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Effect of the herbicide prometryn on metabolic activities of two Fusarium wilt fungi
[ 35 1 Tram Br. myco/. Soc. 90 (3), 351-358 (1988)
]
Primed in Great Britain
EFFECT OF THE HERBICIDE PROMETRYN ON METABOLIC ACTIVITIES OF TWO FUSARIUM WILT FUNGI By M. S.EL-ABYAD, H.ATTABY AND K.M.ABU-AISHA Department of Botany , Faculty of Science, Cairo Uni versity, Giza, Egypt The effect of the herbicide prometryn on the metabolic activities of two formae speciales of Fusarium oxysporum that cause cotton and tomato wilts in Egypt was studied. ·Prometryn at concentrations of 128 and 256 p.p.m . significantly inhibited growth and respiration rates, reduced absorption of both sugar and nitrate and reduced rates of synthesis of carbohydrates and organic nitrogenous compounds by both fungi. A concentration of 16 p.p.m. did not significantly affect the metabolic activities of F. oxysporum f.sp . uasinfectum. Triazine compounds are used widely in agriculture as herbicides. Their side-effects on morphology, growth and metabolism of higher plants are outlined by Gunter (1976). The main phytotoxic effect of chlorotriazines is to decrease chlorophyll content. The phytotoxicity of symtriazine herbicides is mainly attributed to the reduction in assimilation rate and that of S-methyl thiotriazines to inhibition of photosynthesis (Eshel & Bani, 1975). Degradation rates of these herbicides seem to increase with temperature and soil moisture content (H olly & Roberts, 1963). The herbicide prometryn is used in Egypt for selective weed control in various crops . However, this triazine herbicide is reported to cause toxicity in many crop plants (T hompson, Truelove & Davis, 1969). The inhibitory effects of triazine herbicides on growth and disease incidence by Fusarium oxyspo rum f.sp . ua sinfectum and F . oxysporum f. sp. lycopersici have been studied by many workers (Cowley & Lichtenstein, 1970; Rodriguez-Kabana & Curl, 1970; Tang, Curl & Rodriguez-Kabana, 1970; Grinstein et al., 1984; Macedo, Blanco & Chiba, 1984). In Egypt, several studies have been made on the effect of prometryn on incidence of cotton wilt caused by F. oxysporum f.sp. uasinfectum (Youssef & Heitefuss, 1983; EI-Khadem, EIKazzaz & Hassan, 1984; Youssef, Amr & Heitefuss, 1985). Zayed et al. (1982) found that trifluralin decreased the rates of respiration in F. oxy sporum and Trichoderma viride. EI-Abyad, Ismail & AIMeshhadani (1983) found that prometryn, among other biocide s studied, significantly inhibited macroconidial germination, germ-tube elongation, growth and sporulation of F . oxysporum f.sp. uasinfe ctum and F. oxysporum f.sp. lycopersici especially at the higher concentrations studied (256 and 384 p.p.m.). Thus it appears that substantial information is
already available concerning the mechanisms of inhibition by prometryn on higher plants, but its effects on the pathogenic fungi under consideration require further investigation. MATERIALS AND METHODS
Sources and culture of fungi Two pathogenic formae speciales of Fusarium oxysporum were used in this study : F. oxysporum f.sp. uasinfectum (Atk .) Snyder & Hansen and F. oxysporum f.sp. lycopersici (Sacc.) Snyder & Hansen as previously described by EI-Abyad et al. (1983). Both were maintained on modified CzapekDox agar which has the following composition (g 1 1 ) : sucrose, 20 ; KN0 3 , 4; NaH 2P0 4 , 2 ; MgS0 4 , 1; KCl, 0'5 ; FeS0 4 , 0'1; agar, 15. Macroconidia were produced on slants of modified Czapek-Dox agar incubated at 25 °C for 7 d. Suspensions were obtained by gentle shaking with distilled water . Conidial suspensions from different cultures were mixed to ensure thorough homogeneity. The herbicide prometryn (2A-bis-isopropylamino)-6-methylthio-S-triazine) was licensed by the Ministry of Agriculture to control weeds in cotton cultivation, and is produced by Ciba-Geigy under the commercial name 'Gesagard 50 Wp' . Stock solutions were prepared in sterile distilled water. Three concentrations were used: 16, 128 and 256 p.p.m . Growth experiment s Prometryn was added, aseptically, to modified Czapek-Dox medium to give final concentrations of 16, 128 or 256 p.p.m. This medium was dispensed in 100 ml aliquots, in 250 ml Erlenmeyer flasks. Each flask was inoculated with 1 ml of a spore suspension of the required fungus and incubated at 25° for 9 d. Fifteen flasks were
352
Effect of prometryn on Fusarium wilt fung i
prepared for each treatment, five of which were removed every three days , filtered under suction and the mycelium produced was dried to constant weight at 80°. CO 2 produced from respiration was estimated by using a Warburg constant volume respirometer as described by Umbreit, Burris & Stauffer (1959).
Carbohydrate analysis Samples of medium were neutralized to pH 7 by 1 N-NaOH and cleared by adding 1-2 ml of basic lead acetate. Excess lead was precipitated by Na 2H 2P0 4 containing 1 ml conc. H 2SO)1 (Said & Naguib, 1964 ). The lead phosphate was filtered off and the solution was made up to 50 m!. Mycelial samples were prepared for analysis by the method of Naguib (1963). Mycelial mats were oven-dried to constant weight at 80°. The soluble sugars were extracted from finely-powdered samples using 85 % ethyl alcohol in Soxhlet apparatus for 3 h. Alcohol was then evaporated to least volume, and the sample treated as described above. The residue was dried to constant weight for estimation of polysaccharides. Carbohydrate fractions were estimated photometrically using a Perkin-Elmer spectrophotometer Model 35 (C 033-0020). A modification of Nelson's solution (N aguib, 1965 ) was used in which sodium sulphate was replaced by potassium oxalate and the boiling period was extended to 15 min. Monosaccharides were determined by estimation of the direct reducing value (D RV). Samples (5 ml ) were mixed with 2 ml modified Nelson's solution, heated to tOOO for 15 min and rapidly cooled. Arsenomolybdate solution (3 ml ) was added and the mixture was shaken till effervescence stopped and absorbance was measured at 700 nm. Free monosaccharides and sucrose (to tal reducing value; TRV) were estimated by hydrolysing samples (5 ml ) with 5 ml 1 N-HCI at 60° for 30 min. The mixture was neutralized to pH 7 and TRV was determined using modified Nelson's solution. Data are expressed in terms of glucose. The difference between TRV and DRV gives an estimate of disaccharides in terms of glucose equivalents. For polysaccharide analysis, samples were refluxed in 5 ml of 1'5 N sulphuric acid for 4 h, neutralized to pH 7, cleared with basic lead acetate, deleaded by NaH 2P0 4 and the reducing value determined using Nelson 's solution. All data are expressed in terms of glucose equivalents .
Nitrogen analysis Samples of medium were diluted, mixed with an equal volume of 20-25 % trichloroacetic acid
(T CA) to precipitate soluble peptides and then centrifuged to eliminate the interference during the estimation of ammonia and amino nitrogen . Mycelial samples (50 mg ) were prepared by homogenization in 5 ml borate buffer at pH 8. The homogenates were transferred using small amounts of distilled water to small beakers and kept standing overnight before being filtered by suction. The residue was dried at 80° and the filtrate was made up to 50 m!' Soluble peptides were eliminated as described above and the oven-dry residue was used for estimation of total protein. Ammonia was determined using the Bertholet reaction (Chaney & Marbach, 1962) following neutralization of samples . The optical density was determined at 630 nm. Amino-N was measured using the method of Russel (1944) and amide-N as described by Naguib (1964). The difference between the value of ammonia-N and that obtained for free ammonia and amino-N gives an estimate of the amide-No According to Naguib, acyclic-N compounds are not hydrolysed under these conditions. Nitrate-N was measured as described by Taha (1964), nitrite-N by the method of Paech & Tracy (1956) and peptide-N according to Lowry et al. (1951). Total soluble nitrogen was determined according to Naguib (1969 b). The values obtained by this procedure provided estimates of all soluble nitrogen components except nitrate or nitrite nitrogen which were added to obtain a true estimate of the total soluble nitrogen. Other soluble nitrogen fractions, including the amide and acyclic forms (purines and pyrimidines) were calculated by subtracting the sum of peptide, amino, amide, nitrite, nitrate and ammonia nitrogen from the value obtained for total soluble nitrogen. Protein contents of mycelial mats were determined by refluxing with 5 ml 5 N-NaOH in a boiling water bath for 5 h (N agu ib, 1964). The hydrolysate was made slightly acidic using 2'5 ml of 10 N-H 2S04 before being filtered and made up to 50 m!' The amino acid content of the hydrolysate was estimated as above. All the measurements described in this work were carried out four times and the results obtained were analysed statistically by applying the LSD at 1 % and 5 %. RESUL TS
Growth and CO 2 production Both fungi were very sensitive to the herbicide prometryn when applied at high concentrations (128 and 256 p.p.m.), causing reductions in dryweight yields which were most obvious after 6 and 9 d incubation (Table 1). A concentration of 16 p.p.m. did not significantly inhibit growth of
Table
1.
Effect oj prometryn on dry weight yield (mg) oj mycelium and CO . outputs (mg/g dry wt ) pr oduced du ring growth oj F. oxysporum [.sp , vasinfcctum and F . oxysporum j .sp . lycopersici at 25° LSD
Concn of prometryn (p .p .rn. )
- - _ ._ - - _. Incubation period (d ays)
° (control) Dry w t
16 Dry wt
CO 2
--
CO 2
128
- - -- - Dr wt
C O,
256
- - - -- - Dry wt
Dry wt
CO 2 output
CO,
1 ~{J
5O ,I0
l
0"
5 ~/~l
4 2'7 49'9 62 '7
t 7'1 22'4 25'3
12,8 15'1 16'5
14'1 10'9 12'3
9'35 7,84 8'45
, 0
~
F , oxysporum f.sp, uasinfec tum 19° 274 306
3 6 9
7'5 8 5'0 1
LSD 1 % 5 01 /0
112 15° 17 6
18 4 277 3 15
12'5 8'27
7'59 5'01
9 1'8 110 139
117 164 182 10'9 7'20
4 1'7 52 '3 67'9
6'7 1 4'43
68 '1 7 1' 4 85'2
18'7 12'3
7'06 3'99
14'6 9'63
280 3 12 346
LSD 1 % 5%
9'22 6'7 1
168 202 234
2°9 281 295 8,86 5'37
10'4 6'89
138 16 4 179 9'49 6'26
122 158 178
7 8 '6 102 116 12'3 8'12
7'67 5'14
~ I ::t..
~ ~
~
F, oxysporum f.sp, ly copersici 3 6 9
Vl
62'S 72' 1 9°'3
66'3 7 2 'S 80'4
27'5 29 '7 33'S
9'81 11'9 13'8
21 '3 20 '1 24'4
7' 6 4 7'62 8'4 2
...
~
8'4 1 5'6
6'2 4 4'74
~ ::t.. ~ ~
;:: l:l...
~
~
Table
2.
Effect oj prometryn on sucrose inversion by F . oxysporu rn j .sp. vas infectum and F, oxysporurn f .sp . lycopcrsici (mg gluc osef too ml) after 9 d incubation at 25° 1'. oxysporum f. sp . vasinfectum
Concn of prometryn (p .p. rn .)
Uninverted su crose
° (co nt ro l) 16 128 25 6
29 '8 84'3 180 194
LSD 1 % 5%
65'6 45'1
Inverted .sucrose 1850 1850 1690 1680 63 '97 43 '9 8
( %)
99'9 9 8'7 9°'4 89 '6 3'66 2'52
Uninverted sucro se 96'7 117 12 5 133 77'5 35 '3
Inverted sucrose 1780 1760 1720 17 10 77'4 53 '2
<::J-
;;: I
::t.. ~.
F, oxysporum f.sp. ly copersici
Inversion
::t..
~ ~
Inversion (%) 94 '9 93 '8 9 1'9 9 1'S 3'45 2'3 8
UJ
VI
UJ
Effect of prometryn on Fusarium wilt fungi
354
Table 3. Effect of prometryn on sugar uptake (mg glucose j soo ml) by Fusarium oxysporum f.sp, vasinfectum and F. oxysporum f.sp, lycopersici after 9 d incubation at 25° F. oxysporum f.sp, oasinfectum Concn of prometryn (p.p.m.)
DRV
o (control) 16 128 256
70 1 612 1010 1120
F. oxysporum f.sp, lycopersici ----_._------------
LSD 1%
55'2 34'3
5 ~/~)
DRV TRV
TRV
Sugar uptake
DRV
TRV
Sugar uptake
731 696 1190 1310
12 70 13°0 807 686
4 63 557 995 1160
559 674 1120 1290
144 0 1320 87 8 707
50'8 34'9
1°4 7 1"4
56'9 39'2
42'5 29'2
77"7 62'9
= Direct Reducing Value, = Total Reducing Value.
Table 4, Effect of prometryn on soluble sugars and polysaccharides (mg glucosefg dry wt biomass) of Fusarium oxysporumj.sp. vasinfectum and F. oxysporumj.sp. lycopersici produced after 9 d at 25° F. oxysporum f.sp. uasinfectum Concn of prometryn (p.p.rn.)
DRV
TRV
o (control) 16 128 25 6
3"44 2-83 4-51 9-63
8-14 7-73 13'7 15-9
LSD 1% 5 0/0/
0-55 0'40
0-76 0-54
F. oxysporum f.sp, lycopersici Soluble sugars
Soluble sugars -----
DRV TRV
Polysaccharides 412 497 3 26 309 33 23-5
DRV
TRV
1'63 3-12 10'2 14
7-73 8-84 18 25'6
1-06 0-76
0'64 0-45
Polysaccharides 594 49 1 275 161 3 8'5 27"4
= Direct Reducing Value. = Total Reducing Value.
Table 5- Effect of prometryn on nitrate uptake (mg Noa/g dry wt) by mycelia of Fusarium oxysporum f.sp, vasinfectum and F. oxysporumj.sp. lycopersici after 9 d incubation at 25° Concn of prometryn (p.p.rn.)
Nitrate uptake
(mgjg Dry wt)
F. oxysporum f.sp. vasinfeetum
F. oxysporum f.sp, lycopersici
° (control) 16 128
47'7 48 40"4
53-8 48"4 42'9
~6
~
~
LSD
1
%
5 °''0
F. oxysporum f.sp. vasinfectum but did inhibit growth of F. oxysporum f.sp. lycopersici. With the exception of 16 p.p.m. for F. oxysporum f.sp. vasinfectum, all concentrations of prometryn sig-
nificantly decreased CO 2 production by the two fungi (Table 1) and the amounts of CO 2 produced were always correlated with mycelial dry weight. Maximum inhibition at the highest concentration (256 p.p.m.) occurred after 6 d for both fungi,
10'2 7"39
Carbohydrate metabolism
In the presence of 0 and 16 p.p.m. prometryn, F. oxysporum f.sp. vasinfectum inverted 99'9 % of the sucrose originally present within 9 d incubation at 25° (Table 2). Inversion was significantly reduced at concentrations of 128 and 256 p.p.m. Sucrose inversion by F. oxysporum f.sp, lycopersici was not significantly reduced by concentrations of pro-
M. S. EI-Abyad, H . Attaby and K. M . Abu-Aisha
355
metryn used . Sugar uptake by F. oxysporum f.sp. oasinfe ctum in the presence of 16 p.p.m . prometryn was only significantly higher than the control at LSD 5 % (T able 3) and was significantly inhibited at concentrations of 128 and 256 p.p.m., although high proportions of monosaccharides resulting from sucrose inversion were still retained in the medium. Sugar uptake by F . oxysporum f.sp . lycopersici was significantly inhibited at all herbicide concentrations and inhibition at the higher doses was accompanied by significant increases in the amounts of monosaccharides in the medium. It may also be noted that the processes of sucrose inversion and sugar uptake by both fungi appeared to be operating independently. In the presence of 16 p.p.m. prometryn, the amount of soluble sugars retained in the mycelium of F . oxysporum f.sp. vasinfectum after 9 d incubation was not significantly different from the control, but was significantly increa sed at concentrations of 128 and 256 p.p .m . prometryn (Table 4). However, the synthesis of polysaccharides significantly increased at 16 p .p.m. prometryn (497 mg glucose /g dry wt compared with 412 in the control) while higher concentrations (128 and 256 p.p.m.) decreased polysaccharide synt hesis. A similar trend was found for F. oxysporum f. sp. ly copersici (T able 4), but it appears that thi s forma specialis was more efficient in th e biosynthesis of polysaccharides than F. oxy sporum f.sp . v asinf ectum especially in the absence of herbicide. It also appears that the inhibitory effect of prometryn on polysaccharide formation was greater for F . oxysporum f.sp . ly copersici than F . oxysporum f.sp . v asinf ectum , especiall y at the higher concentrations.
:::;
Nitrogen metabolism
·E
Nitrate absorption by the mycelia of both fungi was not affected by 16 p.p.m. prornetryn, but was significantly decreased at higher concentrations, particularly in F. oxysporum f.sp.lycopersici (T able 5). Of the several nitrogen fractions measured, only the amino and peptide nitrogen were excreted in appreciable amounts with increasing herbicide concentration. Ammonia, amide and nitrite were present in trace amounts only , and showed significant increases with increasing herbicide concentration (T able 6). The presence of 16 p.p.m. prometryn had little or no effect on the different fractions recorded. For F. oxysporum f.sp . lycopersici (T able 6) the amide-N fraction was not detected in the culture medium and all other fractions increased significantly in the presence of prometryn . Amino and peptide-N fractions were greater for both fungi . Although the amounts of total soluble N excreted by F . oxy sporum f.sp .
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Effect of prometryn on Fusarium wilt fungi
356
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vasinfeetum at all concentrations except 16 p.p.m. were higher than those produced by F. oxysporum f.sp. lycopersici, the percentages of amino and peptide-N in relation to the total soluble N were similar for both fungi. The nitrogen content of mycelium of F. oxysporum f.sp. vasinfeetum was unaffected by 16 p.p.m. prometryn but in all other treatments there were significant decreases in peptide-N and protein-N (Table 7). Ammonia, amino, other soluble N, and total soluble N increased significantly with increasing prometryn concentration and there was a highly significant decrease In the total nitrogen contents (total soluble N + protein N) at 128 and 256 p.p.m. prometryn. For F. oxysporum f.sp. lycopersici (Table 7) prometryn significantly decreased peptide-N and protein-N contents, increased amino-N and ammonia-N (traces) at higher concentrations and significantly increased other forms of soluble-No As with F. oxysporum f.sp. vasinfectum, amounts of total nitrogen of mycelia were significantly decreased. Nitrate and nitrite nitrogen were absent from treated and untreated mycelia, even In highly concentrated samples. A summary is presented in Table 8 giving estimates of the percentages of total soluble Nand protein-N in relation to the total N content. It is evident that the percentage of PNj TN for both fungi decreased with increasing prometryn concentration except at 16 p.p.m. for F. oxysporum f.sp. vasinfeetum, where little or no effect was produced. On the other hand, the percentage of TSNjTN increased significantly at the higher concentrations for both fungi. The maximum values recorded were 60'7 for F. oxysporum f.sp. vasinfeetum and 64'3 for F. oxysporum f.sp. lycopersici at 256 p.p.m. DISCUSSION
'0 '1""4
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boo.:::t
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Concentrations of 128 and 256 p.p.m. of the herbicide prometryn significantly inhibited respiration and growth of F. oxysporum f.sp . vasinfeetum and F. oxysporum f.sp. lycopersici, Inhibition of respiration and mycelial growth by other herbicides has been recorded by several workers (Naguib, 1969a; Harhash, 1976; Wayne-Beam, Curl & Rodriguez-Kabana, 1977; Kurtz, Wayne Cole & Salin, 1982; Zayed et al., 1982). It has also been recognized that low concentrations of toxicants often stimulate respiration (McCallan, Miller & Weed, 1964). It seems likely that membrane permeability to substrate is increased under such conditions, although other explanations are possible (Kurtz et al., 1982). The observed decreases in sugar uptake by the two fungi studied, especially at the higher con-
M. S. El-Abyad, H. Attaby and K. M. Abu-Aisha
357
Table 8. Effect of prometryn on the percentage of total soluble nitrogen (TSN) and protein nitrogen (PN) in relation to total nitrogen content (TN) as determined in mycelial mats of 9-d cultures of F. oxysporum j.sp. vasinfectum and F. oxysporum f.sp. lycopersici Conen of prometryn
F. oxysporum f.sp. uasinfectum
F. oxysporum f.sp. lycopersici
TSNjTN
PNjTN
TSNjTN
(p.p.rn.)
(%)
(0,,)
(0,,)
(~,,)
o (control)
36"1
16 128 25 6
634
55'2 60'7
35"3
64'7 448 39'3
34'8 46'9 59'8 64'3
65'2 53"1 4°"2 35"7
centrations of prometryn, could account for inhibition of mycelial growth and although most of the applied sucrose was inverted the data suggest slight inhibition of the enzymic system of sucrose inversion. The accumulation of a large amount of the sucrose inverted by the two fungi as monosaccharides in the external medium might contribute to the reduction in respiration rates at higher concentrations, suggesting inhibition of glycolysis. High concentrations of prometryn also reduced biosynthesis of polysaccharides by both fungi. Naguib (1969a) showed that 125 p.p.m. sevin decreased sugar and phosphorus absorption by Rhizoctonia solani. This was associated with low carbohydrate and protein accumulation together with low organic phosphorus, keto acid and sulphydryl content. Harhash & Heikal (1976) showed that the herbicide diuron was mainly effective on the carbohydrate metabolism of F. oxysporum f.sp. vasinfectum. This herbicide caused low sugar absorption, low oxygen uptake and low carbohydrate accumulation by the fungus. Thus the observed inhibition of growth of the two fungi at the high concentrations of prometryn might be attributed to the lowered rate of sugar absorption, accumulation of carbohydrates in the medium and reduced rates of polysaccharide formation. The slight stimulatory or neutral effect of the low concentration of prometryn (16 p.p.m.) on F. oxysporum f.sp. vasinfectum might suggest that the herbicide acts as a growth promoter at this concentration. Data on nitrogen metabolism suggest that high concentrations of prometryn may affect the permeability of the fungal mycelia, increasing excretion of the different nitrogen fractions into the media. The detection of nitrite-N in the media may indicate stimulation of nitrate reduction to nitrite with further reduction to ammonia. The increased accumulation of nitrite-N following prometryn treatment suggests inhibition of nitrite reduction. Micro-analysis of mycelia at different concentrations of prometryn revealed, except for 16
PNjTN
p.p.m. and F. oxysporum f.sp. vasinfectum, inhibition of protein synthesis at the expense of increased total soluble nitrogen that existed mainly in the form of amino-No It also appears that the process of inactivation of protein synthesis induced successive accumulation of amino-N, as well as increased rate of excretion of the accumulated amino acids and peptides. The absence of nitrate in all treated mycelia might suggest that all the absorbed nitrate was completely reduced to nitrite and ammonia which reacted with organic acids producing the accumulated amino and peptide nitrogen as well as other soluble nitrogen forms detected in the mycelia. We are very grateful to Professor M. Kassas for reading the manuscript. REFERENCES
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Effect of prom etryn on Fusarium wilt fungi Influence of diur on in soil on RUSSEL, J A. (1944). Colorimetric
HARHASH, A. W . (1976). growth acti vities of Fusarium oxys porum. B ulletin of the Faculty of S cience, Cairo University S0, 1--9. HARHASH,A. W . &HEIKAL,N. (1976). The role of diu ron on the metabolism of Fusarium oxysporum. Bulletin of the Faculty of S cience, Cair o Uni versity 47, 1-16 . HOLLY, K. & ROBERTS, H . A. (1963)· Per sisten ce of phytotoxic residues of triazine herbicides in soil. We ed R esearch 3, 1-10 . KURTZ, M . E. , WAYNE COLE, A. & SALIN, M . L. (1982). Some metabolic responses of Rhizo ctonia solani to napropamide. W eed Scien ce 30, 49 1-494 . LOWRY, O. H ., ROSEBROUGH, N . J., FARR, A. L. & RANDALL, R. J. (1951)· Protein measurement with th e Folin phenol reagent. Journal of B iological Chem istry 193, 256-275 . MACEDO, E . c., BLANCO, H . G. & CHIBA, S. (19 84). Effect of herbicides on growth of Fusarium oxysporum f. uasinfectum. Bioldgico S0, 103- 113. MCCALLAN, S. E. A., MILLER, L. P . & WEED, R. M. (1954)· Comparative effect of fungicides on oxygen uptake and germination of spores . Cont ributions from Boyce Thompson Institute 18, 39-68 . NAGUIB, M . I. (1963). Colorimetric estimation of plant polysaccharides. Zucker 16, 15· NAGUIB, M . I. (1964). Effect of sevin on th e carb ohydrate and nitrogen metabolism during the germination of cotton seeds. Indian Journal of Exp eriment al Biology 2, 149- 152. NAGUIB, M. I. (1965). Effect of ascorbic acid on respiration and carbohydrate metab olism of Cunningham ella sp . Folia M icrobiologiy a 10, 215-223 . NAGUIB, M . I. ( 1969 a) . Effect of sevin on the metabolism of Rhizoctonia solani. Journal of Botany of the United Arab R epublic 11, 7-18 . NAGUIB, M. 1. (1969 b). On the colorim etr y of nitrogen components of plant tissues. Bulletin of the Faculty of Scienc e, Cairo Univ ersity 43, 1-5 · PAECH, K . & TRACY, M . V. (1956). Modern M ethods of Plant Analysis . Berlin : Springer Verlag . RODRIGUEZ-KABANA, R. & CURL, E. A. (1970). Effect of atrazine on growth of Fusarium oxys porum f.sp. vasinfectum , Phytopathology 60, 65-69.
detection of amin o nitrogen. J ournal of B iological Chemistry 56, 467. SAID, H . & NAGUIB, M . I. (1964). Sucrose determinat ion as a means of estimation of the dra wback tax on exported Halawa Tehinia. Bulletin of the Faculty of S cience, Ca iro University 39, 207-216. TAHA, M. S. (1964). The effect of the hydr ogen ion concentration in the medium and of temperature on the growth and nitrogen fixation by blue-green algae. Microbiologiya 32,968--972 . TANG, A., CURL, E. A. & RODRIGUEZ-KABANA, R. (1970). Effect of trifluralin on inoculum den sity and spo re germ inati on of Fusarium oxysporum f. sp. uasinf ectum in soil. Phytopathology 60, 1082-1086. THOMSON, O. c., TRUELOVE, B. & DAVIS, D . E. (1969). Effect of the herbicide prometryne (2,4-(bis isopropylamino j-o-methylthio-y-triazine) on mitochondria. Journal of Agricultural Food Chemistry 17, 997--998. UMBREIT, W . W.) BURRIS, R. H. & STAUFFER, J. F . (1959). Manometric T echniques. Minneapolis: Burgeff. WAYNE-BEAN, H . W., CURL, E. A. & RODRIGUEZKABANA, R. (1977). Effects of the herbicides fluometuron and prometryn on Rhizoctonia solani in soil cultures . Canadian Journal of Microbiology 23, 617-7 23. YOUSSEF, B. A. & HEITEFUSS, R. (1983). Side effects of herbicides on cott on wilt caused by Fusarium oxysporum f.sp. uasinfe ctum. 3. Microbiological studies. Z eitschrift fur Pfianzenkrankheu en und Pflanzenschutz 90, 160172. YOUSSEF, B. A., AMR, A. M . & HEITEFUSS, R. (198 5). Interactions between herbicides and soil-borne pathogens and cotton under greenhouse conditions. Zeitschrift fur Pfian z enkrankheiten und Pftan z enschut z 92, 55-63. ZAYED, S. M. A. D ., MOSTAFA, I. Y., ATTABY, M . S. H. & ADAM, YOUSR, M . (1982). The use of radi orespirometry for evaluating the effect of trifluralin on the microbiological activity of Fusarium oxy sporum and Tr ichoderma vi ride. Isotope and Radiation Research 14, 117- 12 1.
(Re ceived for publication
12
June 1987 )
]
Primed in Great Britain
EFFECT OF THE HERBICIDE PROMETRYN ON METABOLIC ACTIVITIES OF TWO FUSARIUM WILT FUNGI By M. S.EL-ABYAD, H.ATTABY AND K.M.ABU-AISHA Department of Botany , Faculty of Science, Cairo Uni versity, Giza, Egypt The effect of the herbicide prometryn on the metabolic activities of two formae speciales of Fusarium oxysporum that cause cotton and tomato wilts in Egypt was studied. ·Prometryn at concentrations of 128 and 256 p.p.m . significantly inhibited growth and respiration rates, reduced absorption of both sugar and nitrate and reduced rates of synthesis of carbohydrates and organic nitrogenous compounds by both fungi. A concentration of 16 p.p.m. did not significantly affect the metabolic activities of F. oxysporum f.sp . uasinfectum. Triazine compounds are used widely in agriculture as herbicides. Their side-effects on morphology, growth and metabolism of higher plants are outlined by Gunter (1976). The main phytotoxic effect of chlorotriazines is to decrease chlorophyll content. The phytotoxicity of symtriazine herbicides is mainly attributed to the reduction in assimilation rate and that of S-methyl thiotriazines to inhibition of photosynthesis (Eshel & Bani, 1975). Degradation rates of these herbicides seem to increase with temperature and soil moisture content (H olly & Roberts, 1963). The herbicide prometryn is used in Egypt for selective weed control in various crops . However, this triazine herbicide is reported to cause toxicity in many crop plants (T hompson, Truelove & Davis, 1969). The inhibitory effects of triazine herbicides on growth and disease incidence by Fusarium oxyspo rum f.sp . ua sinfectum and F . oxysporum f. sp. lycopersici have been studied by many workers (Cowley & Lichtenstein, 1970; Rodriguez-Kabana & Curl, 1970; Tang, Curl & Rodriguez-Kabana, 1970; Grinstein et al., 1984; Macedo, Blanco & Chiba, 1984). In Egypt, several studies have been made on the effect of prometryn on incidence of cotton wilt caused by F. oxysporum f.sp. uasinfectum (Youssef & Heitefuss, 1983; EI-Khadem, EIKazzaz & Hassan, 1984; Youssef, Amr & Heitefuss, 1985). Zayed et al. (1982) found that trifluralin decreased the rates of respiration in F. oxy sporum and Trichoderma viride. EI-Abyad, Ismail & AIMeshhadani (1983) found that prometryn, among other biocide s studied, significantly inhibited macroconidial germination, germ-tube elongation, growth and sporulation of F . oxysporum f.sp. uasinfe ctum and F. oxysporum f.sp. lycopersici especially at the higher concentrations studied (256 and 384 p.p.m.). Thus it appears that substantial information is
already available concerning the mechanisms of inhibition by prometryn on higher plants, but its effects on the pathogenic fungi under consideration require further investigation. MATERIALS AND METHODS
Sources and culture of fungi Two pathogenic formae speciales of Fusarium oxysporum were used in this study : F. oxysporum f.sp. uasinfectum (Atk .) Snyder & Hansen and F. oxysporum f.sp. lycopersici (Sacc.) Snyder & Hansen as previously described by EI-Abyad et al. (1983). Both were maintained on modified CzapekDox agar which has the following composition (g 1 1 ) : sucrose, 20 ; KN0 3 , 4; NaH 2P0 4 , 2 ; MgS0 4 , 1; KCl, 0'5 ; FeS0 4 , 0'1; agar, 15. Macroconidia were produced on slants of modified Czapek-Dox agar incubated at 25 °C for 7 d. Suspensions were obtained by gentle shaking with distilled water . Conidial suspensions from different cultures were mixed to ensure thorough homogeneity. The herbicide prometryn (2A-bis-isopropylamino)-6-methylthio-S-triazine) was licensed by the Ministry of Agriculture to control weeds in cotton cultivation, and is produced by Ciba-Geigy under the commercial name 'Gesagard 50 Wp' . Stock solutions were prepared in sterile distilled water. Three concentrations were used: 16, 128 and 256 p.p.m . Growth experiment s Prometryn was added, aseptically, to modified Czapek-Dox medium to give final concentrations of 16, 128 or 256 p.p.m. This medium was dispensed in 100 ml aliquots, in 250 ml Erlenmeyer flasks. Each flask was inoculated with 1 ml of a spore suspension of the required fungus and incubated at 25° for 9 d. Fifteen flasks were
352
Effect of prometryn on Fusarium wilt fung i
prepared for each treatment, five of which were removed every three days , filtered under suction and the mycelium produced was dried to constant weight at 80°. CO 2 produced from respiration was estimated by using a Warburg constant volume respirometer as described by Umbreit, Burris & Stauffer (1959).
Carbohydrate analysis Samples of medium were neutralized to pH 7 by 1 N-NaOH and cleared by adding 1-2 ml of basic lead acetate. Excess lead was precipitated by Na 2H 2P0 4 containing 1 ml conc. H 2SO)1 (Said & Naguib, 1964 ). The lead phosphate was filtered off and the solution was made up to 50 m!. Mycelial samples were prepared for analysis by the method of Naguib (1963). Mycelial mats were oven-dried to constant weight at 80°. The soluble sugars were extracted from finely-powdered samples using 85 % ethyl alcohol in Soxhlet apparatus for 3 h. Alcohol was then evaporated to least volume, and the sample treated as described above. The residue was dried to constant weight for estimation of polysaccharides. Carbohydrate fractions were estimated photometrically using a Perkin-Elmer spectrophotometer Model 35 (C 033-0020). A modification of Nelson's solution (N aguib, 1965 ) was used in which sodium sulphate was replaced by potassium oxalate and the boiling period was extended to 15 min. Monosaccharides were determined by estimation of the direct reducing value (D RV). Samples (5 ml ) were mixed with 2 ml modified Nelson's solution, heated to tOOO for 15 min and rapidly cooled. Arsenomolybdate solution (3 ml ) was added and the mixture was shaken till effervescence stopped and absorbance was measured at 700 nm. Free monosaccharides and sucrose (to tal reducing value; TRV) were estimated by hydrolysing samples (5 ml ) with 5 ml 1 N-HCI at 60° for 30 min. The mixture was neutralized to pH 7 and TRV was determined using modified Nelson's solution. Data are expressed in terms of glucose. The difference between TRV and DRV gives an estimate of disaccharides in terms of glucose equivalents. For polysaccharide analysis, samples were refluxed in 5 ml of 1'5 N sulphuric acid for 4 h, neutralized to pH 7, cleared with basic lead acetate, deleaded by NaH 2P0 4 and the reducing value determined using Nelson 's solution. All data are expressed in terms of glucose equivalents .
Nitrogen analysis Samples of medium were diluted, mixed with an equal volume of 20-25 % trichloroacetic acid
(T CA) to precipitate soluble peptides and then centrifuged to eliminate the interference during the estimation of ammonia and amino nitrogen . Mycelial samples (50 mg ) were prepared by homogenization in 5 ml borate buffer at pH 8. The homogenates were transferred using small amounts of distilled water to small beakers and kept standing overnight before being filtered by suction. The residue was dried at 80° and the filtrate was made up to 50 m!' Soluble peptides were eliminated as described above and the oven-dry residue was used for estimation of total protein. Ammonia was determined using the Bertholet reaction (Chaney & Marbach, 1962) following neutralization of samples . The optical density was determined at 630 nm. Amino-N was measured using the method of Russel (1944) and amide-N as described by Naguib (1964). The difference between the value of ammonia-N and that obtained for free ammonia and amino-N gives an estimate of the amide-No According to Naguib, acyclic-N compounds are not hydrolysed under these conditions. Nitrate-N was measured as described by Taha (1964), nitrite-N by the method of Paech & Tracy (1956) and peptide-N according to Lowry et al. (1951). Total soluble nitrogen was determined according to Naguib (1969 b). The values obtained by this procedure provided estimates of all soluble nitrogen components except nitrate or nitrite nitrogen which were added to obtain a true estimate of the total soluble nitrogen. Other soluble nitrogen fractions, including the amide and acyclic forms (purines and pyrimidines) were calculated by subtracting the sum of peptide, amino, amide, nitrite, nitrate and ammonia nitrogen from the value obtained for total soluble nitrogen. Protein contents of mycelial mats were determined by refluxing with 5 ml 5 N-NaOH in a boiling water bath for 5 h (N agu ib, 1964). The hydrolysate was made slightly acidic using 2'5 ml of 10 N-H 2S04 before being filtered and made up to 50 m!' The amino acid content of the hydrolysate was estimated as above. All the measurements described in this work were carried out four times and the results obtained were analysed statistically by applying the LSD at 1 % and 5 %. RESUL TS
Growth and CO 2 production Both fungi were very sensitive to the herbicide prometryn when applied at high concentrations (128 and 256 p.p.m.), causing reductions in dryweight yields which were most obvious after 6 and 9 d incubation (Table 1). A concentration of 16 p.p.m. did not significantly inhibit growth of
Table
1.
Effect oj prometryn on dry weight yield (mg) oj mycelium and CO . outputs (mg/g dry wt ) pr oduced du ring growth oj F. oxysporum [.sp , vasinfcctum and F . oxysporum j .sp . lycopersici at 25° LSD
Concn of prometryn (p .p .rn. )
- - _ ._ - - _. Incubation period (d ays)
° (control) Dry w t
16 Dry wt
CO 2
--
CO 2
128
- - -- - Dr wt
C O,
256
- - - -- - Dry wt
Dry wt
CO 2 output
CO,
1 ~{J
5O ,I0
l
0"
5 ~/~l
4 2'7 49'9 62 '7
t 7'1 22'4 25'3
12,8 15'1 16'5
14'1 10'9 12'3
9'35 7,84 8'45
, 0
~
F , oxysporum f.sp, uasinfec tum 19° 274 306
3 6 9
7'5 8 5'0 1
LSD 1 % 5 01 /0
112 15° 17 6
18 4 277 3 15
12'5 8'27
7'59 5'01
9 1'8 110 139
117 164 182 10'9 7'20
4 1'7 52 '3 67'9
6'7 1 4'43
68 '1 7 1' 4 85'2
18'7 12'3
7'06 3'99
14'6 9'63
280 3 12 346
LSD 1 % 5%
9'22 6'7 1
168 202 234
2°9 281 295 8,86 5'37
10'4 6'89
138 16 4 179 9'49 6'26
122 158 178
7 8 '6 102 116 12'3 8'12
7'67 5'14
~ I ::t..
~ ~
~
F, oxysporum f.sp, ly copersici 3 6 9
Vl
62'S 72' 1 9°'3
66'3 7 2 'S 80'4
27'5 29 '7 33'S
9'81 11'9 13'8
21 '3 20 '1 24'4
7' 6 4 7'62 8'4 2
...
~
8'4 1 5'6
6'2 4 4'74
~ ::t.. ~ ~
;:: l:l...
~
~
Table
2.
Effect oj prometryn on sucrose inversion by F . oxysporu rn j .sp. vas infectum and F, oxysporurn f .sp . lycopcrsici (mg gluc osef too ml) after 9 d incubation at 25° 1'. oxysporum f. sp . vasinfectum
Concn of prometryn (p .p. rn .)
Uninverted su crose
° (co nt ro l) 16 128 25 6
29 '8 84'3 180 194
LSD 1 % 5%
65'6 45'1
Inverted .sucrose 1850 1850 1690 1680 63 '97 43 '9 8
( %)
99'9 9 8'7 9°'4 89 '6 3'66 2'52
Uninverted sucro se 96'7 117 12 5 133 77'5 35 '3
Inverted sucrose 1780 1760 1720 17 10 77'4 53 '2
<::J-
;;: I
::t.. ~.
F, oxysporum f.sp. ly copersici
Inversion
::t..
~ ~
Inversion (%) 94 '9 93 '8 9 1'9 9 1'S 3'45 2'3 8
UJ
VI
UJ
Effect of prometryn on Fusarium wilt fungi
354
Table 3. Effect of prometryn on sugar uptake (mg glucose j soo ml) by Fusarium oxysporum f.sp, vasinfectum and F. oxysporum f.sp, lycopersici after 9 d incubation at 25° F. oxysporum f.sp, oasinfectum Concn of prometryn (p.p.m.)
DRV
o (control) 16 128 256
70 1 612 1010 1120
F. oxysporum f.sp, lycopersici ----_._------------
LSD 1%
55'2 34'3
5 ~/~)
DRV TRV
TRV
Sugar uptake
DRV
TRV
Sugar uptake
731 696 1190 1310
12 70 13°0 807 686
4 63 557 995 1160
559 674 1120 1290
144 0 1320 87 8 707
50'8 34'9
1°4 7 1"4
56'9 39'2
42'5 29'2
77"7 62'9
= Direct Reducing Value, = Total Reducing Value.
Table 4, Effect of prometryn on soluble sugars and polysaccharides (mg glucosefg dry wt biomass) of Fusarium oxysporumj.sp. vasinfectum and F. oxysporumj.sp. lycopersici produced after 9 d at 25° F. oxysporum f.sp. uasinfectum Concn of prometryn (p.p.rn.)
DRV
TRV
o (control) 16 128 25 6
3"44 2-83 4-51 9-63
8-14 7-73 13'7 15-9
LSD 1% 5 0/0/
0-55 0'40
0-76 0-54
F. oxysporum f.sp, lycopersici Soluble sugars
Soluble sugars -----
DRV TRV
Polysaccharides 412 497 3 26 309 33 23-5
DRV
TRV
1'63 3-12 10'2 14
7-73 8-84 18 25'6
1-06 0-76
0'64 0-45
Polysaccharides 594 49 1 275 161 3 8'5 27"4
= Direct Reducing Value. = Total Reducing Value.
Table 5- Effect of prometryn on nitrate uptake (mg Noa/g dry wt) by mycelia of Fusarium oxysporum f.sp, vasinfectum and F. oxysporumj.sp. lycopersici after 9 d incubation at 25° Concn of prometryn (p.p.rn.)
Nitrate uptake
(mgjg Dry wt)
F. oxysporum f.sp. vasinfeetum
F. oxysporum f.sp, lycopersici
° (control) 16 128
47'7 48 40"4
53-8 48"4 42'9
~6
~
~
LSD
1
%
5 °''0
F. oxysporum f.sp. vasinfectum but did inhibit growth of F. oxysporum f.sp. lycopersici. With the exception of 16 p.p.m. for F. oxysporum f.sp. vasinfectum, all concentrations of prometryn sig-
nificantly decreased CO 2 production by the two fungi (Table 1) and the amounts of CO 2 produced were always correlated with mycelial dry weight. Maximum inhibition at the highest concentration (256 p.p.m.) occurred after 6 d for both fungi,
10'2 7"39
Carbohydrate metabolism
In the presence of 0 and 16 p.p.m. prometryn, F. oxysporum f.sp. vasinfectum inverted 99'9 % of the sucrose originally present within 9 d incubation at 25° (Table 2). Inversion was significantly reduced at concentrations of 128 and 256 p.p.m. Sucrose inversion by F. oxysporum f.sp, lycopersici was not significantly reduced by concentrations of pro-
M. S. EI-Abyad, H . Attaby and K. M . Abu-Aisha
355
metryn used . Sugar uptake by F. oxysporum f.sp. oasinfe ctum in the presence of 16 p.p.m . prometryn was only significantly higher than the control at LSD 5 % (T able 3) and was significantly inhibited at concentrations of 128 and 256 p.p.m., although high proportions of monosaccharides resulting from sucrose inversion were still retained in the medium. Sugar uptake by F . oxysporum f.sp . lycopersici was significantly inhibited at all herbicide concentrations and inhibition at the higher doses was accompanied by significant increases in the amounts of monosaccharides in the medium. It may also be noted that the processes of sucrose inversion and sugar uptake by both fungi appeared to be operating independently. In the presence of 16 p.p.m. prometryn, the amount of soluble sugars retained in the mycelium of F . oxysporum f.sp. vasinfectum after 9 d incubation was not significantly different from the control, but was significantly increa sed at concentrations of 128 and 256 p.p .m . prometryn (Table 4). However, the synthesis of polysaccharides significantly increased at 16 p .p.m. prometryn (497 mg glucose /g dry wt compared with 412 in the control) while higher concentrations (128 and 256 p.p.m.) decreased polysaccharide synt hesis. A similar trend was found for F. oxysporum f. sp. ly copersici (T able 4), but it appears that thi s forma specialis was more efficient in th e biosynthesis of polysaccharides than F. oxy sporum f.sp . v asinf ectum especially in the absence of herbicide. It also appears that the inhibitory effect of prometryn on polysaccharide formation was greater for F . oxysporum f.sp . ly copersici than F . oxysporum f.sp . v asinf ectum , especiall y at the higher concentrations.
:::;
Nitrogen metabolism
·E
Nitrate absorption by the mycelia of both fungi was not affected by 16 p.p.m. prornetryn, but was significantly decreased at higher concentrations, particularly in F. oxysporum f.sp.lycopersici (T able 5). Of the several nitrogen fractions measured, only the amino and peptide nitrogen were excreted in appreciable amounts with increasing herbicide concentration. Ammonia, amide and nitrite were present in trace amounts only , and showed significant increases with increasing herbicide concentration (T able 6). The presence of 16 p.p.m. prometryn had little or no effect on the different fractions recorded. For F. oxysporum f.sp . lycopersici (T able 6) the amide-N fraction was not detected in the culture medium and all other fractions increased significantly in the presence of prometryn . Amino and peptide-N fractions were greater for both fungi . Although the amounts of total soluble N excreted by F . oxy sporum f.sp .
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Effect of prometryn on Fusarium wilt fungi
356
0'
-._p
0 ("f) 0 \0
'"
b b
\0 ['--.\0 N l"f"lO\f\I("f)
bN~i--
Oi
,S 0)
c
o~ V\
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§
vasinfeetum at all concentrations except 16 p.p.m. were higher than those produced by F. oxysporum f.sp. lycopersici, the percentages of amino and peptide-N in relation to the total soluble N were similar for both fungi. The nitrogen content of mycelium of F. oxysporum f.sp. vasinfeetum was unaffected by 16 p.p.m. prometryn but in all other treatments there were significant decreases in peptide-N and protein-N (Table 7). Ammonia, amino, other soluble N, and total soluble N increased significantly with increasing prometryn concentration and there was a highly significant decrease In the total nitrogen contents (total soluble N + protein N) at 128 and 256 p.p.m. prometryn. For F. oxysporum f.sp. lycopersici (Table 7) prometryn significantly decreased peptide-N and protein-N contents, increased amino-N and ammonia-N (traces) at higher concentrations and significantly increased other forms of soluble-No As with F. oxysporum f.sp. vasinfectum, amounts of total nitrogen of mycelia were significantly decreased. Nitrate and nitrite nitrogen were absent from treated and untreated mycelia, even In highly concentrated samples. A summary is presented in Table 8 giving estimates of the percentages of total soluble Nand protein-N in relation to the total N content. It is evident that the percentage of PNj TN for both fungi decreased with increasing prometryn concentration except at 16 p.p.m. for F. oxysporum f.sp. vasinfeetum, where little or no effect was produced. On the other hand, the percentage of TSNjTN increased significantly at the higher concentrations for both fungi. The maximum values recorded were 60'7 for F. oxysporum f.sp. vasinfeetum and 64'3 for F. oxysporum f.sp. lycopersici at 256 p.p.m. DISCUSSION
'0 '1""4
tr.NOO 0\ O"''O'''NO~
boo.:::t
V)r---. .....
~
tiJc:io
("f)
... N
"""
("I')
00"'''''"'''''''"00 bb\~~V,f-..-N ... N
"""
Concentrations of 128 and 256 p.p.m. of the herbicide prometryn significantly inhibited respiration and growth of F. oxysporum f.sp . vasinfeetum and F. oxysporum f.sp. lycopersici, Inhibition of respiration and mycelial growth by other herbicides has been recorded by several workers (Naguib, 1969a; Harhash, 1976; Wayne-Beam, Curl & Rodriguez-Kabana, 1977; Kurtz, Wayne Cole & Salin, 1982; Zayed et al., 1982). It has also been recognized that low concentrations of toxicants often stimulate respiration (McCallan, Miller & Weed, 1964). It seems likely that membrane permeability to substrate is increased under such conditions, although other explanations are possible (Kurtz et al., 1982). The observed decreases in sugar uptake by the two fungi studied, especially at the higher con-
M. S. El-Abyad, H. Attaby and K. M. Abu-Aisha
357
Table 8. Effect of prometryn on the percentage of total soluble nitrogen (TSN) and protein nitrogen (PN) in relation to total nitrogen content (TN) as determined in mycelial mats of 9-d cultures of F. oxysporum j.sp. vasinfectum and F. oxysporum f.sp. lycopersici Conen of prometryn
F. oxysporum f.sp. uasinfectum
F. oxysporum f.sp. lycopersici
TSNjTN
PNjTN
TSNjTN
(p.p.rn.)
(%)
(0,,)
(0,,)
(~,,)
o (control)
36"1
16 128 25 6
634
55'2 60'7
35"3
64'7 448 39'3
34'8 46'9 59'8 64'3
65'2 53"1 4°"2 35"7
centrations of prometryn, could account for inhibition of mycelial growth and although most of the applied sucrose was inverted the data suggest slight inhibition of the enzymic system of sucrose inversion. The accumulation of a large amount of the sucrose inverted by the two fungi as monosaccharides in the external medium might contribute to the reduction in respiration rates at higher concentrations, suggesting inhibition of glycolysis. High concentrations of prometryn also reduced biosynthesis of polysaccharides by both fungi. Naguib (1969a) showed that 125 p.p.m. sevin decreased sugar and phosphorus absorption by Rhizoctonia solani. This was associated with low carbohydrate and protein accumulation together with low organic phosphorus, keto acid and sulphydryl content. Harhash & Heikal (1976) showed that the herbicide diuron was mainly effective on the carbohydrate metabolism of F. oxysporum f.sp. vasinfectum. This herbicide caused low sugar absorption, low oxygen uptake and low carbohydrate accumulation by the fungus. Thus the observed inhibition of growth of the two fungi at the high concentrations of prometryn might be attributed to the lowered rate of sugar absorption, accumulation of carbohydrates in the medium and reduced rates of polysaccharide formation. The slight stimulatory or neutral effect of the low concentration of prometryn (16 p.p.m.) on F. oxysporum f.sp. vasinfectum might suggest that the herbicide acts as a growth promoter at this concentration. Data on nitrogen metabolism suggest that high concentrations of prometryn may affect the permeability of the fungal mycelia, increasing excretion of the different nitrogen fractions into the media. The detection of nitrite-N in the media may indicate stimulation of nitrate reduction to nitrite with further reduction to ammonia. The increased accumulation of nitrite-N following prometryn treatment suggests inhibition of nitrite reduction. Micro-analysis of mycelia at different concentrations of prometryn revealed, except for 16
PNjTN
p.p.m. and F. oxysporum f.sp. vasinfectum, inhibition of protein synthesis at the expense of increased total soluble nitrogen that existed mainly in the form of amino-No It also appears that the process of inactivation of protein synthesis induced successive accumulation of amino-N, as well as increased rate of excretion of the accumulated amino acids and peptides. The absence of nitrate in all treated mycelia might suggest that all the absorbed nitrate was completely reduced to nitrite and ammonia which reacted with organic acids producing the accumulated amino and peptide nitrogen as well as other soluble nitrogen forms detected in the mycelia. We are very grateful to Professor M. Kassas for reading the manuscript. REFERENCES
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