Abstract: An improved method of seed health testing was developed to detect lurked pathogens on fenugreek which coexist in low percentage. Blotting papers were soaked in solutions of NaOH and KOH 0.3 and 0.2 M, respectively instead of tap water which is used in standard blotter and deep freezing method. Thirty two samples of fenugreek seed collected from Egyptian commercial market were examined. Using 400 seeds of each sample, 25 seeds were distributed on 3 layers of blotting paper soaked in 0.3 M NaOH or 0.2 M KOH in 9 cm diameter sterilized Petri dishes. A comparable set was kept in which sterile water was used as control. The dishes were incubated at 20±2 for 7 days under cool white fluorescent light with alternating cycles of 12 h light and 12 h darkness as recommended by ISTA. KOH and NaOH treatments respectively showed the presence of 6.5 and 7.5% of Verticillium dahliae as compared to 0.3% in the standard blotter and 1% in the deep-freezing method. KOH treatment also showed the presence of Fusarium moniliforme and F. solani which were detected at 5.4 and 0.7% compared to 0.5 and 0.2% in the standard blotter method and 2.9, 0.4%, in the deep freezing method, respectively. Sodium hydroxide stimulated the growth of F. semitectum and Curvularia sp., which were detected at 1.4 and 0.7%, respectively as compared to SBM and DFB which showed the presence of 0.3 and 0% in case of SBM and 0.5 and 0.3% in case of DFB. No difference was observed in different treatments for detecting Cercospora traversiana.
Introduction
Fenugreek (Trigonella foenum-graecum Linn.) is an indigenous annual herb of southern Mediterranean shores of Egypt and Morocco. It is widely cultivated in India (Wallis, 1967) and has an economic nutritive value to human as well as to livestock as cattle-fodder for increasing breast milk (Tomar et al., 1996). It is used in China as a medicinal treatment of peptic ulcers (Al-Meshal et al., 1985). Moreover, its alkaloid contents of trigonelline, cholesterin, lecithin and choline are known as tonic and digestive substances (Wallis, 1967; Pandey, 1996).
Several reports of seed-borne mycoflora attacking fenugreek-seeds have been given (Richardson, 1990). Cercospora traversiana is considered the major seed-borne pathogen of fenugreek. (Leppik, 1960; Nagy et al., 1972; Zimmer, 1984; Ryley, 1989; Bobev et al., 1999). It affects leaves, stems, young-buds causing blights and damping off. Hashmi (1988) studied the seed-borne mycoflora in fenugreek samples collected from 7 different countries by using standard moist blotter method. Alternaria alternata and Fusarium moniliforme were found preponderant with low occurrence of Botrytis cinerea, Curvularia inaequalis, C. lunata, Drechslera tetramera, Epicoccum purpurascens, F. oxysporum, F. semitectum, F. solani, Phoma sp. Stemphylium botryosum, Ulocladium sp. and Verticllium albo-atrum. He also reported that F. moniliforme, F. oxysporum and F. solani causes seed rot and wilting in tested seedlings. Sinha and Prasad (1989) using standard blotter and agar plate methods recorded a different number of seed mycoflora on fenugreek seeds viz., Aspergillus niger, A. flavus, A. tamarii, Rhizopus arrhizus, F. equiseti, Macrophomina phaseolina, Alternaria alternata and Curvularia lunata. Mushtaq et al. ( 1996) reported some new seed-borne mycoflora of fenugreek seeds using standard blotter and deep freezing methods viz., Aspergillus candidus, A. fumigatus, A. parasiticus, A. versicolor, Cephalosporium acremonium, Chaetomium bostrychodes, C. globosum, Cladosporium sp., Fusarium proliferatum, Paecilomyces sp. and Scopulariopsis sp. Of the standard moist blotter (SBM) and deep-freezing blotter (DFB) as recommended by the International Seed Testing Association (ISTA) for detecting the seed-borne fungi, the SBM develops saprophytes which often seriously impaired recording the growth of parasitic fungi while deep-freezing blotter enhances the growth and development of saprophytic bacteria and yeasts on seeds (Neergaard, 1979) and also inhibit the spore-germination of some important seed-borne fungi. For the above reasons it is difficult to isolate and identify some important seed-borne pathogens from seed. Some of these important pathogens have slow growth rate during cultivation on blotter e.g., Cephalosporium and Verticillium produce poor mycelial growth and have never been observed in their imperfect state on seeds (Neergaard, 1979). In order to detect the slow growing seed-borne fungi, experiments were carried out to evaluate the ability of alkaline treatments on the recovery of seed-borne fungi of fenugreek.
Materials and Methods
Source of seed samples: Thirty-two seed samples of fenugreek collected from commercial markets in different regions of Egypt were used in the present study.
Seed health testing (SHT): Detection of seed-borne mycoflora was carried out following the rules stated by International Seed Testing Association (ISTA, 1996). Four hundred seeds of each sample were tested using the standard blotter, deep freezing methods, as well as the alkaline treatment method as proposed in the present study.
Standard blotter method (SBM): Twenty-five seeds were plated in a 9-cm diameter Pertri dish containing three layers of moist blotter paper using sterilized tap water (pH 7.2). The plates were incubated at 20±2°C for 7 days under cool white fluorescent light with alternating cycles of 12 hours light and 12 hours darkness.
Deep freezing blotter method (DFB): After plating seeds as described in the blotter method, the dishes were incubated at 20°C for 24 hours and then transferred to a –20°C freezer for 24 hours followed by 5 days incubation at 20±2°C under cool white fluorescent light with alternating cycles of 12 hours light and 12 hours darkness.
Alkali treatments: A simple modification in standard blotter method was applied by soaking the 3 layers of blotters in alkaline solutions viz., 0.2 M potassium hydroxide (KOH) or 0.3 M sodium hydroxide (NaOH) and then placed in the Petri-dish where 25 seeds were distributed according to the described system of ISTA. Both conc. of the alkaline solution showed the maximum value on the pH meter scale (pH 14). The plates were incubated at 20±2°C for 7 days under cool white fluorescent lights with alternating cycles of 12 hours light and 12 hours darkness.
The incubated seeds were examined after 7 days under stereoscopic microscope (6-50X magnification) for the presence of the seed-borne fungi and their morphological characteristics. Whenever necessary, a compound microscope was used to confirm the identification by examining the morphology of conidia and conidiophores. The seed-borne fungi were identified using the Commonwealth Mycological Institute, Kew, Surrey, England (CMI) description sheets, Danish Government Institute of Seed Pathology (DGISP) publications, and after reference to Booth (1985), Burrges et al. (1988), Chidambaram et al. (1973), Ellis (1971), Raper and Fennel (1965), Singh et al. (1991), Domsch et al. (1980) and Moubasher (1993).
By the aid of stereoscopic microscope, the hyphal-tips of the fungi were transferred on potato dextrose agar (PDA) using tips of heat-stretched capillary tubes. Pure cultures of the fungi were obtained and all isolates were maintained on potato-carrot agar (PCA) slants for further studies.
Data was analyzed with the statistical analysis system (SAS Institute, 1988). All multiple comparisons were first subjected to analysis of variance (ANOVA). Comparisons among means were made using least significant differences (LSD) at p=0.05 and Duncan’s multiple range test.
Results
Thirty five species of fungi belonging to 22 genera were identified from fenugreek seeds (Table 1). Alternaria alternata, Aspergillus flavus, A. niger, Cladosporium sp. and Penicillium sp., were found to be preponderant. A significant difference was observed between standard moist blotter (SBM), deep-freezing blotter (DFB) and alkaline soaking method (0.2 M KOH or 0.3 M NaOH). A higher number of fungi were recovered with deep freezing method (DFB) than with blotter method (BM) or the two alkaline treatments.
Standard moist blotter method (SBM) significantly enhanced the saprophytes viz., A. niger (35.2%) and Rhizopus sp., (4.7%) while DFB method enhanced Aternaria alternata (10.3%), Aspergillus carnus (1.2%), A. clavatus (1.5%), A. nidulans (6.9%), Cladosporium, (18%), Drechslera tetramera (1%), Mucor (1.2%), Myrothecium sp. (1.3%), Nigrospora sp. (1%) and Penicillium sp. (31.5%).
Sodium hydroxide (NaOH) and potassium hydroxide (KOH) treatments significantly showed their potential to enhance the recovery of some important seed-borne fungi as compared to SBM or DFM. Blotters soaked in (0.2 M) KOH enhanced the recovery of Fusarium moniliforme (5.4%), F. solani (0.7%) as well as Epicoccum spp. (0.5%) as compared to 0.5 , 0.2 and 0.3% in the standard method and 2.9, 0.4 and 0.3%, in the deep freezing method respectively. On the other hand, 0.3 M NaOH treatment showed the recovery of both F. semitectum (1.4%) and Curvularia sp. (0.7%) as compared to 0.3 and 0.0% in the SBM and 0.5, 0.3%, respectively in DFB methods.
Percentage of recovery of Verticillium dahliae associated with fenugreek seeds was 6.5% in KOH and 7.5% NaOH as compared to 0.3% in the SBM and 1% in the DFB. The method proved to be more sensitive as being capable of revealing even minor infections. The sporulation was heavy, particularly in Fusarium moniliforme and Verticillium dahliae.
| Table 1: | Efficiency of three detection methods for detecting lurked seed-borne mycoflora of fenugreek seeds |
| Thirty-two samples were tested for fungal assay by 3 different methods. Values between brackets represent the range of infection percentage from lower (L) to maximum (M). Statistical analysis for data is represented by means of 32 replicates of samples. Values of means within a row followed by the same letter(s) are not significantly different (P=0.05) according to Duncan’s multiple range test. | |
Both alkali-methods and DFB were equally effective in detecting the seed-borne Cephalosporium, F. oxysporum, Stachybotrytis and Trichothecium spp. (Table 1). On the other hand, KOH and DFB-methods showed an equal sensitivity in detection of F. equiseti, Drechslera spp. and A. versicolor. No difference between SBM, DFB and KOH was observed in the detection of Aspergillus flavus. All the treatments showed no differences in the recovery of Aspergillus ochraceus, A. tamarii, Cercospora traversiana and Stemphylium sp. In the meanwhile, the presence of saprophytes in most samples impaired the identification of important seed-borne fungi. Their incidences were significantly lower when using the alkaline treatments, especially in detection of Rhizopus sp. Since the presence of Phoma sp., Rhizoctonia solani, Sclerotinia scleotiorum and Trichoderma sp., in the tested samples were extremely low, they were neglected in this investigation.
Discussion
A significant difference between the standard blotter, deep freezing and the alkaline treatment methods was obsreved. Of these KOH showed a potential to enhance the growth of F. moniliforme, F. solani and Epicoccum sp. whereas NaOH showed an enhanced growth of F. semitectum and Curvularia sp. At the same time, the treatments provide an effective control of the saprophytes, which impair the detection of the colonized pathogenic fungi in seeds.
It is interesting to note that the presence of saprophytes during the course of seed examination compel the analyst to use high magnification of the stereoscopic microscope (X 50) which is distressful to the eyes. The lower magnifications are not befitting to the detection of slow growing pathogens, which always are covered by the saprophytes. Alakline treatment could therefore help avoiding such complications.
The effect of alkaline media in the recovery of lurked seed-borne fungi may probably be due to the effect of the alkaline ions (K+ or Na+) which replace H+ in the fungal cell. This explanation is in support with the finding of Horikoshi and Akiba (1982) who indicated that Na+ increase the uptake of nutrients in the cells of some Bacillus strains. Abo-Ellil (1999a-b) found a positive relation between Na+ ion in the medium and the production of α-amalyse in Verticillium lateritium and that the uptake of sugars in the fungal cell was accomplished with the increase of alkalinty of the medium. Since alkaline chemicals showed there competence in coming out with the lurked Verticillium dahliae, the results presented there suggested that the alkaline seed-bed technique is a sensitive method for detecting such fungi in fenugreek.
Acknowledgment
We thank Prof Dr. Abdul Ghaffar, Professor (Retd.) Department of Botany, University of Karachi, Karachi-75270, Pakistan as well as Dr. Conrad J. Krass, Primary State Plant Pathologist, California Department of Food and Agriculture, Sacramento, CA. USA, for helpful insights and critical reviews of the manuscript.