Month: February 2025

There is precedent in the human cognitive neuroscience literature suggesting noisier representations in aging underlie performance decrements. Using fMRI, Samanez-Larkin et al. revealed that in older adults, greater temporal variability in nucleus accumbens BOLD response was directly related with suboptimal performance on a financial risk-taking task. Variability has been shown to be reproducible within subject across time and tasks , and has been linked to individual differences in dopamine . In aging, declines in D1 receptors have been associated with increased intraindividual variability in reaction times during an executive function task . Generally, there is consensus that intraindividual variability in neural and behavioral responses is a valuable measure for understanding the underlying neural basis of impaired performance , though it is important to note that in some cases neural variability may be beneficial . One central component of this line of research will be to distinguish between variability that is driven by imprecision in signaling versus variability that simply reflects a healthy dynamic range in neural responses that are not muted by, for example, disease processes. Important research by Garrett, Kovacevic, McIntosh, and Grady has demonstrated that, overall, procona buckets older adults show less variability in BOLD signal than younger adults, and that this reduced variability is associated with poorer cognitive performance.

Interestingly, they found subcortical structures including hippocampus and regions in the striatum that were more variable in older adults than young adults . Together, these findings suggest variability in subcortical responses may be an important, age-sensitive measure in human imaging studies that warrants further investigation to establish possible relationships with dopamine. For example, future studies could test whether individuals with highly variable cortical responses also have highly variable subcortical response, or whether these are dissociable measures potentially reflecting different underlying sources.PET imaging provides spatial information that allows for the assessment of regional differences in dopamine function. This provides unique opportunities to test hypotheses about the differential influence of region-specific measures of dopamine on discrete cognitive operations. For example, PET can be useful for specifying a role of PFC dopamine signaling in decision-making. Spatial information may be particularly relevant for studying dopaminergic mechanisms of decision-making in aging as there is growing evidence that dopamine receptor densities may decline at different rates across the brain . Here, we describe ways in which the spatial information afforded by PET imaging can be leveraged to probe the role of dopamine in agerelated changes in reliance on striatal versus extrastriatal brain regions during decision making. Evolving research has developed our understanding of complementary learning mechanisms that may trade off or interact with model-free reinforcement learning processes to affect decision-making.

These include, but are not limited to, processes for the building of deliberative internal models to guide choices , as well as processes for learning and planning that more heavily rely on prefrontal processes including working memory or medial temporal lobe episodic memory . The degree to which these processes interact, and the nature of these interactions is an area of active research . However, the multiprocess view of decision-making incorporates roles for multiple neural systems that include frontoparietal, medial temporal lobe, and limbic structures. There is general agreement that most value-based decision-making tasks accommodate multiple strategies. In some cases, these strategies can be distinguished from one another using computational modeling approaches . These lines of research have revealed profound individual differences in the extent to which people adopt one strategy over another. Age differences in the adoption of task strategy has been identified as a critical factor in considering discrepant findings in the aging literature. For example, older adults may show better or worse performance than young adults depending on whether a given task favors win-stay lose-shift strategies . Research examining how reliance on specific strategies is affected by regional differences in dopamine function is only beginning, but holds promise for informing findings in aging. Influential learning models have distinguished between model-free processes and model based processes, which can be dissociated from one another computationally using the two state Markov decision task . Briefly, this task involves two decision phases . In the first step, participants choose between two stimuli, and this selection determines a second set of choices with differing reward probabilities. Similar to single phase tasks, learning can occur slowly and incrementally via model-free mechanisms. Task performance may also rely on goal-directed, model-based strategies, for which subjects develop an internal model of the task structure. At the first decision phase, subjects may prospectively consider future reward probabilities that would occur after the second phase.

This strategy is more computationally demanding than model-free strategies, but supports rapid and flexible learning. While links between model-free reinforcement learning and striatal dopamine have been long established, model-based strategies are also powerfully modulated by dopamine. Pharmacological manipulations provide general evidence that elevating dopamine tone increases reliance on model-based processes, though do not give information about the spatial specificity of dopamine’s role. Enhancing dopamine in young adults shifts bias in task strategy toward model-based processes . Consistent with this, Parkinson’s disease patients tested off medication show selective impairment in model-based learning that is remediated by dopaminergic medication . Complementing these findings in Parkinson’s patients, model-based processing is reduced in people with disorders characterized by alteration in dopamine function such as addiction , obsessive compulsive disorder , and schizophrenia . To date, there has been only limited investigation of the spatial specificity of dopamine’s influence on model-based learning. Dopamine may modulate PFC via direct inputs from the ventral tegmental area or by indirect effects in striatum where dopamine affects PFC function via fronto-striato-thalamic loops . Pointing to a role of striatal dopamine, PET evidence in healthy young subjects demonstrated greater striatal dopamine synthesis capacity was associated with preferential reliance on model-based learning and was correlated with fMRI activation in PFC . Pointing to a role of PFC dopamine, people with genetically inferred reductions in COMT enzymatic activity show greater reliance on model-based processes . Additional research is needed to understand how direct dopamine signaling in PFC may influence model-based strategies.Though model-based processing has been linked to striatal activity , it is also associated with increased reliance on prefrontal systems. Supporting this view, higher working memory capacity is associated with greater propensity to engage model-based strategies . Further, manipulations that increase cognitive load , stress , or perturb PFC function via rTMS reduce reliance on model-based processing, particularly in participants with low working memory capacity. In aging, there is evidence of reduced reliance on model-based processing . These findings are generally consistent with age-related changes in PFC function though, somewhat perplexingly, propensity to adopt model-based rather than model-free appears to be dissociated from working memory capacity . Other studies have identified subgroups of older adults demonstrating an over reliance on model-free learning . However, the extent to which age-related shifts from model-based to model-free learning mechanisms are explained by alterations in PFC dopamine function is unknown. Accounting for alteration in dopamine function in PFC and striatum may be valuable for understanding the neural basis of shifts away from model-based processes in aging. In aging, loss of PFC D2/3 dopamine receptors may outpace losses in ventral striatum . Further, rates of D1 receptor losses may differ between nigrostriatal pathways that innervate dorsal striatum versus mesocortical/mesolimbic pathways that innervate PFC and ventral aspects of striatum, respectively . Together, such changes may alter the weighting of PFC versus striatal influences on task performance and the coherence of dorsal versus ventral striatal contributions to processes for learning, procona florida container updating and integration that support value-based decision-making. Future studies pairing task performance with neurochemical PET measures could directly test how individual differences in receptor densities and dopamine release in PFC versus ventral striatum influence variability on the adoption of model-based versus model-free strategies in aging.

Such studies may identify those subgroups of older adults most reliant on model-free processes are those with most marked reductions in PFC dopamine measures. Such lines of research are ripe for cross-species comparisons which could assess relationships between age and performance while providing critical information as to the temporal dynamics of dopamine signaling in PFC versus striatum using microdialysis techniques and voltammetry. Together, such studies hold promise for promoting our basic understanding of PFC dopamine’s involvement in goal-directed decision-making while characterizing age differences in the recruitment of distinct dopamine pathways underlying specific task strategies.Models of complex decision-making support the view that multiple systems for learning, memory, and attention interact to shape our choices. We have argued that in vivo dopamine imaging is a powerful tool for understanding the neural basis of individual differences in performance in aging. This line of research can illuminate the extent to which neurochemical traits affect decision-making. However, it is important to also consider how neurochemical influences on behavior interact with other attentional or state measures to affect decision making. A recent study by Kircanski et al. illustrates the mutual influence of these factors on value-based choices. First, they modified the monetary incentive delay task by including trials in which participants unexpectedly gained or lost relatively large amounts of money . They found monetary loss and gain modifications induced negative and positive affective arousal respectively, which they assessed with self-report rating scales. Following the affective arousal manipulation, participants performed a separate decision-making task in which they chose whether or not to purchase items with misleading advertisements. They found deficits in choice performance following both arousal conditions relative to a neutral condition. Together, these findings demonstrate the capacity for financial reward and punishment manipulations to impact self-reported affective state, which in turn modulates the quality of subsequent value-based decisions. This study found no evidence for differences in the induction of arousal between young and older adults or in the detrimental effect of arousal on later choice performance. Other studies have shown differential age effects such that following positive affect induction , older adults, but not young adults, are more likely to make risky decisions than when in neutral states . In the following section, we describe possible systematic changes in affective attention over the life span that may influence decision-making performance. We consider how these attentional changes may interact with influences of dopamine on performance, and suggest strategies for empirically examining these interactions. Older adults report reduced levels of negative affective experience as they age, but preserved levels of positive experience . These observations have been linked specifically to memory and attention where older adults are more likely to remember positive events than young adults . It is possible that there are age-related changes in neurophysiology and neurochemistry that drive these effects, though there has been little direct investigation of this possibility model first described by Mather, Clewett, Sakaki, & Harley, 2016). Functionally, there appears to be relative preservation of the networks supporting emotional processing in aging , though in some cases the engagement of these networks by older adults may be more effortful . The dominant interpretation is that at the end of life there is a shift in motivational goals derived from changes in the perception of time horizons . This view is supported by evidence that positivity effects have been reported in young people diagnosed with terminal illness, and people on death row, where systematic neurophysiologic and neurochemical changes mirroring those that occur in aging are unlikely . Further, positivity can be enhanced in young subjects experimentally in task scenarios in which they are encouraged to think about a limited future . The positivity effect has been implicated in fMRI findings that responses associated with reward anticipation are intact in aging, but that responses associated with the anticipation of monetary losses in the insula are muted in older adults . Other studies have examined relationships between subjective ratings of emotional stimuli and fMRI responses in PFC and amygdala. Behaviorally, older adults report lower arousal for negative stimuli than young adults , report less unpleasantness for “lowarousal” negative stimuli, and greater pleasantness for “low-arousal” positive stimuli in a study that manipulated the levels of stimulus valence . These behavioral differences are accompanied by age-group differences in amygdala responsivity. Older adults consistently show greater enhancement of amygdala activation for positive relative to negative stimuli . Further, there is evidence that the relative suppression of amygdala activation for negatively valenced stimuli in aging is associated with greater recruitment of rostral anterior cingulate cortex when viewing unpleasant stimuli .

Our metagenomic data analysis predicts genes in GB-CYN1 involved in the Calvin cycle, TCA cycle, and photosystems I and II . Sequence identities for these genes in GB-CYN1 and published Chroococcales genomes were high, ranging from 75 to 99% . The observation of photosystem II genes in GB-CYN1 indicates the metabolic potential for oxygenic photosynthesis, unlike UCYN-A which lacks photosystem II, RuBisCo and the TCA cycle. These findings confirm that GB-CYN1 resembles free-living Chroococcales of the clades B and C rather than the metabolically streamlined, endosymbiotic UCYN-A clade . Related Chroococcales species are known to produce copious quantities of exopolysaccharides , and EPS produced by Crocosphaera watsonii has been linked to the formation of transparent exopolymeric particles in pelagic environments . A cassette of genes required for EPS production has been identified by comparative genomic analysis of related Crocosphaera watsonii strains ; however, we did not recover homologs of these genes in our metagenomic sequence . We conclude that the absence of these sequences is most likely due to our incomplete sampling of the GB-CYN1 genome, though it could also indicate either an unknown pathway for EPS synthesis in GB-CYN1, or an alternate source of the green berry exopolymeric matrix . Consistent with the dynamic oxygen conditions within the green berries, plastic plant pot we found sequences in the metagenomic data that suggest the metabolic potential for aerobic and anaerobic respiration, and anaerobic fermentative pathways assigned to several different phyla of bacteria .

Abundant fixed nitrogen from diazotrophy and transient anoxia presents an ecological opportunity for dissimilatory nitrogen metabolism, a process that could cause concomitant nitrogen fixation and loss over rapid spatiotemporal scales. Indeed, previous studies of diazotrophic Nodularia spumigena aggregates measured significant rates of both nitrification and denitrification . We specifically investigated potential marker genes for dissimilatory nitrogen metabolism within the green berries. Metagenomic sequences homologous to the nitrite reductase gene nirK were found in six unassembled reads and on a corresponding 753 bp long contig . A single read homologous to nitrous oxide reductase nosZ was also found suggesting the presence of a denitrification pathway . The top database matches to these nirK and nosZ sequences belonged to marine phytoplankton epiphytes from the Alphaproteobacteria and Flavobacterales . In the case of the nirK metagenomic sequences, the best match was to Roseibium sp. TrichSKD4, an alphaproteobacterial species isolated from a nitrogen-fixing Trichodesmium aggregate in the Atlantic Ocean . Homologs to the napA periplasmic nitrate reductase and the nirB nitrite reductase were identified from six and five unassembled reads, respectively, and were most similar to database sequences from marine heterotrophs in the Alphaproteobacteria and Gammaproteobacteria . We did not detect sequences supporting the presence of nitrifying bacteria or archaea, though given our limited sequence depth, this could be a function of missing data.

Cyanobacterial nitrogen fixation in the global oceans is frequently aggregate-associated, as with Trichodesmium sp. colonies and rafts , filamentous heterocystous cyanobacterial colonies , or Crocosphaera watsonii associated with TEP . The green berries of the Sippewissett Salt Marsh are nitrogen-fixing macroscopic consortia of unicellular cyanobacteria , diatoms and heterotrophic bacteria. While nitrogen and carbon fixation mediated by the green berries is unlikely to play a major role in the overall marsh ecosystem due to their low abundance and patchy distribution , these consortia provide an interesting comparative system to investigate the dynamics nitrogen flux within diazotrophic cyanobacterial aggregates. More broadly, studies of coastal marine estuarine sediments have indicated complex dynamics and close spatial coupling of co-occurring nitrogen fixation and denitrification processes . In other oceanic diazotrophic cyanobacterial aggregates, similarly rapid respiratory rates create transient anoxic zones within the aggregate core , and create a heterogeneous microenvironment where both aerobic and anaerobic metabolisms co-exist. This parallel is not merely conceptual: the green berry heterotrophic bacteria were similar, both phylogenetically and in their metabolic marker genes, to those observed to colonize other marine phytoplankton aggregates. The recovery of denitrification marker genesequences in metagenome suggests that there might be heterotrophic denitrifiers in the green berries with the potential to exploit this suboxic niche. However, further studies including rate measurements and better genome reconstructions are needed to clarify the importance of denitrification in the berries. The presence of such a pathway for nitrogen loss in the green berries is speculative, given the fragmented metagenome and absence of activity measurements, but remains an interesting direction for future work.

The existence of closely coupled nitrogen cycling within diazotrophic cyanobacterial aggregates has been explored previously, though initial studies demonstrating the association of heterocystous cyanobacterial aggregates with key bacterial species and marker genes for nitrification and denitrification measured only low to negligible rates . However, more recent investigations of in vitro rates and in situ marker gene transcription indicates that denitrification within anoxic cyanobacterial aggregates could contribute significantly to nitrogen loss in hypoxic waters, where bulk oxygen concentrations would normally inhibit such activity . In the light of modeling studies showing tight spatiotemporal coupling of nitrogen fixation and denitrification processes in the global ocean , we postulate that this coupling may be occurring at the microscale within ephemeral, aggregate associated niches. The potential for such interactions emphasizes the need to examine bio-geochemical cycles from the microbial perspective in the spatially heterogeneous marine environment.Green berries were sampled in June and July of 2010 or July and August 2014 from a single intertidal pool formed in the Little Sippewissett Salt Marsh, Falmouth, MA United States . These aggregates, however, have also been observed at other locations throughout Little and Great Sippewissett marshes. Berries were collected from the sediment-water interface by sieving and were washed three times in 0.2 µm filter sterilized marsh water. Imaging of the berries was conducted using a Zeiss Axio IMAGER MZ epifluorescence microscope equipped with a color camera and a Zeiss LSM 710 spectral confocal scanning laser microscope .The MG-RAST 3.3 pipeline was used to classify unassembled metagenomic sequence reads as ribosomal RNA and protein coding sequences using the M5RNA and M5NR databases to provide a diversity description shown in Figure 2 . Metagenomic sequence data was mined using MG-RAST. Functions of interest were mined using the hierarchical function assignment and were assigned to GB-CYN1 when the best hits were to sequenced Chroococcales genomes. Marker genes were further investigated via profile HMM and BLASTX searches . PCR amplified SSU rRNA gene sequences were aligned to the SILVA 115 database using SINA and curated using ARB . 16S rRNA phylogenies were inferred using the GTRGAMMA rate approximation. For functional genes of interest that were found to have frame shift sequencing errors , the sequences were corrected using Framebot . The metagenomic nifH amino acid sequence was aligned to references sequences from the nifH database available from the Zehr research group using the ARB software package . ProtTest3 was used to select the fixed WAG model of amino acid evolution with an inverse gamma rate approximation for the nifH phylogeny, plastic planter pot though similar topologies were recovered using related models. Phylogenetic reconstruction for both 16S rRNA and nifH genes were conducted as follows: maximum likelihood phylogeny was constructed with RAxML 7.2.8 with 1000 rapid bootstrap inferences and Bayesian phylogeny with MrBayes 3.3 . For Bayesian tree inferences, MCMC was run with default parameters and convergence was assessed when the reported average standard deviation of split frequencies fell below 0.01. Hidden Markov model profiles of 40 phylogenetic marker genes for Bacteria and Archaea were used to search the green berry metagenomic peptide sequence database using the trusted cutoffs. For each of the 34 markers that have green berry hits, green berry peptide sequences were aligned with all the bacterial and archaeal references sequences by hmmalign in HMMER3 . A maximum likelihood tree was built by Fastree2 for each alignments, and the alignments and trees were examined. As a result, 33 green berry peptide sequences were selected for further analysis because they branched with Cyanobacteria with good alignments. These sequences were further filtered to include only those 29 phyeco gene families with a single copy, cyanobacterial green berry hit. Single-copied reference sequences from 126 Cyanobacteria genomes of the 29 markers and the green berry cyanobacterial metagenomic sequences were retrieved from the alignments built in the previous step and were concatenated into a large alignment. A maximum likelihood tree was built using PHYML3.0 with the LG substitution model .

Tree topology and branch lengths were optimized by the program and SH statistics was used for branch support estimation.Whole-aggregate in vivo acetylene reduction assays were conducted on two 30 mL serum bottles of five washed green berries each. Acetylene, generated by the hydration of calcium carbide in an evacuated 150 mL serum-vial, was added to 10% of the headspace in a 30 mL serum-vial containing the berries in 5 mL of anoxic 0.2 µm filtered in situ marsh water under an 90% N2-CO2 atmosphere . Experiments were incubated on a 14 h light, 10 h dark cycle with full spectrum illumination at 30◦C for 2 days. Acetylene and ethylene were quantified using a Varian 2400 series gas chromatograph with an H2 flame ionization detector, as described previously . Rates were calculated over the 2 days incubation period with the assumption that nitrogen fixation was restricted to the dark hours of incubation, as previously described for unicellular cyanobacteria .Surface mulches are widely used in the production of strawberries and certain high value vegetable crops. Polyethylene mulch is used on virtually all tomato and strawberry production in Florida and is also widely used in the production of other crops such as peppers, eggplant, and melons throughout much of the southern United States. Researchers at the University of Florida estimate that more than 100,000 acres of vegetable crops in that state currently use plastic mulches annually, making Florida the national leader in this production system . In California, the majority of strawberry and staked tomato production uses polyethylene mulch. Peppers, eggplant, and melons also use mulches in certain situations, especially when earliness is desired. Field management and research related to plastic mulches in these production regions is now quite developed. Potential benefits as well as drawbacks of polyethylene mulches for vegetable crop production are given in table 1. The use of inter crop cover crop residues as surface mulches is a more recent and far less widely used production practice. It has recently gained considerable interest in a number of commercial vegetable crop production regions in the United States. The use of organic mulches in smaller-scale gardening contexts has had a very long history. Potential advantages and disadvantages of this vegetable crop production technique are summarized in table 2. Reflective plastic and some cover crop mulches share similar features relative to crop production: insect and disease management, weed management, fertilizer availability, and water conservation. In order for production practices using either polyethylene or cover crop mulches to be successfully adopted in California, specific production goals must be carefully matched with specialized management know how and experience.Plastic mulches have been commonly used for commercial vegetable crop production for more than 30years. Excellent sources of information on specific characteristics and applications of plastic mulches are available on the World Wide Web, and several of these are provided in the references to this publication. Most plastic mulch materials are made of either high- or low-density polyethylene, ranging from 0.30 to 0.79 inches in thickness, are 5 to 6 feet wide, and are available on rolls 555 to 1,338 yards long, depending on the thickness of the mulch . Black, white, and clear plastic mulches are most commonly used in commercial production, with black being the dominant color used for vegetables. Black plastic mulch is typically used for spring-seeded crops because it increases soil temperatures about 5ºF at a depth of 2 inches and 3ºF at 4 inches , compared to those of bare soil . Recently, a mulch has been introduced that consists of a strip of black plastic 30 cm wide down the center that is flanked on either side by metalized reflective plastic. This plastic combines the advantages of black plastic over the seed row, to help heat the soil, with the reflective characteristics of metalized plastic for insect and disease management. Black mulches have also recently been shown to reduce weed growth.

The dietary intake of Z is lower than L in all age groups and ethnicities in the U.S.. Dietary intakes of L and Z are strongly associated with their serum levels, as well as with MPOD. Previous studies have shown that high intakes of these carotenoids from dietary sources or supplements can increase plasma L and Z, and MPOD. Once early AMD has progressed to the intermediate stage, dietary supplements are indicated, but no clinical evidence yet exists for interventions thatcan address the prevention of small-intermediate drusen with pigmentary changes, the initial clinical signs of macular disruption . Goji berry , also termed wolfberry or Go Chi Zi, has been used in traditional Chinese medicine for more than 2000 years. The bright red berry contains the highest amount of Z among all known dietary sources and is mainly present in a dipalmitate form. The intake of zeaxanthin dipalmitate extracts from goji berry increases plasma Z to a greater extent than non-esterified Z supplementation. The berries also contain unique carbohydrates that are present as conjugates with peptides or proteins, which are often referred to L. barbarum polysaccharides . These have shown anti-inflammatory and neuroprotective effects in animal and cell culture studies. The typical adult human eye has approximately 2.4 times more Z than L in the central fovea of the macula, plastic pots large making goji berry intake a prime candidate for increasing MPOD.

Nevertheless, there is a paucity of clinical evidence on goji berry and MPOD particularly for the prevention or delay of progression from early to intermediate AMD. In individuals from China with signs of early AMD, 25 g of daily consumption of goji berries for 90 days significantly increased both serum Z and MPOD. However, this study had a broad age range , some participants smoked, and others had certain preexisting medical conditions. Additionally, the authors only reported central MPOD values up to 0.5 retinal eccentricity , whereas macular pathology and visual dysfunction in AMD may extend beyond that central region. Therefore, to provide a more complete understanding of the influence of goji berry intake on the progression AMD, data is needed on for different population groups that measures MPOD at eccentricities over the entirety of the macula. In the current study, we prospectively evaluated if the daily intake of 28 g of goji berries or a commercially available supplement providing 6 mg of L and 4 mg for 90 days can improve MPOD and skin carotenoid levels, an index of total carotenoid intake, among healthy middle-aged adults, 45 to 65 years old, with no signs of drusen or early AMD.Eighty-eight volunteers, ages from 45 to 65 years old, were recruited from an online website and public advertisements in the area of greater Sacramento, California. Participants provided informed consent and were screened with a questionnaire. Inclusion criteria were being generally healthy , having a normal macular condition as verified by an optometrist, and if relevant, being prescribed the same medication regimen for at least 6 months that was not related to carotenoid metabolism and was approved by the study physician.

Exclusion criteria were a dislike of, or allergy to goji berries, diseases of the eye, malabsorption problems, substance or alcohol abuse, smoking, drugs for management of lipids, glucose, or blood pressure, use of dietary supplements other than multivitamins and minerals that provided greater than 100% of the U.S. Dietary Reference Intake, or any supplement containing L or Z. The intervention was registered on ClinicalTrials.gov , with the first posted date of 6 December 2019, complied with the tenets of the Declaration of Helsinki, was approved by the Institutional Review Board of the University of California , Davis and was conducted at the UC Davis Ragle Human Nutrition Research Center.Twenty-eight grams of goji berries is considered a single serving size. The berries in this study were USDA-certified organic goji berries grown in the Ningxia region of northern China and provided by Navitas Organics, Novato CA, USA. The goji berries were portioned into clean, single-serving plastic bags and provided in 45-day allotments. The commercially available supplements were purchased online, contained 6 mg of L and 4 mg of Z per serving and were repackaged into 45-day supplies in clean plastic bottles. Compliance was monitored by a self-administered log. Habitual dietary information was collected with the Automated Self-Administered 24 h dietary assessment web-based tool once between day 0 and 45, and once again between day 45 and 90. The MPOD was assessed by the psychophysical method of customized heterochromatic flicker photometry using a macular densitometer . After participants viewed a 5-minute video detailing the measurement procedures, they were dark-adapted for 7 minutes and then began the test. The light intensity of each relevant wavelength was calibrated with a photodiode.

The flicker frequency was selected based on a preliminary test of the participant’s sensitivity. The task was to eliminate or minimize the flicker in the visual field three times by turning a dial that changed the intensity of a 460 nm light. Each participant performed the test while looking directly at the flickering light at 0.25, 0.5, 1, and 1.75 RE degrees, representing the MPOD level from the center to the periphery of the macula. Skin carotenoid content was measured by reflection spectroscopy . After cleaning, the tip of the right index finger was inserted into the spectrophotometer and three measurements were collected. A skin carotenoid score was calculated by the system software. Carotenoids that exist in human plasma, including β-carotene, lycopene, L, Z, and their isomers have been successfully detected in toto and quantified by this device, which has been validated to reflect fruit and vegetable consumption.Sample size was based on a study that assessed the impact of a Z supplement on MPOD in 24 healthy people. Statistical analyses were performed with JMP version 16 . Two-tailed t-tests evaluated potential between group differences at baseline. The MPOD and skin carotenoid data were analyzed with mixed-effects models using time and treatment as the main factors, with age and sex as the covariates, and participant ID as the random effect. For main effects, student t-tests determined significance within group pairs. p-Values of 0.05 or less were considered statistically significant. Correlation coefficients between the outcome measures were determined via Spearman’s method. The mean values of the dietary intake data were compared by two-tailed t-tests, which were log-transformed when necessary, and presented as the mean ± S.E.M. or the back-transformed mean with 95% confidence intervals .Thirty-one healthy, middle-aged adult males and females met the inclusion criteria between May 2019 and Jan 2020. The participants consumed either goji berries or the LZ supplement 5 days per week for 90 days. Twenty-eight individuals completed the intervention, after which two in the goji berry and one from the LZ group were excluded from the data analysis due to measurement errors. Furthermore, data from one was subsequently removed after learning of a major change in dietary patterns that included a low intake of macronutrients between days 45 and 90 .Ninety days of 28 g of goji berry intake significantly increased the optical biomarker MPOD in healthy adults at 0.25 and 1.75 REs. These results suggest that even in a healthy population with no evidence of small drusen or early AMD, goji berry intake can improve eye health. Our results are consistent with data of improved MPOD after a similar amount and intake period of goji berry in a Chinese population at risk for intermediate AMD. Moreover, our trial is consistent with reports of protection against macular hypopigmentation and drusen development in a population of generally healthy and older individuals who were provided Z at approximately a third of the amount of Z provided in the current trial. Our findings suggest that a higher intake of Z relative to L may be useful in reducing the risk of AMD. This is consistent with increased MPOD levels after 4 months of supplementation with 20 mg Z or 26 mg Z with 8 mg L plus 190 mg of mixed omega-3 fatty acids by young healthy adults. Interestingly, black plastic nursery pots we observed a significant increase in MPOD at 1.75 RE, but not at 0.5 or 1 RE, in the goji berry group. A possible explanation for this trend is the relatively low macular pigment at 1.75 RE compared to the other REs, which may increase the potential for improved MPOD in this peripheral area of the macula.

Our results are also consistent with data from 11 randomized controlled trials where supplementation with at least 10 mg of the macular carotenoids was effective at increasing MPOD. Significant correlations were observed between the overall skin carotenoid score and MPOD, which is consistent with clinical results of carotenoid supplementation. Further analysis demonstrated that L and Z, but not goji berry intake, was significantly influencing this trend. Previous work has shown an association between serum L and Z in skin and blood with macular pigment carotenoid accumulatio. Data from the current trial are consistent with this observation as goji berry intake was significantly associated with the skin carotenoid score. However, in contrast to data with L and Z supplements, MPOD score was not correlated with changes in skin carotenoids with goji berry intake. The skin photometer detects overall carotenoid content, and as goji berries are also rich in β-carotene, neoxanthin, and cryptoxanthin, these carotenoids likely influenced the skin measurements, and would not reflect the selective carotenoid accumulation of L and Z in the macula. Other goji berry components such as taurine, vitamin C, zinc, and LBP may influence the results by lowering oxidant stress and improving eye health. For example, studies in animals and cell lines suggest that LBP can protect against AMD by reducing oxidative stress and cell apoptosis in retinal pigment epithelium. Taken together, under the conditions tested, it is reasonable that MPOD may not fully correlate with skin carotenoids in the goji berry group. To our knowledge, the impact of goji berry intake on MPOD in healthy middle-aged people has not been previously reported. While others have noted improved MPOD after LZ supplementation among people with low MPOD baseline levels, our findings suggest that even in populations with normal MPOD values, a significant increase can be detected after goji berry consumption at the most central part of the macula . A meta-analysis regarding the effects of L, Z, and meso-Z supplementation noted that the MPOD at baseline was inversely associated with macular responses, suggesting individuals with a relatively lower macular pigment status may receive more benefit with higher amounts of L or Z. The Age-Related Eye Disease Study 2 trial assessed the impact of dietary supplements containing 10 mg of L, 2 mg of Z, 500 mg of vitamin C, 400 IU of vitamin E, 80 or 25 mg of zinc, 2 mg of copper, and/or 350 mg of docosahexaenoic acid plus 650 mg of eicosapentaenoic acid. The results showed a significantly reduced rate of progression from intermediate- to late-stage AMD after 5 years. Secondary analyses of the study indicated protective roles of L and Z. We did not use the AREDS2 supplement for the comparison group because this formula has only been shown to be effective for those with intermediate AMD, and no clinical evidence exists for its efficacy in our study population of healthy people. In addition, we note that 80 mg of zinc in the AREDS2 supplement is twice the upper limit of recommended daily intakes for zinc. In epidemiological studies, L and Z intakes have been inversely associated with the development of AMD. In the current study, the reported dietary intake of L plus Z, not including the berries or supplement, was 3.1 and 1.9 mg/d in the goji berry and supplement groups, respectively, which is higher than the typical estimated intakes in the US of 1.6–1.86 mg/d. Three to five mg/d of L and Z have been recommended to help support normal macular function, although no recommended dietary allowance values yet exist. Daily consumption of one Hass avocado containing 0.5 mg of L over 6 months was associated with a significant increase in MPOD in healthy adults. In contrast, no increase in MPOD was observed after consuming one Hass avocado daily for 3 months. Daily consumption of egg yolks providing 1.38 mg L and 0.21 mg Z resulted in a significant increase in MPOD and other measures of visual acuity in older adults with signs of early stage AMD after 12 months.

The tool could also be effective in performing model-based vineyard designs in which the optimal design is determined under a set of constraints such as slope aspect or minimum row spacing. Although this means that the optimal design is likely to be case-specific, the tool was used to examine general trade-offs in various designs, which are summarized below. While the N-S row orientation on flat terrain was effective in balancing daily berry light interception and heat accumulation between opposing sides of the vine, it is also susceptible to temporally localized berry temperature spikes on the west side that could be managed by applying dense shade cloth. For cases with no shade cloth, the NE-SW orientation was likely the best compromise between berry temperature reduction and balance between opposing sides of the vine, although it still had significant imbalance in heat accumulation and extreme berry temperatures. Addition of sloped terrain tended to exacerbate berry temperature extremes and imbalance when the slope was facing south or west, which in several cases could not be well managed using shade cloth. The shade cloths were more effective in reducing berry temperatures in cases with greater row spacing relative to plant height because adjacent rows could potentially provide their own shade. The simulation experiment in this work used the new modeling tool to examine general trends in berry temperature and light interception as vineyard architecture and shade cloth density were varied.

Because of the challenge in concisely presenting results of a very large number of simulated cases, growing raspberries in container results are limited to a limited number of architectures, a narrow time period, a single latitude, and site-specific weather conditions. As such, care should be taken in direct application of the simulated values for vineyard design, as they may change for a certain site or design. However, the model itself provides a tool that could be used to provide quantitative guidance for vineyard design or management at a specific site.Together, Chapters 2-4 motivated and carried out the development of a modeling tool that can be used to identify strategies for mitigating the effect of excess sunlight and unfavorable temperatures on grape berries. The tool was then used to study how vineyard design and management strategies related to berry shading interacted to influence berry temperature and light interception. Chapter 2 evaluated widely used assumptions when modeling solar radiation interception in plant canopies, namely assumptions of vegetation homogeneity and isotropy. Because of their simple, tractable form, one-dimensional turbid medium models of radiation interception that assume homogeneity or isotropy are used across a broad range of disciplines. However, it is relatively well-known that with varying levels of vegetation sparseness and preferential leaf orientations , the implicit assumptions of vegetation homogeneity and isotropy in this simple class of 1D models are frequently violated. Yet it is not well-known how this assumption violation translates into model errors in a given situation.

Results of this work provided quantitative guidance for when a simple 1D model can be appropriately used to estimate light interception. For complex canopies like grapevines, the results highlighted the need to use a 3D radiation model because these models can represent the vertical and horizontal variability in the canopy and their effect on light interception accurately. Chapter 3 developed and validated a three-dimensional model for grape berry temperature to understand whether the effect of excess sunlight on grape production can be mitigated by designing and managing vineyards in a way that effectively creates a favorable berry microcli-mate. For the first time, a 3D vine-resolving structural model was coupled with a high-resolution energy balance model for providing accurate and spatially-explicit predictions of berry temperature dynamics. The developed 3D model accurately simulated the spatial and temporal temperature fluctuations of grape berries in vineyards with different climate, topographies, and trellises. Chapter 4 explored different scenarios to mitigate the effect of excess sunlight and temperature. Previously developed models for grape temperature have not been used to evaluate the interacting effects of different management strategies to reduce grape berry temperature. This chapter measured the effect of vineyard design and shade cloth on berry temperature and quantified trade-offs between the different management strategies to maintain optimal berry heat balance and reduce the elevated berry temperatures. Altering light interception by using shade cloths affected the reduction of elevated berry temperatures depending on the vineyard design. A great effect of the shade cloths was found in clusters exposed to direct sunlight for prolonged hours, which tended to be in cases where the vines had greater plant spacing relative to plant height, and where rows were oriented NS and NW-SE in either a flat or sloped terrain facing W or SW, respectively.

While changing the row orientation to NE-SW can be an effective long-term practice to reduce the effect of elevated temperature, for cases where changing the vineyard design is not possible, the shade cloths presented an alternative to reduce unfavorable berry temperatures in many cases. The variables that determine cluster exposure to direct sunlight, such as topography, trellis systems, row orientation, and shade cloths should be considered carefully to develop management strategies for optimizing grape quality. The primary novel outcome of this dissertation that advances the current state-of-the-art is the development of a new tool that could be used to address the role of agricultural management in climate adaptation. Technologies that increase the rate of adaptation to climate change are of great value to farmers, the wine industry, policymakers, and the scientific community. In future studies, the 3D model could be used to evaluate the effect of vineyard designs on berry temperature under changing environmental conditions, to assess the effect of different irrigation strategies on grape production, and to relate the spatial and temporal variability in berry temperature to variations in berry quality traits.Drosophila suzukii , also known as the spotted wing drosophila , is a vinegar fly originating from Southeast Asia. SWD was first detected in North America in August 2008 in Santa Cruz County, California, where it was observed infesting strawberries and caneberries.1,2 In 2009, SWD was detected in Washington, Oregon, and Florida. By 2010, SWD was detected in Utah, Mississippi, North Carolina, South Carolina, Wisconsin, and Michigan in the United States, and Alberta, Manitoba, Ontario, and Quebec in Canada.3 Recent trapping indicates that SWD can be found in virtually any region of North America where host fruit are available. A coincidental invasion of SWD with a genetically distinct population has also been observed in Europe, with initial detections in both Spain and Italy in 2008, followed by its spread throughout the continent.2,4,5 In North America, SWD is primarily a pest of berries and cherries. In Europe, it is reported to also damage a number of stone fruits and grapes. Unlike native vinegar flies in North America and Europe, female SWD possess a serrated ovipositor that can pierce the skin of healthy, soft-skinned fruits to lay eggs. These eggs quickly develop into larvae, which consume the fruit and render it unmarketable. The only other Drosophila species known to oviposit in sound, marketable fruit is Drosophila pulchrella Tan. This species is native to Japan.1 Growers have attempted to mitigate crop damage risk by applying additional insecticide, harvesting more frequently, performing field sanitation, and implementing trapping programs to detect SWD populations. These management practices are costly and many growers still face significant yield losses from SWD infestations. We examine the economic impact of SWD infestations in the California raspberry industry. Raspberry producers are perhaps the most affected by SWD’s invasion amongCalifornia commodities, although producers of blueberries and cherries have experienced substantial losses too. Strawberry producers have experienced lower damage rates and primarily on the lower-value fruit produced for processing. SWD-related losses in these industries vary by year and crop depending on management practices, weather conditions, time of the year, raspberry container size and geographic location. A primary motivation for focusing on the California raspberry industry is that California accounts for the majority of raspberry production in the U.S. and the raspberry industry accounts for the majority of economic losses due to SWD among berry crops. 6 A second motivation is the magnitude of change in pest management practices; few of the SWD control practices used by raspberry producers were needed to prevent injury from other pests prior to its establishment. Economic losses in the California raspberry industry include the cost of managing SWD and the value of the fruit lost due to SWD infestations despite management efforts. First, we compute the cost of the chemical management programs and the labor-intensive sanitation practices implemented to mitigate SWD-related yield losses. Second, we calculate the industry level yield losses due to infestation. These components form an estimate of the full economic cost of SWD’s invasion into California raspberry production.

In 2013, raspberries were estimated to be the twenty-seventh largest crop in California by value of production. California accounted for 74% of all raspberry production in the United States. The United States is the third largest producer of raspberries in the world, producing 91,300 tonnes, after the Russian Federation and Poland, which produce 143,000 and 121,040 tonnes,respectively. Across all counties, California’s raspberry production was worth an estimated $239 million according to the United States Department of Agriculture’s National Agricultural Statistics Service , and $437 million according to California County Agricultural Commissioners’ Reports.  The difference in these estimates reflects that the NASS data report cash receipts to producers while the Agricultural Commissioners’ Reports estimate the total value of production. Figures 1, 2, 3, and 4 plot California raspberry hectares, production, yield per hectare, price per kilogram, and the total cash receipts between 2004 and 2013. Note that raspberry hectares multiplied by yield per hectare is equivalent to production, and production multiplied by price per kilogram is equivalent to total cash receipts. Four counties account for virtually all commercial raspberry production in California: Ventura, Santa Cruz, Santa Barbara, and Monterey. In 2014, Ventura County produced approximately 52% of California’s raspberry crop by value, $241 million, on 1,873 hectares. Raspberries are the third most valuable crop in Ventura County. Santa Cruz County produced approximately 28% of California’s raspberry crop by value, $131 million, on 979 hectares. Raspberries are the second most valuable crop in Santa Cruz County. Santa Barbara County produced approximately 10% of California’s raspberry crop by value, $45.2 million, on 591 hectares. Raspberries are the ninth most valuable crop in Santa Barbara County. Monterey County produced approximately 10% of California’s raspberry crop by value, $45 million, on 316 hectares. Raspberries are the sixteenth most valuable crop in Monterey County. Table 1 summarizes California raspberry production by county. Counties are listed from north to south along the Pacific Coast. Figure 5 identifies these berry-producing regions with a stylized map of California.Most commercial raspberry plantings in California have had an 18-month lifespan. The crop is planted in the winter and then harvested twice, first in the fall following planting and then in the subsequent summer. Both harvest seasons last approximately three months, with crews harvesting fruit every three days on average. Variations in harvest frequency depend on yields and pest management activities. Yields are low at the beginning and end of a harvest season, and peak near the middle of a season. Pesticide applications may require an interval of time, depending on the particular pesticide, before normal harvesting activities can resume. This period is known as the pre-harvest interval , and it is determined by the U.S. Environmental Protection Agency. Occasionally, low yields are realized during the harvest season due to crop damage resulting from weather, pest activity, or other external factors. The summer harvest is typically larger than the fall harvest. Organically produced raspberries represent a significant share of total California raspberry production. In 2008 and 2011, California’s organic raspberry production was valued at $11.4 million and $8.98 million, respectively, according to the USDA-NASS. In 2012, 408 hectares of California raspberries were organically managed according to the University of California Agricultural Issues Center. Raspberry prices vary throughout the year, but on average organic raspberries are sold at a price premium. In 2015, the national average retail price of organic raspberries over the entire year was $3.52 per six ounce tray according to the USDA Agricultural Marketing Service. The average retail price of conventional raspberries over the same period was $2.55 per tray. The average California terminal market prices for organic and conventional raspberries were $3.29 and $1.97 per tray, respectively. California raspberries are a major export crop. In 2013, the combined category of raspberry, blackberry, mulberry, and loganberry exports was the twentieth largest export crop category by value in California.

These results highlighted the need to use a 3D radiation model to account for complex canopies because these models can represent the vertical and horizontal variability in the canopy and its effect on light interception accurately. Chapter 3 develops a 3D model that can accurately resolve spatial and temporal heterogeneity in berry temperature. The spatially-explicit nature of the model allows for robust representation of varying canopy architectures and their effect on berry temperature. The high model complexity is afforded by performing calculations in parallel on the computer’s graphic processing unit . This ability to resolve the geometry of the vineyard is critical in this particular study because it means the model is robust to changes in row spacing, trellis system, row height, etc. To generate data for validation of the 3D grape berry temperature model, field and laboratory experiments were conducted. Validation results demonstrated that by accurately representing the 3D vine structure, the model was able to closely replicate measured spatial and temporal fluctuations in berry temperature. Chapter 4 aims to explore whether elevated berry temperature can be mitigated by designing and managing vineyards in a way that effectively creates a favorable microclimate for berry development. Identifying strategies that have the potential to reduce elevated temperatures in a warming climate is of great interest to grape growers. However, raspberry container growing given the extremely large number of interacting variables that determine berry temperature it is not feasible to independently vary all of these parameters in field experiments. Thus, to study the interactions between these variables that might yield favorable results, Chapter 4 expands the model developed and validated in Chapter 3 by incorporating the effects of shade cloth on berry temperature.

The model was used to ultimately predict the efficacy of potential mitigation strategies for high berry temperature. The results of this study provided new insights into the effect of fruit zone shading to control berry temperature for the establishment of new vineyards and targeting the management of existing vineyards.Light interception in plant canopies is most commonly estimated using a simple one dimensional turbid medium model . Inherent in this class of models are assumptions that vegetation is uniformly distributed in space and in many cases that vegetation orientation is uniformly distributed . It is known that these assumptions are violated in a wide range of canopies, as real canopies commonly have heterogeneity at multiple scales and almost always have highly anisotropic leaf angle distributions. However, it is not quantitatively known under what conditions these assumptions become problematic given the difficulty of robustly evaluating model results for a range of canopy architectures. In this study, assumptions of vegetation homogeneity and isotropy were evaluated under clear sky conditions for a range of virtually-generated crop canopies with the aid of a detailed three-dimensional, leaf-resolving radiation model. Results showed that Beer’s law consistently over predicted light interception for all canopy configurations. For canopies where the plant spacing was comparable to the plant height, Beer’s law performed poorly, and over predicted daily intercepted sunlight by up to 115%. For vegetation with a highly anisotropic leaf inclination distribution but a relatively isotropic leaf azimuth distribution, the assumption of canopy isotropy resulted in relatively small errors. However, if leaf elevation and azimuth were both highly anisotropic, the assumption of canopy isotropy could introduce significant errors depending on the orientation of the azimuthal anisotropy with respect to the sun’s path.

Solar radiation is a primary driver of most plant biophysical processes, including energy transfer, turbulent transport, evapotranspiration, photosynthesis, and phenology. Fluxes of absorbed radiation in plant canopies have strong gradients in the vertical direction, and potentially in horizontal directions in the case of heterogeneous canopies. Capturing these high gradients through direct measurement is often challenging, and therefore models are frequently used to predict absorbed radiative flux distributions. For practical purposes, relatively simple models are frequently used to estimate light interception in plant canopies. For example, crop models have become important tools for studying agricultural system, yet they commonly utilize relatively simple models for light interception given the frequent lack of detailed architectural inputs. The most commonly used approach for estimating light interception treats the canopy as a homogeneous medium of unresolved vegetation , which allows for the use of a simple exponential model for radiation attenuation commonly know as Beer’s law, Beer-Lambert law, or Beer-Lambert-Bouguer law. Beer’s law calculates the probability of radiation interception as an exponentially increasing function of the leaf area projected in the direction of radiation propagation and the distance travelled through the canopy.The form of Beer’s law given in Eq. 2.1 functions under two main assumptions. The first assumption is that leaves are randomly distributed both vertically and horizontally in a continuous medium where leaves are relatively small. The second assumption is that leaves absorb all incident radiation, which may be reasonable for photosynthetically active radiation bands where leaves absorb roughly 90% of incident radiation, but is likely a poor assumption in other bands such as the near-infrared where absorption is low.

Equation 2.1 also requires specification of G, which is most commonly set to be equal to 0.5 based on the assumption that leaves are isotropic. Leaf area density typically varies sharply in the vertical direction. Many natural plant canopies have considerable horizontal heterogeneity such as savannas, or heterogeneity due to natural or man-made disturbances. Crop canopies also commonly have a sparse, row-oriented configuration that creates high heterogeneity in light interception. Furthermore, it is rare to find canopies with isotropic leaf angle distributions, as this is typically not the most efficient configuration for light interception. Despite the known limitations of Beer’s law in the above cases, it is still frequently applied in these systems due to its simple, tractable form. However, there is a general lack of quantitative understanding of the errors resulting from the application of these simplified models in various canopy architectures, primarily because it is difficult to quantify light interception from field measurements for a range of architectures. The objective of this study is to better understand and quantify errors in modeled radiation absorption under assumptions of vegetation heterogene-ity or isotropy for various canopy configurations. The authors’ hypothesis is that Beer’s law will perform well for relatively dense, closed canopies provided that G is specified appropriately. For sparse canopies, it is hypothesized that assumptions of vegetation homogeneity will result in significant model errors, thus necessitating a more complicated model. Since accurately measuring the distribution of absorbed radiation in space and time is often unfeasible using traditional experimental approaches, we used a sophisticated 3D radiation model along with virtually-generated canopies to evaluate Beer’s law under different simplifying assumptions. Virtual canopies with varying levels of heterogeneity, sparseness, and leaf orientation distributions were generated to evaluate assumptions of vegetation homogeneity or isotropy in terms of absorption of direct solar radiation. A considerable advantage of using virtually generated canopies is that the input parameters in Eq. 2.1 can be calculated exactly from the virtual canopies. When combined with a detailed 3D radiation model, this resulted in a robust means for evaluating the performance of simplified models for a range of canopy architectures.For simulating plant light interactions, blueberry plant pot detailed 3D geometric models were used to describe the architecture of the canopy. Agricultural crops were chosen for the plant types because: many 3D models are readily available, they have sufficient yet regular heterogeneity that limited the degrees of freedom when generating the canopies, and they represent an economically important practical application of the use of Beer’s law. The chosen crop canopies were grape, almond, potato, and corn, which were represented in the 3D model using a mesh of rectangular and triangular elements.

To minimize the number of elements needed to describe their complex geometries, images with a transparency channel could be overlaid on these basic elements, where the transparency channel is used to remove a portion of the element’s surface. Virtually-generated plants were either read from a polygon file , or created using the procedural plant generator described by Weber and Penn .Parameters used to quantify canopy architecture are given in Table 2.1. The procedural model used to generate the grape and almond plants had a random component to each architectural parameter, making each plant unique. Each corn and potato plant was identical, therefore a random azimuthal rotation was applied to each plant to decrease regularity of the canopy. Plants were placed in a marked row structure to form a canopy. Two grape canopy cases were considered: one with a North-South row orientation and one with an East-West row orientation . Two potato canopies were considered in which plants were arranged in either a East-West row-oriented pattern , or a uniformly spaced planting pattern . In all cases, the size of the 3D scene was chosen such that further increasing the total number of plants did not have an impact on results. To test the model in the case of homogeneous and isotropic vegetation, a set of canopies were created with uniformly distributed leaves in space with three different leaf area index values: L =1.5, which consisted of 100,000 leaves; L =3.1, which consisted of 200,000 leaves; and L =6.2, which consisted of 400,000 leaves. The surface area of each leaf was 0.006 m2. Each leaf angle was set by randomly drawing from a spherical distribution. To characterize the plant geometry, L and the leaf inclination angle probability density function were calculated for all five generated canopies, and the leaf azimuthal angle PDF was calculated for the Grape N-S and Grape E-W cases . The L was calculated by summing the one-sided area of all leaves in the canopy and dividing by the total canopy footprint area. The leaf inclination angle and leaf azimuthal angle were calculated for each of the elements from the surface normal of the leaf, and a PDF was formed by weighting each element’s contribution to the PDF by its surface area, then normalizing such that the PDF integrates to unity. The corn model had predominantly vertically oriented leaves, while the almond and potato models had leaves closer to horizontal on average . Grape leaf inclination skewed toward vertical, and leaf azimuth tended to be oriented parallel with the row , which is supported by previous manual and LiDAR measurements. The gap fraction was calculated from the 3D models by computing the fraction of direct sunlight not intercepted when qs = 0 . Gap fraction values ranged from 80% in the grape canopy cases down to 21% for the corn canopy case. Although both potato canopy cases had the same L, their gap fractions were 22% for uniformly spaced plants and 36% for row-oriented plants.The incoming radiation data used to drive the radiation absorption simulations in this study was generated following the REST-2 model of Gueymard. The hourly incoming radiation was calculated based on the assumed virtual site longitude , latitude , offset from UTC , atmospheric pressure , air temperature near ground level , atmospheric turbidity coefficient , relative humidity , and Julian day of the year . It is noted that the precipitable water in the REST-2 model was specified using the model of Viswanadham. The direction of the sun for any time of day at the virtual site was calculated following the approach outlined by Iqbal. In cases where scattering was included, two radiative bands were considered – one character-istic of efficient absorption by leaves such as the photo synthetically active band , and another characteristic of high scattering such as the solar near-infrared band . The total incoming solar flux was partitioned as 47% in the PAR band and 53% in the NIR band. In the PAR band, r was set to 0.056 and t to 0.042, while in the NIR band, r was set to 0.425 and t to 0.334.Results for the daily total light interception on Julian days 153, 232, and 305 are listed in Table 2.2, and shown graphically in Fig. 2.5. For the homogeneous canopy cases, very close agreement was found between the 1D and 3D models regardless of L, which indicated that the approach used to compare the 1D and 3D models was consistent and that leaf-scale heterogeneity created by discrete leaf surfaces did not create significant errors.

Several genes were captured in this interval, including NADH:Ubiquinone oxidoreductase subunit A8 ; mucin-5AC-like, a gene putatively related to human mucin-5AC, and two hemomucin genes involved in hemocyte adhesion and innate immunity in insects. The PBS hotspot on contig 15 contained cryptochrome-2 , encoding a key component of the circadian clock . Finally, the large hotspot on contig 22 contained 69 genes, including multiple copies of juvenile hormone binding protein, ribosomal proteins, gustatory response genes, rhodopsins, andacetylcholinesterase , a gene implicated in organophosphate insecticide resistance . Interestingly, based on alignment data it appears the Central and Andes populations may have two copies of ache, while North populations only have one. We looked for known mutations conferring organophosphate resistance and found moderate frequencies in all three clusters as well .Using whole genome sequencing data, we found that Tuta absoluta samples collected from 11 locations in Latin America clustered into three basic regions, comprised of a North, Andes, and Central group. In addition, we see that Spanish populations likely originated from a Central Chilean source based on their low level of Fst with the Andes and location on the ML tree. Previous analyses with mitochondrial sequences were unable to differentiate populations ; however, large pots plastic analyses using microsatellite data was able to identify these same three clusters and suggest a Central Chilean source for the European migration as well .

In agreement with this conclusion, looking at fresh tomato export data we see Chile is a worldwide exporter, shipping 12 tons of tomatoes an average distance of 11,700 km in 2018, while most other countries in South America tend to export within the continent . The Andes Mountains represent an obvious geographic barrier that would separate the Central population from the North and Andes populations. Population structure between Andes and North populations may be due to factors related to the changing latitude, including temperature and daylength. While PCA groups our Ecuador samples with Andes populations, admixture analysis and Treemix both provided some evidence that Ecuador may represent an admixture zone between the two regions. The weighted Fst between the North and Andes is also lower than between North and Central, suggesting that North and Andes are indeed more closely related. This general Fst pattern was also observed based on microsatellite analyses . Sequencing of more samples fromPeru and Ecuador might be needed to further elucidate the extent of an admixture zone between these clusters. While Tuta absoluta was first discovered in Peru in 1917, its native range is not well established. One hypothesis is that Tuta absoluta migrated out of the Andes region and across South America through the 1960s-80s because of human transport by agricultural shipping. This aligns with the surge in domestic tomato agriculture in South America at the same time . However, based on the similar nucleotide diversity levels between clusters, as well as high levels of Fst, we hypothesized it might be more likely that this migration across South America may have happened prior to tomato commercialization, with populations of Tuta absoluta later adapting to the appearance of commercial tomato agriculture.

Based on our simple 3 population model, it appears the ancestral population diverged twice tens of thousands of years ago. Relative to the ancestral population size, the combined effective population size is roughly three times larger, although this is heavily weighted toward a very large Andes population, relative to the North and Central regions. The fact that the estimated Andes population size is nearly 10 times larger than that of Central or North populations, ,as well as its slightly higher level of genetic diversity, could suggest that the Andes cluster represents the ancestral population range. Given that the wild ancestors of tomatoes and potatoes are also native to the Andes region , the Andes region would be an ideal place to search for native parasitoids for biocontrol. To date, biocontrol methods in South America have relied on non-natives or generalist parasites, with only a handful of native specialists identified in the literature and none that are commercially available . Thus, knowledge of Tuta absoluta’s native range may help focus efforts to identify more natural parasitoids. Using the PBS, we found multiple genomic windows under apparent selective forces. Not surprisingly, one of the highest PBS windows contained the para gene, which encodes a sodium ion channel that is targeted by pyrethroids. The extremely low allele diversity in the Andes and Central populations relativeto the North suggest a hard selective sweep occurred here. Heavy pyrethroid use in Brazil led to the appearance of resistant strains starting in the 1990s . We found that Central and Andes populations were completely fixed for the L1014F mutation, one of the most common causes of knock-down-resistance to pyrethroids , while the North had an intermediate frequency. A study looking at Brazilian populations found a similar pattern of fixed L1014 , while another study looking at multiple populations in South America also found the same pattern of L1014F fixation in Central and Andes populations but not in the North .

While both studies also found M918T and T929I at elevated frequencies in all populations, we additionally detected the resistance allele V1016G in the North. We also found L925M in Central and I254T in Andes. While these have not been characterized as resistance alleles, mutations at these same positions have been shown to confer resistance in Drosophila melanogaster and Bemisia tabaci . Other regions under selection were less obvious. We found one region in contig 2 containing Ndufa8 and several hemomucin/mucin genes with a similar low genetic diversity in the Central and Andes populations and high PBS in North, indicating a hard selective sweep. Ndufa8 produces a nuclearencoded subunit of the NADH dehydrogenase complex I, part of the electron transport chain in the mitochondria used to generate ATP. Mutations here are known to cause mitochondrial complex I deficiency in humans , although a few studies have found evidence of positive selection occurring in other species, potentially related to metabolism . The hemomucin genes are a component of the insect immune system that are involved in endocytosis . Selection here could be in response to the increased use of parasitoids and predators such as Trichogramma evanescens and Nesidiocoris tenuis as an biological control alternative to insecticides .The elevated PBS region in contig 22 was relatively large at approximately 1Mb in size, containing over 60 genes. Interestingly, we noticed two copies of ache contained within this window, which codes for acetylcholinesterase, a gene which encodes a protein which degrades the neurotransmitter acetylcholine . As this enzyme is the main target of organophosphates and carbamate insecticides, alleles in ache have been documented to confer resistance. Using the amino acid numbering scheme based on T. california , the resistance allele A201S has previously been reported to be present in European populations , and we found this allele to be at moderate to high frequency in all three regions. We also found moderate frequencies of the mutation F290V and F290N. The F290V resistance allele has been documented in mosquitoes and moths , while other mutations such as F290Y have been documented in Drosophila and M. domestica . Interestingly, based on mapping read depth the North population only contained a single copy of ache. As duplication of ache has been implicated in improved organophosphate resistance , square planter pots this large structural duplication may be the reason for elevated PBS levels across such a large interval. Follow-up work with long-read methods or higher sequencing coverage will be needed to confirm the presence of structural duplication at this locus. We expect that addition of a new contiguous genome assembly with annotations will be of benefit to the Tuta absoluta and Lepidopteran research community. Previous studies have worked to develop potential RNA interference strategies to use as an alternative to traditional pesticides . Work is also being conducted to develop Cas9 gene-editing techniques for Tuta absoluta to facilitate future genetics studies . These developments in combination with an accurate assembly and gene annotations will allow for accelerated research towards understanding Tuta absoluta biology and methods to contain its economic impacts and spread.The tomato plant represents a massive economic industry worldwide, with an estimated 252 million metric tons of tomatoes harvested in 2020 .

This production is concentrated in a few major producing countries, with 70% of the world’s production currently accounted for by China, the European Union, India, the USA, and Turkey . However, this industry is quickly becoming threatened by the invasive gelechiid moth Tuta absoluta Meyrick, commonly known as the tomato leafminer. T. absoluta is an agricultural pest of the nightshade family, including peppers, eggplants, and potatoes, but primarily represents a serious threat to tomatoes. Left untreated, the larvae will consume leaf tissue, bore into flower buds, or burrow into fruit with infestations causing crop losses as high as 80 to 100% . While T. absoluta was first identified in Peru in 1917 , it was not considered an agricultural pest until the 1960s when T. absoluta was detected in farms in Argentina, causing significant crop loss . From there it rapidly spread throughout South America, causing severe agricultural losses everywhere it was found. In 2006, T. absoluta was identified in a greenhouse in Spain, marking the first intercontinental migration of the pest, likely due to anthropological transportation . From Spain, it rapidly spread throughout Europe, SubSaharan Africa, the Middle East, and most recently into Asia . Without rapid identification and quarantine strategies, T. absoluta will likely continue to spread to North America. Preventing initial establishment of an invasive species in a risk ecosystem can be an effective strategy but requires vigilant monitoring of imported products as well as an ability to rapidly detect the presence of the target . In the United States, policies such as federal orders issued by the Animal and Plant Health Inspection Service have been used to prevent accidental introduction of T.absoluta by regulating import of tomato fruit or propagation material from infested countries . Correctly detecting and identifying the insect, however, remains challenging due to the presence of other gelechiid moths such as P. operculella and Keiferia lycopersicella in the United States, which look nearly identical to T. absoluta and are also commonly found on tomato and potato crops . Distinguishing these species morphologically requires dissection of adult male genitalia, which requires entomological expertise and increases waiting times for identification. Molecular diagnostics, on the other hand, would allow for accurate and rapid identification without entomological expertise. To date, several molecular diagnostics have been developed, all using Polymerase Chain Reaction to amplify target DNA presence before detecting signal either by fluorescence, gel electrophoresis, or mass spectrometry . A potential limitation of these PCR-based diagnostics is the requirement for a thermocycler, as well as specialized equipment to visualize results. For field detection, a system that requires minimal specialized equipment would be ideal. The CRISPR-Cas system has recently become an attractive option for molecular diagnostics . CRISPR-Cas originated as a bacterial immunity system, in which the Cas protein complexes with a CRISPR RNA that is complementary to a target DNA sequence . When the Cas-crRNA complex binds to its target it cleaves it, causing a double-strand break in the DNA. While Cas9 is the most commonly used Cas protein for its genomic editing capabilities, the Cas12a enzyme was discovered to exhibit indiscriminate single-stranded DNA nuclease activity upon binding to its target DNA . If a single-strand oligonucleotide probe modified with a fluorophore and quencher is present when Cas12a-crRNA binds its DNA target, Cas12a will proceed to also cleave the single-strand probe, separating the fluorophore from the quencher and allowing a fluorescent signal to be measured. As ssDNAse activity is proportional to the number of target DNAmolecules and can occur isothermally between 15℃ to 50℃, using an isothermal DNA amplification method such as Recombinase Polymerase Amplification to amplify target DNA allows for sensitive detection with Cas12a at constant temperature, eliminating the need for an expensive thermocycler. Additionally, detection of probe cleavage is flexible as several methods have been published on alternative visualization methods, including using flow strip assays, gold-conjugated DNA probes, and naked-eye fluorescence detection . In this study, we leveraged prior sequencing data to develop two molecular diagnostic assays.

Even if honey bees displace wild insects at the flower scale , this is unlikely to scale up to the field, as indicated by our data, if mass flowering crops provide floral resources in excess of what can be exploited by local pollinator populations. Therefore, although insect pollinators appear not to be limited by crop floral resources, yield was commonly pollen limited, as crops set more fruit in fields with more visitation by pollinators . Contrary to the fourth prediction, fruit set increased with flower visitor richness independently of honey bee visitation . Correspondingly, the CVs of fruit set decreased with richness; in contrast, evenness did not affect the mean or CV of fruit set . Visitation by wild insects increased strongly with richness , and improved model fit , even when richness was included in the model . However, richness did not enhance model fit when added to a model with wild-insect visitation , suggesting that the effects of richness on fruit set reflect increased wild-insect visitation . Like wild-insect visitation , richness did not correlate with honey bee visitation . Previous studies have shown that agricultural intensification reduces both species richness of pollinator assemblages and wild-insect visitation . Our results for multiple crop systems further demonstrate that fields with fewer species experience less visitation by wild insects and reduced fruit set, independent of species evenness or honey bee visitation. Globally, plastic gutter wild-insect visitation signals both species richness and pollination services, and is a relatively inexpensive indicator that can be standardized easily among observers in field samples .

Large, active colonies of honey bees provide abundant pollinators that can be moved as needed, hence their appeal for pollination management in most animal-pollinated crops . By comparison, methods for maintaining diverse wild insects for crop pollination are less developed, and research on such pollination services is more recent . Although honey bees are generally viewed as a substitute for wild pollinators , our results demonstrate that they neither maximize pollination, nor fully replace the contributions of diverse, wild-insect assemblages to fruit set for a broad range of crops and agricultural practices on all continents with farmland. These conclusions hold even for crops stocked routinely with high densities of honey bees for pollination, such as almond, blueberry or watermelon . Dependence on a single species for crop pollination also carries the risks associated with predator, parasite and pathogen development . Our results support integrated management policies that include pollination by both wild insects as ecosystem service providers, and managed species, such as honey bees, bumble bees , leaf cutter bees , mason bees , and sting less bees as agricultural inputs . Such policies should include conservation or restoration of natural or semi-natural areas within croplands, promotion of land use heterogeneity , addition of diverse floral and nesting resources, and consideration of pollinator safety as it relates to pesticide application . Some of these recommendations entail financial and opportunity costs, but the benefits of implementing them transcend the supply of pollination services alone and extend to, for example, mitigation against soil erosion, and improved pest control, nutrient cycling and water use efficiency .

Without such changes, the on-going loss of wild insects is destined to compromise agricultural yields worldwide.Half of all fruit and vegetables produced globally are lost each year . While the causes of losses vary by region and commodity, fungal phytopathogens have a widespread role, as 20–25% of all harvested fruit and vegetables are lost to rotting caused by such fungi . In fleshy fruits, this issue is exacerbated because, in general, fruit become more susceptible to fungal pathogens as they ripen . Ripening-associated susceptibility has been demonstrated in multiple commodities including climacteric fruits such as tomato, stone fruit, banana, apple, and pear, as well as non-climacteric fruits such as strawberry, cantaloupe, citrus, and pineapple . The most devastating post harvest pathogens in fruit are those with necrotrophic lifestyles, which deliberately kill host tissue, resulting in rotting. Example pathogens include the model necrotrophic fungi Botrytis cinerea and Sclerotinia sclerotiorum as well as Monilinia spp., Alternaria spp., Rhizopus spp., Penicillium spp., and Fusarium spp. . Plant immune responses against necrotrophic fungi are multilayered, involving recognition of pathogen-associated molecular patterns, such as chitin or chitosan, by pattern recognition receptors, intracellular signaling through mitogen-activated protein kinase cascades, induction of downstream defenses by coordinated activity of phytohormones, particularly ethylene and jasmonic acid , cell wall fortifications, and production of various secondary metabolites and antifungal proteins . However, most defense strategies have been studied in leaves, and their utilization and effectiveness in fruit have been assessed only with single pathogens .

The outcome of any fruit–necrotroph interaction relies on the balance between the presence or induction of defenses and the contributions of susceptibility factors. Though induced defenses are heavily studied in plant immunity, the impact of preformed defenses and susceptibility factors are less researched . Preformed defenses include structural barriers, such as the cell wall and cuticle, and the accumulation of secondary metabolites , while susceptibility factors include the abundance of simple sugars and organic acids or activity of host cell wall modifying proteins . A sufficient understanding of ripening-associated susceptibility requires a characterization of the ripening program’s impact on the ability of the host to express necessary defense genes upon pathogen challenge, the integrity of preformed defenses, and the abundance of susceptibility factors. In this study, we first applied a transcriptomic approach to characterize core tomato fruit responses to three fungal pathogens and changes in gene expression that occur during ripening to promote susceptibility. To identify core responses that are not merely pathogen-specific, we used three pathogens with necrotrophic infection strategies: B. cinerea, Rhizopus stolonifer, and Fusarium acuminatum. Using well-established defense gene classifications, we developed profiles of host defense gene expression responses in unripe and ripe fruit. We then determined the susceptibility phenotypes of three non-ripening mutants: Colorless non-ripening , ripening inhibitor , and non-ripening , which have unique defects in ripening features . After demonstrating that each mutant has distinct susceptibility to disease, we identified ripening genes whose expression changes may impact the disease outcome. By integrating our transcriptomic data and mutant analyses, we found preformed defenses and susceptibility factor candidates associated with B. cinerea infections. Using CRISPR-based mutants, we established that one candidate, the pectin-degrading enzyme pectate lyase, is indeed a disease susceptibility factor in ripe tomato fruit.To characterize tomato fruit responses to fungal infection at unripe and ripe stages, we inoculated fruit with B. cinerea, F. acuminatum, or R. stolonifer spores. Each pathogen successfully infected RR fruit, producing visible water-soaked lesions and mycelial growth by 3 dpi, whereas MG fruit remained resistant and, except in samples inoculated with R. stolonifer, had a dark, necrotic ring around the inoculation sites , a feature of the pathogen response that did not appear in wounded fruit. Thus, MG fruit resistance and RR fruit susceptibility are a feature common to multiple necrotrophic infections. We hypothesized that these susceptibility phenotypes are the result of differences in immune responses at each ripening stage and developmental processes during ripening that alter the levels of preformed defenses and susceptibility factors. First, we assumed that, compared with arobust immune response in MG fruit, RR fruit have a weaker response, consisting of fewer genes induced, less diverse functionality, and absent expression of critical genes. Additionally, blueberry container we predicted that ripening may decrease the expression of preformed defenses and increase the expression of susceptibility factors, which create a more favorable environment for infection.

To test if immune responses to fungal pathogens are compromised in RR compared with MG fruit, we sequenced mRNA from B. cinerea-, F. acuminatum-, and R. stolonifer-inoculated fruit at 1 dpi, an early time point at which either a resistant or a susceptible phenotype becomes apparent. A principal component analysis of the mapped normalized reads for all tomato genes revealed that the major driver separating sample data was the ripening stage , while inoculation status accounted for less of the separation . The one exception to this pattern was the R. stolonifer-inoculated MG samples, which clustered with the healthy and wounded MG samples, suggesting that unripe fruit did not display strong responses to this pathogen and yet remained resistant. However, quantification of normalized pathogen reads confirmed that all three pathogens were detectable at 1 dpi even in MG samples. To identify the responses for each ripening stage common to all three pathogens, we performed a differential expression analysis between inoculated and healthy samples for MG and RR fruit. We chose the healthy samples as controls for these comparisons in order to capture responses to necrotrophic infection, which may share features with mechanical wounding. Of all 34 075 protein-coding genes found in the tomato transcriptome, 9366 were found to be differentially expressed in response to inoculation in fruit at 1 dpi in at least one comparison . Of these, 475 genes were significantly up-regulated in MG fruit in response to all three pathogens, corresponding to the MG core response , whereas 1538 genes formed the RR core response . The MG core response overlapped substantially with the wounding response in MG fruit , which suggests that unripe fruit activate similar functions when responding to pathogen attack and mechanical damage. However, this large overlap is also due to the similarity between the gene expression profiles of wounded and R. stolonifer-inoculated samples as seen in the PCA . In contrast, the lack of a strong wounding response in RR fruit indicates that nearly all RR core response genes were strictly pathogen-related . Downregulated genes in response to infection were largely unique to each pathogen, with only 57 and 225 down-regulated across all three pathogens in MG and RR fruit, respectively, and thus we decided to continue our analysis only on the up-regulated core response genes. Complete lists of gene set intersections of up-regulated and down-regulated genes are in given in Supplementary Table S5. We then assessed the MG and RR core responses for the presence of various well-established gene classifications relatedto pathogen defense, including selected GO terms, KEGG pathways, transcription factor families, hormone biosynthesis, signaling and response genes, and receptor-like kinase genes . For each category, we performed enrichment analyses to identify classifications of particular importance in both MG and RR core responses. A total of 70 defense genes were identified in the MG core response. Interestingly, these were enriched in only two categories: chitin catabolic process and RLK genes. The RR core response was enriched in 13 defense categories, including the plant–pathogen interaction and MAP kinase signaling pathways , secondary metabolite biosynthesis pathways , WRKY and ethylene responsive factor transcription factors, RLKs, and JA biosynthesis. Altogether, 302 defense genes were identified among the RR core response. Thus, in contrast to their respective susceptibility phenotypes, RR fruit appear to mount a more robust and diverse immune response than MG fruit early during inoculation, demonstrating that, contrary to our initial hypothesis, weakened immune responses in RR fruit are not a contributor to ripening-associated susceptibility. However, it is possible that tomato fruit resistance to necrotrophs could be determined by a small number of genes that were exclusive to the MG core response. Out of the 70 defense genes in the MG core response, 27 were not found in the RR core response . These 27 genes are heterogeneous, representing 12 different defense categories. Notable genes in this category include a three-gene cluster of PR-10 family proteins , a chitinase previously identified during infections of tomato with Cladiosporum fulvum , and an ERF active at the onset of ripening . Although these 27 genes were not in the RR core response, most of them were induced during RR infections by one or two of the pathogens studied. Only seven were not up-regulated by any of the three pathogens in RR fruit, including the ERF mentioned above , as well as three RLK genes, two glutaredoxin genes involved in the response to oxidative stress, and a cysteine protease. Given that each of these genes belongs to a large family of genes whose members are often functionally redundant, and their average expression levels in infected MG fruit were fairly low , we consider it unlikely that the lack of these genes in the RR core response contributes heavily to susceptibility.

Achenes are small single-seeded fruit, whereas the receptacle is considered to be anatomically equivalent to floral meristem tissue . F. × ananassa is an allo-octoploid that originated as a synthetic hybrid between the octoploid species Fragaria chiloensis and Fragaria virginiana . Strawberry is affected by several pathogens including fungi, bacteria, viruses and nematodes. The most economically impactful pathogens of strawberry are fungi, which can infect all parts of the plant and cause severe damage or death . Amongst the fungal pathogens, the ascomycete Botrytis cinerea is considered the primary pathogen of harvested strawberries in the world leading to impactful economical losses to the strawberry industry. B. cinerea causes grey mould in fruit and senescing organs but can also affect vegetative tissues . Under wet conditions, more than 80% of strawberry flowers and fruits can be lost if plants are not sprayed with fungicides .B. cinerea has no apparent host specificity and can infect more than 1000 plant species . The pathogen is found worldwide and causes disease in many fruit, flower and leafy vegetable crops . B. cinerea is classified as a necrotroph, meaning that it prefers to infect and grow on damaged or senescing tissues, eventually causing tissue death. The inoculum of the fungus is highly abundant and ubiquitous and usually comes from infected plant tissues . B. cinerea mainly enters the host via wounds or natural openings . Infections of non-senescing or unripe plant organs usually lead to limited damage and quiescent infections .

Different types of quiescence have been described: delay of conidia germination or growth arrest after germination , square plastic plant pot endophytic symptomless growth in the apoplast , colonization of abscising flower organs followed by growth into ovaries or receptacles where growth arrests . Independent of the type of infection, the pathogen generally enters a short asymptomatic, biotrophic phase at the beginning of the disease cycle . An aggressive necrotrophic phase commonly succeeds the quiescent or asymptomatic phase once plant organs start to senesce or ripen, during which B. cinerea causes rapid decay of the infected tissues . B. cinerea’s infection mechanisms have been studied in model organisms and further characterized thanks to the availability of high-quality reference genome sequences . The fungus is known to actively promote plant susceptibility by employing a variety of virulence factors . In early stages, B. cinerea deploys sRNAs and effector proteins to suppress premature host cell death and immune responses, which enables the fungus to establish inside the host and accumulate biomass prior to the necrotrophic phase . It was demonstrated that B. cinerea Dicer-like proteins DCL1 and DCL2 produce sRNAs that are secreted from fungal hyphae and translocated to the plant cell where they interfere with the host RNAi mechanisms to silence host immune response genes in Arabidopsis and tomato leaves . Some secreted virulence factors can lead to host cell death, like effector proteins, toxins and enzymes involved in reactive oxygen species production . B. cinerea can also secrete oxalic acid that lowers the pH of the host tissuesand stimulates the production and activity of fungal enzymes like pectinases, laccases and proteases . Furthermore, oxalic acid accumulation leads to Ca2+ chelation, which in turn weakens the pectin structures of plant cell walls and inhibits the deposition of callose . Other virulence factors are cell wall degrading enzymes that enable B. cinerea to cause plant cell lysis and loosen walls to facilitate tissue penetration .

The fungus is known to produce plant hormones or hormone analogues that may disturb the host’s cellular metabolism and immune responses. The relevance of these mechanisms for the capacity of B. cinerea to infect strawberry remains unknown.Grey mould in strawberries can result from B. cinerea infections of open flowers or by penetration of fruit receptacle tissues . In primary infections, B. cinerea infects flower organs during or right after flowering, allowing hyphae to grow into the receptacle . The sources of primary inoculum range from overwintering sclerotia to conidia or mycelium from infected neighbouring plants . Infected senescent petals, stamens and calyxes can facilitate primary infections in fruit . Histological studies have shown that even though styles are frequently infected, fungal growth appears to be strongly inhibited and never reaches the receptacle. In contrast, fungal growth in colonized stamens can reach the receptacle in some cultivars . Following infection of the unripe receptacle by B. cinerea, fungal growth is usually arrested and a symptomless quiescent phase occurs. The mechanisms that lead to quiescent infections are not yet fully understood. Proanthocyanins appear to induce B. cinerea quiescence in unripe fruit by restricting the activity of fungal enzymes like polygalacturonases that are necessary for aggressive infection of hosts . Even though PA content in fruit remains constant during ripening, increasing polymerization of PAs leads to lower inhibitory activity in ripe fruit . Similarly, anthocyanins might delay B. cinerea infections or cause quiescence . For instance, strawberries illuminated with white fluorescent light showed increased anthocyanin content and delayed development of grey mould . Reduced fruit decay has also been observed in raspberries with high pigmentation and in transgenic tomatoes that accumulate anthocyanins .

Other small phenolics, especially catechins, may have a role in quiescence. High levels of catechins inhibit fungal growth, and a decrease in catechins is correlated with a reduction of other anti-fungal compounds such as lipoxygenases . Interestingly, young and ripe fruit have low catechin concentration, suggesting that initial infections of young receptacles are possible because they do not yet accumulate enough catechins to stop colonization . B. cinerea quiescence is complex and involves additional factors besides the accumulation of phenolic compounds. It has been proposed that quiescence in unripe fruit is initiated by: lack of nutrients such as sugars from the host, presence of preformed anti-fungal compounds, unsuitable environment for fungal virulence factors . In unripe strawberries, factors from all three categories are present, including lack of available sugars , preformed anti-fungal compounds , and high activity of PG-inhibiting proteins . Induction of the necrotrophic phase in ripe strawberries could be triggered by changes in biochemical composition of the host tissues associated with the ripening process, such as increased sugar content, volatile production and alteration of plant defences . These modifications promote not only fungal growth but also host susceptibility, e.g. via the release of oxalic acid and efflux of toxins . During secondary infections, the fungus initiates the necrotrophic phase without quiescence . The sources of conidia for secondary infections can also be diverse, from senescent leaves to infected fruit . Conidia from B. cinerea-infected flower parts are major sources of secondary inoculum . It has been estimated that more than 64% of the strawberry infections result from organic fragments that are in contact with the fruit, such as petals and stamens . Contrary to other fruit , senescent flower parts often adhere to strawberries long enough to retain water films for at least 8 h, which is the time needed for B. cinerea conidia germination . Secondary infections can also result from nesting, which corresponds to direct penetration of mycelia growing on neighbouring plant organs such as infected leaves and fruit . Generally, secondary infections proceed rapidly and B. cinerea can complete its germination and infection as fast as 16 h post-inoculation with a rapid increase in fungal biomass at 48 hpi . Early responses of strawberries to infection include higher expression of the defence genes FaPGIP and FaChi 2-1 , 25 liter square pot whereas lower expression of the reference gene DNA Binding Protein – FaDBP indicates extensive cell death induced by B. cinerea at late stages of infection .Fruit ripening influences the susceptibility of strawberry fruit to B. cinerea . Strawberries are mostly resistant to infection in their unripe stage, where they restrict fungal growth by causing quiescence. However, in the ripe stage, strawberries are highly susceptible and decay rapidly. Fruit susceptibility to fungal disease increases as ripening progresses; hence, B. cinerea appears to promote susceptibility in unripe fruit by activating specific ripening-related processes . In tomato fruit, master transcriptional regulators of ripening have been shown to have different roles in disease susceptibility. For example, the activity of the tomato transcription factor NON-RIPENING favours B. cinerea infection . Strawberries are non-climacteric fruit with a ripening programme different from that of climacteric tomatoes. Thus, a deeper understanding of strawberry ripening regulation and how B. cinerea may modulate particular ripening events are pivotal to characterize the dynamics of the strawberry-B. cinerea pathosystem.

Recent transcriptomic studies of developing strawberries point out that ripening events start between the ‘large green’ and ‘white’ stages, and involve changes in cell wall composition, sugar metabolism, hormone biosynthesis and responses, pigmentation and antioxidant levels . Moreover, a general decrease of oxidative phosphorylation processes has been observed during strawberry ripening . Normal strawberry ripening involves a variety of biochemical and physiological processes, some of which are discussed below in the context of B. cinerea interactions.Ripening is associated with the disassembly of the fruit cell walls, which leads to tissue softening. Cell wall degradation benefits B. cinerea as it reduces mechanical barriers to infection and spread, increases the possibilities of bruising and provides the fungus with access to simple sugars as a carbon source . In strawberry, cell wall solubilization occurs early in fruit development when the walls start to swell . Cell wall solubilization correlates with an increase in fruit sugar content, resulting from polysaccharide breakdown. A decrease of acid-soluble pectins and the alcohol-insoluble fraction of cell walls occur during ripening, whereas the water-soluble content increases . The degree of pectin solubilization and depolymerization is highlyrelated to strawberry fruit firmness . Silencing of an endogenous pectin lyase gene in strawberry resulted in fruit with higher external and internal firmness, mostly due to low pectin solubilization, stiffer cell walls, and increased cell to cell adhesion . Besides PL, other enzymes that may have affected strawberry firmness include PGs, β-galactosidases, endoglucanases, α-arabinofuranosidases and β-xylosidases . In addition to the fruit endogenous cell wall disassembly, B. cinerea secretes an extensive array of CWDEs that target most polysaccharides in the fruit cell walls, particularly pectins . These CWDEs include fungal PGs, such as Bcpg2, a gene that is mainly active in the early penetration stage . The expression of B. cinerea PGs is dependent on the host species, the plant tissue, temperature and the stage of infection .During ripening, the content of sugar in strawberries increases and therefore can serve as nutrients for B. cinerea. In unripe strawberries, the main sugars are glucose and fructose with low concentrations of sucrose. Sucrose levels increase rapidly during de-greening and red colouring . In tomato, it has been shown that the Cnr mutant, which does not accumulate high levels of sugars is still highly susceptible to B. cinerea infection . This observation suggests that even though sugars may serve as a susceptibility factor, high sugar concentrations are not essential for B. cinerea infection. However, sugar content could still influence susceptibility to B. cinerea as specific sugars may serve as ripening initiation signals. For instance, sucrose regulates abscisic acid levels in strawberries, which are necessary for normal ripening and could influence fruit susceptibility as described below . Like other ripening-related events, B. cinerea can alter neutral sugar and sugar acid levels in the infected host tissues, mainly by degradation and depolymerization of cell walls. This was reported for infections in tobacco and Arabidopsis leaves, where the fungus degrades pectins to release the monosaccharide galacturonic acid . ABA biosynthesis during fruit ripening is triggered by a decrease in pH, turgor changes, sugar accumulation, and the switch of sugars from mainly glucose and fructose to sucrose . Effects of ABA on strawberry susceptibility to fungal disease have not been extensively studied, but down regulation of the ABA biosynthetic gene β-glucosidase FaBG3 has been reported to result in fruit with limited ripening and higher B. cinerea resistance . In tomato, ABA accumulation is related to higher pathogen susceptibility, probably via activation of senescence . During strawberry ripening, the increase of ABA is correlated with a decrease of auxin, which induces early fruit growth and expansion but is known to inhibit ripening processes . The role of auxin in fruit susceptibility seems to depend on the plant species, as indole acetic acid treatment in Arabidopsis leads to susceptibility, whereas IAA-treated tomato leaves and eggplant fruit show lower infection severity . Ethylene has a secondary organ-specific role in strawberry ripening, particularly in achenes and green and white receptacles .

These values are higher than for the majority of domesticated fruits commonly reported, closest to very sweet fruits like pineapple and papaya ,access date 19 April 2010. The mean value for °Brix increased 22% from wild to cultivated fruits, from 13.6 to 16.6. It is interesting to note that even the wild fruits are very sweet, which may have predisposed this species to early selection as a cultivar. The reduction we saw in levels of phenolics in the pulp is consistent with a human preference for fruits with less bitterness . However, while a reduction in phenolics may indicate direct selection for palatability, it may also reflect an indirect effect of selection on the production of larger, sweeter fruits. Rosenthal and Dirzo suggest that selection for yield may result indirectly in the reallocation of energy away from defense, because of physiological constraints and tradeoffs. While all these differences were significant, the Discriminant Analysis showed that the great majority of the variance distinguishing wild from cultivated classes was driven by fruit size, with sugar concentration also contributing. This suggests that humans may have selected primarily for increased fruit size in this species, a hypothesis that could be tested with ethnobotanical studies. ENVIRONMENTAL VS. GENETIC CONTRIBUTIONS Many of the traits we measured, black plastic plant pots such as fruit size or sugar content, could be influenced by environmental conditions. Cultivated trees are likely to experience less competition for light, water, and nutrients than do wild trees.

However, several lines of evidence suggest that there is a large genetic component to the traits that distinguish wild from cultivated trees. First, both classes of trees, but especially the wild individuals, came from a wide range of environmental conditions. Some of the wild trees are now in open areas because the forest was cut around them, or are on the edges of forest fragments and therefore have access to more light and possibly nutrients than wild trees in the center of the forest. When we categorize the wild trees for their location, we see no trend toward differences in °Brix between trees on edges or in the open vs. trees in closed-canopy forests . We do see evidence suggesting that edge fruits areslightly bigger, a difference of about 10 g; this is substantially lower than the 73 g difference between the wild and cultivated means. Second, despite year-to-year variation in environmental conditions such as timing and intensity of the dry season, we saw very high correlations between years in trait values, especially for traits related to fruit size. We have recently planted a broad sample of wild and cultivated genotypes in two field plantations in Panama, which in 10 to 20 years should provide a better estimate of the genetic contribution to variation in this species.For most traits, we found no difference in trait variance between wild and cultivated samples. However, we did find significantly greater variance among cultivated trees than among wild trees for fruit mass and seed number . In contrast, there was lower variance among cultivated trees for the concentration of phenolics in pulp.

Through the process of domestication, we expect to see genetic variation strongly reduced for “domestication genes,” those traits that are critical for bringing the species into cultivation, while genetic variation should increase for “crop diversification genes,” reflecting a range of preferences during selection . Domesticated plants often show more intraspecific variation than their wild relatives for traits of interest to people , even in cases where there is an overall reduction in genetic diversity due to a domestication bottleneck . The reduced variance we found for phenolics may indicate consistent selection against bitterness, while the increased variance for fruit size could possibly reflect a range of preferences by the distinct cultural groups that utilize this species in central Panama, including Emberá and Wounaan communities, as well as people of Hispanic and Antillean descent. As mentioned earlier, caimito in this region may also include some genotypes transported from the Antilles. Ethnobotanical studies aimed at understanding cultural preferences, selection targets, and selection intensity could help elucidate this issue. However, it seems more likely that the broad range in fruit size is generated by a lack of fixation of alleles associated with domestication due to a contemporary or historical influx of wild-type genes, as well as recombination among cultivated genotypes. Zohary and Spiegel-Roy have suggested that most fully domesticated tree crops are asexually propagated, at least in the Old World. This is thought to increase uniformity and reduce the production of unwanted intermediate forms, particularly as many tree crops are highly heterozygous and show an outcrossing sexual system. Chrysophyllum cainito is not propagated asexually in Panama.

The mating system of C. cainito has not been studied, but the species has small flowers that are pollinated by small bees , and we expect that it is primarily outcrossing like similar tropical lowland tree species . We suggest that the extremely wide range of variation in fruit size among cultivated phenotypes is what might be predicted for a fruit tree species in the early stages of domestication. This is consistent with the findings of Leakey et al. for the semidomesticated fruit trees Dacryodes edulis and Irvingia gabonensis. However, unlike those species, the distributions of traits in our cultivated samples of C. cainito did not show statistically significantly more skew than wild populations .We found a strong positive correlation between fruit size and both seed number and seed size. A positive correlation between fruit size and seed number has also been reported in sweet pepper and tomato , and for these species the genetic basis is known. The correlations that we observe in C. cainito may be due to pleiotropic effects, tight linkage of loci, or a combination of these factors. From a physiological perspective, fruit size may be directly related to seed number because the developing seeds are a source of auxin for the developing fruit; therefore, an increase in number of seeds results in a larger fruit . In addition, a general “gigas effect” in domesticates can produce an increase in organ size through increased cell number and/or cell size reviewed in Pickersgill 2007, which could also explain the positive correlation of fruit size and seed size/number observed in caimito. Therefore, even if human preferences were in the direction of smaller seeds, as is the case in tempesquistle , or fewer seeds, genetic and physiological factors may constrain seed size and number in cultivated C. cainito.Nearly one-third of the world’s food production is lost or wasted through the food supply chain, according to the Food and Agriculture Organization. Fungal pathogens are responsible for causing diseases like rots and molds that result in reduced product quality, shelflife, and market value, leading to significant losses of harvested fruits and vegetables in post harvest. Fungal pathogens can gain access to the fruit tissues in different ways: by establishing latent infections of flowers, exploiting wounds and natural openings , or by directly penetrating the host cuticle. Vector insects can also cause damage, which is a common entry point for pathogens. Additionally, mishandling and physical damage of the products during harvest, sorting, packing, transportation, cold storage, and retailing contribute to a higher incidence of disease. The germination of fungal spores requires moisture and stimulation from host solutes that diffuse into the initial penetration site. After germination, specific signals from the plant surface trigger the formation of specialized fungal structures that enable the pathogen to penetrate the plant cell walls and establish infection. Once the disease is well established in one fruit, black plastic planting pots it spreads quickly to adjacent healthy fruit, a process known as nesting. A well-known nesting pathogen is Botrytis cinerea, which releases airborne conidia that readily nest on damaged or senescent fruits, initiating decay and facilitating further spread. Rhizopus stolonifer, the causal agent of Rhizopus rot in various fruits and vegetables, is another prominent example of a nesting pathogen. Following spore germination, R. stolonifer produces mycelial stolons that attach to the host surface, enabling it to colonize healthy fruits and initiate infections. Penicillium spp. are commonly considered wound-dependent pathogens; however, it is commonly observed that if the fungi are initially established in a rich food source like a decaying fruit, the mycelium can readily invade the tissues of an adjacent healthy fruit. This phenomenon is how an initial low incidence of green or blue mold in a packinghouse storage facility can develop into major losses after prolonged fruit storage. Integrated pest management strategies have been developed to reduce or eliminate fruit infections, including synthetic fungicides before and after harvest, biological control agents, essential oils, cold storage, and modified atmosphere packaging.

To test the effectiveness of these strategies, reliable laboratory or field-based inoculation methods are required to obtain quantitative data that goes beyond subjective ordinal rating scales that may be influenced by human bias. While natural infections can provide insights into disease dynamics in real-world scenarios, they can be unpredictable and impacted by environmental factors, making it difficult to control and replicate experimental conditions. Therefore, the study of plant-pathogen interactions generally relies on pathogen inoculation techniques. Dip and spray are two common inoculation methods where fruits are covered in a fungal spore suspension by submersion or application with an atomizer. These methods allow for uniform, whole-fruit inoculation but may result in lower disease incidence and severity due to the challenges in standardizing the process. Another method is wound inoculation, which involves creating artificial entry points on the fruit surface before applying the fungal spore suspension or mycelial plug. This method simulates wounds and enables precise and reproducible experiments. Still, it may not accurately represent the natural infection process as it bypasses the initial steps of adhesion and penetration on the plant tissues. As of present, our comprehensive literature review has not revealed any reported methods that faithfully replicate the post harvest nesting phenomenon. This study presents a novel methodology for assessing post harvest infections of persistent fungal pathogens through contact-based inoculation of fruits. We optimized this protocol using four impactful fungal pathogens and post harvest commodities that are commonly affected by fungal disease: Botrytis cinerea and Penicillium expansum in tomato and apple, respectively, and Penicillium italicum and Penicillium digitatum in orange. B. cinerea is the causal agent of gray mold, a devastating disease that causes billion-dollar losses on fruit commodities worldwide. P. italicum and P. expansum, causal agents of blue mold, and P. digitatum, causal agent of green mold, are significant post harvest diseases. Our protocol unveils new possibilities for testing disease management strategies and studying the nesting behavior of post harvest fungal pathogens.We conducted several trials to determine the best source of fruit tissues for contact-inoculation. The developed inoculation method involves producing the source fruits through wound inoculation, and preparing the non-wounded target fruits. The selected source and target fruits ideally should not have any surface imperfections such as scars, wounds, or bruises, and apples should have an intact pedicel. Fruits were first disinfected in 10% sodium hypochlorite, rinsed twice in sterile Milli-Q, and dried with sterile tissue paper before inoculation. Source fruits were wounded multiple times on the stem end. For oranges, three equidistant wounds were created using a sterile nail following the protocol developed by Vilanova et al.. For tomatoes and apples, equidistant wounds were created in four locations with a sterile pipette tip . Oranges were inoculated with P. italicum or P. digitatum, while tomatoes and apples were inoculated with B. cinerea and P. expansum, respectively. Source fruits were incubated under high relative humidity at 10 °C for 10 and 13 days for orange, 20 °C for 4 days for tomato, and 10 days for apple. By then, the source fruits should have developed lesion sizes of about 30, 10, and 20 mm for oranges, tomatoes, and apples, respectively. In initial attempts, we used culture media plugs with well-established fungal mycelial growth as the inoculum source. However, this method was not always successful and did not accurately mimic how fungal infections occur while handling and storing fresh produce.Two different inoculum sources were explored for the contact inoculation: whole fruits and fruit tissue sections. When using whole fruits as the source, target fruits were placed on a plastic boat lying on the equatorial region.