University of Kentucky College of Agriculture

The Vaillancourt Lab

Selected Publications and Abstracts

University of Kentucky

Department of Plant Pathology

Last updated: September 2009

To obtain a PDF copy of any of the publications listed here, contact Lisa Vaillancourt at

Colletotrichum graminicola/Corn Anthracnose Papers from the Vaillancourt Laboratory

Venard, C., Kulshrestha, S., Sweigard, J, Nuckles, E., Vaillancourt, L. 2008. The role of a fadA ortholog in the growth and development of Colletotrichum graminicola in vitro and in planta. Fungal Genetics and Biology 45: 973-983

A transposon-based split-marker protocol was used to produce insertional mutations in the fadA ortholog of the maize anthracnose pathogen Colletotrichum graminicola. The mutants grew more slowly in culture, produced fewer oval spores, produced fusiform rather than falcate phialospores, lost their normal clockwise spiral growth pattern in culture, and were significantly reduced in their pathogenicity to maize stalks and leaves. The differential effect of the fadA mutation on oval spore versus phialospore production suggests there are differences in the signaling pathways that regulate these two types of sporulation. It has been suggested that oval spores function in anthracnose lesion extension.  In maize stalks, production of oval spores appeared to be relatively unaffected in the mutant strains, but production of vegetative hyphae and elongation of primary lesions were both reduced.  This suggests that vegetative hyphae play a more important role than oval spores in primary lesion development.  However, production of discontinous secondary lesions in maize stalks infected by mutant strains did not appear to be seriously affected, and thus oval spores may play a more important role in that process.

Venard, C., Vaaillancourt, L., 2007. Penetration and colonization of unwounded maize tissues by the maize anthracnose pathogen Colletotrichum graminicola and the related nonpathogen C. sublineolum. Mycologia 99(3): 368-377

The maize anthracnose stalk-rot fungus Colletotrichum graminicola infects its host primarily through wounds in the stalks that are caused by insects.  However, it also can cause stalk-rot disease without wounding. It is not known how the pathogen enters stalks in the absence of wounds. Studies have suggested that direct invasion through the highly lignified rind tissues is not a viable means of entry.  A cytological approach was used to investigate the ability of C. graminicola to penetrate and colonize intact maize stalks. The pathogen had a significant capacity for direct penetrtion, but this mechanism of infection was much slower and less efficient than penetration through wounds. The fungus breached the lignified rind fibers by passing through small openings in the cell walls via narrow hyphal connections. Epidermal and rind fiber cells did not appear to become rotted. Rotting only occurred once the pathogen had penetrated into the pith parenchyma cells.  To our surprise the closely related fungus C. sublineolum, which is not normally a pathogen of maize, also was capable of infecting intact maize stalks, although to a lesser degree than C. graminicola. The two species also were observed on intact roots and leaves, and C. sublineolum was incapable of infecting those tissues, whereas C. graminicola efficiently colonized both.  This suggests the interesting possibility that nonhost resistance to C. sublineolum is conditional and perhaps also tissue-specific.

Venard, C., Vaillancourt, L. 2007. Colonization of fiber cells by Colletotrichum graminicola in wounded maize stalks. Phytopathology 97: 438-447

Colonization of wounded maize stalks by a wild-type strain of Colletotrichum graminicola was compared with colonization by a C. graminicola mutant that is avirulent on maize leaves, and by a wild-type srain of C. sublineolum that is normally a pathogen of sorghum but not maize. Local infection by all strains at the wound site resulted in formation of primary lesions consisting of disintegrated parenchyma cells beneath an intact rind and epidermis.  However, subsequent rapid longitudinal expansion of the primary lesion occurred only in infections with the wild-type C. graminicola strain, and proceeded specifically through the fiber cells associated with the vascular bundles and the rind. Hyphae emerged from the fiber cells to produce discontinuous secondary lesions. There was no evidence that C. graminicola is a vascular wilt pathogen. Resistance of wounded cv. Jubilee maize stalks to the mutant strain of C. graminicola and to C. sublineolum was associated with restriction of colonization and spread of the pathogen through the fibers, as well as with the limitation of localized destruction of parenchyma cells at the wound site.

Flowers, J.L., Vaillancourt, L.J. 2005. Parameters affecting the efficiency of Agrobacterium tumefaciens-mediated transformation of Colletotrichum graminicola. Current Genetics 48: 380-388

We have developed an Agrobacterium tumefaciens-mediated transformation (ATMT) protocol for the plant pathogenic fungus Colletotrichum graminicola, the cause of anthracnose leaf blight and stalk rot of corn. The ATMT results in higher transformation efficiencies than previously available polyethylene glycol-mediated protocols, and falcate spores can be used instead of protoplasts for transformation. Various experimental parameters were tested for their effects on transformation efficiencies. The parameters with the greatest influence were the A. tumefaciens strain used and the Ti-plasmid it carried, the ratio of the bacterium to fungus during cocultivation, and the length of cocultivation. Southern analysis demonstrated that most transformants (80%) contained tandem integrations of plasmid sequences, and at least 36% had integrations at multiple sites in the genome. In a majority of cases (70%), the whole Ti-plasmid, and not just the T-DNA, had integrated as a series of tandem repeats. Tandem integrations, especially of the whole plasmid, make it difficult to rescue DNA from both flanks of the integrations with standard PCR-based approaches. Thus, ATMT may be unsuitable for insertional mutagenesis of C. graminicola without further modification. 

Du, M., Schardl, C.L., Nuckles, E.M., Vaillancourt, L.J. 2005. Using mating-type gene sequences for improved phylogenetic resolution of Colletotrichum species complexes. Mycologia 97(3): 641-658

Colletotrichum species are defined primarily on the basis of host preference and morphology of the organims in planta and in culture. However, the genus contains several species complexes that encompass such a broad range of morphological and pathological variation that the species name is of relatively little use either to the taxonomist or plant pathologist. Phylogenetic analyses, primarily based on variable regions of the ribosomal DNA (rDNA) sequences, have indicated that these species complexes comprise a variable number of identifiable monophyletic clades. However, rDNA sequences often are insufficiently diverse to fully resolve such closely related lineages. A group of isolates representing three species complexes (C. graminicola, C. gloeosporioides, and C. acutatum) were analyzed by using the high mobility group (HMG)-encoding sequence of the MAT1-2 mating type sequence, which has been shown in other fungi to be especially suitable for distinguishing relationships among closely related groups. Results were compared with those obtained from analysis of variable regions of the rDNA as well as from standard morphological classification methods. Results achieved through analysis of MAT1-2 sequences correlated well with those obtained by analysis of rDNA sequences but provided significantly better resolution among the various lineages. Morphological traits, including hyphopodia size, colony appearance, spore size, appressorial shape and size, and host preference, frequently were unreliable as indicators of phylogenetic association. Spore shape and hyphopodia shape more often were useful for this purpose.

Mims, C.W., Vaillancourt, L.J. 2002. Ultrastructural characterization of infection and colonization of maize leaves by Colletotrichum graminicola, and by a C. graminicola pathogenicity mutant. Phytopathology 92: 803-812

Observations were made of the ultrastructure of infection and colonization of leaves of a susceptible maize inbred by Colletotrichum graminicola and by a C. graminicola pathogenicity mutant. The mutant causes no symptoms on either maize leaves or stalks. Prior evidence suggested that it is deficient in production of signal peptidase, responsible for cleavage of signal peptides from proteins destined for transport through the endoplasmic reticulum. There was no significant difference in the process of infection or colonization by the mutant and wild-type strains up to 48 h after inoculation. Both the mutant and the wild type produced globose, melanized appressoria within 24 h after inoculation on the host surface. By 36 h, both strains had penetrated the host epidermal cells directly. The host cells frequently formed papillae in response to appressoria, but these were not usually successful in preventing fungal ingress in either case. Penetration was followed by formation of irregularly shaped, swollen infection hyphae. Infection hyphae of both strains grew biotrophically for a relatively short time (less than 12 h). One or more hyphal branches was produced from each infection hypha, and these invaded adjacent mesophyll cells. Both strains of the fungus grew cell-to-cell, setting up new biotrophic interactions in each cell, between 38 and 48 h after inoculation. Papillae were frequently formed by the mesophyll cells, but these were not successful in preventing fungal ingress. The first noticeable difference between the mutant and the wild type was related to their interaction with the mesophyll cells. Cells invaded by the wild type died relatively quickly, whereas those infected by the mutant appeared to survive longer. The most dramatic difference between the mutant and the wild type occurred when the mutant completely failed to make a transition to necrotrophic growth, while the wild type made that switch at 48 to 72 h after inoculation. The mutant may be unable to secrete sufficient quantities of one or more proteins that are necessary to support the switch between biotrophy and necrotrophy.

Thon, M.R., Nuckles E.M., Takach J.E., Vaillancourt, L.J. 2002. CPR1: A gene encoding a putative signal peptidase that functions in pathogenicity of Colletotrichum graminicola to maize. MPMI 15: 120-128

Colletotrichum graminicola causes anthracnose leaf blight and stalk rot of maize. We used restriction-enzyme mediated insertional (REMI) mutagenesis to identify a gene in this fungus that is required for pathogenicity to both stalks and leaves. The predicted polypeptide encoded by this gene, which we have named CPR1, is similar to a family of proteins that comprise one subunit of the eukaryotic microsomal signal peptidase. The nonpathogenic CPR1 REMI mutant contains a plasmid integration in the 3' untranslated region of the gene, 19bp downstream of the stop codon. The result is a significant decrease in transcript levels in comparison to the wild type, perhaps as a result of increased transcript instability. We were unable to knock out the CPR1 gene and it may be essential for viability. Microscopic examination of the REMI mutant on maize leaves revealed that it is fully capable of penetrating and colonizing host cells during the initial biotrophic phases of the disease interaction, but, unlike the wild type, it appears to be unable to switch to a necrotrophic mode of growth. We suggest that the CPR1 REMI mutant may be unable to secrete sufficient quantities of degradative enzymes to support that transition. The CPR1 REMI mutant provides us with a useful tool for future studies of the role of fungal protein transport in this important stalk rot disease of maize.

Chaky, J., Anderson, K., Moss, M., Vaillancourt, L. 2001. Surface hydrophobicity and surface rigidity induce spore germination in Colletotrichum graminicola. Phytopathology 91: 558-564

We investigated the relationship between physical characteristics of artificial surfaces, spore attachment, and spore germination in Colletotrichum graminicola. Surface hydrophobicity and surface rigidity were both signals for breaking dormancy and initiating spore germination, but spore attachment was not an important inducing signal. The presence of a carbon source overrode the necessity for a rigid, hydrophobic substrate for spore germination. Spore attachment was typically stronger to more hydrophobic surfaces, but certain hydrophilic surfaces also proved to be good substrates for spore attachment. In contrast to spore germination, appressorial induction was more dependent on attachment to a rigid substrate than it was on surface hydrophobicity. Appressorial formation was induced efficiently on hydrophilic surfaces, as long as there was significant conidial attachment to those surfaces.

Thon M.R., Nuckles, E.M., Vaillancourt, L.J. 2000. Restriction-enzyme mediated integration (REMI) used to produce pathogenicity mutants of Colletotrichum graminicola MPMI 13: 1356-1365

We have developed a restriction enzyme-mediated insertional mutagenesis (REMI) system for the maize pathogen Colletotrichum graminicola. In this report, we demonstrate the utility of a REMI-based mutagenesis approach to identify novel pathogenicity genes. Use of REMI increased transformation efficiency by as much as 27-fold over transformations using linearized plasmid alone. Ninety-nine transformants were examined by Southern analysis, and 51% contained simple integrations consisting of one copy of the vector integrated at a single site in the genome. All appeared to have a plasmid integration at a unique site. Sequencing across the integration sites of six transformants demonstrated that in all cases the plasmid integration occurred at the corresponding restriction enzyme recognition site. We used an in vitro bioassay to identify two pathogenicity mutants among 660 transformants. Genomic DNA flanking the plasmid integration sites was used to identify corresponding cosmids in a wild-type genomic library. The pathogenicity of one of the mutants was restored when it was transformed with the cosmids.

Vaillancourt, LJ, Du, M, Wang, J, Rollins, J, Hanau, RM. 2000. Genetic analysis of cross fertility between two self-sterile strains of Glomerella graminicola. Mycologia 92 (3) pp. 430-435

The ascomycete Glomerella cingulata has an unusual and complex mating system which is controlled by multiple, multiallelic loci. Cross fertility between different isolates occurs via complementation of mutated fertility genes, a process known as unbalanced heterothallism. We have examined the heritability of cross fertility among progeny of crosses between two self-sterile strains of G. graminicola to determine if unbalanced heterothallism also occurs in this species. Both random spore and tetrad progeny were analyzed. One-half of the progeny mated with one parent, one-fourth mated with the second parent, and one-fourth did not mate with either parent. None mated with both parents, and none were self fertile. The data support a model in which cross fertility between strains is regulated by two unlinked loci. One locus may be a component of the pathway for self fertility. The other locus appears to be involved in self/nonself recognition, because alternate functional alleles must be present in the parents. However, this second locus is unlikely to be a typical ascomycete MAT idiomorph, since the HMG box of a putative MAT2 idiomorph from G. graminicola was found in both parent strains at a locus which is not linked the self/nonself recognition locus identified in this study. The data reported here suggest that cross fertility in G. graminicola is genetically complex, and that it functions at least in part via unbalanced heterothallism.

Vaillancourt, LJ, Hanau RM. 1999. Sexuality of self-sterile strains of Glomerella graminicola. Mycologia 91(4) pp. 593-596

The mating system in the ascomycete Glomerella graminicola is unlike that in heterothallic ascomycetes where mating is controlled by a single locus with two alternate specificities. G. graminicola has multiple mating types that are determined by more than one genetic locus. Crosses between a melanin-deficient (Mel-) strain and a wild type (Mel+) strain of G. graminicola produced both darkly pigmented and lightly pigmented perithecia. In heterothallic ascomycetes, it is the ascogonial parent that produces the perithecial wall and the cytoplasm of the ascospores. Given the unusual nature of the mating system in G. graminicola, it was important to test the hypothesis that perithecium color is an indicator of parentage in matings of this fungus. Although Mel+ and Mel- progeny were recovered from both types of perithecia, mitochondrial DNA RFLPs were correlated with the color of the perithecium from which the progeny were derived. Crosses involving the Mel- strain and a second Mel+ strain produced Mel+ and Mel- progeny, but only one type of mitochondrial DNA which was like that of the Mel- parent, indicating that this Mel+ strain was female sterile. The putative female sterile strain, which displays normal sensitivity to potassium chlorate (ChlS), was further examined by crossing it with a Mel+, female fertile, chlorate resistant (ChlR) strain. Among the progeny, the ChlR and ChlS phenotypes segregated 1:1 but only the type of mitochondrial DNA found in the ChlR parent was detected. These results indicate that sexual differentiation and cytoplasmic inheritance in matings of G. graminicola are like those processes in heterothallic fungi, in spite of the fundamental differences between the genetic mechanisms that regulate mating specificity in these two groups.

Anthracnose papers by Vaillancourt, and others, from the Hanau Laboratory:

Fang, G-C., Hanau, R.M., Vaillancourt, L.J. 2002. The SOD2 gene, encoding a manganese-type superoxide dismutase, is up-regulated during conidiogenesis in the plant-pathogenic fungus Colletotrichum graminicola. Fungal Genetics and Biology 36: 155-165

The SOD2 gene, encoding a manganese-type superoxide dismutase (MnSOD), was identified from Colletotrichum graminicola among a collection of cDNAs representing genes that are up-regulated during conidiogenesis. The SOD2 gene consists of a 797-bp open reading frame that is interrupted by three introns and is predicted to encode a polypeptide of 208 amino acids. All conserved residues of the MnSOD protein family, including four consensus metal binding domains, are present in the predicted SOD2 protein. However, the predicted protein does not appear to contain a signal peptide that would target it to the mitochondria. Northern hybridizations revealed that expression of the ~900-bp SOD2 transcript is closely associated with differentiation of both oval and falcate conidia. Southern analysis indicated that there is only a single copy of the gene. SOD2 disruption strains were morphologically and pathogenically indistinguishable from wild-type strains. The dispensability of the MnSOD enzyme may be due to the activities of two other SOD enzymes, a highly-expressed iron-type superoxide dismutase and a much less abundant copper/zinc type, that were also detected in C. graminicola.

Vaillancourt, LJ, Hanau, RM. 1994. Nitrate-nonutilizing mutants used to study heterokaryosis and vegetative compatibility in Glomerella graminicola (Colletotrichum graminicola) Exp Mycol 18: 311-319

Nitrate-nonutilizing (nit) mutants of Glomerella graminicola were recovered by selecting chlorate-resistant sectors. Heterokaryons were formed by complementation between two different classes of nit mutants. Complementation groups were distinguished in nitrogen feeding tests and segregated as two, unlinked genes among random progeny of sexual crosses. The two genes are comparable to those encoding the nitrate reductase enzyme and one of a series of molybdenum cofactors in Aspergillus nidulans and Neurospora crassa. Heterokaryon tests were reliable indicators of allelic and dominance relationships between mutations with similar phenotypes. Vegetative compatibility (VC) between two strains of G. graminicola appeared to be regulated by approximately five unlinked VC loci, analogous to those described for other fungi.

Vaillancourt LJ, Hanau RM. 1994. Cotransformation and targeted gene inactivation in the maize anthracnose fungus, Glomerella graminicola. Applied and Environmental Microbiology 60: 3890-3893

Cotransformation of Glomerella graminicola was achieved with the G. graminicola genes TUB1R1 (encoding a b-tubulin which confers resistance to the fungicide benomyl) and PYR1 (encoding orotate phosphoribosyl transferase, which confers pyrimidine prototrophy. The cotransformation frequency was about 30% when selection was for pyrimidine prototrophy (Pyr+) and 87% when selection was for benomyl-resistant (Bmlr) transformants. Southern blots confirmed that both transforming DNAs had integrated into the genomes of transformants which were expressing both Pyr+ and Bmlr phenotypes. A plasmid, p23, which contained a truncated 500-bp segment representing the central region of the PYR1 gene was constructed. The plasmid was introduced with pCG7, containing TUB1R1, into G. graminicola M1.001 (Pyr+ Bmls) and Bmlr transformants were selected. The Bmlr transformants were screened on medium which did not contain uridine in order to identify Pyr- mutants created by integration of p23 at the PYR1 locus. None of the primary transformants were Pyr-, but 0.2% of uninucleate conidia collected from the pooled primary transformants gave rise to Pyr- auxotrophs. Southern blots representing two of these Pyr- mutants confirmed that they had the expected homologous integration of p23 at the PYR1 locus. This suggested that the integration resulted in production of two nonfunctional copies of the gene, one lacking the 5' sequences and the other lacking the 3' sequences. This study demonstrates the feasibility of using cotransformation to perform targeted gene disruptions in G. graminicola.

Vaillancourt LJ, Hanau RM. 1992. Genetic and morphological comparisons of Glomerella (Colletotrichum) isolates from maize and from sorghum. Exp Mycol 16: 219-229

Various morphological and genetic characteristics were compared among six isolates of Colletotrichum from maize and six from sorghum. For the first time, a teleomorph was induced in sorghum isolates by pairing them on autoclaved sorghum leaves in a humidity chamber. The sorghum teleomorph was morphologically similar to Glomerella graminicola and Glomerella tucumanensis, the teleomorphs of Colletotrichum isolates from maize and from sugarcane, respectively. Mating tests demonstrated that Glomerella isolates from maize and sorghum were not interfertile. Several small but consistent differences in the morphologies of the isolates from maize and from sorghum were observed which agreed with earlier reports. DNA fingerprints detected as restriction fragment length polymorphisms of mitochondrial DNA and random polymorphic DNA (RAPD) produced from nuclear DNA by the polymerase chain reaction could be used to reliably and unambiguously distinguish members of the two groups of isolates. Results of a statistical analysis of similarity of the RAPD fingerprints suggested that maize and sorghum isolates of Colletotrichum are only about 45% similar (+/- 10%) and represent two distinct and separate genetic lineages. We conclude that isolates of Colletotrichum from maize and sorghum are sibling species, since they are morphologically very similar but reproductively completely isolated.

Vaillancourt LJ, Hanau RM. 1991. A method for genetic analysis of Glomerella graminicola (Colletotrichum graminicola) from maize. Phytopathology 81: 530-534

Strains derived from nine different isolates of Colletotrichum graminicola from maize participated in the production of perithecia when incubated on pieces of autoclaved corn leaves in a humidity chamber. Matings occurred between self-fertile and self-sterile strains, and also between certain self-sterile strains. As many as 200 ascospore progeny were recovered easily from individual perithecia. Characterization of progeny showed that sexual recombination and Mendelian segregation of distinct traits could be detected. Segregation of markers for chlorate resistance (ChlR), benomyl resistance (BmlR) and melanin deficiency (Mel-) approximated a 1:1 ratio and defined three separate linkage groups. Crosses involving a pyrimidine auxotroph (Pyr-) showed 2:1 segregation (Pyr+/Pyr-) and linkage between markers for Pyr- and ChlR. Attempts to combine multiple markers resulted in successful construction of a Mel- Pyr- self-fertile strain that was crossed with a BmlR strain to produce offspring with a triple-mutant Mel- Pyr- BmlR phenotype.

Panaccione, D.G., Vaillancourt, L.J., Hanau, R.M. 1989. Conidial dimorphism in Colletotrichum graminicola. Mycologia 81(6): 876-883

Colletotrichum graminicola produced two types of conidia in culture and during infection of corn leaves. One was lunate to falcate and was produced blastically from morphologically distinct conidiogenous cells. The second type was oval to elliptic, variable in size but smaller than falcate conidia, and was produced blastically from hyphae that lacked distinct conidiogenous cells. Falcate conidia each contained one nucleus, whereas the oval type contained one to four nuclei. A difference in cell wall structure was suggested by greater sensitivity of oval conida to cell-wall degrading enzymes. Oval conidia were the only type produced in shake culture grown in the dark. Both types of conidia were produced by cultures on agar media and by standing cultures in small volumes of liquid medium but, unlike oval conidia, development of falcate conidia was light-dependent. When used to inoculate susceptible corn seedlings, both types of conida caused symptoms comparable over a range of inoculum concentrations. Regardless of the type of conidia used to inoculate seedlings, both were found in lesions of infected leaves.

Colletotrichum higginsianum/Arabidopsis Anthracnose Papers, a collaboration with the Kachroo Laboratories

Venugopal, S.C., Chanda, B., Vaillancourt, L., Kachroo, A., Kachroo, P. 2009. The common metabolite glycerol-3-phosphate is a novel regulator of plant defense signaling. Plant Signaling and Behavior 4: 8, 1-4

Conversion of glycerol to glycerol-3-phosphate (G3P) is one of the highly conserved steps of glycerol metabolism in evolutionary diverse organisms. In plants, G3P is produced either via the glycerol kinase (GK)-mediated phosphorylation of glycerol, or via G3P dehydrogenase (G3Pdh)-mediated reduction of dihydroxyacetone phosphate (DHAP). We have recently shown that G3P levels contribute to basal resistance against the hemibiotrophic pathogen, Colletotrichum higginsianum. Since a mutation in the GLY1-encoded G3Pdh conferred more susceptibility compared to a mutation in the GLI1-encoded GK, we proposed that GLY1 is the major contributor of the total G3P pool that participates in defense against C. higginsianum.

Chanda, B., Venugopal, S.C., Kulshrestha, S., Navarre, D.A., Downie, B., Vaillancourt, L.J., Kachroo, A., Kachroo, P. 2008. Glycerol-3-phosphate levels are associated with basal resistance to the hemibiotrophic fungus Colletotrichum higginsianum in Arabidopsis. Plant Physiology 147: 2017-2029

Glycerol-3-phosphate (G3P) is an important component of carbohydrate and lipid metabolic processes. In this article, we provide evidence that G3P levels in plants are associated with defense to a hemibiotrophic fungal pathogen Colletotrichum higginsianum. Inoculation of Arabidopsis (Arabidopsis thaliana) with C. higginsianum was correlated with an increase in G3P levels and a concomitant decrease in glycerol levels in the host. Plants impaired in utilization of plastidial G3P (act1) accumulated elevated levels of pathogen-induced G3P and displayed enhanced resistance. Furthermore, overexpression of the host GLY1 gene, which encodes a G3P dehydrogenase (G3Pdh), conferred enhanced resistance. In contrast, the gly1 mutant accumulated reduced levels of G3P after pathogen inoculation and showed enhanced susceptibility to C. higginsianum. Unlike gly1, a mutation in a cytosolic isoform of G3Pdh did not alter basal resistance to C. higginsianum.  Furthermore, act1gly1 double-mutant plants were as susceptible as the gly1 plants. Increased resistance or susceptibility of act1 and gly1 plants to C. higginsianum, respectively, was not due to effects of these mutations on salicylic acid- or ethylene-mediated defense pathways. The act1 mutation restored a wild-type-like response in camalexin-deficient pad3 plants, which were hypersusceptible to C. higginsianum. These data suggest that G3P-associated resistance to C. higginsianum occurs independently or downstream of the camalexin pathway. Together, these results suggest a novel and specific link between G3P metabolism and plant defense.

Diplodia (Sphaeropsis) sapinea/Pine Tip Blight Papers, a collaboration with John Hartman.

Bateman, A., Hartman, J., Vaillancourt, L. 2009. The role of shearing in the management of Diplodia tip blight.  American Christmas Tree Journal 53(4): 20-24

Diplodia tip blight, caused by the fungus Diplodia pinea, has been increasing in importance as a disease of Scots pine Christmas trees in Kentucky and the surrounding regions.  It was suggested that D. pinea propagules can be acquired on tools during shearing of diseased trees, and that this inoculum contributes to disease transmission. Propagules were detected on tools after shearing diseased trees on two farms in 2005 and 2006.  However, results of this study did not support the hypothesis that spore transmission was occurring on the farms via shearing. Infections occurred, instead, via needle bases of elongating unsheared shoots. In greenhouse studies, application of at least 5000 D. pinea spores to sheared shoot tips was necessary for consistent production of symptomatic infections. Allowing sheared shoot tips to heal for at least six hours before applying inoculum reduced subsequent disease development. Results of this study suggest that disinfesting shearing tools may not always be necessary, and that shearing can actually be beneficial in some cases for management of tip blight on Christmas trees.

Hartman, J.R., Vaillancourt, L.J., Flowers, J.L., Bateman, A.M. 2009. Managing Diplodia tip blight of landscape Austrian pines. Arboriculture and Urban Forestry 35(1): 27-32

A long-term survey of 449 Austrian pines growing on the University of Kentucky campus revealed that Diplodia tip blight disease killed 84% of the trees during a 15-year period and that the pines developed increasing levels of disease as they aged. Treatments consisting of the fungicides oxycarboxin, debacarb, or tebuconaole or water were injected into the lower trunk and root flare of individual trees in a block of mature, diseased pines for 4 years. The fungicide treatments did not significantly affect disease levels. The causal fungus could be isolated readily from diseased and symptomless shoots regardless of the treatment.  Diplodia pinea was very sensitive to tebuconazole and debacarb in invitro fungicide tests. Basal drenches with paclobutrazol affected shoot growth but did not significantly reduce tip blight disease levels or tree mortality.  Under midsouth United States climate conditions, Diplodia tip blight of Austrian pines is destructive and very difficult to manage.

Flowers, J.L., Hartman, J.R., Vaillancourt, L.J. 2006. Histology of Diplodia pinea in diseased and latently infected Pinus nigra shoots. Forest Pathology 36: 447-459

Diplodia tip blight is a serious disease of >30 conifer species worldwide. Symptoms are particularly severe on non-native, two-needled Pinus species, and typically include stunted, necrotic needles and shoots and a general decline of the tree. Latent Diplodia pinea infections occur in current-year shoots of some symptomless pines, and in some apparently healthy current-year shoots of diseased pines. Latent infections also occur in symptomless terminal buds. A histological approach was used to investigate the nature of latent infections in shoot stems and terminal buds of landscape Pinus nigra. Fungal colonization was compared in healthy, diseased, and latently infected tissues. A nested-PCR technique that is specific for D. pinea was used to differentiate latently infected tissues from uninfected ones. Latent D. pinea infections were localized in the outer stem cortex, usually in the vicinity of needle scales at leaf axes. In contrast, pathogenic D. pinea infections were characterized by fungal colonization throughout the shoot stem tissues, even very early in symptom development. The presence of necrophylatic periderms in two of the latently infected samples suggests that host defences play a role in the production and maintenance of latent stem infections. Latent infections of terminal buds appeared to originate from the distal bud scales of axillary buds in the terminal bud cluster, and not from the subtending shoot. Fungal tissues were never observed inside asymptomatic, PCR-negative shoots.

Flowers, J., Hartman, J., Vaillancourt, L. 2003. Detection of latent Sphaeropsis sapinea infections in Austrian pine tissues using nested-polymerase chain reaction. Phytopathology 93: 1471-1477

Sphaeropsis sapinea is the causal agent of Sphaeropsis tip blight disease of pines. Past surveys of diseased and symptomless Austrian and Scots pines revealed that latent infections of symptomless shoots by S. sapinea are common. The role of these latent infections in the tip blight disease is unknown. A sampling technique and nested-polymerase chain reaction (PCR) protocol were developed to detect latent S. sapinea in symptomless pine shoots. The sampling protocol was designed to be minimally destructive to the shoot so it could be preserved for further studies.  The primers that were developed were specific for S. sapinea DNA and did not amplify DNA from any of 13 other endophytic fungal species that were commonly isolated from symptomless pine shoots. The PCR primers also amplified DNA of Botryosphaeria obtusa, which was, however, rare in symptomless Austrian pine tissues. The protocol detected as little as 0.93 pg of S. sapinea DNA in terminal bud samples and 10.4 pg of DNA in bark samples.  Correlation (chi-square) analyses indicted that the nested-PCR protocol detected latent S. sapinea infections in both bud and bark samples with an efficiency that was statistically equivalent to isolating the fungus from the tissue. The nested-PCR protocol will make it possible to more quickly identify latent S. sapinea infections in symptomless pine shoots and should be useful in future studies of the latency phenomenon.

Flowers, J., Nuckles, E., Hartman, J., Vaillancourt, L. 2001. Latent infection of Austrian and Scots pine tissues by Sphaeropsis sapinea. Plant Disease 85: 1107-1112

This study documents latent infection of Austrian and Scots pine tissues by Sphaeropsis sapinea, the causal agent of Sphaeropsis tip blight disease. Symptomless shoots from diseased or apparently healthy Austrian and Scots pine trees were cultured and analyzed for the presence of S. sapinea. Nearly half of the sampled Austrian and Scots pine trees that appeared to be completely healthy had detectable latent S. sapinea infections. More than half of the symptomless shoots on visibly diseased Austrian and Scots pine trees were also latently infected with S. sapinea. S. sapinea was isolated from symptomless shoot stems, needles, buds, immature cones, and male flowers. In symptomless infected shoots, the fungus was primarily associated with bark and phloem tissues. Six isolates of S. sapinea from symptomless Austrian and Scots pine shoot tissues were pathogenic on Austrian pine seedlings in the greenhouse. The presence of S. sapinea in symptomless tissues of tip-blight diseases trees has important implications for disease management.

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