**1. Introduction**

Mycotoxins, i.e., aflatoxins, are a type of fungal polyketide secondary metabolite that are produced mostly by *Aspergillus*, including *Aspergillus flavus* [1,2]. Currently, there are 18 types of aflatoxin produced by *Aspergillus* spp., of which the four principal kinds are Aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), aflatoxin G1 (AFG1), and aflatoxin G2 (AFG2) [3]. AFB1 is the most common mycotoxin in nature and is functionally carcinogenic in animal models as well as mammals if the toxicity level exceeds a certain threshold [4]. Aflatoxigenic fungi can cause damage loss in seeds growth, preservation, or viability [5]. Aflatoxin contamination primarily affects dried fruits (such as nuts and peanuts), cereal grains (maizes, etc.), some spices, and oils [6,7]. The primary sources of the world's exposure to aflatoxins are maize and peanuts because of their high consumption [8]. Consequently, solutions for the control of aflatoxigenic *A. flavus* in maize grains and food during storage are in demand worldwide [9]. Chemical treatments can effectively control aflatoxins, but they cannot be used on grains, cereals, or other food materials due to hazardous residues, teratogenicity, carcinogenicity, spermatotoxicity, and hormonal imbalances, as well as the development of resistance microbes against antimicrobial agents [10–12]. Currently, the use of plant-based natural antifungal agents is considered a beneficial and healthy practice in this regard [13,14]. Plant extracts could be employed as antimicrobial agents or for improved food storage and preservation due to their high activity, the simplicity of their production and utilization, their reliability, and their biocompatibility [15,16].

The release of large quantities of agro-by-product wastes such as peels and seed husks is one of the biggest problems facing society, as they are grave threats to the environment [17,18]. As a result, researchers continue to assess into the possibility of reusing these wastes. Such wastes encompass a wide range of compositions, including high levels of proteins, carbohydrates, and minerals. It was reported that many fruit peels offer a range of biological and medicinal properties and are known to contain them [19,20]. Pomegranate peels, lemon peels, and green walnut husks have been reported to be effective natural antimicrobials in various investigations [21–23]. Pomegranate peel extracts are high in functional molecules, such as flavones, phenylpropanoids, and alkaloids, which feature potent antioxidant properties [24,25]. Many investigators have reported the significant antifungal activity of pomegranate and eggplant peel extracts against many phytopathogens [26–29]. Linoleic acid is known to feature antifungal activity in larger plants as a substrate for producing a series of trihydroxy oxylipins [30]. The growth and biomass production of *Rhizoctonia solani* was reduced by 74% and *Pythium ultimum* by 65% when 1000 μM linolenic acid and allylphenol were applied together [31]. Several studies have reported that such natural compounds, including essential oils or extracts such as monoterpenoids and sesquiterpenes could suppress *A. flavus* growth and AFB1 formation by downregulating the transcription of genes involved in AFB1 synthesis [32,33].

Generally, AFB1 is produced from a complicated biosynthetic pathway, including at least 28 enzymatic steps. The structural genes encoding these enzymes are grouped in one gene cluster while two cluster-specific regulators, aflR and aflS, mainly regulate their expression [34,35]. It was reported that the decrease in the transcription levels of aflatoxin genes was associated with a reduction in AFB1 production. The ability of many plant-derived substances to stop AFB1 production or inhibit its expression has been observed [36–38]. Several investigations have shown that various doses of different plant extracts suppress the expression of 25 of the 27 studied genes in the AFB1 biosynthesis pathway [39,40]. The purpose of this study was to evaluate the effectiveness of different extracts of pomegranate, sugar apple, and eggplant peels to inhibit *A. flavus* growth; to test the ability of the peel extracts to suppress the expression of AFB1 biosynthesis genes in maize grains compared with Topsin fungicide; and to identify the different bioactive compounds of the best extracts using the GC-MS analysis technique.

### **2. Materials and Methods**

### *2.1. Fungus Isolation and Identification*

Aspergillus isolate was isolated from local maize grains, purified, characterized morphologically, and assessed for its ability to produce AFB1. The aflatoxigenic isolate was identified by sequencing the amplified ITS region [41,42].

### *2.2. Peel Extracts Preparation*

The pomegranate (*Punica granatum* L.), sugar apple (*Annona squamosa* L.), and eggplant (*Solanum melongena* L.) edible fruits were purchased from local markets in Alexandria Governorate, Egypt. All the fruits were washed and surface sterilized; the peels obtained, air-dried, and pulverized to a fine powder [43]. Twenty grams of the fine powder for each fruit peel was mixed with 100 mL of each of the four solvents: ethanol, diethyl ether, methanol, and acetone, with three concentrations, of 25%, 50%, and 75% (solvent/water, *v*/*v*). The preparations were left overnight on an orbital shaker (Heidolph, Schwabach, Germany) at 200 rpm. All the mixtures were filtered using Whatman No. 1 and stored in a refrigerator (at 5 ◦C) until further use.

### *2.3. Total Phenolics Content*

The Folin–Ciocalteau reagent (FCR) assay (Sigma-Aldrich, Taufkirchen, Germany) [44], was used to determine the total polyphenols content (TPC), with slight modifications. A total of 0.1 mg/mL of extract was dissolved in distilled water. Next, 0.5 mL FCR (1 mol/L) and 1.5 mL of sodium carbonate (10% *w*/*v*) were added to 0.5 mL of each extract. The final mixture was kept for 30 min in the dark, and the absorbance values at a wavelength (λ) = 725 nm were measured. The TPC was calculated according to a standard curve using gallic acid prepared in methanol with 12.5, 25, 50, 75, and 100 μg/mL concentrations. The concentrations of TPC were expressed in milligrams of gallic acid equivalents per gram of dry extract weight (mg GAEs/g DW) [45].

### *2.4. DPPH Radical Scavenging Ability*

The 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical (Sigma-Aldrich, Taufkirchen, Germany) was used to test antioxidant activity, and the capacities of the extracts to scavenge free radicals were determined as described by Asnaashari et al. [46]. The calculation equation was: (DPPH) % = [(Ab − Abs)/Ab] × 100 where Ab is the blank absorbance value and Abs is the sample absorbance value.

### *2.5. Gas Chromatography-Mass Spectroscopy Analysis*

Thermo Scientific ISQ Quadrupole GC-MS with Trace GC Ultra (Thermo Fisher Scientific, Waltham, MA, USA) equipped with a capillary column TG–5MS (30 m × 0.25 mm × 0.25 μm film thickness) was used as previously described [47,48]. The separation conditions were performed as outlined by Okla et al. [49]. For the identification of the different compounds in the fruit peel extract samples, the retention times and mass spectra databases were compared to those of authentic standards.

### *2.6. Effect of Fruit Peel Extracts on Fungal Biomass and Aflatoxin Production* 2.6.1.FungalBiomassDetermination

Fifteen mL of potato dextrose agar (PDA) media was poured into a petri dish, and after the solidification, a 5 mm disc of the aflatoxigenic fungus was placed in the center of the PDA petri dish and incubated for 7 days at 30 ◦C. In total, 1 mL of each fruit peel extract, previously prepared as described in Section 2.2, was added to 50 mL yeast extract sucrose (YES) broth in a conical flask. Next, a fungus disc was placed in each conical flask and set for 15 days at 30 ◦C. Each treatment's fungal mat was oven-dried. The wet and dry weights (g) of the fungal mat were recorded in all the treatments, and the filtrates were

maintained at 4 ◦C for later aflatoxin B1 analysis. The following equation was used to calculate the aflatoxin inhibition (AI) percentage ratio [50]:

$$(\text{AI})\% = \left[\frac{\text{AFB1 control} - \text{AFB1 treatment}}{\text{AFB1 control}}\right] \times 100\%$$

### 2.6.2. Maize Storage Experiment

Fifty grams of maize grains were treated with the fruit peel extracts, yielding the most significant results, as in the previous section. The treated grains were placed in sterilized-glass bottles. The fungicide treatment (2.5 mg/mL) was treated with Topsin (Thiophanate methyl, 70% wettable powder, United Phosphorus, Inc., King of Prussia, PA, USA), which was used as a control. Subsequently, each bottle was inoculated with a 5 mm disc of the aflatoxigenic fungus and kept for 30 days at 30 ◦C. The shape and odor of the maize grains were assessed after the storage period, using the scale developed by Youssef et al. [15]. All of the analyzed grains were crushed and refrigerated at 4 ◦C until they were used for further aflatoxin studies.

### 2.6.3. Aflatoxin B1 Extraction

Aflatoxin B1 (AFB1) was extracted by mixing 2 mL of fungal filtrate YES broth medium with chloroform (1:1 *v*/*v*). The mixture was centrifuged at 10,000 rpm for 5 min; a total of 2 mL of the bottom layer was transferred to a fresh glass vial. After evaporating under a moderate air stream, the dried chloroform extracts were re-dissolved with 1 mL methanol [51]. To extract the AFB1 from the contaminated maize grains, about 20 g of crushed grains was mixed with 100 mL of methanol and 12 mL of 4% potassium chloride (*w*/*v*), according to Hoeltz et al. [52], with some adjustments. The samples were filtered after a spin for 2 min at 10,000 rpm. The filtrate was then added to 100 mL of 10% ( *w*/*v*) CuSO4, mixed, and filtered. To extract the AFB1, 15 mL of an equal volume of chloroform and distilled water (1:1 *v*/*v*) was mixed with the filtrate in the separating funnel; this process was repeated twice. The solvent extracts were collected and evaporated. Before high-performance liquid chromatography (HPLC) analysis, all the samples were filtered into HPLC vials using a 0.2 m syringe filter (Thermo Fisher Scientific, Waltham, MA, USA).

#### 2.6.4. Preparation of AFB1 Standard and HPLC Conditions

To prepare the AFB1 standard (Merck, MO, USA), 1 mg was dissolved in 100 mL of toluene: acetonitrile (9:1, *v*/*v*) to obtain a final concentration of 10 μg/L. A working standard solution was prepared with a sample diluent (7% methanol + 92% 0.01 phosphate-buffered saline + 1% dimethylformamide) at concentrations of 5, 2, 1, 0.5, 0.2, and 0 μg/L [53]. The limit of detection and quantification for AFB1 as detected by the UV detector were 0.01 μg/L and 1 μg/L, respectively. Agilent HPLC (Santa Clara, CA, USA) was used to analyze the AFB1 using a Zorbax Eclipse Plus C18 column (4.6 mm 150 mm, 3.5 m) and a UV 365 nm detector. The mobile phase ratios were water, methanol, and acetonitrile (50:40:10, *v*/*v*/*v*). The flow rate was 0.8 mL/min, at ambient temperature, and the injection volume was 10 μL, with a concentration of 0.044 mg/mL [51].

### 2.6.5. RNA Extraction, cDNA Synthesis, and qRT-PCR Assay

The guanidium isothiocyanate technique was used to isolate whole-plant RNA, with certain modifications [54]. As previously described, the reverse transcription procedure was carried out [55,56]. The real-time PCRs (Qiagen Rotor-Gene Q2, Qiagen, Hilden, Germany) were carried out with designated primers targeting the aflatoxin biosynthesis pathway (Table 1). For normalization, the β-tubulin gene was served as an internal reference. As previously indicated, 20 μL SYBR Green qPCR reactions were performed [57,58]. The 2−ΔΔCT method was used to calculate the relative gene expression levels from the threshold cycle [59].

**Table 1.** Primer sequences were used in this study.


**2. Materials and Methods**

A questionnaire was developed to collect information from ornamental horticulture businesses. It consisted of 40 questions pertaining to businesses' online new-media marketing practices (we used the term "online" in the questionnaire, which was likely more familiar to the respondents than "new media," but we use the terms interchangeably), including their relationships with customers. Questions were formulated around four factors: (1) Business characteristics, (2) overall marketing practices, (3) online marketing practices, and (4) respondent demographics. The questionnaire was described in the introductory email as covering business characteristics and marketing practices, requesting respondents to collaborate with colleagues, if needed, to complete the questionnaire.

The questionnaire was designed to account for three types of respondents: Those not using any new-media marketing; those using some new-media marketing, but not social-media marketing; and those using new- and social-media marketing. After collecting information about their scope of business, a question asked what the frequency of use of various marketing venues was, including "print advertisements" (newspapers, store circulars, and postal mailings), "personal interactions" (phone calls, emails, and visits), "television/radio," "fairs/trade shows/garden shows," and "online marketing" (websites, blogs, social media, and e-newsletters). Those who indicated that they never used online marketing were routed to answer reasons for their non-use. Those who indicated they had used online marketing at least once proceeded to answer additional questions about their new-media marketing practices. Then, a question asked for the frequency of reaching their customers through different online marketing tools, including "websites," "HTML newsletters" (e.g., Constant Contact and MailChimp), "blogs," and "social-media platforms" (e.g., Facebook and Twitter). Those who indicated some use of social-media platforms proceeded to answer questions related to their experience with social-media marketing, while those who never used any social-media platforms were diverted to answering questions related to their reasons for not using social media.

The questionnaire was designed and distributed using Qualtrics software (Qualtrics, LLC, Salt Lake City, UT, USA), which was compatible for access on computers and mobile devices [14]. Following Dillman et al. [14], respondents could return to previous questions, and forced responses were imposed on 23 key questions, including marketing expenses, annual gross sales, and perceived importance of social media, to ensure that responses were provided. Responses to multiple-choice questions were randomized to minimize order effects [15]. The questionnaire was pre-tested by a nursery-marketing specialist and two other people with no relationship to ornamental horticulture industries. Suggestions made by these respondents were considered for the final version of the questionnaire.

According to the 2012 Economic Census, there were 13,928 establishments classified as nursery, garden center, and farm supply stores (NAICS code 444220) nationwide, and 634 in the North Plains region, including Kansas [16]. (The Northern Plains region is one of twelve regions defined by the USDA National Agricultural Statistics Service and includes North Dakota, South Dakota, Nebraska, and Kansas.) Without a comprehensive directory of these businesses coupled with the exploratory nature of the study, convenience sampling was adopted. Distribution of the questionnaire was planned with a goal to reach as many ornamental horticulture businesses, including nurseries, garden-center businesses, and landscape businesses, as possible in the 48 contiguous states of the United States, in both rural and urban areas. Businesses did not have to be new-media users to participate.

After obtaining approval from the Institutional Review Board at Kansas State University, data were collected in two waves in March and September of 2015. The questionnaire link was distributed through 87 regional and national ornamental horticulture associations and trade publications or magazine email lists. Instructions to obtain a paper copy of the questionnaire were included in the email invitation to participate in the study. The link was also emailed to email addresses for ornamental horticulture businesses that could be collected from publicly-available directories of "live plant dealer licensees" in the North Central United States region, followed by two reminders sent at weekly intervals [14]. Participants were invited to enter into a drawing for two \$50 Amazon (www.amazon.com, Seattle, WA) gift cards as an incentive to take the survey, as recommended by Dillman et al. [14]. At the beginning of the second wave, those with postal addresses received a postcard with the link, followed by two email reminders sent at weekly intervals to those with email addresses.

#### **3. Results**

#### *3.1. Sample Characteristics*

Of the 192 responses obtained, 161 were complete and were included in the subsequent analysis at a 95% confidence rate, which indicated a confidence interval of 7.68. Responses were obtained from all USDA National Agricultural Statistics Service regions, with the largest number of responses (40.5%) from the Northern Plains states where the survey was administered, followed by 15.7% from the Northeastern states. Sixty-five responses from the Northern Plains region would represent 10.3% of the establishments identified by the 2012 Economic Census. There were also four responses from Canada. Based on the zip codes of the business location, 42.9% were located in communities with less than 10,000 people.

Respondents represented businesses of various sizes, with a disproportionate number of businesses grossing sales over \$500,000 annually (Table 1), compared to the distribution of horticultural specialty operations in the 2014 Census of Horticultural Specialties [17] across the sales categories. More than half (57.1%) of the businesses in the sample sold \$500,000 or more in 2014, with the median response category of sales being between \$500,000 and \$1 million. In comparison, 8.7% sold less than \$25,000. For reference, the average market value of products sold by nurseries, greenhouses, and floriculture farms, according to the 2012 Census of Agriculture, was \$353,788 [3]. While the Census data are likely skewed to the right, suggesting the average would exceed the median, our sample was skewed to the left.

**Table 1.** Total gross business sales in 2014 of the ornamental horticulture industry respondents in the study.

Most businesses (87.6%) in the sample were well established, having been in operation for more than 10 years. Overall, response categories were represented almost uniformly in the sample, with a small number of businesses having been in operation for more than 100 years (5.0%). Nearly two-thirds (64.0%) of businesses were open year-round.

The primary marketing channel was retail to consumers, accounting for 90% or more of total sales for half of the respondents (Table 2). The second most popular marketing channel was wholesalers to landscapers, other garden centers, and re-wholesalers. "Re-wholesalers" generally do not own production facilities, but instead buy products wholesale from producers to sell at a wholesale price to allied horticulture industry businesses, such as landscapers and garden centers. In contrast, 95.6% of respondents did not sell any of their products through the mass merchandisers' channel. Respondents also reported selling up to 10% of their products through channels not listed in the questionnaire including construction and maintenance firms, municipalities, universities, and non-profit organizations.

**Table 2.** Distribution of 2014 business sales, across marketing channels, of ornamental horticulture industry respondents in the study questionnaire.

Adopting the description of ornamental horticulture-industry products and services by Hall et al. [1], the questionnaire asked respondents to identify products and services their business offered. Consistent with the marketing channels, retail product offering was the most prevalent (Table 3). Within the retail product category, bedding and nursery stock was offered by 73.3% of respondents, followed by lawn and garden products (54.7%), general merchandise (54.0%), and landscape materials (42.2%). Bedding and nursery stock and landscape materials were the most common products among those who wholesaled. According to the 2012 Agricultural Census, nursery stock crops and bedding and garden plants were the highest valued (\$5 billion and \$3.6 billion, respectively) in ornamental horticulture industries [3]. Respondents mentioned various other activities including pottery, gift and jewelry retail, herbs, vegetables, pet shop, agritourism, educational services, and vocational training for individuals with disabilities.

Individuals who responded to the questionnaire on behalf of the businesses were on average 50 years of age, with slightly fewer female respondents (48.5%) than male respondents. More than half of the respondents held a baccalaureate degree (67.1%), with most (88.8%) attending some amount of college. Nearly two-thirds (63.4%) of the respondents were business owners, while 23.6% were managers. Thirteen respondents (8.1%) were marketing managers. This low representation of

marketing managers might suggest that either owners or managers conduct their own marketing activities, including social media, or contract their marketing services to third-party consultants. Other respondent roles included extension master gardener, office manager, sales manager, and search engine optimizer. The majority (62.7%) of respondents had worked at the business for 10 years or more. Only 3.1% of the respondents had joined or owned the business within one year. Most owners (76%) had worked at their business for at least 10 years.

**Table 3.** The percentage of ornamental horticulture industry respondents that indicated they carry these general categories of items or provide these services.


*2.3. Perilla*

Perilla (*Perilla frutescens* L. Briit) belongs to the Lamiaceae family (formely Labiateae) which consists of 235 genera and more than 700 species [76]. Perilla is an edible herb widely consumed in Asian countries such as China, Korea, Japan, and India. Similar to spinach, perilla leaves are also characterized by a high concentration of carotenoids. In fact, according to Müller-Waldeck et al. [77], perilla may contain high contents of carotenoids, especially up to five-fold higher lutein than other carotenoid-rich leafy vegetables. The leaves of perilla contain a range of bioactive phenolic molecules such as caffeic acid, catechin, chrysoeriol, ferrulic acid, luteolin, quercetin, and rosmarinic acid [78,79]. In particular, secondary metabolites such as rosmarinic acid and perillaldehyde (an essential oil constituent) have demonstrated potential to prevent a wide range of diseases particularly owing to their anti-diabetic, anti-depressant, anti-bacterial, anti-cancer, and antimicrobial properties [76]. Thus the concentrations of these two phytochemicals are crucial for their clinical and culinary applications. It is worth noting that these two secondary metabolites are produced in perilla by two different biosynthetic pathways, namely the monoterpene and phenylpropanoid pathways, and may increase independently in relation to the perilla chemotype and abiotic environmental stress conditions [80]. Perilla is present in nature in two main chemical-varietal phenotypes: (i) The red-pigmented cultivar *P. frutescens* var. *crispa*, known as "Zi-So" and widely grown in China where it is used as a spicy herb, leafy vegetable, and medicinal plant, and (ii) the non-pigmented green cultivar *P. frutescens* var. *frutescens* known in Japan as "Shisoyo" or "Shiso" and mainly

used as an oil crop but also as ingredient of skin creams and food products [81,82]. According to Meng et al. [78,83], three cinnamic derivates (caffeic acid, coumaroyl tartaric acid, and rosmarinic acid) ranged from 0.1 to 11 mg/g; six flavonoids (apigenin 7-*O*-caffeoylglucoside, apigenin 7-*O*-diglucuronide, luteolin 7-*O*-diglucuronide, luteolin 7-*O*-glucuronide, scutellarein 7-O-diglucuronide, and scutellarein 7-*O*-glucuronide) ranged from 3.5 to 18.5 mg/g; and six anthocyanins (0.7–2 mg/g) including cis-shisonin, cyanidin 3-*O*-(*E*)-caffeoylglucoside-5-*O*-malonylglucoside malonylshisonin and shisonin were detected on eight tested cultivars of perilla. Concerning the health effects of perilla extract, Narisawa et al. [84] reported that perilla leaves carry anti-tumor properties. In their work, the authors showed that treatment of female rats with a 12% fat diet based on perilla extract and safflower oil in 1:3 or 1:1 ratio effected better protection against colon cancers as compared to safflower oil alone [84].

Comparing green (Korean cultivar) and red-pigmented (Japanese cultivar) perilla, Rouphael et al. [82] observed that green perilla produced exclusively perilla ketone (PK), whereas the red perilla contained perillaldehyde (PA). Similar results were reported by Martinetti et al. [81] in a study profiling two red-leaf ("Aka Shiso" and "Purple Zi Su") and three green-leaf cultivars ("Ao Shiso", "Qing Su", and "Korean perilla") with the later containing PK instead of PA. The terpenoid component present in green-pigmented perilla has been demonstrated to be toxic for cattle and horses, since PK is considered a potent lung toxin [77,85], but the health-effect as well as the toxic dose/concentration to humans is still controversial, therefore Müller-Waldeck and co-workers [77] concluded that some Korean genotypes (green-pigmented cultivars) are not suitable/recommended for fresh consumption. Interestingly, several authors showed that PA and PK present in red and green perilla can stimulate the TRPA1 (Transient Receptor Potential) cation channels which are actively involved in multiple biological mechanisms such as pain perception and their functional role in the prevention of certain types of tumor has been proved [86,87].