Arts and Sciences, Irving K. Barber School of (Okanagan)
Biology, Department of (Okanagan)
DSpace
UBCO
Holland, Taylor Craig
Doctor of Philosophy - PhD
University of British Columbia
Arbuscular mycorrhizal (AM) fungi are often incorporated into growing systems in hopes of increasing nutrient uptake and alleviating stress for plants in exchange for photosynthetic carbon. However, the benefits provided by AM fungi are inconsistent and it is important we understand the factors surrounding this symbiosis to determine when their addition may be beneficial and economical for agricultural production systems. I used three greenhouse experiments and one field study to answer questions revolving around the timing of inoculation, the provenance of AM fungi used, and plant stresses. AM fungi did not consistently benefit plants, but some advantages were found. Overall, AM fungi were shown to be variable in their effect on vine growth. In a greenhouse study, a natural AM fungal community provided physiological benefits to hosts when compared to a commercial AM inoculant. In contrast, adding commercial AM fungi to grapevines prior to field transplant increased the survival of vines when entering stressful soil conditions. When testing the ability of AM fungi to protect young vines against a weak pathogen no benefits were observed, but instead AM fungi increased pathogen levels in hosts, though without any symptoms. Lastly, I tested how pruning of roots at the time of transplant may affect the symbiosis, finding that the symbiosis persisted likely due to the abundance of propagules in older, untrimmed roots. Thus, growers can pre-inoculate vines without compromising the symbiosis during planting. The range of results displayed in this thesis confirm how inconsistent AM fungal additions can be. Nevertheless, AM fungal inoculants may have a place in viticulture practices. In particular, they may be useful prior to transplanting into the field. In general, AM fungi should be tested on a site-basis prior to large scale applications
TESTING THE EFFICACY OF ARBUSCULAR MYCORRHIZAL FUNGAL INOCULANTS ON GRAPEVINE ESTABLISHEMENT by Taylor Craig Holland M.Sc., University of British Columbia, 2013 B.Sc., Simon Fraser University, 2010 A THESIS SUBMITTED IN PARTIAL FULLFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE COLLEGE OF GRADUATE STUDIES (Biology) THE UNIVERSITY OF BRITISH COLUMBIA (Okanagan) July 2019 © Taylor Craig Holland, 2019 ii The following individuals certify that they have read, and recommend to the College of Graduate Studies for acceptance, a thesis/dissertation entitled: TESTING THE EFFICACY OF ARBUSCULAR MYCORRHIZAL FUNGAL INOCULANTS ON VINE ESTABLISHEMENT Submitted by Taylor Craig Holland in partial fulfilment of the requirements of the degree of Doctor of Philosophy in Biology. Dr. Miranda Hart, Irving K. Barber School of Arts and Sciences Supervisor Dr. Pat Bowen, Agriculture and Agri-Food Canada, Summerland BC Supervisor Dr. Jose Urbez-Torres, Agriculture and Agri-Food Canada, Summerland BC Supervisory Committee Member Dr. Andrew Walker, Faculty of Viticulture and Enology, University of California, Davis California Supervisory Committee Member Dr. Craig Nichol, Irving K. Barber School of Arts and Sciences University Examiner Dr. Deborah Henderson, Institute for Sustainable Horticulture, Kwantlen Polytechnic University, Langley BC External Examiner iii Abstract Arbuscular mycorrhizal (AM) fungi are often incorporated into growing systems in hopes of increasing nutrient uptake and alleviating stress for plants in exchange for photosynthetic carbon. However, the benefits provided by AM fungi are inconsistent and it is important we understand the factors surrounding this symbiosis to determine when their addition may be beneficial and economical for agricultural production systems. I used three greenhouse experiments and one field study to answer questions revolving around the timing of inoculation, the provenance of AM fungi used, and plant stresses. AM fungi did not consistently benefit plants, but some advantages were found. Overall, AM fungi were shown to be variable in their effect on vine growth. In a greenhouse study, a natural AM fungal community provided physiological benefits to hosts when compared to a commercial AM inoculant. In contrast, adding commercial AM fungi to grapevines prior to field transplant increased the survival of vines when entering stressful soil conditions. When testing the ability of AM fungi to protect young vines against a weak pathogen no benefits were observed, but instead AM fungi increased pathogen levels in hosts, though without any symptoms. Lastly, I tested how pruning of roots at the time of transplant may affect the symbiosis, finding that the symbiosis persisted likely due to the abundance of propagules in older, untrimmed roots. Thus, growers can pre-inoculate vines without compromising the symbiosis during planting. The range of results displayed in this thesis confirm how inconsistent AM fungal additions can be. Nevertheless, AM fungal inoculants may have a place in iv viticulture practices. In particular, they may be useful prior to transplanting into the field. In general, AM fungi should be tested on a site-basis prior to large scale applications. v Lay Summary It is important to look for ways to improve crop performance during transplant. One way of doing this is by adding arbuscular mycorrhizal (AM) fungi, root associated fungi that improve host plant’s uptake of nutrients and water, while buffering plants from stresses. To determine their efficacy as a transplant addition I conducted various field and greenhouse experiments surrounding the plant-fungal association. My research revealed a wide range of effects, from providing benefits during field transplant, to increasing the amount of pathogen found within roots. I showed that pre-inoculating vines prior to transplant provided the most benefit and root pruning did not eliminate the symbiosis. The source of inoculum was important, as a more diverse natural community provided more benefit in some cases, but commercial isolates were better at colonizing roots. Overall, AM fungal inoculants are likely most advantageous in the presence of stressful growing conditions. vi Preface A version of Chapter 1 has been published. Holland TC, Vukicevich E, Thomsen C, Pogiatzis A, Hart M, Bowen P. 2018. Arbuscular mycorrhizal fungi in viticulture: should we use biofertilizers? Catalyst discovery into practice. 2: 59-63. I wrote the review paper presented as Chapter 1 with guidance and edits from Dr. Miranda Hart and Dr. Pat Bowen, along with input and feedback from Eric Vukicevich, Antreas Pogiatzis and Corrina Thomsen. A version of Chapter 2 has been published. Holland TC, Hart MM, Bogdanoff C, Bowen P. 2016. Response of Grapevine Rootstocks to Soil Inocula from Different Sources. American Journal of Enology and Viticulture 69:1 94-100 The greenhouse study presented in Chapter 2 was performed at Agriculture and Agri-food Canada’s Summerland Research and Development Centre (AAFC-SuRDC) with materials and assistance being provided by the labs of Dr. Pat Bowen and Carl Bogdanoff. The experiment was designed with consultation from Dr. Miranda Hart and Dr. Pat Bowen, while Rob Brownlee assisted in setting up and maintaining this experiment. A version of Chapter 4 has been accepted for publication. Holland TC, Kokkoris V, Urbez-Torres JR, Bowen P, Hart MM. 2019. Timing of AM fungal inoculation influences the abundance of a common grapevine rootstock pathogen Ilyonectria. Horticulturae vii Chapter 4 was performed in the greenhouse at the University of British Columbia, Kelowna BC Canada, with design consultation by Dr. Miranda Hart and Dr. Pat Bowen. Rootstock wood was collected by the lab of Dr. Pat Bowen, from the rootstock collection block at the AAFC-SuRDC. The lab of Dr. Jose Ramon Urbez-Torres supplied the Ilyonectria isolates and consulted in procedures surrounding the inoculation and isolation of the pathogen. Tuddi Clark volunteered in setting up and harvesting the experiment, while Daniel Rosa helped with experiment maintenance and harvest. Vasilis Kokkoris and Daniel Rosa assisted with AM fungal quantification via digital droplet PCR. A version of Chapter 5 has been submitted for publication review. Holland TC, Kokkoris V, Rosa D, Richards A, Bowen P, Hart MM. 2019. The impact of root pruning transplant practices on the arbuscular mycorrhizal symbiosis within grapevine rootstocks. Chemical and Biological Technologies in Agriculture Chapter 5 was performed in the greenhouse at the University of British Columbia, Kelowna BC Canada, with design consultation by Dr. Miranda Hart. Rootstock wood was collected by the lab of Dr. Pat Bowen, from the rootstock collection block at the AAFC-SuRDC. Daniel Rosa and Sarah Bird helped with experiment setup and maintenance. Vasilis Kokkoris, Daniel Rosa, and Andrew Richards assisted with AM fungal quantification via staining and digital droplet PCR. viii Table of Contents Abstract ................................................................................................................................ iii Lay Summary ........................................................................................................................ v Preface .................................................................................................................................. vi Table of Contents ............................................................................................................ viii List of Tables ...................................................................................................................... xi List of Figures ................................................................................................................... xii Acknowledgements ........................................................................................................ xiv Dedication .......................................................................................................................... xvi 1 Introduction ...................................................................................................................... 1 1.1 The global trend in AM fungal biofertilizers. ............................................................... 1 1.2 Are AM fungi developed for annuals suitable for perennials?.............................. 2 1.3 Grapevines: A case study. ................................................................................................. 4 1.3.1 The mycorrhizal effect is highly context dependent. ..........................................................4 1.3.2 Rootstocks react differently to inoculation ................................................................................5 1.4 Do perennial systems benefit from AM fungal inoculants? ................................... 6 1.5 AM fungal inoculants must be suitable to vineyard site conditions. .................. 7 1.6 Research objectives ............................................................................................................. 8 1.6.1 Does the source of AM fungi influence the growth of different grapevine rootstocks? ...............................................................................................................................................................9 1.6.2 Does inoculation with AM fungi improve vine establishment in a field having poor soil conditions and does this depend on the timing of inoculation or source of inoculum? ..................................................................................................................................................................9 1.6.3 Will AM fungal inoculation reduce fungal infection of grapevine rootstocks and does the timing of AM fungal addition determine the strength of this effect?............... 10 1.6.4 Do standard transplant root trimming practices affect the mycorrhizal symbiosis? ............................................................................................................................................................. 11 2 Response of grapevine rootstocks to soil inocula from different sources ................................................................................................................................................ 12 2.1 Background .......................................................................................................................... 12 2.2 Materials and methods ..................................................................................................... 15 2.2.1 Study site and experimental design .......................................................................................... 15 2.2.2 AM colonization ...................................................................................................................................... 18 2.2.3 Plant response ........................................................................................................................................ 19 2.2.4 Statistical analyses .............................................................................................................................. 20 2.3 Results ................................................................................................................................... 20 2.3.1 AM fungal root colonization ............................................................................................................ 20 2.3.2 Plant response ........................................................................................................................................ 21 2.3.3 Rootstock effects ................................................................................................................................... 22 2.4 Discussion ............................................................................................................................ 24 2.4.1 AM fungal colonization ...................................................................................................................... 24 2.4.2 Rootstock growth response ............................................................................................................ 25 2.4.3 Inoculum source .................................................................................................................................... 27 ix 2.4.4 Rootstock cultivars ............................................................................................................................... 29 3 Arbuscular mycorrhizal fungi impact transplant of grapevines into vineyard with poor soil conditions. ........................................................................... 31 3.1 Background .......................................................................................................................... 31 3.2 Materials and methods ..................................................................................................... 33 3.2.1 Study site ................................................................................................................................................... 33 3.2.2 Experimental design............................................................................................................................ 33 3.2.3 Locally sourced inocula ..................................................................................................................... 34 3.2.4 Pre-transplanting plant preparation and treatment .......................................................... 35 3.2.5 Field planting and management .................................................................................................. 36 3.2.6 Vine performance.................................................................................................................................. 36 3.2.6.1 Vine establishment .......................................................................................................................... 36 3.2.6.2 Vine growth ......................................................................................................................................... 36 3.2.7 Analysis for AM fungi .......................................................................................................................... 37 3.2.7.1 Inoculant abundance and persistence .................................................................................... 37 3.2.7.2 Threshold determination and data processing..................................................................... 38 3.2.8 Statistical analyses .............................................................................................................................. 39 3.2.8.1 Vine survival ....................................................................................................................................... 39 3.2.8.2 Vine growth ......................................................................................................................................... 39 3.3 Results ................................................................................................................................... 40 3.3.1 Vine survival ............................................................................................................................................. 40 3.3.2 Vine performance.................................................................................................................................. 40 3.3.3 Commercial inoculant presence and persistence ............................................................. 40 3.4 Discussion ............................................................................................................................ 41 3.4.1 Vine survival ............................................................................................................................................. 41 3.4.2 Vine performance.................................................................................................................................. 44 3.4.3 Commercial AM fungal establishment ..................................................................................... 45 4 Timing of AM fungal inoculation influences the abundance of a common grapevine rootstock pathogen Ilyonectria. ............................................................. 46 4.1 Background .......................................................................................................................... 46 4.2 Materials and methods ..................................................................................................... 49 4.2.1 Experimental design............................................................................................................................ 49 4.2.2 Rootstock preparation ........................................................................................................................ 49 4.2.3 Pathogen isolate preparation......................................................................................................... 50 4.2.4 Application of treatments, and plant culture ......................................................................... 51 4.2.5 Harvest and measurements ........................................................................................................... 52 4.2.5.1 Vine growth response ..................................................................................................................... 52 4.2.5.2 Pathogen isolation and assessment of necrosis ................................................................. 53 4.2.5.3 Digital droplet PCR quantification.............................................................................................. 54 4.2.5.3.1 AM fungi ...................................................................................................................................... 54 4.2.5.3.2 Ilyonectria spp. ......................................................................................................................... 55 4.2.6 Statistical analysis ................................................................................................................................ 56 4.3 Results ................................................................................................................................... 57 4.3.1 Plant response ........................................................................................................................................ 57 4.3.2 Molecular quantification .................................................................................................................... 57 4.3.2.1 AM fungi ............................................................................................................................................... 57 4.3.3 Necrosis ...................................................................................................................................................... 59 4.3.3.1 Ilyonectria spp. .................................................................................................................................. 59 4.4 Discussion ............................................................................................................................ 60 4.4.1 Vine growth ............................................................................................................................................... 61 x 4.4.2 Pathogen isolation and necrosis.................................................................................................. 61 4.4.3 Ilyonectria and AM fungal quantification ................................................................................. 63 5 The impact of root pruning transplant practices on the arbuscular mycorrhizal symbiosis within grapevine rootstocks........................................... 66 5.1 Background .......................................................................................................................... 66 5.2 Materials and methods ..................................................................................................... 68 5.2.1 Rootstock preparation and treatment ....................................................................................... 68 5.2.2 Staining and visual quantification................................................................................................ 70 5.2.3 DNA extraction and molecular AM fungal quantification .............................................. 71 5.2.4 Statistical Analysis................................................................................................................................ 72 5.3 Results ................................................................................................................................... 72 5.3.1 Root trimming effects on AM fungi and rootstocks........................................................... 72 5.3.2 High versus low roots ......................................................................................................................... 74 5.4 Discussion ............................................................................................................................ 75 5.4.1 The impact of root trimming on the mycorrhizal symbiosis......................................... 75 5.4.2 Distribution of AM fungal structures between old and new roots ............................ 78 5.4.3 Root trimming and vine growth ..................................................................................................... 78 6 Conclusions.................................................................................................................... 80 6.1 Does the source of AM fungi influence the growth of different grapevine rootstocks? ................................................................................................................................. 81 6.2 Does inoculation with AM fungi improved vine establishment in a field having poor soil conditions and does this depend on the timing of inoculation or source of inoculum? ........................................................................................................... 82 6.3 Will AM fungal inoculation reduce fungal infection of grapevine rootstocks and does the timing of AM fungal addition determine the strength of this effect? ......................................................................................................................................................... 83 6.4 Do standard transplant root trimming practices affect the mycorrhizal symbiosis? .................................................................................................................................. 84 6.5 Concluding remarks .......................................................................................................... 85 Bibliography....................................................................................................................... 89 Appendices ..................................................................................................................... 102 Appendix A: Supplementary material for Chapter 3 ................................................... 102 Appendix B: Supplementary material for Chapter 4 ................................................... 105 xi List of Tables Table 2.1 Details of inocula treatments applied to roots of Riparia Gloire, 101-14 Mgt and SO4 rootstock plants……..…………………………………………………17 Table 2.2 Effects of microbial inoculum and rootstock cultivar on characteristics of vine growth and photosynthesis. …..……………………………………………..23 Table 4.1. Inoculation treatments applied to Riparia gloire grapevine rootstocks. The fungal inoculants consisted of an AM fungus R. irregularis, and black foot pathogens Ilyonectria macrodidyma and I. liriodendra…………………………….50 Table 4.2 Means for plant growth chracteristics and abundance of fungal isolates, six months after inoculations with AM fungi and Ilyonectria isolates. F-values and P-values are the result from ANOVA for inoculation treatment effects (n= 40). Copy numbers of mtDNA (AM fungi) and beta tubulin (Ilyonectria) were obtained using molecular-based quantitative digital droplet PCR.……………….58 Table 5.1 Linear mixed-effect model results for different fungal and plant responses measured after vines were either trimmed or not, and from different areas of the root system. Bolding indicates significance at p=0.05……………..73 Table A. 1 Soil conditions of the experimental block, which had the top soil removed prior to planting the new grapevines, along with the adjacent block where soil communities originated from. Numbers represent averages across five samples taken throughout each block………………………………………...102 Table A. 2 The p-values are presented for pairwise comparisons in survival, using Fisher-Exact Test, among different inoculation treatments; non-mycorrhizal vines receiving no treatment, vines receiving commercial inoculant in greenhouse, vines receiving commercial inoculant in the field, natural spore treatment inoculated in greenhouse and a natural root treatment inoculated in greenhouse……………………………………………………………………………102 Table A. 3 Results of Mixed Model tests (Formula: Response ~ Treatment + (Treatment| Year)), performed on vine growth data collected from the 2016 and 2017 field seasons following transplant……………………………………………103 Table A. 4 Molecular digital droplet PCR data used to quantify the AM fungal isolate that is used in the inoculum. This illustrates the absence of commercial arbuscular mycorrhizal in vineyard soils before vines were planted……………104 Table B. 1 A lineage report resulting from specificity testing for the new primer-probe set, using the blast tool in NCBI. First a primer blast was performed using the newly developed primer set. The generated accession numbers were subsequently nucleotide blasted using the probe sequence. The organism, score, average e-value and number of hits for each match are reported.…………….106 xii List of Figures Figure 2.1. Arbuscular mycorrhizal (AM) fungal colonization of roots in response to five inoculum treatments (untreated control, Biosyneterra, MycoGrow, locally sourced roots, and locally sourced AM fungal spores) applied to three rootstocks (101-14 Mgt, Riparia Gloire and SO4). Letters indicate differences in AM colonization among inoculum treatments detected using Tukeys honest significant difference post-hoc test at α = 0.05. Colonization was not affected by rootstock. ………………………………………………………………………………21 Fig. 3.1 Surviving vine count in 2016 and 2017 in response to inoculation treatments: no-mycorrhizal treatment control (NM), locally sourced grapevine roots pre-inoculated (RP), locally sourced spores pre-inoculated (SP), commercial inoculant pre-inoculated (CP) and commercial inoculant field-inoculated. (CF). Letters indicate significant differences based on Fisher Exact Tests, which was statistically similar for both years of data………………………41 Fig. 4.1 Abundance of AM fungi within vine roots (ddPCR, copies mtDNA/μL) after harvest (6 mo after the second inoculation). Treatments: AMF/Ctrl, mycorrhizal control inoculated only with AM fungi; AMF/Ily, pre-inoculated with AM fungi followed by Ilyonectria inoculation; Ctrl/AMF+Ily, co-inoculated with AM fungi and Ilyonectria; Ctrl/Ctrl, non-microbial control; Ctrl/Ily, pathogen control inoculated with only Ilyonectria. Differences determined using one-way ANOVA (F5,41 = 5.135, p < 0.001), with differences at p < 0.05 indicated by letters above bars (Tukey’s multiple comparison via general linear hypothesis)..……………..58 Fig. 4.2 Ilyonectria beta-tubulin copy number per μL in vine roots in response to inoculation treatments. . Treatments: Ctrl/Ctrl, non-microbial control; Ctrl/Ily, pathogen control inoculated with only Ilyonectria; AMF/Ctrl, mycorrhizal control, inoculated with only AM fungi; AMF/Ily, pre-inoculated with AM fungi followed later by Ilyonectria inoculation; Ctrl/AMF+Ily, co-inoculation with AM fungi and Ilyonectria. Values were obtained by digital droplet polymerase chain reaction and differences determined using a one-way analysis of variance (ANOVA; F5,41 = 5.586, p < 0.001), with differences at p < 0.05 indicated by letters above bars (Tukey’s multiple comparison via general linear hypothesis)……………………..60 Fig. 5. 1 The quantity of AM fungal mitochondrial LSU gene copies in root systems that were trimmed (Trim) or not (Non-Trim), and from either the High and Low roots. An interaction was detected between trimming and root location (p = 0.033). The * indicates a difference in AM fungal mtDNA between the Non-Trimmed High and Trimmed High roots, detected using a Welch Two Sample t-test (t = 2.97, p = 0.006) …………………………………………………………...…74 Fig. 5. 2 Total (light grey), Vesicle (dark grey) and Arbuscule (white) percent colonization of vine roots collected from the ‘High’ and ‘Low’ roots of 48 rootstocks. The quantity of all three structures……………………………………..75 xiii Fig B.1 Specificity test using the newly designed primer probe in a digital droplet PCR assay. Droplets above the threshold represent positive amplification to the primer/probe, with a manual threshold amplitude set at 3700. From left to right the isolates tested in each well are: A01 – PARC393, A02 – PARC349, B02- PARC100, C01 - PARC60, C02 – NTC, D01 – PARC340, D02 – Rhizophagus irregularis (DAOM197198), E01 – PARC72, G01 – PARC398, H01 – PARC345. Rhizophagus irregularis DNA originated from spores that were extracted from the AGTIV® product..…………………………………………………………………….105 xiv Acknowledgements I am so grateful that I had the opportunity to work Dr. Miranda Hart and Dr. Pat Bowen. I would not have survived the last five to eight years without your belief in me and your grounding influences. I have learned so much from each of you, both academically and in life, and I know I have become a better professional and person because of you. Thank you for all the encouragement, guidance and support throughout my journey. Many people at Agriculture and Agri-Food Canada and the surrounding viticulture community have had a positive impact on the last five years of my life. I shared many insightful doorway discussions with Carl Bogdanoff, Dr. Peter Toivenen, Dr. Tom Lowery and Dr. Jose Urbez-Torres. They provided great professional opinion throughout my degree, and wonderful advice for what comes next. The amazing AAFC-SuRDC staff were instrumental in my studies, offering amazing knowledge and expertise, in particular I must thank Steve Marsh, Brad Estergaard, Emmanual Jean, Rob Brownlee, Paula Haag and Julie Boule. I must also thank Dr. Andy Walker from UC Davis for serving on my committee and providing thoughtful viticulture insight throughout my thesis. This work would not be possible the without funding and support received by various councils including UBC Okanagan, NSERC, the Killam Trust, the American Society for Enology and Viticulture, and the BC Wine and Grape Council. Also, I must also give gratitude to Summerhill Estate Winery and Willem Semmelink for the materials and opportunity they provided me with at their vineyard. xv A degree is not complete without peers to share in experiences and ideas. I was lucky be part of an amazing lab and graduate program at the University of British Columbia, including but not limited to: Eric Vukicevich, Vasilis Kokkoris, Antreas Pogiatzis, Andrew Richards, Corrina Thomsen, Mehrbod Estaki, and Sydney Morgan. There are many other professors and students that I am happy to now call friends, but special thanks must go out to Barb Lucente. In addition, my academic brothers Cameron Egan and Aaron Godin, who were always there to remind me who I am outside of campus. Lastly, I must give all my family. My parents and grandparents for all the love and support they have provided me. And especially my love, Tuddi Clark, when times are tough she has always pointed me in the right direction and I could not have asked for a better partner in life. xvi Dedication To all our furry friends, where would we be without their love? 1 1 Introduction 1.1 The global trend in AM fungal biofertilizers. The importance of soil biota in cropping success is becoming increasingly clear. Decades of research attest to the role of soil microbial communities in increasing nutrient supply for plants (Marschner and Dell 1994, Gosling et al. 2006) and providing protection from soil-borne pathogens (Borowicz 2001). Biofertilizers are marketed to capitalize on these services. Comprised of commercially propagated rhizosphere microbes (bacterial and/or fungal), they are applied to soil with the goal to establish highly effective strains. Whether or not these microbial inoculants are beneficial or cost effective for growers is not yet clear (Ryan and Graham 2002, Ceballos et al. 2013). For perennial crops, the use of biofertilizers is virtually unexamined. Arbuscular mycorrhizal (AM) fungal inoculants represent a quickly growing class of biofertilizers marketed for almost all cropping systems including perennial crops. These fungi act as an extension of the functional root surface area, giving their host plant greater access to nutrients (Marschner and Dell 1994, Entry et al. 2002) and other benefits such as pathogen protection (Borowicz 2001) and tolerances to drought, salinity, nutrient deficiencies and other adverse soil conditions (Augé 2001, Entry et al. 2002). This symbiosis often leads to increased growth and fitness for the host plant, which is termed the “mycorrhizal effect” (Smith and Read 2008). Because AM fungi form a symbiosis with 74% of angiosperm species (Smith and Read 2008) including many 2 important crops, they are easily marketable as amendments to horticultural and agricultural production systems (Gianinazzi and Vosátka 2004, Berutti et al. 2016). Although the production and use of AM fungal inoculants has increased as part of the sustainable movement (Gianinazzi and Vosátka 2004), there is scant evidence supporting their efficacy in crop production. Rodriguez and Sanders (2015) suggest that the motivation to apply AM fungal inoculum can be too simplistic and that ecological principals should be recognized in ensuring inoculum establishment and benefits. Even in annual cropping systems, studies show that while AM fungal inoculants can provide modest increases in yield of grains (Mohammad et al. 1998), legumes (Canizares et al. 2016), lettuce, zucchini (Colla et al. 2015), potato (Hijri 2016) and tomato (Douds et al. 2016), there are numerous studies that result in no change of yield (Cordiki et al. 2004, Klironomos 2003) or to yield declines (Graham and Eissenstat 1998, Klironomos 2003). 1.2 Are AM fungi developed for annuals suitable for perennials? For perennial crops, there have been few studies that directly look at the effect of AM fungal inoculants on yield in field settings. AM inoculum has been shown to be beneficial in the production of tubers such as cassava (Ceballos et al. 2013) resulting in higher yields, whereas in woody perennial plants such as grapevines results have been less consistent (Camprubí et al. 2008, Nogales et 3 al. 2009). Given the variability of results from a small number of studies, we are far from ready to predict the utility of AM fungal biofertilizers in perennial crops. It is difficult to predict whether the benefits AM inoculants provide to annuals can be extrapolated to woody perennial crops due to fundamental differences in annual versus woody perennial plants. Ostensibly, woody perennials are harder to evaluate because studies must span the period from inoculation at planting to crop production (about 3 years for grapevines). More fundamentally, differences in perennial anatomy and physiology mean that they may not respond to the AM symbiosis in the same way as do annual plants. Woody perennials develop permanent, complex root systems that are seasonally dynamic (Alsina et al. 2011), with annual turnover of fine roots (Schreiner 2005). It is not fully understood how AM symbiosis functions in these root systems, and whether or not it contributes to yield or affects fruit quality consistently over years. It is possible that the benefits from AM fungal inoculations are expressed over a longer time frame in perennials than annuals. In environments dominated by woody perennial plants where litter is recalcitrant and nutrient turnover is mediated by a fungal-dominated soil nutrient cycling system (Orwin et al. 2010, de Vries et al. 2012), AM fungi play a significant role increasing the nutrient availability to plant hosts (Smith and Read 2008). In contrast, annuals are better adapted to environments where resources are made readily available from fast cycling of nutrients and are often bacterial-dominated (Orwin et al. 2010, de Vries et al. 2012). Perennial crops could realize increasing benefits over time as the 4 AM fungal extraradical mycelial network develops throughout the soil, which would rarely occur in annual systems which are disturbed by frequent tillage. It remains unclear if the addition of fungal biofertilizers such as AM fungi would benefit perennial crops over their production lifespan. 1.3 Grapevines: A case study. Grapevines are an ideal model plant for studying AM symbiosis in woody perennial production systems. They are highly mycorrhizal-dependent, and their growth is stunted in the absence of AM fungi (Menge et al. 1983, Linderman and Davis 2001). Such dependency may stem from stressful growing conditions that prevail in the native ranges of grape rootstock species (e.g., rocky stream banks, droughty calcareous soils (Morano and Walker 1995, Ocete et al. 2015)). AM fungal associations likely are key to stress tolerance and plant survival in these native habitats. Similar stressors can exist in viticulture to some degree (i.e. salinity (Belew et al. 2010, Walker et al. 2014), extreme pH (Bavaresco and Poni 2003, Reyes et al. 2006), or low water availability(Chaves et al. 2010)). While the importance of AM fungal symbiosis for grapevine growth is clear (Trouvelot et al. 2015) there is no research yet supporting (or discouraging) the use of AM fungal inoculants in viticulture. In this thesis, I discuss the outstanding issues surrounding the use of AM fungal inoculants in viticulture. 1.3.1 The mycorrhizal effect is highly context dependent. The extreme context dependency of the grapevine-AM symbiosis may obviate any predictable response to fungal inoculants. A single fungal genotype 5 can produce variable effects depending on host identity (Klironomos 2003), soil environment (Hetrick et al. 1992, Entry et al. 2002) and climatic conditions (Hayman 1974). Considering also the variety of management strategies in viticulture, it may be unlikely that a commercial inoculant will benefit all grape plantings in all situations. 1.3.2 Rootstocks react differently to inoculation Grapevines are grown on rootstocks which are selected to enhance the tolerance of plants to environmental conditions, or to induce particular developmental or physiological characteristics, such as influencing vigor (Pongrácz 1983). However, because different rootstocks evolved under a varying range of stressful environments, there are large physiological differences among rootstocks that can impact the mycorrhizal symbiosis in both the abundance of AM fungal associations (Schreiner 2003) and the influence they have on plant physiology (Eftekhari et al. 2012). For example, Linderman and Davis (2001) showed that the plant growth response of different rootstocks to AM infection ranged from 69% to 200%. Such differences are likely related to root architecture and environmental tolerances that are known to vary among rootstocks (Pongrácz 1983). The AM symbiosis itself may result in changes to root architecture: differences among rootstocks in AM-induced production of first and second order lateral roots may lead to increased nutrient uptake (Aguin et al. 2004). In the decision of whether to apply AM fungal inoculum, it is important to consider that the results may vary among rootstocks. 6 1.4 Do perennial systems benefit from AM fungal inoculants? Regardless of local conditions or site history, there is good reason for growers to believe that their perennial crop is already colonized by AM fungi before planting. AM fungi are excluded from roots of plants propagated in hydroponic or sterile media, and grown under sterile greenhouse conditions. However, woody perennial nursery stock grown in natural field soil will have been exposed to AM fungi and likely contain the symbiosis. If grapevines originate from a sterile system, it is likely that they will be colonized soon after transplant. Thus in many perennial systems, AM fungal inoculation may be redundant over the long term. Inoculation may also be ineffective in highly fertile production systems whose nutrient levels may suppress mycorrhizal functioning. Perennial systems may have more robust AM fungal communities compared to annual systems due to reduced disturbance levels and higher plant diversity. However, in some circumstances arising from historical land use practices, or in stressful natural soil conditions, the use of periodic inoculation may be beneficial. Conditions when an AM fungi inoculant may be a good investment; and potential factors that should be considered when contemplating AM fungal additions include: i) Low inoculum potential: Following non-mycorrhizal crops (Schreiner and Koide 1993), pre-planting fumigation (Menge 1982), or removal of the surface soils (Oehl et al 2005), AM fungal propagules may be in low numbers or absent. An inoculant may give vines a 7 competitive advantage over non-mycorrhizal weeds (Rinaudo et al. 2010). This would also be the case in artificial growing systems (i.e. hydroponics, tissue culture, sterile greenhouse soils, peat, etc.), which lack soil biota. ii) Marginal sites: Fields with a history of high pathogen load, or poor soil characteristics such as extremely high or low pH, excessive salinity, or extremely low nutrient availability may require rehabilitation, including microbial additions (Belew et al. 2010, Nogales et al. 2009, Herrera et al. 1993). Similarly, high intensity agriculture may result in reduced or impoverished AM fungal communities (Verbruggen et al. 2010). Inoculation may promote later successional plants and discourage more ruderal weedy species (Rinaudo et al. 2010, Sýkorová et al. 2012), thereby promoting greater AM functioning in the soil. 1.5 AM fungal inoculants must be suitable to vineyard site conditions. The benefits of inoculation are only realized if inoculants not only colonize, but establish and persist in their new environment. Few studies, however, have tested whether inoculants persist. While some studies suggest inoculants can persist in the short term, such as for a single season (Pellegrino et al. 2012, Sýkorová et al. 2012, Köhl et al. 2016), none were able to provide evidence that the fungi become permanent fixtures in soil and root communities. However, with advancements in molecular techniques it is easier to determine whether 8 foreign inoculum establish in ecosystems after being applied. More study is needed to establish the economic benefit of AM fungal applications. Whether AM fungal inoculants successfully establish on crops depends on the ability of applied propagules to germinate, contact hosts and colonize. Commercially available inocula may not be well suited to the target crop or site conditions. A variety of inoculant formulations are available (Faye et al. 2013), many of which contain relatively few species and have been selected for their ability to quickly infect the roots of annual crops (Gianinazzi and Vosátka 2010). Using inoculants developed for grasses and herbaceous forbs may not be best suited for woody perennial crops, as it has been shown there are clear differences in the AM fungal communities established within these different plant types (Holland et al. 2014). Early evidence suggests that AM fungi, like plants, have distinct abiotic and biotic tolerances and may not thrive under broader conditions, which explains why many commercial inocula have been shown to fail at colonizing host roots (Cordiki et al. 2004, Tarbell and Koske 2007). Much remains to be learned about the ecology of AM fungi, which will help predict which fungi will proliferate and provide benefits in specific crops and specific conditions. 1.6 Research objectives A combination of vineyard and greenhouse studies were used to evaluate the use of AM fungal inoculants with grapevines. This was done to illustrate various factors surrounding the symbiosis such as the provenance of the AM 9 fungi and timing of application, along with the ability of the fungi to alleviate particular stresses. This thesis addresses four main questions surrounding the AM fungal – grapevine symbiosis: 1.6.1 Does the source of AM fungi influence the growth of different grapevine rootstocks? This question is the focus of chapter 2 of this dissertation and was evaluated using a greenhouse study. AM fungal inoculum sources, in the form of soil, roots and spores were gathered from a natural habitat adjacent to a vineyard and compared to commercial products. These were applied to three rootstocks and the plants’ physiology was monitored, along with root colonization. Due to differences in host preferences among microbes, I hypothesized that more diverse inocula originating from naturally growing woody perennial plants would establish more readily and provide more beneficial effects than commercial inocula. Further, I predicted that rootstock cultivars would vary in their response to inoculation due to the innate differences in their rooting characteristics and physiology. 1.6.2 Does inoculation with AM fungi improve vine establishment in a field having poor soil conditions and does this depend on the timing of inoculation or source of inoculum? Chapter 3 is a field experiment that tested how transplanted vines responded to different inoculation treatments. In particular it examined whether the timing of inoculation influences vine establishment, with vines being 10 inoculated prior to, or at the time of transplanting into a field that had the top soil removed. In addition, the effect of inoculum source was examined, comparing commercial AM fungi to an addition of vine roots originating from a vineyard block adjacent to the one being transplanted. Vine establishment was monitored for two growing seasons, including measures of growth and mortality. At the end of the trial the persistence of the commercial AM fungi within vine roots was measured. I predicted that vines pre-inoculated with an AM fungus would have improved transplant success compared to those infected with AM fungi at transplanting in the field. I also expected that locally sourced inoculum would be more beneficial compared to a commercial product. 1.6.3 Will AM fungal inoculation reduce fungal infection of grapevine rootstocks and does the timing of AM fungal addition determine the strength of this effect? This question is addressed in Chapter 4, attempting to further understand if AM fungi are a suitable method to mitigate the infection of pathogens, and whether the timing of application impacts this interaction. A greenhouse study was utilized, in which the timing of AM fungal and pathogen exposure was compared. Vines were either pre-colonized with AM fungi before exposure to a common grapevine pathogen, exposed to both fungal entities at the same time, or received only one of the fungi. The growth and pathogenesis responses of these vines were compared after six months of exposure to the pathogen. I predicted that grapevine rootstocks pre-colonized with AM fungi would have 11 reduced pathogen infection and symptom severity compared to rootstocks inoculated at the time of transplant. 1.6.4 Do standard transplant root trimming practices affect the mycorrhizal symbiosis? This question is addressed in Chapter 5 as it is important to understand whether trimming roots would eliminate or alter the symbiosis of pre-colonized vines. A greenhouse study was used in which vines were colonized with commercial AM fungi, then had all active roots trimmed before being potted in new sterile soil. Vines were grown for another 3 months before their growth and root system was examined. The persistence of AM fungi was measured both by visually detecting different mycorrhizal structures via microscopy and using digital droplet PCR of the mitochondrial DNA. This was completed for both newly developed roots and older roots to determine if the trimming influenced the functionality and distribution of the mycorrhiza. I hypothesized that root trimming would either eliminate the active mycorrhiza from grapevine roots or influence the distribution of the mycorrhizal functional structures found within the roots. That is, the amount of mycorrhizal structures and activity within the lower and upper roots would be altered by root trimming. 12 2 Response of grapevine rootstocks to soil inocula from different sources 2.1 Background Grapevines are an economically and culturally important crop in many regions of the world, but local conditions can inhibit their establishment. Selecting rootstocks with characteristics that are well adapted to local conditions can be challenging, but may be important for reducing the susceptibility of vines to environmental stress. Selection based on physiological characteristics and environmental tolerances can ensure that grafted vines have the best chance of producing high-quality grapes. Often overlooked, however, are the soil microbes which interact with rootstocks and can affect plant growth and possibly the establishment of newly transplanted vines. While grapevine roots interact with many soil organisms that affect establishment and physiology, an important association occurs with arbuscular mycorrhizal (AM) fungi. AM fungi (Phylum Glomeromycota) are obligate root endophytic fungi that associate with most land plants (Smith and Read 2008). AM fungi benefit their hosts through improved nutrition (Latef and He 2011), pathogen protection (Petit and Gubler 2006) and abiotic stress tolerance (Augé et al. 2001, Latef and He 2011), all of which can influence plant establishment. Although grapevines are known to be mycorrhizal (Linderman and Davis 2001) and have been shown to have increased nutrient uptake compared to non-mycorrhizal vines (Cheng and Baumgartner 2006), the role and importance of AM fungi in vine establishment is not well understood. 13 Because of their benefits to crop plants, AM fungi have been developed and applied as inoculants or biofertilizers for several decades, primarily as a means to improve crop nutrition and yield in grain and horticultural crops (Ruehle and Marx 1979, Berruti et al. 2016). However, little is known about the effects of using AM fungi inoculants in viticulture, including their ability to colonize grapevine rootstocks and influence vine growth. In order to make informed decisions about inoculating grapevines with AM fungi, many factors should be explored, including the influences of fungal and rootstock identity on young vine growth and establishment. Commercial inoculants developed for agriculture often consist of a single or few AM fungal isolates grown in plant culture or greenhouse conditions with annual grasses or forbs (Gianinazzi and Vosátka 2004). While Glomeromycota were often considered generalists with respect to hosts, there is increasing evidence for host preference among these fungi (Torrecillas et al. 2012, Holland et al. 2014). Different AM fungal communities have been found in association with grapevines compared to adjacent forbs and grasses (Holland et al. 2014). Thus, it is therefore possible that commercial inoculants adapted to generalist grasses and forbs might not establish on woody grapevines that have different ecosystem preferences. An alternative to using a commercial inoculum is the use of AM fungi and other microbes sourced from natural sites near, and with similar edaphic conditions to, the vineyard site to be transplanted. This allows AM fungi to be isolated from plants of a similar functional group (i.e. woody perennials) and 14 enables a diversity of microbes adapted to local conditions to be included. Mycorrhizal fungi are vital in the uptake of nutrients made available by other associated soil microbes (Cheng and Baumgartner 2006). Rhizosphere functioning can be enhanced by adding a mixture of mycorrhizal fungi and associated microbes, which can improve fungal establishment in roots, increase nutrient uptake and improve growth of young vines (Dodd and Thomson 1994, van der Heijden et al. 1998). Rootstock genotype could also influence the effectiveness of mycorrhizal inoculation. Because rootstocks are selected cultivars of Vitis species and hybrids which differ in rooting architecture, environmental stress tolerance, and other physiological traits (Pongracz 1983, Christensen 2003), they respond differentially to AM fungi (Newsham et al. 1995). Studies involving other plant genera have shown that both plant and fungal identity contribute to mycorrhizal responsiveness (Klironomos 2003). For example, differences in rooting patterns can contribute to the magnitude of mycorrhizal responsiveness of the host plant, as root systems that are less branched and coarser often benefit more from mycorrhizal colonization (Newsham et al. 1995), as do less stress tolerant rootstocks (Belew et al. 2010). This suggests that multiple factors may contribute to the differential responsiveness of rootstocks to AM fungi. The goal of this study was to answer the question, Does the source of AM fungi influence the growth of different grapevine rootstocks? Because host preferences differ among microbes, I hypothesized that diverse inocula sourced from naturally-growing woody perennials would establish better and 15 provide more benefits than commercial inocula. I also predicted that the response of rootstock cultivars to inoculation would vary, due to the innate differences in their rooting characteristics and physiology. 2.2 Materials and methods 2.2.1 Study site and experimental design This glasshouse study was established at the Summerland Research and Development Centre in Summerland, B.C. in June 2014. Treatments included a factorial combination of five soil inocula and three rootstocks. The rootstocks Riparia Gloire, 101-14 Mgt and SO4 were selected based on ancestry, range of rooting characteristics, and high degree of usage by local grape growers. Rootstock plants were grown from three-bud cuttings obtained from a rootstock collection block at the Summerland Research & Development Centre, Summerland, B.C., Canada. The cuttings were calloused and rooted over 21 days in moist perlite in a rooting bed with ambient air temperature at 4°C and the rooting medium temperature of 22°C. In total, 150 plants were included in five inoculation treatments, with 10 replicates of Riparia Gloire, 12 for 101-14 Mgt and eight for SO4. The inoculation treatments were a non-microbial control, two locally sourced inocula (root fragments and isolated spores) and two widely available commercial inocula (Table 2.1). The commercial inocula were Biosyneterra MykoΣyn Mikro-VAM GR, (Biosyneterra Solutions Inc.) which contains Glomus intraradices isolate (~3.75 propagules/g); and MycoGrow™ for Vegetables (Fungi 16 Perfecti LLC) which contains G. intraradices, G. mosseae, G. aggregatum and G. etunicatum (~ 55 propagules/g). Local root and spore inocula were isolated from rhizosphere soils at an undeveloped site (49°30’57.0”N 119°34’42.4”W) inhabited with locally native AM shrubs (sagebrush (Artemisia tridentata Nutt.), rabbit brush (Ericameria bloomeri Gray) and antelope brush (Purshia tridentata Pursh)). This site was located in the Naramata Bench growing region between Penticton and Naramata, B.C. In total, 1080 cm3 of soil was collected, thoroughly homogenized and sieved (500 μm) to remove large roots, rocks and organic matter. Remaining roots were washed with distilled water, air dried, and cut into fragments (~1 cm long). AM fungal spores were isolated from 100 mL of homogenized root-free soil, which was agitated in 10 L of water for 2 minutes and allowed to settle over 18 hrs. The supernatant was poured through a series of sieves (500 μm, 250 μm, and 45 μm) and the 45μm fraction was collected and washed in distilled water. The number of AM spores in 1 mL of five fractions were counted under a dissecting microscope and averaged to determine spore concentration. More than five spore morphotypes were observed based on their size, shape and color. 17 Table 2.1 Details of inocula treatments applied to roots of Riparia Gloire, 101-14 Mgt and SO4 rootstock plants. aSoil samples collected from the rhizosphere of an undeveloped shrub-steppe site at 49°30’57.0”N 119°34’42.4”W, inhabited with sagebrush (Artemisia tridentata Nutt.), rabbit brush (Ericameria bloomeri Gray) and antelope brush (Purshia tridentata Pursh). bManufacturer’s information Treatment Source Content Propagule density Application method Roots Isolated from soil collected locally.a Root fragments that may have contained microbes other than mycorrhizae, such as fungal endophytes, bacteria, and pathogens. Diversity of AM fungi unknown. Estimated at 20 propagules/g, based on two/root fragment, and 10 fragments/gram. 2.5 g of 1-cm root fragments spread evenly over vine roots before planting. Spores Isolated from soil collected locally.a More than five morphotypes of mycorrhizae spores based on size, shape and color. 20 spores/mL; unknown viability. 50 mL of spore suspension poured over of vine roots before planting. Biosyneterra Commercial granular product. Rhizophagus irregularisb (label claim) and additives including sand and perlite. 3.75 propagules/gb; unknown viability. 3 g of inoculum, half applied directly to roots before planting, and half mixed with surface planting medium. MycoGrow Commercial dry powder product. G. intraradices, G. mosseae, G. aggregatum and G. etunicatumb, and unknown soluble powder additives. 55 propagules/gb; unknown viability. 4 g of inoculum, half applied directly to roots before planting, and half mixed with surface planting medium. 18 Rooted plants were planted in 2-L pots containing a mixture of sterilized sand and Turface® (1:1 by mass). Turface is an inert montmorillonite clay often used in mycorrhizal greenhouse studies (Caron et al. 1985), because of its physiochemical properties including high porosity and water-holding capacity, low-to-moderate nutrient availability, and high cation exchange capacity. The inocula were applied directly to the plant roots before planting, and the dry commercial products were additionally applied to the surface of the growing medium (Table 2.1). Commercial inocula were applied according to the manufacturer’s suggested application rate. Locally sourced inocula additions were applied to provide propagule numbers comparable to the commercial additions. The plants were grown for 25 wks on greenhouse benches in a completely randomized layout with pots spaced at 18 ´ 18 cm. Once per week, 100 mL of a low-P fertilizer solution (12.5-2-14) was provided. The concentration of fertilizer was increased throughout the growing season as follows: 50 mg/L N (weeks 1-4), 75 mg/L N (weeks 5 and 6) and to 100 mg/L N (week 7 onward). The plants were provided 100 mL of water every second day. Dominant shoots were staked vertically. Artificial lighting was used to ensure at least 12 hours of light exposure per day. 2.2.2 AM colonization Percent root colonization was assessed for five vines of each rootstock/treatment combination according to Holland et al. (2014). Briefly, 1-cm 19 roots were washed with distilled water, cleared by autoclaving for 30 min in 10% KOH, bleached in 3% H2O2 at room temperature for 30 min, acidified in hot 10% HCl for 10 min, and stained in trypan blue by autoclaving for 15 min. Roots were destained in lactoglycerol overnight and mounted in lactoglycerol with 10 roots per slide. Root AM fungal colonization was measured using a magnified intersection method and scanning 100 root intersects per plant using the magnified intersection method. 2.2.3 Plant response At 12 wks post-inoculation, leaf greenness was measured using a SPAD meter (SPAD 502 Plus, Konica Minolta), and leaf photosynthesis rate was measured at mid-day using a LI-6400XT photosynthesis system (LI-COR Biosciences). The measurements were taken on, and averaged from, the third leaf from shoot base and fifth leaf from the growing tip. The vines were harvested 25 wks after planting. Roots were washed with water to remove media and dried before weighing. Fresh weight of aboveground biomass was measured at harvest. Total shoot length was determined by measuring all stems, including branches. Average area per leaf was determined for the fourth and fifth leaves from the growing tip and for the basal two leaves of each plant, using a scanning leaf area meter (Li-3000C and LI-3050C, LI-COR Biosciences). Whole-plant photosynthetic capacity was estimated as the product of photosynthesis rate, average area per leaf, and the number of leaves. 20 2.2.4 Statistical analyses All statistical analyses were conducted for a completely randomized design using R v2.8.1 (R Development Core Team, 2008). Differences in percent colonization were determined using two-way analysis of variance (ANOVA), with fungal inocula and rootstock as treatment factors. Differences among treatment means were detected using Tukey’s post-hoc test. Differences in overall vine growth and physiology were detected using a two-way multivariate analysis of variance (MANOVA) with fungal inocula and rootstocks as treatment factors. The MANOVA response variables were root biomass, aboveground biomass, cane length, leaf area, leaf chlorophyll content, and photosynthesis rate. Pairwise MANOVAs were used to determine treatment differences. Effects of inocula on growth characteristics, leaf greenness, and estimated photosynthetic capacity were determined in a series of ANOVA tests followed by Tukey’s post-hoc tests to compare treatment means. 2.3 Results 2.3.1 AM fungal root colonization Percent root mycorrhizal colonization differed among inoculum treatments but not rootstocks (Figure 2.1). Biosyneterra resulted in the highest rate of colonization (averaging 62.9%), followed by the local root inoculum (averaging 19.0%). Addition of MycoGrow and local spores averaged colonization rates of 2.73% and 0.67 %, respectively, similar to the control which averaged 0.40 % colonization, so these inocula were omitted from further evaluation. 21 Figure 2.1. Arbuscular mycorrhizal (AM) fungal colonization of roots in response to five inoculum treatments (untreated control, Biosyneterra, MycoGrow, locally sourced roots, and locally sourced AM fungal spores) applied to three rootstocks (101-14 Mgt, Riparia Gloire and SO4). Letters indicate differences in AM colonization among inoculum treatments detected using Tukeys honest significant difference post-hoc test at α = 0.05. Colonization was not affected by rootstock. 2.3.2 Plant response Colonization was associated with an overall difference in plant growth and leaf functional characteristics (MANOVA for the combined response of root biomass, aboveground biomass, cane length, leaf area, leaf chlorophyll content, and leaf gas exchange: F12,146 = 2.841, p = 0.002; Pillai’s Trace = 0.379). Pairwise comparisons showed differences between vines inoculated with Biosyneterra and control vines (F6, 45 = 2.986, p = 0.015; Pillai’s Trace = 0.285), and between vines inoculated with Biosyneterra and local root inocula (F6, 48 = 7.021, p = 2.11e-5; Pillai’s Trace = 0.467). Colonization of vines treated with local 101-14 R.gloire SO4 101-14 R.gloire SO4 101-14 R.gloire SO4 101-14 R.gloire SO4 101-14 R.gloire SO4 Control Biosynterra Mycogrow Roots Spores c a c b c 22 root inocula did not differ from that of control vines (F6, 46 = 1.096, p = 0.379; Pillai’s Trace = 0.125). Analyzed separately, growth characteristics and leaf greenness, but not photosynthesis, were found to be affected by inoculum treatments (Table 2.2). Leaf greenness was the only parameter that differed between inoculated and control vines. SO4 vines inoculated with locally sourced roots or with Biosyneterra had greener leaves compared to control vines. Compared to Biosyneterra, the local root inoculum was associated with greater aboveground biomass and total shoot length in Riparia Gloire and 101-14 Mgt, and increased root biomass in 101-14 Mgt. 2.3.3 Rootstock effects As expected, the rootstocks showed different growth characteristics, including root and aboveground biomass, and average leaf area. The only interactive effect of rootstock and inoculation treatment occurred for leaf greenness (only SO4 responded significantly to inoculation) (Table 2.2). However, Tukey’s pairwise comparisons showed that growth of Riparia Gloire and 101-14 Mgt vines responded differently to the local-root and Biosyneterra inocula (growth was enhanced by the local inoculum); growth of SO4 vines did not respond to the inoculation treatments. 23 Table 2.2 Effects of microbial inoculum and rootstock cultivar on characteristics of vine growth and photosynthesis. Rootstock/ Inoculum Root dry mass (g) Above-ground fresh mass (g) Shoot length (cm) Leaf greenness (spad units) Area per leaf (cm2) Photosynthesis (µmol/m2/sec) Plant photosynthetic capacity (µmol/sec) R. Gloire(n=50) Control 8.78 19.3 aba 154 ab 19.1 46.7 5.11 1.06 Biosyneterra 7.57 15.7 b 136 b 22.1 49.2 5.80 1.13 Root 8.34 22.6 a 181 a 20.4 49.5 5.41 1.31 101-14 Mgt (n=60) Control 10.5 ab 24.3 ab 200 ab 18.0 44.8 5.88 0.867 Biosyneterra 8.60 b 21.2 b 181 b 18.3 40.9 4.99 0.706 Root 10.7 a 25.9 a 230 a 16.8 42.5 4.95 0.812 SO4 (n=40) Control 5.19 23.4 192 14.1 b 51.8 4.59 0.837 Biosyneterra 5.83 20.0 159 19.4 a 55.8 5.58 1.04 Root 6.91 19.8 165 20.8 a 45.8 4.90 0.852 Effectsb Inoculum * ** ** * ns ns ns Rootstock *** ** *** * ** ns *** I*R ns ns ns * ns ns ns aWithin rootstocks, letters indicate differences among inoculum treatments based on Tukey’s post-hoc tests at α = 0.05. b*, ** and ns indicate significant main or interactive effects at p = 0.05, p = 0.01, and not significant, respectively. 24 2.4 Discussion 2.4.1 AM fungal colonization In this study, the locally sourced mycorrhizal fungi did not consistently establish better than commercial inoculants. Establishment varied widely among inocula, from almost no root colonization by locally sourced spores or MycoGrow, to 15 to 20% colonization by local root inocula, and 40 to 90% by Biosyneterra. Colonization by the Biosyneterra inoculum was comparable to that observed in previous greenhouse and field studies (60-80 %) (Linderman and Davis 2001, Belew et al. 2010, Holland et al. 2014). The large difference in colonization between the two commercial inocula was unexpected. It is not clear why the MycoGrow inoculum did not establish well; it was specified as having a higher density of propagules, more AM fungal species, and was applied per the manufacturer’s instructions. Mycogrow might have been incompatible with my host plants, although it is marketed as being appropriate for a diverse range of plant types. Commercial inoculants also failed to establish in other studies (Rowe et al. 2007, Berruti et al. 2013), which illustrates the importance of verifying the suitability of an inoculum for a particular plant species and environment before application in commercial operations. Another potential reason for the poor colonization by Mycogrow is its fine powder formulation, which might have washed out of the coarse growing medium. The low AM fungal colonization resulting from the spore inoculum could have been due to preparation and application. While the use of spores instead of 25 soil or roots can help prevent the introduction of pathogens in the inoculum, the risk of inoculation failure appears to be greater compared to inoculation with root fragments, which produced better colonization. It is possible that the spores were washed out of the coarse growing-medium during irrigation. The use of field spores may be useful in other situations, such as with finer textured media, but the technique requires more development before it can be recommended. Berruti et al. (2016) argued that the best practice for achieving AM infection is to apply trap cultures that contain a mixture of different types of infective propagules. This would be advantageous because AM fungi have differing abilities to colonize plant roots, and colonization is influenced by the form in which propagules are applied (as spores, infected roots or extraradical hyphae; Klironomos and Hart 2002). 2.4.2 Rootstock growth response Although AM fungal colonization was associated with changes in rootstock growth, I did not detect a direct association between colonization and plant growth, which is commonly observed in mycorrhizal studies. The only differences in growth occurred between the Biosyneterra and natural root inoculum, with the latter enhancing growth in Riparia Gloire and 101-14 Mgt rootstocks. Leaf greenness in SO4 plants was the only individual trait that was positively affected by mycorrhizal colonization, when compared to non-mycorrhizal plants. Leaf greenness is associated with leaf chlorophyll content and photosynthetic ability (Markwell et al. 1995), but I did not detect an association between enhanced leaf greenness and increased photosynthesis. 26 The increase in leaf greenness is consistent with other studies in which mycorrhizal plants had greener leaves or higher chlorophyll content, as observed in tomato plants (Latef and He 2011) and young orange seedlings (Ezz and Newar 1994) grown under saline soil conditions. Those studies emphasized the role of AM fungi in improving nutrient acquisition under harsh conditions. Because I found this benefit only for SO4, it may be related to the adaptive traits of this genotype, such as its lower salt tolerance compared to 101-14 Mgt and Riparia Gloire (Christensen 2003). The mycorrhizal response needs to be further explored under a range of conditions and in association with different rootstocks to make further conclusions. Although previous studies reported increased growth in grapevines inoculated with AM fungi (Linderman and Davis 2001, Belew et al. 2010), mycorrhizal symbioses are dynamic associations that can result in a continuum of growth responses (Johnson et al. 1997, Klironomos 2003). Here, the lack of growth responses in infected compared to non-inoculated plants could be due to the conditions and duration of my study. Depressed growth in response to AM infection is commonly observed in greenhouse studies (Johnson et al. 1997, Klironomos et al. 2003). Benefits of the AM association may more commonly manifest under highly stressful conditions, which did not occur in my study, such as extremely low nutrient or water availability, high salinity, or pathogen exposure (Johnson et al. 1997, Augé et al. 2001, Petit and Gubler 2006). Schreiner (2007) found that mycorrhizal response was highly dependent on the soil in which vines were grown; vines grown in phosphorus-deficient soils had enhanced growth, 27 which was linked to an increase in phosphorus uptake due to mycorrhizal inoculation. In addition, mycorrhizal responsiveness can change over time (van der Heijden et al. 2006); fungi can behave parasitically or as mutualists during different stages of plant development or under different conditions. My study may have been too short, or the conditions were insufficiently stressful to elicit a mycorrhizal response. 2.4.3 Inoculum source Physiological differences between vines treated with local root inoculum and commercial Biosyneterra inoculum supported my hypothesis that locally sourced inocula would be more beneficial than commercial inocula. This could be important in selecting AM fungal inocula to improve establishment of transplanted vines. Although naturally sourced inocula have been shown to be more beneficial than commercial inocula in other cropping and restoration systems (Rowe et al. 2007, Ortas and Ustner 2014), grapevines can be less responsive to native AM fungal inoculum than to commercial strains (Linderman and Davis 2001, Camprubí et al. 2008). However, the differences observed here compared to previous studies may arise from differences in how the inocula were prepared. Other studies have used trap cultures, similar to cultures used to produce commercial inocula, which filter the AM fungal community to only the fungal isolates that are compatible with trap culture plants. In comparison, my locally sourced root inoculum consisted of root fragments that contained other fungi and bacteria, likely both as endophytes and on root surfaces. Depending on whether 28 these organisms are beneficial or pathogenic, they could influence plant growth as well as have mycorrhizal effects. Synergistic effects of combined microbial species can result in greater growth compared to AM fungi alone. Singh et al. (2013) found that apple rootstocks treated with a combination of AM fungi, Azobacter and Azospirillum grew better than rootstocks treated with the individual organisms. Cheng and Baumgartner (2006) found that AM fungi were associated with higher nutrient uptake that also coincided with higher microbial biomass detected in the rhizosphere. That study showed that AM fungi are vital for nutrient acquisition and that they work in parallel with other soil microbes that break down organic material and increase nutrient availability. The natural root inoculum in my study may have contained beneficial non-Glomeromycota taxa that enhanced growth, in contrast to the commercial inoculum that contained only AM fungi. These results indicate that application of a more complex assemblage of microbes may be more beneficial to young vines because of interactions between different microbial groups. To better understand these interactions, studies should examine interactions within microbial communities and the role of individual components in affecting grapevine performance and establishment. The greater diversity of AM fungi in the local inoculum could also explain the enhanced growth in vines that received that inoculum. Naturally sourced inocula typically contain multiple AM fungal species; the natural root additions that originated from native perennials A. tridentate, E. bloomer and P. tridentate likely contained numerous AM fungal species (Carter et al. 2014). Greater AM 29 fungal diversity can lead to higher productivity for plant communities (van der Heijden 1998) and greater functional diversity in host vines, as different AM fungal species vary in developmental and functional traits (Hart and Reader 2002a, 2002b). It may be best to source AM fungi from near local vineyards, which should include a wide diversity of fungi adapted to local conditions and increase the probability of obtaining a suitable and beneficial fungal partner (Dodd and Thomson 1994) 2.4.4 Rootstock cultivars The rootstocks had similar colonization but differed in growth responses to the different inocula. These results were not surprising because rootstocks have differing physiology and environmental sensitivity based on their parentage (Pongracz 1983, Christensen 2003), which should influence the mycorrhizal interaction. Compared to previous grapevine studies (Linderman and Davis 2001, Belew et al. 2010), I found similar variation in the mycorrhizal response among rootstocks, but of less magnitude. My experimental conditions may have limited the effects of interactions between rootstocks and mycorrhizal infection. These conditions include the short duration of the experiment, the use of rootstocks with physiological and genetic similarities (V. riparia is a common parent), and environmental conditions that were not stressful enough to elicit a strong mycorrhizal response. My results, in addition to previous findings (Linderman and Davis 2001, Belew et al. 2010) indicate that variation in mycorrhizal response among rootstocks is common. The interaction between rootstock genotype and AM fungi needs to be further explored if inoculants are to 30 be used for commercial grapevine propagation and cultivation, as the economic value of applying AM fungi is uncertain. 31 3 Arbuscular mycorrhizal fungi impact transplant of grapevines into vineyard with poor soil conditions. 3.1 Background Whether grapevines survive transplanting and establish in the field depends on many factors including biotic, abiotic and anthropogenic influences. While substantial attention has been paid to rootstock selection (Pongracz 1983, Christensen 2003), the role of beneficial microbes in vine establishment has not been well investigated. Arbuscular mycorrhizal (AM) fungi are symbiotic fungi that associate with over 74% of angiosperm plant species (Brundrett 2009) including grapevines (Linderman and Davis 2001). Through the symbiosis the fungi receive plant derived photosynthates in exchange for nutrients and water that may otherwise be unavailable to the plant (Smith and Read 2008). Evidence from the grapevine-mycorrhizal fungal association indicates the symbiosis enhances a wide array of physiological and developmental characteristics such as tolerance to poor soil conditions, water use efficiency and growth (Linderman and Davis 2001, Camprubi et al 2008, Nogales et al 2009, Belew et al. 2010, Nicolás et al. 2015, Trouvelot et al 2015). Although it is accepted that AM fungi are an important component of grapevine ecophysiology, there is limited evidence on how to best utilize AM fungi in viticulture. Most grapevines are transplanted into vineyards with little consideration of their mycorrhizal status. Inoculating vines with AM fungi before transplanting may help the vines tolerate vineyard conditions during the critical establishment 32 phase (Holland et al. 2018a). Grapevines with AM established prior to transplant may better tolerate stressors they encounter in the vineyard by having enhanced disease resistance (Pozo and Azcón-Aguilar, 2007), greater root branching (Gutjahr and Paszkowski 2013) and a higher tolerance to salinity (Belew et al. 2010, Latef and He 2011), low nutrient availability (Johnson et al. 1997, Latef and He 2011) and drought (Augé et al. 2001). The source and composition of AM fungal inoculants may influence the buffering effect of AM fungi on vine transplants. Most commercial inoculants are composed of a single or few AM fungal isolates, greenhouse-grown in plant tissue culture or with annual grasses or forbs (Gianinazzi and Vosátka 2004). While AM fungi are often considered to be generalists with the ability to infect many plant genera, there is increasing evidence for host preference (Torrecillas et al. 2012, Holland et al. 2014). For instance, Holland et al. (2014) found that grapevines preferentially associated with different AM fungal communities compared to co-occurring forbs and grasses. It is possible that commercially available inoculants may not be best suited to woody perennials such as grapevines. Thus, it may be better to use AM fungi sourced from actively growing vines, as they will likely be more compatible and better adapted to local conditions. This idea was tested in greenhouse study (Holland et al. 2018b), which found that although commercial AM fungi colonized grapevines to a much higher degree, a naturally sourced AM fungal community resulted in greater plant growth and leaf greenness in comparison to the commercial fungi. 33 In this study, I asked does inoculation with AM fungi improve vine establishment in a field having poor soil conditions and does this depend on the timing of inoculation or source of inoculum? I predicted that vines pre-inoculated with an AM fungus would have improved transplant success compared to those infected with AM fungi at transplanting in the field. I also compared the effects for commercial and locally sourced AM fungal inoculants. 3.2 Materials and methods 3.2.1 Study site The experiment was conducted in in a new vineyard block at Summerhill Estate Winery, in southwest Kelowna, British Columbia, Canada, 49.8064 °N, 119.5014 °W. Topsoil had been removed prior to the experiment. Compared to the adjacent vineyard block the exposed subsurface soil was low in organic matter, nitrogen, phosphorus and had an elevated pH naturally buffered by its high carbonate content (Table A1). The remaining soil was sampled before planting to confirm, via digital droplet PCR, that the fungal isolate contained in the commercial inoculum was not present. 3.2.2 Experimental design The experiment tested the effects of four AM fungal inoculants applied to grapevine (Pinot Noir 115 on SO4) transplant roots six weeks before planting: a non-mycorrhizal control (NM), a commercially produced inoculum (CP) Biosyneterra MykoΣyn Mikro-VAM GR (Biosyneterra Solutions Inc.) containing Glomus intraradices at ~3.75 propagules/g, locally sourced AM fungal spores 34 (SP), and locally sourced grapevine root fragments colonized by local fungi (RP) (see details on inoculum preparation below). In addition, the commercial inoculum was applied at transplanting (CF) to test the hypothesis that inoculation timing was important for vine establishment. I did not apply field treatments of the locally sourced spore and root inoculants at planting because it was assumed that the fungi would already be present in the soil. There were 10 replicate plants of each treatment which were arranged as single-vine plots in a completely randomized design in the field. The row x vine spacing was 4m x 8m 3.2.3 Locally sourced inocula The locally sourced inoculants were prepared using 40 L of bulk soil collected from the base of Pinot Noir 115 on SO4 grapevines in the vineyard block adjacent to experiment block. The soil was first homogenized and sieved (1 mm) to remove vegetation and rocks. A 0.5 mm sieve was used to remove roots, which were cut into fragments roughly 1 cm long. These roots fragments were homogenized and then separated into 25 mL aliquots that comprised individual root (RP) treatments. The sieved soil was further processed to produce the spore inoculant (SP). Soil (250 mL) was mixed with 1 L of water and homogenized in a blender for 30 seconds. Spores were extracted from the resulting slurry following a modified method from INVAM (International Culture Collection of Vesicular Arbuscular Mycorrhizal Fungi). In short, the blended slurry was poured through 500 μm and 50 μm sieves. The collection from the 50 μm sieve was then put into 35 a 50 mL falcon tube with 50% glucose : water (w/w), agitated and centrifuged at 4500 rpm. The supernatant containing spores was placed in a 50 μm sieve, rinsed with RO water and then collected in a 15 mL falcon tube. Subsamples of the suspension were observed using microscopy to determine spore density. The suspension was diluted to 50 glomeromycotan spores/mL. 3.2.4 Pre-transplanting plant preparation and treatment Six weeks prior to transplanting the bottom 5-10 cm of all vine roots were root trimmed, as per normal grower practice, leaving 10 cm of root mass on each plant. The plants were hydrated overnight by submerging the roots in water. Each vine was then planted in a 750 mL plant pot filled to a depth of 20 cm with a sterile medium consisting of a 1:1 ratio of sand:Turface® MVP. Turface is an inert montmorillonite water-absorptive clay often used in mycorrhizal greenhouse studies (Koide and Li 1989). The inoculation treatments were each applied directly to the exposed roots of 10 vines using the following amounts of each inoculant: 40 g of CP, 25 mL of RP, and 25 mL of SP. After treatment, the sterile medium was added to fill the pots. The potted vines were arranged in a completely randomized design on a greenhouse bench and grown for six weeks during which water was applied as needed. Vine roots were inspected for inoculum establishment. To detect colonization a staining protocol was used from Holland et al. (2014), which consisted of a 15-minute autoclave in 10% KOH, 10-minute soak in 3% H2O2, 5-minute wash in 10% HCl and an overnight stain in a 5% ink : vinegar solution. 36 Roots were viewed at 200x magnification to determine the presence of mycorrhizal structures (spores, hypha and/or arbuscules). All non-control plants showed signs of fungal establishment by 6 weeks, so plants were prepared for field planting. 3.2.5 Field planting and management The vines were planted in the field to a depth of 15 cm. For the field-applied CF treatment 40 g of the inoculant was applied directly to the roots at planting. After transplanting the vines were irrigated using a drip system as required. Nutrients were managed organically, as per the grower’s standard practices, which includes the addition of an in-house prepared compost composed of horse manure, harvest waste, grass clippings and leaves. This was applied during the second year of growth (2017). The soil surface at the base of the vines was covered with 10-15 cm of mulch and planted with clover inoculated with rhizobium. 3.2.6 Vine performance 3.2.6.1 Vine establishment Vine survival was determined when growth measurements were taken in Oct 2016 and Oct 2017. 3.2.6.2 Vine growth Shoot measurements consisted of total shoot length, shoot diameter, and internode length, the latter two were measured on five internodes evenly 37 distributed along the canes starting at the fifth internode from the growing tip, and averaged. At the end of the second growing season, surviving vines were sampled to determine whether the commercial inoculant (the only inoculant for which molecular tools existed) had established from the CF or CP treatments. From each vine, 3 soil cores (20-30 cm deep) were obtained and sieved (0.5 mm) to separate roots from the soil. The resulting roots were frozen and used for DNA extraction. 3.2.7 Analysis for AM fungi To detect the presence of the commercial AM fungal inoculant prior to transplanting, DNA was extracted from a subsample of 0.25 g of soil, following the protocol outlined in the MoBio PowerSoil kit (MoBio Laboratories Inc). The same procedures were used for extracting root DNA from samples collected at the end of the experiment, except 0.1 g of roots, cut into 5 mm sections, were used for the analysis. 3.2.7.1 Inoculant abundance and persistence The abundance of the commercial AM fungal inoculant was determined using ddPCR (digital droplet polymerase chain reaction). This was used to determine its abundance within vineyard soils pre-planting, and within roots to determine abundance after year two years of growth. I measured copy number for the mtDNA of the isolate from the commercial inoculum using a probe-based ddPCR assay using the probe developed by Kokkoris et al. (2019) 38 Each ddPCR reaction contained 10 μL ddPCR Supermix for probes 1X (Bio-Rad Inc), 1 μL of primer-probe mix (Integrated DNA Technologies) 20X (500 nM primers and 250 nM probe in final reaction), and 2 μL of undiluted root DNA and 7 μL of DNase free water, for a total volume of 20 μL. Bio-Rad reagents were used, along with the reaction mixes, to create 40 μL droplets were generated as per manufacturers guidelines. Thermal cycling conditions were 95°C for 10 min, followed by 40 cycles of 94°C for 30 sec and 59°C for 30 sec, followed by a single step at 98°C for 10 min; the ramp rate was increased by 2°C/sec at every step. Droplet analysis was carried out on the BioRad QX100 and raw data was collected using Quantalife software ver. 1.7.4. (Bio-Rad). In each plate three non-template controls (NTC) and three environmental NTC were used. The former was used to eliminate the possibility of contamination and the latter to determine the fluorescence amplitude threshold. 3.2.7.2 Threshold determination and data processing The fluorescence amplitude threshold was set based on an experimental negative control (eNTC). All droplets below the negative control threshold were considered to be negative. Wells with less than 10000 droplets were not considered in any analysis. After threshold determination and droplet quantification, the values (copies per μl of reaction) originated from Quantalife software ver. 1.7.4. (Bio-Rad) were back calculated to copies per gram of tissue (root) using the following formula: 39 !"#$ = ('()*+),-./ 1),(/ ∗ 3456'7 ∗ 8')/'4 Cpgr: target copies per gram of root Quantalife value: Copies per μl of reaction given by the Bio-Rad software (copies/ μL) RV: Volume of the reaction (μL) SQ: Sample DNA quantity used in the reaction (μL) EQ: Quantity of DNA extraction (uL) QR: Quantity of root tissue used for the extraction (g) 3.2.8 Statistical analyses 3.2.8.1 Vine survival A Chi-Square test was used to determine whether survival of vines differed among the inoculation treatments. To compare survival between treatments a series of Fisher Exact Tests were used. Both the Chi-Square and Fisher Exact tests were performed in R (v3.5.1 R Core Development Team). 3.2.8.2 Vine growth Differences among established vine growth over the two growing seasons were detected using the lmer function from the lme4 package (Bates et al. 2015) in R (v3.5.1, R Core Development Team). A linear mixed model (Formula: Response ~ Treatment + (Treatment| Year)) was fit by the Restriction Maximum Likelihood method followed by a Type III Analysis of Variance using Satterthwaite’s method. This was done separately for each of the vine response variables (total cane length, internode length and internode width). 40 3.3 Results 3.3.1 Vine survival Grapevines treated with different AM fungal inoculants varied greatly in their mortality rate (χ2= 17.8, df = 4, p = 0.001) (Figure 3.1). The vines pre-treated with the commercial inoculum (CP) had 80% survival by year 2, compared to no survival of the non-treated vines (p = 0.003), and 10% (p = 0.02) survival of those pre-treated with root inoculum 10% (p = 0.02). No differences in survival were detected among vines inoculated with CP, locally sourced spores (SP, 40%), and commercial inoculant applied in the field (CF, 30%) (Figure 3.1, Table A2). 3.3.2 Vine performance For surviving vines, there were no differences among inoculation treatments for any growth characteristic measured (Average Internode length = 3.7 cm, Average Internode Width = 4.7 mm, Total Cane Length = 151 cm). (Table A3). 3.3.3 Commercial inoculant presence and persistence At the start of the trial the commercial inoculum was not found within the vineyard soils (Table A4). Two years after transplanting , the commercial inoculant could be detected in only one of 12 vines treated. 41 Fig. 3.2 Surviving vine count in 2016 and 2017 in response to inoculation treatments: no-mycorrhizal treatment control (NM), locally sourced grapevine roots pre-inoculated (RP), locally sourced spores pre-inoculated (SP), commercial inoculant pre-inoculated (CP) and commercial inoculant field-inoculated. (CF). Letters indicate significant differences based on Fisher Exact Tests, which was statistically similar for both years of data. 3.4 Discussion 3.4.1 Vine survival As predicted, there was a clear benefit from inoculating vines with a commercial inoculant 6 wk before transplanting into poor soil conditions, when compared with applying no inoculant or the locally sourced root inoculant. Less clear were the benefits of the pre-transplant inoculation with locally sourced AM spores, or of applying the commercial inoculant at transplanting, which both 012345678910 b b ab a ab 2016 2017 42 resulted in reduced vine survival compared to the commercial pre-inoculated vines. Previous studies have shown that infection with AM fungi increases vine health and growth (Aguin et al. 2004, Camprubi et al. 2008), but they did not test the effects of inoculation timing relative to field transplanting. Instead, most AM studies have focused on the effects of field inoculations, or inoculations of greenhouse grown plants under otherwise near-sterile conditions. The only report on AM inoculant effects on grapevine field establishment as an indicator of vine performance is that by Thomsen (2018). In that study, similar commercial treatments (applied in either greenhouse or field) found no significant difference in survivorship. It is interesting to note however, that the vineyard conditions in that study were less stressful. Compared to the study by Thomsen (2018), the soils in this experiment are significantly more stressful, illustrated by the experimental block having less available nutrients, and organic material and a high pH compared to the adjacent established vineyard block (Table A1). Further, in my study extremely hot and dry weather during transplanting created stressful conditions for the newly planted vines. This stress may have contributed to the death of many vines, and possibly accentuated differences in survivorship among treatments. This study provides evidence that inoculating vines prior to transplanting into a field having poor soil and stressful climate conditions, a common challenge for commercial producers, can help increase vine survivorship Exactly how inoculation improved survivorship is not clear. Mycorrhizal vines have the advantage of extraradical hyphae which act as extensions of the 43 roots and grow quickly into regions where water is present (Augé et al. 2001). The hypha can alter soil properties such as increasing soil water retention (Augé et al 2001) and hydraulic conductivity, allowing plants to access more water (Bitterlich et al. 2018). In addition, AM fungi have been shown to improve the water use efficiency of plants, allowing them to maintain higher levels of leaf water potential, transpiration and photosynthesis when under water stress (Wu and Xia 2006, Nicolas et al 2015). These mechanisms may have contributed to the better survival of the pre-inoculated vines upon their sudden exposure to stressful outdoor conditions particularly high temperatures, low humidity and low soil water availability. The type of inoculant applied was also important to establishment success. Regardless of application timing, survival of vines exposed to the commercial inoculant was higher or similar to that of locally sourced inoculants. Previous studies have been inconsistent on whether commercial or local sources are best suited for grapevine applications (Linderman and Davis 2001, Camprubi et al. 2008, Pellegrino et al 2011, Holland et al. 2018b), with results often depending on the experimental growing system. Hart and Reader (2002) found evidence that isolates from three different AM families had different colonization strategies, with the Glomaceae (containing the isolate used in the commercial inoculant), colonizing roots rapidly and extensively. In respect to this, the locally sourced inoculants may have needed more time to colonize vine roots to the same degree as the commercial product. It would be interesting to see whether a longer colonization period for the root and spore treatments would allow for 44 greater colonization and a detectable mycorrhizal effect, as found in a previous greenhouse experiment using similar treatments (Holland et al. 2018b). While my study indicates that the commercial inoculant is more suitable for both greenhouse and field applications where transplant survival is a concern, the extent of colonization rather than its presence/absence, as used in this study, should be taken into account when comparing suitable mycorrhizal inoculum. 3.4.2 Vine performance I failed to detect treatment differences in growth of the surviving vines in this study (Table A3). While field studies have shown evidence of inoculation providing plant growth benefits (Camprubi et al 2008, Nogales et al 2009), there is also evidence for no growth differences (Klironomos 2003, Corkidi et al 2004), and even depressed growth under adequate soil phosphorus levels (Koide 1985, Graham and Eissenstat 1998). Because such a broad range of outcomes exist for mycorrhizal symbiosis, the lack of growth differences is not unprecedented. However, why the treatments influenced survival but not growth may provide an insight on the duration of the effect. AM infection by the commercial inoculant failed to persist beyond 2 years after the vines were transplanted and exposed to vineyard conditions. Once eliminated, the commercial AM would have little or no influence on plant growth. It may be that commercial inoculants are valuable only during establishment. 45 3.4.3 Commercial AM fungal establishment The lack of persistence of the commercial AM infection was consistent for 11 of 12 plants inoculated. Verbruggen et al. (2012), discussed the factors that could limit the success of inoculum persisting over time, which include species compatibility, priority effects and the lack of niche that may exist due to resident populations of AM fungi. Because the inoculum successfully colonized the vines, it is not likely that there are compatibility issues between the vines and the commercial inoculant. However, lack of niche and priority effects may both be at play in this experiment. If the AM fungi resident to the vineyard soils had a competitive advantage such as adaptation to the poor soil conditions resulting from topsoil removal, they would satisfy both occupation of the niche and having the priority effect advantage. In that case, they could have outcompeted the exotic (commercial) fungi for photosynthetic carbon (Kiers et al. 2011), displacing them within roots. In this case, this result could be considered beneficial as an exotic mycorrhizal species is not establishing within the vineyard. Regardless of the mode and speed of inoculum displacement, the infection of these vines by the commercial inoculum during establishment was still advantageous as previously discussed. 46 4 Timing of AM fungal inoculation influences the abundance of a common grapevine rootstock pathogen Ilyonectria. 4.1 Background Black foot disease (BFD) is a grapevine trunk disease contributing to poor growth or death of primarily young grapevines. Black foot is caused by several soil-borne fungal species belonging to the genera Campylocarpon (Halleen, Schroes & Crous), “Cylindrocarpon” (Wollenw) , Cylindrocladiella (Boesew) and Ilyonectria (Chaverri & Salgado) (Úrbez-Torres et al. 2014, Agusti-Brisach and Armengol 2013). Black foot impacts young grapevines during field establishment by infecting root vascular tissues at the basal end of the rootstock or scion in self-rooted vines and restricting the movement of water and nutrients (Halleen et al. 2006, Úrbez-Torres et al. 2014, Agusti-Brisach and Armengol 2013). These BFD causal agents can also be found dormant within agricultural soils. Because of this, young vines transplants are likely to encounter these pathogens in the vineyard. Additionally, materials coming from nursery can be contaminated. The severity and spread of BFD is enhanced by environmental stress, and while management practices can improve the performance of diseased grapevines, there are no known practices that can fully eliminate BFD (Gramaje and Armengol 2011). Arbuscular mycorrhizal (AM) fungi (phylum Glomeromycota (Walker and Schüssler)) are obligate root symbionts found in the majority of plant species, including grapevine rootstocks (Linderman and Davis 2001, Smith and Read 47 2008). These fungi enhance plant nutrient uptake, water use efficiency and overall growth (Smith and Read 2008, Trouvelot et al. 2015). They are also known to provide protection against many grapevine fungal pathogens in the genera, Armillaria (Fr. Vahl) (Nogales et al. 2009), Fusarium (Link), Phytophthora (de Bary), Pythium (Pringsh), Rhizoctonia (DC), Sclerotium (Tode), and Verticillium (Nees) (Azcón-Aguilar and Barea, 1996). In previous studies, AM fungi were shown to suppress the effects of BFD (Petit and Gubler 2006) and white root rot (Nogales et al. 2009) in grapevine rootstocks. However, an additional factor that could influence this interaction, the timing of AM fungal inoculation, has not yet been explored and could have important consequences on the bio-protection mechanism of AM fungi. There are several proposed mechanisms for how AM fungi may provide pathogen protection. There is evidence that when AM fungi colonize roots, infection by other fungi is limited (Cordier et al. 1996). Thus, colonization by AM fungi within vine roots may occlude pathogen infection via competition for space (Dehne 1982, Azcón-Aguilar and Barea, 1996; Cordier et al. 1996). Another mechanism is via activating systemic host defense responses. AM fungi may suppress pathogen infection and symptoms throughout a plant by stimulating host immune response (Azcón-Aguilar and Barea 1996), otherwise known as induced systemic resistance. In this mechanism, AM fungal colonization results in an array of defense compounds produced by the plant’s natural defense pathways (Pozo and Azcón-Aguilar 2007, Azcón-Aguilar and Barea 1996) but also early-defense mechanisms including stomatal closure, reactive oxygen 48 species accumulation and cell wall reinforcement (Cameron et al., 2013). Through these means it is possible that inoculating young grapevines with AM fungi could reduce BFD in newly planted vineyards. Regardless of the mechanism, the ability of AM fungi to mediate pathogen susceptibility may be determined by the timing of infection by AM fungi. For example, if AM fungi inhibit pathogen establishment via competition for root space (Cordier et al. 1996), then it would be advantageous for AM fungi to be introduced to grapevine rootstocks in advance of pathogen exposure, such as at the nursery. Similarly, if AM fungal disease suppression is via induced systemic resistance, then exposure to AM fungi before pathogens would be necessary to active these defences. In a greenhouse study by Petit and Gubler (2006) it was found that pre-inoculating grapevines could reduce BFD symptoms in V. rupestris cv. St. George rootstocks. However, in practice, commercial AM fungal inoculum is typically applied in the field at the time of planting as per manufacturers’ instructions. This approach is likely suboptimal for preventing pathogen infection and suppressing disease progression. To improve grapevine rootstock resistance to fungal pathogens it may be more effective to introduce AM fungi early (Nogales et al. 2009, Petit and Gubler 2006), shortly after rooting in the greenhouse or nursery, prior to transplanting into the field. In this study I asked Will AM fungal inoculation reduce fungal infection of grapevine rootstocks and does the timing of AM fungal addition determine the strength of this effect? I predicted that grapevine rootstocks 49 pre-colonized with AM fungi will have reduced BFD infection and symptom severity compared to rootstocks inoculated at the time of transplant. 4.2 Materials and methods 4.2.1 Experimental design The experiment was conducted in a greenhouse at the University British Columbia Okanagan Campus in Kelowna, BC. Potted Riparia gloire rootstocks were inoculated with an AM fungus and or the BFD causal agents Ilyonectria liriodendra (PARC60, PARC72, PARC340 and PARC393), previously isolated from BFD symptomatic vines in the Okanagan Valley, BC (Úrbez-Torres et al. 2014). These were applied either alone, or with AM applied as a pre-inoculant or a co-inoculant with the pathogens (Table 4.1). The AM inoculant was a common commercial product (AGTIV® wettable powder for specialty crops, 12,000 spores per g) containing Rhizophagus irregularis. Each treatment was applied to eight replicates of rootstocks and treatments were arranged in a completely randomized design on a greenhouse bench. 4.2.2 Rootstock preparation Dormant Riparia gloire rootstock canes were collected from a 3 year-old field planting at the Summerland Research and Development Centre (Summerland, BC, Canada) in early April, 2017. The canes were soaked in 10% bleach for 30 s before they were sectioned into 2-bud cuttings, which were dipped in Stimroot No. 2 (Plant-Prod Inc.) and planted to an approximate depth of 15 cm in a rooting box containing damp perlite. The root zone was kept at 21 °C 50 to stimulate root development, while the buds were cooled at 4 °C to slow bud break. After 4 wk, root development was apparent, and the rooted cuttings received inoculation treatments in May (Table 4.1, and below) before transplanting into 1 L cardboard sleeves filled with a peat-based growing medium (Sunshine Mix #1, AGTIV® wettable powder for specialty crops). Table 4.1. Inoculation treatments applied to Riparia gloire grapevine rootstocks. The fungal inoculants consisted of an AM fungus R. irregularis, and black foot pathogens Ilyonectria macrodidyma and I. liriodendri. Treatment May, 2018 Aug, 2018 Description Ctrl/Ctrl No addition No addition Non-microbial control, receiving neither AM fungi nor Ilyonectria Ctrl/Ily No addition Pathogen Pathogen positive control, receiving only Ilyonectria in August AMF/Ctrl AM fungi No addition Mycorrhizal pre-inoculation control, receiving AM fungi at the first time point, but no pathogen AMF/Ily AM fungi Pathogen Pre-inoculated pathogen treatment, first receiving AM fungi in May, then exposed to Ilyonectria in August. Ctrl/ AMF + Ily No addition Pathogen + AM fungi Co-inoculation treatment, applying both AM fungi and Ilyonectria concurrently in August. 4.2.3 Pathogen isolate preparation Six cultures of Ilyonectria liriodendra were obtained from the Plant Pathology fungal collection at the Summerland Research and Development Centre, Summerland BC and activated on 4 % (w/w) potato dextrose agar (PDA) 51 on April 3, 2018. After 23 days (April 26), four isolates were chosen that had signs of sporulation, which was based on visual microscope confirmation of conidia on cultures. These were I. liriodendri isolates PARC60, PARC72 PARC340 and PARC393 (Úrbez-Torres et al. 2014). Viable cultures were transferred to new plates of 4 % (w/w) PDA containing ampicillin and maintained until the time of pathogen addition (16 weeks). At this point conidia plus mycelia were scraped off the plates using a 1% tween solution, with the concentration of conidia measured using a hemocytometer. All isolates were diluted to a concentration of 1.5x106 conidia per mL so that when combined, the total concentration of pathogen was 6x106 conidia per mL. 4.2.4 Application of treatments, and plant culture The AM pre-inoculation treatments AMF/Ctrl and AMF/Ily (Table 4.1) consisted by dipping the grapevine roots in a slurry of commercial AM fungi (12.5 g of inoculum in 25 mL RO water) before planting. All other grapevines were dipped in RO water prior to planting. The ambient environment in the greenhouse was set at 26 °C, with 16 hours of light and 8 hours of darkness. Watering was every Monday, Wednesday and Friday to soil saturation and visible runoff. Plants were provided 5 g of low phosphorus of Miracle-gro fertilizer (24-8-16) every two weeks, which was spread over the soil surface and watered in via hand irrigation. In mid-Aug 2018, 2 months after planting, roots from each plant were stained using the protocol from Holland et al. (2014) to visually determine AM infection. Briefly, 52 root fragments, one cm in length were rinsed in RO water, soaked in 10 % KOH at 80 °C for 2 hours, 1 hour in a 1:1 (v/v) mixture of 3 % H2O2 and 10 % KOH, and then stained overnight in 5 % ink in vinegar. Roots were then de-stained in RO water for 24 hours and visualized under 200x magnification for the presence of mycorrhizal hypha, vesicles or arbuscules. After detection of AM fungi colonization, the root systems were pruned to 15 cm of length to mimic the common grower practice root trimming at transplanting, and to increase the probability of pathogen infection. The root systems were submerged in the Ilyonectria fungal suspensions overnight, approximately 12 hours, before planting into the peat based medium (Sunshine Mix #1). At this point the stems were pruned to four growing nodes to standardize new growth from the time of inoculation. 4.2.5 Harvest and measurements 4.2.5.1 Vine growth response At the time of plant harvest, 6 mo after the Ilyonectria treatment, leaf greenness was measured using a SPAD meter (SPAD 502 Plus, Konica Minolta), taking an average measure of 5 leaves which were selected evenly along the length of the vine shoots. Additional measurements were taken to determine total stem length, average internode length (total stem length divided by number of nodes), and internode width (averaged for three internodes between the fifth node from the base and fifth node from the growing tip). The base (approximately 4 cm) of each stem was removed and used for pathogen 53 isolation and stem necrosis quantification. To preserve these until use, the isolated stem sections were stored at 4 °C. The remaining stems and leaves were dried and weighed. Roots were removed from the pots, washed clean of soil using RO water, blotted using paper towels to remove surface water, and weighed. A 5 g sample was taken randomly from the root system and stored at 4 °C, to be used for DNA extractions and pathogen re-isolation. 4.2.5.2 Pathogen isolation and assessment of necrosis To determine the establishment of the pathogen, 10 1-cm root segments and 10 1-cm stem slices (from the plant base) were plated on 4% PDA containing 0.25 % (w/w) ampicillin. The root segments were submerged in bleach (10 % v/v) for 15 seconds, ethanol (10 % v/v) for 30 seconds and finally rinsed in RO water before being placed on the plates. The stem slices were dipped in ethanol and flamed to disinfect the outer surface before the outer bark was removed and slices of the inner tissue (<1mm thick and <5 mm in width) were sectioned from the cortex region, targeting darkened areas that showed signs of necrosis. These were plated evenly around the PDA plates. Both root and stem plates were grown for up to 6 weeks with regular monitoring for the growth of colonies, which were used to confirm the presence of Ilyonectria. To determine the amount of necrosis within the stem cortex, a cross-section was taken from the bottom section of the stem, where roots emerged, and necrosis is commonly most evident. A one-centimeter cross section was cut 54 and brushed with mineral oil and scanned using an Epson Expression 11000XL scanner. Images were imported into ImageJ (Rasband 1997) to measure the relative proportion of the cross section with necrotic staining. To do this, first the necrotic region (indicated by darkening and browning of the tissue) was outlined and measured using the ‘area’ tool. The same was done for the entire cortex, excluding the pith. The necrotic region was then divided by cortex area to provide percent of necrosis. 4.2.5.3 Digital droplet PCR quantification To perform molecular quantification of both the AM fungal and Ilyonectria isolates, DNA was first extracted from a subsample of 0.1 g of roots per plant, cut into <1 cm fragments., according to manufacturer’s protocol (MPBio FastDNA ™ SPIN kit (MP Biomedicals LLC). This was followed by digital droplet (dd)PCR, which allows for quantification of the number of copy numbers within a sample. 4.2.5.3.1 AM fungi To quantify AM fungi, I targeted a segment of the mtDNA specific to the commercial isolate using a probe-based reaction in ddPCR (197198F 5’- AGCAAATCTAAGTTCCTCAGAG -3’ (Providencia et al., 2013); Reverse 5’- ACTTCTATGGCTTTGTACAGG - 3'; and probe 5’- FAM/ CCCACCAGG/ ZEN/ GCAGATTAATCTTCCTT /3IABKFQ -3’ (Kokkoris et al. in press). In short, each ddPCR reaction contained: 10 μL of 1X ddPCR SensiMix (supermix for probes by Bio-Rad Inc), 1 μL of primer-probe mix (Integrated DNA Technologies) 20X (500 nM primers and 250 nM probe in final reaction), and 2 μL of undiluted root DNA 55 and 7 μL of DNase free water, for a total volume of 20 μL. The total volume was used to generate droplets, along with 70 μL of Bio-Rad Droplet Generator Oil for Probes, using the Bio-Rad QX100 Droplet Generator producing 40 μL of droplets. Thermal cycling conditions were 95°C for 10 min, followed by 40 cycles of 94°C for 30 sec and 59°C for 30 sec, followed by a single step at 98°C for 10 min; the ramp rate was increased by 2°C/sec. Droplet analysis was carried out on the BioRad QX100 and raw data was collected using Quantalife software (Version 1.7.4.0917, Bio-Rad Laboratories Inc.). For each run, three non-template controls (NTC) were included, containing no DNA and three environmental controls which were collected from an area that would contain no commercial inoculum. The former to eliminate the possibility of contamination and the latter to determine the fluorescence amplitude threshold. 4.2.5.3.2 Ilyonectria spp. A new primer pair and probe was developed to quantify Ilyonectria, targeting a portion of the beta-tubulin region; (5’ – CGAGGGACATACTTGTTTCCAGAG – 3’ Tm 61, GC 60%,), and (Reverse 5’ – TCAACGAGGTACGCGAAATC – 3’ Tm 62, GC 50%). In addition, a probe (5’ –TGTCAAACTCACACCACGTAGGCC – 3’ Tm 68, GC 52%) was included to increase specificity to Ilyonectria. To check for specificity to Ilyonectria I performed two tests. First the primer-probe set was used with each of the four Ilyonectria isolates (PARC60, PARC72, PARC340 and PARC393), along with other fungal DNA to ensure amplification (Appendix Figure B1). The Ilyonectria 56 ddPCR probe reaction was performed using the same method as the AM fungal reaction previously described, except for the following differences: the reaction thermal cycling conditions were 95°C for 10 min, followed by 60 cycles of 95°C for 10 sec and 60°C for 10 sec, followed by a single extension step at 72°C for 30 sec. Second, I used the blast and nucleotide tools from NCBI to check the primer-probe specificity against the entire NCBI database. For this, a blast was initially performed with the two primers to obtain accession numbers. These were then cross-referenced with the nucleotide tool using the probe sequence to determine which species would amplify and provide match scores and e-values (Appendix Table B1). 4.2.6 Statistical analysis All statistical analyses were conducted as appropriate for a completely randomized design using R v2.8.1 (R Development Core Team, 2008). Differences in stem necrosis, ddPCR copy number and vine growth responses were determined among the treatments using a one-way analysis of variance (ANOVA). Differences among the treatment means were detected using a general linear hypothesis within the multcomp R-package (Hothorn et al. 2008). This was done with the multiple comparison function (mcp) defined for Tukey linear function testing, as follows: Tukey comparison = glht(aov, linfct = mcp(Treatment = "Tukey")). Multivariate analysis of variance (MANOVA) was used to further detect overall differences in vine growth responses, with root biomass, dry stem weight, 57 dry leaf weight, internode width, internode length and leaf greenness included in the analysis. A Pearson’s correlation in R v2.8.1 (R Development Core Team, 2008)) was used to detect any correlation between the AM fungal and Ilyonectria abundance. 4.3 Results 4.3.1 Plant response Inoculation by AM fungi and Ilyonectria isolates did not affect plant response. This was consistent both when looking at individual traits using an ANOVA (Table 4.2) or when looking at total overall vine response using MANOVA. 4.3.2 Molecular quantification 4.3.2.1 AM fungi The presence of AM fungi was visually confirmed in all three treatments which were inoculated. However, the quantities differed among the treatments: Rootstocks that received both AM fungi and Ilyonectria at the same time (Ctrl/AMF+Ily) had a higher AM fungal copy number (F5,41 = 5.135, p < 0.001) compared to both rootstocks not inoculated with AM fungi (Ctrl/Ctrl and Ctrl/Ily) and those pre-inoculated with AM fungi but not inoculated with Ilyonectria (AMF/Ctrl) (Table 4.2, Figure 4.1). 58 Table 4.2 Means for plant growth chracteristics and abundance of fungal isolates, six months after inoculations with AM fungi and Ilyonectria isolates. F-values and P-values are the result from ANOVA for inoculation treatment effects (n= 40). Copy numbers of mtDNA (AM fungi) and beta tubulin (Ilyonectria) were obtained using molecular-based quantitative digital droplet PCR. * Significant at P = 0.05. Measure F-value P Average Dry Stem Mass 1.24 0.308 11.6 g Dry Leaf Mass 0.651 0.662 12.2 g Fresh Root Mass 1.53 0.201 333.5 g Internode Width 1.52 0.206 223 mm Internode Length 0.188 0.965 2.3 mm Leaf Greenness 0.594 0.705 23.6 spad units % Stem Necrosis 0.577 0.717 25 % Ilyonectria copy # 5.59 <0.001* See Fig 2. AM fungal copy # 5.15 <0.001* See Fig 1. Fig. 4.1 Abundance of AM fungi within vine roots (ddPCR, copies mtDNA/μL) after harvest (6 mo after the second inoculation). Treatments: AMF/Ctrl, mycorrhizal control inoculated only with AM fungi; AMF/Ily, pre-inoculated with AM fungi followed by Ilyonectria inoculation; Ctrl/AMF+Ily, co-inoculated with AM fungi and Ilyonectria; Ctrl/Ctrl, non-microbial control; Ctrl/Ily, pathogen control inoculated with only Ilyonectria. Differences determined using one-way ANOVA (F5,41 = 5.135, p < 0.001), with differences at p < 0.05 indicated by letters above bars (Tukey’s multiple comparison via general linear hypothesis). AMF/Ctrl AMF/Ily Ctrl/AMF+Ily Ctrl/Ctrl Ctrl/Ily0e+001e+072e+073e+07TreatmentAM fungal mtLSU (copies/g) b ab a b b 59 4.3.3 Necrosis There were no differences among the treatments in percent stem necrosis (F5,41 = 0.577, p = 0.717). Visually, the root systems appeared healthy with no signs of necrotic development. 4.3.3.1 Ilyonectria spp. Ilyonectria was isolated from all rootstocks inoculated with the pathogens, but also from 4 of 24 rootstocks not inoculated with Ilyonectria. Rootstocks inoculated with Ilyonectria and AM fungi at the same time had higher number of copies of Ilyonectria beta-tubulin genes ((F5,41 = 5.586, p < 0.001), Table 4.2, Figure 4.2). 60 Fig. 4.2 Ilyonectria beta-tubulin copy number per μL (ddPCR) in vine roots. Treatments: Ctrl/Ctrl, non-microbial control; Ctrl/Ily, pathogen control inoculated with only Ilyonectria; AMF/Ctrl, mycorrhizal control, inoculated with only AM fungi; AMF/Ily, pre-inoculated with AM fungi followed later by Ilyonectria inoculation; Ctrl/AMF+Ily, co-inoculation with AM fungi and Ilyonectria. Values were obtained by digital droplet polymerase chain reaction and differences determined using a one-way analysis of variance (ANOVA; F5,41 = 5.586, p < 0.001), with differences at p < 0.05 indicated by letters above bars (Tukey’s multiple comparison via general linear hypothesis). 4.4 Discussion Contrary to my prediction, AM fungal inoculation did not decrease Ilyonectria abundance or disease, instead leading to increased abundance of the fungal pathogen within grapevine rootstock. This has not been previously reported, with the generally reported trend being that AM fungal pre-inoculation decreases the presence and expression of plant diseases. AMF/Ctrl AMF/Ily Ctrl/AMF+Ily Ctrl/Ctrl Ctrl/Ily050000150000250000350000TreatmentIlyonectria beta-tub (copies/g)b ab a b b 61 4.4.1 Vine growth Surprisingly, neither the Ilyonectria nor AM fungi affected vine growth during the experiment. While AM fungi are generally thought to increase vine physiological performance (Linderman and Davis 2001, Camprubi et al 2008, Nogales et al 2009, Belew et al. 2010, Nicolás et al. 2015, Trouvelot et al 2015), this is not always the case. Holland et al. (2018) using a similar commercial product found no effects on growth in both greenhouse and field studies. There are many situations where AM fungal inoculation has had no influence on plant growth, (Klironomos 2003, Corkidi et al 2004, Thomsen 2018,), or even growth depression (Koide 1985, Graham and Eissenstat 1998). Mycorrhizal benefits are more commonly realized when plants are grown under stressful conditions, such as poor soil field conditions (Holland et al. 2018). 4.4.2 Pathogen isolation and necrosis Surprisingly, inoculation with the Ilyonectria pathogens did not influence vine growth. While the abundance of beta tubulin genes (Figure 4.2) indicates the pathogen was present, I observed no expression of disease symptoms. This lack of disease symptoms is contrary to other studies that applied similar Ilyonectria treatments to young grapevines, which regularly resulted in leaf chlorosis and growth reductions. (Petit and Gubler 2005, 2006, Úrbez-Torres et al. 2014). That my pathogen control did not have higher levels of pathogen and necrosis was surprising, as even rootstocks that had higher levels of Ilyonectria did not differ in their levels of necrotic streaking or show foliar symptoms. This 62 indicates that infection was successful, but that environmental conditions, such as stress factors, may not have been significant enough to influence vine growth. Ilyonectria is a weak pathogen that can live asymptomatically within grapevines without causing visual signs of disease until stress occurs (Halleen et al. 2006, Agusti-Brisach and Armengol 2013, Úrbez-Torres et al. 2014). For example, when grapevines are first transplanted into a vineyard they will be subjected to a new soil environment that could include stressors that trigger the Ilyonectria to become pathogenic. In particular, it is understood that BFD has a greater effect on grapevines exposed to high soil compactness, poor drainage or low water availability (Halleen et al. 2006, Agusti-Brisach and Armengol 2013, Úrbez-Torres et al. 2014). Lack of stressors could have been a factor in my experiment. In this study I kept the medium well-watered to mimic poor drainage which is known to enhance Ilyonectria proliferation. However, due to the mode of disease (vascular tissue occlusion) it is possible that once the disease had proliferated within stem tissues, limiting the available moisture would have caused more pronounced disease expression, as upper plant tissues would have suffered water stress. Further, the rootstock Riparia gloire is well adapted to moist soils found alongside rivers and streams (Pongracz 1983). It would have been better to test this hypothesis with a rootstock that is more susceptible to diseases and maladapted to saturated soils. 63 4.4.3 Ilyonectria and AM fungal quantification Contrary to my hypothesis, Ilyonectria abundance within grapevine rootstocks did not decrease with the addition of AM fungi. There are numerous studies that provide evidence that AM fungi causes the activation of both local and systemic defenses, leading to a reduction in pathogen levels (Azcón-Aguilar and Barea, 1996, Pozo et al. 2002, Pozo and Azcón-Aguilar 2007). For instance, in similar greenhouse experiments challenging annual plants with cultured pathogens, AM fungi have been found to reduce fusarium solani f. sp haseoli in bean (Filion et al. 2003) and Aphanomyces euteic in pea (Kjoller and Rosendahl 1996). Similarly in perennial systems, mycorrhizal infections in papaya (cv. Surya) (Sukhada et al. 2011) and banana (cv. Neypoovan) (Mohandas et al. 2010) led to lower levels of Phytopthora parasitica var. nicotianae and Fusarium oxysporum f. sp cubense, respectively. These studies found a reduction in pathogen abundance and also observed suppressed disease symptoms in mycorrhizal plants, neither of which occurred in the present study. As previously discussed, it is possible that various parameters associated with my experiment did not facilitate the proliferation of Ilyonecrtria or the associated disease symptoms. For instance, a longer duration and more stressful growing conditions may be required for the pathogen to further proliferate. That I did not observe this reduction is both unique and important as it signifies that mycorrhizal effects can be variable and context dependent. While AM fungi generally provide disease suppression, treatments that received AM fungi in this study tended to contain more Ilyonectria. A synergistic 64 effect could have occurred between the two entities, as has been described when multiple pathogens are introduced into plants (Lamichhane and Venturi 2015; Abdullah et al. 2017). While AM fungi are not pathogenic, they could co-exist or even provide indirect benefits to a weak pathogenic endophyte such as Ilyonectria. For instance, AM fungi could allow Ilyonectria to further proliferate as a result of the increased carbon sink (Staddon et al. 1999, Wright et al. 1998) that the beneficial AM fungi often induce. It has been shown that AM fungi can create a carbon sink within roots, causing mycorrhizal roots to contain more sugar than non-mycorrhizal counterparts (Staddon et al. 1999, Wright et al. 1998). This has been seen in multi-species AM fungal systems, where low-quality AM fungi persist within roots, but only due to the benefits provided by another beneficial AM fungal counterpart (Hart et al. 2013). Similar effects could occur in AM fungal – pathogen systems; as fungi depend on exogenous carbon for growth, increased carbon caused by beneficial AM fungi could subsequently lead to rootzones supporting higher fungal loads as evidenced with increased exudate levels (Broeckling et al. 2008). If this were the case, the benefit that AM fungi may be providing could be negated by also allowing higher levels of pathogenic fungi to persist. It was interesting that rootstocks only inoculated with AM fungi (AMF/Ctrl treatment) had very low, if not negligible levels of AM fungi in roots, compared to rootstocks that received both AM fungi and Ilyonectria. The low levels of AM fungi in response to AMF/Ctrl may have been due to the lack of stress. In the other treatments that included the pathogen (Ctrl/AMF+Ily and AMF/Ily) this 65 stress was likely provided by Ilyonectria. While the presence of Ilyonectria did not result in visual stress, it still could have caused limited water and nutrient status for which the AM fungi may have still have provided protective benefits. This could result in carbon flow to the AM fungi, mediating fungal proliferation in the root system. In contrast, grapevines without Ilyonectria would have less need for the AM fungi symbionts and therefore the AM fungi would not benefit the grapevines, limiting their establishment in vine roots. This effect may be similar to the influence of phosphorus found in experimental systems in which plants are exposed to adequate levels of phosphorus have lower AM fungal colonization levels (Graham et al. 1997, Treseder 2004, Gosling et al. 2006, Schreiner 2007). 66 5 The impact of root pruning transplant practices on the arbuscular mycorrhizal symbiosis within grapevine rootstocks. 5.1 Background Grapes are an important global crop grown for both wine and fruit production. Perhaps the most vulnerable part of the growing cycle is transplant from nursery to vineyard. When plants are transitioned from the nursery to the field they are exposed to unfamiliar abiotic conditions and soil pathogens, often acting as stressful factors affecting vine growth (Bavaresco and Poni 2003, Nogales et al. 2009, Belew et al. 2010, Urbez-Torres et al. 2014). An important research goal is to find sustainable methods to improve vine establishment during transplant into the field. One cultural method used to improve vine establishment during transplant is trimming roots. This practice reduces the incidence of roots growing upward because of limited space in the planting hole, resulting in a “J-rooting” pattern. J-rooting can lead to root death and has been associated with incidence of root and trunk disease (Urbez-Torres et al. 2014). To avoid J-rooting, roots are aggressively pruned before planting. Such trimming practices are associated with increased proliferation and growth rate of roots (Geisler and Ferree 1984). Although pruning reduces the incidence and severity of J-rooting, it is not without drawbacks. Pruning wounds root tissues, making them vulnerable to infection (Gramaje and Armengol 2011, Agusti-Brisach and Armengol 2013). Additionally, trimming reduces the vigor of young vines, likely due to a smaller 67 root system and subsequent reduced water uptake and transpiration (Poni et al. 1992, Ferree et al. 1999). One consequence of root pruning rarely considered is the impact it has on beneficial root endophytes, such as arbuscular mycorrhizal (AM) fungi. AM fungi are obligate root symbionts that associate with the roots of most plants (Brundrett 2009). They grow from root cortical cells into the surrounding soil, greatly increasing resource access and uptake by plants (Smith and Read 2008). Soil resources are exchanged for plant derived carbon within specialized fungal structures called arbuscules, which are highly branched hypha enveloped by a root cell. The AM hyphal network significantly increases the uptake of resources and improves stress tolerance. For instance, AM fungi help maintain vine growth in saline soils (Belew et al. 2010), reduce the impact of pathogens (Petit and Gubler 2006) and improve plant water status and water use efficiency (Nicolás et al. 2015). How root pruning affects the mycorrhizal symbiosis is not known. It is likely that the distribution of AM fungal structures through the root system will influence the impact of root pruning on the symbiosis. For instance, arbuscules are often found in young absorptive roots near the growing root tips, whereas vesicles and spores are more abundant in older roots (Barker et al. 1998, Resendes et al. 2008, Vukicevich et al. 2019). Depending on the severity of root pruning the entire mycelium may be eliminated. Alternatively, trimming only the young growing roots may remove the hyphae in the root tips, leaving only fungal storage structures (i.e. vesicles) within the older root system – perhaps serving 68 as an inoculum source for new roots. If the mycorrhiza cannot re-establish, the young grapevines will be at a significant disadvantage due to the reduced root area and the loss of symbiotic benefits. Thus trimming, while useful to prevent J-rooting, may inhibit a functional AM symbiosis that could be critical for the survival of the grapevine during the transition to nursery to field. The goal of this experiment was to determine how root trimming practices affect the mycorrhizal symbiosis. I asked, Do standard transplant root trimming practices affect the mycorrhizal symbiosis? I hypothesized that root trimming would either reduce or eliminate the active mycorrhiza from grapevine roots. 5.2 Materials and methods 5.2.1 Rootstock preparation and treatment Rootstock canes were collected from the rootstock collection block at the Summerland Research and Development Centre, Summerland BC, Canada in May of 2017. Twelve canes of similar diameter were collected for each of four rootstocks (5C, 3309C, 101-14Mgt, and Ramsey). The canes were soaked in 10% bleach for one minute and then rinsed with reverse osmosis treated water. The basal end was cut within 5 mm below a node leaving three or four viable buds above the cut. The basal ends of the canes were soaked in liquid indole-3-butyric acid (5000 ppm) for four minutes and then allowed to dry before planting in a rooting box filled with damp perlite. The rooting box was placed in a cooler 69 at 4 °C to delay bud break of apical buds, while a heating pad underneath maintained a rootzone temperature of 22 °C to promote rooting. Once roots had established, the rooted canes were transplanted into 4 L pots, in a 1:2 mixture of Turface : Sand. Each vine was inoculated with 15 mL of AgTiv® Specialty Crops which was distributed over the roots, covering them prior to filling the pot with media. These were grown in the Summerland Research and Development Centre greenhouse for one year, provided supplemental lighting to ensure a 16-hour photoperiod, watered two or three times a week to saturation, and fertigated bi-weekly with a solution of 15 mL of Miracle-gro (24-8-16) dissolved in 6 L of water. After one year the plant roots were checked for mycorrhizal colonization using staining and microscopy, explained in the following section. Having confirmed colonization, the root trimming treatment was applied to half of the plants in June of 2018 at the University of British Columbia Okanagan campus, Kelowna BC, Canada. Before treatment, all the vines were removed from their pots and rinsed of planting medium. Six vines of each rootstock had the actively growing lower zone of the root mass removed leaving only <10 cm of root tissue and the remaining six vines were left untrimmed. All plants were repotted into 8 L pots with new sterile medium composed of sand. All vines were arranged in a completely randomized design on a greenhouse bench. The vines were grown for four months to allow new root growth. During this time plants were irrigated via drip emitters, providing 300 mL of water every 70 day. Plants were grown with supplemental lighting to ensure they received a 16-hour photoperiod. At the end of four months, aboveground biomass was harvested, weighed, dried in an oven (50 °C) for 48 hours, and re-weighed. Roots were removed from pots and washed free of any sand, and two 5-g samples were taken from the root mass and stored at 4 °C for visual analysis and molecular quantification. One root sample was taken from the actively growing roots within the bottom 5 cm of the roots. The other was taken from the older roots, no longer actively growing, within 5 cm of the stem base. The remaining roots were dried 50 °C in an oven for 48 hours and weighed. 5.2.2 Staining and visual quantification The sampled roots were stained and visually inspected for the abundance of arbuscular mycorrhizal structures. A staining protocol was used, modified from Vierheilig et al. (1988). Roots were soaked in 10 % KOH, at 90 °C, for 3-hours, followed by soaking overnight in room-temperature 10 % KOH. Roots were then rinsed with RO water and stained for 15 minutes in a 90 °C, 5 % ink : vinegar solution. Lastly the roots were de-stained for 20 minutes in 0.01 % vinegar : RO water. The roots were mounted in PVLG and baked at 60 °C until the media hardened, then viewed at 200x magnification to quantify abundance of mycorrhizal structures (vesicles, arbuscules and AM fungal presence/absence) using the method of McGonigle et al. (1990). 71 5.2.3 DNA extraction and molecular AM fungal quantification To perform molecular quantification of the AM fungal isolate, DNA was first extracted from the two 0.1-g subsamples of roots per plant (one from the upper root-system sample, one from the lower active root zone), according to manufacturer’s protocol (MPBio FastDNA ™ SPIN kit (MP Biomedicals LLC). This was followed by digital droplet (dd)PCR, which quantifies the number of a specific gene segment within a sample. To quantify AM fungi, I targeted a segment of the mitochondria (mtDNA) specific to the commercial isolate using a probe-based reaction (197198F 5’- AGCAAATCTAAGTTCCTCAGAG -3’ (Providencia et al., 2013); Reverse 5’- ACTTCTATGGCTTTGTACAGG - 3'; and probe 5’- FAM/ CCCACCAGG/ ZEN/ GCAGATTAATCTTCCTT /3IABKFQ -3’ (Kokkoris et al. 2019). Each ddPCR reaction contained: 10 μL of 1X ddPCR SensiMix (supermix for probes by Bio-Rad Inc), 1 μL of 20X primer-probe mix (Integrated DNA Technologies) (500 nM primers and 250 nM probe in final reaction), 2 μL of undiluted root DNA and 7 μL of DNase free water, for a total volume of 20 μL. The total reaction volume in addition with 70 μL of Bio-Rad Droplet Generator Oil for Probes was used to produce 40 μL of droplets, via the Bio-Rad QX100 Droplet Generator. Thermal cycling conditions were 95°C for 10 min, followed by 40 cycles of 94°C for 30 sec and 59°C for 30 sec, followed by a single step at 98°C for 10 min; the ramp rate was increased by 2 °C/sec. 72 Droplet analysis was carried out on the BioRad QX100 and raw data was collected using Quantalife software (Version 1.7.4.0917, Bio-Rad Laboratories Inc.). For each run, three non-template controls (NTC) were included containing no DNA and three negative controls which contained no commercial inoculum DNA. The former was used to eliminate the possibility of contamination and the latter to determine the fluorescence amplitude threshold. 5.2.4 Statistical Analysis A linear mixed model was used, to compare the abundance of mtDNA (ddPCR) and mycorrhizal structures. The lme4 package was used with the formula lmer(Response ~ Treatment * Region + (1 | Rootstock)), where Treatment and Region are main effects and Rootstock is a random factor. For plant responses (dry root mass and dry aboveground mass) a similar model was used with the formula lmer(Response ~ Treatment + (1 | Rootstock)), where Treatment is main effect and Rootstock is a random factor. Welch’s Two Sample t-test were used in some instances to compare groups after significant interactions were detected between treatments. 5.3 Results 5.3.1 Root trimming effects on AM fungi and rootstocks Trimming did not affect mtDNA concentration within root systems (Table 5.1) but an interaction was found between trimming and region of root system sampled (p = 0.035), indicating that trimming reduced AM fungal mtDNA abundance in the older root zone only (t = 2.97, p = 0.006) (Figure 5.1). The 73 abundance of mycorrhizal structures was not influenced by trimming (Table 5.1). Vines had lower aboveground biomass when roots were trimmed (p = 0.039), but root biomass was not affected by the trimming treatment (p = 0.176) (Table 5.1). There was no interaction between rootstock identity and root trimming in regard to AM fungal abundance or plant biomass. Table 5.1 Linear mixed-effect model results for different fungal and plant responses measured after vines were either trimmed or not, and from different areas of the root system. Bolding indicates significance at p=0.05. Effect Factor Trimming Treatment Upper vs. Lower Roots Interaction mtDNA Concentration 0.222 0.204 0.036 Arbuscules 0.765 0.004 0.979 Vesicles 0.302 <0.001 0.997 Total Colonization 0.629 0.004 0.721 Aboveground Mass 0.176 N/A N/A Root Mass 0.039 N/A N/A 74 Fig. 5.1 The quantity of AM fungal mitochondrial LSU gene copies in root systems that were trimmed (Trim) or not (Non-Trim), and from either the High and Low roots. An interaction was detected between trimming and root location (p = 0.033). The * indicates a difference in AM fungal mtDNA between the Non-Trimmed High and Trimmed High roots, detected using a Welch Two Sample t-test (t = 2.97, p = 0.006). 5.3.2 High versus low roots There was no difference in fungal mtDNA abundance between older and newer roots (p=0.204) (Table 5.1). There were, however, more arbuscules in the newer roots (p = 0.004) but vesicle abundance (p < 0.001) and total colonization (p = 0.004) was higher in the old roots (Figure 5.2) . AMF/Ctrl AMF/Ily Ctrl/AMF+Ily Ctrl/Ctrl Ctrl/Ily0e+001e+072e+073e+07TreatmentAM fungal mtLSU (copies/g) b ab a b b AM fungal mtDNA (copies/g) 75 Fig. 5.2 Total (light grey), Vesicle (dark grey) and Arbuscule (white) percent colonization of vine roots collected from the ‘High’ and ‘Low’ roots of 48 rootstocks. The quantity of all three structures differed between zones (total, p = 0.004; vesicles, p < 0.001; arbuscules, p = 0.004). 5.4 Discussion Contradictory to my hypothesis, root trimming did not reduce the abundance of AM fungi in roots. However, root trimming decreased mtDNA concentrations within the upper roots. 5.4.1 The impact of root trimming on the mycorrhizal symbiosis. I predicted that a trimmed root system would have less AM fungal abundance due to the removal of mycorrhizal resources. Instead, the AM fungi in this study tolerated the trimming treatment, with both new and old roots exhibiting High Low Root System 20406080100Structure and Region% Colonization 76 high levels of AM fungal mtDNA and colonization 15 weeks post trimming. In general, root damage has been found to reduce fungal abundance. For instance, disrupting roots via soil tillage has been shown to have significantly decreased the abundance and slowed the development of mycorrhizae (McGonigle and Miller 1993, Lekberg and Koide 2005, Bowles et al. 2017). Similarly, early root herbivory can decrease the ability of mycorrhiza to form (Bennett et al. 2003), but herbivory effects on AM fungi will vary on a per study basis (Johnson and Rasmann 2015). In my study, because only the youngest roots are removed by root trimming, it is likely that the mycelium in older roots served as inoculum for new root growth. Whether or not this resistance to disturbance is true for all AM fungi remains to be seen. Due to physiological differences among AM fungal species, such as propagule production (Hart and Reader 2002, Klironomos and Hart 2002), different AM fungi will vary in their response to disturbance such as root trimming or disruption (Kabir 2005). For instance, tillage in agricultural studies has led to shifts in AM fungal community composition, resulting in decreased diversity (Kabir 2005, Bowles et al. 2017). Species that grow rapidly or produce more vesicles and spores are likely to become the dominant species in these situations (Johnson and Pflegr 1992, Douds et al 1995, Jansa et al. 2003). Trimming represents a specialized form of disturbance compared to tillage. Because the mycelium is only retained within the roots – and not in the soil – only fungi capable of regrowth from root colonization may tolerate root trimming. Taxa that rely on spores or soil hyphae to initiate new infection units (ie. 77 Gigasporaceae and Scutellospora (Klironomos and Hart 2002)) may be unable to regrow following trimming. Similarly, the vines will be subjected to a resident fungal community in the field, unlike my system where they were trimmed and potted into sterile media. It may be that the residual mycelium in trimmed roots may not be competitive with resident fungi and may fail to persist in field conditions. This remains to be tested. Trimming lowered the abundance of AM fungi within the older roots. My measure of fungal abundance, mtDNA concentration, is also associated with metabolic activity. This difference in mtDNA, together with the lack of difference in colonization (arbuscules, vesicles and total), indicates less mycorrhizal activity within the older roots of trimmed root systems. Why does trimming the root system lower the mycorrhizal activity within the older roots? It could be that trimming roots causes a greater need for the regeneration of new roots and mycorrhizal symbiosis. Further, it has been shown that plants with a loss of root area may depend more upon the mycorrhizal symbiosis (Graham et al. 1997, Tawaraya 2003). If trimming causes increased root production and a greater dependency on the mycorrhizal symbiosis, more mycorrhizal development would likely be concentrated in newer roots, pulling resources and activity away from the older root system potentially resulting in the decreased mtDNA in the lower, younger roots. While this is speculative, it indicates that a better understanding is on how root disturbance, such as root damage, can influence both plant carbon and biomass allocation, and the subsequent effects on mycorrhizal development. 78 5.4.2 Distribution of AM fungal structures between old and new roots Older roots harbored more vesicles and overall colonization of AM functional structures than newer roots, which had more arbuscules. Similar results have been found previously (Rosewarne et al. 1997, Vukicevich et al. 2019). The lower roots are smaller, unbranched, and responsible for accessing and absorbing nutrients and water (Eissenstat et al. 2000). Arbuscules are short-lived structures with highest abundance in young roots, declining with age (Rosewarne et al. 1997, Barker et al. 1998). Therefore, in the present study it was expected that arbuscules would be more abundant in these lower roots, allowing for more nutrient exchange in this zone, and that arbuscular degradation would occur in older roots. The abundance of vesicles in the older roots suggests that they were primarily a source of carbon for the fungus, since these structures are important in providing resources to new mycorrhizal growth (Brundrett 1991, Klironomos and Hart 2002). It is likely that the vesicle and hypha contained within older roots served as a source of inoculum for the trimmed roots. 5.4.3 Root trimming and vine growth The reduction of vine shoot biomass due to root trimming has been seen in previous studies (Hipps et al. 1996, Dong et al. 2003). This response may be from a combination of nutrient limitations due to reduced root biomass, plus an allocation of plant resources to root growth (Geisler and Ferree 1984). This initial loss of aboveground biomass may reduce the first year’s growth after transplant, 79 but whether this has long term effects on vine establishment, such as years until fruit set, is unknown. That no difference in root biomass was observed between trimmed and untrimmed root systems provides evidence that root trimming increased the allocation of biomass towards root development and regeneration. This redistribution of biomass in response to root trimming has been previously observed (Poorter and Nagel 2000, Poorter et al. 2012). This increase in root production is important to growers as it indicates that the root system will regenerate after root pruning, while also being provided the benefits of avoiding J-rooting. While aboveground biomass production may be reduced, the allocation towards root may be more beneficial in the establishment of young vines, an area that needs to be tested further in the field. 80 6 Conclusions Throughout this thesis I aimed to elucidate factors and consequences of the symbiosis between AM fungi and grapevines, focusing on three common themes: 1) how the timing of inoculation impacts the symbiosis 2) whether the provenance of the fungi affects the efficacy of the symbiosis, and 3) how inoculant effects relate to environmental stresses. Further, throughout the course of conducting the experiments included in this thesis there were many instances where microbial treatments were unable to infect plants, which will be outlined on a per chapter basis. This both illustrates that soil microbes are not straightforward additions and often do not behave as expected, even in controlled conditions. While AM fungi exist throughout nature and agricultural soils, this thesis provides evidence that the use of mycorrhizal inoculants is justified in certain instances. In general, my results indicate that AM fungal inoculants can be beneficial to grapevines, but that these effects are not consistent. Rather, symbiotic effects depend on environmental factors, including exposure of the plant to stress, as well as the plant and fungal identity. For instance, the origin of AM fungi is emphasized, as commercial and natural inoculations infected grapevines to different severity, leading to different physiological effects on the plants. 81 6.1 Does the source of AM fungi influence the growth of different grapevine rootstocks? This study demonstrated that the growth response and colonization of grapevines by AM fungi can vary widely. This variation was based on whether the fungi were a commercial product or composed of a natural community, and it also depended on the grapevine rootstock identity. Using AM fungal inocula from different sources resulted in a range of colonization levels within grapevine roots - spanning full colonization to none. A commercial product and naturally isolated spores were unable to colonize roots, this may have been due to the media used, even though it is commonly used in mycorrhizal studies. However, this highlights that inoculum may not always be able to establish within host roots. The natural inoculum originating from the roots of woody perennial shrub-steppe plants resulted in moderate levels of root colonization. This root inoculant was more beneficial than commercial strains in the short term of this study, with growth responses also dependent upon rootstock identity. An important note for this chapter, along with the Chapter 3, is that although the focus was on AM fungi as the inoculant, the natural root inoculant also likely contained other organisms, such as nematodes, bacteria and fungi. This is discussed in Chapter 2 as the benefits of adding an entire microbial community potentially outweighing those of just inoculating with AM fungi. My results from Chapter 2 show that the commercial inoculum is more aggressive colonizer but may not provide the benefits of a natural inoculum. To 82 fully understand the differences local and commercial inocula may have on grapevine establishment, similar studies need to be performed in vineyards under varying environmental conditions. Given the differences among vineyards in land use history, cropping practices and biogeography, some sites and regions may benefit more than others from inoculation. 6.2 Does inoculation with AM fungi improved vine establishment in a field having poor soil conditions and does this depend on the timing of inoculation or source of inoculum? The field study in this chapter indicates that AM fungi may be a useful tool for stressful vineyard transplant situations. Mycorrhizal inoculation significantly increased vine survivorship when planted into a vineyard that had the topsoil removed. Vines inoculated with a commercial inoculum prior to transplanting had 80% survival, compared 10% for those inoculated with locally occurring AM fungi. Non-inoculated vines did not survive. These survival results are likely due to a harsh soil environment that was present during transplant. Despite the increase in survival, the commercial inoculant did not persist in vines, indicating that the benefits may be short term. AM fungal applications may be useful in the nursery production of plants, as transplant quality would be increased by having an AM infection that improves survival and establishment during stressful transplants. The cost to vineyard managers of poor stand establishment can be significant as it includes purchase and planting of replacements and dealing with uneven plant development for 83 subsequent years. Overall this study illustrates that pre-inoculating vines with AM fungi prior to transplanting may be the best strategy for avoiding transplant shock into stressful conditions. To determine if my results are robust across contexts, it is important to replicate this study across different abiotic conditions. In addition, there were multipe iterations of this experiment, both in the field and greenhouse. In the first attempt 1800 grapevines were grafted, composed of a Riesling cultivar and four different rootstocks. However, it was found that a bacterial infection had occurred during the propagation phase resulting in crowngall disease, caused by Agrobacterium strains. Subsequently a greenhouse experiment was designed to test the influence AM fungi have on growing vines in high pH soils. However, after inoculating rootstocks with the same Biosyneterra product that was used in Chapters 2 and 3, and using a similar growing system, colonization of vine roots never occurred. This indicates the efficacy of AM fungi products is not guaranteed and unpredictable in nature, as are all microbes even when applied in controlled experiments. 6.3 Will AM fungal inoculation reduce fungal infection of grapevine rootstocks and does the timing of AM fungal addition determine the strength of this effect? I found that co-inoculation with AM fungi and Ilyonectria actually led to abundance of Ilyonectria, which contrasts with findings of previous pathogen studies (Azcón-Aguilar and Barea 1996, Petit and Gubler 2006). For growers, this indicates that AM fungi are not consistently beneficial. It may be that a 84 further stress such as drought, or a longer incubation period was needed to realize the interactive effects of AM fungi and Ilyonectria. While AM fungi may still be suitable to stressful growing conditions, for example when exposed to pathogens such as Ilyonectria, my study found no positive result of pre-inoculating grapevine rootstocks with the ‘beneficial’ fungal addition. This experiment had to be replicated due to the lack of infection by Ilyonectria and Biosyneterra. In the first attempt at this experiment Turface® media was used instead of peat-based media, and instead of soaking the vine roots in Ilyonectria, the fungus was applied to potted vines via pipetting directly onto the rootzone. It is likely that the well-draining soil combined with the less intense method of pathogen application led to the fungi not infecting the vine roots. It is important to note that this experiment also had a lack of colonization by the AM fungi, even though the same commercial product that was successful in Chapter 2 was applied in the same manner. 6.4 Do standard transplant root trimming practices affect the mycorrhizal symbiosis? This chapter arose due to a question discussed at my original thesis proposal: if AM fungi are introduced into vine roots at the nursery stage, will the AM fungi be ‘cut off’ when roots are trimmed during transplant? I found no negative effects of root trimming on the AM symbiosis. AM fungi persisted in the roots, regardless of root trimming. It is probable that older roots with an abundance of vesicles serve as a source for new mycorrhizal development after 85 root trimming. It is also likely that after trimming vine roots, more resources are allocated to the root system to recover lost biomass, and mycorrhizal are more active in the lower roots of trimmed roots. Whether these findings are true for all AM fungi remains to be seen. The high vesicle colonization and re-colonization of newly growing roots observed in this study indicates that the commonly used commercial isolate is a suitable choice for pre-colonizing vines, especially when it comes to regenerating after a disturbance such as root trimming during transplant. 6.5 Concluding remarks The most important finding from this thesis is that inoculation by AM fungi does not always benefit grapevines. To the contrary, only two of the four experiments found benefit resulting from AM fungal inoculation. It is imperative that growers understand the limitations of AM fungi and biofertilizers and that inoculating agricultural systems will not always result in increased growth or yields. The AM fungal literature has shown a large amount of variation in plant growth responses to inoculation (Koide 1985, Klironomos 2003, Corkidi et al 2004), and it could be that perennial plants are less responsive to AM fungi under optimal growing conditions. Not discussed previously was the rate of colonization, or lack thereof, that regularly occurred in conducting this thesis. This negatively impacted or made for the need to repeat multiple experiments. For instance, in Chapters 2 and 4 commercial products failed to infect vine roots, while in Chapter 3 the slow rate of infection (taking upward of 2 months) delayed planting until June. Further, two pH experiments did not move forward due to the 86 lack of AM fungal establishment. This raises the issue of how consistent commercial products are at effectively colonizing woody perennials such as grapevines, providing further evidence that growers should be cautious when investing in these applications. A common discussion point throughout this thesis is that plant stress is likely important for realizing inoculation benefits. Chapter 2 showed that non-mycorrhizal plants did not survive vineyard transplant into harsh soils under hot, dry conditions. In comparison, the greenhouse experiments in Chapters 3 and 4 were maintained at relatively good growing conditions and showed no effect of inoculation by AM fungi. It is possible that under stressful conditions the symbiosis becomes more important to maintaining plant health and if the experiments had included more stress, mycorrhizal effects may have manifested. Future work should focus on what common field stresses AM fungi are capable of alleviating and understanding the balance of when plants invest more into AM fungi for these purposes. For instance, AM fungi are known to maintain plant growth when in the presence of common soil stresses, but what level of stress is needed to realize mycorrhizal benefits, and can this be detected pre-emptively in certain plant traits? With a better understanding in this area it would be possible to educate growers on when mycorrhizal use will likely be beneficial in agricultural settings. One surprising outcome was that AM fungi did not mediate tolerance to the pathogen Ilyonectria in Chapter 4. While Ilyonectria was found within grapevine tissues, it did not cause disease symptoms or impact vine growth. 87 Because Ilyonectria is a weak pathogen (Agusti-Brisach and Armengol 2013) it is possible that the conditions were not correct for pathogenesis to occur. Because AM fungal abundance increased when applied in combination with Ilyonectria it is possible that there was an interaction between AM fungi and the pathogen, but conditions were not severe enough to generate a response. Lastly, when using AM fungal inoculants, timing of inoculation is important, as was observed in Chapter 3. I showed that inoculating during the rooting phase, before transplant, may lead to the benefits of inoculation to be realized right from the time of transplant. To further understand how commercial AM fungal inoculants can be effectively incorporated into viticulture practices, it may be important to include microbial community aspects into field studies. It may be important to understand what resident AM fungal isolates are already present in vineyard soils communities of vineyards, and further, how the root community may change after transplant due to the incorporation of resident fungi. For instance, Chapter 3 saw the loss of the commercial inoculum from within pre-colonized roots and it would be interesting to understand whether these were replaced by AM fungi originally present within soils. By understanding these community dynamics there will be a better comprehension of whether inocula can have long-term impacts or if they are a short-term solution requiring re-application. Overall, this thesis shows that AM fungal inocula may be best incorporated into viticulture practices when stressful soil conditions limit vine performance. However, AM fungi exist in many agricultural landscapes and in 88 many instances the further addition of inoculants may be redundant. 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Vukicevich E, Lowery DT, Eissenstat D, Hart M. 2019, Changes in arbuscular mycorrhizal fungi between young and old Vitis roots. Symbiosis doi.org/10.1007/s13199-019-00598-3 Walker RB, Blackmore DH, Clingeleffer PR, Emanuelli D. 2014. Rootstock type determines tolerance of Chardonnay and Shiraz to long-term saline irrigation. Aust J Grape Wine Res 20: 496-506. Wright DP, Read DJ, Scholes JD. 1998. Myorrhizal sink strength influences whole plant carbon balance of Trifolium repens L. Plant Cell and Environment. 21:9 881-891. Wu QS, Xia RX. 2006. Arbuscular mycorrhizal fungi influence growth, osmotic adjustment and photosynthesis of citrus under well-watered and water stress conditions. J of Plant Physiol. 163:4 417-425. 102 Appendices Appendix A: Supplementary material for Chapter 3 Table A.1 Soil conditions of the experimental block, which had the top soil removed prior to planting the new grapevines, along with the adjacent block where soil communities originated from. Numbers represent averages across five samples taken throughout each block. Sample Calcium (mg/kg) Potassium (mg/kg) Magnesium (mg/kg) Sodium (mg/kg) Phosphorus (mg/kg) pH Total Organic Carbon (%dry) Nitrogen (Kjeldahl) (%dry) Experiment Block 969 1799 542 306 177.038 7.6 2.98 0.02 Adjacent Block 672 2105 456 272 615.16 6.7 5.06 0.2 Table A.2 Pairwise comparisons in survival, using Fisher-Exact Test, among different inoculation treatments; non-mycorrhizal vines receiving no treatment, vines receiving commercial inoculant in greenhouse, vines receiving commercial inoculant in the field, natural spore treatment inoculated in greenhouse and a natural root treatment inoculated in greenhouse. Fisher-Exact Test comparison P-value Treatment Non - Mycorrhizal Greenhouse - Commercial Field Commercial Greenhouse - Spore Non-Mycorrhizal Greenhouse – Commercial 0.003 Field Commercial 0.21 0.17 Greenhouse - spore 0.087 0.17 1 Greenhouse - root 1 0.02 0.582 0.303 103 Table A.3 Results of Mixed Model tests (Formula: Response ~ Treatment + (Treatment| Year)), performed on vine growth data collected from the 2016 and 2017 field seasons following transplant. Mixed Model by Restricted Maximum Likelihood Measure Num df, Den df Sum Sq. Mean Sq. F-value Pr (>F) Total Cane Length 3, 2 6909 2303 0.3627 0.791 Average Cane Length 3, 2 1025.7 342 0.351 0.797 Internode Length 3, 2 0.323 0.108 0.080 0.965 Internode Width 3, 2 2.376 0.792 0.365 0.790 104 Table A.4 Molecular digital droplet PCR data used to quantify the AM fungal isolate that is used in the inoculum. This illustrates the absence of commercial arbuscular mycorrhizal in vineyard soils before vines were planted. Sample ID Droplets Negative Droplets Positive Droplets Concentration Row 2 Sample 1 16004 16004 0 0 Row 3 Sample 1 16562 16562 0 0 Row 2 Sample 2 16535 16535 0 0 Row 3 Sample 2 17699 17699 0 0 Row 2 Sample 3 14467 14467 0 0 Row 3 Sample 3 15836 15836 0 0 Row 2 Sample 4 16198 16198 0 0 Row 3 Sample 4 16074 16074 0 0 Row 2 Sample 5 15529 15529 0 0 Row 3 Sample 5 14482 14482 0 0 105 Appendix B: Supplementary material for Chapter 4 Figure B1. Specificity test using the newly designed primer probe in a digital droplet PCR assay. Droplets above the threshold represent positive amplification to the primer/probe, with a manual threshold amplitude set at 3700. From left to right the isolates tested in each well are: A01 – PARC393, A02 – PARC349, B02- PARC100, C01 - PARC60, C02 – NTC, D01 – PARC340, D02 – Rhizophagus irregularis (DAOM197198), E01 – PARC72. Rhizophagus irregularis DNA originated from spores that were extracted from the AGTIV® product. 106 Table B1. A lineage report resulting from specificity testing for the new primer-1 probe set, using the blast tool in NCBI. First a primer blast was performed using 2 the newly developed primer set. The generated accession numbers were 3 subsequently nucleotide blasted using the probe sequence. The organism, score, 4 average e-value and number of hits for each match are reported. 5 6 Organism Score Average e-value Number of hits ..Ilyonectria liriodendri 185 3.47E-43 53 ..Ilyonectria destructans 185 9.71E-25 35 ..Ilyonectria strelitziae 163 8.00E-37 10 ..Ilyonectria palmarum 158 4.00E-35 6 ..Ilyonectria lusitanica 147 8.00E-32 1 ..Ilyonectria rufa 137 4.40E-28 5 ..Ilyonectria sp. 132 2.00E-27 8 ..Ilyonectria robusta 132 2.00E-27 1 ..Ilyonectria cyclaminicola 126 1.00E-25 2 ..Ilyonectria sp. CAB-2017a 126 1.00E-25 3 ..Ilyonectria protearum 126 1.00E-25 3 ..Ilyonectria vredenhoekensis 126 1.33E-24 4 ..Ilyonectria panacis 126 1.00E-25 1 ..Ilyonectria mors-panacis 121 5.00E-24 18 ..Ilyonectria crassa 121 5.00E-24 7 ..Ilyonectria leucospermi 121 5.00E-24 2 ..Ilyonectria pseudodestructans 121 5.00E-24 3 ..Ilyonectria sp. CBS 120370 121 5.00E-24 1 .Cylindrocarpon sp. cy22 121 5.00E-24 1 .Cylindrocarpon sp. cy20 121 5.00E-2 1 7 8 9 10
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Testing the efficacy of arbuscular mycorrhizal fungal inoculants on grapevine establishment
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