Tag: maceta cuadrada plastico

Generalized linear hypotheses was used to perform pairwise comparisons in the multcomp . P values were adjusted using the p.adjust command. Alpha diversity data was analysed using a negative binomial generalized linear models at a sequence depth of 3000 sequences/sample to normalize data to the highest number where all sample mosquitoes were present. The alpha level for all tests was 0.05.PCA demonstrates that various instars separate from each other in the four treatment groups with third and fourth instar loading similarly on the first dimension and all three instars loading distinctly in the second dimension. Bacterial families Oxalobacteraceae and Aeromonadaceae closely follow the separation pattern in the first dimension and Cryomorphaceae follows in the second . In the antibiotic treatments second instar loaded separately from third and fourth on the second dimension . The bacterial family Propionibacteriaceae also follows this trend and was the only family with at least 85 % correlation in either of the first two dimensions . For hormone treated mosquitoes, second instars loaded separately from fourth instars on the first dimension . The bacterial families, which follow this trend, are Enterobacteriaceae and Pseudomonadaceae. As shown in Figure 3.2 and Table 3.2, control treatments’ bacterial families changed with instar; starting with Cytophagaceaein second instars, changing to Enterobacteriaceaein third instar and finally the control fourth instars’ most predominant family was Microbacteriaceae . There were a total of eight bacterial families with proportionalities greater then 1% in at least one instar of the control treatment. In fourth instars there is a resurgence of bacterial families from second instars,macetas cuadradas which were overshadowed by the third instar bacterial families . Interestingly, the family Rickettsiaceae was the second most predominant family in all control instars.

Operational taxonomic units assigned to the family Rickettsiaceae were found in most treatments and instars, although they were reduced in many hormone treated samples relative to the controls and increased in the antibiotic treatments. Notably, antibiotic and mixture treatment groups’ most predominant family was Rickettsiaceae over all instars. The second most predominant family in antibiotic and mixture treatment groups was Sphingobacteriaceae in all instars. Hormone treatments changed bacterial communities between instars but not as drastically as the control group. Second instars exposed to hormones predominately contained Oxalobacteraceae, which changed to Microbacteriaceae and Rickettsiaceae in the third instar. The predominant family of fourth instar hormones was Microbacteriaceae, although some proportion of Rickettsiaceae was still present.In the alpha diversity analysis, richness was examined as mean observed species and evenness was measured by mean Shannon’s index . For mean observed species at a sequencing depth of 3000 sequences/sample there was a significant difference between treatments , instars and a significant interaction of treatment and instar . Mosquitoes treated with antibiotics had lower richness and fewer total sequences per sample than all other treatments with the richness decreasing as larvae age. This is evident in Fig. 3.2, as there are proportionally fewer bacterial families outside of Rickettsiaceae and Sphingobacteriaceae than in other treatments. In contrast, mosquitoes reared in the mixture of hormones, antibiotics and the common contaminants, acetaminophen and caffeine; demonstrate a relatively constant richness over time .

The control groups and hormone treatments fluctuate more than the mixture and antibiotic treatment groups but demonstrate consistently higher richness . The mean Shannon’s diversity index suggests that antibiotics alone substantially reduced diversity. The mixture treatments also display reduced diversity, however, they are more diverse than their antibiotic treatment counterparts. The control groups display a greater diversity than both the antibiotic alone and mixture treatments when compared by increasing instar, where as the hormone treatment group, had no discernable pattern. The mixture also displays no discernable pattern compared by instar, which is likely due to the effects of the hormones added to antibiotics. Finally, it is notable that in some treatments, the mean species number failed to reach an asymptote. Here we have demonstrated that the microbiome of larval Culex mosquitoes changes throughout development, and variation between instars is affected when exposed to various PPCPs. It has previously been demonstrated that mosquitoes rely on their microbiomes to aid in development and that removing certain symbionts can significantly slow larval development. Pennington et al. demonstrated that PPCPs at environmentally relevant concentrations, which are significantly lower than those used in most laboratory studies, can alter the microbiome of mosquitoes and slow their development. In the field, Duguma et al. showed pooled Culex species’ microbiomes will change from early to late instars. Coon et al. and Wang et al. showed that the microbiome of mosquitoes will change as the insects advance from fourth instar larvae, to pupae, to the adult stage, and after adults fed on a blood meal. We have shown that the microbiome of early instars’ will change from one instar to the next even without exposure to PPCPs .

In the second, third, and fourth instars, predominant families change from Cytophagaceae to Enterobacteriaceae and finally to Microbacteriaceae. However similar to Pennington et. al , third and fourth instar were not significantly different and our findings also correlate to what Duguma et al. found in their laboratory reared Culex tarsalis late instars. However, as their third and fourth instars were pooled, only the Enterobacteriaceae family predominates. These families were all removed in the antibiotic and mixture treatments. Fourth instar larvae in the control group match what was described in Aedes aegypti by Coon et al. . Coon et al. also described the microbiome of two other mosquito species during the fourth instar. Their microbiomes had different proportions of familial microbiota between each other and both were different from the findings in our C. quinquefasciatus fourth instars. To our knowledge ours is the first study to look at the microbiome changes of individual early instars in mosquitoes. This suggests the possibility of a new strategy for mosquito control targeting the critical microorganisms essential for development at specific stages. Specifically, additional research targeting key symbionts found in earlier instars would determine if the younger larvae can be controlled more effectively, as has been seen with pesticides such as Bti . A number of mosquitoes are common carriers of the bacterial genus Wolbachia, which usually acts as a reproductive parasite in the ovaries of the females, and is suspected to be in at least 20% of all insect species . As in Pennington et al. , Rickettsiaceae, the family containing Wolbachia pipientis, continuously holds the majority count of the antibiotic and mixture treatments’ microbiome. When the OTUs mapped to the family Rickettsiaceae was examined at the level of genus the predominant and sole genus detected was Wolbachia. Rickettsiaceae is vertically transmitted from mother to offspring ; however, for many of the other bacterial families present, it is difficult to discern the source or how they are incorporated into the insects’ microbiome. This is made further complicated since these traits may vary by species or genus and mapping OTUs to finer taxonomic levels was generally not possible. Similarly, it may be possible to determine some origins via comparison with the water in rearing pans over time, although there was no DNA found in water at the start of the experiments. However, our focus was not on the origin of bacterial species in these mosquitoes,maceta cuadrada plastico and we do not have these data. Analyses or the microbial community in such pans would be an interesting follow-up study. Interestingly, Enterobacteriaceae, which includes the genus Buchnera and other common endosymbionts, is the predominant family of the third instars in the control treatment. For example, the gut symbiont of the plataspid stinkbug is phylogenetically similar to Buchnera species . In potato psyllids various genera of the family Enterobacteriaceae have been reported in the life stages and faeces accounting for at least 21% of the microbiome. Enterobacteriaceae is one of, if not the most important family of endosymbionts in the pea aphid , and is commonly used in research regarding the effects of antibiotics on insect-symbiont interactions.

Similarly, Chouaia et. al described a slowing of larval development in Anopheles mosquitoes when they removed Asaia bacteria from the family Acetobacteraceae. However this family has one of the lowest proportionalities in all of the mosquitoes including control treated. This suggests it is not an endosymbiont of this Culex mosquito specie. However, reports of the effects of other PPCPs on insect-symbiont interactions are rare. It is interesting to note that the hormones found in wastewater from treatment plants are all mammalian female sex hormones and would not be expected to affect bacteria. We would not expect an effect of these hormones on bacteria, as there is no endocrine system; nonetheless, substantial changes in the microbiome occurred in response to exposure to these hormones . Similarly, caffeine and an antihistamine, would not be expected to effect biofilms, but were shown to repress respiration in stream biofilms 51. We think there may be some influence the hormones have on bacterial gene expression however that is not in the scope of this paper and thus, specific effects for each PPCP or combination of contaminants will need to be determined from more experimental data. The increased bacterial diversity during mosquito ontogeny could result from either bacterial replication during development or by acquisition through ingestion. In the hormone and control treatment groups, we are unsure if bacteria are lost during development, or if the change in bacterial diversity was caused by differential growth among taxa. Interestingly, in pairwise comparisons of the hormone treated mosquitoes , the majority of the significant differences were between second and fourth instar larvae. The hormone treated mosquitoes also had the most families that were correlated to the first principal component at a minimum of 85%. Combined this suggests that the mosquitoes exposed to hormones had the most diverse microbial communities and that this diversity increases over time. Mammalian hormones change the microbiome of C. quinquefasciatus mosquitoes and it is possible they are responsible for the increased richness and diversity seen in the mixture treatments compared to antibiotics alone; however more studies will need to be conducted to confirm this conclusion. Regardless, our results indicate that reclaimed wastewater has the potential to impact mosquito ecology. Considerably more research will be required to discern how mixtures of PPCPs could affect bacterial microbiomes for important medical pests. If similar results are found for agriculturally important insects exposed to these emerging contaminants, additional research documenting the effects of increasing use of reclaimed water and associated changes to the insect microbiome will become even more important. Similarly, because insects are a critical food source for higher trophic level organisms in terrestrial surface waters, releases of PPCPs in aquatic environments have the potential to modify the ecology of these ecosystems.Pharmaceuticals have been increasingly prescribed for the past 30 years, and prescription rates have almost tripled in the past 14 years. In food-producing animals alone, there were 9.1 million kg of medically important antibiotics used in 2013. Of those 9.1 million kg used, 73.6% was used for the purpose of increasing production of the animals, and this use continues to increase. Many antibiotics and other common Contaminants of Emerging Concern , are excreted by both humans and animals with little change in their chemical structure . It is no surprise pharmaceuticals have been appearing in wastewater, and in some cases tap water, over the past few years .Many pharmaceuticals are released during heavy storms in the untreated wastewater, due to overflow, which then flows directly to the environment 46. These pharmaceuticals are now found at biologically active concentrations in surface waters around the world . In addition to runoff, there is an increasing effort to use reclaimed wastewater in drought affected areas, such as Southern California. In agriculture/livestock operations, pharmaceuticals are also found in manure that is then used as fertilizer, effectively compounding the pharmaceutical concentrations. Current research shows these chemicals tend to be both long lived in soil and detrimental to soil microbes . Recent studies on the effects of pharmaceuticals on aquatic insects show that at environmentally relevant concentrations they can alter development of the mosquito Culex quinquefaciatus, its susceptibility to a common larvicide, and its larval microbial communities. Watts et al. 11 showed alterations and deformities in the midge Chironomus riparius after treatment with a common birth control agent, 17α- ethinylestradiol, and a common plasticizer, Bisphenol-A.

In 1994 the SWP project contractors and operators met to renegotiate the conditions for water sales among contractors and the allocation of cuts in water deliveries during drought periods. The resulting Monterey agreement also enabled contractors who overlie a state operated groundwater storage project to exchange the control of the project for surface water entitlements; these entitlements could then be transferred to urban contractors. Finally, the agreement sanctioned the permanent transfer of 130 thousand acre-feet of water from agricultural to urban users. The CVP parallels the SWP and delivers 4.6 million acre-feet of water to both urban and agricultural contractors. Urban contractors receive 10 percent of total water deliveries while the remaining 90 percent of water is diverted to agricultural contractors. The CVP was operational in 1965, but by 1992 there was considerable political pressure to modify the operation of the project to reduce environmental damage to different fish populations in the Sacramento River Delta. The resulting Central Valley Project Improvement Act reallocated water to environmental uses by cutting water deliveries by 1 million acre-feet in normal rainfall years and by 804 thousand acre-feet in critical rainfall years. The CVPIA mandated that 800 thousand acre-feet of water be reallocated to in stream uses to protect the salmon runs, while 400 thousand acre-feet of water be reallocated to wildlife refuges . Water markets in the CVP districts are limited to local sales among agricultural contractors. These sales are short in duration and are generated by differences in the water allocations between farm regions and years. Due to institutional constraints,macetas cuadradas CVP water is still largely used for agricultural irrigation despite a three-fold difference between the value of water in nearby urban sectors and agricultural sectors.

In recent years, State and Federal law have mandated a set of modifications that affect both the state and federal water projects in California. In 1996 and 1997 California developed the 4.4 Plan that aims to reduce diversions from the Colorado River to 4.4 million acre-feet over a period of 15 years. Moreover, in 2000 the Environmental Water Account was implemented by the state and federal governments. The purpose of the EWA is to regenerate the fisheries of the San Francisco Bay-Delta system while simultaneously securing water supplies to both urban and agricultural users. Both these developments have encouraged water trading.Figure 3 plots both actual transfers and regression predictions of water transfers in California between 1985 and 2001. The regression fitted to water transfer data confirms that rainfall levels have a significant effect on annual water transfers . The data also confirms a positive correlation between the time trend and water transfers. When expressed as a percentage of the mean level of water transfers, the regression time trend shows an annual growth rate of 1.26 percent over the period. We can conclude that the current data shows a steady growth in water markets despite the recent predominance of relatively wet years. In spite of the active and growing water market, Hanak points out that California’s water market only accounts for 3 percent of total annual water use. Hanak estimates that Central Valley farmers have accounted for approximately three-quarters of all water sales, while the rest of the water has been supplied from Imperial and Riverside Counties. According to Hanak, environmental regulations, rather than urban agencies, have been the major sources of the increased demand for water. Direct purchases for in stream uses and wildlife reserves constituted over one third of increased water trades since 1995, while agricultural activities in the San Joaquin valley accounted for over half of the increase in water purchases. This increase in agricultural demand for water stems from the reduction in contractual water deliveries under environmental regulations. However, municipal agencies are the principal purchasers of long-term and permanent water contracts, which constitute approximately 20 percent of total water trades.

The 2001 legislation that requires that local governments ensure adequate water supplies for development is likely to increase the urban demand for long-term water transfers.Within California there is considerable resistance to water trading which stems from communities in the source regions. These communities are concerned that water sales will generate significant “third-party” effects; i.e. trades may have an adverse impact on both local groundwater users and the local economy. These concerns have arisen from communities’ perception of the impacts of short-term water transfers in the early 1990’s, which involved the implementation of fallowing contracts by the state to purchase water for the 1991 drought water bank. Water transfers, which were accompanied by land fallowing, slightly reduced the demand for labor and other farm inputs and also decreased the supply of raw materials to local processors. Howitt estimated that losses in county income in two counties that transferred water ranged between 3.2 percent in Solano County, where 8 percent of the acreage was fallowed for transfers, to 5 percent in Yolo County, where 13 percent of the irrigated acres were fallowed. Those farmers who replaced the surface water they had sold by pumping additional groundwater were accused of reducing both the quantity and quality of water available to other users. Because groundwater resources are not regulated by the state, the implementation of the Californian water market has sparked concerns that aquifers will be subject to uncontrolled mining. The experience of the 1990’s has exacerbated another source of anxiety: local officials fear that once water has been transferred elsewhere, local communities will have insufficient money and political influence to retrieve these water entitlements . Currently, state approval is only required for water transfers pertaining to surface water entitlements that were acquired since 1914, certain types of groundwater banking and any water that is conveyed through a publicly owned facility.

The state only actively safeguards against negative economic impacts on source counties when water is conveyed through these publicly owned facilities. In the other two cases, traders are obligated not to harm other surface water rights-holders, fish and wildlife. Rural counties have attempted to protect their water interests by implementing local restrictions on water marketing in the form of local ordinances . By late 2002, 22 of the state’s 58 counties had put ordinances into effect . These ordinances mandate the acquisition of a permit before exporting groundwater or extracting groundwater to substitute for exported surface water. Individuals who wish to obtain a permit have to undergo an environmental review process. According to Hanak, the very low number of permit applications indicates that this process acts as a deterrent to water trades, rather than as a screening mechanism. Statistics for 1990 to 2001 suggest that the implementation of groundwater export restrictions reduced a county’s water trades by 14,300 acre-feet and transferred 2,640 acre-feet of water purchases to in-county buyers. Since 1996 total groundwater exports were reduced by 932,000 acre-feet or 19 percent and total water sales were reduced by 787,000 acrefeet or 14 percent .While the 1994 appellate court decision favoring Tehama County sanctioned the implementation of groundwater ordinances, counties do not have the legal authority to ban crop fallowing, although several counties have implemented such policies. According to Hanak, these counties tend to have boards that are elected by the general community,maceta cuadrada plastico as opposed to boards that only permit landowners to vote. In general, landowners are more likely to fallow land for the water market, especially when crop prices are low. Section 1745.05 of the Water Code mandates that any fallowing proposal that exceeds 20 percent of the local water supply must undergo a public review. Hanak found that water districts that implement fallowing programs tend to include restrictions in these programs that ensure that the viability of idled land is maintained and that landowners who engage in land idling are not solely engaged in selling water. In summary, a well functioning water market is seen as essential to California’s ability to adapt its restricted developed water supplies to changing demands for water. Over the past seventeen years the water market has evolved different forms and has shown steady growth despite relatively good water years. However in recent years, local resistance to water markets has taken the form of local ordinances. These ordinances need to reflect both the interests of local communities and state water users to enable the development of effective markets without imposing undue costs on local communities.Over the course of a year, some 35,000 of the state’s 750,000 employers hire a total 800,000 individuals to work on the state farms, so that about 5 percent of California’s 16 million workers are “farm workers” sometime during a typical year.

Agriculture is a seasonal industry, hiring a peak 455,000 workers in September 2002 and a low of 288,000 in February 2002. Since most farm workers are employed for fewer hours than manufacturing workers, and earn lower hourly wages, they have lower than average annual earnings. Average hourly earnings in California agriculture are about half of average manufacturing wages, $7 to $8 an hour versus $14 to $15 per hour,1 and farm workers average about 1,000 hours a year, so that farm workers have annual earnings of $7,000 to $8,000 a year, a fourth of the $30,000 to $35,000 average for factory workers.Since 1975, farm workers have had organizing and bargaining rights, but there have been elections on only about 5 percent of the state’s farms, and there are contracts on only about 1 percent. Farm worker unions have about 30,000 farm worker members; the organizing and bargaining activities of the dominant union, the United Farm Workers, have increased since founder Cesar Chavez died in 1993. Beginning in 2003, the state can require mandatory mediation that results in an imposed contract if employers and unions cannot negotiate a first agreement. During the 1990s, the percentage of unauthorized farm workers increased along with the market share of farm labor contractors and other intermediaries who, for a fee, bring workers to farms. Wages and fringe benefits generally declined in the 1990s, and farmers, fearing losses if unauthorized workers were to be removed suddenly, have lobbied in Congress since the mid-1990s for an employer-friendly guest worker program. They have not yet succeeded in winning such a program, and the debate in 2003 is whether surging Mexico-U.S. illegal migration is best managed with guest workers, legalization, or a combination of the two, so-called earned legalization, under which unauthorized foreigners in the U.S. would obtain a temporary legal status that could be converted to an immigrant visa with continued U.S. employment.Food and fiber is produced on farms, which are defined in the U.S. Census of Agriculture as places that sell at least $1,000 worth of farm commodities a year. Most of the 2.2 million U.S. farms are considered family farms, a term that is not defined officially, but a common definition is that a family farm uses less than 1.5 person-years of hired labor. Most family farms are diversified crop and livestock operations that provide work for farmers and family members year-round, and the mechanization of many farm tasks has enabled most farm families to include one or more persons employed in non-farm jobs. California farms are different because of specialization, size, and the presence of hired workers. Instead of combining crops and livestock, most California farms specialize, producing only lettuce, peaches or grapes. These FVH crops—fruits, nut and berries, vegetables and melons, and horticultural specialties that range from nursery and greenhouse crops to Christmas trees, mushrooms, and sod—require large amounts of labor for short periods of time, so large FVH farms can require hundreds of workers for 3 to 6 weeks, and only a handful the rest of the year. In California, FVH commodities occupy a third of the state’s irrigated crop land and account for half of the state’s farm sales. Producing FVH commodities with hired workers in California fields is often compared to manufacturing products on factory assembly lines. Like factories, the farms bring together people, land, water, and machines to transform seeds into crops, with agriculture’s biological production process marked by risks that do not arise in manufacturing production processes governed by engineering relationships. FVH commodities are considered “labor-intensive:” labor costs range from 20 percent to 40 percent of total production costs—higher than labor’s 20 percent share of average production costs in manufacturing, but less than labor’s 70 to 80 percent share of costs in many service industries.

High GA-like concentrations in midsummer reduce generative bud development, and the highest GA-like substance in fruits occurred in June and July . These collective results suggest that gibberellins in developing seeds of fruits on one year old growth suppress development of fruit buds on the apical current year’s shoot growth, resulting in alternate bearing. Thus, alternate bearing is due to inhibition of floral induction , and floral induction in the current year is inhibited by fruit load from the previous year. There is a negative relationship between the current year’s flowering intensity and the previous year’s production . In ‘Manzanilla de Sevilla’ cultivar, 58% of the variance in the number of flowers was explained by production in the previous year. The current year’s yield depends on flowering intensity, which depends on the previous year’s production. This relationship was also found in pistachio. The yield of the previous-year harvest is most strongly and negatively correlated with the yield of current year . However, the specific mechanism of suppressing floral bud induction and development is unknown. The role of ethylene in precipitating plant organ abscission, including floral organs, is clearly defined .It increased to a maximum seven days after FB and this peak coincided with massive flower shedding . Inhibition of ethylene results in longer-lived carnation flowers . In the ethylene biosynthesis pathway, methionine is catalyzed to SAM by SAM synthetase, SAM is then converted to 1-aminocyclopropane-1-carboxylic acid by ACC synthase,macetas cuadradas and ACC is further converted to ethylene by ACC oxidase . The ACC synthase is the targeted compound in limiting ethylene synthesis in tomatoes .

The compound aminoethoxyvinylglycine competitively inhibits ACC synthase activity by binding to the substrate’s active site, preventing ethylene synthesis . AVG is now used on multiple fruit crops. AVG applications to apple trees during harvest slowed ripening, effectively extending the harvest period . AVG inhibited ethylene biosynthesis, increasing peach fruit quality . Similarly, AVG decreased ethylene generation and increased fruit firmness in plums post harvest . AVG treatment delayed cocoa flower abscission and decrease ethylene generation in pear flowers, increasing fruit set and yield . Similar results were found in apples . Whole-tree applications of AVG on ‘Regina’ and ‘Kordia’ cherry trees significantly improved fruit set and yield . AVG application in walnuts increased yield because the high ethylene concentrations in female flowers caused of pistillate flower abortion . The effect of AVG on ethylene suppression has been confirmed multiple times. For example, AVG prevented ethylene generation in rapeseed and sunflower plants . AVG applications inhibited fruit ethylene production in ‘Golden Supreme’ apples . The primary role of AVG is inhibition of ethylene biosynthesis . Extensive research of AVG applications in walnut demonstrated that ReTain™ applied to pistillate flowers successfully decreased ethylene production and resulted in reduced flower abortion . Subsequent orchard trials confirmed these findings. The AVG treated walnut trees produced higher yields than control trees because the AVG reduced pistillate flower abortion, improving fruit set . We hypothesized the short longevity of olive pistils is the primary reason for the low fruit set and yield in olives. We further hypothesized that AVG applications, as ReTain™, during bloom can potentially decrease ethylene generation by olive inflorescences, extend the pistil and ovule longevity for a longer effective pollination period, and increase yield.

Therefore, we tested the ability of AVG, as ReTain™, applied at 25 to 50% bloom to decrease ethylene production by olive flowers, extend the pistil viability, EPP, and potential successful fertilization, and increase fruit set and oil yield in Arbequina olives. The experimental site was a 12 year-old Oleae europeae cv. ‘Arbequina’ orchard located at 38.07°N, -121.21°W, farmed commercially by Lodi Farming Inc. The orchard comprises 59 rows oriented on a north-south axis with ~ 220 trees in each row. Trees are spaced at 5 feet inrow x 13 feet between rows, for 670 trees per acre. We designated this orchard as Orchard A. A randomized complete block design was used in the experiment, with six, eight-row blocks containing two, three-row sets within a block, separated by two buffer rows. The ReTain™ treatments were applied once in every block to three contiguous rows . The center row of the three contiguous rows was used to collect samples and yield data.In 2019, after spraying on May 13th, 2019 , sampling of ethylene evolution started and was repeated daily for 13 days, until there was no difference in ethylene generation between control and treatment trees. It rained on 10 of the 13 sampling dates. From each treatment in each block, two trees, each from the north and south ends of the row, were selected for ethylene measurements. From each tree, two uniform shoots, similar in floral load, facing two directions , and at equal position in the canopy were collected daily for 13 days. They were placed on ice and transported to the lab immediately. In the lab, ~ 0.6 g, or three to five inflorescences were cut from each shoot, weighed on a Mettler balance and placed in a 15 cm airtight test tube. The tubes were placed in a controlled temperature room at 20°C to equilibrate for 1 hour. After equilibration, 10 mL of air was withdrawn from each sealed tube with a 10 mL syringe and injected into Series 400, AGCCarle Gas chromatograph for ethylene measurement. Ethylene production was expressed as µL/.In 2019, olive inflorescence samples were collected and preserved in FAA solutions .

They were further dyed with Aniline Blue and observed using a fluorescence microscope. Pictures of olive ovaries were taken, and pollen tube widths were measured using ImageJ. The pollen tube width was used as a measure of pollen tube growth, which reflected the effect of ReTain™. The treated and control rows were individually harvested on November 15th, 2019, by a Vinestar canopy contact parallel bow rod harvester with a single detached fruit bin traveling at a ground speed of 1.5 mph. The weight of the detached fruit bin was determined before harvest. The fruit weight per row was then determined using a digital in-ground scale with the bin tare entered. After weighing, a 5 kg sample was collected and put on ice for transport to the UC Olive Center Laboratory of Dr. Selina Wang at UC Davis for oil quality analyses.The same experiment was not repeated in 2020 due to strong alternate bearing and a lack of flowers. However, a grower trial was performed; selected rows in two orchards were sprayed on with the same ReTain™ treatment and control, and ethylene emission was measured from inflorescences collected from the sprayed and unsprayed rows. The sprayed and control rows in Orchard B and C were harvested and yields compared on November 15th, 2020, in both orchards. The yield from Orchard A of the 2019 experiment was measured for comparison with the yield of 2019. In 2019, the six treated rows in Orchard A produced an average yield of 3320 lb, with a standard deviation of 408, while the 6 control rows produced an average of 2970 lb, with a standard deviation of 98. With a p-value of 0.067, the difference in yield between ReTain™ treatment and control rows was not significant when the alpha level equaled 0.05. However, it was significant when the alpha level equaled to 0.1 . In 2020, the six treated rows in Orchard A produced an average yield of 1160 lb, with a standard deviation of 290,maceta cuadrada plastico while the six control rows had an average of 1185 lb, with a standard deviation of 369. The p-value was 0.9, showing no significant difference in yield between ReTain™-sprayed and unsprayed rows . The yields across the orchards in 2019 and 2020 were also determined . In 2019, the significant difference in ethylene generation between treated and control trees suggested that ReTain™ worked as assumed: it decreased ethylene generation. Day 0 was denoted as the ReTain™ application date. The confidence bands for ethylene generation were very narrow before day 3 and after day 12 . During those days, some ethylene measurements appeared to be zero and were omitted, because it is impossible to have zero ethylene generation . Fewer ethylene measurements before day 3 and after day 12 resulted in narrow confidence bands. The primary finding was not affected: from day 3 to day 12, the ethylene generation from ReTain™-treated trees was significantly lower than that from control trees. We hypothesized that delayed floral senescence was a consequence of decreased ethylene generation; however, the visual inflorescence rating data suggested the opposite. The senescence ratings of treated trees were significantly higher than the control trees, indicating ReTain™ increased the rate of flower senescence. Throughout the experiment, the first-opened flowers senesced first.

The blocks on the edge of the orchard senesced earlier than blocks in the center of the orchard, east sides of the rows than the west, and the south end of the rows earlier than the north. The flowers in the row on the edge of the orchard and the ones on the south end of the rows also bloomed first. This could be potentially explained by that those flowers were at locations to receive the most heat. The visual evaluation of inflorescence senescence was based on petal color change and petal drop. It might not indicate the ovule viability throughout the experiment. Therefore, it is possible that the petal drop was not correlated with the effective pollination period and ovule viability. Aniline blue fluorescence was argued to be an accurate method to measure ovule senescence in olives The findings on pollen tube growth supports this possibility. There was no significant difference between treated and control flowers in pollen tube width. This suggested both treated and control flowers were pollinated equally, even though the treated flowers senesced more rapidly than the control flowers. The non-significant difference in pollen tube width suggested that pollination an ovule viability were not affected by ReTain™. It is possible that pollen tube width is not a good indicator of pollination. The pollen tube width is primarily a measurement of the amount of pollen deposited on the stigma that germinated and produced a pollen tube. We selected this measurement reasoning that a longer effective pollination period provides more time for pollen grains to land on the stigmas. Determining whether the pollen tube has reached the ovary might be a better indicator of successful pollination . However, after reaching the ovary, the pollen tube was unrecognizable . The tissue in the ovary was too thick to observe under a fluorescence microscope. In Arabidopsis, both ethylene-dependent and ethylene-independent pathways are required to initiate and progress through floral senescence . It is possible that the floral senescence in olives is not regulated primarily by ethylene. Pollination induces a series of post-pollination developmental events, including petal senescence . Pollination-triggered senescence has multiple advantages. Once sufficient pollen has been set on the stigma, additional pollen deposition is wasteful, and excess pollen tubes may compete for nutrients. In addition, maintenance of floral structures is costly . In 2020, the ReTain™ treatment was applied to two different orchards at the same concentration. However, no ethylene was detected from either treated or control flowers, which may be due to reduced number of flowers in an “off” year. In 2019, heavy rain during bloom could have prolonged the flowering time. The heavy rain and low temperature combined with the spray of ReTain™, while the control rows were not sprayed, could potentially explain the earlier senescence of the treated rows. The lowest temperature during the bloom reached 8°C, while the optimal temperature for olive pollen germination and pollen tube growth is 20 to 25°C . The low temperature during the bloom may have negatively impacted pollination in both ReTain™-treated and control trees. In 2019, the difference in yield between ReTain™-treated and control rows was not significant at 0.05 but was significant at 0.1. However, yield fluctuated greatly in treated rows, while the yield among control rows was stable. ReTain™ strongly improved yield in five out of six rows, but the increase was not consistent and the effect of ReTain™ was not uniform among rows .