Cultural Characteristics of Two Eutypa lata Isolates on D

Srđan Aćimović’s Senior Project i.e. Diploma Work on a Department for Plant and Foodstuff Protection, Faculty of Agriculture, University of Belgrade

- part two -

Cultural Characteristics Examination of Two Eutypa lata Isolates from France and One Unknown Isolate from Serbia on Different Nutrient Media

In order to examine cultural and morphological characteristics of Eutypa lata on different nutrient media, two isolates were recieved from the National Institute for Research in Agriculture (INRA) - France, thanks to Mr. Pascal Lecomte.

In order to examine appearance (colour and shape), growth and sporulation i.e. fructification of colonies, 6 different nutrient media have been made.

Nutrient media* (substrates) used for examination were:

1. PDA - potato dextrose agar

2. V8 - vegetable 8, in this case tomato agar

3. OA - oat agar

4. GWA - grapevine wood agar

5. MALT - maltose agar

6. WA - water agar

__________________

* Used materials for the preparation of this media are listed in appendix at the end of this paper.

After sterilisation these substrates have been smeared aseptically in a Petri dishes which are 9 cm in diameter. After cooling, media were seeded in sterile conditions with Eutypa lata and then placed in thermo-chamber on constant temperature of 25 ºC.

Every second day from seeding measurements and visual observations of colonies have been performed - on all media. In order to gain more precise measuring results and visual observations, every isolate has been seeded three times on one nutrient medium type. By measuring the diameter of colonies in all three Petri dishes - for every medium type, it was enabled to calculate the average colony growth value for every isolate.

Cultural Characteristics of Two Eutypa lata Isolates from France and One Unknown Isolate from Serbia on Different Nutrient Media

Appearance (shape and colour) of the colonies. Eutypa lata forms roundish colonies with fibrous brim and at first, white cottony aerial mycelium (the upper side of the colony) (Fig. 1 i 2). In reverse colonies are cream-colored. Later, on PDA medium, and the most often in the same time with sporulation, mycelium can change colour in light-grayish to pale olive-green - only in a form of ring situated in a medium range from the colony center. In the same time, substrate part of the colony - only under sporulation sites, becomes dark-gray or black. The rest is white, cream or pale-grayish. On the other nutrient media colour-changes of the aerial mycelium are not detected. Colour-changes of the substrate part of the colony - only under sporulation sites, are clearly visible on all nutrient media except on WA medium (water agar).

Growth of the colonies. On potato dextrose agar (PDA medium), Eutypa lata colonies had the fastest development. Isolate F1 has covered the whole Petri dish surface in 8, and F2 in ten days (Table 1). On tomato medium (V8 i.e. vegetable 8), both isolates have covered the whole Petri dish surface also pretty fast - after 10 days (Table 2). On OA medium (oat agar), isolate F1 has covered the whole surface also in 10 days, and isolate F2 in 12 days (Table 3). On GWA medium (grapevine wood agar), both isolates have covered the whole surface also in 12 days (Table 4).

The slowest growth of both isolates has been reported on MALT medium (maltose agar). Isolate F1 has covered the whole Petri dish surface after 16 days. Extremely slow growth of isolate F2 on this medium has stopped after 24 days and thereat isolate F2 has not even covered the whole Petri dish surface (Table 5). On WA medium, isolate F1 has covered the whole Petri dish surface after 14 days, and isolate F2 not until 20 days have passed (Table 6).

Sporulation (fructification). On PDA medium, two E. lata isolates have sporulated in different times. Small, untotally formed black pycnidia have appeared after 10 days at isolate F1, and after 14 days at isolate F2 (Table 1). On V8 medium only isolate F1 has sporulated - small, untotally formed black pycnidia have appeared after 12 days. On V8 medium isolate F2 has not sporulated at all (Table 2). On MALT medium isolate F1 has started to sporulate after 14, and F2 after 18 days from seeding (Table 5). On GWA medium small, untotally formed black pycnidia have appeared after 18 days at isolate F1, and two days earlier - after 16 days at isolate F2 (Table 4). Small, untotally formed black pycnidia and completely formed black pycnidia - with conidia which are exuded in a droplet, can be seen on Fig. 3.

The earliest appearance of small, untotally formed black pycnidia is reported on OA medium, respectively. Isolate F1 has sporulated already after 8 days, and isolate F2 after 10 days from seeding (Table 3). Totally oposite from this but unexpected too, is complete absence of sporulation of both isolates on WA medium (Table 6).

KDA

∆R - average diameter of colonies in [mm] and sporulation after days

18,6

90(P)

37,3

87,6

90(P)

35,3

46,3

54,6

69,6

82,3

86,3

90(P)

Table 1. Legend: PDA - potato dextrose agar; F1 and F2 - two isolates of Eutypa lata; D - unknown

isolate from Serbia; P - Petri box surface of medium totally covered with colony (∆R = 90 mm);

S - sporulation i.e. fructification; ■ - stopped growth and sporulation

∆R - average diameter of colonies in [mm] and sporulation after days

90(P)

■

15,3

36,6

59,6

82,3

90(P)

15,3

52,6

72,6

85,3

86,3

■

Table 2. Legend: V8 - vegetable 8, in this case tomato agar; F1 and F2 - two isolates of Eutypa lata;

D - unknown isolate from Serbia; P - Petri box surface of medium totally covered with colony

(∆R = 90 mm); S - sporulation i.e. fructification; ■ - stopped growth and sporulation

∆R - average diameter of colonies in [mm] and sporulation after days

15,6

54,6

90(P)

■

63,3

81,6

90(P)

■

15,3

31,3

52,6

73,3

81,6

90(P)

■

Table 3. Legend: OA - oat agar; F1 and F2 - two isolates of Eutypa lata; D - unknown isolate

from Serbia; P - Petri box surface of medium totally covered with colony (∆R = 90 mm);

S - sporulation i.e. fructification; ■ - stopped growth and sporulation

GWA

∆R - average diameter of colonies in [mm] and sporulation after days

14,6

35,3

56,3

90(P)

■

68,6

85,3

90(P)

■

12,5

27,1

48,6

58,6

75,6

76,3

Table 4. Legend: GWA - grapevine wood agar; F1 and F2 - two isolates of Eutypa lata; D - unknown

isolate from Serbia; P - Petri box surface of medium totally covered with colony (∆R = 90 mm);

S - sporulation i.e. fructification; ■ - stopped growth and sporulation

MALT

∆R - average diameter of colonies in [mm] and sporulation after days

29,3

43,3

57,6

88,3

90(P)

13,3

21,3

43,3

54,6

71,6

76,3

79,6

83,3

84,3

23,3

30,2

43,3

49,6

56,6

75,6

78,3

80,3

Table 5. Legend: MALT - maltose agar; F1 and F2 - two isolates of Eutypa lata; D - unknown

isolate from Serbia; P - Petri box surface of medium totally covered with colony (∆R = 90 mm);

S - sporulation i.e. fructification; ■ - stopped growth and sporulation

∆R - average diameter of colonies in [mm] and sporulation after days

26,3

60,3

75,3

88,3

90(P)

9,3

36,3

54,3

81,3

85,6

87,3

88,6

90(P)

12,3

25,6

34,3

42,3

49,6

57,6

64,3

67,6

69,3

■

Table 6. Legend: WA - water agar; F1 and F2 - two isolates of Eutypa lata; D - unknown isolate

from Serbia; P - Petri box surface of medium totally covered with colony (∆R = 90 mm);

S - sporulation i.e. fructification; ■ - stopped growth and sporulation

Figure 1. Eutypa lata isolates (F1 - first column, F2 - second column) and isolate D (third column) - on all examined media except on water agar medium (WA). Colonies are 8 days old. Legend: F1 and F2 - isolates of E. lata; D - unknown domestic isolate; PDA - potato dextrose agar (first row); V8 - vegetable 8, in this case tomato agar (second row); OA - oat agar (third row); GWA - grapevine wood agar (fourth row); MALT - maltose agar (fifth row).

Figure 2. Eutypa lata isolates (F1 - first row, F2 - second row) and isolate D (third row) on water agar medium (WA). Photographed 8 days after seeding. In left column, colonies of the same isolates, from the same Petri dishes, are showed with better visibility.

(a) (b)

(c)

(d)

Figure 3. Sporulation of Eutypa lata - small, black, untotally formed pycnidia - (a) and (b), and completely formed black pycnidia - (c) and (d) of Libertella blepharis anamorph. Showed on oat agar medium (OA). Photographed after 14 days from seeding. Small, black, shiny spheres represent droplets rich with conidia (pycnospores) which are exuded from a cracked moisturized pycnidia.

Isolate D included in measurements is of domestic origin and belongs to division BASIDIOMYCOTA. Identification to the level of species has not been done. This fungus is most probably saprofitic organism which inhabits the dead grapevine wood as its decayer. From that reason this isolate will not be the subject of further consideration.

appendix

Needed materials for the preparation of all nutrient media:

1) PDA - potato dextrose agar medium

- peeled and in cubes cut

potato................................... 200 gr

- dextrose ................................ 20 gr

- agar ....................................... 18 gr

- distilled water ..................... 1000 ml

2) V8 - vegetable 8, tomato agar medium

- tomato extract .................... 250 ml

- calcium carbonate ................... 3 gr

- agar ...................................... 18 gr

- distilled water .................... 1000 ml

3) MALT - maltose agar medium

- maltose extract (1 - 10 %) ....... 30 gr

- agar ........................................ 18 gr

- distilled water ...................... 1000 ml

4) GWA - grapevine wood agar medium

- choped grapevine wood ......... 300 gr

- agar ......................................... 18 gr

- distilled water ....................... 1000 ml

5) OA - oat agar medim

- oat flakes ................................ 100 gr

- agar .......................................... 18 gr

- distilled water ........................ 1000 ml

6) WA - water agar medim

- agar .......................................... 18 gr

- distilled water ........................ 1000 ml

For more information about cultural characteristics of Eutypa lata contact corresponding author :

Srdjan Acimovic, Mailing (post) address: Teodora Drajzera 16/3, 11040 Belgrade, Serbia and Montenegro.

Phone: +381 11 660 114; Cell phone (mob): +381 64 23 86 528; E-mail: srdacix@EUnet.yu