A Comprehensive Characterization of Ecological and Epidemiological Factors Driving Perennation of Podosphaera macularis Chasmothecia on Hop (Humulus lupulus).

Hop powdery mildew, caused by the ascomycete fungus Podosphaera macularis, is a consistent threat to sustainable hop production. The pathogen utilizes two reproductive strategies for overwintering and perennation: (i) asexual vegetative hyphae on dormant buds that emerge the following season as infected shoots; and (ii) sexual ascocarps (chasmothecia), which are discharged during spring rain events. We demonstrate that P. macularis chasmothecia, in the absence of any asexual P. macularis growth forms, are a viable overwintering source capable of causing early season infection two to three orders of magnitude greater than that reported for perennation via asexual growth. Two epidemiological models were defined that describe (i) temperature-driven maturation of P. macularis chasmothecia; and (ii) ascosporic discharge in response to duration of leaf wetness and prevailing temperatures. P. macularis ascospores were confirmed to be infectious at temperatures ranging from 5 to 20°C. The organism's chasmothecia were also found to adhere tightly to the host tissue on which they formed, suggesting that these structures likely overwinter wherever hop tissue senesces within a hop yard. These observations suggest that existing early season disease management practices are especially crucial to controlling hop powdery mildew in the presence of P. macularis chasmothecia. Furthermore, these insights provide a baseline for the validation of weather-driven models describing maturation and release of P. macularis ascospores, models that can eventually be incorporated into hop disease management programs.

Core RNA Interference Genes Involved in miRNA and Ta-siRNA Biogenesis in Hops and Their Expression Analysis after Challenging with Verticillium nonalfalfae.

RNA interference is an evolutionary conserved mechanism by which organisms regulate the expression of genes in a sequence-specific manner to modulate defense responses against various abiotic or biotic stresses. Hops are grown for their use in brewing and, in recent years, for the pharmaceutical industry. Hop production is threatened by many phytopathogens, of which Verticillium, the causal agent of Verticillium wilt, is a major contributor to yield losses. In the present study, we performed identification, characterization, phylogenetic, and expression analyses of three Argonaute, two Dicer-like, and two RNA-dependent RNA polymerase genes in the susceptible hop cultivar Celeia and the resistant cultivar Wye Target after infection with Verticillium nonalfalfae. Phylogeny results showed clustering of hop RNAi proteins with their orthologues from the closely related species Cannabis sativa, Morus notabilis and Ziziphus jujuba which form a common cluster with species of the Rosaceae family. Expression analysis revealed downregulation of argonaute 2 in both cultivars on the third day post-inoculation, which may result in reduced AGO2-siRNA-mediated posttranscriptional gene silencing. Both cultivars may also repress ta-siRNA biogenesis at different dpi, as we observed downregulation of argonaute 7 in the susceptible cultivar on day 1 and downregulation of RDR6 in the resistant cultivar on day 3 after inoculation.

Rheological and Microbiological Characteristics of Hops and Hot Trub Particles Formed during Beer Production.

During the production of beer, and especially beer wort, the main wastes are spent grain and hot trub, i.e., the so-called "hot break." Combined with yeast after fermentation, they represent the most valuable wastes. Hot trub is also one of the most valuable by-products. Studies on the chemical composition of these sediments and their rheological properties as waste products will contribute to their effective disposal and even further use as valuable pharmaceutical and cosmetic raw materials. So far, hot trub has been studied for morphology and particle distribution depending on the raw material composition and beer wort extract. However, there are no preliminary studies on the rheological properties of hot trub and hops. In particular, no attention has yet been paid to the dependence of these properties on the hop variety or different protein sources used. The aim of this study was to examine the effect of different hopping methods on hot trub viscosity and beer wort physicochemical parameters. Additionally, the hop solutions were measured at different temperatures. A microbiological analysis of hop sediments was also performed to determine the post-process survival of selected microorganisms in these wastes. For manufacturers of pumps used in the brewing industry, the most convenient material is that of the lowest viscosity. Low viscosity hot trub can be removed at lower velocities, which reduces costs and simplifies washing and transport. The sediments also had similar equilibrium viscosity values at high shear rates.

Isolation and identification of Pantoea agglomerans from the inflated bag with dried hop pellets stored under a modified atmosphere.

AIMS: Isolation, characterization and identification of possible microbial contaminant(s) in the inflated foil bag containing hop pellets packed and stored in a modified atmosphere. METHODS AND RESULTS: Package gas of the inflated foil bag containing hop pellets was analysed by gas chromatography. Compared with the reference modified atmosphere, containing about 16 vol.% of CO2 , the inflated bag atmosphere contained 53 vol.% CO2 , suggesting possible microbial contamination. Therefore, several standard and mineral media, with added hop pellets or hop infusion, were used for cultivation at different temperatures under an anaerobic atmosphere. Cultivation in mineral medium with hop pellets yielded a bacterial isolate that was identified by MALDI-TOF mass spectrometry and verified by partial 16S rRNA gene analysis as Pantoea agglomerans, a known plant epiphyte. CONCLUSIONS: A novel strain of P. agglomerans (designed as DBM 3696) was found to be suspicious of causing inflation of the foil bag containing dried hop pellets packed in modified atmosphere. SIGNIFICANCE AND IMPACT OF THE STUDY: This study suggests that P. agglomerans, probably hop epiphyte, could cause sporadic inflation of bags with hop pellets packed in modified atmosphere causing logistical problems during bags transport.

Downy mildew resistance is genetically mediated by prophylactic production of phenylpropanoids in hop.

Downy mildew in hop (Humulus lupulus L.) is caused by Pseudoperonospora humuli and generates significant losses in quality and yield. To identify the biochemical processes that confer natural downy mildew resistance (DMR), a metabolome- and genome-wide association study was performed. Inoculation of a high density genotyped F1 hop population (n = 192) with the obligate biotrophic oomycete P. humuli led to variation in both the levels of thousands of specialized metabolites and DMR. We observed that metabolites of almost all major phytochemical classes were induced 48 hr after inoculation. But only a small number of metabolites were found to be correlated with DMR and these were enriched with phenylpropanoids. These metabolites were also correlated with DMR when measured from the non-infected control set. A genome-wide association study revealed co-localization of the major DMR loci and the phenylpropanoid pathway markers indicating that the major contribution to resistance is mediated by these metabolites in a heritable manner. The application of three putative prophylactic phenylpropanoids led to a reduced degree of leaf infection in susceptible genotypes, confirming their protective activity either directly or as precursors of active compounds.

Different dry hopping and fermentation methods: influence on beer nutritional quality.

BACKGROUND: Nowadays, the craft beer market is booming and the consumer trend for trying something new is increasing. Here, nine different treatments of a craft beer were realized in a pilot plant, studying fermentation and dry-hopping types. Quality parameters of the beer such as polyphenols, antioxidants, bitterness, colour and alcohol were analysed. In addition, an electronic nose was used to distinguish beer types. RESULTS: Results showed that dry hopping in maturation with warm temperature increased the bitterness from 33 to 40 IBUs. The treatment using two yeasts and two fermentation temperatures resulted in the highest antioxidant capacity of the beer (around 92%). Antioxidant activity was increased by late dry hopping using ale yeasts for fermenting. Principal component analysis performed with electronic nose data explained up to 97% of the total variability of the compounds in the study. CONCLUSIONS: Combined use of ale and lager yeasts seems to increase the antioxidant capacity and total polyphenol content of beer. Antioxidant activity is increased by late dry hopping. An electronic nose is a suitable device for discriminating the volatile profile complexity in beer. © 2020 Society of Chemical Industry.

Temporal and spatial assessment of defence responses in resistant and susceptible hop cultivars during infection with Verticillium nonalfalfae.

Hop (Humulus lupulus L.) is an important industrial plant providing ingredients for brewing and pharmaceutical industry worldwide. Its intensive production is challenged by numerous diseases. One of the most lethal and difficult to control is verticillium wilt, a vascular disease caused by the fungal pathogen Verticillium nonalfalfae. The disease can be successfully controlled by the host resistance. Despite various studies that already researched resistance mechanisms of hops, only limited number of resistance genes and markers that could be utilized for efficient resistance breeding has been identified. In this study we aimed to follow fungus colonization pattern and the differential expression of selected genes during pre-symptomatic period of susceptible (Celeia) and resistant (Wye Target) hop cultivars. Results of gene expressions and fungal colonisation of compatible and incompatible interactions with V. nonalfalfae suggest that the hop plant is challenged already at the very early fungal colonisation stages. In total, nine out of 17 gene targets investigated in our study resulted in differential expression between inoculated and control plants of susceptible and resistant cultivars. The difference was the most evident in stems at an early stage of colonisation (6 dpi), showing relatively stronger changes in targeted gene expression to infection in the resistant cultivar than in the susceptible one. Analysed gene targets are involved in the overall defence response processes of nucleic acid binding, signalling, protein ubiquitination, cell oxidative burst, hydroxylation, peroxidation, alternative splicing, and metabolite biosynthesis. The up-regulation of some genes (e.g. glycine-rich RNA-binding family protein, protein phosphatase, cysteine-rich receptor-like protein kinase, zinc finger CCCH domain-containing protein 40, cinnamic acid 4-hydroxylase, class III peroxidase, putative MAPK2, peroxiredoxin-2F) upon infection in incompatible interactions might reflect defence activation, restriction of disease spreading throughout the plant and successful response of resistant genotype.

RNA interference core components identified and characterised in Verticillium nonalfalfae, a vascular wilt pathogenic plant fungi of hops.

The conserved RNA interference mechanism (RNAi) in the fungal kingdom has become a focus of intense scientific investigation. The three catalytic core components, Dicer-like (DCL), Argonaute (AGO), and RNA-dependent RNA polymerase (RdRP), and their associated small interfering RNA molecules (siRNAs) have been identified and characterised in several fungal species. Recent studies have proposed that RNAi is a major contributor to the virulence of fungal pathogens as a result of so-called trans-kingdom RNA silencing. In the present study, we report on the existence of three core RNAi proteins in the pathogenic plant fungus Verticillium nonalfalfae, which is a soilborne plant pathogen that causes severe wilting disease in hops (Humulus lupulus L.). Two DCL proteins, two AGO proteins, and two RdRP proteins were identified, and their conserved RNAi domains were characterised. Our phylogeny results confirm the existing taxonomic relationships in the Ascomycete fungal phylum and show that the fungi of the Hypocreomycetidae subclass of the Sordariomycetes class have high amino acid sequence similarity. The expression analysis revealed a potential role of RNAi in the pathogenicity of the fungi, since all the RNAi genes were highly upregulated in the highly virulent isolate T2 and were also differentially expressed in the V. nonalfalfae-susceptible Celeia and V. nonalfalfae-resistant Wye Target cultivars.

Cooperation and Coordination in Plant Disease Management.

Scaling of management efforts beyond the boundaries of individual farms may require that individuals act collectively. Such approaches have been suggested several times in plant pathology contexts but rarely have been implemented, in part because the institutional structures that enable successful collective action are poorly understood. In this research, we conducted in-depth interviews with hop producers in Oregon and Washington State to identify their motivations for and barriers to collective action regarding communication of disease levels, coordination of management practices, and sharing of best management practices and other data for powdery mildew (caused by Podosphaera macularis). Growers were generally open to and engaged in communication with neighbors and others on disease status in their hop yards and some evidence of higher levels of information sharing on management practices was found. However, growers who had developed extensive knowledge and databases were reluctant to share information viewed as proprietary. Relationships, trust, and reciprocity were facilitating factors for communication and information sharing, whereas lack of these factors and social norms of independence and pride in portions of the grower community were identified as impediments. Given the heterogeneity of trust, lack of confidence in reciprocity, and weak shared norms, communication of disease risk and coordinated management may be most successful if directed at a smaller scale as a series of neighborhood-based partnerships of growers and their immediate neighbors. Developing a disease reporting system and coordinated disease management efforts with more producers and at larger spatial extents would require formalized structures and rules that would provide assurance that there is consistency in disease data collection and reporting, reciprocation, and sanctions for those who use the information for marketing purposes against other growers. Given the analyses presented here, we believe there is potential for collective action in disease management but with limitations on the scope and nature of the actions.

Genotyping-by-Sequencing Reveals Fine-Scale Differentiation in Populations of Pseudoperonospora humuli.

Pseudoperonospora humuli is the causal agent of downy mildew of hop, one of the most important diseases of this plant and a limiting factor for production of susceptible cultivars in certain environments. The degree of genetic diversity and population differentiation within and among P. humuli populations at multiple spatial scales was quantified using genotyping-by-sequencing to test the hypothesis that populations of P. humuli have limited genetic diversity but are differentiated at the scale of individual hop yards. Hierarchical sampling was conducted to collect isolates from three hop yards in Oregon, plants within these yards, and infected shoots within heavily diseased plants. Additional isolates also were collected broadly from other geographic regions and from the two previously described clades of the sister species, P. cubensis. Genotyping of these 240 isolates produced a final quality-filtered data set of 216 isolates possessing 25,227 variants. Plots of G'ST values indicated that the majority of variants had G'ST values near 0 and were scattered randomly across contig positions. However, there was a subset of variants that were highly differentiated (G'ST > 0.3) and reproducible when genotyped independently. Within P. humuli, there was evidence of genetic differentiation at the level of hop yards and plants within yards; 19.8% of the genetic variance was associated with differences among yards and 20.3% of the variance was associated with plants within the yard. Isolates of P. humuli were well differentiated from two isolates of P. cubensis representative of the two clades of this organism. There was strong evidence of linkage disequilibrium in variant loci, consistent with nonrandom assortment of alleles expected from inbreeding and/or asexual recombination. Mantel tests found evidence that the genetic distance between isolates collected from heavily diseased plants within a hop yard was associated with the physical distance of the plants from which the isolates were collected. The sum of the data presented here indicates that populations of P. humuli are consistent with a clonal or highly inbred genetic structure with a small, yet significant differentiation of populations among yards and plants within yards. Fine-scale genetic differentiation at the yard and plant scales may point to persistence of founder genotypes associated with planting material, and chronic, systemic infection of hop plants by P. humuli. More broadly, genotyping-by-sequencing appears to have sufficient resolution to identify rare variants that differentiate subpopulations within organisms with limited genetic variability.

First Evidence of the Cysteine and Glutathione Conjugates of 3-Sulfanylpentan-1-ol in Hop ( Humulus lupulus L.).

After evidence of the cysteinylated precursors of 3-sulfanyl-4-methylpentan-1-ol (Cys-26) and 3-sulfanylhexan-1-ol (Cys-23) in hop, S-glutathione precursors (G-23 and G-26) were recently discovered in different dual-purpose hop varieties. Because free 3-sulfanylpentan-1-ol (21) has also been detected in hop, the present work aimed to identify its potential precursors. The compounds S-3-(1-hydroxylpentyl)cysteine (Cys-21) and S-3-(1-hydroxylpentyl)glutathione (G-21) were first synthesized and characterized by nuclear magnetic resonance and high-resolution mass spectrometry. High-performance liquid chromatography-positive electrospray ionization-tandem mass spectrometry evidenced both for the first time in hop. Both S conjugates were further quantitated in six hop samples: the well-known Saaz, Amarillo, Citra, Hallertau Blanc, Nelson Sauvin, and Polaris. Similar to G-23, G-21 appeared ubiquitous to all varieties. Of all of the samples investigated here, Citra (harvest 2017) emerged as the richest in G-21, with 18 mg/kg of dry matter. Cys-21 was found in all samples at a much lower concentration (up to 0.2 mg/kg of dry matter in Polaris, harvest 2017). Model media spiked with Cys-21 or G-21 allowed for the confirmation that brewing yeast is able to release free compound 21 from them.

Risk Factors for Bud Perennation of Podosphaera macularis on Hop.

The hop powdery mildew fungus Podosphaera macularis persists from season to season in the Pacific Northwestern United States through infection of crown buds because only one of the mating types needed to produce the ascigerous stage is presently found in this region. Bud infection and successful overwintering of the fungus leads to the emergence of heavily infected shoots in early spring (termed flag shoots). Historical data of flag shoot occurrence and incidence in Oregon and Washington State during 2000 to 2017 were analyzed to identify their association with the incidence of powdery mildew, growers' use of fungicides, autumn and winter temperature, and other production factors. During this period, flag shoots were found on 0.05% of plants evaluated in Oregon and 0.57% in Washington. In Oregon, the incidence of powdery mildew on leaves was most severe and the number of fungicide applications made by growers greatest in yards where flag shoots were found in spring. Similarly, the incidence of plants with powdery mildew in Washington was significantly associated with the number of flag shoots present in early spring, although the number of fungicide applications made was independent of flag shoot occurrence. The occurrence of flag shoots was associated with prior occurrence of flag shoots in a yard, the incidence of foliar powdery mildew in the previous year, grower pruning method, and, in Washington, winter temperature. A census of hop yards in the eastern extent of the Oregon production region during 2014 to 2017 found flag shoots in 27 of 489 yards evaluated. In yards without flag shoots, 338 yards (73.2%) were chemically pruning or not pruned, whereas the remaining 124 (26.8%) were mechanically pruned. Of the 27 yards with flag shoots, 22 were either chemically pruned or not pruned and 4 were mechanically pruned in mid-April, well after the initial emergence of flag shoots. The prevalence of yards with flag shoots also was related to thoroughness of pruning in spring (8.1% of yards with incomplete pruning versus 1.9% of yards with thorough pruning). A Bayesian logistic regression model was fit to the data from the intensively assessed yards in Oregon, with binary risk factors for occurrence of a flag shoot in the previous year, occurrence of foliar mildew in the previous year, and thoroughness of pruning in spring. The model indicated that the median and 95% highest posterior density interval of the probability of flag shoot occurrence was 0.0008 (0.0000 to 0.0053) when a yard had no risk factors but risk increased to 0.0065 (0.0000 to 0.0283) to 0.43 (0.175 to 0.709) when one to all three of the risk factors were present. The entirety of this research indicates that P. macularis appears to persist in a subset of chronically affected hop yards, particularly yards where spring pruning is conducted poorly. Targeted management of the disease in a subset of fields most at risk for producing flag shoots could potentially influence powdery mildew development regionwide.

Investigations on the Impact of the Special Flavor Hop Variety Huell Melon on the Odor-Active Compounds in Late Hopped and Dry Hopped Beers.

Bottom-fermented and top-fermented beers, both either late or dry hopped with Huell Melon hops, and respective reference beers without late or dry hopping were subjected to a comparative odorant screening by aroma extract dilution analyses. On the basis of differences in the FD factors, 14 odorants were identified as hop-derived. Among them were ethyl 2-methylpropanoate, methyl 2-methylbutanoate, ethyl 2-methylbutanoate, propyl 2-methylbutanoate, myrcene, linalool, and geraniol. Differences between late hopped, dry hopped, and reference beers were substantiated by quantitation. Results showed minimal transfer of myrcene from hops into beer. Moderate transfer was observed for propyl 2-methylbutanoate, geraniol, and linalool. Process-induced changes of ethyl 2-methylpropanoate, ethyl 2-methylbutanoate, and methyl 2-methylbutanoate were beyond a direct transfer from hops into beer, suggesting a formation from the corresponding hop-derived carboxylic acids by yeast. Spiking experiments revealed that linalool and propyl 2-methylbutanoate contributed particularly to the characteristic aroma of beers flavored with Huell Melon hops.

Evidence of Dextrin Hydrolyzing Enzymes in Cascade Hops ( Humulus lupulus).

Dry-hopping, the addition of hops to beer during or after fermentation, is a common practice in brewing to impart hoppy flavor to beer. Previously assumed to be inert ingredients, recent evidence suggests that hops contain biologically active compounds that may also extract into beer and complicate the brewing process by altering the final composition of beer. Experiments described herein provide evidence of microbial and/or plant-derived enzymes associated with hops ( Humulus lupulus) which can impact beer quality by influencing the composition of fermentable and nonfermentable carbohydrates in dry-hopped beer. Fully attenuated and packaged commercial lager beer was dry-hopped at a rate of 10 g hops/L beer with pelletized Cascade hops, dosed with 106 cells/mL of ale yeast, and incubated at 20 °C. Real extract of the treated beer declined significantly within several days with a reduction of 1 °P (% w/w) after 5 days and then slowly to a total reduction of approximately 2 °P after 40 days. When fully fermented, this was equivalent to the production of an additional 4.75% (v/v) of CO2 and an additional 1.3% (v/v) of alcohol. The refermentation of beer driven by dry-hopping was attributed to the low but persistent activities of several starch degrading enzymes present in Cascade hops including amyloglucosidase, α-amylase, ß-amylase, and limit dextrinase. The effect of hop-derived enzymes on beer was time, temperature, and dose-dependent. Characterizing bioactive enzymes in hops will help hop suppliers and brewers to address the unexpected quality and safety issues surrounding hopping practices in beer.

Comprehensive analysis of Verticillium nonalfalfae in silico secretome uncovers putative effector proteins expressed during hop invasion.

The vascular plant pathogen Verticillium nonalfalfae causes Verticillium wilt in several important crops. VnaSSP4.2 was recently discovered as a V. nonalfalfae virulence effector protein in the xylem sap of infected hop. Here, we expanded our search for candidate secreted effector proteins (CSEPs) in the V. nonalfalfae predicted secretome using a bioinformatic pipeline built on V. nonalfalfae genome data, RNA-Seq and proteomic studies of the interaction with hop. The secretome, rich in carbohydrate active enzymes, proteases, redox proteins and proteins involved in secondary metabolism, cellular processing and signaling, includes 263 CSEPs. Several homologs of known fungal effectors (LysM, NLPs, Hce2, Cerato-platanins, Cyanovirin-N lectins, hydrophobins and CFEM domain containing proteins) and avirulence determinants in the PHI database (Avr-Pita1 and MgSM1) were found. The majority of CSEPs were non-annotated and were narrowed down to 44 top priority candidates based on their likelihood of being effectors. These were examined by spatio-temporal gene expression profiling of infected hop. Among the highest in planta expressed CSEPs, five deletion mutants were tested in pathogenicity assays. A deletion mutant of VnaUn.279, a lethal pathotype specific gene with sequence similarity to SAM-dependent methyltransferase (LaeA), had lower infectivity and showed highly reduced virulence, but no changes in morphology, fungal growth or conidiation were observed. Several putative secreted effector proteins that probably contribute to V. nonalfalfae colonization of hop were identified in this study. Among them, LaeA gene homolog was found to act as a potential novel virulence effector of V. nonalfalfae. The combined results will serve for future characterization of V. nonalfalfae effectors, which will advance our understanding of Verticillium wilt disease.

Hop stunt viroid: Effect on Host (Humulus lupulus) Transcriptome and Its Interactions With Hop Powdery Mildew (Podospheara macularis).

Viroids are the smallest known plant pathogens that exploit host systems for their replication and cause diseases in many hosts. In this study, the host response of hop plants to Hop stunt viroid (HSVd) infection was studied through transcriptome analysis. RNA sequence analysis of hop leaves infected with HSVd revealed dynamic changes in hop gene expression. Defense-related genes and genes involved in lipid and terpenoid metabolism are the major categories that showed differential expression due to HSVd infection. Additionally, the effect of HSVd on development of hop powdery mildew (Podospheara macularis) (HPM) was studied. Transcriptome analysis followed by quantitative reverse transcription-polymerase chain reaction analysis showed that transcript levels of pathogenesis-related (PR) genes such as PR protein 1, chitinase, and thaumatin-like protein genes are induced in leaves infected with HPM alone. The response in these genes to HPM is significantly down-regulated in leaves with HSVd-HPM mixed infection. These results confirm that HSVd alters host metabolism, physiology, and plant defense responses. Nevertheless, in detached leaf assays, HPM consistently expanded faster on HSVd-negative leaves relative to HSVd-positive leaves. Although HSVd infection suppresses elements associated with the host immunity response, infection by HSVd is antagonistic to HPM infection of hops.

Progress in Brewing Science and Beer Production.

The brewing of beer is an ancient biotechnology, the unit processes of which have not changed in hundreds of years. Equally, scientific study within the brewing industry not only has ensured that modern beer making is highly controlled, leading to highly consistent, high-quality, healthful beverages, but also has informed many other fermentation-based industries.

Identification of Novel Virulence-Associated Proteins Secreted to Xylem by Verticillium nonalfalfae During Colonization of Hop Plants.

Plant pathogens employ various secreted proteins to suppress host immunity for their successful host colonization. Identification and characterization of pathogen-secreted proteins can contribute to an understanding of the pathogenicity mechanism and help in disease control. We used proteomics to search for proteins secreted to xylem by the vascular pathogen Verticillium nonalfalfae during colonization of hop plants. Three highly abundant fungal proteins were identified: two enzymes, α-N-arabinofuranosidase (VnaAbf4.216) and peroxidase (VnaPRX1.1277), and one small secreted hypothetical protein (VnaSSP4.2). These are the first secreted proteins so far identified in xylem sap following infection with Verticillium spp. VnaPRX1.1277, classified as a heme-containing peroxidase from Class II, similar to other Verticillium spp. lignin-degrading peroxidases, and VnaSSP4.2, a 14-kDa cysteine-containing protein with unknown function and with a close homolog in related V. alfalfae strains, were further examined. The in planta expression of VnaPRX1.1277 and VnaSSP4.2 genes increased with the progression of colonization, implicating their role in fungal virulence. Indeed, V. nonalfalfae deletion mutants of both genes exhibited attenuated virulence on hop plants, which returned to the level of the wild-type pathogenicity in the knockout complementation lines, supporting VnaPRX1.1277 and VnaSSP4.2 as virulence factors required to promote V. nonalfalfae colonization of hop plants.

Detection of acid and hop shock induced responses in beer spoiling Lactobacillus brevis by MALDI-TOF MS.

Due to the harsh environment, microorganisms encounter in beer, spoilage bacteria must be able to customise their metabolism and physiology in an order to master various kinds of perturbations. Proteomic approaches have been used to examine differences between various beer spoilage bacteria and between different stress conditions, such as acid and hop (Humulus lupulus) stress. However, these investigations cannot detect changes in low molecular weight (lmw) proteins (<150 amino acids). Therefore, for the first time, we herein present data from a proteomic study of lmw proteins for two Lactobacillus (L.) brevis strains exposed to acid stress or, respectively, two different qualities of hop induced stress. We used MALDI-TOF MS as analytical tool for the detection of lmw stress response proteins due to its high sensitivity and low throughput times. Comparing a hop-sensitive and a hop-tolerant strain, detection of the fatty acid biosynthesis-associated acyl carrier protein varied between different stress conditions and incubation times. The findings coincide with previous studies of our group regarding the fatty acid cell membrane composition of beer spoiling L. brevis. It is demonstrated that MALDI-TOF MS is a fast tool to detect and characterise stress situations in beer spoiling bacteria along the lmw sub-proteome.

Molecular mechanisms behind the antimicrobial activity of hop iso-α-acids in Lactobacillus brevis.

The main bittering component in beer, hop iso-α-acids, have been characterised as weak acids, which act as ionophores impairing microbial cells' function under acidic conditions as present in beer. Besides medium pH, divalent cations play a central role regarding the efficacy of the antimicrobial effect. The iso-α-acids' non-bitter derivatives humulinic acids can be found in isomerised hop extracts and can be generated during hop storage. Therefore, they have been under investigation concerning their influence on beer sensory properties. This study sketches the molecular mechanism behind iso-α-acids' antimicrobial activity in Lactobacillus (L.) brevis regarding their ionophore activity versus the dependence of the inhibitory potential on manganese binding, and suggests humulinic acids as novel tasteless food preservatives. We designed and synthesised chemically modified iso-α-acids to enhance the basic understanding of the molecular mechanism of antimicrobial iso-α-acids. It could be observed that a manganese-binding dependent transmembrane redox reaction (oxidative stress) plays a crucial role in inhibition. Privation of an acidic hydroxyl group neither erased ionophore activity, nor did it entirely abolish antimicrobial activity. Humulinic acids proved to be highly inhibitory, even outperforming iso-α-acids.