Myrtle Rust Screening in Lemon Myrtle Provenance Plantings

by D. Lee, J. Doran, G. Pegg, D. Lea, P. Macdonell and F. Giblin

September 2016

RIRDC Publication No 16/012 RIRDC Project No. PRJ-008883

ISBN 978-1-74254-858-6 ISSN 1440-6845

Myrtle Rust Screening in Lemon Myrtle Provenance Plantings Publication No. 16/012 Project No. PRJ-008883

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Researchers Contact Details

Associate Prof David Lee Dr John Doran Forest Industries Research Centre 8 Ferrier Place University of the Sunshine Coast Kambah ACT 2902 Locked Bag 4, Maroochydore DC, QLD 4558 Phone: 0262962428 Phone: 07 54565759 Email: [email protected] Fax: 07 5430 2887 Email: [email protected] Dr Fiona Giblin Dr Geoff Pegg Forest Industries Research Centre Department of Agriculture and Fisheries University of the Sunshine Coast, GPO Box 46, Brisbane QLD 4001 Locked Bag 4, Maroochydore DC, QLD 4558

Phone: 07 3255 4381 Phone: 0438 043 891 Email: [email protected] Email: [email protected]

In submitting this report, the researcher has agreed to RIRDC publishing this material in its edited form.

RIRDC Contact Details

Rural Industries Research and Development Corporation Level 2, 15 National Circuit BARTON ACT 2600

PO Box 4776 KINGSTON ACT 2604

Phone: 02 6271 4100 Fax: 02 6271 4199 Email: [email protected]. Web: http://www.rirdc.gov.au

Electronically published by RIRDC in September 2016 Print-on-demand by Union Offset Printing, Canberra at www.rirdc.gov.au or phone 1300 634 313

Foreword

The Backhousia citriodora (lemon myrtle) industry is in jeopardy due to the impact of the recently introduced rust fungus Puccinia psidii, commonly known as myrtle rust.

This research indicates that the entire genetic base of lemon myrtle is susceptible to myrtle rust but there are opportunities for the industry to select and plant cultivars that have greater tolerance to the disease than the currently used commercial varieties. With this in mind the lemon myrtle gene-pool planting at Beerburrum which is at risk from development has been saved by re-establishing it on two sites, one on a commercial lemon myrtle property in northern New South Wales near Dunoon and the second on crown land near Traveston, Queensland. In addition, the genetic base of the species has been broadened by new collections from the wild at Woondum and Ubobo in Queensland.

The findings and recommendations in this report will help to inform RIRDC as it plans its future research on myrtle rust control and mitigation in the lemon myrtle industry.

This report is an addition to RIRDC’s diverse range of over 2100 research publications and it forms part of our New and Emerging Plant Industries R&D program, which aims to facilitate the development of new rural industries based on plants or plant products that have commercial potential for Australia.

Most of RIRDC’s publications are available for viewing, free downloading or purchasing online at www.rirdc.gov.au. Purchases can also be made by phoning 1300 634 313.

John Harvey Managing Director Rural Industries Research and Development Corporation

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Acknowledgments

Thanks are due to Alan House, Grant White and other former staff of the Queensland Forestry Research Institute, Gympie who were involved in establishing the Beerburrum Backhousia gene bank planting. Stephen Midgley former leader of the research program in CSIRO Forestry and Forest Products including the Australian Tree Seed Centre approved the funding that supported the initial range wide seed collections of the species. Reg Lockyer and his wife are thanked for identifying the interesting Silver Valley population and striking cuttings of the ten clones included in the gene bank. We acknowledge the great contribution of Gary Mazzorana for his support and assistance with the plantings in New South Wales. We also thank Tracey Menzies, David Osborne, John Oostenbrink and Anthony Burridge for their work on the propagation and management of the trials. Bruce Hogg of the Department of Agriculture and Forestry (Queensland) is thanked for his critical review of this report.

Abbreviations

ARP Australian Rainforest Products

ATSC Australian Tree Seed Centre of CSIRO National Research Collections Australia (NRCA)

CSIRO Commonwealth Scientific and Industrial Research Organisation

DAF Department of Agriculture and Fisheries

Is Island

MD Missing Data

N-P-K nitrogen (N), phosphorus (P), potassium (K)

NSW New South Wales

QLD Queensland

Qld DAF Department of Agriculture and Fisheries, Queensland

USC University of the Sunshine Coast

Contents

Foreword ...... iii Acknowledgments ...... iv Abbreviations ...... iv Executive Summary...... vii Introduction ...... 1 Objectives ...... 3 Methodology ...... 4 Existing material ...... 4 Establishment of new gene bank plantings of lemon myrtle ...... 7 Glasshouse myrtle rust screening ...... 9 Wild material collection ...... 10 Results ...... 12 Capture of existing germplasm ...... 12 Rust Resistance Screening ...... 16 Collection of Wild Material ...... 20 Discussion ...... 22 Implications ...... 24 Recommendations...... 25 Appendix 1. Trial 1 (Dunoon Site) ...... 26 Appendix 2. Trial 2 (Traveston Site) ...... 27 Appendix 3. Trial 3 (Dunoon Site) ...... 28 Appendix 4. Susceptibility of clones to myrtle rust ...... 29 References ...... 37

Tables

Table 1 Details of the genetic material securely captured in new field trials (gene bank plantings) from the existing gene bank at Beerburrum...... 13

Table 2 Backhousia citriodora provenances evaluated for Puccinia psidii resistance (means of all clones) in the glasshouse trial in order of most to least susceptible, based on disease incidence...... 17

Table 5 Susceptibility of individual clones of Backhousia citriodora to Puccinia psidii ranked most to least susceptible within the provenance in the glasshouse trial...... 29

Figures

Figure 1 Juvenile tip cuttings of the Backhousia citriodora clones set in the University of the Sunshine Coast glasshouse...... 6

Figure 2 View of the two Backhousia citrodora gene conservation plantings...... 8

Figure 3 Location of Backhousia citriodora in Woondum National Park...... 14

Figure 4 Location of Backhousia citriodora along Koolkurrum Creek near Ubobo...... 15

Figure 5 Sowing of Backhousia citriodora seed and germination in the USC glasshouse...... 21

Figure 6 Root coppice under Backhousia citriodora plants along Koolkurrum Creek...... 21

Executive Summary

What the report is about

This report documents research activities undertaken to reduce the risks and costs of myrtle rust on the lemon myrtle industry. This was achieved by:

 Capturing the genetic material from the Backhousia citriodora (lemon myrtle) gene bank planting at Beerburrum in southern QLD (currently on land proposed for future development) and the subsequent out planting of this material at more secure sites in southern QLD and northern NSW.

 Screening B. citriodora for susceptibility to Puccinia psidii (myrtle rust). This follows on from research by Doran et al. (2012) which indicated that it may be possible to select cultivars of B. citriodora with greater myrtle rust resistance than the moderately to highly susceptible cultivars that are currently in use by the Native Food, Essential Oil and Horticultural Industries in Australia. Based on Doran et al (2012) research, cultivars were selected, propagated and screened for myrtle rust susceptibility under both glasshouse and field conditions. All material was susceptible to attack from myrtle rust.

Who is the report targeted at?

This report is targeted at the Australian Native Food, Essential Oil and Horticultural Industries that utilise B. citriodora as a source of citral-rich leaves and as an ornamental plant.

Where are the relevant industries located in Australia?

The Australian Native Food Industry is the industry most likely to benefit most from this research. Central QLD and northern NSW are the main locations where lemon myrtle leaf is produced. The largest planting (1.2 million plants) is at Palm Grove near Proserpine in central QLD complemented by multiple smaller plantings (± 10 000 plants) in northern NSW and south-eastern QLD (RIRDC 2012). Accurate production figures are unavailable; however, RIRDC (2012) estimates a total farm gate value at between $7 million and $23 million for dried leaf and essential oil.

Background

Lemon myrtle is one of several native food species under threat from P. psidii (commonly known as myrtle rust), a well-known rust fungus of international importance first found in Australia in April 2010. Most current commercial plantings of lemon myrtle are based on two main Limpinwood varieties (clones) which are proving to be moderately to highly susceptible to the disease. Myrtle rust attacks young leaves and shoots sprouting after harvesting creating unsightly lesions that produce masses of yellow spores. The long-term impact of myrtle rust on the quantity and quality of lemon myrtle dried leaf supply is not yet known but it could jeopardise the industry.

Aims/objectives

The principal aim of the project was to capture and relocate clonally the majority of the existing ex- situ gene bank planting of B. citriodora at the former Beerburrum Nursery (61 families from 11 provenances) to two secure sites in QLD and NSW.

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Secondary aims were to:

 assess for rust resistance at a commercial lemon myrtle industry site the most rust resistant individuals observed in the ex-situ gene bank population of B. citriodora at Beerburrum. This work had the objective of identifying rust tolerant clones for immediate deployment by industry in the place of the rust susceptible clones currently in use.

 test the rooted clones against different isolates of myrtle rust to validate the field observations of plant susceptibility and tolerance.

 expand the gene bank by introducing new families of B. citriodora (seedlots) from Woondum and Ubobo. In assessment of the planting at Beerburrum, (Doran et al. 2012) the Woondum provenance had the most myrtle rust tolerant individuals of the provenances initially evaluated and it is considered important that this provenance be more widely sampled. The Ubobo provenance was not represented in the Beerburrum gene bank planting, so it was not included in the original analysis of growth and rust scores and warranted further sampling.

Methods used

Germplasm capture and trial establishment

Juvenile tip cuttings from 437 B. citriodora clones representing the entire genetic diversity in the Beerburrum planting from all 11 provenances were set in the glasshouse at the University of the Sunshine Coast during September 2013. All cutting material was collected using secateurs, then placed in labelled plastic bags prior to chilling and transported to the glasshouse for setting. Cuttings were dipped in 3% IBA gel and then set in labelled 93 cc hiko trays with crack pot inserts in potting medium consisting of 75% coarse perlite and 25% composted pine bark fines The cuttings were then placed in a temperature controlled glasshouse under a misting watering environment.

The recalcitrate rooting clones (with less than 50% rooting) from the September 2013 setting were re- set during February 2014 to ensure that a wide genetic base of the Beerburrum material was captured. Methodology for setting of cuttings in February 2014 was the same as that used in the September 2013 setting. At the same time 120 cuttings each of the twenty most rust resistant clones identified by Doran et al. (2012) were also set to facilitate establishment of a replicated block trial.

Once rooting occurred in the glasshouse environment, the clones were moved to an external stand down area under shade cloth with an overhead watering system. Shade was removed several weeks prior to planting to harden the plants. In total 433 clones were captured as rooted cuttings of the 437 attempted and 418 clones had enough ramets (minimum of three) to allow them to be planted in at least one field trial. Three field trials were planted during the project. Two of these trials served as ex- situ conservation stands for B. citriodora. The first of these trials was established in northern NSW site on land owned by Australian Rainforest Products at Dunoon and the second trial was planted on land owned by the Queensland Government at Traveston in southern QLD. The third trial was a replicated block planting of B. citriodora planted in January 2015 on land owned by Australian Rainforest Products at Dunoon in northern NSW.

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Glasshouse myrtle rust screening

Rooted cuttings from the range of clonal material set at the USC glasshouse were transferred to the Department of Agriculture and Fisheries glasshouse to undergo extensive testing for resistance to myrtle rust. The plants were inoculated with myrtle rust spores suspended in distilled water and ‘Tween 20’. Immediately after inoculation, the clones were covered with a plastic sheet for 24 hours to maintain high humidity levels and leaf wetness in a controlled environment room set between 18 and 20oC in the dark. After 24 hours plastic sheeting was removed and plants grown in a shade-house and hand watered as required. Disease symptom progression was monitored daily and assessed 12 to 14 days after inoculation using a five category disease rating score on new shoots and expanding leaves. In addition the percent leaf area with pustules (sori) was visually assessed to indicate the severity of the infection.

Results/key findings

The principal aim of the project has been met, with the recapture of the existing gene bank population of B. citriodora from Beerburrum (61 families from 11 provenances) and the establishment of two new gene banks (trials) to ensure the industry had secured the germplasm. One of these trials was established in NSW (Dunoon site, planted May 2014) and the other in southern QLD (Traveston site, planted February 2015). Across these two trials 418 clones, comprising 58 families from 10 provenances, the ten clones from the Silver Valley provenance and 18 unknown clones from unlabelled rows in the Beerburrum gene bank, had sufficient numbers of explants (ramets) to allow them to be established in the gene banks. In addition, a replicated block planting consisting of 20 clones and a commercial control was established at Dunoon (planted January 2015).

P. psidii resistance screening in the glasshouse showed significant differences between provenances for the disease rating score, with all provenances tested being susceptible to the disease. The other method used to assess the severity of the disease was to assess the percentage of leaves affected by the disease. This method again indicated that there were significant differences between provenances: range from 15.9% for the Silver Valley clones to 35.3% for the Cathu clones. Disease incidence was also significantly different at the family level with a wide variation in disease incidence between families. The lowest incidence of leaf infection was recorded at 7.5% for family 1465 from the Woondum provenance, while the highest was 60% for family 1381 from Carlisle Island.

Provenance mean data evaluated for P. psidii resistance in the field trials also indicated significant differences between provenances for both the disease rating and the disease incidence assessments, with a similar trend to that observed in the glasshouse trial with the unknown clones (those without any family or provenance information in the Beerburrum gene bank), Woondum and Silver Valley having the lowest (best) disease incidence and disease severity ratings.

The final aim of the project has also been met with seed collected from 14 trees (12 trees in the Woondum National Park and two trees in the Koolkurrum Creek / Dawes National Park area). This seed was sown at the USC glasshouse and germination is now occurring. In addition, cuttings were collected from root coppice of 19 trees found along Koolkurrum Creek near Dawes National Park. These cuttings were set in the USC glasshouse and at least one cutting has survived for all of the plants so we have successfully broadened the genetic base of the species. All of this new material will be planted in beside the ex-situ conservation stand at Traveston.

This is the first time that a commercial Limpinwood clone has been evaluated for susceptibility to myrtle rust in a trial along with other sources of B. citriodora. On a provenance basis it ranked as the fifth best based on disease incidence and equal worst for disease severity. Based on this finding it would appear that the lemon myrtle industry could reduce the impact of myrtle rust by moving to more tolerant provenances, families or varieties of B. citriodora identified in this study.

Implications for relevant stakeholders

If the lemon myrtle industry is to survive and strive to be organic, then the use of rust resistant / tolerant cultivars is essential. Similarly use of myrtle rust tolerant or resistant varieties would be beneficial for any industry growing myrtaceous species including the plantation sector growing eucalypts, the essential oil industry and nursery trade. All industries impacted by myrtle rust will need on-going disease screening and breeding programs based on broad genetic bases to combat the current incursion of myrtle rust and to prepare for incursions of potentially more virulent strains of the disease.

This study has shown that no clone of lemon myrtle found thus far is resistant to the disease, however, there are clones that appear more tolerant to the disease. If these clones are reasonably productive and they have suitable oil profiles, they could offer the industry an immediate lift relative to the current Limpinwood varieties that are moderately susceptible to the disease.

Recommendations

It is recommended that in a follow-on project:

 Continued screening of the field plantings should be undertaken to confirm the results from the glasshouse study that varieties could be selected that show higher levels of tolerance to the disease than the current commercial clones. This screening should include oil profile screening of varieties before they are made available to the industry for commercialisation;

 That the possibility of hybridising B. citriodora with other less susceptible Backhousia species should be investigated to see if it would be possible to breed myrtle rust resistant hybrid varieties;

 That the industry could consider moving to drier irrigated environments where the impact of the disease should be lower. Trials in these lower rainfall environments should include material from all provenances to ensure adapted varieties are identified.

Introduction

Backhousia citriodora F. Muell. (Myrtaceae) (lemon myrtle) is a small to medium sized rainforest tree that occurs in several disjunct populations in tropical and subtropical eastern QLD. Altitude varies from 40m near Eumundi in the south to 700m at Lead Creek in Silver Valley near Ravenshoe in far north QLD. The most southerly reported (in 1931) occurrence of the species from North Pine River near Brisbane can no longer be located. While the species occurs over a wide geographical area, populations are very small and disjunct. Clearing for settlement and agriculture has significantly affected the size and occurrence of the species, particularly in the Brisbane/Sunshine Coast region, where several populations have disappeared or are locally-threatened.

The lemon-scented leaves of B. citriodora and its snowy-white flowers are the basis of the commercial interest in the species. The leaves are either dried as a spice for culinary applications and flavouring for beverages, or steam distilled to extract an essential oil for medical, cosmetic and aromatherapy purposes. The Australian lemon myrtle industry is the industry likely to benefit most from this research. Central QLD and northern NSW are the main locations where lemon myrtle leaf is produced. The largest planting (1.2 million plants) is at Palm Grove near Proserpine in central QLD complemented by multiple smaller plantings (± 10 000 plants) in northern NSW and south-eastern QLD (RIRDC 2012). Accurate production figures are unavailable. RIRDC (2012) estimates a total farm gate value range from $7 million to $23 million for dried leaf and essential oil.

Lemon myrtle is one of several native food species under threat from P. psidii (commonly known as myrtle rust), a well-known rust fungus of international importance first found in Australia in April 2010. This disease is also commonly referred to as guava or eucalyptus rust. Most current commercial plantings of lemon myrtle are based on two main Limpinwood varieties (clones) which are proving to be moderately to highly susceptible to the disease. Myrtle rust attacks young leaves and shoots sprouting after harvesting creating unsightly lesions that produce masses of yellow spores. These leaves become deformed, with leaf- and shoot- die back occurring in the most susceptible plants (Pegg et al. 2014a). The long-term impact of myrtle rust on the quantity and quality of lemon myrtle dried leaf supply is not yet known but it could jeopardise the industry. Before the disease reached Australia, most major producers were organically certified so the use of chemical fungicides to control the disease was problematic. Since the rust incursion all growers have to use chemical fungicides and hence they have lost their organic status.

In 1995, CSIRO and DAF (formerly Queensland Forestry Research Institute) commenced the establishment of an ex-situ conservation and breeding population of B. citriodora in the grounds of the Beerburrum Nursery. The January 1995 planting comprised 16 open-pollinated families of four south- eastern QLD provenances (Eumundi, Maroochy, Noosa and Woondum). Later (August and December 1995 and October 1997) the ex-situ conservation/breeding population was extended with plantings of representatives of all known populations of the species, including families from populations in central and northern QLD including the Whitsunday Islands and an unusual, stunted form of the species from Lead Creek in Silver Valley near Ravenshoe. Intensive studies of the 1995 trials during 1996 and 1997 produced the first estimates of genetic parameters for commercial traits (Chen 1997). Recent research undertaken in the Beerburrum gene bank (Doran et al. 2012) has shown variation in myrtle rust incidence and severity amongst the diverse range of germplasm present in the planting. Some individual plants of one, open pollinated family from Woondum in southern QLD appeared resistant to attack when assessed in April 2012. There appeared from these preliminary findings to be an opportunity to reduce the impact of myrtle rust on the lemon myrtle industry by replacing susceptible cultivars with rust tolerant cultivars, although the authors have stressed caution until further trials have been undertaken on multiple sites of the most resistant lines.

The valuable collection of B. citriodora germplasm at Beerburrum serves, therefore, as a gene bank for the preservation of a locally-threatened species and provides a genetic base for industry selection and breeding activities aimed at addressing the impacts of myrtle rust on the Australian lemon myrtle

industry. However, planned realignment of roads and railway lines in the vicinity of the B. citriodora gene bank by the Queensland government means that the gene bank will be cleared in the not too distant future with significant implications in terms of conservation of the species and its utilization. This process has been initiated and the Beerburrum Nursery is now closed and dismantled in expectation of this development.

The obvious solution is to move the gene bank to secure sites. This would be best achieved by clonally propagating up to 10 individuals from each family across the 11 provenances at Beerburrum and re- establishing them on government and / or Native Food Industry sites as proposed in this project. This activity also provides the opportunity to expand the genetic resources in the gene bank by introduction of new families from two poorly sampled provenances (Woondum and Ubobo) and further screening of the most rust-resistant genotypes identified in the April 2012 assessment (Doran et al. 2012) of the Beerburrum gene bank. All of the lemon myrtle trees captured as clones will be screened in the glasshouse with myrtle rust isolates, to ensure that we are detecting tolerance or resistance rather than escapes in the field.

Objectives

The principal aim of the project was to:

(a) capture and relocate clonally the majority of the existing ex-situ conservation/breeding population of B. citriodora at Beerburrum Nursery (61 families from 11 provenances) to secure sites (government and industry) in QLD and NSW. This will ensure that this valuable array of germplasm will not be lost to efforts to conserve the species and develop the best possible myrtle-rust tolerant clones as a basis for the lemon myrtle and essential oil industries.

Secondary aims were to:

(b) assess for rust resistance / tolerance at a commercial lemon myrtle industry site the most rust resistant individuals observed in the ex-situ conservation/breeding population of B. citriodora at Beerburrum. This work has the objective of identifying rust tolerant clones for immediate deployment by industry in the place of the rust susceptible clones currently in use.

(c) screen the rooted clones against myrtle rust isolates in the glasshouse to validate the field observations of plant susceptibility and tolerance observed by Doran et al. (2012).

(d) expand the gene bank by introducing new families of B. citriodora (seedlots) from Woondum and Ubobo. In assessment of the planting at Beerburrum, the Woondum provenance had the most myrtle rust tolerant individuals of the provenances evaluated and it is considered important that this provenance be more widely sampled. The Ubobo provenance was poorly represented in the Beerburrum planting, so it was not included in the analysis of growth and rust scores but again it warrants further sampling.

Methodology

The methods detailed in this report will be treated in two sections (1) Existing material – work pertaining to the re-capture, relocation and testing of existing B. citriodora material from the existing gene bank planting at Beerburrum (objectives a, b and c) and (2) Wild material – the capture of new B. citriodora material from natural sources at Woondum and Ubobo (objective d). Existing material

Plant Materials

The plant material used for this research was from a gene bank planting of B. citriodora provenances, families and clones established near Beerburrum in south-eastern QLD in 1995–96 by CSIRO Forestry and Forest Products (now part of CSIRO NRCA) and Queensland Forestry Research Institute (now part of Qld DAF). Maps of this planting are given in a previous RIRDC Publication No. 12/098 (Doran et al. 2012). The B. citriodora gene bank planting consists of plant material propagated mainly from seed, but also comprising ten clones from one remote population in Silver Valley. Most of the known natural range (16 families from four south eastern QLD provenances [521 plants surviving], forty-one families from six northern QLD provenances [229 plants surviving] and ten clones [175 ramets surviving] from one far north QLD population) of the species is represented in the trial. Seven rows (rows 7 to 13: 567 plants surviving) and plots of 4 families (14 plants surviving) include plants that are not currently identified by either family or provenance (called ‘unknown clones’ in this report). The geographic location of the provenances represented in the Beerburrum planting (Doran et al. 2012) ranges from Maroochy in the south to Silver Valley in the north and includes families from two Whitsunday Islands (Carlisle Island and Saint Bees Island). One family from Ubobo was planted but there were no surviving plants of this provenance in 2012.

The planting had been left untended by the collaborators for several years because of lack of interest by the industry in the genetic material it contained. By 2011 the trees were about 4 m tall and suffering the competitive effects of close spacing (1 m between rows and 0.5 m between plants within rows) and lack of tending. RIRDC was made aware of the availability of the planting in 2011 and recognised its potential importance in studying variation in susceptibility to the recently introduced disease myrtle rust (Carnegie et al. 2010) They funded separately the cutting back of the plants to about a 30–50 cm stump height in September 2011.

Initial Site Work

In May 2013 J. Doran and D. Lea arrived at the B. citriodora gene bank at Beerburrum nursery and:

1. Assessed phenotypes and clones for growth and myrtle rust susceptibility using a previously developed (Doran et al. 2012) methodology,

2. Selected phenotypes and clones for propagation based on their vigour and susceptibility to myrtle rust attack,

3. Coppiced the entire planting and removed all felled material from site to avoid any possible contamination effects. Side shoots were also removed from stools to promote epicormic growth,

4. Fertilised all selected plants to assist in the production of coppice material.

Full details of this work are reported in the internal project document ‘First Progress Report – May 2013’.

Setting of Cuttings

In August 2013, D. Lee and technical staff inspected the B. citriodora gene bank at Beerburrum nursery. Rows were clearly pegged and numbered and all plants selected for propagation were clearly labelled to ensure that all cuttings material collected was correctly defined. These hedge plants were trimmed to stimulate coppice growth and any material between plants was removed to minimise the chance of collection of incorrect material. Hedges were then sprayed with the fungicide Bayfiden to ensure all material captured into the glasshouse was free of myrtle rust. The hedged plants were also fertilised with Nitrosol (10.5% N, 2.3% P, 6.8% K, and some trace elements; Yates Pty Ltd.) and Seasol (primarily trace elements; Seasol International Pty Ltd.) liquid fertilizer at 2 ml/litre to promote healthy shoot production for the setting of cuttings.

Juvenile tip cuttings (Figure 1) were taken from 61 families of B. citriodora and set in the glasshouse at the University of the Sunshine Coast during the 16–18 September 2013. All cutting material was collected using secateurs, then placed in labelled plastic bags prior to chilling and transported to the University of the Sunshine Coast for setting. Cuttings were dipped in 3 % IBA gel and then set in labelled 93 cc hiko trays with crack pot inserts in potting medium consisting of 75% coarse perlite and 25% composted pine bark fines (0 to 10 mm and a mix of 12- to 14-month slow-release Osmocote (N- P-K, 17.9:0.8:7.3) at a rate of 4 kg/m3, gypsum at 1 kg/m3, Micromax at 1 kg/m3, and granular wetting agent Hydroflow at 1 kg/m3. They were then placed in a temperature controlled glasshouse under a misting watering environment. The misting regime used for the lemon myrtle cuttings was 10 seconds every 15 minutes during the day (0600 H to 1800 H) and 10 second every 20 min at night (1800 H to 0600 H). In the September 2013 setting, ten cuttings were set of 437 clones representing the entire genetic diversity in the Beerburrum planting from all 11 provenances. Rooting ranged from 0 to 100% across the 437 clones set with 374 clones having 50+% rooting when assessed at age three months.

The recalcitrant rooting clones (with less than 50% rooting) from the September 2013 setting were re- set on the 14 February 2014 to ensure that a wide genetic base of the Beerburrum material was captured. Methodology for setting of cuttings in February 2014 was the same as that used in the September 2013 setting. Rooting performance of these recalcitrant clones was still poor (average 45% rooting for the 73 clones – range 0 to 100% rooted) at the three month rooting assessment with seven clones having zero rooting across both settings.

One hundred and twenty cuttings of the twenty rust resistant clones identified by Doran et al. (2012) were also set in February 2014. Sixteen of these clones initially rooted relatively well (mean 66%, range 25 to 93% rooted). However, four of these clones (each with five ramets) from the Silver Valley provenance had poor rooting (mean, 20%, range 0 to 38%).

Once rooting occurred in the glasshouse environment, plants were moved to an external stand down area under shade cloth with an overhead watering system. Shade was removed several weeks prior to planting to harden the plants.

In total 433 clones were captured as rooted cuttings of the 441 attempted and 418 clones had enough ramets (minimum of three) to allow them to be planted in at least one field trial.

Figure 1 Juvenile tip cuttings of the Backhousia citriodora clones set in the University of the Sunshine Coast glasshouse. 1A. Typical cutting material set. 1B. The overall view of the 4370 cuttings set in September 2013.

Establishment of new gene bank plantings of lemon myrtle

Three field trials were planted in 2014 and 2015.

Clonal Relocation Plantings

Field Trial 1 – Ex-situ conservation of B. citriodora – northern NSW site – Planted in May 2014 (Figure 2A) on land owned by Australian Rainforest Products at Dunoon. The trial comprises 280 clones in an incomplete block design with 3 replicates. See Appendix 1 for a precis of the Establishment Report.

Field Trial 2 – Ex-situ conservation of B. citriodora – southern Qld site – Planted in February 2015 on land owned by the Queensland Government at Traveston in southern QLD (Figure 2B and 2C). Trial comprises 404 clones in an incomplete block design with 3 replicates. See Appendix 2 for a precis of the Establishment Report.

Screening Trial

Field Trial 3 – Field-replicated block planting of B. citriodora planted in January 2015 on land owned by Australian Rainforest Products at Dunoon in northern NSW. The trial comprises 21 clones in a randomised incomplete block design with five replicates; however two of these replicates were substantially damaged by crows digging up scarab beetle grubs, leaving three effective replicates.

This screening trial was initially intended to be planted in May 2014. Unfortunately, by the time the February setting of cuttings were field ready to plant (this includes the post-setting glasshouse and standing down periods) the field site was regarded by both the industry partner (site owner) and the researchers to be too dry for successful establishment. The trial was therefore held over in the stand down area at the USC glasshouses and was not planted until January 2015.

No myrtle rust screening has been carried out on this trial because the field plants needed to be fully established and then have flushes of new growth before accurate assessment of the growth and disease impacts could be assessed. While the plants have established, due to the cooler weather through winter, adequate flushing has not occurred. It is intended that assessment will be undertaken as soon as conditions are favourable.

See Appendix 3 for a precis of the experimental Establishment Report.

A B

Figure 2 View of the two Backhousia citrodora gene conservation plantings. 2A. The trial at Dunoon in New South Wales at age 12 months. 2B. Clonal replanting trial at Traveston in Queensland at age six months. 2C. Six month old clonal cutting flushing after spring rain in the trial at Traveston.

Glasshouse myrtle rust screening

Inoculum

A single lesion isolate of P. psidii (BRIP# 57793) was collected from Rhodamnia sessiliflora growing in the Chapel Hill suburb of Brisbane, QLD, Australia. Urediniospores from a single pustule were collected using a fine bristled paint brush and washed into 5ml sterile distilled water to which one drop of the surfactant ‘Tween 20’ was added. This solution was then applied onto Syzygium jambos and Rhodamnia rubescens seedlings to further propagate spores for screening.

These plants were then covered with black plastic bags, which were sealed to maintain a high humidity level, and placed into an incubator at 18–20oC for 24 hours. After 24 hours, plants were transferred to a shade-house and watered by hand as required. Urediniospores production was monitored and spores collected every two to three days until spore production ceased. These urediniospores were then inoculated back onto other S. jambos and R. rubescens seedlings, a process repeated until sufficient numbers of spores had been collected for inoculation. Urediniospores were then placed in a desiccator for seven days before being placed into Nunc tubes and stored at -80oC until required.

Inoculation

Urediniospores were removed from -80oC storage and allowed to warm to room temperature prior to being added to sterile distilled water (SDW). The surfactant ‘Tween 20’ was added at a rate of two drops per 100ml of SDW and the spore suspension stirred to reduce clumping. Spore counts were then conducted using a haemocytometer and the suspension adjusted to a concentration of 1 x 105 spores/ml for use in the subsequent inoculation.

The plants were inoculated using a fine mist spray (2.9 kPa pressure), generated by a compressor driven spray gun (Iwata Studio series 1/6 hp; Gravity spray gun RG3, Portland, USA), applied to the upper and lower leaf surfaces of the clonal explants ensuring all leaves were coated with a fine mist but runoff of the spore suspension was avoided as described by Pegg et al. (2014b). Ten seedling of S. jambos, R. rubescens and Melaleuca quinquenervia were used as susceptible controls. These susceptible seedling controls were placed randomly within replicates and were inoculated in the same manner as those of the test species.

Trays of the clonal explants were placed into solid plastic tubs and then onto a metal bench layered with a plastic sheet. Immediately after inoculation, the clones were covered with a plastic sheet for 24 hours to maintain high humidity levels and leaf wetness in a controlled environment room set between 18 and 20oC in the dark. Hot tap water (60°C) was applied to the lower plastic sheet immediately after inoculation to ensure high humidity levels were achieved rapidly. After 24 hours plastic sheeting was removed and plants grown in a shade-house and hand watered as required. Disease symptom progression was monitored daily.

Disease assessments and rating system

The B. citriodora clones were monitored for symptom development and assessed 12 to 14 days after inoculation for disease severity on new shoots and expanding leaves using a 1 to 5 disease rating scale according to Junghans et al., (2003): 1 = no symptoms evident or presence of clear flecking; 2 = presence of a hypersensitive reaction (HR); 3 = small pustules, <0.8 mm diameter, with one or 2 uredinia; 4 = medium-sized pustules, 0.8 to <1.6 mm diameter with about 12 uredinia; 5 = large pustules, >1.6 mm diameter, with 20 or more uredinia on leaves, petioles and/or shoots. Plants without new growing flush were not rated and were therefore included in the analysis as missing data (MD).

Repeat inoculations were conducted on plants rated as ‘1’ on the 1 to 5 disease rating scale to ensure they were not escapes.

In addition to the disease rating scale, the percent leaf area with pustules (sori) was visually assessed to indicate the severity of the infection. This was done as a way of determining potential tolerance levels within populations as many plants scored 3 to 5 in the Junghans et al. (2003) scale but only had a few large pustules.

Assessment of myrtle rust in the field trials

The three field trials established during the project have been assessed on a monthly basis for damage caused by myrtle rust. The only trial with assessable damage was Field Trial 1 – the Ex-situ conservation of stand of B. citriodora established at Dunoon (northern NSW). The other trials were too young and not flushing sufficiently to be impacted by the disease. Field trial 1 was assessed twice; first using a simple incidence of flush showing symptoms. The second assessment including an incidence assessment and a severity score based on a 0–6 rating scale developed to assess impact on tree species in native ecosystems (Pegg unpublished):

0. No evidence of pustules on new shoots/leaves 1. Minor infection only (pustules/lesions small in size – >1mm diameter) and on only a few new leaves/shoots. 1-2 pustules/infected leaf. No evidence of infection on green stems 2. Mostly minor infection only (pustules/lesions generally small in size – >1mm diameter) on up to 50% of new flush. Multiple pustules/leaf. No evidence of infection on green stems. 3. Infection present on the majority of new leaves and shoots; mix of minor (>1mm diameter) and moderate sized pustules/lesions (> 1mm diameter) infection, multiple pustules per leaf; evidence of infection/pustules on some green stems; no evidence of stem dieback/death 4. Infection present on all new leaves/shoots; mix of moderate (> 1mm diameter) and severe (> 1.6mm diameter); coalescing lesions present; foliage dieback and death present on some infected stems; stem infection common; some evidence of stem dieback and occasional stem death 5. Foliage/shoot dieback/death evident on most stems throughout tree/shrub 6. Tree/shrub dead as a result of repeat infection by myrtle rust

Data Analysis

As the provenances had unequal numbers of families and clones and of trees per plot and there was variation in survival rates and flushing of the clones; differences between taxa were tested by fitting general linear mixed models by residual maximum likelihood (REML) with replicate, plot and tree as random terms following the approach of Lee et al. (2011). Fisher’s protected least significant difference test was applied if significant effects were detected by the REML analysis. Means are reported, and treatment differences or interactions were regarded as significant at P < 0.05. Wild material collection

Seed and cutting collection

A detailed report is held by D. Lee at USC on the seed collections undertaken. A summary is supplied in this report.

Permits for collection of B. citriodora seed and plant material from crown estates were applied for and permit Numbers WITK13683813, WISP13683913 and TWB/40/2013 were issued by the Department of Environment and Heritage Protection on the 11th November 2013. The first permit number authorised the collection of biological material from Woondum National Park and Dawes National Park, and the other two from Degalgil State Forest. Collections did not occur in Degalgil State Forest

as upon further investigation the previous herbarium records for B. citriodora (BC) have been re-zoned to Dawes National Park.

Woondum National Park

The population was regularly monitored to assess the state of seed development and to determine when seed would be suitable to collect. During 2014 no flowering of native lemon myrtle trees was observed at this site, possibly due the extremely dry previous summer (December 2013 to February 2014) where only 18.3% of the average summer rainfall was recorded at Gympie (approximately 10 km from Woondum National Park). The rainfall over the next summer season (December 2014 to February 2015) was more typical (118.2% of the long term average) and widespread flowering of the wild lemon myrtle trees was observed. This allowed collection of 12 trees from across the range of the species in Woondum National Park (Figure 3). During the seed collection leaf samples were also collected to allow future DNA testing if required (not part of this project). The seed and leaves were dried in a cool location out of the direct sun to minimise any effects on seed viability from drying the green seed in direct sunlight.

Dawes National Park

A single visit to Dawes National Park on the 4-7 May 2015 was undertaken to investigate the population of lemon myrtle along Koolkurrum Creek that had not been sampled as part of the 1994 collections. Timing of this was guided by the flowering of the species in the Woondum National Park, as the Dawes National Park region also experienced a very dry summer (December 2013 to February 2014). There was only a small seed crop available for collection during May 2015 and seed was collected from two trees. For one of these trees the seed was collected from the ground and the other the seed was picked off the tree. Upon breaking open the fruits, no fully developed seed could be seen. Leaf samples were collected from 19 of the 20 trees observed. The species occurrence is described as occasional with the majority of the trees occurring on private land (Figure 4) and not the National Park as indicated by the previous herbarium records.

The trees frequently occurred in clusters and are likely to be clones of each other. Beneath almost all the lemon myrtle trees a carpet of root suckers mostly 10-20 cm tall with occasionally larger stems >1 m were observed. Due to the isolation of the trees it was considered likely that it mainly reproduces clonally and not via seed at this location. Root coppice cuttings were collected from 19 trees from the private land section of the population. These have been captured at the University of the Sunshine Coast nursery.

Results

Capture of existing germplasm

Establishment of new gene bank plantings of lemon myrtle

The principal aim of the project (objective a) has been met with the establishment of plantings of the existing conservation/breeding population of B. citriodora (61 families from 11 provenances) at two secure sites in NSW (Dunoon site, planted May 2014) and southern QLD (Traveston site, planted February 2015).

The collection and setting of material for this out planting from the Beerburrum gene bank was carried out in September 2013 and February 2014. Cuttings material was set at the USC glasshouse area using methods described above. The initial setting produced a wide range of rooting across the various families and clones and therefore a second setting was initiated in February 2014 to ensure enough plants were available for out planting at both the northern NSW and the southern Qld sites. As indicated by Kibbler et al. (2004) rooting of this species was slow with root production occurring over a period of six to 12 weeks. In total 430 clones were propagated of the 437 attempted. Of these, 418 clones, comprising 58 families from 10 provenance, the ten clones from the Silver Valley provenance and 18 unknown clones from unlabelled rows in the Beerburrum gene bank, had sufficient numbers of suitable ramets allowing inclusion in the new gene banks (Field Trial 1 and 2). Details of the number of clones per family and provenance are provided in Table 1.

Establishment of Field Trial 1 – northern New South Wales – Dunoon

As detailed above, this trial was established in May 2014 on land owned by Australian Rainforest Products at Dunoon in northern NSW. This trial comprises 280 clones in an incomplete block design with 3 replicates. Survival of the material planted at the Dunoon site in May 2015 has been very good with 91% survival (accounting for 276 clones) at age one year.

Establishment of Field Trial 2 – Queensland – Traveston

As detailed above, this trial was established in February 2015 on land owned by the Queensland Government at Traveston in southern QLD. This trial comprises 404 clones in an incomplete block design with 3 replicates. Survival of the material planted at the Traveston site in February 2015 has been very good with 92% survival across the trial and all 404 clones surviving at age three months.

Table 1 Details of the genetic material securely captured in new field trials (gene bank plantings) from the existing gene bank at Beerburrum.

Provenance Number of families Number of clones per captured per provenance provenance Carlisle Island 12 38 Cathu 2 10 Conway 9 37 Dryander 5 43 Eumundi 6 61 Maroochy 3 19 Mt Elliot 3 14 Noosa 2 19 Silver valley (clones) 10* 38 (ramets) St Bees 5 23 Unknown clones 18* 18 Woondum 11 98 Total 58 families + 28 clones 418 * clones

Figure 3 Location of Backhousia citriodora in Woondum National Park.

Figure 4 Location of Backhousia citriodora along Koolkurrum Creek near Ubobo.

Establishment of Field Trial 3 the replicated block planting – Northern New South Wales – Dunoon

As detailed above, this trial was established in January 2015 on land owned by Australian Rainforest Products at Dunoon in northern NSW. The trial comprises 21 clones in a randomised incomplete block design with five replicates; however two of these replicates were damaged by crows digging up scarab beetle grubs, leaving three effective replicates. Survival of the three effective replicates at age three months was 79%. Rust Resistance Screening

Glasshouse trial

Objective (c) was to test the rooted clones against different isolates of myrtle rust to validate the field observations of plant susceptibility and tolerance. Machado et al. (2015) evaluated 104 isolates of P. psidii collected from across the diseases range in Australia, using microsatellite analysis. They found that there was no variation in isolates of P. psidii even though there are mutations occurring in the wild. Based on this we have screened the lemon myrtle against only one isolate, following the procedures detailed in Pegg et al. (2014b) and Lee et al. (2015) using P. psidii from a single lesion collected on Rhodamnia sessiliflora.

Material was assessed for disease resistance on an individual plant basis allowing analysis of the data at both a clonal, family and at a provenance level. The symptoms, red-brown or chlorotic leaf spots, were first observed six days after inoculation.

Provenance mean data evaluated for P. psidii resistance are presented in Table 2. These data show that in both the disease rating and the disease incidence there are some significant differences between provenances. It should be noted that all provenances tested are susceptible to P. psidii infection and that even in the best provenance (Noosa) only 39% of the plants were resistant to the disease. The worst source was the commercial clone from Limpinwood that had only 11% of the plants being assessed as resistant. The severity of the disease was rated on the Junghans et al. (2003) ratings system, where plants that obtain scores under 3 are regarded as resistant while numbers above 3 indicate higher levels of susceptibility to infection. All provenances are well over 3 with only Noosa at 3.7 and Woondum at 3.9 under 4 on a scale of 1 to 5, indicating a high level of disease susceptibility. The other method used to assess the severity of the disease was to score the percentage of leaves affected. This method again indicated that there were significant differences between provenances with the range from 15.9% for the Silver Valley clones to Cathu which was the worst at 35.3% of leaves infected by the disease under glasshouse conditions.

Provenance data were also analysed at the family level and these data are presented as Table 3. The disease incidence was again significant at the P<0.05 level with a wide variation in disease incidence between families. The lowest incidence of leaf infection was recorded at 7.5% for family 1465 from the Woondum provenance, while the highest was 60% for family 1381 from Carlisle Island. Nine sources from four provenances had low disease incidence with ≤15% of the leaves affected by myrtle rust.

Table 2 Backhousia citriodora provenances evaluated for Puccinia psidii resistance (means of all clones) in the glasshouse trial in order of most to least susceptible, based on disease incidence.

Source / Disease Incidencea Number plants Disease severity Resistant a b Provenance (% leaves infected) /severity class rating (1-5 scale) plants (%) (1:2:3:4:5) Cathu 35.3 a 0:1:5:4:13 4.3 abc 26.1 St Bees Island 33.8 a 0:0:10:14:32 4.4 a 17.9 Carlisle Island 29.9 ab 2:4:19:30:44 4.1 ab 25.3 Conway 29.9 ab 4:2:19:29:40 4.1 bcd 26.6 Maroochy 29.3 abcd 0:1:9:13:11 4 abcd 29.4 Mt Elliot 29.0 abcde 1:1:5:10:10 4 abcd 25.9 Eumundi 27.7 abc 2:4:30:56:78 4.2 ab 21.2 Dryander 25.3 bcde 4:5:26:42:46 4 bcd 28.5 Limpinwood 22.2 bcdef 0:0:2:7:9 4.4 a 11.1 Unknown clones 21.8 cdef 0:2:16:13:20 4 bcd 35.3 Woondum 21.8 de 4:8:70:79:70 3.9 cd 35.5 Noosa 19.9 ef 1:4:16:20:13 3.7 d 38.9 Silver Valley 15.9 f 3:1:20:48:37 4.1 bc 22 a values followed by the same letter shows means that do not differ significantly (pairwise comparison P<0.05) b resistant plants belong to severity classes 1 to 3 following Junghans et al. (2003)

Table 3 Backhousia citriodora provenances evaluated at the family level for Puccinia psidii resistance in the glasshouse trial in order of most to least susceptible, based on disease incidence. Number plants Total Source / Disease Incidencea Disease Family /severity class b Family Provenance (% leaves affected) rating (1:2:3:4:5:MDc) Count Carlisle Is 1381 60 a 0:0:0:0:3:0 5 ns 3 St Bees Is 1457 58.3 ab 0:0:0:0:3:0 5 ns 3 Conway 1464 51 abcd 0:0:1:1:1:0 4 ns 3 Carlisle Is 1453 46 abcde 0:0:1:0:4:1 4.6 ns 6 Carlisle Is 1452 39.7 abcef 0:1:5:4:8:0 4.1 ns 18 Conway 1342 38.7 abcdefg 0:0:3:4:11:6 4.4 ns 24 Woondum 1413 37.1 abcdefghijk 0:0:1:3:3:5 4.3 ns 12 Conway 1352 36.7 abcdefghijkl 0:0:1:2:3:0 4.3 ns 6 Cathu 1428 36.4 abcdefghijk 0:1:1:2:3:2 4 ns 9 Maroochy 1013 36.3 abcdefghijk 0:1:0:3:4:10 4.3 ns 18 Mt Elliot 1329 35 abcdefghijk 1:0:1:2:5:3 4.1 ns 12 Cathu 1424 34.8 abcdefgh 0:0:4:2:10:5 4.4 ns 21 Mt Elliot 1433 34 abcdefghijklmno 0:1:0:2:2:4 4 ns 9 St Bees Is 1461 33.9 abcdefghij 0:0:3:2:9:4 4.4 ns 18 Conway 1356 33.1 bcdefghijkl 1:0:2:4:6:5 4.1 ns 18 Eumundi 1027 33 cdefgh 0:0:6:10:17:6 4.3 ns 39 St Bees Is 1458 32.6 cdefghijl 0:0:4:4:10:9 4.3 ns 27

Number plants Total Source / Disease Incidencea Disease Family /severity class b Family Provenance (% leaves affected) rating (1:2:3:4:5:MDc) Count Carlisle Is 1454 31.9 cdefghijl 0:0:1:10:10:3 4.4 ns 24 St Bees Is 1462 31.8 cdefghijkl 0:0:3:6:9:3 4.3 ns 21 Eumundi 1024 31.6 cdefghj 0:2:4:7:16:1 4.3 ns 30 Eumundi 1025 30.6 cdefghijl 0:1:9:7:16:3 4.2 ns 36 Conway 1341 30 abcdefghijklmnopqr 0:0:0:2:1:0 4.3 ns 3 Conway 1353 30 abcdefghijklmnopqr 0:0:1:1:0:1 3.5 ns 3 Conway 1355 29.6 cdefghijkln 1:0:3:3:7:4 4.1 ns 18 Maroochy 1014 29 cdefghijkln 0:0:8:7:7:5 4 ns 27 St Bees Is 1459 28.3 abcdefghijklmnopqr 0:0:0:2:1:0 4.3 ns 3 Woondum 1029 27.1 cdefghijklmn 0:1:4:8:12:5 4.2 ns 30 Dryander 1468 26.9 cdefghijklmno 1:1:3:3:9:4 4.1 ns 21 Silver Valley RL9 26.3 cdefghijklmnopqr 0:0:0:2:2:2 4.5 ns 6 Carlisle Is 1337 25.7 bcdefghijklmnopqr 0:0:2:1:0:0 3.3 ns 3 Dryander 1331 25.7 dghijklmnoqr 0:1:2:4:10:7 4.4 ns 24 Dryander 1470 25.6 dghijklmnoqr 0:1:4:10:5:4 4 ns 24 Dryander 1471 25.3 hijklmnoq 1:2:5:12:8:2 3.9 ns 30 Carlisle Is 1455 25.1 dghijklmnopqr 0:2:4:3:7:8 3.9 ns 24 Carlisle Is 1447 25 cdefghijklmnopqr 1:0:3:1:3:1 3.6 ns 9 Silver Valley RL3 25 cdefghijklmnopqr 0:0:1:3:5:0 4.4 ns 9 Woondum 1028 24.4 hijklmnopqr 0:1:8:6:10:8 4 ns 33 Woondum 1012 24.3 hijklmnopqr 0:0:7:10:6:7 4 ns 30 Woondum 1430 24.3 hijklmnopqr 0:0:5:8:8:9 4.1 ns 30 Dryander 1330 24.2 hijklmnoq 2:0:12:13:14:4 3.9 ns 45 Eumundi 1023 24.2 hijklmnopqr 1:0:1:9:9:4 4.3 ns 24 Woondum 1009 23.4 hijklmnopqr 1:0:10:10:6:9 3.7 ns 36 Eumundi 1016 23.2 hijklmnopqr 0:1:6:13:11:2 4.1 ns 33 Mt Elliot 1431 22.9 hijklmnopqr 0:0:4:6:3:8 3.9 ns 21 Woondum 1466 22.8 cdefghijklmnopqr 1:1:1:1:2:0 3.3 ns 6 Carlisle Is 1449 22.5 fgijklmnopqr 1:0:1:2:4:1 4 ns 9 Limpinwood 9999 22.2 hijklmnopqr 0:0:2:7:9:0 4.4 ns 18 Silver Valley RL10 22.1 egijklmnopqr 0:1:1:2:3:2 4 ns 9 Unknown UC 21.8 iklmnopqr 0:2:16:13:20:9 4 ns 60 clones Carlisle Is 1451 21.7 egijklmnopqr 0:0:1:2:3:2 4.3 ns 8 Silver Valley RL8 21.5 hijklmnopqr 1:0:3:4:2:2 3.6 ns 12 Conway 1343 21 jklmnopqr 2:2:6:12:10:4 3.8 ns 36 Eumundi 1026 20.2 iklmnopqr 1:0:4:10:9:9 4.1 ns 33 Noosa 1030 20 iklmnopqr 0:3:7:8:7:5 3.8 ns 30 Noosa 1031 19.8 kmnopqr 1:1:9:12:6:7 3.7 ns 36 Silver Valley RL4 19.4 hijklmnopqr 0:0:2:3:3:1 4.1 ns 9 Conway 1345 18.3 cdefghijklmnopqr 0:0:2:0:1:0 3.7 ns 3

Number plants Total Source / Disease Incidencea Disease Family /severity class b Family Provenance (% leaves affected) rating (1:2:3:4:5:MDc) Count Woondum 1011 18 mnopqr 0:3:8:7:8:4 3.8 ns 30 Maroochy 1015 17.5 fgijklmnopqr 0:0:1:3:0:2 3.8 ns 6 Woondum 1010 17 opqr 1:2:26:23:11:23 3.6 ns 86 Carlisle Is 1450 16.5 glmpqr 0:1:1:1:1:2 3.5 ns 6 Carlisle Is 1456 15 fgijklmnopqr 0:0:0:2:1:0 4.3 ns 3 Woondum 1467 15 hijklmnopqr 0:0:0:1:4:1 4.8 ns 6 Silver Valley RL1 14.3 lmpqr 0:0:1:1:5:5 4.6 ns 12 Silver Valley RL7 13.3 prs 1:0:3:10:9:1 4.1 ns 24 Silver Valley RL2 12 mpq 1:0:2:5:3:1 3.8 ns 12 Carlisle Is 1379 11.7 hijklmnopqr 0:0:0:3:0:0 4 ns 3 Silver Valley RL5 11.7 ps 0:0:4:11:3:0 3.9 ns 18 Silver Valley RL6 11 qs 0:0:3:7:2:3 3.9 ns 15 Woondum 1465 7.5 hijklmnopqr 0:0:0:2:0:1 4 ns 3 a values followed by the same letter shows means that do not differ significantly (pairwise comparison P<0.05) b resistant plants belong to severity classes 1 to 3 following Junghans et al. (2003) c MD = Missing Data – clones not flushing at time of assessment and therefore not susceptible to inoculation d Note - Limpinwood was represented by a single commercially planted clone.

In contrast to the general provenance data we found that disease rating once analysed at the family within the provenance level was no longer significant.

Within provenance clonal data was also collected and analysed for both disease rating (Junghans et al. 2003) and disease incidence (percentage leaves affected). Of the 13 sources only 12 could be analysed because Limpinwood has only one clone. These data are presented as Appendix 4. There are no significant differences evident in the disease rating scores within provenances and there were only significant differences (P<0.05) in the disease incidence scores for two provenances (Silver Valley and Woondum). Silver Valley clones had a range of disease incidence from 11.6 to 25% while Woondum, which has a much larger number of clones had a range from 2 to 95%.

Rust assessment in a field trial

The intention of the second project objective (b) was to screen for rust resistance at a commercial lemon myrtle site the most rust resistant individuals observed in the ex-situ conservation/breeding population of B. citriodora at Beerburrum. As detailed above, Field Trial 3, the replicated block planting of B. citriodora was only established in 2015 due to extremely dry field conditions during 2014. While the plants have now successfully established in the field, the cooler weather through winter has not provided adequate flushing of the plants and therefore infection conditions have been poor. No screening has yet been carried out on this trial. It is intended, however, that assessment will be undertaken as soon as conditions are favourable and these data should assist in identifying the more rust tolerant clones for immediate deployment by industry.

Field trial 1 the first of the ex-situ conservation stands was also planted on the commercial property at Dunoon in NSW. This trial has been assessed by using the 0–6 disease severity rating scale developed to assess impact on tree species in native ecosystems (Pegg unpublished) as described above. In addition the percent damaged of the most mature expanding leaf was determined to indicate the severity of the infection to be consistent with the glasshouse study. These results are presented in Table 4.

The material that was most resistant to the disease in the field had a similar trend to that observed in the glasshouse with the unknown clones (those without any family or provenance information in the Beerburrum gene bank), Woondum and Silver Valley having the lowest (best) disease incidence and disease severity rating. The one difference was the St Bees Island provenance material appeared to be more tolerant to myrtle rust in the field than indicated by its performance in the glasshouse study where it was the second most susceptible.

Table 4 Backhousia citriodora sources evaluated for Puccinia psidii resistance (means of all clones) in the Dunoon Field trial 1, the ex-situ conservation stand established in 2014 in order of most to least susceptible, based on disease incidence. Source / Disease Incidencea Number plants Disease severity Provenance (% last expanding /severity class rating (1–6 scale)a leaf infected) (1:2:3:4:5:6)

Cathu 96.7 ab 0:1:4:4:3:0 3.8 a

Conway 92.5 a 1:16:15:12:9:0 3.2 abc

Mt Elliot 91.2 abc 0:4:6:6:4:0 3.5 ab

Carlisle Island 89.8 ab 2:20:17:16:13:0 3.3 abc

Maroochy 86.5 abc 0:8:6:9:0:0 3.1 abcd

Silver Valley 86 abcd 0:9:8:3:1:0 2.8 cef

Dryander 84.1 abc 6:19:10:8:12:0 3 bcd

Noosa 83.3 abcd 1:6:7:8:3:0 3.2 abcd

St Bees Island 82.9 bcd 1:20:6:6:3:0 2.7 def

Eumundi 80.7 cd 5:31:29:17:8:0 2.9 de

Woondum 75.8 de 20:46:44:10:11:0 2.6 f

Unknown clones 70.4 e 6:19:8:2:0:0 2.2 g a values followed by the same letter shows means that do not differ significantly (pairwise comparison P<0.05).

Collection of Wild Material

Objective (d) aimed to expand the gene bank by introducing new families of B. citriodora (seedlots) from Woondum National Park and Ubobo (Dawes National Park).

Seed was collected from 14 trees (12 trees in the Woondum National Park and two trees in the Koolkurrum Creek / Dawes National Park area). This seed has been sown at the USC glasshouse and germination is now occurring but is very slow and intermittent. This is consistent with the finding of House et al. (1996) who found that germination was slow because the fruits do not dehisce with the seed germinating through the hypanthium (fruit wall). They also found that seed viability was low averaging 3%. We have found a very similar seed viability results in this study as indicated in Figure 5.

Figure 5 Sowing of Backhousia citriodora seed and germination in the USC glasshouse. 5A. Untreated seed was sown directly into vermiculite in germination trays. 5B. Four Backhousia citriodora seedlings from one of the seedlots from Koolkurrum Creek / Dawes National Park area. In addition cuttings were collected from root coppice (Figure 6) of 19 of the 20 B. citriodora trees found along Koolkurrum Creek near Dawes National Park. These cuttings were set in the USC glasshouse and at least one cutting has survived for all of the plants so we potentially have material that can be used for extension of the ex-situ clone banks.

Figure 6 Root coppice under Backhousia citriodora plants along Koolkurrum Creek.

Discussion

The primary focus of this work was to secure the genetic base of B. citriodora that is under threat from redevelopment of the site where the current gene bank is planted at Beerburrum in QLD. This has been achieved with 433 clones captured as rooted cuttings of the 441 attempted and 418 clones having sufficient number of plants to allow them to be planted in at least one of the two new gene banks established (one on a commercial property near Dunoon, NSW and the other on secure crown land near Traveston, QLD). In addition seed has been collected from 14 wild trees from two populations: Woondum National Park (seed from 12 trees) and Ubobo (Koolkurrum Creek; seed collected from two trees). This seed has been sown and is germinating slowly in the USC glasshouse. We have also captured cuttings from root coppice of 19 trees from Ubobo area along the Koolkurrum Creek. All of this recently captured material (seedlings and root coppice clones) will be planted in the Traveston gene bank trial during the 2015–2016 wet season. This has ensured the lemon myrtle industry’s access to the broadly based genetic resources, needed to underpin the ongoing development of the species.

The glasshouse myrtle rust screening shows there is variation in the susceptibility of provenances and families as indicated by Doran et al. (2012), who found that some families appeared to be immune to the disease. However, in contrast, our study indicates all the lemon myrtle germplasm is somewhat susceptible to the P. psidii when screened under conditions that were optimal for the disease. This is not surprising as the gene bank planting at Beerburrum where the Doran and co-workers study was undertaken, is isolated from any surrounding myrtaceous vegetation, by at least 100m, hence, plants could escape infection and the overall infection rate at the site was low.

Based on the rating system of Junghans et al. (2003), all of the germplasm tested in the glasshouse would be considered highly susceptible to myrtle rust (mean provenance severity rating of over 3; Table 2). When the disease incidence (% leaves infected) was considered, it appeared that material from the Silver Valley, Noosa and Woondum provenances were the most tolerant of the disease (Table 2), a similar finding to that of Doran et al. (2012). This variation in myrtle rust susceptibility should be further tested in new replicated block plantings of the most resistant provenances and clones identified in this study. For example, if families were selected that had ≤20% of their leaves infected by the myrtle rust in the glasshouse study, then six provenances (Carlisle Island, Conway, Maroochy, Noosa, Silver Valley and Woondum) comprising 17 families (Table 3) would be included in new block plantings.

This is the first time the commercial Limpinwood clone has been evaluated for susceptibility to myrtle rust in a trial along with other sources of B. citriodora. On a provenance basis it ranked as the 5th best (22.2% of leaves infected) based on disease incidence and equal worst for disease severity (score of 4.4) based on Junghans et al. (2003) 1 to 5 rating scale (Table 2). Based on this finding it would appear that the industry could reduce the impact of myrtle rust by moving to more tolerant varieties of lemon myrtle that have been identified in this study.

The evaluation for myrtle rust susceptibility of the germplasm in Field Trial 1 at Dunoon, had a similar trend to that observed in the glasshouse with the unknown clones (those without any family or provenance information in the Beerburrum gene bank), Woondum and Silver Valley having the lowest (best) disease incidence (% of the last expanding leaf affected by the disease) and disease severity rating (Table 4). The one difference was the St Bees Island provenance material appeared to be more tolerant to myrtle rust in the field than indicated by its performance in the glasshouse study where it was the second most susceptible. In the field the expanding leaves were more severely damaged (range 70.4-96.7%) by the disease than in the glasshouse (range 15.9–35.3%). The cause for this is unknown but may reflect the increased spore load and repeat inoculations of the disease from other lemon myrtle plantings in the area near the field trial. Conversely comparing the 1–6 disease severity rating scale developed to assess impact on tree species in native ecosystems (Pegg unpublished) with the 1–5 disease rating scale of Junghans et al. (2003) highlighted that the same material had lower disease severity scores under field conditions (range 2.2 to 3.8) than for the same material when it was

screened in the glasshouse (range 3.7 to 4.4). This needs further investigation but may indicate the host physiology when growing in a commercial production system results in the plants being less susceptible to myrtle rust.

A possible issue with the rust susceptibility assessments based on the material at Beerburrum is that the physiological age of the hedges at Beerburrum vary due to differences in time since planting, management and source (seedling versus clonal) of the planting stock. For instance the Silver Valley provenance material is clonal material from wild plants of unknown age. These clones were planted in October 1997 whereas rows 1 to 7 at Beerburrum were derived from seedlings and were planted in January 1995. As noted by Ferreira (1983) the susceptibility of plants to myrtle rust in eucalypts is linked to the trees age, with the younger the tree the more susceptible it is to myrtle rust. The foliage of the Silver Valley clones looks more adult than the seedling derived plants (e.g. rows 1 to 7) and this may explain why we are seeing reduced susceptibility in this material.

A PhD student (Ms Emily Lancaster), studying at the University of Queensland has recently commenced working on the epidemiology of myrtle rust on lemon myrtle and will continue the screening work in the field trials and investigate the impact of plant age on disease susceptibility.

Implications

If the lemon myrtle industry is to survive and strive to be organic then the use of rust resistant / tolerant cultivars is essential. Similarly use of myrtle rust tolerant or resistant varieties would be beneficial for any industry growing myrtaceous species including the plantation sector growing eucalypts, the essential oil industry and nursery trade. All industries impacted by myrtle rust will need on-going disease screening and breeding programs based on broad genetic bases to combat the current incursion of myrtle rust and to prepare for incursions of potentially more virulent strains of the disease. This project has ensured that a broad genetic base of lemon myrtle will be available for future development of the industry. The new germplasm collected during this project further broadens the domesticated genetic base of the species and will facilitate on-going testing of the species for disease and agronomic traits.

This study has shown that no clone of lemon myrtle found thus far is resistant to the disease, however, there are clones that appear more tolerant to the disease. If these clones are reasonably productive and they have suitable oil profiles (high citral [neral and geranial] content), they could offer the industry an immediate lift relative to the current Limpinwood varieties that are moderately susceptible to the disease.

Recommendations

Continued screening of the field plantings should be undertaken to confirm the results from the glasshouse study. These results indicated that, even though no resistant clones, families or provenances exist, there are clones, families and provenances that show higher levels of tolerance to the disease (as indicated by low percentage of leaf damage).

Other initiatives that could be considered by industry include:

 The Limpinwood clones have an abundant essential oil that is very high in citral (neral plus geranial) and this is what the market requires (pers. comm. Gary Mazorrana 2012). The Beerburrum clones have not been screened for oil concentration and citral content using an appropriate extraction method. Therefore, the oil concentration and citral content of the 20 clones planted in the replicated block trial, and any other clones we have identified as displaying higher levels of tolerance to myrtle rust, should be assessed.

 The possibility of hybridising B. citriodora with other less susceptible Backhousia species (e.g. B. bancroftii, B. gundara (Prince Regent), B. sciadophora or B. subargentea) that have been found to be relatively resistant to myrtle rust (Pegg et al. 2014a) should be investigated to see if it would be possible to breed myrtle rust resistant hybrid varieties.

 Screening of the newly captured germplasm (the 14 seedlots and 19 root coppice clones) should be undertaken to evaluate their disease tolerance relative to the germplasm already screened for disease susceptibility.

 The industry could consider moving to drier irrigated environments where the impact of the disease should be lower. Demonstration plantings should be established to investigate the potential for this to be achieved. These planting should include material from all provenances to ensure adapted varieties are identified.

 The impact of growing older physiological aged lemon myrtle plants with more mature foliage should be investigated to see if this reduces the impact of the disease.

 The screening of the germplasm against races of P. psidii not currently in Australia should be investigated to ensure the industry is prepared for any future incursions of this disease. This would also apply to other industries using Australian myrtaceous species including the essential oil producers, the nursery industry and forestry.

 Growing lemon myrtle plants under different nutrient regimes and assessing the influence of rates of leaf expansion on disease severity should be investigated to see if varying the production system can reduce the impact of the disease.

Appendix 1. Trial 1 (Dunoon Site)

A full Establishment Report is held by D. Lee at USC.

Plant materials

 Details of the 280 clones from the Beerburrum gene bank available for planting at Dunoon in May 2014 are given in the full report.

 Propagation took place in University of the Sunshine Coast glasshouses during 2013/14.

Site details

Owner Australian Rainforest Products (Gary Mazzorana) Location 106 The Channon Road, Lismore NSW 2480 Site Gently to moderately sloping block (northerly and north-easterly aspects) on Red, Ferrosol soils amongst basalt boulders. This was formerly rough grazing country cleared many years previously. Climate is humid, subtropical with high and evenly distribute rainfall. There may be a rare winter frost. Annual rainfall is 1592 mm. Design

Type Randomised incomplete block (each ICB comprises 4 rows of 5 trees) No. of replicates 3 No. of treatments 280 clones Plot details Single-tree plots Buffering No buffer Spacing 3.2 m between rows and 0.5 m within rows General

Planting date 18 to 24 May 2014 Planting Planting lines were ripped before planting and a thick layer of B. citriodora mulch applied to the planting lines. Maintenance Each plant received c. 1.5 L of water on the day it was planted Weed control Weed growth is to be controlled by slashing down the rows, and spraying weeds that emerge in the mulch. Large camphor laurel regrowth along the western boundary that shades the experimental planting in the afternoons poisoned by ARP.

Appendix 2. Trial 2 (Traveston Site)

A full Establishment Report is held by D. Lee at USC.

Site details

Owner Queensland Government Location Mary Valley Research Facility, Traveston Rd, Traveston, QLD Latitude – 26.331º south, Longitude – 152.711º east Site Westerly with a slope of <10% on Red Ferrosol (kraznozem) soils. Formerly closed sclerophyll forest. Eucalyptus. tereticornis on lower slopes, E. intermedia on mid slopes, E. longirostrata and E. intermedia on upper slopes with an understory of Acacia aulacocarpa and rainforest species. Cleared about 25 years prior to trial establishment and used as improved pasture and grazing country. The climate is humid, subtropical with the odd winter frost. Mean annual rainfall 1932–2014 is 1271 mm (source BOM 2015). Site has predominantly summer rainfall with approximately 70% falling between November and the end of April. Design

Experimental area 40 m x 80 m. Layout 33 line plots each with 40 tree spaces. Plots are consecutive 1–33 and are defined at the start with a wire and labelled cattle ear tag. Plots are numbered from the south-east corner of the block and tree 1 for each plot was planted on the plot locating wire. Type Randomised incomplete block design No of replicates 3 No of treatments 404 clones Plot details single-tree plots Buffering no buffer Spacing 2 m between rows and 1 m along row General

Preparation 4/2/2015: Site overall herbicide treatment using a boom spray with Roundup and Brushoff 24/2/15: Rows ripped with a tractor and tine to 600 mm Planting 26/2/2015: Hand planted using grubbers along rip lines. Planting was carried out within the week of a >250 mm rainfall event. Soil moisture was excellent and all plants were soaked in a water and Seasol mixture prior to planting. Weed control Weed growth is to be controlled as required.

Appendix 3. Trial 3 (Dunoon Site)

A full Establishment Report is held by D. Lee at USC.

Plant materials

 Details of the 21 clones (4 of these are from Silver Valley and each of these consists of 5 bulked ramets) from the Beerburrum gene bank available for planting at Dunoon in May 2014 are detailed in the full report.

 Propagation took place in University of the Sunshine Coast glasshouses during 2013/14.

Site details

Type 3 incomplete blocks in an Alpha design No. of replicates 5 No. of treatments 21 Replicate size 3 incomplete blocks, 7 families per block and 21 families per replicate Plot details 10 tree plots (2 rows X 5 trees) Spacing 3.2 m between rows and 0.75 m within rows (4166 st/ha) General

Planting date 20 to 22 January 2015. Note: this stock was from the setting in February 2014 and due to unfavourable planting conditions had to be held until January 2015 to be planted. Planting Planting lines were ripped before planting and a thick layer of B. citriodora mulch was applied to the planting lines. Maintenance Each plant received c. 1.5 L of water on the day it was planted Weed control Weed growth is to be controlled by Australian Rainforest Products as required.

Appendix 4. Susceptibility of clones to myrtle rust

Table 5 Susceptibility of individual clones of Backhousia citriodora to Puccinia psidii ranked most to least susceptible within the provenance in the glasshouse trial. Provenance Clone Disease Ratinga Leaf area affected (%) Carlisle Island 301 5.0 ns 66.7 ns 137 5.0 ns 60.0 ns 385 5.0 ns 50.0 ns 389 4.3 ns 50.0 ns 323 5.0 ns 50.0 ns 371 5.0 ns 47.5 ns 324 4.3 ns 43.3 ns 376 4.5 ns 40.0 ns 300 4.3 ns 38.3 ns 306 4.3 ns 38.3 ns 308 4.5 ns 37.5 ns 314 4.7 ns 36.7 ns 384 4.7 ns 35.0 ns 307 4.3 ns 35.0 ns 313 4.3 ns 35.0 ns 392 4.0 ns 35.0 ns 381 4.0 ns 33.3 ns 391 3.0 ns 28.3 ns 388 4.4 ns 27.5 ns 379 3.7 ns 26.7 ns 311 4.3 ns 26.7 ns 203 3.3 ns 25.7 ns 312 5.0 ns 22.5 ns 383 4.5 ns 22.5 ns 390 3.7 ns 20.0 ns 373 4.3 ns 18.3 ns 305 4.0 ns 17.5 ns 321 4.3 ns 15.0 ns 377 2.7 ns 12.3 ns 179 4.0 ns 11.7 ns 382 3.0 ns 10.0 ns 387 4.0 ns 10.0 ns 374 3.0 ns 10.0 ns 380 3.0 ns 10.0 ns 386 3.0 ns 5.3 ns 375 3.5 ns 2.0 ns 378 3.0 ns 2.0 ns Cathu 436 5.0 ns 45.0 ns 437 5.0 ns 62.5 ns 438 5.0 ns 31.7 ns 356 4.5 ns 57.5 ns 355 4.0 ns 31.7 ns

Provenance Clone Disease Ratinga Leaf area affected (%) 358 4.0 ns 25.0 ns 435 4.0 ns 26.0 ns 439 4.0 ns 36.7 ns 357 3.5 ns 22.5 ns 434 3.0 ns 7.5 ns Conway 141 *b ns * ns 192 5.0 ns 15.0 ns 140 5.0 ns 75.0 ns 202 5.0 ns 25.0 ns 142 4.7 ns 53.3 ns 191 4.7 ns 50.0 ns 198 4.7 ns 40.0 ns 138 4.7 ns 50.0 ns 177 4.5 ns 30.0 ns 143 4.5 ns 37.5 ns 152 4.5 ns 10.0 ns 170 4.3 ns 31.7 ns 180 4.3 ns 30.0 ns 189 4.3 ns 28.3 ns 154 4.3 ns 28.3 ns 199 4.3 ns 26.7 ns 153 4.3 ns 65.0 ns 139 4.0 ns 23.3 ns 155 4.0 ns 16.7 ns 169 4.0 ns 30.0 ns 178 4.0 ns 22.5 ns 197 4.0 ns 28.3 ns 293 4.0 ns 51.0 ns 145 4.0 ns 25.0 ns 200 4.0 ns 25.0 ns 147 4.0 ns 15.0 ns 190 3.8 ns 36.4 ns 166 3.7 ns 11.7 ns 181 3.7 ns 18.3 ns 146 3.7 ns 50.0 ns 151 3.7 ns 12.8 ns 167 3.5 ns 15.0 ns 204 3.5 ns 30.0 ns 157 3.0 ns 29.0 ns 156 3.0 ns 6.7 ns 201 2.0 ns 25.0 ns Dryander 163 5.0 ns 25.0 ns 168 5.0 ns 60.0 ns 173 5.0 ns 50.0 ns 295 5.0 ns 35.0 ns 296 5.0 ns 48.3 ns 297 5.0 ns 37.5 ns 161 4.8 ns 46.0 ns 193 4.7 ns 23.3 ns

Provenance Clone Disease Ratinga Leaf area affected (%) 175 4.7 ns 40.0 ns 361 4.7 ns 40.0 ns 395 4.7 ns 28.3 ns 172 4.5 ns 30.0 ns 401 4.5 ns 22.5 ns 182 4.3 ns 11.7 ns 194 4.3 ns 33.3 ns 397 4.3 ns 26.7 ns 364 4.3 ns 15.0 ns 160 4.2 ns 22.5 ns 196 4.0 ns 35.0 ns 174 4.0 ns 50.0 ns 298 4.0 ns 28.3 ns 366 4.0 ns 36.7 ns 398 4.0 ns 40.0 ns 363 4.0 ns 25.0 ns 367 4.0 ns 30.0 ns 164 3.8 ns 9.4 ns 396 3.8 ns 31.0 ns 149 3.7 ns 11.7 ns 184 3.7 ns 25.0 ns 294 3.5 ns 15.0 ns 365 3.5 ns 17.5 ns 362 3.3 ns 15.0 ns 310 3.3 ns 15.0 ns 359 3.3 ns 47.5 ns 360 3.3 ns 7.3 ns 162 3.2 ns 9.5 ns 195 3.0 ns 5.0 ns 158 3.0 ns 17.5 ns 159 3.0 ns 7.5 ns 399 3.0 ns 7.3 ns 400 3.0 ns 5.0 ns 309 2.5 ns 3.5 ns Eumundi 39 5.0 ns 25.0 ns 93 5.0 ns 20.0 ns 113 5.0 ns 37.5 ns 134 5.0 ns 25.0 ns 280 5.0 ns 25.0 ns 369 5.0 ns 28.3 ns 7 5.0 ns 50.0 ns 21 4.7 ns 15.0 ns 64 4.7 ns 40.0 ns 12 4.7 ns 51.7 ns 17 4.7 ns 40.0 ns 20 4.7 ns 23.3 ns 95 4.7 ns 17.5 ns 110 4.7 ns 31.7 ns 130 4.7 ns 38.3 ns

Provenance Clone Disease Ratinga Leaf area affected (%) 276 4.7 ns 35.0 ns 277 4.7 ns 40.0 ns 285 4.7 ns 43.3 ns 368 4.7 ns 63.3 ns 89 4.5 ns 47.5 ns 36 4.5 ns 35.0 ns 106 4.5 ns 32.5 ns 278 4.5 ns 15.0 ns 13 4.3 ns 18.3 ns 14 4.3 ns 31.7 ns 33 4.3 ns 36.7 ns 35 4.3 ns 31.7 ns 37 4.3 ns 35.0 ns 52 4.3 ns 13.3 ns 55 4.3 ns 28.3 ns 66 4.3 ns 25.0 ns 88 4.3 ns 23.3 ns 100 4.3 ns 23.3 ns 279 4.3 ns 33.3 ns 284 4.2 ns 28.8 ns 34 4.0 ns 8.3 ns 40 4.0 ns 16.7 ns 54 4.0 ns 28.3 ns 56 4.0 ns 21.7 ns 65 4.0 ns 18.3 ns 92 4.0 ns 40.0 ns 112 4.0 ns 42.3 ns 119 4.0 ns 56.7 ns 131 4.0 ns 30.0 ns 19 4.0 ns 27.5 ns 273 4.0 ns 25.0 ns 274 4.0 ns 12.5 ns 282 4.0 ns 20.0 ns 299 4.0 ns 22.5 ns 105 3.8 ns 17.5 ns 370 3.7 ns 12.3 ns 18 3.5 ns 17.5 ns 275 3.5 ns 15.0 ns 283 3.5 ns 15.0 ns 414 3.5 ns 35.0 ns 11 3.3 ns 30.0 ns 53 3.3 ns 11.7 ns 94 3.3 ns 12.5 ns 281 3.3 ns 17.3 ns 118 3.0 ns 16.7 ns 38 1.0 ns 0.0 ns Limpinwoodc Maroochy 127 5.0 ns 62.5 ns 129 5.0 ns 25.0 ns

Provenance Clone Disease Ratinga Leaf area affected (%) 102 4.7 ns 36.7 ns 103 4.7 ns 68.3 ns 87 4.5 ns 42.5 ns 104 4.5 ns 50.0 ns 101 4.0 ns 18.3 ns 128 4.0 ns 25.0 ns 287 4.0 ns 25.0 ns 78 4.0 ns 25.0 ns 124 4.0 ns 10.0 ns 48 4.0 ns 20.0 ns 286 3.7 ns 15.0 ns 49 3.3 ns 18.3 ns 126 3.0 ns 5.7 ns 125 3.0 ns 25.0 ns 79 2.0 ns 5.0 ns Mt Elliot 347 * ns * ns 185 5.0 ns 52.5 ns 348 4.5 ns 37.5 ns 341 4.5 ns 42.5 ns 349 4.3 ns 21.7 ns 350 4.0 ns 15.0 ns 187 4.0 ns 17.5 ns 188 4.0 ns 25.0 ns 340 4.0 ns 20.0 ns 186 3.7 ns 41.7 ns 342 3.5 ns 32.5 ns 351 3.5 ns 19.3 ns 352 3.5 ns 25.0 ns Noosa 32 * ns * ns 22 4.7 ns 25.0 ns 63 4.7 ns 15.0 ns 23 4.5 ns 30.0 ns 30 4.5 ns 20.0 ns 61 4.3 ns 33.3 ns 9 4.2 ns 8.2 ns 31 4.0 ns 28.3 ns 51 4.0 ns 10.0 ns 83 4.0 ns 42.5 ns 50 3.7 ns 13.3 ns 82 3.7 ns 26.7 ns 4 3.5 ns 22.5 ns 135 3.5 ns 30.0 ns 136 3.3 ns 11.7 ns 3 3.2 ns 15.0 ns 24 3.0 ns 10.0 ns 8 2.7 ns 15.0 ns 62 2.5 ns 10.0 ns Silver Valley RL1 4.5 ns 13.1 bd RL3 4.4 ns 25.0 a

Provenance Clone Disease Ratinga Leaf area affected (%) RL9 4.4 ns 23.0 abcd RL7 4.1 ns 13.3 cd RL4 4.1 ns 19.4 abcd RL10 4.0 ns 24.2 ab RL5 3.9 ns 11.7 d RL6 3.9 ns 11.6 cd RL2 3.8 ns 12.0 cd RL8 3.6 ns 21.5 abc St Bees Island 317 5.0 ns 55.0 ns 322 5.0 ns 58.3 ns 332 5.0 ns 50.0 ns 290 5.0 ns 50.0 ns 302 5.0 ns 75.0 ns 315 5.0 ns 50.0 ns 330 5.0 ns 10.0 ns 334 4.7 ns 20.0 ns 320 4.5 ns 25.0 ns 331 4.5 ns 20.0 ns 337 4.5 ns 30.0 ns 319 4.4 ns 37.0 ns 335 4.3 ns 41.7 ns 327 4.3 ns 56.7 ns 328 4.3 ns 20.0 ns 336 4.3 ns 25.7 ns 338 4.3 ns 40.0 ns 304 4.3 ns 28.3 ns 316 4.0 ns 10.0 ns 289 4.0 ns 30.0 ns 333 3.7 ns 18.3 ns 329 3.5 ns 30.0 ns 318 3.3 ns 7.0 ns Woondum 402 * ns * a 27 5.0 ns 17.5 efghijklmn 28 5.0 ns 50.0 abcdefghij 405 5.0 ns 15.0 defghijklmn 57 5.0 ns 50.0 bcd 58 5.0 ns 47.5 bcdegh 74 5.0 ns 95.0 a 81 5.0 ns 30.0 cdefghijklmn 108 5.0 ns 50.0 abcdefghij 325 5.0 ns 17.5 efghijklmn 418 5.0 ns 75.0 ab 424 5.0 ns 75.0 ab 426 5.0 ns 25.0 cdefghijklmn 428 5.0 ns 25.0 cdefghijklmn 429 5.0 ns 25.0 cdefghijklmn 431 5.0 ns 15.0 efghijklmn 26 4.7 ns 50.0 bcd 76 4.7 ns 58.3 abc

Provenance Clone Disease Ratinga Leaf area affected (%) 326 4.7 ns 13.3 fijklmn 67 4.7 ns 50.0 bcd 29 4.5 ns 17.5 efghijklmn 47 4.5 ns 22.5 defghijklmn 345 4.5 ns 50.0 bcde 423 4.5 ns 17.5 efghijklmn 16 4.3 ns 25.0 defghijklmn 91 4.3 ns 12.5 hijklmn 116 4.3 ns 30.0 cdefghijklmn 427 4.3 ns 33.3 cdefghijklm 2 4.0 ns 45.0 bcdeg 6 4.0 ns 17.5 efghijklmn 42 4.0 ns 40.0 bcdefgh 43 4.0 ns 8.0 fijklmn 46 4.0 ns 17.5 efghijklmn 70 4.0 ns 18.3 efghijklmn 71 4.0 ns 16.7 ghijklmn 72 4.0 ns 30.0 cdefghijklmn 80 4.0 ns 11.7 fijklmn 99 4.0 ns 6.0 kmn 109 4.0 ns 15.7 hijklmn 120 4.0 ns 26.7 defghijklmn 123 4.0 ns 18.3 efghijklmn 303 4.0 ns 10.0 defghijklmn 344 4.0 ns 10.0 jkmn 346 4.0 ns 35.0 bcdefghijkl 403 4.0 ns 28.3 defghijklmn 407 4.0 ns 25.0 cdefghijklmn 408 4.0 ns 2.0 fijklmn 409 4.0 ns 18.3 efghijklmn 410 4.0 ns 10.0 defghijklmn 413 4.0 ns 26.7 defghijklmn 415 4.0 ns 7.5 jkmn 59 4.0 ns 16.7 ghijklmn 117 4.0 ns 25.0 defghijklmn 430 4.0 ns 17.5 efghijklmn 432 4.0 ns 21.7 defghijklmn 433 4.0 ns 25.0 cdefghijklmn 440 4.0 ns 25.0 defghijklmn 394 3.8 ns 18.3 fijklmn 60 3.8 ns 11.0 lmn 44 3.7 ns 7.3 lmn 98 3.7 ns 8.3 kmn 292 3.7 ns 25.7 defghijklmn 393 3.7 ns 24.2 efghijklmn 420 3.7 ns 12.0 ilmn 5 3.7 ns 7.5 jkmn 75 3.7 ns 4.0 n 96 3.7 ns 11.7 fijklmn

Provenance Clone Disease Ratinga Leaf area affected (%) 121 3.7 ns 10.0 jkmn 122 3.7 ns 35.0 bcdefghijkl 115 3.5 ns 26.0 defghijklmn 416 3.5 ns 15.0 efghijklmn 69 3.5 ns 22.5 defghijklmn 288 3.5 ns 11.0 fijklmn 425 3.5 ns 30.0 cdefghijklmn 107 3.4 ns 33.4 cdefghjk 422 3.4 ns 9.4 lmn 1 3.3 ns 4.7 mn 90 3.3 ns 23.3 defghijklmn 343 3.3 ns 23.3 defghijklmn 421 3.3 ns 13.3 fijklmn 419 3.3 ns 16.3 fijklmn 10 3.0 ns 5.0 ghijklmn 41 3.0 ns 6.0 kmn 45 3.0 ns 5.0 ghijklmn 68 3.0 ns 25.0 cdefghijklmn 114 3.0 ns 7.5 jkmn 412 3.0 ns 6.0 kmn 15 3.0 ns 7.3 lmn 291 3.0 ns 20.0 efghijklmn 25 2.5 ns 10.0 fijklmn 404 2.3 ns 10.0 jkmn 73 2.0 ns 25.0 defghijklmn 411 2.0 ns 2.5 mn a within clone ratings followed by the same letter show means that do not differ significantly (pairwise comparison P<0.05) while ns indicates no significant difference at P<0.05 b *Indicates missing values and therefore no estimate c Limpinwood has only 1 clone and was therefore not able to be analysed

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