Micromycetes on highbush blueberry Vaccinium corymbosum L. leaves

e study aimed at the identification of micromycetes found on the leaves of high-bush blueberries Vaccinium corymbosum . e study yielded 23 fungal species (261 colonies) isolated from living and fallen leaves with symptoms of disease (necrosis and spots). e dominant species were Pestalotiopsis sydowiana , Alternaria alter-nata , Epicoccum nigrum , Trichoderma viride , T. harzianum , Pestalotia spp., Penicillium expansum , P. jensenii , Mortierella spp., Truncatella spp., and Dichotomopilus spp.


Introduction
Highbush blueberry Vaccinium corymbosum L. is wild, growing on bogs and peat bogs in the eastern part of North America, reaching up to two m high.It is also commonly cultivated due to the tasty dessert fruits.Every year the crop of this plant takes on importance.According to data of the National Research Institute of Agricultural and Food Economics in Poland, the area of blueberry cultivation in 2018 was 11.8 thousand hectares.
For many years this species was considered free of diseases and pests.According to Kukuła et al. (2017), several pathogens (viruses, fungi, and bacteria) and pests of this plant species have been identified and characterized in the cultivated highbush blueberry Poland.
e study aimed was to determine the quantitative composition of micromycetes inhabiting leaves of highbush blueberries in two localities in Poland: (1) the Botanic Garden of the Jagiellonian University in Cracow, and (2) the Rogów Arboretum of the Warsaw University of Life Sciences (WULS-SGGW) in Rogów.

Materials and methods
Observations of the highbush blueberry (V.corymbosum) health status were conducted in the years 2012-2014 on the collection of ericaceous plants of the Botanic Garden of the Jagiellonian University and the Rogów Arboretum of the WULS-SGGW.Infected green leaves (showing necrotic symptoms) and fallen leaves were sampled three times a year (25th May, 25th July, and 25th September).Twenty leaves (10 living and 10 fallen) were collected from each plant at each site.Leaf fragments were cut out from the border of healthy and necrotic tissues from single spots.ey were surface sterilized in 70% ethanol for one minute, then thoroughly rinsed three times for one minute in sterile water before placing on a Petri dish with a 2% PDA medium.Cultures were incubated for seven days at 21-22 °C.Subsequently, young fungal colonies were transferred on slant agar with a 2% PDA medium.Macro-and microscopic comparisons of fungal colonies were made, and representative colonies were selected.e microscope used for observation was a Delta Optical microscope (Evolution 300).e species identification was based on the mycological keys and monographs (Domsch et al., 1980;Ellis & Ellis, 1987;Guba, 1961;Sutton, 1980).e taxonomic system proposed by Kirk and co-workers (2008) was adopted, while the names of taxa follow the Index Fungorum database accessed in July 2020.Participation of individual species in the total number of species was defined as the dominants (constituting >5% of the entire community), influents (1-5%), and accessory taxa (<1%).e coefficient of similarity S o (Sörensen number) was calculated according to Kowalik (1993).

Results
As the results of the mycological analysis of plant material 261 fungi colonies belonging to the genera: Alternaria, Ascochyta, Coleophoma, Dichotomopilus, Epicoccum, Fusarium, Gilmaniella, Humicola, Juxtiphoma, Mortierella, Nigrospora, Penicillium, Pestalotia, Pestalotiopsis, Phialophora, Trichoderma, Truncatella and Wardomyces were obtained.e community of fungi isolated from green and fallen leaves with spots and necroses from highbush blueberries grown in the Botanic Garden and the RogowArboretum was different in colony number and species diversity (Table 1, Table 2).
From affected green and fallen leaves of highbush blueberry in the Botanic Garden, 134 colonies representing 16 species (14 genera) were isolated.27% of the total number of colonies represented Pestalotiopsis sydowiana, referred to as the dominant species.e dominant role of Alternaria alternata and Epicoccum nigrum (together over 34% of all isolates) in inhabiting leaves of highbush blueberry was observed.Species of the genera: Trichoderma (T.viride, T. harzianum), Pestalotia, Penicillium (P.expansum, P. jensenii), Mortierella, Truncatella, and Dichotomopilus occurred with lower abundance and were classified as influents.e accessory group, accounting for less than 1% of the isolated colonies is constituted by Coleophoma rhododendri, Fusarium chlamydosporum, Nigrospora oryzae (syn.Khuskia oryzae), and Phialophora cinerescens.
From affected green leaves of highbush blueberry in the Botanic Garden, 59 colonies representing 14 species were isolated.Over 35% of the total number of colonies represented A. alternata, referred to as the dominant species.Genera: Pestalotiopsis, Pestalotia, Epicoccum, and Penicillium, with the predominance of P. sydowiana, P. rhododendri, E. nigrum, P. expansum, and P. jensenii were also inhabited by affected highbush blueberry leaves.e living leaves of V. corymbosum in the Rogów Arboretum were inhabited by 12 species of micromycetes.Almost 55% of the total number of colonies represented P. sydowiana, referred to as the dominant species.Genera: Penicillium, Alternaria, and Humicola were also numerous, accounting for more than 28% of all isolates.
From the necrotic tissues of fallen leaves in the Botanic Garden, nine species representing eight genera were isolated.Species P. sydowiana and E. nigrum were the dominant species.Genera Gilmaniella and Trichoderma, with the predominance of G. humicola, T. harzianum, and T. viride also inhabited the fallen leaves in this location.Species: A. alternata, P. expansum, T. truncate, and M. parvispora have sporadically colonized leaves, with small frequency.
From the fallen leaves in the Arboretum, 74 colonies of fungi were isolated.e dead tissue of the plant was dominated by P. sydowiana (almost 23%).e fungi belonging to the genera Trichoderma, Alternaria, Truncatella, Juxtiphoma, and Ascochyta with the predominance of T. koningii, T. harzianum, A. alternata, T. truncata, J. eupyrena (syn.Ph. eupyrena) and A. medicaginocola (syn.Ph. medicaginis), also inhabiting to fallen leaves.Also, other filamentous fungus complexes with the advantage of E. nigrum and Ph.cinerescens caused spots on the leaves.Six species: A. alternata, C. rhododendri, G. humicola, P. jensenii, P. sydowiana, and T. truncata were common species that inhabited green leaves of highbush blueberries in the Botanic Garden in Cracow and the Rogów Arboretum.e main species living on fallen and dead leaves in these locations were: A. alternata, E. nigrum, P. sydowiana, T. viride, and T. truncata.
For the calculation of similarity coefficients (S o ) of micromycetes on leaves of V. corymbosum in the Botanic Garden and Arboretum, total values for the years 2012-2014 were used.For the micromycetes community on alive leaves in Botanic Garden and Arboretum, the highest similarity coefficient calculated was in 2012 (75%).e lower similarity coefficient was found in 2013 and 2014 (33.33% and 15.38%).
For the micromycetes community on fallen leaves in 2012 the similarity coefficient was calculated at 100%.e lower (58.82%)similarity coefficient was found in 2013.e lowest similarity coefficient (44.44%) was found in 2014.

Discussion
261 colonies of micromycetes belonging 23 species were identified from the green and fallen leaves of highbush blueberries in the Botanic Garden of the Jagiellonian University in Cracow and the Rogów Arboretum of the WULS-SGGWs.Among them, species from the genera Botrytis, Colletotrichum, Valdensina, and Godronia, which, according to numerous sources (Dzięcioł, 2008;Dzięcioł et al., 2014;Kukuła et al., 2017;Łabanowski et al., 2001;Wright et al., 2004Wright et al., , 2005;;Shivas & Tan, 2009), are the main causative agents of highbush blueberry diseases in commodity crops, were not found.
Pestalotiopsis sydowiana and Alternaria alternata predominated in the total number of isolates.Both species were also earlier isolated from Ericaceae plants in the Botanic Garden of the Jagiellonian University in Cracow and the Rogów Arboretum of the WULS-SGGW by Duda and Bonio (2014) and Drzewiecka et al. (2016).e frequent occurrence of P. sydowiana on the leaves of the heath plants was also recorded by Keith et al. (2006) and Kowalik et al. (2011), who attributed to this species a causal role of numerous discolorations and spots.Hopkins and McQuilken (2000) reported that P. sydowiana and Pestalotia species can cause spots and necrosis of the heath plants, leading to their deaths, while Łabanowski et al. (2001) ascertained the fungi of these species do not have a pathogen attribute.
According to Greco et al. (2012), Alternaria alternata was isolated from round or irregular, greyish spots with a red rim, occurring on leaves, fruits, and other organs of highbush blueberry.Kowalik et al. (2010) reported that this toxin-producing species plays a dominant role in the development of disease lesions and increased leaf defoliation and fall.In the current research, this species was one of the dominants and accounted for almost 16% of the total number of isolates.
Fungi belonging to the Fusarium genus were isolated from necrosis and spotted leaves.Species of this genus were isolated by Wright et al. (2004Wright et al. ( , 2005) ) from the same blueberry host in Argentina.According to Werner et al. (1998), species of the genus Fusarium and Gibberella infect the roots and the collarroot (F.avenaceum (Fr.)Sacc.) and destroy the fiber-vascular bundles (F.oxysporum Schltdl.), which leads to the gradual dieback of the shrubs.Kuzdraliński et al. (2014) reported that the symptoms of infestations by Fusarium species include: not fully developed inflorescences and flower petals, yellowing and falling leaves, which may lead to a decrease in not only the decorative value of plants but also the yield.
In addition, the species of the following fungal genera were isolated for the first time: Coleophoma, Dichotomopilus, Epicoccum, Gilmaniella, Nigrospora, Mortierella, Phialophora, Trichoderma, and Wardomyces.ey were isolated from leaves with discolorations, spots, and necrosis.Species from the genera were recorded in samples of atmospheric air and leaves of Ericaceae plants in both the Botanic Garden of the Jagiellonian University in Cracow and the Rogów Arboretum of the WULS-SGGW (Bonio & Drzewiecka, 2016;Bonio & Duda, 2014;Kowalik et al., 2014aKowalik et al., , 2014b;;Kowalik et al., 2015).
the coefficient of similarity (Sörensen number), a -number of fungal species on the leaves of V. corymbosum in the Botanic Garden of the Jagiellonian University in 2012-2014, b -number of fungal species on the leaves of V. corymbosum in the Rogow Arboretum of the WULS-SGGW in 2012-2014, c -number of fungal species recorded at both research sites.

Table 1
Micromycetes on leaves of Vaccinium corymbosum in the Botanic Garden of the Jagiellonian University in Cracow.

Table 2
Micromycetes on leaves of Vaccinium corymbosum in the Rogów Arboretum of the Warsaw University of Life Sciences -SGGW.