Abstract: The aim of the present study was to check the deterioration of Jatropha curcas L. seeds during storage. Agar plate and standard blotter methods were used to study the seed-borne mycoflora of Jatropha curcas L. Both surface sterilized and unsterilized seeds were taken for isolation of fungi. Surface sterilization was done by 0.1% mercuric chloride (HgCl2) solution. A significant contamination with fungal genera was detected in analyzed stored seeds. Sixteen fungal species with two strains of Aspergillus flavus i.e., brown and green were isolated from physic nut seeds during one year of storage. Fungi isolated and identified were Alternaria alternata, Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger, Aspergillus terreus, Cephaliophora irregularis, Chaetomium globosum, Cladosporium cladosporioides, Curvularia lunata, Fusarium moniliforme, Fusarium roseum, Penicillium citrinum, Penicillium rubrum, Rhizopus stolonifer, Dark sterile mycelium and White sterile mycelium. This is the first report of Cephaliophora irregularis on Jatropha curcas L. seeds. Agar plate method showed better results for isolation of Chaetomium globosum, Cladosporium cladosporioides, Curvularia lunata and Fusarium moniliforme.
INTRODUCTION
The oil plant Jatropha curcas L., a multipurpose drought resistance, perennial plant belonging to Euphorbiaceae family is gaining a lot of importance for the production of bio-diesel. The seeds of physic nut are a good source of oil which can be used as a diesel substitute. “Bio-diesel” is well known chemically as the mono-alkyl esters of long-chain fatty acids. It is produced from several types of conventional and non-conventional vegetable oils and animal fats including those of used oils from the frying industry, soyabean oil, rapeseed oil, rubber seed oil and palm oil (Tomasevic and Siler-Marinkovic, 2003; Shah et al., 2004; Ramadhas et al., 2005; Ahmad et al., 2007). In the situation of rapidly growing energy requirements, the contribution of new and especially some non-food oils have to play a significant role (Chitra et al., 2005; Rashid and Anwar, 2008; Chakrabarti and Ahmad, 2008; Harun and Ahmed, 2009). Augustus et al. (2002) have reported that Jatropha curcas seeds contain 20-40% oil. The use of biomass to provide energy has been fundamental to the development of civilization. Biomass contributes a significant share of global primary energy consumption and its importance is likely to increase in future world energy scenarios (Vasudevan et al., 2005). Most of the research and technology has been utilized in stepping up crop production in agriculture but all efforts to produce more will not be of much avail until similar of agricultural products.
Seeds are regarded as highly effective means for transporting plant pathogens over long distances (Agarwal and Sinclair, 1996). Seed-borne mycoflora have been found to affect the growth and productivity of crop plants. A seed-borne pathogen present externally or internally or associated with the seed as contaminant, may cause abortion, seed rot, seed necrosis, reduction or elimination of germination capacity as well as seedling damage. This resulting in development of diseases at later stages of plant growth by systemic or local infection (Bateman and Kwasna, 1999; Khanzada et al., 2002).
Jatropha seeds are constantly subjected to deterioration which implies an irreversible degenerative change in the quality of seeds after it has reached its maximum quality of seeds after it has reached its maximum quality level (Worang et al., 2008; Dharmaputra et al., 2009). The fungi associated with seeds at the harvest stage and under storage bring about several undesirable changes and degradation of seed constituents, thus making the seed unfit for oil extraction, export purpose, consumption or sowing. Chelkowski (1991) reported that in many cases, fungi infecting seeds are seed-borne pathogens. They play an important role in the transmission of numerous pathogenic fungal species to seedlings as well as to the soil.
The objective of this study was to analyze the effect of duration of seed storage on fungal population of physic nut seeds.
MATERIALS AND METHODS
The experiment was conducted in the laboratory of Department of Mycology and Plant Pathology, Institute of Agricultural Sciences, Banaras Hindu University, Varanasi from October, 2008 to September, 2009. Seed samples of Jatropha curcas L. was collected from Varanasi district were used for the isolation and identification of seed-borne fungi. Agar plate method (Muskett, 1948) and Blotter method (De Tempe, 1953) recommended by International Seed Testing Association (Anonymous, 1966) were used for the isolation of fungi. An agar plate is a Petri dish that contains a growth medium (typically agar plus nutrients) used to culture microorganisms. The blotter method is one of the incubation methods where the seeds are plated on well water soaked filter papers and incubated usually for 7 days at 25±2°C under 12 hours alternating cycles of light and darkness. After incubation, fungi developed on each seed are examined under different magnifications of a stereomicroscope and identified. Both surface sterilized and unsterilized seeds were taken for isolation of fungi. Surface sterilization was done by 0.1% mercuric chloride (HgCl2) solution (Ramakrishna et al., 1991).
Agar plate technique: The seeds were externally sterilized by 0.1% mercuric chloride solution to 1 to 2 min then washed by sterilized distilled water (Habib et al., 2007). The isolated fungi were identified using light microscope after slides were stained lactophenol (Anonymous, 1994; Henselov and Hudecov, 2001; Gwary et al., 2006).
Blotter test: The blotter test (Limonard, 1966; Lantos et al., 2002) was used to isolate the fungal pathogens associated with the seeds during storage.
Identification of fungi: After incubation the growth characters as well as percentage of infection were recorded. In order to isolate these fungi into pure culture, Potato Dextrose Agar (PDA) was prepared and the fungi were inoculated onto the sterile PDA and incubated for 7 days at the end of which the fungi were identified based in their colour, spore morphology and mycelia growth using the light microscope (Begum et al., 2004; Chuku et al., 2007; Al-Sheikh, 2009). The fungi were identified by using the reference of Thom and Raper (1945), Raper and Thom (1949), Booth (1971), Ellis (1971) and Barnett and Hunter (1972).
Statistical analysis: Mean value with standard error was calculated to check the variation of isolated seed mycoflora from seeds and kernels of Jatropha curcas L. by agar plate method and blotter method under sterilized and unsterilized conditions during one year of storage. The term ‘Standard Error’ of any estimate is used for a measure of the average magnitude of the difference between the sample estimate and the population parameter taken over all possible samples of the same size, from the population (Chandel, 2002).
RESULTS
From stored seeds of Jatropha curcas L., 11 genus and 16 species of fungi viz., Alternaria alternata, Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger, Aspergillus terreus, Cephaliophora irregularis, Chaetomium globosum, Cladosporium cladosporioides, Curvularia lunata, Fusarium moniliforme, Fusarium roseum, Penicillium citrinum, Penicillium rubrum, Rhizopus stolonifer, Dark sterile mycelium and White sterile mycelium were isolated and identified (Table 1-8).
Table 1 representing seed mycoflora isolated from sterilized Jatropha seeds by Agar plate method. In which Aspergillus niger and Aspergillus flavus (Green) were found dominant. Alternaria alternata, Fusarium roseum and Penicillium rubrum were found common. Aspergillus flavus (Brown), Aspergillus fumigatus, Aspergillus terreus, Cephaliophora irregularis, Chaetomium globosum, Cladosporium cladosporioides, Curvularia lunata, Fusarium moniliforme, Penicillium citrinum, Rhizopus stolonifer, Dark sterile mycelium and white sterile mycelium were found rare.
Table 2 representing seed mycoflora isolated from sterilized Jatropha kernels by Agar plate method. In which Aspergillus niger, Aspergillus fumigatus, Alternaria alternata, Fusarium roseum were found dominant.
| Table 1: | Seed mycoflora isolated from sterilized Jatropha seeds by agar plate method |
| +: Present, -: Absent, *: No recognition as fungal species, #: Dominant fungal species and other are rare | |
| Table 2: | Seed mycoflora isolated from sterilized Jatropha kernels by agar plate method |
| +: Present , -: Absent, *: No recognition as fungal species, #: Dominate fungal species and other are rare | |
Aspergillus flavus (Brown), Aspergillus flavus (Green), Aspergillus terreus, Penicillium rubrum were found common. Cephaliophora irregularis, Chaetomium globosum, Cladosporium cladosporioides, Curvularia lunata, Fusarium moniliforme, Penicillium citrinum, Rhizopus stolonifer, Dark sterile mycelium and white sterile mycelium were considered to be rare mycoflora.
Table 3 representing seed mycoflora isolated from sterilized Jatropha seeds by Blotter method. In which Aspergillus niger, Fusarium roseum and Rhizopus stolonifer, were found dominant. Alternaria alternata, Aspergillus flavus (Green and Brown), Penicillium rubrum and white sterile mycelium were found common. Aspergillus fumigatus, Aspergillus terreus, Cephaliophora irregularis, Penicillium citrinum and Dark sterile mycelium were rare. Where as Chaetomium globosum, Cladosporium cladosporioides, Curvularia lunata, Fusarium moniliforme were absent.
Table 4 representing seed mycoflora isolated from sterilized Jatropha kernels by Blotter method. In which Aspergillus flavus (Green and Brown), Aspergillus niger were found dominant. Alternaria alternata, Aspergillus fumigatus, Fusarium roseum, Penicillium rubrum, Rhizopus stolonifer and white sterile mycelium were found common and Aspergillus terreus, Cephaliophora irregularis, Chaetomium globosum, Curvularia lunata, Penicillium citrinum and Dark sterile mycelium were found rare while Cladosporium cladosporioides and Fusarium moniliforme were absent.
Table 5 representing seed mycoflora isolated from unsterilized Jatropha seeds by Agar plate method. In which Alternaria alternata, Aspergillus flavus (Green and Brown), Aspergillus fumigatus, Aspergillus niger, Penicillium rubrum and Rhizopus stolonifer were found dominant. Cladosporium cladosporioides, Curvularia lunata, Fusarium roseum, Penicillium citrinum and Dark sterile mycelium were found common and Aspergillus terreus, Chaetomium globosum, Fusarium moniliforme and white sterile mycelium were found rare.
Table 6 representing seed mycoflora isolated from unsterilized Jatropha kernels by Agar plate method. In which Alternaria alternata, Aspergillus flavus (Green), Aspergillus fumigatus, Aspergillus niger and Rhizopus stolonifer were found dominant.
| Table 3: | Seed Mycoflora isolated from sterilized Jatropha seeds by blotter method |
| +: Present, -: Absent, *: No recognition as fungal species, #: Dominant fungal species and others are rare | |
| Table 4: | Seed Mycoflora isolated from sterilized Jatropha kernels by blotter method |
| +: Present, -: Absent, *: No recognition as fungal species, #: Dominant fungal species and others are rare | |
Aspergillus flavus (Brown), Aspergillus terreus, Cladosporium cladosporioides, Fusarium roseum, Penicillium citrinum, Penicillium rubrum and *Dark sterile mycelium were found common. While Cephaliophora irregularis, Chaetomium globosum, Curvularia lunata, Fusarium moniliforme and white sterile mycelium were found rare.
Table 7 representing seed mycoflora isolated from unsterilized Jatropha seeds by Blotter method. In which Alternaria alternata, Aspergillus flavus (Green and Brown), Aspergillus fumigatus, Aspergillus niger, Penicillium rubrum and Rhizopus stolonifer were found dominant.
| Table 5: | Seed Mycoflora isolated from unsterilized Jatropha seeds by agar plate method |
| +: Present, -: Absent, *: No recognition as fungal species, #: Dominant fungal species and others are rare | |
| Table 6: | Seed Mycoflora isolated from unsterilized Jatropha kernels by agar plate method |
| +: Present, -: Absent, *: No recognition as fungal species, #: Dominant fungal species and others are rare | |
Only Penicillium citrinum were found common and Aspergillus terreus, Cephaliophora irregularis, Chaetomium globosum, Curvularia lunata, Fusarium moniliforme, Fusarium roseum and Dark sterile mycelium were found rare. While Cladosporium cladosporioides found completely absent.
Table 8 representing seed mycoflora isolated from unsterilized Jatropha kernels by Blotter method. In which Alternaria alternata, Aspergillus flavus (Green and Brown), Aspergillus fumigatus, Aspergillus niger and Rhizopus stolonifer were found dominant. Penicillium rubrum and white sterile mycelium were found common.
| Table 7: | Seed Mycoflora isolated from unsterilized Jatropha seeds by blotter method |
| +: Present, -: Absent, *: No recognition as fungal species, #: Dominant fungal species and other are rare | |
| Table 8: | Seed Mycoflora isolated from unsterilized Jatropha kernels by blotter method |
| +: Present, -: Absent, *: No recognition as fungal species, #: Dominent fungal species and others are rare | |
Aspergillus terreus, Cephaliophora irregularis, Chaetomium globosum, Cladosporium cladosporioides, Curvularia lunata, Fusarium roseum and Penicillium citrinum were rare. While Fusarium moniliforme and Dark sterile mycelium were completely absent.
Data presented in Table 1 to 8 reveals that of the fungi isolated Alternaria alternata, Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger, Fusarium roseum and Penicillium rubrum were found to be dominant. There does not appear to be any previous report of Cephaliophora irregularis on Jatropha curcas L. seeds. At the beginning the some fungi isolated were field fungi i.e., Cladosporium cladosporioides, Fusarium moniliforme and Fusarium roseum. Their population decreased with the increase of storage duration and then storage fungi replaced them. Agar plate method showed better results for isolation of Chaetomium globosum, Cladosporium cladosporioides, Curvularia lunata and Fusarium moniliforme than blotter method. It was also observed that sterilized seeds yielded less population of seed-borne fungi than the unsterilized seeds indicating partial elimination of some contaminating fungi.
Data presented in Table 9 reveals that the seeds of Jatropha curcas L. were mainly deteriorated by Mitosporic fungi of class-Hyphomycetes (75.00%).
Data presented in Table 10 reveals that annual percent occurrence of species of Aspergillus was found to be maximum i.e., from 25% to 100% followed by Alternaria alternata (50-75%), Fusarium roseum (41.67-66.67%) and Penicillium citrinum (25-58.33%).
| Table 9: | Class wise occurrence of fungi and percent occurrence of various classes colonizing the Jatropha seeds during storage |
| Table 10: | Percent occurrence (Annual) of seed Mycoflora of sterilized and unsterilized Jatropha seeds by using agar plate and blotter method |
| *: No recognition as fungal species | |
| Table 11: | Percentage frequency# of fungi in agar plate method and blotter method |
| *: No recognition as fungal species, Value are calculated as Mean±SE | |
Data presented in Table 11 reveals that the occurrence of all fungi which were frequently encountered, is recorded in terms of mean value with standard error. The maximum variation was showed by Cladosporium cladosporiodes i.e., 25.00 isolated from unsterilized Jatropha seeds followed by the same fungus isolated from unsterilized Jatropha kernels by Curvularia lunata and Rhizopus stolonifer isolated from sterilized Jatropha seeds and by white sterile mycelium isolated from sterilized Jatropha kernels by showing standard error 20.84. Fusarium roseum and Penicillium citrinum shown constant value of standard error i.e., 4.17 under both sterilized and unsterilized condition of Jatropha seeds and kernels. Less variation were observed in Alternaria alternata, Aspergillus flavus (brown and green), Aspergillus fumigatus, Aspergillus niger, Cephaliophora irregularis, Chaetomium globosum, Penicillium rubrum, Dark sterile mycelium and White sterile mycelium which showed the dominance of these fungal species.
DISCUSSION
The fungi isolated from stored Jatropha seeds were the main cause of deterioration of seeds during storage (Worang et al., 2008; Dharmaputra et al., 2009). The sterilized seeds show less population of seed-borne fungi than the unsterilized seeds are in close conformity with those of Limnord (1968) who reported that chloral disinfection effectively reduced the microbial contamination. Surface sterilization also has the advantage of minimizing competition among fungi on the seed (Kaur, 2010). Seed surface disinfection with HgCl2 usually suppresses the growth of saprophytic and other superficial fast growing fungi Limnord (1968); Bhutta (1988). It was also observed by Ramakrishna et al. (1991) that surface sterilization with 0.1 or 0.2% (w/v) HgCl2 for 3 min significantly decreased Alternaria alternata, Fusarium sp. and Epicoccum purpurascens but Niaz and Dawar (2009) observed that surface disinfection of seed with 1% Na(OCl)2 reduced the incidence of Aspergillus spp., Chaetomium spp., Cladosporium spp., Rhizopus spp., Cephalosporium spp. Reduction of frequency rate of fungi from sterilized sunflower seeds was also found by Sharfun-Nahar et al. (2005) and Bhutta et al. (1998). Of the two methods used, the agar plate method yielded the highest number of fungi as compared to blotter method. Saprophytic fungi like Aspergillus species, Cladosporium species, Curvularia species, Rhizopus species, Trichoderma species were isolated in higher percentage by agar plate method. The agar plate method was found most suitable for the isolation of saprophytic fungi (Niaz and Dawar, 2009). It was also proved by Mathur and Neergaard (1970) and Khan et al. (1988) that the use of agar plate was preferred over the blotter method for the isolation of mycoflora but Gowdar et al. (2007) observed that standard blotter method was better for isolation of large number of fungal species. The blotter method was more effective for the isolation of Alternaria alternata, Mucor sp., Chaetomium sp. and Stemphylium sp. (Elwakil and El-Metwally, 2001). At the beginning of storage, some of the fungi that infected the seeds were classified as field fungi and their population decreased with the increase of storage duration (Worang et al., 2008; Dharmaputra et al., 2009). Consequently, fungi infecting physic nut seeds grew and developed better, especially the fungi which have abundant spores such as Aspergillus and Penicillium (Dharmaputra et al., 2009).
CONCLUSION
From the present study, it was concluded that Jatropha seeds are constantly subjected to deterioration which implies an irreversible degenerative change in the quality of seeds.