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

Stem canker of brassicas, also known as blackleg is the most damaging disease of many Brassicaceae. The disease is caused by Leptosphaeria maculans (Desm.) Ces et de Not. and L. biglobosa sp. nov., Shoemaker & Brun, which coexist in plants and resulting in disease symptoms and decreased yield, quantity and quality of cultivated vegetables and oilseed rape. The paper presents taxonomic relationships between these coexisting pathogen species, describes particular stages of their life cycles, summarizes the differences between the species, and reviews methods for their identification.


INTRODUCTION
Stem canker or blackleg is one of the most destructive diseases of Brassicaceae worldwide.The disease attacks numerous forms of cabbage (Brassica oleracea), spring and winter forms of canola or oilseed rape (B.napus L. forma annua and f. biennis), as well as Crambe sp., Eruca sp., Erysimum sp., Lepidium sp., Raphanus sp., Sisymbrium sp., Thlaspi sp. and others (J ę d r y c z k a , 2006).Disease symptoms are attributed to two pathogens: Leptosphaeria maculans (Desm.)Ces et de Not. and L. biglobosa sp. nov.(S h o e m a k e r and B r u n , 2001), with the former being more damaging than the latter (P e t r i e , 1978).The pathogenicity of the isolates of L. maculans may differ considerably (K u t c h e r et al. 2007).This is one of the reasons why L. maculans is becoming a model system for the study of genetic relationships between host and pathogen (P l u m m e r et al. 1994; K u t c h e r et al. 2010b).In numerous countries, including Poland, both pathogen populations co-exist, and they can jointly lead to severe disease symptoms as well as substantial yield losses of vegetable and agricultural crops ( B r a c h a c z e k et al. 2010).Both species are found in numerous, but not all countries where stem canker has been reported (R o u x e l et al. 2004).Moreover, in L. maculans the race composition of each local fungus population greatly depends on the specific resistance genes present in cultivated plants, such as canola (K u t c h e r et al. 2010a).
The order Ascomycota contains the most numerous (>30,000 species) and also the most versatile group of fungi.In most cases they have septated mycelium, divide using asexual mechanisms such as budding, fragmentation of mycelium or vegatative spores such as conidia, and they produce resting spores such as chlamydospores.However, they mainly reproduce in a generative way, using sexual reproduction.The sexual spores are formed in asci, which is indicated by their classification into the taxonomic order, Ascomycota (F i e d o r o w et al. 2006).
Pezizomycotina forms the biggest suborder of Ascomycota.It contains endophytic fungi, lichens and the pathogens of plants and animals (K i r k et al. 2001).The majority of representatives of this group produce fruiting bodies in the form of asci that are closed with the top, called operculum.The representatives of some genera of Pezizomycotina lost the ability to form such fruiting bodies and their phylogenetic relationship was established based on molecular studies (S p a t a f o r a et al. 2006).
Most fungi belonging to the class Dothideomycetes form pseudothecia with double layers of asci in the stroma.These two layers play different roles in ascospore release: the inner layer is stiff, whereas the outer layer is flexible, which allows high velocity ejection of spores.The asci are usually cylindrical and have thick cell walls.Ascospores are usually multicellular, with longitudinal and horizontal septa, and are transparent to dark brown.Members of the Dothideomycetes are mainly endo-or epiphytes of living plants; pathogens and saprophytes of plants or wood.The class contains twelve genera, including Pleosporales (B i s b y et al. 2009).
Pseudothecia formed by Pleosporales are usually formed as single fruiting bodies, but sometimes can be produced in groups, usually on the substrate surface or shallowly submerged in mycelium.The asci are cylindrical and are separated by pseudoparaphyses.Ascospores of this class are usually hyaline and may be partly coverd by slime.The fungi of this genus are mostly ubiquitous saprophytes living on dead fragments of plants or the pathogens of living plants (K o c h m a n and W e b e r , 1997).
The family Leptosphaeriaceae usually forms transparent, hyaline ascospores with vertical septa.The most typical representatives of this family are fungi from the genus Leptosphaeria, including L. maculans and L. biglobosa (F i e d o r o w et al. 2006).These fungi commonly inhabit the ecosystems with moderate climates.

SIMILARITIES OF THE LIFE CYCLES OF LEPTOSPHAERIA MACULANS AND L. BIGLOBOSA
Both L. maculans and L. biglobosa have a similar life cycle (Figs 1, 2).In Australia, Canada and Europe ascospores are primarily responsible for plant infection.They are formed in pseudothecia that are produced on plant residues from the previous season (P e t r i e , 1995; W e s t et al. 2001; A u b e r t o t et al. 2006).The fruiting bodies of L. maculans and L. biglobosa are mainly formed on plants of the Brassicaceae family.It was found that pseudothecia on the stubble of oilseed rape can survive over five years, and for the first three years they can be a very efficient source of inoculum (P e t r i e , 1986).In this phase of saprophytic growth, the fungus L. maculans can produce phytotoxic metabolites from the sirodesmin family, such as sirodesmin PL (F e r e z o u et al. 1977).This metabolite is not produced by L. biglobosa, more-over -sirodesmins can retard the growth of this fungal species, similar to several other microorganisms (E ll i o t t et al. 2007).
The maturation of pseudothecia is influenced by air temperature and humidity (T o s c a n o -U n d e rw o o d et al. 2003).At temperatures lower than 10˚C the fruiting bodies of L. maculans mature faster than those of L. biglobosa, which is why ascospores of L. biglobosa are observed later in the season (F i t t at al. 2006).The experiments done by D a w i d z i u k et al. (2010) did not prove differences in pseudothecia maturation of L. maculans and L. biglobosa, but the ratio between the species was favourable to the latter one, which could mask differences between these species.Monitoring of ascospores in air samples, combined with molecular detection of L. maculans and L. biglobosa, using Real-Time PCR method, showed no distinct pattern for time as concerns the earliness of spore release of these two pathogens.
The presence of spores in aeroplankton is related to meteorological factors (G r i n n -G o f r o ń , 2009).B u r g e (1986) found more Leptosphaeria ascospores in rainy days.In Crete the ascospores of the genus Leptosphaeria were the most numerous among all spores of Ascomycotina, and they constituted nearly 7% of the air mycoflora and nearly half of all ascospores present in the air.It was suggested that such abundance of Leptosphaeria ascospores is connected with the microclimate of this island.The relationships between meteorological parameters and air spora can be studied with numerous methods, including neural-networks (G r i n n -G o f r o ń and S t r z e l c z a k , 2008).In the case of the L. maculans and L. biglobosa species complex studies resulted in the elaboration of mathematical models (S a l a m et al. 2003 and 2007; D aw i d z i u k et al. 2011).To quantify the ratio between these fungal pathogens in air samples the technique of Real-Time PCR proved useful (K a c z m a r e k et al. 2009 and 2011).The genus Leptosphaeria showed allergenic properties and has been implicated in respiratory allergic diseases (G r i n n -G o f r o ń , 2008), similar to the spores of Alternaria and Cladosporium (S t ę p a l s k a et al. 1999; K a s p r z y k and W or e k 2006.In the UK, the spores of Leptosphaeria spp.produced positive skin-prick test reactions (L a c e y , 1996).It means that the described fungal species are not only pathogenic to plants, but may also be allergenic to humans.
After their release, ascospores can survive dry conditions at 5 o C to 20 o C for as much as 30 days (H u a n g et al. 2003a) and they can be transmitted by air currents for 5 km (H a l l , 1992).However, most spores are deposited on plants within 500 metres of the source (A u b e r t o t et al. 2006).The spores   The symptoms on leaves depend on the fungal species causing infection.Generally symptoms caused by L. biglobosa are dark brown or grey, surrounded by a dark margin.In the case of L. maculans, most disease symptoms are light green to pale beige, with no margin.J ę d r y c z k a ( 2006) reported that the identification of the particular species responsible for stem canker of brassicas from observation of disease symptoms can result in misdiagnosis because the symptoms are not as clear-cut as suggested in the literature (B r u n et al. 1997, F i t t et al. 2006).
Small dark spots formed within leaf lesions are pycnidia -the fruiting bodies of the asexual stage of the pathogen.They contain pycnidiospores, which are transmitted by rain splash.In this anamorphic stage, the pathogen is referred to as Phoma lingam (Tode ex Fr.) Desm.(D e s m a z i ì r e s , 1849), both for L. maculans and L. biglobosa, which is misleading, because it does not indicate which fungal species (L.maculans or L. biglobosa) is responsible for the disease.Pycnidiospores are numerous and regarded as the secondary inoculum of the pathogen.Compared to ascospores, pycnidiospores are transmitted in droplets of rain to short distances, usually from 2 to 40 cm (T r a v a d o n et al. 2007) and cause infection of the leaves, as do ascospores (H a l l , 1992).It was reported, that pycnidiospores take longer to germinate than ascospores under the same environmental conditions (L i et al. 2004).According to W e s t and F i t t (2005) pycnidiospores are much less important than ascospores in the epidemiology of stem canker of brassicas in Europe.A much greater role has been attributed to pycnidiospores in Australia (B a r b e t t i , 1976; H o w l e t t et al. 2001) and Canada (G u o and F e r n a n d o , 2005), although it is almost exclusively the spring form of oilseed rape (Brassica napus L. forma annua) that is grown in these countries.
The mycelium is at first restricted to a small leaf area, but then gradually expands and accesses the leaf petiole, through the veins.This phase of disease development is latent; there are no macroscopic symptoms on plants.With time, the symptoms of plant infection became visible on outer parts of plants.On stems dark spots surrounded by grey or brown margin are observed.Inside the plant, the fungus can partially or totally block the veins, which retards or inhibits the transport of water and nutrients.This destructive process results in premature ripening of oilseed rape plants (H a m m o n d et al. 1985, H a m m o n d and L e w i s , 1987; W e s t et al. 2001).The infection of stems may lead to the infection of siliques, which also results in pod spots with dark pycnidia inside.This process may result in direct contamination of the seeds.Infected seeds may lead to disease symp-toms observed on plants that develop from infected seeds.In Australia, the transmission of the pathogen by infected seeds is regarded as an important source of plant infection (S a l i s b u r y et al. 1995; L i et al. 2003).It may bring new races of the pathogen to new area (G u g e l and P e t r i e , 1992).In Poland the movement of the pathogen on seed was reported to be negligible (G u o p i n g , 1999, G w i a z d o ws k i , 2004) and the spread of the pathogen was mainly by air and rainfall dissemination of spores produced on infected plant residues.Small plant parts of oilseed rape stubble, fragmented by harvesters, remain on the soil surface and give rise to the production of primary inoculum of these pathogens the following season.Gradually, L. maculans and L. biglobosa colonize previously uninfected stem fragments.With time, the fruiting bodies of the pathogen are found on many stubble fragments (G l a d d e r s and M u s a , 1980).
Infected stubble provides a good environment for the generative stage of pathogen development, particularly when the stubble is untilled (S a l a m et al. 2003).Similar to other ascomycetes, both species form asci with eight ascospores.These are long, straight or slightly curved cylindrically shaped spores.The size of L. maculans ascospores is on average 6-7 x (45)50-60(68) μm.The length to the width ratio is approximately 8:1.The spores of L. biglobosa are slightly smaller: 6-7 x 42-48(60) μm and the ratio of length to width is in most cases 7:1.The ascospores of L. maculans usually contain 5 septa, and each cell has one to several droplets of fat.Ascospores of L. biglobosa have 3-5 septa and the biggest are their central cells, usually containing one or two droplets of fat.Ascospores formed by both species have a smooth surface (W i ll i a m s , 1992; S h o e m a k e r and B r u n , 2001).

DESCRIPTION OF THE CONIDIAL STAGE -PHOMA LINGAM
Pycnidia of the fungus P. lingam are black, smooth and round (200 x 200 μm).The beak of a pycnidium is in the centre and is cylindrically shaped.These fruiting bodies contain thick walls (15-20 μm), composed of 6-8 layers of polygonal, pseudoparenchymatic cells with dimensions of 2-4 μm.Pycnidia do not contain paraphyses but are filled with pycnidiospores.In contrast to ascospores, which are composed of 6 cells of approximately 60 x 6,5 μm, pycnidiospores are composed of single cells of 4-5 x 1,5-2 μm.They are hyaline to light brown, cylindrical, but blunt at both ends.They contain two droplets of fat, which refract light.In contrast to ascospores, which are long and narrow, the length to width of a single pycnidiospore is 5:2.A detailed description of pycnidospores was reported by S h o e m a k e r and B r u n (2001).

DIFFERENCES BETWEEN LEPTOSPHAERIA MACULANS AND L. BIGLOBOSA AND METHODS OF SPECIES IDENTIFICATION
Until 2001, L. maculans and L. biglobosa were classified as the same fungal species, in spite of numerous studies indicating substantial differences between these two pathotypes/groups.The first reports on the diversity of the population of the fungus L. maculans were published in 1927 in New Zealand.C u nn i n g h a m ( 1927) described two separate forms of L. maculans, differing by morphological characteristics observed on agar media.Subsequent work confirmed these studies.
Slow growing isolates that could form abundant pycnidia were termed type 'A'.Type 'B' included fast growing isolates with more aerial mycelia and less pycnidia (W i l l i a m s and F i t t , 1999).Due to differences in pathogenicity the isolates were also characterized as aggressive (A) and nonaggressive (NA) (K o c h et al. 1989) or highly virulent (HV) and weakly virulent (WV) (S i p p e l and H a l l , 1995).Isolates belonging to these two groups differed greatly in their ability to produce secondary metabolites, especially nonspecific phytotoxins called sirodesmins (K o c h et al. 1989; P e d r a s and S e g u i n -S w a r t z , 1992).This is why isolates forming sirodesmins were also called Tox + (B a l e s d e n t et al. 1992).K a c h l i c k i et al. (1996) demonstrated that although the isolates of type 'B' did not produce sirodesmins, they did produce other metabolites, some of which had phytotoxic properties.This is why H o w l e t t et al. (2001) proposed the more appropriate term: Siro + instead of Tox + and Siro 0 instead Tox 0 .The common taxonomic affiliation of these two forms of L. maculans was questioned by many scientists.In 2001, S h o e m a k e r and B r u n , suggested the existence of two species L. maculans and L. biglobosa, based on morphological differences of pseudothecia.The morphology of the fruiting bodies is not the only feature differentiating the two pathogens, but usually is required to distinguish species diversity.W i l l i a m s and F i t t (1999) reported that the fungi L. maculans and L. biglobosa differ in pathogenicity, with L. maculans responsible for the majority of yield loss. Disease symptoms caused by Leptosphaeria species are apparent at the early stages of plant development.Usually lesions form on leaves and subsequently necrosis may occur at the base of the stem or root collar.The symptoms at the soil level often result in disruption of the vascular system and decay of the adult plant may be observed as early as the flowering stage.L. biglobosa usually results in more superficial symptoms on the stem than L. maculans (G l a d d e r s and M u s a , 1980; W e s t et al. 2002).However, analysis of fungi present on the stem base of oilseed rape with symptoms of dry rot of brassica carried out in Poland by J ę d r y c z k a (2006) has frequently demonstrated the presence of L. biglobosa.She also suggested that description of symptoms to differentiate the species of Leptosphaeria was oversimplified.The species L. maculans and L. biglobosa can be distinguished on the basis of growth rate and morphology of colonies cultured in vitro, as already described in 1927 by C u n n i n g h a m .Leptosphaeria maculans is characterized by slow growth, less abundant mycelium and extensive sporulation on agar media.Leptosphaeria biglobosa produces abundant aerial mycelium, grows rapidly, but produces fewer pycnidia than L. maculans (K o c h et al. 1989; W i l l i a m s and F i t t , 1999).
Secondary metabolites are another criteria differentiating L. maculans and L. biglobosa (K o c h et al. 1989).Chromatographic studies allow accurate diagnosis of chemotypes.Sirodesmin PL is the predominant metabolite produced in culture filtrate of L. maculans (P e d r a s and S e g u i n -S w a r t z , 1990; K a c h l i c k i , 2004).The analysis of secondary metabolites produced by L. biglobosa revealed the presence of numerous compounds, with wasabidienon B prevalent (P e d r a s et al. 1995; P e d r a s and B i e s e n t h a l , 2001; K a c h l i c k i , 2004).In the first phase of growth of L. biglobosa phomaligin A was produced (P e d r a s et al. 1995).Brown colored culture filtrates or dark exudates present on mycelia grown on agar media indicate the development of melanins.Populations of isolates of L. biglobosa show considerable variation in the profiles of secondary metabolites (J ę d r y c z k a et al. 1999a).They may serve as chemotaxonomic markers, allowing identification of individual isolates of the fungus (K a c h l i c k i , 2004).
Until recently, reports on the effect of fungicides on fungal growth of L. maculans and L. biglobosa were ambiguous.The research conducted by E c k e r t et al. (2004, 2010) and K a r o l e w s k i (1998) indicated that inhibition of growth of L. maculans required lower doses of fungicides than L. biglobosa.In a study published by G w i a z d o w s k i ( 2008) there were no statistically significant differences in the rate of fungal growth of L. maculans and L. biglobosa after treatment with fungicides, but the doses used were very high, which may have masked species-specific differences among isolates.Recently, K a c z m a r e k and J ęd r y c z k a (2010) clearly demonstrated differences in susceptibility of L. maculans and L. biglobosa to flusilazole and the flusilazole-containing fungicide, and they proved that L. biglobosa requires higher doses of these chemical compounds, to obtain fungal cultures comparable to L. maculans.The study took into account the different growth rate of both fungal species (J ę d r y c z k a , 2006).
Molecular studies provided further evidence of the existence of two biotypes or populations in the taxon identified as L. maculans.Isozyme diversity was high between these two sub-groups of isolates.Differences between the biotypes in the migration rate of phosphoglucoisomerase (PGI) in starch gels was developed as a diagnostic tool (S i p p e l and H a l l , 1995; S o m d a et al. 1996; B r u n et al. 1997).Similarly, pulsed field gel electrophoresis, which separates whole chromosomes, also showed substantial differences between L. maculans and L. biglobosa.These species differed for the number and size of particular chromosomes (M o r a l e s et al. 1993).The isolates of L. maculans had a greater number of large chromosomes, the largest of which exceeded 3 Mbp, and L. biglobosa isolates possessed more small size chromosomes of about 1 Mbp (J ę d r y c z k a and K a c h l i c k i , 1996; B a t l i ń s k a , 2004; B i a ł k o w s k a , 2004).
Due to the significant genetic differences between L. maculans and L. biglobosa, identification of these species was also possible using methods that detect polymorphisms of nucleic acids.Restriction fragment length polymorphism (RFLP) analysis confirmed the existence of genetic differences between the species and showed more or greater variation among isolates of L. biglobosa than among isolates of L. maculans (V o i g t et al. 1998).On the basis of RFLP analysis and the profile of isoenzymes of the non-aggressive (NA) isolates, three subgroups were reported -NA1, NA2 and NA3 (K o c h et al. 1991; G a l l et al. 1994).Random Amplified Polymorphic DNA and rep-PCR methods also distinguished L. maculans from L. biglobosa and revealed the intraspecific differences of both groups (P l u m m e r et al. 1994; M a h u k u et al. 1997; J ę d r y c z k a et al. 1999b).Sequencing of the ITS ribosomal DNA fragment helped to illuminate the phylogenetic relationship of these species (M e nd e s -P e r e i r a et al. 2003).The above-mentioned molecular methods gave rise to indirect proof of the distinctness of L. maculans and L. biglobosa.
In spite of numerous trials (V e n n , 1979; P et r i e and L e w i s , 1985; G a l l et al. 1994; S o md a et al. 1997) it was not possible to obtain any fertile pseudothecia from matings between these two species.In contrast, the same research teams obtained hybrid isolates within L. maculans (M e n g i s t u et al. 1993; G a l l et al. 1994) and within L. biglobosa (S o m d a et al. 1997).Fertile pseudothecia of both species were frequently observed in natural environments (J o h ns o n and L e w i s , 1990).., 2008. Artificial neural network models of relationships between Cladosporium spores and meteorological factors in Szczecin (Poland).Grana, 47: 304-314.G u g e l R .K ., P e t r i e G .A . , 1992. History, occurrence, impact, and germinate on the cotyledons and young leaves of oilseed rape plants over a temperature range of 5 o C to 20 o C (H u a n g et al. 2003b) and they cause infection due to the penetration of plant tissues through stomata or directly through wounds caused by biotic (insects) or abiotic factors (H a m m o n d et al. 1985, C h e n and H o w l e t t , 1996).Fungal infection results in large intercellular spaces between the mesophyll cells, but the mycelium of L. biglobosa grows much faster in vitro (J ę d r y c z k a , 2006) and in planta than L. maculans (H u a n g et al. 2003b).As a result of infection, small lesions are formed on plants; they usually become paler and form larger symptoms as the disease develops.Symptoms can be seen even a few days after infection occurs (S e x t o n and H o w l e t t , 2001).Experiments conducted under controlled environments indicated that temperature has an impact on the time of incubation.For example, this parameter requires 5 days at 20 o C and 14 days at 8 o C (B i d d u l p h et al. 1999).

Fig. 1 .
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.

Fig. 2 .
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.
Pseudothecia of L. maculans and L. biglobosa are round to oval and flattened at the bottom.The diameter of the fruiting bodies of L. maculans ranges from 300 to 400 μm, occasionally up to 500 μm, and are formed on the epidermis of the stem.The diameter of L. biglobosa pseudothecia ranges from 280 to 350 μm and are located under the epidermis of the stem (T o s c a n o -U n d e r w o o d et al. 2003).In both species the opening is in the centre of the fruiting body, although the size of this orifice is smaller in L. maculans (90 to 100 μm) than in L. biglobosa.It is formed from 5 to 8 layers of scleroplectenchymatic cells of 3-5(10) μm.S h o e m a k e r and B r u n (2001) observed that pseudothecia of L. biglobosa have a longer beak (200-400 μm tall by 200-300 μm wide) than L. maculans.It contains 8-10(15) layers of cells, of 5-8 μm diameter.The pseudothecial surface of both species is formed of cells with thick, melanised walls.The fruiting bodies of L. biglobosa contain paraphyses of 2-3 μm, distributed every 20-25 μm.The pseudothecia of L. maculans contain numerous, bitunicate asci of 100-120(150) x (12)18-21(22) μm, whereas asci of L. biglobosa are less numerous.
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