ACTA AGROBOTANICA Vol. 64 (2): 3–14 2011 CHARACTERIZATION OF TWO COEXISTING PATHOGEN POPULATIONS OF Leptosphaeria spp., THE CAUSE OF STEM CANKER OF BRASSICAS Joanna Kaczmarek, Małgorzata Jędryczka Institute of Plant Genetics, Polish Academy of Sciences, Strzeszyńska 34, 60-479 Poznań, Poland e-mail: [email protected] Received: 15.01.2011 Abstract Kutcher et al. 2010b). In numerous countries, in- Stem canker of brassicas, also known as blackleg is cluding Poland, both pathogen populations co-exist, the most damaging disease of many Brassicaceae. The disease and they can jointly lead to severe disease symptoms is caused by Leptosphaeria maculans (Desm.) Ces et de Not. as well as substantial yield losses of vegetable and ag- and L. biglobosa sp. nov., Shoemaker & Brun, which coexist in ricultural crops ( Brachaczek et al. 2010). Both plants and resulting in disease symptoms and decreased yield, species are found in numerous, but not all countries quantity and quality of cultivated vegetables and oilseed rape. where stem canker has been reported (Rouxel et al. The paper presents taxonomic relationships between these co- 2004). Moreover, in L. maculans the race composition existing pathogen species, describes particular stages of their of each local fungus population greatly depends on the life cycles, summarizes the differences between the species, and specific resistance genes present in cultivated plants, reviews methods for their identification. such as canola (Kutcher et al. 2010a). Key words: ascospore, stem canker, Brassica napus, Lepto- TAXONOMY OF LEPTOSPHAERIA sphaeria maculans, Leptosphaeria biglobosa, MACULANS AND L. BIGLOBOSA oilseed rape, pseudothecium According to the latest taxonomy, the fun- gi Leptosphaeria maculans and L. biglobosa belong INTRODUCTION to the order Ascomycota, suborder Pezizomycoti- na, class Dothideomycetes and genus Pleosporales Stem canker or blackleg is one of the most de- (www.index-fungorum.org, 2011). structive diseases of Brassicaceae worldwide. The The order Ascomycota contains the most nume- disease attacks numerous forms of cabbage (Brassica rous (>30,000 species) and also the most versatile oleracea), spring and winter forms of canola or oil- group of fungi. In most cases they have septated my- seed rape (B. napus L. forma annua and f. biennis), celium, divide using asexual mechanisms such as bud- as well as Crambe sp., Eruca sp., Erysimum sp., Lepi- ding, fragmentation of mycelium or vegatative spores dium sp., Raphanus sp., Sisymbrium sp., Thlaspi sp. such as conidia, and they produce resting spores such and others (J ę dryczka, 2006). Disease symptoms as chlamydospores. However, they mainly reproduce are attributed to two pathogens: Leptosphaeria macu- in a generative way, using sexual reproduction. The lans (Desm.) Ces et de Not. and L. biglobosa sp. nov. sexual spores are formed in asci, which is indicated (Shoemaker and Brun, 2001), with the former by their classification into the taxonomic order, Asco- being more damaging than the latter (Petrie, 1978). mycota (Fiedorow et al. 2006). The pathogenicity of the isolates of L. maculans may Pezizomycotina forms the biggest suborder of differ considerably (Kutcher et al. 2007). This is Ascomycota. It contains endophytic fungi, lichens one of the reasons why L. maculans is becoming a and the pathogens of plants and animals (Kirk et al. model system for the study of genetic relationships 2001). The majority of representatives of this group between host and pathogen (Plummer et al. 1994; produce fruiting bodies in the form of asci that are 4 Joanna Kaczmarek, Małgorzata Jędryczka closed with the top, called operculum. The representa- over – sirodesmins can retard the growth of this fungal tives of some genera of Pezizomycotina lost the ability species, similar to several other microorganisms (E l - to form such fruiting bodies and their phylogenetic re- liott et al. 2007). lationship was established based on molecular studies The maturation of pseudothecia is influenced by (Spatafora et al. 2006). air temperature and humidity (Toscano-Under- Most fungi belonging to the class Dothideomy- wood et al. 2003). At temperatures lower than 10˚C cetes form pseudothecia with double layers of asci in the fruiting bodies of L. maculans mature faster than the stroma. These two layers play different roles in as- those of L. biglobosa, which is why ascospores of cospore release: the inner layer is stiff, whereas the outer L. biglobosa are observed later in the season (Fitt at layer is flexible, which allows high velocity ejection of al. 2006). The experiments done by Dawidziuk et spores. The asci are usually cylindrical and have thick al. (2010) did not prove differences in pseudothecia cell walls. Ascospores are usually multicellular, with maturation of L. maculans and L. biglobosa, but the longitudinal and horizontal septa, and are transparent to ratio between the species was favourable to the latter dark brown. Members of the Dothideomycetes are main- one, which could mask differences between these spe- ly endo- or epiphytes of living plants; pathogens and cies. Monitoring of ascospores in air samples, com- saprophytes of plants or wood. The class contains twelve bined with molecular detection of L. maculans and genera, including Pleosporales (Bisby et al. 2009). L. biglobosa, using Real-Time PCR method, showed Pseudothecia formed by Pleosporales are usu- no distinct pattern for time as concerns the earliness of ally formed as single fruiting bodies, but sometimes spore release of these two pathogens. can be produced in groups, usually on the substrate The presence of spores in aeroplankton is related surface or shallowly submerged in mycelium. The asci to meteorological factors (Grinn-Gofroń , 2009). are cylindrical and are separated by pseudoparaphy- Burge (1986) found more Leptosphaeria ascospo- ses. Ascospores of this class are usually hyaline and res in rainy days. In Crete the ascospores of the genus may be partly coverd by slime. The fungi of this genus Leptosphaeria were the most numerous among all spo- are mostly ubiquitous saprophytes living on dead res of Ascomycotina, and they constituted nearly 7% fragments of plants or the pathogens of living plants of the air mycoflora and nearly half of all ascospores (Kochman and Weber, 1997). present in the air. It was suggested that such abundan- The family Leptosphaeriaceae usually forms ce of Leptosphaeria ascospores is connected with the transparent, hyaline ascospores with vertical septa. The microclimate of this island. The relationships between most typical representatives of this family are fungi meteorological parameters and air spora can be studied from the genus Leptosphaeria, including L. maculans with numerous methods, including neural-networks and L. biglobosa (Fiedorow et al. 2006). These (Grinn-Gofroń and Strzelczak, 2008). In fungi commonly inhabit the ecosystems with moderate the case of the L. maculans and L. biglobosa species climates. complex studies resulted in the elaboration of mathe- matical models (Salam et al. 2003 and 2007; D a - SIMILARITIES OF THE LIFE CYCLES widziuk et al. 2011). To quantify the ratio between OF LEPTOSPHAERIA MACULANS these fungal pathogens in air samples the technique AND L. BIGLOBOSA of Real-Time PCR proved useful (Kaczmarek et Both L. maculans and L. biglobosa have a si- al. 2009 and 2011). The genus Leptosphaeria showed milar life cycle (Figs 1, 2). In Australia, Canada and allergenic properties and has been implicated in respi- Europe ascospores are primarily responsible for plant ratory allergic diseases (Grinn-Gofroń , 2008), infection. They are formed in pseudothecia that are similar to the spores of Alternaria and Cladosporium produced on plant residues from the previous season (S t ę palska et al. 1999; Kasprzyk and W o - (Petrie, 1995; West et al. 2001; Aubertot rek 2006. In the UK, the spores of Leptosphaeria spp. et al. 2006). The fruiting bodies of L. maculans and produced positive skin-prick test reactions (Lacey, L. biglobosa are mainly formed on plants of the Bras- 1996). It means that the described fungal species are sicaceae family. It was found that pseudothecia on the not only pathogenic to plants, but may also be allerge- stubble of oilseed rape can survive over five years, nic to humans. and for the first three years they can be a very efficient After their release, ascospores can survive source of inoculum (Petrie, 1986). In this phase dry conditions at 5oC to 20oC for as much as 30 days of saprophytic growth, the fungus L. maculans can (Huang et al. 2003a) and they can be transmitted produce phytotoxic metabolites from the sirodesmin by air currents for 5 km (Hall, 1992). However, family, such as sirodesmin PL (Ferezou et al. 1977). most spores are deposited on plants within 500 metres This metabolite is not produced by L. biglobosa, more- of the source (Aubertot et al. 2006). The spores Characterization of two coexisting pathogen populations of Leptosphaeria spp., the cause of stem canker of brassicas 5 germinate on the cotyledons and young leaves of oil- L. maculans (Huang et al. 2003b). As a result of in- seed rape plants over a temperature range of 5oC to fection, small lesions are formed on plants; they usually 20oC (Huang et al. 2003b) and they cause infection become paler and form larger symptoms as the disease due to the penetration of plant tissues through stomata develops. Symptoms can be seen even a few days after or directly through wounds caused by biotic (insects) infection occurs (Sexton and Howlett, 2001). or abiotic factors (Hammond et al. 1985, Chen Experiments conducted under controlled environments and Howlett, 1996). Fungal infection results in indicated that temperature has an impact on the time large intercellular spaces between the mesophyll cells, of incubation. For example, this parameter requires but the mycelium of L. biglobosa grows much faster 5 days at 20oC and 14 days at 8oC (Biddulph et al. in vitro (J ę dryczka, 2006) and in planta than 1999). Fig. 1. The life cycle of Leptosphaeria maculans on winter oilseed rape in Europe.