First report of Neoramularia bidentis for Ukraine and notes on several rare Ramularia species (Ascomycota)

Several noteworthy species of the genera Neoramularia and Ramularia (Mycosphaerellaceae) were recorded in August 2018 in the territory of Prypyat’-Stokhid National Nature Park (Volyn Region, Liubeshiv District, Ukraine). Neoramularia bidentis, a species so far only known from South Korea and Poland, was collected on withering leaves of Bidens frondosa (a new host species). Ramularia crepidis on Crepis tectorum was recorded only for the second time in Ukraine since the 1950th and R. gnaphalii was found on a new host plant, Laphangium luteoalbum. The latter species was so far known from Ukraine only on Omalotheca sylvatica (Gnaphalium sylvaticum) and Gnaphalium uliginosum from Kyiv Region collected about 45 years ago. The plant pathogenic fungus R. lysimachiae on leaves of Lysimachia vulgaris was found to be abundant in Volyn Region. All species are described, illustrated with original scanning electron microscope micrographs, supplemented with host range, distribution data and comments.


Introduction
The examination of the diversity of phytopathogenic micromycetes is important as it enables one to estimate the fungal interactions with plants and the environment, as well as to predict changes in ecosystems. The family Mycosphaerellaceae comprises about six thousand species and is amongst the largest groups of Ascomycota. These micromycetes are predominantly plant pathogens that can cause leaf lesions, fading and dying of leaves and whole plants, besides some pathogens of fungi, endophytes and saprotrophs (Vasil'evsky, Karakulin, 1937;Tomilin, 1979;Braun, 1998;Videira et al., 2016). The diversity of mycosphaerellaceous fungi is in general huge and still under the investigation in Ukraine. At the same time, fluctuations in climatic factors have led to drier and hotter summer seasons in forest and foreststeppe zones, provoking the migration and invasions of new and rare species of fungi.

Materials and methods
Infected plants with lesions were collected in the north of Ukraine, on the territory of Prypyat'-Stokhid National Nature Park near Buchyn, Liubyaz', Selisok and Svalovychy villages (Volyn Region, Liubeshiv District) in August 2018. This protected area is a part of the Ukrainian Polissya (Western Polissya) and stretches along the Prypyat' River and a part of its tributary, the Stokhid River.
The fungal specimens were examined under a dissecting microscope (DM) and by standard light microscopy (LM), finely cut and mounted in water or 5% aqueous lactic acid solution and 1% cotton blue in lactophenol, respectively. Samples for scanning electron microscopy (SEM) were coated with a thin layer of gold and palladium by ion beam sputtering coater JFC-1100. Images were obtained under the scanning electron microscope JEOL JSM-6060 LA.
Analyses of the general distribution of the studied fungi were based on the data from various bibliographic sources and publications, as well as databases available through the Internet: USDA Fungal Database (Farr, Rossman, 2019) and Mycology Collections Portal (MyCoPortal, 2019).
The specimens are deposited in the Mycological Collection of the M.G. Kholodny Institute of Botany, National Academy of Sciences of Ukraine (KW-M).

Results and discussion
During the course of field studies in Prypyat'-Stokhid National Nature Park, Neoramularia bidentis Shin & U. Braun, a fungal species new to Ukraine, was found. This species had been supposed to be allied to the genus Cercospora Fresen. (Shin, Braun, 1993). Recently it is provisionally placed in Ascomycota, Incertae sedis, according the Index Fungorum (Index Fungorum, 2019). The phylogenetic position of N. bidentis and the whole genus Neoramularia U.Braun in general is still unknown. Moreover, several noteworthy species of the genus Ramularia Unger (Mycosphaerellaceae, Capnodiales, Ascomycota) were collected in the park as well.
Detailed descriptions and notes on micromorphological characters and the distribution of Neoramularia bidentis, Ramularia crepidis Ellis & Everh., R. gnaphalii (P.Syd.) Karak. and R. lysimachiae Thüm. are provided, supplemented by the photographs of specific symptoms (under the dissecting microscope) and micrographs of morphological structures (under the scanning electron microscope).

Neoramularia bidentis Shin & U. Braun,
Mycotaxon 49: 352. 1993. (Fig. 1) On living and fading leaves. Leaf spots oblong to angular, irregular, 2-3 mm across, brown to hazel, small, scattered and without distinct margins; older lesions dry, irregular, scattered, developing along the veins and limited by main veins, 3-5(-6) mm, tan to brown, with dark brown to reddish brown margins, 0.5 mm wide, without halo; sometimes older lesions more delicate, parchment-like and depressed in the center of initially infected tissues, lesions on the lower surface of leaf concolorous, with convex, dark brown margins; lesions bearing greyish white or yellowish white caespituli on both sides of the leaves. Mycelium immersed, hyphae colorless, septate, 1-2 μm wide; subepidermal hyphae growing into leaf stomata, bearing outward projecting conidiophores or erumpent through collapsed epidermal cells, single or in small, loose groups, mainly hypophyllous. Conidiophores colorless, cylindrical, (10-)12-25(-30) × 3.5-4.5(-6) μm, straight or slightly sinuous, smooth, simple or rarely branched, usually with a single septum or unicellular and hence functioning as a conidiogenous cell; with swollen base and slightly tapering toward a rounded apex, provided with slightly papillate scars. Conidiogenous cells colorless, holoblastic, terminal, subcylindrical, showing minimal percurrent enteroblastic proliferation, followed by replacement apical wall-building, collarettes small, or sometimes with several sympodial proliferations and inconspicuous conidiogenous loci where conidia have been seceded (scars); conidiogenous loci unthickened (viewed under the LM), slightly prominent, 0.2 -0.5 (-1.0) μm high and 0.8-1.0 μm diam., with a collarette where the walls of conidiogenous cell and conidium were joined prior to secession, looking like a rupture of the conidiogenous cell wall, with initially fine crenulated edge or with an involute sinuous edge in older loci, up to 0.2 μm wide, forming a shallow central crater (viewed under the SEM). Conidia colorless, solitary or catenate, often in unbranched connected chains, where conidia may also function as conidiogenous cells while still attached, producing further conidia, resulting in long chains of spores; single conidia subcylindrical to cylindrical, fusiform, 12-24(-32) × 3.0-5.0 μm, smooth to sparsely verruculose, rough (LM), with sparsely granulate protuberances, 0.1-0.3 μm diam. (viewed under the SEM); unicellular or 1-2-septate, not narrower at the septa; apex rounded and minutely tapered at place of conidial junction (terminal hilum), basal end tapered and truncate; hila (scars) inconspicuous or raised up to 0.1-0.2 μm (SEM). The fungus Neoramularia bidentis was described on Bidens tripartita L. from Asia, South Korea (Shin, Braun, 1996) and found in various localities of this country (Kim, Shin, 1999). The species was collected on the same host plant in Poland about a decade later (Ruszkiewicz-Michalska, Wołczańska, 2008). Observed short conidiophores and catenate conidia of N. bidentis in the Polish specimens were similar to characteristics of reproduction structures in the Korean ones, and the finding was supported by one of the species authors, U. Braun. Since then this fungus has not been observed in other countries and on other host plants of the genus Bidens L.

Distribution in
In a recent survey of more than 80 pathogens invading cosmopolitan weeds B. pilosa L. and B. subalternans DC., N. bidentis has not been discovered amongst them (Guatimosim et al., 2015).
Collected specimens of this species on B. frondosa (an alien species of North American origin) in Ukraine are morphologically similar to the descriptions and drawings in Shin & Braun (1996) as well as Braun (1998) and Ruszkiewicz-Michalska & Wołczańska (2008). Study of micromorphological structures under the scanning electron microscope showed that the conidia are not just smooth but have sparsely verruculose, rough walls ( Fig. 1D, E) and the observed conidiogenous scars (loci) have low, crenulate to sinuous edges and a small crater in their center (Fig. 1F, H), which distinguish this species from the genus Ramularia. The features of N. bidentis conform well with the current concept of the genus Neoramularia that is characterized by straight conidiophores with terminal, polyblastic, percurrent and sympodial conidiogenous cells, not thickened or darkened conidiogenous loci, solitary and catenate conidia (Videira et al., 2016). The studied fungus N. bidentis belongs to a group of Neoramularia species characterized by producing catenate conidia, such as N. esfandiarii (Petr.) U. Braun and N. phragmitis (Nagorny) U. Braun. On living and fading leaves. Leaf spots on both sides of living leaves, at first orbicular or irregular, 1-2 mm across, greenish grey, small, scattered and without distinct margins; later becoming dry, angular, sometimes orbicular, scattered, developing along the small veins and limited by them, 3-5 mm, tan to light brown, the same lesions on lower surface of the leaf appearing as small diffuse dark greenish or brownish areas; sometimes surrounded by a narrow, dark brown halo; when numerous leaf lesions causing irregular, mottled, pale yellowish brown necrotic patches with some darker areas, bearing greyish white caespituli on both sides of leaf. Mycelium immersed, hyphae colorless, septate, 2-3 μm wide, forming stromatic, colorless, small, subglobose hyphal aggregations in leaf tissues, 16-25 μm diam., aggregations subepidermal or partly superficial, gradually widen the leaf stomata apart and slightly protruding, giving rise to upward-projecting conidiophores in small to large tufts, loose or dense, mainly hypophyllous. Conidiophores colorless, subcylindrical to cylindrical, straight or sinuous, without or with a few geniculations in the apical part, (10-)15-40 × 2.5-4.0(-4.5) μm, smooth, simple, with up to 2 septa. Conidiogenous cells terminal, colorless, holoblastic, with one or a small number of sympodial proliferations and conspicuous conidiogenous loci (scars), thickened, pigmented (viewed under the LM), 0.8-1.2 μm diam., each with a low, smooth periclinal rim, 0.1-0.2 μm wide, where the walls of the conidiogenous cell and conidium are joined prior to secession, with a slightly sinuous edge, and a slightly bulging dome in the central part of delimiting septum, with a barely noticeable crater between the periclinal rim and dome; older scars with a flatter central dome (viewed under the SEM). Conidia colorless, single or catenate, in simple, short chains, produced holoblastically; individual conidia ovate-oblong, fusiform-ellipsoid to fusiformcylindrical, (8-)11-23(-33) × (3.0-)3.5-4.5 μm, smooth or verrucose to sometimes delicately echinulate (viewed under the LM) and sparsely muricate to granulate with projections 0.1-0.3 μm long and 0.1-0.2 μm wide, older and longer conidia sometimes smooth (viewed under the SEM); unicellular or 1(3)-septate, sometimes narrower at the septa, cells not differing in size or apical cell smaller than the basal ones; ends rounded or sometimes basal end tapered and truncate, apical end rounded and slightly attenuated; conidial hila (scars) pointed, thicker, darker, raised to 0.1-0.2 μm (viewed under the SEM). This species is a typical hemibiotroph causing small leaf spots and then developing diffuse necrotic lesions, finally leading to leaf fading and withering. It was first described from New Mexico (USA) as Ramularia crepidis (Ellis, Everhart, 1888) and later under the synonymous name R. eximia from Montenegro (Bubák, 1903). In Europe, this species occurs in central and southern part of the continent (Braun, 1998;Mułenko et al., 2008), although it has been collected rather rare. This fungus may undoubtedly also survive under dry and hot climatic conditions in central Asia and it was recorded from the south of eastern Russia as well (Domashova, 1960;Shvartsman et al., 1973;Osipyan, 1975;Nakhutsrishvili, 1986;Koshkelova, 1977). Ramularia crepidis was observed only once in Ukraine, in the steppe zone, during early autumn of 1954 (Morochkovsky, 1958) without any additional reports since then. The present collection of this species in summer 2018 is the second one from Ukraine and the first one from Western Polissya on the same host plant, Crepis tectorum. The sultry summers and mild winters of the last decade may have resulted in an increase in distribution of R. crepidis in the northern part of Ukraine and possibly facilitated infections.
Studies of micromorphological structures of this fungus by the methods of scanning electron microscopy revealed the presence of sparsely muricate to granulate conidial walls, becoming smooth with ageing and the presence of characteristic (ramularioid) conidiogenous loci (material collected in Ukrane, KW-М71177, KW-М71184). Observed scars on conidiogenous cells are composed of a low periclinal rim and a minutely projected central dome delimited by a barely noticeable crater (Fig. 2F, H). These features of R. crepidis conform well to the current concept of the genus Ramularia (Braun, 1998;Kirschner, 2009;Videira et al., 2016 Bubák, in Vestergren J.T.C., Botaniska Notiser 170. 1902. (Fig.3) On living and withering leaves. Leaf spots at first orbicular or angular, irregular, 1-2 mm across, yellowish to hazel, small, scattered and without distinct margins, closer to leaf edges; developed spots dry, orbicular to irregular, scattered along the central veins, 3-4(-6) mm, tan to brown, sometimes light reddish brown, slightly pressed in the centers of initially infected tissue, with an indistinct reddish brown halo, delimitation from the healthy tissue not clearly visible; the same lesions on lower surfaces of the leaf sometimes paler; small leaf lesions becoming confluent when numerous, resulting in irregular reddish brown to brown areas on the leaf; lesions bearing greyish white to greyish pink caespituli, mainly, mainly hypophyllous. Mycelium immersed, hyphae colorless, septate, 1-3 μm wide, forming stromatic, colorless, subglobose hyphal aggregations in the leaf tissues under the stomata, (15-)20-35 μm diam., the aggregations gradually widening leaf stomata apart and emerging through the stomata, bearing small to large fascicles of projecting conidiophores, mainly hypophyllous. Conidiophores colorless, cylindrical, variously oriented, 20-60(-70) × 3.0-3.5(-4.0) μm, smooth, straight or flexuous, geniculate in the apical part, unbranched, with a few septa. Conidiogenous cells terminal or intercalary, colorless, holoblastic, with one or a small number of sympodial proliferations, conidiogenous loci (scars) thickened, pigmented, distinctly prominent (viewed under the LM), 0.9-1.2 μm diam., each with a periclinal rim, less than 0.1 μm wide and raised up to 0.3 μm high at the point of attachment of the conidium, where the walls of the conidiogenous cell and conidium are joined prior to secession, with a slightly crenulated edge, and a bulging conical central dome formed at the delimiting septum, up to 0.2 μm over the rim and tending to flatten in a central part, sometimes slightly granulated at the periphery, with a noticeable, deep crater between the periclinal rim and dome (viewed under the SEM). Conidia colorless, single or catenate, chains short, produced holoblastically; individual conidia ovate-oblong to obpyriform, ellipsoidal, subcylindrical or cylindrical, 9-16(-22) × 4.5-5.5(-6.5) μm, echinulate or verruculose (viewed under the LM); muricate to densely spinulose with projections 0.2-0.3 μm long and 0.1-0.2 μm wide (viewed under the SEM), younger single conidia with dense, digitate wall projections up to 0.6 μm long; usually unicellular or 1-septate, not narrower at the septa, apical and basal cells of similar size; ends rounded or apex rounded, base tapered and truncate; hila (conidial scars) pointed, thickened, darkened, raised, 0.1-0.  of Pseudognaphalium Kirp. and Helichrysum Mill., and a more distant relationships with species of Gnaphalium (Acosta-Maindo, Galbany-Casals, 2018). The report of R. gnaphalii on Dahlia pinnata Cav. from China (Zhang, 2006) is rather controversial and in need of further studies although Ramularia species with wider ranges on different host genera from the family Asteraceae cannot be excluded.
The fungus R. gnaphalii occurs in forests of the temperate zone in Europe and was also reported from Asia and North America. In Ukraine, it has only been known from Kyiv Region since 1958 (Morochkovsky et al., 1971). The collection of this species in Volyn Region dated August 2018 is the third record from Ukraine and is the most recent one since 1973. It is the first report from Western Polissya.
The conidial wall ornamentation and the structure of conidiogenous loci of R. gnaphalii (Fig. 3E, F, H) agree with the current concept of the genus Ramularia (Braun, 1998;Kirschner, 2009;Videira et al., 2016 (Fig. 4) On living leaves. Leaf spots suborbicular, oblong or irregular, 3-4 mm across, hazel or yellowish brown, scattered and without distinct margins; older lesions orbicular to irregular, scattered, limited by the central leaf vein, 3-5(-8) mm, bright tan to light brown, with indistinct, thin, brown margins, without a halo, the same spots are paler on lower leaf surface, with a greyish tint, fuzzy, bearing clusters of hypophyllous conidiophores; leaf spots later becoming confluent, causing irregular, pale tan to greyish tan patches that cover about half of the leaf surface, bearing greyish white caespituli on both sides of the leaf. Mycelium immersed, hyphae colorless, septate, 2-3 μm wide, branched, forming stromatic, colorless, small, subglobose hyphal aggregations in the leaf tissues, 10-25 μm diam., subepidermal and epidermal, sometimes in the substomatal chambers, gradually slightly protruding and erumpent through the leaf cuticle or rupturing the leaf stomata apart and emerging strait, upward-projecting conidiophores, usually in small fascicles, dense to loose in apical part or forming synnemata which are mostly erect and straight, tightly joined at the base and in the middle, and free, slightly bent outwards at the apex. Conidiophores colorless, in clusters of 10-20, cylindrical, (10-)20-50(-60) × (2.5-)3.0-4.0 μm, smooth, straight or sinuous, geniculate in apical part, unbranched, thinwalled, unicellular or 1-2-septate, sometimes apical part sparsely spinulose near the conidiogenous loci (viewed under the SEM). Conidiogenous cells terminal, colorless, holoblastic, with one or few sympodial proliferations and conspicuous, thickened, darkened scars of conidiogenous loci (viewed under the LM), 1.2-1.4 μm diam.; each scar with a periclinal rim, about 0.1 μm wide and raised to 0.1-0.3 μm, where the walls of the conidiogenous cell and conidium were joined prior to secession, with a slightly sinuous to fine crenulated edge and granulated central dome rising up to 0.2 μm over the rim edge and formed by minute bulging of the delimiting septum, granules up to 0.1 μm diam., with a minute crater between the periclinal rim and dome (viewed under the SEM). Conidia colorless, formed singly or catenate, chains short, produced holoblastically; individual conidia ellipsoidal, fusiform to subcylindrical or cylindrical, (10-)15-25(-30) × 3.0-4.0(-4.5) μm, verrucose to echinulate (viewed under the LM) and with muricate to spinulose projections, 0.15-0.30 μm long and up to 0.20 μm wide (viewed under the SEM), unicellular or 1-septate, not narrower at the septa, apical and basal cells of similar size; ends rounded and slightly attenuated, obtuse; hila (scars) pointed, thickened, darkened, raised up to 0·3μm (viewed under the SEM).