We created a multiplexed probe-based quantitative PCR assay able to identify whether a sample contains T. absoluta, K. lycopersicella, or P. operculella DNA based on single nucleotide polymorphisms between species. In addition, we created RPA-Cas12a assays to detect the presence of T. absoluta DNA, using either a fluorescent reader or a simple UV illuminator paired with a cell phone camera. This will allow for more rapid field detection of T. absoluta.We assessed 21 single nucleotide polymorphism markers differentiating T. absoluta, K. lycopersicella and P. operculella identified in Tabuloc et al. 2019 for qPCR genotyping potential using RealTimeDesign software . Our analysis resulted in seven potential qPCR assays, each containing a set of universal amplifying primers and two distinguishing fluorescent oligonucleotide probes. We used a serial dilution qPCR on all seven sets of amplifying primers on all three species to assess primer efficiency. Reactions were performed with 300nM of forward and reverse primers with SsoAdvanced Universal SYBR Green Supermix on a CFX96 real time PCR machine . We checked for off-target products when multiplexing pairs of assay primers using melt curve analysis as well as gel electrophoresis. From the seven loci tested, square pot we selected two to develop assays and ordered single-strand BHQplus probes unique to each of the four alleles .
Each probe was tagged with a distinct 5′ fluorophore and 3’ quencher. Assay 1 distinguishes T. absoluta from K. lycopersicella and P. operculella, while assay 2 distinguishes P. operculella from K. lycopersicella and T. absoluta.When considering which method is best suited for T. absoluta identification, a key consideration is sensitivity. Based on our analyses, we find that our qPCR diagnostics can reliably identify species down to 0.02 ng of input DNA, while our RPA-Cas12a assay was capable down to 0.1 ng input DNA. Assuming a haploid genome size of 564Mb from flow cytometry estimates , this represents approximately 35 copies of target DNA for qPCR detection and approximately 170 copies of target DNA for RPA-Cas12a detection. Both should be sufficient for scenarios where DNA is extracted from a single insect or small pool of insects. However, in situations where target DNA concentrations are extremely low, such as the case would be with eDNA collections, the more sensitive qPCR method is preferred.signal in the RPA-Cas12a assay as measuring fluorescence with a qPCR machine. Compared to the qPCR diagnostic, the RPA-Cas12a diagnostic does require additional user time, as amplification and detection occur in two distinct steps, although a one-pot reaction variant has been described . While we used an oligonucleotide with a Cal Red 610 fluorophore paired with the appropriate quencher as the substrate for Cas12a-mediated ssDNAse activity, the fluorophore/quencher molecules can easily be swapped for other fluorophores that absorb and emit at different wavelengths, making this protocol adaptable to the equipment available to the user.
It is possible in the future to replace the fluorometric detection method with a colorimetric detection by using biotin and FAM-labeled oligonucleotides applied to a lateral flow assay. This flexibility has been implemented in other detection assays and lets Cas12a-based detection systems stand out from qPCR methods, despite the slight reduction in sensitivity . While T. absoluta has not yet been detected in North America, demographic modeling suggests low to moderate levels of invasion are possible in Mexico, the California Central Valley, and the southeastern USA . Rapid reliable detection of an invasive pest is one of the key components of a successful eradication program, meaning it is crucial that molecular diagnostics are available . Even if eradication is not possible, molecular diagnostics reduce the need for expert entomologists to hand identify specimens, allowing detection to be done faster, cheaper, and more accurately . This is especially relevant for T. absoluta testing as tomato crops in the United States already contain the presence of P. operculella and K. lycopersicella, which are both nearly identical morphologically. Once detected, agencies can decide between strategies including eradication, quarantine, or continued monitoring . We expect that the molecular diagnostics presented here will add to the toolkit available to institutions to rapidly monitor for the appearance of T. absoluta.Tomato fruit are highly perishable, and sensitive to handling techniques and storage conditions after harvest .
Post harvest losses in quantity and quality commonly occur in fresh market tomatoes . The irreversible and continuous changes after fruit harvest leads to cell death and senescence, which makes some post harvest losses unavoidable . However, some losses are caused by improper post harvest practices, such as mechanical injury or pathological breakdown . Appropriately applying post harvest techniques could slow fruit senescence and therefore, maintain fruit quality from vine to consumers. While the specific recommendations for post harvest handling are dependent on the desired fruit shelf-life and vary among the produced and consumed regions of tomatoes, some examples of commonly applied tomato post harvest practices are discussed below.Fruit maturity at harvest plays an important role in its post harvest quality. Tomato fruit could be harvested between the stages of mature green and fully ripe, depending on the demand for fruit shelf-life. For the tomato produced for local markets, producers harvest fruit close to the fully ripe stage. Tomato destined for sale in distant markets, may be harvested earlier , followed by off-the-vine ripening to effectively satisfy the market need for an extended shelf-life. Tomato fruit that is harvested early, i.e., ‘Mature green’ fruit is able to fully ripen, but it loses its carbon and energy source from the mother plant, resulting in quality loss . Reduced fruit quality including loss of sugar, a lower ratio of TSS/TA, and changes in volatiles have been reported in off-the-vine ripening fruit compared with optimal harvest .Storage of the fresh market tomato after harvest provides stability to the supply chain. Precise temperature management is powerful in fruit storage, and the prescribed temperature may differ by fruit cultivar, harvesting stage, expected storage period, or, transportation . Before storage, precooling of freshly harvest fruit is used to remove ‘field heat’, ameliorating the rise in metabolic activities in fruit . For short-periods of storage, tomato fruit can be placed at room temperature under good ventilation, while, for longer storage time, early harvesting followed by low-temperature storage is a possible strategy, but this will negatively reduce the fruit flavor . When sensitive produce are exposed to low, non-freezing temperature for long periods, post harvest chilling injury may occur . PCI symptoms in tomato fruit include a failure to ripen,uneven ripening, surface pitting, decay, and high rates of ethylene and respiration upon rewarming . There are other approaches to calibrate the rate of fruit ripening during storage. Applying controlled and modified atmospheres could delay fruit ripening during post harvest storage. Controlling relative humidity , and modifying the relative proportion of oxygen and carbon dioxide in the atmosphere can extend the shelf-life of tomato fruit without a significant loss of flavor . For greater flexibility, ripening can be further controlled by regulating post harvest ethylene. When fruit are exposed to ethylene, ripening would speed up . Conversely, reducing ethylene production will maintain fruit quality and prolong shelf-life, blueberries in containers and several commonly examples are as following: precooling or heat treatment is efficient in slowing down ethylene production during storage ; high rates of CO2 is able to inhibit autocatalytic ethylene biosynthesis ; applying 1-Methylcyclopropene would delay fruit ripening by binding to the ethylene receptors , however use of these chemicals may affect fruit quality ; calcium chloride is successfully used in inhibiting ethylene production after harvest ; ethylene biosynthesis and sensitivity are affected by low-temperature post harvest storage .Consumers have been complaining about the quality of store-bought, conventional varieties of tomatoes over the last 30 years .
Understanding the physiological basis of fruit quality has therefore become the main focus of tomato researchers, as this information would support the breeding of new tomato cultivar with better quality . Fruit quality characteristics depend on the fruit developmental and physiological pathways which are dynamically regulated in the ripening process . The quality of the tomato fruit is mainly determined by its color, texture and flavor. Tomato fruit color is not only an appealing eating quality for consumers, but also a commonly used indicator of the fruit ripening stages. During ripening in most tomato cultivars, chloroplasts are converted to chromoplasts accompanied by chlorophyll breakdown and carotenoids accumulation, resulting in fruit color transition from green to red . The color of the ripe fruit is red primarily due to lycopene and the accumulation of a smaller amount of the orange or yellowish β-carotene . The dynamic interplay of carotenoid and chlorophyll metabolism dictates tomato fruit color .Fruit texture is a sensory, multi-parameter characteristic that can be perceived through the consumer’s senses, i.e., vision, touch, and taste . Tomato fruit texture is commonly described by the attributes of firmness, juiciness, and mealiness . There are various methods of measuring fruit texture, in which puncture and compression are commonly used destructive methods, and non-destructive approaches may include ultrasonic and optical techniques . Firmness of tomato fruit is considered as the main determinant of texture, and the decline of firmness leads to the fruit softness . Fruit softening is a natural phenomenon in ripening, and critical to seed dispersal and attracting animals . A certain degree of fruit softening is desirable in fresh market tomatoes, but too much softening is unwanted , as it limits fruit shelf-life and increases susceptibility to decay. Tomato fruit softening is associated with disassembly of the cell wall matrix, involving depolymerization and solubilization of the cell wall polysaccharides components, i.e., cellulose, hemicellulose and pectin . This event is mainly controlled by the activity of many cell wall degrading enzymes, such as polygalacturonase, pectin methylesterase , galactanase, or remodeling proteins, like expansin . Tomato fruit flavor is determined by a complex interaction of many biochemical compounds, including sugars, acids, amino acids, with aroma volatiles . Tomato aroma volatiles are often complex secondary metabolites, and they are derived from carotenoids and simpler primary metabolites precursors such as amino and fatty acids . There are more than 400 volatiles discovered in tomato, but only a limited proportion influences tomato flavor . The non-volatile compounds determine fruit taste. Sugar, mainly the reducing sugars glucose and fructose, makes the largest contribution to tomato taste. There are also organic acids in tomato fruit, predominantly citric and malic acids. It is well established that tomato flavor is highly interdependent on the relative levels of sugars, acids, and key volatiles, and no single chemical controls quality .Carbohydrate metabolism is important in determining tomato fruit post harvest quality . Fruit are “sink” tissues , that require carbohydrates supplied by photosynthetic “source” tissues . The chloroplasts in green tomato fruit have active photosynthetic ability , thus, green tomato fruit is able to fix carbon . The carbon dioxide from respiration is the major source of green fruit photosynthesis . Fruit fixed carbon accounts for 10-15% of that used by the fruit, and may be critical for seed development , and maximal carbohydrate and carotenoid accumulation . The carbohydrate metabolic pathway in developing tomato fruit is mediated by the activities of numerous enzymes and sugar transporters . Sucrose, the main translocated assimilate, is taken up by tomato fruit . In early fruit development, starch synthesis is the main flux; fruit starch level reaches its peak at the mature green stage, and it is then degraded during ripening . Starch synthesis and breakdown coexists throughout fruit development, with net synthesis occurring prior to the mature green stage, and net degradation to sugars occurring during ripening . Starch degradation therefore contributes to sugar accumulation in ripening fruit .Epigenomic modifications, especially DNA methylation of gene promoter regions, are considered to play a critical role in triggering tomato fruit ripening . DNA methylation is characterized by the addition of a methyl group to the 5’ position of cytosine. The cytosines that are methylated occur in either a symmetrical CHG and CG, or an asymmetrical CHH context . Symmetrical and asymmetrical DNA methylation are regulated by different genes . DNA demethylases are able to remove methyl groups from cytosines, a process termed DNA demethylation. In plants, DEMETER-Like demethylases are a group of enzymes that can activate gene promoter demethylation, by the activity of DNA Glycosylase-Lyases . There are four DML isoforms in tomato, of which SlDML2 is most important in fruit .