Non-invasive assessment and visualization of Phytophthora cactorum infection in strawberry crowns using quantitative magnetic resonance imaging.

Phytophthora cactorum is an oomycete species that causes enormous losses on horticultural crops, including strawberries. The purpose of this work was to investigate the alterations caused by P. cactorum inoculation in hydroponically grown strawberry plantlets (Fragaria × ananassa Duch.) using quantitative magnetic resonance imaging (qMRI). It was observed that with MRI, spatial and temporal progression of the infection could be observed in the crown using quantitative MR parameters, namely relaxation time maps. Relaxation times are numeric subject-specific properties that describe the MR signal behavior in an examined anatomical region. Elevated [Formula: see text] relaxation time values were observed inside the infected plant crowns with respect to the healthy references. The [Formula: see text] and [Formula: see text] values of healthy plants were small in the crown region and further diminished during the development of the plant. Furthermore, elevated [Formula: see text] relaxation time values were seen in regions where P. cactorum progression was observed in corresponding plant dissection photographs. Quantitative susceptibility maps (QSM) were calculated to estimate the local magnetic field inhomogeneities. The QSM suggests magnetic susceptibility differences near the center of the pith. This study provides novel non-invasive information on the structure and development of strawberry plants and the effects caused by the P. cactorum infection.

Terpenoid and lipid profiles vary in different Phytophthora cactorum - strawberry interactions.

Specialized metabolites are essential components in plant defence systems, serving as signalling molecules and chemical weapons against pathogens. The manipulation of plant defence metabolome or metabolites can thus be an important virulence strategy for pathogens. Because of their central role, metabolites can give valuable insights into plant-pathogen interactions. Here, we have conducted nontargeted metabolite profiling with UPLC-ESI-qTOF-MS to investigate the metabolic changes that have taken place in the crown tissue of Fragaria vesca L. (woodland strawberry) and Fragaria × ananassa (Weston) Duchesne ex Rozier (garden strawberry) during 48 h after Phytophthora cactorum challenge. Two P. cactorum isolates were compared: Pc407 is highly virulent to F. × ananassa and causes crown rot, whereas Pc440 is mildly virulent. In total, 45 metabolites differentially accumulated between the treatment groups were tentatively identified. Triterpenoids and various lipid compounds were highly represented. The levels of several triterpenoids increased upon inoculation, some of them showing distinct accumulation patterns in different interactions. Triterpenoids could either inhibit or stimulate P. cactorum growth and, therefore, triterpenoid profiles might have significant impact on disease progression. Of the lipid compounds, lysophospholipids, linoleic acid and linolenic acid were highly accumulated in the most compatible Pc407 - F. × ananassa interaction. As lysophospholipids promote cell death and have been linked to susceptibility, these compounds might be involved in the pathogenesis of crown rot disease. This metabolite analysis revealed potential factors contributing to the outcome of P. cactorum - strawberry interactions. The information is highly valuable, as it can help to find new breeding strategies and new solutions to control P. cactorum in strawberry.

RPA-PCR couple: an approach to expedite plant diagnostics and overcome PCR inhibitors.

DNA extraction can be lengthy and sometimes ends up with amplification inhibitors. We present the potential of recombinase polymerase amplification (RPA) to replace plant DNA extraction. In our rapid 'RPA-PCR couple' concept, RPA is tuned to slower reaction kinetics to promote amplification of long targets. RPA primers amplify target and some flanking regions directly from simple plant macerates. Then PCR primers exponentially amplify the target directly from the RPA reaction. We present the coupling of RPA with conventional, TaqMan and SYBR Green PCR assays. We applied the concept to strawberry Phytophthora pathogens and the Phytophthora identification marker atp9-nad9. We found RPA-PCR couple specific, sensitive and reliable. The approach may also benefit other difficult samples such as food, feces and ancient samples.

Expression of the GAF Sensor, Carbohydrate-Active Enzymes, Elicitins, and RXLRs Differs Markedly Between Two Phytophthora cactorum Isolates.

The phytopathogen Phytophthora cactorum infects economically important herbaceous and woody plant species. P. cactorum isolates differ in host specificity; for example, strawberry crown rot is often caused by a specialized pathotype. Here we compared the transcriptomes of two P. cactorum isolates that differ in their virulence to garden strawberry (Pc407: high virulence; Pc440: low virulence). De novo transcriptome assembly and clustering of contigs resulted in 19,372 gene clusters. Two days after inoculation of Fragaria vesca roots, 3,995 genes were differently expressed between the P. cactorum isolates. One of the genes that were highly expressed only in Pc407 encodes a GAF sensor protein potentially involved in membrane trafficking processes. Two days after inoculation, elicitins were highly expressed in Pc407 and lipid catabolism appeared to be more active than in Pc440. Of the carbohydrate-active enzymes, those that degrade pectin were often more highly expressed in Pc440, whereas members of glycosyl hydrolase family 1, potentially involved in the metabolism of glycosylated secondary metabolites, were more highly expressed in Pc407 at the time point studied. Differences were also observed among the RXLR effectors: Pc407 appears to rely on a smaller set of key RXLR effectors, whereas Pc440 expresses a greater number of RXLRs. This study is the first step toward improving understanding of the molecular basis of differences in the virulence of P. cactorum isolates. Identification of the key effectors is important, as it enables effector-assisted breeding strategies toward crown rot-resistant strawberry cultivars.

Reprogramming of Strawberry (Fragaria vesca) Root Transcriptome in Response to Phytophthora cactorum.

Crown rot (Phytophthora cactorum) causes significant economic losses in strawberry production. The best control strategy would be to use resistant cultivars, but polygenically inherited resistance makes the breeding of the garden strawberry (Fragaria × ananassa) challenging. The diploid wild strawberry Fragaria vesca Hawaii 4 genotype was shown previously to have resistance against crown rot. To explore the resistance mechanisms, we inoculated the roots of Hawaii 4 with P. cactorum in a novel in vitro hydroponic system to minimize interference caused by other microbes. Major reprogramming of the root transcriptome occurred, involving 30% of the genes. The surveillance system of the plant shifted from the development mode to the defense mode. Furthermore, the immune responses as well as many genes involved in the biosynthesis of the defense hormones jasmonic acid, ethylene and salicylic acid were up-regulated. Several major allergen-like genes encoding PR-10 proteins were highly expressed in the inoculated plants, suggesting that they also have a crucial role in the defense responses against P. cactorum. Additionally, flavonoids and terpenoids may be of vital importance, as several genes involved in their biosynthesis were up-regulated. The cell wall biosynthesis and developmental processes were down-regulated, possibly as a result of the down-regulation of the key genes involved in the biosynthesis of growth-promoting hormones brassinosteroids and auxin. Of particular interest was the expression of potential resistance genes in the recently identified P. cactorum resistance locus RPc-1. These new findings help to target the breeding efforts aiming at more resistant strawberry cultivars.

De Novo assembly and annotation of the freshwater crayfish Astacus astacus transcriptome.

We generated RNA-seq data to assemble the transcriptome of the noble crayfish (Astacus astacus) from four combined tissues (abdominal muscle, hepatopancreas, ovaries, green glands). A total of 194 million read pairs with a length of 100 bp were generated. The transcriptome was assembled de novo using Trinity software, producing 158,649 non-redundant transcripts. Lowly expressed transcripts were filtered out leaving 45,415 transcripts of which 14,559 were found to contain open reading frames with predicted gene function. The Transrate software revealed that 91% of the total reads were realigned to the assembly. Furthermore, BUSCO analysis indicated that our assembly is 64% complete. A total of 13,770 transcripts were assigned at least one GO term. This first de novo transcriptome assembly is an important foundation for future genomic research on the noble crayfish and adds to the general knowledge and further characterization of transcriptomes of non-model organisms.