Horticulture

White or coextruded white-on-black mulches can slightly lower surface soil temperatures by about 1ºC at 2 cm depth or 0.4ºC at 10 cm relative to bare soil because they reflect most incoming radiation . These mulches are used when lower soil temperatures may be desirable for planting vegetables in particular summer production windows. Clear mulches effectively retain much of the heat normally lost to the atmosphere by bare soil, increasing daytime soil temperatures from 4º to 8ºC at a depth of 5 cm , and 3º to 5ºC at 10 cm relative to bare soil . These clear plastics are the choice for soil solarization. Clear plastics, however, do not control weeds and require other weed management practices such as fumigation and herbicide application. Plastic mulches also influence nutrient levels and uptake. Wien and Minotti found plastic mulching increased shoot concentrations of nitrogen , nitrate , phosphorus , potassium , calcium , magnesium , copper and boron in transplanted tomatoes. Bhella , also working with tomatoes, 30 litre plant pots found higher levels of ammonium , nitrate , and magnesium in plastic mulched soils.

Hassan et al. found higher levels of nitrogen, phosphorus, potassium, and calcium in leaf tissue of chilies grown over plastic reflective mulch compared to those grown over bare soil. A wide variety of other colored plastic mulches, including red, yellow, silver, blue, gray, and orange, have been used in various efforts to achieve specific production goals. Each of these colors has distinct spectral reflectivity characteristics and thus modifies the radiation balance in and below a crop canopy. These colored mulches affect not only the microclimate around a crop but have also been shown to influence insect behavior. Yellow, for example, is generally highly attractive to insects and has been shown to increase green peach aphid and striped and spotted cucumber beetle populations compared to plants grown over bare soil . White at times repels aphids and at other times attracts them, depending on the physiological state of the insect . Orange has been shown to repel various aphids and white flies , while pink and green attract aphids . Red has been shown to attract both aphids and white flies . Highly reflective or shiny aluminum plastic mulches have been shown to repel certain aphids and thereby reduce or delay the onset of aphid-vectored mosaic viruses in zucchini squash and melons and tomato spotted wilt virus in tomatoes . These are the mulches of choice when insect and disease management are the principal objective. On balance, other than UV-metalized reflective mulches, repeated and consistent benefits in managing insects with most colored mulches have not been documented.

Colored plastic mulches have also been used in many parts of the United States to enhance yields. These mulches, however, have produced mixed results . Mahmoudpour and Stapleton noted that “the influence of mulch colour on growth and productivity has been postulated to be highly specific, and may vary with plant taxa, climate, and seasonal conditions.” As is the case with insect management, aluminum mulches have provided the most positive and consistent findings on crop production .Using cover crops as mulches is a relatively recent management strategy that is currently being refined and evaluated in a wide range of vegetable production systems. The winter annual legume hairy vetch , for example, has been used successfully as both a cover crop and as a mulch in fresh market tomato production systems in the southeastern United States. As a cover crop, the vetch fixes nitrogen, recycles nutrients, reduces soil erosion, and adds organic matter to the soil. When mowed and converted to a mulch, the vetch reduces weed emergence, lowers soil temperatures during the hot summer months, reduces water loss from the soil, and acts as a slow-release fertilizer . This system, developed by USDA ARS researchers, eliminates tillage, reduces the need for applying synthetic fertilizers and herbicides, and reportedly adapts to both large- and small-scale tomato production in a low-input, no-tillage system. Recent work in Florida by Chellemi et al. has shown that although a cover crop surface residue mulch production system had lower yields than the standard black polyethylene plastic, the overall profitability of the alternative system was actually higher.

Work in California’s Central Valley has shown that cover cropping increases water infiltration and reduced winter runoff and increases soil carbon . Additionally, cover crops, when cut and dried, have been shown to delay and reduce the incidence of aphids and white flies as well as the incidence of aphidborne viruses. Burton and Krenzer observed a reduction in green bug populations where surface residues of a previous wheat crop existed. Summers et al. found wheat straw mulch significantly delayed and reduced the incidence of alate aphids and several aphidborne cucurbit viruses in zucchini squash. The incidence of silver leaf whitefly and squash silver leaf was also significantly reduced. Similar results were obtained with cantaloupe grown over wheat straw residue. A number of other examples of the successful use of cover crop mulches have been reported in Georgia, Virginia, North Carolina, and Pennsylvania, but their potential in California’s vegetable crop production is only now beginning to be investigated, evaluated, and refined. Combining the potential benefits of surface residue cropping alternatives with those of conservation tillage is becoming increasingly attractive to row crop producers in many of California’s agricultural regions as shown by the following case studies.Beginning in 1995, a series of studies and demonstrations were initiated in the Central Valley to evaluate and develop conservation tillage and cover crop mulch production systems for tomato crop rotations. While the immediate goal of these efforts was to reduce production costs, a longer-term objective was to develop information on the potential of reduced tillage to improve soil quality, store carbon in the soil, and conserve resources. Initial studies, conducted at the University of California West Side Research and Extension Center in Five Points, at the UC Davis campus, and in commercial production fields in Tracy, Vernalis, and Le Grand, evaluated the use of winter cover crops as surface mulches, the feasibility of no-till and strip-till transplanting, and options for in-season weed management. No-till transplanting requires the use of coulters or some form of residue manager to cut surface residues ahead of the transplanter shoe. Strip-till is a form of CT in which a set of coulter or shank implements tills a narrow band of soil 15 to 20 cm wide to a depth of about 8 to 36 cm only in the line into which transplants will be placed. Results from these preliminary evaluations have indicated that planting and harvesting both processing and fresh market tomatoes is possible and that yields comparable to those attained using standard winter fallow techniques may be achieved with certain reduced-till approaches that do not result in excessive cover crop regrowth or weed competition with the tomato crop. On-farm strip trial data for demonstrations conducted in 1999 in Tracy and in 2000 in Vernalis are given in table 3. This early work, and other experiments summarized by Herrero et al. 2001b, also revealed that in-season weed control by a surface cover crop mulch alone is not adequate. The authors of this publication have subsequently investigated and refined the use of a high-residue cultivator that is able to effectively slice through residues while cultivating weeds. Cover crop mulch species selection and mulch management must be optimized if organic mulch tomato production is to expand in California. Care must be taken to avoid the use of certain cover crops such as sorghum-Sudan hybrid as mulches because they are highly allelopathic to tomatoes and several other vegetable crops . More efficient and low-risk production protocols for managing cover crop mulches in vegetable crop rotations must also be developed.Aphid-transmitted virus diseases cause significant economic losses to California’s multi-million-dollar vegetable crop industries annually.

Over the past few years, 25 liter pot plastic production of fall melons , squash , peppers , and tomatoes has been extremely difficult in certain regions of the San Joaquin Valley due to extensive virus epidemics and severe infestations of silverleaf whitefly . Spring crops, while affected to a lesser extent, have also suffered significant losses by aphid-transmitted viruses and whitefly infestations.Several plant viruses are responsible for these epidemics, and most are capable of infecting all of the crops mentioned above. Among the most important viruses are cucumber mosaic virus , zucchini yellows mosaic virus , potato virus Y , and watermelon mosaic virus . These viruses are transmitted by aphids in a styletborne, nonpersistent manner. They are acquired and transmitted in as few as 15 seconds, and are transmitted by a large number of aphid species, all of which are abundant throughout California. Due to the rapidity with which the viruses can be acquired and transmitted, insecticides are of little value in preventing virus spread and under some circumstances may actually increase the rate of virus transmission and spread. This has not, however, dissuaded a large number of growers and PCAs from attempting to control the spread of these viruses by using insecticides. UV-reflective mulches consist of a polyethylene base to which a thin coat of aluminum ions has been adhered. The mulches are collectively referred to as metalized mulches. These mulches reflect UV wavelength , which confuses and repels incoming alate aphids, adult whiteflies, and leaf hoppers , reducing their incidence of alighting on plants . UV-reflective plastic mulches have been used successfully to reduce the incidence of aphidborne virus diseases in squash and other crops . Brown et al. found silver plastic mulch superior to white, yellow, or black with yellow edges in repelling aphids in yellow crookneck summer squash. Plants grown on silver mulch produced significantly higher yields of marketable fruit than did those grown on bare soil. Mulches applied to the planting beds before seeding were effective in repelling alate aphids and delaying the onset of several virus diseases as well as the onset of silver leaf white fly colonization and the appearance of squash silver leaf in spring and fall-planted zucchini squash in California’s San Joaquin Valley . Disease symptoms in plants growing over these mulches appeared 10 to 14 days later than in plants growing on unmulched beds. In spring-seeded squash, approximately 30 percent of the plants on unmulched beds were infected with one or more viruses by the first harvest, while only 10 to 15 percent of those grown over the metalized mulches showed virus symptoms. In fall-planted trials, 100 percent of the plants grown on unmulched beds, with and without insecticide applications, were virus-infected by the first harvest. Metalized mulches were generally more effective in repelling aphids and delaying virus onset than were white-pigmented mulches . Although plants grown over the metalized mulched plots eventually became infected, they continued to produce a significantly higher percentage of marketable fruit throughout the season than did the unmulched controls. In addition, squash, cantaloupes, cucumbers, and corn grown over reflective mulch produced marketable fruit 7 to 10 days earlier that plants growing over bare soil. Stapleton and Summers also showed than cantaloupe grown over reflective mulches yielded over 500 cartons of marketable fruit per acre compared to less than 50 cartons per acre from plants grown on bare soil. A delay of 4 to 6 weeks in infection by CMV in plants growing over reflective mulch allowed them time to mature and set a good crop of melon fruit before becoming infected. Even though the plants eventually became infected, the delay in infection permitted the harvest of a highly profitable crop. Summers and Stapleton have shown that tomatoes grown over reflective mulches averaged approximately 7 percent virus-infected plants, while plants grown over bare soil averaged in excess of 50 percent infection with the same viruses. This approach is currently the only viable means of managing virus disease in these production systems. Growers are cautioned to use only metalized reflective mulches when insect and 5 ANR Publication 8129disease management is the primary objective. Other colors lack the high degree of UV reflectance necessary to repel incoming insects. Also, the mulch must be applied prior to seedling emergence. Plants may be inoculated with aphidborne viruses in the cotyledon stage, and any delay in applying the mulch could lead to an extensive infection. Some crops are more susceptible to injury when planted over metalized mulches.

Another study giving older adults two egg yolks/day for 5 weeks, followed by four egg yolks/day for 5 weeks, reported increases in MPOD, but only among those with low baseline MPOD value. The addition of either spinach or corn , or the combination, for 14 months significantly increased the MPOD among the majority of healthy individuals. Our study has some limitations. Choice of a control is always a challenge in whole food studies, since masking is an issue. A commercially available LZ supplement was used, rather than an inert capsule, since our research design was intended to compare options available to consumers and explore the role of goji berries over and above the intake of purified L and Z. The actual amount of L and Z in the supplement was not confirmed. A previous report noted that the carotenoid content of some powder-based supplements tested in 2017 did not meet label claims, while oil-based supplements did. Since L and Z are preferentially deposited at different eccentricities in the retina, 30 plant pot the different amounts of Z in the goji berries and supplement may not be ideal. Volunteers were not screened for low MPOD as an inclusion criterion.

Although the relatively modest number of participants in each group may raise some concerns, these numbers are similar to those reported by Obana et al. and are consistent with an initial probe study. Finally, although MPOD was the primary outcome measure, other ocular measurements such as contrast sensitivity and best corrected visual acuity were not assessed. Future studies on goji berry intake and eye health ideally should combine functional and anatomic measurements.Viruses are obligate intracellular pathogens that require living host cells to replicate and spread in the infected plant. During compatible interactions, viruses overcome the plant immune system and hijack host cellular processes to establish active infections . Viruses disrupt the plant cell cycle, inhibit cell death pathways, restrict macromolecular trafficking, alter cell signaling, protein turnover, and transcriptional regulation, and suppress defense mechanisms. The interference with these processes in the host leads to a wide range of plant developmental and physiological defects . Cultivated grapevines are highly susceptible to a variety of viruses and viroids, which cause significant crop losses and shorten the productive life of vineyards. More than 65 different viral species classified in at least 15 families have been reported to infect grapevines, which represents the highest number of viruses so far detected in a single cultivated plant species .

Although these viruses are generally transmitted by plant-feeding insects or soilborne nematodes, they can also be spread through infected propagation material . Grapevine red blotch is a viral disease discovered in northern California in 2008 that has become a major economic problem for the wine industry in the USA . This disease is caused by the Grapevine red blotch-associated virus , a circular ssDNA virus with resemblance to geminiviruses, which infects wine grape cultivars with significant detrimental effects on productivity . The incidence and severity of the red blotch symptoms vary depending on the grape cultivar, environmental conditions, and cultural practices . In red-skinned varieties, GRBaV infections result in the appearance of red patches on the leaf blades, veins, and petioles; in white-skinned varieties, they manifest as irregular chlorotic regions on the leaf blades. GRBaV also affects berry physiology, causing uneven ripening, higher titratable acidity, and lower sugar and anthocyanin content, among others . Consequently, must and wine produced from infected berries present altered flavor and aroma. To date, there is limited information on how GRBaV infections affect grape metabolism. Comprehensive analyses to study specific cellular processes that GRBaV exploits to promote infections in berries are still needed, in particular those that relate to changes in berry chemical composition during fruit development. Grape berry development exhibits a double sigmoid growth pattern with three distinct phases: early fruit development, lag phase, and berry ripening.

Most metabolic pathways that promote desired quality traits in grape berries are induced during ripening. The onset of ripening is accompanied by significant changes in berry physiology and metabolism, including softening, sugar accumulation, decrease in organic acids, and synthesis of anthocyanins and other secondary metabolites that define the sensory properties of the fruit . Berry ripening is controlled by multiple regulatory pathways, and occurs in an organized and developmentally timed manner. Interactions between transcriptional regulators and plant hormones regulate the initiation and progression of ripening processes . Like other non-climacteric fruit, grape berries do not display a strong induction of ethylene production and respiration rate at véraison, and the activation of ripening events does not depend primarily on ethylene signaling. Even though the hormonal control of grape berry development is not completely understood, it is established that abscisic acid , brassinosteroids, and ethylene are positive regulators of ripening processes, while auxin delays the initiation of ripening . In the context of virus–grape berry interactions, dissecting the mechanisms that regulate ripening and plant defenses may provide new opportunities to develop vineyard management strategies to control viral diseases and ameliorate the negative effects on berry quality. In this study, we integrated genome-wide transcriptional profiling, targeted chemical and biochemical analyses, and demonstrated that grapevine red blotch disrupts ripening and metabolism of red-skinned berries. We sampled berries at different ripening stages from vines infected with GRBaV and healthy vines in two vineyards. We identified grape metabolic pathways that were altered in ripening berries because of the viral infection. We determined that GRBaV-induced pathways that are normally associated with early fruit development in berries at late stages of ripening, and suppressed secondary metabolic pathways that occur during normal berry ripening and/ or in response to stress. Using targeted metabolite profiling and enzyme activity analyses, we confirmed the impact of GRBaV on phenylpropanoid metabolism. We identified specific ripening-related processes that were disturbed in GRBaV-infected berries. Remarkably, these processes included alterations in ripening regulatory networks mediated by transcriptional factors, post-transcriptional control, and plant hormones, which lead to berry developmental defects caused by red blotch.To determine the impact of grapevine red blotch on berry physiology, we studied naturally occurring GRBaV infections in distinct wine grape-growing regions in northern California . We sampled red-skinned grape berries from two different vineyards, one in Oakville and one in Healdsburg . We used multiple vineyard sites to focus on observations consistently made across environments and, thus, to exclude factors associated with specific environmental or cultural conditions. Prior to sampling, vines were screened for the presence of GRBaV and other common grapevine viruses. The appearance of red blotch symptoms on leaves of GRBaV-positive vines and not on those of healthy controls confirmed the initial viral testing. We sampled grape berries from vines that tested positive for GRBaV and negative for other common grapevine viruses. At the same time, we also collected berries from vines that tested negative for all viruses and included them in the study as healthy controls. In order to determine the impact of the disease on berry development and metabolism, we collected GRBaV-positive and control berries at comparable developmental stages: pre-véraison , véraison , post-véraison , and harvest . This sampling strategy also aimed to limit confounding effects due to differences in the progression of ripening between berry clusters of GRBaV-positive and healthy vines. In some cases, we observed that GRBaV-positive vines presented grape clusters with evident uneven ripening . Comparisons between berries from GRBaV-positive vines and healthy controls indicated that, at equivalent stages of development, grow raspberries in a pot berries affected by red blotch had reduced soluble solids and total anthocyanins in agreement with previous reports on red-skinned wine grapes .

Sampled berries were used for genome-wide transcriptional profiling of viral and grape genes. RNAseq was performed using 3–4 biological replicates of each ripening stage, infection status, and vineyard. We first confirmed the presence of the virus in the berries of GRBaV-positive vines by qPCR amplification of viral DNA . Viral activity in the berries was also assessed by quantifying plant-derived mRNA transcripts of GRBaV genes in the RNAseq data. Plant expression of five out of the six predicted genes in the GRBaV genome was detected in all berry samples obtained from GRBaV-positive vines but not in berries collected from the control vines . The most expressed GRBaV genes in the berries corresponded to V1, which encodes a coat protein, and V3 with unknown function. Expression levels of the GRBaV genes appeared to change as berries ripened. However, we could not determine to what extent the progression of ripening or other environmental factors influenced the plant’s transcription of viral genes because their pattern of variation between ripening stages differed in the two vineyards . Expression of 25 994 grape genes was detected by RNAseq across all berry samples. Principal component analysis was carried out with the normalized read counts of all detected genes. The two major PCs, which together accounted for 42.97% of the total variability, clearly separated the samples based on ripening stage, regardless of the vineyard of origin or their infection status . These results indicated that the intervineyard variation was smaller than the ripening effect, and the overall progression of ripening was similar between berries from GRBaV-positive and control vines. Therefore, we hypothesized that GRBaV infections in berries have altered the expression of particular grape genes and/or molecular pathways, which could subsequently have led to developmental and metabolic defects.While the PCA described above indicated that overall transcriptome dynamics associated with berry ripening were not perturbed by the infection, the lower levels of soluble solids and anthocyanins in GRBaV-positive berries,particularly later in development, suggested that red blotch may affect specific primary and secondary metabolic processes. We therefore focused the RNAseq analyses to identify grape molecular pathways that were differentially regulated as a result of GRBaV infections. We identified grape genes with significant differential expression due to red blotch by comparing GRBaV-positive and GRBaV-negative berries at each ripening stage and independently for each vineyard. We then looked at the intersection of differentially expressed genes between the two vineyards to identify common responses to red blotch. A total of 932 grape DE genes were found to be consistently down- or up-regulated in infected berries in both vineyards at a given ripening stage, and were classified as GRBaV-responsive genes . On average these GRBaV-responsive genes showed 0.49 ± 0.22-fold changes compared with the healthycontrols. Comparing berries at similar ripening stages may have contributed to exclude more dramatic changes in gene expression associated with more pronounced ripening delay due to GRBaV. Key metabolic processes that were suppressed or induced as a consequence of red blotch in ripening berries were identified by enrichment analyses of the functional categories defined by Grimplet et al. in the set of GRBaV-responsive genes . Amino acid bio-synthetic pathways were repressed in GRBaV-positive berries, while amino acid catabolic pathways were induced. Changes in carbohydrate metabolism were also observed; in particular, genes involved in glycolysis/gluconeogenesis and starch metabolism had reduced expression in GRBaV-infected berries.Genes involved in nucleic acid metabolism, including RNA processing and surveillance, showed higher expression in GRBaV-infected berries. These pathways coincided at véraison with the induction of genes involved in stress responses to virus . RNA metabolic pathways are commonly altered in plants during viral infections and have been related to resistance or susceptibility depending on the particular plant–virus interaction . Red blotch also impacted the transcription of several abiotic stress response pathways. In particular, berries after véraison showed lower expression of genes encoding hypoxia responsive proteins and heat stress transcription factors, among others . A prominent feature of the GRBaV-positive berries was the transcriptional suppression of secondary metabolic pathways, in particular the biosynthesis of phenylpropanoids, stilbenoids, and lignin . Because the lower anthocyanin content observed in the GRBaV-positive berries may have resulted from reduced metabolic flux in the core phenylpropanoid pathway and alterations in flavonoid and anthocyanin biosynthesis, we pursued a deeper evaluation of these pathways using an integrated approach of transcriptional and metabolite profiling coupled to enzymatic analyses.Most enzymes involved in phenylpropanoid metabolism are encoded by large gene families. There is also high redundancy among these genes, which ensures the functional integrity and plasticity of the phenylpropanoid-related pathways . Therefore, to test the hypothesis that the red blotch-induced transcriptional changes had an actual impact on phenylpropanoid metabolism, we measured the activity of key enzymes and the abundance of compounds involved in these pathways .

Raspberries account for the majority of the production volume and the total value of this category. This California export category was valued at $157 million, and accounted for approximately 85% of total US fresh and processed raspberry, blackberry, mulberry, and loganberry exports. 84% of these exports are received by Canada, 6% by Japan, and 5% by the European Union. The presence of SWD has clearly increased production costs and caused yield losses for California raspberry producers through a variety of channels. Three previous studies have attempted to quantify the economic cost of the SWD invasion. However, these studies occurred within one or two years of the first SWD infestations in North America when information on the pest was still sparse and management techniques were rapidly evolving. We can improve on these original estimates now that much more is known about SWD biology, risks, and management. We briefly review these original studies before establishing new estimates of the economic cost of SWD in the California raspberry industry. Walsh et al. 1 and Bolda et al. are the first studies to estimate the economic cost of SWD.

These studies utilize yield loss estimates and observations for strawberries, blueberries, raspberries plant container, blackberries, and cherries in California, Oregon, and Washington in conjunction with production data to calculate revenue loss estimates for each state and crop pairing. Walsh et al. assume a yield loss of 20% for all the listed crops in these states. As a result, the study estimates a total of $511 million in potential damages annually due to SWD. Bolda et al. continue the analysis by assuming the maximum reported yield losses of 40% for blueberries, 50% for blackberries and raspberries, 33% for cherries, and 20% for processing strawberries. The study concludes that potential revenue losses across these states and crops could be as large as $421.5 million given current prices. Goodhue et al. 18 refine these estimates of lost revenue for the California raspberry and strawberry industries by including potential price responses into their estimates. This additional assumption reflects that as the production of raspberries and strawberries decreases, the prices of these products may increase in response. The interaction between production and price is quantified with the inverse own-price elasticity of demand for each crop. The elasticity predicts the percentage change in price of a good in response to a 1% increase in quantity demanded. Drawing upon elasticity estimates established in prior studies, the authors conclude that SWD-induced yield losses could decrease California raspberry and processed strawberry revenues by up to 37% and 20%, respectively.

The authors also evaluate the cost of different SWD-targeting insecticide applications and the cost of a specific conventional raspberry pest control program in California’s Central Coast region. The insecticide material and application costs are estimated to be $825.33 per hectare. However, these chemical applications may also provide incidental control of other pests. This implies that the estimate represents an upper bound of the potential chemical control costs associated with SWD.18 The revenue loss and management cost estimates in these prior studies can be substantially improved using current information about SWD-induced yield losses and management practices. Fruit losses due to SWD and SWD management costs have decreased over time as researchers and producers have developed and implemented better techniques for reducing crop losses. We can also more accurately estimate historic yield losses now that more is known about SWD biology, its spread, and the efficacy of different management techniques.Lastly, we can now incorporate increases in labor costs into these SWD management cost estimates.Prior estimates of SWD-induced revenue losses were based on the maximum observed yield losses in different industries where SWD infestations occurred. These estimates provide information about SWD’s damage potential, but do not yield an accurate estimate of actual SWD crop damage. Actual crop damage is useful for estimating revenue losses due to SWD and will differ by year and production style. This analysis incorporates field trial results and expert opinions to estimate SWD-induced revenue losses for the California raspberry industry. SWD infestations directly reduce raspberry yields in two ways. First, fruit infested by SWD decay more quickly. These yield losses are difficult to attribute to SWD because the initial infestation is difficult to detect, and the accelerated decay has a similar appearance to decay caused by fungal diseases, bacteria, and yeasts. Second, raspberry shippers that detect SWD infestations may reject the entire delivery from the grower.

Fresh fruit are held to rigorous quality standards. The risk of rejection of an entire delivery incentivizes growers to eliminate all visible defects in harvested fruit. SWD infestations are more prevalent late in the year as the population grows until winter weather reduces the population. Further, raspberry production is fairly concentrated geographically and the leftover, overripe fruit from nearby fields’ summer harvest acts as a breeding ground for SWD. SWD infestations are also more prevalent in fruit destined for the processing market, where the price is lower than in the fresh market. Fruit intended for processing are harvested later in the season, tend to be riper because they are harvested less frequently, and receive less frequent pesticide treatments. SWD damage rates could change significantly in the future due to pesticide resistance development and the introduction of new SWD management practices, including introducing biological control agents. Recent studies in the US and Europe found that indigenous parasitoids had limited effect on SWD populations. However, in Asia, where SWD originates, several endemic parasitoids attack and develop from SWD. We begin by examining SWD-induced yield losses in California’s conventional raspberry industry. The original reports of SWD damage in the raspberry industry indicated that as much as 50% of production could be lost if SWD was left unmanaged. Yield losses of this magnitude occurred as raspberry producers first learned how to manage SWD, but are now uncommon due to implementation of extensive academic research and industry experience. According to private communications with conventional raspberry producers, they have managed to reduce SWD induced yield losses to less than 3% of production. In recently published reports, conventional raspberry producers that employ effective chemical management programs face virtually no yield losses due to SWD. This substantial reduction in yield losses is primarily attributable to two factors. First, conventional raspberry producers have access to cheap and effective chemical management options. Second, these producers are harvesting their crop more frequently in order to reduce the amount of time raspberries are susceptible to infestation. These observations of actual SWD-induced yield losses are consistent with field trial observations as well. Entomologists Kelly Hamby and Frank Zalom monitored traps and evaluated fruit samples for damage between October 2010 and December 2012 in both organically- and conventionally-managed raspberry sites. Analyzing the 40-fruit samples collected from these fields resulted in estimated yield loss observations for raspberry producers employing standard management practices at the time. SWD-induced yield losses for conventional producers in the study were estimated to be approximately 10% of production in 2011 and less than 1% in 2012. These estimated yield losses are consistent with those observed by De Ros et al. in Italy between 2011 and 2013. De Ros et al. estimated container raspberry losses of 11.5% prior to i and 3.24% after the implementation of an integrated strategy. The yield losses observed in the UC Davis study were concentrated in the fall harvest. The summer harvest is hypothesized to experience less SWD pressure because the population grows throughout the year until cold weather arrives and lack of host fruit in the winter significantly reduces population levels. SWD biology and infestation intensity is affected by climatic conditions and the availability of host fruit, implying that different climatic conditions and influences of neighboring crops could significantly impact SWD-related yield losses. On the other hand, organic raspberry producers still face significant SWD-induced yield losses. Private communications with raspberry producers indicate that these producers experience yield losses between 5% and 15% of production due to a lack of efficacious chemical treatments approved for organic use, and the efficacy and high cost of other labor-intensive SWD management practices.

Once again, these field observations are consistent with the yield losses measured in field trials. SWD-induced yield losses for organic raspberry producers in the study were estimated to be approximately 12% of production in both 2011 and 2012. We calculate yearly estimates of industry-level revenue losses using these observed yield losses due to SWD and a procedure similar to Goodhue et al. . First, we assume an ownprice elasticity of demand for raspberries of -1.66. This elasticity value is the value estimated for fresh raspberries by Sobekova, Thomsen, and Ahrendsen . Second, we assume that actual yield losses in the California raspberry industry correspond to the yield losses observed in the field trials. Specifically, we assume that SWD-induced yield losses between 2009 and 2011 correspond to the yield losses observed in 2011, and that losses after 2011 correspond to the yield losses observed in 2012. Raspberry production and price data are obtained from the U.S. Table 2 provides the resulting revenue loss estimates organized by production practice and year grouping. California’s conventional raspberry producers faced a total of $36.1 million in revenue losses due to SWD between 2009 and 2011. These estimated revenue losses are equivalent to 4.62% of realized revenues over the same period. After 2011, effective SWD management techniques in conventional production eliminated virtually all revenue losses. Revenue losses due to SWD between 2011 and 2014 are estimated to be $277 thousand, which is less than 1% of realized revenues over the same period. In total, California’s conventional raspberry producers faced $36.4 million in revenue losses due to SWD between 2009 and 2014. California’s organic raspberry producers faced a total of $3.43 million in revenue losses due to SWD between 2009 and 2014. These estimated revenue losses are equivalent to 5.74% of realized revenues over the same period. Revenue losses of this magnitude are expected to continue in organic raspberry production until more effective chemical, cultural, or biological management programs are discovered. Furthermore, revenue losses incurred by organic raspberry producers could potentially increase dramatically if SWD populations develop greater resistance to the current, limited set of chemical controls approved for organic use.SWD management is multifaceted. In addition to yield losses, managing SWD has significantly increased production costs for raspberry producers. Raspberry growers increase the number of insecticide applications and use additional labor to harvest their crop in response to SWD infestation pressure. These necessary insecticide applications require additional chemical purchases and access to sprayers and specialized equipment through custom application or purchase. Overuse of pesticides can lead to rejections of shipments if residues exceed legal tolerances for the chemicals; however, producers who adhere to mandatory label rates should, theoretically, never encounter this problem. Conventional raspberry producers have access to a variety of insecticides that provide excellent control for SWD populations at present. Raspberry growers observed in the UC Davis study discussed earlier applied SWD-targeting insecticides four to six times for both the fall and spring harvests. The most commonly used insecticides for this purpose were spinetoram, zetacypermethrin, and malathion. Assuming these chemicals are applied at their maximum label rates and with generic purchase prices observed in 2015, the per hectare material costs of these insecticide applications are $179.40, $7.22, and $29.78, respectively. Using a conventional raspberry grower observed in the UC Davis study as a point of reference, an example chemical management program included two applications of spinetoram and a combined application of zeta-cypermethrin and malathion in both the fall and spring harvest seasons. Each application is estimated to have labor and equipment costs of $61.78 per hectare. In 2015, such a program would cost an estimated $581.14 per hectare in both the fall and spring harvests for a total cost of $1,161.28 per hectare for a single planting. This is consistent with the per hectare treatment program cost of $825.33 observed in Goodhue et al in 2011.Even though conventional raspberry producers have developed effective chemical management programs that virtually eliminate fruit losses due to SWD, organic producers still experience non-trivial yield losses due to more expensive and less effective insecticide options. Most California organic raspberry producers used only two SWD-targeting insecticides, spinosad and pyrethrin, during the time of this study. Of these two insecticides, only the organic formulation of spinosad has efficacy comparable to conventional insecticides. 

Cases with relatively high ground cover fractions and uniformly arranged plants showed good agreement between the 1D and 3D models regardless of whether the assumption of leaf isotropy was made. As the canopies became more heterogeneous in space, agreement between the models generally declined. Although Potato-Uniform and Potato-Row had identical leaf area indices and leaf angle distributions, the regular distribution of plants within the canopy in Potato Uniform resulted in better agreement between the 1D and 3D models compared to Potato-Row. Despite all cases having highly anisotropic leaf inclination distributions, the assumption of leaf anisotropy had relatively small impact for all cases except for the Grape E-W case. Any effects of heterogeneity or anisotropy tended to decrease as the day of year became further away from the summer solstice. Toward the end of the year , the 1D and 3D models were in fairly good agreement for all canopy cases.The diurnal flux of radiation intercepted by the canopy for an hourly time step on Julian day 153 is shown in Fig. 2.6, plastic gardening pots with corresponding fractions of total radiation intercepted by the canopy shown in Fig. 2.7. The fraction of total radiation intercepted on Julian day 253 and 305 are shown in Figs. 2.8 and 2.9, respectively.

For the homogeneous canopy cases, the assumptions of vegetation homogeneity and isotropy were closely satisfied, and therefore, the 1D model was in very good agreement with the 3D model regardless of leaf density . For the crop canopy cases, the 1D model consistently over estimated light interception as compared to the 3D model, except for Grape E-W and Potato-Row on Julian day 305. For all but the grape cases, eliminating the isotropic assumption resulted in little improvement of agreement between the 1D and 3D models, indicating that errors arose primarily from heterogeneity in these cases. For the Grape N-S, Almond, and Potato cases, errors between the 1D and 3D models were largest near midday when sunlight could most readily reach the ground by penetrating through gaps in vegetation. For Grape E-W, the largest discrepancies occurred at early and late times of the day. The effect of row orientation on diurnal interception patterns for the grape cases was dramatic, as this completely changed the character of interception at different times of the year . The potato cases also illustrated the pronounced effect of heterogeneity in planting pattern on diurnal interception patterns.Figure 2.10 depicts vertical profiles of the absorbed radiation flux at 8:00, 10:00, and 12:00 hours on Julian day 153 for Grape N-S, Grape E-W, Almond, and Corn. Errors in absorbed fluxes for Grape N-S were relatively consistent with height, where errors at a given height were most closely related to the magnitude of the absorbed flux at that height. This was also roughly the case for Almond, except that there was the potential for some under estimation of absorption in the lower canopy when the 1D model was used, which was most pronounced for larger solar zenith angles.

For Grape E-W, the 1D model tended to shift the peak in the absorbed flux deeper into the canopy, which was most pronounced for larger solar zenith angles. In the corn canopy, the vertical pattern in radiation interception differed significantly between the 1D and 3D models. There were up to 50% differences between 1D and 3D fluxes at a given vertical level, with irregular patterns of over or under estimation. In the lower canopy, there was a peak in absorption in the 3D model, which was largely absent in the 1D model, leading to under prediction of absorption by the 1D model in the lower canopy. This is likely due to the substantial over prediction of absorption by the 1D model in the upper canopy, which removes the necessary energy needed to produce a secondary peak in absorption in the lower canopy.If leaf azimuth is uniformly distributed, this effectively reduces the impact of anisotropy in leaf inclination on the projected area fraction G. Since a leaf with a certain elevation angle could be parallel to the sun at one azimuth and perpendicular to the sun at another, an integration over all azimuths can smear out the effects of leaf inclination alone. As in the virtual canopies of this study, field measurements have shown that leaf inclination distributions are usually highly anisotropic. The azimuthal distribution of leaves may be strongly anisotropic within a single plant, but for relatively dense canopies, the azimuthal distribution is often fairly isotropic. In these cases, the assumption of leaf isotropy is likely to result in minimal errors. However, sparse, row-oriented crops such as vineyards may have highly anisotropic azimuthal distributions, in which case it may be necessary to explicitly calculate G based on measurements.

These types of canopies are becoming increasingly prevalent in agricultural applications, due in part to the improved access to mechanical harvesters that a trellised or hedgerow canopy provides.Plant spacing and the resulting heterogeneity had the most pronounced effect on errors resulting from the use of Beer’s law. For the Grape N-S case, the assumption of heterogeneity resulted in an overestimation of the total daily absorbed radiation by 28%, 30%, and 36% on Julian days153, 232, and 305, respectively, with larger instantaneous over estimation near midday. For the Grape E-W case, the assumption of heterogeneity also resulted in overestimating the total daily absorbed radiation by 74%, 51%, and 5% on Julian days 153, 232, and 305, respectively. This was not simply an effect related to L, as was illustrated by the two potato cases. By simply rearranging the potato plants from a uniformly spaced into a row-oriented configuration, errors in the 1D model increased substantially. It is possible that the effect of horizontal heterogeneity can vary in the vertical direction, which appeared to be the case with the Corn canopy. This significantly altered the performance of the 1D model at any given height, although the canopy was dense enough overall that the 1D model performed well when predicting whole-canopy radiation absorption. This could have important implications if the radiation model is coupled with other biophysical models such as a photosynthesis model. The response of photosynthesis to light is nonlinear and asymptotic, so although whole-canopy absorption may be well-represented in some cases by a 1D horizontally homogeneous model, it is unclear if that will result in significant errors in total photosynthetic production given the non-linearity of its response to light. A limitation of this study is that results are only applicable under clear sky conditions. However, results can provide some insight regarding diffuse sky conditions by simultaneously considering all canopy geometries and simulated sun angles. Under a uniformly overcast sky, equal energy originates from all directions. A particular combination of sun angle and leaf orientation bias was required in order to observe a pronounced effect of leaf anisotropy. Thus, for diffuse solar conditions, it is speculated that the impact of leaf anisotropy will be decreased. Sun angle had an important effect on the instantaneous impact of leaf heterogeneity, blueberry pot size and most commonly it was observed that low sun angles resulted in a decreased impact of heterogeneity. Therefore, it is likely that highly diffuse conditions will reduce the impact of heterogeneity near midday because a significant fraction of incoming radiation will originate from directions nearer to the horizon. Estimating light interception with Beer’s law is based on the assumption that canopies are homogeneous. This inherently means that the rate of radiation attenuation along a given path is linearly related to the flux at that location. As the canopy becomes sparse, there are pathways for radiation propagation that allow radiation to penetrate the entire canopy without any probability of interception, which fundamentally violates the assumptions behind Beer’s law or a turbid medium. Therefore, the non-random leaf dispersion in canopies limits the ability of Beer’s law to link light interception to simple bulk measures of plant architecture.

It is well-known that this heterogeneity or “clumping” of vegetation usually results in decreased radiation interception as compared with an equivalent homogeneous canopy. A common means of dealing with this problem without significantly increasing model complexity is to add a “clumping coefficient” W to the argument of the exponential function in Beer’s law. While this is a simple and practical means of reducing the amount of radiation attenuation predicted by Beer’s law, the challenge in applying the clumping coefficient approach is that W is a complex function of nearly every applicable variable, and thus is it is difficult to mechanistically specify. Another approach is to use a model that explicitly resolves plant-level heterogeneity, as it may not be necessary to explicitly resolve every leaf if within-plant heterogeneity is small.Row orientation played an important role when estimating light interception from Beer’s law, particularly when the rows were widely spaced. For sparse, row-oriented canopies, the effective path length of the sun’s rays through vegetation can change dramatically with changes in sun azimuth. For East-West rows, absorption is significantly reduced early and late in the day because the rows are close to parallel with the sun’s rays, whereas North-South rows are perpendicular to the sun at this time. As the day of year progresses further from the summer solstice, the sun spends more time closer to the horizon and thus the impact of heterogeneity in an East-West row orientation increased. For the East-West row configuration, G and light interception were surprisingly constant throughout much of the day, which resulted in 41% and 36% less absorption on Julian days 153 and 232, respectively, compared to North-South rows. In some climates, it may be desirable to maximize sunlight interception, whereas in others it may be desirable to mitigate effects of excess sunlight to reduce temperatures and water use.Despite the simplified assumptions in Beer’s law regarding scattering, there was good agreement between predicted radiation interception using the 1D and 3D models in the PAR band. Scattering did not significantly influence light interception in this band because most of the incident radiation received by individual leaves was absorbed. However, in the NIR band, scattering introduced significant over estimation of absorption using the standard 1D model, since leaves are poor absorbers in this band. Using an ad hoc correction to account for reflection only reduced this over estimation of absorption. An additional correction to account for both reflection and transmission resulted in over correction, and a net under prediction of total radiation absorption.The objective of this work was to evaluate common assumptions used in estimating radiation absorption in plant canopies, namely assumptions of homogeneity or isotropy of vegetation. Our results demonstrated that for relatively dense canopies with azimuthally symmetric leaves, a 1D model that assumes homogeneity and isotropy of vegetation generally produced relatively small errors. As plant spacing became large, the assumptions of homogeneity break down and model errors became large. In the case of a vineyard with rows oriented in the East-West direction, errors in daily intercepted radiation were up to ⇠ 70% due to heterogeneity alone, with much larger instantaneous errors occurring during the day. If leaves were highly anisotropic in the azimuthal direction, there was also the potential for large errors resulting from the assumption of vegetation isotropy which had the potential to increase errors above 100%. Day of year had an impact on model errors, which was that overall errors tended to decline with time from the summer solstice. In cases of canopies where the plant spacing starts to approach the plant height, it is likely necessary to use a plant-resolving radiation model in order to avoid substantial over prediction of absorbed radiative fluxes. Additionally, if vegetation is highly anisotropic in terms of both elevation and azimuthal angle distributions, it is also likely necessary to explicitly calculate the projected area fraction G based on measurements and the instantaneous position of the sun.Recent shifts in climatic patterns have influenced the frequency, timing, and severity of heat waves in many wine grape growing regions, which has introduced challenges for viticulturists. Growing the same varieties under these altered climatic conditions often requires mitigation strategies, but quantitative, generalized understanding of the impacts of such strategies can be difficult or time consuming to determine through field trials. This work developed and validated a detailed three-dimensional model of grape berry temperature that could fully resolve spatial and temporal heterogeneity in berry temperature, and ultimately predict the impacts of potential high berry temperature mitigation strategies such as the use of alternative trellis systems.

We created a multiplexed probe-based quantitative PCR assay able to identify whether a sample contains T. absoluta, K. lycopersicella, or P. operculella DNA based on single nucleotide polymorphisms between species. In addition, we created RPA-Cas12a assays to detect the presence of T. absoluta DNA, using either a fluorescent reader or a simple UV illuminator paired with a cell phone camera. This will allow for more rapid field detection of T. absoluta.We assessed 21 single nucleotide polymorphism markers differentiating T. absoluta, K. lycopersicella and P. operculella identified in Tabuloc et al. 2019 for qPCR genotyping potential using RealTimeDesign software . Our analysis resulted in seven potential qPCR assays, each containing a set of universal amplifying primers and two distinguishing fluorescent oligonucleotide probes. We used a serial dilution qPCR on all seven sets of amplifying primers on all three species to assess primer efficiency. Reactions were performed with 300nM of forward and reverse primers with SsoAdvanced Universal SYBR Green Supermix on a CFX96 real time PCR machine . We checked for off-target products when multiplexing pairs of assay primers using melt curve analysis as well as gel electrophoresis. From the seven loci tested, square pot we selected two to develop assays and ordered single-strand BHQplus probes unique to each of the four alleles .

Each probe was tagged with a distinct 5′ fluorophore and 3’ quencher. Assay 1 distinguishes T. absoluta from K. lycopersicella and P. operculella, while assay 2 distinguishes P. operculella from K. lycopersicella and T. absoluta.When considering which method is best suited for T. absoluta identification, a key consideration is sensitivity. Based on our analyses, we find that our qPCR diagnostics can reliably identify species down to 0.02 ng of input DNA, while our RPA-Cas12a assay was capable down to 0.1 ng input DNA. Assuming a haploid genome size of 564Mb from flow cytometry estimates , this represents approximately 35 copies of target DNA for qPCR detection and approximately 170 copies of target DNA for RPA-Cas12a detection. Both should be sufficient for scenarios where DNA is extracted from a single insect or small pool of insects. However, in situations where target DNA concentrations are extremely low, such as the case would be with eDNA collections, the more sensitive qPCR method is preferred.signal in the RPA-Cas12a assay as measuring fluorescence with a qPCR machine. Compared to the qPCR diagnostic, the RPA-Cas12a diagnostic does require additional user time, as amplification and detection occur in two distinct steps, although a one-pot reaction variant has been described . While we used an oligonucleotide with a Cal Red 610 fluorophore paired with the appropriate quencher as the substrate for Cas12a-mediated ssDNAse activity, the fluorophore/quencher molecules can easily be swapped for other fluorophores that absorb and emit at different wavelengths, making this protocol adaptable to the equipment available to the user.

It is possible in the future to replace the fluorometric detection method with a colorimetric detection by using biotin and FAM-labeled oligonucleotides applied to a lateral flow assay. This flexibility has been implemented in other detection assays and lets Cas12a-based detection systems stand out from qPCR methods, despite the slight reduction in sensitivity . While T. absoluta has not yet been detected in North America, demographic modeling suggests low to moderate levels of invasion are possible in Mexico, the California Central Valley, and the southeastern USA . Rapid reliable detection of an invasive pest is one of the key components of a successful eradication program, meaning it is crucial that molecular diagnostics are available . Even if eradication is not possible, molecular diagnostics reduce the need for expert entomologists to hand identify specimens, allowing detection to be done faster, cheaper, and more accurately . This is especially relevant for T. absoluta testing as tomato crops in the United States already contain the presence of P. operculella and K. lycopersicella, which are both nearly identical morphologically. Once detected, agencies can decide between strategies including eradication, quarantine, or continued monitoring . We expect that the molecular diagnostics presented here will add to the toolkit available to institutions to rapidly monitor for the appearance of T. absoluta.Tomato fruit are highly perishable, and sensitive to handling techniques and storage conditions after harvest .

Post harvest losses in quantity and quality commonly occur in fresh market tomatoes . The irreversible and continuous changes after fruit harvest leads to cell death and senescence, which makes some post harvest losses unavoidable . However, some losses are caused by improper post harvest practices, such as mechanical injury or pathological breakdown . Appropriately applying post harvest techniques could slow fruit senescence and therefore, maintain fruit quality from vine to consumers. While the specific recommendations for post harvest handling are dependent on the desired fruit shelf-life and vary among the produced and consumed regions of tomatoes, some examples of commonly applied tomato post harvest practices are discussed below.Fruit maturity at harvest plays an important role in its post harvest quality. Tomato fruit could be harvested between the stages of mature green and fully ripe, depending on the demand for fruit shelf-life. For the tomato produced for local markets, producers harvest fruit close to the fully ripe stage. Tomato destined for sale in distant markets, may be harvested earlier , followed by off-the-vine ripening to effectively satisfy the market need for an extended shelf-life. Tomato fruit that is harvested early, i.e., ‘Mature green’ fruit is able to fully ripen, but it loses its carbon and energy source from the mother plant, resulting in quality loss . Reduced fruit quality including loss of sugar, a lower ratio of TSS/TA, and changes in volatiles have been reported in off-the-vine ripening fruit compared with optimal harvest .Storage of the fresh market tomato after harvest provides stability to the supply chain. Precise temperature management is powerful in fruit storage, and the prescribed temperature may differ by fruit cultivar, harvesting stage, expected storage period, or, transportation . Before storage, precooling of freshly harvest fruit is used to remove ‘field heat’, ameliorating the rise in metabolic activities in fruit . For short-periods of storage, tomato fruit can be placed at room temperature under good ventilation, while, for longer storage time, early harvesting followed by low-temperature storage is a possible strategy, but this will negatively reduce the fruit flavor . When sensitive produce are exposed to low, non-freezing temperature for long periods, post harvest chilling injury may occur . PCI symptoms in tomato fruit include a failure to ripen,uneven ripening, surface pitting, decay, and high rates of ethylene and respiration upon rewarming . There are other approaches to calibrate the rate of fruit ripening during storage. Applying controlled and modified atmospheres could delay fruit ripening during post harvest storage. Controlling relative humidity , and modifying the relative proportion of oxygen and carbon dioxide in the atmosphere can extend the shelf-life of tomato fruit without a significant loss of flavor . For greater flexibility, ripening can be further controlled by regulating post harvest ethylene. When fruit are exposed to ethylene, ripening would speed up . Conversely, reducing ethylene production will maintain fruit quality and prolong shelf-life, blueberries in containers and several commonly examples are as following: precooling or heat treatment is efficient in slowing down ethylene production during storage ; high rates of CO2 is able to inhibit autocatalytic ethylene biosynthesis ; applying 1-Methylcyclopropene would delay fruit ripening by binding to the ethylene receptors , however use of these chemicals may affect fruit quality ; calcium chloride is successfully used in inhibiting ethylene production after harvest ; ethylene biosynthesis and sensitivity are affected by low-temperature post harvest storage .Consumers have been complaining about the quality of store-bought, conventional varieties of tomatoes over the last 30 years .

Understanding the physiological basis of fruit quality has therefore become the main focus of tomato researchers, as this information would support the breeding of new tomato cultivar with better quality . Fruit quality characteristics depend on the fruit developmental and physiological pathways which are dynamically regulated in the ripening process . The quality of the tomato fruit is mainly determined by its color, texture and flavor. Tomato fruit color is not only an appealing eating quality for consumers, but also a commonly used indicator of the fruit ripening stages. During ripening in most tomato cultivars, chloroplasts are converted to chromoplasts accompanied by chlorophyll breakdown and carotenoids accumulation, resulting in fruit color transition from green to red . The color of the ripe fruit is red primarily due to lycopene and the accumulation of a smaller amount of the orange or yellowish β-carotene . The dynamic interplay of carotenoid and chlorophyll metabolism dictates tomato fruit color .Fruit texture is a sensory, multi-parameter characteristic that can be perceived through the consumer’s senses, i.e., vision, touch, and taste . Tomato fruit texture is commonly described by the attributes of firmness, juiciness, and mealiness . There are various methods of measuring fruit texture, in which puncture and compression are commonly used destructive methods, and non-destructive approaches may include ultrasonic and optical techniques . Firmness of tomato fruit is considered as the main determinant of texture, and the decline of firmness leads to the fruit softness . Fruit softening is a natural phenomenon in ripening, and critical to seed dispersal and attracting animals . A certain degree of fruit softening is desirable in fresh market tomatoes, but too much softening is unwanted , as it limits fruit shelf-life and increases susceptibility to decay. Tomato fruit softening is associated with disassembly of the cell wall matrix, involving depolymerization and solubilization of the cell wall polysaccharides components, i.e., cellulose, hemicellulose and pectin . This event is mainly controlled by the activity of many cell wall degrading enzymes, such as polygalacturonase, pectin methylesterase , galactanase, or remodeling proteins, like expansin . Tomato fruit flavor is determined by a complex interaction of many biochemical compounds, including sugars, acids, amino acids, with aroma volatiles . Tomato aroma volatiles are often complex secondary metabolites, and they are derived from carotenoids and simpler primary metabolites precursors such as amino and fatty acids . There are more than 400 volatiles discovered in tomato, but only a limited proportion influences tomato flavor . The non-volatile compounds determine fruit taste. Sugar, mainly the reducing sugars glucose and fructose, makes the largest contribution to tomato taste. There are also organic acids in tomato fruit, predominantly citric and malic acids. It is well established that tomato flavor is highly interdependent on the relative levels of sugars, acids, and key volatiles, and no single chemical controls quality .Carbohydrate metabolism is important in determining tomato fruit post harvest quality . Fruit are “sink” tissues , that require carbohydrates supplied by photosynthetic “source” tissues . The chloroplasts in green tomato fruit have active photosynthetic ability , thus, green tomato fruit is able to fix carbon . The carbon dioxide from respiration is the major source of green fruit photosynthesis . Fruit fixed carbon accounts for 10-15% of that used by the fruit, and may be critical for seed development , and maximal carbohydrate and carotenoid accumulation . The carbohydrate metabolic pathway in developing tomato fruit is mediated by the activities of numerous enzymes and sugar transporters . Sucrose, the main translocated assimilate, is taken up by tomato fruit . In early fruit development, starch synthesis is the main flux; fruit starch level reaches its peak at the mature green stage, and it is then degraded during ripening . Starch synthesis and breakdown coexists throughout fruit development, with net synthesis occurring prior to the mature green stage, and net degradation to sugars occurring during ripening . Starch degradation therefore contributes to sugar accumulation in ripening fruit .Epigenomic modifications, especially DNA methylation of gene promoter regions, are considered to play a critical role in triggering tomato fruit ripening . DNA methylation is characterized by the addition of a methyl group to the 5’ position of cytosine. The cytosines that are methylated occur in either a symmetrical CHG and CG, or an asymmetrical CHH context . Symmetrical and asymmetrical DNA methylation are regulated by different genes . DNA demethylases are able to remove methyl groups from cytosines, a process termed DNA demethylation. In plants, DEMETER-Like demethylases are a group of enzymes that can activate gene promoter demethylation, by the activity of DNA Glycosylase-Lyases . There are four DML isoforms in tomato, of which SlDML2 is most important in fruit .

Additionally, the induction of defense genes in the RR core response could be ineffective if their expression levels were too weak compared with those seen in resistant MG fruit. We evaluated the levels of gene expression in inoculated RR fruit via a differential expression comparison to inoculated MG fruit. Of all the RR core defense genes identified above, 269/302 were expressed at equal or greater levels in inoculated RR fruit compared with inoculated MG fruit for all three pathogens. Conversely, 33/302 of these defense genes were expressed at higher levels in MG fruit compared with RR fruit for at least one of the three pathogens . These genes are diverse, representing 15 different defense categories. Prominent genes in this category include TAP1 and TAP2 , two peroxidases associated with defensive suberization in tomato ; CHI3 and CHI17 , two chitinases associated with C. fulvum infection ; and the JA biosynthesis gene OPR3 . While it is possible that resistance may be determined by these genes, growing blueberries in containers these results indicated that the differences in immune responses observed between MG and RR fruit are not likely solely responsible for differences in susceptibility, and, therefore, we considered the alternate hypothesis.

We explored the possibility that the increase in susceptibility to fungal pathogens is heavily influenced by a decline of preformed defenses and accumulation of susceptibility factors that occur during fruit ripening prior to pathogen challenge. Due to the complexity of the ripening program, we utilized isogenic non-ripening tomato mutants as tools to identify specific developmental features that are integral to fruit resistance or susceptibility. The Cnr, rin, and nor mutants produce fruit that lack most of the characteristic changes associated with normal ripening, such as color, texture, acidity, sugar accumulation, and ethylene production, but yet are phenotypically different from one another . All three mutant lines likely result from spontaneous gain-of-function mutations in transcription factors with key roles in the regulation of ripening . We inoculated fruit of these mutant genotypes at comparable stages to MG and RR wild-type fruit with B. cinerea, F. acuminatum, and R. stolonifer and measured disease incidence and severity up to 3 dpi . For all three pathogens at both MG-like and RR-like stages, only nor fruit were consistently resistant to infection. MG-like fruit of Cnr were the only unripe fruit susceptible to any pathogen, with both B. cinerea and F. acuminatum able to produce lesions on a significant number of these fruit. Consistent with this, Cnr RR-like were more susceptible than wild-type RR fruit to B. cinerea, with average disease severity nearly twice as great at 3 dpi .

The fruit of rin at both MG-like and RR-like stages showed similar or slightly lower susceptibility to all pathogens when compared with wild-type, with the exception of a significant reduction in disease incidence to F. acuminatum at the RR-like stage. Because some ripening processes may promote susceptibility, others may maintain resistance, and others may have no impact, we hypothesized that the Cnr, rin, and nor mutations differentially affect ripening-associated genes or pathways that are critical to tip the balance towards either susceptibility or resistance. We sequenced mRNA from B. cinerea-inoculated and healthy fruit from the non-ripening mutants at MG-like and RR-like stages at 1 dpi. We chose B. cinerea inoculations because this pathogen showed the clearest differences in susceptibility phenotypes between these genotypes. We first characterized transcriptional responses of mutant fruit to pathogen challenge by using enrichment analysis of defense-related processes to determine if differences in immune responses could explain the distinct susceptibility phenotypes . In most cases, the mutant fruit exhibited similar patterns of defense classification enrichments as wild-type fruit in both stages, with some notable exceptions. Compared with the other genotype–stage combinations, Cnr MG-like responses were deficient in the expression of genes from several prominent defense classifications, including chitin catabolic process , the plant–pathogen interaction and glutathione metabolism pathways, ERF and WRKY transcription factors, and RLK and CAMK genes.

Given that Cnr fruit were the only genotype at the MG-like stage to display susceptibility to B. cinerea infection, it can be suggested that these defense processes may be necessary for resistance in unripe fruit. However, these processes were enriched in the susceptible RR-like fruit of Cnr and rin, as well as wild-type RR fruit, which clearly indicates that they are not sufficient to result in a resistant outcome.For example, the responses of resistant nor fruit in both MG-like and RR-like fruit were noticeably less enriched in ethylene-associated pathways and more enriched in JA-associated pathways. These results suggest that JA-mediated defenses may contribute to tomato fruit resistance in the absence of ethylene, and that the nor mutation may activate JA-associated resistance. In support of this observation, levels of JA in healthy fruit appeared to be linked to resistance: they were highest in RR-like nor fruit, and only nor fruit experienced an increase in JA in the transition from MG-like to RR-like . Ethylene levels increase dramatically during ripening in wild-type fruit, but they remain low in all three non-ripening mutants . However, both Cnr and rin mutants produce ethylene in response to B. cinerea inoculation, with ethylene production in inoculated Cnr MG-like fruit reaching levels nearly three times greater than wild-type MG fruit by 3 dpi. Moreover, ethylene signaling/response genes are highly enriched in Cnr MG-like fruit responses . In contrast, healthy nor fruit did not produce substantial amounts of ethylene at either stage, and inoculation in nor fruit did not appear to induce significant ethylene production as in rin and Cnr fruit. These results indicate that high levels of ethylene are not required for B. cinerea resistance and most likely promote susceptibility. Regardless, the combination of hormone activity and defense gene enrichment suggests that, with the exception of Cnr MG-like fruit, resistance or susceptibility in the non-ripening mutants cannot be merely explained by the presence and/or magnitude of immune responses.

To identify genes that are involved in resistance or susceptibility that change during tomato fruit ripening, we used a differential expression analysis comparing healthy RR/RR-like to healthy MG/MG-like fruit for each wildtype and mutant line. In wild-type fruit, 6574 genes were significantly down-regulated in RR fruit compared with MG, while 5674 genes were significantly up-regulated . We used the susceptibility phenotypes and the transcriptional profiles of the mutant fruit to filter these ripening-associated genes and identify critical preformed defense mechanisms or susceptibility factors. Of the four genotypes, square pots all except nor experience an increase in susceptibility in the transition from MG/MG-like to RR/RR-like fruit. Thus, we selected ripening-associated genes that showed the same expression pattern in wild-type, Cnr, and/or rin, but not nor. This filtering resulted in 2893 down-regulated and 2003 up-regulated genes, respectively. We assumed that effective preformed defenses will decrease during ripening. Thus, the set of filtered down-regulated genes, being those that are highly expressed in healthy MG fruit compared with healthy RR fruit, should contain key genes related to preformed defenses. The filtered down-regulated genes contained 251 defense genes, while up-regulated genes included only 171 defense genes, indicating a net loss of about 80 genes in the transition from MG/MG-like to RR/RR-like susceptible fruit. Furthermore, the 251 defense genes from the filtered down-regulated set were over-represented by functional categories involved in reactive oxygen species response and detoxification, proteolysis, and the biosynthesis of secondary metabolites . These down-regulated ROS-related genes spanned several subfamilies including thioredoxins, glutaredoxins, glutathione S-transferases, and peroxidases. Among the down-regulated proteolytic genes were several subtilisin-like proteases, including SBT3 . Lastly, in addition to several genes involved in the methylerythritol 4-phosphate pathway of terpenoid biosynthesis, two copies of the lignin biosynthesis gene CCoAOMT were also among the filtered down-regulated class, suggesting that cell wall fortification could be inhibited upon infection. These results indicate that ripening involves a loss of multiple defense genes, and that the pre-existing levels of genes involved in ROS regulation, proteolysis, and secondary metabolite biosynthesis may be critical for resistance. Finally, we evaluated filtered up-regulated genes that are highly expressed in healthy RR fruit compared with healthy MG fruit, as they may include potential susceptibility factors. Since there is little scientific literature on classes of genes that may constitute susceptibility factors in plants, we focused on the up-regulated genes that were highly expressed in the RR/ RR-like fruit of the susceptible genotypes. Such genes may have disproportionate impacts on susceptibility due to their high expression. To identify these genes, we calculated average normalized read count values for each gene across WT, Cnr, and rin RR/RR-like fruit. The distribution of these values over the filtered up-regulated genes is a notably long-tailed one with a range of 2.43 to 179 649.29 and an average of 1295.

We identified genes with abnormally high expression values by selecting outliers from a log10-transformed distribution of the data. This resulted in a list of 16 genes . They include several genes previously discovered to be active during tomato fruit ripening, including the flavor volatile biosynthesis gene ADH2 , the carotenoid biosynthesis gene Z-ISO , the pectin-degrading enzymes PG2a and PL , and the ethylene receptor ETR4 , among other genes involved in carbohydrate metabolism. While any of these genes has the potential to impact susceptibility, genes for cell wall-degrading enzymes, such as PL and PG2a, which facilitate fruit softening during ripening, represent especially good candidates given both the importance of cell wall integrity in defense against fungal pathogens and previous research on RNAi-developed mutants in tomato . To validate the impact of PG2a and PL expression in wild-type RR fruit on susceptibility to B. cinerea, we utilized CRISPR-based mutants in each of these genes . RR fruit from these lines are similar in regards to soluble solids content, titratable acidity, and juice pH, but CRISPR-PL fruit are nearly 30% firmer than fruit from the CRISPR-PG2a and azygous WT control lines . In conjunction with these firmness differences, RR fruit of the CRISPR-PL line, but not the CRISPR-PG2a line, demonstrated reduced susceptibility to B. cinerea compared with the azygous line . At 3 dpi, disease incidence in the CRISPR-PL fruit was 56% lower than that in azygous fruit. We conclude that the ripeningassociated pectate lyase enzyme is a major susceptibility factor for B. cinerea infection in tomato fruit.During ripening, fruit may gradually lose either the ability to activate or the effectiveness of components of the plant immune system, defensive hormone production and signaling, and downstream transcriptional responses. Alternatively, ripening processes such as cell wall breakdown, simple sugar accumulation, changes in pH and secondary metabolite composition, and, in climacteric fruit, increased levels of ethylene may impact the fruit’s capability to resist fungal attack . The widespread nature of this phenomenon in diverse fruit pathosystems suggests that ripening-associated susceptibility is likely to be mediated by combinations of the above factors. In tomato, ripening-associated susceptibility has been demonstrated not only for the model necrotrophic pathogen B. cinerea, but for other fungal pathogens including Colletotrichum gloeosporioides , R. stolonifer, and F. acuminatum . Here, for the first time, we identified specific host responses in both resistant unripe and susceptible ripe fruit that are common to multiple pathogens and thus represent core responses to fungal infection. Most prominently, these core responses featured RLKs, WRKY and ERF transcription factors, JA biosynthesis, and chitin catabolism . Some genes that appear in both the MG core and the RR core responses were previously studied components of plant immunity in tomato, including the JA biosynthesis gene LoxD , the subtilisin-like protease SBT3 , the peroxidase CEVI-1 , and the chitinase CHI9 . Though response to inoculation overlaps somewhat with response to wounding in MG fruit , transcriptional profiles , and ethylene measurements indicate that the bulk of inoculation responses are a direct result of fungal attack. This is also evident by the presence of a necrotic ring only in inoculated MG fruit and not in the wounded controls or the inoculated RR fruit, indicating that the unripe fruit is capable of inducing an oxidative burst in response to the pathogen presence . However, most defense genes uncovered were found solely in the RR core response. These included several well-known defense genes that were only expressed in RR fruit, such as WRKY33 , the ERF PTI5 , the RLK TPK1b , and the MAP kinase MPK3 . While the MG core response did contain some defense genes that were not present in the RR core response , expression of most of these genes was also identified in the RR response to one or two pathogens.

Ethylene increases the susceptibility of tomato to B. cinerea by inducing ripening; however, its functions during strawberry infections are yet to be fully characterized. ABA, IAA and ethylene accumulation are altered by polyamine levels, which are positively correlated with fruit susceptibility to B. cinerea during strawberry ripening . Other hormones, such as brassinosteroids and jasmonic acid are present at lower levels during strawberry ripening. BR positively regulates vitamin C levels, sugar and anthocyanin biosynthesis during ripening, while negatively regulating acidity and concentration of other phenolic compounds . JA acts synergistically with ethylene by activating its biosynthesis in strawberries . Endogenous JA levels are modulated by methyl jasmonate and the JA carboxyl methyltransferase that lead to high levels in white fruit and a decline during ripening, antagonistically to ABA . In strawberry, JA appears to be involved in defence responses against B. cinerea. For example, gallon pot strawberries treated with MeJA had a delayed and much slower progression of B. cinerea infections .

As indicated previously, B. cinerea releases enzymes and metabolites that act as virulence factors but may also induce plant responses that are beneficial for fungal infection . A relevant example of the manipulation of physiological processes in the host by B. cinerea is the interference with specific developmental processes. In tomato plants, B. cinerea infections modified host gene expression to increase susceptibility, such as the induction of senescence in leaves . Moreover, infected unripe tomato fruit show premature expression of genes involved in ethylene synthesis during tomato ripening . These findings suggest that B. cinerea could initiate ethylene production and thereby stimulate early ripening. As strawberries are non-climacteric fruit, ethylene production of B. cinerea may not have substantial effects on strawberry ripening; however, the fungus was also shown to induce genes involved in the biosynthesis of other plant hormones such as ABA. Moreover, B. cinerea can synthesize and secrete ABA that functions as a virulence factor . Besides hormones, increased oxidative reactions caused by the pathogen may influence ripening progression .Defence mechanisms can be divided into preformed and induced defences. In strawberries, preformed defence compounds are especially abundant in the unripe stage, as reviewed in the section on quiescence of B. cinerea.

Even though plants accumulate defence compounds, B. cinerea has mechanisms to cope with these metabolites by efflux and detoxification of inhibitory substances. ATP-binding cassette transporters are used by B. cinerea to facilitate the efflux of anti-fungal compounds, such as stilbenes . B. cinerea is capable of detoxifying inhibitory substances, like epicatechin by secretion of laccases . Active B. cinerea infections can result in a reduction of specific secondary metabolites. It has been reported that levels of flavan-3-ol, benzoic acid and phenylpropanoids drop in B. cinerea-infected strawberries . Strawberries respond to B. cinerea infection by triggering defences. In some cases, preformed and induced defences can overlap such as in the case of PGIPs. An endogenous PGIP appears to be constitutively expressed in fruit from various strawberry cultivars . However, this PGIP and six additional ones show higher expression levels upon infection with B. cinerea . Overexpression of FaPGIP1a and FaPGIP2a in cisgenic plants conferred enhanced resistance to grey mould . Other enzymes induced by B. cinerea infections are chitinases. Expression of the chitinases FaChi2-1 and FaChi2-2 peaked 16 hpi in B. cinerea-infected strawberries . Furthermore, heterologous expression of Phaseolus vulgaris chitinase cH5B in strawberry resulted in higher resistance to infection . Another study demonstrated that application of heat-inactivated cells of the yeast Aureobasidium pullulans promoted tolerance to B. cinerea in strawberries . This primed resistance is probably due to the fruit’s perception of chitin from the yeast leading to induction of chitinases or other plant immune responses. Moreover, fruit defence responses may be primed using mechanical stimulation as it was reported for strawberry leaves .

Induced defences include accumulation of secondary metabolites and ROS. For instance, strawberries accumulate proanthocyanins around infection zones possibly to restrict fungal growth . The surroundings of infection sites generally display higher ROS production . ROS can serve as an effective defence against pathogens but also can lead to cell death, which is considered beneficial for necrotrophic fungi . B. cinerea itself produces ROS to induce host cell death, deplete plant antioxidants and increase lipid peroxidation . It is therefore interesting that, in unripe tomato fruit ROS production leads to resistance against B. cinerea, whereas in ripe fruit it seems to promote susceptibility . Future research will likely shed more light on the role of ROS in induced defences of strawberry fruit. Basal immunity is activated upon fungal infection. Degradation of fruit cell wall pectins can produce demethylated oligogalacturonides that trigger basal immune responses . Expression of the F. x ananassa pectin methylesterase 1 FaPE1 in Fragaria vesca resulted in reduced methyl-esterification of oligogalacturonides in fruit. This reduced esterification activated basal defences via the salicylic acid signalling pathway that led to a higher resistance to B. cinerea . Involvement of SA signalling in responses against B. cinerea was previously suggested when strawberry plants and fruit treated with SA showed decreased post harvest decay . B. cinerea can suppress the expression of plant defence responses by hijacking the host sRNA regulatory pathways . In strawberry fruits, B. cinerea infections can alter the expression of microRNAs involved in the regulation of defence genes, including the plant intracellular Ras group-related LRR protein 9-like gene . Interestingly, B. cinerea can also take up plant sRNAs during its interaction with the host. For instance, transgenic plants expressing sRNA that targets B. cinerea DCL1 and DCL2 show significantly reduced fungal growth in strawberries . The suppression of fungal growth via host sRNA is not well understood, and it is yet to be demonstrated that this mechanism of defence naturally occurs in plants.The diverse arsenal of infection mechanisms employed by B. cinerea explains its extremely wide-host range. It is therefore not surprising that entirely resistant strawberry genotypes do not exist . Several authors have analysed field resistance of strawberries to B. cinerea by quantifying disease development without artificial inoculation. A multi-year study of three strawberry cultivars found a significant effect of year, cultivar and cultivar by year interaction on the incidence of B. cinerea infections . Moreover, there was a positive correlation between row density and disease. Other studies investigated field resistance in annual winter production systems and found that variation of B. cinerea incidence between years was larger than genotype differences within years . Even though field resistance assessments investigate conditions similar to commercial production, considerable variability between environmental conditions and years can interfere with the detection of genotype differences.Due to the confounding effects of different non-genetic variables in field studies, gallon nursery pot assessment of post harvest resistance to B. cinerea infections has been pursued to determine genotype differences between strawberry cultivars or species. A large study of grey mould development during post harvest storage of non-inoculated fruit reported variation in disease incidence and speed of progression amongst cultivars, but no complete resistance was observed . Another approach to reducing environmental effects in disease tests is to inoculate fruit with B. cinerea conidia suspensions. Bestfleisch et al. tested quantitative resistance in 107 accessions of wild and cultivated strawberry. In this study, two wild ecotypes of F. virginiana showed high resistance to B. cinerea infections and slow disease progression. Such high tolerance in wild species was also reported in B. cinerea-inoculated leaves and fruit of F. chiloensis accessions from Chile . In these wild accessions, B. cinerea grew much slower. Comparative studies of disease progression indicated that fruit from the cultivar Chandler developed lesions at 24 hpi, while fruit from an F. chiloensis ecotype developed symptoms at 72 hpi . Fruit were entirely covered with mould at 6 days post-infection for the cultivar Chandler and at 9 dpi for the F. chiloensis ecotype. Considering that some accessions, particularly wild ecotypes, show reduced grey mould incidence and progression, there might be genetic sources of resistance against B. cinerea that could be used to increase resistance in strawberry. However, information about resistance mechanisms is mostly based on assumptions or empirical data. Differences in ripening patterns have been suggested as a potential explanation for resistance. For instance, some strawberries ripen from inside to outside, leaving the skin, which is the entry point of infections, unripe and thus resistant for a longer time .

Some more tolerant cultivars remain white or unripe around the calyx , which is where many B. cinerea infections tend to initiate. Another mechanism of resistance could be the presence of fungal inhibitors or the induction of PR proteins. FcPR5 and FcPR10 are highly induced in resistant F. chiloensis accessions when compared to commercial F. x ananassa cultivars . Based on sequence homology, FcPR5 probably possesses antifungal activity, and FcPR10 is likely a ribonuclease. These findings reflect that even though efforts have been made to explore resistance mechanisms of strawberry to B. cinerea, very little is known. Therefore, more research is necessary to better understand the biology of strawberry interactions with B. cinerea infections using diverse germplasm accessions.Many disease management strategies have been implemented for the control of B. cinerea in strawberry as further described below. However, even combined approaches are only capable of reducing disease incidence and severity but cannot completely prevent or eliminate grey mould in strawberries .Historically, B. cinerea infections in strawberry production have been managed by agronomic and horticultural practices, such as removal of senescent plant material to avoid inoculum buildup . Preventing contact of fruit with soil is another common practice to avoid B. cinerea infections, as most of the inoculum is present on the ground and soil moisture promotes conidia germination . Selecting the right irrigation system could help reduce grey mould incidence; mainly, the use of drip irrigation and micro-sprinklers results in limited inoculum spread and reduction of water films on the fruit . As canopy characteristics influence microclimates , nitrogen fertilization can lead to dense canopies and favour grey mould . Similarly, shorter plant spacings promote higher incidence of B. cinerea in the field . Additionally, plastic tunnels can avoid airborne inoculum and B. cinerea incidence is lower in non-fungicide treated tunnels than in fungicide treated fields , but tunnels favour powdery mildew and complicate harvest. In summary, cultural practices are essential to limit preharvest B. cinerea infections of strawberries, especially in organic agriculture.In modern production, pesticide applications are the most common management practice for B. cinerea control . In the previous two decades, the main pesticides used in strawberry production against B. cinerea belonged to the Fungicide Resistance Action Committee Groups 1 and 2, as well as captan . However, due to increasing fungicide resistance and new legal restrictions, producers have been forced to diversify their fungicide regimen . One application of fenhexamid at anthesis can be as efficient as multiple weekly applications . Additionally, alternation and combination of different fungicides with different modes of action are recommended . Resistance of B. cinerea to fungicides is a real challenge in horticulture and fungicide resistance profiles can shift considerably even within a single season . A screen of 13 B. cinerea isolates in Louisiana showed that all were partial to full resistance to FRAC 1 fungicides, and several of the isolates also had different levels of resistance to FRAC 2 fungicides . A larger survey of 1890 B. cinerea isolates revealed that 7 isolates from different locations were resistant to all single-action site FRAC fungicides groups that are registered for B. cinerea control . B. cinerea resistance to fungicides is usually associated with over expression of efflux transporters or with modification of fungicide targets. These resistance mechanisms are acquired via mutations and recombination that occur frequently in B. cinerea due to heterokaryosis, sexual reproduction and the presence of abundant transposable elements in its genome . Efflux of fungicides or accumulation of altered fungicide targets has also been shown to lead to multi-resistances . The presence of resistant isolates against the most common multi-action site fungicides reinforces the need for innovative management practices. A new generation of RNA-based fungicides has been proposed, which relies on the application of sRNA or dsRNAs that target B. cinerea virulence genes to reduce fungal infections in strawberries . However, these RNA-based fungicides remain far from commercialization, which is why fungicide resistance management such as mixture and rotation of different fungicides or testing local isolates for resistance is necessary .

Source fruits were then placed on top of the target fruits so that the inoculated stem end region would be in contact with the equatorial region of the target. Using the whole fruits was the most effective way to contact-inoculate oranges. Source fruits with visible mycelium and with visibly macerated tissue radially spread from the initial wound but no mycelium were placed on top of the equatorial side of three target oranges, with each infection site of the source orange in direct contact with one of the target oranges. The contact point between the source and target oranges was pre-wetted by spraying sterile water containing 0.01% Tween-20. Successful infections occurred when using oranges that did not have visible mycelium, while those with external mycelium failed to infect the target fruit. For tomatoes and apples, the contact inoculation procedure failed when using whole fruits due to leakage and accumulation of juices from the source fruits during incubation, leading to off-target infections in noncontact points. We then decided to use tissue sections instead of whole fruits to ensure a successful and uniform inoculation process. For tomatoes, a 3.5×3.5 cm, black plastic pots for plants square shaped pericarp section containing healthy/asymptomatic and symptomatic tissue in a 1:1 ratio was used.

Similarly, for apples, a cross-cut 1.5 cm thick was performed below and above the limit between symptomatic and asymptomatic tissue. The resulting disc was then cut into four 3×3×5.5 cm triangles containing decayed and macerated tissue in the middle and asymptomatic tissue on the sides. Source tissue sections of tomato and apple were placed on plastic boats, with the endocarp facing upwards for the tomato. Individual target tomatoes and apples with the equator side as the contact point were placed on top of their respective source tissue sections.The contact inoculation time was determined as the minimum time needed for successful disease development in the target fruits once the source fruits were detached. For control oranges and fungicide-treated oranges , the contact time was reduced to two days when using P. italicum WT and 1.5 days when using P. digitatum WT due to their advanced infection rates and aggressiveness on control and fungicide treated oranges. In all cases, contact inoculation was performed at room temperature, and target and source fruits were stored in high-humidity chambers .

Following the contact inoculation, the source whole fruits or tissue sections were removed, and target fruits were stored at 20 °C under high relative humidity until mycelium reached the equatorial region, or until evaluations were completed. Target oranges inoculated with P. italicum were stored at 20 °C for four days and at 15 °C for 12 days; while oranges inoculated with P. digitatum were stored at 15 °C for 15 days. Negative control samples were included for each trial. Control source fruits underwent the same steps as inoculated samples but were not inoculated with a fungal spore suspension.After contact inoculation, disease incidence, and severity were measured daily for tomatoes and every two days for apples and oranges. Disease incidence was calculated as the percentage of fruits displaying visual signs of tissue maceration or soft rot. Disease severity was obtained by measuring lesion area from pictures taken at each time point using a Nikon D5100 DSLR Camera with 18–55 mm f/3.5–5.6 and a custom-made macro in the ImageJ software.High-resolution multispectral images were taken using a VideometerLab 4 and processed with the VideometerLab software version 3.22.29. This equipment includes a sphere that uses strobe light-emitting diode technology to provide uniform and diffuse illumination. Reflectance images were taken at 19 wavelengths , including the long pass filters for a total of 50 spectral bands of the electromagnetic spectrum.

Multispectral images for all target fruits were taken with the stem end pointed to the side and the equator in the center before and after contact-based inoculation. For image analysis, pixels representing healthy and infected tissues were collected from a subset of fruit images from the total oranges. A normalized canonical discriminant analysis transformation based on the reflectance of each pixel was created to minimize the distance within classes and to maximize the distance among classes. A region of interest was obtained from all images by applying a mask to segment the fruits from the background. All fruits were collected in a blob database, and the healthy and infected areas were extracted based on the previously created nCDA transformation. Shape and spectral features were extracted from individual blobs, including area and tristimulus components of color, such as hue and saturation. The SpectralMean feature extracts the reflectance mean of each fruit for the 50 spectral bands. Region MSI_Mean calculates a trimmed mean of transformed pixel values within the blob , and MSIThreshold measures the percentage or area of the blob region with a transformation value higher than the threshold, based on the nCDA model .After choosing the appropriate source inoculum type and incubation periods, we tested the reliability and reproducibility of the contact-based inoculation method using different fungal species and strains on target fruits with and without fungicide treatments . In all the tests, the fungi spread from the contact points between the source and target fruit, causing tissue maceration and, in some cases, extensive mycelium growth . No symptoms were evident when non-infected fruits or tissues were used as source inoculum to control for secondary or unintended infections. Although we noticed some variation across different fruit commodities, fruit treatments, and fungal species and strains, most of our trials yielded disease incidence values of at least 80%, indicating the high performance of the contact-based inoculation method . Additionally, we observed that established lesions in the target fruits continued to expand over time, confirming that the fungal pathogens tested in this study could colonize and complete their life cycle once they penetrated the fruits . Wild-type strains of P. italicum and P. digitatum reached a disease incidence of 80% and 96.7% after 10 and 8 dpci, respectively, in untreated target oranges. In contrast, when using fungicide-treated oranges, a low overall disease incidence was observed for P. italicum and P. digitatum WT strains after 8 dpci and 14 dpci, respectively . On the other hand, disease incidence in fungicide and wax treated oranges contactinoculated with a P. digitatum fungicide-resistant strain was found to be 95.5%, which was similar to the untreated control oranges infected with P. digitatum WT . Lesion expansion in oranges varied depending on the target orange treatment and fungal isolate used . Overall, although lesion sizes were smaller for the FT oranges when using WT strains, the increase in lesion size across time points shows that the fungal pathogens tested in this study can colonize and complete their life cycle once they penetrate the target fruit. Field-grown tomatoes of the ‘Celebrity’ hybrid cultivar showed a high disease incidence, drainage pot reaching 90% across all evaluated time points when contact-inoculated with a B. cinerea WT strain . Meanwhile, other field grown tomatoes from the ‘Rutgers’ variety and ‘Shady Lady’ hybrid cultivar achieved the maximum disease incidence at 4 dpci and 6 dpci, with 80% and 94.4%, respectively. Commercial, greenhouse-grown, hybrid ‘Beefsteak’ tomatoes showed 100% disease incidence at 2 dpci when contact-inoculated with a B. cinerea FR strain. Tomatoes that did not show any disease incidence remained uninfected throughout the trial. Similarly to oranges, lesion size development varied across tomato cultivars and strains used, but shows the fungal strains used can colonize the target fruits . In apples, P. expansum FR infected 72.5% of the apples at 4 dpci and 90% at 6 dpci. The number of infected target apples remained unchanged until the last recorded time. Seven out of 40 apples never showed infection or lesion development from P. expansum and were considered as not infected during this study .

For the infected apples, lesion sizes steadily increased throughout the duration of the evaluation period .In oranges, Penicillium spp. growth showed minimal visual progression and seemed limited to the contact point until 12 dpci . However, using multispectral imaging we detected disease progression on the surface of target fruits, which was not apparent to the naked eye. Changes in the reflectance profiles of contact-inoculated oranges show that lesions appeared as early as 8 dpci and continued to expand even when mature lesions and visible mycelium were only obvious until 14 dpci and 18 dpci, respectively . On the other hand, practically no changes in the reflectance profile were observed for control fruits . Furthermore, normalized canonical discriminant analysis transformation, which combines all wavelengths, could detect changes in areas where lesions were to be developed in earlier time points. These areas were calculated using a threshold in the nCDA transformed scale ranging from healthy tissues to infected tissues . The highest separation potential in the nCDA transformation was obtained when combining about ten wavelengths with minor gain with additional wavelengths . Using MSI to monitor pathogen growth revealed the early onset of infection in oranges. It also can be used for sensitive quantification of lesion area before the disease is visible to the naked eye.Consistent and reliable inoculation methods that mimic natural conditions and industry scenarios are key for the study of plant-pathogen interactions and the development of post harvest control methods. Here, we established a non-wounding, contact inoculation protocol that recreates the infections naturally occurring in the post harvest supply chain through nesting. We produced whole fruits or tissues that were infected and served as inoculum sources for spreading the disease to healthy fruit. In all fruits, except for fungicide-treated oranges contact inoculated with wild-type pathogen strains, disease incidence rates of 80% or higher were observed by the final evaluation time point, showing the effectiveness of the proposed methodology. Disease severity measurements were used to evaluate disease progression and growth behavior of fungal pathogens, confirming successful infections beyond the initial contact point in each fruit commodity. Even though disease incidence was lower in fungicide-treated oranges contact-inoculated with fungicide-sensitive pathogens, having successful infections in fungicide-treated oranges showed that the pathogens were capable of causing disease through this protocol, even in disadvantageous conditions. Furthermore, visualizing the fruits using MSI allowed us to confirm that, although lesions were not visible to the naked eye until 14 days post-contact inoculation with P. digitatum, the fungus was established in the fruit tissue and growing by 8 dpci. While successful B. cinerea infections were observed in all tomato trials, differences in disease incidence and severity values were observed across varieties. ‘Rutgers’ tomatoes exhibited greater variation between infection rates and a lower disease incidence than other tomato varieties. These differences could be due to surface characteristics, such as cuticle thickness and permeability, which influence the generation of pathogen-induced signals that activate defense responses. The ‘Rutgers’ tomatoes are more similar to processing types. Furthermore, thicker fruit cuticles provide higher resistance to initial B. cinerea infections. Also, differences in pathogen behavior were observed in this study, particularly between Penicillium spp. when contact inoculated in oranges and apples. P. italicum WT showed an earlier mycelium appearance as compared to P. digitatum WT, although it was the latter that covered the fruits completely in mycelium first. In apples, P. expansum showed a slower disease incidence and severity progression than the other commodities. This could be due to differences in infection mechanisms between fungal species, as well as due to the fact that blue mold develops better at cooler temperatures compared to green mold. For all three fruit commodities tested, a percentage of the fruits did not get infected, even after several weeks of monitoring. This could be because the fruit was able to halt pathogen infection at the contact point, preventing it from spreading throughout the tissues. Another possibility is that, despite homogeneous incubation conditions, fruit-specific microclimates were not always conducive to disease development in all fruits, even though we ensured consistency of the technical aspects of the protocol. The overall results of this study confirm that the non-wounding, contact-based inoculation method was effective in all fruit-pathogen interactions tested. Although an initial step of wound-inoculating the source fruit material is required, spread of the disease to healthy target fruits is done solely through contact between the tissues, and the target fruits remain unwounded throughout the entire procedure. This method holds promise for further application in other pathosystems by focusing on several key aspects. First, it is crucial to recognize the importance of the homogeneity of the fruits used as source inoculum and the initial 24 h during contact inoculation, as both play a significant role in the establishment and spread of fungal infections. For example, it is recommended that, if possible, fruits should come from the same location and supplier, and transportation-storage conditions should remain constant. Also, the incubation should be done at high humidity with some level of gas exchange , especially after the first day of contact. Second, investigating the contact time between source and target fruits is essential to ensure the accuracy and reproducibility of the inoculation method. Third, exploring the position of infected fruits or tissues, which serves as the source of inoculation, will help identify the most favorable conditions for efficient pathogen transfer between fruits through contact.

A strong positive relationship between crop size and the number of seeds dispersed and in the number of seeds being dispersed long distance is expected in abiotic dispersal modes such as anemochory and hydrochory and in the biotic dispersal mode epizoochory . However, the expectation is less clear with endozoochory, synzoochoryand myrmecochory, where animal dispersers make foraging decisions in resource-heterogeneous environments where these dispersers can be satiated or prematurely leave feeding trees in order to mix diets with complementary resources . Nonetheless, expected patterns with respect to crop size have been proposed for endozoochory. Howe and Estabrook developed two models based on specialized versus opportunistic frugivore/seed dispersal systems. They suggested that the number of seeds dispersed should increase with fruit availability for both types of species, although the number dispersed should plateau for model 1 plant species that depend on specialized dispersers that tend to involve relatively few species and become satiated. They further predicted that the effect of crop size on the proportion of the available seeds dispersed would differ for model 1 and model 2 species.

For model 1 species, pot with drainage holes the proportion of seeds dispersed was expected to initially increase with crop size but would reach a peak at some intermediate crop size due to disperser satiation and then drop with ever larger fruit crops. In contrast, for model 2 species they predicted that the proportion of the seed crop dispersed would increase with increasing crop size, perhaps stabilizing at a constant proportion at larger crop sizes, but not decreasing. However, the dichotomy between specialized and opportunistic dispersal systems is not generally accepted at this point , leading other authors to propose a general expectation that the number of seeds dispersed should increase with increasing crop size. In fact, this is considered one major driver of the development of frugivory hubs, where hub individuals in the network receive more dispersal services than expected, leaving non-hub individuals with little dispersal services . This last prediction appears to be supported by studies mostly of endozoochory that demonstrate that as crop size increases, visitation rate by avian and mammalian dispersers increases, which translates into an increased quantity of seeds dispersed . For example, Prunus mahaleb fruit crop size explained 80 % of seeds dispersed in a population in southern Spain . With respect to the proportion of seeds dispersed, results to date show no consistent relationship . These patterns suggesting a general increase in the number but not the proportion of seeds dispersed with increasing crop size are supported by a meta-analysis that found positive bird-mediated selection on fruit crop sizes as measured by both visitation rate and the quantity of seeds dispersed, but no selection on the proportion of seeds dispersed .

Crop size can also affect the quality component of SDE and the probability of LDD. Increasing crop size lifts the entire dispersal kernel, resulting in more seeds in the tail of the distribution and thus more LDD and increased population spread and gene flow . Increasing crop size also results in more seeds dispersing farther in a local dispersal context, increasing the chances of reaching suitable sites and surviving distance- and density-dependent mortality . Although empirical evidence is limited, crop size can also affect the quality of endozoochorous dispersal by altering disperser behaviour and disperser assemblages of individual plants. For example, as Vassobia breviflora crop size increased, disperser residence time in the canopy decreased, increasing the probability of seed dispersal away from the parent rather than seeds processed in situ . Increasing crop size also increased fruit consumption by legitimate dispersers without affecting consumption by pulp consumers, altering the realized dispersal assemblage and increasing overall dispersal quality . Lastly, with a population-wide increase in Fagus sylvatica crop size , there was an increase in survival of seeds cached by Apodemus flavicollis, a clear increase in the quality of dispersal . On the other hand, seed survival in caches can be lower under trees that produced large seed crops ; thus, spatial variation in crop size might have different effects than temporal variation .

Fruit and seed size variation is likely the second most widely studied driver of inter individual variation in seed dispersal. Fruit and seed size vary within individuals, but also among individuals, years and populations . In a study of 39 species from 46 populations, on average 62 % of seed size variation was within individuals while 38 % was among individuals, though individual species varied substantially . Thus, fruit and seed size variation can influence animal disperser decisions regarding which plants to forage in and then which fruits to consume . Furthermore, mean fruit size of individuals can be highly heritable, indicating potential selection response . Many studies have demonstrated size-based fruit or seed selection by dispersers, suggesting a potentially important role for fruit/seed size in driving inter individual variation in the quantity component of SDE, although actual patterns of selection are not consistent and appear to depend on the plant and animal species involved . It is generally thought that fruit/seed size-based selection is a function not so much of fruit/seed size, but rather by the fruit/ seed size relative to the disperser size. For example, for endozoochorous birds that swallow fruits whole, it is widely believed that fruit selection is driven by fruit diameter and bird gape width . Similarly, it is thought that seed size selection by synzoochorous seed dispersers is related to the ratio of seed to disperser size . The extent to which fruit/seed size selection contributes to inter individual differences in the quantity of seeds dispersed is unclear. Dispersers may select among individual plants based on mean traits or among individual fruits independent of the mother plant. While some studies demonstrate that frugivores select among fruiting plants based on mean fruit or seed size , others demonstrate that at the population level, individual fruits are selected based on their sizes . Thus, even strong selection of fruits based on size need not lead to differential selection of individual plants based on fruit size. For example, dispersers of P. mahaleb strongly selected fruits based on size, but this was almost entirely driven by selection of smaller fruits within an individual plant’s fruit crop, while there was inconsistent and weak selection among individual plants based on fruit size . Because multiple traits associated with selection by dispersers may be correlated with seed size, the degree to which selection is driven by fruit or seed size, rather than a correlated trait is unclear. For example, the four main avian dispersers of Rubus ulmifolius in southern Spain differed in the distribution of seed sizes dispersed, but seed size, seed number, pulp/seed ratio and percent pulp co-varied, making it difficult to determine which trait or traits were being selected . Fruit and seed size can also drive intraspecific variation in the quantity and quality of dispersal in other ways. For gape-limited, endozoochorous birds, large pot with drainage intra- and inter individual variation in fruit diameter can affect the proportion of a plant’s fruit crop that a disperser can swallow. For instance, in a Myrtus communis population in southern Spain, some individuals produced large fruits that only Turdus merula and T. philomelos could swallow and disperse. However, other individuals in the same population produced smaller fruits that were completely available to these species and partially available to Sylvia atricapilla, Erithacus rubecula and, in the case of one individual, the smallest disperser, S. melanocephala . Thus, the realized disperser assemblages of individual plants varied from two to five species. Moreover, realized disperser assemblages of individual plants varied across years due to changes in fruit size.

Such among-individual and among-year variation in realized disperser assemblages can affect inter individual variation in dispersal outcomes. First, variation in the number of animal species feeding on an individual plant likely affects the quantity of seeds dispersed . Second, inter individual variation in realized disperser assemblages is expected to drive inter individual variation in LDD, gene flow and the quality of dispersal because disperser species differ in their dispersal kernels, treatment in the mouth and gut, and microhabitat destination of seeds . Species-specific preferences in microhabitat and fruit/seed size can also result in microhabitats accumulating different seed size distributions .Lastly, seed size affects whether a seed is swallowed and passed through the digestive system versus being dropped, spat out or regurgitated, which affects both treatment in the mouth and gut and dispersal distances . In general, larger seeds tend to be dispersed more rapidly and farther, and are more likely to be cached than smaller seeds; in contrast, no obvious pattern links seed size and the probability of surviving in a cache. The actual outcome of the interaction may be more related to the ratio of seed to disperser size rather than seed size alone . However, three species of rodents varying 4-fold in mass all preferentially selected and dispersed larger fruits of the Chilean desert shrub Myrcianthes coquimbensis . While there is abundant evidence that synzoochorous dispersers select and handle individual seeds based on size, there are fewer studies documenting dispersers selecting on mean seed size among individual plants. The large Japanese wood mouse preferentially dispersed seeds of individual Q. serrata trees with larger mean acorn size . Similarly, Apodemus spp. disproportionately dispersed and cached seeds from Pinus armandii individuals with larger mean seed mass , although the greater probability of their seeds being consumed cancelled the benefits of increased dispersal. By contrast, mean seed size of the Queen palm had no influence on tree selection by squirrels , and Garrulus glandarius preferentially fed on Quercus ilex trees with smaller acorns . Thus, although evidence is limited, there is potential for seed size to contribute to inter individual variation in the quantity and quality of seed dispersal by synzoochorous dispersers. Although the consequences of within-individual variation in plant traits have not been considered frequently in ecology , in addition to selection based on individual or mean fruit/seed size, mutualistic dispersers may select among individual plants based on the extent of intra individual variation in fruit or seed size. In a latitudinal study of Crataegus monogyna seed dispersal by Turdus spp. in Europe, birds selected against intra individual fruit size variation in populations with lower variation and selected for intra individual fruit size variation in populations with higher variation . Similarly, A. speciosus not only selected individual Q. serrata trees with larger acorns, but also selected individual trees with a greater variability in acorn weight . Seed size also affects abiotic seed dispersal. In the seagrass Zostera marina, settling rate increases with seed size, suggesting smaller seeds disperse farther . When grazed by the specialist herbivore Ophraella communa, the riparian weed Ambrosia artemisiifolia produces lighter, more buoyant seeds, demonstrating a clear mechanism for inter individual variation in dispersal . In ballistically dispersed species, both seed and fruit size can affect patterns of seed dispersal. In Oxalis acetosella and O. corniculata , dispersal distances increased with seed mass, while in Mercurialis annua, dispersal distances decreased with increasing seed mass . In the only study on fruit size and ballistic dispersal of which we are aware, dispersal distance increased with fruit length in Erodium cicutarium . It is generally believed that dispersal distances of anemochoric species will decrease as seed mass increases, and this expectation appears to be well-supported, although the variance explained is generally low. This general pattern has been reported in both tropical and temperate environments as well as across trees, shrubs and herbaceous species , although there are exceptions . Given that seed mass varies both among and within individuals , seed mass variation may contribute to inter individual variation in dispersal distances. For example, under highly competitive conditions, plants of the wind-dispersed desert annual Dithyrea californica produce smaller, lighter seeds that are dispersed farther . Given the typical heterogeneous distribution of individuals in populations, it is likely that D. californica individuals vary continuously in competitive environments and thus potentially in dispersal ability. Finally, the actual pattern of wind dispersal is driven not simply by seed mass, but by the relationship between seed mass and the dispersal structure . Interspecific studies of the effect of plant height on seed dispersal suggest plant height is a major correlate of dispersal distances and is considerably more important than seed size .

The enlarged biding sites were then mapped to the CLV3 genomic sequence, tolerating up to 2 mis-matches in the flanking regions. In order to account for the presence of transcription factors whose cis-motifs are not currently known, MEME analysis were employed to identify motifs shared between genes that are co-expressed with CLV3.Overall, 231 potential cis-motifs and transcription factor binding sites were identified. Most were randomly distributed over the entire CLV3 genomic sequence, but irregular clusters could be recognized near the coding region. The largest cluster occurred in the upstream 500bp of the 5’ promoter, while up to three smaller clusters occurred in the 3’ enhancer region . The list of potential factors was then filtered to include those found inside the previously identified CLV3 regulatory regions, which left just 157 predictions . Many of the remaining predictions were found to have overlappingsequences, though it is unclear how well this might predict their actual function in-vivo. One notable example of this phenomenon is a predicted MYB-like binding site located at -155bp, square pot which was predicted by four different databases. In other cases, two structurally different transcription factors were predicted to have overlapping cis-motifs, such as the bZIP/homeodomain pair Opaque-2/ALFIN-1 in the 3’ enhancer region. Interestingly, the data also revealed four partial miR414 targets, three of which overlapped with the DNA/Mariner family transposable element At2gTE50670 in the 3’ enhancer , and the fourth occurred in the 3rd exon.

In an alternative approach to identify unknown cis-motifs, phylogenetic footprinting was used to compare CLV3 orthologous sequences from different species. In this method, functional regulatory structures can be identified by their conservation over evolutionary time, which often requires little more than performing a sequence alignment. The method is also quite robust, as previous studies found that the identified footprints matched 80 and 85% of known transcription factor binding sites. To begin this analysis, three CLV3 orthologs were identified by their syntenic relationships within the Brassicaceae using the tools in the Brassica Genome.org database. Their cDNA sequences were aligned with 27 CLE family paralogs identified in A. thaliana  in order to identify features that were unique to CLV3 orthologs, before expanding the search to additional species. This analysis revealed three potentially unique traits that might be used to distinguish orthologs from the multitudes of closely related CLE genes. These included three consecutive histidines at the C terminal end of the CLE motif, a C-terminal oligo extension, and a 3-exon gene structure, all of which had been previously identified in the CLV3 sub-group. Additional orthologs werethen identified using tBlastn searches against the AtCLV3 protein, for which nine species which met the criteria described above: Brachypodium distachyon, Oryza sativa, Ricinus communis, Glycine max, A. thaliana, Arabidopsis lyrata and Brassica rapa, Capsella grandiflora, and Camelina sativa. No AtCLV3 orthologs were identified in the gymnosperms, basal angiosperms, or the Asteriids using these search parameters.

The Euphorbiaceae and Fabaceae each contributed one species in the closely related Eurosiids I, while the monocots are represented by two species in the Poaceae. As a result, this sampling is heavily biased towards the Brassicaceae family , which provide more than half of the total number of species. In order to footprint the promoter regions, initial sequence alignments were performed using 8kb genomic fragments, containing up to 5kb of upstream and downstream sequences on either side of the coding region. However, little or no homology was found when all nine orthologs were aligned simultaneously. This was not improved by removing monocot clade, as the two grass orthologs failed to align with each other. Repeating this pattern, both R.communis and G. max also failed to alignment with each other, or with any of the remaining orthologs. In contrast, conserved regions became clearly visible when the five Brassicaceae species were aligned separately . This result appears to reflect the optimum degree of sequence divergence for this gene, as previous studies have found that orthologs outside of the Brassicaceae were less informative due excessive divergence, whereas sequences obtained entirely within the Brassicaceae have been found to have too little divergence. Three of the remaining species had complete genomic sequences, while the other two consisted of two contigs separated by a gap of unknown size. In the B. rapa ortholog, the gap was located in the 3’ region, and was flanked by 256 and 452 base pair sequences that did not align with any of the other Brasssicaceae orthologs, despite strong sequence conservation in the surrounding regions.

This indicates the recent insertion of a large DNA fragment, potentially >700bp in size. Attempts to locate the source of the two end-fragment sequences in the B. rapa genome with BLAST searches, unexpectedly found that each was present in multiple copies, and were distributed across several different chromosomes. No evidence of transposable element sequences were found, so the flanking regions were here interpreted to be contaminating scaffold sequences from the original genome assembly. A similar gap of unknown size occurred in C. grandiflora, where one contig aligned with the CDS and 3’ UTR, while the entire 5’ upstream contig failed to align with any other ortholog. In both cases, the non-aligning sequences were removed from the analysis, providing a final alignment consisting of four orthologs in the 5’ promoter region, and five orthologs spanning the CDS and 3’ UTR. Overall, the five orthologs shared between 27% and 65% sequence similarity, and grouped into two closely related pairs. One pair contained C. grandiflora and C. sativa, and the othercontained A. thaliana and A. lyrata. In contrast, B. rapa was found to be distinct from all other Brassicaceae orthologs, which accurately recapitulates its predicted evolutionary relationship with the rest of the family. Upon closer inspection, the coding regions were found to be 79-93% similar, which dropped to just 14-34% in regions with no significant alignments. The initial alignment was considerably fragmented, with many insertions, deletions, and isolated nucleotides. In many cases, the position of these features varied with the settings in the alignment software, and were here interpreted to be artifacts of the alignment procedure. To correct such artifacts, isolated nucleotides were manually adjusted left or right to maximize local sequence alignments within ±5bp. Where variation in the length of tandem repeats was apparent, gaps were introduced into one or more ortholog sequences to accommodate the largest number of repeats present. Conserved regions were then identified by using a 5bp sliding window to identify regions with more than 60% identity. This window is unusually small compared to previous studies that have used 15-50bp sliding windows, but was chosen here to more accurately reflect the minimum size of known transcription factor binding sites. Where large contiguous conserved regions were found, drainage collection pot the presence of small 1-3bp indels within their sequences were used to break them into smaller fragments, as disruption of these sites indicates that they do not contain functional cis-motifs. scattered in the 3’ UTR. Several predicted transcription factor sites were found within the coding regions, but these were interpreted to be non-functional, as previous GUS-reporter systems did not reveal any significant regulatory elements within this region. Among other notable features was a predicted signal peptide in the first exon, identified with signal P 4.0, which was almost entirely conserved and is consistent with the secretion of the mature CLV3 oligopeptide. In addition, the second exon was found to be completely conserved with no In all, 42 conserved regions were identified, ranging in size from 5 to over 111bp long. Fourteen footprints were found in the coding sequence, of which nine of were clustered around the three exons.

Only one footprint was found entirely within in the 5’ UTR, and the remaining four were intervening gaps. The second exon also completely overlapped with several predicted transcription factors, including HOX2a, as well as cytokinin and gibberellic acid responsive motifs. This suggests as-yet unrecognized functional role for the second exon, which might explain why it has been retained in a family that consists largely of single exon genes. The 3rd exon was also highly conserved, although curiously the most conserved region only partially overlapped with the CLE motif and instead included part of the C-terminal extension. In the 3’ UTR, the footprints were found to overlap with potential zinc-finger and MYB binding sites, as well as a cytokinin responsive ARR10 site. In the upstream regulatory region, the 5’ promoter contained ten conserved footprints, eight of which formed a large and nearly contiguous block near the TSS. The two isolated footprints were located at -204bp and -167bp upstream, corresponding to the palindromic Motif#2 and the redundantly predicted MYB binding site, respectively. In the remaining footprints, additional predictions were found for an overlapping AGL15/CBF site, an auxin response element, overlapping GT1 and AGAMOUS sites, and one prediction for a TATA-less promoter. The latter may be related to the position of the only recognizable TATA box-like sequence, which at – 68bp upstream, which is more than double the usual 25-35bp described for other TATA-based promoters. In contrast, the 3’ enhancer region contained seventeen footprints arranged in roughly three clusters, spanning a region nearly 600 bp long. Two of these clusters closely corresponded with the previously noted clusters of predicted transcription factor sites, while the third was distinctly isolated and had no predicted transcription factors. Together, the footprints contained one of the three known WUS binding sites , two predicted AtHB1 binding sites, a cytokinin responsive element , several bZIP motifs, a KNOX-like site, and a predicted cis-motif forNPR1. Strikingly, the majority of the footprints also overlapped with a DNA transposable element in A. thaliana, At2TE50665 . It has previously been implied that WUS controls CLV3 expression in a concentration dependent manner, which is consistent with the close proximity of two demonstrated WUS binding sites . The region around these two sites also contains several other TAAT cores within a single stretch about 100bp long, much of which is represented by four conserved footprints, which together might form a WUS binding site cluster. However, only the +970 WUS binding site was found to be perfectly conserved, while the other TAAT cores displayed mutations or were interrupted by indel sequences in one or more orthologs. Instead, when the region around the known WUS binding sites was examined in more detail with a 5bp sliding window, a strikingly periodic pattern was observed, where four different conserved motifs were found to be regularly spaced about 15 bp apart. In order from 5’-3’, these motifs were identified as CCGTTGGG, AGTAC, TTGTCAA, and TAATTAATGG , the latter two of which correspond to a predicted W-box motif, and the +970 WUS binding site. In addition, a perfectly conserved sequence was found just 25-36 bp downstream in all orthologs, which consisted almost entirely of tandem repeats containing ATG. The ATG repeats also overlapped with a predicted ALFIN-1 homeodomain/Opaque-2 binding site, suggesting that this sequence may actually represent a modified bZIP motif, or perhaps an atypical homeodomain binding site containing a TGAT core motif. It is not clear how many potential binding sites are present in these ATG repeats, but in consideration of the size of the conserved region, it seems likely that they could accommodate up to three transcription factor proteins simultaneously. The potential functional role of the TGAT motifs is further supported by the observation that they are 4x over-represented in the surrounding 124 bp conserved region, while the TAAT cores actually are 5x under-represented. In addition, pair-wise distance measurements between the two cores revealed a skewed distribution, where few sites were found closer together than the median value of 5bp. When several median-length pairs were aligned, this corresponded to the 13bp motif TAATnnWnnTGAT. When this motif was subjected to Patmatch searches of the A. thaliana genome, it was found to be 26x over represented among the genes directly targeted by WUS. Multiple copies of the 13bp motif were also found in several target genes, including two in the 3’ enhancer of AtCLV3. Together, this evidence suggests the presence of a larger cis-regulatory module that may include up to five other transcription factors besides WUS. Slightly further downstream, another conspicuous feature of the 3’ enhancer is the presence of two large and perfectly conserved sequence blocks, spanning 42 and 32 bp, respectively . Both the size and the degree of sequence conservation in these two regions were exceptional in that they exceeded those found in the coding region of AtCLV3.