Current status of grapevine trunk disease pathogens on asymptomatic nursery-produced grapevines in Türkiye

Summary. Good health of grapevine plants is important for productivity and sustainability of newly established vineyards, and accurate detection of bacterial and fungal pathogens is a prerequisite for managing the diseases they cause in nurseries. This study screened marketable, bare-rooted grapevine plants, obtained from different geographical regions of Türkiye, for fungal pathogens associated with grapevine trunk diseases (GTDs). In 2021, 43 grapevine nurseries located in eight provinces were surveyed to reveal the status of GTD pathogens on asymptomatic marketable plants. Fungal pathogens isolated from the roots and basal parts of asymptomatic dormant grapevines were identified using with morphological characteristics and molecular markers, and were subjected to pathogenicity tests. Six species; Cytospora viticola, Diaporthe ampeli-na, Diplodia seriata, Lasiodiplodia brasiliensis, Neofusicoccum parvum, and Truncatella angustata (associated with dieback), and six species; Cadophora ferruginea, Cadophora luteo-olivacea, Cadophora malorum, Phaeoacremonium minimum, Phaeoacremonium tuscanicum and Phaeomoniella chlamydospora (associated with Petri disease) were identified based on DNA sequencing of ITS and TEF1-α genes. GTD pathogens were detected in 12 and 14 of the 43 nurseries, respectively. Pathogenicity tests on 1103P vines revealed that all species were pathogenic ( N. parvum and C. luteo-olivacea being the most virulent), and caused significant wood necroses when compared to non-inoculated experimental controls. This is the first report of C. ferruginea, C. malorum, L. brasiliensis, and P. tuscanicum associated with GTDs in Türkiye.


INTRODUCTION
Grapevine nurseries became important early in the 20 Century, when grafted vines began to be planted in many regions.According to data from the Turkish Ministry of Agriculture and Forestry, more than 60 establishments currently produce grapevine saplings, and over three million standard grafted grapevine plants (registered) are produced annually (Anonymous, 2019).Grafted vines are often in demand for establishment of each new vineyard, for planting to replace dying grapevines, or for small scale retail sales.Although vineyard areas in Türkiye decreased by 9.8% between 2012 and 2017, demand remains high for grafted grapevine saplings (Söylemezoğlu et al., 2020).
In newly established vineyards, plants may die due to factors related to physiological issues and cultivation techniques, such as unfavourable climatic conditions, poor planting practices, nutritional disorders, and the quality of propagation material.Nematodes, insects, soil-borne fungi, and grapevine trunk pathogens also cause serious plant losses in nurseries (Gramaje and Armengol, 2011).
Most of the previously described GTD pathogens were also identified in Türkiye in mature vineyards, regional grapevine nurseries and young vineyards.Poyraz and Onoğur (2013) carried out a survey targeting just Petri Disease and Esca pathogens in the nurseries and mature vineyards in the Aegean Region, where Akgül et al., (2015) identified fungal trunk pathogens in 10-30-year-old Sultana Seedless plants.Akgül and Ahioğlu (2019) screened GTD pathogens associated with young grapevine decline in 1-3-year-old vineyards in Southern Türkiye.A survey 3 years later revealed occurrence and diversity of black foot pathogens in nurseries (Akgül et al., 2022).However, no substantial information is available on the current status of other GTD pathogens in marketable dormant plants in Turkish grapevine nurseries.Determining the latent fungal pathogens in each nursery may help to develop appropriate plant protection technology for producing healthy plants.
The objectives of the present study were: (i) to assess the presence of GTD pathogens on asymptomatic marketable plants produced in Turkish grapevine nurseries; (ii) to identify the associated fungal species based on molecular characterization; and (iii) to determine the virulence of representative isolates using pathogenicity tests on dormant grapevine cuttings.

Survey and isolation of dieback and Petri Disease pathogens
During January 2021, a total of 450 dormant grapevine plants were sampled from the nurseries (10-12 plants from each nursery), located in eight provinces Türkiye (Adıyaman, Bursa, Denizli, Manisa, Mersin, Şanlıurfa, Tekirdağ, and Tokat) (Figure 1).Roots and rootstocks were washed under running tap water, and were then superficially disinfected with sodium hypochlorite solution (>5% active chlorine, and diluted in sterile distilled water (1:1 v/v)) for 3 min.The tissues were then rinsed with sterile distilled water, briefly blotted on sterile paper towels, and allowed to dry under a sterile cabinet.Root hairs and inner tissues of the rootstocks (3-4 mm long) were then placed (six to seven fragments per Petri dish) onto PDA (Potato Dextrose Agar, Conda Lab) amended with streptomycin sulfate (250 mg×L -1 ).Petri dishes (10-12 dishes per nursery) were incubated at 24°C in the dark for 7-10 d.Resulting fungal colonies were examined under a light microscope (Olympus BX51), and were sub-cultured to fresh PDA plates for further studies.Altogether, 285 fungal colonies were isolated from the samples.Based on microscopic features and colony morphologies specified in relevant publications (Barnett and Hunter, 2003;Essakhi et al., 2008;Trouillas and Gubler, 2010;Phillips et al., 2013;Lawrence et al., 2017;Maciá-Vicente et al., 2020), Botryosphaeriaceae, Cadophora, Cytospora, Diaporthe, Phaeomoniella, Phaeoacremonium, and Truncatella spp.were considered as probable GTDs pathogens.As well, Acremonium, Alternaria, Aspergillus, Aureobasidium, Cladosporium, Clonostachys, Entoleuca, Epicoccum, Fusarium, Mortierella, Petriella, Penicillium and Trichoderma spp.were also determined, but they were considered as endophytic species.Isolation frequencies (%) of Petri disease and other trunk pathogen fungi was calculated as proportions of 70 tissue fragments from 10-12 vines in each nursery (Table 1).The overall isolation frequency (%) of each fungus was calculated from isolation frequencies in each nursery.The prevalence of each species (%) was calculated as the proportion of the nursery numbers (pathogen detected) to the total nursery number.The overall disease prevalence (%) was calculated as the proportion of nursery numbers (pathogen detected) to the total nursery number.

MOLECULAR IDENTIFICATION OF FUNGI
To obtain pure cultures and provide genetic purity, single spores or hyphal tips of the fungi were isolated under a light microscope.Twenty-six representative isolates were used for identification with molecular markers.Approx.50 mg of aerial mycelia was harvested from each pure culture (grown on PDA, incubated at 25°C, in the dark, for 7-10 d).Total DNA was extracted following the CTAB (2%) protocol (O'Donnell et al., 1998).Genomic DNA from each isolate was diluted with 80 µL of PCR grade water, then stored at -18°C for further use.For sequencing, ITS1, 5.8S, and ITS2 regions of the rDNA, and partial TEF 1-α (translation elongation factor) genes, were amplified with PCR reactions using the ITS4/ITS5 and EF688F-EF1251R primers (White et al., 1990;Alves et al., 2008).The PCR reaction mixtures each contained 5 μL of buffer (10× Green Buffer, DreamTaq Green DNA Polymerase; Thermo Scientific), 2 μL of the dNTPs mixture (10 mM each, Thermo Scientific), 1 μL of forward and reverse primers (stock concentration, 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 (approx.100 ng•μL −1 ).PCR amplifications were using a Simpli-Amp A24811™ Thermal Cycler (Applied Biosystems), with the following conditions; 95°C for 3 min.(initial denaturation), followed by 35 cycles each of denaturation at 95°C for 1 min, annealing at 52°C (for ITS) or 53°C (for TEF1-α) for   The ITS and TEF1-α sequences were submitted to the NCBI GenBank, and accession numbers were obtained (Table 2).

Pathogenicity tests
All the 26 isolates were subjected to pathogenicity tests (two replicates in a year) on potted 1103 Paulsen rootstock plants in a controlled climate room (26°C, 80% relative humidity, and 12 h illumination).Dormant cuttings (each 30 cm long, with three buds) were superficially disinfected with sodium hypochlorite solution (2.5%) for 3 min.They were then rinsed once with sterile distilled water and blotted on sterile paper towels.The ends of the cuttings were then cut with a sterile pruning shear, and their bases were dipped in gibberellic acid solution (2000 µg•mL -1 ) for 10 sec, to induce root formation.The apical ends were inoculated with mycelium agar discs (5 mm diam.) of fungal isolates and wrapped with parafilm (Curwood ® ) to allow colonization.For non-inoculated controls, sterile agar discs were placed on the apical ends of the cuttings (Ayres et al., 2011).After inoculation, the cuttings were planted in plastic bags (one cutting per bag) each containing 1 L of the potting mix (peat moss, perlite, and sawdust in equal volumes) and watered.Twelve plants per isolate (one plant per pot and four replicates with three plants per replicate) were inoculated with each of the isolates.The plants were supplied with Hoagland solution twice each month for 4 months to provide balanced nutrition.At the end of the period, the plants were uprooted, and their shoots and roots were removed with a pruning shear.The inoculation points were split with a knife, and the lengths of necrotic wood tissues were measured with a caliper and recorded.The pathogenicity of the isolates was confirmed by following Koch's postulates.The inoculated pathogens were re-isolated from the symptomatic wood chips but not from the non-inoculated controls.Mean lesion lengths were analyzed separately as Petri diseases or other trunk pathogens, because the growth rates of the fungi causing the diseases were not the same either in PDA cultures or in wood tissues.

Statistical analyses
Analysis of variance (ANOVA) was carried out on data of lengths of wood necrosis (mean lengths of two experiments), and the data were checked for normality.Means were compared using Fisher's least significant difference (LSD) test (at P = 0.05) (Gomez and Gomez, 1984).

Isolation of the fungi involved in disease prevalence in the nurseries
According to morphological/microscopic examination, some of the fungi related to Petri diseases and other GTD pathogens, referred to here as trunk pathogens, were detected in marketable plants in the surveyed grapevine nurseries (Table 1).These pathogens were isolated from 23 (54%) of the 43 surveyed nurseries.No pathogens were detected in 20 nurseries.One or more fungal species associated with both diseases were isolated from the roots and rootstocks of asymptomatic dormant vines.When the fungal pathogens were grouped separately, the isolation frequencies of Petri diseases were 27.9%, and other trunk pathogens 32.5%.Phaeoacremonium spp.and P. chlamydospora were the most prevalent fungi isolated (25.9%), followed by Botryosphaeriaceae (13.9%),Cadophora (11.6%),Diaporthe and Truncatella spp.(7.0%).

Molecular identification of the isolates
Identification of 26 isolates was performed using partial sequencing of ITS and TEF 1-α genes, and nucleotide sequences were deposited in the GenBank with accession numbers OP412792 to OP412817, OP508220 to OP508233, and OP550034 to OP550044 (Table 2).According to nucleotide BLAST searches (with ≥99 similarities), nine isolates were associated with Petri disease, and 17 isolates were detected as trunk pathogens.and C. malorum (Kidd & Beaumont) W. Gams)) predominated (six isolates; 66.7%) the fungi related to Petri disease.Only one isolate each (5.9%) of Phaeoacremonium minimum, P. tuscanicum, and P. chlamydospora belonged to Togniniaceae and Phaeomoniellaceae.Among these, C. ferruginea, C. malorum, L. brasiliensis, and P. tuscanicum had not been previously recorded in vineyards in Türkiye.

Pathogenicity tests
After 4 months in of 1103 Paulsen cuttings, all isolates caused blackish-brown vascular discolourations (of mean lengths from 8.2 to 40.0 mm) below inoculation points (Figure 2).No visible symptoms were observed on the green shoots or leaves of the plants.When the inoculated fungal groups (Petri disease pathogens and other GTD pathogens) are examined separately, wood lesions caused by Petri disease pathogens were less extended than those caused by other GTD pathogens.Among the dieback fungi, N. parvum produced the most extended lesions (from 36.8 to 40.0 mm), and was the most virulent pathogen (Table 3).It was followed by L. brasiliensis (mean lesion length 24.7 to 32.6 mm), T. angustata (22.5 to 29.9 mm), and D. ampelina (9.7 to 13.2 mm) (Table 3).Average lesion lengths produced by C. viticola were slightly longer (8.2 mm) than non-inoculated control (6.2 mm).Similarly, the isolates associated with Petri disease did not cause shoot or leaf symptoms, but their mean internal lesion lengths were longer (P ≤ 0.05) than that for the control.Cadophora isolates were more virulent (mean lesion length 9.4 to 9.7 mm), compared to Phaeoacremonium spp.and P. chlamydospora (8.5 to 8.9 mm), but no statistical difference was found between mean lesion lengths of Cadophora spp.(Table 4).The re-isolation experiment also confirmed severe colonization of the plant wood tissues.Both dieback and Petri disease fungi were recovered, with isolation rates from 18.6 to 72.4%.Th e virulence variability between the species was found by averaging the lesion lengths produced by isolates within each species (Figures 3 and 4).According to this evaluation, Cadophora luteo-olivacea was the most virulent fungus causing Petri disease and N. parvum was the most virulent fungus causing other GTDs.

DISCUSSION
Grafted grapevine production is a critical sector of Turkish viticulture.Screening the plants for health and determining related pathogens are important steps for preventing the distribution of diseases.Th e present study was conducted in Turkish grapevine nurseries to determine prevalence of GTD pathogens on asymptomatic marketable plants.Th e results showed that dieback (in 30% of surveyed nurseries) and Petri disease pathogens (33% of nurseries) were moderately prevalent.Previously, black foot pathogens were found to be very common in most of surveyed nurseries (Akgül et al., 2022).Nine of 43 nurseries producing non-graft ed Sultana Seedless grapevines were examined in this survey, and dieback and Petri disease pathogens were identi- fied only in three nurseries, from asymptomatic plants.These results indicate that most of the rootstock mother plant plots obtained latent infections during vegetative or propagation stages in the infested nurseries.Almost all grapevine rootstock mother fields in Türkiye are irrigated by flooding or furrow irrigation.Plants are also not grown on trellises, so the shoots of rootstock mother grapevines are sprawled on the soil surfaces.Soil-borne inocula of Cadophora, Phaeomoniella, and Phaeoacremonium may have caused shoot infections of the rootstock mother vines, resulting in greater prevalence of Petri disease than GTDs in the surveyed nurseries.
Rootstock/scion mother plants used for grapevine propagation may harbour trunk pathogens without showing disease symptoms.Therefore, these pathogens can be disseminated over large areas within young infected vines, and disease symptoms may appear 8-10 years later (Gramaje and Armengol, 2011).This has been confirmed in several studies using conventional fungal isolation and next-generation sequencing methods.Aroca et al. (2010) evaluated plant health in grapevine nurs- In Türkiye, we have also detected many of the species listed above in some of the surveyed nurseries, and these fungi were moderately prevalent.Incidence of Botryosphaeriaceous fungi was calculated as 19% in rootstocks and 17% in scion cuttings.These rates are greater than those (11.4%)determined in the present study.Cadophora ferruginea, C. malorum, L. brasiliensis and P. tuscanicum were detected for the first time in Türkiye.Some Cadophora spp.(including C. luteo-olivacea, C. malorum, C. melinii, C. orientoamericana, C. novieboraci, and C. spadicis) have been previously detected in grapevine nurseries, in California, Canada, Germany, South Africa, and Uruguay (Rooney-Latham, 2005;Halleen et al., 2007;Navarrete et al., 2011;Gramaje et al., 2011;Úrbez-Torres et al., 2014;Gierl and Fischer, 2017;Travadon et al., 2015).These fungi have been suggested as having important roles in the occurrence of Petri disease in young grapevines.The thesis of Özben (2020) reported C. luteo-olivacea and T. angustata (identified morphologically and microscopically) in 12 grapevine nurseries in Türkiye, but no pathogenicity or molecular identification data were reported.Cadophora ferruginea has been reported on grapevine in the United States of America (Travadon et al., 2022), so our isolate (AFP159) would be the second of this fungus.Lasodiplodia brasiliensis was first reported on table grapes in Brazil (Correia et al., 2016), then in Mexico (Rangel-Montoya et al., 2021).Phaeoacremonium tuscanicum was first reported in Spain (Essakhi et al., 2008), and then in the other countries, including New Zealand (Graham et al., 2009), Algeria (Berraf-Tebbal, 2011), Iran (Mohammadi, 2012), andCanada (Úrbez-Torres et al., 2014).We carried out an additional search to find the occurrence of these fungi in scientific journals and the Mycobank databases, but no record was found aside from the above countries.
Regarding to virulence of the isolates, fungal pathogens associated with Petri disease were found to be more virulent than the other GTD pathogens on 1103P rootstock plants (4 months after incubations) in pathogenicity tests.In previous studies, Lasiodiplodia and Neofusicoccum were shown to be highly virulent on grapevines when compared to other Botryosphaeriaceous fungi (Luque et al., 2009;Úrbez-Torres and Gubler, 2011).Neofusicoccum parvum was the most virulent species among 17 dieback-related isolates evaluated, followed by L. brasiliensis, T. angustata, and D. ampelina in the present study.Correia et al. (2016) studied the phylogeny, distribution, and pathogenicity of Lasiodiplodia species on table grapes in Brazil, and L. brasiliensis was the most virulent species, followed by L. theobromae, L. pseudotheobromae and L. hormozganensis on detachedgreen shoots of the Isabel grape cultivar, after 10 d incubation.Similarly, Rangel-Montoya et al. (2021) tested the pathogenicity of L. brasiliensis, L. gilanensis, L. exigua, and L. crassispora, and L. brasiliensis was the most virulent, causing average lesion lengths of 5.1-5.5 mm on Cabernet Sauvignon cuttings after 2 months incubation.Akgül et al. (2015) tested pathogenicity of several grapevine trunk disease pathogens, obtained in the Aegean Region of Türkiye.These tests were for approx.4 months on potted 1-year old Sultana Seedless plants.Among these fungi, N. parvum was the most virulent, producing lesions of average length 79.1 mm, followed by L. theobromae (59.8 mm), D. ampelina (34.6 mm), Phaeomoniella chlamydospora (27.5 mm), Togninia minima (25.5 mm), B. dothidea (24.8 mm) and D. seriata (21.4 mm).
The pathogenicity test results from the present study were similar to the previous studies but not to all.Some inconsistencies can also be seen among previous pathogenicity test results, probably due to differences in cultivar susceptibility, incubation conditions, the virulence of fungal isolates, and the inoculation methods used.
This research has demonstrated the current status of GTD pathogens on asymptomatic, marketable, nurseryproduced grapevines in Türkiye.Although these pathogens were rare, the results indicate that appropriate disease control methods, such as screening of pathogens, hot water treatments, and use of biological pesticides, should be adopted in rootstock plots of grapevine nurseries.

Table 2 .
Petri disease and other grapevine trunk disease pathogens identified in this study, their locations, hosts, and GenBank accession numbers.

Table 3 .
Mean lesion lengths and proportions (%) of re-isolations for species of fungi associated with grapevine trunk diseases (except Petri disease) on 1103 Paulsen rootstock plants.
*Means accompanied by the same letter are not significantly different (P = 0.05), according to LSD tests.Each mean is the average for 24 cuttings (12 per experiment).

Table 4 .
Mean lesion lengths resulting from inoculations of grapevine plants with different fungal species associated with Petri disease on 1103 Paulsen grapevine rootstock plants.