1 Introduction

Pakistan is mainly dependent on its agriculture sector. About half of Pakistan’s population is dependent upon agriculture. Different researchers recommended that agricultural development is directly related to the national economy of the country (Ali and Iqbal, 2004). In addition to other agriculture products, livestock is also thought to be an important contributor to Pakistan’s GDP because it adds almost 37% value-addition to the national GDP. According to the Food and Agriculture Organization (FAO) reports, 25% of the cereals in the world are destroyed by mycotoxins (Klopfenstein,2000).

Mycotoxins are diverse types of molecules that are injurious to living things. They are generally present in cereals and grains during the pre or post-harvesting stage and due to their varied toxicity, they are considered injurious for the users (Yiannikouris and Jouany,2002). Many species of fungi account for the production of mycotoxins in animal feedstuff (Yiannikouris and Jouany,2002; Salari et al., 2012). The threat of mycotoxin contamination especially aflatoxin in cereals, grains and other field crops is a serious safety concern for the world (Reddy et al.,2009).

In olden times, many techniques were used to inhibit the progression of mycotoxins i.e. microorganisms, absorbents, chemicals and ionizing radiations (Sinha, 1998). Nevertheless, natural plants and plant extracts have expanded the attention of researchers due to their less toxic effects. Natural compounds from higher plants are cheap and ecofriendly and are considered as antimicrobial agents (Reddy et al., 2009; Gonçalez et al., 2003).

Plants possessing powerful antimicrobial properties can be used to protect food from different types of toxins. Natural compounds from the plant can also be served as antifungal agents (Abid et al., 2022). Additionally, citrus plants are reported to have medicinal importance. Crude extracts from these plant's families can be used as natural remedies to cure many diseases i.e. diarrhea, constipation and vomiting, etc (Gonçalez et al., 2003). Citrus fruits have an ample amount of carotenoids, minerals, limonoids, essential oils, flavonoids, acridone alkaloids, and vitamin B and C complex. Limonoids, glycosides, flavones and polymethoxyflavones are the flavenoids naturally produced by citrus plants. (Gonçalez et al., 2003; Sinha, 1998). They have many health regulating activities i.e. anti-viral, anti-cancerous, anti-fungal, anti-bacterial, anti-oxidant, and anti-allergic potencies. Lemon juice is employed as a diuretic agent, antiscorbutic and also used against the common cold (Salari et al., 2012). Moreover, the antifungal and anti-aflatoxigenic efficacy of Aloe vera, Zizyphusspina, Cassia italica, Lavandula vera, Olea europaea, Ricinus communis, Lawsonia inermis, Prosopisjuliflora, Datura stramonium and Eucalyptus globulus is also documented to be used as inhibitors of aflatoxin production (Reddy et al.,2009). The current study aimed to isolate the fungal species and also analyze their mycotoxins in the samples of animal feeds available in the local markets of Peshawar. Also, it was aimed to test the antifungal activity of different plant extracts against those fungal species. The synergistic effects of toxin inhibition and mycoflora promotion were also aimed in the present study.

2 Methods and materials

3 Results

3.1

3.1 Quantification of fungus in animal feed

All the samples were found to have a variable number of fungal colonies that were presented as cfu/g in Fig. 1. The highest TPC value of 4x10²¹ was shown by sample C while A was found to have the lowest TPC value of 5x10^14. All other samples showed moderate TPC values.

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Fig. 1

All samples showing different TPC values.

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3.2

3.2 Fungal flora of animal feed samples

The plating technique was used for the investigation of feed samples. A representative figure of a couple of isolates is shown in Fig. 2 and Fig. 3. Table 3 shows the list of isolated fungi samples. Seven species of Aspergillus i-e; A. parasiticus, A. flavus, A. niger, A. ochraceus, A. clavatus, A. fumigatus, A. carbonarius and three species of Penicillium i-e; P. citrinum, P. verrucosum and P. notatum were obtained from feed samples. All ten samples were found positive for Aspergillus flavus while only one sample was positive for Aspergillus notaum i-e; sample G.

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Fig. 2

Growth of Aspergillus flavus on PDA medium.

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Fig. 3

Growth of Aspergillus ochracheus on PDA medium.

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Table 3 Identification of fungal flora.

S. No	Sample ID	Identified Fungus
01	A	Aspergillus niger
		Aspergillus flavus
		Aspergillus parasiticus
		Aspergillus carbonarius
02	B	Aspergillus niger
		Aspergillus flavus
		Aspergillus ochraceus
03	C	Aspergillus niger
		Aspergillus flavus
		Aspergillus parasiticus
		Aspergillus carbonarius
04	D	Aspergillus flavus
		Aspergillus parasiticus
05	E	Penicillium citrinum
		Aspergillus parasiticus
		Aspergillus carbonarius
		Aspergillus flavus
06	F	Aspergillus niger
		Aspergillus flavus
		Aspergillus parasiticus
		Aspergillus carbonarius
07	G	Aspergillus notatum
		Aspergillus flavus
		Aspergillus parasiticus
		Aspergillus carbonarius
08	H	Aspergillus flavus
		Aspergillus parasiticus
		Penicillium citrinum
09	I	Aspergillus niger
		Aspergillus flavus
		Aspergillus parasiticus
		Aspergillus carbonarius
		Aspergillus ochraceus
		Penicillium citrinum
10	J	Aspergillus niger
		Aspergillus flavus
		Aspergillus parasiticus
		Aspergillus carbonarius

3.7

3.7 Percent mycelial inhibition of A. flavus

The antifungal activity of the aqueous extract of each plant was determined against A. flavus by poison food technique for the determination of percent mycelial inhibition. The result is presented in Fig. 4. Citrus aurantium showed the highest (62.3 ± 0.33) mycelial inhibition of A. flavus, while Citrus limon showed the lowest mycelial inhibition (46.5 ± 0.46).

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Fig. 4

% Mycelial inhibition of A. flavus by selected plant extract. Data given are the mean of four replicates ± standard error.

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4 Discussion

The present study was carried out to check the antifungal effect of the plant leaves aqueous extract of specific plants to inhibit or lower the fungal growth and their mycotoxins in animal feed samples. In the current study, six medicinal plants (listed in Table 1) were used to investigate their antifungal and antimycotoxinigenic capacity. The highest activity among six plants was shown by Myrtus communis against aspergillus clavatus. It has been previously reported that Myrtus communis has significant antifungal ability against dermatophytic fungi. The research has confirmed that methanolic extract of M.communis leaves inhibits Mycosporum canis (Mehrabani et al., 2013). In another study, the growth of R. solani was inhabited by 60% through essential oils of Myrtus communis at a concentration of 1600 ppm (Curini et al., 2003). The difference between the results of our study and Curini et al., (2003) may be due to the techniques used to calculate antifungal activity and selection of test fungi.

Numerous studies exposed the noteworthy biological activity of Aloe vera against bacteria, viruses, and fungi. Ethanol and aqueous extract of Aloe vera had shown exciting results against a number of bacteria and fungi. (Nidiry et al., 2011). In the current study aqueous, extracts of aloe vera leaves were tested that inhibited the growth of different species of Aspergillus and Penicillium.

Shireen et al., (2015) documented that Aloe vera extract showed strong antifungal activity against Candida albicans (Shireen et al., 2015). In the present study, an aqueous extract of Aloe vera leaves was also subjected to the percentage (%) mycelial inhibition of Aspergillus flavus and results showed that about 51 ± 0.37% growth was inhibited. Coopoosamy and Magwa et al., (2007) documented the antifungal activity of Aloe vera and reported that Aloe vera restricts the growth of Aspergillus niger to 24.29%, 9.26% of Aspergillus flavus growth and 6.24% of Penicillium digitatum growth (Coopoosamy and Magwa et al., 2007). Moreover, Kawai and his group (1998) showed that crude extracts of Aloe vera controlled the production of aflatoxin (Kawai et al., 1998).

Sharma and Tripathi, 2009 reported that citrus essential oils comprise a complex mixture of volatile compounds having a potential antifungal activity that reduces the growth of fungi in a dose–response manner or inhibits fungal growth. In the present research, the Citrus sinensis leaves were found to be an effective inhibitor against Penicillium and Aspergillus. The highest antifungal activity of Citrus sinensis was recorded against Aspergillus parasiticus whereas MIC and MFC were recorded as 140 mg/ml and 160 mg/ml respectively and their results showed MIC 4.09.0 µl/ml against the Trichoderma harzianum and Verticillium fungicola.

In the present research, the C. sinensis leaves extract was also subjected to the percentage (%) mycelial inhibition of Aspergillus flavus. (Chutia et al., 2009) studied C. sinensis essential oil for the percentage (%) growth inhibition of diverse fungal species, they found that C. sinensis inhibited Fusarium oxysporum Alternaria alternate (84%) (42%), Curvularia lunata (93.25%) and Helminthosporium oryzae (54%). The variation of our results in comparison with earlier studies is possible because of the selection of different fungal species and methods.

In our study, Citrus aurantium presented high antifungal efficiency. The highest activity was found in the case of Aspergillus ochraceus, the zone of inhibition was calculated as 17 ± 1 mm, where MIC and MFC were noted to be 110 mg/ml and 130 mg/ml respectively. (Hsouna et al., 2013) found that the antifungal potency of Citrus aurantium against the growth of, Fusarium, Alternaria alternata and Aspergillus. According to this research, Citrus aurantium essential oil inhibited the growth of Fusarium graminearum and Aspergillus flavus with a zone of inhibition of 22 mm and MIC value of 78 µg/ml respectively. Also, Citrus aurantium showed high antifungal potential against Aspergillus niger. Fusarium oxyporum and Aspergillus flavus. The zone of inhibition ranged 14–34 mm and MIC was 0.078–1.25 mg/ml.

In the current investigation, Citrus limon showed modest antifungal activity. The most important activity was recorded in the case of Aspergillus clavatus and its zone of inhibition was 11 mm. In the present research, the percentage (%) of mycelial inhibition of A. flavus was also done. Citrus limon inhibited the growth of A. flavus up to 46.5 ± 0.46%. Similarly, the percentage (%) of mycelial inhibition of A. flavus was also done by (Chutia et al., 2009). Furthermore, Citrus limon essential oil also restricted the growth of A. flavus up to 44.0 ± 0.07%. Fisher and Phillips, (2006) found that the spread of foodborne microbes can be controlled by using essential oils of different citrus plants such as C. sinensis, C. limon, C. bergamia and their constituents. Also, (Hosni et al., 2010) reported that three monoterpenes are effective antifungal components that are 1,8-cineole, thymol, and (S)-limonene. (Hosni et al., 2010) found that essential oils from C. limon showed antifungal activity against Glomerella cingulata and Fusarium oxysporum.

5 Conclusions

Despite the existence of plentiful agricultural-based food resources for livestock in Pakistan, some pathogenic fungal species are accountable for polluting livestock feed by making mycotoxins. Moreover, mycotoxin contamination, mainly aflatoxins, is the main distress for the safety of grains and other field crops around the world. Natural plants and plant extracts, on the other hand, have captured the attention of the scientific community due to their less toxic effects. Natural products derived from higher plants are environmental friendly, biodegradable, substantial, and have antifungal and antimycotoxigenic properties. Results of the present study showed that all the aflatoxins i.e., G1, G2, B1 and B2 were present in varying amounts depending on the plants. The highest quantity was present of B1 in all ten samples while G2 was in a lower amount as compared to other samples. Relating to the antifungal properties of the samples Citrus aurantium and Myrtus communis were absorbed to have antifungal potency against all test fungi. Citrus aurantium extracts were found to inhibit the growth of Aspergillus flavus. Future research is needed to investigate the proper genomic understanding of these mycotoxins as an effective treatment therapy. Furthermore, good surveillance and proper hygienic measures should be implemented to reduce the likelihood of pathogenic contaminants in animal feed.

CRediT authorship contribution statement

Raza Ullah: Conceptualization, Methodology. Isfahan Touseef: Conceptualization, Methodology, Supervision. Rameesha Abid: Data curation, Writing – review & editing, Visualization, Validation. Arshad Farid: Writing – review & editing, Visualization, Formal analysis, Supervision. Sohail Ahmad: Writing – review & editing, Formal analysis. Hesham Ali El Enshasy: Writing – review & editing, Resources, Validation, Formal analysis. Adil Aksoy: Resources, Investigation, Supervision. Nada H. Aljarba: Funding acquisition. Tahani Mohamed Al–Hazani: Funding acquisition. Muhammad Muzammal: Resources, Investigation, Supervision. Shakira Ghazanfar: Validation, Formal analysis, Writing – original draft, Writing – review & editing.