Detection of Fungi in Irrigation Water Sources using Different Isolation Methods

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%).


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
Khaimi 9 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

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.
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%.
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.
The pH values ranged from 5.6-7.8. Acidic pH had an adverse effect on Candida at and acidic 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.

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 ramifications 18 and well water 19 , 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 Johnson 20 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 Nimura 22 reported that a pH range of 3-10 was appropriate for the majority of fungi to grow in aquatic environments. However, Mehrotra and Gupta 23 found that pH had a minor effect on the presence of fungi. Khaimi 9 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.

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.

dAtA AVAilABilitY
All datasets generated or analyzed during this study are included in the manuscript.