BIOTROPIA No. 9, 1996: 15 - 25 FUNGI ISOLATED FROM GROUNDNUTS IN SOME LOCATIONS OF WEST JAVA O.s. DHARMAPUTRA SEAMEO BIOTROP, P.O. Box 116, Bogor, Indonesia; and Department of Biology, Faculty of Mathematics and Natural Sciences, Bogor Agricultural University, Bogor, Indonesia I. RETNOWATI SEAMEO BIOTROP, P.O. Box 116, Bogor, Indonesia ABSTRACT One hundred and ninety eight groundnut samples were collected from freshly harvested groundnuts (FHG), farmer storage systems (FSS), middlemen warehouses (MW), whole salers (WS) and retailer sample (RS) during the dry and wet seasons from Cidolog, Cianjur, Sukabumi and Bogor, West Java, Indonesia, in 1990/1991. The moisture content (m.c.), intactness of kernels, and the percentages of groundnut kernels infected by each species of fungi were analyzed. In genera), the m.c. of the samples collected during the dry season was lower than of those collected during the wet season. Also, the m.c. of samples collected from FHG, FSS and MW was higher than of those collected from WS and RS. The m.c. of samples collected from FHG was the highest (12.5-45.75%), but the percentages of damaged kernels were the lowest (2.5-13.8%), because the samples were shelled manually. A total of 25 species of fungi were isolated from samples collected from the 4 localities. They were Acremonium strictum, Aspergillus candidus, A. flavus, A. niger, A. ochraceus, A. tamarii, A. wentii, Botryodiplodia theobromae, Cladosporium cladosporioides, C. sphaerospermum, Eumtium chevalieri, E. repens, E. rubrum, Fusarium equiseti, F. longipes, F. oxysporum, F. semitectum, Mucor sp., Papulaspora sp., Pestalotia sp., Penicillium aethiopicum, P. citrinum, Rhizapus sp., R. stolonifer and Syncephalastrum sp. The predominant fungi in samples collected from Cidolog and Sukabumi during the dry season were Aspergillus wentii, while those collected from Cianjur and Bogor were A. niger. The percentages of kernels infected by A. wentii in samples collected from Cidolog and Sukabumi were between 30-100% and 36-100%, respectively, while those of kernels infected by A. nigerin samples collected from Cianjur and Bogor were between 34-93% and 14-98%, respectively. The predominant fungi in samples collected from each location during the wet season were A. flavus. The percentage of kernels infected by the fungus in samples collected from Bogor was the highest (83-100%). Key words: Indonesia/West Java/Stored product pests/Groundnut/Fungi/Acremonium strictum/Aspergillus sp./Botryodiplodia theobromae/Cladosporium sp./Eurotium sp./Fusarium sp./Mucor sp./Papulaspora sp./Pestalotia sp./SPenicillum sp/Rhizopus sp./Syncephalastrum sp./Moisture content. 15 BIOTROPIANo. 9, 1996 INTRODUCTION Groundnut is a major food legume and an important secondary crop after maize and soybean in Indonesia. Since Indonesia has a humid tropical climate, this commodity could be easily infected by fungi during storage when freshly harvested or even before harvest. There are various practices of post-harvest processing and storage that could affect the moisture content and the intactness of the kernels and thus their susceptibility to fungal infection. Aspergillus and Penicillium are the two common genera of fungi found on stored products. They can cause weight loss, seed discolouration, heating and mustiness, and production of mycotoxins, specially aflatoxins. The latter are toxic metabolic substances produced by A. Flavus and A. parasiticus and are known to be carcinogenic agents (Butler 1974). In Indonesia, very few studies have been conducted on storage fungi of groundnuts and their ability to produce aflatoxin. This study shows the presence of fungi infecting goundnuts. Information on the percentages of groundnut kernels infected by each species of fungi, moisture content and damaged kernels of freshly harvested, post-harvest, stored and marketed materials during dry and wet seasons are also reported. MATERIALS AND METHODS Sample collection A total of 198 groundnut samples were collected in Cidolog, Cianjur, Sukabumi and Bogor, West Java, Indonesia, in 1990/1991. Of the total, 100 were collected during the dry season (August, September and October, 1990) from farmer storage systems (FSS), middlemen warehouses (MW), wholesalers (WS) and retailer samples (RS), while 98 samples were collected during the wet season (February and March 1991) from freshly harvested groundnuts (FHG), FSS, MW, WS and RS. The samples consisted of shelled and unshelled groundnuts with the latter shelled manually. About 1 kg of each sample was then divided into 4 subsamples for (1) moisture content analysis, (2) damage kernels analysis, 3) fungal analysis and 4) reserve sample. 16 Fungi isolated from groundnuts - OS. Dharmaputra & I. Retnowati Moisture content analysis Percent moisture content of kernels (on the basis of water loss) was determined by the oven method (ISO 1968). Three replicates were used for each sample. The kernels were ground and dried in the oven at 130 C for 2 hours. The moisture content was determined using the formula: 100 Moisture content = (Mo - Ml) x ———— Mo where : Mo is the initial mass, in gram, of the test portion Ml is the mass, in gram, of the dry test portion Damaged kernel analysis The kernels were classified as either intact or damaged. Damaged kernels are those cracked, broken, porous, shrivelled or insect- damaged. Three replicates were used for each sample. The percentage of damaged kernels was determined using the formula: weight of damaged kernels Damaged kernels = ———————————————————— x 100 % weight of the kernels analyzed for intact and damaged kernels Fungal analysis Fungi were isolated using direct plating methods on Dichloran 18% Glycerol Agar (DG 18). According to Hocking and Pitt (1980), DG 18 was especially used for the isolation of xerophilic fungi. Before plating, samples were individually disinfected using 1% sodium hypo-chlorite for 2 minutes. One hundred kernels were then plated (10 kernels/plate) on the medium and incubated at 25 C for 7 days. The fungi were identified using the publications of Samson et al. (1984), and Pitt and Hocking (1985) as the main references. The percentage of kernels infected by fungal species on each medium was determined. 17 BIOTROPIA No. 9, 1996 RESULTS AND DISCUSSION Moisture content Moisture content (m.c.) is the most important factor determining the development of storage fungi in seeds (Christensen & Kaufmann 1975; Neergaard 1979). According to WHO (1979) the minimum m.c. of groundnuts for the development of Aspergillus flavus was 9.0 - 10.0%. The m.c.'s of samples collected from different localities during the dry season are presented in Table 1. The m.c.'s of samples from Cidolog, Cianjur, Sukabumi and Bogor were between 9.9 - 14.1%, 7.9 - 10.5%, 7.5 - 11.2% and 6.1 - 10.7%, respectively. It seems that the m.c.'s of samples collected from the same location were not affected by their source (FSS and MW from Cidolog, WS and RS from Cianjur, Sukabumi and Bogor). The m.c.'s of samples collected from different localities during the wet season are presented in Table 2. The m.c.'s of the samples from Cidolog were between 12.5 -45.7% (FHG) and 10.8 - 18.1% (samples from FSS and MW), while those from Cianjur, Sukabumi and Bogor were between 8.7 - 13.9%, 7.5 - 10.3% and 7.7 - 11.1%, respectively. Table 1. Moisture contents and percentages of damaged kernels of groundnuts collected during the dry season 18 Fungi isolated from groundnuts - OS. Dharmaputra & I. Retnowati Table 2. Moisture content and percentages of damaged kernels of groundnuts collected during the wet season In general, the m.c.'s of the samples collected during the dry season were lower than of those of the wet season. The moisture content of samples collected on 30 March 1991 was the highest (12.5 - 45.7%), because they were not yet dried. According to BULOG (1981) the maximum moisture content of stored groundnuts should be 7%. In this study, the moisture contents of the samples collected from farmers storage systems and middlemen warehouses were higher than 7%. Damaged kernels Damaged kernels gave a chance for fungi to infect the seeds. According to Christensen (1980) physical damage of seeds is one of the factors that affects fungal infestation. The percentages of damaged kernels of samples collected from different localities during the dry season are presented in Table 1. The percentages of damaged kernels from Cidolog, Cianjur, Sukabumi and Bogor were between 12.0 - 51.8%, 10.2 - 38.0%, 8.7 - 27,9%, and 1.5 - 26.0%, respectively. 19 BIOTROPIA No. 9, 1996 The percentages of damaged kernels of samples collected from different localities during the wet season are presented in Table 2. The damaged kernels from Cidolog were between 2.5 - 13.8% (FHG) and 9.9 - 31.7% (samples from FSS and MW), while those from Cianjur, Sukabumi and Bogor were between 13.0 - 61.0%, 18.0 - 55.0%, and 6.5 -56.0%, respectively. The percentage of damaged kernels of samples collected on 30 March 1991 was the lowest because they were shelled manually, while the other samples were shelled either manually or using wooden/zinc sheller. It was assumed that handshelling prevented the occurrence of damaged kernels, but this method is time consuming. Proper tools should be used for shelling groundnuts to minimize the occurrence of damaged kernels. Fungal analysis Twenty three species of fungi were isolated from samples collected from Cianjur, Sukabumi and Bogor during the dry and wet seasons. The isolated fungal species belonged to the field fungi (i.e. Botryodiplodia theobromae, Cladosporium cladospo-rioides, C. sphaerospermum, Fusarium equiseti, F. longipes, F. oxysporum, F. semitectum and Papulaspora sp.), the storage fungi (i.e. Aspergillus Candidas, A. flavus, A. niger, A. ochraceus, A. tamarii, A. wentii, Eurotium chevalieri, E. repens, E. rubrum, Penicillium aethiopicum and P. citrinum), and fungi belonging to the Mucorales (i.e. Mucor sp., Rhizopus sp., R. stolonifer and Syncephalastrum sp.). Aspergillus candidus, A. flavus, A. niger, A. ochraceus, A. tamarii, A, wentii, Botryodiplodia theobromae, Eurotium chevalieri, E. repens, Papulaspora sp. and Penicillium citrinum were always isolated from samples collected in each location during the dry season, while those isolated from samples collected during the wet season were always A. flavus, A. niger, A. tamarii, A. wentii, B. theobromae, E. chevalieri, Papulaspora sp., Penicillium citrium and R. stolonifer. According to Dharmaputra and Rahayu (1988) the predominant fungal species isolated from groundnut samples collected from a market in Bogor, West Java, Indonesia belonged to the genera Aspergillus, Eurotium and Penicillium, i.e. A. candidus, A. flavus, A. niger, A. restrictus, Eurotium spp. and Penicillium spp. The fungal species and the percentage of kernels infected by each species of fungi from samples collected from the three localities are presented in Table 3. The percentages of kernels infected by the field fungi i.e. F. longipes (collected from Cianjur during the dry season), C. cladosporioides and F. semitectum (collected from Bogor during the dry season), F. equiseti and F. oxysporum (collected from, Cianjur during the wet season) and C. cladosporioides, C. sphaerospermum and F. semitectum (collected from Sukabumi during the wet season) were very low. They were between 0 - 4%, 0 - 8%, 0 - 5%, 0 - 1%, 0 - 9%, 0 - 3%, 0 - 5% and 0 - 2%, respectively. 20 Fungi isolated from groundnuts OS. Dharmaputra & I. Retnowati 21 BIOTROPIA No. 9,1996 Among the storage fungi, A. niger was the predominant species of fungi infecting kernels collected from Cianjur and Bogor during the dry season. The percentage of kernels infected by the fungus in samples collected from both localities was between 34- 93% and 14 - 98%, respectively. The percentages of kernels infected by A. flavus in samples collected from both localities were between 9 - 98% and 8-95%, respectively. It was assumed that the moisture contents of the samples collected from the two localities were still favourable for their growth. They were between 7.9 - 10.5% and 6.1- 10.7%, respectively. The predominant species of fungi infecting kernels collected from Sukabumi during the dry season was A. wentii. The percentages of kernels infected by the fungus in samples collected from that location were between 36 - 100%, while those of A. flavus were between 10 - 85%. The m.c. of the samples was between 7.5 - 11.2%. Aspergillus flavus was the predominant species of fungi infecting kernels collected from Cianjur, Sukabumi and Bogor during the wet season. The percentages of infected kernels collected from the three localities were between 41 - 100%, 36 - 96% and 83 -100%, respectively. Pitt et al. (1993) reported that the predominant fungus infecting groundnut collected from farmer storage, middlemen and retailer in Thailand was A. flavus, followed by A. niger. The percentages of kernels infected by R. stolonifer and Syncephalastrum sp. could not be determined, because their growth was very vigorous. Twenty species of fungi were isolated from samples collected from Cidolog during the dry and wet season. They were Acremonium strictum, Aspergillus candidus, A. flavus, A. niger, A. ochraceus, A. tamarii, A. wentii, B. theobromae, C. cladospo-rioides, E. chevalieri, E. repens, F. equiseti, F. longipes, F. semitectum, Papulaspora sp., Penicillium aethiopicum, P. citrinum, Pestalotia sp., Rhizopus stolonifer and Syncephalastrum sp. (Table 4). The predominant species of fungi infecting kernels collected from Cidolog during the dry season was A. wentii, with an infected percentage bertween 30 - 100%, while the percentage of kernels infected by A. flavus was between 0 - 91%. The m.c. of the samples collected from that location was between 9.9 - 14.1%. During the wet season, the predominant species of fungi was A. flavus. The percentages of kernels infected by the fungus in samples collected from FSS and MW were between 17 - 96%, while those in FHG were between 51 - 99%, and their m.c's were between 10.8 - 18.1% and 12.5 - 45.7%, respectively. According to Christensen and Kaufmann (1969) A. flavus can grow in seeds which have a moisture content of at least 18%. On the other hand Martin and Oilman (1976) reported that optimum moisture contents of natural substrate, which permitted the growth of A. flavus, were between 15 -25%. 22 Fungi isolated from groundnuts - OS. Dharmaputra & I. Retnowati Table 4. The fungal species and range of precentage of infected kernels of groundnut samples collected from Cidolog in different storage systems The percentages of kernels infected by A. flavus in FHG were higher than in samples collected from FSS and MW, because it was assumed that A. flavus infected the pods of groundnut before harves. According to Kozakiewicz (1989) A. flavus is cosmopolitan in distribution. It is found soil, and decomposes vegetation in stored cereals and other seeds as well as variety of food products. Dickens (1977) and Pitt (1989) reported that the invasion of groundnut by A. flavus take place before groundnuts are harvested. Damage of peanut kernels caused by insects and other small animals forces soil into close contact with kernels, and so facilitates the invasion of the fungus. 23 BIOTROPIA No. 9, 1996 CONCLUSION Fungi isolated from groundnuts collected during the dry and wet season from the farmer storage system (FSS), middlemen warehouses (MW), wholesalers (WS), retailer samples (RS), and freshly harvested groundnut (FHG) in Cidolog, Cianjur, Sukabumi and Bogor showed the presence of a large number of species. A total of 25 fungal species were isolated from the 4 locations. The total fungal species isolated from samples collected from Cianjur, Sukabumi and Bogor during the dry and wet season were 23, while those from Cidolog were 20. There were variations in the percentage of kernels infected by each fungal species from different sources and localities. The predominant fungi in samples collected from Cidolog and Sukabumi during the dry season were Aspergillus wentii, while those collected from Cianjur and Bogor were A. niger. The predominant fungus in samples collected from the 4 locations during the wet season was A. flavus. The moisture contents of groundnuts collected from FHG, FSS and MW were higher than of those collected from WS and RS. There were also variations in the percentages of damaged kernels of samples collected from different sources and localities. ACKNOWLEDGEMENT The authors gratefully acknowledge the Australian Centre for International Agricultural Research for their financial support. The authors are also thankful to Dr. J.I. Pitt, Leader of ACIAR Project 8806; Dr. Ruben C. Umaly and Dr. Gloria L. Enriquez, the former Deputy Director and Deputy Director of SEAMEO BIOTROP, respectively, who gave advice and suggestions; Mr. Sunjaya, scientist of the Tropical Pest Biology (TPB) Programme, SEAMEO BIOTROP; the technicians of the Laboratory of Plant Pathology, TPB Programme, SEAMEO BIOTROP; and to Dr. A.D. Hocking, for the confirmation of fungal identification. REFERENCES BULOG, 1981. BULOG procurement on quality and purchasing cost of secondary crop for FY 1981/1982. Letter of Appoinment, Head of National Logistics Agency, No. KEP-311/KA/10/-1981. BUTLER. W.H. 1974. Aflatoxin. In Purchase, I.F.H. (ed.). Mycotoxins. Elsevier Scientific Publishing Company, Amsterdam: 1- 28. 24 Fungi isolated from groundnuts - OS. Dharmaputra & I. Retnowati CHRISTENSEN, C.M. 1980. Needed: Research on storage molds in grains, seeds and their products. Plant Disease 64(12): 1067- 1070. CHRISTENSEN, C.M. and H.H. KAUFMANN. 1969. Grain storage; the role of fungi in quality loss. 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