THE DIVERSITY OF FUNGI COLONIZING NECROTIC INFLORESCENCE BUDS OF RHODODENDRON (Rhododendron L.)

The infection of rhododendron (Rhododendron L.) inflorescence buds caused by pathogenic fungi induces its browning, withering, and dieback. The identification of fungi causing the infection of rhododendron inflorescence buds can be a reason for creating new improved cultivars with genetically determined resistance to pathogens. The investigations were carried out in 2010–2011 on the collection of ornamental plants of the Faculty of Horticulture, University of Agriculture in Kraków. The material comprised infected inflorescence buds collected from nine newly bred taxa and one botanical species of rhododendron. 596 colonies of fungi belonging to 31 species were isolated from infected rhododendron inflorescence buds. The dominant species were: Pestalotiopsis sydowiana, Truncatella truncata, Alternaria alternata, Phialophora asteris, and Trichoderma viride, which constituted almost 74% of the isolated fungi population. Boeremia exigua var. exigua, Epicoccum nigrum, Fusarium poae, Mammaria echinobotryoides, Paraphoma chrysanthemicola, Phialophora cyclaminis, Phoma eupyrena, Talaromyces wortmannii, Umbelopsis isabellina, and other fungi were isolated in a lower number. The results of mycological analysis confirm the diversity of species colonizing necrotic inflorescence buds of rhododendron.


INTRODUCTION
Due to the decorative qualities of its flowers, rhododendron (Rhododendron L.) is a popular shrub in home gardens.The infection of rhododendron inflorescence buds by pathogenic fungi causes their browning, withering and dieback.
The main cause of dieback of inflorescence buds is the fungus Pycnostysanus azaleae.Other pathogens involved in the process of dieback of inflorescence buds are as follows: Alternaria alternata, Botrytis cinerea, Fusarium avenaceum, Epicoccum nigrum, Aureobasidium pullulans, Sclerotinia sclerotiorum, and fungus-like organisms Phytophthora spp.(C z e k a ls k i and F r u ż y ń s k a -J ó ź w i a k, 1995; W e r n e r et al. 1996, 1998; F r u ż y ń s k a -J ó ź w i a k and W e r n e r , 2000; G a r i b a l d i et al. 2002).
According to K r y c z y ń s k i and W e b e r (2010), in breeding practice resistance to diseases is the trait taken into consideration while constructing the cultivar ideotype, therefore the identification of the agents causing the infection of rhododendron inflorescence buds may be contributive to creating new improved cultivars revealing genetically determined resistance.
The present study aimed to identify fungi causing dieback of rhododendron (Rhododendron L.) inflorescence buds.

MATERIALS AND METHODS
The investigations were conducted in 2010-2011 on the collection of ornamental plants of the Faculty of Horticulture, University of Agriculture in Krakow, using ten Rhododendron L. taxa.These were newly bred hybrids: R. brachycarpum x R. brachycarpum, R. brachycarpum x R. purdomii, R. aureum x R. brachycarpum, R. aureum x Koichiro Wada, Koichiro Wada x R. aureum, Koichiro Wada x R. brachycarpum, R. purdomii x Koichiro Wada, R. aureum x Catharine van Tol, R. yakushimanum x Catharine van Tol, and R. purdomii species.
The study material comprised necrotic inflorescence buds, 20 pieces collected from 4-5 shrubs of individual taxa.A total of 400 fragments of buds were analyzed.
Mycological analysis was performed by means of standard methods used in phytopathology.Taxonomic identification of fungi species was conducted using the keys of: G u b a (1961), D o m s c h et al. (1980), E l l i s and E l l i s (1987), R i f a i (1987).The basis of classification was the system of K i r k et al. (2008) and the authors' epithets by the fungal names were verified according to I n d e x F u n g o r u m (2012).
In terms of the percentage of species in the whole community, the division was made, after K o w al i k (1993), into dominants, influents, and accessory fungi.

RESULTS
569 fungi colonies were isolated from rhododendron (Rhododendron L.) inflorescence buds with symptoms of browning and withering.The fungi belonged to 31 species within 20 genera (Table 1).A comparison of the number of fungal colonies and species on necrotic rhododendron inflorescence buds did not reveal any considerable quantitative differences between growing seasons.In the first year of the study, 275 fungi colonies represented by 28 species were isolated from the inflorescence buds and in the second year 294 colonies within 23 species.
The fungi P. sydowiana, A. alternata, T. truncata, T. viride and E. nigrum infested buds of all analyzed taxa.The fungus F. poae infected buds of the hybrids R. brachycarpum x R.brachycarpum and R. aureum x Catharina van Tol, while B cinerea infected R. aureum x R. brachycarpum.
When comparing the number of colonies and species of isolated fungi, it was observed that rhododendron inflorescence buds were characterized by varying susceptibility to infection.Between 21 and 94 colonies belonging to from 3 to 15 species were isolated from the buds of individual taxa (Table 2).The largest number of fungal colonies was isolated from buds of the hybrids R. brachycarpum x R. brachycarpum and R. purdomii x Koichiro Wada.Infestation of inflorescence buds by fungi in great numbers (expressed by the number of isolated colonies) was correlated with the fungal species diversity, since 14 and 10 species, respectively, were isolated from infected buds.On the other hand, 21 colonies represented only by 3 fungi species were isolated from inflorescence buds of R. purdomii.The obtained results are evidence of the diversity of fungi colonizing necrotic inflorescence buds of rhododendron.

DISCUSSION
As reported in the literature data, the agent causing dieback of rhododendron inflorescence buds is the fungus P. azaleae syn.Brosia azaleae, identified for the first time in the 19 th century in the south-eastern part of the United States, whereas in Poland it was found for the first time in 1994 by Prof. Mieczysław Czekalski.According to numerous descriptions, infected inflorescence buds become brown, take on a silvery tinge, and several millimetre long black synnemata are visible on their surface.The buds shrink, wither and do not open (C z e k a l s k i and F r u ż y ń s k a -J ó źw i a k , 1995; G l a w e and H u m m e l , 2006).On the bud coats, particularly at the base, there are visible brownish necroses, whereas the number of flowers in the infected bud is reduced (G a r i b a l d i et al. 2002).Necrotic buds do not fall off, so there may be between 3 to 5 generations of withered buds on a shrub (F r uż y ń s k a -J ó ź w i a k and W e r n e r , 2000; G l a w e and H u m m e l , 2006).
C z e k a l s k i and F r u ż y ń s k a -J ó ź w i a k (1995) found that top buds become infected earliest, then side buds, including vegetative ones, leaves and shoots.S t a r k (1985,1994) reports that the pathogen poses a serious threat to rhododendrons in Germany where it causes dieback of between 50-75% of buds.Catharine van Tol (also discussed in this paper), on which over 75% of buds died, was among the most infected varieties.
The research conducted by K o w a l i k ( 2009) in 2004-2007 on the collection of the Jagiellonian University Botanical Garden demonstrated the occurrence of P. azalea on rhododendron leaves.
The fungus P. azalea was not identified in the present study.It did not infest necrotic inflorescence buds.Therefore, the community of pathogenic and saprophytic fungal colonies with the following fungi occurring in large numbers: P. sydowiana (syn.Pestalotia sydowiana), A. alternata, T. truncata (syn. Pestalotia truncata), Ph. asteris (syn. Phialophora fastigiata), E. nigrum (syn. Epicoccum purpurascens), T. viride and Phialophora cyclaminis, can be regarded as the cause of dieback of inflorescence buds.These fungi constantly live in great numbers on fallen and necrotic leaves of azaleas and rhododendrons (K o w a l i k , 2008, 2009; K o w a l i k et al. 2010, 2011, 2012), so they can be a source of primary infection for inflorescence buds.Infestation of buds by A. alternata should be regarded as the most dangerous due to generated mycotoxins causing symptoms of necrosis (P ł a ż e k , 2011).The role of this necrotrophic fungus in causing necrosis and leaf spot diseases was described, among others, by K o w a l i k (2009).
The papers by W e r n e r et al. (1996, 1998) as well as F r u ż y ń s k a -J ó ź w i a k and W e r n e r (2000) documented the occurrence of pathogens and saprophytes of: Fusarium, Sclerotinia, Botrytis, Ovulinia, Aureobasidium, Epicoccum, and Pestalotiopsis, on rhododendron buds and flowers.The research conducted by K o w a l i k (2008,2009) and K o w a l i k et al. (2011, 2012) emphasized the role of E. nigrum, P. sydowiana and T. truncata as necrophytes causing dieback of rhododendron leaves.
W e r n e r et al . (1996) found that the cause of spot disease of flower petals was the fungus B. cinerea, abundantly isolated from the infected organs, whereas F r u ż y ń s k a -J ó ź w i a k and W e r n e r (2000) wrote about its devastating effect on buds and inflorescences.Under the conditions of high air humidity, the fungus infested rhododendron leaves (K o w a l i k , 2009; K o w a l i k et al. 2012), whereas in the present investigations only single cases of occurrence of this pathogen were noted.
The fungi isolated only sporadically from dead inflorescence buds of the analyzed rhododendron taxa were as follows: Mortierella, Umbelopsis, Trichoderma, Phoma, and Paraphoma.According to K ow a l i k et al. (2010, 2011, 2012), these fungi exist on leaves and also greatly increase the necrotization process.
Fungi living on inflorescence buds may cause infections at the flowering stage, which leads to withering and dieback of inflorescences and as a result reduces the decorative qualities of these plants.
The assessment of fungal infection of inflorescence buds in newly bred rhododendron taxa allows us to conclude that they reveal genetically determined resistance to pathogens.The results of mycological analysis can be useful for breeders, because they indicate parental forms with high susceptibility to infection by fungi causing dieback of inflorescence buds.Różnorodność grzybów zasiedlających obumarłe pąki kwiatostanowe różanecznika zawsze zielonego (Rhododendron L.)

Table 2
Number of fungal colonies and species on inflorescence buds of particular rhododendron taxa