Potential role of Fusarium spp. in grapevine decline

Summary. Despite studies associating Fusarium spp. with grapevine decline since late 1970s, no consensus has been reached regarding the roles these fungi play in grapevine health. Recent studies in British Columbia, Canada, assessed prevalence of grapevine trunk diseases (GTDs) in young and mature vineyards, and the presence of GTD fungi in ready-to-plant nursery material sold in Canada. This study characterized the Fusarium spp. isolated from grapevines in BC by sequencing part of the translation elongation factor 1-alpha ( TEF1 ) gene, and carried out pathogenicity studies to determine whether Fusarium plays a role in grapevine decline. Fusarium spp. were isolated from 9.8% of samples collected from young vines (≤ 8-year-old), and 7.3% from mature vines (> 8-year-old), showing decline symptoms in commercial vineyards. Fusarium was also isolated from 43.9% of ready-to-plant dormant grapevines analyzed from four nurseries. Fusarium incidence varied between plants within the same nursery and between plants from the different nurseries. DNA sequences of TEF1 allowed identification of Fusarium oxysporum , F. proliferatum , F. ramigenum , and a Fusarium sp. Path-ogenicity studies were conducted in 1-year-old dormant rooted ‘Chardonnay’ plants grafted onto ‘3309C’ rootstock, and treatments included: i) whole plant, ii) trimming of roots, iii) cut at rootstock basal ends, and iv) trimming of roots plus cut at rootstock basal ends. Plants were inoculated using standardized methods, and were then planted in a greenhouse. Fusarium was compared with Dactylonectria macrodidyma , D. pauci-septata and Ilyonectria liriodendri used as positive controls. The Fusarium spp. caused necroses in rootstock roots and basal ends that were similar to those caused by black-foot fungi. Fusarium spp. and black-foot fungi reduced root and shoot dry weights when compared with non-inoculated controls, but no statistically significant differences were recorded for most treatments. This study is the first in Canada to identify Fusarium spp. from grapevines. Though Fusarium was common in these grapevines, pathogenicity tests suggest that the identified Fusarium spp. were weakly pathogenic to ‘3309C’ rootstock grapevines.


INTRODUCTION
Grapevine trunk diseases (GTDs) are major causes of grapevine decline, resulting in economic losses to grape and wine production industries (Wicks and Davies, 1999;Siebert, 2001;Kaplan et al., 2016).These complex diseases are associated with many fungal species, belonging to more than 30 genera, and include black-foot and Petri disease, responsible for grapevine decline and mortality in young vines, and Botryosphaeria dieback, Eutypa dieback, Esca, and Phomopsis dieback, which reduce yields and limit the lifespan of vines in mature vineyards (Gramaje et al., 2018).Most GTD fungi infect grapevines through pruning wounds, but those causing black-foot are soilborne (Agustí-Brisach and Armengol, 2013).Several of these fungi are also introduced into vineyards via contaminated grapevine nursery material (Gramaje and Armengol, 2011;Billones-Baaijens et al., 2013;Hrycan et al., 2023).Some GTD fungi are known to naturally occur in the vascular systems of asymptomatic grapevines, possibly as latent pathogens (González and Tello, 2011;Kraus et al., 2019;Hrycan et al. 2020).
Fusarium is a species-rich genus, which includes important plant pathogens causing root and crown rots, wilts, blights, and/or cankers in a wide range of annual and perennial crops (Gordon, 2017).Marais (1979) reported Fusarium spp. to be commonly isolated from roots and rhizosphere soil of stunted, dying or dead grapevines in South Africa.Grasso (1984) associated death of young grapevines with Fusarium oxysporum and 'Cylindrocarpon' destructans in Sicily, Italy.Highet and Nair (1995) reported F. oxysporum to be prevalent in roots of declining 'Semillon' grapevines in the Hunter Valley, Australia.Omer et al. (1995), and Granett et al. (1998) isolated F. oxysporum, F. roseum and F. solani in high frequencies from roots damaged by grape phylloxera in self-rooted 'Chardonnay' and 'AXR#1' rootstock vines collected from commercial vineyards in California (CA), United States of America.Luque et al. (2009) isolated Fusarium spp.from wedge-shape cankers, central necroses and wood decay in declining vines from mature vineyards in Spain.Studies from different countries have continued to report Fusarium spp. as prevalent in roots, cordons, and/or trunks of grapevines showing decline symptoms (Ziedan et al., 2011;Abdullah et al. 2015;Chehri, 2017;Bustamante et al., 2022;Zhang et al., 2023).
Fusarium spp.have been associated with declining grapevines from young and mature vineyards and in grapevine nursery plants.Halleen et al. (2003) reported Fusarium spp. as the most frequently isolated fungi from asymptomatic rooted cuttings collected 3 months after planting from different commercial nurseries in South Africa.A survey in South Africa by van Coller (2004) identified up to ten Fusarium spp.from roots and crowns of declining nursery grapevines.Studies across several nurseries in Spain reported six different Fusarium spp.isolated from plants showing decline or vascular wilt symptoms (Pintos et al., 2018;Astudillo-Calderon et al., 2019).Akgül et al. (2023) confirmed Fusarium spp. to be commonly found in asymptomatic nursery-produced plants from different geographical regions in Türkiye.
Grapevine trunk diseases and consequent vine decline and mortality have been identified as one of the main threats to long-term economic sustainability of the grape and wine industries in British Columbia (BC), Canada.To date, over 40 different fungal species have been identified from young and mature vines showing GTD symptoms (Úrbez-Torres et al., 2014a;2014b;2015a).These studies have routinely isolated Fusarium spp.from declining grapevines in the field or from ready-to-plant nursery vines.Accordingly, the objectives of this study were: i) to characterize the different Fusarium spp.isolated from grapevines from commercial vineyards and nursery plants in BC using molecular methods; and ii) to complete pathogenicity studies to determine the role of Fusarium spp. in grapevine health.

Fusarium isolates used in this study
Fusarium isolates were obtained from grapevine samples collected from young and mature commercial vineyards in BC during field surveys conducted between 2010 and 2013 (Úrbez-Torres et al., 2014a;2014b).In total, 215 and 248 vine samples (entire or part of the vine) showing decline symptoms were collected from 90 young vineyards (≤ 8-years-old) and 101 mature vineyards (> 8-years-old).Fungal isolations were conducted as described by Úrbez-Torres et al. (2014a).In addition, 362 ready-to-plant dormant rooted vines, including different scion-rootstock combinations and self-rooted cultivars, were collected between 2014 and 2021 from four nurseries selling grapevines in Canada.Isolations to identify different fungi were conducted from plant roots, basal ends of the rootstocks, graft unions, and scions, with the exception of self-rooted plants, from which samples were taken from roots, scion basal ends, and scions, as described by Hrycan et al. (2023).Wood pieces from the different host sections were plated on potato dextrose agar (PDA) (Difco) in Petri plates, amended with tetracycline hydrochloride (0.01%) (Sigma-Aldrich) (PDA-tet), and then incubated at 22°C in the dark until fungal colonies were observed.Fusarium-like colonies obtained from sample wood were sub-cultured on PDA.Hyphal tip subculturing onto PDA was then conducted from actively growing colonies, and these pure subcultures were held at 22°C in the dark.Based on colony morphology, a number of isolates representing different geographical locations, host cultivars and nurseries were chosen for molecular and pathogenicity studies.Fifteen to 20 mycelium plugs (0.5 cm diam.) were obtained from each selected pure culture isolate and placed in clear screw-cap glass vials containing autoclaved micropore filtered water.The vials were then maintained at 4°C in the Plant Pathology fungal collection at the Summerland Research and Development Centre (SuRDC), Summerland, BC, Canada until used.

Molecular identification of Fusarium isolates
Fusarium isolates selected for DNA extraction were retrieved from the SuRDC fungal collection and revived on PDA at 22°C in the dark.DNA was extracted from actively growing colonies using the PowerSoil DNA Isolation Kit (MO BIO Laboratories Inc.).Oligonucleotide primers EF1 and EF2 were used to amplify part of the translation elongation factor 1-α (TEF1) (O'Donnell et al., 1998).Each polymerase chain reaction (PCR) contained 2 μL of 10× PCR buffer, containing 15 mM MgCl 2 , 1.6 μL of 25 mM MgCl2, 1 μL of 10 mM dNT-Ps, 0.5 μL of 0.5 mM of each primer, 0.1 μL of Ultra Therm DNA polymerase (BocaScientific), and 1 μL of DNA template, adjusted with micropore filtered water to a final volume of 20 μL.Amplification reactions were carried out on a GeneAmp 2700 thermal cycler (Applied Biosystems), with an annealing temperature (T) of 53°C (Geiser et al., 2004).Amplified products were purified using a QIAquick PCR purification Kit (QIAGEN Inc.), and both forward and reverse strands of the TEF1 were sequenced using a 8-capillary 3500 Genetic Analyzer (Applied Biosystems) at the SuRDC.Sequences were edited and assembled using DNASTAR SeqMan™ Ultra version 17.4.1 (DNASTAR Inc.).Consensus sequences were then subjected to BLASTn queries in the GenBank database to determine species identifications.Fusarium sequences collected in this study were deposited into GenBank, and representative isolates are maintained in the SuRDC fungal collection.

Pathogenicity study
Four isolates representing the Fusarium spp.identified in this study were selected to inoculate dormant rooted 'Chardonnay' vines grafted onto '3309C' rootstocks.Dactylonectria macrodydima (SuRDC-1207), Dactylonectria pauciseptata (SuRDC-1248) and Ilyonectria liriodendri (SuRDC-1203) isolates, previously identified from black-foot symptomatic vines in BC (Úrbez-Torres et al., 2014a), were included in the study as positive controls.Conidium suspensions from the different Fusarium spp.and black-foot fungi were prepared as described by Úrbez-Torres et al. (2014a).Plants were inoculated by immersion of the roots and basal end of each plant in a suspension (10 5 conidia mL -1 ) of each fungus in a separate bucket, and left overnight.Four different treatments were included in this study.These were: 'whole plant' (WP, no cut roots and basal end not cut), 'roots cut' (RC), 'basal end cut' (BC), and 'roots and basal end cut' (RBC).Six plants per fungus/treatment combination were used.Six plants for each treatment were immersed in separate buckets containing noninoculated autoclaved distilled water as negative controls.After incubation (for approx.18 h), the plants were retrieved from the buckets and planted into pots (22 L capacity) containing doubled-autoclaved standard potting mix soil, in a greenhouse.All plants were watered and fertilized equally and as needed.Six months after planting, the plants were uprooted and a small portion of roots and rootstock basal end of each plant were collected for fungus re-isolations.These were carried out from the roots (R), basal end (BE) and 1 cm above the basal end (1-BE) of each plant.Fungal isolations and shoot and root dry weights were recorded from each plant using the methods of Úrbez-Torres et al. (2014a).
Data analyses were carried to determine effects of fungal isolate and experimental treatments on shoot and root dry weights.These data were square root transformed to normalize residuals.Normality was confirmed using inspection of residuals and Shapiro-Wilk tests.Shoot and root weights were analyzed using analysis of variance and Tukey's Honest Significant Difference test in R Software version 4.3.0(R Core Team, 2023).

RESULTS
Incidence of Fusarium in young and mature vineyards in BC, and in nursery grapevines.Fusarium was detected in 21 of 215 samples (9.8%) collected from young grapevines (≤ 8 years old) showing decline symptoms in commercial vineyards, and Fusarium isolates were mostly obtained from necrotic roots and vascular necroses observed around the stem pith at the bases of rootstocks or self-rooted cultivars (Figure 1).Fusarium was detected from 18 of 248 of samples (7.3%) collected from mature vines (> 8 years old) showing dieback in commercial vineyards, and was isolated from different host symptoms, including necrotic roots, wedge-shape cankers, vascular necroses and wood decay in cordons and trunks (Figure 1).Incidence of Fusarium in ready-to-plant nursery grapevines is shown in Table 1.Fusarium was isolated from 159 of 362 (43.9%) of the ready-to-plant dormant grapevines analyzed from all the assayed nurseries.Overall, the greatest infection was recorded from the graft-union sections of plants, followed by the base of the rootstocks or self-rooted cultivar roots and scions.Nursery C-2021 had the greatest level of infections, with 86.7% of plants yielding Fusarium.Nurseries A-2019 and D-2021 followed with, respectively, 70% and 78% of their plants infected with Fusarium.Nurseries A-2014 (31.8%) and B-2014 (20%) had the least Fusarium infection proportions (Table 1).Fusarium incidence also varied depending on the assessment year.Nursery A was sampled in 2014 and 2019.Though the number of plants analyzed in 2019 was less than in 2014, Fusarium incidence in 2019 was more than double than in 2014 (Table 1).Incidence of Fusarium in the different parts of the plants varied between plants within each nursery, and between nurseries.For example, roots and rootstock bases yielded, respectively, the greatest (73.1%) and least (15.4%)Fusarium incidence in nursery C-2021.In contrast, roots and rootstock bases yielded, respectively, the least (4.3%) and greatest (44.9%)Fusarium incidence nursery A-2014 (Table 1).

Molecular identification of Fusarium isolates
In total, 311 Fusarium isolates were obtained in this study, 39 from commercial vineyards and 272 from ready-to-plant dormant rooted nursery plants.Eight isolates from commercial vineyards and 49 isolates from nursery plants were selected for molecular identifications.PCR amplification of the TEF1 gave products between 650 and 750 bp.BLASTn analyses of the consensus sequences identified four species, including F. oxysporum, F. proliferatum, F. ramigenum, and a Fusarium sp.(Table 2).All four species were identified from samples collected from declining vines in commercial vineyards, and F. oxysporum and F. proliferatum were identified from nursery plants.Fusarium proliferatum was the most commonly isolated species from nursery plants (39 of 49 isolates).All F. oxysporum and F. proliferatum isolates from BC had 100% identity (100% query cover) with previously identified and published sequences in GenBank.Similarly, F. ramigenum isolates PARC425 had 99.84% similarity (100% query cover) to GenBank sequences.Isolate PARC428 had 95.40% similarity to Fusarium napiforme Genbank, so these isolates were classified as Fusarium sp.(Table 2).

Pathogenicity studies
Fusarium spp.caused root necroses in '3309C' in all treatments, including when roots were not cut, and the symptoms were similar to those caused by the blackfoot fungi used as positive controls.Statistical analyses showed that effects on mean root dry weights, of isolate (F = 5.813, P = 7.24E -06 ), treatment (F = 2.784, P = 0.04354), and the isolate by treatment interaction (F = 2.069, P = 0.00728) were statistically significant.The effect of isolate on mean shoot dry weight (F = 4.868, P = 6.81E -06 ) was significant, but this was not the case for treatment (F = 0.582, P = 0.628) or the isolate by treatment interaction (F = 1.35,P = 0.156).Though not significantly different from the control plants, most inoculated isolates reduced plant root dry weights compared with the non-inoculated controls (Table 3).Ilyonectria liriodendri was the only fungus that increased mean shoot dry weight for inoculated plants with the basal ends cut (Table 3).Among all the isolates and treatments, only D. macrodidyma and F. proliferatum significantly reduced root dry weight when inoculated onto plants with cut roots or basal ends.Similarly, Fusarium sp.isolate PARC428 significantly reduced root dry weight when the inoculated plants had cut ends (Table 3).A similar trend was observed for shoot dry weights.With few exceptions, where the inoculated plants had greater shoot dry weights than the non-inoculated controls, most isolates reduced mean shoot dry weights after all of the treatments, but none of these were statistically different from the controls (Table 3).Re-isolation proportions of the different inoculated fungi depended on experimental treatment (Table 4).None of the fungal pathogens were re-isolated from the non-inoculated controls.Fusarium solani, Ilyonectria robusta and I. torresensis were isolated from roots of some control plants, from the 'whole plant' and 'roots trimmed' treatments.Black-foot pathogens and Fusarium spp.were re-isolated from between 20 and 100% from the roots of 'whole plants'.Re-isolation percentages from roots were greater for all fungi in the 'roots trimmed' treatment (60 to 100%), and were even greater (80 to 100%) from the 'roots and basal end cut' treatment (Table 4).Only I. liriodendri, D. macrodidyma and Fusarium sp.PARC428 were re-isolated from the basal ends of 'whole plants', though at low proportions (20 to 40%).Re-isolation percentages were greater from the basal ends from the 'roots trimmed' (20 to 60%), 'basal end cut' (40 to 100%) and 'roots and basal end cut' (80 to 100%) treatments (Table 4).No fungal pathogens were re-isolated from 1 cm above the basal ends from the 'whole plant' treatment, and very re-isolation was recorded for D. pauciseptata, F. oxysporum and F. proliferatum from the 'roots trimmed' treatment (Table 4).Re-isolation of all fungi from 1 cm above the basal ends increased from the 'basal end cut' (20 to 60% isolation) or 'roots and basal end cut' (20 to 80%) treatments (Table 4).

DISCUSSION
Seventeen different Fusarium species have been isolated and identified from asymptomatic and symptomat-ic grapevines (Highet and Nair, 1995;van Coller, 2004;Chehri, 2017;Kraus et al., 2019;Bustamante et al., 2022;Li et al., 2023).The present study identified F. oxysporum, F. proliferatum, F. ramigenum, and a potentially novel Fusarium sp., from young and mature grapevines with GTDs symptoms, from vineyards in BC, Canada.Previous studies have identified more than 40 fungal species belonging to 14 genera, from declining vines in BC (Úrbez-Torres et al., 2014a;2014b;2015a).The present study adds Fusarium spp. to the group of fungi associated with GTD symptoms in Canada.
Molecular identification of Fusarium spp. in this study was achieved using amplification of part of the TEF1 using primers EF1 and EF2 (O'Donnell et al., 1998), since this approach has been reported to be the most informative for identification and discrimination among Fusarium spp.(Geiser et al., 2004).In addition, the use of short sequences obtained from other TEF1 primers has been shown wrongly identify Fusarium spp.(Torres-Cruz et al., 2022).Other loci, such as the RNA polymerase largest (RPB1) and the second largest subunit (RPB2) have been shown to effectively discriminate among Fusarium spp. in single or multi-loci phylogenetic analyses (O'Donnell et al., 2013).Identification of isolates in the present study as belonging to the F. oxysporum species complex and as F. proliferatum and F. ramigenum using the TEF1 is likely to be accurate, but further research including the RPB1 and/or RPB2 would allow further characterization of isolate PARC428, and determining if it is a novel Fusarium sp. or belong to an already known species.
Fusarium was isolated from 8.4% of samples collected from young and mature grapevines showing different vascular symptoms in roots, trunks, cordons, and spurs.Previous studies investigating fungi associated with GTDs symptoms or diversity of fungal communities in grapevine wood have identified Fusarium spp. at different levels of abundance.Marais et al. (1979), Granett et al. (1998), Abdullah et al. (2015), Moreno-Sanz et al. (2013), andLi et al. (2023) found Fusarium spp. to be among the most prevalent fungi isolated from mature grapevines showing decline symptoms.In contrast, other studies have reported incidence of Fusarium to be low in grapevines with characteristic GTD symptoms (Luque et al., 2009).The incidence of Fusarium isolated from vines with GTD symptoms in vineyards in BC was low, and these results were similar to those reported by Luque et al. (2009).Since Fusarium spp.are primarily soilborne, the low incidence of Fusarium in the present study could be because most of the samples analyzed were symptomatic aerial plant parts, such as cordons and spurs.Incidence of Fusarium in the present study was greater in young vines (≤ 8 years old) primarily showing characteristic black-foot symptoms, including root rot and vascular necrosis surrounding pith tissues at the rootstock bases and in self-rooted plants.This agrees with most studies that have shown high abundance of Fusarium in samples from symptomatic roots and basal areas of trunks (Marais et al., 1979;Grannet et al., 1998;Highet and Nair, 1995).Therefore, in order to better understand the associating between Fusarium spp.and vine decline in BC, further research should include assessments of the belowground status of symptomatic plants.
Fusarium incidence has been reported to be usually greater in ready-to-plant nursery grapevines than in young and/or mature vines from commercial vineyards.The present study showed that 43.9% of all analyzed nursery plants were infected by Fusarium, and in one nursery up to 87% incidence of infection was detected.The high prevalence of Fusarium spp.isolated from nursery material sold in Canada agrees with previous research in other countries (Halleen et al., 2003;van Coller, 2004;Pintos et al., 2018;Astudillo-Calderon et al., 2019;Akgül et al., 2023).
Only two species, F. oxysporum and F. proliferatum (i.e.low species diversity) were identified from nursery stocks in the present study.These results are of pertinent because the nursery plants came from different geographical regions, so greater species diversity was expected.Previous studies have identified greater Fusarium species diversity from nursery plants, including F. oxysporum and F. proliferatum (van Coller, 2004;Pintos et al., 2018).In contrast and similarly to the present study, other investigations have identified one Fusarium sp. from surveyed nursery plants (Astudillo-Calderon et al., 2019;Zhang et al., 2023).Several factors could explain these differences, but since morphological identification of Fusarium spp. is challenging, possible loss of diversity may have occurred in the present study as a result of the initial morphological classifications.Previous studies have shown molecular detection to be more informative than traditional culturing when determining the incidence of GTD fungi from grapevine nursery material (Úrbez-Torres et al., 2015b;Hrycan et al., 2023).Only traditional plating was conducted in the present study, so Fusarium incidence in nursery plants may have been greater if a molecular method was used.Fusarium incidence varied between plants within each nursery, between sections within the individual plants, and between plants from the different nurseries.These results were similar to those of Hrycan et al. (2023).
Results from the present study add to those that have reported high incidence of Fusarium spp. in ready-toplant nursery material (Halleen et al., 2003;Pintos et al., 2018;Astudillo-Calderon et al., 2019;Akgül et al., 2023).Previous studies have investigated GTD fungi during nursery propagation processes, and the health status of nursery plants.(Gramaje and Armengol, 2011;Billones-Baaijens et al., 2013;Pintos et al., 2018;Hrycan et al., 2023;Akgül et al., 2023).Therefore, the high incidence of Fusarium confirmed in nursery material in several countries should be further investigated, to determine the main sources of infections during nursery propagation processes.Furthermore, the role of Fusarium as a common grapevine endophyte should be investigated.
Despite the numerous reports of Fusarium spp. as fungal pathogens of grapevines, there is no consensus regarding the role of Fusarium in grapevine health and its association with GTD symptoms.This could be due to lack of consistency in results from pathogenicity tests conducted with Fusarium.Marais et al. (1979) reported that three isolates of Fusarium (species unknown) caused root rot when inoculated onto three different grape rootstocks.That study also showed no statistically significant differences on root or shoot masses between Fusarium inoculated plants and non-inoculated controls.These results convinced the authors that Fusarium was not a pathogen of grapevines, but was an endophyte, so Fusarium was not included in pathogenicity studies investigating effects of other soilborne fungi on grapevine health (Marais et al., 1980).In contrast, Highet and Nair (1995) (Zhang et al., 2023), but no other plant health parameters were considered in these studies.
In the present study, all four assessed Fusarium spp.caused root necroses in '3309C' plant, from all the experimental treatments including when roots were not cut.These symptoms were similar to those caused by the black-foot isolates used as positive experimental controls.Plants inoculated with Fusarium and black-foot isolates had reduced root and shoot dry weights when compared with negative controls, no matter the experimental treatment.However, with very few exceptions, data analyses gave no statistically significant differences between inoculated and non-inoculated plants.The results are similar to those reported by Marais (1979), and similar results were obtained with the well-known pathogens D. macrodidyma, D. pauciseptata, and I. liriodendri.Fusarium and black-foot fungi were re-isolated from roots from the 'whole plant' treatment, suggesting that these fungi do not need wounds for entry into host root tissues.However, results from the present study showed that fungal colonization of roots and rootstock basal ends increased when plants were wounded.Fungi were also capable of colonizing up to 1 cm above the basal ends when wounded.These results are similar to those reported by Grannett et al. (1998), where Fusarium spp.were recovered in greater incidence from roots damaged by the grape-phylloxera in CA, than from undamaged roots.
Fusarium may be a secondary pathogen on grapevines, as reported by Marais (1979), and wounded host tissues would facilitate vascular colonization.This is an important result, because poor quality nursery material with weak roots or poor callusing at the rootstock bases could be susceptible to Fusarium colonization.The differences observed among pathogenicity studies could be due to differences in host susceptibility to Fusarium.Omer et al. (1999) assessed effects of Fusarium on eight grapevine rootstocks, and concluded that rootstock type played a significant role in infection.'Self-rooted' 'Carignan' and AXR#1 rootstocks were the most susceptible to root necrosis, while Fusarium infections did not cause necroses on '3309C', '420A', '5C', and 'Freedom'.In the present study, Fusarium spp.caused root necrosis on '3309C'.Based on available studies, including the present one, Fusarium spp.were more successful at causing root necrosis and plant wilt on 'self-rooted' V. vinifera than on rootstocks.This could explain why Fusarium, though present in grapevines, may not be a primary cause of grapevine decline, as most vineyards have vines grafted onto phylloxera resistant rootstocks.Self-rooted vines from nursery B-2014 showed the least Fusarium infection when compared with the rootstock material from the other nurseries.This could have given low Fusarium inoculum levels in nursery B-2014 during the propagation process, or could have resulted from effective sanitation strategies applied by that nursery (Gramaje and Armengol, 2011).However, since graft unions were the plant parts in the other three nurseries from which Fusarium was most isolated, lack of graft-union wounding in self-rooted material most likely resulted in the low incidence observed.
It is well-known that several GTD fungi occur in asymptomatic grapevines, so it has been suggested that these fungi may be latent pathogens transitioning from endophytic to pathogenic states under abiotic and/or biotic host stress conditions.Initial inoculum concentrations of these fungi may also affect this transition (Hrycan et al., 2020).Results from the present study showed Fusarium to occur in declining field-grown vines, and at high incidence in ready-to-plant nursery material.However, the pathogenicity studies indicated that Fusarium was an opportunist or weak pathogen of grapevines.The same conclusion could be made from the pathogenicity tests with the three black-foot fungi used as positive controls.Abiotic and biotic factors have been associated with disease severity and fungal growth in other pathosystems.Light soil types increased severity of soybean Fusarium root rot symptoms compared with symptoms developing in sandy loam and silt loam soils (Yan and Nelson, 2022).Fusarium pseudograminearum biomass increased in drought stressed barley seedlings (Liu and Liu, 2016).For biotic factors, Li et al. (2023) reported that Fusarium spp.were more prevalent in GTD-symptomatic grapevine roots and rhizospheres than in for asymptomatic plants.That study also showed that disease indices were increased when Fusarium spp.were co-inoculated with D. macrodidyma than with individual inoculations, suggesting that Fusarium could enhance disease severity when in the presence of other GTD fungi.Further research should investigate Fusarium inoculum thresholds in ready-to-plant nursery grapevine material and determine correlation of this information with studies assessing effects of abiotic and/or biotic factors on Fusarium infected grapevines.Also, there is no information from Fusarium pathogenicity studies conducted under natural field conditions.These types of research will increase understanding of the roles of Fusarium spp. in grapevine health.

Figure 1 .
Figure 1.Grapevine decline symptoms from which Fusarium spp.were isolated in British Columbia, Canada.A, Decline symptoms and collapse observed in a young 'Merlot' vine in a commercial vineyard.B, Perennial canker, necrosis and soft wood symptoms observed at the base of the trunk of a mature 'Siegerrebe' vine in a commercial vineyard.C, Young vines showing poor shoot growth and root development.D, Vascular necrosis at a rootstock basal end.E and F, Vascular necrosis around the pith of a ready-to-plant nursery vine.G, Root necrosis and discolouration in a young vine from a commercial vineyard.

Table 1 .
Total numbers (and percentages) of plants and plant parts infected with Fusarium, that were analyzed from four grapevine nurseries.Different letters correspond to different nurseries, and different numbers indicate the years when samples were analyzed from each nursery.
a b Number (and percentage) of the total analyzed plants that were infected with Fusarium.c Number (and percentage) of different plant parts infected with Fusarium.
a b f: isolate obtained from commercial vineyard.n: isolate obtained from nursery .c TEF1: Translation elongation factor 1-α.

Table 3 .
Mean root and shoot dry weights for 'Chardonnay' grapevine plants grafted onto '3309C' rootstock and inoculated with spore suspensions of different Fusarium and black-foot fungi, in four experimental treatments.Means in each column accompanied by the same letter are not significantly different (P = 0.05), Tukey's Honest Significant Difference test.a WP: 'whole plant' untouched roots and basal end of the rootstock.b RC: roots cut and basal end of the rootstock untouched.c BC: basal end of the rootstock cut and roots untouched.d RBC: roots cut and basal end of the rootstock cut.

Table 4 .
Percentage reisolations of Fusarium and black-foot fungi from different parts of grapevine plants after inoculations with different pathogen isolates.