Many post harvest strategies are designed to extend fruit shelf life, but often result in loss of fruit quality, and may unintentionally contribute to post harvest waste. While the relationship between DNA methylation and tomato fruit ripening is well understood , it was not known what effect early harvest and storage temperature, which often disrupt the ripening program, would have on DNA methylation and the expression of the key genes in this process. Our work demonstrated that early harvest and post harvest storage temperatures greatly influenced the speed of fruit ripening, fruit quality and DNA methylation levels, but that the relationship was not linear. During tomato fruit development and especially during the transition from green fruit to red that occurs during ripening, many genomic demethylation events trigger the expression of ripening-related genes . We found more demethylation events in Turning fruit compared to Mature green fruit, consistent with data from cv. Alisa Craig . This suggests that ripening-induced DNA demethylation is conserved in tomato cvs. ‘MicroTom’ and ‘Alisa Craig’, even though ‘Micro-Tom’ has comparatively more methylated regions . The chilling-induced inhibition of demethylation detected in this study, potentially explains why post harvest chilling inhibits ripening in Mature green fruit . Similar to chilled red fruit , chilling green fruit inhibited the DNA demethylation that occurs during normal ripening. The expression of these genes may be regulated by RIN, blueberries in containers growing but methylation also inhibits RIN’s actions, and would delay ripening.

Rewarming the chilled Mature green fruit gave rise to DNA demethylation observed in the MSAP analysis, which is a prerequisite for ripening . However, not all sites influenced by chilling were demethylated during rewarming, explaining why ‘5T’ fruit were of poorer quality compared to other Turning fruit. This is in agreement with the growth rate and methylation pattern of chilled and rewarmed cucumber radicles . By incorporating all measurements, the rank in fruit quality at Turning from best to poorest, was: ‘FHT’ > ‘20T’ > ‘12.5T’ > ‘5T’ , opposite to changes in cytosine methylation levels generated by MSAP where ‘20T’ < ‘12.5T’ < ‘5T’ when compared to ‘FHT’. Vine-ripened fruits import nutrients until harvest, while post harvest-ripened fruit are prematurely removed from their source of nutrients. Low temperature storage further disrupts the ripening program of these harvested fruit. It may be inferred that changes in methylation events are integral to how these anthropogenic factors affect fruit biological processes and influence quality. The multivariable analysis indicated that DNA methylation is influenced by post harvest handling and fruit ripening stage. Fruit quality correlated strongly with ripening. The exception was ‘5T’ fruit, where there was a distinct DNA methylation state and quality characteristics, implying a strong regulatory mechanism between chilling, ripening and methylation. Broader analyses of the methylome and transcriptome by whole genome bisulfite-sequencing and RNA-Seq , may provide a more comprehensive picture of how early-harvest and low-temperature storage influence tomato ripening at the molecular level. We proposed a model connecting post harvest strategies and its induced changes in fruit ripening .

Post harvest handling modulates SlDML2 expression, which in turn influences fruit global DNA methylation. Changes in methylation status may have consequences for a subset of ripening genes , even if RIN expression remained robust in ripened fruit regardless of storage treatment . RIN is therefore not a reliable proxy for informing on the endogenous or physiological conditions that influence ripening, only that the stage was attained.This model is based on variables studied in this work and how they are related. The red arrows indicate positive relationships between variables, and the black arrow indicates no change based on this work. Genes and post harvest conditions are shown in blue and red font respectively. Early harvest and lowtemperature storage changed ripening time, resulting in differences in the transcriptional levels of SlDML2 and changes in global DNA methylation status. However, the relationship between SlDML2 expression and DNA methylation was not linear . RIN expression remained the same regardless of the different post harvest handling and DNA methylation levels verified in this work but was related to the ripening stage attained by the fruit. Our post harvest practice widened the fruit ripening/developmental window which influenced SlDML2 expression. There are many strategies for prolonging the shelf-life of tomato fruit, but they often reduce flavor. The post harvest treatment used in this study negatively influenced fruit sensory attributes and this may be mediated in part through DNA methylation. In support of this, Zhang et al., recently found that exogenous ethylene stimulated SlDML2 transcripts and DNA demethylation. There are also additional epigenetic regulatory mechanisms that may indirectly influence tomato fruit ripening and quality, and it would be of interest to determine how they are affected by post harvest methods . Greater efforts are needed to help to unravel the complex regulatory ripening network in tomato at the epigenetic and transcriptional level. For example, it will be important to explore the effect of anthropogenic post harvest environments on the timing and dynamics of DNA demethylases and DNA methylation.Pests and diseases cause high losses in crop yields worldwide that can reach approximately 45% loss annually. Due to the rapid growth of world population, increase in the agricultural productivity is urgent to meet rising food needs. Chemical pesticides are considered the main component in protecting agricultural products in the field and store to maintain crop yield and quality. Pesticides usage in Egypt has increased, according to the Food and Agriculture Organization report, from 4931 tons in 2000 to 13,178 tons in 2019.

Globally, the total pesticides use in agriculture was 4.12 million tons in 2018. The worldwide application of pesticides was 2.63 kg ha−1 in 2018, which showed a more than doubled increase in pesticide usage in the 2010s compared with the 1990s. Since banning of organochlorides, other groups , carbamates, and synthetic pyrethroids were the most widely used classes of insecticides due to their high activity and relatively low persistence. New groups of chemical insecticides have been also introduced in agriculture, including neonicotinoids, spinosyns, avermectins, and diamides. They are recommended to be eaten fresh, unpeeled, and unprocessed for their high nutritional value and content of minerals, vitamins, fibers, and antioxidants. On the other hand, food is one of the main ways through which humans are exposed to pesticides, at a rate five times higher than other methods such as air andwater. Accordingly, efforts to ensure a sustainable use of chemical pesticides to avoid the increase of pesticide levels in the environment and food commodities are necessary. Pesticide residues in fruit and vegetable samples have been reported in many countries including Croatia, South America, Turkey, Poland, China , Jordan, UAE, Kenya, and South Korea=. The chronic effects of exposure from contaminated food intake are mostly unknown. Studies have demonstrated that exposure to pesticides has dose-related chronic and acute toxicity in humans through different mechanisms including deregulation of transporters or enzymes involved in xenobiotic metabolism. This has effects on cell processes such as growth, differentiation, and survival, including reactive oxygen species, cell damage through subsequent oxidative stress, and DNA damage. There is growing evidence of carcinogenicity and genotoxicity as well as endocrine disruption capacity attributed to the ingestion of contaminated food or direct exposure to pesticides. Despite the fact that the use of certain organochlorides, Ops, and carbamates are prohibited in many countries, some of these compounds have been detected in the environment worldwide due to their persistent nature or illegal use of the banned chemical pesticides. A large number of programs are being implemented to address this issue. For instance, planting blueberries in containers to protect the Brazilian population from severe risks associated with food contaminated with pesticides, the Brazilian National Sanitary Agency has initiated a nationwide monitoring program for pesticide residues in fruits and vegetables since 2001. In 2009, 20 types of fruits and vegetables were analyzed and the results indicated that 23.2% were positive for insecticide residues, and 14.3% of the samples exceeded the European Union maximum residue levels. In contrast, there is lack of data on contamination of the food available in the Egyptian market. Only a few studies have been published on this subject over the past 20 years, such as Tchounwou et al.. Constant evolution of the pesticide industry requires closer surveillance and better assessment of factors including pesticides bio-accumulation, stability, widespread usage, and food quality and safety that impact directly on human health. Hence, the aim of this study was to determine pesticide residues in vegetable and fruit samples in local markets in Egypt and to show the differences and frequencies in pesticides detection. The most common pesticides and the type of crops with the highest number of pesticide residues are also shown.

This study will help understanding of the most applied pesticides on vegetables and fruits as well as the most common polluted crops locally. Risk assessment of pesticides exceeding MRLs in vegetable and fruit samples was also determined.For pesticide residues in vegetables, 66 samples belonging to 13 types of vegetables collected from the farmer markets of Sharkia Governorate were analyzed. Pesticide residues were detected in 44 samples and 22 samples showed no pesticides detection. Regarding pesticide residues in fruits, it was found that out of 54 samples analyzed, 33 samples were positive for the presence of pesticides and 21 samples had no pesticide residues. The number of pesticides that were detected in each vegetable sample ranged from 1 to 15 pesticides. Carrot was the sample that showed the lowest number of pesticide residues while cucumber was the highest sample with 15 pesticide residues. Pesticide residues in each fruit sample ranged from 1 to 20 pesticides. One pesticide residue appeared in banana while 20 pesticide residues were found in apples . A total of 40 different pesticides were detected in the tested vegetable and fruit samples belonging to classes of insecticides, fungicides, and herbicides. It was shown that 12 pesticide residues and 21 pesticide residues were detected one time only. The number of pesticides that were detected two times or more was 16 pesticides and 16 pesticides . The total number of pesticides detected in vegetable and fruits were 28 and 37, respectively .The results presented in Table 3 show the assessment of the acute and chronic risks of pesticide residues detected in vegetables or fruits that exceed the permissible MRLs, using two rates of consumption for all the tested samples. Acute and chronic risks were determined for children, teenagers, and adults. The results showed existing acute risk with fipronil, lambad-cyhalothrin, dimethoate, and omethoate in the case of children consuming okra, zucchini, apples, guava , spinach , kaki , and strawberries . It was also found that acute risks appear in teenagers consuming spinach , kaki , and strawberries , while the presence of acute risks appears in adults consuming kaki and strawberries contaminated with both dimethoate and omethoate, respectively. Regarding chronic risks, they appear in children consuming zucchini, spinach, kaki, and strawberries containing residues of fipronil, lambada-cyhalothrin, dimethoate, and omethoate, respectively, while chronic risks appear for teenagers when consuming kaki contaminated with dimethoate.Taking into consideration that pesticides play a major role in increasing the production of agricultural products with high quality when moderately and safely applied in the control of crop pests, diseases, and weeds, their misuse may cause severe health problems. Pesticide residues’ determination in food is an important action for monitoring contamination and ensuring food safety. This might help farmers and stakeholders in the proper handling of pesticides in terms of the applied dose, times of application, as well as the permissible level locally in each type of food for the health and safety of consumers. Our results showed that pesticide-free samples were 36% for both vegetables and fruits, while 64% of samples contained from one to 20 pesticide residues. Cucumber, pepper, zucchini, and tomato showed 15, 12, 12, and 10 pesticide residues, respectively. In fruit samples, apple, grapes, and apricot recorded 20, 18, and 14 pesticide residues, respectively . In agreement with our results, pesticide residue analyses in apples carried out by Pirsahib et al. reported 26% of free-pesticide samples, 74% contained at least one pesticide, and 54%, 46%, and 26% of the samples had diazinon, chlorpyrifos, and both diazinon and chlorpyrifos residues, respectively. Fruits and vegetables with multiple pesticide residues are widely observed globally, including 26% from Italy, 25% from China, 48% from Brazil, and 39% from Argentina; the fruit and vegetable monitoring surveys found that carbendazim, pyrimethanil, imidacloprid, and procymidone had high detection frequency and showed wide use in fruits and vegetables in Colombia.