MATERIALS AND METHODS

The experiment was carried out in the laboratory of the Department of Plant Pathology, Patuakhali Science and Technology University, Bangladesh. The experiment was conducted by Completely Randomized Design with three replications.

Isolation and identification of seed borne fungi: Isolation ofseed-borne fungi was done using recommended techniques by the ISTA¹⁹ namely, Standard moist Blotter (SB) methods. A total number of 100 seeds from each treatment were used.

Different fungal colonies grown on chilli seeds were observed under stereo-binocular microscope (Carl Zeiss Axio vision). All the fungi were identified using keys and manuals^7,20-22. In case of confusion, temporary mounts were prepared and examined under compound microscope (Carl Zeiss ) for identification of the associated fungi. Prevalence of fungi was expressed in percentage based on total number of seeds plated. The % incidence of fungi of particular species within a genus of fungi was calculated²³:

In vitro evaluation of different conc. of H₂O₂: Performance of 3 different conc. of H₂O₂ (1, 2 and 3% H₂O₂) were used to evaluate in vitro against seed borne fungi, by Poisoned food technique²². Each treatment was replicated thrice. When medium in the untreated control plates was fully covered with mycelial growth of the test fungus, radial mycelial growth was measured in all the treatment plates. Percent inhibition of mycelial growth (%) in treated plates was calculated by applying the formula given by Sundar et al.²⁴:

Germination and vigor test: One hundred and fifty seeds of each treatment were counted. Plastic tray was filled with fine sand moistened with distilled water. Fifty seeds were sown in each tray and considered as a replication and there were three such replications of each treatment. After 7, 10 and 15 days of sowing each seedling was evaluated in accordance with the general principles laid down in ISTA rules¹⁹. The Germination Index (GI) was calculated by using the formula as suggested by the AOSA²⁵:

For Vigor Test root and shoot length of randomly taken 10 seedlings per replicate were measured after 15 days. The seedling vigor was determined following the formula²⁵:

Statistical analysis: Data were analyzed using one-way ANOVA with the Web Agri Stat Package 2.0 (WASP). Means were compared by the Duncanʼs multiple tests and statistical significance was determined at 5% level using WASP.

RESULTS AND DISCUSSION

Prevalence of seed borne fungi: A total of 6 different fungi belonging to 6 genera were isolated from chilli seeds. Aspergillus flavus, Rhizopus stolonifer, Colletotrichum capsici, Curvularia lunata, Alternaria alternata and Fusarium monilliforme were found to be associated with chilli seeds. Among them the most predominant fungi was Aspergillus flavus followed by Colletotrichum capsici, Curvularia lunata, Fusarium moniliforme, Alternaria alternata and Rhizopus stolonifer (Fig. 1). Alam et al.², Chigoziri and Ekefan²⁶ and Solanke et al.²⁷ also reported about the association of seed borne fungi.

In vitro effect of hydrogen peroxide treatment on radial mycelial growth of seed borne fungi: The inhibition (%) of the growth of the fungus with different percent concentration of hydrogen peroxide over control was calculated and presented in (Table 1).

At 3% H₂O₂ treatment the highest mycelial growth inhibition of Aspergillus flavus was obtained. The growth of Rhizopus stolonifer was poorly inhibited by H₂O₂ treatment compare to other isolated fungi. The highest inhibition of colony growth of Colletotrichum capsici was observed by the treatment of 3% H₂O₂ followed by 2 and 1% H₂O_2. The mycelial growth of Curvularia lunata was highly inhibited by 1% H₂O₂ compare to 2% H₂O₂ and 3% H₂O₂. In case of Alternaria alternata. The highest inhibition of colony growth was obtained at 3% H₂O₂ concentration. The mycelial growth of Fusarium moniliforme was highly inhibited by 2% H₂O₂. The results of this study indicated that all the treatments reduced the seed borne fungi over the control. Among them, 3% H₂O₂ showed the best performance in terms of reducing percent seed-borne infection followed by 1% H₂O₂. Similar findings also observed by Kotchoni et al.⁸.

Fig. 1:	Percent fungi obtained from different sources of chili seeds

Table 1:	In vitro effect of Hydrogen peroxide on inhibition of mycelial growth of seed borne fungi
Means followed by same letter in a column did not differ significantly at 5% level by LSD

Table 2:	Mean effect of seed treatment on germination of chilli seed at different days after sowing
Means followed by same letter in a column did not differ significantly at 5% level by LSD

Table 3:	Mean effect of seed treatment on root and shoot length of chilli seedling
Means followed by same letter in a column did not differ significantly at 5% level by LSD

They showed that H₂O₂ inhibited pathogen growth in seeds and seedlings. Similar results were also obtained by Szopinska²⁸. He stated that, treatment with hydrogen peroxide, regardless of concentration, positively affected the health of the seeds, significantly increasing (at 5% level of significance) the percentage of seeds free from fungi.

In the present investigation, 1, 2 and 3% H₂O₂ were tested against six fungi viz., Aspergillus flavus, Rhizopus stolonifer, Colletotrichum capsici, Curvularia lunata, Alternaria alternata and Fusarium moniliforme isolated from chili seeds by Poison food technique. The results revealed that all concentration inhibited the mycelial growth of all fungi tested in comparison with control having large colony diameter. It is may be due to antimicrobial properties of hydrogen peroxide⁶. Among them, 1% H₂O₂ was superior in inhibition of mycelia growth of all fungi tested.

Effect of seed treatment on germination of chili seeds collected from different sources: There was significant difference (at 5% level of significance) among different conc. of Hydrogen peroxide in respect of germination % at different DAS (Table 2). The germination percentages were 55.47, 60.53 and 84.80% at 7, 10 and 15 DAS, respectively were obtained from 1% hydrogen peroxide treated seeds. The minimum germination percentages were 4.26, 5.60 and 5.86% at 7, 10 and 15 DAS, respectively were found at 3% Hydrogen peroxide treated seeds. This might be due to the reason of higher oxidative reactivity of hydrogen peroxide.

In this experiment higher percentage of germination was found in chili seeds when treated with 1% H₂O₂. Similar results were obtained by Szopinska²⁸. He reported the negative influence of higher concentration of hydrogen peroxide in germination of seed.

Vigor index: The root length, shoot length and vigor index was significantly influenced (at 5% level of significance) by different concentrations of hydrogen peroxide (Table 3). The maximum root length (4.767 cm) was found in seedling raised from seeds treated with 1% H₂O₂. Lower root length (2.401 cm) was recorded in seedlings raised from water soaked seeds which is statistically similar to 3% H₂O₂ treated seeds. Significantly higher shoot length was obtained from the seedlings raised from 1% H₂O₂ treated seeds. Significant lower value of shoot length 1.812 and 1.704 cm were obtained from seedlings raised from the seeds treated with 2 and 3% H₂O₂, respectively_. The highest 640.7% vigor index was recorded in seedlings obtained from 1% H₂O₂ treated seeds and significantly the lowest vigor index 23.20% was recorded from seedlings obtained from 3% H₂O₂ treated seeds.

Higher vigor index also found in seeds treated with 1% H₂O₂ compared to other concentrations of hydrogen peroxide over the untreated control seeds of each seed source. This could have resulted from the ability of the chemical to eliminate most of the seed-borne pathogens from the seed. Similar findings also reported by Ogawa and Iwabuchi¹⁸ and Narimanov²⁹. Ogawa and Iwabuchi¹⁸ discussed the possibility that endogenously generated H₂O₂ functions as a promoter of zinnia seed germination by oxidizing germination inhibitors. Narimanov²⁹ observed that short seed treatment in H₂O₂ solution promoted the early appearance of sprouts and accelerated the development of barley, maize, haricot, melon, vegetable marrow, garden radish and carrot.

CONCLUSION

It can be safely concluded that the hydrogen peroxide seed treatment is highly effective, economical and easily applicable as it can reduce the seed-borne mycoflora, improve seed germination and vigor. Based on the findings of the present study, the treatment of chili seeds with hydrogen peroxide, regardless of concentration of H₂O₂, significantly reduced seed infestation with seed borne fungi in all types of seed sources and improved health of chilli seeds. Among the concentrations of H₂O₂, 1% H₂O₂ is more effective in increasing seed germination percentage, vigor index and percent inhibition of mycelial growth.

SIGNIFICANCE STATEMENT

This study discovers the efficacy of H₂O₂ against different mycoflora associated with chilli seeds and enhancement of seed germination and vigor. This study will help the researcher to uncover the mystery of H₂O₂ efficiency.

ACKNOWLEDGMENTS

For this study, laboratory assistance was provided by "Plant Disease Clinic" of the department of Plant Pathology, Patuakhali Science and Technology University, Patuakhali, Bangladesh.