FUNGAL PARASITISM OF THE CYSTS AND EGGS OF THE GLOBODERA ROSTOCHIENSIS

A survey of Globodera rostochiensis infested field in Velingrad region (South-West Bulgaria) revealed the spread of a black egg disease in the old cysts. The fungus mycelium infected the embryos within the egg shell. The highest egg parasitization by fungi was observed during June (22.2%). The fungus destroyed the new progeny in September. Four species of nematophagous fungi were isolated from cysts and eggs: Botryotrichum piluliferum Sacc. And March, Scolecobasidium constrictum Abbott, Gliocladium roseum and Phoma fineti Brun.

Data on the mycology of globoderid nematodes of the world is limited.R o e s s s n e r (1987) isolated Clodosporium herbarum, Preussia sp. and two of F. oxisporum from G. rostochiensis eggs in Germany.Septocylindrium -like fungus Exophila spp.and Cylindrocarpon spp.were isolated from G. rostochiensis at three localities in Sweden (D a c k m a n , 1990).Several fungi were associated with G. rostochiensis in Peru where the potato and these cysts are native (R o d r i g u e s -K a b a n a et al., 1988).
Casual observations of populations of G. rostochiensis revealed the presence of black eggs in some cysts in Velingrad region in Bulgaria.The purpose of the present investigation was to examine the extent of fungal parasitism of G. rostochiensis at one localitiy in Velingrad and isolate fingi from cysts and eggs.

Materials and Methods
Soil samples were collected from one field in Velingrad region (South-West Bulgaria).At the time of sampling the field had been grown for several years in a crop rotation for 3 years of susceptible potatoes.Cores were taken to 20 cm depth on a zig -zag pattern over the field and mixed to give one composite sample of 1 kg.Monthly samples were taken from the field for two years (September 1999 to October 2000).The cysts were extracted from 100 g soil in 3 replications by wet -sieve decantation technique (S o u t h e y , 1986) and egg numbers were determined by separating the eggs from the cysts by crushing with hand.The healthy and diseased eggs were counted with the aid of a microscope (40 x).
Part of the soil collected from the field was used in pot experiment.24 (11cm -diam) pots were filled with infested -field soil and planted with potatoes, S. tuberosum L. (cv.Focal).They were then placed in glasshouse.During August (when the plants matured) the shoots were removed.Three pots were removed at monthly intervals and cysts were extracted.Ten white/brown cysts were randomly chosen from each pot, rinsed several times in sterile water and preserved at room temperature in sterile sand in glass vials.During December cysts were separated from sand, crushed in water and the numbers of black and healthy eggs were recorded.
Part of the soil collected from Velingrad village was steam-sterilized and put in 24 pots.A second set of 24 pots was filled with naturally -infested field soil (containing 45 healthy and five black eggs per g soil).Potato seeds were placed and one week after germination seedlings in the pots containing sterilized soil were inoculated with healthy eggs and juveniles (1500, in cysts).From June onward the pots were examined more frequently and when the white females emerged, these were examined at higher magnification (400 x) to ascertain if there was any microbial infection.Weekly counts of healthy and diseased females were recorded in both of the sets in three replications until the end of July when no more females were formed.
Fungi were isolated from field -grown cysts and eggs of G. rostochiensis.Ten diseased cysts were surface sterilized by agitation for 3 min.in 5 ml NaOCL (0.1%), then removed by washing the cysts twice with sterile destilled water (DW).They were carefully crushed in DW and suspension was placed in Petri dishes containing oat agar.The eggs were released by carefully crushing the diseased cyst (or females) so as not to break the eggs' shells.Cyst debris was separated from eggs by passing the suspension through a 200 μm-pore nylon sieve and collected the eggs on 20 μm -pore sieve.Eggs were washed three times with sterile water and then transferred to plates containing oat agar.The plates were incubated at room temperature for 14 days to allow fungi to grow.Fungal colonies were identified to species by B a r n e t t (1960), B a r r o n (1968) and D o r e n b o s c h (1970).

Results and Discussion
Under mountain conditions of Bulgaria, potatoes are sown from mid-April to the beginning of May.White cysts of G. rostochiensis are present during June and turn brown from July to August.These brown cysts remaining then in the soil can easily be distinguished by their full contents and elasticity from those of the previous season still present in the soil.Thus the egg stage of the nematode is exposed to parasities/predators for as long as 6-7 months, although protected within the cyst wall.Our study began in September when the eggs within the cysts had already been in the soil for several months.
Irrespective of the season, diseased eggs were recorded in all the field samples, although the number of diseased eggs varied.From September (1999) to October (2000) the percentage of black eggs was from 4.3 % to 22.2 % only in the old cysts.The field from which the bulk soil sample was obtained had a long time infestation of G. rostochiensis.Black eggs comprised 8.1 % of the total egg population in September (1999).As the juveniles from healthy eggs emerged and started penetrating the potato roots, the percentage of black eggs increased from 14.27 % in May to 22.20 % in June when most of the eggs had hatched.In June, as the females started laying eggs, the population of healthy eggs increased from 42.0 to 61.33/g in July.The population of black eggs decreased from 22.20 % in June to 5.23 % in July (Table 1).
The new population of eggs did not show any sign of fungus infection.Black egg disease was noticed for the first time in the new progeny of eggs from September and their population increased during September -October.The availability of soil moisture and conducive temperature during this period may be responsible for the activity the fungus.K e r r y at al. (1980) observed that parasitism of fungal antagonists was greater in H. avenae during prolonged periods of higher rainfall.Between 3.8 -22.2 % of eggs were destroyed by the fungus in these study.The survey clearly showed the incidence of black egg disease in G. rostochiensis -infested area of Velingrad region.The pot experiment confirmed these findings.Newly formed cysts removed from pots containing naturally -infested soil from May to August and preserved in sterile sand had not developed disease eggs by December.Cysts removed from September onward however contained 1.7 -4.7 % black eggs by December (Table 2).Thus fungus becomes active from September onwards.In the second pot experiment females emerging on plants grown in naturally infested soil were attacked by fungal parasites during the second half of June (Table 3).Presumably, the infection might have occurred at the time of white cyst stage.A total of 6.63 % of the females were destroyed due to fungal attack in naturally infested soil.Younger eggs are generally more susceptile to fungal attack than older ones (C a y r o l et al., 1982; I r v i n g et al. 1986).The physiological state of the eggs probably also influences their susceptibility to fungal colonization (I r v i n g et al. 1986; N i g h et al., 1980) and although some aspects of the biology of fungi infesting the eggs of cyst nematodes are known, their mode of action is not yet fully understood.
The investigations showed that four species of nematophagous fungi were isolated from eggs and cysts of G. rostochiensis: Scolecobasidium constrictum Abbott, Gliocladium roseum and Phoma fineti Brun was isolated from the eggs, and Botryotrichum piluliferum Sacc.and March from the inside of cysts.Presumably, these 4 species decreased cysts population of G. rostochiensis.
The significance of G. roseum and Scoleccobasidium spp. as a destructive parasites was studied by B a r n e t t and L i l l y (1962), U p a d h y a y (1966).According R o d r i g u e s -K a b a n a et al. (1984) isolates of G. roseum differ in their pathogenicity from phytonematodes.Species of Phoma and allied genera, although unable to physically disrupt and destroy eggs of heteroderid nematodes, produce pigmented diffusible metabolites that penetrate egg shells, affected eggs become pigmented and fail to hatch (R o d r i g u e s -K a b a n a et al. 1988).
B e r n a r d and S e l f (1987) recently showed that cultural practices can affect the population dynamics of several egg -destroying fungi.Fild studies by S o s n o w s k a et al. (2000) showed that organic fertility can affect significantly the level of egg parasitism by fungi.These limited data suggest that soil mycofloras may be manipulated through cultural practices or with appropriate crop production systems to stimulate development of species antagonistic to phytonematodes.
Mycoflora in individual fields is probably determined by such factors as plant host, cropping history, soil properties and climatic conditions.Cropping systems, other than monocultures, may enhance the level of parasitism of phytonematodes by fungi, however, the ecology and population dynamics of mycoflora associated with phytonematodes are least understood.Without knowledge of the factors that influence field dynamics of these fungi, we cannot hope to assess their importance in regulating nematode populations.This is the first report of identification of fungi infected G. rostochiensis cysts and eggs in Bulgaria.Pathogenicity tests in the future will be required in order to establish the aggressiveness of these fungi as egg parasites and their suitability as biocontrol agents.

C o n c l u s i o n
The studies made it possible to draw the following conclusions: 1.Four fungi were isolated and identified from the eggs and cysts of G. rostochiensis: Botryotrichum piluliferum; Scolecobasidium constrictum; Gliocladium roseum and Phoma fineti.

2 .
Infection of the new progeny of cysts occurred during September.3. Maximum diseased eggs in the old cysts was observed in June (22.2%).4. Fungus becomes active from September onwards.R E F E R E N C E GLJIVIČNI PARAZITIZAM CISTA I JAJA GLOBODERA ROSTOCHIENSIS Zlatka Trifonova 1 i Jordanka Karadjova 1 T a b. 1. -Survey on black egg disease of Globodera rostochiensis at Velingrad region ± 3.05 5.0 ± 0.1 54.33 9.1 ± 1.65