Biological control of grapevine trunk diseases using bacterial endophytes from grapevines
Jennifer Millera Niem BSc Agriculture (Plant Pathology) University of the Philippines Los Baños, Philippines MSc Plant Pathology Washington State University, USA
A thesis submitted in fulfilment of the requirements for the degree of
Doctor of Philosophy
National Wine and Grape Industry Centre School of Agricultural and Wine Sciences Faculty of Science Charles Sturt University April 2020
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Table of Contents
Table of contents...... 3
List of figures ...... 8
List of tables ...... 11
Certificate of Authorship ...... 12
Acknowledgements...... 13
Publications arising from the research ...... 16
Author contribution...... 17
Abstract ...... 19
List of abbreviations ...... 23
Chapter 1: Introduction and literature review ...... 24
1.1. Importance of grapevine trunk diseases… ...... 24
1.2. Symptoms and causal pathogens… ...... 25
1.3. Current management practices ...... 31
1.3.1. Chemical control ...... 31
1.3.2. Cultural methods…………………………………………………………………..33
1.3.3. Biological control agents………………………………………………………….35
1.4. Grapevine endophytes ...... 38
1.5. Potential of Pseudomonas spp. as biocontrol agents ...... 39
1.5.1. Mechanisms for biocontrol activity exhibited by Pseudomonas spp ...... 40
1.6. Summary and project aims ...... 44
1.7. Research objectives ...... 45
3 Chapter 2: Journal paper ...... 47
Niem, J., Billones-Baaijens, R., Savocchia, S., Stodart, B. (2020). Diversity profiling of grapevine microbial endosphere and antagonistic potential of endophytic Pseudomonas against grapevine trunk diseases. Front. Microbiol. 11:477. doi:
10.3389/fmicb.2020.00477.
Chapter 3: Biocontrol potential of endophytic Pseudomonas against Neofusicoccum luteum, a fungal pathogen causing Botryosphaeria dieback, and its persistence in grapevine tissues ...... 67
3.1. Introduction ...... 67
3.2. Materials and Methods ...... 69
3.2.1. Plant materials ...... 69
3.2.2. Bacterial strains, culture conditions, inoculum preparation ...... 69
3.2.3. Pathogenicity tests of Pseudomonas poae BCA17 strain ...... 70
3.2.4. Suppression of Neofusicoccum luteum infection in planta
by strains of Pseudomonas ...... 71
3.2.4.1 Fungal isolates ...... 71
3.2.4.2 Challenge inoculations using detached canes ...... 72
3.2.4.3 Challenge inoculations using potted grapevines ...... 73
3.2.4.3.1 Assessment of N. luteum infections by isolations ...... 74
3.2.4.3.2 Assessment of N. luteum infections by quantitative PCR ...... 75
3.2.5 Pseudomonas colonisation and establishment in grapevine tissues ...... 76
3.2.5.1 Development of rifampicin-resistant strain ...... 76
3.2.5.2 Pre-planting application of RifMut strain ...... 77
3.2.5.3 Post-planting application of RifMut strain ...... 79
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3.2.6 Data analysis… ...... 80
3.3. Results… ...... 81
3.3.1. Pathogenicity tests on grapevine tissue ...... 81
3.3.2. Suppression of N. luteum infection in planta by Pseudomonas strains ...... 83
3.3.2.1. Challenge inoculation using detached canes ...... 83
3.3.2.2. Challenge inoculation using potted grapevine plants… ...... 84
3.3.2.2.1. Assessment of N. luteum infection by pathogen isolation ...... 84
3.3.2.2.2. Assessment of N. luteum infections by qPCR ...... 85
3.3.3. Pseudomonas colonisation and establishment in grapevine tissues… ...... 86
3.3.3.1. Pre-planting application of Pseudomonas RifMut strain… ...... 86
3.3.3.2. Post-planting application of Pseudomonas ...... 87
3.4. Discussion ...... 88
Chapter 4: Elucidating the mechanism of action of Pseudomonas in the control of grapevine trunk disease pathogens ...... 94
4.1. Introduction ...... 94
4.2. Materials and Methods ...... 98
4.2.1 Bacterial strains...... 98
4.2.2 Fungal isolates ...... 98
4 2 3. Elucidation of antagonistic mechanisms of
Pseudomonas poae BCA17 against GTD pathogens…...... …..99
4.2.3.1. Siderophore production ...... 99
4.2.3.2. Bioactivity of the Pseudomonas poae culture filtrate on
mycelial growth and spore germination of GTD pathogens… ...... 99
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4.2.3.2.1 Preparation of culture filtrates ...... 99
4.2.3.2.2 Effect of culture filtrates on mycelial growth ...... 100
4.2.3.2.3 Effect of culture filtrates on spore germination ...... 101
4.2.3.3 Test for production of bioactive volatile compounds… ...... 102
4.2.3.4 Test for production of the antibiotics 2,4 diacetylphloroglucinol
and phenazine thru thin layer chromatography (TLC) ...... 102
4.2.3.5 Identification of biocontrol genes… ...... 104
4.2.3.6 Detection of bioactive lipopeptides… ...... 104
4.2.3.7 Statistical analysis… ...... 105
4.3 Results… ...... 105
4.3.1 Test for siderophore production ...... 105
4.3.2 Bioactivity of the Pseudomonas poae culture filtrate on mycelial growth and
spore germination of GTD pathogens… ...... 106
4.3.2.1 Effect of culture filtrate on mycelial growth ...... 106
4.3.2.2 Effect culture filtrate on spore germination ...... 107
4.3.3 Test for production of bioactive volatile compounds… ...... 109
4.3.4 Test for production of the antibiotics 2,4 diacetylphloroglucinol (DAPG) and
phenazine………………………………………………………………………... 109
4.3.5 Identification of biocontrol genes… ...... 110
4.3.6 Detection of bioactive lipopeptides ...... 114
4.4 Discussion ...... 117
Chapter 5: General discussion...... 126
References ...... 136
Appendix A: Other publication arising from this research ...... 170
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Appendix B: Presentations and award ...... 174
B.1. Oral presentations ...... 174
B.2. Posters… ...... 174
B. 3. Award… ...... 174
Appendix C: Media preparation ...... 175
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List of figures
Chapter 1
Figure 1.1: Symptoms of Esca………………...... 26
Figure 1.2: Symptoms of Eutypa dieback… ...... 28
Figure 1.3: Symptoms of Botryosphaeria dieback of grapevines ...... 30
Chapter 2
Figure 2.1: Diversity analysis of bacterial endophytic communities sampled from GTD symptomatic and asymptomatic vines, in the Hilltops and Hunter Valley regions ...... 53
Figure 2.2: Genera of grapevine endophytic bacteria from Hilltops, NSW and their relative abundance to grapevines that were: (A) asymptomatic; and (B) symptomatic to grapevine trunk diseases ...... 54
Figure 2.3: Genera of grapevine endophytic bacteria from the Hunter Valley, NSW and their relative abundance to grapevines that were: (A) asymptomatic; and (B) symptomatic to grapevine trunk diseases ...... 55
Figure 2.4: Diversity analysis of fungal endophytic communities sampled from GTD symptomatic and asymptomatic vines, in the Hilltops and Hunter Valley regions………...... 56
Figure 2.5: Genera of grapevine endophytic fungi from Hilltops, NSW and their relative abundance to grapevines that were: (A) asymptomatic; and (B) symptomatic to grapevine trunk diseases ...... 57
Figure 2.6: Genera of grapevine endophytic fungi from the Hunter Valley, NSW and their relative abundance to grapevines that were: (A) asymptomatic; and (B) symptomatic to grapevine trunk diseases ...... 58
Figure 2.7: Mycelial growth inhibition of representative GTD pathogens by antagonistic Pseudomonas BCA strains ...... 59
Figure 2.8: Antagonistic effect of Pseudomonas BCA strains on Esca/Petri disease pathogens ...... 61
Figure 2.9: Antagonistic effect of selected Pseudomonas BCA strains on Phaeomoniella chlamydospora and Phaeoacremonium minimum ...... 61
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Figure 2.10: Neighbor joining tree obtained from the 16S rRNA gene sequence data of fluorescent bacteria isolated from inner grapevine tissues ...... 62
Chapter 3
Figure 3.1: Dormant grapevine canes (cv Shiraz) dipped in: A) ¼ strength Ringer’s solution; and B) RifMut bacterial suspension for 24 h prior to rooting ...... 78
Figure 3.2: Inoculation of the Pseudomonas poae BCA17 RifMut strain in potted vines ...... 79
Figure 3.3: Schematic diagram of a vine inoculated with the RifMut strain showing the inoculation point, the positions and sizes of the tissue samples collected for re-isolations ...... 80
Figure 3.4: Pathogenicity test of Pseudomonas poae strain BCA 17 on grapevine tissues: A) berries; B) leaves, and C) canes… ...... 82
Figure 3.5: Recovery of the pathogen, Neofusicoccum luteum, from detached canes treated with different strains of Pseudomonas ...... 83
Figure 3.6: Recovery of the pathogen, Neofusicoccum luteum from glasshouse potted vines treated with Pseudomonas poae strains BCA13, BCA14 and BCA17 prior to pathogen inoculation ...... 84
Figure 3.7: Number of Botryosphaeriaceae β-tubulin gene copies detected by quantitative PCR analysis of DNA extracted from potted vines treated with BCA strains prior to Neofusicoccum luteum inoculation ...... 85
Figure 3.8: Recovery of the rifampicin-resistant mutant (RifMut) strain of Pseudomonas poae BCA17 from dormant canes dipped in bacterial suspensions for 24 h and rooted for 8 weeks…...... 87
Figure 3.9: Re-isolation of rifampicin-resistant mutant (RifMut) strain of Pseudomonas poae BCA17 from inoculated potted vines at three different sampling periods… ...... 88
Chapter 4
Figure 4.1: Antagonistic Pseudomonas spp. in Chrome Azurol S (CAS) medium showing change in colour of the medium from blue to orange indicating production of siderophores (A), and Chrome Azurol S medium in the absence of siderophore-forming Pseudomonas (B)… ...... 106
9 Figure 4.2: Effect of culture filtrates derived from Pseudomonas poae BCA17 on mycelial growth of a) Diplodia seriata; b) Neofusicoccum parvum; c) N. luteum; and d) Eutypa lata ...... 107
Figure 4.3: Effect of culture filtrates derived from Pseudomonas poae BCA17 on spore germination of: a) Diplodia seriata; b) Neofusicoccum parvum; c) N. luteum; and d) Eutypa lata ...... 108
Figure 4.4: Mycelial growth of Diplodia seriata after exposure to P. poae BCA17 volatiles… ...... 109
Figure 4.5: Thin layer chromatography profile of organic compounds extracted from Pseudomonas poae BCA17 filtrate visualised by 254 nm UV light ...... 110
Figure 4.6: MALDI-TOF spectrum for the antagonistic Pseudomonas poae BCA17 ...... 115
Figure 4.7: MALDI-TOF spectrum for the non-antagonistic Pseudomonas poae JMN1 indicating the absence of the lipopeptide at 2096.297 m/z (1+ ion)… ...... 116
Figure 4.8: Fragmentation pattern (MS/MS) of lipopeptide from P. poae BCA17 sample ...... 116
10 List of tables
Chapter 2
Table 1: Primers and PCR conditions for 16S and ITS amplicons ...... 51
Table 2: Fluorescent bacterial strains with antagonistic activity against GTD pathogens Diplodia seriata, Neofusicoccum parvum, N. luteum, and Eutypa lata ...... 51
Table 3: Fungal strains used for in vitro assays to test the antagonistic activity of Pseudomonas strains ...... 51
Table 4: Mycelial growth inhibition of Botryosphaeria dieback pathogens by Pseudomonas strains as potential biocontrol agents (BCAs) ...... 59
Table 5: Mycelial growth inhibition of Eutypa dieback pathogens by Pseudomonas strains as potential biocontrol agents (BCAs) ...... 60
Table 6: Mycelial growth inhibition of Esca/Petri disease pathogens by Pseudomonas strains as potential biocontrol agents (BCAs) ...... 60
Chapter 4
Table 4.1: Secondary metabolites of P. poae BCA strains 13, 14, 17, and JMN1 and their corresponding biosynthetic gene clusters… ...... 111
11 Certificate of Authorship
“I hereby declare that this submission is my own work and to the best of my knowledge and belief, understand that it contains no material previously published or written by another person, nor material which to a substantial extent has been accepted for the award of any other degree or diploma at Charles Sturt University or any other educational institution, except where due acknowledgement is made in the thesis. Any contribution made to the research by colleagues with whom I have worked at Charles Sturt University or elsewhere during my candidature is fully acknowledged.
I agree that this thesis be accessible for the purpose of study and research in accordance with normal conditions established by the Executive Director, Library Services, Charles Sturt University or nominee, for the care, loan and reproduction of thesis, subject to confidentiality provisions as approved by the University.” Signed:
Name: Jennifer Millera Niem
Signature:
Date: April 2020
12 Acknowledgements
I would like to share this piece of work with the following people who have either directly contributed for the culmination of this degree or inspired me to plough through this academic journey. This thesis becomes a reality with the unwavering support from these individuals.
I am forever indebted to my principal supervisor, Associate Professor Sandra
Savocchia for accepting me in her project and giving me the opportunity to pursue a PhD degree at CSU. Thank you for your expert and valuable direction as I navigate my way through this somewhat circuitous PhD journey and for always making time for me no matter how busy you are. It is a genuine pleasure to be under the supervision of Dr. Ben
Stodart, whose ingenuity has always been a valuable contribution to my supervisory team.
Without his sound advice and generous guidance, this dissertation would not have come to this wonderful fruition. I am extremely grateful to Dr. Reggie Baaijens, who is not only a mentor, but also a friend who genuinely cares. Thank you for your unceasing support and for pushing me further than I thought I could go. Your conscientious guidance and meticulous ways often drove me at my wit’s end but it effectively kept me on track and made the completion of this study possible.
I would like to acknowledge the financial support I received from Charles Sturt
University for the Postgraduate Research Scholarship and International Tuition Program,
Wine Australia for the top-up funding, and the National Wine and Grape Industry Centre
(NWGIC) through the centre director Prof. Leigh Schmidtke for the conference/travel fund.
My gratitude also goes to the UPLB Museum of Natural History through its director, Dr.
13 Juan Carlos T. Gonzalez and to UPLB Chancellor, Dr. Fernando C. Sanchez, for allowing me to go on study leave to pursue graduate studies in Australia and for again accommodating my request to extend my leave. Special thanks also to my favourite mentor in Plant
Patholology, Dr. Marina P. Natural for always being supportive of all my academic endeavors and generously recommending me, along with Dr. Edna Y. Ardales, in my pursuit of graduate studies and also to Dr. Lina L. Ilag who gave me my first break in plant pathology research.
I am grateful to all those I have had the pleasure to meet and work with for the 3.5 years duration of my PhD. To Helen Pan, thank you for the friendship and for providing me personal and professional assistance in research and life in general. My gratitude also goes to the ever reliable Mei Liu for her innumerable help in the lab; Aude Gourieroux for helping me out in processing my plant samples and for the chit chat that made the long hours in the plant processing lab bearable; and Robyn Harrington, for the valuable technical support and for her help in office concerns. My sincere appreciation goes to
Pierluigi Reveglia for his generous advice with the chemistry aspect of my work and for the countless favours; Sashika Yalage for our shared fandom of Korean drama which kept us sane during this arduous PhD; Dilhani Perera, my office bosom buddy, for the good and the bad times that we shared for 3.5 years; and to Jessica Rabelo Amaral, who never failed to show her support and to cheer me on, for the friendship that even the distance cannot part. I am immensely thankful to Stella Antony, whom I have only known a short time, yet she generously opened her home to me when I needed a temporary abode even if she barely knew me.
To these friends whom I have the good luck of crossing paths with, thank you for
14 being my second family away from home. My heartfelt thanks to Cathy Wu and PG
Reveglia, my naughty but awesome roommates for making my sojourn in Oz surprisingly
fun and interesting. I will always look back to those Friday movie nights and international
cuisine Sunday special with fond memories. My gratitude also goes to my Pinoy friends,
Ate Ivy Zia, Michelle Navato-Galam, and Jhoana Opeña for bringing in the Filipino
camaraderie that I miss from home. To my UPPS brod and sis, Kuya Dante and Ate Encar
Adorada, for taking me under your wing and ‘adopting’ me as your eldest child in my first
few months in Wagga.
Nobody and nothing have been more important to me than my family. They have always been my bulwark and their unparalleled love and steadfast support empowered me to finish this journey. To Inay and Tatay, my first teachers who have been exacting in their ways as they inculcated in me the value of a good education, thank you for believing in my capacity when I often doubted myself. All that I was, all that I am, and all that I am going to be, it was because of you and is for you. It has always been my dream to make you proud of me… I hope I have done that. To my best and only sister Jhona, my brother- in-law Froy, and my nephews - Biboy, Kobe and Kyrie --- our LDR video calls kept me sane and attuned to the reality outside the gazillion things that I stressed myself with.
Thank you, my babies, for keeping Tita Mommy grounded and for inspiring me to carry on so I can be home with you again. Last but not the least, I give the highest praise to my
Lord and savior Jesus Christ, for giving me this miracle and victory. Indeed, I would not have finished this race without your amazing grace. I give all the glory and honor back to
You.
15 Publications arising from the research
Refereed journal articles
Paper 1
Niem, J., Billones-Baaijens, R., Savocchia, S., Stodart, B. (2019). Draft genome sequences of endophytic Pseudomonas spp. isolated from grapevine tissue and antagonistic to grapevine trunk disease pathogens. Microbiol. Resour. Announc. 8: e00345–19.
Paper 2
Niem, J., Billones-Baaijens, R., Savocchia, S., Stodart, B. (2020). Diversity profiling of grapevine microbial endosphere and antagonistic potential of endophytic Pseudomonas against grapevine trunk diseases. Front. Microbiol. 11:477. doi:
10.3389/fmicb.2020.00477.
16 Author Contribution Statements
The candidate contributed to the above-mentioned publications as follows.
Research paper published in Microbiology Resource Announcements:
The candidate carried out the experiments, data collection and analysis, preparation of the tables and figures, and the first draft of the full manuscript. The revisions recommended by the reviewers were also implemented by the candidate.
Research paper published in Frontiers in Microbiology:
The candidate carried out the experiments and data collection; contributed to data analysis, preparation of the tables and figures; and wrote the first draft of the full manuscript. The candidate helped with the revisions of the manuscript as per recommendation of the reviewers.
17 “I declare that the author contribution statements above are accurate, and I give permission for the candidate to include the papers and manuscripts in this thesis.”
Co-author name Signature Date
Regina Billones-Baaijens 05/04/2020
Benjamin Stodart 17/04/2020
Sandra Savocchia 03/04/2020
18 Abstract
Grapevine trunk diseases (GTDs) such as the Esca complex, Eutypa dieback
and Botryosphaeria dieback are considered major constraints to grapevine production.
These diseases can cause dieback, cankers and wood decay affecting the long-term
sustainability and productivity of vineyards. Current management of these diseases is
through protection of grapevine propagation materials in nurseries, remedial surgery
and fungicide treatment of pruning wounds. However, registered fungicides for GTDs are
limited and some only offer short term protection. Thus, there is a need to find alternate
solutions to combat these diseases for the wine grape industry.
Endophytes are plant-associated microorganisms that reside within plant tissues.
These may be beneficial, commensal or pathogenic to their host plant. In recent years,
the use of beneficial endophytes in plant disease control has gained popularity as the
application of fungicides raises concerns for human and environmental safety. This
research explored the diversity of the grapevine endomicrobiome with the aim of
identifying endophytes that could potentially be used as biocontrol agents (BCAs) for
GTDs.
The diversity of microbial endophytes associated with grapevine wood was investigated using next-generation sequencing. Characterisation of the grapevine endophytic community was undertaken using DNA extracted from both asymptomatic and GTD symptomatic vines collected from the Hilltops and Hunter Valley regions of
New South Wales (NSW), Australia. Both the bacterial 16S rRNA gene and ITS region of fungal rRNA genes were sequenced. The analysis determined that Pseudomonas
19 spp. predominated the bacterial community in asymptomatic grapevine wood from both regions, comprising 56-75% of the total population. In contrast, the Pseudomonas population in GTD symptomatic tissues were significantly lower representing 29% and
2% of the bacterial community in Hilltops and Hunter Valley, respectively. The fungal community was dominated by the genus Phaeomoniella in both asymptomatic and symptomatic grapevines, representing 74-89% and 59-78% of the total fungi, respectively. The other two abundant genera were Phaeoacremonium spp. and
Inonotus spp. Phaeomoniella chlamydospora and Ph. minimum(syn. Ph. aleophilum) and some Inonotus spp. have been implicated in Petri and Esca disease of grapevines.
The abundance of Pseudomonas spp. in asymptomatic grapevines led to further
investigation of its potential antagonistic activity against GTD pathogens. Isolation of
fluorescent Pseudomonas from grapevine wood resulted in 47 isolates for examination.
In dual-culture assays, 10 of these were antagonistic towards GTD pathogens (a testing
panel consisting of nine species of Botryosphaeria dieback, three species of Eutypa
dieback, and two Esca/Petri disease pathogens). Identification through 16S rRNA gene
sequencing determined that nine Pseudomonas isolates represented strains of P. poae
and one isolate, a strain of P. moraviensis.
The ability of the antagonistic strains of Pseudomonas to suppress GTD infection in grapevine plants were investigated using Neofusicoccum luteum as a model pathogen. The 10 antagonistic strains of Pseudomonas were screened for their ability to protect grapevine pruning wounds from infection by N. luteum using detached canes.
Re-isolations from canes treated with P. poae strains BCA13, 14, 16, 17, 18, and 19 showed 67-78% reduction in the recovery of N. luteum while all inoculated control canes
20 were 100% positive. Wounds created by pruning glasshouse potted vines were treated with P. poae strains BCA13, BCA14 and BCA17 prior to inoculation with N. luteum.
BCA17 significantly reduced the recovery of N. luteum by 80% relative to the control, while BCA13 and BCA14 were not effective in reducing pathogen recovery. A quantitative assay (qPCR) developed for N. luteum further elucidated the efficacy of
BCA17. The qPCR analysis indicated a 40-fold reduction in the amount of pathogen
DNA in vines that were treated with BCA17 compared to the positive control.
Pathogenicity tests using BCA17 showed this strain was not pathogenic to detached grapevine leaves, berries or single node canes.
A rifampicin-resistant mutant (RifMut) strain of BCA17 was generated to assess
its establishment in grapevine tissue. Re-isolations of RifMut from inoculated vines
showed that this strain can persist in the host tissue for up to 6 months. The RifMut
strain could also be re-isolated from dormant canes dipped in RifMut bacterial
suspension, indicating this strain was potentially transmissible in propagation material.
Investigation of the underlying mechanisms involved in the inhibition of GTD
pathogens and suppression of GTD infection in grapevine plants by BCA17 was
conducted. All 10 antagonistic Pseudomonas spp. produced siderophores in chrome
azurol S (CAS) media. The cell-free filtrate of BCA17 inhibited mycelial growth and spore
germination of all four GTD pathogens tested (Diplodia seriata, Neofusicoccum parvum,
N. luteum, Eutypa lata). The lipopeptide profile of the antagonistic (BCA17) and the non-
antagonistic (JMN1) strains were compared using MALDI-TOF-MS analysis. A
dominant lipopeptide was detected in a crude filtrate of BCA17 but not in that of JMN1.
However, due to the lack of a comprehensive lipopeptide database with specific mass
21 spectra, this lipopeptide remains to be identified. Draft genomes of the four strains
(BCA13, BCA14, BCA17, and JMN1) were generated and analysis revealed putative gene clusters that may impart biocontrol activity against plant pathogens including: lipopeptide antibiotics, siderophores, proteases, detoxification, lipopolysaccharide, multidrug resistance, microbe-associated molecular proteins (MAMPS), biofilms, and a number of nonribosomal proteins.
This research identified Pseudomonas spp. as abundant in healthy grapevine wood and further showed that they may provide some protection against GTDs. From this, the research characterised an antagonistic strain of Pseudomonas (BCA17) that could potentially be used as a BCA to manage GTD pathogens.
22 List of Abbreviations
AGRF Australian Genome Research Facility ANOVA Analysis of Variance APAF Australian Proteome Analysis Facility APE aryl polyenes BCA biological control agent BD Botryosphaeria dieback BLAST basic local alignment search tool CAS chrome azurol S CTAB cetyl trimethyl ammonium bromide CLP cyclic lipopeptide DAPG 2,4-diacetylphloroglucinol DNA deoxyribonucleic acid ED Eutypa dieback EDTA ethylenediaminetetraacetic acid gDNA genomic deoxyribonucleic acid GTDs grapevine trunk diseases HCN hydrogen cyanide ITS internal transcribed spacer LSD least significant difference MALDI-TOF Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight NA nutrient agar NB nutrient broth NRPS non-ribosomal peptides NSW New South Wales NWGIC National Wine and Grape Industry Centre PCA phenazine-1-carboxylic acid PCR polymerase chain reaction PDA potato dextrose agar PDAC potato dextrose agar-chloramphenicol qPCR quantitative PCR RF retention factor SA South Australia SDW sterile distilled water RNA ribonucleic acid rpm revolutions per minute sp. species (singular) spp. species (plural) TLC thin layer chromatography UV ultraviolet VOC volatile organic compound WLIP white-line-inducing principle
23
Chapter 1
Chapter 1.
Introduction and Literature Review
1.1. Importance of grapevine trunk diseases
Grapevine trunk disease (GTD) is a disease complex that can cause Esca, Petri
and Black foot diseases, and Eutypa and Botryosphaeria dieback, with symptoms
more often overlapping among different GTDs (Gramaje et al., 2018). Once
established, they are often difficult to control and can cause establishment problems to
young vines and decline of established vines (Somers et al., 2005).
Trunk diseases of grapevines present an ongoing economic issue for grape
producers and wine industries and occur in all grape growing regions worldwide
(Úrbez-Torres, 2011; Gramaje et al., 2018). For example, Eutypa and Botryosphaeria
dieback are two major diseases responsible for grapevine decline in Australia,
affecting both vineyard productivity and longevity. Eutypa dieback affects more than
60% of vineyards in South Australia (SA) and has also been reported in some
vineyards in New South Wales (NSW) (Pitt et al., 2010a). Botryosphaeria canker, on
the other hand, was found to infect 54% of grapevines in a survey of NSW vineyards (Pitt
et al., 2009). These diseases threaten the $8.3 billion Australian wine industry,
persisting in affected vines from season to season and causing loss in productivity. It
was estimated that Eutypa dieback in SA cost growers up to $2800/ha through
production loss in vineyards where at least 47% of the vines were infected (Wicks and
Davies, 1999). In California, management of Botryosphaeria dieback in vineyards with
severe infections is estimated to be $4.20 per vine or approximately $US2, 200 per
acre (~$AUD 7,000 per ha) (Epstein et al., 2008). This entails the cost of cutting vines
above the graft union, painting the wound, removing and reinstalling a new cordon 24
Chapter 1
wire, retraining and tying the vine, repairing trellises and other vineyard operations.
Chemicals to protect pruning wounds also incur additional expenses in managing
these diseases.
1.2. Symptoms and causal pathogens
Petri Disease and Esca. Petri disease also known as “black goo” is a vascular
disease causing decline and dieback of young vines (Fourie and Halleen, 2004). The
disease is primarily associated with Phaeomoniella chlamydospora (Gubler et al.,
2015) although several species of Phaeoacremonium, Pleurostoma, and Cadophora
have recently been associated with the disease (Gramaje et al., 2018). Symptoms of
Petri disease include reduced trunk diameter, shortened internodes, stunted shoot
growth, shoot dieback, and chlorotic, and/or necrotic leaves (Gubler et al., 2015;
Gramaje et al., 2018). Localised symptoms in wood cross sections include black dots
or streaks, black rings around the pith, and black goo oozing from cut vessels. It can
be transmitted through planting materials resulting in slow growth and reduced yield of
young vineyards, or may enter via wounds in mature vines (Somers et al., 2005;
Gramaje and Armengol, 2011).
Esca is a complex disease that originally pertains to a disease in grapevine that
appeared as a sudden, lethal wilting of the entire vine (Mugnai et al., 1999; Gubler et
al., 2015). The disease is generally associated with mature vines more than 10 years
old. The fungi commonly associated with the disease often includes the Petri disease
pathogens Ph. chlamydospora and Phaeoacremonium spp., and the basidiomycetes
belonging to the genus Fomitiporella, Fomitiporia, Inocutis, Inonotus, Phellinus, and
Stereum (Martin et al., 2012; Gramaje et al., 2018). Symptoms of the disease include
black borders around a soft, white heart rot in infected trunks, interveinal chlorosis or
discolouration instead of chlorosis in red varieties, shrivelling of berries, and gradual 25
Chapter 1 decline of vine productivity and vigour (Figure 1.1; Mugnai et al., 1999; Somers et al.,
2005). The aetiology of Esca disease is still not fully understood. Although a number of unrelated microorganisms have been isolated from Esca-infected vines, their role and interaction with each other are still unclear (Gramaje et al., 2018).
A B
Figure 1.1. Symptoms of Esca showing: A) interveinal chlorosis (red arrow); B) leaf blight (red arrow) and shriveled berries (yellow arrow). Photos by A. Deloire.
26
Chapter 1
Euypa dieback. Eutypa dieback is one of the most destructive trunk diseases of grapevines. It is also known as “dying arm” of grapevines (Tey-Rulh et al., 1991;
Rolshausen et al., 2015). The disease is caused by Eutypa lata and other members of the Diatrypaceae including Anthostoma, Cryptosphaeria, Cryptovalsa, Diatrype,
Diatrypella, and Eutypella (Pitt et al., 2010a; Trouillas et al., 2011; Luque et al., 2012).
Symptoms of the disease are observed in mature vines that are at least 10 years old, and are more pronounced in the spring when healthy new shoots are growing (Pitt et al., 2010a; Trouillas et al., 2011; Rolshausen et al., 2015).
Eutypa dieback infection is initiated when ascospores enter into the open ends of xylem vessels that are exposed through pruning wounds (Rolshausen et al., 2015).
Characteristic foliar symptoms of Eutypa dieback on infected young leaves include small size, chlorosis, cupping and necrosis (Figure 1.2A; Moller and Kasimatis, 1980;
Pitt et al., 2010a; Trouillas et al., 2011). Foliar symptoms progress through the years until all cordons fail to produce new shoots in the spring and ultimately, the vine dies.
These foliar symptoms are attributed to the production of the toxin, eutypine (Tey-Rulh et al., 1991; Molyneux et al., 2002). Symptomatic cordons and trunks reveal cankers surrounding pruning wounds (Figure 1.2B) and a zone of brown, wedge-shaped necrosis are formed (Figure 1.2C; Rolshausen et al., 2015).
27
Chapter 1
Figure 1.2. Symptoms of Eutypa dieback. A) Mature vine displaying stunted shoots (arrow); B) cordon with external canker (arrow); C) cross section of the wood with necrosis (arrow). Photos by R.B. Baaijens.
28
Chapter 1
Botryosphaeria dieback. Botryosphaeria dieback, formerly Botryosphaeria canker, are caused by species of fungi in the Botryosphaeriaceae family. Most species belonging to the Botryosphaeriaceae are saprophytic and are non-pathogenic on many hosts (Slippers and Wingfield, 2007). However, there is increasing evidence that some species are pathogenic and infect wounded and stressed plants. The primary mode of entry in grapevines is through pruning wounds, and wounds from mechanical injury
(Castillo-Pando et al., 2001; Úrbez-Torres, 2011). The pathogen can also enter through cracks and natural openings. Many species associated with Botryosphaeria canker were grouped within the genus Botryosphaeria, hence, the disease is called
Botryosphaeria dieback. However, current reclassification now include the genera
Botryosphaeria, Diplodia, Dothiorella, Lasiodiplodia, Neofusicoccum, Diplodia,
Neoscytalidium, Phaeobotryosphaeria and Spencermartinsia (Pitt et al., 2010b; Úrbez-
Torres, 2011; Gramaje et al., 2018).
29
Chapter 1
Figure 1.3. Symptoms of Botryosphaeria dieback of grapevines. Lack of vegetative growth from single or multiple spur positions of infected vines (A); trunk with external canker (B); trunk sections showing necrosis in the form of wedge (C) and central stain (D). Photos by R.B. Baaijens.
30
Chapter 1
The most distinguishing external symptoms associated with Botryosphaeria dieback is the absence of spring growth from single or multiple spur positions of infected vines, dead spurs and cordons (Figure 1.3A; Úrbez-Torres, 2011; Billones- Baaijens and
Savocchia, 2019). Other symptoms of Botryosphaeria dieback include perennial cankers
(Figure 1.3B), and internal wood necrosis (Figure 1.3 c-d) which can only be differentiated from those caused by Eutypa after isolation and/or DNA sequencing of causal organisms.
The disease is typically observed on mature vines although cankers have also been observed to occur on young vines (Úrbez-Torres et al., 2015).
1.3. Current management practices
Currently there is no single curative measure that provides 100% protection against GTDs, although different cultural techniques have shown partial beneficial effects
(Gramaje et al., 2018). GTD pathogens are generally cosmopolitan in nature and can be found in a broad range of hosts that surrounds vineyards (Rolshausen et al., 2015; Úrbez-
Torres et al., 2015). Once the pathogen enters through pruning wounds or mechanical injury, symptoms occur internally and take months to years to develop (Carter, 1988;
Lehoczky, 1988), making eradication difficult. As eradication is impossible, disease control is mainly through disease prevention and mitigation (such as remedial surgery). As with most plant diseases, there is no silver bullet method that can solely control GTDs. Different control measures should be integrated to attain maximum effectiveness.
1.3.1. Chemical control
Management of GTDs is a challenge as information on effective disease control measures is limited and varies between geographical locations. However, there is a general
31
Chapter 1 agreement among researchers from all over the world that infection can be prevented by protecting pruning wounds (Munkvold and Marois, 1995; van Niekerk et al., 2006; Epstein et al., 2008; Sosnowski et al., 2008; Rolshausen et al., 2010; Pitt et al., 2010c; Amponsah et al., 2012; Pitt et al., 2012; Úrbez-Torres et al., 2015). Painting pruning wounds with
10,000 ppm benomyl was found to afford a high degree of protection against E. lata infection of grapevine (Moller and Kasimatis, 1980). Munkvold and Marois (1993a) confirmed the effectiveness of benomyl as a pruning wound protectant against E. lata when applied using a paintbrush or pneumatic sprayer-pruning shear. However, benomyl was removed from the market in 2006 and therefore suitable alternatives required testing for efficacy (Mondello et al., 2018). Rolshausen and Gubler (2005) demonstrated that boric acid effectively inhibited both mycelial growth and the germination of ascospores of E. lata, in vitro. Furthermore, disease incidence in the field was reduced when boric acid was used to treat wounds as either a liquid spray or paste, providing 72-97% disease control as compared with untreated control, boron-free paste, and Cladosporium herbarum. The combination of the biocontrol agent (BCA), C. herbarum with boric acid did not improve disease control (Rolshausen and Gubler, 2005).
Various reports indicate the effectiveness of hand-applied fungicide sprays against grapevine canker pathogens. Munkvold and Marois (1993a) found that flusilazole was found to be effective against E. lata reducing the percentage of infected wounds by over
90%. Sosnowski et al. (2013) reported carbendazim and tebuconazole to effectively reduce colonization of pruning wounds inoculated with E. lata. However, carbendazim has already been banned for use on grapevines in Australia (Sosnowski et al., 2013).
The protection of pruning wounds has also been attempted utilising durable paint
32
Chapter 1 as a means to minimise trunk disease infection. Paints form an impenetrable physical barrier on grapevine and pruning wounds and serve as wound protectants. Compared to fungicides, paint is long-lasting, will not wash off during repeated rains and is not phytotoxic. The disadvantage, however, is that painting is labour-intensive for large vineyards (Epstein et al., 2008).
Only limited studies have been conducted on the management of Botryosphaeria canker in grapevines. Results from an in vitro study and bioassays in South Africa indicated that benomyl, tebuconazole and prochloraz are the most effective pruning wound protectants against B. obtusa, Neofusicoccum australe, N. parvum and L. theobromae
(Bester et al., 2007). Evaluation of 20 fungicides registered for use in Australian viticulture to manage other diseases revealed carbendazim (Bavistin), fluazinam (Shirlan), tebuconazole (Folicur), Garrison and acrylic paint (ATCS tree wound dressing) to be the most effective in reducing infection by Diplodia seriata and D. mutila. Infection was reduced by 41 to 65% when these fungicides were applied to fresh pruning wounds (Pitt et al., 2012).
In New Zealand, flusilazole, carbendazim, tebuconazole, thiophanate-methyl and mancozeb were found to reduce infection by Botryosphaeriaceae pathogens in field vines by 100, 93, 87, 83 and 80%, respectively (Amponsah et al., 2012).
1.3.2. Cultural methods
Many growers renew GTD infected vines using remedial surgery to remove the infected tissues. In spring, symptomatic cordons are tagged and affected vines are cut back during winter until no discoloured wood remains. Where both cordons are removed, water shoots are trained to replace the lost canopy (Creaser and Wicks, 2004). Remedial surgery is widely recommended for the management of grapevines infected with E. lata (Creaser
33
Chapter 1 and Wicks, 2004; Sosnowski et al., 2011) but has also been found to be effective for grapevines infected with Botryosphaeria (Epstein et al., 2008). Remedial surgery to restore infected vines to productivity can be a labour intensive and costly process. Infected tissue must be cut out, wounds treated to prevent new infections, and water shoots trained to replace the lost canopy. In most cases, trellises must also be re-wired (Creaser and Wicks,
2004).
Weber et al. (2007) introduced a control method for E. lata that requires two pruning passes through the vineyard which was coined “double pruning.” This method involved mechanical pruning in midwinter using a tractor mounted saw blade, followed by hand pruning in late winter or early spring where the vines are pruned to two-bud spurs. Double pruning ensured that final wounds were made when pathogens were least likely to successfully colonise exposed tissue. However, double pruning is not applicable in areas where risk of infection remains high after the second pruning as in the case in Catalonia,
Spain (Luque et al., 2014).
A management strategy that has also been widely recommended to prevent or reduce infection of pruning wounds by Botryosphaeriaceae is the removal of pruning debris from the vineyard (van Niekerk et al., 2006). Ground sanitation through burial or removal of prunings is an effective way to reduce the source of inoculum as prunings and deadwood on the vineyard floor can produce large quantities of conidia (Epstein et al., 2008).
Timing of pruning to avoid rainfall (i. e. delaying pruning until late winter or early spring) have been reported to reduce dieback infection (Petzoldt et al., 1981; Munkvold and
Marois, 1995). Wound susceptibility was found to be highest when vines are pruned early in the dormant period and lower when vines are pruned later in dormancy. Conversely, van
34
Chapter 1
Niekerk et al. (2011) reported that late winter pruning wounds were more susceptible to infection than wounds made earlier in the season in South Africa. Elena and Luque (2016) reported that susceptibility of wounds to D. seriata depended on the pruning season, with a decrease in cane infection of 35% at two weeks after an early pruning. However, a comparable decrease was not recorded until 8 weeks after a late pruning (Elena and
Luque, 2016). Pruning in late winter or early spring is recommended because the number of potential pathogenic airborne fungal spores are low around this time. Wounds are also believed to be less susceptible and the period of susceptibility is shorter during this time due to reduction in the number of susceptible sites as a result of vessel healing (Petzoldt et al., 1981). Increased temperature later in the dormant period resulted in a higher microbial population which, in effect, reduced the ability of E. lata to infect due to competition or other types of antagonism (Munkvold and Marois, 1995).
1.3.3. Biological control agents
The use of BCAs to manage plant diseases has increased in popularity as it offers a viable alternative to chemical methods. Specifically, biocontrol strategies against GTDs are being developed to replace or supplement chemical control methods.
Several studies report successful results for the biological control of E. lata on grapevines, particularly with the application of Trichoderma spp. (John et al., 2005; John et al., 2008; Kotze et al., 2011; Mutawila et al., 2011). Trichoderma-based products are now commercially available for the control of Eutypa dieback of grapevines, among other diseases of various crops; Eco-77® (Plant Health Products, South Africa), Vinevax
(Agrimm Pty Ltd, Moonee Ponds, Victoria, Australia; Agrimm Technologies, Christchurch,
New Zealand), Trichoseal (Agrimm Technologies, Christchurch, New Zealand), and
35
Chapter 1
Biotricho (Agro-Organics Pty Ltd., South Africa) (Hunt, 2004; John et al., 2008). Esquive®
WP (Agrauxine Lesaffre Plant Care), Remedier WP (Italy), and Vintec (Belgium) are some of the Trichoderma-based products that have been registered across several European countries (Woo et al., 2014). Aside from E. lata, Trichoderma spp. have been demonstrated to inhibit growth of Botryosphaeriaceae species (i.e. Neofusicoccum australe, N. parvum,
Diplodia seriata, Lasiodiplodia theobromae) and other GTD pathogens (Ph. chlamydospora, Diaporthe ampelina, and Phaeoacremonium sp.) in growth media (Kotze et al., 2011; Mutawila et al., 2011). However, these studies from South Africa have demonstrated varying levels of success in the laboratory and little success in the field.
Mycelial growth inhibition of E. lata was postulated to be due to production of the volatile metabolite, 6-n-pentyl-2H-pyran-2-one, and unidentified non-volatile metabolites produced by T. harzianum. However, 6-n-pentyl-2H-pyran-2-one was found to have fungastatic effect only to E. lata, reducing mycelial growth but not completely inhibiting
(John et al., 2004). Complete inhibition was only observed with the diffusible non-volatiles which were assumed to be antibiotics or enzymes produced by T. harzianum (John et al.,
2004). Co-inoculation of the pathogen with T. harzianum resulted in hyphal malformation leading to collapsed, shrivelled and abnormal swellings, parallel growth, and coiling of the hyphae (John et al., 2004; Kotze et al., 2011).
Fusarium lateritium Nees has also been reported as a successful pruning wound treatment against E. lata (Munkvold and Marois, 1993b; John et al., 2005). Treatment of fresh pruning wounds with spores of F. lateritium was found to significantly reduce recovery of E. lata from infected vines. In both studies, a delay of 14 days between wounding and inoculation with ascospores of E. lata reduced recovery of the pathogen compared with inoculation on the day after wounding, reducing infection by as much as 70%. In contrast, 36
Chapter 1
Gendloff et al. (1983) found that F. lateritium did not effectively control E. lata in Michigan vineyards. Even though F. lateritium consistently became established in more than half of the pruning wounds to which it was inoculated, no significant control of ascospore infection by E. armeniacae was observed when pruning wounds on 2-yr-old wood were inoculated either on the day of pruning and spraying or 14 days later.
Cladosporium herbarum (Pers.) Link was also found to inhibit E. lata infection of grape wood when applied as a pruning wound protectant (Munkvold and Marois, 1993b;
Rolshausen and Gubler, 2005). Munkvold and Marois (1993b) reported C. herbarum to be as effective as benomyl in reducing infection by E. lata (71% reduction in infection) when the pathogen was inoculated 14 days after application of the protectant.
Biological control of E. lata on grapevines has likewise been demonstrated using bacterial antagonists, such as Bacillus subtilis (Ehrenberg) Cohn (Ferreira et al., 1991;
Schmidt et al., 2001; Kotze et al., 2011). A strain of B. subtilis originally isolated from the pruning wound of a grapevine with symptoms of Eutypa dieback was found to inhibit mycelial growth of E. lata by 91% on culture medium, ascospore germination by 100%, and infection of pruning wounds by 100% (Ferreira et al., 1991). In dual cultures with B. subtilis,
E. lata was inhibited, showing very limited mycelium growth and clear inhibition zones. At a microscopic level, malformations of the hyphae occurred where hyphal swelling, coiling, adhesion and disintegration were observed (Kotze et al., 2011). Treatment of pruning wounds with B. subtilis significantly reduced incidence of E. lata by 90% following inoculation (Kotze et al., 2011). Working with other bacterial organisms, Schmidt et al.
(2001) reported that a cell-free culture filtrate derived from Erwinia herbicola resulted in close to 100% inhibition of infection by E. lata on autoclaved grape wood, over a period of
37
Chapter 1 four weeks. This inhibition was suggested to be due to the production of herbicolin and pyrrholnitrin, broad-spectrum antifungal substances known to be produced by the bacterium (Schmidt et al., 2001).
To date, field experiments in which biological control agents have been investigated have yielded erratic results (Gendloff et al., 1983; Munkvold and Marois, 1993b; John et al., 2005; Mutawila et al., 2011). Also, unlike chemical applications which have an immediate effect, maximum protection from BCAs that are used as pruning wood protectants appear to require colonisation of the wounded surface. Thus, there is a period of tissue susceptibility after treatment until the BCA becomes well established, in which the development of the pathogen is possible. Therefore, feasible and effective methods for the application of BCAs need to be explored.
1.4. Grapevine endophytes
The increasing popularity of BCAs as an alternative method to combat plant diseases sparked interest in exploring the potential of plant endophytes as BCAs.
Endophytes may be ideal BCAs as they are ubiquitous in their host plant and can actively colonise plant tissues (Hallmann et al., 1997). Endophytes may be commensals that reside within the plant tissue without any effect to the plant; symbionts with mutualistic relationships with the plant, providing benefits to their host (Hallmann et al., 1997; Iniguez et al., 2004; Ryan et al., 2008; Rybakova et al., 2015). However, some endophytes may be pathogenic when conditions become favourable (Hardoim et al., 2015; Brader et al., 2017).
Several studies have been conducted on the composition of bacterial endophytes in grapevine tissues (Bell et al., 1995; Bulgari et al., 2009; West et al., 2010; Bulgari et al.,
2011; Compant et al., 2011; Campisano et al., 2014; Campisano et al., 2015; Andreolli et
38
Chapter 1 al., 2016). Use of DNA-based, culture- independent methods in the study of the grapevine endophytic population has become popular in the recent years (Bulgari et al., 2009; West et al., 2010; Bokulich et al., 2013; Faist et al., 2016; Deyett et al., 2017). This has become important so as not to discount non-culturable microbial communities (Bulgari et al., 2009;
West et al., 2010). DNA sequencing technology using an Illumina MiSeq was employed by
Deyett et al. (2017) in studying the microbial composition of the grapevine endosphere.
This technique revealed a decrease in the relative abundance of the bacteria Xylella fastidiosa, responsible for Pierce’s disease in grapevine, when Achomobacter xylosoxidans and Pseudomonas fluorescens were also present in grapevine tissues. The presence of grapevines asymptomatic for Pierce’s disease in vineyards with high disease pressure was attributed to these beneficial bacteria inhabiting the grapevine vascular endosphere (Deyett et al., 2017). This presents the potential for exploring the resident microflora of grapevines for beneficial microorganisms capable of inhibiting GTD pathogens and suppressing subsequent infection.
1.5. Potential of Pseudomonas spp. as biocontrol agents
In recent years, the use of Pseudomonas spp. as BCAs has been widely studied within various pathosystems, including soil-borne diseases such as take-all disease of wheat (Weller, 1982; Keel et al., 1992; Ownley et al., 1992; Raaijmakers and Weller, 1998);
Rhizoctonia root rot on bean (D'aes et al., 2011); damping-off in cotton (Howell and
Stipanovic, 1979); damping-off, root rot and bacterial wilt in tomato (Solanki et al., 2014); potato scab (Arseneault et al., 2013), and black root rot of tobacco (Voisard et al., 1989;
Keel et al., 1992). Commercial formulations based on strains of Pseudomonas are already available in the market. P. fluorescens A506, marketed as BlightBan® A506 (Plant Health 39
Chapter 1
Technologies, Idaho, USA), was released in 1996 for the control of fire blight and frost injury in apple and pear (Wilson, 1997).
In grapevines, Pseudomonas spp. have been shown to suppress Botrytis cinerea, the causal agent of grey mould (Ait Barka et al., 2002; Trotel-Aziz et al., 2008). In vitro studies demonstrated a zone of inhibition resulted when the fungus was inoculated two days after introduction of Pseudomonas sp. The bacteria appeared to inhibit the growth of B. cinerea by disrupting cellular membranes and inducing cell death, with microscopy analysis determining the disruption of fungal mycelium and leakage of protoplasm (Ait Barka et al.,
2002). Trotel-Aziz et al. (2008) demonstrated the ability of P. fluorescens PTA-268 to protect grapevine leaves against grey mould disease through the induction of plant resistance via release of defense-related compounds such as lipoxygenase, phenylalanine ammonia-lyase and chitinase. The reduction in incidence and severity of Fusarium root rot in grapevines, and increased fruit yield by 36%, has also been reported when P. fluorescens was applied as soil treatment (Ziedan and El-Mohamedy, 2008). P. aureofaciens B-4117 and P. fluorescens CR 330D were reported to significantly reduce the incidence and severity of crown gall caused by Agrobacterium tumefaciens on grapevine during seedling root production and grafting (Khmel et al., 1998). The disease incidence on root cuttings of grapevine was reduced by 56 to 80% and the disease severity index was decreased by 75 to 86% while the number of healthy rooted grafts increased by
2 to 3.5-fold (Khmel et al., 1998).
1.5.1. Mechanisms for biocontrol activity exhibited by Pseudomonas spp.
The mechanisms by which Pseudomonas spp. suppress disease has been well
40
Chapter 1 studied for soil-borne pathogens, and includes the production of antibiotics (Raaijmakers et al., 2002; Chin-A-Woeng et al., 2003; D’aes et al., 2011; Arseneault et al., 2013;
Hernández-León et al., 2015), siderophores (Kloepper et al., 1980; Leong, 1986; Neilands and Leong, 1986; Schippers et al., 1987), volatiles (Cordero et al., 2014; Hernández-
León et al., 2015), bioactive metabolites (Begum et al., 2014), hydrolytic enzymes
(Fridlender et al., 1993), the competition for nutrients (Paulitz, 1990; Wilson, 1997), and induced systemic resistance (Haas and Défago, 2005).
Production of antibiotics and other related antimicrobial compounds. The antibiotics identified as being produced by putative BCAs within Pseudomonas include
2,4-diacetylphloroglucinol (DAPG) (Keel et al., 1992; Défago, 1993; Rosales et al., 1995;
Raaijmakers and Weller, 1998), hydrogen cyanide (Schippers et al., 1987; Voisard et al., 1989; Hernández-León et al., 2015), phenazine and its derivatives ( Schippers et al.,
1987; Hamdan et al., 1991; Ownley et al., 1992; Pierson and Thomashow, 1992;
Rosales et al., 1995; Chin-A-Woeng et al., 2003; Hernández-León et al., 2015), pyoluteorin (Howell and Stipanovic, 1980), pyrrolnitrin (Howell and Stipanovic, 1979;
Rosales et al., 1995), and cyclic lipopeptides (Nielsen et al., 2002; de Souza et al., 2003;
Begum et al., 2014). Mutant strains of bacteria that were deficient in the production of antibiotics have been reported to lose their ability to suppress plant disease
(Thomashow and Weller, 1988; Thomashow et al., 1990; Vincent et al., 1991; Mazzola et al., 1992; Pierson and Thomashow, 1992; Shanahan et al., 1992; Bangera and
Thomashow, 1999). The endophytic strain P. fluorescens FPT 9601 has been reported to synthesise DAPG and deposit DAPG crystals in the roots of tomato, indicating that an endophyte has the capacity to produce antibiotics in planta (Compant et al., 2005).
41
Chapter 1
Inhibition of wheat root infection by Gaeumannomyces graminis var. tritici by P. fluorescens 2-79 and P. aureofaciens 30-84 was attributed to the production of the antibiotic phenazine and its derivative, phenazine-1-carboxylic acid (Thomashow and
Weller, 1988; Thomashow et al., 1990). The role of these antibiotics in disease suppression was established using Tn5 transposon mutants of the bacterial strains, which were deficient in the production of the antibiotic (Thomashow and Weller, 1988).
When used to challenge the fungal pathogen, phenazine deficient mutants lost their ability to reduce symptoms of take- all disease in wheat (Thomashow and Weller, 1988).
When phenazine production was restored by wild-type DNA introduced as a cosmid clone, the strains regained the ability to prevent the fungal infections (Thomashow and
Weller, 1988).
Production of siderophores. Compounds other than antibiotics are also involved in the biocontrol of pathogens by other microorganisms. The most widely studied of these are the siderophores, which are extracellular, low-molecular-weight compounds with a very high affinity for ferric iron. The ability to sequester iron provides a competitive advantage to microorganisms, and there is evidence that siderophores can play an active role in the inhibition of one microorganism by another (Fravel, 1988). Mutants that lost the ability to produce siderophores in vitro also lost the ability to suppress plant diseases (Simeoni et al., 1987; Weller et al., 1986). Aside from their biocontrol ability, many species of Pseudomonas are also known to promote plant growth. Specific strains of the P. fluorescens-putida group known as plant growth-promoting rhizobacteria
(PGPR), are found to rapidly colonise plant roots of potato, sugar beet and radish, and cause statistically significant yield increases of up to 144% in field experiments. This
42
Chapter 1 plant growth-promoting activity was reported to be due to the deprivation of iron for the microbial community.
Production of volatile compounds. Volatile organic compounds (VOCs) emitted by certain strains of Pseudomonas were also reported to be responsible for both the control of plant disease and plant growth promotion (Cordero et al., 2014; Hernández-León et al., 2015). P. fluorescens strains UM16, UM240, UM256, and UM270, which were highly antagonistic against the phytopathogen B. cinerea and reduced stem disease symptoms and root browning in Medicago truncatula, were found to produce dimethyl disulfide which has known antifungal properties (Hernández-León et al., 2015). In addition, P. fluorescens strain UM270 was further found to produce the volatile compound, dimethylhexadecylamine which is known to have antifungal and plant growth promoting activities (Hernández-León et al., 2015). Volatiles have also been implicated in the growth inhibition of F. proliferatum which causes head blight of cereal grains (Cordero et al.,
2014). The antifungal volatiles benzothiazole, cyclohexanol, n-decanal, dimethyl trisulfide, 2-ethyl 1- hexanol, and nonanal produced by Pseudomonas spp., have been reported to inhibit sclerotia and ascospore germination and mycelial growth of Sclerotinia sclerotiorum that cause economically important diseases in western Canada, including stem rot of canola, head, stem and basal rot of sunflower, white mould of beans, and crown and stem rot of alfalfa (Fernando et al., 2005).
Production of enzymes. Some BCAs have been reported to produce hydrolytic enzymes which degrade the cell wall of pathogenic fungi. Extracellular chitinase and laminarinase, synthesized by P. stutzeri, were demonstrated to digest and lyse mycelia of F. solani, the causal agent of root rot of many plants. The degraded hyphae showed
43
Chapter 1
abnormal swelling and the presence of a penetration hole which caused leakage of
protoplasm (Lim et al., 1991). Fogliano et al. (2002) reported that control of Fusarium and
Botrytis species is attributed to the synergistic activity between lipodepsipeptides and cell
wall–degrading enzymes produced by P. syringae pv. syringae strain B359. Addition of
purified glucanase or chitinase from P. syringae enhanced the biocontrol activity of the
lipodepsipeptide, syringopeptin (Fogliano et al., 2002). Some bacteria like Pseudomonas
spp. trigger induced systemic resistance which is believed to have a role in disease
suppression. Biochemical investigations have shown that accumulation of callose and
lignin along with synthesis of hydrolytic enzymes such as chitinases occurred during
bacterial- mediated induced resistance in plants (Benhamou, et al., 1996). Biochemical
changes that occur in plants during induced resistance also include induced accumulation
of pathogenesis-related proteins, chitinases, peroxidases, phenylalanine ammonia lyase,
phytoalexins, polyphenol oxidase, chalcone synthase, among many others (Compant et
al., 2005).
1.6. Summary and project aims
The current management practices to reduce incidence of GTDs are mainly through
various cultural methods and the application of fungicide to protect pruning wounds. A
serious constraint to management is the limited number of commercially available
fungicides, and the compounding issue that several of the more effective chemistries
have been banned in the market due to concerns for human and environmental safety.
Another downside with fungicide usage is that some options provide short term
protection; when fungicides are applied to pruning wounds their effectiveness quickly
deteriorates. 44
Chapter 1
There is the potential for endophytic bacteria to be exploited as BCAs either as an alternative or as a supplement to fungicides, and these can be incorporated in an integrated trunk disease management strategy. There are several products available commercially based on both fungal and bacterial organisms. However, efficacy can be variable and such products are yet to become mainstream. At the consumer level, markets are being driven to accept lower thresholds of pesticides, and this has in turn driven an increased interest in alternative disease management strategies. Bacterial endophytes may offer an untapped resource of potential BCAs, and identifying strains specific to grapevines may have several advantages for the industry. Strains isolated from their host may have better adaptive and competitive potential than organisms isolated from other plant species due to their natural residence and could therefore provide better control of GTDs.
Preliminary studies at the National Wine and Grape Industry Centre revealed the ability
of endophytic Pseudomonas spp. isolated from grapevine wood to inhibit the growth of
several Botryosphaeria dieback pathogens (R.B. Baaijens, personal communication). To
our knowledge, studies on the use of grapevine endophytic Pseudomonas spp. to control
GTDs have not been conducted to date. Therefore, there is a need to investigate the
potential of these bacteria as BCAs for GTDs. Pruning wound protection by Pseudomonas
spp. may provide more sustainable and long-term protection from the impact of GTD
pathogens. This could potentially provide an alternate solution to a major problem in the
wine industry without adversely affecting humans and the environment.
1.7. Research objectives
The literature review presented in this chapter has provided some evidence on the
45
Chapter 1
potential of grapevine endophytic bacteria as BCAs against GTDs. Although numerous
studies on the use of Pseudomonas spp. in the control of various plant pathogens and
their corresponding diseases has been reported, only a single study has been reported
regarding its application against GTDs. Wicaksono et al. (2017) identified 10 endophytic
bacteria from mānuka (Leptospermum scoparium) with inhibitory activity against N.
luteum and N. parvum that cause GTDs in New Zealand. Seven of these antagonistic
bacteria are strains of Pseudomonas spp. These bacteria also reduced lesion lengths
caused by the two Neofusicoccum species (Wicaksono et al., 2017).
Therefore, the overall aim of the research was to investigate the potential of bacterial endophytes from grapevines as BCAs to manage GTDs with the following specific objectives:
(i) to identify and compare the microbiome inhabiting symptomatic and asymptomatic
grapevine tissue using next generation sequencing. This is based on the hypothesis
that endophytic bacteria such as Pseudomonas spp. that are inherent in grapevines
may provide some level of protection against GTD pathogens if their populations are
abundant.
(ii) to select, isolate, and screen candidate microorganisms for their biocontrol potential
against GTD pathogens, particularly Esca, Eutypa and Botryosphaeria dieback
pathogens;
(iii) to elucidate the mechanism of action of the BCA that is involved in the control of GTD
pathogens; and
(iv) to determine the persistence of BCAs within living grapevine tissue.
46
Chapter 2
Chapter 2. Research Paper
Diversity profiling of grapevine microbial endosphere and antagonistic potential of endophytic Pseudomonas against Grapevine trunk diseases
Niem JM, Billones-Baaijens R, Stodart B and Savocchia S. (2020). Frontiers in Microbiology.
11:477. doi: 10.3389/fmicb.2020.00477.
Submitted to Frontiers in Microbiology: 24 October 2019
Accepted: 04 March 2020
Published online: 26 March 2020
47 fmicb-11-00477 March 24, 2020 Time: 16:2 # 1
ORIGINAL RESEARCH published: 26 March 2020 doi: 10.3389/fmicb.2020.00477
Diversity Profiling of Grapevine Microbial Endosphere and Antagonistic Potential of Endophytic Pseudomonas Against Grapevine Trunk Diseases
Jennifer Millera Niem1,2*, Regina Billones-Baaijens1, Benjamin Stodart2,3 and Sandra Savocchia1,2*
1 National Wine and Grape Industry Centre, Charles Sturt University, Wagga Wagga, NSW, Australia, 2 School of Agricultural and Wine Sciences, Charles Sturt University, Wagga Wagga, NSW, Australia, 3 Graham Centre for Agricultural Innovation (Charles Sturt University and NSW Department of Primary Industries), School of Agricultural and Wine Sciences, Charles Sturt University, Wagga Wagga, NSW, Australia
Grapevine trunk diseases (GTDs) are a serious problem of grapevines worldwide.
Edited by: The microbiota of the grapevine endosphere comprises prokaryotic and eukaryotic David Gramaje, endophytes, which may form varied relationships with the host plant from symbiotic Institute of Vine and Wine Sciences (ICVV), Spain to pathogenic. To explore the interaction between grapevine endophytic bacteria and Reviewed by: GTDs, the endomicrobiome associated with grapevine wood was characterized using Stéphane Compant, next-generation Illumina sequencing. Wood samples were collected from grapevine Austrian Institute of Technology (AIT), trunks with and without external symptoms of GTD (cankers) from two vineyards in Austria Ales Eichmeier, the Hunter Valley and Hilltops, NSW, Australia and metagenomic characterization of Mendel University in Brno, Czechia the endophytic community was conducted using the 16S rRNA gene (341F/806R) *Correspondence: and ITS (1F/2R) sequences. Among the important GTD pathogens, Phaeomoniella, Jennifer Millera Niem [email protected] Phaeoacremonium, Diplodia and Cryptovalsa species were found to be abundant in Sandra Savocchia both symptomatic and asymptomatic grapevines from both vineyards. Eutypa lata and [email protected] Neofusicoccum parvum, two important GTD pathogens, were detected in low numbers
Specialty section: in Hilltops and the Hunter Valley, respectively. Interestingly, Pseudomonas dominated This article was submitted to the bacterial community in canker-free grapevine tissues in both locations, comprising Plant Microbe Interactions, 56–74% of the total bacterial population. In contrast, the Pseudomonas population in a section of the journal Frontiers in Microbiology grapevines with cankers was significantly lower, representing 29 and 2% of the bacterial Received: 24 October 2019 community in Hilltops and the Hunter Valley, respectively. The presence of Pseudomonas Accepted: 04 March 2020 in healthy grapevine tissues indicates its ability to colonize and survive in the grapevine. Published: 26 March 2020 The potential of Pseudomonas spp. as biocontrol agents against GTD pathogens was Citation: Niem JM, Billones-Baaijens R, also explored. Dual culture tests with isolated fluorescent Pseudomonas against mycelial Stodart B and Savocchia S (2020) discs of nine Botryosphaeria dieback, three Eutypa dieback, and two Esca/Petri disease Diversity Profiling of Grapevine pathogens, revealed antagonistic activity for 10 Pseudomonas strains. These results Microbial Endosphere and Antagonistic Potential suggest the potential of Pseudomonas species from grapevine wood to be used as of Endophytic Pseudomonas Against biocontrol agents to manage certain GTD pathogens. Grapevine Trunk Diseases. Front. Microbiol. 11:477. Keywords: Pseudomonas poae, microbiome, endophyte, biocontrol, Botryosphaeria dieback, Eutypa dieback, doi: 10.3389/fmicb.2020.00477 Esca/Petri disease
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INTRODUCTION density, remedial surgery wherein the infected parts of the vines are excised, and appropriate timing of pruning and Grapevine trunk diseases (GTDs) are economically important training of vines (Gramaje et al., 2018). In Spain, anecdotal in all grape growing regions and significantly impact grape evidence has been presented for the use of copper nails placed production and the wine industry worldwide. These diseases are in the scion parts of grapevine trunks, which slowed the a major threat to the Australian wine industry and affect the long- progress of wood rot in infected plants, but this is yet to be term sustainability and productivity of many vineyards. Esca, scientifically validated (Mondello et al., 2018). Fungicides such Petri and black foot diseases, and Eutypa and Botryosphaeria as the benzimidazole carbamate group, tebuconazole, flusilazole, diebacks are the major constraints in Australian vineyards pyrimethanil, pyraclostrobin, and fluazinam are also used as potentially causing considerable economic loss to the industry pruning wound protection against Botryosphaeria and Eutypa (Wicks and Davies, 1999; Edwards and Pascoe, 2004; Whitelaw- dieback pathogens (Gramaje et al., 2018). For Esca and Petri Weckert et al., 2013; Billones-Baaijens and Savocchia, 2019). disease pathogens, thiophanate-methyl and boron are found to Once established, these diseases are often difficult to control be more effective pruning wound protectants (Rolshausen et al., and can cause establishment problems for young vines and the 2010). However, the downside with fungicide usage is that some decline of established vines (Gramaje et al., 2018). The general fungicides may only provide short-term protection. This may be symptoms of these diseases are wood necrosis characterized problematic, since pruning wounds remain susceptible to E. lata by brown streaking or cankers, foliar discoloration and drying. for 2–4 weeks and up to 16 weeks to species of Botryosphaeriaceae Symptoms of GTDs usually take several years to develop, (Munkvold and Marois, 1995; Úrbez-Torres and Gubler, 2011; making early detection difficult. Pruning wounds are the main Ayres et al., 2016). Furthermore, there are only limited fungicides point of entry for fungal spores (Slippers and Wingfield, 2007; that are commercially available to manage GTDs. Gramaje et al., 2018). Protection of pruning wounds is deemed Biological control agents (BCAs) may be a potential strategy essential to reduce infections from GTD pathogens (Mondello to supplement chemical methods. In recent years, the use of et al., 2018). The causal organisms grow, decay the wood and endophytic BCAs in the management of plant disease has gained eventually kill the vines. popularity as an alternative to chemical application, driven in part Botryosphaeria canker and dieback are caused by species of by concerns for human and environmental safety with the use of fungi in the Botryosphaeriaceae family. Many species associated fungicides (Chen et al., 1995; Ryan et al., 2008; Cabanás et al., with Botryosphaeria canker were grouped within the genus 2014; Rybakova et al., 2015; Aydi Ben Abdallah et al., 2016; Hong Botryosphaeria, hence, the disease is commonly referred to and Park, 2016). Endophytes are microorganisms that spend at as Botryosphaeria dieback. However, current reclassification least parts of their life cycle inside the plant (Hardoim et al., 2015). now includes the genera Fusicoccum, Neofusicoccum, Diplodia, They are ubiquitous in their host plant and may reside latently Lasiodiplodia, Dothiorella, Spencermartinsia, and Sphaeropsis or actively colonize plant tissues (Hallmann et al., 1997). Most (Úrbez-Torres et al., 2015). Eutypa dieback is caused by Eutypa endophytes are commensals which reside within the plant tissues lata and other Diatrypaceae species including Anthostoma, and live on metabolites produced by their host, often without any Cryptosphaeria, Cryptovalsa, Diatrype, Diatrypella, and Eutypella known effect on the plant (Hardoim et al., 2015; Brader et al., (Trouillas et al., 2011; Luque et al., 2012). The etiology of Esca 2017). Another group of endophytes has mutualistic relationship disease is still not fully understood. Although a number of with plants (Hardoim et al., 2015; Brader et al., 2017) and unrelated microorganisms have been isolated from Esca-infected provide benefits to their host though promotion of plant growth, vines, their role and interaction with each other are not clear biocontrol of plant pathogens, enhancement of plant nitrogen (Gramaje et al., 2018). However, Phaeomoniella chlamydospora fixation and phosphate solubilization (Hallmann et al., 1997; and/or species of Phaeoacremonium are commonly associated Iniguez et al., 2004; Ryan et al., 2008; Rybakova et al., 2015). Some with the disease. Although some authors claimed several endophytes may also exhibit pathogenicity when conditions basidiomycetous species belonging to the genera Fomitiporia, become favorable (Hardoim et al., 2015; Brader et al., 2017). Phellinus, Stereum, Fomitiporella, Inonotus, and Inocutis to be Limited studies have been conducted on the grapevine requisites for the development of classic Esca symptoms (Cloete endophytic microbial community, with most research focusing et al., 2015; Gramaje et al., 2018), others suggested that young esca on the microbial composition of the grapevine phyllosphere is induced by Phaeomoniella chlamydospora alone (Edwards et al., (Leveau and Tech, 2011; Bokulich et al., 2013; Perazzolli et al., 2001). Petri disease or black goo decline is primarily associated 2014; Pinto et al., 2014; Portillo et al., 2016). Moreover, most of with P. chlamydospora (Gubler et al., 2015) although several the studies conducted on grapevine microbiota are focused on species of Phaeoacremonium, Pleurostoma, and Cadophora have bacterial endophytes (Bell et al., 1995; Bulgari et al., 2009, 2011; recently been linked to the disease (Gramaje et al., 2018). West et al., 2010; Compant et al., 2011; Campisano et al., 2014, Currently there are no reliable curative control measures 2015; Andreolli et al., 2016) and much less on endophytic fungi. against GTDs and there are no means, chemical or other, to Deyett et al.(2017) conducted a comprehensive study of both eradicate the organisms once they become established within a the bacterial and fungal assemblages in the grapevine endosphere vine. As eradication is problematic, disease control is mainly of a Californian vineyard and found a negative correlation though disease prevention and mitigation (Úrbez-Torres, 2011). between the Pierce’s disease pathogen, Xylella fastidiosa and the Management of GTDs is mainly though employment of cultural endophytic bacteria Pseudomonas fluorescens and Achomobacter practices that include vineyard sanitation to reduce the inoculum xylosoxidans. In Spain, Elena et al.(2018) characterized the fungal
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and bacterial composition and diversity in GTD asymptomatic microorganisms, while the remaining half was stored at −80◦C and symptomatic vines and found that there were no differences prior to DNA extraction. in the fungal communities between the healthy and diseased vines, while differences in the bacterial composition in non- DNA Extraction From Grapevine Wood necrotic and necrotic tissues were evident. The lone attempt to Samples study the grapevine microbial profile in Australia was conducted DNA was extracted from 100 mg of ground wood shavings from by West et al.(2010), characterizing the diversity of culturable both asymptomatic and symptomatic inner grapevine tissues and non-culturable bacterial endophytes in grapevines. However, following the methods of Pouzoulet et al.(2013) with slight microbial distribution patterns may differ within and between modifications. Frozen samples were lyophilized for 24 h in a geographical locations due to differences in environmental freeze drier (John Morris Scientific, United States) and ground at conditions (Bokulich et al., 2013). In Australia, Eutypa dieback 20 Hz for 2 min in a TissueLyser II (Qiagen, United States) within and Botryosphaeria dieback are two of the most important 10 ml stainless-steel grinding jars, containing 20 mm stainless GTDs (Scholefield and Morison, 2010; Billones-Baaijens and steel balls (Qiagen, United States). Total DNA was extracted Savocchia, 2019). To our knowledge, studies on the use of from pulverized freeze-dried wood samples using the CTAB grapevine endophytes to control Eutypa and Botryosphaeria extraction buffer of Doyle and Doyle(1987). Pulverized wood diebacks in grapevines have not been conducted. There is samples were incubated in CTAB buffer at 65◦C for 1 h, and a need therefore to investigate the potential of grapevine then extracted using chloroform/isoamyl alcohol (24:1) (Sigma- endophytic organisms as BCAs for dieback diseases, providing Aldrich, United States) on ice for 5 min. The mixture was then environmentally sustainable solutions to a major problem in centrifuged at 4◦C for 10 min at 2300 × g. The lysate was the Australian wine industry. This study was conducted to then transferred to a QIAshredder spin column provided in the identify the microbiome inhabiting the inner grapevine tissues DNeasy Plant Mini Kit (Qiagen, United States). Following this and to examine the interaction between grapevine endophytic procedure, the subsequent steps indicated in the DNeasy Plant bacteria and GTD pathogens. Furthermore, the biocontrol Mini Kit were followed. DNA samples were eluted to a final potential of the most dominant endophytic bacterium against volume of 100 µl using AE buffer (Qiagen, United States), and GTD pathogens was explored. Beneficial microorganisms that stored at −20◦C. DNA concentration was determined using a occur naturally within plant tissues have potential as effective Quantus Fluorometer (Promega, United States). The quality of BCAs as they are adapted to the plant host they were originally DNA was further verified though gel electrophoresis and DNA isolated from and may have greater potential to be sustained samples (∼70 ng) were used for microbial diversity profiling within the host. analysis. Raw sequence reads are available from the NCBI’s Sequence Read Archive (Bioproject PRJNA605898). MATERIALS AND METHODS Sequencing of the 16S and ITS rRNA Plant Sample Collection Genes Samples were collected during the spring of 2016 from PCR amplification and sequencing was performed at the commercial vineyards in Hilltops and the Hunter Valley, Australian Genome Research Facility (AGRF, Sydney, NSW, which are the major grape-growing regions in New South Australia). Amplicons for both 16S and ITS were generated using Wales, Australia. These vineyards were selected as GTDs were the primers and conditions outlined in Table 1. Thermocycling previously reported to occur in these areas (Baaijens, personal was completed with an Applied Biosystem 384 Veriti and using communication). Vineyard surveys identified Botryosphaeriaceae AmpliTaq Gold 360 (Life Technologies, Australia). Illumina and Diatrypaceae species as being present in these regions indexing of the amplicons was achieved in a second PCR utilizing (Pitt et al., 2010; Qiu et al., 2011; Trouillas et al., 2011) while TaKaRa Taq DNA Polymerase (Clontech). Indexed amplicon Esca/Petri disease pathogens were found in the Hunter Valley libraries were quantified by fluorometry (Promega Quantifluor) and inland NSW. For the Hunter Valley, 10 asymptomatic and normalized. An equimolar pool was created and adjusted (no visible cankers) and 10 symptomatic (exhibiting cankers) to 5 nM for sequencing on an Illumina MiSeq (San Diego, CA, × Verdelho vines (18 years old) were sampled. For Hilltops, 20 United States) with a V3, 600 cycle kit (2 300 base pair, years old Shiraz vines that had previously undergone remedial paired-end reads). surgery (Savocchia et al., 2014) were sampled. These vines (10 each) exhibited internal necroses or were necroses-free when Isolation of Fluorescent Pseudomonas trunks were cut during surgery. The selected vines were debarked From Inner Grapevine Tissues with a surface-sterilized knife and wood shavings (∼1 g) were As the next generation sequencing data for microbial profiling obtained using an 8 mm auger bit driven by a CDL-018 cordless revealed Pseudomonas to be the predominant endophytic drill drive (Ozito Industries Pty. Ltd., Victoria, Australia). The bacterium in grapevine tissues, these were targeted for isolation drill bit was sprayed with ethanol for each vine sampled to from the collected grapevine wood samples. Approximately prevent cross contamination. Wood shavings were placed in 0.5 g of wood shavings per sample were suspended in 6 ml sterile plastic containers and transported to the laboratory on ice. of 3 × Ringer’s solution (Surette et al., 2003). Samples were One half of each sample was stored at 4◦C prior to isolation of homogenized by incubating in a rotary shaker for 2 h at
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TABLE 1 | Primers and PCR conditions for 16S and ITS amplicons.
Target Cycle Initial Disassociate Anneal Extension Finish
ITS1F–ITS2 35 95◦C for 7 min 94◦C for 30 s 55◦C for 45 s 72◦C for 60 s 72◦C for 7 min 16S: V3–V4 29 95◦C for 7 min 94◦C for 30 s 50◦C for 60 s 72◦C for 60 s 72◦C for 7 min
240 rpm under ambient temperature. A dilution series of up to TABLE 2 | Fluorescent bacterial strains with antagonistic activity against GTD 10−6 was prepared using 3 × Ringer’s solution and plated on pathogens Diplodia seriata, Neofusicoccum parvum, N. luteum, and Eutypa lata. nutrient agar (NA, Oxoid Ltd., Hampshire, United Kingdom). Bacterial strains Identification based GenBank accession no. Well separated and distinct bacterial colonies were subcultured on 16S rRNA gene onto King’s B medium (King et al., 1954). Additional bacterial strains were further isolated from pruning stubs collected from BCA11 Pseudomonas poae MN480480 Cabernet Sauvignon vines from the vineyard of Charles Sturt BCA12 P. moraviensis MN480481 University, NSW, Australia. BCA13 P. poae MN480482 The pure cultures obtained from the vineyards were observed BCA14 P. poae MN480483 for fluorescence on King’s B media, under UV light at 365 nm BCA15 P. poae MN480484 BCA16 P. poae MN480485 after 3 days of incubation at 25◦C in the dark. Fluorescent BCA17 P. poae MN480486 bacterial strains from single colonies were stored in 20% BCA18 P. poae MN480487 glycerol at −80◦C. BCA19 P. poae MN480488 In vitro Screening for Bacterial BCA20 P. poae MN480489 Antagonists Against GTD Pathogens Bacterial Strains TABLE 3 | Fungal strains used for in vitro assays to test the antagonistic activity of Pseudomonas strains. A total of 57 fluorescent bacterial strains from inner grapevine wood tissues were tested for their antagonistic activity against Fungal species Isolate Herbarium References a the GTD pathogens: (a) Diplodia seriata; (b) Neofusicoccum accession parvum; (c) Neofusicoccum luteum; and (d) Eutypa lata, which Botryosphaeria BMV14 DAR79239 Pitt et al., 2010 are the most prevalent and virulent GTD pathogens in Australia. dothidea From this initial screen, 10 strains (Table 2) demonstrating Diplodia mutila FF18 DAR79137 Pitt et al., 2010 inhibition of the GTD pathogens were further tested for D. seriata A142a DAR79990 Qiu et al., 2011 their antagonistic activity against 14 GTD pathogens that are Dothiorella L5 DAR78993 Pitt et al., 2013 associated with Botryosphaeria dieback, Eutypa dieback, and vidmadera Esca/Petri disease (Table 3). Lasiodiplodia W200 DAR81024 Wunderlich et al., 2011 theobromae Fungal Strains Neofusicoccum SDW4 DAR79505 Pitt et al., 2010 australe Fourteen fungal strains associated with GTDs were obtained from N. luteum BB175-2 DAR81016 Wunderlich et al., 2011 the National Wine and Grape Industry Centre (Charles Sturt N. parvum B22a DAR80004 Qiu et al., 2011 University, Wagga Wagga, NSW, Australia) culture collection Spencermartinsia L19 DAR78870 Pitt et al., 2010 (Table 2). Each of the selected strains had been stored on viticola − ◦ agar discs in 20% glycerol at 80 C prior to testing. All Cryptovalsa KC6 Trouillas et al., 2011 Botryosphaeriaceae strains were associated with Botryosphaeria ampelina dieback symptoms in Australian vineyards (Pitt et al., 2010; Qiu Eutypa lata WB052 Billones-Baaijens et al., 2018 et al., 2011; Wunderlich et al., 2011), while all Diatrypaceae Eutypella citricola WA06FH Trouillas et al., 2011 strains were associated with Eutypa dieback symptoms (Trouillas Phaeoacremonium SMA 056 Billones-Baaijens et al., 2018 et al., 2011). The Phaeoacremonium minimum and Phaeomoniella minimum chlamydospora strains were obtained from the culture collection Phaeomoniella SMA 006 Billones-Baaijens et al., 2018 of the South Australian Research and Development Institute chlamydospora (SARDI, Adelaide, Australia). All strains were previously aDAR – Agricultural Scientific Collections Unit, NSW DPI, Orange, Australia. identified to species level by DNA sequencing of the ITS region of the rRNA and β-tubulin genes (Pitt et al., 2010, 2013; Qiu et al., 2011; Wunderlich et al., 2011; Billones-Baaijens et al., 2018). 6 mm in diameter were placed on opposite sides of potato dextrose agar (PDA, Difco Laboratories, Maryland, United States) Dual Culture Assays plates. A 10 µl Pseudomonas suspension (1 × 108 CFU/ml), To study the ability of fluorescent Pseudomonas spp. (Table 1) grown for 24 h in nutrient broth (NB, Oxoid Ltd., Hampshire, to inhibit growth of the GTD pathogens (Table 2), dual plating United Kingdom), was then pipetted in the centre of the PDA assays were performed. For each pathogen, mycelial plugs plates. Petri plates containing mycelial plugs of the pathogens
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only served as controls. Three replicate plates were used for each GenBank1 using the Basic Local Alignment Search Tool (BLAST) bacteria-fungal pathogen treatment combination, and incubated (Altschul et al., 1990) to determine the most probable identity at 25◦C under continuous darkness. Mycelial growth of the fungi for the 21 isolates examined. For phylogenetic analysis, published was measured after 7, 10, and 30 days to evaluate inhibition reference sequences for the 16S rRNA gene were obtained from of the Botryosphaeria dieback, Eutypa dieback and Esca/Petri the NCBI database and aligned with the 21 generated sequences, disease pathogens, respectively, by the antagonistic Pseudomonas by Clustal W within MEGA 7 (Kumar et al., 2016). The aligned species. Average fungal growth was determined by measuring the sequences were tested for their phylogeny (Tamura et al., 2004) perpendicular diameter of the mycelial growth of the respective and a neighbor joining tree was generated using MEGA 7 to GTD pathogens. Percentage mycelial growth inhibition was determine the relationships between the reference and unknown calculated using the formula: sequences (Saitou and Nei, 1987).
(C − T) Data Analysis Percentage mycelial growth inhibition = × 100 C Paired-ends reads were assembled by aligning the forward and reverse reads using PEAR version 0.9.5 (Zhang et al., 2014). Where C = colony diameter (mm) of the control Primers were identified and removed, with trimmed sequences T = colony diameter (mm) of the test plate being processed using Quantitative Insights into Microbial In the case of Ph. minimum, the bioassays were conducted Ecology (QIIME 1.8) (Caporaso et al., 2010), USEARCH (version using a spore suspension of the fungi in the well dual culture. 7.1.1090) (Edgar, 2010; Edgar et al., 2011), and UPARSE (Edgar, Ph. minimum readily produces abundant spores in PDA and 2013) software. Within USEARCH, sequences were quality spores were harvested from a 7 days old culture of the fungus. filtered, full length duplicate sequences removed, and reads Spore concentration was adjusted to 1 × 106 spores/ml with a sorted by abundance. Singletons or unique reads in the data set hemocytometer and 100 µl of the spore suspension was spread were discarded. Sequences were clustered followed by chimera onto PDA plates. Four equidistant holes were created in the filtering using “rdp_gold” database as the reference. To obtain media using a flame-sterilized cork borer (4 mm) and 10 µl the number of reads in each OTU, reads were mapped back to of the bacterial suspension was pipetted into each well. For the OTUs with a minimum identity of 97%. Taxonomy was assigned control plates, sterile distilled water was pipetted into the wells. by QIIME using the Greengenes database (version 13_8 Aug Three replicate plates per treatment combination were used. The 2013) (DeSantis et al., 2006). In the case of ITS sequences, these diameter of the zone of inhibition was measured around each well were clustered followed by chimera filtering using the “Unite” after 7 days of incubation under the conditions described above. database as reference. To obtain number of reads in each OTU, reads were mapped back to OTUs with a minimum identity Identification of Selected Bacterial Strains by 16S of 97%. Taxonomy was assigned by QIIME using the Unite rRNA Gene Sequencing database (Unite Version7.1 2016-08-22) (Kõljalg et al., 2005). The 10 fluorescent bacteria that significantly inhibited the Unless mentioned, default settings were used for all procedures. mycelial growth of the GTD fungal pathogens and 11 additional Microbial diversity profiling data were generated from nine non-antagonistic fluorescent bacterial strains were selected for and eight samples each from asymptomatic and symptomatic molecular identification. The bacterial growth conditions and vines from Hilltops and the Hunter Valley, respectively. Only DNA extraction method were as described by Niem et al.(2019), those taxa that represent more than 1% of the bacterial and using the Gentra Puregene Bacterial DNA Extraction Kit (Qiagen, fungal operational taxonomic units (OTUs) were included in United States) following the manufacturer’s instructions. the data set (Deyett et al., 2017). Data for 0.01–0.9% of the The bacterial species were identified though PCR fungal OTUs were included as an inset to take into account amplification of 16S rRNA gene using the universal primers fungal genera that were obscured by high relative abundance of 8F and 1492 R (I) (Turner et al., 1999). PCR amplification was the dominant genus. Chloroplast and mitochondrial sequences performed with 1 µl of genomic DNA template in a 25 µl reaction or OTUs with unidentified taxa were removed. Percent mixture containing 1× PCR buffer (Bioline, United Kingdom), relative abundance of the bacterial and fungal taxa inhabiting 1.25 U of My Taq DNA polymerase (Bioline, United Kingdom) the grape inner tissues was calculated. The compositional and 0.4 µM of each primer. PCR was performed with a thermal diversity of the fungal and bacterial endophytic communities cycler (C100 Thermal Cycler, BIO-RAD Laboratories Pty. were considered separately. Diversities were compared with Ltd., United States) under the following conditions: initial alpha (Chao1 and Simpson) and beta (Bray-Curtis index) denaturation for 3 min at 95◦C; 35 denaturation cycles of 15 diversity statistics within the Marker Data Profiling module s at 95◦C, annealing for 1 min at 56◦C, extension for 1 min at of MicrobiomeAnalyst (Dhariwal et al., 2017), implementing 70◦C; and a final extension of 8 min at 72◦C. PCR products were R version 3.6.1 (2019-07-05). Abundance data for bacteria purified with a FavorPrep Gel/PCR purification kit (Favorgen and fungi were normalized by data transformation using Biotech Corp, United States) and sequenced at the AGRF. centered log ratio, as recommended by Gloor et al.(2017). To identify the bacterial species, all DNA sequences and Differences in the Chao1 and Simpson index among factors chromatographs were analyzed and trimmed using Chromas Lite (location and presence/absence of symptoms) were determined Version 2.6.2 (Technelysium Pty. Ltd.) and DNAMAN Version 6.0.3.99 (Lynnon Biosoft) and compared with sequence data in 1www.ncbi.nlm.nih.gov
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via ANOVA when considering all factors, or T-tests for account the differences between asymptomatic and symptomatic factors individually. For the beta diversity, differences were samples from within each location were determined. For visualized using non-metric multidimensional scaling (NMDS). both Hilltops and the Hunter Valley asymptomatic samples Permutational multivariate analysis of variance (PERMANOVA) were lower in terms of OTU richness than symptomatic, was used to test which factors best explained the NMDS. however, the differences were not significant. For richness For the dual culture assay, all data were analyzed within and evenness (Simpson) there were no significant differences GenStat (18th edition). Since mycelial growth rate differed between factors. between Botryosphaeria dieback, Eutypa dieback and Esca/Petri In regards to the composition of bacterial communities, non- disease pathogens, assessments for each pathogen group were metric multidimensional scaling and PERMANOVA analysis done at different periods and analyzed separately. Percent indicated that communities could be separated when considering mycelial diameter inhibition for each pathogen group was all factors (R-squared = 0.24, P ≤ 0.05; Figure 1B). This was initially tested for homogeneity using Levene’s test at P ≤ 0.05. attributable to compositional differences between location (R- Effects of treatments were determined by two-way analysis of squared = 0.14, P < 0.01) rather than expressed symptomology variance (ANOVA).Pairwise comparison of treatment means was (R-squared = 0.07, P > 0.05). conducted using least significant differences (LSD) at P ≤ 0.05. When considering abundance at the phylum and genus level, in all samples, the dominant phylum among the prokaryotic community was Proteobacteria, accounting for 51–96% of the RESULTS total reads for the given taxa followed by Actinobacteria with 3–44%. The phyla Bacteroidetes, Firmicutes, and Chloroflexi Microbial Profile of Grapevine were detected in less than 10% of the reads. At the genus Endosphere level, with the exception of symptomatic samples from the A total of 48,461 bacterial 16S rRNA gene sequences and Hunter Valley at 2% relative abundance, the most abundant 1,567,896 fungal ITS sequences were generated after removing genus within the phylum Proteobacteria was Pseudomonas mitochondrial and chloroplast sequences. These sequences with 75 and 29% in asymptomatic and symptomatic samples, were assigned to 573 bacterial and 244 fungal operational respectively, from Hilltops, and 56% in asymptomatic vines from taxonomic units (OTUs). the Hunter Valley. It is also noteworthy that in both locations, Pseudomonas had higher relative abundance in samples Bacterial Community asymptomatic for GTDs (Figures 2A, 3A) as compared to Alpha diversity analysis indicated that significant differences samples from symptomatic vines (Figures 2B, 3B). Symptomatic existed in terms of richness (Chao1) within communities vines harbored higher diversity of the most abundant genera of bacteria considering all factors (F = 7.13, P ≤ 0.05). accounting for 19 and 15 bacterial genera from Hilltops and Comparison between location suggested that the Hilltops region the Hunter Valley, respectively. In contrast, asymptomatic had significantly greater diversity in terms of OTU richness than samples host a total of nine genera in Hilltops and 10 in the Hunter Valley (t = 3.07, P ≤ 0.05; Figure 1A). Taking this in to the Hunter Valley. The genera Pseudomonas, Sphingomonas,
FIGURE 1 | Diversity analysis of bacterial endophytic communities sampled from GTD symptomatic and asymptomatic vines, in the Hilltops and Hunter Valley regions. Abundance of OTUs was determined following the sequencing of the V3–V4 region of 16s rRNA genes: (A) Alpha-diversity measure using Chao1 at the OTU level, with each boxplot representing the diversity distribution of each factor (location and symptomology); and (B) Ordination based upon beta diversity analysis using the Bray Curtis index. Closed circles = Hilltops, asymptomatic; closed squares = Hilltops, symptomatic; X = hunter Valley, asymptomatic; O = Hunter Valley, symptomatic.
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FIGURE 2 | Genera of grapevine endophytic bacteria from Hilltops, NSW and their relative abundance to grapevines that were: (A) asymptomatic; and (B) symptomatic to grapevine trunk diseases.
Agrobacterium, Deviosa, Friedmanniella, and Pseudonocardia which causes crown gall in grapevines, was identified in 1–9% of were present in both asymptomatic and symptomatic samples the total bacterial reads. from Hilltops. The composition of bacterial genera from the Hunter Valley appeared to be more unique with only Fungal Community Pseudomonas, Rhodoplanes, Coprococcus, and Deviosa shared In terms of the fungal community, the richness of OTUs were between asymptomatic and symptomatic samples. Other significantly different across all factors (F = 1.3, P ≤ 0.05), being agriculturally important bacterial genera that occurred driven by location with Hilltops greater than the Hunter Valley in greater than 1% relative abundance were Streptomyces (t = 2.12, p = 0.05; Figure 4A). When richness and evenness and Agrobacterium. Streptomyces, which was detected in were considered, no significant differences at the OTU level asymptomatic samples from Hilltops (6%), is implicated in plant were present. The compositional analysis of fungal communities, disease control and plant growth promotion. Agrobacterium, analyzed for beta diversity, indicated that while communities
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FIGURE 3 | Genera of grapevine endophytic bacteria from the Hunter Valley, NSW and their relative abundance to grapevines that were: (A) asymptomatic; and (B) symptomatic to grapevine trunk diseases.
could be clustered, these were not strict and no significant P. chlamydospora), representing 59–89% of the total fungi differences were evident (R = 0.21, P > 0.05; Figure 4B). detected (Figures 5, 6). Phaeomoniella was more abundant in The eukaryotic microbiome was mostly characterized by asymptomatic tissues accounting for 89 and 74% in Hilltops and a preponderance of the phylum Ascomycota with greater the Hunter Valley (Figures 5A, 6A), respectively as compared than 99% relative abundance from both asymptomatic and to symptomatic tissues with 78 and 59% relative abundance symptomatic samples from Hilltops and asymptomatic tissues (Figures 5B, 6B). This was followed by Phaeoacremonium from the Hunter Valley. The relative abundance of Ascomycota species, specifically Ph. iranianum (5–21%) save for a divergence in symptomatic vines from the Hunter Valley was 83% while in asymptomatic vines from the Hunter Valley where Inonotus phylum Basidiomycota was 17%. At the genus level, the fungal from the phylum Basidiomycota came in second at 18% relative community was predominated by Phaeomoniella (primarily abundance. Phaeomoniella and Phaeoacremonium species are
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FIGURE 4 | Diversity analysis of fungal endophytic communities sampled from GTD symptomatic and asymptomatic vines, in the Hilltops and Hunter Valley regions. Abundance of OTUs was determined following the sequencing of the ITS regions of rRNA genes: (A) Alpha-diversity measure using Chao1 at the OTU level, with each boxplot representing the diversity distribution of each factor (location and symptomology); and (B) Ordination based upon beta diversity analysis using the Bray Curtis index. Closed circles = Hilltops, asymptomatic; closed squares = Hilltops, symptomatic; X = hunter Valley, asymptomatic; O = Hunter Valley, symptomatic.
GTD pathogens that are widely associated with Petri and 12.0 to 43.1% with BCA11 and BCA13 having the highest Esca diseases of grapevines. Inonotus has also been isolated inhibition (Table 4). BCA15 was the least inhibitory of the in some grapevines exhibiting Esca symptoms. Other trunk isolates examined. Sensitivity to BCAs was also significantly disease pathogens, Cryptovalsa, Diplodia, and Neofusicoccum different (P ≤ 0.05) among the nine Botryosphaeria dieback were also detected in low abundance (0.01–1%). Some fungi pathogens that were tested, with Dothiorella vidmadera implicated with various diseases of grapes and grapevines such being the most sensitive (52.3% average) (Figures 7A,B) as Alternaria, Stemphylium, Phomopsis, Phyllostica, Penicillium, and N. parvum and N. luteum (15.4–18.5%) being the Phoma, Phialophora, and Aspergillus were also noted in 0.01– least sensitive. 2% of the eukaryotic population. Fungi with known biocontrol potential, including Aureobasidium, Epicoccum, and Arthobotrys Antagonism Against Eutypa Dieback Pathogens were also found to inhabit the inner grapevine tissues. All 10 Pseudomonas BCA isolates showed antagonistic activity against Eutypa dieback pathogens, however, mycelial growth Isolation of Fluorescent Pseudomonas inhibition differed significantly between bacterial strains and pathogens. Average mycelial inhibition caused by the From Inner Grapevine Tissues different bacteria ranged from 8.3 to 64.6% with BCAs A total of 200 bacterial endophytes from Hilltops and the 11, 13, 14, 18, 19, and 20 having the greatest inhibition Hunter Valley samples and 10 bacterial strains from the CSU (Table 5). BCA15 was the least inhibitory among the vineyard were isolated. From these, 57 isolates resembling BCAs. Sensitivity to BCAs was also significantly different Pseudomonas fluoresced under UV light at 365 nm when (P ≤ 0.05) among the three Eutypa dieback pathogens that grown in King’s B medium. From among the 57 bacteria, 10 were tested, with Cryptovalsa ampelina (Figures 7C,D) and isolates exhibited antagonistic activity against the selected GTD Eutypella citricola being more sensitive (53.8–51.0%) than pathogens (data not shown). E. lata (49.9%).
In vitro Screening for Bacterial Antagonism Against Esca/Petri Disease Pathogens The 10 Pseudomonas BCA isolates showed varying degrees Endophytes Antagonistic Toward GTD of antagonistic activity against Esca/Petri disease pathogens. Pathogens Mycelial growth inhibition was significantly affected by both Antagonism Against Botryosphaeria Dieback BCAs and pathogens. Average mycelial inhibition caused by the Pathogens different bacteria ranged from 9.7 to 44.9% with BCA11 and The 10 Pseudomonas isolates showed varying degrees of BCA13 having the highest inhibition (Table 6). BCA12 was antagonistic activity against Botryosphaeria dieback pathogens. the least inhibitory among the BCAs. Sensitivity to BCAs was Inhibition of mycelial growth was significantly affected by also significantly different (P ≤ 0.05) between the Esca/Petri both BCAs and pathogens. Average mycelial inhibition disease pathogens that were tested, with P. chlamydospora caused by the different isolates of bacteria ranged from being more sensitive (55.0% average) (Figures 7E,F) than Ph.
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FIGURE 5 | Genera of grapevine endophytic fungi from Hilltops, NSW and their relative abundance to grapevines that were: (A) asymptomatic; and (B) symptomatic to grapevine trunk diseases.
minimum (14.5%). However, although inhibition of mycelial 21 mm and were significantly different from the control at growth by the BCAs was not as pronounced in Ph. minimum, P ≤ 0.05 (Figure 9). an apparent change in the color along the edges of the hyphal colonies was observed (Figures 7G,H). Co-inoculation of Ph. minimum with the BCAs resulted in the disappearance Identification of Selected Bacteria by of mycelial pigmentation along the margin indicating spore 16S rRNA Gene Sequencing production was inhibited by the BCAs. Further tests on Querying the Genbank database with the 16S rRNA gene spore suspensions of Ph. minimum in a well dual culture sequences generated from the 12 selected bacterial isolates, assay showed the presence of zones of inhibition when and those from the CSU vineyard, indicated these belonged to cell suspensions of BCAs 13, 14, and 17 were present. the genus Pseudomonas. Phylogenetic analysis of the aligned Among the three BCAs, BCA11 produced the largest zone of sequences from the bacterial strains and reference sequences inhibition at 21 mm (Figure 8). Average zones of inhibition obtained from Genbank resulted in a neighbor joining tree with produced by the three bacterial strains ranged from 19 to two major branches (Figure 10). The first main branch was
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FIGURE 6 | Genera of grapevine endophytic fungi from the Hunter Valley, NSW and their relative abundance to grapevines that were: (A) asymptomatic; and (B) symptomatic to grapevine trunk diseases.
occupied by eight strains from CSU, five from Hilltops, and clustered with P. azotoformans. The second main branch was one from the Hunter Valley. This was further divided into two occupied by one strain from CSU, three from Hilltops, and three sub-branches. The first sub-branch consisted of eight strains from the Hunter Valley and was divided into two sub-branches. from CSU and one strain each from Hilltops and the Hunter The first sub-branch indicates a close relationship to P. koreensis. Valley, which clustered with P. poae reference strains. The second The second sub-branch is composed of one strain from CSU, sub-branch was composed of four strains from Hilltops, which two from Hilltops, and three from the Hunter Valley, which
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TABLE 4 | Mycelial growth inhibition of Botryosphaeria dieback pathogens by Pseudomonas strains as potential biocontrol agents (BCAs).
BCA Percent mycelial growth inhibition (%)d
BDa DMa DSa DoVa LTa NAa NLa NPa SVa BCA meanb
11 70.4 a 26.3 abc 45.5 ab 66.5 ab 41.0 a 46.8 a 30.3 a 20.4 ab 40.9 b 43.1 A 12 58.1 a 0.0 d 47.6 a 0.0 d 38.5 a 0.0 d 0.0 c 10.1 ab 62.0 a 24.0 F 13 68.9 a 25.9 abc 37.6 abcd 53.5 bc 44.4 a 36.0 ab 29.5 a 22.2 a 58.1 a 41.7 A 14 64.1 a 29.4 ab 39.2 abc 45.8 c 23.5 b 37.8 ab 25.0 ab 21.0 ab 42.7 b 36.5 B 15 12.6 b 14.1 c 12.6 e 11.0 d 11.5 b 12.3 cd 12.0 bc 8.5 b 13.1 c 12.0 G 16 25.4 b 19.7 bc 28.1 cd 69.4 a 12.4 b 17.6 c 14.2 b 12.0 ab 40.9 b 26.6 EF 17 19.5 b 24.1 abc 25.5 de 72.8 a 13.3 b 15.6 c 14.9 b 12.6 ab 43.1 b 26.8 EF 18 22.9 b 29.2 ab 29.5 cd 77.6 a 13.8 b 17.8 c 12.6 bc 13.5 ab 44.6 b 29.0 DE 19 22.9 b 35.5 a 32.2 cd 79.1 a 22.0 b 17.6 c 20.8 ab 13.1 ab 45.0 b 32.0 CD 20 59.7 a 22.6 abc 33.8 bcd 47.6 c 24.0 b 32.4 b 25.4 a 21.0 ab 38.5 b 33.9 BC Pathogen meanc 42.3 W 22.7 Y 33.1 X 52.3 V 24.4 Y 23.4 Y 18.5 Z 15.4 Z 42.9 W
aMeans within a column followed by different letters are significantly different at P ≤ 0.05 LSD = 13.055 for BCA x pathogen interaction. bBCA means within a column followed by different letters are significantly different at P ≤ 0.05 LSD = 4.352 for BCA means. cPathogen means within a row followed by different letters are significantly different at P ≤ 0.05 LSD = 4.128 for pathogen means. dBD, Botryosphaeria dothidea; DM, Diplodia mutila; DS, D. seriata; DoV, Dothiorella vidmadera; LT, Lasiodiplodia theobromae; NA, Neofusicoccum australe; NL, N. luteum; NP, N. parvum; SV, Spencermartinsia viticola.
FIGURE 7 | Mycelial growth inhibition of representative GTD pathogens by antagonistic Pseudomonas BCA strains: (A) Dothiorella vidmadera + BCA19, (B) Do. vidmadera only, (C) Cryptovalsa ampelina + BCA14, (D) C. ampelina only, (E) Phaeomoniella chlamydospora + BCA11, (F) P. chlamydospora only, (G) Phaeoacremonium minimum + BCA13, (H) Ph. minimum only.
clustered with P. moraviensis. The fluorescent bacterial strains are asymptomatic wood tissues of grapevines in Australia, and the all from the P. fluorescens complex (Garrido-Sanz et al., 2017). outcomes suggest that sampling of a number of agroecological P. aeruginosa was included in the tree as an outgroup. Nine out zones would be beneficial to improve our understanding of of the 10 fluorescent bacterial strains (BCA11, BCA13–BCA20) endophytic communities. The microbial diversity data revealed that inhibited the mycelial growth of the GTD pathogens in the a preponderance of the genus Pseudomonas in grapevine tissues dual culture assay belong to the P. poae group. The remaining that were asymptomatic to GTD in both locations, accounting antagonistic strain, BCA12, is closely related to P. moraviensis. for 56–75% relative abundance and in symptomatic tissues from Hilltops with 29% abundance. This concurs with the results of Bell et al.(1995), Campisano et al.(2015), Faist et al.(2016), DISCUSSION and Deyett et al.(2017) who reported Pseudomonas to dominate the bacterial assemblage in grapevines in separate This study investigated the diversity of the endomicrobiome studies conducted in California, Germany, Italy, and Canada, associated with grapevines with and without symptoms of GTDs. respectively. Interestingly, in a study conducted in Australia, Overall results showed that diversity of bacterial and fungal West et al.(2010) reported Bacillus to be the most frequently communities of grapevines with and without GTD symptoms isolated bacterium in grapevine canes, comprising 55% of the differed between the regions examine, suggesting potential total. Pseudomonas spp., Curtobacterium spp., Burkholderia spp., interactions between the beneficial and pathogenic organisms and Streptomyces spp. accounted for 34% of the total endophytic within a grapevine. To our knowledge, this study is the first bacteria isolated. Bacillus was not detected in the current samples attempt to investigate the microbiome of GTD symptomatic and even though these were also collected from grapevines in
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TABLE 5 | Mycelial growth inhibition of the Eutypa dieback pathogens by endophytes in grapevines, as well as genus richness, with greater Pseudomonas strains as potential biocontrol agents (BCAs). genus richness observed in younger vines. The results obtained in BCA Percent mycelial growth inhibition (%) this current research indicated higher diversity of bacterial genera in symptomatic vines, hosting 15–19 genera as compared with Cryptovalsa Eutypa lataa Eutypella BCA meanb 9–10 for asymptomatic vines. While these were not found to be ampelinaa citricolaa significant differences, these were used to provide the indicator
11 70.3 ab 54.8 abc 63.7 a 62.9 AB for identifying organisms contributing to the effect. Other factors 12 6.5 d 49.8 c 27.3 c 27.9 D may also influence the composition of the grapevine microbiome. 13 70.6 ab 50.9 c 60.2 ab 60.6 ABC Campisano et al.(2015) found bacterial endophytic communities 14 77.1 a 51.8 c 65.0 a 64.6 A to be affected by the pest management strategy employed in 15 6.2 d 1.8 d 16.8 d 8.3 E the vineyard, and revealed differences in microbial composition 16 56.5 c 53.4 bc 55.4 ab 55.1 C in grapevines cultivated using organic production as opposed 17 59.3 c 57.8 abc 56.6 ab 57.9 BC to those using integrated pest management (IPM). The health 18 61.6 bc 62.6 ab 56.0 ab 60.1 ABC status of the vine (Bulgari et al., 2011; Bruez et al., 2014; Faist 19 59.3 c 65.2 a 53.1 b 59.2 ABC et al., 2016), the growing region, cultivar and climate (Bokulich 20 70.8 ab 51.1 c 55.0 ab 58.9 ABC et al., 2013; Bruez et al., 2014; Portillo et al., 2016) may also Pathogen meanc 53.8 X 49.9 Y 51.0 XY shape the community of microorganisms within the grapevine. The results presented in this current research concur with past aMeans within a column followed by different letters are significantly different at P ≤ 0.05 LSD = 10.462 for BCA × pathogen interaction. bBCA means within a findings as location was the influencing factor for differences in column followed by different letters are significantly different at P ≤ 0.05 LSD = 6.04 endophytic diversity. for BCA means. cPathogen means within a row followed by different letters are Higher relative abundance of Pseudomonas in asymptomatic significantly different at P ≤ 0.05 LSD = 3.308. tissues observed in this study may impart a suppressive effect on GTD pathogens, and suggests a plausible antagonistic role TABLE 6 | Mycelial growth inhibition of the Esca/Petri disease pathogens by of Pseudomonas in inhibiting symptoms of GTDs. In a similar Pseudomonas strains as potential biocontrol agents (BCAs). study, Deyett et al.(2017) found a decrease in the relative BCA Percent mycelial growth inhibition of abundance of Xylella fastidiosa, a xylem-limited bacterium Esca/Petri disease pathogens (%) causing Pierce’s disease in grapevines, in the presence of Achomobacter xylosoxidans and P. fluorescens. The presence of Phaeomoniella Phaeoacremonium BCA meanb grapevines asymptomatic for Pierce’s disease in vineyards with chlamydosporaa minimuma high disease pressure was attributed to the microbial community 11 70.3 a 17.3 abc 43.8 A that inhabits the grapevine vascular endosphere. 12 19.3 f 0.0 d 9.7 E This study also showed that the eukaryotic community was 13 64.0 ab 25.8 a 44.9 A dominated by the genus Phaeomoniella in both asymptomatic 14 53.7 cde 17.5 abc 35.6 BCD and symptomatic grapevines, representing 74–89% and 59– 15 60.7 abc 8.3 cd 34.5 BCD 78% of the total fungi, respectively. Phaeoacremonium came in 16 59.7 bcd 12.6 bc 36.1 BCD second, with 5–21% relative abundance except in asymptomatic 17 64.0 ab 13.6 bc 38.8 ABC vines from the Hunter Valley where Inonotus had a higher 18 50.3 de 15.0 bc 32.7 C relative abundance at 18%. P. chlamydospora and Ph. aleophilum 19 47.7 e 15.2 bc 31.5 D (syn. Ph. minimum) have been implicated in two GTDs: Petri 20 60.7 abc 19.3 ab 40.0 AB disease (previously known as black goo decline) and Esca Pathogen meanc 55.0 X 14.5 y (Crous and Gams, 2000; Mostert et al., 2006b). Inonotus has also been reported in grapevines exhibiting white heart rot, a aMeans within a column followed by different letters are significantly different at P ≤ 0.05 LSD = 10.462 for BCA x pathogen interaction. bBCA means within symptom that is also generally associated with Esca (Edwards a column followed by different letters are significantly different at P ≤ 0.05 and Pascoe, 2004; Fischer et al., 2005; González et al., 2009; LSD = 6.604 for BCA means. cPathogen means within a row followed by different White et al., 2011). Occurrence of these three dominant fungi letters are significantly different at P ≤ 0.05 LSD = 3.308 for pathogen means. in grapevines showing symptoms of Petri disease and Esca has previously been reported in Australian vineyards. Petri disease New South Wales, Australia. The difference in the microbial was found to be expressed in 82% of the diseased samples composition may be explained by the difference in the age of (from a total of 124 symptomatic samples) while only 18% were the vines as West et al.(2010) sampled from relatively younger diagnosed with Esca (Edwards and Pascoe, 2004). Moreover, vines (10 years old) as compared to the ≥20 years old grapevines P. chlamydospora was found to be widely distributed in Australia sampled in this study. Similar observations were reported by and was detected in Western Australia, South Australia and Bruez et al.(2015) where Bacillus sp. and Pantoea agglomerans Victoria in 98% of the samples while Ph. minimum was isolated were the most commonly isolated bacteria from 12 years old in only 15% of the samples. The current fungal diversity profile grapevine wood samples either symptomatic or asymptomatic for indicates P. chlamydospora to be the dominant fungal species but esca-foliar symptoms. Andreolli et al.(2016) identified age of the Phaeoacremonium is from the species Ph. iranianum in contrast vine as an important factor affecting the composition of bacterial to Ph. minimum that was previously reported in Australia.
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FIGURE 8 | Antagonistic effect of Pseudomonas BCA strains on Esca/Petri disease pathogens: (A) Phaeomoniella chlamydospora + BCA17, (B) spores of P. chlamydospora only, (C) Phaeoacremonium minimum + BCA17, (D) spores of Ph. minimum only.
FIGURE 9 | Antagonistic effect of selected Pseudomonas BCA strains on Phaeomoniella chlamydospora and Phaeoacremonium minimum.
Ph. iranianum, which has been reported to be isolated from when the grapevines are stressed, after which Petri disease starts Vitis vinifera in Iran, Italy and South Africa (Mostert et al., to manifest. Furthermore, symptoms of Esca are erratic and may 2006a; White et al., 2011) is phylogenetically and morphologically not show every year (Edwards et al., 2001). Hofstetter et al. (2012) close to Ph. minimum and can only be differentiated by the also found the same taxa of presumed Esca-associated fungal predominance of type III phialides and by its subcylindrical pathogens, including P. chlamydospora and Phaeoacremonium type II phialides (Mostert et al., 2006a). Its presence indicated spp., in both diseased and healthy grapevines that were 15– here by diversity profiling would need to be confirmed by these 30 years old. This indicates that these fungi are part of the conventional techniques and molecular methods using β-tubulin normal mycota associated with adult grapevines. Ferreira et al. and ITS genes. (1999) reported water stress to be a predisposing factor for The diversity profiling revealed a higher abundance of P. chlamydospora infection and slow dieback of grapevines in Phaeomoniella in asymptomatic grapevines than in the South Africa. Plant genotype (Marchi, 2001), age (Mugnai et al., symptomatic tissues. In France, Bruez et al. (2016) was able to 1999), and climate (van Niekerk et al., 2011) are also factors that recover the Esca pathogens, Ph. minimum and P. chlamydospora may affect the susceptibility of the grapevines to the disease. and the white rot fungi, Fomitiporia mediterranea and Stereum Environmental changes may also elicit various plant responses hirsutum, consisting 50% of all the fungal taxa isolated, from 42 (i.e., development of thicker wax layers on leaves or changes in years old vines that did not express symptoms of grapevine trunk stomatal densities) that could affect infection and expression of disease. Edwards and Pascoe (2004) were also able to isolate symptoms (Fischer and Peighami Ashnaei, 2019). P. chlamydospora in symptomless grapevines, as did Mugnai Abundance of the Pseudomonas spp. in asymptomatic et al. (1999). They postulated that the fungus may initially behave grapevines led to the further investigation of the antagonistic as an endophyte or latent pathogen, only becoming pathogenic
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FIGURE 10 | Neighbor joining tree obtained from the 16S rRNA gene sequence data of fluorescent bacteria isolated from inner grapevine tissues. The red dotted line indicates the main branches of the tree. Strains with red symbols are from the CSU vineyard, green from Hilltops, and yellow from the Hunter Valley. Reference sequences obtained from the NCBI data base are indicated by their accession numbers. The tree is rooted to the outgroup P. aeruginosa.
activity of these bacteria against GTDs. The results of the Pseudomonas spp. are capable of producing potent antifungal dual culture assays revealed 10 strains of grapevine endophytic agents that leached into the agar and inhibited the growth of Pseudomonas with antagonistic activity against Botryosphaeria the fungal pathogens. Of the Pseudomonas strains identified, dieback, Eutypa dieback and Esca/Petri disease pathogens nine out of the 10 were closely related to P. poae. P. poae, based on their ability to inhibit mycelial growth in culture. originally isolated from the phyllosphere of grasses, has been Pseudomonas spp. also appeared to affect spore production reported as an endorhiza of sugar beet (Behendt et al., 2003; of Ph. minimum as demonstrated by the presence of a zone Müller et al., 2013; Xia et al., 2019). It is used as a BCA of inhibition between the bacterium and the spores of the and is applied as a seed treatment to suppress late root rot pathogen in a well dual culture assay. This suggests that the in sugar beet (Zachow et al., 2010). The other antagonistic
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bacterium, BCA12, belongs to the same clade as P. moraviensis DATA AVAILABILITY STATEMENT which has been reported as a soil bacterium implicated in the suppression of Rhizoctonia diseases of potato (Tvrzová et al., The datasets generated for this study can be found in 2006; Mrabet et al., 2013; Miller et al., 2016). However, the in vitro the Genbank Accession numbers: MN480480-MN480489. Raw assay conducted in the current study indicated that BCA12 sequence data for the diversity profiling is available through was less inhibitory compared to the other nine BCAs from the BioProject PRJNA605898. P. poae group. Although P. poae has been identified to have biocontrol potential, the exact mechanism of control is yet to be elucidated. AUTHOR CONTRIBUTIONS Unlike other species of Pseudomonas that are more popularly known to produce 2,4 diacetylphloroglucinol, phenazine 1- JN conducted the research, analyzed the data, and wrote the carboxylic acid and its derivatives, and the siderophores manuscript. RB-B, SS, and BS contributed to the direction of the pyoluteorin and pyrrolnitrin, which are antifungal metabolites research and advised on analysis and interpretation of the data. identified with Pseudomonas, P. poae lacks the ability to All co-authors revised the manuscript. produce these compounds (Mavrodi et al., 2012). Instead, it was found to be a good rhizosphere colonizer with exoprotease activity (Mavrodi et al., 2012). Niem et al.(2019) identified putative biocontrol gene clusters in the genomes of BCAs 13, FUNDING 14, and 17 (P. poae) responsible for the biosynthesis of the secondary metabolites Poaeamide, Rhizomide and Rhizoxins. This work was supported by Charles Sturt University, and Gene clusters for production of lipopeptide antibiotics, Australia’s grape growers and winemakers though their siderophores, proteases, detoxification, lipopolysaccharide, investment body Wine Australia, with matching funds from the multidrug resistance, microbe-associated molecular proteins Australian Government. (MAMPS), and biofilms were likewise detected (Niem et al., 2019). Production of other antifungal compounds, possibly not previously reported in other Pseudomonas species, requires ACKNOWLEDGMENTS further investigation. Furthermore, the efficacy of P. poae to suppress trunk disease infection in planta should be investigated We acknowledge the financial support provided by Charles to ascertain its efficacy as a biocontrol agent. Sturt University’s Faculty of Science in the publication of the The results of this study indicate that some grapevine manuscript. Our gratitude also goes to Australia’s wine grape endophytic bacteria such as P. poae may be potential BCAs growers for allowing the collection of samples from their which could be used to control GTD pathogens. Further tests vineyards. Special thanks also to the Museum of Natural History, are currently being conducted to assess the bioactivity of these University of the Philippines Los Baños. We further acknowledge BCAs in planta. the editorial assistance of Mark Filmer.
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Xia, Y., DeBolt, S., Ma, Q., McDermaid, A., Wang, C., Shapiro, N., et al. (2019). Conflict of Interest: The authors declare that the research was conducted in the Improved draft genome sequence of Pseudomonas poae A2-S9, a strain with absence of any commercial or financial relationships that could be construed as a plant growth-promoting activity. Microbiol. Resour. Announc. 8:e00275-19. doi: potential conflict of interest. 10.1128/MRA.00275-19 Zachow, C., Fatehi, J., Cardinale, M., Tilcher, R., and Berg, G. (2010). Strain-specific Copyright © 2020 Niem, Billones-Baaijens, Stodart and Savocchia. This is an open- colonization pattern of Rhizoctonia antagonists in the root system of sugar access article distributed under the terms of the Creative Commons Attribution beet. FEMS Microbiol. Ecol. 74, 124–135. doi: 10.1111/j.1574-6941.2010.00 License (CC BY). The use, distribution or reproduction in other forums is permitted, 930.x provided the original author(s) and the copyright owner(s) are credited and that the Zhang, J., Kobert, K., Flouri, T., and Stamatakis, A. (2014). PEAR: a fast and original publication in this journal is cited, in accordance with accepted academic accurate Illumina Paired-End reAd mergeR. Bioinformatics 30, 614–620. doi: practice. No use, distribution or reproduction is permitted which does not comply 10.1093/bioinformatics/btt593 with these terms.
Frontiers in Microbiology| www.frontiersin.org 19 March 2020| Volume 11| Article 477
Chapter 3
Chapter 3.
Biocontrol potential of endophytic Pseudomonas against Neofusicoccum luteum, a fungal pathogen causing Botryosphaeria dieback, and its persistence in grapevine tissues
3.1. Introduction
In recent years, there has been an increasing interest in biocontrol of plant
pathogens, particularly in the use of resident antagonists which are better known as
endophytes (Ryan et al., 2008; Cabanás et al., 2014; Rybakova et al., 2015; Hong and
Park, 2016). The environmental pollution caused by excessive use and misuse of
agrochemicals, and the hazard that these chemicals pose to human health and the
ecosystem, has led to strict regulations on chemical pesticide use and banning of the
most hazardous from the market. This has paved the way for research efforts to discover
alternative methods for controlling plant diseases. Among those alternatives is biological
control. Biological control as defined by Baker (1987) is the “decrease of inoculum or the
disease- producing activity of a pathogen accomplished through one or more
organisms”.
Application of biocontrol agents (BCAs) for the control of grapevine trunk
diseases (GTDs) has been explored previously due to the limited number of
management options available. The use of Trichoderma spp. for the control of Eutypa
lata on grapevines have been extensively studied (John et al., 2005; John et al., 2008;
Kotze et al., 2011; Mutawila et al., 2011) and is now available in commercial formulations
in South Africa, New Zealand, Australia, Europe, USA, and South America. Biological
control of E. lata using other microorganisms, including Bacillus subtilis (Ferreira et al.,
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1991; Schmidt et al., 2001; Kotze et al., 2011), Erwinia herbicola (Schmidt et al., 2001),
Fusarium lateritium (Munkvold and Marois, 1993b; John et al., 2005) and Cladosporium herbarum (Munkvold and Marois, 1993b; Rolshausen and Gubler, 2005), has demonstrated varying levels of success in the laboratory, and subsequently little success in the field. In a limited number of laboratory studies, the biocontrol efficacy of
Trichoderma spp. towards other GTDs have shown inhibition of mycelial growth of E. lata, Botryosphaeriaceae species (i.e. Neofusicoccum australe, N. parvum, Diplodia seriata, Lasiodiplodia theobromae), Phaeomoniella chlamydospora, Phomopsis viticola and Phaeoacremonium spp. (Kotze et al., 2011; Mutawila et al., 2011).The use of
Pseudomonas spp. as BCAs has been widely studied within various pathosystems including potato scab (Arseneault et al., 2016) and late blight (Hultberg et al., 2010b);
Rhizoctonia root rot on bean (D’aes et al., 2011); damping-off and root rot in tomato
(Solanki et al., 2014); anthracnose in beans (Bardas et al., 2009); postharvest diseases of stone fruit (Aielloa et al., 2019); and take-all disease of wheat (Weller and Cook, 1983;
Thomashow and Weller, 1988; Keel et al., 1992). In grapevines, Pseudomonas spp. were shown to suppress Botrytis cinerea (Ait Barka et al., 2002; Trotel-Aziz et al., 2008), root-rot pathogens (Ziedan et al., 2008) and Rhizobium vitis which causes crown gall
(Khmel et al., 1998). Pseudomonas spp. have been found to be present in the grapevine endosphere (Bell, 1995; West et al., 2010; Deyett et al., 2017). The presence of
Pseudomonas spp. in the inner grapevine tissues may be an indication of their ability to colonise and survive in grapevine tissues.
In Chapter 2 it was demonstrated that 10 endophytic strains of Pseudomonas from grapevines exhibited antagonistic activity against the pathogens associated with
Botryosphaeria and Eutypa dieback, and Esca/Petri disease. In vitro inhibition of these
GTD pathogens may imply that these antagonistic Pseudomonas strains are potential
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Chapter 3
BCAs against GTDS. However, in vitro inhibition does not always translate to effective biocontrol in the plant against colonisation by the pathogen. The only way to ascertain the efficacy of the BCAs is via in planta assays wherein they should be able to demonstrate their ability to control or diminish plant disease. A successful BCA must also be able to survive in the host plant and efficiently colonise the plant tissues for a sufficient period of time to provide protection (Weller, 1988; Arseneault et al., 2016). Most importantly, it must not cause disease or damage to any of the host tissues.
This study aims to determine the potential of Pseudomonas spp. as BCAs against
GTDs. The objectives of this research are: 1) To establish that the candidate BCA is not pathogenic to grapevines; 2) To investigate the ability of Pseudomonas spp. to suppress
N. luteum in detached canes and potted grapevines; 3) To demonstrate the endophytic colonisation of the grapevine tissues by Pseudomonas in pre-planted cuttings and potted vines.
3.2. Materials and Methods
3.2.1. Plant materials
Vitis vinifera (cv. Shiraz) was used for all in planta experiments. One-yr old dormant canes with no GTD symptoms were collected from disease-free commercial vineyards in the Hilltops and Riverina regions, New South Wales (NSW), Australia in the winter of 2017 and 2019, respectively. All canes were stored at 4°C for 4-6 weeks until rooting or use in the detached cane assays.
3.2.2. Bacterial strains, culture conditions, inoculum preparation
The 10 strains of Pseudomonas that inhibited mycelial growth of GTD pathogens in the dual culture test in Chapter 2, Table 2 were selected for in planta
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assays. Nine strains were from the P. poae group while one strain was closely related
to P. moraviensis (Chapter 2, Fig. 10). Bacterial cells were obtained from single
colonies of each antagonistic strain and stored in 20% glycerol at -80°C.
To prepare the bacterial inoculum, a loopful of the bacterial suspension in
glycerol was streaked onto nutrient agar (NA, Oxoid Ltd., Hampshire, England) and
incubated at 25°C for 2 days. A loopful of the subsequent bacterial growth was then
transferred to nutrient broth (NB, Oxoid Ltd) and incubated for 24 h in an OM15 orbital
shaking incubator (RATEK Instruments Pty. Ltd., Victoria, Australia) at 25°C, 150 rpm.
The optical density of the bacterial broth culture was measured at 600 nm using an
UNICAM 8625 UV/VIS spectrometer (UNICAM Limited, Cambridge, UK). The bacterial
10 concentration was adjusted to 10 CFU which was further confirmed by dilution
plating.
3.2.3. Pathogenicity tests of Pseudomonas poae BCA17 strain
Prior to all in planta experiments, one representative strain of P. poae was
tested for its pathogenicity to ensure that it was not pathogenic to grapevines. P. poae
strain BCA17 was inoculated on different grapevine tissues: a) leaves; b) berries; and
c) single node dormant canes. The bacterial inoculum was prepared following the
methods described in Section 3.2.2 and 20 µl of the bacterial suspensions (estimated to
10 be 10 CFU) were used to inoculate each of the different tissues. For the control
treatments, each grapevine tissue type was inoculated with 20 µl of sterile deionised
water (SDW). Ten replications per treatment were used for each tissue type.
For leaf inoculation, medium sized grapevine leaves from the fifth youngest
node of glasshouse-grown Shiraz vines were selected. Leaves were surface-sterilised
in 0.5% sodium hypochlorite solution for 30 s and rinsed twice with SDW. Prior to 70
Chapter 3
inoculation, the central part of each leaf was marked (~1 cm diameter) with a permanent
pen and a wound was created inside the circle by puncturing the tissue with a sterile
hypodermic needle (10 cc/ml). The leaves were incubated at 25°C on Petri plates lined
with moist filter paper. Ripe single grape berries (cv. Thompson seedless) were
likewise surface-sterilised, marked and wounded as described for the leaves before
inoculation and incubated in polystyrene boxes with holes.
For cane inoculations, one-year old dormant Shiraz canes (~40 cm) were collected
from a commercial vineyard in the Hilltops regions in winter 2017. Canes were surface-
sterilised with 0.5% sodium hypochlorite solution for 1 minute and rinsed twice with tap
water before cutting into single nodes (~10 cm). Fresh wounds at the apical end were
then inoculated with bacterial suspensions. Canes were placed in 5 ml plastic
containers which were regularly refilled with tap water up to the brim.
All inoculated tissues were incubated at 25°C under 12 h/12 h dark/light regime for
7 days, in the case of leaves and berries, and for 30 days for detached canes. All tissue
samples were observed for the presence or absence of GTD symptoms.
3.2.4. Suppression of Neofusicoccum luteum infection in planta by strains of
Pseudomonas
3.2.4.1. Fungal isolates
Neofusicoccum luteum, a virulent Botryosphaeria dieback pathogen in
Australia (Wunderlich et al. 2011) was used as a model pathogen for all the challenge
inoculation experiments. This species produces abundant pycnidia, conidia and a
yellow pigment in culture media which assists in identification. Grapevine isolates of
N. luteum (DAR 80983, 81013, 81016) were used for inoculating both detached canes
and potted vines. All isolates were previously identified by sequence analyses of the
ITS, EF1-α and β-tubulin genes (Wunderlich et al., 2011). The three isolates that were
stored as mycelial plugs on SDW were sub-cultured onto fresh potato dextrose agar 71
Chapter 3
(PDA, Difco Laboratories, Maryland, USA) amended with chloramphenicol (PDAC,
100 mg/L of agar). Plates were incubated at 25°C overnight in the dark before being
transferred under UV light (Phillips 20w UVB) and exposed to 12 h light-dark regime
at 18-22°C for 4-5 weeks until fruiting bodies developed. To harvest the conidia,
pycnidia were scraped off the surface of the agar media with a sterile scalpel after
adding 5 ml of SDW with a drop of Tween 80. The pycnidial suspension was then
transferred into a sterile mortar and pestle and ground to release the conidia. The
conidial suspension was passed through two layers of sterile Miracloth (Merk
Millipore) to filter out the mycelial fragments and agar. Conidial suspensions from
each of the three isolates were combined and the mixed suspension was
standardised to 1x104 conidia/ml using a haemocytometer. For succeeding
inoculations, 20 µl of the conidial suspension was used, equivalent to 200 conidia per
-3 sample. The viability of the spores was determined by plating 100 µl of a 10 dilution
of the conidial suspension onto PDA amended with Triton X-100 (Sigma-Aldrich,
USA) at three replicate plates. The plates were incubated at 25°C for 5-7 days and
the resulting colonies were counted.
3.2.4.2. Challenge inoculation using detached canes
The 10 antagonistic strains of Pseudomonas were initially screened for their ability to suppress GTD infection using detached canes. Dormant one-yr old Shiraz canes from the same source as in Section 3.2.1 were surface-sterilised and processed into single node cuttings (~10 cm) as described in Section 3.2.3. The fresh wound at the apical
10 end of each cane was inoculated with 20 µl of bacterial suspension (10 CFU) for each of the 10 antagonistic strains (Table 1, Chapter 2). One hour after inoculation, when the bacterial suspension was absorbed by the plant tissues, the wounds were subsequently
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Chapter 3 inoculated with conidia (200 conidia) of N. luteum. Canes inoculated with conidia only served as the positive control, while the negative control canes were inoculated with
SDW. Each cane was placed in a 5 ml tube containing water as previously described
(Section 3.2.3) and incubated for 4 weeks at 25°C under 12:12 dark-light regime. For each treatment, three replicate canes were used.
After 4 weeks, isolations were performed to assess the colonisation of the cane
sections by N. luteum. Cane tissue above the node was peeled and the top 2 cm
sections from the inoculation point were collected aseptically. The wood sections were
then surface-sterilised by soaking in 2.5% sodium hypochlorite solution for 2 min and
then rinsed twice with SDW for 2 min each time. After the last rinse, surface-sterilised
wood samples were transferred onto sterile paper towel and air dried inside a
biohazard cabinet. Each cane section was cut in half longitudinally with flame-sterilised
secateurs and each section was then transferred onto opposite ends of PDAC. The
plates were incubated at 25°C in total darkness and assessed for yellow mycelial
growth typical of N. luteum for 7-14 days.
3.2.4.3. Challenge inoculation using potted grapevines
The three strains identified as P. poae (BCA13, BCA14, and BCA17) which
resulted in the lowest pathogen recovery in the detached cane assay were further used
for challenge inoculation using glasshouse potted vines. One-yr-old dormant canes
(cv. Shiraz, ~40 cm) from the same source as in Section 3.2.1 were rooted in plastic
tubs filled with perlite and placed on heat pads at 28-30°C for 6-8 weeks during winter
2018 until rooting. Healthy-looking rootlings were then transferred to 10 L pots that
were filled with commercial garden soil (compost, 60%; washed sand, 20%;
screened loam, 20%). All vines were maintained in the glasshouse (17-27°C) and
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Chapter 3
watered every 12 h for 5 min with an automatic dripper system (8 L/h).
In the summer of 2018, inoculations were performed on fresh wounds that were
created by cutting the top node of each vine using a sterile secateur. Bacterial
suspensions were prepared as previously described in Section 3.2.2 and 20 µl of the
10 BCA suspension (10 CFU) was pipetted onto the wounds and left for ~1 h until
absorbed by the plant tissue. Conidial suspensions of three N. luteum isolates (DAR
80983, 81013, 81016) were prepared as described in Section 3.2.4.1 and 20 µl of the
conidial suspension (200 conidia) from the combined suspensions of three N. luteum
isolates was applied onto the same inoculation point. The tip of the grapevine plants
was wrapped with Parafilm® to prevent inoculum runoff. Positive control vines were
inoculated with conidia of N. luteum only while the negative control vines were
inoculated with SDW. Ten replicate vines per treatment were used.
3.2.4.3.1. Assessment of N. luteum infections by isolations
The efficacy of BCA13, BCA14 and BCA17 to supress N. luteum colonisation in
potted vines was initially assessed by fungal isolations. At 3 months after inoculation,
the top end for each vine (~2 cm) where the inoculation point was located, was
collected and stored at 4°C for 5-7 days until processed.
Each section was peeled and surface-sterilised as previously described before
being aseptically cut longitudinally into quarters so that each quarter contained a
portion of the inoculation point. Two of the quarters were transferred onto PDAC and
incubated at 25°C in total darkness and assessed for growth of N. luteum for 5-7 days.
The remaining two quarters were placed in sterile 2 ml tubes and stored at -80°C until
required for DNA extractions and quantitative PCR (qPCR) analysis.
3.2.4.3.2. Assessment of N. luteum infections by quantitative PCR (qPCR)
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The efficacy of BCA13, BCA14 and BCA17 to suppress N. luteum colonisation in potted vines was further assessed by qPCR. DNA was extracted following the methods of Pouzoulet et al. (2013) as described in the Methods section of Chapter 2.
All DNA samples were quantified using a Quantus™ Fluorometer (Promega, USA) prior to qPCR. The qPCR assay was performed with the RotorGene 6000 system
(Corbett Life Science, Qiagen, USA) using Botryosphaeriaceae multi-species primers
Bot-BtF1 (5′- GTATGGCAATCTTCTGAACG-3′) and Bot-BtR1 (5′-
CAGTTGTTACCGGCRCCAGA-3′), and a hydrolysis probe, Taq-Bot probe (5′-/56-
FAM/TCGAGCCCG/ZEN/GCACCATGGAT/3IBkFQ/-3′) (Billones-Baaijens et al.,
2018). The PCR amplification was carried out in a 20 µl reaction containing 10 µl of 2×
GoTaq® Master Mix (Promega, USA), 500 nm each of the primers Bot-BtF1 and Bot-
BtR1, 250 nm of Taq-Bot probe, 5 µl of template DNA and nuclease-free water
(Qiagen, USA) with four technical replicates per sample. Standard Bot-Btub gBlock
(500 pg) and a non-template control were included in the assay as positive and negative controls, respectively. The qPCR analysis was performed following the conditions optimised by Billones-Baaijens et al. (2018) with the following thermal cycling conditions: 2 min at 98°C, followed by 40 cycles of 98°C for 10 s, 60°C for 30 s and fluorescence detection at 60°C for 30 s.
The amount of amplified pathogen DNA was interpolated from a previously developed standard curve (Billones-Baaijens et al., 2018). The imported standard curve was calibrated using the standard Bot-Btub gBlock (500 pg) (Integrated DNA Technologies,
Inc., USA). The number of Botryosphaeriaceae β-tubulin gene copies in each inoculated wood sample was calculated using the formula:
N=g(d ˟ c)/t ˟ c
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Chapter 3
Where:
N = calculated number of β-tubulin gene copies in 100 mg of wood sample, g = the mean number of gene copies detected by qPCR; d = total gDNA extracted from 100 mg of wood (100 µl), c = DNA concentration (µl) t = the amount of DNA template (5 µl) in one reaction.
The amount of pathogen detected in wood sections that were inoculated with
BCA13, BCA14, BCA17, pathogen only (positive control), and SDW (negative control),
were compared.
3.2.5. Pseudomonas colonisation and establishment in grapevine tissues
3.2.5.1. Development of rifampicin-resistant strain
The endophytic colonisation of grapevine tissues by Pseudomonas was studied
using a laboratory-induced rifampicin-resistant mutant (RifMut) strain developed from
strain BCA17 (P. poae). The RifMut strain was developed following the methods of
Adorada et al. (2015) and West et al. (2010). A single colony of BCA17 was streaked
onto NA amended with 1 ppm rifampicin (Sigma Chemical Co., St. Louis MO USA).
After 3 days, the resulting colonies of BCA17 were transferred onto NA containing 5
ppm rifampicin and incubated for 3 days. The resulting single colonies were then
sequentially sub-cultured onto NA containing increasing concentrations of rifampicin
(10, 50, and 100 ppm). The final mutant strain, now referred to as RifMut, was re-
inoculated onto NA with 100 ppm rifampicin on five subsequent occasions to ensure
stability of the resistance. Colonisation and movement of RifMut within the grapevine
plants was evaluated by plating wood sections onto NA amended with 100 ppm
rifampicin.
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Chapter 3
3.2.5.2. Pre-planting application of RifMut strain The potential for transmission of strains of Pseudomonas in nursery propagation materials was tested by treating the grapevine canes with the RifMut strain prior to rooting. Dormant canes (cv. Shiraz) collected from the same source as in
Section 3.2.1 were removed from the 4°C cold room 24 h prior to inoculation to acclimatise at room temperature. All canes were surface-sterilised using the methods described in Section 3.2.3.
To prepare the bacterial suspension, a 2-day-old RifMut culture grown on NA was suspended in 10 ml ¼ strength Ringer’s solution (Sigma Aldrich, USA) and dislodged with a sterile L-shaped plastic hockey stick. The resulting suspension was poured into a 1 L Schott bottle, bringing the total volume to 500 ml by adding ¼ strength
Ringer’s solution. The suspension was incubated in an orbital shaker as described in
Section 3.2.2 and the bacterial concentration was determined after 24 h prior to cane treatment.
Surface-sterilised canes were trimmed (5 mm) at the apical and basal ends to create fresh wounds prior to inoculations. The canes were then placed in a 500 ml beaker containing 250 ml RifMut suspension (Figure 3.1) with the basal ends of the canes (~ 4 cm) immersed in the bacterial suspension. Control canes were placed in a
500 ml beaker containing 250 ml ¼ Ringer’s solution (Sigma Aldrich, USA) only.
Fifteen replicate canes per treatment were used. All treatments were incubated at 25°C under 12:12 dark:light regime. A 500 ml beaker containing 250 ml ¼ Ringer’s solution without canes was also incubated to account for water loss due to evaporation. After
24 h, the canes were removed from the beakers and the amount of inoculum absorbed by the canes was calculated, after accounting for loss to evaporation, by deducting the volume of suspension remaining from the initial volume of 250 ml.
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Chapter 3
Prior to rooting, five canes per treatment were randomly selected and used for re-isolation to determine if the RifMut strain was successfully transmitted into the canes. Tissue samples (~1 cm) were cut from the base, middle and top sections of each cane and were surface-sterilised as described in Section 3.2.4.2. Each section was cut longitudinally and plated onto NA amended with 100 ppm Rifampicin and observed for bacterial growth following incubation at 25°C in the dark for 5-7 days. The
10 remaining canes were rooted in perlite as described in Section
3.2.4.3. After eight weeks, when roots had developed, the plants were uprooted, washed and roots were removed. Attempted re-isolation of the RifMut was performed by cutting ~1 cm sections from the base, middle, and top sections of the canes as described previously.
A B
Figure 3.1. Dormant grapevine canes (cv Shiraz) dipped in: A) ¼ strength Ringer’s solution; and B) RifMut bacterial suspension for 24 h prior to rooting.
78
Chapter 3
3.2.5.3. Post-planting application of RifMut strain The ability of the strain of Pseudomonas to colonise and establish in potted grapevines was further investigated using RifMut. Dormant cuttings (cv. Shiraz) from the same source as in Section 3.2.1 were rooted and planted in pots in the spring of
2017 as previously described in Section 3.2.4.3.
For inoculation, fresh wounds were created by drilling through the second internode of the grapevine plant using a sterile 3 mm auger bit driven by a CDL-018 cordless drill drive (Ozito Industries Pty. Ltd., Victoria, Australia; Figure 3.2A). A 20 µl suspension of RifMut (estimated to be 1010 CFU), prepared as described in Section
3.2.2, was pipetted onto the fresh wounds (Figure 3.2B) and wrapped with Parafilm® to prevent inoculum runoff. The potted vines were maintained at ambient temperature
(17-27°C) inside the glasshouse for up to 6 months. The translocation of the RifMut strain within the grapevine tissue was assessed at 1, 3, and 6 months post-inoculation.
A) B)
Figure 3.2. Inoculation of Pseudomonas poae BCA17 RifMut strain in potted vines. A) Creating a wound using a cordless drill; B) inoculation of the bacterial suspension into the wound using a sterile pipette.
For each assessment, five replicate vines were uprooted, washed and roots removed before storage at 4°C for 5-10 days until processed. Each vine was cut into
1 cm sections from the apical tip down to the crown (Figure 3.3) and processed as
79
Chapter 3 described in in 3.2.4.2. At 6 months, further isolations were conducted from the lateral shoots above the inoculation point to determine the lateral movement of the RifMut strain. All wood sections were plated onto NA amended with 100 ppm rifampicin and observed for bacterial growth as previously described.
6 months
1, 3, and 6 months
Figure 3.3. Schematic diagram of a vine inoculated with the RifMut strain showing the inoculation point, the positions and sizes of the tissue samples collected for re- isolations. Sampling period for different parts of the vine are indicated by brackets. Diagram by P. Reveglia.
3.2.6. Data analysis
Data for pathogen recovery from detached canes and glasshouse potted vines, and qPCR quantification of N. luteum DNA from BCA-treated glasshouse potted vines were analysed using GenStat (18th edition) (VSN International Ltd., UK). All data were tested for homogeneity using Levene’s test at P ≤0.05. Effects of treatments for all the above-mentioned experiments were separately analysed by one-way ANOVA.
Treatment means were separated by pairwise comparison using least significant
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Chapter 3
difference (LSD) at P ≤0.05. Standard error of the mean was also obtained to estimate
the standard deviation of the sampling distribution of the mean.
3.3 Results
3.3.1. Pathogenicity tests on grapevine tissues
Pathogenicity tests of the representative antagonistic strain of P. poae, BCA17,
on different grapevine tissues indicated that this strain was not pathogenic to
grapevines. No symptoms of infection were observed for the berries inoculated with
BCA17, which was similar to those berries inoculated with SDW only (Figure 3.4A).
Similarly, no infection was observed on BCA17-inoculated leaves (Figure 3.4B). Lastly,
all inoculated detached canes did not produce external or internal symptoms and were
similar to the non-inoculated control canes (Figure 3.4C).
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Chapter 3
A
BCA17 SDW
B
BCA17 SDW
C
BCA17 SDW
Figure 3.4. Pathogenicity test of Pseudomonas poae strain BCA 17 on grapevine tissues: A) berries; B) leaves, and C) canes. Inoculation treatments were P. poae strain BCA 17 and sterile deionised water (SDW). The inoculation points for berries and leaves are marked with a circle while inoculation points for canes are indicated by arrows.
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Chapter 3
3.3.2. Suppression of N. luteum infection in planta by Pseudomonas strains
3.3.2.1. Challenge inoculation using detached canes
Of the 10 antagonistic strains of Pseudomonas inoculated on detached canes, seven significantly reduced the recovery of N. luteum (P ≤0.005). Of the seven effective strains, BCA17 showed the lowest pathogen recovery at 0% which was similar to the negative control canes inoculated with SDW (Figure 3.5). However, these results were not significantly different from BCA13, BCA14, BCA16, BCA18, and
BCA19 which resulted in 22-33% pathogen recovery. Pathogen recovery in canes treated with BCA12, BCA15, and BCA20 (56-78%) were not significantly different from the positive control canes (100%) that were inoculated with the N. luteum only.
120 e e 100 de
cde
80 bcd
recovery(%) 60 abc abc abc ab ab 40
N. luteumN. 20 a a
Figure 3.5. Recovery of the pathogen, Neofusicoccum luteum, from detached canes treated with different strains of Pseudomonas. Bars with different letters indicate significant differences in pathogen recovery with the strains of Pseudomonas at P ≤0.05 LSD. Error bars represent standard error of the means.
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Chapter 3
3.3.2.2. Challenge inoculation using potted grapevine plants
3.3.2.2.1. Assessment of N. luteum infection by pathogen isolation
At 3 months after challenge-inoculations, none of the vines exhibited external symptoms and all showed normal development. Re-isolation from inoculated potted grapevines showed significant difference (P ≤0.001) in pathogen recovery between the three strains of Pseudomonas examined (Figure 3.6). The pathogen recovery was lowest (20%) for those vines treated with BCA17 and this was significantly lower than for vines treated with BCA13 and BCA14- (P≤0.001), where N. luteum recovery was
70% and 90%, respectively. Pathogen recovery for BCA14 was not significantly different from the positive control (N. luteum only) with 100% pathogen recovery. N. luteum was not recovered from the negative control vines that were inoculated with
SDW.
120 bc c 100 b
80
60
recovery(%) a eum eum 40
lut N. 20
a SDW BCA17 BCA13 BCA 14 N. luteum
Figure 3.6. Recovery of the pathogen, Neofusicoccum luteum from glasshouse potted vines treated with Pseudomonas poae strains BCA13, BCA14 and BCA17 prior to pathogen inoculation. Bars with different letters indicate significant differences in pathogen recovery upon challenge inoculation with the selected BCA strains at P ≤0.05 LSD. Error bars represent standard error of the means.
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Chapter 3
3.3.2.2.2. Assessment of N. luteum infections by qPCR
A quantitative PCR assay of tissue samples treated with strains of
Pseudomonas prior to N. luteum inoculation confirmed the results of the isolations
(Section 3.3.2.1.1). The amount of Botryosphaeriaceae DNA detected in vines treated
with BCA17 was significantly lower in comparison with that of the control vines
inoculated with N. luteum. The qPCR analysis of inoculated vines indicated a 40-fold
reduction in the amount of pathogen DNA when vines were treated with BCA17, with
an average of 300 copies of Botryosphaeriaceae DNA detected, compared to an
average of 12,000 copies of the pathogen DNA in vines inoculated with N. luteum only
(Figure 3.7). Grapevine plants treated with BCA13 and BCA14 did not significantly
reduce the pathogen infection compared to the positive control, with an average of
3,000-14,000 copies of Botryosphaeriaceae DNA detected. Pathogen DNA was not
detected in any of the un-inoculated vines, indicating that there was no background
infection due to Botryosphaeriaceae species.
b bb b
a a a a
Figure 3.7. Number of Botryosphaeriaceae β-tubulin gene copies detected by quantitative PCR analysis of DNA extracted from potted vines treated with BCA strains prior to Neofusicoccum luteum inoculation. Each treatment is represented by 100 mg of dried wood from 10 replicate vines, with four technical replicates per DNA sample. Bars with different letters indicate significant differences in the number of Botryosphaeriaceae β-tubulin gene copies detected by qPCR at P ≤0.05 LSD. Error bars represent standard error of the means. 85
Chapter 3
3.3.3. Pseudomonas colonisation and establishment in grapevine tissues
3.3.3.1. Pre-planting application of Pseudomonas RifMut strain
The analysis of canes treated with the rifampicin-resistant mutant (RifMut) strain
of P. poae BCA17 prior to rooting demonstrated that Pseudomonas spp. can be
artificially-inoculated on propagation materials and subsequently establish in
grapevine tissues. Following 24 h incubation of canes, a decrease in volume of 54 ml
for the bacterial suspension (1.6 x 107 CFU/ml) and 56 ml for the Ringer’s solution (250
ml) was recorded. For the control check (Ringer’s solution without canes), a 22 ml
decrease in volume was recorded. This indicates that approximately 22 ml of the
dipping solution was lost due to evaporation while the remaining 32-34 ml were
absorbed by the dormant canes. Re-isolations of the RifMut strain from five randomly
selected canes revealed the bacterial strain was translocated into the grapevine canes.
The RifMut strain was reisolated in all five wood pieces obtained from the base and
middle sections (approximately 13-15 cm distance) of the canes while only two wood
pieces of the top end of the canes were positive to RifMut (Figure 3.8). No bacteria
were re-isolated from canes dipped in Ringer’s solution only.
Subsequent re-isolations from the remaining 10 canes that were rooted for a
further 8 weeks showed the RifMut strain was persistent in grapevine tissues. The
RifMut strain was present in all the 10 rooted vines from the base and middle sections,
while the RifMut strain was only re-isolated from three vines of the top sections (Figure
3.8). No bacteria were recovered from the control canes in Ringer’s solution at post-
rooting.
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Base Middle Top 30
25
20 3
15 10
10 2 Incidenceof RifMut frominoculated canes 5
5 10 5 0 0 0 RifMut Control RifMut Control
Pre-rooting Post-rooting (N=5) (N=10)
Figure 3.8. Recovery of the rifampicin-resistant mutant (RifMut) strain of Pseudomonas poae BCA17 from dormant canes dipped in bacterial suspensions for 24 h and rooted for 8 weeks.
3.3.3.2. Post-planting application of Pseudomonas
The assessment of potted grapevine plants inoculated with RifMut provided evidence that the bacteria were able to colonise and establish within the plant for a period of time. As early as one month after inoculations, acropetal and basipetal movements of up to 2.4 and 2.8 cm below and above the inoculation point, respectively, were recorded from all sub- sampled vines (Figure 3.9). Acropetal and basipetal movements of the RifMut strain was further determined at 3 months after inoculations. At 6 months, the entire plant of all the five sampled vines was colonised by the RifMut strain. The bacterial strain was reisolated from the apical tip of the living tissue down to the base of the trunk below the soil line. Subsequent re-isolation on
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Chapter 3 lateral shoots indicate lateral movement of the bacteria with the RifMut strain being recovered up to a distance of 3 cm in the shoot sections. No bacteria were recovered from the control plants that were inoculated with SDW only.
30 Basipetal Acropetal Lateral 2.6 25 3.4
20
(cm) 15 19.2 10 1.6 2.8 4.6 2.4 0
5 0 0 Bacterial Bacterial movementthe from inoculationpoint
0 RifMut Control RifMut Control RifMut Control 1 month 3 months 6 months
Figure 3.9. Re-isolation of rifampicin-resistant mutant (RifMut) strain of Pseudomonas poae BCA17 from inoculated potted vines at three different sampling periods.
3.4. Discussion
The main aim of this study was to investigate the antagonistic ability of strains belonging to P. poae and P. moraviensis against the GTD pathogen, N. luteum, in planta. This study also investigated the transmission and persistence of these bacterial strains when artificially inoculated in living hosts. The results demonstrated that particular strains of P. poae could suppress N. luteum infection in planta, indicating that they have the potential to be used as BCAs against Botryosphaeria dieback. To our knowledge, this study is the first attempt to investigate Pseudomonas spp., endophytic to grapevine, for their potential to control the GTD pathogen, N. luteum.
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Pathogenicity tests using P. poae strain BCA17 showed that this strain did not produce any symptoms on detached leaves, berries and canes, indicating that this species is not pathogenic toward grapevines. The foremost requisite for a BCA is that it must not be recognised by the host tissues as a pathogen itself. Some species of
Pseudomonas are known pathogens of plants. P. syringae, for instance, has been implicated in bacterial inflorescence rot (Whitelaw-Weckert et al., 2011) and bacterial leaf spot (Hall et al., 2002) on wine-grape cultivars in Australian vineyards. Lack of symptom development in any of the grapevine tissues upon inoculation with P. poae strain BCA17 affirmed that it can be a good BCA candidate, being a non-pathogen of grapevine.
Further in planta experiments affirmed the efficacy of an antagonistic P. poae strain as a BCA. Recovery of the pathogen was nil to low in the presence of BCA17 in both detached cane and potted vine inoculations. Challenge inoculation of N. luteum with Pseudomonas spp. using detached canes showed an absence of cane infection upon treatment with P. poae strain BCA17. This was further confirmed by challenge inoculation on potted vines which resulted in 80% reduction in pathogen recovery when canes were treated with BCA17. The qPCR analysis further corroborated these results where a significant decrease in the amount of pathogen DNA (40-fold reduction) was detected when the vines were treated with BCA17. Pseudomonas poae strain DSM
14936 was first reported and described by Behrendt et al. (2003) and was isolated from the phyllosphere of grasses (Poa spp.). Another strain isolated from the internal root tissue of sugar beet, P. poae RE*1-1-14, was found to inhibit the sugar beet root pathogens Phoma betae, Rhizoctonia solani and Sclerotium rolfsii (Zachow et al.,
2010; Müller et al., 2013). Field trials using this strain of antagonistic bacteria demonstrated control of late root rot caused by R. solani (Müller et al., 2013).
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Chapter 3
A recent study in New Zealand reported endophytic Pseudomonas obtained from stem tissues of Leptospermum scoparium (mānuka) demonstrated strong inhibitory activity against N. luteum in a dual culture assay (Wicaksono et al., 2016).
This strain of Pseudomonas also reduced the lesion lengths and colonisation of the host by N. luteum by up to 40% (Wicaksono et al., 2017). The bacterial endophyte, P. poae strain BCA17 from the inner grapevine tissues from this study was shown to have similar antagonistic characteristics to the mānuka strain reported by Wicaksono et al.
(2017). The challenge inoculations showed that BCA17 was able to suppress N. luteum, in planta, using detached canes and potted vines. However, due to the limited time available for conducting this PhD research, the efficacy of BCA17 as a wound protectant was only conducted on potted vines using one GTD pathogen, N. luteum.
Thus, it is important to test these strains of Pseudomonas in vineyards to determine their stability and persistence under field conditions. Furthermore, this study only used
N. luteum as a model pathogen, thus, there is a need to test the efficacy of this BCA in planta using a range of GTD pathogens.
Pseudomonas poae strain BCA17 was determined to be an efficient coloniser, capable of multiplying and establishing itself within the grapevine. The RifMut, a mutant derivative generated from P. poae strain BCA17 resistant to the antibiotic rifampicin, was able to endophytically colonise grapevine tissues regardless of whether it was inoculated basally as a pre-planting treatment on grapevine cuttings, or apically, being applied on established glasshouse potted vines. The RifMut strain was recovered from the base and middle sections of all the canes that were dipped for 24 h in the bacterial suspension and were recorded to persist 8 weeks post- inoculation in rooted vines. The bacteria was re-isolated in 40% of the apical tip sections at 24 h after inoculation and in 30% of the tip sections after rooting for 8 weeks. The upward movement of bacteria
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Chapter 3 in grapevines has previously been documented for the pathogenic Agrobacterium tumefaciens which has been reported to move up to 30 cm within 24 h when freshly cut basal ends of the shoots were dipped in a bacterial suspension (Tarbah and Goodman,
1987). In grapevine, rapid bacterial colonisation via the primary xylem is possible due to the presence of numerous and long vessels which can be up to 1 m long which facilitate ease in bacterial movement within the stem (Thorne et al., 2006). Thorne et al. (2006) further postulated that there are open, continuous vessels from the stem to the leaf lamina of grapevine. As BCA17 has shown promise as a BCA for N. luteum in grapevine, there may be potential for this to be used in nurseries where the grapevine cuttings can be pre-treated with the bacterial BCA. These findings may be important especially with the emerging issue of propagation materials being a source of inoculum of GTDs in new vineyards (Billones-Baaijens et al., 2012).
Inoculation of three-mo-old potted vines also resulted in the successful colonisation and persistence of the bacterial BCA. Upward and downward movement of the RifMut strain was noted at 1 month post inoculation. Complete colonisation of the grapevine plant was achieved 6 months post inoculation with the RifMut strain being recovered from the tip of the living tissue down to the basal end of the trunk, below the soil line, and in the lateral shoots. In one-yr-old and three-yr-old potted vines,
West et al. (2010) demonstrated movement of the bacteria Bacillus cereus from inoculated leaves down to the vine shoots, and up to 13 cm distance from the inoculated leaves, 1 week after inoculation. Downward translocation of the bacteria (up to 13 cm from the inoculated shoot) within the grapevine plant was also detected after
1 week. However, after 4 weeks, B. cereus was found only at 6.5 cm distance from the inoculation point. No further sampling was conducted beyond 4 weeks. Wicaksono et al. (2017) also inoculated a strain of Pseudomonas from mānuka into the wound of a
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Chapter 3 one-yr-old Sauvignon Blanc vine and found this strain was able to colonise the wound site for 6 months. However, the distance travelled by this endophytic Pseudomonas within the vine was not reported (Wicaksono et al., 2017). BCA17 was able to persist within the grapevine for greater than 4 weeks and its ability to colonise the grapevine tissue 6 months after introduction implies that it may be a promising BCA. Furthermore, it appears that the bacterial population can be maintained in the host plant in sufficiently high amount, and therefore capable of providing protection to the host plant for longer periods of time. Host colonisation by the BCA is necessary for suppression of plant diseases. Essentially, all the disease-suppressive mechanisms exhibited by the potential BCAs are of no real value unless the bacteria can successfully establish themselves in the host plant. Many Pseudomonas spp. are aggressive colonisers and when inoculated onto seeds, roots and other plant tissues, they cause substantial increase in plant growth and yield (Suslow and Schroth et al., 1982; Weller, 1982).
However, not all strains showing pathogen inhibition in vitro provide disease control.
In rhizosphere-inhabiting Pseudomonas spp., biocontrol efficacy of pseudomonads is often related to their density in the rhizosphere (Weller, 1988). To assess the long- term establishment and persistence of BCA17 in the grapevine, further studies are required using mature vines in vineyard conditions.
In summary, all controlled experiments in the laboratory and glasshouse provided empirical evidence that P. poae strain BCA17 has potential as a biocontrol candidate against GTD pathogens. Further investigations still need to be undertaken to ascertain its ecological competence in competing and surviving in nature. There were reports that repeated culturing of fluorescent pseudomonads in vitro can result in loss of field efficacy, as a consequence of changes in cell and colony morphology, loss of cell surface structures, or reduction in antibiotic and siderophore production (Weller,
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Chapter 3
1988). Hence, prior to adoption of biocontrol as a method of controlling disease in a large-scale vineyard setting, a greater understanding of the complex interactions between the BCA, pathogen, host and the environment is of utmost importance.
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Chapter 4 Chapter 4.
Elucidating the mechanism of action of Pseudomonas in the control of grapevine trunk disease pathogens
4.1. Introduction
Many bacteria with biocontrol potential have been reported to promote growth or protect plants by producing compounds that are antagonistic to pathogens, as well as inducing plant defense or host resistance responses (Thomashow and Weller, 1988;
Keel et al., 1992; Hultberg et al., 2010a, Zachow et al., 2010). The ability of fluorescent
Pseudomonas spp. to promote plant growth, either by directly stimulating the plant or by suppressing fungal root pathogens, has been the focus of research for many years
(Kloepper et al., 1980; Leong, 1986; Mazzola et al., 1992; Raaijmakers and Weller,
1998; Nielsen et al., 2003; Pierson and Pierson, 2010; Arseneault et al., 2016; Aielloa et al., 2019). Concurrently, the production of a wide variety of bioactive secondary metabolites by different strains of Pseudomonas has also been studied extensively
(Thomashow and Weller, 1988; Nielsen et al., 1999; Keel et al., 1992; Loper et al.,
2012; Arseneault et al., 2013).
The dual culture assays presented in Chapter 2 demonstrated the antagonistc activity towards grapevine trunk disease (GTD) pathogens of different strains of
Pseudomonas collected from grapevine inner tissues. Some of these strains suppressed N. luteum infections in detached canes and potted vines, as presented in
Chapter 3. Therefore, there is a need to investigate the mechanism of action involved in the control of GTD pathogens by these strains. Further understanding of their mode of action may help determine their capacity to suppress disease in natural environments.
Plant protection against pathogen infection via the application of fluorescent
94
Chapter 4 Pseudomonas spp. is mediated by a variety of secondary metabolites especially diffusible antibiotics. Antibiotics are low-molecular weight compounds produced by microorganisms which produce deleterious effect at low concentrations to the growth and metabolism of other microorganisms, including plant pathogens (Fravel, 1988;
Thomashow et al., 1996; Raaijmakers et al., 2002). Among the most important antibiotics produced by several strains of Pseudomonas is 2,4-diacetylphloroglucinol
(DAPG) (Keel et al., 1990; Keel et al., 1992; Raaijmakers and Weller, 1998; Ramette et al., 2003). Keel et al. (1992) postulated that DAPG may play a role in the induction of plant defense mechanisms. Kwak et al. (2011) demonstrated that DAPG affected membrane permeability, regulation of reactive oxygen, and cell homeostasis. DAPG caused membrane damage to Pythium spp. and inhibits production of zoospores (de
Souza et al., 2003).
Another well-known antibiotic associated with many species of antagonistic
Pseudomonas is phenazine and its derivatives (Bull et al., 1991; Mazzola et al., 1992;
Raaijmakers et al., 2002; Arseneault et al., 2016). Phenazines exist in several chemical forms, including hydroxyphenazine, pyocyanine, and phenazine-1-carboxylic acid
(PCA), all of which possess redox activity although they differ in their properties (Price-
Whelan et al., 2006). Phenazines may be important in cell signalling, biofilm formation and bacterial survival (Pierson and Pierson, 2010). The main antibiotics responsible for suppression of take-all disease in wheat caused by Gaeumannomyces graminis var. tritici by P. fluorescens 2-79 are phenazine and its derivatives (Thomashow and
Weller, 1988; Mazzola et al., 1992).
Beneficial fluorescent Pseudomonas spp. also enhance plant growth and exert antagonistic activity against plant pathogens by producing siderophores (Scher and
Baker, 1982; Ahl et al., 1986; Becker and Cook, 1988). Siderophores are low molecular weight, high affinity iron (III) chelators that transport iron into bacterial cells. Those 95
Chapter 4 produced by strains of Pseudomonas are strong iron chelators and these result in reduced iron availability for other microorganisms, including plant pathogens, with less efficient iron-sequestering systems (Kloepper et al., 1980; Weller, 2007).
Cyclic lipopeptides (CLPs) are a class of compounds also produced by many strains of Pseudomonas spp. (Nielsen et al., 2003; Martinez and Osorio, 2007; Tran et al., 2007; Hultberg et al., 2010a). Some CLP act as a biosurfactant, providing the organism improved surface colonisation and utilisation of surface-bound substrates
(Nielsen et al., 1999). In addition, CLPs are able to insert into membranes and perturb their function, which result in broad antibacterial and antifungal activities (Stanghellini and Miller, 1997). Pseudomonas fluorescens DR54, antagonistic to plant pathogenic
Rhizoctonia and Pythium, has been found to produce the CLP viscosinamide that can cause reduced hyphal growth and abnormal hyphal morphology (Nielsen et al., 1999).
Volatile organic compounds (VOCs) may also play a role in the antagonistic capability of some strains of Pseudomonas toward plant pathogens (Voisard et al.,
1989; Ramette et al., 2003; Lo Cantore et al., 2015; Rojas-Solís et al., 2018). The antagonistic activity of P. stutzeri E25 against Botrytis cinerea was reported to be due to its ability to produce antimicrobial sulphur-containing compounds (Rojas-Solís et al.,
2018). In the case of P. fluorescens, hydrogen cyanide (HCN) has been implicated in the suppression of black root rot of tobacco (Voisard et al., 1989).
Stimulation of the plant’s own defense mechanisms through the induction of systemic acquired resistance has also been reported for Pseudomonas spp. (Smith et al., 1991; Van Peer et al., 1991; Benhamou et al., 1996; M’Piga et al., 1997; Audenaert et al., 2002; Iavicoli et al., 2003; de Vleesschauwer et al., 2008). In addition, niche exclusion, competition for nutrients, production of lytic enzymes have all been implicated in plant protection by different strains of Pseudomonas (Suslow et al., 1982;
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Chapter 4 Elad and Baker, 1985; Lorito et al., 1994; Aielloa et al., 2019; Barry and Challis, 2009;
Berg, 2009; Santoyo et al., 2012).
The contribution or importance of a given bacterial trait may be strain-specific.
Pseudomonas fluorescens DR54 originally isolated from sugar beet was found to produce viscosinamide and chitinase which effectively control the sugar beet pathogens Pythium ultimum and R. solani (Nielsen et al., 1999). Pseudomonas
fluorescens CHA0 strain from tobacco produce a wide array of biocontrol mechanisms
(DAPG, HCN, ISR, pyoluteorin, pyoverdine, and salicylate) which enable the bacteria to control Thielaviopsis basicola, G. graminis var. tritici, and Pythium ultimum and their corresponding diseases in tobacco, wheat, and cucumber, respectively (Ahl et al.,
1986; Keel et al., 1992; Ramette et al., 2003;) Common scab of potato, caused by
Streptomyces scabies was controlled by Pseudomonas fluorescens LBUM223 through phenazine and phenazine-1-carboxylic acid production (Arseneault et al., 2013;
Arseneault et al., 2016). P. fluorescens Pf-5, now known as P. protegens, also produces a very broad range of antimicrobial compounds including pyoluteorin, pyrrolnitrin, DAPG, HCN, and pyoverdine (Loper et al., 2012). This strain provided protection to cotton against damping-off by Pythium ultimum or R. solani through a mechanism involving production of the antibiotics pyoluteorin and pyrrolnitrin (Howell and Stipanovic, 1979; Howell and Stipanovic, 1980).
Strains of Pseudomonas poae with plant growth-promoting activity in sugar beet and switch grass have been reported by Zachow et al. (2010) and Xia et al.
(2019), respectively. Further, the endophytic strain P. poae RE*1-1-14 from sugar beet, exhibited antagonistic activity against R. solani via production of the lipopeptide poaeamide (Zachow et al., 2015) and the bacteria was successfully applied as a seed treatment to protect sugar beet from late root rot (Zachow et al., 2010). The strain P. poae 88A6, isolated from Missouri soil, was reported to have exoprotease activity but
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Chapter 4 did not produce DAPG, phenazine, siderophores, CLP or any other antifungal metabolites associated with antagonistic species of Pseudomonas (Mavrodi et al.,
2012).
The aim of this study is to elucidate the underlying mechanism that is employed by the grapevine endophyte P. poae BCA17 in the control of GTD pathogens and disease. Specifically, objectives of this chapter are: 1) Test the ability of P. poae BCA17 to produce antimicrobial compounds, including siderophores, phenazine, VOC, DAPG, and lipopeptides; 2) Evaluate the bioactivity of the diffusible compounds in P. poae
BCA17 culture filtrate on mycelial growth and spore germination of GTD pathogens; and 3) Identify biosynthetic gene clusters with biocontrol implication against plant diseases.
4.2. Materials and Methods
4.2.1. Bacterial strains
A total of 10 antagonistic strains and one non-antagonistic strain of
Pseudomonas that were identified by sequencing of the 16S rRNA gene (Chapter 2,
Table 2) were used in testing for the production of siderophores, while P. poae strain
BCA17 was used to investigate the mechanisms of biocontrol for all the other studies.
These bacterial strains isolated from the inner wood tissue of grapevines were stored in 20% glycerol at -80°C and maintained in the National Wine and Grape
Industry Centre (NWGIC) culture collection at Charles Sturt University, Australia prior to assays.
4.2.2. Fungal isolates
Four GTD fungal pathogens: D. seriata DAR79990, N. parvum DAR80004, N. luteum DAR81016, and E. lata WB052 were used to study the antagonistic mechanisms of P. poae. The fungal pathogens were originally isolated from grapevines
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Chapter 4 exhibiting symptoms of GTD in Australian vineyards and maintained in the culture collection at the NWGIC. All fungal isolates were previously identified by sequencing part of the ribosomal RNA gene (Qiu et al., 2011; Wunderlich et al., 2011; Billones-
Baaijens et al., 2018).
4.2.3. Elucidation of antagonistic mechanisms of Pseudomonas poae BCA17 against GTD pathogens
4.2.3.1. Siderophore production
The ability of the antagonistic strains of Pseudomonas to produce siderophores was investigated using Chrome Azurol (CAS) agar plates (Schwyn and Neilands,
1987; Louden et al., 2011). Siderophores are strong iron chelators, providing an organism the capability to efficiently sequester iron (Louden et al., 2011). The chrome azurol S (CAS) assay is a universal test to detect siderophores, particularly in Gram- negative bacteria, providing a simple blue to orange colour change in the presence of siderophores (Louden et al., 2011).
Single colonies from 48-h-old cultures of the 11 strains of Pseudomonas examined (Chapter 2, Table 2) were streaked onto CAS media, while SDW was used as negative control. The plates were incubated for 3 days and were observed for change in the colour of the media. Four replicates were made per bacterial strain.
4.2.3.2. Bioactivity of Pseudomonas poae derived culture filtrates on mycelial growth and spore germination of GTD pathogens
4.2.3.2.1 Preparation of culture filtrates
The ability of P. poae BCA17 to produce bioactive diffusible compounds against the selected GTD pathogens was investigated. The strain was streaked on NA, incubated for 24 h, and subsequently a loopful of bacterial cells from a single colony 99
Chapter 4 transferred into McCartney bottles containing 10 ml NB with and without the mycelial plug (4 mm) from either D. seriata DAR79990, N. parvum DAR80004, N. luteum
DAR81016, or E. lata WB052. BCA17 was grown with and without the fungal pathogens to assess whether bioactive compounds are induced by the presence of the pathogen. All McCartney bottles were incubated for 24 h in an orbital shaking incubator at 25°C, 150 rpm. The resulting bacterial suspensions (10 ml) were passed twice through a 0.22 µm Minisart syringe filter (Sartorius Stedim Biotech, Goettingen,
Germany) to remove bacterial cells, creating a cultural filtrate. The filtrate was plated onto NA to ensure that it was free from bacterial cells. The cell-free P. poae BCA17 culture filtrate was used to investigate the effect of diffusible compounds on mycelial growth and spore germination of selected GTD pathogens.
4.2.3.2.2. Effect of culture filtrates on mycelial growth
The bioactivity of the culture filtrate derived from P. poae BCA17 was evaluated on the mycelial growth of four representative GTD pathogens: D. seriata DAR79990,
N. parvum DAR80004, N. luteum DAR81016, and E. lata WB052.
Mycelial plugs of the selected fungal pathogens were subcultured on PDA amended with chloramphenicol (PDAC, 100 mg/L of agar). The plates were incubated at 25°C for 5 days in total darkness. Mycelial discs were obtained from the margin of an actively growing fungal colony using a 4 mm cork borer and were used as fungal inocula.
The culture filtrates (10 ml) derived from P. poae BCA17 with and without co- incubation with a GTD pathogen was transferred into 55 mm Petri plates. A mycelial plug (4 mm) of each of the four pathogens were added to individual Petri plate containing the culture filtrates, and to plates containing only NB which acted as negative controls. Three replicate plates represented each treatment. All plates were 100
Chapter 4 incubated at 25°C in the dark. After 7 days, the mycelium was harvested and placed on a sterile filter paper (Whatman™, GE Healthcare Life Sciences). The agar plug was removed and the mycelia was allowed to air dry for 30 minutes before being weighed.
4.2.3.2.3. Effect of culture filtrates on spore germination
The effect of culture filtrates derived from P. poae BCA17 on spore germination of the selected GTD pathogens was evaluated. For production of conidia, mycelial plugs of
D. seriata DAR79990, N. parvum DAR80004, and N. luteum DAR81016, were transferred onto prune extract agar (Appendix A). The plates were incubated at 25°C overnight under continuous darkness before transferring to under UV light (Phillips 20w
UVB) in a 12 h light-dark regime for 4 weeks at 18-22°C or until fruiting bodies developed. To harvest the conidia, plates were flooded with 5 ml of SDW and fruiting bodies were scraped off and ground with a sterile mortar and pestle before sieving using two layers of sterile Miracloth (Merk Millipore). An ascospore suspension of E. lata isolated from infected grapevine wood was obtained from the South Australian
Research and Development Institute, Australia (SARDI). The spore concentrations for conidia and ascospores were determined using a haemocytometer and adjusted to
5 3x10 spores/ml. An aliquot (200 µl) of spore suspension from each of the pathogens were transferred into a sterile 1.5 ml tube and an equal volume of the BCA17 culture filtrate without co-inoculation with fungal pathogen was added. For the control treatment, an equal volume of NB was added to the spore suspension instead of the culture filtrate. The assay was performed with three replicate tubes per treatment. All tubes were incubated for 24 h at 25°C. A loopful of the spore suspension was transferred into a microscope slide. The first 100 spores in the microscope field of view at 40 x magnification were counted and the number of spores that germinated were recorded. A spore was considered to have germinated if the germ tube was half the length of the spore.
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Chapter 4 4.2.3.3. Production of bioactive volatile compounds
The ability of P. poae BCA17 to produce bioactive volatile compounds with antagonistic activity to the four selected GTD pathogens was assessed in vitro using the double plate technique. In this assay, the mycelial discs (4 mm) of the fungal pathogens were inoculated onto PDA plates while 100 µl of P. poae BCA17 (1010 CFU ml-1) was evenly spread onto NA plates with an L-shaped hockey stick. The NA plate with bacteria had the PDA plate with fungi inverted over it. This was then sealed with parafilm to trap the volatiles. The GTD pathogens were exposed to the same day, 24 h, 48 h and 72 h culture of P. poae BCA17. Control plates had the mycelial discs of the pathogens exposed to NA only. The assays were performed at three replicates per treatment. Colony diameter was measured following incubation at 25°C for 3-7 days.
4.2.3.4. Thin layer chromatography (TLC) for the production of antibiotics 2,4 diacetylphloroglucinol (DAPG) and phenazine
The role of the antibiotics 2,4 diacetylphloroglucinol and phenazine in the biocontrol of GTD was examined. A preliminary test on the ability of P. poae BCA17 to produce DAPG and phenazine was conducted with thin layer chromatography (TLC).
Single colonies of BCA17 were streaked onto 12 NA plates, covering the entire surface of the media. After 3 days of incubation at 25°C in darkness, 10 ml SDW was added to each plate and bacterial cells were dislodged with a sterile L-shaped hockey stick.
Bacterial suspensions were collected in sterile 500 ml Schott bottles and homogenised using a vortex mixer. The bacterial suspension (20 ml) was then pipetted into a Schott bottle containing 1 L NB for a total of six bottles. The bacterial cultures were incubated at 30°C in a rotary shaker (150 rpm) for 3 days. Bacterial suspensions were centrifuged at 3500 rpm for 5 min to separate the bacterial cells from the filtrate. The volume of the filtrate was then reduced to 1 L from an intial volume of 6 L using a rotavapor. The
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Chapter 4 organic compounds from cell-free filtrate were then extracted three times with ethyl acetate (3 x 400 ml) until a colourless extract was obtained. MgSO4 salts were added onto the colourless organic extracts to absorb the remaining water. The extract was concentrated to near dryness in a Buchi Rotavapor R-114 (Artisan Technology Group,
IL, USA) after passing the extract through a filter paper (Whatman #1, 90 mm diameter,
0.18 mm thick). The dried sample was dissolved by adding a small volume of methanol and chloroform (98:2, v/v) onto the sample flask, swirling the flask to dissolve the sample adhering to its wall. The dissolved concentrated extract was transferred into pre-weighed small vials using a Pasteur pipette. An aliquot of the extract (100 ml) was also taken and acidified to pH2 after concentration of the filtrate in rotavapor.
A 100 µl aliquot of the extract was applied onto TLC plates (Merck, Darmstadt,
Germany) coated with an adsorbent layer of silica gel. 2,4 diacetylphloroglucinol
(Santa Cruz Biotechnology, Inc., Heidelberg, Germany) and phenazine (Sigma-Aldrich
Pte. Ltd., Singapore) standards were also spotted in the same TLC plates. Separation was achieved by dipping the plates into the solvent mixture. Different solvent systems were tested: chloroform:isopropanol (9:1), chloroform:isopropanol (9.5:0.5), chloroform:methanol (9.5:0.5), acetonitrile:methanol:water(1:1:1), isopropanol:Ammonia:Water (8:1:1). Spots were viewed under UV light at 254 nm. The plates were also dabbed with cotton balls soaked in sulphanilic acid (10% in ethanol, oxidizing solution) and then dried for 5 min in an oven at 90°C for improved visualization of the spots. The retention factor (Rf) of the standard and the samples were computed, using the formula:
Rf = distance travelled by the compound distance travelled by the solvent
RF values of the sample was compared with the standard.
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Chapter 4 4.2.3.5. Identification of biocontrol genes
The genomes of four strains of Pseudomonas poae (BCA13, BCA14, BCA17 and JMN1) were sequenced, as described by Niem et al. (2019; Appendix 1), by the
Australian Genome Research Facility (AGRF, Australia). Strains BCA13, BCA14 and
BCA17 were antagonistic to GTD pathogens (Chapter 2) and reduced Botryosphaeria dieback infection in grapevine plants (Chapter 3) while P. poae JMN1 did not exhibit any antagonistic activity. Draft genomes were assembled and annotated as described by Niem et al. (2019). Genome contigs were submitted to the antiSMASH 4.0 web- server (Blin et al., 2017) to identify gene clusters which may play a role in the control of plant pathogens.
4.2.3.6. Detection of bioactive lipopeptides
Detection of antifungal lipopeptides was performed using Matrix-Assisted Laser
Desorption/Ionization Time-Of-Flight mass spectrometry (MALDI-TOF MS) analysis.
Crude extraction of lipopeptide was achieved following the methods of Sajitha et al.
(2016). Two strains, P. poae BCA17 (antagonistic) and P. poae JMN1 (non- antagonistic), were analysed for the presence of bioactive lipopeptides. A bacterial suspension (10 µl) of each strain was streaked on one side of a PDA plate while a mycelial plug of the GTD pathogen, D. seriata, was placed on the opposite side. After
72 h of incubation at 25°C, one loopful of the bacterial cells from the interface of the dual culture was suspended in 500 µl acetonitrile with trifluoroacetic acid (0.1 %) for 2 min. The mixture was vortexed to obtain a homogenised suspension. Bacterial cells were separated from the supernatant by centrifugation at 5000 rpm for 10 min. The cell-free supernatants were transferred to new microcentrifuge tubes and were sent to
Australian Proteome Analysis Facility (APAF) for MALDI-TOF-MS analysis.
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Chapter 4 Samples were diluted 1:10 with 50% acetonitrile 0.1% formic acid and directly infused into a QExactive Plus mass spectrometer. MS1 scan indicated the detection of intact lipopeptides, and MS/MS and subsequent de novo sequencing was performed on the ion at 1049.6654 m/z to assess its sequence.
4.2.3.7. Statistical analysis
All data from the culture filtrate assays were analysed within Genstat, 17th edition. The data were tested for homogeneity of variance using Levene’s test at
P≤0.05. Since the pathogen growth rates differ, the effects of BCA17 culture filtrate on mycelial biomass for each pathogen were determined separately using one-way
ANOVA, with significance at P ≤0.05. Means were separated by pairwise comparison using LSD (P ≤0.05 significance level). Differences on the effect of BCA17 culture filtrate on spore germination for each species were determined using t-tests at P ≤0.05 level.
4.3. Results
4.3.1. Test for siderophore production
Each of the 10 strains of Pseudomonas that exhibited antagonistic ability in vitro
(as described in Chapter 2, Table 2) as well as the non-antagonistic strain (JMN1), produced siderophores in Chrome azurol S (CAS) medium. This was evidenced by the change in colour of the medium from blue to orange 3 days after the inoculation of the media with each strain. These results indicate all strains of Pseudomonas examined were capable of sequestering the iron.
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A B
Figure 4.1. Antagonistic Pseudomonas spp. in Chrome Azurol S (CAS) medium showing change in colour of the medium from blue to orange indicating production of siderophores (A), and Chrome Azurol S medium in the absence of siderophore-forming Pseudomonas (B).
4.3.2. Bioactivity of the Pseudomonas poae culture filtrate on mycelial growth and spore germination of GTD pathogens
4.3.2.1. Effect of culture filtrate on mycelial growth
The cell-free culture filtrates derived from P. poae BCA17 were inhibitory to the mycelial growth of GTD pathogens D. seriata DAR79990, N. luteum DAR81016, N. parvum DAR80004, and E. lata WB052, regardless of whether these were obtained from cultures co-inoculated with the pathogen or not. Mycelial weight of D. seriata
DAR79990 grown in NB only was significantly higher (76.26 mg; (P ≤0.01) when compared to mycelia grown in the culture filtrate of P. poae BCA17, where mycelial biomass of 5.23 mg (filtrate obtained from BCA17 + pathogen co-culture) and 1.87 mg
(filtrate obtained from BCA17 only culture). E. lata WB052 was typically slow growing and the mycelial weight in NB was 15 mg which was still significantly different from the biomass of the pathogen that was grown in the culture filtrate of P. poae BCA17 (P
≤0.01) where no growth of the pathogen was observed after 7 days. A similar pattern was observed with N. luteum DAR81016 with181.7 mg mycelial biomass, and N. parvum DAR80004 with 190.63 mg in filtrates containing NB only. N. luteum 106
Chapter 4 DAR81016 and N. parvum DAR80004 did not grow in the culture filtrate of P. poae
BCA17. No significant differences in fungal biomass (P ≥ 0.05) were observed between culture filtrates derived from broth cultures with and without the pathogen.
Figure 4.2. Effect of culture filtrates derived from Pseudomonas poae BCA17 on mycelial growth of Diplodia seriata; b) Neofusicoccum parvum; c) N. luteum; and d) Eutypa lata. The treatments were filtrates from the BCA17 strain nutrient broth culture with and without the pathogen. Bars with different letters indicate significant differences in mycelial biomass at P ≤0.05 LSD.
4.3.2.2. Effect of culture filtrate on spore germination
Spore germination of the GTD pathogens D. seriata DAR79990, N. luteum
DAR81016, N. parvum DAR80004, and E. lata WB052 were inhibited by the culture filtrates of P. poae BCA17 at varying degrees 24 h after incubation. The BCA17 culture filtrate reduced D. seriata DAR79990 spore germination to 1%, which was significantly lower (P ≤0.0001) than the untreated control at 60% germination (Figure 4.3a). The 107
Chapter 4 BCA17 culture filtrate also reduced N. parvum DAR80004 (Figure 4.3b) and N. luteum
DAR81016 (Figure 4c) spore germination with 13 and 27% germination, respectively, which was significantly lower than the untreated control at 47 and 48% spore germination, respectively. E. lata WB052 was the least sensitive to the culture filtrate, resulting in 86% germination in the control treatment and 77% in the P. poae BCA17 culture filtrate. However, mean difference between these treatments was still significantly different from each other at (P ≤0.002).
Figure 4.3. Effect of culture filtrates derived from Pseudomonas poae BCA17 on spore germination of: a) Diplodia seriata; b) Neofusicoccum parvum; c) N. luteum; and d) Eutypa lata. Bars with different letters for each species indicate significant differences in spore germination at P ≤0.002 using t-tests.
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4.3.3. Test for production of bioactive volatile compounds
Tests to assess the ability of P. poae BCA17 to produce inhibitory volatile compounds yielded negative results in the double plate assay. There was no difference in the rate of growth or mycelial diameter between fungi exposed to volatiles produced by BCA17 and those of the negative control (Figure 4.4a and 4.4b. The pathogens grew profusely with and without P. poae BCA17 on the opposite plate. There were instances when the replicates exposed to volatiles produced by BCA17 had larger mycelial diameter. This indicates that the antagonistic and suppressive activity of P. poae BCA17 was not directly due to volatile compounds produced by the bacterial strain.
Actual measurements and statistical analysis were not continued since the absence of treatment effect was very evident, regardless of the age (same day, 24 h,
48 h, and 72 h) of P. poae culture.
A) B)
Figure 4.4. Mycelial growth of Diplodia seriata after exposure to P. poae BCA17 volatiles: A) Control treatment – without P. poae BCA17; B) with P. poae BCA17.
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Chapter 4 4.3.4. Test for production of the antibiotics 2,4 diacetylphloroglucinol (DAPG) and phenazine
The TLC analysis revealed the presence of DAPG in the organic extracts obtained from the culture filtrate of P. poae BCA17 under unmodified pH condition
(Figure 4.5, Lane 1). The Rf value of the extract from the culture filtrate was the same as the DAPG standard (Rf = 0.64; Figure 4.5, Lane 2). However, DAPG was not detected when the culture filtrate was extracted at pH2 (Figure 4.5, Lane 3) as evident by the absence of corresponding spots that migrated to the same distance as the
DAPG standard. Phenazine was not detected in the chromatographic profile of the organic extract in both unmodified and acidified pH conditions as indicated by the absence of spots with the same Rf as the phenazine standard (Figure 4.5, Lanes 4-6).
1 2 3 4 5 6
Figure 4.5. Thin layer chromatography profile of organic compounds extracted from Pseudomonas poae BCA17 filtrate visualised by 254 nm UV light. Lane 1, BCA17 culture filtrate unmodified (pH 8); 2, 2,4 DAPG standard; 3, BCA17 culture filtrate (pH2); Lane 4, BCA17 culture filtrate unmodified (pH 8); 5, phenazine standards; 6) BCA17 culture filtrate (pH 2).
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Chapter 4 4.3.5. Identification of biocontrol genes.
The secondary metabolites and their corresponding biosynthetic gene clusters are summarised in Table 4.1. The genome features of the antagonistic P. poae strains
BCA13, BCA14, and BCA17 and the non-antagonistic P. poae strain JMN1 were very similar (Appendix A, Table 1). Genome analysis using antiSMASH 4.0 (Blin et al., 2017) showed consistent presence of gene clusters coding for the synthesis of the siderophore pyoverdine and APEs (aryl polyenes) Vf and Ec, which comprised the largest biosynthetic gene cluster. The four P. poae BCA strains also displayed a notable array of gene clusters associated with the production of various cyclic lipopeptides (CLPs), including rhizoxin, viscosin, orfamide, putisolvin, poaeamide, bananamide, entolysin, anikasin, white-line-inducing principle (WLIP), cichopeptin, tolaasin, sessilin, xantholysin, arthrofactin (Table 4.1). Some of these CLP have been previously described to have antifungal property.
Table 4.1. Secondary metabolites of P. poae BCA strains 13, 14, 17, and JMN1 and their corresponding biosynthetic gene clusters.
Secondary Metabolite Gene Location Subject cluster
Pyoverdine PFL_4178 AAY93434.1 MbtH-like_protein PFL_4179 AAY93435.2 2,4-diaminobutyrate_4-transaminase PFL_4180 AAY93436.1 sensor_histidine_kinase PFL_4081 AAY93437.1 DNA-binding_response_regulator PFL_4082 AAY93438.1 thiol:disulfide_interchange_protein_DsbD PFL_4083 AAY93439.1 antioxidant,_AhpC_family PFL_4084 AAY93440.1 thiol:disulfide_interchange_protein_DsbG PFL_4089 AAY93445.1 non-ribosomal_peptide_synthetase_PvdL RNA_polymerase_sigma- PFL_4190 AAY93446.1 70_factor,_ECF_subfamily,_PvdS PFL_4191 AAY93447.1 pyoverdine_biosynthesis_protein
Viscosin PFLU_2552 CAY48788.1 putative_non-ribosomal_peptide_ synthetase PFLU_2553 CAY48789.1 putative_non-ribosomal_peptide_ synthetase 111
Chapter 4
PFLU_2555 CAY48790.1 putative_drug-efflux_protein PFLU_2556 CAY48791.1 macrolide-specific_ABC-type_efflux_ carrier PFLU_2557 CAY48792.1 putative_LuxR-family_regulatory_protein PFLU_2558 CAY48793.1 methionine_gamma-lyase PFLU_2559 CAY48794.1 AsnC_family_regulatory_protein
Orfamide PFL_2145 AAY91419.3 non-ribosomal_peptide_synthetase_OfaA PFL_2146 AAY91420.2 non-ribosomal_peptide_synthetase_OfaB
PFL_2147 AAY91421.3 non-ribosomal_peptide_synthetase_OfaC PFL_2148 AAY91422.3 efflux_transporter,_RND_family,_MFP_subunit
PFL_2149 AAY91423.3 efflux_ABC_transporter,_permease/AT P- binding_protein
PFL_2150 AAY91424.1 transcriptional_regulator,_LuxR_family
Putisolvin ABW17375.1 ABW17375.1 PsoA ABW17376.1 ABW17376.1 PsoB ABW17377.1 ABW17377.1 PsoC ABW17378.1 ABW17378.1 MacA ABW17379.1 ABW17379.1 MacB ABW17380.1 ABW17380.1 LuxR-like_protein
Poaemide H045_07035 AGE25479.1 putative_non-ribosomal_peptide_synthetase H045_10940 AGE25480.1 amino_acid_adenylation_protein H045_10945 AGE25481.1 putative_drug-efflux_protein H045_07050 AGE25482.1 macrolide-specific_ABC-type_efflux_carrier H045_10955 AGE25483.1 putative_LuxR_family_regulatory_protein
Anikasin A8O26_RS1 3955 WP_06411855 7.1 MacB_family_efflux_pump_subunit A8O26_RS1 3960 WP_06411855 8.1 macrolide_transporter_subunit_MacA A8O26_RS1 3965 WP_06411855 9.1 non-ribosomal_peptide_synthetase A8O26_RS1 3970 WP_06411856 0.1 non-ribosomal_peptide_synthetase A8O26_RS1 3975 WP_06411856 1.1 non-ribosomal_peptide_synthetase A8O26_RS1 3980 WP_06411856 2.1 non-ribosomal_peptide_synthetase
Rhizoxin CAL69886.1 CAL69886.1 RhiI_protein CAL69887.1 CAL69887.1 RhiG_protein CAL69888.1 CAL69888.1 RhiA_protein CAL69889.1 CAL69889.1 RhiB_protein CAL69890.1 CAL69890.1 RhiC_protein CAL69891.1 CAL69891.1 RhiD_protein CAL69892.1 CAL69892.1 RhiH_protein
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CAL69893.1 CAL69893.1 RhiE_protein CAL69894.1 CAL69894.1 RhiF_protein WLIP AFJ23825.1 AFJ23825.1 WLIP_synthetase_B AFJ23826.1 AFJ23826.1 WLIP_synthetase_C AFJ23827.1 AFJ23827.1 MacA-like_protein AFJ23828.1 AFJ23828.1 MacB-like_ABC_transporter_protein
Cichopeptin AHZ34239.1 AHZ34239.1 CipB AHZ34242.1 AHZ34242.1 CipE AHZ34248.1 AHZ34248.1 MacA AHZ34249.1 AHZ34249.1 MacB
Tolaasin CCJ67637.1 CCJ67637.1 TaaB CCJ67640.1 CCJ67640.1 TaaE CCJ67641.1 CCJ67641.1 macrolide-specific_efflux_protein_macA CCJ67642.1 CCJ67642.1 macrolide_export_ATP- binding/permease_protein_MacB
Sessilin AFH75320.1 AFH75320.1 nonribosomal_peptide_synthetase AFH75321.1 AFH75321.1 nonribosomal_peptide_synthetase AFH75323.1 AFH75323.1 macrolide-specific_efflux_protein AFH75324.1 AFH75324.1 macrolide_export_ATP- binding/permease_protein_MacB
Bananamides AOA33121.1 AOA33121.1 nonribosomal_peptide_synthetase AOA33122.1 AOA33122.1 nonribosomal_peptide_synthetase AOA33123.1 AOA33123.1 nonribosomal_peptide_synthetase AOA33124.1 AOA33124.1 RND_family_efflux_transporter_MFP_subunit AOA33125.1 AOA33125.1 ABC_transporter_ATP-binding_protein AOA33126.1 AOA33126.1 LuxR_family_DNA-binding_response_regulator
Entolysin PSEEN3042 CAK15812.1 macrolide_ABC_efflux_protein_MacB PSEEN3043 CAK15813.1 macrolide_efflux_protein_MacA PSEEN3044 CAK15814.1 putative_non- ribosomal_peptide_synthetase,_terminal_comp o nent PSEEN3045 CAK15815.1 putative_non_ribosomal_peptide_synthetase
Xantholysin AGM14933.1 AGM14933.1 xantholysin_synthetase_B AGM14934.1 AGM14934.1 xantholysin_synthetase_C AGM14935.1 AGM14935.1 MacA-like_protein AGM14936.1 AGM14936.1 MacB-like_ABC_transporter_protein
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Syringafactin PSPTO_282 9 AAO56328.1 non-ribosomal_peptide_synthetase_SyfA PSPTO_283 0 AAO56329.1 non-ribosomal_peptide_synthetase_SyfB PSPTO_283 1 AAO56330.1 syringafactin_efflux_protein_SyfC PSPTO_283 2 AAO56331.1 syringafactin_efflux_protein_SyfD
Arthrofactin BAC67534.2 BAC67534.2 arthrofactin_synthetase_A
BAC67535.1 BAC67535.1 arthrofactin_synthetase_B BAC67536.1 BAC67536.1 arthrofactin_synthetase_C
BAC67537.1 BAC67537.1 putative_periplasmic_protein APE Vf VF_084155 AAW85336.1 dehydrogenase VF_084662 AAW85341.1 acyl_carrier_protein VF_084753 AAW85342.1 acyl_carrier_protein VF_085154 AAW85346.1 predicted_acyltransferase VF_085263 AAW85347.1 phenylalanine_and_histidine_ammonia-lyase VF_085346 AAW85348.1 esterase VF_085751 AAW85352.1 3-oxoacyl-(acyl_carrier_protein)_synthase VF_086061 AAW85355.1 3-oxoacyl-(acyl_carrier_protein)_synthase
APE Ec c1186 AAN79648.1 Putative_beta-ketoacyl-ACP_synthase c1187 AAN79649.1 3-oxoacyl-[acyl-carrier_protein]_reductase c1189 AAN79651.1 Putative_3-oxoacyl-[ACP]_synthase c1193 AAN79655.1 Hypothetical_protein c1194 AAN79656.1 Putative_enzyme c1199 AAN79661.1 Putative_acyl_carrier_protein c1200 AAN79662.1 Putative_acyl_carrier_protein
4.3.6. Detection of bioactive lipopeptides
Due to the abundance of genes associated with the synthesis and regulation of
CLP identified within the strains of P. poae, an analysis was undertaken to verify if lipopeptides are involved in the biocontrol activity of the antagonistic P. poae BCA17.
An assessment of possible lipopeptides by MALDI-TOF MS detected the 1+ ion at
2098.2913 m/z in P. poae BCA17 (Figure 4.6) but not in P. poae JMN1 (Figure 4.7). A comprehensive assessment of literature articles indicated that the 1+ ion at 2098.2913 m/z is likely a new lipopeptide, however the sequence of the compound could not be confirmed with MS/MS analysis. Lack of a comprehensive lipopeptide database with 114
Chapter 4 specific MS detail limited the assessment of the potential identity of this lipopeptide.
From de novo sequencing, the partial sequence was confirmed to have the sequence leu-val-gln-leu-val-val-gln-leu-val (LVQLVVQLV) (C48H87N11O12) (Figure 4.8), which has a monoisotopic mass of 1009.654. This data does not match that in the literature
(Nielsen et al., 2002; de Souza et al., 2003; Raaijmakers et al., 2006; Zachow et al.,
2015; Geudens and Martins, 2018). Further ring opening reaction with ammonia confirmed that the unknown lipopeptide is cyclic in nature and this was not present in
JMN1 sample. From the literature, the new lipopeptide is likely related or belongs to the group of tolaasin. The partial identified sequence shares 8 out of 9 amino acids with L4VSLVVQLV12 sequence of tolaasin with the only difference of Glu instead of Ser in tolaasin (Geudens and Martins, 2018; Bassarello et al., 2004).
Figure 4.6. MALDI-TOF spectrum for the antagonistic Pseudomonas poae BCA17. Red box indicates the presence of a dominant lipopeptide at 2098.2913 m/z (1+ ion).
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Figure 4.7. MALDI-TOF spectrum for the non-antagonistic Pseudomonas poae JMN1 indicating the absence of the lipopeptide at 2096.297 m/z (1+ ion).
Figure 4.8. Fragmentation pattern (MS/MS) of lipopeptide from P. poae BCA17 sample. Amino acid sequence information and key ions are labelled on the spectrum.
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Chapter 5 4.4. Discussion
The research described in this chapter attempted to elucidate the underlying mechanisms involved in the inhibition of GTD pathogens, and the suppression of GTD infection in grapevines, by the grapevine endophytic strain P. poae BCA17. Preliminary tests indicated that the antibiotic DAPG was produced in the culture filtrate of P. poae
BCA17. Furthermore, we found a putative novel CLP that may contribute to the biocontrol ability of P. poae BCA17.
All the strains of Pseudomonas, be it antagonistic or non-antagonistic, produced siderophores as determined by the change in colour of the CAS media following inoculation. The change from blue to orange is an indication of the uptake of iron from the dye complex component of the media by the fluorescent Pseudomonas. This is presumably made possible by the ability of all the fluorescent Pseudomonas strains to produce a strong iron chelator such as siderophores (Schwyn and Neilands, 1987;
Louden et al., 2011). This result shows that siderophores may not be directly involved in the antagonistic activity of P. poae strains against the GTD pathogens due to the non-antagonistic Pseudomonas strains also producing siderophores. However, it is possible that siderophore production may provide a competitive advantage to the endophytic Pseudomonas spp. tested over the GTD pathogens. There are also other possible indirect role of siderophores in disease control that could be considered.
Leeman et al. (1996) postulated that Fe3+ chelating siderophores produced by P. fluorescens WCS417 at low iron availability, is involved in the induction of systemic resistance to Fusarium wilt of radish. Disease suppression was observed even in the absence of direct interaction between the pathogen and bacteria after application of
Pseudomonas sp. at separate locations on the plant (Leeman et al., 1996). Aside from direct antagonism of the pathogen, siderophores may also indirectly stimulate the
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Chapter 5 biosynthesis of other antimicrobial compounds by increasing the availability of minerals to the bacteria (Duffy and Défago, 1999). Siderophores may also have an indirect contribution to the biocontrol ability of Pseudomonas via its role in biofilm formation (Saha et al., 2012). Biofilm formation and its development depends on the availability of nutrients, specifically, intracellular iron (Saha et al., 2012). The production of biofilms enhances the ecological competence of the bacteria by providing a shield against harsh environmental condition. Thus, the siderophores produced by the BCAs inadvertently increases the competitiveness of the bacteria through its role in biofilm formation.
Production of bioactive diffusible compounds by P. poae BCA17 was also established. This was made evident by the reduction in biomass produced by the GTD pathogens D. seriata DAR79990, N. luteum DAR81016, N. parvum DAR80004, and
E. lata WB052 when incubated in the cell-free culture filtrate of P. poae BCA17. No significant difference in the mycelial biomass was observed in cultrate filtrates derived from BCA17 broth cultures with and without the pathogen, indicating that the bioactive fraction in the culture filtrate was innately produced by the bacteria and does not need to be induced by the presence of the pathogen. The P. poae BCA17 culture filtrate had a similar inhibitory effect on spore germination of the above mentioned GTD pathogens with 10-99% reduction in spore germination when the spores were incubated in the cell-free culture filtrate of the bacteria. The highest inhibition of spore germination was observed with D. seriata with 99% reduction in spore germination indicating this pathogen was highly sensitive to the diffusible compounds produced by strain BCA17.
For the four pathogens tested, E. lata WB052 was the least sensitive with only 10% reduction in spore germination although this was significantly different than the control.
Sexual reproductive structures are generally produced by fungi when food supply is diminishing or when environmental factors are not favourable to their growth (Agrios,
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Chapter 5 2005). Thus, it is possible that the lower efficacy of BCA17 culture filtrate on E. lata ascospores may be due to the sexual structures that are potentially more resistant to external factors compared to conidia. General observations also suggest ascospores of E. lata have thicker cell walls compared to the conidia of Botryosphaeriaceae, which may explain their differences in sensitivity to the diffusible compound. However, this hypothesis needs further investigation. It is possible that species of
Botryosphaeriaceae are more susceptible to the diffusible compounds produced by
BCA17 than E. lata.
Several strains of antagonistic Pseudomonas genus with biocontrol ability against a wide range of fungal pathogens have been reported to produce various anti- fungal compounds (Howell and Stipanovic, 1980; Thomashow and Weller, 1988; Keel et al., 1992; Mazzola et al., 1992; Lorito et al., 1994; Ramette et al., 2003; Santoyo et al., 2012; Loper et al., 2012; Arseneault et al., 2013; Arseneault et al., 2016). Some produce known diffusible compounds including hydrolytic enzymes, such as proteases, cellulases, chitinase and ß-glucosidase; antibiotics like DAPG, pyrrolnitrin, pyoluteorin, phenazines, siderophores; and a number of CLPs, (Raaijmakers et al.,
2010; Calderón et al., 2015). For instance, a study conducted by Zachow et al. (2015), demonstrated that when Phytophthora capsici, P. infestans, Pythium ultimum, and
Rhizoctonia solani were grown in direct contact with the lipopeptide poaeamide produced by the sugar beet endophyte Pseudomonas poae RE*1-1-14, an inhibitory effect was present in regards to the fresh and dry weights of the mycelial biomass for all tested pathogens. Increased hyphal branching was also observed for Rhizoctonia after exposure to a culture filtrate of the CLP-producing Pseudomonas CMR12a (D’aes et al., 2011). Subjecting hyphae of Pythium ultimum and R. solani to viscosinamide resulted in increased branching, hyphal swelling and increased septation. Changes in the esterase activity, intracellular pH and mitochondrial organization and activity, and
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Chapter 5 decrease in hydrophobicity of cellular membranes and cell walls were also observed
(Thrane et al., 1999). This effect was extended to the zoospores of Phytophthora capsici and P. infestans. Cell-free extracts of Pseudomonas poae RE*1-1-14 and purified poaeamide were reported to cause lysis and immobilisation of Phytophthora capsici and P. infestans zoospores but cell-free culture supernatant of the poaeamide- deficient mutants did not (Zachow et al., 2015).
Although volatile compounds have been implicated in disease control by some species of Pseudomonas, P. poae BCA17 did not appear to produce volatile compounds inhibitory to GTD pathogens. All the GTD pathogens grew profusely in the double plate technique which exposed the pathogens to the trapped bacterial volatiles.
Hydrogen cyanide from P. fluorescens has been involved in the protection of tobacco against T. basicola that causes black root rot (Voisard et al., 1989). Alteration of mitochondrial activity and cell death of mushroom mycelia have been reported when
Pleurotus spp. and Agaricus bisporus were exposed to Pseudomonas tolaasii volatiles methanethiol and dimethyl disulfide. In vitro tests exposing the GTD pathogens to the volatiles produced by P. poae BCA17 did not show any apparent inhibition of the pathogens.
DAPG and phenazines are two of the more well-known and studied antibiotics associated with the biocontrol ability of antagonistic Pseudomonas spp. Preliminary testing using TLC for detection of DAPG and phenazine showed possible production of DAPG by P. poae BCA17, but not phenazine. This is not uncommon, since as previously mentioned, different strains of Pseudomonas are equipped with different mechanisms which enable them to exert antagonistic activity to pathogens. The role
DAPG in disease control has been reported in many studies. It has been implicated in the suppression of take-all in wheat (Raaijmakers and Weller, 1998) and control of damping-off in sugar beet (Nielsen et al., 1999), Fusarium crown and root rot in tomato,
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Chapter 5 (Sharifi-Tehrani et al., 1998), black root rot in tobacco (Keel et al., 1992), to name a few. Involvement of DAPG in the induction of resistance has been suggested by Keel et al. (1992). De Souza et al. (2003) further proposed that DAPG could cause accumulation of secretion vesicles that are closely associated with the rough endoplasmic reticulum. This would subsequently result in the inhibition of hyphal elongation. Although TLC results showed that a detectable amount of DAPG was produced by P. poae BCA17, this is only a preliminary analysis and thus warrants a more in depth investigation.
The draft genomes of P. poae strains BCA13, BCA14, BCA17 and JMN1 revealed that all strains possess the same set of putative biocontrol genes. Pyoverdines
(also known as pseudobactins) are a defining characteristic of the fluorescent
Pseudomonads, and so pyoverdine gene clusters are a component of the core genome common to all species. With the exception of Azotobacter vinelandii, pyoverdines are siderophores exclusively produced by Pseudomonads (Gross and Loper, 2009).
Together with APE Vf and APE Ec, they comprised the largest biosynthetic gene cluster in the four strains examined here. Pyoverdines are important for the acquisition of Fe3+ ions from the environment and may serve as intracellular signalling compounds controlling gene expression (Gross and Loper, 2009). The other roles of siderophores in biocontrol have been discussed previously. In general, APEs play a role in protecting bacterial cells from exogenous oxidative stress by reducing the concentration of free radicals that would otherwise cause damage to other cellular lipids, proteins, or nucleic acids (Cimermancic et al., 2014). Analysis of the secondary metabolite encoding genes present in the genome of P. poae strains BCA13, BCA14,
BCA17, and JMN1 revealed the presence of biosynthetic genes that could be involved in the production of antifungal compounds. Gene clusters for the synthesis of different
CLPs were abundant in the genomes of the four strains. CLPs are produced by non-
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Chapter 5 ribosomal peptide synthetases (NRPSs) in bacteria, mostly Pseudomonas, Bacillus, and Streptomyces spp. (Raaijmakers et al., 2010). They are made up of a fatty acid tail linked to a short oligopeptide, which is cyclised to form a lactone ring between two amino acids in the peptide chain (Raaijmakers et al., 2006). CLPs are a structurally diverse group owing to differences in the length and composition of the fatty acid tail and to variations in the number, type and configuration of the amino acids in the peptide moiety, which in turn cause diversity in their biological activity (Raaijmakers et al., 2006). A common trait for CLPs is their efficiency to lower the surface tension in a medium, indicating that the compounds were very powerful surfactants. One likely role of the CLP compounds, therefore, is an enhancement of surface motility, which in turn induces swarming motility in fluorescent Pseudomonas spp. (Nielsen et al., 2002).
This characteristic is important for bacterial colonisation. CLPs are able to insert into membranes and perturb their function, leading to broad antibacterial and antifungal activities (Raaijmakers et al., 2006).
Among the many secondary metabolites produced by P. poae strains BCA13,
BCA14, BCA17, and JMN1, the CLPs viscosin, orfamide, sessilin, bananamide, white- line- inducing principle (WLIP), anikasin, and poaemide are regarded with interest in this chapter as they have been suggested to play a role in biological control of plant pathogens. The CLP viscosin plays a role in motility and biofilm formation by the biocontrol strain P. fluorescens SBW25, and has lytic activity against zoospores of
Phytophthora infestans (De Bruijn et al., 2007). Viscosin has also been ascribed to the colonisation efficiency and microbial fitness of Pseudomonas fluorescens SBW25 in the plant environment due to its role in surface motility and efficiency of surface spreading over the plant root (Alsohim et al., 2014). Viscosin further protects germinating sugar beet seedlings in soil infected with the plant pathogen Pythium
(Alsohim et al., 2014). Mutants impaired in the ability to produce viscosin were found
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Chapter 5 to be less inhibitory to G. graminis var. tritici and R. solani in vitro and has reduced ability to suppress take-all (Yang et al., 2014). A viscosin-like lipopeptide from
Pseudomonas parafulva JBCS1880 is implicated for the control of Xanthomonas axonopodis pv. glycines and Burkholderia glumae which cause bacterial pustule in soybean plants (Kakembo and Lee, 2019). Disruption of viscosin-like lipopeptide via random transposon (Tn) mutation suppressed antagonism, swarming motility and biofilm formation of P. parafulva JBCS1880 (Kakembo and Lee, 2019). Suppression of damping-off disease on Chinese cabbage and root rot on bean was attributed to the ability of Pseudomonas sp. CMR12a to produce the CLPS orfamides and sessilins
(Olorunleke et al., 2015). In vitro microscopic assays revealed that mutants that only produced sessilins or orfamides inhibited mycelial growth of R. solani when applied together, but were ineffective on their own (Olorunleke et al., 2015). Sessilin and orfamide production in CMR12a are coregulated by two of the three luxR-type genes namely ofaR1 and ofaR2 and either OfaR1 can regulate the biosynthesis of these two
CLPs, while the function of SesR is not yet clear (Olorunleke et al., 2017).
Pseudomonas sp. COW3 displayed antagonistic activity against a soil-borne pathogen of cocoyam Pythium myriotylum and a foliar rice pathogen Pyricularia oryzae due to its ability to produce bananamide (Omoboye et al., 2019). Purified extract of this CLP inhibited the growth of Pythium myriotylum and Pyricularia oryzae and caused hyphal distortion. The CLP WLIP produced by Pseudomonas putida RW10S2 has been implicated in the inhibition of phytopathogenic Xanthomonas species that infects the rice rhizosphere (Rokni-Zadeh et al., 2012). WLIP is a member of the viscosin group of cyclic lipononadepsipeptides and is referred to as such due to their capacity to reveal the presence of a nearby colony of P. tolaasii by inducing the formation of a visible precipitate (“white line”) in agar between both colonies (Rokni- Zadeh et al., 2012).
Anikasin has been reported to display amoebicidal activity and inhibited the amoeba
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Chapter 5 Polysphondylium violaceum while mutants deficient for this compound did not have such ability (Götze et al., 2017). Poaemide produced by Pseudomonas poae RE*1-1-
14 has been reported to be the determinant factor in the biocontrol ability of the bacteria against sugar beet pathogens (Zachow et al., 2015). Suppression of R. solani both in vitro and in the field is attributed to the production of this lipopeptide. Like other
CLPs, this lipopeptide is also found to be essential for surface motility and swarming ability of P. poae RE*1-1-14 and enhances root colonisation of sugar beet seedlings
(Zachow et al., 2015).
While all the protein coding genes for the reported antifungal CLPs were present in both the antagonistic and non-antagonistic P. poae strains, it remains unknown if these genes are expressed similarly by the four strains. As part of the continuing effort to understand the basis for the biocontrol ability of BCA17, an attempt to identify the
CLP profile of the antagonistic and non-antagonistic Pseudomonas strains was undertaken using MALDI-TOF analysis.
An unknown cyclic lipopeptide produced by the antagonistic strains of P. poae gave a 1+ ion peak at 2098.2913 m/z with the molecular formula (C48H87N11O12) and amino acid sequence leu-val-gln-leu-val-val-gln-leu-val (LVQLVVQLV). The CLP poaemide from P. poae RE*1-1-14 appears to be distinct from the lipopeptide produced by the four P. poae strains used in this study. The major compound poaeamide A of P. poae RE*1-1-14 gave an [M+Na]+ peak at m/z 1275.7805 in the high-resolution electrospray ionisation mass spectrometry (ESI-MS), with a molecular formula C61H108N10O17 and the peptide sequence Xle-Glu-Thr-Xle-Xle-Ser-Xle- Xle-
Ser-Xle (Xle = Leu or Ile) (Zachow et al., 2015).
The results of the study indicate that P. poae BCA17 produce a combination of compounds that may serve different roles in the biocontrol activity of the strain.
However, the role of the potentially novel CLP and that of DAPG in the biocontrol ability
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Chapter 5 of P. poae BCA17 needs further confirmation by in vitro testing of the purified compounds, and should include in planta assays to assess the effect on disease infection or development. This can only be done after careful identification and purification of the biocontrol compounds. Isolation and purification of the compound followed by a more sensitive and specific analytical technique such as a high resolution
LC/MS-MS may enable identification of the metabolite by comparison of the annotated high resolution m/z value with those reported in the database.
However, this can prove to be challenging as a BCA may employ more than one mechanism to suppress a pathogen, and the relative importance of a particular mechanism may be affected by many factors including nutrient availability, stress factors, or signal molecules of microbial or plant origin that can affect the expression of biocontrol traits. Gene knockouts targeting the biosynthetic genes of these biocontrol compounds will confirm the importance of these compounds in the biocontrol ability of P. poae BCA17. Inactivation of their production through mutagenesis should result in reduced ability to control the GTD pathogens or GTD infection.
The full potential of any BCA can only be achieved if there is a better understanding of the mechanisms that govern their biocontrol ability. Such knowledge can be useful in providing optimum conditions that will favour production of these biocontrol compounds.
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Chapter 5
Chapter 5.
General Discussion
Grapevine trunk diseases (GTDs) are considered major problems of grapevine worldwide. The widespread occurrence of these diseases threaten the sustainability of vineyards, and the use of fungicides to control these diseases only offers a short term solution. Concerns due to environmental problems and the development of resistance by pathogens to fungicides further limit their use. In recent years, there has been increased interest in the use of endophytes as biocontrol agents (BCAs) to potentially manage plant diseases. The main aim of this project was to identify grapevine endophytes with potential as BCAs for the control of GTD and its pathogens.
To understand the interactions between bacterial endophytes and GTDs, the microbial profile of wood collected from GTD asymptomatic and symptomatic grapevines were examined. The dominance of the genus Pseudomonas inhabiting the inner grapevine tissues of asymptomatic wood led to further investigation of isolates from this genus. This entailed targeted isolations of Pseudomonas from grapevine tissues, in vitro screening for their ability to control a range of GTD pathogens, and identification of the most effective BCAs based on molecular tools. In planta assays were conducted to: a) establish that the most promising candidate BCA was not pathogenic to grapevine berries, leaves, or canes; b) ascertain the efficacy in controlling GTD infection in detached canes and intact glasshouse plants; c) assess efficiency in colonising grapevine tissues and; d) investigate the potential of the BCA as a nursery treatment when applied prior to rooting. Finally, the mechanism of control employed by the most promising candidate BCA was investigated in an attempt to understand the mode of action of the BCA in inhibiting the pathogen or suppressing the 126
Chapter 5 disease. These studies were in line with the objectives of the project, which were as follows: a) To identify and compare the microbiome inhabiting symptomatic and asymptomatic GTD grapevine tissue; b) To select, isolate, and screen candidate microorganisms for their biocontrol potential; c) To elucidate the mechanism of action involved in the control of the GTD pathogens by the candidate BCAs; d) To determine the persistence of BCAs within living grapevine tissue. The significant findings in each of the experimental chapters are summarised and presented below along with their implications or recommendations for possible studies in the future.
As described in Chapter 2, the microbiome associated with the grapevine wood was characterised using next-generation Illumina sequencing. Comparison of the microbial composition in GTD asymptomatic and symptomatic vines revealed the dominance of the genus Pseudomonas in asymptomatic grapevine tissues, comprising
56-74% of the bacterial community. In contrast, a lower abundance of Pseudomonas was observed in symptomatic wood samples, accounting for 2-29% of the bacterial population. For the eukaryotic community, this was characterised by the abundance of the genus Phaeomoniella, representing 59-89% of the total fungi detected, followed by Phaeoacremonium. These two fungal genera are associated with GTDs, being implicated in Esca and Petri disease (Mostert et al., 2006b; Crous and Gams, 2000).
Higher abundance of Phaeomoniella in asymptomatic tissues as compared to symptomatic implies that this fungus may initially exist as an endophyte and possibly become pathogenic when the grapevines become stressed (Mugnai et al., 1999;
Edwards and Pascoe, 2004). This was further supported by the studies of Hofstetter et al. (2012) who identified these presumed Esca-associated fungal pathogens in both diseased and healthy 15 to 30-yr-old grapevines, indicating that these fungi are part of the normal mycota associated with adult grapevines.
The abundance of the Pseudomonas genus in asymptomatic grapevine tissues
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Chapter 5 prompted further investigation on the possible role of these bacteria in the control of
GTDs. In vitro assays provided evidence for the inhibition of mycelial growth of D. seriata, N. luteum, N. parvum, E. lata, and Phaeomoniella chlamydospora, or spore production in the case of Ph. minimum by the 10 strains of Pseudomonas. Molecular identification targeting the 16S rRNA gene identified nine strains as P. poae and one strain as P. moraviensis. These results suggested that P. poae displayed a wide spectrum of activity against different GTD pathogens. Its ability to inhibit D. seriata, N. parvum, N. luteum, E. lata, and Phaeomoniella chlamydospora and Ph. minimum may be considered an additional advantage in terms of practical application, and may potentially aid in the management of a wide range of pathogens, not just those associated with GTDs. Bacterial endophytes are commonly present inside the vascular system of plants (Tontou et al., 2015). These are initially rhizobacteria, which first inhabit the endorhiza and then the above ground part of the plant, and have been reported to aid in promoting development and health of their host (Hardoim et al., 2008;
Ryan et al., 2008; Compant et al., 2010). The interaction of endophytes with their environment is influenced by the secondary metabolites that they produce (Brader et al., 2014). These secondary metabolites contribute to biofilm formation and act as antimicrobial agents to protect plants from pathogens (Raaijmakers and Mazzola,
2012). Therefore, it was first necessary to demonstrate that the BCA strains obtained in this study were capable of inhibiting GTD infection in planta.
The results from a range of in planta assays presented in Chapter 3 experiments aimed at validating the efficacy of the antagonistic strains of Pseudomonas to suppress
GTD infection. Based on the pathogenicity tests conducted on leaves, berries and canes using the most effective bacterial strain, P. poae BCA17, it was established that
P. poae BCA17 was not pathogenic to grapevines. Therefore, the absence of disease symptoms on different grapevine tissues upon inoculation of the bacterial strain
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Chapter 5 indicated that P. poae strain BCA17 may be an effective BCA candidate.
This study demonstrated the effectiveness of P. poae as a BCA against N. luteum, one of the species of the Botryosphaeriaceae family causing Botryosphaeria dieback. Challenge inoculation on detached canes and established potted vines affirmed the efficacy of P. poae strain as a BCA to N. luteum. N. luteum was not recovered from detached canes while an 80% reduction in pathogen recovery was observed in potted vines upon challenge inoculation with P. poae BCA17. This result was supported by analysis utilising qPCR where a 40-fold decrease in the amount of pathogen DNA was recorded when vines were pre-treated with P. poae BCA17. It is important to note that the in planta assays only used N. luteum as a model pathogen, thus, further tests on the efficacy of this BCA in planta using a range of GTD pathogens is essential. An endophytic P. poae strain, RE*1-1-14, originally isolated from internal root tissue of sugar cane has been reported to display antagonistic activity against the sugar beet pathogen Rhizoctonia solani (Zachow et al., 2010). Similarly, the grapevine endophyte P. poae BCA17 that was isolated from inner grapevine wood tissues has demonstrated biocontrol activity against GTD pathogens. To my knowledge, studies on the use of Pseudomonas spp. endophytic to grapevine to manage GTD have not been conducted.
This study demonstrated that the Pseudomonas strain was able to endophytically colonise the grapevine tissues via generation of a rifampicin-mutant
(RifMut). In planta assays demonstrated this capability regardless of whether the
RifMut was inoculated basally as a pre-planting treatment, or was apically applied on established potted vines. In potted vines, the RifMut was recovered from the tip of the living tissue down to the basal end of the trunk below the soil line, and on lateral shoots
6 months after inoculation. Inoculations of the RifMut on pre-rooted plants also resulted in recovery of the bacteria from the top, middle, and base sections of all the canes that
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Chapter 5 were dipped for 24 h in a bacterial suspension. The bacteria were still recovered from all the sections after 8 weeks of plant growth. This result has potential application as a nursery treatment to prevent grapevine cuttings from being infected with GTDs prior to planting. This may address the problem presented by Billones-Baaijens et al. (2012) where nursery planting materials were found to act as a source of inoculum for GTDs infections.
The persistence of P. poae BCA17, as shown by its ability to survive within the grapevine tissues for up to 6 months with just a single inoculation, potentially indicates a high adaptive capacity of P. poae BCA17. Biological control depends upon the establishment and maintenance of a threshold population of bacteria on planting material or in soil, and a drop in viability below that level may eliminate the biological control capability (Xu and Gross, 1986; Weller, 1988). Rhizosphere bacteria with biocontrol potential which establish on the plant become a partial sink for nutrients in the rhizosphere. They inhibit sporulation of fungal pathogens, and subsequent colonisation of the roots, by reducing the amount of carbon and nitrogen available. The ability of Pseudomonas spp. to grow rapidly and thrive under a variety of environmental conditions, and their nutritional versatility, are traits that make members of the genus good colonisers (Weller, 1988; Khmel et al., 1998, Chin-A-Woeng et al., 2003; Weller,
2007). Additional traits involved in the ecological competence of introduced biocontrol strains include quorum sensing, biofilm formation, production of antifungal metabolites, attachment to roots, chemotaxis, site-specific recombinase activity, and uptake and catabolism of root exudates (Lugtenberg et al., 2001; Mavrodi et al., 2012).
The findings presented in Chapter 4 demonstrated the mechanisms of action employed by P. poae BCA17 in the inhibition of GTD pathogens, in vitro, and suppression of GTD infections in planta. The results from in vitro assays indicated that diffusible compounds produced by P. poae BCA17 played a major role in the
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Chapter 5 antagonistic activity, while siderophores and volatile compounds appear to not be involved. Siderophores were produced by both the antagonistic and non-antagonistic strains of Pseudomonas. This result implied that siderophores may not be directly involved in the antagonistic activity of P. poae against GTDs, with the caveat that a qualitative rather than quantitative assessment was undertaken. However, siderophores may be indirectly involved in the disease control by stimulating the biosynthesis of other antimicrobial compounds via their role in increasing the availability of minerals to the bacteria (Duffy and Défago, 1999), inducing systemic resistance (Leeman et al., 1996), and having a role in biofilm formation (Saha et al.,
2012). Future studies on the siderophore production by P. poae strains will further clarify its role in the disease suppression by these bacterial strains. Similarly, the lack of inhibition of mycelial growth when GTD pathogens were exposed to P. poae strains in the double plate assay indicated volatile compounds were not involved in the bacterial strains’ antagonistic activity. It is possible that the volatiles produced by P. poae BCA17 were not bioactive or the strain does not produce any volatile compounds.
This was not assessed further based on the recorded outcome.
Results from additional in vitro assays using cell-free culture filtrates of P. poae
BCA17 strongly indicated the involvement of bioactive diffusible compounds in the inhibition of GTD pathogens. The cell-free culture filtrate of P. poae BCA17 significantly reduced mycelial biomass production and spore germination, although inhibition varied among pathogen species. These diffusible compounds were shown to be produced by the bacterial strain in the presence or absence of the pathogen. The differences in efficacy against pathogen species was most likely due to the pathogens’ inherent growth characteristic, with Neofusicoccum species producing dense mycelia while E. lata is generally slow growing. Spore germination by D. seriata was also greatly inhibited by the culture filtrate while E. lata was less sensitive. These differences may
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Chapter 5 be related to their lifecycle and cell structure. Microscopy studies including scanning electron microscopy would assist to further clarify the differences in cell structures between pathogen species that influence their sensitivity or resistance to diffusible compounds produced by P. poae. An attempt to identify the bioactive diffusible compound in the culture filtrate of P. poae BCA17 revealed the presence of an antibiotic that is associated with the biocontrol activity of strains of Pseudomonas.
Using TLC, DAPG was detected in the organic fraction extracted from the broth culture of P. poae BCA17. However, this is only a preliminary investigation and further studies still need to be done to ascertain its role in the biocontrol ability of P. poae BCA17.
Phenazine was not detected in the organic extract from the culture filtrate of P. poae
BCA17.
The genome sequencing of the P. poae antagonistic strains BCA13, BCA14,
BCA17 and the non-antagonistic JMN1 revealed that all strains possessed a similar set of putative biocontrol genes. This included gene clusters that code for the synthesis of
CLPs with reported biocontrol activity including viscosin, orfamide, sessilin, bananamide, white- line-inducing principle (WLIP), anikasin, and poaemide. However, presence of these genes in the four Pseudomonas strains does not guarantee that they are all equally expressed. Biochemical studies using MALDI-TOF further detected a putative novel compound from the culture filtrate of P. poae BCA17. Further analysis of the lipopeptide profile of the cell-free culture filtrate of P. poae BCA17 revealed a dominant cyclic lipopeptide which gave a 1+ ion peak at 2098.2913 m/z. This putatively new CLP has the molecular formula (C48H87N11O12) and amino acid sequence leu-val- gln-leu-val- val-gln-leu-val (LVQLVVQLV). This novel compound may contribute to the bioactivity of the filtrate. However, due to lack of a comprehensive lipopeptide database, this novel compound could not be identified during the course of this study.
The results presented in this chapter indicate that P. poae BCA17 produced a
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Chapter 5 combination of compounds that may provide the capability for the biocontrol activity.
Future studies that can characterise and identify this novel compound will further clarify its mode of action and role in the antagonistic activity of the bacterial strain.
The genes for DAPG biosynthesis have already been cloned and sequenced, and the biosynthetic pathway has been proposed (McSpadden Gardener et al., 2001).
A gene knockout approach may help identify the role of DAPG in the biocontrol ability of P. poae BCA17. Development of mutants that are deficient in DAPG production could result in a reduced ability to control the GTD pathogens or suppress GTD disease. The dominant CLP found in P. poae BCA17 needs to be isolated and purified before testing its bioactivity on GTD pathogens. The culture filtrates and the purified
CLP must provide similar levels of control. Again, inactivation of CLP production should result in the loss of biocontrol ability. DAPG or CLP must also be detected in situ when producing strains are inoculated in actual plants. A better understanding of the mechanisms of suppression of the pathogen by the BCA is imperative to effective biocontrol of GTD. Ultimately, this may lead to the development of more effective and ecologically sound applications of biological control of plant pathogens.
The challenge for the in field application of a BCA is how it responds to different environmental factors. Carbon substrates and availability, access to N and P, iron limitation, and plant phenology and growing conditions, may affect antibiotic production
(Nielsen et al., 2002). The frequency of application for BCAs is also another factor to look at before implementing these as a control strategy in the field. There is general consensus that for a BCA to effectively control plant disease, it must establish and maintain a threshold population density in the host plant after application. By doing so, the potential exists for the prevention or limitation of infection by pathogens via production of antifungal metabolites (Bull et al., 1991; Raaijmakers et al., 1995). Most introduced rhizobacteria initially establish high population density in the host
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Chapter 5 rhizosphere after inoculation, but decline significantly with time and distance from the inoculum source (Haas and Défago, 2005). Arsenault et al. (2016) found that effective biocontrol of potato common scab was only achieved when the P. fluorescens
7 LBUM223 population was 10 bacteria/g of soil, and this was maintained with biweekly reapplication of the bacteria. Under field conditions, reapplying P. poae BCA17 after pruning may be necessary to maintain a sufficiently high population of bacteria to achieve effective control of GTD. An application of a strain mixture or mixture of microorganisms may also be more effective as this might maximize the synergistic effect of multiple antagonistic traits in the inoculum. Different BCAs may serve complementary and multiple purposes, potentially producing a suit of antifungal compounds that will likely act in synergism with each other. This multiple activity may be useful under natural conditions in which grapevines may be infected simultaneously with different GTD pathogens. Combining of strains or microorganisms may also have the advantage in that they can work under different environmental conditions (Mavrodi et al., 2012).
In comparison with chemical pesticides, BCAs are typically viewed as: a) generally safe to use; b) having reduced environmental damage and potentially smaller risk to human health; c) displaying targeted activity; d) capable of multiplying themselves but are controlled by the plant as well as by the indigenous microbial populations; (e) easily degradable; (f) durable, in terms of decreased likelihood of target resistance; and (g) applicable to conventional or integrated pest management systems (Berg, 2009). Biocontrol metabolites are also designed so that they do not have any adverse effects on host plants or on indigenous microflora (Dowling and
O'Gara, 1994; Dunne et al., 1996).
Pseudomonas, along with Bacillus, are among the most widely studied bacteria with biocontrol potential against a wide range of plant pathogens and the diseases
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Chapter 5 these cause (Raaijmakers and Weller, 2001). Pseudomonas is known for its rapid growth rate and ability to colonise in different environments. It can survive in different conditions that would be stressful for other bacteria and can use various substrates as nutrients. In addition, it is known to promote plant growth and can synthesise a wide range of metabolites or antibiotics against diverse kind of plant pathogens
(Raaijmakers and Weller, 2001; Santoyo et al., 2012). These characteristics made
Pseudomonas a desirable prospective BCA, and thus P. poae BCA17 has the potential to be further developed as a biological control application. Depending on efficacy of a commercial product, this may lead to use either alone or in combination with chemicals to lower the doses or decrease the frequency of chemical application.
This study comprehensively investigated the potential for strains of P. poae from inner tissues of grapevines as antagonists to pathogenic fungi. Sufficient evidence was generated to indicate that P. poae BCA17 had strong bioactivity against GTD pathogens both in vitro and in planta. It was further demonstrated that the strain could be artificially-inoculated onto grapevines and colonise the vine for a certain period of time. While these studies provided compelling evidence that indicate P. poae BCA17 is a promising BCA for GTDs, further work is needed to assess its applicability in a large- scale vineyard setting, and under the variable environmental conditions in which vineyards are established. Inoculation methods that can be adapted to fit the technology of modern agriculture represents a significant challenge in the application of the BCA. Considerable work must be done in developing a highly concentrated inoculum that can be applied commercially. The development of an effective microbial product that is cost-effective is an area of investigation that can be explored in the future. The challenge is to develop inexpensive, easily applied formulations that can remain viable under less than optimal conditions.
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Appendix A
Appendix A:
Other publication arising from this research:
Niem, J., Billones-Baaijens, R., Savocchia, S., Stodart, B. (2019). Draft genome sequences of endophytic Pseudomonas spp. isolated from grapevine tissue and antagonistic to grapevine trunk disease pathogens. Microbiol. Resour. Announc. 8: e00345–19.
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Appendix A
GENOME SEQUENCES
Draft Genome Sequences of Endophytic Pseudomonas spp. Isolated from Grapevine Tissue and Antagonistic to Grapevine Trunk Disease Pathogens
Jennifer Niem,a,b Regina Billones-Baaijens,a Sandra Savocchia,a,b Benjamin Stodartc
Downloaded froDownloaded aNational Wine and Grape Industry Centre (NWGIC), Charles Sturt University, Wagga Wagga, NSW, Australia bSchool of Agricultural and Wine Sciences, Charles Sturt University, Wagga Wagga, NSW, Australia cGraham Centre for Agricultural Innovation (Charles Sturt University and NSW Department of Primary Industries), School of Agricultural and Wine Sciences, Charles Sturt University, Wagga Wagga, NSW, Australia
ABSTRACT Endophytic strains of Pseudomonas were isolated from grapevine tissues and exhibited antagonistic activity against several grapevine trunk disease patho- gens. The m draft genome sequences of the four strains revealed the presence of puta- tive gene http://mra.a clusters that may impart biocontrol activity against plant pathogens.
pecies within Pseudomonas may be beneficial or detrimental to plant production
Ssystems. Efficacy has been established for Pseudomonas spp. as biocontrol agents against sm.org/ late blight and scab of potato (1, 2), Rhizoctonia root rot on bean (3), damping- off and root rot in tomato (4), black root rot of tobacco, and take-all disease of wheat (5, 6). In grapevines, Pseudomonas spp. are found in the phyllosphere (7–9) and inner tissues (10– 12) and are known to suppress Botrytis cinerea (13, 14) and Rhizobium vitis on July by11, 2019 guest (15). Pseudomonas isolates BCA13, BCA14, and BCA17 were obtained from grapevine canes exhibiting Botryosphaeria dieback (BD) in Wagga Wagga, New South Wales (NSW), Australia. The canes were stripped of bark, surface sterilized, and placed on nutrient agar. Emerging bacteria were streaked onto King’s B medium to obtain single colonies. All isolates inhibited BD and Eutypa dieback (ED) pathogens in culture and Citation Niem J, Billones-Baaijens R, reduced BD infection in planta (our unpublished data). A fourth isolate, JMN1, was Savocchia S, Stodart B. 2019. Draft genome obtained from an asymptomatic vine in Harden (NSW, Australia) by suspending internal sequences of endophytic Pseudomonas spp. isolated from grapevine tissue and antagonistic trunk wood shavings in Ringer’s solution. Single colonies were selected by streaking on to grapevine trunk disease pathogens. Microbiol King’s B medium at 25°C. JMN1 was not antagonistic to BD and ED pathogens. The four Resour Announc 8:e00345-19. isolates were identified by amplification and sequencing of the 16S rRNA and rpoD https://doi.org/10.1128/ MRA.00345-19. Editor David A. Baltrus, University of Arizona genes. Gene sequences were subjected to BLASTn searches of the NCBI database, and Copyright © 2019 Niem et al. This is an reference sequences were selected for phylogenetic analyses. Sequence alignment was open- access article distributed under the completed with Clustal W, and a neighbor-joining tree was constructed within MEGA 7 terms of the Creative Commons Attribution 4.0 International license. (16). The four isolates were found to be closely related to Pseudomonas poae. Address correspondence to Jennifer Niem, Each isolate was grown in nutrient broth for 24 h at 25°C and then harvested for DNA [email protected], or Benjamin Stodart, extraction using the Gentra Puregene bacterial DNA extraction kit (Qiagen), following the [email protected]. Received 11 April 2019 manufacturer’s specifications. Shotgun library preparation and Illumina sequencing (HiSeq Accepted 3 June 2019 2500 platform) were conducted by the Australian Genome Research Facility, resulting in Published 27 June 2019 12,569,718 reads (150-bp paired ends; Table 1). Data were gener- ated with the Illumina bcl2fastq pipeline version 2.20.0.422. Draft genomes were assembled using the Unicycler assembler, implementing an optimizer for SPAdes 3.13.0 (17). k-mer lengths between 0.2
171
Appendix A and 0.95 of total read length were examined, and contigs of <200 bases were removed. Annotation was completed with the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) 4.7 (18) and the Rapid Annotations
Volume 8 Issue 26 e00345-19 mra.asm.org 1
Niem et al. TABLE 1 Genome information and accession numbers of four Pseudomonas strains isolated from grapevine tissue G+C No. of No. of RAST SRA No. of Assembly No. of N50 value content coding subsystems Strain accession no. reads size (bp) contigs (bp) (%) genes represented BCA13 SRX5463364 3,189,991 6,318,228 34 1,278,996 60.18 5,171 370 BCA14 SRX5463365 2,798,878 6,322,821 34 562,240 60.18 5,666 403 BCA17 SRX5463366 3,028,714 6,318,257 28 760,227 60.18 5,559 403 JMN1 SRX5463367 3,552,135 6,322,966 29 691,035 60.18 5,564 401
using Subsystems Technology server, implementing RASTtk (19). Default parameters for all software programs were used, unless otherwise specified. Gene clusters which may play a role in the control of plant pathogens were identified. Queries of the Plant-bacteria Interaction Factors Resource (PIFAR) (20) fromDownloaded found that each strain of Pseudomonas contains a remarkable number of putative biocontrol gene clusters, including those responsible for lipopeptide antibiotics, siderophores, proteases, detoxification, lipopolysaccharides, multidrug resistance, microbe- associated molecular proteins (MAMPs), and biofilms. antiSMASH 4.0 (21) was implemented to detect gene clusters responsible for the biosynthesis of secondary metabolites, http://mra.asm.org/ Data availability. The genome sequences for BCA13, BCA14, BCA17, and JMN1 are available under NCBI BioProject number PRJNA522029, with annotated assemblies available under accession numbers SGWK00000000, SGWJ00000000, SGWI00000000, and SGWH00000000, respectively. The Sequence Read Archive (SRA) accession num- bers are listed in Table 1. Sequence reads were deposited in the NCBI SRA under the accession numbers SRR8667294 to SRR8667297.
ACKNOWLEDGMENTS
This work was supported by Charles Sturt University and Australia’s grape growers on July by11, 2019 guest and winemakers through their investment body, Wine Australia. We thank the Museum of Natural History, University of the Philippines Los Baños.
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guest
Appendix B
Appendix B:
Presentations and award
B. 1. Oral presentation
J. Niem, R. Billones-Baaijens, B. Stodart and S. Savocchia. (2018). Bacterial antagonists to control trunk diseases. CRUSH Grape and Wine Science Symposium. Adelaide, South Australia. Sept. 25-26, 2018.
JM Niem, RB Billones-Baaijens, B Stodart, and S Savocchia. (2019). Exploring the interactions between bacterial endophytes and trunk disease pathogens of grapevine. 50th Annual APPS Conference. Melbourne, Victoria. Nov. 25-28, 2019.
B. 2. Poster
Jennifer Niem, Regina Billones-Baaijens, Benjamin Stodart and Sandra Savocchia. Exploring endophytic bacteria as potential biocontrol agents against grapevine trunk disease pathogens. Poster presented at 10th International Workshop on Grapevine Trunk Diseases. Reims, France. July 4-7, 2017.
J. Niem, R. Billones-Baaijens, B. Stodart and S. Savocchia. (2017). Biological control of grapevine trunk diseases using endophytic bacteria. Poster presented at Science Protecting Plant Health Conference. Australasian Plant Pathology Society (APPS). Brisbane, Queensland. Sept. 26-28, 2017.
B. 3. Awards
“Innovation and Science” Award. Oral presentation Title: Bacterial antagonists to control trunk diseases. CRUSH Grape and Wine Science Symposium. Adelaide, South Australia. Sept. 25-26, 2018.
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Appendix C Appendix C:
Media preparation
Prune extract / Prune extract agar
To make prune extract:
1. 25 g prunes per 500 mL distilled water. 2. Boil at 100°C in free steam for 30 minutes. 3. Filter prunes using Whatman filter paper no. 3 (or 2-3 layers of Miracloth). 4. Refrigerate.
To make prune extract agar:
Sucrose 5 g Yeast extract 1 g Technical Agar 30 g Prune extract 100 mL Distilled water 900 mL Autoclave at 120°C for 15 minutes.
Crome Azurol S (CAS) media
A. Blue Dye: a. Solution I: i. Dissolve 0.06 g of CAS (Fluka Chemicals) in 50 ml of ddH2O. b. Solution 2: i. Dissolve 0.0027 g of FeCl3-6 H2O in 10 ml of 10 mM HCl. c. Solution 3: i. Dissolve 0.073 g of HDTMA in 40 ml of ddH2O. d. Mix Solution 1 with 9 ml of Solution 2. Then mix with Solution 3. Solution should now be a blue color. Autoclave and store in a plastic container/bottle.
B. Mixture solution: a. Minimal Media 9 (MM9) Salt Solution Stock i. Dissolve 15 g KH2PO4, 25 g NaCl, and 50 g NH4Cl in 500 ml of ddH2O. b. 20% Glucose Stock i. Dissolve 20 g glucose in 100 ml of ddH2O. c. NaOH Stock i. Dissolve 25 g of NaOH in 150 ml ddH2O; pH should be ~12.
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Appendix C
d. Casamino Acid Solution
ii. Extract with 3% 8-hydroxyquinoline in chloroform to remove any trace iron. iii. Filter sterilize. C. CAS agar Preparation: a. Add 100 ml of MM9 salt solution to 750 ml of ddH2O. b. Dissolve 32.24 g piperazine-N,N’-bis(2ethanesulfonic acid) PIPES. i. PIPES will not dissolve below pH of 5. Bring pH up to 6 and slowly add PIPES while stirring. The pH will drop as PIPES dissolves. While stirring, slowly bring the pH up to 6.8. Do not exceed 6.8 as this will turn the solution green. c. Add 15 g Bacto agar. d. Autoclave and cool to 50oC. e. Add 30 ml of sterile Casamino acid solution and 10 ml of sterile 20% glucose solution to MM9/ PIPES mixture. f. Slowly add 100 ml of Blue Dye solution along the glass wall with enough agitation to mix thoroughly. g. Aseptically pour plates.
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