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Article

A Novel Approach for Fungal Control with Ethyl Formate in Barley and Its Impact on Germination

by
Eman Barkat
1,†,
Xin Du
1,2,*,†,
Jun-Ran Kim
3,
Zhengyan Wang
2,4,
Byung-Ho Lee
5,
Abrar Uaraboa
6,
Esra Uaraboa
7 and
Bong-Su Kim
3,*
1
Harry Butler Institute, Murdoch University, Murdoch, WA 6150, Australia
2
School of College of Environmental and Life Sciences, Murdoch University, South Street, Murdoch, WA 6150, Australia
3
Department of Plant Quarantine, Animal and Plant Quarantine Agency (APQA), Gimcheon 39660, Republic of Korea
4
School of Food and Strategic Reserves, Henan University of Technology, Zhengzhou 450001, China
5
Institute of Agricultural and Life Science, Gyeongsang National University, Jinju 52828, Republic of Korea
6
School of Biomedical, Science, Curtin University, Kent St, Bentley, WA 6102, Australia
7
School of Pharmacy, Curtin University, Kent St, Bentley, WA 6102, Australia
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Agriculture 2025, 15(7), 748; https://doi.org/10.3390/agriculture15070748
Submission received: 15 February 2025 / Revised: 12 March 2025 / Accepted: 28 March 2025 / Published: 31 March 2025
(This article belongs to the Special Issue Biosecurity Innovations for Pest Management in Agricultural Products)
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Versions Notes

Abstract

:
Fungi can degrade grain quality, produce harmful mycotoxins, and hinder germination in the post-harvest stage, resulting in significant economic losses. Ethyl formate (EF) is an efficient and eco-friendly fumigant for controlling pest insects in grains, horticulture, and quarantine treatments. However, there is a lack of research on the antifungal activity of EF and its degradation products on barley seeds. In this study, fifteen fungal species, predominantly Alternaria infectoria, were isolated and identified from seven Australian barley samples. Efficacy results indicated that EF significantly inhibited fungal growth at a commercial concentration of 2.4 mmol/L, except for Penicillium sp. 2, Fusarium chlamydosporum, and Rhizopus arrhizus. To control these EF-tolerant fungal species, the EF concentration was increased to 5 mmol/L, which achieved a 100% inhibition rate. The degradation product of EF, formic acid, effectively inhibited all EF-tolerant fungi, requiring only 0.08 mmol/L in the in vitro study. There were no significant differences in vigor and germination rates in barley treated with EF at concentrations of 2.5, 3.7, and 5 mmol/L. Additionally, EF treatments led to a significant increase in the barley root length from an average of 9.1 cm in the control group to 13.2, 13. 6, and 12.3 cm at 2.5, 3.7, and 5 mmol/L. The findings suggest that EF could be a suitable alternative fumigant to safeguard grain from fungal infestation, particularly in the context of advancing agricultural practices and improving seed germination quality. The degradation compound, formic acid, may contribute significantly to the overall antifungal function of ethyl formate fumigation, particularly in high-humidity environments.

1. Introduction

Barley (Hordeum vulgare) is considered to be one of the most economically and environmentally important crops in food products and fermentation industries [1]. After harvesting, barley crops may become infected with many different pests, such as bacteria, fungi, rodents, and insects, which in turn affects the germination of seeds and may affect the metabolic processes of the seeds [2]. Previous studies by Hao et al. [3] and Cao et al. [4] indicated that the main fungus genera associated with barley grain include Fusarium, Aspergillus, Microdochium, Alternaria, and Epicoccum. The most common genera present during storage were Epicoccum, Alternaria, Bipolar, Cladosporium, Fusarium, and Aspergillus [5,6]. Alternaria species are the most common strain in barley grains during storage [7]. This genus is considered to be a pathogenic fungus that produces some mycotoxins, such as alternariol (AOH), alternariol monomethyl ether (AME), tentoxin (TEN), altenuene (ALT), and tenuazonic acid (TeA) [8]. Fusarium is a fungus that infects wheat and barley grains, causing Fusarium head blight (FHB), and produces deoxynivalenol (DON) during infection, resulting in grain contamination and a virulence factor that promotes FHB [9]. Therefore, controlling the growth of such fungi in stored grains is critical for healthy and quality grains.
Fumigation is a process used to control pests in grains and food production [10,11,12]. It has been used to control insects, rodents, and fungi or to disinfest stored products, food, wheat, timber, structures, and soils because of its low cost and high effectiveness. Fumigation with different chemicals, such as ozone gas, phosphine, propionic acid, and essential oils, to suppress fungal growth in stored grains has been investigated [2,13,14,15,16]. However, due to residual toxicity, limited antifungal action, and excessive cost, these fumigants cannot be used to prevent and control fungal spoilage in grain storage [17]. Phosphine has been used as an antifungal to control the growth of fungi, but it has no complete effect on fungal growth and is only useful in delaying fungal spoilage during short-term storage. Observation studies on the use of ozone treatment were not able to adequately reduce fungi growth in the silo, and some fungi strains such as Penicillium sp. remained resistant to treatment [18,19].
Ethyl formate (EF) is deemed safe and environmentally friendly, and the Food and Drug Administration assessed the use of EF as a flavoring ingredient in 1984 and labeled it as generally recognized as safe (GRAS), which marks a product as a likely bio-resource of eco-friendly antifungal agents [20,21]. It has been used as a fumigant for preserved goods and is currently registered as a fumigant for dried fruit [22]. Moreover, EF does leave undesired residues when broken down into ethanol and formic acid (FA) [23]. Formic acid, the other product of the decomposition of EF, has higher antifungal capability than ethanol to inhibit the fungal growth of A. parasiticus isolated from barley [24,25]. Ethyl formate and its byproducts exhibit low toxicity and high decomposability, which make them very appealing for use in natural goods [26].
Ethyl formate provides a human-safe and environment-friendly solution to replace existing toxic fungicides. The present study identified the research gap of understanding and mitigation of the fungicidal efficacy of EF and its degradation product FA on Australian barley and germination quality. This research aimed to explore the potential of the application of EF as a green fungicide for the global grain industry. As EF has been commercially used to control stored grain pest insects, EF treatment can reduce overall cost by eliminating pest insects and fungi together.

2. Materials and Methods

2.1. Grain Sample Collection

Seven Australian standard barley samples harvested (2020/2021 (S7), 2021/2022 (S6), and 2022/2023 (S1, S2, S3, S4, and S5)) without chemical treatment and gathered from six distinct regions in Western Australia Merredin (S1, S2), Kwinana (S3), Albany (S4), Esperance (S5), Black seeds (S6), and Forrestfield (S7) were purchased from local farmers and donated by the CBH group. The locations chosen for the sample were geographically diverse, covering almost all the major barley-growing areas. The barley samples (1 kg) were collected from farm bins with capacities ranging from 50 to 150 t. The sampling points were located 1 and 2 m below the grain surface, and the two sets of samples were combined and stored in a 2 kg metal sample can. Within two days of collection, all samples were delivered to the laboratory and stored at −4 °C.

2.2. Chemical Reagents

Food grade EF (purity > 97%, balanced with ethanol and water) was purchased from Ingredient Solution, Perth, WA, Australia. The analytical standards of EF (purity > 99.5%) and FA (purity > 98%) were purchased from Merck, Rahway, NJ, USA.

2.3. Isolation and Identification of Fungi

A total of 10 healthy grains were placed with one centimeter between each grain on full-strength potato dextrose agar (PDA; Becton Dickinson and Company, Sparks, MD, USA) and were incubated in the dark at room temperature (23 ± 2 °C). After 1 to 14 days of incubation, 5 mm2 plugs were taken from emerging colonies in each replicate, deposited on fresh PDA, and cultured at 23 ± 2 °C in the dark. Three replication plates of ten grains were utilized for each location.
The taxonomic identification of fungi was conducted according to Booth [27,28], Pitt [29,30], Moubasher [31], Samson [32], and Summerell [33]. Selected isolates from each group were chosen based on morphological and cultural similarities for further identification using molecular approaches.
The molecular identification of all species was accomplished by cultivating cultures on PDA plates for roughly 1 week at 20–30 °C. Mycelium from selected individual isolates was extracted and freeze-dried in 1.5 mL sterile Eppendorf tubes. Genomic DNA was extracted using a hexadecyl trimethyl ammonium bromide technique modified by the addition of 100 g/mL of Proteinase K and 100 g/mL of RNAse A to the extraction solution, as described by Andjic et al. [34]. The ribosomal DNA operon’s internal transcribed spacer (ITS) region was amplified in all isolates using the primers ITS-1F [35] and ITS4 [36]. Sakalidis et al. [37] detailed how PCR products were purified and sequenced. To compare the isolates utilized in this investigation to associated species, sequences of known vouchered isolates were retrieved from GenBank (www.ncbi.nlm.nih.gov/Genbank accessed on 27 February 2025) and confirmed by phylogenetic analysis using Geneious Prime v2023 (Table 1).

2.4. Fumigation

A four-liter airtight glass jar equipped with a rubber septum on the lid was used as the fumigation chamber. EF and FA were introduced via the septum using a liquid syringe (Agilent, Melbourne, VIC, Australia). The 24 h fumigations were conducted in a fume hood at 23 ± 2 °C. The gas fumigation method was used to assess the effect of fumigants on fungal mycelial growth both in vitro and in vivo [22].
In vitro, 5 mm2 plugs from emerging fungal colonies were placed in the center of 9 cm-diameter Petri dishes lined with sterile filter paper strips and were opened inside the chamber. After 24 h of ethyl formate and formic acid fumigation at various concentrations, the plugs were replanted onto potato dextrose agar, and fungal growth was measured and compared to the control after 5 days of incubation at 20–30 °C, with three biological repetitions for each fungus. The efficacy of EF treatment against various fungal strains was assessed at a commercial suggestion dosage of 90 mg/L (2.4 mmol/L). The width, colony size length (cm), and area (cm2) were measured.
In vivo, ten grams of barley were fumigated once with various concentrations of EF (2.5, 3.7, and 5 mmol/L) for 24 h, and then were replanted onto potato dextrose agar, with fungal growth measured and compared after 5 days of incubation, also featuring three biological repeats per sample. Controls without any treatment were employed for both in vitro and in vitro tests. The fumigated barley samples were also used for the barley germination tests.
The inhibition rate was calculated as the percentage of the inhibition zone area to the area of a control colony in a 9 cm Petri dish without treatment.
C o l o n y   A r e a = L e n g t h × W i d t h × π
I n h i b i t i o n   R a t e = ( 1 − ( C o l o n y   A r e a   o f   S a m p l e / C o l o n y   A r e a   o f   C o n t r o l ) ) × 100 %

2.5. Chemical Analysis of Fumigants

The fumigant gas sample containing ethyl formate and its degradation products, ethanol and formic acid, was introduced into an Agilent 7890 gas chromatograph (GC) with an Agilent FFAP 30 m × 0.25 mm I.D. (0.25 µm film) fused silica capillary column for separation. The ethyl formate and formic acid were detected at retention times of 1.361 min and 3.226 min with an Agilent 5977 mass spectrometry (MS) detector (Agilent, Melbourne, VIC, Australia), respectively (Supplementary Figure S1). The GC column oven was set to 120 °C with a He carrier flow of 1 mL/min.
The gas samples were injected using a 100 µL syringe (SGE, Melbourne, VIC, Australia) with duplicates. Ethyl formate concentrations were calculated based on peak areas compared to external gas standards. Gas standards were prepared by injecting a known volume of liquid ethyl formate into 1 L flasks with glass beads and a half-hole septa. A 60 µL fumigant sample was injected into the GC, and the concentrations were determined using the calibrated gas standards [38]. There were three calibration standards prepared at 1.4, 2.4, and 10.8 mmol/L for ethyl formate and 0.01, 0.04, and 0.18 mmol/L for formic acid GC analysis.

2.6. Barley Germination Test

The germination tests were performed using the between-paper (BP) method of international seed testing [1,2]. Five hundred and thirty seeds (ten replicates of 53 seeds) were used in each treatment. The surface of the bench on which the steel template was placed was sterilized before starting the experiment and was also sterilized between each treatment. A large sheet of filter paper was saturated with 60 mL of distilled water and was folded in half. A steel template (290 × 580 mm) was sterilized with 70% ethanol and was placed over this wet paper, with holes in the stencil allowing barley seeds to be placed 30 mm apart. Once the seeds were positioned correctly, the upper half of the filter paper was folded over the seed area. All treatments were incubated for 7 days at 25 ± 1 °C in the dark. The vigor rate was detected after 4 days. The morphological characteristics were tested, including the length of the shoot and root (cm), and the ratio of the root to shoot length (%) on day 7. The tests were repeated ten times, and the averages were recorded. Non-treated grain was employed as a control.

2.7. Statistical Data Analysis

The Student’s t-test is used to determine if the difference between the two means is statistically significant or due to random sampling variations at α = 0.05. Data visualization and one-way analysis of variance (ANOVA) and Tukey’s Honestly Significant Difference (HSD) test were applied to evaluate the significant difference between the control and treatments using MetaboAnalyst 6 (https://www.metaboanalyst.ca/MetaboAnalyst/home.xhtml accessed on 2 January 2025).

3. Results

3.1. Isolation and Identification of Barley Fungi

A total of 22 isolates from the seven field samples collected from Western Australia were subjected to molecular analysis to confirm their identity. In a comparison of internal transcribed spacer (ITS) sequences based on 16S rDNA sequence data and phylogenetic analysis, 22 fungal genera were isolated and classified as belonging to four fungal classes: Dothideomycetes, Eurotiomycetes, Mucoromycetes, and Sordariomycetes (Figure 1). Four of the Alternaria isolates (G01, G02, G03, and G04) appeared to be most closely related to A. infectoria. Two isolates were closely related to Penicillium sp. 1 and sp. 2 (G05, G06). In addition, three isolates of Epicoccum were closely related to E. nigrum (G07, G10, and G19). Furthermore, two isolates of Fusarium were closely related to F. chlamydosporum (G08, G09) and one to Fusarium sp. (acuminatum/tricinctum, G11). Three isolates of Aureobasidium aligned to Aureobasidium sp. 1 and sp. 2 (G12, G14, and G17) (Table 1).
The phylogenetic tree showed that one isolate each of Pyrenophora, Cladosporium, Pleiochaeta, Curvularia, Didymella, Rhizopus, and Nigrospora closely related to P. teres/graminea, P. setosa, Cladosporium sp., Curvularia sp., D. prosopidis, R. arrhizus, and N. oryzae (G15, G18, G20, G21, and G22), respectively (Figure 2).
A. infectoria had the highest percentage of contamination (69.0%), followed by R. arrhizus and E. nigrum at 6.2% and penicillum sp. 2 at 3.1%. Aureobasidium sp. 1 and N. oryzae had the same percentage of 2.3%, and the lowest contamination was 1.5% and 0.77%, respectively (Supplementary Table S1).

3.2. Inhibition of Ethyl Formate and Formic Acid Against Fungal Strains

The efficacy of EF treatment against various fungal strains was assessed at a commercial suggestion dosage 90 mg/L (2.4 mmol/L) [38], based on the colony size (width, length (cm), and area (cm2), Table 2). The results indicated that an 100% inhibition level of fungal isolates was observed in the following species: A. infectoria (all groups G01-G04), Penicillium sp. 1 and 2 (G5 and G6) E. nigrum (G07,G10 and G19), F. chlamydosporum (G08 and G09), Fusarium sp. (G11), Aureobasidium sp. 1(G12)and sp. 2(G14 and G17), P. teres, graminea (G13), P. setosa (G15), Cladosporium sp. (G16)., C. sp. (G18), D. prosopidis (G20), R. arrhizus (G21) and N. oryzae (G22) (Table 2).
In contrast, ethyl formate exhibited no inhibition activity against Penicillium sp. 2 (G06) and F. chlamydosporum (G09). Notably, R. arrhizus (G21) displayed a partial inhibition rate of 50%, indicating resistance to EF treatment compared to the other fungi tested (Table 2).
Three EF-resistant fungal species isolated from Australian barley, Penicillium sp. 2, Fusarium chlamydosporum, and R. arrhizus, were further investigated to determine their tolerance of EF and FA. The EF-susceptible A. infectoria was tested as a control. The treatment with 1.4 mmol/L EF resulted in 26%, 0%, 16%, and 0% inhibition rates of A. infectoria, Penicillium sp. 2, F. chlamydosporum, and R. arrhizus, respectively. The treatment with 2.4 mmol/L EF resulted in a 100% inhibition rate of A. infectoria. F. chlamydosporum showed an increasing inhibition rate from 63% to 100% when the concentration of EF ranged from 2.4 to 5.4 mmol/L (Table 3). The minimal concentration for EF to Penicillium sp. 2 and R. arrhizus was 10.8 and 5.4 mmol/L, respectively.
The inhibition rates of A. infectoria, Penicillium sp. 2, F. chlamydosporum, and R. arrhizus after exposure to serial dosages of FA (0.01, 0.02, 0.04, 0.08, and 0.18 mmol/L) showed cross-resistance and species-dependent variation. The treatment with 0.01 and 0.02 mmol/L of FA resulted in 17% and 16% inhibition rates for A. infectoria, respectively. F. chlamydosporum showed higher inhibition rates of 31% and 26% when treated with 0.01 and 0.02 mmol/L of FA, respectively. In contrast, Penicillium sp. 2 and R. arrhizus were not influenced at these levels. The treatment with 0.04 mmol/L of FA resulted in the complete inhibition of A. infectoria and R. arrhizus and a 55% inhibition rate of F. chlamydosporum. Penicillium sp. 2 remained resistant after exposure to 0.04 mmol/L of FA but reached a 100% inhibition rate after exposure to 0.08 mmol/L of FA. However, F. chlamydosporum required the highest dose of 0.18 mmol/L to completely inhibit fungi growth (Table 4).

3.3. Effect of EF on Barley Germination

3.3.1. Vigor and Germination Rate of Barley

The vigor rates of barley seeds treated by EF with dosages of 2.5, 3.7, and 5 mmol/L (90, 140, and 180 mg/L) were 91.5%, 97.2%, and 94.3%, respectively, compared with untreated barley seeds with a 100% rate. The germination rates of barley seeds treated using EF with dosages of 2.5, 3.7, and 5 mmol/L (90, 140, and 180 mg/L) were 90.6%, 97.2%, and 92.5%, respectively, compared with untreated barley seeds with a 96.2% rate. The germination rate remained above 97.2% for all treatments, demonstrating no significant difference in seed viability, even at the highest EF concentration of 5 mmol/L (Figure 3). All seeds with root and plumule lengths of less than 2 cm were classified as ungerminated seeds, with percentages of 0.9%, 4.0%, 2.0%, and 4.0% of such seeds found in the control and EF-treated groups at 2.5, 3.7, and 5 mmol/L EF, respectively.

3.3.2. Root and Plumule Lengths

The effects of EF fumigation at concentrations of 2.5, 3.7, and 5 mmol/L on barley root and plumule length were evaluated and compared to control group samples. For root growth, EF treatments resulted in a significant increase in the length of barley roots, from an average of 9.1 cm in the control group to 13.2, 13.6, and 12.3 cm at 2.5, 3.7, and 5 mmol/L, respectively (Figure 4a). A one-way ANOVA analysis indicated there was a statistically significant difference p-value < 0.01 in the root length between the treatment groups and the control. However, at the highest concentration, 5 mmol/L, a slight reduction in root growth was observed by approximately 1 cm compared to the lower concentrations. According to plumule growth, EF treatments did not show significant variations compared to the control. The mean plumule length across all treatment groups remained relatively stable at 11.6 cm, with no significant differences observed between the control and the EF-treated groups (Figure 4b).

4. Discussion

In the isolated Australian barley, Alternaria incidence outweighed that of other fungi, and the findings are consistent with previous research on barley and other small grains [39]. When commodities are stored in unsuitable conditions, Alternaria species can develop a range of mycotoxins in the field. Other species observed in low counts from almost all the barley grain samples included R. arrhizus, E. nigrum, penicillum sp. 2, Aureobasidium sp. 1, N. oryzae, Fusarium sp., Curvularia sp., P. teres/graminea, P. setosa, Cladosporium sp., Aureobasidium sp. 2, Curvularia sp., and D. prosopidis. These results concur with Sultan et al. [40], who detected Alternaria, Cladosporium, Nigrospora, Aureobasidium pullulans, E. nigrum, Penicillium, and Fusarium in grains, which typically require high moisture content. Members of other genera, such as Pyrenophora, Curvularia, and Drechslera, are present in barley grain and are also causal agents of common foliar diseases of barley in Canada [41]. Isolates belonging to the genus Didymella present on Western Australian grain have been isolated from the leaves of the youngest plants [42]. P. setosa isolated from barley grain has the lowest account and has been identified as the most critical disease of lupins, causing considerable yield losses in cultivated lupin species worldwide [43]. Animal and human health are seriously threatened by exposure to these substances, which can cause a variety of acute or chronic mycotoxicoses [44].
Ethyl formate demonstrated its capablity to control the majority of the fungal strains isolated from barley, including A. infectoria, Penicillium sp. 1, E. nigrum, F.chlamydosporum, Fusarium sp., Aureobasidium sp. 1 and 2, P. teres/graminea, P. setosa, Cladosporium sp., Curvularia sp., E. nigrum, and D. prosopidis, after exposure to 2.4 mM (90 g m−3) of EF. However, three fungi species, Penicillium sp. 2, F. chlamydosporum, and R. arrhizus, exhibited no inhibition, highlighting significant resistance to EF treatment. These EF-resistant fungi required a significantly high concentration of EF for 100% inhibition. However, Penicillium sp. 2 was highly tolerant to EF, even though the chemical level was raised up to 10.8 mM. Ryan et al. [23 stated that the mechanisms of the antifungal action of EF against mycelia growth comprise membrane disruption, protein and enzyme inhibition, and DNA damage. Therefore, the various morphological and physio-chemical characteristics of barley fungi play an important role in ethyl formate tolerance.
In this study, we found that EF fumigation does not affect the germination of barley seeds. Even more, EF improved the development of barley roots. These results are consistent with Waterford et al. [45] and Ren et al. [46], who found that a high concentration of EF (120 mg/L, 3.24 mmol/L) with 72 h of exposure did not affect the germination or plumule length of stored products such as wheat, paddy, barley, maize, sorghum, safflower, cotton seeds, soya beans, and mung beans. Also, Noots et al. [47] reported that microbial metabolism influences the barley quality through chemical and biochemical interactions; this effect results from interferences with barley respiration and the secretion of metabolites and enzymes. Furthermore, the enhancement of barley root development due to ethyl formate fumigation was first reported.
EF can decompose into ethanol and formic acid, which may further contribute to its antifungal efficacy. Ethanol is less effective against microorganisms than EF at the same concentration level [48]. FA was also recognized as a safe antimicrobial agent [49]. FA was more efficient than EF in controlling fungi isolated from barley. To achieve a 100% inhibition rate for EF-resistant fungi, a minimum EF concentration of 10.8 mmol/L was required, whereas only 0.18 mmol/L could meet this demand, which is six times lower. Revis et al. [50] also reported that FA strongly inhibited every bacterial and fungal hypha examined. EF and FA exhibit distinct fungicidal activity with different mechanisms, targeting microbial structures and metabolic processes differently. Ethyl formate primarily acts as a fumigant, exerting its antimicrobial effects through cell membrane disruption, protein denaturation, oxidative stress induction, and enzyme inhibition [51]. Additionally, the action for EF resistance in Penicillium sp. 2 and Rhizopus arrhizus has been explained by molecular studies due to the contamination of the fungicide with gene promoter duplications paired with specific amino acid substitutions [52]. On the other hand, FA functions primarily through pH reduction and acidification, creating an inhospitable environment for fungal growth [14,24,48,49]. This fact is due to the hydrophobic feature of most organic acids, which permits free diffusion of the protonated form over the cell membrane [24]. Penicillium and Rhizopus species were resistant to FA at low concentrations due to the ability of the microbes to consume the acid; this process is called deacidification or the formation of alcohols [23]. However, both species were successfully controlled under the examined FA dosages in this study.

5. Conclusions

Grain stored in high moisture content environments is prone to various pathogens such as Fusarium, Penicillium, Rhizopus, Aspergillus, and Alternaria, known for their mycotoxin-producing abilities, which pose a significant threat to human health due to the opposing effects of these toxins. The contamination of stored grains with bacteria is also an important consideration for consumers. Conditioning grain to increase the moisture content and temperature to a high level can also increase the counts of bacteria, yeasts, and molds. Hence, the key to managing high-moisture grain is to keep it cold (to reduce mold growth) and to dry the grain before delivery. The appropriate aeration of grain can help to control temperature and moisture in storage facilities. The methods needed to prevent these fungi are advancing given the increasing prevalence of fungicide resistance, narrowing controls of chemical use, and market trends causing new food-preservation challenges. The use of fungicides or preservatives can reduce the spoilage of grains during storage. These considerations are considered in the context of existing challenges for food preservation, focusing on pre-harvest and after-harvesting fungal control.
Creating a sustainable strategy to prevent contamination and the proliferation of fungi in stored grains is of great importance. It is essential to control the factors responsible for fungi contamination and growth. In addition, the chemical-free strategies used to control fungal explosion, such as drying and aeration, hermetic storage, dielectric heating, cold plasma treatment, ozonation, irradiation, ultra-superheated steam treatment, and the application of vegetable oil and plant derivatives, are also reflected, consequently that a strategy for sustainable fungal management practices can be developed with domestic and international industrial agreement.
Overall, the findings of this study highlight the potential of EF as a safe and effective treatment for barley seeds with no detrimental effects on germination rates. The degradation product, FA, is attributed to highly effective fungi inhibition. The observed increase in root growth at 2.5 mM and 3.7 mmol/L suggests that EF could be beneficial in promoting root development, potentially improving seedling establishment. Further research is needed to understand EF and FA’s impact on seed development, optimal concentrations, and molecular resistance mechanisms. Exploring FA fumigation, EF-FA combinations, and synergistic treatments will enhance fungal control in agriculture.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agriculture15070748/s1, Figure S1: Gas chromatography-mass spectrometry (GCMS) total ion chromatography (TIC) of ethyl formate and formic acid; Table S1: Species and numbers of fungal isolates obtained from barley samples isolated from WA (seven different locations) and the percentage of fungal species across different locations.

Author Contributions

Conceptualization, E.B. and X.D.; methodology, validation, and sample analysis, E.B., X.D., A.U. and E.U.; writing—original draft preparation, E.B. and X.D.; writing—review and editing, Z.W., B.-H.L. and X.D.; supervision, B.-H.L., B.-S.K. and X.D.; funding acquisition, B.-S.K., J.-R.K. and X.D. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the Animal and Plant Quarantine Agency South Korea (APQA) ‘Ethyl Formate-base Systems Approaches that Maintain Commodity Quality and Control of Arthropod Pests’ in 2022–2024 (Project Number: I-1543086-2021-23-01).

Data Availability Statement

The authors confirm that the data supporting the findings of this study are available within the article and the Supplementary Materials.

Acknowledgments

We sincerely thank Yonglin Ren for his knowledge contributions and providing research resources to facilitate the study.

Conflicts of Interest

The authors state that they have no conflicts of interest. The funders were not involved in the study’s design, data collection, analysis, interpretation, article preparation, or decision to publish the results.

References

  1. Dong, X.; Sun, L.; Agarwal, M.; Maker, G.; Han, Y.; Yu, X.; Ren, Y. The effect of ozone treatment on metabolite profile of germinating barley. Foods 2022, 11, 1211. [Google Scholar] [CrossRef] [PubMed]
  2. Xiao, Y.; Gu, C.; Yu, J.; Shan, C.; Ren, Y. A novel method for analysis of phosphine (PH3) residues in canola and cottonseed. Curr. Microw. Chem. 2018, 5, 155–159. [Google Scholar]
  3. Hao, G.; McCormick, S.; Usgaard, T.; Tiley, H.; Vaughan, M.M. Characterization of three Fusarium graminearum effectors and their roles during Fusarium head blight. Front. Plant Sci. 2020, 11, 579553. [Google Scholar]
  4. Cao, D.; Lou, Y.; Jiang, X.; Zhang, D.; Liu, J. Fungal diversity in barley under different storage conditions. Front. Microbiol. 2020, 13, 895975. [Google Scholar]
  5. Braga, R.M.; Padilla, G.; Araújo, W.L. The biotechnological potential of Epicoccum spp.: Diversity of secondary metabolites. Crit. Rev. Microbiol. 2018, 44, 759–778. [Google Scholar]
  6. Woudenberg, J.H.C.; Groenewald, J.Z.; Binder, M.; Crous, P.W. Alternaria redefined. Stud. Mycol. 2013, 75, 171–212. [Google Scholar]
  7. Nguyen, T.T.; Kim, J.; Jeon, S.J.; Lee, C.W.; Magan, N.; Lee, H.B. Mycotoxin production of Alternaria strains isolated from Korean barley grains determined by LC-MS/MS. Inter. J. Food Microbiol. 2018, 268, 44–52. [Google Scholar]
  8. Khan, M.K.; Pandey, A.; Athar, T.; Choudhary, S.; Deval, R.; Gezgin, S.; Hamurcu, M.; Topal, A.; Atmaca, E.; Santos, P.A.; et al. Fusarium head blight in wheat: Contemporary status and molecular approaches. 3 Biotech 2020, 10, 172. [Google Scholar]
  9. Ons, L.; Bylemans, D.; Thevissen, K.; Cammue, B.P. Combining biocontrol agents with chemical fungicides for integrated plant fungal disease control. Microorganisms 2020, 8, 1930. [Google Scholar] [CrossRef]
  10. Köhler, H.R.; Triebskorn, R. Wildlife ecotoxicology of pesticides: Can we track effects to the population level and beyond? Science 2013, 341, 759–765. [Google Scholar]
  11. Mostafalou, S.; Abdollahi, M. Pesticides and human chronic diseases: Evidences, mechanisms, and perspectives. Toxicol. Appl. Pharmacol. 2013, 268, 157–177. [Google Scholar] [PubMed]
  12. Handford, C.E.; Elliott, C.T.; Campbell, K. A review of the global pesticide legislation and the scale of challenge in reaching the global harmonization of food safety standards. Integr. Environ. Assess. Manag. 2015, 11, 525–536. [Google Scholar]
  13. Allen, B.; Wu, J.; Doan, H. Inactivation of fungi associated with barley grain by gaseous ozone. J. Environ. Sci. Health Part B 2003, 38, 617–630. [Google Scholar]
  14. Mylona, K. Fusarium Species in Grains: Dry Matter Losses, Mycotoxin Contamination and Control Strategies Using Ozone and Chemical Compounds. Ph.D. Thesis, Cranfield University, Wharley End, UK, 2012. [Google Scholar]
  15. Plumier, B.M.; Schramm, M.; Ren, Y.; Maier, D.E. Modeling post-fumigation desorption of phosphine in bulk stored grain. J. Stored Prod. Res. 2020, 85, 101548. [Google Scholar]
  16. Bluma, R.; Amaiden, M.R.; Etcheverry, M. Screening of Argentine plant extracts: Impact on growth parameters and aflatoxin B1 accumulation by Aspergillus section Flavi. Int. J. Food Microbiol. 2008, 122, 114–125. [Google Scholar]
  17. Zhang, S.; Zheng, M.; Zhai, H.; Lyu, Y.; Hu, Y.; Cai, J. Effects of hexanal fumigation on fungal spoilage and grain quality of stored wheat. Grain Oil Sci. Technol. 2021, 4, 10–17. [Google Scholar]
  18. Hocking, A.D.; Banks, H.J. Effects of phosphine fumigation on survival and growth of storage fungi in wheat. J. Stored Prod. Res. 1991, 27, 115–120. [Google Scholar]
  19. Savi, G.D.; Gomes, T.; Canever, S.B.; Feltrin, A.C.; Piacentini, K.C.; Scussel, R.; Oliveira, D.; Machado-de-Avila, R.A.; Cargnin, M.; Angioletto, E. Application of ozone on rice storage: A mathematical modeling of the ozone spread, effects in the decontamination of filamentous fungi and quality attributes. J. Stored Prod. Res. 2020, 87, 101605. [Google Scholar]
  20. Agarwal, M.; Ren, Y.; Newman, J.; Learmonth, S. Ethyl formate: A potential disinfestation treatment for eucalyptus weevil (Gonipterus platensis) (Coleoptera: Curculionidae) in apples. J. Econ. Entomol. 2015, 108, 2566–2571. [Google Scholar]
  21. Aharoni, Y.; Stewart, J.K.; Guadagni, D.G.; Mon, T.R. Thrips mortality and strawberry quality after vacuum fumigation with acetaldehyde or ethyl formate. J. Am. Soc. Hortic. Sci. 1980, 105, 926–929. [Google Scholar]
  22. Ryan, R.; Dominiak, B.C. Ethyl formate: Review of a rapid acting fumigant. In Proceedings of the 11th International Conference on Controlled Atmosphere and Fumigation in Stored Products (CAF2020), Winnipeg, Canada, 23–28 August 2020; Jayas, D.S., Jian, F., Eds.; CAF Permanent Committee Secretariat: Winnipeg, MB, Canada, 2021; pp. 269–275. [Google Scholar]
  23. Abo-El-Saad, M.M.; Badawy, M.E.; Shawir, M.S.; Hegazi, Y.H.; Mohammed, Y.M. In vitro and in vivo antimicrobial activity of the fumigant, ethyl formate, against bacteria and fungi of date fruits in postharvest phase. J. Food Meas. Charact. 2024, 18, 5791–5803. [Google Scholar] [CrossRef]
  24. Hassan, R.A.; Sand, M.I.; El-Kadi, S.M. Effect of some organic acids on fungal growth and their toxins production. J. Agric. Chem. Biotechnol. 2012, 3, 391–397. [Google Scholar] [CrossRef]
  25. Holmberg, T.; Kaspersson, A.; Larsson, K.; Pettersson, H. Aflatoxin production in moist barley treated with suboptimal doses of formic and propionic acid. Acta Agric. Scand. 1989, 39, 457–464. [Google Scholar] [CrossRef]
  26. Ren, Y.L. Natural levels of ethyl formate in stored grains determined using an improved method of analysis. J. Stored Prod. Res. 2004, 40, 77–85. [Google Scholar]
  27. Booth, C. The Genus Fusarium; Commonwealth Mycological Institute: Kew, UK, 1971. [Google Scholar]
  28. Booth, C. Fusarium. Laboratory Guide to the Identification of the Major Species; Commonwealth Mycological Institute: Kew, UK, 1977. [Google Scholar]
  29. Pitt, J.I. The Genus Penicillium and Its Teleomorphic States Eupenicillium and Talaromyces; Academic Press Inc. Ltd.: Cambridge, MA, USA, 1979. [Google Scholar]
  30. Pitt, J.I. A Laboratory Guide to Common Penicillium Species; Commonwealth Scientific and Industrial Research Organization, Division of Food Processing: Sydney, NSW, Australia, 1991. [Google Scholar]
  31. Moubasher, A.H. Soil Fungi in Qatar and Other Arab Countries; The Centre for Scientific and Applied Research, University of Qatar: Doha, Qatar, 1993. [Google Scholar]
  32. Samson, R.A.; Hoekstra, E.S.; Frisvad, J.C. Introduction to Food-and Airborne Fungi, 7th ed.; Centraalbureau voor Schimmelcultures (CBS): Utrecht, The Netherlands, 2004. [Google Scholar]
  33. Summerell, B.A.; Salleh, B.; Leslie, J.F. A utilitarian approach to Fusarium identification. Plant. Dis. 2003, 87, 117–128. [Google Scholar]
  34. Andjic, V.; Barber, P.A.; Carnegie, A.J.; Hardy, G.S.J.; Wingfield, M.J.; Burgess, T.I. Phylogenetic reassessment supports accommodation of Phaeophleospora and Colletogloeopsis from eucalypts in Kirramyces. Mycol. Res. 2007, 111, 1184–1198. [Google Scholar]
  35. Gardes, M.; Bruns, T.D. ITS primers with enhanced specificity for basidiomycetes-application to the identification of mycorrhizae and rusts. Mol. Ecol. 1993, 2, 113–118. [Google Scholar]
  36. White, T.J.; Bruns, T.; Lee, S.J.W.T.; Taylor, J. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. PCR Protoc. A Guide Methods Appl. 1990, 18, 315–322. [Google Scholar]
  37. Sakalidis, M.L.; Hardy, G.E.S.; Burgess, T.I. Endophytes as potential pathogens of the baobab species Adansonia gregorii: A focus on the Botryosphaeriaceae. Fungal Ecol. 2011, 4, 1–14. [Google Scholar]
  38. Coetzee, E.M. Exploration of Ethyl Formate + Nitrogen as a Fumigant for Shipping Containers and Their In-Transit Fumigation. Ph.D. Thesis, Murdoch University, Murdoch, WA, Australia, 2020. [Google Scholar]
  39. Castañares, E.; da Cruz Cabral, L.; Dinolfo, M.I.; Andersen, B.; Stenglein, S.A.; Patriarca, A. Alternaria in malting barley: Characterization and distribution in relation with climatic conditions and barley cultivars. Int. J. Food Microbiol. 2021, 357, 109367. [Google Scholar]
  40. Sultan, A.; Frisvad, J.C.; Andersen, B.; Svensson, B.; Finnie, C. Investigation of the indigenous fungal community populating barley grains: Secretomes and xylanolytic potential. J. Proteom. 2017, 169, 153–164. [Google Scholar] [CrossRef] [PubMed]
  41. Chen, W.; Turkington, T.K.; Lévesque, C.A.; Bamforth, J.M.; Patrick, S.K.; Lewis, C.T.; Chapados, J.T.; Gaba, D.; Tittlemier, S.A.; MacLeod, A.; et al. Geography and agronomical practices drive diversification of the epiphytic mycoflora associated with barley and its malt end product in western Canada. Agric. Ecosyst. Environ. 2016, 226, 43–55. [Google Scholar] [CrossRef]
  42. Oksińska, M.P.; Magnucka, E.G.; Kmieć, A.; Pietr, S.J. Molecular identification of ascomycetes from American cranberry (Vaccinium macrocarpon Aiton) grown in plantation in Poland. Appl. Sci. 2024, 14, 4328. [Google Scholar] [CrossRef]
  43. Vipin, C.A. Identification of Loci Associated with Pleiochaeta Setosa in White Lupin. Master’s Thesis, Charles Sturt University, Bathurst, NSW, Australia, 2013. [Google Scholar]
  44. Babič, J.; Tavčar-Kalcher, G.; Celar, F.A.; Kos, K.; Knific, T.; Jakovac-Strajn, B. Occurrence of Alternaria and other toxins in cereal grains intended for animal feeding collected in Slovenia: A three-year study. Toxins 2021, 13, 304. [Google Scholar] [CrossRef]
  45. Waterford, C.J.; Ren, Y.; Lee, B.H.; Mahon, D.; Ryan, R.F. Carbonyl sulphide and ethyl formate update. In Proceedings of the 2004 Annual International Research Conference on Methyl Bromide Alternatives and Emissions Reductions, Orlando, FL, USA, 31 October–3 November 2004; Volume 31, pp. 60–61. [Google Scholar]
  46. Ren, Y.L.; Lee, B.H.; Padovan, B. Penetration of methyl bromide, sulfuryl fluoride, ethanedinitrile and phosphine into timber blocks and the sorption rate of the fumigants. J. Stored Prod. Res. 2011, 47, 63–68. [Google Scholar] [CrossRef]
  47. Noots, I.; Delcour, J.A.; Michiels, C.W. From field barley to malt: Detection and specifi cation of microbial activity for quality aspects. Crit. Rev. Microbiol. 1999, 25, 121–153. [Google Scholar] [CrossRef]
  48. Utama, I.M.S.; Wills, R.B.; Ben-Yehoshua, S.; Kuek, C. In vitro efficacy of plant volatiles for inhibiting the growth of fruit and vegetable decay microorganisms. J. Agric. Food Chem. 2002, 50, 6371–6377. [Google Scholar] [CrossRef]
  49. Lastauskienė, E.; Zinkevičienė, A.; Girkontaitė, I.; Kaunietis, A.; Kvedarienė, V. Formic acid and acetic acid induce a programmed cell death in pathogenic Candida species. Curr. Microbiol. 2014, 69, 303–310. [Google Scholar] [CrossRef]
  50. Revis, H.C.; Waller, D.A. Bactericidal and fungicidal activity of ant chemicals on feather parasites: An evaluation of anting behavior as a method of self-medication in songbirds. Auk 2004, 121, 1262–1268. [Google Scholar] [CrossRef]
  51. Yan, D.; Liu, J.; Wang, X.; Fang, W.; Li, Y.; Cao, A.; Wang, Q. A review on the mechanisms of fumigant action. New Plant Prot. 2025, 2, e27. [Google Scholar] [CrossRef]
  52. Islam, T.; Danishuddin Tamanna, N.T.; Matin, M.N.; Barai, H.R.; Haque, M.A. Resistance mechanisms of plant pathogenic fungi to fungicide, environmental impacts of fungicides, and sustainable solutions. Plants 2024, 13, 2737. [Google Scholar] [CrossRef]
Agriculture 15 00748 g001
Figure 1. The morphological characteristics of fungal species on PDA isolated from barley grain: (a–d) A. infectoria; (e) Penicillium sp. 1; (f) Penicillium sp. 2; (g) E. nigrum; (h) F. chlamydosporum; (i) F. chlamydosporum; (j) E. nigrum; (k) Fusarium sp.; (l) Aureobasidium sp. 1; (m) P. teres; (n) Aureobasidium sp. 2; (o) P. setosa; (p) Cladosporium sp.; (q) Aureobasidium sp. 2; (r) Curvularia sp.; (s) E. nigrum; (t) D. prosopidis; (u) R. arrhizus; and (v) N. oryzae.
Figure 1. The morphological characteristics of fungal species on PDA isolated from barley grain: (a–d) A. infectoria; (e) Penicillium sp. 1; (f) Penicillium sp. 2; (g) E. nigrum; (h) F. chlamydosporum; (i) F. chlamydosporum; (j) E. nigrum; (k) Fusarium sp.; (l) Aureobasidium sp. 1; (m) P. teres; (n) Aureobasidium sp. 2; (o) P. setosa; (p) Cladosporium sp.; (q) Aureobasidium sp. 2; (r) Curvularia sp.; (s) E. nigrum; (t) D. prosopidis; (u) R. arrhizus; and (v) N. oryzae.
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Agriculture 15 00748 g002
Figure 2. A phylogenetic tree of fungal species isolated from Australian barley constructed using internal transcribed spacer (ITS) sequence data.
Figure 2. A phylogenetic tree of fungal species isolated from Australian barley constructed using internal transcribed spacer (ITS) sequence data.
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Agriculture 15 00748 g003
Figure 3. Bar charts of vigor and germination rates (%) of barley seedlings treated with ethyl formate (EF) at concentrations of 2.5, 3.7, and 5 mmol/L (90, 140, and 180 mg/L). Except for ungerminated seeds, seeds with either root or plumule lengths of less than 2 cm were also classified as ungerminated. These differences between the control and treatments are considered to not be statistically significant with a p-value < 0.05.
Figure 3. Bar charts of vigor and germination rates (%) of barley seedlings treated with ethyl formate (EF) at concentrations of 2.5, 3.7, and 5 mmol/L (90, 140, and 180 mg/L). Except for ungerminated seeds, seeds with either root or plumule lengths of less than 2 cm were also classified as ungerminated. These differences between the control and treatments are considered to not be statistically significant with a p-value < 0.05.
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Figure 4. Violin plots of ethyl formate on barley root (a) and plumule (b) lengths at concentrations of 2.5, 3.7, and 5 mmol/L (mM/L) compared to the untreated control group. Yellow points represent the mean values, and black dots indicate individual measurements.
Figure 4. Violin plots of ethyl formate on barley root (a) and plumule (b) lengths at concentrations of 2.5, 3.7, and 5 mmol/L (mM/L) compared to the untreated control group. Yellow points represent the mean values, and black dots indicate individual measurements.
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Table 1. Putative identification of fungal species isolated from barley with full-strength potato dextrose agar (PDA) media. Identification was based on 16S rDNA sequence similarity obtained from heuristic search against sequences in NCBI GenBank.
Table 1. Putative identification of fungal species isolated from barley with full-strength potato dextrose agar (PDA) media. Identification was based on 16S rDNA sequence similarity obtained from heuristic search against sequences in NCBI GenBank.
NamesBlast Sequences with the Highest Identity (%)GenBank Accession Number
G01Alternaria infectoria (99.83%)PP429847.1
G02Alternaria infectoria (100%)MW446954.1
G03Alternaria infectoria (100%)PP429847.1
G04Alternaria infectoria (100%)LN809032.1
G05Penicillium sp. 1 (99.95%)MK450671.1
G06Penicillium sp. 2 (99.95%)MH861314.1
G07Epicoccum nigrum (100%)MK460967.1
G08Fusarium chlamydosporum (99.82%)ON074988.1
G09Fusarium chlamydosporum (100%)ON074991.1
G10Epicoccum nigrum (100%)AJ279486.1
G11Fusarium sp. (100%)MF509746.1
G12Aureobasidium sp. 1 (99.82%)ON074960.1
G13Pyrenophora teres (100%)MK450003.1
G14Aureobasidium sp. 2 (100%)JK984782.1
G15Pleiochaeta setosa (100%)EU167563.1
G16Cladosporium sp. (100%)MN313285.1
G17Aureobasidium sp. 2 (100%)JK984782.1
G18Curvularia sp. (99.82%)MN215655.1
G19Epicoccum nigrum (99.81%)OM363478.1
G20Didymella prosopidis (100%)ON682327.1
G21Rhizopus arrhizus (100%)MW821588.1
G22Nigrospora oryzae (100%)JN207354.1
Table 2. The effect of 90 mg/L (2.4 mmol/L) of ethyl formate on the growth of 22 fungal isolates.
Table 2. The effect of 90 mg/L (2.4 mmol/L) of ethyl formate on the growth of 22 fungal isolates.
Fungi NamesEthyl Formate Treatment * Colony Size of Control Group
Width (cm)Length (cm)Area (cm2)
A. infectoria (G01)-2.72.722.9
A. infectoria (G02)-2.7217.0
A. infectoria (G03)-32.725.4
A. infectoria (G04)-2.21.510.4
Penicillium sp. 1 (G05)-9.09.0254.5
Penicillium sp. 2 (G06)+9.09.0254.5
E. nigrum (G07)-2.82.219.4
F. chlamydosporum (G08)-2.72.218.7
F. chlamydosporum (G09)+2.52.015.7
E. nigrum (G10)-3.23.333.2
Fusarium sp. (G11)-2.01.59.4
Aureobasidium sp. 1 (G12) -2.01.811.3
P. teres/graminea (G13)-1.71.58.0
Aureobasidium sp. 2 (G14)-2.32.417.3
P. setosa (G15)-2.62.419.6
Cladosporium sp. (G16)-1.32.08.2
Aureobasidium sp. 2 (G17)-1.71.79.1
Curvularia sp. (G18)-3.03.331.1
E. nigrum (G19)-4.74.059.1
D. prosopidis (G20)-3.03.028.3
R. arrhizus (G21)+9.09.0254.5
N. oryzae (G22)-9.09.0254.5
* -: 100% complete inhibition. +: Non-complete inhibition.
Table 3. The effect of 1.4, 2.4, 4.1, 5.4, and 10.8 mmol/L ethyl formate on the growth of four selected fungus species of A. infectoria, penicillum sp. 2, F. chlamydosporum, and R. arrhizus isolated from barley grain.
Table 3. The effect of 1.4, 2.4, 4.1, 5.4, and 10.8 mmol/L ethyl formate on the growth of four selected fungus species of A. infectoria, penicillum sp. 2, F. chlamydosporum, and R. arrhizus isolated from barley grain.
Selected FungiInhibition Rate %
1.4 mmol/L 2.4 mmol/L4.1 mmol/L5.4 mmol/L10.8 mmol/L
A. infectoria26 ± 9%100%100%100%100%
Penicillium sp. 2 0%0%0%0%98 ± 2%
F. chlamydosporum16 ± 12%63 ± 4%85 ± 12%100%100%
R. arrhizus0%0%0%100%100%
Table 4. The inhibition rate of A. infectoria, Penicillium sp. 2, F. chlamydosporum, and R. arrhizus after exposure to 0.01, 0.02, 0.04, 0.08, and 0.18 mmol/L of formic acid.
Table 4. The inhibition rate of A. infectoria, Penicillium sp. 2, F. chlamydosporum, and R. arrhizus after exposure to 0.01, 0.02, 0.04, 0.08, and 0.18 mmol/L of formic acid.
Selected FungiInhibition Rate %
0.01 mmol/L0.02 mmol/L0.04 mmol/L0.08 mmol/L0.18 mmol/L
A. infectoria17 ± 9%16 ± 14%100%100%100%
Penicillium sp. 20%0%0%100%100%
F. chlamydosporum31 ± 10%26 ± 1%55 ± 6%88 ± 21%100%
R. arrhizus0%0%100%100%100%
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Barkat, E.; Du, X.; Kim, J.-R.; Wang, Z.; Lee, B.-H.; Uaraboa, A.; Uaraboa, E.; Kim, B.-S. A Novel Approach for Fungal Control with Ethyl Formate in Barley and Its Impact on Germination. Agriculture 2025, 15, 748. https://doi.org/10.3390/agriculture15070748

AMA Style

Barkat E, Du X, Kim J-R, Wang Z, Lee B-H, Uaraboa A, Uaraboa E, Kim B-S. A Novel Approach for Fungal Control with Ethyl Formate in Barley and Its Impact on Germination. Agriculture. 2025; 15(7):748. https://doi.org/10.3390/agriculture15070748

Chicago/Turabian Style

Barkat, Eman, Xin Du, Jun-Ran Kim, Zhengyan Wang, Byung-Ho Lee, Abrar Uaraboa, Esra Uaraboa, and Bong-Su Kim. 2025. "A Novel Approach for Fungal Control with Ethyl Formate in Barley and Its Impact on Germination" Agriculture 15, no. 7: 748. https://doi.org/10.3390/agriculture15070748

APA Style

Barkat, E., Du, X., Kim, J.-R., Wang, Z., Lee, B.-H., Uaraboa, A., Uaraboa, E., & Kim, B.-S. (2025). A Novel Approach for Fungal Control with Ethyl Formate in Barley and Its Impact on Germination. Agriculture, 15(7), 748. https://doi.org/10.3390/agriculture15070748

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