Horticulture

An automated translation process converts plain old Java objects directly into Corfu objects through both runtime and compile-time transformation of code. This allows programmers to quickly adapt existing code to run on top of Corfu. The Corfu runtime also provides strong support for transactions, which enable multiple applications to read and modify objects without relaxing consistency guarantees. We show that with Stream materialization, Corfu can support storing large amounts of state while supporting strong consistency and transactions. In Chapter 6, we describe our experience in both writing new applications and adapting existing applications to Corfu. We start by building an adapter for Apache Zookeeper clients to run on top of Corfu, then describe the implementation of Silver, a new distributed file system which leverages the power of the vCorfu stream store. We then conclude the chapter by describing our efforts to retrofit a large and complex application: a software defined network switch controller, and detail how the strong transaction model and rich object interface greatly reduce the burden on distributed system programmers. Finally, Chapter 7 summarizes the findings from the previous chapters and concludes the dissertation. Before the cloud era,hydroponic vertical garden system designers focused on building systems which provided increasingly stronger guarantees.

Problems such as consensus and byzantine fault tolerance were at the forefront of distributed systems research. Stronger guarantees simplify programming complex, unreliable distributed systems, and few would consider relaxing those guarantees. Indeed, it was often possible to avoid distributed algorithms altogether by strong all data on a single, centralized server. Feature-rich databases could coordinate the data needs of an entire system, and support for transactions and complex queries made it easy for multiple clients to safely and concurrently operate on data.The cloud era and “big data”, however, changed the landscape. Suddenly, system designers had to deal with a workloads that they never dealt with before – workloads that no longer fit within the confines of a single machine. The focus became scalability, and the quickest way to do that was to split the data that used to fit in a single database across multiple machines, a process known as sharding or partitioning. With partitioning, the system can utilize the aggregate throughput of all the machines that data has been partitioned across to service requests. Unfortunately, partitioning was not a panacea. Splitting data across multiple nodes made the features programmers started to depend on, such as complex queries, transactions and consistency difficult to support. Strong consistency was seen as the enemy, and the NoSQL movement sought to relax consistency as much as possible in order to achieve maximum scalability. Consistency guarantees were greatly relaxed, and support for transactions and queries across multiple partitions were often dropped entirely.

Key-value stores, which have a greatly simplified interface rose to prominence, providing unparalleled scalability but placing the burden on the programmer to maintain consistency across partitions.Migrating applications from SQL databases to early NoSQL stores was difficult and bug-prone for all but the most simple applications. In order to build feature-rich, reliable distributed applications, programmers really needed the capabilities of a traditional database with strong guarantees. System designers began to make compromises, trading off some scalability in order to provide some guarantees. Relaxed consistency models such as eventual consistency and causal consistency emerged to fill the gap, but these models increased the burden on the programmer further by forcing them to reason about an unconventional consistency model. Yet other systems used multi-version concurrency control on key-value stores with protocols such as two phase locking or specialized hardware to ensure consistency, with a significant performance penalty and added system complexity. ince the beginning of the cloud era, developers have thought of consistency and scalability as mutually exclusive: that scalable systems must sacrifice consistency, and the strongly consistent systems must not be scalable. This line of thought surfaced when system designers were pushed to a corner to deliver as much performance as possible. Shedding consistency did indeed make their systems more scalable, but programmers paid the price, sometimes with catastrophic results. System designers reacted by retrofitting consistency back on, which resulted in performance losses and complexity as protocols had to work around the strict partitioning which was added for scalability.

In the next chapters, we will explore Corfu, which takes a clean slate approach. Instead of tacking on consistency, Corfu addresses scalability and consistency at its core, providing a strongly consistent yet scalable fabric, the Corfu distributed log,vertical vegetable tower which applications leverage through the use of an object-oriented interface. One motivation has changed significantly, however. Corfu was originally designed for flash memory, and the shared log was cast as a distributed flash translation layer for networked devices which exposed raw flash. Flash memory has a unique idiosyncrasy: although random reads are fast, the ideal write pattern is sequential due to the cost of garbage collection. The Corfu log takes all the writes across the data center and transforms them into sequential writes, enabling Corfu to consume the full write throughput of flash devices. It turns out Corfu works well on disk as well: due to the nature of the log, random reads on the log are rare and clients typically read the tail of the log. In the next chapters, we will see that the Corfu log works well irrespective of whether the underlying storage devices are flash or disk.The Corfu storage unit is now referred to as logging unit, and the projection is now a component of an layout which describes the entire Corfu system. In this chapter, we will use the older terminology. We begin by introducing the Corfu distributed log and describing its interface and implementation in Section 3.3. Next, in Section 3.4 we describe an hardware-accelerated implementation of the log on a FPGA, and then conclude in Section 3.5 with an evaluation of the performance of the log. Traditionally, system designers have been forced to believe that the only way to scale up reliable data stores, whether on-premise or cloud hosted, is to shard the database. In this manner, recent systems like Percolator, Megastore, WAS and Spanner are able to drive parallel IO across enormous fleets of storage machines. Unfortunately, these designs defer to costly mechanisms like two-phase locking or centralized concurrency managers in order to provide strong consistency across partitions. At the same time, consensus protocols like Paxos have been used as a building block for reliable distributed systems, typically deployed on a small number of servers, to provide a lever for a variety of consistent services: virtual block devices, replicated storage systems, lock services, coordination facilities, con- figuration management utilities, and transaction coordinators. As these successful designs have become pillars of today’s data centers and cloud back-ends, there is a growing recognition of the need for these systems to scale in number of machines, storage-volume and bandwidth. Internally, the Corfu distributed shared log is distributed over a cluster of machines and is fully replicated for fault tolerance, without sharding data or sacrificing global consistency. The Corfu distributed shared log allows hundreds of concurrent client machines in a large data-center to append to the tail of a shared log and read from it over a network.

Nevertheless, there is no single I/O bottleneck to either appends or reads, and the aggregated cluster throughput may be utilized by clients accessing the log. Historically, shared log designs have appeared in a diverse array of systems. Quick Silver and Camelot used shared logs for failure atomicity and node recovery. LBRM uses shared logs for recovery from multicast packet loss. Shared logs are also used in distributed storage systems for consistent remote mirroring. In such systems, Corfu fits the shared log role perfectly, pooling together the aggregate cluster resources for higher throughput and lower latency. Moreover, a shared log is panacea for replicated transactional systems. For instance, Hyder is a recently proposed high-performance database designed around a shared log, where servers speculatively execute transactions by appending them to the shared log and then use the log order to decide commit/abort status. In fact, Hyder was the original motivating application for Corfu and has been fully implemented over our code base. Interestingly, a shared log can also be used as a consensus engine, providing functionality identical to consensus protocols such as Paxos. Used in this manner, Corfu provides a fast, fault-tolerant service for imposing and durably storing a total order on events in a distributed system. From this perspective, Corfu can be used as a drop-in replacement for existing Paxos implementations, with far better performance than previous solutions.So far, we have argued the power and hence the desirability of a shared log. The key to its success is high performance, which we realize through a paradigm shift from existing cluster storage designs. In Corfu, each position in the shared log is projected onto a set of storage pages on different storage units. The projection map is maintained – consistently and compactly – at the clients. To read a particular position in the shared log, a client uses its local copy of this map to determine a corresponding physical storage page, and then directly issues a read to the storage unit storing that page. To append data, a client first determines the next available position in the shared log – using a sequencer node as an optimization for avoiding contention with other appending clients – and then writes data directly to the set of physical storage pages mapped to that position. In this way, the log in its entirety is managed without a leader, and Corfu circumvents the throughput cap of any single storage node. Instead, we can append data to the log at the aggregate bandwidth of the cluster, limited only by the speed at which the sequencer can assign them 64-bit tokens, i.e., new positions in the log. The evaluation for this chapter was done with a user-space sequencer serving 200K tokens/s, whereas our more recent user-space sequencer is capable of 500K tokens/s. Moreover, we can support reads at the aggregate cluster bandwidth. Essentially, Corfu’s design decouples ordering from I/O, extracting parallelism from the cluster for all IO while providing single-copy semantics for the shared log. Naturally, the real throughput clients obtain may depend on application workloads. However, we argue that CORFU provides excellent throughput in many realistic scenarios. Corfu’s append protocol generates nearly perfect sequential write-load, which can be turned into a high throughput, purely sequential access pattern at the server units with very little buffering effort. Meanwhile, reads can be served from a memory cache as in, or from from a cold standby as in. This leaves non-sequential accesses which may result from log compaction procedures. Because we design for large clusters, compaction need not be performed during normal operations, and may be fulfilled by switching between sets of active storage drives. Finally, we have originally argued in for the use of nonvolatile flash memory as CORFU storage units, which alleviates altogether any performance degradation due to random-accesses. Indeed, our present evaluation of CORFU is carried on servers equipped with SSD drives. The last matter we need to address is efficient failure handling. When storage units fail, clients must move consistently to a new projection from log positions to storage pages. Corfu achieves this via a reconfiguration mechanism capable of restoring availability within tens of milliseconds on drive failures. A challenging failure mode peculiar to a client-centric design involves ‘holes’ in the log; a client can obtain a log position from the sequencer for an append and then crash without completing the write to that position. To handle such situations, Corfu provides a fast hole-filling primitive that allows other clients to complete an unfinished append within a millisecond.The system setting for Corfu is a data center with a large number of application servers and a cluster of storage units. Our goal is to provide applications running on the clients with a shared log abstraction implemented over the storage cluster. Our design for this shared log abstraction aims to drive appends and random-reads to/from the log at the aggregate throughput that the storage cluster provides, while avoiding bottlenecks in the distributed software design.

The proportion of the crystalline to amorphous cellulose for the glucose carbon 1 to carbon 6 transfer appears to decrease more for the 2-minute milling period to 80.28 ± 1.36% and 74.65 ± 1.39%, respectively.The signal intensity for the crystalline cellulose and amorphous cellulose appear to be nearly equal for the glucose carbon 1 to carbon 6 after 15 minutes of milling.In the future, dephasing curves could be applied to specifically probe cellulose fiber substructures with the 2D CP-PDSD experiment similar to previous studies on the plant cell walls.Dephasing curves would give more specific mixing times for the experiment between 30 ms and 1500 ms mixing times of the 2D CP-PDSD experiments.However, for the purpose of exploring the recalcitrant implications of milling where cellulose fibrils are disrupted to accommodate particle sizes smaller than the fibril width 1 μm or length 100 μm, other experiments may be also explored.Future use of the 30 ms CP-PDSD experiment may be more suitable for cellulose morphology based on carbon 4 as sizes of cellulose fibrils are reduced and long range CP mixing times becoming more inefficient.After milling,vertical garden indoor system cellulose fibrils are still present in the morphology of the sample and there is not a reduction in the crystalline cellulose cross-peaks in the 30 ms 2D CP-PDSD.This means that the crystalline morphology of cellulose within the fibril is not directly converted to amorphous cellulose as suggested by analysis milling pure cellulose fibril.

The atomic structure of the cellulose fibrils appears to be maintained after milling based on the 30 ms CP-PDSD cellulose one carbon correlations, for example the crystalline cellulose to amorphous cellulose signal intensity ratio was maintained for carbons 4 and 6.Cellulose carbon 4 signals are particularly important because it can offer insight into the cellulose 1-O-4 polymer backbone of cellulose and chemical shifts which indicate the environment interior through exterior of the cellulose fibril.Signals in the 2D CP-rINADEQUATE arise from highly immobilized polymers in the secondary plant cell wall structural hemicellulose and cellulose.Interpretation of these signals is less straightforward due to resonance overlap for these carbohydrates, but the signals pertaining to the carbon 4 chemical shifts of glucose within cellulose are well resolved.The CP-rINADEQUATE shows directly bonded nuclei and is useful for identifying specific subtypes of prominent monomers within the secondary plant cell wall.The chemical shifts of the bonded nucleisum to another peak in the indirect dimension shown in the y-axis.The single quantum chemical shifts were assigned using previously established values of sorghum secondary plant cell wall polymers and were verified using cross peaks.Monomers were traced out using cross peaks because the y-axis double quantum coherence is assigned by a chemical shift from the sum of chemical shifts from two directly bonded 13C spins.Note, the monomers found by the rINADEQUATE are contained within the polymers within the secondary plant cell wall and shifted downfield compared to literature of dry solid monomers and monomers in water.Many of the unambiguous signals of cellulose all reduced in signal intensity : after 2 minutes of milling the signals reduced by 15% for all unambiguous amorphous cellulose peaks.

After 15 minutes of milling the crystalline cellulose signal associated with interior crystalline cellulose within the dehydrated space around 84 ppm of the cellulose fibril decreased nearly to the noise.The signal reduction of the crystalline and amorphous cellulose appeared to be equal suggesting crystalline cellulose did not convert to amorphous cellulose when milled, contrary to pure cellulose as found in Ling et al.2019.However, the remaining crystalline cellulose carbon 4 does not appear to significantly differ in the proportions of decreasing amorphous cellulose 4 for the interior and exterior positions, as noted in Gao et al.2020.In Figure 9, additional subscripts i to iii refer to the least to the most dehydrated cellulose i.e.the interior to exterior polymers in the cellulose fibers based on previous studies.Results of the current study show overall signal reduction of the amorphous cellulose after 2 minutes is only around 3% and by 75% after the 15 minutes of milling.Beyond the carbon 4 area of the CP-rINADEQUATE spectrum, cellulose signals often overlap with rigid hemicellulose.The total amount of cellulose estimated by the glucose carbon 1 peak at 105.2 ppm overlaps with many of the hemicellulose carbon 1 peaks including structurally relevant 2-fold xylanhemicellulose at 105.1 ppm.The carbon 1 of cellulose can be less quantitative in the CPrINADEQUATE compared to the CP-PDSD experiments discussed here.The slice from the carbon 1 region of the CP-rINADEQUATE supports an overall cellulose signal reduction in Figure 9G which is apparent in the overlay of the control, stems milled for 2 minutes, and stems milled for 15 minutes in Figure 9D.To summarize, there is an overall signal reduction and the proportion of amorphous and crystalline cellulose appear to be maintained after milling in the CP based experiments CP-rINADEQUATE, 30 ms and 1500 ms 2D CP-PDSD experiments.

Additional evaluation of the signals using normalized intensities of carbon 1 for the 30 ms CP-PDSD experiment could offer further statistical insight on cellulose within the sample based on changes in monomers, without handling as much signal overlap as the CP-rINADEQUATE.As the cellulose structure is further milled beyond the 1 μm particle size, the amount of information which will be available in the 1500 ms CP-PDSD experiment is expected to decrease and may be removed from the experiment set for more informative experiments.CP becomes less efficient for more liquid like samples and the 1500 ms CP-PDSD has a mixing time suited for distances potentially exceeding cellulose fibril lengths as they are broken down.Overall, the argument of increased recalcitrance due to conversion of crystalline to amorphous cellulose using milling is not supported by the CP-PDSD and CPrINADEQUATE spectra.However, understanding the available digestible cellulose may be more informative especially as hemicellulose and lignin could sit on the surface of cellulose fibrils and reduce available surface area for digestion.Signal intensities from arabinose all significantly decrease by at least 30% after ball-milling while 3-fold xylancarbon 4 and carbon 5 signal intensities increase after 2 minutes.For the stems milled for 15 minutes,mobile vertical grow racks the arabinose intensities decrease by >87%, but are still more prominent than cellulose.After 15 minutes of milling, xylan peaks remained consistently 10% above the relative intensity of other peaks in the CP-rINADEQUATE , while cellulose signals frequently dropped below 10% of the relative signal intensity compared to the control.Arabinose serves as a common substitution and a defining feature dictating some xylan interactions with cellulose and is known for facilitating cross linking between hemicellulose and lignin.The hemicellulose signals appear to remain intact in the 2D CP-rINADEQUATE, although reduce in signal intensity after 15 minutes of ball-milling, meaning that polymers maintain their rigid conformations while the cellulose signals area reduced in the spectra.Hemicellulose signals appear to be more preserved when contrasting overall spectra of the control to the stems milled for 15 minutes , supporting the structural importance of hemicellulose.As highlighted in Figure 9C, the arabinose substitutions on rigid hemicellulose signals persist with decreasing intensity in the spectra as presented in the 2D CP-rINADEQUATE overlay.Observation of xylan hemicellulose in the CP-rINADEQUATE has the same issue of signal overlap as cellulose and only a select number of unambiguous chemical shifts , but tracing the full monomers within the spectra helps with some deconvolution.Another note is the predominant xylancellulose interaction between the 3-fold xylan and the amorphous cellulose surfaces in sorghum: it is not necessarily expected to increase as amorphous cellulose did not appear to increase.The 2fXylan that interacts with crystalline cellulose surfaces exhibits heavy signal overlap and has a lower prominence in sorghum secondary plant cell walls.Findings from this study appear to confirm that the use of filtered DP-rINADEQUATE could assist in detecting semirigid hemicellulose structures relative to cellulose.It is inconclusive whether hemicellulose is creating recalcitrant interactions in the current 2D CP-rINADEQUATE, however remaining structural hemicellulose is more rigid than cellulose in the secondary plant cell wall.

The line shapes are generally narrower for the dynamic polymers captured in the 1H-13C 2D rINEPT experiment.To better understand how this is possible, note that spins have an equilibrium aligning their precession along the applied magnetic field and realign their precessions through longitudinal and transverse relaxation to the applied magnetic field after RF pulses.In solid-state NMR this relaxation of spins back to equilibrium is typically limited by T2 relaxation.In the experiment dynamic peaks have a greater signal intensity thanks to the long periods between pulses which captures the long relaxation dynamic spins in liquid-like samples and the signals arising from short T2 relaxations predominating rigid spins are not captured.So, some heterogeneous line broadening from rigid signals and dynamic signals overlap at similar chemical shifts in the rINEPT.And as the more dynamic signals are captured, narrower lines are also observed because the random motion of the spins allows some proton chemical shifts to be canceled out.Directly bonded 13C and 1H are probed through J-coupling delays between pulse excitations for each nucleusand Matlahov and van der Wel.The assignment of the 1H-13C 2D rINEPT was possible given the chemical shifts of polymers identified in the sorghum plant cell wall by Gao et al.2020and previous work examining pectic hemicelluloses in Wang et al.2014.It is worth noting that in this work, the arabinose at 108 ppm was confirmed using the control DP-rINADEQUATE experiment.Historically, the monomer chemical shifts are up field of the polymer chemical shifts, computational modeling in silico was used to confirm chemical shifts of xylan and its symmetry, and polymer chemical shifts tend to remain consistent between solid state NMR experiments even if line broadening needs to be considered.Some challenges are associated with identifying hemicellulose in the carbon 1 region of the 2D rINEPT as the peaks aside from Arabinose coincide with chemical shifts assigned for pectin and hemicellulose found in the primary plant cell wall, which is disputed in the secondary plant cell wall and it is challenging to distinctly separate polymers thus far between regions of the primary and secondary plant cell wall.For these reasons, full interpretation of the 2D-rINEPT beyond a fingerprint requires further research.Using general chemistry knowledge of 1H chemical shifts information the carbon 1 region was supported by the ether chemical shifts typical around 5 ppm consistent with sugar backbones.As seen in Figure S3 for the freeze-thaw experiment, the protons of the secondary plant cell wall range from aliphatic shifts to protons in conjugated aromatic systems in the 1H range of 3 ppm to 10 ppm for the plant cell wall polymer 13C chemical shift range of 170 to 60 ppm.Perhaps future work with 13C-13CrINADEQUATE or more dynamic J-coupled experiments could extract information on the number of sub environments monomers, polymers, and substitutions of hemicellulose.This information would be particularly useful in deconstruction to assess the changes in 1H chemical shifts after preprocessing methods such as oxidation and acid treatment used in plant cell wall samples to solubilize cellulose.Cross peaks are not assigned in the 2D rINEPT for 1H because they are continuing to be assigned for the plant cell wall and the correlation of 1H to 13C peaks only accounts for directly bonded nuclei, not double quantum coherences as seen in the CP-rINADEQUATE Figure 9C.The 1H-13C 2D rINEPT fingerprintof highly dynamic hemicellulose generally changes over the milling period.Carbon 1 of arabinose hemicellulose substitutions demonstrate a change in the number of proton environments over the milling process and a reduction in signal intensity.The carbon 1 of arabinose substitutions of hemicellulose decreases to 39.35 ± 3.92% and pectic hemicellulose to 29.62 ± 1.62% after 2 minutes of milling.Arabinose carbon 1 of arabinose substitutions on structural hemicellulose 14.81 ± 2.23% and pectic hemicellulose 5.69 ± 0.92% after 15 minutes of milling.This interpretation of lost signals in Figure 10 suggests that some highly dynamic lignin and hemicellulosecould become less dynamic.Further, the reduction of arabinose hemicellulose substitution signals in the CP-rINADEQUATE and in the rINEPT of polymers together suggest that arabinose, and arabinose substituted hemicellulose takes on a more intermediate dynamic in the plant cell wall.Tracking arabinose substitutions may show a potential marker for rigid and dynamic hemicellulose changes related to recalcitrance.Additionally, the recalcitrance hypothesis based on structural hemicellulose maintaining their rigidity is supported as signals are stronger for arabinose and 3f,AXylan remain in the CP-rINADEQUATE experiment.Reduction in lignin signals would indicate a loss of mobility associated with lignin condensation and emerging lignin cross-linkages are noted in the rINEPT experiment.

The FAO, in collaboration with the Ministry of Water Resources and Irrigation and the Ministry of Agriculture and Land Reclamation , has recently launched the project “Support sustainable water management and irrigation modernization for newly reclaimed areas.” This project will increase the efficient use of resources to achieve high productivity at low input level, while minimizing adverse external factors.They also focus on managing the ecological, social, and economic risks associated with production systems in the agricultural sector, including disease and climate change.The project will also focus on identifying and increasing the role of ecosystem services, especially regarding their effects on resources utilization, risk response and preserving the environment.As the freshwater availability from the Nile river is decreasing, farmers are using low-quality groundwater for irrigation instead.It results in increased soil salinization thereby negatively impacting crop yields and quality.Therefore, it is essential that Egypt regulates the reuse of drainage water to control soil salinization.This will require a robust salinity monitoring program that can provide updated information on the quality and quantity of drainage water and groundwater.Most importantly, these data are vital in developing strategies for the safe use of these waters.Like many other countries, Egypt needs to prepare comprehensive guidelines for the use of poor-quality drainage and groundwater for irrigation,indoor vertical farming considering soil types, climatic conditions, and crops to be grown.

For the coastal areas where salinity levels are very high, the use of salt-tolerant crops and halophytes must be encouraged.Despite vast salt-affected areas in Ethiopia, research and development projects that address salinity are mostly absent.Consequently, the current and future extent of salt-affected soils are unknown, whereas economic implications are not brought to the attention of policy makers.No country organization monitors, evaluates, and permits for expanding irrigation or to discontinue existing irrigated farms.Available information is limited and is based on preliminary studies that are incomplete in most cases or comes from outside Ethiopia.The country lacks a systematic analysis of salt-affected areas and its strategic plan that addresses soil salinization and sodification.Such a project should lead to sustained funding of soil salinity research that assesses the quantification of its extent and damage, as well as the development of technologies and management practices that reclaim and prevents further expansion of soil salinity in the country.Specifically, the introduction of adequate drainage systems must be considered, and irrigation water conveyance channels should be lined to reduce water losses, especially in areas of saline groundwater.In addition, the selection of salt-tolerant forages, crops and legumes could largely improve the productivity of salt-affected lands.In summary, Ethiopia must develop a long-term national policy and strategic plan that leads to lasting solutions for its irrigated agriculture.Another significant development in this region is the construction of the world’s largest dam on the Nile River by Ethiopia.The Grand Ethiopian Renaissance Dam , on the River Nile near the Sudan border will have a reservoir capacity of 70Bm3and an electricity generation capacity of 6000MW.It is estimated that GERD will irrigate 1680 km2 forest land in the northwest of Ethiopia.

Ethiopia claimed that this dam would also benefit the downstream countries mainly Sudan and Egypt by removing 86% of their silt and sedimentation load and conserving water by regulating flow that will allow reliable all-season water supply to Sudan and Egypt.Although Ethiopia claims that there will be no consequences for downstream users such as Egypt.There are concerns that GERD will reduce 12–25%of Nile flow into Egypt especially during the dam filling period of 5–7 years.This will have severe consequences for optimal crop production and management of soil salinization in Egypt.Therefore, the cooperation between the Nile water-sharing countries is essential for the management and protection of this vital water resource to ensure future food security and livelihood of the 280 million people living in the Nile basin.Irrigated agriculture in Pakistan is mainly confined to the Indus plains where it has been developed by harnessing major water resources available to the country.The agriculture in the arid and semi-arid areas of Pakistan largely depends on sustained irrigation supplies, as the evapotranspiration demand is high, and rainfall is either inadequate or unreliable.The contiguous Indus basin irrigation system irrigates an area of about 16 million ha , diverting annually 131 billion m3of surface water to 43 main canal systems.The perennial water supply is available to 8.6Mha while the remaining area receives water only during the summer season.About 93% of the total water withdrawal is allocated to the agricultural sector, 4% is used for domestic purposes and the rest 3% goes to industrial use.The large-scale irrigation development in the Indus Basin was initiated in the second half of the 18th century to expand the settlement opportunities, avoid crop failure and famine.At that time, the groundwater levels were below 30m from the soil surface, and therefore drainage needs were not considered.Due to persistent seepage from unlined canals and percolation from irrigated fields, the groundwater table rose to within 1.5m of the soil surface, creating waterlogging and, consequently, soil salinity problems.The problems of soil salinity became more noticeable in areas where groundwater was saline.

Most of the soil salinity in the Indus basin comes from primary salinization.However, secondary salinization using poor-quality groundwater for irrigation has further compounded the problem.The Indus basin is faced with a considerable salt balance problem.The average annual salt inflow by the Indus river water is estimated to be 33 million tons , while the outflow to the sea is only 16.4Mt.The average annual salt storage of around 16.6 million tons is equivalent to 1ton of salts per hectare of irrigated land.Therefore, saline soils have become an important ecological conundrum with 4.5Mhaalready afflicted.As illustrated in Fig.35, the salinity problems in Sindh are most severe where about 50% of the irrigated area is affected.This is mainly due to poor drainage conditions, shallow saline groundwater, and the use of poor-quality groundwater for irrigation, as surface water supplies are far less than the actual crop water requirements.In addition to total soil salinity in the Indus basin, sodicity is a major problem because 70% of all groundwater wells in the basin pump sodic water, affecting soil structure and infiltration rates.Salt-affected soils of the Indus basin are usually classified into four types as shown in Table 6.The combined threats of water logging and soil salinization were recognized as early as 1870, and since then various remedies have been undertaken to overcome this twin menace.These include engineering solutions, reclamation strategies,hydroponic vertical farming and biological interventions.These are briefly discussed below.Engineering solutions—The first detailed survey of groundwater table depth and salinity was conducted in the 1950s with the collaboration of the US Geological Survey.It formed the basis for public sector vertical drainage program through Salinity Control and Reclamation Projects.As a result, in both fresh and shallow groundwater areas, 14,000 tube wells with an average capacity of 80 L s 1 were constructed between 1960 and 1970, covering 2.6Mha of irrigated land with an estimated cost of US$ 2 billion.This program was aimed at lowering the groundwater table and increasing irrigation supplies at the farm gate by mixing pumped groundwater with fresh canal water.The SCARPs were partially successful in arresting water logging and salinity by lowering groundwater tables below 1.5m in 2.0Mha and below 3m in 4.0Mha.As a result, areas with soil salinity decreased from 42% in 1960 to about 32% in 1977–79, and improved irrigation supplies allowing increased cropping intensities from 84% to 125% in most SCARP areas.In the 1970s, one realized that circulating salt-contaminated water through vertical drainage aggravated the salinity problem, thereby shifting to constructing horizontal drainage systems that were 10 times more expensive.The main argument in favor of horizontal drainage was that drainage water quality would improve over time, allowing more of it to be used for irrigation as well as reducing disposal problems.Since then, about 10 major horizontal drainage projectshave been completed in different parts of Pakistan.The major bottleneck in the successful operation of these drainage systems was the safe disposal of saline drainage effluent.To overcome this, Pakistan constructed a 2000 km long surface drain on the East side of the Indus River, moving drainage waters of more than 500,000 ha of land to the sea.

Reclamation strategies—The salinity management in Pakistan remained focused on lowering of groundwater table and leaching of salts, without a national action plan for reclaiming sodic and saline-sodic soils.Efforts by local governments were mainly confined to supporting field-level research and providing subsidies to the farmers for gypsum application.The use of gypsum, acids, and farmyard manure, in combination with surface scarping and deep plowing were extensively applied.Agricultural and industrial wastes such as farmyard manure and sugar industry byproducts have also been used to improve sodic soils.A large range of acid materials was tested in Pakistan including sulfur, sulfuric acid, and aluminum sulfate.However, due to their cost and management complexities, farmers deemed these less attractive.Instead, gypsum was considered the most cost-effective additive for the reclamation of sodic soils and is heavily subsidized by the government.Biological interventions—The biological approach emphasizes the use of highly saline water and lands on a sustained basis through the profitable and integrated use of the genetic resources embedded in plants, animals, and improved agricultural practices.In Pakistan, a considerable amount of work has been done to use highly saline waters for growing salt-tolerant crops.This includes the planting of salt-tolerant plants, bushes, trees, and fodder grasses.Plants, particularly trees, are commonly referred to as biological pumps and play an important role in the overall hydrological cycle for a given area.In Pakistan, bioremediation was promoted as a valuable tool for controlling rising water tables and salinity, through enhanced evapo-transpiration.During the last 20 years or so, many salt-tolerant species and varieties have been developed in Pakistan, such as poplar, eucalyptus, tamarix, maskit and acacia.Similarly, non-woody plants such as bushes, sedges, grasses, and herbs can develop deep-rooted systems that can use shallow groundwater.However, their ability to maintain low water tables is expected only when these plants occupy a sufficiently large area.During the last 2 decades, Pakistan has made significant efforts to control soil salinization, which has reduced the saline area from over 6Mha in 1970s to 4.5Mha in 2007.Despite these massive investments over the last 3 decades, soil salinization remains the biggest challenge for the Indus basin.It continues threatening the sustainability of its agricultural system and the capacity of Pakistan to feed its growing population.Much discussion is focused on future water shortages and the need for adequate drainage of the Indus basin.The salt management issues in Pakistan are complex, and therefore an integrated approach is a key for sustainable irrigated agriculture.Irrigation and drainage are closely linked because excess irrigation is the main cause of water logging while the level of irrigation management dictates the amount of effluent disposal.Drainage water disposal will remain a major issue for effective salinity management in Pakistan.Disposal of saline effluent in rivers merely transports the salts to irrigated lands at the tail end of the irrigation system.It is therefore neither a practical nor environmentally friendly long-term solution.Due to the siltation of main reservoirs, the water storage capacity of Pakistan is expected to reduce by 57% by the year 2025 and to meet the future water requirements, 22Bm3 of more water will be needed.Furthermore, due to climate change effects, future unmet water demand is likely to reach 134 million m3 by 2050.Consequently, unless Pakistan significantly increases its freshwater use efficiencies, it will have to use more poor-quality irrigation water in the future.Also, it will need to seek sustainable re-use of drainage water to minimize drainage effluent.Timely availability of farm inputs such as salt-tolerant germplasm and promotion of saline agriculture through crop diversification can improve the capacity of individual farmers as well.Most importantly, farmers will need to have access to new information about improved irrigation management and reclamation approaches.The case studies of Section 15 illustrate the need for application of improved management practices for the major irrigated regions of the world.In this final section, we will synthesize the identified research priorities with these region-specific challenges and needs.Despite the large research and developmental efforts on salt-affected soils in the past, knowledge gaps remain, for new and innovative research and tools that will provide increasing resilience to salt-affected agriculture.Water moves through hydrologic cycles and always carries salts and other elements with it naturally, as it moves through the landscape into the oceans.It is therefore that salinity and water logging have impacted agricultural production in arid areas for more than 2000 years.

Drainage and salinity problems on the Valley’s West side were exacerbated by the construction of the San Luis Unit.The San Luis Unit authorized by the Luis Act of 1960 is part of both the federal Central Valley Project and the State Water Project.The primary purpose of the San Luis Unit was to supply irrigation water for over 400,000 ha of prime farmland.The Luis Act of 1960 as part of a comprehensive basin salinity management plan required that drainage be constructed either as a master drain constructed by the state of California to serve the entire valley or an interceptor drain constructed by the federal government to serve the San Luis Unit service area.The idea was that either of these two drainage systems would convey brackish water northward in a concrete canal into the Sacramento-San Joaquin delta.In mid-1960s the federal and state governments started planning for a master drain that would drain and transport salts out of the entire valley from Bakersfield on the southern end of the valley to the delta.However,plant benches in the early stages of the project the state of California withdrew after failing to get assurances from irrigators that they would pay state expenditures for the project.

In 1968 the federal government through the Bureau of reclamation started construction of a drain system to collect and transport subsurface drainage water from the San Luis Unit service area to the Sacramento-San Joaquin Bay-Delta.However, of the planned 302 km of drain, only 140 km were completed from Kettleman City, near Fresno County, to Kesterson Reservoir in Merced County.Construction was halted in 1975 because of mounting costs and water quality concerns.The main water quality issue was selenium in the Kesterson National Wildlife Refuge which caused various ecological concerns including wildlife birth defects and other toxicities.These instances had a major impact on irrigated agriculture in California.To date the project has stalled due in part to ecological and environmental concerns.In terms of salinity management, the failure to complete the drainage system resulted in reduced agricultural productivity on many farmlands particularly in the Western San Joaquin due to shallow water tables and evapo-concentration of salts in the root zone.Lack of a system to export drainage water and salts out of the valley has stimulated innovative management practices to reduce drainage waters and to find “in valley” solutions for disposal.The threat that salinity poses to California’s economy is widely acknowledged by both public and private stakeholders.For example, a study by Howitt et al.reported that Central Valley salinity accumulations would cause an estimated loss of $2.167 billion in California’s value of goods and services produced by 2030, if they remain unmanaged.

Incomes would decline by $941 million while employment would reduce by 29,270 jobs.Potential benefits of implementing salinity management strategies in the Central Valley were estimated at over $10 billion.It is reasonable to assume that improved salinity management could bring economic benefits to other regions of California that experience salt problems including the Imperial Valley and the Central Coast, while neglecting this problem would bring dire consequences.In California current efforts to address salinity management have included both traditional and contemporary strategies.Traditional salinity management strategies have included source control , dilution, and displacement.While contemporary strategies have included salinity management such as treatment , storage, export, real-time management, and recycling as described in a 2016 inter-agency report by the California Department of Water Resources.The following sections describe recent salinity management case studies in California ranging from on-farm to basin-wide efforts.On-farm salinity management—In California providing environmentally and politically acceptable disposal of drainage water from irrigated agricultural lands is a major challenge for growers.Ayars and Soppe reported to have successfully used a technique called Integrated On-Farm Drainage Management to significantly reduce drainage water to 0.7% of field-applied irrigation water, eliminating the need for evaporating ponds.

IFDM was demonstrated on four 65-ha fields located at Red Rock Ranch on the West side of the San Joaquin Valley of California, by sequentially using saline drainage water for supplemental irrigation.In this study three of the 65 ha blocks were used to grow salt sensitive crops and drainage from these blocks was used to irrigate a salt-tolerant crop.IFDM has been successfully used on other farms in the San Joaquin Valley, e.g., Andrews Ag farm, located in Kern County where IFDM was implemented on 486 ha.At Andrews Ag, salt-sensitive crops were irrigated using drip and sprinkler irrigation.A subsurface drainage system collected the drainage water that was subsequently used to irrigate salt-tolerant crops such as cotton.The salt grass volatilizes selenium as it grows, removing it from the drainage water and rendering it harmless.By 2005 the farm reported that it was able to reduce drainage by 90% and selenium by 80%.Eliminating the need for an evaporating basin provides several benefits, including minimizing the size of land taken out of production and need to mitigate environmental impacts associated with evaporating basins such as leaching of salts to groundwater.However, it is worth noting that management practices such as IFDM only provide short term solutions, and that long-term sustainable irrigation requires exporting salts out of the basin to maintain a salt balance, for example through a brine line.Regional salinity management—A major regional initiative to address the salinity problem in California is the Central Valley Salinity Alternatives for Long-term Sustainability.CV-SALTS is focused on sustainable salinity management.CV-SALTS is a collaborative effort that was initiated in 2006 to find solutions to the salt problem in the Central Valley.It includes several stakeholders such as the State Water Resources Control Board, the Central Valley Regional Water Quality Control Board, agricultural coalitions, cities and municipalities, growers, academics, and environmental justice groups.The goals of CV-SALTS are multifaceted and include sustaining the Central Valley’s lifestyle, support regional economic growth, sustain agricultural economy, maintain a reliable and high-quality urban water supply, and protect and enhance the environment.Because of the seriousness of salt and nitrate issues in the Central Valley, the California State Water Resources Control Board voted in 2019 to approve a Central Valley-wide Salt and Nitrate Control Program that was submitted by CV-SALTS.Subsequently, the Regional Central Valley Water Board started sending out Notices to Comply for the Nitrate Control Programin late May 2020.The Salt and Nitrate Control Program includes both short- and long-term strategies to address salt issues in the central valley.Dischargers can participate in the program as individuals or as a part of a group of dischargers organized in form of a management zone.This is significant because the Salt and Nitrate Control Program provides a framework for the Central Valley Water Board to regulate salt and nitrate discharges for an area covering 46,619 km2.

Issues of salinity in California have tremendous consequences, as there is a lot at stake in terms of economic losses,rolling bench environment degradation and livelihood disruptions.Therefore, to not proactively address salinity is not an option.In California salt moves statewide through the interconnected waterways across different basins.Salinity management should carefully integrate water flows and salt loadings.Sustainable salinity management decisions in any basin involves a wide range of stakeholders such as water managers, regulators, facility operators, policy makers, landowners, growers, agricultural coalitions, environmental justice groups and others.To successful manage salinity in California these entities must strive to coordinate their efforts to use resources efficiently, develop solutions to local and regional problems, optimize funding opportunities, and seek to achieve a salt balance in any given basin.Sustainable salinity management in California will require collaborative efforts to build consensus on scientifically proven solutions that meet multiple objectives for its diverse regions.Both short and long strategies will need to be considered, for example, to achieve a salt balance in a closed basin such as the Tulare Lake basin, discussions must include options for exporting salts out of the basin using a brine line.Water conservation in the Salton Sea basin should be integrated with salinity management.Integrated approaches should be pursued to mitigate sea saltwater intrusion including substituting groundwater pumping in agricultural regions along the central coast with recycled water.Salt-affected soils have a broad distribution and a rich variety of types in China, totally accounting for approximately 100 Mha, or about 1/10th of the entire land area of the country.Climatic conditions, landform and geomorphology, and agricultural practices are key factors influencing soil salinization in this country.The ratio of evaporation to precipitation is often more than one in most regions of Northern China.According to the formation characteristics and geological distribution, salt-affected soils are clarified into seven major zones.In Northwest China, the closed inflow basins , provide the physical base for the development of soil salinization, which associated with the localized arid and hot climate conditions eventually result in the formation of salt-affected soils.In Northeast China and North China Plain, controlled by the monsoon climate, 60–70% of the precipitation occurs in summer, resulting in a cycle of summer water logging and spring drought.Therefore, salt is frequently exchanged between the soil and groundwater.China also has a large area of coastal low plains distributed with salt-affected soils that are mainly due to the seawater encroachment.

Modern integrated investigations on land resources at large-scales in China were started in the 1950s, which have provided an important foundation for improved understanding of the geographical and genetic classifications of salt-affected soils.From then on, research and management practices have been focusing on the reclamation, improvement and sustainable utilization of salt-affected soils regionally, typically located in Xinjiang, Ningxia, Inner Mongolia, and the Songnen plain.Key measures to control soil salinity included artificial salt leaching, paddy rice sowing, fodder rotation, and application of drainage and irrigation systems.Relying on these measures, China has promoted agricultural development in some of its salt-affected soil zones, especially in Northwest China.However, the inadequate irrigation and drainage systems resulted in a dramatic rising of the groundwater table across these regions, and eventually led to secondary soil salinization.Nevertheless, agricultural development led to research and use of water-conserving agricultural technologies, including those that control groundwater depth, prevent water losses of irrigation canals, and building of open ditch and subsurface drainage systems.Additional engineering and agronomic practices developed during this period, including land leveling, flooding sedimentation, green manure planting, organic manure application, and salt-tolerant crop selection.After mid-1970s, key state research projects were launched aiming for integrated management of drought, water logging, and soil salinization.Typically, a national project was initiated in Huang-Huai-Hai Plain in 1978.To systematically study the interrelation and regularities of drought, water logging and soil salinization, a system for monitoring and predicting the regional water and salt was developed.The focus of soil management in this region was on shallow groundwater exploitation.The shallow groundwater water was extracted from tube wells and used for irrigation, which simultaneously lowered the groundwater table.In addition, the low-pressure water transport technique, deep ditches, optimized fertilizer, and shelter belt were used to improve the basic conditions of agricultural production.By 1995, the agricultural total output value in this region was raised by 20–56%.Meanwhile, important progresses were made in the other regions, such as drainage-based rice sowing in Xinjiang and Ningxia, soda-saline soil improvement in Jilin Province , coastal salt marsh development, and improved agricultural drainage systems in Inner Mongolia.Food security is a long-lasting challenge for China because it needs to feed 20% of the world’s population, relying on only 7% of the world’s arable land.The implementation of the integrated salt-affected soil management projects has improved nearly 1.67 Mha of saline-alkali land and has increased nearly 4 million tons of grain production since 2000.Since 2000, China has prioritized the application of water-saving irrigation techniques, especially in arid and semi-arid regions, including the use of pressurized irrigation such as drip and sprinkler irrigation, as well as subsurface irrigation.By the end of 2015, the total area of water-saving irrigated cropland was about 31 Mha in China, including 9 Mha with sprinkler and drip irrigation.In addition, China has initialized multiple policies to facilitate intensive implementations of WSI technologies, e.g.to mobilize local governments by providing additional funds for WSI investment, and to promote the Water Users Associations to take on the irrigation management responsibilities in rural areas.Furthermore, China has launched a comprehensive water management plan in 2006 to improve agricultural water use efficiency.By the end of 2015, it was reported that the average agricultural water-use efficiency increased from 0.53 to 0.58.On the other hand, the agricultural water consumption estimated in 2017 accounted for more than 62% of China’s total annual water consumption, announcing limited potential for continued application of high-quality irrigation water to meet food demand.

The same authors also showed that, while development of wireless networks has focused mostly on integrating sensor technologies, there is limited research done on integrating control systems with sensor data acquisition, aimed at automating smart valve systems at the plant or tree scale.Much of this is required for advanced PI systems, allowing for high resolution control of water and nutrient application such as presented by Coates et al..In addition to ground-based sensors, there is great potential for use of airborne instruments, with the development of commercial airplane based remote sensing and UAVs for agricultural applications.Especially non-contact platforms such as Electro Magnetic Induction could potentially be used with drones for soil salinity monitoring.In addition, hyperspectral and thermal cameras can be used for plant monitoring of water or salinity stress and diseases.In addition to using wireless and new sensor technologies for improved soil salinity management and control, integrating real-time sensor and control data with soil and crop growth simulation models allows for real-time management at the plant/tree scale,potted blueberries when combined with visualization tools and decision support systems.Recent examples of such an approach that integrates sensor information with a combined irrigation application and biophysical crop simulation model was presented by Sperling et al.and Gonzales Perea et al..

However, other applications have shown the successful application of machine-learning, ANN and AI algorithms, training the DDS system using past information to improve forecasting of soil and plant status, as well as for calibration and validation.Advancing PI even further, is to combine sensing and modeling information in a single DDS system , to allow for adaptive irrigation water and soil salinity management.With specific attention to soil salinity, such an integrated management system would allow real-time and plant-scale or zone control of water and fertilizer application, minimizing crop water and salinity stress and optimizing yield and water use efficiency.Summary: The general absence of intensive soil salinity measurements and monitoring prohibits development of improved soil salinity management practices that maintain crop productivity while minimizing soil and water degradation.Knowledge gaps to advance PI are mostly associated with the need for cost-effective technologies that integrate soil moisture, salinity, and nutrient measurements within a cloud-based multi-sensor platform.When included to an IoT cloud-based system that integrates a wireless monitoring and control network with real-time computer simulations of soil water, salinity and crop growth, PI will allow for real-time adaptive management close to the individual plant/tree or zone scale.Soil moisture and salinity monitoring networks are cost prohibitive, as sensors are still too expensive for application at small management zones.Crops vary widely in their tolerance to soil salinity.The physiological response of crops to salinity is related to two processes: osmotic and specific ion effects.

These processes are dependent on each other and often impact the crop collectively.Salinity reduces the osmotic potential of the soil solution thereby requiring the plant to osmotically adjust by concentrating solutes inside their cells to readily extract water via osmosis.This concentration process requires metabolic energy , but its ultimate cost to plant growth depends on ion transport efficiencies across membranes and energy requirements to synthesize organic solutes, which differs among species and varieties within a species.As such, the efficiency of transport processes involving specific ions will affect the overall osmotic response.As a result, salt-stressed plants are stunted, even though they may appear healthy in all other regards.Both adjustment processes, i.e., accumulation of ions and synthesis of organic solutes occur but the extent by which one process dominates over the other is dependent on plant type and level of salinity.At the plant cell level, compartmentalization is critical to keep toxic ions away from locations of sensitive metabolic processes in the cytoplasm.Such compartmentation is controlled by transport processes across the plasma membrane and the tonoplast , as explained in Section 10.Specific ion effects can be directly toxic to the crop, due to excess accumulation of Na, Cl or B in its tissue, or cause nutritional imbalances.While specific ions reduce the osmotic potential of the soil solution, ion toxicities are rarely observed in annual crops grown in the field , provided the ion ratios are not extreme or salinity is too high.However, when Na+ dominates the cations or Cl
concentrations are sufficiently high, these constituents can accumulate in older leaves and produce plant injury.Specific ion toxicities are particularly prominent in tree and vine crops and injury becomes more prevalent over the years, sam but can be controlled by root stock selection.Specific ions can also induce nutritional disorders due their effect on nutrient availability, competitive uptake, transport, and partitioning within the plant.

For example, excess Na+ can cause sodium-induced Ca2+ or K+ deficiency in many crops.As indicated earlier, soil salinity adversely affects plants by a combination of mechanisms, including osmotic influences, toxic ion effects and nutritional imbalances.The most relevant one depends on the crop, its growth stage, duration of salinity exposure and environmental conditions , so that salt tolerance is difficult to quantify.For example, ion toxicity in tree and vine crops becomes more pronounced over the years with foliar injury particularly prominent later in the season.Because of the many factors affecting soil salinity tolerance, there is considerable uncertainty regarding the yield threshold values, as they lack physiological justification.Despite investigators controlling salinity and minimizing all other stresses that could affect yield, the standard errors associated with the ‘threshold’ values can be 50–100%.Obviously, these large percentages represent considerable uncertainty and suggest that true “threshold” values do not exist.Instead, it has been suggested by van Straten et al.to substitute it with a soil salinity parameter, ECe90, that equates to 90% yield.Others have developed non-linear expressions to improve on the physiological response of plants to salinity stress.Crops in most these studies were irrigated frequently, using high leaching fractions to avoid crop water stress.This was done intentionally to create a uniform soil salinity profile across the rooting zone that remained approximately constant during the growing season.In this way, one could compare salinity tolerances among crop species and rank their sensitivity and explains why most salt tolerance models fit such data very well.While creating uniform, steady state root zones experimentally, such uniform profiles are uncharacteristic for an irrigated field.Field soils develop characteristic salt distribution patterns that vary with soil depth and irrigation type.These patterns are a result of water movement via gravitational and capillary action and subsequent root water extraction and soil evaporation.Under sprinkler or border irrigation, the salinity increases with soil depth while under furrow or drip, salinity increases horizontally in the direction of water flow in addition to their increases in the depth direction.Furthermore, soil salinity is affected by rainfall patterns during the growing season,square plastic pot whereas crop salt tolerance can be affected by soil structural changes due to sodic conditions.Under such conditions, three-fold variations in wheat yield were determined for similar soil profile.The accumulation of salts vis-a`-vis their osmotic effects is further modified as a function of soil texture, agro-climatic conditions, ionic constituents of salinity, and soil-irrigation-crop management strategies which impact salt tolerance limits of crops under field conditions.

The current salt tolerance data are based on crop response to saturated soil extract measurements, whereas the crop is responding to the salinity of the soil water in situ, which is continuously changing over space and time.Over the past several decades it is noted that agricultural irrigation is increasingly shifting from conventional surface irrigation methods to pressurized systems that are more efficient.Studies have shown that crops with high-frequency irrigation are more tolerant to salinity than using conventional irrigation methods.Though the wetted root zone is typically much smaller than for low frequency surface irrigation, under high frequency drip irrigation, the salinity of the soil water near the dripper is close to that of the irrigation water with the water content close to field capacity.Therefore, the roots are exposed to a lower soil salinity than for conventional irrigation practices.While the wetted soil volume is smaller, high frequency irrigation allows the crop to maintain its crop water needs.The more recent change to pressurized irrigation puts into question the current validity of historical soil salinity tolerance data using the concepts of Eq.developed for conventional surface irrigation systems , as the root-accessible soil water is near that of the irrigation water salinity using high frequency irrigation.Alternatively, one may think about measuring real-time salinity in situ at multiple soil depths based on the depth-dependent root distribution.The non-uniform conditions of the irrigated soil complicates how best to characterize the root zone in their response to soil salinity, as the roots are exposed to changes in soil water content and salinity in different parts of the profile.It has been recognized for decades that the major root activity is found in the least saline portions of the soil profile.Consequently, it has been shown that shoot biomass can be 3–10-fold higher in heterogeneous soil profiles than under equivalent homogeneous salinity conditions, equal to the average root zone salinity of the heterogeneous soil.Experiments with alfalfa indicated that root water uptake rate reacts to soil salinity, but that additional factors such as root activity and evaporative demand can become more important in controlling uptake patterns.Roots will grow and develop in the most favorable portions of the root zone considering factors such as salinity, water content, nutrients, pH, oxygen availability, soil strength, and disease pressure.For example, soil salinity may be low in the upper portion of the soil profile but soil water content will vary widely due to higher root length density there.In the lower portion of the soil profile the salinity can be substantially higher but water content is higher and fluctuates less due to lower root activity.Other experimental and modeling studies have shown that the sensitivity of plants to salinity depends on the evaporative demand.When multiple stresses occur simultaneously, the dominant stress largely controls crop growth and response.Likewise, release of the most dominant stress will promote the most growth.The root’s developmental response to a combination of variable stresses is remarkable , yet there is considerable uncertainty how the plant integrates multiple stresses over space and time and it remains a huge knowledge gap.More research is needed to better understand the physiological mechanisms underlying plant water relations and shoot ion regulation in plants under heterogenous salinities and how roots can adapt over the growing season with changing soil conditions.While there will likely be complex interactions, it is nonetheless an important area of future research.Summary: Crop salt tolerance data are urgently needed for micro-irrigated crops, rather than using historical information developed for surface irrigation.Though of tremendous value in the past, soil saturation extracts do not necessarily represent in-situ root zone salinity.In addition, there is considerable uncertainty how the plant integrates multiple stresses across the rooting zone and during its growing season and it is a huge knowledge gap.As new cost-effective sensor technologies are being developed, they may be applied across field trials, thereby much better representing real-time and in situ information on the plant’s response to soil salinity, together with other relevant abiotic and biotic soil and plant measurements.Drought and salinity are the two most common abiotic stressors in agricultural crops and their simultaneous occurrence is relatively common in irrigated fields.In addition, the use of saline or brackish water or the reuse of treated effluents for irrigation is expanding , particularly in arid and semi-arid regions with an increased pressure on water resources.Although the proper understanding of crop response to combined water and salinity stressors is a key question for hydrological and crop modeling, little is known about how the combination of these two stressors affects plant health and crop development, transpiration, dry matter accumulation and yield.In this section we will focus primarily on plant root water uptake, whereas the associated impacts of soil salinity on crop yield is treated in Section 10.Plant root water uptake is controlled by potential gradients across the soil-root interface, and is generally described by a Darcy-type flow equation, with flow into the roots driven by a combination of matric potential and osmotic potential gradients , multiplied by a conductance coefficient.Water fluxes into the plant root will reduce both because of decreasing potential gradients, for example due to salt accumulation in the rhizosphere, and by decreasing soil and plant conductance, for example, as the soil dries.At low soil matric potentials, Gardnerand Cowan showed that water uptake is reduced by a local drop in soil hydraulic conductivity at high water potential gradients.

It is estimated that the area of salinized soils is expanding with a rate of about 1.0–2.0Mha/year.However, recent data are scarce, and reported data are greatly outdated.As freshwater resources become more scarce, alternative irrigation waters are tapped into, further threatening soil degradation in many arid regions.Furthermore, climatic change is causing sea level rise and more rapid saltwater intrusion in coastal areas, whereas increased evaporative demands require larger irrigation water amounts.Examples of soil salinization by ancient societies are documented widely, caused by over irrigation, flooding and associated rising water tables, specifically in Iraq across the Euphrates and Tigris, but also in Pakistan and India along the Indus plains and in the Americas.In most if not all of these cases, salts have accumulated in the soil rooting zone over hundreds to thousands of years, because of capillary transport from the rising water tables invading the crop’s rootzone, thereby necessitating cultivating increasingly salt-tolerant crops , eventually leading to hunger and wars, ending those early agricultural civilizations.More recently in the last 50 years or so, salinization has degraded lands in the Aral Sea basin in Central Asia, the Yellow River basin in China, the Murray-Darling Basin in Australia, and the San Joaquin Valley in California at a much faster pace.

Although estimates vary widely, salinized lands are growing at an approximate rate of 10%/year or about, mostly by human-induced agricultural practices ,stacking pots according to Szabolics.The intent of writing this synthesis paper was triggered after a literature review on soil salinity over the past few decades.It was found that most recent publications are applied and hardly added new basic research, because most presented concepts dated back to before 2000, when large-scale irrigation projects largely expanded the world’s irrigated area.Moreover, we believe that funding for salinity research has declined as soil research priorities changed.Though not comprehensive but believed to be indicative, a search using Google Scholar on publications that have soil salinity in their title showed that such publications have stagnated over the past 10 years.Similar conclusions were presented in Li et al.for China,that summarizes our mutual findings and discusses a future perspective on the sustainability of irrigated agriculture in the context of societal issues of water and food security.By identifying the most critical knowledge gaps in soil salinity, we intent to accelerate new research funding to generate new knowledge and innovative solutions.We further want to inspire the science community in developing new directions of salinity research that addresses the identified knowledge gaps presented.The purpose of this section is to comprehensively review the soil’s literature on soil salinity and its relevance, and to present proven soil salinity management practices.We will do this through a review of established handbooks and articles.

Specifically, we note the following key references: Salinization of Land and Water Resources , the ASCE Manual on Agricultural Salinity Assessment and Management with revision by Wallender and Tanji , Soil Salinity under Irrigation , and Saline and Sodic Soils.Other references that are relevant are Kamphorst and Bolt and Sposito.Soil salinity issues occur under a wide range of climatic conditions, both under natural and human-induced conditions, but are especially widespread in arid and semi-arid climates where rainfall is inadequate to leach accumulated salts below the plant’s rooting zone, whether irrigated or rainfed.The key factors associated with soil salinity are geology and its chemistry, climate, and local hydrology.It is the main source of salt in seawater and irrigation water taken from streams, lakes, and groundwater.Salts in seawater arrive on land, via atmospheric deposition either by rain or wind, or via seawater intrusion such as by tsunamis or hurricane winds along coastal areas.When formed, soils may already contain high amounts of salts, due to the parent rock material from which it is derived, such as through the weathering of carbonate mineralsor feldspars.Sedimentary rocks typically contain high amounts of carbonates and sulfates, so that their weathering leads to high alkaline soils containing significant amounts of gypsum and/or calcite.In contrast, weathering of granitic rock dominated by primary minerals such as quartz, feldspars, and micas result in more acidic soils.

Climate dictates the rate of both chemical and physical weathering through its temperature regimes, dissolution, and precipitation of salts, leaching of dissolved ions/salts or accumulation of salts.One distinguishes between primary and secondary, human-induced salinization.Primary salinization occurs by natural processes, such as by atmospheric deposition through rainfall or wind or by rock weathering, accumulating soluble minerals in soils, geological deposits and ground waters.For example, fossil ground waters originate from marine depositions, from which salts become available through seepage to near the land surface or through groundwater pumping.Natural soil salinization occurs widely in seawater-submerged soils and geologic formations and in coastal areas with shallow saline ground waters.For example, much of the salinity in US Northern Great Plains is associated with saline seepage through marine shales and derived weathered regolith, originating from a shallow ocean overlying the region some 100 million years ago.The changing salinity of the Plains in recent times is largely attributed to the change in land-use from prairie grassland to cropland and changing weather patterns with extreme summer rains and associated flooding.In the Netherlands, saline seepage from rising seawater in their coastal areas below sea levelal ways threatens its freshwater availability and are causing main concerns for their agricultural land use.Secondary salinization is caused by human activities, principally by irrigation of agricultural crops under poor drainage conditions and while using marginal irrigation waters.In addition, soil salinity can be caused by removal of deep-rooted vegetation and thereby increasing groundwater recharge ,sawtooth greenhouse and by addition of chemicals to soils such as through fertilizers and waste waters.The specific cause of soil salinization depends on local soil and groundwater transport processes relevant to the landscape and thus varies with climate, landscape type, agricultural activities, irrigation method and associated soil and water management practices.Groundwater related salinity occurs when saline ground waters rise to reach close to the plant rooting zone, followed by upwards transport into the near-surface soil through capillary forces that are triggered by soil evaporation and plant transpiration.This can occur through both primary and secondary salinization, for example, through seepage in low-lying areas or when irrigation-induced by rising groundwater tables.In the latter case, either through excess irrigation or native perennial deep-rooted vegetation removal in dryland agriculture.Non-groundwater associated salt accumulation occurs in landscapes with ground waters that are too deep for upwards capillary action to the plant rooting zone.It is prevalent when drainage of rain or irrigation waters is limited, such when largely controlled by soil textural variations in the landscape or with soil depth.Specifically, coarse-textured soils allow for adequate drainage and salt leaching, whereas soils containing low-permeable soil textural layers restrict deep percolation such as in sodic soils, causing water-logging conditions and shallow water tables.Approximately 6% of the world’s terrestrial land is believed to be salinized by primary salinization.In addition, some 20% of all cropland and between 1/4 and 1/3 of irrigated land is salinized by secondary salinization, totaling about 1Bha globally.To quantify soil salinity, one commonly estimates the concentration of total soluble salts through the electrical conductivity or EC, expressed in dS/m or mmho/cm.We note that 1 dS/m corresponds to a salt concentration of approximately 680mg/L of total dissolved solidsin soil solution.However, the effective concentration will depend on the ion activity coefficients, as affected by many factors such as the presence of ion pairs, other complex formations, and temperature.Whereas field measurements of EC represent the bulk soil, the more accepted measurement of soil salinity is using the EC of the extracted solution of a saturated soil paste, defined by ECex.This is so, because plants are dominantly affected by soil salinity through the concentration of salts in the soil’s solution.

While other extraction methods may quantitatively be more reproducible and have shown good correlations with the chemistry of the saturated paste for Cl
dominated systems , the US Salinity Laboratory promoted using ECex becausethe chemistry of the saturated soil extract is close to that of the soil waterandthe chemistry could vary due to dissolution and precipitation of sulfate and carbonate minerals, should larger soil water dilutions be employed.The widely accepted classification of what constitutes different levels of soil salinity was defined by the US Laboratory Staff , with ECex values smaller than 2 dS/m classified as non-saline soils, whereas ECex values between 2 and 4, 4–8, 8–16 dS/m are defined as slightly, moderately, and strongly saline soils, respectively.Though widely accepted, there are limitations to its use.Firstly, it is a laboratory measurement that underestimates the in situ salinity for unsaturated soils.Second, soil wetting in the laboratory will often lead to dissolution of precipitated salts , thereby overestimating EC of the natural soil.The other relevant soil salinity property is related to the amount of sodiumin soils, as expressed by the Exchangeable Sodium Percentage or the Sodium Adsorption Ratio.The weathering of primary rock minerals results in the generation of individual soil particles that are negatively charged, thereby leading to the electrostatic adsorption of cations from soil solution to counterbalance the total charge along the particle’s interstitial hydrated surfaces.Much of the ability to adsorb cations will depend on soil mineral type and varies widely between clay minerals.However, all soils do adsorb ions at a certain level with the type of cations absorbed largely controlled by the composition of the soil solution.The magnitude of adsorptive capacity and level of negative charge is quantified by the soil’s Cation Exchange Capacity and varies between near zero for pure sands to 100meq/L or larger for smectite clay soil minerals.Much of the impact by on soils is caused by relatively high exchangeable sodium levels , through its adsorption from soil solution thereby largely affecting soil physical properties such as the bulk density and the water retention and hydraulic conductivity characteristics.As compared with divalent cations such as Ca and Mg, the sodium ionis less strongly adsorbed to soil particle surfaces.When hydrated, soil particles surrounded with sodium dominated water film tend to repel each other, thereby leading to soil dispersion.This causes soil aggregates to break down into individual soil particles, thereby clogging interstitial pore spaces and forming depositional soil crusts upon drying.Particle dispersion is further accelerated by soil swelling, driven by osmotic gradients, forcing pore water into the interlayers of clay minerals, especially pronounced at low salinity.Upon soil drying, these soils will shrink, creating soil cracks that can go very deep into the soil profile.When wetting, these types of soil structural degradation will largely reduce water infiltration and soil drainage, causing water logging and flooding and making soil prone to water and wind erosion.Much more detail will be presented in Section 12.Plants—Increasing levels of total salinity in the soil water solution will reduce the ability of plants to take up water from the soil because of osmotic effects, whereas specific ions such as Na, Cl, or B negatively impact plant physiological processes and can become toxic when adsorbed by the plant.In addition, saline soils can reduce plant nutrient uptake or cause ion imbalances as specific ions such as Na can compete with other essential plant nutrients, causing mineral nutrition disorders and further the plant’s ability to survive and produce.Any of these effects vary among plant species and crops.For that reason, empirical crop salt tolerance response functions have been developed , defining yield reduction as a function of total soil solution salinity based on ECex data.However, such data for developing salt tolerance parameters were collected assuming constant and high soil moisture conditions, both during the growing season and with crop rooting depth.However, in real field conditions soils wet and dry depending on irrigation frequency so that soil salinity conditions are typically non-uniform with time and soil depth.These factors along with the exclusion of specific ion effects on plant stress and yield limits their applicability.Yet, more detailed additional information is often not available.The osmotic effect on crop growth, as quantified by the soil water osmotic potentialis often considered simultaneously with soil water stress, as determined by soil water matric potential.Both are abiotic stresses that can be added, such as illustrated in Fig.2 , to reflect the combined additional energy required for plant root water uptake , as a function of soil water content and soil solution salinity.

By examining how researchers, experts, and officials frame and design their projects and set up responses to problems, anthropology can open up new spaces for insight into the complexities of environmental politics, and the idea of islands by design helps me to do that in the purposefully particular place that is The Bahamas today.Bagged greens in the market are often labeled “pre-washed,” “triple-washed,” or “ready-to-eat,” and look shiny and clean. But are they really “clean” of harmful microbes? We cannot be so sure. Food safety has been threatened by contamination with human pathogens including bacteria, viruses, and parasites. Between 2000 and 2008, norovirus and Salmonella spp. contributed to 58 and 11% of forborne illnesses, respectively in the United States . In those same years, non-typhoidal Salmonella alone was ranked as the topmost bacterial pathogen contributing to hospitalizations and deaths . In 2007, 235 outbreaks were associated with a single food commodity; out of which 17% was associated with poultry, 16% with beef, and 14% with leafy vegetables that also accounted for the most episodes of illnesses . Apart from the direct effects on human health, enormous economic losses are incurred by contaminated food products recalls. The 8-day recall of spinach in 2006 cost $350 million to the US economy .

It should be realized that this is not the loss of one individual, but several growers, workers,grow strawberry in containers and distributors. This is a common scenario for any multistate food borne outbreak. Additionally, the skepticism of the general public toward consumption of a particular food product can lead to deficiencies of an important food source from the diet.Economic analysis shows that money spent on prevention of food borne outbreak by producers is much less than the cost incurred after the outbreak . Contamination of plants can occur at any step of food chain while the food travels from farm to table. Contaminated irrigation water, farm workers with limited means of proper sanitation, and fecal contamination in the farm by animals can expose plants to human pathogens before harvest of the edible parts . After harvest, contamination can occur during unclean modes of transportation, processing, and bagging . Mechanical damage during transport can dramatically increase the population of human pathogens surviving on the surface of edible plants . Control measures to decrease pathogen load on plant surfaces have been defined by the Food Safety Modernization Act and Hazard Analysis and Critical Control Point system . Using chlorine for post-harvest crop handling has been approved by US Department of Agriculture under the National Organic Program. However, some studies indicated that internalized human pathogens escape sanitization . Thus, understanding the biology of human pathogen-plant interactions is now crucial to prevent pathogen colonization of and survival in/on plants, and to incorporate additional, complementing measures to control food borne outbreaks.

We reasoned that as plants are recognized vectors for human pathogens, enhancing the plant immune system against them creates a unique opportunity to disrupt the pathogen cycle. In this cross-kingdom interaction, the physiology of both partners contribute to the outcome of the interactions . Bacterial factors important for interaction with plants have been discussed in recent, comprehensive reviews . Plant factors contributing to bacterial contamination is much less studied and discussed. In this review, we highlight current knowledge on plants as vectors for human pathogens, the molecular mechanisms of plant responses to human bacterial pathogens, and discuss common themes of plant defenses induced by phytopathogens and human pathogens. We have focused on human bacterial pathogens that are not recognized plant pathogens such as Salmonella enterica and Escherichia coli , but yet are major threats to food safety and human health.The leaf environment has long been considered to be a hostile environment for bacteria. The leaf surface is exposed to rapidly fluctuating temperature and relative humidity, UV radiation, fluctuating availability of moisture in the form of rain or dew, lack of nutrients, and hydrophobicity . Such extreme fluctuations, for example within a single day, are certainly not experienced by pathogens in animal and human gut. Thus, it is tempting to speculate that animal pathogens may not even be able to survive and grow in an environment as dynamic as the leaf surface. However, the high incidence of human pathogens such as S. enterica and E. coli O157:H7 on fresh produce, sprouts, vegetables, leading to foodborne illness outbreaks indicate a certain level of human pathogen fitness in/on the leaf.

The plant surface presents a barrier to bacterial invaders by the presence of wax, cuticle, cell wall, trichomes, and stomata. All except stomata, present a passive defense system to prevent internalization of bacteria. Nonetheless, several bacteria are able to survive on and penetrate within the plant interior. The surface of just one leaf is a very large habitat for any bacteria. The architecture of the leaf by itself is not uniform and provides areas of different environmental conditions. There are bulges and troughs formed by veins, leaf hair or trichomes, stomata, and hydathodes that form microsites for bacterial survival with increased water and nutrient availability, as well as temperature and UV radiation protection . Indeed, distinct microcolonies or aggregates of S. enterica were found on cilantro leaf surfaces in the vein region In addition, preference to the abaxial side of lettuce leaf by S. enterica may be is an important strategy for UV avoidance . Conversion of cells to viable but non-culturable state in E. coli O157:H7 on lettuce leaves may also be a strategy to escape harsh environmental conditions . Hence, localization to favorable microsites,hydroponic nft channel avoidance of harsh environments, and survival by aggregation or conversion to non-culturable state may allow these human pathogens to survive and at times multiply to great extent on the leaf surface. As stomata are abundant natural pores in the plant epidermis which serve as entrance points for bacteria to colonize the leaf interior , several studies addressed the question as to whether human bacterial pathogens could internalize leaves through stomata. Populations of E. coli O157:H7 and S. enterica SL1344 in the Arabidopsis leaf apoplast can be as large as four logs per cm2 of leaf after surface-inoculation under 60% relative humidity suggesting that these bacteria can and access the apoplast of intact leaves. Several microscopy studies indicated association of pathogens on or near guard cells. For instance, S. enterica serovar Typhimurium SL1344 was shown to internalize arugula and iceberg lettuce through stomata and bacterial cells were located in the sub-stomatal space . However, no internalization of SL1344 was observed into parsley where most cells were found on the leaf surface even though stomata were partially open . Cells of S. enterica serovar Typhimurium MAE110 , enteroaggregative E. coli , and E. coli O157:H7 were found to be associated with stomata in tomato, arugula leaves, and baby spinach leaves, respectively. In the stem E. coli O157:H7 and Salmonella serovar Typhimurium were found to be associated with the hypocotyl and the stem tissues including epidermis, cortex, vascular bundles, and pith when seedlings were germinated from contaminated seeds . The plant rhizosphere is also a complex habitat for microorganisms with different life styles including plant beneficial symbionts and human pathogens. Nutritionally rich root exudate has been documented to attract S. enterica to lettuce roots .

Although bacteria cannot directly penetrate through root cells, sites at the lateral root emergence and root cracks provide ports of entry for S. enterica and E. coli O157:H7 into root tissues , and in some instances between the epidermal cells . High colonization of S. enterica has been observed in the root-shoot transition area . Once internalized both bacterial pathogens have been found in the intercellular space of the root outer cortex of Medicago truncatula . Salmonella enterica was found in the parenchyma, endodermis, pericycle, and vascular system of lettuce roots and in the inner root cortex of barley . A detailed study on the localization of E. coli O157:H7 in live root tissue demonstrated that this bacterium can colonize the plant cell wall, apoplast, and cytoplasm . Intracellular localization of E. coli O157:H7 seems to be a rare event as most of the microscopy-based studies show bacterial cells in the intercellular space only. Bacterial translocation from roots to the phyllosphere may be by migration on the plant surface in a flagellum-dependent manner or presumably through the vasculature . The mechanism for internal movement of enteric bacterial cells from the root cortex to the root vasculature through the endodermis and casparian strips and movement from the roots to the phyllosphere through the vascular system is yet to be demonstrated. Several outbreaks of S. enterica have also been associated with fruits, especially tomatoes. Salmonella enterica is unlikely to survive on surface of intact fruits raising the question: what are the routes for human pathogenic bacteria penetration into fruits? It has been suggested that S. enterica can move from inoculated leaves , stems, and flowers to tomato fruits. However, the rate of internal contamination of fruits was low when leaves were surface-infected with S. enterica . The phloem has been suggested as the route of movement of bacteria to non-inoculated parts of the plant as bacterial cells were detected in this tissue by microscopy . Figure 1 depicts the observed phyllosphere and rhizosphere niches colonized by bacteria in/on intact plants and probable sources of contamination.Plants possess a complex innate immune system to ward off microbial invaders . Plants are able to mount a generalized step-one response that is triggered by modi- fied/degraded plant products or conserved pathogen molecules. These molecules are known as damage or pathogen associated molecular patterns . In many cases, conserved PAMPs are components of cell walls and surface structures such as flagellin, lipopolysaccharides, and chitin .PAMPs are recognized by a diverse set of plant extracellular receptors called pattern-recognition receptors that pass intracellular signals launching an army of defense molecules to stop the invasion of the pathogens. This branch of the immune system known as pathogen-triggered immunity is the first line of active defense against infection. Human pathogen on plants is an emerging field that only recently has caught the attention of plant biologists and phytopathologists. A few studies have been reported in the last 5–10 years, which focused on the most well studied PAMPs, flagellin and lipopolysaccharide , in the interaction of human pathogens with plants. Table 1 lists the plants, bacterial strains, and method details for such studies.Flagellin, the structural component of flagellum in bacteria, is involved in bacterial attachment and motility on the plant , is recognized by plant through the FLS2 receptor , and induces plant defenses . Similar to the well-studied PTI elicitor flg22 , the flg22 epitope of S. enterica serovar Typhimurium 14028 is also an effective PAMP and elicitor of downstream immune responses in Arabidopsis , tobacco, and tomato plants . Flagellum-deficient mutants of S. enterica serovar Typhimurium 14028 are better colonizers of wheat, alfalfa, and Arabidopsis roots as compared to the wild type bacterium further suggesting that the Salmonella flagellum induces plant defenses that may restrict bacterial colonization of several plant organs. However, the Salmonella flflg22 peptide is not the only PAMP for elicitation of plant immune response as flfls2 mutant of Arabidopsis still shows a low level of PTI activation in response to this PAMP . Purified flagellin or derived epitopes of E. coli O157:H7 has not been used to induce plant defenses. However, flagellum-deficient mutant of this strain does not activate the SA-dependent BGL2 gene promoter as much as the wild type strain and shows larger population in Arabidopsis than the wild type strain further suggesting that surface structures in the bacterial cell are perceived by plants. The differences in responses observed could be attributed to the presence of other microbial signatures eliciting plant defense. Variations in plant response to S. enterica flagellin could be owed to host-strain specificity as well. Although flagellin sequences from S. enterica strains and other bacteria are highly conserved, even a minor change of five amino acids in the flflg22 epitope leads to reduced activation of PTI in Arabidopsis, tobacco, and tomato plants .

Given that JEDI–PDF genes are JA-inducible, this signaling molecule may also be important for the induction of basal defense against Hpa. No other studies on silencing or disruption of the JEDI–PDF genes have been reported on in the literature. Future studies will have to address this possibility. A set of RNAi transgenic lines was developed which can be used to elucidate roles the JEDI-PDF genes play in plant defense. Pathogen assays indicated that these genes may, in fact, play a role in basal defense against HpaNoco2. Since a defense phenotype was observed in some PDF-RNAi lines indicated that this technique may have overcome the problem of redundancy previously limiting the study of these genes. These observations, together with this experimental data suggested a potential role of the JEDI-PDF genes in plant defense against Hpa. The PDF-RNAi lines could prove valuable tools for the further study of the JA/ET signaling network and plant defense responses.Even within different Arabidopsis accessions variation exists in the level of cross-talk between SA and JA signaling, indicating that variation in downstream signaling of plant hormones contributes to natural variation in basal resistance .

SA and JA are also known to contribute differently to defense based upon the invading pathogen . While a large body of research indicates that SA and JA pathways are mutually antagonistic ,rolling benches more recent studies have found indications of synergism between these pathways . Current methods providing resistance against viral infections in plants often involve transgenic plants, which are often not widely accepted by consumers . Additionally, pesticides are being used but are often toxic to humans and the environment or are only useful for a small range of plant-species or virus-species . One recent study performed by Shang et al., 2011 observed that SA accumulates one day after JA during immune responses of Nicotiana benthamiana against viruses. This group also found that the stimulation of JA accumulation followed by external application of SA reduced viral replication by 80–90% . This regime of phytohormone application produced broad-spectrum and effective resistance in Arabidopsis, N. benthamiana, Nicotiana glutinosa, Nicotiana tabacum, Capsicum frutescens , and Solanum lycopersicumagainst Cucumber mosaic virus, Turnip crinkle virus, Tobacco mosaic virus and Tobacco necrosis virus, respectively . PDF1.2 is known as a JA-inducible and SA-repressible gene ; however, its expression level was higher in SA-pretreated virus-infected seedlings than in the virus-infected seedlings without any treatment. This may be due to the complex cross-talk between JA and SA.

Other studies also found that the timing and/or order of pathway induction matters when trying to induce plant defense . During a long-term plant pathogen interaction , PDF1.2a may become SA-inducible, although it is negatively regulated by SA at early time points during pathogenic interactions . To have a complete picture of what is taking place during these interactions, longer time points should be evaluated. This is especially important since much work on pathway crosstalk has been performed at early time points. This data highlights the fact that the use of synthetic elicitors targeting SA and/or JA-dependent defense signaling mechanisms to induce broad spectrum resistance is environmentally friendly, safe, easy to perform, and did not affect agronomical traits in a negative manner. Unfortunately, the cost of JA for the use in agriculture is prohibitively high, while that of MeJA is lower but not low enough for widespread use. However, the cost of SA or BTH is low enough for these compounds to be used in crop protection. Cheaper synthetic elicitors would be beneficial for the agricultural industry. The identification of synthetic elicitors targeting both the SA and JA branch of the defense network may lead to novel agrochemicals that induce effective broad-spectrum diseases resistance but are not toxic to the surrounding biosphere. Plant innate immunity depends on a network of genes that regulate and execute defense reactions.

Using chemical genetics, small molecules can be identified that induce plant immune responses, but are structurally distinct from natural defense elicitors. I have initiated a chemical genomics-based approach to identify, characterize and utilize new types of synthetic elicitors for the dissection of the plant immune network as well as the development of novel types of pesticides. A cluster of genes were identified that are coordinately up-regulated after treatment with the synthetic elicitors, DCA or INA. The ACID cluster is composed of 137 genes, which are upregulated during time-points when defense is also on. The ACID genes were found to be enriched for protein kinases, which I hypothesized play key roles in plant defense signaling. Based on this knowledge, ACID genes that are kinases were selected first for study. Each ACID gene was also selected based upon the availability of two independent insertion lines. I showed that of the 16 ACID genes examined, 10 are required for Arabidopsis basal defense against HpaNoco2. Seven of the 10 ACID genes are unknown components of the plant immune system. While important for basal defense, I found that these genes are not essential for immunity mediated by the two R-genes RPP7 or RPP4 . I next determined if DCA-mediated immunity was compromised in the ACID mutant lines. Although they are transcriptionally activated by DCA, they are not required for DCA-mediated resistance. Additionally, RT-PCRs showed that the up-regulation PDF1.2 transcripts are abolished in many of these mutants. Based on these results I hypothesize that these ACID genes may play a role in later Arabidopsis defense responses and/or have roles in the cross talk between defense pathways. By high-throughput chemical screening we previously identified 114 synthetic elicitors that activate expression of a pathogen-responsive reporter gene in transgenic Arabidopsis. Here I described the characterization of nine novel synthetic elicitors identified in the screen performed by Knoth et al., 2009.

Notably, I identified a synthetic elicitor that has a substantially lower active concentration than DCA. I report in depth on the characterization of one of these compounds, 2–thiazolidine-4-carboxylic acid . CMP442 is able to quickly and transiently induce disease resistance against Hpa in Arabidopsis, has a distinct mode-of-action from DCA, and is structurally unique from previously identified synthetic elicitors. CMP442 can be synthesized quickly, easily, and inexpensively with a high degree of purity. During my efforts to develop a screen for mutants expressing altered sensitivity to CMP442 a surprising root phenotype was identified. I found that at low doses, CMP442 and other synthetic elicitors enhanced growth of roots and aerial parts of Arabidopsis thaliana and tomato , while high concentrations were toxic or inhibited plant growth. The effect of these synthetic elicitors on root growth is suggestive of a hormetic response. Hormesis is characterized by low dose stimulatory or beneficial effects, as evidenced by the increase in root length and plant weight,ebb and flow bench and a high dose inhibitory effect. The ability of CMP442 and other elicitors to beneficially affect both plant immunity and development points to crosstalk between both types of biological processes and may allow for the design of novel types of multi-functional agrochemicals. These agrochemicals would not only induce defense but increase crop yield at the same time. CMP442 may also allow us to uncover fundamental causes of the general phenomenon of hormesis. The ease and inexpensiveness of CMP442 synthesis show great promise for the use of this synthetic elicitor on larger studies. As previously mentioned synthetic elicitors make it possible to dissect and study the plant defense network, which advancing our goal of developing novel “green” pesticides. Here I report on the development of a screen to identify synthetic elicitors that activate the JA/ET branch of the defense network.

Towards this end a set of genes was identified showing a SA-independent upregulation in response to infection of Hpa. Four out of the five genes are PDFs, and one was the JA pathway molecular marker, PDF1.2a. While the resulting transgenic lines may not be appropriate for a high-throughput chemical screen they still are an additional asset for the study of plant-pathogen interactions. Additionally, I describe the creation of transgenic plants with a RNA silencing transgene able to knock down the transcripts of this highly related family of PDFs. Using the PDF-RNAi silenced plants I performed HpaNoco2 defense assays. These pathogen assays indicate that the RNA silencing was a success and that these genes may, in fact, play a role in basal defense against HpaNoco2. The defense phenotype indicates that the JEDI-RNAi lines overcame the problem of redundancy previously limiting the study of these genes. These observations, together with my experimental data suggest a definitive role of the JEDIs in plant defense against Hpa. The previous observations stating that the JA/ET branches of the defense network have no role in resistance against Hpa are challenged by these results. The JEDI-RNAi lines could prove invaluable tools for the further study of the JA/ET signaling network and plant defense responses. Through the use of a synthetic elicitor such as DCA, we have discovered novel components of the plant defense network. My work has firmly established the ACID genes as significant aspects of the defense signaling network. While their roles in plant defense are still yet to be defined it is clear that our synthetic elicitors are powerful tools to dissect plant defense responses. I have identified nine additional synthetic elicitors which show great potential, two of which were studied further here. One of these CMP442 shows a distinct mode of action from DCA and thus proves that a single screen can be used to identify functionally distinct synthetic elicitors. We have not yet been able to identify any targets of our synthetic elicitors. Although, a screen has been developed that could be used to identify mutants that show altered responses to our synthetic elicitors. Any mutants discovered in these screens with altered sensitivity to these compounds could identify novel features of the defense network or the target of our synthetic elicitor. In addition, these synthetic elicitors show great potential for their use in other plant systems which may facilitate the study of homologous processes across species barriers. These elicitors have already proven to be invaluable tools for the study of plant defense and may prove useful in systems not readily accessible by current molecular techniques. There is also great potential for some of these compounds to pave the way for the development of novel pest control regimes. Application of blends of non-toxic defense activators could be a viable alternative to environmentally hazardous toxic pesticides. These elicitors will be exceedingly valuable tools for further dissection of the plant immune system.Plant innate immunity depends on a network of genes that regulate and execute defense reactions. Two major branches of this network have previously been characterized and are known to involve either SA or JA and ET. Using chemical genetics, small molecules can be identified and characterized that induce plant immune responses, but are structurally distinct from natural defense elicitors. These synthetic elicitors can be used to dissect the different branches of the defense network. I have initiated a chemical genomics based approach to identify, characterize and utilize new types of synthetic elicitors for the dissection of the plant immune network as well as the development of novel types of pesticides. Previous work by our lab identified a cluster of Arabidopsis thalianagenes that are coordinately up-regulated after treatment with the synthetic elicitors, DCA or INA . The ACID cluster is composed of 137 genes, which are up-regulated during time-points after DCA or INA treatment when each of these synthetic elicitors triggers disease resistance against Hyaloperonospora arabidopsidis. The ACID genes were found to be enriched for protein kinases, which I hypothesized are likely to play key roles in plant defense signaling. Based on the knowledge that kinases are important for plant defense, a selection of ACID genes predominantly encoding protein kinases were subject to reverse genetics-based functional studies. I showed that of the 16 ACID genes examined, 10 are required for Arabidopsis basal defense against HpaNoco2 . Seven of these 10 ACID genes are so far unknown components of the plant immune system. While important for basal defense, I found that these genes are not required for immunity mediated by the two R-genes, RPP7 or RPP4. I further determined if DCA-mediated immunity is compromised in the ACID mutant lines.

One possible explanation was that the experimental conditions varied between the assays for basal defense and DCA-mediated immunity. In contrast to the analysis of DCA-mediated immunity , there was no pre-treatment with compound or mock solution before being sprayed with pathogen in the basal defense assays . It was observed in other experiments that this type of pretreatment affected the experimental outcome and indicated that pre-treatment of Arabidopsis plants with mock solution reduced the extent of susceptibility to Hpa. To examine if any of these synthetic elicitors induced phytotoxicity, trypan blue staining of seedlings after saturation treatment , where seedlings were grown and treated in liquid medium, was used. Dark blue staining, indicating cell death, was prevalent in 100% of the seedlings treated for 24 h with 300 µM of DCA, CMP144, CMP994, CMP202, CMP199, and CMP384 . Cell death was evident starting at treatments of 100 µM for CMP199. Therefore, the absence of GUS expression at 100 µM CMP199 was likely due to compound-induced phytotoxicity. For all synthetic elicitors cell death was not observed at concentrations showing effective GUS reporter activation ,blueberry grow pot indicating that cell death was not responsible for the GUS reporter activation.

Based on these preliminary assays, these synthetic elicitors were clearly potent inducers of GUS expression in the pCaBP22::GUS lines. Additional study was warranted to scrutinize their unique properties and ascertain their potential for future research use. To accurately quantify the defense-inducing activity of these eight novel synthetic elicitors, a dose-response analysis measuring the inhibition of HpaNoco2 spore development in three-week-old Col-0 plants was performed . For this experiment Col-0 seedlings were sprayed with the respective compound or mock solution and challenged with HpaNoco2 24 h later. Hpa spore formation was evaluated 7 dpi. Each synthetic elicitor behaved similarly during either Hpa defense or the reporter gene assays. CMP199 showed nearly 70% inhibition of spore development at 1 µM and 100% at 10 µM. CMP199 which showed a high activity in GUS saturation treatments, proved to be a more efficient defenseinducer compared to DCA at every concentration tested . This finding was important since this was the first time that a synthetic elicitor more potent than DCA had been discovered by our program. CMP199 showed great potential for future use. In the defense assays CMP199 and DCA provided most efficient protection against HpaNoco2, in contrast CMP384 was only able to induce significant levels of immunity at the highest tested concentration . The weakest of the examined new synthetic elicitors, CMP384 and CMP608 mediated only a 30% inhibition of spore development at 100 µM. The remaining five compounds exhibited intermediate levels of defense-inducing activity, with 55-75% inhibition of spore development at 100 µM. Different inducers of the same biological response may act antagonistically, additively, or synergistically. Synergistic activities produce a greater effect than the sum of their independent effects on the system.

A suite of synthetic elicitors with distinct modes of action, acting synergistically, may have great potential as environmentally safe pesticides, as they may trigger strong defense responses at extremely low concentrations when applied in combination. In addition, synergistically acting synthetic elicitors may be useful for plant defense network studies by facilitating the discovery of cooperative crosstalk mechanisms linking distinct compound-triggered pathways. Two redundantly acting compounds should not induce pCaBP22-333::GUS expression when each is applied at concentrations below their minimally active concentration. Additively acting compounds should activate the reporter when combined at concentrations equaling the sum of their minimally active ones. However, these compounds should not activate the reporter when applied at lower combined concentrations. If two compounds activate reporter expression at combined concentrations lower than the sum of their minimally active concentrations, their effects are synergistic. Finally, if two compounds act antagonistically the reporter gene response triggered by one compound should be reduced by application of the second compound. Antagonistically acting compounds would be tested at concentrations guaranteed to induce pCaBP22- 333::GUS expression. Here, the synergy of DCA, CMP442 , or CMP199 under saturation treatment conditions was tested. Consistent with previous observations, CMP199 proved to be the most potent of these three synthetic elicitors . CaBP22-333::GUS expression was scored visually based upon intensity of color. The minimal concentration of CMP199 for inducing the pCaBP22-333::GUS reporter was 100 nM, whereas that of DCA was 250 nM, and that of CMP442 was 1 µM. When any two of the synthetic elicitors DCA, CMP442 or CMP199 were combined the resulting responses were neither synergistic nor antagonistic .

GUS expression was weakly evident after the mixture of 50 nM each of DCA and CMP199 . This combination does not indicate synergism because if it was, GUS expression should have been stronger resulting in a more intense blue color. Instead, it appeared that this interaction was additive given that they activated the reporter at concentrations equaling the sum of their minimally active ones. Further experimentation will be necessary to determine more precisely the minimally active concentration of CMP199 to confirm that the tested compounds do not act synergistically. A concentration below 250 nM of DCA must be used in conjunction with CMP442 to distinguish between these two options. However, it is certain that they did not act strongly synergistic, as 50 nM of each, which is below their minimally active concentration, did not induce GUS expression. Finally, CMP199 and CMP442 appeared to act additively when used together. If they acted synergistically, a mix of 50 nM of each compound would have induced GUS expression. Instead 50 nM CMP199 and 250 nM CMP442 induced GUS in an additive manner. Saturation treatments were revealing about the role each synthetic elicitor plays in the activation of defense. Nonetheless, a more comprehensive defense assay may be necessary to confirm these observations while testing a wider range of concentrations. The complex responses associated with the activation of the plant immune system can be dissected and studied with the aid of synthetic elicitors activating the system at distinct points. These synthetic elicitors may serve as powerful tools to complement traditional genetics used to study plant defense networks. The versatility and reversibility of many synthetic elicitors makes the identification of a wider range and spectrum of these compounds a priority. Building on the success experienced by Knoth et al. in their identification of DCA, eight more synthetic elicitors were reported on here. Experiments demonstrated that most of these elicitors, excluding CMP608 and CMP384, have the ability to induce pCaBP22-333::GUS expression at concentrations of at least 10 µM. These low concentrations increase the probability of high target specificity,hydroponic bucket decreasing the likelihood of off-target or unwanted side effects . At synthetic elicitor concentrations that induced defense, no herbicidal effects were observed, while all compounds proved to be phytotoxic at higher concentrations. Each new synthetic elicitor discussed here adheres to Lipinski’s rule of five suggesting they may all be readily absorbed by plant cells. These rules state that properties that favor bioactive compounds include a molecular weight of less than 500 g/mol, a lipophilicity value of more than five, less than five hydrogen-bond donors, and less than 10 hydrogen-bond acceptors . CMP60, CMP994, and CMP202 share a phenyl-1H-pyrazol-1-ylmethanone skeleton as a common substructure.However, several closely related analogs act as potent inhibitors of human neutrophil elastase, antiinflammatory, analgesic, anticonvulsant, anticholinergic, antihistaminic, antibacterial, antiepileptic, and antifungal agents . CMP384 analogs, but not CMP384 itself, were reported to have antimicrobial activities . Of the remaining compounds, the exact structures or closely related analogs of CMP144, CMP174, and CMP199 are not referenced to in any biological studies. The wide range of structures and their apparent uniqueness offers many promising candidates for potential synthetic elicitors. In HpaNoco2 defense assays, the novel synthetic elicitor CMP199 outperformed DCA with a lower active concentration. A lower active concentration may reflect a higher degree of target specificity. Also, a major goal of this project was the identification of compounds with different modes-of-action. A suite of functionally distinct synthetic elicitors is likely to allow for more control and flexibility when studying the plant defense network and is likely to facilitate the creation of novel environmentally friendly pesticides. CMP199 will be further examined by other lab members to determine if its mode of action is distinct from that of DCA.

To determine if the available synthetic elicitors acted synergistically, dual combinations of DCA, CMP199, and CMP442 were tested at a range of different concentrations for each compound. This experiment demonstrated that these synthetic elicitors are likely to act in an additive, but not synergistic, manner. As a confirmation of this data, additional assays should be performed testing disease resistance inducibility with these combinations of synthetic elicitors. These experiments are necessary since it is possible that the GUS assay is not of sufficient sensitivity to display synergistic effects since. Pathogen assays would provide quantitative data instead of the qualitative data provided by the visually read GUS assays. The plant immune system is a highly complex web of interactions that allow plants to modulate their responses to the type of infecting pathogen. Currently, only a small number of defense network components have been identified and characterized. The identification of additional elements that compose this web will lead to a better understanding of plant defense and new strategies to combat diseases. In microarray experiments performed by Knoth et al. , the ACID cluster was identified as a set of genes upregulated at time points corresponding to synthetic elicitor-mediated disease resistance. These 137 ACID genes were hypothesized to be important for plant defense. More than one quarter of the 137 ACID members encode kinases . Of the 16 ACID genes examined in this study, 12 encoded protein kinases. Protein kinases often act as regulatory elements controlling plant defense responses. To increase the probability of success in the functional analysis of ACID members, genes encoding protein kinases were preferentially chosen for study. ACID3 encoded a TIR-NBS-LRR class disease resistance protein and ACID8 a LRR-receptor like protein kinase. ACID9 and ACID13 encoded LRR transmembrane protein kinases and ACID12 was a receptor like protein kinase. Additionally, ACID1, ACID7, ACID11, ACID10, ACID14, ACID15, and ACID16 were genes that encoded for proteins with potential kinase activity. The remaining four ACID genes did not encode protein kinases. Of the remaining genes: ACID2 is an armadillo/beta-catenin repeat family protein, ACID4 a remorin family protein, ACID6 a member of EXO70 gene family, and ACID5 is an ankyrin repeat family protein. These four genes were included based on the availability of two independent homozygous T-DNA insertion lines. For ACID1, ACID4, ACID6, ACID7, ACID8, ACID9, ACID10, ACID12, ACID13, and ACID15 both tested T-DNA mutant lines exhibited enhanced Hpa susceptibility. This indicated that the mutations in/near the genes themselves were likely responsible for the respective defense phenotypes, as opposed to unknown mutations. Only one of the two tested T-DNA insertion lines for ACID5, ACID11, and ACID16 exhibited reduced basal defense to Hpa. It is likely that in each case the second line contained its T-DNA insertion in a position not capable of reducing gene function. For example, the insertion in acid11-1 is located in an exon and caused enhanced susceptibility to HpaNoco2. The insertion in acid11-2 did not affect HpaNoco2 resistance; this T-DNA located in the promoter of ACID11 did not to significantly reduce ACID11 expression. Neither the acid16-2 nor acid16-3 line displayed a defense phenotype. The insertions in these two mutants were intronic and might be spliced out of the ACID16 pre-mRNA . In contrast, the insertion in acid16-1, which is located in an exon, caused enhanced susceptibility to HpaNoco2. The insertion in the promoter in acid5-2, but not the exonic insertion in acid5-1, resulted in a defense-related phenotype. One possible explanation for this is that the insertion in the promoter abolished proper expression of this gene, while the exonic insertion results in a truncated, but functional, protein. Alternately, the insertion loci predicted by TAIR may not be accurately annotated . Among the acid mutants lines that exhibited reduced basal defense phenotype, only acid4-1, acid4-2, acid6-1, acid6-2, acid7-1, acid7-2, acid8-1, acid13-1, acid13-2, acid15-1, and acid15-2 displayed reduced levels of the respective transcripts. The observation that some T-DNA insertions did not affect endogenous ACID levels was not entirely unexpected.

Several transcription factors were differentially expressed in this study. GO enrichment showed the molecular function GO term ‘DNA-binding transcription factor activity’ was significantly enriched. In addition to the MYB77 transcription factor gene described earlier, a GRAS transcription factor gene, HAM3, was DE in this study. GRAS transcription factors were previously found to play a role in berry development and ripening in grapes, tomato, and citrus. This transcription factor showed increased expression later in the season when fruit were grown on trifoliate root stock, suggesting the root stock influences its role in improved citrus fruit quality. The largest phenotypic differences seen in mature fruit grown on trifoliate compared to rough lemon root stock were in the levels of total soluble sugar and titratable acid in ripe fruit. The levels of sugars and acids and their ratio in fleshy fruits is one of the most important determinants of sensory traits such as taste and flavor. Two genes were identified as differentially expressed that could play a role in the accumulation of these compounds. Firstly, a P-type ATPase was DE in fruit growing on trees grafted onto trifoliate versus rough lemon.

This gene was down-regulated at time two,hydroponic nft system but upregulated at time three . Studies have proposed a number of ATPases as proton pumps that are responsible for organic acid accumulation in citrus fruit. The reduced expression of this ATPase gene later in the season in fruit grown on rough lemon root stocks could contribute to the lower accumulation of titratable acid levels in these fruits. This ATPase gene identified in this study was not identified in the previous citrus studies, but the regulation of acid accumulation is a complex, as can be seen in other fruits, such as papaya and apple. It is possible this is a graftinduced effect observed with these specific root stocks, which were not examined in the previous studies. Secondly, a homolog of Arabidopsis BETAFRUCT4 was down-regulated in fruit of trees grown on trifoliate root stock compared to rough lemon at time three . This gene encodes a vacuolar invertase. Decreased expression of vacuolar invertases has been associated with increased sucrose content and accelerated ripening. Interestingly, by using an antisense acid invertase gene in transgenictomato to reduce acid invertase activity, fruit displayed higher levels of sucrose, as well as smaller fruit. We see similar trends in sugar accumulation and alterations in fruit size in this study. Klann et al. suggested that the water influx that drives fruit expansion is closely related to the concentration of osmotically active soluble sugars and therefore, all genotypes accumulate water until they reach a similar threshold of soluble sugar concentration. This could also contribute to the increased size of fruit grown on rough lemon fruit compared to trifoliate root stocks.

This study did not identify any statistically significant differentially expressed miRNAs from our fruit small RNA seq data. Therefore, potential miRNAs that target DEGs were predicted. An in-house R-script was used to select for miRNA-mRNA interaction pairs with an expected negative correlation in gene expression. These pairs were identified for the ten genes described above. All ten miRNA genes and their target mRNAs were detected by qRT-PCR. Pearson correlation coefficient value between the relative expression level detected by qRT-PCR and by RNA-sequencing was highly significant with r = 0.94. Of the ten interaction pairs, eight followed expected fold changes between time points . Therefore, it is likely that these eight target mRNAs are being regulated to some extent by their respective miRNA. Only two pairs do not follow the expected inverse relationship between time points, suggesting those mRNAs are not being regulated post transcriptionally by their miRNAs. This has been observed in previous integrated miRNA-mRNA studies. There have also been reports of target genes having a negative or positive feedback regulation on their respective miRNA, which could be another explanation for the inconsistent correlations seen in this study.Citrus is now grown in more than 140 countries in tropical, subtropical and Mediterranean regions. It is one of the most economically important crops in the world.This reduces the juvenile phase, allowing for the trees to produce fruit many years earlier than would trees grown from seed. Due to the large variation in growing conditions and climate in the regions where citrus is grown, different citrus root stocks are required to improve yield and fruit quality in numerous diverse climates, as well as resist various pests and diseases.

Root stocks impart certain traits to the scion and the effects of root stocks can be large. The most significant impacts are on growth, vigor and yield, tree nutrition, stress resistance, and fruit quality. The root stock effects on various aspects of tree growth and fruit development are well documented, but the molecular mechanisms underlying most of these differences are unknown. Previous studies have shown changes in the transcriptome of various root stock genotypes, especially in response to biotic and abiotic stressors. These types of changes have been seen in Arabidopsis, corn, mulberry, tomato, and poplar. In citrus, gene expression profiling has been used to understand root stock effects and responses to biotic and abiotic factors. In another study, expression studies of leaves from mandarin grafted onto various root stocks were analyzed in order to explain root stock effects on the growth of scions. There is extremely limited tissue-specific transcriptome knowledge in citrus, especially for root tissue. A small number of studies have evaluated trifoliate, trifoliate hybrid, and mandarin root transcriptomes in response to citrus diseases, but these studies each assessed only one genotype. Only recently has an RNA-seq based approach been used to establish a reference transcriptome for citrus and of the 28 samples used in the study,nft channel only two were obtained from roots. The root samples collected for this study were sour orange and trifoliate genotypes, but samples were grouped by organ to perform differential expression and subsequent analyses. To our knowledge, there are no comparative studies of citrus root transcriptomes between genotypes. In plants, the root system is critical for plant growth and development. It serves the functions of anchorage, nutrient and water uptake, and is the main boundary between the plant and its soil environment. Root growth relies on a specific set of signals that involves hormone signaling, availability of nutrients and carbon supply. There is a large degree of genotypic variation in crop plant root systems that can influence the plants growth and production including root length, root density, root angle, lateral root number, and root:shoot ratio. These parameters can impact the plant’s size, tolerance to biotic and abiotic stressors, and ability to uptake water and nutrients. For this reason, grafting, a process which connects the roots of one plant to the scion of another, has been widely using in plant breeding programs in order to improve vigor, alter plant architecture, enhance tolerance to disease and abiotic stress, and contribute to the quality of crops. In citrus, root stocks are bred for a variety of traits that are imparted to scions, such as tree size, yield, tolerance to salt, cold, and drought, tolerance to various pests and diseases, and improved fruit quality. Many studies in citrus have been conducted to assess the impacts of genetically differing root stocks on these traits. However, the study of molecular mechanisms behind root attributes lags far behind above ground tissues in plants, especially in perennial crops. Understanding the genetics of how root systems develop, and the regulatory controls of these processes will help optimize the improvement of yield and quality in citrus. Root system length, growth, and architecture control the ability of plants to respond to various stress conditions. The development of the root system and its architecture is determined by genetic factors interacting with numerous environmental factors. Plants must adapt to their environment by controlling their physiological reactions and morphogenesis. This can create complex root system architectures. For example, different root types can produce lateral roots that significantly extend the elaborate root system and allow the plant to search the soil for water and nutrient-rich areas. The increase in lateral root formation allows plants to more easily uptake these essential molecules in order to survive in unfavorable conditions.

Root stock genotypes exhibiting higher abilities to adapt to stress and create more extensive root systems improve nutritional status and water uptake, which can increase marketable yield. A clear example of this was seen in grafted mini-watermelon and tomato plants. Studies in Arabidopsis, rice, and corn have identified several genes that influence root development and root system architecture. In citrus, transcriptomic studies have been performed to understand the effect of root stocks on growth and in response to cold, nutrient deficiency, and fungal inoculation. Besides these studies, little effort has been invested into studying the effects of citrus root stocks at the molecular level, especially genotype-specific effects and their relation to fruit quality. Plants have evolved to cope with a constantly changing environment, modifying the root system architecture in response to nutrient availability and soil microorganisms. This flexibility requires fine tuning of gene expression. Among the molecules that control root development, small RNAs play a vital role in regulating genes at the post transcriptional level in plants. The most well-studied class of sRNAs are microRNAs , which are approximately 21 nucleotides in length and are produced from noncoding transcripts. Mature miRNAs have been shown to negatively regulate gene expression at the post-transcriptional level by specific binding and subsequent cleavage of their target mRNAs, or by the repression of target translation. Increasing evidence demonstrates that plant miRNAs play critical roles in almost all biological and metabolic processes. A review of miRNAs in roots indicated that they participate in root development, the modulation of root architecture, and root biotic interactions. This review focuses on numerous studies using Arabidopsis and legume plants as models. In citrus, miRNAs profiling has revealed their involvement in adaptation to nutrient deficiency, drought and salinity stress, and pathogen infection. However, no information, to date, is available about the role of miRNAs underlying differences in fruit quality observed between citrus root stocks. In the present study, trees grafted onto four root stocks were chosen from a root stock trial at the University of California, Riverside to assess for various fruit quality traits; Argentina sweet orange, Schaub rough lemon, Carrizo citrange, and Rich 16-6 trifoliate orange. Generally speaking, rough lemon root stocks produce the highest yield and fruit size, but this fruit is often of lower quality . Trifoliate orange root stock, when well adapted, produces high quality fruit, with high yield on smaller trees. Carrizo citrange root stocks produce intermediate yield with good fruit quality. Sweet orange root stocks produce good quality fruit but are very susceptible to various citrus diseases, and therefore are rarely used as root stocks for commercial trees. An RNA-seq approach was used to assess differences in gene expression between root stocks that produce fruit with varying quality traits with the aim of identifying genes that could potentially play a role in improvement of fruit quality. Moreover, miRNA expression profiles were obtained for each of the root stocks to identify potential regulatory mechanisms associated with their target genes. The grafted trees were part of a root stock trial conducted at the Citrus Research Center and Agricultural Experiment Station at the University of California, Riverside that included 28 root stocks. Trees were planted in 2011 in a randomized block design with ten replications. Trees were planted on berms, irrigated with mini sprinklers according to soil moisture sensors, and treated with fertilizers and pesticides according to standard commercial practices. Plastic mulch was used to cover the berms to suppress weeds and retain soil moisture. Trees were grown in a fine sandy loam and surface soil pH was 7.3 at the time of planting. It is important to note that no trees with Huanglongbing have been identified at UCR. For sequencing, roots from two biological replicate trees were harvested at four time points throughout the 2014-2015 growing season . Young, newly growing fibrous root tissue was collected from the first 6 inches of soil along the outer edge of the canopy.