Individual weed biomass for A. palmeri and D. sanguinalis, however, was lower for all weed densities when grown in the presence of sweet potato compared with weeds grown without sweet potato. The reduced individual biomass and biomass per meter of row for both weeds, when grown with sweet potato, indicate that interspecific interference is occurring between sweet potato and weeds. Crop biomass reductions are generally associated with increased weed competition and yield losses . However, in this study, although weed biomass was lower when grown with sweet potato, increased weed density did not reduce sweet potato biomass, despite the reduction in sweet potato yield at the same densities.Individual dry biomass of each weed species growing without sweet potato decreased as weed density increased . In the absence of sweet potato, individual dry biomass of both weeds was fit to a linear-plateau model. Individual weed biomass was greatest for both weeds at the lowest density. Amaranthus palmeri and D. sanguinalis individual plant biomass decreased 71% from 1 to 3 plants m−1 of row and 62% from 1 to 4 plants m−1 of row, respectively, and remained unchanged at densities above 4 plants m−1 row for both weeds . This finding was similar to the trend observed in peanut for A. palmeri. We believe that the reduction in individual weed biomass for A. palmeri and D. sanguinalis at lower weed densities when grown without sweet potato is due to increasing intraspecific competition as weed density increases. At the higher densities of both weeds,dutch bucket hydroponic the impact of intraspecific competition has limited effect on further decreasing individual weed biomass. The established threshold is the density at which all weeds achieve maximum accumulated biomass before intraspecific competition begins.

Further biomass increases would require densities resulting in weed mortality due to intraspecies competition, and such densities were not evaluated in this study. This study demonstrates that A. palmeri and D. sanguinalis have the ability to reduce yield at densities as low as 1 to 2 plants m−1 row. Sweetpotato competes with A. palmeri or D. sanguinalis, resulting in reduced weed biomass. This observation suggests that sweet potato with rapid canopy establishment and dense growth habit may provide additional competition with weeds and reduce yield loss, as proposed by Harrison and Jackson . Future studies should establish critical weed-free periods for these weeds in sweet potato, investigate competitiveness of resistant weed biotypes with sweet potato, and determine weed interference with sweet potato under varying management practices .The Salinas and Pajaro Valleys of coastal central California are among the most important lettuce-producing regions in the United States. One of the top disease concerns for lettuce in the area is Verticillium wilt caused by the fungus Verticillium dahliae, which is a soilborne pathogen with a wide host range that also includes artichoke, cotton, eggplant, hops, potato, sunflower, tobacco, and tomato. Two races of V. dahliae occur in coastal central California based on their differential virulence on cultivar La Brillante; however, race 1 is more prevalent and economically important than race 2. In tomato, race 1 of V. dahliae carries Ave1 that is recognized by Ve1 in resistant genotypes. Ve genes encode receptor-like proteins with extracellular leucine-rich repeats; such RLPs are widespread in land plants. In addition to Ve1, tomato also contains the closely linked paralog Ve2; their encoded RLPs work antagonistically to confer resistance to V. dahliae race 1. Several Ve paralogs also confer resistance in otherwise V. dahliae-susceptible species including cotton, potato, hops, and wild eggplant, but it is unknown whether they function analogously to the tomato Ve genes in conferring V. dahliae race 1resistance. In lettuce, resistance to V. dahliae race 1 was originally identified in the Batavia-type cultivar, La Brillante, as conferred by a single dominant locus located on chromosomal linkage group 9. The lettuce Vr1 locus contains several genes with sequence similarity to the Ve genes of tomato; it is very likely that one or more of these LsVe homologs are functional resistance genes.

The goals of this study were to identify the lettuce Ve allele that play a role in resistance to V. dahliae race 1 and to develop PCR-based assays for marker-assisted selection. For this purpose, we analyzed the genome sequences of cultivars La Brillante and the previously published Salinas. Subsequently, we sequenced and/or used allele-specific PCR screens of 150 additional lettuce accessions to identify the allele of the LsVe genes that are exclusively present in resistant phenotypes.Cultivar La Brillante is a Batavia type lettuce with a small, round head that is less dense than those of modern iceberg cultivars. Because of the certain phenotypic similarities in the shape of heads, fewer back crosses are usually needed to develop true to type iceberg cultivars when introgressing desirable genes from Batavia accessions than would be needed if those genes were introgressed from non-heading types of lettuces. Our current analyses showed that besides cultivar La Brillante, another Batavia cultivar can also be used for a relatively rapid development of iceberg cultivars with resistance to V. dahliae race 1. Both of these cultivars contain the same combination of LsVe alleles . Only two out of 36 romaine accessions were resistant to the disease in field experiments. One of the resistant accessions, cultivar Annapolis, is a dark red lettuce with a relatively small and light head that is usually grown for baby leaf production and is therefore harvested at early stages of development. The other resistant cultivar was Defender, which is green. Origin of resistance in this cultivar is unknown because it was developed through open pollination. A high frequency of resistance to the disease was found in Latin type accessions thatphenotypically resemble a small romaine lettuce with more pliable and oily leaves. Because of the phenotypic similarity between romaine and Latin types, Latin-type accessions may also be used for a relatively rapid development of romaine cultivars with resistance to V. dahliae race 1. Both romaine cultivars and three sequenced Latin cultivars that are resistant to the disease contain an identical combination of LsVe alleles . Substantially different frequencies of LsVe1L alleles and resistant phenotypes in different horticultural types of lettuce are not unexpected considering that comparable differences were previously described for other monogenically inherited traits, such as resistance to lettuce dieback and sensitivity to triforine.

Differences in the frequency of specific alleles among horticultural types are likely caused by the breeding approach that is used to develop lettuce cultivars. Only a few elite progenitors or founder lettuce cultivars have given rise to most of the modern commercial cultivars. Each of these progenitors is frequently found in pedigrees of cultivars of the same horticultural type. Additionally, new cultivars are mainly developed by recurrent breeding within small pools of closely related germplasm of the same type. Therefore, alleles present in an original progenitor of a certain type are found in high frequency in cultivars of the same type, but may be absent or present in low frequency in cultivars of other horticultural types. Our data are consistent with the LsVe1 gene identified in the cultivar La Brillante being involved in resistance to V. dahliae race 1 in lettuce. Among the 152 accessions included in this study, 21 were resistant to V. dahliae race 1 and all 21 contained the LsVe1L allele; this allele was not present in any of the susceptible accessions. The other La Brillante Ve alleles, LsVe3L and LsVe4L,blueberry grow pots were also present in all the resistant accessions, but they also occurred in two and twelve susceptible accessions, respectively. Therefore, LsVe1L is the strongest candidate as being required for resistance to V. dahliae race 1 in lettuce, although our data do not exclude LsVe3L or LsVe4L from also being involved similarly as in tomato. Complementation and knock-out studies are still required to determine the functional basis of LsVe-mediated resistance to V. dahliae race 1. The function and the significance of the differences between the LsVe1L and LsVe1S alleles remains to be investigated. The proteins encoded by LsVe1L and LsVe1S have the same domain organization, including the 37 extracellular, leucine-rich repeats separated by a short spacer region, as in previously characterized functional Ve proteins in other species. However, in addition to sequence diversity in the extracellular LRR domain, LsVe1L has an additional Cterminal transmembrane domain as compared to Ve1 and Ve2 in tomato, suggesting that maybe LsVe1L crosses the membrane three times and terminates with anon-cytoplasmic domain instead of a cytoplasmic domain. The distribution of disease incidence in susceptible accessions and across horticultural types indicates a possible presence of a modifying factor or factors that affect disease incidence. Our data do not exclude the possibility of interactions between two or more Ve genes, similar to those reported in tomato. A more detailed study of accessions with different frequencies of disease incidence and allelic compositions is needed to elucidate the basis of variation in disease incidence.Experiments were conducted in a field infested with V. dahliae race 1 located at the USDA-ARS station in Salinas, California. One hundred and fifty accessions were direct-seeded in a randomized complete block design with three replications. The original seed batches of previously sequenced cultivars Salinas and La Brillante were not available for field tests; therefore, seed batches used in field tests are shown as separate entries . Each plot was 7 m long and consisted of two seed lines on 1 m wide beds standard for lettuce production in coastal California.

Plant spacing was approximately 28 cm between seed lines and 30 cm between plants within a seed line. All field experiments were maintained using standard cultural practices for coastal California lettuce production.Unless indicated otherwise, ten plants from each plot were uprooted and visually evaluated. Disease incidence was assessed by cutting taproots longitudinally and recording the number of plants exhibiting the yellowish-brown discoloration of root vascular tissues that is typical of Verticillium wilt. Absence of V. dahliae race 1 in cultivars with race 1-resistant genotype was confirmed by plating surface-sterilized symptomatic root tissue on NP-10 semi-selective agar medium and PCR screening any resulting isolates with Ave1-specific primers. Three additional experiments were performed in the same field to confirm phenotypic observations. These experiments comprised only a subset of accessions that were either symptomless in the first experiment or were used as susceptible checks. Disease incidence values from all four experiments were combined and used for statistical analyses with JMP 14.2 .Intracellular transport of plant viruses during infection operates via unique interactions between viral proteins and selected host cytoskeletal and membrane elements. Microtubules and/or actin filaments are required for host cytoskeleton functions that are involved in virus replication and movement . Virus infections often result in modification of these systems and mechanisms affecting these processes have become special topics of enquiry over the past decade . For example, the roles of cellular remodeling in formation of replication factories and intracellular movement of several viruses has been reviewed recently . Since these reviews, the Potato virus X triple gene block 1 movement protein has been shown to remodel actin and endomembranes to form X-bodies that function in replication, and to recruit the TGB2 and TGB3 proteins to the X-bodies . Also, the endoplasmic reticulum and Golgi apparatus have been shown to be extensively remodeled as a consequence of Turnip mosaic virus infection . Modified actin filaments and the endomembrane system are involved intracellular and intercellular movement of several plant viruses . The plasma membrane and ER are continuous between cells, and form desmotubule conduits for intercellular movement of macromolecules between adjacentcells that are regulated by actin filaments, myosin-motor associations, and/or poorly understood membrane flow mechanisms . Virus studies over the past decade have shown that plasmodesmata connections forming cellular symplast boundaries are remarkably plastic and are involved in numerous complex interactions required for virus transit . Considerable variation is now apparent in mechanisms of virus intercellular movement, and this is most evident in viruses in which nucleoprotein complexes move through enlarged PD, versus viruses in which remodeled tubular PD function in cell-to-cell transit of whole virions . All plant viruses encode movement proteins that function in cell-to-cell movement, but these proteins and the pathways involved in movement vary enormously in their complexity and host component interactions . The most intensively studied MPs are Tobacco mosaic virus 30K protein, TGB proteins encoded by hordeiviruses and potexviruses, the Closterovirus movement complex proteins, the Nepovirus 2B protein, and the P6 Cauliflower mosaic virus . Each of these MP complexes interact in a variety of ways with endomembrane components, cytoskeleton networks and PD .

Hydroponics can promote faster growth in plants compared to traditional soil-based cultivation for several reasons:

1. Optimized Nutrient Delivery: In hydroponics, plants receive a carefully balanced and readily available nutrient solution directly to their roots. The nutrient solution contains all the essential elements plants need for growth, eliminating the need for them to search and extract nutrients from the soil. This allows plants to access nutrients more efficiently, leading to faster growth rates.
2. Enhanced Oxygenation: Hydroponic systems provide excellent oxygenation to plant roots. The absence of soil allows for increased oxygen levels in the root zone, ensuring better respiration and nutrient uptake. This oxygen-rich environment promotes robust root development and faster nutrient absorption, contributing to accelerated plant growth.
3. Controlled Environmental Conditions: Hydroponic systems allow growers to have precise control over environmental factors such as temperature, humidity, light intensity, and photoperiod. By optimizing these conditions, growers can create an ideal environment for plants to grow, maximizing their growth potential and accelerating their development.
4. Efficient Water Usage: Hydroponics is known for its efficient water usage. The use of closed-loop systems and recirculating nutrient solutions significantly reduces water consumption compared to traditional soil-based farming. With less energy spent on searching for water, plants can allocate more resources towards growth and development.
5. Reduced Competition and Disease Risk: In soil-based systems, plants often compete for limited resources, such as water and nutrients. In hydroponics, plants are grown individually or in controlled groups, eliminating competition. This reduces stress on plants and allows them to focus their energy on growth. Additionally, the absence of soil can minimize the risk of soil-borne diseases, providing a healthier environment for plant growth.
6. Faster Germination and Root Development: With precise control over environmental conditions, hydroponics can create an optimal environment for seed germination. The controlled moisture, temperature, and oxygen levels can lead to faster and more uniform germination rates. Additionally, the absence of physical barriers like soil allows for unimpeded root growth, facilitating faster establishment and nutrient uptake.
7. Year-round Production: Hydroponic grow systems can be set up indoors or in greenhouses, enabling year-round cultivation regardless of external weather conditions. With consistent and favorable growing conditions available throughout the year, plants can grow continuously without seasonal limitations, resulting in faster overall growth rates.

It’s important to note that while hydroponics can promote faster growth, it requires careful monitoring, proper system setup, and nutrient management to ensure optimal plant health and productivity.

Growing vegetables in an NFT (Nutrient Film Technique) hydroponic system can be an efficient and productive method. NFT growing systems provide a continuous flow of nutrient-rich water to the plant roots, promoting optimal nutrient uptake and water efficiency. Here’s an overview of growing hydroponic vegetables using an NFT system:

1. System Design: Plan the layout and design of your NFT hydroponic system based on the space available and the number of plants you intend to grow. Consider factors such as the size of the system, the number of channels, the distance between channels, and the overall capacity.
2. Channels and Growing Troughs: Select appropriate channels and growing troughs for your NFT system. PVC pipes or specially designed NFT channels are commonly used. Ensure the channels have a slight downward slope (around 1:30) to allow a thin film of nutrient solution to flow over the plant roots.
3. Growing Medium: In NFT systems, a growing medium is typically not used. Instead, the roots of the plants come into direct contact with the thin film of nutrient solution flowing through the channels, maximizing nutrient absorption.
4. Nutrient Solution: Develop a balanced nutrient solution specific to the vegetables you are growing. The nutrient solution should contain essential macro and micronutrients. Monitor and adjust the pH, electrical conductivity (EC), and nutrient levels of the solution regularly to meet the plants’ needs.
5. Pump and Reservoir: Install a submersible pump in a nutrient solution reservoir. The pump will circulate the nutrient solution through the channels, providing a thin film of solution over the roots. Ensure the pump operates consistently and efficiently.
6. Irrigation Schedule: Set up a timer to control the irrigation cycles in your NFT system. The nutrient solution should flow continuously but at a slow rate. Aim for a flow rate that provides a thin film of solution over the roots without causing excessive waterlogging.
7. Lighting: Provide adequate lighting to support vegetable growth. If growing in an indoor or low-light environment, supplement with high-quality, full-spectrum LED grow lights. Adjust the light intensity and duration based on the specific light requirements of the vegetables you are growing.
8. Temperature and Humidity Control: Maintain optimal environmental conditions within the growing area. Most vegetables thrive at temperatures between 65-80°F (18-27°C) during the day and slightly cooler at night. Control humidity levels to prevent disease development.
9. Pruning and Training: Depending on the type of vegetables you are growing, pruning and training may be necessary to manage growth and maximize productivity. Remove excess foliage or suckers to redirect the plant’s energy toward fruit or vegetable production. Provide support, such as trellises or stakes,vertical hydroponic nft system for climbing or vining vegetables.
10. Pest and Disease Management: Implement an integrated pest management (IPM) program to monitor and control pests and diseases. Regularly inspect plants for common pests such as aphids, whiteflies, or spider mites, as well as diseases like powdery mildew or fungal infections. Use biological controls, organic insecticides, or fungicides as needed.
11. Harvesting: Monitor your plants regularly and harvest vegetables at the appropriate stage of maturity for optimal flavor and quality. Harvesting in a timely manner also encourages continuous production.

Regularly monitor the nutrient solution levels, pH, and EC in your NFT system. Ensure the system is operating properly, and make adjustments as needed. With proper management, an NFT hydroponic system can provide an efficient and controlled environment for growing a variety of vegetables with high yields and quality harvests.

Growing hydroponic tomatoes can be a rewarding and efficient way to produce fresh, high-quality tomatoes. Here are the general steps to grow hydroponic tomatoes:

1. Choose a Hydroponic System: There are different types of hydroponic systems you can use for growing tomatoes, such as deep water culture (DWC), nutrient film technique (NFT), or drip irrigation systems. Select a system that suits your space, budget, and skill level.
2. Set up the Growing Environment: Create an indoor or controlled environment with appropriate temperature, humidity, and lighting conditions for tomato growth. Tomatoes require around 8-12 hours of direct light per day, so consider using high-intensity grow lights if natural light is insufficient.
3. Select Tomato Varieties: Choose tomato varieties that are well-suited for hydroponic cultivation. Look for varieties that are known for their compact growth, disease resistance, and high-yield potential.
4. Start with Seeds or Seedlings: You can start with tomato seeds or purchase seedlings from a reputable source. If starting from seeds, germinate them in a suitable growing medium, such as Rockwool cubes or peat pellets.
5. Transplanting: Once the seedlings have developed a strong root system and several true leaves, transplant them into the hydroponic system. Place them in net pots or growing media that allow the roots to come into contact with the nutrient solution.
6. Nutrient Solution: Prepare a well-balanced nutrient solution suitable for tomato plants. Monitor and adjust the nutrient levels regularly to ensure the plants receive the necessary macro and micronutrients for healthy growth. Follow the instructions provided by the nutrient solution manufacturer.
7. Watering and Feeding: Depending on the hydroponic system, you’ll need to regularly provide the plants with the nutrient solution. Ensure that the roots have constant access to oxygen by maintaining appropriate water levels in the system.
8. Training and Pruning: As the tomato plants grow, train them by gently tying the main stem to a stake or trellis to provide support. Remove any suckers that develop in the leaf axils to encourage better airflow and direct energy towards fruit production.
9. Pollination: Tomatoes are self-pollinating, but in an indoor environment, you may need to assist with pollination. Gently shake the plants or use a small brush to transfer pollen between flowers.
10. Harvesting: Harvest the tomatoes when they have reached the desired size, color, and ripeness. The harvesting time varies depending on the tomato variety.

Remember to regularly monitor the pH and nutrient levels in the hydroponic system, maintain appropriate lighting and temperature, and provide adequate airflow to prevent diseases. With proper care and attention, you can enjoy a bountiful harvest of hydroponic tomatoes.

An indoor NFT (Nutrient Film Technique) hydroponics system is a great option for growing plants in a controlled environment, such as a grow room or indoor garden. Here’s a general guide to setting up an indoor NFT hydroponics system:

1. Choose the location: Select an indoor space that provides adequate lighting, temperature control, and ventilation. Ensure there is enough room for the NFT system, including space for the grow bed, NFT channels, and access for maintenance.
2. Set up the grow bed: Install the grow bed at an elevated position, such as a table or raised platform. Ensure it is stable and can support the weight of the plants, grow medium, and water.
3. Install the NFT channels: Place the NFT channels or gutters on top of the grow bed, ensuring they are level and positioned to allow proper water flow. You can use PVC or food-grade plastic channels, and connect them securely to maintain stability.
4. Set up the water pump and reservoir: Position the water pump in a water reservoir or container. Submerge the pump to ensure it is completely covered in the nutrient solution. Connect the pump to the NFT channels using tubing and fittings, allowing the nutrient-rich water to flow through the system.
5. Choose a growing medium: Select a suitable growing medium for your NFT system, such as rockwool cubes, grow plugs, or coco coir. Place the growing medium in the NFT channels, ensuring it supports the plant roots and allows the nutrient solution to flow through.
6. Planting: Plant your chosen crops in the growing medium, placing the root system directly in the NFT channels. Ensure the plants are securely positioned and the roots have access to the flowing nutrient solution.
7. Nutrient solution and pH management: Mix the appropriate hydroponic nutrient solution according to the needs of your plants. Monitor and adjust the pH level to the optimal range for the specific crops you are growing. Regularly check and maintain the nutrient solution levels in the reservoir to ensure proper nutrient delivery.
8. Lighting: Provide adequate lighting for your indoor NFT system. Depending on the plants you are growing, you may use natural sunlight, LED grow lights, or other types of horticultural lighting systems. Position the lights at an appropriate distance from the plants to ensure proper coverage and intensity.
9. Climate control and ventilation: Maintain suitable temperature and humidity levels in the indoor space. Use fans, air conditioners, humidifiers, or dehumidifiers to regulate the environment and provide optimal growing conditions for your plants. Proper ventilation is essential for air exchange and preventing excessive heat or humidity buildup.
10. Maintenance and monitoring: Regularly check the NFT channels for any clogs, obstructions, or nutrient solution flow issues. Prune and maintain the plants as they grow, removing any dead or overcrowded growth. Monitor the overall health of the plants, including leaf color, growth rate, and signs of pests or diseases.

Remember to research the specific requirements of the plants you are growing, as different crops may have different needs regarding light, temperature, and nutrient levels. Regular maintenance and monitoring, as well as proper nutrient and pH management, are key to the success of your indoor NFT hydroponics system.

However, the concentration of Liberibacter DNA has to be high enough to allow for confirmation by conventional PCR followed by cloning and sequencing. By the time titers are high enough in plants for this, the infection has already spread to adjacent trees—especially if Asian Citrus Psyllid are present. This scenario is completely inadequate if the California citrus industry is to keep HLB out of groves. Several early detection techniques are being evaluated by the California Citrus Research Board. These include both direct measurement of elicitor proteins produced by the bacterium and the detection of small interfering and messenger RNAs, proteins, metabolites, and volatile organic compounds produced by the citrus tree in response to infection. Many of these approaches measure systemic responses. This holds a huge advantage over any PCR technique which is significantly limited by sampling since Liberibacter are not uniformly distributed in an infected tree. Nevertheless, a PCR protocol is urgently needed to validate these early detection methods that would provide acceptable evidence of their efficacy to federal and state regulatory agencies. Droplet digital polymerase chain reaction amplification is such a protocol. The technique utilizes the same qPCR primers and probes and amplified products can be isolated, cloned, and sequenced from ddPCR as with conventional PCR. We have evaluated 87 transect/risk-based survey site samples from around the Hacienda Heights positive with ddPCR that have been evaluated by several of the early detection protocols. Correlation of ddPCR results with those of these methodologies will determine which protocol show the most promise for detecting HLB early enough in the infection process to make tree removal, and therefore elimination of inoculum, feasible.

Huanglongbing in Florida is caused by Candidatus Liberibacter asiaticus , a phloem-limited fastidious α-proteobacterium,10 liter drainage pot which is transmitted by Asian citrus psyllids . HLB is causing an unprecedented crisis for citrus industry in Florida and poses a severe threat to citrus production in California, Texas, and Arizona. Currently, no effective HLB management is available. We aim to control HLB by targeting critical traits of Las to break down its infection cycle. Interestingly, Las contains the General Secretory Pathway , which is important for the viability and secretion of putative Sec dependent effectors of Las. SecA, an ATPase, is vital for the function of the Sec pathway and a good target to develop antimicrobials. We have identified multiple SecA inhibitors with high antibacterial activity to Liberibacters and their relatives. We will represent our recent progress in controlling HLB using SecA inhibitors and other antimicrobial compounds. In addition, our study indicates Las contains a functional salicylic acid hydroxylase which breaks down SA and its derivatives. SA and its derivatives play a central role in plant defenses, e.g., systemic acquired resistance , and are exogenously applied on plants as SAR inducers to control plant diseases. Here, we will present our recent progress in controlling HLB by nullifying SA hydroxylase of Las. Finally, breaking down the interactions of virulence factors and their targets in planta has been suggested to be one strategy to generate disease resistant plants. We will present our recent progress in identifying SDEs and their putative targets. We aim to generate HLB resistant or tolerant plants by disrupting the interaction between SDEs and their targets in citrus. Conventional PCR and real-time PCR methods had been developed and systematically validated for detection of citrus Huanglongbing before the disease was detected and confirmed in Miami, Florida, in 2005. Since then, the two rapid and sensitive multiplex qPCR assays have been used to screen field samples for HLB as part of an on-going survey conducted by state, industry, and/or USDA-APHIS regional laboratories. The two screening qPCR assays can detect all three known species of HLB bacteria: ‘Ca. L. asiaticus’ , ‘Ca. L. africanus’, and ‘Ca. L. americanus’. Since 2005, over 200,000 samples of HLB host plants and Asian citrus psyllids collected mainly in Florida, Texas, South Carolina, Louisiana, Georgia, and California have been tested using the screening qPCR assays. Confirmatory tests which include three multiplex qPCR , three cPCR assays , and sequence analysis of the cPCR amplicons are done exclusively at USDA CPHST Beltsville laboratory on samples that test suspect positive in the screening qPCR assays.

The first regulatory detections of HLB-Las by the states using the screening assays were confirmed by the federal confirmatory assays in new quarantine areas. We combined the two screening qPCR assays and validated them as a single assay . The performance of the new combined assay remained unchanged; however, the cost was reduced by half. We have supplemented our confirmatory assays by developing and validating two additional qPCR assays that target two different conserved genes of Las. Although the current qPCR assay is at least 100-fold more sensitive than cPCR, which in 1996 proved useful for pre-symptomatic detection of the disease, in the future we will evaluate several emerging technologies and/or platforms such as digital PCR and digital sequencing for advanced HLB diagnosis. Huanglongbing , associated with Candidatus Liberibacter asiaticus , is the most devastating disease of citrus and threating the citrus industry in Florida. Early root infection has been suggested to play a central role in HLB disease development and of significance to tree health. Therefore, introduction of beneficial bacteria to roots to promote root health might be an alternative approach to management of HLB. Our recent investigations showed that three beneficial Bacillus and closely relative isolates were able to promote citrus plant development with stronger root systems and delay the development of HLB symptoms and Las populations in greenhouse assays. Here, we characterized the rhizosphere competence of these three isolates in both greenhouse and natural environment. Using culture-dependent and – independent approaches, bacterial populations of these isolates on roots of citrus and in rhizospheric soil were determined following soil inoculation. The bacterial populations on the roots of citrus and in soil one month after inoculation were approximately 5.0 × 104 CFU/g and 5.0 × 103 CFU/g , respectively, for the three isolates. The assays revealed a similarity in rhizosphere competence with survival rates ranging from approximately 0.05% to 0.5% for the three isolates. Further analysis revealed that the rhizosphere competence of these isolates may be associated with various phenotypic traits, including substrate utilization, nitrogen dissimilation, siderophore mediated iron acquisition, stress tolerance, copper resistance, and the production of antimicrobial substances.Globalization has increased long-distance human-mediated pathways for invasive disease introduction. Detection of initial introductions of exotic pathogens/pests is challenging because they occur in very low incidence. Optimal probability of eradication/mitigation depends on early detection prior to spread. The earlier the detection, the more likely the pathogen can be eliminated or the epidemic slowed,plant pot with drainage lessening impact over multiple years. To find point introductions across a broad geographic landscape of mixed agricultural/residential areas requires substantial manpower and fiscal resources. Point introductions often go undetected for prolonged periods until incidence exceeds the lower threshold of sampling sensitivity. The Census/Travel model utilizes probable pathways, parses regions into smaller areas , and predicts the most likely locations in a given geographic area for introduction. The model’s geospatial method uses US census and international travel data combined with a pathosystem’s epidemiological characteristics, i.e., latency; detection sensitivity, reliability of confirmation, reproductive rate, environmental suitability, dispersal rate, ease of control, etc. Combining existing foreign population habitat and international pathway data, the model generates a risk index map to identify locations with the highest introduction potential. The risk map is linked to a survey optimizer that calculates the number of samples to be taken in a given area based on risk, and estimates manpower and fiscal requirements. It also ranks foreign countries by their relative contribution to risk of disease introduction. Risk maps were generated for HLB and other pathogens/pests. For prior disease introductions in Florida with identified distributions, the model performed well and validated known points of introduction. The census/travel model is being integrated into existing risk-based model platforms to optimize early detection surveys in California, Texas, Arizona, in Florida to optimize disease intervention/control.

The model is independent of pathosystem, and can be extend to all States to predict introductions of human, animal, or plant diseases/pests. Citrus Health Management Areas facilitate the coordinated control of ACP populations, the clean-up of abandoned groves, and the removal of infected trees. Growers cooperating within a CHMA have been effective in suppressing ACP populations and slowing the spread of HLB in FL, indicating CHMAs are a viable management strategy. It has been confirmed that ACP populations are decreasing where coordinated spray efforts have been implemented in commercial groves in FL. However, the boundaries of CHMAs in FL are not optimized in size, but constructed primarily on arbitrary boundaries with an attempt to combine resources and garner neighbor participation. There are other various relevant factors, such as urban population size, abandoned groves’ acreage, ratio between commercial citrus and residential area, will have a significant contribution to CHMA performance. Improving from CHMA design deficiency in FL, we intend to construct CHMA boundaries for Central Valley based on estimated HLB/ACP risk level under 1-mile2 grid resolution. Where mixed landscapes exist, an optimal mix of residential and commercial landscape is considered so that regional disease management decisions can be implemented more effectively. Through cluster analysis and spatial statistics, we have developed maps that organize plantings based on the spatial pattern and dynamics of ACP populations and HLB risk. K-means clustering method is preferred to construct CHMA as homogenous as possible for risk. The CHMA size and locations are also optimized for cost-effective management. Through thousands of simulations and optimization, 28 CHMAs are currently proposed for California Central Valley, which explains more than 55% of the total risk variance. For super large CHMAs, sub-CHMAs can be further constructed with approximate balanced between acreage and ownership. A similar methodology can be applied for CHMA construction in other citrus producing areas or states .Citrus Huanglongbing , spread by the psyllid vector, is a devastating disease threatening nearly every citrus producing area worldwide with the exception of Australia and Mediterranean countries. The recent finds of ACP in California central valley emphasize the urgency for regulatory intervention and disease control, as this would pose a major threat to the viability of the citrus industry. Increasing ACP incidence and risk are considered inevitable for the Central Valley. A spatial explicit ACP simulation model is developed with attempt to understand the dynamic of ACP population changes, in particular, how ACP spread in a mixture of residential and commercial citrus landscapes in the Central Valley. A 16 mile2 area of Porterville, CA is used as the baseline for ACP spread simulation. The mathematical model considers the following parameters for ACP progression: psyllid life span and mortality, net reproduction rate in natural environment, psyllid dispersal distance, citrus host type and density, new flush production, and effect of different spray schemes.We then modeled factors influencing ACP population variation and interaction with new flush availability in relation to temperature for a period of 3 years using a daily time step. ACP spread occurs more frequently and faster within commercial citrus clusters, but comparatively slower for low density or well separated residential areas. This study also evaluates the behavior and distribution of ACP by subjected to different spray strategy scenarios. A comparison between simulation outputs confirms that the synchronize rate for coordinated spray plays an important role in slowing ACP epidemic development. Besides justifying the benefit of large-scale disease management, the outcome of this simulation model can also quantify the influence of input epidemiological parameters on ACP development, and can assist management decision-making by running specified scenario-based analyses. Powerful computational tools have enabled us to learn/extract patterns of residential host plant distribution. To a large extent, residential citrus biodiversity/choice is influenced by both the physical environment and preferences of household for specific host types. An understanding of social preferences for dooryard citrus tree is critical to residential citrus host density mapping and modeling. Dooryard citrus preferences are heterogeneous and far from random, where certain factors may be able to explain part of the variation in preference. In addition to local climatic and environmental factors, we postulate that a range of demographic and socioeconomic characteristics can also affect the residential preferences for citrus types.

L-PEACH has been implemented using the general-purpose plant modeling software. This makes it possible to keep the model code compact by delegating generic issues to the general functionality of. As a result, L-PEACH is easy to maintain and conducive to simulated experimentation. Furthermore, it may serve as a template for constructing other functional–structural models that involve solving complex systems of equations in growing plant structures. In a comprehensive review of carbon-based tree growth models, Le Roux et al. pointed out that three critical issues have not been adequately addressed by most models: adequate representation of the dynamic and feedback aspects of carbon allocation on tree structure and carbon acquisition; explicit treatment of carbon storage reserves and remobilization over multiple years; and integration of below ground processes and tree water and nutrient economies into whole-plant function. Although the work is far from complete, the L-PEACH model provides a platform for addressing all these critical issues. By combining the sink-driven carbon partitioning concepts of the original PEACH model into a distributed network of architecturally explicit sources and sinks, factors such as the proximity of individual sinks to other sinks and sources, as well as the transport resistances between these entities, can be accounted for and become involved in growth and carbon allocation outcomes. However, although there are some experimental data to indicate the functional nature of these relationships , more data will be required before the model is fully calibrated. Considerable conceptual experimental research will be necessary to provide quantitative data required for this calibration. This model also explicitly addresses carbohydrate storage in stems and roots during the growing season,growing blueberries in pots and remobilization of stored carbohydrates during the spring growth flush.

In the process of developing this model, we became increasingly aware of the lack of information about the quantitative dynamics of carbohydrate reserves in trees. As pointed out by Le Roux et al. , the lack of knowledge of the mechanisms driving reserve deposition and remobilization is a major obstacle for evaluating the carbon available at any given time, or for relating reserve dynamics with internal and external variables in tree growth models. Based on preliminary data on root starch concentrations in peach trees we have chosen to treat the starch reserve sinks in stems and root segments as compartments that have sink capacities proportional to their annual growth increment. These reserves then become carbon sources during the spring flush . Current model functions related to carbohydrate reserves are based on preliminary data, and it is our intention to test more fully and quantify these aspects of the model in the near future. The inclusion of the water stress/interaction component in the model is an attempt to demonstrate how root function can be incorporated into a dynamic L-system model of this type. As with carbon storage, the relationships between developing water stress and physiology are based more on published conceptual relationships than on precise quantitative data collected for the purpose of calibrating the model. Nevertheless, the potential of this model to simulate functional interactions between root and shoot processes is readily apparent. Similarly, there is clearly the potential to incorporate additional root processes such as nutrient uptake into the model to more fully capture the functional dynamics of root– shoot interactions. Future developments could also involve the integration of existing architecturally based models of carbon transport and partitioning in roots , in order to model root function more explicitly.The Ranunculaceae is a large and complex plant family, including approximately 59 genera and 2,500 species . Pulsatilla Miller, first described in 1753, consists of about 40 species that are restricted to temperate subarctic and mountainous areas in the Northern Hemisphere .

Plants of Pulsatilla species are often covered with long, soft hairs. Their flowers are solitary and bisexual, with three bracts forming a bell-shaped involucre. The tepal number is always six, and stamens are generally numerous, with the outermost ones resembling degenerated petals . Most authors have treated Pulsatilla as a subgenus or section of the genus Anemone s.l. . However, Miller , Adanson , and Wang et al. have supported a model that separates Pulsatilla from Anemone as an independent genus. Recent phylogenetic studies have shown that all species within Pulsatilla are clustered in a monophyletic group, which is nested within Anemone . Morphologically, Pulsatilla can easily be distinguished from Anemone s.s., since species of the former have a long, plumose beak on the achenes formed by the persistent style and stamens whereas species of the latter do not. Because the primary goal of the present study is to test the use of DNA barcodes for species in the Pulsatilla clade, we here follow the treatment of Wang et al. and Grey-Wilson , regarding Pulsatilla as a distinct genus. There are eleven species of Pulsatilla found in China, most of which are found primarily in the northern part of the country . Some species of Pulsatilla have been used in traditional Chinese medicine for many years for “blood-cooling” or “detoxification” . In particular, the root of Pulsatilla chinensis Regel is a well-known ingredient included in the Chinese Pharmacopoeia . Many species used in folk medicine have been found to contain pharmacologically useful chemical components, including those with anti-cancer and anti-inflammatory activities . The contents of these components differ in various species, resulting in different clinical pharmacological effects. Thus, in cases where target species can be easily confused with their close relatives, undesired species can be inadvertently collected, resulting in negative effects on drug efficacy and patient safety, as has been shown in other plant groups of medicinal importance in China . Pulsatilla is an especially challenging, complex group. In all treatments published to date, the genus has been treated as comprising two to four subgenera: subgenus Miyakea, which contains only one species, P. integrifolia; subgenus Kostyczewianae, which has only one species, located in Central Asia and northwestern China; subgenus Preonanthus, which includes six species; and the largest subgenus Pulsatilla, which comprises 29 species.

However, Pulsatilla shows a frustratingly complicated pattern of intrageneric morphological variability . The recognition and identification of wild Pulsatilla species based on traditional approaches is difficult due to transitional intraspecific morphological characteristics in many Pulsatilla species. For instance, P. turczaninovii and P. tenuiloba were considered to be two separate species that could be told apart by the number of pairs of lateral leaflets . After carefully checking specimens and population investigation, we found that the leaflet numbers of P. turczaninovii and P. tenuiloba are overlapping,drainage gutter and some individuals have both 4 and 5 pairs of lateral leaflets. Flowers nodding before anthesis is recorded as a diagnostic character of P. campanella, but this character was also found in P. ambigua, P. cernua and P. dahurica, and their flower colors show continuous transitional shades of blue . Thus, these characters are not reliable and make Pulsatilla difficult to identify. DNA barcoding aims to achieve rapid and accurate species recognition by sequencing short DNA sequences or a few small DNA regions . This technology was first developed to identify animal species; for example, Hebert et al. argued that “the mitochondrial gene cytochrome c oxidase I , can serve as the core of a global bio-identification system for animals”. Studies have continued to demonstrate that the COI gene fragment efficiently discriminates among animal species, including amphibians , birds , fish , and insects . In plants, however, frequent recombination and low mutation rates restrict the utility of mitochondrial barcode markers . The search for suitable candidates has therefore focused on chloroplast and nuclear DNA markers , although such markers are not always easy to amplify and sequence in all plant taxa using universal primers. Numerous studies have suggested that four standard barcodes — three from the chloroplast genome [the ribulose-bisphosphate/ carboxylase Large-subunit gene , the maturase-K gene , and the trnH-psbA intergenic spacer and the nuclear ribosomal internal transcribed spacers ] — should be used as core barcode markers for the molecular identification of plants . Significant progress has been made in DNA barcoding in plants . However, the discrimination of closely related species using only molecular data is still a major challenge in some genera . Morphological characters, including the shape of nutritive and reproductive organs, remain highly valuable for plant identification and studies of plant evolution . Micromorphological characters have been shown to have great value for species identification and systematics , and these have rarely been considered by previous barcode studies. However, the combination of morphological data and DNA barcodes may be essential for species discrimination, especially in closely related species . Previous molecular phylogenetic studies have included few species from the genus Pulsatilla . In a recent phylogenetic study of Pulsatilla, few species were from Asia and few individuals were collected for one species . Obtaining DNA barcode data from a dataset created by comprehensive sampling of a taxonomically difficult genus such as Pulsatilla should contribute to understanding the discriminatory potential of barcodes in morphologically complex clades. The establishment of an available barcoding system for Pulsatilla may also facilitate further utilization of these taxa, as well as further research into their taxonomy. In this study, four DNA barcode regions were assessed in 19 species of Pulsatilla. Approximately 50% of the accepted species of Pulsatilla found in Europe and the Americas were included, as were 90% of the species found in China.

Our objectives were to: test the effectiveness of common core DNA barcodes in Pulsatilla, evaluate the resolution of these four barcodes, and use 2- to 4-region combinations to correctly identify individuals. We also aimed to develop a protocol that could effectively discriminate among closely related species, primarily for species discrimination of medicinal plants. In addition, we added micro-morphological analyses of leaf tissue obtained using scanning electronic microscopy to reveal the taxonomic relationships among Pulsatilla.In total, 52 accessions representing 19 Pulsatilla species were involved in this study . This sample covered each of the three subgenera from Asia, Europe, and America. Nine samples were sourced from herbarium specimens, while 43 samples were newly collected. All samples were taxonomically identified using published floras, monographs, and references. In total, one to five individuals per species were sampled from different populations in the wild. Fresh leaves were dried in silica gel upon collection and the longitude, latitude, and altitude of each collection site were recorded using a GPS unit . Voucher specimens were stored in the Herbarium of Northwest A&F University and the US National Herbarium . Singleton species were only used as potential causes of failed discrimination, and were not included in the calculation of the identification success rate. Three members of Anemone, two of Clematis, one of Anemoclema, and one of Hepatica were selected as outgroups for tree-based analyses.In this study, the short DNA sequences ITS and trnH–psbA had the best performance in PCR amplification and sequencing among the four barcode markers . Moreover, successful sequencing rates for sequences ITS and trnH–psbA were over 90% for silica-dried samples but lower for herbarium specimens. These findings are consistent with many previous studies . In addition, the varying lengths of insertions/deletions found at the trnH–psbA loci for different species provide important phylogenetic information and species discrimination power . Thus, sequence alignments of this region must be performed with great care to avoid overestimating substitution events. The rbcL and matK genes are approximately 1,428 bp and 1,570 bp in length, respectively . The greatest problem with rbcL and matK was that it was difficult to amplify them from the degraded DNA isolated from old herbarium specimens, since the short lengths of remaining fragments hampered the extension phase of the PCR for these longer genes. Although some problems may be alleviated by using additional pairs of primers, the amplification and sequencing success rate of the old herbarium samples remained poor. Thus, we were not able to obtain all sequences for all herbarium samples.An ideal DNA barcode should be universal, reliable, cost effective, and show considerable discriminatory power. Because none of the proposed single-locus barcodes perfectly meets all these criteria.Multilocus barcodes can often improve the resolution rate of species identification . In the present study, when evaluated alone, the species resolutions based on tree-building for the three chloroplast regions rbcL, matK, and trnH-psbA were 48.78, 14.63, and 9.30%, respectively.

In fleshy fruits, soluble sugars, including sucrose, fructose, and glucose, are not only essential for fruit growth and development but also central to fruit quality. Fruit taste and flavor is closely related to the composition and concentration of sugars and their balance with acids. As the composition and concentration of sugars at fruit maturity is determined by metabolic and transport processes during fruit development, understanding these processes and their regulation is important for fruit quality improvement. At the center of sugar metabolism in sink cells is the Sucrose cycle, previously named the Sucrose–Sucrose cycle or the futile Sucrose recycle, which consists of the breakdown of sucrose by invertase and sucrose synthase, the phosphorylation of the resulting hexoses and the interconversion between hexose phosphates and UDP-glucose, and the re-synthesis of sucrose via sucrose-6-phosphate synthase and sucrose-6- phosphate phosphatase. This metabolic system connects sugar metabolism with many other metabolic pathways such as glycolysis and tricarboxylic acid cycle, starch synthesis, and cellulose synthesis, and its coordination with the sugar transport system on the tonoplast is expected to determine the partitioning of sugars between metabolism in the cytosol and accumulation in the vacuole. In fleshy fruits, the concentration and distribution of sugars in parenchyma cells are affected via this cycle by developmental processes and environmental factors. However,plastic pots for planting the biochemical regulation of the cycle and the associated transport system is not fully understood.

In apple and many other tree fruit species of the Rosaceae family, sorbitol is a primary end product of photosynthesis and a major phloem-translocated carbohydrate, accounting for 60–80% of the photosynthates produced in apple leaves and transported in the phloem. In source leaves, sorbitol is synthesized from glucose-6-phosphate in a two-step process: G6P is first converted to sorbitol-6-phosphate via aldose-6-phosphate reductase , then followed by dephosphorylation of S6P to sorbitol via S6P phosphatase. The loading of both sorbitol and sucrose into the companion cell-sieve element complex in the phloem is passive and symplastic in apple, but their phloem unloading in fruit involves an apoplastic step.Once released from the SE-CC complex of the phloem in apple fruit, sorbitol is taken up into the cytosol of parenchyma cells by plasma membrane-bound sorbitol transporters and then converted to fructose by sorbitol dehydrogenase ; sucrose is either directly taken up into parenchyma cells by sucrose transporters , or first converted to glucose and fructose by cell wall invertase and then transported into the parenchyma cells via hexose transporters. Compared with plants that transport and utilize only sucrose, such as Arabidopsis, tomato , and poplar , apple is unique in that both sorbitol and sucrose are transported in the phloem and are metabolized in sink organs. It is estimated that >80% of the total carbon flux goes through fructose in apple. Once taken up into parenchyma cells of fruit, both sorbitol and sucrose feed into the Sucrose cycle to meet the carbon requirement for fruit growth and development while excess carbon is converted to starch for storage in plastids or transported into vacuole by sugar transporters for accumulation. Although we have characterized the genes and proteins involved in sugar metabolism and accumulation in apple, it remains unclear how apple trees adjust the Sucrose cycle and the transport system in response to altered supply of sorbitol and sucrose from source leaves.

In transgenic apple trees with antisense suppression of A6PR, leaf sorbitol concentration is dramatically decreased, whereas sucrose concentration is significantly elevated in the source leaves, but neither leaf CO2 assimilation nor plant vegetative growth is altered. The decreased sorbitol synthesis leads to significant changes in the expression profile of key genes in leaf starch metabolism and many stress response genes. In addition to being a key metabolite in carbohydrate metabolism, sorbitol also acts as a signal regulating stamen development and pollen tube growth and resistance to Alternaria alternata in apple. In the shoot tips of the A6PR transgenic plants, both the activity and transcript level of SDH are downregulated, whereas those of sucrose synthase are upregulated in response to a lower sorbitol but higher sucrose supply. Teo et al.reported that fruit of the transgenic apple trees accumulated a higher level of glucose and lower levels of fructose and starch at maturity, but no significant difference was detected in the activity of key enzymes in sugar metabolism, CWINV, neutral invertase , fructokinase , hexokinase , or SPS between the transgenic lines and the untransformed control . Considering that antisense suppression of A6PR has drastically decreased leaf sorbitol level and increased sucrose level, leading to less sorbitol but more sucrose being transported in the phloem; and both transcript levels and activities of SDH and SUSY responded to the altered sorbitol and sucrose supply in the shoot tips of the transgenic plants, we predicted that the decreased supply of sorbitol and increased supply of sucrose would lead to down regulation of sorbitol metabolism and upregulation of sucrose metabolism in the transgenic fruit as well. The discrepancy between the data obtained by Teo at al. and our predicted responses on the activities of sucrosemetabolizing enzymes in the transgenic fruit has prompted us to re-evaluate sugar metabolism and accumulation in the fruit of these transgenic plants to better understand how the Sucrose cycle and the sugar transport system respond to an altered supply of sorbitol and sucrose.

Antisense suppression of A6PR significantly decreased sorbitol concentration but increased sucrose concentration while largely maintaining fructose and glucose concentrations in source leaves throughout fruit development in the two transgenic lines relative to the untransformed CK . Sorbitol concentration in the source leaves of antisense line A27 was decreased to ~70% initially and 13% at harvest of that detected in CK. For antisense line A04, sorbitol concentration was decreased to 32% initially and 10% at harvest of the CK level. By contrast, sucrose concentration in the source leaves of A27 and A04 was much higher than in CK throughout fruit development, with larger differences detected at later developmental stages . Concentrations of sorbitol and sucrose were also measured for source leaves, leaf petioles, and fruit pedicels at 75 days after bloom . Compared with CK, antisense lines A27 and A04 had lower concentration of sorbitol, higher concentration of sucrose, and lower molar ratio of sorbitol to sucrose in the source leaves,strawberries in a pot leaf petioles, and fruit pedicels. The abundance of sorbitol followed the order of source leaves > leaf petioles > fruit pedicels .Average fruit fresh weight did not differ significantly between the two antisense lines and CK during fruit development except for about a 10% lower value detected for A27 and A04 at 108 DAB and at harvest . Average fruit dry weight did not show any significant difference throughout fruit development . Dark respiration was ~1.5–1.9-fold higher in A27 and A04 fruits than in CK fruits between 40 and 108 DAB during fruit development, but no significant difference was detected at harvest . Fruit yield per tree was significantly lower in the two antisense lines than in CK, largely due to lower average fruit weight at harvest as fruit number per tree was not significantly different between the two antisense lines and CK .Suppression of sorbitol synthesis in source leaves led to a significant decrease in sorbitol concentration in the fruit of two antisense lines A27 and A04 throughout fruit development, particularly in A04 . However, sucrose concentration was similar in the fruits of the two antisense lines and CK during fruit development with a higher level detected in the transgenic fruit only at 74 DAB. Fructose concentration showed no difference between the transgenic fruit and CK except being slightly lower at 108 DAB in the transgenic fruit. Compared with CK, concentrations of glucose and galactose were much higher throughout fruit development, with larger differences detected at later developmental stages. Concentrations of G6P and fructose-6- phosphate decreased during fruit development and were significantly lower in A27 and A04 than in CK from40 to 108 DAB . At fruit maturity , total soluble solids concentration was significantly higher in A27 and A04 than in CK . Fruit starch concentration did not show obvious difference between the transgenic lines and CK before 74 DAB but was slightly lower in A27 and A04 than in CK after 108 DAB .SDH activity decreased during fruit development and was significantly lower in both A27 and A04 than in CK at each developmental stage . CWINV activity dropped dramatically from 40 to 74 DAB and then remained fairly constant to maturity, but no significant difference was detected between the two antisense lines and CK.

NINV activity decreased throughout fruit development and was ~1.5–2.0-fold higher in A27 and A04 than in CK from 74 DAB to fruit maturity . Vacuolar acid invertase activity showed no significant difference between the two antisense lines and CK except a slightly higher activity detected in A27 and A04 than in CK at 108 DAB. SUSY activity declined during fruit development and was significantly higher in A27 and A04 than in CK from 40 to 108 DAB. FK activity decreased during fruit development and was significantly lower in A27 and A04 than in CK at 40 and 74 DAB. HK activity decreased during fruit development and was significantly higher in both A27 and A04 than in CK from 74 to 134 DAB. SPS activity increased slightly from 40 to 108 DAB and then dramatically to fruit maturity, with a significantly lower activity detected in both A27 and A04 from 40 to 108 DAB.Our data clearly showed that sorbitol concentration was significantly lower, whereas sucrose concentration was significantly higher in the source leaves of 5-year-old transgenic “Greensleeves” apple trees with antisense suppression of A6PR compared with the untransformed CK throughout fruit development. These results are consistent with those reported for the 1-year-old transgenic trees. The higher sucrose concentration in the source leaves is an indication that a larger proportion of the photosynthetically fixed carbon ends up in sucrose over a 24-h period because most of the starch accumulated during the day breaks down for sucrose synthesis at night in the transgenic plants although no difference in the carbon flux to sucrose during the day was detected. As both sorbitol and sucrose diffuse into SE-CC complex from mesophyll cells via plasmodesmata,accumulation of a higher level of sucrose in leaves is expected to facilitate the transport of sucrose in the phloem when less sorbitol is translocated in the transgenic plants. The lower concentration of sorbitol and higher concentration of sucrose in both leaf petiole and fruit pedicel and a smaller ratio of sorbitol to sucrose indicate that significantly less sorbitol but much more sucrose is translocated from leaves to fruit in the transgenic trees, which is consistent with a lower sorbitol but a higher sucrose concentration in the phloem exudates collected from fruit pedicels of these plants. The total amount of carbon translocated to fruit is expected to be very similar between the transgenic lines and the CK because all the trees had a very similar cropload and no significant difference was detected in average fruit dry weight between the transgenic lines and the CK at fruit maturity . These data clearly demonstrate that, when sorbitol synthesis is decreased in the source leaves, more sucrose is synthesized in the leaves and translocated to the fruit, thereby largely maintaining fruit growth and development. This is also consistent with the homeostasis of vegetative growth observed in the transgenic lines. The transgenic trees with decreased sorbitol synthesis grown under our experimental conditions were only slightly smaller after 5 years of growth than those of the untransformed CK . This is consistent with comparable photosynthetic rates measured in the transgenic lines and the untransformed CK throughout the growing season , with the lower rates detected only at fruit harvest being largely related to the leaf brown spots caused by Alternaria alternata in the transgenic lines. However, Teo et al.found that the transgenic trees were much smaller than the CK trees. This discrepancy is likely due to differences in growing conditions between the two locations. As sorbitol is implicated in drought-stress tolerance in apple, these trees might have experienced more drought stress under warm and dry conditions in California than under cool and humid conditions in upstate New York.

Similar to the findings for BrpHMA2, our results suggest that these TF genes may respond to Cd. The coding sequences of the three NAC TFs and three AREB TFs listed above were cloned and submitted to the NCBI database. The last three or four numbers of each gene’s full name was used as the gene name. MEGA5 was used to create a phylogenetic tree of these NAC TF or AREB TF genes and Arabidopsis NAC or AREB genes using the neighbor-joining method. The results revealed that the BrpNAC4584 and BrpNAC895 sequences were closer to those of Arabidopsis ANAC046 and ANAC087, respectively ; in addition, the BrpABI227 and BrpABI678 sequences were closer to that of AtABF4, and the BrpABI449 sequence was more comparable to that of AtABF3 .Our results reveal that BrpHMA2 could be activated by Cd2+ , which is similar to the results found for HMA2 in Arabidopsis. Results suggest that BrpHMA2 is involved in the Cd response of plants. BrpHMA2 was also found to be expressed explicitly in the vascular tissues of roots, stems, leaves, flowers, siliques, and carpopodia, and its protein was localized in the plasma membrane . These results are consistent with previous findings for HMA2 in Arabidopsis, OsHMA2 in rice, and TaHMA2 in wheat. The protein plasma membrane localization and the vascular-specific expression pattern of the genes revealed that HMA2 might function as a membrane transporter in long-distance transport in plants. In recent years,best indoor vertical garden system some studies have investigated the function of HMA2. Most of these studies demonstrated that HMA2 is involved in Zn2+ and Cd2+ transmembrane transport and influences root-to-shoot Zn/Cd translocation.

For example, HMA2 in Arabidopsis is thought to be involved in the outward transport of Zn2+ and Cd2+ from the cell cytoplasm, and HMA2 mutants are more sensitive to Cd stress and exhibit higher Zn or Cd accumulation than wild-type plants in the presence of high levels of Zn2+ or Cd2+ 14,15. The over expression of OsHMA2 in wheat, rice, and Arabidopsis improves root-to-shoot Zn/Cd translocation. In addition, the transformation of TaHMA2 in yeast enhances the resistance of cells to Zn/Cd. In rice, the suppression of OsHMA2 decreases the Zn and Cd concentrations in leaves, increases the retention of Zn in roots and reduces the translocation of Cd and Zn from roots to shoots compared with the results obtained with wild type plants. According to the literature, HMA2 is responsible for Zn2+/Cd2+ efflux from cells, plays roles in Zn and Cd loading to the xylem, and participates in the root-to-shoot translocation of Zn/Cd. However, Yamaji et al. found that OsHMA2 is localized at the pericycle of the roots and in the phloem of enlarged and diffuse vascular bundles in the nodes. Their insertion lines of rice showed decreased concentrations of Zn and Cd in the upper nodes and reproductive organs. The study revealed that the heterologous expression of OsHMA2 in yeast is associated with the influx transport of Zn and Cd. These researchers suggested that OsHMA2in the nodes plays an important role in the preferential distribution of Zn and Cd through the phloem to the developing tissues. Our results also revealed that, in the presence of Cd2+, transgenic Arabidopsis seedlings and yeast over expressing BrpHMA2 showed higher concentrations of Cd and enhanced Cd2+ sensitivity compared with the controls . Thus, we propose that BrpHMA2 functions in Cd2+ transport in the phloem tissue of vascular systems through influx into cells, and the efflux from phloem cells during long-distance transport may be performed by other transporters. The differential function of HMA2 from various plants might come from the tiny difference in amino acids in their function domains; this puzzle requires further investigation.

In this study, we identified the NAC TF gene BrpNAC895, a homolog of Arabidopsis ANAC087 , which could be induced by Cd2+ stress . We confirmed that BrpNAC895 has a role in the response of B. parachinensis to Cd2+ stress by upregulating BrpHMA2 expression through direct binding to the BrpHMA2 promoter using EMSA, ChIP–qPCR, and the transient transformation method with B. parachinensis protoplasts . Previous studies have demonstrated that Arabidopsis ANAC087 is associated with plant programmed cell death . It functions along with the TF ANAC046 to show partial redundancy in coregulating the expression of some PCD genes in the root columella, including ZEN1, BFN1, and RNS3. Whether ANAC087 could participate in regulating Cd transporters in plants has not been reported. Our findings on BrpNAC895 show that this NAC TF has a novel role in upregulating BrpHMA2 expression in response to Cd2+ stress. We also identified the Cd-responsive AREB TF BrpABI 449 , which is a homolog of Arabidopsis ABF3 and can bind to the promoter of BrpHMA2 . ABF3 modulates the response to drought, salt, and other osmotic stresses as a master component in ABA signaling. This TF can also regulate the expression of multiple genes, such as the AGAMOUSlike MADS-box TF family gene SOC1, which is a floralintegrator regulating flowering in response to drought, and the AREB TF ABI5, which is a core component in the ABA signaling pathway in the regulation of seed germination and early seedling growth during exposure to ABA and abiotic stresses. In general, ABF3 can form protein complexes with other TFs. For example, ABF3 forms homodimers or heterodimers with AREB1/AREB2 and acts cooperatively to regulate ABRE dependent gene expression. ABF3 forms a complex with NF-YC3 to promote the expression of the SOC1 gene and thus accelerate flowering and drought-escape responses; ABF3 interacts with NAC072 to regulate RD29A and RD29B expression in response to ABA. Thus, complex formation might be the important functional mechanism by which ABF3 regulates gene transcription.

Using EMSAs and ChIP–qPCR assays, we found that BrpABI449 could directly bind to regions of the BrpHMA2 promoter . The interaction of BrpABI449 and BrpNAC895 was further confirmed by pull-down and BiFC assays . The inhibition of BrpABI449 on the transcriptional regulatory role of BrpNAC895 was detected in the B. parachinensis protoplast transient system . The inhibition by BrpABI449 of the transcriptional regulatory role of BrpNAC895 complex, likely interferes with BrpNAC895’s activity in the transcriptional activation of BrpHMA2 in response to Cd stress. It has also been reported that Cd stress can induce a stress response via ABA signaling. Our results showing that BrpNAC895 and BrpABI449 are upregulated by Cd stress also support this point. The uptake or homeostatic regulation of heavy metals needs proper modulation to ensure plant health. Previous studies have shown that Cd stress induces the MYB TF gene MYB49 in Arabidopsis. This TF may further positively regulate the downstream TF gene bHLH38 and bHLH101 by directly binding to their promoters, and activate iron-regulated transporter 1 to enhance Cduptake. In contrast,growing strawberries vertically Cd stress upregulates the expression of ABI5. ABI5 interacts with MYB49, prevents its binding to the promoters of downstream genes, and functions as a negative regulator to control Cd uptake and accumulation. Our present results also demonstrate a mechanism for controlling the expression of the heavy metal transporter gene BrpHMA2 under Cd stress. We propose that Cd2+ induces the expression of BrpNAC895 and BrpABI449, which might be mediated by ABA signaling. The activation of BrpHMA2 enhances Cd2+ uptake and may induce cell damage. Negative regulation of BrpHMA2 is then achieved by the upregulation of another AREB TF, BrpABI449, which interacts with BrpNAC895 and forms BrpNAC895-BrpABI449 protein complexes to inhibit the BrpHMA2 transcription activated by BrpNAC895 . BrpABI449 could also bind to the promoter of BrpHMA2 directly to compete with BrpNAC895 in binding to the BrpHMA2 promoter. This negative regulation may play a supplementary role in the uptake and transport of Cd.Many plant species of Brassicaceae, including Arabidopsis, turnip, and oil seed rape, can be genetically modified, but the creation of transgenic B. parachinensis remains difficult. Therefore, we over expressed BrpHMA2 in Arabidopsis to investigate the function of BrpHMA2 and established a transient transformation system in B. parachinensis protoplasts to perform gene regulatory network analysis. Protoplasts have been widely used for subcellular protein localization and gene regulation analyses. In this study, the transient transformation of B. parachinensis protoplasts was demonstrated to be a powerful system for ChIP–qPCR analysis. Previous studies have applied a similar approach to Populus trichocarpa and Brassica napus. Although the transient transformation system of B. parachinensis protoplasts was successfully used in this study of molecular mechanisms, the system cannot be easily used for phenotype and physiological analyses. The lack of BrpNAC895 and BrpABI449 transgenic B. parachinensis is a problem that severely limits research on this plant.

New techniques, such as the transient reprogramming of plant traits via the transfection of RNA based viral vectors using Agrobacterium and gene editing combined with fast-treated Agrobacterium coculture, may be useful approaches for comprehending gene function concerning physiology and for the further application of modifications of gene function to effectively control the accumulation of Cd in B. parachinensis.Reduced pod shattering is an important breeding target in many crops, including common bean . In the wild, many legumes benefit from seed dispersal mediated by explosive pod dehiscence, known as pod shattering. During the domestication process, the trait has been strongly reduced across most legume taxa . Despite this, some market classes of common bean have persistently high levels of pod shattering, leading to reduced yields and a constrained harvest window. This issue is particularly problematic in semiarid environments, which cause pods to become brittle and fracture more easily . Common bean is a vital source of protein and micro-nutrition for hundreds of millions of people globally . The crop was independently domesticated in Middle America and the Andes , leading to the species’ two major domesticated gene pools. These are additionally subdivided into several ecogeographic races, each with a long history of adaptation to specific environmental conditions . In particular, members of the Middle American ecogeographic race Durango are adapted to the semiarid highland environments of northern Mexico and the southwestern USA, whereas the Middle American raceMesoamerica inhabits humid lowland regions of Mexico, Central America and lowland South America. Useful alleles from any major gene pool can readily be moved into others, and crosses between races have major untapped potential for breeders . Seven independent domestication events occurred in the Phaseolus genus, including close relatives of common bean such as Lima bean , runner bean year bean and tepary bean . An improved genetic understanding of pod shattering in common bean will be useful for improvement of numerous other domesticated legumes that suffer from pod shattering . Several genes are known to influence resistance to pod shattering in common bean , and the genes involved vary by gene pool. In the Middle American domesticated beans, the locus Phaseolus vulgaris Pod dehiscence 1 on chromosome Pv03 is associated with a major reduction in pod shattering . The shattering resistance allele is found at high frequency in race Durango, but is nearly absent in market classes belonging to race Mesoamerica or the Andean gene pool . This is a major target for improvement in these classes. Orthologs of this Pv03 gene may also regulate pod shattering in other species, such as cowpea , chickpea and soybean , where the orthologous locus plays a role in adaptation to arid climates by modifying the extent of twisting in pod valves . A possible second locus on chromosome Pv08 in Middle American beans has been proposed to reduce pod shattering , but a relatively small sample size of these individuals has hindered the study of this allele. The Pv08 QTL is also believed to have a major effect in Andean beans, so a deeper investigation of this QTL could provide insight on whether it has evolved in parallel between domestication events. A recently discovered QTL on Pv05, in immediate vicinity of PvMYB26, is associated with a loss of dehiscence in the Andean gene pool . This locus was mapped in detail in a biparental recombinant inbred population , which also found significant QTLs on Pv04 and Pv09 in the same population. The role of the Pv05 and Pv09 loci were identified in parallel in a diversity panel of Andean beans , which also identified significant loci on Pv03 and Pv08. PvMYB26 was subsequently found to be differentially expressed between dehiscent and non-dehiscent individuals, leading to major differences in development of cell walls in the suture . Other loci, including St and To , control strong fiber development in pod sutures and pod walls , respectively, and the mutant variants are found only in snap beans grown as a vegetable. St has been mapped to Pv02, and To has been mapped to Pv04 .