This study was conducted to assess the incidence of seed-borne fungi and their
... Standard blotter method was used to isolate seed-borne fungi, whereas the.
Pak. J. Phytopathol., Vol. 21(1): 55-60, 2009. SEED BORNE FUNGAL PATHOGENS ASSOCIATED WITH PEARL MILLET (PENNISETUM TYPHOIDES) AND THEIR IMPACT ON SEED GERMINATION Azhar Hussain, Safdar A. Anwar1, G. M. Sahi1, Q. Abbas and Imran Department of Food, Agriculture and Chemical Technology, Karakoram International University, Northern Areas, Gilgit 1 Department of Plant Pathology, University of Agriculture, Faisalabad-38040, Pakistan. Corresponding author’s e-mail:
[email protected] ABSTRACT This study was conducted to assess the incidence of seed-borne fungi and their impact on seed germination of pearl millet (Pennisetum typhoides). Standard blotter method was used to isolate seed-borne fungi, whereas the impact of seed associated fungal pathogens on seed germination was determined by rolled paper method. The fungal pathogens incidence, their frequency of occurrence and impact on germination varied from cultivar to cultivar. The fungi belonging to nine genera were isolated and identified from the seeds of eight pearl millet cultivars collected from National Agricultural Research Centre (NARC), Islamabad. The highest fungal incidence (35%) was observed on seeds of pearl millet cv. MC-94-11-RM, followed by cv. KMV-96772 (34.5%), and the lowest incidence (15%) on cv. RARI-Comp-II. Whereas, the seeds of remaining five cultivars had intermediate fungal incidence. Most frequently isolated fungi were Alternaria alternata (35.5%), Fusarium semitectum (33.5%), and Curvularia lunata (23.5%). The presence of other associated mycoflora was less than 20%. Germination test of these naturally infected seeds of pearl millet cultivars showed above 80% germination with variable number of normal and abnormal seedlings for each cultivar. The mycoflora associated with seeds reduced germination ability of the seeds. Our findings necessitate the use of fungicide treated seeds before sowing. Key words: Seedborne fungi, frequency, germination of pearl millet seeds, incidence. vital component of food in the developing world (FAO and ICRISAT, 1996). This crop is best suited to harsh climate of seasonally hot drought prone semi-arid region of Africa and Indian sub-continent. Pearl millet is an important coarse grain summer cereal crop of Pakistan that is planted in rainfed areas located in Attock, Bahawalnagar, Bahawalpur, Bunnu, Chakwal, Dadu, D. I. Khan. Gujrat, Hyderabad, Jhelum, Karak, Khairpur, Khuzdar, Lorali, Mianwali, Nawabshah, Rawalpindi, Sanghar, and Sibbi. Several diseases are responsible for low productivity but fungal diseases are the most important. However little information is available on the fungal pathogens of millet seeds in Pakistan. It suffers from many seed borne pathogens that cause reduction in germination at initial and foliage stage. At the maturity stage Alternaria alternata, Aspergillus flavus, A. niger and Fusarium semitectum deteriorate the quality and quantity of developing floral parts that reduces grain yield at maturity (Williams and McDonald, 1983). Pearl millet pathologists working at International Crop Research Institute for the Semi Arid-Tropics estimated global yield losses of 45%, 32%, 9%, 3%, and 1% due to downey mildew, striga, smuts, rusts,
INTRODUCTION Seeds are regarded as highly effective means for transporting plant pathogens over long distances. Numerous examples exist in literature for the international spread of plant diseases as a result of the importation of seeds that were infected or contaminated with pathogens (Agarwal and Sinclair, 1996). Seed-borne pathogens have been involved in seed rots during germination and seedling mortality leading to poor crop stand (Khalid et al., 2001); reduction in plant growth and productivity of crops (Williams and McDonald, 1983; Kubiak and Korbas, 1999; Dawson and Bateman, 2001.). The seed-borne pathogens associated with seeds externally or internally may cause seed abortion, seed rot, seed necrosis, reduction or elimination of germination capacity, as well as seedling damage resulting in development of disease at later stages of plant growth by systemic or local infection (Bateman and Kwasna, 1999; Asif et al., 2001; Ijaz, et al., 2001; Khanzada et al., 2002). Pearl millet (Pennisetum typhoides), an important staple food for millions of people inhabiting the semi-arid tropics, is a major source of calories and a
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and viruses, respectively. (FAO and ICRISAT, 1996). The present study was conducted to identify and investigate the association of seed borne mycoflora with eight pearl millet cultivars and their effects on seed germination in eight seed samples of various cultivars of pearl millet, collected from National Agricultural Research Centre, Islamabad-Pakistan
and recorded using ISTA rules (1993). Fungi associated with abnormal seedlings were recorded. The emerged seedlings were graded as normal or abnormal as defined by Anwar et al. (1994). Normal seedlings: Seedlings with well-developed root and shoot; free of disease symptoms. Abnormal seedlings: Seedlings with under developed either root or shoot or both and exhibiting disease symptoms. The fungi on abnormal seedlings and rotted seeds were examined under stereobinocular microscope. The infected portions were cut and plated on PDA for fungal growth and confirmation of fungal association.
MATERIALS AND METHODS Collection of seed samples: This study was carried out on sixteen seed samples of eight cultivars of pearl millet including MC-94-11-RM, 18BY, GhanaWhite, K-8206, Bulk-7704, RARI-Comp-I, RARIComp-II and KMV-96772 which were collected from National Agricultural Research Center, Islamabad, Pakistan.
RESULTS During this study, a total of nine genera and twelve species of fungi were isolated and identified from seeds of eight pearl millet cultivars. The percentage of seedborne fungal infection ranged from 15 to 35% (Table.1). The seed samples of two millet cultivars including MC-94-11-RM and KMV – 96772 exhibited highest infection of 35% and 34.5%, respectively. Whereas, other five cultivars namely; Ghana-White, K-8206, 18BY, Bulk-7704, RARIComp-I had intermediate infection of 28.5%, 27.5%, 25.5%, 19%, 18.5%, respectively. Millet cv. RARIComp-II exhibited the lowest (15%) infection. The healthy seed percentage ranged from 65% to 85%. The fungal pathogens found associated with seeds were, Alternaria alternata, Aspergillus alba, A. flavus, A. niger, Bipolaris spp, C. lunata, Drechslera spp., F. semitectum, F.moniliforme, Helminthosporium spp, Penicillium spp., and Rhizopus spp.. Three fungal pathogens including A. alternata, F. semitectum and C. lunata were dominant among the fungal pathogen population with an incidence of 35.0%, 33.5% and 23.5%, respectively. The incidence of Aspergillus alba and F. moniliforme were same, but higher than that of Bipolaris spp.. The fungal incidence of A. flavus (14%), A. niger (19%), Drechslera spp. (10.5%), Penicillium spp. (7.5%), Rhizopus spp. (13.5%), and Helminthosporium spp. (8.5%) was also recorded (Table 2). The highest range of fungal infection on seeds was1.0-4.0 of Rhizopus spp., lowest (0.5-2.0) of A. alba and F. moniliforme, and intermediate of other fungal pathogens (Table 2).
Seed health testing: Seeds were examined for seedborne mycoflora by using standard blotter paper method (ISTA, 1985). A working sample of four hundred seeds was randomly taken from composite sample of each cultivar to isolate and identify the fungal pathogens. A total of forty sterilized Petri dishes each of 9-cm diameter were used to plate the seeds of each cultivar. Ten seeds of each cultivar were platted in each Petri-plate containing three layers of sterilized water-soaked blotter paper and incubated at 25 ± 2ºC in 12 h alternating cycles of light and darkness for eight days. Each seed was examined under stereobinocular microscope at 40X to record the association of fungi based on their habit characteristics. Fungi were isolated from seeds and cultured on Potato Dextrose Agar (PDA) for further identification with the help of various keys (Raper and Fennel, 1965; Booth, 1971; Barnett & Hunter, 1972; Ellis, 1980).The results have been expressed in percentages. Seed germination testing: Seeds were evaluated for germination by using the standard rolled paper method (ISTA, 1985).One hundred untreated seeds of each cultivar drawn randomly, were allowed to germinate between two layers of autoclaved blotter paper at 25+ 2OC for 12 days under fluorescent light to investigate the influence of seedborne fungi on seed germination. After incubation, number of germinated and ungerminated (including rotted) seeds, normal and abnormal seedlings were counted
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Table 1. Fungal pathogens isolated from seeds of eight cultivars of pearl millet. Cultivars tested
Associated fungal pathogens
MC-94-11-RM
Seed percentage Healthy Infected 65 35
18BY
74.5
25.5
A. alternata, Bipolaris spp., C. lunata, Helminthosporium spp., Penicillium spp., Rhizopus spp.
Ghana-White
71.4
28.5
A. flavus, A. niger, Bipolaris spp., C. lunata, Penicillium spp., Rhizopus spp.,
K-8206
72.5
27.5
A. alba, A. niger, Drechslera spp., F. semitectum, F. moniliforme, Helminthosporium spp.
Bulk-7704
81
19
A. alba, A. flavus, C. lunata, Drechslera spp,
RARI-Comp-I
81.5
18.5
A. alternate, A. niger, C. lunata, F. semitectum Helminthosporium spp.
RARI-Comp-II
85
15
A. alternata, Bipolaris spp., C. lunata, F. moniliforme Penicillium spp., Rhizopus spp
KMV-96772
65.5
34.5
A. alba, A. alternate, A. niger, Bipolaris spp.,Drechslera spp., F. moniliforme, F. semitectum.
Alternaria alternata, Aspergillus flavus, Bipolaris spp., Curvularia lunata, Drechslera spp., Fusarium semitectum, Helminthosporium spp.
Table 3. Percentage seed germination, number of normal and abnormal seedlings and fungal pathogens isolated from abnormal seedlings emerged from naturally infected seeds of eight cultivars of pearl millet. Condition of seedlings Cultivars Germinated (%) Fungal pathogens isolated from abnormal seedlings tested seed (%) Abnormal Normal MC-94-11-RM
81
65
16
18BY
89
72
17
Alternaria alternatea Aspergillus flavus, Bipolaris spp., Curvularia lunata, Drechslera spp., Fusarium semitectum, Helminthosporium spp. A. alternata, Bipolaris spp., Penicillium spp..
Ghana-White
88
60
28
A. flavus, C. lunata, Rhizopus spp.,
K-8206
87
59
28
A. alba, A. Niger, Drechslera spp., F. semitectum, F. moniliforme, Helminthosporium spp.
Bulk-7704
90
72
19
A. alba, A. flavus, C. lunata, Drechslera spp,
RARI-Comp-I
90
70
18
A. alternate, C. lunata, F. semitectum,
RARI-Comp-II
91
65
16
A. alternata, C. lunata,
KMV-96772
84
62
25
A. alba, A. alternata A. niger, Drechslera spp., F. semitectum.
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Table 2. Incidence and range of infection of fungal pathogens associated with seeds of eight cultivars of pearl millet Fungal pathogens Incidence [%] Range of infection* Alternaria alternata 23.5 1-3 Aspergillus alba 06.5 0.5-2 A. flavus
14.0
1.0-1.3
A. niger
19.0
1.0-3.5
Bipolaris spp.
05.0
1.0-3.0
Curvularia lunata
23.5
3.0-3.5
Drechslera spp.
10.5
1.5-3.0
Fusarium moniliforme
06.5
0.5-2.0
F. semitectum
33.5
2.0-2.5
Penicillium spp.
07.5
1.0-2.5
Rhizopus spp.
13.5
1.0-4.0
Alternaria alternata
35.0
3.0-3.5
Helminthosporium spp. *0 to 5 scale
08.5
1.0-3.0
leading to poor crop stand as well as foliage and inflorescence diseases at the adult stage resulting in both qualitative and quantitative loss in grain yield. Seedborne pathogens such as A. alternata, A. flavus, A. niger, Bipolaris spp., C. lunata and F. semitectum affect plant growth at seedling, foliage, and flowering stages (Williams and McDonald, 1983). The association of seedborne pathogens including A. alternata, C. lunata and Fusarium spp has been reported on various pearl millet cultivars causing deterioration in seed germination (Elisabeth et al., 2008). The high population of A. alernata, F. semitectum, and C. lunata on seeds might be responsible for deteriorating the seed quality and had adverse effects on plants growth and development. Ingle and Raut (1994) reported that A. alternata, Bipolaris spp, C. lunata and Drechslera spp cause grain failing to the physiological maturity stage. Fusarium semitectum and F. moniliforme were frequently isolated with seeds and abnormal seedlings. Fusarium is a highly pathogenic fungus and its different species have been reported to cause seed rot, seedling blight and wilt in a number of crops (Karim, 2005). Similarly among Drechslera spp., state of Cochlibolus spicifer and D. halodes showed seed rot and brown rot symptoms in seedlings. Alternaria alternata and Curvularia lunata caused delay or reduction in seed germination due to decay of seeds. Similar results have been reported by Mishra and Prakash (1975). The high incidence of field fungal pathogens of seed suggests that the seed got contaminated in the field during harvest. The association of field and storage fungal pathogens with millet seed has been reported. Mishra and Daradhiyar (1991) reported that A. flavus and A. parasiticus were the predominant species of millet seeds during the rainy season, whereas the population of Aspergillus, Penicillium, Fusarium, Rhizopus, Helmithosporium and Curvularia was low. Fungal pathogens including Aspergillus niger, and species of Penicillium, Fusarium, Penicillium, Rhizopus, and Helminthosporium. Isolated from rotted seeds and abnormal seedlings have been reported as cause of seed decay and damping-off seedling of millet (Makun et al., 2007). The occurrence of storage fungal genera namely Aspergillus and Penicillium with seed indicates that the seed become contaminated during storage. Presence of A. niger and A. flavus on seeds, abnormal seedlings, rotted and ungerminated seeds confirmed the findings that species of Aspergillus though occur as saprophytes may cause low germination in seeds (Shakir and Mirza, 1992; Dawar, 1994). Aspergillus flavus produces toxic metabolites that result in reduction of shoot and root elongation (Jain
Although, the germination of naturally infected seeds of all pearl millet cultivars was above 80% but the number of abnormal seedlings were higher over that of normal ones (Table 3). The highest number (72%) of abnormal seedlings emerged from seeds of two millet cv.18BY and Bulk-7704 while the lowest number (59%) came from seeds of cv. K 8206. Whereas, other five cultivars had intermediate number of abnormal seedlings DISCUSSION Good seed is recognized as an important input in any agricultural production system. One of the important aspects of good seed, beside high germination and purity, is that the seed should be free from pathogens. Pearl millet suffers from many seedborne pathogens that cause reduction in germination at initial stage
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and Pathak, 1996). Aspergillus niger is damaging storage fungus that deteriorates the seed quality and reduces seed germination (Ijaz, et al., 2001). Moulds associated with seeds of pearl millet cultivars have been reported pathogenic to human beings due to their ability to produce toxins, which might be responsible for causing respiratory diseases. Hough, (1991) found that A. fumigatus caused lungs disease to field workers due to inhalation of the fungal spores. Whereas, Rhizopus spp. caused food spoilage and occasionally caused fatal infections in humans (Badau. 2006). The effect of seedborne fungi on seed germination was evident. The field fungus A. alternata was predominantly associated with seeds of seven cultivars. This fungus caused pre-emergence decay of seeds and leaf spot at the foliage stage of millet (Mishra and Prakash, 1975). The seed germination was reduced by fungi (Table 3). The fungi had been reported to cause loss of seed germinability leading to (Haikal. 2008), reduction in seed germination and seedling diseases (McGee et al, 1980). Fusarium moniliforme and A. alternata reduced germination and induced seedling blight (Konde et al., 1980; Karim, 2005). Curvularia species have been found frequently associated with seed, which cause leaf spots leading to development of abnormal seedlings (Sivanesan, 1990). The association of fungal pathogens with millet seeds demonstrates that the seeds are a major source of transmission of pathogens, which might have adverse effects at seedling and adult stage of plants. The emergence of abnormal seedling from naturally infected seeds and isolation of same fungal pathogens from such seedlings as that of seeds suggest the involvement of fungal pathogens in causation of abnormal seedlings. Our finding has demonstrated that associated fungal pathogens reduce the germination ability of seeds, which causes poor crop stand, a major constraint of low harvested crop yield. These results strongly suggest the use of seed treatment with fungicide before planting to escape the infection by seed/soil borne fungal pathogens of seedlings. Breeding material must be tested for their health status for development of new varieties.
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