Few studies have conducted a thorough evaluation of below ground vine biomass in vineyards, although Elderfield did estimate that fine roots contributed 20–30% of total NPP and that C was responsible for 45% of that dry matter. More recently, Brunori et al. studied the capability of grapevines to efficiently store C throughout the growing season and found that root systems contributed to between 9 and 26% of the total vine C fixation in a model Vitis vinifera sativa L. cv Merlot/berlandieri rupestris vineyard. The results of our study provide a utilitarian analysis of C storage in mature wine grape vines, including above and below ground fractions and annual vs. perennial allocations. Such information constitutes the basic unit of measurement from which one can then estimate the contribution of wine grapes to C budgets at multiple scales— fruit, plant or vineyard level—and by region, sector, or in mixed crop analyses. Our study builds on earlier research that focused on the basic physiology, development and allocation of biomass in vines. Previous research has also examined vineyard-level carbon at the landscape level with coarser estimates of the absolute C storage capacity of vines of different ages, as well as the relative contribution of vines and woody biomass in natural vegetation in mixed vineyard-wild land landscapes. The combination of findings from those studies, together with the more precise and complete carbon-by-vine structure assessment provided here, mean that managers now have access to methods and analytical tools that allow precise and detailed C estimates from the individual vine to whole-farm scales. As carbon accounting in vineyard landscapes becomes more sophisticated, blueberry in container widespread and economically relevant, such vineyard-level analyses will become increasingly important for informing management decisions.

The greater vine-level measuring precision that this study affords should also translate into improved scaled-up C assessments . In California alone, for example, there are more than 230,000 ha are planted in vines. Given that for many, if not most of those hectares, the exact number of individual vines is known, it is easy to see how improvements in vine-level measuring accuracy can have benefits from the individual farmer to the entire sector. Previous efforts to develop rough allometric woody biomass equations for vines notwithstanding, there is still a need to improve our precision in estimating of how biomass changes with different parameters. Because the present analysis was conducted for 15 year old Cabernet vines, there is now a need for calibrating how vine C varies with age, varietal and training system. There is also uncertainty around the influence of grafting onto rootstock on C accumulation in vines. As mentioned in the methods, the vines in this study were not grafted—an artifact of the root-limiting duripan approximately 50 cm below the soil surface. The site’s location on the flat, valley bottom of a river floodplain also means that its topography, while typical of other vineyard sites per se, created conditions that limit soil depth, drainage and decomposition. As such, the physical conditions examined here may differ significantly from more hilly regions in California, such as Sonoma and Mendocino counties. Similarly, the lack of a surrounding natural vegetation buffer at this site compared to other vineyards may mean that the ecological conditions of the soil communities may or may not have been broadly typical of those found in other vineyard sites. Thus, to the extent that future studies can document the degree to which such parameters influence C accumulation in vines or across sites, they will improve the accuracy and utility of C estimation methods and enable viticulturists to be among the first sectors in agriculture for which accurate C accounting is an industry wide possibility.

The current study was also designed to complement a growing body of research focusing on soil-vine interactions. Woody carbon reserves and sugar accumulation play a supportive role in grape quality, the main determinant of crop value in wine grapes. The extent to which biomass production, especially in below ground reservoirs, relates to soil carbon is of immediate interest for those focused on nutrient cycling, plant health and fruit production, as well as for those concerned with C storage. The soil-vine interface may also be the area where management techniques can have the highest impact on C stocks and harvest potential. We expect the below ground estimates of root biomass and C provided here will be helpful in this regard and for developing a more thorough understanding of below ground C stores at the landscape level. For example, Williams et al. estimated this component to be the largest reservoir of C in the vineyard landscape they examined, but they did not include root biomass in their calculations. Others have assumed root systems to be ~30% of vine biomass based on the reported biomass values for roots, trunk, and cordons. With the contribution of this study, the magnitude of the below ground reservoir can now be updated.Grapes are the most valuable fruit crop in the United States, valued at over $6.5 billion annually , but climate change is projected to reduce grape production and quality . Climate affects grape quality by impacting the concentration of sugars, organic acids, and secondary compounds . The climatic conditions producing the highest quality wine cause the berries to reach optimal ratios between sugar and acid concentrations and maximum concentrations of pigment, aroma, and flavor compounds simultaneously . Hot temperatures accelerate sugar accumulation, forcing growers to harvest earlier, before berries reach optimal flavor development, to avoid the high alcohol content and insipid wine flavor from excessive sugar to acid ratios .

Harvest dates have shifted earlier historically, and climate models predict further acceleration of ripening . Growers can partly compensate through management practices, such as trimming canopies or using shade clothes to reduce the ratio of sugar supply to demand , though these practices are costly and increasingly ineffective in the face of climate change . Planting existing cultivars or developing new cultivars with slower sugar accumulation are promising alternative strategies to mitigate these climate change impacts, but these efforts have been hindered by uncertainty around the plant traits controlling sugar accumulation . Grape cultivars vary in berry maturation and sugar accumulation rates, and in their response to abiotic stress, but the main anatomical and physiological mechanisms driving these differences remain unknown . Multiple physiological processes influence berry sugar accumulation and its responses to climate, including photosynthesis, long-distance sugar transport, and local transport and metabolism in the berries . However, the relative importance of these factors in regulating sugar concentrations and fruit growth is debated . Photosynthetic responses to heat and water stress could impact cultivar differences in accumulation rates by affecting the sugar supply for ripening . Further, sugar is transported from the photosynthesizing leaves to the berries through the sugar-conducting vascular tissue – the phloem. At the onset of ripening , the berries significantly accelerate sugar accumulation by initiating active sugar unloading from the phloem, making the phloem the primary pathway for water and resource influx into the berries . The importance of phloem transport to ripening suggests that phloem traits could be important drivers of cultivar differences in sugar accumulation, and that modifying phloem traits to slow sugar accumulation under hot conditions could help mitigate the impacts of climate change on wine quality. However, plastic planters bulk the main traits controlling sugar accumulation in grape remain unclear . The rate of phloem transport is determined by both the hydraulic resistance to the flow of sugar sap, and the activity and kinetics of water and sugar transporters in the sources, sinks, and along the transport pathway . Modeling studies suggest that increasing the hydraulic resistance of the phloem reduces sugar export to the sinks . Therefore, selecting grape cultivars with lower total phloem conductance could decelerate sugar accumulation and improve the synchronization of sugar accumulation with flavor development under hotter conditions. However, a higher hydraulic resistance can make the phloem more susceptible to declines or even complete failures in transport under severe water stress . Thus, we expect cultivars that produce high-quality wine in hot, dry conditions to exhibit phloem hydraulic resistances that slow berry sugar accumulation while avoiding phloem failure. The phloem transport pathway is composed of individual sugar-conducting cells with porous end walls stacked to form conduits . The anatomy of the transport pathway, including the total cross-sectional area of sieve tubes in plant organs, lumen area of individual sieve tubes, and porosity of the sieve plates, significantly impacts pathway resistance . Plants with a greater cross-sectional area dedicated to phloem , sieve tubes with wider lumen areas , and larger and more abundant pores in the sieve plates are expected to have a lower hydraulic resistance .

Total phloem cross-sectional area in the shoots has been found to vary between several grape cultivars , and a greater cross-sectional phloem area has been linked to faster sugar accumulation in the fruit in other crop species . However, the variation of phloem structural traits across cultivars adapted to a diverse range of climatic conditions and the relationship of these traits to sugar accumulation is largely unknown for grapevines. Establishing these anatomical links could allow breeders to modify sugar accumulation by selecting for phloem traits, instead of management practices that can negatively impact the fruit zone environment or yield . In this study, we used a common garden experiment to evaluate the links between phloem anatomy and sugar accumulation across 18 winegrape cultivars typically grown in climatically diverse grape growing regions. We assessed phloem and xylem vascular anatomy in leaf petioles and midveins and berry pedicels, to capture hydraulic resistance along the long-distance transport pathway. We also measured maximum berry sugar accumulation rates in the post-veraison ripening period to capture the greatest capacity for sugar transport . We predicted that traits that reduce hydraulic resistance, including larger total cross-sectional phloem areas, larger mean lumen areas for individual sieve tubes, and more porous sieve plates would increase maximum sugar accumulation rates. We also predicted that cultivars typically grown in hotter wine regions would have traits that increase hydraulic resistance, as an adaptation to increase wine quality by reducing the rate of sugar accumulation. In addition, we measured photosynthesis and vine water stress to compare the impacts of phloem anatomy, vine carbon supply, and vine water status on sugar accumulation rates. Overall, our goals were to determine the most influential traits for sugar accumulation in grape berries and evaluate the role of phloem anatomy in adapting grape cultivars to a wide range of different climates.After 7 days in FAA, the light microscopy samples were soaked in 50% ethanol for 5 mins and then stored in 70% ethanol in preparation for paraffin embedding. Samples were first infiltrated with paraffin by using an Autotechnicon Tissue Processor to treat samples with the following sequence of solutions: 70%, 85%, 95%, 100% ethanol, 1 ethanol:1 toluene, 100% toluene , and paraffin wax , each for 1 hour. The infiltrated samples were then embedded into paraffin blocks with a Leica Histo-Embedder , and allowed to cool. A rotary microtome was then used to make 7μm-thick cross-sections for leaf laminas, petioles, and berry pedicels. Pedicel cross-sections were sampled from the receptacle and petiole and midvein cross-sections were sampled near the interface of the lamina and petiole. After the cross-sections were imaged, pedicels for four cultivars were remelted from their wax molds, oriented longitudinally and sectioned again at 7μm to obtain sieve element lengths. Sections were stained using a 1% aniline blue and 1% safranin solution following a modified staining procedure . Sections were then viewed under bright field or florescence microscopy using a Leica DM4000B microscope and a DFC7000T digital camera . Each pedicel , midvein , and petiole section was then measured for total phloem and xylem cross-sectional area using ImageJ software, by manually selecting relevant tissue areas. Safranin stained the secondary cell walls of the xylem red and phloem cell walls were stained blue by aniline blue. The phloem area measurements included sieve tubes and phloem fibers and parenchyma , and xylem area measurements included xylem vessels, fibers, and parenchyma.The pedicel electron microscopy samples were processed following Mullendore . Briefly, samples were thawed at room temperature, washed in DI water, and cut into 1 mm cross sections with a fresh double-sided razor blade.

Shade cloths can be used to cover the entire canopy and thus affect a large-scale change in vineyard microclimate, or they can be applied directly to the fruiting zone to localize their impacts to berry microclimate. Previous studies have reported that shade cloths are effective in reducing maximum berry temperatures, though they have used different methods for quantifying these effects. Mart´ınez-Luscher et al. ¨ reported that 40% black shade cloths covering the fruiting zone of the canopy reduced cluster temperature by 3.7◦C during the warmest time of the day, while Greer found that 70% shade cloths covering the grapevines reduced canopy temperature by an average of 4.6◦C throughout the day. Similarly, thin shade cloths and plastic films covering the grapevines were shown by Rana et al. to reduce midday berry temperatures by 2◦C and 6◦C below air temperature, respectively. While it is clear that shade cloth can be effective in reducing overall berry temperature, a higher degree of control of berry temperature may be desired. In certain instances, it may be beneficial to reduce berry temperature by a defined margin to avoid negative trade-offs, while also balancing temperatures between opposing sides of the vine. However, many interacting variables are likely to influence the efficacy of shade cloth, such as row orientation, row spacing, trellis type, and topography. Due to the large number of important variables affecting berry temperature, it can be difficult to generalize the relatively small number of experimental results that are only able to explore a few variable combinations in order to predict the effect of shade cloth for a given vineyard system. Crop models provide the potential for generalizing the results of field experiments to predict the outcomes of proposed management strategies for a specific site or climate scenario. Such models could allow for optimization of the design or management of vineyards to mitigate elevated berry temperatures under current or future climates, growing berries in containers given that a large number of simulations can be efficiently performed to cover a wide parameter space.

Previous work has developed models of spherical fruit temperature, including one study that simulated the effect of hail nets on apple temperature. In a recent study, a 3D model was developed and validated that accurately simulated the spatial and temporal temperature fluctuations of grape berries in vineyards with different climates, topographies, and trellises.However, the current version of this model is not able to represent the effects of shade cloth on canopy and berry temperature. The overall goal of this work was to enable model-based evaluation and optimization of strategies for grape berry temperature control using fruit zone shade cloth. With this goal in mind, specific objectives of this study were to: 1) develop a physically-based 3D model of grape berry temperature that incorporates the effect of shade cloth, 2) generate an experimental data set against which the model can be validated, and 3) quantify the interacting effects of different strategies for excessive berry temperature mitigation such as altered row orientation, row spacing, topography, and shade cloth density.The model of grape berry temperature was based on the 3D model described in Ponce de Leon´ and Bailey, and modified to include the effects of shade cloth. A brief description of the overall model is provided below, with a focus on novel additions associated with shade cloth. The model was developed within the Helios modeling framework and has been validated based on field measurements of berry temperature between veraison to harvest. The computational domain consists of 3D geometric elements that fully resolve the spatial structure of the plants, berries, and shade cloth . The berries were represented by 3D tessellated spheres composed of triangular elements, the ground surface by a planar grid of rectangular elements, the woody tissues by a cylindrical mesh of triangular elements, and the leaves by planar rectangles masked to the shape of leaves using the transparency channel of a PNG image.

The shade cloth was formed by rectangles masked to the shape of a grid using the transparency channel of a PNG image, where “holes” in the shade cloth were created based on a grid of transparent pixels.Radiation transfer was simulated using a backward-ray-tracing approach that ensures each geometric element is adequately sampled for both short-wave and long-wave radiation. The model launches a large number of rays from each geometric element to simulate the various modes of radiation transfer, including emission, reflection and transmission based on the radiative properties of each element. When a ray encounters a transparent pixel on an element masked by a PNG image, the ray continues with no interaction. This allows for an efficient fully-resolved representation of shade cloth with a large number of holes. To eliminate domain edge effects, periodic lateral boundaries can be enabled that effectively creates an infinitely repeating vine geometry in the horizontal. For berries, the latent flux term was assumed to be zero since latent cooling is typically considered negligible after veraison. For this study, heat released from metabolic activity ´ within the fruit was assumed to be low enough to be neglected. The heat transfer coefficient from fruit, ground, and leaf surfaces to the atmosphere, h, are specified as described in Ponce de Leon and Bailey . Previous field tests suggested an important role of berry heat storage in accurately representing temperature dynamics, and laboratory and field tests have shown that the chosen values for Cp and rhoA result in berry dynamic temperature responses in close agreement with measurements.To calculate Rn, the model requires specification of the incoming radiation flux, which was generated following the REST2 model of Gueymard, which itself requires site longitude, latitude, offset from UTC, atmospheric pressure, air temperature, atmospheric turbidity coefficient, relative humidity, and Julian day of the year.

The incoming radiation was split into short-wave radiation and long-wave radiation. The short-wave radiation was assumed to be partitioned between PAR and NIR bands, 47% and 53%, respectively. The incoming diffuse long-wave radiation flux from the sky was estimated using the model of Prata. The input parameters to calculate Rn are the surface reflectivity and transmissivity for PAR and NIR bands, and the surface emissivity for long-wave radiation for each element in the domain. The measured values for berries, leaves and ground are reported in Ponce de Leon´ and Bailey and listed in Table 4.1. For all simulations, ambient diffuse solar radiation was neglected and all solar energy was collimated in the direction of the sun. The number of direct rays sampled on each element was set to 500 rays. The radiation scattering depth was chosen to be 3, and it was verified that using larger values would have a negligible impact on results.To evaluate the model accuracy, the time series of experimental measurements and simulated temperatures for berries furthest west were compared. Model agreement with field measurements was quantified using the error metrics normalized root mean squared error – normalized by the difference between maximum and minimum measured data, the coefficient of determination , and the index of agreement. To analyze the modeled berry temperature results in the “simulation experiment”, we took the average temperature of five berries in each of five exterior clusters on both sides of the vine. The selected number of berries and clusters represent a typical experimental sampling strategy . For comparison purposes, the total canopy and cluster daily light interception was calculated by integrating the light interception fluxes over the day. The daily light interception was calculated per vine area for the canopy and per berry surface area for the clusters To compare the effect of shade cloth density on berry temperatures in the simulated vineyard designs, we used indices based on the duration of elevated temperature and the intensity . The 35◦C temperature threshold was chosen because it has been reported to correspond to the approximate onset of unfavorable effects of elevated temperature on grape composition. Furthermore, to identify conditions that effectively balance berry temperature between opposing sides of the vine, we constructed a conditional inference tree with the variables listed in Table 4.2. The conditional inference tree was constructed using the “party” package of R statistical software. The conditional inference tree is a tree-based classification algorithm that performs binary recursive partitioning of data into groups containing observations with similar values. Conditional inference trees are similar to decision trees, blueberry containers the main difference being that each node in the conditional inference trees uses a significance test of independence to select a predictor variable rather than selecting the predictor variable that maximizes the information measure. In the conditional inference tree, predictor variables are circled and ranked and the ones at the top have the highest correlation with the response variable. The algorithm stops if the null hypothesis of independence is not rejected, however, for visualization purposes, the maximum depth of the decision tree was set to 4.

The magnitude of the wind speed varied across the sampling period, but overall Unilateral tended to have greater wind speeds compared to Goblet . During the day, the wind in the fruiting zone tended to come from the east while at night the wind tended to come from the west for both trellis systems . Since the row orientation of Goblet and Unilateral was N-S and the vines were on an east-facing slope, the wind coming from the east tended to be of greater magnitude. For both Goblet and Unilateral, the relative humidity significantly decreased on 25 Sept. 2019, likely due to the increase in air temperature. Overall, during the sampling period, Unilateral tended to be slightly less humid compared to Goblet. The relative reduction in maximum berry temperatures due to the shade cloth in Goblet and Unilateral was consistent across the sampling period, which included a wide range of ambient air temperatures . Pulp temperatures of more than 40◦C were measured on 25 Sept. 2019 for unshaded berries in both trellis systems. For both Goblet and Unilateral, the berry temperature under the shade cloth was close to the air temperature in the morning, while air temperature differences between berries under the shade cloths increased in the afternoon. The peak in measured elevated temperatures happened at different times in Goblet and Unilateral , primarily due to the influence of the ratio between plant height and row spacing on berry exposure. In the evening, under low-light conditions, the During the sampling period, average daily maximum temperatures of the west-facing berries under the shade cloths were 1.6◦C and 2.7◦C higher than the daily maximum air temperature in Goblet and Unilateral, respectively. The control west-facing berries with no shade cloth were up to 8.2◦C and 5.9◦C above the daily maximum air temperature in Goblet and Unilateral, respectively . The relative reduction in maximum berry temperatures due to the shade cloth in Goblet and Unilateral was consistent across the sampling period, which included a wide range of ambient air temperatures . Pulp temperatures of more than 40◦C were measured on 25 Sept. 2019 for unshaded berries in both trellis systems. For both Goblet and Unilateral, the berry temperature under the shade cloth was close to the air temperature in the morning, while air temperature differences between berries under the shade cloths increased in the afternoon. The peak in measured elevated temperatures happened at different times in Goblet and Unilateral , primarily due to the influence of the ratio between plant height and row spacing on berry exposure. In the evening, under low-light conditions, the berry temperatures were similar to the air temperatures, while at night, radiative cooling likely caused the pulp temperature to fall below the air temperatures. Under low light and at night, the temperature differences among treatments were small .The model was validated by using the experimental measurements to determine whether the modeled berry temperatures appropriately responded to the shade cloth relative to the control . The model reproduced the magnitude of the berry temperature increase over air temperature reasonably well for both Goblet and Unilateral. The largest source of error appeared to be due to the transition period when berry sun exposure began, where the time of the simulated maximum temperature increase tended to happen earlier than that of the measurement . This mismatch could be due to slight inaccuracies in determining the position of each berry and leaf.Figures 4.7, 4.8, and 4.6 summarize the results of the simulation experiment in which the effect of different row orientations, row spacing, and slope aspects on berry temperature were evaluated.

A survey in Poland and China found that strawberries had the highest frequency of multiple pesticide residues. In the current study, insecticides were highly prevalent in vegetables and fruits . Some insecticides appeared one time and others were detected several times. Chlorantraniliprole is one of the insecticides detected one time only in apple. This insecticide is one of the diamide insecticides that are widely used against a variety of insect pests due to their selectivity and low mammalian toxicity. Tian et al. determined diamide insecticides in mushrooms and found that these insecticides can be effectively analyzed using HPLC-MS/MS with LOD and LOQ of 0.05 and 5 ug kg−1 , respectively, and recovery rates ranging from 73.5–110.2%. On the other hand, chlorpyrifos is an insecticide that was detected several times in the tested vegetable and fruit samples. Although this insecticide is recommended in Egypt against almond worms in cotton and termites in buildings according to the approved recommendations for agricultural pest control , it was detected in vegetables and fruits collected from farmers’ markets . This insecticide is no longer approved by European Commission due to harmful effects on different organs. In spite of that, it is still detected in a high percentage in many samples of fruits and vegetables, which is consistent with the results obtained in this study. Fungicides were detected in fruits in a higher percentage than in vegetables . The fungicide pyraclostrobin was detected in grapes only . The dissipation rate of this fungicide was studied in strawberry in Egypt when treated with the recommended field rate. It was found that 82% of this fungicide degraded within 14 days of treatment with a half-life of 5 days. In contrast, container growing raspberries the fungicide thiophanate-methyl was detected in four vegetable samples and in six fruit samples as recorded in Tables 1 and 2.

As this fungicide is widely used in the control of a variety of pathogens pre- and post-harvest, it was detected in many vegetable and fruit samples, herbal medicine, raisins, salmon, beebread, and also in cow and human milk. Samples of cucumber and apples were found to have ≥15 pesticide residues . Chlorpyrifos and lamda-cyhalothrin were detected in more than 15 samples with some values higher than MRLs. In our study, pesticide residues exceeding MRLs in vegetables and fruits were 41 and 39%, respectively. Other studies showed the same results, i.e., in Mwanja et al., pesticide residues were detected in 63.3% of the tested vegetable and fruit samples with residue levels exceeding MRLs of the codex Alimentarius in cabbage, tomato, and orange samples. Further, in the study of Hamed et al., residues of pesticides in apples and grapes from Egypt were determined and they reported that 12.7 and 16.4% of pesticide residues exceeded the MRLs, which was slightly lower than what we found in the current study . Consistent with our findings, a study conducted by Parveen et al. in Pakistan reported that pesticide residues in apple and grape samples exceeded MRLs with 28 and 20%, respectively. Estimation of pesticide residues in imported food is necessary to know about food safety. A study in the United Kingdom for monitoring levels of pesticide residues in imported foods from different countries showed that 51.3% of Egypt samples, compared to 77% , 68.3% , 55.1% , 46.1% , and 45.7% contained detectable pesticide residues. They recorded that India, Kenya, Brazil, Egypt, Chile, and the United States were countries with residue levels exceeding MRLs in 18.1%, 11.4%, 7.8%, 5.1%, 3.2%, and 2%, respectively. In the same context, Osaili et al. determined pesticide residues in samples of imported vegetables to the United Arab Emirates.

They found that 30.5% pesticide residues higher than MRLs in total imported samples and found 14% of the Egyptian samples compared to 47%, 33%, 13%, and 43% from India, United Kingdom, China, and Sri Lanka, respectively, contained residues higher than MRLs. The results of monitoring pesticide residues in fruits and vegetables showed that some samples had residues that exceed the MRL standard, which may lead to risks when consuming food contaminated with these pesticides. In addition, some pesticides do not have corresponding residual limits, which make it difficult for farmers to safely use these pesticides and for the government to monitor their use. Therefore, identification of acute and chronic dietary risks is necessary to assess the risks associated with consuming vegetables or fruits that contain pesticide residues above the MRLs. In this regard, Chu et al. evaluated the risks of food exposure to 26 insecticides on strawberries and found that despite the presence of high detection rates for these residues, they showed risks of acute and chronic exposure at a level of less than 100%. In our results of risk assessment, residues of lambda-cyhalothrin, fipronil, dimothoate, and omethoate were found to have acute or chronic risks in consumers in the case of consuming 100 or 200 gm day−1 of spinach, zucchini, kaki, and strawberry, respectively . In line with our findings, the results of Tao et al., 2021 showed that the fungicide carbendazim had a risk quotient value of 2.9 in wheat flour samples, indicating an unacceptable dietary risk. Furthermore, Tankiewicz and Berg showed that pesticides of lambda-cyhalothrin in courgettes, captan in apples and cucumbers, dimethoate in cour-gettes, and linuron in carrots exceeded the MRLs and pose a health risk. In an Indian study conducted by Sinha et al., they stated that excessive application of pesticides on grapes cause adverse health effects in developing countries as grapes and apples are contaminated with different classes of pesticides including organophosphate, which cause high health risks for consumers.

In this context, Javeres et al. showed that the prolonged exposure to insecticides could lead to physiological disorders including high blood pressure, hyperglycemia, overweight or dyslipidemia, which may cause metabolic syndrome and other chronic diseases. For these adverse effects, it is important in each country to monitor pesticide residues in food for food safety and human health.Tomato is the world’s second largest vegetable crop rich in nutrients. Tomato fruit development includes three stages. The first stage is characterized by an increase in cell number and starch accumulation, followed by cell enlargement with starch degradation and soluble sugar accumulation in the second stage. Fruit ripening is the last stage, associated with the accumulation of soluble sugars, carotenoids, organic acids, and volatile organic compounds in fruits. The chlorophyll accumulation and photosynthetic activity of green fruits influence the nutritional components and flavor of ripening tomato fruits. Some genes have been reported to affect chlorophyll accumulation, chloroplast development and fruit quality. As negative regulators, DE-ETIOLATED 1/high pigment 2 and UV-DAMAGED DNA-BINDING PROTEIN 1/ high pigment 1 are involved in chloroplast formation and chlorophyll accumulation in tomato fruits. The tomato GOLDEN2-LIKE transcription factors SlGLK1 and SlGLK2 play an important role in chloroplast formation and chlorophyll accumulation.Evidence suggests that the SlGLK2 gene is predominantly expressed in fruits and that the latitudinal gradient of SlGLK2 expression influences the production of unevenly colored tomato fruits. Over expression of the APRR2- LIKE gene, the closest homolog of SlGLK2, increased the size and number of chloroplasts and enhanced chlorophyll accumulation in green tomato fruits. TKN2 and TKN4, two Class I KNOTTED1-LIKE HOMEOBOX proteins, act as transcriptional activators of SlGLK2 and APRR2-LIKE genes to promote chloroplast development in tomato fruits. BEL1-LIKE HOMEODOMAIN11 also plays an important role in chlorophyll synthesis and chloroplast development in tomato fruits. The ripening of tomato is mainly regulated by the ethylene pathway and many transcription factors. In the ethylene bio-synthetic pathway, S-adenosylmethionine synthetase catalyzes the reaction of ATP and methionine to form S-adenosyl-L-methionine. 1-Aminocyclopropane-1-carboxylic acid synthase and ACC oxidase catalyze the conversion of SAM to ACC and of ACC to ethylene, respectively. The MADS box gene RIPENING INHIBITOR controls the early phase of ripening and ethylene production via transcriptional regulation of ACSs and ACOs. The other ripening regulators affecting ethylene production also include the NAC transcription factor NOR, the SQUAMOSA PROMOTER BINDING protein CNR, blueberries in pots the ethylene response factor ERF B3, the AP2/ERF member AP2a, and several MADS box proteins, such as TDR4/SlFUL1, SlFUL2, SlMADS1, TAGL1, and TAG1.

Auxin is an important phytohormone involved in flower fertilization, fruit setting, fruit initiation and development. Auxin is also essential in the regulation of cell division and expansion, controlling final fruit size. Auxin modulates plant development through transcriptional regulation of auxin-responsive genes, which is primarily mediated by two gene families: the short-lived nuclear protein Aux/IAA family and auxin response factors. Most ARFs have an N-terminal DNAbinding domain required for transcriptional regulation of auxin response genes, a middle region functioning as a repression domain or activation domain , and a C-terminal dimerization domain involved in the formation of homodimers or heterodimers. ARFs can act as either an activator or a repressor of the transcription of auxin-responsive genes. Numerous studies have indicated that ARFs are involved in many tomato developmental processes. SlARF4 negatively regulates chlorophyll accumulation and starch biosynthesis in tomato fruit. Our previous study showed that SlARF10 positively regulated chlorophyll accumulation via direct activation of the expression of SlGLK1. Downregulation of ARF6 and ARF8 by over expression of Arabidopsis microRNA167 results in the failure of pollen germination on the stigma surface and/or growth through the style in tomato. However, the function of SlARF6 in the regulation of fruit development is still not well understood. In this study, SlARF6A was found to be involved in photosynthesis, sugar accumulation and fruit development in tomato. Our data demonstrate that SlARF6A plays an important role in the regulation of fruit quality and development.The SlARF6A gene has an open reading frame of 2608 bp encoding a putative protein of 869 amino acids. Amino acid sequence analysis revealed that, like SlARF7 and SlARF8, which have typical conserved ARF domains, SlARF6A protein also contained B3-DNA, ARF, and AUX/IAA binding domains . A phylogenetic tree was constructed to gain insight into the phylogenetic relationship among ARF proteins in Arabidopsis and tomato. ARFs were divided into four major classes: I, II, III, and VI. SlARF6A along with SlARF6B and AtARF6 were grouped into subclass IIa and are closely related to AtARF8 and SlARF8 , indicating possible functional similarity among them. To determine the expression pattern of SlARF6A in planta, a transcriptional fusion was constructed between the SlARF6A promoter and the GUS reporter gene. GUS staining in the transgenic tomato plants was detected in leaves, stems, buds, flowers, and fruits at different developmental stages, an indication of the ubiquitous expression of SlARF6A in all tissues tested. The GUS staining was weak in the early fruits at 2 and 4 days post anthesis but became strong at 8, 30 and 45 DPA , suggesting possible roles of SlARF6A in the development of tomato fruits. To examine its subcellular localization in plants, SlARF6A was fused to GFP and transferred into tobacco protoplasts. Fluorescence microscopy analysis revealed that SlARF6A was specifically localized in the nuclei . A GAL4-responsive reporter system in yeast was employed to reveal the transcriptional activity of SlARF6A. SlARF6A was fused to GAL4-BD to form a pGBKT7-SlARF6A fusion plasmid and subsequently transformed into yeast. Yeast transformants harboring the pGBKT7-SlARF6A construct grew well in the medium lacking Trp, His, and Ade , while the yeasts transformed with pGBKT7 vector alone could not . Assessing transcriptional activity revealed that SlARF6A is a transcriptional activator.To elucidate the physiological significance of the SlARF6A gene in fruit development, upregulated and downregulated transgenic lines corresponding to independent transformation events were generated in tomato plants. qRT-PCR was used to evaluate the expression level of SlARF6A in all transgenic lines. Compared with thelevel in the wild type , the expression level of SlARF6A was decreased in RNAi 2 and 6 plants but increased in OE-4 and 6 plants . It is noteworthy that altered SlARF6A expression led to a dramatic change in chlorophyll accumulation in transgenic lines. Compared with WT plants, the OE-SlARF6A plants had dark-green fruits at the green fruit stage, whereas the RNAi-SlARF6A plants had light-green fruits . The impact of altered SlARF6A expression on chlorophyll accumulation was analyzed by measuring the chlorophyll content in fruits and leaves. The SlARF6A overexpression lines possessed greater accumulation of chlorophyll in the fruits at immature green, mature green, breaker, and orange stages, whereas the RNAi lines had lower chlorophyll accumulation in the fruits at immature green and mature green stages than the WT plants .

However, using 9 migration edges Treemix reported the reverse direction; as F3 and F4 statistics cannot easily infer directionality, more heavily sampling of the Asian populations or alternate methods may be needed to determine whether flow is occuring in both directions.To determine if invasive populations have experienced loss in genetic diversity, we used the software ANGSD to estimate average pairwise nucleotide diversity in 20 kb increments across the 20 largest contigs of genome for each population. Invasive populations can sometimes exhibit reduced levels of diversity early on in their history due to a founder effect , while ancestral populations tend to have the greatest amount of diversity as they have had many generations to accumulate mutations. A Welch one-way test found a significant difference in mean pairwise nucleotide diversity between clusters. We then used pairwise Games-Howell tests and found each cluster to be significantly different , except for the Eastern U.S., Brazil, and Italy when compared to each other. As Asia is the ancestral home of D. suzukii, it is no surprise that South Korean wild populations exhibit the highest diversity levels . Similarly, the lab populations from Japan and Hawaii have half as much pairwise diversity as the wild South Korean population, consistent with as mall lab population size. The invasive populations display an intermediate level between these extremes. To assess whether invasive populations may have experienced a bottleneck or population shrinkage, we also estimated Tajima’s D in the same genomic intervals. Extremely positive values can indicate a loss of rare alleles, plant pots with drainage which can occur during a population shrinkage, while extremely negative values can indicate a recent bottleneck followed by rapid expansion .

A Welch one way test again indicated significant differences in mean Tajima’s D between clusters , and pairwise Games-Howell tests found all clusters to be statistically different except for Western U.S. against Brazilian flies. Strains from Hawaii and Japan both had high genome-wide levels of Tajima’s D, indicative of a loss of rare alleles that can occur during a population shrinkage . The remaining populations had neutral values of D, except for Ireland’s relatively high value. Based on this, we conclude there are no strong signals for a recent bottleneck, although the high genome-wide D value for Ireland suggests a recent population shrinkage. As our Irish samples were collected in 2016 only one year after its discovery in Ireland, we could be observing the founder’s effect in action .Based on population allele frequencies, we have shown that D. suzukii exhibit population structure based on region and invasion history. In the New World populations, we find that Eastern and Western U.S. samples appear to be distinct populations. While this could be the result of continuous population variation from East through Central to the West coast, it is more likely the case that the two populations experience little gene flow due to strong geographic barriers such as the Sierra Nevada or Rocky Mountain ranges, and the fact that key target fruit crops such as cherries, raspberries, blueberries, and strawberries are primarily grown in states that we sampled . Any genetic exchange between these regions would likely be the result of human activity, such as could be the case with samples collected from Alma Research Farm, Bacon County, Georgia clustering with the Western U.S. populations. As other nearby collections failed to share this signal, the Alma research population could represent a recent and isolated migration event. Otherwise, we see little evidence of migration events or admixture between the Eastern and Western U.S., which is somewhat surprising as the country’s supply of fresh blueberries, cherries and caneberries are concentrated in a few states and shipped across the country .

However, recent changes to cultural management such as more frequent harvesting and post-harvest chilling may be responsible for disrupting the D. suzukii lifecycle and limiting cross-country transport . While we were able to detect population structure between eastern and western locations in the U.S., we were surprised to discover a lack of structure on a finer scale, either based on latitude or simple geographic distance, given the large number of loci analyzed. In a similar study using 3,484 SNPs in 246 Hawaiian D. suzukii samples, researchers were able to identify three distinct populations roughly seperated by islands . The fact that D. suzukii has been present in Hawaii since 1980, in addition to the isolation by island, are likely the strongest factors in providing enough genetic drift to create such differentiation. As the continental U.S. D. suzukii have only been present since 2008, it may be too early for finer structure to have developed. Alternatively, continual dispersion and transportation of D. suzukii around the U.S. may be hindering the development of more local structure. Several studies have reported a low probability of D. suzukii surviving when exposed to freezing temperatures, based on cold survival assays of wild-caught specimens , suggesting that flies collected in cold-winter regions such as Washington, Michigan, Maine, and New York could be annual migrants to the area from nearby warmer locations. The lack of north south population structure supports the hypothesis that flies are regularly re-migrating into colder climates after the harsh winters have ended. Alternatively, flies could be tolerating winters by surviving inside human structures , or by having evolved resistance to freezing temperatures. Studies using D. suzukii collected from different locations have reported different levels of rapid cold-hardening response, suggesting there could be regional selection present .

If populations in northern regions undergo strong seasonal fluctuations in allele frequencies, such as has been demonstrated in wild D. melanogaster populations collected in Pennsylvania , by only sampling sites in the summer we may be missing signals of population differentiation between the north and south. Likely, some combination of these factors is responsible for the success of D. suzukii in these regions, and further studies will be needed to identify the causes. North-south clines in specific traits such as diapause and circadian rhythms have been previously identified in drosophilids and could be at play here as well . Further analyses using methods such as those recently used to detect SNPs correlated with invasive success could be applied to this dataset to find signals of selection. Fst values between populations from the U.S., Brazil, and South Korea were low and agree with previously published Fst estimates based on Pool-Seq data; Olazcuaga et al. 2020 observed that Fst between U.S., European, Asian, and Brazilian populations varied between 8.86% to 9.02%. However, we were surprised to see that our Italian and Irish samples had much higher values of Fst compared to the other populations, and even to each other. This discrepancy could be due to the small sample sizes we had from Europe; in this scenario, pooling larger number of samples can improve power to estimate Fst, and we instead rely on comparing the relative Fst values between populations for our analysis. High Fst values between our Japanese and Hawaiian populations were expected, however, as these have likely experienced strong drift during their time in captivity. In general, plastic plants pots we find that our treemix and migration results largely coincide with the proposed invasion pathway inferred from microsatellites , as well as a recent pre-print that reanalyzed invasion pathways with pooled sequencing data . We see that European and U.S./Brazilian populations form two distinct clades, emphasizing these regions were invaded by two independent migrations from Asia. Hawaii is the first population to diverge in the Americas, followed by the Western U.S., then the Eastern U.S. and Brazil. Additionally, in the Western U.S., we detected a strong signal of admixture from Hawaii, which could be due to multiple or ongoing migration events. We also detected signals of admixture from the Eastern U.S./Brazil to Ireland, which matches the predicted initial invasion pathway and suggests multiple migration events. Unique to our analysis, we recover support for admixture of Western U.S. samples in Asia, suggesting that migrations could be ongoing in both directions. Invasive species transport is strongly associated with international trade of live plants and plant products , and indeed agricultural export data supports the possibility of this migration as Japan receives almost 15% of all U.S. blueberry exports, and Oregon recently became the first state to begin shipping blueberries to South Korea in 2012 . It should be noted that while Treemix infers direction of migration, the model can occasionally infer the incorrect direction, particularly when populations are closely related without an available outgroup . In conjunction with evidence of this widespread ongoing migration, we observed nucleotide diversity levels of all invasive populations to be only moderately below that of the wild South Korean population, a trend also observed in Fraimout et al. . Typically, recent invasion events are characterized by reduced diversity relative to the ancestral populations due to founder or bottleneck effects . However, successive invasion events can provide relief from any initial bottlenecks by providing increased genetic diversity. This has been observed to occur in multiple animal studies , and could lead to increased ability to adapt and evolve to new climates.

Correspondingly, in our analysis we did not find populations with broadly low values of Tajima’s D, suggesting little bottleneck effect. As measures to reduce impacts ofinvasive species are often hindered by repeated migrations , it will be important to enforce that fruits being exported and imported internationally are free of live D. suzukii as required by the U.S. Department of Agriculture, even though this species is already internationally established. We anticipate that the genomic data provided here will prove useful in many fields of biology beyond the scope of this study. Knowledge of genetic variation and alternate alleles present within a species can be informative for the design of probes and micro RNAs , such as for the purpose of creating gene drives to control invasive species. Gene drive mechanisms to eliminate D. suzukii have been experimentally tested on multiple lines to ensure the miRNAs are broadly effective , but a large dataset of wild population sequencing allow researchers to more confidently select target sites that are non-variable and thus susceptible to Cas9 targeting . Drury et al., demonstrated that minor natural polymorphisms in target sites reduce gene drive effectiveness in flour beetles, and tools have been developed to help researchers design gRNAs accounting for population variation . Similarly, with the recent development of a CRISPR-Cas9 editing and RNAi knockdown protocols for D. suzukii , prior knowledge of allelic variation will allow researchers to design targeting oligonucleotides more precisely to avoid loci with variability. Most recently, our dataset has been used to study sensory receptor evolution in D. suzukii, giving insights into its evolution toward becoming a major agricultural pest . Other future uses of this trove of genomic data could involve insecticide resistance studies or the development of diagnostic assays for rapid detection in the field.Tuta absoluta is a worldwide economic pest of tomatoes and other solanaceous crops. A member of the gelechiid family, this moth lays eggs on the above-ground portion of the plant, where the hatched larvae will spend their lives creating “mines” throughout the plant tissue before pupating and emerging as adults . At a reproduction rate of up to 10 generations per year, untreated infestations will eventually result in complete death of the plant, leading to up to 100% agricultural loss. Although a large effort has been made to develop and implement integrated pest management programs across different world regions , typical treatments have included heavy use of a variety of insecticides , leading to the rapid appearance of insecticide resistance. As tomatoes represent a massive economic industry, with an estimated 252 million metric tons of tomatoes harvested in 2020 , there is a serious need to understand the invasive biology of this insect and to develop tools for detection and prevention. Tuta absoluta was originally detected in Peru in 1917 but was not recorded as an agricultural pest until the 1960s and 70s when it was discovered in tomato fields in Chile, Argentina, and Venezuela; by the 1990s it was widespread across South America. In 2006, Tuta absoluta appeared in Spain ; since then it has rapidly colonized Europe, Asia, and Africa. It is generally believed that the Peruvian highlands is the ancestral home of T. absoluta, and that the rapid colonization to the rest of Latin America was due to the introduction of Tuta absoluta by human transport of contaminated fruit, although few studies have confirmed this .

Although rare events of dispersal to distant islands would probably lead to population divergence and eventual speciation , population genetic data suggested that most species with FFs readily overcome oceanic barriers and show weak population genetic structure across islands . Extensive gene flow across islands associated with FFs suggests that this fruit type represents a trait favouring species cohesion in fragmented landscapes . Our conclusions drawn from population genetic data seem to be at odds with studies on Hawaiian lineages for which high species diversity has been linked to limited dispersal of FFs . These contradictory results are most probably explained by the different habitats in which Hawaiian vs. Galápagos and Canary Island lineages of FF species evolved. According to the previous studies, Hawaiian FF lineages that have undergone extensive diversification mostly occur in moist forests, where limited dispersal by sedentary birds, in some instances because of increased seed or fruit size , could have promoted speciation . Hawaiian tropical moist forests currently cover an area of 6700 km2 , which is substantially larger than the estimated potential area for densely forested zones in the drier Canary Islands and Galápagos , especially if we take into consideration the fact that human impact has significantly reduced the original area of moist forests in Hawai‘i. Thus, high species number as a consequence of limited bird dispersal in forested areas is a more plausible explanation for Hawaiian lineages than for the other archipelagos. In addition to the high availability of forested areas on the Hawaiian Islands, growing raspberries in pots other factors may account for the substantial proportion of ‘woodyFF’ ancestors inferred for this archipelago .

For instance, trait evolution on other Pacific islands prior to the colonization of Hawai‘i may have promoted the acquisition of a woody habit under insular conditions, as suggested for Tetramolopium Ness . In summary, differences in patterns of character evolution and speciation among archipelagos can be attributed to context-dependent conditions for lineage diversification , although common trends across archipelagos also occur .Phylogenetic studies have demonstrated that oceanic archipelagos have promoted the in situ diversificationof many plant lineages. In this sense, oceanic islands could also represent a framework in which to address questions about the opposite pattern: i.e. what factors are responsible for a lack of speciation in certain lineages? Excluding the Hawaiian lineages mentioned previously, our analyses suggest that FFs could be one intrinsic factor involved in species cohesion. FFs are generally linked to high colonization ability and extensive gene flow among islands , which may, at least in part, explain why this type of fruit is often displayed by monotypic lineages . Nevertheless, lack of speciation in extant oceanic lineages is obviously not limited to FF species. DF species also represent a substantial proportion of monotypic lineages . Several factors, most also applicable to FF, monotypic lineages, may account for this fact. Because the colonization of oceanic archipelagos could have been constrained to discrete temporal windows , some of these lineages may have found opportunities for colonization only in recent times, so that there has not been sufficient time for recurrent speciation. In turn, it is also plausible that earlier colonizers had more opportunities for in situ diversification. In line with this argument, niche pre-emption by earlier colonizers may have hindered adaptation to novel environments , limiting opportunities for speciation.

Such a possibility could be further tested by the analysis of habitat differentiation among populations of monotypic lineages with DFs showing widespread distributions . Another complementary explanation is that some DFs are frequently dispersed over large distances , and thus recurrent gene flow among populations would be expected to reduce the likelihood of speciation, as discussed previously for FFs. Molecular studies on Phylica arborea Thouars, for example, indicate that this tree species overcame distances of up to 8000 km during the colonization of multiple oceanic islands around southern Africa despite its DF condition . A final point for consideration is that the actual number of monotypic lineages might be conditioned by taxonomic uncertainty in those plant groups in which limited taxonomic research or cryptic speciation obscures the real pattern of diversification. Further interdisciplinary research, including taxonomic, molecular and ecological data, is needed to identify the most important factors related to lack of speciation in monotypic lineages. Lastly, although poorly studied on islands, other intrinsic factors affecting patterns of diversification include those characters related to habitat colonization and population persistence that are induced by environmental cues . For instance, the architectural organization of trees and shrubs favours phenotypic plasticity across canopy layers, allowing persistence and reproduction even under stressful conditions . In addition, resprouting ability is thought to be another key trait for population persistence . Recent ecological studies on widespread island taxa, such as Pinus canariensis C.Sm. ex DC. , Olea cerasiformis Rivas-Mart. & del Arco and Croton scouleri Hook.f. , have indicated that phenotypic plasticity and resprouting ability play a significant role in habitat colonization across broad environmental gradients in Macaronesia and Galá- pagos. These studies support the idea that not only fruit dispersal traits, but also specialized life history strategies involved in population establishment and persistence, may be important in the successful colonization of remote areas and subsequent patterns of diversification of colonizing lineages .

Further studies using a plant trait perspective may help us to understand the implications of intrinsic factors for lineage diversification.Although the present study suffers from typical constraints of meta-analyses , it constitutes, to our knowledge, the first attempt to summarize the inferences drawn from molecular studies on three paradigmatic oceanic archipelagos. Despite clear differences in physiography and geographical isolation, our analyses revealed some similar patterns among these island systems. Thus, phylogenetic reconstructions of character states suggest that ancestors of species-rich lineages in these archipelagos were predominantly herbaceous and with DFs. Island environmental conditions apparently selected for a shift from an herbaceous to a woody habit in most lineages. In contrast, fruit type appears to be strongly phylogenetically constrained, which may have promoted speciation in DF lineages because of generally limited dispersal ability compared with FF lineages. The complexity of the evolutionary processes and biotas considered here, including diversity in fruit morphologies relevant to dispersal ability and dispersal vectors, places limits on the applicability of these patterns. For instance, our analyses suggest that the relationship between high species diversity and FFs indicated by previous studies could be a particular outcome for plant groups that evolved in moist forests, such as those found in Hawai‘i. Traits related to frequent dispersal , in combination with those favouring population persistence , probably play a signifi- cant role in species cohesion and thus contribute to prevent speciation in some lineages. Although further phylogenetic research with new molecular markers is resolving complex evolutionary patterns in radiating lineages , ecological and population genetic studies on islands, particularly those focusing on monotypic lineages, are still critically needed to allow a broader picture to be developed of how evolution works on oceanic archipelagos.In recent years, plant pot with drainage sustainable irrigation has become a crucial aspect of orchard management to reduce inputs in agricultural systems. In the current global warming and desertification scenario, both environmentally and economically oriented reasons provide the basis for a water saving approach, which has become paramount in irrigated orchards. Automated irrigation management is evenmore important in high-density systems in which growers tend to increase orchard productivity and reduce management costs by mechanizing operations. In the past, irrigation management was commonly based on soil water status or environmental indices. However, tree water status provides the most precise drought stress indices, in spite of soil and environmental conditions. Indeed, plants represent the intermediate component of the soil plant-atmosphere continuum, and their physiological responses are the result of an integration of both soil and environment. This implies an advantage of plant-based over soil-based methods for an accurate irrigation scheduling . Plant water requirements differ among species and even cultivars, making irrigation scheduling and management a complex task for growers. The physiological responses of plants to decreasing water availability are various and depend on evolutionary adaptation and acclimation to new climatic conditions. The olive species has a very wide genetic pool, and includes genotypes that can respond to drought using different mechanisms of leaf dehydration tolerance and leaf morphological and structural adaptations .

Gucci et al. and Lo Bianco and Scalisi found different leaf stomatal regulation among olive cultivars. Connor suggested that olive genotypes use avoidance and tolerance mechanisms under drought. Midday stem water potential is considered a very sensitive parameter of plant water status for irrigation management . However, 9 stem is mostly measured by the Scholander pressure chamber, which does not allow for continuous monitoring and automated irrigation. Recently, plant-based sensing technologies are taking hold for the continuous plant water status monitoring in fruit trees. In most of cases, sensors are mounted on above ground organs such as stem, fruit and leaves . In olive, trunk dendrometers have been associated with tree water stress thresholds and proposed for irrigation management due to their relatively easy installation and stability across the season . In the last few years, emphasis has been given to the use of leaf patch clamp pressure probes for the continuous assessment of olive leaf water status . The output of LPCP probes is expressed as attenuated pressure of leaf patches , which is inversely related to cell turgor pressure . Therefore, the highest values of pp occur around solar noon, as that is the moment in which leaf cell turgor is the lowest. Ben-Gal et al. found an inversion of the pp curve in severely drought stressed olive trees. Thereafter, Fernández et al. classified water deficit states based on the degree of inversion of the curve. State I represented no drought stress and leaves with a non-inverted curve, state II grouped leaves experiencing partial inversion of the curve and mild water deficit, and state III enclosed all leaves experiencing severe water deficit and full inversion of the curve. Fruit-based probes based on linear variable displacement transducers can provide good information on fruit growth, which on a diel scale is mostly dominated by water in- and out-flows, rather than carbon gain; thus, fruit diameter variations respond to water deficit . Fernandes et al. studied olive FD dynamics in response to water deficit, suggesting the appropriateness of fruit gauges for continuous plant water status monitoring. Although FD and pp are strictly related to soil water availability and plant water status, they are also influenced by environmental variables, crop load, genetic factors and phenology. The derived values of FD and pp represent good indicators of the rate at which water enters and exits leaf or fruit, respectively. The reasoning behind this assumption resides in the fact that they both represents rates of changes – pressure and diameter, respectively – from an initial state, which, in the short term are mainly driven by tissue water exchanges. Plants modulate water movements to and from the two main transpiring organs using several strategies such as osmotic adjustments, stomatal closure or cell-wall elasticity regulation. As a result, we hypothesized that either leaf or fruit water status might be privileged under increasing water deficit in olive genotypes with different drought tolerance/avoidance mechanisms. This work aimed to study olive fruit and leaf water dynamics in relation to tree water status. Our hypothesis was that, similarly to what found in nectarine , the combination of RR fruit and RRleaf might provide an even more accurate identification of plant water status, rather than monitoring each parameter independently. In addition, this study aimed to identify cultivar-specific RR fruit/RR leaf relationships to determine whether the genotypes under study preserve leaf or fruit water exchanges under increasing water deficit, as sink power for water might differ among genotypes.After removing the three-day buffer period from FD, pp, RRfruit, and RRleaf data, a 5-day interval in stage II was obtained . Trends of FD did not highlight different fruit growth dynamics among irrigation levels in stage II, for both NB and MN. FI induced sharper fruit shrinkage than deficit irrigation at 222 and 223 DOY, as fruit with an optimal water status are likely to exchange more water in the warmest hours of the day.

In MG fruit, all three fungi were able to grow on the surface, but none of the pathogens was able to cause rot. In contrast, ripe tomato fruit represented a compatible system for infections as all three fungi induced lesions that spread rapidly. This contrasting ability to cause disease in fruit has been previously reported for a variety of fungal pathogens, particularly those displaying necrotrophic behavior . The tomato gene SlWRKY33 has been shown in leaves to be expressed in response to B. cinerea inoculation, and deletion of this gene leads to increased susceptibility, indicating its role in defense response . We demonstrated that, as in leaves, B. cinerea is capable of inducing SlWRKY33 in MG and RR fruit. Furthermore, F. acuminatum also induced SlWRKY33 in MG and RR fruit, and R. stolonifer did so substantially in RR fruit. These findings indicate that all three pathogens triggered disease responses in the host and that the strength of the response was reflective on the success of the infection process. During interactions with tomato fruit, B. cinerea, F. acuminatum, and R. stolonifer employed a variety of pathogenicity and survival strategies that involved redox processes, drainage collection pot carbohydrate catabolism, and proteolysis. Moreover, the degree to which particular strategies were used varied according to the ripening stage of the fruit, as certain processes were emphasized in either MG or RR fruit . These observations suggest that the fungi can sense the physiological environment of the fruit and react accordingly with suitable infection, growth, or quiescence strategies.

Though these fungi are incapable of causing disease symptoms in MG tomato fruit, this study demonstrates that they do make attempts to either establish infections or create a suitable environment in fruit for fungal growth and do not merely die on the host tissues. However, when the conditions in fruit are highly unsuitable , the infection strategy of the fungal pathogen is often insufficient to cause successful infections. In many cases, when fungal pathogens encounter incompatible conditions, like in unripe fruit, they enter a quiescent phase with limited growth and activity . During ripening, the physicochemical properties of the fruit tissues change, resulting in compatible conditions for the fruit-pathogen interaction and the reactivation of quiescent pathogens . In this context, it would also be interesting to investigate the strategies employed by the three fungi during inoculations of other plant organs such as leaves. Our initial tests, however, indicated that both F. acuminatum and R. stolonifer are incapable of infecting tomato leaves even when leaves were senescing. This observation may suggest that the isolates of these two fungi are exclusive fruit pathogens and lack the molecular toolset to grow on leaves. The redox environment of the plant-pathogen interface influences the outcome of the interaction. Upon pathogen detection, ROS are rapidly produced by the host, triggering a downstream signaling of various defense responses . The enzymatic agents of this oxidative burst are respiratory burst oxidative homologs , which generate superoxide O − 2 in the apoplast . This oxidative burst has been previously reported in incompatible tomato-Botrytis interactions , including MG fruit, in which the appearance of a necrotic ring is associated with resistance to B. cinerea . However, necrotrophic pathogens can exploit this ROS response by overwhelming the host with their own ROS production . In leaves of French bean , B. cinerea has been shown to produce ROS as virulence factors by activating the NADPH oxidases BcnoxA and BcnoxB , coupled with the regulatory protein BcnoxR .

Although we did not detect strong upregulation of these genes during inoculation of fruit, other ROS producing systems, including laccases and glucose oxidases were upregulated during inoculations of tomato fruit. In F. acuminatum, a BcnoxA homolog FacuDN4838c0g1i1 and BcnoxB homolog FacuDN3221c0g1i1 were induced in specific treatments. A BLAST search did not reveal anyhomologs of BcnoxA or BcnoxB in R. stolonifer, nor were any homologs of Bclcc8 or BcGOD1 detected in either F. acuminatum or R. stolonifer. In addition to ROS generation machinery, fungal pathogens must protect themselves against the oxidative stress of the infection site. Methods of ROS scavenging in phytopathogenic fungi include enzymatic and non-enzymatic mechanisms . SODs catalyze the conversion of O− 2 produced by RBOHs into the less reactive hydrogen peroxide . B. cinerea mutants lacking the BcSOD1 gene have been shown to have reduced virulence on tomato leaves . In tomato fruit, BcSOD1 is upregulated for both MG and RR ripening stages, which suggests it is also a critical gene for fruit colonization. H2O2 can be converted to water by either catalases or peroxidases such as GPXs or PRXs. All three pathogens demonstrated upregulation of specific mechanisms of catabolizing H2O2, but only F. acuminatum showed enrichment of genes involved in the H2O2 catabolic process. The usage of these H2O2 catabolizing systems varied between the pathogens. While B. cinerea utilized catalases in MG fruit at 1 dpi, F. acuminatum and R. stolonifer produced more catalases and peroxidases in RR fruit at 1 dpi. In each pathogen, multiple genes involved in protein degradation were found to be upregulated during fruit inoculations. The strong enrichment of proteolysis-related genes may indicate that protein degradation is important for pathogenicity of F. acuminatum and R. stolonifer but not B. cinerea. Some pathogen-derived proteases, such as Sep1 and Mep1 in Fusarium oxysporum, are known to serve as suppressors of host-immune response in plant-pathogen interactions .

Even though their specific roles in pathogenesis are not fully characterized, several aspartic proteinases in B. cinerea have been described . Three of the B. cinerea aspartic proteinases that we found to be induced in tomato fruit were also found to be upregulated during infection of grape berries . Aspartic proteinases were also found to be among the upregulated proteinases in F. acuminatum and R. stolonifer , though all three pathogens appeared to utilize a diverse suite of proteinases of different families. Especially prominent in F. acuminatum and R. stolonifer were proteins with similarity to subtilisin-like proteases. This family of enzymes is mostly associated with plants and particularly plant defense, but subtilisin-like proteases involved in pathogenicity have been described for fungi as well . Since these inhibitors possess sequence similarity to the proteases themselves, the enzymes identified in F. acuminatum and R. stolonifer may be inhibitors, proteases, or a mixture of both. Additionally, proteases can help with host tissue decomposition by breaking down cell wall structural proteins or can serve in degradation of proteins to provide a source of nutrition for fungal growth . For example, the saprotrophic fungal species Verticillium albo-atrum and V. dahliae were described to secrete proteases to break down structural proteins that stabilize the plant cell walls . High proteolytic activity resulting in the degradation of proteins into free amino acids was also reported during fermentation of tempeh by several Rhizopus species . Botrytis cinerea, F. acuminatum, and R. stolonifer also make use of a variety of CAZymes during interactions with the host. Several CAZyme families are involved in the breakdown of physical barriers present in the host tissues, namely the various cell wall components , cell wall reinforcements , and the waxy fruit cuticle. Many of these enzymes, such as polygalacturonases, pectin methylesterases, pectate lyases, and endo-β-1,4-glucanases, square plastic pot mirror the activities of host enzymes active during the ripening-related softening of the fruit . Others, such as cellulases, cutinases, and lipases, degrade components that are not typically degraded during ripening. Production of cellulases is also coupled with enzymes involved in degradation of cellobiose, the disaccharide product of cellulose breakdown. Both B. cinerea and F. acuminatum appear to focus on production of these latter CAZyme families in MG fruit more than in RR fruit. This may be due to the greater strength and integrity of the cell wall in MG fruit, which requires the fungus to mount a larger attack on the physical barriers in order to penetrate into the cells. Degradation of pectin is a hallmark feature of B. cinerea infection of plant tissues .

The principal enzymes responsible for this process are polygalacturonases , pectin methylesterases , and pectate lyases . Both PGs and PLs cleave the α- 1,4-linkages in the homogalacturonan backbone of pectins. PMEs catalyze the removal of methylester groups on the C6 carbons of galacturonan, which allows for further degradation by PGs. Although overexpression of PME inhibitors in Arabidopsis leaves has been shown to increase resistance to B. cinerea , mutations in Bcpme1 and Bcpme2 do not appear to affect virulence in tomato leaves . In B. cinerea, all three classes of enzymes appear to be highly expressed in MG fruit but not as prominently in RR fruit. Not only do the GH28, PL1-7, and PL3-2 families constitute a greater fraction of upregulated CAZymes in MG fruit, but for PGs, PLs, and PMEs that are commonly upregulated in MG and RR fruit, upregulation is consistently greater in MG fruit over RR fruit. Additionally, although no F. acuminatum PGs were detected in MG, the two upregulated PMEs, FacuDN5818c0g1i1 and FacuDN10179c0g1i1, were only active in MG fruit. Moreover, PL1-7 and PL3-2 genes were strongly expressed in MG fruit, with one PL3- 2 gene, FacuDN8473c0g1i1, showing a log2FC of 10.29 at 1 dpi, the highest of any plant CWDE in this treatment. Only one R. stolonifer PG, RstoDN2036c0g1i1, was detected in MG fruit. However, given that this single R. stolonifer PG was one of only two CAZymes found in 1 dpi MG fruit, it is reasonable to believe PG activity in R. stolonifer isbeing underestimated due to low sequence coverage of fungal transcripts in this treatment. The absence of upregulation of any R. stolonifer pectate lyases in any fruit further underscores this point. Given the prominence of pectin degradation in B. cinerea and F. acuminatum, a more targeted analysis of R. stolonifer pectin degradation, especially in MG fruit, is warranted. Degradation of the host cell wall in MG fruit by pathogen enzymes may accelerate ripening and in turn facilitate a more favorable environment for colonization. Pectinderived oligosaccharides have been shown to induce ethylene production in tomato fruit , which further upregulates expression of host CWDEs, including PG. B. cinerea can synthesize its own ethylene via the α-keto-γ-methylthiobutyric acid pathway , though it is still unknown whether the pathogen produces ethylene during interactions with the fruit. Ethylene production during plant infection has also been reported via the KMBA pathway for species of Fusarium , but not, to our knowledge, for R. stolonifer. However, the specific genes involved in the KMBA pathway in B. cinerea or Fusarium spp. have yet to be elucidated. As colonization proceeds, sugar substrates become available due to degradation of cell wall polysaccharides as well as increased access to stored sugars in the fruit. As a consequence, fungi actively infecting RR tomato fruit induced enzymes that metabolize simple sugars. Sugar metabolism is accompanied by expression of CAZyme families involved in the production and modification of chitin, the structural component of fungal cell walls. Chitin production is known to be a hallmark of growth for fungal pathogens . Interestingly, chitin production and modification appear to be prominent not only in RR fruit for each pathogen, but also in MG fruit inoculated with F. acuminatum, where a much greater amount of mycelia growth was observed compared to the other two pathogens. The equal representation of CE4 enzymes in MG and RR fruit inoculated with F. acuminatum is reflective of the ability of this fungus of producing hyphae at either fruit ripening stage. The abundance of polysaccharide-building glycosyltransferases in RR infections with R. stolonifer is also likely connected to the abundant mycelial growth. Other CAZyme families represent more specialized roles in the infection process. Production of enzymes in the AA7 family may be related to the production of polyketide toxins in B. cinerea and R. stolonifer. B. cinerea is known to produce botcinic acid, a polyketide mycotoxin, during infection . However, the AA7 genes detected to be upregulated in fruit infection here are not known members of the botcinic acid pathway, suggesting that B. cinerea may produce additional uncharacterized polyketide mycotoxins during fruit infection. Even though upregulated F. acuminatum genes involved in toxin production are not annotated as members of the AA7 family, fumonisins are products of polyketide metabolism . The observed upregulation of fumonisin biosynthesis related genes indicates that F. acuminatum also produces polyketide mycotoxins during infection of unripe and ripe tomato fruit.

For wind dispersal, modelling suggests that even moderate topographic variability can have large impacts on variation in dispersal distances and directionality . Finally, slope steepness influences dispersal distances of heavy seeds, which are more likely to roll down downhill , and the likelihood of seed dispersal via runoff . Individual plants might also differ in the quantity and quality of animal-mediated seed dispersal due to the actions of thirdparty players. Some of these effects are mediated by vegetation structure, with plants in more open and risky places receiving fewer visits by dispersers . In other situations, animals respond to olfactory, visual or acoustic predator cues, leading to reduced seed removal rates in frugivorous birds , bats and granivorous rodents . In addition, rodents are sensitive to ungulate presence because of trampling risk or disturbance by rooting ; in Q. ilex, the presence of ungulates was associated with lower quality seed dispersal by rodents and changes in caching sites . Finally, responses to predators and competitors can interact with other traits, such as the presence and concentration of deterrents . Insects frequently infest fruit pulp, seeds or dispersal structures, which can affect seed dispersal. Howler monkeys preferentially feed in Ocotea diospyrifolia trees with high fruit infestation by curculionids and low fruit infestation by moths . The seed parasitoid wasp Macrodasyceras hirsutum reduces attractiveness of Ilex integra berries to frugivorous birds through ‘colour manipulation’; infested fruits are less likely to ripen and turn red, decreasing the risk that the fruits will be eaten and wasps killed .

In synzoochorous dispersal, seed infestation can increase the probability of rejection or of immediate consumption , round plastic plant pot but generally reduces caching rates , thus decreasing dispersal quality. However, not all scatter hoarders discriminate between infested and sound seeds, particularly before insect emergence . Note that these synzoochorous examples are based on responses to individual seeds and it is unknown to what degree they translate into selection among trees differing in infestation levels. Insect attack also affects anemochory. For example, Rhinocyllus conicus larvae feeding on C. nutans receptacles induce callus formation, resulting in inhibited seed release, shortened pappus filaments and reduced dispersal distances . In turn, insect infestation is often affected by masting , thereby creating another, indirect pathway through which temporal variation in seed output can affect seed dispersal.Most of what we know about intraspecific variation in seed dispersal represents a snapshot in time—a frame or two in a potentially long movie of life. While these frames might accurately represent the fitness outcomes for an annual plant, the majority of plants discussed in this review are long-lived perennials that are interacting with an extremely dynamic world where both intrinsic and extrinsic factors vary through time. Although we are not in a position to evaluate the overall consequences of this variation, it is important to acknowledge the variation exists. Fruit crop sizes vary between years. Sometimes this variation is relatively subtle and driven by such factors as resource availability or climatic conditions . Sometimes the variation is extreme, as seen in masting species . Different dispersal kernels are necessary to capture mast versus non-mast years , with potentially greater LDD when acorn density is low . The fitness impacts of this variation should depend at least partially on how synchronous fruit crop size variation is in the population and community. Fruit crop size also varies over longer, ontogenetic time scales; crop sizes increase with perennial plant age and size, often plateauing at some point and remaining relatively constant until death, sometimes showing declines with senescence late in life .

Many other intrinsic traits relevant to intraspecific variation in dispersal are temporally dynamic. Fruit/seed size, and most likely such traits as pulp:seed ratio, vary across years . Plant height increases ontogenetically . Rewards and deterrents can change from year to year and in some cases even seasonally. Temporal variation in extrinsic factors, or the ecological context, is perhaps even more extreme. Fruiting neighbourhoods can change from year to year as different individuals and species respond differently to changing resources and climate . Other aspects of habitat structure around individual plants can change through time due to successional processes and demographic processes , as well as anthropogenic impacts . Lastly, interactions with non-disperser animal communities can vary greatly from year to year as a function of, among other drivers, changes in individual crop sizes and in fruiting neighbourhoods, and population fluctuations of other interacting animal species .Although many exceptions exist, much work on intraspecific variation in seed dispersal has taken a more or less univariate approach; for example, the impact of fruit crop size, fruit size or plant height on dispersal. Alternatively, some address multiple traits affecting dispersal and quantify the relative importance of each and the presence or absence of interactions. In a recent study using an individual-plant-based network analysis of frugivory, locations of individual H. succosa trees within the network were determined by a combination of plant height, fruit size and sugar concentration, with shorter individuals with larger fruits and intermediate sugar concentration being most central . Nonetheless, the true complexity of dispersal is often overlooked. In this review we have also taken primarily a univariate approach, which we argue has value, especially at our early stage of understanding the drivers of inter individual variation in seed dispersal. However, it is critical to understand that we do not believe that this is really how the world exists.

We noted the difficulties of knowing what animal seed dispersers base their harvesting decisions on when so many potentially important traits co-vary: fruit size, absolute and relative quantity of reward, seed number and size, nutrients, toxins and more . For example, do frugivores select fruits to harvest based on size per se or on the underlying variation in pulp:seed ratio ? Such complexities surely exist in other dispersal systems as well. For example, in anemochorous plants, the size of the dispersal structure increases with seed mass, but generally not sufficiently to maintain a constant wing loading . Co-variation of seed release height and seed terminal velocity , and of abscission force and terminal velocity have also been reported. It is highly likely that co-variation of traits relevant to seed dispersal is as extensive with wind dispersal as with frugivory. Complexity also arises in animal-dispersed species because foraging animals often make foraging decisions hierarchically . For example, foraging frugivores must first select the foraging patch, then choose the individuals to feed in, and then choose which fruits to harvest from that plant. In addition, multiple cues may be used hierarchically at any single stage of this process. For example, experiments with the large fleshy-fruited shrub C. monogyna elegantly demonstrated hierarchical selection by Turdus migratorius of individual trees in which to forage. First, birds preferred trees with larger crop sizes, but if crop sizes were constant, they preferred plants with larger fruits, and, finally, if fruit size was constant, they preferred plants with greater pulp:seed ratios . Understanding variation in seed dispersal is further complicated by the concomitant inter individual variation in seed dispersers, including sexual dimorphism, ontogenetic changes, inter individual variation in specialization and unique animal personalities . For example, our discussion of fruit size variation in M. communis and its effect on fruit availability to different seed dispersers was based on measured intra individual and inter individual variation in fruit diameters but only mean gape width for the dispersal agents . Interpretations could be different if inter individual variation in the seed disperser species was also incorporated. More generally, 25 liter rount pot inter individual variation in plants and dispersers interact and it might be difficult to understand one without understanding the other . Plants almost certainly respond at the individual level to variation in how seed dispersers interact with them; these eco-evolutionary feedbacks mean that intraspecific variation is important in both sides of the interaction, perhaps even intensifying the individual-level variation in both players . Further complexity is likely in particular dispersal systems, such as for example with diplochorous dispersal, where dispersal is accomplished by a sequence of steps that involve different dispersal agents such as primary dispersal by a frugivorous bird and secondary dispersal by a rodent . We predict that, all else being equal, diplochorous dispersal systems would have even greater inter individual variation in seed dispersal success than non-diplochorous systems given that variability arising during the second phase of dispersal is building on variability created during the first phase of dispersal.

For example, as discussed previously, intraspecific variation in seed size can affect selection by both frugivorous birds and rodents, sometimes in the same way and sometimes not.While we show substantial evidence that drivers of intraspecific variation in seed dispersal are diverse and pervasive, we also reveal large gaps in our understanding, partly due to a paucity of research directly addressing intraspecific, especially inter individual, variation in seed dispersal, and partly due to the complexity of interactions among drivers. Our understanding is limited further by the existing empirical work’s focus on the quantity of seed dispersal, with much less consideration of the quality of dispersal or LDD. Of particular interest are the intrinsic trait-based drivers that can respond to natural selection. The best-supported and best-understood intrinsic driver of inter individual variation in seed dispersal is crop size; with more seeds produced, more seeds are dispersed. Crop size is also likely the most widespread driver, being relevant to most if not all forms of dispersal. Though less well supported and less well understood, fruit/seed size is likely the second most widespread intrinsic driver. Again, it seems to be relevant to a broad range of seed dispersal modes. However, when it comes to animal-mediated dispersal we do not have a good understanding of the ultimate cause of size-based fruit or seed selection—is it fruit/seed size per se, or some co-varying trait such as pulp:seed ratio? Remaining intrinsic drivers are even more poorly understood, though apparently range from widespread but weak, such as plant height, to sporadic and variable in strength, such as colour polymorphism. For extrinsic drivers, a variety of studies have addressed the impact of fruiting neighbourhoods on inter individual variation in seed dispersal, but we do not understand well when to expect competition for dispersers and when to expect facilitation of dispersal. With respect to habitat structure, much relevant work has been from the perspective of anthropogenic impacts of habitat fragmentation and degradation on seed dispersal rather than from the perspective of interspecific variation in seed dispersal. Beyond limited empirical work, we are further hindered by an even greater lack of theory related to the drivers of intraspecific, especially inter individual variation in seed dispersal. While there have been some theoretical developments around fruit crop size and seed dispersal success , we are aware of no other developed theory that can guide our understanding of the drivers of inter individual variation in dispersal and potential demographic and evolutionary responses to such variation. Looking forward towards potential research directions, in Box 2 we highlight a selection of outstanding questions concerning intrinsic drivers of intraspecific variation in seed dispersal that we personally believe to be especially informative and intriguing to answer. We present these questions as a starting point to advance our understanding of intraspecific drivers of seed dispersal. One promising approach to answer these questions and disentangle the complexity inherent in intraspecific seed dispersal is a frugivore-centred modelling approach . This approach advocates parameterizing field data on intrinsic animal factors and behaviour, as well as extrinsic landscape factors, to test and quantify the strength of the variables affecting the spatially explicit deposition of seeds across the landscape . Mechanistic simulations can be used in a hierarchical manner to test the effect of multiple factors one at a time, to quantify their relative influence on patterns of seed deposition . Studies using this approach have successfully quantified the impact on seed dispersal of edge-following behaviour in a fragmented landscape , fruiting neighbourhoods and drivers of reduced LDD . Although primarily envisioned to study endozoochory, similar methods have been applied to epizoochory and other dispersal modes by considering relevant intrinsic and extrinsic factors . Additionally, a powerful molecular approach that matches individual seeds or seedlings to maternal plants across dispersal modes is also promising for studying individual variation in seed dispersal and may compliment simulation modelling approaches. D

The work presented in this chapter is thus an attempt to narrow down the most likely mechanism that cytokinin uses to affect WUS transcription, translation, and protein distribution. Surprisingly, the results found that elevated levels of cytokinin did not directly affect WUS transcription, nuclear localization, or stability, nor did cytokinin have any significant effect on CLV3, eliminating a possible indirect mechanism. Instead, a novel absence of cytokinin response was identified in the CZ, and evidence suggests that this zone is maintained both by the lack of transcription, and by an unknown repressive mechanism that can affect B-type ARR proteins.Cytokinin responses were also correlated with WUS protein stability, starting roughly 12 hours after exogenous treatment. Auxin however, dramatically reduced WUS protein levels within just 4 hours, suggesting that this hormone has a more direct effect on protein stability. This suggests a model where auxin responses in the CZ and PZ cells stimulate protein degradation pathways that confine WUS proteins to the RM, where cytokinin responses may favor protein stability.In order to identify which part of the WUS-CLV3 feedback loop is affected by cytokinin responses, this work began by crossing the pTCSn1:mGFP5-ER reporter was crossed into wus-1 and clv3-2 mutant backgrounds. In untreated plants, both clv3-2 and WT meristems were found to have strong cytokinin responses in the RM, with a faint fluorescent signal extending deep into the pith and provascular tissue . The wus-1 mutant was similar, though its fluorescent signal did not become faint in the deeper tissue layers, presumably because this mutant meristem did not produce pith or provascular tissue. Treatment with exogenous 6- benzylaminopurine for 24 hours did not change the location of the cytokinin response in WT meristems, or in the meristems of either mutant. Instead, 25 liter plant pot the strength of the fluorescent signal was more than tripled in all three backgrounds, suggesting that endogenous cytokinin response mechanism is able to function over a wide range of concentrations.

The enhanced signal was most easily detected in the weakly fluorescent pith cells, but interestingly, the immature leaves, young anlagen, and the apex of the SAM all failed to produce any fluorescent response at all. In both WT and clv3-2 mutants, the absence of cytokinin responses occurred in a circular patch at the apex of the Central Zone and extended two cell layers deep. In wus-1 homozygousmutants, a similar response-free zone was found to be variable, but was detected in 84% of sectioned meristems, and extended only one cell layer deep . The near-complete lack of cytokinin responses in the CZ was unexpected, though presence of this function indicates a previously unrecognized feature of meristem organization involving two opposite and adjacent cytokinin response fields. As exogenous cytokinin applications were not able to induce pTCSn1:mGFP5-ER expression in the response-free zone to any significant degree, the two-component system pCLV3:GR-LhG4 x p6xOP:ARR1ΔDDK-GR was used to ectopically stimulate cytokinin responses specifically in the CZ tissue. As previously described, the ARR1ΔDDK-GR construct is a modified version of ARABIDOPSIS RESPONSE REGULATOR 1 , which activates cytokinin response genes following exposure to dexamethasone. Plants containing both constructs were then crossed to three different lines containing the fluorescent reporters: pTCSn1:mGFP5-ER, pCLV3:mGFP5-ER, and pWUS:eGFP-WUS. Surprisingly, when observed over a 48 hour time course of dexamethasone treatment, the pTCSn1:mGFP5-ER reporter did not immediately occur in the middle of the CZ as expected. For comparison, the pCLV3:mGFP5-ER reporter uses an identical promoter, indicating that the induced cytokinin response should occur in a pattern similar to the middle row in Figure 3.2. Instead, the pTCSn1:mGFP5-ER signal first appeared at the extreme edges of the peripheral zone, where it progressively appeared in adjacent cells in a centripetal manner, slowly constricting the cytokinin response-free zone until it disappeared between 24 and 48 hours later.

The centripetal pattern was visible in both L1 and L2 cells, though the L2 signal was weaker and lagged behind the L1 by 1-3 cell diameters. By 48 hours, the response-free zone was completely lost, and pTCSn1:mGFP5-ER expression become nearly homogenous throughout the SAM.The pCLV3:mGFP5-ER reporter in contrast, was expressed in the apical pattern as expected for the CLV3 promoter. The fluorescent pattern occurred in conical patch of cells at the apex of the SAM, and extended up to four cells deep. The fluorescence levels were mostly uniform, though the L2 frequently had significantly less expression than the other layers. The expression pattern was already fully formed in the absence of dexamethasone treatment, and remained unchanged through at least 24 hours. The fluorescent pattern became broader in proportion to the size of the meristem at 48 hours, but the longitudinal pattern did not significantly change. Deep inside the SAM tissues however, a faint pCLV3:mGFP5-ER signal could be detected, which produced a central hourglass-shaped column more than 20 cell layers deep. At the beginning of the time course, the pWUS:eGFP-WUS reporter produced a nuclear-localized pattern centered on the RM, with a radial concentration gradient spreading into all adjacent cells as expected. This pattern did not change after 6 hours of dexamethasone treatment, but by 12 hours a subtle increase in the number of cells displaying the pWUS:eGFP-WUS fluorescent reporter was apparent. The number of small meristem-like cells also began to increase over time, accumulating in a rootward direction at a rate directly proportional to the loss of the underlying large pith cells. The pWUS:eGFP-WUS expression pattern followed the downward appearance of the new cells, eventually producing a brightly visible fluorescent column extending more than 20 cells deep.

Elongate voids with no fluorescence were visible on either side, similar to the faint column produced by pCLV3:mGFP5-ER. Interestingly, long term ectopic induction of the pCLV3:GR-LhG4 x p6xOP:ARR1ΔDDK-GR system did not significantly change the volume of the SAM over the first 24 hours, but by 48 hours the SAM volume had quadrupled. This exponential growth pattern continued in plants subjected to prolonged 120 hour treatments, eventually producing a spherically swollen SAMs 1- 2mm in diameter, with frequent superficial cracks . Curiously, while the change in cell proliferation appeared to be abrupt in the pTCSn1:mGFP5-ER and pCLV3:mGFP5-ER reporter backgrounds, the proliferation rate in the pWUS:eGFP-WUS background was more gradual, beginning at least 12 hours earlier than in the other two lines. This precocious behavior may be related to the concentration of WUS proteins in this line, as the presence of the pWUS:eGFP-WUS construct can complement wus-1 mutants, and likely double doubles the concentration of WUS proteins in the presence of the WT copy of WUS.While CLV3 does not appear to induce or respond to cytokinin to any significant extent, WUS proteins displayed a more complicated pattern as shown by pWUS:eGFP-WUS reporter in Figure 3.4: Part of the WUS pattern overlaps with the cytokinin-response-free zone, and typically no WUS was found in either the deep RM or the PZ, where cytokinin responses were clearly present at comparable time points. The failure of WUS to activate cytokinin responses in the CZ is somewhat surprising, as 24 hours of exogenous 6-bap treatment moderately increased pWUS:eGFP-WUS fluorescent levels in both WT and clv3-2 mutant backgrounds . On the other hand, if cytokinin is required to activate WUS transcription, the presence of WUSexpression in tissues that lack a clear cytokinin response is equally difficult to explain. When cytokinin responses are eliminated with the cytokinin receptor triple mutant ahk2/3/4, only trace amounts of pWUS:eGFP-WUS fluorescent signal could be detected in seven day old plants. The lack of cytokinin responses was further confirmed by treating pWUS:eGFP-WUS x ahk2/3/4 plants with exogenous 6-bap, which did not significantly change the fluorescent pattern . However, the ahk2/3/4 mutant was quite variable, as 74% of examined SAM tissues displayed no fluorescence, while the remaining 26% ranged from faint GFP patterns to nearly full WT-like patterns . To deplete native cytokinin in WT meristems without the physical defects of the ahk2/3/4 mutant, the dex-inducible construct p35S:GR-LhG4::p6xOP:CKX3 was used to over-expresses CYTOKININ OXIDASE 3 , which degrades native cytokinin molecules. Following 24 hours of dexamethasone treatment in this background, the pCLV3:mGFP5-ER reporter showed no significant change in expression . Parallel attempts to study pWUS:eGFPWUS in the p35S:GR-LhG4::p6xOP:CKX3 background produced extremely variable results during the first 24 hours, black plastic plant pots ranging from the complete absence of fluorescent signal, to near-WT patterns, but became consistent by 48 hours of dex treatment.When WUS transcription was checked with RT-PCR however, both WT and ahk2/3/4 mutants background were found to have detectable WUS transcription localized to the RM .

The expression pattern of WUS also largely unchanged in ahk2/3/4 mutant RNA in-situ’s, suggesting that cyokinin responses primarily affect WUS protein. Further RNA in-situ’s following the time course treatment of the pCLV3:GR-LhG4 x p6xOP:ARR1ΔDDK-GR system found that cytokinin did not significantly increase WUS transcription in the CZ cells .This indicates that the pWUS:eGFP-WUS fluorescence observed in CZ cells is a product of protein movement, not local transcription. The RNA in-situ’s further revealed that WUS transcription patterns also expanded in a rootward direction, similar to the pWUS:EGFP-WUS pattern shown in Figure 3.2. By 48 hours, WUS expression was clearly found throughout the entire volume of the enlarged RM, with the exception of L1 and L2, which had little or no WUS transcripts. In many cases, large elliptical voids appeared in the peripheral zone, which corresponded to the presence of lateral anlagen. When two voids were present simultaneously , the central RNA expression pattern is reminiscent of the central column displayed by the pWUS:eGFP-WUS reporter in Figure 3.2. Though WUS is known to be non-cell autonomous, the close correlation between RNA and GFP patterns suggests that WUS proteins has a short mobile range, here estimated at 3 cell diameters.Previous research has shown that the non-cell autonomous movement of WUS protein does not have tissue-specific patterns , suggesting that the plasmodesmata are unlikely targets of cytokinin regulation. In order to explore other possible means of protein movement regulation, this study began by performing hand-cut longitudinal sections of pWUS:eGFP-WUS plants, providing an un-biased view of the WUS concentration profile in the deeper layers of the SAM, thereby avoiding the loss of signal associated with tissue depth. Special care was taken to avoid saturating the pWUS:eGFP-WUS reporter during imaging, so that semi-quantitative analysis might reveal subtle patterns . In untreated plants, the pWUS:eGFP-WUS reporter revealed a nearly symmetrical concentration profile, with a triangular peak centered on the RM, tapering off over 3-4 cell diameters . The location of the peak varied between L3-L5 in different sections, which likely reflects error introduced by tangential or oblique cuts. Above the peak, the fluorescent gradient was strongly linear, tapering to near undetectable levels in L1 cells. In the deep meristem tissues, the rootward gradient was equally linear and symmetric for the first 2-3 cell diameters, but then began to flatten out into a low but relatively constant background signal. It is not clear how much of the deep-layer signal reflects the presence of WUS, as the pWUS-eGFP-WUS reporter did not display its characteristic nuclear-localized pattern in these cells. Instead, the fluorescent signal largely co-localized with the developing chloroplasts in deepest cells layers, suggesting that this background signal is at least partially derived from chlorophyll auto-fluorescent noise. However, no such noise can be detected in the absence of pWUS-eGFP-WUS, or when histone or ERtagged fluorescent proteins are used , strongly implying that this background signal reflects the actual WUS protein distribution. When pWUS:eGFP-WUS plants were treated with exogenous 6-bap for 48 hours, no significant changes were observed in the upper gradient , either in the slope or in the total fluorescent concentration. The signal started to diverge by L4 however, where fluorescent signal became as much as 2x brighter down through at least L10 .When cytokinin responses were ectopically induced in the pCLV3:GR-LhG4 x p6xOP: ARR1ΔDDK-GR background, the pWUS:eGFP-WUS reporter produced patterns very similar to the exogenous cytokinin treatments. The upper gradient remained unchanged, while the deeper cell layers starting at roughly L5 doubled their fluorescent signal. A time-course analysis further revealed that the deep-meristem signal began to appear after 12 hours, and was fully formed by 24 hours. Interestingly, by 48 hours the gradient in L1-L3 cells suddenly increased their fluorescent amplitude by 140%, yet the slope of the gradient in these cells remained unchanged.When cytokinin responses were reduced with the p35S:GR-LhG4::p6xOP:CKX3 construct, a slightly different pattern emerged.

The declining trend began before significant numbers of senescent leaves were observed, and continued unperturbed even after most leaves had been lost In comparison, rosette leaf initiation rates revealed a nearly constant production rate at about 1.1 leaves per day, which is about the same as the initial rate of flower bud production. Meristem growth stopped between 26-28 days after induction, indicated by the simultaneous plateau in both immature green bud numbers and total flower numbers . In many cases the buds around the arrested meristem remained green for several days, during which time they can be induced to resume growth by removing the fruits or following the senescence of the old fruits. Flowers produced during the transition to this quiescent phase were often small, infertile, and had petals that did not exceed the sepals. The visible symptoms of senescence eventually appeared as an abrupt color change, proceeding from pale green, yellow, dark red in less than 48 hours, and eventually became brown as the tissue dried out. Senescence affected multiple tissues within the apical region simultaneously, including the SAM, 1-2 mm of the subtending stem, and all attached flower buds within that region, while tissues immediately below remained green . No abscission layer was detected. One possible trigger for such apical senescence may come from the leaves, which showed an increasing trend of senescence prior to meristem arrest . To study the possibility that apical senescence might be triggered by mobile signal produced by senescent leaves, growing strawberries vertically a further experiment was performed that removed leaf tips from adult-phase leaves before they began to display senescent-related color changes.

Removal was timed to coincide with the maximum leaf length growth rate, well before senescence could occur. The results however, show that this actually had the opposite effect, as meristem arrest occurred on average 4.6 buds, or about 1.8 days sooner in leaf-tipped plants than it did in the controls . Alternatively, the fruits might be the source of a growth inhibitor, as predicted by the alternate bearing inhibitor hypothesis. To eliminate this possibility, the flowers were removed on a daily basis in order to observe changes in meristem activity and the time of growth arrest. Because previous results had shown that the rate of immature bud production and total stem height were closely correlated during the flowering period , internode lengths were measured as a proxy for meristem activity in A. thaliana. The resulting data revealed that both the controls and de-fruited plants displayed a similar declining trend of meristem activity that eventually reached zero growth . However, de-fruited plants clearly offset the time of meristem arrest through an extended period of almost linear bud production rates. This linear trend began a day before the control meristems slowed to zero growth, and lasted foral most a week before resuming the declining trend that the control plants had already completed. To verify the effects of fruit load in Avocados, SAM tissue was collected from branches with and without subtending fruit, four months after anthesis. To test the dominance and inhibitor hypothesis, the expression profile of four genes were measured with qPCR, yet this analysis found no significant patterns related to the presence of the fruit .In the carbohydrate competition model, the fruits are thought to consume the majority of available nutrients, leaving little if any for the rest of the plant. The remaining parts must either reduce their growth in direct proportion to the limiting nutrient, or trigger starvation responses in order to survive short-term depletions.

In most plants, the two most commonly encountered nutrient depletions involve carbohydrates and nitrogen, both of which have well-characterized response patterns that also show a significant degree of overlap. Under carbohydrate starvation conditions, for example, the plant tissues typically suppress respiration and growth while consuming their starch reserves. Eventually both proteins and lipids are degraded, releasing free amino acids and nitrogen in the form of ammonia. Some nitrogen is recovered in glutamine or asparagine biosynthesis, while the rest either diffuses into the media or is consumed by the nearest available sink tissue. Under prolonged conditions, large portions of the cytoplasm are consumed by autophagy, in which even organelles are degraded in lytic vacuoles containing cysteine/serine proteases. These vacuoles eventually coalesce until the cell consists of little but the nucleus and a large vacuole, which is followed by cell death under extreme cases. This pattern of responses is also strongly reflected in the present study. Starch breakdown is predicted both by trehalose signaling and direct digestion by AMY1. Although an increase in asparagines biosynthesis was not detected, the methionine gamma-lyase enzyme is known to release ammonia, and two nitrate transporters were upregulated, perhaps reflecting a decrease in free nitrates. Autophagy is consistent with the upregulation of an array of cystein/serine proteases, the near-lack of DNA laddering, and the presence of MC3. Carbohydrate starvation has also been reported to reduce osmolarity and membrane permeability, which parallels the observation of a weak plasmembrane, and the up-regulation of desiccation responses found by the present study.

Nitrogen starvation in contrast, is associated with the suppression of most chloroplast and photosynthesis related enzymes, loss of starch reserves, and the increase in uptake transporters and various storage compounds, including asparagine, glutamine, and various organic acids, while reducing losses that occur through degradation. Prolonged shortages typically result in anthocyanin production. Autophagy also seems to be an important part of nitrogen recycling in senescent leaves, though this may not have been detected in published studies that rely on short term starvation experiments.In the present data, the symptoms of nitrogen starvation are equivocal. Two photosystem subunits were reduced , which is broadly consisted with the degradation of the chloroplasts predicted by FZL and ELIP2. Autophagy signals do occur in the data, but the collected tissues were not obviously senescent, suggesting that autophagy may be more closely related to carbohydrate starvation. The increase of PAP1 is consistent with the biosynthesis of flavonols and other red pigments under nitrogen stress, though the much stronger increases in GL3, PAP2, and MYB12 reported by were not detected in the present study. The nitrate importer NRT1.2 was increased, but this conflicts with the earlier interpretation of amino acid catabolism in the present data. Thus the IM does not appear to be synthesizing storage proteins as predicted by nitrogen starvation studies. In consideration that the growth conditions used in this study included supplemental nitrogen fertilizer, the existence of a nitrogen shortage is unlikely. Instead, the relatively weak nitrogen starvation signal found here is potentially a consequence of the much more significant carbohydrate starvation response.Among the candidate induction genes that might be inhibited by high fruit loads, the results found that none of the floral induction pathways were reduced. Instead the expression of FT was strongly increased , as were several downstream targets including SOC1, AGL71, and perhaps also AGL44. One possible explanation for this pattern might be found in the expression pattern of FT, which based on microarray data is also produced by the ovaries and immature fruits, in addition to the leaf and stem vasculature. The failure to remove all immature flower buds during tissue collection might then bias the results in favor of these induction genes. In avocado trees, the TFL1 and DAM1 paralogs showed no significant response to fruit load . The bio-synthetic enzymes that produce phloridzen are currently unknown, so the presence of this compound cannot be evaluated with the present data.The fruit dominance hypothesis also does not seem to be strongly supported by the present study, drainage planter pot as all detected parts of the auxin response pathway were suppressed by high fruit load conditions. However, Arabidopsis is among the minority of species that do not have strong apical dominance responses, which could imply that this finding is an artifact. The failure to abscise immature fruits like many alternate bearing trees, for example, may indicate the lack of a functional fruit dominance system. However, the observation that isolated nodes of Arabidopsis can suppress axillary bud growth following auxin treatment , and that the lower portion of Arabidopsis branches initiate growth in a basipetal pattern, suggest that the dominance mechanism has not been lost, but is instead suppressed. A likely candidate for this suppression is the plant hormone strigolactone, which is able to suppress branching in the shoot when expressed in the roots.

In A. thaliana, the widespread expression of the strigolactone biosynthesis gene MAX1 in the vasculature is consistent with the broad suppression of dominance pathways in the species. Given that strigolactones appear to inhibit bud growth by blocking their ability to export auxin, this system has interesting parallels with the 2nd fruit drop, king fruit dominance, and even the number of flowers produced by axillary inflorescences. Immature fruits are already known to export auxin, suggesting that the plant may control their numbers by secreting root or shoot derived strigolactone into the inflorescence. The up regulation of strigolactone production under starvation conditions is certainly consistent with the increase in MAX1 expression levels found in the present microarray data. Such a role may explain the different alternate bearing amplitudes that were found when avocado trees were grafted to different rootstocks. Expression of ARF2 and RMS2 paralogs in Avocado trees however, did not reveal a significant response to fruit load .Another potential candidate for fruit inhibition might also include the trehalose signaling pathway. Although the precise role of this signaling sugar is not well understood, biosynthesis mutant are lethal, and high concentrations of trehalose-6-phosphate do seem to be correlated with starch biosynthesis. Accumulating evidence however, suggests that trehalose may have a central role in controlling the broad details of cellular metabolism. Several TPP genes are upregulated in response to nitrate treatments , and also by carbohydrate starvation. This mirrors the findings of the two trehalose phosphate phosphatases that were identified in this study, and in a recent profiling experiments with alternate bearing trees. Trehalose has also been shown to inhibit the kinase activity AKIN10/11, which has broad effects throughout the cell, and may explain how trehalose can regulate pathways such as cell size and stress tolerance. Intriguingly, the vegetative-adult phase change has also been implicated to be a product of carbohydrate supply, which also involves trehalose signaling.Although the data supports the existence of a massive senescence response, much of this signal disappears when the symptoms of carbohydrate starvation are considered. This is consistent with observations of the tissue during collection, which was often still green and displayed no external symptoms of senescence. The only remaining sign of biologic stress is the slight increase of AtRLP54 expression levels.The inflorescence meristem of Arabidopsis is a determinate growth, known to stop functioning after producing a predictable number of flowers in the Ler ecotypes. Under the growth conditions used by this study for the Col-0 ecotypes, the time of meristem arrest was slightlymore variable, but otherwise displayed a nearly identical pattern. Growth arrested meristems however, do not immediately terminate their activities, but instead exists in a quiescent state for several days, during which they can resume growth when the subtending fruits are removed. The present data suggests that this behavior is driven to a large extent by the re-allocation of carbohydrate resources. Interpreted in this way, the quiescent state is comparable to the survival phase exhibited by excised maize root tips, where growth could be resumed by adding supplemental sugar to the media. However, starvation isn’t sufficient to explain all of the behaviors of the inflorescence meristem. In contrast to previous report of a linear rate of anthesis, a closer examination of meristem activities revealed a number of subtle trends. When measured in terms of anlagen/day, the vegetative meristem is found to have a nearly constant rate of production that does not change with the juvenile/adult phase transition, which occurred between leaves 6-8 . After induction, the inflorescence meristem revealed a rapid rise in flower buds/day, a period of time that corresponds to an enlarged SAM diameter, and an increase in gibberellic acid biosynthesis. As anlagen production requires a finite surface area in order to develop, the increase rate of bud production may be related to the larger diameter of the meristem during this time.

The differences in fungal spore concentration were adjusted to ensure uniform and comparable development of lesions in tomato fruit. In the case of R. stolonifer inoculations of MG fruit, we also tested a concentration of 1,000 spores/µl but no differences in fruit responses or fungal growth between this concentration and 30 spores/µl were observed. Inoculated fruit were incubated at RT in high humidity chambers. For mock inoculations, the same procedure was followed but without the addition of the inoculum. The pericarp and epidermis of the blossom end were collected at 1 and 3 days post-inoculation , immediately frozen in liquid nitrogen, and stored at −80◦C until use. One biological replicate consisted on average of eight fruit, and five biological replicates per treatment were obtained.Tomato fruit tissues were ground using a Retsch R Mixer Mill MM 400 and RNA was extracted from 1 g of fine-powdered tissue according to the procedure described in Blanco-Ulate et al. . Fungal RNA from the in vitro cultures was extracted using TRIzol and purified using the Quick-RNA MiniPrep Kit following the procedure described in MoralesCruz et al. . The RNA concentration and purity were assessed with the Qubit 3 and the NanoDrop One Spectrophotometer , respectively. Gel electrophoresis was used to confirm the RNA was not degraded. Barcoded cDNA libraries were prepared using the Illumina TruSeq RNA Sample Preparation Kit v2 . Quality control of the cDNA libraries was performed with the High Sensitivity DNA Analysis Kit in the Agilent 2100 Bioanalyzer . 50-bp single-end libraries were sequenced on the Illumina HiSeq 4000 platform in the DNA Technologies Core of the UC Davis Genome Center. In total, 18 libraries were sequenced for B. cinerea , 17 libraries were sequenced for F. acuminatum , and 17 libraries for R. stolonifer .

Quality trimming of the raw reads was performed with sickle v1.331 and adapter sequences were removed with scythe v0.9912 . The basic quality measurements were assessed with FastQC3 . To generate transcriptome assemblies for F. acuminatum and R. stolonifer, stacking flower pot tower reads from samples infected with each of these pathogens were mapped to the tomato genome using STAR 2.6 . Reads that failed to map to the tomato gene were pooled with the reads from the in vitro samples and used for de novo transcriptome assembly. Transcriptomes were assembled with Trinity 2.5.1 using default parameters . Quality of the assemblies was assessed with BUSCO 3.0.2 using the “fungi odb9” dataset, while basic assembly metrics were obtained with Transrate 1.0.3 . Potential contaminanttranscripts were identified via BLAST using both the blast nr database and the UniProt database. Functional annotations for transcriptomes of all three fungi were created using Trinotate 3.0.1 . The default Trinotate parameters were used to retrieve the best BLAST hits as well as annotations for Gene Ontology terms, Pfam families, Kyoto Encyclopedia of Genes and Genome pathways, EggNOG predictions, and SignalP sequences. Custom BLAST databases were incorporated according to the Trinotate manual for the Transporter Classification Database and the Pathogen-Host Interactions database . Custom HMMER alignment results for HMM profiles from dbCAN7 and fPoxDB8 were incorporated similarly.To determine if F. acuminatum and R. stolonifer show similar patterns of infections in tomato fruit as B. cinerea , we did side-by-side inoculations of fruit at two developmental stages: unripe and ripe . As displayed in Figure 1A, we confirmed that all fungi were unable to cause rotting in MG fruit but aggressively colonized RR fruit. These results were further validated by quantifying fungal biomass based on relative expression of fungal reference genes via qRT-PCR .

At 3 dpi, RR fruit inoculated with B. cinerea and F. acuminatum showed water-soaked lesions of approximately 15 mm covered by dense mycelia, whereas RR fruit inoculated with R. stolonifer were almost decomposed and entirely covered by mycelia. Although no lesions were observed in MG fruit when inoculated with any of the pathogens, some differences in fungal growth and tomato responses were observed. Inoculations with B. cinerea and R. stolonifer did not show any visible mycelia, whereas F. acuminatum inoculations showed limited hyphal growth without disease symptoms. All three fungi induced a necrotic ring surrounding the inoculation sites during the incompatible interaction with MG fruit, yet F. acuminatum inoculations caused dark and wide rings while fruit infected with R. stolonifer developed a weaker response. Because we were not able to visually detect any hyphal growth of B. cinerea and R. stolonifer inMG fruit, we used a microscope to observe whether the spores germinated in the inoculated wounds. At 1 dpi, B. cinerea spores were mainly ungerminated or in the process of germination . By contrast, F. acuminatum and R. stolonifer already showed active hyphal growth, indicating that spores of these fungi germinate earlier on MG fruit. At 3 dpi, some hyphal growth was also observed for B. cinerea. Together, these observations suggest that the incompatibility of the interaction between these fungi and MG tomato fruit occurs after spore germination. To provide initial support that both F. acuminatum and R. stolonifer are capable of inducing disease responses in the host, like B. cinerea, and do not merely behave as saprotrophs , we evaluated the expression of the host gene SlWRKY33 , which is well-known to be pathogen-responsive but is not induced by abiotic stresses . To test that the induction of this gene occurred only as a result of inoculation and not wounding, we included a mock-inoculated control in our analyses. The expression patterns of SlWRKY33 measured by qRT-PCR reflected the accumulation of fungal biomass and the presence of lesions in each of the treatments . At 1 dpi, expression of SlWRKY33 was induced by inoculation with both B. cinerea and F. acuminatum but not with R. stolonifer or mock inoculation in MG fruit. In RR fruit, pathogen-induced SlWRKY33 was detected for all three pathogens at greater levels than found in MG fruit.Our observations of lesion development, fungal biomass, and activation of pathogen responses led to the hypothesis that F. acuminatum and R. stolonifer display a similar necrotrophic behavior in tomato fruit as B. cinerea. Therefore, to discover pathogenicity or virulence factors in these fungi that are important for necrotrophic infections, we performed a genomewide transcriptomic analysis of inoculated fruit at both time points as well as in vitro cultures. Due to the lack of publicly available genomic data for F. acuminatum and R. stolonifer, we assembled de novo transcriptomes for both of these pathogens from our cDNA libraries following the Trinity pipeline . Using the fungal ortholog dataset of the Benchmarking Universal Single-Copy Orthologs tool , we determined that our assemblies presented high completeness, with 88.2 and 90.3% of F. acuminatum and R. stolonifer matches being complete, respectively. Our F. acuminatum transcriptome contained 20,117 unique transcripts, danish trolley while our R. stolonifer transcriptome contained 19,754 . We then used homology-based annotation to obtain information on gene functions for each of the transcriptomes, including the B. cinerea B05.10 ASM83294v1 . We annotated transcripts based on nine separate functional classifications, including GO , Pfam domains , Pathogen-Host Interaction , membrane transporters , Carbohydrate-Active Enzymes , and fungal peroxidases .

Each type of functional annotation was represented by a similar percentage of annotated transcripts across all pathogens . The specialized enzyme classifications of peroxidases and CAZymes made up a relatively small fraction of the annotated transcripts, whereas general functional classifications such as GO, Pfam, and KEGG descriptions were available for at least 70% of the annotated transcripts for all pathogens. Annotations for all three transcriptomes can be found in Supplementary Table S3. Although the F. acuminatum and R. stolonifer transcriptomes are preliminary and may require further curation and validation, we consider that they are a valuable resource to perform gene expression analyses and to shed light on the infection strategies utilized by these fungi.First, we performed principal component analysis to determine if the fungal-inoculated and in vitro samples could be discerned based on the expression of the fungal transcripts. The PCAs revealed that all samples clustered by treatment . In most cases, the first component clearly differentiated the MG fruit from the RR fruit inoculations and the in vitro samples. Then, we determined DEGs between inoculations of MG or RR fruit and in vitro cultures for each pathogen. Across all comparisons, we detected 6,488 B. cinerea DEGs , 6,154 F. acuminatum DEGs , and 8,777 R. stolonifer DEGs . The number of DEGs for R. stolonifer were mainly identified in the RR fruit comparisons, as the low amount of fungal biomass in MG fruit samples did not allow for an in-depth sequencing coverage of the fungal transcripts. To confirm the accuracy of the DEG analysis, we selected a subset of genes for each pathogen to validate their expression using a qRT-PCR approach . Our results confirmed that the gene expression values were consistent, showing significant Pearson correlation coefficients and between the RNA-seq and the qPCR expression data . We further evaluated the fungal DEGs based on whether they were commonly or uniquely expressed under specific treatments, which can provide insight on particular sets of genes that are relevant during incompatible or compatible interactions . For each pathogen, genes uniquely upregulated in RR fruit constituted a sizable fraction of upregulated genes . This result may be influenced by the fact that RR fruit samples had more coverage of fungal transcripts in the RNA-seq experiment than MG fruit samples, which is a technical limitation of this type of study. Nevertheless, the comparisons of common and unique DEGs among treatments for each of the pathogens support the results of the PCAs, indicating that these fungi display a specific behavior in each of the fruit stages at early and late time points after inoculation. We also identified upregulated DEGs shared across categories that are likely to represent core pathogenicity factors during fruit infections.To gain insight into key biological processes that are relevant during compatible or incompatible fruit infections, we performed GO enrichment analyses of the upregulated DEGs in all combinations of ripening stage and dpi for each pathogen . We mainly focused on GO terms of the “biological process” class that were significantly enriched and appeared to be involved in pathogenesis or fungal growth in the host tissues . Upregulated DEGs from all comparisons, except for R. stolonifer MG inoculations, were enriched in oxidation reduction processes . A closer inspection of these DEGs revealed functions that are likely to be involved with pathogenicity, such as catabolism of ROS [e.g., superoxide dismutases , catalases , peroxidases] and breakdown of cell wall molecules such as cellobiose and lignin . In B. cinerea, the SOD BcSOD1 was induced in both MG and RR fruit at 1 and 3 dpi. Additionally, BcSOD3 was upregulated only in MG fruit at 1 dpi, and BcSOD2 is upregulated only at 1 dpi in MG and RR fruit. Although two potential SODs, FacuDN9613c0g1i1 and FacuDN4275c0g1i2, were employed by F. acuminatum in all treatments except 1 dpi MG, none of the seven putative SODs identified in R. stolonifer were upregulated in any of the treatments. To further identify enzymatic scavengers of hydrogen peroxide , we examined the upregulated DEGs of each pathogen which showed significant similarity to members of the Fungal Peroxidase Database. This analysis revealed differences both in the classes of enzymes used in each pathogen and the treatments in which they were used. For example, in B. cinerea, only two known catalases, BcCAT2 and BcCAT4 , were found to be upregulated during tomato fruit interaction. Both of these were only active in MG fruit. In contrast, F. acuminatum exhibited very strong induction of two predicted CATs, FacuDN12367c0g1i1 and FacuDN13048c0g1i1, at 1 dpi in RR fruit but not in MG fruit, although a handful of CATs and catalase-peroxidases were upregulated less strongly across both MG and RR fruit. In all F. acuminatum-inoculated samples, there was also an enrichment of DEGs involved in hydrogen peroxide catabolism , further highlighting the importance of fungal responses to oxidative stress during fruit colonization. In R. stolonifer, peroxidases were only upregulated at 1 dpi in RR fruit and included two 2-cysteine peroxiredoxins , one cytochrome C peroxidase, and one glutathione peroxidase . Additionally, in all B. cinerea-inoculated samples, DEGs annotated with the oxidation-reduction process GO term included enzymes in the biosynthetic pathways for the phytotoxins botrydial and botcinic acid.