Control of Mint Rust on Peppermint Epidemiology and Chemical Control

A report for the Rural Industries Research and Development Corporation by J. Edwards University of Melbourne

ISBN 0 642 579954 ISSN 1440-6845

Control Of Mint Rust of Peppermint - Epidemiology And Chemical Control Publication No. 99/122 Project No. UM-16A

The views expressed and the conclusions reached in this publication are those of the author and not necessarily those of persons consulted. RIRDC shall not be responsible in any way whatsoever to any person who relies in whole or in part on the contents of this report.

This publication is copyright. However, RIRDC encourages wide dissemination of its research, providing the Corporation is clearly acknowledged. For any other enquiries concerning reproduction, contact the Publications Manager on phone 02 6272 3186.

Researcher Contact Details Jacqueline Edwards Institute of Land and Food Resources The University of Melbourne PARKVILLE VIC 3052

Phone: 03 9344 6458 Fax: 03 9349 4518 email: [email protected]

RIRDC Contact Details Rural Industries Research and Development Corporation Level 1, AMA House 42 Macquarie Street BARTON ACT 2600

PO Box 4776 KINGSTON ACT 2604

Phone: 02 6272 4539 Fax: 02 6272 5877 Email: [email protected] Internet: http://www.rirdc.gov.au/

Published in October 1999 Printed on environmentally friendly paper by Canprint

Foreword

Essential oil production is an emerging new industry in the river valleys of north east Victoria. Commercial sales of peppermint oil from this region began in 1991, and have increased annually since that time. The long term viability of the industry, however, is dependent upon production of an oil of consistent quality and reliable yield.

The disease mint rust, caused by the fungus Puccinia menthae, has caused major problems for the peppermint growers. If uncontrolled, mint rust reduces oil yield by 50% or more and also reduces oil quality. Control measures to date have been based on those effective in the USA, yet little is known about the behaviour of the disease under local conditions.

This publication considers the epidemiology of the disease as it occurs in north east Victoria. It examines the environmental requirements of the uredinial stage of the fungus. An action threshold for timely initiation of chemical control is developed, and several fungicides are screened for their effectiveness. The use of flaming for mint rust control is investigated. Variation within the pathogen population is examined, and two distinct groups are revealed suggesting that the taxonomy of Puccinia menthae needs to be re-examined.

This project is part of RIRDC’s Essential Oils and Plant Extracts Program which aims to obtain efficiency in production and to improve Australia’s presence in and share of world markets.

Most of our diverse range of over 400 publications are available for viewing, downloading or purchasing online through our website: • downloads at www.rirdc.gov.au/reports/Index.htm • purchases at www.rirdc.gov.au/pub/cat/contents.html

Peter Core Managing Director Rural Industries Research and Development Corporation

iii Acknowledgements

The work presented here is part of a PhD study undertaken at The University of Melbourne. I would like to thank my PhD supervisors Dr. Doug Parbery, Dr. Gerald Halloran and Dr. Peter Taylor for their valuable advice. Many people contributed to the successful completion of this study. At the Ovens Research Station, Myrtleford, I would particularly like to thank the Director, Mike Morgan, for allowing me to collaborate with his staff, Fred Bienvenu, Helen Morgan and Brendan Ralph, and to use the facilities of the Research Station. In the Institute of Land and Food Resources at The University of Melbourne, Dr. Glyn Rimmington allowed me use of his computer, Dr. Paul Taylor, Rebecca Ford, Allison Croft, Dr. Peter Ades and Dr. Pauline Gere provided assistance with the genetic analysis of Puccinia menthae. Jocelyn Carpenter of the Botany School, The University of Melbourne, carried out the scanning electron microscopy. Dr. Robert Beresford, HortResearch, New Zealand, gave me permission to use his disease assessment key developed specifically for peppermint rust.

iv Contents

Foreword iii Acknowledgements iv Executive Summary vi

Introduction 1 Objectives 2 I. Epidemiology of Mint Rust on Peppermint 3 A. The Field Disease Cycle 3 B. The Uredinial Cycle 7 C. Studies of Mint Rust under Field Conditions 19 2. Effect of Mint Rust Infection on Peppermint Growth and Yield 23 3. Investigation of Methods for Control of Mint Rust 32 A. Development of an Action Threshold for Effective Chemical Control 32 B. The Use of Flaming in Peppermint Crops 37 C. The Effect of the Herbicide Paraquat on the Viability of Puccinia menthae Urediniospores 40 D. Evaluation of Fungicides to Control Mint Rust 43 4. Variation in the Pathogen, Puccinia menthae 51 Conclusions 61 Bibliography 66

v Executive Summary

Mint rust, caused by Puccinia menthae, is a disease limiting the development of a successful essential oil industry in north east Victoria. This publication documents a study of the disease on peppermint, Mentha x piperita, from 1994 to 1998, and methods for control.

This study reports a four-year study of the disease cycle on peppermint growing at the Ovens Research Station, Myrtleford, north east Victoria, and it showed that the full life cycle of the rust does not occur on peppermint growing in this region. No spermogonia or aecia were ever seen on peppermint, whereas urediniospores persisted all year round.

A study of the infection process of urediniospores on peppermint found that, under artificial conditions, the minimum, optimum and maximum temperatures for infection were <5, 20 and 27°C respectively, and the minimum leaf wetness duration was six hours. This explained the persistence of urediniospores over the winter months in this region. These findings suggested that control measures would be best targeted during October - November, when temperatures are still less than optimum for the fungus, and the crop canopy is still open. Once the canopy is dense and closed, the fungus is protected from dessication and high temperatures.

An investigation of the effect of rust infection on the growth and yield of peppermint showed that mint rust significantly increased leaf loss, and significantly reduced leaf area, leaf fresh weight, oil content, stem and root dry weights, and the numbers of stolons per plant. These effects were seen to have implications for the long-term health of the crop, and a plot of peppermint where mint rust was allowed to develop without control died out after three and a half years.

Under field conditions, daily maximum temperatures above 35°C caused disease levels to fall. A strong relationship was found to exist between disease incidence and disease severity for mint rust, and it was proposed that this may be used to develop an action threshold for the initiation of chemical control of the disease. The incidence - severity relationship indicated that when the disease reached a mean disease incidence of 60% or

vi less infected leaves per shoot, fungicide should be applied. This was borne out in the field, when applications of the fungicide propiconazole when disease incidence was 60% eradicated from the crop, but applications when disease incidence was 80% merely slowed disease progress.

Autumn flaming of peppermint resulted in increased oil yields, but spring flaming did not. Six currently available fungicides were tested for mint rust control on M. x gracilis cv. Scotch spearmint over two years. Bitertanol and tebuconazole were identified as significantly more efficient than the other fungicides in controlling the disease, which resulted in higher oil yields.

Variation within the pathogen population was examined by comparing host ranges, teliospore morphology and RAPD-PCR profiles. It became evident that there are two types of Puccinia menthae found in Victoria which differ in morphology, host range and disease cycles, and they appear to be genetically isolated from each other. One type infects peppermint and related hosts, and the other type infects spearmint and its relatives. These results raise doubts about the current taxonomic classification of this rust as a single species.

vii

Introduction

Peppermint is grown in north east Victoria for essential oil production. Research into essential oil production began at the Ovens Research Station (ORS), Agriculture Victoria, near Myrtleford, in 1976 when rootstock of the main varieties of peppermint and spearmint were imported from the USA through the Australian Government Quarantine Station, arriving at ORS twelve months later (Bienvenu 1993). The peppermint cultivar Todd’s Mitcham was chosen as the one best-suited for the region and commercial plantings were established in 1990. Two and a half tonnes of Victorian peppermint oil were sold for export in 1991, increasing annually to 16 tonnes by 1996, at $43,000/tonne (Mr. Fred Bienvenu, pers. comm. 1997).

The economic viability of the peppermint oil industry depends upon its capacity to expand the area of plantings and maintain high average yields of top quality oil. A limiting factor has been inadequate control of the disease mint rust (Peterson 1995). In the absence of mint rust, growers in north east Victoria can achieve yields in excess of 90 kg/ha of top quality oil, but unfortunately the disease has commonly reduced it to less than 40 kg/ha of an oil of inferior quality (Bienvenu 1992). This is comparable with yield losses reported from the USA and the USSR (Fletcher 1963). An example of the devastating effects that inadequate control of the disease can have was given in New Zealand. The New Zealand peppermint oil industry began in 1970 and was rust-free until 1978. After the first epidemic during the season of 1978/79, Harvey (1979) predicted that unless effective control measures were found, the emerging industry would not survive. Unfortunately his prediction was correct; by the mid-1980s production ceased, mainly because of mint rust (Dr. Robert Beresford, HortResearch NZ, pers. comm.).

Mint rust is caused by the fungus Puccinia menthae Pers., which attacks many members of the herb family Lamiaceae, including peppermint. Control measures in the USA aim to break the life cycle of the fungus before the destructive urediniospore stage is reached. Non-chemical means, such as cultivation or the use of flame, have been used, but generally the use of fungicides, the simplest procedure, is reasonably effective. Fungicides are also used against infection that may develop during the growing season. Currently, Victorian growers use recommendations adapted from those developed in the USA, but the reliance

1 on chemicals for control is restricted by its high cost, concern over contamination of the oil and concern that resistance will develop to the chemicals. There is also growing awareness internationally that Australia is striving for a “clean and green” agriculture for marketing its produce. An understanding of how the rust fungus interacts with its mint host under local conditions, and how such infection affects oil yield, is important to facilitate the efficient use of a range of control measures.

Objectives

Until the present investigation was undertaken, no work on the biology and pathology of mint rust had been done in Australia. The objectives of this study, therefore, were:

1. To determine and demonstrate an effective strategy for the control of peppermint rust based on minimal use of chemical sprays;

2. To identify fungicides which will effectively control Puccinia menthae without contamination of distilled oil;

3. In conjunction with (1) and (2) to conduct detailed studies of the epidemiology of mint rust eg. life cycle and the influence of temperature and humidity on its capacity for growth and reproduction.

2 1. Epidemiology of Mint Rust on Peppermint

A. The Field Disease Cycle

Introduction Puccinia menthae is a rust fungus with a complicated life cycle, involving five spore stages produced in sequence on its host (Figure 1). During winter, the fungus survives on debris as tough-walled black teliospores. These teliospores germinate in spring, producing tiny colourless basidiospores which infect young host tissue as it emerges through the soil surface. Spermagonia result from this infection, and if cross-fertilisation occurs, yellow aeciospores are produced. These become foci of disease in the field, infecting nearby mint leaves, and resulting in the production of brown urediniospores, which are aerially dispersed and can travel large distances. Urediniospores infect mint leaves through the stomata and within two weeks, if conditions are favourable, thousands of new urediniospores are released from uredinia (rust pustules) formed on the undersides of the leaves. The urediniospore stage is the very destructive summer phase of the disease.

The sequence of these events and how they are influenced by the seasons of the year is known as the disease cycle. The disease cycle as it occurs in the field under north-east Victorian conditions was unknown when this project began, therefore a study was initiated to monitor the spore stages as they developed on peppermint growing at the Ovens Research Station near Myrtleford.

Methodology One of the tobacco seedbed plots (1m x 15m) at the Ovens Research Station, Myrtleford, was planted with peppermint cv. Todd’s Mitcham in September 1993. This seedbed plot of peppermint was regularly watered and fertilised, but no measures were taken to control mint rust. Sampling was done by cutting 20 mint stems at ground level along a longitudinal transect of the plot. This sample size was determined to be adequate for statistical analysis by comparison of the mean and standard deviation with sample size, as described by Kranz (1988) and Neher and Campbell (1997).

3 Sampling was carried out weekly during the growing season (September to February) and fortnightly from autumn through to spring (March to August) from November 1993 to December 1997.

Summer Urediniospore cycle repeated many times

Spring Aeciospores infect mint leaves and produce brown urediniospores Autumn-winter Uredinia convert to telia producing dark-brown teliospores

Spring Red 'blisters' on the stems Late winter - early spring break open releasing large numbers Teliospores germinate to produc of yellow aeciospores colourless basidiospores which infect the emerging mint shoots

Spring Red 'blisters' containing spermogonia appear on leaves and petioles

4 Figure 1 Diagram of the disease cycle of Puccinia menthae as reported for mint-growing areas of the USA.

The peppermint samples were examined for the presence of P. menthae using a Wild Leitz dissecting microscope (x16 - x50). The spore stages observed were recorded, and the percentage of shoots bearing each spore stage was calculated.

Results Urediniospores were always present, although less abundant over the winter months (June to August). Some teliospores were produced in autumn and winter, but no spermogonia or aecia were ever observed (Figure 2). The exact timing of teliospore production varied from year to year, but was between April and August.

100 75 50 Telia

% telia 25 0 N DJ F M A M J J A S O N DJ F M A M J J A S O N DJ F M AM J J A S O N D J F M A M J J A S O N D

80 60 Spermagonia 40 20 0 N DJ F M A M J J A S O N DJ F M A M J J A S O N DJ F M AM J J A S O N D J F M A M J J A S O N D % spermagonia

40 30 20 Aecia

% aecia 10 0 N DJ F M A M J J A S O N DJ F M A M J J A S O N DJ F M AM J J A S O N D J F M A M J J A S O N D

100 Uredinia 75 50 25

% uredinia 0 N DJ F M A M J J A S O N DJ F M A M J J A S O N DJ F M AM J J A S O N D J F M A M J J A S O N D 19941995 1996 1997

Figure 2 Disease cycle of Puccinia menthae on peppermint growing in the Ovens Valley, north east Victoria.

Urediniospores were collected fortnightly during the winter months of 1994 to 1997, plated onto water agar and incubated for 24 hours at 20°C to test for viability. The percentage of germinating urediniospores was never less than 30%, indicating that viable urediniospores

5 are present all year round and that they, rather than teliospores, carry the disease from one growing season to the next.

Discussion Prior to this study, the disease cycle of Puccinia menthae in the field in north-east Victoria was unknown, but was assumed to be similar to that reported in the USA. As a consequence, control measures were being recommended to local growers that aimed to eliminate the aecial stage in spring. The results presented here show that the full life cycle does not occur on peppermint grown under local conditions. This is not an unexpected phenomenon, as the cereal rust Puccinia graminis also persists as urediniospores all year round in Australia, despite being macrocyclic in the cold climate conditions of the northern hemisphere wheat-growing regions (Brown 1997).

At the same time, however, spermogonia and aecia were observed every spring on both commercial spearmint species (Mentha spicata cv. Native and M. x gracilis cv. Scotch speamint) also growing at the Ovens Research Station (data not shown). In the case of the spearmints, therefore, the teliospores are the main source of overwintering inoculum as reported in other mint growing areas, but on peppermint the urediniospores carry the disease through the winter.

6 B. The Uredinial Cycle

Introduction Because viable urediniospores were present throughout the year, a series of controlled environment experiments was conducted to investigate environmental influences on the uredinial cycle. Each experiment was conducted at least twice, unless otherwise stated.

Methodology i. Effect of temperature on urediniospore germination Fresh urediniospores were collected by tapping infected leaves over petri dishes containing 2% water agar, and were spread evenly over the surface of the agar using a sterile bent glass rod. The petri dishes were placed in darkness at temperatures of 5, 10, 15, 20, 25, 30 and 35°C, and left for 24 hours to ensure maximum germination. The petri dishes were then examined to determine urediniospore germination. There were six replicates (petri dishes) per treatment (temperature) in each of which 100 urediniospores were inspected. ii. Effect of temperature on the rate of urediniospore germination Urediniospores were collected, plated on water agar and incubated as described above. In this case, germination was determined hourly for the first six hours of incubation to follow the kinetics of the germination process. There were three replicates for each combination of incubation time and temperature and, as previously, 100 urediniospores were examined per replicate. iii. Effects of leaf wetness duration and temperature on germination and penetration processes Three-week-old peppermint cuttings were inoculated by spraying the leaves to run-off with a spore suspension containing 104 urediniospores / ml. They were then randomly divided into five groups of 24 cuttings, and each group placed into a plastic box with a tight-fitting lid. Immediately after sealing, each box was placed in the dark at one of five different temperatures (5, 10, 15, 20 and 27°C). Four plants were randomly removed from each box after 0, 3, 6, 9, 12 and 24 hours respectively, but kept at the same temperature in the dark for 24 hours after inoculation. All moisture had evaporated from the leaves within 10

7 minutes of removal from the boxes. There were four replicates (plants) for each combination of leaf wetness duration and temperature. After the 24 hour period, all plants were returned to the glasshouse.

Disease severity (a visual estimate of the percent leaf area per plant covered by lesions) was assessed 17 days after inoculation using a peppermint rust key developed by Beresford and Mulholland (1987). Each leaf was assessed and the mean of the leaf values was taken as the disease severity value for the plant. Absolute disease severity values differed between repeats of the experiment, so were normalised by taking the highest value in each repeat as 1.00 and expressing the other values as proportions of 1.00. These normalised values did not allow valid statistical analysis as they are not independent of each other. iv. The infection process under optimum conditions A 12-week-old plant, growing in a 10 cm pot in the glasshouse, was inoculated and kept in a sealed plastic box for 24 hours at 20°C. It was assumed that these conditions would produce the highest levels of infection. At intervals (1, 2, 4, 6, 12 and 24 hours) after inoculation, a young fully-expanded leaf was detached and immediately submerged in Chlorazole Black E clearing and staining solution. After seven days, the leaves were washed with deionised water and transferred to a saturated solution of chloral hydrate for a further two days. This procedure stains the fungal structures grey-black and clears the leaf tissue (Keane et al. 1988). Leaves were then mounted in lactic acid and examined using a Dialux 20 microscope (x100, x400 and x1000). At least 50 spores (where available) were examined per leaf, and the percentage of spores producing germ tubes, appressoria, substomatal vesicles, infection hyphae or haustoria was calculated for each time interval. This experiment was not repeated as considerable numbers of spores were examined for each time period. v. Effect of temperature on the latent period of infection Peppermint cuttings were inoculated and kept at 20°C for 24 hours to ensure maximum penetration. The plants were then placed into six controlled environment cabinets, five at constant temperatures of 5, 10, 15, 20, 27°C and one at 24°C day/20°C night. There were six replicates for each of the six temperature treatments. Plants were inspected daily and the

8 number of days taken to reach flecking (the first visible symptoms of disease) and the first opening of uredinia were recorded. Daily counts of the number of open uredinia per leaf were made until all uredinia had opened.

The definition of the ‘latent period’ of rust infection cycles varies between research workers (Teng and Close 1978). In this experiment, the latent period is defined as the time from inoculation to when 50% of uredinia had opened. This is in accord with previous latent period studies on P. menthae (Beresford and Mulholland 1987, Johnson 1995).

Disease severity was assessed 23 days after inoculation when all uredinia had opened, with the exception of the 5°C treatment which was recorded as zero. vi. Effect of temperature on urediniospore production Plants were inoculated and maintained in the glasshouse until the start of uredinium opening. Leaves were then detached and single leaves were placed, abaxial surface upwards, in petri dishes containing 2% water agar, amended with 0.5 gL-1 benzimidazole, to postpone senescence of the leaf tissue (Chandrashekar 1982, Shtienberg and Vintal 1995). The petri dishes were sealed and incubated at six different temperatures (5, 10, 15, 20, 25 and 27°C). There were three replicates (leaves) per treatment (temperature). After five days, the number of uredinia per leaf was recorded and each leaf was carefully transferred to a glass test tube. Two mls of sterile deionised water containing a surfactant (1% Tween 20) was added to each leaf. The tubes were shaken for 24 hours in darkness at 4°C to dislodge the mature spores, following which the leaves were discarded. The number of spores in each tube was counted using a haemocytometer and the mean number of spores produced per uredinium per day was calculated. This experiment was repeated twice. The second repeat, however, was slightly modified, with six replicates (leaves) per treatment (temperature), and incubation for seven instead of five days. In this case, the benzimidazole was omitted from the medium with no deleterious effect, as it had not been effective in postponing leaf senescence in the earlier repeats. vii. Data analysis Appropriate curves were fitted to the data sets using the curve-fitting options of both software packages, Sigma Plot and Cricket Graph. Differences between treatments were

9 assessed, where applicable, using analysis of variance within the statistical software package Minitab.

Results i. Effect of temperature on urediniospore germination The effect of temperature on urediniospore germination was best described by a 3rd-order polynomial regression (Figure 3). The optimum temperature was 20°C and the maximum 30°C. Maximum germination occurred at 20°C and there was little difference between 10, 15 and 25°C, but was low at 5°C. Only three of 600 spores germinated at 30°C, and there was no germination at 35°C. After completion of the germination counts, plates from 5, 30 and 35°C were incubated for a further two hours at 20°C and then re-examined. Most of the ungerminated spores from the 5°C treatment germinated during this period, but those from 30 and 35°C did not.

80 y = 7.44 + 3.1x+ 0.28x2-0.01x3 R2 = 0.97

40 Germination (%) 20

0 0 5 10 15 20 25 30 35

Temperature (°C)

10 Figure 3 Effect of temperature on P. menthae urediniospore germination after 24 hours incubation on water agar.

11 ii. Effect of temperature on the rate of urediniospore germination Most germ-tube emergence occurred during the first three hours of incubation, reaching a maximum within four hours (Figure 4). The rate of urediniospore germination was highest at 15 and 20°C, reaching a maximum of 63%, while at 10 and 25°C the maximum was 45%. The germination pattern was the same at 10 and 25°C, and similar at 15 and 20°C. Maximum germination at 5°C (11%), however, was reached after two hours incubation. At 30°C, only one spore out of 1,800 germinated (0.06%) and this was noted after one hour of incubation. No spores germinated at 35°C. Once again, plates from the 5, 30 and 35°C treatments were removed to 20°C and re-examined after two hours, and again most ungerminated spores from the 5°C treatment germinated during this period, but those from 30 and 35°C did not.

Figure 4 Effect of temperature on the rate of P. menthae urediniospore germination.

12 iii. Effects of leaf wetness duration and temperature on germination and penetration processes No infection occurred under three hours of leaf wetness, regardless of temperature. Trace infection (1 - 2 uredinia) on some individuals at each temperature was noticed after six hours of leaf wetness. After nine hours, the wetness effect could be ranked 10>15>20>5>27°C.

At all temperatures except 27°C, disease severity increased as the duration of leaf wetness increased (Figure 5). At 27°C, maximum severity was reached following nine hours of leaf wetness. The optimum temperature range was 10 - 20°C, the minimum temperature was less than 5°C and the maximum was 27°C.

Figure 5 Effects of leaf wetness duration and temperature on disease severity (data have been normalised) of P. menthae on peppermint.

13 iv. The infection process under optimum conditions Urediniospores began to germinate on the leaf surface within two hours, but appressoria and sub-stomatal vesicles were not observed until six hours after inoculation (Table 1). The percentage of spores producing appressoria and sub-stomatal vesicles continued to increase with time until observations ceased at 24 hours. At this time infection hyphae and haustoria inside the leaf tissue were observed. The mean germ-tube length continued to increase over the 24 hour period.

Table 1 Percentage of Puccinia menthae urediniospores that had germinated and produced infection structures (appressoria, substomatal vesicles, infection hyphae and haustoria) on peppermint leaves at time intervals after inoculation.

Infection Time after inoculation (h) structure 1 2 4 6 12 24 Ungerminated 100 73 14 14 15 5 urediniospores Germ tube - 27 86 86 85 95 Appressorium - - - 3.6 7.0 20.0 Substomatal - - - 1.2 2.4 17.4 vesicle Infection hypha - - - - - 14 Haustorium - - - - - 6 Mean germ tube - 29.0 56.6 71.1 192.6 254.0 length (µm)

The study of the infection process was undertaken to gain a better understanding of the host-parasite relationship and to help place other observations into context. Urediniospores began germinating within two hours of inoculating the leaf surface, and no more germination occurred after four hours, corresponding to observations of urediniospore germination on water agar. Appressoria and substomatal vesicles began to form within six

14 hours, indicating that the fungus was penetrating inside the leaf and was no longer vulnerable to the external environment, and six hours was shown to be the minimum period of leaf wetness allowing infection to occur. The steady increase in disease severity observed as the duration of leaf wetness increased can be attributed to the continued elongation of the germ tubes over 24 hours, which increases the chances of the fungus finding an open stoma through which to enter. The exception to this was 27°C, when the percentage of leaf area affected was negligible and did not increase significantly with an increased duration of leaf wetness. It is evident that germ-tubes exposed to extended periods of high temperature did not survive, whereas mycelium within the leaf survived a little better. v. Effect of temperature on the latent period of infection Temperature had a significant effect (P<0.001) on latent period which decreased as temperature increased to 20°C. This relationship can be described by the hyperbolic equation LP = 253.291temp-1.056 (Figure 6).

y = 253.291x-1.056 R2 = 0.98

40 Latent period (days) period Latent 20

0 0 5 10 15 20 25

Mean temperature (°C)

Figure 6 Effect of mean temperature on the latent period of P. menthae on peppermint.

15 No difference was evident between the constant 20°C and the alternating 24/20°C (mean 22°C) treatments. No infection developed at 27°C. The response curve between severity and temperature was a 3rd-order polynomial, with a maximum at 20°C and no disease at 27°C (Figure 7). Although no infection was evident at the time severity assessments were made, plants growing at 5°C developed sori after a latent period of 50 days, at which point many teliospores were present amongst the urediniospores. Teliospores were also observed in sori 42 days after inoculation on the plants growing at 10°C.

1 y = 0.160 - 0.091x + 0.015x2 - 0.000x3 R2 = 0.96 0.8

0.6

0.4

0.2 Disease severity (normalised)

0 0 5 10 15 20 25 30 Mean temperature (°C)

Figure 7 Effect of post-penetration temperature on the disease severity of P. menthae on peppermint after 23 days. Data have been normalised.

16 1200

y = 8.771 + 12.691x + 7.815x2 - 0.295x3 2 1000 R = 0.92

800

600

400 Urediniospores / sorus day

200

0 0102030 Temperature (°C)

This data point was assumed and added to the analysis Figure 8 Effect of temperature on the daily production of P. menthae urediniospores per uredinium. vi. Effect of temperature on urediniospore production

Urediniospore production data were transformed for normality using a log10 transformation prior to analysis. Temperature had a significant effect (P<0.001) on urediniospore production, with the response curve again being 3rd-order polynomial (Figure 8). Daily urediniospore production increased by 353% as the temperature increased from 5 to 20°C, and was inhibited significantly at 27°C.

Discussion In summary, the optimum temperature for the development and function of the uredinial stage of P. menthae is 20°C, with a near-optimum range of 10 - 25°C and a maximum temperature around 27°C. The lower temperature threshold, however, is below 5°C, which explains the presence of viable urediniospores on peppermint growing during winter in north-east Victoria, when daily average temperatures are between 5 - 10°C. It can be seen from these results that low temperatures merely slow down infection and sporulation processes, allowing the fungus to persist until conditions become favourable.

17 In order to produce high-quality peppermint oil, a photoperiod response (long days and short nights) is required to induce flowering and a diurnal temperature fluctuation in the month preceding harvest is essential, restricting production to climates characterised by clear hot days and clear cool nights in the summer (Bienvenu 1993). The river valleys of north-east Victoria are particularly suitable for essential oil production, with plenty of irrigation water available during summer. These conditions, however, also favour the development of rust epidemics. The maximum daily temperature in summer is often above 30°C, but it is usually accompanied by overnight minima of 8 - 14°C. Overhead irrigation of the crop late in the day or at night provides conditions that are conducive to infection, and there will most certainly be areas within the dense peppermint canopy that remain shaded, relatively cool and moist even during the hottest part of the day. It would appear from this study, therefore, that control measures would be best applied during spring growth when temperatures are between 5 - 15°C, before the canopy has closed over and conditions have become ideal for rapid generation of the fungus.

18 C. Studies of Mint Rust under Field Conditions

Introduction In order to develop and implement a successful plant disease management strategy, it is necessary to understand as much as possible about the factors that contribute to the development of the plant disease epidemics. A common method for quantifying the progressive changes in disease levels in the field is to plot the amount of disease present at consecutive time intervals. This then serves as a summary of the disease present in a particular crop during a particular season, and can be compared with other plots of the same disease in other regions or seasons, or with concurrent variation in temperature and rainfall, in order to identify the effects of climatic variables, fungicide treatments, etc. on the course of the epidemic.

The amount of disease present is usually assessed by either or both of two methods: (1) measurement of the incidence of disease and (2) measurement of the severity of disease.

Disease incidence is calculated as the percentage of the total leaves on a stem with one or more uredinia. It is quick and simple, while being objective with little opportunity for user- error. The drawback, however, is that once all leaves are infected, no further increase in disease can be measured, limiting its value for investigating crop losses or disease management strategies.

Disease severity is a measure of the leaf area loss due to the disease. Estimations of how much leaf area is affected by mint rust are made by comparing leaves with a visual disease assessment key. Disease severity in this study was calculated as the mean percentage loss of leaf area per mint stem, using a visual assessment key adapted for peppermint rust by Beresford and Mulholland (1987). Unless otherwise stated, one leaf was assessed from each third of the main stem and the mean of the three measurements was taken as the disease severity value of the stem. Missing leaves were ignored.

As far as can be determined, there is no such published information for disease epidemics of mint rust. The following experiment was undertaken to study the influence of climate on the progress of the disease on peppermint growing in north east Victoria.

19 Methodology Samples were collected weekly from the peppermint seedbed plot, Ovens Research Station, during September to February, 1994 to 1998. Twenty shoots were taken at each sampling time and assessed for disease incidence and disease severity as previously described, and the mean values were calculated for each sample. Disease progress was monitored by plotting the means of the disease measurements (incidence and severity) against time.

Meteorological data were collected using the Hardi Metpole installed at the Ovens Research Station. The minimum and maximum daily temperatures were measured at 20 cm. above ground level, which was within the crop canopy. Minimum and maximum daily relative humidity was measured at the same place as temperature, and total daily rainfall, total daily solar radiation and maximum daily wind speed were also recorded.

Results and Discussion

Disease incidence: Disease incidence began at less than 10% in all seasons (Figure 9A). During 1994/5 and 1995/6, disease incidence increased at a linear rate from early October to 80% by the end of December, but in 1996, disease incidence only reached 55% when the combination of hot summer weather and three years of continual rust infection severely checked the growth of the plants and they did not recover. The following season, 1997/8, there were very few shoots of peppermint to be found and no rust was observed. Weekly sampling had to be abandoned due to lack of shoots.

Disease severity: Disease severity was negligible until the beginning of November in each season (Figure 9B). In 1994/5, it increased rapidly to 15% by the end of November 1994, then fluctuated between 5 and 10% during December 1994, increasing to 15% again by February 1995. In 1995/6, disease severity increased in steps, reaching a plateau of 5% in December 1995. In 1996/7, disease severity was again 5% by December 1996, but then the rust disappeared as the peppermint died off, as explained when discussing disease incidence, and no rust was observed on the meagre growth of peppermint in 1997/8.

20 100

80 A

Disease incidence (% leaves infected) 20

15 B

5 Disease severity (% leaf area affected) 0 Sept Oct Nov Dec Jan Feb

94/5 95/6 96/7 97/8

Figure 9 Changes in disease levels for peppermint growing in the seedbed plot - A disease incidence, B disease severity.

The influence of climatic variables on disease There were no obvious correlations between relative humidity, solar radiation, rainfall and wind speed and disease. Temperature, however, had a direct effect on the levels of disease, particularly disease incidence (Figure 10). The peppermint plants died out during the summer of 1996, so there is no 1997/8 graph. On 5th September 1995, a severe frost (-5°C) caused high levels of defoliation in all the crops and reduced the amount of disease present at the beginning of the season. In late spring and summer, days with maximum temperatures above 35°C caused an immediate reduction in the amount of rust present, but

21 the levels rose again if the maximum temperature dropped below 35°C for a few days eg late January 1994/5.

100 100 1994/5 90 80 80 70 60 60 50 max T 40 40 30 20 20 10 Temperature (°C) Disease incidence (% leaves infected) 0 min T 0 (10) Sept Oct Nov Dec Jan Feb

100 100 1995/6 90 80 80 70 60 60 max T 50 40 40 30 20 20

10 Temperature (°C) Disease incidence (% leaves infected) 0 0 min T (10) Sept Oct Nov Dec Jan Feb

100 100 1996/7 90 80 80 70 60 60 max T 50 40 40 30 20 20 Temperature (°C)

Disease incidence (% leaves infected) min T 10 0 0 Sept Oct Nov Dec Jan Feb

Figure 10 Effect of temperature on the incidence of mint rust on peppermint in the seedbed plot, Ovens Research Station.

22 2. Effect Of Mint Rust Infection On Peppermint Growth And Yield

Introduction It is well established that mint rust reduces the quantity of essential oil produced by commercially-grown peppermint, but there is little information on its impact on the growth of the plant itself. The objective of the following experiments, therefore, was to quantify the effects of P. menthae on the growth and yield of peppermint.

Methodology On 1st September 1995, 84 rooted tip cuttings of Todd’s Mitcham peppermint were transplanted singly into 10 cm pots containing potting mix. The plants were kept in an outdoor area of the System Garden at The University of Melbourne. On 22nd September 1995, they were divided randomly into two groups, each of 42 plants. One group was inoculated with 5 x 103 urediniospores/ml. The plants were returned to the outdoor area for the duration of the experiment and maintained under natural temperature and photoperiod with supplementary daily watering. They were fertilised monthly with the complete slow- release fertiliser, Osmocote. The control group was sprayed with propiconazole (as Tilt 250EC) at a rate of 0.4 ml/L as soon as rust was detected (spray dates: 21-11-95, 27-11-95, 21-12-95 and 4-1-96).

The experiment was repeated in 1997/8, with slight modifications. On 12th September 1997, 40 rooted tip cuttings of Todd’s Mitcham peppermint were transplanted singly into 10 cm pots as previously, and on 8th October 1997 the resultant plants were divided randomly into two groups of 20 plants each. One of the groups was inoculated as described above, but with a higher inoculum dose of 105 spores/ml. In 1995/6, the pots were watered daily but unavoidably dried out between watering during periods of hot weather. In 1997/8, the pots were placed in plastic trays to retain the run-off water, ensuring that the plants remained free from any water stress. The control group was again kept ‘rust-free’ with applications of propiconazole as described above (spray dates: 13-10-97, 21-11-97, 23-12- 97 and 30-12-97).

The first experiment was harvested during April 1996, and the second experiment was harvested during March 1998. At harvest, the following measurements were made per

23 plant: oil content, shoot number, stolon number, number of leaf nodes per shoot and number of missing leaf pairs per shoot, total leaf area, leaf fresh weight, stem fresh weight and root fresh weight. Stem and root samples were oven-dried at 80°C for one week, then reweighed to obtain dry weights. Leaf samples were frozen immediately after weighing and stored at -20°C until the harvest was complete. The frozen leaf samples were then steam-distilled, the oil content measured and the oil analysed. The protocol for oil extraction prevented measurements of leaf dry weight. Total leaf area was measured using a LI-3100 Area Meter (LICOR, Lincoln, Nebraska, USA).

Disease incidence was measured as the percentage of leaves that were infected per plant, disease severity was visually assessed as previously described, and defoliation was measured as the percentage of leaf nodes per stem that had no leaves.

Oil content was determined by steam-distillation of all leaves from each plant. The extracted oil was bulked into four samples, two control samples and two rusted samples, and sent to the Ovens Research Station for oil quality analysis. A Shimadzu Gas Chromatograph 9A fitted with a 30 mm x 0.25 mm free fatty acid phase column was used to measure the percentage of the five major oil components (menthone, menthol, menthyl acetate, menthofuran and isomenthone) in each oil sample, according to the standard procedure used by peppermint oil producers in the region. The bulking of the samples was necessary as the apparatus could not analyse quantities of oil less than 1ml.

The data were analysed using the statistical software package Minitab. Where the assumptions for normality were met, the two-sample t-test was applied to test for significant differences between the two treatments at the 5% level. In the cases of stem and root dry weights (1995/6) and oil content, stem dry weight, leaf fresh weight and leaf area

(1997/8), the data were transformed using a log10(x+1) transformation before analysis by two-sample t-tests. In the case of stolon number (1995/6), no transformation could be found that would overcome the problem of unequal variances, so differences between the control and rusted treatments were assessed using the non-parametric Mann-Whitney U-test, which allows for unequal variances between the two groups, and does not assume normality.

24 Regression analysis was used to examine relationships between the disease measurements and oil yield of the plants. Curves were fitted using the graphical software package Cricket Graph.

25 Table 2 Effects of rust infection on growth and yield components of peppermint cv. ‘Todd’s Mitcham’. Means with 95% confidence intervals are presented, with P-values calculated from the two sample t-test, except a which are medians, with P-values calculated from the Mann-Whitney U-test.

1995/6 1997/8 Control Rusted P- Change due Control Rusted P-value Change value to rust due to rust Oil/plant (µl) 64.4 ± 7.5 36.1 ± 4.1 <0.01 −44% 131.0 ± 15.7 80.1 ± 8.4 <0.01 −39% Stem number/plant 53.9 ± 2.5 30.0 ± 3.1 <0.01 −44% 54.9 ± 9.2 51.6 ± 5.6 0.53 n.s Stolon number/plant 5.0 a 0.0 a <0.01 a −100% 24.7 ± 3.8 8.7 ± 1.9 <0.01 −65% Total nodes/stem 14.9 ± 0.5 11.1 ± 0.6 <0.01 −26% 15.2 ± 1.2 15.5 ± 1.1 0.65 n.s Leaf fresh wt. (g) 17.43 ± 1.27 11.19 ± 1.27 <0.01 −36% 46.64 ± 5.98 21.03 ± 3.01 <0.01 −55% Stem dry wt. (g) 8.46 ± 0.96 3.63 ± 0.43 <0.01 −57% 47.75 ± 7.00 18.28 ± 2.45 <0.01 −62% Root dry wt. (g) 6.08 ± 0.92 2.41 ± 0.51 <0.01 −61% 21.08 ± 3.32 10.58 ± 2.45 <0.01 −50% Leaf area (cm2) 826.3 ± 56.8 620.6 ± 69.4 <0.01 −25% 1922.1 ± 229.9 1031.8 ± 141.7 <0.01 −46% Defoliation (%) 50.0 ± 1.9 45.5 ± 3.7 0.043 −9% 37.3 ± 3.8 62.4 ± 3.5 <0.01 +67% Disease incidence (%) 13.2 ± 6.4 63.5 ± 8.7 <0.01 55.2 ± 9.1 89.4 ± 4.2 <0.01 Disease severity (%) 0.8 ± 0.4 7.8 ± 1.7 <0.01 3.6 ± 1.0 22.6 ± 2.4 <0.01

Results A small amount of rust was observed in the control group by the end of harvest, but because the plants were free of rust for most of the experiment, such infection was accepted as insufficient to invalidate the comparisons. Disease severity and incidence were low in the control compared to the rusted group.

There were significant differences (P<0.05) between the rusted and control groups in each of the variables measured (Table 2). Mint rust significantly reduced leaf nodes per shoot, shoot number, stolon number, total leaf area, leaf, stem and root weights, and oil content per plant. During a period of very hot weather in January 1996 many older shoots on the rusted plants wilted irreversibly and died, and this leaf loss is not accounted for in the defoliation measurements. Therefore it appears as if defoliation was higher in the control plants than the rusted plants in 1995/6, but this is an aberration of the data. There was no difference between treatments in oil content per leaf fresh weight (data not shown).

Table 3 Effect of rust infection on the relative proportions of the major oil components (measured by gas chromatography). Means for each treatment are presented.

Oil constituents Experiment 1 (1995/6) Experiment 2 (1997/8) (% total oil) Control Rusted Control Rusted Typicala Menthone 13.7 15.8 6.3 11.3 18-22 Menthol 39.1 46.6 54.9 53.6 43-46 Menthyl acetate 16.3 9.4 7.8 5.8 3-6 Menthofuran 11.8 9.9 8.3 11.7 2-4 Isomenthone 1.7 2.0 1.4 1.9 2-3 atypical: the range expected for oil of this type, grown and harvested under optimum conditions.

Results of the gas chromatography analysis of the extracted oil are shown in Table 3. None of them conformed to the range typical of oil produced under optimum conditions at

Myrtleford, but the oil composition from the rusted plants indicated that these plants had not reached the same level of maturity as the control plants.

27 0.25 A y = 0.063 - 0.024x R2 = 0.29, p < 0.01

0.2 B y = 0.201 - 0.088x R2 = 0.42, p < 0.01

0.15

Oil yield (ml) 0.1 B 0.05

A 0 0.000 0.500 1.000 1.500 2.000 Disease incidence (transformed)

0.25 A y = 0.061 - 0.077x R2 = 0.26, p < 0.01

0.2 B y = 0.162 - 0.158x R2 = 0.50, p < 0.01

0.15

0.1 Oil yield (ml)

0.05 B

A 0 0.000 0.100 0.200 0.300 0.400 0.500 0.600 Disease severity (transformed)

Figure 11 Relationships between oil yield and measures of disease incidence and severity. Original disease values were transformed using an arsine transformation. Open circles (o) and equations A refer to 1995/6, and closed circles (•) and equations B refer to 1997/8.

28 The disease incidence and severity levels were plotted against oil yields from both experiments and although the actual levels of disease were higher in 1997/8, the same trend of decreasing oil yield with increasing disease levels was evident in each (Figure 11). The disease data for each experiment were normalised using an arcsine transformation and linear regression was applied. The equations were significant (P<0.05), but the coefficients of determination (R2) were low in 1995/6.

0.25

0.20 y = 0.203 - 0.002x R2 = 0.60, p < 0.01 0.15

this data point assumed and 0.10 added to the analysis Oil yield (ml) 0.05

0.00 0 20406080100 Defoliation (%)

Figure 12 Relationship between oil yield per plant and amount of defoliation (1997/8 data only).

Defoliation measurements in 1995/6 were confounded by adverse hot weather and associated moisture stress. Consequently, only defoliation data from 1997/8 were examined for a relationship with oil yield. The data did not need to be transformed prior to analysis. It was assumed that if defoliation was 100%, oil yield would be zero, and this point was added to the analysis. There was a strong negative linear relationship (R2 = 0.60) between oil yield and the amount of defoliation (Figure 12). Defoliation exhibited a positive linear relationship (R2 = 0.55) with disease incidence (Figure 13), and positive logarithmic relationship (R2 = 0.69) with disease severity (Figure 14).

29 80

70 y = 15.166 + 0.479x R2 = 0.55 60

Defoliation (%) 30

0 10 20 30 40 50 60 70 80 90 100 Disease incidence (%)

Figure 13 Relationship between amount of defoliation and disease incidence per plant (1997/8 data only).

Defoliation (%) 30 y = 26.868LOG(x) + 24.846 20 r2 = 0.692

0 0 5 10 15 20 25 30 35 Disease severity (%)

Figure 14 Relationship between amount of defoliation and disease severity per plant (1997/8 data only).

30 Discussion Excessive leaf drop has been observed in the field in north east Victoria during years of severe rust infection. In the present study, the comparison of defoliation in healthy and rusted peppermint was confounded in 1995/6 by the death of a high proportion of shoots in the rusted plants caused by water stress during hot weather. In 1997/8, however, with unlimited water availability, leaf drop was increased by 67% in the plants infected with mint rust compared to the control. Since the essential oil of peppermint is produced in glands on the surfaces of the leaves, the increased defoliation explains the significant reduction in oil yield observed, as the number of shoots per plant and the number of leaf nodes per shoot were not affected by mint rust infection. The negative correlation between oil yield and level of defoliation was adequately described by a simple linear equation, and the effect of rust infection on the level of defoliation could also be quantified. Leaves were shed rapidly as disease severity increased from zero to approximately 5% leaf area affected, reaching 40% shoot defoliation, but further increases in disease severity had less effect on the rate of defoliation. These results indicate that even a small amount of rust infection will cause peppermint cv. Todd’s Mitcham to shed its leaves, emphasising the importance of effective control of the disease.

Infection courts of biotrophic parasites, such as rust fungi, are known to act as sinks for assimilate in competition with the infected host plant, resulting in reduced plant growth rate, size and weight compared to healthy individuals (Parbery 1996). In the present study, mint rust caused reductions of 50-60% in stem and root dry weights, and stolon production was severely affected. The reduction in growth accompanying such infection, particularly that of roots and storage organs such as rhizomes and stolons, is likely to have profound effects on the ability of a perennial crop such as peppermint to yield well and persist over several years. This was demonstrated in the peppermint seedbed plot where mint rust was allowed to develop and progress unhindered by any control measures (see previous section). Within four years, the peppermint plants had died out. Peppermint crops in north east Victoria are currently replanted every three to five years, but growers see economic advantage in extending this period to possibly ten years. The implications from the results of the present study are that unless adequate rust control can be developed, the likelihood of longer cropping periods seems remote.

31 3. Investigation Of Methods For Control Of Mint Rust

A. Development of an Action Threshold for Effective Chemical Control

Introduction At the present time, no chemical is registered for control of mint rust in Australia. Local growers, however, currently use the fungicide propiconazole (as Tilt 250EC, manufactured by Novartis) during the growing season to control rust on peppermint, as it is known to be effective against other rust fungi in many agricultural and horticultural crops. Growers wait until the disease can be readily observed, usually mid to late November, and then apply two to three spray treatments of the fungicide at fortnightly intervals.

Effective integrated disease management depends upon a system of crop monitoring and recognition of a critical level of disease as an ‘action threshold’ for the initiation of fungicide treatment, resulting in minimal yet effective use of chemicals for control. In the past, such action thresholds have relied upon recognition of a particularly critical level of disease severity (Brown and Holmes 1983, Zadoks 1985, Royle 1994, Shtienberg 1995, Brown and Keane 1997), but visual estimates of severity vary considerably between assessors (Zadoks 1985, Nutter and Schultz 1995, Parker et al. 1995, Brown and Keane 1997, Nutter 1997). If it is possible to replace the assessment of disease severity with the more objective assessment of disease incidence, the use of an action threshold is more likely to be adopted (Zadoks 1985).

To do this, it is necessary to first establish whether there is a quantifiable relationship between disease incidence and disease severity for the disease in question. Therefore it was decided to investigate whether an incidence - severity relationship exists for mint rust, and if so, whether it might be exploited to develop an action threshold for peppermint growers to use as a decision tool for the initiation of fungicide applications.

32 Methodology Part 1 The disease incidence and severity measurements from both the field experiments (p.17) and the pot experiments (p.21) were plotted against each other using the graphical software package Cricket Graph, and any relationship between them was examined both visually and by using the coefficient of determination, as proposed by Fowler and Cohen (1990).

Part 2 Samples of peppermint were collected weekly as previously described. The samples were taken from:

(i) a farmer’s paddock (O’Sullivan) near Myrtleford, Ovens Valley, September 1994 to February 1995. Three applications of propiconazole (‘Tilt 250EC’ at a rate of 500 ml/ha) were applied on November 20th, December 4th and December 11th 1994. Treatment was initiated when disease incidence had reached 80%.

(ii) a research plot on the Ovens Research Station, September 1996 to February 1997. Propiconazole (‘Tilt 250EC’ at a rate of 500 ml/ha) was applied on October 23rd, November 7th, December 9th and January 9th. Treatment was initiated when disease incidence had reached 60%.

(iii) a research plot on the Ovens Research Station, September 1997 to February 1998. Propiconazole was applied on November 7th, November 28th and December 12th. Treatment was initiated when disease incidence had reached 80%.

Twenty shoots were taken at each sampling time and assessed for disease incidence and disease severity as previously described and the mean values were calculated for each sample. Disease progress was monitored by plotting the means of the disease measurements (incidence and severity) against time.

33 Results Part 1 Visual examination of the disease incidence and severity data for each of the experiments revealed a relationship that was very similar for each data set. Therefore, the data for all experiments were combined and plotted as one (Figure 15). The curve could be described by the exponential equation: Y = 0.264 x 100.02X (R2 = 0.82) where Y = disease severity (%) and X = disease incidence (%). The relationship between the two measurements appeared to be linear at levels of incidence below 60%.

40 0.021x field experiment data y = 0.264 * 10 R2 = 0.82 30 pot experiment data

10 Disease severity (% leaf area affected)

0 0 102030405060708090100 Disease incidence (% leaves infected)

Figure 15 Incidence - severity relationship for mint rust of peppermint. Data from field experiments and pot experiments were pooled together.

34 Part 2 The fungicide treatments applied to the peppermint crops in 1994/5, 1996/7 and 1997/8 all reduced disease incidence and severity considerably, and the time of application had an effect on disease progress (Figure 16). The earlier the applications began, the less disease developed. The earliest treatment, applied in October 1996 when disease incidence was 60%, resulted in eradication of rust from the crop, lasting for two months until inoculum was carried in from elsewhere. There were some strong winds in late December 1996 which could have blown in fresh urediniospores.

90 A 80 70 60 50 40 30 20 Disease incidence (% leaves infected) 10 0

30 25 B 20 15 10 5 Disease severity (% leaf area affected) 0 Sept Oct Nov Dec Jan Feb

paddock 96/7 paddock 97/8 O'Sullivan 94/5

Figure 16 Changes in disease levels for peppermint cv. Todd’s Mitcham growing in the ORS paddock plot and O’Sullivans farm - A disease incidence, B disease severity. Tilt was applied on 20th November, 4th and 11th December 1994; 23rd October, 7th November, 9th December 1996 and 9th January 1997; and 7th and 28th November and 12th December 1997.

35 Discussion In the present study an incidence - severity relationship was found for mint rust on peppermint that could be described by an exponential equation, with a linear phase at low levels of disease. Furthermore, the relationship was consistent across experiments carried out in different years, in pots and in the field. Such an exponential relationship, with a linear phase at the lower incidence levels, has been reported for other diseases (James and Shih 1973, Seem 1984, Dillard and Seem 1990a, 1990b, Beresford and Royle 1991). Dillard and Seem (1990b) successfully used this relationship to identify a level of disease incidence that could be recommended to maize growers as an action threshold for the initiation of fungicide treatment to prevent the onset of common maize rust epidemics. In the light of this, therefore, the preliminary finding of a strong incidence - severity relationship for the mint rust pathosystem was encouraging, and possibly helpful in determining a critical time for the initiation of fungicide applications to control the disease. The action threshold recommended by Dillard and Seem (1990b) for common maize rust was the level of disease incidence reached at the point where the relationship stops being linear. After this point, disease severity increases very rapidly and the disease becomes increasingly difficult to control. In the case of mint rust, this point is at 60% disease incidence, when disease severity is approximately 5%.

Does this theory work in practice in a peppermint crop? With this question in mind, data collected over three years in the Ovens Valley was closely examined. In 1996/7, fungicide treatment was initiated when disease incidence was approximately 60%, and resulted in complete control of mint rust until late in the season. A preliminary recommendation, therefore, could be made to peppermint growers that they monitor the disease level in their crops by regular sampling of stems, and as the percentage of infected leaves per shoot reaches 60%, begin their fungicide applications. This could be expected to be in middle to late October, when the canopy is still relatively open and average temperatures are cooler than the optimum for the uredinial cycle of the rust (see section IB). This would be earlier than the current practice, and the indications from this study so far are that it could lead to more effective control of the disease.

36 B. The Use of Flaming in Peppermint Crops

In the major peppermint growing areas of the USA, flaming is a recommended practice in programs for controlling mint rust (Lewis McKellip, Wm. Leman Company, Bremen, Indiana, USA, personal communication 1996). Spring flaming of peppermint crops in the USA is used to destroy the aecial stage of the fungus (Horner 1965, Maloy and Skotland 1969, Hardison 1976) and has been recommended for the same purpose in Australia (Small 1986). Autumn flaming is used as a post-harvest clean-up measure to destroy regrowth and debris.

Control ‘Tilt’ - autumn Flamed - autumn

no treatment early and late spring ‘Tilt’ no treatment

late spring flame early spring ‘Tilt’ late spring flame late spring flame early and late spring flame early and late spring flame early and late spring flame early and late spring flame early and late spring flame early and late spring flame

late spring flame early spring ‘Tilt’ late spring flame late spring flame

no treatment early and late spring ‘Tilt’ no treatment

Figure 17 Plot layout, showing where the autumn and spring treatments were applied. Autumn treatments were applied down (columns in table), and spring treatments were applied across (rows in table), the trial area.

The use of flaming technology as a potential control method for mint rust in Victorian peppermint crops was investigated during 1994/5 in conjunction with another RIRDC- funded project, DAV71A. Autumn flaming, spring flaming, and combinations of both were compared with the use of propiconazole (as Tilt 250EC) for their effect on disease levels

37 and oil yields of peppermint growing under local conditions (Figure 17). The methodology and results have already been reported in ‘Evaluation of a chemical free method of controlling pest and diseases in Australian peppermint crops’ RIRDC Report Project Number DAV 71A, so only a discussion of the findings will be presented here.

Spring flaming caused considerable disruption to plant growth, resulting in large bare patches of soil and much weed infestation, and therefore produced comparatively poor yields of oil, even though the levels of disease and defoliation were low compared to the other treatments. Effective spring flaming requires a knowledge of the life cycle of Puccinia menthae under local climatic conditions, which was not available for north east Victoria until the present study was conducted, when it was discovered that the aecial stage is not produced. The effectiveness of spring flaming for rust control in Australian peppermint crops as recommended by Small (1986) was therefore unsubstantiated. In addition, Grey and Welty (1995) reported that in the USA, while the frequency of rust was considerably reduced by spring flaming, the peppermint crop subsequently suffered sporadic plant death and reduced plant vigour. In view of the absence of the aecial stage on peppermint grown in north east Victoria, and the adverse effects on peppermint growth and yield, it would be inadvisable to recommend the use of spring flaming for control of mint rust under north east Victorian conditions.

The autumn-flamed treatments produced significantly higher oil yields than the other treatments despite carrying high levels of mint rust, and it is important to elucidate the reasons for its effect. The reduced impact on plant assimilates and shoot damage by pests and diseases over the subsequent winter may have allowed more shoots to develop to maturity, therefore increasing biomass. Quadrat counts of plant numbers or measurements of fresh weight would have indicated whether this was the case. In order to propose any hypothesis with confidence, it would be necessary to repeat the experiment and include some measurements of biomass. This was not possible during the present study as the flamer was not available for a second trial.

There have been several reports over the years recommending autumn flaming as a clean- up measure for use in peppermint crops (Drummond-Goncalves 1943, Murray 1961, Horner and Dooley 1965, Maloy and Skotland 1969, Fox 1995, Whitten 1997).

38 Unfortunately, there are two serious impediments to this recommendation in north east Victoria. First, there is the legal barrier to cross before flaming can be used on a commercial scale in Australia. It is currently illegal to transport filled bulk liquid petroleum gas (LPG) tanks as seen in static industrial situations. Such tanks must be transported empty. The exception is where specially designed recessed valves and controls are fitted to the trucks. The current sizes of these tanks which are available are far larger than required for a flamer. The second impediment is that autumn flaming in north east Victoria would coincide with the high fire risk period, and the special permission which would be required from the Country Fire Authority would almost certainly be denied.

The application of flaming on peppermint at the Ovens Research Station, October 1994.

39 C. The Effect of the Herbicide Paraquat on the Viability of Puccinia menthae Urediniospores

Introduction Several peppermint growers in north east Victoria currently use the contact desiccant herbicide paraquat (1.5L/ha as Gramoxone, supplied by Crop Care Australasia) as an autumn clean-up measure and the question is raised as to whether this is an effective alternative to flaming. The previous experiment established that autumn flaming killed above-ground insect pests and weeds, and incinerated foliage and debris that served as a reservoir of overwintering rust inoculum. Paraquat is equally effective against weeds, but would not affect the insect population or remove debris in the same manner. There have been some reports of the use of contact herbicides to control mint rust (Campbell 1954, 1955, 1956), including a study by Horner (1965) that compared the effectiveness of such a herbicide with propane gas flaming, but the focus was always to kill developing aecia and thus break the life cycle of the fungus. The study of the disease cycle under local conditions showed that urediniospores carry the disease through winter in north east Victoria where aecia have not been observed. The objective of this experiment was to examine whether urediniospore viability was affected by treating peppermint foliage with paraquat.

Methodology Sixteen 12-week-old peppermint cuttings were inoculated with 5x104 spores/ml and maintained in the glasshouse for a further six weeks, by which time the leaves were well- covered with sporulating uredinia. The plants were then randomly divided into four groups, each containing four plants, and the following treatments were applied: Treatment C - no treatment (control); Treatment S - the shoots were cut off at the base of the stem and placed on aluminium foil on a glasshouse bench; Treatment L - the individual leaves were cut off the shoots and placed on aluminium foil on a glasshouse bench; Treatment P - paraquat (2.5ml/L as Gramoxone) was sprayed with a hand-held spray unit on to the plants until run-off.

40 The rate of paraquat used was equivalent to that applied in the field (250ml/100L as Gramoxone).

Urediniospores were collected from each plant by lightly pressing the abaxial surface of an infected leaf onto 1.5% water agar, then, once the leaf was removed, remaining groups of urediniospores were spread uniformly across the surface of the agar with a bent glass rod. The agar plates were incubated for 24 hours in darkness at 20°C, then examined to determine the percentage of spores which had germinated. At least 400 spores per replicate were examined, and the mean percentage germination was calculated for each treatment. This was done immediately prior to application of the treatments and then daily until 20 days after treatment. The experiment was repeated. Since the results were similar for both, the data sets were combined and the daily means for each treatment were calculated. As the daily fluctuation of urediniospore viability per se was not of interest in the present study, the urediniospore germination for each treatment was expressed as a percentage of the control.

Results The leaves from Treatments S and L shrivelled and dried within a couple of days. The paraquat-treated leaves (Treatment P) wilted and died within three days, but did not fall off the stems. Eight days after the paraquat treatment, the plants began to recover, with the emergence of new shoots and also new leaves from the nodes of the existing shoots. These new leaves had to push past the dead leaves still hanging on the stems as they grew and expanded.

Urediniospore viability remained similar for all treatments for the first six days (Figure 18). After that, the viability of the paraquat-treated spores was less than that of the other treatments. By 15 days, the paraquat-treated spores were only 20% as viable as the control spores, and the viability remained at this level to the end of the experiment (Figure 19). Spores from dead leaves which had not been killed with paraquat (Treatments S and L) began to die 15 days after treatment, and then dropped to 60% of the control by 20 days.

41 240% Treatment C - control 220% Treatment S - cut stems 200% Treatment L - cut leaves

180% Treatment P - paraquat

160%

140%

120% C 100%

80% L 60% Urediniospore viability (expressed as % control) S 40% P 20%

0% 0 2 4 6 8 101214161820 Days after treatment

Figure 18 Effect of the treatments on the level of urediniospore viability. Data are expressed as a percentage of the control treatment.

140%

120%

100%

80%

60%

40% Urediniospore viability (expressed as % control) -0.041x 20% y = 1.231 * 10 R2 = 0.80

0% 0 2 4 6 8 101214161820 Days after treatment

Figure 19 Effect of paraquat on the level of urediniospore viability, expressed as a percentage of the control treatment.

42 Discussion These results indicate that the use of paraquat is not equivalent to the use of autumn flaming. Although urediniospore viability was reduced by paraquat compared to the control, new shoots and leaves were beginning to emerge within eight days of herbicide application while the spore viability was still 60%. It is quite possible that as the new foliage grew passed the dead leaves remaining on the stems, viable urediniospores could be transferred and infect them or be blown on to them.

As already discussed, however, autumn flaming is not an option due to current legal constraints concerning bushfire risk. In this experiment, urediniospores taken from paraquat-treated foliage were less viable than those taken from foliage which had died after cutting, such as may be left lying on the ground after harvest. Therefore using paraquat as a clean-up measure, while not as effective as flaming, would substantially reduce the inoculum level that would be carried over the winter into the next season, and may reduce its infection potential even more.

D. Evaluation of Fungicides to Control Mint Rust

Introduction Research is currently being carried out at the Ovens Research Station to evaluate the potential of Scotch spearmint as a complementary essential oil crop for the region (RIRDC Project Number DAV 101A ‘Economic feasibility of Native and Scotch Spearmint production in Tasmania and Victoria’). An objective of this current project (UM 16A) was to identify fungicides which will effectively control Puccinia menthae without contamination of distilled oil. Due to the lack of available land at the Ovens Research Station, however, it was not possible to use field-grown peppermint for a fungicide screening trial, so the decision was made to collaborate with RIRDC Project DAV 101A and use rust-infected Scotch spearmint for the fungicide screening trial. The aim, therefore, was to compare six commercially available fungicides for use against mint rust on Scotch spearmint grown at the Ovens Research Station across two seasons.

43 Methodology Scotch spearmint rhizomes were transplanted from a seedbed plot on the Ovens Research Station to the research plot area in May 1996. A randomised complete block design with seven treatments and four replicates per treatment was applied to the trial site. The treatments were:

- control (no fungicide),

- bitertanol (1.7ml/L as Baycor 300EC, manufactured by Bayer),

- triadimenol (0.7ml/L as Bayfidan 250EC, manufactured by Bayer),

- fluquinconazole (30g a.i./100L as Castellan 250WP, manufactured by Agrevo),

- tebuconazole (300ml/ha as Folicur 250, manufactured by Bayer),

- myclobutanil (1.2g/L as Systhane 400WP, manufactured by Hoescht),

- propiconazole (500ml/ha as Tilt 250EC, manufactured by Novartis).

Each row contained seven 1m x 5m plots, and the four rows were separated from each other by an untreated row of Scotch spearmint, one metre wide. This was to ensure that there was no drift of fungicide across the rows, and to keep inoculum pressure high. The trial was not inoculated with urediniospores prior to treatment as the Scotch spearmint was already well infected with rust from field inoculum.

In May 1997, rhizomes from the first year’s trial site were transplanted to an adjacent area of paddock, and again a randomised complete block design was used with the same plot sizes as described above.

The fungicide treatments were applied using a 15L knapsack sprayer with the addition of 1ml/L wetting agent (as Agral 600, manufactured by Crop Care Australasia) and the dates of application were:

trial 1: 25th November, 12th and 27th December 1996;

trial 2: 1st, 14th and 28th December 1997.

Ten samples per plot were assessed for disease incidence and severity as described previously. Pre-treatment assessments of each plot in both seasons confirmed that there

44 were no significant plot differences in disease levels before the fungicides were applied (data not shown).

Assessment of the final disease levels and defoliation levels was undertaken in the week prior to harvest. At this time, however, the plants were large and bushy, and cutting the stems at ground level would have been very destructive, removing a large proportion of the biomass in the plots. In trial 1, twenty samples were taken per plot as ten side-shoots from the upper half of the plants and ten side-shoots from the lower half of the plants. Mean disease severity measures were calculated for the upper and lower side-shoot groups both separately and combined, and the three different measures were compared against the respective final oil yields per treatment. The disease severity levels on the upper side- shoots showed the strongest negative relationship with oil yield, and therefore only data from the upper shoots were analysed further. In trial 2, ten upper side-shoots per plot were sampled at the final assessment date.

Although the plots were 1m x 5m, only one m2 was harvested from each, which was taken near the centre of each plot to minimise interplot interference. Measurements of fresh weight per m2 and oil yield, converted to kg/ha, were made for each plot.

Disease incidence, severity, shoot defoliation, fresh weight and oil yields per treatment were compared using one-way analysis of variance, and differences were compared using Fisher’s least significance difference (P < 0.05). Disease severity data were transformed using a log10 transformation prior to analysis in order to satisfy the requirement for normality.

Results Bitertanol and tebuconazole were consistently the most effective fungicides and fluquinconazole was consistently the least effective in each trial (Table 4 and Figure 20), even though the level of disease was much higher in the first trial.

Environmental conditions in the first year (trial 1) were extremely favourable for disease development. Under the consequent high inoculum level, bitertanol significantly reduced the disease incidence, but in trial 2 with less inoculum potential, disease incidence was the same across all treatments (Table 4). Disease severity was significantly reduced by all of

45 the fungicides, except fluquinconazole, in both trials, with the most effective, bitertanol, reducing severity to approximately 5% (Figure 20).

Under conditions conducive to severe disease (trial 1), all fungicides reduced the level of shoot defoliation compared to the control, but under less disease pressure (trial 2) fluquinconazole was not effective. Once again, bitertanol was significantly better than all the rest over both years.

Fresh weights did not differ (P < 0.05) across treatments in trial 1, but there were significant differences between oil yields, with twice as much oil produced under the bitertanol and tebuconazole treatments than under the fluquinconazole treatment or the control. In trial 2, treatments with bitertanol and tebuconazole resulted in significantly higher yields both in terms of biomass and oil production. The other fungicides were not significantly better than the control in that year.

There was a clear negative correlation between the final level of disease severity on the upper side-shoots and the oil yield (Figure 20). The performance of the fungicides was fairly consistent for both years, although triadimenol was more effective and myclobutanil less effective when the conditions were conducive to severe disease (trial 1).

46 Table 4 Effects of a range of fungicides on fresh weight, oil yield, disease severity, incidence and shoot defoliation of Scotch spearmint infected with mint rust.

Fresh weight Oil Disease Disease Defoliation TRIAL 1 (1996/7) (mg) (kg/ha incidence severity (%) ) (%) (%) Control 2526a 49c 99.0a 66.9e 57.6d Bitertanol 3609a 104a 54.9b 5.3a 24.2a Triadimenol 3459a 86b 96.7a 51.2bc 38.6b Fluquinconazole 2485a 54c 97.9a 65.9e 49.4c Tebuconazole 3516a 100a 96.8a 27.1b 35.6b Myclobutanil 3057a 63bc 96.8a 67.9d 42.1bc Propiconazole 3414a 78ab 98.1a 56.1cd 38.8b

Fresh weight Oil Disease Disease Defoliation TRIAL 2 (1997/8) (mg) (kg/ha incidence severity (%) ) (%) (%) Control 1868bc 64b 59.5a 26.6d 31.0d Bitertanol 3294a 135a 56.7a 5.6a 1.1a Triadimenol 2016bc 64b 68.9a 18.3bc 15.5bc Fluquinconazole 1744c 59b 62.7a 24.7d 24.6d Tebuconazole 3248a 113a 60.3a 13.7b 6.4b Myclobutanil 2560ab 85b 63.9a 20.2c 19.7c Propiconazole 2512b 82b 61.1a 17.6c 11.8c Values in the same column sharing a common letter are not significantly different at P = 0.05.

47 120 70 Trial 1 (1996/7)

oil yield 60 100 disease severity

50 80

30 Oil (kg/ha)

40 20 Disease severity (% leaf area) 20 10

0 0 control fluquinconazole myclobutanil propiconazole triadimenol tebuconazole bitertanol

140 30 Trial 2 (1997/8)

120 oil yield 25 disease severity

100 20

60 Oil (kg/ha)

10 40

5 Disease severity (% leaf area) 20

0 0 control fluquinconazole myclobutanil propiconazole triadimenol tebuconazole bitertanol

Figure 20 Oil yields and final disease severity levels of Scotch spearmint infected with mint rust after different fungicide treatments. The data presented is the mean of the replicates.

48 Discussion The fungicide bitertanol gave the most effective control of mint rust and resulted in the highest oil yields of the six fungicides evaluated on Scotch spearmint at the Ovens Research Station. This was consistent over two seasons with different levels of disease in each. Bitertanol had shown potential in 1989/90 on peppermint and in 1990/91 on Scotch spearmint (Bienvenu 1992) and was nominated as a fungicide that should be investigated further. It has also been reported as effective against mint rust on peppermint and corn mint (Mentha arvensis) in Bulgaria (Margina and Zheljazkov 1994).

Tebuconazole was also effective, its use resulting in high levels of disease control and good oil yields, particularly under the high inoculum potentials of the first year. It was identified as a cheaper alternative to propiconazole for use on peppermint crops in Tasmania in 1995 (Warner and Bailey, unpublished data), and has been reported as effective on peppermint in the USA (Grey and Welty 1995).

The other two fungicides nominated by Bienvenu (1992), propiconazole and myclobutanil, were not as effective at controlling mint rust as expected. Propiconazole is the most commonly used fungicide on several mint species and is the currently used fungicide in both Victorian and Tasmanian peppermint crops. In this experiment, while it was more effective than the control, it was not as effective as the fungicides discussed above. Myclobutanil was also not very effective, which was surprising in view of it being the best fungicide in the 1990/91 screening trial (Bienvenu 1992) and was reported as being particularly effective against mint rust on peppermint in the USA (Grey and Welty 1995, 1997). In the present study, its effectiveness broke down in trial 1 when the environment was especially favourable for disease development.

The practice for controlling rust among north east Victorian peppermint growers is to rely on a single fungicide, and currently it is propiconazole. This practice carries the risk of developing a fungicide-resistant pathogen population (Skylakakis 1987). The results of this trial show that several readily-available fungicides are more effective at controlling mint rust on Scotch spearmint than propiconazole, with a corresponding increase in oil yield. For example, bitertanol reduced rust severity from 67% to 5% in the first year and from 27% to 6% in the second year, compared to propiconazole which reduced it from 67% to 56% in

49 year 1 and 27% to 18% in year 2. In addition, bitertanol increased oil yield by 112% and 111% respectively, whereas propiconazole increased it by only 57% and 28% respectively. In view of this, it could be recommended that growers use a combination of fungicides, alternating their use to reduce the likelihood of fungicide resistance arising in the local pathogen population. The fungicides identified in this study that could be used in such a manner were bitertanol and tebuconazole, with triadimenol in seasons particularly favourable to the development of the disease. Unfortunately, they are all demethylation- inhibiting (DMI) fungicides which inhibit C-14 sterol demethylation ie. they all have a similar mode of action, and changes in pathogen sensitivity to one may affect sensitivity to the others (Heaney 1988). In practice, however, the risk of resistance building up in response to DMI fungicides is considered moderate to low, as DMI fungicides have been widely used since the 1970s yet few cases of resistance have been clearly identified (Georgopoulos 1987, Scheinpflug 1988a, 1988b). Scheinpflug (1988b) recommends that to minimise the development of DMI fungicide resistance the following management strategies should be followed:

(1) DMI fungicides should not be used continuously throughout the growing season. The number of foliar applications should be limited to a maximum of four to eight sprays per season;

(2) a minimum of two to three consecutive (block) treatments is necessary to achieve the maximum performance benefit from DMI fungicides. If more than this is required, two or more blocks of two to four treatments is the preferred method.

Clearly, further screening trials should be undertaken to investigate fungicides with other modes of action. Incorporation of two or more fungicides from different chemical groups into the growers’ mint rust control programme should be promoted to ensure that their effectiveness is not broken down.

50 4. Variation in the Pathogen, Puccinia menthae.

Introduction Although outside the objectives of this project, a study was also included to investigate the extent of variation in the Puccinia menthae population present in Victoria. Samples of mint rust were collected from mint species growing in Melbourne and north east Victoria, and 18 single-spore isolates were established and maintained in the glasshouses at the University of Melbourne. These isolates of rust were compared on the basis of host range differences, teliospore morphology, and genetic differences as revealed by a molecular biology technique known as the random amplified polymorphic DNA (RAPD) assay.

Methodology Host range differences Host range differences between P. menthae isolates were examined in the glasshouse by inoculating 30 clones from 13 Mentha species, and recording the host reactions.

Inoculations were made by spraying sets of three-week-old rooted tip-cuttings with urediniospore suspensions (104 spores/ml) of each isolate and incubating in darkness at 100% humidity and 20°C for 24 hours. Inoculations were made only during April to October, when the glasshouse temperatures could be consistently maintained at 14/22°C, as temperatures above this could lead to unreliable symptom expression. Each isolate was tested on at least two separate occasions by inoculating sets of plants containing three cuttings per host clone. Each time, a control set inoculated with water and wetting agent only was maintained to check for contamination by foreign rust spores. Uredinia appeared on susceptible hosts after 10 to 12 days, and results were recorded after 18 to 20 days.

Host reactions were recorded on a scale of 0-4 based on that used by Baxter and Cummins (1953) and Fletcher (1963), and adapted by Beresford (1982), for P. menthae:

0 Immune to extreme resistance. No visible reaction, or small necrotic spots but no uredinia formed. 1 Highly resistant. Uredinia few, small, in necrotic spots most failing to rupture the epidermis.

51 2 Moderately resistant. Uredinia fairly abundant, some failing to rupture the epidermis; in necrotic spots. 3 Moderately susceptible. Uredinia abundant of moderate size, surrounded by chlorotic spots which later turn necrotic. 4 Highly susceptible. Uredinia abundant, large, with little or no chlorosis. Uredinia often produced on the upper leaf surface as well as on the lower leaf surface.

‘+’ was used to record reactions varying between two adjacent reaction types. Reaction types 0 to 2 were considered resistant reactions, and types 3 to 4 were considered susceptible reactions.

Teliospore morphology Teliospores were collected from four Mentha species in 1994, 1996 and 1997. Five collections were from M. x piperita, host of peppermint rust, and five were from M. spicata, M. x cordifolia and M. suaveolens Ehrh. (applemint), all hosts of spearmint rust. Teliospores were lifted from the sori with a fine needle, mounted in lactic acid and examined under bright field magnification (x1000).

From each collection, 50 mature teliospores that had broken off from their points of attachment were measured for the following characteristics:

- apical cell width and length, measured to the outside of the wall; - basal cell width and length, measured to the outside of the wall; - range of wall thickness, measured at the thickest and the thinnest parts; - septum thickness, measured at the centre of the septum; - pedicel length; - apical papilla thickness, measured from the centre of the pore in the spore wall to the top of the papilla; - apical papilla width at the base.

Scanning electron microscopy (SEM) was used to examine teliospore wall ornamentation. Fragments of leaves from M. x piperita cv. ‘Todd’s Mitcham’ and M. spicata cv. ‘Native’ on which teliospores were being produced were air-dried, mounted on an aluminium stub with a carbon sticky tape and sputter-coated with gold in an Edwards Sputter-coater 5150B.

52 The teliospores were then examined using a Philips XL30 FEG scanning electron microscope over a range of magnifications from x750 to x 3600.

RAPD analysis DNA was extracted using the adapted CTAB method of Taylor et al. (1995). 113 decamer oligonucleotide primers were tested for amplification of P. menthae genomic DNA.

Reaction volumes of 25 µl were optimised to contain 40 ng of DNA template, 0.8 units of Taq DNA polymerase (Boehringer Mannheim Biochemica, Germany), 0.24 mmol each of dATP, dCTP, dGTP and dTTP, 0.2 µM of primer, and PCR buffer with a final concentration of 0.01 M Tris-HCl / 3 mM MgCl2 / 0.05 M KCl / 0.1 mg/ml gelatin, pH 8.3 (Boehringer Mannheim Biochemica, Germany). PCR was performed with a PTC-100 thermocycler (MJ Research, Inc., USA) programmed as follows: initial denaturation step at 94°C for 1 min followed by 35 cycles of 94°C for 10 sec, 40°C for 30 sec and 72°C for 1 min, with a final extension at 72°C for 5 min. Amplification products were separated by electrophoresis on 1.4% agarose gel in TAE buffer, stained with ethidium bromide and visualised with UV illumination. The molecular weight marker used was Lambda-DNA digested with EcoRI and HindIII (Promega Inc., USA). The PCR with each primer was performed at least twice to check consistency of results.

PCR products from each DNA sample were scored as either present (1) or absent (0). Only distinct, reproducible bands were scored. Data analysis was conducted using SAS (SAS Institute 1996) to compute simple matching distances (Sneath and Sokal 1973). UPGMA cluster analysis was carried out using Proc Cluster in SAS Release 6.12 and a dendrogram was produced from the output of Proc Cluster using Proc Tree.

Results Host range differences The reactions of the 30 host genotypes to the rust isolates differed (Table 5). Some isolates produced only slight differences in infection types but others produced distinctly different infection types on several Mentha clones. Fifteen races were separated within the 18 isolates on the basis of resistant or susceptible reactions. There was a clear demarcation between ‘peppermint rust’ races and ‘spearmint rust’ races, according to the host from

53 which the isolate had originated. Among 12 isolates from M. x piperita and M. x piperita ssp. citrata, nine ‘peppermint rust’ races were differentiated, and the six isolates from M. x cordifolia, M. x gracilis and M. spicata each differentiated into separate ‘spearmint rust’ races.

54 Ta ble 5 Ho st rea ctions (0-4 scale) producedby18isolates o f Pucciniamenthae. Isolates f ol low e d bythe s ameletter a to missing data isolates 93-03 and 93-06 could not be conclusively differentiated and could be either a or b. Isolates of P. me nt hae [DNA sample] (host from which original urediniospore sample was collected) 93-08a 93-03ab 93-06ab 93-09b 95-02c 95-21c 93-01d 93-02e 93-05f 95-16g 95-17h 95-14i 93-12j [Mp-04] [Mp-01] [Mp-10] [Mp-05] [Mp-07] [Mp-09] [Mp-08] [Mp-11] [Mpc] [Ms-02] Hosts inoculated (M. x piperita) (M. x piperita (M.sp i s sp. cit rat a) M. aquatica 00001100000 00 M. arvensis 4 3+ 4 3+ 3 3 3+ 3+ 3+ 3 3 3+ 3 M. x cordifolia I00000000011 24 II----1--0-01+03 M. di emeni ca 00000000000 10 M. x gr aci li s I443+4444323+4 44 I I 3+ - - 3+ 4 3 4 3 3 3 3+ 4 4 III2- - 33342- 33 34 IV 0 - - 0 0 0 0 0 - 0 0 1 4 M. laxiflora 3- - 23303+-33 1+0 M. x maximilianea 0- - 00000000 00 M. x pi per it a I44444444444 41 II44444444444 41 III44444444444 41 IV44444444444 40 V44444444444 40 VI1+1+11+1201104 10 M. x pi per it a s sp. ci trat a I 3+ 3+ 3 3+ 1 1 2+ 4 2+ 2 1 2 0+ II 0 - - 0 1 0 0 1 - 1+ 0+ 0+ 1 III443411+2+2421 21 M. pulegium I21022+210133 2+0 II343+44444244 40 M. rubra raripila 1 1+ 1 1+ 1 2 0 1+ 0 1+ 0 0 0 M. spicata I10002110011 04 II11+212131122 03 III44444444443 44 M. spicata ssp. crispa 00100001000 34 M. suaveolens I00000000000 00 II 0 - - 0 0 0 0 0 - 0 0 0 0 M. xverticillata 0- - 0000000- -- Me li ssa of fi ci nali s 00000- - - 0- - -- Origanum vulgare 00000- - - 0- - -- Sat ur ej a mont ana 00000- - - 0- - -- - = no inoculation made; 0 = immune/extreme resistance; 1 = highly resistant; 2 = moderately resistant; 3 = moderately susceptibl e ; 4 = hig + = react ion varying bet we e n tha t shown and t he next hi gher react io n type.

55 M. x gracilis cv. ‘Scotch spearmint’ was susceptible to all isolates tested except 93-05. There were also differences between the reactions of weed relatives found growing in the region. Mentha x cordifolia was susceptible to ‘spearmint rust’ races, M. pulegium (II) was susceptible to ‘peppermint rust’ races and M. spicata (III) was susceptible to all the isolates tested.

Teliospore morphology

The teliospores could be separated into two distinct groups, peppermint rust and spearmint rust, according to the host they were taken from (Table 6). Peppermint rust teliospores have thicker cell walls and septa, longer pedicels and wider apical papillae than the spearmint rust teliospores. They also have two unequally-sized cells, the apical cell being larger and thicker-walled than the basal cell, while the spearmint rust teliospores have equally-sized cells with uniform wall thickness.

Table 6 Comparison of teliospore morphology of peppermint and spearmint rust.

Teliospore character Peppermint rust (µm) Spearmint rust (µm) P-value Range Mean Range Mean Width of spore 21 - 28 23.8 19 - 26 22.1 0.40 Length of spore 24 - 37 31.2 25 - 34 29.1 0.07 Wall thickness - Thickest part 1.4 - 3.2 2.4 0.9 - 2.8 1.8 0.00 - Thinnest part 0.5 - 1.8 1.0 0.5 - 1.4 0.7 0.00 Width of apical cell 20 - 28 23.6 17 - 25 21.3 0.01 Length of apical cell 14 - 23 18.0 12 - 18 15.6 0.00 Width of basal cell 16 - 26 21.2 18 - 25 21.4 0.82 Length of basal cell 6 - 16 12.8 10 - 18 13.7 0.18 Pedicel length 3 - 64 23.1 4 - 28 14.4 0.05 Septum thickness 0.5 – 2.0 1.1 0.5 – 0.9 0.7 0.00 Apical papilla - Width at base 8 – 13 10.4 8 – 11 9.7 0.05 - Thickness 2 - 5 3.5 3 - 5 3.6 0.59

56 Differences in teliospore wall ornamentation between the two rust groups were most apparent under SEM (Figures 21 and 22). The spearmint rust teliospores are uniformly verrucose, while the peppermint rust teliospores have smooth basal cells and mostly smooth apical cells.

Figure 21 Wall ornamentation of peppermint rust teliospores revealed by SEM (bar=10µm).

Figure 22 Wall ornamentation of spearmint rust teliospores revealed by SEM (bar=10µm).

RAPD analysis

Six out of 113 primers amplified distinct and reproducible marker profiles from the 18 P. menthae DNA samples. Each primer amplified between nine and 18 scoreable DNA markers, which ranged in size from 100 bp to 3000 bp. In total, 83 bands were scored, of which 58 were polymorphic (70%).

The dendrogram constructed using the UPGMA algorithm (Figure 23) clearly differentiated two non-overlapping clusters. One cluster contained the isolates from M. x piperita and the other, those from M. spicata, M. x gracilis and M. x cordifolia.

Mp -0 1

Mp -0 4 Mp -0 5

Mp -0 7

Mp -0 8 Mp -1 0

Mp -1 1

Mp c Peppermint rust Mp -0 9

Mg Ms -02

Mc-03 Mc-02

Mc-01 Spearmint rust Ms -01

0.3 2 0.24 0. 16 0. 08 0 .0 0

Figure 23 Dendrogram constructed using UPGMA cluster analysis from simple matching distances (Sneath and Sokal 1973), using RAPD data from 15 P. menthae isolates. Isolates Mp-01 to Mp-11 were from M. x piperita, Mpc was from M. x

58 piperita ssp. citrata, Mg was from M. x gracilis, Ms-01 and Ms-02 were from M. spicata, and Mc-01 to Mc-03 were from M. x cordifolia.

Discussion

Fifteen physiologic races were identified from 18 isolates of mint rust examined in this study, indicating the existence of a great deal of variation present within mint rust in Australia. The lack of internationally accepted differential host clones precludes detailed comparisons with overseas races, but certain comparisons can be made with the results of workers in other countries. In France, Cruchet (1907, in Beresford 1982) found eight races from 10 collections of P. menthae; Niederhauser (1945) identified six races out of six isolates from the north east USA; in the north western states of the USA, Baxter and Cummins (1953) found 15 races from 22 isolates and Johnson (1995) differentiated 11 races among 17 collections; Fletcher found nine races in 15 collections from around England, and Beresford (1982) identified three races in New Zealand. The 15 races found in the present study can constitute two distinct groups dependent upon the Mentha species from which they originated. This was expected, as so-called ‘peppermint rust’ and ‘spearmint rust’ have been reported as distinct on numerous occasions in the literature. Of particular interest in this case, however, was that this separation was also clearly evident at the morphological and molecular levels.

Two distinct groups were revealed by RAPD analysis, one containing the isolates from M. x piperita, and the other, isolates from M. spicata, M. x gracilis and M. x cordifolia. The two groups corresponded to peppermint and spearmint rusts as mentioned previously. The distinct clustering of the two rust types revealed by the RAPD profiles, with no evidence of intermediates, is a strong indication that the two groups are genetically isolated.

The two rust groups are also clearly different based on the more traditional comparison of teliospore morphology. Although the range of values for any one trait may overlap, the means are usually different, and when all the traits are considered together the two groups are quite distinct. As presently defined, P. menthae encompasses many varieties and forms based on teliospore morphology and host range. Sydow (1904, cited in Fletcher 1963) and Grove (1913) considered P. menthae to be a collective species and proposed division into several independent species on the basis of such morphological differences as the presence

59 or absence of warts on the teliospores, variations in the apical papillae, and lengths of the pedicels. The present study shows significant differences between the two groups in each of these teliospore characters. The spearmint rust teliospores described in the present study fit within the description of P. menthae var. menthae (Baxter 1959), but the peppermint rust teliospores described do not.

Differences in host range alone generally characterise formae speciales, whereas morphological differences can indicate varietal distinction. Until now, the morphological differences observed within P. menthae have not been deemed sufficient to warrant further separation of the species. With the addition of the molecular data, there is now a strong case for some degree of taxonomic separation of these two rusts, but the question posed is at what taxonomic level? In view of the consistent pattern of difference in the suite of characters ie. RAPD profile, teliospore morphology and host specificity, in the mint rusts of Victoria, it appears that peppermint rust deserves at least separate varietal, or specific, rank from spearmint rust. If the biological definition of a species is genetic isolation, then the molecular data points in that direction. Given the insights now possible using molecular- based techniques, it appears that the taxonomy of P. menthae as a whole should be re- examined.

60 Conclusions

At the outset of the present investigation, there was little known about the behaviour of mint rust in south eastern Australia, about the extent of variation within Puccinia menthae in the region, or about the effectiveness of the various control options either already being adopted or available but untested. The objectives of the present investigation were to determine and demonstrate an effective strategy for the control of peppermint rust based on minimal use of chemical sprays, to identify fungicides which will effectively control Puccinia menthae without contamination of distilled oil, and to conduct detailed studies of the epidemiology of mint rust eg. life cycle and the influence of temperature and humidity on its capacity for growth and reproduction. To a large extent these objectives have been met.

Results of a four-year study showed the full life cycle of the rust does not occur on peppermint grown locally. No spermogonia or aecia were ever seen on peppermint where urediniospores persisted all year round. In view of this, considerable emphasis was placed on the effects of environmental factors on components of the uredinial cycle of P. menthae on peppermint. Under artificial conditions, urediniospore germination required at least six hours leaf wetness and lasted four to six hours at temperatures of 5-25°C. Appressoria, substomatal vesicles and haustoria were produced six, six and 24 hours after inoculation, respectively, at 20°C. The latent period of infection ranged from 50 days at 5°C to 10 days at 22°C. Sporulation occurred over a wide range of temperatures (5-27°C), the optimum being 15-20°C. The minimum, optimum and maximum temperatures for infection were <5, 20 and 27°C respectively.

Infection with mint rust substantially reduced peppermint growth and oil yield. Leaf loss was significantly increased, which as a consequence significantly reduced leaf area, leaf fresh weight and oil content per plant. Apparent retention of synthate by the infection courts was associated with significantly reduced stem and root dry weight and numbers of stolons. The results suggested that infection with mint rust was likely to reduce the persistence of peppermint as a perennial crop, which was borne out by the death of the

61 seedbed plot of peppermint after three and a half years of continuous rust infection. Under disease-free conditions such beds would be expected to persist indefinitely.

Field studies of rust on peppermint crops showed that during summer, the level of disease was influenced considerably by temperature. Whenever the daily maximum temperature reached 35°C, the level of disease in crops fell within a few days. This correlated with the results from the controlled environment experiments on the urediniospore cycle, where it was seen that high temperatures adversely affected all stages of the process.

There has been recent interest in the disease incidence - severity relationship of several rust diseases (Seem 1984). This relationship was quantified for common maize rust as an exponential curve (Dillard and Seem 1990a). The point of inflection in the curve was determined to indicate a critical level of disease incidence, above which disease severity increased rapidly. It was considered that when disease incidence in the maize crop reached this critical level (indicated by the point of inflection in the incidence - severity curve), chemical control should be initiated. This ‘action threshold’ was recommended to maize growers, who were then able to successfully control common maize rust under field conditions (Dillard and Seem 1990b). The incidence - severity relationship for mint rust was examined in the present study where it was found that it could be described by a single equation for both field- and pot-grown peppermint. The relationship was exponential, the point of inflection in the curve occurring when disease incidence reached 60%. This information provides a basis for developing an action threshold for determining the most propitious time to apply chemical sprays in Victoria, as was described for common maize rust in the USA (Dillard and Seem loc. cit.).

The only mint rust control measure currently employed by local peppermint growers is spraying with the fungicide propiconazole. Spraying is begun when growers perceive that they have a rust problem, which is usually late in November. They know that they cannot use the fungicide within 14 days of harvest because of the risk of oil contamination, so that peppermint crops usually receive three applications of fungicide separated by 14 days, each at the recommended rate of 500 ml/ha as ‘Tilt 250EC’, during November and December each year. The investigation of the effectiveness of propiconazole in controlling mint rust demonstrated that if propiconazole treatment was applied before the disease incidence

62 exceeded 60%, rust was eradicated from the crop, but if applied after the incidence had exceeded 60%, the level of rust was reduced, but it was not eradicated. This confirmed the importance and value of 60% disease incidence as an action threshold point for initiating control programs.

Effective control of mint rust on peppermint crops in the USA depends on breaking the life cycle of Puccinia menthae at the aecial stage in early spring. This is commonly achieved by burning the new mint growth with a tractor-drawn propane-gas flamer which destroys any aecia present and stimulates a second flush of growth into a subsequently rust-free field. This method of control was investigated for use in north east Victoria, but was found to cause a severe setback to the spring growth of the peppermint, resulting in reduced oil yield and quality. The study of the disease cycle also showed that no aecia are produced on peppermint growing in north east Victoria, which meant that there was no point in targeting this stage of the life cycle for control purposes.

Flaming the plots in autumn, however, resulted in high yields of peppermint oil in the following growing season. It is evident from this that if peppermint is allowed to overwinter free from pests and disease, healthy regrowth results in the subsequent spring. Unfortunately it is not possible to recommend autumn flaming to growers in north east Victoria because of the risk of bushfire which has resulted in legal constraints being placed on gas transportation in addition to legislated fire restrictions.

The use of a contact desiccant herbicide, paraquat, as an early autumn treatment for controlling the disease was investigated but, although it reduced the viability of urediniospores on the treated peppermint foliage, the shoots recovered and produced new leaves while many urediniospores were still viable on the dead foliage. It was concluded, therefore, that the use of paraquat in autumn was not an effective rust control measure. Many growers, however, use paraquat in late autumn to ‘clean’ the fields of peppermint regrowth and weeds prior to winter. This practice would certainly reduce the inoculum load carried over winter and should therefore slow the onset of rust in the following season.

Six fungicides, namely bitertanol, fluquinconazole, myclobutanil, propiconazole, tebuconazole and triadimenol, were compared for effectiveness at controlling mint rust on Scotch spearmint. Two of them, bitertanol and tebuconazole, proved particularly effective

63 against the rust. Both are readily available and cheaper than propiconazole, the currently used fungicide. Although it is evident that their immediate use in preference to propiconazole is warranted, it is unfortunate that they are both demethylation-inhibiting (DMI) compounds, as is propiconazole. Continued use of them poses a slight risk that fungicide-resistant strains of mint rust may evolve in time (Heaney 1988). It is important, therefore, that fungicidal compounds with different chemical modes of action are also evaluated for their effectiveness against mint rust and incorporated into a rotational spray regime.

Isolates of mint rust collected from four different Mentha species from different regions of Victoria allowed comparisons to be made of host range, morphology and molecular differences in the rust. Isolates clearly differentiated into two distinct groups, ‘peppermint rust’ and ‘spearmint rust’, according to the host species from which they had been collected. Two groups of mint rust have often been mentioned in the literature, but are both widely considered to belong to one variety of rust, P. menthae var. menthae described by Baxter (1952, 1959), which also contains the type specimen for the species. The evidence presented here strongly suggests that the two groups in fact represent separate species of rust. While there is overlap of the host ranges on a common host, M. x gracilis cv. ‘Scotch spearmint’, for the most part they occur on separate groups of hosts. Even on the common hosts they behave differently and at the molecular level, there is little evidence of gene flow between them. The two groups exhibit different disease cycles under the same field conditions, with spearmint rust undergoing annual sexual reproduction while peppermint rust remains asexual. In addition to these differences, teliospore morphology is consistently distinct between the two groups.

The taxonomic status of P. menthae has been controversial for many years. Several species were initially described, and subsequently incorporated into the one species, P. menthae, making it a species with one of the widest host ranges in the Uredinales. In the 1950s, Baxter (1952, 1959, 1960) subdivided the species into varieties on the basis of host range differences and teliospore morphology. The teliospore morphology of the spearmint rust described herein is consistent with Baxter’s description of P. menthae var. menthae, but the morphology of the peppermint rust teliospore is outside any existing descriptions, and has

64 only been noted in passing by Walker and Conroy (1969). In view of this, it appears that peppermint rust should be separated from P. menthae var. menthae, but should it be regarded as a new variety of P. menthae or as a new species altogether? A comparison of Victorian isolates of peppermint rust with isolates collected from peppermint grown in regions where the full life cycle occurs, such as in the USA and the UK, would be desirable before a decision is made.

65 Bibliography

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