American Journal of Plant Sciences, 2010, 1, 138-146
doi:10.4236/ajps.2010.12018 Published Online December 2010 (http://www.SciRP.org/journal/ajps)
Maintenance of Ephedra alata Seeds Viability via
Storage Containers
Abdulaziz A. Al-Qarawi, Elsayed F. Abd_Allah*
1Plant Production Department, College of Food & Agriculture Sciences, King Saud University, Riyadh, Saudi Arabia.
Received October 22nd, 2010; revised November 19th, 2010; accepted November 22nd, 2010.
ABSTRACT
Sequential incubation of seed samples yielded 20 fungal species belonging to 13 genera. The prevalent genera were
Aspergillus (A. flavus and A. parasiticus), Fusarium (F. moniliforme and F. oxysporum) and Penicillium (Penicillium
sp.). Such seedborne fungi differ in their coloniza tion in different parts of seeds with most of them are colon ized in seed
coat, endosperm, and embryo of the seed. The usage of different storage containers for storing seeds indicated that the
cotton cloth bags were the most favorable ones as they maintain seed moisture content (SMC) below the critical level
resulting in minimum seed deterioration compared with other seed storage containers.
Keywords: Seedb orne Fungi, Ephedra alata, Sto ra ge C ontainers, Saudi Arabia
1. Introduction
Ephedra alata Decne, a gymnosperm belong to family
Ephedraceae, is one of the oldest range and medicinal
herb known in Saudi Arabia as well as in different
rangelands in the world [5]. It was noted as it is accom-
panied with sand dunes formation in Saudi Arabia espe-
cially the mobile ones and therefore it is a very effective
sand-binder and resistant to desertification [3]. The foli-
age of E. alata have acceptable aroma and used as food-
stuff for animals especially camels, cattle and sheep. The
deterioration of plant community had occurred in Saudi
Desert due to abiotic factors such as soil salinity [7], ed-
aphic factors of the soil [29] and soil drought [6]. On
other hand, the biotic stresses such as seedborne fungi
play an important and vital role in deterioration of seed
quality [1,2]. Seedborne fungi use different mechanisms
to deteriorate seeds such as production of both mycotox-
ins [2] and enzymes [26] which have attracted much at-
tention of our investigations.
Application of prophylactic fungicides is not the pre-
ferred choice in range seeds especially in the sheltered
areas. This raised the need to study safe alternative
strategies to control seedborne fungi that attack range
plants and might be transmitted to the aerial parts of the
plants [4]. Seed storage containers play an important and
considerable role in the production of healthy and vital
seeds [21, 25; 27]. The successfulness of the storage con-
tainers restricted with surrounding factors such as seed
nature, storage period, temperature, relative humidity,
and seed moisture content. Consequently, the production
of healthy and vital range seeds should managed through
integrated approach.
The present study was designed to investigate the
seedborne fungal flora of E. alata with special reference
to their incidence in different seed parts. Furthermore,
the effect of different storage containers on seed moisture
content (SMC), seed vigor (SV), aflatoxins accumulation,
and nutritional value of storage seeds (E. alata) was
studied.
2. Materials and Methods
2.1. Seed Samples Collection
Seed samples of Ephedra alata Decne (approximately
100 g seeds per sample, each in replicates) were col-
lected from King Khalid Center for Wildlife Research
and Development at Thumama which belonging to Ri-
yadh Region, Saudi Arabia during 2009. The samples
were collected in sterile cellophane bags and held at 2℃
until analyzed according to the International Seed Test-
ing Association [20].
2.2. Enumeration of Seedborne Fungi
From each seed sample, 400 seeds were surface-disin-
fected in Na-hypochlorite for two minutes followed
washing with several changes of sterile saline water (8.5
gm NaCl in 1000 ml distilled H2O). The disinfected

Maintenance of Ephedra alata Seeds Viability via Storage Containers
139
seeds were then incubated aseptically on potato dextrose
agar (PDA, Difco Laboratories, Detriot MI). Rose Ben-
gal (33 mg/ml, w/v) and Streptomycin (30 mg/ml, w/v)
were added as bacteriostatic agents. Surface-disinfected
seeds were spaced on Petri dishes (9 cm in diameter) and
incubated at 28 ± 2℃ for 10 days. Similarly, sur-
face-disinfected seeds were incubated on sterile moist
filter paper with cellulose wadding as blotters and incu-
bated as above. The fungal colonies developing around
the seeds incubated on both agar plates and filter papers
were examined and the fungi were identified micro-
scopically [15] and the level of incidence were recorded.
To enumerate seedborne fungi in different seed parts,
surface disinfected seeds were soaked in sterile water for
four hours and then dissected aseptically into different
parts (coat, endosperm, and embryo). Each seed part
aseptically used for investigation of seedborne fungi as
described above.
2.3. Determination of Seed Moisture Content
(SMC)
Each seed sample (100 gm) was grounded in a blender
and known weight of the resultant powder was dried in
an oven for 24 hours at 105℃, cooled in a desiccators
and reweighed. The moisture content (MC) is expressed
as percentage of the wet weight.
2.4. Storage Experiment
Seed samples (100 g each) were stored in four storage
containers namely polyethylene bags, cotton cloth bags,
tin cans and paper bags for six months at room tempera-
ture (25 ± 1℃) in the dark.
2.5. Seed Analysis
Vigor index (VI) of E. alata seeds were calculated Vigor
index (VI) for each treatment was determined according
to the following formula: VI = [mean of root length (cm)
+ mean of shoot length (cm)] X percent seed germination.
Nutritional values (total lipids, total nitrogen, ash content,
and fiber content) of stored seeds were determined ac-
cording to AOAC [11]. Aflatoxins (B1, B2, and G1) were
extracted and cleaned up from storage seeds using
chloroforme and cleaned using column chromatography
according to AOAC [10]. Quantitative estimation of
aflatoxins were carried spectrophotometrically [24] using
standard aflatoxins (Sigma) as reference.
2.6. Statistical Analysis
For each experiment, the data were statistically analyzed
using the analysis of variance procedure for completely
randomized design. Treatment means were compared
using the protected least significant difference (LSD)
analysis according to Daniel [14].
3. Results and Discussions
In the present investigation, 31 seed samples of E. alata
were analyzed to investigate their seedborne fungal flora
by means of standard blotter and agar plate methods (Ta-
ble 1 and Figure 1). As suggested by Abd_Allah and
Hashem [2] the comprehensive outcome recommended
Table 1. Incidence (%); case of isolations and occurrence remarks of seedborne fungal flora of E. alata following incubation
on agar plate and blotter.
Incidence (%) of fungal species
Fungal species
Blotter test Agar plate
Cases of isolation (No.) OccurrenceZ
Alternaria alternata 3.87 2.35 1 R
Alternaria sp. 4.36 2.21 3 R
Aspergillus flavus 14.65 16.37 42 H
Aspergillus nidulans 2.75 0 4 L
Aspergillus niger 8.36 12.75 13 M
Aspergillus parasitcus 18.52 21.18 37 H
Aspergillus sp. 16.32 12.16 7 L
Aspergillus terreus 7.15 8.13 6 L
Chaetomium globsum 0.95 1.15 1 R
Cladosporium sp. 1.78 0 2 R
Drechslera sp. 1.07 0 1 R
Epicoccum sp. 0.54 0.63 1 R
Fusarium moniliforme 3.27 3.85 7 L
Fusarium oxysporum 2.96 2.56 6 L
Penicillium sp. 7.36 8.81 7 L
Pythium sp. 0.62 0.87 1 R
Rhizoctonia solani 2.45 2.65 2 R
Rhizopus sp. 0.61 0.83 4 R
Sclerotium bataticola 1.54 1.35 2 R
Trichoderma sp. 1.27 1.75 3 R
Z: Out 31 E. alata seed samples. H = High occurrence (> 24 case); M = Moderate occurrence (from 12 to 24 cases); L = Low occurrence (from 6 to 11 cases)
and R = Rare occurrence (< 6 cases).

Maintenance of Ephedra alata Seeds Viability via Storage Containers
140
Figure 1. Detection of seedborne fungi associated with E. alata. A) Blotter and B) agar plate techniques used for enumeration
of seedborne fungi. C-H) Stereo-microscopy of E. alata seeds acquaint the incidence of seedborne fungi on different seed
parts.
that agar plate method was more conformable than moist
paper (standard blotter) method in yielding more fungal
flora (Table 1). The sequential incubation of seed sam-
ples yielded twenty fungal species belonging to thirteen
genera, which are new to mycoflora of E. alata in Saudi
Arabia (Table 1). The genes Aspergillus was the most
predominant and represented by 6 species namely A.
flavus, A. nidulans, A. niger, A. parasiticus, A. terreus
and Aspergillus spp.. Aspergillus was followed by Fusa-
rium (F. mon iliforme and F. oxysporum) and Penicillium
(Penicillium spp.), respectively. The other fungal genera
(Alternaria, Chaetomium, Cladosporium, Drechslera,
Epicoccum, Pythium, Rhizoctonia, Rhizopus, Sclerotium,
Trichoderma) were rare in their occurrence (Table 1).
Up to our knowledge, this is the first investigation for
seedborne mycoflora of E. alata especially in Saudi Ara-
bia although the contamination of Saudi herbs with toxi-
genic mycoflora was reported [8]. Nevertheless, similar
mycological studies for many other seeds showed that
Aspergillus and Fusarium were the most common genera
as seedborne fungi [2; 16]. The component plating of E.
alata seeds showed that most prevalent fungi colo-

Maintenance of Ephedra alata Seeds Viability via Storage Containers
141
nized seed coat (testa) followed by endosperm and em-
bryo, respectively (Table 2). The highest colonization of
storage aflatoxigenic molds such as A. flavus and A.
parasiticus were in seed coat (21.97 and 11.53%, respec-
tively) followed by endosperm (12.93 and 10.37%) and
embryo (12.93 and 6.47%) (Table 2). Similar mycologi-
cal investigations showed the colonization of A. flavus
and A. parasiticus in different seed parts involving em-
bryo [2; 28]. Therefore, the role of seedborne fungi (es-
pecially aflatoxigenic) as one of the major source of seed
deterioration during storage should be studied throughout
integrated management to minimize the chances of fur-
ther storage losses and field infection [30]. It was re-
ported the alteration in moisture content (MC) of stored
seeds depends up on the hygroscopic nature of storage
containers [9]. Similarly, in our study both polyethylene
bags and tin cans caused no any significant alteration in
SMC (compared with the initial SMC), however cloth
and paper bags caused significant decrement in SMC
(Table 3). The retention of superior SMC recorded here
by both polyethylene bags and tin cans is probably at-
tributed to impervious nature of previous storage con-
tainers compared with either cloth or paper bags [21].
There was a strong relationship between both storage
periods and type of seed storage containers with seed
health expressed as root depth, shoot height, percentage
germination (Tables 4(a-d)). Prolonged storage periods
were accompanied with decrease in vigor index ranged
between slight and significant reduction depending upon
the nature of storage containers. Such recorded decrease
in vigor index (Seed germination, root depth and shoot
height) strongly agrees with Basay et al., [12] and corre-
lated with the alteration in SMC (Table 3) which act as a
key factor influencing seed physical properties [27] and
effectiveness of naturally seedborne fungal flora [2;9]
which play vital role in diminution of viability and vigor
of seeds [13]. The decrease in vigor index was significant
with polyethylene storage container followed by tin cans,
papers bags and cloth bags respectively (Table 4(d)). In
the same connection, the polyethylene as impermeable
storage container followed by tin cans, paper bags and
cloth bags, respectively caused significant alteration in
concentration of both O2 and CO2, which are the main
cause of deterioration of agricultural products [18].
Table 2. Detection of seedborne fungi (Incidence [%] of fungal species) in different seeds parts of E. alata using standard
blotter method.
Incidence (%) of fungal species
Fungal species Surface disinfected seeds Seed Coat Endosperm Embryo
Alternaria sp. 12.16 3.56 2.78 5.82
Aspergillus flavus 42.71 12.93 6.35 21.97
Aspergillus parasitcus 28.37 11.53 10.37 6.47
Fusarium moniliforme 3.45 1.86 0.93 0.66
Fusarium oxysporum 4.63 2.06 2.17 0.4
Sclerotium bataticola 4.36 1.89 1.75 0.72
Penicillium sp. 0.8 0.61 0.19 NDZ
Chaetomium sp. 1.62 0.86 0.76 ND
Drechslera sp. 0.86 0.51 0.24 0.11
Trichoderma sp. 0.12 0.09 0.03 ND
Pythium sp. 0.92 0.61 0.31 ND
ZND: Not detected under the experimental conditions.
Table 3. Effect of various storage containers on seed moisture contentZ (SMC) [%] of E. alata stored for different storage
periods (months).
Seed moisture content (SMC) [%] of E. alata stored for different storage periods (months)
Storage container
1 2 3 4 5 6 LSD at: 05
Polypropylene bag 9.58 9.54 9.53 9.51 9.44 9.41 0.2512
Cotton cloth bags 8.20 8.11 7.91 7.50 6.67 6.07 0.1947
Tin cans 9.63 9.57 9.50 9.45 9.43 9.39 0.1484
Paper bags 9.13 8.82 8.60 8.15 7.71 7.41 0.1955
L. S. D. at: 05 0.2337 o.11750.20670.19350.23780.2566
Z: Initial seed moisture content was 9.72 (%).

Maintenance of Ephedra alata Seeds Viability via Storage Containers
142
Table 4-a. Effect of various storage containers on root depth (cm) of germinating seeds of E. alata stored for different storage
periods (months).
Root depth (cm) of germinating seeds of E. alata stored for different storage periods (months)
Storage container
1 2 3 4 5 6 LSD. at: 05
Polypropylene bag 15.30 14.33 12.77 11.73 9.97 8.27 0.4398
Cotton cloth bags 18.60 18.43 17.97 17.50 15.67 15.27 0.7383
Tin cans 17.53 17.03 16.17 15.90 15.07 13.80 0.8323
Paper bags 18.27 17.60 17.03 16.53 16.00 15.07 0.6134
LSD at: 05 0.4280 0.4892 0.8117 0.4175 0.8044 1.0651
Table 4-b. Effect of various storage containers on shoot height (cm) of germinating seeds of E. alata stored for different stor-
age periods (months).
Shoot height (cm) of germinating seeds of E. alata stored for different storage periods (months)
Storage container
1 2 3 4 5 6 LSD at: 05
Polypropylene bag 9.63 8.97 8.40 7.30 6.37 4.83 0.5204
Cotton cloth bags 10.17 9.97 9.70 9.20 8.33 7.73 0.4836
Tin cans 8.97 8.67 8.07 7.50 6.40 4.90 0.4926
Paper bags 9.67 9.10 8.67 8.13 7.30 6.67 0.4438
L. S. D. at: 05 0.3689 0.4104 0.321 0.4644 0.6221 0.7590
Table 4-c. Effect of various storage containers on percentage germination of E. alata stored for different storage periods
(months).
Percentage germination of E. ala ta stored for different storage periods (months)
Storage container
1 2 3 4 5 6 LSD at: 05
Polypropylene bag 61.30 59.93 57.20 53.70 51.20 43.33 1.9266
Cotton cloth bags 70.17 69.00 65.03 63.00 59.00 55.20 1.2275
Tin cans 65.23 63.20 60.57 56.87 52.27 46.53 1.3697
Paper bags 69.30 65.27 61.70 57.13 53.73 51.07 1.6294
LSD at: 05 0.5040 0.9993 1.4174 2.1543 1.8938 2.2118
Table 4-d. Effect of various storage containers on seed vigor indexZ of E. alata stored for different storage periods (months).
Vigor index of E. alata stored for different storage periods (months)
Storage container
1 2 3 4 5 6 LSD at: 05
Polypropylene bag 1528.41 1396.45 1210.73 1022.09 836.27 567.67 60.3480
Cotton cloth bags 2018.46 1959.60 1799.26 1682.10 1416.00 1269.60 83.1640
Tin cans 1728.68 1624.24 1467.73 1330.68 1121.99 870.17 73.3190
Paper bags 1935.78 1742.62 1585.69 1409.29 1251.99 1109.85 49.980
L. S. D. at: 05 32.05 60.769 65.249 72.736 92.857 91.266
Z: Vigor index = Seed germination X [mean Root depth (cm) + mean Shoot length (cm)].
The effect of seed storage containers on aflatoxins pro-
duction was investigated for seeds of many crops how-
ever, this approach does not provide a comprehensive
view of the impact of range seeds. In our results, afla-
toxins production were found to be inferior with em-
ployment of cloth bags followed by paper bags, tin cans
and polyethylene bags respectively (Tables 5(a,d)). Such
inhibition agrees with the findings of Paramawati et al.,

Maintenance of Ephedra alata Seeds Viability via Storage Containers
143
Table 5-a. Effect of different storage containers and storage periods (month) on the natural contamination of E. alata seeds
with aflatoxin B1 (µg/Kg seed).
Natural contamination with aflatoxin B1 (µg/Kg seed) of E. alata seeds stored for different storage periods (months)
Storage container
1 2 3 4 5 6 LSD at: 05
Polypropylene bag 42.07 38.70 34.20 29.77 24.23 19.27 2.4825
Cotton cloth bags 14.53 12.00 6.20 2.70 0.00 0.00 5.8834
Tin cans 36.00 27.20 23.13 20.93 17.30 11.20 3.0896
Paper bags 27.70 24.93 22.93 14.50 9.17 0.00 3.2088
L. S. D. at: 05 4.3322 2.6583 5.9746 5.7786 2.1357 2.1357
Table 5-b. Effect of different storage containers and storage periods (month) on the natural contamination of E. alata seeds
with aflatoxin B2 (µg/Kg seed).
Natural contamination with aflatoxin B2 (µg/Kg seed) of E. alata seeds stored for different storage periods (months)
Storage container
1 2 3 4 5 6 LSD at: 05
Polypropylene bag 92.17 85.30 77.80 73.27 69.03 63.37 4.1764
Cotton cloth bags 45.07 34.93 28.63 18.50 11.17 9.50 3.5914
Tin cans 71.50 65.10 58.30 51.30 38.10 27.50 6.7281
Paper bags 77.17 66.27 55.67 46.87 37.43 24.27 4.9676
L. S. D. at: 05 6.8605 4.6379 3.6884 6.4785 5.8013 3.2707
Table 5-c. Effect of different storage containers and storage periods (month) on the natural contamination of E. alata seeds
with aflatoxin G1 (µg/Kg seed).
Natural contamination with aflatoxin G1 (µg/Kg seed) of E. alata seeds stored for different storage periods (months)
Storage container
1 2 3 4 5 6 LSD at: 05
Polypropylene bag 35.87 30.03 26.77 21.50 18.40 11.23 2.5326
Cotton cloth bags ND ND ND ND ND ND 0.00
Tin cans 23.10 18.10 12.10 10.70 ND ND 4.3153
Paper bags 26.07 19.57 10.97 9.80 5.73 ND 2.6836
L. S. D. at: 05 5.0457 1.5258 1.3135 1.1019 4.6876 1.2105
ND: Not detected under the experimental conditions.
Table 5-d. Effect of different storage containers and storage periods (month) on the natural contamination of E. alata seeds
with total aflatoxinsZ (µg/Kg seed).
Natural contamination with total aflatoxinsZ (µg/Kg seed) of E. alata seeds stored for different storage periods (months)
Storage container
1 2 3 4 5 6 LSD at: 05
Polypropylene bag 170.11 154.03 138.77 124.53 111.67 93.87 7.8536
Cotton cloth bags 59.60 46.93 34.83 21.20 11.17 9.50 7.0388
Tin cans 139.23 113.03 89.77 77.60 60.47 35.47 10.7660
Paper bags 105.17 79.00 62.40 47.80 29.93 11.13 5.5776
L. S. D. at: 05 12.8030 7.2977 9.0200 8.8850 6.5909 3.6033
Z: Total aflatoxins (Sum. of B1 + B2 + G1).
[25]. It can explain in terms of the decrement alteration
in SMC inferior requisite level for growth and aflatoxins
production by seedborne fungi [2;9]. Data in our investi-
gation (Tables 6 (a-d)) shows that prolongation of stor-
age periods was accompanied with gradual deterioration in
the biochemical aspects of seed such as lipids, ash, total
nitrogen, and fiber contents. The employment of cloth
bags followed by paper bags, tin cans and polyethylene
bags respectively diminished such as sharpness in the
deterioration of seed biochemical aspects (Tables 6 (a-d)).

Maintenance of Ephedra alata Seeds Viability via Storage Containers
144
Table 6(a). Effect of different storage containers and storage periods (month) on nitrogen content (mg/g dry weight) of E.
alata seeds.
Nitrogen content (mg/g dry weight) of E. alata seeds stored for different storage periods (months)
Storage container
1 2 3 4 5 6 LSD at: 05
Polypropylene bag 32.48 30.06 28.6 28.37 27.48 26.50 0.1773
Cotton cloth bags 48.65 45.54 43.97 43.67 42.08 40.83 0.1002
Tin cans 39.65 35.86 32.88 31.31 30.76 31.073 0.1273
Paper bags 44.21 42.55 41.30 39.89 38.62 36.68 0.1119
L. S. D. at: 05 0.1609 0.0981 0.1092 0.1084 0.0853 0.2270
Table 6(b). Effect of different storage containers and storage periods (month) on fiber content (% of dry weight) of E. alata
seeds.
Fiber content (% of dry weight) of E. alata seeds stored for different storage periods (months)
Storage container
1 2 3 4 5 6 LSD at: 05
Polypropylene bag 5.03 4.49 4.25 5.02 3.84 3.48 1.1738
Cotton cloth bags 5.36 5.09 4.96 4.84 4.76 4.61 0.8601
Tin cans 5.20 4.39 4.07 3.77 3.38 3.13 0.1723
Paper bags 5.27 4.93 4.66 4.37 4.94 4.12 0.3679
LSD at: 05 0.2849 0.2246 0.2517 1.5158 1.1231 0.2852
Table 6(c). Effect of different storage containers and storage periods (month) on lipids content (% of dry weight) of E. alata
seeds.
Lipids content (% of dry weight) of E. alata seeds stored for different storage periods (months)
Storage container
1 2 3 4 5 6 LSD at: 05
Polypropylene bag 6.03 5.12 4.44 3.51 3.02 2.40 0.4421
Cotton cloth bags 7.63 7.21 7.03 6.86 4.29 6.50 0.4052
Tin cans 6.35 5.97 5.41 4.97 4.28 3.81 0.5560
Paper bags 7.20 6.83 6.40 6.06 5.83 5.50 0.3554
LSD at: 05 0.5457 0.4259 0.4288 0.3882 0.3366 0.6253
Table 6(d). Effect of different storage containers and storage periods (month) on ash content (% of dry weight) of E. alata
seeds.
Ash content (% of dry weight) of E. alata seeds stored for different storage periods (months)
Storage container
1 2 3 4 5 6 LSD at: 05
Polypropylene bag 4.69 4.35 4.11 3.92 3.59 3.45 1.6900
Cotton cloth bags 6.52 6.40 6.25 6.20 6.09 5.89 3.5538
Tin cans 5.70 4.94 4.23 3.87 3.71 3.56 1.9654
Paper bags 6.22 5.921 5.67 5.40 5.18 4.91 0.7454
LSD at: 05 4.1092 3.1294 1.3800 1.5175 0.6931 1.4413
Such results were in agreed with our data recorded pre-
viously concerning soybean seeds [17]. In this regard, the
alteration in SMC due to the employment of different
storage containers (Tabl e 3) was the main cause of seed-
borne fungal activities [2; 9] including production of
hydrolytic enzymes such as proteinase [26], lipase [23]
and lignocellulolytic enzymes [19] which were responsi-
ble for the biotic degradation of seed contents of protein,
lipids and fiber [22]. Our results indicate that these bio-
chemical events were correlated with maintenance of

Maintenance of Ephedra alata Seeds Viability via Storage Containers
145
high germination rate during storage. The success of
storage container to preserve seed viability recorded in
this investigation is still limited and more studies through
integrated seed management program to maintain healthy
range plants in the grassland is needed hence production
vital seeds are necessary for desert ecological mainte-
nance. These will be considered in the forthcoming in-
vestigation.
4. Acknowledgment
We acknowledge Dr. Abeer Hashem, Bot. & Microbiol.
Department, Faculty of Science, King Saud University,
Riyadh, Saudi Arabia for her excellent assistance in fun-
gal identification that contributed in this success of this
survey.
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