Abstract: A method suitable for detecting the slow growing seed-borne fungi of peanut was developed. Twenty-five seed samples collected from commercial markets in Egypt were used in this investigation. With the Standard Moistened Blotter Method (SBM) and Deep-Freezing Blotter method (DFB) recommended by the International Seed Testing Association, saprophytes developed quickly and often impaired the detection of parasitic fungi and inhibited the germination of some important seed-borne fungi. Moistening the blotter disks used for seed germination with an alkaline solution at pH 12.5 using NaOH (0.8%) or KOH (0.4%) enhanced the growth and recovery of the slow growing seed-borne pathogens Cephalosporium sp. and Verticillium sp. The treatments also were effective in suppressing the growth of saprophytes which impair the detection of pathogenic fungi on seed. We recommend using the alkaline blotter method for seed health testing when searching for slow growing seed-borne fungi.
INTRODUCTION
Seeds, pods and seedlings of peanut are susceptible to the attack of several soil-borne fungi, including Rhizoctonia sp., Fusarium sp., Pythium sp., Rhizopus sp., Penicillium sp., Aspergillus sp., Trichothecium sp., Macrophomina phaseolina, Alternaria sp., Botrytis cinerea, Helminthosporium sp., Mucor sp., Curvularia sp., Cladosporium sp., Botryodiplodia theobromae, Chaetomium sp. (Porter et al., 1990; Richardson, 1990; Baird et al., 1993a, b; Dharmaputra and Retnowati, 1996; Shim et al., 1996), Sclerotium rolfsii and S. bataticola (Porter et al., 1990; Hollowell et al., 1998). Verticillium sp., the cause of floury rot disease, was isolated from peanut pods at a later stage of maturity (Mathur et al., 1975; Melouk et al., 1983). Rhizoctonia solani, the major causal organism of seedling damping off and Fusarium sp. were isolated from seeds.
An improved method of seed health testing was developed by Elwakil and Ghoneem (2002) to detect lurked pathogens on fenugreek, which coexist at a low percentage. Blotting papers were soaked in solutions of NaOH and KOH, 0.3 and 0.2 Mole, respectively, instead of tap water, which is used in the standard blotter and deep-freezing methods. KOH and NaOH treatments detected Verticillium dahliae at 6.5 and 7.5%, respectively, compared to 0.3% in the standard blotter and 1% in the deep-freezing method. KOH treatment also detected Fusarium moniliforme and F. solani at 5.4 and 0.7%, respectively, compared to 0.5 and 0.2% in the standard blotter method and 2.9 and 0.4%, in the deep-freezing method. They also found that sodium hydroxide stimulated the growth of F. semitectum and Curvularia sp. Since little information about methods for detecting the slow-growing seed-borne fungi of peanut is available, the present research was planned to create a seed health testing method to detect the slow growing seed-borne fungi of peanut and suppress the growth of saprophytes which impair the microscopic examination of seeds.
MATERIALS AND METHODS
Twenty-five seed samples of mixed peanut cultivars (Giza 4 and 5) representing peanut producing areas in Egypt were used in this study.
The conventional technique for the detection of seed-borne mycoflora was carried out following the procedures published by ISTA (1996). Two hundred seeds of each sample were tested using the standard blotter and deep freezing methods.
The innovative method (Alkaline blotter method): Several trials were carried out by soaking blotters in one of two alkaline solutions, potassium hydroxide solution (KOH) or sodium hydroxide solution (NaOH). The blotters were placed in a Petri-dish, where 10 seeds were distributed. NaOH (0.4, 0.8 and 1%) and KOH (0.2, 0.4 and 0.8%) were used. The plates were incubated at 20±2°C for 7 days under cool white fluorescent light with alternating cycles of 12 h light and 12 h darkness. Seeds were then ready for examination under a stereoscopic binocular microscope (6-50 X) for the presence of seed-borne fungi and to study their growth characteristics. When necessary, a compound microscope was used for confirming the identifications after having examined the morphology of conidia and conidiophores. The fungi were micro-photographed.
Fungi present on seeds were identified by utilizing the description sheets of the Commonwealth Mycological Institute (CMI) Kew, Surrey, England, Danish Government Institute of Seed Pathology (DGISP) publications and publications of (Raper and Fennel, 1965; Ellis, 1971; Chidambaram et al., 1973; Moubasher et al., 1977; Booth, 1985; Burrges et al., 1988; Singh et al., 1991).
Measuring the linear growth and determining fungal sporulation: The following pathogenic fungi were investigated; Cephalosporium sp., Fusarium verticillioides, F. oxysporum, F. solani and Verticillium sp. NaOH or KOH were dissolved in 10 mL distilled water. The solutions were added to Czapek’s agar medium in 9 cm Petri-dishes. The final concentrations of NaOH and KOH in the medium reached 0.8 and 0.4%, respectively, while the pH was 12.5 in both cases.
A mycelial disk (0.4 cm diameter) was cut from 7 day-old cultures grown on PDA and placed in the center of the agar plate. Three replicates were used per treatment. All cultures were incubated at 25±2°C for 7 days in the dark. The linear growth and number of spores produced by each fungus was recorded 7 days after incubation. The means of three replicates were calculated.
To quantify spore production, the fungus was gently scraped from the Petri-dish with a sharp spatula and washed several times with a total volume of 100 mL of sterilized water. The fungal suspension was filtered through a plastic sieve to separate the spores from the mycelium. The number of spores mL-1 was determined using a haemocytometer slide.
RESULTS
Detection of the slow growing seed-borne fungi
Alkaline blotter method: The blotters soaked in a solution of NaOH
(0.4, 0.8 and 1%) or KOH (0.2, 0.4 and 0.8%) instead of water showed a decrease
in the growth of fast growing saprophytes, viz: A. flavus, A. niger,
Rhizopus sp. and Penicillium sp. and an increase in the growth
of the slow growing seed-borne fungi, viz; F. solani (15, 30 and 10%
in NaOH and 11.7, 25 and 20% in KOH). F. oxysporum (13.3, 21.7 and 10%
in NaOH and 10, 15 and 11.7% in KOH) and Cephalosporium sp. (3.3, 11.7
and 3.3% in NaOH and 3.3, 6.7 and 0% in KOH). F. verticillioides was
detected at a rate of 3.3, 10 and 6.7% in NaOH and 3.3, 3.3 and 0% in KOH. Verticillium
sp. was detected at a rate of 6.7, 8.3 and 0% in NaOH and 0, 6.7 and 0%
in KOH as shown in (Table 1).
| Table 1: | Incidence of seed-borne fungi of peanut using the innovative method of NaOH or KOH solutions |
| *: The method represents 25 seed samples of peanut; Values having different letter(s) are significantly different at p<0.05 | |
| Table 2: | Incidence of seed-borne fungi of peanut using the innovative method of NaOH or KOH solutions |
| *: The method represents 25 seed samples of peanut; Values having different letter(s) are significantly different at p<0.05 | |
| Table 3: | Effect of alkaline treatments on the linear growth and sporulation of five fungi isolated from peanut seeds 7 days after incubation under 25±2°C |
| Diameter of the colony (cm) grown on Czapek's`gar supplemented with the test compound. Number of spores x106 mL-1. Statistical analysis for data are the means of 3 replicates. Values of means within a column followed by the same letter(s) are not significantly different (p = 0.05) according to Duncan (1995) multiple range test | |
Alkaline treatment followed by deep-freezing treatment: In this method, the application was carried out as applied in the standard Deep-Freezing Method (DFM) but the blotters were soaked in solutions of NaOH (0.4, 0.8 and 1%) or KOH (0.2, 0.4 and 0.8%) instead of water. Results showed a decrease in the growth of fast growing saprophytes, including A. flavus, A. niger, Rhizopus sp. and Penicillium sp. and an increase in the slow growing seed-borne ones. F. solani was detected at rates of 20, 50 and 28.3% in NaOH, while 23.3, 40 and 15%, respectively, in KOH. F. oxysporum was detected at 25, 33.3 and 18.3% in NaOH and 20, 26.7 and 11.7%, respectively, in KOH. F. verticillioides was detected at 6.7, 13.3 and 10% in NaOH and 10, 13.3 and 10%, respectively, in KOH. Cephalosporium sp. was detected at 6.7, 16.7 and 1.7% in NaOH and 5, 8.3 and 3.3%, respectively, in KOH. Verticillium sp. was detected at 5, 10 and 3.3% in NaOH and 3.3, 10 and 0%, respectively, when KOH was used as sown in (Table 2).
Effect of alkaline solution on the linear growth and sporulation: To verify the effect of alkaline media on the enhancement of slow growing fungal pathogens, a number of seed-borne fungi attacking peanut seeds were grown on Czapek’s alkaline medium and the linear growth and sporulation were recorded.
Alkaline treatments enhanced the linear growth and sporulation of Cephalosporium
sp., Fusarium verticillioides, F. oxysporum, F. solani
and Verticillium sp. as shown in Table 3 and Fig.
1.
| Fig. 1: | Effect of NaOH or KOH on the linear growth of seed -borne fungi of peanut; 1: Check; 2: NaOH 0.4%; 3: NaOH 0.8%; 4: NaOH 1.0%; 5: KOH 0.2%; 6: KOH 0.4%; 7: KOH 0.8% |
Cephalosporium sp. grew 4.5 cm and produced 21.9x106 spores in NaOH 0.8% and 4.4 cm and 22.5x106 spores in KOH 0.4%. Fusarium verticillioides grew 9 cm and produced 169.7x106 spores in NaOH 0.8% and 8 cm and 162x106 spores in KOH 0.4%. F. oxysporum grew 9 cm and produced 207.3x106 spores in NaOH 0.8% and 9 cm and 191.3x106 spores in KOH 0.4%. F. solani grew 9 cm and produced 80.6x106 spores in NaOH 0.8% and 9 cm and 81.3x106 spores in KOH 0.4%. Verticillium sp. grew 8 cm and produced 39.4x106 spores in NaOH 0.8% and 7.9 cm and 35.9x106 spores in KOH 0.4%. The check grew (2.8 cm and 14.1x106 spores), (7.4 cm and 54x106 spores) (6.5 cm and 134.2x106 spores), (7 cm and 74.2x106 spores) and (5.4 cm and 13.4x106 spores), respectively.
Both NaOH (0.8%) and KOH (0.4%) had a pH of 12.5 and gave almost the same results for linear growth and sporulation.
DISCUSSION
A number of standard methods for seed health testing are utilized to test seeds for the presence of seed-borne fungi. The (SBM) and (DFB) are recommended by the ISTA (1996). It is known that with SBM saprophytes develop quickly and often impair the detection of parasitic fungi, while with DFB the growth of saprophytic bacteria and yeasts is enhanced, which may inhibit spore germination of some important seed-borne fungi (Neergaard, 1979).
With these methods, it is difficult to detect some seed-borne pathogens due to their slow growth rates, e.g., Cephalosporim and Verticillium. This research was designed to develop a method suitable for detecting the slow growing seed-borne fungi. The results presented here show that moistening the blotter disks with an alkaline solution enhanced the growth of the slow growing seed pathogens Cephalosporim sp. and Verticillium sp.
The alkali treatments also proved to be an effective means to suppress the growth of saprophytes which impair the detection of pathogenic fungi on seed. Examination of seed-testing blotters overgrown by saprophytic fungi required the use of high magnification (X50) and additional time. Lower magnifications (X6 and 10) were not suitable to detect slow growing pathogens overgrown by saprophytes.
On conclusion, the alkaline blotter method is an effective method for the detection of some important seed-borne pathogenic fungi (Cephalosporium sp., Fusarium verticillioides, F. oxysporum, F. solani and Verticillium sp.) on peanut seeds. The two alkaline treatments were effective for the recovery of lurked Verticillium sp. from peanuts.
Elwakil and Ghoneem (2002) stated that the effect of alkaline media on the recovery of lurked seed-borne fungi may be due to the presence of the alkaline ions K+ or Na+, which replace H+ in the fungal cell. This explanation agrees with the findings of Horikoshi and Akiba (1982), who indicated that Na+ increases the uptake of nutrients in the cells of some Bacillus strains. Abo-Ellil (1999a, b) found that the 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 increased with the alkalinity of the medium. Since alkaline chemicals have previously shown their competence in detecting the lurked Verticillium sp., the results presented here verify that the alkaline blotter method is a sensitive method for detecting the slow-growing seed-borne fungi of peanut.
Intensive seed health testing research has shown that pH plays an important role in the recovery of seed-borne fungi from seed.
The results of this research clearly indicate that the regular seed-testing methods recommended by ISTA are less effective for detecting both Cephalosporium spp. and Verticillium spp. than our innovative alkaline blotter method.
Based on present research, it is expected that seeds grown in alkaline soils may be affected by the above fungi, as the soil environment is ideal for their growth. For that reason, we recommend using the alkaline blotter method for seed testing in regions where peanut is grown in alkaline soil.
ACKNOWLEDGMENT
The authors thank Dr. Conrad J. Krass, Primary State Plant Pathologist, California. Department of Food and Agriculture, Sacramento, CA, USA (retired) for critical review of the manuscript.
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