TX_1~ABS:AT/ADD:TX_2~ABS:AT 32 http://journals.cihanuniversity.edu.iq/index.php/cuesj CUESJ 2025, 9 (1): 32-35 ReseaRch aRticle Fungal Contamination of Automobile Air Conditioner System with Reference to Aqueous Extract Effect of Dodonaea viscosa on Aspergillus fumigatus Salah M. S. Al-Bader1, Zean Zefenkey2, Muhsin H. Ubeid3 1Department of Community Health Nursing, Cihan university-Erbil, Kurdistan Region, Iraq, 2Department of Medical Laboratory Science, Knowledge University, Kurdistan Region, Iraq, 3Department of Anesthesia Technologies, Cihan University-Erbil, Kurdistan Region, Iraq ABSTRACT One of the indoor environments where people spend a lot of time these days is public and private transportation, and there is more and more attention to explaining the correlation between human health and indoor air quality in transportation. The present investigation aims to identify the fungus species in car cooling dispensers and quantify the antifungal efficacy of an aqueous crude extract derived from Dodonaea viscosa leaves and seeds against Aspergillus flavus. Fifty swab samples were obtained in 8 h and then they were cultivated on Sabourad’s dextrose agar medium. The emerging fungi were Aspergillus spp., Penicillium spp., Cladosporium spp., Ulocladum spp., Alternaria spp., and white and black mycelia. The occurrence, frequency, and importance value index were counted. The predominant isolate was A. fumigatus (importance value index = 36.2). In vitro, fungal growth suppression was evaluated using the hot water extract of D. viscosa seeds and leaves. 20% and 40% (w/v) concentrations were made. The results showed that seed extract (40%) presented the highest activity against the predominant isolate Aspergillus fumigatus. Keywords: Automobile, Air conditioner, antifungal activity, Dodonaea viscosa, water extract INTRODUCTION Automobiles are one of the most environmentally polluting machines and the toxic compounds released by cars and other transportation types are well highlighted. On the other hand, the closed environment of land transport modes did not receive enough attention and needed further investigations. As probable sources of bio- pollution, there were 947 million passenger automobiles and 335 million commercial vehicles on the planet Just in Europe, there are 256 million registered passenger cars. (https://theicct.org/wp-content/uploads/2021/06/ ICCT_Pocketbook). The poor interior air quality of cabs, buses, lorries, and private vehicles poses a risk to both drivers and passengers, particularly for professional drivers who spend long hours on the road.[1-3] Actually, because of the surface bioaerosols and increasing automobile indoor air humidity car air conditioning systems frequently operate as active sources of hazardous biological agent emissions (mostly fungi and bacteria) and microorganisms develop ducts and depressors as a result. Certain microorganisms can readily form a biofilm in the air conditioning ducts, which allows them to spread throughout the other installation components and the automobile interior.[4,5] Fungi attached to car’s air conditioners (AC) filters were recorded by Al-Bader et al.,7 genera were recorded Penicillium spp., Alternaria spp., and Aspergillus spp. the predominant, while Acremonium, Rhizopus, Stachybotrys, and Rhodotorula represented low O%.[6] Aquino examined fungal contamination in air conditioning filters from 21 automobiles. The study discovered that 17 fungal genera were present in all samples (100%), including toxic fungi, such as Fusarium, Aspergillus, and Penicillium. This suggests that indoor air quality can harm certain groups, including professional drivers.[7] Corresponding Author: Salah M.S. Al-Bade, Department of Community Health Nursing, Cihan University-Erbil, Kurdistan Region, Iraq. E-mail: salah.saleem@ cihanuniversity.edu.iq Received: November 26, 2024 Accepted: February 26, 2025 Published: March 20, 2025 DOI: 10.24086/cuesj.v9n1y2025.pp32-35 Copyright © 2025 Salah M.S. Al-Bader, Zean Zefenkey, Muhsin H. Ubeid. This is an open-access article distributed under the Creative Commons Attribution License. Cihan University-Erbil Scientific Journal (CUESJ) https://creativecommons.org/licenses/by-nc-nd/4.0/ https://creativecommons.org/licenses/by-nc-nd/4.0/ Al-Bader, et al.: Antifungal Activity of Dodonaea viscosa Oil against Fungi from Car Air conditioner 33 http://journals.cihanuniversity.edu.iq/index.php/cuesj CUESJ 2025, 9 (1): 32-35 Jo and Lee examined the overall colony-forming unit both before and after turning on the car’s air conditioning. They discovered that the levels of bioaerosols in cars before they used air conditioning were either lower or comparable to those in the outside air. Elevated amounts often happened 5–15 min after driving and over time, these concentrations dropped.[2] Numerous physical and chemical methods have been used to clean air conditioning systems,[1] and as a result, a significant impact on the quality of air inside cars. Unfortunately, the eco- friendly application had less attention in this field. Antifungal activity of five essential oils in liquid and vapor phases against fungi isolated from automobile air conditioner filters was examined in Erbil city.[6] The present study was conducted to investigate fungi that inhabit ducts of automobile AC, as well as estimate the antifungal efficacy of the aqueous extract of Dodonaea viscosa seeds and leaves on Aspergillus fumigatus. MATERIALS AND METHODS Samples Collection Fifty swab samples were collected from the air conditioner duct of 50 privet cars. After that swabs were cultured by streaking method on Sabouraud’s Dextrose Agar (SDA) supplemented by Chloramphenicol (15 mg/L), then plates were incubated at (25 ± 2°C), and the cultures were checked daily from the 3rd day over 2 weeks. To create a pure culture for further steps, the growing fungi were immediately transferred to SDA plates. The identification was done through morphological characteristics based on.[8,9] Analysis of Fungal Community After the identification of isolated fungi, the frequency% (F%), occurrence% (O%), and important value index (IVI) for distinguishable genera and species were determined. The following equations were used for this purpose: [10,11] TO No of timesappear No of collected samples % . . � �100 (1) TF%= No.of fungal isolates No.of total fungal isolates ×100 (2) IVI = TO+TF 2 11[ ] (3) Aqueous Extract Preparation D. viscosa materials were collected from the public garden in Erbil city including fresh/green leaves, and dry flowers with seeds, they were washed thoroughly by tape then sterile distilled water SDW and left to dray at laboratory temperature until no change in their weight were occurred. An electric grinder machine was used to ground the leaves and seeds into a powder. 20 mL of SDW was added to five grams of powdered plant material to reach a concentration of 40% and the mixture was shaken for 24 h. The mixture was filtered using a metallic mesh (50 microns), and the resulting filtrate was stored at 4°C in a sterile conical flask as a stock solution (40%).[12] From this stock extract, a diluted extract (20%) was prepared for the antifungal test. Antifungal Activity Test The modified agar disc diffusion method was used[13] and sterilized filter paper (no. 1) disks with a diameter of 10 mm were prepared. Conversely, a water spore suspension of active A. fumigatus was prepared at a concentration of 1 × 10-6, and 0.250 mL of the suspension was spread on the SDA surface. The filter paper disks were carefully saturated by lemongrass oil extracts and were fixed 1 cm from the Petri dish edge. Disk with SDW was done as control. All work was completed inside a laminar flow cabinet, with three replicas. An average of two perpendicular diameters of each oil was calculated to determine the antifungal activity. Finally, paraffin tape was used to seal the Petri dishes, and they were then incubated at 25°C. Four days later, the results were recorded. RESULTS AND DISCUSSION Fungal community structure 101 isolates related to 5 fungal genera were recorded beside white and black mycelium [Table 1 and Figure 1]. They included 4 anamorphic Aspergillus (3 spp.), Cladosporium, Penicillium, and Stemphylium beside the zygomycotic Rhizopus. Aspergillus species, which are cosmopolitan fungi that can grow on a variety of substrates in a wide range of temperatures, exhibited the highest level of O%, F%, and IVI%. As airborne fungi, Aspergilli are the most common isolate worldwide.[14,15] Previous studies for airborne fungi in Erbil City exhibited the predominance of Aspergillus.[16,17] The indoor air fungal community is affected by its external counterpart, as well as internal environmental factors, one of the most important factors that interact with the indoor environment is the air conditioning systems. Fungal formations can spread into indoor air environments through air conditioning systems. The indoor air environment of automobiles is no exception to this. Fungi that were recorded Figure 1: Fungal colonies developed on Sabouraud’s Dextrose Agar medium Al-Bader, et al.: Antifungal Activity of Dodonaea viscosa Oil against Fungi from Car Air conditioner 34 http://journals.cihanuniversity.edu.iq/index.php/cuesj CUESJ 2025, 9 (1): 32-35 in the present study [Table 1] were commonly isolated from outdoor air samples; moreover, they were identified in air conditioning systems of types of buildings such as hospitals and schools.[18,19] The Antifungal Test The aqueous extract of D. viscosa seeds and leaves was scanned [Table 2 and Figure 2] it was found that the 40% seed extract exhibited the highest inhibitory effect (11 mm). This was followed by the 40% leaf extract and 20% seed extract, which showed an inhibitory effect of 6 mm and 2 mm, respectively while, the 20% leaf extract showed no activity. In addition to its antimicrobial activity, different types of extracted tissues contain a variety of compounds with diverse insecticidal and pharmaceutical properties.[20,21] Our results were consistent with previous studies that were conducted to use different solvents to extract the bioactive compound in D. viscosa, different tissues of the plant represented antifungal compounds against Aspergillus niger, Aspergillus fumigatus, Fusarium, and Rhizopus.[22,23] CONCLUSION Based on the results, it is clear that there was a clear relationship between outdoor air-borne fungi and interior automobile air fungi. The car air conditioner system may be a source of allergy since Aspergillus, Cladosporium, and Penicillium, the most fungal allergens are predominant in AC dispensers. An allergy may arise due to a car air conditioner depending on the time of exposure and tidiness of the AC system. Given that D. viscosa shrubs are plentiful and common in the area, have a pleasant scent, and are highly effective as antifungal, they could be the source of successful natural antiseptics of the future. REFERENCES 1. M. Gołofit-Szymczak, A. Stobnicka-Kupiec and R. L. Górny. Impact of air-conditioning system disinfection on microbial contamination of passenger cars. Air Quality, Atmosphere & Health, vol. 12, pp. 1127-1135, 2019. 2. W. K. Jo and J. H. Lee. Airborne fungal and bacterial levels associated with the use of automobile air conditioners or heaters, room air conditioners, and humidifiers. Archives of Environmental & Occupational Health, vol. 63, no. 3, pp. 101-107, 2008. 3. R. P. Vonberg, P. Gastmeier, B. Kenneweg, H. Holdack-Janssen, D. Sohr and I. F. Chaberny. The microbiological quality of air improves when using air conditioning systems in cars. BMC Infectious Diseases, vol. 10, p. 146, 2010. 4. R. B. Simmons, J. A. Noble, L. Rose, D. L. Price and D. G. Ahearn. 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Figure 2: Inhibition zones (I H) of aqueous seeds and leaves extracts of Dodonaea viscosa 1-seeds (concentration [conc.] = 40%, I H = 11 mm), 2-Leaves (conc. = 40%, I H= 6 mm), 3-seeds (conc. = 20%, 2 mm), 4- leaves (conc.=20%, I H = 0) Table 2: Inhibition ratio of Dodonaea extracts Concentration Seed’s extraction Leave’s extraction 40% 11 mm 6 mm 20% 2 mm 0 Table 1: Occurrence%, Frequency%, and important value index of isolated fungi (IVI) S. No. Fungi No. of colonies Occurrence% Frequency% IVI 1. Aspergillus fumigatus 82 32 40.5 36.2 2. Aspergillus niger 54 17 26.7 21.8 3. Aspergillus flavus 28 11 13.8 12.4 4. Cladosporium spp. 16 6 7.9 6.95 5. Penicillium spp. 10 3 4.9 3.95 6. Stemphilium spp. 4 2 1.9 1.95 7. Rhizopus spp. 2 1 0.99 0.99 8. White mycelia 4 1 1.9 1.45 9. 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