germinability and consequently on fruit-set and yield were investigated mostly on pear, apple,
raspberries Redalen, 1980; Fell et al., 1983; Mar- cucci and Filiti, 1984, as fungicides are frequently
used in orchards during flowering time. The great diversity of published results is partly due to
non-comparable levels of the presented data in vitro, in vivo on stigmas, just after treatment or
ex post by means of fruit crop, respectively. Taking the case of fruit trees as a model, a direct
effect of fungicides on fruit crop was not detected.
The study of fungicides at cellular level showed the influence of triazoles on sterol metabolism due
to inhibition of ergosterol synthesis. The effect is non-specific, in that fungicides inhibit sterol syn-
thesis in host plant as well as in fungus hyphae Fuller et al., 1990; Tomlin, 1994. The accumula-
tion of steroid precursors lanosterol after propi- conazole
treatment led
to cholesterol
and phytosterol inhibition Vanden et al., 1987. Tria-
zole fungicides decreased the activity of peroxi- dase Lebedev et al., 1989, while carbendazim
inhibited aldehyde dehydrogenase Wiegand-Rosi- nus et al., 1988. The quantitative estimation of
the phytotoxic activity of pesticides carbendazim is based on specific enzyme inhibition in photo-
synthetic active leaf discs from cotyledons of Sinapis alba Petzold et al., 1988.
Little information is available as yet concerning the effect of field applications of fungicides on
pollen development,
subsequent germination,
andor leaf morphology in any species. He and Wetzstein 1994 showed for the first time that the
application of some early season fungicides caused degeneration of developing pollen and re-
tarded growth of catkins and leaves.
Many papers have focused on testing xenobi- otics for example fungicides for germination of
pollen grains He et al., 1995. Studying the in vitro germination of pollen treated with fungicides
showed usually a decrease in pollen grain germi- nation, deformation of pollen tubes and their
bursting, but also an increase in pollen germina- tion at very low concentrations of fungicides.
Authors used a great range of concentrations sometimes without relation to actual doses ap-
plied in field. Papers focused on pollen in vitro tests used frequently 1 mg of testing pollen in 1 ml
of medium during experiments Kappler and Kristen, 1987; Strube et al., 1991. Calculation of
doses for tests in vitro from doses using in tests in vivo used results from tests in vitro. Therefore the
aim of this work is to verify calculation of dose of fungicides for tests in vitro from doses using in
field conditions and toxicity of minimum tested doses of fungicides on limited quantity of tested
pollen. The evaluation of ecotoxicity of a given dose is possible when optimal conditions for pol-
len germination of donor plants are known Jan- durova´ and Pavlı´k, 1995. If the presence of
xenobiotic compound is the only stress factor in the experiment, then any significant difference
between control optimal conditions and treat- ment should be explained as a direct reaction to
the stress factor.
2. Material and methods
Description of the tested fungicides and their doses added in treatments are given in Table 1.
We used pollen from the rapid-cycling cultivar Toria turnip rape of Brassica campestris L.
subsp. oleiferae DC., genome descriptor
2
n is aa.o Williams and Hill, 1986. All plants were
cultivated under greenhouse conditions on soil without
chemical treatment.
Stamens from
opened flowers were hand collected and pollen grains were released finely from six anthers from
different flowers into the nutrient solution. The pollen was germinated in petri dishes diameter 40
mm in 1 ml of Roberts medium Roberts et al., 1983 at 20°C. There were 8 ml Roberts medium
without fungicides as control K and other treat- ments were prepared as mixtures of 8 ml of
Roberts medium with the appropriate volumes of fungicide. Doses of fungicides and their active
ingredients were added into the germination medium for every treatment A, B, C, D, E, F, G,
H and K. Treatments A and B were prepared from 8 or 2 ml of the stock solution Z Table 2.
The other treatments C, D, E, F, G, and H were prepared from treatment A by ten times dilution.
The range of concentrations of every tested fungi- cide was prepared according to the field — ap-
plied doses for one flower six anthers as shown in Table 2.
The change of pollen shape and tube emergence were microscopically observed magn. 700 × af-
ter 60 and 120 min of incubation under labora- tory conditions. Grains with a round shape with
the ratio of axes approaching one were scored as hydrated; grains with tubes longer than the di-
ameter of hydrated pollen were considered as germinated. The rest was aborted as non-hydrated
pollen grains. Pollen grains with bursting tubes were excluded from evaluation. Thousand 10 ×
100 pollen grains were evaluated in every treat- ment, with three replications per treatment.
Histochemical staining methods were used to detect activities of alkaline phosphatase APH,
peroxisomal catalase PER, succinate dehydroge- nase SDH and non-specific esterases EST, after
2 h and incubation of germinated pollen with investigated fungicides. Pollen was incubated
at treatments A, B and C by dropping 100 ml of pollen suspension into the slides’ chamber.
The germination medium with the different fungi- cides was carefully removed and replaced with
appropriate cultivation medium with specific sub- strate.
Alkaline phosphatase was detected by azocou- pling technique based on reaction between naph-
thol and diazonium salt Lojda et al., 1979. The specific substrate was naphthol AS BI phosphate,
and Tris buffer was used to keep the pH between 8.2 and 9.2. The same principle was used in
non-specific esterases where naphthol was liber- ated from a-naphthyl acetate and forms azodye
Table 1 Doses of active ingredients of fungicides and their characterization
Chemical formula Active
Commerc. name Classified
Contents CAS RN
ingredients 100 g l
− 1
Systemic Alto
Cyproconazole a
-4-chlorophenyl- 113096-99-4
a -1-cyclopropylethyl-
1H-1,2,4-triazol-1-ethanol Contact and
9 -a-2-fluorophenyl- Flutriafol
76674-21-0 125 g l
− 1
Impact 125 EC systemic
a -4-fluorophenyl-
1H-1,2,4-triazole-1-ethanol Systemic
Flusilazole 85509-19-9
Punch 40 EC 40
1-[[bis4-fluorophenylmethylsilyl] methyl]-1H-1,2,4-triazole
250 g l
− 1
60207-90-1 Systemic foliar
Propiconazole Tilt 250 EC
9 -1-[2-2,4-dichlorphenyl- 4-propyl-1,3-dioxolan-2-yl]methyl]-
1H-1,2,4-triazole Systemic
a
Carbendazim 10605-21-7
a
Tilt CB FW
b
25; 12.5 Methyl 1H-benzimidazol-2-
ylcarbamate
a
Doses of active ingredients Treatments
Propiconazole Flusilazole
Flutriafol Carbendazim;
Cyproconazole propiconazole
80 A mg ml
− 1
50 320
200 200; 100
B mg ml
− 1
20 12.5
80 50
50; 25 8
5 C mg ml
− 1
32 20
20; 10 D ng ml
− 1
800 500
3200 2000
2000; 1000 320
50 80
E ng ml
− 1
200; 100 200
8 F ng ml
− 1
20; 10 5
20 32
G pg ml
− 1
500 3200
800 2000
2000; 1000 320
50 H pg ml
− 1
200; 100 80
200
a
Only for carbendazim.
b
Tilt CB FW is a mixture of two active ingredients, carbendazim and propiconazole.
Table 2 Calculation of fungicides doses for in vitro testing theoretical maximum doses
Field dose per 1 flower nl Number of plants
Field dose Fungicides
Number of flowers Concentration
per m
2
per m
2
ml m
− 2
ml ml
− 1
Z
d
II.
b
I.
a
A
c
II.
b
B
c
I.
a
II.
b
I.
a
200 5
7 250
1050 800
200 100
Alto 100
5 7
250 Impact
1050 400
100 50
5 100
200 800
200 1050
Punch 250
7 200
5 7
250 Tilt 250
1050 800
200 100
200 Tilt
5 7
250 1050
800 200
100
a
Minimum number of plants flowers per m
2
for optimum yield.
b
Maximum number of plants flowers per m
2
for optimum yield.
c
A, B, Basic treatments of doses, which were taken from basic stock solution.
d
Z, ml of fungicides per 1 ml of basic stock solution.
with diazonium salt Davis and Ornstein, 1959. The reduction of different tetrazolium salts and
formasan formation is routinely used for dehydro- genase detection Stanley and Linskens, 1966. In
our experiments the substrate was sodium succinate and the hydrogen acceptor was nitro blue tetra-
zolium. Activities of peroxisomal catalases were detected with diaminobenzidine that produced
brown coloured product after oxidation Roels, 1976. The evaluation of enzyme activity was made
after appropriate incubation microscopically. Ten times 100 pollen grains were counted and percent-
age of stained grains represented the level of enzyme activity. No fixation step was needed as the
samples were observed immediately after the incu- bation period.
Programs Quattro Pro 3.01 for DOS, Statgraph- ics 4.0 for DOS and Delta Graph Professional 2.0
for Windows were used for calculation. It is not possible to include extreme measured values de-
pendent variable y
i
= 0 or y
i
= 1 for calculating the
regression equation that follows from single doses independent variable x
i
. The Y is the probability of pollen germination in the interval 0;1. Y = 1
corresponds to probability of pollen germination for control treatment. The X is the scale of logarith-
mic value of fungicide doses in the interval X ] 0. Units are determined as the minimum value of
dilution of fungicide, which has a negative effect on the probability of pollen germination, for which it
is valid, that log x
i
] 0 or x
i
] 1. Function I is given
as y = fx for X {0; x
min
}, for which it is valid, that Y = 1 if the germination of the pollen treated
by fungicide was identical with the control 100. Value x
min
is the dose of fungicide, which has no effect on pollen germination. It is not significantly
different from the control. Function II y = fx is by polynome nth of level of common of form:
fz = a +
a
1
z
1
+ a
2
z
2
+ … + a
n
z
n
, where fz = flog x, for X {x
min
; x
max
}, for which it was valid, that Y 1; 0.001. Because the range x
i
was over more than six orders, it was necessary to make a
logarithmic transformation of value x
i
to log x
i
. Value x
max
is the dose of fungicide, when pollen germination is 0.1. Function III is function y =
fx for X {x
max
; 100}, for which it valid, that Y = 0. Pollen treated by fungicides, does not germi-
nate. The calculation was performed by the t-test.
3. Results
