SEED-BORNE FUNGI OF SOYBEANS ( GLYCINE MAX [ L . ] MERR ) IN THE GUINEA SAVANNAH AGROECOLOGY OF NIGERIA

Seed health testing of fifteen soybean cultivars obtained from five locations in the Guinea Savannah agro-ecology of Nigeria was carried out using two main seed health testing methods described by the International Seed Testing Association (ISTA); the standard blotter and two variants of agar plate incubation method (Potato Dextrose Agar and Czapeck Dox Agar plate methods) to determine the seed infection by fungi. Seed component plating was also carried out to determine the most active site of infection of the seeds by the fungi. Five fungal species were isolated from the cultivars tested. The percentage of seeds infected with Fusarium sp. and Penicillium sp. was significantly different (p<0.05) among the cultivars, with the highest value standing at 15.20 percent of Fusarium sp. and 8.54 percent of Penicillium sp. in cultivars MSJ and FSSBu, respectively. The difference in the methods of isolation was also significant (p<0.05) and the agar (PDA) plate method was the most efficient in the isolation of Aspergillus sp., Penicillium sp. and Phomopsis sp. The efficiency of the other methods of isolation however varied for the different organisms. The cotyledon was observed to be the most active site of infection with thirteen of the fifteen tested cultivars showing 40–100% of infection of the cotyledons. The need for soybean seed health testing before their distribution to farmers is made manifest in this study.


Introduction
Soybean is one of the world's most important grain legumes.It is a source of high quality protein, vegetable oil and other important industrial food-derived products (milk, fibre and phytochemicals) for human and animal consumption (Lusas and Riaz, 1995;Fukushima, 2000;Kerley and Allee, 2003;Palacios et al., 2004).The protein content of soybean is rated as having a protein digestibilitycorrected amino acid score (PDCAAS) of 0.99 (the maximum value for individual protein is 1.0 or 100%) (WHO/FAO/UNU, 2007).It is, therefore, a promising substitute for animal protein source supply which is usually inadequate and expensive for poor households in a developing country like Nigeria.The oil is also cholesterol-free, making it one of the healthiest cooking oils.
Soybean is cultivated to a very limited extent in the sub-Saharan Africa, and Nigeria ranks first in the list of producing countries in the region (Laswai et al., 2005;Shurtleff and Aoyagi, 2007).The major soybean producing areas in Nigeria fall within the Guinea and Derived Savannah agroecological zones with Kaduna, Benue, Plateau and Niger states dominating production.Other areas of production include Nasarawa, Kebbi, Kwara, Oyo, Jigawa, Borno, Bauchi, Sokoto, Taraba, Zamfara and Federal Capital Territory (Omotayo et al., 2007).FAO (2009) soybean production record showed that Nigeria contributed 437,000 MT (about 0.3%) of the produce to the total output (about 0.6%) from Africa in 2003.This level of production is very low compared to the one obtained in Brazil, Argentina, USA and China in the same production year.
Disease is one of the factors contributing to the low productivity of soybean in Africa and Nigeria in particular (Osho and Dashiel, 1995).More than 135 microorganisms have been described on soybeans with about 30 species belonging to the group of economically important pathogens (Roy et al., 2000).Some of the pathogens are now known to be transmitted through seeds (Shovan et al., 2008).Seed-borne pathogens represent a major threat to crop establishment and yield.When soybean seeds are sown, seed-borne pathogens may cause disease or death of plants, resulting in crop losses.When used for direct consumption, seed-borne organisms may cause chemical changes, deterioration in seed content or mycotoxin release with the potentially harmful effect on humans and livestock resulting in food waste or famine (Chiarapa and Gambogi, 1986).A knowledge of the type and quantity of pathogens present in a seed sample will be an indication of the risk of disease development in the crop should the seeds be sown under conditions which allow the pathogens to launch a successful attack.Also, if the seeds are to be disinfected to prevent disease establishment, knowledge of the maximum extent of contamination will help to decide how effective such disinfection is likely to be or whether it would be wise to reject the sample for sowing (Malone and Muskett, 1997).
This study was therefore carried out to isolate, quantify and identify seedborne fungi associated with the seeds of soybean cultivars obtained from selected locations in the Guinea Savannah agro-ecology of Nigeria and to evaluate the relative efficiency of two main seed health testing methods (blotter and agar plate methods) and to locate the site of infection of the soybean seeds in the seed components.
Seed-borne fungi of soybeans in the Guinea Savannah agroecology of Nigeria 59

Sources of seeds
Soybean seeds were sourced from five locations within the Guinea Savannah agroecological zone of Nigeria as follows: 3 locations from Southern Guinea Savannah (SGS) located at latitudes 8°4′ and 11°3′ N and longitudes 2°41′ and 13°33′ E and 2 locations from Northern Guinea Savannah (NGS) located at latitudes 9°10′ and 11°59′ N and longitudes 3°19′ and 13°37′ E. The three locations in the Southern Guinea Savannah agro-ecology and the sample zones include: Benue (Buruku, Gboko, Guma and Vandelkya), Kwara (Ilorin and Shonga) and Niger (Bidda and Lapai).The two locations in the Northern Guinea Savannah agroecology and the zones include: Adamawa (Gire and Jimeta) and Kaduna (Mai Lafia, Makarfi and Zaria) (Plate 2).Seeds collected belong to three categories: storage seeds, farmer-saved seeds and market seeds.Some of the seeds are shown in Plate 1.The seeds were bulked in labelled muslin bags to allow an exchange of air during transportation and while seeds await laboratory tests.

Seed inspection
The collected seed samples were examined when dry for evidence of the production of fungal fruiting structures or the effect of fungal pathogens on the physical appearance of the seeds.
Plate 1. Appearance of some seed samples used for the study.

Sterilization
Soybean seeds used were surface sterilized using 0.5% sodium hypochlorite (NaOCl) for 30 seconds and later rinsed in several changes of sterile water.A sterile blotter was prepared by first moistening Whatman filter paper #1 with distilled water.The filter papers were then wrapped in aluminum foil for sterilization in the autoclave before arranging them aseptically into sterile Petri dishes.

Isolation and quantification of fungal pathogens from the seeds
Two main methods were used to isolate the soybean seed-borne fungi, the standard blotter and agar plate methods as described by the International Seed Testing Association in the ISTA (1996) procedures.In the Standard Blotter method, ten seeds pre-treated with 0.5% sodium hypochlorite were placed equidistantly on sterile moistened Whatman filter paper #1 in Petri dishes (90mm diameter).Three replicates were prepared per soybean variety.The plates were incubated at 25 ± 2 o C under alternating cycles of 12 hours of light and darkness for 7 days.Potato dextrose agar (39g/litre) and Czapeck Dox agar (49g/litre) were used as replacements for the blotter in the agar plate method.The media were amended with streptomycin to suppress bacterial growth.Incidence of the seed-borne fungi was recorded as percentage of the total population for each of the soybean cultivar.

Component plating
Component plating was done in accordance with the method described by Maden et al. (1975).Soybean seeds were soaked for about three hours in sterile water after which the seeds were separated into testa, cotyledon and embryo using sterile forceps and a dissecting knife.These three components were later surface sterilized for about 30 seconds with 0.5% NaOCl solution and then separately arranged and equally spaced on PDA medium in 90mm Petri dishes.The setup was incubated as described earlier.
Preparation of cultures from the isolated fungi Hypha or spores were transferred aseptically unto fresh sterile media with an ethanol flamed inoculating needle to prepare the pure cultures from each of the fungal isolates.The plates were later incubated for seven days at 25 o C ± 2 o C. The pure cultures are required for identification of the fungal isolates.

Identification of the fungal isolates
Morphological characteristics of the isolates on plates were observed.Temporary slides were also prepared for each of the isolates.The slides were then observed under a compound microscope.The morphological and microscopic features were recorded.These features were matched with those described in standard references (Kulwant et al., 1994;Malone and Muskett, 1997;Mathur and Kongsdal, 2003) for identification of the isolates.The identities of the isolates were confirmed at the plant pathology laboratory of the International Institute for Tropical Agriculture (IITA), Ibadan, Nigeria.

Data collection and analysis
The data collected during the exercise were the incidence of seed-borne fungi recorded as percentage of the population in the different soybean cultivars using different incubation methods and incidence of seed-borne fungi in the seed components of the different cultivars.The collected data were subjected to analysis of variance using SPSS Version 21 statistical package and the means were separated using Duncan's new multiple range test at the 5% level of significance.

Results and Discussion
Seed cultivars and their sources A total of fifteen soybean cultivars were collected from the thirteen zones in the five locations selected for the study (Table 1).

Seed inspection
Table 1 shows the results of dry seed inspection carried out on the collected seeds.The samples collected from markets showed the highest proportion of broken and shrivelled seeds followed by those collected by farmers.The largest proportion of undamaged seeds was found in those obtained from storage.Isolation, identification and quantification of seed-borne fungi Five fungal species were isolated from the fifteen soybean cultivars tested.They included Aspergillus sp., Curvularia sp., Fusarium sp., Penicillium sp. and Phomopsis sp.(Table 2).The morphology of the organisms on plates and their microscopic features are shown in Plate 2.
Fusarium sp. and Penicillium sp.showed the significantly different (p=0.05)percentage occurrence in the different soybean cultivars tested.Cultivar MSJ with the significantly high percentage occurrence (15.2) was obtained from the market.All the fungal isolates appeared on all the soybean cultivars.
The results also showed that PDA performed better than the other methods in isolating Aspergillus sp., Penicillium sp. and Phomopsis sp. and blotter performed significantly (p=0.05)better than the other methods in isolating Fusarium sp.(Table 3).

Infection of the seed components
The percentage infection of the soybean seed components is shown in Table 4.It was observed that the cotyledon was the most active site of infection of the seed components.The data showed the highest infection percentage of the cotyledons for thirteen of the fifteen soybean cultivars with mean values ranging from 40 to 100.
A high proportion of broken and shrivelled market seeds observed in this study indicate that the sellers care less about the status of the seeds they sell to farmers.This might be due to their poor education and awareness of the implication of poorly handled seeds meant for planting.On the other hand, farmers are aware of the risk involved in poor handling of their buffer stocks, so they take adequate care to secure them for planting in the coming season.The same goes for seeds in the research stations and other agricultural establishments.This study provides an assessment of the diversity of soybean seed-borne fungi within the Guinea Savannah agroecology of Nigeria.The results showed a varying degree of infection of the different soybean cultivars by the fungi with fairly high frequencies observed in regions with a long history of high productive capacity of the crop in Nigeria (Benue and Kaduna states).This might be attributed to favourable weather conditions for their survival in the region.All the fungal isolates observed in this study were listed among the eight species isolated from ten soybean cultivars with agar (PDA) plate and blotter methods in India (Khayum et al., 2006) and the thirty-nine species isolated from J one cultivar of soybean using four methods of isolation in Pakistan (Nasreen, 2003).Both studies also found the agar plate method of isolation to perform better than the blotter method in isolating the fungi.This is in partial agreement with the findings in this study which show that the agar plate method supported better isolation of Curvularia sp., Penicillium sp. and Phomopsis sp. while the blotter method promoted better isolation of Fusarium sp.Michail et al. (1981) however found both methods to be equally good for detecting species of fungi other than Fusarium, Myrothecium and Cephalosporium among the isolated fungal species.The isolation of these three fungi (Fusarium, Myrothecium and Cephalosporium) was better on blotter.The performance of PDA and Czapeck Dox agar can be attributed to the fact that they contain substances essential for the growth of the fungi.
The results of soybean seed component plating show that the cotyledon is the most infected component by the fungi.These results are in line with the findings of Ahmed et al. (2006), but for cowpea seeds.The cotyledon of soybean contains food reserve and may have been the reason for a higher degree of colonization of the seed components.

Conclusion
The seeds of soybean cultivars obtained from the Guinea Savannah agroecology of Nigeria were found to be associated with five fungal species namely Aspergillus sp., Curvularia sp., Fusarium sp., Penicillium sp. and Phomopsis sp.The associated fungal flora was most predominant in the cotyledons of most of the soybean cultivars.The agar (PDA) plate isolation method supported the isolation of more of the fungi than the other methods of isolation.

Figure 1 .
Figure 1.Map of Nigeria showing the selected locations for a collection of soybean seeds used for the study.

Plate 2 .
Fungi isolated from soybean seeds: A-E are the pure cultures of Phomopsis sp., Aspergillus sp., Penicillium sp., Fusarium sp. and Curvularia sp.F -Phomopsis conidia, G -Macroconidia of Curvularia sp.; H -Conidial head of Aspergillus sp.;I -Conidiophores of Penicillium sp.; J -Micro and macroconidia of Fusarium sp.; K -A close up view of the fruiting bodies of Aspergillus sp. as seen in situ on soybean seeds.

Table 1 .
Categorization of sources of collected seeds into storage, market and farmer-saved and their status.
Values are means of three replicates (each of which is made up of one hundred seeds).Storage seeds were obtained from agricultural establishments and research stations.Market seeds were bought from sales points where farmers buy their planting seeds (MSGi Market Soybean from Gire, MSGu Market Soybean from Guma also known as Local, MSJ Market Soybean from Jimeta, MSMl Market Soybean from Mai Lafia, MSMk Market Soybean from Makarfi, MSZ Market Soybean from Zaria).Farmer-saved soybean seeds (FSSB Farmer Saved Soybean from Bidda, FSSBu, Farmer Saved Soybean from Buruku also known as Agric, FSSLa Farmer Saved Soybean from Lapai).

Table 2 .
Occurrence (%) of fungi on seeds of different soybean cultivars.
CultivarsAspergillus sp.Curvularia sp.* Fusarium sp.* Penicillium sp.NS (Not significantly different at the 5% level of probability).*Significantly different at the 5% level of probability.Values in the same column followed by the same letter(s) are not different significantly at p=0.05 by Duncan's multiple range test.

Table 3 .
Relative effectiveness of the methods of isolation of soybean seed-borne fungi.
Methods of isolationAspergillus sp.* Curvularia sp.* * Fusarium sp.* Significantly different at the 5% level of probability.Values are percentages of isolated fungi.Mean values in the same column followed by the same letter (s) are not significantly different at p=0.05.

Table 4 .
Mean percentage infection of soybean seed components.