The effect of dietary fiber has long been proposed to contribute to human health through prebiotic enhancement of certain beneficial microbes that produce butyrate,absorb bile acids,decrease colon pH,112 and promote GI motility via shortening of the mean transit time.However, not all dietary fibers have the same effect, which is dependent on their physicochemical characteristics.The prebiotic effect of indigestible polysaccharides on gut microbiota has previously been broadly discussed. Several human dietary intervention studies have shown that intake of certain types of dietary fibers can significantly modify the gut microbiota observed in feces. For example, consumption of whole-grain breakfast cereal for 3 weeks significantly increased fecal bifidobacteria and lactobacilli compared to wheat bran alone; however, no difference was observed in fecal short-chain fatty acids .Introduction of barley β-glycan in the diet elevated fecal Bifidobacterium and Bacteroides counts in older healthy human subjects , whereas only the Clostridium perfringens count increased in the younger group.Typically, consumption of nondigestible carbohydrates such as wheat bran arabinoxylan oligosaccharides, short-chain fructooligosaccharides , and soybean oligosaccharides and galactooligosaccharides induces enrichment of human fecal bifidobacteria.Inulin has been shown to stimulate the growth of Bifidobacterium adolescentis, Bifidobacterium longum, Bifidobacterium bifidum and the butyrateproducing bacteria Faecalibacterium prausnitzii and Roseburia inulinivorans. Similarly, Bifidobacterium spp. levels significantly increased upon consumption of biscuits containing partially hydrolyzed guar gum and FOS for 21 days, whereas Bacteroides spp., Clostridium spp., and Lactobacillus−Enterococcus spp. remained at similar levels.

GOS alone or combined with FOS are often supplemented in infant formula to favor the growth of bifidobacteria spp.,drainage planter pot and the bifidogenic response of GOS has been shown to be dosedependent.Interestingly, Rossi et al. reported that only 8 of the total 55 Bifidobacterium strains were able to grow on longchain inulin in vivo,suggesting that not all bifidobacteria species benefit in the same way from the presence of these substrates as their energy source. Indeed, the specificity of polysaccharide use by the gut microbiota supports the underlying cross-feeding interaction between gut microbiota . Various types of resistant starch demonstrate substrate specialization of the gut microbiome. For example, the impact of type 2 resistant starch is associated with enrichment of Ruminococcus bromii, whereas type 3 resistant starch elevates both E. rectale and R. bromii in healthy130 and overweight subjects.Type 4 resistant starch significantly differs from types 2 and 3 and has been shown to induce a profound phylum-level change and elevate B. adolescentis and Parabacteroides distasonis. Furthermore, the variability observed in these studies suggests that the host-specific environment affects the composition of the gut microbiome. The fermentation profile depends on the glycosidic linkage type of the dietary substrate as well as the functional capability of the gut microbiota . Metatranscriptome analysis revealed a functional enrichment of genes associated with carbohydrate uptake and metabolism in the small intestine and feces.A fecal metaproteomic analysis from three healthy subjects over a period of 6−12 months revealed a common functional core enriched in carbohydrate transport and degradation.In particular, the ability to degrade complex polysaccharides has been identified in a range of bacteria.In particular, the Bacteroides phylum contains a large repertoire of genes that exhibit broad capacities to degrade a great diversity of plant-derived polysaccharides.Microbial sequencing projects revealed that starch utilization systems are highly abundant and conserved in the phylum Bacteroidetes .

In contrast, members from the Firmicutes phylum exhibit a wide range of functionalities; for example, Ruminococcus, which is in the order of Clostridiales, can degrade cellulose and pectin,whereas E. rectale and Eubacterium eligens have fewer polysaccharide degrading enzymes and are enriched with more ATP binding cassette transporters and phosphotransferase systems than the Bacteroidetes.Although many microbes have the ability to ferment undigestible dietary components, diet-induced microbial changes seem to favor the groups that have a stronger survival advantage and perhaps specifically depend on host-derived factors such as pH and bile acid profiles.The human colon has not often been considered to be a site of fat absorption; however, the small intestine absorbs only approximately 95% of dietary lipids after consumption of a typical Western diet.Furthermore, some studies have suggested that colonic absorption of medium-chain fatty acids takes place in dogs,rats,and humans and that glycerol accumulates in the colon when fat absorption is disturbed in the small intestine.Accumulation of glycerol has been shown to alter the Lactobacillus and Enterococcus communities in the gut.A diet high in animal fat and low in dietary fiber stimulates the synthesis and enterohepatic circulation of primary bile acids.Although the majority of bile acids are recycled in the ileum, some of them escape the enterohepatic circulation in the intestine and become substrates for microbial metabolism in the colon.Bile acids restrict the growth of several microbes.Accordingly, only the microbes that are able to tolerate the physiologic concentrations of bile acids survive in the gut; thus, bile acids appear to exert strong selective pressure on gut microbial structure and function. For example, administration of cholic acid to mice induces phylum-level population shifts of the relative abundance of Firmicutes and Bacteroidetes that resembles microbial changes observed by feeding a high-fat diet alone.Gut microbiota detoxify primary bile acids via deconjugation, in which well-conserved bile salt hydrolases release the amino acids glycine and taurine.

The free primary bile acids are then converted into various types of secondary bile acids such as deoxycholic acid and lithocholic acid by the 7α- dehydroxylation reaction.A detailed list of bacteria with genes encoding bile salt hydrolase activity is reviewed by Ridlon et al.In general, the conjugated bile acid profile is heavily dependent on microbial activity. The bile acid distribution profile in multiple compartments of germ-free animals shows less diversity and is smaller in size compared with conventional counterparts.Dietary lipid composition can also modulate bile acid profile, in particular, increasing taurocholic acid that, as a consequence, promotes the growth of Bilophila wadsworthia, a Gram-negative opportunistic pathogen.B. wadsworthia utilizes taurine as a source of sulfite,which serves as the terminal electron acceptor for the respiratory chain.The concentrations of bile acids and their conjugation status to glycine or taurine between individuals may be influenced by diet, as people eating a meat-rich diet tend to have more taurine-conjugated bile acids in their bile acid pool than those eating a vegetarian diet.It has been recognized that the production of secondary bile acids is pH dependent. The proximal colon is more acidic than the distal colon,which results in an elevated activity of 7α-dehydroxylase in the cecum versus the left colon.Subjects consuming diets high in resistant starch showed a significantly decreased stool pH compared with subjects consuming a low resistant starch diet. A decrease in pH is associated with an elevated production of SCFAs, which selectively regulate the intestinal microbial community, with a tendency to suppress Bacteroides spp.and promote butyrate-producing Gram-positive bacteria such as E. rectale. Similarly, subjects on a vegan or vegetarian diet showed significantly more acidic stool pH89 and significantly lower fecal secondary bile acid production83 than omnivores. Higher consumption of animal protein is one possible explanation of higher fecal pH value in an omnivorous diet, as proteolytic putrefactive bacteria are able to increase stool pH by producing alkaline metabolites. Thus, increases in SCFAs result in a more acidic colonic pH, a decreased solubility of bile acids, an indirect increased absorption of minerals, and a reduction of ammonia absorption, which indirectly alters the composition of gut microbiota.Importantly, 50−70% of acetate taken up by the liver becomes the primary substrate for cholesterol synthesis, whereas propionate inhibits cholesterol synthesis in hepatic tissue . Therole of cholesterol on gut microbiota was first elucidated using germ-free rats. Danielsson et al. demonstrated that germ-free rats exhibit a higher serum cholesterol level than their conventional counterparts.More recently, the characterization of the gut microbiota in a hamster model of hypercholesterolemia showed that dietary intervention with grain sorghum lipid extract180 modulated the gut microbial composition, with bifidobacteria being positively associated with increases in HDL cholesterol level and the family Coriobacteriaceae being associated with non-HDL cholesterol.Together, high intake of dietary fat, in particular animal fat, and cholesterol not only changes the composition of bile acids and neutral sterols in the colon but also modifies the gut microbiota,plant pot with drainage which consequently transforms these compounds into secondary bile acids and cholesterol metabolites.Polyphenols present in a wide range of plant-based foods have received great interest owing to their antioxidant capacity and potential protective effect in reducing cardiovascular disease risk through improvement in vascular function and modulation of inflammation. The interpretation of the influence of polyphenols on cardiovascular health in dietary intervention studies can be complicated due to dynamic bio-availability during the processes of absorption, metabolism, distribution, and excretion. Generally, the absorption of dietary polyphenols is widely dependent on the type and structure of the compound and is often slow and largely incomplete in the small intestine. Therefore, significant quantities of polyphenols are retained in the colon. In addition, the nonabsorbed polyphenols are subjected to biotransformation via the activity of enzymes from various microbial groups . Consequently, the microbiota-derived metabolites of polyphenols are better absorbed in the gut,which then become an important factor in the health effect of polyphenol-containing foods. Important plant polyphenols and their microbial derivatives are listed in ref.

Many of the studies that assess bio-availability and effects of polyphenols have evaluated the balance between the enterohepatic circulating levels, residence time in plasma, and urine excretion rate of the parent phenolic compounds and their microbial-derived metabolites using metabolomic techniques.Importantly, although endogenous enzyme and transporter activities in the small intestine as well as transformation of polyphenols are subject to a wide inter individual variability, the functional capability of the gut microbiota is important to partially explain the variation of bio-availability among the population.Assessing the properties of a single dietary constituent from the polyphenol family alone without dietary fiber is difficult due to the complex dietary food matrix present in a flavonoid-rich diet. For example, regular consumption of apples increased the numbers of fecal bifidobacteria and decreased the C. perf ringens count.Similarly, the concomitant dietary presence of apple polyphenols and FOS increased SCFA production.In contrast, compared to consumption of an inulin-containing diet alone, including a grapefruit flavonoidrich extract decreased both production of SCFAs and the bifidobacteria population.Furthermore, a randomized crossover intervention study in which subjects consumed high levels of cruciferous vegetables for 14 days revealed an alteration of the fecal microbial community profile compared with a lowphytochemical, low-fiber diet, including a higher abundance of Eubacterium hallii, Phascolarctobacterium faecium, Burkholderiales spp., Alistipes putredinis and Eggerthella spp.The observed changes could also be explained by the increase in dietary fiber that is enriched in cruciferous vegetables.Overall, the direct effects of fiber blur the ability to judge the specific effects of individual dietary ingredients on gut microbiota. These dietary ingredients may act in an additive or a synergistic manner, exerting their effects on gut microbiota. Six week consumption of a wild blueberry drink that was high in polyphenols was shown to increase the proportion of Bifidobacterium spp. compared to the placebo group;however, a high interindividual variability in response to the dietary treatment was also observed.Similarly, the daily consumption of a high-cocoa flavonol drink for 4 weeks significantly enhanced the growth of fecal bifidobacterial and lactobacilli populations, but decreased the Clostridia histolyticum counts relative to those consuming a low-cocoa flavonol drink .Furthermore, unabsorbed dietary phenolics and their metabolites selectively inhibit pathogen growth and stimulate the growth of commensal bacteria. For example, grape pomace phenolic extract increases Lactobacillus acidophilus CECT 903 growth in liquid culture media.In addition, upon bacterial incubation, tea phenolics were shown to suppress the growth of potential pathogens such as Clostridium spp. and Gram-negative Bacteroides spp., whereas commensal anaerobes such as Bifidobacterium spp. and Lactobacillus spp. were less affected.Similarly, the flavanol monomer -catechin significantly increases the growth of the Clostridium coccoides−Eubacterium rectale group, Bifidobacterium spp., and E. coli and significantly inhibits the growth of C. histolyticum group in vitro.To date, there is a wide range of phenolic compounds that have been demonstrated to have antimicrobial properties,and many have been previously reviewed.Although many of the studies highlighting the beneficial role of plant polyphenols through regulation by gut microbiota appear promising, there are limitations in the results that can be drawn regarding the ability of flavonoids to influence the growth of selected intestinal bacterial groups using a batch-culture model.

To make foods safe, cyanogenic glycosides are frequently hydrolyzed through processes including grinding, pounding, boiling, steaming or during soaking or fermentation in water.Thermal treatments have also been used to effectively decrease levels of cyanogenic glycosides in foods.Commercial elderberry juice is thermally processed to inactivate enzymes, improve microbiological stability, and to degrade the potentially toxic cyanogenic glycosides found in this berry which include amygdalin, dhurrin, linamarin, and sambunigrin . The range and content of cyanogenic glycosides in the blue elderberry have not yet been identified. However, there was an occurrence of people getting sick after consuming raw blue elderberry juice at a at a religious gathering in Monterey County, California in 1983.Therefore understanding levels of cyanogenic glycosides in this subspecies is critical if it is going to be considered for use in commercial products. In European elderberry, sambunigrin is the main CNG in all parts of the plant, with the highest levels in the leaves and the lowest levels in the ripe berries .6 Sambunigrin is a diastereoisomer of prunasin, containing L-glucose instead of D-glucose.In the American subspecies, amygdalin, dhurrin, linamarin, and sambunigrin were identified in the skin, seed, and juice of the berry as well as in the stem.Juice of the American elderberry contained the lowest levels of CNGs relative to the skin, seeds and stems, averaging about 4 µg g- 1 , while the stems had the highest concentrations in the two cultivars evaluated.A recent study demonstrated that the total phenolic content of the blue elderberry is similar to the European and American subspecies,danish trolley however this subspecies has significantly lower levels of anthocyanins.

The dominant phenolic compounds identified in the blue elderberry include the flavonols rutin and isorhamnetin glucoside, the phenolic acid chlorogenic acid, a novel phenolic compound tentatively identified as 5-hydroxypyrogallol hexoside , and the anthocyanins cyanidin 3-glucoside and cyanidin 3-sambubioside .At the same time, anthocyanidins are desired by consumers for their potential health promoting bioactivity.Unfortunately, heat processing and pasteurization can lead to the degradation of anthocyanins resulting in a loss in color or a formation of brown polymers, possibly impacting the acceptability of the final product. Elderberry juice and extracts have been evaluated for their thermal stability, which have shown that anthocyanins degrade following first-order reaction kinetics.The stability of anthocyanidins can be reinforced via intra- and inter-molecular interactions with protective structures and flavonoids through a phenomenon termed copigmentation.Notably, acylated anthocyanins, such as those found in American elderberry like cyanidin 3-coumaroylsambubioside, are sometimes more stable during thermal processing due to protective properties of the coumaroyl group folding over the flavylium ion.Like the European elderberry, the blue elderberry does not contain acylated anthocyanins.The thermal stability of anthocyanidins in blue elderberry juice has not yet been evaluated. However, as elderberry juice and extracts are frequently thermally processed to make products, such as jam, syrup, or gummies, it is important to understand the thermal degradation of anthocyanidins in the juice from blue elderberry.

The purpose of this study was to determine the stability of the cyanogenic glycosides and main phenolic compounds in blue elderberry juice cooked at 72 °C and 95°C for two hoursto elucidate the kinetics of degradation of these important compounds. HPLC-grade methanol , and LCMS grade acetonitrile, methanol, and formic acid were purchased from Fisher Scientific . Ultrapure water was obtained from a Milli-Q water system . Prunasin was also obtained from Millipore Sigma. HPLC-grade acetonitrile , rutin , isorhamnetin 3-O-glucoside, caffeic acid, chlorogenic acid, -catechin, protocatechuic acid, ammoniumformate, and amygdalin were purchased from Sigma-Aldrich . Cyanidin 3-Osambubioside chloride and cyanidin 3-O-glucoside were purchased from ExtraSynthese Ripe berries were harvested in July 2019 from a farm in Winters, CA at latitude and longitude coordinates of 38.634884, -122.007502. Fruit was selected from all sides of the plant at a variety of heights to obtain a representative sample of berries from each plant. Only fully ripe berries . About 5 kg were harvested from each of shrub in three hedgerows. The plant material was transported to University of California, Davis on ice in plastic gallon bags within two hours of harvest and stored at -20 °C until analysis. A hot water bath was prepared using an immersion circulator , set to the desired cooking temperature . The temperature was also monitored with a thermometer in the water bath. Vials of juice randomized and place into the hot water bath and the bath was covered during cooking. Duplicate vials were removed at the following times: 15, 30, 45, 60, 75, 90, 105, and 120 min. An extended processing time was used to observe degradation of more heat-stable compounds as well as to make comparisons to other studies that processed juice for multiple hours at these temperatures. Once removed from the hot water bath, vials were placed immediately into an ice bath for 15 min.

Then from each vial, 1 mL of juice was placed in a microcentrifuge tube and centrifuged at 4 °C, 15,000 rpm for 15 min . Next, the supernatant was diluted 1:10 with 1% formic acid in water, filtered with 0.2 µm PTFE filter, and placed in an HPLC vial for analysis. Five replicate juice samples were prepared and cooked at both temperatures. Composite juice samples were prepared by combing equal aliquots of the time points 0, 15, 30, 60, and 120 minutes of thermal processing for both temperatures for each juice prepared. To extract CNGs for blue elderberry juice, 0.500 mL of juice was mixed with 2.00 mL of methanol in a 5 mL centrifuge tube, then sonicated at 30 °C for 30 min. After sonication, 1.00 mL of extract was transferred to a 1 mL microcentrifuge tube and centrifuged at 15,000 rpm at 4 °C for 15 min. The supernatant was collected and filtered through 0.22 µm PTFE into an HPLC vial and used for analysis. Five replicate extractions were made of raw elderberry juice and triplicate extractions were made for the other time/temperature juices. CNGs were analyzed via ultra-high performance liquid chromatography with electrospray ionization and triple quadrupole tandem mass spectrometry using an Agilent 1290 Infinity HPLC and 6460 mass spectrometer . The UHPLC was equipped with a binary pump with an integrated vacuum degasser , an autosampler with thermostat , and a thermostated column compartment . CNGs were separated using a Kinetex F5 column at 40.0 °C. The mobile phase consisted of a linear gradient of 1 mM ammonium formate in water and 1 mM ammonium formate in 650:50 MeOH:ACNe as follows: 5% B, 0–5 min; 100%B 5-5.50 min, 95% A 5.60-6.50 min. The flow rate was 0.400 mL/min, and the injection volume was 5.0 μL. The CNGs were analyzed using negative ESI mode. The drying gas temperature was 300 °C and the flow rate was 8.0 L min-1 . The sheath gas temperature and flow rate were 350 °C and 11.0 L min-1 , respectively. The nebulizer gas pressure and capillary voltage were 45 psi and 3.5 kV, respectively. The fragmentor voltage was 160 V for amygdalin and 100 V for sambunigrin. The dwell time was 100 ms for amygdalin and 200 ms for sambunigrin. The collision energy was set to 12 V for amygdalin, 0 V for sambunigrin. The multiple reaction monitoring mode was utilized to analyze amygdalin and sambunigrin. Quantification of CNGs was performed using external calibration curves using standard addition at levels of 500, 100, 50, and 5 ng L-1 . For amygdalin, vertical aeroponic tower garden the area of m/z 456.2 to m/z 323.1 was measured. For sambunigrin, the area of m/z 340.3 to m/z 294.2 was measured. Half-life values of phenolic compounds were calculated by plotting the natural log of C0/C ratio vs heating time t, where C0 is the initial concentration of a compound, C is the concentration of the same compound at time t in hours. The slope calculated from the figure is k . Longitudinal Analysis of variance was performed with Tukey’s post-hoc test with p value at 0.05. Excel used for average and standard deviation and R Studio was used for ANOVA and post hoc analysis . CNG data was analyzed using MassHunter Quantitative Analysis to obtain peak areas for the targeted compounds. Microsoft Excel was used to create the calibration curves and determine CNG concentrations in samples . The concentrations of cyanogenic glycosides were quantified in raw and cooked blue elderberry juice for the first time. Results indicate that neoamygdalin , sambunigrin and prunasin are the primary CNGs in blue elderberry . Concentration of neoamygdalin were significantly higher than sambunigrin and prunasin . Neoamygdalin has been measured in raw bitters almonds in concentrations lower than amygdalin.133 In studies of American and European elderberry, sambunigrin is typically major CNG identified. Levels of total CNGs in blue elderberry are lower than American and European elderberry. European elderberry CNG levels range from 0.08 ± 0.01 to 0.77 ± 0.08 µg g-1 depending on the elevation and growing location.6 CNG levels in American elderberry juice range from 0.29 to 2.36 µg mL-1 .

Differences between the subspecies may be due to genetic variation, impact of growing environment such as altitude, or methodology used to extract and analyze CNG content in the fruit and fruit juice, including how berries were handled prior to juice and juicing method. The degradation of neoamygdalin > sambunigrin > prunasin was observed during cooking and the rate of degradation was faster at 95 °C as compared to 72 °C . However, degradation in juice at 72 °C was not linear, such that sambunigrin levels in juice cooked at significantly increased during the final timepoint measured . This may be attributed to neoamygdalin breaking down resulting in sambunigrin and a glucose molecule, an equivalent pathway to amygdalin degrading to prunasin and a glucose molecule. However, some of the resulting sambunigrin from that reaction would also have to be degrading since the decrease in concentration of neoamygdalin did not cause an equivalent increase in sambunigrin. An increase in sambunigrin at the end of the processing time was not observed in the juice cooked at 95 °C. In the juice processed at 95 °C, the combined concentration of neoamygdalin and sambunigrin decreased at each measured time point and did not increase at any time points like the juice processed at 72 °C. Prunasin levels did not significantly change in the elderberry juice cooked at 72 °C but prunasin did degrade significantly when processed at 95 °C . It appears that sambunigrin is more stable than prunasin in the elderberry juice; retaining about 70% of the original concentration in the juice heated to 95 °C. Neoamygdalin levels decreased significantly in the elderberry juice at both processing temperatures, with increased degradation at 95 °C as compared with the treatments at 72 °C. As previously mentioned, sambunigrin is the expected breakdown product from neoamygladin, but sambunigrin levels did not have concomitant increase due to thermal degradation of sambunigrin as well. Thermal processing has been seen to degrade CNGs in elderberry, flaxseed, and almond in previous studies.Furthermore, studies have seen enzymatic activity contributing to the breakdown of CNGs in nuts to reduce after exposure to heat.If the β-glucosidases for the CNGs present in blue elderberry are similar, they would also be inactivated during thermal processing at 72 and 95 °C, indicating thermal degradation is the main contribution to CNG levels decreasing in the present study. Because enzymatic degradation of CNGs was not measured during the thawing and juicing steps, the impact of the enzymes before the thermal processing cannot be evaluated here. The presence of neoamygdalin instead of amygdalin is unexpected. Amygdalin can convert to neoamygdalin with heat and in alkaline conditions. However, herein the raw elderberry juice had significantly higher levels of neoamygdalin as compared to amygdalin. In a study of amygdalin content in almond varieties, amygdalin was found to convert to neoamygdalin during extraction , but the addition of acetic acid prevented the conversion.Blue elderberry naturally contain citric and malic acids, with an average titratable acidity of 0.60 ± 0.10 to 0.65 ± 0.07 g citric acid per 100 g FW.The average pH value of the juices in the present study was 3.76 ± 0.11. Therefore, there may not be enough acid in the matrix to prevent the conversion. In contrast, another study of amygdalin and derivatives in almonds found that heat of cooking caused neoamygdalin and amygdalin amide to convert to amygdalin, which was not observed in the present study.Further analysis of conversion of amygdalin to neoamygdalin in the blue elderberry could uncover why this epimer is dominant.

Electrophoresis was run at 80 V and then the gel was visualized with a UVP High Performance Ultraviolet Transilluminator . Genomic DNA was also amplified with primer pairs pSA3_Cm_F/pSA3_CmR for the chloramphenicol resistance gene of plasmid pSA3 and gfp_qPCR_F/gfp_qPCR_R for the egfp gene of plasmid pIGSAF. Primers nifH_qPCR_F/nifH_qPCR_R for the nifH nitrogenase gene from the F. alni genome were used as a positive control for the quality of DNA extracts. Additionally, purified plasmid pDiGc from E. coli DH5α was used as a DNA template for egfp amplification as a positive control. PCR products were separated on an agarose gel and extracted as in Plasmid Synthesis, above, and sequenced by the UCDNA Sequencing Facility . Forward and reverse sequences were combined to make fulllength amplicon sequences. These were aligned with MUSCLE with published reference sequences for the egfp and camR genes. Additionally, the sequences were compared by BLAST against gene sequences obtained from the original plasmids . Confocal microscopy was used to visualize expression of egfp in F. alni hyphae and vesicles. egfp was expressed both constitutively, from plasmid pIGSAF, and differentially between N and N media under control of the F. alni nif promoter using plasmid pIGSAFnif. Cultures were imaged after on1 week of incubation from the previous transfer, 25 weeks after transformation, as described in Plasmid Maintenance on Selective Media, below. For imaging, F. alni cultures were grown either in N or N BAPP medium as in Culture Conditions, above,blueberry grow pot and immobilized on glass slides with a drop of 3% molten agarose solution maintained in a water bath at 50◦C. Slides were pre-heated on a slide warmer at 50◦C. Fifteen microliter of Frankia hyphal suspension was then pipetted onto each slide and covered with 35 µl of 3% molten agarose.

A #1.5 coverslip was added to the Frankia cells in agarose, and then the slide was allowed to cool to room temperature. The Frankia preparations were visualized on a Leica TCS SP8 STED 3X confocal microscope with either a 20X objective, or a 100X oil-immersion objective, using either bright field or fluorescence with a HyD detector, in the Advanced Imaging Facility, University of California, Davis. For fluorescence imaging, samples were excited with 488 nm light. The emission wavelengths were collected from 500 to 550 nm. Images were stored as .lif files from Leica LAS X and then viewed in FIJI . For imaging of individual hyphae and vesicles, the 100X objective lens was used and images were taken in Z-stacks with a step size of 0.1 µm. To visualize F. alni colonies at low magnification, Z-stack images were taken with the 20X objective in five steps of 2 µm each and then combined using the highest fluorescent intensity of each pixel . Three separate transformation experiments were carried out, from August 2017 through March 2019, as outlined in Table 2. Transformed cultures were maintained with weekly subculturing into fresh selective media and used in experiments over the course of several months following each transformation. Initial transformations of F. alni with plasmid pIGSAF were performed in August of 2017; the cultures were maintained by subculturing and were visualized by confocal microscopy from December 2017 through March 2018 . Transformation of F. alni with plasmid pIGSAFnif was performed in January 2018, selected as above, and imaged by confocal microscopy in April 2018 . These cultures were maintained by subculturing in selective media for RNA extraction for qPCR through July 2018 . These cultures were transformed in September 2018 and subcultured weekly in selective media through March 2019. The presence of the plasmid was confirmed by DNA extraction and PCR amplification of the egfp gene in March 2019 as described in Frankia Transformation, above.

To test the persistence of plasmid pIGSAF in transformed F. alni without selection, a time-course of growth in media without antibiotics was performed. Fresh cultures were transformed with plasmid pIGSAF and initially selected as described in Frankia Transformation, above. After this 4-week selection process, cultures were then grown in non-selective media: cells were first pelleted and re-suspended in fresh BAPP media, then subcultured without the addition of chloramphenicol into six-well plates and incubated for 1 week. A separate set of cultures from the same stock was maintained in selective media and subcultured at the same time points as a control. Each week both sets of cultures were pelleted and re-suspended in 500 µl fresh BAPP media. The suspensions were homogenized by passage through a 21G needle twice. 250 µl of each homogenate was transferred to 4 ml fresh BAPP media without chloramphenicol in each well and the remaining 250 µl was used for total genomic DNA extraction as described in DNA Extraction above. This process was repeated once per week for 4 weeks. At each sampling point, the relative amount of plasmid in each sample was quantified by qPCR, performed in duplicate for each of three biological replicates, using egfp primers GFP_qPCR_F and GFP_qPCR_R . Fold-change of plasmid between each time point was calculated using the 11 Ct method . The infC gene , amplified from the same DNA extracts with primers infC_qPCR_F and infC_qPCR_R , was used as a control to normalize the amount of DNA in each sample. To determine significant changes in plasmid abundance, twotailed Welch’s t-tests were performed in R on normalized 1 Ct values . Cultures grown with and without selection for 4 weeks were also imaged with fluorescence according to methods in Confocal Microscopy, above. After electroporation F. alni cells formed visible hyphae in culture after about 10 days. When subcultured into chloramphenicolselective media, F. alni cultures transformed with unmethylated plasmid pIGSAF were able to grow, whereas untransformed cultures and those transformed with methylated plasmid were not. When analyzed on the gel, purified plasmid pIGSAF from E. coli formed bands representing linear , circular , and supercoiled plasmid .

Presence of plasmid pIGSAF in transformed cultures was verified by a band present in DNA extracts corresponding to the linear form of plasmid pIGSAF that was not present in extracts from wild-type F. alni . PCR amplification and sequencing of the egfp gene from DNA extracts of transformed F. alni cultures confirmed the identity of the plasmid . Absolute copy number quantification with qPCR estimated the plasmid was present at 12.5 copies of plasmid per molecule of genomic DNA; relative quantification gave a similar estimate of 11.8 copies per genome . Each experiment was performed on cultures maintained with routine weekly subculture for 15–32 weeks post-transformation, as described in the Methods. When imaged under 488 nm wavelength of excitation in the confocal microscope, green fluorescence typical of GFP was observed in hyphae as shown in Figure 4. Wild-type F. alni hyphae displayed autofluorescence around 575 nm as observed by Hahn et al. , but no autofluorescence was observed in the 500–550 nm range . When grown in N culture, transformants carrying the pIGSAFnif plasmid showed significant up-regulation of the egfp gene conjugated to the nif cluster promoter, approximately 100- fold relative to N culture, or approximately 8.5-fold per copy of plasmid . The F. alni nifH gene , used as a positive control for nitrogen fixation, was similarly significantly up-regulated approximately 8.5-fold in N media compared with N cultures. Expression of the F. alni rpoD housekeeping gene , used as a negative control, was not significantly different between N and N cultures . F. alni containing pIGSAFnif grown in N media fluoresced predominantly in the vesicles,square plastic pot observed at 100X magnification . Little to no fluorescence was observed in hyphae. Fluorescence in the vesicles was present both in the spherical portion as well as in the stalk connecting the vesicle to the hyphae. No fluorescence was observed in hyphae or vesicles of wild-type F. alni grown in N media . Due to the step size of 0.1 µm bright fluorescence was only observed when vesicles were in the plane of focus . Few vesicles were present in images likely since the cells were observed after 1 week of culture in N medium. We have shown that F. alni can be stably transformed with an unmethylated replicating plasmid introduced by electroporation. The methods presented here circumvent two major transformation barriers in Frankia. First, the lack of methylation avoids restriction of the plasmid by type IV methyl-directed restriction enzymes. Second, the use of a plasmid replicated and maintained outside the genome does not rely on the reduced homologous recombination rate in actinobacteria . Plasmids pIGSAF and pIGSAFnif were stably maintained in F. alni culture and used to perform routine experiments. Three independent transformations were performed over the course of 2 years and the resulting transformants were maintained on selective media by repeated subculture for at least 7 months .

The presence and stability of the plasmids were confirmed with gels of whole genome DNA extracts , PCR and qPCR amplification of plasmid-bound genes , and visualization of GFP expressed from plasmids . The copy number of plasmid pIGSAF per genome in F. alni was determined to be about 12 copies per cell , in line with findings for other plasmids derived from plasmid pIP501 that have been estimated to be maintained at approximately 10 copies per cell . In addition, plasmid pIGSAF was determined to be stable in non-selective media for a period of at least 3 weeks . Of the restriction enzymes identified in Frankia genomes our analysis indicated that the type IV enzymes posed the most likely barrier to transformation. Types I and II enzymes recognize specific sequence motifs of generally six to eight nucleotides and hence are statistically highly unlikely to be a broad-range barrier to transformation or horizontal transfer . Additionally, in the genome of F. alni ACN14a the majority of types I and II genes showed very low expression in both N-culture and symbiosis . A type IV homolog of an mrr type methyladenine-targeting restriction gene was highly expressed in F. alni in culture , suggesting that DNA with methylated adenine bases is degraded in this organism. Actinobacteria, especially Frankia, express type IV methyl-directed restriction enzyme genes more highly in culture than do proteobacteria and firmicutes , a finding that correlates with previous reports of higher transformation efficiencies with unmethylated plasmids than methylated in Corynebacterium and Streptomyces spp. . Genomes of the majority of actinobacteria are missing homologs of the dam methyltransferase gene whose product is used to mark parent DNA strands during replication, and mutS and mutL that form a complex for the removal and repair of mismatched bases on the daughter strand determined by the methylation of adenine residues . Together, these factors suggest a preference for unmethylated over methylated DNA among most of the actinobacteria. Type IV restriction enzymes have been suggested to have evolved as a counter to phage methylation systems that themselves evolved to evade host restriction systems through the methylation of restriction target sites . Phage genomes adopt the methylation patterns of their previous host thus increasing the likelihood of digestion by actinobacterial enzymes if replicated in a dam + host. The expression of type IV restriction enzymes in actinobacteria therefore could represent an adaptation to prevent infection by phages based on the methylation state of their genomes. Differences in methylation patterns between actinobacteria and other bacterial phyla potentially constitute a barrier to horizontal gene transfer between these groups, including phage-mediated gene transfer. Of particular interest to the evolution of root nodule symbioses is the possibility of transfer of relevant genes between Frankia and the rhizobia, and vice versa. It has been suggested that the nodA gene involved in Nod factor biosynthesis evolved in the actinobacteria, including some Frankia, and was then horizontally transferred to the rhizobia . If type IV restriction enzymes create a barrier to horizontal transfer into actinobacteria from dam + bacteria including proteobacteria, it would seem that horizontal transfer from actinobacteria to other phyla would be more likely than the reverse. However, F. alni was observed to down-regulate its type IV mrr gene substantially in symbiosis . As roots contain much lower concentrations of bacteriophage than the surrounding soil this could represent a decreased necessity for restriction enzymes as a defense mechanism during symbiosis. A potential side-effect of this down-regulation, however, is that the barrier to horizontal transfer posed by type IV enzymes is likely lowered during symbiosis. In plants, the endophytic compartment is dominated by actinobacteria, with specific taxa of other phyla including proteobacteria and bacteroidetes .

Similarly, Mohaisen and Ma developed and simulated asolar assisted liquid dehumidification air conditioning system using LiCl solution on Matlab Simulink platform. Their result has been validated with experimental results by Fumo and Goswami. The simulation results based on three consecutive sunny summer days in Sydney show daily COP of 0.5-0.55 by the proposed system and 73.4% of thermal energy required for thermal regeneration was provided by the solar collectors. Croffot and Harrisson experimentally evaluated the performance of a solar driven system installed in Canada. The system includes a low-flow parallel plate liquid desiccant air conditioner, and a 95m2 evacuated tube solar collector array. Results from five test days have shown an overall solar collector efficiency of 56%, solar fraction of 63% and a thermal COP of 0.47. The average total cooling was 12.3kW and average latent cooling was 13.2kW. Peng et al.studied a solar air pre-treatment collector/regenerator. Their simulation results showed that the storage capacity of proposed system could be improved by 50%, when the regeneration temperature was 60℃, and the inlet air specific humidity was 2.33kg/kg. In another study, Lychnos and Davies performed experimental and theoretical simulation for a solar powered liquid desiccant system using MgCl2. The theoretical model was developed and verified with the experimental results. Compared with conventional evaporative cooler,dutch buckets system the proposed system could further lower the average daily maximum temperature by 5.5-7.5oC. Alizadeh fields tested implementing polymer plate heat exchanger into LDAC in Australia summer weather condition. Lithium chloride with 43% was used in the experiments and a scavenger air regenerator concentrates the dilute solution from the dehumidifier using hot water from flat plate solar. Alizadeh experimentally analyzed the effects of various air flow rate and desiccant flow rate on the system performance.

The experimentally obtained data was compared with a developed model. The comparison showed a good agreement between the experiments and model predictions. The results showed that the proposed system could provide cooling capacity up to 20kW with dehumidification efficiency up to 72%. Qi et al.simulated yearly system performance of solar assisted LDAC for commercial application in five cities . In this study, the effects of various solar collector areas and monthly solar radiations, ambient air conditions on the electricity consumption saving and monthly average COP were theoretically analyzed.Results shows that for buildings located in humid areas with low sensible-total heat ratio, the electricity energy reduction of the system was high and about 450MWh in Houston and Singapore and payback was approximately 7 years. For building in dry climate, although the total cooling load was low, up to 45% of electricity of AC can be saved because of significant improve in chiller COP during more than 70% of operation time and the payback was around 22 years. However, for the buildings with mild outdoor humidity, such as those in Beijing and Los Angeles the electricity energy saved only around 100MWh and the cost payback period was more than 30 years, and the application of LDAC was not that suitable. Li and Zhang investigated a solar energy driven hollow fiber membrane-based desalination system. The system consists of a membrane-based humidifier, a fin-and-tube type dehumidifier and a solar heating unit, which consists of a U-tube evacuated solar collector and a heat storage tank. The hollow fiber membrane-based humidifier is similar to a shell-and-tube heat mass exchanger. Through numerical modelling and experimental tests, they found that the COP of the system can reach about 0.75 and electric COP of 36.13 is achievable, which means electrical energy consumption is much less due to solar energy reclamation. Results show that solar accounts for 92.0% of the electric energy consumption by the entire system.

Sensible heat losses account for most of the energy losses from the system. Chen et al. proposes solar assisted liquid desiccant dehumidifier and regenerative indirect evaporative cooling system for fresh air treatment. The hot and humid fresh air is firstly dehumidified by LDD and then sensibly cooled by RIEC. The thermal energy captured by solar collectors is used for desiccant solution regeneration. In this study, they have looked into the influences of solar collector area and inlet air conditions and optimizing the extraction air ratio of RIEC. The energy saving ratio is quantitatively evaluated with respect to a conventional A/C system. Results shows that the energy saving ratio ranges from 22.4% to 53.2% under various inlet air conditions. This characteristic of liquid desiccant dehumidification system that the dilute liquid desiccant can be regenerated by low grade heat makes it attractive option for integrating with thermal sources. Many studies have been done on integration of liquid desiccant system with solar, vapor compression, heat pump, and CHPs. But only two research has been done integrating liquid desiccant with SOFCs. The typical SOFC system exhaust temperature matches very well with the required temperature for liquid dehumidification. Elmer has looked into design, development and proof of concept combined generation of power, cooling and heating based on SOFC and Liquid desiccant air conditioning technology for building application. A 1.5kW SOFC integrated with liquid desiccant has been proposed for providing heating, cooling and electricity to low carbon buildings. Elmer et al.used empirical SOFC and liquid desiccant component data for an energetic, economic and environmental analysis. They have used a simple 0D model in Engineering Equation Solver platform for regeneration, dehumidifier and fuel cell. The model is based on dimensionless vapor pressure and temperature difference ratio designed for packed bed liquid desiccant. The results show the moisture removal of the supply rate is mainly controlled by desiccant temperature and cooling water temperature in constant flow rates. 

The air inlet condition has a large effect on cooling output performance and the unit performs better in a hot and humid climate. By increasing the regeneration heat source temperature more water vapor vaporizes from the weak solution. The model they used have limitation such as not including the effectiveness of heat and mass exchanger effectiveness, geometry of the contactor, and desiccant carry over. For desiccant air conditioning system Elmer developed an Integrated Desiccant air Conditioning System . This system combines regenerator, dehumidifier and evaporative inter-cooler into a single membrane-based heat and mass exchanger to reduce the size, complexity and leakage. The IDCS operates with a KCOOH desiccant working fluid. The liquid desiccant is sprays through nozzle from the top where regenerator is located and flows downward due to gravity. In this design instead of preheating desiccant before regeneration and precooling it before dehumidification, thermal energy is supplied to the regenerator through airstream and an evaporative inter-cooler is designed. This is because all desiccant flow is contained within one complete Heat and Mass Exchanger the solution cannot be extracted for prior heating and cooling. This feature is weakness of this design as precooling and preheating of liquid desiccant increases the performance of the system.Their design was supposed to significantly reduces the number of heat exchangers, pipes, and ducting in liquid desiccant air conditioning systems. The main issue with this integrated system was imbalance between moisture removal rate in the dehumidifier and moisture addition rate in the regenerator. This mass imbalance is primarily due to the available surface area for heat and mass exchange in the regenerator being too small and an insufficient vapor pressure differential between the air and desiccant solution. In order to regenerate the desiccant solution back to its original condition following the dehumidification process,dutch buckets the regenerator needs to operate for extended time periods. Across the variables investigated there is a greater instantaneous moisture removal rate in the dehumidifier than moisture addition rate in the regenerator. Desiccant solution leakage/carry-over on the dehumidifier side of the HMX has been noticed during the experiment as well. In response to the highlighted shortcomings of the novel IDCS Theo Elmer developed a Separated liquid Desiccant air Conditioning System for trigeneration system integration. SDCS consist of three separate cores including dehumidifier, regenerator, and an evaporative cooler. The SDCS uses a semi-permeable, microporousmembrane-based cross flow contactor, operating with KCOOH desiccant solution. The solution channel consists of polyethylene sheet, with fiber membrane attached on either side to allow diffusion of water but prevent the liquid desiccant entering the air side. In evaporative cooler, air and water come into contact in a cross flow HMX. Before liquid desiccant entering the dehumidifier, a plate heat exchanger is used to precool the desiccant to increase its potential. After dehumidifying the air, the weak solution enters the dehumidifier tank.

Desiccant is preheated before entering the dehumidifier. The strong desiccant flows to regenerator tank after regeneration. In this design, because the entire SDCS is placed within the environmental, the water for the evaporative cooler and desiccant for dehumidifier are at the ambient temperature which has an impact on moisture absorption capacity of the desiccant and the sensible cooling. This design causes little to no sensible cooling achievable. Also, the evaporative cooler only provides around 80–150W of cooling over their study range. As a result, the evaporative cooling provided is not enough to produce a sufficient solution temperature decrease and to provide sensible cooling to the supply air in the dehumidifier. Elmer et al. experimental results on SDCS show effective instantaneous balancing of the dehumidifier and regenerator across a range of environmental and operational values, operation of the dehumidifier is dictated, to some degree by the available SOFC thermal exhaust, COP values in the range of 0.4–0.66 are achievable with a low-grade thermal input typical of an SOFC CHP system and potential for non-synchronous operation in a tri-generation system context, bringing about improvements to peak cooling output and total system efficiency. Elmer et al. used empirical SOFC and liquid desiccant component data for an energetic, economic and environmental analysis. The 1.5kW BlueGen SOFC systems is electrically connected to the grid to import or export as required. The waste heat recovery circuit is connected to a home’s 300L hot water cylinder which also includes an auxiliary gas boiler. Experimental results shows that the electrical efficiency of SOFC system increases from 14% at 200W to maximum of 60% at 1500W and then decreases to 56% as 2000W capacity. Thermal output from fuel cells varies linearly from 320Wth at 200We to 540Wth at 1500W. The calculated water heat recovery temperature for 2l/min flow based varies between 47 C at 100We to 52℃ at 2000We. Be Power Tech, Inc. developed BeCool, a natural gas-powered air-conditioning system that produced electricity as by product on the site. The innovative idea shifts some grid powered electricity for cooling demand to natural gas which would substantially reduce peak summer power demand and help to reduce the need for costly peaking power plants. Their studies showed that their 5ton BeCool unit eliminates ~ 10kW of peak electricity demand, generates 43MWh/yr electricity and saves ~ 10MWh/yr compared to conventional air conditioning. They build a prototype and the test results showed that the electrical power efficiency was 45% to 60%. The primary potential energy saving is 4.1 Quads, and the technology has the potential to reduce 222 million metric tons of CO2. Their analysis for California climate 7 and 14 shows 53% CO2 reduction in a 50% electrical efficiency for fuel cell and 90% combined heat and power efficient fuel cell system is used. BeCool uses exhaust heat from the fuel cell and heat from the burner to increase the concentration of LiCl to 42% mass fraction in the regeneration process. Be Power Tech designed and built an experimental prototype BeCool system integrating a 2.5kW SOFC system , a boost natural gas burner, and a custom-made liquid desiccant air conditioner designed to produce from 2.5-tons to 4-tons of cooling. The system was tested under multiple outdoor air conditions. Their study showed that, in all cases the heat supplied by the fuel cell was not sufficient to provide the heat required for the air conditioning process, since these isolated tests could not rely on storage from daily continuous regeneration. For this reason, the supplemental natural gas burner was used to supply the heat required [80]. Integrated fuel cell dehumidification systems have never previously been studied for data center application. The only two studies that have looked at integration of SOFC with dehumidification systems were focused on building applications of the technology.

White or coextruded white-on-black mulches can slightly lower surface soil temperatures by about 1ºC at 2 cm depth or 0.4ºC at 10 cm relative to bare soil because they reflect most incoming radiation . These mulches are used when lower soil temperatures may be desirable for planting vegetables in particular summer production windows. Clear mulches effectively retain much of the heat normally lost to the atmosphere by bare soil, increasing daytime soil temperatures from 4º to 8ºC at a depth of 5 cm , and 3º to 5ºC at 10 cm relative to bare soil . These clear plastics are the choice for soil solarization. Clear plastics, however, do not control weeds and require other weed management practices such as fumigation and herbicide application. Plastic mulches also influence nutrient levels and uptake. Wien and Minotti found plastic mulching increased shoot concentrations of nitrogen , nitrate , phosphorus , potassium , calcium , magnesium , copper and boron in transplanted tomatoes. Bhella , also working with tomatoes, found higher levels of ammonium , nitrate , and magnesium in plastic mulched soils. Hassan et al. found higher levels of nitrogen, phosphorus, potassium, and calcium in leaf tissue of chilies grown over plastic reflective mulch compared to those grown over bare soil. A wide variety of other colored plastic mulches, including red, yellow, silver, blue, gray, and orange, have been used in various efforts to achieve specific production goals. Each of these colors has distinct spectral reflectivity characteristics and thus modifies the radiation balance in and below a crop canopy. These colored mulches affect not only the microclimate around a crop but have also been shown to influence insect behavior. Yellow, for example, is generally highly attractive to insects and has been shown to increase green peach aphid and striped and spotted cucumber beetle populations compared to plants grown over bare soil .

White at times repels aphids and at other times attracts them, depending on the physiological state of the insect . Orange has been shown to repel various aphids and whiteflies , while pink and green attract aphids . Red has been shown to attract both aphids and whiteflies . Blue has been shown to be repellent in some studies and attractive in others . Highly reflective or shiny aluminum plastic mulches have been shown to repel certain aphids and thereby reduce or delay the onset of aphid-vectored mosaic viruses in zucchini squash and melons and tomato spotted wilt virus in tomatoes . These are the mulches of choice when insect and disease management are the principal objective. On balance, other than UV-metalized reflective mulches, repeated and consistent benefits in managing insects with most colored mulches have not been documented. These mulches, however, have produced mixed results . Mahmoudpour and Stapleton noted that “the influence of mulch colour on growth and productivity has been postulated to be highly specific, and may vary with plant taxa, climate, and seasonal conditions.” As is the case with insect management, aluminum mulches have provided the most positive and consistent findings on crop production .Using cover crops as mulches is a relatively recent management strategy that is currently being refined and evaluated in a wide range of vegetable production systems. The winter annual legume hairy vetch , for example, has been used successfully as both a cover crop and as a mulch in fresh market tomato production systems in the southeastern United States. As a cover crop, the vetch fixes nitrogen, recycles nutrients, reduces soil erosion, and adds organic matter to the soil. When mowed and converted to a mulch, the vetch reduces weed emergence,grow strawberry in containers lowers soil temperatures during the hot summer months, reduces water loss from the soil, and acts as a slow-release fertilizer .

This system, developed by USDA ARS researchers, eliminates tillage, reduces the need for applying synthetic fertilizers and herbicides, and reportedly adapts to both large- and small-scale tomato production in a low-input, no-tillage system. Recent work in Florida by Chellemi et al. has shown that although a cover crop surface residue mulch production system had lower yields than the standard black polyethylene plastic, the overall profitability of the alternative system was actually higher. Work in California’s Central Valley has shown that cover cropping increases water infiltration and reduced winter runoff and increases soil carbon . Additionally, cover crops, when cut and dried, have been shown to delay and reduce the incidence of aphids and whiteflies as well as the incidence of aphidborne viruses. Burton and Krenzer observed a reduction in green bug populations where surface residues of a previous wheat crop existed. Summers et al. found wheat straw mulch significantly delayed and reduced the incidence of alate aphids and several aphidborne cucurbit viruses in zucchini squash. The incidence of silver leaf whitefly and squash silver leaf was also significantly reduced. Similar results were obtained with cantaloupe grown over wheat straw residue. A number of other examples of the successful use of cover crop mulches have been reported in Georgia, Virginia, North Carolina, and Pennsylvania, but their potential in California’s vegetable crop production is only now beginning to be investigated, evaluated, and refined. Combining the potential benefits of surface residue cropping alternatives with those of conservation tillage is becoming increasingly attractive to row crop producers in many of California’s agricultural regions as shown by the following case studies.Beginning in 1995, a series of studies and demonstrations were initiated in the Central Valley to evaluate and develop conservation tillage and cover crop mulch production systems for tomato crop rotations.

While the immediate goal of these efforts was to reduce production costs, a longer-term objective was to develop information on the potential of reduced tillage to improve soil quality, store carbon in the soil, and conserve resources. Initial studies, conducted at the University of California West Side Research and Extension Center in Five Points, at the UC Davis campus, and in commercial production fields in Tracy, Vernalis, and Le Grand, evaluated the use of winter cover crops as surface mulches, the feasibility of no-till and strip-till transplanting, and options for in-season weed management. No-till transplanting requires the use of coulters or some form of residue manager to cut surface residues ahead of the transplanter shoe. Strip-till is a form of CT in which a set of coulter or shank implements tills a narrow band of soil 15 to 20 cm wide to a depth of about 8 to 36 cm only in the line into which transplants will be placed. Results from these preliminary evaluations have indicated that planting and harvesting both processing and fresh market tomatoes is possible and that yields comparable to those attained using standard winter fallow techniques may be achieved with certain reduced-till approaches that do not result in excessive cover crop regrowth or weed competition with the tomato crop. On-farm strip trial data for demonstrations conducted in 1999 in Tracy and in 2000 in Vernalis are given in table 3. This early work, and other experiments summarized by Herrero et al. 2001b, also revealed that in-season weed control by a surface cover crop mulch alone is not adequate. The authors of this publication have subsequently investigated and refined the use of a high-residue cultivator that is able to effectively slice through residues while cultivating weeds. Cover crop mulch species selection and mulch management must be optimized if organic mulch tomato production is to expand in California. Care must be taken to avoid the use of certain cover crops such as sorghum-Sudan hybrid as mulches because they are highly allelopathic to tomatoes and several other vegetable crops . More efficient and low-risk production protocols for managing cover crop mulches in vegetable crop rotations must also be developed.Aphid-transmitted virus diseases cause significant economic losses to California’s multi-million-dollar vegetable crop industries annually. Over the past few years, production of fall melons , squash , peppers , and tomatoes has been extremely difficult in certain regions of the San Joaquin Valley due to extensive virus epidemics and severe infestations of silver leaf whitefly . Spring crops, while affected to a lesser extent, have also suffered significant losses by aphid-transmitted viruses and whitefly infestations.Several plant viruses are responsible for these epidemics,hydroponic nft channel and most are capable of infecting all of the crops mentioned above. Among the most important viruses are cucumber mosaic virus , zucchini yellows mosaic virus , potato virus Y , and watermelon mosaic virus . These viruses are transmitted by aphids in a stylet borne, non-persistent manner. They are acquired and transmitted in as few as 15 seconds, and are transmitted by a large number of aphid species, all of which are abundant throughout California. Due to the rapidity with which the viruses can be acquired and transmitted, insecticides are of little value in preventing virus spread and under some circumstances may actually increase the rate of virus transmission and spread. This has not, however, dissuaded a large number of growers and PCAs from attempting to control the spread of these viruses by using insecticides. UV-reflective mulches consist of a polyethylene base to which a thin coat of aluminum ions has been adhered. The mulches are collectively referred to as metalized mulches. These mulches reflect UV wavelength , which confuses and repels incoming alate aphids, adult whiteflies, and leaf hoppers , reducing their incidence of alighting on plants .

UV-reflective plastic mulches have been used successfully to reduce the incidence of aphidborne virus diseases in squash and other crops . Brown et al. found silver plastic mulch superior to white, yellow, or black with yellow edges in repelling aphids in yellow crookneck summer squash. Plants grown on silver mulch produced significantly higher yields of marketable fruit than did those grown on bare soil. Mulches applied to the planting beds before seeding were effective in repelling alate aphids and delaying the onset of several virus diseases as well as the onset of silver leaf whitefly colonization and the appearance of squash silver leaf in spring and fall-planted zucchini squash in California’s San Joaquin Valley . Disease symptoms in plants growing over these mulches appeared 10 to 14 days later than in plants growing on unmulched beds. In spring-seeded squash, approximately 30 percent of the plants on unmulched beds were infected with one or more viruses by the first harvest, while only 10 to 15 percent of those grown over the metalized mulches showed virus symptoms. In fall-planted trials, 100 percent of the plants grown on unmulched beds, with and without insecticide applications, were virus-infected by the first harvest. Metalized mulches were generally more effective in repelling aphids and delaying virus onset than were white-pigmented mulches . Although plants grown over the metalized mulched plots eventually became infected, they continued to produce a significantly higher percentage of marketable fruit throughout the season than did the unmulched controls. In addition, squash, cantaloupes, cucumbers, and corn grown over reflective mulch produced marketable fruit 7 to 10 days earlier that plants growing over bare soil. Stapleton and Summers also showed than cantaloupe grown over reflective mulches yielded over 500 cartons of marketable fruit per acre compared to less than 50 cartons per acre from plants grown on bare soil. A delay of 4 to 6 weeks in infection by CMV in plants growing over reflective mulch allowed them time to mature and set a good crop of melon fruit before becoming infected. Even though the plants eventually became infected, the delay in infection permitted the harvest of a highly profitable crop. Summers and Stapleton have shown that tomatoes grown over reflective mulches averaged approximately 7 percent virus-infected plants, while plants grown over bare soil averaged in excess of 50 percent infection with the same viruses. This approach is currently the only viable means of managing virus disease in these production systems. Growers are cautioned to use only metalized reflective mulches when insect and disease management is the primary objective. Other colors lack the high degree of UV reflectance necessary to repel incoming insects. Also, the mulch must be applied prior to seedling emergence. Plants may be inoculated with aphidborne viruses in the cotyledon stage, and any delay in applying the mulch could lead to an extensive infection. Some crops are more susceptible to injury when planted over metalized mulches.

The first is an on-farm loss caused by cluster thinning to reduce berry variability during ripening, a practice presumed to improve wine quality. Cluster thinning at veraison is a common practice performed in California wine grape cultivation, and the timing is dependent on variety, vineyard location, and cultivation practices. The second by-product is grape marc, which consists of grape skins, stems, seeds, and pulp accumulated either after extracting the juice to ferment white wine or after fermentation and pressing for red wine . Several studies on red and white wine grape varieties have shown that concentrations of total phenolic content, -catechin, -epicatechin, procyanidin dimers and trimers, and proanthocyanidins of wine grapes would generally decrease and then stabilize between veraison and harvests . Thus, thinned at veraison are considered rich sources of flavanols. Nonetheless, thinned clusters are traditionally made into verjus, sour grape sauce, and unripe grape syrup in European and Middle East countries or simply left behind in many vineyards as soil fertilizers in spite of the increased production costs and lost yields . The health-promoting properties of flavanol monomers, an important subgroup of the dietary polyphenols, particularly -catechin and -epicatechin, and their oligomeric procyanidins, have been extensively studied in cocoa and chocolate . Epidemiology studies have suggested that the consumption of flavanol-rich diets and plant extracts is associated with reducing risk of cardiovascular disease , lowering blood pressure, especially in pre-hypertensive and hypertensive individuals, and enhancing their physical activity . Cocoa oligomeric procyanidins were found to be effective at preventing weight gain, development of glucose intolerance, and insulin resistance during a long-term high-fat feeding study by Dorenkott et al. . Lotito and Fraga demonstrated in an in vitro study, dutch bucket for tomatoes that the addition of -catechin and -epicatechin would delay lipid oxidation, and α-tocopherol and β-carotene depletion in human oxidized plasma induced by a radical generator.

The biological activities of -epicatechin and -epicatechin-containing foods are of particular interests in recent years because -epicatechin causes multiple actions that may provide beneficial synergy for cardiovascular and neuropsychological health . It is also worth mentioning that a predominance of -catechin, rather than the more bio available -catechin , was found in commercial chocolate samples. -Catechin is the naturally occurring form in foods such as berries and cacao beans, however, the Dutching process converts cocoa flavanols to the -catechin enantiomer . Grapes are known for their rich polyphenols including abundant -catechin, -epicatechin, and procyanidins. In an evaluation of anti-platelet activity of grape marc extracts, the ethanolic extract with catechin, epicatechin, and quercetin being the most abundant phenolic compounds was capable of inhibiting platelets aggregation in a wide range of agonist concentrations . Another recent study on the digested fractions of seedless red and white wine grape marc revealed that all digested fractions prevented the hyperglycemic actions in the cell viability and nitric oxide /reactive oxygen species balance, suggesting the protective effects of grape marc products on the vascular endothelial barrier function . Holt et al. also conducted a comprehensive literature review on using chardonnay marc, a plant-based natural product, as a new model for upcycled co-products in food applications and comparing with considerable clinical data generated from cocoa products on human cardiometabolic health in the context of healthy dietary patterns. Thus, it is logical to explore upcycling solutions for flavanol-rich thinned clusters in food applications, especially in cocoa-based products that have been perceived as flavanol-rich by consumers. The upcycling of wine-making by-products in food applications has been generating more interest in recent years with the main focus on using dried grape marc as a bulking agent to change textural and rheological properties in foodstuffs such as cookies , soft candies , and chocolate spread . In a chocolate ice cream feasibility study conducted by Soukoulis and Tzia , grape molasses was used as a potential sucrose substitution that exhibited the best chocolate color and mouth-coating ability in the final product.

Bolenz and Glöde evaluated the processing aspects and the impact on chocolate properties like particle size, total phenolic content, and sensory perception of milk chocolate enriched with grape marc and grape seed flour. However, concentration data on bio active flavanols such as -epicatechin are beyond the study scope. When cocoa-based products are analyzed for their flavanol content, individual ingredients, such as thinned clusters in various forms , are generally not separated from the matrix to gain insights on the individual contribution from each ingredient. Our study utilized improved ultra-performance liquid chromatography-fluorescence detector methods that provided new reference information on health-promoting flavanol monomers -catechin and -epicatechin, and procyanidins of thinned clusters alongside cocoa flavanols. The idea of incorporating grape thinned grape clusters into cocoa-based food products as functional ingredients opens new doors to upcycle underutilized grapevine by-products and to improve cocoa-based products with potentially more health-promoting and positive sensory properties with fewer calories.Fresh Chardonnay and Pinot noir thinned clusters were collected at veraison from the Los Carneros American Viticultural Areas in Sonoma County in mid-August 2019. The clusters were then categorized into light and dark for Chardonnay, and red and green for Pinot noir, respectively, based on berry colors. To gain more in-depth understanding of the flavanol composition of the thinned clusters, the seed and seedless fractions were manually separated, freeze-dried, and milled as described in Sinrod et al. .Flavanols and procyanidins of grape thinned grape clusters and cocoa powders were analyzed and reported following a single laboratory validated UPLC-FLD method by Bussy, Hewitt, et al. and Bussy, Ottaviani, and Kwik-Uribe with modifications. Cocoa powders were defatted three times by sonicating 5 g powder in ~45 mL hexane at 50°C for 5 min each time and completely dried in a fumehood prior to extraction. Defatting was skipped for grape thinned clusters due to low-fat content of seedless samples and concerns of phenolic degradation during extensive hot hexane extraction for seed fractions.

For subsequent extraction of flavanols and procyanidins, 0.05 g defatted Acticoa™ cocoa powder, 0.2 g defatted Mullica™ cocoa powder, and 0.2 to 0.25 g thinned clusters were dissolved in 5–10 mL acetone:water:acetic acid , sonicated at 50°C for 5 min, and centrifuged at 5000 rcf for 10 min, respectively. Supernatants were further cleaned on Oasis PRiME MCX cartridges , diluted, and filtered through PTFE filters for UHPLC-FLD analysis. A Waters™ Torus diol column kept at 50°C was used for separation on an Agilent 1290 Infinity UPLC coupled with a 1260 Infinity FLD. Mobile phase A was acetonitrile: acetic acid and mobile phase B was methanol:water:acetic acid . The gradient was following 0% B for 0.37 min to 45% B in 10.03 min to 95% B in 0.25 min and held for 2.35 min. The flow rate was 1 mL min−1 with a sample injection volume of 2 μL. The FLD condition was optimized at a photo multiplier tube gain at 11 with an excitation wavelength of 230 nm and an emission wavelength of 321 nm. The quantification was determined using NIST RM 8403 as external standards.Flavanol enantiomers – and -epicatechin as well as – and -catechin were characterized using a modified method from Machonis et al. For cocoa powders, 0.05 and 0.1 g of defatted Acticoa™ and Mullica™ samples were dissolved in 5 mL methanol:water:acetic acid , respectively,blueberry grow pot while for grape thinned clusters, 0.1 to 0.15 g freeze-dried samples were dissolved in 5 mL MWAA. All mixtures underwent sonication at 50°C for 5 min and centrifuged at 5000 rcf for 10 min to obtain supernatants. Supernatants were further diluted and filtered for UPLC-FLD analysis. An Astec® Cyclobond® I2000 RSP kept at 35°C was used for separation on an Agilent 1290 Infinity UPLC-FLD. The isocratic mobile phase was 20 mM ammonium acetate buffer :methanol . The flow rate was 1 mL min−1 for a total run time of 30 min with a sample injection volume of 10 μL. The FLD was set at an excitation wavelength of 276 nm and an emission wavelength of 316 nm. Five-point calibration curves of each enantiomer were plotted for quantification.The moisture content of freeze-dried thinned grape clusters was determined according to Thakur et al. for reporting TPC, and flavanols and procyanidins of thinned clusters in DW.The total phenolic content was measured in triplicate for thinned cluster fractions. All data are expressed as mean ± standard deviation . One-way analysis of variance followed by Tukey & Dunnett multiple comparisons were applied using XLSTAT to evaluate significant differences among thinned clusters and cocoa powders flavanol content. For all comparisons, differences were considered statistically significant at a value of p< .05.The two wine grape varieties, Chardonnay and Pinot noir, were categorized into four different thinned cluster fractions based on berry color. Each variety that was analyzed in the current study included: Chardonnay seed and seedless dark , Chardonnay seed and seedless light , Pinot noir seed and seedless red , and Pinot noir seed and seedless green . To help illustrate the potential of using Chardonnay and Pinot noir grape thinned clusters as functional ingredients in cocoa-based products, two commercial cocoa powder samples were also analyzed and used as reference samples for their monomeric and oligomeric flavanols and procyanidins content. The total phenolic content determination was the starting point for the thinned grape cluster characterization because it provided important information, although unspecific, about the relative composition of the thinned grape cluster sample .

The composition and concentration of phenolics in grapes vary greatly due to variety and degree of ripeness , and environmental and vineyard management factors such as soil type and irrigation strategy . Thus, it is not surprising to see a 2.1-fold and 1.7-fold variations of TPC in Chardonnay and Pinot noir fractions, respectively. As shown in Figure 1, Chard_S_Light, which was considered the least mature among all fractions, had the highest TPC at 70.1 ± 1.1 mg g−1 in dry weight . The highest TPC for Pinot noir was detected in its green seedless fraction at 62.4 ± 1.1 mg g−1 DW. Within the Chardonnay fractions, the less mature fractions had higher TPC compared to the more mature fractions in both seed and seedless forms although no significant difference was found in two seedless fractions . The same trend was also observed in Pinot noir fractions as the less mature green fractions for both seed and seedless had higher TPC compared to the more mature red fractions. Pantelić et al. also found that TPC values were significantly higher in seeds than in skins and pulps in seven red and six white grapevine varieties grown in Serbia; our Chardonnay Dark and Pinot noir Green fractions were on the opposite where the seedless fractions yielded higher TPC for both varieties. Typically, the amount of TPC in the white grape varieties is lower than that of red grapes due to lack of anthocyanins synthesis , yet, previous studies have found that the TPC of grapes mainly depends on the varietal differences, not on grape skin color . Furthermore, in this study, thinned clusters for Pinot noir were collected at the beginning of veraison before anthocyanins accumulation reached its peak. Variations among grape fractional sample preparation , TPC extraction , and data reporting format could result in significant TPC differences from study to study for the same varieties . Flavanol monomers and procyanidins are abundant in cocoa and grapes. In cocoa, procyanidins are the major flavanols which consist of oligomers as well as polymers of catechins and epicatechins commonly bonded through a C4-C6 or C4-C8 linkage . Grapes are also rich sources for oligomeric and polymeric procyanidins with studies showing oligomeric procyanidins such as dimers and trimers being easier to be absorbed in vivo . Although cocoa-based products can be perceived as “healthy” among consumers because cocoa flavanols exhibited numerous health-promoting properties , the amount of bioactive and bioavailable cocoa flavanols, mainly -epicatechin, -catechin, and procyanidin dimers and trimers, vary greatly among cocoa-based products thus are unlikely to achieve the same level of healthpromoting effects in vivo. Therefore, it is crucial to quantify the flavanol monomers and oligomeric procyanidins content in the grape thinned clusters to determine their bioactive potential when compared with cocoa ingredients such as cocoa powder. A new cocoa extract reference NIST RM8403 was used as an external standard in the recently approved 2020.05 AOAC official method which allows more accurate and reliable determination of cocoa flavanols and procyanidins using UPLC-FLD . The current study successfully applied this method to the Chardonnay and Pinot noir thinned cluster fractions as well as two commercial cocoa powders in the context of catechin, epicatechin, and their oligomeric procyanidins . An example of chromatogram is shown in Figure 2.

These overt statements of support for Lewis’s misogynistic motif may appear starkly different from scholarly assessments, but I will argue that a similar acceptance of the novel’s attitude toward women emerges in scholarly treatments of The Monk if we look closely enough. For instance, in some of the pioneering gothic studies, though scholars do not express approval of the way the novel’s female characters are brutalized, they evoke a similarly brutal attitude toward femininity when they write about the novel’s genre. Even today, scholars traditionally discuss Lewis and Ann Radcliffe together because of the common narrative that The Monk responded to The Mysteries of Udolpho and The Italian responded to The Monk, and revisiting some of the earliest instances of this comparison reveals that implicit in it can be a misogynistic attitude. The gendered genre binaries that scholars utilize when contrasting Radcliffe and Lewis sometimes cast the two authors as a sheltered, timid woman and a wild, bold man. For instance, in 1978 Howells writes that “Lewis is shocking and subversive in a way that Mrs Radcliffe never was in his exploration of the dark irrational hinterland of the human mind,” as if he is an intrepid explorer.More troublingly, some early scholars create scenarios in which the Lewisian man menaces the Radcliffean woman. Montague Summers writes that “Mrs. Radcliffe shrank from the dark diablerie of Lewis,” and Michael Sadleir narrates, “Into the firelit refuge of the Radcliffian novelist the follower of Lewis would fain intrude, haggard and with water streaming from his lank hair, shrieking . . . then,nft growing system when he had struck the company with silent fear, he would wish to vanish once again into the howling darkness.”45 The seemingly inoffensive idea that Lewis’s novel unsettled the status quo looks not quite so inoffensive when coupled with imagery that recalls the way his male characters violate his female ones.

Even scholars who object to the novel’s treatment of female characters can end up aligning themselves with the perspective they censure through their own interpretive methods. Feminist critic Judith Fetterley, writing in 1978 about the masculine tradition of American authorship, argues that “a female reader is co-opted into participation from which she is explicitly excluded; she is asked to identify with a selfhood that defines itself in opposition to her; she is required to identify against herself.”Building on this description of coercive reading, she writes, “Powerlessness is the subject and powerlessness the experience,” as female readers are forcibly alienated from the classic literary plots of men dominating women.Using a similar framework to Fetterley’s, feminist critics have repeatedly called attention to the novel’s tendency to objectify and show contempt for women,but some suggest that readers have no choice but to inhabit a masculinized, heterosexual, lascivious, dehumanizing point of view while reading, as if the novel forces readers to take a position they find abhorrent. Under the control of Lewis’s narrative, they imply that there is no possibility of reading his abused female characters sympathetically. In describing the rape scene, Howells observes that it reads like pornography, and though she clarifies that its carnal account is not sexually exciting, she maintains that “there is for us the voyeuristic fascination of watching how far conventional limits can be transgressed,” as if the depiction of a taboo compels all readers to become mesmerized spectators and leaves no option of withdrawing in disgust.Elizabeth Napier concurs, arguing that Lewis’s writing inspires “a kind of prurient curiosity that depends upon withdrawal and distance.”Though the novel appears to advocate for compassion, she claims, the way it aestheticizes sex and violence makes pity impossible for readers.

She writes that Lewis “puts the reader in the novel’s most crucial moments in the position of a voyeur” and makes his victimized characters “undergo punishments so extreme as to excite horror rather than pity,” and she hints that Antonia desires the man who rapes and kills her and thus prevents readers from having “[a] correct response to this purest of characters.”Napier disallows the possibility of readers having a simultaneously aesthetic and emotional response, or being capable of feeling both revolted at the violence and sympathetic for the victim, or reading Antonia’s warmth toward Ambrosio as that of a sibling, or being capable of a “correct” response to a victim even if that victim is not irreproachable, or even reading against Lewis’s authorial prompts in order to feel into fictional suffering. These scholars’insistence that Lewis’s distancing techniques allow for no emotional connection with his female characters ends up sounding like an acceptance of the novel’s voyeurism and misogyny rather than the objection that they likely intend. Close reading, which could provide an antidote to the distance and disconnection that many feminist critics identify, can end up performing an uncomfortable sympathy with the text as well. Fredric Bogel recently championed close reading as a practice of what he calls “true infatuation”—intense attention to the text that is comparable to erotic obsession.This erotic textual obsession, when applied to The Monk, can become unseemly. When scholars take time to analyze the passages in which they find Lewis’s writing most effective, their careful literary analysis begins to sound like they are reveling in the sensational violence and sex. Robert Kiely observes Lewis’s tendency toward abstract, indirect writing and descriptions of Ambrosio’s sensible bewilderment, in contrast to which “the encounters with female corpses are, in addition to their intrinsic unpleasantness, stylistically striking.”

KIiely meditates on the power of Lewis’s specificity and simplicity of style in these scenes where women are dead or dying, writing that passages like the one where Ambrosio chokes and suffocates Elvira or the one where a mob tramples the prioress interrupt the conventional and “possess an energy and realism for which the reader is not fully prepared.”Though Kiely means to make the point that the directness of Lewis’s violent writing makes the violence more awful and the victims more human, his appreciation of forceful style sounds like an appreciation of violent action in this context. George Haggerty similarly venerates Lewis’s skill with violent scenes, analyzing how his combination of abstractions and vivid carnage in his description of the prioress’s mutilation makes the scene conceptually and viscerally effective. He goes so far as to claim that this philosophical brutality is the only effective technique Lewis could have used to make readers feel this scene without having another character’s reactions to prompt them: “Without the filter of a responding consciousness, there is no other way to assure affective success. If we are disgusted, Lewis has succeeded.”The tendency of literary scholars to write as if their own response is the only possible one can be jarring when they linger appreciatively over scenes of violence or sex that obviously provoke very different responses in other readers. Writing in this mode, Ahmet Süner delves into the sexuality of a moment that I will revisit shortly, in which Matilda bares her breast, contending that Lewis’s language enhances the erotic effect of the scene even as it appears to be modestly euphemistic. He writes, “The sensation . . . overtakes the metaphor, taking over the object; consequently, the object is surrendered to the bare mechanics and bare metaphoricity of sensation . . . all these clichéd metaphors are already chewed and devoured so that they may unobtrusively give way to the free flow of desire.”His own language as he analyzes Lewis’s sounds like a sexual encounter, or a sexual assault: “overtakes,” “taking over,” “surrendered,” “bare,” “devoured,” “free flow of desire.” Süner likely intended this sexualized analytical language to reinforce his point about the passage’s prurience, but it ends up seeming as if Süner has been absorbed into the voyeurism and sensual ecstasy he was trying to parse. When scholars analyze Lewis’s sensational writing,nft hydroponic system whether they chafe at it or embrace it, they often implicate themselves. Because of the ways that normative scholarly methodologies shape discussions of sensation, critiques or close readings of The Monk’s depictions of violent rage and transgressive desire can implicate scholars, but they are also limited in the ways they can address Lewis’s portrayals of other feelings. The suspicious critical mood informs the way scholars approach Lewis’s less embodied descriptions of emotion, which they tend to find false. For instance, scholars have written that the hero Raymond’s horror upon encountering a ghost has “the behaviouristic detail of a theatrical performance,” that Ambrosio’s frequent confusion of mixed emotions makes him “flutter and reel” like a sensible heroine, that Agnes’s grief over her dead child is part of an “over-the-top tableau” typical of Lewis’s “theatrical excess,” and that the sadness of Antonia’s reunion with her beloved and her mother’s ghost as she dies “is undercut by Lewis’s parodic manipulation of Sentimental conventions.”Whether these scholars assume that Lewis is forced to write in the style of drama or sentimental novels because of a lack of available fictional options or that he is performing a critique of these modes, they generally do not find these emotional moments worthy of sustained critical attention.

What options exist for scholars who want to write about the novel’s emotions but don’t have an acceptable scholarly method for doing so without either dismissing its portrayals of fear, confusion, and sadness as ineffective or analyzing its portrayals of lust and rage in ways that become disturbingly aligned with those feelings? This is the bind I found myself in after rereading The Monk with the voices of these scholars in my head. A lay critic’s comparatively broad range of affective choices do not fit comfortably within a scholarly role. What ended up working for me was to approach emotional scenes not through feeling but through seeing, by first considering how artists have illustrated them. My next section will show how this kind of consideration can reveal a variety of affective and interpretive possibilities in a single textual moment, and my final section will explore what these possibilities can open up in the text itself.In this section, I will return to visual analysis, focusing on illustrations rather than book covers. Rachel Schmidt writes that illustrations of narratives require that the artist engage in a process of interpretation that will enhance certain effects of the text for readers.In the case of Don Quixote, Schmidt argues that early illustrations were involved in shaping the novel’s critical reception. Many of the illustrated editions of The Monk that I could find actually appear to have little in common with the novel’s critical reception, unlike its twentieth- and twenty-first-century book covers. Whereas the book covers represent extremes of sensationalism or abstraction and thus illuminate the mixture of extremes that I have argued are operating uncomfortably in certain scholarly treatments of the novel, the illustrations present a wider range of feelings, and many are indeterminate in their affect. In contrast to book covers, which govern readers’ impressions of the book as a whole early in the reading experience, illustrations represent carefully chosen moments and provide visual cues for understanding them. The moments that are chosen for illustrations and the ways particular artists interpret them provide multiple possibilities for contextualizing an emotional situation at a particular point in the text. After viewing every illustration I could locate, from soon after the novel’s publication to the 1980s, I discovered that the moment in which Matilda exposes her breast and threatens to end her life is by far the most common visual representation from the main plot of the novel.This is perhaps in part because it allows artists to titillate with nudity, though what is arguably the most sensual scene, when Ambrosio spies on Antonia bathing, was illustrated in only one of the editions I found. The moment of threatened suicide, occurring in the scene where Rosario reveals herself to be Matilda, is also likely a subject of fascination because the scene is when the stable ground of gender identity gives way in the novel, which some artists emphasize by portraying both figures in their entirety, so viewers can see that they are dressed identically in monks’ robes. Interdependent with Lewis’s portrayal of sexuality and gender in the scene, though, is his portrayal of power. As I will examine in my close readings of the next section, the drawn-out scene in the novel features complex, shifting power dynamics . The illustrations I will compare all freeze the scene in the urgent standoff before Ambrosio surrenders his control over Matilda’s fate, and the choice to represent this moment of possibility implies that this is one of the key decisions that determines the course of the novel.

The modern edition states that it reproduces the 1793 version exactly, except for what the editors deem typographical errors, including “seemingly random and nonsensical commas.”This omitted comma is far from random, though, as it makes Matilda’s temporal and experiential situation either restrictive or nonrestrictive . Thus, the recent version implies that the Matilda who has suffered is a changed Matilda—her situation has been absorbed into her identity. The original version implies that Matilda is not that closely identified with her circumstances, just as it refuses to state something like “she was sad.” In the syntax of the Parsons’s emotional curriculum, situations can supplant a person, but they cannot become subordinate or equal to her. In this happy moment, Matilda’s travails remain, temporarily minimized but independent of her. It is the fact of these travails that matter most in the novel’s world—the relatable information, not the verbal expression of a sufferer’s response to them, which is too personal to be expressed or sympathized with. Indeed, words may be not only insufficient but also inappropriate, as suggested by a poem that Matilda finds scratched on the window of the room where the Countess of Wolfenbach was imprisoned by her husband after the murder of her lover and theft of her baby. The verse is necessarily brief, reading, “I am dumb, as solemn sorrow ought to be; / Could my griefs speak, my tale I’d tell to thee.”This passage of Wolfenbach is the one most often analyzed by scholars: Gillian Beer uses these lines as an example of the rhetorical force of incapacity,Karen Morton asserts the effectiveness of the window poetry as a mediated articulation of grief,grow bag for blueberry plants and Angela Wright quotes another of the verses to illustrate the novel’s powerful critique of marriage.

Deidre Shauna Lynch writes that when Matilda encounters this verse and others along with a handprint on the blood-stained floor of the room without knowing the Countess’s history, the writing “accentuates the other disappearances that this site of inscription has witnessed . . . The unknownness of that hand and the inaccessibility of the backstory behind the words are arresting.”In addition to way the discovered poetry makes the scene comparable to Radcliffe’s more reputable work, this is one situation in the novel where Parsons’s emotional writing can meet with approval from a modern audience, because the fact that Matilda encounters these words in writing, apart from the mysterious poet, removes the pressure of the fictional writer or the actual author to make some greater attempt at articulating emotion.In the context of these disembodied, departicularized, ghostly words, we do not expect a modern articulation of physical sensation or precise emotion from the composer or the narrator, so in this instance the lack of information can be as effective for us as it always is for the inhabitants of Parsons’s school of affliction. Many people today believe that the powerful and personal feelings that arise in response to extreme situations can never truly be explained to those outside that experience. In real life, we rarely expect someone who has lost a child to wrap up every feeling involved in that excruciating event and its aftermath in language and hand it to an uninvolved person. We are more likely to accept that the horrific nature of the circumstance makes the sufferer worthy of sympathy or aid even without knowing the details, and to believe that the feelings a person in that situation would experience are unimaginable, only knowable to those who have experienced the same event. In this way, Parsons’s style more accurately reflects our current beliefs than our own fiction, which routinely enters experiential territory that its readers and even its authors have not traversed firsthand. In this rare moment in the Parsons’s novel, our ethical convictions and aesthetic sensibilities are in accord with Wolfenbach’s school of affliction. Despite the inadequacy of language in Parsons’s emotional curriculum, stories can supplement the firsthand experiences of suffering by educating the audience about the types and proportions of human misery.

Soon after meeting Matilda, Mother Magdalene tells her, “Another time you shall know my sad story, and will then confess, of the two, I have been most wretched.”When she does elaborate on her various distresses, Mother Magdalene asks Matilda the seemingly rhetorical question, “[H]ave your troubles ever equalled mine?”Mother Magdalene’s storytelling helps teach Matilda how to measure and rank suffering, to subordinate her own troubles to the superior affliction of another. Though Santos argues that the stories in the novel perform a similar didactic function for readers, “as a mediating device that anticipates and regulates the reader’s response to intense feeling,” the form does not necessarily result in the intended effect. Napier, for example, criticizes the fact that the most dramatic moments in the novel reach the reader at a distance, through these stories of pain and woe, which she reads as the mistaken prioritizing of a moral message over vivid action.Both these readings disregard the fact that apart from any intended or actual effect on a reader, within the world of the novel the stories themselves play their own essential role in plot and character development. Deidre Lynch writes that Wolfenbachelevates books as “essential props for its protagonists’ projects of memory and mourning,” as “the pages the characters behold are often conceptualized as their legacies, either familial or cultural.”and I would argue that in the case of oral stories in the novel, this phenomenon is doubly true. Over the course of the novel, Matilda gains wisdom and perspective in part by listening to several tales of woe. The climactic scene even focuses on the telling of a sad story, as Matilda’s uncle slowly spins the tale of how he wronged her parents and deprived her of her birthright. The resolution of the novel consists not only of the typical restoration of Matilda’s status, mother, and lover, but also, and essentially, her learning more of the story of suffering that shaped her life. It is a story that gives her identity, and it also completes the process of educating her about the extreme forms of affliction others experience, to the point where she can see her own troubles in the proper proportions taught by the school of affliction and become a more altruistic person. Through all these messages and the techniques Parsons uses to convey them, the novel itself acts as its own school of affliction, with Parsons teaching her readers how to properly respond to her stories of suffering. The novel’s frequent assertions that emotions are indescribable mark off the narrative territory that the novel will not cover and attempts to recalibrate readers’ expectations, if they are not already primed to read incapacity for speech as a sign of deep feeling.

Instead of attempting to enter into the particulars of her characters’ feelings, Parsons encourages her readers to learn a culturally appropriate response to these types of situations—one that she models in the novel through her listening characters. Santos’s observation about the modeling of sympathetic response in the novel is correct as far as the intention built into the convention, if not correct about its actual effect on all readers. However, an essential part of this modeling is the way Parsons’s listening characters do not make a sympathetic response conditional to details; the telling and hearing of stories is not only conventional in itself but also conventional in its reliance on types alone for effect. In responding to Matilda’s story of evading her lustful uncle, the Countess of Wolfenbach is a perceptive and experienced reader who recognizes the character type of the damsel in distress and responds with the expected attention and sympathy. As these scenes in which a sufferer relates her sad story and a listener responds with compassion recur, Parsons uses repetition to teach her readers how to recognize generic categories and respond appropriately—to feel with the formula. Whereas The Castle of Wolfenbach represents a school of affliction that teaches a method of feeling with the formula of stories of suffering, its reception teaches a method of feeling against that formula. This is a different kind of literary emotional training, but one that still teaches that we do not need to know particulars in order to respond correctly. As I discussed in the first section of this chapter,blueberry grow bag eighteenth-century discourse about novels, and especially Minerva novels, created an atmosphere in which a work like Wolfenbach could be perceived as a part of a broader threat—one of a mass of new gothic fiction that appeared to cater to a public appetite for extreme emotion and to dull readers’ powers of aesthetic discrimination, and even, some feared, to encourage immoral behavior by portraying vice. Eighteenth-century critics considered Parsons’s writing to be somewhat better than most of the novels published during the growth of disreputable circulating libraries, and publishers reprinted it until the mid-nineteenth century in editions alongside reputable novels, but after this time it was forgotten. By the twentieth century, scholars knew Wolfenbach only as one of Northanger Abbey’s Horrid Novels, a label that implies it is both exhilarating and bad. It is this latter quality that scholars and amateur critics tend to focus on when they approach Wolfenbach through Northanger, and they often treat Jane Austen as a model for how to condescend to the emotional and aesthetic excesses of Wolfenbach and other novels of its kind. Scholars of the twentieth and twenty-first centuries rarely mention Wolfenbach apart from its context as a Northanger Horrid Novel. In this context, scholars sometimes characterize Austen as a book critic who uses Northanger as a venue for teaching her readers to discriminate between Ann Radcliffe’s worthwhile gothic novels and her imitators, including Eliza Parsons. For example, Bette Roberts argues that the list of seven novels that Isabella recommends to Catherine “enumerates the negative extremes to which the reader may contrast what Austen considers to be higher expressions of the Gothic in the notably superior novels of Ann Radcliffe.”These novels fall far short of Radcliffe, Roberts explains, because although they “pretend” to advocate for morality, rationality, and self-improvement, the “disastrous stories” they include are not truly meant for edification of the “already perfect” characters but merely to “appeal to the readers’ emotions.”This critique overlooks the way that in Wolfenbach, Matilda demonstrates her growth not only by gaining her identity through the disastrous story that relates to her origins but also through learning to subordinate her own disasters to those of others and access universal wisdom.

Other scholars have argued that Austen’s novel teaches not only proper literary taste but also life lessons through aesthetic means. Robert Kiely demonstrates how Henry Tilney challenges Catherine’s indiscriminate usage of words as a means to enable her to transcend her own naïve perspective and exercise better judgment of people and situations, similar to how I argue Parsons uses stories of suffering in Wolfenbach. For Kiely, however, Austen’s un-gothic restraint and distinction are the key pedagogical tools, as he finds that “as a display of the disciplined mind and the well-chosen word [Northanger Abbey] does more than all the hysterical criticism of the periodicals to deflate some of the poses and excesses of Romanticism.”Jacqueline Howard argues that Kiely’s reading misses the ways that Austen not only ridicules gothic novels but also skewers the criticism of novels, making Henry an object of critique by styling his instruction as the masculine condescension “of those authoritative, male-authored pronouncements addressed to young women in conduct books and reviews.”Kiely’s analysis is the sort of scholarship on Jane Austen that Eve Kosofsky Sedgwick describes as being sadistically concerned with “the spectacle of a Girl Being Taught a Lesson.”This critical fascination with the way Austen’s protagonists become chastened by experience has likely placed even more emphasis than Austen does on the problematic reading habits of the young Northanger women, underscoring the idea that Wolfenbach and the other Horrid Novels are pleasures that should not be indulged. Wolfenbach’s own pedagogical narrative appears to register as insufficiently harsh to these scholars, perhaps because Matilda does not experience the kind of shame that Catherine does when she gains perspective on her youthful feelings, or perhaps because Parsons herself indulges in too many aesthetic and emotional excesses in Wolfenbach.Emotional involvement of the sort that Wolfenbach portrays and encourages appears to some scholars as the fault that Austen intends to educate readers out of with Northanger.

The scholarly distrust that Kramnick mentions and that Lynch complicates has become an object of scholarly concern in the last two decades. In her 2003 book Touching Feeling, Eve Kosofsky Sedgwick reintroduces Paul Ricoeur’s “hermeneutics of suspicion,” observing that this style of criticism resembles paranoia and that the use of it is now “widely understood as a mandatory injunction rather than a possibility among other possibilities.”Perhaps because many of us consider it mandatory, scholars have had difficulty defining this mode of scholarly critique or describing what other methods readers employ. Michael Warner, prompted by Sedgwick’s essay, attempts to delineate what critical reading is and what it is not, concluding that our immersion in critical reading has meant that “whatever worlds are organized around frameworks of reading other than critical protocols remain, for the most part, terra incognita.It is this largely unmapped territory that I explore in my dissertation. I will compare responses to the same novels—and often the same features of those novels—from scholars working in the last few decades, earlier scholars, periodical critics, and amateur critics, which will make their different techniques and affective protocols apparent. The current affective protocols of the hermeneutics of suspicion have been most thoroughly demonstrated by Rita Felski, whose 2015 book The Limits of Critique arrived after I began this project but has been essential in helping me explain some of the problems I see with today’s critical practices. Felski uses the term “critical mood” to describe the suspicious orientation of most scholars toward texts that has pervaded otherwise disparate methodologies and become so dominant that it obscures or invalidates other attitudes and methods.

Felski writes that in the opinion of many humanistic scholars, “To refuse critique . . . is to sink into the mire of complacency, credulity,grow bag gardening and conservatism.”She acknowledges that this critical mood allows for pleasure of the disciplinary sort that Lynch describes, but she objects to the way it disallows receptivity to “the multifarious and many-shaded moods of texts,” as a suspicious critic is “fearful of being tricked or taken in. Locked into a cycle of punitive scrutiny and self-scrutiny, she cuts herself off from a swathe of intellectual and experiential possibility.”It is worth emphasizing that for groups that are routinely attacked by ideologies and policies, suspicion is self-preservation, especially considering the ways that literature informs our awareness of the world. But Felski’s point that this affective orientation limits scholars is important. In my dissertation, by devoting attention to the emotional qualities of numerous responses, both professional and nonprofessional, older and newer, I hope to suggest alternatives to the dominant scholarly attitude. For reasons I will address later, I am not attempting to offer a historicist account of emotion and criticism in this dissertation. However, I will briefly discuss some of the ideas of eighteenth-century philosophers and critics that influenced the reception of gothic novels. British eighteenth-century writers were actively engaged in defining and describing both emotions and taste, which they often treated as subjective phenomena that could nonetheless be assessed according to standards of appropriateness. For some, these standards were universal, deriving from God and humans’ common sensory makeup For others, they were social, established by what was beneficial to society or what had been consistently judged by others over hundreds of years .Many eighteenth century British writers shared an interest in moderation and were invested in disciplining excess, which helped create a cultural atmosphere in which many gothic novels would be judged as inappropriate for their extremes of character and situation, and even more so for their extravagant portrayals of feeling. To illustrate how these eighteenth-century judgments of appropriateness in art and emotion have been applied to literature and the slipperiness of this application, we can consider Adela Pinch’s analysis of a passage from Henry Home, Lord Kames.

Kames, the author of a thorough eighteenth-century treatment of aesthetics, discusses the need for writers to consider propriety when representing a characters’ feelings. For Pinch, this is an issue specific to eighteenth-century conceptions of emotion, but a similar problem in judging literary emotion continues beyond that historical period. For example, in 1968 critic Northrop Frye writes of Radcliffe’s heroines, “We may wonder why any literary convention should have produced these absurd creatures, drizzling like a Scotch mist and fainting at every crisis in the plot.”And in 2009, a Goodreads reviewer writes to Radcliffe’s Emily, “STOP CRYING YOU STUPID WHINY BITCH.”When critics judge the failure of emotional portrayals, these judgments often sound like indictments of the emotional people portrayed. This sort of judgment of literary feeling was vigorously exercised in the eighteenth century, as periodical critics, moralists, authors, and the general public argued about the effects of new fictional forms. William Warner writes that in the early debate about novels, “the novel reader is characterized as a susceptible female whose moral life is at risk” due to the dangerous pleasures she supposedly received from early amatory fictionBy the mid eighteenth century, Warner argues, the works of Henry Fielding and Samuel Richardson participated in a project of elevating the status the novel, and as the perceived cultural threat of novels in general diminished, critics and authors shifted focus to the emotional and moral value of particular kinds of novels.Those who wrote about gothic fiction in its early years often drew on this discourse to make claims for the emotional and moral benefits of these works or to condemn the novels for their dangers. In 1773, poet Anna Letitia Barbauld and her husband, John Aiken, describe the emotional conflict of reading narratives of terror, in which the pain induced by the miseries described is rivaled by the pain of suspense. However, they find that the exceptions are “well-wrought scenes of artificial terror which are formed by a sublime and vigorous imagination.”These sorts of fictional terror, of which The Castle of Otranto is “a very spirited modern attempt,” “elevate the soul” and provide pleasure through the combination of the terrible and the marvelous.Similarly, these writers recommend works that evoke pity with a delicate hand, in which authors lighten scenes of suffering with “strokes of pleasantry and mirth,” as excessive exposure to distress can “render us insensible to every thing” and even make people incapable of feeling pity for real people.

The novels of Ann Radcliffe, published later, would accord to these rules, but Matthew Lewis’s Monk would violate them, which helps explain their very different contemporary reception. Barbauld and Aiken’s concern about desensitization would take a more urgent form in William Wordsworth’s 1802 preface to Lyrical Ballads. Wordsworth decries the emotional danger of modern life, which “blunt[s] the discriminating powers of the mind” and “produces a craving for extraordinary incident” that is satisfied by “frantic novels” that many scholars assume to be gothic.The horror that early critics express at the proliferation of poorly written, formulaic gothic novels in the 1790s, which I will discuss inchapter 1, is thus in part horror over the power of literature to shape and be shaped by the baser feelings of readers.E. J. Clery identifies 1797 as “the year in which reviewers and critics began to put a name to the category of fiction we now call Gothic or the fantastic, although the name varied: ‘modern Romance’; ‘the terrible school’; ‘the Terrorist System of Novel Writing’; ‘Terrorist Novel Writing’; ‘the hobgoblin-romance’.James Watt argues against the critical tendency to unify the genre because “‘Gothic’ fiction was far less a tradition with a generic identity and significance than a domain which was open to contest from the first, constituted or structured by the often antagonistic relations between different writers and works.”Though I contest Watt’s own contestation in my second chapter, I am less invested in judging the legitimacy of the generic categorization of gothic novels than in examining what this categorization implies for critics, especially in terms of quality and feeling. Thus, what matters most for the selection of books in this study is that critics have labeled them as gothic. Scholars of gothic fiction have argued that, above all, gothic is a genre defined by its emotional characteristics. Coral Ann Howells names “feeling as the distinctive attribute of Gothic—feeling as it is explored and enacted in the fictions themselves, and feeling as the primary response elicited from the reader.”George Haggerty even claims that “Gothic form . . . is affective form. It almost goes without saying that these works are primarily structured so as to elicit particular responses in the reader. Perhaps, however, plastic grow bag it is not so obvious that Gothic fiction therefore cannot have specific meaning . . . it is central to the nature of Gothic fiction that differing interpretations will seem equally valid.”Haggerty’s argument here inspires my approach in this dissertation, as I try to determine how readers’ situated interactions with the texts allow for different affective, evaluative, and interpretative possibilities. For example, a twenty-first-century reader who picks up The Monk because Stephen King recommended it will have a different experience than an eighteenth-century reader who saw an advertisement suggesting the novel would be like The Mysteries of Udolpho, and the audience for which either of those readers would write a review would shape their responses differently as well. Haggerty himself, however, turns his argument to different ends, writing that “Gothic works only become fully intelligible when we understand the extent of their affective rationale.”Haggerty and most other scholars of gothic and similarly emotionally dependent fiction turn either to the text itself or to its contemporary context in order to determine a novel’s “affective rationale,” investigating the formal, structural, verbal, and conceptual ways that the text prompts certain responses. Barbara Benedict elucidates some of this affective rationale in her examination of how sentimental fiction “frames” feeling in a way that simultaneously encourages and discourages it. She argues, “The conventional language, pictorial diction, tonal instability, structural fragmentation, and multiple narrative voices work to externalize these interior experiences, to deprive them of authority, and to subordinate them within a social frame.”For example, she writes that Ann Radcliffe’s “disjunctive styles and structures . . . aestheticize emotional reactions with the discourse of spectatorship while they also exhibit control of sentimental excess.”Stephen Ahern thoroughly explores the contradictions that Benedict identifies, explaining that amatory, sentimental, and gothic fiction are all rooted in the unstable concept of sensibility and thus share “an ambivalence toward excess” which “arises from two fundamental yet conflicted imperatives: sensibility is an idealist discourse that is ineradicably rooted in physiological response, and sensibility is coded according to unstable categories of class and gender difference.”The effects of the portrayals of extreme emotion in sensibility narratives are unpredictable, he writes, “for overwrought descriptions of pathos can quickly descend into bathos if the audience . . . is unconvinced of the authenticity of the emotion expressed. Theatricality is a necessary condition of literary forms that find their truth in scenes of heightened emotion, but it is also a destabilizing force.”These scholars and many others have done valuable work in clarifying the emotional functions of gothic novels, but even though they acknowledge readers’ volatile responses to the mechanisms they describe, they still suggest that these novels evoke a fairly small range of emotions. For example, Napier’s analysis of gothic form leads her to conclude, “A successful response to the Gothic is based on instability: one must be pleased by what one dreads, take pleasure from distress, luxuriate in terror.”When scholars attempt to explain what kinds of responses the emotional conventions of fiction dictate, even when they allow for instability, they can end up suggesting that there are only a certain number of correct responses. Most scholarly accounts of feeling in literature do not recognize actual readers’ idiosyncrasies because they focus on what the construction of concepts and conventions enable. Even when the construction is built with internal contradictions, actual responses always exist in a more complicated world of influences beyond the text, and the cultural norms that may have caused contemporary readers to feel a certain way do not necessarily obtain for readers distant from the circumstances in which the text was written. Various scholars have explained the origins of the English gothic genre, its popularity, and its early critical reception as being attributable to a range of political, economic, philosophical, and social forces: anxieties about the French Revolution, foreign influences, or consumerism; dissatisfaction with Enlightenment rationality or women’s position in society; and worries about the preservation of middle-class sensibilities or the growing power of the lower classes, among others.This historical research allows us to situate gothic fiction in its contemporary context in several different ways, which is vital for my dissertation and scholarship on the gothic in general.

The plants for our experiments were artificially inoculated with race 1 or 2 isolates of F. oxysporum f. sp. fragariae using previously described protocols . The AMP132 isolate originated in California, whereas the MAFF727510 isolate originated in Japan . To produce spores, the pathogen was grown on potato dextrose agar or Kerr’s broth under continuous fuorescent lighting at room temperature, as previously described . Crude suspensions were passed through two layers of sterilized cheesecloth to remove hyphae. Spore densities were estimated using a haemocytometer and diluted with either sterile DI water or 0.1% water agar to a final density of 5 × 106 spores/ml. Seedling and bare-root plants were inoculated by submerging their root systems up to the crown in the spore suspension for 7–8 min prior to planting. The individuals in these studies were visually phenotyped for resistance to Fusarium wilt over multiple post-inoculation time points using an ordinal disease rating scale from 1 to 5 . For our field studies, individual plants were phenotyped once per week for four to eight consecutive weeks beginning in early June. Symptoms were observed on plants 26- to 36-weeks post-inoculation. For greenhouse and growth chamber studies, entries were phenotyped weekly for 6 to 12 weeks post-inoculation. For field, greenhouse,square black flower bucket and growth chamber experiments, the onset and progression of disease symptoms among resistant and susceptible checks were used as guides for initiating and terminating phenotyping.Our race 1 resistance screening experiments were conducted over a three year period at the UC Davis Plant Pathology Farm. The plants for these experiments were artiificially inoculated with the AMP132 isolate of the pathogen. Strawberries had not been previously grown in the fields selected for our studies.

The fields were tilled and disked prior to fumigation and were broadcast fumigated in October of each year with a 60:40 mixture of chloropicrin:1,3-dichloropropene at 560.4 kg/ha. The entire field was sealed with an impermeable plastic film for one-week post-fumigation before shaping 15.3 cm tall × 76.2 cm center-to-center raised beds. Sub-surface irrigation drip tape was installed longitudinally along the beds followed by black plastic mulch with a single row of planting holes spaced 30.5 cm apart. Artifcially inoculated plants were transplanted in mid-November both years. The fields were fertilized with approximately 198 kg/ha of nitrogen over the growing season and irrigated as needed to prevent water stress. For the 2016–17 field experiment, 344 germplasm accessions were screened for resistance to AMP132 and were part of a study that included 565 germplasm accessions developed at UC Davis, which is hereafter identified as the ‘California’ population. The resistance phenotypes for the latter were previously reported by Pincot et al. . Collectively, 981 germplasm accessions were screened in the 2016-17 field study. These were arranged in a square lattice experiment design with four single-plant replicates per entry . The experiment design and randomizations of entries within incomplete blocks were generated with the R package agricolae . For the 2017-18 and 2018- 19 field experiments, 144 ‘host diferential panel’ individuals were screened for resistance to AMP132 . These individuals were arranged in a 12 × 12 square lattice experiment design with four single-plant replicates per entry as described above. Guardian, Wiltguard, and Earliglow S1 and 61S016P006 S2 populations were screened for resistance to AMP132 in the 2016-17 field study. Ninety-nine Guardian S1 and 98 Wiltguard S1 individuals were phenotyped and genotyped and 85 Earliglow S1 and 77 61S016P006 S2 individuals were phenotyped. Nine-month-old S1 or S2 plants started as seedlings and asexually multiplied bare-root plants of the parents were artificially inoculated with AMP132, transplanted to the field in March 2018, and visually phenotyped weekly for six to 11 weeks post-inoculation.

The 12C089P002 × PI602575 , PI552277 × 12C089P002 , and PI612569 × 12C089P002 full-sib families, 17C327P010 S1 family, and parents of these families were screened for resistance to AMP132 in greenhouse experiments at UC Davis. Two- to four-month-old seedlings of the progeny and bare-root plants of the parents were artificially inoculated with AMP132 and planted in February 2019 , June 2019 , or November 2019 into 10.2 × 10.2 × 15.2 cm plastic pots filled with 3 parts coir : 1 part perlite and phenotyped weekly for six to 12 weeks post-inoculation. Four uninoculated and four inoculated single-plant replicates of the parents were arranged in completely randomized experiment designs. The plants were irrigated with a dilute nutrient solution as needed to maintain adequate soil moisture. The 12C089P002 × PI602575 and PI552277 × 12C089P002 populations were genotyped with a 50K Axiom SNP array .We screened a host diferential panel for resistance to the MAFF727510 isolate of Fof race 2 in a growth chamber at the UC Davis Controlled Environment Facility in 2018-19. Two single-plant replicates/individual were arranged in a randomized complete block experiment design. The entire experiment was repeated twice, resulting in four clonal replications/individual. The bare-root plants for these experiments were produced in high-elevation nurseries, preserved in cold storage, artificially inoculated with the MAFF727510 isolate, transplanted into 10.2 × 10.2 × 15.2 cm plastic pots filled with a 4 parts sphagnum peat moss : 1 part perlite , and phenotyped weekly for six to 12 weeks post-inoculation. The plants were grown under a 12-hour photoperiod with a 20 °C night temperature and 28 °C day temperature and irrigated with a dilute nutrient solution as needed to maintain adequate soil moisture. Because these experiments utilized a non-California isolate of the pathogen, the experiments were quarantined and conducted in compliance with federally-mandated bio-safety regulations .

DNA was isolated from newly emerged leaves harvested from field grown plants using a previously described protocol . Leaf samples were placed into 1.5 ml tubes or coin envelopes and freeze-dried in a Benchtop Pro . Approximately 0.2 g of dried leaf tissue/sample was placed into wells of 2.0 ml 96-well deep-well plates. Tissue samples were ground using stainless steel beads in a Mini 1600 . Genomic DNA was extracted from powdered leaf samples using the E-Z 96®Plant DNA Kit according to the manufacturer’s instructions. To enhance the DNA quality and yield and reduce polysaccharide carry-through, the protocol was modifed by adding Proteinase K to the lysis bufer to a final concentration of 0.2 mg/ml and extending lysis incubation to 45 min at 65 °C. Once the lysate separated from the cellular debris, RNA was removed by adding RNase A. The mixture was incubated at room temperature for 5 min before a final spin down. To ensure high DNA yields, the sample was incubated at 65 °C for 5 min following the addition of elution bufer. DNA quantification was performed using Quantifor dye on a Synergy HTX . The individuals phenotyped in these studies were genotyped with either 50K or 850K Axiom® SNP arrays . SNP markers on the 50K Axiom array are a subset of those on the 850K Axiom array. The probe DNA sequences for SNP markers on both arrays were previously physically anchored to the ‘Camarosa’ and ‘Royal Royce’ reference genomes . The ‘Camarosa’ genome assembly has been deposited in the Genome Database for the Rosaceae and Phytozome . The ‘Royal Royce’ genome assembly has been deposited in the Genome Database for the Rosaceae and Phytozome . The assemblies for each ‘Royal Royce’ haplotype have been deposited in a Dryad repository . The physical addresses for the SNP markers are provided in our online resources . We utilized both reference genomes as needed to cross-check and compare statistical findings and search genome annotations. The results presented in this paper utilized the haplotype-resolved ‘Royal Royce’ reference genome FaRR1 unless otherwise noted. SNP genotypes were called using the Affymetrix Axiom Suite . Samples with call rates exceeding 89-93% were included in genetic analyses. The haplotypes for 71 50K Axiom array-genotyped SNPs within a 1.60 Mb haploblock on chromosome 2B were imputed and phased using BEAGLE software version 5.3 for 651 individuals phenotyped for resistanceto Fusarium wilt race 1. The individuals were classified as resistant or susceptible ,square black flower bucket wholesale where ̄y is the estimated marginal mean for resistance score calculated from replicates. We ran BEAGLE with 10 burnin iterations for imputation and 25 subsequent iterations for phasing on sliding windows of 5 Mb with a window overlap of 2 Mb.The R package lme4 was used for linear mixed model analyses of the germplasm screening experiments . LMMs for square lattice experiment designs were analyzed with entries as fixed effects and incomplete blocks, complete blocks, years, entries × years, and residuals as random effects . LMMs for randomized complete block experiment designs were analyzed in parallel to estimate the relative efficiency of the square lattice to the randomized complete block experiment designs .

We did not observe an increase in efficiency by using incomplete blocks; hence, the statistics reported throughout this paper were estimated using LMMs for randomized complete block experiment designs . Estimated marginal means for entries were estimated using the R package emmeans . Genome-wide assocation study analyses were carried out to search for the segregation of loci affecting resistance Fusarium wilt races 1 and 2 among individuals genotyped with either the 50K or 850K Axiom SNP array . GWAS analyses were applied to estimated marginal means for resistance phenotypes using physical positions of SNP markers in the ‘Camarosa’ and ‘Royal Royce’ reference genomes . SNP marker genotypes were coded 1 for AA homozygotes, 0 for heterozygotes, and -1 for aa homozygotes, where A and a are the two SNP alleles. GWAS analyses were performed using the GWAS function in the R package rrBLUP. The genomic relationship matrix was estimated from SNP marker genotypes for each population using the rrBLUP A.mat function . The genetic structure of the GWAS population was investigated using hierarchical clustering and principal components analysis of the GRM as described by Crossa et al. . To correct for population structure and genetic relatedness, a Q + K linear mixed model was used where Q is the population stratification structure matrix and K is the GRM . The first three principal components from eigenvalue decomposition of the GRM were incorporated into the Q + K model. Bonferroni-corrected significance thresholds were calculated for testing the hypothesis of the presence or absence of a significant effect. GWAS was repeated in the California population by ftting a SNP marker in LD with FW1 as a fixed effect using the rrBLUP::GWAS function .SNP markers with ≤ 5% missing data, high quality codominant genotypic clusters, progeny genotypes concordant with parent gentoypes, and non-distorted segregation ratios were utilized for genetic and quantitative trait locus mapping analyses. The linkage phases of the SNP markers were not known a priori. The arbitrarily coded SNPs in the original data were in mixed coupling and repulsion linkage phases. The linkage phases of the SNP markers were ascertained using pair-wise recombination frequency estimates, and recoded so that the 100% of the SNP markers were in coupling linkage phase. This was only necessary in the S1 populations. The recoded SNP markers were genetically mapped in S1 populations using phase-known F2 mapping functions. SNP markers were genetically mapped in full-sib populations using phase-known back cross mapping functions from the subset of SNPs that were heterozygous in the resistant parent and homozygous in the susceptible parent. Genetic maps were constructed using the R packages onemap and BatchMap and custom PERL scripts for binning co-segregating SNP markers, calculating pairwise recombination frequencies, and grouping markers using LOD threshold of 10 and maximum recombination frequency threshold of 0.05. The custom PERL scripts are available in the Dryad repository for this paper . Linkage groups were aligned and assigned to chromosomes using inter-group linkage disequilibrium statistics and percent-identity against the reference genome . Marker orders and genetic distances were estimated in parallel using the RECORD algorithm in Batchmap with a 25-marker window, window overlap of 15 markers, and ripple window of six markers . For smaller linkage groups, the window size was reduced incrementally by five to ensure at least two overlapping windows. We used the checkAlleles, calc.errorlod, and top.errorlod functions of the R package qtl and custom R scripts to identify and eliminate spurious SNP markers and successively reconstruct linkage groups as described by Phansak et al. . Genetic distances were estimated from recombination frequencies using the Kosambi mapping function .