concentrations decreased sharply from 418 on February 1 to 81 spores/m 3 on Feb. 5. At Kindred, ascospores were also trapped throughout the sampling period from sowing to harvest of the beans (Fig ). Daily average spore concentration was larger and with nearly half of the trapping days (42 out of 92) spore concentrations exceeded 5 spores/m 3. Three peak periods were found. The major one occurred from February 1 to February 7, 22 during which period the spore concentration exceeded 1 spores/m 3. The highest concentrations were found on February 3, 4 and 5, 22, reaching 479, 598 and 448 spores/m 3 respectively. Two other smaller peak periods were respectively on December 23-26, 21 with spore concentrations ranging from 22 to 271 spores/m 3 and on February 2 to February 4, 22, with spore concentrations from 147 to 167 spores/m Kindred Mersey Lea Concentration (/m 3 ) /11/21 5/12/21 12/12/21 19/12/21 26/12/21 2/1/22 9/1/22 16/1/22 23/1/22 3/1/22 6/2/22 13/2/22 2/2/22 27/2/22 Fig : Seasonal variations of daily average spore concentrations of Sclerotinia sclerotiorum during the sampling period at Kindred and Mersey Lea in 22. The larger dot with the hollow centre indicates the date when beans started flowering at each site. Harvest break is marked with an arrow. 18

22 Diurnal pattern of ascospore dispersal For the Mersey Lea site, hourly average concentration of ascospores were large between 2: and 6: h and the smallest, with spore concentrations less than 15 spores/m 3, between 7: and 19: h (Fig ). Ascospore concentration increased from about 19: h and peaked at 5: h, just before sun rise. After that, spore dispersal decreased rapidly, reaching concentrations below 15 spores/m 3 at 7: h. At Kindred, hourly average concentrations of ascospores were large between : and 8: h and the smallest, with spore concentrations less than 15 spores/m 3, between 9: and 23: h (Fig ). Ascospore concentration increased from about : h and peaked at 2: and 6: h respectively before declining gradually with the concentrations below 15 spores/m 3 at 9: h. 4 Concentration (/m 3 ) Mersey Lea Kindred : 2: 4: 6: 8: 1: 12: 14: 16: 18: 2: 22: Time of day Fig : Diurnal variation of hourly average concentration of Sclerotinia sclerotiorum ascospores over 24 days at Mersey Lea and 42 days at Kindred where 24h average spore concentration exceeded 5 spore/m3 in 22 Temperature and ascospore trapping Ascospores were caught at Mersey Lea at a wide range of temperatures recorded from 2.7 to 28ºC (air temperature) or 12 to 23ºC (soil temperature). Hourly concentrations exceeding about 5 spores/m 3 were usually associated with hourly air temperature from 6 to 19ºC and soil temperature 14-21ºC (Fig ). Correlation analyses of spore counts and hourly temperatures show that they were significantly correlated. Spore dispersal slightly increased with the decrease of both air temperature (r=-.14, P<.1) and soil temperature (r= -.17, P<.5) (Table ). When examining the relationship between daily average spore concentrations and daily average, maximum and minimum air temperatures, no significance (P>.5) was found for any of these parameters (Table ). However, spore dispersal appeared to increase slightly with the increase of all of these temperature parameters, although the major peak 19

23 period of spore dispersal was more or less coincident with a decline of daily minimum air temperatures (Fig ). In common with air temperature, there were no significant correlations between daily average spore concentrations and daily average, maximum and minimum soil temperatures at Mersey Lea (Table ). In contrast to air temperature, however, spore dispersal tended to decrease when all of these soil temperature parameters increased (Table , Fig ) Concentration (/m 3 ) Concentration (/m 3 ) Air temperature ( C) Air temperature ( C) 35 Concentration (/m 3 ) Concentration (/m 3 ) Soil temperature ( C) Soil temperature ( C) Fig : Hourly concentration of Sclerotinia sclerotiorum ascospores plotted against hourly air and soil temperature at Mersey Lea (left) and Kindred (Right) in 22 At Kindred temperatures were recorded ranging from 5 to 25ºC with air temperatures) and 9 to 24ºC with soil temperatures. Ascospores were caught almost throughout the entire ranges from 5 to 23ºC (air temperatures) or 12 to 23ºC (soil temperatures). Hourly concentrations exceeding about 5 spores/m 3 were found when hourly air temperature ranged from 7 to 18ºC and soil temperature from 13 to 17ºC (Fig ). Correlation analyses of hourly spore concentrations and hourly temperatures showed that they were negatively correlated at 99.99% significance (r=-.43 for air temperature and r=-.16 for soil temperature) (Table ). At Kindred unlike at Mersey Lea, significant correlations were found between daily average spore concentrations and daily average, maximum and minimum air and soil temperatures (Table ). They were all negatively correlated at 99.99% or 95% significance with correlation coefficients ranging from -.27 to -.38 (Table ). Three peak periods of spore dispersal were coincident with three peak periods of low air temperatures and also more or less with low soil temperatures (Fig ). 2

24 Temperature ( C) Daily average Daily max Daily miniimum Temperature ( C) Mersey Lea daily means Minimum Maximum Kindred 1/12/21 8/12/21 15/12/21 22/12/21 29/12/21 5/1/22 12/1/22 19/1/22 26/1/22 2/2/22 9/2/22 16/2/22 23/2/22 8/12/21 15/12/21 22/12/21 29/12/21 5/1/22 12/1/22 19/1/22 26/1/22 2/2/22 9/2/22 16/2/22 23/2/22 2/3/ Temperature ( C) /12/21 8/12/21 15/12/21 22/12/21 29/12/21 5/1/22 12/1/22 19/1/22 Daily average Daily max Daily miniimum Mersey Lea Temperature ( C) 26/1/22 2/2/22 9/2/22 16/2/22 23/2/ daily means Minimum Maximum Kindred 8/12/21 15/12/21 22/12/21 29/12/21 5/1/22 12/1/22 19/1/22 26/1/22 2/2/22 9/2/22 16/2/22 23/2/22 2/3/22 Fig : Daily air temperature (top) and soil temperature (bottom) measured at Mersey Lea and Kindred sites in 22 Humidity/leaf wetness and ascospore trapping Seasonal spore dispersal at Mersey Lea occurred throughout the range of the relative humidity recorded from 31% to 1%, and appeared to increase with the increase of relative humidity (Fig ). A significant positive correlation (r=.45, P<.1) was found between hourly spore concentrations and hourly relative humidity (Table ). There were no significant correlations between daily average spore concentrations and maximum and minimum relative humidity (Table ), but significantly correlated with daily average (r=.3, P=.7). Spore dispersal increased along with the increase of daily average humidity (Fig ), although the spore dispersal coincidently reached its high intensities on the days when the day minimum relative humidity declined (Fig ). Ascospores were caught throughout the entire trapping period from -1% of leaf wetness (Fig ). In common with relative humidity, seasonal spore dispersal was also highly correlated with leaf wetness. A significant correlation (r=.43, P<.1) was detected between hourly spore concentrations and hourly leaf wetness (Table ). When examining the relationship between daily average concentrations and leaf wetness, spore dispersal was found not to be correlated with daily average, maximum and minimum leaf wetness (Table ). Relative humidity at Kindred was recorded ranging from 35% to 1%. In common with Mersey Lea, seasonal spore dispersal at Kindred occurred throughout the entire recorded range of humidity, and increased with the increase of relative humidity (Fig ). A 21

25 significant positive correlation (r=.35, P<.1) was found between hourly spore concentrations and hourly relative humidity (Table ). There were no significant correlations between daily average spore concentrations and daily average, maximum and minimum relative humidity (Table ). No recorded data of leaf wetness for Kindred were available for the growth season. Humidity (%) Humidity (%) /12/21 8/12/21 15/12/21 7/12/21 14/12/21 21/12/21 28/12/21 Daily average Daily miniimum 22/12/21 29/12/21 5/1/22 12/1/22 19/1/22 26/1/22 daily means Minimum Mersey Lea 2/2/22 9/2/22 16/2/22 23/2/22 2/3/22 Kindred 4/1/22 11/1/22 18/1/22 25/1/22 1/2/22 8/2/22 15/2/22 22/2/22 1/3/22 Concentration (/m 3 ) Concentration (/m 3 ) Mersey Lea Relatvie humidity (% ) Kindred Relative humidity (% ) Wetness (%) /12/21 8/12/21 Daily average 15/12/21 22/12/21 29/12/21 5/1/22 12/1/22 19/1/22 26/1/22 2/2/22 9/2/22 16/2/22 23/2/22 Concentration (/m 3 ) Leaf wetness (%) Fig : Daily relative humidity, leaf wetness and hourly concentrations of Sclerotinia sclerotiorum ascospores plotted against hourly relative humidity and leaf wetness at Mersey Lea and Kindred in 22 The diurnal pattern of spore dispersal at Mersey Lea was apparently dictated by the humidity pattern and leaf wetness (Fig ). Hourly average spore concentrations were 22

26 highly significantly correlated with hourly average humidity (r=.78, P<.1) and hourly average wetness (r=.82, P<.1) (Table ). As at Mersey Lea, the diurnal pattern of spore dispersal at Kindred was also determined by the humidity pattern (Fig ). Hourly average spore concentrations were highly and significantly correlated with hourly average humidity (r=.95, P<.1) (Table ). 1 8 Concentration (/m3) Leaf wetness (%) Humidity (%) : 2: 4: 6: 8: 1: 12: 14: 16: 18: 2: 22: Time of day Fig : Diurnal variation of hourly average concentration of Sclerotinia sclerotiorum ascospores, hourly average leaf wetness and relative humidity over 24 days measured at Mersey Lea in 22 23

27 Table : Spearman Rank correlation coefficients for the relationships between daily average (da), hourly (h) or hourly average (ha) concentrations A of Sclerotinia sclerotiorum ascospores and temperature ( C), relative humidity (%) and leaf wetness (%) for Mersey Lea and Kindred sites measured in the growth season Correlation coefficient (r) Mersey Lea (df) E Kindred (df) B Daily average air temperature (versus da).11 (79) (P=.31) -.36**(76) (P=.1) Daily maximum air temperature (versus da).2 (79) (P=.89) -.27* (76) (P=.15) Daily minimum air temperature (versus da).1 (79) (P=.38) -.38**(76) (P=.1) C Hourly air temperature (versus h) -.14**(1942) (P<.1) -.43**(187) (P<.1) Daily average soil temperature (versus da) -.1 (79) (P=.36) -.3**(76) (P=.8) Daily maximum soil temperature (versus da) -.5 (79) (P=.69) -.27* (76) (P=.17) Daily minimum soil temperature (versus da) -.16 (79) (P=.18) -.28* (76) (P=.12) Hourly soil temperature (versus h) -.17* (1942) (P=.3) -.16**( (187) (P<.1) Daily average relative humidity (versus da).3**(79) (P=.7) -.14 (76) (P=.22) Daily maximum relative humidity (versus da).45 (79) (P=.24) -. (76) (P=.45) Daily minimum relative humidity (versus da) -.18 (79) (P=.11) -.18 (76) (P=.12) Hourly relative humidity (versus h).45**(1942) (P<.1).35**(187) (P<.1) D Hourly average relative humidity (versus ha).78**(22) (P<.1).95**(22) (P<.1) Daily average leaf wetness (versus da) Daily maximum leaf wetness (versus da) Daily minimum leaf wetness (versus da) Hourly leaf wetness (versus h) Hourly average leaf wetness (versus ha).2 (71) (P=.1).23 (71) (P=.53).26 (71) (P=.45).43**(175) (P<.1).82**(22) (P<.1) A Daily average (da) concentration is an average of ascospores counted each day over 24 hours, ie. the total numbers of spore counts of each day are divided by 24); hourly (h) concentration is a total counts of each hour and hourly average (ha) concentration is an average of each hour (from : to 23: h) over the trapping days where daily average spore concentration exceeded 5 spore/m 3, (eg. 24 days for Mersey Lea site, 42 for Kindred). B Daily average air temperature is an average of 24h readings each day; same for daily average soil temperature, daily average humidity and daily average leaf wetness. C Hourly air temperature is a average of readings of four quarters within an hour, same for hourly soil temperature, hourly humidity and hourly leaf wetness. D Hourly average humidity is an average of each hour (from : to 23: h) over the days corresponding to those used for the calculation of hourly average concentrations (eg. 24 days for humidity at Mersey Lea, 42 for Kindred) E Degrees of freedom * Significant value, 95%. **Significant value, 99%. Not significant. 24

28 Ascospore dispersal and disease incidence For both spore trapping trial sites sampling transects were conducted through the crop to assess the number of infected plants. A total of eight 5-metre-long transects were undertaken at each site with an average of 7 plants/transect being examined for evidence of S. sclerotiorum infection. At the time of assessment, individual plants showing typical symptoms of white mould at stems and on pods were spotted (Fig ). The infection level of transects at Mersey Lea ranged from 2.5 to 29.6% with a mean of % of plants assessed, and from 1.1 to 8.49% at Kindred with a mean of 3.67% (Table ). There was no disease problems reported by the processor at Simplot Ltd. for either site. B A C Fig : Symptoms of white mould caused by Sclerotinia sclerotiorum on green beans (Phaseolus vulgaris L.) at Mersey Lea, showing stem infection of a plant (A), close-up view of white fluffy mycelium on the surface of infected stem (B) and infected pod with black sclerotia (C). 25

29 Table : Disease incidence of Sclerotinia sclerotiorum at Kindred and Mersey Lea during the growth season Kindred Mersey Lea Number of Transect Infected plants Total Plants in 5m transect % Sclerotinia Infection for each transect Infected plants Total Plants in 5m transect % Sclerotinia Infection for each transect Mean

30 Spore release patterns of the growth season Seasonal pattern of ascospore dispersal At Mersey Lea, ascospores were caught in spore traps in the 23 growth season continually from pre-emergence of seedlings through after harvest. Daily average spore concentrations in 23 were found exceeding 5 spores/m 3 during most of the trapping days (47 out of 63 days). Dispersal of spores fluctuated periodically. There were three peak periods found for the 23 season (Fig ). The highest concentrations were found in the peak period from February 9 to 16 on the first three consecutive days (9-11 th ), reaching 51, 494 and 461 spores/m 3 respectively. For the other five days of the peak period, the spore concentrations ranged from 314 to 415 spores/ m 3. Two other smaller peak periods were respectively on January and January 3 to February 3, having spore concentrations between 19 and 33 spores/m 3. In resemblance with the Mersey Lea site, ascospores were found throughout the sampling period at Kindred. Daily average spore concentrations were smaller and only nearly half of the trapping days had concentrations exceeded 5 spores/m 3. Two peak periods were found, with the major one occurring from February 1 to 18, 23 during which period the spore concentration exceeded 2 spores/m 3 (Fig ). The highest concentrations occurred on February 12 and 13, 23 reaching 687 and 676 spores/m 3 respectively. After that, concentrations decreased gradually, reaching a minimum concentration of 177 spores/m 3 on February 19, the last sampling day before the harvest break. The small peak period occurred from January 25 to February 2, 23, having concentrations ranging from 115 to 26 spores/ m 3. Concentration (/m 3 ) Kindred Mersey Lea 1/1/23 17/1/23 24/1/23 31/1/23 7/2/23 14/2/23 21/2/23 28/2/23 7/3/23 14/3/23 Fig : Seasonal variations of daily average spore concentrations of Sclerotinia sclerotiorum during the sampling period at Kindred and Mersey Lea in 23. The larger dot with the hollow centre indicates the date when beans started flowering at each site. Harvest break is marked with an arrow. 27