Eur J Plant Pathol (2016) 145:89–102 DOI 10.1007/s10658-015-0817-2 Interactive physiological potential of Peyronellaea curtisii (=Phoma narcissi) strains for enzymatic attack and defence capabilities against phytoalexins Katarzyna Hrynkiewicz & Paweł Deka & Małgorzata Ruszkiewicz-Michalska & Dominika Thiem & Adriana Szmidt-Jaworska Accepted: 9 November 2015 /Published online: 18 November 2015 # The Author(s) 2015. This article is published with open access at Springerlink.com Abstract Phytopathogenic fungi use a variety of strat- alone. Tested cellulolytic, pectolytic and amylolytic ac- egies to infect hosts and have a diverse sensitivity to the tivities were strain specific. A high level of amylase natural defence mechanisms of the host plant, e.g. syn- activity was positively correlated with cellulase activity thesis of phytoalexins. Many important pathogens of but negatively correlated with pectolytic activity. crops belong to the problematic genus Phoma,including Moreover, some strains suppressed phytoalexin produc- species recently reallocated to Peyronellaea. The work tion in Hippeastrum scales and were resistant to various presents: (1) molecular identification of five pathogenic concentrations of phytoalexin added to the culture me- strains of Peyronellaea (syn. Phoma) based on two loci, dium. Based on these results, we suggest that pathoge- actin (ACT) and β-tubulin (TUB), and morphological nicity in P. curtisii strains is complex and that it may be observations; (2) differentiation of these strains associated with their interactive physiological potential based on their enzymatic activity; and (3) their effect for enzymatic attack and defence capabilities against on biosynthesis of phytoalexin in Hippeastrum scales. phytoalexin. Phylogenetic analysis showed a close relationship be- tween our strains and Peyronellaea curtisii (syn. Phoma Keywords Phytopathogenic fungi . Enzymatic activity. narcissi) species. This was also supported by morpho- Phytoalexins . β-tubulin (TUB) . Actin (ACT) . anatomical analysis although its results were less con- Dothideomycetes . Hippeastrum disease clusive. Some phenotypic traits of the strains overlapped with characteristics of other Phoma s.l. species, allowing for misidentification based on morphology Introduction : : K. Hrynkiewicz (*) P. Deka D. Thiem Genus Peyronellaea (Dothideomycetes), valildy intro- Department of Microbiology, Faculty of Biology and Environment Protection, Nicolaus Copernicus University, duced in 1952 by Goidànich, was synonymized with Lwowska 1, PL-87-100 Torun, Poland Phoma and included in section Peyronellaea by e-mail: [email protected] Boerema and co-workers in 1960s (Boerema et al. 2004). Recently Peyronellaea genus has been re- A. Szmidt-Jaworska Chair of Plant Physiology and Biotechnology, Faculty of Biology established as an effect of molecular studies of Phoma and Environment Protection, Nicolaus Copernicus University, s.l. phylogeny (Aveskamp et al. 2010). The half of the Lwowska 1, PL-87-100 Torun, Poland species previously accommodated in Peyronellaea sec- tion as well as a dozen of taxa from other Phoma M. Ruszkiewicz-Michalska Department of Algology and Mycology, Faculty of Biology and sections (including Ph. narcissi = Peyronellaea curtisii) Environmental Protection, University of Lodz, Banacha 12/16, have been recombined into the newly re-instated genus. PL90-237 Lodz, Poland Currently, Peyronellaea genus comprises of 66 taxa, 90 Eur J Plant Pathol (2016) 145:89–102 including 24 taxa accepted in the genus by Aveskamp Peyronellaea) taxonomy should be based on a combi- et al. (2010) and two plant-associated species nation of data from phylogenetic and morphological newly described by Crous et al. (2013)aswellas studies. 40 unverified species (Index Fungorum 2015). In the present paper, the authors present physiologi- Peyronellaea groups a small part of members of the cal properties of Peyronellaea strains that are associated coelomycetous genus Phoma s.l. that is one of the with phytopathogenicity. Fungal pathogens synthesise largest fungal genera, with more than 3000 infrageneric large amounts of cell wall degrading enzymes (CWDE), taxa described (Monte et al. 1991). In course of future including pectinases, xylanases, cellulases, and prote- studies the number of Peyronellaea species is likely to ases, which are capable of depolymerising various com- increase enlarged by other Phoma s.l. species. Majority ponents of the host cell wall (Tonukari 2003). Pathogens of Peyronellaea members were isolated from plants differ in terms of quantity and diversity of enzyme (including pathogens) but some species were also re- secretion. Fungi causing plant cell death release a large ported from plant products, soil and animal or inorganic quantity of cell wall degrading enzymes, whereas substrata (Boerema et al. 2004; Aveskamp et al. 2010). pathogens that require a living host to provide Approximately 50 % of Phoma s.l. species are phy- nutrients minimise the production of such enzymes topathogens (Boerema et al. 2004; Aveskamp et al. (Horbach et al. 2011). Therefore, differences in physio- 2008). However, Phoma s.l. strains have also been logical activity can be a key factor in differentiating isolated from soil, aerial and aquatic environments, Peyronellaea strains according to their pathogenicity. and even from humans and animals, where they cause The interaction between plants and their pathogens is opportunistic infections (Irinyi et al. 2009). Plant dis- complex and vary depending on the plant and the fun- eases caused by Phoma spp. can occur under favourable gus. The defence strategies of plants against their path- environmental conditions and result in leaf and stem ogens are many fold and include the synthesis of phy- spots and significant yield losses (Aveskamp et al. toalexins. Phytoalexins are a group of chemically di- 2008). Phoma s.l. is one of the most ubiquitous fungal verse broad-spectrum antimicrobial compounds that genera, characterised by its great ecological diversity plants synthesise in response to wounding and pathogen and difficult identification (Aveskamp et al. 2010). The attack. Many studies have shown that phytoalexins play large number of described species is due to the common a role in plant disease resistance (Dixon 2001). Most practice of host associated nomenclature, paucity in the phytoalexins, which are an important element of plants’ micro-morphological features, and a high variability in active defence mechanisms, have been reported to be culture characteristics. Because of these problems, the highly fungitoxic. For example, potato (Solanum systematics of the Phoma s.l. genus has never been fully tuberosum) tubers produce lubimin (a sesquiterpene understood (Aveskamp et al. 2008). To solve this prob- phytoalexin) upon infection, which inhibits the growth lem, a comprehensive taxonomic system is required of a variety of microorganisms (Kuć 1982). Malusfuran, based on DNA techniques (Aveskamp et al. 2010)or the phytoalexin that is produced in apple (Malus DNA barcodes that are used in the rapid detection of domestica), can inhibit spore germination and the potentially serious plant pathogens (Armstrong and Ball growth of Venturia inaequalis at millimolar concentra- 2005). In recent years actin (ACT) and β-tubulin (TUB) tions (Hrazdina et al. 1997). The phytoalexin resvera- genes have been proposed as standard loci for use in trol, such as medicarpin, can inhibit the hyphal growth DNA barcoding in selected genera of fungi (Aveskamp of the alfalfa fungal pathogen Phoma medicaginis et al. 2009a, 2009b). These genes are so-called house- (Dixon 2001). Pisatin from pea inhibits mycelial growth keeping genes that encode for proteins required for the of several fungi, including both pathogens and non- basic functioning of every cell. Moreover, the integra- pathogens of pea (Delserone et al. 1999). The same is tion of morphological and culture features with DNA true for momilactones A and B and phytocassanes A sequence data can resolve the genetic limits of the taxa from rice (Hasegawa et al. 2010). The ethanol extract of that have been placed in Didymellaceae.DeGruyter wyerone acid could significantly reduce Botrytis fabae et al. (2009)foundaseriesofgeneraclusteringinthe spore germination with increased concentration, al- Didymellaceae that were not clearly defined or were though phytoalexin had no effect on in vitro germ tube morphologically distant from each other. Therefore, growth of the fungus (Nawar and Kuti 2003). In this the further development of Phoma s.l. (including work, the ornamental bulbous plant of the genus Eur J Plant Pathol (2016) 145:89–102 91 Hippeastrum and its Peyronellaea pathogens were used (Difco) in 9 cm Petri dishes at 24–26 °C in complete as a model for phytoalexin - fungal interaction. Various darkness for 8 days. Next, the cultures were exposed to organs of Hippeastrum that are Peyronellaea-infected or UV light (dark interval of 11:13, 12 days) to induce mechanically wounded produce red pigment on the sporulation. The colonies were characterized based on surface of injured tissues. Chemical analysis revealed growth rate, pigment formation, colony pattern and that red pigment produced upon wounding of outline, NaOH test on OA and MEA. The micro- Hippeastrum x hortorum bulbs is a mixture composed morphological studies included the light microscope of an orange-coloured chalcone and 3 flavans (Wink and determination of features of hyphae, pycnidia, conidia Lehmann 1996). After mechanical injuries,