Alkaline Seed-Bed: An Innovative Technique for Manifesting Verticillium dahliae on Fennel Seeds
Khalid M. Ghoneem,
Wesam I.A. Saber
Mohamed A. Elwakil
Verticillium dahliae attacks a wide range of
plants including fennel causing a wilt disease. The fungus grows slowly
on seeds when tested at the seed health laboratories. This habit character
allows saprophytes to impair the fungal growth and interfere the identification
on both Moist Blotters (MB) and the Deep-Freezing Blotters (DFB). Since,
these two techniques are not efficient enough to detect this fungus, the
researchers planned to search for an alternative technique for detecting
this fungus. Soaking three layers of blotters used as seed-beds in water
solutions alkalined with KOH or NaOH at pH 10 presents the optimum seed-bed
condition for manifesting the fungus on seed. This seed-bed condition
also suppress the growth of saprophytes, so as the fungus was transparently
shown on seeds. The in vitro study presents pH 9.5 as the optimum
condition for the growth, sporulation and maximum glucose coefficient
of the fungus. So far, it is recommended to use the alkalined seed-bed
when searching for V. dahliae on fennel seed.
Verticillium wilt is a worldwide disease caused by the soil and seed-borne
fungi, Verticillium dahliae and Verticillium albo-atrum.
V. dahliae attacks seeds of more than 120 plant species including
parsley and fennel (Blum et al., 2006). The fungus survives in
forms of mycelium or conidia in or on seed`s surface (Xu, 2000; Huang
et al., 2004).
The Moist Blotter (MB) and Deep-Freezing Blotter (DFB) are recommended
techniques by the International Seed Testing Association (ISTA, 1999)
for detecting seed-borne fungi. MB technique develops saprophytes which
often seriously impaired with the growth of parasitic fungi. DFB technique
enhances the development of saprophytic bacteria and yeasts on seeds and
inhibits the spore-germination of some important seed-borne fungi as well.
Subsequently, it is difficult to isolate and identify the slow growing
seed-borne fungi including Acremonium sp. and Verticillium sp.
on seed when using such techniques. These fungi produce poor mycelium
and do not show their imperfect stage on seeds (Neergaard, 1979).
The research presents the role of seed-bed pH on manifesting the slow
growing seed-bone fungi on fennel seeds and establish an efficient technique
for transpiring V. dahliae when seed health test is carried out.
MATERIALS AND METHODS
Samples: Twenty fennel seed samples collected from various regions
of Egypt including Alexandria, Domitta, Gharbia, Dakhlia, Cario and Assuit,
were used in the present study.
Seed health testing: The conventional technique for the detection
of seed-borne fungi on fennel seeds was carried out following two techniques
recommended by the International Seed Testing Association (ISTA, 1999)
i.e., MB and DFB. The proposed method by the researchers was applied.
The percentages of the recovered fungi in each method were tabulated.
The alkalined proposed seed-bed: Three layers of blotter (filter
paper) were soaked in tap water at pH ranged from 5 to 11. Blotters moistened
in sterilized tap water were used as check treatment (MB and DFB). An
acidic water was prepared by adding drops of HCl to the tap water and
adjusted to give different acidic pHs whereas; the alkalined water was
prepared by using KOH or NaOH. The soaked blotters in a wide range of
pH (4.5-12.5) were placed in 9 cm diameter Petri-dish, where 25 seeds
were distributed on each seed-bed as described by ISTA (1999). The plates
were incubated at 20 ± 2 °C under cool white fluorescent lights
with alternating cycles of 12 h light and 12 h darkness.
Seven days latter, the incubated seeds were examined under a stereoscopic
microscope at 6-50X magnification to detect fungi on seeds and to study
their morphological characteristics. The compound microscope was used
to confirm the identification. Hyphal-tip from each fungus was transferred
onto Potato Dextrose Agar (PDA) plates using tip of heat-stretched capillary
tube. Pure cultures of the isolated fungi were obtained and all isolates
were maintained on slants of potato-carrot agar for further studies.
The fungal isolates were identified in consultation with the Commonwealth
Mycological Institute description sheets, Danish Government Institute
of Seed Pathology publications, Raper and Fennel (1965), Ellis (1971),
Chidambaram et al. (1973), Domsch et al. (1980), Booth (1985),
Burrges et al. (1988) and Moubasher (1993).
In vitro growth, sporulation and glucose utilization of V.
dahliae at different pHs: Verticillium dahliae was grown
on plates of Czapek`s Agar medium for 10 days at 25 °C in dark. The
fungal growth was scraped gently from the medium surface by using a glass
rod and suspended in sterile distilled water. The collected spores was
regulated to about 4x106 spores mL-1, while 0.5
mL adjusted to inoculate 50 mL of Drews liquid medium (Drews, 1983) in
250 mL Erlenmeyer flasks. The growth media presented different pH values
ranged from 4.5 to 12.5. The cultures were incubated at 20 ± 2
°C for 14 days under cool white fluorescent light with alternating
cycles of 12 h light and 12 h darkness.
The final culture pH of V. dahliae was measured at the end of
incubation period. Spore formation per 1 mL of culture was determined
using hemocytometer. Cultures were then, filtered though filter paper
(Whatman No. 1) and dried at 80 °C till constant weight.
Glucose residue in the cultural supernatant was estimated at the end
of the incubation period following o-toluidine method (Sasaki et
Statistical analysis: The statistical analysis software CoStat
6.311 was used to compare means using Duncan`s multiple range test, as
well as to estimate the correlation coefficient (r), at p ≤ 0.05.
Optimum seed-bed pH condition for manifesting V. dahliae on
fennel seeds: A wide range of pH was used for soaking the blotters
to detect the slow growing fungus; V. dahliae on fennel seeds.
Among the wide range of blotters pH, the alkalined seed-bed condition
was suitable for detecting V. dahliae in compare with MB and DFB
(Table 1). These condition also suppress the growth
of the saprophytic fungi. Blotters of pH 10 presented the optimum condition
for manifesting V. dahliae abundantly on fennel seeds. This alkaline
seed-bed at pH 10 condition was studied in details.
|| Effect of blotter pH in detection of lurked seedborne
fungi of fennel
|*Mean of fungal presence in the tested samples. Three
samples each of 400 seeds were investigated. **The pH of standard
methods is approximately 7. Values within a raw followed by the same
letter(s) are not significantly differed at p ≤ 0.05
|| Comparison of MB and DFB and alkaline blotters (pH
10) for manifesting fungi on fennel seeds
|*Percentage of positive samples for the specific fungus.
Twenty samples, each of 400 seeds, were tested for fungal assay. **Mean
within the positive samples containing the specific fungus. Values
of means within a row followed by the same letter(s) are not significantly
different (p ≤ 0.05)
Significant differences in detecting seed-born fungi of fennel among
MB, DFB and the proposed alkalined seed-bed. A total of 30 species belongs
to 20 genera of fungi were isolated from fennel seeds by using the above
mentioned techniques (Table 2).
MB enhanced the recovery of the fast growing saprophytes i.e., Rhizopus
sp. and Nakataea sp. as well as the pathogenic Fusarium equiseti.
On the other hand, DFB showed a significant reduction in the presence
of most saprophytes including Aspergillus sp., Chaetomium
sp.; the commonly growing fungi on the non-germinated seeds.
The Alkalined Blotters (AB) technique used in this investigation enhanced
the recovery of the slow growing fungi and proved to be a sensitive method
in manifesting the slow growing seedborne fungi including Acremonium
sp. and V. dahliae. It also, increased the detection of Alternaria
radicina and F. verticillioides on seeds. Sporulation of
V. dahliae was also increased.
Optimum culture pH for growth, sporulation and glucose utilization
of V. dahliae: V. dahliae was able to grow in a wide
range of pH on drews liquid medium (4.5-12.5) during the tested intervals
(6, 10 and 14 day) (Fig. 1). The cultural pH of 9.5
enhanced the fungal growth while it reached 7.59 g L-1 after
14 days of incubation at 20 ± 2 °C. In all treatments the final
culture pH was reduced to the direction of acidic side. After 14 days
of incubation, the final culture pH was kept within a narrow pH range
(4.5-5.5). The sporulation gradually increased by increasing the initial
pH and decreased at the lower pHs. When the fungus was grown at pH 9.5,
it recorded the highest glucose coefficient. At the end of incubation
period, the statistical analysis reveals a significant positive correlation
between the glucose coefficient and both mycylial dry weight (r = 0.908,
p ≤ 0.01) and sporulation (r = 0.938, p ≤ 0.01). The increment in
growth and sporulation of V. dahliae was directly related to the
utilized glucose and glucose coefficient.
||Effect of initial culture pH on (a) final culture pH,
(b) mycelial dry weight during various growth periods of V. dahlia,
(c) sporulation and (d) glucose utilization and coefficient at the
end of incubation period (14 days). *Initial glucose in the medium
was 20 g L-1, **Glucose coefficient = Mycelial dry weight
(g L-1)/utilized glucose (g L-1)
Fennel seeds used in preparing medicine should be free from toxins which
produced by several seed-borne fungi including V. dahlia. The fungus
produces protein-lipopolysaccharide complexes toxins in nutrient-limited
culture filtrates (Meyer and Dubery, 1993). These toxins are reported
also to be a cause of wilt and dehydration symptoms on many plants (Pegg
and Brady, 2002; Wang et al., 2004).
The methods used for seed health testing may be suitable for detecting
particular fungus at high percentage; other(s) may be good for detection
other pathogens. The recommended seed-health techniques by ISTA (1999)
i.e., MB and DFB are not-efficient in detecting the slow growing fungi.
They allow the saprophytes to impair the identification of pathogenic
fungi and the annalists find difficulties in manifesting the pathogens
on seeds under the stereoscopic microscope. This research recorded an
innovative technique for manifesting V. dahliae, as it is classified
as one of the slow growing fungi on fennel seeds.
The results presented here proved that the alkalined seed-bed condition
is optimum for detecting the slow growing fungi from fennel seeds including
V. dahliae. Soaking blotters in alkalined tap water at pH 10 is
recommended for the detection of V. dahliae (Table
Moistened blotters in alkaline solution of pH 10 show a significant reduction
in the saprophyte growth (Table 2). The presence of
saprophytes on seed compels the analyst to use high magnification of the
stereoscopic microscope (X50) which is distressful to eyes. The lower
magnifications (X6 and 10) are not suitable for the detection of slow
growing pathogens which are always covered by the saprophytes.
These results were confirmed with that shown in Fig. 1.
Although most fungi grow in initial acidic conditions, V. dahliae was
able to grow abundantly media having initial pH 9.5. However, when the
final culture pH was measured, 6, 10 and 14 days after incubation, it
turned to be acidic. The acidic final pH of the medium may be the reason
of the abundant growth of V. dahliae on alkaline pH. It seems likely
that the physiological role of this process is to bring the pH of the
medium to a range favorable for the fungal growth. The glucose coefficient
reached its maximum at pH 9.5 and that reflect the ability of such fungi
to survive in alkaline conditions. Abo-Ellil (1999a, b) found a positive
relationship between Na+ ion in the medium and the production
of α-amylase in Verticillium lateritium and the uptake of
sugars in the fungal cell was accomplished with the increase of alkalinity
of the medium.
Although, the proposed alkalined technique proved to be more sensitive
in up growing the slow growing seed-borne fungus V. dahlia, it
also, efficient in detecting the pathogenic fungi i.e., Alternaria
radicina and F. verticillioides (Table 2).
The presence of the alkaline ions (K+ or Na+) replace
H+ in the fungal cell (El-Wakil and Ghoneem, 2002). They increase
the uptake of nutrients in the cells of some Bacillus strains (Horikoshi
and Akiba, 1982). Another explanation is referred only to the pH of the
growth media, which may be suitable to the growth and sporulation of such
seed-borne fungi (El-Wakil et al., 2007).
The over all benefit of the results in this research is recording an
innovated technique to help Seed Health Analysts at seed-health laboratories
to manifest V. dahliae and other slow growing fungi on fennel seeds.
More research on the habit character of other slow growing fungi on different
seeds under different pH conditions is needed.
The authors thank Dr. Conard J. Krass, Primary State Plant Pathologist,
California. Department of Food and Agriculture, Sacramento, CA, USA (retired)
for critical review of the study.
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