MPMI Vol. 10, No.2, 1997, pp. 147-152. Publication no. M-1997-0109-01O. "".II :-,• nV 8·. Current Review Supplied by U.S. Dept. of Agriculture Mycotoxins in Plant Pathogenesis National Center for [\nr!t'nihi,t,~l Utiiization Research p Peoria r aW,,§\.YL) Anne E. Desjardins1 and Thomas M. Hohn2 lBioactive Agents Research and 2Mycotoxin Research, National Center for Agricultural Utilization Research, USDAIARS, 1815 N. University Street, Peoria IL 61604 U.S.A. Received 11 November 1996. Accepted 13 December 1996. The study of fungal toxins in plant pathogenesis has made Mycotoxins are defined as low molecular weight fungal remarkable progress within the last decade. Prior to the mid metabolites that are toxic to vertebrates. Mycotoxins can have 1980s there was indeed a long history of research on fungal dramatic adverse effects on the health of farm animals and toxins. Fungal cultures provided a bewildering array of low humans that eat contaminated agricultural products. Myco­ molecular weight metabolites that demonstrated toxicity to toxicology has not been a traditional field of plant pathologi­ plants. But although it was easy to demonstrate that fungal cal research. Mycotoxin research has historically been per­ cultures contained toxic substances, it proved far more diffi­ formed by natural product chemists, mycologists, animal cult to establish their causal role in plant disease (Yoder toxicologists, and human disease epidemiologists. The appar­ 1980). Critical analysis of the role of toxins in pathogenesis ent lack of specificity of mycotoxins has hindered the accep­ had to wait for the development of laboratory methods to spe­ tance of a role for mycotoxins in plant pathogenesis. In addi­ cifically eliminate a toxin from a biological system. The de­ tion, mycotoxin contamination was perceived to be a post­ velopment of DNA-mediated transformation of fungal species harvest problem of stored grain. But it is now well established during the 1980s provided the essential tool to rigorously test that many mycotoxin-producing fungal species cause plant the role of toxins, and other factors, in plant pathogenesis. disease under field conditions. It thus becomes logical to ask Beginning in the 1960s, biochemical and classical genetic whether mycotoxins themselves play a role in plant patho­ analyses provided strong evidence that toxins produced by genesis in addition to their role in animal diseases. three Cochliobolus spp. are important in plant pathogenesis. A wide variety of fungal metabolites are both mycotoxic These classic systems are (l) HC-toxin, a cyclic tetrapeptide, (toxic to animals) and phytotoxic (toxic to plants). This paper and C. carbolllllll, which causes Northern leaf blight of maize; will focus on four classes of mycotoxins of continuing impor­ (2) T-toxin, a linear polyketide, and C. heterostrophus, which tance in animal and human diseases worldwide: ergot alkaloids, causes Southern leaf blight of maize with Texas male-sterile aflatoxins, trichothecenes, and fumonisins. For each mycotoxin cytoplasm; and (3) victorin, a chlorinated cyclic pentapeptide, class, we will present a brief toxicological history, and an update and C. victoriae, which causes Victoria blight of oats. Re­ on the current status of toxin pathway genetic analysis and its cently, mutants of C. carbollum and C. heterostrophus with application to the role of each toxin in plant pathogenesis. gene disruptions that block the biosynthesis of their charac­ teristic toxins have been produced by DNA-mediated trans­ Ergot alkaloids. formation (panacccione et al. 1992; Yang et al. 1996). These The most notorious mycotoxicosis in human history is er­ toxin-nonproducing mutants were greatly reduced in viru­ gotism, which is caused by consumption of grain, usually rye, lence, thus firmly establishing the importance of HC-toxin and contaminated with sclerotia of Claviceps pUlpurea. Ergotism T-toxin in pathogenesis on susceptible genotypes of maize. has been known for more than 2,000 years, and was responsi­ The success of these pioneering studies of Cochliobolus spp. ble for numerous epidemics of the disease called St. An­ has encouraged the application of gene disruption techniques thony's Fire, which included gangrene of the extremities, con­ to other, less well-established, fungal systems. In this paper vulsions, psychoses, and death, in Europe during the Middle we will discuss recent progress toward applying gene disrup­ Ages. Outbreaks of ergotism are now rare in human popula­ tion to testing the role of mycotoxins in plant pathogenesis. tions, largely because modem grain-cleaning procedures re­ move most sclerotia. In 1918, the alkaloid ergotamine was isolated from sclerotia of C. purpurea and proven to be a po­ Corresponding author: Anne E. Desjardins tent vasoconstrictor. Sclerotia can contain a complex mixture E-mail: [email protected] of biologically active alkaloids, which are the principal causa­ Names are necessary to report factually on available data; however, the tive agents of ergot poisoning (Marasas and Nelson 1987; USDA neither guarantees nor warrants the standard of the product, and Beardall and Miller 1994). the use of the name by the USDA implies no approval of the product to Ergot alkaloids represent a large family of mycotoxins that the exclusion of others that may also be suitable. are derived from both amino acid and isoprenoid precursors, and include clavines, simple derivatives of lysergic acid, and This article is in the public domain and not copyrightable. It may be freely reprinted with customary crediting of the source. The American Phytopa­ structurally complex ergopeptines such as ergotamine. The thological Society, 1997. core structural feature of ergot alkaloids is the ergoline nu- Vol. 10, No.2, 1997/147 cleus, which is formed from 4-(y,y-dimethylally1)tryptophan. 18,000 men in Shanghai, China, have provided the strongest Synthesis of 4-(y,y-dimethylallyl)tryptophan, the branch point evidence to date that aflatoxins themselves increase the risk of step in ergot alkaloid biosynthesis, is catalyzed by the prenyl­ human liver cancer and that aflatoxins interact synergistically transferase, 4-(y,y-dimethylallyl)tryptophan (DMAT) synthase, with hepatitis B virus (Scholl and Groopman 1995). A par­ from dimethylallyl diphosphate and tryptophan. The gene en­ ticularly critical aspect of these studies was the measurement coding DMAT was recently isolated from C. purpurea (Tsai et of aflatoxin adducts in urine and serum as a means of accu­ aI. 1995), but no information is available concerning the pos­ rately relating aflatoxin metabolism to an individual's risk of sibility that additional ergot alkaloid pathway genes may be developing hepatocellular carcinoma. The biologically effec­ closely linked, as occurs in other fungal toxin pathways. tive dose of aflatoxins is determined by aflatoxin metabolism Biosynthesis of the ergopeptines is catalyzed by a non­ as well as by dietary intake, because the aflatoxin B J parent ribosomal peptide synthetase that employs D-Iysergic acid, D­ compound is not harmful prior to metabolic activation. The proline, and two additional amino acids as substrates. For er­ liver Phase I detoxification pathway forms aflatoxin BJ-8,9­ gotamine, these unspecified amino acids are L-alanine and L­ epoxide, which is believed to cause site-specific mutations in phenylalanine. Genes encoding specific peptide synthetases the tumor suppressor gene p53 that lead to carcinogenesis involved in ergopeptine biosynthesis have not been identified (Scholl and Groopman 1995). in C. purpurea. However, portions of genes for putative pep­ Aflatoxins are produced by A. flavus and A. parasiticus; tide synthetases have been amplified by polymerase chain re­ whereas a wide range of Aspergillus spp. produce the afla­ action from C. purpurea and from the closely related species toxin precursor sterigmatocystin, which also is an animal Acremonium coenophialum (Panaccione 1996). toxin and carcinogen. The aflatoxinlsterigmatocystin path­ Outbreaks of ergotism occur in animals that eat grain con­ ways of Aspergillus spp. are perhaps the most thoroughly taminated with C. purpurea and other Claviceps spp. Similar studied fungal polyketide pathways. The first step in the bio­ mycotoxicoses occur in livestock that graze on pastures of synthesis of sterigmatocystinlaflatoxin is catalyzed by a type I certain fescue and ryegrass species that are infected with vari­ polyketide synthase (Chang et aI. 1995; Feng and Leonard ous Acremonium endophytes. These endophytic fungi appear 1995). In contrast to most polyketide synthases, which utilize to enhance growth, disease resistance, and drought tolerance acetate as a starter unit, the starter unit for the aflatoxinl of their grass hosts, but also contaminate them with ergot al­ sterigmatocystin enzyme is hexanoate (Brobst and Townsend kaloids that produce gangrene, convulsions, and other neuro­ 1994). The synthase reaction product and first stable interme­ logical disorders in animals that graze infected pastures. En­ diate in the pathway is norsolorinic acid, which undergoes a dophyte-free pastures of fescue and ryegrass can be estab­ complex series of modifications to yield sterigmatocystin and, lished to control mycotoxicoses, but such pastures show en­ finally, aflatoxin. Studies of the sterigomatocystin pathway in hanced susceptibility to insect damage (Marasas and Nelson A. nidulans have shown that the gene encoding the polyketide 1987; Scott and Schardl 1993). Little is known