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Enteric pathogens navigate distinct regional microenvironments within the intestine that cue important adaptive behaviors. We investigated the response of Citrobacter rodentium, a model of human pathogenic Escherichia coli infection in mice, to regional gastrointestinal pH. We found that small intestinal pH (4.4-4.8) triggered virulence gene expression and altered cell morphology, supporting initial intestinal attachment, while higher pH, representative of C. rodentium's replicative niches further along the murine intestine, supported pathogen growth. Gastric pH, a key barrier to intestinal colonization, caused significant accumulation of intra-bacterial reactive oxygen species (ROS), inhibiting growth of C. rodentium and related human pathogens. Within-host adaptation increased gastric acid survival, which may be due to a robust acid tolerance response (ATR) induced at colonic pH. However, the intestinal environment changes throughout the course of infection. We found that murine gastric pH decreases postinfection, corresponding to increased serum gastrin levels and altered host expression of acid secretion-related genes. Similar responses following Salmonella infection may indicate a protective host response to limit further pathogen ingestion. Together, we highlight interlinked bacterial and host adaptive pH responses as an important component of host-pathogen coevolution.

Intestinal parasites are part of the intestinal ecosystem and have been shown to establish close interactions with the intestinal microbiota. However, little is known about the influence of intestinal protozoa on the regulation of the immune response. In this study, we analyzed the regulation of the immune response of germ-free mice transplanted with fecal microbiota (FMT) from individuals with multiple parasitic protozoans (P) and non-parasitized individuals (NP). We determined the production of intestinal cytokines, the lymphocyte populations in both the colon and the spleen, and the genetic expression of markers of intestinal epithelial integrity. We observed a general downregulation of the intestinal immune response in mice receiving FMT-P. We found significantly lower intestinal production of the cytokines IL-6, TNF, IFN-γ, MCP-1, IL-10, and IL-12 in the FMT-P. Furthermore, a significant decrease in the proportion of CD3+, CD4+, and Foxp3+ T regulatory cells (Treg) was observed in both, the colon and spleen with FMT-P in contrast to FMT-NP. We also found that in FMT-P mice there was a significant decrease in tjp1 expression in all three regions of the small intestine; ocln in the ileum; reg3γ in the duodenum and relmβ in both the duodenum and ileum. We also found an increase in colonic mucus layer thickness in mice colonized with FMT-P in contrast with FMT-NP. Finally, our results suggest that gut protozoa, such as Blastocystis hominis, Entamoeba coli, Endolimax nana, Entamoeba histolytica/E. dispar, Iodamoeba bütschlii, and Chilomastix mesnili consortia affect the immunoinflammatory state and induce functional changes in the intestine via the gut microbiota. Likewise, it allows us to establish an FMT model in germ-free mice as a viable alternative to explore the effects that exposure to intestinal parasites could have on the immune response in humans.

ABSTRACTQuorum Sensing (QS) is a form of cell-to-cell communication that enables bacteria to modify behavior according to their population density. While QS has been proposed as a potential intervention against pathogen infection, QS-mediated communication within the mammalian digestive tract remains understudied. Using an LC-MS/MS approach, we discovered that Citrobacter rodentium, a natural murine pathogen used to model human infection by pathogenic Escherichia coli, utilizes the CroIR system to produce three QS-molecules. We then profiled their accumulation both in vitro and across different gastrointestinal sites over the course of infection. Importantly, we found that in the absence of QS capabilities the virulence of C. rodentium is enhanced. This highlights the role of QS as an effective mechanism to regulate virulence according to the pathogen’s spatio-temporal context to optimize colonization and transmission success. These results also demonstrate that inhibiting QS may not always be an effective strategy for the control of virulence.

ABSTRACTEnterohemorrhagic Escherichia coli (EHEC) is a major cause of severe bloody diarrhea, with potentially lethal complications, such as hemolytic uremic syndrome. In humans, EHEC colonizes the colon, which is also home to a diverse community of trillions of microbes known as the gut microbiota. Although these microbes and the metabolites that they produce represent an important component of EHEC’s ecological niche, little is known about how EHEC senses and responds to the presence of gut microbiota metabolites. In this study, we used a combined RNA-Seq and Tn-Seq approach to characterize EHEC’s response to metabolites from an in vitro culture of 33 human gut microbiota isolates (MET-1), previously demonstrated to effectively resolve recurrent Clostridioides difficile infection in human patients. Collectively, the results revealed that EHEC adjusts to growth in the presence of microbiota metabolites in two major ways: by altering its metabolism and by activating stress responses. Metabolic adaptations to the presence of microbiota metabolites included increased expression of systems for maintaining redox balance and decreased expression of biotin biosynthesis genes, reflecting the high levels of biotin released by the microbiota into the culture medium. In addition, numerous genes related to envelope and oxidative stress responses (including cpxP, spy, soxS, yhcN, and bhsA) were upregulated during EHEC growth in a medium containing microbiota metabolites. Together, these results provide insight into the molecular mechanisms by which pathogens adapt to the presence of competing microbes in the host environment, which ultimately may enable the development of therapies to enhance colonization resistance and prevent infection.

Whole genome sequencing is emerging as a first-line test for rare genetic diseases. In this study, authors maximise diagnoses by supplementing existing semiautomated analyses with clinically driven reevaluation of genomic data by a specialist multidisciplinary team.

Our understanding of solar system evolution is closely tied to interpretations of asteroid composition, particularly the M-class asteroids. These asteroids were initially thought to be the exposed cores of differentiated planetesimals, a hypothesis based on their spectral similarity to iron meteorites. However, recent astronomical observations have revealed hydration on their surface through the detection of 3 μ m absorption features associated with OH and potentially H _2 O. We present evidence of hydration due mainly to OH on asteroid (16) Psyche, the largest M-class asteroid, using data from the James Webb Space Telescope (JWST) spanning 1.1–6.63 μ m. Our observations include two detections of the full 3 μ m feature associated with OH and H _2 O resembling those found in CY-, CH-, and CB-type carbonaceous chondrites, and no 6 μ m feature uniquely associated with H _2 O across two observations. We observe 3 μ m depths of between 4.3% and 6% across two observations, values consistent with hydrogen abundance estimates on other airless bodies of 250–400 ppm. We place an upper limit of 39 ppm on the water abundance from the standard deviation around the 6 μ m feature region. The presence of hydrated minerals suggests a complex history for Psyche. Exogenous sources of OH-bearing minerals could come from hydrated impactors. Endogenous OH-bearing minerals would indicate a composition more similar to E- or P-class asteroids. If the hydration is endogenous, it supports the theory that Psyche originated beyond the snow line and later migrated to the outer main belt.

A near-infrared spectrum of nova V1716 Scorpii (PNV J17224490-4137160), a recent bright ( ${V}_{\max }=7.3$ mag), Fermi-LAT detected γ -ray source, was modeled using the photoionization code CLOUDY. Abundances were estimated for He, C, N, O, Si, Al, Mg, Fe, Ne, S, Ca, and P. Notably, P (a factor of 120) and N (a factor of 248) are highly overabundant. It was necessary to assume the ejecta consist of two components (with a cylindrical geometry): a dense component from which the bulk of the H, He, and neutral O i and N emission arises and a more diffuse component from which most of the coronal lines arise. Some of the coronal lines are found to originate from both the dense and diffuse components. The mass of the ejecta, including neutral and ionized gas, is ≃4.19 × 10 ^−4 M _⊙ . Our analysis indicates that in the case of V1716 Sco (which has a carbon–oxygen white dwarf), a fraction of 25% white dwarf matter rather than 50% is favored for the mixing between a white dwarf and the accreted envelope before the outburst. This mixing ratio is like that found for oxygen–neon novae where a 25% mixing fraction is also indicated. Helium hydride—the first molecule to form after the Big Bang—may have formed in the ejecta of V1716 Sco based on photoionization modeling. This prediction suggests that novae may be potential formation sites of this important molecular ion.

We report on studies of classical nova (CN) explosions where we follow the evolution of thermonuclear runaways (TNRs) on oxygen–neon (ONe) white dwarfs (WDs). Using NOVA, a 1D hydrodynamic computer code, we accrete solar matter until the TNR is ongoing and then switch to a mixed composition. This approach is guided by the results of multidimensional studies of TNRs in WDs, which find that sufficient mixing with WD core material occurs after the TNR is well underway, and levels of enrichment of the CNONeMg elements are reached that agree with observations of CN ejecta abundances. Because the amount of accreted material is inversely proportional to the oxygen abundance, by first accreting solar matter, the amount of accreted material is larger than in those simulations with an initially enriched composition. We vary the mass of the WD (from 0.6 M _⊙ to 1.35 M _⊙ ) and the composition of the mixed materials. Our results show large enrichments of ^7 Be in the ejected gases, implying that ONe CNe and CO CNe may be responsible for a significant fraction (∼100 M _⊙ ) of the Galactic ^7 Li (∼1000 M _⊙ ). The production of ^22 Na and ^26 Al in CN explosions and the γ -ray emission predicted by our simulations are discussed. The WDs in all our simulations eject less material than they accrete and we predict that the WD is growing in mass as a consequence of the CN outburst. ONe CNe, therefore, may be an important channel for accretion-induced collapse events.

We report the detection of carbon monoxide (CO) and dust, formed under hostile conditions, in recurrent nova V745 Sco about 8.7 days after its 2014 outburst. The formation of molecules or dust has not been recorded previously in the ejecta of a recurrent nova. The mass and temperature of the CO and dust are estimated to be T _CO = 2250 ± 250 K, M _CO = (1–5) × 10 ^−8 M _⊙ , and T _dust = 1000 ± 50 K, M _dust ∼ 10 ^−8 –10 ^−9 M _⊙ , respectively. At the time of their detection, the shocked gas was at a high temperature of ∼10 ^7 K as evidenced by the presence of coronal lines. The ejecta were simultaneously irradiated by a large flux of soft X-ray radiation from the central white dwarf. Molecules and dust are not expected to form and survive in such harsh conditions; they are like snowflakes in a furnace. However, it has been posited in other studies that, as the nova ejecta plow through the red giant's wind, a region exists between the forward and reverse shocks that is cool, dense and clumpy where the dust and CO could likely form. We speculate that this site may also be a region of particle acceleration, thereby contributing to the generation of γ -rays.

Background. Studies suggest that early-life gut microbiota composition and intestinal short-chain fatty acids (SCFAs) are linked to future asthma susceptibility. Furthermore, infancy offers a critical time window to modulate the microbiota and associated metabolites through diet-microbe interactions to promote infant health. Human milk oligosaccharides (HMOs), nondigestible carbohydrates abundant in breast milk, are prebiotics selectively metabolized by gut microbiota that consequently modify microbiome composition and SCFA production. Methods. Using a house dust mite mouse model of allergy, we investigated the impacts of early oral treatment of pups with biologically relevant doses of 2′-fucosyllactose (2′-FL) and 6′-sialyllactose (6′-SL), two of the most abundant HMOs in human milk, in amelioration of allergic airway disease severity. Results. We found that administration of 2′-FL and 6′-SL during early life reduced lung histopathology scores, circulating IgE, cytokine levels, and inflammatory cell infiltration, all hallmark symptoms of allergic asthma. HMO supplementation also increased the relative abundance of intestinal Bacteroidetes and Clostridia, known SCFA producers within the gut. Indeed, we detected increased SCFA concentrations in both the intestine and blood of adult mice who received HMOs prior to weaning. Conclusion. We propose a model in which orally administered HMOs delivered during early life shift the microbiota toward increased production of SCFAs, which dampens the allergic immune responses behind allergy and asthma. Overall, these data suggest the potential for HMO supplementation to protect infants against asthma development later in life, with possible benefits against additional atopic diseases such as eczema and food allergies.

V445 Puppis, the only known Galactic helium nova, is a unique test bed to verify supernova (SN) theories in the single-degenerate channel that involve a white dwarf (WD) accreting matter from a helium-rich donor. An estimate of the mass of the helium shell on the WD is crucial to deciding whether or not it will undergo an SN detonation. In this context, this study estimates the dust and ejecta masses in the 2000 November eruption of V445 Pup. Subsequent to its outburst, the star became cocooned in a dust envelope. An analysis of the spectral energy distribution (SED) of the dust using infrared data shows that V445 Pup produced at least 10 ^−3 M _⊙ of dust, which is unprecedented for a classical or recurrent nova. The SED can be explained by a combination of a cold dust component at 105 ± 10 K, mass (1.9 ± 0.8) × 10 ^−3 M _⊙ , and a warm dust component at 255 ± 10 K, mass (2.2 ± 1.2) × 10 ^−5 M _⊙ . For a conservative choice of the gas-to-dust mass ratio in the range 10–100, the mass of the ejecta is 0.01–0.1 M _⊙ . Such a high mass range raises the question: why did V445 Pup not detonate as a Type Ia SN as is predicted in certain double-detonation sub-Chandrasekhar supernovae formalisms? We reexamine the nature of V445 Pup and discuss its role as a potential SN progenitor.

A number of fungi have been shown to have negative effects on plant-parasitic nematodes. Most of these fungi have been isolated from soil, plant roots, or nematodes themselves. Fungi associated with crops can provide a diverse pool of candidates to test for antagonistic effects against plant parasites and other stressors. We used a hierarchical two-tiered approach to evaluate the efficacy and repeatability of 55 strains of fungi originally isolated as foliar facultative endophytes from upland cotton (Gossypium hirsutum) along with one commercial isolate of Beauveria bassiana for in planta antagonistic effects on root-knot nematodes (Meloidogyne incognita). All fungi were inoculated to cotton using a seed treatment. The number of root galls was quantified 3 weeks after egg inoculation of cotton seedlings. The majority of the fungi tested reduced the number of root galls relative to those on untreated control plants. To assess repeatability, 22 strains that exhibited the strongest reductions in gall numbers were further tested in replicate follow-up assays. Ninety-five percent (21/22) of these retested strains significantly reduced galling in the follow-up assay. Strains that reduced galling the most belonged to the genera Alternaria, Chaetomium, Cladosporium, Diaporthe, Epicoccum, Gibellulopsis, and Purpureocillium. On the contrary, three strains in the genera Alternaria and Curvularia significantly increased gall numbers. Our results indicate that a large proportion of the fungal strains originally isolated from cotton as naturally occurring foliar facultative endophytes are capable of reducing root-knot nematode infection when applied back to the plant as a seed treatment. These findings help establish a rich pool of candidate fungi for further evaluation as novel biological control tools against root-knot nematodes in cotton and other plants.

As comets journey into the inner solar system, they deliver particulates and volatile gases into their comae that reveal the most primitive materials in the solar system. Cometary dust particles provide crucial information for assessing the physicochemical conditions in the outer disk from which they formed. Compared to the volatiles and soluble organics, the refractory dust particles are more robust and may be traceable to other small bodies. Using data from the Spitzer Heritage Archive, we present thermal dust models of 57 observations of 33 comets observed spectroscopically with the NASA Spitzer Space Telescope. This comet spectral survey offers the opportunity to study comets with data from the same instrument, reduced by the same methods, and fitted by the same thermal model using the same optical constants. The submicron dust tends to be dominated by amorphous carbon, and the submicron silicate mass tends to be dominated by amorphous silicate materials. We discuss the implications of these findings as they relate to Mg-rich crystalline silicates, which are high-temperature condensates, as well as to potential ion irradiation of amorphous Mg:Fe silicates prior to their incorporation into comets. These results impact our understanding of the protoplanetary disk conditions of planetesimal formation. Lastly, we cannot definitively conclude that a distinct difference exists in the dust composition between the Oort cloud and Jupiter-family comet dynamical population as a whole.

JWST Near Infrared Camera (NIRCam) observations at 1.5–4.5 μ m have provided broadband and narrowband imaging of the evolving remnant of SN 1987A with unparalleled sensitivity and spatial resolution. Comparing with previous marginally spatially resolved Spitzer Infrared Array Camera (IRAC) observations from 2004 to 2019 confirms that the emission arises from the circumstellar equatorial ring (ER), and the current brightness at 3.6 and 4.5 μ m was accurately predicted by extrapolation of the declining brightness tracked by IRAC. Despite the regular light curve, the NIRCam observations clearly reveal that much of this emission is from a newly developing outer portion of the ER. Spots in the outer ER tend to lie at position angles in between the well-known ER hotspots. We show that the bulk of the emission in the field can be represented by five standard spectral energy distributions, each with a distinct origin and spatial distribution. This spectral decomposition provides a powerful technique for distinguishing overlapping emission from the circumstellar medium and the supernova ejecta, excited by the forward and reverse shocks, respectively.

Bacterial members of the infant gut microbiota and bacterial-derived short-chain fatty acids (SCFAs) have been shown to be protective against childhood asthma, but a role for the fungal microbiota in asthma etiology remains poorly defined. We recently reported an association between overgrowth of the yeast Pichia kudriavzevii in the gut microbiota of Ecuadorian infants and increased asthma risk. In the present study, we replicated these findings in Canadian infants and investigated a causal association between early life gut fungal dysbiosis and later allergic airway disease (AAD). In a mouse model, we demonstrate that overgrowth of P. kudriavzevii within the neonatal gut exacerbates features of type-2 and -17 inflammation during AAD later in life. We further show that P. kudriavzevii growth and adherence to gut epithelial cells are altered by SCFAs. Collectively, our results underscore the potential for leveraging inter-kingdom interactions when designing putative microbiota-based asthma therapeutics.

ABSTRACT Nonalcoholic fatty liver disease (NAFLD), largely studied as a condition of overnutrition, also presents in undernourished populations. Like NAFLD, undernutrition disrupts systemic metabolism and has been linked to gut microbiota dysbiosis. Indeed, chronic exposures to fecal microbes contribute to undernutrition pathology in regions with poor sanitation. Despite a growing prevalence of fatty liver disease, the influence of undernutrition and the gut microbiota remain largely unexplored. Here, we utilize an established murine model (C57BL/6J mice placed on a malnourished diet that received iterative Escherichia coli/Bacteroidales gavage [MBG mice]) that combines a protein/fat-deficient diet and iterative exposure to specific, fecal microbes. Fecal-oral contamination exacerbates triglyceride accumulation in undernourished mice. MBG livers exhibit diffuse lipidosis accompanied by striking shifts in fatty acid, glycerophospholipid, and retinol metabolism. Multiomic analyses revealed metabolomic pathways linked to the undernourished gut microbiome and hepatic steatosis, including phenylacetate metabolism. Intriguingly, fatty liver features were observed only in the early-life, but not adult, MBG model despite similar liver metabolomic profiles. Importantly, we demonstrate that dietary intervention largely mitigates aberrant metabolomic and microbiome features in MBG mice. These findings indicate a crucial window in early-life development that, when disrupted by nutritional deficiency, may significantly influence liver function. Our work provides a multifaceted study of how diet and gut microbes inform fatty liver progression and reversal during undernutrition. IMPORTANCE Nonalcoholic fatty liver disease (NAFLD) remains a global epidemic, but it is often studied in the context of obesity and aging. Nutritional deficits, however, also trigger hepatic steatosis, influencing health trajectories in undernourished pediatric populations. Here, we report that exposure to specific gut microbes impacts fatty liver pathology in mice fed a protein/fat-deficient diet. We utilize a multiomics approach to (i) characterize NAFLD in the context of early undernutrition and (ii) examine the impact of diet and gut microbes in the pathology and reversal of hepatic steatosis. We provide compelling evidence that an early-life, critical development window facilitates undernutrition-induced fatty liver pathology. Moreover, we demonstrate that sustained dietary intervention largely reverses fatty liver features and microbiome shifts observed during early-life malnutrition.

The understanding of molecular biology in neurocritical care (NCC) is expanding rapidly and recognizing the important contribution of neuroinflammation, specifically changes in immunometabolism, towards pathological disease processes encountered across all illnesses in the NCC. Additionally, the importance of individualized inflammatory responses has been emphasized, acknowledging that not all individuals have the same mechanisms contributing towards their presentation. By understanding cellular processes that drive disease, we can make better personalized therapy decisions to improve patient outcomes. While the understanding of these cellular processes is evolving, the ability to measure such cellular responses at bedside to make acute care decisions is lacking. In this overview, we review cellular mechanisms involved in pathological neuroinflammation with a focus on immunometabolic dysfunction and review non-invasive bedside tools that have the potential to measure indirect and direct markers of shifts in cellular metabolism related to neuroinflammation. These tools include near-infrared spectroscopy, transcranial doppler, elastography, electroencephalography, magnetic resonance imaging and spectroscopy, and cytokine analysis. Additionally, we review the importance of genetic testing in providing information about unique metabolic profiles to guide individualized interpretation of bedside data. Together in tandem, these modalities have the potential to provide real time information and guide more informed treatment decisions.

The molecular basis of cytochrome c oxidase (COX, complex IV) deficiency remains genetically undetermined in many cases. Homozygosity mapping and whole-exome sequencing were performed in a consanguineous pedigree with isolated COX deficiency linked to a Leigh syndrome neurological phenotype. Unexpectedly, affected individuals harbored homozygous splice donor site mutations in NDUFA4, a gene previously assigned to encode a mitochondrial respiratory chain complex I (NADH:ubiquinone oxidoreductase) subunit. Western blot analysis of denaturing gels and immunocytochemistry revealed undetectable steady-state NDUFA4 protein levels, indicating that the mutation causes a loss-of-function effect in the homozygous state. Analysis of one- and two-dimensional blue-native polyacrylamide gels confirmed an interaction between NDUFA4 and the COX enzyme complex in control muscle, whereas the COX enzyme complex without NDUFA4 was detectable with no abnormal subassemblies in patient muscle. These observations support recent work in cell lines suggesting that NDUFA4 is an additional COX subunit and demonstrate that NDUFA4 mutations cause human disease. Our findings support reassignment of the NDUFA4 protein to complex IV and suggest that patients with unexplained COX deficiency should be screened for NDUFA4 mutations.

Mutations in the nuclear gene POLG (encoding the catalytic subunit of DNA polymerase gamma) are an important cause of mitochondrial disease. The most common POLG mutation, A467T, appears to exhibit considerable phenotypic heterogeneity. The mechanism by which this single genetic defect results in such clinical diversity remains unclear. In this study we evaluate the clinical, neuropathological and mitochondrial genetic features of four unrelated patients with homozygous A467T mutations. One patient presented with the severe and lethal Alpers-Huttenlocher syndrome, which was confirmed on neuropathology, and was found to have a depletion of mitochondrial DNA (mtDNA). Of the remaining three patients, one presented with mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS), one with a phenotype in the Myoclonic Epilepsy, Myopathy and Sensory Ataxia (MEMSA) spectrum and one with Sensory Ataxic Neuropathy, Dysarthria and Ophthalmoplegia (SANDO). All three had secondary accumulation of multiple mtDNA deletions. Complete sequence analysis of muscle mtDNA using the MitoChip resequencing chip in all four cases demonstrated significant variation in mtDNA, including a pathogenic MT-ND5 mutation in one patient. These data highlight the variable and overlapping clinical and neuropathological phenotypes and downstream molecular defects caused by the A467T mutation, which may result from factors such as the mtDNA genetic background, nuclear genetic modifiers and environmental stressors.

Field studies were conducted in 2013 and 2014 in south Texas near Yoakum and from 2008 to 2011 in central Texas near Stephenville to evaluate various fungicides for foliar and soilborne disease control as well as peanut yield response under irrigation. Control of Sclerotinia blight caused by Sclerotinia minor Jagger with penthiopyrad at 1.78 L/ha was comparable to fluazinam or boscalid; however, the 1.2 L/ha dose of penthiopyrad did not provide consistent control. Peanut yield was reduced with the lower penthiopyrad dose when compared with boscalid, fluazinam, or the high dose of penthiopyrad. Control of early leaf spot, caused by Cercospora arachidicola S. Hori or southern blight, caused by Sclerotium rolfsii Sacc., with penthiopyrad in a systems approach was comparable with propiconazole, prothioconazole, or pyraclostrobin systems and resulted in disease control that was higher than the nontreated control. Peanut yield was also comparable with the penthiopyrad, propiconazole, prothioconazole, or pyraclostrobin systems and reflects the ability of the newer fungicides to control multiple diseases found in Texas peanut production.

Field studies were conducted from 2007 to 2010 to evaluate the response of peanut cultivars to different fungicides, application timings, and methods. Overall, fungicides reduced Sclerotinia blight incidence and increased pod yields when applied to susceptible and partially resistant cultivars. Disease suppression was greater when full fungicide rates were applied preventatively; however, yields between fungicide treated plots were similar. Lower levels of disease and higher yields were achieved with the partially resistant cultivar Tamrun OL07 compared to the susceptible cultivars Flavor Runner 458 and Tamrun OL 02. Despite possessing improved resistance Tamrun OL07 responded to all fungicide applications. While similar levels of disease control were achieved with broadcast or banded applications made during the day or at night, the yield response for the different application methods was inconsistent among years. A negative relationship (slope = −73.8; R2=0.73; P