Journal of Fungi Brief Report First Report of the Production of Mycotoxins and Other Secondary Metabolites by Macrophomina phaseolina (Tassi) Goid. Isolates from Soybeans (Glycine max L.) Symptomatic with Charcoal Rot Disease Vivek H. Khambhati 1, Hamed K. Abbas 2,* , Michael Sulyok 3 , Maria Tomaso-Peterson 1 and W. Thomas Shier 4 1 Department of Biochemistry, Molecular Biology, Entomology, and Plant Pathology, Mississippi State University, Mississippi State, MS 39762, USA; [email protected] (V.H.K.); [email protected] (M.T.-P.) 2 Biological Control of Pests Research Unit, US Department of Agriculture, Agricultural Research Service, Stoneville, MS 38776, USA 3 Institute of Bioanalytics and Agro-Metabolomics, Department of Agrobiotechnology, IFA-Tulln, University of Natural Resources and Life Sciences, Vienna (BOKU), Konrad-Lorenz-Str. 20, Tulln 3430, Austria; [email protected] 4 Department of Medicinal Chemistry, College of Pharmacy, University of Minnesota, Minneapolis, MN 55455, USA; [email protected] * Correspondence: [email protected]; Tel.: +1-662-686-5313 Received: 15 October 2020; Accepted: 1 December 2020; Published: 3 December 2020 Abstract: Macrophomina phaseolina (Tassi) Goid., the causal agent of charcoal rot disease of soybean, is capable of causing disease in more than 500 other commercially important plants. This fungus produces several secondary metabolites in culture, including (-)-botryodiplodin, phaseolinone and mellein. Given that independent fungal isolates may differ in mycotoxin and secondary metabolite production, we examined a collection of 89 independent M. phaseolina isolates from soybean plants with charcoal rot disease using LC-MS/MS analysis of culture filtrates. In addition to (-)-botryodiplodin and mellein, four previously unreported metabolites were observed in >19% of cultures, including kojic acid (84.3% of cultures at 0.57–79.9 µg/L), moniliformin (61.8% of cultures at 0.011–12.9 µg/L), orsellinic acid (49.4% of cultures at 5.71–1960 µg/L) and cyclo[L-proline-L-tyrosine] (19.1% of cultures at 0.012–0.082 µg/L). In addition, nine previously unreported metabolites were observed at a substantially lower frequency (<5% of cultures), including cordycepin, emodin, endocrocin, citrinin, gliocladic acid, infectopyron, methylorsellinic acid, monocerin and N-benzoyl-L-phenylalanine. Further studies are needed to investigate the possible effects of these mycotoxins and metabolites on pathogenesis by M. phaseolina and on food and feed safety, if any of them contaminate the seeds of infected soybean plants. Keywords: moniliformin; kojic acid; mellein; orsellinic acid; cyclo[L-proline-L-tyrosine]; cordycepin; phaseolinone; botryodiplodin; natural products; phytotoxins; secondary metabolites; fungi 1. Introduction Soybean (Glycine max L.) is an economically valuable crop due to its high protein and oil content and wide variety of uses in food, agricultural pharmaceutical and industrial applications [1]. The United States produces 32.5% of the world’s soybeans (120 million tons/year valued at $31.2 billion), making J. Fungi 2020, 6, 332; doi:10.3390/jof6040332 www.mdpi.com/journal/jof J. Fungi 2020, 6, x FOR PEER REVIEW 2 of 10 J. Fungi 2020, 6, 332 2 of 10 Soybean (Glycine max L.) is an economically valuable crop due to its high protein and oil content and wide variety of uses in food, agricultural pharmaceutical and industrial applications [1]. The it the second most valuable US crop [2]. In the southern US, high temperatures and dry conditions, United States produces 32.5% of the world’s soybeans (120 million tons/year valued at $31.2 billion), alongmaking with it poorthe second management, most valuable can lead US to crop drought-stressed [2]. In the southern plants US, that high are temperatures susceptible to and infection dry byconditions, opportunistic along soil- with and poor seed-borne management, pathogens, can lead such to drought-stressed as Fusarium virguliforme plants thatO’Donnell are susceptible & T. Aoki, to Cercosporainfection sojinaby opportunisticHara, Phomopsis soil- and longicolla seed-borneT.W. pathogens, Hobbs and suchMacrophomina as Fusarium virguliforme phaseolina (Tassi) O’Donnell Goid., resulting& T. Aoki, in significant Cercospora economicsojina Hara, impact Phomopsis due tolongicolla yield loss T.W. and Hobbs reduced and seedMacrophomina quality [3 phaseolina–10]. M. phaseolina(Tassi) infectsGoid., over resulting 500 species in significant of commercially economic impact important due to plants, yield loss causing and redu variousced seed dry-weather quality [3–10]. wilts M. and rots,phaseolina including infects charcoal over rot500 disease species in of soybean commercially [6–10]. important In many years,plants, charcoal causing rotvarious disease dry-weather is the major causewilts of and soybean rots, including crop yield charcoal loss and rot seed disease quality in soybean deterioration [6–10]. in In the many southern years, UScharcoal states rot of Arkansas,disease Louisianais the major and cause Mississippi of soybean [11– 13crop], although yield loss soybean and seed cyst quality nematode deterioration is the major in the cause southern of crop US states damage inof most Arkansas, parts of Louisiana the country. and ItMississippi is not understood [11–13], whatalthough factors soybean specific cyst to nematode the region is arethe responsiblemajor cause for theof relatively crop damage greater in most pathogenicity parts of the of country.M. phaseolina It is notthere understood [14]. what factors specific to the region areOne responsible plausible for explanation the relatively for greater the greater pathogenicity frequency of M. of phaseolina charcoal there rot disease [14]. in soybeans in this regionOne is that plausibleM. phaseolina explanationisolates for the endemic greater to frequency the region of charcoal produce rot unusual disease levels in soybeans or diversity in this of mycotoxins.region is that Therefore, M. phaseolina as an isolates approach endemic to exploring to the region the possible produce role unusual of mycotoxin levels or productiondiversity of in themycotoxins. increased pathogenicityTherefore, as an of approachM. phaseolina to explorin, theg present the possible study role was of undertakenmycotoxin production to investigate in the the increased pathogenicity of M. phaseolina, the present study was undertaken to investigate the diversity of known mycotoxins and other secondary metabolites produced in culture by isolates from diversity of known mycotoxins and other secondary metabolites produced in culture by isolates from regional soybean plants symptomatic for charcoal rot using liquid chromatography tandem mass regional soybean plants symptomatic for charcoal rot using liquid chromatography tandem mass spectrometry (LC-MS/MS) with culture medium filtrates. M. phaseolina exhibits extensive genetic spectrometry (LC-MS/MS) with culture medium filtrates. M. phaseolina exhibits extensive genetic diversity as assessed by DNA markers and differences in pathogenicity [15–18]. The genetic diversity diversity as assessed by DNA markers and differences in pathogenicity [15–18]. The genetic diversity M. phaseolina of of M. phaseolinamay may extend extend to to mycotoxins, mycotoxins, given that (-)-b (-)-botryodiplodinotryodiplodin (1) (1) and and not not phaseolinone phaseolinone (3) (3) waswas found found [19 [19–21]–21] in in soybeans soybeans with with charcoal charcoal rot rot (Figure (Figure1), 1), whereas whereas phaseolinone phaseolinone (3) (3) (Figure (Figure1) 1) but but not (-)-botryodiplodinnot (-)-botryodiplodin was isolatedwas isolated from from the culture the culture medium medium of an ofM. an phaseolina M. phaseolinaendophyte endophyte from from mung beanmung [22 ].bean Similarly, [22]. Similarly, mellein (Figuremellein1 (Figure) has been 1) has observed been observed in some butin some not allbut isolates not all ofisolatesM. phaseolina of M. fromphaseolina soybean from plants soybean symptomatic plants symptomatic of charcoal of rot charcoal in Mississippi, rot in Mississippi, U.S.A. [23 U.S.A.]. [23]. FigureFigure 1. 1. StructuresStructures of some some previous previouslyly reported reported toxins toxins from from MacrophominaMacrophomina phaseolina phaseolina cultures,cultures, (-)- (-)-botryodiplodinbotryodiplodin (1 (),1 ),mellein mellein (2 ()2 and) and phaseolinone phaseolinone (3). ( 3). 2.2. Materials Materials and and Methods Methods 2.1.2.1. M. M. phaseolina phaseolina Culture Culture Collection Collection andand SourcesSources ForFor this this study, study, 89 isolates89 isolates of M. of phaseolina M. phaseolinawere obtainedwere obtained from soybean from soybean plants exhibiting plants exhibiting symptoms ofsymptoms charcoal rot of diseasecharcoal inrot various disease fieldsin various in Mississippi fields in Mississippi and the surroundingand the surrounding areas using areas theusing method the describedmethod bydescribed Mengistu by etMengistu al. [9], or et they al. [9], were or providedthey were by provided other investigators by other investigators in the same in areas the same [20,21 ] (seeareas Figure [20,21]2). Cultures(see Figure were 2). Cultures maintained were on mainta potatoined dextrose on potato agar dextrose (PDA) untilagar use.(PDA) until use. 2.2. Preparation of Cell-Free Culture Extracts M. phaseolina isolates were cultured in Czapek-Dox broth (CDB) prepared by dissolving 2.0 g NaNO3, 1.0 g K2HPO4, 0.5 g KCl, 0.5 g MgSO4, 0.01 g FeSO4 and 30 g sucrose in 1 L water. Aliquots