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 al., 1972).

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.

RESULTS

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.

Table 1:	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

Table 2:	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.

Fig. 1:	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)

DISCUSSION

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 1).

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.

ACKNOWLEDGMENT

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.