Lensless imaging for continuous CHO viable cell density monitoring in bioreactors

During suspension cell cultures in bioreactors, traditional measure for cell count and cell viability still rely on sampling and staining protocol where the Trypan Blue exclusion method is performed once a day. While automatic cell counters have reduced the statistical error of the original manual method, sampling the bioreactor is a risk for contamination and is prohibiting the use of such method for process control as the sampled volume becomes significant. Lensless Imaging Technology is a new breakthrough method for accurately and precisely determine cell concentration and viability without staining. This technique has the unique capability of acquiring microscopy images without any objective, lens or focus settings. A light source illuminates the cells, the interference between the light diffracted by the cells and the incident light coming from the same light source generates holograms that are directly recorded onto a CMOS sensor. A holographic reconstruction algorithm retrieves in real time the objects that have created the holograms and reconstructs the microscopy image. Living and dead cells have significant diffraction properties creating very different hologram patterns that are distinguished by the algorithm. Our contribution reports the comparison of cell counts and viability determination between the Trypan Blue reference method and our Lensless Imaging device. Cell counts is performed once a day on samples from 12 separated bioreactors, starting from the inoculation to the end of a 13 days CHO culture. The Lensless Imaging prototype is immersed in a measurement chamber, manually receiving the bioreactor samples, to both reproduce an in situ measure and avoid steam sterilization at this time of the experiment. With a concentration range from 0,43x106 up to 20x106 cells /ml and viability from 100% declining to 75% at the end of the cultures, we obtained a correlation factor of 0.986 (figure1). A specific study reports on the repeatability of the Lensless Imaging measurement method. The very large field of view (30m2), allows the analyze of several thousand cells within a single image, keeping the statistical variability of the measure as low as 3% in low as well as high cell concentration range. We are now working at designing a steam strerilizable probe, and we envision Lensless Imaging to become the future method of choice for on-line and in-situ monitoring of suspension cells and a perfect tool for process control in fed-batch or perfusion mode

Lensless imaging for continuous CHO viable cell density monitoring in bioreactors

During suspension cell cultures in bioreactors, traditional measure for cell count and cell viability still rely on sampling and staining protocol where the Trypan Blue exclusion method is performed once a day. While automatic cell counters have reduced the statistical error of the original manual method, sampling the bioreactor is a risk for contamination and is prohibiting the use of such method for process control as the sampled volume becomes significant. Lensless Imaging Technology is a new breakthrough method for accurately and precisely determine cell concentration and viability without staining. This technique has the unique capability of acquiring microscopy images without any objective, lens or focus settings. A light source illuminates the cells, the interference between the light diffracted by the cells and the incident light coming from the same light source generates holograms that are directly recorded onto a CMOS sensor. A holographic reconstruction algorithm retrieves in real time the objects that have created the holograms and reconstructs the microscopy image. Living and dead cells have significant diffraction properties creating very different hologram patterns that are distinguished by the algorithm. Our contribution reports the comparison of cell counts and viability determination between the Trypan Blue reference method and our Lensless Imaging device. Cell counts is performed once a day on samples from 12 separated bioreactors, starting from the inoculation to the end of a 13 days CHO culture. The Lensless Imaging prototype is immersed in a measurement chamber, manually receiving the bioreactor samples, to both reproduce an in situ measure and avoid steam sterilization at this time of the experiment. With a concentration range from 0,43x106 up to 20x106 cells /ml and viability from 100% declining to 75% at the end of the cultures, we obtained a correlation factor of 0.986 (figure1). A specific study reports on the repeatability of the Lensless Imaging measurement method. The very large field of view (30m2), allows the analyze of several thousand cells within a single image, keeping the statistical variability of the measure as low as 3% in low as well as high cell concentration range. We are now working at designing a steam strerilizable probe, and we envision Lensless Imaging to become the future method of choice for on-line and in-situ monitoring of suspension cells and a perfect tool for process control in fed-batch or perfusion mode

Phylogeography of the Solanaceae-infecting Basidiomycota fungus Rhizoctonia solani AG-3 based on sequence analysis of two nuclear DNA loci

<p>Abstract</p> <p>Background</p> <p>The soil fungus <it>Rhizoctonia solani </it>anastomosis group 3 (AG-3) is an important pathogen of cultivated plants in the family Solanaceae. Isolates of <it>R. solani </it>AG-3 are taxonomically related based on the composition of cellular fatty acids, phylogenetic analysis of nuclear ribosomal DNA (rDNA) and beta-tubulin gene sequences, and somatic hyphal interactions. Despite the close genetic relationship among isolates of <it>R. solani </it>AG-3, field populations from potato and tobacco exhibit comparative differences in their disease biology, dispersal ecology, host specialization, genetic diversity and population structure. However, little information is available on how field populations of <it>R. solani </it>AG-3 on potato and tobacco are shaped by population genetic processes. In this study, two field populations of <it>R. solani </it>AG-3 from potato in North Carolina (NC) and the Northern USA; and two field populations from tobacco in NC and Southern Brazil were examined using sequence analysis of two cloned regions of nuclear DNA (pP42F and pP89).</p> <p>Results</p> <p>Populations of <it>R. solani </it>AG-3 from potato were genetically diverse with a high frequency of heterozygosity, while limited or no genetic diversity was observed within the highly homozygous tobacco populations from NC and Brazil. Except for one isolate (TBR24), all NC and Brazilian isolates from tobacco shared the same alleles. No alleles were shared between potato and tobacco populations of <it>R. solani </it>AG-3, indicating no gene flow between them. To infer historical events that influenced current geographical patterns observed for populations of <it>R. solani </it>AG-3 from potato, we performed an analysis of molecular variance (AMOVA) and a nested clade analysis (NCA). Population differentiation was detected for locus pP89 (Φ_{<it>ST </it>}= 0.257, significant at P < 0.05) but not for locus pP42F (Φ_{<it>ST </it>}= 0.034, not significant). Results based on NCA of the pP89 locus suggest that historical restricted gene flow is a plausible explanation for the geographical association of clades. Coalescent-based simulations of genealogical relationships between populations of <it>R. solani </it>AG-3 from potato and tobacco were used to estimate the amount and directionality of historical migration patterns in time, and the ages of mutations of populations. Low rates of historical movement of genes were observed between the potato and tobacco populations of <it>R. solani </it>AG-3.</p> <p>Conclusion</p> <p>The two sisters populations of the basidiomycete fungus <it>R. solani </it>AG-3 from potato and tobacco represent two genetically distinct and historically divergent lineages that have probably evolved within the range of their particular related Solanaceae hosts as sympatric species.</p

Analysis of mycobiomes to uncover biodiversity: a case study between soil fungi and orchid species in Sweden

Abstracts from the April 12-14, 2019 MASC Conferenc

Editorial: The Use and Understanding of Style Differences to Enhance Learning

Comparison of Molecular Function annotations of R. solani genes between the induced (log2FPKM > 0) and repressed (log2FPKM < 0) genes when challenged with S4 Serratia proteamaculans. (FPMK = fragments per kilobase of exon per million fragments mapped

Characterization of a Basidiomycete Fungus from Stored Sugar Beet Roots

Eighteen isolates sampled from sugar beet roots associated with an unknown etiology were characterized based on observations of morphological characters, hyphal growth at temperatures ranging from 4 to 28 C, production of phenol oxidases, and sequence analysis of internal transcribed spacer (ITS) and large subunit (LSU) regions of the ribosomal DNA (rDNA). The isolates did not produce asexual or sexual spores, had binucleate hyphal cells with clamp connections, grew from 4 to 22 C with and estimated optimal growth at 14.5 C, and formed a dark brown pigment on potato dextrose or malt extract agar amended with 0.5% tannic acid. Color changes observed when solutions of gum guiac, guiacol, and syringaldzine were applied directly to mycelium grown on these media indicated that all isolates produced phenol oxidases. Sequences of ITS and LSU regions on the rDNA gene from 15 isolates were 99.2 to 100% identical and analysis of sequence data with Maximum Likelihood and Maximum Parsimony suggest that the isolates from sugar beet roots are phylogenetically related to Athelia bombacina, Granulobasidium vellereum, and Cyphella digitalis. High statistical support for both loci under different criteria confirmed that Athelia bombacina was consistently the closest known relative to the sugar beet isolates. Additional taxonomic investigations are needed before species can be clarified and designated for these isolates

Draft genome sequence of the plant-pathogenic soil fungus Rhizoctonia solani anastomosis group 3 strain Rhs1AP

The soil fungus Rhizoctonia solani is a pathogen of agricultural crops. Here, we report on the 51,705,945 bp draft consensus genome sequence of R. solani strain Rhs1AP. A comprehensive understanding of the heterokaryotic genome complexity and organization of R. solani may provide insight into the plant disease ecology and adaptive behavior of the fungus

Population structure of Sclerotinia subarctica and Sclerotinia sclerotiorum in England, Scotland and Norway

Sclerotinia species are important fungal pathogens of a wide range of crops and wild host plants. While the biology and population structure of Sclerotinia sclerotiorum has been well-studied, little information is available for the related species S. subarctica. In this study, Sclerotinia isolates were collected from different crop plants and the wild host Ranuculus ficaria (meadow buttercup) in England, Scotland, and Norway to determine the incidence of Sclerotinia subarctica and examine the population structure of this pathogen for the first time. Incidence was very low in England, comprising only 4.3% of isolates while moderate and high incidence of S. subarctica was identified in Scotland and Norway, comprising 18.3 and 48.0% of isolates respectively. Characterization with eight microsatellite markers identified 75 haplotypes within a total of 157 isolates over the three countries with a few haplotypes in Scotland and Norway sampled at a higher frequency than the rest across multiple locations and host plants. In total, eight microsatellite haplotypes were shared between Scotland and Norway while none were shared with England. Bayesian and principal component analyses revealed common ancestry and clustering of Scottish and Norwegian S. subarctica isolates while English isolates were assigned to a separate population cluster and exhibited low diversity indicative of isolation. Population structure was also examined for S. sclerotiorum isolates from England, Scotland, Norway, and Australia using microsatellite data, including some from a previous study in England. In total, 484 haplotypes were identified within 800 S. sclerotiorum isolates with just 15 shared between England and Scotland and none shared between any other countries. Bayesian and principal component analyses revealed a common ancestry and clustering of the English and Scottish isolates while Norwegian and Australian isolates were assigned to separate clusters. Furthermore, sequencing part of the intergenic spacer (IGS) region of the rRNA gene resulted in 26 IGS haplotypes within 870 S. sclerotiorum isolates, nine of which had not been previously identified and two of which were also widely distributed across different countries. S. subarctica therefore has a multiclonal population structure similar to S. sclerotiorum, but has a different ancestry and distribution across England, Scotland, and Norway

"Jumping Jack": Genomic Microsatellites Underscore the Distinctiveness of Closely Related Pseudoperonospora cubensis and Pseudoperonospora humuli and Provide New Insights Into Their Evolutionary Past

Downy mildews caused by obligate biotrophic oomycetes result in severe crop losses worldwide. Among these pathogens, Pseudoperonospora cubensis and P. humuli, two closely related oomycetes, adversely affect cucurbits and hop, respectively. Discordant hypotheses concerning their taxonomic relationships have been proposed based on host-pathogen interactions and specificity evidence and gene sequences of a few individuals, but population genetics evidence supporting these scenarios is missing. Furthermore, nuclear and mitochondrial regions of both pathogens have been analyzed using microsatellites and phylogenetically informative molecular markers, but extensive comparative population genetics research has not been done. Here, we genotyped 138 current and historical herbarium specimens of those two taxa using microsatellites (SSRs). Our goals were to assess genetic diversity and spatial distribution, to infer the evolutionary history of P. cubensis and P. humuli, and to visualize genome-scale organizational relationship between both pathogens. High genetic diversity, modest gene flow, and presence of population structure, particularly in P. cubensis, were observed. When tested for cross-amplification, 20 out of 27 P. cubensis-derived gSSRs cross-amplified DNA of P. humuli individuals, but few amplified DNA of downy mildew pathogens from related genera. Collectively, our analyses provided a definite argument for the hypothesis that both pathogens are distinct species, and suggested further speciation in the P. cubensis complex

Genome sequencing and comparative genomics of the broad host-range pathogen Rhizoctonia solani AG8

Rhizoctonia solani is a soil-borne basidiomycete fungus with a necrotrophic lifestyle which is classified into fourteen reproductively incompatible anastomosis groups (AGs). One of these, AG8, is a devastating pathogen causing bare patch of cereals, brassicas and legumes. R. solani is a multinucleate heterokaryon containing significant heterozygosity within a single cell. This complexity posed significant challenges for the assembly of its genome. We present a high quality genome assembly of R. solani AG8 and a manually curated set of 13,964 genes supported by RNA-seq. The AG8 genome assembly used novel methods to produce a haploid representation of its heterokaryotic state. The whole-genomes of AG8, the rice pathogen AG1-IA and the potato pathogen AG3 were observed to be syntenic and co-linear. Genes and functions putatively relevant to pathogenicity were highlighted by comparing AG8 to known pathogenicity genes, orthology databases spanning 197 phytopathogenic taxa and AG1-IA.We also observed SNP-level “hypermutation” of CpG dinucleotides to TpG between AG8 nuclei, with similarities to repeat-induced point mutation (RIP). Interestingly, gene-coding regions were widely affected along with repetitive DNA, which has not been previously observed for RIP in mononuclear fungi of the Pezizomycotina. The rate of heterozygous SNP mutations within this single isolate of AG8 was observed to be higher than SNP mutation rates observed across populations of most fungal species compared. Comparative analyses were combined to predict biological processes relevant to AG8 and 308 proteins with effector-like characteristics, forming a valuable resource for further study of this pathosystem. Predicted effector-like proteins had elevated levels of non-synonymous point mutations relative to synonymous mutations (dN/dS), suggesting that they may be under diversifying selection pressures. In addition, the distant relationship to sequenced necrotrophs of the Ascomycota suggests the R. solani genome sequence may prove to be a useful resource in future comparative analysis of plant pathogens