Nature Agriculture

Calling UCSC’s programs “near and dear to my heart,” Farr said there was no place he would rather have been than on campus among the pioneers and contemporary leaders of sustainable agriculture. Farr hailed UCSC’s spirit of innovation and ability to accomplish a lot with minimal resources, and he credited UCSC leaders with fighting for agroecology, even when it meant taking on vested interests within the University of California system who wanted to confine agriculture programs to the Davis and Riverside campuses. Reading from remarks he entered into the Congressional Record on October 4 to honor the 40th anniversary of sustainable agriculture programs at UCSC, Farr called the Center for Agroecology and Sustainable Food Systems “one of the most prominent centers of agricultural research and education in the world.” In Congress, Farr has helped secure more than $3 million to support CASFS research and extension projects. Farr has been a proponent of organic farming since his service in the Peace Corps in Colombia in the 1960s, when he saw the importance of helping people improve their ability to grow food. In UCSC’s programs, he recognized the relevance of developing small-scale, intensive, organic food production systems. Over the years, Farr has made several landmark contributions to UCSC’s programs, and CASFS director Patricia Allen thanked him for his commitment to sustainable food and agriculture research and education. As host of the program, Allen read from remarks prepared by environmental studies professor Stephen Gliessman, who was unable to attend the ceremony because he was teaching a class. “We owe the existence of agroecology and CASFS to Sam, and it is up to us to continue to carry his vision of sustainable food systems forward,” Allen read on behalf of Gliessman, who was the first director of agroecology and who holds the Alfred E. Heller Chair in Agroecology.

Farr also authored the 1990 state law that established standards for organic food production and sales in California, square plastic plant pots which became the basis for recent federal organic food standards. In Congress, he has insisted that U.S. Department of Agriculture research stations include a focus on organic agriculture. Former CASFS director Carol Shennan, a professor of environmental studies, thanked Farr for his recent support of research efforts, including UCSC’s Central Coast water monitoring project that has helped farmers reduce pesticide runoff into the Monterey Bay National Marine Sanctuary, and for research on regional food systems. As part of the celebration, Farr and garden manager Christof Bernau planted an heirloom climbing rose—the first in a new rose garden established in Farr’s honor. An adjacent plaque honors Farr for his “visionary support of sustainable food and agriculture research and education.”The second national conference on Facilitating Sustainable Agriculture Education, held July 11–14 at Cornell University in New York, brought together nearly 200 educators and students to share ideas for improving sustainable agriculture education at colleges, universities, apprenticeship programs, and other settings. Center for Agroecology and Sustainable Food Systems graduates, students, and staff have played key roles in developing this new organization, whose mission is to “. . . promote and support the development, application, research, and exchange of best teaching and learning practices in sustainable agriculture education and curricula through communication, training, development, and collaborative activities for teachers and learners.”

The burgeoning interest in sustainable agriculture education prompted the founding of the SAEA. “We did a survey of stakeholders around the U.S. before moving forward with developing the association,” says Albie Miles, former curriculum editor at CASFS who helped organize the first national conference, held at Asilomar in 2006. “It was clear from that survey that there was a lot of interest in creating an organization that would help promote educational opportunities and move sustainable agriculture forward as a mainstream academic discipline,” he says. Although the SAEA was originally envisioned as a group that would promote sustainable agriculture programs at post-secondary schools, this year’s conference attendees broadened that scope to include education at a variety of levels. “I was impressed by how enthusiastic everyone was about not defining ourselves too narrowly,” says Katie Monsen, a UCSC graduate student in Environmental Studies who has helped develop the new organization. “We had people there from universities, but also growers and others with educational programs. For example, people are really interested in getting high school students involved in order to increase enrollment in college agronomy programs; other say we need to start sustainable agriculture education even earlier—this opens us up to work with a broad range of folks.” That range was reflected in this year’s group of conference participants, who came from throughout the country to share resources, discuss teaching methods, and create new networks. Monsen notes that despite the geographic differences, attendees found common ground in their educational goals. “We didn’t have a lot of conversations about ‘what is sustainable agriculture,’” she says. “Although there are certainly differences in regional cropping systems, people from across the country had very similar interests when it comes to creating educational opportunities.” In contrast to the typical “top down” approach to education, students have had a major voice in SAEA’s development. UC Davis graduate student Damian Parr, a graduate of UCSC and the CASFS apprenticeship program, is a leading advocate for giving students an equal voice in developing sustainable agriculture programs.

That goal is reflected in SAEA’s statement of values, which include, “A focus on learning and the development of communities of co-learners,” and “The democratization of knowledge and learning.” Next steps for the new organization include developing an online resource directory. “We want to make the web a place where people will be able to put up curricula, ask questions, post ideas and resources,” says Miles. That effort is already underway, with resources shared at this summer’s conference now available online .SAEA subcommittees are also working on student outreach, fundraising, and developing plans for next summer’s conference, which will take place in the Midwest.In Together at the Table, Patricia Allen provides a comprehensive and critical analysis of the potential for alternative agrifood movements to create substantial change in the entire food system. She looks in particular at the sustainable agriculture and community food security movements as examples, weighing the strengths and weaknesses of each. The UC Sustainable Agriculture Research and Education Program was, happily, accurately showcased in the discussion of sustainable agriculture programs. Then, in the final chapter , she suggests specific strategies for how we can all work together to improve and build the capacity of these movements for creating lasting changes that address all aspects of sustainability—ecological, economic viability, and social equity for all people in the food system. In her conclusions, Allen points out two key areas we, in the sustainable food systems research/practitioner communities need to work on: • Develop a broad-based vision for an alternative agrifood system that goes beyond the traditional ideological framework, and • Continue to broaden constituencies and engage them in democratic processes that can provide political power to move us toward significant change in the agrifood system. Developing a broad-based vision. In Chapter 4, Allen articulately describes the dominant epistemological approach that guides research and education in sustainable agriculture programs. In a nutshell, this is a focus on natural sciences, production innovations, and farm-level projects, mostly at the expense of resources devoted to social equity issues . She points out that sustainable, square pot plastic agrifood systems research and education must be larger in scope and more truly interdisciplinary than the current involvement of mostly production-oriented natural science disciplines. Broadening constituencies. Allen also makes the case that we clearly need constituents that participate in local food system actions, and those that help us link local efforts in larger movements that involve national and international politics. Some of this is happening already through sustainable food and farm advocates that are working on the Farm Bill. We also need to be vigilant about enlarging our “circles” to include those left out of the discussion. The new Agricultural Sustainability Institute at UC Davis is one new promising organization for addressing the above concerns in California. Through its strategic planning process, the ASI is developing a broad-based vision that includes diverse stakeholders and encourages participation at all levels. Its vision, core values and operating principles attest to its commitment to be inclusive . This may be one example of what Allen is saying in Together at the Table—that we need to work at many levels in the food system simultaneously from the local to the international.

To do that well, we need many kinds of people with different expertise and local knowledge to be involved. Moreover, we need to acknowledge the importance of each of our contributions and communicate with each other effectively if we really want to make a lasting difference in changing our food system to one that is more sustainable and equitable.Conventional strawberry growers rely on multiple applications of pesticides per year to control lygus bugs , a pest capable of damaging berries badly enough to make them unacceptable for fresh market sale. According to CASFS entomologist and extension specialist Sean Swezey, typical control programs entail 6–8 biweekly calendar applications of insecticide per season, with costs capable of exceeding $500/acre. Yet even these efforts are beginning to fall short of controlling the pest, as lygus has started to display resistance to commonly applied insecticides in California. Swezey and Charlie Pickett of the California Department of Food and Agriculture’s Biological Control Program recently received a two-year grant from the USDA’s Pest Management Alternatives Program to extend trap crop techniques developed in organic systems to conventional strawberry operations in an effort to reduce the need for insecticide applications. Trap crops of alfalfa offer lygus a preferred “host” plant: by establishing strips of alfalfa in strawberry plantings, growers can concentrate the pest in one place and control it with either a vacuum system or conventional sprays. The PMAP grant will fund efforts by Swezey, Pickett, and CASFS research associates Janet Bryer and Diego Nieto to fine tune techniques they’ve developed over the past several years in organic operations using alfalfa trap crops combined with periodic trap crop vacuuming, supplemented by an introduced lygus parasitoid. In the organic research site, the team will determine the number of vacuum passes over an alfalfa trap crop that will optimize lygus removal. “We want to find out whether a significant or economically important number of lygus bugs are removed after each successive vacuuming pass,” says Swezey. “We may find that the first pass or two removes the majority of the lygus present, and that further passes don’t make enough difference in decreasing the remaining number of lygus to be justified.” Establishing an optimal ratio of lygus bug reduction-to-tractor expense will help prevent unnecessary passes, thereby lowering labor costs and tractor operation expenses. The research team will also determine the extent to which an introduced parasitoid of lygus, the braconid wasp Peristenus relictus, is helping control lygus populations in strawberry rows planted between strips of alfalfa. Introduced into a Central Coast organic strawberry operation in 2004, the wasp is now established at the site. Research efforts over the next two years will focus on the percent of lygus parastized by P. relictus in strawberry rows 1, 5, 10, 15, 20, and 25 . In the 40-acre conventionally managed strawberry system, the researchers want to determine whether treating a trap crop with insecticides will control the pests effectively enough to decrease or eliminate the need to spray the crop itself. They also hope to determine the best way to manage a trap crop in a conventional system. “We want to optimize the way alfalfa plantings are managed to make them effective ‘traps’ for lygus throughout the season,” says Swezey. This will entail mowing the crops to stimulate new growth and flowering during the summer as a way to enhance the alfalfa’s attractiveness to lygus. Alfalfa plantings have already been established for the upcoming field season at the organic and conventional research sites. Results of the study will appear in future issues of The Cultivar, and will be presented at grower field days planned for late 2009 and 2010.As interest in farm-to-college programs designed to bring local, sustainably produced food into college cafeterias grows, there is also a growing need to understand how to best address the interests and needs of the consumers served by such programs.

And after the Forest Service absorbed range research a few years later, the interests and priorities of forestry would dominate the fledgling field of range science for decades to come.Frederick Vernon Coville was the son of a bank director in upstate New York. After graduating from Cornell, he was hired as Assistant Botanist in the USDA in 1888, and five years later he succeeded George Vasey as Chief Botanist and honorary Curator of the National Herbarium. He is most famous for his work as a botanist, especially in connection with the Death Valley Expedition of 1891, and for his path-breaking research on the blueberry, which helped make it a commercial crop in the northeastern US. Although less well known, his role in charting the course of rangeland research and administration must be considered among his most enduring accomplishments. For Roosevelt’s Public Lands Commission, he wrote the blueprint for the grazing lease system subsequently adopted by the Forest Service , and his fieldwork and reports in the decade after 1897 set the terms of engagement for public range grazing research. As Pinchot succinctly put it years later, “Until the Forest Service developed a body of experts of its own, Frederick V. Coville was the first and the earliest authority on the effect of grazing on the forest” .Struggling to define and assert management authority over the forest reserves, the General Land Office had banned all livestock grazing in 1894, believing it necessary to protect forest regeneration and reduce fires. The move provoked resistance from sheep and cattle owners throughout the West, and at the end of June 1897, new policies were announced rescinding the ban for cattle nationwide but retaining it for sheep, blueberry grow bag size except in the reserves of Oregon and Washington, where “the continuous moisture and abundant rainfall…make rapid renewal of the herbage and undergrowth possible” .

A week before announcing the partial rescission, the Secretary of Agriculture instructed Coville “to make an investigation of the alleged damage to forests by grazing of live stock [sic], more especially the effects of sheep herding in the Cascade Forest Reserve of Oregon,” which had been created in September 1893. His subsequent report clearly articulated the empirical and analytical framework from which the CoyoteProof Pasture Experiment would later be designed. Coville’s field methods were both ecological and ethnographic,8 and he came to see fencing, herding and predators as tightly inter-related elements of the problem of the western range.Coville firmly rebutted two widespread claims about sheepherders: first, that they were “aliens” or non-citizens who represented “a comparatively low class of humanity” ; second, that they were responsible for starting forest fires. These were politically volatile arguments to make, and they stood in sharp contrast to the prevailing views of many prominent figures, such as John Muir, who famously described sheep as “hooved locusts” and whose vision of wilderness erased and excluded both non-whites and laborers of all kinds . Coville sidestepped the polemics, apparently considering both of his claims to be settled by the facts on the ground. Instead, he focused his analysis elsewhere, detailing the herders’ management techniques, their knowledge of the value of different plants for sheep growth, and the variation in their skills. He noted that “The acreage per sheep required for grazing throughout the summer is exceedingly variable, depending on the kind and character of the vegetation” .Overgrazing in the Cascades had occurred, according to Coville, but it was neither ubiquitous nor longstanding, and it had not yet altered the composition of vegetation in the reserve. He attributed the damage he observed not to the herbivory of the sheep but to their physical movements. “The principal bad effects of overgrazing are to be attributed rather to trampling than to actual close cropping” . This could be prevented, he believed, by securing sheep owners’ access to the range resource.

Open access for all made each herder rush to use the range ahead of the others, “to get all the grass possible without reference to the next year’s crop, for he [the herder] is never certain that he will be able to occupy the same range again. Where the competition is close the difficulty of insufficient forage is increased by the haste of the herder in forcing his sheep too rapidly over a grazing plot, the result being that they trample more feed than they eat. So year after year each band skins the range” . A system of permits to graze specified areas would relieve this competition, and also enable the government to impose other terms, such as stocking rates and rules regarding fires.Coville conducted similar fieldwork in Arizona in 1900 at the invitation of Pinchot. The conflict there also concerned sheep grazing, but in relation less to timber and fires than to watershed conditions above the Salt River, whose waters were being developed for irrigation in the Phoenix basin below. The trip was proposed and hosted by Alfred F. Potter, a prominent local sheep owner and secretary of the Arizona Wool Growers’ Association. After a three-week wagon tour of the Mogollon Rim country, Coville and Pinchot concluded, as in Oregon before, that sheep grazing should be permitted but carefully regulated. Shortly thereafter, Pinchot recruited Potter to come to Washington as Assistant Forester in charge of the Branch of Grazing. Potter and Coville would later author key parts of the Public Land Commission’s 1905 report, making the case for exclusive leases to graze fenced allotments of the Western range. The trip also cemented a long-lasting friendship between Coville and Pinchot.In late March of 1907, Pinchot and Potter met with C. V. Piper, Chief Agrostologist in the BPI, to discuss “the range investigations which they would like to have undertaken by this Bureau.” In a memo the following day, Piper reported to Bureau Chief B. T. Galloway that the Forest Service had reduced stocking on northwestern sheep ranges by about 25 percent, “by agreement with the stockmen. It is however the desire of the Forest Service to increase the carrying capacity of these summer ranges and consequently the allotment of stock to each district as rapidly as possible,” and “to permit as many stock as possible to run on these summer ranges.”

Experiments were needed to determine whether degraded ranges could be reseeded, and to determine “what system of range management both with cattle and with sheep will best permit the more valuable native grasses to re-seed themselves and thus increase the carrying capacity and maintain it at a maximum.The investigations will not only be necessary to the best administration of these range lands but will result in enormous benefit to the live stock interests of the West.” Piper recommended that J. S. Cotton, who had studied range conditions in central Washington be put in charge of the experiments. It appears Pinchot overrode this suggestion in favor of Coville. An inter-bureau agreement was reached that the BPI would cover Coville’s salary and expenses, while the Forest Service paid the rest of the costs. In mid-May, Coville headed west to set up the experiments. From his hotel in Lincoln, Nebraska, he wrote to Pinchot: “After a conference with Professor [Charles] Bessey and Professor [Frederic] Clements, and with the prospective appointee himself, I have secured one of the men we want for the forest grazing experiments. He is Mr. Arthur W. Sampson, a resident of the state of Nebraska, and a graduate student of the University. He is an expert in plant ecology and should be appointed as such.” Sampson’s training under Bessey and Clements was critical for Coville because it meant that the other men he hired would not need expertise in botany or ecology; its importance for the future of range science was even greater, because it helped install Clementsian ecological theory as the foundation for the discipline. Five dayslater, in Logan, Utah, Coville hired Jardine, describing him thus to Pinchot: “Mr. Jardine is twenty-five years old, a graduate of Utah agricultural college, and now an instructor in that institution. He was brought up on a ranch in southern Idaho and is familiar with the handling of cattle, horses, and sheep. He is well qualified both by his personal characteristics and his training to take part in the forest grazing investigations.” En route from Lincoln to Logan, Coville stopped in Laramie, Wyoming, blueberry box to interview a prominent sheep owner by the name of Francis S. King. An immigrant from England, King and his brothers owned or leased some 120,000 acres of private land in Wyoming and were famous for their prize-winning purebred Merino, Rambouillet, and Corriedale rams and ewes . Coville’s notes from the meeting suggest that King’s ideas and opinions played a significant role in refining the details of the imminent experiments in Oregon. King saw two advantages to protecting sheep within a predator-proof enclosure: reduced labor costs and improved animal performance.On June 3, Coville reported to Pinchot that he had selected a site with good forage, ready access by wagon, and “an abundance of coyotes, wild cats, and bear, with an occasional cougar and lynx.” “The stockmen” in the area, he wrote, “are greatly interested in the experiment, the consensus of opinion being that under a pasture system the carrying capacity of the range will be doubled.” He had found a “thoroughly efficient hunter” to hire for the project, and he recommended some modifications to the fence design based on his conversations in Wyoming . He had also met with the press to promote the experiment. One reporter wrote: “It is believed that the same amount of grass will support many more sheep when they are pastured than when they are herded. Should this theory be proved, the result will revolutionize the entire policy of the department in regard to the reserves and will have a great bearing upon the stock industry.”10 .By mid-June, Jardine and Sampson had reported for duty, the fence was under construction, and Coville soon returned to Washington. The fence—an imposing and elaborate combination of barbed wire, wire mesh, and stout posts —was completed too late for a full season’s research, but a band of sheep was placed in the new pasture for one month and observations were made both of their behavior and of the effectiveness of the fence at repelling predators. As for the latter, “the fence proved successful as a protection against coyotes, not successful as a protection against grizzly bears, doubtfully successful against black and brown bears, still problematic against cats, and not successful against badgers” . The hunter and his hounds patrolled the perimeter each day, recording signs of predators and, when possible, pursuing them . But just as Merriam had feared, one coyote was trapped inside the completed fence, and two sheep were lost to predators—one to a bear and another to the coyote . These predators interfered with efforts to observe “the action of sheep when they are allowed perfect freedom in an inclosure [sic] and protected from annoyance by animals” , but Jardine nonetheless judged that “the test was very satisfactory. They [the sheep] retained, more or less, the natural instinct to mass, but from the first day the tendency to open, scattered grazing, with little or no trailing, increased” . While conceding that solid data had yet to be collected, Jardine concluded: “That the experimental system will materially increase the carrying capacity of the range is not to be doubted” .The following summer, after repairs had been made to the fence, a band of 2,209 sheep were turned into the coyote-proof pasture without a herder. The hunter resumed his daily patrols, and the behavior of the sheep was monitored all day, every day from June 21st to September 24th. Only 15 sheep died during the 96 days, none due to predation. The objective was to induce “open grazing,” as Jardine termed it. “Whether sheep are in large or small bunches it is essential for the protection of the range that they be well scattered and graze quietly. Close-bunched grazing, massing, running, and trailing one after another should be prevented if possible, not only for the good of the range but for the good of the sheep. In this respect there was marked change during the season” . Here, the predators were not the only culprits: herders themselves, and especially herders’ dogs, were like predators in that they could provoke fright in sheep and cause them to bunch, mass, and run.

Using the OPEX value calculated and the mass of knotweed rhizomes required each year for 100 MT production of resveratrol, the cost of knotweed rhizome is determined to be $0.19 per kg. To give a better assessment of the CAPEX required for the upstream production, farm equipment cost was also calculated alongside the annual operating cost. Table 2.5 list the equipment deemed necessary for harvesting Japanese knotweed rhizomes. Values for the equipment cost were again retrieved from the UC Davis Agriculture and Resource Economics Center. Since the report published by the UC Davis Agriculture and Resource Economics Center was released in 2015, an inflation/cost adjustment of 1.125 was used to estimate the cost of the same farming equipment in 2021. Here, the total farming equipment cost was multiplied by a factor of 0.6 since some equipment purchased may be used rather than new equipment. Additionally, it is assumed that the equipment requirements scaled with cultivation acreage, thus estimating the number of each equipment required was multiplied by 7.4 to account for the larger cultivation acreage than use in the report. Using all this information, the total cost of equipment was to be $3.6 million.The utilization of natural plants as a source for chemical compounds is an ever-growing field. To date, pe grow bag there have been a variety of compounds which have been extracted from plants, including but not limited to, tetrahydrocannabinol from hemp 1 , phenolic acids from purple corn 2 , and flavonoids from chamomile flowers 2 .

Although natural plants offer a reduction of complexity in upstream bioprocessing in comparison to using genetically engineered microbes, the downstream and purification methods remain just as rigorous. A standardized process for the extraction of resveratrol from Japanese knotweed remains unestablished as novel technologies and methods are continuously being developed and researched. The most common unit operations seen utilized in current patent and scientific journal articles are shown in Figure 3.1 as a block flow diagram.The procedure needed for purification of resveratrol is understood, however, there is no consensus on what the best methods to use are since there remain numerous options for each step, each consisting with their own advantages and disadvantages. Notably, a crucial step during the downstream process is the deglycosylation and hydrolysis of polydatin performed after crushing and shredding the rhizome. Two methods can be employed to hydrolyze the polydatin compound found beside resveratrol, either using strong acids or enzymes. Performing either method has demonstrated conversion yields of polydatin to resveratrol above 90% 4,5. However, the use of acid hydrolysis may produce additional adducts in the reaction, thus decreasing the content of resveratrol present and prompting concerns for additional purification procedures. Furthermore, acid hydrolysis has been reported to require harsh processing conditions, often causing pollution as a result. Nevertheless, the large quantity and high cost of industrial cellulase enzymes serves as the biggest deterrent to the latter approach. Currently, there exists numerous published techno-economic analysis studies of plant based production focusing on bio-fuels, recombinant therapeutic proteins, industrial enzymes, and antimicrobial proteins for food safety.

Here, this chapter will describe the techno-economic analysis performed on the downstream processing required for the plant-based production of Japanese knotweed for the extraction of the biopolymer precursor, resveratrol, which has not been demonstrated before. This study will aim to establish a framework for more informed decisions on the development of a domestic production route for such polymer precursors.The simulation model and economic analysis was performed using a computer-aided process modeling and design software, SuperPro Designer® Version 12, Build 3 Special Build 1600. SuperPro was used to determine equipment sizing, specify equipment process parameters, and determine operations scheduling and raw material requirements. The economic analysis was used to determine the total capital expenditure , the total annual operating expenditure , and the cost of goods sold . A further detailed analysis provides a breakdown of all costs, e.g., raw materials, consumables, utilities, labor, and waste disposal- with the goal being to identify the materials and process steps which contribute most significantly to the total cost of production. Pricing for raw materials, consumables, and equipment within the model were calculated using publicly available commercial prices, unpublished personal communications with manufacturers of large-scale bio-processing equipment, previous SuperPro design files, and in some cases, SuperPro default values.The base case model was developed to process 100 metric tons of resveratrol per year, with the facility operating 330 days a year. A single batch duration is estimated to be 45.6 hours, totaling 1,295 batches per year with a cycle time of about 6 hours. Due to the short cycle time relative to the annual operating time, the process may be assumed to be operating under pseudo-continuous conditions. To attain the quantity of 100 MT of resveratrol, roughly 7.3 million kg of knotweed rhizomes a year are required for downstream processing.

The model was scaled using publicly available patent literature, scientific journal articles and working process knowledge. Bioprocessing operations and conditions for certain unit operations within the simulation were adopted from scientific literature focusing on resveratrol production from various plant sources . Table 3.1 and Table 3.2 list the various processing techniques utilized for resveratrol purification from Japanese knotweed in patents and scientific journals, respectively.In the design of the downstream production process model, certain key bioprocessing parameters were extrapolated from publicly available information on resveratrol production. In particular, the quantity of enzymes per batch required to hydrolyze polydatin to resveratrol was tuned to match production methods found in patents. Patent literature detailing the extraction of resveratrol have suggested amounts of 2 – 4 weight % of enzymes per knotweed per batch should hydrolyze polydatin to resveratrol effectively for processing. However, scientific literature focused on optimizing the enzymatic hydrolysis process for resveratrol in knotweed have reported using enzyme concentrations closer to 10% of the total processed knotweed13. An average of these values resulted in 6.5 weight % of enzymes per the total knotweed rhizomes per batch; this percentage was used in the process simulation. Another parameter which was adjusted in the model to match resveratrol production methods described in patents was the percent recovery of resveratrol after undergoing the extraction process. Multiple sources have denoted the use of the ultrasonic technology and Ultrasonic Assisted Extractions to remove resveratrol from knotweed rhizomes but fall short by failing to provide key parameters such as percent recovery. UAEs are becoming a common operation for extracting polyphenols from plant biomass. Its application has already been utilized to extract resveratrol from other plant sources such as grape stems and grape leaves18. These two studies demonstrate the capability of UAE technology in extraction of resveratrol while noting a percent recovery of 78.8% and 80%, respectively. A conservative approach was taken, and the former of these values was used to define the resveratrol recovery in the UAE operation used in the process model. In an effort to accurately model the UAE operation in SuperPro, the other parameters associated with UAE such as power, temperature, and duration of agitation were aligned to Japanese knotweed roots processing conditions. Traditional methods of purification involve using silica gel resins in chromatography columns. However, silica gel resins are predominantly used for smaller scale production and there is often complication scaling up for large scale manufacturing. More recently, growing bags macroporous resins have been utilized to serve as a replacement as they hold several advantages over their silica gel counterparts. First, silica gel resins utilize mixtures of acids, such as chloroform and methyl alcohol, to serve as their eluents, whereas macroporous resins only require mixtures of ethanol and water. Additionally, the cost of using silica gel resins is higher compared to using macroporous resins while the recovery of using silica gel remains lower. In attempt to accurately model an industrial chromatography unit in SuperPro, an unpressurized vessel filled with macroporous resin was initialized to operate as an adsorption mixing tank. The resin is exchanged with fresh resin every 100 batches. All process flow specifications and assumptions used in the development of the downstream facility model is shown in Table 3.3 along with its source.

A detailed downstream process flow sheet depicting the purification of resveratrol from Japanese knotweed is shown in Figure 3.2. Each batch begins with the harvested knotweed rhizomes being transported from a designated storage warehouse to a silo bin using a conveyor belt . Approximately 5,635 kg of knotweed rhizomes are retrieved from the silo bin and transferred to a washer where they are washed with water at a 1:1 w/w ratio. Next, the knotweed rhizomes are mixed with water at a 1:1 w/w ratio andare grounded into a slurry solution using an industrial grinder operating at a throughput of 11,327 kg/hr. The slurry is pumped down the process line where 100 kg of citric acid is mixed with the solution to adjust the pH value down to 5.0. Following the pH adjustment step, a stream of enzymes consisting of cellulase and ß-glucosidase at an 80:20 ratio enters the process line where it is mixed with the solution before being pumped into a batch vessel reactor . Within the reactor , the solution is agitated for fifteen minutes and heated to 55 C to allow the enzymatic deglycosylation and hydrolysis of polydatin to occur, converting 90% of existing polydatin to resveratrol. To deactivate the enzymes and halt the reaction, the reactor is heated to 85 C using steam. Once the deactivation step is complete, the slurry is transferred to the ten ultrasonic assisted extractor units running in parallel . The ultrasonic assisted extraction units are first charged with ethanol at a 1:1 mass ratio with the plant biomass where it is then agitated for sixty minutes, allowing for efficient polyphenol extraction from the plant biomass . The slurry solution is then sent to a belt press filtration unit where the plant biomass can be separated from the liquid solution and disposed of properly . An additional separation unit in the form of a plate and frame filter is used to capture and separate any residual plant biomass within the solution before being sent for further processing. The filtrate is then pumped into a batch adsorption vessel containing NKA-II, a macroporous adsorption resin. After the binding step occurs, the resin is first rinsed with water to remove any impurities which may have been captured. Following the washing step, the adsorption vessel is set to charge in a stream of ethanol to elute the resveratrol from the macroporous resin . After the elution step, the eluate is then pumped into a crystallization column for further processing. The crystallizer operates under reduced pressure, an evaporation temperature of 98 C, and a crystallization temperature of 60 C, removing most of the residual ethanol and water present from before and yielding solids crystals . These crystals undergo a mixing step where they are resuspended in ethanol . This liquid-solid mixture is then sent to another crystallizer unit operating under similar conditions as before, but this time yielding 77.3 kg of resveratrol at 99% purity.A summary of the economic analysis results from the base case model set to produce 100 MT resveratrol is presented in Table 3.4. The CAPEX is the sum of the direct fixed capital , working capital , and startup and validation cost . The DFC for the facility was calculated by using a distributed set of purchase cost factors shown in Table 3.5 to estimate the facility direct costs , indirect costs , and other costs . Here, the purchase cost is the sum of all major equipment purchase costs shown in the SP flowsheet and the unlisted equipment purchase cost is assumed to be 20% of the PC. A list of all the major equipment, their composition, and purchase prices can be found in Table 3.6. The price of unlisted equipment was also included in Table 6 for reference. The working capital was estimated to two percent of the total DFC. The startup and validation cost combined were estimated to be about 1% of the DFC. Most of the equipment purchase costs were estimated using SuperPro default values. The composition of a majority the equipment are carbon steel except for the UAE, which is composed of Stainless steel 316L and the silo bin which is composed of concrete. The justification for using equipment fabricated using carbon steel instead of more costly stainless steel is recognizing that the process is being designed for polymer applications and not for a more regulated pharmaceutical application.

Our results indicate that the evolutionary trajectories of both the core and the pan-genomes allow for a bacterial species with an extensive host range to specialize many times over a broad array of plant hosts. We see this system as an example of one that “leaps”, with host genera seemingly changing not via phylogenetic signal to related plant hosts, but switching across large regions of plant host phylogenies . Prior to this study, we have not been able to trace a pattern of underlying genetic origins of host specificity in X. fastidiosa. In this way, our study shows that the phylogeny and gene gain/loss are connected to the adaptations that diversify host specificity in X. fastidiosa. Phylogenies for MLST, pan-genome, core-genome, and non-recombinant core-genome data were topologically similar, but not identical. While the subspecies relationships are not important to predicting host range, they are frequently used in management decisions and our ability to converse about outbreaks, so we are including our findings alongside our data on host use. In terms of taxonomic subspecies, there are differences between the four trees in whether the two debated subspecies, X. fastidiosa subsp. morus and subsp. sandyi, are contained within subsp. fastidiosa or subsp. multiplex, or if they should be considered their own subspecies. While there are pairs of strains that are consistently close to each other like the morus strains MulMD and Mul0034, the uncertainty in their position from phylogeny to phylogeny likely reflects large gaps in diversity that we have not yet sequenced or horizontal gene transfer more intensely affecting the pan-genome and particular genes used for MLST than the core genes, plastic plant pot leading to issues recreating the vertical descent we aim for in a phylogeny . The subspecies morus has been documented to have up to 15.30% of its core genome undergoing intersub species homologous recombination, which could account for its uncertain placement in the four phylogenies .

The two strains that have been described as sandyi-like, CO33 and CFBP8356, both clustered within subspecies fastidiosa, not with the other potential sandyi strains Ann1 and RAAR8_XF70, supporting previous work showing that there is not a strong distinction between subspecies sandyi and fastidiosa . The core genome tree also has very low bootstrap support for subsp. pauca, which is the most diverse and oldest of the three main subspecies that could be potentially due to conflicting histories between horizontal and vertical descent, or alternatively reflect that this group is simply not well supported as one subspecies . In terms of the poor resolution in the OQDS clade, an analysis has recently been conducted to increase resolution within these strains . Given the diversity of subspecies pauca, the Hib4 strain, the outgroup of the subspecies, could be a potentially inTheresting strain in terms of both function and evolutionary history . It is difficult to know which phylogenies are more accurate than others, however we assume that the core genome is the most accurate at depicting the descent of this bacterial species and the topology should be robust to even high levels of recombination . While the non-recombinant core-genome might reduce some issues with horizontal gene transfer, the lack of resolution because of too many identical sequences makes it difficult to use. While more data are not intrinsically better, there are known issues with the MLST genes used for X. fastidiosa phylogenetics and having a larger set of unbiased homologous regions should be able to lend data to support nodes that are difficult to differentiate using the smaller MLST dataset . Using the core genome phylogeny, the most likely ancestral host was inferred from the phylogeny. These results show us that the phylogenetic history of X. fastidiosa is significantly correlated with the agricultural plant host that the strains were isolated from. While the coregenome phylogeny depicts mainly vertical descent within this bacterial species, the pan-genome phylogeny likely combines vertical descent with horizontal gene transfer. This is due to the pangenome’s inclusion of the accessory genome, which are genes not shared by all members of the group .

Based on this, we speculate that there is both adaptation and convergence depicted in these results. Potentially, both convergent horizontal descent via gene gain and loss as well as vertical descent in the core leads to our modern distribution of traits. While the ancestral state reconstruction did not show a classic host-parasite story of cospeciating or phylogenetically conserved host specificity, the phylogeny and gene presence/absence are predictive of the hosts from which the strains were isolated from, and thus hypothetically, host specificity as well. While the four ancestral state reconstructions do not show identical histories, they all infer high likelihood of ancestral hosts at many key branch points of the three subspecies. The pan and core-genome reconstructions predict the genus Vaccinium as the most likely ancestral host of the subspecies multiplex, which supports the overall reliability of the reconstruction as blueberry, like subsp. multiplex, is native to the eastern North America . Subsp. pauca, subsp. multiplex, and subsp. fastidiosa all exhibit host shifts from another genus to Prunus, suggesting potential for increased vulnerability in this genus toinfection from varied alternative hosts. All four reconstructions also support the genus Coffea as the most likely ancestor of the introduced subsp. fastidiosa strains from Central American to California. This supports a previous hypothesis made by Nunney et al. wherein coffee plants that were imported from Central America to southern California in the mid 1800s might have brought X. fastidiosa subsp. fastidiosa along with them. Given the potential role of imported Coffea in devastating global outbreaks of disease caused by X. fastidiosa , it should be much more carefully monitored or restricted in global trade. Given the current policy emphasis on eradication, trade restrictions, it is vital to identify genera such as Coffea that are especially relevant to global outbreaks and that should be monitored carefully. The relationship between X. fastidiosa and Coffea should be further explored as a model host to aid our understanding of the molecular mechanisms of this complex interaction. A potential alternative hypothesis for these nodes could also be that Coffea and Vaccinium are permissive hosts. From a parsimony perspective, they could be akin to ‘universal hosts’ so that it takes very little change for X. fastidiosa strains to switch to Coffea or Vaccinium from other infected plants. This could be investigated by further interrogating the genes shown to be uniquely absent in Coffea-infecting strains. Phylogenetically, this would reflect deep homology in which the underlying genetic framework of the pathogens make it easy to shift from other plant hosts to Coffea or Vaccinium . The two plant genera with genes significantly correlated with them, Vitis and Coffea, had 179 and 20 whole genome sequences from diverse sampling regions. The larger clades of Proteales, Asterid, and Rosid were also used to look for convergent gene presence absence and again the two groups with the majority of samples, Asterid and Rosid had genes correlated with them, while Proteales did not. Unfortunately, out of these 23 genes, 20 are hypothetical proteins, the ones with known functions could have very interesting implications for host range. fitB_1 has been known to be involved in in-host migration and metal binding, similar genes are also frequently gained and lost in other Xanthomondaceae and are hypothesized to affect both gene regulation and resistance mechanisms . vhbT is an interbacterial effector protein, facilitating bacterial conjugation, another process with potential for large genomic and functional changes . Another significant gene contains a helix-turn-helix region, a DNA binding-domain that has been found to control metal resistance bacteria generally and biofilm growth in X. fastidiosa specifically . These genes should be explored further through fitness tests with the presence and absence of these non-essential accessory genes in multiple host environments to further evaluate if their presence and absence is adaptive or due to drift. Future research pertaining to host range should focus on both convergent gene gain and loss as well as the adaptive vertically descended genetics underlying host range. As both genomic assays have identified the pan-genome to be linked to host association, it would be beneficial to our understanding of host specificity to pursue this further. This study has identified a group of candidate genes associated with particular hosts, nursery pots and they can be tested in the lab to determine if they are significantly linked to fitness in their particular hosts.

The study has also identified Coffea as an especially relevant host in global plant trade in terms of spreading infection across borders and oceans. Using these data, we can start identifying patterns of likely host shifts that can help make decisions on when eradication and quarantine is necessary based on the historical likelihood of host shifts. However, we should also carry out further whole genome sequencing of strains outside of the classic agricultural settings. To truly understand abiological system, we not only need to understand the relevant biological components but also how they interact both inside and outside of agricultural landscapes.The first step in creating all phylogenies was building a nucleotide or gene alignment of the genomic regions of interest. Four alignments were created all using the same set of taxa : a core genome alignment, non-recombinant core alignment, a multilocus sequence type alignment, and a pan-genome alignment. The core genome was built with Roary to identify nucleotide regions shared by at least 99% of all taxa. We ran Roary with the parameters -s -ap to cluster paralogs and allow them in the core genome. The non-recombinant core alignment was based on the core genome, but recombinant sites identified with ClonalFrameML were removed from the alignment using an in-house R script . The MLST alignment was based on a nucleotide alignment of the 7 MLST housekeeping genes commonly used for X. fastidiosa with reference sequences acquired from the X. fastidiosa MLST database . We then searched each MLST reference sequence against all whole genomes using the Basic Local Alignment Search Tool at an E value of 10-3 in BLAST +, with a database created for each whole genome . We concatenated all MLST gene sequences for individual taxa and aligned them to all other taxa using MAFFT v. 7 . The pan-genome alignment was made using Roary’s gene presence-absence output by constructing a matrix of all genes as characters with binary presence or absence of that gene in a strain as the character state. As each character represented a known genetic region and there were no gaps in this matrix, no additional alignment algorithm was used. In total, this alignment contained 17,024 characters, representing the 17,024 total genes that make up the pan-genome of Xylella spp. sequences. The outgroup used for all trees was Xylella taiwanensis strain Wufong1 isolated in Taiwan in 2014 from Pyrus pyrifolia . We constructed four maximum likelihood phylogenies using RAxML v8.2.11 under a generalized time reversible model. Node support was measured with 1,000 bootstrap replicates . Trees were visualized in FigTree v1.4.4 and the Interactive Tree of Life . Phylogenetic diversity was calculated as the summation of total branch lengths for each phylogeny using the R package adephylo .To conduct ancestral state reconstructions, we used an extant distribution of characters , in this case, the genera of plants from which we isolated the bacteria. Using that distribution, we constructed the most likely history of hosts across the phylogeny at all internal nodes. We are in essence seeking parameter values that maximize the probability of the data given the hypothesis . Based on available data, the identity of the host plant from which each strain was isolated in the field is identifiable to at least the genus level. This value is used as a point proxy for the true state of inTherest – potential host range. Since host range must be experimentally determined, in this study we use the host each strain was isolated from as a point representative of an unknown range of susceptibility. Due to this, any subsequent results cannot infer specificity to a given host but imply the ability to infect said host. Because sampling is heavily biased towards symptomatic agricultural crops in the case of X. fastidiosa, we interpret each ancestral state as the most likely agriculturally relevant host that the pathogen would have been isolated from. All taxa were coded based on plant host genus and super order/order . This included two super orders , one order and 26 genera that were potential hosts for X. fastidiosa’s hypothetical ancestors at each internal node of the phylogenies.

Furthermore, there is substantial research illustrating that many ecosystem processes are a function of biodiversity levels and that ecosystem process change is often less marked when biodiversity levels are high, due to the potential functional redundancy among species and response diversity . Thus by maintaining high population-level genetic diversity and community-level species diversity, restorations can provide potential safeguards against future ecosystem alterations from climate and other environmental stressors. Furthermore, it is possible to take the mitigation strategies discussed above and adapt them to the specific situations predicted in global climate model change scenarios. One potential result based on these scenarios is a temporal or spatial shift in species flowering or foraging, which would be likely given that species exhibit varying responses to climate . As discussed in the previous section, one pollinator network study suggests that one way to make pollinator restoration habitats more resilient to future climate conditions is to extend the flowering season of existing restoration areas . While this study provides insight into how potential phenological changes could be incorporated into existing restoration plans, there are a vast number of restoration strategies that require further research in the context of climate change. Specifically, future work is needed to examine the role of nesting resources and flowering species density in altered climate conditions.While conservation biologists are often focused on taking measures to adapt existing management strategies to whatever climate plausibly could occur in the future or to create new strategies that are more resilient, plastic flower buckets wholesale the goal to develop policies that minimize all human climate forcing needs to be a high priority.

The next line of defense is to ensure that conservation management strategies, both existing and in planning, are designed with climate effects in mind. Many of the recommended strategies for making protected areas more resilient to climate are also those long recommended as best practices for conservation, such as ensuring connectivity between reserves, encompassing latitudinal and elevational gradients within reserves or reserve networks, creating buffer zones around reserves, and ensuring that land use practices in the matrix are favorable to biodiversity. Given the enormous existing challenges of implementing conservation action successfully , the concordance between recommended conservation measures for adapting to climate, including plausible changes in local and regional climatic conditions, and other environmental threats is indeed a welcome relief. This concordance also signals how important it is, in the face of climate risks, to enact a full suite of multiscale conservation measures on the ground to deal with the multiple, synergistic effects of interacting drivers of local and global extinction . Thus, rather than repeating this ground, we focus here on the maintenance and conservation of pollination function, rather than pollinators per se, and recommend five key focal points for policy and management, at local and regional scales.Hotter and drier climates globally, coupled with periodic drought, often necessitate large quantities of irrigation water to maintain visual quality, growth, and development of landscape plants . Approximately 60–90% of household water is used for urban landscape irrigation in the western United States . However, due to the increasing water demand of a growing population, designing landscapes with drought-tolerant adaptive plants or plants native to arid and semiarid areas is important for long term water conservation in the western United States.

In addition, landscape plants are threatened by increasingly common droughts and heatwaves in the western United States because they are largely reliant on irrigation . A recent drought caused urban vegetation coverage in downtown Santa Barbara, California, to decline from 45 to 35% . Hence, landscape plants characterized by morphological and physiological plasticity, which can better acclimate to water and heat stresses, are desirable for future landscapes. Unfortunately, drought responses of landscape plants are seldom investigated, and drought tolerance studies have largely been conducted based on local precipitation rates, rather than well-controlled inputs . Reduction in soil water availability causes cell dehydration, resulting in leaf wilting and degrading aesthetic appearance . Cell dehydration then prevents chlorophyll production and photosynthesis, which reduces leaf greenness and plant growth . For instance, Orthosiphon aristatus exhibited wilted leaves and reduced leaf and root biomass when no irrigation was applied . Water stress also inhibits leaf expansion, reducing light-capture area and may indirectly induce heat stress in plants because of reduced transpirational cooling to counter absorbed radiation . Gaillardia aristata and Penstemon barbatus , for example, showed over 50% of the leaves burned when water was limited . High temperatures may disrupt plant metabolism and protein stability, leading to leaf burn and necrosis . Plant acclimation involves changes in morphology and physiology without genetic modification . Under drought conditions, plants may acclimate to drought by decreasing water loss and reducing heat load and leaf temperature . Root growth may be promoted to increase water uptake, leading to a greater root-to-shoot ratio .

Water loss may be minimized via stomatal closure, leaf senescence, and reduced leaf size . For instance, Stromberg found that xeric species growing in the southern United States have greater root-to-shoot ratios, but smaller leaves, than mesic species. In hot and arid environments, plants gradually reduced their stomatal conductance and transpiration along with increasing leaf temperatures and higher leaf-to-air vapor pressure deficit to prevent excessive water loss . Minimizing stomatal conductance when solar radiation and air temperature are greatest at midday can protect plants from xylem dysfunction and maintain water status . Plant leaf temperature may be regulated by adjusted leaf size, orientation, and trichome density . For example, small leaves are advantageous for increasing sensible heat loss. The leaves of native plants in the western United States, such as Artemisia tridentata and Cercocarpus montanus , are less than 2.5 cm wide, helping to reduce plant heat load more efficiently . Leigh et al. reported that plants in hot and dry environments of Australia, such as Banksia grandis , Grevillea agrifolia , and Telopea speciosissima have leaves covered by dense trichomes and vertical leaf orientation, which reduces the interception of solar radiation. Trichome density has been found to be affected by soil water content, air temperature, and VPD . For instance, the trichome density of Lotus creticus increased when the amount of irrigation water decreased by 70% . Shibuya et al. discovered that Cucumis sativus had 255 trichomes per cm2 of leaf area at a vapor pressure deficit of 0.4 kPa which increased to 463 trichomes per cm2 at 3.8 kPa. Ehleringer observed that trichome density of Encelia farinosa grown in California positively correlated to the mean maximum air temperature of the growing habitat. However, the effect of water stress on plant trichome development has not been widely studied. Early research suggested leaf trichome production was promoted under water deficit . However, this finding contradicts the fact that plant cell division is inhibited under drought stress conditions . Brodribb et al. reported that changes in cell size provided a substantial means to modify leaf function without disturbing other tissue/organ functions. Murphy et al. found that epidermal cell expansion facilitated the decrease of stomatal density under shade, where large leaves had low stomatal density. Stomata and trichomes are both epidermal appendages and their development occurs prior to cell expansion. Hence, changes in cell size may modify trichome density under water stress. Shepherdia ×utahensis ‘Torrey’ is an interspecific hybrid between Shepherdia argentea and Shepherdia roundifolia . Shepherdia argentea tolerates a wide range of growing conditions from wet to dry soil , while S. roundifolia is extremely resistant to hot and arid conditions . Xeric S. roundifolia has denser leaf trichomes as compared to riparian S. argentea , which indicates trichome density of Shepherdia species may be influenced by water availability. Shepherdia ×utahensis has leaf trichomes and grows well in a variety of substrates . However, the effects of soil moisture level on trichome density have rarely been investigated. The hypotheses of this research are the morphology and physiology ofS. ×utahensis change at different substrate water contents, and leaf trichome density is affected by cell size under drought. To test these hypotheses, black flower buckets the objectives of this research were to evaluate the morphological and physiological responses of S. ×utahensis under various substrate volumetric water contents in a greenhouse and to quantify the relationship between trichome density and water deficit.On 12 January 2021, images of the upper surface of leaves of plants at substrate volumetric water content of 0.10 and 0.40m−3 ·m−3 were recorded using a dissecting microscope before plants were destructively harvested.

Three plants at substrate volumetric water content of 0.10, 0.20, 0.30, or 0.40m3 ·m−3 were randomly chosen and three mature leaves were sampled from the third to fifth nodes counting downward from the tip of the main shoot of each plant. Leaf size was also recorded. Leaves were stored in Petri dishes containing wet germination paper. A disk from each leaf was sampled using a #12 cork borer with an area of 7cm2 to study the leaf reflectance using a spectroradiometer . The mean reflectance of photosynthetically active radiation was calculated using the wavelengths from 400 to 700nm. The reflectance of blue, green, and red light was calculated using the wavelengths of 450, 530, and 660nm, respectively . Following leaf reflectance measurements, leaf disks were immediately sent to the USU Microscopy Core Facility . A sample was collected from each leaf disk using a hole punch . Nine fields of view at ×300 magnification were photographed from the upper surface of each leaf punch using an environmental SEM . Fine-scale morphological traits were determined following the method of Murphy et al. . Trichome density , uncovered stomata , trichome radius , trichome coverage fraction, epidermal cell size , and epidermal cell density were quantified in each field of view using ImageJ . The values of fine-scale morphological traits from the nine fields of view were averaged for each leaf, and the mean value of three leaves was recorded for each plant. The numbers of epidermal cells and trichomes per leaf were calculated using the density and leaf size, and the ratio between trichomes and epidermal cells of each leaf was determined.The experiment was arranged in a randomized complete block design with eight treatments and three blocks. A mixed model analysis was performed to test the effects of substrate volumetric water contents on all measured parameters. Trend analyses were conducted for all data to test the nature of the relationship between plant responses and substrate volumetric water contents. Correlation analyses were performed to study the relationships between trichome density and leaf size, epidermal cell size, epidermal cell density, or light reflectance; between leaf size and epidermal cell size or epidermal cell density; and between stem water potential and epidermal cell size. All statistical analyses were performed using PROC MIXED or PROC REG procedure in SAS Studio 3.8 with a significance level specified at 0.05.Plant morphology and physiology in this study changed along with decreasing substrate matric potential that resulted from reduced substrate volumetric water contents . As substrate volumetric water content decreased, S. ×utahensis leaves and stems dehydrated, and the proportion of visibly wilted leaves increased . In addition, plant growth indices, relative chlorophyll content , numbers of shoots and leaves, total leaf area and dry weight, and photosynthesis were impaired . These results are in line with previous studies that reported negative effects of water stress on aesthetic appearance, plant growth, and net assimilation rate of ornamental plants . In this case, decreased stem water potential is best interpreted as a passive response resulting from the effects of decreased soil water potential and higher leaf evaporative demand . Similarly, Rosa ×hybrid and Nerium oleander decreased stem water potential in response to low substrate or soil water potential under drought conditions . Decreased substrate volumetric water contents also inhibited nodule formation in S. ×utahensis , which suggested that infection of symbiotic actinobacteria was affected by water availability. Actinobacteria move with water in the soil, and the process of reaching and infecting the roots of host plants slows down when soil water content decreases . Plant morphological and physiological acclimations were observed in this study. In response to drought, S. ×utahensis reduced midday stomatal conductance to a value close to 0 when substrate volumetric water content decreased . Midday stomatal conductance is positively correlated to stomatal opening and plant water status .

The harvest period for ‘Star’ began the first week of May and ended after the third harvest. Most other cultivars required five or more harvests, 1 week apart. Based on the berry size , the cultivars studied would be separated into large berry and medium berry . The cultivars studied have an erect plant stature, except for ‘Misty’, which has a spreading stature that makes hand-harvest difficult. Fruit quality. Quality attributes such as soluble solids concentration, titratable acidity, soluble-solids-totitratable-acidity ratio and firmness were significantly different among cultivars and seasons . There was wide variability in soluble solids concentration among cultivars. ‘Reveille’ had the highest average value of the 2005 to 2007 seasons, followed by ‘Misty’ , ‘Emerald’ and ‘Star’ . ‘Jewel’ and ‘O’Neal’ had the lowest soluble solids concentration within this group. Titratable acidity within cultivars was less variable, and only ‘O’Neal’ had a significantly lower average value than the rest of the tested cultivars. Titratable acidity varied from 0.70% to 0.80% within this group with the exception of ‘O’Neal’. Cultivars segregated into three groups based on their soluble-solids-to-titratable-acidityratio. Because of its low titratable acidity, ‘O’Neal’ had the highest ratio, while ‘Jewel’ had the lowest ratio due to its high titratable acidity. The rest of the cultivars formed an intermediate group in which the soluble-solids-to-titratableacidity ratio ranged from 17 to 20.3. ‘Jewel’ and ‘O’Neal’ also had the lowest firmness , while ‘Reveille’ and ‘Misty’ had the highest . ‘Emerald’ and ‘Star’ were significantly different than these two groups, forming an intermediate group . Quality attributes were also significantly affected by the season. Soluble solids concentration across all cultivars was highest in 2007 and lowest in 2006, procona valencia buckets while titratable acidity was highest in 2006. Soluble-solids-to-titratableacidity ratio and firmness were significantly higher in 2007 than the other years. There was a significant interaction between cultivar and season for all these quality attributes .

The lowest soluble solids concentration was 10.8% in 2006 for ‘O’Neal’ and the highest was 15.8% for ‘Reveille’ in 2007. During this 3-year period, all of the cultivars yielded soluble solids concentrations higher than 10%, which has been proposed as a minimum quality index for blueberries . Titratable acidity was similar among cultivars in these three seasons except for ‘O’Neal’ in 2007, which reached 0.3%, and ‘Jewel’ and ‘Emerald’ in 2006 with about 1.0%. ‘O’Neal’ and ‘Reveille’ had the highest soluble-solids-to-titratableacidity ratio, followed by the rest of the cultivars with ratios from 11.4 to 20.6. During this 3-year period, ‘Jewel’ and ‘O’Neal’ were the softest cultivars, and ‘Misty’ and ‘Reveille’ the firmest. Antioxidant capacity was significantly different among the cultivars but not between seasons . There was a wide variability of TEAC within cultivars. ‘Misty’ had the highest average TEAC followed by ‘Reveille’ and ‘Emerald’ . ‘Star’ , ‘O’Neal’ and ‘Jewel’ had the lowest TEAC within this group. Like the rest of the quality attributes, there was a significant interaction between cultivars and seasons for antioxidant capacity . Storage of the six blueberry cultivars at 32°F for 15 days did not affect either antioxidant capacity or firmness, except for ‘O’Neal’ and ‘Misty’, whose firmness was reduced slightly but not significantly . Consumer acceptance. During the 2006 season, our in-store test results indicated that consumers liked the three tested cultivars slightly to moderately, with an acceptance range of 73.3% to 80%. There were no significant differences in degree of liking between ’Jewel’, ‘O’Neal’ and ‘Star’. In these three cultivars the percentage of consumers disliking these fruit reached about 17% . During the 2007 season, there were significant differences in degree of liking between the six cultivars tested . In this test, degree of liking varied from liking slightly to moderately. ‘Reveille’ had the highest and ‘O’Neal’ the lowest degree of liking with an acceptance of 92.1% and 67.3%, respectively. Degree of liking of ‘Misty’ and ‘Jewel’ was significantly lower than ‘Reveille’, but higher than ‘Star’ and ‘Emerald’. Acceptance was near 80% for ‘Jewel’, ‘Misty’ and ‘Star’, while only 67% for ‘O’Neal’ and 72% for ‘Emerald’. The percentage of consumers that disliked these cultivars varied from 5.9% to 19.8%; ‘Reveille’ and ‘Misty’ had the lowest dislike percentage and ‘O’Neal’ the highest. Degree of liking for ‘Jewel’ and ‘Star’ were similar during the two seasons. For ‘O’Neal’, the degree of liking decreased from like slightly-moderately to like slightly.

This reduction in consumer acceptance can be explained by the change of titratable acidity from 0.6% to 0.8% in previous years down to 0.3% in 2007 that only occurred in ‘O’Neal’. This reduction of titratable acidity for ‘O’Neil’ was independent of soluble solids concentration, which remained between 10.8% and 11.8% for the 2005 to 2007 seasons. These results indicated that blueberries with very low titratable acidity , despite soluble solids concentrations between 10% and 12%, are not acceptable to consumers. A similar situation has been observed in white and yellow flesh peaches and nectarines with very low acidity . This reduction in consumer acceptance also points out that the ratio of soluble solids to titratable acidity is not a good indicator for blueberry taste when titratable acidity is low. We are not sure of the reasons for the low titratable acidity in 2007 of ‘O’Neal’ fruit, which appears to be independent of other cultivars. The 2007 season was characterized by high chilling accumulation and a hotter than normal spring, which could have affected ‘O’Neal’ ripening. Choosing a variety The six southern highbush blueberry cultivars studied growing in the San Joaquin Valley had soluble solids concentration levels above the 10% proposed for a minimum quality standard. Blueberries with very low titratable acidity, despite acceptable soluble solids concentration, had lower consumer acceptance and degree of liking, indicating that the solublesolids-to-titratable-acidity ratio is not a good indicator of consumer acceptance for blueberries. For San Joaquin Valley conditions, these cultivars are all good options for our fast-growing, early fresh blueberry market.Agricultural managed aquifer recharge is a recharge technique for groundwater replenishment, in which farmland is flooded during the winter using excess surface water in order to recharge the underlying aquifer . In California, for example, Ag-MAR is currently being implemented as part of the efforts to mitigate California’s chronic groundwater overdraft . Ag-MAR poses several risks for agricultural fields and groundwater that may influence its future adoption. This includes crop tolerance to flooding, soil aeration, biogeochemical transformations, long-term impact on soil texture, leaching of pesticides and fertilizers to groundwater, and potential greenhouse gas emissions. Some of these issues have been addressed in recent studies of Ag-MAR, including soil suitability guidelines , nitrate leaching to groundwater , crop suitability and soil aeration . In the current study, we focused solely on the question of “how long can water be applied for Ag-MAR with minimal crop damage?”, while ignoring some of the above-mentioned challenges involving Ag-MAR implementation. Preferably, Ag-MAR flooding is done during fallow or dormant periods, when crop damage is potentially minimal, so agricultural lands can serve as spreading basins for groundwater recharge.

Root zone residence time is defined as the duration that the root-zone can remain saturated during Ag-MAR without crop damage . RZRT is a crucial factor in Ag-MAR, as long periods of saturated conditions in the root-zone can damage crops due to oxygen deficiency or complete depletion of oxygen, which ultimately may result in yield loss . However, flood tolerance among crops varies considerably due to biotic and abiotic conditions , therefore only appropriate crops under specific conditions may be suitable for Ag-MAR application. For example, Dokoozlian et al. have found that grapevine during dormancy can be flooded for 32 days each year without yield loss. Dahlke et al. recently investigated the effect of different Ag-MAR flooding schemes on established alfalfa fields. Results suggest a minimal effect on yield when dormant alfalfa fields on highly permeable soils are subject to winter flooding. On the other hand, some crops are sensitive even to short-period flooding. Kiwi vines for example, are highly sensitive to root anoxia with reported yield lost and vines death due to extreme rainfalls and/or shallow groundwater levels . In a study on peach trees, flood cycles of 12 h per day with 5 cm ponding, applied for two months, resulted in branches with lower diameter and length growth, as well as smaller, low-quality, fruits, compared to the control trees . The above examples demonstrate the need for an RZRT planning tool that can estimate Ag-MAR flood duration with minimal crop damage. Usually, when Ag-MAR water application starts, aeration of the rootzone will be quickly suppressed by a water-layer covering the soil surface, as it prevents oxygen transport to the root-zone in the gas phase. When water application ceases, re-aeration of the root-zone will depend on the soil’s drainage rate that controls the formation of connected air pores between the root-zone and atmosphere . Hence, proper estimation of the planned flood duration during Ag-MAR requires prior knowledge of both crop characteristics and soil texture. Only a few attempts for estimating RZRT during Ag-MAR were made, as Ag-MAR is a relatively new MAR technique. O’Geen et al. used a fuzzy logic approach to rate the RZRT during Ag-MAR, procona buckets based on the harmonic mean of the saturated hydraulic conductivity of all soil horizons, soil drainage class, and shrink-swell properties. Their RZRT rating was combined with other factors generating a Soil Agricultural Groundwater Banking Index . Flores-Lopez et al. proposed a root-zone model that includes crop type, soil properties, and recharge suitability to estimate water application, flooding duration, and the interval between water applications. Their model was integrated with a Groundwater Recharge Assessment Tool to optimize Ag-MAR water application. Here, we propose a simple model to estimate the planned water application during Ag-MAR based on the following parameters: soil texture; crop saturation tolerance; effective root-zone depth; and critical water content. The concept of critical water content was proposed by several authors as it indicates a percolation threshold where the gas transport path is blocked by pore-water, which results in gas diffusivity and permeability of practically zero. Hence, when the water content is either below or above this threshold, gaseous oxygen transport into the soil is blocked or opened, respectively . As opposed to the previous Ag-MAR models mentioned above, our proposed model is physically based and includes explicitly the soil water content, that is used to infer the soil aeration status. Yet, thanks to its simplicity, this model can be integrated easily into various existing Ag-MAR assessment tools such as SAGBI or GRAT . In the following, we first describe the theory of the model and the methods used to test the model performance. Next, we present the model predictions and compare them with observations and numerical simulations. Last, we present an example of how to calculate Ag-MAR water application duration and we discuss the applicability of the model and its limitations.The potential for warmer temperatures to expand pathogen ranges and alter epidemiology is an important consequence of global climate change for human populations and the environment . Plant pathogens influence large-scale forest mortality events, so understanding their future range and impacts will assist conservation planning . Plant pathogens also impact agricultural production, meaning their response to climate change threatens global and regional food security . Pathogens are sensitive to multiple climatic and environmental factors, as reflected in the ‘disease triangle’ , a conceptual model that states that disease is the outcome of the presence of a virulent pathogen, a susceptible host, and suitable environmental conditions. Theoretical and empirical studies addressing climate change impacts on plant disease tend to focus on individual environmental factors such as temperature , elevated atmospheric carbon dioxide concentrations and water availability , despite the likelihood that climate change will alter temperature, precipitation, potential evaporation, and ecological regimes simultaneously. Retrospective analyses show that multiple environmental drivers and their interactions influence expansion of disease ranges . Understanding these interactions remains an area of outstanding research need . Climatic and edaphic factors could limit Phytophthora cinnamomi range in several ways. First, Pc is sensitive to cold temperatures . Temperatures warm enough to permit survival of Pc may still be cold enough to suppress its ability togrow, reproduce, and cause disease to hosts . Previous modeling studies considering temperature effects on Pc range in Europe suggest the potential for considerable expansion in warming climates . However, Pc growth rates also display a threshold-like response to soil moisture in laboratory conditions.

However, at Bruntland Burn cryogenic and equilibration methods gave similar results for peaty soils, and reasonable agreement with xylem water. Extraction focusing on small-scale moisture isotope dynamics at the root – soil interface may be needed, including scalable methods to explore the phase change/mycorrhizal mechanisms suggested above. Our findings, based on bulk soil field measurements, underline the major difficulties associated with relating potential water sources to plant water stable isotope compositions. Even under controlled laboratory conditions, Orlowski et al. could not confidently link relate the soil water to root crown isotopic compositions, but reported similar 2 H depletion as we found in Dandelions growing on sandy soils.Differences between angiosperms vs gymnosperms: A clear finding of our study is that the extracted xylem waters of angiosperms and gymnosperms have a very different isotopic composition at most sites, with gymnosperms generally showing a greater degree of fractionation. In this regard, several hypotheses could be tested. Firstly, root networks and root-mycorrhizal networks of different species may be able to access different pore sizes. For example, gymnosperms may have greater potential to mobilize water that has undergone some fractionation during the interactions among water, gas, and solid phases of the soil. Secondly, storage and mixing of water within plant tissues may be greater in softwood gymnosperms, as suggested in recent modelling work . The generally slower metabolism and transpiration rates for gymnosperms might exacerbate this mechanism. InTherestingly, Amin et al. showed little difference between angiosperms and gymnosperm xylem waters for cold and temperate environments in their meta-analysis, plastic planters wholesale whereas angiosperms in arid regions were offset in δ2 H compared to gymnosperms.

We sampled xylem water in conjunction with soil water at five well-instrumented sites across northern cold landscapes. At all sites except Krycklan, water sources of angiosperms could be associated with soil water. At all sites except Dry Creek, the sources of water uptake by gymnosperms were much less easily explained. Whereas the isotopic composition of xylem water for angiosperms generally overlapped that of soil water for a range of antecedent periods, overlap did not occur for gymnosperms . This suggests that the xylem water of angiosperms was influenced by the isotopic composition of water retained in the soil weeks or months prior to plant sampling, whereas gymnosperms generally did not exhibit such a memory effect. The isotopic offset between soil and xylem samples was generally greatest during the growing season for the wetter sites . However, at the drier two sites xylem and soil water isotopes tended to be similar, showing the effects of evaporation. We attribute this dry site anomaly to the relatively rare occurrence of mobile water during the growing season. There simply are not many choices of water sources form plants in dry areas, so soil water and xylem water trend towards similarity, and typically have a strong evaporation signal. Our study also raised questions that will need to be addressed in future research: Which biophysical processes at the root – soil interface contribute to isotopic fractionation in uptake that affects the composition of xylem water? What are the internal dynamics of water storage, mixing and release within vegetation and how does this relate to the degree of synchronicity between phenology and soil water availability? What reservoirs are sampled during cryogenic extraction – only xylem water or does this include water from other plant cells? And finally, why are angiosperms and gymnosperms at the same sites so isotopically different? Addressing some or all of these questions will contribute to our understanding of soil-plant-atmosphere interactions in northern landscapes.Understanding the mechanisms of adaptive evolution in pathogenic bacteria is central to long-term disease control.

One major focus of research into adaptive bacterial evolution has been lateral gene transfer , usually defined as the transfer of genes across species boundaries . Until recently, discussions of LGT focused on the transfer of novel genes, as exemplified by the discovery of the plasmid-mediated transfer across species of the genes coding for penicillin resistance ; however, with the increasing availability of genomic sequence data, it has become apparent that the transfer of homologous gene copies is also widespread . These two kinds of exchange, the transfer of novel genes or novel alleles, are fundamentally different. The acquisition of novel genes can result in the acquisition of a completely new trait that has already been refined in other taxa by natural selection . It can determine critical traits such as virulence, antibiotic resistance, and ecological niche , even though most of the material transferred appears to be evolutionarily transient . In contrast the acquisition of novel alleles is analogous to the effect of sexual reproduction in eukaryotes: it increases the genetic variance that natural selection can act on but does not, in itself, result in a qualitative change in the ecology of the recipient . Due to these fundamental differences, we favor reserving the term “LGT” for the transfer of novel genes, using the term “interspecific” or “intersubspecific homologous recombination” for the transfer of alleles; however, both processes, if successful, lead to genetic “introgression,” a term commonly used to describe the spread of genetic material across taxonomic boundaries in plants and animals and now increasingly used to describe the analogous process in bacteria . Homologous recombination is almost ubiquitous among bacteria, although the degree to which it occurs varies widely among species . It involves the replacement of a stretch of DNA sequence in one individual’s genome by a homologous sequence from another individual of the same species following any of the 3 mechanisms of DNA transfer . It typically involves short pieces of DNA . Given the prevalence of homologous recombination, it is generally assumed that it is beneficial, in some cases enabling bacteria to enhance their resistance to antibiotics and avoid host defenses or perhaps promoting adaptation to novel environments . Analogy with the assumed benefits maintaining sexual recombination in metazoans strongly supports this view. Documenting the adaptive benefit of homologous recombination in bacteria has proved difficult. This is to be expected even if the benefits are large and common. Homologous recombination typically falls off rapidly with genetic distance , so a well-established population will usually reflect the mixing of relatively similar alleles. This mixing can be easily detected by the lack of clonality between genes and quantified using evolutionary models ; however, detection of recombination breaks within genes is more problematic. The approaches currently used have very limited power; although the introgression test has improved this situation . Another approach is to test loci sequenced from 2 or more taxa and use the genetic partitioning program STRUCTURE . Alleles that cannot be confidently allocated to one or more of the taxa are likely to be mosaics generated by recombination . To link recombination to adaptive change, it is useful to study a system in which recombination is limited, recognizable, plastic plant pot and likely to lead to novel adaptation. Arnold et al. recently made an inTheresting link between the acquisition of novel adaptations in bacteria via LGT and that via hybridization in metazoans. Excellent examples of how interspecific introgression can result in adaptation to new environments in higher plants are given in the work of Rieseberg and colleagues on the effects of introgression in sunflower species . However, it is not only metazoans that hybridize: bacterial homologous recombination can sometimes result in interspecific introgression . Interspecific hybridization of this kind is likely to be relatively rare, suggesting that the ideal study system is one with a significant frequency of homologous recombination between well-defined groups within a species . This level of study appears most likely to provide valuable insights into recombination-related adaptive change in pathogens. For example, Didelot et al. showed that two human-pathogenic forms of Salmonella enterica are relatively dissimilar across about 75% of their genomes but show marked convergence across the rest.

They concluded that this similarity reflects adaptation to the human host, driven by homologous recombination and selection. Similarly, Sheppard et al. proposed that human activity has probably led to an increase in recombination between Campylobacter jejuni and Campylobacter coli and may have also created novel environments that have favored the evolution of hybrids. Another species in which homologous recombination between closely related but distinct taxa has been documented is the plantpathogenic bacterium Xylella fastidiosa . X. fastidiosa is a xylem-limited bacterium that is transmitted by xylem-feeding insects, typically leafhoppers, and is divided into four subspecies: fastidiosa, sandyi, multiplex, and pauca . These subspecies have diverged genetically by 1 to 3%, apparently due to their geographical isolation over about the last 20,000 to 50,000 years . This isolation has now broken down, due presumably to human activity . The cooccurrence of the previously allopatric subspecies has resulted in intersubspecific homologous recombination , recombination that can be detected relatively easily due to the preexisting genetic divergence of the subspecies . Consistent with these observations, recent experimental work has confirmed that X. fastidiosa is transformationally competent and that some isolates carry a conjugative plasmid . X. fastidiosa is known to infect a wide range of hosts, causing scorch and dwarfing diseases . In citrus, it causes citrus variegated chlorosis , a disease restricted to South America, and in grapevines in the United States and Central America, it causes Pierce’s disease. In the United States, it also causes disease in almond, apricot, plum, peach, alfalfa, pecan, and blueberry. However, individual X. fastidiosa strains are not generalists. The different subspecies infect a characteristic and largely nonoverlapping range of plant hosts, and even within subspecies, different genotypes show differences in host specificity . For example, in the South American X. fastidiosa subsp. pauca, citrus isolates do not typically grow in coffee and vice versa , and in X. fastidiosa subsp. multiplex, Nunney et al. found associations between the genotype and host plant. In their study of X. fastidiosa subsp. multiplex, Nunney et al. used the multilocus sequence typing protocol of Yuan et al. to categorize 143 isolates. The MLST protocol is valuable for gaining insight into the evolutionary history and genetic diversity of taxa . MLST groups isolates into sequence types , where each ST defines a unique set of alleles across the loci used . Based on 8 loci, 31 of these isolates were identified as IHR forms , and 2 isolates were considered “intermediate” , while the remaining 110 non-IHR isolates showed no evidence of introgression. The IHR and intermediate types together were considered to define the “recombinant” group of X. fastidiosa subsp. multiplex isolates . Most were observed more than once, and 5 were found in two different U.S. states or districts . The analysis of Nunney et al. was focused on the evolution and host range ofX. fastidiosa subsp. multiplex. For this purpose, it was necessary to identify and exclude isolates whose recent evolution was influenced by intersubspecific recombination. As such, once the 23 non-IHR STswere identified, there was no further analysis of the remaining recombinant group STs. In particular, no evidence was presented for classifying some alleles as atypical of X. fastidiosa subsp. multiplex beyond the observation that they were never found in the non-IHR group . Nunney et al. did observe one intriguing pattern when they compared their results to those of Parker et al. . Of the 143 isolates, 13 were also used in the study by Parker et al. , in which typing was based on a different set of 9 loci. Unexpectedly, these 13 isolates maintained the same grouping with the IHR and non-IHR types corresponding, respectively, to the clade A and clade B groupings . This highly statistically significant concordance strongly suggested that IHR is not distributed randomly across all X. fastidiosa subsp. multiplex isolates but instead is restricted to a small subset, while the remainder is little influenced by IHR. However, Parker et al. failed to find evidence of intersubspecific recombination within any of the X. fastidiosa subsp. multiplex isolates, despite applying a series of 9 tests designed to detect recombination contained within the RDP4 program and the PHI program . This result presented a strong argument against our hypothesis that clade A members cluster because they are recombinant types carrying alleles derived from IHR . Here we reexamined the sequence data obtained in their study by using the more sensitive introgression test to determine if their tests missed evidence of IHR and, if so, whether it was confined to clade A.

We compiled and analysed a dataset of 17 functional traits with a sufficient number of records in the TRY database to characterize the main ecoregions of the world , that is, environmentally homogeneous areas with distinct biota . The dataset is based on 225,206 georeferenced observations comprising records of 20,655 species. The trait data were complemented with 21 climate variables and 107 soil variables . Trait–environment relationships were analysed for species medians aggregated to ecoregions using ridge regression , a robust method suitable to deal with high-dimensional, unbalanced and collinear predictors in combination with hierarchical partitioning .The rationale is that species presence indicates how the trait space can be realized in a given environment. Spatial aggregation is a suitable means to increase the detectability of global trait patterns , as described in earlier studies, where traits have been binned by temperature classes or for different altitudinal ranges . Extreme outliers, for instance towering trees such as the Californian Sequoia , may still exist far away from the equator, where precipitation is sufficiently high but their influence is outweighed in our approach by an increasing fraction of small-statured herbaceous species from tropical to temperate and boreal regions.To understand whether the axes of variation identified for the grouping of six traits also hold for the extended set of 17 traits, we cluster their trait–trait correlations and further represent these relations on the basis of their principal components . This analysis supports the clear distinction of size versus economics traits identified by Díaz and colleagues . The group of size traits contains two subclusters. The first includes height and seed size traits: plant height , seed mass, 10 plastic plant pots seed length and dispersal unit length . The second subset contains traits that are linked through plant hydraulic scaling relationships and contrasts high conduit density with high leaf area and leaf fresh mass .

Economics traits represent dry mass and nutrient investments in plant tissues, and the rate and duration of returns on those investments . They are represented by leaf nitrogen content per leaf area , leaf nitrogen , phosphorus and carbon content per dry mass, leaf N to P ratio and SLA. This study shows that the proposed global spectrum of plant form and function fits well to a substantially extended trait space compared to the original study , with seven traits that capture the whole-plant size spectrum and seven traits that capture the leaf economic spectrum and only three traits that do not fall along these dimensions . One explanation could be that the varying fraction of woody and non-woody species would drive these patterns. However, we showed that these two main trait groups remain clearly identifiable when the analysis is conducted separately, yet with fewer samples, for woody and non-woody species . However, we cannot discard the possibility that additional traits may add relevant axes of trait variation. For example, our study does not include carbon fixation rates or fire adaptation traits , nor does it include any root traits—representing an essential gap to be filled at the global scale . If such data were available they would have the potential to fundamentally change our perception of global plant form and function, and their relation to ecosystem functioning. Variation in size traits, represented by PC1 in Fig. 1b, shows a clear latitudinal gradient . In contrast, variation in economics traits does not show a latitudinal trend. Only a dip is apparent at around 35° , in addition to a decrease at high latitudes above 60° where available data become increasingly limited. However, comparison to a recent arctic dataset indicates that this decrease in variation at high latitudes reflects available observations . These patterns might represent a response to nutrient limitation and drought in water-scarce and nutrient-scarce deserts and Mediterranean regions or boreal and arctic areas characterized by short growing periods slowing down mineralization.

The dip at ~35° indeed can be related to low water availability . At high latitudes, cold winters and short growing seasons constrain plant height and require on average more conservative nutrient-use strategies and protection against frost damage than the global mean, despite the high functional diversity in economics traits observed at these latitudes . Additional datasets may shed more light on specific conditions, for example see Bjorkman et al. . Future studies should quantify how individual stressors, for example radiative stress or water stress, relate to global patterns of trait variation. The climate and soil factors used in this analysis explain up to 77% of observed trait variation—a high fraction given that trait variation is widely known to be determined also by other factors such as biotic interactions and anthropogenic effects or disturbances and local effects such as those of microclimate . Recent findings on how different trait groups vary with the environment indicate that size and economics traits vary differently and in particular respond differently to climate and soil . Our analyses reveal a dominant joint effect of climate and soil drivers on trait variation—as already suggested by a number of earlier studies but not yet quantified globally. The orthogonality of the two main dimensions of plant trait variation suggests that different aspects of climate and soil variables are relevant to explain plant trait patterns at the global scale . While latitude-related variables explain size traits, variables that share less explanatory power with latitude explain economics traits . The RDA presented in Fig. 4 provides some insight on the nature of these climate–soil interactions. The first RDA axis, which describes variation in size traits, resembles a latitudinal gradient. On one extreme end, ample water supply from high and frequent precipitation, abundant water vapour and constant rates of high solar radiation meet the fundamental requirements of plant physiology—water, sunlight and warm temperatures.

Additionally, these conditions promote weathering of soil minerals but also microbial activity, contributing to fast turnover rates of organic matter supporting nutrient provisioning ; in brief, they represent conditions that allow plants to grow fast and tall in the race for light. Large vessels supporting large leaves promote high rates of water transport and thus growth, which is only possible because of the small risk of embolism under these benign water conditions . The high carbon gains can be invested in large fruits and seeds . Further along this gradient, the above-mentioned plant requirements become limited: water supply and temperatures are reduced and slow metabolic rates above ground and below ground. In ecoregions of the boreal and desert biomes, conduit diameter is constrained by the risk of cavitation during freeze–thaw cycles and water scarcity, amplified by little water holding capacity of gravel-rich soils. Our analysis thus indicates that size traits appear to be related to a latitudinal gradient of climatic favorability for plant growth determined by water and light availability. Important correlates of water and nutrient availability are associated with the second RDA axis, describing variation in economics traits. Traits associated with an acquisitive strategy are related to indicators of soil fertility, most importantly silt and organic matter concentration as well as pH . Soil pH is intermediate between the two axes, as might be expected given that pH reflects both broad-scale climate variation and a variety of processes related to nutrient availability and soil microbial communities . Silt forms the substrate of our most fertile soils as its structure is able to retain water against gravitation but renders it accessible to plants under drought conditions . The high fertility is associated with a high concentration of organic matter, which has a high cation exchange capacity especially under high pH . On the opposite end of the gradient, sandy soils require adaptations to both water and nutrient limitation. The trait configuration at the conservative end of the economics traits represents an adaptation to both . Various processes exist that lead to variation in the soil characteristics underlying the second RDA axis independent of latitude —for example, sandstone as a geological substrate giving rise to sandy soils exists from the tropics to the arctic . However, different climate variables related to solar radiation, temperature and precipitation, plastic pots large which influence long and short-term soil development processes directly and indirectly via soil biology , are related to this axis. Variation in economic traits is most probably the evolutionary response to exploiting this partly climate-independent edaphic niche axis. Size traits are on average explained better than economics traits by the environmental variables considered in this study. The lower fraction of explained variance for economics traits could have several causes. Firstly, data on soil factors that are likely to be very important, such as soil nitrogen and phosphorus availability , are not yet available at a global scale. Secondly, economics traits show relatively more within-site variation than across-site variation in comparison to size traits , probably because economics traits vary more than size traits within one plant; for example, leaf N per area and SLA vary with age and light availability . Thirdly, soil heterogeneity within ecoregions—both abiotic and biotic—may weaken the relationship between economics traits and environmental variables .

Reasons for small-scale soil variation are, for example, topography, soil age and thus fertility but also abundance of microbial communities and mycorrhiza that interact with climate, pH, soil properties and also plant traits . Trait–environment relationships due to smaller scale variation require well-resolved soil data. However, we note that soil physics and chemistry explain a large portion of variance along the trait PC axis three ; Supplementary Figs. 5, 6 and 38). We expect that with improved soil datasets and a higher resolution, the joint control of climate and soil on trait variation will probably appear even stronger and more evenly distributed between the two groups of driver variables. Our analysis can serve as reference for model developments that increasingly consider plant functional traits as part of vegetation dynamics under climate change . Individual plants and their trait syndromes are considered to be viable only within specific environmental conditions2 . Therefore trait–environment relationships should be scale-independent. However, different plant strategies can be successful under given environmental conditions, which in addition are often confounded by small-scale variation. In analyses to date, trait–environment relationships become more apparent for aggregations higher than the community scale , where most of the small-scale variation is averaged out. In addition the difference between potential and actual vegetation is suggested to explain some of this gap . Dynamic global vegetation models predict individual plant processes well but fail to produce reliable forecasts with a changing environment . Deciphering at which spatial and temporal scale, or conditions, actual vegetation is representative of potential vegetation may advance our understanding of community assembly and necessary model complexity. Yet, the ubiquitous importance of climate variables for explaining current differences in trait expression at ecoregion scale, suggests that trait shifts will occur with climate change. Trait shifts are constrained by available trait combinations in addition to other constraints such as species dispersal. For example, our results indicate that plant size increases with temperature so long as sufficient water is available , in line with the finding that species become larger and large species are more prevalent at warmer and wetter sites in the tundra . Global change is also reflected by soil degradation. Changes in soil parameters can be considered to also correspond with trait shifts, especially for economics traits. Human-induced soil degradation has many facets: often fertile topsoil is lost or toxic substances accumulate; rooting is impeded and alThered by artificial fertilizers; while soil formation takes millenia . The trait shifts may thus be similarly complex and depend on the extent and type of soil degradation. For example, in areas of wind and water erosion, species that tolerate lower nutrient availability may be more successful and this may be reflected in lower leaf nutrient contents . The fertilization of nutrient-poor grasslands, for example resulting from agricultural run-off, may shift these areas from more conservative to more competitive species with higher leaf nutrient contents. Plants as a whole need to balance both size and economics traits. To sustain human livelihoods, it may be important to understand the local expression of trait shifts and their global consequences for biodiversity when viable trait combinations change. In conclusion, the insights extracted here advance our understanding of broad-scale plant functional patterns.

Unfortunately, low-cost sequencing was unavailable at that time, so the study required post-preservation plating of organisms to distinguish, which severely limited the number of organisms that could be studied. With such little information for comparison, it is difficult for us to infer whether the patterns observed in the preservation experiments originated simply from the differential responses of oral microbes or from some mutually beneficial relationships among these microbes. Our plan to investigate composition-based differential responses to preservation will certainly help discriminate between these two sources, but we may need to supplement our planned approach with experiments on the viability of single organisms.Figure 32 outlines the experimental scheme for this phase. All organisms were purchased commercially from either ATCC or DMSZ as sealed plates or pellets in sealed ampules. Compositions of the inocula were designed based on the results from the preservation experiments, where we observed a dichotomy between the Veillonella and Streptococcus taxa and a simultaneous possible affiliation between the Veillonella and Prevotella taxa. Controls 1 and 2 represent the two compositional boundaries, one with a high abundance of the Veillonella dispar and the other with a high abundance of Streptococcus salivarius. The other four compositions were constructed as a gradient between the two boundaries, blueberry container size with the proportion of Streptococcus oralis held constant so that the effect of Prevotella salivae on the community can be studied.

These six compositionally defined microcosms will be anaerobically incubated for 72 hours at 37°C in conditions identical to those in the preservation experiment, refrigerated for one day with minimal physical, chemical and biological disturbance, and harvested without propagation. Harvested cells will be extracted with the PowerSoil kit and quantified with the PicoGreen method detailed in the Section 4.2.2. After quantification, we will perform qPCR on the samples with organism-specific rpoB primers to quantify the number of cells from each strain. We will also perform qPCR on cultures before preservation to gain an understanding of how the “baseline” composition changes in response to refrigeration.All organisms were cultured in sealed tubes with sterile SHI medium at pH 7.0, which consisted of proteose peptone, trypticase peptone, yeast extract, KCl, and pig mucin III, as well as low concentrations of menadione, L-arginine, urea, hemin, and Nacetylmuramic acid. Components that were robust against high heat and high pressure were added to the media prior to autoclaving; components sensitive to heat or pressure, such as sucrose and sheep blood, were added after autoclaving, by passing through 0.2µm filters. Sterile and complete media was degassed by passing filtersterilized anaerobic gas through the media for 15 or more minutes. Single colonies of organisms were selected with colony pickers and gently resuspended in degassed medium in a culture tube. The tube was then sealed with a rubber cap, flushed and filled with the aforementioned anaerobic gas, and incubated at 37°C without shaking. Approximately every 24 hours, we briefly flushed and refilled the tubes with anaerobic gas to maintain positive pressure.

The growth of the organisms was periodically monitored by optical density measurements at 600nm, and growth curves constructed were based on these measurements. After organisms reached their growth phases, we inoculated blood-agar plates with loopfuls of the liquid cultures and incubated the plates at 37°C in a sealed chamber under an anaerobic atmosphere. All genomic DNA used in this phase of the project was extracted with the PowerSoil DNA Isolation kit according to manufacturer’s instructions.For each organism, two sets of rpoB primers with Tm values between 58°C and 62°C and PCR product length between 100bp and 170bp were designed in silico. Table 3 details the characteristics and sequences of the forward and reverse primers. All sequences are written 5’ to 3’. “Name” represents the name of the primer, an abbreviation of the strain name plus a number; temperatures are in units of degrees Celsius; “PL” is the PCR product length, in units of base pairs. The primer names will be used in subsequent visualizations of results. Upon receiving the primers, we dissolved them in water and determined the concentrations using the Qubit 2.0 instrument with the high-sensitivity single-stranded DNA assay kit . We then performed colony PCR in duplicate, one colony per reaction, to test the efficiencies of the primers. For each PCR reaction, we use a total volume of 20µL: 1µL each of the forward and reverse primers, 1µL extracted gDNA or colony, and 17µL SsoAdvanced Universal SYBR Green Supermix . We used the following general PCR protocol: initial step at 95°C for 2 minutes, then 35 cycles consisting of 30s at 95°C, 30s, 30s of gradient temperature, and 60s at 72°C, followed by extension at 72°C for 10 minutes.

For the P. salivae PCR, we used 45s instead of 60s for the 72°C step, and for the V. dispar PCR, we used the same protocol as for P. salivae but with a lower cycle number of 30. After selecting the better primer set for each organism, we optimized the annealing temperature for that organism and checked for crosstalk between the primer set and the three off-target organisms. We also attempted to reduce the P. salivae cycle number. PCR products were mixed with gel loading dye, denatured at 95°C for 3 minutes, and loaded onto 8% urea gels. Gels were run at 215V, 100V, or a mix of the two until the fastest markers in the ladders almost reached the bottom. For more accurate reference, we used both the Low Molecular Weight DNA Ladder and the TriDye Ultra Low Range ladder . Gels were stained for 20 – 30 minutes in SYBR Gold diluted 1:10,000 in 1X TBE buffer, and then visualized using short-range ultraviolet Radiation.The growth curves of three of the four organisms in liquid SHI under anaerobic atmosphere are shown in Figure 33, as estimated by optical density measurements at 600nm. Each data point represents the average of OD600 values for two tubes. The baseline of the optical measurement consisted of a culture tube with degassed sterile SHI medium, incubated at 37°C under anaerobic conditions simultaneously with the inoculated media. Unlike conventional bacterial growth curves, these curves appeared somewhat jagged, with a dip around 13.5 hours and another dip around 34.5 hours. These dips did not fully correspond to the intermittent flushing and refilling of the tubes with anaerobic gas, which occurred approximately every 24 hours. In addition, all three organisms grew somewhat slowly, and their growths seemed to plateau starting around the 25-hour mark. It is possible that the nutrients in the media had been depleted at 25 hours, though we would need to extend the incubation time for a more complete curve to be sure. Interestingly, none of the curves showed a lag phase at the start of incubation. With the time available, we were unable to grow Veillonella dispar successfully.Accurate predictions of time-to-death are important in bio-medicine to allow patients to better consider their future, for health professionals to make more informed medical decisions, and for family members to have realistic expectations . Consequently a robust literature exists in the medical sciences concerned with predicting survival time of persons suffering from one or more of any number of different fatal diseases . This literature extends to the veterinary sciences and basic biology, as well to papers on predicting survival times for terminally-ill pets including dogs , cats and laboratory rodents . One of the common threads across this than atological research on humans, pets and rodents is that the predictions are informed, growing raspberries in container not only by extensive knowledge of the causes, origins and natural courses of the underlying diseases but, at least for humans, also by access to deep databases containing the outcome of tens of thousands, if not millions, of previous cases at different disease stages that end in the individual’s death. Remarkably, even with access to this extensive information on disease progress and outcomes, the vast majority of models in bio-medicine used for predicting survival times are both imprecise and inaccurate, with short-term survival usually overestimated and long-term survival often underestimated .

The inaccuracy of sophisticated models designed to predict human survival time that are based on extraordinary amounts of data and extensive biomedical information on both the disease and the patient lays bare the challenges of predicting survival time in non-human species for which virtually none of this information available. Inasmuch as there is no practical need for developing models for predicting time to death in the overwhelming majority of non-domesticated organisms, it is unsurprising that the literature in this area is extraordinarily scarce. With the exception of the papers by Rauser, Mueller and their colleagues in which these researchers classify Drosophila melanogaster females according to whether or not they were in a death spiral stage , and the paper by Papadopoulos and his colleagues who showed that supine behavior was predictive of impending death in medflies, we are aware of no other papers that are expressly concerned with predicting the timing of death in fruit flies or other non-domestic group of organisms.However, there are two groups of studies whose results are related to our research. The first group involves end-of-life egg laying patterns in the context of aging including: Novoseltzev and his colleagues on the senescent stage of D. melanogaster and the medfly as exponentially-decreasing rate of reproduction, Curtsinger and his colleagues on working versus retired who characterized the end-phase degree of “roughness” of individual egg laying using the fractal concept of lacunarity ; and Rogina and her colleagues who, by manipulating the timing of mating in D. melanogaster females, discovered reproductive patterns that were conditional on when females mated. All experiments in her and her colleague’s studies revealed characteristics suggesting that longer-lived flies passed through three stages, the last of which they labeled “declining terminal”. The second group of studies related to our work, albeit more tangentially, includes research concerned with the timing of reproduction relative to their death. One study representative of this group involves research concerned with the length of remaining life in individual flies relative to others based on the rate of decrease in reproduction after the peak. For example, Müller and his colleagues showed that the exponential rate of decrease in egg laying by female medflies predicted the remaining life spans of individuals. The other area within this second group involves research on the cost of reproduction in which increments of reproduction in early life result in decrements in survival later in life including papers by Harshman and Zera on mechanisms and modeling papers by Mangel and Heimpel , Fletcher and his coworkers and Rosenheim concerned with foraging strategies relative to both remaining reproduction and lifespans. There are at least three reasons why developing predictive models of impending death or that can be used to distinguish terminal periods in non-human species such as fruit flies is potentially important. First, accurate predictive models can affirm patterns associated with the transition from early stages of aging to the late stages in which the manifestations of increasing frailty are catastrophic, which is to say, ends in death. This same “staging” process might have the potential to be used to identify parse other stages of the aging process. Second, predictive models for death could be used in intervention trials in which flies showing early signs of impending death could be identified and subjected to treatments designed to test death-postponing interventions. Third, the model-building required to identify different patterns of reproduction that identify impending death in flies, in some form, may be relevant to the model-building process predicting the timing of death in humans. Indeed the model-building process itself might shed light on the classification process involved in separating the non-dying and dying as well as in calibrating survival time . In light of the paucity of information on end-of-life patterns of life history phenomena in non-human species as well as the potential importance to basic biology of these types of studies, the overarching goal of our study was to determine whether it was possible to distinguish across three fruit fly species a set of general patterns of egg laying in individual females who were near death from the egg-laying patterns in individual who were not near death . Our specific aims were to: Visualize and summarize the egg-laying patterns in individuals from all species with respect to both their chronological and thanatological ages; create subsets of these data in containing egg laying sequences from both the end-of-life and mid-life ; and test hypotheses that the patterns of egg laying between the two categories of egg-laying segments have distinct patterns.

One factor was already mentioned: the number of OTUs in the sample. In this sense, adding another OTU would affect evenness if the sample contained few OTUs. The other factor is the number of individuals in the OTU added relative to the numbers of individuals in the other OTUs in the sample. If the new OTU contains many more members than the existing ones, then its addition will decrease the evenness and therefore decrease the overall diversity, as we observed in the effect of E. coli spike-in on the low-biomass samples in the preliminary experiments. If the new OTU has fewer members than the existing ones, its addition may not affect evenness much at all, as in the case of E. coli spike-ins relative to the preliminary cultures. In either case, rarefaction to a large enough depth would help retain biological differences, in other words, differences inherent to the samples and not as consequences of mathematical manipulations. As already pointed out in literature, a major drawback of rarefaction would be losing rarer OTUs because of the lack of sampling depth, i.e. discarding rarer sequences as large samples are reduced to smaller ones. Fortunately, that was not a major concern in the preliminary experiments, as we did not design the procedure as a way to discover rare microbes or uncover whether our methods support the growth of as-of-yet uncultivated organisms. Our goals, plastic potting pots combined with the culturing methodology as well as the rarefaction threshold we chose, meant that, ultimately, we did not lose exclude crucial or even important information from the data set.It is interesting to note that streptococcal species tend to attach to a surface first and provide a suitable colonization environment for other species.

Previous studies have shown that several streptococcal species produce adhesins that preferentially bind to different substrates in human saliva and cells. For instance, S. salivarius, an OTU prevalent in our model, expresses amylase-binding proteins that interact with both salivary amylase and surface lectins that bind to extra parotid glycoproteins. S. salivarius also expresses antigen C fibrillar glycoprotein that binds to the epithelial cells on the human cheek, and fibrillar antigen B that binds to Veillonella parvula. Similarly, S. oralis expresses surface lectins and antigens that bind to salivary glycoproteins. These surface proteins facilitate streptococcal attachment to the enamel surface on one hand, and on the other help bacterial cells from other phyla immobilize in preferred environmental niches and subsequently proliferate. These proteins are doubtless a large part of the reason that streptococcal cells act as early colonizers of the dental surface. The results from the preliminary experiments indirectly support this colonization order, by the relative abundances of Streptococcus OTUs in the cultures . Incidentally, the Streptococcus OTUs were only the second most abundant in the preliminary cultures; members from the Veillonella OTUs were the most abundant. Not as much research has been done for the attachment processes of Veillonella, though members of this genus are known to be early colonizers in dental plaque as well, coaggregating with streptococcal species and using the lactic acid produced by Streptococcus oralis. Clearly, our results indicate that the methods in this phase of the project properly support some best known early colonizers in the human dental plaque bacterial community.In this phase, three volunteer hosts were used as sources of dental plaque.

Sample collection took place under protocols 3-18-0189 and 3-19-0119, approved by the UCSB Human Subjects Committee. Prior to plaque collection, hosts abstained from food, nonwater liquids, and dental hygiene for 12 hours. At the time of collection, supragingival plaque of five molar teeth was obtained from each host using a sterilized Gracey curette. Collected plaque was immediately suspended in sterile centrifuged SHI medium, gently mixed, and divided equally among wells in a sterile surface modified 24-well plate such that each well received 1.98mL of the mixture. Prior to receiving inoculated medium, wells were conditioned with an artificial pellicle formed by clarified human saliva , which was supplied as frozen fractions pooled from healthy human volunteers. Saliva was stored at -20°C until clarification, at which point we defrosted the saliva on ice. Clarification of defrosted saliva was performed on site by centrifuging at 6,000 x g for 3 minutes at 4°C, mixing with 1X PBS in a 1:1 ratio, and passing the mixture through 0.2µm filters. Unused clarified saliva was stored at 4°C for no more than 3 days before being used or discarded. The artificial pellicle in each well was formed by adding 150µL clarified saliva to the bottom of the well and air drying at 37°C for 60 minutes. The plate was then sterilized with short-wave UV light for 60 minutes. At this point, wells were considered conditioned and ready for media. For this set of experiments, cultures derived from each host received a separate 24- well plate. We divided the plates into sections to facilitate harvesting at the specific time points of 12, 24, 48, 96, and 168 hours, in order to investigate the effects of increased incubation time on culture composition.

Each plate was divided into 6 sections of 4 wells , consisting of 2 controls in the top row with the medium and pellicle and 2 cultures in the bottom row with host-plaque-inoculated medium and pellicle. After receiving sterile or inoculated medium, each well also received 20µL of 0.5% sucrose. Plates were then incubated in a sealed vessel at 37°C in an anaerobic atmosphere composed of 85% nitrogen, 10% hydrogen, and 5% carbon dioxide. At designated feeding times , all wells were supplemented with 5µL of 0.5% sucrose and specified volumes of SHI such that a constant well volume of 2.0mL was kept throughout the incubation process. If the feed time coincided with the harvest time for any particular section of wells, then feeding did not occur. At the five designated times, we harvested the designated section of wells by first aspirating the liquid, and then mixing the sedimented cells gently but well. We pipetted 900µL of properly mixed sedimented cultures into sterile microfuge tubes and added 100µL of 10% diluted E. coli cultures with OD600 values of 0.8. The resulting mixtures were pelleted, flash-frozen in liquid nitrogen, and kept at -80°C until further processing at the UC Davis Host-Microbe Systems Biology Core . To compare the culture composition with original host plaque and track the temporal development, we pelleted and froze plaque samples from all hosts at the start of incubation. In addition, we pelleted and froze 500µL aliquots of all E. coli cultures used for spike-ins at the times of the spike-ins, with the intentions of observing OTUs in pure E. coli cultures and retaining the ability to remove these OTUs from spiked cultures. In this phase of the project, we elected to keep the E. coli spike-in step such that we could continue checking the biomass of the controls as well as gain an understanding of the biomass in cultures and the potential differences in biomass across the cultures derived from different hosts.DNA extraction and sequencing were performed at the UC Davis Host Microbe Systems Biology Core , in a manner similar to the sequencing procedures from the preliminary experiments . Sequencing of properly amplified and diluted libraries was performed on the Illumina MiSeq platform using the paired-end method with a length of 253bp. Quality control of the raw sequences was first performed in QIIME by HMSB , and then formally reperformed in R with the mothur software. Briefly, 4,298,748 contigs were constructed from raw reads that were size-selected to be in the range of 240 to 275 bp. Constructed contigs were trimmed to eliminate ambiguous reads, and the resulting reads were screened for homopolymers with an upper threshold of 8 and pre-clustered. Chimeric sequences were then removed with VSearch, and non-bacterial sequences were removed based on the full-length SILVA database . The resulting 3,132,678 contigs were clustered into operational taxonomic units based on the full-length SILVA reference database at 97% level of sequence identity, approximating species-level taxa. These OTUs were constructed into a dense BIOM table for bio-informatics analysis.Bio-informatics analysis was first performed in QIIME at HMSB, and then formally reperformed in R with version 1.38.0 of the phyloseq software package. In this analysis, raspberry container growing we examined the sequencing depth, the number of OTUs, and the inverse Simpson’s index values of the samples.

As in the preliminary experiments, we considered the prevalence of organisms at the phylum level to gain an understanding of the general community structures. Then, we examined the correlation between sequencing depth and diversity using rarefaction curves and plots of read counts vs. diversity indices. To standardize sample sizes and minimize the presence of potentially spurious OTUs, we rarefied samples to a depth 30,000 and reexamined the number of OTUs and inverse Simpson’s index values. We also studied the absolute counts and relative abundances of the controls and cultures, inferring the biomass in controls and cultures relative to the E. coli spikes, to verify that there was minimal contamination throughout the culturing, extraction, amplification, and sequencing processes. After characterizing the samples as a whole and confirming minimal contamination, we focused on the culture and plaque samples. We removed the most prominent spike-in OTU reads from the rarefied read counts, converted read counts to relative abundances, and then examined the 12 most prominent OTUs in the plaque and culture samples. With the relative abundances, we performed Principal Coordinate Analysis using the Bray-Curtis dissimilarity metric, to investigate patterns in sample clustering according to incubation times and sample types. From the clustering patterns, we identified the OTUs that likely played the most important roles in sample differences. Lastly, we used the stats package for the relative abundances and the mixOmics package for the centered-log-ratio-transformed relative abundances to perform Principal Component Analysis .Sequencing of the 16S rRNA V4 region yielded a total of 4,298,748 raw contigs. Using the mothur software and release 132 of the full-length SILVA database, we trimmed and filtered out ambiguous bases, retaining 3,144,467 of the raw reads. From these, 122,617 unique sequences were found. Screening for homopolymers led to the retention of 99.6% and 98.1% of total reads and unique sequences, respectively. Pre-clustering to remove likely pyrosequencing errors with a tolerance of two mismatches resulted in 3,132,678 reads and 26,757 unique sequences, and chimera removal using VSearch led to the retention of 98.3% and 76.7% of total reads and unique sequences, respectively, from the previous step. Subsequent temoval of non-16S-rRNA sequences resulted in 20,508 unique sequences and 3,077,896 total reads. With the same full-length SILVA database, we then generated OTUs based on 97% sequence similarity and constructed the BIOM table from these OTUs.Temporal samples with spike-ins yielded high read counts across sample types and incubation times, except for one that failed to sequence well . Fewer than 200 OTUs were found in all samples regardless of rarefaction . As expected, negative controls and pure E. coli samples contained the fewest OTUs out of all sample types; the number of OTUs for negative controls and that for pure E. coli were comparable to each other. Interestingly, 4 out of 6 plaque samples contained fewer OTUs than many cultures while 2 plaque samples clearly yielded many more OTUs than all other samples. This difference deserves a much closer look, which we provide later in this section. A quick scan of the prevalence at the phylum level shows that Firmicutes and Proteobacteria were the most prevalent and the most abundant taxa, followed by Actinobacteria, Bacteriodetes, and Epsilonbacteraeota. These five taxa numbered among the most prevalent phyla in the preliminary experiments as well, indicating that the conditions we used favored members of these phyla and that the conditions were appropriate for promoting the growth of bacteria from key phyla of the oral microbiome. We then examined the rarefaction curves and plots of sequencing depth vs. diversity indices. The rarefaction plot of plaque and cultures shows that the number of OTUs discovered begins leveling around 30,000 reads for all samples. In other words, below 30,000 reads, new OTUs would still be discovered with increasing sampling depth, so rarefaction thresholds below 30,000 reads run the risk of artificially reducing true diversity in the samples. Furthermore, plots of read counts vs. diversity indices show that a somewhat weak but statistically significant negative linear correlation exists between sequencing depth and the Shannon index , and between sequencing depth and the inverse Simpson’s index , though the correlations are not immediately apparent on the plots.