Cutter Assembly for Microscope and Related Method

A low-profile cutter assembly for use on a microscope and related cutting method are provided. The cutter assembly includes a support subassembly having a mounting ring for receiving and engaging the objective of a microscope. A cutter subassembly carried by the support subassembly is displaceable between a home and a forward position in response to actuation. Upon reaching the forward position, additional actuation causes the front end of the cutter subassembly to pivot toward the stage, thereby placing a cutter adjacent to or in contact with the specimen. By selectively actuating the cutter subassembly, multiple cuts may be performed, as necessary or desired for cutting a spore or isolating a particular specimen

Relative efficiency of split-marker versus double-crossover replacement protocols for production of deletion mutants in strain PH-1 of Fusarium graminearum

The split-marker (SM) protocol has become a popular method for production of knockout mutations in fungi. We used Southern hybridization to compare the performance and efficiency of the SM protocol with the more traditional double-crossover intact marker (IM) method for creating deletions of the mating type genes in Fusarium graminearum. Both methods successfully produced knockouts at a rate of between 24 and 75%: the SM method produced mutants more efficiently for larger constructs (\u3e1 kb), but it was similar to IM for a smaller construct that was 865 bp. Both methods also produced strains with additional ectopic integrations at a similar rate of approximately 10%, but on average the SM produced a higher number of independent integrations in those strains. Ectopic integrations produce off-site mutations, and strains with multiple integrations are less desirable since it is more difficult to remove them by backcrossing. Southern hybridizations will be generally superior to PCR to identify strains with fewer ectopic integrations for experimental purposes

Characterization of \u3cem\u3eGlomerella\u3c/em\u3e Strains Recovered from Anthracnose Lesions on Common Bean Plants in Brazil

Anthracnose caused by Colletotrichum lindemuthianum is an important disease of common bean, resulting in major economic losses worldwide. Genetic diversity of the C. lindemuthianum population contributes to its ability to adapt rapidly to new sources of host resistance. The origin of this diversity is unknown, but sexual recombination, via the Glomerella teleomorph, is one possibility. This study tested the hypothesis that Glomerella strains that are frequently recovered from bean anthracnose lesions represent the teleomorph of C. lindemuthianum. A large collection of Glomerella isolates could be separated into two groups based on phylogenetic analysis, morphology, and pathogenicity to beans. Both groups were unrelated to C. lindemuthianum. One group clustered with the C. gloeosporioides species complex and produced mild symptoms on bean tissues. The other group, which belonged to a clade that included the cucurbit anthracnose pathogen C. magna, caused no symptoms. Individual ascospores recovered from Glomerella perithecia gave rise to either fertile (perithecial) or infertile (conidial) colonies. Some pairings of perithecial and conidial strains resulted in induced homothallism in the conidial partner, while others led to apparent heterothallic matings. Pairings involving two perithecial, or two conidial, colonies produced neither outcome. Conidia efficiently formed conidial anastomosis tubes (CATs), but ascospores never formed CATs. The Glomerella strains formed appressoria and hyphae on the plant surface, but did not penetrate or form infection structures within the tissues. Their behavior was similar whether the beans were susceptible or resistant to anthracnose. These same Glomerella strains produced thick intracellular hyphae, and eventually acervuli, if host cell death was induced. When Glomerella was co-inoculated with C. lindemuthianum, it readily invaded anthracnose lesions. Thus, the hypothesis was not supported: Glomerella strains from anthracnose lesions do not represent the teleomorphic phase of C. lindemuthianum, and instead appear to be bean epiphytes that opportunistically invade and sporulate in the lesions

Anthracnose: The sophisticated rot

The mold fungus Colletotrichum graminicola causes anthracnose, one of the most economically damaging corn diseases worldwide. Anthracnose can occur either as a stalk rot (ASR), or a leaf blight (ALB) (4; 27). The leaf blight phase is generally insignificant in North America as a cause of yield loss, although in the tropics and subtropics it is much more important. Resistance to ASR is usually not correlated with resistance to ALB, complicating efforts to breed resistant corn varieties (2; 4). Resistance to ASR and ALB is mostly quantitative, although sources of major gene resistance have been described (10; 29). Hybrids containing some of these major-gene resistance sources are likely to become available for management of ASR in the near future

A \u3cem\u3eColletotrichum graminicola\u3c/em\u3e Mutant Deficient in the Establishment of Biotrophy Reveals Early Transcriptional Events in the Maize Anthracnose Disease Interaction

Background: Colletotrichum graminicola is a hemibiotrophic fungal pathogen that causes maize anthracnose disease. It progresses through three recognizable phases of pathogenic development in planta: melanized appressoria on the host surface prior to penetration; biotrophy, characterized by intracellular colonization of living host cells; and necrotrophy, characterized by host cell death and symptom development. A “Mixed Effects” Generalized Linear Model (GLM) was developed and applied to an existing Illumina transcriptome dataset, substantially increasing the statistical power of the analysis of C. graminicola gene expression during infection and colonization. Additionally, the in planta transcriptome of the wild-type was compared with that of a mutant strain impaired in the establishment of biotrophy, allowing detailed dissection of events occurring specifically during penetration, and during early versus late biotrophy. Results: More than 2000 fungal genes were differentially transcribed during appressorial maturation, penetration, and colonization. Secreted proteins, secondary metabolism genes, and membrane receptors were over-represented among the differentially expressed genes, suggesting that the fungus engages in an intimate and dynamic conversation with the host, beginning prior to penetration. This communication process probably involves reception of plant signals triggering subsequent developmental progress in the fungus, as well as production of signals that induce responses in the host. Later phases of biotrophy were more similar to necrotrophy, with increased production of secreted proteases, inducers of plant cell death, hydrolases, and membrane bound transporters for the uptake and egress of potential toxins, signals, and nutrients. Conclusions: This approach revealed, in unprecedented detail, fungal genes specifically expressed during critical phases of host penetration and biotrophic establishment. Many encoded secreted proteins, secondary metabolism enzymes, and receptors that may play roles in host-pathogen communication necessary to promote susceptibility, and thus may provide targets for chemical or biological controls to manage this important disease. The differentially expressed genes could be used as ‘landmarks’ to more accurately identify developmental progress in compatible versus incompatible interactions involving genetic variants of both host and pathogen

A Comparative Genomic Analysis of Putative Pathogenicity Genes in the Host-Specific Sibling Species \u3cem\u3eColletotrichum graminicola\u3c/em\u3e and \u3cem\u3eColletotrichum sublineola\u3c/em\u3e

Background: Colletotrichum graminicola and C. sublineola cause anthracnose leaf and stalk diseases of maize and sorghum, respectively. In spite of their close evolutionary relationship, the two species are completely host-specific. Host specificity is often attributed to pathogen virulence factors, including specialized secondary metabolites (SSM), and small-secreted protein (SSP) effectors. Genes relevant to these categories were manually annotated in two co-occurring, contemporaneous strains of C. graminicola and C. sublineola. A comparative genomic and phylogenetic analysis was performed to address the evolutionary relationships among these and other divergent gene families in the two strains. Results: Inoculation of maize with C. sublineola, or of sorghum with C. graminicola, resulted in rapid plant cell death at, or just after, the point of penetration. The two fungal genomes were very similar. More than 50% of the assemblies could be directly aligned, and more than 80% of the gene models were syntenous. More than 90% of the predicted proteins had orthologs in both species. Genes lacking orthologs in the other species (non-conserved genes) included many predicted to encode SSM-associated proteins and SSPs. Other common groups of non-conserved proteins included transporters, transcription factors, and CAZymes. Only 32 SSP genes appeared to be specific to C. graminicola, and 21 to C. sublineola. None of the SSM-associated genes were lineage-specific. Two different strains of C. graminicola, and three strains of C. sublineola, differed in no more than 1% percent of gene sequences from one another. Conclusions: Efficient non-host recognition of C. sublineola by maize, and of C. graminicola by sorghum, was observed in epidermal cells as a rapid deployment of visible resistance responses and plant cell death. Numerous non-conserved SSP and SSM-associated predicted proteins that could play a role in this non-host recognition were identified. Additional categories of genes that were also highly divergent suggested an important role for co-evolutionary adaptation to specific host environmental factors, in addition to aspects of initial recognition, in host specificity. This work provides a foundation for future functional studies aimed at clarifying the roles of these proteins, and the possibility of manipulating them to improve management of these two economically important diseases

Le Forum, Vol. 41 No. 1

https://digitalcommons.library.umaine.edu/francoamericain_forum/1090/thumbnail.jp

Development of aggression subtypes from childhood to adolescence:a group-based multi-trajectory modelling perspective

The persistence of elevated subtypes of aggression beginning in childhood have been associated with long-term maladaptive outcomes. Yet it remains unclear to what extent there are clusters of individuals following similar developmental trajectories across forms (i.e., physical and indirect) and functions (i.e., proactive and reactive) of aggression. We aimed to identify groups of children with distinct profiles of the joint development of forms and functions of aggression and to identify risk factors for group membership. A sample of 787 children was followed from birth to adolescence. Parent and teacher reports, and standardised assessments were used to measure two forms and two functions of aggressive behaviour, between six and 13 years of age along with preceding child, maternal, and family-level risk-factors. Analyses were conducted using a group-based multi-trajectory modelling approach. Five trajectory groups emerged: non-aggressors, low-stable, moderate-engagers, high-desisting, and high-chronic. Coercive parenting increased membership risk in the moderate-engagers and high-chronic groups. Lower maternal IQ increased membership risk in both high-desisting and high-chronic groups, whereas maternal depression increased membership risk in the high-desisting group only. Never being breastfed increased membership risk in the moderate-engagers group. Boys were at greater risk for belonging to groups displaying elevated aggression. Individuals with chronic aggression problems use all subtypes of aggression. Risk factors suggest that prevention programs should start early in life and target mothers with lower IQ. Strategies to deal with maternal depression and enhance positive parenting while replacing coercive parenting tactics should be highlighted in programming efforts

Genetic manipulations of Glomerella graminicola (anamorph Colletotrichum graminicola)

Glomerella graminicola is a plant-pathogenic fungus which in its anamorphic stage (Colletotrichum graminicola) causes anthracnose disease on maize and numerous other grasses. The objective of my thesis research was to characterize various aspects of sexual reproduction in G. graminicola, with the intention of developing practical methods for conducting a classical genetic analysis of the fungus. The results of my study included the development of a technique for conducting and analyzing crosses among isolates of Glomerella from maize. A Glomerella stage produced by sorghum isolates of the fungus was discovered, and methods for conducting crosses with it were also developed. For the maize isolates, methods were developed for tetrad analysis and for study of the inheritance of cytoplasmic factors. Maternal inheritance of mitochondria was demonstrated by studying segregation of mitochondrial restriction fragment length polymorphisms. Techniques were developed for genetic linkage mapping of G. graminicola with molecular markers, specifically random amplified polymorphic DNA (RAPDs). Preliminary maps were generated, and it was tentatively estimated that G. graminicola has nine linkage groups. Maize and sorghum isolates of Glomerella, although generally fertile among themselves, were not interfertile. Further comparisons of the morphologies of their anamorphic and teleomorphic stages, mitochondrial DNA restriction fragment length polymorphisms, and RAPD fingerprints led me to conclude that the two groups are sufficiently different to deserve separate species designation. I have proposed the name Glomerella sorghi for the sorghum species. I also established a method for cotransformation and characterized its performance, and I used the technique to generate a pyrimidine auxotrophic mutant by producing a targeted gene disruption at the PYR1 locus by homologous integration of a copy of the PYR1 gene which lacked the 3\sp\prime and 5\sp\prime ends. The ability to perform a sexual genetic analysis, as well as to use cotransformation to make targeted gene disruptions, should expedite future studies of molecular determinants of pathogenicity in this fungus