Introduction

Seed is the principal key for the production of crop and are the prerequisite for healthy crop and good harvest. There are large number of plant diseases occurring due to seed borne pathogens which leads to massive crop damage by affecting growth and productivity of crops (Williams and McDonald 1983; Kubiak and Korbas 1999; Dawson and Bateman 2001). Pulses are easily available and huge reservoir of proteins for the mankind. But out of the several factors responsible for their low production, seed borne diseases are one of the most common factors (Nine 1986; Pal 1996). There are various reported cases where plant diseases spread as a result of the transport of infected seeds (Agrawal 1976) and therefore seeds are considered as prevalent source for transporting pathogens from one place to another (Agrawal and Sinclair 1996). A pathogen infecting the seed can cause abortion of the seed, seed rot, necrosis of the seed thereby reducing its ability to germinate (Khanzada et al. 2002).

Healthy seed is the key for developing a healthy crop and an essential requirement for the maximum yield (Diaz et al. 1998). Neergard (1977) stated that, the grain germplasm is damaged chiefly by the activity of fungi than other microorganism. Of the many types of plant pathogens, seed borne fungi is one of the most crucial pathogen determining the quality of seed. In a world facing problem of malnutrition, protein rich leguminous crops are of prime importance. The legumes are next to cereals as the most important source of food for humans and animals (Rathod et al. 2012; Ghangaokar and Kshirsagar 2013). The aim of the present study was to isolate and identify seed borne fungal pathogen which can affect the seed during storage condition and germination capacity.

Materials and Method

Collection of seed sample

Seed samples from eight leguminous crops namely, Macrotyloma uniflorum, Vigna radiata, Vigna mungo, Vigna unguiculata, Arachis hypogea, Phaseolus vulgaris, Cicer arietinum and Glycine max were purchased from local market of Hudco, Bhilai, India. The collection and testing of samples were done as per the norms of the International Seed Testing Association (ISTA 1993).

Detection of seed borne fungi

Detection of seed borne fungi from selected legume seeds were carried out by agar plate method. There were two types of seeds taken- treated and untreated. The treated seeds are the one sterilized with 0.1% sodium hypochlorite for 10 minutes. Both types of seeds were then placed on Petriplate containing Potato dextrose Agar medium (PDA) by using flame sterilized forceps, 5 seeds per petridish. The inoculated plates were then incubated at 25± 2°C for 8 days under 12 hrs of alternating cycles of light and darkness. Thereafter, on the subsequent day seeds were examined under binocular microscope for the presence of fungi. The numbers of fungi found on the seeds were recorded and percentage of seeds infected and germination of seeds with different fungi was calculated.

Identification of fungi

The isolated fungi were identified with the help of the keys, monograph and available literature (Raper and Fennell 1965; Booth 1971; Ellis 1971; Barnett and Hunter 1972) whereas untreated seeds were used as control.

Fungal species found growing on the surface of seeds, type and frequency of occurrence of identified fungal species was recorded. Percentage Frequency (PF) of occurrence of fungal was calculated by using the following formula:

PF = (Number of seeds on which fungus appear / Total number of seeds) X 100

Percentage of Germination (PG) of seeds varieties are determined as proportion of germinated seeds over the total number of seeds and computed by using the following formula:

PG = (Number of seeds germinated / Total number of seeds used for germination) X 100

Results and Discussion

In the present study, total 12 fungal species viz. Aspergillus niger,  A. flavus, A. fumigatus, A. nidulans, Alternaria alternata, Rhizopus sp. 1, Rhizopus sp. 2, Penicillium sp., Mucor sp., Fusarium sp., Macrophomina sp. and Curvularia sp. were isolated from eight leguminous seeds. Percentage frequency of occurrence of fungi of both treated and untreated seeds shows 100% of occurrence because in all seeds fungal growth occurs. Percentage of germination shows the proportion of seed germinated over the total number of seed taken into consideration of both treated and untreated seed. Treated seed shows high percentage of germination and was perhaps due to surface sterilization with HgCl₂ solution which kills most of the pathogenic fungi (Table 1 and 2).

A = number of seeds germinated, B = number of seed taken into consideration, PG = percentage of germination

A = number of seeds germinated, B = number of seed taken into consideration, PG = percentage of germination

The fungal infection of the stored seeds was the main cause of drop of seed quality during storage. Surface sterilization minimizes the fungal growth on the seeds (Bhutta 1988). Whereas, the untreated seeds were associated with high number of seed borne fungi. Seed surface sterilization with mercuric chloride usually suppresses the growth of saprophytic and other specific growing fungi (Ramakrishna et al. 1991).  It was also observed that surface sterilization of seed with 0.1% sodium hypochlorite significantly decreased Alternaria alternata and Fusarium sp. amongst the species Aspergillus, A. flavus was the most dominant. Fungi such as species of Aspergillus sp., Curvularia sp. occur in higher percentages on untreated seeds compared to those of the treated seeds, because they were removed by surface sterilization. The dominant fungi and fungal growth depend on period of storage and environmental condition. In this study significant numbers of fungi were isolated from these seeds. It was found that Vigna unguiculata seeds yielded the highest number of fungal species while Cicer arietinum and Glycine max both shows the least number of fungi. In the current study, the order of highest to lowest yielding of fungal species in selected seed was Vigna unguiculata > Vigna mungo > Arachis hypogeal = Vigna radiate = Macrotyloma uniflorum > Phaseolus vulgaris > Glycine max = Cicer arietinum. The results of present investigation in terms of seed borne fungus of leguminous plants showed resemblance of result with various research papers taken into consideration, who reported same fungi on leguminous seed (Abdulwehab et al. 2015, Hussain et al.  2009). Seed borne myco-flora is the most important component determining the quality and longevity of seeds.  Microbial invasions can lead to the rotting and loss of seed viability, vigour, germination and oil quality (Nagaraja and Krishnappa 2009; Saxena et al. 2015).

Conclusion

Based on the present study it is evident that seeds of selected leguminous crop plants are infected with various fungi. Among all the seeds Aspergillus niger is most dominant fungi. This fungus implies an irreversible degenerative change in the quality of seed, and germination capacity.

Acknowledgement

We express our gratitude towards Department of Botany for providing necessary resources during the work.

Conflict of interest

Authors had no conflict of interest

References

Abdulwehab SA, El-Nagerabi SAF, El-Shafie AE (2015) Leguminicolous fungi associated with some seed of Sudanese legumes.  Biodiversitas, 16(2):269-280.

Agrawal  PK (1976)  Identification  of  suitable  seed  storage  places  in  India  on  the  basis  of  temperature  and  relative humidity condition. Seed Research, 4(1):6-11.

Agarwal VK, Sinclair JB (1996) Principles of Pathology. 2^nd edition, CRC Press, Inc., Boca Raton, pp 539.

Barnett HL, Hunter BB (1972) Illustrated genera of imperfect fungi: Burgess. Pub. Co., Minnesota.

Bhutta AR (1988) Comparison of cotton seed health testing method and their economics. Pakistan Cotton, 32: 146-153.

Booth C (1971) The  genus  Fusarium.   Commonwealth   Mycological Institute, Kew.

Dawson WAM, Bateman GL (2001) Fungal communities on roots of wheat and barley and effects of seed treatments containing fluquinconazole applied to control take-all. Plant Pathology, 50:575-582.

Diaz C, Hossain M, Bose ML, Mercea S, Mew TW (1998) Seed quality and effect on rice yield: findings from farmer participatory experiment in Central Luzon, Philippines. Philippine Journal of Crop Science 23(2):111-119.

Ellis MB (1971) Dematiaceous hyphomycetes, Commonwealth Mycological Institute, Kew pp 608

Ghangaokar NM, Kshirsagar AD (2013) Study of seed borne fungi of different legumes. Trends in Life Science, 2(1):2319–4731.

Hussain A,  Anwar SA, Sahi GM,  Abbas Q,  Imran (2009) Seed  Borne  Fungal  Pathogens  Associated  with  Pearl  Millet (Pennisetum typhoides)  and  their  impact  on  seed  germination.   Pakistan Journal of Phytopathology, 21(1): 55-60.

ISTA: International Seed Testing Association (1999) International Rules for Seed Testing. Seed Science and Technology, 29:1-127.

Khanzada KA, Rajput MA, Shah GS, Lodhi AM, Mehboob F (2002) Effect of seed dressing fungicides for the control of seed borne mycoflora of wheat. Asian Journal of Plant Sciences, 1(4):441-444.

Kubiak K, Korbas M (1999) Occurrence of fungal diseases on selected winter wheat cultivars. Postepy Ochronie Roslin, 39 (2):801-804.

Nagaraja O, Krishnappa M (2009) Seed borne mycoflora of Niger (Guizotia abyssinica Cass,) and its effect on germination. Indian Phytopathology, 62(4): 513-517.

Neergard P (1973) Detection of seed borne pathogens by culture tests. Seed Science and Technology, 1:217-254.

Nine YL (1986) Opportunities for research on diseases of pulse crops. Indian Phytopathology, 39(3):333-342.

Pal M (1996). Pulse disease scenario. Indian Phytopathology, 49 (2):129-131.

Ramakrishna N, Lacey J, Smith JE  (1991) Effect of surface sterilization, fumigation and gamma irradiation on the microflora and germination of barley seeds. International Journal of Food Microbiology, 13(1):47-54.

Raper KB, Fennell DI (1965) The Genus Aspergillus. Williams and Wilkins, Philadelphia, pp 1-686.

Rathod LR, Jadhav MD, Mane SK, Muley SM, Deshmukh PS (2012) Seed borne mycoflora of legume seeds. International Journal of Advanced Biotechnology and Research, 3(1):530-532.

Saxena N, Shiva Rani SK, Deepika M (2015) Biodeterioration of Soybean (Glycine max L.) seeds during storage by fungi. International Journal of Current Microbiology and Applied Sciences, 4(6):1118-1126.

Williams RJ, McDonald D (1983) Grain molds in the tropics: Problems and importance. Annual Review of Phytopathology, 21:153-178.