New Phoma species on Leonurus cardiaca

Two species of Phoma obtained from motherwort Leonurus cardiaca L., during mycological analyses attendant upon three-years study connected healthiness of the plants. Isolates of Phoma capitulum were obtained from roots, whereas Phoma septicidalis from roots and leaves. Description in vitro, photos of morphological structures and distribution of Phoma spp. are given. This is the first report of P. capitulum and P. septicidalis on motherwort in Poland.


InTrODUcTIOn
Phoma spp.(Sphaeropsidales) constitute a group of fungi with very differentiated biological specialization (M a r c i n k o w s k a 1995).They include saprophytic species, developing on different substrata, secondary pathogens of plants with polyphagous character of parasitism and specific pathogens of cultivated plants.The group of plants from which fungi from genus Phoma were isolated includes for example herbaceous plants (M a c h o w i c z -S t e f a n i a k et al. 2002; M a c h o w i c z --S t e f a n i a k et al. 2004).Phoma dictamnicola Boerema, de Gruyter et Noordel., P. glaucii Brun., P. multirostrata var.microspora (Allescher) Boerema from the leaves, stems and roots of common thyme, while P. eupatorii Died.was obtained from creemocarps (M a c h o w i c z -S t e f a n i a k , Z i m o w s k a 2000; M a c h o w i c z --S t e f a n i a k et al.Considering the key importance of Phoma spp.morphology, with the proper differentiation of taxa, the present paper deals with the identification of the enumerated two species of fungi made on the basis of thorough studies of macro-and microscopic properties of these organisms in cultures in vitro.

MATerIAL AND MeTHODS
The study material consisted of isolates Lc 186, Lc 259, Lc 368, Lc 544 and Lc 56 P. capitulum obtained from the roots and isolates Lc 238, Lc 79, Lc 139 and Lc 225 P. septicidalis from the roots and leaves of 2-year-old motherwort.The fungi cultures used were obtained as a result of the mycological analysis (Z i m o w s k a , M a c h ow i c z -S t e f a n i a k 2004) accompanying the three-year-long studies (2004)(2005)(2006) on plants' healthiness that were conducted on production plantation in the communes of Fajsławice and Dziecinin in the Lublin district (Z i m o w s k a unpublished).
Identification of the obtained isolates and the taxa they belong to was carried out on the basis of a thorough analysis of morphological structures, macroscopic properties of colonies and biochemical features conducted in vitro on standard media (maltose MA, oat agar OA and cherry agar CA) after 7 and 14 days of incubating the cultures in a thermostat at the temperature of

rESUlTS
The studies conducted on the growth of the analyzed isolates of P. capitulum on standard media showed that the cultures of the fungus on MA after 7 days reached the diameter of 38-40 mm, while after 14 days 75 mm.The hyphae of the air mycelium formed a flocky structure of white or light olive-grey colour.The reverse was peach-salmon colour.The edge of cultures of all the studied isolates was characterized by regular growth.The growth of P. capitulum isolated on OA was slightly poorer than on MA since the diameter of the colony measured after 7 days was 36 mm, while after 14 days it ranged from 65 to 70 mm.The colonies were white-creamy with a salmon reverse, while the hyphae of the air mycelium formed a velvet-flocky structure.The smallest diameter of colonies was observed on medium CA.After 7 days it ranged from 27 to 29 mm, while after 14 days -from 53 to 58 mm.In the case of most isolates, on this medium the edge of a colony was characterized by irregular growth.The hyphae of the air mycelium were white and they formed a flocky structure.The reverse of the colony was of light salmon colour.
Numerous pycnidia on OA centered in concentric circles or sectors.They were round, olive to olive-black, mostly smooth or covered with scarce hyphal outgrowths (Fig. 1).From 1 to 3 ostioles, from which grey conidial exudates, were observed on the surface of the pycnidia.The latter were mostly formed on the surface of agar or they were partly immersed in it.Their size was from 50 to 100 μm, on average (Tab.1).The conidia were hyaline, round or slightly ellipsoidal, and they contained 1 to 2 guttules, their size ranging from 3.7 to 2.5 μm (Fig. 2, Tab. 1).In the case of all the studied isolates of P. capitulum, the reaction with 1N NaOH was negative.Besides, none of the isolates formed chlamydospores.
After 7 days of growth P. septicidalis isolates on MA medium formed colonies with the diameter ranging from 20 to 38 mm, while after 14 days the diameter ranged from 41 to 70 mm.The hyphae of the air mycelium formed a compact felt-like structure of rosy-wine colour and the reverse of the same colour.A slightly bigger diameter was observed in the case of the growth of the colonies of P. septicidalis isolates on OA medium.After 7 days it ranged from 21 to 40 mm, while after 14 days -from 50 to 57 mm.The structure of the air mycelium was the same as on MA medium.The central part of the colony was olive-grey, while the edge had rosy-wine colour.On CA medium the fungus colonies reached the diameter from 20 to 29 mm after 7 days, while after 14 days it ranged from 40 to 60 mm.The structure of the air mycelium, the colour of the obverse and the reverse was similar to the colonies growing on OA medium.The edge of the studied isolates of P. septicidalis on all the three media was characterized by irregular growth.Detailed observations on OA medium showed that the pycnidia of the studied fungus isolates got formed individually or in small groups.They were most frequently round, honey-olive to black-olive one, and their walls were covered by very numerous setae with the length ranging from 180 to 210 μm (Fig. 3, Tab.1). 1 to 2 ostioles, from which white drops of conidial exudates, were observed on the surface of the pycnidia.The size of the pycnidia ranged from 65 to 160 μm (Tab.1).The conidia were hyaline, 1-cell, ellipsoidal, with a few gutteles with the dimensions of 4.4 x 2.2 μm (Fig. 4, Tab. 1).In the case of all the studied isolates of P. septicidalis, the studies observed a positive reaction with 1N NaOH on MA and OA media.The edge of the colonies was of violet-purple.Besides, fragmentation of the mycelium cells characteristic of this species was visible on the hyphae of the mycelium, especially in older cultures (Fig. 5).None of the studied isolates of the fungus formed chlamydospores.

DIScUSSIOn
Thanks to the studies in vitro on the morphology of pycnidia and conidia and on the character of the growth of the analyzed cultures it was possible to distinguish two discussed species of Phoma and classify them into the proper sections.results of the studies as compared to the descriptions contained in the monographs of fungi from the genus Phoma made it possible to place P. capitulum isolates in part I of Phoma section since they include studies on taxa with very small dimensions of conidia, with the length not exceeding 5.
2002).P. exigua var.exigua Desm.and P. glomerata (Corda) Wollenw.et Hochapf.were isolated from the roots, stems and leaves of common thyme, common balm and St. John's wort (Z i m o w s k a , M a c h o w i c z -S t e f a n i a k 2004; M a c h o w i c z -S t e f a n i a k et al. 2004).Isolates of P. strasseri Moesz.were obtained from the stems and rhizomes of peppermint showing the signs of black rot (Z i m o w s k a , M a c h o w i c z -S t e f a n i a k 2005).In recent years the species of Phoma capitulum Pawer, Mathur et Thirumalachar (P.ostiolata var.ostiolata Pawer, Mathur et Thirumalachar, P. ostiolata var.brunnea Pawer, Mathur et Thirumalachar) as well as Phoma septicidalis Boerema (Pyrenoacheta telephii Alesch., Phoma telephii (Vestergr.)Kestern), which have not been described earlier, on this plant, have been isolated from Leonurus cardiaca L. (d e G r u y t e r , N o o r d e l o o s 1992; d e G r u y t e r , B o e r e m a 2002).In accessible literature these species are enumerated as commonly occurring saprotrophs developing on different substrata, and P. septicidalis can also cause necrosis of the leaves of plants from the family Gramineae (d e G r u y t e r , N o o r d e l o o s 1992; d e G r u y e r , B o e r e m a 2002).
22 o C (d e G r u y t e r , N o o r d e l o o s 1992; Z i m o w s k a , M a c h o w i c z -S t e f a n i a k 2005).The studied fungi cultures were marked on the basis of a monograph by d e G r u y t e r and N o o r d e l o o s (1992) and d e G r u y t e r and B o e r e m a (2002).
5 μm (d e G r u y t e r , N o o r d e l o o s 1992).Although the dimensions of the conidia in fungi from genus Phoma play a secondary diagnostic importance (D o r e n b o s c h 1970), in the case of the species belonging to Phoma section this property acquires a special significance due to the division of this section into three parts performed on the basis of the conidia dimensions (d e G r u y t e r , N o o r d e l o o s 1992; d e G r u y t e r et al. 1993; d e G r u y t e r et al. 1998).On the basis of a detailed analysis of morphological structures, especially pycnidia, the studied isolates of P. septicidalis were classified into the section of Paraphoma.A characteristic feature of the species described by the Dutch mycologists is the formation of pycnidia with the walls covered with setae, which are formed on their whole surface or they occur only around the ostiole (d e G r u y t e r , B o e r e m a 2002).The conducted studies confirmed the correctness of the assumption presented in the 1930's and 1940's by Woollenweber, Hochapfel and Dennis (D e n n i s 1946), and later continued by the mycologists from a Dutch Station of Plant Protection in Wageningen (B o e r e m a , B o l l e n 1975) that defining the taxa within Phoma spp. is possible only on the basis of constant morphological features that are observed in vitro in the cultures developing in standard conditions.