Do flower-colonizing microbes influence floral evolution? A test with fast-cycling Brassica.

Pollinators are thought to be the main drivers of floral evolution. Flowers are also colonized by abundant communities of microbes that can affect the interaction between plants and their pollinators. Very little is known, however, about how flower-colonizing microbes influence floral evolution. Here we performed a 6-generation experimental evolution study using fast-cycling Brassica rapa, in which we factorially manipulated the presence of pollinators and flower microbes to determine how pollinators and microbes interact in driving floral evolution. We measured the evolution of 6 morphological traits, as well as the plant mating system and flower attractiveness. Only one of the 6 traits (flower number) evolved in response to pollinators, while microbes did not drive the evolution of any trait, nor did they interact with pollinators in driving the evolution of morphological traits. Moreover, we did not find evidence that pollinators or microbes affected the evolution of flower attractiveness to pollinators. However, we found an interactive effect of pollinators and microbes on the evolution of autonomous selfing, a trait that is expected to evolve in response to pollinator limitations. Overall, we found only weak evidence that microbes mediate floral evolution. However, our ability to detect an interactive effect of pollinators and microbes might have been limited by weak pollinator-mediated selection in our experimental setting. Our results contrast with previous (similar) experimental evolution studies, highlighting the susceptibility of such experiments to drift and to experimental artefacts.

Low prevalence of secondary endosymbionts in aphids sampled from rapeseed crops in Germany.

Peach-potato aphids, Myzus persicae Sulzer (Hemiptera:Aphididae), and cabbage aphids, Brevicoryne brassicae Linnaeus (Hemiptera:Aphididae), are herbivorous insects of significant agricultural importance. Aphids can harbour a range of non-essential (facultative) endosymbiotic bacteria that confer multiple costs and benefits to the host aphid. A key endosymbiont-derived phenotype is protection against parasitoid wasps, and this protective phenotype has been associated with several defensive enodsymbionts. In recent years greater emphasis has been placed on developing alternative pest management strategies, including the increased use of natural enemies such as parasitoids wasps. For the success of aphid control strategies to be estimated the presence of defensive endosymbionts that can potentially disrupt the success of biocontrol agents needs to be determined in natural aphid populations. Here, we sampled aphids and mummies (parasitised aphids) from an important rapeseed production region in Germany and used multiplex PCR assays to characterise the endosymbiont communities. We found that aphids rarely harboured facultative endosymbionts, with 3.6% of M. persicae and 0% of B. brassicae populations forming facultative endosymbiont associations. This is comparable with endosymbiont prevalence described for M. persicae populations surveyed in Australia, Europe, Chile, and USA where endosymbiont infection frequencies range form 0-2%, but is in contrast with observations from China where M. persicae populations have more abundant and diverse endosymbiotic communities (endosymbionts present in over 50% of aphid populations).

DArTseq-Based, High-Throughput Identification of Novel Molecular Markers for the Detection of Blackleg (Leptosphaeria Spp.) Resistance in Rapeseed.

Blackleg disease, caused by Leptosphaeria spp. fungi, is one of the most important diseases of Brassica napus, responsible for severe yield losses worldwide. Blackleg resistance is controlled by major R genes and minor quantitative trait loci (QTL). Due to the high adaptation ability of the pathogen, R-mediated resistance can be easily broken, while the resistance mediated via QTL is believed to be more durable. Thus, the identification of novel molecular markers linked to blackleg resistance for B. napus breeding programs is essential. In this study, 183 doubled haploid (DH) rapeseed lines were assessed in field conditions for resistance to Leptosphaeria spp. Subsequently, DArTseq-based Genome-Wide Association Study (GWAS) was performed to identify molecular markers linked to blackleg resistance. A total of 133,764 markers (96,121 SilicoDArT and 37,643 SNP) were obtained. Finally, nine SilicoDArT and six SNP molecular markers were associated with plant resistance to Leptosphaeria spp. at the highest significance level, p < 0.001. Importantly, eleven of these fifteen markers were found within ten genes located on chromosomes A06, A07, A08, C02, C03, C06 and C08. Given the immune-related functions of the orthologues of these genes in Arabidopsis thaliana, the identified markers hold great promise for application in rapeseed breeding programs.

Supplementation of lactobacillus-fermented rapeseed meal in broiler diet reduces Campylobacter jejuni cecal colonization and limits the l-tryptophan and l-histidine biosynthesis pathways.

BACKGROUND: Campylobacter jejuni (C. jejuni), a widely distributed global foodborne pathogen, primarily linked with contaminated chicken meat, poses a significant health risk. Reducing the abundance of this pathogen in poultry meat is challenging but essential. This study assessed the impact of Lactobacillus-fermented rapeseed meal (LFRM) on broilers exposed to C. jejuni-contaminated litter, evaluating growth performance, Campylobacter levels, and metagenomic profile. RESULTS: By day 35, the litter contamination successfully colonized broilers with Campylobacter spp., particularly C. jejuni. In the grower phase, LFRM improved (P < 0.05) body weight and daily weight gain, resulting in a 9.2% better feed conversion ratio during the pre-challenge period (the period before artificial infection; days 13-20). The LFRM also reduced the C. jejuni concentration in the ceca (P < 0.05), without altering alpha and beta diversity. However, metagenomic data analysis revealed LFRM targeted a reduction in the abundance of C. jejuni biosynthetic pathways of l-tryptophan and l-histidine and gene families associated with transcription and virulence factors while also possibly leading to selected stress-induced resistance mechanisms. CONCLUSION: The study demonstrated that LFRM inclusion improved growth and decreased cecal Campylobacter spp. concentration and the relative abundance of pivotal C. jejuni genes. Performance benefits likely resulted from LFRM metabolites. At the molecular level, LFRM may have reduced C. jejuni colonization, likely by decreasing the abundance of energy transduction and l-histidine and l-tryptophan biosynthesis genes otherwise required for bacterial survival and increased virulence. © 2024 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Persistence of Escherichia coli in the microbiomes of red Romaine lettuce (Lactuca sativa cv. 'Outredgeous') and mizuna mustard (Brassica rapa var. japonica) - does seed sanitization matter?

BACKGROUND: Seed sanitization via chemical processes removes/reduces microbes from the external surfaces of the seed and thereby could have an impact on the plants' health or productivity. To determine the impact of seed sanitization on the plants' microbiome and pathogen persistence, sanitized and unsanitized seeds from two leafy green crops, red Romaine lettuce (Lactuca sativa cv. 'Outredgeous') and mizuna mustard (Brassica rapa var. japonica) were exposed to Escherichia coli and grown in controlled environment growth chambers simulating environmental conditions aboard the International Space Station. Plants were harvested at four intervals from 7 days post-germination to maturity. The bacterial communities of leaf and root were investigated using the 16S rRNA sequencing while quantitative polymerase chain reaction (qPCR) and heterotrophic plate counts were used to reveal the persistence of E. coli. RESULT: E. coli was detectable for longer periods of time in plants from sanitized versus unsanitized seeds and was identified in root tissue more frequently than in leaf tissue. 16S rRNA sequencing showed dynamic changes in the abundance of members of the phylum Proteobacteria, Firmicutes, and Bacteroidetes in leaf and root samples of both leafy crops. We observed minimal changes in the microbial diversity of lettuce or mizuna leaf tissue with time or between sanitized and unsanitized seeds. Beta-diversity showed that time had more of an influence on all samples versus the E. coli treatment. CONCLUSION: Our results indicated that the seed surface sanitization, a current requirement for sending seeds to space, could influence the microbiome. Insight into the changes in the crop microbiomes could lead to healthier plants and safer food supplementation.

MicroRNA319a regulates plant resistance to Sclerotinia stem rot.

MicroRNA319a (miR319a) controls cell division arrest in plant leaves by inhibiting the expression of TCP (TEOSINTE BRANCHED 1/CYCLOIDEA/PCF) family genes. However, it is unclear whether miR319a influences infection by necrotrophic pathogens and host susceptibility. In this study, we revealed that miR319a affects plant resistance to stem rot disease caused by Sclerotinia sclerotiorum. In Brassica rapa plants infected with S. sclerotiorum, miR319a levels increased while the expression levels of several BraTCP genes significantly decreased compared with those of uninfected plants. Overexpression of BraMIR319a in B. rapa increased the susceptibility of the plants to S. sclerotiorum and aggravated stem rot disease, whereas overexpression of BraTCP4-1 promoted plant resistance. RNA sequencing data revealed a potential relationship between miR319a and pathogen-related WRKY genes. Chromatin immunoprecipitation, electrophoretic mobility shift, and reporter transaction assays showed that BraTCP4-1 could bind to the promoters of WRKY75, WRKY70, and WRKY33 and directly activate these pathogen-related genes. Moreover, the expression levels of WRKY75, WRKY70, and WRKY33 in plants overexpressing BraMIR319a decreased significantly, whereas those of plants overexpressing BraTCP4-1 increased significantly, relative to the wild type. These results suggest that miR319a and its target gene BraTCP4 control stem rot resistance through pathways of WRKY genes.

The transcriptional response to salicylic acid plays a role in Fusarium yellows resistance in Brassica rapa L.

KEY MESSAGE: Fusarium yellows resistant and susceptible lines in Brassica rapa showed different salicylic acid responses; the resistant line showed a similar response to previous reports, but the susceptible line differed. Fusarium yellows caused by Fusarium oxysporum f. sp. conglutinans (Foc) is an important disease. Previous studies showed that genes related to salicylic acid (SA) response were more highly induced following Foc infection in Brassica rapa Fusarium yellows resistant lines than susceptible lines. However, SA-induced genes have not been identified at the whole genome level and it was unclear whether they were up-regulated by Foc inoculation. Transcriptome analysis with and without SA treatment in the B. rapa Fusarium yellows susceptible line 'Misugi' and the resistant line 'Nanane' was performed to obtain insights into the relationship between SA sensitivity/response and Fusarium yellows resistance. 'Nanane's up-regulated genes were related to SA response and down-regulated genes were related to jasmonic acid (JA) or ethylene (ET) response, but differentially expressed genes in 'Misugi' were not. This result suggests that Fusarium yellows resistant and susceptible lines have a different SA response and that an antagonistic transcription between SA and JA/ET responses was found only in a Fusarium yellows resistant line. SA-responsive genes were induced by Foc inoculation in Fusarium yellows resistant (RJKB-T23) and susceptible lines (RJKB-T24). By contrast, 39 SA-induced genes specific to RJKB-T23 might function in the defense response to Foc. In this study, SA-induced genes were identified at the whole genome level, and the possibility, the defense response to Foc observed in a resistant line could be mediated by SA-induced genes, is suggested. These results will be useful for future research concerning the SA importance in Foc or other diseases resistance in B. rapa.

Expression and Role of Biosynthetic, Transporter, Receptor, and Responsive Genes for Auxin Signaling during Clubroot Disease Development.

Auxins play a pivotal role in clubroot development caused by the obligate biotroph Plasmodiophora brassicae. In this study, we investigated the pattern of expression of 23 genes related to auxin biosynthesis, reception, and transport in Chinese cabbage (Brassica rapa) after inoculation with P. brassicae. The predicted proteins identified, based on the 23 selected auxin-related genes, were from protein kinase, receptor kinase, auxin responsive, auxin efflux carrier, transcriptional regulator, and the auxin-repressed protein family. These proteins differed in amino acids residue, molecular weights, isoelectric points, chromosomal location, and subcellular localization. Leaf and root tissues showed dynamic and organ-specific variation in expression of auxin-related genes. The BrGH3.3 gene, involved in auxin signaling, exhibited 84.4-fold increase in expression in root tissues compared to leaf tissues as an average of all samples. This gene accounted for 4.8-, 2.6-, and 5.1-fold higher expression at 3, 14, and 28 days post inoculation (dpi) in the inoculated root tissues compared to mock-treated roots. BrNIT1, an auxin signaling gene, and BrPIN1, an auxin transporter, were remarkably induced during both cortex infection at 14 dpi and gall formation at 28 dpi. BrDCK1, an auxin receptor, was upregulated during cortex infection at 14 dpi. The BrLAX1 gene, associated with root hair development, was induced at 1 dpi in infected roots, indicating its importance in primary infection. More interestingly, a significantly higher expression of BrARP1, an auxin-repressed gene, at both the primary and secondary phases of infection indicated a dynamic response of the host plant towards its resistance against P. brassicae. The results of this study improve our current understanding of the role of auxin-related genes in clubroot disease development.

A Gain-of-Function Screen Reveals Redundant ERF Transcription Factors Providing Opportunities for Resistance Breeding Toward the Vascular Fungal Pathogen Verticillium longisporum.

Verticillium longisporum is a vascular fungal pathogen leading to severe crop loss, particular in oilseed rape. Transcription factors (TF) are highly suited for genetic engineering of pathogen-resistant crops, as they control sets of functionally associated genes. Applying the AtTORF-Ex (Arabidopsis thaliana transcription factor open reading frame expression) collection, a simple and robust screen of TF-overexpressing plants was established displaying reduced fungal colonization. Distinct members of the large ethylene response factor (ERF) family, namely ERF96 and the six highly related subgroup IXb members ERF102 to ERF107, were identified. Whereas overexpression of these ERF significantly reduces fungal propagation, single loss-of-function approaches did not reveal altered susceptibility. Hence, this gain-of-function approach is particularly suited to identify redundant family members. Expression analyses disclosed distinct ERF gene activation patterns in roots and leaves, suggesting functional differences. Transcriptome studies performed on chemically induced ERF106 expression revealed an enrichment of genes involved in the biosynthesis of antimicrobial indole glucosinolates (IG), such as CYP81F2 (CYTOCHROME P450-MONOOXYGENASE 81F2), which is directly regulated by IXb-ERF via two GCC-like cis-elements. The impact of IG in restricting fungal propagation was further supported as the cyp81f2 mutant displayed significantly enhanced susceptibility. Taken together, this proof-of-concept approach provides a novel strategy to identify candidate TF that are valuable genetic resources for engineering or breeding pathogen-resistant crop plants.

Agrobacterium-mediated vacuum infiltration and floral dip transformation of rapid-cycling Brassica rapa.

BACKGROUND: Rapid-cycling Brassica rapa (RCBr), also known as Wisconsin Fast Plants, are small robust plants with a short lifecycle that are widely used in biology teaching. RCBr have been used for decades but there are no published reports of RCBr genetic transformation. Agrobacterium-mediated vacuum infiltration has been used to transform pakchoi (Brassica rapa ssp. chinensis) and may be suitable for RCBr transformation. The floral dip transformation method, an improved version of vacuum infiltration, could make the procedure easier. RESULTS: Based on previous findings from Arabidopsis and pakchoi, plants of three different ages were inoculated with Agrobacterium. Kanamycin selection was suboptimal with RCBr; a GFP screen was used to identify candidate transformants. RCBr floral bud dissection showed that only buds with a diameter less than 1 mm carried unsealed carpels, a key point of successful floral dip transformation. Plants across a wide range of inflorescence maturities but containing these immature buds were successfully transformed, at an overall rate of 0.1% (one per 1000 T1 seeds). Transformation was successful using either vacuum infiltration or the floral dip method, as confirmed by PCR and Southern blot. CONCLUSION: A genetic transformation system for RCBr was established in this study. This will promote development of new biology teaching tools as well as basic biology research on Brassica rapa.

Activity Improvement and Vital Amino Acid Identification on the Marine-Derived Quorum Quenching Enzyme MomL by Protein Engineering.

MomL is a marine-derived quorum-quenching (QQ) lactonase which can degrade various N-acyl homoserine lactones (AHLs). Intentional modification of MomL may lead to a highly efficient QQ enzyme with broad application potential. In this study, we used a rapid and efficient method combining error-prone polymerase chain reaction (epPCR), high-throughput screening and site-directed mutagenesis to identify highly active MomL mutants. In this way, we obtained two candidate mutants, MomLI144V and MomLV149A. These two mutants exhibited enhanced activities and blocked the production of pathogenic factors of Pectobacterium carotovorum subsp. carotovorum (Pcc). Besides, seven amino acids which are vital for MomL enzyme activity were identified. Substitutions of these amino acids (E238G/K205E/L254R) in MomL led to almost complete loss of its QQ activity. We then tested the effect of MomL and its mutants on Pcc-infected Chinese cabbage. The results indicated that MomL and its mutants (MomLL254R, MomLI144V, MomLV149A) significantly decreased the pathogenicity of Pcc. This study provides an efficient method for QQ enzyme modification and gives us new clues for further investigation on the catalytic mechanism of QQ lactonase.

AhlX, an N-acylhomoserine Lactonase with Unique Properties.

N-Acylhomoserine lactonase degrades the lactone ring of N-acylhomoserine lactones (AHLs) and has been widely suggested as a promising candidate for use in bacterial disease control. While a number of AHL lactonases have been characterized, none of them has been developed as a commercially available enzymatic product for in vitro AHL quenching due to their low stability. In this study, a highly stable AHL lactonase (AhlX) was identified and isolated from the marine bacterium Salinicola salaria MCCC1A01339. AhlX is encoded by a 768-bp gene and has a predicted molecular mass of 29 kDa. The enzyme retained approximately 97% activity after incubating at 25 °C for 12 days and ~100% activity after incubating at 60 °C for 2 h. Furthermore, AhlX exhibited a high salt tolerance, retaining approximately 60% of its activity observed in the presence of 25% NaCl. In addition, an AhlX powder made by an industrial spray-drying process attenuated Erwinia carotovora infection. These results suggest that AhlX has great potential for use as an in vitro preventive and therapeutic agent for bacterial diseases.

A New Subclade of Leptosphaeria biglobosa Identified from Brassica rapa.

Blackleg (Phoma stem canker) of crucifers is a globally important disease caused by the ascomycete species complex comprising of Leptosphaeria maculans and Leptosphaeria biglobosa. Six blackleg isolates recovered from Brassica rapa cv. Mizspoona in the Willamette Valley of Oregon were characterized as L. biglobosa based on standard pathogenicity tests and molecular phylogenetic analysis. These isolates were compared to 88 characterized L. biglobosa isolates from western Canada, 22 isolates from Australia, and 6 L. maculans isolates from Idaho, USA using maximum parsimony and distance analysis of phylogenetic trees generated from the ITS rDNA (internal transcribed spacer rDNA) sequence, and the actin and ß-tubulin gene sequences. The L. biglobosa isolates derived from B. rapa collected in Oregon formed a separate subclade based on concatenated gene sequences or a single gene sequence, regardless of the analyses. Pathogenicity tests showed that these isolates failed to infect either resistant or susceptible B. napus cultivars, but caused severe symptoms on three B. rapa cultivars (Accession number: UM1113, UM1112, and UM1161), a B. oleracea var. capitata (cabbage) cultivar (Copenhagen Market), and two B. juncea cultivars (CBM, a common brown Mustard, and Forge). These findings demonstrated that the L. biglobosa isolates derived from a B. rapa crop in Oregon were genetically distinct from existing species of L. biglobosa, and constitute a new subclade, herein proposed as L. biglobosa 'americensis'.

Activity of a novel succinate dehydrogenase inhibitor fungicide pyraziflumid against Sclerotinia sclerotiorum.

Pyraziflumid is a novel member of succinate dehydrogenase inhibitor fungicides (SDHI). In this study, baseline sensitivity of Sclerotinia sclerotiorum (Lib.) de Bary to pyraziflumid was determined using 105 strains collected during 2015 and 2017 from different geographical regions in Jiangsu Province of China, and the average EC50 value was 0.0561 (±0.0263)µg/ml for mycelial growth. There was no cross-resistance between pyraziflumid and the widely used fungicides carbendazim, dimethachlon and the phenylpyrrole fungicide fludioxonil. After pyraziflumid treated, hyphae were contorted with offshoot of top increasing, cell membrane permeability increased markedly, oxalic acid content significantly decreased and mycelial respiration was strongly inhibited. But the number and dry weight of sclerotia did not change significantly. The protective and curative activity test of pyraziflumid suggested that pyraziflumid had great control efficiency against S. sclerotiorum on detached rapeseed leaves, and protective activity was better than curative activity. These results will contribute to us on evaluating the potential of the new SDHI fungicide pyraziflumid for management of diseases caused by S. sclerotiorum and understanding the mode of action of pyraziflumid against S. sclerotiorum.

Pharmacological characteristics of the novel fungicide pyrisoxazole against Sclerotinia sclerotiorum.

Pyrisoxazole is a pyridine compound of demethylation inhibitor fungicides. In this study, baseline sensitivity of Sclerotinia sclerotiorum to pyrisoxazole was determined using 166 strains from the oilseed rape fields in 2014, 2015 and 2016. The EC50 values for mycelial growth inhibition ranged from 0.0214 to 0.5443 µg mL-1, with a mean EC50 value of 0.2329 ±â€¯0.1048 µg mL-1 and were normally distributed. The EC50 values had no significant difference among three populations from 2014, 2015, and 2016. There was no correlation with sensitivity between pyrisoxazole and carbendazim or iprodione. After treated with pyrisoxazole, we observed increased cell membrane permeability, and decreased exopolysaccharide and oxalic acid production, which can contribute to reduced virulence of S. sclerotiorum and lead to failure of disease infection. Protective and curative activity tests showed that pyrisoxazole exhibited excellent protective and curative activity against S. sclerotiorum in oilseed rape, and protective activity was better than curative activity. Compared with the currently used fungicides, pyrisoxazole not only exhibited excellent control efficacy on Sclerotinia stem rot, but also dramatically reduced the doses of fungicides in the field trials. Overall, these data provide more references for revealing pharmacological effect of pyrisoxazole against S. sclerotiorum and managing Sclerotinia stem rot on oilseed rape caused by benzimidazole- and dicarboximide-resistant populations.

A Six-Year Investigation of the Dynamics of Avirulence Allele Profiles, Blackleg Incidence, and Mating Type Alleles of Leptosphaeria maculans Populations Associated with Canola Crops in Manitoba, Canada.

Blackleg, caused by the fungal pathogen Leptosphaeria maculans, is one of the most economically important diseases of canola (Brassica napus, oilseed rape) worldwide. This study assessed incidence of blackleg, the avirulence allele, and mating type distributions of L. maculans isolates collected in commercial canola fields in Manitoba, Canada, from 2010 to 2015. A total of 956 L. maculans isolates were collected from 2010 to 2015 to determine the presence of 12 avirulence alleles using differential canola cultivars and/or PCR assays specific for each avirulence allele. AvrLm2, AvrLm4, AvrLm5, AvrLm6, AvrLm7, AvrLm11, and AvrLmS were detected at frequencies ranging from 97 to 33%, where the AvrLm1, AvrLm3, AvrLm9, AvrLepR1, and AvrLepR2 alleles were the least abundant. When the race structure was examined, a total of 170 races were identified among the 956 isolates, with three major races, AvrLm-2-4-5-6-7-11, AvrLm-2-4-5-6-7-11-S, and Avr-1-4-5-6-7-11-(S) accounting for 15, 10, and 6% of the total fungal population, respectively. The distribution of the mating type alleles (MAT1-1 and MAT1-2) indicated that sexual reproduction was not inhibited in any of the nine Manitoba regions in any of the years L. maculans isolates were collected.

Stimulatory Effects of Flusilazole on Virulence of Sclerotinia sclerotiorum.

Flusilazole, a member of the demethylation inhibitor fungicides, is highly efficacious for control of Sclerotinia sclerotiorum. To achieve judicious applications of flusilazole, its hormetic effects on virulence of S. sclerotiorum were investigated. Flusilazole sprayed at concentrations from 0.02 to 0.5 µg/ml caused statistically significant (P < 0.05) stimulatory effects on virulence of S. sclerotiorum to potted rapeseed plants, and the maximum stimulation magnitudes were 11.0 and 10.7% for isolates GS-7 and HN-24, respectively. Studies on the time course of the infection process showed that a stimulatory effect on virulence could be discerned at 18 h postinoculation, indicating a direct stimulation mechanism rather than an overcompensation for initial inhibitions. In order to determine whether the stimulations were caused mainly by effects of flusilazole on S. sclerotiorum or on rapeseed plants, mycelia grown on flusilazole-amended potato dextrose agar (PDA) media were inoculated on leaves of rapeseed plants without spraying the fungicide. Mycelium radial growth on PDA supplemented with flusilazole at concentrations from 0.005 to 0.16 µg/ml was inhibited by 10.11 to 48.7% for isolate GS-7 and by 4.1 to 24.9% for isolate HN-24. Observations with a scanning electron microscope showed that flusilazole in PDA at 0.04 and 0.08 µg/ml caused slightly deformed mycelia and twisted mycelial tips. Nevertheless, after inoculating on leaves of potted rapeseed plants, virulence of the inhibited mycelia was statistically significantly (P < 0.05) greater than that of the nontreated control, and the maximum stimulation magnitudes were 16.2 and 19.8% for isolates GS-7 and HN-24, respectively. Studies on a physiological mechanism for virulence stimulations showed that tolerance to hydrogen peroxide did not increase significantly for mycelia grown on flusilazole-amended PDA, thus excluding the possibility of tolerance to reactive oxygen species as a potential mechanism for virulence stimulations.

Micro-organisms growing on rapeseed during storage affect the profile of volatile compounds of virgin rapeseed oil.

BACKGROUND: Micro-organisms populate on rapeseed after harvest during storage depending on the growing conditions. The composition of the bacterial colonization is unknown, although its contribution to the profile of volatile aroma-active compounds determines the sensory quality of virgin cold-pressed rapeseed oil. RESULTS: From four rapeseed samples, 46 bacterial strains were isolated. By DNA-sequencing, the identification of four bacteria species and 17 bacteria genera was possible. In total, 22 strains were selected, based on their typical off-flavors resembling those of virgin sensory bad cold-pressed rapeseed oils. The cultivation of these strains on rapeseed meal agar and examination of volatile compounds by solid phase microextraction-gas chromatography-mass spectrometry allowed the identification of 29 different compounds, mainly degradation products of fatty acids such as alkanes, alkenes, aldehydes, ketones and alcohols and, in addition, sulfur-containing compounds, including one terpene and three pyrazines. From these compounds, 19 are described as aroma-active in the literature. CONCLUSION: Micro-organisms populating on rapeseed during storage may strongly influence the sensory quality of virgin rapeseed oil as a result of the development of volatile aroma-active metabolic products. It can be assumed that occurrence of off-flavor of virgin rapeseed oils on the market are the result of metabolic degradation products produced by micro-organisms populating on rapeseed during storage. © 2017 Society of Chemical Industry.

Identifying the Active Microbiome Associated with Roots and Rhizosphere Soil of Oilseed Rape.

RNA stable isotope probing and high-throughput sequencing were used to characterize the active microbiomes of bacteria and fungi colonizing the roots and rhizosphere soil of oilseed rape to identify taxa assimilating plant-derived carbon following 13CO2 labeling. Root- and rhizosphere soil-associated communities of both bacteria and fungi differed from each other, and there were highly significant differences between their DNA- and RNA-based community profiles. Verrucomicrobia, Proteobacteria, Planctomycetes, Acidobacteria, Gemmatimonadetes, Actinobacteria, and Chloroflexi were the most active bacterial phyla in the rhizosphere soil. Bacteroidetes were more active in roots. The most abundant bacterial genera were well represented in both the 13C- and 12C-RNA fractions, while the fungal taxa were more differentiated. Streptomyces, Rhizobium, and Flavobacterium were dominant in roots, whereas Rhodoplanes and Sphingomonas (Kaistobacter) were dominant in rhizosphere soil. "Candidatus Nitrososphaera" was enriched in 13C in rhizosphere soil. Olpidium and Dendryphion were abundant in the 12C-RNA fraction of roots; Clonostachys was abundant in both roots and rhizosphere soil and heavily 13C enriched. Cryptococcus was dominant in rhizosphere soil and less abundant, but was 13C enriched in roots. The patterns of colonization and C acquisition revealed in this study assist in identifying microbial taxa that may be superior competitors for plant-derived carbon in the rhizosphere of Brassica napusIMPORTANCE This microbiome study characterizes the active bacteria and fungi colonizing the roots and rhizosphere soil of Brassica napus using high-throughput sequencing and RNA-stable isotope probing. It identifies taxa assimilating plant-derived carbon following 13CO2 labeling and compares these with other less active groups not incorporating a plant assimilate. Brassica napus is an economically and globally important oilseed crop, cultivated for edible oil, biofuel production, and phytoextraction of heavy metals; however, it is susceptible to several diseases. The identification of the fungal and bacterial species successfully competing for plant-derived carbon, enabling them to colonize the roots and rhizosphere soil of this plant, should enable the identification of microorganisms that can be evaluated in more detailed functional studies and ultimately be used to improve plant health and productivity in sustainable agriculture.

Comparison of transcriptome profiles by Fusarium oxysporum inoculation between Fusarium yellows resistant and susceptible lines in Brassica rapa L.

KEY MESSAGE: Resistant and susceptible lines in Brassica rapa have different immune responses against Fusarium oxysporum inoculation. Fusarium yellows caused by Fusarium oxysporum f. sp. conglutinans (Foc) is an important disease of Brassicaceae; however, the mechanism of how host plants respond to Foc is still unknown. By comparing with and without Foc inoculation in both resistant and susceptible lines of Chinese cabbage (Brassica rapa var. pekinensis), we identified differentially expressed genes (DEGs) between the bulked inoculated (6, 12, 24, and 72 h after inoculation (HAI)) and non-inoculated samples. Most of the DEGs were up-regulated by Foc inoculation. Quantitative real-time RT-PCR showed that most up-regulated genes increased their expression levels from 24 HAI. An independent transcriptome analysis at 24 and 72 HAI was performed in resistant and susceptible lines. GO analysis using up-regulated genes at 24 HAI indicated that Foc inoculation activated systemic acquired resistance (SAR) in resistant lines and tryptophan biosynthetic process and responses to chitin and ethylene in susceptible lines. By contrast, GO analysis using up-regulated genes at 72 HAI showed the overrepresentation of some categories for the defense response in susceptible lines but not in the resistant lines. We also compared DEGs between B. rapa and Arabidopsis thaliana after F. oxysporum inoculation at the same time point, and identified genes related to defense response that were up-regulated in the resistant lines of Chinese cabbage and A. thaliana. Particular genes that changed expression levels overlapped between the two species, suggesting that they are candidates for genes involved in the resistance mechanisms against F. oxysporum.