Molecular Identification and Pathogenicity of Fusarium Species Associated with Wood Canker, Root and Basal Rot in Turkish Grapevine Nurseries

Fusarium species are agriculturally important fungi with a broad host range and can be found as endophytic, pathogenic, or opportunistic parasites in many crop plants. This study aimed to identify Fusarium species in bare-rooted, dormant plants in Turkish grapevine nurseries using molecular identification methods and assess their pathogenicity. Asymptomatic dormant plants were sampled from grapevine nurseries (43) in different regions of the country, and fungi were isolated from plant roots and internal basal tissues. The Fusarium strains were identified by performing gene sequencing (TEF1-α, RPB2) and phylogenetic analyses. Pathogenicity tests were carried out by inoculating mycelial agar pieces of strains onto the stem or conidial suspensions into the rhizosphere of vines (1103 Paulsen rootstock). Laboratory tests revealed that Fusarium species were highly prevalent in Turkish grapevine nurseries (41 out of 43). Gene sequencing and phylogenetic analyses unraveled that 12 Fusarium species (F. annulatum, F. brachygibbosum, F. clavum, F. curvatum, F. falciforme, F. fredkrugeri, F. glycines, F. nanum, F. nematophilum, F. nirenbergiae, F. solani, and Fusarium spp.) existed in the ready-to-sale plants. Some of these species (F. annulatum, F. curvatum and F. nirenbergiae) consistently caused wood necrosis of seedling stems, rotting of the basal zone and roots, and reduced root biomass. Although the other nine species also caused some root rot and root reduction, their virulence was not as severe as the pathogenic ones, and they were considered opportunistic parasites or endophytic species. This study suggests that Fusarium species might play an important role in root-basal rot, wood canker symptoms, and young vine decline in Turkish grapevine nurseries and that these species need to be considered for healthy seedling production.


Introduction
Grapevine sapling production is one of the most important agricultural sectors in Türkiye, and many young vines (2.5 to 3 million plants) are produced yearly in different geographical regions in the country [1].The need for grapevine seedlings in the domestic market is relatively high, and this production needs to be increased to meet the demand for grapevine seedlings in Türkiye.
In grapevine nurseries, abiotic factors (unfavorable weather conditions, nutritional disorders, use of poor-quality production materials, rootstock-scion incompatibility, etc.), nematodes, insects, and fungal pathogens cause the death of plants, and these factors bring about low productivity and economic losses every year.Fungal grapevine trunk disease (GTD) pathogens, which often settle on young seedlings with infected propagation materials, belonging to Botryosphaeriaceae, Diaporthaceae, and Diatrypaceae families, Cadophora, Cytospora, Phaeomoniella, Phaeoacremonium Seimatosporium genera, and soilborne fungi (Armillaria, Cylindrocarpon-like anamorphs, Fusarium, Macrophomina, Phytophthora, Rhizoctonia, and Verticillium sp.) are considered to be the main actors of plant mortality in the nurseries [2,3].
The genus Fusarium has an exceptional place in plant pathology, medical mycology, and the food industry as they are both plant and human pathogens and threaten humananimal health by producing mycotoxins in foods.To date, more than 400 Fusarium species have been identified, which nested in 23 different species complexes [4].Most Fusarium species are soil-borne and are also called one of the ubiquitous fungal genera in mycology due to their endophytic, saprophytic, hemibiotrophic, or parasitic forms and strong competitive ability.Plant pathogenic species may result in significant crop damage and economic losses in some years by causing root and basal rots, damping-off, seed-tuber-fruit rots, wilt, and head blight diseases.According to the American Phytopathological Society, 83 out of 108 plant species in the field and horticultural crops are affected by one or more Fusarium diseases [5].Many species in the Fusarium genus are true plant pathogens, while others are opportunists waiting for soil and environmental conditions to turn unfavorable for plants.
It has been pointed out that root rot-associated fungi considerably reduce young vine health and marketable sapling yield; Fusarium and Cylindrocarpon-like fungi were the main actors affecting plant vigor, and quality in grapevine nurseries.These fungi cause necrosis in the roots and basal tissues, leading to a reduction in hairy roots, retarded growth, and the death of seedlings or young vines in later stages [6].Research on the pathogenic roles and diversity of Fusarium species on plant death in grapevine nurseries and young vineyards has intensified in recent years.Highet and Nair [7] proved the infection of grapevine hairy roots by Fusarium oxysporum through transmission electron microscopy and pathogenicity tests and suggested it would be considered as one of the fungi associated with root rot and decline in the nurseries.Reveglia et al. [8] revealed that the phytotoxins of Fusarium oxysporum (such as fusaric acid) and other potential metabolites have a critical role in the occurrence of these symptoms in seedlings and young vines in Australia.Vilvert et al. [9] claimed that Fusarium oxysporum f.sp.herbemontis is an important species responsible for decline and plant death in Brazilian grapevine nurseries, and it would be possible to control this pathogen using mycorrhizal fungi.Úrbez-Torres et al. [10] stated that Fusarium species were common in British Columbia (Canada) vineyards, but the most frequently isolated species might be secondary pathogens on grapevine rootstock 3309C.Similarly, Bustamente et al. [11] suggested that Fusarium species isolated from grapevine nurseries and young vineyards in California (USA) are opportunistic pathogens attacking plants under stress.In contrast, Li et al. [12] reported that when the Fusarium strains were inoculated into grapevine seedlings, they caused necrosis in the xylem vessels and basal regions of the plants resembling the infections of Dactylonectria macrodidyma (a black-foot disease pathogen).Zhang et al. [13] reported for the first time that Fusarium commune was a pathogen in grapevines causing leaf yellowing, stunting and root rot in Beijing Region, China.These studies indicate that Fusarium species on grapevines are a potential threat to nurseries and newly established vineyards and should not be underestimated.Furthermore, since these species can be found in the latent phase in plants, it is possible to spread them over large areas with marketable grapevine seedlings.Akgül and Ahio glu [14] detected some Fusarium species in young vineyards, along with fungal pathogens associated with grapevine trunk diseases, in southern Türkiye and confirmed the pathogenicity of these species.However, a nationwide study on the diversity and pathogenicity of Fusarium species in marketable grapevine saplings is needed.Therefore, this study aimed to identify Fusarium species in dormant marketable plants in Turkish grapevine nurseries and to reveal their pathogenicity.

Survey and Isolation of Fusarium Species
The survey was conducted in January 2021 in 43 grapevine nurseries in different geographical regions of Türkiye (in Adıyaman, Bursa, Denizli, Manisa, Mersin, Tekirda g, Tokat, and Urfa provinces).Ten dormant, commercially ready-for-sale seedlings from each nursery were randomly sampled and transported to the laboratory.The root and basal parts of the seedlings were washed under tap water and disinfected superficially with sodium hypochlorite solution (including >5% active clorine) for 3 min.Root and internal basal tissues (3-4 mm) were placed onto PDA (Potato dextrose agar, CondaLab; Madrid, Spain) containing streptomycin-sulfate (250 mg × L −1 ), and the Petri plates were kept at 25 • C in dark, for ten days to promote fungal colony growth.According to the colony morphology and microscopic characteristics detailed by Leslie and Summerell [15], a single spore was taken from the Fusarium colonies and purified on PDA for further stages.Ten Petri plates (containing seven tissue fragments in each) were used, and the isolation frequency of Fusarium colonies was calculated by proportioning the tissue number (Fusarium detected) to the total number (n = 70).

Molecular Identification and Phylogenetic Analyses
Strains were categorized according to colony morphology and microscopic features, and 60 Fusarium strains were selected for molecular identification.They were grown on PDA at 25 • C in the dark for seven days, and mycelium (56-60 mg) was harvested for DNA extraction.The genomic DNA was obtained following the CTAB protocol recommended by O'Donnell et al. [16] and diluted with 100 µL PCR grade water (Lonza) and stored at −18 • C for further use.Translation elongation factor (TEF1-α) and the second largest protein subunit of RNA polymerase II (RPB2) genes were amplified using the primers, EF1/EF2 and RPB2-5f2/fRPB2-7cr, respectively [17].The PCR reaction mixture contained 5 µL of buffer (10X Green Buffer, DreamTaq Green DNA Polymerase, Thermo-Scientific, Waltham, MA, USA), 2 µL of dNTPs mixture (10 mM each, Thermo Scientific, Waltham, MA, USA), 1 µL of forward and reverse primers (10 pmol•µL −1 ), 0.25 µL of Taq polymerase (DreamTaq Green DNA Polymerase, Thermo-Scientific), 39.75 µL PCR grade water and 1 µL genomic DNA (100 ng•µL −1 ).PCR amplifications were conducted in SimpliAmp A24811™ Thermal Cycler, Applied Biosystems, (Waltham, MA, USA) with the conditions detailed in the publications of O'Donnell et al. [16][17][18].The PCR products were separated using gel electrophoresis in 1.5% agarose (Invitrogen, Waltham, MA, USA) gel under 55V DC voltage, 250 mA current for 90 min.and were checked for DNA quality visually.After that, PCR products were sequenced bidirectionally via Sanger sequencing, derived chronogram files were trimmed from 3 and 5 prime with CLC main Workbench 5.5, and manual editing was carried out where necessary.Cleaned sequences were compared with those deposited in the NCBI GenBank database using the NCBIblastn suite (National Center for Biotechnology Information).Nucleotide sequences of TEF1-α and RPB2 genes were submitted to the NCBI GenBank, and the accession numbers were obtained.According to the nucleotide BLAST search results of TEF1-α and RPB2, a representative sequence dataset was used from the NCBI nucleotide database to perform the phylogenetic study.Constructed datasets for TEF1-α and RPB2 sequences were aligned individually via the ClustalW alignment tool in Geneious Prime 2019.1.3software.After the alignment step, TEF1-α and RPB2 sequences were concatenated from end to end via Geneious Prime 2019.1.3software for the multi-gene phylogenetic tree.Phylogenetic analyses were based on maximum likelihood (ML).The ML analysis was performed with IQ-TREE on the Galaxy Europe platform [19].Model Finder was used to determine the best-fit model for the ML tree [20].ML tree construction was performed under the TIM2e model with equal base frequencies and Invariable + Gamma with four categories (TIM2e + I + G4) in the nucleotide substitution model according to the Bayesian information criterion scores and weights (BIC and w-BIC).For the pseudoreplications of the ML tree, a 1000 ultrafast bootstrap parameter was used [21].The alignment and the phylogenetic tree were deposited in TreeBASE under the study number S31385 (http://purl.org/phylo/treebase/phylows/study/TB2:S31385,accessed on 23 April 2024).

Pathogenicity Tests
Based on the identification results, 38 Fusarium strains were selected for pathogenicity tests, and two types of inoculation methods were followed in order to evaluate from different aspects.In the first, the bark of the dormant cuttings was removed with a sterile cork-borer (3 mm), fresh mycelial agar discs of the strains (10-day old) were placed on these wounds, and these points were wrapped with parafilm™.The cuttings were planted in the pots and grown in greenhouse conditions for four months.The inoculation points were scraped with a scalpel, and necrosis lengths in the wood tissues were measured and recorded [11].Plants inoculated with an Ilyonectria liriodendri (a black-foot disease pathogen) strain (AFP115, obtained from the fungal culture collection of Mycology Lab. in Dept. of Plant Protection in Çukurova University, Adana, Türkiye) were set as positive, and sterile agar-inoculated plants were set as healthy controls.The fungi were re-isolated from the internal tissues at the inoculation points to confirm the pathogenicity by culturing wood chips on a PDA medium.For each seedling, five Petri dishes (seven tissues in each) were used, and the frequency of re-isolation was calculated by proportioning the number of Fusarium colonies to the total number of wood chips.In the second trial, rootstock cuttings (cv.1103 Paulsen) were planted in the plastic pots (0.85 L) containing sterile rooting mix (equal volumes of peat moss and perlite), and the pots were kept in lath house conditions (natural temperature, relative humidity and illumination).The Fusarium strains were grown on PDA at 25 • C for 15 days, and conidial suspensions (in sterile distilled water at 10 6 conidia•ml −1 concentration) were prepared using a haemocytometer.Following root formation, conidial suspensions were poured into the root zone of the plants (20 mL per pot), and plants were grown in lath house conditions for four months.The pathogenicity of the strains was assessed based on root dry weight and necrosis length in the plants' basal zone (in wood tissues).The seedlings were uprooted from the pots, the roots were gently washed under tap water, and were harvested using a pruning shear.After briefly blotting with paper towels, the roots were held in a drying chamber at 65 • C for 48 h, then weighed using a precision balance, and weights were recorded [22].Nevertheless, the bark of the cuttings was carefully peeled off with a knife, and the length of the necrosis in the wood tissues was measured with a caliper.The Fusarium strains were re-isolated from the internal tissues of the basal parts by culturing the tissues on a PDA medium amended with streptomycin-sulfate (250 mg•L −1 ), and the re-isolation frequency was calculated as mentioned above.Pathogenicity tests were arranged according to the design of the randomized plot with six replications (two plants in a replicate), and twelve plants were used for each strain.The trials were repeated once (2022 and 2023 years), and the data were subjected to statistical analysis.To clarify the virulence of each Fusarium species, an analysis of variance (ANOVA) was performed again on the mean values of all strains belonging to the same species.

Statistical Analyses
ANOVA was performed on lengths of wood necrosis in basal parts and internodes of the stems and root dry weights.The data were checked for normality, and root square transformation was applied.Means were compared using Fisher's least significant difference (LSD) test at the 5% significance level [23].

Fungal Isolation and Prevalence of Fusarium Species
The first Fusarium colonies were aroused on the internal basal tissues and hairy roots of marketable, dormant grapevine plants after 5-6 days of incubation (at 24 • C in the dark) in PDA media.Through colony morphology (colony reverse color, appearance of mycelia), 779 Fusarium colonies were detected in 3010 plant tissues (in 430 Petri dishes) plated for 43 grapevine nurseries.Their colony morphology is shown in Supplementary Materials.The isolation frequency of Fusarium species in these nurseries is shown in Table 1.As shown in Table 1, Fusarium species were detected in 41 out of 43 grapevine nurseries, and the prevalence of these species in Turkish grapevine nurseries was calculated at 95.3%.The isolation frequency in nurseries ranged between 2.9 and 65.7%, while the overall average was 24.9%.Nevertheless, black foot, Petri disease pathogens, Botryosphaeriaceae fungi, Cytospora, Diaporthe, Truncatella species, and soil-borne plant pathogenic fungi (Macrophomina and Rhizoctonia) were also found.Considering different geographical regions and morphological-microscopic characteristics, 121 Fusarium colonies were pre-selected for molecular-phylogenetic analyses.

Molecular Identification and Phylogenetic Analyses
Using the primers EF1/EF2 and RPB2-5f2/fRPB2-7cr, TEF1-α and RPB2 gene regions of the Fusarium strains were amplified by conventional PCR, and agarose gel electrophoresis revealed DNA bands with sizes ranging from 680 to 1600 bp (respectively).The initial approach for the identification of strains relied on a blastn search of partial sequences of the TEF1-α and RPB2 gene.NCBIblastn search was performed with the nucleotide sequences of these regions, and the strains were 99.2-100% similar to other Fusarium species in the GenBank.Afterwards, these sequences were aligned with the closest matching and nearly closest references obtained from GenBank (Table 2) for resolve the ambiguities.However, the NCBIblastn search results from the two gene regions of all strains were not parallel, and the second gene region in some strains matched with different Fusarium species.Yet, phylogenetic analyses conducted with concatenated nucleotide sequences clarified the ambiguity observed in the blastn results.To construct the phylogenetic tree, we used a dataset comprising 150 taxa, including 60 local strains, 89 reference Fusarium strains, and one strain from the Dactylonectria genus.The dataset contained 3040 nucleotide sites.The phylogenetic tree was rooted using the Dactylonectria torresensis strain CBS 129086.In the aligned final dataset, out of the 3040 nucleotide sites, 1787 were identified as constant, and 1001 were identified as parsimony informative.Additionally, 1598 nucleotide sites were found to have distinct site patterns.Model finder analysis of the aligned dataset indicated that the TIM2e + I + G4 model was the most appropriate for constructing the maximum likelihood (ML) tree, with a BIC score of 43,876.5755and a w-BIC score of 0.785.The constructed ML tree showed a sum of branch lengths of 2.5465 and a sum of internal branch lengths of 1.2670 (in Figure 1).Table 2. GenBank accession numbers of partial sequence of TEF1-α and RPB2 of references species used in the phylogenetic analyses.

Pathogenicity of Fusarium Strains and Species
In four-month pathogenicity tests, some Fusarium strains inoculated on the plants' stems (considering the possibility of contamination to the vines through pruning wounds) produced lesions ranging from 5.9 to 12.0 mm (Figure 2).The lesion lengths of 10 of the 38 strains inoculated on the plants in the first year were longer than those of the control and other strains and statistically different.
produced lesions ranging from 5.9 to 12.0 mm (Figure 2).The lesion lengths of 10 of the 38 strains inoculated on the plants in the first year were longer than those of the control and other strains and statistically different.The majority of these strains belonged to the following species: F. annulatum (seven strains), F. brachygibbosum (one strain), F. nirenbergiae (one strain), and I. liriodendri.In the second year, only nine strains had lesions longer than other Fusarium strains and control statistically.They were F. annulatum (five strains), F. nirenbergiae (two strains), F. curvatum (one strain) and I. liriodendri (Table 4).In both years, lesions caused by other Fusarium strains were not significantly longer than those of the control.However, these strains could be re-isolated from the point of inoculation (except from the non-inoculated control) at rates ranging from 15.2% to 53.8%.The majority of these strains belonged to the following species: F. annulatum (seven strains), F. brachygibbosum (one strain), F. nirenbergiae (one strain), and I. liriodendri.In the second year, only nine strains had lesions longer than other Fusarium strains and control statistically.They were F. annulatum (five strains), F. nirenbergiae (two strains), F. curvatum (one strain) and I. liriodendri (Table 4).In both years, lesions caused by other Fusarium strains were not significantly longer than those of the control.However, these strains could be re-isolated from the point of inoculation (except from the non-inoculated control) at rates ranging from 15.2% to 53.8%.In the second type of pathogenicity test, 38 strains increased basal rot and significantly reduced root dry weight compared to the non-inoculated control.Due to the large number of strains and replicates, there was a large variance among the means of strains, resulting in many statistical groups.The Fusarium strains, and I. liriodenri caused basal rot of the wood tissues in the basal region of the seedlings (Figure 3), and their lengths ranged from 3.6 to 37.0 mm in the first year and from 3.7 to 7.8 mm in the second year (Table 5).As in the case of stem necrosis, F. annulatum, F. curvatum, and I. liriodendri species were found to cause the most extended necrose length in basal rot formation.However, the wood necrosis induced by the other species was not as severe and consistent as that of these three species.The Fusarium strains and I. liriodendri could be re-isolated from the basal necroses (except from the non-inoculated control) at rates ranging from 10.3% to 36.8%.In the second type of pathogenicity test, 38 strains increased basal rot and significantly reduced root dry weight compared to the non-inoculated control.Due to the large number of strains and replicates, there was a large variance among the means of strains, resulting in many statistical groups.The Fusarium strains, and I. liriodenri caused basal rot of the wood tissues in the basal region of the seedlings (Figure 3), and their lengths ranged from 3.6 to 37.0 mm in the first year and from 3.7 to 7.8 mm in the second year (Table 5).As in the case of stem necrosis, F. annulatum, F. curvatum, and I. liriodendri species were found to cause the most extended necrose length in basal rot formation.However, the wood necrosis induced by the other species was not as severe and consistent as that of these three species.The Fusarium strains and I. liriodendri could be re-isolated from the basal necroses (except from the non-inoculated control) at rates ranging from 10.3% to 36.8%.In parallel to basal rot, the Fusarium strains and I. liriodendri decreased hair root formation and root dry weight in the inoculated plants compared with the non-inoculated control plants.In 30-35% of the plants inoculated with F. annulatum, F. curvatum, and I. liriodendri, shoots dried up, and plants died after one month.The average root dry weight recorded per plant in the first year varied between 0.022 and 0.344 g, while in the second year, these values were recorded between 0.599 and 1.463 g (Table 6).The average values (necrosed lengths in stem and basal part, and dry root weights) of the strains (belonging to the same species) were considered, and ANOVA was performed on the means to clarify the pathogenicity of each Fusarium species.When the pathogenicity of Fusarium species was evaluated according to the length of necrosis at the inoculation point, it was found that F. annulatum, F. brachygibbosum, F. nirenbergiae and Ilyonectria liriodendri caused wood necrosis.In contrast, the others did not show the same influence (Figure 4).Regarding the effects of Fusarium species on basal rot formation, it was determined that the three species causing the most extended necrosis in the first year were F. annulatum, I. liriodendri, and F. glycines; in the second year, I. liriodendri, F. nirenbergiae, and F. annulatum, respectively (Figure 5).oculation point, it was found that F. annulatum, F. brachygibbosum, F. nirenbergiae and Ilyonectria liriodendri caused wood necrosis.In contrast, the others did not show the same influence (Figure 4).Regarding the effects of Fusarium species on basal rot formation, it was determined that the three species causing the most extended necrosis in the first year were F. annulatum, I. liriodendri, and F. glycines; in the second year, I. liriodendri, F. nirenbergiae, and F. annulatum, respectively (Figure 5).oculation point, it was found that F. annulatum, F. brachygibbosum, F. nirenbergiae and Ilyonectria liriodendri caused wood necrosis.In contrast, the others did not show the same influence (Figure 4).Regarding the effects of Fusarium species on basal rot formation, it was determined that the three species causing the most extended necrosis in the first year were F. annulatum, I. liriodendri, and F. glycines; in the second year, I. liriodendri, F. nirenbergiae, and F. annulatum, respectively (Figure 5).The effect of Fusarium species on root dry weight reduction was almost parallel to basal rot; when the results of both years were generally evaluated, F. annulatum, I. liriodendri, and F. nirenbergiae were found to be the most effective species (Figure 6).The effect of Fusarium species on root dry weight reduction was almost parallel to basal rot; when the results of both years were generally evaluated, F. annulatum, I. liriodendri, and F. nirenbergiae were found to be the most effective species (Figure 6).

Discussion
In this study, Fusarium fungi were found to be relatively common (95.3% of the nurseries) in bare-rooted plants ready for sale in Turkish grapevine nurseries.This rate is considerably higher than that found in North America and Canada, but it is close to that in the nurseries in France and Spain.Garnett et al. [24] investigated the fungal species associated with root rot of grapevines in two different vineyards in California and isolated a high proportion of Fusarium species (together with Rhizoctonia, Pythium, Macrophomina, Phytophthora fungi) in the sampled vines.Torres et al. [10] found Fusarium species in 43.9% of the seedlings ready for sale in four grapevine nurseries in Canada and determined that these species were isolated from the plants between 20.0 and 86.7%.Bustamente et al. [11] determined that the incidence of Fusarium species was 36.7% in young vineyards and 31.7% in nursery plants in California.However, Pintos et al., [25]

Discussion
In this study, Fusarium fungi were found to be relatively common (95.3% of the nurseries) in bare-rooted plants ready for sale in Turkish grapevine nurseries.This rate is considerably higher than that found in North America and Canada, but it is close to that in the nurseries in France and Spain.Garnett et al. [24] investigated the fungal species associated with root rot of grapevines in two different vineyards in California and isolated a high proportion of Fusarium species (together with Rhizoctonia, Pythium, Macrophomina, Phytophthora fungi) in the sampled vines.Torres et al. [10] found Fusarium species in 43.9% of the seedlings ready for sale in four grapevine nurseries in Canada and determined that these species were isolated from the plants between 20.0 and 86.7%.Bustamente et al. [11] determined that the incidence of Fusarium species was 36.7% in young vineyards and 31.7% in nursery plants in California.However, Pintos et al., [25] detected 92% to 98% of Fusarium fungi, among other GTD pathogens, from plants sampled from two commercial grapevine nurseries in Spain and one in France.
In the current study, 12 distinct Fusarium species were found in six Fusarium species complexes in the grapevine nurseries, with the most common species complexes being F. oxysporum (38.3%),F. fujikuroi (20.0%), and F. solani (18.3%).The results revealed more diversity of Fusarium species than previous studies conducted in Canada and United States.Urbez-Torres et al. [10] reported that Fusarium species diversity was very low in four nurseries in British Columbia (Canada) and found only two species from two different species complexes (F.oxysporum and F. fujikuroi).However, Bustamente et al. [11] reported a high diversity of Fusarium in young vines in California (total, nine Fusarium species in six species complexes) and found species (F.annulatum, Fusarium sp., F. solani, F. keratoplasticum, F. nirenbergiae) belonging to these complexes in the nurseries.The fungal isolation results in our study, the high Fusarium species diversity in ready-to-sale grapevine seedlings, and the presence of joint species (F.annulatum, F. brachygibbosum, F. clavum, F. nirenbergiae, F. solani) in the plants were consistent with the findings of Bustamente et al. [11].However, in the abovementioned studies, F. avenaceum, F. ramigenum, F. culmorum, F. keratoplasticum, F. oxysporum, and F. proliferatum were not found in grapevine nurseries in Türkiye.Interestingly, although F. oxysporum is a large species complex, including 21 species [26], and F. oxysporum has an essential place in this complex, we could not detect F. oxysporum among the Fusarium species we isolated from vines.In contrast to our findings, Zeidan et al. [27] reported that F. oxysporum strains obtained from different grape varieties (cv.Crimson Seedless, Flame, King Robi, Superior, and Thompson), showing wilt and root rot in Egypt were pathogenic by producing polygalacturonase and cellulase enzymes.These strains were associated with wilt in Crimson Seedless but with root rot in other cultivars.When phylogenetic analyses were performed, it was revealed that many strains similar to this species were F. curvatum, F. glycines, and F. nirenbergiae.Similarly, although F. proliferatum has been reported as an important root rot pathogen in maize, soybean, tomato, and grapevine [10,[28][29][30], we could not detect F. proliferatum among the available grapevine Fusarium strains.These differences may have been made possible by detailed phylogenetic analyses using concatenated genes such as TEF1-α and RPB2, which are highly recommended to identify Fusarium.O'Donnell et al. [4] suggested that when identifying Fusarium species, the TEF1α and RPB2 gene regions should be amplified and concatenated to perform phylogenetic analyses if possible, and in case of financial limitations, the sequence of the TEF1α region might be sufficient.
Based on the pathogenicity results of Fusarium strains inoculated on grapevine stems, F. annulatum, F. brachygibbosum, F. curvatum, and F. nirenbergiae were found to produce more considerable wood necrosis in comparison to the control and other Fusarium species.Reveglia et al. [7] widely isolated F. oxysporum strains from grapevines showing young vine decline symptoms (in Italy) and investigated their phytotoxic metabolites.The fusaric acid purified from these strains caused severe necrosis when injected into tobacco leaves, and they suggested that this metabolite may also cause root and basal rot in grapevines.Akgül and Ahio glu [14] inoculated F. brachygibbosum strains (obtained from three-year-old young grapevines) on the stems of grapevine seedlings and determined it to be a highly virulent species in woody tissues.Rajput et al. [31] investigated the pathogenicity of F. equiseti strains isolated from the trunks of grapevines in the Kunar province of Afghanistan and reported that tissue necrosis occurred when this species was inoculated on woody shoots of three-year-old plants.Bustamente et al. [11] inoculated F. annulatum, F. nirenbergiae, and F. solani strains from young grapevines on the stems of one-year-old vines and revealed that after seven months; these species produced longer necroses in the wood tissues of plants when compared to non-inoculated controls.The results of these studies support the view that the wounds occurring via disbudding of cuttings or basal cuts in the seedlings or wounds by removing vine suckers on trunks (in the vineyards) may be susceptible to Fusarium infections and that Fusarium species may be involved in young vine decline or trunk diseases.
Another outcome from the pathogenicity tests was that some Fusarium species (F.annulatum, F. curvatum, F. nirenbergiae, F. solani) significantly increased root-basal rot and reduced root biomass in the inoculated plants.Highet and Nair [7] investigated the effect of Fusarium oxysporum infections on root rot development in grapevines (cv.Semillon 5-25 years old) in New Zealand.When plants were inoculated with F. oxysporum, they observed the disintegration of bark cells (by transmission electron microscopy) and determined that Fusarium-infected root cells lacked cytoplasm compared to uninfected cells.Vilvert et al. [9] stated that F. oxysporum f.sp.herbemontis was an important fungal pathogen in Brazilian grapevine nurseries, causing basal rot, reduction in root biomass, and root rot symptoms in infected vines.Zhang et al. [13] detected several Fusarium species from young grapevines Red Globe) showing decline and leaf yellowing in vineyards in Beijing, China and found that F. commune was pathogenic among these species and associated with these symptoms.Li et al. [12] revealed that Fusarium strains inoculated on grapevine seedlings caused not only a reduction in root biomass, root rot but also interveinal discolorations and coalescent necrosis on the leaves of the plant.When these strains were inoculated together with Dactylonectria macrodidyma, the severity of the disease was further increased.
Regarding the pathogenicity of other Fusarium species, F. annulatum has also been reported to cause fruit-corm and root rot in crop plants such as melon and onion, as well as medicinal-aromatic plants such as Blettila striata L. and saffron, in addition to vine [32][33][34][35].These studies indicate that F. annulatum is pathogenic in many hosts.F. nirenbergiae and F. curvatum were other virulent species in the pathogenicity tests on the grapevine seedlings.When we reviewed the studies on this subject, we found only one study [11] in which F. nirenbergiae was detected as a pathogen in grapevine.However, in other studies, it has been reported to be pathogenic in crop plants such as maize, passion fruit, almond (in Portugal and Spain), and maple.Sanna et al. [29] investigated Fusarium species associated with post-emergence damping-off and root rot in maize and found that F. nirenbergiae caused a disease index of over 50% in some maize areas of Italy, as did F. verticilloides, F. annulatum and F. commune.Aiello et al. [36] identified F. nirenbergiae as the cause of root rot and wilt in passion fruit plants.Zhao et al. [37] identified it as the cause of wilt in maple trees (in China), and Moral-Lopez et al., [38] in almonds (in Portugal and Spain).Another virulent species, F. curvatum, which we identified in pathogenicity tests, has previously been found to be associated with dieback disease in Dendrobium officinale [39] and leaf spot of cherry [40] in China.
In this study, although other Fusarium species (F.clavum, F. falciforme, F. fredkrugeri, F. glycines, F. nanum, F. nematophilum, F. solani) from Turkish grapevine nurseries reduced root biomass in grapevine seedlings compared to the control, their virulence was not as consistent as F. annulatum, F. brachygibbosum, F. curvatum, and F. nirenbergiae.These results support the view that most of the Fusarium species may be present in grapevine seedlings as parasites in the current study.However, it has been determined that these species with low virulence here were highly virulent in other crop plants, causing root and fruit rots in these plants and thus resulting in yield losses.Medeiros-Araújo et al. [41] characterized Fusarium species (F.falciforme, F. kalimantanense, F. pernambucanum, and F. sulawesiense) associated with fruit rot of muskmelon in Brazil.From these species, F. falciforme and F. sulawesiense were found to be more aggressive than others.Zhang et al. [42] detected that F. nanum strains obtained from muskmelon fields in China were also associated with fruit rot.In another study conducted in Vietnam, F. falciforme was found to be an aggressive species causing wilt in chrysanthemum [43].F. clavum was reported as a plant pathogen causing leaf spot and fruit rot on tomato, and petal brown spot on rose in Italy [44,45].These studies indicate that different Fusarium species are pathogenic in different plants, while they have no significant effect in others.It is conceivable that host-pathogen interactions may be important in these events.Environmental factors (temperature, precipitation, relative humidity, soil structure-microbiome, etc.), the physiology and biochemistry of host plants, and gene contents of Fusarium fungi are also suggested to influence the availability of metabolites and toxins necessary for fungal growth and pathogenicity, which in turn influence the host-pathogen interaction [46,47].Some studies also suggest that Fusarium species provide various benefits to plants by enhancing plant growth, triggering production of secondary metabolites, and protection against pathogens [48].Jelenic et al. [49] found endophytic F. solani and F. subglutinans to reduce gray mold incidence (caused by Botrytis cinerea) on grape clusters, increase yield, and provide plants with more robust development under unfavorable weather conditions.However, no detailed biochemical or transcriptomics explanation underlying these positive effects could be suggested.Such studies reveal that much remains unknown about the interactions of Fusarium species in plants and their function in agriculture in phytopathological aspects.

Figure 1 .
Figure 1.Multi gene (TEF1-α and RPB2) Maximum Likelihood tree of Fusarium strains.Circles of different sizes show bootstrap support values from 1000 replicates which are indicated at the nodes.Bootstrap values less than 50% are not shown.The bold characters represent the Turkish strains.Dactylonectria torresensis CBS 129086 was used for rooting the ML tree.

Figure 1 .
Figure 1.Multi gene (TEF1-α and RPB2) Maximum Likelihood tree of Fusarium strains.Circles of different sizes show bootstrap support values from 1000 replicates which are indicated at the nodes.Bootstrap values less than 50% are not shown.The bold characters represent the Turkish strains.Dactylonectria torresensis CBS 129086 was used for rooting the ML tree.

Table 1 .
Location of surveyed grapevine nurseries, rootstock/cultivars, and isolation frequency of Fusarium species in Türkiye.
N/A: Data not available.

Table 3 .
Location of surveyed grapevine nurseries, cultivars, and the species found with their TEF1α and RPB2 gene sequence numbers.

Table 3 .
Location of surveyed grapevine nurseries, cultivars, and the species found with their TEF1-α and RPB2 gene sequence numbers.

Table 4 .
Mean wood lesion lengths caused by Fusarium species in the inoculation points of 1103 Paulsen rootstock plants after four months.

Table 4 .
Mean wood lesion lengths caused by Fusarium species in the inoculation points of 1103 Paulsen rootstock plants after four months.
AFP (Asma Fusarium Projesi in Turkish).* Means accompanied by same letter are not significantly different (p = 0.05) according to LSD tests.

Table 5 .
Mean basal necrose lengths caused by Fusarium species in 1103 Paulsen rootstock plants after four months.

Table 6 .
Mean root dry weights of 1103 Paulsen rootstock plants inoculated with Fusarium species after four months.
F. ann F. bra F. cla F. cur F. fal F. fre F. gly F. nan F. nem F. nir F. sol Fus.
detected 92% to 98% of Fusarium fungi, among other GTD pathogens, from plants sampled from two commercial grapevine nurseries in Spain and one in France.