Characterization of Phoma negriana Thüm., a new species from grapevine canes

c S t e f a n i a K r ó l E.: Characterization of Phoma negriana Thüm., a new species from grapevine canes. Acta Mycol. 42 (1):113-117, 2007. The occurrence and elements of morphology of P. negriana Thüm . were studied. The fungus cultures were isolated from grapevine canes cultivated in South–East Poland. Grapevine stems from which the cultures of P. negriana were obtained showed symptoms of necrosis and bark crashes. The species of the fungus was identified on the basis of pycnidia and conidia morphology, the character of colonies growth and biochemical features of the studied isolates.


INTRODUCTION
Fungi from the genus Phoma are represented by numerous species grouped into nine sections, selected on the basis of pycnidia structure, especially their cells wall, the presence of chlamydospores including dictiochlamydospores and the ap- Considering manner of life and trophism within Phoma spp.saprotrophs, opportunistic pathogenes and specific pathogenes of plants can be distinguished.Among opportunistic pathogenes numerous species occur on above ground parts of fruit-trees and bushes.One of them is P. pomorum Thüm., section Peyronella, causing necrotic lesions on the leaves and fruit of apple tree (B o e r e m a 1993; B l e c h t o v a 1995).Phoma macrostroma Mont., sec.Phyllostictoides, belongs to the complex of fungi colonizing decayed stems and buds of walnut, ash and beech (P i n t e r et al. 2000; G r i f f i t h , B o d d y 1990; To t i et al. 1993).Phoma exigua var.populi de Gruyter et Scheer, sec.Phyllostictoides was recognized as opportunistic pathogen of poplar seedling, P. exigua var.viburni (Roum.ex Sacc.)Boerema as pathogen of various cultivars of viburnum and P. exigua var.lilacis (Sacc.)Boerema of lilac stems (d e G r u y t e r , S c h e e r 1998).Phoma herbarum Westend., sec.Phoma, in addition to Phomopsis viticola and Cytospora sp. was isolated from grapevine stems with symptoms of bark necrosis (S t o j a n o v i č 1086).Recently, Phoma negriana Thüm., sec.Phoma has been recognized as opportunistic pathogen of grapevine in the Netherlands (d e G r u y t e r et al.1998).This species was noted in the vineyards of South Europe earlier, causing disease symptoms on the leaves, fruit and stems (d e G r u y t e r et al. l.c.).In Poland P. negriana has not been observed so far.

MATERIAL AND METHODS
Four isolates of P. negriana i.e.W 1205, W 1308, W 1075, W 1432 from a collection of this species isolates in our possession, were randomly chosen for the study.These cultures were isolated from grapevine canes cv.Schuyler, Iza and RF-16 with symptoms of necrosis and cultivated in South-East Poland in the years 2000-2003.The occurrence of the fungi was determined on the basis of etiological symptoms present on the surface of diseased canes and on the basis of mycological analysis according to the artificial culture method.The isolation was carried out from superficially disinfected canes using maltose medium (bioMerieux) according to M a c h o w i c z -S t e f a n i a k and K u r o p a t w a (1993).The obtained isolates of P. negriana were identificated on the standard media  2004), and the measurment of their diameter were performed after 7 and 14 days of colonies incubation in the thermostat.After 2 weeks of incubation on OA the measurment of 400 conidia (4 isolates x 100 conidia) and 200 pycnidia (4 isolates x 50 pycnidia) were made and the presence of chlamydospores was tested.

RESULTS
The studies indicated that on the surface of grapevine canes in the place of necrotic lesions the pycnidia with conidia with features typical of Phoma species were present.A big number of Phoma spp.cultures were isolated from these canes.The share of Phoma negriana isolates among fungi isolated from grapevine canes in the study years was: 0,4% in 2000; 1,4% in 2001; 0,3% in 2002 and 0,5% in 2003 (0,65% in average).
The studies of selected isolates on the standard media indicated that 7-day-old fungus colonies on the CA medium were rather regular and had the diameter from 28 to 35 mm.In the beginning, they formed soft floccose, white-gray aerial mycelium with cream reverse.After 4-6 days of incubation in the centre of colony the pycnidia appeared and after 7 days conidia were formed.After 14 days of growth, the margin of colonies was irregular and their diameter ranged from 44 to52 mm (Fig. 1).The aerial mycelium was dark-green or gray.On the whole surface of the colony abundant pycnidia were formed.They were partly immersed in the substrate and secreted the yellow-saffron drops containing conidia.The reverse of 14-day-old colonies was dark-green to almost black.
On OA medium, 7-old-days colonies had a less regular margin and colony diameter from 25 to 28 mm.The aerial mycelium was white-gray and floccose with a rather compact structure.First pycnidia were formed after 4 -6 days of incubation.After 14 days the colony's diameter was from 35 to 48 mm.The mycelium structure was slightly velvety, green-olivoceous with white margin.Pycnidia were formed on the whole surface of the colony.
On MA medium, the diameter of 7-day-old colonies was from 18 to 22 mm.The colonies were regular with floccose, white-gray surface.The diameter of 14-day-old colonies was from 23 to 35 mm.The colonies were with more or less regular growth, rather compact, of floccose structure, gray-green or gray-black.The reverse was olivaceous-black with a somewhat brighter margin.The formation of pycnidia was poorer than on OA and CA media.
A detailed observation carried out on OA medium allowed to determine that pycnidia of the studied isolates of fungus were formed as singular or with small groups.They were thin-walled, globose or sometimes oval, honey-brown with papillate ostiole (Tab. 1, Figs 2, 3).The majority of pycnidia had 1 ostiole.The surface of the walls was smooth with some hyphal outgrowths.The diameter of the pycnidia was from 72 to 250 μm.Conidia aseptate, oblong with 2 or more guttules (Tab. 1, Figs 4, 5).The conidia had length from 4.2 to 7.88 μm with some reaching 9.88 μm (Tab.1).The width of conidia ranged from 1.97 to 3.9 μm.Chlamydospores and crystals were not observed in the cultures of the studied isolates of P. negriana.4.5 -8.5 (10.5) x 2 -4 In the case of 3 isolates no changes were observed in colony coloration on the media MA and OA after reaction with 1N NaOH.Only in the case of isolate W 1432 on MA medium the change of coloration into red-brown was noted.

DISCUSSION
The obtained results concerning characterization of colonies growth of the studied isolates, the morphology of pycnidia and conidia, compared with a description in a monograph of Phoma section (d e G r u y t e r et al.The fact of isolation of P. negriana isolates from grapevine canes contributs to an increased a number of species described on the stems of this plant, cultivated in South-East Poland. Considering earlier recognition of this fungus as opportunistic pathogen of the above ground parts of grapevine (d e G r u y t e r et al. 1998), the possible pathogenicity of the fungus towards this plant in climatic conditions of our country require more scrupulous tests.
pearance of pycnidiospores (B o e r e m a 1997; d e G r u y t e r , N o o r d e l o o s , B o e r e m a 1998; d e G r u y t e r , B o e r e m a , Va n D e r 2002).Phoma spp.occur commonly in different geographical regions of the world on the plants from va rious botanical groups (S u t t o n 1980; M a r c i n k o w s k a 1995).
according to a monograph by d e G r u y t e r et al. (l.c.), with regard to the present principles of taxonomy (B o e re m a 1976; M a r c i n k o w s k a 1995; B o e r e m a et al. 2004).The inoculum of chosen isolates of P. negriana was put in the centre of Petri dishes with solidified media: malt-extract agar (MA), cherry agar (CA) and oatmeal mealagar (OA) (d e G r uy t e r , N o o r d e l o o s 1992).The cultures were incubated in thermostat for 7 days at temperature 22°C, without light and during a second week at 13 hours in UV light and 11 hours in the darkness.A description of the colonies morphology, with regards to the principles of B o e r e m a et al. ( l.c.; B o e r e m a et al. 2004) allowed to identify the studied species as Phoma negriana.Macroscopic features of colonies of the studied isolates of P. negriana, especially the growth rate, colouration and mycelium structure showed similarity to a description by d e G r u y t e r et al. ( l.c.).The observation indicated that the mentioned feature and character of the colonies growth should have great importance in identification of the species from the genus Phoma which corresponds to earlier information of other authors (B o e r e m a 1976; 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. l.c.).Considering great morphological similarity of conidia within Phoma species, their dimension should be an important but additional diagnostic feature (B o e r e m a 1997; M a r c i n k o w s k a 1995; 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).According to the opinion by d e G r u y t e r et al. (l.c.) the reaction of P. negriana isolates with 1N NaOH is not specific, which confirms the results of the present study.
Characterization of pycnidia and conidia of Phoma negriana (mean for 4 isolates)