**Mycotoxins in Feed and Food Chain Present Status and Future Concerns**

Printed Edition of the Special Issue Published in *Toxins* Filippo Rossi Edited by

www.mdpi.com/journal/toxins

## **Mycotoxins in Feed and Food Chain**

## **Mycotoxins in Feed and Food Chain Present Status and Future Concerns**

Editor

**Filippo Rossi**

MDPI • Basel • Beijing • Wuhan • Barcelona • Belgrade • Manchester • Tokyo • Cluj • Tianjin

*Editor* Filippo Rossi Catholic University Italy

*Editorial Office* MDPI St. Alban-Anlage 66 4052 Basel, Switzerland

This is a reprint of articles from the Special Issue published online in the open access journal *Toxins* (ISSN 2072-6651) (available at: https://www.mdpi.com/journal/toxins/special issues/mycotoxins feed food chain).

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LastName, A.A.; LastName, B.B.; LastName, C.C. Article Title. *Journal Name* **Year**, *Article Number*, Page Range.

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## **Contents**

Reprinted from: *Toxins* **2019**, *11*, 640, doi:10.3390/toxins11110640 .................. **185**

## **About the Editor**

**Filippo Rossi** was born in Piacenza (1963) and graduated in Agricultural Sciences in 1989 (Catholic University, of Piacenza, Italy). In 1992, the Italian Ministry of Research awarded him with a Ph.D. in Molecular Biotechnology. Since 1993, he has been working at the Department of Food Science and Nutrition of the Faculty of Agricultural Sciences in Piacenza, where he is in charge of the Human Nutrition course in the Food Science and Technology degree. His research interests cover the role of food in the prevention of noncommunicable disease, particularly Mediterranean Diet and breast cancer, mycotoxins and liver cancer, dairy foods and hypertension, and starch digestion and obesity. Since 2004 (EU fundend project "Safe Food"), he has been involved in research regarding the prevention of mycotoxins contamination in the feed and food chain.

### *Editorial* **A Long Road to Safer Food**

#### **Filippo Rossi**

Section of Food Science and Nutrition, Department of Animal Sciences, Food and Nutrition, Faculty of Agricultural, Food and Environmental Sciences, Catholic University of Sacred Heart, Via Emilia Parmense 84, 29122 Piacenza, Italy<EMAIL_ADDRESS>
Received: 17 June 2020; Accepted: 10 July 2020; Published: 14 July 2020

As a side effect of food production, mycotoxins have always accompanied humanity, even if the danger posed by these molecules has only recently been understood and new research has begun to identify and study ways to reduce their presence in food.

This Special Issue of *Toxins* includes papers on new findings concerning well-known mycotoxins, results of studies regarding emerging mycotoxins, such as alternaria and botryodiplodin, and new techniques to reduce mycotoxin contamination in processed cereals.

Reliable data on the presence of mycotoxins in food is very important in the toxicological evaluation of the risk associated with these toxic fungal compounds. Two papers cover this subject: Quevedo-Garza et al. [1] analyze Mexican infant formula food for aflatoxin M1 and Zentai et al. [2] determine the fumonisins in Hungarian maize-based food.

*Fusarium* spp., together with *Aspergillus* spp., are the most relevant fungi genus responsible for mycotoxin production. Researchers have focused their attention on cereals, while neglecting other crops. A paper from a Chinese group reports on the identification of the *Fusarium* species causing sweet pepper fruit rot and on the kinds of mycotoxins produced by these microorganisms [3].

A new toxic molecule produced by a fungal parasite of soybean is the focus of two papers from Abbas et al. who investigate the production of botryodiplodin [4] and its toxicity [5], while another contribution [6] considers secalonic acids, which are the main ergot ergochromes in overall ergot toxicity.

We have observed not only the appearance of new or emerging mycotoxins, but also of new foods, such as insects, that can also be contaminated by mycotoxins. On this topic, a paper in this Special Issue studies the metabolism of aflatoxin B1 in the larvae of the black soldier fly (*Hermetia illucens*) [7].

The reduction of mycotoxin contamination can be obtained by intervening during the cultivation or storage of products. Research carried out by Giorni et al. [8] tested the efficacy of the fungicide azoxystrobin on fungal parasites of rice and obtained a strong reduction (−67%) of sterigmatocystin while deoxynivalenol remained unaffected.

A clear reduction in *Fusarium*-produced toxins can be observed in the paper of Brodal et al. [9], by sieving oat grains and removing broken kernels, which are more contaminated than intact ones.

The last research article of the Special Issue describes an analytical method for the detection of 19 mycotoxins and three phytoestrogens in fish feed and fish meat [10]. The reduction of the risk posed to human health by mycotoxins requires the development and validation of reliable methods to monitor mycotoxins in feed and food.

The three reviews included in the Special Issue cover as many topics. Issues related to the use of lactic acid bacteria as aflatoxin binders in developing countries are discussed in the review of Ahlberg et al [11]. Kamle et al. [12] summarize the effect of fumonisin on human health and the strategies to reduce the level of this toxin in food. A group of emerging mycotoxins, those produced by *Alternaria*, is the focus of Crudo et al [13], who analyze "the most relevant data concerning the occurrence and toxicity of mycotoxins produced by *Alternaria* spp., ( ... .) alone or in combination with other mycotoxins and bioactive food constituents".

In conclusion, all the contributions to this Special Issue expand our current knowledge and, as Guest Editor, I am happy and proud to present this issue to the community of scientists involved in research on mycotoxins.

All research and review articles proposing novelties and overviews, respectively, were successfully and carefully selected for this Special Issue after rigorous revision by the expert peer reviewers. As the Guest Editor, I would like to express my deep appreciation to all the selfless and fair reviewers.

**Acknowledgments:** The editor would like to thank all the authors who contributed to this Special Issue and the reviewers for their evaluation work. The editor is also grateful to the MDPI management team for their valuable support.

**Conflicts of Interest:** The author declares no conflict of interest.

#### **References**

© 2020 by the author. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

## *Article* **Aflatoxin M1 Determination in Infant Formulae Distributed in Monterrey, Mexico**

**Patricia A. Quevedo-Garza 1, Genaro G. Amador-Espejo 2, Rogelio Salas-García 1, Esteban G. Ramos-Peña 1 and Antonio-José Trujillo 3,\***

Received: 27 December 2019; Accepted: 31 January 2020; Published: 4 February 2020

**Abstract:** The occurrence of aflatoxin M1 (AFM1) in infant formulae commercialized in the metropolitan area of Monterrey (Nuevo León, Mexico) was determined by using immunoaffinity column clean-up followed by HPLC determination with fluorimetric detection. For this, 55 infant formula powders were classified in two groups, starter (49 samples) and follow-on (6 samples) formulae. Eleven of the evaluated samples (20%) presented values above the permissible limit set by the European Union for infant formulae (25 ng/L), ranging from 40 to 450 ng/L. The estimated daily intake (EDI) for AFM1 was determined employing the average body weight (bw) of the groups of age in the ranges of 0–6 and 6–12 months, and 1–2 years. The results evidenced high intake values, ranging from 1.56 to 14 ng/kg bw/day, depending on the group. Finally, with the EDI value, the carcinogenic risk index was determined, presenting a high risk for all the evaluated groups. Based on these results, it is a necessary extra effort by the regulatory agencies to reduce the AFM1 presence in infant formulae consumed in Mexico.

**Keywords:** AMF1; infant formulae; estimated daily intake; carcinogenic risk index; Monterrey (Mexico)

**Key Contribution:** Aflatoxin B1 can be metabolized by mammals to aflatoxin M1 (AFM1), a form that retains potent carcinogenicity and which can be excreted into milk. There is scarce information on the occurrence of AFM1 in milk and dairy products, and no data are available in Mexico concerning infant formulae contamination by this mycotoxin. The results of the present study further demonstrate the potential risk for the infant population associated with the AFM1 presence in the infant formulae marketed in Monterrey (Mexico).

#### **1. Introduction**

Aflatoxins' presence in food products is one of the major health concerns of the regulatory agencies around the world. These toxins include around 20 metabolites produced by molds such as *Aspergillus flavus* and *A. parasiticus*, which is the most important of the aflatoxin B1 (AFB1) and is normally found in foods, especially those having high carbohydrate and/or fat contents [1]. Its occurrence has been reported in numerous food and feedstuff, including cereals and cereal-derived products [2].

Cattle feed with contaminated crops of AFB1 may lead to the formation of a hydroxylated metabolite named aflatoxin M1 (AFM1), which is excreted in the milk of lactating animals and whose name is due to the source detected [3]. Numerous researchers have reported a linear relationship of about 0.3–6.2% between the amount of AFM1 detected in milk and AFB1 in feed consumed by the animals [4]. Nevertheless, the percent of AFM1 excreted depends on various factors, including concentration of AFB1 in feed, milk yield, stage of lactation and breed [5].

Even though AFM1, the main monohydroxylated derivate of AFB1, presents less carcinogenic and mutagenic activity than AFB1, it exhibits a high level of genotoxic activity and certainly represents a health risk because of its elevated possibility of accumulation and binding to DNA [6]. Based on this, different health agencies such as the World Health Organization and the International Agency for Research on Cancer (IARC) have published articles in which AFM1 is a strong genotoxic and hepatotoxic agent [7]. Therefore, AFM1 has been evaluated as a possible human carcinogen agent, and although until 2002 it was classified in the 2B Group, with a tolerable daily intake (TDI) of 2 ng/kg bw [8], based on numerous scientific evidence that demonstrated carcinogenic and other (teratogenic, genotoxic and immunosuppressive) effects, it was reclassified into the first group [7].

Hence, the elimination of risk sources represents a major assignment for government agencies and food processors, not only for the contaminated products directly consumed by humans but also in feeding cattle that consume contaminated crops, whose products can reach the human being. Government regulations around the world concerning AFM1 limits differ from one other. The lowest AFM1 concentration was approved by the European Union (EU) and the Codex Alimentarius, fixing a maximum admissible level of 50 ng/L in fluid milk and dried or processed milk products [9,10]. On the contrary, higher AFM1 concentrations (500 ng/L) are permitted in the United States of America (USA) and some Latin American countries (such as Mexico and the MERCOSUR agreement), and China allows a maximum limit of 62.5 ng/L [11–14]. However, because of the higher susceptibility of infants to AFM1, the EU and the Codex Alimentarius fixed the maximum admissible level of 25 ng/L for infant formulae, follow-on formulae and dietary foods for medical purposes intended specifically for infants [9].

Another major problem concerning the presence of AFM1 in milk is the different dairy products it's included in (e.g., liquid milk, yogurt, cheese, milk powder, ice cream, regular cream, among others) and the fact that the aflatoxin cannot be eliminated by regular heat treatments such as pasteurization or ultra-high temperature processing [15]. Besides, one of the most important products manufactured from milk are the infant formulae, in which there is significant risk of AFM1 intoxication because small amounts of this toxin in the product may represent an important portion of aflatoxin intake [16].

Despite the danger associated to the AFM1 presence in milk, only a few articles are available regarding the presence of this toxin in milk and dairy products in Mexico [17,18], and no studies have been published regarding its presence in infant formulae or intake assessment for AFM1 in the country. Based on this, the aim of this study was to evaluate the AFM1 occurrence in infant formulae and to estimate the exposure of infant milk consumers to AFM1 by means of a sampling of the infant formulae brands distributed in Monterrey (Nuevo León, Mexico).

#### **2. Results and Discussion**

#### *2.1. Occurrence of AFM1 in Infant Formulae*

Table 1 shows the results obtained from the analyzed samples, with 20% of them being positive for the toxin in a range of 40 to 450 ng/L, and an average AFM1 concentration of 40 ± 99 ng/L for all analyzed samples, which is higher than the limit established for AFM1 in infant formulae by the Codex Alimentarius (25 ng/L) [11]. Nevertheless, when the infant formulae were evaluated separately (starter and follow-on groups), it can be observed that the AFM1 values increased from one group to another. In the starter formulae, the percentage of samples exceeding the AFM1 limit was 14%, remarkably lower than the percentage of samples above the limit in the follow-on formulae (67%). Furthermore,

the media in the starter formulae (20 ± 67 ng/L) was below the EU or Codex Alimentarius AFM1 limit (25 ng/L), compared to the follow-on formulae, with an average (180 ± 185 ng/L) exceeding the AFM1 limit. Although the AFM1 levels in starter formulae were significantly (*p* < 0.05) lower than those in follow-on formulae, it is important to notice the small number of samples evaluated in the follow-on formulae, compared to the infant formula evaluated in the starter group.

**Table 1.** Aflatoxin M1 presence in infant formulae.

\* Value in parentheses indicates the samples percentage above the limit set by the Codex Alimentarius (25 ng/L) with respect to the total. a,b Different online letters indicate significant mean differences among the different types of infant formulae (*p* < 0.05).

Regarding legislation about AFM1 limits in infant formulae, most of the countries do not have an established limit, which is the case of most of the Latin-American countries (including Mexico), which tends to apply the limit established by the Codex Alimentarius or the EU regulation (25 ng/mL) [11,19].

The occurrence of AFM1 in infant formulae varies in different countries. Gomez-Arranz and Navarro-Blasco [20] evaluated the presence of AFM1 in infant formulae in Spain, testing 69 samples and detecting the presence of AFM1 in 26% of them. In this case, all the detected samples were below the EU established limit. More recently, Akhtar et al. [21] determined the AFM1 presence in infant formulae in Pakistan, evaluating 13 samples, in which 53.84% of the samples were positive to the toxin presence and 30.76% exceeded the EU limit. Kanungo and Bhand [22] evaluated the AFM1 presence in infant formulae in India, determining that in 72 evaluated samples, all of them were above the EU permitted limit (25 ng/kg) and 75% of the samples exceeded the USA and Indian Food regulation limit (500 ng/kg). Er et al. [4] published a study evaluating the AFM1 presence in infant formula in Turkey, evaluating 84 samples with only one sample positive for the toxin. In this sense, Li et al. [14] detected the presence of AFM1 in powder base for infant formulae in China, evaluating a total of 1207 samples, with 56 samples being positive for the toxin without passing the Chinese limit (62.5 ng/kg). Awaisheh et al. [23] determined the AFM1 content in infant formulae (120 samples; 48 starter and 72 follow-on formulae) distributed in Jordan, with 58 positive samples for the toxin presence, with a media of 69 and 84 ng/kg for the starter and follow-on formulae, respectively.

#### *2.2. Infant Formulae Daily Intake by Age Group*

The present study is the first evaluation of the daily intake by Mexican minors, based on average consumption and body weight (Table 2). The Mexican Standard NOM-031-SSA2-1999 [24] classifies infants in two groups of infant formulae consumption: i) minor lactating (0–12 months), and ii) major lactating (one to two years). The consumption in these groups is starter and follow-on formulae for the first and the second year, respectively.

Based on the occurrence of AFM1 in infant formulae and the body weight of infants, the estimated daily intake (EDI) for AFM1 was in a range of 1.56 to 14 ng/kg bw per day, which represents the values estimated for one year-old infants when they are fed with starter or with follow-on formulae, respectively. However, when major lactating groups gain weight and reduce the follow-on formula intake (i.e., two years old), the EDI is reduced up to 4.28 ng/kg bw/day. Awaisheh et al. [23] have evaluated the infant formulae consumed in Jordan, presenting an EDI of 1.57 and 1.55 ng/kg bw/day for infants aged six and 12 months, respectively. On the other hand, Ismail et al. [25] reported an EDI value of 4.1 ng/kg bw/day for children aged one to three years in Pakistan. It is considerable the work developed by the food agencies seeking to reduce the presence of AFM1 in milk and infant

formulae. In this sense, Oliveira et al. [26] published an article evaluating the presence of AFM1 in infant formulae in Brazil with a daily intake of 22 ng/kg bw/day. In contrast, almost 20 years later, Ishikawa et al. [27] determined the AFM1 presence in infant formulae in the same country, presenting an important reduction in EDI values (0.078–0.306 ng/kg bw/day). Likewise, lower EDI values than the present study were detected in infant formulae consumed in Spain (n = 69) (0.02–0.13 ng/kg bw/day) [4]. Further, Ruangwises et al. [28] evaluated AFM1 presence in milk powder distributed in Thailand (90 samples) showing EDI values of 0.16 ng/kg bw/day in milk consumed by infants up to three years.

**Table 2.** Estimated aflatoxin M1 daily intake by average body weight and carcinogenic risk index (CRI) in children population based on the ENSANUT (2012).

\* According to the Kuiper-Goodman equation [8].

Comparing the results of AFM1 occurrence in infant formulae and in breast milk in Mexico, the results are quite similar. Thereby, Cantú-Cornelio et al. [29] evaluated the presence of AFM1 in breast milk of nursing mothers in central Mexico (112 samples), with an EDI value of 2.35 ng/kg bw/day, comparable results to the values obtained in the present study. These results show the importance of evaluating the presence of AFB1 in different products consumed by nursing mothers in order to reduce the toxin that may be transformed into AFM1 and reach infants by breast milk.

Table 2 also presents the result of the carcinogenic risk index (CRI) for the evaluated population. At this day, up to our knowledge, no CRI study evaluating the infant population of Mexico has been published. The AFM1 ingestion obtained in this study was greater than the TDI value (2 ng/kg bw/day) calculated by Kuiper-Goodman [8] dividing the TD50 by the safety factor 5000, indicating that there is a potential high risk for liver cancer due to the consumption of infant formulae in Mexican consumers groups studied.

#### **3. Conclusions**

The results of the current study have shown a high presence of AFM1 in infant formulae distributed in the Monterrey (Mexico) metropolitan area. From fifty-five samples evaluated, 20% exhibited a toxin content above the EU and Codex Alimentarius limit (25 ng/L), presenting a range of 40–450 ng/L. Further, in classifying the samples by the type of infant formulae and infant age for consumption (starter formula for minor infants up to one year, and follow-on formula for major infants between one and two years), different levels of AFM1 were obtained (20 ng/L for starter and 180 ng/L for follow-on formulae). Besides, based on the average body weight of the evaluated groups, the EDI value was calculated, with values in the range of 1.56–14 ng/kg bw/day. Finally, with the EDI data, the CRI was determined, obtaining a result of risk in all the evaluated groups. Based on these results, an important effort should be carried out by the regulatory agencies and milk producers in order to reduce AFM1 levels in milk in general, and, in particular, in batches that will be employed for infant formulae elaboration because of the high cancer risk associated with AFM1 presence and the infant consumers' vulnerability.

#### **4. Materials and Methods**

#### *4.1. Sample Collection*

Fifty-five infant formula samples from drug stores and supermarkets sold in Monterrey (Nuevo León, Mexico) were obtained. From these, 49 were starter formulae (0–12 months) and 6 were follow-on formulae (1–2 years). Among the starter formulae, 6 were pre-term formulae (formulated for prematurely born, regurgitation episodes by immature esophageal sphincter, or low birth weight infants), 11 were hypoallergenic formulae (specialized formula based on casein, whey or soy protein hydrolysates) and 9 were lactose free formulae (designed for lactose intolerant infants based on lactose hydrolysis by β-galactosidase or formulated from soy protein isolates).

All formulae were supplied as powder milks. Infant formula containers (cans or bags) were stored in dark at room temperature until analyses were performed.

#### *4.2. Sample Preparation*

Powder-based formula samples were suspended in deionized warm water according to the manufacturer instructions. The method used for sample preparation and AFM1 determination was that specified by the method ISO 14,501 [30]. Suspended infant formula samples were centrifuged at 4200 × *g* for 15 min to separate and remove the milk fat. Aliquots of skimmed milk (50 mL) were filtered (Whatman no. 4 filter paper) and slowly passed (1–2 drops/s) through an immunoaffinity column (AflaM1 HPLC, VICAM, Milford, MA, USA) fitted on a vacuum manifold, and washed twice with 10 mL of distilled water. Thereafter, the AFM1 was eluted with 4 mL of acetonitrile, allowing a time contact of at least 60 s. The eluate was collected in amber vials, the solvent was evaporated in a water bath at 40 ◦C with nitrogen, and the residue reconstituted in water:acetonitrile (67:33) and filtered by Millipore filters (0.45 μm) in amber vials.

#### *4.3. HPLC Analysis*

The HPLC analysis was carried out in a Varian HPLC model 9012 (Agilent Technologies, Santa Clara, CA, USA) connected with a fluorescence detector Varian ProStar (Agilent Technologies Santa, Clara, CA, USA). The separation column was a Phenomenex C18 with 4.5 × 250 mm and 5 μm of particle size (Phenomenex, Torrance, CA, USA). Water and acetonitrile mixture were used as a mobile phase in a proportion of 67:33 (v/v), at a flow rate of 1 mL/min, and an injection volume of 100 μL. Fluorometric detection was achieved at 360 nm excitation and 440 nm emission wavelength.

To assess the performance of the analytical method, linearity, limits of detection (LOD) and quantification (LOQ), recovery and precision (repeatability) were studied. Linearity was evaluated using standard calibration curves that were constructed by plotting the peak area versus the analyte concentration. The calibration curves were established using eight levels of concentrations from LOQ to 100 times LOQ. The regression curve obtained was y = 287.78 x + 75.10 giving appropriate value for the linearity (R2 = 0.998). LOD (2 ng/L) and LOQ (5 ng/L) were calculated as the sample blank value plus 3 and 10 times its standard deviation, respectively. In order to determine the recovery, reconstituted milk was added with 3 levels of AFM1 concentrations (50, 100 and 200 ng/L). The obtained values of recovery were between 83% and 104%. The precision (15.18%) was calculated as repeatability by means of triplicates in each of the levels analyzed in the recovery assay.

#### *4.4. Determination of AFM1 Exposure in the Population*

The determination of the exposure level or estimated daily intake (EDI) in the population of Monterrey to the AFM1 due to the consumption of infant formulae was carried out by combining data on the average daily consumption of milk by groups of age, with the average concentration of AFM1 found in this work, as well as the average body weight (bw) of the population by age groups. For this, Equation (1) was applied:

$$\text{Estimated AFM daily intake} \left(\frac{\text{ng}}{\text{kg bw}} / \text{day}\right) = \frac{\text{Milk intake (L)} \times \text{AFM1} \left(\frac{\text{ng}}{\text{L}}\right)}{\text{Body weight (kg)}} \tag{1}$$

where: Milk intake is the average amount of milk that the infant population ingests daily, expressed in liters. AFM1 is the average concentration of AFM1 contained in the analyzed samples, expressed in ng/L. Body weight is the bw average in the population by age groups in kilograms.

The data corresponding to the daily milk consumption by age groups was obtained from the National Survey of Health and Nutrition of Mexico (ENSANUT) [31], in the section corresponding to Nuevo León State.

In order to obtain the daily intake of AFM1 in the infant population, it was necessary to separate the population, as indicated by Mexican Standard NOM-031-SSA2-1999 [24] in: (1) minor lactating (newborn up to 6 months), at this stage of the infant's life, their diet is only based on breast milk or infant formulae for initiation; (2) minor lactating (from 6 to 12 months), at this stage, ablactation occurs, and the starter infant formulae and dairy infant formulae containing cereals and honey are taken as the infant diet at this stage of life; (3) major lactating (from 12 to 24 months), at this stage the dairy intake is determined by the follow-on formulae and those containing cereals and honey. From the ENSANUT [31] survey, the average weights of the infant population (minor and major lactating) were obtained.

Likewise, the CRI was estimated based on the proposal of Kuiper-Goodman [8], which estimates the TDI of AFM1 by dividing the TD50 (threshold dose by body weight; 10,380 ng/kg bw per day for AFM1) by the safety factor 5000, to give an estimated value of 2 ng/kg bw per day. A CRI of AFM1 higher than 2 ng/kg bw indicates liver cancer risk to consumers [8,32].

#### *4.5. Statistical Analysis*

All infant formulae were analyzed in duplicates. Collected data were statistically evaluated using the nonparametric Wilcoxon rank sum test with continuity correction of R Core Team (Vienna, Austria) [33]. AFM1 concentrations were expressed as mean ± standard deviation in order to show the occurrence of the toxin in infant formulae.

**Author Contributions:** Research concept, design and supervision: P.A.Q.-G. and A.-J.T.; HPLC method validation: P.A.Q.-G. and R.S.-G.; AFM1 analysis in infant formulae: P.A.Q.-G. and E.G.R.-P.; writing and correcting of the manuscript: P.A.Q.-G.; A.-J.T.; R.S.-G.; E.G.R.-P.; G.G.A.-E. All authors have read and agreed to the published version of the manuscript.

**Funding:** The authors appreciate the funding provided by the Universidad Autónoma de Nuevo León, Monterrey, México, through the PAICYT – UANL program, and by the CIRTTA of Universitat Autònoma de Barcelona, Bellaterra, Spain, for this study.

**Acknowledgments:** The authors are very grateful to Jesús Piedrafita (UAB, Spain) for his support in statistical analysis.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **Abbreviations**

#### **References**

1. Prandini, A.; Tansini, G.; Sigolo, S.; Filippi, L.; Laporta, M.; Piva, G. On the occurrence of aflatoxin M1 in milk and dairy products. *Food Chem. Toxicol.* **2009**, *47*, 984–991. [CrossRef] [PubMed]

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

## **Occurrence and Risk Assessment of Fumonisin B1 and B2 Mycotoxins in Maize-Based Food Products in Hungary**

#### **Andrea Zentai 1, Mária Szeitzné-Szabó 2, Gábor Mihucz 2, Nóra Szeli 2, András Szabó 2,3,\* and Melinda Kovács 2,3**

Received: 24 October 2019; Accepted: 3 December 2019; Published: 5 December 2019

**Abstract:** Fumonisins are toxic secondary metabolites produced mainly by *Fusarium verticillioides* and *Fusarium proliferatum.* Their toxicity was evaluated, and health-based guidance values established on the basis of both Joint FAO/WHO Expert Committee on Food Additives (JECFA) and European Food Safety Authority (EFSA) recommendations. This study presents the results of fumonisin analyses in different maize- and rice-based food products in Hungary and the potential health risk arising from their dietary intake. In total, 326 samples were measured in 2017 and 2018 to determine fumonisins B1 and B2 levels. Three-day dietary record data were collected from 4992 consumers, in 2009. For each food category, the average concentration values were multiplied by the relevant individual consumption data, and the results were compared to the reference values. With respect to the maximum limits, one maize flour, two maize grits, and two samples of other maize-based, snack-like products had total fumonisin content minimally exceeding the EU regulatory limit. The mean daily intake for all maize-product consumers was 0.045–0.120 μg/kg bw/day. The high intake (95 percentile) ranged between 0.182 and 0.396 μg/kg bw/day, well below the 1 μg/kg bw/day tolerable daily intake (TDI) established by EFSA. While the intake calculations resulted in comforting results, maize-based products may indeed be contaminated by fumonisins. Therefore, frequent monitoring of fumonisins' levels and evaluation of their intakes using the best available data are recommended.

**Keywords:** fumonisin; human exposure; maize products

**Key Contribution:** Fumonisin intake of Hungarian consumers from maize-based products is below the health-based guidance values. Maize-based products may be contaminated by fumonisins

#### **1. Introduction**

Fumonisins are secondary metabolites produced mainly by *Fusarium verticillioides* and *Fusarium proliferatum* [1]. Maize and maize-based products are most commonly contaminated by fumonisins, but fumonisins can be detected in several other cereal grains, such as rice, wheat, barley, rye, and oat [2,3]. More than 15 fumonisin homologues have been described, including fumonisin A, B, C, and P, and, among them, fumonisin B1 (FB1), FB2, and FB3 are the most frequent naturally occurring fumonisins [1,4]. FB1 typically accounts for 70%–80% of the total fumonisin produced, while FB2 usually makes up 15%–25% and FB3 3%–8% when cultured on maize, rice, or in liquid medium [1].

Among fumonisins, FB1 is the most toxic compound and has been shown to promote tumour growth in rats as well as equine leukoencephalomalacia [5] and porcine pulmonary oedema [6]. It was classified by the International Agency for Research on Cancer (IARC) in Group 2B (possibly carcinogenic in humans) [7]. FB1 also causes chronic liver and kidney toxicity when administered in repeated doses to rodents.

Fumonisin B toxins, as structural analogues of sphingoid bases, inhibit ceramide synthases, causing the disruption of the sphingolipid metabolism and leading to sphinganine (and sphingosine) accumulation in cells and tissues [8]. Toxicity studies have mainly focused on the effects of FB1, but FB2–4 appear to have similar toxicological profiles. Acute toxicity is not relevant for fumonisins.

The Scientific Committee on Food (SCF) as well as the European Food Safety Authority (EFSA) in Europe and the Joint FAO/WHO Expert Committee on Food Additives (JECFA) evaluated the dietary risk of fumonisin intakes [9–15].

The SCF established a tolerable daily intake (TDI) of 2 μg/kg bw/day for FB1 in 2000, based on an overall level of no observed adverse effect (NOAEL) of 0.2 mg/kg bw for liver and kidney in rodents [9]. This TDI was expanded into group TDI in relation to the total amounts of fumonisin B1, B2, and B3, alone or in combination [10]. JECFA published a risk assessment on FB1, FB2, FB3 in 2001 [11]. The assessment was essentially based on FB1 data, and the other toxins were considered as having similar toxicological profiles. A group provisional maximum tolerable daily intake (PMTDI) of 2 μg/kg bw/day per day was allocated based on a NOAEL of 0.2 mg FB1/kg bw per day for renal toxicity in a subchronic and a chronic rat study [11]. The PMTDI established by JECFA was retained in 2011 and in 2016 as well [12,13].

EFSA discussed food safety issues of mycotoxins, including fumonisins, in several documents. The chemical structure of mycotoxins can be altered by the defense reaction of plants, rendering them extractable conjugated and/or non-extractable bound mycotoxins or mycotoxin metabolites. Since these modified toxins are usually not detected during the analysis of mycotoxins, they are commonly termed "masked" or "bound". EFSA issued a scientific opinion in 2014 regarding certain modified mycotoxins in food and feed [14].

More recently, EFSA published a scientific opinion on the appropriateness to set a group health-based guidance value for fumonisins and their modified forms in 2018 [15]. For the establishment of the TDI, the benchmark dose lower confidence limit (BMDL10) of 0.1 mg/kg bw per day for induction of megalocytic hepatocytes in mice was used. Taking into account an uncertainty factor (UF) of 100 for intra- and interspecies variability, the TDI was established at 1.0 μg FB1/kg bw per day. FB2, FB3, and FB4 were included in the TDI, based on structural similarity and the limited available data indicating similar mode of action (MoA) and toxic potencies.

In Europe, Commission Regulation (EC) No 1881/2006, setting the maximum levels for certain contaminants in foodstuffs, established the maximum limits for fumonisins (sum of B1 and B2) in different commodities, including unprocessed maize, maize intended for direct human consumption, maize-based foods for direct human consumption, maize-based breakfast cereals, maize-based snacks, processed maize-based foods and baby foods for infants and young children, different milling fractions of maize, and other maize milling products not used for direct human consumption. Table 1 presents the specified maximum limits by commodities.

This article presents the results of fumonisin analyses in different maize- and rice-based food products in Hungary and, consequently, the potential health risk arising from their dietary intake.

**Table 1.** Maximum limits (μg/kg) for fumonisins established by Commission Regulation (EC) No 1881/2006.

#### **2. Results and Discussion**

#### *2.1. An Overview of the Measured Fumonisin Content*

Altogether, 326 samples were measured for fumonisins B1 and B2 mycotoxins levels. The types of samples were from the food categories of maize flour, maize grits, corn flakes, canned maize, other maize-based, snack-like products, white and brown rice, and other rice-based products. The limit of detection (LOD) and the limit of quantification (LOQ) for FB1 were 0.031 and 0.093 mg/kg, while those for FB2 were 0.051 and 0.154 mg/kg.

In total, 70 and 256 samples were analyzed in 2017 and 2018, respectively, and were considered together in our assessment.

We measured 64 maize flour samples, of which 33 (51.6%) had detectable FB1 content, and 6 (9.4%) had detectable FB2 content. The highest FB1 value was 1.46 mg/kg. The average FB1 and FB2 concentrations were 0.17–0.20 mg/kg for FB1 and 0.05–0.10 mg/kg for FB2. In no instance was FB2 detected if FB1 was undetected. Only in six cases, both FB1 and FB2 were detected at a measurable level (above LOQ), while FB2 was never detected alone.

Then, 62 maize grits were analyzed; 26 samples (41.9%) presented detectable FB1, and 4 (6.5%) detectable FB2. The highest concentrations found were 1.96 mg/kg for FB1 and 0.58 mg/kg for FB2. The average FB1 content was 0.13–0.16 mg/kg, while the average FB2 content was 0.03–0.08 mg/kg. Four samples contained both FB1 and FB2 above the LOQ.

Altogether, 8 of the 64 corn flakes samples (12.5%) had measurable FB1 content, whereas FB2 was not detectable in any of them. The average fumonisin B1 content ranged between 0.03 and 0.07 mg/kg, and the highest measured value was 0.46 mg/kg.

Only one of the 18 canned maize samples contained measurable FB1, but none of them contained FB2. The relevant FB1 concentration was 0.20 mg/kg.

Fumonisin B1 was measured in 20% of the other maize-based, snack-like products (17 of the 85 samples), and FB2 in only 2 samples. The average FB1 content ranged between 0.07 and 0.10 mg/kg, with a maximum content of 1.1 mg/kg.

Regarding white rice and brown rice samples and other rice-based products, FB1 and FB2 contents were in all cases below the LOQ. These commodities were therefore not included in our further risk assessment. The most important parameters of the analysis results for each food categories are summarized in Tables 2 and 3.

**Table 2.** Classification of the samples analyzed in this study in relation to fumonisins' limit of detection (LOD), limit of quantification (LOQ) \*, and the regulatory limit.

\* LOD and LOQ for FB1: 0.031 and 0.093 mg/kg, LOD and LOQ for FB2: 0.051 and 0.154 mg/kg. Nr: number of samples.

\* Method of mean calculation: Results below the LOD and between LOD and LOQ were taken into account in two ways. In the lower-bound (LB) scenario, 0 and LOD were inserted for values below LOD and between LOD and LOQ, respectively. In the upper-bound (UB) scenario, LOD and LOQ were inserted for values below LOD and between LOD and LOQ, respectively. \*\* There were only two measured values for the concerned food category and mycotoxin; na: not applicable. Nr: number of samples.

Considering these results in light of the current maximum limits, one maize flour, two maize grits, and two samples of the other maize-based, snack-like products (mexicorn and a maize wafer) had total fumonisin contents minimally exceeding the regulatory limit (the sum was calculated according to the upper-bound (UB) scenario in case of a non-detectable value of FB2).

Our results were also compared with fumonisin contents measured and published in the previous decades in Hungary. Fazekas et al. [16] measured considerably high fumonisin concentrations in maize collected during storage and harvesting in 1993 and 1994. Of the moldy maize samples collected in the period of storage, 70.8% contained fumonisin B1 (0.05–19.8 mg/kg; average concentration: 2.6 mg/kg). Fumonisin B1 content measured in maize ears more or less affected by molds (affected

sample), collected in the period of harvesting, ranged between 0.095 and 52.4 mg/kg, with an average content of 6.64 mg/kg in 70% of the samples. Of the "average samples", 30% were contaminated with fumonisin B1 (0.06–5.1 mg/kg; average: 1.52 mg/kg). Fumonisin concentrations were determined by high-performance liquid chromatography methods.

Tóth et al. [17] investigated *Aspergillus* and *Penicillium* species and their mycotoxins in maize in Hungary in two consecutive years after harvest. Mycotoxin concentrations were measured with HPLC–MS technique. Fumonisins (B1 + B2) were observed in quantities exceeding the EU limit in some samples collected in different regions (4.66 mg/kg; 10.15 mg/kg; 5.13 mg/kg; 7.55 mg/kg) in 2010.

The IARC report cites contamination data in maize for Europe, including Hungary. Fumonisin B1 was detected in 248 out of 714 maize samples, at a concentration range of 0.007–250 mg/kg [7]. Similarly, the WHO series of Environmental Health Criteria dealt with fumonisin B1 in 2000 [18]. The report specifically cites the results of the Hungarian authors Fazekas et al. [19], measuring 0.05–75.10 mg/kg fumonisin B1 in 56 out of 92 maize samples.

Comparing our results with those of the above reports, fumonisin contamination in Hungary in recent years seems to be lower than that measured in previous decades. However, our measurements focused on processed food products (targeting the end consumer), which obviously have lower fumonisin contents than unprocessed maize samples.

#### *2.2. Correlation between FB1 and FB2 Levels*

FB2 content was always lower than FB1 content in our samples and was detected only in those samples also containing FB1. The relationship between fumonisin B1 and B2 contents was further analyzed, to understand whether a possible correlation coefficient could be set up.

The commodity groups of at least one sample containing measurable quantities of FB1 and FB2 together were maize flour (six samples), maize grits (four samples), and the other maize-based snacks (two samples). The correlation coefficient calculated for the maize flour commodity group based on the numerical concentrations was 0.95, indicating a strong correlation.

Taking into account all 35 samples where, beside FB1, FB2 was also detected but not measurable (i.e., between LOD and LOQ), the correlation coefficients were 0.79 and 0.77 in the lower-bound (LB) and UB scenarios, respectively. Considering only the pooled maize flour and maize grits samples (26 samples), the correlation coefficient values were 0.86 and 0.82 in the LB and UB scenarios, respectively.

These results suggest a possible correlation between the levels of fumonisins B1 and B2; however, a higher number of samples with measured fumonisin B1 and B2 concentrations would be necessary to draw further conclusions.

#### *2.3. Risk Assessment*

The resulting intake values—both mean and high percentile—were well below the reference values established by EFSA and JECFA. Table 4 presents the calculated population mean and 95 percentile intakes for the five commodity groups (maize flour, maize grits, corn flakes, canned maize, and other maize-based, snack-like products) concerned.

**Table 4.** Calculated mean and 95 percentile (P) for fumonisin intakes (μg/kg bw/day). The percentage of European Food Safety Authority (EFSA) tolerable daily intake (TDI) is included in brackets.

The mean daily intake for all maize-product consumers based on the LB and UB scenarios was 0.045–0.120 μg/kg bw/day. In addition, the high intake (95 percentile) ranged between 0.182 and 0.396 μg/kg bw/day, well below 1 μg/kg bw/day.

Regarding children (aged 0–18 years), the mean intake was 0.056–0.167 μg/kg bw/day, and the high intake (95 percentile) was 0.244–0.537 μg/kg bw/day.

Figure 1 presents the relative and cumulative frequencies of the resulting distributions of total fumonisin intakes for both total consumer population and children. The figure shows that most intakes cumulated below 0.5 μg/kg bw/day.

**Figure 1.** Relative and cumulative frequencies of total fumonisin intakes derived from maize-based products.

The results were compared to those of the exposure assessment conducted by EFSA in 2014 on the occasion of a derogation request for the maximum levels of several mycotoxins, including fumonisins [20]. On the basis of French contamination data of 2013, the mean exposure levels in children groups ranged between 0.17 and 1.52 μg/kg bw/day in the LB scenario and between 0.47 and 2.11 μg/kg bw/day in the UB scenario. The high (95 percentile) exposure levels ranged between 0.54 and 3.44 μg/kg bw/day and between 1.09 and 4.39 μg/kg bw/day in the LB and UB scenarios, respectively. In adult groups, the mean exposure levels were between 0.03 and 0.81 μg/kg bw/day in the LB scenario and between 0.15 and 1.19 μg/kg bw/day in the UB scenario. The 95th percentile, however, ranged between 0.08 and 1.76 μg/kg bw/day in the LB scenario and between 0.31 and 2.30 μg/kg bw/day in the UB scenario.

Our present results are in the same range or—especially in the case of children—considerably lower than reported results (Table 5).

Although the estimated mean and high intakes remained below both the JECFA and the EFSA reference values in all scenarios, it is worth noting that the maximum and some high values (over the 95 percentile) exceeded the 1 μg/kg bw TDI set by EFSA in 2018. In the case of all consumers, these high values amounted to 0.97% of the population, whereas in the case of children, they amounted to 2.36%. The maximum estimated intake value was 1.81 μg/kg bw. These specific high values were predominantly children's intake values, derived mainly from the consumption of canned and sweet maize and other maize-based snack-like products.

Considering that these intake results are based on the actually registered consumptions, representing only 4.8% of the total population and 7.6% of children consumers, the consequent health risk is probably negligible.

**Table 5.** Summary of estimated intakes (μg/kg bw/day) in comparison with EFSA estimations and health-based guidance values.

\* All (adult + children) consumers included in our calculations. JECFA: Joint FAO/WHO Expert Committee on Food Additives, PMTDI: provisional maximum tolerable daily intake.

#### *2.4. Commodity Contributions*

The contributions of different commodities to the summed intake estimated from all maize-based foods are presented in Table 6.

**Table 6.** Contribution of different commodities to total fumonisin intake from maize-based products.

\* Data are shown, but conclusions cannot be made due to extremely low registered consumption.

Considering the LB scenarios, maize-based, snack-like products contributed the most to the fumonisin intake of the total (all consumers) population (43.3%), followed by maize flour (29.1%) and corn flakes (15.1%). In the case of children, the main contributors in the LB scenario were, similarly, maize-based, snack-like products (60.4%), corn flakes (20.2%), and canned maize (11.2%) (see Figure 2).

**Figure 2.** Contribution of food commodities to total fumonisin intake from maize-based products.

#### *2.5. Uncertainty Considerations*

It should be mentioned that this assessment focused only on the intake of fumonisins B1 and B2 from five different maize-based commodity types. Other types and the modified or masked forms of fumonisins were not analyzed. Total fumonisin intake of the population could be somewhat higher, if all relevant (including also non-maize-based) commodity types were considered. However, given that maize is the focal commodity in relation to fumonisin contamination, the contribution of other food products to total fumonisin intake is considered low.

The effect of household food processing on fumonisin content (relevant only for maize flour and grit) was not taken into account in our calculations. While the change of fumonisin content as a result of processing operations was studied by several authors [21–25], and heating was reported to lead to some losses of the toxin, the results from different studies are variable [13]. Our approach might have led to a slight overestimation of exposure, taking into account that the effect of heating would lower the calculated intakes; however, this would not change our conclusions, considering that our results do not indicate serious health concern.

The fact that we took into account only those consumption days for which actual consumptions were registered also adds uncertainty. Given that maize-based commodities are non-staple commodities in Hungary, consumed only occasionally by the majority of the population, averaging the occasionally registered consumption values would be misleading. Similarly, including the zero-consumptions in our assessment would "dilute" the results.

However, it needs to be mentioned that current trends indicate an increase in gluten-free foods consumption, which is not strictly linked to the number of consumers intolerant to gluten. Regular consumers striving for healthy diets may as well choose maize-based foods. These facts highlight the importance of focusing more attention on these kinds of food products, considering that they also tend to be the focal commodities most highly contaminated with fumonisins.

As the consumption data were collected in 2009, certain changes might have occurred since then. In the case newer/more recent consumption data are published, repeating these evaluations would be of great value. In this regard, the consumption of different maize-based products could be studied in more detail. In our calculations, we linked the concentration data of an aggregated "maize-based, snack-like products" group to the consumption of an aggregated maize-based products group, including popped maize or extruded corn flakes. These calculations, however, could be refined by separately studying the consumptions of these specific products. Our measurement results indicate a relatively high contamination rate in this kind of commodity category.

#### **3. Conclusions**

Our calculations based on recent fumonisin analyses in maize-based foods and consumption data from a Hungarian survey produced comforting results. The calculated fumonisin intakes of the total population and of children consumers were well below the reference values established by JECFA and EFSA. The values were also in the same range or lower than the European exposure rates estimated by EFSA in 2014.

However, the recent trend of increasing the consumption of alternative, "healthy" foods, including maize-based commodities, needs to be monitored. Our results suggest that maize-based products may indeed be contaminated by fumonisins. Therefore, monitoring of fumonisins' levels and the frequent re-evaluation of their dietary intakes with the best available data are recommended.

#### **4. Materials and Methods**

#### *4.1. Sampling*

Maize-based products were purchased from the Hungarian market in three metropolitan regions, i.e., Kaposvár (n = 276), Budapest (n = 29), and other cities, e.g., Debrecen, Keszthely, Székesfehérvár (n = 21). Commercial products were collected from supermarkets, retail shops, and pharmacies. A total

amount of 326 samples purchased in 15 months (from August 2017 to November 2018) included maize flour (64), maize grits (62), corn flakes (64), canned maize (18), and other maize-based, snack-like products (85, extruded corn bread, tortilla chips, popcorn, nacho, maize chips, etc.). Beside these, 16 white and 10 brown rice and 7 rice-based products were also sampled. All information about the samples (i.e., producer, distributor, country of origin) was obtained from the products' labels and recorded. Samples were randomly selected, collecting as many as possible leader and minor brands available on the market.

#### *4.2. Laboratory Analysis*

#### 4.2.1. Chemicals

Fumonisin B1 (FB1) and B2 (FB2) were purchased from Merck-Sigma Aldrich (St. Louis, LO, USA). HPLC–MS-grade acetonitrile and water were purchased from Carl Roth GmbH (Karlsruhe, Germany), HPLC–MS-grade acetic acid was purchased from Merck (Darmstadt, Germany).

#### 4.2.2. Sample Preparation

Dry solid samples were ground using an ETA® Vital Blend II blender (ETA a.s., Praha, Czech Republic). Then, 5 g of sample was vortexed for 1 min with 20 mL of acetonitrile/water (50:50) on a VELP ZX-3 desktop vortex (Velp, Usmate, Italy) and 0.1% acetic acid and extracted for 60 min at 420 rotations/min speed on a horizontal desktop shaker (Edmund Bühler SM30A model, Bodelshausen, Germany). The supernatant of the extracted sample was centrifuged for 10 min at 14,000 rpm, and 4 ◦C. Aliquots of 10 μL internal standard solutions (13C-FB1, 6 μg/mL) were added to 970 μL aliquots of the supernatant of the centrifuged sample. The mixture was analyzed with LC–MS.

#### 4.2.3. High-Performance Liquid Chromatography

Liquid chromatography and mass spectrometry (LC–MS) analysis were performed with a Shimadzu Prominence UFLC separation system equipped with an LC–MS-2020 single quadrupole (ultra-fast) liquid chromatographer–mass spectrometer (Shimadzu, Kyoto, Japan) with electrospray source. Optimized mass spectra were obtained with an interface voltage of 4.5 kV and a detector voltage of 1.05 kV in negative mode and 1.25 kV in positive mode. Samples were analyzed on a Phenomenex Kinetex 2.6 μm XB-C18 100 Å column (100 mm × 2.1 mm, Phenomenex, Torrance, CA, USA). The column temperature was set to 40 ◦C; the flow rate was 0.3 mL/minute. Gradient elution was performed using LC–MS-grade water (Carl Roth GmbH, Karlsruhe, Germany) (eluent A) and acetonitrile (Carl Roth GmbH, Karlsruhe, Germany) (eluent B), both acidified with 0.1% acetic acid (Merck, Darmstadt, Germany). Then, 5 μL of each samples were analyzed with the gradient: (0 min) 5% B, (3 min) 60% B, (8 min) 95% B, followed by a holding time of 3 min at 95% eluent B and 2.5 min column re-equilibration with eluent 5% B. FB1 (diluted from 10 mg/L) standard solutions were used as references. MS parameters: source block temperature 90 ◦C; desolvation temperature 250 ◦C; heat block temperature 200 ◦C; drying gas flow 15.0 L/minute. Detection was performed using selected ion-monitoring (SIM) mode.

Detection (LOD) and quantification (LOQ) limits were 31 and 93 μg/kg for FB1 and 51 and 154 μg/kg for FB2.

For the calculation of LOD and LOQ, nine calibration points (0.1 μg/kg; 0.5 μg/kg; 1 μg/kg; 5 μg/kg; 10 μg/kg; 50 μg/kg; 100 μg/kg; 500 μg/kg; 1000 μg/kg) were measured, and the LOD and LOQ were calculated using the STHIBAYX function in Microsoft® Excel (Version 2013, Microsoft Corporation, Redmond, WA, USA). The slope of the calibration curve was determined using the nine calibration points.

*LOD* = (Peak area 1, Peak area 2, ... .; Concentration 1, Concentration 2, ... .)· 3,3333 Slope of calibration curve *LOQ* = (Peak area 1, Peak area 2, ... .; Concentration 1, Concentration 2, ... .)· 10 Slope of calibration curve

Ms Excel 2010 was used for the evaluation of the results.

#### *4.3. Analysis of the Measurements and Correlation between FB1 and FB2 Concentrations*

Main descriptive statistics (mean, maximum, 95th percentile) of the measured fumonisin contamination of the analyzed commodities were used. The measurement results were also characterized regarding the number of non-detected/not measurable values and the samples with fumonisin content exceeding the regulatory limit.

To take into account the uncertainty derived from the non-detected (<LOD) and detected but not measurable values (<LOQ), two scenarios were considered. First, to account for the worst-case option, assuming the highest possible concentration of these non-numerical values, LOD was inserted for values <LOD, and LOQ was inserted for values <LOQ, for both fumonisin B1 and B2 results. This was termed the upper-bound scenario. To illustrate with numbers, 0.031 mg/kg and 0.051 mg/kg were substituted for values of FB1 and FB2 <LOD, respectively, and 0.093 mg/kg and 0.154 mg/kg were substituted for values of FB1 and FB2 <LOQ, respectively.

To account for an optimistic scenario, assuming the lowest possible concentration, values <LOD were replaced with 0, and values <LOQ were replaced with the relevant LOD. This scenario was termed the lower-bound scenario. To illustrate with numbers, 0 was inserted for values of both fumonisins <LOD, and 0.031 mg/kg and 0.051 mg/kg were inserted for values of FB1 and FB2 <LOQ, respectively. Obviously, in the case of values >LOQ, the measured numerical values were used directly in all scenarios.

The possible correlation between fumonisin B1 and B2 contents in the samples was also analyzed, calculating the correlation coefficients. Besides considering only the corresponding numerical values of FB1 and FB2, we also analyzed a larger sample set, including those samples for which a numerical FB1 value was accompanied by a detected but not measurable (i.e., between LOD and LOQ) FB2 result. Lower- and upper-bound scenarios were calculated for these sample results as well.

#### *4.4. Food Consumption Data*

Consumption data were obtained from a survey carried out jointly by the Hungarian Food Safety Office (HFSO) and the Hungarian Central Statistical Office in 2009. Three-day dietary record data were collected from 4992 consumers, providing overall 14,976 daily food consumption data, including those of 934 children (aged below 18).

Relevant consumptions of maize products were recorded specifically for maize flour, maize grits, corn flakes, sweet maize, canned maize, frozen maize, extruded corn flakes, popped maize (with and without oil), and cheese-flavored popped maize. These products, and consequently their consumptions, were linked to the analyzed products, in order to perform intake calculations based on the concentration and consumption data of these specific commodities. Table 7 presents the commodities analyzed in relation to those consumed.

**Table 7.** Linking of analyzed values to consumed maize commodity categories by commodity name.

The maize-based, snack-like products measured mainly consisted of different types of snacks produced from maize, including nacho, tacoshells, corn flips, tortilla chips, extruded maize snack, etc. Although they had different compositions, they were dealt with in one aggregated commodity group called maize-based, snack-like products, as their compositions were not specified in the consumption data.

The effect of processing was not taken into account for two reasons. First, the effect of milling was not relevant, as the analytical measurements and consumptions were both recorded for milled maize products, enabling a direct linkage between them. On the other hand, the effect of heating was relevant for maize flour and maize grits; however, further studies would be necessary to conclude on the quantitative effect of heating, based on the literature.

We considered only the consumption days for which consumption of the selected foods was reported. Given that maize-based products are not consumed daily, including those individuals who did not report any consumption of these foods would unrealistically dilute our data. The main statistical parameters of the consumption data are summarized in Table 8.

#### *4.5. Risk Assessment Approach*

Risk assessment was performed by semi-probabilistic means, by considering the consumption values as a distribution, since there were exact individual food consumption data available. The concentration values of FB1 and FB2 were summed in each sample and considered accordingly in further calculations.

For each food commodity category, the average concentration was calculated for both the LB and the UB scenarios. These values were then multiplied by the relevant individual consumption data one-by-one, resulting in the relevant calculated fumonisin intake values for each individual consumption of each commodity.

The daily individual intakes calculated from each commodity category were then summed for each individual, resulting in the summed individual daily fumonisin intake from all the selected foods. The resulting distribution of individual total daily fumonisin intakes could then be further studied on a population level. Average and high (95 percentile) values were calculated to determine the fumonisin intake of average and high consumers. These calculations were also applied for the children population of consumers. Finally, the resulting values were compared to the reference values established by JECFA [11] and EFSA [15].

To estimate the commodity contributions to the summed intake estimated from the analyzed commodities, the population average intake from each commodity was calculated separately and then compared to the average summed intake, resulting in the proportion of contribution of each commodity.

**Author Contributions:** Study design, M.S.-S., M.K. and A.Z.; Methodology, A.Z. and M.S.-S.; Analytics, G.M., N.S. and A.S.; Formal Analysis, A.Z.; Resources, M.K.; Original Draft Manuscript Preparation, A.Z.; Review & Editing, A.Z., M.S.-S. and A.S.; Supervision, M.K. and M.S.-S.

**Funding:** This research was funded by the project GINOP-2.3.2.-15-2016-00046 and the EFOP-3.6.3.-VEKOP-16-2017-00005 programs.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

#### **References**

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

#### *Article*

## **Species Composition and Toxigenic Potential of** *Fusarium* **Isolates Causing Fruit Rot of Sweet Pepper in China**

**Jianhua Wang 1, Shuangxia Wang 2, Zhiyong Zhao 1, Shanhai Lin 1,3, François Van Hove 4 and Aibo Wu 2,\***

Received: 28 October 2019; Accepted: 21 November 2019; Published: 24 November 2019

**Abstract:** Apart from causing serious yield losses, various kinds of mycotoxins may be accumulated in plant tissues infected by *Fusarium* strains. Fusarium mycotoxin contamination is one of the most important concerns in the food safety field nowadays. However, limited information on the causal agents, etiology, and mycotoxin production of this disease is available on pepper in China. This research was conducted to identify the *Fusarium* species causing pepper fruit rot and analyze their toxigenic potential in China. Forty-two *Fusarium* strains obtained from diseased pepper from six provinces were identified as *F. equiseti* (27 strains), *F. solani* (10 strains), *F. fujikuroi* (five strains). This is the first report of *F. equiseti*, *F. solani* and *F. fujikuroi* associated with pepper fruit rot in China, which revealed that the population structure of *Fusarium* species in this study was quite different from those surveyed in other countries, such as Canada and Belgium. The mycotoxin production capabilities were assessed using a well-established liquid chromatography mass spectrometry method. Out of the thirty-six target mycotoxins, fumonisins B1 and B2, fusaric acid, beauvericin, moniliformin, and nivalenol were detected in pepper tissues. Furthermore, some mycotoxins were found in non-colonized parts of sweet pepper fruit, implying migration from colonized to non-colonized parts of pepper tissues, which implied the risk of mycotoxin contamination in non-infected parts of food products.

**Keywords:** *Fusarium* species; mycotoxin; toxigenic profile; mycotoxin migration; sweet pepper; fungal disease

**Key Contribution:** *Fusarium* species on sweet pepper in China is different from those in Canada and Belgium, and *F. equiseti*, *F. solani*, and *F. fujikuroi* were first reported causing pepper fruit rot in China. Toxigenic potential of *Fusairum* strains were analyzed in inoculated pepper fruit and diffusions of FA (fusaric acid), FB1 (fumonisin B1), FB2 (fumonisin B2), and MON (moniliformin) from lesions into the surrounding sound tissues were observed. This is the first report about the migration of FA and MON in sweet peppers.

#### **1. Introduction**

Sweet pepper or bell pepper (*Capsicum annuum* L.) is highly appreciated in the fresh vegetable markets worldwide due to its unique taste, aromas, and the multiple culinary uses. It represents one of the most important vegetables for the high content of phytochemicals, such as ascorbic acid and soluble phenols [1,2], having potential positive effects on human health. For example, ascorbic acid is an essential dietary nutrient in the human body with its vital biological function as an antioxidant. Sweet pepper is an economically important vegetable crop and widely used for direct consumption and manufacturing of sauce worldwide, where the global production reached 34.6 million tons of fresh fruit and 3.5 million tons of dried pods [3]. China is the largest producer and exporter of sweet pepper in the world, with a total fresh pepper production per year of almost 23 million tons. Sweet pepper is often grown in commercial greenhouses, which is favorable for the growth and survival of phytopathogenic fungi and the infection of pepper plants [4]. Thus, the disease poses a serious limitation to pepper cultivation, resulting in yield reduction or complete crop loss, as reported previously in the literature [4–6].

Several fungal diseases have caused economic losses in sweet pepper production in Canada, United Kingdom, and Belgium [4,7,8]. *Fusarium* infection on the stem- and blossom-end of pepper fruit caused by *F. solani* was first reported in Ontario and British Columbia in 1991 and caused approximately 5% fruit-yield loss [8–10]. Since the 1990s, this rot disease has been an increasing problem in pepper production in both Europe and North America [7,11,12]. More seriously, a severe outbreak of this disease resulting in a 50% yield loss in a greenhouse was reported in 1990 in Ontario [13].

In addition to external fruit rot, internal rot of sweet pepper fruit is also a big problem in pepper production [14]. In contrast to the external pepper fruit rot, the fruit is infected internally by a fungus. Unless severely infected and rotten, most infected fruits are difficult to cull before delivery to the market as the symptoms are not readily visible [4,8,10]. A comprehensive histopathology analysis performed by Yang et al. [10] indicated that internal fruit rot of greenhouse sweet pepper caused by *F. lactis* was initiated through the infection of the stigma and style during anthesis. Symptomless seed infection may contribute to disease spread, and air and insects also play an important role as intermediates in fungal spores spreading [15]. Based on the surveys to date, *F. lactis* is the principal causal agent of internal fruit rot of pepper, although the closely related *Fusarium* species belonging to the *Fusarium fujikuroi* species complex (FFSC), such as *F. proliferatum*, *F. subglutinans,* and *F. verticillioides*, have also been implicated in this disease [4,6,8].

Apart from causing significant yield losses, *Fusarium* species can produce fumonisins (FBs), trichothecenes (TCs), zearalenone (ZEN), and other mycotoxins in infected plant tissues which are harmful to consumers. For example, B-series fumonisins (Figure 1), which are mainly produced by *Fusarium* species from FFSC, are the most frequently detected mycotoxins in maize, and are involved in animal and human diseases by interfering with sphingolipid metabolism. Moreover, fumonisins have been associated epidemiologically with esophageal cancer in humans in some regions of the world [16,17]. On the basis of available toxicological evidence, the International Agency for Research on Cancer (IARC) has classified fumonisins as possibly carcinogenic (Group 2B) [18]. Mycotoxin contamination in grains, vegetables, and fruit poses a serious threat to food safety.

Mycotoxins can diffuse into tissues surrounding the pathogen-infected site. The water content of fresh sweet pepper is usually higher than 90%, which contributes to the dissolution of polar compounds produced by pathogens in pepper tissues. Fumonisins, fusaric acid (FA), and moniliformin (MON) are the most common mycotoxin contaminants produced by *Fusarium* species, and migration of some of these toxins has been reported in pepper and fruit previously [19,20]. A migration study of beauvericin (BEA) and fumonisins was reported by Monbaliu et al. [19], and the results indicated the migration of fumonisins into healthy parts of the sweet pepper, while beauvericin was not detected in the tissue surrounding the lesion. Mycotoxin contaminations originating from mycotoxin-producing *Fusarium* species in pepper should be considered as an importantly sensitive food safety concern. Therefore, it is of great necessity to assess the types and levels of mycotoxin contamination in pepper and its derived products [4].

**Figure 1.** Structural formulas of B-series fumonisins.

Effective management of mycotoxin contamination in sweet pepper relies on the control of the fungal infection and requires a better understanding of *Fusarium* biology and epidemiology. To our best knowledge, there is little information on the causal agent, etiology, toxigenic potential, and geographic distribution of the *Fusarium* species involved in pepper disease in China. The objectives of this current work were: (i) To isolate and identify the causal organism from *Fusarium* genus on sweet pepper in China; (ii) to assess the potential mycotoxin profiling of the fungal pathogens; (iii) to estimate the migration behavior of mycotoxins in sweet pepper.

#### **2. Results and Discussion**

#### *2.1. Isolation and Identification of Fusarium Isolates*

Sweet pepper fruits were sampled from Hainan, Heilongjiang, Hunan, Shanghai, Shandong, and Zhejiang provinces in China. Following isolation from diseased pepper tissues, a total of forty-two single-spore isolates were obtained and identified as belonging to *Fusarium* genus by morphology. Two of theses strains were isolated from Hainan, one strain was isolated from Heilongjiang, five strains were isolated from Hunan, twenty-one strains were isolated from Shanghai, three strains were isolated from Shandong, and ten strains were isolated from Zhejiang (Table 1).

The purified isolates were first identified to species with morphological characteristics [21], and this was subsequently confirmed by nucleotide sequences analysis of the translation elongation factor 1-α (*TEF*-1α) genes with partial of representative strains. Of the forty-two strains, twenty-seven were identified as *F. equiseti*, ten as *F. solani*, and five as *F. fujikuroi* (Table 1). Previously, a new disease on pepper caused by *F. concentricum* was reported by our group, and the strain MUCL54697 was isolated from Hunan province, China [22].

Eleven representative strains belonging to different species were selected for *TEF-1*α gene sequencing to further confirm the morphological identification results as described before [23–25]. Obtained sequences were subjected to alignment analysis using the network service tool BLASTn of the National Center for Biotechnology Information (NCBI) database. The sequence analysis of the portion of the *TEF-1*α genes of representative strains confirmed that all the strains belonged to *Fusarium* genus. The results indicated that nucleotide sequences of three strains (Q12002, Q12003 and Q12005) showed the highest identity (>99%) with the sequence of *F. equiseti*, three (Q12029, Q12030 and Q12034) showed the highest identity (>99%) with the sequence of *F. solani,* and five (Q12038-Q12042) showed the highest identity (>99%) with the sequence of *F. fujikuroi* in the NCBI database. The molecular identification results were all identical with the morphological results. The *TEF-1*α gene sequences generated in this study were deposited in GenBank, NCBI (http://www.ncbi.nlm.nih.gov/genbank/), under accession numbers KF208617–KF208627, which is presented in Table 1.

**Table 1.** *Fusarium* strains isolated in this study.

Among the forty-two *Fusarium* strains, all the ten *F. solani* (23.81%) strains were isolated from Shanghai, and the five *F. fujikuroi* (11.90%) strains were isolated from Hainan (one strain), Hunan (two strains), and Zhejiang (two strains). *F. equiseti* was the only species isolated from all six sampled provinces in China, and accounting for 64.29% of all *Fusarium* strains isolated (Table 1). According to our survey, *F. equiseti* was the predominant pathogen of sweet pepper fruit rot in China.

*Fusarium* strains were isolated from both external and internal rotten pepper fruits in this study. Of the forty-two strains, thirty-four were isolated from external rotten pepper fruits and eight strains were isolated from internal rotten pepper fruits (Table 1). For the twenty-seven *F. equiseti* strains, twenty-two were associated with external rot disease of pepper fruits and five (Q12002, Q12005, Q12008, Q12018, and Q12022) were isolated from internal rotten pepper fruits. For the ten *F. solani* and five *F. fujikuroi*, two (Q12034 and Q12035) and one strain (Q12041) were isolated from internal rotten pepper fruit, respectively. As such, *F. equiseti*, *F. solani*, and *F. fujikuroi* were the causal agents of external fruit rot of pepper in China, with *F. equiseti* being the predominant, while *F. equiseti*, *F. solani*, and *F. fujikuroi* were associated with internal fruit rot of pepper, with *F. equiseti* being the predominant. To our best knowledge, this is the first report of *F. fujikuroi*, *F. equiseti,* and *F. solani* associated with external and internal pepper fruit rot in China.

*F. solani* was reported as the predominant causal agent of external fruit rot of pepper in Europe and North America [7–13], which is different from China. However, it worth noting that *F. solani* is a common pathogen causing pepper fruit rot worldwide. *F. solani*, *F. lactis*, *F. proliferatum*, *F. subglutinans*, and *F. verticillioides* were reported to cause external or internal pepper fruit rot in Belgium, Canada, and United Kingdom, with *F. lactis* being the principal one [4,6,8]. In this study, *F. equiseti*, *F. solani*, and *F. fujikuroi* were found to be associated with internal pepper fruit rot, and with *F. equiseti* being the predominant. In light of the above, it is obvious that the population structure of *Fusarium* species associated with pepper fruit rot (external or internal) in China is quite different from those in surveys from Canada and Belgium [4,8,10]. Although the underlying factors for species distribution are unknown, climatic conditions (such as the annual temperature weather and humidity), hosts and their rotation, and adaptive evolution have been reported to influence the distribution of *Fusarium* species [14,26–28]. In view of the population genetic diversity and dispersal difference of the *Fusarium* pathogens, procedures for effective management of these pathogens on pepper are urgently needed.

#### *2.2. Molecular Phylogenetics*

Phylogenetic analyses were conducted on partial sequences of *TEF-1*α genes. Figure 2 shows the phylogenetic tree constructed with MEGA 5.10 [29]. As several *Fusarium* species have been reported to be the causal agents of pepper fruit rot, in addition to the nucleotide sequences obtained in this study (Table 1), corresponding sequences available in GenBank for the strains belonging to *F. concentricum*, *F. equiseti*, *F. fujikuroi*, *F. lactis*, *F. proliferatum*, *F. solani*, *F. subglutinans*, and *F. verticillioides* were retrieved and served as references.

**Figure 2.** Phylogenetic tree inferred from alignments of *TEF-1*α sequences of *Fusarium* species by the Neighbor-Joining method with program MEGA 5.10. The numbers beside branches are the percentages of congruent clusters in 1000 bootstrap trials. Bootstrap values higher than 75% are shown.

As shown in Figure 2, bootstrap analyses of the *TEF-1*α gene partial sequences clearly separated the thirty-six *Fusarium* isolates into three major clades with a bootstrap value of 100%; the *F. equiseti* clade (seven isolates), the *F. solani* clade (eight isolates), and the third clade that was composed of the remaining twenty-one isolates belonging to *F. concentricum*, *F. fujikuroi*, *F. lactis*, *F. proliferatum*, *F. subglutinans*, and *F. verticillioides*. The six *Fusarium* species mentioned above in the third clade are all from the *F. fujikuroi* species complex (FFSC) [30], and they are relatively closer species among the species analyzed.

It is obvious that six distinct subclades were formed with >97% bootstrap support in the third clade which separated the twenty-one isolates to *F. concentricum* (three isolates), *F. fujikuroi* (ten isolates), *F. lactis* (two isolates), *F. proliferatum* (two isolates), *F. subglutinans* (two isolates), and *F. verticillioides* (two isolates) (Figure 2). Thus, the *TEF-1*α sequences can efficiently differentiate these *Fusarium* species. The *TEF-1*α sequence BLASTn and phylogenetic analysis results strongly supported the identification results of *Fusarium* strains isolated.

#### *2.3. Multi-Mycotoxin Analysis in Pepper Fruit*

For large-scale screening of mycotoxin production capabilities in pepper by the sampled *Fusarium* isolates, the well-established multi-component LC-MS/MS method was used for scanning of the 36 mycotoxins reported in agricultural products. In this multi-mycotoxin analysis, two *F. equiseti* isolates (Q12002 and Q12004), three *F. solani* isolates (Q12029, Q12034 and Q12037), four *F. fujikuroi* isolates (Q12038, Q12039, Q12040 and Q12041), and the *F. concentricum* strain MUCL54697 [22] were selected to do the inoculation experiment and assess their mycotoxin production in different sites of sweet pepper fruits (Figure 3).

**Figure 3.** Inoculated pepper with the indication of inoculation site (red dot) and healthy site (black oval) for mycotoxin analysis.

Mycotoxin detection results from lesions (Figure 3, red dot) were used to do the mycotoxin profile analysis, and the results from the healthy parts (Figure 3, black oval) were used for the migration study. Mycotoxin profiles of the ten strains selected were summarized in Table 2. The multiple mycotoxin analysis resulted in the detection of BEA, FA, fumonisin B1 (FB1), fumonisin B2 (FB2), MON, and nivalenol (NIV) in pepper tissues (Figures 1 and 4). In one out of the ten isolates inoculated, none of the thirty-six mycotoxins investigated was detected. FA was the most frequently detected metabolite which was produced by nine isolates in concentrations that greatly varied from 41.44 to 10,662.36 μg/kg. BEA was produced by eight isolates with average concentrations varying between 5.20 and 1019.60 μg/kg. Both FB1 and FB2 were produced by the same four isolates ranging from 43.64 to 39,326.60 μg/kg and 26.96 to 3734.16 μg/kg, respectively. MON was produced by five isolates in greatly varying amounts (35.80–2439.48 μg/kg), while NIV was produced by only one isolate at a concentration of 184.16 μg/kg.

**Table 2.** Mycotoxins produced by selected *Fusarium* strains in sweet pepper samples.

1 IS, Inoculation site; HS, Healthy site.

**Figure 4.** Structural formulas of moniliformin (**A**), nivalenol (**B**), fusariuc acid (**C**), and beauvericin (**D**).

As shown in Table 2, significantly different toxigenic profiles were observed among different species in inoculated pepper fruits. For the investigated *Fusarium* species, the most abundant of toxic metabolites were produced by *F. fujikuroi*, and all the four *F. fujikuroi* isolates can produce BEA, FA, FB1, FB2, and MON. With regard to the individual mycotoxin, relatively higher contents of FB1 were generated compared to FB2 by the same strain, which were consistent with the previously reported studies [31,32]. The ratios between the two fumonisins for individual *F. fujikuroi* strain were in the range of 1.05–1.83 in pepper lesions. Meanwhile, significant differences in fumonisin production capacities of *F. fujikuroi* strains were observed. For example, strain Q12040 produced FB1 and FB2 in concentrations of 43.64, 26.96 μg/kg, respectively, while large amounts of FB1 and FB2 were produced by isolate Q12041 (3926.60, 3734.16 μg/kg, respectively) which were about 90 and 139 times as higher than those produced by Q12040 under the same conditions. Note that similar or higher amounts of FA and MON were produced by the four *F. fujikuroi* strains when compared with FBs. For example, the amount of FA is higher than the total fumonisins (FB1 + FB2) produced by three out of four isolates. Strain Q12002 produced NIV, a kind of type B trichothecene, at a concentration of 184.16 μg/kg, while trichothecenes were not observed with the other strain Q12004. These two *F. equiseti* isolates showed a considerable intraspecies variation in profiles of trichothecene production, similar to results reported by Hestbjerg et al. [33]. Similarly, intraspecies variation in toxigenic profiles was also observed in

*F. solani*. Regarding the three *F. solani* strains, only FA was produced by strain Q12029, compared to BEA and FA (94.84, 78.98 μg/kg, respectively) that were produced by strain Q12034, while none of the thirty-six mycotoxins were detected with strain Q12037.

Large variation among strains, both in terms of their toxigenic profiles and the quantity of mycotoxins produced in pepper, was found in this study. Based on the mycotoxin profile results, it could be concluded that interspecies toxigenic profile variation appears to be a species-specific characteristic, while the intraspecies quantity variation appears to be a strain-specific characteristic.

The results of the migration study are summarized in Table 2. Among the mycotoxins detected in this study, diffusion phenomenon of FA, FB1, FB2, and MON from a moldy area to healthy tissues was observed in pepper fruit, while no detectable BEA and NIV were found in unaffected parts.

As shown in Table 2, FA was the most frequently detected mycotoxin in unaffected parts, with concentrations varying from 13.80 to 874.04 μg/kg. As a phytotoxin [34], the ratios of FA detected from lesions and healthy parts were in the range of 5.54–51.87. FB1 was detected in unaffected parts with concentrations varying from 41.08 to 183.04 μg/kg. The ratios of FB1 detected from lesions and healthy parts were in the range of 21.45–37.95. FB2 was detected in unaffected parts with concentrations varying from 24.30 to 144.92 μg/kg and the ratios of FB2 detected from lesions and healthy parts were similar to FB1 (25.77–37.58). MON was detected in unaffected parts with concentrations varying from 172.13 to 253.12 μg/kg, and the ratios of the compound from lesions and healthy parts were in the range of 7.61–12.59. Migration of FB1 and FB2 in sweet pepper was reported by Monbaliu et al. [19], and this is the first report about the diffusion of FA and MON in sweet pepper.

Mycotoxins can persist in infected plant tissues, and depending on physical or chemical properties (solubility, polarity, hydrophilicity, molecular weight, concentration, etc.) and tissue components, might also transfer from a rotten part of the plant tissues into the surrounding sound tissues, even in the absence of fungal growth. In this study, no NIV was detected in healthy pepper tissues maybe due to the low concentration, while BEA was not detected even at a high concentration (1019.60 μg/kg). Similar results were reported by Monbaliu et al. [19] that BEA can not be detected in surrounding tissues even with an extremely high concentration, 73,800 μg/kg, in pepper lesions. As shown in Figure 4, BEA is a cyclic hexadepsipeptide that contains three D-hydroxyisovaleryl and three N-methylphenylalanyl residues in an alternating sequence [35]. As an organic and non-polar compound, BEA is insoluble in the aqueous environment. Vegetable, and sweet peppers in particular, contain >90% water, which is probably why BEA was not detected in the surrounding tissues. These results demonstrated the possible risk of mycotoxin contamination in non-infected parts of food products, and some mycotoxins can diffuse into sound tissues. Since second-quality vegetables and fruits may be used to produce derivatives such as juices, jam, etc., further studies on migration behaviors and affecting factors of different mycotoxins in various vegetables and fruits are very important to establish suitable means of protecting consumers from exposure to toxic substances [20].

#### **3. Conclusions**

*Fusarium* species causing pepper fruit rot (external and internal) were analyzed in this study. Altogether, forty-two isolates belonging to *F. equiseti* (27 isolates), *F. solani* (10 isolates), and *F. fujikuroi* (five isolates) were identified with *F. equiseti* being the predominant species. To our best knowledge, this is the first report of *F. fujikuroi*, *F. equiseti* and *F. solani* associated with pepper fruit rot in China. Toxigenic profiles of ten pathogens were determined in sweet peppers, and six toxic metabolites (BEA, FA, FB1, FB2, MON, and NIV) were detected in total. Significantly different toxigenic profiles were observed among the three *Fusarium* species. Diffusions of FA, FB1, FB2, and MON from lesions into the surrounding sound tissues were observed in sweet peppers, and this is the first report about the migration of FA and MON in sweet peppers. Further studies on migration behaviors and affecting factors of different mycotoxins in various vegetables and fruit should be conducted.

#### **4. Materials and Methods**

#### *4.1. Isolation and Purification of Fusarium Strains*

During the growing season, pepper fruit samples were collected from different regions in China, including Hainan, Heilongjiang, Hunan, Shandong, Shanghai, and Zhejiang provinces (Table 1). Pepper fruits with visible symptoms (external or internal when cut open) were selected for pathogen isolation. Fungi were isolated using conventional methods as follows: Symptomatic tissues (3 × 3 cm) were surface-sterilized in 0.1% HgCl2 for 1 min, transferred into 70% ethanol for 30 s, then rinsed three times in sterilized distilled water, dried, and plated on 90 mm Petri dishes containing potato dextrose agar (PDA). After incubation for 3–5 days at 28 ◦C in the dark, colonies resembling morphologically to *Fusarium* were transferred onto new PDA. Plates were incubated at 28 ◦C in the dark until colonies developed, and then purified through serial transfers. No more than one strain per fruit was isolated. For each strain, a single spore culture was obtained by single-sporing as described before [36]. The pure cultures were used for the morphological and molecular characterization. Monoconidial strains were cryopreserved and maintained in tubes on PDA in the lab.

#### *4.2. Nucleotide Maniplation*

Mycelia plugs from 3-day-old PDA cultures were transferred to 50 mL of potato dextrose broth (PDB) medium and incubated with shaking (100 rpm) at 28 ◦C in the dark for 3 days. After incubation, mycelium was filtered through two-layered cheesecloth, washed with sterile water, then freeze-dried and ground to a fine powder using a TissueLyser II system (Qiagen Tissuelyser II, Retsch, Haan, Germany). Genomic DNA was extracted and purified using a Cetyl Trimethylammonium Bromide (CTAB) protocol as described before [37]. In brief, after homogenization, the ground power was suspended with 600 μL of CTAB lysis buffer, mixed well by shaking, and incubated at 65 ◦C for 1 h. After incubation, the solution was cooled at room temperature for 5 min, cellular debris were pelleted by centrifugation at 12,000 rpm for 10 min, and 500 μL of supernatant was transferred into a new tube. The supernatant was extracted with 500 μL of chloroform:isoamyl alcohol mixture (24:1, *v*/*v*). After centrifugation at 12,000 rpm for 10 min, 400 μL of aqueous phase was transferred to a fresh tube, then 400 μL of ice-cold isopropyl alcohol and 40 μL of sodium acetate (3 M, pH 5.2) were subsequently added to the samples. The tubes were mixed by gentle inversion. After incubation for 1 h at −20 ◦C, DNA was precipitated by centrifugation at 12,000 rpm for 10 min. The DNA sample was washed with 800 μL of pre-chilled 70% ethanol and air-dried before resuspension in 50 μL TE buffer (10 mM Tri-HCl, 0.1 mM EDTA, pH 8.0). DNA quantification and quality analysis were carried out by agarose gel electrophoresis with known DNA marker as standard.

#### *4.3. Fusarium Strain Identification*

The purified *Fusarium* strains were identified to species by morphological characteristics, and this was confirmed by *TEF-1*αgene sequence analysis of the representative strains. Methods for determining phenotypic characters and mycelial growth of *Fusarium* strains were from published protocols [21].

In order to verify the identity of *Fusarium* strains collected, DNA sequence comparisons were made for a subset of the strains using the *TEF-1*α gene, known as one of the most pertinent gene for determining the species rank in the *Fusarium* genus [23–25]. Portions of the *TEF-1*α gene were amplified with primer pair EF-1 (3 -ATGGGTAAGGA(A/G)GACAAGAC-5 ) and EF-2 (3 -GGA(G/A)GTACCAGT(G/C)ATCATGTT-5 ) in a thermal cycler. Polymerase chain reaction (PCR) was performed in a 50 μL reaction system afterwards [4], with minor modifications. PCR reaction mixtures contained 1× TransStar FastPfu Fly PCR SuperMix (TransGen Biotech, Beijing, China), 0.2 μM of each primer, and 50 ng of genomic DNA template. A negative control omitting the DNA template was used in every set of reactions. The thermal cycler (T100 Thermal Cycler, Bio-Rad, Hercules, CA, USA) conditions consisted of an initial denaturation step at 94 ◦C for 2 min, followed by 30 cycles of denaturation at 94 ◦C for 20 s, annealing at 58 ◦C for 20 s and extension at 72 ◦C for 30 s, then

a final extension of 72 ◦C for 5 min. Amplified products (50 μL) were separated by electrophoresis on 1.5% (*w*/*v*) agarose gels. Gels were stained with ethidium bromide and photographed under UV light in the Bio-Imaging system (Bio-Rad, Hercules, CA USA). Fragments were excised and extracted from the gel using the QIAquick gel extraction kit (QIAGEN, Hilden, Germany) according to the manufacturer's instructions. Purified amplicons were sequenced in both directions using an ABI3730XL DNA sequencer (Applied Biosystems, Foster City, CA, USA) for each strain, and contigs were assembled with Sequencher version 4.1 program (Gene Codes Corporation). The *TEF-1*α gene sequences generated in this study were subjected to similarity searches with the BLASTn network service of NCBI nucleotide database.

#### *4.4. Phylogenetic Analysis*

Portions of the *TEF-1*α gene sequences from eleven *Fusarium* strains were generated for phylogenetic analysis in this study (Table 1). All sequences were compared with sequences of *Fusarium* species available in the GenBank database through BLASTn searches for similar sequences.

Phylogenetic analyses were conducted using MEGA v. 5.10 [29] to characterize the genetic diversity and evolutionary relationships of the strains. *TEF-1*α sequences of twenty-five fungal strains belonging to *Fusarium* genus retrieved from the NCBI database were also used as references for constructing a phylogenetic tree. In total, thirty-six sequences were analyzed, including seven *F. equiseti* strains, ten *F. fujikuroi* strains, eight *F. solani* strains, three *F. concentricum* strains, two *F. lactisi* strains, two *F. proliferatum* strains, two *F. subglutinans* strains, and two *F. verticillioides* strains. The *TEF-1*α sequences of these *Fusarium* strains were all available in NCBI nucleotide database, and detailed information about their strain code, geographic origin, and host/substrate is listed in Table 3.

All sequences were aligned initially with ClustalX software [44] and the alignments manually edited. Phylogenetic analyses of the sequences were performed with MEGA5.1 for Neighbor-joining (NJ) analysis, and Kimura-2 parameter model and pairwise deletion option for gaps were used. The reliability of the tree topologies was evaluated using bootstrap support with 1000 pseudoreplicates of the data.

#### *4.5. Mycotoxin Production Analysis in Pepper Fruits via LC-MS*/*MS*

Fungal strains were initially grown on PDA in 90 mm diameter Petri dishes for 7 days at 28 ◦C in the dark, after which they were used to inoculate pepper fruits. Mature pepper fruits were inoculated with mycelium plug as described by Van Poucke et al. [4]. After incubation, the fruit tissues from different positions, including the inoculation site and healthy site, were collected, homogenized separately, and processed for mycotoxin detection analysis. Mycotoxin detection results from lesions were used to do the mycotoxin profile analysis, and the results from the healthy parts were used for the migration study.

For each sample, 2 g of the grounded material was extracted with 8 mL extraction solvent (acetonitrile:water = 84:16, *v*/*v*). Multi-component analysis of mycotoxins in the inoculated samples was performed as described previously [45,46]. In total, 36 mycotoxins were included in the multi-mycotoxin analyses: Aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), aflatoxin G1 (AFG1), aflatoxin G2 (AFG2), aflatoxin M2 (AFM2), aflatoxin M1 (AFM1), beauvericin (BEA), citrinin (CIT), cyclopiazonic acid (CPA), deoxynivalenol (DON), 3-acetyldeoxynivalenol (3-ADON), 15-acetyldeoxynivalenol (15-ADON), deoxynivalenol-3-glucoside (DON-3G), deepoxydeoxynivalenol (Deep-DON), diacetoxyscirpenol (DAS), fusaric acid (FA), fumonisin B1 (FB1), fumonisin B2 (FB2), fusarenon-X (FUSX), gliotoxin, HT2 toxin (HT2), moniliformin (MON), neosolaniol (NEO), nivalenol (NIV), ochratoxin A (OTA), patulin (PAT), penitrem A (PenA), sterigmatocystin (SMC), T-2 toxin (T-2), verruculogen (VER), zearalenone (ZEN), α-zearalanol (α-ZOL), β-zearalanol (β-ZOL), α-zearalenol (α-ZAL), β-zearalenol (β-ZAL), and zearalanone (ZAN). HPLC or analytical grade of acetonitrile, hexane, and other chemical agents were purchased from Merck (Darmstadt, Germany). Deionized water purified by a Milli-Q water system (Millipore, Billerica, MA, USA) was used throughout the experiments.

**Author Contributions:** S.W. and J.W. collected the samples; Z.Z. performed the mycotoxin analysis; S.W., S.L. and J.W. performed the experiments and analyzed the data; F.V.H. provided some protocols for isolation; writing—original draft preparation, J.W.; writing—review and editing, A.W.; supervision, A.W.

**Funding:** This research was funded by the National Key Research and Development Program of China (2017YFC1600304), National Natural Science Foundation of China (31871896, 31401598, and 31602124), Shanghai Agriculture Commission Basic Research Programs (2014NO.7-3-7 and 2011NO.4-3).

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

#### *Article*

## **Toxin Production in Soybean (***Glycine max* **L.) Plants with Charcoal Rot Disease and by** *Macrophomina phaseolina,* **the Fungus that Causes the Disease**

#### **Hamed K. Abbas 1,\*, Nacer Bellaloui 2, Cesare Accinelli 3, James R. Smith 2 and W. Thomas Shier 4,\***

Received: 13 September 2019; Accepted: 29 October 2019; Published: 6 November 2019

**Abstract:** Charcoal rot disease, caused by the fungus *Macrophomina phaseolina*, results in major economic losses in soybean production in southern USA. *M. phaseolina* has been proposed to use the toxin (-)-botryodiplodin in its root infection mechanism to create a necrotic zone in root tissue through which fungal hyphae can readily enter the plant. The majority (51.4%) of *M. phaseolina* isolates from plants with charcoal rot disease produced a wide range of (-)-botryodiplodin concentrations in a culture medium (0.14–6.11 μg/mL), 37.8% produced traces below the limit of quantification (0.01 μg/mL), and 10.8% produced no detectable (-)-botryodiplodin. Some culture media with traces or no (-)-botryodiplodin were nevertheless strongly phytotoxic in soybean leaf disc cultures, consistent with the production of another unidentified toxin(s). Widely ranging (-)-botryodiplodin levels (traces to 3.14 μg/g) were also observed in the roots, but not in the aerial parts, of soybean plants naturally infected with charcoal rot disease. This is the first report of (-)-botryodiplodin in plant tissues naturally infected with charcoal rot disease. No phaseolinone was detected in *M. phaseolina* culture media or naturally infected soybean tissues. These results are consistent with (-)-botryodiplodin playing a role in the pathology of some, but not all, *M. phaseolina* isolates from soybeans with charcoal rot disease in southern USA.

**Keywords:** fungi; mycotoxins; phaseolinone; LC/MS; soybean; charcoal rot disease; root infection mechanism

**Key Contribution:** This is the first report of a toxin being found in the tissues of soybean plants naturally infected in the field with charcoal rot disease, specifically finding (-)-botryodiplodin and not phaseolinone. This is also the first report of results consistent with some isolates of *M. phaseolina* using different toxins, other than (-)-botryodiplodin, to facilitate root infection in soybean.

#### **1. Introduction**

The fungus *Macrophomina phaseolina* (Tassi) Goidanich [1], also known by the teleomorph *Sclerotium bataticola* Taub. [2], is the cause of charcoal rot disease, and other named diseases, in soybeans and about 500 other crop and ornamental species in the United States and internationally [3–5]. Charcoal rot

disease, also known as summer wilt, dry weather wilt, or black root disease, results in crop yield loss and seed quality deterioration in soybeans and other crops [6–11]. Charcoal rot disease is more prevalent in heat- and drought-stressed conditions [12,13]. *M. phaseolina* can spread to adjacent plants with interdigitating roots through the soil, infecting the roots and spreading throughout the infected plant through the vascular system [14,15]. *M. phaseolina* forms black spore-like mycelial structures called microsclerotia, which allow the fungus to survive over winter. These microsclerotia are the grey and black dots in the stems and roots of soybean plants that give charcoal rot disease its name [16]. Common agricultural practices such as managing planting dates, fungicide applications, and biological control have been ineffective in controlling this disease [17–23]. Despite extensive efforts to control charcoal rot disease by developing resistant soybean genotypes [24–26], currently available genotypes are still not sufficiently resistant to prevent the disease in the field, although moderately resistant genotypes have been shown to have lower levels of *M. phaseolina* in plant tissues [27–29].

The mechanism used by *M. phaseolina* to infect plants with charcoal rot disease is not yet understood, in part because of the diversity in *M. phaseolina* isolates [30–33]. *M. phaseolina* has been reported to produce toxins, including (-)-botryodiplodin and phaseolinone [34–37]. It has been proposed that a toxin may play a role in an early step of the mechanism used by *M. phaseolina* to infect susceptible plants through the roots from the soil reservoir, where the fungus normally lives, particularly over the winter [7,36].

The objective of the present study is to investigate the involvement of toxins, particularly (-)-botryodiplodin, in the charcoal rot disease of soybeans. Soybeans are selected as the subject for these studies because charcoal rot disease causes major economic losses for soybean production in the midsouthern USA (Mississippi, Arkansas, and Louisiana) [10,11,38–40]. The role(s) of toxins in root infection is investigated in these studies by assessing the production of (-)-botryodiplodin, phaseolinone, and other toxins in cell-free culture filtrates of charcoal rot disease-causing *M. phaseolina* isolates and in roots and other tissues from soybean plants naturally infected with charcoal rot disease in the field. Studies on the culture filtrates of charcoal rot disease-causing *M. phaseolina* isolates resulted in the discovery that some, but not all, isolates produce (-)-botryodiplodin, but not phaseolinone, and some isolates that do not produce (-)-botryodiplodin do produce another as yet unknown toxin(s). Studies on toxins present in soybean plant tissues provided the first demonstration of a mycotoxin known to be produced by *M. phaseolina* in soybean plant tissues naturally infected with charcoal rot disease, specifically (-)-botryodiplodin, but not phaseolinone.

#### **2. Results and Discussion**

#### *2.1. Toxin Production in Culture by M. Phaseolina Isolates from Plants with Charcoal Rot Disease*

Toxin production in culture by *M. phaseolina* isolates from many USA sites and numerous types of plant sources were examined as the toxicity of cell-free culture medium filtrates in soybean leaf disc cultures from two soybean genotypes, DS97-84-1 and DT97-4290 (Table 1). Toxicity assessments with the two genotypes exhibited a similar rank order with no substantive difference between the two, whether assessed at 50% strength or at full strength. The same cell-free culture filtrates from *M. phaseolina* isolates were also assayed by LC/MS for levels of (-)-botryodiplodin, the toxin previously [35] found associated with culture filtrates of a *M. phaseolina* isolate from a soybean plant in Mississippi with charcoal rot disease. Observed concentrations of (-)-botryodiplodin ranged from not detectable to 6.11 μg/mL (Table 1). The majority of isolates (51.4% of isolates studied) produced quantifiable levels of (-)-botryodiplodin in culture filtrates, while 37.8% of isolates studied produced trace levels (i.e., above the limit of detection (1 × 10−5 ng/μL), but less than the limit of quantitation (1 × 10−2 ng/μL), and 10.8% of isolates studied produced no detectable level of (-)-botryodiplodin in culture filtrates.

 *Macrophomina phaseolina, Mp*; Mississippi, MS; Kentucky, KY; Arkansas, AR; Louisiana, LA; Dakota, SD; Tennessee, TN; Texas, TX; Dakota, ND; Minnesota,MN; Oklahoma, OK; Kansas, KS; Nebraska, NE; North Carolina, NC; Michigan, MI; Florida, FL; Arizona, AZ; Georgia, GA; light, l; dark, d. a Toxicity score measured in soybean leaf disccultures of two soybean genotypes: (i) DT97-4290, which is moderately resistant to charcoal rot disease and (ii) DS97-84-1, which is susceptible to charcoal rot disease. Toxicity wasassessed qualitatively according to the following symptom rating scale: healthy tissue < a little browning around the edges of the leaf disc, + < moderate browning around the edgesthe leaf disc, ++ < browning of the whole leaf disc, +++ < browning of the leaf disc with some photobleaching, ++++ < photobleaching of the whole leaf disc, +++++. b Color densitywas assessed qualitatively according to the following color density scale: whitish < light yellow < light tan < light grey < tan < beige or amber < dark tan < dark brown or dark grey black. c (-)-Botryodiplodin concentrations in culture medium filtrates were measured quantitatively by LC/MS.

 of <

Whether *M. phaseolina* isolates were from trees, soybeans, melons, or other plant sources, cell-free culture filtrates were toxic in soybean leaf disc cultures, and toxicity levels varied from not detectable to very toxic (Table 1). Culture filtrates from *M. phaseolina* isolates that contained high levels of (-)-botryodiplodin (>1 μg/mL) were all very toxic in soybean leaf disc cultures, resulting in maximal or near maximal toxicity with both DT97-4290 and DS97-84-1 soybean leaf discs at 100% and 50% strength. Culture filtrates that contained intermediate levels of (-)-botryodiplodin (0.2–1.0 μg/mL) were moderately toxic in soybean leaf disc cultures. However, some other *M. phaseolina* isolate culture filtrates that contained only trace levels or even no detectable (-)-botryodiplodin were highly toxic in soybean leaf disc cultures. This observation is consistent with some disease-inducing isolates of *M. phaseolina* producing one or more toxins other than (-)-botryodiplodin. This is the first report of results supporting the hypothesis that different isolates of *M. phaseolina* may use different toxins to facilitate root infection in soybeans. Further studies are needed to determine if any of those isolates use the other toxin(s) to facilitate root infection by a mechanism analogous to the one by which (-)-botryodiplodin might facilitate root infection. Some culture filtrates from the disease-inducing isolates of *M. phaseolina* contained very little toxicity in soybean leaf disc cultures, despite the isolate being able to cause charcoal rot disease. Explanations for this observation include the possible presence of a toxin-production regulatory mechanism that suppresses toxin production by those isolates under the culture conditions used in this study, or the possibility that charcoal rot disease in soybeans may be caused by a seed-borne *M. phaseolina* endophyte that would not need a root infection mechanism or any toxins associated with it [14]. The *M. phaseolina* isolate from which phaseolinone was originally isolated [34,37] was a seed-borne endophyte.

Also included in Table 1 is an assessment of the color of week-old cultures of *M. phaseolina*. Dunlap and Bruton [41] reported that a *M. phaseolina* isolate formed pigment in an infected muskmelon (*Cucumis melo*) and in liquid culture media containing glycine and some other amino acids. Some *M. phaseolina* isolates that cause charcoal rot disease in soybeans have been observed to form large numbers of black microschlerotia under the same culture conditions that induce the production of (-)-botryodiplodin [42]. In the data in Table 1, the rank order of pigment production, as assessed qualitatively according to the color density scale used, differed substantially from the rank order of toxicity in cell-free culture filtrates as assessed in soybean leaf disc cultures at either full strength or 50% dilution and from the relative amount of (-)-botryodiplodin present as measured by LC/MS. Thus, pigment production as assessed in this study appeared to be unrelated to toxin production, consistent with the previously identified correlations not being a general phenomenon when larger numbers of *M. phaseolina* isolates are examined.

#### *2.2. Analysis of Toxin Levels in Tissue Samples from Soybean Plants Naturally Infected with Charcoal Rot Disease*

If the hyphae of a *M. phaseolina* strain that causes charcoal rot disease use a toxin(s) to create a necrotic area in the root and thereby facilitate entry into soybean plant roots from a soil reservoir, those hyphae are expected to produce a toxin(s) at least from the time the fungus detects the root in the soil until the fungal hyphae inside the plant have detected that a stable infection has been established there. Fungi that spread from plant to plant through interdigitating roots, as *M. phaseolina* does in the charcoal rot disease of soybeans, may also secrete a toxin(s) inside the roots of fully infected plants in order to create a necrotic area within the root from which hyphae may exit the plant to spread to adjacent plants. Thus, soybean plants exhibiting the symptoms of charcoal rot disease may contain a chemically and metabolically stable toxin in tissues at a level detectable by standard analytical methods such as LC/MS. If the *M. phaseolina* strain causing charcoal rot disease in a soybean plant is a constitutive (continuous) producer of the toxin, comparable levels of the toxin may be expected in all the affected tissues of diseased plants. Therefore, naturally infected soybean plants exhibiting symptoms of charcoal rot disease were collected from different infected areas in commercial soybean production fields in Mississippi in the 2004 growing season. Control soybean plants not exhibiting symptoms of charcoal rot disease were also collected. Samples of roots, leaves, stem pulp, branches, twigs, and seeds were individually extracted and analyzed by LC/MS for levels of (-)-botryodiplodin, phaseolinone, phomenone, and gigantenone (Table 2). Only (-)-botryodiplodin was detected and only in the roots of soybean plants exhibiting symptoms of charcoal rot disease, not in other tissues of diseased plants and not in the roots or any other tissues of control soybean plants not exhibiting symptoms of charcoal rot disease. This is the first report of a toxin being found in infected plant tissues associated with charcoal rot disease in soybeans. This observation would be expected if *M. phaseolina* used (-)-botryodiplodin in its mechanism for (i) initial root infection and (ii) to exit heavily infected plants in order to spread to and infect adjacent plants. However, additional studies are needed to establish a role for (-)-botryodiplodin in either the initial root infection or the root exit mechanism. No phaseolinone, phomenone, or gigantenone was found in any tissue of soybean plants with charcoal rot disease in this study. As shown in Table 3, these observations are confirmed by a similar study conducted in 2007, in which root tissue was collected from naturally infected soybean plants from commercial production fields in Mississippi and Kentucky, USA, and analyzed by LC/MS for levels of (-)-botryodiplodin, phaseolinone, phomenone, and gigantenone. As observed in the first study (Table 2), only (-)-botryodiplodin was detected in diseased roots, not phaseolinone, phomenone, or gigantenone. Again, (-)-botryodiplodin levels varied from traces to 3.14 μg/g, that is, greater than a 1000-fold concentration range. The wide range of (-)-botryodiplodin levels in charcoal rot-diseased soybean roots (Tables 2 and 3) paralleled the wide range of (-)-botryodiplodin production levels in cell-free culture filtrates of *M. phaseolina* isolates from plants with charcoal rot disease. In both experimental systems, there were a substantial number of cases in which (-)-botryodiplodin production was too low for it to be a toxin that could play a role in the pathology caused by those *M. phaseolina* strains, whether by facilitating root infection or any other mechanism.

**Table 2.** Mycotoxin levels in root and other tissues of soybean plants collected from soybean fields in Mississippi in 2004.

aIdentification and quantification of toxins in samples by LC/MS were based on one standard due to the limited amount of standards available. b Soybean plants of the Saline cultivar with no detectable sign of charcoal rot disease were collected from commercial fields in Mississippi. Samples of the same six tissues were taken, pooled, and extracted in the same way as tissues from diseased plants and the extracts were assayed by LC/MS in the same manner. Extracts of all undiseased soybean tissues, including roots, contained no detectable (-)-botryodiplodin or other toxin.

**Table 3.** Toxins in the roots of soybean plants exhibiting charcoal rot disease properties collected from commercial soybean fields in Kentucky and Mississippi in 2007.

\* Soybean plants exhibiting symptoms of charcoal rot disease were collected in the indicated commercial field numbers in the indicated states, brought to the laboratory, tissues harvested and stored at −20 ◦C until assayed. Soybean root samples had symptoms of charcoal rot and were run by LC/MS. Determination and quantification of these mycotoxins was by LC/MS based on one standard because a limited amount of these standards were available.

#### **3. Conclusions**

A wide range of (-)-botryodiplodin levels were observed in both cell-free culture medium filtrates from *M. phaseolina* isolates from plants with charcoal rot disease and in the roots, but not in the aerial parts, of soybean plants naturally infected with charcoal rot disease. Cell-free culture medium filtrates from some *M. phaseolina* isolates from plants with charcoal rot disease were strongly phytotoxic, despite containing only traces or no (-)-botryodiplodin. No phaseolinone was detected in either cell-free culture medium filtrates from *M. phaseolina* isolates or in tissues from soybean plants naturally infected with charcoal rot disease. The results of this study are consistent with some, but not all, isolates of *M. phaseolina* associated with charcoal rot disease in soybean-producing (-)-botryodiplodin. Some isolates of *M. phaseolina* cultured from soybean plants with charcoal rot disease produce no detectable (-)-botryodiplodin in culture, but do produce other unknown toxins. Further research is needed to determine what role, if any, (-)-botryodiplodin and other toxins produced by *M. phaseolina* isolates play in the root infection mechanism of the charcoal rot disease of soybeans.

#### **4. Materials and Methods**

#### *4.1. Soybean Plant and Greenhouse Conditions*

The soybean genotype DT97-4290 [28] was selected as an example of a genotype that is moderately resistant to charcoal rot disease, and the soybean genotype DS97-84-1 [43] was selected as an example of a genotype that is susceptible to charcoal rot disease. Plants were germinated in trays with vermiculite, and the seedlings of each genotype were transplanted into six soil-filled 9.45 L pots, each containing four plants of the same genotype. During the growth period, the soil water potential of the plants was maintained at approximate field conditions, 15 to −20 kPa. Six pots, each containing four plants, were used for each genotype. The greenhouse temperature was set to 34 ◦C for the day cycle and 28 ◦C for the night cycle. Light intensity ranged from that of sunny to cloudy days. Plants were harvested during the vegetative stage.

#### *4.2. M. phaseolina Culture Sources*

The collection locations and plant hosts of the 37 cultures of *M. phaseolina* used in the study are presented in Table 1. Some *M. phaseolina* cultures were isolated from infected plant tissues in the Abbas laboratory in 2013 using the method of Mengistu et al. [4,25], while other cultures were provided by colleagues from their collections, particularly G.L. Sciumbato, Mississippi State University.

#### *4.3. Preparation of Cell-Free Culture Extracts*

Potato dextrose broth (PDB) was prepared by boiling 200 g of peeled potatoes, straining them through a cheesecloth, and adding 20 g of dextrose per liter of water. PDB (150 mL) was placed in 500 mL Erlenmeyer flasks, covered with cotton plugs, autoclaved for 15 min, and allowed to cool to room temperature. Upon cooling, each flask was inoculated with three to four plugs of *M. phaseolina* isolate and placed on an Innova 40 Benchtop Incubator Shaker (New Brunswick Scientific Co., Inc., Edison, NY, USA) for seven days at 128 rpm, 28 ◦C. The color change of each culture after one week of incubation was observed and recorded according to the following color density scale: whitish < light yellow < light tan < light grey < tan < beige or amber < dark tan < dark brown or dark grey < black.

After seven days of incubation, the culture medium was passed through Whatman No.1 filter paper into a plastic beaker. The filtrate was then filtered through an 0.45 μm membrane filter in a disposable filter unit (Nalgene Company, Rochester, NY, USA, Size 250 mL cellulose nitrate CN Filter Unit) using a laboratory vacuum to achieve a cell-free filtrate that was stored at −20 ◦C until used.

#### *4.4. Toxicity of Cell-Free Filtrates of M. phaseolina Culture Media in Soybean Leaf Disc Cultures*

The toxicity of *M. phaseolina* culture filtrates was assessed by rating the appearance of soybean leaf discs from two genotypes (DT97-4290, which is moderately resistant to charcoal rot, and DS97-84-1, which is susceptible) after four to five days in half (50%) and full strength (100%) cell-free culture filtrates, *M. phaseolina* isolates were grown on potato dextrose agar (PDA) for seven days at 28 ◦C. True mature leaves with no signs of damage were harvested from 3- to 4-week-old soybean plants, and 4 mm discs were cut from the leaves using a sterile cork borer (No.4). Three leaf discs were placed in each well of sterile 24-well tissue culture trays with low evaporative lids (Becton Dickinson and Company, Franklin Lakes, NJ, USA) containing 1.5 mL of culture filtrate in triplicate at two concentrations (50% and 100%). The trays were then incubated in a growth chamber at 25 ◦C under continuous light for 96 h. The discs were observed for signs of toxic effects after 24, 48, 72, and 96 h. Toxicity was assessed qualitatively according to the following symptom rating scale: healthy tissue < a little browning around the edges of the leaf disc, + < moderate browning around the edges of the leaf disc, ++ < browning of the whole leaf disc, +++ < browning of the leaf disc with some photobleaching, ++++ < photobleaching of the whole leaf disc, +++++.

#### *4.5. Toxin Standards for LC*/*MS Analyses*

The structures of toxins measured in this study are presented in Figure 1. (±)-Botryodiplodin was synthesized, as described in the accompanying manuscript [44], as a white powder with purity over 98%. A stock solution of (±)-botryodiplodin (1000 ng/μL) was prepared in chloroform. Working standards were prepared in the concentration range 1.0 × 10−5 ng/μL to 40 ng/μL in ethyl acetate. Gigantenone and phomenone were gifts from Gary A. Strobel, Montana State University, Bozeman, MT. Phaseolinone was synthesized (Figure 2) from a sample of the phomenone (6.5 mg, 0.0246 mmole) dissolved in 1 mL chloroform and mixed with a 1.2 molar excess of m-chloroperoxybenzoic acid (Acros Organics, 0.029 mmole, 7.15 mg of 70% pure material) and pyridine (4.7 μL, 4.6 mg, 0.058 mmole) dissolved in 200 μL chloroform. The mixture was incubated for 1 h at −10 ◦C with stirring and then allowed to warm to room temperature overnight. The reaction mixture was diluted with ether, extracted twice with water, once with 1N HCl to remove pyridine, twice with saturated sodium bicarbonate-brine solution to remove product m-chlorobenzoic acid and unreacted m-chloroperoxybenzoic acid, dried over anhydrous sodium sulfate, and evaporated in vacuo. The product (7.5 mg) gave a single peak at *m*/*e* 281 (phaseolinone + H+) in LC/MS analysis under the conditions described below, with no detectable phomenone starting material at *m*/*e* 265. A single peak was observed in LC/MS for the phaseolinone preparation, even though the reaction conditions would be expected to produce a mixture of phaseolinone and epi-phaseolinone, presumably because the two forms were not resolved under the liquid chromatography conditions used.

**Figure 1.** Chemical structures of the toxins measured by LC/MS in *M. phaseolina* culture media and soybean root tissues.

**Figure 2.** The chemical reaction used in the semi-synthesis of the LC/MS standard phaseolinone from the natural toxin phomenone. MCPBA = meta-chloroperoxybenzoic acid.

#### *4.6. Preparation of Plant Tissue and M. phaseolina Culture Medium Extracts for LC*/*MS Analyses*

Soybean root and other tissue samples were cleaned of adherent earth, dried in an oven at 45 ◦C for two to three days, and ground to the consistency of flour using a Stain Laboratory Mill Grinder, Model M-2 (Fred Stein Laboratories, INC., Atchison, Kansas, USA). Ethyl acetate (10 g) was added to 50 g of each sample, shaken for 1 h, filtered through filter paper (Whatman No.1), and transferred to vials for analysis by LC/MS as described below. *M. phaseolina* culture medium cell-free filtrate samples were extracted with ethyl acetate in a 1:1, *v*:*v* ratio on a vortex mixer for 1 min and allowed to separate into two distinct layers. The ethyl acetate layer was transferred to vials for analysis by LC/MS.

#### *4.7. LC*/*MS Analysis*

LC/MS analyses of toxin samples obtained prior to 2007 were conducted on a Thermo Finnigan LCQ Advantage instrument coupled to a Thermo Finnigan Surveyor MS and a Thermo Finnigan Surveyor MS Pump (Thermo Electron Corporation, West Palm Beach, FL, USA). After 2007, a more advanced and upgraded LTQ XL Ion Trap Mass Spectrometer, Finnigan Surveyor Autosampler, and Finnigan Surveyor MS Pump (Thermo Scientific, West Palm Beach, FL, USA) were used. Analyses were carried out in positive scan mode at ambient temperature using a Waters Nova-Pak C18 column, a 10 μL partial loop injection, and mobile phases (A) 1% acetic acid in methanol, (B) water, and (C) methanol at a flow rate of 500 μL/min. The analysis occurred over 25 min using a gradient of 20% A and 80% B for 12 min, then 20% A, 5% B, and 75% C for 3 min, and then back to 20% A and 80% B for the duration of the 25 min. The analysis utilized the following scan events of a full scan from *m*/*e* 100 to 300. The confirmation of (-)-botryodiplodin used three masses: *m*/*e* 127, 145, and 109. The limit of

detection (LOD) was 1 × 10−5 μg/mL and the limit of quantitation (LOQ) was 0.01 μg/mL. The LOQ was based on the regression of the standards used for analysis. The full scan run of phomenone, gigantenone, and phaseolinone was from *m*/*e* 100 to 500, and their confirmations were identified by using *m*/*e* 265, 265, and 281, respectively.

#### *4.8. Statistical Analysis*

The analysis of variance was performed on toxin concentration data using the PROC GLM procedure in SAS Version 9.22 (Cary, NC, USA, 2010). Means were separated by Fisher's Least Significant Difference test with *p* ≤ 0.05 level of significance.

**Author Contributions:** Conceptualization, H.K.A., N.B., C.A., J.R.S., W.T.S.; methodology, H.K.A., W.T.S.; validation, H.K.A.; investigation, H.K.A., W.T.S.; resources, H.K.A., W.T.S.; data curation, H.K.A., W.T.S.; writing—original draft preparation, H.K.A., W.T.S.; writing—review and editing, H.K.A., N.B., C.A., J.R.S., W.T.S.; visualization, W.T.S.; supervision, H.K.A.; project administration, H.K.A.; funding acquisition, H.K.A., W.T.S.

**Funding:** This research was funded by the Mississippi Soybean Promotion Board, grant numbers 34-2016 and 34-2017.

**Acknowledgments:** The authors are grateful to Alemah Butler, Bobbie J. Johnson (retired), Jeremy Kotowicz, and Vivek H. Khambhati for their technical assistance. Also, the authors are grateful for G.L. Sciumbato (retired) for supplying cultures and plant samples. Trade names are used in this publication solely for the purpose of providing specific information. The mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the United States Department of Agriculture.

**Conflicts of Interest:** All authors declare no conflict of interest.

#### **References**

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

#### *Article*

## **Phytotoxic Responses of Soybean (***Glycine max* **L.) to Botryodiplodin, a Toxin Produced by the Charcoal Rot Disease Fungus,** *Macrophomina phaseolina*

**Hamed K. Abbas 1,\*, Nacer Bellaloui 2, Alemah M. Butler 1, Justin L. Nelson 3, Mohamed Abou-Karam 3 and W. Thomas Shier 3,\***

Received: 13 September 2019; Accepted: 18 December 2019; Published: 1 January 2020

**Abstract:** Toxins have been proposed to facilitate fungal root infection by creating regions of readily-penetrated necrotic tissue when applied externally to intact roots. Isolates of the charcoal rot disease fungus, *Macrophomina phaseolina*, from soybean plants in Mississippi produced a phytotoxic toxin, (−)-botryodiplodin, but no detectable phaseolinone, a toxin previously proposed to play a role in the root infection mechanism. This study was undertaken to determine if (−)-botryodiplodin induces toxic responses of the types that could facilitate root infection. (±)-Botryodiplodin prepared by chemical synthesis caused phytotoxic effects identical to those observed with (−)-botryodiplodin preparations from *M. phaseolina* culture filtrates, consistent with fungus-induced phytotoxicity being due to (−)-botryodiplodin, not phaseolinone or other unknown impurities. Soybean leaf disc cultures of Saline cultivar were more susceptible to (±)-botryodiplodin phytotoxicity than were cultures of two charcoal rot-resistant genotypes, DS97-84-1 and DT97-4290. (±)-Botryodiplodin caused similar phytotoxicity in actively growing duckweed (*Lemna pausicostata*) plantlet cultures, but at much lower concentrations. In soybean seedlings growing in hydroponic culture, (±)-botryodiplodin added to culture medium inhibited lateral and tap root growth, and caused loss of root caps and normal root tip cellular structure. Thus, botryodiplodin applied externally to undisturbed soybean roots induced phytotoxic responses of types expected to facilitate fungal root infection.

**Keywords:** botryodiplodin; root infection mechanism; root toxicity; *Macrophomina phaseolina*; hydroponic culture

**Key Contribution:** Botryodiplodin was observed to be phytotoxic in cultured leaf discs from soybean genotypes susceptible or resistant to charcoal rot disease, but the phytotoxic response was greatest in susceptible genotypes. Botryodiplodin was shown to be phytotoxic when applied externally to intact *Lemna pausicostata* plantlets. Botryodiplodin treatment of undisturbed soybean seedling roots in hydroponic culture resulted in loss of root tips, creating a lesion of a type that may facilitate root infection.

#### **1. Introduction**

Charcoal rot is a plant disease caused by the fungus, *Macrophomina phaseolina* (Tassi) Goid [1], in over 500 commercially-important plant species ranging from ornamental plants to trees to major food

and fiber crops, including soybean (*Glycine max* L. (Merr.)). An example of the impact of charcoal rot disease on agriculture was provided by attempts to establish commercial natural rubber production with guayule (*Parthenium argentatum* Gray) in the arid southwest region of the US as an alternative to imported material from the rubber tree (*Hevea brasiliensis*) [2]. Guayule rubber production was only competitive when plants were grown close enough together that the roots interdigitated, under which conditions charcoal rot could spread from plant to plant destroying the crop [3]. Because charcoal rot is favored by hot, dry conditions [4], it is a climate-impacted plant disease that is predicted to be an increasingly important agronomic problem going forward, given that climate change is predicted to result in hotter, drier conditions in the majority of the world [5].