1. Isolation trials on the tissue culture Murashige and Skoog’s (MS-) medium
and other media resulted in different non-sporulated mycelial growths but all
except one isolate could be sporulated will on PDA medium. On PDA medium
Alternaria sp., Botrytis sp., Cladosporium sp., Epicoccum sp., Stemphylium sp.,
Tuberculina sp. could be identified.
2. However some isolated fungal growths were grown only on MS-medium
producing whitish colonies 25-40 mm in diameter with septate hyphae and
dicaryotic cells. Some dicaryotic enlarged, rounded cells like-spores were also
observed. The latter isolates formed like-rust fungus pustule on glass slides in
vitro and subjected to further studies. These fungal growths produced scarcely,
pale, very week and not sporulated colonies when inoculated onto honeybee
agar medium and produced brownish, superficially, profusely branched mycelial
growth with very dense sporulation on honeybee agar medium supplemented
with pollen grains. These characters were improved with increasing amount of
pollen grains up to 6.0 g/l. Enormous numbers of Uredospores and few of
teletospores like-structures were clearly seen on the latter medium. Based on
measurements and characters of uredospore and teletospore, the obtained
isolates could be identified as Uromyces fabae.
3. The basal MS-medium containing pollen grains alone or combined with
inisitol were better for Uromyces fabae linear growth. The media diluted to 1N,
½N or ¼N was best for growth of Uromyces fabae than the full strength of the
basal medium (2N). However, the highest rate of linear growth of Uromyces
English Summary
11 8
fabae was obtained by adding both pollen grains and myo-inositol to basal
medium diluted to ¼N. After 11 days of incubation, the lowest Uromyces fabae
linear growth was observed on the 2N strength basal medium supplemented with
myo-inisitol.
4. The addition of myo-inisitol alone to most of tested dilutions of the basal
medium decreased formation of Uromyces fabae-uredospores, while, basal
medium with pollen grains only or combined with myo-inisitol produced the
highest numbers of uredospores. The number of uredospores on medium with
1N strength was relatively larger than that produced on media diluted to ½N or
¼N strength. In general, the non diluted (2N) basal medium supplied with pollen
graines only, 1N supplied with both pollen grains and myo-inisitol followed by the
same dilution provided with pollen grains only were promising for formation of
Uromyces fabae-uredospores in axenic culture. In contrast, the lowest number
of Uromyces fabae-uredospores was produced on the non-diluted (2N) basal
medium supplemented with myo-inisitol only.
5. The wall thickness of Uromyces fabae- uredospore produced in vitro was
ranged between 2.15 and 2.88 μm. However the highest wall thickness was
associated with dilution’s of ½N and ¼N provided with both pollen grains and
myo- inositol compared with the non diluted medium (2N) provided with same
amendments. The length and width of these Uromyces fabae uredospore were
ranged between 20.56-23.23 μm and 19.14 - 21.20 μm, respectively. The higest
length and width were produced on the non diluted (2N) and ¼N strength of the
basal medium, respectively, while the lowest values for both mesurements were
associated with uredospores formed on ¼N strength of the basal medium
provided with both pollen grains and myo-inisitol. The average of width/length
ratio was ranged between 0.895 in case of ½N diluted medium provided with
myo-inisitol only to 0.994 in case of ¼N diluted medium supplemented with both
pollen grains and myo-inisitol.
English Summary
11 9
6. The obtained results proved that both Qalubia and Beheira isolates (i.e. A
and B, respectively) of U. fabae were significantly varied. Isolate A produced the
highest values of growth rate and number of uredospores. Removing stock
solution D (contained CaCl
2
.2H
2
O) from composition of the normal MS- medium
improved rate of growth and uredospores production in both isolates A and B
compared with the complete (control) medium. On the other hand, removing of
the stock solution E” (contained MnSO
4
.4H
2
O, ZnSO
4
.7H
2
O, and CuSO
4
.5H
2
O)
resulted in the highest reduction in both rate of growth and uredospores
production especially in isolate A while removing stock solutions E, G, H or
Inositol showed intermediate effects in this respect. It was interested to state that
the uredospores production was very tiny and was not affected by removing of
any stock solution if compared with the normal MS- medium in case of isolate B.
7. On MS-medium containing NH4NO3 at rate of 1.65 g/L , the linear growth
was significantly increased with increasing KNO3 from 1.9 g/L to 5.9 g/L and
myo-inisitol from 0.1 g/L to 0.2 g/L. However, MS-media contained NH4NO3 at
rate of 0.825 g/L ,in most cases, produced the highest linear growth particularly
when KNO3 was added at rate of 6.85 g/L. When myo-inisitol was added at rate
of 0.2 g/L to MS-media contained NH4NO3 and KNO3 at rates of 0.825 and 6.85
g/L, respectively both linear growth and number of uredospores produced were
significantly lower than the corresponding values in case of the same media with
0.1 g/L of myo-inisitol. From these results it could be concluded that the MS-7
medium (which contained 0.825; 6.85 and 0.1 g/L of NH4NO3; KNO3 and amyoinisitol,
respectively) was the best for linear growth and production of
uredospores of U. fabae.
8. The linear growth of isolate B was greatly enhanced on MS-7 medium
provided with broad bean leaf extracts compared with the same medium without
broad bean leaf extracts. As for isolate A the same trend was observed with 10
days-old culture only, while no variations were detected in both 15 and 21 daysEnglish
Summary
12 0
old cultures. The mycellial dry weight of both isolates A and B was increased
with aging and greatly stimulated by broad bean extracts added to MS-7
medium. Uredospores production was similarly affected, however, the highest
numbers of uredospores was produced by 21 days-old cultures of both isolates
particularly in MS-7 medium containing broad bean leaf extracts.
9. U. fabae-uredospores production was greatly enhanced on the modified
MS-7 medium supplemented with broad bean leaf extracts compared with MS-7
medium alone particularly in case of isolate B which produced few numbers of
uredospores when grown on the latter medium. Percentage of germination of
uredospores was inversed proportionally with spore concentration. It was
conspicuously increased as uredospores concentration was decreased and
higher in case of uredospores produced on MS-7 medium alone than those
produced on the same medium provided with broad bean leaf extract, however
the opposite behavior was noticed in case of isolate B. In most cases percentage
of uredospores germinated was time elapsed up to 24 hours from incubation.
10. Percentages of U. fabae-uredospores germination were sharply
decreased with increasing age of cultures. The highest % uredospores
germination was associated with 5 dyas old cultures then decreased with aging
of cultures up to 21 days particularly in case of uredospores produced from
cultures grown on MS-7 medium with broad bean leaf extract. At different ages
of cultures, % spore germination was higher in isolate B than isolate A.
11. The highest percentage of uredospores germination were obtained when
2% sucrose or glucose solutions were used as substrate for germination.
Germinability of uredospores of both isolates A and B was slightly decreased
with increasing concentration of both sucrose and glucose up to 6% particularly
in case of uredospores produced on MS-7 medium containing broad bean
extract. In contrast, percentage of germination of uredospores formed on MS-7
English Summary
12 1
medium without broad bean extract, was higher on 4 and 6% glucose solution
than the same concentrations of sucrose.
12. When distilled water was used as a substrate for uredospore germination
the resultant germ tubes as well as mycellial fragments changed to yeast like
sprouted mycelium. The cell of this mycelium were fragmented and large
numbers of one-celled structures like oidiospores were formed. When these
structures germinated in sucrose or glucose solution, germ-tube like
conidiophores wereproduced.
13. Both linear growth and uredospores formation were significantly affected
by kind of the tested carbon sources (xylose, arabinose, glucose, galactose,
fructose, maltose, lactose, sucrose, starch and mannitol and citric acid). Except
citric acid (not utilized) all other tested carbon sources were utilized better by
U.fabae isolate A than isolate B. In this regard the best linear growth and
uredospores production were obtained when sucrose and starch were used as
sole sources of carbon for U. fabae isolae A and B, respectively.
14. The linear growth of both isolate A and B of U. fabae was increased
significantly by increasing concentration of sucrose in MS-7 medium up to 30 g/l
then decreased by elevating concentration up to 60 g/l., however production of
uredospores in case of isolate A only was significantly affected. Adding sucrose
to MS-7 medium at rate of 30, 45 or 15 g/l resulted in the highest numbers of
uredospores /ml without significant differences in between, however uredospores
production in case of isolate A only was decreased significantly by increasing
sucrose up to 60 g/l. Uredospores production in case of isolate B was not
significantly affected by sucrose concentrations added to MS-7 medium.
15. The isolates A and B of U. fabae responded differently against tested
nitrogen sources (Sodium nitrate, Potassium nitrate, Ammonium nitrate, Sodium
nitrite, Ammonium sulfate, Urea, Aspargine , Gelatin, Yeast extract, Beef extract,
Casin and Peptone). The isolate A could utilize most tested nitrogen sources
English Summary
12 2
while isolate B could utilize asparagine, yeast and beef extracts only. The highest
linear growth and uredospores production of isolate A was obtained in control
MS-7 medium (contained potassium nitrate + ammonium nitrate) followed by
media contained beef extract, yeast extract, peptone, or asparagine as sole
source of nitrogen. However, the highest linear growth of isolate B was
produced on MS-7 media containing beef extract, yeast extract, or asparagine
but yeat extract was the best nitrogen source for its uredospores production.
16. The linear growth and sporulation of U. fabae isolate A was significantly
affected by wavelengths. Under controlled temperature (25 °C) the continues
hyaline light produced the highest linear growth followed by red, blue, yellow and
green light conditions. Meanwhile under normal daylight and room temperature
conditions the blue followed by red and hyaline wavelengths, in respective, gave
the best results. Darkness (black) in the first case and green light and yellow light
in the later case produced the lowest values of linear growth. As for uredospores
production, the red, blue and hyaline wavelengths under continues illumination
and blue wave length followed by darkness, red and hyaline light waves under
discontinues light (daylight) conditions produced the highest numbers of
uredospores
17. The temperature range required for linear growth and uredospores
formation was wider in isolate A of U. fabae (17-32 °C) than isolate B (17-23 °C).
However, the best linear growth of isolate A grown on MS-7 medium without.
The range of temperature regime required for uredospores production was wider
(17 – 30 °C) in case of isolate A than isolate B (17-25 °C). The highest number
of uredospores was produced at 25 and 23 °C for isolates A and B, respectively.
18. Growing colonies of U. fabae isolate A under controlled relative humidity
conditions (14-100% R.H.) resulted in significantly lower values of both linear
growth and uredospores production compared with colonies grown under
uncontrolled relative humidity conditions (Control). In case of isolate B the
English Summary
12 3
highest values of linear growth was obtained in control treatment followed by
treatments of 74 and 50% R.H.%, respectively, however treatments of 14 and
100% R.H. produced the lowest linear growth values of this isolate.
19. The best linear growth of isolates A and B of U, fabae was produced at
pH values of 8.6 and 9.0, respectively, however, linear growth of isolate B was
significantly decreased by pH values below 6.6 and approximately stopped at 4.6
and 4.0 pH values. As for uredospores production, similar trend was also
observed particularly in isolate A.
20. Both isolates A and B can hydrolize starch (Amylase test) and liquefy
gelatin (gelatinase test). Isolate A secret starch hydrolytic and gelatin liquefying
enzymes faster than isolate B. Amylase and gelatinase enzymes activities
expressed in term of percentage of halo-zone was slightly decreased with aging
of culture in case of isolate A, however the opposite trend was observed in case
of isolate B.
21. The activities of the induced pectulolytic (PG) and cellulolytic (Cx)
enzymes were higher in U. fabae isolate A than isolate B. Activities of these
enzymes were slightly increased by increasing pH values in substrate reaction
from 4.0 to 6.0 for PG enzymes and from 4.o to 6,6 for Cx enzymes in both
isolates A and B of U. fabae then decreased again by raising pH values above
these limits. From these results it could concluded that the pH values 6.0 and 6.6
were the best for highest activitiese of PG and Cx enzymes, respectively.
22. The activities of both PG and Cx enzymes were steadily progressed by
increasing time reaction from 5 to 240 minutes. The Cx enzyme activity was very
higher in isolate A after 5 minutes than isolate B.
23. Both constitutive and inductive PG and Cx enzymes were more active in
cultural filtrates of isolate A than isolate B. Activities of these enzymes were
steadily increased by increasing age of cultures. However, increasing age of
cultures from 14 to 21 days resulted in sharp increase in Cx enzymes activity
English Summary
12 4
from 26.1% to 75.6% expressed as loss in viscosity of substrate reaction. The
ratios between activity of inductive and constitutive PG enzymes of both isolates
A and B were narrow in filtrates of 14 and 21 days old cultures compared with
filtrates of 7 days old cultures. Regarding with ratios between activity of inductive
and constitutive Cx enzymes, the same trend was noticed also in case of isolate
A only but reversed manner was associated with isolate B.
24. The uredospores of both isolates A and B of U. fabae which produced in
axenic cultures were able to infect callus tissues, detached and intact leaves of
broad bean plants. In callus tissues, intercellular mycelium and oidiospores like
structures were clearly observed. The oidiospore like structures were also
observed in epidermal tissues of inoculated detached leaves. The typical rust
pustules were formed on leaves of inoculated broad bean plants which grown
under greenhouse conditions. |
