- Email: [email protected]
Germination of Botrytis cinerea conidia in the presence of quintozene, tecnazene and dichloran
[ 4°5 ] Trans. B,. mycol. Soc. 51 (3 and 4),405-410 (1968) Printed in Great Britain
GERMINATION OF BOTRYTIS CINEREA CONIDIA IN THE PRESENCE OF QUINTOZENE, TECNAZENE AND DICHLORAN By O. F. ESURUOSO, T. V. PRICE AND R. K. S. WOOD Department of Botany and Plant Technology, Imperial College, London, S. W. 7 (With Plate 25) Various substances alter the activity of quintozene, tecnazene and dichloran towards Botrytis cinerea. The toxicity of tecnazene and dichloran is increased in agar gels, and in o· 1 % solutions ofagar, sodium carboxymethylcellulose, sodium polypectate and gelatine; there are no similar effects with quintozene. A glucose/casein hydrolysate solution enhances the activity of dichloran, but not that of the other two fungicides. Less striking, but still pronounced effects are caused by tecnazene and dichloran in the presence of these high molecular weight substances, and nutrients.
A characteristic of the fungicides quintozene, tecnazene and dichloran is their inactivity in the standard spore germination test in water. They may, however, cause the germ tubes of susceptible fungi to grow more slowly and abnormally. Thus, Hewlett (1955) found that quintozene and tecnazene vapours were more effective in nutrient than water agar in reducing growth of germ tubes and in causing abnormal hyphae to develop. In 1961, Clark & Hams reported thatdichloran, added to nutrient agar, retards growth of B. cinerea, and Sharples (1961) stated that B. cinerea responds differently to dichloran in agar and in liquid media. He also showed that dichloran causes hyphae to burst in media containing nutrients. More recently, Weber & Ogawa (1963, 1965) report that dichloran affects the protein metabolism of Rhi:::;opus arrhizus. The effects of agar and certain other substances in altering the activity of these three fungicides against B. cinerea became apparent during a study by Esuruoso (1964) of resistant strains. This work was repeated later and our results are summarized below. MATERIALS AND METHODS
The test fungus was a freely sporing isolate of Botrytis cinerea from a diseased lettuce plant. The fungicides were quintozene (pentachloronitrobenzene), tecnazene (2,3,5,6-tetrachloronitrobenzene) and dichloran (2,6-dichloro-4-nitroaniline). Each was recrystallized from acetone solutions of commercial products. Relatively stable suspensions were obtained by dissolving 0'3 g in 20 ml acetone containing 0'5 % Tween 80, a non-toxic wetting agent.
406
Transactions British Mycological Society
This solution was slowly added to 150 ml water with constant stirring and the fine suspension obtained was centrifuged, washed three times with water and finally suspended in 25 ml ofwater containing 0'05 % Tween 80. The final concentration was determined with 0'5 ml samples dried at 35 ° to constant weight. To prepare fungicide-impregnated agar, aqueous suspensions of fungicide were autoclaved at 15lb/inz (103'4 kN/mZ) for 5 min. and suitable volumes added to molten 1'5 % agar at 40° just before pouring plates. The following substrates were used for spore germination: (I) Plain agar (Davis): 1'5 % in water. (2) Plain agar: extracted twice for 2 h with five times its weight of pyridine with constant stirring, collected on a filter, extracted twice for 2 h in twelve times its weight of water with constant stirring, collected and dried at 70° for 24 h, then used at 1'5 % in water. (3) 0'1 % solutions in water of: agar; powdered gelatine (Oxoid); carboxymethylcellulose D.S. 0'7 Premium Low (Hercules Powder Company); sodium polypectate (California Fruit Growers Exchange). (4) Glucose/casein hydrolysate solution: glucose, 1'0 %; casein hydrolysate (Oxoid), 0'4 %; KHzP04, 0'1 %; MgS04.7HzO, 0'05 %, in water. (5) Glucose/casein hydrolysate agar: as 4 with 1'5 % agar. Spores were obtained from 7- to 14-day-old cultures on a mass inoculated glucose/casein hydrolysate agar by adding water containing 0'05 %Tween 80. The crude suspension was filtered through muslin and washed by centrifuging three times in water. Final suspensions were prepared in sterile water. Germination tests were done in 0'05 ml films ofliquid spread evenly over 12 mm coverslips attached to 2'5 x 7'5 ern glass slides by thin films of Vaseline, and incubated at c. 100 % R.H. at 25° for 24 h. They were then stained with cotton blue. The germination of 100 spores randomly distributed on each of three coverslips was recorded for each treatment; a spore was counted as germinated when the germ tube exceeded the spore length, Results are given as mean inhibition ofgermination after angular transformation ofpercentages. EXPERIMENTAL
Germination in aqueous suspensions of thefungicides The films on the coverslips contained 5 x 10 4spores/ml, and the fungicide at concentrations in the range 1-4000 ppm. Quintozene and tecnazene did not reduce germination even at 4000 ppm. Below 1000 ppm., over go % of spores were unaffected by dichloran. At higher concentrations 15-20 % of spores did not germinate. Germination on agar impregnated with fungicides Plain agar, pyridine-extracted plain agar, and glucose/casein hydrolysate agar were mixed with aqueous suspensions of the three fungicides to concentrations of 0, I, 2, 5, 25, 50, 100, 250 and 500 ppm before plates were poured. Acetone solutions of the fungicides (employed by earlier workers) were not used because we found that residual acetone retarded growth of the fungus.
Botrytis cinerea. O. F. Esuruoso et al, Table
I.
Effect of quintozene, tecnazene and dichloran in agar on germination of spores of Botrytis cinerea Inhibition of germination (transformed percentages) Fungicide (ppm)
Quintozene Tecnazene Dichloran
Agar
0
PA* PPA* GCA* PA PPA GCA PA PPA GCA
0 0
0 0 I
0 I
0 0
5 0 2 0 17 5 71 4 5 8
25
100
500
5 0 0
2 0 0 71 65 83 7 6 84
0
39 50 80 13 4 71
3 0
7473 79 32 25 83
Least difference significant at P = 0·05 5 3 13 8 6 8 14 16
* PA, plain agar; PPA, pyridine extracted agar; GCA, glucose/casein hydrolysate agar.
Table 2. Effect of quintozene, tecnazene and dichloran on germination of spores of Botrytis cinerea in solutions of different substances Substance in solution (0·1%) Quintozene
Agar Gelatine CMC* NaPP* GCA* Agar Gelatine CMC NaPP GCA Agar Gelatine CMC NaPP GCA
Inhibition of germination (transformed percentages) Fungicide (ppm) 0 5 II
2 0 0
5 8 10 16 5 0 15 13 14 0 2 26 17 25 6 3
25 8 15 12 4 0 22 17 13 19 0 42 21 40 9 49
100
500
8
4 0 0 0 0 85 78 90 74 13 37 37 53 39 64
II
8 0 0 23 28 29 34 4 41 26 42 21 64
Least difference significant at P = 0'05 7 6 6 5
10 4 10 6 0 6 0 7 6 Dichloran 5 6 7 6 4 6 3 0 5 • CMC, Sodium carboxymethy1cellulose; NaPP, sodium polypectate; GCA, glucose/casein hydrolysate. Tecnazene
7
II
Drops (0-05 ml) of spore suspension (5 x J04/ml) were put on the agar surface and incubated at 25°. Germination after 24 h is given in Table I. Quintozene at 500 ppm and lower concentrations did not affect spore germination. Tecnazene reduced germination by 50 % when suspended in plain agar at 25 ppm, and almost completely prevented germination at 500 ppm. In glucose/casein hydrolysate agar, 5 ppm fungicide was sufficient to reduce germination by 88 %. Surprisingly, therefore, extraction of agar with pyridine did not reduce activity of the fungicide. Dichloran
408
Transactions British Mycological Society
caused 28 %inhibition at 500 ppm in plain agar, and 92 % in nutrient agar at 25 ppm. These results showed that tecnazene and dichloran which are almost inactive in water become highly active when suspended in an agar gel and that this effect is enhanced by nutrients. In contrast, the activity of quintozene was not affected by ag ar or nutrients.
Germination in solutions of various substances In these experiments, spores and fungicides were suspended in solutions of the substances listed in T able 2 . Glucose/casein hydrolysate solution was used because of the striking results obtained with the corresponding agar. The pH was adjusted to 4'0 with H'Cl to prevent bacterial growth. Agar Table 3. Effect
of quintorene, tecnazene and dichloran on growth ofgerm tubes on agar Mean germ tube length (x spore length) Fungicide (ppm)
.
Agar Quintozene
PA* GCA*
Tecnazene
PA*
° 5° (16 h ) So (14 h) 50 (20h)
5
25
100
3°
25
20
5 00 IS
25
20
15
IS
3
3
3
Ab
Ab
Ab
10 IS 3° 10 Ab 3° • PA , Plain agar ; eCA, glucosefcasein hydrolysate agar. tAb, short, mu ch.branched tubes, abnormally swollen.
5 Ab
GCA
60
35
Ab']
(14 h ) Dichloran
PA* GCA
60
> 100
was used at o· I % to give a solution instead of a gel. Gelatine, carboxymethylcellulose and sodium polypectate were taken as examples of high molecular weight substances for comparison with agar. Drops (0'05 ml) of mixtures of spores, fungicide and solution were placed on 12 mm coverslips on glass slides and incubated at c. 100 % R.H. for 24 h at 250 • The fungicides were used at the same concentrations as in the experiments with agar and statistical analyses were based on the complete data from all tests. Selected but typical results are shown in Table 2. Spore germination in the presence of quintozene was not materially affected by any of the substances. Inhibition of germination by tecnazene was increased by agar, gelatine, sodium polypectate and carboxymethylcellulose to give ED 50 values between 100 and 500 ppm compared with no effect of 4000 ppm in water. Activity of dichloran was also increased by these substances but to a lesser extent. In contrast, glucose/casein hydrolysate solution, which markedly increased the activity of dichloran, had little effect on tecnazene, an anomalous result compared with the one obtained with agar, and one that was confirmed in a second experi men t.
Botrytis cinerea.
o. F. Esuruoso et al.
409
A similar series in which pyridine-extracted agar was used gave similar results. Effect offungicides on growth ofgerm tubes The fungicides also altered growth of germ tubes. Some results for plain and glucose/casein hydrolysate agar are given in Table 3 where the figures relate to 24 h incubation unless stated otherwise. They are similar to those for a parallel series done with pyridine extracted agar. On plain agar, increasing concentrations of quintozene reduced linear growth; in the nutrient agar the germ tubes were also much more swollen and branched than in the controls. There was hardly any germination on plain agar containing over 5 ppm tecnazene. Germ tubes, when present, were very short. The effect of nutrient agar was to increase the swelling and branching of the germ tubes. Similar results were obtained with dichloran. In nutrient agar, there was local lysis of cell walls, followed by release of the cell contents as an amorphous mass. This is illustrated in PI. 25 and confirms an earlier observation by Sharples (1961). When spores were suspended in mixtures of the fungicides and o· 1 % solutions of substances shown in Table 2, it was found that quintozene up to 500 ppm did not alter growth of germ tubes. Germ tubes were shorter with increasing concentration of tecnazene and above 100 ppm rarely exceeded 10 times the spore length. At 250 ppm germ tubes were very swollen and proliferated. Dichloran also reduced germ-tube length and caused lysis of cell walls in glucose/casein hydrolysate solution. DISCUSSION
It is difficult to assess quantitatively the significance of the above results because the fungicides are insoluble in water. But it is clear that tecnazene, and to a lesser extent dichloran, which have little effect on spore germination in water, reduce it greatly when suspended in an agar gel, or in o· 1 % solutions ofagar, gelatine, carboxymethylcellulose and sodium polypectate. This means that agar and each of the higher molecular weight substances somehow alter the susceptibility of the spores to the fungicides or make the fungicides more readily available to the spore. Nutrients as a glucose/casein hydrolysate solution also increase the activity of dichloran but not of tecnazene, a result which needs further study. Another surprising but well substantiated finding is that none of the treatments effective with tecnazene or dichloran increase the activity of quintozene in spore germination tests. Quintozene did have some effect on growth of germ tubes in agar but not so striking as that produced by tecnazene which grossly distorted growth, or by dichloran which caused characteristic lysis of the cell walls. Whether our results can be obtained with other high molecular weight substances and nutrients remains to be established. Further work would seem to be justified if only because of the practical significance of any system that increases the activity of a fungicide towards a parasite.
410
Transactions British Mycological Society REFERENCES
CLARK, N. G. & HAMS, A. F. (1961). Antifungal activity of substituted nitroanilines and related compounds. ]. Sci. Fd Agric. I2, 751-757. ESURUOSO, O. F. (1964). Adaptation of Botrytis species to certain fungicides. Ph.D. Thesis University of London. HEWLETr, M. A. (1955). Studies on the fungicidal activity of certain chlorinated nitrobenzene compounds. Ph.D. Thesis, University of London. SHARPLES, R. O. (196 I). The fungitoxic effects of dichloran on Botrytiscinerea. Proc. British Insecticide and Fungicide Conference, 2, 327-336. WEBER, D.]. & OGAWA,]. M. (1963). Mode of action of 2,6-dichloro-4-nitroaniline. Phytopathology 53, 893· WEBER, D.]. & OGAWA, ]. M. (1965). The mode of action of 2,6-dichloro-4-nitroaniline on Rhizopus arrhizus. Phytopathology 55, 159-165. EXPLANATION OF PLATE
25
Botrytis cinerea Germination of conidia of Botrytis cinerea on glucose/casein hydrolysate agar containing 10 ppm DCNA. A, Normal cell; B, early stage of lysis of cell; C, later stage of lysis with cytoplasm discharged from cell.
(Accepted for publication 4 May 1967)
Trans. Br. mycol. Soc.
Vol. 51.
Plate 25
(Facing p. 410)
GERMINATION OF BOTRYTIS CINEREA CONIDIA IN THE PRESENCE OF QUINTOZENE, TECNAZENE AND DICHLORAN By O. F. ESURUOSO, T. V. PRICE AND R. K. S. WOOD Department of Botany and Plant Technology, Imperial College, London, S. W. 7 (With Plate 25) Various substances alter the activity of quintozene, tecnazene and dichloran towards Botrytis cinerea. The toxicity of tecnazene and dichloran is increased in agar gels, and in o· 1 % solutions ofagar, sodium carboxymethylcellulose, sodium polypectate and gelatine; there are no similar effects with quintozene. A glucose/casein hydrolysate solution enhances the activity of dichloran, but not that of the other two fungicides. Less striking, but still pronounced effects are caused by tecnazene and dichloran in the presence of these high molecular weight substances, and nutrients.
A characteristic of the fungicides quintozene, tecnazene and dichloran is their inactivity in the standard spore germination test in water. They may, however, cause the germ tubes of susceptible fungi to grow more slowly and abnormally. Thus, Hewlett (1955) found that quintozene and tecnazene vapours were more effective in nutrient than water agar in reducing growth of germ tubes and in causing abnormal hyphae to develop. In 1961, Clark & Hams reported thatdichloran, added to nutrient agar, retards growth of B. cinerea, and Sharples (1961) stated that B. cinerea responds differently to dichloran in agar and in liquid media. He also showed that dichloran causes hyphae to burst in media containing nutrients. More recently, Weber & Ogawa (1963, 1965) report that dichloran affects the protein metabolism of Rhi:::;opus arrhizus. The effects of agar and certain other substances in altering the activity of these three fungicides against B. cinerea became apparent during a study by Esuruoso (1964) of resistant strains. This work was repeated later and our results are summarized below. MATERIALS AND METHODS
The test fungus was a freely sporing isolate of Botrytis cinerea from a diseased lettuce plant. The fungicides were quintozene (pentachloronitrobenzene), tecnazene (2,3,5,6-tetrachloronitrobenzene) and dichloran (2,6-dichloro-4-nitroaniline). Each was recrystallized from acetone solutions of commercial products. Relatively stable suspensions were obtained by dissolving 0'3 g in 20 ml acetone containing 0'5 % Tween 80, a non-toxic wetting agent.
406
Transactions British Mycological Society
This solution was slowly added to 150 ml water with constant stirring and the fine suspension obtained was centrifuged, washed three times with water and finally suspended in 25 ml ofwater containing 0'05 % Tween 80. The final concentration was determined with 0'5 ml samples dried at 35 ° to constant weight. To prepare fungicide-impregnated agar, aqueous suspensions of fungicide were autoclaved at 15lb/inz (103'4 kN/mZ) for 5 min. and suitable volumes added to molten 1'5 % agar at 40° just before pouring plates. The following substrates were used for spore germination: (I) Plain agar (Davis): 1'5 % in water. (2) Plain agar: extracted twice for 2 h with five times its weight of pyridine with constant stirring, collected on a filter, extracted twice for 2 h in twelve times its weight of water with constant stirring, collected and dried at 70° for 24 h, then used at 1'5 % in water. (3) 0'1 % solutions in water of: agar; powdered gelatine (Oxoid); carboxymethylcellulose D.S. 0'7 Premium Low (Hercules Powder Company); sodium polypectate (California Fruit Growers Exchange). (4) Glucose/casein hydrolysate solution: glucose, 1'0 %; casein hydrolysate (Oxoid), 0'4 %; KHzP04, 0'1 %; MgS04.7HzO, 0'05 %, in water. (5) Glucose/casein hydrolysate agar: as 4 with 1'5 % agar. Spores were obtained from 7- to 14-day-old cultures on a mass inoculated glucose/casein hydrolysate agar by adding water containing 0'05 %Tween 80. The crude suspension was filtered through muslin and washed by centrifuging three times in water. Final suspensions were prepared in sterile water. Germination tests were done in 0'05 ml films ofliquid spread evenly over 12 mm coverslips attached to 2'5 x 7'5 ern glass slides by thin films of Vaseline, and incubated at c. 100 % R.H. at 25° for 24 h. They were then stained with cotton blue. The germination of 100 spores randomly distributed on each of three coverslips was recorded for each treatment; a spore was counted as germinated when the germ tube exceeded the spore length, Results are given as mean inhibition ofgermination after angular transformation ofpercentages. EXPERIMENTAL
Germination in aqueous suspensions of thefungicides The films on the coverslips contained 5 x 10 4spores/ml, and the fungicide at concentrations in the range 1-4000 ppm. Quintozene and tecnazene did not reduce germination even at 4000 ppm. Below 1000 ppm., over go % of spores were unaffected by dichloran. At higher concentrations 15-20 % of spores did not germinate. Germination on agar impregnated with fungicides Plain agar, pyridine-extracted plain agar, and glucose/casein hydrolysate agar were mixed with aqueous suspensions of the three fungicides to concentrations of 0, I, 2, 5, 25, 50, 100, 250 and 500 ppm before plates were poured. Acetone solutions of the fungicides (employed by earlier workers) were not used because we found that residual acetone retarded growth of the fungus.
Botrytis cinerea. O. F. Esuruoso et al, Table
I.
Effect of quintozene, tecnazene and dichloran in agar on germination of spores of Botrytis cinerea Inhibition of germination (transformed percentages) Fungicide (ppm)
Quintozene Tecnazene Dichloran
Agar
0
PA* PPA* GCA* PA PPA GCA PA PPA GCA
0 0
0 0 I
0 I
0 0
5 0 2 0 17 5 71 4 5 8
25
100
500
5 0 0
2 0 0 71 65 83 7 6 84
0
39 50 80 13 4 71
3 0
7473 79 32 25 83
Least difference significant at P = 0·05 5 3 13 8 6 8 14 16
* PA, plain agar; PPA, pyridine extracted agar; GCA, glucose/casein hydrolysate agar.
Table 2. Effect of quintozene, tecnazene and dichloran on germination of spores of Botrytis cinerea in solutions of different substances Substance in solution (0·1%) Quintozene
Agar Gelatine CMC* NaPP* GCA* Agar Gelatine CMC NaPP GCA Agar Gelatine CMC NaPP GCA
Inhibition of germination (transformed percentages) Fungicide (ppm) 0 5 II
2 0 0
5 8 10 16 5 0 15 13 14 0 2 26 17 25 6 3
25 8 15 12 4 0 22 17 13 19 0 42 21 40 9 49
100
500
8
4 0 0 0 0 85 78 90 74 13 37 37 53 39 64
II
8 0 0 23 28 29 34 4 41 26 42 21 64
Least difference significant at P = 0'05 7 6 6 5
10 4 10 6 0 6 0 7 6 Dichloran 5 6 7 6 4 6 3 0 5 • CMC, Sodium carboxymethy1cellulose; NaPP, sodium polypectate; GCA, glucose/casein hydrolysate. Tecnazene
7
II
Drops (0-05 ml) of spore suspension (5 x J04/ml) were put on the agar surface and incubated at 25°. Germination after 24 h is given in Table I. Quintozene at 500 ppm and lower concentrations did not affect spore germination. Tecnazene reduced germination by 50 % when suspended in plain agar at 25 ppm, and almost completely prevented germination at 500 ppm. In glucose/casein hydrolysate agar, 5 ppm fungicide was sufficient to reduce germination by 88 %. Surprisingly, therefore, extraction of agar with pyridine did not reduce activity of the fungicide. Dichloran
408
Transactions British Mycological Society
caused 28 %inhibition at 500 ppm in plain agar, and 92 % in nutrient agar at 25 ppm. These results showed that tecnazene and dichloran which are almost inactive in water become highly active when suspended in an agar gel and that this effect is enhanced by nutrients. In contrast, the activity of quintozene was not affected by ag ar or nutrients.
Germination in solutions of various substances In these experiments, spores and fungicides were suspended in solutions of the substances listed in T able 2 . Glucose/casein hydrolysate solution was used because of the striking results obtained with the corresponding agar. The pH was adjusted to 4'0 with H'Cl to prevent bacterial growth. Agar Table 3. Effect
of quintorene, tecnazene and dichloran on growth ofgerm tubes on agar Mean germ tube length (x spore length) Fungicide (ppm)
.
Agar Quintozene
PA* GCA*
Tecnazene
PA*
° 5° (16 h ) So (14 h) 50 (20h)
5
25
100
3°
25
20
5 00 IS
25
20
15
IS
3
3
3
Ab
Ab
Ab
10 IS 3° 10 Ab 3° • PA , Plain agar ; eCA, glucosefcasein hydrolysate agar. tAb, short, mu ch.branched tubes, abnormally swollen.
5 Ab
GCA
60
35
Ab']
(14 h ) Dichloran
PA* GCA
60
> 100
was used at o· I % to give a solution instead of a gel. Gelatine, carboxymethylcellulose and sodium polypectate were taken as examples of high molecular weight substances for comparison with agar. Drops (0'05 ml) of mixtures of spores, fungicide and solution were placed on 12 mm coverslips on glass slides and incubated at c. 100 % R.H. for 24 h at 250 • The fungicides were used at the same concentrations as in the experiments with agar and statistical analyses were based on the complete data from all tests. Selected but typical results are shown in Table 2. Spore germination in the presence of quintozene was not materially affected by any of the substances. Inhibition of germination by tecnazene was increased by agar, gelatine, sodium polypectate and carboxymethylcellulose to give ED 50 values between 100 and 500 ppm compared with no effect of 4000 ppm in water. Activity of dichloran was also increased by these substances but to a lesser extent. In contrast, glucose/casein hydrolysate solution, which markedly increased the activity of dichloran, had little effect on tecnazene, an anomalous result compared with the one obtained with agar, and one that was confirmed in a second experi men t.
Botrytis cinerea.
o. F. Esuruoso et al.
409
A similar series in which pyridine-extracted agar was used gave similar results. Effect offungicides on growth ofgerm tubes The fungicides also altered growth of germ tubes. Some results for plain and glucose/casein hydrolysate agar are given in Table 3 where the figures relate to 24 h incubation unless stated otherwise. They are similar to those for a parallel series done with pyridine extracted agar. On plain agar, increasing concentrations of quintozene reduced linear growth; in the nutrient agar the germ tubes were also much more swollen and branched than in the controls. There was hardly any germination on plain agar containing over 5 ppm tecnazene. Germ tubes, when present, were very short. The effect of nutrient agar was to increase the swelling and branching of the germ tubes. Similar results were obtained with dichloran. In nutrient agar, there was local lysis of cell walls, followed by release of the cell contents as an amorphous mass. This is illustrated in PI. 25 and confirms an earlier observation by Sharples (1961). When spores were suspended in mixtures of the fungicides and o· 1 % solutions of substances shown in Table 2, it was found that quintozene up to 500 ppm did not alter growth of germ tubes. Germ tubes were shorter with increasing concentration of tecnazene and above 100 ppm rarely exceeded 10 times the spore length. At 250 ppm germ tubes were very swollen and proliferated. Dichloran also reduced germ-tube length and caused lysis of cell walls in glucose/casein hydrolysate solution. DISCUSSION
It is difficult to assess quantitatively the significance of the above results because the fungicides are insoluble in water. But it is clear that tecnazene, and to a lesser extent dichloran, which have little effect on spore germination in water, reduce it greatly when suspended in an agar gel, or in o· 1 % solutions ofagar, gelatine, carboxymethylcellulose and sodium polypectate. This means that agar and each of the higher molecular weight substances somehow alter the susceptibility of the spores to the fungicides or make the fungicides more readily available to the spore. Nutrients as a glucose/casein hydrolysate solution also increase the activity of dichloran but not of tecnazene, a result which needs further study. Another surprising but well substantiated finding is that none of the treatments effective with tecnazene or dichloran increase the activity of quintozene in spore germination tests. Quintozene did have some effect on growth of germ tubes in agar but not so striking as that produced by tecnazene which grossly distorted growth, or by dichloran which caused characteristic lysis of the cell walls. Whether our results can be obtained with other high molecular weight substances and nutrients remains to be established. Further work would seem to be justified if only because of the practical significance of any system that increases the activity of a fungicide towards a parasite.
410
Transactions British Mycological Society REFERENCES
CLARK, N. G. & HAMS, A. F. (1961). Antifungal activity of substituted nitroanilines and related compounds. ]. Sci. Fd Agric. I2, 751-757. ESURUOSO, O. F. (1964). Adaptation of Botrytis species to certain fungicides. Ph.D. Thesis University of London. HEWLETr, M. A. (1955). Studies on the fungicidal activity of certain chlorinated nitrobenzene compounds. Ph.D. Thesis, University of London. SHARPLES, R. O. (196 I). The fungitoxic effects of dichloran on Botrytiscinerea. Proc. British Insecticide and Fungicide Conference, 2, 327-336. WEBER, D.]. & OGAWA,]. M. (1963). Mode of action of 2,6-dichloro-4-nitroaniline. Phytopathology 53, 893· WEBER, D.]. & OGAWA, ]. M. (1965). The mode of action of 2,6-dichloro-4-nitroaniline on Rhizopus arrhizus. Phytopathology 55, 159-165. EXPLANATION OF PLATE
25
Botrytis cinerea Germination of conidia of Botrytis cinerea on glucose/casein hydrolysate agar containing 10 ppm DCNA. A, Normal cell; B, early stage of lysis of cell; C, later stage of lysis with cytoplasm discharged from cell.
(Accepted for publication 4 May 1967)
Trans. Br. mycol. Soc.
Vol. 51.
Plate 25
(Facing p. 410)