Journal of Phytopathology and Pest Management 9(1): 41-47, 2022
pISSN:2356-8577 eISSN: 2356-6507
Journal homepage: http://ppmj.net/
∗
Corresponding author:
Mohamed A. M. Hussein,
E-mail: mmon3m@gmail.com
41
Copyright © 2022
Control certain associated fungi of Date fruits
under Aswan conditions, Egypt
Mohamed A. M. Hussein*
Department of Plant Pathology, Faculty of Agriculture and Natural Resources, Aswan
University, Egypt
Abstract
Key words: date fruit molds, Rhizopus stolonifer, Aspergillus niger, ascorbic acid, fungal pathogens control.
This study aimed to identify fungi associated with mold development in date fruits. The
analysis revealed that Rhizopus stolonifer, Aspergillus niger, and Aspergillus flavus were the
most prevalent fungal species isolated from the samples. Specifically, six isolates of R.
stolonifer, nine isolates of A. niger, and five isolates of A. flavus were found in association
with palm date fruits. The application of ascorbic acid significantly reduced the incidence of
these fungal pathogens. These associated fungi pose a substantial health risk, as they can
produce hazardous metabolites that affect human health. Given the nutritional value of dates
and their importance for human wellness, the findings highlight the urgent need for effective
biological control methods to manage fungal diseases in edible fruits.
Hussein, 2022
42
1. Introduction
In 2019, the world's annual production of date
fruits exceeded 7,600,315 tonnes
(Anonymous, 2019). With about 1,465,030
tonnes of dates produced, Egypt is the world's
largest date producer (Anonymous, 2019),
The date palm is a dioeciously plant with
hundreds of variants, technically known as
Phoenix dactylifera of the monocot family
(Kacem-Chaouche et al., 2013). Soft dates,
semi–dry dates, and dry dates are all found in
Egypt's Nile valley, oases, and desert.
Furthermore, due to sexual reproduction,
there is a large number of seedling date
palms, some of which are invasive. Fruit
quality and multiplication of offshoots are
both highly desirable (Ragab, 2011). Fresh
fruits are susceptible to fungal contamination
in the field, during harvest, transportation, and
marketing, as well as with the client. Fungi
are important in the rotting of fruits because
of their pathogenicity to the collected
products. Fungi like Aspergillus spp. and
Rhizopus spp. flourish in high-moisture
conditions, particularly when gathered after
rain or high humidity (Ibrahim & Rahma,
2009). fungal studies of palm date fruits
studied previously (Bokhary, 2010; Alghalibi
& Shater, 2004; Gherbawy, 2001). A. flavus
has been found in date palm fruits by several
researchers (Ragab et al., 2001; Alghalibi and
Shater, 2004). Fungi such as Aspergillus
ochraceus and Aspergillus niger have also
been found in date fruits (Ferracin et al.,
2009; Heenan et al., 1998). The goal of this
study was to identify the related fungi and
assess the potential to reduce the occurrence
of the fungal diseases.
2. Materials and methods
2.1 Isolation and identification fungi
Dates were collected from the Aswan
governorate and gently washed, dried,
sprayed with a 1 percent sodium hypochlorite
solution, cleaned with sterile distilled water,
and dried on sterile filter paper sheet.
Samples were cut into small pieces and
aseptically put in Petri dishes on medium
Potato dextrose agar PDA. Petri dishes were
incubated at 27°C and were monitored every
day. Fungal colonies were purified on new
PDA media Petri dishes using the hyphal tip
technique. The morphological properties of
the isolated fungi were used to identify them
(Leslie and Summerell, 2008). The identity
was confirmed by the Plant Pathology
Department of Aswan University's Faculty of
Agriculture Sciences and Natural Resources
in Aswan, Egypt.
2.2 In vitro assessment
On PDA medium, in vitro experiments on the
mycelial growth of related fungal strains by
Ascorbic acid at three doses were conducted.
The plates were inoculated with 5 mm
diameter 7-day-old mycelia discs of various
strains, four petri dishes per replicate,
incubated at 25°C, and compared to the
control. The length of mycelial growth
diameter was measured, as was the reduction
in growth diameter. El-Nasr Company for
Intermediate Chemicals in Egypt provided all
of the tested chemicals.
2.3 In vivo assessment
Uninfected date fruits were sprayed with a
spore suspension of various fungal strains at a
concentration of approximately 1 × 10⁵ spores
/mL. The treated fruits were packed in
polypropylene punnets and stored at 25 °C
with 80–90% relative humidity for 48 hours.
The disease severity was evaluated after 6
days, following the methodology described
Hussein, 2022
43
by Romanazzi et al. (2000).
2.4 Total phenol contents (TPC)
The total phenol concentration was quantified
using the Folin-Ciocalteau method with
absorbance monitoring at 765 nm after the
phenolic components were isolated using the
extraction methods described in the preceding
section (Ough & Amerine, 1988). The
spectrophotometric measurement was repeated
twice for each extract, with the average value
interpolated onto the gallic acid calibration
curve and represented as g of gallic acid per
kilogram of material.
2.5 Statistical analysis
For statistical analysis, MSTAT-C version
2.10 was employed (1991). The least
significant difference (L.S.D.) was used to
compare the means of treatments, according
to Gomez & Gomez (1984).
3. Results and Discussion
3.1 Isolation and identification
During a survey of fungi associated with mold
disease of dates in Aswan governorate, Egypt,
six isolates of Rhizopus stolonifer, nine
isolates of Aspergillus niger, and five isolates
of Aspergillus flavus were identified and
isolated. These isolates were recovered from
naturally infected date fruits exhibiting typical
mold symptoms. The genus Aspergillus,
comprising five species, was the most
frequently isolated group, detected in 45% of
the samples. This aligns with findings by
Abdel-Sater and Saber (1999), who reported
Aspergillus as the predominant genus isolated
from dates in Egypt. Similarly, Gherbawy et
al. (2012) identified A. niger and A. flavus as
the most common species within this genus.
Further supporting this, Abu-Zinada and Ali
(1982) demonstrated the association of A.
flavus with various date fruit cultivars in
Saudi Arabia. In another study, Nassar (1986)
isolated three Aspergillus species, including
A. niger, from dates in Aswan, Egypt. These
findings collectively highlight the widespread
occurrence of Aspergillus species, particularly
A. niger and A. flavus, as significant fungal
pathogens associated with date mold diseases
in Egypt and neighboring regions.
3.2 In vivo assessment
Data in Table (1) reveal that R. stolonifer, A.
niger, and A. flavus demonstrated the ability
to produce mold symptoms on date fruits.
The highest disease incidence was observed
with R. stolonifer 1 at 37.5%, followed by R.
stolonifer 2 at 32.4%, while R. stolonifer 4
exhibited the lowest incidence at 12.6%.
Among the Aspergillus species, A. niger 1
caused a disease incidence of 18.6%, while A.
flavus 1 showed an incidence of 22.2%,
followed by A. flavus 2 with 10.8%. These
findings are consistent with those of Shenasi
et al. (2002), who reported the presence of A.
flavus during the early maturation stages of
ten date fruit cultivars from the United Arab
Emirates, thereby supporting the current
results. Similarly, Alghalibi and Shater
(2004) identified Aspergillus as the most
frequently isolated genus from Yemeni date
samples, occurring in 75% of the tested
samples, with Rhizopus stolonifer being the
second most commonly isolated fungus.
These findings corroborate the significant
role of both Aspergillus and Rhizopus species
as key pathogens associated with mold
development in date fruits.
Hussein, 2022
44
Table 1: Disease incidence (%) of fungal strains isolated from naturally infected dates.
Isolates
Disease incidence (%)
R. stolonifer 1
37.5
R. stolonifer 2
32.4
R. stolonifer 3
22.4
R. stolonifer 4
12.6
R. stolonifer 5
0.0
R. stolonifer 6
0.0
A. niger 1
18.6
A. niger 2
0.0
A. niger 3
0.0
A. niger 4
0.0
A. niger 5
0.0
A. niger 6
0.0
A. niger 7
0.0
A. niger 8
10.4
A. niger 9
6.2
A. flavus 1
22.2
A. flavus 2
10.8
A. flavus 3
4.2
A. flavus 4
0.0
A. flavus 5
0.0
L.S.D. P ≥ 0.05
1.9
3.3 In vitro assessment with different
concentrations of ascorbic acid
Data in Table (2) indicate that none of the
tested concentrations of ascorbic acid (AA)
demonstrated significant inhibitory activity
against the causal strains of mold (R.
stolonifer 1, A. niger, and A. flavus 1). The
evaluation was based on growth diameter
measurements, which served as an indicator
of fungal growth reduction. However, the
results showed no notable decrease in
growth diameter across all tested
concentrations.
Table 2: In vitro assessment of growth diameter for date mold causal strains
under different ascorbic acid (AA) treatments.
Treatments
Growth diameter (mm)
R. stolonifer 1
A. niger 1
A. flavus 1
AA 1000 ppm
98
100
94
AA 500 ppm
96
100
96
AA 250 ppm
100
100
100
Control
100
100
100
L.S.D. P ≥ 0.05
-
-
2.04
3.4 In vivo assessment with different
concentrations of ascorbic acid on date mold
Ascorbic acid (AA) significantly suppressed
mold incidence on dates, as shown in Table
(3). The highest concentration of AA (1000
ppm) reduced the incidence of R. stolonifer 1
to 12.4%, while the lowest concentration (250
Hussein, 2022
45
ppm) resulted in a suppression of 22.2%,
compared to the control, which showed an
incidence of 40.6%. For A. niger 1, AA at
1000 ppm reduced the incidence to 6.8%,
whereas 250 ppm showed lower suppression
at 14.6%, compared to the control, which had
an incidence of 34.8%. Similarly, AA at 1000
ppm achieved the highest suppression of A.
flavus 1, reducing its incidence to 2.8%, while
250 ppm resulted in a suppression of 4.4%,
compared to the control, which exhibited an
incidence of 18.4%. These results align with
findings from previous studies, which suggest
that ascorbic acid can enhance plant resistance
mechanisms, improving their ability to combat
fungal infections. For instance, Khan et al.
(2011) and Singh et al. (2020) reported that
ascorbic acid treatments can activate resistance
responses in plants, such as strawberries,
making them more resilient to fungal pathogens.
Table 3: In vivo disease incidence of date mold under different ascorbic acid
(AA) treatments.
Treatments
Disease incidence (%)
R. stolonifer 1
A. niger 1
A. flavus 1
AA 1000 ppm
12.4
6.8
2.8
AA 500 ppm
16.4
18.4
3.2
AA 250 ppm
22.2
14.6
4.4
Control
40.6
34.8
18.4
L.S.D. P ≥ 0.05
0.0
0.0
0.0
3.5 Biochemical analysis evaluation under
different treatments of ascorbic acid
3.5.1 Total phenolics estimation of dates
Results in Table (4) show that total phenolic
content was estimated for both control and
treated samples of date fruits after six days of
treatment with specific concentrations of
ascorbic acid (AA). Significant differences
were observed among treatments. For R.
stolonifer 1, the control exhibited the highest
total phenolic content at 7.24, followed by
treatments with AA at 250 ppm (6.34) and
500 ppm (5.82). For A. niger 1, the control
also had the highest total phenolic content at
6.14, followed by AA at 250 ppm (5.2) and
500 ppm (4.94). In contrast, potassium
phosphite at 500 mg/L resulted in the lowest
total phenolic content at 4.35. For A. flavus 1,
the control recorded the highest phenolic
content at 8.68, while AA at 1000 ppm
resulted in the lowest phenolic content at
1.86. The increase in phenolic content,
observed in some treatments, is consistent
with the hypothesis that AA stimulates a
defensive metabolic shift in date fruits.
Additionally, previous studies have shown
that nanoparticle treatments, such as Cr NPs,
can activate plant metabolism to enhance
phenolic synthesis, providing stronger
antioxidant and defense responses in conjunction
with increased PPO activity (Lipșa et al., 2020;
Bayat et al., 2019; Xie, 2016). These findings
highlight the complex biochemical interactions
influencing phenolic accumulation under
different treatment conditions.
Hussein, 2022
46
Table 4: In vivo disease incidence of date mold under different ascorbic acid
(AA) treatments.
Treatments
Total phenolic content (mg/g)
R. stolonifer 1
A. niger 1
A. flavus 1
AA 1000 ppm
3.22
2.98
1.86
AA 500 ppm
5.82
4.94
2.34
AA 250 ppm
6.34
5.2
7.66
Control
7.24
6.14
8.68
L.S.D. P ≥ 0.05
1.64
1.64
1.64
4. Conclusion
The findings of this study demonstrate that
specific fungi are associated with mold
development on date fruits, and experimental
evidence confirmed their role in causing
mold. The application of ascorbic acid
directly to date fruits proved highly effective
in reducing the incidence and spread of mold
caused by these fungi. These results highlight
the importance of employing safe and
effective resistance agents, such as ascorbic
acid, to combat microorganisms associated
with mold on date fruits. This approach not
only helps preserve the quality of the fruits
but also plays a crucial role in safeguarding
human health.
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