Research Article | Open Access
Al-Meshal Areej Suliman
Department of Biology, College of Science and Humanities in Al-Kharj, Prince Sattam bin Abdulaziz University, Al-Kharj 11942, Saudi Arabia.
J Pure Appl Microbiol. 2022;16(2):1330-1336 | Article Number: 7094
https://doi.org/10.22207/JPAM.16.2.61 | © The Author(s). 2022
Received: 10/06/2021 | Accepted: 25/04/2022 | Published online: 01/06/2022
Issue online: June 2022
Abstract

This study was conducted over a 6-month period (2018-2019) to study the inherent fungal contaminants of irrigation water in Al-Kharj farms. Water samples were collected from three sites of the wells (the north and south sides and the plastic housing) at the farms. Sewage samples were also collected. One hundred and sixty-seven fungal isolates were obtained from the wells and the sewage. These isolates belonged to 16 genera, of which 13 were deuteromycetes, one was a zygomycetes and one was an oomycete. The results showed that Pythium, Aspergillus, Rhizoctonia, Rhizopus, and Fusarium genera were found throughout most months of the year. The results for each month were similar in terms of the frequency of fungi detected (99.96–100%) and the fungi isolation frequency (average, 6.247–6.25%). Pythium, Aspergillus, Rhizoctonia, Penicillium, and Diplococcium were identified in the water samples taken from the wells and the plastic housing. Alternaria and Fusarium were identified in water samples taken from the wells, while Candida and Macrophoma were also found in water samples taken from the well, while Cladosporium was found in the water samples and the sewage samples. Rhizopus was found in water samples taken from all of the wells near the plastic housing. Ulocladium was found in water samples taken from the wells and the plastic housing, while Thielaviopsis was found in water samples taken from the wells near the housing and the sterile fungus was found in water samples taken from the wells and the plastic housing. Moreover, we found that the seed trap method was the best method for fungi isolation (isolation frequency, 11.17%) and was better than the direct method of isolation (isolation frequency, 8.53%).

Keywords

Well-Farm, Deuteromycete, zygomycetes, oomycete, seed traps

Introduction

Water is a vital resource for all known living organisms, as there is no life without it.1 The Earth’s surface is 71% water and fresh water comprises 3% of the total amount water of on the Earth’s surface.2 This small percentage of fresh water plays an essential role in human life, as it is the main source of water for human consumption and it is suited to human needs.3 It is estimated that 80% of water use in the Middle East is for irrigation purposes.4 There are many sources of water pollution, but the most notable source is microbial contamination. Sewage or untreated wastewater releases a large amount of dissolved organic material, suspended materials, and harmful microbiological organisms.5

Fungi are one of the most important biological sources of water contamination. These fungi may originate from the soil. Several studies have been conducted to detect the presence of fungi in water. Muhsin et al.6 isolated several fungi from well water, including Alternaria alternate Aspergillus sp., Trichoderma viride, Penicillium sp., Fusarium oxysporum, and Cladosporium sp. Moreover, Rajanaika et al.7 isolated Pythium, Allomyces, Rhizopus, Achyla, Alternaria, Aspergillus, Saprolegnia, Chaetominm, Cladosporium, Fusarium, Trichoderma, and Penicillium from the Tunga River in India. Kraume and Awad8 found 60 species of fungi belonging to 27 genera in wastewater samples, including 8.8% Geotrichum, 75% Penicillium, 65.7% yeasts, and 55.5% Trichoderma.

The aim of the present study was to identify pathogenic and other contaminating fungi in water samples collected from wells and sewage, which is used for irrigation at some Al-Kharj farms due to the lack of irrigation water. The fungal genera present in irrigation water samples and the physical and chemical properties of the water samples were analyzed.

Materials and Methods

Sample Collection
Water samples were collected from three sites of wells (the north and south sides and the plastic housing) at Al-Kharj farms and from the sewage. Samples were collected in clean, sterile 600-mL plastic bottles that were opened under water and filled with the water sample. The water temperature was also measured directly using a thermometer, and an additional clean, sterile 600-mL plastic bottle was filled to measure mineral element concentrations.

Fungi Isolation
1. Direct Isolation
Khaimi9 used the plate-pouring method, in which 1 mL of each water sample was placed on a sterile Petri dish with a 9-cm diameter. Five replicates were plated per sample. Potato dextrose agar (PDA) was added and sterilized using steam autoclave sterilization. Chloramphenicol was added to the medium at a concentration 250 mg/L. The plates were rotated to achieve homogenization between the sample and culture media and were then incubated at 25°C for 7-21 days. The number of growing fungal colonies was determined and the fungi were purified by transferring a small part of the outer fungal growth of the colony to new plates containing PDA. These plates were used for identification using taxonomic keys. The frequency of isolated fungal species was calculated using the following equation:

frequency (%) = number of fungal isolates/total number of isolates × 100. Moreover, the prevalence was calculated using the following equation: prevalence (%) = number of samples containing fungi/total number of samples × 100.

2. Seed Traps
The seed trap method described by Hussain et al.10 was used to isolate fungi from water samples. Fifty chard seeds were sterilized using an autoclave and placed in 15 mL of each water sample (three replicates for each source) for 24 hours. The seeds were dried with sterile blotting paper and then transferred to a sterile 9-cm-diameter Petri dish containing PDA, with 10 seeds per plate. Chloramphenicol was added to the medium at a concentration of 250 mg/L and the plates were incubated at 25°C for 7-21 days. The fungi were purified and the number of the growing fungal colonies, the frequency of isolated fungal species, and the appearance of the fungi were determined as described for the direct isolation method.

Chemical Analyses
The temperature of the water samples was measured directly using a thermometer. pH was also measured using a pH meter, as described by Jackson.11 Electrical conductivity and the concentrations of dissolved positive and negative ions were measured using the method described by Richard.12

RESULTS

Fungi Isolation
One hundred and sixty-seven fungal isolates were obtained from well water and sewage water samples collected from Al-Kharj. These fungi belonged to 16 genera and included 13 deuteromycetes, one zygomycete, and one oomycete. The frequency of fungi isolation was equal between different samples (99.96–100%), with an isolation rate of 6.247–6.25%. Pythium, Rhizopus, Rhizoctonia, Aspergillus, and Fusarium were the most frequently isolated genera, as shown in Table 1.

Table (1):
The percentage of the fungi appearance at different water sources in six months.

FebruaryMarchAprilMay

Isolated Fungi
December
January
Aspergillus
0
17.39
7.5
2.85
29.62
32.25
Alternaria
18.18
0
0
28.57
0
0
Candida
6.81
0
0
0
0
0
Cephalosporium
0
0
27.5
0
0
0
Cladosporium
0
0
12.5
0
1.85
9.67
Diplococcium
29.54
0
0
0
0
0
Fusarium
6.81
0
0
5.71
5.55
3.22
Macrophoma
0
0
0
0
1.85
0
Mycelia sterilia
0
0
0
0
0
4.83
Penicillium
0
10.86
0
0
1.85
0
Pythium
0
4.34
50
45.71
35.18
27.41
Rhizoctonia
6.81
23.91
0
8.57
11.11
22.58
Rhizopus
13.63
32.6
2.5
0
12.96
0
Thielaviopsis
0
0
0
8.57
0
0
Trichoderma
0
10.86
0
0
0
0
Ulocladium
18.18
0
0
0
0
0
Total
99.96
99.96
100
99.98
99.97
99.96
Rate
6.247
6.247
6.25
6.248
6.248
6.247


Direct Isolation Method
As shown in Table 2, the prevalence of fungi in the samples was 8.53%. Pythium, Aspergillus, Cladosporium, Rhizopus, Rhizoctonia, Candida, Penicillium, Alternaria, Fusarium, Macrophoma, Diplococcium, Trichoderma, and Mycelia sterilia were isolated, but Ulocladium, Thielaviopsis, and Cephalosporium were not detected.

Table (2):
The percentage of the appearance of fungi, isolated by direct isolation- per site.

Isolated Fungi
The North well
The South well
The Plastic houses well
Sewage Water
Appearance %
Aspergillus
27.77
27.77
27.77
11.11
22.216
Alternaria
5.55
16.66
0
0
6.664
Candida
0
16.6
0
0
3.332
Cephalosporium
0
0
0
0
0
Cladosporium
16.66
16.66
0
22.22
11.108
Diplococcium
0
0
0
0
1.11
Fusarium
16.66
0
0
0
8.886
Macrophoma
22.22
0
0
0
4.444
Mycelia sterilia
0
0
5.55
0
1.11
Penicillium
5.55
16.66
0
0
5.552
Pythium
33.33
38.88
38.88
33.33
35.55
Rhizoctonia
44.44
16.66
16.66
11.11
21.106
Rhizopus
16.66
16.66
0
16.66
13.328
Thielaviopsis
0
0
0
0
0
Trichoderma
0
0
0
0
2.222
Ulocladium
0
0
0
0
0
Total
188.84
166.61
88.86
94.43
136.6
Rate
11.80
10.41
5.55
5.90
8.5375

Well water samples had the highest percentage of fungal isolates, at 11.80%, and the plastic housing samples were the least polluted with fungi, at 5.90%.

Seed Trap Method
Fungal isolation using the seed trap method resulted in an overall fungal prevalence of 11.17% (Table 3). Pythium, Rhizoctonia, Aspergillus, Rhizopus, Cephalosporium, Ulocladium, Diplococcium, Thichoderma, Alternaria, Fusarium, Mycelia sterilia, Penicillium, Cladosporium, and Thielaviopsis were isolated, but Candida and Macrophoma were not detected using this method.

Table (3):
The percentage of the appearance of fungi, isolated by seeds traps- per site.

Isolated Fungi
The North well
The South well
The Plastic houses well
Sewage Water
Appearance %
Aspergillus
11.11
22.22
27.77
38.88
31.107
Alternaria
16.66
33.33
0
0
13.33
Candida
0
0
0
0
0
Cephalosporium
16.66
16.66
16.66
11.11
12.218
Cladosporium
0
0
0
11.11
2.222
Diplococcium
16.66
16.66
16.66
0
13.328
Fusarium
0
5.55
0
0
2.22
Macrophoma
0
0
0
0
0
Mycelia sterilia
11.11
0
0
0
2.22
Penicillium
0
0
5.55
0
1.11
Pythium
8.883
838.8
33.33
72.22
46.662
Rhizoctonia
27.7
27.77
33.33
5.55
19,994
Rhizopus
16.66
22.22
16.66
16.66
18.884
Thielaviopsis
11.11
0
0
0
3.332
Trichoderma
0
0
0
0
3.332
Ulocladium
16.66
0
11.11
0
8.886
Total
183.28
183.29
161.07
155.53
178.847
Rate
11.455
11.455
10.66
9.720
11.177

The frequent appearance of Pythium, Rhizoctonia, Aspergillus, and Rhizopus was clear using both the direct isolation and seed trap methods.

Chemical Analysis of Water Samples
As shown in Table 4, the temperature range was from 11.9-35.3°C during the study period. A high temperature had adverse effects on Alternaria, Candida, Ulocladium, Trichoderma, and Cephalosporium, while temperatures above 25°C had a direct effect on Macrophoma and Thielaviopsis. The effect of temperature on the rest of the fungi ranged from a direct effect to the opposite effect.

Table (4):
Temperature, pH, EC and mineral elements of the water of the sites under study during a period of six months.

Location Factors December January February March April May
The North well ° C 18 12.8 17.5 32.3 27.3 30
Ph 6.65 6.81 7.2 7,6 7.6 7.2
EC 3.66 4.6 3.22 2.14 1.87 1.59
Na 24.5 11.84 22.8 17.52 307.2
K 4.9 5.83 0.09 0.48 3.62
Ca 2.2 2.8 2.5 9.5 4.6
CL 22.5 10.8 13 8.5 5
Mg 21.8 22.4 9 8.5 16.5
HCO3 4.4 5.2 6 1 4
The South well ° C 14.6 13.4 14.5 25.6 30.9 31.2
Ph 6.66 6.8 7.3 6.9 7.4 7
EC 2.49 3.11 2.1 2.66 2.1 1.97
Na 16.5 10 28.6 35.4 801.4
K 3.20 4.96 0.09 0.32 5.44
Ca 1.3 1.6 2 8 6.5
CL 14.5 8.9 16.5 13.5 8.5
Mg 14 13.4 20 14.5 12
HCO3 3.2 2.8 3.5 1.5 9
The Plastic houses well ° C 15.3 18.2 22 35.3 25.6 26.3
Ph 6.82 6.67 7.4 7.5 7.6 7.2
EC 1.12 3.22 2.77 2.97 1.66 1.96
Na 12.75 9.78 26.3 12.8 400.71
K 3.10 4.45 0.06 0.32 3.62
Ca 2.4 2.3 2.5 7.5 6.5
CL 10.30 8.6 22 9.5 11
Mg 15.8 20.4 18.5 10 10.5
HCO3 4.4 4 2.5 2 6
Sewage Water ° C 14 13.2 19 28.1 25.3 25.6
Ph 5.98 6.58 6.8 6.7 7.1 6.1
EC 2.34 3.08 1.89 1.46 1.58 2.35
Na 11.85 8.36 17.16 13.78 398.48
K 25 8.55 0.97 3.54 81.66
Ca 0.5 0.6 1 5.4 6
CL 9.8 7.51 16 16.5 21
Mg 14.4 10.6 11 11.6 8
HCO3 10.8 9.3 2.3 1 13.5

The pH values ranged from 5.6-7.8. Acidic pH had an adverse effect on Candida at and acidic and alkali pH had adverse effects on Thielaviopsis. Direct effects on Diplococcium, Ulocladium, Cephalosporium, and Alternaria were observed at acidic and alkali pH. Trichoderma and Fusarium were affected by alkali pH. The effect of pH on the other fungi varied.

The sodium concentration ranged from 4.56–801.4 mL/L, the potassium concentration from 0.06–81.66 mL/L, the calcium concentration from 0.3–9.5 ml/l, the chlorine concentration from 1–22 mL/L, the magnesium concentration from 1–22.4 mL/L, and the bicarbonate concentration from 1–13.5 mL/L. These elements had adverse effects on Trichoderma for the duration of the study, while potassium and bicarbonate had adverse effects on Penicillium. Moreover, sodium, calcium, chlorine, magnesium, and bicarbonate had positive effects on Cephalosporium, while potassium had negative effects on Cephalosporium and Alternaria. Sodium, potassium, and bicarbonate had direct effects on Alternaria. Sodium, calcium, potassium, and chlorine had adverse effects on Fusarium, while sodium, potassium, chlorine, and magnesium had adverse effects on Thielaviopsis, and calcium had a direct effect on Thielaviopsis. Sodium and calcium also had positive effects on Penicillium. Bicarbonate, chlorine, and magnesium had positive effects on Penicillium, while sodium and potassium had adverse effects on Mycelia sterilia. Moreover, magnesium and bicarbonate had a direct effect on Mycelia sterilia throughout the study period.

DISCUSSION

Fungi Isolation
As shown in Table 1, the reason for the presence of Rhizoctonia, Pythium, and Fusarium during the study period may be attributed to the fact that these fungi are soil fungi that may be transmitted during irrigation processes. This is supported by several studies showing the presence of soil fungi in water sources.13,14 The reason for the abundant presence of Aspergillus may be attributed to the fact that this fungus is able to produce large numbers of asexual breeding units, it is able to survive in different environments, and it secretes enzymes that enable it to benefit from different food sources.15 Rhizopus is a widespread fungus that may exist in water. Candida, which is a genus of pathogenic fungi, was also isolated, indicating the serious public health impact of water from these wells if it was used for drinking or domestic purposes.

The fungi isolated in this study were similar to those isolated from other water sources, such as the Nile River,16 drinking water,17 polluted water from the Shatt Al-Arab River and its ramifications18 and well water19, but at different frequencies.

Direct Isolation Method
The results presented in Table 2 show that well water had the highest incidence of fungi, at 11.80%. The plastic housing well water was the least polluted, as the fungi incidence was 5.90%. The lack of fungi in the plastic housing may be attributed to the frequent use of chemical pesticides near the water of the plastic housing.

Seed Trap Method
The results presented in Tables 2 and 3 show that the seed trap method was the most efficient at isolating pathogenic and non-pathogenic fungi, such as Ulocladium, Thielaviopsis, and Cephalosporium, which were not isolated using the direct isolation method.

The results showed that the lowest incidence of fungi was in sewage water (9.72%), and this may be due to the lack of oxygen in sewage water and the high percentage of pollutants, resulting in an inappropriate environment for these fungi.

Chemical Analysis of Water Samples
The difference in the effect of temperature on the fungi confirmed that each fungus has a certain temperature range at which it can grow. Borut and Johnson20 found that there is a lack of response of fungi to physical and chemical factors. Moreover, Nakagiri et al.21 reported that temperature changes affect the geographical distribution of fungi.

The difference observed in the effect of pH values on fungi confirmed that each fungus has a certain pH range within which it can grow. Suzuki and Nimura22 reported that a pH range of 3-10 was appropriate for the majority of fungi to grow in aquatic environments. However, Mehrotra and Gupta23 found that pH had a minor effect on the presence of fungi. Khaimi9 isolated Aspergillus niger, A. flavus, A. terms, Penicillium sp., Cladosporium herbarum, Aspergillus sp., Cladosporium sp., Ulocladium botrytis, A. foetidus, Phomopsis, Phoma exigua, Pythium, and yeasts and showed that they were able to grow at pH 6.5–8. However, salt concentrations from 0.63–6.81 had adverse effects on Trichoderma, Candida, and Thielaviopsis and direct effects on Ulocladium, Cephalosporium, Macrophoma, and Alrernaria. The effects of salt concentration on the rest of the fungi were varied.

The difference in the effect of salts on different fungi confirmed that each fungus has a sustainable range of salt concentrations at which it can grow. Abdel-Fattah et al.24 found that the total number of fungi is significantly affected by the total concentration of dissolved salts, especially sodium and calcium. We found that the effects of metallic elements on fungi were varied, resulting in the stabilization of some fungi, as shown in Table 4.

Declarations

ACKNOWLEDGMENTS
The author would like to thank Prince Sattam bin Abdulaziz University for their scientific contributions and the laboratory in the Department of Biology for allowing to perform various experiments.

FUNDING
None.

ETHICS STATEMENT
This article does not contain any studies with human participants or animals performed by the author.

AVAILABILITY OF DATA
All datasets generated or analyzed during this study are included in the manuscript.

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