Phenolic metabolites of Ceratocystis ulmi

The C₁₀ acid 2,4-dihydroxy-6-(1-hydroxyacetonyl) benzoic acid, together with the 6-acetonyl- and 6-pyruvyl-analogues, has been identified as a metabolic product of Ceratocystis ulmi, the causative agent of Dutch elm disease. In a comparison of aggressive “fluffy” and non-aggressive “waxy” strains of C. ulmi, the C₁₀ acids were produced more rapidly and in greater yield by the former group.     

Analysis of the Italian Dutch Elm Disease Fungal Population

Sixty‐two Ophiostoma ulmi sensu lato strains have been collected from symptomatic trees in seven areas of Central Italy. Isolates were compared with 10 reference strains, belonging to the species O. ulmi and to the two subspecies of O. novo‐ulmi, in order to establish the genetic variability within the Italian population of this fungal pathogen. The structure of the population has been analysed by means of morpho‐physiological features and of the direct amplification of minisatellite‐region DNA polymerase chain reaction (DAMD‐PCR) by using the M13 core sequence. The DNA profiles have been compared with taxonomic parameters (growth rate, culture aspect and fertility barriers). Taxa could thus be well separated. None of the isolates collected was recognized as O. ulmi. Isolates assigned to the two subspecies of O. novo‐ulmi (novo ulmi and americana) by means of the fertility test, showed short genetic distances with the respective reference strains and they constituted subgroups according to their geographical origin. The high level of variation detected indicates a postepidemic situation in Italy. Some inconsistency was found within the subspecies clusters. Several isolates, assigned to subspecies americana using fertility test, were in the novo‐ulmi cluster and vice versa. A possible explanation is that these isolates are americana–novo‐ulmi hybrids.     

Non-Mendelian segregation influences the infection biology and genetic structure of the African tree pathogen Ceratocystis albifundus

The African fungal tree pathogen, Ceratocystis albifundus, undergoes uni-directional mating type switching, giving rise to either self-fertile or self-sterile progeny. Self-sterile isolates lack the MAT1-2-1 gene and have reduced fitness such as slower growth and reduced pathogenicity, relative to self-fertile isolates. While it has been hypothesized that there is a 1:1 ratio of self-fertile to self-sterile ascospore progeny in relatives of C. albifundus, some studies have reported a significant bias in this ratio. This could be due to the fact that either fewer self-sterile ascospores are produced or that self-sterile ascospores have low viability. We quantified the percentage of self-sterile and self-fertile ascospores from ascospore masses in C. albifundus using real-time PCR. Primers were designed to distinguish between spores that contained the MAT1-2-1 gene and those where this gene had been deleted. A significant bias towards the self-fertile mating type was observed in all single ascospore masses taken from sexual structures produced in haploid-selfed cultures. The same result was observed from a disease outbreak situation in an intensively managed field of cultivated native trees, and this was coupled with very low population diversity in the pathogen. This was in contrast to the results obtained from ascospore masses taken from the crosses performed under laboratory conditions or ascomata on native trees in a non-disease situation, where either self-fertile or self-sterile ascospores were dominant. The results suggest that reproductive strategies play a significant role in the infection biology and genetic structure of C. albifundus populations.     

Two novel phytotoxic substances from Leucas aspera

Leucas aspera (Lamiaceae), an aromatic herbaceous plant, is well known for many medicinal properties and a number of bioactive compounds against animal cells have been isolated. However, phytotoxic substances from L. aspera have not yet been documented in the literature. Therefore, current research was conducted to explore the phytotoxic properties and substances in L. aspera. Aqueous methanol extracts of L. aspera inhibited the germination and growth of garden cress (Lepidum sativum) and barnyard grass (Echinochloa crus-galli), and the inhibitory activities were concentration dependent. These results suggest that the plant may have phytotoxic substances. The extracts were then purified by several chromatographic runs. The final purification was achieved by reversed-phase HPLC to give an equilibrium (or inseparable) 3:2 mixture of two labdane type diterpenes (compounds 1 and 2). These compounds were characterized as (rel 5S,6R,8R,9R,10S,13S,15S,16R)-6-acetoxy-9,13;15,16-diepoxy-15-hydroxy-16-methoxylabdane (1) and (rel 5S,6R,8R,9R,10S,13S,15R,16R)-6-acetoxy-9,13;15,16-diepoxy-15-hydroxy-16-methoxylabdane (2) by spectroscopic analyses. A mixture of the two compounds inhibits the germination and seedling growth of garden cress and barnyard grass at concentrations greater than 30 and 3 μM, respectively. The concentration required for 50% growth inhibition (I₅₀) of the test species ranges from 31 to 80 μM, which suggests that the mixture of these compounds, are responsible for the phytotoxic activity of L. aspera plant extract.     

Synthesis of tryptophol and O-acetyltryptophol from tryptophan by Ceratocystis fagacearum

Tryptophol (TOL) and O-acetyltryptophol (OAcTOL) were identified as tryptophan metabolites of C. fagacearum; OAcTOL was the only metabo     

A highly virulent pathogen, Aeromonas hydrophila, from the freshwater crayfish Pacifastacus leniusculus

Aeromonas spp. are characteristic bacteria of freshwaters and many of them can be components of the bacterial flora of aquatic animals and may become pathogens on animals including humans. In this study Aeromonas hydrophila was isolated from the freshwater crayfish, Pacifastacus leniusculus, and was found to be a highly pathogenic bacterium among many isolated bacteria. Mortality reached 100% within 6 h when 200 μl of 1.24 × 10⁷ CFU/ml was applied by injection. Histopathological studies of moribund crayfish showed that extensive necrotic nuclei and clump-infiltrated hemocytes were found in observed tissues including gill, heart, hepatopancreas and the circulatory system. To verify how crayfish are susceptible to this bacterium, crude extracellular products (ECPs) obtained from culture supernatant of A. hydrophila was studied either in vivo or in vitro. ECPs (200 μl) were able to kill crayfish by injection. In an in vitro study, ECPs induced cytotoxicity of hemocytes as well as hematopoietic cells in a dose- and time-dependent manner after 30 min post inoculation. Two genes coding for endotoxins were also found in this isolate of A. hydrophila. This indicates that the bacterial endotoxins are the causative agents of crayfish mortality. Moreover, the effect of temperature on the infectivity of A. hydrophila to crayfish was also studied. At 4 °C, all crayfish survived, whereas at 20 °C the animals died rapidly after bacterial challenge. At this low temperature A. hydrophila did not replicate or replicated at a very low degree and hence crayfish could probably mount effective cellular reactions towards A. hydrophila.     

The structure of the glycopeptides from the fish pathogen Flavobacterium columnare

Proteolytic digestion of the phenol-water extraction product of the fish pathogen Flavobacterium columnare afforded a mixture of glycopeptides in which the oligosaccharide moiety was an unusual hexasaccharide composed of 4-O-methyl-2-acetamido-2-deoxy-D-glucuronic acid (GlcNAcA), D-glucuronic acid (D-GlcA), 2,3-di-O-acetyl-D-xylose (D-Xyl), 2-O-methyl-D-glucuronic acid (D-GlcA), D-mannose (D-Man), and 2-O-methyl-L-rhamnose (L-Rha). By the application of high-resolution 1D and 2D NMR, mass spectrometry, and chemical analysis, the hexasaccharide structure was determined to be: [carbohydrate structure--see text] where all monosaccharides have the D-configuration except for 2-O-methyl-L-rhamnose; and were in the pyranose form. Only one carbohydrate structure was found. The peptide part was represented by tri- to hepta-peptides with a minimal common tripeptide fragment Asp-Ser-Ala, extended with Ala and Val.     

Elm bark beetle boring and feeding deterrents from Phomopsis oblonga

The two groups of Phomopsis oblonga which invade the phloem of stressed elm trees have been investigated for the production in vitro of boring/feeding deterrents for elm bark beetles. The secondary metabolites of this fungus are compared with those of closely-related Phomopsis spp. associated with ash and sycamore. Active compounds isolated from P. oblonga include a novel norsesquiterpene γ-lactone, the tiglic esters of two novel 5,6-dihydro-5-hydroxy-2-pyrones, nectriapyrone, 4-hydroxyphenylethanol, 5-methylmellein, 2-furoic, orsellinic and 3-nitropropanoic acids and mellein-5-carboxylic acid; portensterol and thymine, from P. oblonga, were inactive. (+)-Mellein and furan-2,5-dicarboxylic acid were obtained as minor metabolites of the ash and sycamore Phomopsis strains, which also produced the norsesquiterpene γ-lactone and one of the tiglic esters.     

Analysis of a high frequency transformation system for Ophiostoma ulmi, the causal agent of Dutch elm disease

Summary A transformation system for Ophiostoma ulmi (Buism.) Nannf. was developed and analyzed. Protoplasts were generated from actively budding yeastlike cells by digestion with NovoZym 234 in MgSO₄ after pretreatment with 2-mercaptoethanol. Protoplast regeneration was most efficient when 0.6 M sucrose was used as the osmoticum. Several plasmids containing fusions between fungal promoters and a bacterial gene for hygromycin phosphotransferase successfully transformed O. ulmi to hygromycin resistance. One of these vectors, pPS57, which contains a promoter for isopenicillin N synthetase from Penicillium chrysogenum, consistently conferred the greatest resistance to hygromycin. Linearization of the vector and inclusion of 2-mercaptoethanol in the transformation reaction resulted in enhanced transformation efficiency. Approximately 4 × 10³ transformants/g DNA per 10⁷ protoplasts were obtained using the optimized procedure. Southern hybridization after alternating field and standard electrophoresis suggested random insertion of tandem repeats (some greater than 250 kb) into the fungal chromosomes. Antibiotic resistance was stable through mitosis. However, expression of the transforming DNA after meiosis was highly variable.     

Ultrastructure of the nematode pathogen, Bacillus penetrans

The ultrastructure and development of Bacillus penetrans in root-knot nematodes, Meloidogyne spp., was studied with a transmission electron microscope. Host infection was by a germ tube from the cup-shaped sporangium containing the endospore. The prokaryotic vegetative cells contained septa formed by an ingrowth of the inner layer of the trilaminate cell wall and were associated with mesosomes. Structure of the endospore was similar to other bacteria with a spore protoplast enclosed within two cortical layers and three spore coats. An exosporium which may function in attachment and host specificity surrounded the endospore. Ultrastructural changes accompanying sporulation were similar to those reported for other endospore-forming bacteria but with some parasite specialization. The filamentous vegetative growth was characteristic of some Actinomycetales. Endospore development at the apices of dichotomously branched filaments of the thallus resembled the genus Actinobifida.     

Methylation analysis of cell wall glycoproteins and glycopeptides from Chlamydomonas reinhardii

Cell wall glycoproteins from Chlamydomonas reinhardii and the glycopeptides produced by the action of thermolysin were subjected to standard methylation analysis. GC-MS of the methylated alditol acetates revealed short oligosaccharides some of which show branching. O-glycosidically linked galactofuranosyl residues are present. The asymmetric distribution of the major O-glycosidic linkages is also reported.     

Sequencing of the Dutch Elm Disease Fungus Genome Using the Roche/454 GS-FLX Titanium System in a Comparison of Multiple Genomics Core Facilities

As part of the DNA Sequencing Research Group of the Association of Biomolecular Resource Facilities, we have tested the reproducibility of the Roche/454 GS-FLX Titanium System at five core facilities. Experience with the Roche/454 system ranged from <10 to >340 sequencing runs performed. All participating sites were supplied with an aliquot of a common DNA preparation and were requested to conduct sequencing at a common loading condition. The evaluation of sequencing yield and accuracy metrics was assessed at a single site. The study was conducted using a laboratory strain of the Dutch elm disease fungus Ophiostoma novo-ulmi strain H327, an ascomycete, vegetatively haploid fungus with an estimated genome size of 30–50 Mb. We show that the Titanium System is reproducible, with some variation detected in loading conditions, sequencing yield, and homopolymer length accuracy. We demonstrate that reads shorter than the theoretical minimum length are of lower overall quality and not simply truncated reads. The O. novo-ulmi H327 genome assembly is 31.8 Mb and is comprised of eight chromosome-length linear scaffolds, a circular mitochondrial conti of 66.4 kb, and a putative 4.2-kb linear plasmid. We estimate that the nuclear genome encodes 8613 protein coding genes, and the mitochondrion encodes 15 genes and 26 tRNAs.     

Putaminoxin, a phytotoxic nonenolide from Phoma putaminum

Phoma putaminum, the causal agent of leaf necrosis of Erigeron annuus, a common weed of field and pasture, produced toxic metabolites when grown in liquid culture. The main phytotoxin, named putaminoxin, was isolated and characterized using spectroscopic and chemical methods as (5S)5-hydroxy-9-propyl-6-nonen-9-olide, a new 10-macrolide. When assayed on leaves of host and non-host plants, putaminoxin showed a wide range of toxicity, with leaves of E. annuus being most sensitive.     

Isolation and growth of the grasshopper pathogen, Entomophthora grylli

Entomophthora grylli was isolated and grown in pure culture in protoplast form. Cultures were obtained by germinating conidia collected from naturally infected adult Carolina grasshoppers, Dissosteira carolina, in Grace's tissue culture medium supplemented with 5% fetal bovine serum and an aqueous extract of grasshopper tissue. Grasshopper extract was not necessary for subsequent growth and subculture of the protoplasts. Healthy adult grasshoppers could be reinfected with the fungus by injection of a protoplast suspension.     

Secondary metabolite profile and phytotoxic activity of genetically distinct forms of Colletotrichum gloeosporioides from yam (Dioscorea spp.)

Highly virulent, slow-growing grey (SGG); moderately virulent, fast-growing salmon (FGS); and avirulent/weakly virulent, fast-growing grey (FGG) forms of Colletotrichum gloeosporioides have been described from yam (Dioscorea spp.), but little is known about their chemodiversity or the role of toxins in their pathogenesis. Secondary metabolite profiles in high performance tlc (hptlc) showed that the pathogenic SGG and FGS forms have a chemotype (A or B) that is distinct from the non-pathogenic FGG form (chemotype C). Crude extracts of 35-d-old Czapek-Dox yeast broth cultures of FGS and SGG isolates caused tissue necrosis on treated yam leaves but not those of FGG isolates. Extracts from uninoculated broth cultures showed no phytotoxic activity. Toxicity of the culture filtrate was not host specific and toxic substances were thermostable. Dioscorea genotypes with varying levels of resistance to anthracnose differed in their sensitivity to crude toxin extract of FGS (Cg33) and SGG (Cg25) isolates, indicating that these extracts may be useful in evaluating host resistance to anthracnose in vitro. Analysis of two toxin fractions unique to the pathogenic FGS and SGG forms using hlpc, mass spectrometry and nuclear magnetic resonance suggested the presence of a low molecular weight amide peptide. However, possibly due to low yield and the presence of impurities, the chemical structure of the compound(s) could not be fully elucidated.     

Microarray analysis of the human antibody response to synthetic Cryptosporidium glycopeptides

Glycoproteins expressed by Cryptosporidium parvum are immunogenic in infected individuals but the nature of the epitopes recognised in C. parvum glycoproteins is poorly understood. Since a known immunodominant antigen of Cryptosporidium, the 17 kDa glycoprotein, has previously been shown to bind to lectins that recognise the Tn antigen (GalNAcα1-Ser/Thr-R), a large number of glycopeptides with different Tn valency and presentation were prepared. In addition, glycopeptides were synthesised based on a 40 kDa cryptosporidial antigen, a polymorphic surface glycoprotein with varying numbers of serine residues, to determine the reactivity with sera from C. parvum-infected humans. These glycopeptides and non-glycosylated peptides were used to generate a glycopeptide microarray to allow screening of sera from C. parvum-infected individuals for the presence of IgM and IgG antibodies. IgG but not IgM in sera from C. parvum-infected individuals bound to multivalent Tn antigen epitopes presented on glycopeptides, suggesting that glycoproteins from C. parvum that contain the Tn antigen induce immune responses upon infection. In addition, molecular differences in glycosylated peptides (e.g. substituting Ser for Thr) as well as the site of glycosylation had a pronounced effect on reactivity. Lastly, pooled sera from individuals infected with either Toxoplasma or Plasmodium were also tested against the modified Cryptosporidium peptides and some sera showed specific binding to glycopeptide epitopes. These studies reveal that specific anti-glycopeptide antibodies that recognise the Tn antigen may be useful diagnostically and in defining the roles of parasite glycoconjugates in infections.