Zentralbl. Mikrobiol. 148 (1993), 298-303

Gustav Fischer Verlag Jena

[Botany Department, Faculty of Science, Assiut University, Assiut, Egypt]

Response of Soil Fungi to the Herbicide Primextra

Reaktion von Bodenpilzen auf das Herbizid Primextra

M. M. K. Bagy and S. K. Hemida

Key words: Herbicide , Primextra , soil fungi

Summary

Primextra 500 FW (acetanilide and triazine combination) was significantly toxic to the total counts of soilfungi after 2, 5 and 60 days of application with the three doses (0.94, 1.88 and 4.70 mg a.iIkg). In the agarmedium, it was significantly inhibited the total counts of the fungi, Aspergillus and A. niger; and promoted thecounts of A. candidus and A. carneus. The mycelial growth of A. niger, A. terreus, A. fumigatus andTrichoderma harzianum was significantly raised by the high dose (390 p.p.m.) .

Zusammenfassung

Primextra 500 FW (AcetanilidITriazin-Kombination) war signifikant toxisch gegeniiber Bodenpilzen(Gesamtkeimzahl) , 2, 5 und 60 Tage nach seiner Applikation in 3 Konzentrationsdosen (0,94 , 1,88 und 4,76rng/kg Boden). 1m Agarmedium wird die "Gesamtkeirnzahl" der Pilze Aspergillus und A. niger gehemmt,wiihrend die Zahl von A. candidus und A. carneus erhoht wird . Das Myzelwachstum von A. niger, A. terreus,A . fumigatus und Trichoderma harzianum wird durch die hohe Dosis (390 ppm) signifikanterhoht ,

Many studies, in field and laboratory have been done on the side effects of triazine andacetanilide herbicides on soil microorganisms (Bartha et a1. 1967, Deshmukh and Shrikhande1974, Chahal et al. 1976, Zidan et a1. 1977, Percich and Lockwood 1978, Mathur et al. 1980,Korpraditskul et al. 1988 and Moorman 1989). The present study was undertaken to evaluatethe effect of Primextra (acetanilide and triazine mixture) on soil fungi .

Material and Methods

The chosen herbicide to study its effect on soil fungi was Primextra 500 FW. It is a combination productbased on an acetanilide and a triazine. This herbicide is used in maize (com). Table 1 shows, the chemicalconstitution, the active ingredient and doses used of Primextra.

Treatment of soil

A clay soil aliquot collected from the Botanical Garden of Assiut University was used in this experiment .Half kg air-dry soil was put in a polyethylene bag and mixed well with the herbicide solution . The bag wasmaintained in a plastic pot. Three doses of Primextra were used (Table 1) the water content of the soil wasadjusted to 28 % of its maximum water holding capacity using Hilgard pan, to permit good aeration. Treatmentswere set up in duplicates in addition to the control. Pots wee incubated at 28DC for 60 days and at intervals thewater content was readjusted . After 2,5, 10,20,40 and 60 days, soil samples were taken for assaying the countsof their fungal colonies .

Response of Soil Fungi to the Herbicide Primextra 299

Table 1. Chemical constitution, percentage of active ingredient and doses added to the soil* or whenincorporated with the agar ar liquid medium** of Primextra

chemical constitution group Common % Dosesname Active

ingredient Soil Agar orliquid medium

Field 2-fold 5-fold Low Me- Highdose FD FD dium(FD)

Combination of N-(l-methyl- Aceta- 30 0.94 1.88 4.70 78 156 3902-methoxy-ethyl)-l-ethyl-6- nilidemethyl-ex chloraectanmilide

+2-chloro-4-ethylamino-6- Triazine Atrazine 20isopropylamino-5-triazine

* calculate as mglkg dry soil active ingredient; ** Calculated as ppm active ingredient in the volume of water tobe added to kg soil to raise its water content to 28 % W.H.C.

Isolation of soil fungi

The dilution plate method was used for the estimation of soil fungi (Johnson and Curl, 1972). ModifiedCzapek's-Dox agar medium in which glucose (10 gIl) was used. Rose bengal (1115000) was employed asbacteriostatic agent (smith and Dawson, 1944). Three sterilized Petri-dishes were used for each treatment andincubated at 28°C for 7-15 days, during which the developing colonies were examined, identified and counted.

Incorporation of Primextra with the agar medium

The herbicide was added to Czapek's agar at low, medium and high doses (calculated as p.p.m., Table 1).Three plates which each contained 1 ml of an appropriate soil dilution were used for each treatment. The plateswere incubated at 28°C for 7-15 days. The developed fungal colonies were examined, identified and counted.

The effect of Primextra on the mycelial growth of some fungi

Cultures of 4 fungal species isolated during this study were used as test organisms. 50 ml protions ofglucose-Czapek's medium were dispensed into each of 250 ml Erlenmeyer conical flasks. Primextra solutionwas added to the sterilized medium at 3 doses as shown in table 1. Five slants of 7-day old cultures of test fungus(Aspergillus fumigatus, A. niger, A. terreus and Trichoderma harzianum) were used in preparing the sporesuspension by adding sterilized distilled water to each slant under aseptic conditions and shaking the sporesuspension. The whole suspension was collected in a sterilized conical flask. One ml aliquots of the sporesuspension were introduced into the flasks of treaments and control. Two flasks were used for each treatment.The flasks were incubated at 28°e for 7 days, then the fungal mycelium was collected by filtration, washed,dried at 105°C overnight and weighed.

The obtained data were statistically analyzed by using a programme of one-way analysis of variance (Davis1973). Such a programme was executed on VME 2900 computer at Assiut University.

Results and Discussion

Table 2 revealed that, when Primextra (Acetanilide-triazine combination) was applied tothe soil in three doses (0.94, 1.88 and 4.70 mg a. i/kg dry soil), it was generally induced aconsiderable reduction all over the 60 days period in the count of the total fungi comparablywith that of untreated soil. This effect was statistically significant, 2, 5 and 60 days afterapplication. This may means that Primextra persisted in soil or its transformation productswere also toxic.

5*

Tab

le2.

Cou

nts

(per

mg

dry

soil)

ofco

mm

onfu

ngal

spec

ies

inso

ilaf

ter

diff

eren

tper

iods

oftr

eatm

entw

ithva

riou

sdo

ses

ofP

rim

extr

a(0

=C

ontr

ol;

1=

Fiel

dd

ose

;2

=2-

Fold

fiel

ddo

se;

5=

5-F

old

fiel

ddo

se)

ongl

ucos

e-C

zape

km

ediu

mat

28°C

VJ

Gen

era

&Sp

ecie

s+

Peri

ods

of

trea

tmen

t0 0

2da

ys5

days

10da

ys20

days

40da

ys60

days

~D

oses

~

aI

25

aI

25

aI

25

aI

25

a1

25

aI

25

?" t:xi

'"(IQ*

****

***

****

****

'<T

otal

coun

t53

.040

.930

.328

.155

.340

.628

.427

.037

.632

.430

.630

.763

.060

.938

.693

.661

.548

.021

.524

.575

.537

.02

8.0

17

.0'"t:I

***

****

****

****

****

**Q

..

Asp

ergi

llus

46.0

36.6

27.0

21.4

48.7

32.3

22.4

23.7

30.3

25.7

20.3

25.3

53.7

53.0

28.1

90.6

46.0

28.0

15.5

12.0

50.5

21.0

17.5

7.5

:.nA

.ca

rneu

s(v

.T

iegh

.)0

.00

.00

.00.

01.

70.

00.

00

.00.

00.

00

.00.

00.

00.

00

.00.

01.

50

.00.

00.

00.

00

.00

.00

.0~

Blo

chw

itz

::t:A

.fl

avus

Lin

k1.

71.

30.

71.

00.

30.

00.

00

.00.

30.

30.

00

.01.

00.

71.

02.

30.

00

.00.

00.

00.

51.

00

.00

.0(I

> El.A

.fr

ucti

culo

sus

1.7

0.7

1.3

0.7

0.0

3.7

2.7

1.0

0.0

1.3

2.3

1.7

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

Q..

Rap

er&

Fen

nell

'"*

*A

.fu

mig

atus

Fre

seni

us0

.00.

30.

70.

32.

01.

00.

71.

71.

73.

72.

72.

029

.029

.311

.777

.316

.513

.510

.55.

56.

52

.012

.56

.0**

****

***

****

***

****

A.

nige

rv

.T

iegh

em31

.326

.715

.312

.719

.317

.09.

313

.320

.013

.77.

316

.317

.713

.38.

75.

311

.58.

04.

56.

022

.57

.01

.00

.0*

***

****

**A

.te

rreu

sT

horn

11.3

7.3

6.7

6.7

18.7

10.3

9.7

7.7

8.3

6.7

7.7

5.3

6.0

9.0

6.0

5.7

15.0

5.5

0.5

0.5

16.5

10.0

4.0

1.5

*E

mer

ieel

la1.

30

.70.

72.

71.

61.

02.

32

.02.

34.

01.

34

.01.

04

.02.

32.

00.

50.

52.

54.

54.

55.

52

.30

.0E

.ni

dula

ns1.

30

.70.

72.

70.

31.

00.

32

.02.

33.

71.

33.

71.

00.

70.

32.

00

.00.

50.

00.

04.

55.

52.

30

.0(E

idam

)V

uill

emin

**

E.

nidu

lans

var.

lata

0.0

0.0

0.0

0.0

0.0

0.0

0.7

0.0

0.0

0.3

0.0

0.3

0.0

3.3

1.7

0.0

0.0

4.5

2.5

4.5

0.0

0.0

0.0

0.0

(Tho

rn&

Rap

er)

**

**N

eetr

iaha

emat

ocoe

ea0.

31.

30.

30.

70.

71.

30.

70

.02.

00.

70

.00.

70.

72.

74

.00.

70

.04.

52

.05.

52.

55

.03

.54.

5B

erk.

&B

r.P

enic

illi

um2.

7**

***

****

2.3

0.0

3.0

2.6

3.7

2.7

0.0

0.3

1.7

8.7

0.7

1.6

0.0

0.0

0.0

9.0

5.0

1.0

0.0

7.0

3.5

1.0

0.0

P.

chry

soge

num

Tho

rn*

1.0

0.0

0.0

0.0

0.0

1.3

1.3

0.0

0.0

1.7

6.0

0.7

I.3

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

P.

cory

loph

ilum

Die

rckx

0.0

0.0

0.0

0.0

I.3

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.5

0.0

0.0

0.0

0.0

0.0

0.0

P.

funi

culo

sum

Tho

rn0

.00.

30

.0**

****

0.0

0.0

I.3

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

4.5

0.5

0.5

0.0

0.0

0.0

0.0

0.0

P.

mar

tens

iiB

iour

ge0

.00

.00

.00

.00.

00.

00

.00.

00.

30.

0*

**0.

00

.00

.00.

00.

00

.00.

50

.00

.00.

05

.03.

51

.00

.0

P.

oxal

ieum

Cur

rie

&T

horn

1.7

2.0

0.0

I.3

0.3

0.0

0.0

***

0.0

0.7

0.7

0.0

2.7

0.0

0.0

0.0

0.0

3.0

2.5

0.5

0.0

2.0

0.0

0.0

0.0

Ste

rile

myc

eliu

m(W

hite

0.0

0.0

0.3

0.0

0.0

0.3

0.0

0.0

0.0

0.0

0.0

0.0

0.3

0.0

1.3

0.0

3.5

2.0

0.0

1.5

3.5

0.5

2.0

3.5

yello

wan

dda

rkco

lour

)

+N

onsi

gnif

ican

tly

resp

onde

dan

dra

rely

enco

unte

red

fung

iw

ere

omm

itte

d,bu

tw

ere

incl

uded

inth

eto

tal

coun

tof

fung

i;*

Mea

nssi

gnif

ican

tdif

fere

nce

com

para

ble

with

the

cont

rol;

**M

eans

high

lysi

gnif

ican

tdi

ffer

ence

com

para

ble

with

the

cont

rol.

Response of Soil Fungi to the Herbicide Primextra 301

This was nearly the picture in case of Aspergillus; in addition to the three doses ofPrimextra were significantly inhibited Aspergillus count after 40 days. Among the Aspergillusspecies, A. niger and A. terreus were the most inhibited species by this herbicide; where A.niger was lowered by the higher doses after each period, and A. terreus after 5, 40 and 60 daysof application. There was a single case of promotion among Aspergillus species and thatdemonstrated by A. fumigatus whose count was significantly raised by the highest dose after20 days of treatment. This promotion effect of Primextra was also observed with Emericellanidulans var. lata (40 days, low and high doses) and Nectria haematococca (20 days, low andmedium doses).

Penicillium population was significantly toxicated by the medium and the high doses after40 days and by the three doses after 60 days of application. Three of Penicillium species werealso inhibited by this herbicide after 40 days (P. funiculosum and P. oxalicum) and 60 days (P.martensii) under the higher doses.

Odeyemi et al. (1988) studied the effect of Dual (acetanilide) on soil fungi; they reportedthat this herbicide seemed to stimulate the fungal growth after 3 days of application.Moreover, Dual at 0.04 ftg/g probably acted as a substrate to increase the fungal density from34000 to 78000. On the other hand, results of Abdel-Kader et al. (1981) indicated that, Dualwas significantly toxic to the total count of soil fungi after 2 days of treatment with three doses(1.18,4.72 and 9.42 ug a.ilg). The triazine herbicide, Gesaprim-Combi showed a depressiveeffect on soil fungi (Abdel-Fattah et al. 1983). Korpraditskul et al. (1988) investigated the sideeffects of three herbicides (Atrazine, Ametryn and Paraquat) in sugar cane fields onpopulations dynamics of beneficial and non-target soil microorganisms. They found that thetotal count of microorganisms decreased during the first week of application. Also Mathur etal. (1980) reported that, application of Prometryne (a triazine herbicide) in carrot field led toincrease in fungal population at 27 days.

In the agar medium, Primextra was of inhibiting effect on the total counts of fungi,Aspergillus and A. niger by the medium and/or high doses (156 and 390 p.p.m.) as shown intable 3. However, the individual fungi responded variously; A. carneus, A. candidus and"sterile mycelium" were significantly activated by the high dose, the low and high doses, andthe medium dose, respectively. Table 3 also revealed that some fungal species (Acremoniumstrictum, Aspergillus candidus, A. flavus, A. ustus, Fusarium nivale, Mucor hiemalis, Nectriahaematococca and Penicillium chrysogenum) were counted in treated medium but not in thecontrol and vice versa (Aspergillus japonicus, Penicillium corylophilum, P. funiculosum andP. verruculosum).

In the liquid medium, it was observed that, when the dose of Primextra increased, themycelial growth of all tested fungi was substantially also increased (Table 4). This effect wassignificant with all tested fungi at the high dose plus the medium dose in case of Aspergillusterreus. The low dose (78 p.p.m.) was significantly induced an inhibition effect on themycelial growth of A. fumigatus only. Its worthmentioning that, despite A. fumigatus and A.terreus showed no significant response when Primextra incorporated with the agar medium,they were contrastablely significantly responded here. Furthermore, A. niger was highlysignificantly toxicated with the high dose in the agar medium whereas the same dose induced apromotion effect in liquid medium.

Abdel-Fattah et al. (1983) reported that Gesaprim-Combi significantly promoted thetotal count of fungi by the three doses used when incorporated with the agar medium. Incontrast with the results presented here, Abdel-Fattah et al. (1983) showed that, the mycelialgrowth of A. fumigatus, A. terreus and A. niger was not responded at any dose. In the agarmedium, the three doses of Dual (10.3, 41.0 and 82.4 p.p.m.) used by Abdel-Kader et al.(1981) were initiative to the total count of soil fungi, but the individual fungi exhibited eitherinitiative or depressive responses. This herbicide inhibited the mycelial growth ofTrichoderma viride, A. niger and Myrotherium verrucaria and stimulated the mycelial growth

302 M. M. K. Bagy and S. K. Hemida

Table 3. Effect of various doses of Primextra when incorporated in glucose agar medium on the counts (per mgdry soil) of common soil fungi (0 - Control; L = Low dose; M = Medium dose; H = High dose)

Genera & species

Total countAcremonium strictumAspergillusA. candidusA. carneusA. flavusA. fumigatusA. japonicusA. nigerA. terreusA. ustusA. versicolorFusarium nivaleMucor hiemalisNectria haematococcaPenicilliumP. chrysogenumP. corylophilumP. funiculosumP. verruculosumSterile mycelium (Yellow and white)

Doses

o

29.30.0

26.00.00.30.02.00.7

14.08.30.00.70.00.00.02.30.00.31.30.71.0

L

24.70.3

22.70.73.0**0.70.00.09.38.30.00.70.00.70.01.01.00.00.00.00.0

M

17.2**0.0

13.9**0.00.00.00.30.06.37.30.00.00.30.00.00.70.70.00.00.02.3*

H

20.0*0.0

18.3*2.0*5.0**0.00.00.02.7**8.00.30.30.00.01.00.70.70.00.00.00.0

* Means significant difference comparable with the control; ** Means highly significant difference comparablewith the control.

Table 4. Effect of various doses of Primextra on the mycelial dry weight (calculated as percentage of thecontrol)

Species

Aspergillus fumigatusA. nigerA. terreusTrichoderma harzianum

Doses

Low Medium High

34.0* 99.8 263.6**91.2 119.8 185.7*

116.7 222.8* 292.6**66.0 80.5 202.5*

* Means significant difference comparable with the control; ** Means highly significant difference comparablewith the control.

of Alternaria alternata (Abdel-Kader et al. 1981). In agreement with the results presented hereAbdel-Mallek (1981) reported that, the mycelial growth of A. niger was significantlyincreased by the high dose of Prometryn (triazine group). Nepomiluev and Kuzyakina (1972)reported that the growth of A. niger or Penicillium tardum was increased by Atrazine andSimazine at 4, 10 and 100 p.p.m.

Rhodes et al. (1980) studied the fungitoxic activity of hexazinone (triazine herbicide) on

Response of Soil Fungi to the Herbicide Primextra 303

soil fungi. They found that at very high treatment rates (l000 p.p.m.), hexazinone was morefungitoxic toward the plant disease-producing fungi (Alternaria, Fusarium, Pythium andRhizoctonia) than the normally saprophytic fungi (Aspergillus niger, A. terreus and Penicil­lium citrinum).

References

Abdel-Fattah, H. M., Abdel-Kader, M. 1. A., Hemida, S. K.: Selective effects of two triazine herbicides onEgyptian soil fungi. Mycopathologia 82 (1983), 143-151.

Abdel-Kader, M. 1. A., Abdel-Fattab, H. M., Hemida, S. K.: Effects of two herbicides from acetanilide andaniline groups on soil fungi. Egypt. J. Bot. 24 (1981),37-47.

Abdel-Mallek, A. Y.: Effect of some pesticides on soil root-surface and leaf-surface fungi. M.Sc. Thesis, Bot.Dept., Fac. of Science, Assiut Univ., Egypt (1981).

Bartha, R., Lanzilotta, R. P., Bramer, D.: Stability and effects of some pesticides in soil. Appl. Microbiol. 15(1967),67.

Chahal, D. S., Bans, 1. S., Chopra, S. L.: Degradation of Alachlor [2-chloro-N-(methoxymethyl)-2,6­diethylacetanilide] by soil fungi. Plant and Soil 53 (1976), 689-692.

Davis, J. C.: Statistics and data analysis in Geology. New York: Wiley (1973).Deshmukh, J. G., Shrikhande: Effect of pr- and post-emergence treatment of herbicides on soil microflora and

two microbial processes. J. Indian Soc. Soil Sci. 22 (1974), 36-42.Johnson, L. F., Curl, E. A.: Method for research on ecology of soil borne pathogens. Burgress Soc. 4 (1972),

97-102.Korpraditskul, V., Jiwajinda, S., Korpraditskul, R., Wicharn, S., Ratanagreetakul, c.: Side effects of three

herbicides on soil microorganisms. Kasetsart J. Natural. Sciences 22 (1988), 54-56.Mathur, S. P., Belanger, A., Hamilton, H. A, Khan, S. U.: Influence on microflora and persistence of field­

applied Disulfoton, Permethrin and Prometryne in an organic soil. Pedobiologia 20 (1980), 237-242.Moorman, T. B.: A review of pesticide effects on microorganisms and microbial processes related to soil

fertility. J. Production Agriculture 2 (1989), 14-23.Nepomiluev, V. F., Kuzyakina, T. 1.: The effect of Atrazine and Simazine on soil fungi and herbicide

decomposition in the soil. BioI. Nauki 15 (1972),127-131.Odeyemi, 0., Salami, A., Ugoji, E. 0.: Effect of common pesticides used in Nigeria on soil microbial

population. Indian J. of Agricultural Sciences 58 (1988), 624-628.Percich, J. A., Lockwood, J. L.: Interaction of Atrazine with soil microorganisms: Population changes and

accumulation. Can. J. Microbiol. 24 (1978), 1145-1152.Rhodes, R. C., Krause, R. L., Williams, M. H.: Microbial activity in soils treated with hexazinone. Soil

Science 129 (1980), 311- 314.Smith, N. R., Dawson, V. T.: The bacteriostatic action of rose bengal in media used for the plate counts of soil

fungi. Soil Science 58 (1944), 467-471.Zidan, Z. H., Rizk, T. Y., Shehata, A. F.: Some factors influencing the persistence of Fluometruron and

Paraquat in soils under laboratory conditions. Proc. 2nd Arab. Pesticides Conf. Tanta Univ. (1977),545-554.

Authors' address: Dr. M. M. K. Bagy and Dr. S. K. Hemida, Dept. of Botany, Faculty of Science,University of Assiut, Assiut, Egypt.