ACTA AGROBOTANICA Vol. 64 (2): 3–14 2011

CHARACTERIZATION OF TWO COEXISTING PATHOGEN POPULATIONS OF spp., THE CAUSE OF STEM CANKER OF

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 (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 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, napus, Lepto- 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 , suborder Pezizomycoti- na, class and 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 (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 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. Ascospores of L. maculans are formed in pseudothecia on infected stubble from the previous growing season(s). The spores land on leaves and cause leaf spots. The secondary infections are caused by pycnidiospores of Phoma lingam, formed in pycnidia. Over winter the fungus grows systemically in veins of leaf blades and petioles (latent phase). The growth rate is 2-3 times slower than this of L. biglobosa. The fungus invades the stem and causes severe stem cankers at root necks and stem bases. 6 Joanna Kaczmarek, Małgorzata Jędryczka

Fig. 2. The life cycle of Leptosphaeria biglobosa on winter oilseed rape in Europe. Ascospores of L. biglobosa are formed in pseudothecia on infected stubble from the previous growing season(s). The spores land on leaves and cause leaf spots. The secondary infections are caused by pycnidiospores of Phoma lingam, formed in pycnidia. Over winter the fungus grows systemically in veins of leaf blades and petioles (latent phase). The growth rate is 2-3 times faster than this of L. biglobosa. The fungus invades the stem and causes profound but superficial upper stem lesions. Characterization of two coexisting pathogen populations of Leptosphaeria spp., the cause of stem canker of brassicas 7

The symptoms on leaves depend on the fungal toms observed on plants that develop from infected species causing infection. Generally symptoms caused seeds. In Australia, the transmission of the pathogen by L. biglobosa are dark brown or grey, surrounded by by infected seeds is regarded as an important source a dark margin. In the case of L. maculans, most disease of plant infection (Salisbury et al. 1995; L i et symptoms are light green to pale beige, with no mar- al. 2003). It may bring new races of the pathogen to gin. J ę dryczka (2006) reported that the identifica- new area (Gugel and Petrie, 1992). In Poland tion of the particular species responsible for stem can- the movement of the pathogen on seed was reported ker of brassicas from observation of disease symptoms to be negligible (Guoping, 1999, Gwiazdow- can result in misdiagnosis because the symptoms are ski, 2004) and the spread of the pathogen was mainly not as clear-cut as suggested in the literature (Brun by air and rainfall dissemination of spores produced et al. 1997, Fitt et al. 2006). on infected plant residues. Small plant parts of oil- Small dark spots formed within leaf lesions are seed rape stubble, fragmented by harvesters, remain pycnidia – the fruiting bodies of the asexual stage of on the soil surface and give rise to the production of the pathogen. They contain pycnidiospores, which are primary inoculum of these pathogens the following transmitted by rain splash. In this anamorphic stage, season. Gradually, L. maculans and L. biglobosa colo- the pathogen is referred to as Phoma lingam (Tode nize previously uninfected stem fragments. With time, ex Fr.) Desm. (Desmaziìres, 1849), both for the fruiting bodies of the pathogen are found on many L. maculans and L. biglobosa, which is misleading, stubble fragments (Gladders and Musa, 1980). because it does not indicate which fungal species Infected stubble provides a good environment for the (L. maculans or L. biglobosa) is responsible for the generative stage of pathogen development, particularly disease. Pycnidiospores are numerous and regarded when the stubble is untilled (Salam et al. 2003). as the secondary inoculum of the pathogen. Com- pared to ascospores, pycnidiospores are transmitted GENERATIVE STAGES OF in droplets of rain to short distances, usually from LEPTOSPHAERIA MACULANS 2 to 40 cm (Travadon et al. 2007) and cause infec- AND L. BIGLOBOSA tion of the leaves, as do ascospores (Hall, 1992). It Pseudothecia of L. maculans and L. biglobosa was reported, that pycnidiospores take longer to ger- are round to oval and flattened at the bottom. The minate than ascospores under the same environmental diameter of the fruiting bodies of L. maculans ranges conditions (L i et al. 2004). According to West and from 300 to 400 μm, occasionally up to 500 μm, and Fitt (2005) pycnidiospores are much less important are formed on the epidermis of the stem. The diame- than ascospores in the epidemiology of stem canker of ter of L. biglobosa pseudothecia ranges from 280 to brassicas in Europe. A much greater role has been at- 350 μm and are located under the epidermis of the stem tributed to pycnidiospores in Australia (Barbetti, (Toscano-Underwood et al. 2003). In both 1976; Howlett et al. 2001) and Canada (Guo and species the opening is in the centre of the fruiting body, Fernando, 2005), although it is almost exclusively although the size of this orifice is smaller in L. macu- the spring form of oilseed rape (Brassica napus L. for- lans (90 to 100 μm) than in L. biglobosa. It is formed ma annua) that is grown in these countries. from 5 to 8 layers of scleroplectenchymatic cells of The mycelium is at first restricted to a small 3-5(10) μm. Shoemaker and Brun (2001) ob- leaf area, but then gradually expands and accesses served that pseudothecia of L. biglobosa have a lon- the leaf petiole, through the veins. This phase of dis- ger beak (200-400 μm tall by 200-300 μm wide) than ease development is latent; there are no macroscopic L. maculans. It contains 8-10(15) layers of cells, of symptoms on plants. With time, the symptoms of plant 5-8 μm diameter. The pseudothecial surface of both infection became visible on outer parts of plants. On species is formed of cells with thick, melanised walls. stems dark spots surrounded by grey or brown margin The fruiting bodies of L. biglobosa contain paraphyses of 2-3 μm, distributed every 20-25 μm. The pseudoth- are observed. Inside the plant, the fungus can partially ecia of L. maculans contain numerous, bitunicate asci or totally block the veins, which retards or inhibits of 100-120(150) x (12)18-21(22) μm, whereas asci of the transport of water and nutrients. This destructive L. biglobosa are less numerous. process results in premature ripening of oilseed rape Similar to other ascomycetes, both species form plants (Hammond et al. 1985, Hammond and asci with eight ascospores. These are long, straight or Lewis, 1987; West et al. 2001). The infection slightly curved cylindrically shaped spores. The size of stems may lead to the infection of siliques, which of L. maculans ascospores is on average 6-7 x (45)50- also results in pod spots with dark pycnidia inside. 60(68) μm. The length to the width ratio is approxi- This process may result in direct contamination of mately 8:1. The spores of L. biglobosa are slightly the seeds. Infected seeds may lead to disease symp- smaller: 6-7 x 42-48(60) μm and the ratio of length to 8 Joanna Kaczmarek, Małgorzata Jędryczka width is in most cases 7:1. The ascospores of L. macu- et al. (1996) demonstrated that although the isolates lans usually contain 5 septa, and each cell has one to of type ‘B‘ did not produce sirodesmins, they did pro- several droplets of fat. Ascospores of L. biglobosa have duce other metabolites, some of which had phytoto- 3-5 septa and the biggest are their central cells, usu- xic properties. This is why Howlett et al. (2001) ally containing one or two droplets of fat. Ascospores proposed the more appropriate term: Siro+ instead of formed by both species have a smooth surface (Wil- Tox+ and Siro0 instead Tox0. The common taxonomic liams, 1992; Shoemaker and Brun, 2001). affiliation of these two forms of L. maculans was qu- estioned by many scientists. In 2001, Shoemaker DESCRIPTION OF THE CONIDIAL and Brun, suggested the existence of two species STAGE – PHOMA LINGAM L. maculans and L. biglobosa, based on morphological Pycnidia of the fungus P. lingam are black, smo- differences of pseudothecia. The morphology of the oth and round (200 x 200 μm). The beak of a pycnidium fruiting bodies is not the only feature differentiating is in the centre and is cylindrically shaped. These fru- the two pathogens, but usually is required to distingu- iting bodies contain thick walls (15-20 μm), composed ish species diversity. Williams and Fitt (1999) of 6-8 layers of polygonal, pseudoparenchymatic cells reported that the fungi L. maculans and L. biglobosa with dimensions of 2-4 μm. Pycnidia do not contain differ in pathogenicity, with L. maculans responsible paraphyses but are filled with pycnidiospores. In con- for the majority of yield loss. Disease symptoms cau- trast to ascospores, which are composed of 6 cells of sed by Leptosphaeria species are apparent at the early approximately 60 x 6,5 μm, pycnidiospores are com- stages of plant development. Usually lesions form on posed of single cells of 4-5 x 1,5-2 μm. They are hyali- leaves and subsequently necrosis may occur at the base ne to light brown, cylindrical, but blunt at both ends. of the stem or root collar. The symptoms at the soil They contain two droplets of fat, which refract light. level often result in disruption of the vascular system In contrast to ascospores, which are long and narrow, and decay of the adult plant may be observed as early the length to width of a single pycnidiospore is 5:2. as the flowering stage. L. biglobosa usually results in A detailed description of pycnidospores was reported more superficial symptoms on the stem than L. ma- by Shoemaker and Brun (2001). culans (Gladders and Musa, 1980; West et al. 2002). However, analysis of fungi present on the DIFFERENCES BETWEEN stem base of oilseed rape with symptoms of dry rot LEPTOSPHAERIA MACULANS of brassica carried out in Poland by J ę dryczka AND L. BIGLOBOSA AND METHODS OF (2006) has frequently demonstrated the presence of SPECIES IDENTIFICATION L. biglobosa. She also suggested that description of Until 2001, L. maculans and L. biglobosa were symptoms to differentiate the species of Leptospha- classified as the same fungal species, in spite of nume- eria was oversimplified. The species L. maculans and rous studies indicating substantial differences betwe- L. biglobosa can be distinguished on the basis of growth en these two pathotypes/groups. The first reports on rate and morphology of colonies cultured in vitro, as the diversity of the population of the fungus L. macu- already described in 1927 by Cunningham. Lep- lans were published in 1927 in New Zealand. Cun- tosphaeria maculans is characterized by slow growth, ningham (1927) described two separate forms of less abundant mycelium and extensive sporulation on L. maculans, differing by morphological characteri- agar media. Leptosphaeria biglobosa produces abun- stics observed on agar media. Subsequent work con- dant aerial mycelium, grows rapidly, but produces fe- firmed these studies. wer pycnidia than L. maculans (Koch et al. 1989; Slow growing isolates that could form abundant Williams and Fitt, 1999). pycnidia were termed type ‘A’. Type ‘B’ included fast Secondary metabolites are another criteria dif- growing isolates with more aerial mycelia and less pyc- ferentiating L. maculans and L. biglobosa (Koch et nidia (Williams and Fitt, 1999). Due to differen- al. 1989). Chromatographic studies allow accurate ces in pathogenicity the isolates were also characteri- diagnosis of chemotypes. Sirodesmin PL is the pre- zed as aggressive (A) and nonaggressive (NA) (Koch dominant metabolite produced in culture filtrate of et al. 1989) or highly virulent (HV) and weakly viru- L. maculans (Pedras and Seguin-Swartz, lent (WV) (Sippel and Hall, 1995). Isolates be- 1990; Kachlicki, 2004). The analysis of secon- longing to these two groups differed greatly in their dary metabolites produced by L. biglobosa revealed ability to produce secondary metabolites, especially the presence of numerous compounds, with wasabi- nonspecific phytotoxins called sirodesmins (Koch et dienon B prevalent (Pedras et al. 1995; Pedras al. 1989; Pedras and Seguin-Swartz, 1992). and Biesenthal, 2001; Kachlicki, 2004). In This is why isolates forming sirodesmins were also the first phase of growth of L. biglobosa phomaligin called Tox+ (Balesdent et al. 1992). Kachlicki A was produced (Pedras et al. 1995). Brown colored Characterization of two coexisting pathogen populations of Leptosphaeria spp., the cause of stem canker of brassicas 9 culture filtrates or dark exudates present on mycelia and the profile of isoenzymes of the non-aggressive grown on agar media indicate the development of me- (NA) isolates, three subgroups were reported – NA1, lanins. Populations of isolates of L. biglobosa show NA2 and NA3 (Koch et al. 1991; Gall et al. 1994). considerable variation in the profiles of secondary me- Random Amplified Polymorphic DNA and rep-PCR tabolites (J ę dryczka et al. 1999a). They may serve methods also distinguished L. maculans from L. bi- as chemotaxonomic markers, allowing identification globosa and revealed the intraspecific differences of of individual isolates of the fungus (Kachlicki, both groups (Plummer et al. 1994; Mahuku et 2004). al. 1997; J ę dryczka et al. 1999b). Sequencing of Until recently, reports on the effect of fungici- the ITS ribosomal DNA fragment helped to illuminate des on fungal growth of L. maculans and L. biglobosa the phylogenetic relationship of these species (Men- were ambiguous. The research conducted by Eckert des-Pereira et al. 2003). The above-mentioned et al. (2004, 2010) and Karolewski (1998) indi- molecular methods gave rise to indirect proof of the cated that inhibition of growth of L. maculans required distinctness of L. maculans and L. biglobosa. lower doses of fungicides than L. biglobosa. In a study In spite of numerous trials (Venn, 1979; P e - published by Gwiazdowski (2008) there were no trie and Lewis, 1985; Gall et al. 1994; Som- statistically significant differences in the rate of fungal d a et al. 1997) it was not possible to obtain any fertile growth of L. maculans and L. biglobosa after treatment pseudothecia from matings between these two species. with fungicides, but the doses used were very high, In contrast, the same research teams obtained hybrid which may have masked species-specific differences isolates within L. maculans (Mengistu et al. 1993; among isolates. Recently, Kaczmarek and J ę - Gall et al. 1994) and within L. biglobosa (Somda dryczka (2010) clearly demonstrated differences in et al. 1997). Fertile pseudothecia of both species were susceptibility of L. maculans and L. biglobosa to flu- frequently observed in natural environments (John- silazole and the flusilazole-containing fungicide, and son and Lewis, 1990). they proved that L. biglobosa requires higher doses of these chemical compounds, to obtain fungal cultures ACKNOWLEDGEMENTS comparable to L. maculans. The study took into ac- count the different growth rate of both fungal species The authors thank Dr. Randy Kutcher, the As- (J ę dryczka, 2006). sociate Professor at the University of Saskatchewan, Molecular studies provided further evidence of Saskatoon, Canada for a thorough revision of the man- the existence of two biotypes or populations in the ta- uscript. xon identified as L. maculans. Isozyme diversity was high between these two sub-groups of isolates. 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