From Colletotrichum Graminicola, a Systemic Vascular Pathogen of Maize
Total Page:16
File Type:pdf, Size:1020Kb
mycological research 113 (2009) 1433–1442 journal homepage: www.elsevier.com/locate/mycres Antifungal metabolites (monorden, monocillins I, II, III) from Colletotrichum graminicola, a systemic vascular pathogen of maize Donald T. WICKLOWa,*, Annalisa M. JORDANb, James B. GLOERb aMycotoxin Research Unit, Agricultural Research Service, National Center for Agricultural Utilization Research, USDA, Peoria, IL 61604, USA bDepartment of Chemistry, University of Iowa, Iowa City, IA 52242, USA article info abstract Article history: Colletotrichum graminicola is a systemic vascular pathogen that causes anthracnose stalk rot Received 18 August 2009 and leaf blight of maize. In the course of an effort to explore the potential presence and Received in revised form roles of C. graminicola metabolites in maize, ethyl acetate extracts of solid substrate 1 October 2009 fermentations of several C. graminicola isolates from Michigan and Illinois were found to Accepted 4 October 2009 be active against Aspergillus flavus and Fusarium verticillioides, both mycotoxin-producing Available online 13 October 2009 seed-infecting fungal pathogens. Chemical investigations of the extract of one such isolate Corresponding Editor: Marc Stadler (NRRL 47511) led to the isolation of known metabolites monorden (also known as radicicol) and monocillins I–III as major components. Monorden and monocillin I displayed in vitro Keywords: activity against the stalk- and ear-rot pathogen Stenocarpella maydis while only the most Anthracnose abundant metabolite (monorden) showed activity against foliar pathogens Alternaria alter- Biotrophy nata, Bipolaris zeicola, and Curvularia lunata. Using LC–HRESITOFMS, monorden was detected Hsp90 inhibitor in steam-sterilized maize stalks and stalk residues inoculated with C. graminicola but not in Necrosis the necrotic stalk tissues of wound-inoculated plants grown in an environmental chamber. Radicicol Monorden and monocillin I can bind and inhibit plant Hsp90, a chaperone of R-proteins. It is hypothesized that monorden and monocillins could support the C. graminicola disease cycle by disrupting maize plant defenses and by excluding other fungi from necrotic tissues and crop residues. This is the first report of natural products from C. graminicola, as well as the production of monorden and monocillins by a pathogen of cereals. Published by Elsevier Ltd on behalf of The British Mycological Society. Introduction stalk rot, leaf blight, or top dieback, the latter occurring when the fungus invades stalk tissues above the ear. Sukno Colletotrichum graminicola (Holomorph: Glomerella graminicola) et al. (2008) have demonstrated that C. graminicola can infect causes anthracnose stalk rot and anthracnose leaf blight of maize roots, forming specialized infection and survival struc- maize (Zea mays) and is considered one of the most common tures (i.e. runner hyphae, hyphodia, and microsclerotia) asso- and economically important vascular stalk rot pathogens of ciated with other root pathogens. Furthermore, root infection maize (reviewed by Bergstrom & Nicholson 1999). The fungus often led to the systemic colonization of the above ground can be isolated from both healthy and anthracnose diseased stems and leaves of asymptomatic plants, the fungus being re- seeds and may produce a seedling blight, crown rot, root rot, stricted to individual vascular bundles. The authors noted that * Corresponding author. Tel.: þ1 309 681 6243. E-mail address: [email protected] 0953-7562/$ – see front matter Published by Elsevier Ltd on behalf of The British Mycological Society. doi:10.1016/j.mycres.2009.10.001 1434 D. T. Wicklow et al. while xylem cells were colonized by hyphae, no blockage of flavus and Fusarium verticillioides. Our objective was to describe the vascular system was observed, nor did the plants exhibit the isolation and characterization of antifungal metabolites symptoms of wilting typically associated with vascular dis- produced by C. graminicola and to compare their in vitro activity eases. C. graminicola can directly penetrate and colonize maize against other fungal endophytes and pathogens of maize. leaves (Mims & Vaillancourt 2002) and can also infect the highly lignified rind of maize stalks, even in the absence of Materials and methods wounds caused by insects (Venard & Vaillancourt 2007). An- thracnose leaf blight of maize occurs on juvenile corn leaves Fungal strains and after pollination on leaves that are beginning to senesce. Typical symptoms appear as semitransparent water-soaked Two maize kernel isolates of Colletotrichum graminicola were spots which become tan with a yellow to reddish brown bor- obtained from the USDA Agricultural Research Service Culture der. Dark immature acervular conidioma, also identified as Collection, Peoria (NRRL). C. graminicola NRRL 47511 was iso- acervuli or stromata, develop on necrotic host tissues, often lated by D.T.W. from a corn seed of the variety ‘Mandan Bride’ in concentric rings. Similar lesions may occur on leaf sheaths, produced at an unspecified field location in Michigan, the seed husks, and shanks (White 1999). The development of stalk an- sample obtained from Seed Saver’s Exchange, Decorah, Iowa thracnose begins during the period just before tasseling and in 2005; NRRL 47509 was isolated by D.T.W. from a white extending through anthesis, appearing earlier than stalk rots corn seed produced commercially near Cerro Gordo, Illinois produced by either Gibberella zeae or Stenocarpella maydis (Kel- in 2005. The cultures were identified based on their micromor- ler & Bergstrom 1988). The initial symptom of stalk infection is phology (Sutton 1980) and subjected to partial sequence anal- a water-soaked discoloration of rind tissue in the lower inter- ysis of the internal transcribed spacer region (ITS) and nodes. Large numbers of immature acervuli give a black ap- domains D1 and D2 of the nuclear large subunit (28S) rDNA pearance to sunken lesions covering the stalk rind. These gene using ITS5 and NL4 as polymerase chain reaction and external lesions are accompanied by a brown or black discol- sequencing primers (O’Donnell 1996; White et al. 1990). oration of the underlying pith tissue which becomes soft or Sequences were deposited in GenBank (National Center for disintegrates resulting in stalk breakage or ‘lodging’ (White Biotechnology Information) as GQ221855 (NRRL 47509) and 1999). The establishment and survival of acervuli within an- GQ221856 (NRRL 47511). Test strains were all isolated from thracnose lesions on overwintered maize crop residues is gen- maize seeds and included: Acremonium zeae NRRL 13540, erally recognized as being of primary importance to the Alternaria alternata NRRL 6410, Aspergillus flavus NRRL 6541, production of an infective inoculum. Falcate, hyaline conidia Curvularia lunata NRRL 6409, Nigrospora oryzae NRRL 6414, are produced from acervuli on crop residues left to overwinter Trichoderma viride NRRL 6418 from North Carolina, Fusarium on the soil surface, providing a source of rain splash-dissem- graminearum NRRL 31250, Stenocarpella maydis NRRL 31249 inated infective inoculum throughout the growing season from Indiana, Bipolaris zeicola NRRL 47238 from Cerro Gordo, (Lipps 1985). Greater numbers of sporulating acervuli were Illinois, F. verticillioides NRRL 25457 from South Carolina. recorded from rind surfaces and vascular bundles of stalk res- idues that overwintered on the surface of Ohio crop fields, in contrast to stalks that were buried. Fermentation conditions Competitive interactions among microbes within parasit- ized and prematurely senescent or non-living necrotic plant Colletotrichum graminicola NRRL 47511 was grown as a potato tissues can impact pathogen growth and survival (Pfender dextrose agar (PDA) slant culture (6 d at 25 C). A suspension 1996). For example, weakly parasitic fungal colonists of leaves of conidia and hyphal cells prepared from these cultures giv- 4 and stems such as species of Epicoccum, Cladosporium, and ing a propagule density of approximately 4 Â 10 ml served Alternaria can extend into senescing leaves and exploit both as inoculum. Fermentations were carried out in two 500 ml easily metabolized and more refractory components of Erlenmeyer flasks each containing 50 g of rice (Botan Brand; a declining nutrient source. Pathogens that produce primary J.F.C. International, Los Angeles, CA) that was soaked over- inoculum on necrotic crop residue are also susceptible to the night in distilled water (50 ml) before being autoclaved at À2 effects of fungal antagonists (Heye & Andrews 1983; Pfender 1.055 kg f cm for 30 min. After the flasks had cooled to et al. 1993). Hymenula cerealis (syn. ¼ Cephalosporium gramineum) room temperature, they were inoculated with 1.0 ml of the hy- which causes a vascular wilt of winter wheat, occupies the phal fragment-spore inoculum, and incubated for 30 d at vascular bundles of wheat stems and reduces competition 25 C. among potential colonists of senescent and necrotic stem tis- sues by producing a broad-spectrum antifungal antibiotic Bioassay of extractable residue (Bruehl et al. 1969). An ability to exclude other stalk rot fungi and colonists of the phylloplane from C. graminicola-parasit- Following incubation, the fermented rice substrate in each ized and senescent/necrotic maize tissues should contribute flask was first fragmented with a large spatula and then to pathogen establishment, survival and repeated production extracted three times with ethyl acetate (50 ml each time). of conidia from acervuli