Introduction

Alfalfa (Medicago sativa L.) is one of the most important perennial livestock forage crops in the world. In Saudi Arabia, alfalfa is the most important routinely cultivated legume. It ranks first in cultivated forage crops and occupies more than 30% of the cultivated area (Abdel-Aziz et al. 2008). The plant is recognized as a widely adapted agronomic crop, an effective source of biological N2 fixation, one of the highest sources of protein yield per hectare, and an attractive source of nectar for honey bees (Stuteville and Erwin 1990).

Seeds play a vital role in the production of healthy crops but can carry some important fungi that result in considerable loss of yield. Some seed-borne fungi have been found to be very destructive, causing seed rot, decreased seed germination and/or pre-/post-germination death (Abul-Hayja et al. 1983; Al-Kassim and Monawar 2000). Several reports of seed-borne mycoflora attacking alfalfa seeds have been published worldwide. Alternaria alternata, Aspergillus spp., Bipolaris setariae, Botrytis cinerea, Cephalosporium spp., Cladosporium spp., Colletotrichum dematium, Curvularia lunata, Epicoccum purpurascens, Fusarium equiseti, F. moniliforme, F. pallidoroseum, Macrophomina phaseolina, Microdochium dimerum, Penicillium spp., Phoma spp., Stemphylium spp., Trichoderma spp., Trichothecium roseum, Ulocladium spp., Verticillium albo-atrum and Verticillium spp. have been detected as the most important seed-borne fungi on alfalfa seed samples collected from different countries (Mathur and Manandhar 2003). In Saudi Arabia, there is no literature available on seed-borne fungi attacking alfalfa; however, efforts are being made in the kingdom to develop high resistance alfalfa cultivars.

A number of abiotic and biotic factors cause seed discoloration that may lower seed quality. Abiotic factors, including environmental stress such as a low temperature (Morrison et al. 1998), dampness and high humidity (especially during storage), and the genetic makeup of a cultivar (Gai et al. 2000) can greatly affect seed coat color and lower seed quality. Biotic stress in the form of seed-borne fungi reflects a direct impact on seeds. Many fungi are serious parasites that attack primordial and mature seeds causing quantitative and qualitative yield reduction. Other fungi, including saprophytes and weak parasites, may lower the quality of seeds by causing discoloration, which may seriously depreciate the commercial value of seeds. Many seed-borne fungi infect the seed coat, causing conspicuous necrotic black, or gray-brown discoloration (Neergaard 1979). The present study was undertaken to detect alfalfa seed-borne fungi in Saudi Arabia, and to determine the cause of seed discoloration in alfalfa.

Materials and methods

Samples

Fifteen seed samples of local alfalfa cultivar (Hegazy) collected from growing fields in different governorates of Riyadh region in Saudi Arabia (Al-Deri'yya, Hraymla, Afeef, Al-Kharj, Al-Zulfi, Al-Dawadmy, Rmah, Al-Hareeq, Hotat Bani Tameem, Al-Aflaj, Al-Quway'iyah, Shaqra and Al-Majma'ah beside Riyadh city) during 2010 were used in this study.

Seed health testing

Conventional seed health testing was carried out to detect fungi associated with alfalfa seeds. Standard moist blotter (SMB) and deep-freezing blotter (DFB) methods were used (ISTA 1999). The proposed alkaline seed-bed method was also applied (Elwakil and Ghoneem 2002). A total of 400 seeds from each sample was used and the percentages of fungi recovered from each method were tabulated.

Standard moist blotter method

Twenty-five seeds were plated in a 9-cm diameter Petri-dish containing three layers of blotter paper moistened with sterilized tap water. The plates were incubated at 20±2°C for 7 days under cool white fluorescent light with alternating cycles of 12-h light and 12-h darkness.

Deep freezing blotter method

After plating seeds as described in the SMB method, the dishes were incubated at 20°C for 24 h and then transferred to a −20°C freezer for 24 h. This was followed by a 5-day incubation at 20±2°C under cool white fluorescent light with alternating cycles of 12-h light and 12-h darkness.

Alkaline seed-bed method

Three layers of blotter paper were soaked in tap water alkalinized with KOH or NaOH at pH 10. The blotters were placed in 9-cm diameter Petri-dishes and 25 seeds were distributed on each seed-bed as described (ISTA 1999). The plates were incubated at 20±2°C under cool white fluorescent light with alternating cycles of 12-h light and 12-h darkness.

Seven days later, the incubated seeds were examined under a stereoscopic microscope at 6–50X magnification to determine the presence and morphological characteristics of any seed-borne fungi. Pure cultures of the examined fungi were obtained using single spore and hyphal tip techniques. The isolated fungi were maintained on slants of potato carrot agar for further studies.

Seed-borne fungi associated with discolored alfalfa seeds

Seed samples were subjected to preliminary inspection for seed discoloration either by the naked eye or under a low power stereoscopic microscope. Alfalfa seed samples that exhibited 20% or more of discoloration symptoms were selected for further study. NaOH alkaline treatment was used to determine the presence of fungi on discolored alfalfa seed. After incubation, seeds were examined and seed-borne fungi were identified.

Identification of fungi

Media used for the identification of fungi were: carnation leaf agar (CLA) (Fisher et al. 1982) [pieces of sterilized carnation leaves; agar (10 g); distilled water (1 L)]; cornmeal agar (CMA) [cornmeal (20 g); peptone (20 g); glucose (20 g); agar (15 g); distilled water (1 L); pH 6.5]; Czepek’s solution agar (CzA) [NaNO3 (3 g); K2PO4 (1 g); KCL (0.5 g); MgSO4⋅7H2O (0.5 g); FeSO4⋅7H2O (0.01 g); sucrose (30 g); agar (20 g); distilled water (1 L); pH 6.5]; malt extract agar (MEA) [malt extract (20 g); peptone (1 g); dextrose (20 g); agar (20 g); distilled water (1 L); pH 6.5]; oatmeal agar (OA) [oatmeal (20 g); yeast extract (0.5 g); agar (20 g); distilled water (1 L); pH 6.5]; and potato dextrose agar (PDA) [potato dextrose (20 g); agar (20 g); distilled water (1 L); pH 6.5].

Fungi were identified according to their cultural properties, morphological and microscopical characteristics as described by Raper and Fennel (1965); Ellis (1971); Chidambaram et al. (1973); Domsch et al. (1980); Booth (1977); Burrges et al. (1988). For determination of morphological structures, portions of fungal growth were mounted in lacto-phenol cotton blue stain on clean slides as proposed by Sime and Abbott (2002). The prepared slide was examined under a light microscope using the 40X and 100X objectives for vegetative mycelium; septation, diameters, conidiophores (sporangiophores) and the reproductive structures: conidia, sporangiospores etc. Fungal colonies were examined under the 10X (low power) objective of the microscope. The colonial characteristics of size, texture and color of the colony were investigated.

Germination percentage

A total of ten collected seed samples, showing more than 20% symptoms of seed discoloration was used in this study. The standard blotter test recommended by the International Seed Testing Association (ISTA 1999) was used to determine the germination percentage of each seed lot. For each sample of normal and discolored seeds, 400 seeds were taken at random, laid out on moistened absorbent paper, which was then wrapped up in a wet cotton towel and placed inside a plastic bag. The paper was kept moist for 10 days at 20 ± 2°C under cool white fluorescent light with alternating cycles of 12-h light and 12-h darkness. The numbers of germinated seeds following incubation for 10 days were counted.

Statistical analysis

Comparison of means was performed using LSD at P = 0.05 and the standard error was calculated using the statistical analysis software “CoStat 6.34” (CoStat 2005) .

Results and discussion

Seed health testing methods

A total of 24 genera and 35 species of fungi were isolated from alfalfa seeds using the techniques mentioned in Materials and methods. Data from identification of the alfalfa seed-borne fungi thus isolated are summarized in Table 1. Considerable differences in the frequency of the presence of alfalfa seed-borne fungi among the SMB, DFB and the alkaline seed-bed techniques were recorded (Table 2). Of the three tested methods used, NaOH alkaline treatment yielded the highest number of fungi (21 genera and 32 species) as compared to KOH alkaline treatment, SMB and DFB methods.

Table 1 Identification data of the isolated alfalfa seed-borne fungi
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Table 2 Occurrence of alfalfa seed-borne fungi using standard moist blotter (SMB), deep-freezing blotter (DFB) and alkaline seed-bed methods
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Data of incidence percentages and occurrence ranges of the isolated fungi revealed that Alternaria alternata, Cladosporium sp., Aspergillus flavipes , A. flavus , A. niger, A. nidulans, A. tamarii, Stemphylium sp. and Penicillium sp. were the most abundant. Fusarium incarnatum was the most frequently isolated dominant Fusarium species, followed by F. equiseti, while F. verticillioides and F. solani were detected at low percentages. The data indicated also that the SMB method enhanced the recovery of fast-growing saprophytes as Cladosporium sp. (100%), A. flavipes (86.7%), A. flavus (86.7%), A. nidulans (80%) and A. tamarii (73.3%), which commonly attack nonviable (dead) seeds, while the DFB method enhanced the recovery of Stemphylium sp. (80%) and Phoma medicaginis (33.3%).

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 legumes crops (Booth 1977). Phoma medicaginis var. medicaginis and Stemphylium botryosum were reported as being responsible for leaf spotting and defoliation, which result in a significant reduction of forage yield and quality (Campbell and Madden 1990; Stuteville and Erwin 1990; Nutter et al. 2002; Guan and Nutter 2002). In addition to quantitative yield losses, some foliar pathogens including Fusarium spp. and Alternaria spp. can lower forage quality by producing mycotoxins (Scudamore and Livesey 1998). In Australia, foliar diseases have been reported to reduce Medicago herbage yield by 16–20%, seed yield by 37% and seed weight by 7–13% (Barbetti 1995).

The alkaline blotter method enhanced the recovery of slow-growing seed-borne fungi beyond that obtained with the SMB and DFB methods. Both treatments of the alkaline seed-bed method were efficient in the detection of F. incarnatium (80%) on alfalfa seeds. Treatment with KOH increased the frequency of the detected Cephalosporium acremonium (73.3%) on the tested seeds, compared to 13.3%, 60% and 60% in SMB, DFB and alkaline NaOH methods, respectively. Both alkaline treatments showed an equal increase in the frequency of F. verticillioides and A. glaucus found on alfalfa seeds compared with the SMB and DFB methods. On the other hand, no differences were observed among the two alkaline seed-bed techniques and the DFB method in the frequency of the detected Cephalosporium sp. on alfalfa seeds, and no differences were observed among all techniques in the frequency of the detected Rhizoctonia solani and Colletotrichum trifolii. On the other hand, the incidence of saprophyte fungi was lower in the case of the alkaline seed-bed method than with the other techniques.

The results presented here show that the alkaline KOH seed-bed proved an efficient method of detecting slow-growing fungi such as Cephalosporium sp on alfalfa seeds. In addition, it was also efficient in detecting other pathogenic fungi, e.g., F. verticillioides. These results are in agreement with the findings of Ghoneem et al. (2009), who reported abundant growth of slow-growing Verticillium dahliae and Cephalosporium sp. on fennel seeds when using alkaline seed-bed techniques at pH 9.5. The ability of such fungi to grow abundantly may be due to the high pH of the medium, which is favorable for the growth of this fungus (Ghoneem et al. 2009). Abo-Ellil (1999a, b) reported that the uptake of sugars in the V. lateritium cell increases when the alkalinity of the medium increases. This enhancement may be attributed to the alkaline ions (K+ or Na+) that replace H+ in the fungal cell and increase the uptake of nutrients in the cells (Horikoshi and Akiba 1982; Elwakil and Ghoneem 2002). It was found also that alkalinized seed-beds help analysts at seed-health laboratories to more easily detect slow-growing fungi compared with other methods.

Germination percentages

Germination of ten normal seed samples obtained from different locations varied significantly from 58.3% to 88.7% depending on the location of the sample (Fig. 1). The highest germination was recorded in seed samples obtained from Al-Quway'iyah (88.7%), followed by shaqra, Hotat Bani Tameem and Rmah (83.6, 82.9 and 72.3%, respectively); while the lowest germination was reported in the sample collected from Al-Dawadmy (58.3%). The data indicated that seed discoloration decreased seed germination significantly (by 26.3–60%). Highest germination was recorded in the discolored seed sample obtained from Al-Quway'iyah (60%), followed by AL-Hareeq and Hotat Bani Tameem (55.8%, for both); while the lowest germination was recorded in the sample collected from Al-Dawadmy (26.3%).

Fig. 1
figure 1

Germination percentages of normal and discolored seed samples from different governorates. Least significant difference (LSD; P = 0.05) = 3.41 for normal seeds and 1.84 for discolored seeds

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Symptoms caused by certain fungi associated with discolored alfalfa seed samples are characteristic and more easily distinguishable by severe infection. The discolored seeds appeared smaller and shriveled, with variable color from light brown to dark brown or black. Infected seeds are usually flattened, elongated and deformed with wrinkled seed coat (Fig. 2).

Fig. 2a,b
figure 2

Alfalfa seeds. a Healthy (x5). b Discoloration symptoms caused by certain fungi (x5)

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Seed-borne fungi associated with alfalfa seed discoloration

Data in Table 3 show a total of 15 genera and 26 species of fungi isolated from normal and discolored alfalfa seeds using the NaOH alkaline method. In general, discolored seeds yield a higher number of fungal species than normal seeds. Cladosporium sp., Alternaria alternata, Aspergillus flavus, A. niger, A. tamarii, followed by A. flavipes and A. nidulans were most commonly isolated fungi. Fusarium incarnatum was the frequently dominant isolated Fusarium species at frequency percentage of 60% and mean intensity percentage of 10.6%. The average frequency and intensity percentage of several fungal species that have pathogenic references were highest on discolored seeds compared to normal seeds. Stemphylium botryosum was recorded at a maximum frequency of 70% and intensity percentage of 2.45%, while the lowest intensity percentages were for F. equiseti (1.05%), Colletotrichum trifolii (0.95%) and Myrothecium verrucaria (0.5%).

Table 3 Occurrence of fungi on normal and discolored alfalfa seeds using the alkaline seed-bed method (NaOH)
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The presence of Aspergillus spp., especially A. niger and A. flavus, on alfalfa seeds in higher frequencies, and their association with ungerminated seeds confirmed the findings that species of Aspergillus occur as saprophytes that may cause low germination in seeds (Christensen 1967; Dawar 1994; Sultana and Ghaffar 2009). Shafie and Webster (1981) reported that these fungi may invade seed pericarp and embryos. Contamination by Alternaria and Cladosporium sp. is common in many kinds of seeds, which may depreciate their market value heavily because of their sooty appearance (Neergaard 1979). Many seed-borne pathogenic fungi, e.g., F. equiseti, F. semitectum and Macrophomina phaseolina were reported to infect the seed coat causing conspicuous necrotic black, brown-to-gray discolorations in mungbean and Phaseolus aureus (Nath et al. 1970). These fungi associated with seeds produce proteolytic enzymes that help in the spread and development of plant pathogens (Aboughania and Faraj 1978). Therefore, discolored seeds are not good for cultivation as this will not only lead to low yields but also contaminate other seeds.