Horticulture

No surfactant production was observed in a ∆gacS mutant, and syfR and syfA transcription are nearly abolished. Additionally, we determined whether SyfR operates independently of SalA, or if SalA is upstream from SyfR function. Surprisingly, the ∆salA deletion mutant also exhibited abolished surfactant production and very low levels of syfR and syfA transcription. This suggests that these genes mediate the baseline expression of SyfR. It also suggests that these pathways are not responsible for the surface-dependent induction of syfA, but rather have an epistatic role in syringafactin production. We were surprised to observe that both ∆salA and ∆gacS mutants exhibited abolished surfactant production. We thus questioned if the strains are still capable of producing and secreting syringafactin or whether pleiotropic effects on cell metabolism that made them incapable of this metabolic process was operative in these mutants. To test this, we constitutively expressed SyfR by introducing plasmid p519n-syfR into both the ∆gacS and ∆salA mutant strains. These strains produced surfactant detectable both by the atomized oil spray as well as water drop collapse in both broth and plate cultures of these strains. 

These mutants, in which syfR is not normally transcribed, thus produce and secrete syringafactin when SyfR is supplied constitutively. This demonstrates that neither GacS nor SalA are necessary for syringafactin transcription,flood and drain table or for supply of necessary intermediates for syringafactin production, but rather exert their influence on syringafactin production solely through their control of the SyfR regulator. We initially suspected that secondary mutations might have been the cause of what looked to be a poorly growing or stressed strain. However, all independently isolated syfA– and syfB transposon mutants as well as the site-directed ∆syfA deletion mutant displayed this same phenotype, suggesting that it is a direct response to the absence of syringafactin. Nonetheless, in order to rule out the possibility of secondary mutations in our syringafactin-deficient strains, we created a syfR– mutation in a ∆syfA deletion strain. Surprisingly, this second mutation abolished the fried egg phenotype normally exhibited by the ∆syfA mutant, and the appearance of this ∆syfA/syfR– double mutant was indistinguishable from that of a syfR– mutant alone. This suggested that SyfR transcriptionally regulates more genes than just those enabling syringafactin production, specifically including genes encoding whatever trait triggers development of the fried egg phenotype.

We initially hypothesized that perhaps SyfR induced both syringafactin production and also a system involved in either its transport or its perception, and we further postulated that syringafactin served not only as a surfactant, but also as a signaling molecule. To additionally support the conjecture that SyfR controls more than just syringafactin production, we introduced the plasmid conferring the constitutive expression of SyfR into a ∆syfA strain. Curiously, this strain also exhibited a fried appearance, but one that appeared to be an exaggerated and earlier-onset version of the rough “white” from a ∆syfA strain. Thus, the strong visual phenotype of this strain provides further evidence that SyfR transcriptionally regulates more than just syringafactin production. In comparison, a wild-type strain capable of syringafactin production and that constitutively expresses SyfR develops a slightly matted appearance, but does not exhibit a fried appearance. Assuming that this rough fried egg appearance is indeed indicative of stress, we might hypothesize that syringafactin normally plays a protective role for the cell, and that its absence makes the cells somehow more susceptible to other factors induced by SyfR in P. syringae itself. In order to identify genes under the control of SyfR, we screened 2,000 transposon mutants in a ∆syfA mutant background for any that had lost the fried egg phenotype. Several such mutants were identified. Prominent among the mutants found were several insertional events in the syringomycin biosynthetic genes and an associated secretion gene , prompting further investigation.

In order to confirm the requirement for syringomycin to initiate the fried egg phenotype, we constructed a site-directed knockout of syrE in a ∆syfA mutant background.One of the most surprising aspects of this finding was the fact that syringomycin and syringopeptin are assumed to have overlapping roles as plant virulence factors, and are typically co-regulated by SyrF which is downstream of SalA. However, a site-directed knockout in the syringopeptin biosynthetic gene sypA did not lead to a loss of the fried egg phenotype. These results strongly suggest that syringomycin has a specific role in this phenoptype that is independent from syringopeptin. However, while syringomycin appeared necessary for the fried egg phenotype, this was not proof that it was a factor regulated by SyfR, which was posited to be required for this phenomenon. We postulated that the production of syringomycin in the absence of syringafactin is altering cell physiology in a way that leads to a production of the fried egg phenotype. In order to confirm that syringomycin expression is under the control of SyfR, we developed a plasmid-based transcriptional reporter of syrB expression. Indeed, the GFP fluorescence indicative of syrB expression was much lower in cells of a syfR– mutant harboring pPsyrB-gfp than in either a ∆syfA mutant or the wild type strain; expression in the ∆syfA mutant was similar to that in the wild type strain. In broth cultures,rolling bench expression of syrB was similarly low in all strains, as further proof that genes downstream of SyfR are not activated in broth conditions. This confirms that syringomycin is induced in cells cultured on plates and is under the regulatory control of SyfR.Because the fried egg phenotype is observed only in colonies older than 3 days, we investigated syringomycin expression in different mutant strains over the course of several days.

Surprisingly, although syringomycin was highly expressed in wild-type and ∆syfA strains after one day of growth, only very low levels of expression were detected at any subsequent time. To further explore this apparent temporal regulation, we measured the GFP fluorescence of cells of a wild-type strain harboring pPsyrB-gfp over the course of 48 hours of growth on plates. In agreement with our initial observations, syrB expression, and thus presumably syringomycin production, is limited to a short period during initial phases of colony development, peaking after about 24 hours and thereafter diminishing. This pattern of expression was seen in both the wild-type and a ∆syfA mutant strain, but not in the syfR- mutant, in which syfA expression was always low. This temporal regulation of syrB is contrary to that of syfA expression, which remains stably induced over this time period. Thus the role of SyfR in stimulating syringomycin expression is distinct from its effect on syringafactin expression. We hypothesized that the strong temporal, and hence cell density-related, regulation of syringomycin synthesis may be due to its suppression by quorum sensing in older cultures. To test this we measured syrB expression in a ∆ahlR mutant incapable of quorum sensing. The temporal expression of syrB in this strain was identical to that in a wild type strain, with peak expression at 24 hours. This suggests that quorum sensing does not mediate temporal regulation of syringomycin production. Also, these results also cast doubt on the model that syringomycin is directly responsible for inducing the fried egg phenotype, since the colony phenotype appears after about four days of growth, while syringomycin production apparently peaks after only 24 hours. Although the biosynthetic pathway for syringomycin and regulation of its expression has been extensively investigated, SyfR has never been implicated in its regulation. We thus questioned if SyfR was, in fact, an overlooked necessary regulatory element for syringomycin production. To test this we measured syrB expression in a wild-type and a syfR– mutant mutant strain on media specifically formulated to induce syringomycin and syringopeptin production. Although the levels of GFP fluorescence exhibited by a syfR– mutant harboring pPsyrBgfp were reduced compared to that in the wild type strain, we still see substantial expression of syrB in the medium conducive to syringomycin production. This suggests to us that SyfR is not absolutely required for the induction of syringomycin production in this medium that mimics the plant environment, but plays a more ancillary role in its production. This moderate effect on syringomycin production might explain why SyfR has not previously been identified as a regulator of syringomycin. By chance, it was observed that the fried egg phenotype in colonies of ∆syfA mutants appeared much earlier when they were grown near colonies of a ∆syfA mutant blocked in any of various steps in the AlgT regulatory pathway. When colonies of a ∆syfA mutant were grown on the same plate with those of a ∆syfA/algT– mutant , the timing of the onset of the fried egg phenotype was directly correlated with the distance from the ∆syfA/algT– mutant. The fried egg phenotype was induced in ∆syfA mutants after as few as 2 days of incubation when cultured near a ∆syfA/algT– mutant.

When cultured by an algT– mutant that was still capable of syringafactin production, this early-onset property was diminished. Premature induction of the fried egg phenotype occurred only in a ∆syfA mutant, while colonies of wild type and syfR– strains did not change their appearance in response to this signal. This observation suggested that the fried egg phenotype must be a response to an extracellular compound that is only sensed by a component of the SyfR regulon. We earlier hypothesized that syringomycin is the compound that induces this fried egg phenotype. If syringomycin directly stresses the cell or otherwise induces this colony phenotype, and if syringomycin is produced in large quantities in an algT– mutant, then we should have seen a restoration of the fried egg phenotype in a ∆syfA/syrE– double mutant strain upon exposure to syringomycin. However, when a ∆syfA/syrE– mutant strain is placed in close proximity to a ∆syfA/algT– mutant, there is no restoration of the fried egg phenotype. Furthermore, a ∆syfA mutant still exhibits a strong fried egg phenotype when placed near a ∆syfA/algT-/syrE– triple mutant. Therefore, it does not appear that syringomycin acts as the direct extracellular signal that invokes this response, but rather is necessary for enabling other factors to induce the response. This evidence, in addition to the finding that syringomycin is only produced during the initial 24 hours of surface growth, lead us to assume that syringomycin instead acts as a signal that primes the colony for the fried egg phenotype that we later observe. We recently observed that an algT– mutant of P. syringae produces high levels of a surfactant termed BRF , whose production requires an rhlA homolog. This surfactant also exhibits a strong temporal pattern of regulation, with production increasing over time. Therefore, we hypothesized that this surfactant could be responsible for inducing the fried egg phenotype in the ∆syfA mutant. Colonies of a ∆syfA/∆brfA double deletion mutant did not express the fried egg phenotype at any age. However, this double mutant regained the fried egg phenotype in the presence of a ∆syfA/algT– mutant strain, suggesting that BRF could be a signal that induces this phenotype. Furthermore, neither a ∆brfA/algT– double mutant, nor a ∆syfA/∆brfA/algT– triple mutant is capable of inducing an early fried egg phenotype in a ∆syfA mutant. However, application of a BRF extract near a colony of a ∆syfA mutant does not induce the early appearance of the fried egg phenotype. It is thus possible that BRF is modified to gain its activity, or that BRF might play a role in delivering an insoluble signal over the long distances that separate colonies. Similar to the other characterized members of LuxR-type regulators, SyfR appears to form multimers in order to initiate transcription. Furthermore, in keeping with observations of LuxR and SalA, SyfR appears to have an autoregulatory role in its own transcription. However, it is unclear how the activation of SyfR is mediated by external conditions. In the case of LuxR, binding of a quorum signal induces dimerization which then allows LuxR to function as a transcription factor; thus, cell density is conveyed to the cell by increased availability and binding of the autoinducer signal, which stimulates increased LuxR activity. However, SyfR belongs to a class of LuxR-type regulators that do not contain characterized small molecule binding domains, and thus there is no factor that has been determined to limit SyfR dimerization and activity other than its own transcription levels.

Vying with OCWD’s GWRS for the title of California’s premier water recycling facility, is the Edward C. Little Water Recycling Facility . Built by the West Basin Municipal Water District in 1995, it has been expanded on three occasions and now is capable of advanced cleaning technologies, micro-filtration, and reverse osmosis . WBMWD received state and federal funding to design and build the 30 million gallon a day treatment facility in the City of El Segundo, south of Los Angeles. The ELWRF is the only water recycling facility in the world that produces five different types or qualities of waters to meet the needs of its customers, from basic irrigation water, to ultra-pure high pressure boiler feed water, to purified water that is injected into the west coast groundwater injection barrier . The facility also has a 60,000 square foot solar power generating system to offset energy production. It currently has over 350 industrial customers for its recycled water and is once again under expansion. Like Factory 21 and GWRS, the facility conducts basic water treatment and technology research . California’s water recycling regulations are spread out among several legal entities – the California Code of Regulations,bato bucket the Water Code, and the Health and Safety Code .

The Federal government’s EPA treats reused water just like water. It has guidelines for water reuse with criteria and design considerations but leaves implementation to the the states . The California water recycling program is the shared responsibility of the California Department of Health Services , under the direction of the California Environmental Protection Agency , and State Water Resources Control Board . The DHS is responsible for establishing criteria that ensures recycled water maintains water quality and protects public health. The SWRCB has water allocation and quality protection authority over all States water resources. The DHS works with the SWRCB—and the nine Regional Water Control Boards— in the drafting of permits and management of contamination . The SWRCB also provides financial assistance for both the planning and construction of water reuse projects. Some funds are also available through the DWR and DHS. The Water Recycling Facilities Planning Grant Program provides up to $75,000 in grants to local agencies for reuse feasibility studies. The Water Recycling Construction Program provides low-interest grants and loans for construction and development. Since the late-1970s, the program has distributed close to $151 million in planning and construction grants and approximately $611 million in low-interest loans for water reuse projects . As pointed out in the discussion of California water market drivers, water reuse has become a cost effective new source of water.

In 2005, California’s Recycled Water Task Force found that the costs of reclaimed water, including the annualized capital and operational costs, would range from $0 to $2000 per acre-foot, but averaged approximately $600 per acre-foot . Operation and maintenance costs alone were also found to vary, with an average projection to be $300 per acre-foot. At the time, this cost was perceived to be very high—MWD water often sold for less then $300 an acre-foot . These costs, however, compared favorably with the costs of other options to increase the water supply, such as reservoirs or desalination plants.. Water reuse has always been controversial. Quite simply, people do not like the idea of drinking sewage. One water manager I interviewed privately disclosed their personal distaste for reused water—even for landscaping purposes. This person trusted the science completely and understood—they simply could not get past the grossness of the idea. Psychologists refer to this phenomenon as “psychological contagion,” essentially the idea that once two entities touch they are forever linked . Studies have found that nearly 60% of people refuse to drink water that has been linked with sewage . So it is not surprising that in California, water reuse has often met with fierce public resistance—often organized at the grassroots . The most famous case occurred in the mid 1990s, the city of Los Angeles launched a reuse program specifically to recharge the groundwater basin with tertiary-treated reused water. It was smeared as a “toilet to tap” program. The negative publicity led the city to abandon the groundwater recharge plan and instead marketed the water to irrigation and industrial customers . This hasn’t been all bad for the city though.

Today, the year round water in the Los Angeles River is actually a result of plants discharging treated water originally meant for the groundwater system. Some activists have claimed that the flowing water has greatly contributed to the to the city’s river revitalizations efforts . It should be noted that the Los Angeles County Sanitation Department is still in the reuse businesses and working with various partners have launched the Century and the Rio Hondo Water Reclamation Programs .In addition to the environmental injustices they often endure by virtue of living in poorer—and often industrial— areas. These communities often come with a history of state or societal sanctioned discrimination and may react very strongly to water reuse proposals . Being asked to drink shit is simply one indignity too many. For this reason, many scholars who have looked into the politics of water reuse have recommended that educational campaigns target these groups, rather than the higher educated business leaders who are likely to be easily converted . One could speculate that the fact that the Southern California region that has been the most successful in the implementation of water reuse, Orange County, enjoys a reputation as a relatively wealthy suburb of Los Angeles may have helped facilitate the development of water reuse. Although to be fair,dutch bucket hydroponic today’s Orange County has roughly the same ratio of minorities and is only a little wealthier then the state as a whole . The best technique psychologists have found to cognitively cleanse the dirty reused water is to “cognitively co-mingle the water with nature” . Indeed, I found this be true with the interviewee that expressed repulsion. The individual was perfectly fine drinking water derived from the Colorado river, despite the fact that it is full of reused water discharged from numerous upstream municipalities. Today reuse projects that augment water supplies create “indirect potable water” that incorporate this psychological technique by piping reused water into lakes or rivers where before being drawn again. For example, Orange County’s GWRS only directly injects half of the water it cleans into the ground, and that is into the saltwater intrusion barrier. The rest is pumped to the Kraemer/Miller recharge basins, essentially large lakes, in Anaheim where it then percolates into the groundwater supply naturally, hence co-mingling with nature. . This strategy requires more land and infrastructure than simply direct groundwater injection—or for that matter, direct potable reuse—and hence results in more expensive projects. It is not only Southern Californians, however, who prefer their reused water to be returned to nature before becoming part of the drinking supply. Globally, only one major city, Windhoek, Namibia practices large scale direct potable reuse . Windhoek happens to be a very poor city located in one of the driest regions of Africa far from sufficient water sources. Another major strategy employed by water reuse advocates is positive marketing campaigns. Burned by pervasive “Toilet to Tap”, media campaigns against reused water utilities have worked hard to brand reused water as a good, “new” source . Like Singapore’s NEW water program, Californian advocates have embraced “new” and “blue” in order to enlist support for recycled water as pure, clean and safe technology. Additionally, campaigns like the New Blue Water Coalition promote educational campaigns filled with associations with safety and scientifically advanced technologies, and environmental protection themes to further reassure the public .

Some water agencies have even cultivated these themes as their personal brand. See for example the logo of OCWD and its partner in the GWRS project, OCSD in figure 5. The two organizations have lead an extensive outreach program in order to build community support for the GWRS. They have given over 1500 Presentations to community groups, organizations, and clubs and conduct daily tours of Water Factory 21, GWRS, and the recharge facilities, . The district also operate an well designed and informative website detailing their project and are happy to send informational packets on demand. OCWD has runs a water education program designed for children and young adults, H20 University . Other local agencies such as WBMWD at the Ed C. Little facility, and LADWP at its Hyperion treatment plant also offer tours and both are very active in working to educate the public OCWD works closely with other water agencies, such as Israel, Singapore, and Australia, in order to exchange knowledge and share best practices . For example the website lists reuse data in both metric and imperial measurement units, as reflects the web traffic it receives from interested people around the world. Water reuse project marketeers throughout the world point to the GWRS as a model. Together,the OCWD team and its global partners are slowly building support for water reuse globally, and consequently building a market for new innovative reuse infrastructure and technologies.Orange County Water District’s Groundwater Replenishment System is one of the most celebrated civil engineering and water reuse projects in the world. The GWRS has received more than 35 local, regional, national, and international awards, including the American Society of Civil Engineers 2009 Outstanding Civil Engineering Achievement Award and the 2008 Stockholm International Industry Water Award . The design and construction of the GWRS was jointly-funded by the Orange County Water District and the Orange County Sanitation District who together run an extensive ongoing public outreach program. Notable for a drinking water reuse project, the project went forward without any major opposition. Large businesses such as Sempra Energy, environmental groups like Surfriders and Coast keepers, community groups such as Kiwanis and Rotary, all voiced support for the project . Several key minority leaders were also strong proponents of the project, most notably Hispanic Congresswoman, Loretta Sanchez. Elected in 1997, Ms. Sanchez has also been instrumental in steering federal support—and dollars—towards GWRS. Most recently, she authorized H.R. 383, which was included in the 2009 Omnibus spending bill. The bill increased federal support of the project by $6 million . The GWRS was designed to produce 70 million gallons of recycled water per day, or a total production of 23.5 billion gallons per year . Today, the project is once again under construction and is being expanded to 100 million gallons a day. OCWD and OCSD together secured $92 million in state, federal and local grants to help fund the $481 million project. In addition, the GWRS also receives a $7.5 million annual subsidy for 12 years from the Metropolitan Water District . OCSD and OCWD also agreed to share the cost to construct the GWRS. OCWD consented to manage and fund the operations of the facility into the future. The annual operating budget for GWRS is approximately $34 million, which includes electricity, chemicals, labor, and maintenance. The replenished water is affordable and even without outside funding the cost of water would still be approximately $800 AF—very close to the 2012 price of tier-1 treated water charged by the MWD . Orange County Water District is a special district formed in 1933 to protect Orange County’s rights to Santa Ana River water and to manage the large groundwater basin that underlies much of Orange County . OCWD encompasses 229,000 acres in the lower watershed of the Santa Ana River. The Orange County groundwater basin provides water for over 2.3 million people. OCWD is governed by a 25-member board of directors comprised of representatives of local agencies and cities within its 470-square-mile service area . OCWD receives revenue from assessments paid by the groundwater basin pumpers, a percentage of the local property taxes, and from investments. Each day, OCSD treats approximately 220 million gallons of water—70% of which is groundwater —with the remainder imported from MWD. Northern and central Orange County receive the majority of their water supply from large groundwater basin managed by the Orange County Water District, while areas to the south are more dependent on imported water from Northern California and the Colorado River.

During this time, PUB was restructured and expanded its duties from simply providing drinking water to also treating all municipal wastewater and managing water infrastructure. PUB manages Singapore’s 15 reservoirs, six water reclamation plants and nine water treatment plants . The PUB administers the federally funded Active Beautiful Clean Waters program, a planning project intended to beautify the island’s drains, canals and reservoirs. Meanwhile, the separate NEA serves as the regulatory enforcement arm of the MOEWR. Commensurate with its investment in water, Singapore has also embarked on an aggressive economic development strategy of building an internationally renowned water technology industry. Singapore set up the Environment and Water Industry Program Office in 2006 and the government committed S$330 million to fund research and workforce development in the water industry. In 2011, the government added another S$140 million, bringing the total amount committed to S$470 million, or US$400 million. The bulk of this money supports research at the two major water research centers, but individual grants such as the innovative technology challenge are also available. EWI also provides PhD Scholarships for both national and international students to study water technologies.

These scholarships are redeemable at not only both national institutions, but also international centers,ebb and flow trays although only native Singaporean are eligible for international scholarships. Complementing the government’s efforts, is the regional trade association, the Singapore Water Association . SWA aggressively markets Singapore as a one-stop center for all water related services and as a water technology hub. The SWA is a government and business partnership with over 220 local members . Many members are local representatives of international water services and technology firms such as MWH Global, Suez, Mitsubishi, CDMH, etc. Singapore budgets S$50 million a year in government subsidies to entice water innovation companies to locate and maintain operations there. The small country also has over 70 local firms . The SWA is active at every scale all the way from the huge annual international conference to small local workforce development workshops. The association holds multiple monthly meetings, produces a monthly newsletter, conducts biweekly workforce development events, provides industry certifications, and even hosts a monthly casual “Singapore Water Industry Nite” . Singapore desires to be more than simply a gateway to neighboring international markets. Singapore’s strategy has been to develop local expertise through its infrastructure investment and research. Towards this aim, it has established numerous partnerships, notably through joint venture agreements used for the construction of the NEW water plants.

Singapore has recently signed a partnership agreement with Siemens Water Technologies, to build a new water treatment plant that uses half the energy of existing technologies . It should be noted that Siemens Water Technologies global research and development center is now in Singapore. Singapore’s PUB has also recently inked a new partnership with the famous Dutch water consulting agency, Delft Hydraulics, to develop a regional center of excellence for water knowledge . Singapore has agreements with the Dutch Kiwa water research center, and the important American trade industry group, American Water Works Association. as well as several others .Vying with Singapore and Israel for the world’s best branded water industry cluster is Australia. Since 2002, the already world’s driest continent, has been in the grips of a severe drought, which some have even called a thousand year drought. A few Australian economists have even estimated that the drought has shaved a percentage point of GDP growth annually . Water governance reforms begun in 1994 have created a more commercial industry and led to major improvements in water productivity and efficiency and growth in the domestic industry . The government’s National Water Initiative program is a partnership among local, state, and federal government that continues the reforms intended to develop a more efficient industry. In 2008, the government created a $13 billion ‘Water for the Future’ investment program to provide funding for water purchases, irrigation modernization, desalination, recycling, and storm water capture. Knowledge creation is a central goal of the efforts and two new research centers dedicated to water technology have been created, the Center of Excellence for Water Recycling and Center of Excellence for Desalination .

Since the turn of the millennium, Australia has experienced major growth in water technology patents . Desalination plants and city-wide wastewater recycling systems are also being pursed. In Australia, the use of recycled water is increasing, and now totals 5% of total consumption . In 2006, the government also implemented a new certification standard, the Watermark, for water supply, sewerage, plumbing and drainage goods . Government entities and the private sector have together recognized the economic growth potential of an export oriented water industry and are working hard to bolster Australian water technology and engineering firms, most actively in neighboring southeast Asian countries. Today Australia is widely recognized for its progressive water management practices. The last decade has seen the rise of multiple industry associations such as Australian Water Association , Water Australia, Water Supply Advocate, and the Australia Water Industry Alliance . For example, Water Australia aims to triple exports of Australian water technologies by 2015 . These various agencies are now working closely with governmental departments such as the department of Innovation, Industry, Science and Research and Australian trade commission to promote Australian water industry expertise throughout the world. That trade promotion has begun to pay off, for example,4×8 flood tray the Australian Water Industry Alliance claims to have grown annual exports of South Australian water technology products and water management services from less than $30 million in 1998 to over $550 million in 2011, over $3 billion in total export revenues . Australian firms such as engineering giant Whorly Parsons, Earth Systems, or SMEC have parlayed their mining and water engineering expertise into a global presence. Smaller and midsize firms with niche products such as specialized pump technologies, have also seen growth in exports . In 1994, well before the current crisis and growing public cognition of the countries vulnerability to global climate change, the Council of Australia Governments agreed to a significant water reform framework. The reforms were intended to make the industry more commercially oriented and responsive to change. Operational and regulatory responsibilities were separated throughout the country and are now administered through state or local bodies. Local utilities were reformed into large integrated state-owned corporate water service providers. Australia has over 300 utilities, but 71% percent of the population is served by the 22 largest ones centered around the major urban centers . Despite its vast size, Australia is heavily urbanized with over 90% of the population connected to a main water supply. Local competition in the procurement of capital items and facility management services was strongly encouraged and has resulted in more economic development . Significantly, water price subsidies were removed from Australia, which has been credited by industry as resulting in much higher investments in efficiency improvements. Water rights were also reformed and today, users purchase entitlements that can be bought and sold, with the result of a water market arising with rural to urban water transfers . The trading market has been very successful for farmers, particularly during dry years. For example in 2009, there were transfers of over $220 million in the south Murray Basin alone. As a result of these reforms, Australia’s water productivity has increased over 50%, and urban efficiency has improved over 25% . Water knowledge creation, at least as measured by Australia water technology patent production, has thrived since the reforms. Australian inventors filed 1,475 patents between 2004-2006, over one third of all water technology patents filed in the country . A large portion of the patents is for residential or one off uses, such as rain collection, rather than industrially oriented products . This is expected to change as industrial producers have rapidly embraced conservation technologies and many water intensive industries are incorporating direct water reuse technologies .

Large investments in desalination and water recycling will also increase research and development opportunities and capabilities. For example, the city of Melbourne is currently building the world’s largest desalination plant which will complement the country’s existing plants at Sydney, Perth, and the Gold Coast. One significant barrier to innovation, however, is the relatively conservative financial sector. Australia has a developed conventional financial industry, but historically it has not been a major source of capital for small technology startups . Nor is there an overly large venture capital presence. Surveys of inventors find that lack of resources is a major impediment to commercialization . Similarly, surveys of the renewable energy sector have also found that financial tools are an impediment to industry growth. The federal government has developed grant and loan programs aimed at overcoming this gap but potentially more is needed . Additionally, various federal and state governments have developed business incubators aimed at connecting innovative start ups with capital. Several niche start ups have prospered, particularly in wastewater products. For example, Biogill Environmental, invented an innovative membrane technology; Aqua Diagnostics, pioneered a wastewater sensor; Environstream Solutions created a new filter technology . As the water reuse industry grows internationally, these firms are well positioned to thrive. Another significant development brought about by the 1994 reforms, is that the federal government’s Bureau of Meteorology now prepares the annual report, the National Water Account, which details volumes of rainfall, water stocks and current flows, water use, water rights, and water market trades . This public information is used to plan water management accordingly. The government has also created the “Water for a Healthy Country Flagship Program” a long-term water urban planning program with a goal of developing integrated urban water resources management approaches that utilize the full water cycle from storm water capture to effluent discharge . This is a very important governance goal that is similar to Singapore and Israel’s strategies of comprehensive water resource management. The Netherlands has also moved decisively in this direction with water management now handled under central ministry of infrastructure and the environment. In Australia, however, implementation of integrated management practices has been somewhat spotty in practice. For example, Benetini and Brown’s study of the drought stricken western Australia city of Perth, found major institutional barriers despite the overwhelming compelling logic of integrated management. As most parts of the water process are managed separately from one another, each player is only focused on their role rather than the complete picture. Information was not easily shared, single use infrastructure continued to be the preferred method, and incentives were misaligned . Findings such as these have potential ramifications for continued reforms. Many water resource managers recognize the logic of integrated water management, particularly as a cost effective new source, yet are cognizant of the structural tension that currently exists. Therefore, many have concerns about additional reforms. In a policy paper on additional possible reforms, the Australia Water Alliance explicitly cautioned that planning must continue to be integrated with service delivery. The AWA further warns that unplanned “disaggregation of the industry” could diminish institutional knowledge capacity and aggravate the problem. . The United States water and wastewater industries are the world’s largest and most developed. In 2011, sales topped $136 billion—over a quarter of the global total. Since 1994 the industry has nearly doubled in size, growing an average of 4.1% every year. . There are hundreds of thousands of water businesses in the United States. Accordingly, there are also numerous regional centers of water technologies. I do not intend to get into a detailed explanation of the workings of the United States water technology innovation system here as I discuss that in much more detail in my analysis of southern California in the chapters that follow. Instead I wish to briefly discuss three regional centers here. Again I would like to note that a case could be made for other regional water technology centers. For example, the Tristate New York, Pennsylvania, New Jersey region is a very strong player in water technologies with US headquarters of Siemens Water Technologies, Dow Chemical, Pall, and the newly water focused ITT spin off Xylem .

Meanwhile, enzymes involved in GSH synthesis, regeneration and transport appeared to work in concert to maintain GSH homeostasis during acetaminophen transformation and detoxification. Detoxification by GSTs is known to play an important role in the biotransformations of a multitude of xenobiotics in plants . When emerging contaminants such as acetaminophen are introduced in agroecosystems, GSTmediated detoxification may serve the purpose to minimize their potential phytotoxicity to susceptible plants. On the other hand, however, the conjugation may effectively conserve the parent compound and its biological activity, if deconjugation occurs, e.g., in the human digestive tract. Conjugates back transformation to other biologically active compounds has been reported for benzotrizole , triclosan , and naproxen . Thus, understanding the toxicological consequence of Phase II conjugates of such emerging contaminants in agricultural plants may improve risk assessment of reuse practices of treated wastewater and biosolids. Moreover, non-food plants capable of such detoxification may be used for removing such trace contaminants, in settings such as storm water basins,vertical grow table wetlands and vegetative buffers.Southern California’s urbanization was accomplished through the technologically-aided harvesting of faraway water sources.

Currently, procuring additional freshwater sources using conventional water delivery infrastructure is expensive, environmentally damaging, and rarely attempted as there are no longer sufficient freshwater supplies within reach. Climate change models predict a long-term decline in Sierra Nevada snow pack, threatening current freshwater sources. In anticipation of this, Southern California utilities have begun shifting to a strategy of conservation and the development of local sources such as groundwater, desalination, or recycled water. Progressive water agencies have built advanced infrastructure projects like Orange County Water District’s Groundwater Replenishment System , the world’s largest and most technologically advanced indirect potable water reuse project. At the same time, the region has been mired in a prolonged multiyear economic downturn leading many observers to advocate economic development through nurturing environmentally sensitive “clean tech,” industries. This thesis investigates the Southern California water industry in general and, specifically, the regional innovation system that has generated major breakthroughs in water reuse technology and infrastructure. It seeks to understand how this technologically innovative system emerged and the prospects for the region’s future economic growth. Using an analytical framework derived from economic agglomeration scholarship, innovation systems literature, and an international case study analysis of the water technology innovation systems of Israel, Singapore, Netherlands, and Australia, this study employs qualitative interviews, industry analysis, and a case study of the GWRS project to investigate the regional water industry. It finds a strong local industry—often led by a forward looking state government—with auspicious growth prospects, but with significant local impediments.

The fact that Southern California has become a global leader in the development of water reuse technologies and infrastructure is notable because water reuse often evokes strong negative psychological reactions. Historically, “toilet to tap” campaigns against water reuse have been very successful, leading agencies and voters to reject reuse as a legitimate water source strategy. Nevertheless, the region has since become a global leader in water reuse. My case study analysis of GWRS found a set of unique circumstances starting with early salt-water intrusion on its wholly owned groundwater basin. As a result, the Orange County Sanitation District began to invest in the water reuse industry in 1976, focused on the GWRS’s groundbreaking predecessor, Water Factory 21. This early investment helped spur major private and public water reuse research and investment leading to a strong local industry in the region. Today the region is home to numerous water technology leaders—particularly in irrigation as well as the membrane technologies that are critical to water reuse and desalination. The region also has a large number of innovative start-ups, as well as many of the world’s leading water infrastructure engineering service firms. Compared to other regional world technology case studies, however, the local industry appears less motivated in banding together and marketing its regional expertise. Water delivery systems are comprised of multiple differing technological systems often with highly individualized components, making it difficult for entrepreneurs to scale or export products to different markets. Water infrastructure systems are also highly capital intensive, and managed by public utilities that are often risk—and publicity—adverse.

In the Southern California region, direct and indirect price subsidies, inefficient water pricing regimes, and water utilities dependent on volume pricing models for economic survival, all conspire to reduce investment in innovative water technologies. Furthermore, the region’s severe fragmentation of markets, customers, and particularly governmental administrative bodies—who often have conflicting missions—greatly complicates private sector decision-making, inhibits the development of return on investment calculations, and retards potential industry growth. Confusing legal institutions regarding ground water rights, pollution responsibilities, and flood water management further complicate the development of innovative solutions to water problems. Despite these multiple and significant impediments, there is a vibrant and innovative regional water industry which is a global leader in water reuse and recycling technologies. Historically, California has been a global leader in progressive water legislation and water related research, which has helped spur these industries. The state has continued this tradition but now often lags more progressive regions. The state should consider fundamentally reorganizing its water governance institutions in order to align incentives, reduce inter-agency competition, and manage limited regional water supplies more effectively. Water subsidies should be lessened and water agencies’ financial viability should be decoupled from volume of water sold. Integrated water management strategies with appropriate groundwater management rules should be implemented, and enforced, by appropriate administrative bodies. Such a reorganization would spur economic growth as the local innovation system adapts. Additionally,mobile vertical grow tables the regional industry should consider working more closely together in order to promote the industry as a force for economic development and maintain the region’s position as a global center of water innovation and technological development. For example, the region was once home to the one of the world’s dominate water technology corporate conglomerations, US Filter, now Siemens Water Technologies with national and international headquarters in Pennsylvania and Singapore respectively. Today Singapore, rather than Southern California is Siemens’s global research and development center. Other regions, even neighboring Northern California are rapidly growing their water technology expertise—occasionally at the expense of Southern California. More effective regional governance and better public/private cooperation would further strengthen the region’s already robust industry and accelerate the local creation of technological innovation. In January 2010 the board of the Metropolitan Water District , the regional agency that supplies over half the water to Southern California, commissioned a Blue Ribbon Committee of experts charged with rethinking the powerful agency’s fifty year business plan, Vision 2060. After a thorough analysis of Vision 2060, the committee made an additional recommendation that MWD should become a global leader in managing water in arid urban environments; furthermore, they should take a leadership role in fostering the economic development of the region through active promotion of innovative technologies.

The committee thought that Metropolitan “could help create an industry cluster of water technologies and management, of potential global significance in Southern California” The Blue Ribbon Committee’s recommendation is well-grounded in regional economic development theory. Economic geographers have long understood that local specialized demand conditions—in this case supplying water to a dry urban region—can spur local innovation in equipment and services. This local innovation can give the business that supply those products and services a competitive advantage over similar businesses from other regions . The businesses that supply those goods and services can use that competitive advantage to profitably export, and thereby grow the local economy. In fact, this is already happening in Southern California’s water industry where a globally significant industry cluster in water reuse technologies and the design and construction of reuse infrastructure is emerging. Furthermore this cluster is a thriving center of innovation, and potentially a vehicle for local economic development. Innovation is an invention of economic significance . Understanding how innovation occurs is absolutely critical to modern economic development. Today’s modern economic growth theories are premised on the idea that growth comes from increasing returns to knowledge. Yet these theories—and traditional neoclassical economic theory as a whole—say surprising little about the creation of knowledge in general, much less how knowledge becomes innovation . Innovation is almost always the result of learning, which occurs through proximity or via social networks. Therefore, innovation scholars have turned to studying propinquity and association as a—if not the—key source of innovation . Understanding how a region promotes—or hinders— innovation is one of the most important questions in regional economic policy today. Accordingly, this thesis seeks to understand how Southern California’s strength in water technology innovation developed, what sustains it, and what factors could transform the region into the BRC’s vision of a global center of water innovation. Arguably, California’s history has been forged through successful innovation in the water sector more than any other region in the modern world. In the twentieth century, California water agencies built the world’s most advanced system of waterworks. This system propelled the state’s growth into the nation’s most populous and prosperous state. Well before then however, California already had a very proud pedigree in water, with numerous technological inventions, engineering feats, and management innovations. The 1849 gold rush spurred the local invention of hydraulic sluicing that felled whole mountainsides in pursuit of yellow riches . This technological innovation enabled infrastructural projects such as the Los Angeles Aqueduct of the Panama Canal to be built, without which the world would be a very different place. The massive mining and lumber operations of the nineteenth century required equally massive water networks to transport goods to market; they are gone today but the legacy of those operations has likely contributed to why California is now home to four of the ten largest engineering firms in the world . When pioneering farmers flocked to the verdant but salty Central Valley of California they not only built wells and canals and water agencies to manage them, but also created new water rights legal systems to prevent disputes and enable effective allocation . California’s technical prowess in water technologies came in handy when oil was discovered and expertise in piping and pressure management was required to bring it forth, spurring the growth of a whole new local industry including the world’s first offshore oil platforms . When Hollywood followed the oil industry to Southern California and the population exploded— along with the real estate market—the state created innovative institutions like the Metropolitan Water District to ensure an ample water supply for regional growth. Growth came shortly as the aeronautics, defense industry, manufacturing, and communications industries, and more followed Hollywood. MWD is the powerful engineering and water distribution organization that built the massive Colorado River aqueduct and now supplies nearly two thirds of all water to the semiarid south coast . California’s economic growth has always followed the innovative water sector. But what is a “water sector”? The procuring of, treating, transporting, collecting, and discharging of water, all utilize different technologies that originate from several very different industries. Indeed our modern taxonomy of SIC codes does not have a unique SIC code for “water sector.” Instead there are a whole range of codes—water utilities, water systems , irrigation, various levels of pipe manufacturing, chemical treatments, and manufacturing—covering the multiple technologies involved in the “water sector.” . Additionally critical service providers, such as engineering consultants and contracting firms not only fall outside of water industry sectorial codes, but they also often work in other sectors as well. Many of the technological goods produced for the water industry, such as piping materials and chemical treatments also have significant overlap with external industries, such as the oil and gas industry or chemical engineering. The lack of a clear definition and obvious distinctions from other industrial codes make water a very difficult “sector” to analyze. For the purposes of this investigation I define the water industry as all of the firms, agencies, contractors, and technologies that are involved in the extraction, transport, distribution, use, collection, and discharge of water. This would include everything from large-scale infrastructure contractors, to pipe manufacturers, to groundwater geologists, to specialized filter manufactures to chemical treatment suppliers to water monitoring device manufacturers. I concede this is perhaps an overly broad definition–certainly too broad to study sectorial trends effectively— however, despite its inelegance this is an accurate description.

The potential beneficiaries of this guide include building owners, designers, energy modelers, users, building developers, building facility managers and operators, building product manufacturers, and other stakeholders. A wide diversity of building costs, services, and comfort levels requires application-specific design for optimizing energy efficiency. A small portion of the office stock consists of unconditioned, lower-cost indigenous buildings; with arguably acceptable low-energy solutions for comfort levels adapted to regional and climatic considerations. The bulk of the existing stock consists of mass-produced business-as-usual office buildings with a typically lower level of services , or fitted with ad hoc air conditioning to provide ostensibly higher level of services. However, the BAU trend is toward the construction of new, air-conditioned, sophisticated buildings that provide international levels of service. With the exponential increase in the floor space of this type of buildings, the projected growth in building cooling demand will be explosive.Several building physical systems have been adopted from western applications without accounting for the regional, climatic, cultural, and economic context. On the other hand, several region specific systems already exist in indigenous buildings that are able to offer higher performance for minimal cost, but the methods used to design such buildings are rapidly disappearing,plastic pots for seedlings because of a lack of visible documentation and analysis of the techniques.

India needs appropriate localization of energy-efficient technologies to meet the needs of various regions, with respect to weather, standards, materials, construction, and technological maturity . Buildings in India were traditionally built with high thermal mass and used natural ventilation as their principal ventilation and cooling strategy. However, contemporary office buildings are energy-intensive, increasingly being designed as aluminum and glass mid- to high- rise towers . Their construction uses resource-intensive materials, and their processes and operations require a high level of fossil fuel use. A large share of existing and upcoming Indian office space caters to high-density of occupancy and multiple shift operations. Whereas the average for U.S. government offices is 20 m2 /occupant and for US private sector offices is 30 m2 /occupant, Indian offices have a typical density of 5–10 m2 /occupant. Business Processing Office spaces have three-shift hot seats—a situation that while conserving space because of its multiple usage also leads to considerably higher EPI levels. . Moreover, with the increased demand for commercial office spaces from multinationals and IT hubs, and the current privileges being accorded to Special Economic Zones , the trend is toward larger buildings with international standards of conditioned spaces, dramatically increasing the energy footprint of Indian offices .Building energy consumption in India has seen an increase from 14% of total energy consumption in the 1970s to nearly 33% in 2004-2005.

The gross built-up area added to commercial and residential spaces was about 40.8 million square meters in 2004-05, which is about 1% of annual average constructed floor area around the world and the trends show a sustained growth of 10% over the coming years, 4 highlighting the pace at which the energy demand in the building sector is expected to rise in India. In 2004– 2005, the total commercial stock floor space was ~516 million m2 and the average EPI across the entire commercial building stock was ~61 kWh/m2 /year. Compare this to just five years later in 2010, when the total commercial stock floor space was ~660 million m2 and the average EPI across the entire commercial building stock almost tripled to 202 kWh/m2 /year . Energy use in the commercial sector is indeed exploding, not just due to the burgeoning of the Indian commercial sector- India is expected to triple its building stock by 2030 , but also through the increase in service-level requirements and intensity of energy use. Thus there are two intertwined effects: an increase in total building area and an increase in the EPI. According to India’s Bureau of Energy Efficiency , electricity consumption in the commercial sector is rising at double the rate of the average electricity growth rate of 5%–6% in the economy. To deliver a sustained rate of 8% to 9% through 2031-32 and to meet life time energy needs of all citizens, India would need to increase its primary energy supply by 3 to 4 times and electricity generation capacity about 6 times.

According to UNEP, approximately 80%–90% of the energy a building uses during its entire life cycle is consumed for heating, cooling, lighting, and other appliances. The remaining 10%–20% is consumed during the construction, material manufacturing, and demolition phases. 6 To manage and conserve the nation’s energy, it is imperative to aggressively manage building energy efficiency in each commercial building being designed and operated in India. By increasing energy efficiency in buildings and other sectors such as agriculture, transportation, and appliances, it is estimated that the total Indian power demand can be reduced by as much as 25% by 2030. 7 To this end, the best practices outlined below identify processes and strategies to boost the energy efficiency in buildings, while also focusing on cost efficiency and occupant comfort.Just as no two buildings are identical, no two owners will undertake the same energy management program. It is also improbable to include all the listed best practices into one building, since some of them will conflict with each other. The practices are presented individually; however, they should not be thought of as an “a la carte” menu of options. Rather, designers and engineers, developers, and tenants need to work together to capitalize on the synergies between systems . From the demand side, this means implementing a suite of measures that reduce internal loads as well as external heat gains . Once the demand load is reduced, improve systems efficiency. Finally, improve plant design. This is illustrated through the Best Practice strategies and Data Points in this guide. The supply side can then add value by provision of renewables, waste heat sources, and other measures that are beyond this guide’s scope .The guide illustrates innovative strategies and technologies across office buildings in India. It focuses on cross-cutting, whole-building strategies, as well as systematic measures for each load type . Tables of quantitative metrics allow for apples to-apples comparisons, and provide hard targets. The “standard” data in the tables reference numbers from ECO III bench marking or the National Building Code of India. This “standard” data is representative of the median or 50th percentile of commercial buildings in India. For “better” practice data,blueberries container either the Energy Conservation Building Code or better performing buildings have been referenced, and are representative of the top quartile. For the “best” practice data , the top 5th percentile, or best-in-class buildings have been referenced. To design and operate an energy-efficient building, focus on the energy performance based on modeled or monitored data, analyze what end uses are causing the largest consumption/waste, and apply a whole-building process to tackle the waste. For instance, peak demand in high-end commercial buildings is typically dominated by energy for air conditioning. However, for IT operations, the consumption pattern is different. In the latter, cooling and equipment plug loads are almost equally dominant loads.

The equipment plug load is mostly comprised of uninterrupted power supply load from IT services and computers, and a smaller load is from raw power for elevators and miscellaneous equipment. Figure 8 shows typical energy consumption end-use pies — energy conservation measures need to specifically target these end uses. By doing so, one can tap into a huge potential for financial savings through strategic energy management. However, a utility bill does not provide enough information to mine this potential: metering and monitoring at an end-use level is necessary to understand and interpret the data at the necessary level of granularity. Energy represents 30% of operating expenses in a typical office building; this is the single largest and most manageable operating expense in offices. As a data point, in the United States, a 30% reduction in energy consumption can lower operating costs by $25,000 per year for every 5,000 square meters8 of office space. Another study of a national sample of US buildings has revealed that buildings with a “green rating” command on an average 3% higher rent and 16% higher selling price. 9 Whether in the US or India, improvements to energy efficiency can often be attained through no-cost or low cost ECMs that lower the first costs of construction and equipment. Optimizing building loads can lead to lower first costs and operating costs. By targeting low-hanging fruit through early-stage ECMs, the first costs saved through these can be applied toward more expensive technology solutions like high-quality glazing or sensors that can further the energy and cost benefits later in the building life cycle. Hence, it is important to decide which measures to prioritize initially, and then what to cross-subsidize eventually. For example, if one is able to save costs by reducing the number of lighting fixtures and taking advantage of high daylight levels in a space, then those savings can be used to install daylight sensors. The latter can provide a large cost benefit with a relatively short payback time by driving down the operational hours for artificial lighting. The ECMs at the whole building level using systems integration can greatly benefit the EPI of a building. Table 1 shows whole building energy use metrics, using Standard , Better , and Best Practices at the whole building level.Optimizing day lighting and lighting can provide better views and improve the visual acuity of the occupants. Well-designed mechanical systems can improve indoor air quality while reducing initial equipment and operating energy costs. Workplace productivity can increase by providing individual controls, and with direct access to daylight. Given that the bulk of working time is spent indoors, a better indoor environment can boost worker performance and reduce sick leave that could equate to monetary benefits to businesses.Displacement ventilationdelivers the air at low speeds using the principle of air stratification. Here, air is delivered at close to floor level for primarily conditioning the occupied volume and extracted at the ceiling height rather than conditioning the unoccupied higher volume first. Well designed DV systems provide better indoor air quality since the air in the occupied zone is generally fresher than that for mixing ventilation. There are no perceived air drafts. Any released pollutants rise rapidly to above the occupied zone. Large cooling energy savings are possible, as it uses a higher supply air temperature at 18°C, which also increases the efficiency of mechanical cooling equipment and lowers equipment requirements. Underfloor Air Distributiontechnology uses the underfloor plenum beneath a raised floor to provide conditioned air through floor diffusers directly to the occupied zone. A thoughtful design can overcome the usually cited challenges of uneven floor surfaces, difficulty in providing added airflow to the perimeter of the building, and perceived control difficulty. The advantages of a well-designed UFAD system are: improved thermal comfort, occupant satisfaction, ventilation efficiency and indoor air quality, reduced energy use and the potential for reduced floor-to-floor height in new construction. Radiant Cooling works on the principle that water can store 3,400 times more thermal energy per unit volume than air. It offers the potential to reduce cooling energy consumption and peak cooling loads when coupled with building thermal mass. Some radiant systems circulate cool water in dedicated panels; others cool the building structure . Because radiant surfaces are often cooled only a few degrees below the desired indoor air temperature, there are many opportunities for innovative cooling energy sources, such as night cooling and ground-coupled hydroponic loops. The heating and cooling supply water temperatures for radiant systems operate at higher set points compared to traditional systems. The radiant cooling system supply water temperature would typically operate at 15°C–18°C for cooling, whereas typical supply water temperatures for a traditional forced air system are around 5.5°C–7.5°C. The central cooling equipment can operate more efficiently at these temperature set points.In a typical office space, the airflow required to cool and ventilate the space can be three to four times greater than that required to just ventilate the space. If the space cooling is decoupled from the ventilation, especially through a hydroponic system, the central air handling system and associated distribution system can be downsized accordingly.

Given this finding, we cultured the strains that had exhibited putative surface-dependent regulation of surfactant production for their ability to induce drop collapse when grown in viscous broth. While P. syringae B728a does not produce a surfactant capable of conferring drop collapse from normal broth cultures, it did so when grown in a viscous broth. A similar induction of surfactant production was induced by growth of other environmental strains of P. syringae, as well as Pantoea strain PB64 in viscous broth. Interestingly, P. fluorescens strain PB59, which produced surfactant only in broth media, still produced abundant biosurfactant detectable by drop collapse when grown in viscous broth , suggesting that its biosurfactant production is regulated by a different mechanism. Although it is tempting to speculate that the P. syringae and Pantoea strains are sensing a surface by directly measuring viscosity, growth in viscous broth could be indirectly stimulating biosurfactant production via alteration of growth patterns such as cell aggregation which was stimulated by the reduced turbulent drag of this culture medium. Vigorous shaking of P. syringae cultures reduced pellicle formation and resulted in a lower induction of syfA. 

Since leaves are a waxy habitat, we hypothesized that the phyllosphere is enriched for biosurfactant-producing bacterial taxa due to the benefits this phenotype may confer. To test this hypothesis we examined the incidence of this trait in bacteria from different habitats including leaf surfaces using the atomized oil assay. Using this assay,garden pots for vegetables we screened over 5,000 bacteria recovered from leaf surfaces, soil, and freshwater samples in close proximity to each other in the early spring, when there were many ephemeral pools of water and streams. To determine the frequency of surfactant production in bacterial populations this trait was assessed in approximately 50 random strains per sample, and at least 30 samples were collected for each environment. The frequencies at which surfactant producers were found in a community from a given sample ranged from zero to close to 90%. Overall, a much lower frequency of surfactant producers was observed in freshwater samples than from leaf surfaces or soil. Student’s t-test with unequal variance comparing the frequencies of surfactant production revealed that leaves and soil harbored significantly higher frequencies of bacteria with this phenotype than water. Interestingly, while soil and leaf surfaces harbored a similar average frequency of surfactant producers, there was a much higher deviation in this frequency between samples of leaves than soil; nearly 30% of the leaf samples harbored no surfactant producers compared to 17% and 6% for water and soil samples respectively.

Conversely, many leaves also harbored very high proportions of surfactant producing bacteria. Several features of leaves were examined in an attempt to account for the substantial sample to sample differences in frequency of biosurfactant-producing bacteria. Given that the leaf surfaces of different plant species differ in hydrophobicity, we addressed whether plant species or the degree of water-repellency of leaves was predicative of the fraction of surfactant-producing bacterial strains recovered. There was no correlation between leaf hydrophobicity, measured as the total area covered by a 10 ul droplet of water applied to the leaf, and the frequency of surfactant producers. Likewise no association between plant species and the frequency of surfactant producers was evident , although more species would need to be examined to rigorously test this conclusion. Overall, our observations suggest that leaf properties are not the dominant factor that leads to the occurrence of surfactant-producing strains on a given plant. However, since our collections were made in early spring, the leaves examined were all at early stages of growth and thus the microbial communities were also in early stages of colonization. The apparent random patterns of occurrence of bacteria on the leaves therefore suggests that colonization can be described by a neutral theory of competition. As such, the abundance of a given bacterial strain on a leaf is reflective of its early time of arrival on that plant, and largely dependent on chance. In comparison to leaf surfaces, a much more uniform frequency of occurrence of surfactant production was observed in bacteria from soil and water.

There was no apparent effect of the source of water on the incidence of surfactant production in these samples, since about 5% of the bacteria in all samples from streams, ephemeral pools and a lake produced biosurfactant. Additionally, the frequency of surfactant-producing bacteria found in a given soil sample was not correlated with that from adjacent plant samples , suggesting that mixing of bacterial members of these two communities was not prominent. The application of the atomized oil assay to a wide variety of environmental bacterial strains and synthetic surfactants revealed it to be both more versatile and sensitive than the more commonly used drop collapse assay. The atomized oil assay confirmed surfactant production in every bacterial strain in which surfactants were detected using the drop collapse assay. More importantly, several bacterial strains were identified that produced either low amounts of surfactant or apparently hydrophobic surfactants that were not detectable using the drop collapse assay. The atomized oil assay readily confirmed biosurfactant production in taxa in which it had previously been described. The majority of the strains that produced surfactants detectable by both tests belonged to the genera Pseudomonas and Bacillus , both of which have been described in the literature to produce biosurfactants that lower the surface tension of water. Likewise, the Pantoea strain PB64 may produce rhamnolipids as do other members of this genera , although this was not verified. While surfactant production has not been previously documented in Staphylococcus, some species of this genus have been observed to be motile on swarming plates , suggesting their production of surfactants.

The identification of such previously recognized surfactant-producing taxa emphasizes that while the drop collapse assay is suitable for finding such biosurfactant producers,flower plastic pots the atomized oil assay may be more readily employed due to its high-throughput capability and higher sensitivity. The atomized oil assay was particularly useful in identifying biosurfactants in taxa in which this trait had not previously been shown. The surface-active compounds that are produced by the seven strains that were detectable only with the atomized oil assay would have escaped attention in most other studies; these compounds may well have unique biological functions and/or potential industrial applications. For example, our assay detected the hydrophobic pumilacidins produced by Bacillus pumilis which have been documented for their potent antibiotic and antiviral properties , although their surfactant activity has previously been ignored due to their low water solubility. Likewise, we detected surfactant production by a Rhizobium strain ; although we have not verified the compound, we suspect it could be similar to the long-chain AHLs produced by Rhizobium etli, which cannot be detected with a drop collapse assay but are documented as surfactants with a dual role in quorum sensing and swarming motility. Furthermore, a biosynthetic gene cluster proposed to synthesize a surface-active lipopeptide virulence factor was identified in the genome sequence of the plant pathogen Xanthomonas axonopodis ; although incapable of imparting drop collapse, both an authentic culture of X. axonopodis pv. glycines as well as a related environmental strain found in this study produced compounds detectable with the atomized oil spray. Biosurfactants detectable only with the atomized oil assay were also observed in a Cedecea strain, a taxon not previously known to produce surfactants; this feature may prove biologically important to its success as an opportunistic pathogen. Therefore it appears that application of the atomized oil assay in environmental surveys might greatly expand our knowledge of novel biosurfactants. While the atomized oil spray assay has many advantages over other assays there are some limitations that could bias the detection of surfactant producers. This assay best identifies bacterial strains that produce “bright” halos around colonies , although we have previously shown that some highly hydrophilic synthetic surfactants can modify oil droplets to appear “dark” due to their flattened nature. 

“Dark” halos are less visibly obvious and no strains that unambiguously exhibited this appearance were found in our survey even though we approached the study with the expectation that we would find biosurfactants of this type. We were surprised that we did not find any biosurfactants that yielded a water drop collapse and such a “dark” halo. Bacteria that produce such compounds must thus be quite uncommon, or it may be that such surfactants are not easily distinguished or detected by either assay. Another limitation of the atomized oil assay, which is shared with any culture-based assay, is that the nutrient medium that we used may have precluded us from detecting production of surfactants by some strains which require specific conditions for surfactant production. Furthermore, our assay is restricted to surfactant production by culturable organisms, although there is evidence that at least on leaves the most common cultured taxa are also among the most prevalent taxa identified by culture-independent methods. Metagenomic investigation into the prevalence of biosurfactant production could be fruitful in expanding our understanding of their prevalence in bacterial communities, although advances will be limited until more genetic determinants for their production are described. An unexpected finding from this study was that the production of surfactants that conferred a reduction of surface tension was very conditional on whether the bacteria were grown on a surface or cultured planktonically. Although a number of studies have connected surface sensing with swarming motility , we are only aware of one report, of Serratia liquefaciens, which has noted increased biosurfactant production in cells grown on a surface. In the current work we have shown that a surprisingly large proportion of bacterial strains restrict biosurfactant production to growth on a surface. Although most of these surface-dependent surfactant producers were strains of P. syringae isolates, this phenomenon was also seen in a Pantoea strain, suggesting that it may be a common trait. Commonly-used methods of screening for biosurfactants by drop collapse employ broth cultures and would likely not identify such strains. On the other hand, two strains were identified that only conferred drop collapse from broth culture and not from cells grown onplates and subsequently suspended in water drops. However, surfactant production was still detectable in these strains as a small halo of de-wetted oil droplets with the atomized oil spray when cells were grown on plates. The small halo size of these two strains indicates that the amount of surfactant produced by cells grown on plates was probably too low in concentration to be detected by the drop collapse assay; therefore surfactant production was not fully blocked at a surface, but rather dramatically reduced. Although we have not yet encountered such strains, there is the potential for us to overlook biosurfactants which are produced only in broth culture. However, such strains must be uncommon based on our extensive survey, and the high sensitivity of the atomized oil assay should enable even very low production on solid surfaces to be detectable. Presumably the strong environmental-dependent regulation of surfactant production at surfaces is linked to its role in the habitat of some strains. For example, surfactants contributing to biofilm growth or movement on a surface would be pointless if produced in an aqueous environment. Thus, it makes sense that bacteria with multiple habitats should survey their growth environment before committing to production of a biosurfactant. The surface trigger for surfactant production and its conservation among bacterial taxa remains an active area of research.A few specific mechanisms for surface sensing have been investigated, such as two-component systems and flagellar inhibition. Once a surface is perceived, there is growing evidence that cyclic-di-GMP levels control genes involved in cell surface features that participate in processes such as biofilm growth. It is intriguing that increases in viscosity led to increases in surfactant production in this study , much as it has been shown to induce production of flagella in Vibrio parahaemolyticus. However, our results lead us to believe that it is not viscosity sensing per se that is inducing surfactant production, but rather perception of a growth pattern such as cell aggregation that perhaps restricts movement of cells which, in turn is induced by the reduced turbulent drag of a viscous medium.

The top three unigenes representing the greatest number of ESTs encoded proteins related to metallothionein, RD22-like BURP domain-containing proteins, and chitin binding heve in-like proteins. All three of these proteins have functions related to biotic or abiotic stress responses.An analysis of biological function indicates that 27% of the unigenes encode proteins with metabolic activity. Unigenes with NCBI matches encoding proteins with unknown function comprise 14% of the total and another 28% are predicted to be involved in various cellular processes such as protein synthesis and protein degradation.The specific biochemical steps leading to THCA are pro posed to begin with a reaction involving a type III PKS enzyme that catalyses the synthesis of olivetolic acid from hexanoyl-CoA and three molecules of malonyl-CoA. Malonyl-CoA is derived from the carboxylation of acetyl-CoA. ESTs encoding acetyl-CoA carboxylase were identified. Hexanoyl-CoA could be produced by more than one pathway in the trichomes. One route to produce hexanoyl-CoA would involve the early termination of the fatty acid biosynthetic pathway, yielding hexanoyl-ACP. The hexanoyl moiety would then be transferred to CoA by the action of an ACP-CoA transacylase or it would be cleaved by the action of a thioesterase, yielding n-hexanol, which would then be converted into n-hexanoyl-CoA by the action of acyl-CoA synthase.

Most of the enzymes needed for this route are represented in the EST database,macetas de 20 litros except for thetransacylase and 2,3-trans-enoyl-ACP reductase. A second route to hexanoyl-CoA would involve the pro duction of hexanol from the breakdown of the fatty acid linoleic acid via the lipoxygenase pathway. A survey of the sequenced ESTs revealed candidate genes encoding the enzymes needed to synthesize linoleic acid from acetyl-CoA by the typical fatty acid biosynthetic pathway in plastids followed by the production of hexanol from linoleic acid via the LOX pathway. An third pathway related to the biosynthesis of branched chain amino acids has been proposed to be involved in the production of short-chain and medium-chain fatty acids. However, the enzymes in this pathway [2-isopropylmalate synthase, 3-isopropylmalate dehydratase, 3-isopropylmalate dehydrogenase, and 2- oxoisovalerate dehydrogenase ] were not repre sented in the Cannabis trichome EST library. After the formation of olivetolic acid, a prenyltransferase is proposed to add a prenyl group derived from geranyl diphosphate to create cannabigerolic acid. GPP is derived from the fusion of two isoprene units. Two different biochemical pathways support the synthesis of isoprenoids in plants. Within the list of unigenes all but one of the enzymatic activities needed to convert pyruvate and glycer aldehyde-3-phosphate into isopentenyl and dimethylallyl diphosphate via the methylerythritol 4-phosphate pathway were represented. This finding is consistent with isotopic studies showing that the GPP cannabinoid precursors are synthesized via this pathway. 

The formation of GPP is mediated by GPP synthase. Several unigenes related to GPP synthase were identified , however, they were more closely related to other terpene synthases. In particular, CAN36 and CAN55, which possibly were de rived from the same gene, and the closely related CAN37, are most similar to hop sesquiterpene synthases HISTS1 and HISTS2 , with an average identity of 56% over the first 160 amino acid residues. CAN41 is most similar to hop monoterpene synthase HIMTS2.The nature of the prenyltransferase is unknown. How ever, previous studies identified a soluble aromatic geranyl pyrophosphate:olivetolate geranyltransferase in the extract of young leaves with the appropriate activity. The only EST encoding a predicted prenyltransferase was CAN121. However, the encoded protein is more closely related to members of the mem brane-bound chloroplast-localized family of prenyltrans ferases than to soluble prenyltransferases. The final step in the pathway is mediated by THCA synthase, which mediates the conversion of cannabigerolic acid to THCA. Two ESTs with sequences identical to the previous reportedly THCA synthase were identified.Whereas the nature of the prenyltransferase responsible for the synthesis of cannabigerolic acid is unknown, three unigenes, CAN24, CAN383, and CAN1069, comprising eight, one, and two ESTs, respectively, could encode the PKS activity needed to synthesize olivetolic acid. These were therefore characterized in more detail. All three unigenes were represented by individual ESTs encoding complete PKS polypetides. These were sequenced and compared to related PKS sequences. CAN1069 was identical to a previously identified Cannabis gene encoding a chalcone synthase, and is the most closely related of the PKS sequences to other known chalcone synthases from hop and Arabidopsis. The relationships of hop phlorisovalerophenone synthase , which mediates the conversion of malonyl-CoA and isovaleryl-CoA to phlorisovalerophenone, to CAN24 and CAN383 is less clear. CAN24 and CAN383 show 64.6% identity and are nearly equally similar to hop VPS at 71.2% and 72.0%, respectively. The enzymatic activities encoded by CAN24 and CAN1069 were explored in detail. The coding regions of the two genes were inserted into the pHis8 vector in frame with a His8 tag.

The tagged proteins were purified on a nickel-containing magnetic bead matrix and were assayed for chalcone and olivetol/olivetolic acid synthase activities. Recombinant protein from CAN1069, but not CAN24, produced reaction products when incubated with coumaroyl-CoA and malonyl-CoA. The reaction products were analysed by LC-MS and peak 2 was found to have a molecular mass and absorption spectrum consistent with naringenin , the major product of chalcone synthases. Both CAN24 and CAN1069 were capable of using malonyl-CoA and hexanoyl-CoA as re action substrates and LC-MS indicated that products of these enzymes were the same,maceta 5 litros but neither molecular mass nor the absorption spectrum of this product matched olivetol or olivetolic acid. Results similar to CAN24 were obtained using protein purified from CAN383.Genes required for THCA production are probably more highly expressed in glands of pistillate inflorescences be cause this is where THCA is most highly concentrated. To test this hypothesis, the relative expression levels in isolated glands versus young inflorescence-associated leaves of selected unigenes were compared using real-time qPCR. The identity of the genes assayed and the differences in relative expression levels are listed in Table 2 and in Supplementary Table 4A at JXB online. Consistent with this hypothesis, THCA synthase expression was 437 times higher in isolated glands than in leaves. CAN24 was expressed 1600 times higher in glands of the inflores cence than in associated leaves. CAN1069 encoding CHS was also more highly expressed in glands than leaves. The expression of a third PKS, CAN383, was expressed at similar levels in glands and leaves. These results are not explained by poor RNA isolation from leaves as unigene CAN219 encoding chlorophyll A/B binding protein showed elevated leaf expression levels. The activities of several housekeeping genes were also tested. A relatively modest increase in levels of histone H2A and beta tubulin expression in glands compared to leaves was detected. The increase in expression levels of these latter two genes might reflect a combination of the heightened metabolic activity and the unique cellular structure of glandular trichomes. Two different pathways could provide the hexanol re quired for olivetolic acid synthesis, as shown in Fig. 2. Expression levels provide support for the de novo pathway as a primary source, given that CAN498, CAN82, and CAN915 were much more highly expressed in glands than leaves , whereas the relative expression of genes encoding enzymes in the lipid breakdown pathway were depressed or modestly elevated in glands.The identities of the most abundant ESTs derived from the glandular trichomes of Cannabis sativa are consistent with the protective function of plant glands. For example, the most abundant ESTs encoded a protein closely related to type II metallothioneins. These proteins bind heavy metals such as Cd, Zn, and Cu, and their proposed primary function is the maintenance of Cu tolerance. The second most abundant class of ESTs encoded an RD22-like BURP domain containing protein.

This class of proteins contains a hydrophobic N terminal signal peptide, and an N-terminal conserved region followed by a series of small repeats. The BURP domain of approximately 230 amino acids is located in the C-terminal region. The function of RD22-like proteins is unknown but some members of this class of genes are induced by dehydration. The third most abundant ESTs encoded a protein containing a heve in domain. Hevein domains contain a conserved 43-amino acid motif that binds chitin and members of this protein class are known for anti-fungal activity. The unique secondary metabolism in Cannabis may also play a role in plant defence. Synthesis of THCA is extracellular and results in hydrogen peroxide production, which has general antimicrobial properties , and a recent report further indicates that THCA may directly inhibit microbial growth. The analysis of gland-derived ESTs has identified nearly all the candidate genes required for THCA synthesis from primary metabolic products. These findings differ from a proteomic study that aimed to identify genes expressed in Cannabis glands but failed to associate any highly expressed proteins with THCA synthesis. This difference reflects the much greater volume of genomic data enabling more robust identification of DNA sequences when compared to proteomics approaches based on the molecular weights of fragmented polypeptides. This is especially true for species such as Cannabis sativa for which there is little amino acid sequence data available to compare with peptide profiles. The present study highlights the utility of using isolated glands as starting material for making EST libraries to study gland metabolism, as was the case in other plant species. In this study more than 50% of the ESTs with NCBI matches were involved in metabolism or cellular activities such as transport and protein translation. Many other cannabinoids, in addition to THCA, have been identified in Cannabis , and it is likely that many of the genes identified in Supplementary Table 2 at JXB online are involved in the production of these other compounds. In addition to cannabinoids, many other classes of secondary compounds have been found in Cannabis. For example, both monoterpenes and sesquiterpenes have been identified and candidate ESTs encoding activities to pro duce these compounds have been identified.Synthesis of olivetolic acid from malonyl-CoA and hex anoyl-CoA represents the first committed step toward the synthesis of THCA. Olivetolic acid synthesis is predicted to be mediated by a member of the type III PKS family through a series of three condensation reactions producing a triketide. CAN24, represented by eight ESTs and one of the most highly expressed unigenes in our analysis, encodes a member of the PKS family. This gene was expressed 1600-fold higher in glands than in leaves. CAN1068, another PKS member represented by two ESTs, corresponds to a previously identified Cannabis CHS gene. A third PKS represented by a single EST, CAN383, was also identified. Analyses of PKS crystal structures indicate that the type III PKS enzymes are composed of a dimer with conserved reaction centres and a hollow reaction cavity. All three Cannabis PKS genes encoded polypeptides containing the conserved amino acids, Cys 167, His 307, and Asn 340, that are believed to constitute the reaction centre. In addition, two of the three amino acids that are important for defining the size of the reaction cavity in chalcone synthases are conserved. The third amino acid, Thr at position 300 that is conserved in all chalcone synthases, was missing in CAN24 and CAN383. Instead CAN24 and CAN383 contained Leu and Iso, respectively, at position 300. Such differences might alter substrate specificity. It has been proposed that either olivetol or olivetolic acid are products of polyketide synthase in the THCA pathway. However assays of plant extracts found that olivetol, the decarboxylation product of olive tolic acid, was not a substrate in the pathway. Products of the three PKS genes identified in this study were tested for olivetolic acid synthesis in vitro. CAN24 and CAN383 yielded identical products according to HPLC analysis. Because CAN24 was more abundant, this PKS gene was analysed in detail, along with CAN1069, which had CHS activity as shown in Fig. 4. The size of the product produced by the CAN24-encoded enzyme was smaller than olivetolic acid. Further, the absorption spectrum did not match olivetol. A sequence identical to CAN24 has been deposited in the NCBI database and was annotated as having olivetol synthase activity but without supporting data. The product generated in our analyses possibly represents a derailment product in which the enzyme catalyses two decarboxylative condensations instead of three. The failure of in vitro conditions to support the complete synthesis of native PKS products is well documented. 

Stresses on the sector where rigid policies exist, might not reverberate across sectors, such that the nexus is able to conform in a sustainable way to existing conditions. Our results indicate that fluctuations in food production are not controlled by water availability in the region but rather, access to water rights, which signals the decoupled response between agricultural markets, especially alfalfa, and climatic changes. In drought years, we observed a slight decrease in irrigation but an overall increase in total active cropland. Similarly, the amount of hydroelectricity produced is dependent on a minimum water level in Lake Mead that is below the level that indicates a period of critical drought. If an extreme drought were to occur, water allocations based on the Law of the River would occur to an extent, but electricity production would not. This is an example of how national, regional, and global objectives of food, water,maceta 7 litros medidas and energy are often in conflict, making win-wins difficult or impossible to achieve. Inherent in each of the ways discussed above that resource governance impacts the nexus are power differentials that are reified by politics and institutions.

Resource governance is inherently political and often results in outcomes based on the larger goals of the government or states involved in policy making. Rigidity and the ability to respond to market and climatic changes are therefore largely determined by the modes of distribution, appropriation, and allocation of water decided by the states, in this case those of the Lower Colorado River Basin managed by the Bureau of Reclamation. These material forms of resource management are also reflected in nexus discursive practices, that Leese and Meisch describe as a reframing of “the conflict between distributional justice and the needs of the world economy under the paradigm of security.” By emphasizing hydraulic and economic variables, the nexus concept risks placing the needs of the global poor as secondary.This analysis, while not a complete picture due to data constraints, gives insight into how the FEW nexus can be used to better understand what drives production or access to food, energy, and water. However, there are many opportunities for future research to improve the accuracy of nexus quantitative research. In order to fully describe and quantify the components of the nexus for any region, consistent data is needed at the watershed level, for yearly irrigation including pumping costs and energy amounts, on the amount of energy needed to produce and encompassed in agricultural chemicals, of water treatment and prices, and export information that details how much product, where it is from, and where it is going.

Consistent data will allow for frequently updated, automated, and dynamic production scenarios, though static snapshots still provide insight to how resource use or production might change. Analysis at the watershed scale, is valuable because of regional ecosystem characteristics; however, it is complicated because of stakeholder conflict and the arbitrary political boundaries that data often conforms to. In order to ground the nexus in a study area such that it lends to conversations about water management to ensure healthy habitat or the value of ecosystem services in a region, analysis should be done at the watershed level. The international border of the Colorado River Basin between the U.S. and Mexico further exemplifies historical geopolitical struggles, where resource needs coincide and conflict with each other as transboundary struggles at the watershed level. While it is clear based on this analysis that the nexus is deeply connected to policy and economics, the way our social, cultural, and technological systems change will also impact the way resources are produced and used. Energy technology, for example, is improving rapidly, requiring those who study the nexus to understand technological advances as they relate to large scale production, conveyance, fuel use, and emissions. Science silos will not help us manage resources for the future, cross-discipline communication is key. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Human pathogen contamination of produce was the leading cause of foodborne illnesses and outbreaks associated with a single-ingredient commodity between 2004 and 2013. Lack of visual evidence that indicates the presence of contamination on produce contributes to the estimated 9.4 million cases of foodborne illness in the United States annually. Various pathogen groups and toxins can be causal agents of foodborne illness associated with produce; however, non-typhoidal Salmonella ranks as the second leading cause of all illnesses associated with consumption of produce. In a pre-harvest setting, enteric pathogenic bacteria are introduced to fresh produce through many routes, including low quality irrigation water, use of contaminated organic fertilizers, close proximity to livestock operations, wildlife intrusions, improper worker hygiene, or contaminated equipment. Once on the leaf surface, bacteria are faced with harsh conditions, such as UV irradiation,macetas de 1 litro low nutrient and water availability, and unfavorable weather. Bacteria may escape these conditions by attaching to the leaf surface and forming biofilms or by transitioning to an endophytic lifestyle through internalization into the leaf extracellular space via natural pores or wounds. While leaf internalization is likely to confer some protection to the bacteria, it is not without some disadvantages. Plants can detect endophytic bacteria in the apoplast through pattern recognition receptors localized at the cell membrane that perceive conserved microbial molecules known as pathogen- or microbe-associated molecular patterns. PRR-PAMP binding leads to initiation of PAMP-triggered immunity , which functions to prevent further internalization of bacteria and to eradicate those that have already entered the apoplast. This suggests that internalization trades one challenge for another , and only bacteria that can cope with these challenges will be able to colonize leaves successfully.

Previous studies have shown that Salmonella spp. interact with plants in a sophisticated manner, although the exact mechanisms are not fully understood. For instance, similar to some plant pathogens, Salmonella enterica serovar Typhimurium SL1344 can modulate stomatal movement in Arabidopsis leaves, where it induces a transient stomatal closure and re-opening at 4 h post incubation. Stomatal closure can diminish bacterial internalization and subsequent contamination of internal leaf tissues. Bacterium induced re-opening of stomata can lead to higher pathogen load in the leaf apoplast. The mechanism for stomatal re-opening by the phytopathogen Pseudomonas syringae pv. tomatois through the action of coronatine , a polyketide phytotoxin. However, the genomes of STm strains LT2 and 14028s do not encode genes for coronatine synthesis. Furthermore, stomatal re-opening is not a ubiquitous response to human pathogens. For instance, Escherichia coli O157:H7 induces a lasting stomatal closure in lettuce and Arabidopsis for at least 4 and 8 h, respectively. Beyond the ability to modulate stomatal movement, STm SL1344 can survive at a higher tier within the apoplast of Arabidopsis leaves than O157:H7 after surface-inoculation and S. enterica serovar Thompson strain RM1987 can survive at high titers on the surface of romaine lettuce leaves. Therefore, S. enterica may either induce a weak plant immune response or can counteract plant immunity and consequently persist on and in leaves. Internalization and persistence within the apoplast are arguably the most important targets for managing contamination of produce by Salmonella, as endophytic populations cannot be removed through typical washing tactics. Here, we provide details of multiple genomic regions required for internalization and persistence of STm 14028s into lettuce leaves. These genomic regions were identified with a genetic screen of multi-gene deletion mutants of STm 14028s to pinpoint proteins and metabolic pathways responsible for stomatal re-opening and apoplastic persistence. Selected MGD mutants were further characterized regarding their ability to survive in the apoplast, induce hallmark plant defenses, and replicate in apoplastic wash fluid. While all mutants induced a prolonged stomatal closure when applied to the leaf surface, the mutants were found to vary in other aspects of phyllosphere survival.We first confirmed that this strain induces a similar stomatal response to that of STm strain SL1344. 

We evaluated changes in the stomatal aperture width in leaves of young lettuce plants by floating leaf pieces onto bacterial inoculum as previously reported. Both STm strains induced an initial stomatal closure at 2 h post inoculation followed by re-opening at 4 hpi , suggesting that the MGD library could be useful to identify STm genomic regions required for successful stomatal re-opening at 4 hpi. Second, to ensure that lack of re-opening was due to deletion of genes required for stomatal re-opening by STm 14028s rather than temporal factors, the circadian movement of lettuce stomata was determined. This analysis indicated that the stomatal aperture was widest at 6 h after first light. We therefore, chose to start the stomatal bio-assay at 2 hafl to ensure that the 4 hpi time point corresponded to a time with maximum expected stomatal aperture width. A primary screen of 303 MGD strains with a single biological replicate indicated that 177 mutants were unable to re-open stomata, suggesting a high rate of false-positives. Thus, we functionally annotated the predicted deleted genes in these 177 mutants. Considering the current knowledge of STm epiphytic behavior , we reasoned that genes involved in secretion, perception of environmental signals, signaling, and regulatory functions could be involved in opening of the stomatal pore. Thus, we selected 51 MGD mutants based on their functional annotation for retesting with at least three biological replicates. The primary functional units missing in these 51 mutants are described in Supplementary Table S2. From this confirmation screen, only eight mutants were unable to reopen lettuce stomata consistently and they were selected for further characterization. Furthermore, previous results indicated that mutants for the Salmonella Pathogenicity Island 1 and 2 were unable to open lettuce stomatal pores. Thus, we also analyzed two MGD strains from our collection that have a predicted deletion of these regions in addition to a few adjacent genes. To confirm that the lack of stomatal re-opening using leaf pieces floating on bacterial inoculum was a reproducible response that can also be observed in leaves still attached to the plant, we designed a stomatal bio-assay that included infiltration of mature lettuce leaves with STm 14028s, Mut3, or Mut9 bacterium suspensions. In this assay, bacteria are placed in the leaf apoplast, including the sub-stomatal chamber, where they can be in contact with the guard cells. All three strains induced a strong stomatal closure at 2 hpi , similar to observations made using surface inoculation of mature, whole plants. Furthermore, the wild type strain STm 14028s, but not the mutant strains, induced stomatal reopening at 4 hpi , suggesting that this response is robust. To rule out the possibility that the infiltration procedure induced an unpredictable stomatal movement, we assessed the circadian stomatal movement in untreated lettuce leaves as well as leaves infiltrated with water , STm 14028s,or Mut9. Mock-treated and untreated leaves showed an almost identical movement pattern, stomata of Mut9-infiltrated leaves remained closed throughout the daylight period, and STm 14028s-infifiltrated leaves showed a transient reduction in stomatal aperture width at 2 hpi that corresponded to 4 haflfl.Upon completion of this screening procedure, the genome position of the deleted region for each mutant was identified at the nucleotide level by whole genome sequencing of the mutant strains. This procedure, which was readily available in a timeand cost-effective matter, allowed us to predict the genotype and the functional units missing in each mutant using the available STm 14028s and LT2 genome annotations. The genomic regions deleted in all ten selected mutants were also confirmed by PCR. Furthermore, each mutant, except Mut5, was able to swim and swarm , confirming the predicted genotype of Mut5 is missing genes involved in flagellar biosynthesis and chemotaxis. Movement and chemotaxis have previously been associated with STm SL1344 internalization through the stomatal pore. Thus, the identification of Mut5 during the Salmonella genetic screening validates our procedure, which identified known and novel features associated with bacterial epiphytic behavior. The 10 MGD strains were further tested for phenotypic traits required for colonization of leaves as described below. As all selected mutants were unable to stimulate stomatal re-opening at 4 hpi , we sought to determine whether each mutation also affected the population dynamics in the lettuce leaf apoplast.

It is also suggested that to prevent the spreadof Phytophthora spp. from nursery stock to previously uninfected orchards, roots of potted trees should be inspected and tested for the presence of Phytophthora spp.. Even after planting healthy nursery stock, the application of fungicides is necessary in part because young, fibrous roots are more susceptible to root rot than older roots and it is suggested that fungicides, together with nematicides or a soil fumigant be used to protect replants during the first two years of growth, especially if the orchard has a history of Phytophthora problems. The buildup of inoculum during root rot outbreaks can quickly grow and during rain or irrigation events. Sporangia or zoospores, sometimes together, may be splashed up to low-hanging fruit and cause outbreaks of brown rot. Brown rot develops as light brown, leathery lesions that expand until the whole fruit turns brown. Diseased fruit have a characteristic pungent odor and at high humidity, the fruit surface is eventually covered by distinct white mycelium. Both the root rot and brown rot phases are damaging, though while fruit brown rot leads to immediate crop loss,maceta 25 litros root rot outbreaks instead results in a slow decline of infected trees.

Trees infected by Phytophthora spp. eventually show reduced vigor and production. Systematic die back of the canopy occurs when uptake of water and nutrients is restricted due to poor root health caused by the decay of feeder roots. Following infection of the roots, the root cortex is eventually degraded, leaving only the inner stele and giving the root system a stringy appearance. This underlines the importance of root rot management in both an integrated brown rot management program to prevent both the build-up of inoculum in citrus orchards and as a means to maintain a productive orchard. Currently the control of root rot and brown rot is addressed through integration of cultural practices and fungicide applications. Among cultural practices, the usage of resistant root stocks has long been utilized for the management of Phytophthora root rot and gummosis issues. Trifoliate and hybrid rootstocks such as C-35 and C-32 citrange are tolerant of root rot and gummosis. Other citrange rootstocks such as Carrizo and Troyer citrange are intermediate in their tolerance to root rot but are tolerant of gummosis. Irrigation management is another important facet to controlling the spread of Phytophthora root rot, brown rot and gummosis. It has previously been shown that irrigation management that avoids over-watering can significantly reduce populations of Phytophthora species in the soil by preventing saturated soil conditions that are conducive to sporangia formation, zoospore release, and zoospore motility. To promote consistent drainage and reduce the presence of saturated soil, trees can be planted on raised berms and micro-sprinkler irrigation systems installed to replace furrow irrigation which easily over saturates the soil.

Additional in-season and preharvest fungicide applications, however, are often still needed to control root rot, especially in the establishment of new orchards, and to manage brown rot during the rainy winter harvest season. Preharvest applications of copper or phosphonates are used to manage brown rot, and phosphonate or phenylamide fungicides are applied to the soil to reduce root rot. The limited number of chemistries available for controlling Phytophthora diseases and few options for fungicide rotations has led to their overuse and resulted in the development of resistance. Several formulations of the phenylamide fungicides metalaxyl and mefenoxam have been used by growers for many years to control root rot and gummosis since their introduction. It is known that Phytophthora species can develop resistance to this fungicide class after repeated exposure. It has also been observed that phenylamide-resistant populations tend to decrease in the absence of selection pressure. This suggests that reducing usage of phenylamide-containing products could eventually make this fungicide class effective again when used strategically in a resistance management program. The new chemistries ethaboxam, fluopicolide, mandipropamid and oxathiapiprolin all possess unique modes of action, all different from one another and from currently registered products. The availability of new modes of action will facilitate the implementation of resistance management strategies using fungicide rotations that will minimize the risk of resistance development.

As a result of our previous work , oxathiapiprolin has since been federally and state registered for usage on citrus, while the remaining fungicides are still pending registrations for field application use. Once these fungicides are registered, the implementation of new fungicide rotations with existing chemistries will help not only to manage Phytophthora diseases but also slow the development of resistance, maximize the lifespan of available chemical treatments, and help ensure continued trade with foreign markets by reducing the prevalence of Phytophthora root rot and brown rot in orchards, effectively lowering the disease incidence to below detection levels. Thus, the objectives of the following studies were to: Identify the efficacy of new fungicides ethaboxam, fluopicolide, mandipropamid and oxathiapiprolin, at inhibiting several different life stages of Phytophthora spp. known to cause root rot, and to determine baseline sensitivity references for future resistance monitoring efforts using isolates of several Phytophthora spp. collected from throughout California citrus production regions; evaluate these new compounds in both greenhouse and field settings to verify their efficacy as potential root rot treatments and determine potential growth promotion benefits of the fungicides and ascertain a better understanding of the properties of oxathiapiprolin in citrus plant systems following application to determine if oxathiapiprolin is capable of systemic translocation for the purposes of refining potential application practices and improve management of Phytophthora diseases common to citrus within California. Phytophthora spp. are plant pathogens in the Kingdom Stramenopila that cause diseases on a wide variety of cultivated and non-cultivated plants. Numerous species have been associated with citrus crops worldwide ,maceta 10 litros and in California, P. citrophthoraLeonian, P. syringaeKleb., P. nicotianae Breda de Haan , and P. hibernalis Carne are present. These species can cause fruit brown rot , root rot , foot rot , and trunk cankers or gummosis. 

Overall, P. hibernalis is the least commonly encountered species in California. P. nicotianae is most prevalent from late spring to early fall, P. citrophthora can be found year-round, whereas P. syringae and P. hibernalis are restricted to the cooler seasons. Recently, P. syringae and P. hibernalis have been designated quarantine pathogens in China after they were detected in citrus fruit shipments from California. This has restricted the California citrus trade with this important export market, causing high economic losses to the state’s citrus industry. The disease phases of Phytophthora species on citrus are closely interrelated. For example, a high incidence of root rot will lead to the buildup of zoospore inoculum in thesoil that may be splashed by rain or irrigation onto low hanging fruit, causing brown rot. Vice versa, infected fruit that fall to the ground will increase the amount of inoculum in the soil. Therefore, management of Phytophthora diseases should be done in an integrated approach, including cultural practices , and preand post harvest fungicide applications. Few fungicides are currently registered in the United States for the control of Phytophthora diseases of citrus. Copper products and phosphonates are used in fruit and foliar applications to manage brown rot and gummosis, whereas phosphonate and phenylamide compounds are applied to the soil to reduce root rot. Potassium phosphite was registered in 2013 as the first post harvest fungicide to manage brown rot. The limited number of fungicides available has led to their over-use, and in subsequent resistance development. Resistance to the phenylamide class of fungicides has been reported in Oomycota pathogens of numerous crops , including citrus where resistant populations of P. nicotianae are established in Florida nurseries. Resistance was recently also reported for potassium phosphite in isolates of P. citrophthora and P. syringae collected from California citrus orchards, and brown rot caused by these isolates could not be controlled using registered rates of potassium phosphite. New fungicides are becoming available to manage Phytophthora diseases of citrus with the pending registrations of four compounds with modes of action different from those of registered fungicides : the thiazole carboxamide ethaboxam, the benzamide fluopicolide, the carboxylic acid amide mandipropamid, and the piperidinyl thiazole isoxazoline oxathiapiprolin. These new chemistries each have unique and different modes of action. Mandipropamid is suspected to target a cellulose synthase gene vital to cell wall biosynthesis. Ethaboxam inhibits mitosis and cell division by disrupting microtubules. Fluopicolide delocalizes spectrin-like proteins, thereby destabilizing plasma membrane formation. Oxathiapiprolin inhibits oxysterol-binding proteins affecting all stages in the asexual life cycle. 

The introduction of four new fungicides with unique modes of action provides an opportunity for designing new effective disease control programs and to implement resistance management strategies that slow the development and spread of resistance while extending the lifespan of existing treatments. To provide a reference for future resistance monitoring and to possibly use the new fungicides against the most sensitive life stages of Phytophthora spp., we determined the in vitro toxicities of these compounds. Thus, the objectives of this study were to evaluate in vitro sensitivities of mycelial growth of P. citrophthora, P. syringae, P. nicotianae, and P. hibernalis to ethaboxam, fluopicolide, mandipropamid, and oxathiapiprolin and compare to mefenoxam, and assess the toxicity of these fungicides to other selected stages in the life cycles of Phytophthora species on citrus in California. The ubiquitous occurrence of Phytophthora species in citrus growing areas worldwide often requires integrated management strategies to successfully establish and maintain orchards in order to produce a high-quality crop. In California, a high level of disease control is also required to minimize detection of any fruit with brown rot upon arrival in export markets where P. syringae and P. hibernalis are quarantine pathogens. Quarantine laws prohibit the movement of diseased commodities containing pathogens not present in the import country. The use of cultural practices such as irrigation methods that minimize wetting of fruit, removal of lower branches, and harvesting above a selected height to exclude fruit exposed to splashing water and soil contamination provide some level of brown rot management. Still, the use of fungicides has become an integral part of citrus production in preventing fruit from developing brown rot during transit to the port of arrival of the export market. This, however, is hampered by the current limited number of treatments available and the development of resistance to the phenylamides and more recently to the phosphonates in Phytophthora spp. populations. The pending registration of the new active ingredients ethaboxam, fluopicolide, mandipropamid, and oxathiapiprolin will be an important step to increase treatment options for improved disease management and to reduce the risk of resistance development due to overuse of any single mode of action. The Fungicide Resistance Action Committee considers the resistance risk of ethaboxam and mandipropamid as low to medium and of oxathiapiprolin as medium to high, whereas the risk for fluopicolide is currently not known. This is the first study comparing in vitro toxicities of these new Oomycota fungicides to important stages in the disease cycles of Phytophthora species from major citrus production areas in California. Mycelial growth responses were evaluated for all isolates included in the study because mycelium is the main somatic structure that is capable of surviving from season to season in infected plant material if climatic conditions allow. Sporangia are readily formed by the four species, and effects of the fungicides on their production were evaluated using P. citrophthora as a representative. Sporangia produce zoospores that are the main infective propagules of Phytophthora species, and abundant production is a major cause of disease epidemics during rain and irrigation events when zoospores are disseminated from the soil or from infected fruit onto low hanging fruit. Therefore, we also evaluated rate effects of the fungicides to determine the response of sporangia to lower concentrations.The effect of the fungicides on their production was investigated using P. nicotianae where these structures are known to occur and are considered critical for persistence during the colder winter months. Oospores are also produced by the homothallic P. syringae and P. hibernalis. Previously, large quantities of oospores in fallen apple leaf litter were found in outbreaks of P. syringae in apple orchards in the United Kingdom, suggesting the importance of oospores in pathogen survival. 

Clients may have multiple views of the same object: for example, a client may have a local view of the present state of the object with a remote view of a past version of the object, enabling the client to operate against a snapshot.Transactions enable developers to issue multiple operations which either succeed or fail atomically. Transactions are a pain point for partitioned data stores since a transaction may span across multiple partitions, requiring locking or schemes such as 2PL or MVCC to achieve consistency. vCorfu leverages atomic multi-stream appends and global snapshots provided by the log, and exploits the sequencer as a lightweight transaction manager. Transaction execution is optimistic, similar to transactions in shared log systems, which leverage the total ordering provided by the log. However, since our sequencer supports conditional token issuance, we avoid polluting the log with transactional aborts. To execute a transaction, a client informs the runtime that it wishes to enter a transactional context by calling TXBegin. The client obtains the most recently issued log token once from the sequencer and begins optimistic execution by modifying reads to read from a snapshot at that point.

Writes are buffered into a write buffer. When the client ends the transaction by calling TXEnd,frambuesa y mora the client checks if there are any writes in the write buffer. If there are not, then the client has successfully executed a read-only transaction and ends transactional execution. If there are writes in the write buffer, the client informs the sequencer of the log token it used and the streams which will be affected by the transaction. If the streams have not changed, the sequencer issues log and stream tokens to the client, which commits the transaction by writing the write buffer. Otherwise, the sequencer issues no token and the transaction is aborted by the client without writing an entry into the log. This important optimization ensures only committed entries are written to the log, so that when a client encounters a transactional commit entry, it may treat it as any other update. In other shared log systems, each client must determine whether a commit record succeeds or aborts, either by running the transaction locally or looking for a decision record. In vCorfu, we have designed transactional support to be as general as possible and to minimize the amount of work that clients must perform to determine the result of a transaction. We treat each object as an opaque object, since fine-grained conflict resolution would either require the client resolve conflicts or a much more heavyweight sequencer. Opacity is ensured by always operating against the same global snapshot, leveraging the history provided by the log.

Opacity is a stronger guarantee than strict serializability as opacity prevents programmers from observing inconsistent state. Since global snapshots are expensive in partitioned systems, these systems typically provide only a weaker guarantee, allowing programs to observe inconsistent state but guaranteeing that such transactions will be aborted. Read-own-writes is another property which vCorfu provides: transactional reads will also apply any writes in the write buffer. Many other systems do not provide this property since it requires writes to be applied to data items. The SMR paradigm, however, enables vCorfu to generate the result of a write in-memory, simplifying transactional programming. vCorfu fully supports nested transactions, where a transaction may begin and end within a transaction. Whenever transaction nesting occurs, vCorfu buffers each transaction’s write set and the transaction takes the timestamp of the outermost transaction.vCorfu supports several mechanisms for finding and retrieving objects. First, a developer can use vCorfu like a traditional key-value store just by using the stream id for object as a key. We also support a much richer query model: a set of collections, which resemble the Java collections are provided for programmers to store and access objects in. These collections are objects just like any other vCorfu object, so developers are free to implement their own collection. Developers can take advantage of multiple views on the same collection: for instance a List can be viewed as a Queue or a Stack simultaneously. Some of the collections we provide include a List, Queue, Stack, Map, and RangeMap.

Collections, however, tend to be very large objects which are highly contended. In the next section, we discuss composable state machine replication, a technique which allows vCorfu to build a collection out of multiple objects.In vCorfu, objects may be composed of other objects, a technique which we refer to as composable state machine replication. The simplest example of CSMR is a hash map composed of multiple hash maps, but much more sophisticated objects can be created. Composing SMR objects has several important advantages. First, CSMR divides the state of a single object into several smaller objects, which reduces the amount of state stored at each materialized stream. Second, smaller objects reduce contention and false sharing, providing for higher concurrency. Finally, CSMR resembles how data structures are constructed in memory – this allows us to apply standard data structure principles to vCorfu. For example, a B-tree constructed using CSMR would result in a structure with O time complexity for search, insert and delete operations. This opens a plethora of familiar data structures to developers. Programmers manipulate CSMR objects just as they would any other vCorfu object. A CSMR object starts with a base object, which defines the interface that a developer will use to access the object. An example of a CSMR hash map is shown in Figure 5.5. The base object manipulates child objects, which usually store the actual data. Child objects may just reuse standard vCorfu objects, like a hash map, or they may be custom-tailored for the CSMR object, like a B-tree node. In the example CSMR map shown in Figure 5.5,frambueso maceta the object shown is the base object and the child objects are standard SMR maps. The number of buckets is set at creation time in the num Buckets variable. Two helper functions, get Child Number and get Child help the base object locate child objects deterministically. In our CSMR map, we use the Luby-Rakoff algorithm to obtain an improved key distribution over the standard Java hashCode function. Most operations such as get and put operate as before, and the base object needs to only select the correct child to operate on. However, some operations such as size and clear touch all child objects. The vCorfu log provides fast access to snapshots of arbitrary objects, and the ability to open remote views, which avoids the cost of playback, enables clients to quickly traverse CSMR objects without reading many updates or storing large amounts of local state. In a more complex CSMR object, such as our CSMR B-tree, the base object and the child object may have completely different interfaces. In the case of the B-tree, the base object presents a map-like interface, while the child objects are nodes which contain either keys or references to other child objects. Unlike a traditional B-tree, every node in the CSMR B-tree is versioned like any other object in vCorfu. CSMR takes advantage of this versioning when storing a reference to a child object: instead of storing a static pointer to particular versions of node, as in a traditional B-tree, references in vCorfu are dynamic. Normally, references point to the latest version of an object, but they may point to any version during a snap shotted read, allowing the client to read a consistent version of even the most sophisticated CSMR objects. With dynamic pointers, all pointers are implicitly updated when an object is updated, avoiding a problem in traditional trees, where an update to a single child node can cause an update cascade requiring all pointers up to the root to be explicitly updated, known as the recursive update problem.

The design of vCorfu relies on performant materialization. To show that materializing streams is efficient, we implement streams using back pointers in vCorfu with chain replication, similar to the implementation described in Tango. For these tests, in order to compare vCorfu with a with a chain replication-based protocol, we use a symmetrical configuration for vCorfu, with an equal number of log replicas and stream replicas. For the back pointer implementation, we use chain replication , with two replicas per chain and two chains. Our back pointer implementation only stores a single back pointer per entry – Tango uses 4 back pointers. Multiple back pointers are only used to reduce the probability that a linear scan will be required due to hole-filling and should not have an effect on our evaluation. The primary drawback of materialization is that it requires writing a commit message, which results in extra messages proportional to the number of streams affected. We characterize the overhead with a micro-benchmark that appends 32B entries, varying the number of streams and logging units. Figure 5.6 shows that writing a commit bit imposes about a 40% penalty on writes, compared to a back pointer based protocol which does not have to send commit messages. However, write throughput continues to scale as we increase the number of replicas, so the bottleneck in both schemes is the rate in which the sequencer can hand out tokens, not the commit protocol. Now we highlight the power of materializing streams. Figure 5.7 shows the performance of reading an entire stream with a varying number of 32B entries and streams in the log. The 100K stream case uses significantly fewer entries, reflecting our expectation that CSMR objects will increase the number of streams while decreasing the number of entries per stream. As the number of streams and entries increase, vCorfu greatly outperforms back pointers thanks to the ability to perform a single bulk read, whereas back pointers must traverse the log backwards before being able to serve a single read. When hole-filling occurs due to client timeouts, back pointers perform very poorly, falling back to a scan because the hole fill does not contain back pointers resulting in a linear scan of the log. Figure 5.8 examines the number of log entries a back pointer implementation may have to read as a result of a hole. To populate this test, we use 256 clients which randomly select a stream to append a 32B entry to. We then generate a hole, varying the number of streams in the log, and measure the number of entries that the client must seek through. The back pointer implementation cannot do bulk reads, and reading each entry takes about 0.3 ms. The median time to read a stream with a hole takes only 210ms with 32 streams, but jumps to 14.8 and 39.6 seconds with 100K and 500k streams, respectively. vCorfu avoids this issue altogether because stream replicas manage holes. Finally, Figure 5.9 shows that vCorfu performance degrades gracefully when a stream replica fails, and vCorfu switches to using the log replicas instead. We instantiate two local views on the same object, and fail the stream replica hosting the object at t = 29.5s.The append throughput almost doubles, since the replication factor has decreased while the read throughput stays about the same, falling back to using back pointers. Since the local view contains all the previous updates in the stream, reading the entire stream is not necessary. If a remote view was used, however, vCorfu would have to read the entire stream to restore read access to the object. Next, we examine the power of remote views. We first show that remote views address the playback bottleneck: In figure 5.10, we append to a single local view and increase the number of clients reading from their own local views. As the number of views increases, read throughput decreases because each view must playback the stream and reade very update. Once read throughput is saturated readers are unable to keep up with the updates in the system and read latency skyrockets: with just 32 clients, the read latency jumps to above one second. With a remote view, the stream replica takes care of playback and even with 1K clients is able to maintain read throughput and millisecond latency.