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United States Department of Agriculture

Agricultural Research Service

Corn and Sorghum Fungal Disease Laboratory
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Research
 


Corn and Sorghum
Pathology Laboratory

Larry D. Dunkle,
Supervisory Research
Plant Pathologist

The primary goal of the research is to obtain information that will expand approaches for controlling fungal diseases of corn and sorghum and lead to new and improved disease management practices. The principal research activities in the lab focus on northern leaf blight of corn, caused by Setosphaeria turcica, and gray leaf spot of corn, caused by Cercospora zeae-maydis and C. zeina. Additional projects concern the biosynthesis and action of phytotoxic metabolites produced by fungal pathogens and their role in pathogenesis. The lab is located within the Crop Production and Pest Control Research Unit of the USDA-Agricultural Research Service, in Lilly Hall on the Purdue University campus in West Lafayette, Indiana. The research is a component of a CRIS project on Molecular and Genetic Mechanisms of Fungal Disease Resistance in Grain Crops.

Current research projects in the laboratory include:

Role of cercosporin in gray leaf spot of corn. Gray leaf spot is caused by two genetically and taxonomically distinct fungi, Cercospora zeae-maydis and C. zeina. Like many species of Cercospora, C. zeae-maydis produces cercosporin, a light-induced, photoactivated perylenequinone that is toxic to a diverse array of organisms. Cercospora zeina elicits identical disease symptoms but does not produce cercosporin in culture, suggesting that cercosporin is not essential for pathogenicity or virulence. To address the role of cercosporin in pathogenesis, we initiated a project to identify, characterize, and disrupt genes associated with cercosporin biosynthesis. Analysis of a cDNA subtraction (SSH) library revealed transcripts that are up-regulated during cercosporin synthesis. We focused on sequences with high homology to genes involved in signal transduction. Northern analyses of gene expression in cercosporin-inducing and cercosporin-suppressing media confirmed the involvement of a MAP kinase kinase kinase (designated CZK3). Targeted gene disruption of CZK3 generated a mutant that is unable to produce cercosporin and does not produce conidia but is unaffected in vegetative growth. Inoculation of corn leaves with mycelia of the czk3 mutant resulted in small chlorotic lesions with minimal colonization by intercellular hyphae following penetration through stomata. The results indicate that CZK3 regulates functions that are important for pathogenesis and suggest that cercosporin is required only after the early stages of infection for extensive colonization and lesion expansion. Current efforts are directed toward understanding the role of cercosporin by analyzing mutants affected in their ability to perceive or transmit light signals and other environmental cues that influence cercosporin synthesis.

Light-regulation of conidiation and pathogenesis in Cercospora zeae-maydis and Setosphaeria turcica.  Conidia of the pathogens causing gray leaf spot and northern leaf blight are the primary inoculum, inciting leaf lesions and initiating the disease epidemic, and are the source of repeated infections via wind dispersal to other plants and fields. Sporulation in these two pathogens is inhibited by growth in constant light and regulated by nutritional factors. We are investigating the physical and nutritional factors as well as the biosynthetic activities that are essential for conidial development and pathogenesis in these fungi. Only wavelengths in the blue light range repress conidiation, which contrasts with numerous other fungal species in which blue light induces or substantially enhances conidiation. In a project led by ARS Research Associate, Dr. Burt Bluhm, our primary objective is to characterize the gene(s) encoding blue-light photoreceptors and identify light-regulated genes that influence conidiation and biosynthesis of cercosporin in order to discover potential targets for disease control. Preliminary results have indicated that mutants of C. zeae-maydis disrupted in the CWC-1 gene encoding white collar-1, a putative blue light receptor in fungi, are light blind in conidiation and cercosporin synthesis but nevertheless are unable to cause disease symptoms beyond small chlorotic spots at the site of penetration.

Molecular analysis of resistance of corn to Setosphaeria turcica. The host-pathogen interaction in northern leaf blight is controlled by single dominant genes in the corn host that condition resistance to specific races of the pathogen. Thus, it presents a rare opportunity to study gene-for-gene interactions in corn and critically evaluate the involvement of defense mechanisms established in model plant species. The approach involves microarray analyses of genes expressed during stages of disease development in near-isogenic lines of corn differing in the presence of a specific gene for resistance. In addition, this genetically defined pathosystem can be exploited to determine the mechanism of virulence in the pathogen. Our project evaluates the potential involvement of phytotoxins with genotype specificity or, alternatively, of race-specific avirulence factors, e.g., extracellular peptides, that interact directly or indirectly with the individual resistance gene products to ascertain the phenotypic consequences of the host-pathogen interaction. ARS Postdoctoral Research Associate, Dr. Burt Bluhm, is leading this project.


Lab Personnel
Corie Shaner Burt Bluhm

Corie Shaner
ARS Biological Science Lab Technician

Burt Bluhm
ARS Postdoctoral Research Associate

Results

Ras2 encoding a GTPase regulates growth and pathogenesis in Fusarium graminearum.  Fusarium graminearum is a ubiquitous pathogen of cereal crops, including wheat, barley, and maize.  Diseases caused by F. graminearum are of particular concern because harvested grains are frequently contaminated with harmful mycotoxins such as deoxynivalenol (DON). In this study, we explored the role of Ras GTPases in pathogenesis. The genome of F. graminearum contains two putative Ras GTPase-encoding genes. The two genes (RAS1 and RAS2) showed different patterns of expression under different conditions of nutrient availability and in various mutant backgrounds. RAS2 was dispensable for survival but when disrupted caused a variety of morphological defects, including slower growth on solid media, delayed spore germination, and significant reductions in virulence on wheat heads and maize silks. Intracellular cAMP levels were not affected by deletion of RAS2 and exogenous treatment of the ras2 mutant with cAMP did not affect phenotypic abnormalities, thus indicating that RAS2 plays a minor or no role in cAMP signaling. However, phosphorylation of the MAP kinase Gpmk1 and expression of a secreted lipase (FGL1) required for infection were significantly reduced in the ras2 mutant. Based on these observations, we hypothesize that RAS2 regulates growth and virulence in F. graminearum by regulating the Gpmk1 MAP kinase pathway. This research was conducted in collaboration with Dr. Jin-Rong Xu and members of his lab at Purdue University.

Mapping of the Pc locus of Sorghum bicolor, a gene controlling the reaction to a fungal pathogen and its host-selective toxin. Milo disease in sorghum is caused by isolates of the soil-borne fungus Periconia circinata that produce peritoxin, a chlorinated peptide-polyketide hybrid molecule with genotype-specific toxicity. Susceptibility to milo disease and sensitivity to peritoxin is conditioned by a single, semi-dominant gene, termed Pc. The susceptible allele (Pc) converts to a resistant form (pc) spontaneously at a gametic frequency of 10-3 to 10-4. A high-density genetic map was constructed around the Pc locus using DNA markers, allowing the Pc gene to be delimited to a 0.9 cM region on the short arm of sorghum chromosome 9. Physically, the Pc-region was covered by a single BAC clone. Sequence analysis of this BAC revealed twelve gene candidates. Several of the predicted genes in the region are homologous to disease resistance loci, including one NBS-LRR resistance gene analogue that is present in multiple tandem copies. Analysis of pc isolines derived from Pc/Pc sorghum suggests that one or more members of this NBS-LRR gene family is the Pc gene that conditions susceptibility. This research was conducted in collaboration with Dr. Jeff Bennetzen and Dr. Ervin Nagy at the University of Georgia.

Identification and expression analysis of regulatory genes induced during conidiation in Exserohilum turcicum.  Light influences numerous developmental and biochemical processes in fungi. The objectives of this research were to characterize the influence of light on growth and conidiation and associated gene expression in the plant pathogenic ascomycete, Exserohilum turcicum. We found that vegetative growth was more extensive in light-dark cycles than in constant light or darkness as measured by analysis of ergosterol content and genomic DNA. Cultures grown under continuous white light or blue light (~465-480 nm) were developmentally arrested after the formation of conidiophores, whereas those grown in continuous darkness or a light/dark cycle produced mature conidia. Incubation of conidiophore-producing cultures in darkness for a minimum of 2 hr was necessary and sufficient to initiate synchronous conidiation. To identify genes that are expressed during dark-induced conidiation, we constructed subtractive cDNA libraries from cultures grown under conidiation-permissive and -repressive conditions. From 816 sequenced EST clones in the conidiation-permissive and 310 in the repressive libraries, 12 putative regulatory genes were chosen for expression analysis by quantitative real-time PCR. The majority of those genes reached maximum expression by 2 hr after initiation of the dark period and then declined to initial levels by 4 to 24 hr in darkness. Expression of two dark-induced genes remained elevated after 24 hr in darkness but was reset to initial levels if cultures were returned to light. This study revealed several genes whose expression increased rapidly after dark induction of conidiation, suggesting that they encode regulators of asexual development in E. turcicum. This research was conducted in collaboration with Dr. Joseph Flaherty, Coker College, Hartsville, SC.

 

Microcycle conidiation by Cercospora zeae-maydis may impact epidemiology of gray leaf spot.  Conidiaof Cercospora zeae-maydis are the primary inoculum causing gray leaf spot of maize. On nutrient-deficient substrates, but not on water on the leaf surface, conidia germinate and develop secondary conidia on conidiophores produced from germ tubes or conidial cells. A population of conidia increases its numbers more than two-fold by two days on the surface of a water droplet and by four-fold on trichomes. This microcycle conidiation is suppressed by hydrogen peroxide and by ammonium compounds but not by nitrate compounds, amino acids, or simple sugars. Microcycle conidiation is sensitive to a-amanitin and cycloheximide, suggesting that new RNA and proteins must be synthesized. Upon transfer from a humid to a dry atmosphere, secondary conidia and conidiophores dehydrate and collapse. Mature, dehydrated secondary conidia are liberated by wind speeds about one-third those required to liberate hydrated conidia. The dispersed secondary conidia can rehydrate and germinate normally. Because this microcycle conidiation occurs at the expense of endogenous reserves, the ability to produce secondary conidia is lost after four successive cycles without a period of growth on nutrient media. This alternative method of maintaining inoculum potential during periods of fluctuating relative humidity may have epidemiological consequences when primary conidia fail to infect.

Two related fungal pathogens cause gray leaf spot of corn. Gray leaf spot was first discovered and the pathogen described and named Cercospora zeae-maydis by Tehon and Daniels in 1925. The disease was considered a minor problem until the late 1970s when the widespread adoption of conservation tillage practices and monoculture of maize contributed to overwintering of the pathogen and build-up of inoculum. Since then, the frequency of occurrence and severity of gray leaf spot have increased throughout the U.S.A. and other parts of the world. Populations of the gray leaf spot pathogen throughout the maize-growing regions of the U.S.A. were found to be comprised of two closely related forms, which could not be readily distinguished by typical morphological criteria such as conidial dimensions and conidiophore characteristics and were therefore designated C. zeae-maydis group I and group II. However, the two groups were shown to be substantially divergent based on analysis of amplified fragment length polymorphism (AFLP) data, which indicated an average genetic distance of 80% between the two groups of isolates, whereas isolates within each of the two groups were >95% similar. In addition to the distinct AFLP profiles, the two groups were distinguished by nucleotide sequences of the internal transcribed spacer (ITS)-5.8S rDNA region. In comparative analyses of ITS nucleotide sequences of Cercospora species and anamorphs of Mycosphaerella, groups I and II clearly represented different species. Recently, Crous and colleagues confirmed the distinct separation of two species and described Cercospora zeina to replace the previous designation of C. zeae-maydis group II.

In the U.S.A., populations of C. zeae-maydis (group I) and C. zeina (group II) are differentially distributed but partially sympatric. Whereas C. zeae-maydis is generally distributed throughout maize-producing regions, C. zeina is restricted to the eastern third of the country. Significantly, the African population of the gray leaf spot pathogen was found to be comprised solely of C. zeina. The considerable genetic divergence between the species, their differential geographical distribution, and the slightly greater haplotype diversity of African isolates together with historical records suggest that C. zeina originated in Africa. Analyses of the gray leaf spot pathogen in South America have indicated that both C. zeae-maydis and C. zeina comprise the pathogen population on that continent, but only C. zeae-maydis has been recovered from a limited number of specimens from China.

Only isolates of C. zeae-maydis produced cercosporin in culture; none of the isolates of C. zeina produced cercosporin under the same conditions. Even on a variety of media, over a range of temperatures, and in different light conditions, C. zeina was unable to produce cercosporin and consistently grew more slowly than C. zeae-maydis. Those results were surprising, given the critical role that cercosporin plays in stages of pathogenesis and given that both species are highly virulent and destructive. The possibility remains that C. zeina produces cercosporin in planta and that the appropriate conditions were not provided in vitro due to a more tightly regulated expression of cercosporin biosynthesis genes. Nevertheless, variation in cercosporin production indicate populations that have diverged to the point of becoming separate species.


Publications

Bluhm, B.H., B. Dhillon, E.A. Lindquist, G.H.J. Kema, S.B. Goodwin, and L.D. Dunkle. 2008. Analyses of expressed sequence tags from the maize foliar pathogen Cercospora zeae-maydis identify novel genes expressed during vegetative, infectious, and reproductive growth. Biomed Central (BMC) Genomics 9: 523. (doi: 10.1186/1471-2164-9-523)
 
Bluhm, B.H., and L.D. Dunkle. 2008. PHL1 of Cercospora zeae-maydis encodes a member of the photolyase/cryptochrome family involved in UV protection and fungal development. Fungal Genet. Biol. 45: 1364-1372.
 
Brunelli, K.R., L.D. Dunkle, C. A. Sobrinho, A.C. Fazza, and L.E.A. Camargo. 2008. Molecular variability in the maize gray leaf spot pathogens in Brazil. Genet. Mol. Biol. 31: 938-942.
 
Bluhm, B.H., X. Zhou, J.E. Flaherty, J.R. Xu, and L.D. Dunkle. 2007. RAS2 regulates growth and pathogenesis in Fusarium graminearum. Mol. Plant-Microbe Interact. 20: 627-636.
 
Nagy, E.D., T.C Lee, W. Ramakrishna, Z. Xu, P.E. Klein, P. SanMiguel, C.P. Cheng, J. Li, K.M. Devos, K. Schertz, L. Dunkle, and J.L. Bennetzen. 2007. Fine mapping of the Pc locus of Sorghum bicolor, a gene controlling the reaction to a fungal pathogen and its host-selective toxin. Theor. Appl. Genet.114: 961-970.
 
Flaherty, J.E., and L.D. Dunkle. 2005. Identification and expression analysis of regulatory genes induced during conidiation in Exserohilum turcicum. Fungal Genet. Biol. 42: 471-481.
 
Shim, W.-B., and L.D. Dunkle. 2005. Malazy, a degenerate, species-specific transposable element in Cercospora zeae-maydis. Mycologia 97: 349-355.
 
Shim, W.-B., and L.D. Dunkle. 2003. CZK3, a MAP kinase kinase kinase homolog in Cercospora zeae-maydis, regulates cercosporin biosynthesis, fungal development, and pathogenesis. Mol. Plant-Microbe Interact. 16:760-768.
 
Lapaire, C.L., and L.D. Dunkle. 2003. Microcycle conidiation in Cercospora zeae-maydis. Phytopathology 93:193-199.
 
Shim, W.-B., and L.D. Dunkle. 2002. Identification of genes expressed during cercosporin synthesis in Cercospora zeae-maydis. Physiol. Mol. Plant Pathol. 61:237-248.
 
Wolpert. T.J., L.D. Dunkle, and L.M. Ciuffetti. 2002. Host-selective toxins and avirulence determinants: What’s in a name? Annu. Rev. Phytopathol. 40:251-285.


Last Modified: 4/13/2009