Mycoflora and Population Dynamics of Some Seedborne Fungi in Relation to the Fungicide Benlate

Zentralbl. Mikrobiol. 148 (1993),570-581 Gustav Fischer Verlag Jena [Botany Department, Faculty of Science, Cairo University, Giza, Egypt]

Mycoflora and Population Dynamics of Some Seedborne Fungi in Relation to the Fungicide Benlate Mycoflora und Populationsdynamik einiger samenbiirtiger Pilze bei Verwendung des Fungizids Benlat S. A. Ouf Key words: Benlate, fungicide, seedborne fungi, seed germination, leachates.

Summary A total of 1478 and 1108 fungal isolates were associated to one gram seeds of Convolvulus arvensis and Rumex dentatus respectively. The dominant genera were Aspergillus and Penicillium followed by Paecilomyces, Rhizopus and Fusarium. Aspergillus niger, A.flavus and A. humicole, were common on both seeds. A. granulosus, Penicillium expansum were restricted to C. arvensis seeds while A. carbonarius and P. lanosum were recovered only from R. dentatus seeds. The dynamic balance of some seedborne fungi fluctuates on soaking of either seeds in the fungicide Benlate, depending on the dose, soaking period and seed sample. The leachable substances which contain sugar, amino acids and phenolics are believed to have antifungicidal effect after prolonged soaking of the seeds in the fungicide. The germination of both seeds stimulated at the lower doses of the fungicide and the stimulation is more pronounced as the soaking period was extended. However, the higher doses were inhibitory to germination of both seeds.

Zusammenfassung Aus einem Gramm Samen von Convolvulus arvensis und Rumex dentatus wurden jeweils 1478 bzw. 1108 Pilze isoliert. Die dominanten Gatrungen waren Aspergillus und Penicilium, gefolgt von Paecilomyces, Rhizopus und Fusarium. A. niger, A.flavus und A. humicola waren auf den Samen am hiiufigsten, A. granulosus, P. expansum waren auf den Samen von C. arvensis schwiicher vertreten, wahrend A. carbonarius und P. lanosum nur von R. dentatus-Samen isoliert wurden. Beim Quellen der Samen zeigen die samenbiirtigen Pilze eine Fluktuationsdynamik beim Einweichen mit Benlat, in Abhiingigkeit von der Dosis, der Quellzeit und der Samenprobe. Die Quellsubstanzen, welche Zucker, Aminosiiuren und Phenole enthalten, diirften einen antifungiziden Effekt bei liingerer Quellzeit im Fungizid haben. Die Keimung der beiden Samenarten wurde bei niedrigen Dosen des Fungizids stimuliert und urn so deutlicher, je langer die Einquellzeit der Samen mit dem Fungizid dauer!. Hahere Fungiziddosen wirken jedoch hemmend auf die Samenkeimung.

Introduction Environmental contamination of air, water and soil with pesticides has become a threat to the continued existence of many plant communities of the ecosystem as well as their associated microorganisms. The massive application of the pesticides to the environment may lead to reduction in the number of species, decrease in frequency and vitality of herbs (Marker, 1974) or difference in composition of vegetation (Young et a!., 1975). In some other cases, the soiltreated pesticides and other plant growth regulating chemicals stimulate germination and emergence of dormant seeds in vitro and in soil (Fawcett and Slife, 1975, Horowitz and Taylorson, 1985, Metzger, 1983 and Saini et a!., 1986).

Mycoflora of Seedbome Fungi

571

On the other hand, the effect of the pesticides on the seedbome fungi was demonstrated by many investigators (Zwatz and Zederbauer, 1988, Cho et aI., 1988, Ilyas and Bashir, 1987). The seedbome fungi may cause deterioration for the seeds through the alterations in the physical and biological properties (Chidichimo and Asbomo, 1986), changing the level of starch and total reducing sugars (Prasad et aI., 1988) or mycotoxin production (Gbodi et aI., 1989). Although the effect of fungicide dressing of the seeds on their germination and their seedbome microorganisms was studied by many investigators (Runion and Bruck, 1988, Forster and Schaad, 1988; Miles, 1987), however, the interactions of seed leachate and the fungicide are rarely met with in the literature. The objective of the study was to evaluate the effect of prolonged fungicide soaking of the seeds of two weed species on seed germination and seed colonization with the mycoflora. Also, the effect of the fungicide on the growth rate of some representative seedbome fungi was studied. Materials and Methods Tested plants Seed samples of two weeds were employed for this study. They are: Convolvulus arvensis L.. glabrous perennial herb with stout rhizome. It has glabrous subglobose capsule, 7 mm, 3-4 seeded; seeds dark brown tuberculate. Rumex dentatus: L., herbs, perianth-segments with a large grain in the centre and a few teeths on each side. The seeds are smooth dark brown, trigonous, ovate or elliptical.

Seed collections Fruits of the two tested species were collected during October 1988 and transported to the laboratory where the seeds were removed under aseptic conditions from the capsules. Seeds were stored at 4 °C for 18 months before use.

Isolation of fungi Three replicates, each contains 100 seeds of C. arvensis and 500 of R. dentatus (about 1 g of each) were used for fungal isolation. The seeds were transferred to about 15 milO % sodium chloride-Potato Dextrose Agar medium in 9 em Petri dishes (two seeds/plate). NaCI was added to the medium to inhibit bacterial growth, retard the development of fast growing fungal species and to prevent seed germination (Mislivec and Bruce, 1976). The Petri dishes were incubated at 25 °C. The development of fungal growth on the seeds were transferred to Czapek-Dox medium and identified using the publications of Raper and Fennell (1965), Nelson et al. (1983), Barnett and Hunter (1972), Ellis (1971) and Watanabe et al. (1986).

Fungicide used The fungicide Benlate (Methyl 1-(butylcarbamoyl-2-benzimidazolecarbamate) was used at ten concentrations ranged from 0.2 to 64 ppm. For each concentration three replicates, each of two sets of sterilized test tubes (lOami capacity) containing 50 ml were prepared. The tubes of one set each receive 100 seeds of C. arvensis while those of the other set each receive 500 seeds of R. dentatus. The tubes were then incubated at 25 °C for different time intervals which were 2, 8, 16 and 24 h. By the end of each time interval, two tubes of each set were removed. After blotting by dry filter paper the seeds were stored for 15 days to determine whether there is any delayed fungicidal effect. Then the seeds of one tube were used to isolate the dominant fungal species and the seeds of the other tube were used to determine the percentage germination.

Seed germination The seeds were germinated on moist filter paper in sterile Petri dishes. Care was taken to maintain the filter paper moist; sterilized distilled water was added when necessary. The dishes were kept in an incubator (in darkness) at constant temp. of 25 0c. A seed was considered to have germinated when the radicle emerged.

572

S. A. Ouf

Counts of germinated seeds were made daily until no seed germinated for seven successive days. The seed count involved the removal of the Petri dish lid which allowed the change of air and probably prevented the accumulation of carbon dioxide from genninated seeds. The seed gennination was calculated as percenage of total seeds used.

Relative growth of fungal mycelium The effect of different concentrations of the fungicide Benalte on the radial growth was measured for the dominant fungi isolated from C. arvensis and R. dentatus seeds. Known volumes of Czapek-Dox agar were autoclaved and appropriate amounts of fungicide stocks were added to the cooled medium to give the desired concentrations of the fungicide. About 15 cm 3 , each of the fungicide incorporated medium were dispensed into 9 em Petri dishes. Each dish was inoculated at its centre with a 3 mm disc cut from the vegetative margin of 2-4 days old cultures growing on Czapek-Dox agar medium. Three replicate plates for each concentration of the fungicide for every fungus were used. The plates were incubated at 25°C and the colony diameters measured at 24 h intervals, were taken as the mean of two diameters at right angles to each other. Rates of growth were calculated only during the linear phase of extension.

Estimation of some leachable constituents The total soluble sugar, amino acids and phenolic compounds were estimated colourimetrically using a Spekol colourimeter. The methods adopted were that of Naguib (1965) for estimation of total soluble sugar, Russell (1944) for amino acids and Swain and Hillis (1959) for phenolic compounds.

A) Total Soluble Sugars Five ml of the leachate were hydrolyzed with 5 ml 1 N HCI at 60°C for 30 minutes. Neutralization to phenol red was followed by completion to volume (25 ml). Two ml of the neutralized sample was then mixed with 2 ml of the modified Nelson's solution. The mixture was kept in boiling water bath for 15 minutes after which it was rapidly cooled under running tap water. Three ml of arsenomolybdate were added and the mixture was shaken till effervescence stopped. The coloured solution was diluted to 15 ml and the intensity of colour was measured colourimetrically using red filter (700 nm).

B) Amino Nitrogen Five ml of leachate were made just alkaline to phenolphthalein using dilute NaOH. One ml of 1 % Borax was added and the mixture was shaken. This was followed by addition of I ml of 0.25 % freshly prepared 1-2 naphthaquinone-4-sulphonic acid (Na salt) and the mixture was kept in a boiling water bath for 15 minutes, rapidly cooled by tap water, and 1 ml of acid formalin was added followed by 1 ml of 0.1 N sodium thiosulphate. The mixture was made up to 15 ml and kept standing for 20 minutes before estimated photometrically using blue filter (450 nm).

C) Total Phenols Half ml of the leachable substance was diluted with distilled water to about 7 ml in a 10 ml test tube. The content was well mixed, then 0.5 ml of Folin-Denis reagent was added, and the tube was thoroughly shaken. Exactly after 3 minutes, 10 ml of saturated sodium carbonate solution was added and the mixture was made up to 10 ml with distilled water. After one hour, absorptivity was determined in I em cell at 725 nm using a blank water and reagent only.

Results Seedborne fungi Table 1 showed that the seed samples of Convolvulus arvensis and Rumex dentatus were contaminated with fungi at the rates of 83 and 54 % respectively. The contamination rate was calculated based on the number of the seeds with fungi divided by total number of seeds assayed.

Mycoflora of Seedborne Fungi

573

Table I. Rate of contamination with fungi and number of fungal isolates and genera on Convolvulus arvensis and Rumex dentatus seeds. Sample

C. arvensis R. dentatus

(%)

Number of fungal isolates g - I

Number of fungal genera g-l

83 54

1478 1108

14

Contamination

13

A total of 2586 isolates, 1478 from C.arvensis and 1108 from R.dentatus seeds were identified, falling into 27 genera all together, 14 genera for former and 13 genera for the latter. The fungi isolated in this study are listed with number of isolates and percent of total population of each species in Table 2. The results revealed that the genus Aspergillus was the most dominant in both seeds and constituted 39.78 and 37.55 % of the total isolates of C. arvensis and R. dentatus respectively. Aspergillus niger, the most common aspergillii was codominated by A. granulosus, A.flavus and A. humicola on C. arvensis seeds and by A. ochraceus, A. carbonarius and A.flavus on R. dentatus seeds. The genus Penicillium ranked second in percent of total population. It constituted 17 .05 and 14.71 of total isolates of C. arvensis and R. dentatus seeds respectively. P.puberulum on C. arvensis and P.lanosum on R. dentatus recorded the bulk of the genus. Penicillium was followed by the genera Paecilomyces, Rhizopus and Fusarium which were isolated in percent of 8.39, 6.36 and 7.65 % of the total isolates of C. arvensis and 8.21, 5.51 and 3.97 % of the total isolates of R. dentatus seeds respectively. Chaetomium was the only of the rest genera isolated from C. arvensis in percent> 5 %. The genera Scopuiariopsis, Cladosporium, Ulocladium, and Trichoderma were isolated from R. dentatus seeds in percent of 7.76, 6.14, 6.86, 5.96 % respectively. The two former genera were not recorded from C. arvensis seeds. The other genera were recorded in percent of population < 5 % of the total isolates of either C. arvensis and R. dentatus seeds.

Growth rate of fungal mycelium Table 3 showed that increasing the concentration of Benlate from 0 to 64 ppm did not cause significant changes in mean growth rates of Rhizopus nodosus, Aspergillus niger and Ulocladium alternariae. However, the other tested fungi were affected variably, Paecilomyces divaricata was the most affected species and its growth rate was gradually reduced on elevating the concentration of the fungicide from 0.2 to 32 ppm and growth was prevented at 64 ppm. The growth rates of Aspergillus granulosus and Penicillium lanosum began to be reduced at 2 ppm of the fungicide. The growth of the latter species was prevented at 8 ppm of Benlate. At 32 ppm A. granulosus was failed to grow.

Seed germination Percentages seed germination of C. arvensis are presented in Table 4. The results revealed that after 2 and 8 h soaking periods, the percentage germination increases with elevation of the fungicide concentration up to 1 ppm. Prolongation of the soaking periods to 16 and 24 h significantly stimulated the germination only at the lower doses (0.2 and 0.5 ppm). At 4 ppm the seed germination decreased and become more remarkable on extension of soaking period reaching a depression of about 65 % of the control value after 24 h. At 8 and 16 ppm, the seed germination decreased after 2 h soaking, however extension of the soaking period led to complete inhibition of germination.

574

S. A. Ouf

Table 2. Fungi isolated from seeds of C. arvensis and R. dentatus (isolates/g seeds). Fungal Species

C. arvensis

number of isolates Total Aspergilli

A. A. A. A. A. A. A.

niger Van Tieghem humicola Oudemans flavus Link granulosus Raper & Thorn carbonarius Bain. funiculosus G. Smith ochraceus Wilhelm

Total Penicillia

P. P. P. P. P. P.

lanosum Westling puberulum Bain. chrysogenum Thorn variable Sopp expansum Link ex Gray verrucosum Dierckx

Total Fusaria F. oxysporum (Schlecht. F. poae (Peck) Wollenw. F. chlamydosporum Wollenw. & Reinking

R. dentatus % of total population

number of isolates

% of total population

588

39.78

416

37.55

210 72 91 173

14.21 4.87 6.16 11.71

142 58 62

12.83 5.23 5.60

69

6.23

26 16

1.76 1.08

85

7.67

252

17.05

163

14.71

111 83

7.51 5.62

102 6 18 9

9.21 0.54 1.62 0.81

56 2

3.79 0.14

28

2.53

113

7.65

44

3.97

73 39

4.94 2.64 0.07

41 3

3.70 0.27

Total Rhizopus

94

6.36

61

5.51

R. nodosus Namyslowski R. nigricans Ehrenb. R. oryza Went & Gerlings

46 16 32

3.11 1.08 2.17

38 12 11

3.43 1.08 0.99

Total Trichoderma

48

3.25

66

5.96

T. viride Presoon T. hamatum (Donorden) Bainier

6 42

0.41 2.84

65

5.96

124

8.39

91

8.21

P. silvatica Bainier P. divaricata Bainier

Total paecilomyces

93 31

6.29 2.10

63 28

5.69 2.53

Rhizoctonia solani Kuhn Cladosporium herbarum (Persoon) Link Cephalosporium curtipes Saccardo Curvularia lunata (Walker) Boedijn Phoma spp. Epicoccum purpurascens Ehrenberg Humicola fuscoata Traaen Thielaviopsis basicola Zopf Scopulariopsis brevicaulis (Sacc.) Bain. Chaetomium globosum Kunze Stemphylium verruculosum Zimmermann Ulocladium alternariae (Cooke) Simmons

32

2.17

18 3

1.22 0.20

68 20

6.14 1.81

12 3

1.08 0.27

2 86

0.18 7.76

76

6.86

Total

13 6

0.88 0.41

88 33 66

5.95 2.23 4.47

1478

100

1108

100

Mycoflora of Seedborne Fungi

575

Table 3. Mean growth rates of seedborne fungi (mm day-I) on Czapek-Dox agar incorporated with fungicide. Fungicide concentration (ppm)

Fungal species

Rhizopus nodosus Aspergillus granulosus Paeci/omyces divaricata Aspergillus niger Ulocladium alternariae Penicillium lanosum

2

L.S.D.

4

8

16

32

64

0

0.2

0.5

32.7 7.0 19.5 5.6 5.8 3.7

32.0 7.0 15.4 5.7 5.8 3.7

33.9 33.5 35.7 35.5 36.0 33.2 32.7 32.5 5.4 3.6 1.9 1.3 0.0 0.0 7.0 7.1 6.5 6.1 2.8 2.5 0.0 14.9 10.9 8.1 7.1 6.2 5.9 5.9 5.9 6.3 6.7 7.1 5.9 5.9 6.1 5.8 5.7 5.4 5.1 4.9 4.1 4.0 2.2 2.2 0.0 0.0 0.0 0.0

1%

5%

4.8 1.8 2.4 1.9 1.9

3.6 1.5 2.1 1.6 1.5 0.8

l.l

Table 4. Percentage germination of C. arvensis seeds soaked in various concentrations of Benlate for different periods. Concentration (ppm)

Soaking period (h)

0

0.2

0.5

2 8 16 24

65.2 68.6 71.7 76.5

68.2 76.5 79.0 8\.8

71.7 80.0 83.4 90.6

L.S.D.

76.7 73.9 71.6 63.3

2

4

8

16

32

64

1%

5%

66.2 62.0 6\.5 50.5

53.1 48.6 46.7 22.5

43.3 0.0 0.0 0.0

26.5 0.0 0.0 0.0

0.0 0.0 0.0 0.0

0.0 0.0 0.0 0.0

6.2 6.1 5.9 5.2

5.1 5.2 4.8 4.3

Table 5. Percentage germination of R. dentatus seeds soaked in various concentrations of Benlate for different periods. Soaking period (h)

2 8 16 24

Concentration (ppm)

0

0.2

0.5

53.4 56.3 58.7 63.0

56.3 57.6 61.9 64.1

61.0 6\.0 64.2 68.0

L.S.D.

65.7 67.7 74.3 79.3

2

4

8

16

32

64

1%

5%

68.8 71.1 76.2 83.1

71.2 73.9 81.8 86.3

76.3 78.7 76.6 73.1

77.2 78.3 66.1 52.1

77.2 72.6 53.3 44.9

51.0 49.1 36.3 26.7

5.8 5.9 6.1 6.1

4.8 4.3 5.6 5.3

Percentages seed gennination of R. dentatus are presented in Table 5. The gennination increased variably on increasing the doses of the fungicide up to 8 ppm. The increase was more pronounced on extensiori of soaking period to 24 h where it had maximum values at 4 ppm. At 16 and 32 ppm, the percentage seed gennination was significantly higher than the control after 2 and 8 h soaking, while extension of soaking period till 24 h led to reduction in percentage germination. At 64 ppm percentage seed gennination was progressively reduced as the soaking periods extended from 8 up to 24 h.

Development of fungi in relation to fungicide-soaked seeds Fig. I shows that prolonged soaking of C. arvensis seeds in absence of the fungicide (control) led to gradual decrease in counts of R. nodosus, A. granulosus and P. divaricata and increase in counts of A. niger and Ulocladium alternariae. On the other hand the fungal counts differed variably on soaking the seeds in different doses of the fungicide.

576

S. A. Ouf

The counts of R. nodosus revealed a trend that is similar to the control but it became less remarkable as the fungicide dose increased up to 8 ppm. At higher doses (32 and 64 ppm), there were no significant changes in the number of isolates at different soaking periods but their values were higher than their respective control. The number of isolates of A. granulosus showed clear reduction by soaking the seeds in fungicide for 8 to 24 h as compared to the counts recorded after 2 h soaking. At higher doses, from 4 to 24 ppm, the number of isolates of A. granulosus started to decrease reaching 85 isolates at 64 ppm. However prolonged soaking caused an increase in the number of isolates which was more observed by increasing the dose of the fungicide. Table 6. Total sugar, amino acids and phenolic compounds (f.lg/g seeds) of leachate collected from C. arvensis and R. dentatus seeds soaked at different time intervals. Seed

Time of soaking (h)

Total soluble sugar

Amino acids

Phenolic compounds

C. arvensis

2 8 16 24

5.2 9.5 36.0 62.0

1.2 10.1 17.2 33.3

0.22 0.61 1.97 3.21

R. dentatus

2 8 16 24

0.0 3.2 10.6 31.3

0.0 0.0 10.3 17.3

0.0 0.0 0.65 0.93

On soaking the seeds for 2 h, the isolated colonies of Paecilomyces divaricata decreased gradually on rising the dose of the fungicide from 0.2 up to 64 ppm. However soaking for 16 and 24 h caused an increase in the number of isolates which was more significant as the fungicide concentration increased. While the number of the colonies produced by A. niger did not show marked change after 2 h at all the concentrations of the fungicide, however extention of soaking caused an increase in number of isolates which was more significant as the concentration was elevated. The isolated colonies of U. alternariae showed a trend similar to that of A. niger when the seeds were soaked in the fungicide up to 4 ppm. Above that concentration the number of isolated colonies started to decrease showing clear depression by extension of soaking period as compared to the corresponding control. Fig. 2 includes the effect of soaking Rumex seeds in different concentration of the fungicide on the development of the seedbome fungi. A. granulosus was not isolated from Rumex seeds. The counts of the other tested developing fungi after 2 and 8 h soaking has had a trend matching with the effect of the fungicide on growth rate (Table 3). Prolonged soaking of R. dentatus seeds for 16 and 24 h caused an increase in the counts of Rhizopus nodosus up to 1 ppm above which the soaking retarded the estimated colonies to values equal or near to the unsoaked seeds. The counts of A. niger and U. alternariae increased only after 24 h soaking in the fungicide up to I ppm. At 2 and up to 8 ppm the counts of the two fungi decreased but still higher than corresponding values recorded after 2, 8 and 16 h. On the other hand the number of isolates of P. divaricata decreased progressively as the fungicide concentration increased up to 16 ppm on soaking the seeds for 16 and 24 h. At 64 ppm the isolate of P. divaricata was missed.

Mycoflora of Seedborne Fungi

SO-r-----==-====------, m.

A:

R. nodosus

_

2 h

h

CJ

10 h

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24 h

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Conc.ntratlon (ppm)

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8163264

Concentration ( ppm)

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Concentration (ppm)

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64

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200-.----=--=====------,

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100

,.. c

o o o

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0 . 20 . 5

1

2

4

8

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Concentration (ppm)

32

64

Fig. 1. Recovery of some seedborne fungi from C. arvensis seeds soaked in different concentrations of the fungicide Benlate for different periods.

Leachable substances: Table 6 shows that leachates of both C. arvensis and R. dentatus seeds contained sugar, amino acids and phenolic compounds. The leachable substances increased by extension of soaking period and were more in C. arvensis than with R. dentatus seeds.

s.

578

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2 h

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8163264

Concenlrallon ( ppm)

Fig. 2. Recovery of some seedborne fungi from R. dentatus seeds soaked in different concentrations of the fungicide Benlate for different periods .

Discussion The seed-surface is a complex habitat in which microbial populations are held in a dynamic balance by interactions between the seedbome microorganisms and between the host seed and the epiphytic microflora. The delicate balance of microbial numbers , composition and activities on the seed-surface can be modified by application of the fungicide and other fungitoxic substances. The results of the present investigation indicate that the seed samples of C. arvensis, were richer with fungal population than R . dentatus ones. A total of 1478 and 1108 isolates were associated with C. arvensis and R. dentatus seeds, respectively. Aspergillus and Penicillium constituted the bulk of the isolated genera with broadest spectra of species. It seems that, occurrence of Aspergillus spp. was less marked than that of Penicillium spp. in both seeds. The predominant species of apergilli on both seeds were A . niger, A .flavus and A. humicola. A. granulosus and P. expansum were restricted to C. arvensis seeds while A . carbonarius and P.lanosum were recovered only from R. dentatus seeds. The genera Paecilomyces, Rhizopus and Fusarium were common fungi collected from both seed samples.

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The ecological determinants of the mould growth in stored seeds are controlled by the interaction of water activity of fungi on seeds, the interaction of water activity and temperature with germination, growth and sporulation of stored seed fungi and the effect of water activity, gas composition and temperature of growth seed fungi (Magan and Lacey, 1988). The quantitative dominances of aspergilli and penicillia in the tested seed samples might be due to the climatic conditions in our region which are optimal for their growth. Also, it is noted that A.flavus and A. humicola are belonging to the primary colonizers and start growth even when moisture content of the seed is low between 12.5 % and 15 %. They produce metabolic water which increase the seed moisture content and improve conditions for other specis of the genera Aspergillus and Penicillium (Christensen, 1972). In in vitro trial to control the seedborne fungi by application of the fungicide Benlate to the growth medium, the growth rates of some isolated fungi showed pronounced differences in sensitivity to the fungicide. A. niger, R. nodosus and U. alternariae were insenitive while the other tested species variably affected. P. lanosum was the most affected followed by A. granulosus and P. divaricata. The effect of seed treatment by fungicides on seedborne mycoflora was studied by many workers. In this connection Ilyas and Bashir (1987) used benlate among 9 fungicides tested for controlling the seedborne fungi of Cicer arietinum. Also Cho et al. (1988) showed that benlate-T applied as seed dressing was effective in controlling Alternaria dauci on carrots, Fusarium oxysporum on Salvia, Helminthosporium poae on bluegrass, Penicillium vermoeseni on Areca catechu and F. moniliforme and Cephalosporium lecanii on palms. Considering germination as the first step or phase of growth, the present research is thus correlated with the effect of the fungicide on seed germination of the tested plants. The present results showed that soaking C. arvensis seeds in lower doses (up to 1 ppm) of the fungicide stimulate the germination, stimulation became more pronounced as the soaking period was extended up to 24 hrs, while the higher doses were inhibitory. On the other hand the germination of R. dentatus seeds was stimulated by application of relatively higher doses (8 ppm) at all the soaking periods. On elevating the concentration to 32 ppm the stimulation is true only up to 8 h soaking. It seems from the results that the germination of R. dentatus seeds were stimulated with doses of the fungicide more than those required for C. arvensis seeds. The variation in seed germination, vitality and seedling emergence on treatment of the seeds with fungicides have been implicated by some researches. Pardeshi et al. (1989) indicated that seed treatment with fungicides mancozeb, Bavistin, Thiram, Captan and Difolatan increased germination percentage and viability of soybeen and Bragg seeds. Similarly Kulshrestha (1988) stated that the seed dressing with Bavistin T, Thiram, Bavistin SD, Difolatan, Mancozeb and Ceresan reduced seedling mortality. On contrary, Tonkin (1988) showed that seed treatment of wheat and barley with Bay tan and Ferrax seriously reduced the germination potential. In fungicide - free soaking medium, prolonged soaking of Convolvulus seeds led to increase in counts of R. nodosus, A. granulosus and P. divaricata and decrease in counts of U. alternariae and A. niger. On Rumex seeds, the counts of A. nodosus, A. niger and U. alternariae increased while P. drivaricata decreased. This alteration in fungal counts is coupled with increase in the amount of leachable sugars, amino acids and phenolics; The role exerted by the leachate seemed to be specific depending on the fungal species and composition and amount of leachable substance. which may be nutritional activating the recovery of the colonyproducing units or inhibitory possessing retarding effect. Addition of the fungicide to the soaking medium caused pronounced change in the picture of the fungal counts of the tested seedborne species depending on the dose, the soaking period and the fungal specis. On C. arvensis seeds, the counts of some species e.g. R. nodosus decreased on extension of soaking period up to 24 h while the counts of others as A. niger increased at the same conditions. On R. dentatus seeds, after 24 h, the count of A. niger as

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example increased up to 8 ppm while that of P. divaricata decreased. The observed results led to the suggestion that on prolonged soaking the leachable substances might exert antagonistic or synergestic effects against the fungicide depending on the species, so they may have antifungicidal effect leading to a drop in the effectiveness of the fungicide. This claim substantiates the suggestion recorded by Dunn et al. (1971). They found that the presence of glucose or exudates reduced the effectiveness of the fungicide against spore germination of the microfungi tested. In conclusion, the reaction between the leachable substances and fungicide must be taken into consideration during the validation of the fungicide against target seedbome fungal species.

Acknowledgements Appreciation is expressed to Ben Mullinix (Coastal Plain Station, University of Georgia, U.S.A.) for his assistance in preparation of figures, and to Dr. A. K. Hegazy (Botany Department, Faculty of Science, Cairo University) for his help in collection of seed samples.

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Naguib, M. I.: Effect of maleic hydrazide on the nitrogen metabolism of starved and sucrose-Fed etiolated barley leaves. Ind. J. Agr. Sci 35 (1965), 179-185. Nelson, P. E., Toussoum, T. A. and Marasas, W. F. 0.: Fusarium specis. Pennsylvania State University Press, University Park (1983), 193. Pardeshi, V. F., Reddy, V. G. and Nalwandikar, P. K.: Effects of different fungicides on seedling vigour and seed viability in soybeen. J. of Mabarashtra Agricultural University 14 (1989),33-36. Prasad, B. K., Prasad, A., Shanker, U. and Kumar, S.: Level of change in carbohydrate in Lablab bean FD5 cv. seed due to seedborne Aspergillus niger. Indian Journal of Mycology and Plant Pathology 17 (1988), 145-149. Raper, K. B. and Fennell, D. I.: The genus Aspergillus. William and Wilkins Company, Baltimore 1965. Runion, G. B. and Bruck, R. I.: Effect of thiabendazole - DMSO treatment of long leaf pine seed contaminated with Fusarium subglutinans on germination and seedling survival. Plant Disease 72 (1988), 872-874. RusselJ, J. A.: Note on the colorimetric determination of amino nitrogen. J. BioI. Chern. 156 (1944), 467-468. Saini, H. S., Bassi, P. K. and Spencer, M. S.: Use of ethylene and nitrate to break seed dormancy of common lambs quarters (Chenopodium album). Weed Sci. 34 (1986), 502-506. Swain, T. and Hillis, W. E.: The quantitative analysis of phenolic constituents. J. Sci. Food Agric. 10 (1959), 63. Tonkin, J. H. B.: Noted effects of some chemical treatments in germination tests on wheat and barley. Monograph - British Crop Protection Council 39 (1988), 113-120. Watanabe, T., Uematsu, S. and Sato, Y.: Fungus isolates from Japanese black and red pine seeds with some taxonomical notes. Bull. For. and For. Prod. Res. Inst. 336 (1986), 1-18. Young, A. L., Thalken, C. E. and Ward, W. E.: Studies of the ecological impact of repetitive aerial application of herbicides on the ecosystem of test Area C-52A, Eglin Air Force Base, Florida. Final Report, May 1973-December 1974. Eglin AFB, Fl. DOC No AFATL - TR-75-142, (1975). Zwatz, B. and Zederbauer, R.: Control of seedborne helminthosporioses in barley and oats by seed dressing. Pflanzenschutz 2 (1988), 4-5. Author's address: Dr. Salama A. Ouf, Botany Department, Faculty of Science, Cairo University, Giza, Egypt.