Micromycetes colonizing and damaging leaves of evergreen rhododendron (Rhododendron L.) in nursery

In May and October 2010–2012, mycological studies were conducted on 10 cultivars of rhododendron bushes growing in containers in the nursery of ornamental plants. Out of 3000 specimens of infested leaf fragments, 2566 fungal colonies belonging to 41 species were isolated. The following species colonizing the leaves and causing their necrosis were extracted in the largest number of colonies: Alternaria alternata, Aspergillus niger, Epicoccum nigrum, Humicola grisea, Pestalotiopsis sydowiana, Phoma pomorum, Sordaria fimicola, Trichoderma koningii, Trichoderma polysporum, Truncatella truncata, Umbelopsis isabellina and others. The research showed that the micromycetes colonies colonizing and damaging rhododendron leaves varied in species composition and number of colonies in different years and at different times. The study determined which rhododendron cultivars were characterized by good health and which had the greatest susceptibility to infection by micromycetes.


Introduction
Evergreen rhododendron (Rhododendron L.) is a valuable plant in modern gardens. Their harmonious, fragrant flowers that vary in color, form and size are the pride of rhododendrons. Outside of the flowering period, however, their evergreen leaves determine the decorative aspects of these plants. Evergreen rhododendrons and other plants are susceptible to pathogenic organisms, such as the most common micromycetes. Currently, more than 200 mycobiota species have been identified in the phyllosphere of rhododendrons that can determine the health of the leaves.
These organisms deteriorate the condition of shrubs, contribute to defoliation and reduce the value of nursery stock in trade, often leading to the exclusion of cultivars from cultivation. To evaluate the usefulness of some cultivars of rhododendron for cultivation, research is carried out on the health status of nursery material [1][2][3][4].
The aim of the study was to identify micromycetes species inhabiting and damaging the leaves of selected cultivars of evergreen rhododendron (Rhododendron L.).

Material and methods
In the years 2010-2012, studies were carried out in May and October on the bushes of 10 rhododendron cultivars: R. forrestii 'Baden Baden' , R. calophytum 'Dominik' , R. brachycarpum 'Flautando' , R. wardii 'Goldbukett' , R. yakushimanum 'Golden Torch' and 'Sneezy' , R. catawbiense 'Nova Zembla' and 'Roseum Elegans' , R. ponticum 'Rasputin' and R. campylocarpum 'Simona' , grown in containers in the nursery of ornamental plants. A total of 3000 affected leaf fragments were collected for mycological analysis. The leaf fragments were decontaminated in 70% ethanol. Micromycete isolation and culture were carried out according to the standard methods practiced in mycology [5].
The keys used in taxonomic identification were as follows: Domsch et al. [6], Ellis and Ellis [7], Guba [8], and Rifai [9]. The basis for the classification was the Kirk et al. [10] system. The researchers' findings were verified using the database of Index Fungorum 2014 [11].
Meteorological data contained in Fig. 1 and Fig. 2 came from weather station Davis Vantage Pro2, located in Kraków Fiołkowa.

Results
In 10 cultivars of 4-6-year Rhododendron L. evergreen rhododendron shrubs grown in the nursery of ornamental plants, light brown stains were mostly visible on the edges of the leaves and the top of the leaf blade as well as oval and irregular spots along the midrib of the leaf. The tissues developed necrotic spots and died. Leaves with necrosis fell off across a large area.
Micromycetes communities inhabiting the phyllosphere of selected evergreen rhododendron cultivars differed in species composition and number of colonies. From the material collected in 2010-2012, 1241 colonies were isolated in the spring study period, including 33 species (Tab. 2), while 1325 colonies of 30 species were isolated in the autumn (Tab. 3). In May the leaves of individual cultivars were colonized by 12 to 18 species, numbering from 76 to 181 colonies. The most micromycetes colonies were found on the leaves of 'Simona' , with fewer found on 'Rasputin' and 'Nova Zembla' . The greatest number of species were found on 'Baden Baden' and 'Rasputin' . Aspergillus niger and Pestalotiopsis sydowiana inhabited the leaves of ten cultivars of rhododendron.
Between 51 and 205 colonies were isolated from the rhododendron leaves collected in October, and the number of colonies ranged from 8 to 19. 'Simona' leaves were colonized by the most micromycetes colonies and species. The fewest fungal colonies inhabited the 'Dominik' cultivar, while the fewest species were found on 'Golden Torch' . The leaves of all of the cultivars were inhabited at different frequencies by the following: A. alternata, H. grisea, Ph. pomorum and U. isabellina.

Discussion
The phyllosphere of evergreen rhododendrons was dominated by P. sydowiana, U. isabellina and A. alternata. P. sydowiana has been cited in many studies as a harbinger of disease symptoms that appears as the brown edges and tips of the leaves [2,4,[12][13][14]. However, Łabanowski et al. [15] do not attribute pathogenic properties to these fungi. Kita and Mazurek [16] write about the occurrence of P. fibricola, P. rhododendri and T. truncata (syn. P. truncata) in the phyllosphere of azaleas with falling leaves and evergreen rhododendrons, whereas Kowalik [2,5] documents the strong participation of P. sydowiana and T. truncata on infected leaves of rhododendron and pontic azaleas in home gardens, arboretums, and natural habitats.
The mass colonization of the living and fallen leaves of evergreen rhododendron by A. alternata is mentioned by the above-named authors, while publications by Kowalik and Muras [17], Kowalik et al. [3,14] have documented the presence and the role of this necrotroph in the process of the death and severe premature fall of leaves. Kozłowska and Konieczny [18] have also written about the role of necrotrophs in these processes. The necrotrophs E. nigrum, H. grisea and S. fimicola played a significant role in the whole community, which has been confirmed by previous studies [2,3,5,13,14,19], whereas the saprotrophs Ch. crispatum, Ch. globosum, H. fuscoatra and M. heterogamus constituted a small percentage of the total fungal communities isolated from the infected leaves.
The high, but uneven colonization of rhododendron leaves by the hygrophilous fungus U. isabellina appears to be associated with a large amount of rainfall in the last year of the study, as this type of fungus prefers moist habitats, such as wet leaves [20].
The results do not confirm the previous studies concerning the participation of A. niger in colonizing and damaging rhododendron leaves [3,14,19].
The significant share of micromycetes of the Trichoderma and Phoma genera confirms that they stimulate progressive necrosis of leaves [5] and contribute to their death [14,19].
Frequent rainfall and continued high humidity were conducive to the intensified process of necrotizing rhododendron leaf tissue in October 2010-2012. The colonization and damage of leaves by micromycetes, especially by P. sydowiana, S. fimicola and E. nigrum, was conducive to the formation of fog and rain dew.
It can be assumed that the intensive sporulation and induction of necrosis by A. alternata, A. niger, P. expansum, P. jensenii, T. koningii, T. polysporum, T. viride and T. truncata in May 2010 was favored by a rapid increase in ambient temperature.
According to Kryczyński and Weber [21], natural environmental factors, such as water and temperature, affect both plants and pathogenic and saprotrophic micromycetes. During the vegetation period when the temperature rises, fungi grow faster, penetrate plant tissues faster and also reproduce faster.
Comparing the species composition and the quantitative properties of mycobiota, including fungi and fungi-like organisms isolated from the leaves of Rhododendron spp., in backyard growing conditions, in the natural environment and in nursery production, it should be noted that the spectrum of micromycetes is much lower in the phyllosphere of the nursery material. (iv) The rhododendron cultivars 'Sneezy' , 'Golden Torch' , 'Flautando' , 'Baden Baden' and 'Goldbukett' were characterized by high health.