Grapevine trunk disease management for vineyard longevity in diverse climates of Australia
Abstract
Grapevine trunk disease (GTD) threatens the sustainability of the Australian wine industry. Research has progressed recommendations for the management of GTD in Australian vineyards. Rapid, low- cost, DNA-based molecular diagnostic tools have been developed to detect pathogens in the vineyard and in spore traps. Pathogen spores were present all year round in association with rain, and dispersal patterns varied between climatic regions. Pruning wounds were most susceptible to infection for the first two weeks, with reduced susceptibility during late winter in the Adelaide Hills. There was no benefit in adding adjuvants (wetters or stickers) to increase coverage and improve efficacy of fungicides, and several tested biocontrols and alternative products did not provide adequate wound protection against GTD pathogens. Short-term control of Botryosphaeria dieback (BD) was achieved with remedial surgery, with grafting being the only method shown to significantly increase shoot return, and spore detection was reduced in the vineyard following remediation. There was no evidence of tolerance to GTD pathogen infection in clones and rootstocks. Microbiome studies detected relatively low abundance of young vine decline and BD pathogens along with beneficial organisms in nursery vines. Water deficit stress increased the susceptibility of young vines to BD pathogen infection, particularly under high disease pressure, but vines with a low level of infection were able to withstand disease for at least three seasons with sufficient water. These outcomes provide new information that has been extended to industry and is leading to adoption of improved strategies for managing trunk diseases, which will increase vineyard longevity in diverse climates of Australia.
Summary
Eutypa dieback (ED) and Botryosphaeria dieback (BD) are major grapevine trunk diseases (GTDs) worldwide, causing significant yield reduction and threatening the sustainability of the $45 billion Australian wine industry. The causal pathogens infect vines through pruning wounds and colonise wood, causing dieback and, in the case of ED, stunting and yellowing of shoots and leaves, eventually killing the vine. Research led by the South Australian Research and Development Institute in collaboration with the National Wine and Grape Industry Centre (Charles Sturt University), with funding from Wine Australia and industry, has focussed on developing grapevine trunk disease management strategies for vineyard longevity in diverse climates of Australia.
Real-time loop mediated isothermal amplification (LAMP) assays were developed to detect ED and BD pathogens in infected plant materials, spore tapes and from fungal mycelia. A low cost and simple DNA extraction protocol was developed for LAMP and was found to be highly suitable for rapid DNA extraction from infected wood and other substrates. Seven real-time LAMP assays were shown to be highly specific and suitable for detecting and discriminating different ED and BD pathogens. The LAMP assays were at least twice as sensitive as conventional fungal isolation but were less sensitive than existing qPCR methods. This may offer cost effective, simple and robust diagnostic tools in the field or in limited resource environments. These assays can be used for commercial diagnostic laboratories and nurseries for the rapid detection of ED and BD pathogens from infected plant materials. Future research aims to further validate LAMP assays in the field for these and other GTD pathogens.
Spore trapping studies revealed sporadic spore dispersal that varied between regions, seasons and years. The number of samples with detection of ED pathogens was generally higher than that for BD pathogens. Rainfall was a primary factor associated with the release of either ED or BD spores, with as little as 0.2 mm of rain resulting in spore release. However, not all rain events resulted in spore release. The detection of spores in the absence of rain suggests that other climatic factors such as relative humidity or dew point may also play a role in spore release. Spores of BD pathogens were often detected throughout spring and summer, so any wounds created as part of canopy management (i.e. sucker removal, shoot trimming) may also be at risk of becoming infected by these spores. Deploying spore traps in different climatic regions will provide localised data that will assist growers in making decisions for optimal pruning time and fungicide treatments of their vineyards. DNA sequencing has identified seven species of ED pathogens, with Eutypa lata being prevalent in most regions. This study has led to the first report of Diatrype stigma and Cryptosphaeria multicontinentalis in Australian vineyards.
Rotorod spore traps were able to detect spores of both ED and BD pathogens, with ED predominant. ED pathogen spores were detected in and around a Shiraz vineyard with substantial dieback symptoms, with an increasing gradient of spore numbers from the north-east corner to the southern edge of the vineyard, consistent with predominant wind from the north. Spores were detected at all heights tested from 40-250 cm, with the greatest numbers at cordon height, and were also detected in lower numbers at 40 cm, the height of the air intake orifice of the Burkard volumetric spore trap. This suggests that data from the Burkard spore may underestimate the number of spores present at cordon height. Rotorod spore traps should be run for 24 h to maximise spore detection. Spore detection was greater from control vines compared to remediated vines, but annual wound protection is still necessary to prevent infection from spores derived from inoculum sources outside of the vineyard. Rotorod spore traps provide an effective research tool for monitoring spore dispersal in vineyards under different management regimes.
Field trials in the Adelaide Hills, SA revealed that wounds were highly susceptible to E. lata and D. seriata immediately following pruning, after which the susceptibility decreased rapidly over the following 14 days. From 21 days post-pruning, susceptibility was generally negligible. This indicates that wounds are most vulnerable to infection for the first 14 days following pruning, supporting results from a previous trial in McLaren Vale, and highlighting the importance of wound protection for the first two weeks after pruning. For E. lata, wounds were generally most susceptible to infection when pruned early and least susceptible when pruned late. Therefore, in the Adelaide Hills, there may be an advantage in delaying pruning to later in the dormant season to minimise the risk of infection by E. lata. Although D. seriata is not prominent in the region, susceptibility of wounds to D. seriata did not vary greatly between pruning times, so there appears to be no real advantage in avoiding the early pruning time. Future research will focus on other regions with varying climates to provide localised recommendations for pruning and wound protection.
Cryptovalsa ampelina was recovered from cane samples in the Adelaide Hills trial with high frequency. Freshly cut canes were free of C. ampelina, indicating that infection occurred post-pruning, and the prevalence of C. ampelina fruiting bodies on dead arms in the vineyard suggested that this species may be playing a greater role in ED than previously thought. The increased prevalence of C. ampelina in Australian vineyards requires further investigation. It will be important to determine its prevalence and distribution in Australia, its pathogenicity and the efficacy of wound protection treatments currently recommended for GTDs.
A wound spray coverage trial indicated that there was no benefit in adding adjuvants (wetters or stickers) to increase coverage and improve efficacy of fungicides. Poor coverage and control of E. lata with the application of two registered fungicides at a low water rate of 200 L/ha, reiterated the importance of applying the recommended minimum water rate of 600 L/ha to achieve sufficient coverage.
A vineyard trial was established to evaluate Vinevax (Trichoderma harzianum) applications followed by inoculation with trunk disease pathogens from 0-28 days. The results showed that application of Vinevax to wounds led to substantial colonisation by T. harzianum in the canes. However, it did not provide adequate protection from infection by ED and BD pathogens, particularly in the first three days after pruning, and was less effective than the registered fungicide Gelseal (tebuconazole). Further investigation is necessary on the application of higher T. harzianum spore concentrations on wounds to speed up colonisation, evaluating other Trichoderma species and products, and screening of local isolates from Australian wine regions.
Alternative products were evaluated for protection of pruning wounds against infection by ED and BD pathogens in a series of detached cane assays. Botector (Aureobasidium pullulans) and chitosan both showed promise for ED, but less so for BD. Botector was subsequently evaluated in the vineyard, and there was no reduction of wound infection by either pathogen. A. pullulans was re-isolated at low incidence from canes, suggesting either poor or temporary colonisation. Again, further investigation is required to evaluate the efficacy of A. pullulans when applied at higher concentrations on wounds following pruning. Chitosan products developed for disease management should also be further evaluated, and the search for other alternative organic wound treatments should continue, to ensure the sustainable management of grapevine trunk disease.
Remedial surgery trials were established, and control of BD was reported in the short term. However, grafted vines had lower watershoot production compared to own-rooted vines, suggesting that remedial surgery may be more successful in own-rooted vines than in grafted vines. Dieback symptoms with low severity re-emerged on cordons after four years, highlighting the importance of annual wound protection to ensure the long-term success of remedial surgery for the control of BD. In a world first vineyard trial, five methods anecdotally reported to improve watershoot growth following remedial surgery were evaluated. Only one method, grafting, significantly increased shoot production by 6 to 19%. Retaining watershoots prior to conducting remedial surgery resulted in a similar incidence of watershoots to grafting.
Visual assessment of symptoms in the vineyard indicated that the severity of dieback varied between clones. However, evaluation of a selection of clones in replicated detached cane assay experiments provided no evidence for any tolerance against E. lata or D. seriata infection between them. Therefore, it is most likely that any differences in dieback observed on vines in the field may be due to other factors such as vine age or variation in the ability of clones to tolerate other causes of dieback, such as water stress or poor soil conditions.
Assessment of established rootstock trials revealed a variation of dieback symptoms between vines planted on rootstocks and on own roots. Like the clone evaluations, rootstocks did not appear to confer tolerance on Shiraz scion canes inoculated with E. lata or D. seriata in the vineyard. Again, differences in dieback between vines on different rootstocks or own roots that were observed in the vineyard may be due to other factors such as their adaption to water stress, different soil conditions or soil-borne pathogens such as nematodes.
Two microbiome studies provided some initial insights on the microbial communities that naturally inhabit young vines from different Australian nurseries. A diverse population of the microbial communities was present in grapevine materials from different nurseries. The majority of the fungal genera identified are considered to be environmental fungi, while the remaining 8% were associated with YVD, bunch rots or fungal species with biocontrol potential that were classified as beneficial. These studies further revealed that short-term (5 min) hot water treatment used for dormant vines in Australia was ineffective in altering fungal populations but caused some variation in abundance of certain bacterial groups. Thus, investigation on the functionality of these bacterial communities is necessary to understand their role in vine health. The results generated from these studies are crucial first steps in understanding the current health status of grapevine planting materials in Australian nurseries. The new knowledge generated from this study may lead to developing new nursery practices that nurture the abundance of key beneficial microorganisms associated with young vines.
Understanding the threshold of infection and the stress conditions that have the potential to trigger disease expression in vineyards will contribute to the development of management strategies to mitigate GTD infections in nurseries and young vineyards. This study initially focused on the evaluation of a published inoculation method for standardising different levels of BD infection. The method was found to be suitable for inoculating different inoculum levels in vines, without significantly impacting plant viability. The qPCR assays further allowed the accurate quantification of BD infection from inoculated vines. These methods enabled the production of the large number of grapevine canes needed for pot experiments and could be adopted for potential future infection threshold experiments in the vineyard. This study provided key evidence that water deficit can increase the susceptibility of young vines to N. luteum infection, particularly when the vines are under high disease pressure. Furthermore, vines with a low level of infection were able to withstand the disease for at least three seasons when given sufficient water.
Extension of project outcomes has been delivered to industry through 10 industry journals and articles, 36 conference papers and 53 workshop presentations and media interviews. In addition, project team members have contributed to 19 scientific publications, a book chapter and a Best Practice Management Guide. We anticipate a further four scientific publications based on results from the current project. Australia remains a world leader of research on management of grapevine trunk diseases.