A Novel Seimatosporium and Other Sporocadaceae Species Associated with Grapevine Trunk Diseases in Cyprus

Abstract

Besides well-known grapevine trunk disease (GTD)-related pathogens, there is an increased interest in wood-colonizing fungi that infect grapevines. During 2017–2018, a survey was conducted in Cyprus and wood samples were collected from vines exhibiting typical GTD symptoms. Based on morphological and multilocus phylogenetic analyses (ITS, LSU, bt2, tef1-a), four species in the Sporocadaceae family were described and typified; two in the genus of Seimatosporium: Seim. cyprium sp. nov. and Seim. vitis-viniferae and two in Sporocadus: Spo. kurdistanicus and Spo. rosigena. The teleomorph of Seim. cyprium sp. nov. was also described. Pathogenicity trials with representative isolates of each species were performed on woody stems of two-year-old potted grapevines for 12 months under field conditions. All isolates were pathogenic, causing dark brown to black vascular discoloration, extending upward and downward from the inoculation point. Sporocadus isolates were significantly more aggressive than Seimatosporium with lesion lengths ranging from 9.24 to 6.90 and 4.13 to 4.00 cm, respectively. Successful re-isolations were also evident for all species and isolates. Seim. cyprium sp. nov. is a newly described species, while Spo. kurdistanicus and Spo. rosigena are reported for the first time in Europe on Vitis vinifera, suggesting the potential role of Sporocadaceae in the GTDs complex.

1. Introduction

Grapevine trunk diseases (GTDs) form an aggregate of fungal diseases that are currently considered the most destructive biotic factor of grapevines globally [1,2,3,4]. Multifaceted adverse effects due to GTDs include reduced longevity and profitable vineyard lifespan [5], cumulative yield losses, increased costs due to applied management practices, and premature replanting of severely affected vineyards [6,7]. Although long considered a deteriorating factor of viticulture, GTDs’ incidence and subsequent consequences have spiked along the last three decades, mainly due to the circulation of potentially contaminated planting material, an industry shift towards cultivation systems that render vines more prone to wood infections, as well as the lack of effective plant protection products [6,8,9,10].

To date, over 140 fungal species (predominately ascomycetous) belonging to 35 genera have been reported to be associated with GTDs worldwide, while numerous grapevine microbiome studies enrich our knowledge on species involved with GTDs; however, the degree of involvement of many GTD-related species remains elusive [6,11,12,13].

Several fungi within the family of Sporocadaceae (Xylariales, Sordariomycetes) have been reported as saprophytes, endophytes, and plant pathogens in a wide host range [14,15,16,17,18,19]. More specifically, species within Neopestalotiopsis Maharachch., K.D. Hyde and Crous, Pestalotiopsis Steyaert, Seimatosporium Corda, Sporocadus Corda, and Truncatella Steyaert have been recently found in association with Vitis vinifera, either as part of its mycobiome [12,20,21,22] or as components of the GTD complex [13,16,23,24,25,26,27,28,29]. The Sporocadaceae family was revived by Jaklitsch et al. (2016) to accommodate coelomycetous fungi, also known as pestalotioid, characterized by multi-septate conidia, bearing appendages at one or both end-cells [30]. Due to the restricted delineation capabilities of morphological features, phylogenetic studies based on multi-locus sequence data have contributed to address controversial ambiguities in Sporocadaceae [30,31,32,33]. More recently, an extensive multigene phylogenetic study combined with morphological data on appendaged coelomycetous by Liu et al. (2019) placed them in Sporocadaceae and recognized 30 monophyletic genera, including Seimatosporium and Sporocadus also [34].

The genus Seimatosporium was erected with Seim. rosae Corda as the type species and is characterized by septate and branched conidiophores with discrete integrated conidiogenous cells grown within acervuli or pycnidia that produce mostly 3-septate (3 to 6) mostly fusiform, ovoid to falcate conidia with brown-colored zed median cells with or without a single apical (centric) and basal (excentric) appendage [34]. Seimatosporium and Sporocadus were initially synonymized [35]; however, the latter was recently resurrected as a distinct genus and lectotypified on Spo. lichenicola Corda, Icon. fung. (Prague); accommodating species with fusoid, ellipsoidal to obovoid, multi-septate (1–7), mostly 3-septate conidia, bearing colorless obconic basal and apical cells with a truncate base and a round apex, respectively, and pale brown to brown median cell [26,34,36]. Notably, the conidia of Sporocadus species are predominately non-appendaged apart from Spo. trimorphus and Spo. rosarum [34].

Currently, nine Seimatosporium and five Sporocadus species have been found in association with V. vinifera worldwide. More specifically, Seim. botan Sat. Hatak. and Y. Harada, Seim. hysterioides (Fuckel) Brockmann, Seim. lonicerae (Cooke) Shoemaker, Seim. luteosporum D.P. Lawr. and Travadon, Seim. marivanicum Abdollahz., Nahvi M. and Khaledi E., Seim. parasiticum (Dearn. and House) Shoemaker, Seim. vitifusiforme D.P. Lawr. and Travadon, Seim. vitis Y.P. Xiao, Camporesi and K.D. Hyde, and Seim. vitis-viniferae F. Liu, L. Cai and Crous [12,13,26,34,37,38] have been reported in Australia, Chile, Iran, Italy, Hungary, USA. Although Seim. fusisporum H. J. Swart and D.A. Griffiths [24] and Seim. macrospermum (Berk. and Broome) B. Sutton have been also associated with grapevine, the former has been re-evaluated to a sister clade and classified as Allelochaeta fusispora (H. J. Swart and D. A. Griffiths) Crous [39], while the latter, given the absence of conidial appendages it has been moved to Sporocadus, as Spo. macrospermus (Berk. and Broome) M. Morelet [34,40], along with Spo. kurdistanicus Abdollahz., Nahvi M. and Khaledi E., Spo. lichenicola, Spo. rhododendri (Schwein.) M. Morelet, and Spo. rosigena F. Liu, L. Cai and Crous [26,27,34], that have also been associated with V. vinifera. However, limited information is available regarding their pathogenic potential, mainly reported as endophytic saprobes of grapevine and other hosts [11,12,18,41,42,43].

Viticulture is essential for the economy and rural development of Cyprus, covering an acreage of 6790 ha with an annual yield of 23,570 tons [44]. However, the severity of GTDs, as well as the identity of the implicated microorganisms and their pathogenic status remains undescribed and needs to be studied in the country [45]. Considering the numerous Sporocadaceae isolations from grapevines exhibiting GTD symptoms in Cyprus, combined with multiple reports around the world, our objectives are: (a) to characterize Sporocadaceae species diversity in the local vineyards, based on morphological and molecular analyses, and (b) to evaluate their pathogenic potential on grapevines.

2. Results

2.1. Molecular Identification and Phylogenetic Analyses

The best-fit nucleotide substitution model per locus based on the Bayesian Information Criterion (BIC) were: Kimura-2 parameter, also allowing for a proportion of invariant sites (I) and gamma distributed rates (G) (K2P + I + G) for the internal transcribed spacer regions and the 5.8 gene (ITS); Tamura-Nei parameter with I and G4 (TNe + I + G) for partial regions of the ribosomal large subunit (LSU); three substitution site model with empirical-based frequencies (F) and I (TPM3 + F + I) for the translation elongation factor 1-a (tef1-a) gene, and Hasegawa-Kishino-Yano (HKY) with F, I, and G4 (HKY + F + I + G) for the β-tubulin (tub2) gene. Phylogenetic trees of single-locus alignments obtained using the models suggested that four major clades belonging to the genera Allelochaeta, Sarcostroma, Seimatosporium, and Sporocadus were obtained for tub2 and tef1-a. More specifically, the tef1-a tree was the only one that enabled the separation of all species with high bootstrap values (Supplementary Figure S1). For tub2, all species could be separated, except Seim. vitifusiforme (CBS 142600) and Seim. marivanicum (CBS 143781) that were clustered in the same clade (Supplementary Figure S2). The ITS and LSU trees could not properly resolve at the species and/or genus level, due to high levels of nucleotide similarities in these regions (Supplementary Figures S3 and S4).

Sequence alignment of the of the four loci (ITS, LSU, tub2, and tef1-a) resulted in a 2375-character dataset, with 1539 being constant, 616 were parsimony informative, and 229 were variable and parsimony uninformative. After 100 ratchet iterations, the maximum parsimony (MP) analysis produced five equally most-parsimonious trees of 696 steps and a consistency index (CI) and retention index (RI) of 0.459716 and 0.754707, respectively. For the maximum likelihood (ML) analysis the ModelFinder algorithm of IQtree indicated a transition model with equal bases frequencies (TIM2e) with gamma distributed rates, allowing a proportion of invariant sites (TIM2e + I + G) nucleotide substitution model as the best fit for the concatenated dataset. ML and MP analyses revealed that all four Sporocadaceae species recovered from symptomatic grapevine cankers in Cyprus and are strongly supported (100 and ≥96%, respectively) as independent phylogenetic lineages (Figure 1).

2.2. Morphological Description

The color of Sporocadaceae colonies ranged from white to grayish and from light brown to light sepia on PDA and MEA, while on OA, they were from grayish white to light sienna or brown. Characters used to distinguish the species included the relative cell lengths of conidia, conidial septation, and the appendage morphology. Based on microscopic observations, all isolates produced septate conidia, which matched the descriptions for Sporocadaceae-like asexual forms. However, Sporocadaceae isolates were separated into two different groups. The first consisted of isolates (n = 6) showing mostly 3-septate, obovoid, thick-walled, pale brown to brown conidia that lacked appendages at both the basal and apical ends. These features matched those described earlier for the genus Sporocadus [34]. The second group (n = 10) included isolates with fusoid, pale brown, mostly 3- up to 6-septate conidia, bearing basal and apical (sometimes absent appendages, which resembled those of the genus Seimatosporium [34]. There were significant differences in conidial length (p [F_{(1, 209)} > 1058] < 0.0001) and width (p [F_{(1, 209)} > 1208] < 0.0001) between Sporocadus species, with Spo. kurdistanicus being on average longer and wider as compared to Spo. rosigena. Similarly, Seim. vitis-viniferae produced, on average, smaller conidia to Seim. cyprium, with significant differences in their conidial length (p [F(_{1, 121)} > 57.41] < 0.0001) and width (p [F_{(1, 121)} > 88.15] < 0.0001). Homothallic crosses were performed to induce sexual reproductive structures in obtained Sporocadaceae isolates. Seim. cyprium was the only species that produced abundant perithecia immersed on grapevine wood segments bearing eight uniseriate, single-septate, ascospores per ascus.

2.3. Effect of Temperature on Mycelial Growth

All Sporocadaceae isolates were able to grow at all tested temperatures and an analyses of variance (ANOVA) indicated no significant differences (p < 0.05) of the mycelial growth among the experiments, thus the data were combined. The relationship between mycelial growth at different temperatures was best described by a cubic response model (y = aT³ + bT² + cT + d). All regression coefficients were significantly different (p < 0.01), and the coefficients of determination (R²) ranged from 0.89 to 0.99 (

Table 1. Temperature-mycelial growth relationship for Sporocadaceae isolates from indigenous grapevine cultivars ^w.

Species	Isolate	Adjusted Model x					Optimum Temperature (°C) y	Growth Rate (mm/day) z
		R2	a	b	c	d
Seimatosporium cyprium	L111	0.98	−0.0003	0.0068	0.1964	−0.0451	24.1 a	4.4 cd
Sei. cyprium	L112	0.98	−0.0003	0.0065	0.2016	−0.0387	23.8 a	4.4 cd
Sei. vitis-viniferae	L34	0.98	−0.0009	0.0288	−0.0163	0.0867	21.1 c	4.1 e
Sei. vitis-viniferae	P60	0.99	−0.0006	0.0182	0.0785	−0.0383	22.2 b	4.1 e
Sporocadus kurdistanicus	L158	0.94	−0.0007	0.0201	0.0996	−0.0123	21.4 c	4.5 bc
Spo. kurdistanicus	L181	0.99	−0.0007	0.0192	0.0848	0.0306	20.3 d	3.8 f
Spo. rosigena	L105	0.99	−0.0007	0.0216	0.0826	−0.0557	22.2 b	4.8 a
Spo. rosigena	L240	0.99	−0.0007	0.0213	0.0849	−0.0446	22.1 b	4.7 ab

^w Data are the mean of six replicates per isolate. Means with the same letter, within a column, are not significantly (p = 0.05) different according to Kruskal–Wallis and the Dunn’s test for multiple comparisons. ^x Mycelial growth of PDA at 5 to 30 °C was adjusted to a quadratic model: y = aT³ + bT² + cT + d, with y = mycelial growth (mm/day); a, b, c, d = regression coefficients; and R² = coefficient of determination. ^y Optimum temperatures per isolate were estimated by the adjusted model. ^z Maximum growth rate per isolate was estimated by the adjusted model.

). Based on the adjusted models derived per isolate, the optimum temperatures of mycelial growth on PDA ranged from 20.3 to 24.1 °C with significant differences detected among them (Table 1). More specifically, optimum temperature ≥ 23 °C included only Seim. cyprium isolates. Isolates with an optimum temperature between 22 and 21 °C were Seim. vitis-vineferae and Spo. rosigena isolates, as well as the Spo. kurdistanicus isolate P158. The only species that had an optimum temperature < 21 °C was the Spo. kurdistanicus L181. The maximum mycelial growth rates also differed significantly (mean = 4.4; median = 4.4). More specifically, all isolates had a maximum growth rate > 4 mm/day, ranging from 4.1 to 4.8, except L181 with < 4 mm/day (Table 1). Furthermore, maximum growth rates on MEA cultures ranged from 2.6 to 5 mm/day (mean = 4.3; median = 4.9), with isolates L181 and the Seim. cyprium L111 bearing the highest and lowest values, respectively, and optimum growth temperatures 20.4–24.4 °C (data not shown). The highest average growth rate for Sporocadaceae isolates was evident on OA ranging from 4.4 to 5.9 mm/day (mean = 5.3; median = 5.5), with P60 and L34 being the slowest- and fastest-growing isolates, respectively, and optimum growth temperatures from 21.4 to 24.3 °C (data not shown).

Morphological comparisons, coupled with phylogenetic analyses of the ITS, LSU, bt2, and tef1-a dataset, identified four distinct and strongly supported lineages, one of which has no apparent species name. Thus, we propose the following new species name to properly circumscribe and typify this unique taxon.

Seimatosporium cyprium Kanetis L. and Makris G., sp. nov., Figure 2 and Figure 3.

MycoBank number: MB 845835.

Etymology: Epithet refers to the country where the species was first found.

Type: CYPRUS: Limassol Province, Chandria; 34.940583°N, 32.997139°E, 1200 meters above sea level (masl); isolated from wood canker of V. vinifera, June 2017, Kanetis L. (holotype L111; ex-type CBS 149019). GenBank accession numbers: ON680684, ITS; ON705769, LSU; ON695856, bt2; ON863790, tef1-a.

Description: Sexual morph: Ascomata perithecial, stromatic, appearing on the surface of grapevine wood segments, dark circular patches, 198–441 μm (av. = 300 ± 63.8 μm, n = 30) diam., solitary, slightly immersed or directly below the wood epidermis, depressed globose, membranous, black, with a centrical ostiole. Asci 80.8–109.1 × 6.5–9.5 μm (av. = 98.6 ± 8.47 × 8.2 ± 0.91 μm (n = 20), 8-spored, unitunicate, cylindrical, straight, or slightly curved, pedicellate, apically rounded. Ascospores 11.5–16.9 × 5.1–6.8 μm (14.5 ± 1.19 × 5.9 ± 0.43 μm, n = 30), uniseriate, or overlapping uniseriate, hyaline, fusiform to ellipsoidal, hyaline, 1-septate, smooth-walled. Asexual morph: Conidiomata in pure cultures, acervular, dark brown, black, scattered, erumpent, superficial or semi-immersed; on grapevine wood segments 265–774 μm (501 ± 118 μm, n = 30) in diameter; conidiophores branched, cylindrical or reduced to conidiogenous cells, colorless, smooth; conidiogenous cells discrete, mostly cylindrical, slightly curved, colorless, smooth, 10.1–26.4 × 1.4–2.2 μm (16.3 ± 4.15 × 1.8 ± 0.28 μm; n = 15). Conidia straight or slightly curved, 3-septate, wall smooth, 15.7–23.1 × 3.9–5.3 μm (av. = 18.5 ± 1.61 × 4.5 ± 0.32 μm; n = 50), bearing appendages; basal cell obconic, with a truncate base, subcylindrical, colorless, 2.7–5.5 μm (av. = 4.3 ± 0.64 μm) long; two median cells, pale brown, ± equal, each 3.9–6.8 μm (av. = 5.2 ± 0.58 μm) long; apical cell obtuse or conical, colorless, 3.1–6.1 μm (av. = 4.1 ± 0.61 μm) long, apical appendage single, unbranched, 5.8–32.2 μm (av. = 19.6 ± 5.05 μm) long; basal appendage single or rarely dual, unbranched or rarely branched, excentric, 2.6–29.0 μm (av. = 16.5 ± 6.32 μm) long; mean conidium length/width ratio = 4.1:1.

Colony of Seim. cyprium isolate L111 (CBS 149019) on PDA growing, slightly raised, greyish white with smooth wooly margin, reaching 53.5 mm in diameter after 14 days at 25 °C in darkness. Colony on MEA slightly raised, wooly white with smooth wooly margin, reaching 64.75 mm in diameter after 14 days at 25 °C in darkness. Colony on OA raised, wooly light sienna to white with smooth margin, reaching 69.75 mm in diameter after 14 days at 25 °C in darkness. The optimum temperature of mycelial growth on PDA was estimated at 23.8 °C with a growth rate of 4.4 mm/day.

Additional specimen examined: CYPRUS: Limassol Province, Kyperounda, 34.944722°N, 32.98725°E, 1,300 masl; isolated from wood canker of V. vinifera, June 2017, L. Kanetis (L112). GenBank accession numbers: OΝ695889, ITS; ON692404, LSU; ON695848, bt2; ON863791, tef1-a.

Ecology and host characteristics: Isolates L111 (=CBS 149019) and L112 belonging to the present species were recovered from vines of the indigenous wine grape cv. Mavro. Isolate L111 was co-isolated with Phaeomoniella chlamydospora.

Distribution: Cyprus.

Notes: Based on multi-locus phylogenetic analyses Seim. cyprium formed a distinct, highly supported clade that is closely located to Seim. marivanicum (ML/MP = 100/99), Seim. vitifusiforme (ML/MP = 100/99), and Seim. luteosporum (ML/MP = 100/100). Seim. cyprium differs from Seim. marivanicum in ITS (one substitution), tub2 (two substitutions), and tef1-a (one insertion or deletion), while their LSU data are identical, however the species can be distinguished in the tef1-a phylogram; with a total of four PWD. Despite their high sequence similarities, both species have distinct morphological differences in terms of conidial dimensions and septation, with a mean length/width ratio of 4.1 vs. 5(-6), septa number of 3 vs. 3(-6), and media cell length of 5.2 ± 0.58 vs. 8 ± 0.7 μm for Seim. cyprium and Seim. marivanicum, respectively. In addition, the apical appendages of Seim. cyprium are distinctly longer compared to the ex-type of Seim. marivanicum (

Table 2. Conidial features of Seimatosporium and Sporocadus species reported on Vitis vinifera ^a.

Species	Conidia			Appendages			References
	Dimensions (μm)	Septa (No.)	L:W Ratio	Type	Apical (μm)	Basal (μm)
Seim. botan (NBRC 104200 T)	16–20 × 5–7 (av. 18 × 6) 16–20 × 4–5 (av. 18 × 4)	3 3	2.6–3.8 (av. 3) 4–5 (av. 4.6)	basal apical and basal	- 4–8 (av. 5.8)	4–8 (av. 6) 4–8 (av. 5.4)	[46]
Seim. cyprium (CBS 149019 T)	15.7–23.1 × 3.9–5.3 (av. 18.5 × 4.5)	3	4.1	apical and basal	5.8–32.2 (av. 19.6)	2.6–29.0 (av. 16.5)	Present study
Seim. hysterioides *	12–16.5 × 5.2–6.5	3	-	often lacking, basal or both	10–15	10–15	[47]
Seim. lonicerae *	9–16 × 3.5–5 (av. 13 × 4.4)	3 (rarely 2)	3	both or only basal	3–7 (av. 5.5)	2–12 (av. 7)	[48]
Seim. luteosporum (CBS 142599 T)	16.7–25.4 × 4.7–5.6 (av. 19.9 × 5.3)	3	-	apical and basal	10.1–24.2 (av. 17.9)	9.8–23.6 (av. 16.7)	[38]
Seim. marivanicum (CBS 143781 T)	16–31 × 3–7 (av. 24 × 3.5)	3–6	5–6	apical and basal	7–20 (av. 15)	5–20 (av. 16)	[26]
Seim. parasiticum *	22–35 × 5–7 (av. 27.5 × 5.5)	3–5 (mostly 5)	5	apical and basal	2–5 (av. 3.5)	2–8 (av. 4.5)	[48]
Seim. vitifusiforme (CBS 142600 T)	18.6–30.3 × 3.7–5.1 (av. 24.9 × 4.2)	3	-	apical and basal	7–12.6 (av. 10)	3.9–16.6 (av. 9.5)	[38]
Seim. vitis (MFLUCC 14-0051 T)	34–40 × 14–17 (av. 37 × 15)	3	-	basal	-	4–8 (av. 5)	[31]
Seim. vitis-viniferae (CBS 123004 T)	13.5–26 × 4.5–6 (av. 16.5 × 5.2)	3–6 (mostly 3)	3.2	basal or both	4–11 (av. 7)	4–10 (av. 7.9)	[34]
Seim. vitis-viniferae (CBS 149016)	13.5–18.6 × 4.6–5.8 (av. 16 × 5.2)	3–5 (mostly 3)	3.1	basal or both	2.5–24.1 (av. 11.2)	3.7–19.7 (av. 10.7)	Present study
Spo. kurdistanicus (CBS 143778 T)	18–24 × 6.5–9.5 (av. 21.5 × 8)	3	3	absent	-	-	[26]
Spo. kurdistanicus (CBS 149022)	20.6–30.1 × 7.5–10 (av. 24.2 × 8.8)	3	2.8	absent	-	-	Present study
Spo. lichenicola (NBRC 32625)	18–25 × 5.5–8 (av. 21.6 × 7.2)	3 (rarely 5)	3	absent	-	-	[34]
Spo. macrospermus *	28–39 × 9–12.5	5	-	absent	-	-	[35]
Spo. rhododendri *	15.5–20 × 6.5–8.5	3	-	absent	-	-	[49]
Spo. rosigena (CBS 466.96)	12–14 × 5–7.5 (av. 13 × 6.5)	3 (rarely 2)	-	absent	-	-	[34]
Spo. rosigena (CBS 149021 T)	13.2–17.9 × 5.3–6.8 (av. 15.4 × 6.2)	2–3 (mostly 3)	2.5	absent	-	-	Present study

^a CBS: Culture collection of the Westerdijk Fungal Biodiversity Institute, Utrecht, the Netherlands; NBRC: Biological Resource Center, National Institute of Technology and Evaluation, Chiba, Japan. T: ex-type strain. * Designated ex-type culture not available; - = not available or not applied.

). Similarly, phylogenetic analyses and estimates of evolutionary divergence between concatenated sequences used herein, support strong species differentiation (ML/MP = 99/100) of Seim. pistaciae (CBS 138865) and Seim. rosae (CBS 139823), given that they exhibit three PWD and discrete morphological characteristics (Supplementary Table S1; Figure 1) [34].

Seim. luteosporum has significant sequence data differences in ITS (two substitutions and two insertions or deletions), LSU (four substitutions), tub2 (eleven substitutions and one deletion or insertion), and tef1-a (twelve substitutions and seven deletions or insertions) from Seim. cyprium and they could be clearly separated in all the single locus phylogenies, except ITS. However, Seim. cyprium cannot be clearly differentiated from Seim. luteosporum by conidial characteristics (Table 2). The LSU sequences of Seim. cyprium and Seim. vitifusiforme are identical, but they differ in ITS (one substitution), tub2 (one substitution) and tef1-a (five substitutions) and can be clearly distinguished only in the tef1-a phylogram. The conidial characteristics of both species are also discrete (Table 2). Unlike Seim. cyprium, no teleomorph has been currently reported for Seim. luteosporum, Seim. marivanicum, and Seim. vitifusiforme.

Seimatosporium vitis-viniferae F. Liu, L. Cai and Crous, Figure 4.

MycoBank number: MB 828397.

Description: Sexual morph: unknown. Asexual morph: Conidiomata on pure cultures acervular, dark brown to black, scattered, semi-immersed or superficial; on grapevine wood segments 187–546 μm (359 ± 96 μm; n = 30) in diameter; conidiophores septate, mostly branched, sometimes reduced to conidiogenous cells, colorless, smooth. Conidiogenous cells discrete or integrated, cylindrical, variable in size, 8.2–40.2 × 1.3–2.7 μm (av. = 18.3 ± 7.39 × 1.9 ± 0.33 μm; n = 15). Conidia fusoid, straight, mostly 3- up to 5-septate, smooth wall, 13.5–18.6 × 4.6–5.8 μm (av. = 16.0 ± 1.35 × 5.2 ± 0.28 μm; n = 50); bearing appendages; basal cell obconic with a truncate base, subcylindrical, colorless or similar to that of median cells, 1.8–3.8 μm (av. = 2.9 ± 0.46 μm) long; two median cells, ± equal, each 3.7–6.1 μm (av. = 4.9 ± 0.51) long; apical cell obtuse or conical, colorless, sometimes similar to that of median cells, 2.2–4.5 μm (av. = 3.4 ± 0.64 μm) long; apical appendage lacking or, when present, single, unbranched, 2.5–24.1 μm (av. = 11.2 ± 2.5) long; basal appendage single, unbranched, excentric, 3.7–19.7 μm (10.7 ± 4.67 μm) long; mean conidium length/width ratio = 3.1:1.

Colony of Seim. vitis-viniferae isolate P60 (CBS 149016) on PDA, slightly raised, wooly, light brown with smooth off-white margin, reaching 48 mm in diameter after 14 days at 25 °C in darkness. Colony on MEA slow-growing, slightly raised, wooly, light brown with undulate wooly, off-white margin, reaching 52.75 mm in diameter after 14 days at 25 °C in darkness. Colony on OA, fast-growing, slightly raised brown and light brown colony with smooth wooly, off-white margin, reaching 62.75 mm in diameter after 14 days at 25 °C in darkness.

Ecology and host characteristics: Eight isolates P46 (=CBS 149018), P56 (=CBS 149017), P57, P60 (=CBS 149016), P210, L33, L34, and L189 belonging to the present species were recovered from vines of the indigenous wine grape cvs. Mavro, Promara, and Xynisteri. All were single isolations from collected wood samples of symptomatic vines, except L33, L189, and P57 that were co-isolated with Ph. chlamydospora and Phaeoacremonium minimum.

Distribution: Cyprus, Iran, Italy, New Zealand, and Spain (V. vinifera).

Notes: Pronounced disparities in conidial size and characteristics can distinguish Seim. vitis and Seim. vitis-viniferae. More specifically, conidia of Seim. vitis ex-type specimen (MFLUCC 14-0051) [31] are reported to be larger than the conidia of the relevant Seim. vitis-viniferae specimen (CBS 123004; Table 2) [34]. Furthermore, the same authors report that Seim. vitis bears 3-septate conidia with basal appendages, while Seim. vitis-viniferae may produce 3-6-septate and appendaged conidia basally or at both ends (Table 2). In the present study, conidial characteristics of obtained isolates matched with those attributed to Seim. vitis-viniferae. However, the abovementioned distinct morphological differences are not supported by phylogenetic analyses of the available sequence data. The only available sequences of the Seim. vitis ex-type (ITS and LSU) are identical to the Seim. vitis-viniferae ex-type and the results are not different when other available datasets of ITS and LSU sequences are compared. Minor differences were evident when the tub2 sequence data obtained in this study (n = 8) and all others currently available in the NCBI database (n = 23) of Seim. vitis and Seim. vitis-viniferae were analyzed. The dissimilarities found (two substitutions and three insertions or deletions) are not consistently represented among the species, suggesting that the tub2 locus is not informative for species separation. Regarding the tef1-a locus, eight substitutions and two insertions or deletions are found within the same set of sequence data. A single, consistent nucleotide substitution in position 257 (T for Seim. vitis and to C for Seim. vitis-viniferae isolates) was detected, while all other nucleotide differences appear to be variably distributed between the species (Supplementary Figure S5). In conclusion, the available sequence data (ITS, LSU, bt2, and tef1-a) cannot strongly support differentiation of these two species.

Sporocadus kurdistanicus Abdollahz., Nahvi M. and Khaledi E., Figure 5.

MycoBank number: MB 838233.

Description: Sexual morph: unknown. Asexual morph: Conidiomata acervular, black, scattered, erumpent, semi-immersed; on grapevine wood segments, 210–636 μm (av. = 386 ± 89 μm; n = 30) in diameter; conidiophores septate, branched, sometimes reduced to conidiogenous cells, cylindrical, colorless, smooth. Conidiogenous cells discrete, mostly cylindrical, 16.3–50.9 × 2.3–4.6 μm (av. = 31.9 ± 8.88 × 2.9 ± 0.51 μm; n = 15). Conidia ellipsoid, straight or slightly curved, pale brown, and becoming mid-brown when mature, 3-septate, wall smooth, 20.6–30.1 × 7.5–10.0 μm (av. = 24.2 ± 1.88 × 8.8 ± 0.50 μm; n = 50), lacking appendages; basal cell obconic, acute or blunt base, occasionally with a narrow truncate base, pale brown or concolorous with median cells, 4.0–7.6 μm (av. = 5.4 ± 0.68 μm) long; two median cells, cylindrical to doliiform, hyaline to pale brown, and becoming mid-brown when mature, ± equal length, each 4.8–7.9 μm (av. = 6.0 ± 0.65 μm) long; apical cell conic with round apex, concolorous with the median cells, 5.7–8.7 μm (6.9 ± 0.75 μm) long; mean conidium length/width ratio = 2.8:1.

The colony of Spo. kurdistanicus isolate L181 (CBS 149022) on PDA growing, slightly raised, white with smooth, wooly, smooth margin, reaching 44 mm in diameter after 14 days at 25 °C in darkness. Colony on MEA growing, slightly raised, wooly, white with wooly, smooth margin, reaching 44 mm in diameter after 14 days at 25 °C in darkness. Colony on OA, fast-growing, slightly raised, greyish-white with wooly, smooth margin, reaching 77.75 mm in diameter after 14 days at 25 °C in darkness.

Ecology and host characteristics: Isolates L158 (=CBS 149023), L164, and L181 (= CBS 149022) belonging to the present species were recovered from vines of the indigenous wine grape cv. Xynisteri. Isolates L105 and L181 were co-isolated with Ph. chlamydospora, while L164 was co-isolated with P. minimum.

Distribution: Cyprus and Iran on V. vinifera.

Notes: Multigene, as well as single locus phylogeny of LSU, bt2, and tef1-a cluster ex-type and Spo. kurdistanicus isolates were collected in this study in a highly supported clade. Spore dimensions of isolates collected in this study are in the same range to the ex-type isolate from Iran [26], sharing similar characteristics (Table 2).

Sporocadus rosigena F. Liu, L. Cai and Crous, Figure 6.

MycoBank number: MB 828418.

Description: Sexual morph: unknown. Asexual form: Conidiomata black, acervular, scattered, erumpent, semi-immersed or immersed; on grapevine wood segments 349–1444 μm (av. = 805 ± 295 μm, n = 30) in diameter; conidiophores septate, branched, often reduced to conidiogenous cells, colorless, smooth. Conidiogenous cells discrete or integrated, mostly cylindrical, 15.8–41.6 × 1.6–3.2 μm (av. = 26.8 ± 7.61 × 2.4 ± 0.45 μm; n = 15). Conidia obovoid, ellipsoid, or subcylindrical, mostly 3- occasionally 2-septate, wall smooth, 13.2–17.9 × 5.3-6.8 μm (av. = 15.4 ± 0.99 × 6.2 ± 0.40 μm; n = 50), lacking appendages; basal cell obconic with acute or blunt base, hyaline to pale brown, or concolorous with median cells, 2.9–4.7 μm (av. = 3.7 ± 0.45 μm) long; two median cells, short-cylindrical to doliiform, hyaline or pale brown, and becoming mid-brown when mature, ± equal length, each 2.8–4.6 μm (av. = 3.7 ± 0.42 μm) long; apical cell conic, concolorous with the median cells, 3.8–7.3 μm (av. = 4.7 ± 0.86 μm) long; mean conidium length/width ratio = 2.5:1.

Colony of Spo. rosigena isolate L106 (CBS 149021) on PDA fast-growing, flat, dark mouse gray to light sepia with smooth margin, reaching 58.25 mm in diameter after 14 days at 25 °C in darkness, conidiomata black, acervular, gregarious or confluent, erumpent. Colony on MEA fast-growing, slightly raised, wooly, grayish-white with smooth dark sienna margin, reaching in 62.25 mm in diameter after 14 days at 25 °C in darkness, conidiomata black, gregarious or confluent. Colony on OA, fast-growing, slightly raised, wooly, white with smooth olivaceous buff margin, reaching 78.75 mm diameter after 14 days at 25 °C in darkness.

Ecology and host characteristics: Isolates L105, L106 (=CBS 149021), and L240 (=CBS 149020) belonging to the present species were recovered from vines of the indigenous wine grape cvs. Xynisteri and Giannoudi. Isolate L105 was co-isolated with Ph. chlamydospora.

Distribution: Cyprus, Iran, and New Zealand (V. vinifera), Latvia (Rhododendron), the Netherlands (Pyrus communis and Rubus fruticosus), and Italy (Rosa canina and Quercus ilex).

Notes: Multigene, as well as single locus phylogeny of LSU, bt2, and tef1-a cluster ex-type and Spo. rosigena isolates were collected in this study in a highly supported clade. The spore dimensions and all other conidial characteristics of the obtained isolates are in the same range to the ex-type (MFLU 15-0782).

2.4. Pathogenicity

After a 12-month incubation period, all Seimatosporium and Sporocadus isolates evaluated were pathogenic to 2-year-old cv. Xynisteri potted grapevines. Dark brown to black vascular discoloration developed on the wood tissue below the bark, extending upward and downward from the point of inoculation, with the mean lengths shown in Figure 7, while foliar symptoms were absent in all treatments. Although all fungal species caused lesions, the virulence varied among the genera and species, with all Sporocadus isolates being more aggressive than Seimatosporium (p < 0.0001; Figure 8). More specifically, there was no significant difference in wood discoloration among the Sporocadus isolates, ranging from 9.24 ± 1.40 to 6.9 ± 0.85 cm (0.103 = p > 0.999). Similarly, the aggressiveness of Seim. vitifusiforme and Seim. vitis-viniferae isolates were not significantly different (p > 0.999), causing lesion lengths from 4.00 ± 0.83 to 4.13 ± 0.71 cm (Figure 8). No symptoms were evident in the non-inoculated (negative control) plants; thus, they were excluded from the statistical analyses. Successful re-isolations were made only from the inoculated vines and the recovery percentages ranged from 35–67% for Seim. vitis-viniferae, 40% for Seim. cyprium, 25–32% for Spo. rosigena, and 28–44% for Spo. kurdistanicus. Retrieved isolates were confirmed with those used in grapevine inoculations based on morphology (culture and conidial characteristics). Furthermore, no fungal isolates were obtained from the negative-control plants.

3. Discussion

This is the first study to explore the diversity and pathogenicity of Sporocadaceae species isolated from grapevines with GTD-related symptoms in the Mediterranean country of Cyprus. Four species of Sporocadaceae were identified by incorporating reference type and non-type sequences in multigene phylogenetic analyses. Seim. cyprium is a newly described and typified species, whereas two species in the genus of Sporocadus: Spo. kurdistanicus and Spo. rosigena constitute new reports from Europe on grapevines. Furthermore, Seim. vitis-viniferae is a new report from Cyprus.

Conidial morphology has been extensively used to dissect coelomyetous genera [35,48]; however, phylogenetic relationships at the species level have been refined in combination with sequence data analyses [31,32,33,50]. Nevertheless, until recently, the delineation of phylogenetic lineages in Sporocadaceae was still ambiguous to a certain extent, especially due to the lack of ex-type-sequence data in public repositories and designated epitypes, as well as the deficit of more-informative loci, such as partial protein-coding regions [26,34,38].

Phylogenetic analysis based on ITS and LSU sequences grouped the collected pestalotioid isolates in the genera Seimatosporium and Sporocadus. However, tef1-a phylogeny, as well as concatenated DNA sequence datasets (ITS, LSU, bt2, and tef1-a) combined with conidial phenology were sufficient for the species identification of obtained isolates as Seim. cyrpium, Seim. vitis-viniferae, Spo. kurdistanicus, and Spo. rosigena.

A combined four-loci phylogenetic analysis showed that Seim. cyprium formed an independent, fully supported clade that was phylogenetically distinct from Seim. marivanicum (CBS 143781), Seim. luteosporum (CBS142599) and Seim. vitifusiforme (CBS 142600). Seim. cyprium can be also distinguished from the phylogenetically most closely related species Seim. marivanicum by distinct conidial morphology (conidial dimensions, number of septa, morphology, and dimensions of conidial appendages), thus it is identified as a new species. Sexual fruiting structures of Seim. cyprium were also produced and described in in vitro homothallic pairings of collected isolates.

All available ITS LSU, tub2 and tef1-a sequence data in the NCBI database, as well as those obtained presently, do not support species differentiation of Seim. vitis and Seim. vitis-viniferae, suggesting that their phylogenetic delineation is erratic. However, based on the conidial morphology of the ex-type cultures of Seim. vitis (MFLUCC 14-0051) and Seim. vitis-viniferae (CBS 123004), the two species can be clearly distinguished, suggesting that the most found Sporocadacea species in Cyprus is considered as Seim. vitis-viniferae. Previous studies [23,38,51] have described Seim. vitis in Iran and the USA, although the conidial descriptions provided do not match that of the ex-type specimen of the species, being closer to Seim. vitis-viniferae [31,34]. Furthermore, another report from Hungary has tentatively identified Seim. vitis based solely on ITS data [52], while a single study from Italy [53] reports Seim. vitis (dimensions are not provided) associated with grapevine states that the conidial morphology of the collected isolates matched the ex-type provided. Due to the abovementioned ambiguities, we recommend that the bt2 and tef1-a sequence data of the Seim. vitis ex-type should be retrieved and analyzed, while its conidial description should be also reassessed.

Two non-appendaged Sporocadaceae species in the genus of Sporocadus associated with GTDs were also recorded in our study. Spo. kurdistanicus and Spo. rosigena isolates from Cyprus formed distinct and fully supported clades (ML/MP = 100/100) that clustered with respective ex-type cultures CBS 143778 and CBS 182.50, while they also shared similar conidial characteristics.

Seimatosporium and Sporocadus consist predominately of endophytes and saprobes of woody plants [17,33,34,43], although some species are pathogens of different plant hosts, such as eucalypt, blackberry, and grapevine [13,54,55]. More specifically, out of the nine Seimatosporium species previously reported from diseased or dead grapevine wood worldwide, only five (Seim. botan, Seim. luteosporum, Seim. vitifusiforme, Seim. vitis, Seim. vitis-viniferae) have been evaluated in wood inoculations of intact grapevines to confirm pathogenicity and Koch’s postulates. Seim. botan was isolated in Chile from mature vines bearing symptoms resembling those of Botryosphaeria dieback and was reported to be pathogenic on detached green shoots and rooted plants [56]. Similarly, Seim. luteosporum and Seim. vitifusiforme were first reported by Lawrence et al. (2018) in California, USA, from mature grapevines exhibiting typical dieback-type trunk disease symptoms, but due to a lack of re-isolation from inoculated vines, their pathogenic status remains unclear [38]. However, Grinbergs et al. (2021), confirmed the pathogenicity of Seim. vitifusiforme in rooted Petit Shyrah plants in Chile [37]. Seim. vitis has currently been the most reported species of the genus, found to be associated with GTD symptoms in California, USA, Hungary, Iran, and Italy, causing lesions and wood discoloration in pathogenicity assays [23,38,53,54]. The pathogenicity of Seim. vitis-viniferae was recently confirmed in Italy causing lesions of up to 24.70 cm on 1-year-old canes in an open field experiment with an 8-month incubation period [13]. Currently, there is a lack of information for Seim. hysterioides, Seim. lonicerae, Seim. marivanicum, and Seim. parasiticum. Similarly, little information is available on the pathogenicity of Sporocadus species associated with V. vinifera. Spo. kurdistanicus was recently isolated from grapevines exhibiting trunk disease symptoms in the Kurdistan province of Iran; however, it was found to be non-pathogenic on > 10-year-old vines. Furthermore, Spo. rosigena was also detected in association with GTDs in New Zealand [57], Spo. rhododendri has been isolated from V. vinifera canes in Australia [27], while Spo. lichenicola has been reported to cause blackberry cane dieback in Serbia and Oregon, USA [56,58]; however, their pathogenicity on grapevines has not been assessed yet. Herein, we present the ability of four Sporocadaceae species to cause vascular necrosis on grapevines, thus, contributing to the expanding knowledge on the agriculturally important group of GTD-related pathogens. In pathogenicity assays, isolates of all species were able to infect and colonize, and were able to produce brown to black vascular discoloration. Both Sporocadus species (Spo. kurdistanicus and Spo. rosigena) were significantly more aggressive compared to the Seimatosporium (Seim. cyprium and Seim. vitis-viniferae), while there was no difference in aggressiveness among isolates of the same genus. High recoveries of all tested isolates from inoculated vines were also evident, confirming Koch’s postulates.

GTDs are a disease complex resulting from interactions between taxonomically unrelated fungi colonizing grapevine wood [1,6,38]. Although limited research has been conducted on endophytes in economically important crops, particularly on their roles as latent pathogens [59], it is evident that GTD pathogens are routinely isolated from apparently healthy vines and found to express relatively long latency times in disease development [6,60,61], suggesting that some of them are likely commensal endophytes and/or latent saprobes that may act as opportunistic pathogens triggered by stressful edaphoclimatic factors or colonization density shifts of the residing microbiome [10,21,62,63,64]. In the present study, Seimatosporium and Sporocadus isolates were retrieved from wood cankers either solely or in combination with other known trunk pathogens, such as Ph. chlamydospora, P. minimum, and B. dothidea, while they have also been recovered from asymptomatic vine tissues [11,12,43,65]. Recent studies, that investigated the effect of dual inoculations between Sporocadaceae from grapevines and known GTD pathogens, report synergistic, as well as antagonistic interactions, confirming their involvement in the GTD complex [38,65]. Since mixed fungal infections are commonly found in vineyards, it is critical to clarify the complex microbial networks and elucidate pathogenesis and symptom development [66]. Understanding GTDs’ complexity will help us to prolong the sustainability and profitability of grape production, via cultural (late or double pruning) or other plant protection practices (application of plant protection products on pruning woods and the use of healthy planting material) [5,6,38,66].

4. Materials and Methods

4.1. Sampling and Fungal Isolation

During 2017–2018, 10 vineyards in the provinces of Limassol and Paphos, Cyprus were surveyed and sampled for trunk diseases (Supplementary Table S2). Wood samples were collected from 3 vines per vineyard of the indigenous wine grape cvs. Giannoudi, Mavro, Promara, and Xynisteri exhibiting different typical GTD symptoms, including cankers, dead cordons and spurs, and tiger-stripe foliar symptoms (Figure 9). For fungal isolation wood, segments (1–2 cm thick) were cut off, debarked, washed in distilled water (dH₂O), and fragmented in 4–5 pieces (5 mm thick). Accordingly, discolored wood pieces were disinfected in 95% ethyl alcohol for 1 min, rinsed with sterile dH₂O, dried off in a laminar flow cabinet, and plated on potato dextrose Agar (PDA; HiMedia) amended with streptomycin sulfate (50 μg/mL). The plates were then incubated at 25 °C in darkness for up to 1–2 weeks and inspected daily to prevent the loss of slow-growing colonies from fast-growers. Hyphal tips of selected isolates were excised and transferred to fresh PDA plates at 25 °C to establish pure cultures that were maintained under the same conditions, and kept in an 40% aqueous glycerol solution in -80 °C. Based on colony characteristics, 16 Sporocadaceae isolates and isolates belonging to Botryosphaeriaceae, Ph. chlamydospora, and Phaeoacremonium, respectively, were used for morphological and molecular characterization [34,67,68].

4.2. DNA Isolation, PCR, and Sequencing

To avoid direct contact of the mycelium to the culture medium and its subsequent interference in the DNA extraction process, fungal cultures were grown on sterile cellophane discs (Sigma-Aldrich, St. Louis, MI, USA), which were placed on the top of the culture medium surface. Developed mycelia (approximately 14-day-old), were scraped off with a sterile spatula, lyophilized for 48 h, and homogenized with plastic micro-pestles into powder in the presence of liquid nitrogen. Total DNA was extracted according to Cary et al. (2009), following nanodrop quantification spectrophotometry [69]. The final DNA concentration of each isolate was adjusted to 20 ng/mL and stored at −20 °C for further use. The initial identification was based on sequences of ribosomal DNA fragments that included the ITS region. Furthermore, partial regions of the LSU, tef1-a, and tub2 genes were amplified and sequenced for all Sporocadaceae isolates to elucidate their phylogenetic status. The sequence data of the tub2 and tef1-a loci were used for species identification of the Botryosphaeriaceae and Phaeoacremonium isolates, while partial actin (act) gene data were also used for the latter group of isolates. PCR reactions were of 30-μL and performed using the KAPA Taq PCR kit (Sigma Aldrich, catalog no. BK1002) in a C1000TM Thermal Cycler (Bio-Rad). The PCR mixture contained 6 μL of KAPA Taq Buffer B (10×), 3 μL Mg²⁺, 0.6 μL of the dNTPs mixture (2.5 mmol/mL for each nucleotide), 1.5 μL of each primer (10 μM), 0.3 μL of Taq DNA polymerase (5 U/μL), 3 μL of DNA template (20 ng/μL), and 14.1 μL of sterile ddH₂O. The primers used were ITS1 and ITS4 [70] for ITS, LROR [71] and Un-Lo28S1220 [72] for LSU, EF1-728F and EF1-986R [73] for tef1-a, T1 [74], Bt2b [75] for tub2, and ACT-512F and ACT-783R for act [73]. ITS amplification was performed using the following program: initial denaturation at 94 °C for 5 min, followed by 40 cycles of denaturation at 94 °C for 30 s, annealing at 57 °C for 30 s and extension at 72 °C for 1 min and a final extension step at 72 °C for 3 min. For LSU amplification, the n conditions were as follows: initial denaturation for 3 min at 95 °C; 35 cycles of denaturation for 30 s at 95 °C, primer annealing for 30 s at 57 °C, and extension for 1 min at 72 °C; and a final extension for 10 min at 72 °C. Cycling conditions for the rest of the loci were as described for LSU, but with at an annealing temperature of 58 °C for act and 60 °C for tef1-a and tub2, respectively. PCR amplicons were resolved on 1.5% agarose gels in Tris-acetate-EDTA buffer with a SYBR Safe DNA gel stain (Invitrogen, Carlsbad, CA, USA) and visualized under UV light. After confirmation by agarose gel electrophoresis, the PCR products were sequenced in both directions using the same primer pairs used for amplification by the Macrogen Europe B.V. (Amsterdam, the Netherlands).

4.3. Molecular Identifications and Phylogenetic Analysis

The isolates morphologically resembling typical trunk pathogens retrieved herein, such as Ph. chlamydospora, and the species belonging to Botryosphaeriaceae and Phaeoacremonium, were identified to the species level via BLASTn searches of the National Center for Biotechnology Information (NCBI), using respective sequences retrieved per taxon. Furthermore, after a literature review and NCBI BLASTn searches, ITS, LSU, tub2, and tef1-a sequences of 37 Sporocadaceae taxa (38 isolates) were retrieved and included in our phylogenetic analyses, along with the 16 Sporocadaceaea isolates obtained in this study (

Table 3. Isolates used in this study with details regarding their geographic origin, host, and GenBank accession numbers ^a.

Species	Isolate c,d	Country	Host	GenBank Accession Number b
				ITS	LSU	tub2	tef1-a
Allelochaeta biseptata	CBS 131116 ET	Australia	Eucalyptus oresbia	MH554075	MH554286	MH554749	MH554510
All. fusispora	CBS 144172 T	Australia	Eucalyptus sp.	MH554094	MH554304	MH554767	MH554528
Beltrania rhombica	CBS 123.58 T	Mozambique	Mangrove swamp	MH857718	MH869260	MH704631	MH704606
Botryosphaeria dothidea	P11	Cyprus	Vitis vinifera	ON679649	…	ON887280	ON887281
Diploceras hypericinum	CBS 143885 ET	Netherlands	Hypericum perforatum	MH554108	MH554316	MH554781	MH554542
Phaeoacremonium minimum	L234	Cyprus	V. vinifera	ON679660	…	ON887279	ON887278
Phaeomoniella chlamydospora	LP262	Cyprus	V. vinifera	ON679662	…	…	…
Sarcostroma diversiseptatum	CBS 189.81 T	Australia	Correa reflexa	MH554016	MH554236	MH554692	MH554450
Sar. grevilleae	CBS 101.71 T	Australia	Grevillea rosmarinifolia	MH553952	MH554175	MH554611	MH554370
Sar. pestionis	CBS 118153 T	S. Africa	Ischyrolepis cf. sieber	DQ278923	DQ278925	MH554650	MH554408
Seimatosporium botan	NBRC 104200 T	Japan	Paeonia suffruticosa	AB594799	AB593731	LC047770	…
Seim. cyprium	L111 = CBS 149019	Cyprus	V. vinifera	ON680684	ON705769	ON695856	ON863790
Seim. cyprium	L112	Cyprus	V. vinifera	OΝ695889	ON692404	ON695848	ON863791
Seim. discosioides	NBRC 104201	Japan	Punica granatum	AB594800	AB593732	LC047771	…
Seim. germanicum	CBS 437.87 T	Germany	…	MH554047	MH554259	MH554723	MH554482
Seim. luteosporum	CBS142599 T	USA	V. vinifera	KY706284	KY706309	KY706259	KY706334
Seim. marivanicum	CBS 143781 T	Iran	V. vinifera	MW361952	MW361960	MW375352	MW375358
Seim parasiticum	NBRC 32682	Japan	Physocarpus amurensis	AB594808	AB593741	…	…
Seim. physocarpi	CBS 139968 T	Russia	Physocarpus opulifolius	MH823022	MH823069	MH554676	MH554434
Seim. pistaciae	CBS 138865 T	Iran	Pistacia vera	KP004463	KP004491	MH554674	MH554432
Seim. rosae	CBS 139823 ET	Russia	Rosa sp.	LT853105	MH823070	LT853253	LT853203
Seim. soli	CBS 941.69 T	Denmark	Soil (Fagus sylvatica)	MH554071	MH554282	…	MH554507
Seim. vitifusiforme	CBS 142600 T	USA	V. vinifera	KY706296	KY706321	KY706271	KY706346
Seim. vitis	MFLUCC 14-0051 T	Italy	V. vinifera	KR920363	KR920362	…	…
Seim. vitis	Napa764	USA	V. vinifera	KY706273	KY706298	KY706248	KY706323
Seim. vitis-viniferae	CBS 123004 T	Spain	V. vinifera	MH553992	MH554211	MH554660	MH554418
Seim. vitis-viniferae	P46 = CBS 149018	Cyprus	V. vinifera	ON679693	ON721334	ON695849	ON863776
Seim. vitis-viniferae	P56 = CBS 149017	Cyprus	V. vinifera	ON679694	ON721335	ON695850	ON863778
Seim. vitis-viniferae	P57	Cyprus	V. vinifera	ON680689	ON692397	ON695841	ON863779
Seim. vitis-viniferae	P60 = CBS 149016	Cyprus	V. vinifera	ON679695	ON721336	ON695851	ON863777
Seim. vitis-viniferae	P210	Cyprus	V. vinifera	ON680688	ON692398	ON695842	ON863780
Seim. vitis-viniferae	L33	Cyprus	V. vinifera	ON680686	ON692399	ON695843	ON863782
Seim. vitis-viniferae	L34	Cyprus	V. vinifera	ON680687	ON692400	ON695844	ON863783
Seim. vitis-viniferae	L189	Cyprus	V. vinifera	ON680685	ON692401	ON695845	ON863781
Sporocadus biseptatus	CBS 110324 T	…	…	MH553956	MH554179	MH554615	MH554374
Spo. cornicola	CBS 143889	Germany	Cornus sanguinea	MH554121	MH554326	MH554794	MH554555
Spo. cornii	MFLUCC 14-0467 T	Italy	Cornus sp.	KT162918	KR559739	…	…
Spo. cotini	CBS 139966 T	Russia	Cotinus coggygria	MH554003	MH554222	MH554675	MH554433
Spo. incanus	CBS 123003 T	Spain	Prunus dulcis	MH553991	MH554210	MH554659	MH554417
Spo. italicus	MFLUCC 14-1196 T	Italy	Crategus sp.	MF614831	MF614829	…	…
Spo. glandigenus	NBRC 32677	Japan	Fagus sylvatica	AB594803	AB593735	…	…
Spo. kurdistanicus	CBS 143778 T	Iran	V. vinifera	MW361950	MW361958	MW375350	MW375356
Spo. kurdistanicus	L158 = CBS 149023	Cyprus	V. vinifera	ON695891	ON853905	ON695852	ON863784
Spo. kurdistanicus	L164	Cyprus	V. vinifera	ON695892	ON697292	ON695847	ON863786
Spo. kurdistanicus	L181 = CBS 149022	Cyprus	V. vinifera	ON695890	ON853906	ON695853	ON863785
Spo. lichenicola	CPC 24528	Germany	Juniperus communis	MH554127	MH554332	MH554800	MH554562
Spo. mali	CBS 446.70 T	Netherlands	Malus sylvestris	MH554049	MH554261	MH554725	MH554484
Spo. microcyclus	CBS 887.68	Netherlands	Ribes sp.	MH554068	MH554280	MH554744	MH554504
Spo. multiseptatus	CBS 143899 T	Serbia	Viburnum sp.	MH554141	MH554343	MH554814	MH554576
Spo. rosarum	CBS 113832	Sweden	Rosa canina	MH553970	MH554189	MH554629	MH554388
Spo. rosigena	CBS 182.50	Netherlands	Pyrus communis	MH554013	MH554233	MH554689	MH554447
Spo. rosigena	L105	Cyprus	V. vinifera	ON680683	ON692428	ON695846	ON863789
Spo. rosigena	L106 = CBS 149021	Cyprus	V. vinifera	ON679667	ON853907	ON695854	ON863787
Spo. rosigena	L240 = CBS 149020	Cyprus	V. vinifera	ON679668	ON853908	ON695855	ON863788
Spo. rotundatus	CBS 616.83 T	Canada	Arceuthobium pussilum	MH554060	MH554273	MH554737	MH554496
Spo. sorbi	CBS 160.25 T	…	…	MH554008	MH554229	MH554684	MH554442
Spo. trimorphus	CBS 114203 T	Sweden	R. canina	MH553977	MH554196	MH554636	MH554395

^a GenBank accession numbers for the sequences of four loci: ribosomal DNA (rDNA) internal transcribed spacer region (ITS), rDNA large subunit (LSU), β-tubulin (tub2), and translation elongation factor 1-a (tef1-a) that were generated in this study (in bold) or from others. ^b Sequences from isolates in our collection are highlighted in bold. ^c CBS: Culture collection of the Westerdijk Fungal Biodiversity Institute, Utrecht, The Netherlands; CPC: Culture collection of Pedro Crous, housed at the Westerdijk Institute; MFLUCC: Mae Fah Luang University Culture Collection; NBRC: Biological Resource Center, National Institute of Technology and Evaluation, Chiba, Japan. ^d Status of the isolates = ET: ex-epitype; T: ex-type strain. Accession numbers in bold indicate isolates collected and characterized in the present study.

).

The consensus sequences of the four individual loci (ITS, LSU, tub2, and tef1-a) were aligned with MAFFT v. 7.490 [76] using default parameters, manually adjusted using Unipro Ugene v. 43.0 [77], and individual gene sequences were concatenated using SeqKit [78]. Single locus and concatenated alignments were then subjected to a maximum-likelihood (ML) analysis using IQtree version 2.0.3 [79]. The most appropriate substitution model was chosen based on the Bayesian Information Criterion (BIC) using the ModelFinder algorithm as implemented in IQtree version 2.0.3 [80]. Moreover, branch support was obtained using the bootstrap approximation option of IQtree [81], performing 1000 bootstrap replicates. Further support to the phylogenetic inference was provided by a maximum-parsimony (MP) analysis using MPBoot [82]. Similarly, to ML, the MP analysis was performed including 1000 bootstrap replicates. Beltrania rhombica (CBS 123.58) was selected as the outgroup taxon for both the ML and MP analyses (Table 3). The number of pairwise differences (PWD) between taxa on the concatenated sequence data was obtained using the distance estimation option implemented in MEGA X (Supplementary Table S1) [83]. New generated sequences were deposited in the NCBI GenBank database (Table 3). The resulting trees were edited in FigTree 1.4.4. The multigene sequences alignment and the trees obtained were deposited in the figshare repository (doi:10.6084/m9.figshare.20530188).

4.4. Morphological Description

Sporocadaceae isolates were morphologically described based on the colony, microscopic and stereoscopic characteristics of cultures grown on PDA, malt extract agar (MEA; Sigma-Aldrich), and oatmeal agar (OA; Sigma-Aldrich) at 25 °C for 2–4 weeks. Furthermore, 1-year-old lignified canes from the indigenous cv. Xynisteri were cut into 5 cm long segments and autoclaved twice (121 °C for 25 min, with a 24 h interval between autoclaves). The autoclaved segments were then placed in Petri dishes and immersed halfway in WA. Accordingly, three mycelial plugs from actively growing cultures were placed among the wood pieces, the cultures were incubated at 5 and 10 °C with a 12 h photoperiod and fruiting bodies formation was monitored weekly for up to 8 weeks [35]. Morphological observations of all reproductive structures were determined at appropriate magnifications using an Olympus BZX16 dissecting microscope and a Zeiss AX10 compound microscope, both equipped with color digital cameras Olympus ColorView I and Zeiss AxionCam MRc 5, respectively, and the minimum, maximum, mean, and standard deviation were calculated. The conidial length was measured from the base of the basal cell to the base of the apical appendage and the conidial width was measured to the widest point of the conidium. Furthermore, the conidial color, shape, and length of each conidial cell were also recorded. The colony morphology per culture medium was also described, while the colony colors were also rated per culture medium following Rayner’s (1970) charts [84].

4.5. Effect of Temperature on Mycelial Growth

Eight isolates belonging to four different Sporocadaceae species detected herein were randomly selected for the estimation of mycelial growth cardinal temperatures. Mycelial plugs (4 mm in diameter) from the margins of actively grown cultures were transferred into Petri dishes on PDA, MEA, and OA and incubated in the darkness from 5 to 30 °C at 5 °C intervals. Two perpendicular measurements of the diameter were recorded after 14 days. Three replicate plates were used per isolate and the experiment was repeated once. The optimum temperatures for mycelial growth and the maximum daily growth rate for each isolate were calculated based on regression curves of the temperature versus daily radial growth.

4.6. Pathogenicity Tests

In May 2020, a field trial was set up to evaluate the pathogenicity of seven isolates, representative of the Seimatosporium and Sporocadus species identified by phenotypic and phylogenetic analyses. Two-year-old vines of the cv. Xynisteri were grown in 10 L pots filled with potting mix (Miskaar; Lambrou Agro Ltd., Limassol, Cyprus) and amended with a slow-release fertilizer (Itapollina 12-5-15 SK; Lambrou Agro Ltd., Limassol, Cyprus). Lignified canes were aseptically wounded by drilling between the third and the fourth internode from their base. Subsequently, a 4 mm mycelial plug from 14-day-old cultures grown on PDA was placed in the wound that was then sealed with Vaseline (Unilever PMT Ltd., Nicosia, Cyprus) and wrapped with Parafilm (Sigma Aldrich) to prevent inoculum desiccation. Negative controls were inoculated with sterile PDA plugs. Five replicates were used per isolate, with an equal number of controls in a completely randomized design and the experiment was repeated once. All plants were placed under shade netting in open-field conditions and were drip-irrigated according to the weather 1–2 times per week for 30 min (0.5 L/h). Throughout the pathogenicity trial, the inoculated stems were routinely inspected for foliar symptoms, and 12 months after inoculation, were excised and transferred to the lab for further analyses. The newly developed green shoots and the bark were removed, and the length of the wood discoloration was measured in both directions from the inoculation point. The woody canes were washed in a sodium hypochlorite solution (5%) for 2 min and then rinsed twice with dH₂O. Accordingly, 10 small, discolored wood pieces from each stem were disinfected in 95% ethanol for 1 min, and after drying, were placed in PDA dishes amended with streptomycin (50 mg/L) and chloramphenicol (50 mg/L). Plates were incubated at 25 °C in darkness for 1–2 weeks and the re-isolated colonies were identified morphologically.

4.7. Statistical Analyses

The effect of temperature on hyphal growth and wood discoloration length for each isolate were analyzed using an ANOVA to test for the normality and homogeneity of variances. Regression curves were fitted over different temperature treatments versus growth rate for each isolate. Data were analyzed using the Kruskal–Wallis test (non-parametric ANOVA), followed by Dunn’s posthoc multiple comparison test of means (p = 0.05). To assess the differences in the extent of the vascular discoloration induced by the different fungal isolates, pathogenicity data were log10 transformed to satisfy the normality requirements of the ANOVA. Bartlett’s test for homogeneity of variances was also performed for repeated experiments. Since variances were homogeneous (p < 0.05), the data were combined, and the means were compared with Tukey’s test at 5% of significance. All the statistical analyses were performed using SPSS (v. 25; IBM Corporation, New York, NY, USA).