Factors affecting arbuscular mycorrhizal colonisation (AMF) in grapevines : A thesis submitted in partial fulfilment of the requirements for the Degree of Doctor of Philosophy at Lincoln University

In New Zealand, the wine industry constitutes 29% of its total horticultural exports and is the second largest horticultural export commodity. Increasing sustainability and reducing the environmental impacts are key drivers for the New Zealand wine industry. To achieve this, part of the focus should be on integrating sustainable strategies to optimise services from beneficial microbes, such as arbuscular mycorrhizal fungi (AMF), to improve grapevine health and production. Due to difficulties in culturing and identifying AMF little is known about the AMF communities associated with grapevines in New Zealand. Further, there is a significant gap in knowledge regarding the potential effect of vineyard management practices on these communities and hence the resilience of the ecosystem services they provide. For these reasons, the goal of this project was to i) identify the AMF communities associated with grapevines in New Zealand and the effect of abiotic and biotic factors on these communities, ii) determine the beneficial impact of these communities on grapevine growth and health, and iii) determine the effect of vineyard management practices on these communities. Denaturing gradient gel electrophoresis (DGGE) and trap cultures were used to characterise the AMF communities colonising different grapevine rootstocks. Root materials from three vineyards were analysed by DGGE and used in trap cultures for AMF recovery. This is the first study to identify the AMF community associated with grapevine in New Zealand and trap cultures allowed the recovery of six AMF spore morphotypes that belonged to Ambispora sp., Funneliformis sp., Glomus sp. and Claroideoglomus sp. Fifty-four of the eighty sequenced DGGE bands from root samples were AMF. The aligned sequences were assigned to Glomus spp., Rhizophagus spp. and Claroideoglomus spp. The two complementary techniques (DGGE and trap culture) also showed that the rootstock cultivar was the main driver of the arbuscular mycorrhizal community colonising the roots, therefore a rootstock can select a particular AMF community to form a mutualism with, from the diverse community present at a site. Site soil factors such as soil type, pH, soil moisture, soil organic matter, carbon and nitrogen content also had a significant (p < 0.05) effect on the AMF community, probably since these factors affect the community present in the soil at that particular vineyard which are then available for a rootstock to form a symbiosis with. This study is the first to investigate the effect of ‘home’ and ‘away’ AMF communities on grapevine rootstock growth parameters using whole communities as inoculum. The findings revealed that overall a grapevine rootstock cultivar grew better with the AMF community selected by that particular rootstock than when it is grown with the AMF community of another rootstock cultivar. This indicated that the rootstock selects an AMF community which is more beneficial to its growth and this is probably related to improved nutrient uptake. This study also showed that AMF spore diversity and the relative abundance of certain species is an important factor as when present in equal abundance competition between species could occur resulting in a reduction in the positive growth outcomes. Moreover, the AMF communities had a significant (p < 0.05) direct effect by increasing plant biomass and nutrient uptake and indirectly by influencing the production of hormones and the chlorophyll content in grapevine leaves through the increase of specific nutrients such as K, Mn and Zn. The findings also indicated that due to the presence of different species in the different communities, some AMF species may deliver particular benefits to grapevine plants. The effect of AMF on black foot disease was also investigated in a ‘home’ and ‘away experiment using commercial grapevine rootstocks. The study produced limited evidence that AMF treatments lowered disease incidence and severity in vines and that the presence of high disease incidence may have limited the effect of AMF community. However, despite the high disease incidence and severity AMF inoculation was seen to result in an increase in vine growth parameters by 60% to 80% compared to the vines inoculated with the pathogen only. This study was also the first to provide a comprehensive analysis of the community composition and structure of AMF associated with grapevine roots in both conventional and organic systems in New Zealand using high-throughput next generation sequencing (Illumina Miseq). The identified AMF species/genera belonged mainly to Glomeraceae and Claroideoglomeraceae followed by Diversisporaceae, Paraglomeraceae, Archaeosporaceae and Gigasporaceae. The outcomes also revealed a significant (p 0.05) difference on AMF beta diversity was observed in any of the studied factors. Overall, this study revealed that by using complementary techniques a comprehensive identification of AMF communities associated with grapevines in New Zealand was done. The major outcome was that rootstock plays a key role in the selection of taxa in their mutualism with AMF. More research on the ‘home’ communities may uncover additional benefits towards the grapevine from the selected AMF community. This study has increased knowledge of the ecosystem services they provide which will benefit the wine growers and the viticulture industry

AMF community diversity promotes grapevine growth parameters under high black foot disease pressure

Black foot disease is one of the main grapevine root diseases observed worldwide and is especially problematic in New Zealand. Arbuscular mycorrhizal fungi (AMF) have been shown to reduce infection and mitigate the effect of black foot disease on grapevine rootstocks. In contrast to prior studies, which have limited their focus to the effect of one, two or a combination of only a small number of AMF species, this study used whole AMF communities identified from 101-14, 5C and Schwarzmann rootstocks sampled from New Zealand vineyards. The effect of AMF on black foot disease was investigated in a ‘home’ and ‘away’ experiment using three commercial grapevine rootstocks. The study produced some evidence that AMF treatments lowered disease incidence at 5 cm and disease severity in vines by 40% to 50% compared to the vines inoculated with the pathogen only. This work also showed that the presence of high disease incidence may have limited the potential disease protective effect of AMF community. However, despite the high disease incidence and severity, AMF inoculation increased vine growth parameters by 60% to 80% compared to the vines inoculated with the pathogen only. This study is the first to provide an understanding on how young grapevine rootstocks inoculated with their ‘home’ and ‘away’ AMF communities would respond to challenge with a black foot pathogen species mixture. Further research is required to understand the mechanistic effect of AMF colonization on the increase of grapevine growth parameters under high black foot disease pressure

Bunch microclimate influence amino acids and phenolic profiles of Pinot noir grape berries

IntroductionThe increase of temperature due to climate change at different phenological stages of grapevine has already been demonstrated to affect accumulation of primary and secondary metabolites in grape berries. This has a significant implication for Pinot noir especially in New Zealand context as these compounds can have direct and indirect effects on wine quality. MethodsThis study investigates how varying bunch microclimate through changes in temperature applied at veraison stage can affect: fresh weight, total soluble solids, the accumulation of anthocyanins, total phenolics and amino acids of the grape berries. This was studied over two growing seasons (2018/19 and 2019/20) with Pinot noir vines being grown at two different temperatures in controlled environment (CE) chambers. The vines were exposed to 800 µmol/m2/s irradiance with diurnal changes in day (22°C or 30°C) and night (15°C) temperatures. This experimental set up enabled us to determine the accumulation of these metabolite at harvest (both seasons) and throughout berry development (second season). Results and discussionThe results showed that berry weight was not influenced by temperature increase. The total soluble solids (TSS) were significantly increased at 30°C, however, this was not at the expense of berry weight (i.e., water loss). Anthocyanin content was reduced at higher temperature in the first season but there was no change in phenolic content in response to temperature treatments in either season. The concentrations of total amino acids at harvest increased in response to the higher temperature in the second season only. In addition, in the time course analysis of the second season, the accumulation of amino acids was increased at mid-ripening and ripening stage with the increased temperature. Significant qualitative changes in amino acid composition specifically the α-ketoglutarate family (i.e., glutamine, arginine, and proline) were found between the two temperatures. SignificanceThis study is the first to provide detailed analysis and quantification of individual amino acids and phenolics in Pinot noir in response to changes in temperature applied at veraison which could aid to develop adaptation strategies for viticulture in the future

In vitro synthesized RNA generated from cDNA clones of both genomic components of Cucurbit yellow stunting disorder virus replicates in cucumber protoplasts.

© 2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC-BY) license (http://creativecommons.org/licenses/by/4.0/).Cucurbit yellow stunting disorder virus (CYSDV), a bipartite whitefly-transmitted virus, constitutes a major threat to commercial cucurbit production worldwide. Here, construction of full-length CYSDV RNA1 and RNA2 cDNA clones allowed the in vitro synthesis of RNA transcripts able to replicate in cucumber protoplasts. CYSDV RNA1 proved competent for replication; transcription of both polarities of the genomic RNA was detectable 24 h post inoculation. Hybridization of total RNA extracted from transfected protoplasts or from naturally CYSDV-infected cucurbits revealed high-level transcription of the p22 subgenomic RNA species. Replication of CYSDV RNA2 following co-transfection with RNA1 was also observed, with similar transcription kinetics. A CYSDV RNA2 cDNA clone (T3CM8Δ) comprising the 5'- and 3'-UTRs plus the 3'-terminal gene, generated a 2.8 kb RNA able to replicate to high levels in protoplasts in the presence of CYSDV RNA1. The clone T3CM8Δ will facilitate reverse genetics studies of CYSDV gene function and RNA replication determinants.Peer reviewe

Vineyard management systems influence on Mycorrhizal fungi

Arbuscular mycorrhizal fungi (AMF) offer significant potential for sustainable agriculture ecosystems, including vineyards. We showed that AMF had a positive influence on vine growth, nutrient uptake, and tolerance to environmental and biological stresses. Understanding how management practices affect AMF diversity and composition is crucial for harnessing their benefits. We surveyed twelve vineyards in the Marlborough region to compare AMF communities in conventional and organic systems. Using grapevine roots from different rootstocks, AMF spores were isolated via trap culture and identified through next-generation sequencing. The AMF belonged to Glomeraceae, followed by Claroideoglomeraceae and Diversisporaceae. Our findings showed differences in the AMF diversity between organic and conventional vineyards, with significant interactions between rootstock and management practice. This indicated that some rootstocks might be more suited to organic systems due to the AMF communities they support under this management practice. This could provide an increased benefit under organic systems supporting higher biodiversity

Vineyard management systems influence on Mycorrhizal fungi

Arbuscular mycorrhizal fungi (AMF) offer significant potential for sustainable agriculture ecosystems, including vineyards. We showed that AMF had a positive influence on vine growth, nutrient uptake, and tolerance to environmental and biological stresses. Understanding how management practices affect AMF diversity and composition is crucial for harnessing their benefits. We surveyed twelve vineyards in the Marlborough region to compare AMF communities in conventional and organic systems. Using grapevine roots from different rootstocks, AMF spores were isolated via trap culture and identified through next-generation sequencing. The AMF belonged to Glomeraceae, followed by Claroideoglomeraceae and Diversisporaceae. Our findings showed differences in the AMF diversity between organic and conventional vineyards, with significant interactions between rootstock and management practice. This indicated that some rootstocks might be more suited to organic systems due to the AMF communities they support under this management practice. This could provide an increased benefit under organic systems supporting higher biodiversity

Harnessing microbes for vine growth, nutrient uptake and disease protection: The benefits of arbuscular mycorrhizal fungi

Viticulture is a major economic activity in may countries and grapes are one of the most widely grown fruit crops. According to the New Zealand Winegrowers report (2020), there are around 40,000 hectares of grapes in production and more being planted in response to strong demand

Grapevine rootstocks drive the community structure of arbuscular mycorrhizal fungi in New Zealand vineyards

Aim: Arbuscular mycorrhizal fungi (AMF) are often regarded as non-specific symbionts, but some AMF communities show host preference in various ecosystems including vineyards. Grapevine plants are very responsive to AMF colonization. Although these fungi have potentially significant applications for sustainable agricultural ecosystems, there is a gap in knowledge regarding AMF–grapevine interactions worldwide and especially in New Zealand. This study focused on identifying AMF taxa colonizing grapevines in New Zealand vineyards and investigated the effect of grapevine rootstocks on AMF community diversity and composition. Methods and Results: Denaturing gradient gel electrophoresis (DGGE) and trap cultures were used to characterize the AMF communities. Grapevine roots from three vineyards and nine rootstocks were analysed by DGGE and used in trap cultures for AMF recovery. Trap cultures allowed the recovery of six AMF spore morphotypes that belonged to Ambispora sp., Claroideoglomus sp., Funneliformis sp. and Glomus sp. Bands excised, reamplified and sequenced from the DGGE were assigned to Glomus sp., Rhizophagus sp. and Claroideoglomus sp. The AMF community analyses demonstrated that rootstock significantly (P < 0·05) influenced the AMF community composition in all sites. Conclusions: The study showed that for a comprehensive identification of AMF, both results from trap culture and molecular work were needed and that the rootstock cultivar was the main driver of the arbuscular mycorrhizal community colonizing the roots. Significance and Impact of the Study: This study provides a firm foundation for future research exploring the beneficial use of AMF in enhancing grapevine production and sustainability

Effect of arbuscular mycorrhizal fungal communities on growth and nutrient uptake by grapevine rootstocks

Aim: Arbuscular mycorrhizal fungi (AMF) have been shown to have potentially significant applications for sustainable agricultural ecosystems. Yet, there is still lack of evidence showing how different AMF community compositions can benefit grapevines. Therefore, this study is the first to investigate the different responses of grapevine rootstock following their inoculation with different AM fungal communities. Methods: AMF communities were initially identified based on spore extracted from trap cultures and DNA extracted from roots. A glasshouse experiment was then set up to determine the interaction between rootstock variety and different AMF communities using AMF communities originating under their own (i.e., ‘home’) soil and other rootstocks’ (i.e., ‘away’) soil. Plant growth and physiological responses were measured and assessed. Results: This study showed that distinct AMF communities had differential effects on grapevine rootstock growth and nutrient uptake. It was revealed that a rootstock performed better in the presence of its “home” AMF community. Moreover, the AMF communities had a significant direct effect by increasing plant biomass and nutrient uptake and indirectly by influencing the chlorophyll content in grapevine leaves through the increase of specific nutrients such as K, Mn, and Zn. Also, the presence of different species in the different communities indicated that some AMF species may deliver particular benefits to grapevine plants. Conclusion: This work has enhanced our understanding of community level AMF-grapevine interaction and provided additional insight of the ecosystem services they deliver which will be of advantage to the wine growers and the viticulture industry