Cercosporoid fungi of Poland

The paper presents characteristics of cercosporoid fungal species from three genera, Cercospora, Passalora, and Pseudocercospora, occurring in Poland and deposited in Polish herbaria or to be expected due to the occurrence of their host plants in the Polish flora. The physiographic literature, but primarily own collections, as well as those gathered in the national herbaria, were the object of the revision. This monograph provides morphological descriptions, lists of hosts and distribution in Poland and worldwide as well as detailed illustrations for 41 species. Dichotomous keys for identification of fungi parasitizing hosts from different plant families were prepared within the individual fungal genera. The present study represents a compilation of 115 cercosporoid species, of which 63 are reported from Poland and another 6 were doubtful and excluded. The fungi parasitize 221 taxa of host plants from 131 genera and 47 families, out of which 158 taxa from 94 genera and 29 families have been actually found in Poland. Fourteen species described in this book belong to Cercospora apii s. l. Nine quarantine species are listed, from which only three have been published so far. This study will be helpful to mycologists, plant pathologist, and Polish plant protection services for identification purposes of the fungi concerned.


Field research
Field studies involved collecting plant specimens infected by cercosporoid fungi as well as conservation and preparation thereof to be deposited in the herbarium. Own collections mainly originated from the Lublin region and the Tatra National Park, where research was conducted under the grant from the Ministry of Science and Higher Education. Traditional methods of research (so-called route method) and modern methods connected with the use of permanent research plots were employed. In both cases, all possible ecological data and frequency of occurrence of both organisms, plants and fungi parasitizing them, were noted.

Laboratory research
Microscopic preparations for identification of fungi were made in the form of surface slides or cross sections through infected leaves. The fragments obtained were stained with a 50% water solution of cotton blue in lactic acid or placed in 50% water solution of lactic acid in order to determine the colour of conidiophores and conidia. Material prepared in this manner was gently heated and then microscopic observations were performed.
External symptoms of plant diseases were described and data on measurements of conidiophores and conidia were prepared based on the analysed specimens and microscopic preparations. Original diagnostic descriptions of fungi were prepared for 82 fungal species, and descriptions for additional 33 species were based on literature data due to the lack of herbarium specimens.
Stereomicroscopes and light microscopes (Olympus BX41 and BX61) equipped with a drawing apparatus and cameras were used for identification purposes of fungi and plants and the iconographic and photographic documentation (drawings and photographs).
An important complementation of diagnosis and morphological descriptions of fungi are molecular studies, which have become an integrated element, especially in the taxonomic research. Their main objective is to determine the phylogenetic relationship of fungi. They are based mostly on the analysis of gene sequences within the ITS-1 5.8S and ITS-2 rDNA regions. These analyses were not performed during the preparation of the present study; however, cercosporoid fungi have been and are still being extensively investigated by other researchers.
Results of a first important analysis were published by Stewart et al. [60], who reported the affinity of fungi from three genera, Cercospora, Passalora, and Pseudocercospora, which belong to the same phylogenetic group. Simultaneously, they confirmed earlier data that   Cercosporoid fungi can produce internal primary and external secondary mycelium. The primary mycelium is usually septate, branched, hyaline, pale olivaceous or olivaceousbrown. The internal mycelium often forms swollen hyphal cells, whose dense aggregations produce stromata [31].
Conidiophores are formed simply or in divergent to dense fascicles and can rarely form synnemata or sporodochia. They emerge through the stomata of an infected leaf or are sometimes erumpent through the cuticle; they are usually simple or sometimes branched, hyaline or variously pigmented (mostly olivaceous, olivaceous-brown or brown); continuous, i.e. one-celled (e.g. some Passalora species) or septate (e.g. Cercospora species) and composed of few cells (Fig. 4, Fig. 5). After liberating conidia, conidial scars are visible on conidiophores. They are usually conspicuous or inconspicuous, thickened, darkened, protuberant or not, distinctly dark coloured or colourless [10,31].
Conidia are usually straight or curved, sinuous or "vermiform", acicular, clavate, obclavate, cylindrical, filiform or fusiform (Fig. 6), aseptate or usually with numerous, distinct or indistinct septa (Fig. 7). Conidia also vary in colour (from hyaline to olivaceous or brown) and size. The length varies from 10 to over 200 µm (especially in acicular conidia of Cercospora species produced under humid conditions). The width can be divided into three ranges: 1-3 µm, 4-6 µm, or 7-10 µm. The surface of conidia is usually smooth, but in some species conidia may be verrucose. The shape of the apical and basal cell plays an important role. In pure culture, growth and sporulation are usually poor [10][11][12]31].
Conidia are formed through budding of conidiogenous cells, but the conidial wall is built of the same layers as the wall of the conidiophore (holoblastic type of conidiogenesis). Conidium, either formed singly or in chains, may arise from a single conidiogenous locus per conidiogenous cells (monoblastic) or usually from two or several per conidiogenous cells (polyblastic). Sympodial proliferation in cercosporoid fungi occurs by lateral formation of a new axis of the conidiophore after conidium production. Conidia secession being schizolitic involves separation of the wall so that a half becomes the conidial wall, while the other half remains on the conidiogenous cell. Usually thickened and darkened conidiogenous loci (scars) and hila are visible on conidiophores and conidia after conidial secession from conidiophores (Fig. 5a, Fig. 8). Cercosporoid fungi parasitize mostly angiosperms, but hyperparasites of rust fungi (Pucciniales) are also known, e.g. Passalora acori (J.M. Yen) U. Braun & Crous parasitizing uredospores of Uromyces sparmanii Clint. & Peck [10]. Optimal conditions for development, which are conducive to the formation of conidia, include high relative humidity and moderate temperature. Cercospora beticola parasitizing leaves of sugar beets prefers temperatures range from 25 to 30°C during the day, temperatures at night above 16°C and high (95-100%) air humidity for optimal development [109]. Light also plays an important role in the pathogenesis. A study that involved shading coffee leaves showed that the number of leaf spots produced as a result of Cercospora coffeicola Berk & Cooke parasitism was definitely lower. Additionally, the symptoms of infection caused by C. beticola were delayed if the sugar beet grew in places with lower light intensity [34].  Anamorphic stages of this fungal group are obligate parasites. Taking into account the whole life cycle, they should be classified as facultative saprotrophs, i.e. parasites with an initial parasitic phase and final saprobic habit, i.e. they finish their life cycles on dead tissue of leaf spots that they have caused themselves. The perfect stage has been documented for a few species of cercosporoid fungi so far. Numerous species, maybe even most of them, are asexual holomorphs, which have lost the ability to form sexual morphs at all, but reliable, scientifically proven results are usually lacking. In Poland, only a single teleomorphic stage is known for a cercosporoid fungus, while worldwide several other cases have been published, but most of them are little reliable, poorely documented and mostly not properly experimentally proven. Mycosphaerella cerasella Aderh., which represents the sexual morph of Passalora circumscissa, has been recorded from Poland [110].

Occurrence
In the present paper, 115 species of cercosporoid fungi are reported. Up to date, 63 species of cercosporoid fungi, including 29 from the genus Cercospora, 30 from Passalora, and 4 Pseudocercospora, have been reported in Poland. They represent only 10% of the taxa known worldwide. The other 46 species are expected to be found in Poland due to the occurrence or cultivation of potential host species in the Polish flora. Nine species represent quarantine fungi included in the regulation of the Minister of Agriculture and Agricultural Reform, regulation of the Minister of Environment, and regulation of the Minister of Agriculture and Rural Development. Additionally, six species (Cercospora curvata, C. fragariae, C. meliloti, Fig. 8 Emergence of conidia. a Holoblastic conidiogenesis (1 -monoblatic; 2 -polyblastic). b Sympodial proliferation. c Schizolitic secession of conidia.
C. ononidis, C. pietrenii, C. violae-sylvaticae) need taxonomical revision and have possibly to be transferred to different genera.
The fungi listed in this study parasitize 221 taxa of host plants from 131 genera and 47 families, of which 158 taxa belonging to 94 genera and 29 families have been found in Poland. Only one species has been published on a member of Pteridophyta -Passalora pteridis, but after revision, the host species was identified as Dryopteris carthusiana on which Cercospora camptosori or C. dryopteridis may occur, which might be found in Poland.
The largest numbers of fungal parasitic species in Poland have been reported from representatives of Apiaceae (8), Leguminosae (7), and Rosaceae (5). The family Poaceae with the largest number of host species (31) was parasitized by two fungal species only. One fungus was found on a single host species within 15 families. A majority of fungal species infects hosts from one genus only, except for P. graminis (31 host species), P. depressa (9), and C. apii, which are polyphagous species.
Cercosporoid fungi are parasites causing diseases of numerous vascular plants. Almost 53% of all Polish hosts of the studied fungi represent usable plants, i.e. wild-growing plants used by humans and cultivated plants (cf. [111]). Among them, the most frequently occurring species include: Cercospora beticola on representatives of the genus Beta, C. armoraciae on Armoracia rusticana, and C. carotae on Daucus carota, C. chenopodii on representatives of the family Amaranthaceae, C. depazeoides on representatives of Sambucus, Passalora circumscissa on representatives of the family Rosaceae, P. concors on Solanum tuberosum, P. ferruginea on species of the genus Artemisia, P. graminis on ture grasses (Poaceae), P. microsora on Tilia, and P. punctum on Anethum graveolens and Petroselinum crispum.

Economic importance and control
Cercosporoid fungi are important economically. Many of them cause significant loss of cultivated plants grown or otherwise used by man. The most important parasites include Cercospora beticola, C. armoraciae, C. carotae, Passalora circumscissa, P. concors, P. graminis, and P. punctum. Short characteristics of three most important diseases -Cercospora leaf spot (CLS) on sugar beet and carrot, and celery early blight are listed below.
Cercospora leaf spots on sugar beet is a disease caused by Cercospora beticola, the most dangerous pathogen of beets (Beta spp.). An increased threat from C. beticola has been noted in our country since the 90's of the 20th century. This is due to introduction of sugar beet varieties with low resistance to this pathogen to the Polish market [112]. The greatest intensity of prevalence of C. beticola (70-98% of infected plants) was noted in Lublin Voivodeship in 1999, especially in the surroundings of Zamość [113,114]. This was a consequence of the changing of harvest technology, limited use of leaf silage as cattle feed, and leaving leaves in the soil as a green fertiliser [109]. At high temperature and humidity, this fungus can destroy up to 50% of crop yield [115]. In the case of severe infections, the foliage is destroyed (Fig. 9), which is then intensively reconstructed by infected plants. This © The Author(s) 2015 Published by Polish Botanical Society Monogr Bot 105 happens at the expense of the distribution of sugar accumulated in the root, which is transported to newly forming leaves. The progressive infection process as well as withering of other leaves and production of new leaves lead to formation of distinctive conical roots. As a result, inhibition of the weight increase occurs, accompanied by a decline in the sugar content and technological quality of juice [109,116,117].
Agrotechnical and chemical methods are used for protection of beet against C. beticola. The agrotechnical method involves selection of an appropriate position for cultivation. Intervals in the cultivation of beet on the same field should not be shorter than four years. In the case of presence of infested leaves in soil, the first infections occur by up to 4 weeks earlier. Direct neighbourhood with fields, where beets were cultivated in the previous year, should also be avoided. For several years, beet varieties with high resistance to the pathogen have also been available [109]. However, the basic and the best means of direct control of the disease being repeated spraying in plantations with fungicides during the season [114]. Cercospora beticola has been placed on the list of pathogenic organisms, causing epidemics among crop plants [regulation of the Minister of the Environment of 29 November 2002 concerning the list of pathogenic organisms and their classification, as well as the measures necessary for the individual degrees of containment, Dziennik Ustaw (Journal of laws of the Republic of Poland) of 16 December 2002, No. 212, item 1798.
Celery early blight is caused by Cercospora apii; it rapidly develops above all in the first half of the growing season of Apium graveolens. The aetiology of the disease is complicated by the fact that another species of the genus Cercospora parasitizing celery leaves, viz. Cercospora apiicola M. Groenew., Crous & U. Braun, has been recently described [118,119]. The development of this disease is very dynamic, especially in the first half of summer. In favourable, provocative conditions, the pathogen can cause crop infections in a devastating degree. All overground parts of the plant, i.e. leaf blade, petiole, and in the case of seed crops shoots, umbels, and fruits may also be parasitized. Severe infections of overground parts lead to a significant decrease in the weight of storage roots, and the reduction can reach as much as 90% [119,120]. The paralysis of foliar celery by C. apii significantly affects its taste value and colour. Diseased plants are characterized by a lower sugar content and less intense colour [121]. All varieties of root and foliar celery studied so far were characterised by a very high susceptibility to infestation by C. apii. The main source of primary infection refers to remnants of paralysed plants [120].
Cercospora leaf blight of carrot is a disease observed in carrot cultivations. Symptoms of the disease are visible as spots (necroses), which are sometimes confluent and cover a significant part of the leaf blade, and even may lead to foliage destruction. As in the case of the species mentioned before, production of new leaves proceeds by the expense of roots. Remnants of infected plants from the previous growing seasons, which remain in soil for up to four years, are also the main source of infection caused by C. carotae. The use of plant protection products, mainly fungicides, reduces the presence of C. carotae [122].
In the case of all the diseases mentioned above, a very important preventive factor is destruction of infected remnants of plants at the end of the growing season and application of fungicides in the case of appearance of the first infection symptoms.

Taxonomy
In the present study, cercosporoid species occurring in Poland and additional species deposited in Polish herbaria and to be expected in Poland are reported. The quarantine species listed in the regulation of the Minister of Agriculture and Agricultural Reform of 19 March 1984, regulation of the Minister of Environment of 29 November 2002, and regulation of the Minister of Agriculture and Rural Development of 21 February 2008, are also included. All genera and particular species are listed alphabetically. This chapter contains a dichotomous key to the genera concerned. Additionally, the doubtful species are described in a separate chapter. Each subsection of the "Taxonomy" chapter provides information about synonymy, type species, and short characteristics of the biology of each genus. Additionally, dichotomous keys to the species based on host plant families are presented.
The following data are provided within the genera: ■ names of species with references ■ synonyms ■ full description ■ hosts and distribution in Poland ■ geographical distribution ■ notes (if necessary).
An asterisk ( ) placed at the end of the name indicates that the species concerned have not yet been detected in Poland but might be found due to the presence of their hosts species in the Polish flora or cultivation in Poland. According to Crous and Braun [11], species included in C. apii s. l. have an additional name "Cercospora apii s. l. " in brackets under the current name. All known synonymy are mostly based on available monographs and single papers describing and dealing with the species concerned. Descriptions of species are primarily based on herbarium material or literature. Host plant species are listed alphabetically. Information about the distribution of fungal species in Poland is based on literature data from physiographic literature and herbarium collections. All listed localities are grouped into geobotanical regions, marked in the text with the appropriate symbols, according to Matuszkiewicz [123]. Herbarium acronyms given in parentheses refer to revised material. Notes about general distribution are based on the monographs of Chupp [13], Braun and Melnik [55], Crous and Braun [11], single published papers, as well as the fungal database website [124]. Countries listed in the general distribution are arranged in alphabetical order, first European, and then, after semicolon from other continents. In the case of species reported from Poland, drawings were made when the amount of material was sufficient.

Notes.
A true Cercospora s. str. close to C. apii s. l., but distinguished by obclavate-cylindrical conidia with obconically truncate bases [11]. The fungus has been reported on many representatives of Malvaceae worldwide. Groenewald et al. [24] suggested that an isolate from Malva sp. represented a different taxon, as indicated by molecular studies. It is possible to find this fungus in Poland on Arum. Cercospora ari is an insufficiently known species with unclear generic affinity [129].

Geographical distribution. Common in Poland and worldwide.
Notes. This fungus occurs across Poland in places of cultivation of most beet varieties (Beta spp.). In the present paper, all varieties of Beta spp. are regarded as Beta vulgaris L.
In Poland, C. beticola is a quarantine species mentioned in the regulation of the Minister of Environment of 29 November 2002.
The combined phylogenetic analyses performed by Groenewald et al. [24] using calmodulin genes (CAL), combined multi-locus data and histone H3 (HIS) have shown that C. apii s. str. and C. beticola are related sibling species, although C. beticola must be retained as a separate species.
Notes. Leszczenko [80] has also reported C. carotae from the "Biała Mał. " locality, but this name does not exist in the list of villages in Poland. The name may refer to the Biała village in Tarnów district (Małopolska Voivodeship).
Wakuliński and Marcinkowska [87] reported C. carotae on Barbarea vulgaris from Wrocław, which is doubtful and needs revision, because according to Crous and Braun [11] C. barbareae and C. nasturtii are the only cercosporoid species on Barbarea (Brassicaceae). The latter two species have recently been reduced to synonymy with C. armoraciae [24].
The fungus was also recorded by Michalski [128] from Nakło on the Noteć River, but after revision it proved to be Pseudocercosporella daucicola Goh & W.H. Hsieh.
Braun [22] transferred Cercospora chenopodii to the Passalora species based on broadly obclavate conidia with visible large loci. The conidia of this fungus are hyaline, and best retained in Cercospora, which has been confirmed by results of molecular sequence analyses in the TEF, ACT and HIS phylogenies by Groenewald et al. [24]. In this paper, a similar, confusable species tentatively referred to as Cercospora cf. chenopodii, was described and illustrated, which is morphologically distinguished from genuine C. chenopodii by its denser fascicles and above all genetically distinct in ACT and HIS phylogenies. Based on additional collections found in Iran, this species has recently been described as Cercospora pseudochenopodii M. Bakhshi, Arzanlou, Babai-ahari & Crous [163].
Notes. The fungus has been collected outside Poland, but material is deposited in a Polish herbarium. The fungus has been noted worldwide on four from eight species from Epipactis genus occuring in the Polish flora; therefore, it is very likely to find this fungus in Poland.
Notes. The molecular studies performed by Groenewald et al. [24] suggested that the name C. fagopyri can only tentatively be applied to other isolates than those from Fagopyrum.
Notes. The fungus has been collected outside Poland, but material is deposited in a Polish herbarium. The host occurs in the Polish flora; therefore it might be possible to find this fungus in Poland. Description. Leaf spots scattedred or confluent, distinct, circular, often zonate, 1-8 mm diam., centre pale brown to dingy grey, with purplish brown or dark brown margin. Caespituli amphigenous, but chiefly hypophyllous; stromata rudimentary or slightly developed, composed of several brown hyphal cells. Conidiophores 4-12 in divergent fascicles, usually pale olivaceous-brown, usually 1-2(-3)-septate, 1-3 times geniculate at the apex, not branched, 30-166 × 3.5-5.5 µm. Conidia solitary, hyaline, acicular, rarely obclavate, straight or slightly curved, subacute at the apex, truncate at the base, 35-220 × 3-4.5 µm. Notes. The fungus has been collected outside Poland, but material is deposited in a Polish herbarium. Only Ipomoea purpurea (L.) Roth occurs in Poland and C. ipomoeae has been reported on this host species outside Poland. Therefore, it might be possible to find this fungus in Poland.

Hosts. On
The fungus was published as C. juncicola, but according to Braun et al. [129] this name has recently been reduced to a synonym with C. juncina.

Hosts.
On Lotus spp., Leguminosae. Lotus corniculatus L.: A1 -Słowiński National Park [156]; Lotus pedunculatus Cav.: A1 -Słowiński National Park -Kluki, leg. I. Adamska, Jul. 2001 (SZPA 4933) [156];  Notes. This species is similar or could be identical with Cercospora apii s. l. [11]. The environmental conditions, especially high temperature and humidity have an influence on the length of conidiophores and conidia. Under favourable conditions, e.g. in a moist chamber, the conidiophores and conidia C. apii s. l. can reach up to about 500 µm, although under normal conditions they rarely achieve a length between 100 and 150 µm.

Notes.
Cercospora maianthemi was also reported on Paris quadrifolia L. from Stanowice [87]. The herbarium material needs revision, because only C. paridis is known on Paris [11]. Owing to hyaline to pigmented passaloroid conidia, the generic affinity of this species is not yet settled and needs to be confirmed by molecular methods [129].  Notes. This species is indistinguishable from C. apii s. l. Young conidia are mostly obclavatecylindrical, but older ones are distinctly acicular [11].
In Poland, it is a quarantine species mentioned in the regulation of the Minister of Environment of 29 November 2002.
Geographical distribution. Czech Republic, Denmark, Estonia, Finland, Germany, Hungary, Italy, Latvia, Norway, Poland, Romania, Russia (European and Asian part), Slovakia, Slovenia, Sweden, Ukraine; China, Japan, Kazakhstan. Cercosporina physalidis ( Description. Leaf spots amphigenous, scattered, sometimes confluent, subcircular to irregular, 2-15 mm diam., on the upper surface yellowish brown with a dark brown margin, on the lower surface brown or greyish brown. Caespituli amphigenous, but mostly hypophyllous; stromata usually small, dark brown to almost black, composed of several dark brown hyphal cells. Conidiophores 2-9 in dense or divergent fascicles, olivaceous-brown or brown, straight or 1-5 times geniculate, not branched, 1-6-septate, 30-192 × 4-5.5 µm. Conidia solitary, pale olivaceous-brown, mature conidia acicular, small and young conidia may be somewhat obclavate or cylindrical, straight to mildly curved, 3-15-septate, 50-195 × 3-4.5 µm [31]. Notes. The fungus has been found on Hyoscyamus sp. from Poland, but the herbarium material has not been revised. In addition, material on Nicotiana sp. from Java, deposited in the Kraków herbarium, was revised. In the Polish flora, three species of the genus Nicotiana occur: N. alata Link & Otto, N. rustica L., N. tabacum L. The fungus has been collected on the latter two host species in other parts of the world and also on 10 other hosts occuring in Poland. Therefore, it is very likely that this species might be found on these plants in Poland. "Cercospora physalidis emend. Braun & Melnik" is undoubtedly an unresolved complex of specialized as well as plurivorous species. Molecular data are currently not sufficient to define and properly circumscribe acceptable species within this complex [24].
Notes. This species has been reported by Danilkiewicz [83] on Plantago major from Derło and by Mułenko [81] on P. lanceolata from Durne Bagno Reserve. These collections have to be referred to as to Cercospora pantoleuca Sacc. after revision. However, Plantago spp. are common in Poland; therefore, the occurrence of C. plantaginis appears to be most likely.
Geographical distribution. Distributed worldwide on different species of the family Violaceae.
Notes. Wakuliński and Marcinkowska [87] wrongly cited the data of Moesz [133] on Viola palustris from Zaklików. The latter author reported the occurrence of C. violae on V. canina and V. reichenbachiana from Włoszczowa. On V. palustris, he published Cercospora ii Trail, which is currently a synonym of Passalora murina. They also wrongly cited the data of Madej [132] on V. tricolor, who did not report this fungus species in his paper.
Notes. The report of Siemaszko [171] published from Białowieża Forest as Ramularia geranii (Westendorp) Fuckel var. geranii is referred to as P. minutissima after revision of herbarium material by Wołczańska [39]. The material has not been available to be examined; therefore, the present description has been based on available literature.