Investigation of the fungi associated with dieback of prickly ( nilotica subsp. indica) in Northern

AHSANUL HAQUE

B.Sc. Ag. (Hons) (Bangladesh Agricultural University, Bangladesh) MS in Pathology (Bangabandhu Sheikh Mujibur Rahman Agricultural University, Bangladesh)

A thesis submitted for the degree of Doctor of Philosophy at The University of in 2015 School of Agriculture and Food Sciences Abstract

Prickly acacia ( subsp. indica), one of the most harmful weeds of the Australian rangelands, has been occasionally observed displaying natural dieback symptoms since 1970’s. More recently in 2010, a prominent widespread dieback event was observed among the growing around Richmond and Julia Creek in north-western Queensland. Affected plants were found with disease symptoms such as; ashy internal staining, defoliation, blackening of shoot tips through to widespread plant mortality. It was hypothesized that a pathogenic /i could be implicated with this phenomenon and a potential for biological control of this might ensue.

Fungi isolated from dieback-affected and healthy stands of prickly acacia were putatively identified by partial sequencing of the Internal Transcribed Spacer (ITS) region of genomic DNA. was the best represented family of fungi associated with dieback. Among the Botryosphaeriaceae fungi, Cophinforma was found to be the most prevalent genus with 60% of the total isolates identified as Cophinforma spp. following BLAST searches. Cophinforma was also isolated from the healthy plants growing in the trial site at Richmond. Natural dieback on prickly acacia was previously observed in the surrounding areas. Phylogenetic analysis of the ITS sequences revealed the potential existence of new species of Cophinforma in Australia. Apart from Cophinforma sp., a range of other Botryosphaeriaceae and non-Botryosphaeriaceae fungi were isolated from both dieback-affected and healthy prickly acacia plants growing at different locations.

Based on a seedling pathogenicity screening, a sub-set of 40 potentially pathogenic isolates across the range of species were selected to challenge the juvenile plants following a stem inoculation technique under glasshouse conditions. In the glasshouse trials, the test-isolates of Cophinforma sp. (isolated from both dieback-affected and healthy prickly acacia) and an isolate of Lasiodiplodia pseudotheobromae strain NT039 (isolated from dieback-affected aculeata) consistently caused significant stem lesions, gummosis and mortality. Cophinforma sp., Fusarium acuminatum, Neofusicoccum parvum and Tiarosporella graminis isolated from healthy prickly acacia also caused similar symptoms in glasshouse inoculation trials. The remaining species tested in the glasshouse trials were mostly non-pathogenic to the juvenile plants.

In another study, given the observation that several fungi are often isolated from a single piece of dieback-affected stem, it was investigated whether typical dieback symptoms in prickly acacia could be replicated with combinations of different fungi. Therefore, the effect of inoculating with

ii two isolates of Cophinforma sp. and one isolate of L. pseudotheobromae singly and in combination under glasshouse and field conditions was tested. In the glasshouse trial, significant stem lesions were observed with each of the test-isolates and their combinations. However, there was no significant increase in lesion length with any Cophinforma-Lasiodiplodia combinations compared to the individual effect of L. pseudotheobromae. An almost similar pattern was observed for leaf mortality. The glasshouse trial was followed by two field trials (experimental repeats) established at Rockhampton, central Queensland and Richmond, north-western Queensland. The test-fungi were delivered using a gelatine capsule delivery system into holes drilled into the stem. In the field trial conducted at Rockhampton, external stem lesions with streaming of sap were observed. However, the lesion length was significantly greater only in presence of L. pseudotheobromae. In another field trial conducted at Richmond, no external stem lesions were visible due to the masking effect of thick dark . No significant dieback symptoms were observed on aerial plant parts during the course of the trial at Rockhampton. In contrast, at Richmond, widespread branch mortality and thinning of leaf cover leading to significant vigour decline was observed at approximately two years after inoculation. However, a similar degree of decline was also observed on the untreated plants neighbouring the trial site suggesting environmental stresses (drought) may have played a major part in decline.

A series of experiments were designed to further investigate whether increased stress to the host might facilitate development of dieback. In the first set of experiments the stress was produced by application of a sub-lethal dose of the glyphosate (N-(phosphonomethyl) glycine) and in the second set controlled application of water to produce osmotic stress was applied. In a glasshouse trial, plants were stem-inoculated with L. pseudotheobromae and co-treated with a pre-determined sub-lethal dose of glyphosate (45g/L a.i @100 µL/plant). Significantly longer external stem lesions, higher levels of leaf mortality and reduced plant height were recorded with glyphosate co-treatment. The glasshouse trial was followed by two field trials (experimental repeats) at Rockhampton, central Queensland and Richmond, north-western Queensland. L. pseudotheobromae was combined with none or either of two sub-lethal doses of glyphosate (180g/L a.i @ 0.5 or 1.0 mL/plant). At Rockhampton, both L. pseudotheobromae and sub-lethal glyphosate caused significant external stem lesions which developed into a stem canker. However, there was no significant glyphosate- fungus interaction effect on canker length. At Richmond, no external stem lesion was visible. At approximately two years after inoculation, a noticeable reduction of plant vigour was observed at both trial sites.

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In the second glasshouse study, the interactions between drought stress and Cophinforma strain AN71 was investigated across three watering regimens. Stem inoculation caused 100% infection with significant increases in lesion lengths in the presence of drought stress. Leaf mortality also increased significantly with stress and its interaction with Cophinforma inoculation. Although Cophinforma caused larger lesions and leaf mortality, which increased with drought stress, there was no significant stress-fungus interaction effect on plant growth as measured by total leaf production, height increase and stem and branch dry-weight.

This thesis has confirmed that the endophytic Botryosphaeriaceae and other fungi were associated with prickly acacia dieback. Of these, species of Cophinforma are the most likely to be the causative fungus. Another native fungus L. pseudotheobromae also caused significant disease symptoms in prickly acacia. However, confirmation of the potential of Cophinforma and L. pseudotheobromae to be utilized as bioherbicide for prickly acacia will require further investigations. Such other natively occurring dieback fungi may also be useful in biocontrol of prickly acacia. This study has also demonstrated that the Botryosphaeriaceae fungi caused more severe disease symptoms on stressed hosts. To confirm whether such stress-fungus interactions were involved in the natural decline observed in prickly acacia will also require further investigations with simulation of other probable stressors and other likely modes of infection by the Botryosphaeriaceae fungi.

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Declaration by author

This thesis is composed of my original work, and contains no material previously published or written by another person except where due reference has been made in the text. I have clearly stated the contribution by others to jointly-authored works that I have included in my thesis.

I have clearly stated the contribution of others to my thesis as a whole, including statistical assistance, survey design, data analysis, significant technical procedures, professional editorial advice, and any other original research work used or reported in my thesis. The content of my thesis is the result of work I have carried out since the commencement of my research higher degree candidature and does not include a substantial part of work that has been submitted to qualify for the award of any other degree or diploma in any university or other tertiary institution. I have clearly stated which parts of my thesis, if any, have been submitted to qualify for another award.

I acknowledge that an electronic copy of my thesis must be lodged with the University Library and, subject to the policy and procedures of The University of Queensland, the thesis be made available for research and study in accordance with the Copyright Act 1968 unless a period of embargo has been approved by the Dean of the Graduate School.

I acknowledge that copyright of all material contained in my thesis resides with the copyright holder(s) of that material. Where appropriate I have obtained copyright permission from the copyright holder to reproduce material in this thesis.

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Publications during candidature

Haque, A, Galea, V, Bisset, A & van Klinken, RD 2012, 'A preliminary investigation of prickly acacia dieback (Acacia nilotica ssp. indica)', in V Eldershaw (ed.), Eighteenth Australasian Weeds Conference, Melbourne, pp. 131-4.

Haque, A, Galea, V, Goulter, K & van Klinken, RD (2013), 'Botryosphaeriaceae fungi as a potential mycoherbicide for prickly acacia', in Abstract and Programme Book: The 19th Australasian Plant Pathology Conference, Auckland, New Zealand, p. 91. Received early career APPS-HAL travel award for one of the best papers presented in the 19th Australasian Plant Pathology Conference.

Haque, A, Galea, V, Goulter, K & van Klinken, RD (2013), 'Interaction between abiotic stress and fungal infection in prickly acacia', in Abstract and Programme Book: The 19th Australasian Plant Pathology Conference, Auckland, New Zealand, p. 168.

Publications included in this thesis

No publications included.

Contributions by others to the thesis

No contributions by others.

Statement of parts of the thesis submitted to qualify for the award of another degree

None.

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Acknowledgements

I am using this opportunity to express my gratitude to everyone who supported me throughout the course of this PhD project at The University of Queensland, Australia. I am thankful for their aspiring guidance, invaluably constructive criticism and friendly advice during the project work. I am sincerely grateful to them for sharing their truthful and illuminating views on a number of issues related to this PhD project.

I express my sincere gratitude and warm regards to my principal advisor Associate Prof. Dr. Victor J. Galea for the continuous support to my PhD research, for his patience, motivation, enthusiasm and immense knowledge. His guidance helped me through the research and writing of this thesis. I could not have imagined having a better advisor and mentor for my PhD study. I also express my warm thanks to my associate advisors Dr. Ken Goulter and Dr. Rieks van Klinken for their kind support and guidance toward the completion of this research project. Special thanks to Dr. Ken for training me on a number of classical and molecular plant pathology techniques and for providing PCR primers and access to the PAUP software package. Thanks again to my advisors for editing my thesis.

Thanks to the members of “Plant Health and Bioherbicide Research” group at The University of Queensland, Gatton for their great support and constructive criticism to my research project. It was a wonderful experience to be a member of such a dynamic group. Special thanks to Ms Aniline Sacdalan and Ms Arslan Jabeen for assisting me to organize the group meetings.

I also thank to Mr Victor Robertson, Mr Lachlan Fowler, Ms Mel Schneemilch and Ms Heidi Robertson for their technical assistance. Thanks to Mr John Swift for advising me on the occupational health and safety regulations. Special thanks to Mr Allan Lisle for his guidance and support during statistical analysis. Thanks to the library staff for their kind assistance throughout my candidature.

Thanks to the Rockhampton Regional Council for access to a field site and to Mr Leonard Milner, Pink Lilly, Rockhampton and Mr Corbett Tritton, “Silver Hills Station”, Richmond, for the generous provision of their properties for research purposes and for kind assistance during the field work. Thanks again to Associate Prof. Galea and Dr. Goulter for their kind assistance throughout my fieldwork. Thanks to Ms Naomi Diplock for her help to establish one of the field trials. Thanks to Ms Subrima Islam for her kind assistance to establish the water stress trial.

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Very special thanks to the Australian Government Department of Education and Training for awarding me the prestigious Endeavour Postgraduate Award to undertake this PhD study at The University of Queensland. Thanks to Ms Leanne Pooley for processing my initial application for admission at The University of Queensland. Many thanks to Ms Kaye Hunt for her continuous administrative support and regular “Communi-Kaye-tion”. Warm regards to Bangladesh Rice Research Institute for approving my study leave.

I also thank the graduate school at The University of Queensland, Meat and Livestock Australia, BioHerbicides Australia Pty Ltd, Rural Industries Research and Development Corporation and UniQuest for their financial support to this research project. Special thanks to my current and former case managers at Scope Global Pty. Ltd. for their continuous assistance during the course of my scholarship.

Thank you.

Keywords

Prickly acacia, fungi, dieback, weed, biological control, bioherbicide.

Australian and New Zealand Standard Research Classifications (ANZSRC)

ANZSRC code: 060704 Plant Pathology, 50% ANZSRC code: 070308 Crop and Pasture Protection (Pests, Diseases and Weeds), 50%

Fields of Research (FoR) Classification

FoR code: 0607 Plant Biology 50%, FoR code: 0703 Crop and Pasture Production 50%

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Table of Contents

Abstract ……...... ii Declaration by author ...... v Publications during candidature ...... vi Publications included in this thesis ...... vi Contributions by others to the thesis...... vi Statement of parts of the thesis submitted to qualify for the award of another degree ...... vi Acknowledgements...... vii Keywords ……………...... viii Australian and New Zealand Standard Research Classifications (ANZSRC) ...... viii Fields of Research (FoR) Classification ...... viii Table of Contents ...... ix List of Figures ……………………………………………………………………………...……...xii List of Tables .. ………………………………………………………………………….………..xviii List of Abbreviations ...... xix

Chapter 1: General introduction, literature review and research objectives ...... 1

1.1 General Introduction ...... 1 1.2 Literature review ...... 1 1.2.1 Prickly acacia ...... 1 1.2.2 Dieback ...... 8 1.2.3 Possible causes of dieback ...... 10 1.2.4 Interaction between drought stress and opportunistic fungi ...... 17 1.2.5 Dieback of prickly acacia and other invasive weeds in Australia ...... 19 1.2.6 Fungi in weed bio-control ...... 21 1.3 Research objectives ...... 27 1.4 References ...... 28

Chapter 2: Identification and pathogenicity to seedlings and juvenile plants of the fungi associated with dieback of prickly acacia ...... 58

2.1 Abstract ...... 58 2.2 Introduction ...... 59 2.3 Materials and Methods ...... 61 2.3.1 Sample collection ...... 61 2.3.2 Isolate acquisition ...... 62 2.3.3 Identification of the fungal isolates ...... 63 ix

2.3.4 Prioritisation of fungal isolates for glasshouse inoculation studies ...... 65 2.3.5 Glasshouse inoculation trials ...... 67 2.4 Results ...... 69 2.4.1 Identification ...... 69 2.4.2 Phylogenetic analysis ...... 72 2.4.3 Seedling bioassay ...... 76 2.4.4 Glasshouse trials ...... 78 2.5 Discussion ...... 89 2.6 References ...... 93

Chapter 3: Glasshouse and field infection studies in prickly acacia using fungi isolated from dieback-affected ...... 103

3.1 Abstract ...... 103 3.2 Introduction ...... 104 3.3 Materials and Methods ...... 106 3.3.1 Glasshouse and laboratory studies ...... 106 3.3.2 Field trials ...... 109 3.4 Results ...... 112 3.4.1 Glasshouse trial ...... 112 3.4.2 Field trial ...... 116 3.5 Discussion ...... 123 3.6 References ...... 127

Chapter 4: Interactions of a pathogenic strain of Lasiodiplodia pseudotheobromae with sub- lethal glyphosate in prickly acacia ...... 133

4.1 Abstract ...... 133 4.2 Introduction ...... 134 4.3 Materials and Methods ...... 136 4.3.1 Glasshouse trial ...... 136 4.3.2 Field trials ...... 138 4.4 Results ...... 141 4.4.1 Glasshouse trial ...... 141 4.4.2 Field trial ...... 145 4.5 Discussion ...... 152 4.6 References ...... 155

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Chapter 5: Interaction between drought stress and Cophinforma sp. in prickly Acacia dieback ……...... 162

5.1 Abstract ...... 162 5.2 Introduction ...... 162 5.3 Materials and Methods ...... 164 5.3.1 Experimental design and treatments ...... 164 5.3.2 Inoculation ...... 165 5.3.3 Trial assessment ...... 165 5.3.4 Confirmation of infection...... 165 5.3.5 Data analysis ...... 166 5.4 Results ...... 166 5.4.1 Stem lesions ...... 166 5.4.2 Leaf production and leaf mortality...... 166 5.4.3 Plant growth ...... 167 5.4.4 Confirmation of infection...... 168 5.5 Discussion ...... 171 5.6 References ...... 172

Chapter 6: General Discussion ...... 177

6.1 Background...... 177 6.2 Major findings ...... 177 6.3 Conclusion and recommendation for future research ...... 182 6.4 References ...... 183

Appendices …...... ………………………………………….190

Appendix 1 List of the fungal isolates identified and tested using a seedling bioassay under laboratory conditions...... 190 Appendix 2 Average temperature recorded near the trial sites at Rockhampton and Richmond over the trial period (Source: Australian Bureau of Meteorology)...... 197 Appendix 3 Rainfall (mm) received by the trial sites at Rockhampton and Richmond from 6 months before inoculation through the trial period (Source: Australian Bureau of Meteorology)……………………………………………………………………………………198

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List of Figures

Fig. 1.1 Prickly acacia: A. ; B. spine; C. ; D. seed pods and E. seed...... 2

Fig. 1.2 Current and potential distribution of prickly acacia under current climatic conditions conditions (Kriticos et al. 2003)...... 4

Fig. 1.3 Climatic suitability of prickly acacia under different climates scenarios A. Current climate; B-D. +2°C temperature, -10% rainfall; E-G. +2°C temperature, +10% rainfall (Kriticos et al. 2003)...... 5

Fig. 1.4 Symptoms commonly associated with dieback of species; A-B. cankerous external and internal lesions; C. necrosis; D. gummosis; E. wilting and F. plant mortality (Munkvold 2001; Jung 2012; Aghighi et al. 2014; Linaldeddu et al. 2014; McKinney et al. 2014). . 9

Fig. 1.5 Symptoms of prickly acacia dieback A-B. widespread plant mortality; C. development of water suckers and D. internal staining (Galea 2011)...... Error! Bookmark not defined.

Fig. 2.1 Map showing the sampling sites () at north-western Queensland...... 61

Fig. 2.2 Maximum parsimony tree based on bootstrap analyses of the ITS sequences from Cophinforma isolates from dieback-affected and apparently healthy samples of prickly acacia. The isolates in bold are of type species (ex or holo) obtained from NCBI database. The tree was rooted to P. stromaticum and all bootstrap support values are shown…………………………………….. 73

Fig. 2.3 Maximum parsimony tree based on bootstrap analyses of the ITS sequences from different Botryosphaeriaceae fungi from dieback-affected and apparently healthy samples of prickly acacia (except L. pseudotheobromae isolated from dieback-affected P. aculeata). The isolates in bold are of type species (ex or holo) obtained from NCBI database. The tree was rooted to M. phaseolina and bootstrap support values over 50% are shown...... 74

Fig. 2.4 Maximum parsimony tree based on bootstrap analyses of the ITS sequences from different fungi (except Botryosphaeriaceae) isolated from dieback-affected and apparently healthy samples of prickly acacia. The isolates in bold are of type species (ex or holo) obtained from NCBI database. The tree was rooted to M. phaseolina and bootstrap support values over 50% are shown...... 75

Fig. 2.5 Different categories of seedlings in the bioassay: A. pre-emergent damping off; B. post- emergent damping off; C-D. diseased (soft rot, tip necrosis) seedlings; E. seedling with necrotic hypocotyl lesion; F. severe stunting and G. a negative control seedling...... 76

Fig. 2.6 Percentage of isolates of individual fungal species showing different pathogenicity reponse to prickly acacia seedlings under laboratory conditions (any species with less than 3 isolates is not presented) ...... 77

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Fig. 2.7 External stem lesions observed with A. negative control; B. Cophinforma sp. and C. L. pseudotheobromae at 8 weeks under glasshouse conditions...... 79

Fig. 2.8 Length of external and internal stem lesions recorded with the isolates of Cophinforma spp. and L. pseudotheobromae under glasshouse conditions at 8 weeks. Columns with different letters on the error bars indicate significant difference among the treatments by Tukey’s test with 95% confidence in GLM ANOVA (n=5). On the X axis, each capital letter represents individual treatment where A = negative control, B = Cophinforma sp. (AN17), C = Cophinforma sp. (AN32), D = Cophinforma sp. (AN49), E = Cophinforma sp. (AN71), F = Cophinforma sp. (AN114), G = Cophinforma sp. (MP290) and H = L. pseudotheobromae (NT039)...... 79

Fig. 2.9 Effect of Cophinforma spp. and L. pseudotheobromae on (A) total leaf production and (B) leaf mortality recorded under glasshouse conditions at 8 weeks. Columns with different letters on the error bars indicate significant difference by Tukey’s test with 95% confidence in GLM ANOVA (n=5). On the X axis, each capital letter represents individual treatment where A = negative control, B = Cophinforma sp. (AN17), C = Cophinforma sp. (AN32), D = Cophinforma sp. (AN49), E = Cophinforma sp. (AN71), F = Cophinforma sp. (AN114), G = Cophinforma sp. (MP290) and H = L. pseudotheobromae (NT039)...... 80

Fig. 2.10 A. internal stem lesion; B. leaf mortality observed with Cophinforma sp...... 82

Fig. 2.11 Length of external and internal stem lesions recorded with the isolates of Cophinforma sp. and L. pseudotheobromae under glasshouse conditions at 8 weeks. Columns with different letters on the error bars indicate significant difference by Tukey’s test with 95% confidence in GLM ANOVA (n=5). On the X axis, each capital letter represents individual treatment where A = negative control, B = Cophinforma sp. (AN42), C = Cophinforma sp. (AN110), D = Cophinforma sp. (AN44), E = Cophinforma sp. (AN16), F = Cophinforma sp. (AN109), G = Cophinforma sp. (AN19), H = Cophinforma sp. (AN26), I = Cophinforma sp. (AN93), J = Cophinforma sp. (AN75), K = Cophinforma sp. (AN68), L = Cophinforma sp. (AN65), M = Cophinforma sp. (AN76) and N = L. pseudotheobromae (NT039)...... 83

Fig. 2.12 Effect Cophinforma sp. (12 isolates) sourced from dieback-affected prickly acacia stem samples and L. pseudotheobromae (1 isolate) on (A) total leaf production and (B) leaf mortality recorded under glasshouse conditions at 8 weeks. Columns with different letters on the error bars indicate significant difference by Tukey’s test with 95% confidence in GLM ANOVA (n=5). On the X axis, each capital letter represents individual treatment where A = negative control, B = Cophinforma sp. (AN42), C = Cophinforma sp. (AN110), D = Cophinforma sp. (AN44), E = Cophinforma sp. (AN16), F = Cophinforma sp. (AN109), G = Cophinforma sp. (AN19), H = Cophinforma sp. (AN26), I = Cophinforma sp. (AN93), J = Cophinforma sp. (AN75), K = Cophinforma sp. (AN68), L = Cophinforma sp. (AN65), M = Cophinforma sp. (AN76) and N = L. pseudotheobromae (NT039)...... 84

Fig. 2.13 Length of external and internal stem lesions recorded with different fungi sourced from dieback-affected prickly acacia stem samples under glasshouse conditions at 8 weeks. Columns with different letters on the error bars indicate significant difference by Tukey’s test with 95% xiii confidence in GLM ANOVA (n=5). On the X axis, each capital letter represents individual treatment where A = negative control, B = Ps. violaceum, C = Ph. citrigena, D = Au. pullulans, E = Paecilomyces sp., F = Cu. pseudorobusta, G = Cu. lunata, H = As. pistaciarum, I = Cy. austromontana, J = Cl. uredinicola, K = Pl. ootheca, L = Al. alternata, M = Py. microspora, N = E. rostratum, O = R. rufulum, P = U. muehlenbergii and Q = L. pseudotheobromae...... 86

Fig. 2.14 Effect of different fungi sourced from apparently healthy prickly acacia stem and root samples on (A) length of stem lesions and (B) amount of dead leaf under glasshouse conditions at 8 weeks. Columns with different letters on error bars indicate significant difference by Tukey’s test with 95% confidence in GLM ANOVA (n=5). On the X axis, each capital letter represents individual treatment where A = negative control, B = F. acuminatum, C = N. parvum, D = Cophinforma sp., E = M. alpina, F = T. graminis and G = L. pseudotheobromae...... 88

Fig. 3.1 External (top row) and internal (bottom row) stem lesions observed with A. control; B. Cophinforma sp. (AN71); C. Cophinforma sp. (AN110) and D. L. pseudotheobromae (NT039) (Scale=1.94 cm)...... 112

Fig. 3.2 (A) Effect of individual fungal isolates on stem lesion length compared to control at 12 weeks under glasshouse conditions; (B) effect of isolate combinations on stem lesion length compared to the individual effect of L. pseudotheobromae (NT039) at 12 weeks under glasshouse conditions. Columns with different letters on error bars indicate significant difference by Tukey’s test with 95% confidence in GLM ANOVA (n=5). On the X axis, each capital letter represents individual treatment where A= control (autoclaved fresh millet grain), B= Cophinforma sp. (AN71), C= Cophinforma sp. (AN110), D= L. pseudotheobromae (NT039), E= Cophinforma sp. (AN71) + Cophinforma sp. (AN110), F= Cophinforma sp. (AN71) + L. pseudotheobromae (NT039), G= Cophinforma sp. (AN110) + L. pseudotheobromae (NT039) and H= Cophinforma sp. (AN71) + Cophinforma sp. (AN110) + L. pseudotheobromae (NT039)...... 113

Fig. 3.3 Effect of individual fungal isolates and their combinations on (A) total leaf production and (B) leaf mortality at 12 weeks under glasshouse conditions. Columns with different letters on error bars indicate significant difference by Tukey’s test with 95% confidence in GLM ANOVA (n=5). On the X axis, each capital letter represents individual treatment where A= control (autoclaved fresh millet grain), B= Cophinforma sp. (AN71), C= Cophinforma sp. (AN110), D= L. pseudotheobromae (NT039), E= Cophinforma sp. (AN71) + Cophinforma sp. (AN110), F= Cophinforma sp. (AN71) + L. pseudotheobromae (NT039), G= Cophinforma sp. (AN110) + L. pseudotheobromae (NT039) and H= Cophinforma sp. (AN71) + Cophinforma sp. (AN110) + L. pseudotheobromae (NT039)...... 114

Fig. 3.4 Effect of individual fungal isolates and their combinations on stem and branch dry-weight. Columns with different letters on error bars indicate significant difference by Tukey’s test with 95% confidence in GLM ANOVA (n=5). On the X axis, each capital letter represents individual treatment where A= control (autoclaved fresh millet grain), B= Cophinforma sp. (AN71), C= Cophinforma sp. (AN110), D= L. pseudotheobromae (NT039), E= Cophinforma sp. (AN71) + Cophinforma sp. (AN110), F= Cophinforma sp. (AN71) + L. pseudotheobromae (NT#039), G=

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Cophinforma sp. (AN110) + L. pseudotheobromae (NT039) and H= Cophinforma sp. (AN71) + Cophinforma sp. (AN110) + L. pseudotheobromae (NT039)...... 115

Fig. 3.5 Mycelial growth rate of L. pseudotheobromae and Cophinforma sp. in single culture. Columns with different letters on error bars indicate significant difference by Tukey’s test with 95% confidence in GLM ANOVA (n=6)...... 116

Fig. 3.6 Stem lesions observed with A. control; B. Cophinforma sp. (AN71); C. Cophinforma sp. (AN110) and D. L. pseudotheobromae (NT039) at 6 months at Rockhampton (Scale=1.94 cm). . 117

Fig. 3.7 (A) Effect of individual fungal isolates on external stem lesion length compared to control and (B) effect of isolate combinations on stem lesion length compared to the individual effect of L. pseudotheobromae recorded in a field trial at Rockhampton at 21 months after inoculation. Columns with different letters on error bars indicate significant difference by Tukey’s test with 95% confidence in GLM ANOVA (n=5). On the X axis, each capital letter represents individual treatment where A= control (autoclaved fresh Millet grain), B= Cophinforma sp. (AN71), C= Cophinforma sp. (AN110), D= L. pseudotheobromae (NT039), E= Cophinforma sp. (AN71) + Cophinforma sp. (AN110), F= Cophinforma sp. (AN71) + L. pseudotheobromae (NT039), G= Cophinforma sp. (AN110) + L. pseudotheobromae (NT039) and H= Cophinforma sp. (AN71) + Cophinforma sp. (AN110) + L. pseudotheobromae (NT039)...... 117

Fig. 3.8 Internal lesions observed with different fungi in field trials conducted at (A) Rockhampton at 21 months after inoculation and (B) Richmond at 23 months after inoculation (Scale=1cm). ... 118

Fig. 3.9 Individual and combination effect of Cophinforma sp. and L. pseudotheobromae on internal stem lesions of prickly acacia recorded in field trials conducted at Rockhampton (at 21 months) and Richmond (at 23 months). Columns with different letters on error bars indicate significant difference by Tukey’s test with 95% confidence in GLM ANOVA (n=5). On the X axis, each capital letter represents individual treatment where A= control (autoclaved fresh millet grain), B= Cophinforma sp. (AN71), C= Cophinforma sp. (AN110), D= L. pseudotheobromae (NT039), E= Cophinforma sp. (AN71) + Cophinforma sp. (AN110), F= Cophinforma sp. (AN71) + L. pseudotheobromae (AN#71), G= Cophinforma sp. (AN110) + L. pseudotheobromae (NT039) and H= Cophinforma sp. (AN71) + Cophinforma sp. (AN110) + L. pseudotheobromae (NT039)...... 119

Fig. 3.10 Effect of different fungal isolates and their combinations on overall vigour of prickly acacia recorded from May 2012 to February 2014 in a field trial conducted at Rockhampton, central Queensland. Columns with different letters on error bars indicate significant difference by Tukey’s test with 95% confidence in GLM ANOVA (n=4). On the X axis, each capital letter represents individual treatment where A= control (autoclaved fresh millet grain), B= Cophinforma sp. (AN71), C= Cophinforma sp. (AN110), D= L. pseudotheobromae (NT039), E= Cophinforma sp. (AN71) + Cophinforma sp. (AN110), F= Cophinforma sp. (AN71) + L. pseudotheobromae (AN#71), G= Cophinforma sp. (AN110) + L. pseudotheobromae (NT039) and H= Cophinforma sp. (AN71) + Cophinforma sp. (AN110) + L. pseudotheobromae (NT039)...... 121

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Fig. 3.11 Effect of different fungal isolates and their combinations on overall vigour of prickly acacia recorded from June 2012 to May 2014 in a field trial conducted at Richmond, north western Queensland. Columns with different letters on error bars indicate significant difference by Tukey’s test with 95% confidence in GLM ANOVA (n=4). On the X axis, each capital letter represents individual treatment where A= control (autoclaved fresh millet grain), B= Cophinforma sp. (AN71), C= Cophinforma sp. (AN110), D= L. pseudotheobromae (NT039), E= Cophinforma sp. (AN71) + Cophinforma sp. (AN110), F= Cophinforma sp. (AN71) + L. pseudotheobromae (AN#71), G= Cophinforma sp. (AN110) + L. pseudotheobromae (NT039), H= Cophinforma sp. (AN71) + Cophinforma sp. (AN110) + L. pseudotheobromae (NT039) and I= Untreated plants. . 122

Fig. 4.1 (A) External stem lesions observed with L. pseudotheobromae, sub-lethal glyphosate and their combinations at 6 weeks (Scale=1.94 cm); (B) Effect of L. pseudotheobromae, sub-lethal glyphosate and their combinations on length of stem lesion at 6 weeks under glasshouse conditions. Columns with different letters on error bars indicate significant difference by Tukey’s test with 95% confidence in GLM ANOVA (n=5). On the X axis, each capital letter represents individual treatment where A= control (autoclaved fresh millet grains), B= glyphosate 45g/L a.i @ 100 µL/plant, C= L. pseudotheobromae, D= L. pseudotheobromae + glyphosate 45g/L a.i @ 100 µL/plant (applied into same hole) and E= L. pseudotheobromae + glyphosate 45g/L a.i @ 100 µL/plant (applied into different holes)...... 142

Fig. 4.2 Effect of L. pseudotheobromae, sub-lethal glyphosate and their combinations on (A) total leaf production and (B) leaf mortality of prickly acacia at 12 weeks under glasshouse conditions. Columns with different letters on error bars indicate significant difference by Tukey’s test with 95% confidence in GLM ANOVA (n=5). On the X axis, each capital letter represents individual treatment where A= control (autoclaved fresh millet grains), B= glyphosate 45g/L a.i @ 100 µL/plant, C= L. pseudotheobromae, D= L. pseudotheobromae + glyphosate 45g/L a.i @ 100 µL/plant (applied into same hole) and E= L. pseudotheobromae + glyphosate 45g/L a.i @ 100 µL/plant (applied into different holes)...... 143

Fig. 4.3 Effect of L. pseudotheobromae, sub-lethal glyphosate and their combinations on (A) stem and branch dry-weight and (B) height increment at 12 weeks. Columns with different letters on error bars indicate significant difference by Tukey’s test with 95% confidence in GLM ANOVA (n=5). On the X axis, each capital letter represents individual treatment where A= control (autoclaved fresh millet grains), B= glyphosate 45g/L a.i @ 100 µL/plant, C= L. pseudotheobromae, D= L. pseudotheobromae + glyphosate 45g/L a.i @ 100 µL/plant (applied into same hole) and E= L. pseudotheobromae + glyphosate 45g/L a.i @ 100 µL/plant (applied into different holes)...... 144

Fig. 4.4 External stem lesions observed with different treatments at 6 months after inoculation in a field trial at Rockhampton, central Queensland- A. control; B. glyphosate; C. L. pseudotheobromae and D. L. pseudotheobromae + glyphosate (Scale=10 cm)...... 145

Fig. 4.5 Stem canker observed with different treatments at 14 months after inoculation in a field trial at Rockhampton, central Queensland- A. control; B. glyphosate; C. L. pseudotheobromae and D. L. pseudotheobromae + glyphosate (Scale=10 cm)...... 146 xvi

Fig. 4.6 Effect of L. pseudotheobromae and sub-lethal glyphosate on external stem lesion in prickly acacia recorded in field trials conducted at Rockhampton, central Queensland. Columns with different letters on error bars indicate significant difference by Tukey’s test with 95% confidence in GLM ANOVA (n=4). On the X axis, each capital letter represents individual treatment where A= control (autoclaved fresh millet grain), B= L. pseudotheobromae, C= glyphosate 180g/L a.i @ 0.5 mL/plant, D= glyphosate 180g/L a.i @ 1.0 mL/plant, E= L. pseudotheobromae + glyphosate 180g/L a.i @ 0.5 mL/plant and F= L. pseudotheobromae + glyphosate 180g/L a.i @ 1.0 mL/plant...... 146

Fig. 4.7 Internal lesions/staining observed with different treatments in field trials conducted at Rockhampton, central Queensland at 21 months after inoculation (Scale=1cm)...... 147

Fig. 4.8 Internal lesions/staining observed with different treatments in field trials conducted at Richmond, north-western Queensland at 23 months after inoculation (Scale=1cm)...... 148

Fig. 4.9 Effect of L. pseudotheobromae and sub-lethal glyphosate on internal stem lesion in stem cross-section in prickly acacia recorded in field trials conducted at Rockhampton, central Queensland and Richmond, north-western Queensland. Columns with different letters on error bars indicate significant difference by Tukey’s test with 95% confidence in GLM ANOVA (n=4). On the X axis, each capital letter represents individual treatment where A= control (autoclaved fresh millet grain), B= L. pseudotheobromae, C= glyphosate 180g/L a.i @ 0.5 mL/plant, D= glyphosate 180g/L a.i @ 1.0 mL/plant, E= L. pseudotheobromae + glyphosate 180g/L a.i @ 0.5 mL/plant and F= L. pseudotheobromae + glyphosate 180g/L a.i @ 1.0 mL/plant...... 148

Fig. 4.10 Effect of L. pseudotheobromae and sub-lethal glyphosate on overall vigour of prickly acacia recorded from May 2012 to February 2014 in a field trial conducted at Rockhampton, central Queensland. Columns with different letters on error bars indicate significant difference by Tukey’s test with 95% confidence in GLM ANOVA (n=4). On the X axis, each capital letter represents individual treatment where A= control (autoclaved fresh millet grains), B= L. pseudotheobromae, C= glyphosate 180g/L a.i @ 0.5 mL/plant, D= glyphosate 180g/L a.i @ 1.0 mL/plant, E= L. pseudotheobromae + glyphosate 180g/L a.i @ 0.5 mL/plant and F= L. pseudotheobromae + glyphosate 180g/L a.i @ 1.0 mL/plant...... 150

Fig. 4.11 Effect of L. pseudotheobromae and sub-lethal glyphosate on overall vigour of prickly acacia recorded from June 2012 to May 2014 in a field trial conducted at Richmond, north-western Queensland. Columns with different letters on error bars indicate significant difference by Tukey’s test with 95% confidence in GLM ANOVA (n=4). On the X axis, each capital letter represents individual treatment where A= control (autoclaved fresh millet grains), B= L. pseudotheobromae, C= glyphosate 180g/L a.i @ 0.5 mL/plant, D= glyphosate 180g/L a.i @ 1.0 mL/plant, E= L. pseudotheobromae + glyphosate 180g/L a.i @ 0.5 mL/plant, F= L. pseudotheobromae + glyphosate 180g/L a.i @ 1.0 mL/plant and G= Untreated plants...... 151

Fig. 5.1 External (top row) and internal (bottom row) stem lesions on prickly acacia observed with Cophinforma sp. under different levels of drought stress (Scale=1.94 cm). Stem lesions in control plants were similar in both stress and non-stress regimens...... 167 xvii

Fig. 5.2 Effect of Cophinforma sp. and drought stress on length of external and internal stem lesion in prickly acacia. Columns with different letters are significantly different from each other by Tukey’s test in GLM ANOVA (n=10)...... 168

Fig. 5.3 Effect of Cophinforma sp. and drought stress on (A) total leaf production and (B) leaf mortality of prickly acacia. Columns with different letters are significantly different from each other by Tukey’s test in GLM ANOVA (n=10)...... 169

Fig. 5.4 Effect of Cophinforma sp. and drought stress on (A) stem and branch dry-weight and (B) plant height (measured as height increment over 10 weeks) of prickly acacia. Columns with different letters are significantly different from each other by Tukey’s test in GLM ANOVA (n=10)...... 170

List of Tables

Table 2.1 Fungi isolated from prickly acacia (identification is solely based on ITS1 sequence comparison). Species names in bold were associated with both dieback-affected and apparently healthy plants. Data in parenthesis indicates percentage of isolates of individual species to the total number of isolates...... 70

Table 2.2 Fungal isolates tested in the Glasshouse trial 1 ...... 78

Table 2.3 Fungal isolates tested in the Glasshouse trial 2 ...... 81

Table 2.4 Fungal isolates tested in the Glasshouse trial 3 ...... 85

Table 2.5 Fungal isolates tested in the Glasshouse trial 4 ...... 87

Table 3.1 Treatments in glasshouse trial ...... 107

Table 4.1 Treatments in glasshouse trial ...... 136

Table 4.2 Treatments in field trial ...... 139

xviii

List of Abbreviations

a.i active ingredients Al Aluminium ANOVA Analysis of Variance A$ Australian Dollar BLAST Basic Local Alignment Search Tool BLR Binary Logistic Regression Cd Cadmium CI Consistency Index Cl Chlorine cm centimetre(s) Cu Copper DNA Deoxyribonucleic Acid EDTA Ethylenediaminetetraacetic Acid EF Elongation Factor Fe Iron Fig. Figure g gram(s) g/L gram per litre GLM General Linear Model ha hectare(s) HI Homoplasy Index ITS Internal Transcribed Spacer K Potassium KPa kilopascal L liter(s) Ltd. limited MEA Malt Extract Agar MgCl2 Magnesium Chloride ml millilitre(s) mm millimeter(s) Mn Manganese N Nitrogen Na Sodium NaOCl Sodium hypochlorite NCBI National Center for Biotechnology Information OA Oatmeal Agar oC degrees celsius P Phosphorus PAUP Phylogenetic Analysis Using Parsimony Pb Lead PCR Polymerase Chain Reaction PDA Potato Dextrose Agar ½ PDA 1/2 strength Potato Dextrose Agar ppm parts per million RO Reverse Osmosis RI Retention Index

xix

S Sulphur sp./spp. species Sr Strontium TBE Tris base, Boric acid and EDTA TBR Tree Bisection and Reconnection TE Tris base and EDTA TEF Transcription Elongation Factor UQ The University of Queensland USA The United States of America UV Ultraviolet Light V volt v/v volume/volume V8A V8 Juice Agar w/v weight/volume Zn Zinc μl microliter(s) α alfa β beta Ψ water potential Ø diameter % percent : ratio & and @ at the rate of ± plus or minus ® registered trade mark

xx

Chapter 1: General introduction, literature review and research objectives

1.1 General Introduction

Prickly acacia (Vachellia nilotica subsp. indica (Benth.) Kyal. & Boatwr) is one of the most harmful weeds in the Australian rangelands with significant environmental and economic impacts (Dhileepan 2009; Palmer et al. 2012). A significant dieback event of this species was observed in the Richmond and Julia Creek regions of north-western Queensland during 2010 (Galea 2011). Affected trees were found with symptoms ranging from ashy internal staining, defoliation, blackening of shoot tips, partial crown death through to widespread mortality of plant populations. Observed symptoms on prickly acacia and findings on dieback of other rangeland weeds suggested the potential association of fungi with the dieback phenomenon (Wilson & Pitkethley 1992; Diplock et al. 2008; Aghighi et al. 2014). However, a number of previous studies also identified the potential association of drought stress with dieback and tree mortality observed in the Australian semi-arid grassland ecosystems similar to the prickly acacia dieback sites in north-western Queensland (Fensham & Fairfax 2007; Fensham et al. 2009; Allen et al. 2010). The studies presented in this thesis were undertaken to investigate the likely association of fungi, abiotic stressors (such as herbicide and drought stress) and their interactions to develop a conceptual understanding of prickly acacia dieback. There are indications that dieback may offer an exciting potential for biological control of such invasive weed species ( & Ginns 2006; Galea & Goulter 2013). Therefore, the potential of the fungi associated with dieback as bio-control mechanisms of prickly acacia was also investigated.

1.2 Literature review

1.2.1 Prickly acacia

1.2.1.1 Origin and morphology

Prickly acacia is native to and the western parts of Asia with the Australian biotype considered to have been introduced from the (Parsons & Cuthbertson 2001; Wardill et al. 2005; Miller & Seigler 2012).

1

A B D

15 mm

C

15

0 mm

500 mm 15 mm E

5 mm

Fig. 1.1 Prickly acacia: A. flowering plant; B. spine; C. flower; D. seed pods and E. seed.

As described by Parsons and Cuthbertson (2001), March (2004) and Palmer et al. (2012), this spiny shrub or small tree (Fig. 1.1 A) can grow up to 5-10 meter in height. Its bipinnately compound have a pair of 10-50 mm long stipular spines at the base (Fig. 1.1 B). are ball-shaped and golden yellow in colour (Fig. 1.1 C). Pods are typically constricted between individual seed (Fig. 1.1 D). Seeds are brownish to blackish, sub-globular to sub-circular with very hard seed coat (Fig. 1.1 E). The root system consists of deep taproot with branched lateral near the soil surface.

1.2.1.2 Ecology, lifespan and reproduction

Prickly acacia is well adapted to semi-arid warm, temperate, subtropical and tropical climates with annual rainfall in the range of 250-1500 mm, temperature -1 to 50oC and soil types ranging from light sand to heavy clay (Adjers & Hadi 1993; Parsons & Cuthbertson 2001; March 2004). However, it is best suited in subtropical and tropical climates and usually occurs in high density near a water source such as bore drains or creeks (Radford et al. 2002; March 2004). It is tolerant of a high level of soil salinity but young plants are susceptible to frost damage (Adjers & Hadi 1993; Mackey 1998). Seeds can germinate within a temperature range of 14-37oC with an optimum temperature of 25oC (Mackey 1998). 2

Prickly acacia is a perennial woody species with an average lifespan of 30-60 years (Carter 1994). It mainly reproduces by seeds and under favourable conditions a medium-sized tree can produce more than 175,000 seeds per year which are primarily dispersed after pods are eaten by and goats (March 2004). Seeds can remain dormant in soil for up to 6-7 years (Bolton et al. 1987). Such prolonged seed dormancy significantly contributes to the persistence of soil seed-bank which usually consists of 5-724 intact seeds per square meter area (Mackey 1998). Seed germination is accelerated by natural aging, fire and biochemical degradation or by passing through digestive tract of cattle and goats (Parsons & Cuthbertson 2001).

1.2.1.3 and diversity

Prickly acacia was initially classified within the genus Acacia, which is a large and polyphyletic genus with a controversial taxonomic history (Miller et al. 2014). Based on the combinations of morphological, palynological and biochemical properties of different species of Acacia, the genus was divided in 1986 into three subgenera Acacia, Phyllodineae and Aculeiferum (Maslin 2001; Orchard & Maslin 2003). However, based on more recent morphological and genetic studies, the genus Acacia was further classified into five different genera Acacia, , Racosperma, Acaciella and Vachellia (Orchard & Maslin 2003). In that classification, the genus Acacia was conserved while Vachellia was erected to comprise many of the economically important species of the previous subgenus Acacia. Prickly acacia was reclassified from Acacia nilotica subsp. indica to Vachellia nilotica subsp. indica (Seigler & Ebinger 2006). However, according to Miller and Seigler (2012), the genus Vachellia could be expanded to include more species of Acacia. Debate on the taxonomic classification of Acacia sp. is still ongoing and most recently Miller et al. (2014) has proposed a new classification for which was composed of six major clades Acacia, Austroacacia, Vachellia, Senegalia, Acaciella and Mariosousa. However, the taxonomic position of Vachellia nilotica was unchanged in that newly proposed classification.

Initial studies based on plant morphological properties and composition of leaf phenolic compounds indicated that the Australian population of prickly acacia comprises a single biotype of the Indian origin (Brenan 1983; Hannan-Jones 1999). However, more recent studies based on DNA sequence analysis found that the Australian population of prickly acacia mostly belong to the Indian indica biotype with a few individuals which were identical to an unidentified genotype from , not previously reported in Australia (Wardill et al. 2004; Wardill et al. 2005).

3

1.2.1.4 Current and potential distribution in Australia

Prickly acacia is one of the most invasive weeds in Australia with thickest infestation in north- western Queensland (March 2004; Palmer et al. 2012). Current infestation of this species was previously estimated as more than 7 million hectares in the Mitchell grasslands of north-western Queensland with the heaviest infestation near the bore drains (Mackey 1997). However, according to more recent reports, it has already spread over 22 million hectares of rangelands in north-western Queensland (Khan et al. 2014). It is also present as a discrete infestation in the coastal areas of Queensland between Rockhampton and Townsville and is also sometimes found in the , South Australian arid lands and rangelands in (March 2004; Senaratne et al. 2006; Palmer et al. 2012).

Fig. 1.2 Current and potential distribution of prickly acacia under current climatic conditions conditions (Kriticos et al. 2003).

4

A

B E

C F

D G

Fig. 1.3 Climatic suitability of prickly acacia under different climates scenarios A. Current climate; B-D. +2°C temperature, -10% rainfall; E-G. +2°C temperature, +10% rainfall (Kriticos et al. 2003).

Prediction on potential infestation based on eco-climatic modelling (CLIMEX) suggests that under present climatic conditions, prickly acacia may spread to areas larger than the current distribution (Fig. 1.2). Furthermore, with predicted future climate changes it may further infest vast areas in Australia (Fig. 1.3) (Kriticos et al. 2003; Kriticos et al. 2006). Modelling also suggests that coastal areas in central Queensland are ecologically more suitable for prickly acacia compared to the inland arid regions in north-western Queensland (Kriticos et al. 2003). Therefore, the apparently discrete current coastal infestations in central Queensland may turn into a dense infestation in future if appropriate control measures are not implemented in a timely manner.

5

1.2.1.5 Economic and environmental significance

Prickly acacia is one of the most harmful weeds with significant economic and ecological impact. It is a major threat to the Australian grazing industry because of its potential to reduce the amount and quality of pasture and increase the mustering cost (Dhileepan 2009). In Australia, prickly acacia is attributed with a reduction of average beef production by about 25.33% (Miller 1996). Its overall economic impact on primary producers was estimated as high as A$ 9 million per year (Dhileepan 2009). Detrimental ecological effects of prickly acacia broadly include soil erosion and reduction of biodiversity (Spies & March 2004; Martin et al. 2006).

1.2.1.6 Current control options

The current control strategy of prickly acacia is largely based on either manual removal using tractor and bulldozer or the application of chemical (Spies & March 2004; Queensland Department of Agriculture, Fisheries and Forestry 2014). A range of chemical herbicides are available for controlling infestations which are applied using different techniques such as basal bark spraying, cut stump treatment, foliar spraying and soil application, depending on the age of tree and density of infestation (Queensland Department of Agriculture, Fisheries and Forestry 2014). However, mechanical and chemical controls are generally more effective against low-density infestation, but often incur higher control costs due to the requirement of repeated follow-up actions (Mooy et al. 1992; Spies & March 2004). Fire is effective against seedlings, but, ineffective against mature plants and may be problematic through breaking the dormancy of remaining soil seed bank, favouring germination and enabling subsequent establishment (Radford et al. 2001). For these reasons, biological control using self-perpetuating agents is generally considered a more economic and sustainable control option for prickly acacia.

1.2.1.7 Current status of biological control

Bio-control is broadly defined as utilization of natural enemies of the target pests to reduce the pest population. In ancient times, the Chinese discovered that increasing ant populations in their citrus groves helped decrease destructive populations of large boring beetles and caterpillars (Smith et al. 1973). That use of a natural enemy to control a pest marked the birth of bio-control. However, weed bio-control is more specifically defined as the use of native or introduced enemies of a targetweed that will reduce the density of the weed to a level that is acceptable and that will maintain the weed density at that level (Julien and White 1997). It has significant implications in the field of weed

6 management and there have been many successes world-wide in the bio-control of weeds. In different countries, forty-one weeds were successfully controlled using introduced agents ( and pathogens) and a further three were controlled using native fungi applied as mycoherbicides (Mcfadyen 2000).

In Australia, the bio-control effort for prickly acacia was initiated in the 1970’s which was followed by intensive surveys in the 1980’s across its native distribution ranges in Pakistan, , Kenya and South Africa to search for suitable biocontrol agents (Mohyuddin 1981; Dhileepan 2009; Palmer et al. 2012). Several species were collected from those surveys, and tested for host specificity and efficacy as potential biocontrol agents. Among those, a few demonstrated host-specificity of which only two, Bruchidius sahlbergi Schilsky, a seed-feeding bruchid and Cuphodes profluens Meyrick, a shoot boring caterpillar were released in Australia during 1981-1985 (Dhileepan 2009; Palmer et al. 2012). Of those two, B. sahlbergi successfully established in Queensland (Willson 1985). However, in spite of widespread establishment, it was found to exhibit low biological control impact as a seed predator and did not significantly lower the prickly acacia population at the release sites (Radford et al. 2001). On the other hand, the caterpillar C. profluens was initially reported to establish at a coastal infestation site near St Lawrence, Queensland. However, it is now considered as “failed to establish” since the experimental plants in that site were accidentally eradicated (Palmer et al. 2012).

Further searches for suitable biocontrol agents during 1996-2001 resulted in release of three more insects Homichloda barkeri Jacoby, a leaf feeding beetle; Chiasmia inconspicua Warren, a leaf feeding caterpillar and Chiasmia assimilis Warren, a leaf feeding geometrid sourced from Kenya but all failed to establish in at the release sites in Australia (Lockett & Palmer 2003; Palmer et al. 2007; Palmer et al. 2012). Again, in 2002-2003 a South African strain of C. assimilis was released in Australia which successfully established and caused occasional defoliation in the coastal areas of Queensland (Palmer et al. 2012). However, the insect was found with very low population at the core prickly acacia infestation sites in western Queensland possibly because of high temperatures and dry weather (Senaratne et al. 2006; Palmer et al. 2007; Palmer et al. 2012).

During 2004-05, another leaf-feeding caterpillar, Cometaster pyrula Hopffer was released in a few sites in Queensland although its establishment has not been confirmed to date (Palmer et al. 2012). However, studies on climatic suitability suggested that the coastal areas in Queensland are suitable and the inland areas in north-western Queensland are apparently unsuitable for its establishment (Palmer & Senaratne 2007).

7

A number of other insects such as Acacidiplosis spinosa Gagné, Isturgia deerraria Walker, Isturgia disputaria Guenée and Acizzia melanocepala Burckhardt and Mifsud were imported in Australia and tested under quarantine conditions. However, none of those were released in the field conditions because of different limitations such as absence of host specificity, difficulties in rearing, genetic erosion during subsequent laboratory culture, poor larval and adult survival rate and lack of climatic suitability (Palmer & McLennan 2006; Palmer & Witt 2006; Palmer et al. 2012).

During 2008-2011, 33 species of leaf feeding insects and eight species of fungi were collected following extensive survey in multiple locations of southern India (Dhileepan et al. 2013). Among those, a scale insect Anomalococcus indicus Ramakrishna was found to cause significant damage to prickly acacia in India, and therefore, imported in Australia for further investigation of its suitability to be released as bio-control agent (Khan et al. 2014).

It is evident from the above discussion that insect-based classical approach has not resulted in any significant success in prickly acacia bio-control. Therefore, it is necessary to explore other potential bio-control options. Fungi are another option. Potential candidates may be found in the native distribution ranges of prickly acacia and introduced into Australia. In addition, endemic fungi capable of attacking this host can also be utilized in bio-control of this invasive weed species. Extensive dieback and plant death was observed among prickly acacia populations growing in north-west Queensland during 2010 (Galea 2011). The opportunity therefore existed to investigate the dieback phenomenon with the objective of determining whether potential biocontrol agent/s could be found. However, first the nature of dieback and the role of biological agents in this phenomenon must be understood.

1.2.2 Dieback

The term ‘dieback’ theoretically describes the progressive death of apical parts of branches leading to gradual reduction of growth and vigour of a shrub or tree. However, practically this term is more appropriate to indicate the deterioration and death of stands of trees or large areas of trees rather than an individual tree (Jurskis & Turner 2002). It is often synonymously used with another term ‘decline’ but some authors differentiate dieback from decline in a way that dieback is the impact of severe stress factor which may recover after elimination of the stress, whereas decline is a prolonged malfunction and often irreversible deterioration of tree health (Manion 1991; Jurskis 2005; Horton et al. 2011). Trees and shrubs irrespective of age may be affected by dieback or decline, however, it is usually associated with plants that have attained some size and maturity (van Niekerk et al. 2004). 8

B A C

E F

D

Fig. 1.4 Symptoms commonly associated with dieback of perennial plant species; A-B. cankerous external and internal lesions; C. root necrosis; D. gummosis; E. wilting and F. plant mortality Fig. 1.3 (Munkvold 2001; Jung 2012; Aghighi et al. 2014; Linaldeddu et al. 2014; McKinney et al. 2014).

Symptoms of dieback are variable in type and expression depending on the affected tree species, ecological features of the occupying habitat, management practices and severity of associated biotic and abiotic stressors. However, the general symptoms are thinning of foliage, leaf chlorosis and necrosis, premature defoliation, stunted growth, cankerous lesion on stem and shoots, vascular staining, wilting, top desiccation, death of whole twigs and branches or their apices and death of whole trees under severe condition (Fig. 1.4) (Kraj et al. 2012; Enderle et al. 2013; Rolshausen et al. 2014).

There are examples where branch canker and gummosis appeared as preliminary symptoms followed by defoliation, twig necrosis, wilting and mortality (Davidson et al. 2003; McDonald & Eskalen 2011).

9

Other symptoms generally associated with dieback include epicormic branching or development of water suckers from the stem, browning of wood, necrotic blotches, reduced twig and stem growth, small leaves, bud necrosis and mortality, delayed budburst, bleached canes, incomplete graft unions, bunch rot, stunted growth and reduction in tree girth (Bush 2009; Webber et al. 2010; Amponsah et al. 2011; Fleurat-Lessard et al. 2013; Adesemoye et al. 2014).

1.2.3 Possible causes of dieback

Perennial trees and shrubs usually encounter a range of biotic and abiotic stressors during their lifespan. Biotic stressors broadly include different insect pests, diseases and mammals whereas the common abiotic stressors are drought, waterlogging, extremely high or low temperature, soil salinity, soil acidification, soil compaction, application of herbicides or other agrochemicals, acid rain and numerous other environmental factors. These biotic and abiotic stressors and their complex interactions have a significant role in dieback of woody perennials.

1.2.3.1 Fungi

Fungi have long been recognised as potential agents of tree dieback and mortality (Hepting 1971). Frequent association of fungi with dieback and decline previously led to a misconception that dieback is solely a fungal disease rather than a condition or outcome of multiple stress factors. Diverse groups of root-degrading and vascular wilt fungi, branch and stem canker fungi and different foliar fungi are generally found to be associated with dieback and mortality of a range of trees and shrubs.

Among the root-degrading fungi, several species of Pythium (Oomycete), Phytophthora (Oomycete), Fusarium (Sordariomycete), Macrophomina (Dothideomycete) and Armillaria () were reported to be implicated with dieback of many economically important tree species (Randles 2010; Akilli et al. 2013; Arif et al. 2013; Hassan et al. 2014; Souli et al. 2014). These fungi are usually soil-borne which following infection cause root rot, extensive fine-root necrosis followed by invasion resulting in reduced water uptake and subsequently crown and shoot dieback and plant mortality under severe conditions (Bakys et al. 2011; Davison 2011; Davison 2014).

Vascular wilt fungi are another group of fungal pathogen causing wilting in plants leading to dieback. The most common genera under this group are Fusarium (Sordariomycete), Ceratocystis (Sordariomycete) and Verticillium (Sordariomycete) which are reported to be implicated with 10 several events of vegetation decline and mortality (Kapoor et al. 2004; Triki et al. 2011; Bal et al. 2013). In woody plants, these fungi generally cause infection through root or stem wounds followed by colonization and blocking of vascular system resulting in restricted water uptake leading to wilt and dieback (Sinclair & Lyon 2005). Fungi under this group are also known to produce phytotoxins causing wood staining and promoting spread of the pathogen in plant tissues (Zhang et al. 1991; Yadahalli et al. 2007).

Of the diverse assemblages of branch canker pathogens, members of the family Botryosphaeriaceae (Dothideomycete) have been reported across the world from more than 1000 plant hosts including commercially important agricultural and native forest trees (Mehl et al. 2013). In Australia, the Botryosphaeriaceae fungi were found to be associated with dieback and canker of a wide range of economically important perennial trees and shrubs such as mango (Mangifera indica L.), Acacia spp., Eucalyptus spp., peppermint (Mentha spicata L.) and grapevine (Vitis vinifera L.) (Mohali et al. 2007; Taylor et al. 2009; Dakin et al. 2010; Amponsah et al. 2014; Trakunyingcharoen et al. 2014). Similar fungi were also found to be implicated with dieback of different rangeland weeds in Australia such as mimosa (M. pigra) and parkinsonia (P. aculeata L.) (Wilson & Pitkethley 1992; Diplock et al. 2006).

Studies on the life cycle and infection mechanism of Botryosphaeriaceae fungi indicate that they produce asexual fruiting structures such as pycnidia or perithecia which generally overwinter in dead or damaged plant parts, seed cones and mummies (Michailides 1991; Epstein et al. 2008). Under favourable environmental conditions, the spores are released from the fruiting bodies and disseminated by rain splash, wind or insects and infect healthy hosts through wounds or natural openings resulting in disease symptoms (Amponsah et al. 2010; Mehl et al. 2013). Botryosphaeriaceae fungi were also reported to cause root infection and suspected to transmit horizontally through intermingling root systems, but this infection process has not yet been experimentally proven (Whitelaw-Weckert et al. 2006; Mehl et al. 2013). Furthermore, a number of Botryosphaeriaceae fungi including many species of (Dothideomycete), Diplodia (Dothideomycete), Dothidotthia (Dothideomycete), Guignardia (Dothideomycete), Lasiodiplodia (Dothideomycete), Neofusicoccum (Dothideomycete) and Pseudofusicoccum (Dothideomycete) have been identified as endophytes causing non-symptomatic latent infection in a range of perennial woody plant species (Petrini & Fisher 1988; Johnson et al. 1992; Crous et al. 2006; Slippers & Wingfield 2007). More recently, the Botryosphaeriaceae fungi are more generally considered to be opportunistic latent pathogens because the disease symptoms they cause are often associated with other biotic and abiotic stressors involved in host weakening (Slippers & Wingfield 2007; Niekerk

11 et al. 2011; Amponsah et al. 2014). In the presence of widespread stressors, these fungi can cause rapid dieback resulting in extensive losses over vast areas (Slippers & Wingfield 2007). Moreover, a number of Botryosphaeriaceae fungi are also well-recognized as plant pathogens having potential to cause significant disease symptoms in the absence of other stressors (Slippers & Wingfield 2007; Lynch et al. 2013; Chen et al. 2014).

The Botryosphaeriaceae have also been characterized as seed-borne agents, although there is little or no evidence on subsequent seed-transmitted infection in seedlings (Slippers & Wingfield 2007). However, seed infections were previously reported to have significant implications in the dispersal of these pathogens around the world (Smith et al. 2000; Burgess et al. 2004). Different species of Botryosphaeriaceae fungi are also known to produce complex phytotoxic chemicals but the role of these in pathogenesis has not been fully elucidated (Martos et al. 2008; Djoukeng et al. 2009; Evidente et al. 2010).

Alongside the Botryosphaeriaceae, members of the family Valsaceae, particularly different species of Cytospora (Sordariomycete) are also known as important canker and dieback causing fungi (Zhang et al. 2013; Hand et al. 2014). Cytospora species generally cause infection through wounds usually leading to cankerous girdling which subsequently kills the plant above the canker (Biggs et al. 1983; Kepley & Jacobi 2000). Similar to the Botryosphaeriaceae, different species of Cytospora were also reported to asymptomatically colonize healthy tissues and express disease symptoms following an onset of stress or injury (Adams et al. 2006; Kaczynski & Cooper 2013).

In addition, a number of foliar pathogens, such as different species of Alternaria (Dothideomycete), Colletotrichum (Sordariomycete), Phomopsis (Sordariomycete), Aulographina (Dothideomycete) and Mycosphaerella (Dothideomycete) were also reported to be associated with deterioration of crown health or dieback of many woody species (Tsahouridou & Thanassoulopoulos 2000; Carnegie 2007; Raj et al. 2013; Urbez-Torres et al. 2013). These fungi generally cause foliar necrosis resulting in reduction of effective photosynthetic leaf area, which under severe conditions may cause dieback or mortality.

1.2.3.2 Insect infestation

Insect infestation can impose significant stress and may cause tree mortality under severe conditions. However, it is often argued that other site-specific stress factors such as poor soil condition, drought, frost and strong wind probably predispose the trees to severe insect attack and subsequent decline (La Spina et al. 2013; Worrall et al. 2013; Perrette et al. 2014). 12

There are two theories in relation to tree stress and insect infestation. Price (1991) proposed “the plant vigour hypothesis” predicting that insects prefer to feed on the most vigorous plant populations, the most vigorous plants within a plant population and/or the most vigorous parts of a plant. On the other hand, White (2007) postulated “the plant stress hypothesis” suggesting that stressed plants are a more suitable food source for insects as stress results in an increase to the tissue amino acid content which attract the herbivores to feed on their host and, thereby, encourage subsequent population build up. These two theories are somewhat in conflict with each other possibly because those were postulated based on the examination of different host-insect interaction systems. However, the consequences of insect infestation is the same- depletion of food reserves, which often make the affected tree unable to cope with further stress (Gandhi et al. 2014; Stursova et al. 2014).

Among the different categories of insects, mass infestation by leaf and root feeding and wood boring caterpillars, grubs and beetles cause severe defoliation and other damage to the trees (Calder & Kirkpatrick 2008; Randles 2010; Montecchio & Faccoli 2014). Severely defoliated and damaged trees usually have a little chance of survival as they become prone to attack by other secondary insects and pathogens (Stursova et al. 2014). Different sap sucking insects can also impose serious stress which can cause or predispose the trees to dieback (Kunca & Leontovyc 2013; Kaczynski et al. 2014; Neumann et al. 2014). Several insects act as vectors to disseminate the virulent strains of different pathogens which also significantly contribute to the dieback process (Kamata et al. 2002; Eskalen et al. 2012; Eskalen et al. 2013). Insect damage also provides entry sites for dieback and canker pathogens (Mehl et al. 2013). In addition, host weakening resulting from insect damage can enable the latent fungi to express disease symptoms (Moral et al. 2010; Mehl et al. 2013).

1.2.3.3 Drought

Drought is conceptually defined as a prolonged period of dryness resulting in extensive damage to plants (Wilhite and Glantz 1985). It is an insidious hazard of nature and its impacts vary from region to region. Drought induced tree mortality is well documented as a common problem in a range of eco-systems across the world mainly as a consequence of climate change and global warming (Allen et al. 2010; Hoffmann et al. 2011; Cailleret et al. 2014). Predicted changes in global climate indicate that drought is going to emerge as a far greater and chronic stress for trees in coming decades which may worsen the dieback situation (Jentsch et al. 2007; Sterl et al. 2008; Gea- Izquierdo et al. 2014). In Australia, a number of perennial shrubs and trees had been reported to

13 suffer from drought induced dieback and mortality (Rice et al. 2004; Fensham & Fairfax 2007; Calder & Kirkpatrick 2008; Fensham et al. 2009).

Drought can directly cause tree mortality or act as a predisposing factor by altering the physiological and anatomic features of affected trees (Desprez-Loustau et al. 2006; La Porta et al. 2008; Fensham et al. 2009; Hentschel et al. 2014). There are two main theories explaining the role of drought in dieback and tree mortality. The first theory as proposed by Manion (1991), is known as the “decline spiral model” and explains the role of drought as a trigger or inciting factor that cause dieback in trees which are already stressed by other factors such as poor soil conditions, pollution, ageing, insect damage and diseases. The second theory proposed by McDowell et al. (2008) is based on hydraulic failure and carbon starvation. According to this theory, drought results in widespread dieback by three possible mechanisms: (a) extreme drought and heat cavitate the water column in xylem vessels which then restricts the transportation of water to the canopy, ultimately resulting in death of plant tissues because of desiccation; (b) drought-induced carbon starvation reduces the ability of plants to defend against attack by insect pests and pathogens and (c) high temperatures that often occur during drought periods encourage the proliferation of biotic agents, allowing them to overwhelm the stressed host. In addition to carbon starvation, low tissue water potential resulting from drought was identified as another factor restricting the production of metabolites involved in host defence against biotic stress (Ryan et al. 2006; Sala & Hoch 2009). Further details on drought stress-fungi interactions in terms of dieback and canker of woody plants are presented later in this chapter.

1.2.3.4 Waterlogging

Waterlogging affects the soil environment and encourages the accumulation of potentially harmful phytotoxic substances such as sulphides and different organic compounds in soil (Koch et al. 1990; Fogli et al. 2002; Borum et al. 2005). It also alters several physiological processes of affected plants which broadly include restricted root aeration; reduction of stomatal conductance, root hydraulic conductance, and resistance against biotic stressors (Kreuzwieser & Rennenberg 2014). Such physiological malfunctions may cause dieback or predispose the affected vegetation to other biotic or abiotic stressors causing dieback and mortality (Barrett et al. 2005; Aghighi et al. 2014; Davison 2014).

14

1.2.3.5 Chilling injury

Chilling temperature can cause significant physiological damage to the affected plants. Chilling alters plant physiology mainly in two ways (a) intracellular freezing: causes disruption of cell membrane leading to cell necrosis and (b) intercellular freezing or ice formation in intercellular spaces: causes vascular cavitation, cell dehydration, cell contraction and finally collapse of the dehydrated and contracted cells (Palta & Weiss 1993; Ristic & Ashworth 1993). Such physiological damage often leads to the expression of a range of physical symptoms including those associated with dieback (Zhu et al. 2000; Hancock 2008; Cech et al. 2010; Strasser 2011; Weber & Entrop 2013). In addition, chilling also predisposes the affected plants to infection by certain low temperature loving fungi and bacteria and also insect infestation which often lead to development of dieback symptoms (Cambours et al. 2005; Pukacki & Przyby 2005; La Spina et al. 2013; Perez- Sierra et al. 2013).

1.2.3.6 Heat stress

Heat stress resulting from exposure to high temperature beyond the tolerance limit may cause severe damage to the affected plants leading to the expression of a range of symptoms including dieback and mortality (McDowell et al. 2010; Brouwers et al. 2013). However, the symptoms associated with heat stress were broadly classified by Campbell (2010) in three main categories: (a) foliage scorch: generally characterized by dry, distorted and discoloured leaves; (b) shoot-tip dieback: where the young shoots become dry, fragile and discoloured due to sudden raise of temperature and large trees are top-killed due to continuous overheating and (c) bark scorch: usually develop when bark and cambium tissues are exposed to high temperature. Such symptoms are also linked with water deficit as high temperature and water deficit are often co-occurring (Allen et al. 2010). High temperature can also predispose the affected plants to other biotic stressors such as fungal infection and insect infestation leading to rapid and severe dieback (Desprez-Loustau et al. 2006; McDowell et al. 2010).

1.2.3.7 Soil salinity

A number of previous studies have identified soil salinity as a potential cause of vegetation dieback and mortality in Australia and overseas (Barrett et al. 2005; Winning & Saintilan 2009; Elmer et al. 2013; Souli et al. 2014). In addition, there are a number of incidences where salinity caused widespread dieback and tree mortality in the presence of other abiotic and biotic stressors such as

15 drought, water logging, fungal infection and insect infestation (Khakdaman et al. 2008; Neko et al. 2008; Mac Nally et al. 2011; Smith et al. 2012). Salinity reduces the availability of some essential micro-nutrients and increases the concentration of water-soluble ions of Na, Cl, S, Fe, Zn, Mn and Cu which results in osmotic stress, nutritional imbalance and toxicity to the affected trees (Mills 2006; Neko et al. 2008; Dmuchowski et al. 2011). As a consequence of osmotic stress and nutritional imbalance, photosynthetic efficacy of the affected trees decline which may subsequently cause or predispose the affected trees to dieback.

1.2.3.8 Soil acidification

Soil acidification is another important abiotic stressor previously reported to be associated with vegetation dieback (Hadas 2007; Ouimet et al. 2008). Under acidic condition, the concentration of different nutrient elements such as Fe, Mn, Al, Cd, Zn, Cu, Pb, Mn and Sr exceed their toxic limit in both soil and plant which may subsequently result in dieback symptoms (Ranasinghe et al. 2007; Watmough 2010). Soil acidification also restricts the availability of different essential macro- nutrients such as Ca, Mg and K leading to nutrient deficiency, root mortality, stunted growth and eventually die-back (Tomlinson 1993, 2003; Watmough 2010). Nutritional imbalance in acidic soil was also previously reported to predispose the affected vegetation to infection by opportunistic fungi leading to dieback symptoms (Jonsson 2004).

1.2.3.9 Soil alkalinization

Soil alkalinity characterised by high soil pH (>7.0) is another soil chemical factor previously reported to be implicated in dieback of different woody species (Messenger 1986; Czerniakowski et al. 2006; Grigg et al. 2009). As with soil salinity and acidification, alkalinity also affects soil fertility and plant nutritional balance by causing deficiency and/or toxicity of different essential plant nutrients (Jones 2012). Such nutritional imbalance may cause or pre-dispose the affected vegetation to dieback. For example, Czerniakowski et al. (2006) investigated the soil properties associated with decline of Eucalyptus spp. in selected areas of South Australia and Victoria. The study found that deficiency of Fe and Mn and excess of Na and Cl in alkaline soil was potentially associated with the decline symptoms.

Grigg et al. (2009) investigated the causes of Eucalyptus marginata Sm. (Jarrah) and Corymbia calophylla (Lindl.) Hill & Johnson (Marri) decline in a parkland of south-western Australia. The study identified Mn deficiency as a key factor implicated in the decline phenomenon. Such

16 deficiency of Mn was attributed to increased soil alkalinity as a result of continuous irrigation with alkaline water.

In addition, soil nutritional imbalance also reduces the population and diversity of beneficial mycorrhizal fungi (Erland & Taylor 2002; Kraigher & Petkovsek 2011). Such reduction of mycorrhizal fungi was also reported to be potentially associated with vegetation decline (Ishaq et al. 2013).

1.2.3.10 Soil compaction

Soil compaction reduces overall soil productivity which subsequently affects tree health (Han et al. 2009). It restricts root growth and predisposes the affected trees to root infecting fungi (Rhoades et al. 2003; Fonseca et al. 2004; Arefipour et al. 2005). It also results in depletion of the population of beneficial soil microbes (Silva et al. 2011). The beneficial soil microbes play an important role to assist tree growth by organic matter decomposition, nutrient cycling and thereby creating a porous environment for tree roots with better access to water and oxygen. Their depletion results in an unhealthy soil condition which may also encourage dieback.

1.2.3.11 Other biotic and abiotic stressors

Other biotic and abiotic stressors usually found to be associated with dieback are bacteria (Valdez et al. 2013), virus (Vogel et al. 2011), phytoplasmas (Abeysinghe et al. 2014), (Baird et al. 2014), mistletoes (Morel 2010), overuse of herbicide (Duke 2008), transplanting shock (Wang et al. 2010), fire (Brando et al. 2012), hail (Zwolinski et al. 1995) and acid rain (Hauck 2003).

1.2.4 Interaction between drought stress and opportunistic fungi

Opportunistic fungi are those that either express or cause more severe disease symptoms once the host is in a weakened state associated with stress or injury (Slippers & Wingfield 2007). This group encompasses a range of fungi including the several members of Botryosphaeriaceae and Valsaceae, well-documented for their potential association with dieback and canker diseases of different woody perennials (Dakin et al. 2010; Diminic et al. 2012). In general, stress assists the opportunistic endophytes to penetrate the host cuticle and also to persist in plant tissue (Arnold & Herre 2003). However, among the different stress factors, drought is most commonly associated with dieback and canker of woody hosts resulting from infection by the opportunistic endophytic fungi (Desprez- Loustau et al. 2006; Mehl et al. 2013). Similar phenomena were also possibly implicated in prickly

17 acacia dieback which was mostly observed at water-deficit sites in north-western Queensland in association with endophytic fungi (Galea 2011).

Earlier studies on stress-fungi interactions have revealed that moisture stress contributes to nutrient stress, alters host physiology and impair host defence against fungal infection (Schoeneweiss 1981; Madar et al. 1995; Paoletti et al. 2001). However, more recently, Desprez-Loustau et al. (2006) have reviewed the earlier theories and findings on drought-fungi interactions in woody species and explained their interactions as: (1) direct effect of drought on fungi, (2) indirect effect of drought on fungi and (3) drought-fungi interactive effect.

Drought can directly affect fungal populations; however, such effect is generally negative as most of the fungi require sufficient moisture for spore germination and subsequent infection (Lacey 1986). However, there are significant variations among different groups of fungi in terms of their tolerance to desiccation. In general, the members of Ascomycetes (including Botryosphaeriaceae, the major group of endophytes associated with dieback and canker) are more tolerant to desiccation compared to the obligate Basidiomycetes and soil-borne Oomycete pathogens such as Pythium and Phytophthora (Boddy 1983; Ma et al. 2001; Niekerk et al. 2011).

Drought also affects the population dynamics of other members in an ecosystem which in turn may indirectly affect fungal population and disease severity. For example, drought may favour the proliferation of insect vectors and assist in widespread dissemination of particular fungi (Rouault et al. 2006). Such insect vectors may also cause wounds or injury to the plants which may provide entry sites for other fungi. There are a number of dieback fungi which usually infect the host through wounds and subsequently develop disease symptoms (Mehl et al. 2013). Drought can also encourage certain opportunistic fungi by eliminating its potential antagonists (Redfern & Stenlid 1998).

Interaction of drought and fungi can be explained by two theories such as “multiple stress concept” and “predisposition concept”. In the “multiple stress concept”, drought and fungi are considered the individual stress component and their combined occurrence is supposed to add a cumulative stress on the host (Ayres 1991; Mitchell et al. 2013). On the other hand, the “predisposition concept” is based on drought-induced physiological alterations and subsequent predisposition of a healthy host to fungal infection which further relies on two major mechanisms: (a) tissues with low water potential provide better substrate for fungal colonization probably due to the changes in carbohydrate and protein metabolism and (b) disruption of disease resistance possibly because of decline in phytoalexin production under water stress (Boyer 1995; Madar et al. 1995).

18

A number of previous studies have experimentally demonstrated that opportunistic fungi could cause more severe disease symptoms in presence of drought stress. However, majority of those studies focused on the interactions of the Botryosphaeriaceae fungi such as different species of Botryosphaeria, Diplodia, Lasiodiplodia and Neofusicoccum with drought stress (Pusey 1989; Madar et al. 1989; Mullen et al. 1991; Ragazzi et al. 1999; Ma et al. 2001; Piskur et al. 2011; Niekerk et al. 2011; Amponsah et al. 2014). In all those studies, the test-fungi were reported to cause higher levels of disease symptoms such as longer stem lesions or cankers, higher degree of gummosis and more severe leaf blight on stressed hosts. Madar et al. (1995) reported that drought stress reduced the production of certain phytoalexins in woody host which subsequently predisposed the plant to infection by certain Botryosphaeriaceae fungi.

In addition to the Botryosphaeriaceae, a number of other fungi, such as Cryphonectria parasitica (Murrill) Barr. (Valsaceae); Sphaeropsis sapinea (Fr.) Dyko & Sutton. (Valsaceae), Biscogniauxia mediterranea (De Not.) Kuntze. (Xylariaceae) and Fusarium acuminatum Ellis & Everh. (Nectriaceae) were also reported to cause higher degrees of disease symptoms once their woody hosts were deliberately inoculated and subjected to lower levels of water availability (Gao & Shain 1995; Johnson et al. 1997; Stanosz et al. 2001; Capretti & Battisti 2007; Marek et al. 2013).

1.2.5 Dieback of prickly acacia and other invasive weeds in Australia

Prickly acacia was reported by landholders to display naturally occurring dieback symptoms since 1970 across many infestation sites throughout north-western Queensland (Queensland Department of Agriculture, Fisheries and Forestry 2014). Its occurrence was not previously investigated in detail, however, there are indications on potential association of water stress, fungal infection and insect infestation on stressed hosts (March 2009; Queensland Department of Agriculture, Fisheries and Forestry 2014). A prominent dieback event was observed more recently in 2010 among the plants growing in the Richmond and Julia Creek regions of north-western Queensland (Galea 2011). Affected trees were found with a range of disease symptoms (Fig. 1.5) such as defoliation, blackening of shoot tips, partial crown death through to widespread mortality of plant populations.

Stem samples collected from dieback sites were found with ashy internal staining and preliminary laboratory and glasshouse studies confirmed the association of potentially pathogenic fungi (Haque et al. 2012). There are indications that the fungi associated with dieback may offer a novel way to biologically control such invasive weeds (Wood & Ginns 2006; Galea & Goulter 2013).

A number of other invasive weeds in Australia such as mimosa (Mimosa pigra L.), parkinsonia (Parkinsonia aculeata L.), athel (Tamarix aphylla L. Karst), bitou bush (Chrysanthemoides 19 monilifera (L.) Norl.) and European blackberry ( fruticosus L.) were also reported to display dieback symptoms with potential association of fungi (Cother et al. 1996; Diplock et al. 2006; Gouldthorpe 2008; Aghighi et al. 2012; Sacdalan et al. 2012). In addition, there is also evidence on likely association of other stressors with the observed symptoms (Aghighi et al. 2014). However, in most cases, the exact cause of dieback is only partially understood.

A B

C D

Fig. 1.5 Symptoms of prickly acacia dieback A-B. widespread plant mortality; C. development of water suckers and D. internal staining (Galea 2011).

Wilson & Pitkethley (1992) investigated the dieback symptoms observed on mimosa, a woody invasive weed in northern Australia. Affected plants in the floodplains of the Adelaide River near Darwin, Northern Territory were found with necrotic branch lesions which subsequently girdled and killed the affected branches above the lesion. A Botryosphaeriaceae fungus, Botryodiplodia (=Lasiodiplodia) theobromae Pat. was found to be associated with the observed symptoms. However, more recently Sacdalan et al. (2013) isolated an almost similar group of Botryosphaeriaceae and other fungi from dieback-affected and healthy stands of mimosa indicating the potential association of endophytic fungi and other factors with the dieback phenomenon.

Parkinsonia is another invasive rangeland weed reported to display naturally occurring dieback symptoms across northern Australia (Diplock et al. 2006; Diplock et al. 2008). A range of fungi, including those under the Botryosphaeriaceae family were found to be associated with the

20 phenomenon which offered an exciting potential for bio-control of this invasive weed species (Galea 2008; Toh et al. 2008; Galea & Goulter 2013). Currently, dieback is a major limiting factor to the spread of this species in northern Australia (van Klinken et al. 2009).

Extensive dieback was also observed on athel pine near Finke River in Northern Territory (Gouldthorpe 2008). Preliminary studies found potential association of fungi which, however, did not manifest any causative relationship to the observed symptoms (Crossley & Galea 2010).

Bitou bush, an invasive weed in eastern Australia was found with dieback symptoms in coastal areas of New South Wales (Cother et al. 1996). Different fungi such as Phomopsis sp., Stemphylium sp. and Sclerotinia sclerotiorum (Lib.) de Bary were found to be associated with the observed symptoms (Cother et al. 1996). Among these, S. sclerotiorum was evaluated for its potential to be utilized as a bio-control agent of this invasive species which, however, was rejected in the early 1990’s for its lack of sufficient host specificity (Cother et al. 1996; Cother 2000). However, recent studies by Morin et al. (2010) found potential association of a leaf spot fungus, Austropleospora osteospermi Shivas & Morin with bitou bush dieback in Australia.

Symptoms of dieback were also observed on European blackberry, a serious invasive weed species in Australia (Aghighi et al. 2012). Recently, Aghighi et al. (2014) has proposed a conceptual model to clarify the factors implicated with the dieback phenomenon. According to that model, waterlogging pre-disposes Rubus fruticosus to different root-infecting Oomycetes such as Phytophthora, Pythium, and Cylindrocarpon. These fungi in association with other contributing and initiating factors such as high grazing pressure, lack of plant genetic diversity, previously released bio-control fungi and competition with other invasive species cause dieback.

1.2.6 Fungi in weed bio-control

1.2.6.1 Concept

There are two basic approaches to utilizing fungi in weed bio-control, the classical and inundative approaches (Charudattan 2005). The classical approach refers to the inoculation of host-specific fungi which are either co-evolved or foreign in origin. On the other hand, in the inundative approach, native fungi are utilized to augment the development of an epidemic of a certain disease on a particular weed. The inundative approach is also known as the bioherbicide approach where the fungi are applied following particular method(s) under certain situation(s) which are almost similar to those for chemical herbicides (Bailey 2014). However, bioherbicides were more broadly 21 defined by Bailey (2014) as the products derived from living organisms such as bacteria, fungi, viruses and nematodes including any natural derivatives they produce during their growth that suppress weed populations. The bioherbicides composed of living fungi or their derivatives such as mycotoxins, enzymes and acids are generally known as mycoherbicides (Masi et al. 2014; Zhang et al. 2014).

Fungi are applied for weed bio-control using different delivery techniques and formulations (Ash 2010). However, in a number of previous studies, stem wounding followed by insertion of encapsulated or gel-based formulations of fungal materials were found to be effective against woody weeds (Dorworth 1995; Rayachhetry et al. 1999; Galea & Goulter 2013; Bellgard et al. 2014). Such a delivery technique assists the potential bioherbicide fungi to establish while overcoming certain restrictions such as non-establishment because of the physical barrier of the host, lack of sufficient moisture and effect of other eco-climatic factors (Auld et al. 2003).

1.2.6.2 Advantages

The use of chemical herbicides for weed control has gained increasing popularity over the last 6 decades particularly because of their ease of application and high efficacy. Currently, they comprise 44% of total pesticides sold in the world (Bailey 2014). A number of chemical herbicides were also found to be effective against the weeds invading pasturelands (Dube et al. 2009; Harrington 2014; McKenzie et al. 2014). However, extensive use of chemical herbicides may have detrimental effects on agriculture and the overall environment. For example, overuse of a specific herbicide may result in the build-up of herbicide-resistant weed populations (Boutsalis et al. 2012; Owen et al. 2014). Some chemical herbicides persist in soil and even in plant parts for long periods affecting both soil productivity and consumer health (Tandon 2008; Raj et al. 2010). In addition, chemical herbicides are also known to contaminate water bodies and affect non-target species (Wolf 2009; Allinson et al. 2014). On the other hand, the bioherbicides including the mycoherbicides being natural in origin are free from such harmful environmental effects. It is generally considered a more economically appropriate option for weed control (Al-Tawaha et al. 2008). Fungi are also effective in the controlling of the weeds that developed resistance to chemical herbicides (Hoagland et al. 2013).

1.2.6.3 Limitations

Although the use of fungi is considered safer and more economically viable compared to other weed control tools, its efficacy is influenced by several factors such as the extent of inherent or elicited host resistance to the potential bioherbicide fungi, desired level of weed control, variation in 22 soil type, topography and other ecological factors such as temperature and moisture availability (Auld & Morin 1995; Hoagland 1996; Ghorbani et al. 2005; Iffat et al. 2011; Bailey 2014). This approach has some other limitations such as non-establishment under field conditions, low and unpredictable impact on the target weed; slow performance; requirement of repeated application, extensive research effort and combinations with other control tools and unavailability of potential bioherbicide fungi for a range of economically important weeds (Al-Tawaha et al. 2008; Widmer & Rayamajhi 2008; Chalak et al. 2011; Bailey 2014).

1.2.6.4 Integration of potential bioherbicide fungi with other weed control techniques

Application of a bioherbicide as a “stand alone” treatment may not result in desired level of control of the target weed species. Under such circumstance, fungi may be required to be combined with other weed control tools to achieve the desired levels of weed control (Müller-Schärer 2007; Yandoc-Ables & Rosskopf 2007). Such integration has particular implication in rangeland ecosystem where most of the weeds being woody perennials are unexpected to be controlled using a single bio-control agent (Julien 2006). However, the success of integration of fungi with other control practices is determined by several factors such as the morphology and ecology of the target weed species, extent of prevailing natural enemies, current control practices and their interaction with the potential bioherbicide fungi, required level of control, etc.

There are cases where integration of fungi with other natural enemies particularly insects resulted in higher levels of control of weeds including those invading rangelands. For example, Caesar (2003) investigated the interactions between soil-borne fungi and root-attacking insects of leafy spurge (Euphorbia sp.), an invasive rangeland weed in the USA. Combinations of either Fusarium oxysporum Schltdl., Rhizoctonia solani Kühn or both fungi with adults and larvae of the flea beetle (Aphthona spp.) were found to cause significantly higher levels of damage to leafy spurge compared to any single agent.

Buccellato (2012) investigated the interactions between Procecidochares utilis Stone, a stem fly and Passalora ageratinae Crous & Wood, a leaf-spot pathogen in terms of bio-control effect on invasive crofton weed ( (Spreng.) King & Rob.). The study found an additive interaction between the fungus and insect leading to enhance their usefulness as bio-control agents of crofton weed. Similar additive effect of potential bioherbicide fungi and other enemies leading to higher levels of control was also previously observed with Canada thistle (Cirsium arvense (L.) Scop.) and melaleuca (Melaleuca quinquenervia (Cav.) Blake), invasive weeds in North America (Rayamajhi et al. 2010; Sciegienka et al. 2011). 23

Increased efficacy of potential bioherbicide fungi was also reported once combined with particular cultural practice. For example, Bailey et al. (2013) investigated the effect of nitrogenous fertilizer on the efficacy of the fungus, macrostoma Mont. to control different broad-leaved weeds in turfgrass. The study found 10–15% improvement in control of dandelion (Taraxacum sp.) when the fungus was combined with a co-treatment of commercial nitrogen fertilizer.

Fungi are also combined with other suitable chemicals or synthetic herbicides to achieve higher levels of bioherbicidal efficacy (Hoagland 1996; Hoagland et al. 2011; Peng & Wolf 2011). However, the dose of chemical herbicide is an important consideration while combining with fungi. As a general rule, a sub-lethal dose of chemical herbicide is applied to increase the susceptibility of the target species, while minimising the damage to non-target plants (DiTomaso 2008; Velini et al. 2010). Among the different chemical herbicides, glyphosate (N-(phosphonomethyl) glycine) was most frequently used for this purpose and found to be compatible with a range of fungi (Boyette et al. 2008; Gressel 2010). As yet such integration of fungi with sub-lethal chemical herbicides is not a common practice in the control of woody weeds compared to the control of herbaceous weeds. For example, Sharon et al. (1992) investigated the interactions between Alternaria cassiae Jurair & Khan and sub-lethal glyphosate to control American sicklepod (Cassia obtusifolia L.), a weedy . Glyphosate was applied as foliar spray and combined with the fungus as co-treatment. The study found suppression of a specific phytoalexin resulting in increased susceptibility of American sicklepod to A. cassiae.

Shabana & Mohamed (2005) investigated the integrated control options of water hyacinth (Eichhornia crassipes Mart.) using Alternaria eichhorniae Nag Raj & Ponnappa and a synthetic chemical. The study found that fungus caused significantly higher levels of damage to water hyacinth once combined with a pre-treatment of 3,4-methylenedioxy trans-cinnamic acid, a host defence weakening synthetic chemical.

Abdel-Kader & El-Mougy (2007) reported that the combinations of mycoherbicide with foliar spray of a lower dose of glyphosate were more effective to control broomrape (Orobanche ramose L.) infestation in tomato (Lycopersicum esculentum Mill.) field.

Mitchell et al. (2008) investigated the interactions of two potential bioherbicide fungi (Colletotrichum graminicola Ces. and Gloeocercospora sorghi Bain and Edg.) with a sub-lethal dose of glyphosate to control shattercane (Sorghum bicolor (L.) Moench. ssp. drummondii (Nees ex Steud.) de Wet ex Davidse). The study found significantly higher levels of loss in weed biomass when each of the fungi was combined with a pre-treatment of sub-lethal glyphosate.

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Ortiz-Ribbing et al. (2011), reported significantly higher levels of disease incidence and severity in Amaranthus weeds (Amaranthus spp.) of soybean (Glycine max L.) when each or both of the potential bioherbicide fungi Microsphaeropsis amaranthi Heiny & Mintz (Ell. & Barth.) and Phomopsis amaranthicola Rosskopf, Charud. & DeVal. were combined with a pre-inoculation foliar spray of sub-lethal glyphosate.

1.2.6.5 Current status of invasive weed bio-control using fungi in Australia

In Australia, fungi were identified as potential weed bio-control agent in early 1950’s when introduced crofton weed (Ageratina adenophora (Spreng.) King & H. Rob.) was found to be naturally infected by a highly host-specific leaf spot fungus, Phaeoramularia eupatorii-odorati (Yen) Liu & Guo resulting in widespread loss of the weed population in Queensland (Dodd 1961). Since then, the continued damage to the weed population by the fungus has resulted crofton weed being much less invasive (McFadyen 2012). However, the fungus was never deliberately released as a bio-control agent for crofton weed (McFadyen 2012).

Following that, there were several attempts at deliberate release of both foreign and native fungi as classical and inundative bio-control agents of different invasive weeds in Australia. However, the majority of the fungi introduced to Australia from overseas as classical weed bio-control agents belong to the genus Puccinia, commonly known as rust fungi. The rust fungi are more effective bio- control agents because they are often highly host-specific. For example, Puccinia chondrillina Bubak & Syd. was introduced from Italy and released in Australia in 1971 as a classical bio-control agent of skeleton weed (Chondrilla juncea), formerly a major weed of wheat throughout the Australian eastern states (Cullen 1978). The fungus successfully established and caused severe rust epidemics resulting in significant decline of skeleton weed populations (Cullen 1978). The current level of skeleton weed infestation in Australia is within tolerable limit (Cullen 2012).

Another rust fungus, Puccinia myrsiphylli (Thuem) Wint. sourced from South Africa was released in New South Wales, Western Australia and South Australia in 2000 as a classical bio-control agent of bridal creeper (Asparagus asparagoides (L.) Druce) (Morin et al. 2006). The fungus established in a majority of the release sites and caused significant damage in coastal sites where climatic conditions were favourable for development of rust epidemics (Morin & Scott 2012). A recent report by Lefoe & Stephenson (2013) indicates that the rust fungus is now also widespread in southern Victoria causing significant damage to bridal creeper infestations.

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In Australia, deliberate release of rust fungi was also found to be moderately successful for controlling rubber vine (Cryptostegia grandiflora (Roxb.) Brown) and lantana (Lantana camara L.) (Day 2012; Palmer & Vogler 2012). However, there are examples where the classical bio-control approach using rust fungi was not successful as a result of non-establishment of the fungus. For instance, a rust fungus, Diabole cubensis Arthur & Johnst. was imported from central America and released in Australia in 1990s as a classical bio-control agent of mimosa (M. pigra). The fungus failed to establish long-term in Australia because of climatic restrictions although field observations in the native range and preliminary evaluations in England suggested its potential to be utilized as classical bio-control agent for mimosa (Hennecke 2004; Heard 2012).

In addition, there are findings where the rust fungus released as classical bio-control established but, resulted in an insignificant impact on the target weed population. For example, the rust, Puccinia abrupta Diet. & Holw. var. partheniicola (Jacks.) Parmelee sourced from Mexico was released in 1990’s at several sites in northern, central and southern Queensland as a classical bio-control agent of parthenium weed (Parthenium hysterophorus L.) (Dhileepan & McFadyen 1997). The fungus failed to establish in north Queensland because of high temperature and lack of sufficient moisture. However, it was well-established in southern Queensland and also in a few sites in central Queensland. In spite of establishment, its overall impact on parthenium population was negligible (Dhileepan & McFadyen 2012). Following that, another rust fungus, Puccinia xanthii var. parthenii-hysterophorae Seier, Evans & Romero was introduced from Mexico and released in 2000 at several parthenium infestation sites in northern and central Queensland (Dhileepan et al. 2006). The fungus rapidly established in most of the release sites causing significant foliar rust. However, the infection was found to be impaired by subsequent prolonged dry periods in 2004-2006, resulting in insignificant impact on parthenium population (Dhileepan et al. 2006).

Apart from the classical approach, a number of native fungi were utilized as inundative bio-control agents of different invasive weeds in Australia. However, similar to the classical approach, the success with inundative utilization of fungi is also variable. For example, ageratinae Barreto & Evans, a smut fungus previously released in , South Africa and New Zealand as a classical bio-control agent of invasive mistflower ( (Regel) King & Rob) was isolated from the same weed near Lamington National Park, Queensland, Australia (Morin et al. 2012). The fungus was deliberately released in the mistflower infestation sites in New South Wales which resulted in severe defoliation within 5-6 months period (Morin et al. 2012). There are indications that the fungus is spreading rapidly in the surrounding mistflower infestation sites in New South Wales.

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An anthracnose fungus, Colletotrichum orbiculare (Berk.) Arx was found to naturally infect bathurst bur (Xanthium spinosum L.), an invasive rangeland weed in eastern Australia (Morin & Auld 2012; Walker et al. 1991). The fungus causes girdling stem lesions and plant mortality and was subsequently formulated as a bioherbicide (Auld et al. 1988). However, the bioherbicide was not commercialized because of its inconsistent field performance (Morin & Auld 2012).

Gilbert et al. (2008) conducted an extensive survey in New South Wales to collect potential bioherbicidal fungi for controlling invasive alligator weed (Alternanthera philoxeroides (Martius) Grisebach). The survey resulted in 100 different species of pathogenic fungi in the genera Colletotrichum, Fusarium, Phoma, Phomopsis, Sphaceloma and Nimbya. Among those, Nimbya sp. causing leaf and stem lesions was reported to show promise as a potential bioherbicide candidate for alligator weed.

Botryosphaeriaceae fungi associated with dieback of parkinsonia (P. aculeata) are showing promise as potential bioherbicide candidate against this invasive rangeland weed (Galea & Goulter 2013). In addition, there are on-going efforts at The University of Queensland, Australia to find suitable bio- control fungi for other invasive weeds such as prickly acacia, mimosa, athel pine, and gamba grass.

1.3 Research objectives

This study broadly aimed to investigate the potential association of fungi and water stress and their interactions with prickly acacia dieback. Another aim was to investigate the scope of using dieback fungi as bio-control agent of prickly acacia. However, specific objectives of this study were-

1. To identify the fungi present in dieback-affected and apparently healthy stands of prickly acacia and to investigate whether the fungi could cause dieback following artificial inoculation under controlled conditions.

2. To investigate whether the fungi could cause dieback following artificial inoculation under field conditions.

3. To investigate whether dieback could be enhanced through addition of an artificial stressor.

4. To investigate the interactions between drought stress and fungi in prickly acacia dieback.

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Chapter 2: Identification and pathogenicity to seedlings and juvenile plants of the fungi associated with dieback of prickly acacia

2.1 Abstract

Prickly acacia is one of the most harmful woody weeds of the Australian rangelands. Characteristic symptoms of dieback were observed on this invasive species in 2010 among the plants growing around Richmond and Julia Creek in north-western Queensland. Affected plants were found with disease symptoms ranging from ashy internal staining, defoliation, blackening of shoot tips through to widespread plant mortality. It was hypothesized that any fungi associated with this phenomenon could be present in the healthy plants as asymptomatic colonizers. Therefore, this study aimed to identify the fungi associated with dieback-affected and healthy populations of prickly acacia and evaluate their pathogenicity.

A set of 166 fungal isolates were recovered. Of these, 135 isolates were sourced from dieback affected stem samples and 31 isolates were extracted from apparently healthy stem and root samples of prickly acacia. All the isolates were putatively identified by partial sequencing of the internal transcribed spacer (ITS) region of genomic DNA. Botryosphaeriaceae was the most represented family of the fungi associated with dieback. Among the Botryosphaeriaceae, Cophinforma was found to be the most prevalent genus with 60% of the total isolates identified as Cophinforma species by BLAST searches. Cophinforma was also isolated from the healthy plants at Richmond. Other Botryosphaeriaceae fungi associated with dieback were Phaeobotryosphaeria citrigena and Pseudofusicoccum violaceum whereas Neofusicoccum parvum was isolated from the healthy plants. Apart from Botryosphaeriaceae, a number of other fungi were associated with prickly acacia dieback and many of those such as, Alternaria sp., Aureobasidium pullulans, Cladosporium uredinicola and Paecilomyces sp. were also isolated from the healthy plants indicating a possible endophytic association.

Following phylogenetic analysis of the ITS sequences, the Cophinforma isolates extracted from prickly acacia did not typically correspond to any known species of Cophinforma previously extracted from the other hosts. Such findings indicate the potential association of new species of Cophinforma with prickly acacia in Australia. However, the ITS sequences of isolates of other species conformed to the sequences of their putative type species as found in GenBank.

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In the seedling bioassay, all fungal isolates found in dieback-affected and apparently healthy plants were screened for pathogenicity to prickly acacia seedlings, and resulted in observations of either no disease or of various disease symptoms such as, pre-emergent damping off, post-emergent seedling mortality, hypocotyl and cotyledon rot, necrotic cotyledon and hypocotyl lesions and severe stunting. The majority of the isolates screened were found to be highly pathogenic to prickly acacia seedlings. A sub-set of 39 isolates representing the majority of the identified species, plant health categories (dieback-affected and healthy) and all collection sites were used to challenge juvenile plants following a stem inoculation technique under glasshouse conditions. In the glasshouse trials, Cophinforma sp. (isolated from dieback-affected and healthy prickly acacia and dieback-affected M. pigra) and Lasiodiplodia pseudotheobromae (isolated from dieback-affected P. aculeata) consistently caused significant stem lesions, gummosis and leaf mortality. However, the glasshouse studies could not reveal any difference in virulence on juvenile plants among the isolates of Cophinforma sourced from different sites and hosts. F. acuminatum, N. parvum and T. graminis isolated from healthy prickly acacia also caused similar disease symptoms in glasshouse inoculation trials. The other species tested in the glasshouse trials were mostly non-pathogenic to juvenile plants.

This is the first investigation of the potential association of fungi with prickly acacia dieback in Australia. It indicates that a number of endophytic fungi might be involved in this phenomenon, and some displaying high pathogenicity may have potential for use as bioherbicides. However, longer- term trials using appropriate field-delivery techniques are required to confirm their potential to be used as bioherbicides.

2.2 Introduction

Tree dieback is a global phenomenon which is also a concern in many parts of Australia, both in native and introduced trees, shrubs and vines (Jurskis 2005; Desprez-Loustau et al. 2006; Ireland et al. 2012). Of the various causes of dieback, fungi were reported to be associated with a number of incidences (Cahill et al. 2008; Sakalidis et al. 2011; Davison 2014). Moreover, there are increasing number of recent reports on the potential association of opportunistic or endophytic fungi with dieback of different perennial plant species (Dakin et al. 2010; Begoude et al. 2011; Crone et al. 2013; Pitt et al. 2013). Such opportunistic endophytes usually colonize host tissues without producing any clear disease symptom unless the host is under a condition of stress (Slippers & Wingfield 2007; Mehl et al. 2013). Fungi including the opportunistic endophytes were also found to be associated with the dieback of several Australia’s worst weeds such as athel pine (Tamarix

59 aphylla L. Karst), bitou bush (Chrysanthemoides monilifera (L.) Norl.), European blackberry (Rubus fruticosus L.), mimosa (Mimosa pigra L.) and parkinsonia (Parkinsonia aculeata L.) (Wilson & Pitkethley 1992; Cother et al. 1996; Diplock et al. 2006; Gouldthorpe 2008; Sacdalan et al. 2013; Aghighi et al. 2014). At present dieback is the major limiting factor to the spread of parkinsonia in northern Australia (van Klinken et al. 2009). A Botryosphaeriaceae fungus Lasiodiplodia pseudotheobromae A.J.L. Phillips, A. Alves & Crous was associated with parkinsonia dieback. The same fungus was found to be highly aggressive to prickly acacia seedlings and juveniles in preliminary studies (unpublished data).

Prickly acacia (Vachellia nilotica subsp. indica (Benth.) Kyal. & Boatwr) is another invasive weed of the Australian rangelands (Palmer et al. 2012). It had long been reported by landholders to display naturally occurring dieback symptoms in several sites across north-western Queensland (March 2009; Queensland Department of Agriculture Fisheries and Forestry 2014). A more prominent dieback event was observed on this species more recently in 2010 among the plants growing at Richmond and Julia creek in north-western Queensland (Galea 2011). Affected plants were found with symptoms ranging from ashy internal staining, defoliation, blackening of shoot tips through to widespread plant mortality.

Based on the symptoms observed on dieback-affected prickly acacia, preliminary laboratory studies and findings on dieback of other invasive rangeland weeds, we hypothesized that fungi are implicated with this phenomenon. If this is true, then it may offer novel ways of managing this species, and they could offer an exciting potential for biological control of many invasive weed species including prickly acacia (Wood & Ginns 2006; Galea 2008; Galea & Goulter 2013). However, to achieve that goal, first the fungi associated with this phenomenon should be identified, their distribution should be understood and pathogenicity as potential bioherbicides must be evaluated.

Therefore, the studies presented in this chapter were undertaken to identify the fungi associated with prickly acacia dieback, to investigate whether similar fungi were also asymptomatically present in the healthy plants as endophytes and to test their pathogenicity under laboratory and glasshouse conditions to prioritize the potentially pathogenic fungi for further testing under field conditions.

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2.3 Materials and Methods

2.3.1 Sample collection

Sampling was conducted in both dieback-affected and healthy stands of prickly acacia. Samples from dieback-affected plants were collected in July 2010 (before commencing this PhD project) by

Fig. 2.1 Map showing the sampling sites () at north-western Queensland. 61

Dr. Victor Galea. Stem samples (roughly 30 cm × 15 cm) were collected by selectively sampling 3- 5 dieback-affected trees (one stem sample per tree) at nine different sites between Richmond and Julia Creek, north-western Queensland (Fig. 2.1). In June 2012, both stem and root samples were collected from apparently healthy trees by sampling at a single site at Richmond, north-western Queensland and another site at Rockhampton, central Queensland where the field inoculation trials were established (Chapter 3-4). At each site, five trees free from any visible symptoms of dieback were selected. From each tree, five stem (roughly 30 cm × 15 cm) and five lateral root (approximately 20 cm × 0.5 cm) samples were collected. All samples were stored at 4oC until being used to isolate fungi.

2.3.2 Isolate acquisition

Stem and root samples were washed under running de-ionized water to remove the soil and other debris followed by drying within a laminar air-flow cabinet. Once dried, the samples were surface sterilized by spraying to run-off with 70% Ethanol and left for five minutes to dry. Wood shavings from stem samples were extracted by drilling into surface sterilised stem pieces with a flame sterilised drill bit and collected into sterile Petri dishes. In addition, isolation was conducted separately from the bark of stem samples collected from apparently healthy plants. Bark was removed from the surface sterilized stem samples using a flame sterilized knife, cut in to 1 cm pieces using a flame sterilized secateurs and collected in sterile Petri dishes.

From the surface sterilized root samples, bark was removed using a flame sterilized sharp scalpel, cut into 1 cm pieces using a flame sterilized scissors and collected in sterile Petri dishes. Debarked root samples were cut into 1 cm discs using a flame sterilized secateurs and collected in sterile Petri dishes.

Wood shavings extracted by drilling the stem samples were aseptically transferred to either ½ strength Potato Dextrose Agar (½PDA), Malt Extract Agar (MEA), V8 Juice Agar (V8A) or Oatmeal Agar (OA) plates amended with Penicillin (Sigma®, Penicillin G sodium salt) at 0.12 g/400 ml (300 ppm) and Streptomycin (Sigma®, Streptomycin sulfate salt) at 0.08 g/400 ml (200 ppm) (Ritchie 2002). The bark of both stem and root and root cortex samples were surface sterilized again by soaking in 2% NaOCl for two minutes followed by rinsing in sterile water twice for five minutes and drying on sterile tissue paper under laminar air-flow cabinet before transferring to the plates containing the above fungal growth media. Plates were sealed with Parafilm M®, put in air- tight plastic bags and incubated in a darkened incubator at 25oC. The plates were observed daily and after 3-7 days incubation the isolates were purified by subculturing onto the same media. Only one 62 sub-culture was taken from the initial isolation plates unless there were distinct morphological differences observed on a plate and then each variant was sub-cultured on the same media. Finally, all the isolates were sub-cultured again on antibiotic-amended ½PDA plates. From the growing margin of each fungal isolate, five sections (6 mm diameter) were cut using a flame-sterilized cork borer and aseptically transferred to sterile McCartney tubes containing sterile water. The tubes with fungal cultures were stored in sealed boxes at 25oC in a darkened cupboard (Smith 2002).

2.3.3 Identification of the fungal isolates

All the fungal isolates were identified by partial sequencing of the Internal Transcribed Spacer (ITS) region followed by comparison to the non-redundant GenBank database. Details of sequencing and identification are outlined below.

2.3.3.1 DNA extraction, purification and quantification

Fungal isolates were cultured for 3-4 days in 1 mL clarified V8 Juice broth amended with Penicillin (Sigma®, Penicillin G sodium salt) at 0.12 g/400 mL (300 ppm) and Streptomycin (Sigma®, Streptomycin sulfate salt) at 0.08 g/400 mL (200 ppm) in wells of 24 well sterile tissue culture plates (Falcon® 3047). Mycelium was harvested and aseptically transferred to sterile 1.5 mL Eppendorf tubes. While transferring, care was taken to avoid any trace of agar from the original mycelial plug. Tubes were stored at -80oC until extraction. Genomic DNA was extracted and purified using the Rapid Fungal Genomic DNA Isolation Kit (Biobasic Inc., Ontario, Canada) following the manufacturer’s instructions (Bio Basic Inc. 2011) except that the samples were used straight from the freezer rather than freezing in liquid nitrogen. For the isolates with visible pigment, the 70% ethanol wash step in the protocol was repeated for three times. DNA in TE buffer was quantified using a micro-volume spectrophotometer (BioSpec-nano, Shimadzu Scientific Instruments, Japan) and stored at -20oC until PCR amplification.

2.3.3.2 PCR amplification and DNA sequencing

A section of the Internal Transcribed Spacer (ITS) of genomic DNA extracted from each of the fungal isolate was amplified by Polymerase Chain Reaction (PCR) using the primers ITS 1 (5ʹ-TCC GTA CGT GAA CCT GCG G-3ʹ) and ITS4 (5ʹ-TCC TCC GCT TAT TGA TAT GC-3ʹ) (White et al. 1990). PCR amplification was conducted using a PCR Express thermal cycler (Hybrid, Ashford, Middlesex, UK). The 50μL PCR reaction mixture contained 2μL DNA template solution

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(approximately 100ng genomic DNA), 1μM each of primer, 5 μL of My Taq Red Reaction Buffer

(5mM dNTPs, 15mM MgCl2) and 0.25U of My Taq DNA Polymerase (Bioline Pty. Ltd, Australia). The PCR cycle consisted of an initial denaturation at 95oC for three minutes, followed by 35 cycles of 95oC for 15 seconds, annealing at 55oC for 15 seconds, 72oC for 30 seconds, with the final extension at 72oC for 6 minutes. Twenty microlitres of each amplification reaction was run on a 0.5% w/v Agarose/TBE buffer gel (Tris, boric acid, EDTA) containing SYBR® Safe DNA gel stain (Invitrogen, Carlsbad, Canada) against a 100bp molecular ladder (Hyper Ladder IV, Bioline Pty. Ltd., Australia) to estimate the product size. Gels were run in 1x TBE at 120V for 45 minutes before visualizing the amplicons on a UV transilluminator and a digital image captured.

Amplicons were purified using PureLink® PCR Purification Kit (Invitrogen, Carlsbad, Canada) and were quantified using a micro-volume spectrophotometer (BioSpec-nano, Shimadzu Scientific Instruments, Japan) (Invitrogen 2011). 50μL (50ng/μL concentration) purified PCR product was prepared in 0.1x TE buffer of each fungal isolate and was forwarded to Macrogen Inc. (South Korea) and Sanger sequenced using the ITS1 primer. The nucleotide sequences were read and edited with FinchTV 1.4.0 (Geospiza Inc. http://www.geospiza.com/finchtv). All sequences were checked manually and nucleotide arrangements at ambiguous positions were clarified. The sequence identity was determined by performing BLAST searches against the database of National Centre for Biotechnology Information (NCBI), USA. For the Botryosphereaceae fungi, species name was assigned only when there was 100% sequence similarity. However, for other fungi, 98% sequence similarity was considered as conspecific.

2.3.3.3 Sequence alignment and phylogenetic analysis

Sequences were aligned using ClustalW2 (http://www.ebi.ac.uk/Tools/msa/clustalw2/) using the following parameters: pairwise alignment parameters (gap opening = 10, gap extension = 0.1) and multiple alignment parameters (gap opening = 10, gap extension = 0.2, gap distance = 5, numiter = 1). Alignments were checked and manually adjusted if necessary. Wherever available, alignments also included the holotype or ex-type sequences of the same and/or other closely related species downloaded from the GenBank database. Phylogenetic analyses for Maximum-Parsimony of the sequence data was conducted using PAUP v. 4.0 b10 (Swofford 2003). The analyses were performed using 1,000 random taxa addition with branches swapped using the Tree Bisection and Reconnection (TBR) algorithm. The heuristic search option was used with unordered characters of equal weight and gaps were treated as missing data. Maxtrees was set to 100 with automatic increase if required and branches of 0 length were collapsed. All multiple equally parsimonious

64 trees were saved. The robustness of the most parsimonious trees was evaluated from 1,000 bootstrap replications. Other measures used were consistency index (CI), retention index (RI) and homoplasy index (HI). A consensus tree rooted to either Pseudofusicoccum stromaticum (Mohali, Slippers & M.J. Wingf.) Mohali, Slippers & M.J. Wingf. or Macrophomina phaseolina (Tassi) Goid. was derived using the majority rule algorithm and was saved and viewed using TreeView (Page 1996). The image was saved as an enhanced metafile (.emf) and imported into Microsoft PowerPoint for editing.

2.3.4 Prioritisation of fungal isolates for glasshouse inoculation studies

A large number of fungal isolates across the range of species were extracted from the samples collected from dieback-affected and healthy prickly acacia plants. All those isolates were tested in the seedling bioassay technique under laboratory conditions to prioritise the potentially pathogenic isolates for further testing under glasshouse conditions. Details of the techniques are outlined below.

2.3.4.1 Seedling bioassay

This initial screening was conducted following a seedling bioassay technique using pre-germinated prickly acacia seeds sourced from dieback-free plants growing at Richmond, north-western Queensland. Seeds were washed repeatedly on a plastic sieve under running RO water to remove all debris and mummies. Clean seeds were surface sterilized by soaking in 2% NaOCl for one minute followed by washing twice for five minutes with sterile RO water followed by drying with sterile tissue paper. To facilitate water absorption and subsequent germination, the hard seed coat of the surface sterilized seeds were removed at the embryo-end using a surface sterilized clipper. Within the sterile working area of a laminar flow cabinet, clipped seeds were again soaked in 2% NaOCl for five minutes followed by washing twice for five minutes with sterile RO water to remove any traces of NaOCl. The surface sterilized seeds were transferred to surface sterilized plastic trays each containing four pieces of moist sterile blotting paper, covered with a lid and incubated in an incubation chamber operating at 25oC with daily 12-hour-light period. Germination trays were checked and watered with sterile RO water if necessary to ensure the presence of sufficient moisture. Most of the seeds germinated within 2-3 days. Germinated seeds with the radicle appearing and extending over half the length of the seed and free from any visible fungal growth were selected and transferred to a sterile glass beaker under a laminar flow cabinet and again soaked in 2% NaOCl for one minute followed by gentle washing twice for ten minutes with sterile RO water to remove any traces of NaOCl. 65

Plastic McCartney tubes (80 mm × 25 mm) with a 4 mm drainage hole in the base were filled with approximately 12 g (dry wet) vermiculite, wetted with RO water and autoclaved. Within the sterile working area under a laminar flow cabinet, one pre-germinated seed was placed in each tube pointing the radicle downward using a flame sterilized forceps. A single mycelial plug (6 mm diameter) taken from the margin of a colony growing on ½ PDA was placed at 2-3 mm away from the radicle. Then the inoculated seed was covered with approximately 10 g of sterile moist vermiculite. Afterwards the tubes were placed on an elevated drainage mesh in the plastic transparent incubation box (Décor®, 2.75 L, with lids) having a 6 mm ventilation hole in each side. Seedlings inoculated with fresh ½ PDA plug served as negative control. In addition, a highly aggressive isolate of L. pseudotheobromae (strain NT039) sourced from parkinsonia dieback was used as a positive control.

Each incubation box contained 16 tubes each containing one pre-germinated seed. Among these, 10 were treated with a single test isolate plus three positive controls and three negative controls. Both positive and negative controls were consistently included in each incubation box as a check that the experimental conditions were appropriate for infection (positive control) and were also appropriate for seedling growth, and to monitor any background levels of infection due to natural contamination of prickly acacia seed (negative control). The incubation boxes were randomly arranged in an incubation chamber operating at 25oC for two weeks with daily 12-hour-light period.

Pathogenicity assessment was conducted once at the end of the incubation period. Seedlings were grouped into following five categories based on visual observation of the disease symptoms:

(1) Pre-emergent damping-off: No seedling emergence, seed was decayed. (2) Post-emergent damping-off: Seedling emerged but died within the incubation period. (3) Diseased seedling: Seedling emerged, showed clear disease symptoms such as severe stunting, tip necrosis, necrotic lesion on cotyledon and hypocotyl, and was alive at the end of the incubation period. (4) Apparently healthy seedling: Seedling emerged from the seed, did not display any clear disease symptoms but was not completely healthy compared to the negative control seedlings contained in the same incubation box. Seedlings under this category were mostly found with visible fungal mycelia on the leaves and roots and slightly stunted growth. (5) Healthy seedling: Seedling completely healthy compared to the negative control seedlings contained in the same incubation box.

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2.3.4.2 Data analysis

Test-isolates were compared for the incidence (%) of different categories of seedling. The effect of negative control (blank ½ PDA plug) placed in different incubation boxes was slightly variable. On the other hand, the positive control (L. pseudotheobromae NT039) placed in different incubation boxes consistently caused significant seedling mortality. Therefore, the test-isolates were compared in a three way comparison where each isolate was compared to the other isolates, to the effect of the negative control contained in individual incubation box and to the pooled effect of the positive control contained in all incubation boxes. The comparison was performed in a Binary Logistic Regression (BLR) model (with 95% confidence interval) using a statistical software package Minitab version 16. No transformation was required to meet the BLR requirements. However, for each test-isolate, the number of seedlings showing pre-emergent and post-emergent damping off were added together to have a total count of damping off or seedling mortality for each isolate.

Based on BLR comparisons, the test-isolates were further grouped in four categories such as (1) Very highly pathogenic: isolates not significantly different from the positive control in terms of seedling mortality; (2) Highly pathogenic: isolates resulted in significantly higher incidence of diseased seedlings compared to the negative control and other test-isolates but caused significantly lower incidence of seedling mortality compared to the positive control; (3) Moderately pathogenic: isolates resulted in significantly higher incidence of apparently healthy seedlings compared to the negative control and other isolates but caused significantly lower incidence of diseased seedlings and seedling mortality compared to other isolates including the positive control (4) Weak or non- pathogenic: isolates not significantly different from the negative control in terms of the incidence of healthy seedling, no significant seedling mortality or disease symptoms compared to the other isolates including the positive control.

2.3.5 Glasshouse inoculation trials

A sub-set of 39 fungal isolates extracted from both dieback-affected and apparently healthy samples of prickly acacia were tested against juvenile plants under glasshouse conditions. Most of these isolates were found to be highly pathogenic in the seedling screening. However, a number of weak or non-pathogenic isolates were also included for testing under glasshouse conditions to check the reliability of the seedling bioassay technique. Because of limited glasshouse space, the isolates were tested in separate trials conducted in different glasshouses at different periods. Methods followed in the glasshouse inoculation trials are outlined below-

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2.3.5.1 Experimental design, treatments and growing juvenile plants

Each glasshouse trial was conducted for eight weeks using a randomized complete block design with 5 replicates for each treatment (individual fungal isolate). L. pseudotheobromae (strain NT039) found to be highly aggressive against prickly acacia seedlings under laboratory conditions was included in each trial as a standard check while autoclaved millet (Panicum milliaceum L.) grains were used as control. Prickly acacia seeds were collected from dieback-free plants growing at Richmond, north-western Queensland and plants were grown from surface sterilized and pre- germinated seeds (Toh et al. 2008) in 1.5 L pots containing potting mix and arranged on elevated benches in a glasshouse with automatic drip irrigation for five minutes at eight hours interval. No fertilizer was applied during the first two months. After two months, 250 ml of 0.16% Aquasol (N: P: K =23: 3.95: 14 + trace elements) was applied in every pot at two weeks interval. Plants in each trial were six months old at inoculation and were randomly allocated to treatments and blocks. There was a slight difference in plant size (height and stem diameter) in different trials. However, plant size was generally uniform within individual trials.

2.3.5.2 Treatment preparation and application

Fungal isolates were cultured on millet grains (French White) following the techniques outlined by Toh et al. (2008). Using an alcohol sterilized 3.5 mm drill bit mounted on a cordless drill a 5-6 mm deep inoculation hole was made in the stem (at a slight downward angle) at 10 cm above the soil level followed by placing two colonized millet grains using sterilized forceps and covering the wound with Parafilm®.

2.3.5.3 Trial assessment

Data were collected on stem lesion length, total leaf production, leaf mortality and stem-and branch dry-weight. Cut stems were longitudinally split with surface sterilized (using 70% Ethanol) secateurs to access the interlan stem lesions. Length of external and internal stem lesions was measured using a ruler. An aluminium foil tray with a drainage hole cut in the centre was placed beneath each plant to collect fallen leaves throughout the trial. Dead leaves attached with the plants were hand removed at the end of the trial. After removing the attached dead leaves, the plants with live (green) leaves were cut into small pieces and put in labelled aluminium trays and left in the glasshouse for 72 hours to commence desiccation. Both dead (fallen leaves plus attached dead leaves) and live leaves were dried separately in a drying oven at 60-65oC for one week. Total leaf production was calculated by adding the dry-weight of dead leaf and live leaves attached to the 68 plant at the end of the trial. Leaf mortality was expressed as percentage of dead leaf to total leaf production (except the glasshouse trial- 4 where the live leaves was lost, and, therefore, leaf mortality was expressed as dry-weight of dead leaves). Dry-weight of stem and branch samples was obtained following oven drying at 60-65oC for 2-3 weeks. Initial and final plant height was measured using a measuring tape and height increment during the course of the trial, was measured by subtracting initial plant height from the final plant height.

2.3.5.4 Confirmation of infection

Confirmation of infection was achieved by isolating the test-fungi from the inoculated plants. Isolation was also conducted form the uninoculated plants. Potential of the test-fungi to asymptomatically colonize healthy tissue of the inoculated plants as endophytes was also investigated. Isolation was conducted from the leading edge of internal stem lesions and healthy tissues adjacent to that following the techniques modified from Diplock et al. (2006). In each trial, stem segments collected from three randomly selected plants with each treatment were surface sterilized by soaking in 2% NaOCl for two minutes followed by rinsing in sterile water twice for five minutes and drying on sterile blotting paper within a laminar air-flow cabinet. Partially dried samples were aseptically transferred to ½ PDA media followed by incubation at 25oC in a darkened incubator for one week with daily observation. After the incubation period, the fungal isolates were purified by subculturing on fresh ½ PDA plates before comparing to original isolates used in the experiment.

2.3.5.5 Data analysis

Treatment effects on stem lesion length, total leaf production, leaf mortality, stem/branch dry weight and plant height was tested using either one way or GLM ANOVA in the statistical software package Minitab version 16. Treatment means were compared following either Fisher’s or Tukey’s test at 95% level of confidence. Wherever required, the data were transformed using Arcsine transformation to meet ANOVA requirements.

2.4 Results

2.4.1 Identification

A set of 166 fungal isolates were prepared. Of these, 135 isolates were sourced from die-back affected stem samples and 31 isolates extracted from healthy stem and root samples of prickly

69 acacia. All the fungal isolates were putatively identified by the ITS1 comparisons and results are summarised in Table 2.1 and described in more detail in Appendix 1.

Table 2.1 Fungi isolated from prickly acacia (identification is solely based on ITS1 sequence comparison). Species names in bold were associated with both dieback-affected and apparently healthy plants. Data in parenthesis indicates percentage of isolates of individual species to the total number of isolates.

Identification Family No. of Isolates From- Dieback Plants Healthy Plants

Cophinforma spp. Botryosphaeriaceae 93 (60) 8 (26) Pseudofusicoccum violaceum Botryosphaeriaceae 9 (7) 0 Phaeobotryosphaeria citrigena Botryosphaeriaceae 4(3) 0 Neofusicoccum parvum Botryosphaeriaceae 0 2 (6) Aureobasidium pullulans Dothioraceae 6 (4) 1 (3) Paecilomyces sp. Trichocomaceae 7 (5) 1 (3) Alternaria sp. 0 6 (19) Mortierella alpina Mortierellaceae 0 4 (13) Asteromella pistaciarum - 3(2) 0 Pleurostoma ootheca Calosphaeriaceae 3 (2) 0 Zetiasplozna acaciae Amphisphaeriaceae 0 3 (10) Alternaria alternata Pleosporaceae 1 (0.7) 1 (3) Cladosporium uredinicola Davidiellaceae 1 (0.7) 1 (3) Rhytidhysteron rufulum Patellariaceae 2 (15) 0 Curvularia lunata Pleosporaceae 1 (0.7) 0 Curvularia pseudorobusta Pleosporaceae 1 (0.7) 0 Cytospora austromontana Valsaceae 1 (0.7) 0 Exserohilum rostratum Pleosporaceae 1 (0.7) 0 Fusarium acuminatum Nectriaceae 0 1 (3) Paraphaeosphaeria angularis Phaeosphaeriaceae 0 1 (3) Pyrenochaetopsis microspora Cucurbitariaceae 1 (0.7) 0 Tiarosporella graminis Leotiomycetidae 0 2 (6) Umbilicaria muehlenbergii Umbilicariaceae 1 (0.7) 0

The Botryosphaeriaceae was the best represented family of the fungi associated with dieback. Among the Botryosphaeriaceae, Cophinforma was found to be the most prevalent genus with 60% of the total isolates identified as Cophinforma spp. Other Botryosphaeriaceae fungi associated with dieback were Phaeobotryosphaeria citrigena A.J.L. Phillips, P.R. Johnst. & Pennycook (four 70 isolates) and Pseudofusicoccum violaceum Mehl & Slippers (nine isolates). Cophinforma sp. was also isolated from apparently healthy stem and root samples collected from Richmond, north- western Queensland in the same region where the natural dieback event was previously observed on prickly acacia in 2010. On the other hand, another Botryosphaeriaceae fungus Neofusicoccum parvum (Pennycook & Samuels) Crous, Slippers & A.J.L. Phillips (two isolates) was associated with apparently healthy root and stem samples of prickly acacia collected from Rockhampton, central Queensland where natural dieback was not previously reported.

The remaining isolates belonged to 19 different texa: Aureobasidium pullulans (de Bary) G. Arnaud (seven isolates), Paecilomyces sp. (eight isolates), Alternaria sp. (six isolates), Alternaria alternata (Fr.) Keissl. (two isolates), Mortierella alpina Peyronel (four isolates), Pleurostoma ootheca (Berk. & M.A. Curtis) M.E. Barr (three isolates), Zetiasplozna acaciae Crous (three isolates), Asteromella pistaciarum Bremer & Petr. (three isolates), Cladosporium uredinicola Speg. (two isolates), Rhytidhysteron rufulum (Spreng.) Speg. (two isolates), Tiarosporella graminis (Piroz. & Shoemaker) Nag Raj (two isolates), Curvularia lunata (Wakker) Boedijn (one isolate), Curvularia pseudorobusta Meng Zhang & T.Y. Zhang (one isolate), Exserohilum rostratum (Drechsler) K.J. Leonard & Suggs (one isolate), Pyrenochaetopsis microspora (Gruyter & Boerema) Gruyter, Aveskamp & Verkley (one isolate), Cytospora austromontana G.C. Adams & M.J. Wingf. (one isolate), Fusarium acuminatum Wollenw. (one isolate), Umbilicaria muehlenbergii (Ach.) Tuck. (one isolate) and Paraphaeosphaeria angularis Verkley & van der Aa (one isolate).

Of the diverse assemblage of fungi isolated from prickly acacia, Alternaria alternata, Aureobasidium pullulans, Cophinforma spp., Cladosporium uredinicola and Paecilomyces sp. were isolated from both dieback-affected and apparently healthy plants. On the other hand, Asteromella pistaciarum, Curvularia pseudorobusta, Curvularia lunata, Cytospora austromontana, Exserohilum rostratum, Pleurostoma ootheca, Phaeobotryosphaeria citrigena, Pyrenochaetopsis microspora, Rhytidhysteron rufulum, Pseudofusicoccum violaceum and Umbilicaria muehlenbergii were exclusively associated with the dieback-affected plants. The other species, such as Neofusicoccum parvum, Alternaria sp., Mortierella alpine, Zetiasplozna acacia, Fusarium acuminatum, Paraphaeosphaeria angularis and Tiarosporella graminis were solely associated with the apparently healthy plants.

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2.4.2 Phylogenetic analysis

ITS sequences from 102 isolates of Cophinforma spp. resulted in 33 clusters following maximum parsimony analysis. The phylogenetic tree with one representative isolate from each of the 33 clusters is presented in Fig. 2.2. There was no apparent relation among the clustering of isolates, their geographic origin or pathogenicity in the seedling bioassay. The aligned ITS dataset of Cophinforma isolates consisted of 548 characters of the ITS including coded alignment gaps. Of these, 406 characters were constant, 79 were variable and parsimony uninformative and 63 were parsimony informative. The heuristic searches resulted in 202 most parsimonious trees (CI = 0.84, RI = 0.92, RC = 0.77 and HI = 0.15). The maximum parsimony tree is presented in Fig. 2.1. It indicates that the Cophinforma isolates sourced from prickly acacia did not typically cluster with any of the three described species of Cophinforma such as C. eucalypti Doilom, J.K. Liu & K.D. Hyde, C. atrovirens (Mehl & Slippers) A. Alves & A.J.L. Phillips and C. mamane (D.E. Gardner) A.J.L. Phillips & A. Alves. although BLAST searches found 99% similarity of all the Cophinforma isolates with C. eucalypti. The isolates of Cophinforma also did not typically cluster with any other species of Botryosphaeriaceae fungi included in the analysis. However, within the parsimony tree (Fig. 2.2) it was possible to identify three major clades (with less than 50% bootstrap support) – one linked with C. mamane, another linked with both C. eucalypti and C. atrovirens and the third clade not linked with any described species of Cophinforma. Such findings indicate the potential association of previously undescribed species of Cophinforma with prickly acacia dieback.

The aligned ITS dataset of other Botryosphaeriaceae fungi consisted of 621 characters of the ITS including coded alignment gaps. Of these, 464 characters were constant, 59 were variable and parsimony uninformative and 98 were parsimony informative. The heuristic searches resulted in 221 most parsimonious trees (CI = 0.89, RI = 0.93, RC = 0.83 and HI = 0.10). The maximum parsimony tree is presented in Fig. 2.3 with bootstrap support values above 50% shown. No significant intra-specific variation was observed following the phylogenetic (maximum parsimony) analysis. Therefore, one representative isolate of each species is presented in the tree (Fig. 2.2). Two isolates of Cophinforma sp. were included to show the genetic distance of Cophinforma from other Botryosphaeriaceae fungi associated with prickly acacia. The tree indicates that the isolates of Cophinforma formed a distinct in-group and did not cluster with any other Botryosphaeriaceae fungi. However, within the other in-groups, the test-isolates of L. pseudotheobromae, N. parvum, P. citrigena and P. violaceum correspond to their type species with 91-100% bootstrap support.

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AN#110 AN#76 AN#109 AN#65 AN#75 C AN#71 L AN#68 A AN#49 D AN#26 E AN#16 3 AN#93 AN#33 AN#19 AN#1 AN#44 AN#4 C AN#37 2 L 2 AN#6 A FJ888476 Cophinforma atrovirens D AN#7 E JX646800 Cophinforma eucalypti 2 ANE#27 ANE#28 ANE#25 AN#8 AN#67 AN#18 AN#9 C AN#64 L AF246929 Cophinforma mamane A AN#11 D AN#50 E AN#17 1 AN#53 12 AN#5 ANE#32 14 HQ332195 Fusicoccum fabicercianum DQ299245 Botryosphaeria corticis 22 AY236949 B. dothidea EU144055 F. ramosum DQ458888 B. tsugae AF241177 Neofusicoccum ribis EU301034 N. occulatum JX513629 N. brasiliense AY236936 B. ribis GU251139 N. parvum EU821900 N. kwambonambiense 2 AY339262 N. australe 2 AF293480 F. luteum 43 DQ131571 B. eucalypticola AY615185 N. mangiferum

10 JX464102 Pseudofusicoccum stromaticum

Fig. 2.2 Maximum parsimony tree based on bootstrap analyses of the ITS sequences from Cophinforma isolates from dieback-affected and apparently healthy samples of prickly acacia. The isolates in bold are of type species (ex or holo) obtained from NCBI database. The tree was rooted to P. stromaticum and all bootstrap support values are shown.

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EF 585505 Macrophomina phaseolina

AN#110 Cophinforma sp. 98 AN#19 Cophinforma sp.

NR111259.1 Pseudofusicoccum adansoniae

100 FJ888463.1 Pseudofusicoccum olivaceum

EU144062.1 Pseudofusicoccum ardesiacum

EU144058 Pseudofusicoccum kimberleyense

FJ888475.1 Pseudofusicoccum violaceum

99 AN#48 Pseudofusicoccum violaceum

AY236943 Neofusicoccum parvum 100 ANE#30 Neofusicoccum parvum

EF622070.1 Lasiodiplodia theobromae 51 97 NR111264.1 Lasiodiplodia pseudotheobromae 92 NT#039 Lasiodiplodia pseudotheobromae 100

JX646802 Phaeobotryosphaeria eucalypti

91 EU673328 Phaeobotryosphaeria citrigena 95 AN#146 Phaeobotryosphaeria citrigena

Fig. 2.3 Maximum parsimony tree based on bootstrap analyses of the ITS sequences from different Botryosphaeriaceae fungi from dieback-affected and apparently healthy samples of prickly acacia (except L. pseudotheobromae isolated from dieback-affected P. aculeata). The isolates in bold are of type species (ex or holo) obtained from NCBI database. The tree was rooted to M. phaseolina and bootstrap support values over 50% are shown.

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EF 585505 Macrophomina phaseolina AN#14 Asteromella pistaciarum 100 FR681904.1 Asteromella pistaciarum 98 ANE#34 Zetiasplozna acaciae 100 50 KJ869149.1 Zetiasplozna acaciae ANE#7 Mortierella alpina 100 KJ921606.1 Mortierella alpina 61 AN#145 Cytospora austromontana 99 AY347361.1 Cytospora austromontana 83 AN#62 Pleurostoma ootheca 100 96 51 HQ878590.1 Pleurostoma ootheca ANE#16 Fusarium acuminatum 99 KP068924.1 Fusarium acuminatum AN#03 Aureobasidium pullulans 100 FJ150956.1 Aureobasidium pullulans AN#118 Curvularia lunata 99 DQ836799.1 Curvularia lunata 99 AN#124 Curvularia pseudorobusta 100 AB453879.1 Curvularia pseudorobusta 82 88 AN#120 Exserohilum rostratum 100 GQ221129.1 Exserohilum rostratum 64 ANE#39 Alternaria sp. (near tenuissima) 100 91 KJ598870.1 Alternaria tenuissima AN#122 Alternaria alternata FJ228163.1 Alternaria alternata 84 60 AN#144 Pyrenochaetopsis microspora 100 HM751085.1 Pyrenochaetopsis microspora 86 ANE#15 Paraphaeosphaeria angularis 100 JX496047.1 Paraphaeosphaeria angularis AN#141 Rhytidhysteron rufulum 99 99 EU020065 Rhytidhysteron rufulum AN#63 Cladosporium uredinicola 100 KF679757.1 Cladosporium uredinicola AN#123 Paecilomyces sp. (near sinensis) 100 EU272527.1 Paecilomyces sinensis 100 93 KC157764.1 Paecilomyces formosus AN#142 Umbilicaria muehlenbergii 100 JQ036216.1 Umbilicaria muehlenbergii ANE#24 Tiarosporella graminis 100 KF531828 Tiarosporella graminis

Fig. 2.4 Maximum parsimony tree based on bootstrap analyses of the ITS sequences from different fungi (except Botryosphaeriaceae) isolated from dieback-affected and apparently healthy samples of prickly acacia. The isolates in bold are of type species (ex or holo) obtained from NCBI database. The tree was rooted to M. phaseolina and bootstrap support values over 50% are shown.

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The maximum parsimony tree of other fungi (not Botryosphaeriaceae) including Alternaria sp., A. alternata, A. pistaciarum, A. pullulans, C. uredinicola, C. lunata, C. pseudorobusta, C. austromontana, E. rostratum, F. acuminatum, M. alpina, Paecilomyces sp., P. angularis, P. ootheca, P. microspora, R. rufulum, T. graminis, U. muehlenbergii and Z. acaciae is presented in Fig. 2.4 with bootstrap support values above 50% shown. The aligned ITS dataset of the isolates consisted of 1072 characters of ITS including coded alignment gaps. Of these, 423 characters were constant, 63 were variable and parsimony uninformative and 586 were parsimony informative. The heuristic searches resulted in 1958 most parsimonious trees (CI = 0.58, RI = 0.79, RC = 0.45 and HI = 0.43). The tree indicates that except A. alternata, the test-isolates of other species correspond to their type species with 99-100% bootstrap support within the in-groups. However, bootstrap support for the test-isolate of A. alternata was less than 50%.

2.4.3 Seedling bioassay

The potentially pathogenic isolates of fungi resulted in a range of disease symptoms such as, pre- emergent damping off, post-emergent seedling mortality, hypocotyl and cotyledon rot, necrotic lesion on cotyledon and hypocotyl and severe stunting in the seedling bioassay whereas the negative control seedlings were almost free from any disease symptoms (Fig. 2.5). Based on the seedling bioassay, a sub-set of 39 isolates across the range of species were prioritised for further testing under glasshouse conditions.

A B C D

E

F G

Fig. 2.5 Different categories of seedlings in the bioassay: A. pre-emergent damping off; B. post- emergent damping off; C-D. diseased (soft rot, tip necrosis) seedlings; E. seedling with necrotic hypocotyl lesion; F. severe stunting and G. a negative control seedling. 76

In the seedling bioassay, majority (80%) of the isolates of Cophinforma sp. were found to be highly to very highly pathogenic (Fig. 2.6). The reminder of the Cophinforma isolates were either weak or moderately pathogenic. A similar trend was observed with P. violaceum. There was some variation among the isolates of M. alpina in terms of their pathogenicity to the seedlings. Half of the isolates of this species were scored as highly pathogenic and the rest were moderately pathogenic. All the isolates of Alternaria sp. were moderately pathogenic to seedlings. Among the isolates of Aureobasidium pullulans, nearly 20% were moderately pathogenic and the rest were weak or non- pathogenic to seedlings. All the isolates of A. pistaciarum, A. pullulans, Paecilomyces sp., P. citrigena, P. ootheca and Z. acaciae were found to be weak or non-pathogenic to seedlings (Fig. 2.5).

Very Highly Pathogenic Highly Pathogenic

Moderately Pathogenic Weak or Non-pathogenic

100% 80% 60%

40% Isolates (%) Isolates 20%

0%

Fig. 2.6 Percentage of isolates of individual fungal species showing different pathogenicity reponse to prickly acacia seedlings under laboratory conditions (any species with less than 3 isolates is not presented).

Other species such as A. alternata, C. uredinicola, C. lunata, C. pseudorobusta, C. austromontana, P. angularis, R. rufulum and U. muehlenbergii had less than three isolates which were mostly moderately pathogenic to non-pathogenic (data not presented in Fig. 2.5). On the other hand, N. parvum (2 isolates in total) and F. acuminatum (1 isolate in total) caused significant seedling mortality and recorded as very highly pathogenic whereas those of P. microspora (1 isolate in total), T. graminis (2 isolates in total) and E. rostratum (1 isolate in total) resulted in significant diseased seedlings and grouped as highly pathogenic (data not presented in Fig. 2.5). The isolate of L.

77 pseudotheobromae (strain NT039) used as positive control consistently caused high level of seedling mortality (not presented in Fig. 2.5).

2.4.4 Glasshouse trials

2.4.4.1 Trial -1

2.4.4.1.1 Trial outline

A set of six isolates of Cophinforma sp. found to be highly pathogenic in the seedling bioassay (Table 2.2) were tested under glasshouse conditions. Of these, five isolates were extracted from dieback-affected stem samples of prickly acacia representing different dieback sites and another isolate was sourced from dieback-affected mimosa. L. pseudotheobromae (strain NT039) was included as positive control. Fresh (uninoculated) autoclaved millet grains were used as negative control.

Table 2.2 Fungal isolates tested in the Glasshouse trial 1

Treatment Identity Isolate Source Host Pathogenicity in seedling code assay

A Negative control - - - B Cophinforma sp. AN17 Prickly acacia Highly pathogenic

C Cophinforma sp. AN32 Prickly acacia Highly pathogenic D Cophinforma sp. AN49 Prickly acacia Highly pathogenic E Cophinforma sp. AN71 Prickly acacia Highly pathogenic F Cophinforma sp. AN114 Prickly acacia Highly pathogenic G Cophinforma sp. MP290 Mimosa Highly pathogenic H L. pseudotheobromae NT039 Parkinsonia Very highly pathogenic

2.4.4.1.2 Stem lesion development

All the test-isolates of Cophinforma spp. and that of L. pseudotheobromae caused extensive stem lesions whereas such lesions in negative control plants were mostly absent or extremely restricted in length. Lesions were black to brownish in colour, necrotic and spreading in both directions from the point of inoculation and were associated with streaming of sap (Fig. 2.7).

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At 8 weeks after inoculation, L. pseudotheobromae had caused significantly longer external (P=0.008) and internal (P=0.00) stem lesions compared to the isolates of Cophinforma sourced from prickly acacia and mimosa dieback (Fig. 2.8). However, there was no significant difference among the isolates of Cophinforma sourced from different sites/hosts in terms of external and internal lesion length.

A

B

C

Fig. 2.7 External stem lesions observed with A. negative control; B. Cophinforma sp. and C. L. pseudotheobromae at 8 weeks under glasshouse conditions.

A

B B B B B b b b b b C

Fig. 2.8 Length of external and internal stem lesions recorded with the isolates of Cophinforma spp. and L. pseudotheobromae under glasshouse conditions at 8 weeks. Columns with different letters on the error bars indicate significant difference among the treatments by Tukey’s test with 95% confidence in GLM ANOVA (n=5). On the X axis, each capital letter represents individual treatment where A = negative control, B = Cophinforma sp. (AN17), C = Cophinforma sp. (AN32), D = Cophinforma sp. (AN49), E = Cophinforma sp. (AN71), F = Cophinforma sp. (AN114), G = Cophinforma sp. (MP290) and H = L. pseudotheobromae (NT039).

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2.4.4.1.3 Leaf production, leaf mortality, stem and branch dry-weight and plant height

At eight weeks, total leaf production was found to be unaffected by the test-isolates (Fig. 2.9 A). However, both Cophinforma spp. (all 6 isolates) and L. pseudotheobromae caused significant (P=0.000) leaf mortality compared to the negative control treatment (Fig. 2.9 B). Leaf mortality was highest with L. pseudotheobromae followed by the isolates of Cophinforma. However, the isolates of Cophinforma sourced from different hosts/sites had an identical effect on leaf mortality. The resulting effects of leaf mortality on stem and branch dry-weight and plant height was insignificant compared to the negative control treatment (data not presented).

(A) a a a a a a a a

(B)

Fig. 2.9 Effect of Cophinforma spp. and L. pseudotheobromae on (A) total leaf production and (B) leaf mortality recorded under glasshouse conditions at 8 weeks. Columns with different letters on the error bars indicate significant difference by Tukey’s test with 95% confidence in GLM ANOVA (n=5). On the X axis, each capital letter represents individual treatment where A = negative control, B = Cophinforma sp. (AN17), C = Cophinforma sp. (AN32), D = Cophinforma sp. (AN49), E = Cophinforma sp. (AN71), F = Cophinforma sp. (AN114), G = Cophinforma sp. (MP290) and H = L. pseudotheobromae (NT039).

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2.4.4.1.4 Confirmation of infection

Test-isolates of Cophinforma spp. and L. pseudotheobromae were successfully retrieved with 100% recovery rate from symptomatic stem tissues of the inoculated plants. However, the fungi were absent in the asymptomatic tissues of the inoculated plants. Negative control plants were free from similar fungi.

2.4.4.2 Trial -2

2.4.4.2.1 Trial outline

Another set of 12 isolates of Cophinforma were tested under glasshouse conditions. These isolates were initially (in 2011) identified as Botryosphaeria spp. (most similar to Botryosphaeria mamane D.E. Gardener) following BLAST searches, therefore, tested in a separate glasshouse trial. However, in 2013 all of these 12 isolates were identified as Cophinforma spp. following repeated BLAST searches. These isolates were extracted from dieback-affected stem samples of prickly acacia collected from different dieback sites and found to be highly to very highly pathogenic in seedling bioassays (Table 2.3). An isolate of L. pseudotheobromae sourced from parkinsonia dieback and found to be extremely aggressive against prickly acacia seedlings was included as positive control. Fresh (uninoculated) autoclaved millet grains were used as negative control.

Table 2.3 Fungal isolates tested in the Glasshouse trial 2

Treatment Identity Isolate Source Host Pathogenicity in seedling code assay

A Negative control - - - B Cophinforma sp. AN42 Prickly acacia Very highly pathogenic C Cophinforma sp. AN110 Prickly acacia Highly pathogenic D Cophinforma sp. AN44 Prickly acacia Very highly pathogenic E Cophinforma sp. AN16 Prickly acacia Highly pathogenic F Cophinforma sp. AN109 Prickly acacia Very highly pathogenic G Cophinforma sp. AN19 Prickly acacia Very highly pathogenic H Cophinforma sp. AN26 Prickly acacia Highly pathogenic I Cophinforma sp. AN93 Prickly acacia Highly pathogenic J Cophinforma sp. AN75 Prickly acacia Highly pathogenic K Cophinforma sp. AN68 Prickly acacia Highly pathogenic L Cophinforma sp. AN65 Prickly acacia Highly pathogenic M Cophinforma sp. AN76 Prickly acacia Highly pathogenic N L. pseudotheobromae NT039 Parkinsonia Very highly pathogenic

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2.4.4.2.2 Stem lesion development

Cophinforma sp. and L. pseudotheobromae caused significant (P=0.00) external and internal stem lesions similar to those observed in Trial-1 (Fig. 2.10 A). Such lesions in negative control plants were either absent or very short in length. Again, at eight weeks after inoculation, L. pseudotheobromae had caused significantly longer stem lesions compared to the test-isolates of Cophinforma sp. (Fig. 2.11). The test-isolates of Cophinforma were mostly identical in terms of external lesion length but formed three slightly overlapping clusters for internal lesions (Fig. 2.11). However, there was no apparent relation of such clustering with the genetic variations among the Cophinforma isolates (Section 2.4.2). With a few exceptions, the fungal isolates found to be “very highly pathogenic” in seedling bioassay also produced longer internal stem lesions in the juvenile plants compared to those recorded as “highly pathogenic”.

A A A

B

Fig. 2.10 A. internal stem lesion; B. leaf mortality observed with Cophinforma sp.

2.4.4.2.3 Leaf production, leaf mortality, stem and branch dry-weight and plant height

At eight weeks, total leaf production was found to be unaffected by the test-isolate (Fig. 2.12 A). With a few exceptions, the test-isolates of Cophinforma and that of L. pseudotheobromae caused significant (P=0.00) leaf mortality. As observed in Trial-1, L. pseudotheobromae caused significantly higher levels of leaf mortality compared to the isolates of Cophinforma (Fig. 2.12 B). However, the resulting effect of leaf mortality caused by Cophinforma sp. and L. pseudotheobromae on stem and branch dry-weight and plant height was insignificant (data not presented). 82

A Cluster-1 Cluster-2

B a BC Cluster-3 BC C C C C

D D D D D b b b bc bc bc bc bc bc bc bc bc c E

Fig. 2.11 Length of external and internal stem lesions recorded with the isolates of Cophinforma sp. and L. pseudotheobromae under glasshouse conditions at 8 weeks. Columns with different letters on the error bars indicate significant difference by Tukey’s test with 95% confidence in GLM ANOVA (n=5). On the X axis, each capital letter represents individual treatment where A = negative control, B = Cophinforma sp. (AN42), C = Cophinforma sp. (AN110), D = Cophinforma sp. (AN44), E = Cophinforma sp. (AN16), F = Cophinforma sp. (AN109), G = Cophinforma sp. (AN19), H = Cophinforma sp. (AN26), I = Cophinforma sp. (AN93), J = Cophinforma sp. (AN75), K = Cophinforma sp. (AN68), L = Cophinforma sp. (AN65), M = Cophinforma sp. (AN76) and N = L. pseudotheobromae (NT039).

2.4.4.2.4 Confirmation of infection

The test-isolates of Cophinforma and that of L. pseudotheobromae were successfully retrieved with 100% recovery rate from symptomatic stem tissues of the inoculated plants. However, the fungi were absent in the asymptomatic tissues of the inoculated plants. Negative control plants were free from similar fungi.

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(A)

a a a a a a a a a a a a a a

(B)

a

b b b b b b b b b b b b c

Fig. 2.12 Effect Cophinforma sp. (12 isolates) sourced from dieback-affected prickly acacia stem samples and L. pseudotheobromae (1 isolate) on (A) total leaf production and (B) leaf mortality recorded under glasshouse conditions at 8 weeks. Columns with different letters on the error bars indicate significant difference by Tukey’s test with 95% confidence in GLM ANOVA (n=5). On the X axis, each capital letter represents individual treatment where A = negative control, B = Cophinforma sp. (AN42), C = Cophinforma sp. (AN110), D = Cophinforma sp. (AN44), E = Cophinforma sp. (AN16), F = Cophinforma sp. (AN109), G = Cophinforma sp. (AN19), H = Cophinforma sp. (AN26), I = Cophinforma sp. (AN93), J = Cophinforma sp. (AN75), K = Cophinforma sp. (AN68), L = Cophinforma sp. (AN65), M = Cophinforma sp. (AN76) and N = L. pseudotheobromae (NT039).

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2.4.4.3 Trial -3

2.4.4.3.1 Trial outline

The following species- Pseudofusicoccum violaceum, Phaeobotryosphaeria citrigena, Aureobasidium pullulans, Paecilomyces sp., Curvularia lunata, Curvularia pseudorobusta, Cytospora austromontana, Asteromella pistaciarum, Cladosporium uredinicola, Pleurostoma ootheca, Alternaria alternata, Pyrenochaetopsis microspora, Exserohilum rostratum, Rhytidhysteron rufulum and Umbilicaria muehlenbergii were tested under glasshouse conditions. These were extracted from dieback-affected stem samples of prickly acacia collected from different sites. Species other than P. violaceum, P. microspora and E. rostratum were found to be mostly weak or non-pathogenic in seedling bioassays (Table 2.4). L. pseudotheobromae (strain NT039) was included as positive control. Fresh (uninoculated) autoclaved millet grains were used as negative control.

Table 2.4 Fungal isolates tested in the Glasshouse trial 3

Treatment Identity Isolate Source Host Pathogenicity in seedling code assay

A Negative control - - - B P. violaceum AN48 Prickly acacia Highly pathogenic C P. citrigena AN146 Prickly acacia Weak or non- pathogenic D A. pullulans AN03 Prickly acacia Moderately pathogenic E Paecilomyces sp. AN123 Prickly acacia Weak or non- pathogenic F C. pseudorobusta AN124 Prickly acacia Weak or non- pathogenic G C. lunata AN118 Prickly acacia Weak or non- pathogenic H A. pistaciarum AN14 Prickly acacia Weak or non- pathogenic I C. austromontana AN145 Prickly acacia Moderately pathogenic J C. uredinicola AN63 Prickly acacia Weak or non- pathogenic K P. ootheca AN136 Prickly acacia Weak or non- pathogenic L A. alternata AN122 Prickly acacia Weak or non- pathogenic M P. microspora AN144 Prickly acacia Highly pathogenic N E. rostratum AN120 Prickly acacia Highly pathogenic O R. rufulum AN141 Prickly acacia Weak or non- pathogenic P U. muehlenbergii AN142 Prickly acacia Weak or non- pathogenic Q L. pseudotheobromae NT039 Parkinsonia Very highly pathogenic

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2.4.4.3.2 Stem lesion development

At eight weeks after inoculation in November 2011, L. pseudotheobromae had caused significant (P=0.00) stem lesions similar to those observed in previous trials. However, none of the other test- isolates sourced from prickly acacia dieback caused any significant external or internal stem lesions compared to the negative control treatment (Fig. 2.13). Although the test-isolates of P. violaceum, P. microspora and E. rostratum were found to be highly pathogenic against prickly acacia seedlings under laboratory conditions, they did not result in any significant disease symptoms in the juvenile plants under glasshouse conditions. The trial was repeated in April-May 2012 and similar results were found (data not presented).

25 External stem lesion Internal stem lesion a

20

15 b

10 Length of stem lesion (cm) lesion stem of Length

5

0 A B C D E F G H I J K L M N O P Q

Fig. 2.13 Length of external and internal stem lesions recorded with different fungi sourced from dieback-affected prickly acacia stem samples under glasshouse conditions at 8 weeks. Columns with different letters on the error bars indicate significant difference by Tukey’s test with 95% confidence in GLM ANOVA (n=5). On the X axis, each capital letter represents individual treatment where A = negative control, B = P. violaceum, C = P. citrigena, D = A. pullulans, E = Paecilomyces sp., F = C. pseudorobusta, G = Cu. lunata, H = As. pistaciarum, I = Cy. austromontana, J = C. uredinicola, K = P. ootheca, L = A. alternata, M = P. microspora, N = E. rostratum, O = R. rufulum, P = U. muehlenbergii and Q = L. pseudotheobromae.

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2.4.4.3.3 Leaf production, leaf mortality, stem and branch dry-weight and plant height

Effect of the test-isolates on leaf production, leaf mortality, stem and branch dry-weight and plant height were not critically assessed in this trial.

2.4.4.3.4 Confirmation of infection

All the test-isolates except that of C. austromontana were successfully retrieved with 100% recovery rate from the leading edge of short internal lesions of the inoculated plants. Moreover, Cladosporium sp. and Alternaria sp. were also found in the negative control plants.

2.4.4.4 Trial -4

2.4.4.4.1 Trial outline

In this glasshouse trial, we tested one isolate from each of the following species- Fusarium acuminatum, Neofusicoccum parvum, Cophinforma sp., Mortierella alpina and Tiarosporella graminis extracted from field-grown healthy plants (Table 2.5). L. pseudotheobromae (strain NT039) was included as positive control. Fresh (uninoculated) autoclaved millet grains were used as negative control.

Table 2.5 Fungal isolates tested in the Glasshouse trial 4

Treatment Identity Isolate Source Host Pathogenicity in seedling code assay A Negative control - - - B F. acuminatum ANE16 Prickly acacia (root) Very highly pathogenic C N. parvum ANE30 Prickly acacia (root) Very highly pathogenic D Cophinforma sp. ANE27 Prickly acacia (stem) Highly pathogenic E M. alpina ANE07 Prickly acacia (root) Highly pathogenic F T. graminis ANE24 Prickly acacia (root) Highly pathogenic G L. pseudotheobromae NT039 Parkinsonia (stem) Very highly pathogenic

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2.4.4.4.2 Stem lesion development

We observed extensive stem lesions with the test-isolates sourced from healthy prickly acacia stem and root samples similar to those observed with the pathogenic fungi extracted from dieback- affected stem samples. At eight weeks after inoculation, longest external and internal stem lesions were recorded with L. pseudotheobromae (strain NT039) which were statistically identical to those recorded with N. parvum (sourced from apparently the healthy root samples at Rockhampton) (Fig. 2.14 A). F. acuminatum extracted from the apparently healthy root samples at Rockhampton also caused significant (P=0.00) external and internal stem lesions (Fig. 2.14 A).

(A)

A

A B B C a C ab C D D D b c D c c c c

(B)

a

a ab b a b c b b c c c c

Fig. 2.14 Effect of different fungi sourced from apparently healthy prickly acacia stem and root samples on (A) length of stem lesions and (B) amount of dead leaf under glasshouse conditions at 8 weeks. Columns with different letters on error bars indicate significant difference by Tukey’s test with 95% confidence in GLM ANOVA (n=5). On the X axis, each capital letter represents individual treatment where A = negative control, B = F. acuminatum, C = N. parvum, D = Cophinforma sp., E = M. alpina, F = T. graminis and G = L. pseudotheobromae.

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2.4.4.4.3 Leaf mortality, stem and branch dry-weight and plant height

The highest amount of dead leaves was recorded with L. pseudotheobromae (strain NT039). In addition, N. parvum, Cophinforma sp. and T. graminis isolated from the apparently healthy root and stem samples collected from Rockhampton and Richmond also caused significant (P=0.001) leaf mortality (Fig. 2.14 B). However, the resulting effects of leaf mortality caused by test-isolates on stem and branch dry-weight and plant height was insignificant compared to the control treatment (data not presented).

2.4.4.4.4 Confirmation of infection

All the test-isolates were successfully retrieved with 100% recovery rate from symptomatic stem tissues of the inoculated plants. However, the fungi were absent in the asymptomatic tissues of the inoculated plants. Control plants were free from similar fungi.

2.5 Discussion

This study represents the first investigation of the potential association of fungi with occasional dieback observed on prickly acacia across north-western Queensland. On the basis of molecular characters, a number of Botryosphaeriaceae and other fungi were found to be associated with this phenomenon. Based on pathogenicity studies, species of Cophinforma were identified as the most likely aetiological agent. Cophinforma sp. along with other fungi was also isolated from the healthy prickly acacia plants growing around the areas where dieback symptoms were previously observed on this weed species. Pathogenic fungi such as F. acuminatum and N. parvum were also isolated from the healthy plants occupying the areas where natural dieback was not previously reported.

Recovery of Cophinforma sp. and other Botryosphaeriaceae fungi from the asymptomatic plants clearly indicated their endophytic relationship to prickly acacia. Cophinforma was previously isolated from asymptomatic plant tissues (Phillips et al. 2013). In addition, a number of other Botryosphaeriaceae fungi such as, many species of Botryosphaeria, Diplodia, Dothidotthia, Guignardia, Lasiodiplodia, Neofusicoccum and Pseudofusicoccum have been reported as endophytes causing non-symptomatic latent infections in a range of perennial woody plant species (Johnson et al. 1992; Petrini & Fisher 1988; Crous et al. 2006; Slippers & Wingfield 2007). Several species within those genera have been previously identified as the potential cause of dieback and canker in a number of woody hosts (Damm et al. 2007; Marques et al. 2012; Boyzo-Marin et al. 2014; Jiao et al. 2014; Billones-Baaijens et al. 2015).

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Apart from Botryosphaeriaceae, a number of other fungi associated with either dieback-affected or healthy prickly acacia such as different species of Alternaria, Curvularia, Paecilomyces and the other species such as A. pistaciarum, A. pullulans, C. uredinicola, spp., C. austromontana, E. rostratum, R. rufulum, T. graminis, U. muehlenbergii and Z. acaciae have also been previously identified as endophytes of other plant hosts (Bremer & Petrak 1947; Sutton & Marasas 1976; Boehm et al. 2009; Martini et al. 2009; Rodriguez et al. 2009; Rosa et al. 2009; Schoch et al. 2010; Abreu et al. 2012; Ek-Ramos et al. 2013; Crous et al. 2014). The exact ecological role of these non- pathogenic endophytes is not clearly understood, however, they are generally considered as “symbionts” which assist the plant communities to thrive against abiotic and biotic stressors (Rodriguez et al. 2009; Amin et al. 2014). Many of these endophytes such as different species of Alternaria, Aureobasidium, Cladosporium and Paecilomyces are particularly known to be antagonists against fungal and bacterial plant pathogens, insects and nematodes (Ek-Ramos et al. 2013; Ladjal et al. 2013; Han et al. 2014; Sharma et al. 2014; Wachowska & Gowacka 2014). Among the other fungi isolated from the healthy prickly acacia plants, F. acuminatum was previously reported to cause disease symptoms such as leaf spot, seedling damping-off and crown rot in other hosts (Lazreg et al. 2013; Murdoch 2013; Parkunan & Ji 2013) and M. alpina is known to be a soil-borne saprophyte (Yadav et al. 2014).

Cophinforma is a newly erected genus within the family Botryosphaeriaceae which is morphologically very similar to, but phylogenetically distinct from the genus Botryosphaeria (Liu et al. 2012). It was initially introduced as a monotypic genus for C. eucalypti found in Thailand on dead branches of Eucalyptus sp. (Liu et al. 2012). However, Phillips et al. (2013) included two other species under Cophinforma, C. atrovirens (=Fusicoccum atrovirens Mehl & Slippers) and C. mamane (=Botryosphaeria mamane D.E. Gardner). Of these, C. atrovirens was reported in South Africa on asymptomatic branches of Pterocarpus angolensis DC (Phillips et al. 2013) and C. mamane was associated with Witch’s brooms symptoms of Sophora chrysophylla (Salisb.) Seem in Hawaii, USA (Gardner 1997). None of these species of Cophinforma was previously reported in Australia. Therefore, this is the first report of the occurrence of Cophinforma in Australia. However, Sacdalan (2012) isolated Cophinforma spp. from dieback-affected stem samples of mimosa (M. pigra) collected from northern Australia (unpublished findings).

The ITS sequences from the isolates of Cophinforma sourced from dieback-affected and healthy prickly acacia and dieback-affected mimosa were found to have found 99% similarity with C. eucalypti. However, following phylogenetic analysis, the test-isolates of Cophinforma did not typically align with or correspond to the type sequences of any of the three known species of

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Cophinforma obtained from the GenBank database. On the other hand, the ITS sequences from the other fungi (including the other Botryosphaeriaceae) isolated from prickly acacia typically corresponded with their type isolates following phylogenetic analysis. Such non-alignment of the Cophinforma isolates with any of its previously described species could be because of low quality sequencing outcomes or the existence of previously undescribed species of Cophinforma. All the sequences used in the analysis were of high quality and free from ambiguity. Therefore, there is high level of possibility of the presence of new species of Cophinforma in Australia. However, it is generally accepted that Botryosphaeriaceae fungi are taxonomically complicated and their precise identification up to the species level often requires sequencing of the other gene regions apart from the ITS (Crous et al. 2006; Liu et al. 2012; Phillips et al. 2013). Therefore, the region of Transcription Elongation Factor (TEF) 1-α gene of two isolates of Cophinforma spp. were amplified (data not presented in this study). Interestingly, those sequences also did not correspond to that previously amplified by Mehl et al. (2011) in C. atrovirens (=Fusicoccum atrovirens). Therefore, a stringent comparison was not possible. Such findings are further indicative of the presence of previously undescribed species of Cophinforma in Australia. However, further phylogenetic investigations such as sequencing of the other gene regions (for example: β-Tubulin gene) are required to confirm such a claim.

In the seedling bioassays, the majority of the test-isolates of different Botryosphaeriaceae fungi were found to cause a range of disease symptoms. Such findings suggest the potential of these native fungi to be utilized for reduction of the prickly acacia seed-bank. Previous studies by Toh et al. (2008) and Wong (2008) also demonstrated that the Botryosphaeriaceae fungi could cause significant damage to the seeds and seedlings of parkinsonia, another invasive woody weed in Australia. However, there was some extent of intra-specific variation among the test-isolates used in this study. Such variation could be due to a number of factors such as genetic variation among the isolates, difference in the source and size of seeds used in the study and statistical tools used to group the isolates. However, this study did not seek the exact cause of the variations. Similar results were also previously reported by Toh et al. (2008) while screening a large number of fungal isolates using parkinsonia seedlings. The seedling inoculation study was conducted for two weeks using a single temperature regimen (25oC). It is possible that some of these test-isolates may have caused higher levels of damage if the trial had been run for longer period or if the trial had been conducted at temperatures optimal for the individual test-isolates (Toh 2009; Hudec & Muchova 2010; Urbez- Torres et al. 2010). With only a few exceptions, the fungal isolates ranked as very highly or highly pathogenic to seedlings under laboratory conditions also caused significant disease symptoms in the juvenile plants under glasshouse conditions although there was some difference between laboratory

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(constant 25oC) and glasshouse (daily mean 30 ± 7.5oC) temperatures. There are indications that the stem inoculation technique used in this study where the stems were wounded, the fungi placed inside and the wound sealed could assist the fungi to cope with the variations in external factors such as temperature and relative humidity (Waller 2002).

In the glasshouse trials, necrotic stem lesions and gummosis appeared as the main disease symptoms following inoculation with the pathogenic isolates across the range of species. Measurement of stem lesions indicated that Cophinforma sp. (isolated from both healthy and dieback-affected samples of prickly acacia and die-back affected sample of mimosa collected from different locations) and L. pseudotheobromae (strain NT039) consistently caused significant stem lesions. Among the other fungi, N. parvum and F. acuminatum isolated from healthy prickly acacia plants also caused similar lesions. Such lesions were also observed on other hosts following deliberate inoculation with the Botryosphaeriaceae and other fungi (Lynch et al. 2013; Amponsah et al. 2014; Rodríguez-Gálvez et al. 2015). L. pseudotheobromae caused significantly longer stem lesions compared to Cophinforma sp. and most of the other species tested in the glasshouse trials. Such findings are indicative of the more aggressive nature of L. pseudotheobromae compared to the other species of fungi. Similar findings were also previously reported by Mehl et al. (2011) in South Africa where L. pseudotheobromae was found to be highly aggressive compared to the other Botryosphaeriaceae fungi associated with the dieback of Pterocarpus angolensis (Kiaat). However, L. pseudotheobromae has yet to be isolated from naturally dieback-affected prickly acacia in Australia.

In general, the test-isolates sourced from dieback-affected or healthy prickly acacia and other hosts causing significant stem lesions also caused significant leaf mortality. Previous plant-fungus interaction studies also found leaf health being significantly affected by cankerous stem lesions (Davidson et al. 2003; Rodrigues et al. 2014). However, in this study, the resulting effects of leaf mortality on plant growth parameters such as total leaf production, plant height and stem and branch dry-weight was not significant. Such results are indicative of compensatory plant growth which was clearly evident as the plants were increasing in height and defoliation and leaf mortality were either followed by or accompanied with active re-shooting from the plant base. Similar growth compensation following defoliation or leaf mortality was also previously observed in other woody species (Jaremo & Palmqvist 2001; Salomon Ballina-Gomez et al. 2010). Re-shooting was also observed in the natural prickly acacia dieback event (Galea 2011).

After harvesting the glasshouse trials, all the test-isolates but that of C. austromontana were successfully recovered from the symptomatic stem tissues thus confirming infection. Different fungi 92

(particularly those under the Botryosphaeriaceae family) were previously isolated from naturally infected asymptomatic host tissues (Slippers & Wingfield 2007; Trakunyingcharoen et al. 2013). However, asymptomatic colonization of healthy tissues by the Botryosphaeriaceae fungi following artificial stem inoculation had rarely been previously reported. Such a mode of infection was also not found in our study since the test-fungi could not be isolated from apparently healthy tissues outside the necrotic area of stem lesions.

Endophytic Botryosphaeriaceae were the major group of fungi found to be associated with prickly acacia dieback in north-western Queensland. This group of fungi are generally considered as slow- acting endophytes (Slippers & Wingfield 2007; Mehl et al. 2013). A number of previous studies also found the potential association of several species of Botryosphaeriaceae fungi with dieback and canker observed on a single host species (Pavlic et al. 2007; Urbez-Torres & Gubler 2009; Lynch et al. 2013). Therefore, it would be interesting to investigate whether typical dieback symptoms such as widespread plant mortality or significant decline in growth and vigour could be replicated on healthy prickly acacia plants following deliberate inoculation with combinations of different species of fungi in longer-term field trials.

The Botryosphaeriaceae fungi are generally considered as opportunistic latent pathogens because the disease symptoms they cause are often associated with other biotic and abiotic stressors involved in host weakening (Slippers & Wingfield 2007; Niekerk et al. 2011; Amponsah et al. 2014). They are also known to cause widespread plant mortality (not observed in this study) in presence of other stressors (Desprez-Loustau et al. 2006; Slippers & Wingfield 2007; Mehl et al. 2013). There are indications on the potential association of a range of biotic and abiotic stressors with prickly acacia dieback (March 2009; Galea 2011; Queensland Department of Agriculture Fisheries and Forestry 2014). Therefore, interactions between abiotic stressors and endophytic fungi in terms of dieback severity could be another potential area of exploration.

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Chapter 3: Glasshouse and field infection studies in prickly acacia using fungi isolated from dieback-affected trees

3.1 Abstract

Prickly acacia is an invasive woody weed in coastal and semi-arid rangelands of Australia. It is a serious threat to the Australian grazing industry. A prominent dieback event was observed on this species in 2010 among the plants growing around Richmond and Julia Creek in north-western Queensland. A Botryosphaeriaceae fungus, Cophinforma sp. was consistently isolated from the symptomatic stem tissues. In preliminary studies, Cophinforma sp. was found to be pathogenic to juvenile plants causing significant stem lesions, leaf mortality and gummosis under glasshouse conditions. An isolate of L. pseudotheobromae sourced from dieback-affected parkinsonia was also tested in preliminary trials and shown to be more pathogenic to prickly acacia causing longer stem lesions and higher degree of leaf mortality compared to the fungi originally isolated from dieback- affected prickly acacia. However, none of the species could cause typical dieback symptoms such as plant mortality or severe decline in plant vigour observed in the natural dieback event in 2010. In this study, we investigated whether typical dieback symptoms could be replicated with combinations of Cophinforma sp. and L. pseudotheobromae. Therefore, two isolates of Cophinforma sp. and one isolate of L. pseudotheobromae were utilized singly and in combinations under glasshouse and field conditions. In the glasshouse trial, inoculations were conducted by placing colonized millet grains into holes drilled into the stems of six month old juvenile plants. Significant stem lesions were observed with Cophinforma sp., L. pseudotheobromae and their combinations. However, there was no significant increase in lesion length with any Cophinforma- Lasiodiplodia combinations compared to the individual effect of L. pseudotheobromae. An almost similar pattern was observed for leaf mortality. However, the resulting effects of stem lesions and leaf mortality on overall plant growth as measured by total leaf production (dry-weight) and stem and branch dry-weight was insignificant. Koch’s postulates were fulfilled with both Cophinforma sp. and L. pseudotheobromae once inoculated in separate plants. However, Cophinforma sp. could not be reisolated when combined with L. pseudotheobromae in the same plant. None of the test- isolates could be retrieved from healthy tissues beyond the necrotic area of stem lesions whether inoculated individually or in combination. The glasshouse trial was followed by two field trials (experimental repeats) established at Rockhampton, central Queensland and Richmond, north- western Queensland using naturally occurring plants. The test-fungi were cultured on millet grains and delivered using a gelatine capsule delivery system into holes drilled into the stem. In the field trial conducted at Rockhampton, we observed external stem lesions with streaming of sap with all 103 treatments including control (blank millet capsule). However, length of stem lesion was significantly greater than the control treatment only in the presence of L. pseudotheobromae. Similar to the glasshouse trial, there was no significant increase in lesion length with any Cophinforma-Lasiodiplodia combination compared to the individual effect of L. pseudotheobromae. In another field trial conducted at Richmond, external stem lesions were not visible being obscured by the thick and darkly pigmented bark. However, internal lesions were observed and recorded in stems harvested from treated plants at both trial sites. At Rockhampton, internal lesions were less prominent and mostly confined to the pith region as observed in stem cross-sections through the point of inoculation. In contrast, internal lesions at Richmond were relatively larger in size, more prominent and darker in colour. No significant aerial dieback symptoms such as branch death and thinning of leaf cover leading to decline in plant vigour was observed during the course of the trial at Rockhampton. In contrast, at Richmond, we observed significant branch mortality and thinning of leaf cover resulting in prominent vigour decline at approximately two years after inoculation. Symptoms were very similar to those previously observed in natural prickly acacia dieback event at other sites around Richmond. However, similar degree of vigour decline was also observed with the control treatment (blank millet capsule) and on the untreated plants neighbouring the trial. Both Cophinforma sp. and L. pseudotheobromae were successfully isolated from the internally symptomatic stem tissues of inoculated plants, although the recovery success varied between two species. L. pseudotheobromae sourced from parkinsonia appeared as more aggressive to prickly acacia with a higher degree of ecological fitness as a potential bioherbicide candidate compared to Cophinforma sp.

Keywords: Prickly acacia, Botryosphaeriaceae fungi, weed, bio-control.

3.2 Introduction

Prickly acacia (Vachellia nilotica subsp. indica (Benth.) Kyal. & Boatwr), one of the most harmful weeds invading the Australian rangelands was found with dieback symptoms in 2010 at Richmond and Julia Creek in north-western Queensland (Galea 2011). Affected trees were found with symptoms ranging from ashy internal staining, defoliation, blackening of shoot tips, partial crown death through to widespread death of plant populations. A range of fungi were found to be associated with the phenomenon and those from the Botryosphaeriaceae family were consistently isolated from dieback affected materials (Haque et al. 2012). Botryosphaeriaceae were previously reported to be associated with a range of disease symptoms including dieback and canker of different woody hosts (Alemu et al. 2014; Linaldeddu et al. 2014; Trakunyingcharoen et al. 2014).

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They were also found to be associated with dieback of other invasive woody weeds in Australia such as mimosa (Mimosa pigra L.) and parkinsonia (Parkinsonia aculeata L.) (Wilson & Pitkethley 1992; Diplock et al. 2006; Sacdalan et al. 2012). At present, dieback is the major limiting factor to the spread of parkinsonia (van Klinken et al. 2009). There are indications that fungi associated with dieback can be potentially utilized as bio-control agents and incorporated into the management strategy of such invasive woody weeds (Wood & Ginns 2006; Galea & Goulter 2013). Therefore, it was important to investigate whether dieback symptoms could be replicated on healthy prickly acacia plants under field conditions using the fungi sourced from previous natural dieback event.

The Botryosphaeriaceae are a cosmopolitan group of fungi having an extremely wide host range (Slippers & Wingfield 2007; Mehl et al. 2011). Initially they were known to infect healthy hosts mainly through wounds or natural openings resulting in disease symptoms (Smith et al. 1996). However, between late 1980’s and early 1990’s, a number of Botryosphaeriaceae species were identified as endophytes causing non-symptomatic latent infections (Petrini & Fisher 1988; Johnson et al. 1992). More recently, the Botryosphaeriaceae fungi are, however, generally considered as opportunistic latent pathogens because the diseases they cause are often associated with other biotic and abiotic stressors (Old et al. 1990; Slippers & Wingfield 2007; Niekerk et al. 2011; Amponsah et al. 2014). Even so, a number of species under this family are well-recognized as plant pathogens having potential to cause significant disease symptoms in the absence of other stressors (Slippers & Wingfield 2007; Lynch et al. 2013; Chen et al. 2014).

In the initial studies (Chapter 2) it was found that the Botryosphaeriaceae fungi such as Cophinforma sp. and Lasiodiplodia pseudotheobromae Phillips, Alves & Crous previously associated with dieback of prickly acacia and parkinsonia, respectively consistently caused significant disease symptoms such as stem lesions, gummosis and leaf mortality in prickly acacia once the juvenile plants were challenged under glasshouse conditions. However, widespread plant mortality or severe vigour decline typical of the natural dieback event could not be replicated with individual fungus in short-term (eight week) glasshouse trials. In a number of occasions, several species of Botryosphaeriaceae fungi were found to be associated with a dieback and canker observed on a single host species (Pavlic et al. 2007; Urbez-Torres & Gubler 2009; Lynch et al. 2013). Therefore, this study was undertaken to investigate whether the typical dieback symptoms could be replicated on healthy plants with deliberate inoculation of the combinations of different Botryosphaeriaceae fungi in longer-term trials.

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Such integration of different potential bioherbicide fungi has significant implications in the field of weed control. In several occasions, co-existence of different bioherbicide fungi was found to be more effective in controlling different weeds as a result of their synergistic or additive effects compared to the use of a single fungus (Paul & Ayres 1986; Hallett et al. 1990). Infection by one bioherbicide fungus can also render the target weed more susceptible to the infection by another fungus once they are used in combinations (Hasan & Ayres 1990).

In this study, it was also investigated whether the deliberately inoculated Botryosphaeriaceae fungi could cause latent infection in field populations of prickly acacia. If so, then widespread dieback may possibly occur following an event of natural stress (Slippers & Wingfield 2007; Mehl et al. 2011). Trials were initially conducted under glasshouse conditions, and then replicated in two different ecological zones; Rockhampton, central Queensland and Richmond, north-western Queensland representing a wet coastal and water-stressed semi-arid climate, respectively.

3.3 Materials and Methods

3.3.1 Glasshouse and laboratory studies

3.3.1.1 Experimental design, treatments and growing juvenile plants

The effect of two isolates of Cophinforma sp. and one isolate of L. pseudotheobromae individually and in combinations on stem lesion length, total leaf production, leaf mortality and stem/branch dry- weight of prickly acacia was examined under glasshouse conditions. Isolates of C. eucalypti were extracted from dieback-affected stem sample of prickly acacia while L. pseudotheobromae came from a dieback affected stem sample of parkinsonia. The 12-week trial was conducted using a randomized complete block design with five blocks. The following treatments (Table 3.1) were randomly allocated within each block.

Plants were six months old at the start of the trial (average height 131.25 ± 4.95 cm; average stem diameter at 10 cm above the soil level 10.77 ± 0.79 mm), and were randomly allocated to treatments and blocks. Plants were grown from surface sterilized seeds following the techniques outlined in Chapter 2 (Section 2.3.4.1). Seeds were sourced from dieback-free plants growing at Richmond, north-western Queensland.

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3.3.1.2 Treatment preparation and application

Fungal isolates were cultured on millet (Panicum miliaceum L.) grains following the techniques outlined by Toh et al. (2008). Using an alcohol sterilized 3.5 mm drill bit mounted on a cordless drill, a 5-6 mm deep inoculation hole was made on the stem (at a slight downward angle) at 10 cm above the soil level followed by placing two colonized millet grains using sterilized forceps and covering the wound with Parafilm®. While inoculating with combinations of fungi, a separate hole for each was drilled 10 cm apart. Fresh (un-inoculated) autoclaved millet grains were used as a control.

Table 3.1 Treatments in glasshouse trial

Code Treatment Composition A Control (autoclaved fresh millet grain) B Cophinforma sp. (AN71) C Cophinforma sp. (AN110) D L. pseudotheobromae (NT039) E Cophinforma sp. (AN71) + Cophinforma sp. (AN110) F Cophinforma sp. (AN71) + L. pseudotheobromae (NT039) G Cophinforma sp. (AN110) + L. pseudotheobromae (NT039) H Cophinforma sp. (AN71) + Cophinforma sp. (AN110) + L. pseudotheobromae (NT039)

3.3.1.3 Trial assessment

Length of external and internal stem lesions was measured using a ruler. An aluminium foil tray with a drainage hole cut at the center was placed beneath each plant to collect fallen leaves throughout the trial. Dead leaves attached with the plants were hand removed at the end of the trial. After removing the attached dead leaves, the plants with live (green) leaves were cut into small pieces and put in labelled aluminium trays and left in glasshouse for 72 hours to commence desiccation. Both dead (fallen leaves plus attached dead leaves) and green leaves were dried separately in a drying oven at 60-65oC for one week. Total leaf production was calculated by adding the dry-weight of dead leaf and live leaves attached with the plant at the end of the trial. Leaf mortality was expressed as percentage of dead leaf to total leaf production. Dry-weight of stem and branch samples was obtained following oven drying at 60-65oC for 2-3 weeks.

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3.3.1.4 Confirmation of infection

Confirmation of infection was achieved by isolating the test-fungi from the plants inoculated with individual isolates and their combinations. The potential of these isolates to asymptomatically colonize healthy tissue as endophytes was also investigated. Isolations were conducted from the leading edges of internal stem lesions and adjacent healthy tissues following the techniques modified from Diplock et al. (2006). Stem segments collected from three randomly selected plants from each treatment were surface sterilized by soaking in 2% NaOCl for two minutes followed by rinsing in sterile water twice for five minutes and drying on sterile blotting paper within a laminar air-flow cabinet. Partially dried samples were aseptically transferred to ½ PDA plates followed by incubation at 25oC in a darkened incubator for one week with daily observation. After the incubation period, the fungal isolates were purified by subculturing on fresh ½ PDA plates before comparing to original isolates used in the experiment. Isolation was also conducted from the control plants following the same techniques.

3.3.1.5 In-vitro competiveness of the fungi

Inter-specific competition between Cophinforma sp. and L. pseudotheobromae was evaluated under in-vitro conditions following dual culture technique slightly modified from Dhingra and Sinclair (1995) following a completely randomized design with six replicates. A mycelial disc of Cophinforma sp. (5 mm diameter) was paired on a ½ PDA plate (85 mm diameter) against a same sized mycelial disc of L. pseudotheobromae, both sourced from seven day old cultures. Both plugs were placed at equal distances (15 mm) from the periphery of ½ PDA plate. The ½ PDA plates inoculated only with either Cophinforma sp. or L. pseudotheobromae served as control (single culture). Plates were sealed with Parafilm®, transferred into airtight plastic bags and incubated in a darkened incubator. Radial mycelial growth was measured in both dual and single culture (control) plates for 4 times at 24 hrs interval using a transparent ruler. Dual culture plates were carefully observed for the presence of any inhibition zone. The study was initially conducted at 25oC (room temperature) and repeated at 35oC (average maximum temperature recorded during the course of the glasshouse trial).

3.3.1.6 Data analysis

The individual effect of the two isolates of Cophinforma sp. and one isolate of L. pseudotheobromae and their combination effect on stem lesion length, total leaf production, leaf mortality and stem/branch dry weight were tested following GLM ANOVA using the statistical 108 software package Minitab version 16. Treatment means were compared following Tukey’s test with 95% level of confidence. Wherever required, data were transformed using appropriate technique (such as Arcsine transformation) to meet ANOVA requirements. Data on mycelial growth recorded in the in-vitro competiveness study were also analysed following similar techniques.

3.3.2 Field trials

3.3.2.1 Experimental design and treatments

Field trials were established at Rockhampton (23°22′54″S 150°28′30″E), central Queensland and Richmond (20°43′50″S 143°08′32″E), north-western Queensland using naturally occurring plants growing on native pasture. The trial sites were 1108 Km apart from each other. Trials were established between late May and early June, 2012 following a randomized complete block design. There were four blocks, each containing six separate treatment plots. In both trial sites, the individual treatment plots were approximately 40 m2 in size. Each treatment plot at Rockhampton trial comprised of six plants whereas at Richmond each plot comprised of eight plants. Plants in each plot were growing in clusters and comprised an individual treatment. Treatment plots were randomly allocated within a block and each block had a complete set of treatments. Mean stem circumference of the plants at 20 cm above the ground level was 18.48 (± 6.24) cm and 22.47 (± 6.25) cm at Rockhampton and Richmond, respectively. Single cattle ear tags bearing the treatment name were wired to the first plant in each treatment plot along with recording of the GPS waypoint. Experimental plants were also marked on exposed woody stems or branches with white spray paint for the ease of identification.

The trials were established with exactly the same set of treatments tested in glasshouse trial (Table 3.1). However, at the final stage of assessment (approximately two years after trial establishment), we included an additional treatment “untreated control (plants were not drilled and no capsule inserted)” comprising the same number of untreated plants neighbouring the original trial site. At Rockhampton, the untreated plants were assessed 4 months after the final assessment point whereas at Richmond, they were assessed on the final data collection point for the trial.

3.3.2.2 Treatment preparation and application

Fungal isolates were cultured on millet grains following the techniques outlined by Toh et al. (2008). Colonized millet grains were dried in a laminar airflow cabinet for 72 hrs followed by

109 encapsulating in gelatine capsules. Capsules were stored at 4oC in an airtight plastic bag with silica gel desiccant. Each capsule approximately contained 0.3 g of millet grains colonized with individual test-isolates. Capsules made of fresh (autoclaved but un-inoculated) millet grains were used as controls. Capsules were delivered following a stem inoculation technique where a hole was made in the stem at 20 cm above the ground level using a 10 mm drill bit mounted on a cordless drill. Each hole was inoculated by inserting a single capsule followed by sealing with a domestic silicone sealant (ParfixTM). While inoculating with combinations of fungi, a separate hole for each was drilled 20 cm apart.

3.3.2.3 Trial assessment

Trials were conducted for approximately two years and assessed four times (including day 0 assessment) at 6-8 months interval. Stem lesions visible externally on the outer bark were photographed and their length was measured. At the end of the trial, stem segments were destructively sampled from one plant with each treatment from each block (four samples in total per treatment) and brought back to the laboratory. Samples were sawn through at the inoculation point and assessed for the presence of internal lesions in the stem cross sections. Internal lesions were photographed and the percentage of surface area in the stem cross section covered by lesions was measured following the techniques outlined by Cook (2014). Whether the internal lesion was present in the vascular area or confined to the pith region was also assessed.

Based on visual observation, a 0-10 rating scale (0=0%, 1=1-10%, 2=11-20%, 3=21-30%, 4=31- 40%, 5=41-50%, 6=51-60%, 7=61-70%, 8=71-80%, 9=81-90% and 10=91-100%) was used to record the amount of healthy branches and foliage cover present on healthy branches. An overall plant vigour rating scale was obtained by combining the individual ratings for branch health and foliage cover using the following formula: Plant vigour rating = (rating for foliage cover + 1) × (rating for healthy branch), which resulted in an overall vigour rating ranging from 0 to 110 (0= dead and 110= completely healthy).

3.3.2.4 Confirmation of infection

Confirmation of infection was achieved by isolating the test-fungi from the inoculated and control plants. Stem segments were destructively sampled from three plants with each treatment at approximately one year after inoculation and brought back to the laboratory. Samples were cut through the inoculation point using a sabre saw and the cross section was surface sterilized by

110 spraying 70% ethanol in a laminar airflow cabinet. Wood tissues from symptomatic and non- symptomatic areas were removed using a flamed 10 mm drill bit mounted on a cordless drill. From the same point the internal shavings were harvested using a flamed 6 mm drill bit. The technique was repeated at three different points per stem cross section. Shavings from different points were collected into separate sterile Petri dishes before aseptic transfer to ½ PDA plates amended with Penicillin (Sigma®, Penicillin G sodium salt) at 0.12 g/400 ml (300 ppm) and Streptomycin (Sigma®, Streptomycin sulfate salt) at 0.08 g/400 ml (200 ppm). Plates were incubated in a darkened incubator at 25oC for one week with daily observation. After the incubation period, the fungal isolates were purified by subculturing on fresh ½ PDA plates. The isolates looking apparently similar to the original isolates of Cophinforma sp. and L. pseudotheobromae used in inoculation were subcultured again and initially identified by visual comparison on culture plate to the original culture of Cophinforma sp. and L. pseudotheobromae used in inoculation. Later, seven isolates of Cophinforma sp. and six isolates of L. pseudotheobromae retrieved from field trials at Rockhampton and Richmond were partially sequenced for the ITS region following the techniques outlined in Chapter 2 (section 2.3.3) and compared to the same sequences the original isolates used in inoculation.

3.3.2.5 Rainfall and temperature data

Monthly total rainfall and average temperature (mean of maximum and minimum temperature) data recorded at weather stations nearest to the trial sites were obtained from the website of Australian Government Bureau of Meteorology (www.bom.gov.au) (Appendix 2 & 3).

3.3.2.6 Data analysis

The individual effect of two isolates of Cophinforma sp. and one isolate of L. pseudotheobromae and their combination effect on stem lesions and plant vigour were tested following GLM ANOVA using the statistical software package Minitab version 16. Treatment plot means of each parameter were compared following Tukey’s test with 95% level of confidence. No transformations were required to meet ANOVA requirements.

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3.4 Results

3.4.1 Glasshouse trial

3.4.1.1 Stem lesion development

Inoculation with Cophinforma sp. and/or L. pseudotheobromae caused external and internal stem lesions. Lesions were black to brownish in colour, necrotic and spreading in both directions from the point of inoculation and were associated with streaming of sap (Fig. 3.1).

A B C D

Fig. 3.1 External (top row) and internal (bottom row) stem lesions observed with A. control; B. Cophinforma sp. (AN71); C. Cophinforma sp. (AN110) and D. L. pseudotheobromae (NT039) (Scale=1.94 cm).

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At 12 weeks after inoculation, significantly longer external (P=0.002) and internal (P=0.001) stem lesions were observed with L. pseudotheobromae compared to the isolates of Cophinforma sp. (Fig. 3.2 A). Both isolates of Cophinforma sp. used in this study had identical effects on stem lesion development. Similar stem lesions were also observed with combinations of Cophinforma sp. and L. pseudotheobromae. However, the effect of their combinations on external and internal lesion length was not significantly different from the individual effect of L. pseudotheobromae (Fig. 3.2 B).

(A) A

a

b B B c C b

(B) AB A A A ab a a a

B b

Fig. 3.2 (A) Effect of individual fungal isolates on stem lesion length compared to control at 12 weeks under glasshouse conditions; (B) effect of isolate combinations on stem lesion length compared to the individual effect of L. pseudotheobromae (NT039) at 12 weeks under glasshouse conditions. Columns with different letters on error bars indicate significant difference by Tukey’s test with 95% confidence in GLM ANOVA (n=5). On the X axis, each capital letter represents individual treatment where A= control (autoclaved fresh millet grain), B= Cophinforma sp. (AN71), C= Cophinforma sp. (AN110), D= L. pseudotheobromae (NT039), E= Cophinforma sp. (AN71) + Cophinforma sp. (AN110), F= Cophinforma sp. (AN71) + L. pseudotheobromae (NT039), G= Cophinforma sp. (AN110) + L. pseudotheobromae (NT039) and H= Cophinforma sp. (AN71) + Cophinforma sp. (AN110) + L. pseudotheobromae (NT039). 113

3.4.1.2 Leaf production, leaf mortality, stem and branch dry-weight

At 12 weeks, total leaf production was found to be unaffected by the test-fungi and their combinations (Fig. 3.3 A). Cophinforma sp. and L. pseudotheobromae caused significant (P=0.000) leaf mortality when inoculated individually and in combinations (Fig. 3.3 B). However, the combination effect of Cophinforma sp. and L. pseudotheobromae on leaf mortality was identical to the individual effect of L. pseudotheobromae. The resulting effect of leaf mortality on stem and branch dry-weight was insignificant compared to the control treatment (Fig. 3.4).

(A)

a a a a a a a a

(B)

ab a a abc bcd cd d e

Fig. 3.3 Effect of individual fungal isolates and their combinations on (A) total leaf production and (B) leaf mortality at 12 weeks under glasshouse conditions. Columns with different letters on error bars indicate significant difference by Tukey’s test with 95% confidence in GLM ANOVA (n=5). On the X axis, each capital letter represents individual treatment where A= control (autoclaved fresh millet grain), B= Cophinforma sp. (AN71), C= Cophinforma sp. (AN110), D= L. pseudotheobromae (NT039), E= Cophinforma sp. (AN71) + Cophinforma sp. (AN110), F= Cophinforma sp. (AN71) + L. pseudotheobromae (NT039), G= Cophinforma sp. (AN110) + L. pseudotheobromae (NT039) and H= Cophinforma sp. (AN71) + Cophinforma sp. (AN110) + L. pseudotheobromae (NT039).

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a a c a a b a b a b b b a a b a

Fig. 3.4 Effect of individual fungal isolates and their combinations on stem and branch dry-weight. Columns with different letters on error bars indicate significant difference by Tukey’s test with 95% confidence in GLM ANOVA (n=5). On the X axis, each capital letter represents individual treatment where A= control (autoclaved fresh millet grain), B= Cophinforma sp. (AN71), C= Cophinforma sp. (AN110), D= L. pseudotheobromae (NT039), E= Cophinforma sp. (AN71) + Cophinforma sp. (AN110), F= Cophinforma sp. (AN71) + L. pseudotheobromae (NT#039), G= Cophinforma sp. (AN110) + L. pseudotheobromae (NT039) and H= Cophinforma sp. (AN71) + Cophinforma sp. (AN110) + L. pseudotheobromae (NT039).

3.4.1.3 Confirmation of infection

Cophinforma sp. and L. pseudotheobromae were successfully retrieved (100% recovery rate) from symptomatic stem tissues of the plants inoculated with their individual isolates. However, Cophinforma could not be retrieved from the coalescing stem lesions when co-inoculated with L. pseudotheobromae at sites 10 cm apart on the stem, whereas the recovery rate of L. pseudotheobromae from those plants was 100%. None of the isolates of Cophinforma sp. and L. pseudotheobromae could be retrieved from asymptomatic tissues whether inoculated individually or in combinations.

3.4.1.4 In-vitro growth rate and competiveness of the fungi

At 25oC, L. pseudotheobromae displayed a significantly (P=0.002) faster mycelial growth rate (mean radial growth 14.3 mm per day) compared to Cophinforma sp. (mean radial growth 7.5 mm per day) in single culture (Fig. 3.5). In the dual culture, the mycelial growth rate of both species was

115 initially identical to those observed in single culture, and no distinct inhibition zone was observed between the colonies suggesting a non-antagonistic interaction. However, after four days of incubation, the colony margin of both species (each placed at 55 mm apart on PDA plate) coalesced together and later (5 to 7 days incubation), L. pseudotheobromae had overgrown Cophinforma.

a a

b b

Fig. 3.5 Mycelial growth rate of L. pseudotheobromae and Cophinforma sp. in single culture. Columns with different letters on error bars indicate significant difference by Tukey’s test with 95% confidence in GLM ANOVA (n=6).

At 35oC, the growth rate of L. pseudotheobromae was slightly slower (mean radial growth 12.5 mm per day) and that of Cophinforma sp. was slightly faster (mean radial growth 8.7 mm per day) than those recorded at 25oC in single culture (Fig. 3.5). However, the competitive/suppressive interaction between L. pseudotheobromae to Cophinforma sp. in dual culture was unaffected by the higher temperature.

3.4.2 Field trial

3.4.2.1 Stem lesion development

External stem lesions with sap streaming identical to the glasshouse trial were observed in the field trial conducted at Rockhampton (Fig. 3.6). However, lesion length was significantly (P=0.020) greater than the control treatment (blank millet capsule) only in presence of L. pseudotheobromae (Fig. 3.7 A). We also observed significant (P=0.035) stem lesions with Cophinforma-Lasiodiplodia combinations. However, the effect of isolate combinations on external lesion length was not 116 significantly different from the individual effect of L. pseudotheobromae (Fig. 3.7 B) as observed in the glasshouse trial. No external stem lesions or cankers were visible on the dark brown bark in another field trial conducted at Richmond.

A B C D

A B C D

Fig. 3.6 Stem lesions observed with A. control; B. Cophinforma sp. (AN71); C. Cophinforma sp. (AN110) and D. L. pseudotheobromae (NT039) at 6 months at Rockhampton (Scale=1.94 cm).

(A)

a

b b b

(B) a

ab b b b

Fig. 3.7 (A) Effect of individual fungal isolates on external stem lesion length compared to control and (B) effect of isolate combinations on stem lesion length compared to the individual effect of L. pseudotheobromae recorded in a field trial at Rockhampton at 21 months after inoculation. Columns with different letters on error bars indicate significant difference by Tukey’s test with 95% confidence in GLM ANOVA (n=5). On the X axis, each capital letter represents individual treatment where A= control (autoclaved fresh Millet grain), B= Cophinforma sp. (AN71), C= Cophinforma sp. (AN110), D= L. pseudotheobromae (NT039), E= Cophinforma sp. (AN71) + Cophinforma sp. (AN110), F= Cophinforma sp. (AN71) + L. pseudotheobromae (NT039), G= Cophinforma sp. (AN110) + L. pseudotheobromae (NT039) and H= Cophinforma sp. (AN71) + Cophinforma sp. (AN110) + L. pseudotheobromae (NT039). 117

Internal lesions were also observed in cross sections of the stem samples collected from trial sites at Rockhampton and Richmond. At Rockhampton, at 21 months, internal lesions were lighter in colour and found mostly confined to the pith region (Fig. 3.8 A). In contrast, at Richmond, at 23 months, internal stem lesions were more prominent and occupied larger areas in stem cross section compared to that observed in Rockhampton (Fig. 3.8 B). However, in both trials, the relative lesion size (measured as surface area (%) in stem cross-section covered by internal lesion) was unaffected by individual or combination effects of the test-fungi compared to the control treatment (Fig. 3.9).

(A) Pith affected

Control Cophinforma sp. (AN71) Cophinforma sp. (AN110)

Vascular area

Pith

L. pseudotheobromae (NT039)

(B)

Control Cophinforma sp. (AN71) C. eucalypti (AN110)

Vascular area affected

L. pseudotheobromae (NT039)

Fig. 3.8 Internal lesions observed with different fungi in field trials conducted at (A) Rockhampton at 21 months after inoculation and (B) Richmond at 23 months after inoculation (Scale=1cm).

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A A A

A A

A

A

A

a a a a a a a a

Fig. 3.9 Individual and combination effect of Cophinforma sp. and L. pseudotheobromae on internal stem lesions of prickly acacia recorded in field trials conducted at Rockhampton (at 21 months) and Richmond (at 23 months). Columns with different letters on error bars indicate significant difference by Tukey’s test with 95% confidence in GLM ANOVA (n=5). On the X axis, each capital letter represents individual treatment where A= control (autoclaved fresh millet grain), B= Cophinforma sp. (AN71), C= Cophinforma sp. (AN110), D= L. pseudotheobromae (NT039), E= Cophinforma sp. (AN71) + Cophinforma sp. (AN110), F= Cophinforma sp. (AN71) + L. pseudotheobromae (AN#71), G= Cophinforma sp. (AN110) + L. pseudotheobromae (NT039) and H= Cophinforma sp. (AN71) + Cophinforma sp. (AN110) + L. pseudotheobromae (NT039).

3.4.2.2 Plant vigour

At Rockhampton, all the plants were in full vigour (vigour rating 110/110) at the time of inoculation (data not presented) and no decline was observed in the next 2 phases of trial assessment at 6 and 14 months after inoculation at November 2012 and July 2013, respectively (Fig. 3.10). At 21 months after inoculation (February 2014), we recorded a slight decline of plant vigour (vigour rating

119 approximately 100/110) in the inoculated plants (Fig. 3.10). However, such marginal decline was insignificant compared to the control treatment. The untreated plants neighbouring the trial site assessed 4 months later (July 2014) were also found with high vigour (vigour rating approximately 95-100/110) (data not presented).

At Richmond, at the time of inoculation, plants included in the trial were in full vigour (vigour rating 110/110) (data not presented) but showed some signs of cattle damage but were free from natural dieback as previously observed at other locations in the same region. No significant vigour decline was observed in the next 2 phases of trial assessment at 6 and 14 months after inoculation in November 2012 and July 2013, respectively (Fig. 3.11). At 23 months after inoculation (May 2014), we recorded promonent branch mortality and thinning of leaf cover leading to substantial decline in plant vigour (vigour rating approximately 45-60/110) compared to previous assessments (Fig. 3.11). However, the individual and combination effects of Cophinforma sp. and L. pseudotheobromae on vigour decline were insignificant compared to the control plants. The untreated plants neighbouring the trial site assessed at 23 months were also found with low vigour (vigour rating approximately 55-60/110) which was identical to the treated plants.

3.4.2.3 Confirmation of infection

L. pseudotheobromae was successfully retrieved with a 100% recovery rate (12 samples/trial site) from the symptomatic internal stem tissues extracted from the inoculated stem samples collected from trial sites at Rockhampton and Richmond. Fungi similar to L. pseudotheobromae were not found in control plants at any trial site.

On the other hand, recovery rate from the inoculated plants was low with Cophinforma sp. in both trial sites. It was retrieved from only 2 out of 18 stem samples collected at Rockhampton and 7 out of 18 stem samples at Richmond. Fungi similar to Cophinforma were not found in the control plants at Rockhampton but were found in the control plants at Richmond. Comparison of the ITS sequences from a set of fungal isolates retrieved from the field trials with those of the original isolates used in field inoculation (similarity tree not presented) confirmed the infection by Cophinforma sp. and L. pseudotheobromae at Rockhampton and by L. pseudotheobromae at Richmond following deliberate inoculations. However, infection by Cophinforma sp. following deliberate inoculation could not be confirmed at Richmond because the same (based on the ITS sequence) fungus was found in the control plants as a pre-existing endophyte.

120

a a a a a a a a

a a a a a a a a

a a a a a a a a

Fig. 3.10 Effect of different fungal isolates and their combinations on overall vigour of prickly acacia recorded from May 2012 to February 2014 in a field trial conducted at Rockhampton, central Queensland. Columns with different letters on error bars indicate significant difference by Tukey’s test with 95% confidence in GLM ANOVA (n=4). On the X axis, each capital letter represents individual treatment where A= control (autoclaved fresh millet grain), B= Cophinforma sp. (AN71), C= Cophinforma sp. (AN110), D= L. pseudotheobromae (NT039), E= Cophinforma sp. (AN71) + Cophinforma sp. (AN110), F= Cophinforma sp. (AN71) + L. pseudotheobromae (AN#71), G= Cophinforma sp. (AN110) + L. pseudotheobromae (NT039) and H= Cophinforma sp. (AN71) + Cophinforma sp. (AN110) + L. pseudotheobromae (NT039).

121

a a a a a a a a

a a a a a a a a

a a a a a a a a a

Fig. 3.11 Effect of different fungal isolates and their combinations on overall vigour of prickly acacia recorded from June 2012 to May 2014 in a field trial conducted at Richmond, north western Queensland. Columns with different letters on error bars indicate significant difference by Tukey’s test with 95% confidence in GLM ANOVA (n=4). On the X axis, each capital letter represents individual treatment where A= control (autoclaved fresh millet grain), B= Cophinforma sp. (AN71), C= Cophinforma sp. (AN110), D= L. pseudotheobromae (NT039), E= Cophinforma sp. (AN71) + Cophinforma sp. (AN110), F= Cophinforma sp. (AN71) + L. pseudotheobromae (AN#71), G= Cophinforma sp. (AN110) + L. pseudotheobromae (NT039), H= Cophinforma sp. (AN71) + Cophinforma sp. (AN110) + L. pseudotheobromae (NT039) and I= Untreated plants.

122

3.5 Discussion

Different species of Botryosphaeriaceae fungi had been reported to be associated with dieback of different woody hosts including invasive weeds (Wilson & Pitkethley 1992; Diplock et al. 2006; Botella et al. 2010; Dakin et al. 2010; Sacdalan et al. 2012). Similar fungi were also found to be implicated in dieback of prickly acacia (Haque et al. 2012). Therefore, the objective of this study was to investigate whether the Botryosphaeriaceae fungi associated with dieback-affected prickly acacia and parkinsonia in northern Australia could result in dieback following deliberate inoculation of healthy prickly acacia plants under field conditions. If so, then the fungi could offer a novel way of managing this invasive weed species. Our study found significant disease symptoms such as stem lesions and gummosis following deliberate inoculation of healthy population of prickly acacia with these Botryosphaeriaceae fungi under glasshouse and field conditions. In addition, typical dieback symptoms such as widespread branch mortality and thinning of leaf cover leading to significant decline in plant vigour identical to those found in natural prickly acacia dieback event were observed in one of our study sites at Richmond which was severely water-stressed throughout the trial period (Appendix 3).

In the glasshouse trial, necrotic stem lesions appeared as the main disease symptom following inoculation with the test-fungi. Measurement of stem lesions indicated that both L. pseudotheobromae and Cophinforma sp. individually caused significant stem lesions in prickly acacia. Similar lesions were also observed on prickly acacia and other hosts following deliberate inoculation with different Botryosphaeriaceae fungi under controlled environment (Haque et al. 2012; Amponsah et al. 2014). L. pseudotheobromae caused significantly longer stem lesions compared to Cophinforma sp. indicating the more aggressive nature of L. pseudotheobromae. Similar results were found in earlier studies with prickly acacia and other hosts (Mehl et al. 2011; Haque et al. 2012). However, the combined effect of L. pseudotheobromae and Cophinforma sp. on stem lesion length could not be distinguished from the individual effect of L. pseudotheobromae. The combination effect was masked by the individual effect of L. pseudotheobromae.

Plant mortality was not observed during the course of the glasshouse trial (12 weeks). Plant mortality might be evident over a longer period. However, the study demonstrated significant leaf mortality with L. pseudotheobromae and its combination with Cophinforma sp. Our study did not investigate the exact cause of increased leaf mortality with L. pseudotheobromae. However, Davidson et al. (2003) reported that stem canker caused by Phytophthora ramorum Werres, De Cock & Man in't Veld in (Quercus spp.) was followed by significant leaf mortality. Rodrigues

123 et al. (2014) also found that stem girlding canker caused by Colletotrichum theobromicola Delacr. resulted in significant defoliation in eucalyptus (Eucalyptus spp.) saplings. Although we observed leaf mortality with L. pseudotheobromae and its combinations with Cophinforma sp., their resulting effect on plant growth parameters such as total leaf production and stem and branch dry-weight was insignificant, indicating compensatory plant growth. Such compensation was clearly observed in the glasshouse trial as defoliation and leaf mortality was followed by significant re-shooting (water sucker) of the plants from the base. New shoots were full of actively growing leaves. Similar growth compensation following defoliation or leaf mortality was also previously observed on other woody species (Jaremo & Palmqvist 2001; Salomon Ballina-Gomez et al. 2010). Re-shooting was also observed in the natural prickly acacia dieback event (Galea 2011).

After harvesting the glasshouse trial, both Cophinforma sp. and L. pseudotheobromae were successfully retrieved from symptomatic stem tissues of the plants inoculated with the individual isolates. However, Cophinforma sp. could not be retrieved when combined with L. pseudotheobromae although at separate locations 10 cm apart. It was an interesting finding which demanded further investigation, and we conducted in-vitro studies to investigate the interactions between Cophinforma sp. and L. pseudotheobromae which found that L. pseudotheobromae was capable of overgrowing Cophinforma. Similar reaction was observed under glasshouse conditions where Cophinforma and L. pseudotheobromae were inoculated at certain distance apart on stem, both initially caused separate external stem lesions which later merged together externally and internally and Cophinforma sp. could not be isolated at the end of the trial. Such findings indicated a competitive effect of the fast growing L. pseudotheobromae to the slow growing Cophinforma. Such competitive or inter-specific interaction was previously reported with other fungi. For example, Wardle et al. (1993) investigated the inter-specific competetion between Trichoderma polysporum (Link) Rifai and Mucor hiemalis Wehmer. The study found that T. polysporum maintained a competitive advantage over M. hiemalis in different natural substrates. Shearer (1995) also reported that different fungal communities were strongly influenced by competitive interactions similar to other microorganisms.

External stem lesions similar to those observed in the glasshouse trial were also observed in the field trial conducted at Rockhampton where plants were young with green and smooth bark. Similar lesions were also recorded on prickly acacia and other woody hosts following inoculation with different Botryosphaeriaceae fungi including Cophinforma sp. and L. pseudotheobromae under field conditions (Mohali et al. 2009; Cook 2014). However, similar to the glasshouse trial, the Lasiodiplodia-Cophinforma combination effect on stem lesion length could not be distinguished

124 from the individual effect of L. pseudotheobromae. Being highly aggressive, L. pseudotheobromae individually developed stem lesions which probably masked the effect of Cophinforma sp. when combined together. No such stem lesion was externally visible in another field trial conducted at Richmond, possibly because the plants in that site were older with rough and dark brown bark which interfered with external lesion development and/or visibility. In a previous study, Yuan and Mohammed (2001) also found that different canker causing fungi caused larger stem cankers in with eucalyptus (Eucalyptus nitens H.Deane & Maiden) plants with smooth bark compared to those with rough bark. Further microscopic investigation suggested that the particular anatomical structure of smooth bark offered less mechanical resistance to hyphal spread in comparison with rough bark. However, the internal stem lesions observed at Richmond were larger in size and darker in colour compared to those observed at Rockhampton. Such difference in internal lesion size observed between two sites was possibly associated with the differences in diversity and pathogenicity of native endophytes and other site-specific climatic factors such as amount of rainfall and temperature. Pre-existing native endophytes extracted from both trial sites were found to cause significant internal stem lesions in prickly acacia following inoculation under glasshouse conditions (Chapter 2, Section 2.4.4.4). Both trial sites were also subjected to almost similar average temperature (Appendix 2). However, the trial site at Richmond was severely water stressed resulting from lower amount of rainfall compared to the Rockhampton site (Appendix 3) which possibly may have influenced the development of larger internal lesions observed at Richmond as found in previous stress-fungus interaction studies (Paoletti et al. 2001; Niekerk et al. 2011; Amponsah et al. 2014). In addition, the plants at Richmond being older were possibly more susceptible to the introduced or native fungi causing larger internal lesions as reported in previous plant-fungi interaction studies (Balci et al. 2008; Amponsah et al. 2014).

As found in the glasshouse trial, Cophinforma sp. could not be retrieved once combined with L. pseudotheobromae under field conditions. As previously explained, establishment and/or survival of Cophinforma sp. was possibly affected by competition with L. pseudotheobromae and/or other native endophytes over the trial period. Cophinforma sp. was retrieved only when inoculated individually. However, the recovery rate of Cophinforma sp. was fairly low and possibly affected by the interference of other fast growing native endophytes such as Aureobasidium, Curvularia, Chaetomium and Paecilomyces frequently encountered on culture plates and previously reported to have antagonistic effect against other fungi (Demirci et al. 2011; Almaraz-Sanchez et al. 2012). On the other hand, L. pseudotheobromae was consistently retrieved from the necrotic stem tissues of the plants inoculated with L. pseudotheobromae individually or in combinations with Cophinforma

125 sp. which was unaffected by the native endophytes. Such findings are indicative of a higher level of ecological fitness of L. pseudotheobromae as a potential bioherbicide candidate for prickly acacia.

Different Botryosphaeriaceae fungi including L. pseudotheobromae were previously isolated from the naturally infected asymptomatic tissues of a range of host plants (Slippers & Wingfield 2007; Trakunyingcharoen et al. 2013). However, such asymptomatic or systemic colonization of healthy tissues following deliberate stem inoculation with the Botryosphaeriaceae fungi had never been previously reported. Such a mode of infection was also not found in our study since the test-fungi could not be retrieved from apparently healthy tissues outside the necrotic area of stem lesions in the inoculated plants. However, Slippers & Wingfield (2007) and Amponsah et al. (2014) reported that in most of the cases, disease expression by Botryosphaeriaceae fungi were associated with other stressors such as drought, high temperature, insect infestation etc. Therefore, it would be interesting to investigate whether the presence of abiotic stressors could initiate systemic infection by Botryosphaeriaceae fungi.

Apart from investigating stem lesions and fungal colonization, the major interest of this field study was to understand the individual and combination effects of different Botryosphaeriaceae fungi on growth, survival and ultimately mortality of prickly acacia under field conditions. At nearly two years after inoculation, we observed significant branch mortality and defoliation leading to substantial decline in plant vigour in the field trial at Richmond. However, the control plants in the trial and the untreated plants neighbouring the trial site were also found with similar levels of decline indicating that the observed symptoms were naturally occurring and potentially associated with other stressors. On the other hand, no such symptoms were observed during the course of another field trial at Rockhampton. Such natural decline observed at Richmond was possibly linked with drought stress resulting from lower amount of precipitation. The wet season monsoon also did not occur at Richmond for 2013. On the other hand, our study site at Rockhampton received significantly higher amounts of rainfall, and was subjected to slightly lower temperatures compared to the Richmond site (Appendix 2 & 3). In previous studies, drought stress was identified as a key factor affecting tree mortality in semi-arid grassland ecosystems in Australia similar to the study site at Richmond (Fensham et al. 2005; Fensham & Fairfax 2007; Fensham et al. 2009). Natural dieback of prickly acacia was also previously reported from this area (Galea 2011). In addition, ecological modelling also suggests that the climatic conditions of north-western Queensland are more stressful for prickly acacia compared to that of coastal sites (Kriticos et al. 2003).

Different pre-existing Botryosphaeriaceae including Cophinforma sp. and other fungi sourced from the study site were found to be pathogenic to prickly acacia causing disease symptoms under 126 laboratory and glasshouse conditions (Chapter 2, Section 2.4.4.4). The interaction effect of those native and deliberately inoculated fungi with drought stress could be another potential reason for the observed decline as different fungi including the Botryosphaeriaceae are known to cause widespread dieback in the presence of other stressors (Desprez-Loustau et al. 2006; Slippers & Wingfield 2007; Mehl et al. 2011; Kaczynski & Cooper 2013). In addition, grazing pressure, sap sucking and leaf feeding insects and their interactions with pre-existing and inoculated fungi could also be implicated in the decline symptoms observed on prickly acacia (Aghighi et al. 2014; Stursova et al. 2014).

It is not clearly evident from this study whether the decline symptoms observed in north-western Queensland were a sole effect of fungus, stress or stress-fungus interactions. Therefore, the stress- fungus interaction effect in terms of prickly acacia dieback was investigated in the next chapters.

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Chapter 4: Interactions of a pathogenic strain of Lasiodiplodia pseudotheobromae with sub- lethal glyphosate in prickly acacia

4.1 Abstract

Lasiodiplodia pseudotheobromae is a potential bioherbicide candidate for prickly acacia, one of the most harmful weeds of the Australian rangelands. We hypothesized that the pathogenic efficacy of L. pseudotheobromae could be enhanced with co-treatment of sub-lethal dose of glyphosate as a source of plant stress. This was tested under glasshouse conditions using six month old juvenile plants and in the field using natural populations of mature trees. Glasshouse grown plants were stem-inoculated with L. pseudotheobromae and co-treated with a pre-determined sub-lethal dose of glyphosate (45g/L a.i @ 100 µL/plant). Significantly longer external stem lesions, reduced leaf production, higher levels of leaf mortality and reduced plant height were recorded with glyphosate co-treatment. However, there was no significant glyphosate-fungus interaction effect on stem and branch dry weight. This experiment was followed by two field trials (experimental repeats) at Rockhampton, central Queensland and Richmond, north-western Queensland to test the hypothesis under field conditions. L. pseudotheobromae was applied using a gelatine capsule delivery system and combined with none or either of two sub-lethal doses of glyphosate (180g/L a.i @ 0.5 or 1.0 mL/plant) applied into a hole drilled into the stem. At Rockhampton, both L. pseudotheobromae and sub-lethal glyphosate caused significant external stem lesions which developed into stem cankers. No significant internal lesions were recorded in stems harvested from treated plants at this site. At Richmond, external stem lesions were not visible possibly being obscured by the darkly pigmented bark, but there were prominent internal lesions observed in stem cross sections. However, there was no significant glyphosate-fungus interaction effect on external or internal lesion size at either trial site. At approximately two years after treatment we observed substantial reduction of plant vigour at both trial sites. However, there was no significant glyphosate-fungus interaction effect on plant vigour. L. pseudotheobromae was successfully isolated from the inoculated plants in both glasshouse and field trials irrespective of glyphosate co-treatment. Although L. pseudotheobromae caused more severe dieback of prickly acacia with glyphosate co-treatment under glasshouse conditions, such interactions were not clearly evident under field conditions.

Keywords: Prickly acacia, Lasiodiplodia pseudotheobromae, bioherbicide, glyphosate, dieback.

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4.2 Introduction

Bioherbicides are broadly defined as the products derived from living organisms such as fungi and bacteria, including any natural compound they produce during their growth that kill or debilitate weeds (Bailey 2014). In recent years, there has been interest in the use of Lasiodiplodia pseudotheobromae Phillips, Alves & Crous to develop a bioherbicide for prickly acacia (Vachellia nilotica subsp. indica (Benth.) Kyal. & Boatwr), one of the serious woody weeds in coastal wet and northern semi-arid rangelands in Australia. L. pseudotheobromae had been previously reported to have bioherbicidal potential against other invasive and non-invasive weeds in Australia and overseas (Toh 2009; Adetunji & Oloke 2013; Charles & Julius 2013; Galea & Goulter 2013). In northern Australia, it was associated with dieback of parkinsonia (Parkinsonia aculeata L.), another invasive woody weed in Australia and the same fungal strain was found to be highly pathogenic to prickly acacia seedlings and juveniles causing dieback symptoms under laboratory and glasshouse conditions (Haque et al. 2012). However, aetiological studies on such other Botryosphaeriaceae fungi consider that these are mostly facultative pathogens and host damage or other stressors can enhance the disease progression (Slippers & Wingfield 2007).

Bioherbicides are considered a safer and more economical approach when compared to other weed control techniques (Auld & Morin 1995; Charudattan 2001; Bailey 2014). However, efficacy of the bioherbicidal approach is determined by a number of factors such as the characteristics of the weed species and the bioherbicide agent, weed-agent interactions, desired level of weed control and variation in soil type, climate, topography and other ecological factors (Kadir et al. 2000; Hetherington et al. 2002; Ghorbani et al. 2006; Iffat et al. 2009). In addition, success with fungi- based bioherbicides is also a function of appropriate inocula formulation and delivery technique (Charudattan 2001; Ash 2010). There are various bioherbicide formulations and delivery techniques depending on the characteristics of both target weed and bioherbicidal agent. However, in previous studies, stem wounding followed by insertion of pelleted or encapsulated fungal material was found to be effective against woody weeds (Dorworth 1995; Rayachhetry et al. 1999; Galea & Goulter 2013).

Apart from the successful use of bioherbicides against a range of weeds including woody species, there are also examples where this approach failed or was constrained by a number of factors resulting in non-establishment or lower impact on the target weed (Auld & Morin 1995; Charudattan 2001; Green 2003; Barreto et al. 2012; Glare et al. 2012; Bailey 2014). Such occurrences have also been observed with other bio-control agents, particularly of woody perennial

134 weeds in the Australian rangelands (Julien 2006; Vitelli & Pitt 2006). Inherent or elicited host resistance in target weeds against the bioherbicide agent was identified as one of the important reasons of non-establishment or lower control impact of bioherbicide fungi (Auld & Morin 1995; Hoagland 1996). Therefore, to render the host more susceptible, bioherbicides have been combined with other compounds particularly sub-lethal rates of chemical herbicides (Sharon et al. 1992; Graham et al. 2006; DiTomaso 2008; Gressel 2010; Hoagland et al. 2011). Similar effects was also reported for controlling invasive woody weeds (Rayachhetry et al. 1999). Among chemical herbicides, glyphosate was frequently used for this purpose (Gressel 2010).

Glyphosate (N-(phosphonomethyl) glycine) is a widely used, broad spectrum chemical herbicide. This systemic herbicide inhibits the specific plant enzyme 5-enolpyruvylshikimic-3-phosphate synthase (EPSPS), resulting in deficiency of essential amino acids particularly in rapidly dividing cells, eventually leading to nutritional stress and plant death (Becerril et al. 1989; Pavlovic et al. 2013). It was also reported to immobilize specific micronutrients involved in disease resistance and interfere with the elicitation of phytoalexins, the low molecular weight antimicrobial compounds involved in a plant’s defence response against pathogens (Sharon et al. 1992; Johal & Huber 2009). Such glyphosate-induced weakening of plant defence was reported to be associated with higher disease severity caused by a range of fungi including the Botryosphaeriaceae (Johal & Rahe 1990; Johal & Huber 2009). Similarly, a number of potential bioherbicide fungi also showed improved efficacy through synergistic interactions with sub-lethal dose of glyphosate (Boyette et al. 2006; Smith & Hallett 2006; Boyette, et al. 2008; Mitchell et al. 2008; Weaver et al. 2009; Gressel 2010). In this study, sub-lethal doses of glyphosate were used as source of stress to the target weed prickly acacia and combined as a co-treatment with bioherbicide fungus L. pseudotheobromae. Combination of a biocontrol fungus with a sub-lethal dose of chemical herbicide also has practical implications in integrated weed management (Hasan & Ayres 1990). Such combination can significantly reduce the amount of chemical herbicide required for effective weed control.

The objective of this study was to investigate whether the severity of dieback caused by L. pseudotheobromae could be increased through addition of the stressor, sub-lethal dose of glyphosate. The study was initially conducted under glasshouse conditions. It was then replicated in two different ecological zones near Rockhampton, central Queensland and Richmond, north- western Queensland representing a wet coastal and semi-arid dry climate, respectively.

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4.3 Materials and Methods

4.3.1 Glasshouse trial

4.3.1.1 Estimation of sub-lethal dose of Glyphosate

Sub-lethal dose of glyphosate was determined from a glasshouse trial where 100 µL of six different concentrations (0, 22.5, 45, 90, 180 and 360g/L a.i) of a commercial glyphosate formulation (Guard & Grow, Mitre 10 Australia Ltd., 360g/L a.i) were tested under glasshouse conditions following a randomized complete block design with five replicates for each glyphosate concentration (30 plants in total). Plants were six months old at the beginning of the trial which were grown from surface sterilized seeds following the techniques outlined in Chapter 2 (Section 2.3.4.1). Seeds were sourced from dieback-free plants growing at Richmond, north-western Queensland. Glyphosate solutions were prepared using sterile RO water and applied using a micropipette into a hole (3.5 mm in diameter) drilled into each stem at 10 cm above the ground level. The trial continued for six weeks and 100 µL of 45g/L a.i glyphosate was selected as a sub-lethal dose where plants showed some early reaction to application of the herbicide and subsequently recovered.

4.3.1.2 Experimental design, treatments and growing juvenile plants

The effect of inoculation with L. pseudotheobromae was assessed by measurement of stem lesion length, leaf mortality and other growth parameters of prickly acacia in the presence and absence of sub-lethal glyphosate. The 12-week glasshouse trial was conducted using a randomized complete block design with five blocks. The following treatments (Table 4.1) were randomly allocated within each block which contained a complete set of treatments.

Table 4.1 Treatments in glasshouse trial

Code Treatment A Control (autoclaved fresh millet grains) B Glyphosate 45g/L a.i @ 100 µL/plant C L. pseudotheobromae (NT039) D L. pseudotheobromae + Glyphosate 45g/L a.i @ 100 µL/plant (applied into same hole) E L. pseudotheobromae + Glyphosate 45g/L a.i @ 100 µL/plant (applied into different holes)

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Plants were grown from surface sterilized seeds following the techniques described in Chapter 2 (Section 2.3.4.1). Plants were six month old at the start of the trial (average height 130.29 ± 2.92 cm; average stem diameter at 10 cm above the soil level 10.79 ± 0.48 mm), and were randomly allocated to treatments and blocks.

4.3.1.3 Treatment preparation and application

An isolate of L. pseudotheobromae (strain NT039) sourced from parkinsonia dieback was cultured on millet (Panicum miliaceum L.) grains following the techniques outlined by Toh et al. (2008). Using an alcohol sterilized 3.5 mm drill bit mounted on a cordless drill, a 5-6 mm deep inoculation hole was made in the stem (at a slight downward angle) at 10 cm above the soil level followed by placing two millet grains using sterilized forceps and covering the wound with Parafilm®. A pre- determined sub-lethal dose (45g/L a.i. @100 µL/plant) of a commercial glyphosate formulation (Guard & Grow, Mitre 10 Australia Ltd., 360g/L a.i) was applied using a micropipette into a hole (3.5 mm in diameter) drilled into stem at 10 cm above the soil level. It was applied either into the same hole as the L. pseudotheobromae inoculation or in a separate hole drilled 5 cm below the inoculation point when combined with L. pseudotheobromae. Fresh (un-inoculated) autoclaved millet grains were used as control.

4.3.1.4 Trial assessment

Length of external and internal stem lesions was measured using a ruler. An aluminium foil tray with a drainage hole at the center was placed beneath each plant to collect fallen leaves throughout the trial. Dead leaves attached with the plants were hand removed at the end of the trial. After removing the attached dead leaves, the plants with live (green) leaves were cut into small pieces and put in labelled aluminium trays and left in glasshouse for 72 hours to commence desiccation. Both dead (fallen plus attached dead leaves) and green leaves were dried separately in a drying oven at 60-650C for 1 week. Total leaf production was calculated by adding the dry-weight of dead and live (still attached) leaves at the end of the trial. Leaf mortality was expressed as a percentage of dead leaf to total leaf production. Dry-weight of stem and branch samples was obtained following oven drying at 60-65oC for 2 to 3 weeks. Initial and final plant height was measured using a measuring tape and height increment during the course of the trial was calculated by subtracting initial plant height from the final plant height.

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4.3.1.5 Confirmation of infection

Confirmation of infection was achieved by isolating L. pseudotheobromae from inoculated and non- inoculated plants. The potential of this species to asymptomatically colonize healthy tissue of inoculated plants as an endophyte was also investigated. Isolations were conducted from leading edges (either distal or proximal to the plant base) of internal stem lesions and healthy tissues adjacent to that following the techniques modified from Diplock et al. (2006). Stem segments collected from three randomly selected plants with each treatment were surface sterilized by soaking in 2% NaOCl (v/v) for two minutes followed by rinsing in sterile water twice for five minutes and drying on sterile blotting paper within a laminar air-flow cabinet. Partially dried samples were aseptically transferred to ½ PDA media followed by incubation at 25oC in a darkened incubator for one week with daily observation. After the incubation period, the fungal isolates were purified by subculturing on fresh ½ PDA plates before comparing to original isolate used in the experiment.

4.3.1.6 Data analysis

The individual effect of L. pseudotheobromae and the sub-lethal doses of glyphosate and their interactions on stem lesion length, total leaf production, leaf mortality, stem/branch dry weight and height increment was tested using GLM ANOVA in the statistical software package Minitab version 16. Treatment means were compared using Tukey’s test with 95% confidence. No transformations were required to meet ANOVA requirements.

4.3.2 Field trials

4.3.2.1 Experimental design and treatments

Field trials were established at Rockhampton (23°22′54″S 150°28′30″E), central Queensland and Richmond (20°43′50″S 143°08′32″E), north-western Queensland using naturally occurring plants growing on native pasture. The trial sites were 1108 Km apart from each other. Trials were established between late May and early June, 2012 using a randomized complete block design. There were four blocks, each containing six separate treatment plots. Each plot consisted of eight plants growing in a cluster and comprised an individual treatment. Treatment plots were randomly allocated within a block and each block had a complete set of treatments. Mean stem circumference of the plants at 20 cm above the ground level was 18.27 (± 8.31) cm and 26.25 (± 5.94) cm at

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Rockhampton and Richmond, respectively. Single cattle ear tags bearing the treatment name were wired to the first plant in each treatment plot along with recording of the GPS waypoint. Experimental plants were also marked on exposed woody stems or branches with white spray paint for the ease of identification.

The trials were established with the following treatments (Table 4.2). However, at the final stage of assessment (approximately two years after trial establishment), an additional treatment “untreated control (plants were not drilled and no capsule inserted)” comprising the same number of untreated plants adjacent to the original trial was included.

Table 4.2 Treatments in field trial

Code Treatment A Control (autoclaved fresh millet grains) B L. pseudotheobromae (NT039) C Glyphosate 180g/L a.i @ 0.5 mL/plant D Glyphosate 180g/L a.i @ 1 mL/plant E L. pseudotheobromae + Glyphosate 180g/L a.i @ 0.5 mL/plant F L. pseudotheobromae + Glyphosate 180g/L a.i @ 1 mL/plant

At Rockhampton, the untreated plants were assessed four months after the final assessment point whereas at Richmond, they were assessed on final data collection point for the trial.

4.3.2.2 Treatment preparation and application

Preparation and application of millet based inoculum of L. pseudotheobromae followed the procedures outlined in Chapter 3 (Section 3.3.2.2). The commercial formulation of glyphosate (Guard & Grow, Mitre 10 Australia Ltd., 360g/L a.i) was diluted using bottled drinking water (Mount Franklin Spring Water) to achieve the desired concentration of 180g/L a.i and applied at 0.5 mL or 1.0 mL per plant into drilled stem using a syringe pump. Glyphosate was applied in a separate (10 mm diameter) hole 20 cm below the capsule delivery point when it was combined with L. pseudotheobromae.

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4.3.2.3 Trial assessment

Trials were conducted for a period of approximately two years and assessed for four times (including 0 day assessment) at 6 to 8 month intervals. Stem lesions visible externally on the outer bark were photographed and their length was measured. At the completion of the trial, stem segments were destructively sampled from four plants with each treatment and brought back to the laboratory. Samples were sawn through the inoculation point and examined for the presence of internal lesions in stem cross sections. Stem sections were photographed and the percentage of surface area in stem cross section occupied by lesions was determined following the techniques outlined by Cook (2014). Whether the internal lesion was present in vascular area or confined to the pith region was also recorded. Based on visual observation, a 0 to 10 rating scale (0=0%, 1=1-10%, 2=11-20%, 3=21-30%, 4=31-40%, 5=41-50%, 6=51-60%, 7=61-70%, 8=71-80%, 9=81-90% and 10=91-100%) was used to record the amount of healthy branches and foliage cover present on healthy branches. An overall plant vigour rating scale was obtained by combining the individual ratings for branch health and foliage cover using the following formula: Plant vigour rating= (rating for foliage cover + 1) × (rating for healthy branch), which resulted in an overall vigour rating ranging from 0 to 110 (0=dead and 110=completely healthy).

4.3.2.4 Confirmation of infection

Confirmation of infection was achieved by re-isolating L. pseudotheobromae from the inoculated plants both treated and untreated with glyphosate co-treatment. Stem segments were destructively sampled from three plants with each treatment at approximately one year after inoculation and brought back to the laboratory. Samples were cut through the inoculation point using a sabre saw and the cross section was surface sterilized by spraying 70% Ethanol in a laminar airflow cabinet. Wood tissues from symptomatic and non-symptomatic areas were removed using a flamed 10 mm drill bit mounted on a battery operated hand drill to remove the surface layer. From the same point the internal shavings were harvested using a flamed 6 mm drill bit. The technique was repeated at three different points per stem cross section. Shavings from different points were collected into separate sterile Petri dishes before aseptic transfer to ½ PDA plates amended with Penicillin (Sigma®, Penicillin G sodium salt) at 0.12 g/400 mL (300 ppm) and Streptomycin (Sigma®, Streptomycin sulfate salt) at 0.08 g/400 mL (200 ppm). Plates were incubated in a darkened incubator at 25oC for one week with daily observation. After the incubation period, the fungal isolates were purified by subculturing on fresh ½ PDA plates. Isolates looking apparently similar to the original isolate of L. pseudotheobromae used in inoculation were subcultured again and initially

140 identified by visual comparison on the culture plate to the original culture of L. pseudotheobromae used in inoculation. To confirm their identity, a sub-set of six isolates (three isolates from each trial site) apparently similar to the original isolate of L. pseudotheobromae were sequenced for a part of the Internal Transcribed Spacer (ITS) region and compared to the same sequence of the original isolate used in inoculation following the similar techniques outlined in Chapter 2 (Section 2.3.2).

4.3.2.5 Rainfall and Temperature data

Monthly total rainfall and average temperature (mean of maximum and minimum temperature) data recorded at weather stations nearest to the trial sites were obtained from the website of Australian Government Bureau of Meteorology (www.bom.gov.au ) (Appendix 2 & 3).

4.3.2.6 Data analysis

The individual effect of L. pseudotheobromae, glyphosate and their interactions on stem lesions and overall plant vigour was tested following GLM ANOVA using a statistical software package Minitab version 16. Treatment plot means were compared following Tukey’s test with 95% confidence. No transformations were required to meet ANOVA requirements.

4.4 Results

4.4.1 Glasshouse trial

4.4.1.1 Stem lesion development

Both L. pseudotheobromae and sub-lethal glyphosate caused stem lesions. Lesions were black to brownish in colour, necrotic and spreading in both directions from the point of inoculation with streaming of sap from the inoculation point (Fig. 4.1 A). At six weeks after inoculation, we observed significantly longer (P=0.000) external stem lesions when L. pseudotheobromae was inoculated with glyphosate co-treatment whether both applied in the same or different hole in the stem (Fig. 4.1 B).

After six weeks, the plants inoculated with L. pseudotheobromae and receiving the glyphosate co- treatment began to change stem colour from green to dark brown which interfered with lesion measurement. However, plants treated only with glyphosate or inoculated only with L. pseudotheobromae continued to display stem lesions until trial completion. At 12 weeks, the length

141 of external and internal stem lesions recorded individually with L. pseudotheobromae and glyphosate were identical but significantly longer (P=0.001) than control plants (data not presented).

(A)

(B)

a a

b c c c

Fig. 4.1 (A) External stem lesions observed with L. pseudotheobromae, sub-lethal glyphosate and their combinations at 6 weeks (Scale=1.94 cm); (B) Effect of L. pseudotheobromae, sub-lethal glyphosate and their combinations on length of stem lesion at 6 weeks under glasshouse conditions. Columns with different letters on error bars indicate significant difference by Tukey’s test with 95% confidence in GLM ANOVA (n=5). On the X axis, each capital letter represents individual treatment where A= control (autoclaved fresh millet grains), B= glyphosate 45g/L a.i @ 100 µL/plant, C= L. pseudotheobromae, D= L. pseudotheobromae + glyphosate 45g/L a.i @ 100 µL/plant (applied into same hole) and E= L. pseudotheobromae + glyphosate 45g/L a.i @ 100 µL/plant (applied into different holes).

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4.4.1.2 Leaf production and leaf mortality

At 12 weeks after inoculation, the individual effect of L. pseudotheobromae and sub-lethal glyphosate on total leaf production was insignificant compared to the control treatment (Fig. 4.2 A). Leaf production declined significantly (P=0.014) when L. pseudotheobromae was applied with the glyphosate co-treatment. However, the effect was insignificant when the glyphosate co-treatment was applied in a different hole on stem.

(A)

a a a a b b

(B)

a b

c d

d

Fig. 4.2 Effect of L. pseudotheobromae, sub-lethal glyphosate and their combinations on (A) total leaf production and (B) leaf mortality of prickly acacia at 12 weeks under glasshouse conditions. Columns with different letters on error bars indicate significant difference by Tukey’s test with 95% confidence in GLM ANOVA (n=5). On the X axis, each capital letter represents individual treatment where A= control (autoclaved fresh millet grains), B= glyphosate 45g/L a.i @ 100 µL/plant, C= L. pseudotheobromae, D= L. pseudotheobromae + glyphosate 45g/L a.i @ 100 µL/plant (applied into same hole) and E= L. pseudotheobromae + glyphosate 45g/L a.i @ 100 µL/plant (applied into different holes).

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Leaf mortality after 12 weeks was unaffected by the sub-lethal glyphosate treatment. However, it was significantly (P=0.000) increased by L. pseudotheobromae which further increased to 70-90% with the glyphosate co-treatment applied either way (Fig. 4.2 B).

4.4.1.3 Stem and branch dry weight

At 12 weeks after inoculation, the individual effect of L. pseudotheobromae and sub-lethal glyphosate treatment on stem and branch dry weight was insignificant compared to the control treatment. Stem and branch dry-weight declined significantly with glyphosate co-treatment compared to the control. (Fig. 4.3 A).

(A) a

a a

b b b c b b

(B)

a

b b

c c

Fig. 4.3 Effect of L. pseudotheobromae, sub-lethal glyphosate and their combinations on (A) stem and branch dry-weight and (B) height increment at 12 weeks. Columns with different letters on error bars indicate significant difference by Tukey’s test with 95% confidence in GLM ANOVA (n=5). On the X axis, each capital letter represents individual treatment where A= control (autoclaved fresh millet grains), B= glyphosate 45g/L a.i @ 100 µL/plant, C= L. pseudotheobromae, D= L. pseudotheobromae + glyphosate 45g/L a.i @ 100 µL/plant (applied into same hole) and E= L. pseudotheobromae + glyphosate 45g/L a.i @ 100 µL/plant (applied into different holes).

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4.4.1.4 Plant height

Plants inoculated with L. pseudotheobromae with a glyphosate co-treatment were significantly (P=0.004) shorter in height compared to those either inoculated with L. pseudotheobromae or treated with only glyphosate (Fig. 4.3 B).

4.4.1.5 Confirmation of infection

L. pseudotheobromae was successfully isolated from the infected stem tissues of inoculated plants both irrespective of glyphosate co-treatment with 100% recovery rate. It was absent in control plants and also not found to asymptomatically colonize healthy tissues in any inoculated plants tested.

4.4.2 Field trial

4.4.2.1 External stem lesion/canker development

We observed dark brown, necrotic stem lesions externally visible on green bark with streaming of sap identical to the glasshouse trial in field trial conducted at Rockhampton (Fig. 4.4). In later stages, stem lesions in all treatments turned into typical stem cankers (Fig. 4.5).

AA B BC D

Fig. 4.4 External stem lesions observed with different treatments at 6 months after inoculation in a field trial at Rockhampton, central Queensland- A. control; B. glyphosate; C. L. pseudotheobromae and D. L. pseudotheobromae + glyphosate (Scale=10 cm).

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A B C D

Fig. 4.5 Stem canker observed with different treatments at 14 months after inoculation in a field trial at Rockhampton, central Queensland- A. control; B. glyphosate; C. L. pseudotheobromae and D. L. pseudotheobromae + glyphosate (Scale=10 cm).

A A a A

a a B ab B b

C c

Fig. 4.6 Effect of L. pseudotheobromae and sub-lethal glyphosate on external stem lesion in prickly acacia recorded in field trials conducted at Rockhampton, central Queensland. Columns with different letters on error bars indicate significant difference by Tukey’s test with 95% confidence in GLM ANOVA (n=4). On the X axis, each capital letter represents individual treatment where A= control (autoclaved fresh millet grain), B= L. pseudotheobromae, C= glyphosate 180g/L a.i @ 0.5 mL/plant, D= glyphosate 180g/L a.i @ 1.0 mL/plant, E= L. pseudotheobromae + glyphosate 180g/L a.i @ 0.5 mL/plant and F= L. pseudotheobromae + glyphosate 180g/L a.i @ 1.0 mL/plant.

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At six months after inoculation, we recorded significantly longer (P=0.000) stem lesions with individual application of L. pseudotheobromae and two doses of glyphosate compared to the control treatment (Fig. 4.6). However, the glyphosate-fungus interaction effect on lesion length was insignificant compared to the individual effect of glyphosate. An almost similar pattern in canker length was recorded at 14 months (Fig. 4.6). No external stem lesion or cankers were visible on the dark brown tree bark in the field trial conducted at Richmond.

4.4.2.2 Internal stem lesion

We observed internal lesions in cross sections of the stem samples collected from trial sites at Rockhampton and Richmond. At Rockhampton, internal lesions were less prominent and mostly confined to pith region (Fig. 4.7). In contrast, internal stem lesions were more prominent at Richmond and found to spread in both pith and vascular areas in stem cross section (Fig. 4.8). Relative lesion size in Richmond was significantly higher with all treatments compared to millet control (Fig. 4.9). However, there was no significant treatment effect on relative lesion size (measured as surface area (%) in stem cross-section covered by internal lesion) at any trial site (Fig. 4.9).

Pith

Vascular area

Control (blank millet) L. pseudotheobromae 0.5 ml glyphosate

L. pseudotheobromae L. pseudotheobromae with 0.5 ml glyphosate with 1.0 ml glyphosate 1.0 ml glyphosate

Fig. 4.7 Internal lesions/staining observed with different treatments in field trials conducted at Rockhampton, central Queensland at 21 months after inoculation (Scale=1cm).

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Pith

Vascular area

Control (blank millet) L. pseudotheobromae 0.5 ml glyphosate Vascular area affected

L. pseudotheobromae with L. pseudotheobromae 1.0 ml glyphosate 0.5 ml glyphosate with 1.0 ml glyphosate

Fig. 4.8 Internal lesions/staining observed with different treatments in field trials conducted at Richmond, north-western Queensland at 23 months after inoculation (Scale=1cm).

A A

A A A

a B

a

a

Fig. 4.9 Effect of L. pseudotheobromae and sub-lethal glyphosate on internal stem lesion in stem cross-section in prickly acacia recorded in field trials conducted at Rockhampton, central Queensland and Richmond, north-western Queensland. Columns with different letters on error bars indicate significant difference by Tukey’s test with 95% confidence in GLM ANOVA (n=4). On the X axis, each capital letter represents individual treatment where A= control (autoclaved fresh millet grain), B= L. pseudotheobromae, C= glyphosate 180g/L a.i @ 0.5 mL/plant, D= glyphosate 180g/L a.i @ 1.0 mL/plant, E= L. pseudotheobromae + glyphosate 180g/L a.i @ 0.5 mL/plant and F= L. pseudotheobromae + glyphosate 180g/L a.i @ 1.0 mL/plant.

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4.4.2.3 Plant vigour

At Rockhampton, all plants included in the trial were healthy at the time of inoculation (data not presented) and no significant decline was observed in the next two phases of trial assessment at 6 and 14 months after inoculation in November 2012 and July 2013, respectively (Fig. 4.10). At 21 months after inoculation (in February 2014), we recorded decline in plant vigour (vigour rating approximately 20-50/110) with all treatments (Fig. 4.10). However, there was no significant treatment effect or any glyphosate-fungus interaction effect on plant vigour compared to the effect of the control treatment (blank millet capsule). The untreated plants neighbouring the trial site assessed 4 months later (in July 2014), were found with high vigour (vigour rating approximately 95-100/110) (data not presented).

At Richmond, all plants were healthy at the time of inoculation (data not presented) but showed some signs of cattle damage but were free from natural dieback as previously observed in the other locations in the same region. At 6 months, plant vigour was found to be significantly (P=0.022) affected by glyphosate when applied at 1.0 ml per plant (Fig. 4.11). At 14 months (July 2013), plant vigour was found to be unaffected by treatments and all plants in the trial were at high vigour (vigour rating 90-106/110) (Fig. 4.11). At 23 months after inoculation (May 2014), we recorded decline in plant vigour (vigour rating approximately 30-55/110) with all treatments (Fig. 4.11). Lowest plant vigour was recorded when L. pseudotheobromae was combined with glyphosate co- treatment applied at 1 mL per plant. However, such glyphosate-fungus interaction effect on plant vigour was insignificant compared to the individual effect of glyphosate. In this stage, the untreated plants neighbouring the trial site were also found with low vigour (vigour rating approximately 40- 55/110) which was statistically identical to the control treatment (Fig. 4.11).

4.4.2.4 Confirmation of infection

Following laboratory analysis of the stem samples collected from trial sites at Rockhampton and Richmond, we could successfully re-isolate L. pseudotheobromae with a 100% recovery rate (3 samples per treatment). When combined with the glyphosate co-treatment, L. pseudotheobromae was also isolated from the stem cross section at the glyphosate application point. The fungus was not found in un-inoculated or glyphosate only treated samples. The fungus also could not be retrieved from asymptomatic tissues of inoculated plants. Partial sequence of the Internal Transcribed Spacer (ITS) region of a set of L. pseudotheobromae isolates retrieved from the inoculated plants were found with 100% similarity to that of the original isolate of L. pseudotheobromae used in inoculation (phylogenetic tree not presented) which further confirmed the infection. 149

Fig. 4.10 Effect of L. pseudotheobromae and sub-lethal glyphosate on overall vigour of prickly acacia recorded from May 2012 to February 2014 in a field trial conducted at Rockhampton, central Queensland. Columns with different letters on error bars indicate significant difference by Tukey’s test with 95% confidence in GLM ANOVA (n=4). On the X axis, each capital letter represents individual treatment where A= control (autoclaved fresh millet grains), B= L. pseudotheobromae, C= glyphosate 180g/L a.i @ 0.5 mL/plant, D= glyphosate 180g/L a.i @ 1.0 mL/plant, E= L. pseudotheobromae + glyphosate 180g/L a.i @ 0.5 mL/plant and F= L. pseudotheobromae + glyphosate 180g/L a.i @ 1.0 mL/plant.

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a ab ab ab ab b

a a a a a a

a ab abc bcd cd bcd d

Fig. 4.11 Effect of L. pseudotheobromae and sub-lethal glyphosate on overall vigour of prickly acacia recorded from June 2012 to May 2014 in a field trial conducted at Richmond, north-western Queensland. Columns with different letters on error bars indicate significant difference by Tukey’s test with 95% confidence in GLM ANOVA (n=4). On the X axis, each capital letter represents individual treatment where A= control (autoclaved fresh millet grains), B= L. pseudotheobromae, C= glyphosate 180g/L a.i @ 0.5 mL/plant, D= glyphosate 180g/L a.i @ 1.0 mL/plant, E= L. pseudotheobromae + glyphosate 180g/L a.i @ 0.5 mL/plant, F= L. pseudotheobromae + glyphosate 180g/L a.i @ 1.0 mL/plant and G= Untreated plants.

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4.5 Discussion

Different species of potential bioherbicide fungi had been reported to cause more severe damage to a range of weed species growing in different eco-systems once combined with sub-lethal rates of chemical herbicides (Leger et al. 2001; Smith & Hallett 2006; Boyette et al. 2008; Gressel 2010). However, such findings on woody weeds, particularly those invading the rangeland eco-system had never been reported. Our study found L. pseudotheobromae, a potential bioherbicide candidate caused significantly higher levels of disease symptoms in prickly acacia when inoculated with glyphosate co-treatment under glasshouse conditions. However, under field conditions, L. pseudotheobromae caused symptoms identical to those observed under glasshouse conditions but the combinations of fungal pathogen and sub-lethal glyphosate did not lead to significantly greater disease severity.

In this study, sub-lethal levels of glyphosate were applied as a source of systemic physiological stress. However, measurement of stem lesions under glasshouse conditions clearly indicated that it caused significant localized stem lesions. L. pseudotheobromae also caused significant stem lesions in the absence of a glyphosate co-treatment as reported on other hosts in previous studies (Begoude et al. 2011; Castro-Medina et al. 2014). Similar stem lesions were observed on prickly acacia inoculated with the Botryosphaeriaceae fungi in previous glasshouse studies (Haque et al. 2012). In the initial stage of the glasshouse study, significantly longer stem lesions visible externally on the bark were recorded when L. pseudotheobromae was combined with the glyphosate co-treatment. Such findings indicated more rapid progression of infection by L. pseudotheobromae in the presence of glyphosate induced stress. In later stages of the trial, stem lesions were not clearly visible on fungus-glyphosate co-treated plants as the stem colour changed from green to dark brown. Longer stem lesions caused by the fungus in the presence of glyphosate co-treatment as observed in the initial stage of our study was not reported in previous glyphosate-fungus interaction studies. However, our results are in line with other stress-fungus interaction studies reporting longer stem lesions caused by fungi in the presence of other abiotic stressor. For example, Paoletti et al. (2001) and Niekerk et al. (2011) experimentally demonstrated that different fungi caused longer stem lesions when their inoculated hosts were subjected to drought stress.

Another objective of the glasshouse study was to understand glyphosate-fungus interaction effect on growth parameters of prickly acacia in a controlled environment. In this study, we observed reduced leaf production and greater leaf mortality when L. pseudotheobromae was inoculated with the glyphosate co-treatment which in turn significantly affected plant height. However, the combination

152 of L. pseudotheobromae and sub-lethal glyphosate did not lead to significantly lower stem and branch dry-weight. Our study did not investigate the exact cause of longer stem lesions, increased leaf mortality and the resulting effect on plant height. However, the mode of action of glyphosate indicates its ability to create nutritional and physiological stress to plants and the ability of the Botryosphaeriaceae fungi such as L. pseudotheobromae to cause more severe disease symptoms on stressed hosts is well-recognised (Slippers & Wingfield 2007; Johal & Huber 2009). On the other hand, sub-lethal glyphosate itself might cause rapid and severe damage to prickly acacia in the presence of L. pseudotheobromae. For example, Levesque & Rahe (1992) reported that a lower dose of glyphosate caused more severe damage to bean (Phaseolus sp.) seedlings in the presence of root infecting fungi.

Significant external stem lesions with both L. pseudotheobromae and sub-lethal glyphosate were observed in the field trial conducted at Rockhampton where plants were young with green bark. Similar lesions or cankers were also observed on other invasive woody weeds following inoculation with different bioherbicide fungi and their combination with other chemical herbicide under field conditions (Dorworth 1995; Rayachhetry et al. 1999). No such stem lesion was externally visible in another field trial conducted at Richmond, possibly because the plants in that site were older with dark brown stem bark which masked lesion visibility. However, larger and more prominent internal lesions were observed at Richmond compared to Rockhampton. Such difference in internal lesion size between two sites is possibly because of the difference in diversity and pathogenicity of native endophytes and other site-specific climatic factors such as amount of rainfall and temperature. Different fungi including the members of Botryosphaeriaceae were asymptomatically present in the healthy prickly acacia plants growing at both trial sites. Many of those fungi were capable to cause significant internal stem lesions in prickly acacia saplings under glasshouse conditions (Chapter 2, Section 2.4.4.4). Therefore, those native fungi could also be involved with the internal lesions observed under field conditions. During the trial period, the average temperature at Richmond was slightly higher than that recorded at Rockhampton (Appendix 2). However, the trial site at Richmond was severely drought-stressed because of significantly lower amount of rainfall (≈1000mm) during the trial period compared to that at Rockhampton (≈3000 mm) (Appendix 3). Such drought stress resulted from lower amount of precipitation was possibly implicated with larger internal lesions observed at Richmond. In a number of previous studies, it was found that different fungi including the endophytic Botryosphaeriaceae were capable to cause larger stem lesions in the presence of drought stress (Paoletti et al. 2001; Niekerk et al. 2011; Amponsah et al. 2014). L. pseudotheobromae was consistently retrieved from the necrotic stem tissues of the plants inoculated

153 with glyphosate co-treatment. However, the combinations of fungal pathogen and sub-lethal glyphosate did not result in significantly larger external or internal stem lesions in any trial site.

Such glyphosate-fungus interaction effect on lesion size was not previously reported with woody species. However, similar results were observed in cropping systems, for example, Lee et al. (2000) demonstrated that glyphosate did not affect the size of leaf lesions caused by Sclerotinia sclerotiorum in soybean (Glycine max L.). In our study, the interaction effect of L. pseudotheobromae and glyphosate on stem lesion size was possibly affected by the pre-existing native fungi such as Cophinforma spp.; Neofusicoccum parvum (Pennycook & Samuels) Crous, Slippers & A.J.L. Phillips and Fusarium acuminatum Ellis & Everh. having potential to cause significant stem lesions (Chapter 2) and their interactions with glyphosate as indicated by Sanyal and Shrestha (2008) that the activity of herbicides may extend beyond their target organisms. L. pseudotheobromae is also known to cause asymptomatic colonization following natural infection in other host(s) (Trakunyingcharoen et al. 2013). Clearly this mode of infection was not simulated in our study as the fungus could not be isolated from healthy tissues outside the necrotic areas of the deliberately inoculated plants.

Apart from investigating stem lesions and fungal colonization, the real interest of this study was to understand fungus-glyphosate interactions effect on growth, survival and ultimately mortality of prickly acacia under field conditions. In our study, no significant decline in plant vigour was observed in the first year of the trial and at nearly two years, we observed significant decline in plant vigour. The severity of vigour decline was almost identical in both trials sites at Rockhampton and Richmond although there were significant differences in climatic factors between two sites.

At Richmond, the untreated plants neighbouring the trial site were also found with low vigour at the end of the trial which was similar to the treated plants which indicates a natural decline of prickly acacia health at this site. Such decline was possibly linked with drought stress as evident from rainfall data (Appendix 3). The normal wet season monsoon also did not occur in Richmond for 2013. In previous studies, drought stress was identified as a key factor affecting tree mortality in semi-arid grassland ecosystems in Australia similar tour study site at Richmond (Fensham et al. 2005; Fensham & Fairfax 2007; Fensham et al. 2009). Natural dieback of prickly acacia was previously reported from this area (Galea 2011). In addition, the Botryosphaeriaceae fungus Cophinforma spp. found to be associated with previous natural dieback event and a number of other native fungi sourced from the healthy plants growing in this study site were found to be highly pathogenic to prickly acacia under laboratory and glasshouse conditions (Chapter 2). An interaction

154 effect of these native fungi with drought stress could be another potential reason of decline (Desprez-Loustau et al. 2006). This hypothesis was investigated in the next chapter (Chapter 5).

On the other hand, the study site at Rockhampton received significantly higher amounts of rainfall and was subjected to slightly lower temperatures compared to the Richmond site (Appendix 2 & 3). However, we also recorded significant vigour decline in that site at approximately two years after inoculation while the untreated plants neighbouring the trial assessed four months later were found with high vigour. There might be different reasons of such decline and the exact cause was not investigated in the course of this study. It might be a seasonal fluctuation of plant vigour as prickly acacia is an alternately deciduous species where some extent of defoliation occurs in dry season (Parsons & Cuthbertson 2001). Such defoliation could affect the plant vigour assessment. Decline might also be associated with inundation stress as this particular study site was reported to remain periodically flooded. A number of pathogenic fungi such as N. parvum and F. acuminatum were isolated from the root system of the healthy plants growing in this site (Chapter 2). Inundation stress and its interaction with pre-existing root infecting fungi were also previously reported to be associated with decline symptoms observed in other invasive species in Australia (Aghighi et al. 2014). However, this hypothesis was not tested within the course of this study.

Although the establishment of L. pseudotheobromae following artificial inoculation was confirmed under field conditions but the combinations of the fungal pathogen and sub-lethal glyphosate did not lead to significantly greater disease severity. Such findings under field conditions are not surprising. There was little control over the trials conducted using naturally growing plants in a rangeland eco-system. The trial was also subjected to a number of uncontrolled abiotic and biotic factors such as variation in temperature, rainfall, soil moisture, ultra-violet radiation, insect infestation and overgrazing which might have significant effect on the study-outcome (Julien 2006).

The role of water stress, high grazing pressure, infestations of sap sucking and leaf feeding insects and their interactions with native fungi could be a potential area of exploration to better understand the decline observed in field trials. Therefore, it is expected to expand the study in those areas.

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Chapter 5: Interaction between drought stress and Cophinforma sp. in prickly Acacia dieback

5.1 Abstract

The Botryosphaeriaceae fungus Cophinforma sp. was found to be associated with dieback of prickly acacia, an invasive weed of the Australian rangeland. It was hypothesized that Cophinforma could cause dieback in the presence of other potential stressors and in this study we investigated its interactions with drought stress in prickly acacia. The effect of Cophinforma sp. on lesion length, leaf mortality and growth of prickly acacia was tested across three watering regimens in a ten week trial established under glasshouse conditions using a randomized complete block design and six month old juvenile plants. The fungus caused significant stem lesions following stem inoculation with colonized millet grains and lesion length increased significantly with drought stress. Leaf mortality also increased significantly with stress and its interaction with the fungus. Although Cophinforma sp. caused larger stem lesions and increased leaf mortality in the presence of drought stress, there was no significant resulting effect on plant growth as measured by stem and branch dry-weight and height increment. Our study in some extents supports the observations in other Botryosphaeriaceae-stress interaction studies that such opportunistic fungi cause more severe disease symptoms such as longer stem lesions and higher leaf mortality on stressed host.

Key words Acacia family, Botryosphaeriaceae, prickly acacia, Cophinforma sp., drought stress and dieback.

5.2 Introduction

Dieback symptoms have been observed in several invasive plant species in Australia including mimosa (Mimosa pigra L.), parkinsonia (Parkinsonia aculeata L.), athel pine (Tamarix aphylla L. Karst) and European blackberry (Rubus fruticosus L.) (Diplock et al. 2008; Aghighi et al. 2012; Sacdalan et al. 2012). At present, dieback is the major limiting factor to the spread of parkinsonia (van Klinken et al. 2009) and there are indications that the factors which promot dieback can be potentially incorporated into the management strategy of other invasive species. A diverse group of fungi were found to be associated with dieback of different invasive weeds (Wilson and Pitkethley 1992; Diplock et al. 2006; Toh et al. 2008; Aghighi et al. 2012). However, most are expected to only be pathogenic in the presence of other stressors (Desprez-Loustau et al. 2006; Slippers and Wingfield 2007). For example, Phytophthora sp. was implicated in blackberry dieback observed in

162 south-western Australia, but only in presence of stressors such as, flooding and high grazing pressure (Aghighi et al. 2014). In most cases the exact cause of dieback in most invasive weeds remains poorly understood.

Prickly acacia (Vachellia nilotica subsp. indica (Benth.) Kyal. & Boatwr), one of the most serious weeds in the semi-arid Australian rangeland had been reported to display naturally occurring dieback symptoms since the 1970’s across many infestation sites in north-western Queensland (March 2009; Queensland Department of Employment, Economic Development and Innovation 2009). An investigation of this phenomenon was made in 2010 around the locations near Richmond and Julia Creek in north Queensland. Affected trees were found with symptoms ranging from ashy internal staining, defoliation, blackening of shoot tips, partial crown death through to widespread mortality of plant populations. A Botryosphaeriaceae fungus Cophinforma sp. was consistently isolated following laboratory analysis of dieback-affected materials. It was also found to asymptomatically colonize the healthy prickly acacia plants growing in the areas where the natural dieback was previously observed. In initial studies, the fungus was found to be pathogenic to prickly acacia causing stem lesions, gummosis and leaf mortality. However, initial findings also suggested that Cophinforma sp. was unable to cause typical dieback symptoms in the absence of other potential stressor(s).

The basic concept of the plant disease triangle implies that the host-pathogen interaction leading to disease development is influenced by the environment (Agrios 2005). Various groups of fungi have been reported to cause more severe disease symptoms on other invasive and non-invasive woody species under stressful environmental conditions (Desprez-Loustau et al. 2006; La Porta et al. 2008; Aghighi et al. 2014; Davison 2014). There are more specific indications regarding the disease symptoms caused by the Botryosphaeriaceae fungi in many woody hosts which have been reported to be closely associated with other potential stressors, such as wind, extreme temperatures and drought (Smith et al. 1994; Ma et al. 2001; Slippers and Wingfield 2007; Niekerk et al. 2011; Amponsah et al. 2014). More specifically, the previous studies by Pusey (Pusey 1989), Ma et al. (2001), Stanosz et al. (2001), Luque et al. (2002), Niekerk et al. (2011) and Amponsah et al. (2014) identified drought stress as a key factor affecting the disease progress caused by various Botryosphaeriaceae fungi.

Drought is conceptually defined as a prolonged period of dryness resulting in extensive damage to plants (Wilhite and Glantz 1985). It is an insidious hazard of nature. Drought is also an important stressor in Australia where it has historically been responsible for wide-spread native tree death in the semi-arid regions (Fensham et al. 2005, Fensham et al. 2009). This also includes in semi-arid 163 ecosystems in north-western Queensland (Allen et al. 2010). However, its interaction with native fungi on dieback or mortality is still partially explored (Desprez-Loustau et al. 2006), and an area of further interest. Therefore, we investigated the interactions between drought stress and Cophinforma sp., a Botryosphaeriaceae fungus associated with prickly acacia dieback.

Severity and impacts of drought vary from region to region depending on a number of factors such as the amount of precipitation, temperature, rate of evaporation, soil properties, density and type of vegetations etc (Mishra and Singh 2010). In addition, the effect of drought on many ecosystems may depend on the amount of water stored in the soil profile and the plants’ ability to extract this water (Lambers et al. 2008). Therefore, in this study, different level of soil water potential (Ψ) was utilized as an index of availability of soil moisture to the test-plants.

5.3 Materials and Methods

5.3.1 Experimental design and treatments

The effect of Cophinforma sp. on stem lesion length, leaf mortality and growth parameters of prickly acacia was tested across three drought stress regimes; no stress (soil Ψ maintained at 0 KPa), moderate stress (soil Ψ maintained at -30 to -40 KPa) and high stress (soil Ψ dropped to -50 to -60 KPa followed by watering to 0 KPa). Soil water potential (Ψ) was measured using tensiometers (Irrometer, USA) installed in the pots throughout the trial period. The 10-week experiment was conducted following a randomized complete block design with two blocks, each containing five separate plots, with six plants per plot. Treatments were randomly allocated within each plot which contained a complete set of treatments. Plants were six months old at the start of the trial (average height was 116.68 ± 4.68 cm; average stem diameter at 10 cm above the soil level was 11.56 ± 1.38 mm), and were randomly allocated to treatments and replicates. Prickly acacia seeds were collected from a single dieback-free plant growing at Richmond, north-western Queensland. Plants were grown from surface sterilized seeds in 4.5L pots each containing 4.25 L of autoclaved media (silty clay field soil : sand = 3 L : 1 L) with automatic drip irrigation under glasshouse conditions at The University of Queensland, Gatton. Each plant was treated fortnightly with 250 ml of 0.16% Aquasol (N: P: K =23: 3.95: 14 + trace elements). No further fertilizer was applied once the trial was established.

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5.3.2 Inoculation

An isolate of Cophinforma sp. (AN71) extracted from a dieback-affected prickly acacia stem sample collected in 2010 from Lindfield Station near Julia Creek, north Queensland was used in this experiment. Inocula were prepared following the techniques outlined by Toh et al. (2008) using millet (Panicum miliaceum L.) grains. At 10 cm above the soil level, an inoculation point was marked with a pencil and bark around that point was surface sterilized by swabbing with 70% ethanol. Using an alcohol sterilized 3.5 mm drill bit mounted on a cordless drill, a 5-6 mm deep, angular inoculation hole was made in the stem followed by placing two colonized millet grains using sterilized forceps in the wound which was covered with Parafilm®. Fresh (un-inoculated) autoclaved millet grains were used as control.

5.3.3 Trial assessment

The effect of drought stress and Cophinforma sp. on lesion morphology was assessed visually on longitudinally split stems. Length of external and internal stem lesions was measured using a ruler. An aluminium foil tray with a drainage hole was placed under each plant to collect fallen leaves throughout the trial. Dead leaves attached to the plants were hand removed at the end of the trial. After removing the attached dead leaves, the plants with their healthy leaves were cut into small pieces and put in labelled aluminium trays and left to dry in the glasshouse for 72 hours. Partially dried healthy leaves were hand-removed by scraping. Leaf dry-weights were obtained following oven drying at 60-65oC for one week. Leaf mortality was expressed as percentage of dead leaf (fallen plus attached dead leaves) to total leaf production. Dry-weight of stem and branch material was also obtained following oven drying at 60-65oC for two weeks.

5.3.4 Confirmation of infection

Re-isolations were made from the upper margin of the internal lesion and from adjacent apparently healthy tissues of the inoculated plants to i) demonstrate the presence of Cophinforma sp. in fulfilment of Koch’s postulates and to ii) investigate whether this fungus could asymptomatically colonize healthy tissue as an endophyte. Isolation was also conducted from the control plants. This was conducted from two plants per treatments per block following the techniques modified from Diplock et al. (2006). Stem segments were surface sterilized by soaking in 2% NaOCl for two minutes followed by rinsing in sterile water twice for five minutes and drying on sterile blotting paper within a laminar air-flow cabinet. Partially dried samples were aseptically transferred to ½

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PDA media followed by incubation at 25oC in a dark incubator. The plates were observed daily and after one week. Any fungal isolates were purified by subculturing on fresh ½ PDA plates before comparing microscopically with the original isolate used in the experiment.

5.3.5 Data analysis

Interaction effects of drought stress and fungal inoculation on external and internal stem lesion length, total leaf production, leaf mortality, stem and branch dry weight and height increment was tested following GLM ANOVA using a statistical software package Minitab version 16. Since each of the 10 plots (five plots in each of two blocks) represented a complete replication, we considered that the blocks and plots within each block had fixed effect on the above parameters. No transformations were required to meet ANOVA requirements.

5.4 Results

5.4.1 Stem lesions

Cophinforma sp. caused significant stem lesions visible externally on the bark and internally on pith and vascular tissues. Lesions were black to brownish in colour, necrotic and spreading in both directions from the point of inoculation (Fig. 5.1). Internal stem lesions were longer than external lesions. At 10 weeks after inoculation, the length of internal lesions was found to be significantly affected by Cophinforma sp. (P<0.01) and its interaction with drought stress (P<0.01). Longer internal lesions were recorded when the inoculated plants were subjected to drought stress (Fig. 5.2). However, both stress levels showed an identical effect on internal lesion length. Short internal lesions were also recorded in all control plants and found to be unaffected by drought stress. A similar pattern was observed for external stem lesions.

5.4.2 Leaf production and leaf mortality

At 10 weeks after inoculation, total leaf production was found to be significantly reduced by drought stress (P<0.01) (Fig. 5.3 A). However, the effect of Cophinforma sp. and its interaction with drought stress on total leaf production was insignificant (P>0.05). Leaf mortality was unaffected by Cophinforma sp. in the absence of drought stress (Fig. 5.3 B). However, drought stress and its interaction with the fungus had a significant (P<0.01) effect on leaf mortality. A

166 higher level of leaf mortality was recorded when the inoculated plants were subjected to moderate drought stress, and it increased significantly at the highest stress level.

5.4.3 Plant growth

Stem and branch dry-weight was significantly reduced by drought stress (Fig. 5.4 A). It was unaffected by Cophinforma sp. in the absence of stress or in presence of the highest level of stress. However, there was significant stress-fungus interaction effect on stem and branch dry-weight when the plants were subjected to a moderate stress level. Plant height (measured as height increment at 10 weeks) was significantly affected by drought stress (Fig. 5.4 B). Both inoculated and control plants in stress regimens were shorter compared to those in non-stress regimen. However, there was no significant effect of the fungus and its interaction with drought stress on plant height.

Control Cophinforma sp. Cophinforma sp. Cophinforma sp.

Stressed/Non-stressed Non-stressed Moderate stress High stress

Fig. 5.1 External (top row) and internal (bottom row) stem lesions on prickly acacia observed with Cophinforma sp. under different levels of drought stress (Scale=1.94 cm). Stem lesions in control plants were similar in both stress and non-stress regimens. 167

10 External Stem Lesion (cm) Internal Stem Lesion (cm)

A A 8

6 a a B B

4 b

Lesion of Stem (cm) Length

2 C C C a c 0 c c Control Cophinforma Control Cophinforma Control CophinformaCophinforma sp.sp. sp. sp.sp.

Non-stressed Moderate stress High stress

Fig. 5.2 Effect of Cophinforma sp. and drought stress on length of external and internal stem lesion in prickly acacia. Columns with different letters are significantly different from each other by Tukey’s test in GLM ANOVA (n=10).

5.4.4 Confirmation of infection

Cophinforma sp. was successfully isolated from the tip of internal stem lesions observed on the inoculated plants in each stress regimens with 100% recovery rate. Cophinforma sp. was absent in uninoculated plants, and also not found to asymptomatically colonize the healthy tissues adjacent the tip of internal stem lesions in inoculated plants irrespective of stress regime. Alternaria sp. and Cladosporium sp. were found to be associated with short stem lesions observed on the control plants. Such stem lesions on the control plants were mostly internal (almost absent on the bark) and were extremely restricted in length.

168

(A)

a a

b

bc bc c

Cophinforma Cophinforma Cophinforma sp. sp. sp. Non- stressed Moderate stress High stress

(B)

a

b

c d

e e

Cophinforma Cophinforma Cophinforma sp. sp. sp. Non-stressed Moderate stress High stress

Fig. 5.3 Effect of Cophinforma sp. and drought stress on (A) total leaf production and (B) leaf mortality of prickly acacia. Columns with different letters are significantly different from each other by Tukey’s test in GLM ANOVA (n=10).

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(A)

Cophinforma Cophinforma Cophinforma

sp. sp. sp.

Non-stressed Moderate stress High stress

(B) a

ab

bc bc bc

c

Cophinforma Cophinforma Cophinforma sp. sp. sp.

Non-stressed Moderate stress High stress

Fig. 5.4 Effect of Cophinforma sp. and drought stress on (A) stem and branch dry-weight and (B) plant height (measured as height increment over 10 weeks) of prickly acacia. Columns with different letters are significantly different from each other by Tukey’s test in GLM ANOVA (n=10).

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5.5 Discussion

Different species of Botryosphaeriaceae have been reported to cause more severe disease symptoms in the presence of drought stress, and the findings were generally explained through reduced growth and disrupted physiological status of hosts leading to impaired host defence against the pathogen (Schoeneweiss 1986; Ragazzi et al. 1999; Ma et al. 2001; Luque et al. 2002; Desprez-Loustau et al. 2006; Niekerk et al. 2011). This study found that Cophinforma sp. caused significantly longer stem lesions and higher degree of leaf mortality in prickly acacia in the presence of drought stress. However, there was little or no effect of such pathogen-stress interaction on plant growth. Reduced plant growth observed in this study was largely an effect of drought stress.

Similarly, many previous studies have focused on point-source infection and lesion or canker length. For example, studies by Ragazzi et al. (1999); Paoletti et al. (2001) and Niekerk et al. (2011) found that different Botryosphaeriaceae fungi caused significant localized stem lesions around the inoculation point in the absence of other stressor. However, those studies also reported significantly larger stem lesions or cankers in the presence of drought stress. In this study, measurement of stem lesion across the irrigation regimens clearly indicated the ability of Cophinforma sp. to cause to cause significant stem lesions in the absence of stress. Similar stem lesions were also observed on prickly acacia following inoculation with Botryosphaeriaceae fungi under field conditions (Haque et al. 2013). As expected from the studies on other Botryosphaeriaceae fungi, length of stem lesion increased significantly with drought stress (Madar et al. 1989; Ragazzi et al. 1999; Niekerk et al. 2011). This study did not investigate the exact cause of longer stem lesions by Cophinforma sp. in the presence of water stress. However, increased defoliation as a result of water stress possibly contributed to the host weakening process by limiting carbohydrate availability leading to a higher degree of fungal colonization (Old et al. 1990; Capretti & Battisti 2007; Mehl et al. 2013; Sprague et al. 2013).

Poor host defence to fungal invasion could be another reason for longer stem lesions under drought stress. A number of previous studies have experimentally demonstrated such drought-induced predisposition of woody hosts to infection by opportunistic fungi leading to more severe diseases symptoms including dieback and canker. For example, studies by Schoeneweiss (1986) and Madar et al. (1995) found that drought stress reduced the amount of phytoalexin production in different perennial plants which subsequently predisposed the affected plants to fungal infection and resulted in larger canker or more severe dieback.

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The real interest of this study was to understand stress-fungus interactions in terms of growth and survival of prickly acacia. In this study, significant leaf mortality with Cophinforma sp. in the presence of drought stress was observed which is in line with the findings of Ma et al. (2001). However, higher leaf mortality by Cophinforma sp. in the presence of stress had little or no effect on overall plant growth and survival. Such findings indicate compensatory growth by the plants. However, a significant effect of Cophinforma sp. on growth and survival of the plants under stress regimens might be evident if trials run for a longer period to overcome the existing storage resources present in these juvenile plants.

Cophinforma has recently been identified as a distinct genus in the Botryosphaeriaceae family and there is little information available on its epidemiological features and mode of infection (Liu et al. 2012). It has been isolated from both symptomatic and asymptomatic tissues of other tree hosts in Asia, Africa and South America (Liu et al. 2012; Phillips et al. 2013). However, the asymptomatic mode of infection was not simulated in this glasshouse trial as the fungus could not be isolated from healthy tissues outside the necrotic areas.

The potential role of other fungi associated with prickly acacia dieback and their interaction with other stressors such as defoliating, stem boring and sap sucking insects would be worthy of further study. Therefore, we aim to expand our study to understand the role other fungi and stressors in prickly acacia dieback. It would also be interesting to investigate whether systemic infection could be achieved using less invasive inoculation techniques.

5.6 References

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Liu, J-K, Phookamsak, R, Doilom, M, Wikee, S, Li, Y-M, Ariyawansha, H, Boonmee, S, Chomnunti, P, Dai, D-Q, Bhat, JD, Romero, AI, Zhuang, W-Y, Monkai, J, Jones, EBG, Chukeatirote, E, Ko, TWK, Zhao, Y-C, Wang, Y & Hyde, KD 2012, 'Towards a natural classification of Botryosphaeriales', Fungal Diversity, vol. 57, no. 1, pp. 149-210.

Luque, J, Parlade, J & Pera, J 2002, 'Seasonal changes in susceptibility of to Botryosphaeria stevensii and Phytophthora cinnamomi', Plant Pathology, vol. 51, no. 3, pp. 338-45.

Ma, ZH, Morgan, DP & Michailides, TJ 2001, 'Effects of water stress on botryosphaeria blight of pistachio caused by Botryosphaeria dothidea', Plant Disease, vol. 85, no. 7, pp. 745-9.

Madar, Z, Solel, Z & Kimchi, M 1989, 'Effect of water-stress in cypress on the development of cankers caused by Diplodia pinea f.sp. cupressi and Seiridium cardinale', Plant Disease, vol. 73, no. 6, pp. 484-6.

Madar, Z, Solel, Z, Riov, J & Sztejnberg, A 1995, 'Phytoalexin production by cypress in response to infection by Diplodia pinea f.sp. cupressi and its relation to water-stress', Physiological and Molecular Plant Pathology, vol. 47, no. 1, pp. 29-38.

March, N 2009, Prickly acacia national strategic plan progress review (2008–2009), National Prickle Bush Management Group.

Mehl, JWM, Slippers, B, Roux, J & Wingfield, MJ 2013, 'Cankers and other diseases caused by the Botryosphaeriaceae', in P Gonthier & G Nicolotti (eds), Infectious forest diseases. pp. 298-317.

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Old, KM, Gibbs, R, Craig, I, Myers, BJ & Yuan, ZQ 1990, 'Effect of drought and defoliation on the susceptibility of eucalypts to cankers caused by Endothia gyrosa and Botryosphaeria ribis', Australian Journal of Botany, vol. 38, no. 6, pp. 571-81.

Paoletti, E, Danti, R & Strati, S 2001, 'Pre and post-inoculation water stress affects Sphaeropsis sapinea canker length in Pinus halepensis seedlings', Forest Pathology, vol. 31, no. 4, pp. 209-18.

Phillips, AJL, Alves, A, Abdollahzadeh, J, Slippers, B, Wingfield, MJ, Groenewald, JZ & Crous, PW 2013, 'The Botryosphaeriaceae: genera and species known from culture', Studies in Mycology, no. 76, pp. 51-167.

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Chapter 6: General Discussion

6.1 Background

The findings presented in this thesis are based on the first-time investigation of the dieback symptoms observed on prickly acacia (Vachellia nilotica subsp. indica (Benth.) Kyal. & Boatwr) in north-western Queensland. A number of previous studies suggested the potential association of fungi with dieback of other invasive woody weeds in Australia (Diplock et al. 2006; Toh et al. 2008; Sacdalan et al. 2012; Aghighi et al. 2014). In addition, there are indications that fungi associated with dieback could be potentially incorporated into the bio-control strategy of such invasive weeds (Toh et al. 2008; Galea & Goulter 2013). Therefore, the study set out to investigate the potential association of fungi with the dieback symptoms. The study has also sought to determine whether the fungi associated with dieback could cause infection resulting in similar symptoms following deliberate inoculation and, therefore, be utilized in biological control of this invasive weed species. If so, then it might suggest novel ways of managing this species including many other invasive weeds. Our findings suggested that the fungi associated with prickly acacia dieback are probably opportunistic in nature and only cause severe disease symptoms once the host is stressed by other biotic or abiotic factors as has been seen with dieback of other species (Slippers & Wingfield 2007; Niekerk et al. 2011; Amponsah et al. 2014). Therefore, investigation of stress- fungus interactions in terms of prickly acacia dieback was considered as an important objective of this study.

6.2 Major findings

The detailed findings of this study are chapter specific and were discussed within the respective research chapters (Chapter 2-5). In this section, the major findings of this study are synthesized, discussed and supported by relevant literature.

The general theoretical literature on dieback of woody trees and shrubs indicate that it has a complex aetiology with potential association of a range of biotic and abiotic factors (Chapter 1). Among the biotic factors, fungi were often reported to be associated with dieback and mortality of a range perennial plant species (Botella et al. 2010; Hernandez-Hernandez et al. 2010; Linaldeddu et al. 2010; Hand et al. 2014). The cause of dieback symptoms observed on prickly acacia since the 1970’s in north-western Queensland have not been previously investigated. A few industry reports,

177 however, suggested the potential association of fungal infection and other stressors with this phenomenon (March 2009; Queensland Department of Agriculture Fisheries and Forestry 2014).

The present study clearly demonstrated that a range of fungi including stem and branch canker pathogens, vascular wilt fungi and foliar pathogens were associated with prickly acacia dieback observed in 2010 around Richmond and Julia Creek in north-western Queensland (Chapter 2). Of the diverse assemblage of fungi associated with this phenomenon, the members of the Botryosphaeriaceae family were found to be most prevalent. The Botryosphaeriaceae are known to cause dieback and cankers in a wide range of agricultural and forest trees and shrubs across the globe (Perez et al. 2010; Mehl et al. 2013). In Australia, this group of fungi were found to be associated with dieback of several woody trees, shrubs and herbs such as mango (Mangifera indica L.), Acacia spp., Eucalyptus spp., peppermint (Mentha spicata L.) and grapevine (Vitis vinifera L.) (Dakin et al. 2010; Sakalidis et al. 2011; Amponsah et al. 2014; Trakunyingcharoen et al. 2014). They were also reported to be implicated with dieback of other rangeland weeds in Australia such as mimosa (Mimosa pigra) and parkinsonia (Parkinsonia aculeata L.) (Wilson & Pitkethley 1992; Diplock et al. 2006; Sacdalan et al. 2012).

Among the members of Botryosphaeriaceae associated with prickly acacia dieback, Cophinforma sp. was the most frequently encountered fungus. Cophinforma was never previously reported in Australia. Other members of Botryosphaeriaceae associated with this phenomenon were identified as Aureobasidium sp., Phaeobotryosphaeria citrigena A.J.L. Phillips, P.R. Johnst. & Pennycook and Pseudofusicoccum violaceum Mehl & Slippers. Of these, Cophinforma spp. was also frequently isolated from stem and root samples of healthy stands of prickly acacia in north-western Queensland. Another Botryosphaeriaceae fungus, Neofusicoccum parvum (Pennycook & Samuels) Crous, Slippers & A.J.L. Phillips was frequently isolated from root samples of healthy prickly acacia plants at Rockhampton, central Queensland where natural dieback symptoms had not been previously observed. Recovery of Botryosphaeriaceae fungi including Cophinforma spp. from both dieback-affected and apparently healthy stands of prickly acacia indicates that these were probably endophytes or latent pathogens. As found in this study, a number of Botryosphaeriaceae fungi such as different species of Botryosphaeria, Diplodia, Dothidotthia, Guignardia, Lasiodiplodia, Neofusicoccum and Pseudofusicoccum had previously been recovered from perennial woody hosts and reported as endophytes or latent pathogens (Petrini & Fisher 1988; Johnson et al. 1992; Crous et al. 2006; Slippers & Wingfield 2007). In addition to Botryosphaeriaceae, a number of root- infecting wilt pathogens (such as Fusarium acuminatum Ellis & Everh.) and leaf spot pathogens (such as different species of Alternaria) were also recovered from apparently healthy prickly acacia

178 plants. Many of these fungi were also previously reported as being endophytes (Teleha & Miller 2007; Pan et al. 2010; Porras-Alfaro et al. 2014; Swett & Gordon 2015).

The presence of different Botryosphaeriaceae fungi including Cophinforma spp. in the root systems of apparently healthy prickly acacia clearly indicated their capacity to colonise the plant with the possibility of causing natural root infections. They were also previously reported to cause root infection in other hosts and are suspected of horizontal transmission through intermingling root systems although such a mode of infection has not yet been experimentally proven (Whitelaw- Weckert et al. 2006; Mehl et al. 2013). Similarly, there is no experimental evidence of systemic above-ground infection by the Botryosphaeriaceae fungi originating from natural root infections. Moreover, it is possible that root infections leading to root dysfunction may be expressed as death (dieback) of above ground plant parts such as branches as observed in Acacia cyclops A. Cunn. ex G. Don infected by the Basidiomycete Pseudolagarobasidium acaciicola Ginns sp. nov. (Wood and Ginns, 2006).

In the glasshouse pathogenicity trials, Botryosphaeriaceae fungi such as Cophinforma sp. and Lasiodiplodia pseudotheobromae Phillips, Alves & Crous consistently caused localized infections resulting in disease symptoms such as stem lesions, gummosis and leaf mortality in initially healthy juvenile plants. Similar symptoms were also observed on other hosts following inoculation of different Botryosphaeriaceae fungi under controlled environment (Piskur et al. 2011; Amponsah et al. 2014). However, in this study, the typical dieback symptoms such as widespread plant mortality and/or severe decline in growth and vigour were not clearly evident with fungal inoculations in the absence of other stressor during the course of the glasshouse trials (8-12 weeks). Such symptoms might develop in trials allowed to continue beyond 8-12 weeks duration. Nonetheless, it is now well-established that the Botryosphaeriaceae fungi are opportunistic pathogens that usually cause significant disease symptoms in the presence of other stressors, such as wind (high velocity can cause physical injury to plants), extreme temperatures and drought (Smith et al. 1994; Slippers & Wingfield 2007; Niekerk et al. 2011; Mehl et al. 2013; Amponsah et al. 2014). Such fungi are known to cause rapid dieback over vast areas in presence of widespread stressors (Slippers & Wingfield 2007). In addition to Botryosphaeriaceae, other groups of fungi were also reported to cause dieback symptoms on a range of invasive and non-invasive woody hosts under stressful environmental conditions (Desprez-Loustau et al. 2006; La Porta et al. 2008; Aghighi et al. 2014; Davison 2014).

In the stress-fungus interaction trial under glasshouse conditions, L. pseudotheobromae caused significantly higher levels of disease symptoms such as longer stem lesions and higher levels of leaf 179 mortality while reducing plant growth when inoculated with sub-lethal glyphosate co-treatment as a source of physiological stress. In another glasshouse trial, drought stress resulted in significant reduction in plant growth whereas Cophinforma sp. caused more severe disease symptoms such as larger stem lesions and increased leaf mortality on stressed plants. Drought stress was previously reported to be associated with dieback observed in Australian semi-arid similar to the prickly acacia dieback sites in north-western Queensland (Fensham & Holman 1999; Fensham et al. 2005; Fensham & Fairfax 2007; Fensham et al. 2009). In addition, a number of previous studies identified drought stress as a key factor accelerating the disease progress caused by various Botryosphaeriaceae fungi (Pusey 1989; Ma et al. 2001; Stanosz et al. 2001; Luque et al. 2002; Niekerk et al. 2011; Amponsah et al. 2014). Our studies indicated that such interaction might also be involved in the natural dieback symptoms observed on prickly acacia.

In the glasshouse trials, the actively growing juvenile plants grew extensively within 8-12 weeks after inoculation. Following that period, collection of dropped leaves was also hampered by plant growth as the lateral branches extended beyond the capture trays placed beneath each plant resulting in data loss. Therefore, it was not practical to continue the glasshouse trials beyound 8-12 weeks and longer-term trials were conducted under field conditions. Disease symptoms such as necrotic stem lesions and gummosis similar to those observed under glasshouse conditions were also observed in the inoculation trials conducted under field conditions (Chapter 3-4). The successful completion of Koch’s postulates with the symptomatic stem samples clearly indicated that the inoculated Botryosphaeriaceae fungi were the cause of such symptoms. Furthermore, systemic infection beyond the necrotic area of stem lesions could not be confirmed under field conditions. The observation of such point-source infections was also previously reported with other Botryosphaeriaceae fungi in different woody hosts under field conditions (Diplock et al. 2006; Mohali et al. 2009). However, such localized stem lesions were never observed in the natural event of prickly acacia dieback. Typical dieback symptoms such as widespread branch mortality and defoliation leading to significant vigour decline were observed in the final stage of the field trials. However, a similar degree of decline was also observed with the control (blank millet capsule) treatment and/or on the untreated plants outside the trial (Chapter 3-4). Such decline was possibly implicated with other stressors such as water stress, insect infestation, grazing pressure and their potential interactions with different pre-existing Botryosphaeriaceae and other fungi (Desprez- Loustau et al. 2006; Slippers & Wingfield 2007; Stursova et al. 2014).

Endophytic Botryosphaeriaceae fungi are known to cause asymptomatic colonization possibly as an outcome of systemic infection in plant tissues (Johnson et al. 1992; Slippers & Wingfield 2007;

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Mehl et al. 2013). Such systemic infection has significant implications in the field of weed bio- control. It allows the potential bio-herbicide fungi to move systemically through the target weed from the point of inoculation to the distant parts to cause widespread vertical infection. However, none of the Botryosphaeriaceae fungi isolated from either dieback-affected or healthy plants and tested in this study caused systemic infection following deliberate inoculation in prickly acacia under glasshouse or field conditions within the timeframe of these studies. The fungi caused localized infections and could not be isolated from healthy tissues outside the necrotic areas of stem lesions in the inoculated plants. Such a mode of infection was unaffected by the presence of other stressors such as drought or herbicide stress. However, there is not enough scientific evidence supporting the systemic mode of infection by the other endophytic fungi. A very recent study by Yan et al. (2015) reported that endophytic fungi rarely grow systemically within plants; rather they produce different metabolites having systemic effects on plants and other microbial plant inhabitants.

Even so, if systemic infection is an attribute of the Botryosphaeriaceae endophytes then the failure to observe it in prickly acacia following artificial inoculation could be due to a number of possible reasons. For example, the inoculation technique used in this study was comprised of stem wounding which caused injury to the plants. Previous studies have demonstrated the formation of physical barriers such as necrophylactic periderm layer and/or lignified impervious tissue in woody plants in response to such mechanical wounding (Hawkins & Boudet 1996; Oven et al. 1999; Robinson et al. 2004; Cleary et al. 2012; Kovalchuk et al. 2013). Such physical barriers can significantly confine the advancement of fungal infection in different woody hosts (Robinson & Morrison 2001; Robinson et al. 2004). In addition, there are indications that activation and accumulation of different chemical barriers such as phytoalexins and pathogenesis-related proteins during host- pathogen interactions can inhibit fungal spread (Barry et al. 2006; Sturrock et al. 2007; Freeman & Beattie 2008; Kovalchuk et al. 2013). Secretions of sap or resin which may also restrict fungal spread (Kovalchuk et al. 2013) were also observed in this study following stem wounding and inoculation with different Botryosphaeriaceae fungi under glasshouse and field conditions (Haque et al. 2012).

The method of inoculation used in this study to some extent simulated the natural mode of infection of the Botryosphaeriaceae fungi as they are known to naturally infect healthy hosts through wounds or natural openings (Amponsah et al. 2010; Mehl et al. 2013). Similar inoculation methods based on artificial wounding were also used in previous studies with different Botryosphaeriaceae fungi (Mullen et al. 1991; Mohali et al. 2009; Begoude et al. 2011; Niekerk et al. 2011; Amponsah et al.

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2014). In addition, similar fungi resulted in significant dieback symptoms in parkinsonia under similar eco-climatic conditions when delivered following the same techniques used in this study (Galea & Goulter 2013). However, none of those studies reported whether systemic infection resulting in significant vertical spread could be achieved with Botryosphaeriaceae fungi following such inoculation methods. This study repeatedly confirmed that systemic infection could not be achieved with Botryosphaeriaceae fungi in prickly acacia following artificial stem wounding. A number of endophytic fungi known to cause asymptomatic infections in their hosts may also switch between endophytic and pathogenic modes, depending on the host and environmental conditions (Stergiopoulos & Gordon 2014). Therefore, it might be possible to demonstrate asymptomatic colonization with the similar fungi in other hosts or under different environmental conditions using similar inoculation techniques.

6.3 Conclusion and recommendation for future research

This study has answered some of the basic questions about the potential association of fungi and stressors with prickly acacia dieback. However, the key success of this investigation was the experimental demonstration that the fungi associated with dieback were capable of causing infection (although localized) resulting in significant disease symptoms following deliberate inoculation under glasshouse and field conditions. Confirmation of infection in the target weed is generally considered as the first line of success in any bioherbicide research project. Therefore, scope does exist to undertake further investigations in this area. It would be worthwhile to investigate whether the systemic mode of infection resulting in widespread vertical dissemination could be deliberately achieved with the Botryosphaeriaceae fungi following other non-destructive inocula delivery techniques such as foliar spray with spore suspensions. It would also be valuable to investigate whether such potential bioherbicide fungi could cause root infection following artificial inoculation leading to systemic vertical infection resulting in dieback symptoms. Horizontal transmission across intermingled root systems would be another important area for future exploration.

There is a great deal of controversy regarding the taxonomy of Botryosphaeriaceae fungi, and their accurate speciation generally requires sequencing of multiple gene regions including the Internal Transcribed Spacer (ITS) region (Crous et al. 2006; Liu et al. 2012; Phillips et al. 2013). However, fungal taxonomy was not the main focus of this project, and because of time and funding constraints, the identification of a large number of Botryosphaeriaceae fungal isolates used in this study was based only on ITS sequencing. Therefore, it would be interesting to sequence other gene regions such as Transcription Elongation Factor (TEF) and Beta Tubulin (β-Tubulin) to ensure 182 more precise identification because it is highly likely that previously undescribed fungal species have been isolated.

The research has made an important contribution to controlling a significant environmental weed of Northern Australia and that could be used in biocontrol approaches for other weeds. It is evident from this study that the Botryosphaeriaceae fungi such as L. pseudotheobromae and Cophinforma sp. have the potential to cause more severe disease symptoms in presence of other stressors (such as drought and herbicide stress). Therefore, these fungi may be utilized as potential biocontrol agents for prickly acacia and other similar woody weeds growing in a stressful habitat. These fungal biocontrol agents may also be combined with stress inducing insects to formulate an integrated management strategy for prickly acacia.

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Appendices

Appendix 1 List of the fungal isolates identified and tested using a seedling bioassay under laboratory conditions.

Species Isolate code* Host Sample Locality Most Similar GenBank Pathogenicity in Seedling Accession No. (ITS) Bio-assay Al. alternata AN122 V. nilotica subsp. indica Stem Richmond, NW. Qld. FJ228163.1 Weak or non-pathogenic Al. alternata ANE46 V. nilotica subsp. indica Stem Richmond, NW. Qld. FJ228163.1 Moderately pathogenic Alternaria sp. ANE39 V. nilotica subsp. indica Stem Richmond, NW. Qld. KJ598870.1 Moderately pathogenic Alternaria sp. ANE41 V. nilotica subsp. indica Stem Richmond, NW. Qld. KJ598870.1 Moderately pathogenic Alternaria sp. ANE50 V. nilotica subsp. indica Stem Richmond, NW. Qld. KJ598870.1 Moderately pathogenic Alternaria sp. ANE52 V. nilotica subsp. indica Root Richmond, NW. Qld. KJ598870.1 Moderately pathogenic Alternaria sp. ANE54 V. nilotica subsp. indica Root Richmond, NW. Qld. KJ598870.1 Moderately pathogenic Alternaria sp. ANE58 V. nilotica subsp. indica Root Rockhampton, C. Qld. KJ598870.1 Moderately pathogenic As. pistaciarum AN12 V. nilotica subsp. indica Stem Richmond, NW. Qld. FR681905.1 Weak or non-pathogenic As. pistaciarum AN14 V. nilotica subsp. indica Stem Richmond, NW. Qld. FR681905.1 Weak or non-pathogenic As. pistaciarum AN15 V. nilotica subsp. indica Stem Richmond, NW. Qld. FR681905.1 Weak or non-pathogenic Au. pullulans AN3 V. nilotica subsp. indica Stem Richmond, NW. Qld. KC253968.1 Moderately pathogenic Au. pullulans AN128 V. nilotica subsp. indica Stem Richmond, NW. Qld. KC253968.1 Weak or non-pathogenic Au. pullulans AN129 V. nilotica subsp. indica Stem Richmond, NW. Qld. KC253968.1 Weak or non-pathogenic Au. pullulans AN130 V. nilotica subsp. indica Stem Richmond, NW. Qld. KC253968.1 Weak or non-pathogenic Au. pullulans AN137 V. nilotica subsp. indica Stem Julia Creek, NW. Qld. KC253968.1 Weak or non-pathogenic Au. pullulans AN143 V. nilotica subsp. indica Stem Julia Creek, NW. Qld. KC253968.1 Weak or non-pathogenic Au. pullulans ANE18 V. nilotica subsp. indica Stem Richmond, NW. Qld. KC253968.1 Weak or non-pathogenic Cophinforma sp. AN1 V. nilotica subsp. indica Stem Richmond, NW. Qld. JX646801.1 Moderately pathogenic

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Cophinforma sp. AN2 V. nilotica subsp. indica Stem Richmond, NW. Qld. JX646801.1 Weak or non-pathogenic Cophinforma sp. AN4 V. nilotica subsp. indica Stem Richmond, NW. Qld. JX646801.1 Moderately pathogenic Cophinforma sp. AN5 V. nilotica subsp. indica Stem Richmond, NW. Qld. JX646801.1 Moderately pathogenic Cophinforma sp. AN6 V. nilotica subsp. indica Stem Richmond, NW. Qld. JX646801.1 Highly pathogenic Cophinforma sp. AN7 V. nilotica subsp. indica Stem Richmond, NW. Qld. JX646801.1 Weak or non-pathogenic Cophinforma sp. AN8 V. nilotica subsp. indica Stem Richmond, NW. Qld. JX646801.1 Highly pathogenic Cophinforma sp. AN9 V. nilotica subsp. indica Stem Richmond, NW. Qld. JX646801.1 Weak or non-pathogenic Cophinforma sp. AN10 V. nilotica subsp. indica Stem Richmond, NW. Qld. JX646801.1 Highly pathogenic Cophinforma sp. AN11 V. nilotica subsp. indica Stem Richmond, NW. Qld. JX646801.1 Highly pathogenic Cophinforma sp. AN16 V. nilotica subsp. indica Stem Richmond, NW. Qld. JX646801.1 Highly pathogenic Cophinforma sp. AN17 V. nilotica subsp. indica Stem Richmond, NW. Qld. JX646801.1 Highly pathogenic Cophinforma sp. AN18 V. nilotica subsp. indica Stem Richmond, NW. Qld. JX646801.1 Highly pathogenic Cophinforma sp. AN19 V. nilotica subsp. indica Stem Richmond, NW. Qld. JX646801.1 Highly pathogenic Cophinforma sp. AN20 V. nilotica subsp. indica Stem Richmond, NW. Qld. JX646801.1 Highly pathogenic Cophinforma sp. AN21 V. nilotica subsp. indica Stem Richmond, NW. Qld. JX646801.1 Highly pathogenic Cophinforma sp. AN22 V. nilotica subsp. indica Stem Richmond, NW. Qld. JX646801.1 Moderately pathogenic Cophinforma sp. AN23 V. nilotica subsp. indica Stem Richmond, NW. Qld. JX646801.1 Highly pathogenic Cophinforma sp. AN24 V. nilotica subsp. indica Stem Richmond, NW. Qld. JX646801.1 Highly pathogenic Cophinforma sp. AN25 V. nilotica subsp. indica Stem Richmond, NW. Qld. JX646801.1 Highly pathogenic Cophinforma sp. AN26 V. nilotica subsp. indica Stem Richmond, NW. Qld. JX646801.1 Highly pathogenic Cophinforma sp. AN27 V. nilotica subsp. indica Stem Richmond, NW. Qld. JX646801.1 Moderately pathogenic Cophinforma sp. AN29 V. nilotica subsp. indica Stem Richmond, NW. Qld. JX646801.1 Moderately pathogenic Cophinforma sp. AN30 V. nilotica subsp. indica Stem Richmond, NW. Qld. JX646801.1 Highly pathogenic Cophinforma sp. AN32 V. nilotica subsp. indica Stem Richmond, NW. Qld. JX646801.1 Highly pathogenic Cophinforma sp. AN33 V. nilotica subsp. indica Stem Richmond, NW. Qld. JX646801.1 Moderately pathogenic Cophinforma sp. AN35 V. nilotica subsp. indica Stem Richmond, NW. Qld. JX646801.1 Highly pathogenic

191

Cophinforma sp. AN36 V. nilotica subsp. indica Stem Richmond, NW. Qld. JX646801.1 Moderately pathogenic Cophinforma sp. AN37 V. nilotica subsp. indica Stem Julia Creek, NW. Qld. JX646801.1 Highly pathogenic Cophinforma sp. AN38 V. nilotica subsp. indica Stem Julia Creek, NW. Qld. JX646801.1 Highly pathogenic Cophinforma sp. AN39 V. nilotica subsp. indica Stem Julia Creek, NW. Qld. JX646801.1 Highly pathogenic Cophinforma sp. AN40 V. nilotica subsp. indica Stem Julia Creek, NW. Qld. JX646801.1 Highly pathogenic Cophinforma sp. AN41 V. nilotica subsp. indica Stem Julia Creek, NW. Qld. JX646801.1 Highly pathogenic Cophinforma sp. AN42 V. nilotica subsp. indica Stem Julia Creek, NW. Qld. JX646801.1 Very highly pathogenic Cophinforma sp. AN43 V. nilotica subsp. indica Stem Julia Creek, NW. Qld. JX646801.1 Highly pathogenic Cophinforma sp. AN44 V. nilotica subsp. indica Stem Julia Creek, NW. Qld. JX646801.1 Very highly pathogenic Cophinforma sp. AN45 V. nilotica subsp. indica Stem Julia Creek, NW. Qld. JX646801.1 Highly pathogenic Cophinforma sp. AN46 V. nilotica subsp. indica Stem Julia Creek, NW. Qld. JX646801.1 Highly pathogenic Cophinforma sp. AN49 V. nilotica subsp. indica Stem Julia Creek, NW. Qld. JX646801.1 Highly pathogenic Cophinforma sp. AN50 V. nilotica subsp. indica Stem Julia Creek, NW. Qld. JX646801.1 Highly pathogenic Cophinforma sp. AN53 V. nilotica subsp. indica Stem Julia Creek, NW. Qld. JX646801.1 Highly pathogenic Cophinforma sp. AN64 V. nilotica subsp. indica Stem Julia Creek, NW. Qld. JX646801.1 Highly pathogenic Cophinforma sp. AN65 V. nilotica subsp. indica Stem Julia Creek, NW. Qld. JX646801.1 Highly pathogenic Cophinforma sp. AN67 V. nilotica subsp. indica Stem Julia Creek, NW. Qld. JX646801.1 Highly pathogenic Cophinforma sp. AN68 V. nilotica subsp. indica Stem Julia Creek, NW. Qld. JX646801.1 Highly pathogenic Cophinforma sp. AN69 V. nilotica subsp. indica Stem Julia Creek, NW. Qld. JX646801.1 Highly pathogenic Cophinforma sp. AN70 V. nilotica subsp. indica Stem Julia Creek, NW. Qld. JX646801.1 Highly pathogenic Cophinforma sp. AN71 V. nilotica subsp. indica Stem Julia Creek, NW. Qld. JX646801.1 Very highly pathogenic Cophinforma sp. AN72 V. nilotica subsp. indica Stem Julia Creek, NW. Qld. JX646801.1 Highly pathogenic Cophinforma sp. AN75 V. nilotica subsp. indica Stem Julia Creek, NW. Qld. JX646801.1 Highly pathogenic Cophinforma sp. AN76 V. nilotica subsp. indica Stem Julia Creek, NW. Qld. JX646801.1 Highly pathogenic Cophinforma sp. AN77 V. nilotica subsp. indica Stem Julia Creek, NW. Qld. JX646801.1 Highly pathogenic Cophinforma sp. AN78 V. nilotica subsp. indica Stem Julia Creek, NW. Qld. JX646801.1 Highly pathogenic

192

Cophinforma sp. AN79 V. nilotica subsp. indica Stem Julia Creek, NW. Qld. JX646801.1 Highly pathogenic Cophinforma sp. AN80 V. nilotica subsp. indica Stem Julia Creek, NW. Qld. JX646801.1 Highly pathogenic Cophinforma sp. AN81 V. nilotica subsp. indica Stem Julia Creek, NW. Qld. JX646801.1 Highly pathogenic Cophinforma sp. AN82 V. nilotica subsp. indica Stem Julia Creek, NW. Qld. JX646801.1 Highly pathogenic Cophinforma sp. AN83 V. nilotica subsp. indica Stem Julia Creek, NW. Qld. JX646801.1 Highly pathogenic Cophinforma sp. AN84 V. nilotica subsp. indica Stem Julia Creek, NW. Qld. JX646801.1 Highly pathogenic Cophinforma sp. AN85 V. nilotica subsp. indica Stem Julia Creek, NW. Qld. JX646801.1 Highly pathogenic Cophinforma sp. AN86 V. nilotica subsp. indica Stem Julia Creek, NW. Qld. JX646801.1 Highly pathogenic Cophinforma sp. AN87 V. nilotica subsp. indica Stem Julia Creek, NW. Qld. JX646801.1 Highly pathogenic Cophinforma sp. AN88 V. nilotica subsp. indica Stem Julia Creek, NW. Qld. JX646801.1 Highly pathogenic Cophinforma sp. AN89 V. nilotica subsp. indica Stem Julia Creek, NW. Qld. JX646801.1 Moderately pathogenic Cophinforma sp. AN90 V. nilotica subsp. indica Stem Julia Creek, NW. Qld. JX646801.1 Moderately pathogenic Cophinforma sp. AN91 V. nilotica subsp. indica Stem Julia Creek, NW. Qld. JX646801.1 Highly pathogenic Cophinforma sp. AN92 V. nilotica subsp. indica Stem Julia Creek, NW. Qld. JX646801.1 Highly pathogenic Cophinforma sp. AN93 V. nilotica subsp. indica Stem Julia Creek, NW. Qld. JX646801.1 Highly pathogenic Cophinforma sp. AN94 V. nilotica subsp. indica Stem Julia Creek, NW. Qld. JX646801.1 Highly pathogenic Cophinforma sp. AN95 V. nilotica subsp. indica Stem Julia Creek, NW. Qld. JX646801.1 Highly pathogenic Cophinforma sp. AN96 V. nilotica subsp. indica Stem Julia Creek, NW. Qld. JX646801.1 Highly pathogenic Cophinforma sp. AN97 V. nilotica subsp. indica Stem Julia Creek, NW. Qld. JX646801.1 Highly pathogenic Cophinforma sp. AN98 V. nilotica subsp. indica Stem Julia Creek, NW. Qld. JX646801.1 Highly pathogenic Cophinforma sp. AN99 V. nilotica subsp. indica Stem Julia Creek, NW. Qld. JX646801.1 Highly pathogenic Cophinforma sp. AN100 V. nilotica subsp. indica Stem Julia Creek, NW. Qld. JX646801.1 Moderately pathogenic Cophinforma sp. AN102 V. nilotica subsp. indica Stem Julia Creek, NW. Qld. JX646801.1 Highly pathogenic Cophinforma sp. AN103 V. nilotica subsp. indica Stem Julia Creek, NW. Qld. JX646801.1 Highly pathogenic Cophinforma sp. AN104 V. nilotica subsp. indica Stem Julia Creek, NW. Qld. JX646801.1 Highly pathogenic Cophinforma sp. AN105 V. nilotica subsp. indica Stem Julia Creek, NW. Qld. JX646801.1 Highly pathogenic

193

Cophinforma sp. AN106 V. nilotica subsp. indica Stem Julia Creek, NW. Qld. JX646801.1 Highly pathogenic Cophinforma sp. AN107 V. nilotica subsp. indica Stem Julia Creek, NW. Qld. JX646801.1 Moderately pathogenic Cophinforma sp. AN108 V. nilotica subsp. indica Stem Julia Creek, NW. Qld. JX646801.1 Highly pathogenic Cophinforma sp. AN109 V. nilotica subsp. indica Stem Julia Creek, NW. Qld. JX646801.1 Very highly pathogenic Cophinforma sp. AN110 V. nilotica subsp. indica Stem Julia Creek, NW. Qld. JX646801.1 Highly pathogenic Cophinforma sp. AN111 V. nilotica subsp. indica Stem Julia Creek, NW. Qld. JX646801.1 Moderately pathogenic Cophinforma sp. AN112 V. nilotica subsp. indica Stem Julia Creek, NW. Qld. JX646801.1 Highly pathogenic Cophinforma sp. AN113 V. nilotica subsp. indica Stem Julia Creek, NW. Qld. JX646801.1 Highly pathogenic Cophinforma sp. AN114 V. nilotica subsp. indica Stem Julia Creek, NW. Qld. JX646801.1 Highly pathogenic Cophinforma sp. AN115 V. nilotica subsp. indica Stem Julia Creek, NW. Qld. JX646801.1 Moderately pathogenic Cophinforma sp. AN116 V. nilotica subsp. indica Stem Julia Creek, NW. Qld. JX646801.1 Highly pathogenic Cophinforma sp. AN117 V. nilotica subsp. indica Stem Julia Creek, NW. Qld. JX646801.1 Highly pathogenic Cophinforma sp. AN119 V. nilotica subsp. indica Stem Julia Creek, NW. Qld. JX646801.1 Highly pathogenic Cophinforma sp. AN138 V. nilotica subsp. indica Stem Julia Creek, NW. Qld. JX646801.1 Highly pathogenic Cophinforma sp. MP290 M. pigra Stem Darwin, NT. JX646801.1 Very highly pathogenic Cophinforma sp. ANE1 V. nilotica subsp. indica Root Richmond, NW. Qld. JX646801.1 Weak or non-pathogenic Cophinforma sp. ANE8 V. nilotica subsp. indica Root Richmond, NW. Qld. JX646801.1 Weak or non-pathogenic Cophinforma sp. ANE25 V. nilotica subsp. indica Root Richmond, NW. Qld. JX646801.1 Highly pathogenic Cophinforma sp. ANE26 V. nilotica subsp. indica Root Richmond, NW. Qld. JX646801.1 Highly pathogenic Cophinforma sp. ANE27 V. nilotica subsp. indica Root Richmond, NW. Qld. JX646801.1 Highly pathogenic Cophinforma sp. ANE28 V. nilotica subsp. indica Root Richmond, NW. Qld. JX646801.1 Highly pathogenic Cophinforma sp. ANE32 V. nilotica subsp. indica Stem Richmond, NW. Qld. JX646801.1 Moderately pathogenic Cophinforma sp. ANE36 V. nilotica subsp. indica Stem Richmond, NW. Qld. JX646801.1 Highly pathogenic Cl. uredinicola AN63 V. nilotica subsp. indica Stem Julia Creek, NW. Qld. KM513616.1 Weak or non-pathogenic Cl. uredinicola ANE20 V. nilotica subsp. indica Root Rockhampton, C. Qld. KM513616.1 Weak or non-pathogenic Cu. lunata AN118 V. nilotica subsp. indica Stem Julia Creek, NW. Qld. DQ836799.1 Weak or non-pathogenic

194

Cu. pseudorobusta AN124 V. nilotica subsp. indica Stem Richmond, NW. Qld. AB453879.1 Weak or non-pathogenic Cy. austromontana AN145 V. nilotica subsp. indica Stem Julia Creek, NW. Qld. JN693510.1 Moderately pathogenic E. rostratum AN120 V. nilotica subsp. indica Stem Julia Creek, NW. Qld. JN711431.1 Highly pathogenic F. acuminatum ANE16 V. nilotica subsp. indica Root Rockhampton, C. Qld. KC477845.1 Very highly pathogenic L. pseudotheobromae NT039 P. aculeata Stem N. Territory KM006458.1 Very highly pathogenic M. alpina ANE4 V. nilotica subsp. indica Root Richmond, NW. Qld. GU319989.1 Highly pathogenic M. alpina ANE5 V. nilotica subsp. indica Root Richmond, NW. Qld. GU319989.1 Moderately pathogenic M. alpina ANE6 V. nilotica subsp. indica Root Richmond, NW. Qld. GU319989.1 Moderately pathogenic M. alpina ANE7 V. nilotica subsp. indica Root Richmond, NW. Qld. GU319989.1 Highly pathogenic N. parvum ANE30 V. nilotica subsp. indica Stem Rockhampton, C. Qld. JN662928.1 Very highly pathogenic N. parvum ANE31 V. nilotica subsp. indica Root Rockhampton, C. Qld. JN662928.1 Very highly pathogenic Paecilomyces sp. AN123 V. nilotica subsp. indica Stem Richmond, NW. Qld. AJ243771.1 Weak or non-pathogenic Paecilomyces sp. AN125 V. nilotica subsp. indica Stem Richmond, NW. Qld. AJ243771.1 Weak or non-pathogenic Paecilomyces sp. AN127 V. nilotica subsp. indica Stem Richmond, NW. Qld. AJ243771.1 Weak or non-pathogenic Paecilomyces sp. AN131 V. nilotica subsp. indica Stem Richmond, NW. Qld. AJ243771.1 Weak or non-pathogenic Paecilomyces sp. AN139 V. nilotica subsp. indica Stem Julia Creek, NW. Qld. AJ243771.1 Weak or non-pathogenic Paecilomyces sp. AN140 V. nilotica subsp. indica Stem Julia Creek, NW. Qld. AJ243771.1 Weak or non-pathogenic Paecilomyces sp. ANE21 V. nilotica subsp. indica Root Rockhampton, C. Qld. AJ243771.1 Weak or non-pathogenic Paecilomyces sp. AN31 V. nilotica subsp. indica Stem Julia Creek, NW. Qld. KC157764.1 Weak or non-pathogenic Par. angularis ANE15 V. nilotica subsp. indica Root Rockhampton, C. Qld. JX496047.1 Moderately pathogenic Ph. citrigena AN146 V. nilotica subsp. indica Stem Julia Creek, NW. Qld. EU673329.1 Weak or non-pathogenic Ph. citrigena AN147 V. nilotica subsp. indica Stem Julia Creek, NW. Qld. EU673329.1 Weak or non-pathogenic Ph. citrigena AN148 V. nilotica subsp. indica Stem Julia Creek, NW. Qld. EU673329.1 Weak or non-pathogenic Ph. citrigena AN149 V. nilotica subsp. indica Stem Julia Creek, NW. Qld. EU673329.1 Weak or non-pathogenic Pl. ootheca AN62 V. nilotica subsp. indica Stem Julia Creek, NW. Qld. AY725469.1 Weak or non-pathogenic Pl. ootheca AN135 V. nilotica subsp. indica Stem Julia Creek, NW. Qld. AY725469.1 Weak or non-pathogenic

195

Pl. ootheca AN136 V. nilotica subsp. indica Stem Julia Creek, NW. Qld. AY725469.1 Weak or non-pathogenic Ps. violaceum AN48 V. nilotica subsp. indica Stem Julia Creek, NW. Qld. FJ888474.1 Highly pathogenic Ps. violaceum AN51 V. nilotica subsp. indica Stem Julia Creek, NW. Qld. FJ888474.1 Highly pathogenic Ps. violaceum AN52 V. nilotica subsp. indica Stem Julia Creek, NW. Qld. FJ888474.1 Highly pathogenic Ps. violaceum AN54 V. nilotica subsp. indica Stem Julia Creek, NW. Qld. FJ888474.1 Highly pathogenic Ps. violaceum AN55 V. nilotica subsp. indica Stem Julia Creek, NW. Qld. FJ888474.1 Moderately Pathogenic Ps. violaceum AN56 V. nilotica subsp. indica Stem Julia Creek, NW. Qld. FJ888474.1 Highly pathogenic Ps. violaceum AN57 V. nilotica subsp. indica Stem Julia Creek, NW. Qld. FJ888474.1 Highly pathogenic Ps. violaceum AN58 V. nilotica subsp. indica Stem Julia Creek, NW. Qld. FJ888474.1 Moderately Pathogenic Ps. violaceum AN61 V. nilotica subsp. indica Stem Julia Creek, NW. Qld. FJ888474.1 Highly pathogenic Py. microspora AN144 V. nilotica subsp. indica Stem Julia Creek, NW. Qld. HM751085.1 Highly pathogenic R. rufulum AN101 V. nilotica subsp. indica Stem Julia Creek, NW. Qld. KJ787018.1 Weak or non-pathogenic R. rufulum AN141 V. nilotica subsp. indica Stem Richmond, NW. Qld. KJ787018.1 Weak or non-pathogenic T. graminis ANE24 V. nilotica subsp. indica Root Richmond, NW. Qld. KC769962.1 Highly pathogenic T. graminis ANE29 V. nilotica subsp. indica Root Rockhampton, C. Qld. KC769962.1 Highly pathogenic U. muehlenbergii AN142 V. nilotica subsp. indica Stem Richmond, NW. Qld. JQ036216.1 Weak or non-pathogenic Z. acaciae ANE13 V. nilotica subsp. indica Stem Rockhampton, C. Qld. KJ869149.1 Weak or non-pathogenic Z. acaciae ANE33 V. nilotica subsp. indica Stem Richmond, NW. Qld. KJ869149.1 Weak or non-pathogenic Z. acaciae ANE34 V. nilotica subsp. indica Stem Richmond, NW. Qld. KJ869149.1 Weak or non-pathogenic

* Isolates in bold were extracted from apparently healthy prickly acacia

196

Appendix 2 Average temperature recorded near the trial sites at Rockhampton and Richmond over the trial period (Source: Australian Bureau of Meteorology).

Average Temperature (°C) at Rockhampton Average Temperature (°C) at Richmond 35

30 C)

° 25 20 15 10 ( Temperature 5 0

Average Temperature (°C) at Rockhampton Average Temperature (°C) at Richmond

35

30

C) ° 25 20

15

Temperature ( Temperature 10 5 0

Average Temperature (°C) at Rockhampton Average Temperature (°C) at Richmond

35

C) 30 ° 25 20 15

( Temperature 10 5

0

197

Appendix 3 Rainfall (mm) received by the trial sites at Rockhampton and Richmond from 6 months before inoculation through the trial period (Source: Australian Bureau of Meteorology).

198