Louise-Marie Dandurand

Ecological Complexity and the Success of Fungal Biological Control Agents

Fungal biological control agents against plant pathogens, especially those in soil, operate within physically, biologically, and spatially complex systems by means of a variety of trophic and nontrophic interspecific interactions. However, the biocontrol agents themselves are also subject to the same types of interactions, which may reduce or in some cases enhance their efficacy against target plant pathogens. Characterization of these ecologically complex systems is challenging, but a number of tools are available to help unravel this complexity. Several of these tools are described here, including the use of molecular biology to generate biocontrol agents with useful marker genes and then to quantify these agents in natural systems, epifluorescence and confocal laser scanning microscopy to observe their presence and activity in situ, and spatial statistics and computer simulation modeling to evaluate and predict these activities in heterogeneous soil habitats.

Colonisation of sclerotia of Sclerotinia sclerotiorum by a fungivorous nematode

ABSTRACT Aphelenchoides saprophilus nematodes fed on sclerotia, mycelium, and alginate-formulated pellets of Sclerotinia sclerotiorum, mycelium of Trichoderma harzianum, and mixed fungal cultures. As many as 500 nematodes were found inside individual sclerotia. Results suggest potential impacts of fungivory on S. sclerotiorum and its ecological interactions with plant hosts and biocontrol fungi.

Assessment of an Organ-Specific de Novo Transcriptome of the Nematode Trap-Crop, Solanum sisymbriifolium

Solanum sisymbriifolium, also known as “Litchi Tomato” or “Sticky Nightshade,” is an undomesticated and poorly researched plant related to potato and tomato. Unlike the latter species, S. sisymbriifolium induces eggs of the cyst nematode, Globodera pallida, to hatch and migrate into its roots, but then arrests further nematode maturation. In order to provide researchers with a partial blueprint of its genetic make-up so that the mechanism of this response might be identified, we used single molecule real time (SMRT) sequencing to compile a high quality de novo transcriptome of 41,189 unigenes drawn from individually sequenced bud, root, stem, and leaf RNA populations. Functional annotation and BUSCO analysis showed that this transcriptome was surprisingly complete, even though it represented genes expressed at a single time point. By sequencing the 4 organ libraries separately, we found we could get a reliable snapshot of transcript distributions in each organ. A divergent site analysis of the merged transcriptome indicated that this species might have undergone a recent genome duplication and re-diploidization. Further analysis indicated that the plant then retained a disproportionate number of genes associated with photosynthesis and amino acid metabolism in comparison to genes with characteristics of R-proteins or involved in secondary metabolism. The former processes may have given S. sisymbriifolium a bigger competitive advantage than the latter did.

Microaspiration of Solanum tuberosum root cells at early stages of infection by Globodera pallida

BackgroundSedentary endoparasitic cyst nematodes form a feeding structure in plant roots, called a syncytium. Syncytium formation involves extensive transcriptional modifications, which leads to cell modifications such as increased cytoplasmic streaming, enlarged nuclei, increased numbers of organelles, and replacement of a central vacuole by many small vacuoles. When whole root RNA is isolated and analyzed, transcript changes manifested in the infected plant cells are overshadowed by gene expression from cells of the entire root system. Use of microaspiration allows isolation of the content of nematode infected cells from a heterogeneous cell population. However, one challenge with this method is identifying the nematode infected cells under the microscope at early stages of infection. This problem was addressed by staining nematode juveniles with a fluorescent dye prior to infection so that the infected cells could be located and microaspirated.ResultsIn the present study, we used the fluorescent vital stain PKH26 coupled with a micro-rhizosphere chamber to locate the infected nematode Globodera pallida in Solanum tuberosum root cells. This enabled microaspiration of nematode-infected root cells during the early stages of parasitism. To study the transcriptional events occurring in these cells, an RNA isolation method from microaspirated samples was optimized, and subsequently the RNA was purified using magnetic beads. With this method, we obtained an RNA quality number of 7.8. For transcriptome studies, cDNA was synthesized from the isolated RNA and assessed by successfully amplifying several pathogenesis related protein coding genes.ConclusionThe use of PKH26 stained nematode juveniles enabled early detection of nematode infected cells for microaspiration. To investigate transcriptional changes in low yielding RNA samples, bead-based RNA extraction procedures minimized RNA degradation and provided high quality RNA. This protocol provides a robust procedure to analyze gene expression in nematode-infected cells.

Prospecting fungal parasites of the potato cyst nematode Globodera pallida using a rapid screening technique

Seven filamentous fungal species were isolated from individual eggs of Globodera pallida cysts collected from infested fields in Shelley Idaho, USA and identified as Chaetomium globosum, Fusarium oxysporum, Fusarium solani, Fusarium tricinctum, Microdochium bolleyi, Purpureocillium lilacinum, and Plectosphaerella cucumerina. Their ability to reduce infection by G. pallida in planta were assessed in simple, reproducible micro‐rhizosphere chambers (micro‐ROCs). All fungi reduced G. pallida infection in potato, but greatest reduction was observed with C. globosum at an average reduction of 76%. Further non‐destructive methods were developed to rapidly assess biological control potential of putative fungal strains by staining the infectious second stage juveniles of G. pallida with the live fluorescent stain PKH26. In comparisons between the standard, invasive acid fuchsin method and use of the live stain PKH26, no significant difference in infection level of G. pallida was observed whether roots were stained with PKH26 or acid fuchsin. For both methods, a similar reduction (77% for acid fuchsin, and 78% for PKH26 stain) in invasion of infectious stage of G. pallida was observed when potato plants were inoculated with C. globosum compared to non‐inoculated potato.