Fungus Colletotrichum Graminicola Research Articles

Functional characterization of a eukaryotic melibiose transporter.

Pathogenic fungi drastically affect plant health and cause significant losses in crop yield and quality. In spite of their impact, little is known about the carbon sources used by these fungi in planta and about the fungal transporters importing sugars from the plant-fungus interface. Here, we report on the identification and characterization of MELIBIOSE TRANSPORTER1 (MBT1) from the hemibiotrophic fungus Colletotrichum graminicola (teleomorph Glomerella graminicola), the causal agent of leaf anthracnose and stalk rot disease in maize (Zea mays). Functional characterization of the MBT1 protein in baker's yeast (Saccharomyces cerevisiae) expressing the MBT1 cDNA revealed that α-D-galactopyranosyl compounds such as melibiose, galactinol, and raffinose are substrates of MBT1, with melibiose most likely being the preferred substrate. α-D-glucopyranosyl disaccharides like trehalose, isomaltose, or maltose are also accepted by MBT1, although with lower affinities. The MBT1 gene shows low and comparable expression levels in axenically grown C. graminicola and upon infection of maize leaves both during the initial biotrophic development of the fungus and during the subsequent necrotrophic phase. Despite these low levels of MBT1 expression, the MBT1 protein allows efficient growth of C. graminicola on melibiose as sole carbon source in axenic cultures. Although Δmbt1 mutants are unable to grow on melibiose, they do not show virulence defects on maize.

Common Motifs in the Response of Cereal Primary Metabolism to Fungal Pathogens are not Based on Similar Transcriptional Reprogramming.

During compatible interactions with their host plants, biotrophic plant–pathogens subvert host metabolism to ensure the sustained provision of nutrient assimilates by the colonized host cells. To investigate, whether common motifs can be revealed in the response of primary carbon and nitrogen metabolism toward colonization with biotrophic fungi in cereal leaves, we have conducted a combined metabolome and transcriptome study of three quite divergent pathosystems, the barley powdery mildew fungus (Blumeria graminis f.sp. hordei), the corn smut fungus Ustilago maydis, and the maize anthracnose fungus Colletotrichum graminicola, the latter being a hemibiotroph that only exhibits an initial biotrophic phase during its establishment. Based on the analysis of 42 water-soluble metabolites, we were able to separate early biotrophic from late biotrophic interactions by hierarchical cluster analysis and principal component analysis, irrespective of the plant host. Interestingly, the corresponding transcriptome dataset could not discriminate between these stages of biotrophy, irrespective, of whether transcript data for genes of central metabolism or the entire transcriptome dataset was used. Strong differences in the transcriptional regulation of photosynthesis, glycolysis, the TCA cycle, lipid biosynthesis, and cell wall metabolism were observed between the pathosystems. However, increased contents of Gln, Asn, and glucose as well as diminished contents of PEP and 3-PGA were common to early post-penetration stages of all interactions. On the transcriptional level, genes of the TCA cycle, nucleotide energy metabolism and amino acid biosynthesis exhibited consistent trends among the compared biotrophic interactions, identifying the requirement for metabolic energy and the rearrangement of amino acid pools as common transcriptional motifs during early biotrophy. Both metabolome and transcript data were employed to generate models of leaf primary metabolism during early biotrophy for the three investigated interactions.

Identification of virulence genes in the corn pathogenColletotrichum graminicolabyAgrobacterium tumefaciens‐mediated transformation

A previously developed Agrobacterium tumefaciens-mediated transformation (ATMT) protocol for the plant pathogenic fungus Colletotrichum graminicola led to high rates of tandem integration of the whole Ti-plasmid, and was therefore considered to be unsuitable for the identification of pathogenicity and virulence genes by insertional mutagenesis in this pathogen. We used a modified ATMT protocol with acetosyringone present only during the co-cultivation of C. graminicola and A. tumefaciens. Analysis of 105 single-spore isolates randomly chosen from a collection of approximately 2000 transformants, indicated that almost 70% of the transformants had single T-DNA integrations. Of 500 independent transformants tested, 10 exhibited attenuated virulence in infection assays on whole plants. Microscopic analyses primarily revealed defects at different pre-penetration stages of infection-related morphogenesis. Three transformants were characterized in detail. The identification of the T-DNA integration sites was performed by amplification of genomic DNA ends after endonuclease digestion and polynucleotide tailing. In one transformant, the T-DNA had integrated into the 5'-flank of a gene with similarity to allantoicase genes of other Ascomycota. In the second and third transformants, the T-DNA had integrated into an open reading frame (ORF) and into the 5'-flank of an ORF. In both cases, the ORFs have unknown function.

Sfp-type 4'-phosphopantetheinyl transferase is indispensable for fungal pathogenicity.

In filamentous fungi, Sfp-type 4'-phosphopantetheinyl transferases (PPTases) activate enzymes involved in primary (alpha-aminoadipate reductase [AAR]) and secondary (polyketide synthases and nonribosomal peptide synthetases) metabolism. We cloned the PPTase gene PPT1 of the maize anthracnose fungus Colletotrichum graminicola and generated PPTase-deficient mutants (Deltappt1). Deltappt1 strains were auxotrophic for Lys, unable to synthesize siderophores, hypersensitive to reactive oxygen species, and unable to synthesize polyketides (PKs). A differential analysis of secondary metabolites produced by wild-type and Deltappt1 strains led to the identification of six novel PKs. Infection-related morphogenesis was affected in Deltappt1 strains. Rarely formed appressoria of Deltappt1 strains were nonmelanized and ruptured on intact plant. The hyphae of Deltappt1 strains colonized wounded maize (Zea mays) leaves but failed to generate necrotic anthracnose disease symptoms and were defective in asexual sporulation. To analyze the pleiotropic pathogenicity phenotype, we generated AAR-deficient mutants (Deltaaar1) and employed a melanin-deficient mutant (M1.502). Results indicated that PPT1 activates enzymes required at defined stages of infection. Melanization is required for cell wall rigidity and appressorium function, and Lys supplied by the AAR1 pathway is essential for necrotrophic development. As PPTase-deficient mutants of Magnaporthe oryzea were also nonpathogenic, we conclude that PPTases represent a novel fungal pathogenicity factor.

Incidência de podridões do colmo, grãos ardidos e rendimento de grãos em híbridos de milho submetidos ao aumento na densidade de plantas

A adequada combinação entre a escolha da densidade de plantas e do híbrido é um dos fatores que contribuem para o aumento da produtividade do milho. O objetivo deste trabalho foi avaliar o efeito do incremento na densidade de plantas sobre a incidência de podridões do colmo, de grãos ardidos e o rendimento de grãos de dois híbridos de milho contrastantes quanto a tolerância ao adensamento. O experimento foi conduzido em Lages, SC, nas safras agrícolas 2002/03 e 2003/04, em área de semeadura direta e monocultura, sob sucessão de cobertura morta constituída de aveia preta+ervilhaca. Estudou-se a combinação de dois fatores: híbrido e densidades, utilizando-se o delineamento experimental de blocos casualizados com parcela sub-dividida. Na parcela principal avaliaram os híbridos: Speed (simples, tolerante ao adensamento) e AG 303 (duplo, intolerante ao adensamento). Nas sub-parcelas testaram-se cinco densidades de plantas: 25, 50, 75, 100 e 125 mil plantas ha-1. O aumento da densidade de plantas, proporcionou incremento linear na incidência das podridões do colmo e grãos ardidos para os dois híbridos e duas safras avaliadas. O fungo Colletotrichum graminicola foi o mais detectado em colmos doentes, seguido do Fusarium graminearum, F. verticillioides e Stenocarpella sp. Nos grãos ardidos, os fungos predominantes foram F. verticillioides, F. graminearum e Penicillium spp. O híbrido AG 303 demonstrou menor resposta no rendimento do que o híbrido Speed com o aumento da população de plantas. Não foi observada associação direta entre o maior rendimento de grãos do híbrido simples em estandes adensados e a menor incidência de doenças de colmo e de grãos ardidos.

A Chitin Synthase with a Myosin-Like Motor Domain Is Essential for Hyphal Growth, Appressorium Differentiation, and Pathogenicity of the Maize Anthracnose Fungus Colletotrichum graminicola

Chitin synthesis contributes to cell wall biogenesis and is essential for invasion of solid substrata and pathogenicity of filamentous fungi. In contrast to yeasts, filamentous fungi contain up to 10 chitin synthases (CHS), which might reflect overlapping functions and indicate their complex lifestyle. Previous studies have shown that a class VI CHS of the maize anthracnose fungus Colletotrichum graminicola is essential for cell wall synthesis of conidia and vegetative hyphae. Here, we report on cloning and characterization of three additional CHS genes, CgChsI, CgChsIII, and CgChsV, encoding class I, III, and V CHS, respectively. All CHS genes are expressed during vegetative and pathogenic development. DeltaCgChsI and DeltaCgChsIII mutants did not differ significantly from the wild-type isolate with respect to hyphal growth and pathogenicity. In contrast, null mutants in the CgChsV gene, which encodes a CHS with an N-terminal myosin-like motor domain, are strongly impaired in vegetative growth and pathogenicity. Even in osmotically stabilized media, vegetative hyphae of DeltaCgChsV mutants exhibited large balloon-like swellings, appressorial walls appeared to disintegrate during maturation, and infection cells were nonfunctional. Surprisingly, DeltaCgChsV mutants were able to form dome-shaped hyphopodia that exerted force and showed host cell wall penetration rates comparable with the wild type. However, infection hyphae that formed within the plant cells exhibited severe swellings and were not able to proceed with plant colonization efficiently. Consequently, DeltaCgChsV mutants did not develop macroscopically visible anthracnose disease symptoms and, thus, were nonpathogenic.

Penetration and colonization of unwounded maize tissues by the maize anthracnose pathogen Colletotrichum graminicola and the related nonpathogen C. sublineolum.

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 penetration, 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 cells 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.

Penetration and colonization of unwounded maize tissues by the maize anthracnose pathogen Colletotrichum graminicola and the related nonpathogen C. sublineolum.

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 penetration, 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 cells 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.

The Application of Laser Microdissection to In Planta Gene Expression Profiling of the Maize Anthracnose Stalk Rot Fungus Colletotrichum graminicola

Laser microdissection (LM) offers a potential means for deep sampling of a fungal plant-pathogen transcriptome during the infection process using whole-genome DNA microarrays. The use of a fluorescent protein-expressing fungus can greatly facilitate the identification of fungal structures for LM sampling. However, fixation methods that preserve both tissue histology and protein fluorescence, and that also yield RNA of suitable quality for microarray applications, have not been reported. We developed a microwave-accelerated acetone fixation, paraffin-embedding method that fulfills these requirements and used it to prepare mature maize stalk tissues infected with an Anemonia majano cyan fluorescent protein-expressing isolate of the anthracnose stalk rot fungus Colletotrichum graminicola. We successfully used LM to isolate individual maize cells associated with C. graminicola hyphae at an early stage of infection. The LM-derived RNA, after two-round linear amplification, was of sufficient quality and quantity for global expression profiling using a fungal microarray. Comparing replicated LM samples representing an early stage of stalk cell infection with samples from in vitro-germinated conidia, we identified 437 and 370 C. graminicola genes showing significant up- or downregulation, respectively. We confirmed the differential expression of several representative transcripts by quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) and documented extensive overlap of this dataset with a PCR-subtraction library enriched for C. graminicola transcripts in planta. Our results demonstrate that LM is feasible for in planta pathogen expression profiling and can reveal clues about fungal genes involved in pathogenesis. The method in this report may be advantageous for visualizing a variety of cellular features that depend on a high degree of histochemical preservation and RNA integrity prior to LM.

Parameters affecting the efficiency of Agrobacterium tumefaciens-mediated transformation of Colletotrichum graminicola

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 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.

Evaluation of genetic mixtures of sorghum lines for anthracnose resistance management

The main objective of this work was to evaluate the diversification of sorghum (Sorghum bicolor) populations as a way to manage resistance to the sorghum anthracnose fungus Colletotrichum graminicola. A total of 18 three-way hybrids were obtained by crossing six single cross male-sterile F1 hybrids, derived by crossing A (non restorer sterile cytoplasm) and B (non restorer normal cytoplasm) lines, with three fertile R (restorer) lines, previously evaluated for their differential reaction to the pathogen. Variation in the level of resistance was observed, as indicated by the values of the area under the disease progress curve (AUDPC) obtained for each hybrid. Lines contributed differently to the level of resistance of each hybrid. All hybrids in which CMSXS169R was the male progenitor were classified as highly resistant. Some hybrids had a level of resistance superior to the maximum levels of each line component individually.

Race Diversity and Complexity in Populations of the Sorghum Anthracnose Fungus Colletotrichum graminicola

Race Diversity and Complexity in Populations of the Sorghum Anthracnose Fungus Colletotrichum graminicola

A class V chitin synthase gene, chsA is essential for conidial and hyphal wall strength in the fungus Colletotrichum graminicola ( Glomerella graminicola)

The Colletotrichum graminicola tagged mutant T30 has conidia that burst and hyphal tips that swell in media with low osmotic pressure. The disrupted gene in T30 was identified as a class V chitin synthase (CSV) “ chsA,” which has an open reading frame of 1783 amino acids and two introns that are 52 and 54 bp. C. graminicola has one copy of chsA and no other highly homologous class V CHSs. Reverse transcriptase PCR indicated that the T30 mutant does not express the chsA transcript fragment in the conserved region in CSVs. Complementation of the mutant with chsA indicates that the encoded protein is responsible for ≈29% of the chitin in conidial walls, is essential for conidial wall strength in media with high water potential and contributes to strength of hyphal tips. Analysis of the aligned deduced amino acid sequences of the 10 fully sequenced CSVs suggests that they are in two subgroups.

The SOD2 gene, encoding a manganese-type superoxide dismutase, is up-regulated during conidiogenesis in the plant-pathogenic fungus Colletotrichum graminicola

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.

Reaction of sorghum genotypes to the anthracnose fungus Colletotrichum graminicola

The reactions of 22 sorghum (Sorghum bicolor) genotypes to six previously identified races of the sorghum anthracnose fungus Colletotrichum graminicola, were evaluated under greenhouse and field conditions. Races were inoculated in separate tests in the greenhouse. In the field, spreader rows of a susceptible genotype were artificially inoculated with a mixture of the six races of the pathogen. In the greenhouse tests, nine genotypes showed resistance to all six races. In the field high levels of dilatory resistance was observed in the sorghum genotypes CMSXS169 and CMSXS373.

Colletotrichum graminicola

We report the first case of an ocular infection with the fungus Colletotrichum graminicola causing keratitis in a 27-year-old man. Twenty-four months after a postoperative course complicated by recurrent fungal keratitis requiring two penetrating keratoplasties, two anterior chamber washouts, a conjunctival flap, and medical treatment with topical natamycin, intracameral amphotericin B, and oral fluconazole. The patient has shown no signs of fungal recurrence despite a failed corneal graft. C. graminicola is a new corneal pathogen and should be included in the differential diagnosis of mycotic keratitis.

A Rank Test for Distinguishing Environmentally and Genetically Induced Disease Resistance in Plant Varieties

Many species of plant pathogens vary genetically in ability to cause disease on a given host plant. Conversely, plant populations often have variability with respect to a gene or series of genes that provide resistance to a given pathogen (Leonard, 1987, in Populations of Plant Pathogens: Their Dynamics and Genetics, 163-181; Christ, Person, and Pope, 1987, in Populations of Plant Pathogens: Their Dynamics and Genetics, 7-21). These dynamics have become apparent, as there have been many cases in which crop plants bred for homogeneous high-level genetic resistance to a disease have lost the resistance, sometimes catastrophically (Day and Wolfe, 1987, in Populations of Plant Pathogens: Their Dynamics and Genetics, 3-7). This has led plant protectionists to seek ways to develop stable resistance to plant pathogens that are variable by nature. A nonparametric procedure that tests for variation among populations of a specific pathogen at different locations is described. A randomization approach is used to compute critical values and p-values. A small simulation study is carried out to examine the performance of the test. The same procedure can be used to determine the reaction of a specific crop variety to a pathogen under different environmental conditions or in different locations. The procedure is demonstrated using data on the fungus Colletotrichum graminicola (Ces.) Wilson, which is a pathogen on the cereal crop Sorghum bicolor (Moench). Disease reactions of genetically different sorghum varieties to specific strains of the fungus in different environments are recorded. From these, varieties are selected that do not exhibit interaction with environment. These varieties are then used to identify which locations have distinct pathogen populations. Once a set of locations has been identified as having distinct pathogen populations, the breeding materials are planted and selected for insignificant genotype by location (pathogen population) interaction, i.e., for genetic stability with respect to a given disease.

Ultrastructure of Conidia and the Conidium Aging Process in the Plant Pathogenic Fungus Colletotrichum graminicola

The influence of age on conidial ultrastructure was investigated in Colletotrichum graminicola. The cytoplasmic volume of young conidia-those harvested from 5-7-d-old cultures-was dominated by lipid bodies. These conidia also were characterized by numerous small vacuoles with electron-dense contents. Other structures present included mitochondria, microbodies, ribosomes, elements of tubular ER, short strands of rough ER, multivesicular bodies, simple cisternal elements, Woronin bodies, microtubules, and large amounts of what appeared to be glycogen. Older conidia harvested from 30-35-d-old cultures exhibited a significantly different ultrastructure. They contained fewer lipid bodies, lacked the large glycogen deposits, were very highly vacuolate, and contained sheetlike strands of ER and extensive aggregations of tubular ER. Structures thought to be autophagic vacuoles also were present. The conidial wall appeared similar in conidia of both ages and consisted of a thicker inner electron-transparent layer ...

Purification and characterization of cyanide hydratase from the phytopathogenic fungus Gloeocercospora sorghi

Previous studies have demonstrated that fungal pathogens of cyanogenic plants produce cyanide hydratase (CHT, EC 4.2.1.66), which converts HCN to formamide. Production of CHT in these fungi is thought to be a means of circumventing cyanide toxicity, and CHT is thus believed to be an important pathogenicity trait. In the present study, 13 species of fungi were assayed for CHT production, and all 7 species that were pathogens of sorghum, a cyanogenic plant, produced this enzyme. CHT was purified to apparent homogeneity from one of these sorghum pathogens, Gloeocercospora sorghi. The enzyme had a K m of 12 m m for KCN. Enzymatically functional CHT was obtained only as a large molecular entity of greater than 300 kDa. However, a polypeptide of approximately 45 kDa was identified as the only component of purified CHT detectable by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The 45-kDa polypeptide band could be resolved into three isozymes of p I 6.1, 6.3, and 6.5. Antibodies raised against the 45-kDa polypeptide inhibited the G. sorghi CHT activity and showed high specificity in Western blots to a polypeptide of approximately the same size. The evidence suggests that functional G. sorghi CHT is an aggregated protein that consists of 45-kDa polypeptides. A CHT with similar properties was also found in the fungus Colletotrichum graminicola, another pathogen of sorghum.

The PYR1 gene of the plant pathogenic fungus Colletotrichum graminicola: selection by intraspecific complementation and sequence analysis.

A spontaneous uridine-requiring auxotroph of Colletotrichum graminicola was recovered by selection for resistance to 5-fluoro-orotic acid. The auxotroph lacked orotate phosphoribosyl transferase (OPRTase) and was complemented with a clone from a cosmid library of C. graminicola DNA. A 3.1 kb HindIII-SalI fragment was subcloned from the cosmid and it could efficiently transform the auxotrophic strain to uridine prototrophy and integrate by site-specific recombination. This DNA fragment contains an open reading frame that is similar to OPRTase genes of the fungi Sordaria macrospora, Trichoderma reesei, Podospora anserina, and Saccharomyces cerevisiae. Based on the sequence similarities and the ability to restore uridine prototrophy, we conclude that the fragment contains the C. graminicola gene for OPRTase, which we have named PYR1. Our results demonstrate that cloning by complementation is feasible in C. graminicola, that the gene for OPRTase from C. graminicola can be useful as a selectable marker in transformation of the fungus, and that the OPRTase gene product is similar to OPRTase from other fungi.