The first records of Bartheletia paradoxa (Bartheletiomycetes, Agaricomycotina) in Ukraine

A new for Ukraine basidiomycete fungus, Bartheletia paradoxa, strictly confined to leaves of Ginkgo biloba, is reported. The species was collected on fallen leaves of G. biloba in November 2016 and 2017 in three localities within Kyiv city. Both conidial and telial stages were found. Morphological descriptions of conidial sori, conidia, secondary conidia, telia and teliospores are provided. More data on phenology of the fungus is added. Information about distribution of B. paradoxa is considered. To date, the species is known from several countries in Europe as well as from Korea and Japan in East Asia. Described from outside of the ancient distribution area of the host plant, B. paradoxa has not yet been reported within the presumptive native range of Ginkgo in China. Moreover, despite wide cultivation of G. biloba globally, this quite conspicuous fungus has not yet been recorded in some mycologically rather well studied regions, like North America or New Zealand. The article is illustrated by original micrographs.


Мікологічні знахідки Mycological Records Introduction
Bartheletia paradoxa G. Arnaud ex Scheuer, R. Bauer, M. Lutz, Stabenth., Melnik & Grube was briefly described in 1954 (Arnaud, 1954) based on collection made by Jean Jules Barthelet on leaves of Ginkgo biloba L. in France in 1932. However, the species was not validly described as the description lacked a Latin diagnosis required for new taxa at that time. The first valid publication of the fungus was provided only in 2008(Scheuer et al., 2008. Bartheletia paradoxa is an enigmatic fungus with a unique set of characteristics. Unlike any other basidiomycete, it has very unusual septal structure. While in most basidiomycetes, the septa dividing cells within the hyphae are perforated by a large central pore, hyphal septa of B. paradoxa exhibit multiple tiny plasmodesma-like pores (Scheuer et al., 2008). Conidia of B. paradoxa are able to produce secondary conidia surprisingly resembling basidiospores of Agaricomycetes (Koukol, Lotz-Winter, 2016). Phylogenetic analyses suggested B. paradoxa as the most basal member of the Agaricomycotina (Scheuer et al., 2008;Mishra et al., 2017), but at the same time its resting spores are very similar to teliospores of the rust fungi. Another distinctive feature is that being apparently saprotrophic, B. paradoxa is a strictly host-specific and widespread fungal associate of Ginkgo biloba. Most probably, just like its host plant, the fungus is also a living fossil, which "apparently used G. biloba as its Noah's Ark" (Scheuer et al., 2008).
Due to its unique combination of characters and unresolved position at the base of the Agaricomycotina, B. paradoxa was first assigned to the family Bartheletiaceae within the Agaricomycotina (Scheuer et al., 2008) and recently the order Bartheletiales and the class Bartheletiomycetes were introduced (Mishra et al., 2017).
Here we report the first for Ukraine records of this remarkable fungus and provide some data on its morphology, occurrence and life cycle.

Abstract.
A new for Ukraine basidiomycete fungus, Bartheletia paradoxa, strictly confined to leaves of Ginkgo biloba, is reported. The species was collected on fallen leaves of G. biloba in November 2016 and 2017 in three localities within Kyiv city. Both conidial and telial stages were found. Morphological descriptions of conidial sori, conidia, secondary conidia, telia and teliospores are provided. More data on phenology of the fungus is added. Information about distribution of B. paradoxa is considered. To date, the species is known from several countries in Europe as well as from Korea and Japan in East Asia. Described from outside of the ancient distribution area of the host plant, B. paradoxa has not yet been reported within the presumptive native range of Ginkgo in China. Moreover, despite wide cultivation of G. biloba globally, this quite conspicuous fungus has not yet been recorded in some mycologically rather well studied regions, like North America or New Zealand. The article is illustrated by original micrographs. Keywords: Ginkgo biloba, basidiomycete, morphology, phenology, distribution (most conspicuous in the epidermis), or conglutinated in compact, erumpent and finally superficial telia ( Fig., b). Telia single or arranged in circular groups, nearly always surrounded by a halo of solitary intraepidermal teliospores, similar to those of rust fungi, often developing from conidial sori, hemispherical or cushion-like to more or less spherical, the largest ones often more irregular and with a conspicuous depression in the centre, single or gregarious to confluent, 150-850(-1200) μm in diam. Solitary intramatrical teliospores in the epidermal cells or deeper in the leaf tissue, dispersed or somewhat agglomerated, spherical or broadly ellipsoidal to somewhat irregular in shape, brown, 25-40 μm in diam. Conglutinated teliospores in the telia thick-walled, dark brown to blackish brown, (35-)50-125(-140) × 12-30 μm, often with a bifid base, and usually with one protruding, conical to cylindrical cap-like wall thickening up to 25 (-30) μm high at the apex ( General distribution. Europe: Austria, Czech Republic, Denmark, France, Germany, Russia, Sweden, The Netherlands, Ukraine (current report), United Kingdom; Asia: Japan, Korea (Scheuer et al., 2008;Braun, 2009;Lotz-Winter et al., 2011;Kirschner, Okuda, 2013;Koukol, Lotz-Winter, 2016).
Bartheletia paradoxa appears to be a saprobe, rather than endophyte, although highly specific to its substrate. Its biology is still unclear but the fungus is assumed by Kirschner & Okuda (2013) as a pioneer colonizer of Ginkgo leaves at the initial stage of leaf litter decomposition.
Recently a new phenotypic phenomenon, formation of secondary conidia in B. paradoxa, was discovered (Koukol, Lotz-Winter, 2016). In our two specimens containing conidial sori, we also observed secondary conidia although they were not as numerous as reported for the specimens from the Czech Republic and Germany. Mature and detached secondary conidia resemble basidiospores of Agaricomycetes and, most probably, their prompt formation is to provide rapid colonization of freshly fallen leaves. Moreover, secondary conidia are formed apically on rather long stalks which specimens were studied under a dissecting microscope, labelled and dried for further treatment. Conidia and teliospores mounted in water or lactic acid were investigated by light microscopy. Photomicrographs were taken under Primo Star microscope, Canon A300 digital camera and AxioVision 4.7 software, used as well for measurements of microstructures. For scanning electron microscopy, samples were covered with an ultrathin coating of gold by ion beam sputtering unit JFC-1100. Images were obtained by scanning electron microscope JEOL JSM-6060 LA.
The specimens are deposited in the Mycological Herbarium of the M.G. Kholodny Institute of Botany, National Academy of Sciences of Ukraine (KW-M). Foliicolous fungus, growing on fallen leaves. Conidial sori on freshly fallen leaves of the current year, slimy, ca 100-400 μm in diam. when dry (Fig.,   a, c, e). Conidiophores branched. Conidiogenous cells thin, holoblastic, terminal, or intercalary with one or two conidiogenous branches, unilocal with percurrent proliferation. Conidia hyaline, one-celled, straight, cylindrical-bacilliform or sometimes slightly broader below the middle, (15-)17-25(-28) × (2.5-)3(-3.5) μm (Fig., f), with a short attenuate base and minutely truncate scar, often with minute guttules. Conidia occasionally produce secondary conidia, superficially resembling basidiospores of agaricoid fungi, ovoid, up to 12 μm long and 6 μm wide, formed on a stalk of subterminal to submedial position, up to 10 μm high (Fig., f). Teliospores either single, immersed in the leaf tissues and more or less evenly dispersed may also contribute to dispersal over greater distances by rain-splash or air current. Secondary conidia enhance reproductive potential in the conditions of very short period of asexual reproduction.

Results and discussion
Phenology of B. paradoxa is very distinctive. Bartheletia infects only freshly fallen leaves of the current year so that infection may happen just after the leaves have dropped. Up to now, no signs of any symptoms were found on living leaves still attached to the tree. It is suggested that spores may be transferred from the remnants of rotten leaves of the preceding year lying on the ground. Once infection does happen, the growth of Bartheletia is extremely rapid. The developing conidial sori erupt through the leaf surface, producing copious conidia and secondary conidia, and very soon they are gradually replaced by teliospores and telia originated from the same basal cushions. The erumpent and finally superficial telia are surrounded by scattered solitary thick-wall teliospores, submerged below the cuticle. The entire cycle from teliospore germination to teliospore maturity can be over in as little as two weeks. The teliospores remain dormant the following winter through summer and eventually germinate to produce basidia in autumn shortly before the leaves are shed. In our first observations made in very early November, only a small part of freshly fallen leaves exhibited some symptoms of colonization while in a few days conidial sori were abundant. Our specimens collected in the first decade of November 2017 contain both conidiomata and telia; those collected in late November -only telia. Interestingly, in our collections of early November 2016 we do not observe any conidiomata as they have already been totally replaced by telia. Thus, development of both stages varies annually depending on climate conditions during the growing season, and more specifically, on the time when a tree sheds its leaves. During November, the fungus was found under each ginkgo tree we observed as they all had accumulated leaf litter of the preceding year.
According to currently known records, Bartheletia paradoxa has very uneven global distribution, although Ginkgo biloba is widely cultivated across the world. Described from outside of the ancient distribution area of the host plant in 1932, the fungus was subsequently recorded only in the 21 st century in ten European countries as well as in Korea and Japan in East Asia. However, the species has not yet been reported within the presumptive native range of Ginkgo in China. Moreover, despite wide cultivation of G. biloba globally, B. paradoxa has not yet been recorded in some mycologically rather well studied regions, like North America or New Zealand. Since the species does not really belong to inconspicuous fungi, particularly due to its long lasting telial stage, it remains unclear why it remained undescribed until the mid-20 th century and is still overlooked despite its widespread nature.