THE OCCURRENCE AND PATHOGENICITY OF PHOMA EXIGUA DESM. VAR. EXIGUA FOR SELECTED SPECIES OF HERBS

P. exigua var. exigua was isolated from underground and aboveground organs of different herb plant species in the years 1998-2006. Pathogenicity tests of three randomly chosen isolates of the fungus T 299, T 261 and T 277 for thyme and of three isolates M 1657, M 1193 and M 1635 for lemon balm were carried out. The effect of water suspension of fungus spores on the germination ability of schizocarps and of infected soil on sprouting and seedling health was studied. The study of microscopic structures of the fungus developing on plants, isolation of the fungus on artifi cial media and macroand microscopic analysis on standard media are essential for the correct identifi cation of P. exigua var. exigua. The polyphagous nature of the fungus is confi rmed by the obtained results. The tested isolates were found to be occasional pathogens of thyme and lemon balm in the studied conditions. The harmfulness of the tested isolates pointed to inhibition of schizocarp germination, sprouting of plants and the ability to cause necrosis of germs, roots and epicotyl.


INTRODUCTION
. Accidental parasites of some species of cultivated plants include, for example, the polyphagous species of P. exigua Desm. var. exigua. This fungus is distinguished from other varieties of this species by its ability to produce a colourless "E" metabolite in cultures. This metabolite enters into reaction with 1N NaOH, what is taken into account in the identifi cation of these fungi (B o e r e m a and H ö w e l e r , 1967; M a r c i n k o w s k a , 1985).
Numerous isolates of P. exigua var. exigua have the ability to infect injured parts of plants from the genera of Cucumis, Glycine, Lupinus, Pisum and Raphanus (M a r c i n k o w s k a , 1984). Only the isolate from the same plant species caused an inhibiting effect of P. exigua on the germination of achenes Celosia cristata and the growth of seedlings (Ł a c i c o w a et al. 1992). On the other hand, a decrease of the ability of Bellis perennis achenes to germinate was caused by P. exigua isolates independently of the origin. The fungus also caused necrosis of the roots of seedlings grown from artifi cially infected achenes (Ł a c i c o w a et al. 1992). Apart from P. exigua var. foveata, P. exigua var. exigua belongs to the fungi causing the socalled gangrene of bulbs and the stem base of potato (B o e r e m a , 1976). Numerous isolates of the fungus classifi ed as the species P. exigua of P. exigua var. exigua were obtained as a result of the authors' own studies on herb diseases conducted since 1998.
As a result of frequent colonization of leaves, stems and roots of garden thyme and lemon balm by P. exigua var. exigua (M a c h o w i c z -S t e f a n i a k et al. 2002a; 2002b), pathogenicity tests were carried out on the isolates of P. exigua var. exigua as well as shizocarps and seedlings of thyme and lemon balm.

MATERIALS AND METHODS
The studies on fungi colonizing plants of different herb species cultivated in the Lublin region were conducted in the years 1998-2006 (Tab. 1). Fungi were identifi ed on the basis of etiological symptoms occurring on plants, and they were isolated from superfi cially disinfected parts of plants, using the artifi cial culture method and a mineral medium (M a c h o w i c z -S t ef a n i a k et al. 2002a; 2002b). One-spore cultures of fungi were identifi ed on the maltose medium or on standard media. The successively obtained isolates of Phoma spp. were identifi ed on the media of MA (maltose), OA (oats) and CA (cherry), based on the current principles of the taxonomy of fungi of the genus Phoma (de G r u y t e r and N o o r d e l o o s , 1992; de G r u y t e r et al. 2002).
Samples of the sowing material of thyme and lemon balm as well as 6 isolates of P. exigua var. exigua, randomly selected from the authors' individual collection of cultures of Phoma spp., were used in the pathogenicity tests. Isolates T 299, T 261 and T 277 were isolated from the thyme plants, while isolates M 1657, M 1193 and M 1635 were isolated from the lemon balm plants.
The pathogenicity tests examined the effect of P. exigua var. exigua on the germinating ability of shizocarps of thyme and lemon balm and the effect of the fungi through the infected soil on emergencies and health of the seedlings. The infection material consisted of conidia obtained from the one-spore cultures of the examined isolates, growing on the maltose medium (bioMérieux), at the temperature of 22 o C, without the access of light, in the form of a water suspension with the density of 3 x 10 5 conidia ml -1 , with an addition of a few drops of , after which 20 shizocarps were placed in each of the sterile moist chambers. 100 shizocarps, i.e. 5 dishes with 20 shizocarps in each, were used for each of the studied isolates and each plant species. The control was the shizocarps soaked for the same period in distilled water. The dishes with the plant material were kept in a thermostat at 30 o C with full lighting during the day (14 hrs) and at 20 o C in darkness at night (10 hrs). Observations were conducted every three days, and after 9 days the number of shizocarps that had and had not germinated as well as the number of germs with necrosis were determined. The shizocarps that had not germinated and those that had germinated and whose germs had died out were submitted to reisolation according to Koch's postulates. Results obtained in the last observation were statistically analyzed using Tukey's confi dence intervals.
The studies on the effect of P. exigua var. exigua on emergencies and health of seedlings considered single isolates of the fungus, i.e. T 299 and M 1657, of high pathogenicity in laboratory tests. The infection material was prepared according to Noll (Ł a c i c o w a , 1964), using compost earth with an addition of 5% ground rice as the culture subsoil. The superfi cially disinfected shizocarps of garden thyme and lemon balm were sown at points into Noll's mixture in pots with the diameter of 14 cm, and they were covered with a 0.3 cm layer of earth. The control was the superfi cially disinfected shizocarps sown into the compost soil with an addition of ground rice. 50 shizocarps, 5 pots with 10 shizocarps in each, were used for the isolate of the fungus as well as the plant and the control. In total, the experiment considered 100 shizocarps of garden thyme and 100 shizocarps of lemon balm. The pots together with the shizocarps were kept for 35 days in a climatic chamber in the conditions of temperature, moisture and irradiation enabling the emergence and growth of plants. The number of the obtained plants was determined every 10 days, and after the experiment was fi nished, i.e. after 35 days, the occurrence of necrosis on the roots and the hypocotyl of plants was established. Next, the plant material was reisolated according to Koch's postulates. The results obtained in the last observation were submitted to statistical analysis using Tukey's confi dence intervals.

RESULTS
The presence of P. exigua var. exigua was found on the underground and aboveground parts of the studied herb species. Pycnidia and spores typical of the genus Phoma were found on the surface of non-specifi c disease symptoms. The mycological analysis fully confi rmed the occurrence of those fungi, including P. exigua var. exigua belonging to the section of Phyllostictoides. After 7 days the cultures of the studied isolates on the maltose medium produced an olive-brown, fl occulent mycelium with a layered and slightly cutin edge and an olive-brown reverse. After 14 days the colour of the airy mycelium and the reverse was more intense. The structure of the colony was delicate, fl uffy, concentrically zoned, with a layered and not very cutin edge. The diameter of the colony after 7 days was 25-38 mm, and after 14 days 50-72 mm. On the cherry medium, the colonies had a fl uffy-fl occulent or fl occulent mycelium after 7 and 14 days. In the central part, the colonies were dark or brown-olive with a white edge. The reverse of the colony was olive-green. The diameter of the colony after 7 days was 30-42 mm, and after 14 days 55-70 mm. On the oat medium, the edge of the colony after 7 and 14 days was irregular, wavy or slightly layered. The structure of the mycelium was fl occulent, brown or olive-brown with a light margin. The diameter of the colony after 7 days was 30-48 mm, and after 14 days 50-75 mm.
Pycnidia on the oat medium produced aggregates in the central part of the colony, with the structure similar to concentric rings. They were found on the surface of the agar in the airy mycelium or they were partly submerged in the agar. The pycnidia were spherical or almost spherical, dark brown or black, with one outlet and without a nodule (Figs 1a, b). Thick, creamy conidal exudate of the fungus fl owed from the ostiole. The size of the pycnidia ranged from 74.2 to 185.5 μm. The conidia were differentiated in their shape and size, most frequently oval, elliptic or roundish, with singular drops of fat at each end, without any transverse divisions or -sporadically -with one dividing wall. The size of 0-division sporules was 3.7-7.4 x 2.5-3.7 μm, and of those with one division 7.4-9.25 x 2.5-3.5 μm (photo 2). After 1N NaOH was sprinkled on the edge of the colony growing on MA medium, the medium became slightly green. The reaction with NaOH on the oat medium was negative. There were no crystals on the reverse of 14-day-old colonies.
The cultures of the examined fungus were isolated in the study years from 11 species of spice and medicinal plants cultivated in the region of Lublin (Tab. 1). Most frequently, they were obtained from superfi cially disinfected roots, stems and leaves of lemon balm and garden thyme as well as from stems and leaves of caraway and dill (Fig. 3). The fungus colonized the underground and aboveground parts of peppermint, parsnip, common motherwort, St. John's wort and garden sage. It was isolated not only from non-disinfected but also from superfi cially disinfected shizocarps of the majority of the studied species of herbaceous plants (Tab. 1).
In the experiment with inoculation of the water suspension of P. exigua var. exigua spor, from 71 to 76% of thyme shizocarps did not germinate till the 9 th day (Tab. 2). The mean number -ranging from 14.2 to 15.2 -of shizocarps that did not germinate was signifi cantly higher than in the control combination (Tab. 3). On the other hand, no signifi cant difference was found in the mean number of the shizocarps that did not germinate and that had contact with particular isolates of P. exigua var. exigua. Despite the contact with the studied isolates of the fungus, progressing necrosis of the germs was observed in the shizocarps that germinated. It occurred in the form of light brown spots graduating into streaks or it was present on the whole surface of the germs (Fig. 4). After 9 days, the percentage of the germs with necrosis ranged from 20 to 23, and the mean number of the germs with necrosis ranged from 4.0 to 4.6 (Tab. 3). The latter values were signifi cantly higher than the control value, while not differing from each other in a signifi cant way (Tab. 3). a -undisinfected shizocarps b -disinfected shizocarps + -frequency up to 5% ++ -frequency from 5% to 15% +++ -frequency from 15% to 30% Table 2 Effect of inoculation with water suspension of Phoma exigua var. exigua conidia on the germination of shizocarps of the studied herbs.  Table 3 Effect of inoculation with water suspension of Phoma exigua var. exigua conidia on the germination of shizocarps of the studied herbs -the mean of 5 replications.
Isolate no. Values marked with the same letter do not differ signifi cantly Table 4 Effect of P. exigua var. exigua on the shooting up of the studied herb cultivars -the mean of 5 replications.    P. exigua var. exigua, with the macroscopic and microscopic features corresponding to those of the cultures used for artifi cial infection, was isolated from the decayed germs and shizocarps that did not germinate. The largest number of P. exigua var. exigua cultures were reisolated after using isolates T 299 and T 277 (Tabs 2, 3).
In the contact with the water suspension of P. exigua var. exigua, from 66 to 70% shizocarps of lemon balm did not germinate till the 9 th day of observation (Tab. 2). The mean number of the shizocarps that did not germinate, which ranged from 13.2 to 14, was signifi cantly higher than in the control combination (Tab. 3). The number of the germinated shizocarps of lemon balm after 9 days ranged from 30 to 34 (Tab. 2). However, more than half of the germs showed the signs of strong necrosis, leading to their decay (Tab. 2). The mean number of germs with necrosis that had contact with the fungus was -independently of the isolate -signifi cantly higher than in the control (Tab. 3). Like in the case of garden thyme, necrosis of the germs was visible in the form of light brown spots graduating into longitudal streaks. The germs were very thin and inhibited in growth. Like in the case of garden thyme, P. exigua var. exigua cultures were isolated from the non-germinating shizocarps of lemon balm and from the germs with the signs of necrosis. Their macroscopic features corresponded to the features of the cultures used for artifi cial infection. The number of P. exigua var. exigua cultures reisolated from lemon balm was the highest as a result of using the isolates M 1657 and M 1635 for inoculation, and it was 43 and 39, respectively, while the mean number of P. exigua var. exigua cultures reisolated from the shizocarps of lemon balm after using those isolates was 14 and 7.8, respectively (Tabs 2, 3).
An average of 3.6 of plants emerged as a result of artifi cial infection of thyme shizocarps with the isolate T 299 of P. exigua var. exigua through the infected soil. That value was signifi cantly lower than in the control combination (Tab. 4). Within this number, 3.1 were the seedlings with necrosis of the germs and the hypocotyl, and that value was signifi cantly higher compared to the control combination (Tab. 4). Cultures of the fungus P. exigua var. exigua used for artifi cial infection were reisolated from the infected seedlings of thyme. On the other hand, this species was not isolated from the control plants (Tab. 4). The seedlings obtained in the experimental conditions were -contrary to the control plants -inhibited in their growth, they had small cotyledons and decayed endings of the roots (Fig. 5). These seedlings died out within 3-4 days.
An average of 5.9 of plants emerged as a result of inoculation of lemon balm shizocarps with the isolate M 1657 of P. exigua var. exigua through the infected soil. That value was signifi cantly lower than the value in the control combination (Tab. 4). The mean number of seedlings with necrosis of the seedlings and the hypocotyl was 4.1, and it was signifi cantly larger than in the control combination (Tab. 4). The mean number of P. exigua var. exigua cultures reisolated from the infected seedlings of lemon balm was 4.4 (Tab. 4). The fungus was not isolated from the control plants. The lemon balm seedlings in the experiment with artifi cial infection with the isolate M 1657 of P. exigua var. exigua, in comparison to the seedlings in the control combination, were inhibited in their growth and had a reduced root system and necrosis of the root endings.

DISCUSSION
The character of the growth of the colonies of the studied fungus isolates, the morphology of the pycnidia and conidia as well as physiological-biochemical properties, corresponding to the description of the S t e f a n i a k, 2004) provide information on the occurrence of P. exigua var. exigua on other studied herb species. The ability of the fungus to colonize the aboveground and underground parts of herbaceous plants and the facultative character of its parasitism, which was shown in the present studies, point to the fact that P. exigua var. exigua easily survives in unfavourable conditions. It can survive on the surface of the sowing material or inside seeds, which was shown in the case of Achillea millefolium, Coriandrum sativum, Carum carvi, Anethum graveolens, Thymus vulgaris and Pastinaca sativa as well as on the after-crop residue of infected plants or roots and lower parts of stems of perennial herb species. The frequent colonization of herbaceous plants by P. exigua var. exigua in natural conditions and positive results of the pathogenicity tests confi rmed by the fungus reisolation point out that the studied isolates can be considered as accidental pathogens of thyme and lemon balm. Their harmfulness consists in inhibiting the germination of shizocarps and emergences of the tested herb species, in causing necrosis of seedlings, roots and hypocotyls of seedlings. Causing necrosis of infected organs is certainly related to the production of the enzyme of ß-glu- The applied methods of inoculation proved useful in testing P. exigua var. exigua pathogenicity towards herbaceous plants as they ensured a direct contact of the fungus with shizocarps, seedlings and seedling roots. The method of soaking the sowing material in a water suspension of the germs, also positively estimated in reference to soybean seeds, proved effective and useful in evaluating the pathogenicity of numerous isolates of the fungus (M a r c i n k o w s k a , 1984). The lack of a signifi cant difference in the harmfulness of P. exigua var. exigua isolates originally obtained from thyme towards this plant and the harmfulness of isolates obtained from lemon balm towards the latter seems to confi rm earlier suggestions on the relationship between the pathogenicity of fungi and the origin of the isolates (Ł a c i c o w a et al. 1992; Z a l e w s k a and M a c h o w i c z -S t e f a n i a k , 2004).