Mycol. Res. 109 (5): 610–618 (May 2005). f The British Mycological Society 610 doi:10.1017/S0953756205002820 Printed in the United Kingdom.

A protective endophyte of maize: Acremonium zeae antibiotics inhibitory to Aspergillus flavus and Fusarium verticillioides1

Donald T. WICKLOW1, Shoshannah ROTH2, Stephen T. DEYRUP2 and James B. GLOER2 1 USDA, ARS, National Center for Agricultural Utilization Research, Peoria, IL 61604, USA. 2 Department of Chemistry, University of Iowa, Iowa City, IA 52242, USA. E-mail : [email protected]

Received 16 June 2004; accepted 25 February 2005.

The maize endophyte Acremonium zeae is antagonistic to kernel rotting and mycotoxin producing fungi Aspergillus flavus and Fusarium verticillioides in cultural tests for antagonism, and interferes with A. flavus infection and aflatoxin contamination of preharvest maize kernels. Chemical studies of an organic extract from maize kernel fermentations of Acremonium zeae (NRRL 13540), which displayed significant antifungal activity against Aspergillus flavus and F. verticillioides, revealed that the metabolites accounting for this activity were two newly reported antibiotics pyrrocidines A and B. Pyrrocidines were detected in fermentation extracts for 12 NRRL cultures of Acremonium zeae isolated from maize kernels harvested in Illinois (4/4 cultures), North Carolina (5/5), Georgia (1/2) and unrecorded locations within the USA (2/2). Pyrrocidine B was detected by LCMSMS in whole symptomatic maize kernels removed at harvest from ears of a commercial hybrid that were wound-inoculated in the milk stage with A. zeae (NRRL 13540) or (NRRL 13541). The pyrrocidines were first reported from the fermentation broth of an unidentified filamentous fungus LL-Cyan426, isolated from a mixed Douglas Fir hardwood forest on Crane Island Preserve, Washington, in 1993. Pyrrocidine A exhibited potent activity against most Gram-positive bacteria, including drug-resistant strains, and was also active against the yeast Candida albicans. In an evaluation of cultural antagonism between 13 isolates of A. zeae in pairings with A. flavus (NRRL 6541) and F. verticillioides (NRRL 25457), A. zeae (NRRL 6415) and (NRRL 34556) produced the strongest reaction, inhibiting both organisms at a distance while continuing to grow through the resulting clear zone at an unchanged rate. Maximum colony diameters for A. zeae (NRRL 6415) and (NRRL 13540), on potato dextrose agar after 14 d, were attained within the range of 25–30 xC, with less growth recorded at 15 x and 37.5 x and no growth at 5 x. Potential interactions between A. zeae and other maize endophytes are considered and the significance of these interactions relative to the aflatoxin and fumonisin contamination of preharvest maize is presented. This is the first report of natural products from Acremonium zeae.

INTRODUCTION fungi are frequently isolated as colonists of the same kernel. Because F. verticillioides grows more rapidly Acremonium zeae (Gams 1971), also reported as from kernels, it can mask the presence of A. zeae and Acremonium sp., A. strictum, Cephalosporium sp., or the infection frequency may be underestimated (Manns Cephalosporium acremonium, which was mistaken by & Adams 1923, Sumner 1968a, King 1981). Manns & Adams (1923) for C. sacchari (syn. Fusarium Histopathological studies and fungal isolations from sacchari), along with F. verticillioides (syn. F. monili- dissections of ‘sound-appearing’ kernels revealed that forme), are the most prevalent colonists of preharvest F. verticillioides was primarily confined to the pedicel maize (Zea maydis), typically producing symptomless and abscission layers of maize while A. zeae was more kernel infections (Reddy & Holbert 1924, Harris 1936, frequently isolated from excised embryos and endo- King 1981). In the following text, the modern names sperm (Sumner 1968a). Might A. zeae and F. verti- A. zeae and F. verticillioides have been substituted for cillioides therefore function as protective endophytes of earlier names assigned to these fungi. The significance maize? Endophyte establishment within the germ and of high levels of infection by A. zeae and F. verti- endosperm would present an immediate defense against cillioides in asymptomatic maize kernels is not well pathogen attack at the seed and seedling stage, where understood (King 1981, Munkvold et al. 1997). These natural enemies of the plant host would have the 1 Dedicated to John Webster on the occasion of his 80th birthday. greatest impact on fitness (Faeth 2002). D. T. Wicklow and others 611

Mycotoxin contamination of maize affects the quality MATERIALS AND METHODS and safety of human food and animal feeds thereby Fungal strains lowering the value of the grain and resulting in sub- stantial economic losses to maize growers, livestock 13 maize kernel isolates of Acremonium zeae were ob- and poultry producers, grain handlers, and food and tained from the USDA Agricultural Research Service feed processors (Council for Agricultural Science and Culture Collection, Peoria (NRRL). Culture collection Technology 2003). Prominent among the mycotoxins records listed these strains as Acremonium strictum or associated with such losses in maize are aflatoxins Cephalosporium: NRRL 6515, NRRL 13540, NRRL produced by Aspergillus flavus and fumonisins pro- 13541, NRRL 34554 (=A-24200), and NRRL 34555 duced by F. verticillioides. Individual kernels infected (=A-24202), identified by D.T.W. as Acremonium with F. verticillioides were less likely to be infected with strictum, were isolated by R. Rogers from maize A. flavus or other fungal pathogens of maize (Wicklow sampled at harvest in North Carolina in 1977, each 1988, Rheeder et al. 1990). Suppression of A. flavus isolate coming from a different crop field; NRRL infection and aflatoxin in preharvest maize by A. zeae 34556 (=A-25103), identified as A. strictum by B. W. and F. verticillioides was first demonstrated by Horn, was isolated by B. W. Horn from maize left Wicklow et al. (1988) who inoculated maize ears pro- standing in a field near Tifton, Georgia until ears duced on plants grown in an environmental chamber. were hand-harvested in October 1981; NRRL 34557 Similar results were obtained by Zummo & Scott (1992) (=A-25141), identified as A. strictum by B. W. Horn, when they inoculated F. verticillioides and A. flavus was isolated by B. W. Horn from a maize ear that was onto maize ears produced by plants growing in field hand- harvested in 1981 from a field near Tifton, plots. Fumonisin and aflatoxin levels in grain samples Georgia, placed on the ground, and retrieved in may also bear a negative relationship to each other, January 1982; NRRL 34558 (=A-25722), identified as suggesting the interaction of fumonisin producers (e.g. Cephalosporium sp. by D. G. White, was isolated by F. verticillioides) and aflatoxin producers (e.g. A. flavus) D. G. White from maize sampled at harvest in 1980 impacts kernel infection and mycotoxin production from a field at Champaign, Illinois; NRRL 34559 (=A- (Yoshizawa et al. 1996, Ono et al. 2001). The presence 21613) and NRRL 34560 (=A-21614), identified as of F. verticillioides in the ear was also negatively cor- Cephalosporium sp. by D. I. Fennell, were isolated by related with Diplodia maydis (Rheeder et al. 1990) and R. J. Bothast from separate ears of maize that were Gibberella zeae (Blaney et al. 1986), leading Rheeder hand-harvested in 1974 from a field in Peoria County, et al. (1990) to suggest that F. verticillioides may have a Illinois; NRRL 34561 (=A-21769), identified as ‘protective effect’ by suppressing the growth of more Cephalosporium sp. by J. J. Ellis, was isolated by destructive pathogens of maize with the potential for R. Rogers from a sample of whole maize kernels use as a ‘biocontrol agent.’ However, because F. verti- that was fed to pigs near Springfield, Illinois; NRRL cillioides may produce symptoms of Fusarium kernel 34562 (A-18709), identified by T. A. Toussoun as rot or ear rot of maize (White 1999) and contaminate Cephalosporium sp., was isolated by R. Rogers from a the grain with mycotoxins, this fungus is considered less kernel of blight-infected hybrid maize, USA; NRRL attractive for use in biocontrol (Rheeder et al. 1990). 34563 (=A-18737), identified by T. A. Toussoun as A. zeae is not recognized as causing ear, kernel or Cephalosporium sp., was isolated by R. Rogers from a storage rots of maize (White 1999) and there have been sample of whole maize kernels received from a corn no reports that A. zeae isolates from maize produce any mill, locality not specified, USA. metabolites toxic to animals or plants. A. zeae (NRRL 6415) strongly interfered with Aspergillus flavus Fermentation conditions (NRRL 6412) colony growth when paired on agar in Petri dishes (Wicklow et al. 1980). Furthermore, ex- Acremonium zeae (NRRL 13540) was grown on several perimental inoculations of maize ears with a mixed slants of potato dextrose agar (PDA) for 14 d (25 xC). conidial inoculum consisting of three cultures of A hyphal fragment-spore suspension (propagule den- Acremonium zeae (NRRL 6415, NRRL 13540, NRRL sity 106 mlx1 of sterile distilled water) prepared from 13541), limited Aspergillus flavus colonization and the PDA slants served as the inoculum. Fermentations aflatoxin contamination of the intact grains removed were carried out in forty 500 ml Erlenmeyer flasks, each from wound-inoculated ears (Wicklow et al. 1988). At containing 50 g of whole maize kernels (Kelly’s Seed, the same time, we could find no reports of research to Peoria). Distilled water (50 ml) was added to each flask, investigate competitive or cooperative interactions and the contents were soaked overnight before being between Acremonium zeae and F. verticillioides. Our autoclaved at 1.055 kgf cmx2 for 30 min. After the objective was to describe the isolation and character- flasks had cooled to room temperature, they were in- ization of antifungal metabolites produced by A. zeae oculated with 1 ml of the hyphal fragment-spore sus- which inhibit growth of Aspergillus flavus and/or pension and incubated for 30 d at 25 x. The remaining F. verticillioides, molds capable of infecting and rotting 12 isolates of A. zeae were grown as above, but maize maize kernels while contaminating the grain with kernel fermentations were limited to two 500 ml flasks mycotoxins harmful to livestock as well as humans. (100 g whole maize kernels) per isolate. Two flasks Antifungal antibiotics in Acremonium zeae 612 containing autoclaved maize kernels provided an reversed-phased high-performance liquid chromato- uninoculated control. graphy (HPLC) (Alltech 8u C-18 column; 250r 10 mm; 20 to 100% CH3CN in H2O in 45 min). Pyrrocidines A and B eluted as two broad, partially Bioassay of extractable residue overlapped peaks with retention times of 47.0 and Following incubation, the fermented whole maize 49.5 min under these conditions, but collection of the kernel substrate in each flask was first fragmented with first part of peak 1 and the latter part of peak 2, fol- a large spatula and then extracted three times with ethyl lowed by evaporation of the solvent, afforded samples acetate (50 ml each time). The combined ethyl acetate of pyrrocidines A (1.4 mg) and B (2.4 mg), respectively. extracts were filtered and evaporated. Following evap- Collection of the valley between the two peaks afforded oration of the ethyl acetate, approximately 5 mg of the another 6.2 mg of a mixture of the two compounds. residue was redissolved in methanol for antifungal Because of the difficulty in separating the two activity assays. The remaining dried extract was stored components in quantity, a slightly altered procedure at x20 x. One mg equivalents of extractable residue, was employed using fractions obtained from a larger- dissolved in methanol, were pipetted onto analytical- scale fermentation extract. In this case, a 400 g sample grade filter paper discs (13 mm diam) in individual Petri of freeze-dried fermented corn substrate was extracted dish lids and dried for 30 min in a laminar flow hood. with ethyl acetate (2.0 l) and partitioned between aceto- Discs were placed on the surface of PDA seeded with nitrile and hexane (50 ml each) as described above. Aspergillus flavus (NRRL 6541) conidia or Fusarium After washing the acetonitrile layer three times with verticillioides (NRRL 25457), each giving a final hexane (50 ml each time), the acetonitrile fraction was conidial/hyphal cell suspension of approximately 100 concentrated to afford 2.0 g of extract that was spores per ml. The bioassay plates were incubated for subjected to column chromatography on silica gel using 4 d at 25 x and examined for the presence of a zone of the same solvent step gradient described above. A inhibition surrounding a disc, which is a measure of 61 mg fraction eluting with 3% methanol in CH2Cl2 fungistatic activity. This bioassay procedure was used clearly consisted mostly of pyrrocidine A accompanied to guide the isolation of those A. zeae metabolites by some pyrrocidine B and other impurities. which accounted for the antifungal activity. Pure Fortuitously, at this stage, pyrrocidine B was found to compounds were evaluated for antifungal activity by be more soluble in CHCl3 than pyrrocidine A. Thus, placing 0.25 mg onto individual paper discs. simple trituration of this fraction with CHCl3 (0.5 ml) and subsequent filtration was found to remove pyrro- cidine B, some of the pyrrocidine A, and other Isolation of pyrrocidines A and B from solid-substrate impurities leaving a much larger pure sample of pyrro- fermentation cultures cidine A (30 mg) as a residual white solid. A 100 g sample of freeze-dried fermented corn The molecular formula of the pyrrocidine B sample substrate was pulverized using a mortar and pestle and obtained from A. zeae was established by high resol- extracted overnight with 1.3 l of ethyl acetate. The ethyl ution fast atom bombardment mass spectrometry acetate solution was then filtered, dried with mag- (HRFABMS). The structure was independently estab- nesium sulfate, and the ethyl acetate was evaporated lished by detailed, two-dimensional (2D) NMR analysis leaving an orange oil. This procedure was repeated two using 1H NMR, 13C NMR, 1H-1H correlation spec- more times and the combined residue (5.8 g) was then troscopy (COSY), heteronuclear single quantum partitioned between equal amounts of acetonitrile and correlation (HMQC), heteronuclear multiple bond hexanes (10 ml each), and the acetonitrile layer was correlation (HMBC), and 2D nuclear Overhauser en- washed twice with hexanes (2r10 ml). The acetonitrile hancement spectroscopy (NOESY) data. The structure partition was then evaporated to afford 0.91 g of crude of pyrrocidine A was assigned by spectral comparison material. to pyrrocidine B. During the course of this work, pyrro- The acetonitrile-soluble material was then subjected cidines A and B were independently reported from a to fractionation by flash chromatography on a silica gel different fungal source by researchers at Wyeth-Ayerst column. The column was eluted successively with who assigned the names pyrrocidines to these metab-

CH2Cl2 (500 ml), 1% methanol in CH2Cl2 (300 ml), olites (He et al. 2002). Comparison of data obtained for

2% methanol in CH2Cl2 (800 ml), 3% methanol in the compounds isolated from Acremonium zeae with

CH2Cl2 (400 ml), 4% methanol in CH2Cl2 (400 ml), those reported for pyrrocidines A and B confirmed the

5% methanol in CH2Cl2 (400 ml), 10% methanol in identities of these metabolites.

CH2Cl2 (600 ml), 20% methanol in CH2Cl2 (600 ml), 50% methanol in CH Cl (600 ml), and finally 90% 2 2 Inoculations of maize ears methanol in CH2Cl2 (400 ml). Bioassay results, together with 1H nuclear magnetic resonance (NMR) Pioneer 3394 maize kernels were harvested in 2001 from analysis suggested that compounds of interest eluted ears that were wound-inoculated with Acremonium with 2% methanol in CH2Cl2. A 12 mg portion of this zeae NRRL 13540 (10 plants) or NRRL 13541 (10 43 mg fraction was further purified by semipreparative plants) in the milk stage to early dough stage of kernel D. T. Wicklow and others 613 maturity (=21 d after mid-silk; 22 July 2001) at the Upon fragmentation, the pyrrocidine A parent ion at University of Illinois River Valley Sand Farm m/z 488 showed a base peak at m/z 382, as well as less Experimental Station, Kilbourne, IL. Ears were in- intense peaks at m/z 470 and 283. The pyrrocidine B oculated at three points separated by 4 cm in a vertical ion at m/z 490 showed a base peak at m/z 283. The base row. A drill bit (4 mm diam.) was inserted through the peaks were used as diagnostic signals for pyrrocidines husk to wound an underlying kernel. The A. zeae co- A and B because these masses were not present in sig- nidial inoculum was applied as a conidial suspension nificant amounts in the background or in blank runs. using sterile pipe cleaners (8 cm) that were inserted into High resolution electron-impact mass spectrometry each wound and left until ears at 15.5% moisture con- (HREIMS) was performed on the 283 ion, and its mol- tent were hand harvested on 18 Sept. 2001. Eight un- ecular formula was determined to be C20H27O, which is damaged whole kernels nearest to each wound site were consistent with its identity as the expected fragment removed from the ears and the pooled kernel samples resulting from cleavage of the C16–C17 and C13–O bonds. stored in plastic bags at x7 x. Several kernels with For LCMSMS analysis of individual samples, crude visible symptoms of A. zeae infection were selected fungal ethyl acetate extracts were partitioned between from each sample bag and examined for the presence of hexane and acetonitrile (5 ml each). The acetonitrile pyrrocidines. phase was collected and washed twice with 5 ml of hexane each time, and the acetonitrile phase was then evaporated to afford an acetonitrile fraction. A portion Detection of pyrrocidines A and B of this fraction was diluted to a concentration of With samples of pyrrocidines A and B and detailed 100 mgmlx1 in acetonitrile, and 20 ml of the resulting NMR data in hand, initial efforts to analyze other solution (i.e. 2 mg of extract) was injected onto the col- Acremonium zeae cultures involved examination of the umn for direct LCMSMS analysis for pyrrocidines proton NMR spectra of the corresponding acetonitrile using the method described above. The results of these extracts. In some cases, pyrrocidines were sufficiently analyses are summarized in Table 1. abundant to enable recognition of their presence based on comparison of the NMR data for these extracts with those for pure pyrrocidines. In other instances, including Evaluation of cultural antagonism cases where efforts were made to analyze maize kernels Cell and conidial suspensions of each fungal isolate, from field plots, this approach was inconclusive. Thus, made from 14 d old slants of PDA, were used as a protocol was developed to enable analysis of extracts inoculum. Cultures were paired on triplicate plates of for pyrrocidines by liquid chromatography-mass PDA in Petri dishes incubated at 25 x (8 d); spectrometry-mass spectrometry (LCMSMS) methods. Acremonium zeae inoculations were performed 48 h As expected, this approach proved to be much more before Aspergillus flavus (NRRL 6541) and Fusarium sensitive, and was very effective at detecting the pres- verticillioides (NRRL 25457). Conidial suspensions, ence of pyrrocidines at low levels in complex extracts. representing a given test pair, were transferred with an First, an improved HPLC protocol was developed in inoculating loop onto the agar surface at points sep- which pyrrocidines A and B were separable, but with arated by 3 cm according to predesignated markings on shorter retention times (22.2 and 22.8 min respectively). the underside of a Petri dish. Acremonium interference This protocol employed an Alltech Prevail C-18 with A. flavus and F. verticillioides was recorded after 4, column (3 mg particle size; 2.1r150 mm) at a constant 6 and 8 d. Colony reaction types were assigned to each flow rate of 200 ml minx1 Elution began with 50% pairing after Wicklow et al. (1980). acetonitrile in water for 2 min, followed by a linear gradient increasing to 100% acetonitrile over 28 min, after which a flow of 100% acetonitrile was maintained Determination of temperature optima for fungal growth for 10 min. A constant concentration of 0.03% formic Multiple hyphal tip transfers, taken from colonies of acid was maintained in the mobile phase throughout Acremonium zeae (NRRL 6415, NRRL 13540) grown the procedure. The eluent was monitored by positive- on plates of PDA for one week at 25 x, were used to ion electrospray ionization mass spectrometry inoculate test plates containing 15 ml of the same (ESIMS). Selected ion monitoring (SIM), a technique medium. Three plates of each fungus selected for study which limits the mass range observed in exchange for were incubated in the dark at each of the following higher selectivity and sensitivity, was performed. The temperatures: 5, 15, 25, 30, and 37.5 x. The diameters of mass range monitored in this study was 486–492. developing colonies were measured at intervals of 3–4 d The appearance of pyrrocidine A in the eluent was over a 2 wk period. monitored by setting the instrument to detect an ion at m/z 488 [(C31H37NO4+H)+], and pyrrocidine B was detected at m/z 490 [(C31H39NO4+H)+]. In order to RESULTS AND DISCUSSION further verify that the peaks observed between 22 and 23 min were the pyrrocidines, the ions at m/z 488 and Chemical studies of the organic extract from maize 490 were trapped and further fragmented using MSMS. kernel fermentations of Acremonium zeae (NRRL Antifungal antibiotics in Acremonium zeae 614

Table 1. Detection of pyrrocidines A and B in organic extracts of corn kernels fermented with cultures of Acremonium zeae and evaluation of cultural antagonism in pairings with Aspergillus flavus and Fusarium verticillioidese.

Reactionf

Straina,b Received as Source Pyrrocidines A. flavus F. verticillioides

NRRL 6415 Acremonium strictum North Carolina A,Bc ‘E’ ‘E’ NRRL 13540 Acremonium strictum North Carolina A,Bc ‘B’ ‘B’ NRRL 13531 Acremonium strictum North Carolina A,Bc ‘B’ ‘B’ NRRL 34554 Acremonium strictum North Carolina A,Bc ‘B’ ‘B’ NRRL 34555 Acremonium strictum North Carolina A,Bc ‘B’ ‘B’ NRRL 34556 Acremonium strictum Tifton, Georgia A,Bd ‘E’ ‘E’ NRRL 34557 Acremonium strictum Tifton, Georgia NDc,d,g ‘B’ ‘B’ NRRL 34558 Cephalosporium sp. Champaign, Illinois Bd ‘E’ ‘B’ NRRL 34559 Cephalosporium sp. Peoria Co., Illinois A,Bd ‘B’ ‘B’ NRRL 34560 Cephalosporium sp. Peoria Co., Illinois A,Bd ‘E’ ‘B’ NRRL 34561 Cephalosporium sp. Springfield, Illinois Bd ‘B’ ‘B’ NRRL 34562 Cephalosporium sp. USA A,Bd ‘B’ ‘B’ NRRL 34563 Cephalosporium sp. USA Bd ‘B’ ‘B’

a Culture reference nos. in the ARS Culture Collection, Peoria, IL (NRRL). b Ethyl acetate extracts of whole corn kernel fermentations (25 x; 30 d). c Detected by NMR analysis of the solvent-partitioned crude extracts. d Detected by LCMSMS analysis of the solvent-partitioned extracts. e Cultures paired on potato dextrose agar in Petri dishes incubated at 25 x (8 d). A. zeae inoculations were performed 48 h before Aspergillus flavus (NRRL 6541) and Fusarium verticillioides (NRRL 25457). f Reaction types (Wicklow et al. 1980): ‘B’ mutual inhibition on contact, the space between the two colonies is small, but clearly marked; ‘E’, inhibition of one organism at a distance, the antagonist continues to grow through the resulting clear zone at an unchanged rate. g ND, not detected.

13540), which displayed significant antifungal activity 5 1 against Aspergillus flavus and Fusarium verticillioides in H conventional paper disc assays, revealed that the 3 9 metabolites accounting for this activity were two newly 6 7 15 reported antibiotics pyrrocidines A and B (Fig. 1). O Pyrrocidine A at 250 mg discx1 and pyrrocidine B at x1 13 H 17 200 mg disc were more strongly inhibitory to the 11 1' H O growth of F. verticillioides, as measured by the zone of O NH inhibition surrounding each disc, than to A. flavus. The 23 19 pyrrocidines were first reported from the fermentation 26 OH broth of an unidentified species of Cylindrocarpon LL- 21 Cyan426, isolated from a mixed douglas dir hardwood 1 forest on Crane Island Preserve, Washington, in 1993 (He et al. 2002, Bigelis et al. 2003). The pyrrolidinone function has been reported in another antifungal com- H pound, talaroconvolutin A produced by Talaromyces convolutus (Suzuki et al. 2000) along with four tetramic acid derivatives including ZG-1494, a compound first H O isolated from Penicillium rubrum (West et al. 1996). P. rubrum also produces rubratoxin B which interferes H H O with cell wall synthesis and leads to hyphal defor- O NH mation in many fungi (Reiss 1972). The presence of pyrrocidines A and B in ethyl acetate OH extracts of maize kernel fermentations was determined for 13 cultures of Acremonium zeae isolated from maize 2 kernels maintained by the ARS Culture Collection (Table 1). This is the first report of natural products Fig. 1. Structures of pyrrocidine A (1) and B (2). from A. zeae. Pyrrocidines were detected in fermen- tation extracts for twelve cultures of A. zeae isolated from maize kernels harvested in Illinois (4 cultures), detected in three cultures. The production of pyrro- North Carolina (5), Georgia (1) and unlisted locations cidines by the majority of A. zeae isolates from maize within the USA (2). Pyrrocidine A and B were detected suggests that the ability to produce these antibiotics in nine of the cultures while pyrrocidine B alone was could be important to fungal endophyte interactions D. T. Wicklow and others 615 with competing microbes and contributes to fungal 80 survival in maize cultivation. Pyrrocidine B was detected by LCMSMS in pick-out 70 kernel samples removed at harvest from ears of a commercial hybrid that were wound-inoculated with 60 A. zeae strains NRRL 13540 or NRRL 13541. The 50 samples consisted of whole symptomatic maize kernels showing some discolouration of the endosperm or the 40 30 ºC presence of white chalky stripes on the surface of the 25 ºC pericarp (Koehler 1942). Our LCMSMS method 30 enabled us to determine the presence or absence of 37 ºC pyrrocidines A and B in an organic extract but not the Average colony diam (mm) 20 15 ºC concentrations of these compounds. Regional aflatoxin outbreaks are commonly ac- 10 5 ºC (no growth) companied by outbreaks in fumonisin (Mubatanhema et al. 2002) and therefore, aflatoxin and fumonisin can 0 2 46810 12 14 16 be present at unacceptable levels in the same grain Days samples at harvest (Chamberlain et al. 1993, Chu & Li Fig. 2. Growth-increment curves for Acremonium zeae 1994). Aflatoxins are primarily associated with hepatic (NRRL 6415). disease and are powerful hepatotoxins, teratogens, mutagens, and carcinogens, while also causing decreased milk and egg production and suppression of temperatures supporting growth, and share the same immunity in animals consuming low dietary con- optimum for fungal growth. F. verticillioides isolates centrations (Council for Agricultural Science and show temperature optima for growth on agar media of Technology 2003). Fumonisins have been reported to 22.5–30 x with a minimum of 2.5–5 x and a maximum of induce several diseases in animals, including leuko- 35–37.5 x with no growth at 40–45 x (Marin et al. 1995, encephalomalacia in horses (Marasas et al. 1988), Samson et al. 1995). Maximum colony diameters for pulmonary edema in swine (Harrison et al. 1990), act as A. zeae isolates NRRL 6415 (Fig. 2) and NRRL 13540, tumour promoters in rats (Gelderbloom et al. 1996) on PDA after 14 d, were attained within the range of and may also be associated with oesophageal cancer in 25–30 x, with less growth recorded at 15 x and 37.5 x, humans (Rheeder et al. 1992, Chu & Li 1994). The co- and no growth at 5 x. We could not determine the occurrence of these mycotoxins may increase the cancer temperature optimum for pyrrocidine production risk of aflatoxin exposure. Fumonisin B1 was recently because there is presently no method for measuring the shown to promote aflatoxin B1-initiated liver tumours concentration of pyrrocidines. when fed to rainbow trout (Carlson et al. 2001). Aflatoxin outbreaks in preharvest maize are as- Therefore, in considering among ‘protective’ maize sociated with high temperatures during grain develop- endophytes as relevant biocontrol agents, consideration ment (Anderson et al. 1975), especially high evening should be given to microbes that can simultaneously temperatures associated with stress to maturing prevent both aflatoxin- and fumonisin-contamination maize kernels (Smart et al. 1990, Wicklow 1994). of the grain at harvest. Experimental maize inoculations in an environmental In an evaluation of cultural antagonism between 13 growth room showed that Acremonium zeae and F. isolates of A. zeae in pairings with Aspergillus flavus verticillioides could prevent the spread of Aspergillus (NRRL 6541) and Fusarium verticillioides (NRRL flavus infection in preharvest maize at 30 x day and 20 x 25457), Acremonium zeae (NRRL 6415 and NRRL night temperatures (Wicklow et al. 1988). Under these 34556) inhibited both organisms at a distance while mild or moderate temperatures Acremonium zeae and continuing to grow through the resulting clear zone at F. verticillioides can compete successfully with an unchanged rate, designated reaction type ‘E’ (Table Aspergillus flavus and limit the frequency of kernel in- 1). A. zeae (NRRL 34558) and (NRRL 34560) also fection and aflatoxin contamination. F. verticillioides produced reaction type ‘E’ in cultural pairings with and F. proliferatum were generally very competitive and Aspergillus flavus, but in cultural pairings with dominant against A. flavus over a range of tempera- Fusarium verticillioides showed mutual inhibition on tures (10, 15, 25, 30 x) and 0.994 to 0.96 aw (water contact, the space between the two colonies being small availability) (Marin et al. 1998). At the same time, but clearly marked as designated for reaction type ‘B’. A. flavus isolates show a higher temperature optima for Cultural pairings with the remaining Acremonium zeae growth on agar media of 35–37 x with a minimum of 6 x isolates and Aspergillus flavus or F. verticillioides pro- and a maximum of 45 x (Schindler et al. 1967, Magan & duced reaction type ‘B’. This is the first demonstration Lacey 1984). Aspergillus flavus may overcome the of Acremonium zeae interference with the faster competitive effects of A. zeae and F. verticillioides when growing F. verticillioides. A. zeae and F. verticillioides temperatures equal or exceed the 35–37.5 x maximum compete with one another over the same range of growth temperatures recorded for these endophytes. Antifungal antibiotics in Acremonium zeae 616

Pyrrocidine A exhibits potent activity against most 1968a). In field grown maize, seeds infected with A. Gram-positive bacteria (He et al. 2002) and could have zeae did not produce plants having a greater incidence an important role in interactions between Acremonium of stalk rot or any other obvious symptoms of disease zeae and bacterial pathogens or endophytes of maize. than uninfected seeds (Harris 1936, Sumner 1968a, Gram-positive bacterial pathogens of maize include King 1981). Stalk rot is a ‘disease complex’ and it is Clavibacter michiganense subsp. nebraskense (syn. often difficult to determine the cause of stalk rot in the Corynebacterium nebraskense), causal agent of Goss’s field as many kinds of fungi and bacteria, both primary bacterial wilt and leaf freckles in maize (Ngong-Nassah pathogens and secondary invaders, can often be iso- et al. 2002). It will be interesting to learn whether pyrro- lated from a small piece of necrotic tissue (Christensen cidine antibiotics are produced by A. zeae in healthy & Wilcoxson 1966). Therefore, it should not be sur- maize tissues and contribute to the suppression of prising that two of the most common systemic fungal bacterial or fungal pathogens. Fisher et al. (1992) were endophytes of maize, A. zeae and F. verticillioides, are the first to establish that symptomless populations of also commonly recorded as colonists of senescent or bacteria exist in healthy maize stem tissues in conjunc- dead maize stalk tissues (Christensen & Wilcoxson tion with the endophytic fungal biota. A. zeae and 1966, Sumner 1968a). Ustilago sp. were the most frequent fungal isolates from A. zeae and F. verticillioides endophytes of commer- stem core tissues. The authors recorded a low incidence cial corn hybrids should escape the negative effects of of fungal infections in the lower parts of the stems pathologist/breeder selection when their infections are which coincided with the tissues yielding the highest symptomless and have no noticeable impact on yield. bacterial counts. The bacterial endophytes isolated A. zeae could represent a confounding variable in by Fisher et al. (1992) were all Gram-negative (e.g. evaluating varietal resistance to bacterial or fungal Enterobacter, Klebsiella, Pseudomonas, Vibrio). diseases. Grain harvested from irrigated fields was However, Zinniel et al. (2002) characterized 300 bac- more heavily contaminated with A. zeae, whereas the terial isolates from 90 maize plants as Gram-positive incidence of F. verticillioides was much higher in corn (164 isolates; 55%) or Gram-negative (136 isolates; grown under dryland conditions (Sumner 1968b, Arino 45%). Furthermore, among 15 genera isolated as & Bullerman 1994). For some locations and years, the endophytes of maize and sorghum three Gram-positive selection of disease resistant lines may be the indirect genera, Bacillus, Corynebacterium, and Microbacterium result of the presence of pyrrocidine-producing A. zeae were most prevalent, having more assigned isolates. endophytes. For example, in a study of maize genotype Might pyrrocidines negatively impact nonpathogenic effect on internal fungal infection, the incidence of bacterial endophytes of maize (Fisher et al. 1992, A. zeae and F. verticillioides differed significantly Zinniel et al. 2002), including Gram-positive actino- (P<0.01) among maize hybrids grown under irrigation mycetes (Araujo et al. 2000) and bacteria which have in test plots at six locations in Nebraska (Arino & been identified for their biocontrol potential in maize Bullerman 1994). Acremonium was more often isolated (e.g. Bacillus subtilis, Chang & Kommedahl 1968; from grain harvested in 1991 from the ‘T1 genetic B. mojaviensis, Bacon & Hinton 2002, Clavibacter xyli group’, while Fusarium was isolated significantly less subsp. cynodontis, Tomasino et al. 1995)? often. The authors suggest that the ‘T1 genetic group’ A. zeae has been associated with stalk rot in mature was more resistant to Fusarium colonization without maize plants, but the damage is not always apparent as considering interference by Acremonium as a potential stalk rot (Christensen & Wilcoxson 1966), and the confounding variable. One might also ask, are maize fungus is not regarded as an aggressive parasite, es- varieties selected for their resistance to F. verticillioides pecially when compared with fungi recognized as the in field inoculation trials (e.g. King 1981, King & Scott, primary cause of stalk rot Diplodia maydis or Gibberella 1981, Ochor et al. 1987), generally more susceptible to zeae (Harris 1936, Sumner 1968a, White 1999). Reddy Aspergillus flavus infection and aflatoxin? & Holbert (1924) proposed that A. zeae is seed-borne in In the present study we have shown that Acremonium maize, beginning its development with the germinating zeae interferes with F. verticillioides and Aspergillus seed, and resulting in a systemic infection of the plant flavus colony growth in cultural pairings and produced through the vascular system, eventually invading the a pyrrocidine antibiotic in preharvest maize kernels ears and seeds of plants that appear outwardly healthy. which was active against F. verticillioides and A. flavus They isolated A. zeae from blackened vascular bundles in disc assays. We have characterized A. zeae as a and identified it as the causal agent of ‘black-bundle ‘protective endophyte’ of maize, thereby bringing disease’ of maize. Even so, the symptoms were incon- attention to the biocontrol potential of A. zeae, which sistent and subsequent research has shown the black- has never been linked to any mycotoxicosis in culti- bundles to be associated with the variety of maize vated cereals. planted and influenced by environmental conditions such as severe drought (Harris 1936). A. zeae grew systematically on maize grown in growth rooms and ACKNOWLEDGEMENT the greenhouse without significantly impacting the Support for this work from the National Science Foundation mean weight of grain produced per plant (Sumner (CHE-0079141 and CHE-0315591) is gratefully acknowledged. D. T. Wicklow and others 617

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