Impact of Additives and Packing Density on Fermentation Weight Loss, Microbial Diversity, and Fermentation Quality of Rape Straw Silage.

To investigate the effects of the combined addition of Lactiplantibacillus plantarum and sucrose on the fermentation weight loss (FWL), fermentation quality, and microbial community structure of ensiled rape straw under varying packing density conditions. After harvesting, the rapeseed straw was collected, cut into 1-2 cm pieces, and sprayed with sterile water to adjust the moisture content to 60%. The straw was then divided into two groups: one treated with additives (1 × 105 CFU/g fresh material of Lactiplantibacillus plantarum and 10 kg/t fresh material of sucrose), and the other sprayed with an equivalent amount of sterile water as the control (CK). The treated materials were thoroughly mixed and packed into silos at densities of 450, 500, and 550 kg/m3. FWL was recorded on days 1, 3, 6, 15, 20, and 45 of fermentation. On day 45, the samples were analyzed for fermentation quality, microbial counts, and microbial diversity. FWL increased significantly (p < 0.05) in both the treated (LS) and control groups during fermentation. The LS group showed higher lactic acid (LA) levels (p < 0.05) and lower ammonia nitrogen levels (p < 0.05) compared to CK. The CK group had significantly higher (p < 0.05) counts of Coliforms and lower bacterial counts (p < 0.05) than LS. The dominant genera in the silage were Xanthomonas, Lactiplantibacillus plantarum, and Lentilactobacillus. In the LS group, the relative abundances of Lactiplantibacillus plantarum and Lentilactobacillus ranged from 16.93% to 20.43% and 15.63% to 27.46%, respectively, with their combined abundance being higher than in CK. At a packing density of 500 kg/m3, the relative abundances of Lactiplantibacillus plantarum and Lentilactobacillus in the LS group were significantly higher (p < 0.05) than in CK. Increasing packing density and applying additives to rape straw silage effectively reduced FWL, improved fermentation quality, boosted the relative abundance of beneficial lactic acid bacteria, and decreased the presence of undesirable bacteria such as Enterobacter and Bacillus.

Single-cell transcriptome profiling reveals the spatiotemporal distribution of triterpenoid saponin biosynthesis and transposable element activity in Gynostemma pentaphyllum shoot apexes and leaves.

Gynostemma pentaphyllum (Thunb.) Makino is an important producer of dammarene-type triterpenoid saponins. These saponins (gypenosides) exhibit diverse pharmacological benefits such as anticancer, antidiabetic, and immunomodulatory effects, and have major potential in the pharmaceutical and health care industries. Here, we employed single-cell RNA sequencing (scRNA-seq) to profile the transcriptomes of more than 50,000 cells derived from G. pentaphyllum shoot apexes and leaves. Following cell clustering and annotation, we identified five major cell types in shoot apexes and four in leaves. Each cell type displayed substantial transcriptomic heterogeneity both within and between tissues. Examining gene expression patterns across various cell types revealed that gypenoside biosynthesis predominantly occurred in mesophyll cells, with heightened activity observed in shoot apexes compared to leaves. Furthermore, we explored the impact of transposable elements (TEs) on G. pentaphyllum transcriptomic landscapes. Our findings the highlighted the unbalanced expression of certain TE families across different cell types in shoot apexes and leaves, marking the first investigation of TE expression at the single-cell level in plants. Additionally, we observed dynamic expression of genes involved in gypenoside biosynthesis and specific TE families during epidermal and vascular cell development. The involvement of TE expression in regulating cell differentiation and gypenoside biosynthesis warrant further exploration. Overall, this study not only provides new insights into the spatiotemporal organization of gypenoside biosynthesis and TE activity in G. pentaphyllum shoot apexes and leaves but also offers valuable cellular and genetic resources for a deeper understanding of developmental and physiological processes at single-cell resolution in this species.

Novel heterogeneous Fe-based catalysts for carbon dioxide hydrogenation to long chain α-olefins-A review.

The thermocatalytic conversion of carbon dioxide (CO2) into high value-added chemicals provides a strategy to address the environmental problems caused by excessive carbon emissions and the sustainable production of chemicals. Significant progress has been made in the CO2 hydrogenation to long chain α-olefins, but controlling C-O activation and C-C coupling remains a great challenge. This review focuses on the recent advances in catalyst design concepts for the synthesis of long chain α-olefins from CO2 hydrogenation. We have systematically summarized and analyzed the ingenious design of catalysts, reaction mechanisms, the interaction between active sites and supports, structure-activity relationship, influence of reaction process parameters on catalyst performance, and catalyst stability, as well as the regeneration methods. Meanwhile, the challenges in the development of the long chain α-olefins synthesis from CO2 hydrogenation are proposed, and the future development opportunities are prospected. The aim of this review is to provide a comprehensive perspective on long chain α-olefins synthesis from CO2 hydrogenation to inspire the invention of novel catalysts and accelerate the development of this process.

Synthesis of Liquid Hydrocarbon via Direct Hydrogenation of CO2 over FeCu-Based Bifunctional Catalyst Derived from Layered Double Hydroxides.

Here, we report a Na-promoted FeCu-based catalyst with excellent liquid hydrocarbon selectivity and catalytic activity. The physiochemical properties of the catalysts were comprehensively characterized by various characterization techniques. The characterization results indicate that the catalytic performance of the catalysts was closely related to the nature of the metal promoters. The Na-AlFeCu possessed the highest CO2 conversion due to enhanced CO2 adsorption of the catalysts by the introduction of Al species. The introduction of excess Mg promoter led to a strong methanation activity of the catalyst. Mn and Ga promoters exhibited high selectivity for light hydrocarbons due to their inhibition of iron carbides generation, resulting in a lack of chain growth capacity. The Na-ZnFeCu catalyst exhibited the optimal C5+ yield, owing to the fact that the Zn promoter improved the catalytic activity and liquid hydrocarbon selectivity by modulating the surface CO2 adsorption and carbide content. Carbon dioxide (CO2) hydrogenation to liquid fuel is considered a method for the utilization and conversion of CO2, whereas satisfactory activity and selectivity remains a challenge. This method provides a new idea for the catalytic hydrogenation of CO2 and from there the preparation of high-value-added products.

Unamplified and Label-Free Detection of HPV16 DNA Using CRISPR-Cas12a-Functionalized Solution-Gated Graphene Transistors.

The persistent infection of high-risk-human papillomavirus type 16 (HPV16) is considered an essential element for suffering cervical cancer. Despite polymerase chain reaction, loop-mediated amplification, and microfluidic chips are used to detect the HPV16, these methods still exist some drawbacks including time-consuming and false positive results. The CRISPR-Cas system is widely used in the region of biological detection due to its precise targeted recognition capability. In this contribution, the novel solution-gated graphene transistor sensor is designed to realize the unamplified and label-free detection of HPV16 DNA. Using the precise recognition of the CRISPR-Cas12a system and the gate functionalization, HPV16 DNA can be precisely identified without need the amplification and labeling. The limit of detection of the sensor can be up to 8.3 × 10-18  m and the detection can be within 20 min. Additionally, the heat-Inactivated clinical samples can be clearly distinguished by the sensor the diagnosis results have a high degree of agreement with q-PCR detection.

Unamplified and Real-Time Label-Free miRNA-21 Detection Using Solution-Gated Graphene Transistors in Prostate Cancer Diagnosis.

The incidence of prostate cancer (PCa) in men globally increases as the standard of living improves. Blood serum biomarker prostate-specific antigen (PSA) detection is the gold standard assay that do not meet the requirements of early detection. Herein, a solution-gated graphene transistor (SGGT) biosensor for the ultrasensitive and rapid quantification detection of the early prostate cancer-relevant biomarker, miRNA-21 is reported. The designed single-stranded DNA (ssDNA) probes immobilized on the Au gate can hybridize effectively with the miRNA-21 molecules targets and induce the Dirac voltage shifts of SGGT transfer curves. The limit of detection (LOD) of the sensor can reach 10-20  M without amplification and any chemical or biological labeling. The detection linear range is from 10-20 to 10-12  M. The sensor can realize real-time detection of the miRNA-21 molecules in less than 5 min and can well distinguish one-mismatched miRNA-21 molecule. The blood serum samples from the patients without RNA extraction and amplification are measured. The results demonstrated that the biosensor can well distinguish the cancer patients from the control group and has higher sensitivity (100%) than PSA detection (58.3%). Contrastingly, it can be found that the PSA level is not directly related to PCa.

Innovative natural antimicrobial natamycin incorporated titanium dioxide (nano-TiO2)/ poly (butylene adipate-co-terephthalate) (PBAT) /poly (lactic acid) (PLA) biodegradable active film (NTP@PLA) and application in grape preservation.

Poly (butylene adipate-co-terephthalate) (PBAT)/polylactic acid (PLA) blended with compatibilizers (polycaprolactone, PCL; poly (ethylene glycol), PEG; titanium dioxide, nano-TiO2) (TP@PLA composites) were developed by melt processing. Natamycin incorporated into TP@PLA blend composites formed NTP@PLA films, which exhibited high tensile strength (24.1-43.5 MPa) and elongation at break (85.8-258.2 %), and exhibited good oxygen permeability, water vapor permeability, surface hydrophobicity and biodegradability. The in vitro results revealed that inhibition of Penicillium expansum cell growth of the NTP@PLA films with addition of 1.0 wt% natamycin reached 95.72 %. The NTP@PLA film with natamycin effectively reduced incidence of decay (1.52 %) on grapes, maintained their quality, and inhibited the growth of pathogenic fungi to up to 0.42 log cfu·g-1. This study generates new insights into the preservation properties of antimicrobial NTP@PLA film, which endow it with great application potential as a novel and eco-friendly packaging material for the food industry.

Rapid and Unamplified Detection of SARS-CoV-2 RNA via CRISPR-Cas13a-Modified Solution-Gated Graphene Transistors.

The disease caused by severe acute respiratory syndrome coronavirus, SARS-CoV-2, is termed COVID-19. Even though COVID-19 has been out for more than two years, it is still causing a global pandemic. Due to the limitations of sample collection, transportation, and kit performance, the traditional reverse transcription-quantitative polymerase chain reaction (RT-qPCR) method has a long detection period and high testing costs. An increased risk of infection is inevitable, since many patients may not be diagnosed in time. The CRISPR-Cas13a system can be designed for RNA identification and knockdown, as a promising platform for nucleic acid detection. Here, we designed a solution-gated graphene transistor (SGGT) biosensor based on the CRISPR-Cas13a system. Using the gene-targeting capacity of CRISPR-Cas13a and gate functionalization via multilayer modification, SARS-CoV-2 nucleic acid sequences can be quickly and precisely identified without the need for amplification or fluorescence tagging. The limit of detection (LOD) in both buffer and serum reached the aM level, and the reaction time was about 10 min. The results of the detection of COVID-19 clinical samples from throat swabs agree with RT-PCR. In addition, the interchangeable gates significantly minimize the cost and time of device fabrication. In a nutshell, our biosensor technology is broadly applicable and will be suitable for point-of-care (POC) testing.

Ultracompact meta-imagers for arbitrary all-optical convolution.

Electronic digital convolutions could extract key features of objects for data processing and information identification in artificial intelligence, but they are time-cost and energy consumption due to the low response of electrons. Although massless photons enable high-speed and low-loss analog convolutions, two existing all-optical approaches including Fourier filtering and Green's function have either limited functionality or bulky volume, thus restricting their applications in smart systems. Here, we report all-optical convolutional computing with a metasurface-singlet or -doublet imager, considered as the third approach, where its point spread function is modified arbitrarily via a complex-amplitude meta-modulator that enables functionality-unlimited kernels. Beyond one- and two-dimensional spatial differentiation, we demonstrate real-time, parallel, and analog convolutional processing of optical and biological specimens with challenging pepper-salt denoising and edge enhancement, which significantly enrich the toolkit of all-optical computing. Such meta-imager approach bridges multi-functionality and high-integration in all-optical convolutions, meanwhile possessing good architecture compatibility with digital convolutional neural networks.

Ultrasensitive Label-Free DNA Detection Based on Solution-Gated Graphene Transistors Functionalized with Carbon Quantum Dots.

Developing highly sensitive, reliable, cost-effective label-free DNA biosensors is challenging with traditional fluorescence, electrochemical, and other techniques. Most conventional methods require labeling fluorescence, enzymes, or other complex modification. Herein, we fabricate carbon quantum dot (CQD)-functionalized solution-gated graphene transistors for highly sensitive label-free DNA detection. The CQDs are immobilized on the surface of the gate electrode through mercaptoacetic acid with the thiol group. A single-stranded DNA (ssDNA) probe is immobilized on CQDs by strong π-π interactions. The ssDNA probe can hybridize with the ssDNA target and form double-stranded DNA, which led to a shift of Dirac voltage and the channel current response. The limit of detection can reach 1 aM which is 2-5 orders of magnitude lower than those of other methods reported previously. The sensor also exhibits a good linear range from 1 aM to 0.1 nM and has good specificity. It can effectively distinguish one-base mismatched target DNA. The response time is about 326 s for the 1 aM target DNA molecules. This work provides good perspectives on the applications in biosensors.

Aptamer-Based Solution-Gated Graphene Transistors for Highly Sensitive and Real-Time Detection of Thrombin Molecules.

Thrombin is an important biomarker for various diseases and biochemical reactions. Rapid and real-time detection of thrombin that quickly neutralizes in early coagulation in the body has gained significant attention for its practical applications. Solution-gated graphene transistors (SGGTs) have been widely studied due to their higher sensitivity and low-cost fabrication for chemical and biological sensing applications. In this paper, the ssDNA aptamer with 29 bases was immobilized on the surface of the gate electrode to specifically recognize thrombin. The SGGT sensor achieved high sensitivity with a limit of detection (LOD) up to fM. The LOD was attributed to the amplification function of SGGTs and the suitable aptamer choice. The ssDNA configuration folding induced by thrombin molecules and the electropositivity of thrombin molecules could arouse the same electrical response of SGGTs, helping the device obtain a high sensitivity. The channel current variation of sensors had a good linear relationship with the logarithm of thrombin concentration in the range of 1 fM to 10 nM. The fabricated device also demonstrated a short response time to thrombin molecules, and the response time to the 1 fM thrombin molecules was about 150 s. In summary, the sensing strategy of aptamer-based SGGTs with high sensitivity and high selectivity has a good prospect in medical diagnosis.

Preparation, characterization and biological evaluation of ß-cyclodextrin-biotin conjugate based podophyllotoxin complex.

Podophyllotoxin is a natural occurring aryltetralin lignin with pronounced cytotoxic activity. However, its clinical application for cancer treatment has been blocked due to its poor water solubility and selectivity. In this work, biotin as a tumor specific ligand was coupled with ß-cyclodextrin and the resulting biotin modified ß-cyclodextrin was used to complex with podophyllotoxin to improve its aqueous solubility and tumor selectivity. The solubility of ß-cyclodextrin was greatly enhanced(>16 times) by conjugating with biotin. podophyllotoxin/ mono-6-biotin-amino-6-deoxy-ß-cyclodextrin inclusion complex was prepared by freeze-drying method and the complex behavior between mono-6-biotin-amino-6-deoxy-ß-cyclodextrin and podophyllotoxin was studied by water solubility, phase solubility, Job's plot, UV spectroscopy, Proton Nuclear Magnetic Resonance, Rotating-frame Overhauser Effect Spectroscopy, Powder X-ray diffraction and Scanning electron microscopy. The solubility of podophyllotoxin/ mono-6-biotin-amino-6-deoxy-ß-cyclodextrin complex was greatly improved(9 times) compared with Podophyllotoxin. The stability constant of podophyllotoxin/ mono-6-biotin-amino-6-deoxy-ß-cyclodextrin complex (Ks= 415.29 M-1) was 3.2 times that of podophyllotoxin/ß-cyclodextrin complex. The possible inclusion mode of podophyllotoxin/mono-6-biotin-amino-6-deoxy-ß-cyclodextrin complex was inferred from the Proton Nuclear Magnetic Resonance and Rotating-frame Overhauser Effect Spectroscopy. The cellular uptake study showed that the introduction of biotin increased the cellular uptake of rhodamine-B/mono-6-biotin-amino-6-deoxy-ß-cyclodextrin complex. Moreover, cell cytotoxicity study showed that the antitumor activity of podophyllotoxin/ mono-6-biotin-amino-6-deoxy-ß-cyclodextrin complex was more potent than podophyllotoxin/ß-cyclodextrin complex and free podophyllotoxin. The superior water solubility and enhanced cytotoxicity suggested that the mono-6-biotin-amino-6-deoxy-ß-cyclodextrin associated inclusion complex might be a potential and promising delivery system for hydrophobic chemotherapeutics such as podophyllotoxin.

Epidemiological and Genomic Analysis of SARS-CoV-2 in 10 Patients From a Mid-Sized City Outside of Hubei, China in the Early Phase of the COVID-19 Outbreak.

A novel coronavirus known as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of the ongoing Coronavirus Disease 2019 (COVID-19) pandemic. In this study, we performed a comprehensive epidemiological and genomic analysis of SARS-CoV-2 genomes from 10 patients in Shaoxing (Zhejiang Province), a mid-sized city outside of the epicenter Hubei province, China, during the early stage of the outbreak (late January to early February, 2020). We obtained viral genomes with >99% coverage and a mean depth of 296X demonstrating that viral genomic analysis is feasible via metagenomics sequencing directly on nasopharyngeal samples with SARS-CoV-2 Real-time PCR Ct values <28. We found that a cluster of four patients with travel history to Hubei shared the exact same virus with patients from Wuhan, Taiwan, Belgium, and Australia, highlighting how quickly this virus spread to the globe. The virus from another cluster of two family members living together without travel history but with a sick contact of a confirmed case from another city outside of Hubei accumulated significantly more mutations (9 SNPs vs. average 4 SNPs), suggesting a complex and dynamic nature of this outbreak. Our findings add to the growing knowledge of the epidemiological and genomic characteristics of SARS-CoV-2 and offers a glimpse into the early phase of this viral infection outside of Hubei, China.

Seawater-Associated Highly Pathogenic Francisella hispaniensis Infections Causing Multiple Organ Failure.

A rare case of Francisella hispaniensis infection associated with seawater exposure occurred in a deep-sea diving fisherman in Zhejiang, China. He had skin and soft tissue infection that progressed to bacteremia and multiple organ failure. Moxifloxacin treatment cleared the infections, but the patient suffered a sequela of heart damage.

Sterol Demethylation Inhibitor Fungicide Resistance in Leptosphaeria maculans is Caused by Modifications in the Regulatory Region of ERG11.

Blackleg is a worldwide disease of canola (Brassica napus), caused by a complex of fungal species in the genus Leptosphaeria, that impacts canola production and seed quality. Demethylation inhibitor (DMI) fungicides that target sterol 14α-demethylase are an integral part of disease control. Here, we report six DMI-resistant isolates of Leptosphaeria maculans and two different types of genetic modification related to the resistance. Analysis of the regulatory region of the DMI target gene ERG11 (also known as CYP51) revealed a 275-bp insertion in two of the isolates and three long terminal repeat retrotransposons (5,263, 5,267, and 5,248 bp) inserted in the promoter region of three resistant isolates. Genetic approaches confirmed that these elements are responsible for DMI resistance in L. maculans and crosses show segregation consistent with a single locus. Reverse-transcription quantitative PCR assays demonstrated that the 275-bp insertion increases ERG11 gene expression, conferring DMI fungicide resistance both in vitro and in planta. Moreover, transformation of a susceptible isolate of L. maculans with ERG11 driven by a promoter containing the 275-bp insertion increased resistance to tebuconazole. A minimal shift of the values of concentration whereby 50% of the mycelial growth is inhibited in vitro was observed in resistant isolates containing long terminal repeat retrotransposons; nevertheless, these isolates were able to develop significant lesions on cotyledons from fungicide-treated seedlings. This is the first report of genetic modifications in L. maculans relating to DMI fungicide resistance.

Real-time Utilization of Metagenomic Sequencing in the Diagnosis and Treatment Monitoring of an Invasive Adenovirus B55 Infection and Subsequent Herpes Simplex Virus Encephalitis in an Immunocompetent Young Adult.

BACKGROUND: Metagenomic sequencing has shown tremendous promise in solving difficult infectious diseases cases. In this study, we utilized this technology to help guide the care of a critically ill patient with severe pneumonia, fever of unknown origin, and subsequent encephalitis in the intensive care unit (ICU). METHODS: Shotgun metagenomic sequencing was performed on the patient's blood, bronchoalveolar lavage (BAL), and cerebral spinal fluid by using an Illumina MiniSeq sequencer. RESULTS: A high load of human adenovirus B55 (HAdV-B55), a highly pathogenic adenovirus associated with numerous recently reported outbreaks and deaths in China, was detected in both blood and BAL, which explained the severity of the condition. The patient was treated with intravenous ribavirin, which cleared the virus after 26 days. Metagenomic sequencing also helped diagnose an unexpected herpes simplex virus-1 encephalitis during hospitalization, which led to timely treatment. CONCLUSIONS: This was the first successful case utilizing metagenomic sequencing to guide diagnosis and treatment in the ICU setting in China. We have proven the concept that metagenomic sequencing can play an important role in determining clinical approaches and ultimately in improving patient outcomes. We also hope to share our successful treatment protocol for the severe pneumonia and viremia caused by HAdV-B55.

Wendan decoction for primary insomnia: Protocol for a systematic review and meta-analysis.

BACKGROUND: Insomnia is a widespread complaint in the general population. Wendan decoction has been widely applied in the treatment of primary insomnia. However, to our knowledge, there has been no systematic review or meta-analysis of randomized controlled trails regarding the effectiveness of this treatment. Here, we provide a protocol to evaluate the efficacy and safety of Wendan decoction for primary insomnia. METHODS AND ANALYSIS: Relevant randomized controlled trials in 5 English databases [EMBASE, the Cochrane Central Register of Controlled Trials (Cochrane Library), PubMed, the Allied and Complementary Medicine Databases (AMED), and the Cumulative Index to Nursing and Allied Health Literature (CINAHL)], 4 Chinese databases [Chinese Biomedical Literature Database (CBM), Chinese Medical Current Content (CMCC), China National Knowledge Infrastructure (CNKI), and Wanfang Database] will be comprehensively searched by 2 researchers in October 2017. The therapeutic effects according to the Pittsburgh Sleep Quality Index (PSQI) will be accepted as the primary outcomes. We will use RevMan V.5.3 software as well to compute the data synthesis carefully when a metaanalysis is allowed. RESULTS: This study will provide a high-quality synthesis of current evidence of Wendan decoction for primary insomnia from several aspects including PSQI, the total scores of the Insomnia Severity Index (ISI), syndrome according to standards for assessing Traditional Chinese medicine and adverse events. CONCLUSION: The conclusion of our systematic review will provide evidence to judge whether Wendan decoction is an effective intervention for patient with primary insomnia. ETHICS AND DISSEMINATION: The outcomes of this systematic review will offer implications of the use of Wendan decoction treatment for primary insomnia patients. This knowledge informing recommendations will be provided by researchers who are interested in the treatment of primary insomnia. The results of this study will be disseminated through presentation at a conference and publication of the data in a peer-reviewed journal. PROSPERO REGISTRATION NUMBER: PROSPERO CRD 42017065664.

Genotyping-by-sequencing (GBS), an ultimate marker-assisted selection (MAS) tool to accelerate plant breeding.

Marker-assisted selection (MAS) refers to the use of molecular markers to assist phenotypic selections in crop improvement. Several types of molecular markers, such as single nucleotide polymorphism (SNP), have been identified and effectively used in plant breeding. The application of next-generation sequencing (NGS) technologies has led to remarkable advances in whole genome sequencing, which provides ultra-throughput sequences to revolutionize plant genotyping and breeding. To further broaden NGS usages to large crop genomes such as maize and wheat, genotyping-by-sequencing (GBS) has been developed and applied in sequencing multiplexed samples that combine molecular marker discovery and genotyping. GBS is a novel application of NGS protocols for discovering and genotyping SNPs in crop genomes and populations. The GBS approach includes the digestion of genomic DNA with restriction enzymes followed by the ligation of barcode adapter, PCR amplification and sequencing of the amplified DNA pool on a single lane of flow cells. Bioinformatic pipelines are needed to analyze and interpret GBS datasets. As an ultimate MAS tool and a cost-effective technique, GBS has been successfully used in implementing genome-wide association study (GWAS), genomic diversity study, genetic linkage analysis, molecular marker discovery and genomic selection under a large scale of plant breeding programs.

Temperature and leaf wetness duration affect phenotypic expression of Rlm6-mediated resistance to Leptosphaeria maculans in Brassica napus.

Near-isogenic Brassica napus lines carrying/lacking resistance gene Rlm6 were used to investigate the effects of temperature and leaf wetness duration on phenotypic expression of Rlm6-mediated resistance. Leaves were inoculated with ascospores or conidia of Leptosphaeria maculans carrying the effector gene AvrLm6. Incubation period to the onset of lesion development, number of lesions and lesion diameter were assessed. Symptomless growth of L. maculans from leaf lesions to stems was investigated using a green fluorescent protein (GFP) expressing isolate carrying AvrLm6. L. maculans produced large grey lesions on Darmor (lacking Rlm6) at 5-25 degrees C and DarmorMX (carrying Rlm6) at 25 degrees C, but small dark spots and 'green islands' on DarmorMX at 5-20 degrees C. With increasing temperature/wetness duration, numbers of lesions/spots generally increased. GFP-expressing L. maculans grew from leaf lesions down leaf petioles to stems on DarmorMX at 25 degrees C but not at 15 degrees C. We conclude that temperature and leaf wetness duration affect the phenotypic expression of Rlm6-mediated resistance in leaves and subsequent L. maculans spread down petioles to produce stem cankers.