fungal biology 115 (2011) 133e142

journal homepage: www.elsevier.com/locate/funbio

Bioactive metabolites from , a stalk and ear rot pathogen of

Donald T. WICKLOWa,*, Kristina D. ROGERSc, Patrick F. DOWDb, James B. GLOERc aBacterial Food Pathogens and Mycology, National Center for Agricultural Utilization Research, Agricultural Research Service, U.S. Department of Agriculture, Peoria, IL 61604, USA bCrop Bioprotection Research Units, National Center for Agricultural Utilization Research, Agricultural Research Service, U.S. Department of Agriculture, Peoria, IL 61604, USA cDepartment of Chemistry, University of Iowa, Iowa City, IA 52242, USA article info abstract

Article history: Stenocarpella maydis is a fungal pathogen of major importance that causes a dry-rot of Received 30 September 2010 maize ears and is associated with a neuromycotoxicosis in cattle grazing harvested maize Received in revised form fields in southern Africa and Argentina. In an effort to investigate the potential roles of 22 November 2010 S. maydis metabolites in the fungal disease cycle, ethyl acetate extracts of solid-substrate Accepted 23 November 2010 fermentations of several S. maydis isolates from maize grown in the United States were Available online 30 November 2010 found to exhibit significant phytotoxic, antifungal, and antiinsectan activity. Chemical in- Corresponding Editor: Marc Stadler vestigations of extracts of S. maydis isolates from Illinois and Nebraska led to the isolation or detection of the known metabolites diplodiatoxin, chaetoglobosins K and L, and (all-E)- Keywords: trideca-4,6,10,12-tetraene-2,8-diol as major components. A culture of Stenocarpella macro- (all-E )-trideca-4,6,10,12-tetraene- spora from maize grown in Zambia produced diplosporin and chaetoglobosins K and L as 2,8-diol major components that were isolated. Diplodiatoxin produced significant lesions in a maize Antiinsectan leaf puncture wound assay. Diplosporin and chaetoglobosin K displayed moderate antiin- Chaetoglobosin K sectan activity in dietary assays against the fall armyworm Spodoptera frugiperda, while Diplodiatoxin chaetoglobosin K exhibited significant antifungal activity against Aspergillus flavus and Diplodiosis verticillioides. Using LC-ESIMS and 1H NMR data, diplodiatoxin was detected as a major component in S. maydis-rotted grain, stalks, and stalk residues. This constitutes the first report of chaetoglobosins K and L from S. maydis, of (all-E )-trideca-4,6,10,12-tet- raene-2,8-diol from Stenocarpella, and the first reported detection of diplodiatoxin, or any other Stenocarpella metabolite, in diseased maize seeds and stalk tissues. Published by Elsevier Ltd on behalf of The British Mycological Society.

Introduction distribution and Stenocarpella is recognized as the most impor- tant ear rot pathogen in nearly all countries where maize is The genus Stenocarpella Syd. & P. Syd. [teleomorph unknown; produced (Rossouw et al. 2009). While S. maydis is reported (Crous et al. 2006)] contains two species that from humid zones wherever corn is grown, S. macrospora is cause a dry-rot of maize ears, Stenocarpella macrospora (Earle) most prevalent in humid subtropical and tropical zones where B. Sutton (Basionym: macrospora), and Stenocarpella plants exhibiting dry-ear rot and stalk rot may also display maydis (Berk.) B. Sutton (Basionym: Diplodia maydis). Maize symptoms of leaf striping (Latterell & Rossi 1983). Severe diseases caused by Stenocarpella are world-wide in their crop infestation can result in significant yield losses from

* Corresponding author. Tel.: þ1 309 681 6243. E-mail address: [email protected] 1878-6146/$ e see front matter Published by Elsevier Ltd on behalf of The British Mycological Society. doi:10.1016/j.funbio.2010.11.003 134 D. T. Wicklow et al.

the presence of light-weight, rotted ears with discolored, Service Culture Collection, Peoria (NRRL) and included S. maydis shriveled, and non-viable seeds. NRRL 43670, NRRL 52415, and NRRL 53563, from white maize Stenocarpella maydis ear rot is of further importance be- seeds, Cerro Gordo, Illinois; NRRL 53565, NRRL 53566, and cause of its association with authenticated field outbreaks of NRRL 53567 from maize seeds, Kilbourne, Illinois; NRRL 31249, diplodiosis, a common nervous disorder (neuromycotoxicosis) from maize seed, Indianapolis, Indiana; NRRL 53560 from white of cattle and sheep grazing on infected maize crop residue in maize seed, Rochester, Indiana; NRRL 13608 (¼ATCC 10235), southern Africa (Kellerman et al. 1991) and more recently in from maize, Kentucky; NRRL 53564, from white maize seed, Argentina (Odriozola et al. 2005). Diplodiatoxin was isolated Hopkinsville, Kentucky; NRRL 53561, and NRRL 53562 from and characterized from S. maydis-fermented maize seed cul- white maize seeds, Crete, Nebraska; NRRL 13609 (¼ATCC ture material in bioassay-guided chemical studies of the 16438), from maize, unreported, R.B. Stevens; NRRL 13615, chloroformemethanol extract which accounted for ca. 10 % from maize seed, unreported, USA S. macrospora NRRL 13610 of the total activity of the maize culture material (Steyn et al. (¼ATCC 36896) from maize, Costa Rica; NRRL 13611 (¼ATCC 1972) based on its toxicity to poultry (reported by Rabie et al. 42808; MRC 143b) from maize, Zambia, and NRRL 13612 (¼ATCC 1985). No further chemistry has been described for S. maydis. 18606) from maize, Georgia, USA. Each of our isolates from Stenocarpella macrospora was recorded as a prevalent fungal maize was cultured to produce pycnidia and the spores were colonist of maize seeds from Zambia, and S. macrospora-fer- examined microscopically and measured to confirm that these mented sterile maize seed culture material, added to either isolates were S. maydis. The cultures were also sequenced and commercial chicken or rat feed, was acutely toxic to ducklings the sequences deposited in GenBank (Naumann & Wicklow and rats (Kriek & Marasas 1979). Culture material from a toxi- 2010) and are listed in Table 1. Test strains were all isolated genic strain of S. macrospora (MRC 143) was extracted with chlor- from maize seeds and included: Acremonium zeae NRRL 13540, oform:methanol and fractionation monitored by bioassay in Alternaria alternata NRRL 6410, Aspergillus flavus NRRL 6541, Bipo- day-old ducklings led to the isolation of diplosporin (Chalmers laris zeicola NRRL 47238, Colletotrichum graminicola NRRL 47511, et al. 1978). Diplodiol (¼diplosporin) and chaetoglobosin K were Curvularia lunata NRRL 6409, Fusarium graminearum NRRL reported as new metabolites of S. macrospora (ATCC 36896) iso- 31250, NRRL 25457, Nigrospora oryzae lated from maize produced in Costa Rica (Cutler et al. 1980a, b). NRRL 6414, Rhizoctonia zeae NRRL 40186, S. maydis NRRL 31249, Both compounds were toxic to day-old-chicks and may have and Trichoderma viride NRRL 6418. contributed to reported losses in poultry in Mexico that were fed maize seeds rotted by Stenocarpella spp. (Cutler et al. 1980a). Fermentation conditions Stenocarpella metabolites have not been investigated for their relevance to the fungal disease cycle, including the bio- Strains of Stenocarpella maydis and Stenocarpella macrospora chemistry and physiology of infection, or an understanding were grown as potato dextrose agar (PDA) slant cultures (6 d, of the interactions between Stenocarpella and other fungal col- 25 C). A suspension of conidia and hyphal cells was prepared onists of maize or maize insects. Surprisingly, there have been from these cultures giving a propagule density of approxi- no reports on the detection or isolation of diplodiatoxin, diplo- mately 4 104 ml 1 served as inoculum. Initial fermentations sporin, or chaetoglobosin K from Stenocarpella-rotted maize were carried out in two 500-ml Erlenmeyer flasks each con- stalks, ears or seeds. An ability to exclude other fungi from taining 50 g of rice (Botan Brand; J.F.C. International, Los Stenocarpella parasitized and senescent/necrotic maize tissues Angeles, CA, USA) that was soaked overnight in distilled water would contribute to pathogen establishment, survival and re- (50 ml) before being autoclaved at 1.055 kgf cm 2 for 30 min. peated production of conidia as infective inoculum, from the After the flasks had cooled to room temperature, they were in- pycnidia that form on maize crop residues (Pfender 1996; oculated with 1.0 ml of the hyphal fragment-spore inoculum, Flett et al. 2001). Preliminary tests for antifungal activity, using and incubated for 30 d at 25 C. A second fermentation of a traditional paper disk assay, revealed that the organic sol- S. maydis NRRL 53562 was carried out as above in 18 500-ml vent extracts of fermented rice and fermented maize seed cul- Erlenmeyer flasks each containing 50 g of rice. tures inoculated with Stenocarpella maydis and Stenocarpella macrospora inhibited the growth of Aspergillus flavus and Fusa- Bioassay of extractable residue rium verticillioides. These extracts also exhibited potent antiin- sectan activity in a dietary assay with the fall armyworm Following incubation, the fermented rice substrate in each flask (Spodoptera frugiperda) and significant phytotoxicity in a maize was first fragmented with a large spatula and then extracted leaf puncture wound assay. Our objective was to isolate and three times with ethyl acetate (100 ml each time). The combined characterize Stenocarpella metabolites accounting for the bio- ethyl acetate extracts from flasks inoculated with each of the activity of these fermentation extracts and to detect the occur- Stenocarpella maydis and Stenocarpella macrospora strains listed rence of these compounds in diseased seeds and stalk tissues. in Table 1 (two flasks each) were evaluated. One-mg equivalents of extractable residue, dissolved in methanol, were pipetted onto analytical grade filter paper discs (13 mm diameter) in indi- Materials and methods vidual Petri dish lids and dried for 30 min in a laminar flow hood. Discs were placed on the surface of PDA seeded with conidia of Fungal strains Aspergillus flavus NRRL 6541 or Fusarium verticillioides (NRRL 25457), or conidia/hyphal cells of nine other test strains (see Maize isolates of Stenocarpella maydis and Stenocarpella macro- ‘Fungal strains’) giving a final conidial/hyphal cell suspension spora were obtained from the USDA Agricultural Research of approximately 100 propagules ml 1. The bioassay plates Bioactive metabolites from S. maydis 135

Table 1 e Bioactivity of organic extracts from fermented-rice cultures of Stenocarpella maydis or S. macrospora isolated from maizea. GenBank Location Antifungalb Antiinsectanc Leaf necrosisd

A. flavus F. verticillioides Fall armyworm B73 Burrus 794

Stenocarpella maydis NRRL 43670 GQ167213 Illinois 12 15 90 0.26 0.78 NRRL 52415 GQ167214 Illinois (e) 14 26 0.40 0.45 NRRL 53563 GQ167215 Illinois 10 6 50 1.41 2.05 NRRL 53565 GQ259129 Illinois 15 15 90 (50 %M) 1.00 1.61 NRRL 53566 GQ167216 Illinois 15 15 90 0.56 1.11 NRRL 53567 GQ167217 Illinois 15 15 90 (10 %M) 0.53 1.10 NRRL 31249 GQ167218 Indiana 10 10 75 2.10 0.50 NRRL 53560 GQ167219 Indiana 12 14 90 1.60 2.00 NRRL 13608 GQ428199 Kentucky (e)(e) 25 1.50 1.74 NRRL 53564 GQ167220 Kentucky 8 7 50 0.56 0.41 NRRL 53561 GQ167221 Nebraska 15 15 90 0.52 1.90 NRRL 53562 GQ259130 Nebraska 15 15 90 (85 %M) 1.01 1.10 NRRL 13609 GQ167222 Unreported, USA (e)(e) 0 1.33 2.21 NRRL 13615 GQ167223 Unreported, USA 12 14 90 0.66 0.81

Stenocarpella macrospora NRRL 13610 GQ167224 Costa Rica (e)(e) 25 0.66 0.50 NRRL 13611 GQ167225 Zambia 15 15 90 1.20 0.46 NRRL 13612 GQ259128 Georgia, USA (e)(e) 50 0.80 0.28

Methanol: Water z0.25 z0.25 Oxalic acid 2.50 2.30

a Cultures grown on 50 g autoclaved rice, 30 d; 25 C. b Agar disc diffusion assay with zone of inhibition measured from disc edge (mm); presenting as a clear zone nearest to the disc transitioning to a zone of partial clearing from restricted fungal growth. c Fall armyworm (Spodoptera frugiperda) neonate larvae: % reduction in growth relative to control and (%mortality). d Leaf puncture wound assay (10 mg/5 ml) recorded as average (six-wnds.) lesion length (mm) compared with methanol:water (50:50) and oxalic acid (5 mg) positive control. were incubated for 4 d at 25 C and examined for the presence of The ethyl acetate extract from two flasks, each containing a zone of inhibition extending from the edge of each disc that 50 g rice inoculated with Stenocarpella macrospora NRRL 13611, was measured in millimeters (full clearing: partial clearing were filtered and evaporated to give 256 mg of crude extract. with tiny restricted colonies), which is a measure of fungistatic A portion (210 mg) of the crude extract was partitioned activity. This bioassay procedure was also used to guide the iso- between acetonitrile and hexanes to afford 189 mg of an aceto- lation of Stenocarpella metabolites accounting for the antifungal nitrile-soluble fraction. A portion of this fraction (16 mg) was activity observed for the crude extracts by placing 0.5 mg of chromatographic fractions (frs) or 0.2e0.3 mg of individual me- O tabolites onto individual paper discs. OH O H H HO2C N H O OH Stenocarpella N O Isolation of metabolites from solid-substrate H H H fermentation cultures O 12

The ethyl acetate extract from two flasks, each containing 50 g OH rice inoculated with Stenocarpella maydis NRRL 43670 were fil- H tered and evaporated to give 741 mg of crude extract. The N OH OH crude extract was partitioned between acetonitrile and hex- O OH N H O anes to afford 500 mg of an acetonitrile-soluble fraction. A H H O portion of this fraction (248 mg) was subjected to Sephadex 34 LH-20 column chromatography using the following solvents: OOH 1:4 hexanes dichloromethane/acetone (solvent A); 3:2 OH dichloromethane/acetone (solvent B); 1:4 dichloromethane/ O acetone (solvent C); and methanol (solvent D) (Cardellina 5 1983). Twenty-four fractions were collected: fractions 1e7, 20 ml solvent A; frs 8e14, 20 ml solvent B; frs 15e23, 20 ml sol- Fig 1 e Structures of diplodiatoxin (1), chaetoglobosin K (2), vent C; 24, 150 ml solvent D. Fraction 13 consisted of 58 mg of chaetoglobosin L (3), (all-E )-trideca-4,6,10,12-tetraene-2,8- the major component diplodiatoxin (1)(Fig 1). diol (4), and diplosporin (5). 136 D. T. Wicklow et al.

chromatographed over an Alltech Altima C18 column Antifungal assays (10 mm 250 mm) at a flow rate of 2.0 ml min 1 with ultravio- let (UV) detection at 220 mm using the following method: iso- To evaluate the fungistatic activity of purified compounds cratic at 30 % acetonitrile/water over 5 min; ramp from 30 to against Aspergillus flavus or Fusarium verticillioides we placed dip- 50 % acetonitrile/water over 10 min; ramp from 50 to 65 % ace- lodiatoxin (0.2 mg), chaetoglobosin K (0.3 mg), and diplosporin tonitrile/water over 5 min; ramp from 65 to 100 % acetonitrile/ (0.2 mg), onto individual paper discs using the bioassay proce- water over 15 min; isocratic at 100 % acetonitrile over 5 min. dure described earlier. A minimum inhibitory concentration 2 ¼ This protocol afforded chaetoglobosin K ( ; tR 31 min; (MIC) was assigned to the smallest treatment concentrations 3 ¼ 1.4 mg), chaetoglobosin L ( ; tR 27 min; 0.7 mg), and diplo- for which no fungal growth was recorded, while a growth inhi- 5 ¼ sporin ( ; tR 12 min; 5.9 mg) (Fig 1). bition (GI)50 was designated when fungal GI exceeded 50 % of The combined ethyl acetate extract from ten flasks, each the growth recorded for methanol control wells. Diplodiatoxin, containing 50 g rice inoculated with Stenocarpella maydis NRRL chaetoglobosin K, (all-E )-trideca-4,6,10,12-tetraene-2,8-diol, 53566, were filtered and evaporated to give 1 g of crude extract. and diplosporin were further evaluated in 96-well plates at con- A portion of the crude extract (834 mg) was partitioned between centrations of 1, 2, 3, 5, 10, 25, and 50 mgml 1. The inhibitory ac- acetonitrile and hexanes to afford 680 mg of an acetonitrile-sol- tivity of the broad spectrum antifungal agent nystatin served as uble fraction. A portion of this fraction (365 mg) was subjected a positive control. Two hundred microliters of potato dextrose to silica gel column chromatography using the following sol- broth (PDB) seeded with each tested were added to ex- vents: hexanes (solvent A); hexanes/ethyl acetate (solvent B); perimental wells, and 10 ml methanol was added to redissolve acetone (solvent C); and methanol (solvent D). Thin-layer chro- the test compound. The 96-well plates containing the fungi matography (TLC) on silica gel was used to analyze each frac- were incubated on a shelf for up to 64 h at 25 C and examined tion and those with similar components were combined to at 8e16 h intervals using a plate reader at a wave length of afford 14 fractions: fraction 1, 200 ml 5 % solvent A, 200 ml 550 nm for evidence of inhibition of fungal growth in the wells, 5 % solvent B, 200 ml 10 % solvent B, 200 ml 20 % solvent B; fr which is a measure of fungistatic activity. 2, 100 ml 30 % solvent B; fr 3, 100 ml 40 % solvent B; fr 4, 100 ml 50 % solvent B, 100 ml 60 % solvent B; fr 5, 50 ml 70 % sol- Antiinsectan assays vent B; fr 6, 50 ml 70 % solvent B; fr 7, 100 ml 80 % solvent B, 50 ml 90 % solvent B; fr 8, 50 ml 90 % solvent B, 100 ml 100 % sol- In an effort to locate Stenocarpella metabolites that might func- vent B; fr 9, 50 ml solvent C; fr 10, 50 ml solvent C; fr 11, 50 ml tion as insect antifeedants or toxins, a feeding assay was solvent C; fr 12, 50 ml solvent C; fr 13, 50 ml solvent D; fr 14, performed against fall armyworm Spodoptera frugiperda (Lepi- 50 ml solvent D. Fraction 4 (10.9 mg) was chromatographed doptera: Noctuidae) neonate larvae in which the ethyl acetate over a Rainin Dynamax-60/A C18 column (21.4 mm 250 mm, extracts (10 mg) from fermented-rice cultures or purified me- 8-mm particle size) at a flow rate of 5.0 ml min 1 with detection tabolites were incorporated into a standard agar-based pinto at 256 nm using the following method: isocratic at 20 % acetoni- bean diet (0.2 % wet wt.) used in rearing the insects (Dowd trile/water over 5 min; ramp from 20 to 100 % acetonitrile/water 1988). Diplodiatoxin, chaetoglobosin K, and diplosporin, were over 40 min; isocratic at 100 % acetonitrile over 5 min. This tested at a dietary level of 1000 ppm, while (all-E )-trideca- protocol afforded (all-E )-trideca-4,6,10,12-tetraene-2,8-diol 4,6,10,12-tetraene-2,8-diol was tested at 100 ppm. 4 ¼ ( ; tR 22 min; 1.0 mg) (Fig 1). A choice analysis was performed with sap beetle Carpophi- Compounds 1e3 and 5 were identified on the basis of lus freemani (Coleoptera, Nitidulidae) adults using the follow- their 1H nuclear magnetic resonance (NMR) and mass spec- ing approach: Healthy seeds from symptomless ears and trometric (MS) data, and comparison with literature values rotted seeds from Stenocarpella maydis NRRL 53565 wound- (Steyn et al. 1972; Cutler et al. 1980a,b;Probst&Tamm inoculated ears were sampled at harvest from commercial 1982). Compounds 1e3 were also shown to be present in maize grown at Kilbourne, IL in 2009. Each of the grain sam- the ethyl acetate extract from rice inoculated with Stenocar- ples (100 g) was first ground using a Stein mill (Steinlite, Atch- pella maydis NRRL 53562. Compound 4 could only be identi- ison, KS). Fifty milligrams (air dried) of S. maydis-rotted and fied by analysis of 1H NMR data and by comparison with control corn powder in separate pre-weighed uncapped 2-ml literature values. The only 1H NMR data reported in the liter- vials. A separate water reservoir was made by cutting 4 ature for were collected using C6D6 as the solvent (Abate a 35-mm film canister in half and using the bottom half and 1 et al. 1997). H NMR data collected using CDCl3 as the solvent the cap. A 4-mm diameter hole was drilled in the film canister are reported here for the first time. C6D6 gives better resolu- cap and cotton was placed in the vial to wick up water the top. tion of the signals, but CDCl3 isamorecommonlyused The canister was filled with sterile, distilled water, which was solvent. sufficient to supply water for the beetles for the duration of (all-E )-trideca-4,6,10,12-tetraene-2,8-diol (4); 1H NMR data the assay. The two vials containing the corn powder were se- d ¼ (CDCl3, 400 MHz) 6.31 (1H, dt, J 17.0, 10.2 Hz, H-12), 6.22 cured in an upright position to the canister with a rubber (1H, dd, J ¼ 15.1, 10.2 Hz, H-6), 6.13 (1H, dd, J ¼ 15.2, 10.7 Hz, band. The vial-canister setup was placed inside a 6-cm diam- H-11), 6.11 (1H, dd, J ¼ 15.1, 10.7 Hz, H-5), 5.68 (1H, dt, J ¼ 15.0, eter 6.5 cm high glass jar. A 4-mm diameter hole was drilled 7.2 Hz, H-10), 5.66 (1H, dt, J ¼ 15.0, 7.4 Hz, H-4), 5.64 (1H, dd, in the jar lid and the hole covered with organdy secured with J ¼ 15.0, 6.6 Hz, H-7), 5.13 (1H, br d, J ¼ 17.0 Hz, H-13), 5.01 hot glue to provide ventilation. A total of 20 adult beetles were (1H, br d, J ¼ 10.2 Hz, H-130), 4.19 (1H, q, J ¼ 6.2 Hz, H-8), 3.84 added to each of 10 jars used in the assays. The feeding cham- ¼ e (2H, br tq, J 6.2, 6.2 Hz, H2-2), 2.15 2.40 (4H, m, H2-3, H2-9), bers containing beetles were incubated for 5 d at 27 1 C, ¼ 1.19 (3H, J 6.2 Hz, H3-1). 40 10 % relative humidity, and a 14:10 h (light:dark) Bioactive metabolites from S. maydis 137

photoperiod. Following incubation, beetles were removed Maize F-2 seeds from Burrus 794sRR were planted in 18 5-in from the vials which were then reweighed. pots containing a pasteurized potting mix and grown in an en- vironmental chamber. The photoperiod was 16 h, with tem- Leaf puncture wound assay peratures at 26 1 C (day) and 21 1 C (night). After 41 d, plants were toothpick wound-inoculated with Stenocarpella Organic extracts of fermented-rice cultures of Stenocarpella maydis NRRL 53562 or NRRL 53565 in each of the first three in- maydis and Stenocarpella macrospora were tested for phytotox- ternodes above the root crown. Living plants displaying symp- icity using a leaf puncture wound assay. A droplet (5 ml) of toms of stalk rotting accompanied by wilting were harvested methanol/water solution (1:1, v/v) containing 10 mgof after 58 d (¼17 dpi), 72 d (¼31 dpi), and 100 d (¼59 dpi). Inter- extracted residue was placed over each of six needle puncture nodal sections were split longitudinally to reveal discolored wounds (z0.25 mm) on the upper surface of a maize leaf blade regions of the pith. A small sample of discolored pith tissue cut from 4-week-old maize seedlings of Burrus 794sRR, grown near each site of wound-inoculation was cultured on PDA con- in the greenhouse. Oxalic acid (5 mg) served as a positive con- taining streptomycin to reveal colonization by S. maydis. trol. The leaf blades were incubated (72 h; 21e23 C) on moist- Freeze-dried stalk segments showing symptoms of pith tissue ened filter paper in a sealed Petri dish. The lengths of the necrosis with brown discoloration were extracted three times necrotic lesions spreading from needle puncture wounds with ethyl acetate. The combined ethyl acetate extracts were were measured under a stereomicroscope. No symptoms filtered and evaporated to yield a crude extract. were observed with 1:1 methanol/water control solutions The green stalk of a maize plant in the dent stage of kernel (Table 1). Diplodiatoxin, chaetoglobosin K, and diplosporin maturity was harvested and cut into segments (7 cm each), were evaluated by placing a droplet (5 ml) of a MeOH/water so- distributed among six humidity chambers and autoclaved lution (1:1, v/v) containing 5 mg of test compound over each of for 30 min. Maize stalk residues displaying no symptoms of six needle puncture wounds. stalk rot were collected following harvest, rinsed under run- ning tap water, cut into 7-cm segments, and distributed Detection of Stenocarpella metabolites among six humidity chambers. Prior to autoclaving, water was added to each humidity chamber to elevate the stalk res- Thirty naturally infected maize ears severely rotted by Stenocar- idue moisture content to approx. 300 % Md (dry basis). The pella maydis were individually hand-harvested in Sep. 2007 steam-sterilized stem segments were individually inoculated from a commercial production field near Kilbourne, IL that by insertion of wooden toothpicks colonized by Stenocarpella was planted to Burrus 794sRR. Immediately following harvest, maydis NRRL 53562 or NRRL 53565 and incubated for 14 d ears were individually shelled and a single rotted seed from (green stalks) or 21 d (stalk residues) at 25 C. Following incu- each ear was selected for isolation of S. maydis. The remaining bation, the heavily molded stem segments were freeze-dried, seeds from each ear were stored separately in the freezer cut into 1e2 cm pieces, and extracted three times with ethyl (20 C). Individual seeds were treated with 2 % NaOCl plus acetate.

0.01 % Triton X-100 for 2 min, rinsed twice in H2O, and then The crude extracts of kernels from naturally infected ears transferred to individual Petri dishes containing 3 % malt ex- (2007), wound-inoculated ears (2009), infected-necrotic stalk le- tract agar. After 4e5 d incubation at 25 C, hyphal tips of S. may- sions, and steam-sterilized maize stalks inoculated with Steno- dis colonies growing from seeds were transferred to slants of carpella maydis were partitioned between hexanes and PDA and incubated at 25 C. Five-gram subsamples of the rotted acetonitrile. The resulting acetonitrile fractions were then sub- kernels from each of the 30 ears were pooled and extracted jected to analysis by liquid chromatographyeelectrospray ion- three times with ethyl acetate to afford a dried extract weighing ization mass spectrometry (LCeESIMS) and 1HNMRto 1497 mg. The remaining rotted seeds (84e130 g) from three determine whether diplodiatoxin (1), chaetoglobosin K (2), of the S. maydis-rotted ears from which we isolated NRRL chaetoglobosin L (3), or (all-E )-trideca-4,6,10,12-tetraene-2,8- 53565, NRRL 53566, and NRRL 53567 were separately extracted diol (4) were present in the samples. 1H NMR data were obtained with ethyl acetate. Seeds removed from symptomless ears with a Bruker DRX-400 MHz instrument using CDCl3 as solvent. were also extracted with ethyl acetate to serve as a control. MS data were acquired on a ThermoFinnigan LCQ Deca mass The extracted residues were then examined for Stenocarpella spectrometer (Thermo Scientific, San Jose, CA, USA). Samples metabolites. were analyzed using positive ion electrospray ionization (ESI). Maize ears of the commercial corn hybrid Burrus ZD51 Data were obtained over the mass range 100e1000 Da with par- grown in Kilbourne, IL (crop year 2009) were wounded by a sin- allel UV detection at 260 nm. The eluting solvents used were gle inoculation with Stenocarpella maydis (NRRL 53565, NRRL 5 % acetonitrile in water with 0.1 % formic acid (solvent B). Sol- 53566, or NRRL 53567) in the late milk to early dough stage of vents used for LCeESIMS were water with 0.1 % formic acid kernel maturity (21 d after mid-silk). Briefly, 20 ears on plants (solvent A) and acetonitrile with 0.1 % formic acid (solvent B). within a single 7-m length of row were inoculated in the mid- All solvents used for LCeESIMS were Optima LCeMS grade ear region by inserting a single wooden toothpick penetrated (Fisher Scientific, Pittsburgh, PA, USA). Samples were chroma- by S. maydis through the husks and developing seeds, as far tographed over a Supelco Discovery reversed-phase C18 column as the rachis (cob). Following natural dry down in the field (2.1 150 mm: 5-mm particle size) at a flow rate of 0.20 ml min 1 (to 16 % moisture content), ears were hand-harvested and with a sample injection volume of 10 ml using the following the visibly diseased seeds from each ear were pooled and method: isocratic at 40 % solvent B over 2 min; ramp from 40 stored in a freezer at 7 C prior to the preparation of solvent to 95 % solvent B over 23 min. A fraction containing diplodia- extracts. toxin (1), chaetoglobosin K (2), and chaetoglobosin L (3)was 138 D. T. Wicklow et al.

e 4 used as a standard for determination of LC ESIMS retention for ( ) collected using CDCl3 solutions were difficult to fully in- 1 ¼ 2 ¼ 3 ¼ times ( : tR 10.54 min; : tR 12.73 min; : tR 9.36 min). At- terpret due to signal overlap. After a substructure search, a close 4 tempts to detect (all-E )-trideca-4,6,10,12-tetraene-2,8-diol ( ) literature match was found, albeit with data reported in C6D6. by ESIMS, electron impact MS (EIMS), and atmospheric chemi- Analysis of the sample using C6D6 as the solvent resolved the cal ionization MS (APCIMS) techniques were not successful, signals and matched the data reported for (4). A useful aspect 1 even for samples where the H NMR spectrum clearly showed of the simplification observed in the spectrum of the C6D6 solu- its presence as a major component. tion was the appearance of the two diastereotopic sp3 methy- lene units as clear triplets.

Results and discussion Bioactivity of fermented-rice cultures

Chemical studies of the ethyl acetate extracts from rice- Fermented-rice cultures inoculated with different strains of fermented cultures of Stenocarpella maydis NRRL 53565, NRRL Stenocarpella maydis or Stenocarpella macrospora commonly dis- 53566, and NRRL 53567 each of which displayed potent antifun- played potent antifungal activity against Aspergillus flavus and gal activity (Aspergillus flavus and Fusarium verticillioides) and po- Fusarium verticillioides in conventional paper disc agar diffu- tent antiinsectan activity (Spodoptera frugiperda), afforded the sion assays on agar plates seeded with conidia of A. flavus known Stenocarpella metabolites diplodiatoxin, chaetoglobosins (NRRL 6541) or F. verticillioides (NRRL 25457) (Table 1). We also K and L, as well as the bisdienediol, (all-E )-trideca-4,6,10,12-tet- evaluated 1-mg equivalents of the organic extract of S. maydis raene-2,8-diol (Fig 1). The presence of diplodiatoxin as well as NRRL 31249 using paper discs placed on the surface of agar chaetoglobosins K and L was also demonstrated for S. maydis plates seeded with conidia and/or hyphal cells of the following NRRL 53562. This constitutes the first report of chaetoglobosins test fungi (¼zone of inhibition measured from the disc edge): and (all-E )-trideca-4,6,10,12-tetraene-2,8-diol being produced Acremonium zeae (7 mm), Alternaria alternata (15 mm), A. flavus by S. maydis. Numerous unidentified minor peaks were found (12 mm), Bipolaris zeicola (11 mm), Colletotrichum graminicola tightly clustered in close chromatographic proximity to chaeto- (13 mm), Curvularia lunata (13 mm), Fusarium graminearum globosins K and L. No attempt was made to isolate these minor (15 mm), F. verticillioides (20 mm), Nigrospora oryzae (20 mm), metabolites which were presumed to be other chaetoglobosin Rhizoctonia zeae (20 mm), S. maydis NRRL 31249 (15 mm), analogues. Diplodiatoxin is the only compound previously Trichoderma viride (0 mm). These results suggest that, with reported from S. maydis (Steyn et al. 1972). The authors did the exception of T. viride, the antifungal metabolites produced not indicate that their culture of S. maydis was deposited. Anal- by S. maydis are broadly active against common fungal endo- ysis of 1H NMR and MS data demonstrated that isolates of phytes and pathogens of maize. Ten of 17 Stenocarpella culture S. maydis produce diplodiatoxin as a major constituent. extracts exhibited potent antiinsectan activity in a dietary as- In the present study, the gamma-pyrone diplosporin (Fig 1) say using neonate larvae of the fall armyworm (Spodoptera fru- and chaetoglobosins K and L were isolated from fermented- giperda) causing a reduction in weight gain of >75 % relative to rice cultures inoculated with Stenocarpella macrospora NRRL the control, with three of the extracts also causing some larval 13611 (¼MRC 143b) isolated from maize grown in Zambia. mortality. Extracts showing potent antiinsectan activity also This is the same culture that afforded diplosporin, as showed potent antifungal activity, while extracts showing monitored by bioassay in ducklings (Chalmers et al. 1978). Nei- limited antiinsectan activity were typically inactive in anti- ther diplodiatoxin nor (all-E )-trideca-4,6,10,12-tetraene-2,8- fungal assays. Culture extracts from two strains of S. maydis diol was detected in extracts of S. macrospora. Cutler et al. (NRRL 13608; NRRL 13609) and two strains of S. macrospora (1980a, b) isolated and characterized chaetoglobosin K and dip- (NRRL 13610; NRRL 13612) exhibited no antifungal activity losporin (¼diplodiol) from a culture of S. macrospora ATCC 36896 and weak antiinsectan activity. Extracts from a replicated ex- (NRRL 13610) isolated from maize in Costa Rica. While both periment produced the same bioassay results. These strains compounds were shown to be toxic to 1-d-old chicks, chaeto- were previously maintained by periodic culture transfer for globosin K was also shown to be a potent inhibitor of wheat multiple years and may have lost their ability to produce coleoptiles (Triticum aestivum)(Cutler et al. 1980b). Chaetoglobo- such metabolites. Rabie et al. (1987) reported that 13 of 16 iso- sins L and M were also isolated and characterized from cultures lates of Diplodia maydis tested were acutely toxic to ducklings of S. macrospora (Probst & Tamm 1982; Spoendlin & Tamm one of the exceptions being ATCC 10235 (¼MRC 684; NRRL 1988). Chaetoglobosin M has since been isolated from two 13608) where deaths did not occur. Several of the extracts pro- strains of Phomopsis leptostromiformis cultured on sterile maize duced significant necrotic lesions when applied to needle (Burlot et al. 2003). puncture wounds of maize leaves although none exceeded The (all-E )-trideca-4,6,10,12-tetraene-2,8-diol (4), was first those produced by the oxalic acid controls. There was no con- isolated and characterized as a minor metabolite from a rice sistent relationship between lesion length and measures of fermentation culture of the wood-decay fungus Xylaria obovata, antifungal or antiinsectan activity, suggesting that different but no biological activity was reported (Abate et al. 1997). There compounds may account for these activities. are no reports of this compound having been isolated from any other source. Analysis of 1H NMR data led to the recognition Bioactivity of Stenocarpella metabolites that many of our U.S. isolates of Stenocarpella maydis produce this compound as a major component encountered in S. maydis We evaluated 200 mg of diplodiatoxin and diplosporin as well as extracts. This compound was difficult to isolate in quantity, ap- 300 mg of chaetoglobosin K in disc assays using a conidial inocu- parently due to a tendency toward decomposition. 1H NMR data lum of Aspergillus flavus and Fusarium verticillioides. Bioactive metabolites from S. maydis 139

Chaetoglobosin K displayed potent antifungal activity in the disc 100 ppm, 19-O-acetylchaetoglobosin A and D, and chaetoglobo- assay while diplodiatoxin and diplosporin were ineffective sin D exhibited potent antiinsectan activity (RGR ¼ 95 %), while (Table 2). In assays performed using 96-well plates with at dietary levels of 300 ppm chaetoglobosin A exhibited potent conidia and/or hyphal cells as inoculum, chaetoglobosin K antiinsectan activity (RGR ¼ 98 %), chaetoglobosin B showed displayed significant activity against A. flavus (MIC z 50 mgml 1; moderate activity (39 % RGR), and chaetoglobosin C was found e m 1 > e m 1 GI50 25 50 gml )andF. verticillioides (GI50 3 50 gml ), to have relatively low oral toxicity (Dowd et al. 1990; Wicklow while diplodiatoxin, diplosporin, and the (all-E )-trideca- et al. 2000). Jarvis et al. (1982) observed that stalk rot infection > > 4,6,10,12-tetraene-2,8-diol were ineffective (MIC 50; GI50 50). by S. maydis had no effect on natural infestation (stalk cavities There have been few reports on the antifungal activity of chaeto- measured as centimeters of damage) by the European corn globosins. Chaetoglobosin A exhibited potent in vitro antifungal borer Ostrinia nubilalis (Lepidoptera: Noctuidae). In the present activity against the rice blast disease pathogen Pyricularia oryzae study S. maydis did not produce chaetoglobosins K and L or (Kobayashi et al. 1996a), while chaetoglobosin D and 19-O-acetyl- any other compounds with demonstrated antiinsectan activity chaetoglobosins A and D were found to be strongly inhibitory to in diseased-necrotic stalks inoculated with S. maydis. A. flavus at 250 mg/disc (Wicklow et al. 2000). Rheeder et al. (1990) Sap beetles (Coleoptera: Nitidulidae) have been implicated examined individual maize kernels and recorded the fungi oc- as vectors of Fusarium spp. (Windels et al. 1976) and were later curring in each kernel, the most prominent negative association shown to vector and Aspergillus flavus to prehar- being between Stenocarpella maydis and Fusarium moniliforme. vest maize ears (Attwater & Busch 1983; Lussenhop & Wicklow However, neither fungus displayed any visible signs of antago- 1990). Bartelt & Wicklow (1999) demonstrated that volatiles nism toward the other in paired culture. A significant negative produced by Fusarium verticillioides attract sap beetles. The association was also revealed between S. maydis and Stenocarpella fungus produced attractive volatiles following ear inoculation macrospora which were rarely isolated from the same kernels. of milk stage field maize as well as on sterile seeds in the lab- When grown in culture a highly specific cultural antagonism oratory. Sap beetles may serve as vectors of Stenocarpella may- with sharp lines of differentiation separated the two fungi dis to maize ears and we wanted to determine if severely (Hoppe 1936; Rheeder et al. 1990). The present study shows that rotted kernels supporting S. maydis pycnidia would attract or both S. maydis and S. macrospora produce chaetoglobosins K deter adult beetles. A 4-d assay choice test was conducted and L which may account for this mutual antagonism. with paired vials in each feeding chamber containing 50 mg Chaetoglobosin K and diplosporin displayed significant of milled powder from seeds rotted by S. maydis NRRL 53565 antiinsectan activity in dietary assays against the fall army- with controls representing 50 mg of milled powder from seeds worm causing 74 % and 50 % reduction in growth rate (RGR) rel- removed from symptomless ears of the same corn hybrid. ative to controls with no larval mortality, while diplodiatoxin Throughout the experimental period, higher numbers of bee- was inactive when tested at 1000 ppm. The bisdienediol tles were consistently observed in vials containing powder (all-E )-trideca-4,6,10,12-tetraene-2,8-diol was inactive when from S. maydis-rotted seeds. At the conclusion of the experi- tested at a dietary level of 100 ppm. Chaetoglobosin K also dis- ment, the weight of powder remaining in each of the vials played significant activity (RGR ¼ 69 %) against corn earworms was recorded. The mean weight of powder removed (presum- (; Lepidoptera: Noctuidae) when tested at a die- ably consumed) by 20 adult beetles in each feeding chamber tary level of 1000 ppm. The antiinsectan activity of the ethyl ac- was 17.9 mg for the S. maydis-rotted seeds and 5.8 mg of pow- etate extracts from Stenocarpella maydis NRRL 53562 (Table 1) der from symptomless control seeds (F ¼ 26.21; P < 0.0001). was attributed to chaetoglobosin K, while the antiinsectan ac- A leaf puncture wound assay revealed that among the tivity of the ethyl acetate extracts from Stenocarpella macrospora Stenocarpella metabolites tested, diplodiatoxin exhibited signif- NRRL 13611 was ascribed to both chaetoglobosin K and diplo- icant phytotoxicity to maize leaves, producing elongated le- sporin. In dietary assays with corn earworms tested at sions (ave. 2.6 mm), extending in both directions along the

Table 2 e Antifungal activity of Stenocarpella metabolites diplodiatoxin (1), diplosporin (5), chaetoglobosin K (2), and (all-E )- trideca-4,6,10,12-tetraene-2,8-diol (4). 1 5 2 4 Nystatin (þcontrol)

Disc assaya,b 200 mg/disc 200 mg/disc 300 mg/disc 200 mg/disc Aspergillus flavus NRRL 6541 (e)(e)ntnt 14mmc Fusarium verticillioides NRRL 25457 (e)(e)13mmd nt 14 mmc

e MIC MIC GI50 MIC GI50 MIC GI50 MIC GI50 MIC GI50 Aspergillus flavus NRRL 6541 >50 >50 >50 >50 z50 >25e50 >50 >50 10 >3e50 Fusarium verticillioides NRRL 25457 >50 >50 >50 >50 >50 >3e50 >50 >50 10 >3e50

a Inoculum was a conidial suspension filtered through sterile cheese cloth. b Agar diffusion assay with zone of inhibition measured from the disc edge (mm). c Presenting as a clear zone extending from edge of disc. d Presenting as a clear zone nearest to the disc transitioning to a zone of partial clearing from restricted fungal growth. m ¼ e Inoculum was a suspension of conidia and/or hyphal cells. Concentrations tested were 1, 2, 3, 5, 10, 25, and 50 g mL. GI50 growth inhibition >50 % relative to the PDB control. 140 D. T. Wicklow et al.

leaves of Burrus 794 and B73 (ave. 2.8 mm), that were equivalent Stemocarpella maydis conidia germinate and colonize stalk, to lesions produced by the oxalic acid positive control (ave. leaf, and shank tissues by directly penetrating the epidermal 2.40 mm). The presence of diplodiatoxin as a major metabolite cell walls and host cytoplasm through the formation of an ap- could explain the somewhat greater lesion lengths produced by pressorium and enzymatic degradation (Bensch et al. 1992). solvent extracts of Stenocarpella maydis than Stenocarpella macro- The fungus may grow undetected in maize stalks and ears un- spora which does not produce this compound (Table 1). This is til several weeks after silking or until the soft dough stage the first reported phytotoxicity of diplodiatoxin to maize. (Latterell & Rossi 1983). Maize stalks become more susceptible At the same time, chaetoglobosin K produced only minor to Stenocarpella stalk rot during the transition from vegetative lesions (ave. 1.10 mm), while needle wounds treated with dip- to reproductive stages and the onset of pith senescence losporin did not exceed those of methanolewater control and (Wysong & Hooker 1966). Diplodiatoxin (1) was detected in would therefore not explain the blighting of entire leaves of wound-inoculated necrotic discolored stalk lesions at 59 dpi plants attributed to Stenocarpella macrospora (Latterell & Rossi for plants inoculated with S. maydis strains NRRL 53562 or 1983). Might chaetoglobosins K and L function as effectors en- NRRL 53565, but was not detected in necrotic discolored le- abling Stenocarpella maydis endophytic-biotrophic infection of sions harvested at either 17 or 31 dpi. The production of diplo- maize plant tissues? Chaetoglobosin K and other cytochala- diatoxin by S. maydis may coincide with the switch from sins block actin filament elongation (Yahara et al. 1982) and cy- biotrophic growth to a necrotrophic growth phase. Explaining tochalasins may function in disrupting alterations in the actin the mode of action of diplodiatoxin in pathogen virulence cytoskeleton associated with a plant cellular defense response awaits further study. to fungal infection (Kobayashi et al. 1996b; Staiger 2000). Cutler Diplodiatoxin (1) was also detected upon LCeESIMS analy- et al. (1980b) reported that chaetoglobosin K inhibited the sis of acetonitrile fractions from extracts of green stalks and growth of wheat coleoptiles at even lower concentrations stalk residues that had been steam-sterilized and inoculated than the plant hormone abscisic acid. This suggests that with either of two isolates of Stenocarpella maydis (NRRL very small amounts of chaetoglobosin K may bring about pro- 53562 or NRRL 53565). The presence of this compound could found effects on effector targets; therefore while chaetoglobo- also be detected by 1H NMR analysis of the crude extract. sin K was not detected in diseased maize tissues, it may be Using 1H NMR or MS techniques, no other Stenocarpella metab- present at levels that are sufficient to support biotrophy. olites were detected in any of the extracts from living maize plants or steam-sterilized stalk residues that had been inocu- lated with isolates of S. maydis strains (NRRL 53562 or NRRL Detection of Stenocarpella metabolites in maize 53565) that had been found to produce these metabolites in fermented-rice culture. Our LCeMS method enabled us to de- Diplodiatoxin (1) was detected in severely rotted grain from termine the presence of 1 in an organic extract, or its absence maize ears inoculated in the late milk stage of kernel matura- above the limit of detection, but did not afford quantitative in- tion with Stenocarpella maydis NRRL 53565, NRRL 53566, or formation about its concentrations. This constitutes the first NRRL 53567. This grain was further distinguished by the pres- reported detection of diplodiatoxin or any other metabolite ence of numerous black pycnidia on kernel surfaces. However, in S. maydis-rotted seeds and diseased stalk tissues. Stenocarpella metabolites (2e4) were not detected by 1H NMR or MS techniques in any of these extracts. In fermented-rice Interpreting diplodiosis culture, each of the fungi we used as inoculum produced com- pounds 1e4 as major components, and numerous undeter- Is diplodiatoxin responsible for field outbreaks of diplodiosis in mined chaetoglobosin analogues as minor metabolites. None cattle and sheep? There are no reports of purified diplodiatoxin of these Stenocarpella metabolites were detected in extracts having been administered to ruminants. The first clinical and of grain from naturally infected ears in 2007 that were the biochemical evidence that diplodiatoxin might be associated source of S. maydis isolates NRRL 53565, NRRL 53566, or NRRL with symptoms of neuromycotoxicosis is offered by Rahman 53567. While these ears showed visible symptoms of Stenocar- et al. (2002). Acute and subacute doses of purified diplodiatoxin, pella ear rot, as evidenced by a white cottony mycelium be- administered to male and female rats, caused symptoms of ir- tween the seeds, no pycnidia were observed and the yellow ritation, dullness, tremors, and convulsions. Furthermore, sig- seed endosperm was only moderately discolored. Chambers nificant inhibition of brain acetylcholinesterase activity was (1988) demonstrated that the opportunity to produce substan- observed in both acute and subacute treated animals indicating tial S. maydis ear rotting decreases sharply with a later an effect on nerve synapses. Thirteen of 20 Stenocarpella maydis inoculation date after mid-silk and is associated with a corre- isolates from maize grown in different regions of south Africa sponding decrease in seed moisture. Furthermore, as maize produced diplodiatoxin when cultured in PDB with some seeds mature they accumulate abundant amounts of two cultures producing a ‘high amount’ of the compound (Rao & class IV chitinases, Chit A and Chit B which have been shown Achar 2001). Diplodiatoxin was dismissed as the ‘main toxic to inhibit the growth of fungi on agar plates and may contrib- principle’ or ‘unknown toxin’ produced by S. maydis based on ute to resistance to ear rot caused by S. maydis (Huynh et al. experiments in which animals received (fed or dosed) culture 1992; Naumann & Wicklow 2010). Late-infected ears may ap- material from S. maydis sterile whole yellow maize seed fer- pear sound until the ears are shelled and evidence of ‘hidden mentations (Steyn et al. 1972; Marasas 1977; Rabie et al. 1977; diplodia’ is found with symptoms of seed infection, ‘darkened Kellerman et al. 1985; Rabie et al. 1985; Kellerman et al. 1991). germs’ associated with a loss in seed viability being restricted We propose that this ‘unknown toxin’ is likely to be a mixture to the tip-end (Rossouw et al. 2009). of chaetoglobosins including chaetoglobosins K and L as major Bioactive metabolites from S. maydis 141

components, with other chaetoglobosin analogues occurring as a major metabolite in Stenocarpella maydis-rotted seeds as minor metabolites. First, an evaluation of the oral toxicity and necrotic stalk tissues at harvest. At the same time, chae- of chaetoglobosin K to 1-d-old chicks gave an LD50 between 25 toglobosins were not detected in these extracts even though and 62.5 mg kg 1 (Cutler et al. 1980b). The presence of chaeto- the plants were inoculated with isolates of S. maydis shown globosins in S. maydis-fermented maize seed culture material to produce chaetoglobosins K and L in fermented-rice culture. would explain its acute toxicity to poultry (Rabie et al. 1977; Chaetoglobosin K and diplosporin isolated from Stenocarpella Rabie et al. 1987). No other studies of vertebrate toxicology using macrospora displayed moderate antiinsectan activity in dietary purified chaetoglobosin K or L have been reported. Chaetoglo- assays against the fall armyworm Spodoptera frugiperda while bosins are metabolites belonging to the family of cytochalasins chaetoglobosin K exhibited significant antifungal activity which include chaetoglobosin K and other compounds that against Aspergillus flavus and Fusarium verticillioides. Evidence have been shown to disrupt actin polymerization (Yahara is offered that chaetoglobosins comprise the ‘unknown toxin’ et al. 1982; Kumagai et al. 1988; Tikoo et al. 1999) and therefore or ‘main toxic principle’ attributed to S. maydis culture mate- can interfere with the myelination process. Disrupting actin po- rial prepared for animal feeding trials. lymerization with cytochalasin D blocked the differentiation This constitutes the first report of chaetoglobosins K and L and myelination of Schwann cells cocultured with dorsal root from Stenocarpella maydis, of (all-E )-trideca-4,6,10,12-tetraene- ganglion neurons (Fernandez-Valle et al. 1997). An ‘unknown 2,8-diol from Stenocarpella, and the first reported detection of toxin’ was responsible for causing diplodiosis in sheep and in- diplodiatoxin, or any other Stenocarpella metabolite, in dis- terfering with the myelination process, the fetus being particu- eased maize seeds and stalk tissues. larly vulnerable in the later part of pregnancy (Kellerman et al. 1991; Prozesky et al. 1994). Stenocarpella maydis-fermented maize culture material also caused mycotoxic peripheral mye- Acknowledgments linopathy in vervet monkeys (Fincham et al. 1991). Odriozola et al. (2005) have since reported histopathological evidence of We thank R. Kemper and J. Brown for theircapable laboratory as- moderate to severe degeneration of myelin shafts in white mat- sistance. Support for this work from the National Science Foun- ter of the cerebellum of heifers that died from an outbreak of dation (CHE 0718315) is gratefully acknowledged. Mention of diplodiosis after consuming harvested maize fields parasitized trade names or commercial products in this publication is solely with S. maydis in Argentina. Further evidence for chaetoglobo- for the purpose of providing specific information and does not sins comprising the ‘unknown toxin’ comes from studies show- imply recommendation or endorsement by the United States ing that the toxin(s) in S. maydis-fermented maize seed culture Department of Agriculture or the National Science Foundation. material is heat labile (Steyn et al. 1972; Rabie et al. 1977; USDA is an equal opportunity provider and employer. Kellerman et al. 1985; Fincham et al. 1991). Chaetoglobosin A in dried solvent extracts decomposes when exposed to 75 C references for 24 h or 100 C for 90 min. (Fogle et al. 2008). We have since de- termined that chaetoglobosins produced by S. maydis in fer- mented-rice culture material are decomposed to a significant extent by autoclaving at 121 C for 45 min, and this was accom- Abate D, Abraham W-R, Meyer H, 1997. Cytochalasins and phy- totoxins from the fungus Xylaria obovata. Phytochemistry 44: panied by losses in antifungal and antiinsectan activity of the 1443e1448. solvent extracts (K.D.R. & J.B.G., unpubl.). Our results suggest Attwater WA, Busch LV, 1983. Role of the sap beetle Glischrochilus that the synthesis of chaetoglobosins K and L is not supported quadrisignatus in the epidemiology of Gibberella corn ear rot. during the necrotrophic phase of infection yielding dry-ear rot, Canadian Journal of Plant Pathology 5: 158e163. so association of these metabolites with symptoms of diplodio- Bartelt RJ, Wicklow DT, 1999. Volatiles from Fusarium verticillioides sis is uncertain. Might chaetoglobosins form in maize crop res- (Sacc.) Nirenb. and their attractiveness to Nitidulid beetles. Journal of Agricultural and Food Chemistry 47: 2447e2454. idues following harvest? Marasas (1977) noted that in southern Bensch MJ, van Staden J, Rijkenberg FHJ, 1992. Time and site of Africa all outbreaks of diplodiosis are recorded in late winter inoculation of maize for optimum infection of ears by e (Jul. Sep.) when farm-stored roughage becomes depleted and Stenocarpella maydis. Journal of Phytopathology 136: 265e269. livestock are turned out to graze harvested maize fields. In Burlot L, Cherton J-C, Convert O, Correia L, Dennetiere B, 2003. this subtropical climate, S. maydis has the potential for contin- New chaetoglobosins from maize infested by Phomopsis ued saprotrophic growth and/or metabolic activity during this leptostromiformis fungi. Production, identification, and semi- 17 e period, which could lead to the biosynthesis of both diplodia- synthesis. Spectroscopy : 725 734. Cardellina II JH, 1983. Step gradient elution in gel permeation toxin and chaetoglobosins. No analytical methods have been chromatography. A new approach to natural products sepa- developed for determining precise levels of Stenocarpella metab- ration. Journal of Natural Products 46: 196e199. olites in rotted seeds or maize stalks, information that could be Chalmers AA, Gorst-Allman CP, Kriek NPJ, Marasas WFO, used to better predict the occurrence of diplodiosis in rumi- Steyn PS, Vleggaar R, 1978. Diplosporin, a new from nants consuming Stenocarpella-rotted ears. Diplodia macrospora Earle. South African Journal of Chemistry 31: 111e114. Chambers KR, 1988. Effect of time of inoculation on Diplodia stalk and ear rot of maize in South Africa. Plant Disease 72: 529e531. Conclusion Crous PW, Slippers B, Wingfield MJ, Rheeder J, Marasas WFO, Philips AJL, Alves A, Burgess T, Barber P, Groenewald JZ, 2006. Our research has shown that diplodiatoxin is a phytotoxin, Phylogenetic lineages in the Botryosphaeriaceae. Studies in and this is consistent with our detection of this compound Mycology 55: 235e253. 142 D. T. Wicklow et al.

Cutler HG, Crumley FG, Cox RH, Cole RJ, Dorner JW, Latterell FM, Naumann TA, Wicklow DT, 2010. Allozyme-specific modification Rossi AE, 1980a. Diplodiol: a new toxin from Diplodia macro- of a maize seed chitinase by a protein secreted by the fungal spora. Journal of Agricultural and Food Chemistry 28: 135e138. pathogen Stenocarpella maydis. Phytopathology 100: 645e654. Cutler HG, Crumley FG, Cox RH, Cole RJ, Dorner JW, Springer JP, Odriozola E, Odeon A, Canton G, Clemente G, Escande A, 2005. Laterell FM, Thean JE, Rossi AE, 1980b. Chaetoglobosin K: Diplodia maydis: a cause of death of cattle in Argentina. New a new plant growth inhibitor and toxin from Diplodia macro- Zealand Veterinary Journal 53: 160e161. spora. Journal of Agricultural and Food Chemistry 28: 139e142. Pfender WF, 1996. Microbial interactions preventing fungal Dowd PF, 1988. Synergism of aflatoxin B1 toxicity with the co- growth in senescent and necrotic aerial plant surfaces. In: occurring fungal metabolite kojic acid to two caterpillars. Morris CE, Nicot PC, Nguyen-The C (eds), Aerial Plant Surface Entomologia Experimentalis et Applicata 47:69e71. Microbiology. Springer, USA, pp. 125e138. Dowd PF, Cole RJ, Vesonder RF, November 27, 1990. Control of insects Probst A, Tamm C, 1982. Chaetoglobosin L, a new metabolite of by fungal tremorgenic . U.S. Patent No. 4,973,601. Diplodia macrospora. Helvetica Chimica Acta 65: 1543e1546. Fernandez-Valle C, Gorman D, Gomez AM, Bunge MB, 1997. Actin Prozesky L, Kellerman TS, Swart DP, Maartens BP, Schultz RA, plays a role in both changes in cell shape and gene expression 1994. Perinatal mortality in lambs of ewes exposed to cultures associated with Schwann cell myelination. Journal of of Diplodia maydis (¼Stenocarpella maydis) during gestation. Neuroscience 17: 241e250. A study of central nervous-system lesions. Onderstepoort Fincham JE, Hewlett R, DeGraaf AS, Taljaard JJF, Steytler JG, Journal of Veterinary Research 61: 247e253. Rabie CJ, Seier JV, Venter FS, Woodruff CW, Wynchank S, 1991. Rabie CJ, Du Preez JJ, Hayes JP, 1987. Toxicity of Diplodia maydis to Mycotoxic peripheral myelinopathy, myopathy and hepatitis broilers, ducklings and laying chicken hens. Poultry Science 66: caused by Diplodia maydis on vervet monkeys. Journal of Medical 1123e1128. Primatology 20: 240e250. Rabie CJ, Kellerman TS, Kriek NP, van der Westhuizen GC, De Flett BC, McLaren NW, Wehner FC, 2001. Incidence of Stenocarpella Wet PJ, 1985. Toxicity of Diplodia maydis in farm and laboratory maydis ear rot of corn under crop rotation systems. Plant animals. Food and Chemical Toxicology 23: 349e353. Disease 85:92e94. Rabie CJ, van Rensburg SJ, Kriek NPJ, Lubben A, 1977. Toxicity of Fogle MR, Douglas DR, Jumper CA, Straus DC, 2008. Heat stability Diplodia maydis to laboratory animals. Applied and Environmen- of chaetoglobosins A and C. Canadian Journal of Microbiology 54: tal Microbiology 34: 111e114. 423e425. Rahman MF, Rao SK, Achar PN, 2002. Effect of diplodiatoxin Hoppe PE, 1936. Intraspecific and interspecific aversion in Diplo- (Stenocarpella maydis) on some enzymatic profiles in male and dia. Journal of Agricultural Research 53: 671e680. female rats. Ecotoxicology and Environmental Safety 52: 267e272. Huynh Q, Hironaka C, Levine E, Smith C, Borgmeyer J, Shah D, Rao SK, Achar PN, 2001. Screening and in vitro production of 1992. Antifungal proteins from plants. Purification, molecular diplodiatoxin from the isolates of Stenocarpella maydis and its cloning, and antifungal properties of chitinases from maize toxigenic effect on bacterial strains. Indian Journal of Experi- seed. Journal of Biological Chemistry 267: 6635e6640. mental Biology 39: 1243e1248. Jarvis JL, Clark RL, Guthrie WD, 1982. Effect of second-generation Rheeder JP, Marasas WFO, Van Wyk PS, 1990. Fungal associations European corn borers on resistance of maize to Diplodia in corn kernels and effects on germination. Phytopathology 80: maydis. Phytopathology 72: 1149e1152. 131e134. Kellerman TS, Prozesky L, Schultz RA, Rabie CJ, Van Ark H, Rossouw JD, Pretorius ZA, Silva HD, Lamkey KR, 2009. Breeding for Maartens BP, Lubben A, 1991. Perinatal mortality in lambs of ewes Resistance to Stenocarpella Ear Rot in Maize. In: Plant Breeding exposed to cultures of Diplodia maydis (¼Stenocarpella maydis)during Review. John Wiley & Sons, Hoboken, NJ, vol. 31, 223e246. gestation. Onderstepoort Journal of Veterinary Research 58: 297e308. Spoendlin C, Tamm C, 1988. Chaetoglobosin M, a new metabolite Kellerman TS, Rabie CJ, van der Westhuizen GC, Kriek NP, of a mutant of Diplodia macrospora, belonging to the family of Prozesky L, 1985. Induction of diplodiosis, a neuromycotoxi- (1H-indol-3-yl)-substituted 10,11-diethyl-10,11-dinorcytocha- cosis, in domestic ruminants with cultures of indigenous and lasans. Helvetica Chimica Acta 71: 1881e1884. exotic isolates of Diplodia maydis. Onderstepoort Journal of Staiger CJ, 2000. Signaling to the actin cytoskeleton in plants. Annual Veterinary Research 52:35e42. Review of Plant Physiology and Plant Molecular Biology 51: 257e288. Kobayashi H, Namikoshi M, Yoshimoto T, Yokochi T, 1996a. A Steyn PS, Wessels PL, Holzapfel CW, Potgieter DJJ, Louw WKA, screening method for antimitotic and antifungal substances using 1972. The isolation and structure of a toxic metabolite from conidia of Pyricularia oryzae. Journal of Antibiotics (Tokyo) 49:873e879. Diplodia maydis (Berk.) Sacc. Tetrahedron 28: 4775e4785. Kobayashi I, Kobayashi Y, Yamada M, Kunoh H, 1996b. The in- Tikoo A, Cutler H, Lo SH, Chen LB, Maruta H, 1999. Treatment of volvement of the cytoskeleton in the expression of nonhost Ras-induced cancers by the F-actin cappers tensin and resistance in plants. In: Mills D, et al. (eds), Molecular Aspects of chaetoglobosin K, in combination with the caspase-1 inhibitor Pathogenicity and Resistance: requirement for signal transduction. N1445. Cancer Journal Scientific American 5: 293e300. APS Press, St. Paul, pp. 185e195. Wicklow DT, Dowd PF, Gloer JB, 2000. Chaetomium mycotoxins Kriek NPJ, Marasas WFO, 1979. Toxicity of Diplodia macrospora to with antiinsectan or antifungal activity Proceedings of Inter- laboratory animals. Food and Cosmetics Toxicology 17: 233e236. national Symposium of Mycotoxicology ’99, September 9e10, Kumagai H, Imazawa M, Miyamoto K, 1988. Unusual morpho- 1999, Chiba, Japan. Mycotoxins: Supplement 99. In: Kumagi S logical changes in cultured oligodendrocytes induced by cy- (ed.), Mycotoxin Contamination: health risk and prevention project. tochalasin B. Developmental Brain Research 27: 270e274. Matsumoto Printing Co., Tokyo, pp. 267e271. Latterell FM, Rossi AE, 1983. Stenocarpella macrospora (¼Diplodia) Windels CE, Windels MB, Kommedahl T, 1976. Association of and S. maydis (¼D. maydis) compared as pathogens of corn. Fusarium species with picnic beetles on corn ears. Phytopa- Plant Disease 67: 725e729. thology 66: 328e331. Lussenhop JL, Wicklow DT, 1990. Nitidulid beetles (Coleoptera: Wysong DS, Hooker AL, 1966. Relation of soluble solids content and Nitidulidae) as vectors of Aspergillus flavus in pre-harvest pith condition to Diplodia stalk rot in corn hybrids. Phytopathology maize. Transactions of the Mycological Society of Japan 31:63e74. 56:26e35. Marasas WFO, 1977. Diplodiosis in cattle. In: Wyllie D, Yahara I, Harada F, Sekita S, Yoshihira K, Natori S, 1982. Corre- Morehouse GL (eds), Mycotoxic fungi, Mycotoxins and Mycotoxi- lation between effects of 24 different cytochalasins on cellular coses: an encyclopedic handbook, vol. 2. Marcel Dekker, New structures and cellular events and those on actin in vitro. York, pp. 163e165. The Journal of Cell Biology 92:69e78.